INTERNATIONAL REVIEW OF CYTOLOGY V3, Volume 3 (v. 3)

INTERNATIONAL

REVIEW OF CYTOLOGY VOLUMEI11

This Page Intentionally Left Blank

INTERNATIONAL

Review of Cytology E D I T E D BY

G. H. BOURNE

J. F. DANIELLI

London Hospital Medical College London, England

Zoology Department King’s College London, England

VOLUME I11

Prepared Under the Auspices of The International Society for Cell Biology

ACADEMIC PRESS INC., PUBLISHERS NEW YORK

1954

Copyright 1954, by ACADEMIC PRESS INC. 125 East 23rd Street, New York 10, New York All Rights Resewed KO PART OF T H I S BOOK M A Y BE REPRODUCED I N A N Y FORM, B Y PHOTOSTAT, MICROFILM, OR A N Y OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM T H E PUBLISHERS.

Library of Congress Catalog Card Number (52-5203)

PRINTED IN THE UNITED STATES OF AMERICA

Contributors to Volume I11 MAX ALFERT,Departwent of Zoology, University of California at Berkeley.

G. ASBOE-HANSEN, Laboratory for Connective Tissue Research, University Institute of Medical Anatomy, Copenhagen, Denmark. R. A. BEATTY, Institute of Animal Genetics, Edinburgh, Scotltxnd.

J. BERTHET,Laboratory of Physiological Chemistry, University of Loumin, B e l g i w .

SVEN-OLOF BRATTGARD, Department of Histology, The University of Goteborg, Sweden. OTTOBUCIIER, Department of Histology and Embryology, University of Lausanne, Sm*tzerland. IVORCORNMAN, The George Wmhington University Cancer Clin&c and the Department of A+iatomy, School of Medicine, Washington, D . C.

CHR. L)E DUVE,Laboratory of Physiological Chemistry, University of Louzwin, Belgium.

EDWARD W. DEMPSEY, Department of Anatomy, Washington University School of Medicine, St. Louis, Missouri. ALEXANDER L. DOUNCE, Biochemistry Department, University of Rochester School of Medicine and Dentistry, Strong Memorial Hospital, Rochester, New York. TRYGCVE GUSTAFSON, The Wenner-Gren Institute for Experimental Biology, University of Stockholm, Sweden. HOLGER HYDEN,Department of Histology, The University of Goteborg, Sweden. ALBERT I . LANSING,Department of Anatomy, Washingtoil University School of Medicine, St. Louis, Missouri. A. G. EVERSON PEARSE, Post-graduate Medical School, Hammersmith, London, England.

CHARITYWAYMOUTH, Roscoe B. Jackson AIemorial Laboratory, Bar Harbor, Maine.

ROY G. WILLIAMS, Department of Anatomy, University of Pennsylvmkn, Philadelphia, Pennsylvania.

This Page Intentionally Left Blank

CONTENTS Contributors to Volume I11

................................................

V

The Nutrition of Animal Cells BY CHARITYWAYMOUTH. Roscoe B . Jackson Memorial Laboratory. Bar Harbor. Maine

I. I1. 111 I V. V VI . VII VIII . IX . X X I. XI1

. . .

. .

Introduction . . . . . . . . . . ...... Biological Media ....................... ...... Synthetic Media ...... ............................... Inorganic Substances ............................................... Carbohydrates and Oxygen ......................................... Amino Acids and Peptides .................. Purines, Pyrimidines, and Nucleic Acids ..... Lipids ...................................... Vitamins .......................................................... Hormones ......................................................... Concluding Remarks . . . . . . . . . . . . . . . . . References .........................................................

1 5 14 21

27 34 42 44 46 53 57 58

Caryometric Studies of Tissue Cultures BY O m BUCHER. Department of Histology and Embryology. University of Lansanne. Sw'taerland I . Introduction ....................................................... 69 I1. Experimental Material and Method of Evaluation .................... 70 111. Statistical Evaluation of Results .................................... 73 IVA . Discussion of Our Experimental Results............................. 93 V . Conclusions ........................................................ 108 V I . References ......................................................... 110

The Properties of Urethan Considered in Relation to Its Action on Mitosis BY IVOR CORNMAN. The George Washington University Cancer Clinic and the Department of Anatomy. School of Medicine. Washington. D . C. I. Introduction ........................................................ I1. Carcinogenic and Carcinoclastic Properties ............................ I11. Cytologic Effects .................................................. I V. Biochemistry ....................................................... V. Summary .......................................................... V I . References .......................................................

113 114

118 123 127 128

Composition and Structure of Giant Chromosomes BY MAX ALFERT.Department of Zoology. University of California at Berketey

I . Introduction

........................................................

131

IT. Recent Advances in Chromosome Chemistry and Structure ............ 132 111. The "Salivary" Chromosome ........................................ 136 IV . The Lanipbrush Chromosome ....................................... 154

.

V The Functional Significance of Giant Chromosomes ; General Discussion 161 VI . References ......................................................... 164

How Many Chromosomes in Mammalian Somatic Cells?

BY R . A . BEATN. Institute of Animal Genetics. Edifiburgh. Scotland I. Introduction ....................................................... I1. Chromosome Number in Germ Cells ................................. 111. Chromosome Number in Somatic Cells in Situ ....................... IV. Chromosome Number in Somatic Cells of Man in Tissue Culture ...... V. Discussion ......................................................... V I . Conclusions and Summary .......................................... VII . Addendum ......................................................... VIII . References .........................................................

177 178 182 183 186 194 195 1%

The Significance of Enzyme Studies on Isolated Cell Nuclei BY ALEXANDER L. DOUNCE.Biochemistry Department. University of Rochester School of Medicine and Dentistry. Strong Memorial Hospital. Rochester. N e w York

I . Introduction ....................................................... I1. Work of Mirsky and Collaborators on Cell Nuclei Isolated by a Modification of the Technique of Behrens .............................. 111. Work of Lang and Collaborators .................................... IV Recent Work of Hogeboom and Schneider on Synthesis of D P N by Nuclear Preparations ............................................. V . The Problem of Oxidative Enzymes in Cell Nuclei .................. VI General Discussion ................................................. VII Summary and Conclusions .......................................... VIII References .........................................................

.

. . .

199 202 208 212 213 218 221 221

The Use of Differential Centrifugation in the Study of Tissue Enzymes

.

BY CHR. DE DUVEA N D J BERTHET.Laboratory of Physiological Chemistry. University of Louvain. Belgium

. .

I Introduction ........................................................ 11. The Technique of Differential Centrifugation ......................... 111 Scope and Limitations of Differential Centrifugation as Revealed by Enzyme Distribution Studies ...................................... IV. Biological Evaluation of the Results of Tissue Fractionation Studies V . Summary and Conclusions .......................................... VI . References .........................................................

225 226 239 259 269 270

Enzymatic Aspects of Embryonic Differentiation BY TRYGGVE GDSTAFSON.The Wenner-Gren Institute for Experimental Biology. University of Stockholm. Sweden

I. Introduction ....................................................... I1. Analytic Data from the Developing Sea Urchin Egg ................. I11. Intracellular Localization of Enzymes in Eggs and Embryos .......... IV Factors Modifying the Enzyme Activity in Homogenates and in vivo V . Cofactors and the Control of Their Formation ........................ VI . On the Genesis of New Mitochondria in Embryonic Differentiation VII . The Primary Pattern of Mitochondria1 Distribution .................. VIII . The Metabolic Background of the Mitochondrial Distribution ......... IX. The Gradual Complication of the Mitochondrial Pattern .............. X . On Qualitative Biochemical Differentiation .......................... XI . The Mode of Operation of Mitochondria in Morphogenesis . . . . . . . . . . . . XI1. On the Mode of Action of Li Ions in the Developing Egg . . . . . . . . . . . . . . XI11. References .........................................................

.

277 277 279 284 291 295 301 305 309 311

311 316 320

Azo Dye Metliods in Enzyme Histochemistry

BY A . G . EVERSON PEARSE, Post-graduate Medical School. Hammersmith. London.. England

I. I1 I11 I V. V. VI . VII . VIII .

. .

Introduction ....................................................... Criteria for Azo Dye Methods ...................................... The Non-coupling Azo Dye Methods ................................ The Simultaneous Coupling Azo Dye Methods ....................... Effects Due to the Nature of the Diazotate .......................... The Post-coupling Azo Dye Methods ................................ Final Conclusions .................................................. References .........................................................

329 330 334 335 349 353 355 357

Microscopic Studies in Living Mammals with Transparent Chamber Methods

BY ROY G. WILLIAMS. B p a r t m e n t of Anatomy. U n i v e r d y o f Pertnsylmmics. Philadelphia. Pennsyimnia

. .

I I1 I11. IV . V. VI . VII . VIII. IX .

Introduction ....................................................... Construction and Installation of Chambers ........................... Blood Vessels ..................................................... Lymphatic Vessels and White Cells ................................. Other Tissues of the Ear ........................................... Grafts ............................................................. Tuberculosis ....................................................... Conclusion ......................................................... References .........................................................

359 360 364 371 373 376 393 394 394

T h e Mast Cell

BY G. ASBOE.HANSEN, Laboratory f o r Connective Tissue Research. Unizersity Institute of Medical Anatomy. Copenhagen. Denmark I. I1. I11. I V. V. V I. VII . VIII.

Introduction ........................................................ Origin ............................................................. Morphology ........................................................ Distribution ........................................................ Cytochernistry ...................................................... Function ........................................................... Physiologic' Variability ............................................. References .........................................................

399 400 402 405 415 422 4% 431

Elastic Tissue

BY EDWARD W . DEWPSEY A N D ALBERT I. LANSING.De,bartnrent of Anutomy.

.

Washington University School of Medicitte. S t Lor&. Missouri

I. II . 111. I V. V. VI .

Introduction ........................................................ Chemistry of Elastic Tissue ......................................... Staining Reactions and Histochemistry of Elastic Tissue .............. Intrafibrillar Architecture of Elastic Tissue ......................... Age Changes and Pathology of Elastic Fibers ........................ References .........................................................

437 438 442 445 451 452

The Composition of the Nerve CeIl Stu’died with New Methods

BY SVEN-OLOF B R A T ~ RAND D HOLCER HYD~N. Department The University of Goteborg. Sweden

of Histology.

I. Introduction ....................................................... I1. Mass Determination ................................................ I11. Quantitative Determination of Ribonucleic Acid in Individual Nerve Cells ............................................................. I V. Changes in the Neurons with Increasing Age and Intracellular Differentiation ......................................................... V. Chemical Changes Induced by Adequate Stimulation .................. VI . Summary .......................................................... VII . References ......................................................... AUTHOR INDEX

SUBJECT INDEX

........................................................ ..........................................................

455 455 467 469 472 474 474

477

496

The Nutrition of Animal Cells CHARITY WAYMOUTH* Roscoe B. Jackson Memorial Laboratory, B w Harbor, Maine

I. Introduction ................................ 11. Biological Media ........................... 111. Synthetic Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Inorganic Substances ...... ..................... VI. Amino Acids and Peptides

Page 1 5 14 21

....................

VIII. Lipids ............................................ IX. Vitamins .......................................... X. Hormones ................................. ...... ........... XI. Concluding Remarks ................................................ XII. References .........................................................

53 57

58

I. INTRODUCTION “Welche Bedeutung vom physiologischen Gesichtpunkt hat es doch z. B., dass wir den Nahrungsbedarf der verschiedenartigen Gewebszellen zu registrieren vermogen I” (Albert Fischer, 1941a).

Our present extensive knowledge of animal nutrition has been reached through investigations with utilitarian and practical economic motives. The available information about nutritional requirements at the tissue and cell levels is, by comparison, very limited. There are mechanisms in the animal body which assure to each tissue its proper nutritive requirements. Some of these mechanisms may be looked upon as relatively passive, in that the body fluids which reach the cells contain the appropriate nutrients ; others are more active, for example the selection of certain nutrients by particular cells or the accumulation of substances against a concentration gradient. The passive mechanisms, by definition, depend purely on the

* On leave of absence from the Chester Beatty Research Institute, Royal Cancer Hospital, London, England. This work has been supported, in London, by grants to the Royal Cancer Hospital and Chester Beatty Research Institute from the British Empire Cancer Campaign, the Jane Coffin Childs Memorial Fund for Medical Research, the Anna Fuller Fund and the National Cancer Institute of the National Institutes of Health, U.S. Public Health Service; and in Bar Harbor, by the American Cancer Society, Inc., through a British-American Exchange Fellowship awarded on the recommendation of the Committee on Growth of the National Research Council. 1

2

CHARITY WAYMOUTH

environment ; on the other hand, certain specific activities of different cell types can modify the immediate environment to satisfy special nutritional demands and can also (by enzymatic activity or by secretion) produce materials which exert their effects in distant parts of the organism. Because modification of the environment is an important function of most cells, the result is an enormously intricate interdependence of every part of the organism nutritionally, as well as in the general physiological sense. On account of this tissue interdependence, the nutrition of isolated colonies of cells from animal tissues has certain complexities which are not met with in the nutrition of higher animals or of bacteria or free-living animal microorganisms. Microorganisms exhibit a wider range of powers of synthesis of complex from simple components than isolated, differentiated tissues with specialized metabolic functions. The value of protozoa in the investigation of animd nutrition has been pointed out by Kidder (1952), who has been a major contributor in this field with his studies on Tetrahynzenu. But, as Kidder implies, the nutrition of this independent unicellular organism, in which there is no division of labor, is to be contrasted on the one hand with multicellular organisms where such a partition of function exists, and on the other with the individual cells of the metazoan organism with their strong mutual interdependence. As Carrel (1938) has said : “the living body is a heterogeneous spatiotemporal continuum of cells and fluids” in which “neither cells nor tissues are mere cellular aggregates.” Careful adaptation of the environment has been shown (Sanford, Earle, and Likely, 1948; Likely, Sanford, and EarIe, 1952) to be necessary, to produce conditions in which a single isolated metazoan cell (in contrast to a fragment of tissue or a population of cells) can survive and multiply i 7 ~vitro. It is of particular interest to know whether the nutritional needs of different tissues for growth and repair differ from those for the maintenance of normal function and to study the possible special nutritive demands of neoplastic cells in comparison with those of normal cells. Quantitative and qualitative differences in nutritive pattern between tissues within an animal are difficult to study in the intact organism, even though it is evident that such differences exist (cf. Spratt, 1950). One of the aims of nutritional investigations at the cellular level, as Haddow (1947) has stressed, is to attempt to disclose these differences by the study of isolated cell colonies and ultimately to integrate the information for the better understanding of the whole process of nutrition in maintenance, development, and growth, and in neoplasia. Much unnecessary confusion exists in the field of cell nutrition because criteria for nutritional adequacy have not been clearly enough defined.

T H E NUTRITION OF ANIMAL CELLS

3

When cells can proliferate uninterruptedly and be carried through a large number of subcultures, the medium in which this is possible must be sufficient in every sense. Dilute fowl serum is nutritionally complete in this respect for fowl macrophages (Carrel and Ebeling, 1922a ; Jacoby, 1937a ; 1938, 1940, 1941, 1945, 1949). For similar, long-continued cultivation of embryonic fibroblasts in a state of active proliferation, a mixture of fowl plasma and chick embryo extract is the standard medium, But dilute fowl serum (or heparinized plasma) alone is not sufficient in the same sense for fibroblasts (Carrel and Ebeling, 1923b, e, f ) , though it permits their survival for a year or more (Parker, 1936a). I t is convenient to make a distinction between nutritional conditions sufficient for the maintenance of cells in a healthy state for more or less prolonged periods ; and the additional conditions, presupposing the former, required for proliferative growth. Such a distinction is useful in assessing the value of individual nutritive substances and of synthetic nutrient mixtures. The most promising method of designing media of known composition is first to devise a medium in which the cells can survive. When the medium is adequate for prolonged survival of cells, the additional conditions which will permit growth can be recognized. This empirical distinction is important because of the seeming paradox that growth stimulation can reduce survival. I t can do this because, if the cells are stimulated to multiply in a nutritionally deficient environment, they will the more rapidly exhaust it. Because the cell and its environment form a changing and a reciprocating system, the problem of defining nutritional adequacy cannot be simply solved by making a neat classification of nutrient substances into welldefined groups according to the needs of a particular tissue for them. Knight (1945) has stated the position for the nutrition of microorganisms, and it can equally well be said of the nutrition of animal cells that “a given substance, required as a compunent of one of the essential metabolic processes, might appear in three different roles . . . (1) as an ‘essential nutrient’, when its rate of synthesis by the cell was so slow as to be insignificant; (2) as a growth stimulant, when its rate of synthesis was somewhat faster but still slow enough to be a limiting factor; or ( 3 ) as a substance not required at ail for nutrition, because the cell could synthesize it so fast that it was not a limiting factor in growth. I t is the metabolic process which is the essential thing.” It has been repeatedly pointed out by numerous authors (eg. Hueper et al., 1933; Cunningham and Kirk, 1942; Fischer, 1946a; Tompkins, Cunningham, and Kirk, 1947; Waymouth, 1949) that the methods most commonly used to measure “growth” in cell populations iH vitro are unsatisfactory. I t is still necessary to emphasize that measurements of

4

CHARITY WAYMOUTH

superficial area of fibroblast cultures can only be used in carefully selected conditions and with an understanding of the relative contributions to the total area made by cell migration and by cell division (cf. also Katzenstein and Knake, 1931). The application of chick embGo extract to fibroblast cultures, as Willmer and Jacoby (1936) recognized, promotes both activities. It was still more clearly demonstrated by Doljanski and Goldhaber (1945) that these two functions are experimentally separable. They irradiated fibroblast cultures with a dose of X-rays such that all mitotic activity was inhibited. Application of embryo extract to these cultures induced cell migration not significantly less in extent than in unirradiated cultures in which mitosis was taking place. B’ecause criteria of growth or nutritional effectiveness are more often than not unsatisfactory, the reader must treat critically the results reviewed below. Some of them are based on assessments of survival, some on measurements of outgrowth, and others on stimulation of mitosis. In many cases the observations were made over very short periods of time (e.g., 48 hours). When it is realized that cells can remain morphologically unaltered and can even undergo mitosis in simple salt solutions during such periods, it will be clear that little of positive value can be learned about cell nutrition from these short-term tests. They are useful only in the negative sense that they demonstrate that the nutrient tested has no toxic effect and is therefore worthy of more extensive study in longer experiments. The longer the cells are kept in an artificial environment, the less will they be able to “live on their reserves,” and the more important does it then become to supply not only the major nutrients but also the trace substances for which a deficiency may not develop or manifest itself for several weeks. The objective for those who would understand the nutrition of individual tissues or their component cells is to reproduce, in chemically defined terms, an environment sufficiently close to the normal “passive” environment for the cells that they can modify it to fulfil completely their peculiar nutritional requirements. The need to develop nutrient media of known chemical composition for animal cells has been recognized for a long time (Lewis and Lewis, 1911a, b). Baker’s (1929) early experiments led her to hope “that it might be possible to synthetize an artificial medium which would prove adequate for the maintenance of cell life and multiplication.” A fully defined nutrient medium was still hardly regarded as a practical possibility when Willmer and Kendal (1932) wrote : “The future of tissue culture as an experimental method depends very largely upon whether a more complete knowledge can be obtained of the requirements of the cells. The goal at which to aim is a completely synthetic medium whose every

THE NUTXITION OF ANIMAL CELLS

5

constituent is under control. This is probably an ideal rather than a possibility.” But only a little later Willmer (1935) had apparently conceded the possibility, though he felt that “there is still . . . a very long road to be travelled before it will be possible to produce a synthetic environment for tissue-culture cells.” The ideal has not yet been fully realized, but with the rapid extension of our knowledge of cellular metabolism, the possibility is now accepted, the practicability of the project has increased, and the desirability of its achievement has by no means diminished. Our present understanding of the nutrition of cells has been derived partly from analysis of media of biological origin which are nutritionally complete, partly from the study of the effects of known substances added to more or less complete biological media, and partly from experience with partly or wholly synthetic media designed on the basis of known biochemical and nutritional principles. It is the intention, in this review, to assemble what is known of the general and special nutritive requirements of animal cells, with particular reference to the nutrition of colonies of cells in tissue culture. The physiology and nutrition of cells in culture have previously been dealt with in the following reviews, books or articles : Anon. (1947, 1949, 1950) ; Cameron (1950) ; Carleton (1923) ; Carrel (1924a) ; Fischer (1930, 1933, 1940, 1941b, 1946a, b, 1947, 1948c) ; Lewis and Lewis (1925) ; Morgan ( 1950) ; Vogelaar and Erlichman (1933) ; Waymouth (1950, 1952) ; White (1946, 1947, 1950) ; Willmer (1928, 1935, 1945), and Winnick (1952). 11. BIOLOGICAL MEDIA Just as the science of bacteriology was developed by the empirical use of meat infusions, the techniques for cultivation of cells from the tissues of higher organisms have been based on the use of biological fluids as culture media. From the use of lymph by Harrison (1907) and plasma by Burrows (1910, 1911 ; Carrel and Burrows, 1911a) for relatively short-term studies of living cells in vitrro, the finding of Carrel (191 1, 1912a, b, c, 1913a, b, c) that the addition of an extract of chick embryo to the plasma clot wouId permit continuous proliferation of chick fibroblasts for long periods, tempted many to use this new tool to study living, growing cells. The fact that cells, in such a medium, could be forced into a high rate of proliferation proved so fascinating that it was sometimes forgotten [though not by Carrel (1938) himself] that cells perform many functions other than reproduction and that surviving cells in an artificial environment are a valuable experimental material. Because of the strong emphasis upon proliferative growth, the information that has been gathered on nutritional requirements of cells in tissue culture pertains very largely to actively growing cells. As has been pointed out elsewhere, in a discussion of stimulation of mitosis

6

CHARITY WAYMOUTH

in relation to cell nutrition, “it appears . . . to be axiomatic that, for cell division to be possible, adequate nutritional conditions for supplying energy and materials must first be fulfilled” (Waymouth, 1952). That it was desirable to attempt to distinguish between substances required for cell nutrition per se and those for cell multiplication, was recognized by Baker and Carrel (1926a, b, e), who showed that chick embryo extract was an excellent source of both kinds of substance. Heaton (1926) and Hueper et al. (1933), for convenience in studying the properties of biological media, further emphasized that different conditions are necessary for survival, for cell multiplication, and for synthesis of protoplasm, and that each cell type has its special nutritive requirements. One of the earliest students of cell physiology (Loeb, 1912) had stressed the point that variations in internal and external environment could separately affect survival, growth, or movement of the cell. Many variations have been made on the classic biological medium of chicken plasma and chick embryo extract, but it remains true that a great variety of cell types can be grown more or less successfully in this mixture. Growth-promoting power is not confined to homologous media. Heterologous plasmas were used by Volpino ( 1910) ; Lambert and Hanes (191 1) ; Lambert (1912) ; Ingebrigtsen (1912a, b) ; Champy and Coca (1914) ; Fischer (1924, 1929), and Chlopin (1930), and it is now common practice to do so. Extracts of chick .embryos were shown to promote growth of duck (Fischer, 1924 ; Kiaer, 1925), rat (Mottram, 1927), and rahhit, guinea pig, and human tissues (Fischer, 1941b). Chick tissues were grown in extracts of rabbit embryos (Carrel and Ebeling, 1923d ; Landsteiner and Parker, 1940). Amphibian tissues have been grown in media composed wholly of chick plasma and embryo extract (Hughes and Preston, 1949 ; Danes, 1949). Bovine embryo extracts have been used for human (Gey and Gey, 1936), rat (Lewis, 1935, 1939), and chick (Fischer, 1941b) tissues. These early demonstrations of the nonspecies-specificity of growth-promoting media established the now general use of heterologous components in tissue-culture media. The strain L mouse cells of Sanford, Earle, and Likely (1948) have been maintained continuously for 11 years in a medium of horse serum and chick embryo extract. The plasma or serum component of the medium has usually been derived from adult animals. The belief became general, after it had been shown to be SO for the fowl, that serum becomes progressively more and more inhibitory with the age of the donor (Carrel and Ebeling, 1921a, b ; 1922b; 1923a, c ; Baker and Carrel, 1926a, c, d, 1927), and therefore the serum of young adult animals has generally been used. There is little more recent

THE NUTRITION O F ANIMAL CELLS

7

comparative information, but a widespread impression that serum or plasma from a young animal, or from the fetus, provides a more favorable medium for cell growth than the corresponding adult serum. There is no clear evidence for inhibition with increasing age of donor in the case of horse serum (Earle, personal communication). Fetal plasma or serum, and especially human umbilical cord serum (Gey and Gey, 1936), is much used, particularly in media for the growth of tumor cells. Some clues to the nutritional demands of growing cell colonies may be found by comparing the chemical differences between adult and fetal sera, where the latter have been shown to be more effective in promoting growth. As examples (others will be noted later, under the different classes of nutrients), it is now well known that fructose occurs in large amounts in the fetal blood of many species (Bacon and Bell, 1948; Barklay et aE., 1949; Hitchcock, 1949, and Goodwin, 1952). The amount of inositol in fetal plasma is higher than in the adult (Nixon, 1952) ; fetal rabbit plasma has a higher bicarbonate and a lower chloride content than maternal plasma (Young, 1952). It has long been supposed that human umbilical cord serum had a higher amino-N content than maternal serum (Morse, 1917), and similar differences were reported for other species before Christensen (1948) and Christensen and Streicher (1948), using more reliable methods, demonstrated a ratio of 1.5 to 2.0 for the fetal : maternal amino acids in human and rabbit plasmas, and a ratio of about 5 for the guinea pig. The ratio for human plasma was confirmed by Crumpler, Dent, and Lindan (1950), who reported a range of 1.03 to 3.00 (fetal :maternal amino acids) in nine cases, with a mean of 1.60. Schreier and Steig (1950) showed that the amounts of individual amino acids in human cord serum varied from 23.7% higher (leucine) to 182.1% higher (lysine) than in adult human serum. The amount of glutamic acid is much increased in fetal blood (average 9.5 mg./100 ml.) compared with normal adult (average 1.0 ( 9 ) and 1.2 ( b ) ) and maternal (4.3) (White, Beaton, and McHenry, 1952). The growth-promoting effects produced by tissue extracts vary with age, even within the embryonic period (Gaillard, 1935, 1942 ; Gaillard and Varossieau, 1938 ; Miszurski, 1939). Adult tissue extracts have, however, been used with varying degrees of success, mostly for short-term experiments (Carrel, 1913a ; Heaton, 1926 ; Hoffman, Goldschmidt, and Doljanski, 1937; Trowel1 and Willmer, 1939; Doljanski and Hoffman, 1939, 1943; Hoffman and Doljanski, 1939; Hoffman, Tenenbaum, and Doljanski, 1939a, b, 1940; Hoffman, 1940; Doljanski, Hoffman, and Tenenbaum, 1939, 1942 ; Hoffman, Dingwall, and Andrus, 1948, 1951; Margoliash and Doljanski, 1950). Leucocytes are another source of growth-promoting extracts (Carrel, 1922, 1924b, c, 1927), and these cells

8

CHARITY WAYMOUTH

were believed to be important in the nutrition of other types of cell (Carrel and Ebeling, 1922c, 1926b, 1928; Fischer, 1925a, b) and to be able to make and secrete “trephones” (Carrel and Ebeling, 1923g, h ; Carrel, 1924b, c) which could act as intermediaries for the nutrition of more differentiated cells (des Ligneris, 1931). Other biological fluids which have been employed at various times in tissue culture media include peritoneal exudate (Baitsell and Sherwood, 1925), allantoic fluid (Moppett, 1927), amniotic fluid (Grossfeld, 1949; Enders, 1953) or a mixture of amniotic and allantoic fluids (Szarski, 1950, 1951), and aqueous humor. (Albrink and Wallace, 1951). A particularly effective biological fluid is ascitic fluid from patients with carcinomatosis peritonei (Bergman and Waterman, 1935; Ivers and Pomerat, 1947; Ivers, Pomerat, and Neidhardt, 1948; Ulloa-Gregori et ab., 1950; Pomerat, Nowinski, and Rose, 1950; de Lustig, 1951 ; Ellis, Nowinski, and Bieri, 1953). Serum ultrafiltrates, introduced by Simms (1936; Simms and Stillman, 1937) for use with adult tissues, are often employed. Chemical analyses have been attempted on few of the medium components except plasma or serum and embryonic extracts. A general survey has recently been made (Waymouth, 1952) of the history of the use of embryonic and adult tissue extracts and of leucocyte extracts as stimulants to cell proliferation. The discussion here will be confined to the attempts which have been made to analyze the commoner medium components. During the period 1920 to 1940, varioys analyses of embryo extracts, using methods which by present-day standards were somewhat crude, were made by Carrel, Baker, and Ebeling and by Fischer. The influence of Carrel in the field of tissue culture at that time led to the general acceptance of his view (Carrel, 1924a) that the whole of the growth-promoting activity of his medium for fibroblasts resided in the embryo extract. Plasma was regarded as an almost inert substratum. This view appeared to be borne out by the later observations of Carrel and Ebeling (1923b, e) ; Carrel (1928) ; Fischer and Parker (1929) ; Olivo (1931) and Parker (1933, 1936b) that, in a medium of diluted serum, fibroblast cultures could be kept in a state of prolonged survival with little or no increase of tissue, whereas addition of embryo extract produced an immediate proliferative response. Olivo’s (1931) cultures doubled in mass in a period of six months ; Carrel (1928) maintained chick embryo heart cultures, with persistent pulsation, for 104 days in a Ringer-washed plasma clot. Addition of embryo extract then induced active proliferation. Carrel (1928) maintained from this experiment that serum has no nutritive function for fibroblasts and that chick embryo extract is a “complete food.” At this time he also recorded that “although attempts at frac-

THE NUTRITION OF AWIMAL CELLS

9

tionation have been made with all possible techniques, no part has been isolated which was endowed with a greater activating power than the whole.” However, whereas in 1926 Carrel and Ebeling (1926a) had stated that “fibroblasts do not feed on plasma, egg albumin, egg yolk, amino acids or broth. They synthetize protoplasm exclusively from substances contained in the juice of chick, mouse, guinea pig and rabbit embryos,” by 1938 Baker had come to the conclusion that embryo juice alone was unable to furnish the substances necessary for normal fibroblast growth, but that serum was needed to provide additional nutriment for continued culture. The result of the twenty-year period of attempts to isolate active “growth hormones” from embryo extracts was summarized by Fischer (1941b) in the discouraging words that “it made practically no difference how an extract was treated, its activity was lowered anyhow.” While it is lamentably true that analysis of biological media by chemical methods, and the attempts to attribute special growth-promoting activity to individual chemical constituents, have not added very greatly to our knowledge of cell nutrition, a somewhat greater measure of success has attended the use of physical methods. By these methods (dialysis, ultrafiltration and ultracentrifugation) it has been possible to eliminate some parts of the complex biological mixture. Fractionation of biological media by dialysis or ultrafiltration has a long history. Wright (1926), by dialyzing embryo extract against a modified Pannett and Compton saline solution, obtained a protein-f ree dialyzate (i.e., a dialyzate in the modern, not the original, sense; vide Pirie, 1947) which, in a medium containing whole plasma, promoted cell division in cultures of chick heart fibroblasts. On the other hand, Baker and Carrel (1926e) reported that dialysis of embryo extract against water, using “very permeable collodion sacks” and adjusting the salt concentration and p H before use, did not entirely remove the growth-promoting activity of the extract. They found, however, that an ultrafiltrate only slightly increased the area of outgrowth of cultures, and permitted survival no longer than Tyrode solution alone. They concluded that “The growthpromoting substances which distinguish embryonic juice from other fluids in its capacity to maintain the life of fibroblasts and epithelial cells indefinitely in vitro are not to be found among its dialyzable components.’’ Jacoby ( 1937b) reached essentially the same conclusion, using dialysis against Tyrode solution and measuring both areas of migration and mitoses in the cultures. Tazima (194Oa, b) , who dialyzed both the embryo extract and the plasma against Tyrode solution, reported that dialyzed extract gave very poor growth of cultures in their second passage; the dialyzate, with whole plasma, gave better growth than the plasma diluted

10

CHARITY WAYMOUTH

with Tyrode solution. Outgrowth of fibroblasts was less in whole embryo extract and dialyzed plasma than in the complete medium, but cultures could be maintained for up to at least 15 passages. Iris epithelium could be carried through 24 passages (Kimura, 1938; Tazima, 1940a). Dialysis of adult chicken heart extract against Tyrode solution was carried out by Margoliash, Tenenbaum, and Doljanski (1948). When fresh heart extract was used in the medium, the growth-promoting effect (area measurement) was greatly reduced by dialysis, and negligible growth-promoting activity was found in the dialyzate. Dialysis of extracts of acetone-dried heart also reduced the growth-promoting power ; in this case the dialyzate was not completely without activity, and complete restoration of activity was achieved by recombination of the dialyzed extract and its dialyzate. Either fraction taken alone is evidently nutritionally deficient. Dialysis of plasma and embryo extract against a Ringer-glucose solution is the basis of Fischer’s approach to the analysis of growth media (Fischer, 1941a, b, 1942a, b ; 1946b, 1947, 19&, b, c, d ; Fischer and Astrup, 1942, 1943; Astrup, Fischer, and Volkert, 1945;Astrup, Fischer and Phlenschlager, 1947; Astrup and Fischer, 1946; Astrup, Ehrensvard, et al,, 1947; Fischer, Astrup, et d., 1948). The underlying hypothesis is that, by dialysis, low molecular weight substances are removed from the classic nutrients, leaving unaffected the high molecular “growth-promoting f actors” or “Embryonin.” The principle active factor in “embryonin” is believed to be a labile nucleoprotein or phosphoprotein with catalytic properties. The deficiency of low molecular “accessory growth factors’’ is made up by supplementation of the dialyzed medium with known compounds. Fischer has been able, by this method, to demonstrate the importance of amino acids and peptides for the survival of chick fibroblasts. Supplementation of the medium of dialyzed pIasma and embryo extract with trypsin-digested serum produced a medium in which growth could take place: further digestion with acid (i.e., to amino acids) and restoration of tryptophan was sufficient for maintenance only (Fischer, 1941a). Similarly, supplementation with synthetic mixtures of amino acids maintained the cells alive but by no means restored full growth. Boiled kidney extract (Fischer and Astrup, 1942) was also a satisfactory supplement to the dialyzed medium. It was, however, shown (Fischer, 1946a) that cultures which required both an amino acid mixture and a boiled kidney extract as supplements during the first stages of growth in vitro, could proliferate at an undiminished, or even a greater, rate from 13 days onwards if one or the other of the supplements was omitted. Extracts of heart tissue were less active supplements to the dialyzed medium than kidney extracts, when used alone, but were highly active when used with a mixture

T H E NUTRITION OF ANIMAL CELLS

11

of nine amino acids, or with addition of cystine, lysine, glutamic acid, tryptophan and arginine (Astrup, Fischer, and Volkert, 1945). There is some question whether a useful distinction between the “catalytic growth-promoting factors” and the “accessory growth factors” (Fischer, Astrup, et d.,1948) can be maintained. One of the most outstanding characteristics of Fischer’s “embryonin” fraction is its great lability. Apart from the fact that its activity is easily lost, such a fraction undoubtedly contains many low molecular components held in combination or adsorption, but nutritionally accessible to the living cells. In other words, it cannot be said that the small molecules provided as such are qualitatively or quantitatively the only ones the cells may use. The validity of the use of dialyzed plasma and embryo extract as a basal medium for the study of “accessory growth factors” was first disputed by White and Lasfargues (1949), who were able to restore some of the growth-promoting activity for chick osteoblasts by simple dilution of the dialyzed medium with Tyrode solution. This effect of dilution has been confirmed by Barski et al., (1951), who also questioned whether biological components could ever be treated as inert basal media. They reviewed some of the earlier work on dialysis, ultrafiltration, and ultracentrifugation of tissue culture media, and added some valuable new observations. It had already been shown by Fischer (1941a) that epithelium could survive in an unsupplemented dialyzed medium ; from Fischer’s laboratory it has now been demonstrated (Landschutz, 1952) that a dialyzed medium can permit a significant amount of outgrowth of fibroblasts, provided that sufficiently large pieces of tissue are used. Cultures which were halved produced only a minimal amount of outgrowth, whereas undivided cultures in a medium of the same composition increased markedly in area. In a careful examination of the properties of dialyzed media, Harris (1951a, b ; 1952a, b) showed tha& an even greater degree of restoration of ability to support growth could be achieved by adjustment of the bicarbonate concentration. His dialysis was more thorough than Fischer’s (eg., his dialyzing fluid was changed daily for eight days), and he found that media so treated were entirely unable to support any outgrowth of fibroblasts (Harris, 1952a). Media dialyzed without change of fluid, as in Fischer’s experiments, could produce a small outgrowth of cells. Growth-stimulating activity could be restored by adding, to the dialyzed plasma and dialyzed embryo extract, dialyzate from the chick embryo extract. Harris made the important observation that there is a marked fall in p H during dialysis against unbuffered Ringer-glucose solution and pointed out that this could not be rectified by treating the medium with a C02-containing gas mixture. Adjustment of the pH to 7.4-7.6 with

12

CHARITY WAYMOUTH

NaHC03 or Na&Os enabled the dialyzed mixture to support sustained outgrowth. That the effect was not due solely to restoration of the correct pH, but to compensation for a bicarbonate deficiency, was shown by the failure of a dialyzed medium neutralized with NaOH to show the same activity. I t may be recalled that Warburg, Posener, and Negelein (1924) had demonstrated, for neoplastic cells, that glycolytic activity depends not only on the presence of a suitable carbohydrate source, but also on the presence of bicarbonate (optimum concentration 2.5 mM.). Adjustment of the p H of the dialyzed embryo extract with carbonate has also been adopted by Landschiitz (1952). This single adjustment seems to restore conditions in which low molecular materials become available to the cells. Tissue cultures can obtain enough sugar by the hydrolysis of polysaccharides to enable them to proliferate in a bicarbonate-adjusted dialyzed medium for several months (Harris, 1952a) (contrast four to five days in most of Fischer’s experiments). Sufficient phosphate is also available in the dialyzed medium ; supplementary phosphate at 1 mM. was found by Harris to confer no advantage and 3 mM. phosphate were inhibitory. Besides the inorganic phosphate of the Gey’s fluid (containing 1 mM. phosphate) used as a component of the medium, most of the phosphate used probably derives from breakdown of phospholipids, which can take place in the medium at 37” C. (Trowell, 1952). Hass, Schweitzer, and Boscia (1950, 1951), measuring radial outgrowth of chick lung fibroblasts in a medium containing guinea pig plasma, reported growthpromoting activity in the part of embryo extract (dialyzed against water) which was non-diff usible and water-insoluble. The non-diffusible and water-soluble fraction was inhibitory. The active water-insoluble fraction, which could be dissolved in Tyrode solution, could be further fractionated by precipitation with acetone to give a fraction which was calculated to induce division of fibroblasts twice in 24 hours. The material was heat labile (Maganini, Schweitzer, and Hass, 1953) and a great deal of the protein could be discarded without loss of activity. No active fraction was obtained entirely free from protein. A strong absorption at 2600 A. and the presence of phosphorus were characteristics of all fractions retaining activity. Harris ( 1952b) found a non-protein, partly heat-stable, acidlabile material, which could correct a deficiency in unsupplemented dialyzed medium, in a dialyzate from an alcoholic extract of 12-day chick embryos. The effect reported by Simms and Stillman (1937), namely that serum ultrafiltrate overcomes the “dormancy” of adult tissue, in the sense that outgrowth occurs sooner from adult tissue incubated in serum or serum ultrafiltrate before planting in a plasma medium than from tissue

THE NUTRITION OF ANIMAL CELLS

13

previously incubated in Tyrode solution, indicates a protective effect of the ultrafiltrate compared with the Tyrode solution. Gey and Gey (1936) have drawn attention to the fact that “. . . prolonged washing in saline has a definite deleterious effect on fresh tissue.” Human placental cord serum has been commonly used in tissue culture media since attention was first drawn to its efficacy by Gey (1929) (cf. Cey and Gey, 1936) and to that of pregnancy serum by Pybus and Fawns (1931). Jacquez and Barry (1951) have studied human cord serum by dialysis against Simms’ X6 balanced saline-glucose solution. This solution contains bicarbonate and 1.5 mM. phosphate. They found that embryo extract actively stimulated cell migration in rat fibroblast cultures but could not alone cause much increase in the volume of tissue, whereas Serum alone promoted an active increase in culture density, though this was somewhat less than in the complete medium of serum and embryo extract. This activity of the serum was traced entirely to the non-dialyzable part. Further fractionation showed that the euglobulin fraction contained most or all of the activity. Serum albumin had no growth-promoting effect, but exercised a detoxifying action, probably by adsorption of fatty acids and other injurious substances (e.g., heavy metal ions). Barski et d . (1951) also clearly demonstrated by dialysis, ukrafiltration, and ultracentrifugation that the main factor, the loss of which results in reduction of growthpromoting and maintenance effects in an embryo extract-plasma medium, was to be found in the high molecular part of the plasma. Hoffman, Dingwall, and Andrus (1951) found that the supernatant fluids from ultracentrifugation of adult sheep heart extracts retained most of their growth-promoting activity. Wolken (1952) also found activity in the supernatant fluid after 87% of the total solid material had been brought down by ultracentrifugation of chick embryo extract; but in his experiments there was no significant difference in effect on the growth of chick fibroblasts between this supernatant, the remaining pellet, an acetone supernatant, or an acetone precipitate from a low speed fractionation. Kutsky and Harris (1952) also found the supernatant active and the resuspended pellet less active than the whole extract. Sanford et d. (1952) have made a study by ultrafiltration of the medium, containing horse serum and chick embryo extract, in which it has been possible to grow strains of mouse cells in continuous culture for a period of years. More active fractions of embryo extract and serum could be obtained by ultrafiltration than by dialysis against water or saline. The effects of ultrafiltered materials on strain L mouse cells again emphasizes the importance of the high molecular fraction of the serum and of the filtrate of the embryo extract. The protein residue of horse serum

14

CHARITY WAYMOUTH

promoted cell multiplication more than did the ultrafiltrate ; the ultrafiltrate of chick embryo extract increased the proliferation rate much more than the non-filtrable part. High molecular complexes corresponding to Fischer’s “embryonin’” could therefore be eliminated from the medium for the growth of strain L cells without affecting the growth rate. This is an important simplification of the classic biological nutrients. Horse serum, embryo extract and their ultrafiltrates, prepared in Earle’s laboratory, have also been tested in a similar way on strain 14pf of normal rat fibroblasts by Ehrmann and Gey (1953). For these cells, it appeared that horse serum contains a non-ultrafiltrable inhibitory material. Ehrmann and Gey confirmed, with their strain of cells, the nutritive effect of chick embryo extract ultrafiltrate, and found that growth was better in a medium containing this nutrient and human placental cord serum than in the embryo extract ultrafiltrate and horse serum. However, the best growth was still obtained with whole embryo extract and whole human cord serum. These cells seem to need more from the embryo extract, for maximum growth, than is provided by the ultrafiltrate. A lyophilized ultrafiltrate of embryo extract was extracted with 70% ethanol and passed through an ion exchange column by Rosenberg and Kirk (1953). The d u e n t contained about 3% of the N of the original ultrafiltrate and was as active as the untreated ultrafiltrate as a supplement to thoroughly dialyzed embryo extract, for the growth of chick embryo fibroblasts. Insofar as any conclusion can be drawn from the analyses of biological media, it is that we have so.far learned from them little of the nutritional needs of any tissue for particular metabolites. Many low molecular components, e.g., coenzymes, vitamins, hormones, and even metallic ions, are, in nature, bound to proteins ; and cells are endowed with a variety of enzyme systems capable of acting on substrates of high molecular weight. It is not, therefore, altogether surprising that the capacity of cells to avail themselves of high molecular components, or substances associated with them, is evidently, as Harris (1952a) has shown, very great. On the other hand, several of the nutrients of low molecular weight are undoubtedly equally well utilized in the free state, a fact which encourages the hope that a complete nutrient medium of known chemical composition and equal nutritive value with the classic biological media will in due course be devised.

111. SYNTHETIC MEDIA Because of the general belief engendered by Carrel, and referred to above, that plasma acts largely as a supporting structure and serum as some kind of buffer against accumulation of toxic products, the early attempts to devise media of known composition were directed to

THE NUTRITION OF ANIMAL CELLS

15

the design of synthetic mixtures to replace the embryo extract. Plasma o r serum were retained. From the supplementation of biological media in this way it early became clear that “the effect of adding many biologically important substances . . . such as vitamines, iron salts and oxides, cysteine, carbohydrates, cholesterol etc.” to a basic medium of plasma, glycine, nucleic acid and protein digest “had no beneficial action under the conditions of the experiments” (Baker, 1929). Baker adopted the view, later expressed by Morgan, Morton, and Parker (1950) in their work with fully synthetic media that “even though no evidence w a s obtained of the nutritive value of these substances, the negative results must not be accepted as final, for it is conceivable that any one of them might contribute to the functional requirements of the cells, but that its effect could not be observed in experiments on growth when some other substance necessary for complete nutrition was absent” (Baker, 1929). I n 1933, Vogelaar and Erlichman designed a feeding solution (Table I) for use with serum in growing human fibroblasts. This medium contained Witte’s peptone, which had already been found by Baker and Carrel ( 1926a) ; Carrel, Baker, and Ebeling (1927), and by Fischer and Demuth (1927-1928) to have stimulatory effects on tissue cultures. Later (Vogelaar and Erlichman, 1938) a more fully defined feeding solution was described containing glycine in place of the peptone (Table I). Baker ( 193541, 1936), following closely the composition of Vogelaar’s medium, but adding ascorbic acid, cysteine, glutathione and vitamin A, introduced two serum-containing nutrient solutions (Table I), one for fibroblasts and epithelial cells and the other for monocytes. Chick fibroblasts in a horse plasma coagulum proliferated two or three times as rapidly in Baker’s medium as in Vogelaar’s first (1933) medium and, whereas in the latter solution the survival time was only 12 to 14 days, in Baker’s medium the cultures were maintained in active proliferation for over six weeks. The medium for monocytes differed chiefly from that for fibroblasts in the concentrations of the components, containing smaller amounts of peptone and salts, more glucose, and additional B vitamins. Monocytes proliferated in this medium for 80 days. Various elaborations on these mixtures were made, and one solution (Table I) was devised by Baker and Ebeling (1939) for use as a maintenance medium, without serum. This was not fully defined, as it contained peptone and a digest of whole blood. Fibroblast cultures were kept in this medium for 30 to 43 days in the absence of serum. A slight modification of Baker’s (1936) medium was used by Wilson, Jackson, and Brues (1942) (Table I) for the growth of chick embryo tissues in a thin plasma clot but without added serum. A comparison was made in this solution of the growth and nitrogen utilization

16 0

Is I

CHARITY WAYMOUTH

000 g$g I I I

I I?

0

M

0

h

000000

0s

0s

OS

0's 09

0'21

Id Id u+u+u+Gl6~

PI

a 21 pr)

2 II

I I I I I I

I I I I I I I I I I I I I I I I ~ 1 1 1 1 1

121 I I I S 4

~

121 I I I2 8 "I1

I IXI I I l 3 Ih ZI I I I I I I I

CI

1121 I I I 2 I g I I I I I I I I I I I I

1181 I Z I 1 I I I I I I I

131 l N2 W Y I I I I I I ;I I I I

15.6 10.4

L-Leucine m-Isoleucine DL-Aspartic acid as as par tic acid DL-Glutamic acid L-Glutamic acid L-Arginine L-Histidine DL-Methionhe DL-Phenylalanine L-Cystine m-Tryptophan L-Tryptophan L-Tyrosine L-Proline L-Hydroxyproline L-Glutamine Cysteine-HC1 Glutathione Ascorbic acid Carotene Vitamin A Calciferol (vitamin D) Menadione (vitamin K) a-Tocopherd phosphate Thiamine Riboflavin hrridoxine

-

7.8

2.6

13.0 5.0 -

4.0

-

9.0

1.0 0.25

-

1.125 0.34 0.085

0.10 0.10 0.05 0.01 0.01 -

900 to 18oou.

-

0.0053U.

0.0001u.

o.Ooo1 0.0034

0.01 0.01 0.05

-

0.05

Calcium pantothenate Biotin Folic acid Inositol p-Alanine Choline

0.01 0.01 0.0001 0.05

0.5

15.6 10.4

-

6.0

-

-

14.0 7.8 2.6 13.0 5.0 1.5

4.0 5.0 I

-

0.10 0.10 0.05

4.0

6.0

15.0

-

7.0 2.0 3 .O 5.0 2.0

2.0 4.0 4.0

1.o 10.0 0.01 0.005 0.005

0.01

-

0.01

0.01 0.01

-

0.001

0.01

0.001 0.001 0.0025 0.0025 0.0025 0.0025 0.001 0.001

-

0.01 0.05

0.05 0.01 0.04 0.005 0.05 0.05 0.1

0.001

0.001

0.005

-

0.05

I I 1 I I I 1 1 I I I

I 1 1 I 1 1

c

+

12

II I I I I I I I IIII I I I I

I I II I I II I II I I II I I

g

lo1

I0I

I l l I I I I I I I I I I I I I I I I I @I

I I I

1 -

mcl

991

aW 88

881 I I I I I I I I I I I I I I I I I l o51

.PI00

ggr I I I I I I I I I I 1 I I I I I I 121

ooi

Of

upplemented Dialysed Plasma for Growth of Chick Fibroblasts. Fischer (19484.

(1939).

~F: :,oafYE:,; (1942).

Modified Baker's

Growth Baker (1936).

g:z$teiSs'

Serum-Containing

Baker (1936).

E:$;s:osr:::Wthof Epithelium.

Serum-Containing

p&saqt;tion. Erlichman (1938).

Glycine-Containing

Feeding Solution for Human Fibroblasts. Vogelaar and Erlichman (1933).

I I I I

1 1 1 1 1

5

I I I I "1111

THE NUTRITION OF ANIMAL CELLS

19

20

CHARITY WAYMOUTH

of chick embryo muscle tissue in the presence of (1) Witte’s peptone and (2) peptone which had been hydrolyzed with acid to amino acids, and supplemented with tryptophan. There were some morphological differences between the cells in the two variants. Nitrogen was well utilized from the amino acid-containing medium and mitotic rates over a period of eight days were similar to those in the unhydrolyzed peptone medium. Undeterred by the tenacity with which tissue culturists retained an unquestioning faith in the Components of the classic nutrients and in high molecular “growth-promoting factors” of the “trephone” or “embryonin” type, and encouraged by notable success in devising a fully synthetic nutrient for plant tissues, White (1946) tackled the problem of designing, from current knowledge of the certain and probable requirements of animal cells, a synthetic nutrient medium. This medium (Table I) contained a variant of the mixture of amino acids found by Rose (1938) 10 be able to maintain nitrogen equilibrium in the rat; a high glucose concentration, a salt mixture similar to the conventional balanced salt solutions but containing a ferric salt, and a number of vitamins. Not all of the individual components were shown to be essential, and indeed to check the absolute and relative concentrations of all the components would be a very formidable task. The results with this mixture were so promising that it warranted much further study and attempts to improve upon it. With the original mixture, chick embryo tissue cultivated in roller tubes, directly on the glass without clot or other source of high molecular or unknown material, could be maintained for about 50 to 60 days. The addition of five more amino acids, and minor changes in some of the other components, resulted in a mixture (White, 1949) (Table I ) which could maintain chick embryo cells for up to 80 days, with several changes of the nutrient fluid. By contrast, cells of the same kinds in a balanced salt-glucose solution will survive about eight to ten days. The original medium of White (1945) was not able, alone, to support the life and growth of fowl niacrophages (Jacoby and Darke, 1948), which can proliferate indefinitely in fowl serun1,diluted with Tyrode solution (1 : l ) . However, 10 to 2O:h of serum in 90 or 80% of the synthetic medium formed a mixture in which these cells could multiply actively and be iriaintained ior up to two months. Stuermer and Stein (1950) reported that when stromal cells of the human decidua were cultivated in a biological medium (human placental cord serum, fowl plasma, and chick embryo extract), fibroblasts and epithelial cells grew out. In White’s (1946) synthetic medium, only fibroblasts migrated; in seven to ten days the explants, originally 1 mni., had produced 3- to 4-mm. wide collars of outgrowth. The second fully synthetic medium was described by Morgan, Morton,

THE NUTRITION OF A N I M A L CELLS

21

and Parker (1950) (Table I). In their earlier experiments, these authors started their cultures in a biological medium €or several days before subjecting them to the synthetic mixture. Later (Morton, Morgan, and Parker, 1951), this practice was discontinued. The medium no. 199 contains a much larger number of components than White’s media, designedly, as it “was felt that the preliminary media should include as many known nutritional factors as possible, even though many of them had no apparent effect.” (Morgan, Morton, and Parker, 1950). Under the best conditions (renewal of medium at weekly intervals after an initial starting period of three days in a biological medium of serum and embryo extract), cultures of chick embryo muscle survived for an average period of fifty days, and a maximum of seventy days. Without the initiaI starting period in a non-synthetic medium, but with a seven-day starting period in a minimal amount of the synthetic medium, the average survival time was thirtyseven days. The synthetic medium of White (1949), and that of Morgan, Morton, and Parker (1950), were tested by Evans et ul. (1953) on replicate cultures of mouse strain L cells. Whereas in the best biological media the cell population increased twelve to sixteen fold in six days, and in Earle’s salt-glucose solution the population fell to 1.5% or less of the initial value, in White’s medium, renewed every two or three days, the final cell number was 21 to 30% of the initial value. In Morgan, Morton, and Parker’s (1950) medium the final cell number was 41 to 67% of the number of cells inoculated, slightly higher counts being obtained with media freshly prepared than with stored media. Both types of synthetic medium are clearly unsatisfactory, as they stand, for this strain of cells and under this regime. Though Evans et ul. discuss the possible role of adaptation of the cells to media, no attempts were made in this investigation to permit gradual adaptation of the strain L cells to the synthetic media. In the sections which follow, evaluation of the nutritional role of the various components of biological and synthetic media and their effects on different cell types, will be attempted.

IV. INORGANIC SUBSTANCES The first attempts to maintain physiological function in isolated tissues and organs were made at a time when the composition of the cell was regarded as much more stable and constant than it is now known to be. It was then thought that the provision of the necessary inorganic salts in the proper proportions and in osmotic equivalence with the interstitial fluids was all that was required. From these studies emerged the wellknown “physiological salt solutions,” with or without glucose, of Ringer

22

CHARITY WAYMOUTH

(1886), Locke (1895, 1901), Tyrode (1910) and others for homoeoand poikilotherm tissues (Table 11). The first studies aiming to establish the optimum concentrations of various ions in fluids for tissue cultures

TABLE 11

BALANCED SALTSOLUTIONS USEDIN TISSUECULTURE. MEDIA mg./100 ml.

u6 a rd

b

u

NaCl 900 900 800 900 480 900 700 800 800 800 680 800 KCl 42 42 20 42 60 42 37 38 20 20 40 40 CaCll 25 24 20 20 15 12 17 13 20 11.1 20 20 Ca(NO8),.HaO - - - - - MgCla.6HsO - - 21.4 - 50 - 21 21 10 20.3 - MgSO4.7HsO - - - - - - 70 - - - 20 20 MgHPO4 - - - - - - - - - CaHs(P04)I - - - 10 10 - - - - - - 12 12 - 21.3 - 10 NalHPOl - - - - - - - - 4.3% 10.8 NaHIP04 - - - - - - 3.0 2.5 - 10 KHsPOI Fe(NOs)a.9H10 - - - - - - - - - - - NaHCO, - 30 100 20 - 53 227 25 - 101 220 34 100 100 100 100 100 200 Glucose -2100 100 - -

_ - - - - -

-

_ _ - - - - -

-

-

700

37.5

-

A.

LI

56.2

5.75

-

2.6 0.13 55.0

850

were made by Lewis and Lewis (1911a). I n spite of their careful investigations, and the subsequent publication of descriptions of balanced salt solutions specifically for use in tissue culture media by Drew (1922, 1923), Pannett and Compton (1924), Roffo (1925), Gey and Gey (1936), Parker (1938), Simms and Sanders (1942), Earle (1943), Hanks (1948, 1949), White (1949), and Osgood et d. (1951) (Table II), the most generally used salt-glucose solution has, until quite recently, been the formula of Tyrode (1910) (with or without modification, e.g. by Willmer and Kendal, 1932), designed for quite another purpose. As the pioneer experiments of Lewis and Lewis (1911a, b ; 1912) demonstrated, survival of tissue cultures in a salt-glucose mixture alone is very short. The inorganic requirements of different types of tissue may be different. For example, Willmer (1927), using various salt-glucose solutions as diluents in biological media, showed that when only NaCl, KCl, CaC12, NaHC03, and glucose were used, outgrowth of chick fibroblasts was optimum in 0.8% NaCl and 0.8% glucose; slightly hypotonic

T H E NUTRITION OF ANIMAL CELLS

23

NaCl stimulated fibroblast migration; 0.9% NaCl was optimum for chick epithelium. This contrasts with the fact that Parshley and Simms’ (1950) 216 solution for adult epithelium contains a lower salt concentration (0.68% NaCl) than their X6 solution for adult fibroblasts (0.8% NaCl). The 216 solution moreover contains lower concentrations of KCI and MgCI2 than the X6 solution, and no calcium or bicarbonate. These investigators found that the growth of adult epithelium is stimulated by a high phosphate concentration (Parshley and Simms, 1946, 1950) and that, in contrast to adult fibroblasts, epithelium does not require a diluent containing bicarbonate. The concentrations of phosphate (usually in the form of a buffer) and of bicarbonate provided in various solutions used in tissue culture media have varied over a wide range. The amounts of phosphorus in the usual salt solutions vary from Tyrode (0.36 mM.), through Pannett and Compton’s (1924) solution (0.57 mM.), White’s (0.58 mM.), Earle’s (0.9 mM.), Gey and Gey’s (1936) (1.0 mM.), Hanks’ (0.86 or 1.44 mM.), Drew’s (1.26 mM.), to Simms’ X6 solution for fibroblasts (1.5 mM.). W r t h (1948) has used for virus culture a modification of Tyrode’s solution containing six times the usual amount of phosphate, i.e., 2.16 mM. Human pIasma inorganic phosphate (as P) is 2.6 to 5.4 mg./100 ml. (average, 3.2 mg./100 ml., i.e. 1 mM.) and the total phosphate (as P ) is 10.0 to 14.1 mg./100 ml. (average 12.1 mg./100 ml., i.e. 3.9 mM.) (Stearns and Warweg, 1933, quoted by Krebs, 1950). For monkey plasma, McKee et d. (1946) give an average phosphate of 2.1 mM. The inorganic P of fowl plasma is 2.45 to 4.25 mg./100 ml. (0.8 to 1.4 mM.), and forms most of the acid-soluble phosphorus fraction (Davidson and Waymouth, 1946). Harris (1951a, b, 1952a) showed that, in a dialyzed medium containing sources of bound phosphate, supplementary inorganic phosphate was not essential and that the addition of 3 mM. phosphate was inhibitory. Phospholipid P is present in amounts of the same order as inorganic P, i.e., 2.2 to 4.0 mg./100 ml. (Davidson and Waymouth, 1946) in fowl plasma, and it is known that phospholipids can break down on incubation at 37”C . and release inorganic P. The phospholipid P of embryo extract, prepared by extraction of embryo pulp with Tyrode solution, is 1.3 to 2.5 mg. P/100 ml. I t seems, therefore, that salt solutions such as Tyrode’s or Pannett and Compton’s, low in phosphate, should be satisfactory diluents for biological media rich in organic phosphate. As a basis for fully synthetic nutrient media, phosphate to a total concentration of at least 2 mM. should be supplied, either entirely as inorganic phosphate or partly as organic phosphorus compounds. In short-term experiments, W. H. Lewis (1929) demonstrated that

24

CHARITY WAYMOUTH

NaHC03 is essential to the maintenance of heart beat in three-day chick embryo heart in Locke’s solution (without sugar or phosphate, neither of which is necessary to the maintenance of this function). KCl and CaC12 were also shown to be essential to maintain heartbeat. Lewis and Lewis (1912), in experimental culture media with varied NaC1, KCl, CaC12, and NaHC03 concentrations, commonly used NaHC03 at 20 mg./100 ml. (2.38 mM.), and in no case did they employ more than 100 mg./100 ml. The original Tyrode (1910) solution contained NaHC03 at 100 mg./100 ml. (11.9 mM.). A later modification of this solution for use in tissue culture (Willmer and Kendal, 1932) contained half this amount of bicarbonate. The solution of Adler (1909, quoted by Tyrode, 1910) contained more phosphate (12.6 rng./loO ml. NaHZP04, 1.05 mM.) and much more bicarbonate (351.0 mg./100 ml. NaHC09, 42 mM.) than any of the other early salt solutions designed to imitate the ionic composition of blood plasma. This solution does not seem to have been much used in tissue culture work. Roffo (1925) obtained good growth of cultures with 30 to 53 mg./100 ml. (3.37 to 6.32 mM.) NaHC03. The original formula of Gey and Gey (1936) for a saline solution for use in roller tubes contains 227 mg./100 ml. (27 mM.) NaHC03, but a solution containing one-ninth of this was recommended for use with slide cultures with their smaller gas phase. The lower concentration has become generally adopted for both purposes (Gey, personal communication, 1953 ; Cameron, 1950; Parker, 1950). Simms and Sanders’ (1942) solution X6 for adult fibroblasts contains 101 mg./100 ml. (12 mM.) NaHC03, and Harris’ (1952b) F6 solution contains 140 mg.Jl00 ml. (16.7 mM.). The salt solutions in common use that contain a high bicarbonate concentration, near to that in human plasma, are Earle’s (1943) saline, with 220 mg./100 ml. (26.2 mM.) and Dubin and Yen’s ( 1950) modified Krebs-Ringer-bicarbonatesolution for macrophages, with 210 mg./100 ml. (25 mM.). The average bicarbonate content of human plasma or serum, as NaHC03 (Hald, 1933, 1947, quoted by Krebs, 1950) is 226 mg.Jl00 ml. (range 205 to 280 mg./100 mi.) and that for monkey plasma (McKee et al., 1946) falls within the same range. Bicarbonate or carbonate is essential for the outgrowth of fibroblasts, and Harris (1952a) obtained in a medium with 200 mg./100 ml. an outgrowth twice that with 100 mg./lOO ml., and standardized on 210 mg./100 ml. (25 mM.). The balanced salt solutions are generally used as diluents for biological media such as plasma, serum, and tissue extracts, and they may form up to 90% of the medium in some cases. Gey and Gey (1936) emphasized the deleterious effect of prolonged contact with saline upon freshly

25

THE NUTRITION OF ANIMAL CELLS

isolated tissue. Roffo (1925) made a study of the effects of varying the proportions of the different cations, especially K and Ca, on the develop ment of normal chick heart and of two tumors (a sarcoma and a carcinoma of rat) in tissue cultures. In agreement with Carrel and Burrows (191Ib), Lamhert (1914), Ebeling (1914), and Pannett and Compton (1924), he found that hypotonic media, (e.g., plasma diluted to contain 0.51% NaCl) are favorable €or the initial outgrowth of cells. H e prepared a series of modified Ringer solutions (Table 111) and tested these as diluents for TABLE 111 ROFFO’S (1925) MODIFIEDRINGERSOLUTIONS mg./100 ml.

NaCl KCI CaCL NaHCOl

900 42 24

30

900 21 24

53

900 42 12 53

900 0

86 30

900 58 0 30

fowl plasma in his media. Normal chick heart grew well in (3) and (5), poorly in (2) and very poorly in the K-free solution (4). The fusocellular sarcoma grew best in the low-Ca medium (5), well in (3) which was better than normal Ringer ( I ) , and gave little or no growth in (2) or (4). The rat carcinoma grew well in ( 3 ) and (5). These experiments demonstrated the necessity for K and the relatively smaller importance of Ca. According to Jazimirska-Krontowska (1930), the outgrowth and sugar consumption of normal tissue were reduced by high concentrations of Ca. A high concentration of K greatly reduced the area of the cultures, but sugar consumption remained high. Brues et al. (1940) found that tissue cultures could tolerate high concentrations of K, up to 400 mg./100 ml. Lymphocytes seem able to withstand similarly high concentrations of K (greater than 300 mg./100 ml., Trowell, 1953). Parshley and Simms (1950) indicate that 1 mM. Mg and a very low Ca are optimal for adult epithelial cultures. After depleting the medium of free cations, Shooter and Gey (1952) found, by supplementation, that Ca, Mg and K were all essential for the growth of a strain of normal rat fibroblasts. Either Ca or Mg alone, in a Na- and K-containing medium, restored outgrowth to some extent; both were necessary for even 24 hours’ continued growth. Restoration of cations was made by means of solutions of Ca(NOa)a, K H 2 P 0 4 , MgS04, and CuSO4. A trace-metal solution containing Fe, Zn, Mn, and Co (as sulfates) and ammonium molybdate, improved survival in Shooter and Gey’s experiments over the use of the Ca-, Mg-, K-, and Cu-containing supplement alone.

26

CHARITY WAYMOUTH

The use of trace metals in media for tissue cultures has been studied only sporadically, and the effects of anions have been even more thoroughly neglected. Lewis and Lewis (1912) incorporated traces of ferric oxide into a medium for the cultivation of sympathetic nerve. The tolerance of chick connective tissue towards CuS04 and NaAsOs were studied by Wilson (1922). H e found that 1.G mg. Cu,/lOO ml. were toxic, while 0.8 mg. Cu/loO ml. was tolerated. These concentrations are much greater than the range used by Roffo and Calcagno (1928) ; they found 0.005 mg. Cu/lOO ml. to be toxic and 0.002 mg.Jl00 ml. to give good growth. For human fibroblasts, Vogelaar and Erlichman (1934) found an optimum Cu concentration (0.75 mg./lOo ml.) in the range indicated by Wilson. Concentrations of 0.37 and 0.56 mg. Cu/100 ml. were less favorable; 1.12 mg. Cu/lOO ml. was distinctly toxic. Shooter and Gey (1952) include 0.1 mg. Cu/lOo ml. in their medium. Uei (1926) reported that small amounts of Fe and Mg increased the growth of a rat sarcoma in vitro: Zn and Cu were inhibitory. Vogelaar and Erlichman (1933), and following them Baker (1936), included hemin in their media. As a source of iron, the amounts they used (0.oooO55 mM.) would probably not add significantly to that present in other components of their media. Ehrmann and Gey (1953) found that hemoglobin had a distinct stimulatory effect on the outgrowth of rat fibroblasts in a 25% serum medium; they used 200 mg./100 ml., which would provide about 0.12 mM. of iron. Iron was supplied as Fe(NOs)s by White (1946, 1949) in his synthetic media, at 0.003 mM. The effects of ferric, cupric, and manganese chlorides on the survival times of chick heart cultures in biological media were studied by Hetherington and Shipp (1935). Ferric chloride (FeCl&H20) at 100 mg.Jl00 ml. (3.7 mM.) prolonged the life of the cultures for eight days longer than the controls (33 days) ; MnC12.4Hz0 at 1.0 mg./lOO ml. prolonged survival by two days, and CuC12.2H20 at 1.0 mg.Jl00 ml. deferred the peak death rate by five days. Small amounts (of the order of 0.005 mM. or less) of the chlorides of Fe, Mn, Cu, Zn, and Co were included by Fischer st al. (1948) in their synthetic supplementary medium V-605. Morgan, Morton, and Parker (1951) found that small quantities of Co were toxic in biological media but that larger amounts could be tolerated in synthetic media. Histidine and purines form complexes with Co, and histidine and some other components of the synthetic medium no. 199 (Morgan, Morton, and Parker, 1950), probably the purines, exert a protective effect. The amounts of Zn, Pb, Cu, Fe, Al, Co, and Mn in various biological and synthetic media have been studied by Healy, Morgan, and Parker (1952) as a “basis for further studies on the mineral requirements of animal cells in tissue culture.” Several trace

THE NUTRITION OF A N I M A L CELLS

27

metals are found in human and chicken plasma in amounts greater than 0.1 mg.Jl00 ml., e.g., Zn (Vikbladh, 1950, 1951; Healy, Morgan, and Parker, 1952) and F e (Healy, Morgan, and Parker, 1952). These and others which are present in smaller amounts deserve closer study to determine their effects in tissue nutrition. Most of the balanced salt solutions contain as anions only chloride, phosphate, bicarbonate, and sometimes sulfate. Vogelaar and Erlichman (1939) found that 80% of the chloride in the medium for human thyroid fibroblasts could be replaced by iodide. Nitrate is included by White (1949) in his synthetic medium. As the nutritional needs of cells become more clearly defined, the role of the various anions will have to be investigated.

V. CARBOHYDRATES AND OXYGEN The majority of the balanced salt solutions used in preparing and diluting tissue culture media have been fortified with glucose. The principal exceptions are Ringer’s (1886) solution, which was not designed for tissue culture but has often been used as a diluent ; and Drew’s (1922, 1923) and Pannett and Compton’s (1924) solutions, which were developed for use in tissue culture media. By far the most commonly used formula has been that of Tyrode (1910). This solution was designed for use in pharmacological experiments on surviving mammalian intestine and contains 100 mg./100 ml. glucose, which is close to the average normal mammalian blood sugar level. Other salt-glucose solutions (Locke, 1901; Simms and Sanders, 1942; Gey and Gey, 1936; and Earle, 1943) also contain this amount, though a modification of Gey’s solution (Cameron, 1950) contains 200 mg./100 ml. Hanks’ solution (1948, 1949) originally described with 200 mg./100 ml. has sometimes been used with 100 mg./100 ml. (e.g., Weller and Enders, 1948) and, for prolonged maintenance, with 400 mg./100 ml. (Hanks, 1948). Willmer (1942) drew attention to the higher glucose content of bird compared with mammalian blood (cf. also Wright, 1928; Hill, Corkill, and Parkes, 1934, and Erlenbach, 1938), and modified the formula for Tyrode solution used in studying carbohydrate metabolism in chick tissue cultures to include 200 mg./100 ml. glucose. Studies on the effects of various concentrations of glucose on cultures of connective tissue were made by M. R. Lewis (1921, 1922). She found, as did Wilson, Jackson, and Brues (1942), that in the complete absence of glucose, cultures of embryonic connective tissue degenerated and died within two to three days. I n 0.25% glucose, the formation of vacuoles within the cells was delayed, but survival was increased by only a few days. Still higher concentrations (0.5, 0.75, and 1.0%) permitted two to

28

CHARITY WAYMOUTH

four weeks’ survival. In 2.0 to 5.0% the cells degenerated rapidly on account of acid formation. Lens epithelium in tissue culture was found by Kirby, Estey, and Wiener (1933) to tolerate 0.478% but to be inhibited by 0.578% glucose. Carrel (quoted by Ebeling, 1936) found “no interference with tissue growth” at 0.3% glucose. According to Willmer (1927) and Demuth (1931), there is a direct relation between the amount of fibroblast migration and glucose concentration up to 1.0% Ebeling (1936) tested the effect of several concentrations on cultures of fibroblasts, leucocytes and iris epithelium. For all these tissues, the optimum results were obtained within the range 0.39 to 1.15% glucose. Concentrations above 2.0% were in general inhibitory to growth or productive of cell granularity. Latta and Bucholz (1939) reported that 1 to 2% glucose inhibited, and 5% stopped, fibroblast growth in vitro without affecting embryonic muscle migration. Friedheim and Roukhelman ( 1930) claimed that fibroblasts could grow in glucose up to 7.5%. It is evident that cells in tissue culture can tolerate, at least for a short time, concentrations of glucose much higher than physiological. However, high concentrations of the order found optimum by Ebeling (1936) have not often been used. Lewis and Nettleship (1932-1933) found a concentration of 500 mg./100 ml. glucose beneficial in a medium (Kendall’s medium) composed of an extract of hog intestine in a buffered saline solution. Kirk (e.g., Signorotti, Hull, and Kirk, 1950; Boyer and Kirk, 1952; and Stewart and Kirk, 1952) has recently adopted a Tyrode solution containing 400 mg./100 ml. glucose (and NaCl reduced to 770 mg./100 ml.) as a component of biological media for the study of the quantitative aspects of growth in tissue culture, and Burt’s (1943b) medium for spinal ganglia also contains this amount. Wilson, Jackson, and Brues (1942) found that a high mitotic rate was slightly longer maintained with 500 mg./lOO ml. than with 100 mg./100 ml. glucose, in cultures of mixed chick embryo tissue. There was, however, no increase in the mitotic rate, and cultures in which all mitoses were blocked by colchicine continued to use glucose at an unaltered rate. The relationship of carbohydrate concentration to the onset of mitosis in adult epithelium, in Vivo and in Vitro, has been examined by Medawar (1947, 1948a, b) and by Bullough (1949, 1950, 1952; Bullough and Johnson, 1951a, b, c), Medawar’s (1948a) medium for adult skin contained 500 mg./100 ml. glucose. H e used, in some cases, a Krebs-Ringer-bicarbonate extract of adult tissue, supplemented with glucose to this relatively high level. B’ullough and Johnson’s (1951b) solution incorporated 400 mg./100 ml. glucose. The requirements of different tissues for carbohydrate may differ in amount and also in kind. Baker’s (1936) serum-containing feeding solu-

THE NUTRITION OF ANIMAL CELLS

29

tion for fibroblasts and epithelium contained 100 mg./100 ml. glucose; the corresponding medium for monocytes contained twice this amount. A later formula (Baker and Ebeling, 1939) for fibroblasts contained, however, 300 mg./100 ml. Fischer’s (1918a) “basic nutrient” and medium V-605 (Fischer et al., 1948) (Table IV) contain a total of 100 mg./100 ml. sugars, made up of 80 mg. glucose, 10 mg. mannose and 10 mg. galactose. The simpler formulas V-612 and V-614 (Table V ) contain 200 mg./100 ml. glucose only. Astrup, Fischer, and $%lenschlager (1947) found, in agreement with Lewis (1921, 1922) that cells were unable to TABLE I V FISCHER’S MIXTURESOF ACCESSORY GROWTHSUBSTANCES (1 Medium V-605 (Fischer el aL, 1948) (2{ “Basic nutrient” (Fischer, 1948a) ~

rng./100ml.

750.0 20.0

NaCl

KCI

10.0 5.0 100.0 0.06 0.02

0.03

0.10 0.001 80.0

10.0 10.0 2.0 1.0 1.o 1.o 1.o

(1)

(2) “Basic nutrient” mg./100 rnl.

1.2 1.4 0.9 1.0

-

Medium V-605 mg./100 ml.

L-Cystine

L-Tryutophan t-P
Cozymase Thiamine Riboflavin Pyridoxine Pantothenate Biotin p-Aminobenzoic acid Choline-HC1 Nicotinic acid Creatine Hypoxanthine Glutathione Ascorbic acid Methyl naphthohydroquinone sulfate Adenosine triphosphate Fructose diphosphate 8-Glyccrophosphate Inosinic acid

20.0

CaCL MgCL Na,HPOI NaHCO, FeCl, CUCll MnCL ZnCL CoClr Glucose Mannose Galactose Tnositol Sodium succinate Sodium fumarate Sodium malate Sodium oxaloacetate

DbThreonine DL-Valine >Leucine DL-Isoleucine L-Aspartic acid L-Glutamic acid ~-Lysine-2HCI L-Arginine-HC1 L-Histidine-HCI DL-Methionine DL-Phenylalanine

mg./100 ml.

-

-

0.59

1.5 0.2

1.51 0.77

-

0.5

0.6 0.7 0.5 0.2

-

25.0

1.41

0.31

0.86

-

0.15 0.50

-

0.5

0.3 0.02 0.03 0.007 0.0007

0.1 1.0 0.03 1.0 10.0

0.5 0.2 0.0005 20.0 10.0 10.0

3.0

30

CHARITY WAYMOUTH

TABLE V FISCHER’S MEDIAV-612

AND

V-614 mg./100 rnl.

750.0

NaCl KCI CaCI, MgClt NatHP04 NaHCO, Glucose DL-Threonine m-Valine DL-Phenylalanine L-Leucine DL-Isoleucine L-Lysine-2HC1 L-Arginine-HC1 ~-Histidine-HCI L-Cystine L-Tryptophan L-Glutamine Glutathione Fructose diphosphate p-Glycerophosphate Inosinic acid

20.0

z0.0 10.0

5.0

100.0 200.0 2.4 2.8 1.4

i .8 2.0 3.0 0.4

i .o 1.o

0.4 25.0 1.0 20.0

20.0 Omitted in 6.0) V-614

-

~~

- ~-

(Fischer et al., 1948)

survive in sugar-free media, but they reported that fructose or mannose could replace glucose ; that galactose and maltose showed some activity ; but that sucrose, lactose, xylose, arabinose, liver glycogen, soluble starch, p-glycerophosphate, pyruvate, lactate, and glucosaniine were ineffective substitutes. Lewis and Lawler (1931) had, under different conditions from those of Astrup ef aE., examined the effect of starch on tissue cultures. In their experiments, chick embryo skin cells multiplied and grew abundantly for two to three weeks in a Locke-bouillon-glucose medium containing 0.5% glucose. Without glucose, the cells died in three to four days. When starch was substituted for glucose, the cells grew at first and survived for seven to ten days. Maltose had already (Lewis and Lewis, 1911a) been shown to be usable as a substitute for glucose in a simple medium containing only saits, amino acids, and polypeptides. In experiments which are relevant to the strictly tissue-culture studies, Warburg, Posener, and Negelein (1924) showed that glucose and mannose were readily, and fructose and galactose less readily, used as carbohydrate sources for glycolysis by tumor cells. Spratt (1949, 1950) compared a number of carbohydrates for their effects in permitting development of early chick embryos in vitro on a basal medium of salts and agar. Mannose and glucose were equally effective ; fructose, galactose and mal-

THE NUTRITION OF ANIMAL CELLS

31

tose were utilizable, but progressively greater concentrations were required. On a molar basis, the relative efficiencies were (glucose 100) : mannose 100; fructose 40; galactose 10, and maltose 10. Pyruvate and lactate (110 to 440 mg./lOO ml.) could also be used (Spratt, 1950). It is interesting to compare, also, these studies with those of Elman and Weichselbaum (1952) on the utilization of fructose as a source of energy for protein synthesis during intravenous alimentation. Both glucose and fructose are effective as sources of calories, but fructose enters the cell more rapidly than does glucose. T o quote Elman and Weichselbaum: “fructose is better than glucose for protein synthesis from infused amino acids, which are often given . . . as a source of protein food. The reason is that after intravenous infusion amino acids enter the cell just as readily as fructose, both being metabolized together, the one for energy, the other for protein synthesis. By contrast, amino acids given with glucose tend to be used for caIories rather than protein synthesis, because energy needs are given first priority and the infused glucose at this time is still outside the cell.” Worzniak (1952) briefly reported the effects of various carbohydrates on explanted cultures of chick heart, muscle, gut, and liver in a medium composed otherwise only of Tyrode solution. H e found that, of the three hexoses tested, mannose was superior to glucose or fructose for maintaining the life of the cultures, and that proliferation of both fibroblasts and epithelium took place. Glucose, and still more so fructose, favored epithelium rather than fibroblasts. Glycogen, lactate, dihydroxyacetone, pyruvic aldehyde, and glucosamine failed to support proliferation, though heart beat persisted for many days in the presence of lactate. Phosphorylated hexoses were not superior to mannose, but were utilized. The triose intermediates, with the exception of dihydroxyacetone, and the Krebs’ cycle group of substances, supported growth almost as well as glucose. Pyruvate was able to support fibroblast proliferation, but there was little or no epithelial growth, and viability was diminished. Snellman (1937) substituted fructose or galactose for glucose in media for cultures of the Jensen rat sarcoma and found a decrease of 40 to 70% in lactic acid production, but normal growth. Hanging-drop cultures of normal chick tissues, in a Tyrode solution containing sodium lactate in place of glucose, ‘‘showed cellular activity comparable to that shown by cultures in ordinary Tyrode solution and far in excess of that shown by cultures in Tyrode solution without either glucose or lactate” (Pomerat and Willmer, 1939). This was confirmed by Wilson, Jackson, and Brues (1942). The medium of Thomas and Borderioux (1948) for the culture of organs of adult Urodeles contains calcium gluconate in place of glucose. The number of alternative carbohydrate sources which have been men-

32

CHARITY WAYMOUTH

tioned makes it appear not particularly surprising that chick fibroblasts can proliferate, in a medium composed of plasma and embryo extract, both of which have been dialyzed against a sugar-free salt solution, without added sugar (Harris, 1951b). Outgrowth can be increased not only by glucose but by several other hexoses or by maltose o r glycogen. In all cases the carbohydrates were depleted in the medium and lactic acid produced. Salisbury (1947), from his experiments with tissue cultures of normal and malignant cells, concluded that tumor cells were permeable to sucrose, but that the normal cells examined were not. Heart beat can be maintained in tissue in a state of reduced metabolic activity in a wholly synthetic medium containing sucrose (optimum concentration 1.7%) instead of glucose (White, 1946). The high glucose, concentration (0.8%) found by Willmer (1927) to be best for outgrowth of fibroblasts in a medium containing only salts (NaCl, KCI, CaClz and NaHC03) and glucose, and the similar high concentration (0.85%) arrived at by White (1946) for maximum survival in the first (and therefore not complete) synthetic medium, support the view that the essential structure and functions of the cells can be better maintained in nutritionally deficient media when high concentrations of carbohydrate are available. In fully adequate nutrient milieux, lower concentrations of carbohydrate (at, or slightly above, “physiological” levels) are sufficient. It is of interest that, in the nutrition of the protozoon Tetrahymenu, Kidder (1952) has found glucose to exert a sparing action on amino acids, though no carbohydrate source is essential for this organism (Manners and Ryley, 1952). It remains to be seen, in tissue culture nutrition, how far amino acids can spare carbohydrate as an energy source, and vice versa. It has been shown (Fischer, Fischer, et al., 1953) that C14-labeledglucose, added to the classic biological medium of plasma and embryo extract, is incorporated into the amino acids of the protein of the growing embryo chick heart tissue. There is, as Willmer (1941, 1942) showed, in general no correlation between high growth rate and high glucose consumption or lactic acid production. H e suggested that glucose consumption and lactic acid production are associated with cell movement, which in turn is related to the incidence of cell division. In tEe early stages of cultivation of chick tissues in vitro, glucose utilization is high (Wilson, Jackson, and Brues, 1942), starting at 2.6 mg./100 mg. wet tissue per day and falling, as the glucose is depleted, to about 0.5 mg./100 mg./day on the third and fourth days. Cultures on a schedule of daily renewal of medium, or replenishment of glucose only, used a total of 22 mg. glucose per 100 mg. tissue in 11 days, i.e., an average of 1.6 mg./day. Of this glucose, 60 to 70% appears as lactic acid. The results of Willmer (1942) on the carbohydrate utilization

T H E NUTRITION OF ANIMAL CELLS

33

of chick osteoblasts in media with or without embryo extract are in general agreement with Wilson, Jackson, and Brues ( 1942). Glucose consumption was high initially when embryo extract was provided. Both Willmer and Wilson, Jackson and Brues concluded from the results on their culture systems that some lactate may be formed from a source other than glucose. Many of the tissues which grow most satisfactorily in tissue culture have a high capacity for anaerobic glycolysis. Early chick embryos can metabolize glucose or mannose to lactic acid anaerobically (Needham and Nowinski, 1937). Glycogen, disaccharides, and phosphorylated hexoses are not attacked. Glycogcnolytic activity in embryo muscle develops after the fifteenth day of incubation. There is, however, a wide variation in the requirements of different tissues for oxygen. Medawar (1947) and Bullough (1952) have shown that fragments of adult mammalian epidermis can survive in complete absence of oxygen for many days. For this type of tissue, mitotic activity increases with increasing oxygen tension over quite a wide range (Bullough and Johnson, 195la). This is a general, but not a universal, characteristic of adult tissues (Parker, 1936a). Krebs’ cycle intermediates (glutamate, fumarate, or citrate), added to a saline medium with an oxygen gas phase, increase the rate of oxygen consumption and the rate of mitosis in adult epidermis by about 25 to 30% (Bullough, 1952). Some of the literature on the oxygen requirements of tissue cultures is reviewed by Hudspeth, Swann, and Pomerat (19.50). Very young embryonic cells can withstand lower oxygen tensions than older embryonic cells. Heart tissue from four- to five-day chick embryos survived many hours in “pure” nitrogen (Burrows, 1921) and, after a latent period of 10 to 24 hours, during a period of a few hours produced a small amount of outgrowth. Mitosis was possible in tumor cells at lower oxygen tensions than were required by chick embryo myoblasts (Wright, 1928). Heart tissue from the ten-day chick embryo requires 1.8% oxygen (Burrows, loc. cit.) or 1.7% (Wright, loc. cit.) ; the fifteenday heart tissue requires 5.4% (Burrows). In accordance with this is the finding of Danes and Leinfelder (1951), that the oxygen consumption of seven-day chick heart tissue cultures could be reduced 16% without affecting cellular activity. Lowering the oxygen consumption further suppressed cell activity progressively, to the point of complete inhibition when the oxygen consumption was reduced by 85%. Other studies of the oxygen requirements of tissue cultures and of the relation between tissue growth and oxygen tension have been made by Ephrussi *etaE. (1929) and by Paulmann (1940). I n general, above the minimum requirements, migration of fibroblasts is favored by low and inhibited by high oxygen ten-

34

CHARITY WAYMOUTH

sion. The reverse is true for nerve fibers ; the character of the outgrowth from explants of chick spinal cord varies with the oxygen concentration. A high oxygen content (%% Oz, 4% COz) stimulated the outgrowth of nerve fibers and suppressed connective tissue. Connective tissue cell migration is stimulated by very low oxygen tensions (e.g. 2%), though the total absence of oxygen prevents all outgrowth (Hudspeth, Swann, and Pomerat, 1950). Very high concentrations of glucose (1 to 2%) suppressed respiration and inhibited outgrowth. At physiological concentrations and upward (e.g., 5.5 to 37 mM.), glucose inhibited respiration in the Ehrlich mouse carcinoma (Brin, 1953 ; McKee and Lonberg-Holm, 1953) ; only at low concentrations is oxygen consumption stimulated. Leucocytes have a high oxygen consumption in vitro, though the metabolic intensity is markedly influenced by the composition of the surrounding medium (Hartman, 1952; Delaunay and Pag+s, 1946; Macleod and Rhoads, 1939). Oxygen deficiency causes giant cell formation in tissue cultures of lymph nodes (Barta, 1925, 1926). Trowel1 (1952) has demonstrated the need for an abundant oxygen supply for the survival of lymph nodes, which consume rather more than their own volume of oxygen per hour. His cultures were maintained in an atmosphere of 100% oxygen. Bone marrow also needs a high oxygen tension. Rosin and Rachmilewitz (1948) found that 12% or less of oxygen was injurious to rabbit bone marrow in vitro; the cells were kept in excellent condition by 50% oxygen, diluted with nitrogen (no carbon dioxide). Parker ( 1936a), keeping the COz uniform at 8% (in a medium containing a high bicarbonate content and 400 mg./100 ml. glucose), varied the 0 2 and Nz in the gas phase for cultures of adult rabbit spleen in a fluid medium. The cells were much better preserved in 80% oxygen than in 21% or 40%. After four days in 2176 oxygen, there was marked necrosis and degeneration. VI. AMINOACIDSAND PEPTIDES Supplementation of a simple saline medium with amino acids and peptides was one of the early steps taken by Lewis and Lewis (1911a) towards the understanding in chemical terms of tissue culture nutrition. With the same aim of simplification, Smyth (1914) described a tissue culture medium of agar and trypsinized peptone. The objective of Burrows and Neymann (1917, 1918) was also explicitly to work toward a “synthetic medium suitable for the growth of tissue cells outside of the animal organism,” and they expressed the opinion, which is still valid, that “since the preparation of such a medium would lead directly to a better understanding of cellular metabolism this problem has stood forth as one of the most important of those presented by the tissue culture method.” It is

T H E NUTRITION O F ANIMAL CELLS

35

unfortunate, therefore, that their categorical report that amino acids and peptides were toxic probably had its influence in driving investigators interested in the nutrition of cells back from the synthetic approach to the analysis of biological media. Carrel, Baker, and Ebeling included in their long series of studies of the effects of biological media and their components on tissue growth, investigations of amino acids, peptides, and protein digests. It was already apparent to Carrel in 1924 that “amino acids under the same concentration as in the blood have no poisonous effect on fibroblasts and epithelial cells in pure cultures, and that some of them increase the rate of cell migration and multiplication” (Carrel, 1924a). Burrows and Neymann had used excessively high concentrations, and their (1918) statement that “low dilutions” of amino acids stimulate contraction of heart fragments seems to have been overlooked. Both Carrel (1924a) and Ebeling (1924) were of the opinion that the amino acids were not used by the cells as a source of nitrogen, and that they were effective only in promoting cell migration, but not cell multiplication. Likewise, the addition of a mixture of sixteen amino acids to a dialyzed embryo extract produced an increase in area of fibroblast cultures, but no increase in the mass of tissue (Baker and Carrel, 1926e). Gerarde, Jones, and Winnick (1952a) found that a mixture of nineteen amino acids in the proportion found in bovine serum albumin, added at 10.0, 50.0, 100.0, or 500.0 mg./100 ml. to Tyrode solution, was not able to prevent autolysis in cultures of chick lung, heart, o r intestine. In this case, however, the medium was not designed to be nutritionally complete. There have been many reports of the effectiveness of peptones (especially Witte’s peptone) in stimulating growth in tissue cultures. Baker (1933), Baker and Carrel (1926a, 1928a), and Carrel and Baker (1926a, b, c, 1927) found that a proteose prepared from fibrin actively promoted cell proliferation and Guillery ( 1930) made similar claims for preparations made in the same way from embryo extract. Fischer and Demuth (1927) separated an active proteose from Witte’s peptone. So did Kuczinski, Tenenbaum, and Werthemann (1925), who mention that their preparation was rich in vitamin B. By this, as in so many studies on partially purified biological materials, is admitted the possibility that the effects observed are not only, or perhaps sometimes even mainly, attributable to the quantitatively preponderant components. Carrel, Baker, and Ebeling (1927) compared the effects, on rat sarcoma cells, of various supplements to Tyrode solution. Digests of egg albumin and of casein gave poor “growth.” Addition of glycine increased the rate of growth by about 70%, and addition of “nucleic acid” [amount and source unspecified, but perhaps the thymus nucleic acid (Levene) referred

36

CHARITY WAYMOUTH

to by Baker and Ebeling (1938) ] plus glycine to a digest of egg albumin increased the area 91%. “Nucleic acid” plus glycine and casein digest gave an even greater increase in area (193%) over the controls. The sarcoma cells responded much better than normal cells to peptic digests (Baker and Carrel, 1928a, b, c). Willmer and Kendal (1932), who used normai chick fibroblasts, prepared a thermostable heteroproteose from Witte’s peptone, which greatly stimulated the cells to migratory activity and cell division. Glycine was examined by Vogelaar and Erlichman (1936b) as a supplement to media for human fibroblasts. At this time, a very high concentration (525 mg./100 ml.) was used, and this was not capable of replacing the Witte’s peptone in the feeding solution. Later, Vogelaar and Erlichman (1938) used a medium (Table I) containing 1/10 of this concentration (52.5 mg./100 ml.), which gave good results in the absence of Witte’s peptone. Glycine was not one of the mixture of nine amino acids (prepared after the analysis of fibrin made by Bergmann and Niemann (1936) ) used by Fischer (1941a) to supplement dialyzed biological media. Later he (Fischer, 1948a) found that a supplementary medium containing no amino acids except cystine, glycine, and glutamine was able to maintain cultures in a condition comparable to that in a supplement containing the nine Bergmann and Niemann amino acids. Omitting single amino acids from the mixture of nine [reported as Rose’s (1938), but apparently actually Bergmann and Niemann’s (1936) mixture], Fischer (1948b) found that lysine could be dispensed with over a four-day growth period without significant reduction in outgrowth. Omission of cystine caused severe reduction, and of arginine, tryptophan, or glutamine a moderate reduction, of the total area at the fourth day. Stimulating effects on tissue cultures have been observed with cystine, with glutathione (Hueper and Russell 1933; B’etker and Wormiak, 1952), and with arginine (Hueper and Russell, 1933; Bach and Lasnitzki, 1947). Cystine is regarded by Fischer (1941a, 1948a, d) as possessing peculiar importance for the cells. The absence of cystine, even when the other amino acids were provided, led to complete inhibition of growth in Fischer’s dialyzed system. A comparison of the effects of amino acid mixtures based on the composition of two proteins (lactoglobulin and bovine serum albumin) with tryptic or peptic digests of the same proteins was made by Fischer (1948a). The digests (it is not clear whether both enzymes were equally effective) increased the area of outgrowth far beyond that obtained in the artificial amino acid mixtures. Thus, With a lactoglobulin digest, the area at fourteen days was about twice that in the amino acid mixture simulating lactoglobulin, and was still increasing, while in the mixture of twenty amino acids the area

THE NUTRITION OF ANIMAL CELLS

37

had reached a maximum at seven days. The persistent idea that peptides have an important stimulating effect was put forward by EhrensvLrd, Fischer, and Stjernholm (1949) in their conclusion “that we have to seek for the components possessing maximal activity in respect to the induction of cell proliferation between some special definite limits of molecular weight, namely the upper limit of trichloroacetic acid precipitability and the lower limit of non-dialyzability.” While it seems to be established that peptides stimulate fibroblast migration, the evidence for the actual utilization of peptides by the cells is very meager and at best circumstantial. It is possible that, for tissue cells, as has been shown (Kihara and Snell, 1952; Kihara, Klatt, and Snell, 1952) for bacteria, small peptides may have advantages over their component-free amino acids. For example, Kihara, Klatt, and SneIl showed that leucyltyrosine and glycyltyrosine are far superior to tyrosine in growth-promoting activity for Streptococcus fueculis, because free tyrosine, but not the peptides, is attacked by a tyrosine decarboxylase present in the cells. This is an example of a principle which may have far-reaching implications in the search for the optimum nutritional requirements of different cell types. The fact that the Ehrlich mouse ascites tumor cells can take up a-glutmylglutamic acid, glycylglycine and triglycine (Christensen and Rafn, 1952) (though the uptake of these peptides by these cells is less than that of the free amino acids), nevertheless indicates that such small peptides can be assimilated. Whether larger peptides, some of which are known to have important physiological functions, e.g., strepogenin as a growth factor for certain bacteria and the leukotaxinlike peptides (eight to fourteen residues in length) in inflammation and capillary permeability (Duthie and Chain, 1939; Spector, 1951), are taken up by the cells or utilized in their nutrition, remains doubtful. According to Winnick (1952), amino acids or peptides can be used by the cells to build new cell protein and “it is even conceivable that whole protein molecules may be assimilated with only minor structural modifications”. Some evidence has been brought forward in support of this hypothesis (Francis and Winnick, 1953). It has been claimed (Bohus Jensen, quoted by Fischer, 1950) that the “presence of large peptides in the medium causes a remarkable increase in the frequency of cell divisions.” Fischer (1942a, 1947) attempted to demonstrate that the peptides from homologous plasma, which could be expected to conform to the pattern of the species-specific protein in the tissues, were more readily used for the growth of fibroblasts than peptides from heterologous plasma. Only one type of tissue (chick embryo fibroblasts) was, however, used, and there was a wide variation in the effects with digests from plasmas from various species. The evidence was hardly sufficient to

38

CHARITY WAYMOUTH

justify the generalization that homologous peptides are nutritionally superior. I n experiments of the same sort, Fischer (1950) tested peptic digests of normal and tumor tissue for their growth-promoting power on normal myoblasts. The increase in area of the cultures treated with the tumor digest (from a methylcholanthrene-induced fowl sarcoma) was in one case slightly, in another markedly less than in controls treated with normal tissue digests. Again the evidence is too slight to warrant generalization. And, as has been pointed out elsewhere in this review, increases in area alone are an uncertain and often entirely misleading guide to the true growth of tissue. The ability of cells to use heterologous or homologous peptides still remains an open question. A great deal of information is now available on two basic phenomena of cell behavior and biochemistry, highly relevant to the mechanism of amino acid utilization in tissue nutrition. These are: (1) the capacity of cells actively and selectively to concentrate amino acids from their environment, and (2) the mechanisms and scope of transamination with its implications for the ability of the cells to modify the amino acid population presented to them. The application of this knowledge to the understanding of tissue nutrition and the design of nutrient media has hardly begun. T o these two should perhaps be added the information which is accumulating, but is still relatively fragmentary, on the incorporation of amino acids into other cell constituents, e.g., glycine and aspartic acid into nucleic acid purines and pyrimidines. The concentrative uptake of amino acids has been studied over a number of years by Christensen and his collaborators. The interest of this phenomenon for tissue growth was commented on by Christensen and Streicher (1948), who speculated on their findings that the concentrations of amino acids in fetal tissues, and in actively regenerating liver, are several times those in the corresponding normal adult tissues. They suggested that “increased protein synthesis and growth may be initiated or promoted by increased concentrations of amino acids”’ and that therefore the “concentrating function of cells for amino acids represents a possible point for the control of growth.” Fetal guinea pig muscle, for example, contains an amount of a-amino acids three times that in the muscle of the maternal guinea pig. The concentrative ability for amino acids of Ehrlich’s mouse carcinoma (ascites form) cells is very great (Christensen and Henderson, 1952; Christensen and Riggs, 1952). Glycine gradients of 60 mM./1. water between cells and suspending fluid can be achieved in glycineenriched media at the end of 2 hours’ incubation. The ratio of concentrations of glycine in cells to ascitic fluid without added glycine is 12.4. The uptake is dependent on the presence of oxygen and is temperature

THE NUTRITION OF ANIMAL CELLS

39

sensitive, the ratio cells-fluid being maximum near the physiological temperature. Concentration of amino acids is closely associated with the maintenance of ion balance, and glycine uptake can be inhibited by 40 meq./l. of potassium, During glycine concentration (Christensen and Riggs, 1952) or tryptophan concentration (Riggs, Coyne, and Christensen, 1953) the cells lose K and take up Na. These are additional instances of the principle of the control of the ionic environment by amino acids, first studied by Krebs and Eggleston (1949) in respiring brain slices in relation to the effects of glutamic acid and glucose on potassium exchange. An appreciation of the implications of these studies is essential to the understanding of the possible ways in which materials are utilized in the system cells plus medium which constitutes the tissue culture. According to Christensen et al. (1952), the cells of higher animals show a characteristic responsiveness to extracellular levels of amino acids. The amino acid content of the cell is adjusted in relation to the environment, the intracelM a r level always remaining higher than the external level. The concentrative activity Of cells for amino acids decreases during embryonic development and in general as cells become specialized. C1*-labeled glycine at 1.33 mM., and DL-alanine and DL-phenyhlanine at 2.66 mM., are taken up by the proteins of embryonic chick lung, intestine and heart cells. The labeled atom of glycine-l-CIJ appears in the serine as well as in the glycine of the heart and lung proteins (Gerarde, Jones, and Winnick, 1952a). The ability of mouse heart in tissue culture to effect transaminations was demonstrated by Jacquez, Barclay, and Stock (1952). Of eleven mouse and four rat tumors examined, nine had negligible transaminating ability. Bach and Lasnitzki (1947) found that arginine (100 mg./100 ml.) significantly increased the growth of tissue cultures of carcinoma 63 ; chick heart fibroblasts and mouse embryo lung were unaffected. Transaminase activity is, however, fairly widespread among different tissues, and the capacity of the cells thus to modify their nutritional environment should be borne in mind in assessing their nutritional needs. The fact that CI4 from labeled glucose is incorporated into aspartic acid and alanine (Fischer, Fischer, et d., 1953) indicates the intervention of a transaminase in embryo chick tissue, able to convert the pyruvate and oxaloacetate formed from the CI4 glucose into these amino acids. Some CI4 is also found in the tissue serine, glycine, glutamic acid, and proline. It ,is significant that the synthetic medium of White (1946), containing a mixture of ten amino acids corresponding to those originally reported by Rose (1938) to be essential for the maintenance of N equilibrium in the rat, was much improved as a maintenance medium for normal chick tissues by the incorporation of glycine, glutamic acid, aspartic acid, proline, and cystine

40

CHARITY WAYMOUTH

(White, 1949). Parallel with this finding in tissue culture, Rose, Oesterling, and Womack (1948) have also shown that rats fed a diet containing nineteen amino acids gain 25% more weight in a 28 day period than those on the original 10 amino acids. Glutamic acid was particularly important. Parshley and Simms (1950) added aspartic acid to their special salt solution designed for use in media for adult epithelium, after finding that this amino acid stimulated the cells of thyroid and skin in tissue culture. Morgan, Morton, and Parker’s (1950) medium no. 199 contains a mixture of nineteen amino acids based on analyses of tissue protein (Block and Bolling, 1945). It is by no means certain that a mixture of amino acids in the proportions found by analysis in a given protein is the best material for the normal synthesis of the same or similar protein in a biological system. There is much to suggest that extensive interconversion can take place, and that therefore smaller numbers of amino acids are sufficient. Much remains to be learned about the interrelations of the amino acids and about the design of the most effective mixtures for tissue nutrition. The total concentrations of amino acids provided must also be suitable, though surprisingly high concentrations of certain individual amino acids (glycine, 1,ooO; phenylalanine, tyrosine, aspartic acid, SO0 ; tryptophan, histidine, 400; arginine, lysine, 200 mg./100 ml.) were found by Brues et al. (1940) to be non-inhibitory to fibroblasts. White (1946) tested a wide range of concentrations of his ten-amino acid mixture (0.15 to 150 mg./100 ml.) and found a regular increase in survival with concentration up to 45.0 mg./100 nil., and a decrease at 150.0 mg./lOO mi. A concentration of 100 mg./100 ml. was taken as optimum. With the addition of the further five amino acids (White, 1949), the total concentration was raised to 136.5 mg./lCQ nil. Morgan, Morton, and Parker (1950) tested a narrower range (50 to 500 mg./100 ml.) of dilutions and found 100 mg.Jl00 ml. of their nineteen-amino acid mixture to be most favorable. These concentrations are of the same order as the amounts of free amino acids in rabbit fetal plasma, rather more than in human cord serum, somewhat less than the concentration in guinea pig serum (Christensen and Streicher, 1948) and about three times the amount in adult human blood (Krebs, 1950). It is remarkable that Bullough and Johnson ( 1 9 5 1 ~ ) found that a relatively very high concentration (340 mg./100 ml. ;0.02 M.) of a single amino acid (glutamic acid) increased the rate of mitosis in fragments of adult mouse ear epidermis in vitro. This is, however, of the same order as the concentration of aspartic acid (300 mg./lOO mi.) used by Parshley and Simms (1950) in their 216 solution for adult epithelium. The concentration of glutathione in chick embryo extract was reported

T H E NUTRITION OF ANIMAL CELLS

41

to be 40 mg.Jl00 ml. (Hueper and Russell, 1933). As their standard medium contained one-fourth embryo extract, this would contribute about 10 mg./lOO nil. glutathione to the medium. The various synthetic and semisynthetic media in which glutathione has been included have usually contained less than this. It has mostly been added with the aim of stabilizing ascorbic acid, rather than for its possible contribution as a peptide or source of amino acids to the nutrition of the cells. Glutathione has lately been shown to be the prosthetic group of a glyceraldehyde-3-phosphate dehydrogenase (Racker and Krimsky, 1952). Baker’s media for fibroblasts and monocytes ( 1936) contained respectively 0.34 and 1.O mg./100 ml., and Baker and Ebeling’s (1939) medium 1.2 mg./lOO ml. glutathione. White’s ( 1946, 1949) media contained 1.0 mg./100 ml. ; Morgan, Morton, and Parker (1950) had only 0.005 rng./100 ml. in mixture no. 199. Fischer’s (Fischer et al., 1948) media V-605 and V-612 contain respectively 0.5 and 0.1 mg./lOO ml. and these media are designed as supplements to dialyzed media, so the final concentrations would be lower. However, considerably more (8 to 10 mg./100 ml.) seems to have had a beneficial effect (Astrup and Fischer, 1946) and Fischer (1948b) states that glutathione plus a dialyzed medium caused “large areal spreading of the cells”; here the concentration appears to have been 200 mg./lOO ml. He (Fischer, 1948c) devised a medium (Table I ) , in which heart fibroblasts could be maintained for ten passages, containing a final concentration of 14.8 mg./100 ml. glutathione. Cysteine was used at 9.0 mg./lOO ml. by Vogelaar and Erlichman (1933) and Erlichman (1935) in their feeding solution for human fibroblasts and at 14.2 mg./100 ml. in their modification (Vogelaar and Erlichman, 1938) containing glycine. Baker’s (1936) media contained 1.125 mg./lM ml. (for monocytes) and 9.0 mg./100 ml. (for fibroblasts), as did Baker and Ebeling’s (1939) medium and Wilson, Jackson, and Brues’ (1942) medium. White (1946, 1949) used 0.10 mg./100 ml. Morgan, Morton, and Parker (1950) included 0.01 mg./lOO ml. cysteine in medium no. 199. Pires Soares (1947), using guinea pig testis and embryonic chick heart, prepared tissue cultures in biological media (60% plasma, 20% embryo extract plus 20% saline with or without added cysteine). I n the range 0.125 to 1.875 mg./100 ml. in the final medium, the highest and lowest concentrations had little effect compared with the optimum of 0.75 mg.Jl00 ml. in increasing the area of the cultures of both types of cells in a forty-eight-hour period. Parallel with the increase in area, he observed an increase in mitotic index and prolongation of the life of the (hangingdrop) cultures, without transfer to fresh medium, from ten days in the controls to fifteen days in the medium containing 0.75 mg./100 ml. cys-

42

CHARITY WAYMOUTH

teine. All phases of mitosis were increased, but most markedly the number of telophases.

Glutatnine is a major contributor to the free =-amino N of animal tissue and plasma (Hamilton, 1945). The normal glutamine concentrations in dog and human plasmas are 6 to 12 mg./100 ml. Heart tissue contains 225 mg./100 ml. which comprises 50 to 60% of the total free a-amino N of the tissue. Fischer’s (1948a, c) media contained 25.0 and 18.5 mg./100 ml. respectively ; in Ehrensvard, Fischer, and Stjernholm’s ( 1949) medium, the final concentration of glutamine was also about 25 mg./100 ml. Morgan, Morton, and Parker’s ( 1950) reported that glutamine increased the life span of their fibroblast cultures, at 10 mg./100 ml.

VII. PURINES, PYRIMIDINES, AND NUCLEIC ACIDS From some of their early fractionation experiments on embryo extracts, Baker and Carrel (1926a) reported that the proteins therein were “a mixture of nucleoprotein and glycoprotein with mucin-like properties’’ ; but they could not attribute to the isolated fractions, or to other nucleoproteins, to sodium nucleate prepared from embryo pulp, or to nucleic acid from thymus, any growth-stimulating effects. However, Baker and Ebeling (1938, 1939) included 20 mg./100 ml. thymus nucleic acid in their synthetic maintenance medium. Fischer (1939) on the other hand isolated from beef embryo extract a “nucleoprotein” fraction which he found to accelerate the growth of his cultures. Both ribonucleic acid and deoxyribonucleic acid were present, but he maintained that the “growthpromoting activity seems in the meantime to follow the fractions containing the ribose nucleotides,” i.e., the fraction precipitated by glacial acetic acid. Later he reported (Fischer, 1940, 1941b) that reprecipitation always caused reduction of activity. Repeated precipitation with dilute HCl at 0” C. gave “more and more a distinct maximum of absorption in ultra-violet at 2600 A characteristic of nucleic acid” but less and less growth-promoting activity. Fischer was therefore forced to consider the possibility that some substance other than the nucleoprotein itself was responsible for the activity of the “nucleoprotein” fraction. The fraction contained more P than could be accounted for as nucleic acid, it contained 2% s, and probably polysaccharides of the chondroitin sulfate type (Fischer, 1940, 1941b). There is no evidence for the utilization of nucleoproteins or nucleic acids as such in cell nutrition, although Tennant, Liebow, and Stern (1941) and Tennant, Stern, and Liebow (1942) suggested that nucleates stimulated migratory activity in mouse fibroblast cultures. Studies on changes in nucleic acids in growing tissues have been made

T H E NUTRITION OF ANIMAL CELLS

43

by Willmer (1941, 1942), Davidson and Waymouth (1943, 1944a, b, 1945, 1946), Davidson, Leslie, and Waymouth (1949), Davidson and Leslie ( 1951), Leslie and Davidson (1951a, b), Hull and Kirk (195Oa, b, c), Boyer and Kirk ( 1952), and Gerarde, Jones, and Winnick (195Zb). The finding that the growth-promoting effect of chick embryo extract was not reduced by treatment with the enzyme ribonuclease (Davidson and Waymouth, 1943) strengthened the probability that free polynucleotides are not important nutrients. Caution in generalizing from one organism to another about metabolic pathways is always desirable but is particularly necessary in the case of the precursors of the nucleic acids. It has been shown (Abrams and Goldinger, 1952) that, in rabbit bone marrow, hypoxanthine can act as a precursor of both adenine and guanine in the nucleic acids. Adenine and guanine themselves can be used by the rabbit cells (Abrams and Goldinger, 1951), but there is not in the rabbit, as in the rat, any extensive interconversion of the two purines. Moreover, the rat cannot use hypoxanthine as a precursor of the polynucleotide purines (Gebler et ad., 1949). There are now many instances to show that an exogenous source of preformed purine is not essential. With labeled molecules, it has been demonstrated that in the rat (Abrams and Goldinger, 1952) and the pigeon (Greenberg, 1948; Valentine, Gurin, and Wilson, 1949; Sonne and Lin, 1952, 1953) the following can act as precursors of various parts of the hypoxanthine molecule : formate, glycine, glutamate, aspartate, and glutamine. Labeled COa is incorporated into both purine and pyrimidine rings, in rat tissues in vivo (Heinrich and Wilson, 1950). Reichard and B'ergstrom (1951) have shown that, in the rat, there is considerable synthesis of both ribonucleic acid and deoxyribonucleic acid purines from glycine, and of pyrimidines from orotic acid. Ribonucleic acid pyrimidines can also incorporate aspartic acid (Lagerkvist, Reichard, and Ehrensvard, 1951). The biosynthetic incorporation of formate and bicarbonate into nucleic acids in mice and rats is dependent upon folic acid (Skipper, Mitchell, and Bennett, 1950; Drysdale, Plaut, and Lardy, 1951) ; that of bicarbonate into ribonucleic acid in the rat is dependent upon biotin (MacLeod and Lardy, 1949). There is therefore no reason to assume that purines or pyrimidines as such, or in the form of nucleosides or nucleotides, are needed as nutrients. There exists a small amount of evidence upon the effects of some of these substances on cells growing in tissue culture. Hopkins and Simon-Reuss (1944) tested the effects of hypoxanthine in 15% embryo extract on the growth of periosteal fibroblasts. The areas were increased over the controls, at 5 mg.Jl00 ml. ; 10 mg./100 ml. was less effective. In similar cultures in Carrel flasks, analyzed by the photo-

44

CHARITY WAYMOUTH

graphic method of Willmer and Jacoby (1936), 5 mg.JlO0 ml. hypoxanthine caused, over a period of 30 to 40 hours, a marked stimulation of mitosis. No similar effect was obtained with adenine. Ehrensvard, Fischer, and Stjernholm (1949) included hypoxanthine in a simple supplement to a dialyzed medium. They found the optimum concentration in the fluid phase in Carrel flasks to be about 1 mg./100 ml. Adenine and guanine were found to be inactive. Rerabek and Rerabek (1952) treated Maximow cultures of fibroblasts with purine and pyrimidine bases. These authors found that adenine at 1.69 mg./lOO ml. caused area and mitotic increases, greater than the increases produced by the four bases (adenine, guanine, cytosine, and uracil) at the same (0.125 mM.) molarity. Cytosine alone gave some effect, guanine alone was inhibitory, and uracil had no distinct effect. Trowel1 (1953) found adenine and adenosine at 2 mM. toxic to lymphocytes ; he quotes evidence of SchiZler that leukemic lymphocytes in vitro rapidly convert purines to allantoin. Concentrations as low as 0.6 mM. of adenosine, adenylic acids, and adenosine triphosphate can cause pre-prophase inhibition of mitosis in chick fibroblasts (Hughes, 1952), and concentrations in this range or a little higher can cause nucleolar fragmentation, though they do no apparent damage to interphase cells. The synthetic medium no. 199 of Morgan, Morton, and Parker (1950) included a number of nucleic acid components, namely adenine at 1.0 mg.JI00 ml., guanine, xanthine, hypoxanthine, thymine, and uracil each at 0.03 mg./100 ml., ribose and deoxyribose each at 0.05 mg.Jl00 ml. This medium also contained muscle adenylic acid at 0.02 mg./100 ml. and adenosine triphosphate at 1.0 mg./100 ml. Fischer’s (19484 supplements V-605 and basic nutrient contained 10 mg./100 ml. hypoxanthine and 3.0 mg./100 ml. inosinic acid. Also using a dialyzed medium, Harris (1952a) found no effect on the increase in area in ten days of his fibroblast cultures with 10 mg./100 ml. ribonucleic acid. The nucleotides and nucleosides ( 1 to 100 mg./100 ml.) were inactive or inhibitory, and so were the seven purine and pyrimidine bases (adenine, guanine, cytosine, uracil, thymine, xanthine, and hypoxanthine) at 1 or 10 mg./100 ml. and the cofactors adenosine triphosphate ( 1 mg./100 ml.) and diphosphopyridine nucleotide (0.1 mM. or 6.6 mg./100 ml.).

VIII. LIPIDS Little has been added to our knowledge of lipid metabolism in tissue culture systems since the chemical events in such systems were reviewed by Fischer in 1933. Carminati (1933) reported that a synthetic (distearyl) lecithin stimulated chick fibroblasts. Davidson and Waymouth ( 1944b, 1945, 1946) showed that fibroblast cultures do not grow well in a medium

THE NUTRITION O F ANIMAL CELLS

45

consisting of defatted plasma and defatted embryo extract, but that defatted sheep embryo extract promoted greater increase in nucleoprotein phosphorus than the untreated extract, in the presence of whole plasma. The enzyme lecithinase A (which dissociates Iecithin to Iysolecithin with release of an unsaturated fatty acid), and lysolecithin itself (50 mg./100 ml.), produced morphological and chemical changes in chick heart cells which suggested that this treatment may favorably improve the capacity of the cells to take up nutrient materials. Leslie and Davidson (1951a) showed that there is an early increase in phospholipid (during the first 24 to 48 hours) in chick heart explants, at the time when carbohydrate metabolism is particularly intense (Willmer, 1942). Insulin increased the phospholipid content of the cultures, but reduced the amount per cell (Leslie and Davidson, 1951b) . I t is now very well established that lipogenesis is intimately linked with active carbohydrate metabolism, and that carbohydrate and acetate, as well as pyruvate, oxaloacetate, lactate, etc., provide carbon sources for fatty acid synthesis. Free fatty acids do not normally accumulate in lipogenetic tissues, but are immediately esterified to glycerides. Cholesterol incorporates acetate or acetoacetate ; Popjik and Beeckmans (1950) have shown that the rabbit fetus is independent of the mother for lipid synthesis and can incorporate C14 acetate into cholesterol and into both glyceride and phospholipid fatty acids. Popjik and Muir (1950) discuss the probability that a- and P-glycerophosphates may be precursors of phospholipid P. Lipid synthesis and metabolism are believed to be affected at various points by almost all of the B vitamins (thiamine, riboflavin, pyridoxine, pantothenate, nicotinic acid, folic acid, biotin, choline, and inositol) (cf. Frazer, 1952; Bloch, 1952). I n vivo, but not with any certainty in vitro, insulin and the hormones of the adrenal and pituitary glands are concerned in fat metabolism. Fischer et aE. (1948) included in their medium V-605 fumarate, malate, oxaloacetate, and succinate, most of the B vitamins and several carbohydrates. It is improbable that fatty acids as such are nutritionally useful. Morton, Morgan, and Parker (1950) studied the effects of a series of “Tweens” (polyoxyethylene sorbitan esters of fatty acids) on myoblast cultures in synthetic media. The monolaurate, monopalmitate, monostearate, monooleate (Tween So), and trioleate esters, and free oleic acid were tested at 0.05 to 500 mg./100 ml. Concentrations of 5 mg./100 ml. and less were not inhibitory. Tween 80, because it contained an unsaturated fatty acid, was incorporated into medium no. 199 at 2.0 mg./lOO ml., partly for its possible intrinsic value, and partly as a vehicle for non-water-

46

CHARITY WAYMOUTH

soluble components (Morgan, Morton, and Parker, 1950). Jacquez and Barry (1951) showed that oleic acid at 1 mg./100 ml. was toxic to rat fibroblasts in a medium containing embryo extract and the globulins of human placental cord serum. Higher concentrations could be tolerated in the presence of serum albumin, which binds the fatty acid.

IX. VITAMINS 1. The Fat-Soluble Vitamins a. Vitmin A . The survival and growth of several types of tissue is Vitro appear to be affected by vitamin A. The life of liver and spleen cultures was prolonged by addition of the vitamin to the medium (Bisceglie, 1926). Baker (1935a) increased the vitamin A content of chicken serum two-hundredfold by allowing the serum to stand overnight in the presence of purified vitamin A. Fibroblast cultures in a horse plasma clot and a semisynthetic nutrient containing chicken serum so fortified formed colonies up to three times as large as control cultures without added vitamin. Large amounts of the hypervitaminotic serum were toxic; the optimum effect was found when it formed 1% of the medium. This would mean slightly more than doubling the normal concentration, allowing for a proportion of normal serum in the medium. The incorporation of vitamin A into the medium prevented fat accumulation and increased the survival time. The semisynthetic media subsequently described by Baker (1936) contained 900 to 1,800 units/100 ml. for fibroblasts and 50 to 100 units/100 ml. for monocytes. Normal fowl plasma contains 200 to 400 units/100 ml. vitamin A and about 300 units/100 ml. carotene; mouse plasma contains only 20 to 60 units/100 ml. vitamin A (Fell and Mellanby, 1952). Gordonoff and Ludwig (1935, 1936) also found that an increase in vitamin A stimulated the growth of chick fibroblasts, and of a mouse carcinoma in culture. Proliferation of both these types of cells was greatly inhibited by the absence of vitamins ( A B1,BP, C, D, and E) from the plasma used in the medium, or by the absence of vitamins A or BI alone. Small amounts of vitamin A Were found by Vollmar (1939) to stimulate the growth of both normal and tumor cultures ; larger amounts were inhibitory, and more strongly so to the tumor cells than to the normal cells. Fell and Mellanby (1952) produced hypervitaminosis A in plasma, both “artificial” (by adding vitamin A alcohol or acetate to normal fowl plasma) and “natural” (by feeding large amounts of the vitamin to birds). Plasma from these birds contained three to four times the normal amount of vitamin A and about half the normal amount of carotene. The vitamin in the “artificial” hypervitaminotic plasma remains solubie in fat solvents ; in the “natural”

THE NUTRITION OF ANIMAL CELLS

47

hypervitaminotic plasma, it is not extractable with petroleum ether unless the proteins have first been denatured. Both types of vitamin-rich plasma produce profound changes in limb bud rudiments of the chick and mouse in &fro, the effects with the free vitamin being more drastic than those of the combined form. Another remarkable effect was produced by Fell and Meflanby (1953) by treating ectoderm from six- to seven-day chick embryos with vitamin A at about 1,ooO to 2,000 units/100 ml. Amounts of vitamin A of this order prevented keratinization and caused the epithelium to differentiate into an actively secreting mucous membrane, often ciliated and histologically similar to normal chick nasal mucosa. On transfer to normal medium, after seven to fourteen days in the medium rich in vitamin A, there was a t first continued rapid development of the ciliated and secretory epithelium for four to five days. Thereafter the basal cell layer multiplied and formed a squamous epithelium similar to that produced in cultures carried throughout in normal medium. One international unit of vitamin A is equivalent to 0.344 pg. vitamin A acetate or 0.6 pg. p-carotene (W.H.O. Tech. Rept. Series, 1950, no. 3). Human plasma contains about 0.025 mg. vitamin A per 100 ml. and has a total carotenoid content of 0.09 mg./100 ml. (Krebs, 1950). The total vitamin A plus carotene (0.01 mg./100 ml. of each) used by White (1946, 1949) and by Morgan, Morton, and Parker (1950) in their synthetic media are therefore of the same order as the amounts in human or mouse plasma and considerably lower than in fowl plasma. b. Vitamin D. Hosono and Narisawa (1931) added vitamin D to the culture medium for chick heart and also used plasma from birds fed vitamin D orally. They found that the “heart tissue grew more than twice as well as in control plasma.’” On the contrary, Gordonoff and Ludwig (1936) found that, in contrast to the unfavorable effects produced by deficiencies of vitamins A and BI, neither avitaminosis D nor addition of vitamin D to normal plasma had any effect on normal or tumor cultures. Vollmar (1939) also obtained no clear effect. Baker’s (1936) media for fibroblasts and monocytes contained, respectively, 15 to 30 and 1 to 4 units per 100 ml. vitamin D. The international unit of vitamin D is equivalent to 0.025 pg. crystalline vitamin D3 (cholecalciferol). The synthetic medium of Morgan, Morton, and Parker (1950) contains a concentration of 0.01 mg./100 ml. calciferol (ergocalciferol) , which is about the amount found in egg yolk. c. Yitartin E. While Gordonoff and Ludwig (1936) found that plasma deficient in vitamin E, or supplemented with an excess, had no effects on their cultures, JuhAsz-Schaffer (1931) and Rossi (1935, 1936) (quoted by Vogelaar and Erlichman, 1937) reported stimulation of

48

CHARITY WAYMOUTH

cultures of chick embryo tissues. According to Vollmar (1939), normal tissues were stimulated by vitamin E at 1 g./100 ml. and were not inhibited even by a concentration as high as 10 g./lOO ml. One gram per 100 ml., however, inhibited the growth of tumor cultures. In their medium no. 199, Morgan, Morton, and Parker ( 1950) included a-tocopherol phosphate at 0.001 mg./100 ml. The total tocopherol concentration in human pIasma is about 1.2 mg./lOO ml. (Krebs, 1950). d. Vitamin K . This vitamin is included in Morgan, Morton, and Parker’s (1950) medium no. 199 at 0.001 mg./100 ml. Nothing is known of its possible influence on the nutrition of cells. 2. The Water-Soluble Vitamins

a. Vitom’fi C. Vogelaar and Erlichman (1937) reported that 0.5 mg./iOO ml. ascorbic acid favored the growth of the Crocker mouse sarcoma 180 in tissue culture. On the other hand, Vollmar (1939) found that 1 mg./lOo ml. stimulated normal tissue but had no effect on the tumor tissues tested. High concentrations (200 to 500 mg./100 ml.) were found by Hengstmann (1938) to inhibit, and low concentrations to have no effect on the growth of human and chick embryonic tissues. Epithelial cells from embryonic guinea pig kidney and parotid remained healthy in a washed coagulum in the presence of a buffered saline containing ascorbic acid, but not without it (Chambers and Cameron, 1943). Plasma from scorbutic guinea pigs also caused deterioration of these tissues. Here, however, the effect might be an indirect or a multiple one. For example, the amounts of the important amino acids glycine and glutamic acid are greatly reduced in scorbutic guinea pig tissues (onethird of normal in liver and muscle, Christensen and Lynch, 1948), and may well also be reduced below the nutritional optimum in the plasma. Messina and Verga (1937) and Nungester and Ames (1948) found that the phagocytic activity of leucocytes was increased by ascorbic acid. Within the range of 0.10 to 0.60 mg./lOO ml., which is well within physiological limits, the phagocytic activity and the fragility of leucocytes in peritoneal exudates of the guinea pig varied with ascorbic acid content. Above 0.60 mg./100 ml., there was no increase in phagocytic activity (Nungester and Ames, loc. cit.). These observations suggest that, apart from its probable metabolic role, ascorbic acid may have an influence on the physical state of the cell surface and so on the exchange of substances between the cell and its environment. A curious effect was produced by a rather high concentration (20 mg./100 ml.) of ascorbic acid on cultures of the Ehrlich adenocarcinoma which had been maintained through 680 passages in a rat and chicken plasma and chick embryo extract medium (Gaillard, 1942).

T H E NUTRITION O F ANIMAL CELLS

49

Three to four days after addition of the vitamin, a thickening of the center of the explant took place, which proved to be a core of cartilage. ,4 concentration of 10 mg./lOO ml. was able to activate cell proliferation and growth in cultures of kidney tubules (Chambers and Cameron, 1944). Concentrations within the physiological range were used by Baker (1936) (0.25 mg./100 ml. for fibroblasts and epithelium, 0.085 mg./100 ml. for monocytes) . B’aker and Ebeling’s (1939) maintenance medium contains 0.3 mg./100 ml. ascorbic acid. Fischer’s (19484 supplement V-605 contains 0.2 mg./lOO ml. The synthetic media of White (1946, 1949) and of Morgan, Morton, and Parker (1950) contain respectively 0.05 and 0.005 mg./100 ml. b. Vitamins of the B Group.

( 1 ) THIAMINE. From the experiments of Gordonoff and Ludwig (1935) and of Rossi (1935, 1936) it appeared that some part of the B complex was important as a component of nutrients for tissue cultures. Plasma from avitaminotic animals was unfavorable and completely inhibited the growth of chick embryo fibroblasts and of mouse tumor cells. Plasma from hypervitaminotic birds stimulated cultures and favored the survival of tissue in Vitm (Gordonoff and Ludwig, 1936). Very high concentrations of a brewers’ yeast preparation of “B vitamin” were inhibitory, but more dilute preparations were stimulatory (Rossi, 1935). Gordonoff and Ludwig (1935, 1936) concluded that thiamine (vitamin B1) was essential to the proliferation of normal chick and of mouse carcinoma cells. Paterson and Thompson ( 1943) also studied avitaminotic plasma as a tissue culture medium. Conditions favorable to growth could be restored by adding a brewers’ yeast extract low in thiamine and in biotin. It was concluded that this supplement provided a source of some essential and unidentified member of the B complex. Added thiamine was not effective. I n contrast to this, and in agreement with Gordonoff and Ludwig, Hengstmann (1938) claimed a stimulating effect of thiamine on chick embryo heart, human embryonic and chicken leucocyte cultures, at optimum concentrations in the range 5 to 10 mg./100 ml. Higher concentrations were inhibitory. The confusion is increased by the report of Vollmar (1939) that 500 mg./lOO ml. thiamine will stimulate chick heart and inhibit mouse tumor cultures. Some of the differences in the ranges of concentrations found effective by different workers may be due to an underestimation of the lability of free thiamine. Thiamine in the natural, protein-bound form is stable to ordinary conditions of storage, but free thiamine, in addition to being labile to heat and alkali, is liable to inactivation by oxidation (Kandutsch and Baumann, 1953). However,

50

CHARITY WAYMOUTH

in short-term hanging-drop cultures, Hetherington ( 1946) found that thiamine at 6.25 to 200 mg./100 ml. had no detectable effect on brain, skin, or heart cultures. Concentrations of thiamine below (1 to 5 mg./100 ml.) and within (8 to 24 mg./100 ml.) the physiological range had no effect on spinal ganglia in tissue culture (Burt, 1943b). Very high concentrations were inhibitory. Plasma from thiamine-deficient birds (Burt, 1943c) inhibits axon growth, but this inhibition, like that found by Paterson and Thompson ( 1943) was not reversed by addition of thiamine to the medium. Avitaminosis-B probably produces indirect as well as direct effects on the composition of the plasma, For example, it is known that severe thiamine deficiency in rats or pigeons results in a marked reduction in the enzymes concerned in transamination (Kritzmann, 1940, 1943, quoted by Braunstein, 1947). Administration of thiamine in Vim rapidly restores the enzyme activity; but addition of thiamine in vtcro to minced tissues from thiamine-deficient animals does not result in reactivation of the transaminase system (Barron et al., 1941). Baker and Ebeling’s (1939) maintenance medium (medium IV) contains O.ooO1 mg./100 ml., which is less than the average normal level in human plasma (0.005 mg./100 ml.) (Krebs, 1950). Fischer’s (19484 medium contains 0.3 mg./100 ml. The synthetic media of White (1946, 1949) and Morgan, Morton, and Parker (1950) contain respectively 0.01 and 0.001 mg./100 ml. thiamine. (2) RIBOFLAVIN.In short-term cultures, riboflavin has little (Gordonoff and Ludwig, 1936) or no effect (Hengstmann, 1938; Hetherington, 1946). Baker and Ebeling ( 1939) incorporated 0.0034 mg./100 ml. into their medium; Fischer (1948a) had 0.02 mg./100 ml.; White (1946, 1949) had 0.01 and Morgan, Morton, and Parker (1950) 0.001 mg./100 ml. Human plasma contains 0.0032 mg./100 ml. total riboflavin (free riboflavin plus flavin nucleotides) (Suvarnakich, Mann. and Stare, 1952). (3) PYRIDOXINE. Hetherington ( 1946) included pyridoxine in the series of B vitamins which he tested on hanging-drop cultures, and he found no effects attributable to this vitamin under the conditions of his experiments. Fischer (1948a) had 0.03 mg./100 ml. in his medium V-605 ; White (1946, 1949) used 0.05 mg./100 ml. in his synthetic media. Morgan, Morton, and Parker (1950) included both pyridoxine (0.0025 mg./100 ml.) and pyridoxal (0.0025 mg./100 ml.) in their medium no. 199. In view of the part played by pyridoxal phosphate as a codecarboxylase, and in transamination reactions (Gunsalus, 1950), it is possible that pyridoxal alone would be a sufficient addition. Transamination reactions are depressed by pyridoxine deficiency, though not so drastically as by thiamine deficiency. Both pyridoxal phosphate and

THE NUTRITION O F A N I M A L CELLS

51

pyridoxamine phosphate are effective coenzymes of transaminase (Cohen, 1951 ; Meister, Sober, and Peterson, 1952) ; pyridoxine is not able, in etitro, to activate transamination in deficient tissues (Schlenk and Fisher, 1947). Pyridoxal phosphate is also a coenzyme of histaminase (Sinclair, 1952) and of desulf urase and transsuliurase (Tarver, 1952). (4) BIOTINAND FOLIC -4~1~ Of. all the €3 vitamins examined, biotin and folic acid are those on which the best evidence exists for positive effects on the growth and survival of tissue cultures. Hamilton and Plotz (1942) recorded stimulation of the growth of mouse and chick epithelium, fibroblasts, muscle, and nerve cultures by biotin at about 0.03 mg./100 ml., though Burt (1943a) did not find that biotin at this concentration had any effect on chick spinal ganglia, or on other types of cells, in tissue culture. Hetherington (1946) reported that the survival time of chick fibroblasts in hanging-drop cultures was doubled by biotin (0.15 to 0.5 mg./100 ml.) and fdic acid (0.015 to 0.5 mg./lOO ml.). Degeneration of nerve cell cultures was prevented by 0.15 mg./100 ml. folk acid; 18.5 mg./100 ml. biotin (saturation) was not toxic to these cells (Painter, Pomerat, and Ezell, 1949). Folk acid at 1.0 to 5.0 mg./100 ml. promoted maturation of megaloblasts from pernicious anemia bone marrow, in a medium containing pernicious anemia plasma. In a medium containing bovine serum ultrafiltrate (not beef plasma, as reported), maturation could be achieved with 0.1 mg./100 ml. folk acid (Thompson, 1952). The synthetic media of White (1946, 1949) contained 0.01 and 0.04 mg./100 ml. biotin and O.OOO1 and 0.005 mg./100 mi. folk acid, and that of Morgan, Morton, and Parker (1950) O.OOO1 mg./100 ml of each vitamin. Fischer’s ( 1948a) medium contained 0.0007 mg./lOO ml. biotin, and no folic acid. The biotin and folk acid contents of human plasma are given by Krebs (1950) as 0.00127 and 0.00175 mg./100 ml., respectively. (5) NICOTINIC ACIDA N D NICOTINAMIDE.Up to 200 mg./100 ml. of nicotinamide (Brues et d.,1940) and 250 mg./100 ml. of nicotinic acid (Hull, Perrone, and Kirk, 1950) could be tolerated by fibroblast cultures without toxic effect. Nicotinamide at 12.5 mg./100 ml. was found to be optimum for chick embryo heart and brain, and 25.0 mg./100 ml. for chick embryo skin (Hetherington, 1946). “AS little as” 300 mg./100 ml. nicotinamide damaged nerve fibers (Painter, Pomerat, and Ezell, 1949) ; 2,500 mg./100 ml. were totally inhibitory. Morgan, Morton, and Parker (1950) included both nicotinic acid and nicotinamide in their mixture no. 199, each at 0.0025 mg./100 ml. White’s (1946, 1949) media had 0.05 mg./100 ml. nicotinic acid, which is of the same order as the amount used by Fischer (1948a) (0.03 mg./100 ml.), and corresponds to the amount of nicotinic acid plus amide in an average human plasma (Krebs,

52

CHARITY WAYMOUTH

1950). The chick embryo synthesizes nicotinic acid, so that the amount in the embryo is finally twenty times that in the unfertilized egg (Snell and Quarles, 1941). (6) PANTOTHENATE. The amount of pantothenate in chicken blood varies significantly with the amount in the diet, but falls within the range 0.02 to 0.05 mg./100 ml. (Pearson, Melass, and Sherwood, 1946) ; this is higher than the average for human plasma given by Krebs (1950), i.e., 0.012 mg./100 ml. Calcium pantothenate was included in the media of Morgan, Morton, and Parker (1950) at 0.001, Fischer (194th) at 0.007, and White (1949) at 0.01 mg./100 ml. (7) P-ALANINE. Fischer (1941a), treating p-alanine as an amino acid inter aliu, but also having in mind its possible use as a pantothenic acid precursor, found that it had no effect, alone, on his fibroblast cultures in dialyzed media, but that it enhanced the effect of cystine when the two together were supplied at 1.7 mg./100 ml. amino-N. Vogelaar (1953) reports that cystine can be dispensed with in a feeding solution for human fibroblasts, but attests the importance of p-alanine. The synthetic medium of White (1949) contained 0.05 mg./100 ml. 8-alanine. (8) INOSITOL.Fetal plasmas contain more inositol than adult (Nixon, 1952). The amounts in human fetal and adult plasmas are 8.9 and 0.68 mg./100 ml. respectively, and in sheep, 29.0 and 1.4 mg./100 ml. The media of White (1946, 1949) contained 0.05 mg./l00 ml. and of Morgan, Morton, and Parker (1950) 0.005 mg./100 ml. (9) ~AMINOBENZOIC ACID. A semisynthetic medium for malaria parasites, containing proteose peptone, could be made fully synthetic by replacing the peptone (150 mg./100 ml.) by p-aminobenzoic acid (0.01 mg./100 ml.). This was the optimum concentration; 1.0 mg./100 ml. was inhibitory to the growth of the parasites (Anfinsen et al., 1946). At 1,500 mg./100 ml. (saturation), p-aminobenzoic acid was not toxic to nerve fibers in vitro (Painter, Pomerat, and Ezell, 1949). The medium of Fischer (1948a) for embryonic fibroblasts contained 0.1, and Morgan, Morton, and Parker’s (1950) medium no. 199 contained 0.005 mg./100 ml. p-aminobenzoic acid. (10) CHOLINE. The free choline in plasma is rather constant at 0.1 to 0.2 mg./100 ml. (Bligh, 1952). Amounts of 500 mg./100 ml. could be tolerated by fibroblast cultures (Brues et d.,1940) without inhibitory effect. Choline was used by Fischer (194th) at 1.0 mg./100 rnl. in his mixture V-605, by White at 0.5 mg./100 ml. (1946) on 0.1 mg./lOO ml. (1949), and by Morgan, Morton, and Parker (1950) at 0.05 mg./100 nil. One of the methods by which fixed tissue cells can be transformed into macrophages is the addition of choline to the culture medium (Thomas,

THE KUTRITION O F A N I M A L CELLS

53

1937). Chivremont (1943, 1945, 1948; Bacq and Chhremont, 1944) has studied this transformation very thoroughly and he found (1943) that the optimum concentration for niuscle cells in hanging-drop cultures was 3 to 4 mM. (40 to SO mg./100 ml.) and in Carrel flasks 1.3 mM. (16 mg./100 ml.). Acetylcholine at similar molarities was also effective. Hepatic cells are morphologically altered in a similar way (Frederic, 1951), but the optimum concentration of choline was found to be higher (10 mM., i.e., 121 mg./100 ml.). Among many amines examined, LettrC and Albrecht (1943) found that choline chloride at 16 to 160 mg./100 ml. did not produce vacuolisation of chick fibroblasts. (1 1 ) VITAMINBiz. Human serum contains an average of 20 mpg./lOO ml. (range 8 to 42) of vitamin BIZ (Rosenthal and Sarett, 1952). Vitamin B12 at 0.001 to 0.01 mg./100 ml. had no effect on the maturation in zritro of megaloblasts from bone marrow of pernicious anemia patients (Thompson, 1952). Evidence for its nutritional value to other cells is not yet available. Chick fibroblasts have been shown to be able to tolerate excessively high concentrations of vitamin I312 (up to 0.5 mg./lOO ml.) (Waymouth, unpublished), but this and very much lower concentrations have no apparent effect on growth in short-term experiments. X. HORMONES With the exception of insulin, thyroxin, and some of the steroid hormones, which have received a good deal of attention, there have been few systematic studies on the effects of hormones on tissue cultures or of their possible roles in cell nutrition. Insulin in a very wide range of concentrations has been incorporated into tissue culture media. Gey and Thalhimer (1924), at a time when insulin had not been very highly purified, used the preparation then available at 1 to 2 g./100 ml., and reported larger and heavier growth of fibroblasts. This was probably something over 20,000 units per 100 ml. ( 1935 International units). Kuczinski, Tenenbaum, and Werthemann (1925) used 0.66 unit/100 ml. to counteract the deleterious effect of a high (0.25%) glucose concentration on guinea pig liver cultures. Roffo (1928a, b ; 1930) studied the effects of insulin on normal and neoplastic tissues in Vitm and Roffo and Ferramola (1930) measured glycolysis in cultures of normal and tumor cells at 0 and 48 hours and found glycolysis greater when insulin was added. Gomes da Costa (1935) found that small concentrations of insulin increased respiration and depressed glycolysis ; high concentrations diminished respiration and stimulated glycolysis. Insulin, at an optimum concentration of slightly less than 0.2 unit/100 ml., was considered by Vogelaar and Erlichman (1933) to be a cause of

54

CHARITY WAYMOUTH

marked improvement in any medium containing glucose. B’aker ( 1936), in her modification of the Vogelaar medium, used 0.09 unit/100 ml. for fibroblasts and 0.012 to 0.024 unit/100 ml. for monocytes. Baker and Ebeling’s (1939) medium, and Vogelaar and Erlichman’s (1938) medium, contain 0.1 unit/100 ml. For leukocyte cultures, Wallbach (1938) employed 1 unit/100 ml. Latta and Bucholz (1939) found that migration and proliferation of chick heart fibroblasts were not affected, in the first and second passages in d r o , by the addition of insulin up to 100 units/lO nil. to the standard medium of heparin-plasma and embryo extract. 333.3 units/100 ml. caused a slight effect and a-marked increase in fat deposition. Fetal heart fibroblasts were stimulated to proliferation and increase in area by 100 units/100 ml. (von Haam and Cappel, 194Ob), and slightly larger amounts (200 to 300 units/100 nil., i.e., approximately 10 to 15 mg./100 ml.) were found by Leslie and Davidson (1951b) to promote proliferation in chick heart explants in a “fully adequate growthpromoting medium.” A rise in ribonucleic acid phosphorus per cell was obtained. In conjunction with cortisone and/or pituitary growth hormone (Leslie, 1952), insulin caused an increase in lipid phosphorus also. Crystalline or protamine zinc insulin at 1,ooO units/100 ml. (45 mg./100 tnl.) did not at first inhibit nerve fiber outgrowth, though it did so at 48 hours (Painter and Pomerat, 1948). Current evidence points to the importance of the function of insulin in reversing thiamine dephosphorylation (Foa et d., 1952) and in increasing cell membrane permeability to glucose and other biologically important sugars and so accelerating their uptake (Ross, 1953). The extent to which insulin exerts any true hormonal effect in tissue cultures is uncertain. It is possible that such effects as are observed are largely unspecific. Fischer (1941a) demonstrated that insulin (when denatured) was among the sulfur-containing compounds that could in part replace cystine in media rendered deficient by dialysis. S m u r a (1931) studied the effects of thyroxin on chick fibroblasts in plasma and embryo extract. Ten milligrams per 100 ml. were inhibitory ; UP to 1 mg./100 ml., growth increased with concentration. Cultures in plasma (without embryo extract) were stimulated by 0.1 or 1.0 mg./100 ml., and degeneration was retarded. Vogelaar and Erlichman (1936a), on the other hand, found that thyroxin over a wide range of concentrations (0.01 to 10 mg./100 ml.) had no obvious effect on the growth of human fibroblasts. In fetal mouse heart cultures, von Haam and Cappel (194Ob) showed that the addition of O.ooO1 to 0.01 mg./loO ml. thyroxin produced a more rapid gain in area and more mitoses, compared with normal controls. Baker (1936) used 0.009 or 0.000113 mg./100 ml. thyroxin

THE NUTRITION O F AXIM A L CELLS

55

respectively in her media for fibroblasts or monocytes. Carrel (quoted by Parker, 1938) was able to produce profound morphological changes in normal fowl leucocytes in vitro by 2.5 mg./100 ml. thyroxin. Adrenaline at 0.01 to 10 mg./100 ml. was inhibitory to mouse heart fibroblasts (von Haam and Cappel, 1940b). This may be due, according to the theory of LettrC and Albrecht (1941), to the effect of its oxidation product, adrenochrome. LettrC believes that tumor cells, in contrast to normal cells, have lost the capacity to oxidize adrenaline and that tumor celIs are therefore not subject to mitotic inhibition in vitro in the presence of adrenaline. The uninhibited growth of tumor cells in viva is likewise ascribed to the failure of this regulatory mechanism. Gaillard and Veer (1948) found that adrenochrome could increase the radial migration of fibroblasts, but it also reduced the number of cells entering mitosis and, at certain concentrations, caused profongation of the metaphase without giving rise to any morphological abnormalities. Baker and Ebeling’s (1939) medium contained adrenaline, adrenal cortical hormone, antuitrin and pitressin. Antuitrin was found by Semura (1931) to be inhibitory to chick fibroblasts at 10 to 1000 mg./100 ml. Trowel1 and Willmer (1939) and Davidson and Waymouth (1943) found that anterior pituitary extracts had no effect on the growth or nucleoprotein content of chick fibroblasts. From ovarian explants derived from rabbits previously treated for three to four days with 100 to 200 units of prolan, or with 15 t o 20 ml. of pregnancy urine, there was a notable growth of fibroblasts, compared with explants from untreated animals (Vercesi and Guercio, 1935). Lactogenic hormone was found not to stimulate the growth of chick connective tissue, epidermis or esophageal epithefium (SalIe and Shechmeister, 1936). Estrone, at an optimum concentration of 0.01 mg./100 ml., stimulated fetal mouse heart fibroblasts to greater outgrowth and slightly increased mitotic activity (von Haam and Cappel, 194Oa). Human malignant ascites cells were not affected by a medium saturated with estrone (666 mg./100 ml.) (Ivers, Pomerat, and Neidhardt, 1948). A medium containing 1.25 mg./100 ml. estrone sulfate had, in one case, a remarkable stimulatory effect on the epithelium of human malignant ovarian tissue (Rose, Townsend, and Pomerat, 1951). Among other steroids, von Mollendorff (1941) found that mitotic disturbances were caused by estrone, estradiol, testosterone, methyltestosterone and diethylstilbestrol at 0.10 to 0.25 mg./100 ml. According to Bullough (1952) the mitotic rate in mouse epidermis is increased by glycogen and androgens, the duration of mitosis (about 2% hours) remaining unaltered. With estrone,

56

CHARITY WAYMOUTH

the number of mitoses is increased and the duration of each mitosis is reduced to less than one hour. Cortisone is said to stimulate the secretory activity of the renal proximal tubules of the chick in tissue culture (Chambers and Cameron, 1944) and to reduce the number of cells which migrate from lymph node cultures (Heilman, 1945). There is, however, as Barski and de Brim (1952) and Trowell (1953) have noted, some conflict among the results of different workers with adrenal cortical hormones on cells in vitro. Ruskin, Pomerat, and Ruskin (1951) found that the toxicity of various cortisone preparations (acetate, sulfate, etc.) varied with the preparation and mode of solubilizing it, but that the toxicity was in no case high. In cultures of adult rabbit subcutaneous tissue, spleen, and kidney, and of embryonic mouse liver and lung, which were carried on for two to three weeks, Barslci and de Brion (1952) observed the effects of various concentrations of cortisone or deoxycorticosterone. At 5 to 10 mg./lOO ml., cortisone had no effect on the growth of various fibroblasts or on renal epithelium, nor did it visibly affect collagen formation in vitro. Fifty milligrams per 100 ml. caused degeneration at fifteen to seventeen days in adult rabbit spleen, kidney, and subcutaneous tissues grown in a medium containing, probably in addition to an embryonic extract and a balanced salt solution, 30% homologous serum, 10% horse serum and 5% Ringer solution containing 0.05% Tween 20 (polyoxyalkylene sorbitan monolaurate) rfl the steroid. After about seventeen days in 50 mg./lOO ml. cortisone, all epithelium in the kidney cultures disappeared, leaving healthy histiocytes. Serum from cortisone-treated rabbits stimulated cell migration. Deoxycorticosterone acetate at 50 mg./100 ml. in spleen cultures completely (but reversibly up to six days) inhibited outgrowth. Cornman ( 1950) showed that deoxycorticosterone reversibly inhibited heart beat in tissue culture, and that this inhibition was more effective in the absence of potassium. It has been suggested (Elliott and Yrarrazaval, 1952) that the adrenal hormones (especially cortisone) govern the permeability of cell membranes. Trowell (1953) has reviewed previous work on the effects of cortisone (and C-ll-oxygenated adrenal steroids in general) on lymphocytes in vitro. Several groups of workers, e.g., Baldridge et ad. (1951) and Delaunay, Delaunay, and Lebrun (1949) had found no effects even with rather high concentrations (e.g., 100 mg./100 rnl. cortisone acetate). Using his method (Trowell, 1952) for the maintenance of whole lymph nodes in culture for relatively short periods, Trowell (1953) estimated lymphocyte viability by counts of pyknotic (dead) cells in fixed and stained films after five hours’ treatment with cortisone. Five hours had been found (Trowell, 1953) to be the time of maximum toxic effect

THE NUTRITION OF ANIMAL CELLS

57

with agents such as cyanide, mercury, and X-rays. I n the range 0.01 to 1.0 mg./100 ml., a statistically significantly toxic effect was noted even with 0.01 mg./lOO ml. Increasing the concentration one-hundredfold increased the effect only slightly. When it was found that the maximal effect was obtained with cortisone, not after five but after forty-six hours, a greater spread of effect with concentration appeared, and at the highest concentration (1.0 mg./lOO ml.), 50% of the lymphocytes were pyknotic after forty-six hours. Under the same conditions, other steroids (ll-deoxycorticosterone, 11-deoxycorticosterone acetate, testosterone, estradiol, and progesterone) had little or no effect compared with that of cortisone.

XI. CONCLUDING REMARKS It cannot be said that a clear picture of the general and special nutritional needs of metazoan cells emerges from the present stock of biochemical information. There are, however, indications that the design may be drawn in bold strokes. The ionic environment has important effects on the balance between cells and nutrient medium. The p H and the oxidation-reduction potential, not perhaps strictly nutritional factors, are nevertheless highly relevant to the maintenance of proper physiological conditions. So is the gas exchange between cells and environment. Sources of energy and of all the materials for the renewal or synthesis of protoplasm must be available to the cells. The needs of the cells for prolonged maintenance may prove fewer in number and simpler in structure than has often been assumed. Of the special requirements of growing cells there is still little precise knowledge. Much of the information which has been reviewed here needs to be rescrutinized and systematically retested. Because embryonic cells have proved such convenient experimental material, our knowledge of cell nutrition relates preponderantly to these cells of high growth potential. Comparative investigations are needed of the nutritional requirements of many tissues, embryonic, adult, and neoplastic, in relation to maintenance, growth, and (where appropriate) function. The development of media of exactly known chemical composition in which cells can survive and function for long periods is most important for controlled physiological and pharmacological studies at the cellular level. The study of growth in relation to cell nutrition has long been handicapped by insufficiently precise quantitative methods for assessing growth. Uniform strains of cells, derived from single cells (Sanford, Earle, and Likely, 1948; Likely, Sanford, and Earle, 1952) are now available, and cell nuclei can be enumerated (Sanford et al., 1951) as a means of determining changes in cell population. These must prove powerful aids to the study of nutrition and growth. The key

58

CHARITY WAYMOUTH

questions to open up the next advance are: (1) How far can a given environment be modified by any particular cell type, so that the cell can maintain full functional and metabolic activity? and (2) With how few and how simple components can this environment be prepared experimentally ? The application and amplification of information from the whole field of biochemistry will be needed to supply the answers. ACKNOWLEDGMENT The author acknowledges gratefully the helpful and constructive criticisms of Dr. Philip R. White and Dr. Wilton R. Earle, who read the manuscript.

XII. KEFERENCES Abrams, R., and Goldinger, J. M. (1951) Arch. Biochrn., SO, 261. Abrams, R., and Goldinger, J. M. (1952) Arch. Biochem., 35, 243. Albrink, W. S., and Wallace, A. C. (1951) Proc. SOC.Exptl. Bhl. Med., 77, 754. Anfinsen, C. B., Geiman, Q. M., McKee, R. W., Ormsbee, R. A., and Ball, E. G. (1946) J. Exptl. Med., 84, 607. Anon. (1947) Nutrition Revs., 6, 189. Anon. (1949) Nutrition Revs.,7, 8. Anon. (1950) Nutrition Revs., 8, 181. Astrup, T., Ehrensvard, G., Fischer, A., and flhlenschlager, V. (1947) Acta Physiol. Scand., 14, 195. Xstrup, T., and Fischer, A. (1946) Acta Phytysiol. Scand., 11, 187. Astrup, T., Fischer, A., and ghlenschragger, V. (1947) Acta Physiol. Scad., 13, 267. Astrup, T., Fischer, A., and Volkert, M. (1945) Act0 Phy&l. Scad., 9, 134. Bach, S. J., and Lasnitzki, I. (19.17) Enynzologia, U,198. Bacon, J. S. D., and Bell, D. J. (1948) Biochern. J., 42, 397. Bacq, Z. M., and Chhvremont, M. (1944) Con@. rercd. mc. bwl., 138, 888. Baitsell, G. A., and Sherwood, M. B. (1925) Proc. SOC.Expfl. Biol. Med., 2S, 96. Baker, L E. (1929) J. Expfl., Med., 49, 163. Baker, L. E. (1933) J. Exptl. &led., 57, 659. Baker, L. E. (1935a) Proc. SOC.Exptl. Bid. Me& 33, 124. Baker, L E. (1935b) Comfit. rend. SOC. bid., lzo, 1160. Baker, L. E. (1936) Scimce, SS, 605. Baker, L. E. (1938) Proc. Soc. Exptl. Biol. Mcd., 39, 369. Baker, L. E., and Carrel, A. (1926a) J . Exptl. Vcd., 44, 387. Baker, L. E., and Carrel, A. (1926b) Compt. rend. S O T . hiol., 95, 157. Baker, L. E., and Carrel, A. (192%) Compt. rend. SOC. biol., 95, 958. Baker, L. E., and Carrel, A. (1926.d) Compt. rmd. soc. h i d , 95, 1014. Baker, L. E., and Carrel, A. (1926e) J . Exptl. .Wed., 44, 397. Baker, I,. E., and Carrel, A. (1927) J . Exptl. M P ~ 45, . , 305. Baker, L. E., and Carrel, A. (1928a) J . Ezptl. Med., 47, 353. Baker, L. E., and Carrel, A. (1928b) J . Exptl. Med., 47, 371. Baker, L. E., and Carrel, A. (192%) J. Exptl. Med.. 48, 533. Baker, L. E., and Ebeling, A. H. (1938) Proc. SOL.Expfl. Bid. Med.. 39, 291. Baker, L. E., and Ebeling, A. H. (1939) J . E z g f l . Med., 69, 365. Baldridge, G. D., Kligman, X. M., Lipnik, M. J., and Pillsbury, D. M. (1951) Arch. Pathol., 61, 593.

T H E NUTRITION OF A N I M A L CELLS

59

Barklay, H., Haas, P., Huggett, A. St G., King, G., and Rowley, D. (1949) I . Physwl., 109, 98P. Barron, E. S. G., Lyman, C. M., Lipton, M. A., and Goldinger, J. M. (1941) J . Biol. Chew., 141, 957. Barski, G., and de Brion, G. (1952) Ann. Inst. Pasteur, 62, 563. Barski, G., Maurin, J., Wielgosz, G., and Upine, P. (1951) Ann. Inst. Pastmr, 61. 9. Rarta, E. (1925) drch. Exptl. Zellforsch. Gewcbeziicht., 2, 6. Barta, E. (1926) Compt. v d . soc. biol., 94, 1182. Bergmann, M., and Niemann, C. (1936) J. Biol. Cltem., 116, 77. Bergman, R. A. M., and Waterman, N. (1935) Acta Brezria N e e d Physiol. Pharmacol. Mkrobiol., 6, 103. Betker, N., and Wormiak, J. J. (1952) Abstract of meeting of Tissue Culture Association. Bisceglie, V. (1926) Wilhelm Roux’ Arch. Entm’cklwngsmech. Orgam., 108, 708. Bligh, J. (1952) J. Physiol., 117, 234. Bloch, K. (1952) A m . Rev. Biochewa., 21, 273. Block, R. J., and Bolling, D. (1945) The Amino Acid Composition of Proteins and Foods, 1st ed. Charles C Thomas, Springfield, Ill. Boyer, H. K., and Kirk, P. L. (1952) J. Ccllular. Comp. Physiol., 99, 95. Braunstein, A. E. (1947) Advances in Prof& Chew., 3, 1. Brin, M. (1953) Federation Proc. 12, 182. Brues, A. M., SubbaRow, Y., Jackson, E. B., and Aub, J. C. (1940) J . Exptl. Mcd., 71, 423. Bullough, W. S. (1949) Nature, 163, 680. Bullough, W. S. (1950) Exptl. Cell Research, 1, 410. Bullough, W. S. (1952) Biol. Revs., 27, 133. Bullough, W. S., and Johnson, M. (1951a) N a t w e , 167, 488. Bullough, W. S., and Johnson, M. (1951b) Exptl. Cell Research, 2, 445. Bullough, W. S., and Johnson, M. (1951~)Proc. Roy. Soc. (London) BlSS, 562. Burrows, M. T. (1910) I. Am. Med. Assoc., 66, 2057. Burrows, M. T. (1911) J. Exptl. Zool., 10, 63. Burrows, M. T. (1921) Proc. Sac. Exptl. Biol. died., 18, 133. Burrows, M. T., and Neymann, C. A. (1917) I. Exftl. Med., 26, 93. Burrows, M. T., and Neymann, C. A. (1918) Proc. SOC.Exptl. Biol. Med., 16, 138. Burt, A. S. (1943a) Proc. SOC.Exptt. Biol. Med., 64, 191. Burt, A. S. (1943b) J. Cellular Comfi. Physiol., 21, 145. Burt, A. S. (1943~)J. Cellular Con@. Physiol., 22, 205. Cameron, G. (1950) Tissue Culture Technique. Academic Press, New York. Carleton, H. M. (1923) Brit. .7. Exfit!. Riot., 1, 131. Carminati, V. (1933) Arch. Exptl. Zellforsch. Gewebeziicht., 19, 661. Carrel, A. (1911) J. Am. Med. Assoc., 67, 1611. Carrel, A. (1912a) I . Expfl. Med., 16, 393. Carrel, A. (1912b) I. E x j t l . Med., 16, 516. Carrel, A. (1912~)I. Exptl. Med., 16, 165. Carrel, A. (1913a) J. Exptl. Med., 17, 14. Carrel, A. (1913b) 1. Exptl. Med., 18, 287. Carrel, A. (1913~)Berlin kliit. Wochschr., W, 1097. Carrel, A. (1922) I. Exptl. Med., 36, 385. Carrel, A. (1924a) PhySiol. Revs., 4, 1.

60

CHARITY WAYMOUTH

Carrel, A. (1924b) J. Ant. Med. Assoc., 82, 255. Carrel, A. (1924~) Compt. rend. SOC. biol., 90, 29. Carrel, A. (1927) Compt. rend. SOC. biol., 96, 603. Carrel, A. (1928) Arch. exptl. Zellforsch. Gewebeziicht., 6, 70. Carrel, A. (1938) In Foreword to R. C. Parker’s Methods of Tissue Culture, 1st ed. Hoeber, New York. Carrel, A., and Baker, L. E. (1926a) Proc. SOC.Exptl. Biol. Med., 23, 627. Carrel, A., and Baker, L. E. (1926b) Compt. rmd. SOC. biol., 911, 359. Carrel, A., and Baker, L. E. (19%~)J . Expfl. Med., 44, 503. Carrel, A., and Baker, L. (1927) Compt. r e d . sac. bid., 36, 685. Carrel, A., Baker, L. E., and Ebeling, A. H. (1927) Arch. exjtl. Zellforsch. Gmebesiicht., 6, 125. Carrel, A., and Burrows, M. T. (1911a) I. Exptl. Med., 13, 387. Carrel, A., and Burrows, M. T. (1911b) J. Exptl. X e d , 13, 562. Carrel, A., and Ebeling, A. H. (1921a) J. ExptE. Med., S4, 317. Carrel, A., and Ebeling, A. H. (1921b) I. Expfl. Med., 34, 599. Carrel, A,, and Ebeling, A. H. (192a) I. ExPtE. Med., 86, 365. Carrel, A., and Ebeling, A. H. (1922~)J. Exptl. Med., 36, 17. Carrel, A., and Ebeling, A. H. (1922~)1. ExpfL Med., 36, 645. Carrel, A., and Ebeling, A. H. (1923a) J. Exptl. Med., 37, 653. Carrel, A,, and Ebeling, A. H. (1923b) J. Exptl. M e d , 37, 759. Carrel, A., and Ebeling, A. H. (1923~)J , Exptl. Med., 38, 419. Carrel, A., and Ebeling, A. H. (1923d) J . Exptl. Med., 38, 499. Carrel, A., and Ebeling, A. H. (1923e) J . Expfl. Med., 38, 487. Carrel, A., and Ebeling, A. H. (1923f) J. Exptl. Med., 38, 513. Carrel, A., and Ebeling, A. H. (1923g) Compt. rend. SOC. biol., 89, 1142. Carrel, A., and Ebeling, A. H. (1923h) Compt. rend. SOC. biol., 89, 1266. Carrel, A., and Ebeling, A. H. (1926a) J. Ex#& Xed., 44, 261. Carrel, A., and Ebeling, A. H. (1926b) J. Exptl. Med., 44,285. Carrel, A., and Ebeling, A. H. (1928) J. Exptl. Med., 48, 105. Chambers, R., and Cameron, G. (1943) A m . 1. PhysioE., 139,21. Chambers, R., and Cameron, G. (1944) Am. J. P h y h L , 141, 138. Champy, C., and Coca, F. (1914) Compt. rend. sol. biol., 77, 238. Chtvremont, M. (1943) Acta biol. Belgica, 1, 60. Chevremont, M. (1945) I. MorphoE., 76, 139. Chhrremont, M. (1948) Bio?. Revs., 23, 267. Chlopin, N. C. (1930) Comfit. rend. SOC. biol., 103, 14. Christensen, H. N. (1948) Federatian Proc., 7, 150. Christensen, H.N., and Henderson, M. E. (1952) Cancer Research, 12,229. Christensen, H.N., and Lynch, E. L. (1948) J. Biol. Chem., 172, 107. Christensen, H.N., and Rafn, M. L. (1952) Cancer Research, 12, 495. Christensen, H. N., and Riggs, T. R. (1952) J. Biol. C h . ,194, 57. Christensen, H. N., and Riggs, T. R., Fischer, H., and Palatine, I. M. (1952) J . Biol. Chm., 198, 1. Christensen, H. N., and Streicher, J. A. (1948) J. Biol. Chent., l.76, 95. Cohen, P. P. (1951) The Enzymes, Vol. 1, Part 2, p. 1040. Academic Press, New York. Cornman, I. (1950) Proc. SOC. ExptI. Biol. Xed., 76, 355. Crumpler, H. R., Dent, C. D., and Lindan, 0. (1950) Biochem. J., 47, 223.

THE NUTRITION OF ANIMAL CELLS

61

Cunningham, B., and Kirk, P. L. (1942) J . CeIluIar Come. Physiol., 20, 343. Danes, B. (1949) J. Exptl. Zool., 112, 417. Danes, B., and Leinfelder, P. J. (1951) 1. Cellular Comp. Physiol., 37, 427. Davidson, J. N., Leslie, I., and Waymouth, C. (1949) B i o c h . f., 44, 5. Davidson, J. N., and Leslie, I. (1951) Exptl. Cell. Research, 2, 366. Davidson, J. N., and Waymouth, C. (1943) B i o c k . J., 37, 271. Davidson, J. N., and Waymouth, C. (1944a) Nutrition Abstr. 6. Revs.,14, 1. Davidson, J. N., and Waymouth, C. (1944b) Quart. J. Expfl. Physiol., 33, 19. Davidson, J. N., and Waymouth, C. (1945) Biochem. J., 39, 188. Davidson, J. N., and Waymouth, C. (1946) Biochem. J., 40, 568. Delaunay, A., Delaunay, M., and Lebrun, J. (1949) Ann. Inst. Pusteur, 76, 203. Delaunay, A., and PagPs, J. (1946) Rev. immwwl., 10, 33. Demuth, F. (1931) Arch. exptl. Zellforsch. Gewebeeiicht., 11, 98. Doljanski, L., and Goldhaber, G. (1945) Proc. SOC.Exptl. Biol. Med., So, 132. Doljanski, L., and Hoffman, R. S. (1939) Compt. rend. SOC. biol., 190, 1246. Doljanski, L., and Hoffman, R. S. (1943) Growth, 7, 67. Doljanski, L., Hoffman, R. S., and Tenenbaum, E. (1939) Compt. rend. SOL. B i d , 13, 432. Doljanski, L,Hoffman, R., and Tenenbaum, E. (1942) Nature, 160, 23. Drew, A. H. (1922) Brit. f. Exptl. Pathol., 3, 20. Drew, A. H. (1923) Lmcet, 204, 785. Drysdale, G. R., Plaut, G. W. E., and Lardy, H. A. (1951) J . Biol. Chem., 199, 533. Dubin, I. N., and Yen, C. K. (1950) Arch. Pathol., 60, 562. Duthie, E. S., and Chain, E. (1939) Brit. 1,Enptl Pafhol., 20, 417. Earle, W. R. (1943) J . Nutl. Culrcer Insf., 4, 165. Ebeling, A. H. (1914) J , Exptl. Med., 20, 130. Ebeling, A. H. (1924) Compt. r e d . SOL. biol., 90, 31. Ebeling, A. H. (1936) Proc. SOC.Exptl. Biol. Med., #, 886. Ehrensvard, G,, Fischer, A., a i d Stjernholm, R. (1949) Acta Physiol. Scand., 18, 218. Ehrmann, R. L., and Gey, G. 0. (1953) 1. N@l. Cancer Inst., 13, 1099. Elliott, K. A. C., and Yrarrazaval, S. (1952) Nature, 169, 416. Ellis, J. P., Nowinski, W. W., and Bieri, J. G. (1953) Texas Repts. Biol. and Med., 11, 231. Elman, R., and Weichselbaum, T. E. (1952) Science, 116, 521. Enders, J. F. (1953) Proc. Sor. Exptl. Biol. Med., 82, 100. Ephrussi, B., Chevillard, L., Mayer, A., and Plantefol, L. (1929) Ann. physiol., Paris, 6, 642. Erlenbach, F. (1938) 2. vergkich. Physiol., 26,121. Erlichman, E. (1935) Am. J. Cancer, W ,393. Evans, V. J., Shannon, J. E., Bryant, J. C., Waltz, H. K., Earle, W. R., and Sanford, K. K. (1953) I. Natl. Cancer Inst., 13, 773. Fell, H. B., and Mellanby, E. (1952) 1. Physiol., 116, 320. Fell, H. B., and Mellanby, E. (1953) J . Phydol., 119, 470. Fischer, A. (1924) J . Exptl. Med., 39, 577. Fischer, A. (1925a) Acta Pathol. Microbial. Scand., 2, 7. Fischer, A. (1925b) Arch. exptl. Zellforsch. Gewcbeziicht., 1, 369. Fischer, A. (1929) Natiinciss., 17, 157. Fischer, A. (1930) Gewebeziichtung. &fuller and Steinicke, Miinchen. Fischer, A. (1933) Ergebn. Physiol., 36, 82.

62

CHARITY WAYMOUTH

Fischer, A. (1939) Natwe, 144, 113. Fischer, A. (1940) Chem. Prodwts, 3, 79. Fischer, A. (1941a) Acta Physiol. Scand., 2, 143. Fischer, A. (1941b) Acta Pfiysiol. S c a d , 3, 54. Fischer, A. (194Za) Acta PfiyySiol. Scand., 4, 207. Fischer, A. (1942b) N a t u m i s . , 44/46, 665. Fischer, A. (1946a) Biology of Tissue Cells. Gyldendal, Copenhagen. Fischer, A. (1946b) Cfiem. Products, 9, 74. Fischer, A. (1947) Biol. Revs.,22, 178. Fischer, A. (1948a) Biochem. I.,43, 491. Fischer, A. (1948b) Acta Anat., 6, 57. Fischer, A. (1948~)Scimtia, 6, 170. Fischer, A. (194M) Natwe, 161, 1008. Fischer, A. (1950) Enzymologia, 14, 15. Fischer, A., and Astrup, T. (1942) Pffiigers Arch. ges. PhyySiol., 246, 633. Fischer, A., and Astrup, T. (1943) Pffiigers, Arch. ges. Physiol., 247, 34. Fischer, A., Astrup, T., Ehrensvard, G., and e)hlenschlager, V. (1948) Proc. SOC. Exptl. Biol. Med., 67, 40. Fischer, A., and Demuth, F. (1927-1928) Arch. exptl. Zellforsch. Cewebesiicht., 6, 131. Fischer, A., Fischer, G., Landschutz, C., Ehrensvard, G., Rafelson, M., and Stjernholm, R. (1953) Acto Physiol. S c a d , 27, 247. Fischer, A., and Parker, R. C. (1929) Proc. SOC.Exptl. Riol. Med., 26, 585. Foa, P. P., Weinstein, H. R., Smith, J. A., and Greenberg, M. (1952) Arch. Biochem. and Biophys., 40, 323. Francis, M. D., and Winnick, T. (1953) J . Biol. C h m . , 202, 273. Frazer, A. C. (1952) Ann. Rev. Biochcm., 21, 245. FrCdCric, J. (1951) Rev. he'inatol., 6, 423. Friedheim, A. H., and Roukhelman, N. (1930) Compt. rend. soc. biol., 105, 10. Gaillard, P. J. (1935) Protoplasma, 23, 145. Gaillard, P. J. (1942) ActwZitis sci. ef id.,923. Gaillard, P. J., and Varossieau, W. W. (1938) Actu. Need. Morphol., 1, 313. Gaillard, P. J., and Veer, W. L. C. (1948) Konink1:Ned. Akad. Wetenschap. Proc., 61, 807. Gebler, H., Roll, P. M., Tinker, J. F., and Brown, G. B. (1949) I. Biol. Cfienr., 178, 259. Gerarde, H. W., Jones, M., and Winnick, T. (195Za) J . Biol. Chem., 196, 51. Gerarde, H. W., Jones, M., and Winnick, T. (1952b) J. Biol. Chem., 196, 69 Gey, G. 0. (1929) Wisconsin Med. J., 28, 11. Gey, G. O., and Gey, M. K. (1936) Am. 1. Cancer, 27, 45. Gey, G. O., and Thalhimer, W. (1924) I. Am. Med. Assoc., 82, 1609. Gomes da Costa, S. F. (1935) Bol. inst. med. exptl. estud. clincer (Buenos Aires), 12, 619. Goodwin, R. F. W. (1952) Nature, 170, 750. Gordonoff, T., and Ludwig, F. (1935) 2. Yitaminforsch., 4, 213. Gordonoff, T., and Ludwig, F. (1936) Sch7crek wwd. Worhschr., 17, 1129. Greenberg, G. R. (1948) Arch. Biochcm., 19, 337. Grossfeld, H. (1949) Proc. Sac. Exptl. Biol. hled., 71, 475. Guillery, H. (1930) Virchuds .4rck. patkol. Anat. u. Physiol., 276, 181. Gunsalus, I. C. (1950) Federation Proc., 9, 560.

THE NUTRITION OF ANIMAL CELLS

63

vonHaam, E., and Cappel, L. (1940a) Am. J. Cancer, 89, 350. vonHaanb E., and Cappel, L. (1940b) Am. 1. Cancer, 39, 354. Haddow, A. (1947) Growth (Sytnp.), 11, 339. Hamilton, H. L,and Plotz, H. (1942) Proc. SOC.Exptl. Biol. Med., 60, 133. Hamilton, P. B. (1945) I . Biol. Chem., 168, 397. Hanks, J. H. (1948) J. Cellular Comp. Pbysiol., Sl, 235. Hanks, J. H. (1949) Proc. SOC.Exptl. Biol. Med., 71, 328. Harris, M. (1951a) Anat. Record, 109, 301. Harris, M. (1951b) AM^. Record, 109, 372. Harris, M. (1952a) J. Cellular Comp. Physiol., 40, 279. Harris, M. (1952b) Growth, 16, 215. Harrison, R. G. (1907) PYOC.SOC.Exptl. Biol. Med., 4, 140. Hartman, J. D. (1952) Proc. SOC.Exptl. Biol. Med, 79, 3. Hass, G. M., Schweitzer, A. W., and Boscia, H. (1950) Federation Proc., 9, 333. Hass, G. M., Schweitzer, A. W., and Boscia, H. (1951) Federatioii PPOC.,10, 358. Healy, G. M., Morgan, J. F., and Parker, R. C. (1952) 1. Biol. Chem., 198, 305. Heaton, T. B. (1926) f . Pathni. BacEterial., 29, 293. Heilman, D. H. (1945) Proc. Staf Meetings Mayo Clinic, 20, 318. Heinrich, M. R., and Wilson, D. W. (1950) I . Biol. Chem., 186, 447. Hengstmann, H. (1938) 2. P’itanrinforsch., 8, 208. Exptl. Bwl. Med., 62, 312. Hetherington, D. C. (1946) Proc. SOC. Hetherington, D. C., and Shipp, M. E. (1935) BioI. Bull., 68, 215. Hill, R. T., Corkill, A. B., and Parkes, A. S. (1934-5) Proc. Roy. SOC.(London), B116, 208. Hitchcock, M. W. S. (1949) J . Physiol., 108, 117. Hoffman, R. S. (1940) Growth, 4, 361. Hoffman, R. S., Dingwall, J. A., and Andrus, W. de W. (1948) Proc. SOC.Exptl. Biol. Mcd., 69, 364. Hoffman, R. S., Dingwall, J. A,, and Andrus, W. de W. (1951) Science, 113, 268. Hoffman, R. S., and Doljanski, L. (1939) Growth, 3, 61. Hoffman, R. S., Goldschmidt, J,, and Doljanski, L. (1937) Growth, 1, 228. Hoffman, R. S., Tenenbaum, E., and Doljanski, L. (1939a) Nature, 145, 764. Hoffman, R. S., Tenenbaum, E., and Doljanski, L. (1939b) Nature, 144, 1092. Hoffman, R. S., Tenenbaum, E., and Doljanski, L. (1940) Growth, 4, 207. Hopkins, F. G., and Simon-Reuss, I. (1944) Proc. Roy. SOC.(London), BlS2, 253. Hosono, S., and Narjsawa, S. (1931) Trans. Japan. Pathol. SOC.,21, 121. Hudspeth, E. R., Swann, H. G., and Pomerat, C. M. (1950) Texas Repts. Biol. mid Med., 8, 341. Hueper, W. C., Allen, A., Russell, M. A., Woodward, G., and Platt, M. (1933) Am. 1. Cancer, 17, 74. Hueper, W. C., and Russell, M. A. (1933) Arch. exptI. Zellforsch. Gewebesicht., 14, 483. Hughes, A. (1952) Ex@ Cell Research, 9, 108. Hughes, A. F. W., and Preston, M. M’E. (1949) 1. Roy. Microscop. Sor., 69, 121. Hull, W., and Kirk, P. L. (1950) J . Got. Physiol., 33, 327. Hull, W., and Kirk, P. L (1950) 1. Gen. Pkyiol., SS, 335. Hull, W., and Kirk, P. L. (1950) J . Got. Physiol., S4, 81. Hull, W., Perrone, J. C., and Kirk, P. L. (1950) J . G a . P h y ~ o l . ,54, 75. hgebrigtsen, R. (1912a) I. Exptl. Med., 16, 397.

64

CHARITY WAYMOUTH

Ingebrigtsen, R. (191231) I. Exptl. Med., 16, 421. Ivers, J. B., and Pomerat, C. M. (1947) Texas Repts. Biol. m d Med., 6, 92. Ivers, J. B., Pomerat, C. M., and Neidhardt, H. W. (1948) Texas Repprs. Biol. and Med., 6, 206. Jacoby, F. (1937a) J. Physiol., 90, 23P. Jacoby, F. (1937b) Arch. exptl. Zellforsch. Gewebesiicht., 19, 241. Jacoby, F. (1938) I. Physiol., 98, 48P. Jacoby, F. (1940) J. Physiol., 96, 6P. Jacoby, F. (1941) J. Physbl., 100, 2P. Jacoby, F. (1945) Brit. J . Exptl. Pathol., 26, 137. Jacoby, F. (1949) Exgtl. Cell Research, Szlppl. 1, 454. Jacoby, F., and Darke, S. J. (1948) Nature, 161, 768. Jacquez, J. A., Barclay, R. K., and Stock, C. C. (1952) J. Exptl. Med, 96, 499. Jacquez, J. A., and Barry, E. (1951) J. Gen. Physiol., 34, 765. Jazimirska-Krontowska, C. (1930) Compt. rend. SOC. biol., lOS, 1182. Juhisz-Schaffer, A. (1931) Virchow's Arch. pathd. Anat. u. Physiol., Bl, 35. Kandutsch, A. A., and Baumann, C. A. (1953) J. Nutrition, 49, 209. Katzenstein, M., and Knake, E. (1931) 2. Krebsforsch., 88, 378. Kiaer, S. (1925) Arch. exptl. Zellforsch. Gewebeziicht., 1, 289. Kidder, G. W., (1952) Intern. Rev. Cytol., 1, 27. Kihara, H., Klatt, 0. A., and Snell, E. E. (1952) J . Biol. Chem., 197, 801. Kihara, H., and Snell, E. E. (1952) J. Biol. C h . , 197, 791. Kirby, D. B., Estey, K. C., and Weiner, R. von E. (1933) Arch. Ophthdmol. (Chicago), 10, 37. Kimura, T. (1938) Kumamofo lgukkai Zasshi, 14, 19; quoted by Tazima (1940a). Knight, B. C. J. G. (1945) Vitamins and Hormones, 8, 105. Krebs, H. A. (1950) A m . Rev. Biochem., 19, 489. Krebs, H. A., and Eggleston, L. V. (1949) Biochem. J., 44, vii. Kuczinski, M. H., Tenenbaum, E., and Werthemann, A. (1925) Virchods Arch. pafhol. Anat. u. Physiol., 258, 687. Kutsky, R., and Harris, M. (1952) Anat. Record, 112, 419. Lagerkvist, U., Reichard, P., and Ehrensviird, G. (1951) Acto Clcem. Scud.., 6, 1212. Lambert, R. A. (1912) Anat. Record, 6, 91. Lambert, R. A. (1914) J . Exptl. Med., 19, 398. Lambert, R. A., and Hanes, F. M. (1911) J. Exptl. Med., 14, 129. Landschiitz, C. (1952) 2. Krebsforsch., 68, 599. Landsteiner, K., and Parker, R. C. (1940) J. Exptl. Med., 71, 231. Latta, J. S., and Bucholz, D. J. (1939) Arch. exptl. Zellforsch. Gmebeziicht. aS, 146 Leslie, I. (1952) Biochem. J., 62, xxi. Leslie, I., and Davidson, J. N4 (1951a) Biochim. et Biophys. Acta., 7 , 413. Leslie, I., and Davidson, J. N. (1951b) Biochem. J., 49, xli. Lettrh, H., and Albrecht, M. (1941) 2. physiol. Chem., 271, 200. LettrC, H., and Albrecht, M. (1943) 2. physiol. Chem., 279, 206. Lewis, M. R. (1921) Proc. Am. Assoc. Anuf., 21, 71. Lewis, M. R. ( 1 9 2 ) J . Esptl. Med., 311, 317. Lewis, M. R., and Lawler, T. G. (1931) Anat. Record, 48, 51. Lewis, M. R., and Lewis, W. H. (1911a) Anat. Record, 6, 277. Lewis, M. R., and Lewis, W. H. (1911b) Bull. Johns Hopkim Hosp., 22, 126. Lewis, M. R., and Nettleship, W. A. (1932-1933) Arch. exptl. Zellforsch. Gmcbe&W., ls, 111.

THE NUTRITION OF ANIMAL CELLS

65

Lewis, W. H. (1929) Carnegie inst. Wash. Pub[. No. 394, Contribs. Embryol., 20, 173. Lewis, W. H. (1935) Carnegie Inst. Wash. Publ. No. 160, Cotttribs. Embryol., 26, 161. Lewis, W. H. (1939) Arch. exptl. Zeliforsch. GewebeziicM., 23, 8. Lewis, W. H., and Lewis, M. R. (1912) Ailat. Record, 6, 207. Lewis, W. H., and Lewis, M. R. (1925) i n General Cytology, Section VII. The Univ. of Chicago Press, Chicago. Likely, G. D., Sanford, K. K., and Earle, W. R. (1952) J. Natl. Cancer Inst., 13, 177. des Ligneris, M. J. A. (1931) Arch. exptl. Zellforsch. Gewebeziicht., 11, 355. Locke, F. S. (1895) Boston Med. Surg. J., 134, 173. Locke, F. S. (1901) Zentr. Phyriol., 14, 670. Loeb, L. (1912) Anat. Record, 6, 109. deLustig, E. S. (1951) Rev. SOC. argarfina bioE., 27, 114. MacLRod, P. R., and Lardy, H. A. (1949) J . Biol. Chem., 179, 733. Macleod, J., and Rhoads, C. P. (1939) Proc. Sac. Exptl. Biol. Med., 41, 268. McKee, R. W., Ormsbee, R. A., Anfinsen, C. B., Geiman, Q. M., and Ball, E. G. (1946) J . ExptI. hied., 84, 569. McKee, R. W., and Lonberg-Holm, K. K. (1953) Federation Proc., la, 244. Maganini, H., Schweitzer, A. W., and Hass, G. M. (1953) Federation Proc., l!2, 395. Manners, D. J., and Ryley, J. F. (1952) Biochem. J., 62, 480. Margoliash, E., and Doljanski, L. (1950) Growth, 14, 7. Margoliash, E., Tenenbaum, E., and Doljanski, L. (1948) Gvuzuth, 3!2, 1. Medawar, P. B. (1947) Quurt. J. Microscop. Sci., 88, 27. Medawar, P. B. (1948a) Qwart. J. Microscop. Sci., 89, 187. Medawar, P. B. (1948b) Quart. J . Microscop. Sci., 89, 239. Meister, A,, Sober, H. A., and Peterson, E. A. (1952) J. Am. Chem. SOC.,74, 2385. Messina, L., and Verga, G. (1937) Giorn. bafteriol. immunol., 19, 850. Miszurski, B. (1939) Arch. exptl. Zellforsch. Gewebesiicht., 23, 80. von MGllendorff, W. (1941) Schzueiz. wed. Wochschr., 71, 329. Moppett, W. A. (1927) Med. J. Australia, 1, 335. Morgan, J. F., Morton, H. J., and Parker, R. C. (1950) Proc. SOC.Exptl. Bwl. Med., 7s, 1. Morgan, J. F., Morton, H. J., and Parker, R. C. (1951) Growth, 16, 11. Morgan, J. F. (1950) I n Methods of Tissue Culture, Chapter VIII, p. 115. 2nd ed. Hoebcr, New York. Morse, A. (1917) Bull. Johns Hopkins Hosp., 28, 199. Morton, H. J., Morgan, J. F., and Parker, R. C. (1950) Proc. Sac. Exptl. Biol. M e d , 74, 22. Morton, H. J., Morgan, J. F., and Parker, R. C. (1951) J . Cellular Comfi. Phytysiol., 38, 389. Mottram, J. C. (1927) Lancef, 213, 1232. Needham, J., and Nowinski, W. W. (1937) Biochenz. J., 91, 1165. Nixon, D. A. (1952) I . Physiol., 117, 70P. Nungester, W. J., and Ames, A. M. (1948) J . Infecfious Diseases, 83, 50. Olivo, 0. M. (1931) Arch. exptl. Zellforsch. Gmebeziicht., 11, 272. Osgood, E.E., Li, J. G., Tivey, H., Duerst, M. L., and Seaman, A. J. (1951) Science, 114, 95. Painter, J . T., and Pomerat, C. M. (1948) Federation Proc., 7, 95. Painter, J . T., Pomerat, C. M., and Ezell, D. (1949) Texas Repts. Biol. and Med., 3, 417.

66

CHARITY WAYMOUTH

Pannett, C., and Compton, A. (1924) Lancet, 206, 381. Parker, R. C. (1933) J. Exptl. Med., 68, 97. Parker, R. C. (1936a) Science, 83, 579. Parker, R. C. (1936b) J. ExptZ. Med., 64, 121. Parker, R. C. (1938) Methods of Tissue Culture. Hoeber, New York. Parker, R. C. (1950) Methods of Tissue Culture, 2nd e d Hoeber, New York. Parshley, M. S., and Simms, H. S. (1946) Anut. Record, 94, 486. Parshley, M. S., and Simms, H. S. (1950) A m . J . Anat., 86, 163. Paterson, E., and Thompson, M. V. (1943) Biochcm. J., 37, 511. Paulmann, F. K. (1940) Arch. exptl. Zellforsch. Gewebeziicht., 24, 115. Pearson, P. B., Melass, V. H., and Sherwood, R. M. (1946) J . Nutrition, 32, 187. Pires Soares, J. M. (1947) Arch. port. sci. biol., 9, 77. Pirie, N. W. (1947) Nature, 160, 198. Pomerat, C. M., Nowinski, W. W., and Rose, G. G. (1950) Texus Repts. Biol. itmi Med., 8, 521. Pomerat, C. M., and Willmer, E. N. (1939) J. Exptl. Biol., 16, 232. Popjkk, G., and Beeckmans, M. L. (1950) B i o c k m . J., 46, 547. Popjhk, G., and Muir, H. (1950) Biochem. J., 46, 103. Pybus, F. C., and Fawns, H. T. (1931) J. Pafhol. Bacteriol., 34, 39. Racker, E., and Krimsky, I. (1952) I. B i d . Chew., 198, 721. Reichard, P., and Bergstrom, S. (1951) Actu C l m . Scopld., 6, 190. Rerabek, E., and Rerabek, J. (1952) 2. Zellforsch. u. mikroskop. Anat., 56, 503. Riggs, T. R., Coyne, B. A., and Christensen, H. N. (1953) Federation Proc., 12, 258. Ringer, S. (1886) J. Physiol., 7, 291. Roffo, A. H. (1925) Ne'oplusmes, 4, 148. Roffo, A. H. (1928a) Prmm med. urgent., 16, 720. Roffo, A. H. (192813) Bol. inst. med. exptl. estrrd. cancer ( B u m s Aires), 4, 391. Roffo, A. H. (1930) BdZ. ussoc. f r a q . ktz& cancer, 19, 700. Roffo, A. H., and Calcagno, 0. (1928) 301.b s t . med. expfl. estud. ccincer (Buenas Aires), 6, 447. Roffo, A. H., and Ferramola, R. (1930) Bol. imf. med. ezptl. e s t d . cancer (Bwrzos Aires), 7 , 612. Rose, W. C. (1938) PhySiol. Revs., 18, 109. Rose, W. C., Oesterling, M. J., and Womack, M. (1948) J . Bid. Chem., 176, 753. Rose, G. G., Townsend, F. M., and Pomerat, C. M. (1951) J. Natl. Cancer Inst., 11, 1223. Rosenberg, S., and Kirk, P. L. (1953) Science, 117, 566. Rosenthal, H. L., and Sarett, H. P. (1952) 1. Biol. Chem., 199, 433. Rosin, A., and Rachmilewitz, M. (1948) Blood, S, 165. Ross, E. J. (1953) Nature, 171, 125. Rossi, F. (1935) Boll. SOC. itd. b i d . sper., 10, 843. Rossi, F. (1936) Boll. sac. ital. biol. sper., 11, 460; Boll. SOC. med.-chir. Pavia, 60, 457; quoted by Vogelaar and Erlichman (1937). Ruskin, B., Pomerat, C. M.,and Ruskin, A. (1951) Tezus Repts. BWI. arid M e d , 9, 786. Salisbury, P. F. (1947) I. Cellular Comp. Physiol., aS. 345. Salle, A. J., and Shechmeister, I. L. (1936) Proc. SOC. Exptl. Biol. Med., 34, 603. Sanford, K. K., Earle, W. R., Evans, V. J., Waltz, H. K., and Shannon, J. E. (1951) J . NatK. Cancer Inst.. 11, 773.

T H E NUTRITION OF ANIMAL CELLS

67

Sanford, K. K., Earle, W. R., and Likely, G. D. (1948) I . Natl. Cancer Ircst., 9, 229. Sanford, K. K., Waltz, H. K., Shannon, J. E., Earle, W. R., and Evans, V. J. (1952) J . Natl. Cancer Iwt., 13, 121. Schlenk, F., and Fisher, A. (1947) Arch. Biuchem., 12, 69. Schreier, K., and Stieg, H. (1950) Z . Kinderheilk., 68, 563. Semura, S. (1931) Arch. exptl. Zellforsch. Gewebeziicht, 10, 329. Shooter, R. A., and Gey, G. 0. (1952) Brit. J. Exptl. Pathol., 88, 98. Signorotti, B., Hull, W., and Kirk, P. L. (1950) J . Gen, Physiol., 88, 315. Simms, H. S. (1936) Science, 83, 418. Simms, H. S., and Sanders, M. (1942) Arch. Pafhol., 88, 619. Simms, H. S., and Stillman, N. P. (1937) I. Gem. Physiol., !M, 649. Sinclair, H. M. (1952) Biochm. J . 61, x. Skipper, H.E., Mitchell, J. H., Jr., and Bennett, L. L., Jr. (1950) Cancer Research. 10, 510. Smyth, H.F. (1914-15) J . Med. Research, 51, 256. Snell, E. E., and Quarles, E. (1941) 1. Nutrition, 23, 483. Snellman, B. (1937) Arch. exptl. Zellforsch. Gewebeziicht., 20, 213. Some, J. C., and Lin, I. (1952) Federation Proc., 11, 290. Some, J. C., and Lin, I. (1953) Fe&ration Proc., 12, 271. Spector, W.G. (1951) J. Pathol. Bactm'ol., 63, 93. Spratt, N. T. (1949) J. Exptl. Zool., 110, 273. Spratt, N. T. (1950) J. Exptl. Zool., 114, 375. Stewart, D. C., and Kirk, P. L. (1952) J . Cellular Comp. Physiol., 40, 183. Stuermer, V. M. and Stein, R. J. (1950) Am. J . Obstef. Gynecol., 60, 1332. Suvarnakich, K., Mann, G. V., and Stare, F. J. (1952) J. Nuttitwa, 47, 105. Szarski, H. (1950) Bull. acad. polon. sci. et lettres, B a n ,331. Szarski, H. (1951) Experientk, 7, 355. Tarver, H. (1952) A m Rev. Biochem., 21, 301. Tazima, M. (194Oa) TBhuku J. Exptl. N e d . , 38, 1. Tazima, M. (1940b) TBhukir J. Exptl. filed., 38, 8. Tennant, R., Liebow, A. A., and Stern, K. G. (1941) Proc. SOC. Erptt. Biol. Med., 46, 18. Tennant, R., Stern, K. G., and Liebow, A. A. (1942) Catuer Research, 2, 218. Thomas, J. A. (1937) Arch. expfl. Zellforsch. Gewcbrziichf., 19, 299. Thomas, J. A., and Borderioux, I. (1948) Arch. anut. microscop. morphol. ezptl., 37, 264. Thompson, R. B. (1952) Blood, 7, 522. Tompkins, E. R., Cunningham, B., and Kirk, P. L. (1947) J . Cellular Comb. Physiol.. so, 1. Trowell, 0. A. (1952) Exptl. Cell Research 3, 79. Trowell, 0.A. (1953) J . Physiol., 119, 274. Trowell, 0.A., and Willmer, E. N. (1939) J. Exptl. Biol., 16, 60. Tyrode, XI. V. (1910) Arch. intent. pharmacodpiamie, 20, 205. Uei, K. (1926) FoIia Phavmerot. Japan., 2, 288. Ulloa-Gregori, O., Blocker, T. C., Nowinski, W. W., and Pomerat, C. M. (1950) Texas Repts. Biol. and Med., 8, 400. Valentine, J. D., Gurin, S., and Wilson, U. W. (1949) Federation Proc., 8, 262. Vercesi, C., and Guercio, F. (1935) Arch. exptl. Zellforsch. Geweheziicht., 18, 210. Vikblacih, I. (1950) Scand. 3. Clin. 6.Lab. Invest., 2, 143.

68

CHARITY WAYMOUTH

Vikbladh, I. (1951) Scand. J . Clin. 6. Lab. Imest., 3, Suppl. 2, 9. Vogelaar, J. P. M. (1953) Personal communication. Vogelaar, J. P. M., and Erlichman, E. (1933) Am. J . Cancer, 18, 28. Vogelaar, J. P. M., and Erlichman, E. (1934) Am. J. Cawer, 22, 66. Vogelaar, J. P. M., and Erlichman, E. (1936a) Am. J . Cawer, 26, 358. Vogelaar, J. P. M., and Erlichman, E. (1936b) Am. J. Cancer, 28, 301. Vogelaar, J. P. M., and Erlichrnan, E. (1937) Am. J . Cancer, 51, 283. Vogelaar, J. P. M., and Erlichman, E. (1938) Am. J, Cuncer, 33, 246. Vogelaar, J. P. M., and Erlichman, E. (1939) Am. J. Cmcer, 37, 242. Vollmar, H. (1939) Arch. exjfl. Zellforsch. Gezorebezeiicht., 23, 42. Volpino, G. (19O9/1910) Pathologica, 2, 495. Wallbach, G. (1938) Arch. exptl. Zellforsch. Geurebeziicht, 21, 373. Warburg, O., Posener, K., and Negelein, E. (1924) Biochem. Z.,162, 309. Waymouth, C. (1949) Expfl. Cell Research, Subpl. 1, 460. Waymouth, C. (1950) In Methods in Medical Research, Vol. 4, p. 201. Yearbook Publishers, Chicago. Waymouth, C. (1952) I f i A. Hughes, The Mitotic Cycle, Chapter 5, p. 163. Butterworth, London. Weller, T. H., and Enders, J. F. (1948) Proc. SOC.Exptl. B i d . Med., 69, 124. White, J. M., Jr., Beaton, J. R., and McHenry, E. W. (1952) I . Lab. Clk Med., 40, 703. White, P. R. (1946) Growth, 10, 231. White, P. R. (1947) Ann. N . 1’. Acad. S d , 49, 111. White, P. R. (1949) J . Cellular Comp. Physiol., 34, 221. White, P. R. (1950) In Methods in Medical Research, Vol. 4, p. 206. Yearbook Publishers, Chicago. White, P. R., and Lasfargues, E. (1949) Proc. SOC.Exfit!. Biol. Med., 71, 479. Willmer, E. N. (1927) Brit. J. Exptl. Biol., 4, 280. Willmer, E. N. (1928) B i d . Revs., 3, 271. Willmer, E. N. (1935) Tissue Culture. Methuen, London. Willmer, E. N. ( 1 N l ) Ann. Rept. 8. E . C. C., 18, 138. Willmer, E. N. (1942) J . Exptl. Biol.,18, 237. Willmer, E. N. (1945) In Essays on Growth and Form, p. 264. Clarendon Press, Oxford. Willmer, E. N., and Jacoby, F. (1936) J. Ezptl. Biol., 13, 237. Willmer, E. N., and Kendal, L. P. (1932) J. Exptl. Bid., 9, 149. Wilson, H., Jackson, E. B., and Brues, A. M. (1942) J. Gen. PhySioZ., I,689. Wilson, J. L. (1922) B d l . / o h m Hopkins Hosp., 95, 375. Winnick, T. (1952) Texas Repts. Biol. and Med., 10, 452. Wirth, J. (1948) La culture des virus in Vitro. Libraire Maloine, Paris. Wolken, J. J. (1952) I. Cellular Comp. Physiol., 40, 243. Worzniak, J. J. (1952) Abstract of meeting of Tissue Culture Association. Wright, G. P. (1926) J. Exptl. Med., 48, 591. Wright, G. P. (1928) J. Pathol. Bwferiol., 91, 735. Young, I. M. (1952) Am. J . Physiol., 170, 434.

Caryometric Studies of Tissue Cultures* OTTO BUCHER Department of Histology and Embryology, University of Lazlsawne, Sm‘tzerland

I. 11. 111. IV. V. VI.

Introduction ......................................................... Experimental Material and Method of Evaluation .................... Statistical Evaluation of Results ...................................... Discussion of Our Experimental Results .............................. Conclusions ......................................................... References ..........................................................

Page 69

70 73 93 108 110

I. INTRODUCTION Since the publication of W. Jacobj’s “On rhythmic growth of cells by the doubling of their volume” (Ueber das rhythmkche W m h s t m der Zellefi durch Verdoppelung ihres Volumens) in 1925, there have appeared, especially in German, a great number of papers on the size of nuclei and the various statistical interpretations of such measurements. Jacobj was the first to show that there not only exists a certain defined variation in the size of the nuclei of a given organ, but that the frequency curve of this variation often contains several maxima, each corresponding to a certain nuclear volume in the ratio 1 :2 :4 :8, etc., thus forming a geometric progression. The classes of nuclear sizes containing these maxima have been named ordinary classes (Regelklasserb) and symbolized as K I , Ka, K p , Ks, etc. Meanwhile, these facts that we have only touched upon here, have been confirmed by a great number of investigations. It has also been shown that the sizes of nuclei within different organs of the same animal (see Jacobj, 1935) and even of different animals (Birkenmaier, 1934; Sauser, 1936; and others) bear a certain constant ratio to one another. Moreover it was established that the appearance and the local or temporal distribution of these different classes of nuclear sizes in an organ (such as the liver) or in a rhythmic function (as in the case of the endometrium; see Hintzsche, 1949) are related to the particular task of the cells in question. With these advances the quantitative analysis of nuclear sizes (caryometry) has opened up new possibilities for the science of experimental biology (Jacobj, 1942 ; Bucher, 1953~).Further study on tumors has also been stimulated by such caryometric investigations as those of Heiberg, 1921, 1933, 1934; Ehrich, 1936a, b ; Schairer, 1936, 1937; Wilflingseder, 1947; and others.

*

This work was supported in part by funds received from the Swiss Academy of Medical Sciences.

69

70

OTTO BUCHER

The vast majority of these experiments have been carried out on histologic sections, first on organs containing spherical nuclei (such as the liver), because this simplified the process of calculating the nuclear volume from the diameter measured. The calculation of the volume of elliptic nuclei is naturally more complicated (Bohm, 1934; Jacobj, 1935 ;Hintzsche, 1936; Meyer, 1937) and, therefore, a special distrust with regard to the nuclei of connective tissue was manifested, Jacobj (1935) did no work on such nuclei precisely “because of the difficulty of effecting an exact measurement.” Pfuhl ( 1932) certainly exaggerated this difficulty, stating that “direct measurements of connective tissue nuclei are unfortunately impossible: owing to the variability of the nuclear form and in the ignorance of the exact size of the third diameter any calculation of nuclear volumes is useless.” Despite the enormous amount of literature currently published on caryometric experiments, it is striking that, prior to 1950, so little work and with such poor success was carried out on cultures in vitro (Dogliotti, 1927; Wermel and Ignatjewa, 1932a, b ; Freerksen, 1933; Wermel and Portugalow, 1935 ; Gaillard and Bakker, 1938; Lewis, 1948). This failure was most unfortunate, since tissue cultures afford us the possibility of studying many interesting biologic phenomena, as well as experimental influences, under relatively simple and known conditions ; in addition, such living cultures permit us to observe directly the growth of nuclei, The most difficult obstacle in effecting caryometric experiments on hangingdrop cultures is that the cell nuclei do not possess a definite geometric form (such as a sphere or an ellipsoid of rotation) which would permit us to calculate their volume, but are instead greatly flattened. Because of this lack of work on nuclear sizes in tissue cultures due to the above-mentioned technical difficulties, we have developed in our laboratory a relatively simple method of investigating the sizes of the flattened nuclei of connective tissue (Bucher and Horisberger, 1950; Bucher, 195Oa, b). Moreover, we have devoted most of our attention to the statistical evaluation of the results obtained (Bucher and Gattiker, 1952 ; Bucher, 1953a). MATERIAL AND METHOD OF EVALUATION 11. EXPERIMENTAL In our experiments we employed fibroblast and osteoblast cultures of different animals (rabbit, guinea pig, mouse, chick) and man; these having been cultivated, as usual, in a hanging drop of blood plasma and tissue extract, in an incubator at body temperature. All cultures were identically treated with Carnoy’s solution as fixative and the von Mollendorffs’ (1926, pp. 517-19) iron hematoxylin lac as colorant. We then projected the

CARYOMETRIC STUDIES OF TISSUE CULTURES

71

flattened nuclei-the greatest diameters of which are parallel to the coverslip-at a linear magnification of 1,500, made drawings, and measured the designs so obtained with a planimeter. In contrast to histologic sections, coverslip cultures have the advantage of permitting the study of whole preparations. Hence we were not obliged to use paraffin embedding or microtomic sections, these procedures often producing distortions in the size of the nucleus as well as involving the risk that only nuclear fragments may be drawn. Our method is much simpler and, moreover, eliminates these risks. In the thin marginal layer of well-stained cultures (Fig. 10) it is comparatively simple to bring the maximum optical surface into focus, and to draw the contours of the nuclei. In this manner we can measure the two greatest diameters, lying in the plane of the coverslip, whereas the third diameter, perpendicular to the coverslip, cannot be determined. In order to calculate this third diameter, which is, however, not necessary in our method, we would have in addition to make sections, which would further complicate the procedure. In the following paragraph we will demonstrate how we can attain our objective without taking into account this third diameter. Employing the method outlined above, we computed the size of nearly 100,OOO nuclei. To have an idea of the error encountered in our method, we drew a certain fibroblast nucleus and its nucleolus twenty times in succession, and then planimetrically measured each of these drawings five times ; from the 100 values so obtained we calculated the standard deviation ($1. For a nucleus whose projected plane has an area of 499.9 mm.2, s = 6.2 mm.2 ( ~ 1 . 2 7 4 ;) for a nucleolus of 57.0 mm2, s = 1.3 rnm2 (= 2.3%). The total error of the method is composed of the variance due to diagramming, and that due to measurement with the planimeter. The latter error can be directly calculated, and we found in our experiments a value of 0.7% for the nucleus (1.6% for the nucleolus). The variance (9)due to diagramming can be obtained by subtracting the square of the standard deviation (variance) due to the planimetric method from the total variance ; it equals 1.0% in the case of the nucleus and 1.2% in the case of the nucleolus. For the cell nucleus the variance due to diagramming is usually greater than that resulting from the utilization of the planimeter (for details see Bucher and Horisberger, 1950, pp. 261-263). Finally, it should be noted that in evaluation of a certain nuclear material (not only for tissue cultures) there always exist individual differences (characteristic of each investigator) which add to the error due to method. This factor must be taken into account in comparing the results of different

72

OTTO BUCHER

workers. Both the experiments themselves and the controls employed therein must be evaluated by the same person. Since in growing tissue cultures we are dealing with nuclear material, which is not completely homogenous, we obtain, if we successively evaluate the same culture, frequency curves which are not identical in their minute aspects. This phenomenon is observed because the chosen random sample of the population involves a certain factor of chance ; the size of this sample, moreover, plays an important role. Especially when there are only small differences in nuclear sizes in differentially treated tissue cultures, it is often necessary to measure thousands of nuclei in order to obtain reliable results. As we can ascertain in considering transverse sections of fibroblast cultures, the nuclei observed do not possess the form of an ellipsoid of rotation but are, indeed, greatly flattened (Fig. 1). I n our experimental

FIG. 1. Schematic representation of a fibroblast nucleus with half axes drawn in. The axes o and c lie in the projected planes, which we have drawn and planimetricdly measured. (From Bucher and Horisberger, 1950.)

procedure we have assumed the hypothesis that in the growth of a nucleus the third diameter 2b (which cannot be measured in total preparations of tissue cultures) increases proportionally to the augmentation of the two other diameters Za and 2c, which, because they are parallel to the coverslip, can be directly measured. Our results have shown that this supposition is correct insofar as fibroblast and osteoblast cultures are concerned (Bucher, 1953a). Between the nuclear volume Y = 4/3~abcand the measured projected plane F = rac, there exists the relationship V = 4/3bF. If the radius b of each nucleus were also known, we would be able to calculate its volume. Because the relative volumes of nuclei interest us much more than their absolutle volumes, we can, in utilizing the supposition that b is proportional to the projected plane F , eliminate this unknown factor b from our equation, as we have mathematically proved (Bucher and Horisberger, 1950). If the nuclear volumes Y , :Vz :Y,:V8 are in the ratio 1:Z :4:S, then the sizes

73

CARYOMETRLC STUDIES O F TISSUE CULTURES

of the projected planes (easily nieasuralle by planimetric method) show

the following ratio :

&

the proportionality factor of this geometric progression equalling = 1.5875 (whereas the factor equals 2 in the case of the geometric progression of nucfear volumes. Since we always have the possibility of working backward from the considered projected planes to the behavior of the nuclear volumes, we can directly utilize the planimetrically determined values (without the necessity of further calculations) in order to establish our frequency curves of nuclear sizes. This procedure, in addition to its striking simplicity, has the great advantage that the error due to the method need not be raised in a higher power. Most investigators have measured from their projections of nuclear surfaces the two reciprocally perpendicular diameters and from these measurements have proceeded to calculate the nuclear volume. Only a few have used the planimeter in measuring the projected planes (Dogliotti, 1927 ; Voss, 1936 ; Muller, 1937; KiSrner, 1937; and others). This latter procedure is undoubtedly more exact and not any more time-consuming. Voss, in his experiments on the nuclei of liver cells, calculated the nuclear radius from the measured projected plane, and from this he computed the spherical volume. Our procedure differs from that of the earlier authors in that we have found a direct firopor#ioH between the different plattiwtrically measured k rhythmic growth of nuclei. projected planes and f

If instead of the volumes we consider the nuclear surfaces 01:Os in a ratio 1:2, we will find that in this case the corresponding projected planes bear the same ratio 1:2, as we have demonstrated in an earlier paper [ Bucher and Gattiker, 1950a). The question of the absolute size of fibroblast nuclei in tissue cultures is in our minds only of secondary interest; more important are the reciprocal relationships aircong the diferent classes of auclew sizes, and the possibilities of experimentally influencing these relationships.

111. STATISTICAL EVALUATION OF RESULTS While the earlier authors were content to calculate the average values for characterizing the behavior of nuclear sizes, Jacobj (1925) was the first to analyze the values statistically, and he obtained most interesting results, as briefly mentioned in the introduction to this paper. For the statistical evaluation of our results we had to group the different values of nuclear sizes into classes of certain definite intervals. Bearing this in mind, we have not worked with classes of constant intervals (principle of numerical classification) as have almost all investigators since the pioneer

74

OTTO BUCHER

work of Jacobj, but have preferred instead, as did Hintzsche (1943, 1946a, b) and Wilflingseder (1948) to utilize the principle of logarithmic classification, which is superior both biologically and statistically. Only if, instead of marking the nuclear sizes themselves on the abscissa, we use their logarithms, will the difference between K 1 and K Z be the same as between K2 and Kq or as between K4 and K 8 ; this method alone is correct from a biologic point of view, since in each case we are dealing with a doubling of the nuclear volume (“rhythmic nuclear growth” of Jacobj ) The logarithmic principle of classification guarantees that together with the increase of the size of the nuclei there exists a corresponding increase in the size of the class. This can be seen in Tables I and I1

.

TABLE I EXAMPLE OF A CLASSIFICATION WHENTHE MIDPOINT K , Ordinary Classes

Intermediate Classes

Kt At-1 B+-1 (= M i - 1 ) Ct-1 Kl 4 - 2

4 - 2

(=M,-,)

Cl-2

K2

4 - 4

B,,,

(==M2-4) cz-4

K4

Midpoints 63.0 70.7 79.4 89.1 100.0 112.3 126.0 141.4 158.8 178.2 200.0 224.5 252.0

-

Class Limits

59.4- 66.7 66.8- 74.9 75.0- 84.0 84.1- 94.3 94.4-1 05.9 106.0-118.9 119.0-133.4 133.5-149.8 149.S168.1 168.2-188.7 188.8-211.9 212.0-237.8 237.9-267.0

100 p*

Class Sizes 7.3 8.1 9.0 10.2 11.5 12.9 14.4 16.3 18.2 20.5 23.1 25.8 29.1

[From Bueber and Horisbcrger, 1950.)

but not in the graphs, in which the real values of nuclear sizes have already been replaced by their logarithms. Starting off with a certain class whose midpoint equals K1, we can easily calculate the midpoint K z of the class whose volume is twice K I , by multiplying K1 by the proportionality factor 1.5875 of our geometric prqgression (see above). That is, we add to the logarithm of k’l the logarithm of 1.5875 (or subtract this latter value from the logarithm of K1 to obtain K + ) . If, in order to produce a finer grouping of our values, we intercalate between each of the classes containing K1, Kz, K1, etc., three intermediate classes (see Fig. 2) (which turns out to be very useful) we will alternatively obtain by either adding or subtracting 1/8 log 1.5875 (= 0.02509) the logarithms of the limits and the midpoints of each class,

TABLE I1 STANDARD CLASSTYPES

102

100

98

104

106

108

110

90-95 100-105 11tk-120 125135 140-155 160-175 180-195

90-95 loall0 115-125 130-140 145-155 160-175 18o-Mo

m-220

205-225

225-245 250-275 280310 315-350 355-390 395-440 44-95

95-100 105-115 120-130 135-145 150-165 170-185 19&rn5 210-230

230-250

95-100 105-110 115-125 130-140 145-160 165-180 185-200 205-225 230-255

~

K,

K,

Kl

85-90

95-100 105-115 120-130 135-145 150-165 176185 1-205 210-230 235-260

K2

K,

Ka

K,,

265-295' 2958330 335-370 375-415 420-465 470-520 525-585 590-660 665-740 745-830 835-930 Y35-1045 1050-1175 1180-1320 1325-1480

85-90 95-10.9 105~115 12C-130 135-145 150-165 170-185 190-210 215-235 24&-265 271wooa

3004-335 340-375 380-420 425-475 480-535 540400 605-675' 675e755 760-845 850-950 955-1070 1075-1200 1205-1345 1350-1510

(From Bucher end Csttiker, 1952.) In actual cases, the designatloria Rt, K

. XI,

1

90-95 100-105 110-120 125-135 140-150 155-170 175-190 195-215 220-240 245-270 27-05 3103454 345-85 390-4354 43548s 490-545

550-610 615-685 690-770 775-865 87W70 975-1090 1095-1225 1230-1375 138&1570

etc. vary depending

500-555

560-625 630-700 705-785 790-4330 885-990 995-1110 1115-1250 1255-1400 1405-1570

255-280 285315

260-285 290-320

320-355 360-400 405-450 455-505 510-565

325-360 365405 410-455 460-510

570-635

58W5

640-7158 715-00 805-900 905-1010 1015-1130 1135-1270 1275-1425 1430-1600

650-725 730-815 820-915 92&1030 1035-1155 1160-1295 13W1455 1460-1635

515-575

35-260 265-299

295-30 33W70 37.5415 42C465 470-520 525-585 590460 665-740 745-830

835-930 935-1045 1050-1175 1180-1320 1325-1480 1485-1665

upon the material employed.

"Half of the nuclear siaes cormponding to these values have been distributed in the CIUMabove and half in the claw below.

0 hl

76

OTTO BUCHER

Ki

a

b

C

FIG.2. Graphic representation of subdivisions into ordinary classes (G and and intermediate classes (u, b and c). (From Bucher and Horisberger, 1950.)

Kt)

If, to illustrate the example of classification we do these calculations with a value of 100 for K 1 , we obtain the values of the midpoints, limits and intervals of the classes as seen in Table I. If we choose a smaller number of intermediate classes, the frequency curves become too simplified. By using more than 3 intermediate classes there will be fewer individual values falling within each class (assuming that the total number of values remains the same), this being especially notable at the extremities of the curve, where the frequencies are naturally much lower (the importance of this disadvantage could be reduced by employing material with a greater number of values). Moreover, if we used a greater number of intermediate classes, the size of the classes in the region of the small nuclei would fall below the value of 5 mm.8 after magnification (1,500 X ) and hence below the limits of accuracy of our method.

W e now know the principle by which we can calculate our system of classification. Some incertitude eventually lies in the fixation of the midpoint of the modal class corresponding to our measured values. By “modal cIass” we mean that group containing the maximum frequency and presenting the starting-point for the calculation of the other classes (see above). For a more or less symmetric distribution of the resulting values, it is possible to calculate the midpoint of the modal class ; this point represents the geometric mean of all the measured nuclear sizes. When this condition is not fulfilled, as in cases in which the number of values is relatively small, or after certain experimental influences, we are obliged to establish the frequency curves of nuclear sizes in another manner. W e have therefore calculated the system of classification for the theoretically accepted values of K I = 98, 100, 102, 104, 106, 108 and 110 mm.2 (sizes of the projected planes of nuclei) and then established tables in which we classified the values planimetrically measured. The class limits for the above seven groups (“standard class types”) are expressed in mm.2, designating the area of projected planes drawn with a linear magnification of 1,500 x ; for example, a nucleus whose projected plane equals 100 p2 will be magnified to 225 mm.2 (= 1,500 X 1,500 $). All these values are represented in Table 11. I n Figure 3 we observe frequency curves of the measured values of 3,000 nuclei taken from the inner and outer zones of chick fibroblast cultures. These curves result from the classification of the values in all our so-called “standard class types” as represented in Table 11. It can be seen that the

CARYOMETRIC STUDIES OF TISSUE CULTURES

77

FIG.3. Nuclear size frequency curves of chick fibroblast cultures (heart explants) resulting from the classification of measured values in our six standard class types 98, 100, 102, 104, 106, 108, and 110. The curves on the left correspond to the inner zone, those on the right to the outer zone of the cultures. On the abscissa, which is a logarithmic scale, we have marked the nuclear sizes, and on the ordinate, the corresponding frequencies in $J (each zone includes 1,500 nuclei).

78

OTTO BUCHER

two types 98 and 110 show curves that are identical but displaced from each other so that the interval is equal to the size of an intermediate class. A glance at Table I1 reveals that the two types 98 and 110 represent two series of class limits, whose values are identical but which are displaced so that the second group of values of the 98 column corresponds to the first group under the 110 heading, and so on. By the classification of a given sample of nuclei in our standard class types 98 to 108 we have used up all the possibilities of classification. One can naturally interpolate still other class types, for example 99, 101, 103, etc. This is, however, superfluous in our experience. In looking at Figure 3 we note immediately that the choice of the system of classification is of great importance to the form of the curves; it is necessary, therefore, to consider this problem. Thus a maximum frequency of nuclear sizes ought not to be situated between two classes (because in this case the maximum would be split and the curve would have a different Character), but it must fall in the middle of a class. By employing a material of definite mathematical values we have studied the question (Bucher and Gattiker, 1952) of how can one avoid the hazards concerning the form of curves due to statistical evaluation (choice of class type). The purpose of these investigations was not only to demonstrate the influence of the manner of classification on the character of the curves, but also to find a means to maintain the qualities of the base curve in spite of the incertitude in choice of class types. Starting from, the. hypothesis that these hazards in our frequency curves resulting from classification in the six different types could neutralize each other, we have superposed the six “standard curves” in such a manner that, in principle, the ordinates of the maxima coincide; then we have calculated for every point on the abscissa (nuclear size) the arithmetic mean of the corresponding ordinates of the six classes. Thus we obtained the so-called “average curve” (Mittelwertskurve) representing the average of the six standard curves. To obtain an accurate result it is naturally very important that one superpose these curves in an appropriate manner, the details of which can be found in the paper cited above. After having experimented with materials of both theoretical and empiric values, we have arrived at the conclusion that the method explained above gives the best results. Practically it is sufficient-and less time consuming-when we calculate the “average curve” with only three standard classes, i.e. from the class types 100, 104 and 108. If we work with material containing definite mathematical values, we could measure the cliscrepancy between the average curve and the fundamental curve in employing,

CARYOMETRIC STUDIES OF TISSUE CULTURES

79

for example, the method of the x'-distribution and thus see whether or not there is a statistically significant difference between the two. On the other hand, wc were 'also interested in knowing if the elcments of the two curves would present differences or not. All comparisons between the fundamental curves and the average curves show that the small differences are not statistically significant. The method of computing x' may be found in any book on statistics; in one of our earlier publications we gave an example of such a calculation (Bucher and Gattiker, 1952, p. 67).

In order to define mathematically the character of our frequency curves and to compare the different curves from this point of view, we can calculate several elements of curve. Moreover, this permits us to ascertain whether or not these curves correspond to normal distributions. Of these elements we are most interested in Pearson's kurtosis, the measure of skewness, the symmetry of the curve and the standard deviation. By the kttrdosis (pz> we mean a measure of the relative flatness of a curve; if this measure is exactly or approximately equal to 3, we are dealing with a normal Gauss-Laplace distribution. The measure of skewness (a) of a distribution represents the distance between the ordinate of the summit (highest frequency) and the zero point (in our case the zero point of our measure of gradients, see Table 111, which we will discuss in greater detail later). In a normal distribution and in all other symmetrical curves, this distance equals zero, since in these cases the ordinate of the summit of the curve coincides with the ordinate passing through the zero point. The displacement to the left or to the right of the axis of symmetry of a curve depends on whether the skewness is respectively negative or positive. To find out whether this value has a relative as well as an absolute meaning, we will form an expression indicating the relationship between it and the value for the standard deviation, hence obtaining a measure for the symmetry a/s of the curve. In rigorously symmetric curves the skewness and consequently the symmetry are equal to zero. The standard deviation (s) is the distance between the symmetric axis and the points of inflexion of the curve, these indicating where the concave part of the curve limb turns into the convex part. In Table I11 we have given an example of the calculation of cuwe elements. In the first column are included the gradients x, which have been introduced in order to simplify the calculations (thus avoiding calculations using the real nuclear sizes). These gradients must be chosen in such a way that the value of zero will coincide with the value of the maximum frequency of the curve (for exceptions to this rule see below). In the system of coordinates the gradients are negative on the left side of the zero point (where the nuclei are smaller; see upper half of Table 111)

80

OTTO BUCHER

T A B U 111 EXAMPLE OF A CALCULATION OF SEYERhL ELELENTS OF A CURVE AS SEEN I N THE FREQUENCY DISTRIBUTTON OF NUCLEAR SIZESFROM THE OUTERZONE OF CHICKFIBROBLAST CULTURES

f

Gradient x -5 -4

-3 -2 -1

,

10.1 29.7 61.2 109.5 175.4

xf

x3f

+f

-1262.5 -1900.8 -1652.4 - 876.0 - 175.4

6312.5 7603.2 4957.2 1752.0 175.4

X*f

-50.5

252.5 475.2 550.8 438.0 175.4

-1 18.8

-183.6 -219.0 -175.4 --

-747.3

192.1 178.2 122.6

0 1 2 3 4 5

64.1

26.5 13.9

N=983.3

h = - -z x f

.v

1L2

=

us--u4 =

- 0.045

-- 3.975 N

N Zx4f

69.5

-

-

178.2 980.8 1730.7 1696.0 1737.5

1782 1961.6 5192.1 6784.0 8687.5

4-791.2

$6323.2

+

-5867.1 456.1

-747.3 43.9

3908.9

measure of asymmetry

+

=PLa2 =

-

Pearson's kurtosis p2 = p4 p2z

measure of skewness a = - 0.464

-= 44.344 N

178.2 490.4 576.9 424.0 347.5

178.2 245.2 192.3 106.0

p23

ZX2f

za8f

-5867.1

-

-

---

a

-0.068

S

1.993

- = ~-

=0.m "1

- 2.806

15.801 P3(82+3)

Pz ( ~ O P ~ - - ~ ~ P , - W

= -0.068

3.975 (28.064.04-18)

symmetry

62.809

44.344 ~

0.464(2.806+3)

standard deviation s =

43603.7 0.215

w2-p12

=

3.973 = 1.W3

- -0.034

and positive on the right side (where the nuclei are larger; see lower half of Table 111). In the second column are the frequencies (f) corresponding to the gradients. In the third, fourth, fifth, and sixth columns are indicated the products of the frequencies and the first, second, third, and fourth powers respectively of the gradients x ( x f , x2f, x'f, x'f). While the products x*f and x'f (containing the powers of x which are even) are positive, the products xf and x3f (containing the powers of x which

CARYOMETRIC STUDIES O F TISSUE CULTURES

81

are odd) are negative. This must be taken into account when the columns are added up. From the sums of the values of the third to sixth colunins divided by the total frequency N (which equals the sum of the values within the second column) result the moments pl, p2, p3, w. We need these moments in order to calculate the elements of the curve as well as (later on) the normal distributions corresponding to the empiric curves. In order to simplify the following calculations, the expressions CL:

and

@I=--

P4

@a=-

pg3

PZZ

were introduced, in which is a measure of asymmetry and /32 represents at the same time the kurtosis. The measure of skewness ( a ) can be calculated by the following formula : ~a(83+3) a=-

lOfi-1281-18)

~ x (

The standard deviation

( J ~ ) equals p2-pI2,

from which

s .= vPr-plz

equals zero and s2 is identical to p2. The quotient a/s mentioned above represents the measure of symmetry. For the mathematical basis of these calculations see Willigens (1932, 1933).

In a normal distribution

p1

If the kurtosis p, becomes larger than 3, the curve corresponds to a supernormal distribution with a greater concentration of values in the region of the summit. In this case the standard deviation has become smaller than in a normal distribution. If the kurtosis is distinctly less than 3, we speak of a subnormal distribution. In a distribution where the asymmetry is too large, the mathematical conditions for the calculation of the kurtosis are no longer filled. Sometimes we placed the zero point of our system of gradients between two size classes, which then became the gradients 4 . 5 and f0.5 (the following classes becoming the gradients k1.5, c 2 . 5 , 5 3 . 5 , etc.). This displacement was made in the cases where the axis of symmetry did not coincide with the ordinate of the summit and when it involved obtaining values for the measure of skewness and symmetry permitting us also the calculation of the kurtosis (since, as mentioned above, in case of abnormally large asymmetry the calculation of the kurtosis is meaningless). However, if it was a question of determining not the kurtosis, but the deviation between the axis of symmetry and the ordinate of the summit, the gradient of the zero point must then naturally coincide with the abscissa of the summit. To obtain from the calculation of the elements of the curve as accurate a result as possible, we have omitted the most peripheral values on either side of the curves, because these values are due to hazards and are statistically uncertain, the number of nuclei here classified being very small. Moreover these values are multiplied by the first to the fourth powers of the gradient, which augments their errors even more. In general, we have employed 98 to 99% of the frequencies for the calculation of the elements of curve.

W N

TABLE IV

ELFXENTSOF DIPP~RENT FREQUENCY CURVES OF NUCLEAR SIZES Kurtosis Tissues

Zone

Human

Inner zone Outer zone Inner zone Outer zone Inner zone Outer zone Inner zone Outer zone Inner zone Outer zonr Inner zone Outer zone Inner zone Outer zone

fibroblasts Fibroblasts of guinea pig Rabbit fibroblasts (Ist/Znd day) Rabbit fiibroblasts (4th day) Mouse fibroblasts Chick fibroblasts (heart explant)

Chick fibroblasts (skin explant) Chick osteoblasts (frontal bone explant) Average values (14,W nuclei in each zone) (Prom Bucher, 1953a.)

B2

2.80

Standard Deviation s 1.97

2.84 2.78 2.97

3.08 2.83

2.03

3.03 3.00

timer zone Outer zone

3.10

Inner zone Outer zone

2.983

2.15

-0.03

-0.03

+0.19

4.13 +O.W

4.02

-0.01

+o.M)9

+0.019

2.228

-0.35 4.08

4.28

2.41 2.078

3.010

4.07 -0.07

2.21

2.81

+0.10

4.74

1.Q!?

3.06

4.01

4.01

-0.17

2.12

+0.10 -0.04

4-0.19

2.09

2.81

3.12

4.10

+0.21

2.36

2.06

4.25

4.11

-0.09

2.01

S

4.21

2.17

2.83

2.85

(1

4.02

4.29

2.27

-

-.051 4.04

2.09

Symmetry

4.09

4.19 2.07

1.88

3.06

Measure of Skewness a

-0.283

-0.127

83

CARYOMETRIC STUDIES OF TISSUE CULTURES

Table I V represents some elements of nuclear size frequency curves of different tissues cultivated in vitru. Here we see in principle (without giving too great importance to individual figures) that the values for the measure of skewness and symmetry are very small, signifying that all the distributions are practically symmetrical. The values of kurtosis being dispersed around 3, all of o w frequency curves in question correspond to norirwl distributions (Bucher, 1953a). For a more exact definition of normal distributions corresponding to our frequency curves of nuclear sizes, the standard deviation (s) is of very great importance : if s, indicating the distance of the points of inflexion from the ordinate of the summit, is large, the curve is wide and flat; if s is small, the curve is narrow and high. In Table IV we notice that in our variation curves the standard deviation s is approximately 2. (For the so-called standard form of normal distribution one takes s = 1.) When discussing the elements of curves we remarked that our frequency curves of nuclear sizes correspond practically to normal distributions. In the following paragraph we would like to deal with the calculation uf the normal distribzLfion corresponding io a material with empiric values. The general formula of a normal distribution is: 1

y=-

e

-4

L) X-

2

4% where x the nuclear size marked on the abscissa, y = the ordinate corresponding to the x-points on the abscissa (frequency of nuclear sizes), s = d p x = standard deviation (see also p. OOO), r 5.14159, e =2.71828 (base of natural logarithms), Q - arithmetic mean of all the x-values. If we employ gradients xi from 0 to fi or 4respectively instead of real values for x (nuclear size), in the same manner as shown above when we calculated the elements of curves, then Q becomes 0 (because a normal distribution is exactly symmetric) and thus disappears from the exponent of the indicated formula. To arrive at the formula of the standard form of normal distribution it is necessary not only to admit a =0, but also x = 1 as is accepted for this special case; the above forrriula therefore becomes simpler in the following manner : = i

-

1 y=-e

v!K

-%S

--r/zX2

= 0.3989 e

For Table V w e have calculated the values of the ordinates corresponding

84

OTTO UL'CHER

VALUESOF

THE

TABLE V STANDAR~ FORMOF NORMAL DISTRIBUTION Ordinate

Abscissa Gradients for S = 1

Absolute Values

In One-thousandths of the Maximum (O/OO)

0 k0.25 kO.50 k0.75 k1.00

0.39894 0.38668 0.35205 0.30113 0.24202 0.18265 0.12952

1Ooo.o 969.3 882.5 754.8 606.6 457.8 324.7 216.3 135.3 79.6 44.1 22.8 11.1 2.2 0.3

k1.25 21.50 k1.75 22.00

0.08628 0.05399 0.03 174 0.01759

22.25 22.50 k2.75 23.00 k3.50 24.00

O.Oo909 0.00443 0.00087 0.00013

(From Bucher and Gattiktr, 1952.)

to x = O , 10.25, r t O . 5 , k0.75, up to x = k4.0, and we have expressed these values in the third column in figures indicating one-thousandths of the maximum frequency. This value is 0.3989 for the gradient 0 of the standard form of normal distribution because, when x 0, the exponent

of the formula becomes 0 also and consequently e y=--

1

- 1, and

-544

- 0.3989

G The maximum of the normal distributions corresponding to our empiric curves is defined by the formula 0.4 N A x

max. = ___ s

where N signifies the total frequency, Ax the size of the classes (= I in our system of gradients) and s the standard deviation of the material. For the example of such a calculation of normal distribution as is given in Table VI, we have determined the maximum frequency as f0llOMYi

: 0.4 983.3 max. =

- 197.35 1.99

To ohtain*.fromthe standard form of normal distribution (where, in

85

CARYOMETRIC STUDIES O F TISSUE CULTURES

TABLE VI VALUESOF THE NORMALDISTRIBUTION CORRESPONDING TO THE FREQUENCY CURVE OF THE NIJCLFARSIZESIN THE OUTER ZONE OF CHICKFIBROBLAST CULTURES (HEART EXPLANTS)

Ordinate

Abscissa

Displacement by a =

For s

=

-4.00 -3.50 -3.00 -2.75 -2.50 -2.25 -2.00 -1.75 -1.50 -1.25 -1 .oo -0.75 -0.50 -0.25 0 4-0.25 +0.50 $0.75 $1.00 4-1.25 4-1.50 $1.75

For s = 1.99

4-0.05

-7.97 -6.98 -5.98 -5.48 -4.98 4.48 3.99 -3.49

-7.92 -4.93 -5.93 -5.43 4.93 -4.43 -3.94 -3.44 -2.94 -2.44 -1.94 -1.44 -0.95 4.45 $0.05 4-0.55 4-1.05 4-1.54 4-2.04 4-2.54 $3.04 $3.54 4-4.04 4-4.53 4-5.03 +5.53 4-6.03 4-7.03 4-8.02

-2.99

+2.00

+2.25 +2.50

4-2.75 +3.00 4-3.50 +4.00

1

'

-2.49 -1.99 -1.49 -1.00 -0.50 0 $0.50 4-1.00 4-1.49 4-1.99 4-2.49 4-2.99 4-3-49 4-3.99 +4.48 +4.98 4-5.48 +5.98 4-6.98 +7.97

In Onethousandths of the Maximum

0.3 2.2 11.1 22.8

44.1 79.6 135.3 216.3 324.7 457.8 606.6

754.8 882.5 969.3 1000.0 969.3 882.5 754.8 606.6 457.8 324.7 216.3 135.3 79.6 44.1 22.8 11.1 2.2 0.3

Absolute Frequency

0.06 0.43 2.19 4.50 8.70

15.71

26.70 42.69 64.08 90.35 119.71 148.% 174.16 191.29 197.35 191.29 174.16 148.96 119.71 90.35 64.08 42.69 26.70 15.71 8.70 4.50 2.19 0.43 0.06

accordance with the above-mentioned definition, u =0, and s = 1) the normal disiribytiotts which correspond t o our empiric c m e s , we must take into consideration the two parameters a and s of our material. a defines the positional relationship of the theoretical curves with respect to the empirical curves, s indicates the standard deviation, which defines the form of the czwves.

86

OTTO BUCHER

With respect to the standard deviation (s) of the normal distribution (in our example 1.99) we must now multiply the gradients of the abscissa (from 0 to k4.0) by s. The intervals between the units of the abscissa are therefore equal to s (which in the case of the standard form of normal distribution 1). In this way we may determine the dispersion corresponding to our empiric curves. I n principle there are two possibilities of determining the position uf the cztrmes within the system of coordinates, as well as that with respect to the empiric curve. If we use the gradients noted above, we can then calculate the displacement of the zero-point of the theoretical curve with regard to the zeropoint of the empiric curve in the following way: we multiply each gradient by the corresponding frequency and calculate the difference between the total of the positive products and that of the negative products (see Table 111, third column, xf). The resulting value divided by the total frequency is simply the arithmetic mean a (with respect to our system of gradients), indicating the value of the displacement of the zero-point of the theoretical curve to the left or to the right, according to the sign of the mean, which may be negative or positive.

-

Though the calculation of the arithmetical mean employing, instead of gradients, the true values of nuclear sizes may be easier to picture to oneself, it is in our case a more time-consuming procedure. This true value of the arithmetical mean would indicate directly the position of the ordinate of the summit of the theoretical curve in the event that we mark on the abscissa the true values of nuclear sizes.

In the first column of Table VI, the values of the abscissa are indicated for s 1. By multiplying these figures by the value of s in our example, we obtained the values listed in the second column. The third column shows the values found for the x-points with respect to the zero-gradient of the empiric curve, obtained by adding or subtracting a ; thus the alternative position of the two curves is also defined. In the section dealing with the ordinates, the figures in thousandths of the maximum frequency are to the left; to the right are noted the absolute values (which were calculated using the values of the preceding column) with respect to our empiric maximum frequency. The same calculations were also made for the inner zone of chick fibroblasts (heart explants). The two empiric curves of the two zones (for details see p. OOO) of these cultures and the corresponding normal distributions are shown in Figure 4, where the almost exact coincidence may be noted. It can be shown that the frequency curves, not only of cultures in vitro of untreated chick fibroblasts, but also of tissue cultures of other animals

-

CARYOMETRIC STUDIES OF TISSUE CULTURES

87

(rabbit, guinea pig, mouse) and of man, correspond to normal distributions (see Bucher 1953a). This conclusion is upheld in the application of the following criteria: first, the behavior of the elements of curves (see Table IV) ; second, the method of X*-distribution, which indicates whether the difference between the empiric curve and the corresponding normal distribution is due to hazard or whether it is statistically significant (the latter not being the case in our experiments) ; third, the representation of the cumulative frequency distribution, with which we shall deal in the following paragraph ; fourth, the tests of departure from normality (see Fisher, 1950, pp. 52-54; Snedecor, 1953, pp. 174-177).

FIG.4. Frequency curves (average curves) of the inner and outer zone of chick fibroblast cultures (heart explants) . The normal distributions corresponding to the empiric material are drawn in continuous lines, around which the measured values (small circles) are dispersed. (From Bucher, 1953a.)

In many cases it is useful to employ not only the normal distribution, but also the cumulative frequency distribution. This type of curve may be obtained by indicating as ordinates not the simple values of the normal distribution, but rather a series of values, each term of which represents the sum of the values of all preceding terms (frequencies are always indicated in %). Figure 5, drawn on ordinary graph paper, shows the cumulative frequency curves corresponding to the empiric frequency curves of the inner and outer zones of chick fibroblast cultures, which are represented in Figure 4 by small circles. With this type of graphic representation on ordinary graph paper not much progress can be made. An important step forward is made if, instead of using a system of coordinates with a linear scale, we turn to a system of coordinates, wherein the ordinate is graduated according to the integral of probability (see Fig. 6). Thus the cumulative curve of a normal distribution becomes a straight line. The degree by which the empiric distribution deviates from the normal now becomes very apparent. It should be noted that two types of probability charts exist: one with linear graduations and the other with logarithmic graduations of the abscissa (in both cases the ordinate is graduated according to the probability integral). Since we have always

88

OTTO BUCHER

classified our values (nuclear sizes) within a logarithmic system, we have used a probability chart with a linear scale of the abscissa. If a normal distribution of the second type is involved (Bucher and Gattiker, 1952, p. 83; Gebelein and Heite, 1951, p. 43), the cumulative frequency distribution becomes a simple straight line only when a probability chart with logarithmic graduations of the abscissa is used.

FIG.5. Cumulative frequency curves resulting from the summation of the individual empiric nuclear size frequencies (for details see text). The curve on the left corresponds to the inner zone and the curve on the right to the outer zone of the same chick fibroblast cultures as employed for Figure 4. (From Bucher, 1953a.) In Figure 6 we have drawn the empiric values as small circles on a probability chart. The nuclear material here (chick fibroblasts) is the same as in Figures 4 and 5. The cumulative curves of the normal distributions corresponding to the nuclear material in question are indicated by a continuous line. We observe now that in the region between 10 and ?O% the empiric and normal distributions practically coincide. In order to judge accurately the regions below 10% and above !90% it would be necessary for us to consider two points. First there is the fact that the units of the scale increase the further away we get in either direction from the midpoint of the ordinate (50%). Thus, for example, the distances between 1% and 5 % or 95% and 99% are approximately the same as the distances between 25% and 50% or between 50% and

CARYOMETRIC STUDIES OF TISSUE CULTURES

89

7570. It is for this reason that the beginning and the end of the curve, which in the linear system of co-ordinates (Fig. 5 ) curve in the direction of the abscissa, straighten out until they form a straight line in the

FIG.6. Cumulative frequency distributions of nuclear sizes of chick fibroblast cultures (same material as in Figs. 4 and 5) drawn on a probability chart. The cumulative curve of a normal distribution now becomes a straight line. The small circles represent the empiric frequencies, which in the significant region between I0 and 90% practically coincide with the theoretical values. (From Bucher, 1953a.)

mid part of the curve. Small deviations in the peripheral regions-where the individual values can be marked very exactly-are greatly emphasized. this is the However, it is just these parts of the empiric curve-and second point-that are least sure, because for these regions we have fewer measurements at our disposal than in the mid part of the curve.

90

OTTO BUCHER

Therefore, if we notice in these peripheral regions certain deviations between the empiric and normal cumulative distributions, we should not be disconcerted. Practically, a good approximation in the part of the curve between 10 and 90% is sufficient. 1Ve can, consequently, draw the conclusion that the empiric curve shown in Figure 6 corresponds to what the biologists call a normal distribution. In Figure 7 we have drawn, again eniploying a probability chart, the

RG.7. Cumulative distributions of the nuclear size frequencies from the outer zone of tissue cultures of different animals and of man drawn on a probability chart. The curves are displaced from left to right in proportion to the augmentation of their average nuclear size (geometric mean). In the significant region between 10 and 90% the curves correspond approximately to straight lines, which are, depending upon their standard deviation (s), of varying slope. Chick

I

Chick osteoblasts, Mouse fibroblasts,

Rabbit

---0 ----- 0 --,l,oOO nuclei -. -. - 0 -. -. 1.050 nuclei - - - - -- - 0 - - - - - - -

skin explants, 1,050 nuclei heart explants, 1,500 nuclei

{

lst/Znd day, 5,100 nuclei 0 4th day, 2,900 nuclei 0

0 0

Guinea pig fibroblasts, 1,000 nuclei - .. - . . - 0 - . . - . . Human fibroblasts, 1,000 nuclei . -0 -

- -.

- .-.

CARYOMETRIC STUDIES OF T I S S U E CULTURES

91

einpiric cumulative curves of our frequency distributions obtained by evaluation of nuclear sizes in the outer zone of different tissues cultivated iiz cifro (chick osteoblasts and fibroblasts, fibroblasts of mouse, rabbit, guinea pig, and man). In order to give a better over-all view, we have spaced the curves slightly. ‘The curves of tissucs with small nuclei are situated on the left, those of tissues with large nuclei on the right. All these curves are more or less straight lines in the region between 10 and W% (therefore representing normal distributions). The cumulative ciirves in Figure 7 are not exactly parallel to each other, but differ slightly in their slope. This slope depends upon the standard deviation (s) : the slope increasing as s decreases. The steepest curve results from the outer zone of the cultures of chick fibroblasts (lieart explant, s = 1.99). The curve which slopes the least is from the outer zone of chick osteoblasts (s = 2.41). Onc can also calculate the theoretical curves correspor~dingto frequency curves oi nuclear sizcs obtained by the lincar division of the abscissa (normal distributions of the second type : see Fig. 9) ; for a more accurate orientation see Bucher and Gattiker, 1952, pp. 83-86. Naturally, for such curves, which are based on a linear classification of nuclear rnatcrial, one call also construct the cumulative distributions. In order to t,btain a straight line from such a normal cumulative distribution. it is necessary to cnip!oy a probability chart with logarithmic graduations of the abscissa.

To end this chapter, we would like to discuss once again the question of the utility of the principle of logarithmic classification that was briefly

touched upon above. For this purpose we have presented in Figures S and 9 the frequency curves of nuclear sizes of human fibroblast cultures (inner and outer zones) resulting f roin logarithmic and linear classification of nuclear material. The frequencies of the einpiric curves are indicated small circles, the correspvnding normal distributions by continuous lines. In employing a lognvithmic graduation of the abscissa (Fig. 8 ) ,

FIG. 8. Nuclear size frequency curves from the inner and the outer zones of human fibroblast cultures drawn on an abscissa, which is a logarithmic scale (for each zone 1,000 nuclei were evaluated). T h e small circles represent the ernpiric values, while the corresponding normal distributions are drawn as continuous lines.

92

OTTO BUCHER

we find that the curves of the inner and outer zones of the cultures have approximately the same height and are practically symmetric. I n employing a linear classification of nuclear sizes (Fig. 9), the two curves are strongly pulled to the right and the maximum of the frequencies of the outer zone

150

-

5 100 u

B

P

50

K1

KZ

4

FIG.9. Nuclear size frequency curves from the same material as used in Figure 8, but drawn on an abscissa which is a linear scale. The small circles represent the empiric values, while the corresponding normal distributions of the second type are drawn as continuous lines.

(on the right) is much smaller than that of the inner zone (on the left), which makes comparisons between the two curves very difficult. This comportment is the logical consequence of the fact that in the linear graduation of the abscissa the distance between KB and Kq is much greater than that between k'l and K 2 , in contrast to the logarithmic graduation, where these two distances are equal. Moreover, in the linear graduation class size does not augment in a manner corresponding to the nuclear size but stays the same (meaning that it diminishes with respect to nuclear size). Thus the probability that a given value of a nuclear size falls in a certain class decreases. Hence, in using the linear scale, the values of the larger nuclei show a greater dispersion around the corresponding normal distribution than do the values of the smaller nuclei. While the accepted size of the class of 20 nim.2 (projected planes of nuclei) may be sufficient for the classification of small nuclei, it is insufficient for the classification of large nuclei. For this reason it is necessary to choose a class size which increases continuously with the increasing nuclear size by the same factor according to a geometric progression. This supposition is satisfied only by the principle of logarithmic classification. T o avoid as far as possible the hazards involved in statistical evaluation, the nuclear size must be in an appropriate relationship to the class size. Another advantage of the logarithmic system is that it allows the possibility of calculating the elements of the curve and thus also enables the distributions

CARYOMETRIC STUDIES O F TISSUE CULTURES

93

to be defined mathematically, which is not the case if the asymmetric curves resulting from a linear classification, are used. While chance plays a considerable role in a numerical classification, an appropriate simplification of the curve is assured by a logarithmic classification, which brings out the typical characters and the essential differences of the curves without losing sight of significant details. All the above-mentioned factors are indicative of the advantages of a logaritlznbc system of classes, zwlaich is the only valid system both from a biologic point of Vim (as has been noted at the beginning of this chapter) and froma statistical point of vim. Unfortunately this fact has up until now not been sufficiently recognized by the majority of authors. OF OUREXPERIMENTAL RESULTS IV. DISCUSSION

We started our experiments by measuring the nuclear sizes of rabbit fibroblast cultures (fixed two days after subculturing) in those parts of the growth area which appeared appropriate. The graphic representation of these first results shows frequency curves that differ considerably and are hence difficult to interpret (Bucher and Horisberger, 1950, Fig. 2, p. 267; Bucher and Gattiker, 1950a, Fig. 1, p. 431) : the summits of these curves fall, on one hand, at the nuclear size of K1 and, on the other hand, at K z (with a volume double that of K I ) . Some curves are M-shaped and show maxima both at K1 and Kz. If the proper care is taken, even ordinary microscopic examination reveals that nuclei located toward the periphery of the growth area are considerably larger than those situated near the initial fragment (Fig. 10). There is a continuous transition between these two zones, which we call imw zone and outer zone. This separation is not always possible in small and dense growth areas. A certain asymmetry in the frequency curve, especially in the case of a small random sample, may be explained hy the fact that on drawing the nuclei of one zone we inadvertently drew nuclei of the other zone or at best these of the transitional zone (for more specific details see Bucher and Gattiker, 195Oa, pp. 432-33). The statistical evaluation of our results reveals the interesting fact that the planimetrically determined planes, which correspond to the two maxima of frequency in the unimodal curves of the inner and outer zones, are in a ratio of 1 :1.5875; the corresponding volumes stand. therefore, iii the ratio of 1 :2 (Figs. 4 and 8). Wermel and Ignatjewa (193Za, b ) have also noted that the cells in the marginai zone, which in their opinion is comprised of only the three or four outermost laycrs of cells, are larger than those in the neighborhood of the initial fragment. However, with the method of evaluation that they used, it was impossible for them to determine

94

W P

OTTO BUCIIER

FIG.10. Photomicrographs : a ) Sector of a rabbit fibroblast culture. The dark section below represents the initial fragment; from this arises the area of growth, in which we have indicated the different zones : I = outer zone; I1 = transitional zone; 111 = inner zone; IV = initial fragment. b) Nuclei of the outer zone shown at a higher magnification. c ) Nuclei of the inncr zone at the same magnification as in lob. (Figs 10b and c from Bucher and Gattiker 195Oaj

CARYOMETRIC STUDIES OF TISSUE CULTURES

95

the relationship between the nuclear sizes corresponding to the two maxima of frequency KI and KP

What, then, is the relationship of the nuclear sizes in the inner and outer zones of our cultures of fibroblasts in vitro and the nuclear sizes in vivo? To answer this question, we used as material thinly stretched films of subcutaneous tissue from rabbits, and evaluated them employing the same method that we established for our tissue cultures in vitro. Here, too, our method of evaluation (measuring the planes of projection with a planimeter) produced equally satisfactory results. Naturally, we drew only those nuclei presenting their largest plane of projection. In applying this method to total preparations of uncultivated tissue new avenues of study were opened. 20

1

FIG.11. Comparisons of nuclear sizes of rabbit fibroblasts in Vivo and in vitro. The upper graph shows the nuclear size frequency curve resulting from thinly stretched films of subcutaneous tissue (1,SOa nuclei). The lower graph shows the nuclear size frequency curves from the inner and outer zones of the cultures in vitro (8,000 nuclei from each zone). Small circles : empiric values ; continuous lines : corresponding normal distributions. The diagram in Figure 11 illustrates that the frequency distribution of 1,500 nuclear sizes of thin subcutaneous tissue films coincides to a high degree with that of the inner zone nuclei of the cultures (on the fourth and fifth days in v i t r o ) of the same tissue from the same animal. The coincidence of these two curves is so great that even the x*-distribution method does not show a statistically significant difference. The nuclei of

96

OTTO BUCHER

the outer zone in vitro manifest on the average a size double that of the corresponding nuclei in wivo. Now another question arises : how can the difference between the nuclear size in vivo and the inner zone of the cultures in vitro, on the one hand, and the nuclear size in the outer zone of the growth area, on the other hand, be explained? We have discussed this problem in a previous publication (Bucher and Gattiker, 195Oa, pp. 451-53), and at this point we shall indicate only the principles involved. Bizzozero (1903) distinguished between tissues with labile elements, which throughout life undergo constant wear and regenerate, and tissues with stable elements ; the stable tissues also include conjunctive tissue once it has terminated its development. With explantation the growing cells of conjunctive tissue resume lability since, at least in the outer zone of the growth area, mitotic division and growth assume a paramount importance. To such an intensified activity corresponds the appearance of larger nuclei (size class K2 instead of K1), as has been noted by many authors working with other cell-types. Parallel with the differentiation in the inner zone, the mitotic coefficient greatly decreases and the cells gradually return to the state of stability, such as existed in vivo, at which time the average nuclear size adapts to that which was found before explantation. It may be noted that in a part of the fibroblast cultures (e.g., rabbit, Bucher, 1951b; mouse, von Arx, 1953), the maxima of the curves do not fall in the ordinary classes K 1or Kz, but midway between them, as shown in Figure 12 ( M = middle class). With respect to the situation of the maximum frequency we may therefore distinguish two types of cultures : “R-types,” with a maximum frequency in K1 or Kz, and “M-types,” with a maximum frequency in the middle class, which precedes either K1 or Kz.

M

M

FIG.12. Nuclear size frequency curves from the inner and outer zones of rabbit fibroblast cultures. The normal distributions corresponding to the R-types are drawn as continuous lines, the empiric values are drawn as black dots. The normal distributions corresponding to the M-types are drawn as broken lines, the empiric values as small circles.

CARYOMETRIC STUDIES OF TISSUE CULTURES

97

The latter are in our fibroblast cultures of rabbit approximately 4 or 5 times less frequently than the former. What we have just said about the doubling of the nuclear volume in the outer zone of the culture itz vitro in relation to the comportment of nuclear sizes in Vivo, concerns the R-types (ordinary class types, Regelklassentypen). Whether to designate the cultures with larger nuclei as R-types or as M-types has a certain importance, because the interpretation of the nuclear growth will be different depending upon the answer. Let us first consider the cultures with the larger nuclei (Fig. 13a) as ordinary class types. The frequency maxima of M-types fall in this case, as we have said above, in the Preceding (and smaller) middle class. In order to obtain this size, a nucleus Kt or KI would only have to be augmented by the faktor instead of doubling, as in the case of R-types. Different authors (e.g., Wermel and Portugalow, 1935; Krantz, 1952) have advanced the hypothesis that the nuclear growth would be effectuated in two stages, each stage consisting of an augmentation by the factor d F In this case the M-types would only undergo the first stage of growth.

&

b

1

K+

I

M+-i

k,

MIL2

KZ

M2-4

FIG.13. Schema explaining the possibilities of the origin of the middle classes: a ) The augmentation in volume of a nucleus K, or K, by the factor d r b) The doubling of the surface of the nucleus K , or K,. (From von Arx, l353.:

A completely different possibility of explanation presents itself if we designate the cultures with the smaller nuclei as R-types (Fig. 13b). In this case the frequency maxima of M-types fall in the follozwing (and greater) middle class. A nucleus that augments in volume from K , or K I to MI-, or M%,, respectively, would have doubled not its volume but its surface (Bucher and Gattiker, 19%). Thus, a rhythmic nuclear growth by doubling the surfaces, as described by Hertwig in 1939 and even earlier by Herbst in 1914, would result. Without discussing this question in detail from the base of our experimental observations, we have tried to demonstrate the different possibilities in principle,

W e shall now compare the nuclear sizes in the fibroblast cultures of different animals and man (Bucher and Gattiker, 1950b). The smallest nuclei are in chick fibroblasts. Then, in order of increasing nuclear size, come chick osteoblasts, fibroblasts of mouse, rabbit, guinea pig, and man (Fig. 7 ) . Thus, in our experimental material, we find the largest nuclei in human fibroblasts. These are on the average twice the size of chick fibroblasts. It is rather interesting to compare these results with those

98

OTTO BUCHER

of the experiments of Jacobj (1931), Birkenmaier (1934) and Sauser (1936) on the nuclei of liver cells. These authors found for the nuclear size in wivo the same augmenting order as found above. The proportions of specific nuclear sizes therefore maintain themselves, in principle, after explantation in vih-0. After having seen that the frequency curves resulting from the inner and outer zones of not experimentally influenced, fibroblast cultures, grown under optimum conditions correspond to normal distributions (Bucher, 1953a), there arises the question of the behavior of the frequency curves of nuclear sizes obtained from cultures under experimental chemical or physical influences. I n principle there are two possibilities. First: The nuclear size frequency curves still correspond on the whole to normal distributions as before, but the parameters of the frequency curves of the influenced cultures can differ in detail from those of the control cultures. For example, the curve can be displaced to the right or the left along the abscissa, indicating that the average nuclear size has become greater or smaller depending upon the direction of the displacement. This displacement can be effected with respect to the geometric progression of a certain factor such as 2 or d x o r else it can be relatively small as in the case of simple nuclear swelling or retraction. The standard deviation ( s ) may change as well and provoke a modification in the form of the curve, but always without deviating from the principle of normal distribution. If s, which corresponds to the distance between the two points of inflexion of the curve, on the one hand, and the ordinate of the maximum, on the other hand, increases] the curve becomes wider and lower. If s decreases, the curve assumes a narrower and higher aspect. In our non-influenced cultures s is approximately 2. Second : A fundamentally different possibility is that the frequency curves of nuclear sizes of influenced cultures no longer correspond to normal distributions. The average nuclear size may either remain stationary or change. The curve may remain more o r less symmetric or lose its initial symmetry, with the possibility of a very complex curve resulting. The biologic interpretation as well as the mathematical definition of such curves is often very difficult. We shall illustrate these theoretical considerations with two examples. To illustrate the first possibility, we shall select experiments in which our cultures were treated for nine hours with urethan (Gattiker, 1952). For the second possibility, we shall choose experiments in which the fibroblast cultures were subjected to prior treatment with colchicine (Bucher, 1951a, b ; 1952a, b ; 1953b).

CARYOMETRIC STUDIES O F TISSUE CULTURES

99

To effect the treatment with urethan, we filled the hollow grounds of the slides, on which the coverslips with rabbit fibroblast cultures were mounted, with a 1% solution of ethylurethan. In Figure 14 it is im-

FIG. 14. Nuclear size frequency curves (normal distribution) resulting from experiments with urethan. Continuous lines: curves from the inner and outer zones of treated cultures (2,000 nuclei in each zone) ; broken lines : curves from control cultures simultaneously cultivated (1,000 nuclei from each zone). (From Gattiker, L 1952.)

mediately apparent that the curves obtained from the influenced cultures show a certain displacement to the left with respect to the control curves in lines. Thus, under the influence of the poison, the nuclei of the inner and outer zones diminished in size. We do not wish in this paper to enter into a discussion of whether this diminution in size is due to an attack by the poison on the metabolism of nuclear acids and hence by a perturbation of growth, or whether it is caused by a change in nuclear membrane permeability and thus by a loss of nuclear sap (toxic nuclear retraction). Aside from the nuclear size, the form of the curve has also changed slightly, in that the standard deviation of the frequency distribution of the treated cultures has become greater, and therefore the maximum frequency has somewhat decreased. Consequently, the cumulative curves of treated cultures, as represented on a probability chart in Figure 15, are slightly less steep than the control curve (naturally, in this representation as well the first are displaced to the left). Figure 15 shows a significant coincidence between the empiric results (based on evaluation of 6,000 nuclei) and the corresponding normal distributions, at least in the sigriificant region, which lies between 10 and 90%.

For the experiments with colchicine, the cultures were previously treated with a solution of 1 :10 million or 1 2 0 million colchicine and were fixed, at the earliest, 24 hours after the washing of the poison (for details see Bucher, 1947; 1951a). The frequency curve resulting from the experiments with colchicine is found to be asymmetric, being strongly pulled to the right (Fig. 16), and we have been able to show (Bucher, 1952a) that

100

O l T O BUCHER

FIG. 15. Clxnulative curves from the experiments with urethan (using Same material as in Fig. 14) drawn on a probability chart. Continuous lines: mrmal distributions corresponding to the material treated with urethan (empiric values : small circles) ; broken lines : normal distributions corresponding to the control material (empiric values : black dots). (From Bucher, 1953b.)

1

15

-

1

c

g

10-

&

p.

5-

FIG.16. Nuclear size frequency curves from the outer zone of rabbit fibroblast cultures previously treated with colchicine. Continuous line : the empiric curve from the cultures previously treated with colchicine ; broken lines : partial curves (normal distributions) resulting from the analysis of the empiric curve, where the asymmetric form results from the superposition of two partial curves. (From Bucher, 1953b.)

CARYOMETRIC STUDIES OF TISSUE CULTURES

101

this wide-based curve originates from the super-position of two curves with the frequency maxima Kz and Kd, respectively. This proves that by prior treatment with colchicine a new nuclear category with double the volume has appeared : in other words, polyploidy has been provoked. This phenomenon could be shown in an excellent manner by caryometric investigation. Let us now present these results in cumulative curves drawn on a probability chart (Fig. 17). The thin line on the left corresponds to the

FIG.17. Frequency distributions from Fig. 16 as cumulative curves drawn on a probability chart. The straight continuous line on the left corresponds to the control cultures, around which the empiric values are dispersed. The two broken line curves - -, represent the calculated partial curves : -. . . ., Krcurve ; &-curve. The theoretical curve, which results from the two partial normal curves, is also drawn as a heavy line. The corresponding empiric values (small circles) practically coincide with the theoretical curve in the significant region. (From Bucher. 1953b.)

---

-- -

normal distribution that was calculated from the measurements of the nuclei from the control cultures; around this line the empiric values (black dots) are dispersed to a slightly greater extent than in the previous

102

OTTO BUCHER

example. The two partial curves are represented by the broken lines: the K2-curve (with its maximum at K 2 ) is on the left, corresponding to the high curve in broken line in Figure 16; the K4-curve, which is less steep, is on the right and corresponds to the low, wide curve in Figure 16. The different slopes of the two curves are caused by their different standard deviations (s 2.11 for the K2-curve ; s = 2.98 for the K4-curve) . The solid curve on the right is the cumulative curve of a distribution, the ordinates of which represent the totals of the corresponding ordinates of the two partial curves drawn in broken lines. In other words, it represents the theoretical curve resulting from the superposition of the two normal distributions corresponding to the Kz- and Ka-curves. W e can see that the empiric values (small circles) coincide very well with this theoretical curve. It also shows that in the representation of the cumulative distribution after prior treatment with colchicine of our rabbit fibroblast cultures, the frequency curve of nuclear sizes no longer corresponds to a normal distribution, but distinctly derives from the straight line in its upper half. The results of these experiments are, therefore, completely different from those of the experiments with urethan and other influencers.

-

The influence of trypaflavine on nuclear size (Gattiker, 19552) is less clear: by employing a concentration of 1:l million of this poison, larger nuclei were found in both zones of the experimental cultures than in those of the control cultures, but after the influence of trypaflavine in concentrations of 1 :600,000 the nuclei of the inner zone became smaller and those of the outer zone larger. I n spite of the influence of trypaflavine, the curves kept their normal distribution character, as we have already seen in the experiments with urethan. Also, the standard deviation (s) of the curves resulting from the treated cultures is slightly larger than that of the control curves.

W e have also used our caryometric method for the study of binuclear cells (Bucher, 1953d ; Bucher and Gattiker, 1953, 1954a). I n 34,100 fibroand osteoblasts of different animals and man, where nuclei were quantitatively evaluated, we found 531 (= 1.6%) binuclear cells. In the chick fibroblast cultures we observed approximately 1.5% of binuclear cells, or somewhat more than Macklin (1916a, b) and Zweibaum and Szejnman (1936), who found in the same tissue only 0.9 and 0.7%, respectively. This difference could be the consequence of different culture conditions. In the rabbit fibroblast cultures, we found nearly the same percentage of binuclear cells (1.4%) as in the chick fibroblast cultures. It would be interesting to determine if differences in the frequency of bi- and multinuclear cells in the fibroblast cultures of different animals are specific, as has been shown for liver cells i~ vivo (for example, by Miinzer, 1923). If one consults the literature concerning the size proportions between the two nuclei of binuclear cells, one finds that most authors agree that

CARYOMETRIC STUDIES OF TISSUE CULTURES

103

the two nuclei are of approximately the same size. This opinion, however, is based in most cases upon simple estimation (Miinzer, 1923; Jacobj, 1925; Wassermann, 1929; Pfuhl, 1930; Clara, 1930; and others). On the basis of our caryometric experiments, we can be more precise, Figure 18 45c

400

350

300

250

200

150

FIG.18. Dispersion of the binuclear cells of rabbit fibroblast cultures. The size of one nucleus is marked on the abscissa, while that of the other nucleus is marked on the ordinate. The nuclear pairs with two nuclei of equal size fall on the bisecting line. The binuclear cells of the second day (black dots) are above the bisector; the binuclear cells of the fourth day (small circles) are below the bisector. (From Bucher and Gattiker, 1953.)

gives us an initial impression. In this figure, we have represented 115 nuclear pairs of rabbit fibroblast cultures in a coordinate system in such a fashion that the abscissa corresponds to the planimetrically determined size of one of the two nuclei and the ordinate to the size of the other. The points representing the nuclear pairs, where the two nuclei are equal, fall on the bisecting line. One notices that the density of points on either side of this line regularly diminishes as the distance from it increases. We have obtained, on the whole, the same results in applying the same method to binuclear cells of other animals (see also Bucher, 1953d) and man.

104

OTTO BUCHER

So as to have a quantitative criterion for the nuclear size differences between the two nuclei of binuclear cells, we have divided the size of the smaller nucleus by that of the larger and calculated the quotient. Because we have obtained the same results in principle in all the material of our fibroblasts, we have combined them (369 nuclear pairs) for statistical evaluation and classified the quotients in classes of 0.1. The resulting curves are represented in Figure 19; the quotients are marked on the

FIG.19. Frequency distribution of the nuclear size quotients (where the larger nucleus is divided into the smaller) of 369 binuclear fibroblasts cultivated itt d r o . The quotients are marked on the abscissa and the corresponding frequencies (in %) are marked on the ordinate. The nuclear size quotients augment following the ascending lib of a normal curve. abscissa and the corresponding frequencies on the ordinate. I t will be recognized that the frequency becomes greater as the quotient approaches 1.0 or, in other words, as the difference in size between the two nuclei diminishes. With the increasing quotient, the frequency augments as following the ascending limb of a normal curve. This mathematically defined result clarifies the belief, prevalent in literature on the subject, that the two nuclear sizes do not, in general, differ substantially from each other. We also considered whether there exists a relationship between the calculated quotient and the nuclear mass of binuclear cells. In order to resolve this problem, we classified the nuclear sizes (sum of the two nuclei in each binuclear cell) with their corresponding qqptients in the classes of our logarithmic system, and we then calculated the mean quotients for each class size. The result is quite striking: for all tissues investigated and for all the class sizes we found approximately the same quotient

CARYOMETRIC STUDIES OF TISSUE CULTURES

105

(Bucher and Gattiker, 1953, Table 111; 1954a, Table 11): the mean quotient equals 0.82. In Figure 20 the planimetrically measured sizes of the two nuclei in the binuclear cells are represented on the abscissa, while the corresponding

40

20

205

230

260

290

325

365

410

460

515

580

650

730

820

920

1030

FIG.20. Nuclear size quotients (ordinate) as a function of nuclear size classes (abscissa). The striking constancy of nuclear size quotients is shown by the fact that the series of points is parallel to the abscissa. (From Bucher and Gattiker, 1954a.)

mean quotients are represented on the ordinate. The connection between the points gives an almost straight line parallel to the abscissa. We have thus been able to demonstrate that the quotient of the nuclear sizes within binuclear cells is constant and independent of the absolute nuclear mass. This is perhaps an observation which could be of general biologic significance. Concerning the nuclear sizes within binuclear cells in comparison with the nuclear sizes within mononuclear cells, we have obtained the following results. The sizes of the individual nuclei of binuclear cells of the outer zone of hanging-drop cultures show an increase of d F i n comparison with the nuclei of mononuclear cells of the inner zone (as well as with those in viva), and the sum of nuclear volumes of binuclear cells is on the average VTgreater than the nuclear volumes of mononuclear cells of the outer zone (Fig. 21). The nuclear surface of binuclear cells is, as 8,

one can easily calculate, d 2 = 1.26 times as large as that of mononuclear cells with the same nuclear volume (Bucher and Gattiker, 1953, 1954a). The total gain of the surface of binuclear cells (the mean volume of which has already increased by dz and the corresponding surface of a not yet divided nucleus of the same volume by i.e., approximately 60%.

is therefore 1.26X1.26=1.59,

A number of authors (e.g., Miinzer, 1923, 1925) have shown that the binuclear cells Vivo appear not only when the work of the cells has increased, but also under unfavorable conditions. In accordance with this is the observation of G. Levi (1934) ilz

106

OTTO BUCHER

that the number of bi- and multinuclear cells increases with the age of the tissue culture in Vitro, because the aging brings with it deteriorated conditions (see also Bucher and Gattiker, 1954d). In all these cases, the appearance of binuclear cells signifies an increase of the total nuclear surface and consequently facilitates the metabolic exchange between nucleus and cytoplasm.

K,

M

icz

M

FIG.21. Normal distributions corresponding to nuclear size' frequency curves from mono- and binuclear chick fibroblasts. Solid lines: inner and outer zones of mononuclear cells. Broken lines: the curve on the left represents the volume of the individual nuclei oi binuclear cells, that on the right the sums of the volumes of the two nuclei of binuclear cells from the outer zone. (From Bucher and Gattiker, 1954b.)

The increased percentage of binuclear cells in fibroblast cultures having been held at room temperature (Table V II), as well as in cultures having been exposed to trypaflavine in doses of 1 :600,000 over a period of 9 hours TABLE VII PERCENTAGE OF BINUCLEAR CELLS I N CHICK FIBROBLAST CULTURES HELDIN INCUBATOR (38-39°C.) AND AT ROOMTEMPERATURE RESPECTIVELY Explant Heart Frontal bone

Exposed to Temperature of rnz.2"0: 38-39" c. 1.31% 1.04%

3.39% 3.25%

AN

Number of Evaluated Cells 4,950 and 4,650 resp. 1,250 and 1,200 resp.

(From Bucher and Gattiker, 1954.).

(2.6% of binuclear cells in comparison with 1.2% in the control cultures), as well as in cultures previously treated with colchicine in concentrations of 1:10 million and 1:20 million (1.2% of binuclear cells in comparison with 0.7% in the control cultures) is interesting. I n all these cases an increase in the number of amitotic figures was observed in the experimentally influenced cultures (Bucher, 1947, 1952a, b; Gattiker, 1952 ; Bucher and Gattiker, 1954~). We have explained the origin of the mitoses, and consequently that of the binuclear cells, as follows (Bucher, 1952b) : the prior treatment of colchicine results, as indicated above, in the polyploidization of a certain number of cells; the changed internal

CARYOMETRIC STUDIES OF T I S S U E CULTURES

107

organization of these cell nuclei is probably the cause of the amitotic divisions, which in turn explains the increase in the percentage of the binuclear cells. In the experiments exposing cultures to room temperature (Bucher and Gattiker, 1954a, c) or employing trypaflavine, practically no mitoses were observed (with the concentrations we used ; for details see Bucher, 1939). A cell nucleus thus inhibited from undergoing mitosis would therefore undergo direct divisions, as might also be the case in the whole organism ; the amitosis, which is least sensitive to harmful influences and from which binuclear cells result, appears therefore as a substitute solution, while normally in tissues cultivated under favorable conditions the mitoses, from which mononuclear daughter cells result, predominate.

The last experiments (Bucher and Gattiker, 1954b,c, d ; Bucher, 1954) have demonstrated that our caryometric method also gives good results for the study of the size of the n.ucleoli. A linear correlation of 30 to 50% exists between the nuclear and the nucleolar sizes, i.e., with the increment of the nuclei, the nucleoli increase in a constant ratio. Figure 22 shows that the average nucleolar sizes corresponding to the different nuclear size classes (on the abscissa) and the average nuclear sizes corresponding to

FIG.22. Graphic representation of the linear correlation between nuclear a n d nucleolar sizes of 1,000 chick fibroblasts. The average nucleolar sizes (black dots) corresponding to the different nuclear size classes (abscissa) as well as the average nuclear sizes (small circles) show only a slight deviation from the two calculated regression lines. The angle between the two straight lines is a measure of the degree of correlation, which is in this case 34% (in case of coincidence of the two lines, there would exist a correlation of 100%; in the case of an angle of No, there would be no correlation). (From Bucher and Gattiker, 19.54~)

108

OTTO BUCHER

the nucleolar classes (on the ordinate) deviate only slightly from the two calculated straight regression lines. Further, of great interest is the quotient “nuclear size divided by nucleolar size” which depends on the intensity of growth (for more specific details see Bucher and Gattiker, 1954b) and can be influenced experimentally (i.e. Bucher, 1954).

V. CONCLUSIONS Tissue cultures in vitro have up to now rarely been used for caryometric studies. This is due to the fact that the nuclei in hanging-drop cultures af conjunctive tissue do not possess a clearly definable geometric form, which would permit us to calculate their volume, but are instead greatly flattened. In order to fill up this lack in the domain of cytology, we have worked out a method by which it is possible to obtain a clear idea of the behavior of nuclear sizes without measuring the third dimension, perpendicular to the coverslip, and without calculating the absolute nuclear volume. This has been possible because the third axis of the nuclei increases proportionally to the two larger axes lying parallel to the coverslip. The procedure developed in our laboratory is in principle the following: the largest projected plane of a nucleus, which in hanging-drop cultures lies parallel to the coverslip (as mentioned above), is drawn under a linear magnification of 1,500 x , and the surface so obtained is measured with a planimeter. The error in this method is relatively small. If two volumes V1 and V 2 of two nuclei are in a ratio of 1 :2, then the corresponding projected planes F1 and F2 are in the proportion 1 :1.5875. This is the proportionality factor of the geometric progression F1 :Fz:Fq etc. Having in this way a direct proportion not only between the different planimetrically measured projected planes, but also between their relative volumes, we are now able to derive the reciprocal relationships among the different classes of nuclear sizes and the results of experimentally influencing these relationships. In order to characterize our frequency curves mathematically, we have calculated several elements of curves, such as the kurtosis of Pearson, the measure of skewness, and the symmetry of the curve, as well as its standard deviation. Generally the kurtosis ,Elz is approximately equal to 3 ; this is always the case if we are dealing with normal distributions. The standard deviation (s) of our frequency curves from non-influenced cultures varies around 2. If we consider a sufficiently large random sample of measured nuclear sizes, the frequency curves correspond to normal distributions, and for each empiric curve of planirnetric values we are able to calculate the

CARYOMETRIC STUDIES O F TISSUE CULTURES

109

corresponding normal curve as we have demonstrated in a concrete example. In many cases it is advantageous to work with a cumulative frequency distribution instead of with the ordinary frequency curve. The cumulative frequency curve of a normal distribution, drawn on a probability chart, is represented by a straight line. In the growth area of hanging-drop cultures of connective tissue, we can distinguish an inner and an outer zone, between which there is a transitional zone. The statistical evaluation of nuclear sizes reveals that the volumes are in a ratio of 1 :2, the nuclei of the outer zone having doubled their volumes with respect to those of the inner zone in d tr o and to the nuclei in Vivo. Studies of the nuclear sizes of fibroblast cultures of different animals and of man showed that the proportions of specific nuclear sizes are maintained after explantation in vitro. While the frequency distributions from our untreated cultures are in principle always normal distributions, there exist two possibilities in the case of experimentally influenced cultures : first, the nuclear-size frequency curves still correspond to normal distributions (as after application of urethan, trypaflavine and arsenic trioxide) . The standard deviation and the average nuclear size may or may not be modified, or second, the frequency curves no longer correspond to normal distributions, as was the case in our experiments of prior treatment with colchicine. Our caryometric method permitted us as well a thorough study of the nuclear behavior of binuclear cells. In fibroblast cultures of different animals and of man, we found an average percentage of 1.0 to 1.5% binuclear cells. I n order to find a quantitative criterion for the size difference between the two nuclei of binuclear cells, we calculated the quotient by dividing the smaller nucleus by the greater one. Most interesting is the fact that the mean quotient is independent of the absolute nuclear volume of the binuclear cells as well as of the species of tissues investigated. The sum of nuclear volumes of binuclear cells is on the average VTgreater than the nuclear volumes of mononuclear cells. A linear correlation exists between the nuclear and the nucleolar size. An infinitely great number of problems is yet to be studied. It is evident that we must be extremely careful and critical in the evaluation and interpretation of the results and that, moreover, some notion of biologic statistics is indispensable. In outlining in this paper some of these problems and some of our results, obtained up to now in the field of caryometric study, not the least of our intention has been to suggest further investigations in this domain.

110

OTTO B U C H E K

VI.

REFERENCES

von Arx, A. (1953) Vkrfeljahrssclrr. m~titrforsch. Ced. Ziirich, 98, 157. i c h f794. e, Rirkenniaicr. 0. (1934) Z.Anaf. ~ n f ~ ~ c k b t n . ~ s ~ r s c h102, Rizzozero, G. (1893 and 1903) cited after G. Levi. B a r n . J. (1934) Z . rnikroskop.-airat. Forsch., 36, 464. Bucher 0. (1939) Z. Zellforsch. u. mikroskop. Anat., 29, 283. Ruchcr, 0. (1947) d c f a Anaf., 4, 60. Bucher, 0. (19504 Verhandl. Anat. * schweiz. Hochschulen, 1949, reported in Schuviz med. Wochschr., 80, 1219. Bucher, 0. (1950b) Mikroskopie, 6, 124. Bucher, 0. (1951a) Arrh. Juliw Klurrs-Sfift. Verfrbicngsforsch. Socidanthropol. it. Rwscnhyg., 26, 177. Bucher, 0. (1951b) Vrrhandl. Anat. Gcs., Suppf. Anat. Ails.. 98, 86. Bucher, 0. (19521) E r p m h t i a , 8, 201. Bucher, 0. (1952b) Verhandl. Anat. Ges., S ~ p p l Anat. . Anz., 99, 41. Bucher, 0. (1953a) 2. Zellforsch. 11. mikroskop. Anat., 38, 455. Bucher, 0. (1953b) 2. Anat., 117, 20. Bucher, 0. (1953~) Bull. microscop. appl., (11) 3, 113. Bucher, 0. (1953d) Verhandl. Anat. Gcs., Suppl. Atrat. Anz., 100, 197. Bucher, 0. (1954) Verhandl. Anat. Ges., Suppl. Anat. Atra., 101 (In press). Bucher, O., and Gattiker, R. (1950a) Acfa Anuf., 10, 430. Bucher, O., and Gattiker, R. (1950b) RPV.suissc Zool., 67, 769. Bucher, O., and Gattiker, R. (1952) 2. Zellforsch. u. mikroskop. Airat., 37, 56. Bucher, O., and Gattiker, R. (1953) Ezptl. Cell Resoarch, 6, 461. Bucher, O., and Gattiker, R. (1954a) 2.mikroskop.-atraf.Forsch., 60, 308. Bucher, O., and Gattiker, R. (1954b) 2. mikroskop.-anat. Forsch., 60 (In press). Bucher, O., and Gattiker, R. (19%) 2. Anat. (In press). Bucher, O., and Gattiker, R. (1954d) Anat. A m . (In press). Bucher, O., and Horisberger, B. (1950) Acta Anut., 9, 258. Clara, M. (1930) 2.mihroskop.-a~zat.Fot-sch., 22, 145. Dogliotti, G. C. (1927) Arch. erptl. ZeUforsch., 3, 242. Ehrich, W. (193Ga) Z . Krtbsforsch., 44, 308. Ehrich, W. (1936b) Am. J . Med. Sci., 192, 772. Fisher, R. A. (1950) Statistical Methods for Research Workers. Oliver and Boyd. London. Freerksen, E. (1933) 2. Zt'llforsch. u. mikroskop. Anut., 18, 362. Gaillard, P. J., and Bakker, J. H. (1938) N e d . Tijdschr. Geneesk., 82, 5. Gattiker, R. (1952) 2. Zellforsch. u. mikroskop. Ailat., 37, 467. Gebelein, H., and Heite, H.-J. (1951) Statistichc Urteilsbildung. Springer Verlag, Berlin. Heiberg, K. A. (1921) Yirclrows Arch. Pathol. Anat. u. Physiol., 2S4, 469. Heiberg, K. A. (1933) Die Grundlage der Geschwulstlehre. Kabitzsch, Leipzig. Heiberg, K. A. (1934) Mass und Zahl im Zellleben. Levin and hfunksgaard, Copenhague. Herbst, C. (1914) Arch. Enhn'ckhngsmech. Orgm., 89, 617. Hertwig, G. (1939) Anat. Anz., 87, Suppl., 65. Hintzsche, E. (1936) Z . mikroskop.-anat. Forsch., 39, 45. Hintzsche, E. (1945) Erperientia, 1, 103.

CARYOMETRIC STUDIES OF TISSUE CULTURES

111

Hintzsche, E. (1946a) Mift. natwforsch. Ges. Bern, (N.S.) 4, 19. Hintzsche, E. (1946b) 2. Volkszerirfschaff u. Sfatistik, 82, 433. Hintzsche, E. (1949) Gynoecologia, 128, 270. Jacobj, W. (1925) Arch. Entwicklungsmech. Organ., 106, 124. Jacobj, W. (1931) Anat. ANZ.,72, Suppl., 236 Jacobj, 1%'. (1935) 2. mikroskop.-uiaut. Forsch., 38, 161. Jacohj, W. (1942) =Irck. Entw*ckliingsiltc.ch. Organ., 141, 584. Korner, F. (1937) 2. mikroskop.-wrat. Forsch., 42, 81. Krantz, H. (1952) Gege-ttbaurs tnorphot. fahr., 92, 29. Levi, G. (1934) Ergcb. Anat. 14. En~'cklungsgeschichfe,31, 125. Lewis, W.H. (1948) Anat. Record, 100,247. Macklin, Ch.C. (1916a) Anat. Record, 10, 225. Macklin, Ch. C. (1916b) Biol. BdI., So, 445. hfeyer, R. (1937) 2. ZeUforsch. 21. naikroskop. Anut., 26, 353. von hiollendorff, W., and von Mollendorff, hl. (1926) 2. Zellforsch. qd. mikroskop. *4ttat., 3, 503. hfiiller, H. G. (1937) 2. mikroskop.-anat. Forsch., 41, 296. Munzer, F. Th. (1925) Arch. mikroskop. Anat. U. Entzkklungmech., 104, 138. Miinzer, F. Th. (1923) Arch. mikroskop. Anat. u. Enfzwkklungsmech., 98, 249. Pfuhl, W. (1930) 2. Irtikroskop.-anaf. Forsch., 22, 557. Pfuhl, W. (1932) 2. mikroskop.-amt. Forsch., 31, 18. Sauser, G. (1936) 2. Zcl[forsch. u. mikroskop. Anat., 23, 677. Schairer, E. (1936) 2. Krebsforsch., 43, 1. Schairer, E. (1937) 2. Krebsforsch., 46, 279. Snedecor, G. W. (1953) Statistical Methods. The Iowa State College Press, Ames. Voss, H. (1936) Anat. Anz., 82, 230. Wassermann, F. (1929) in Handbuch der mikroskopischen Anatomie des Menschen, Vol. I/2. Springer Verlag, Berlin. Werniel E. M., and Ignatjewa, Z. P. (1932a) Z . Zellforsch. u. mitroskop. And., 16, 674. Werrnei, E. M., and Ignatjewa, 2. P. (1932b) 2. Zellforsclt. u. mikroskop. Anof., 16, 689. Wermel, E. M., and Portugalow, W. W. (1935) 2. ZeZlforsch. U. mikroskop Amf., 22, 185. Wilflingseder, P. (1947) Forsch. Tirolm Aerzteschde 1945/47, 343. Wilflingseder, P. (1948) Mikroskopie, 3, 243. \i'illigens, C. (1932) Z . schwek. Stah'stik u. Volkmirtscheft, 68, 445. Willigens, C. (1933) Z . s c h w e t . Sfatistik 21. L'olkrz&fschaff, 69, 125. Zweibaum. J., and Szejnam, M. (1936) Arch. exptl. Zellforsch., 18, 102.

This Page Intentionally Left Blank

The Properties of Urethan Considered in Relation to Its Action on Mitosis IVOR CORNMAN

The George WaFhington University Cancer Clinic and the Department of Anatomy. School of Medicine, Wa.shington, D. C. I. Introduction ......................................................... 11. Carcinogenic and Carcinoclastic Properties ............................ 111. Cytologic Effects .................................................... IV. Biochemistry ........................................................ v. summary ........................................................... VI. References ..........................................................

Page 113 114 118 123 127 128

I. INTRODUCTION There would seem to be a better chance to understand mitosis if we strike at it with chemicals that are well understood, or that at least produce a number of well-defined effects. A shortcoming of colchicine has been that its most striking property, its ability to disorganize the spindle fiber, was about all we knew about it. It has long been used to relieve gout, but only recently has our information been augmented by biochemical and biophysical investigation of the sort that will help us interpret its effects on mitosis. Some of us, in the meantime, turned to other chemicals which, while less spectacular, were known for their several physiologic effects. One such chemical, quite different in its versatility as well as in the nature of its effects on cell division, is urethan. It has long been known as a narcotic and a mitotic poison. (Here the term mitotic poison will be applied to any substance which disrupts division in any cell without killing it. This loose usage is justified, I believe, if one makes clear at the outset that this does not imply that the sole or predominant action of the substance is to affect mitosis.) More recently it has been shown that urethan is a carcinogen. Nettleship and Henshaw in 1943 found lung tumors in mice that had been previously anesthetized with urethan. This experiment has been successfully repeated by skeptics. Those of us accustomed to thinking of carcinogenesis in terms of hydrocarbons with enough benzene rings to make a floor pattern, found it hard to admit to this unholy company a substance made simply of an ethyl, an amino, and a carboxyl radical. Perhaps some more loose terminology Original research by the author reported in this paper was supported in part by an institutional grant to the George Washington University Cancer Clinic from the American Cancer Society.

113

114

IVOR CORNMAN

needs defending here: carcinogenic is used to describe any process producing neoplastic growth, regardless of invasiveness and metastasis. Among the definitions we should also include “carcinoclastic,” the property of destroying or retarding neoplastic growth. Urethan can boast this property also, to the extent that it has been used as a palliative in human malignant disease. This makes a phenomenal list of accomplishments : narcotic, mitotic poison, carcinogen, and carcinoclast ; four diverse categories of information that can be brought to bear on the nature of the mechanism by which urethan acts. Caution and questions are in order here. Do these multifarious manifestations represent phenomena arising from a central cause? Can we use these varied effects to analyze a single series of events, or does urethan bring about each different effect by a different mechanism ?

11. CARCINOGENIC AND CARCINOCLASTIC PROPERTIES First of all, consider the evidence that tumor production occurs separately from interference with - cell division. For this purpose a homologous series of carbamates is useful (Table I ) . Long ago, Warburg

TABLE I HNHCOOCHt

HNHCOOCHsCHKHt HNHCOOCHiCHs CoHaNHCOOCHLHz (CHa) rNCOOCHiCHi

methylcarbamate propylcarbamate ethylcarbamate ethyl-N-phenylcarbamate ethyl-N,N-dimethy lcarbamate

(1910, 1911) showed that higher members of the series such as ethylphenylcarbamate (synonymous with phenylurethan) were more effective than urethan, simple ethylcarbamate, in retarding cell division. Our experiments, also with sea urchin eggs ( 1950a) , supplied the relative mitotic potency for Table 11, in which it is seen that the higher homologues can be 100 times as effective as urethan. But Larsen (1946, 1948) has shown that tumor formation is restricted almost entirely to urethan, with some carcinogenic activity residing in a few simple congeners (Table 11). It is also pertinent to consider that the narcotic potency is another function which increases as one adds to the urethan molecules, whereas carcinogenesis occurs in mice at subnarcotic doses and is not induced by other narcotics (Larsen, 1946). This can be reduced to a simple rule : almost any radical added to the urethan molecule increases its effectiveness as a mitotic poison and decreases or removes its carcinogenic action.

TABLE I1 CAR~AMATES AS MITOTIC INHIBITORS,CARCINOGENS, AND CARCINCKLASTS Antimitotic 1 2 2

5 6

Carbamate Esters Carbamate Forms Ester Tumors ethyl ProPYl isopropyl butyl isoamyl

Antileukemic

Antimitotic

+++

+++

1/2

+

-

0 0

-

0

0

0

1/4 2 2

10

100 100

N-Substituted Urethans N - Subst. Forms Urethan Tumors methyl dimethyl ethyl isopropyl dipropyl dibutyl phenyl

+ + + ++ + 0 -

s

Antileukemic

0 -

++ 0 0

++ +

The relative effectiveness of carbamatei in retarding cell division i n sea urchin eggs ia expressed with urethan as unity (Cornman, 1950). Uretban is the most effective carbamate in forming tumors (Larsen, 1947a, b; 1948) and retarding leukemia in mice (Skipper et d. 1938, 1949). No activity is erprcsacd as (0) and a dash indicater the substance has not been tested for thii property.

(+++)

s FZ 2 % W

F =!

0

Z

cl 0

Y

8

;

116

IVOR CORNMAN

It is highly interesting that the carcinoclastic activity is just as circumscribed. Table I1 includes some of the compounds Skipper and his coworkers (1948, 1949) found effective in decreasing the white blood cell count and prolonging life of mice with transplantable leukemia ; these were, again, a small group in the neighborhood of urethan. These compounds were the most effective also in reducing the white cell count in non-leukemic mice. Narcosis and lethality to mice, on the other hand, increased as one passed to higher and higher homologues. Haddow and Sexton (1946) and Dustin (1947) similarly report that urethan is more effective than phenylurethan or isopropylphenylcarbamate in the treatment of animal tumors and leukemia. Division inhibition, narcosis, and toxicity, then, progressively increase as one goes up the series, but carcinogenic and carcinoclastic activity are scattered sparsely among compounds not much different from urethan. The evidence for carcinogenesis and carcinoclasis is of necessity drawn from experiments with intact animals, but the mitotic studies relied upon so far have been on isolated sea urchin eggs. W e know the carbamates entered the mice, but did they enter the eggs? This penetrating question is the more fair in the light of the high liposolubility of the antimitotically effective carbamates. W e were able to show (Cornman et al., 1951) that urethan penetrates sea urchin eggs (Tripnemtes) phenomenally fast and is even accumulated within the egg. To make this measurement, eggs were removed from urethan-sea water by centrifugation ; they were then frozen, and the urethan concentration was determined by hydrolysis and colorimetric determination of the alcohol. Table I11 shows that within TABLE 111 URETHAN PENETRATION OF FERTILIZED SEAURCHIN Ems CONCENTRATION OF 20 MG./ML. Duration of Exposure

FROM A

Concentration in Eggs

2’ 44” T 59”

28.9

1928”

35.4

4Y 15”

SEA-WATER

25.1 mg./g.

14.7

a few minutes the concentration within the eggs exceeded the concentration of 20 mg. per milliliter in the surrounding sea water. I t continued to accumulate, reaching a concentration nearly double the initial sea water concentration and then declining, presumably because the urethan was

URETHAN I N RELATION TO MITOSIS

117

metabolized. It is imprcssive, and probably significant, that such a high concentration of urethan should be necessary to disrupt mitosis. Allowed that urethan penetrates well, once inside does it do the same thing as the higher congeners? The cytologic evidence, to which we shall give more attention shortly, indicates that it does. The typical carbamate effect, as one observes living eggs under its influence, is a non-specific weakening and slowing of division, not at all like the distinct spindle destruction of the colchicine type (Cornman, 195Uc). The urethan pattern of interference is found with all carbamates. Where several carbamates have been studied on higher animals, the cytologic picture is similar. A less subjective sort of information can be drawn from observation of effects of temperature on the activity of two carbamates. The effectiveness of urethan depends on the temperature ; the effectiveness increases as the temperature is raised or lowered from the temperature at which the urchin spawns (Cornman, 1950b). If one chooses an arbitrary degree of retardation of cell division and plots the amount of carbaniate required to produce that retardation at any temperature, it is found that for Arbacia eggs there is a minimum effectiveness at about 21 O C. [Fig. I ) , Below and above that temperature, less urethan is required to retard cleavage. A carbamate

Temperature, "C.

FIG.1. Uniform retardation of ArbacM cleavage.

118

IVOR CORNMAN

carrying two additional ethyl groups is affected in the same way; it is minimally effective at 21" and increasingly effective as the temperature varies above or below this level. This parallel exists despite the much higher potency of ethyldiethylcarbamate : at any temperature, the diethyl compound retains an eight-fold advantage over urethan. 111. CYTOLOGIC EFFECTS This, then, gives us a fairly good basis for argument. In their effect on cell division, carbamates act as a continuous series, whereas carcinogenesis and carcinoclasis are limited to a few carbamates. What is it then that carbamates do to a dividing cell? Interestingly enough, there is sharp disagreement about the morphology of their effects. It is safe to say, I think, that much of the disagreement arises from the different organisms studied and the different dose levels used. If one observes the living sea urchin egg or sand dollar egg under the influence of urethan, or of most other carbamates at the threshold concentration, one sees cell division essentially normal but retarded. For the Woods Hole sand-dollar at the summer temperature of sea water (about 20" C.), this threshold is about 17mM. of urethan or 0.03 mM. of phenylurethan. From our marine egg studies, we have no data about the exact amount each phase is elongated, but no one phase is excessively affected. The living egg shows, aside from slight irregularities, a picture inside and outside the cell that is quite normal. At a slightly higher concentration, however, there is a considerable deviation from the normal at one point. This is better shown by a graph plotting the progress of cleavage rather than by a picture at any one instant (Fig. 2). Low concentrations retard cleavage, but permit all eggs to divide. At this higher concentration (about 22mM. for urethan and 0.06 mM. for phenylurethan) something critical happens. Most of the eggs are apparently successful in dividing, but the furrows regress. This does not result from a half-hearted attempt on the part of the egg. The furrow is deep, and to all appearances complete. There is some critical, final pinching off which the narcotized egg can not consummate. Nevertheless, the egg later forms two mitotic figures, and divides into four cells when its turn for second cleavage comes around. The division is late, of course, but you are treated to the interesting spectacle of a higher percentage of second cleavages than of successful first cleavages. This is not a statistical mirage. I t merely means that percentage-wise more two-cell become four-cell than one-cell become two-cell. Or, once divided, an egg divides more easily a second time. This nuclear duplication without cleavage can survive two mitotic cycles and successfully divide the eggs into six to eight blastomeres all at once. Species such as Echinurachnius and

URETHAN IN RELATION TO MITOSIS

119

Lytechinus reveal some details of what goes on inside the cell when the dose slightly exceeds that which permits cleavage. Nearly always there is a buildup of the achromatic figure. The asters become visible and the nucleus disappears. The entire process is much retarded, and the

FIG.2. Retardation and regression of Echinarachnius egg cleavage in urethan.

achromatic figure is stunted and distorted. Sections provide more detail. Tripneustes eggs exposed. to a blocking dose of urethan still have an intact nucleus an hour after fertilization, when the controls have already divided. By 80 minutes the nucleus has dissolved, but the only evidence of an achromatic figure is a vesicle or two of spindle material near the irregularly scattered chromosomes. This situation obtains even at 2 hours, when the chromosomes have drifted farther apart, but nothing else has happened. By 3 5 hours, when the controls have gone through four or five mitotic cycles, the blocked eggs finally show some organization. A small achromatic figure forms, organizes a metaphase plate, and takes at least some of the chromosomes in tow. The chromosomes are short and thick. It is quite usual for these delayed figures to be tripolar, or occasionally two separate spindles form. At lower doses these twin figures are responsible for simultaneous division into four cells, but at doses which prevent cleavage, divisions typically revert to an interphase by the usual process of karyomere formation. A single nudeus usually results because the chromosomes never got very far from each other. By 6 hours a few eggs have divided into two or four cells, but under this continuous treatment] none survive 24 hours.

120

IVOR CORNMAN

These cytologic findings with urethan represent nothing new. They agree in every detail with Painter’s observations (1915, 1918) with phenylurethan on other urchin species. His studies in turn are antedated by brief attention to the carbamates by R. S. Lillie in 1914 and Fuhner in 1903. Studies employing urethan or phenylurethan on the eggs of other forms yield these and related abnormalities (Warburg, 1910, 1911 ; Brachet, 1934 ; Tyler and Horowitz, 1938 ; Moment, 1938; Sentein, 1949-1951). Events in other types of cells closely parallel those seen in sea urchin eggs, at least if we confine our attention to isolated cells. In mammalian and avian cells in tissue culture, urethan incites a range of abnormalities. They involve primarily niisdistribution of chromosomes, or binucleation, again a reflection of a general interference with the achromatic figure and the cleavage process (Ludford, 1936 ; Geiersbach, 1939 ; Bucher, 1947, 1949a, b ; Lasnitzki, 1949; Paterson and Thompson, 1949). The weight of evidence indicates, too, that at critical doses metaphases accumulate well beyond their normal proportions, while anaphases and telophases almost disappear, and prophases are fewer (von Mollendorff, 1937a, b ; Bastrup-Madsen, 1949; Bucher, 1947, 194%, b; Hughes, 1950). In the intact animal one or more of these effects are found. The trout blastoderm shows multipolar mitoses, binucleation, and some accumulation of metaphases (Battle and Laing, 1949). The salamander shows a relative increase of metaphases when exposed to phenylurethan (Luther and Lorenz, 1947). In rodents, the cornea shows only a decrease in mitosis (Guyer and Claus, 1947), but wounds show an increased proliferation at about the same dose (Lushbaugh et al., 1948). Marrow shows a good range of abnormalities, such as metaphase block, binucleation, clumping and bridging of chromosomes, and reversion to interphase from other stages (Balduini, 1949; Rosin, 1951). In the crypts of Lieberkiihn there is pycnosis (Dustin, 1947) or a transitory increase in mitosis (Haddow and Sexton, 1946). Maturation also seems to be blocked a t metaphase in the mouse ovary (Fuhrmann, 1950). In human marrow also can be seen blocking of mitosis, along with clumping and scattering of chromosomes without particular effect on any one phase (Schulze et al., 1947; Reimer, 1948; Moeschlin, 1947). Most of these abnormalities fit those seen in isolated cells: general disruption of most aspects of cell division. The exception is the extreme pycnosis in the intestine. This might be regarded as a minor variation or result of the other abnormalities except that much has been made of it. Dustin ( 1947) showed that urethan and isopropyl-N-phenlycarbamate, at least, when injected into a mouse or rabbit, produce severe pycnosis where dividing cells should be. Thus, the intestinal crypts are scenes of

URETHAN I N RELATION TO MITOSIS

121

intense cellular destruction, while normal cell division disappears. The sequence of events and the location of these cells is such as to indicate that the pycnotic nuclei are those which would have divided, From this it is concluded that the prophase or pre-prophase is particularly susceptible to urethan. This follows logically from the observations that cell division decreases and is then followed by pycnosis where karyokinesis should be. Now this disastrous effect is the result of repeated treatment or even a single dose of urethan, which is quickly disintegrated by the body. Isolated cells in sea water or tissue culture are rendered pycnotic only at that excessively toxic dose which destroys the cell. I t seems reasonable to suggest caution, then, regarding conclusions drawn from cytologic effects in the whole animal. There seems to be no lethal prophase susceptibility in the isolated cell. If in the intact animal the urethan-crippled cell is quickly destroyed, it probably is from influences of the organism on the cell. I n the organism the cell has a function to perform. If, partially incapacitated by urethan, it is unable to meet its obligation; that is, if it can neither divide on time nor act as a normal epithelial cell, then a crisis is created which it cannot surmount and from which it cannot retreat. The sea urchin egg, with only division to attend to, can carry on inefficiently for' awhile, and resume cell division-albeit abnormal-when the urethan is removed or destroyed. Dustin's (1947) own experiments showed that the organ is important, for in the thymus, which responds to colchicine with wholesale blocking of mitosis, there was no urethan-induced pycnosis. Far-reaching conclusions have been drawn from the abnormality pattern in Vivo. A mitosis-free period followed by pycnosis typifies a radiation effect on proliferating cells. Accordingly, urethan is included among the radiomimetic drugs, and the more so because of the carcinogenic and carcinoclastic effects we considered earlier, and the chromosome damage we will consider next. Fragmentation of chromosomes, an important aspect of the radiomimetic syndrome, has been described in plant and animal cells, but again we must be on guard. We have already seen that carbamates do different things by different mechanisms. Before we decide that the chromosome alterations are part of the effect on the mitotic mechanisms, we should look more closely at the biochemical aspects. In plants, whether in seedlings or roots from bulbs, in monocots or dicots, the list of urethan-induced abnormalities is the same as for animal cells. Because of the precise orientation of meristematic cells, it is possible to detect slight disorientations such as deviation of the axis of the figure. From this threshold effect, abnormalities range through various degrees of spindle disruption, and end with complete pycnosis, depending on the

122

IVOR COKNMAN

dose (Hohl, 1947; Battaglia, 1949; Deufel, 1951). As in animals, ethyl-N-phenylcarbamate (Simonet and Guinochet, 1939; Lefkre, 1939; Deysson, 1944, 1945 ; Ostergren, 1951 ; Ivens and Blackman, 1949; van Breemen, 1949) or propyl-N-phenylcarbamate (Ennis, 1948, 1949 ; Doxey, 1949) are more effective. Binucleation is common, indicating that as in animal cells, cytokinesis is particularly susceptible. But in plants the metaphase seems to be the genuine weak link. Carbamates, and especially the higher congeners such as ethylphenylcarbamate or isopropylphenylcarbamate, produce a true metaphase block and permit repeated cycles to build a high degree of polyploidy. In some respects this is unfortunate, for plants have been used in many laboratories for the study of mitotic poisons. The greater susceptibility of the plant metaphase has, I think, led to misunderstanding of the mechanisms which affect mitosis. This problem has not gone unnoticed by those working with plant material. Deysson ( 1944) underlined the toxicity of phenylurethan at concentrations that affect mitosis. Hindmarsh (1951) is one of those who have most recently pointed out that when one obtains a metaphase block with these indifferent mitotic poisons (in this case nitrophenols) it is at a concentration which causes other damage, and very near the threshold of toxicity from which the roots do not recover. Now, if the highly specific effect of colchicine, which appears to attack the processes immediately concerned with spindle formation, is acknowledged as separate from these narcotic effects, which almost certainly operate farther in the background, plants become ideal material for the study of narcotic series. Ustergren (1944, 1951) in Sweden, Gavaudan and his colleagues in France (Gavaudan and Poussel, 1944; Gavaudan et al., 1946) and others have shown that the effectiveness of a series of aromatic compounds correlates well with increase in the ratio of oil- to water-solubility. This correlation of thermodynamic activity with narcotic potency has been convincingly presented by Ferguson ( 1939), by Badger (1946), and by Brink and Posternak (1948). For our purpose here it will be adequate simply to say that as the carbon chain is increased in length, and water-solubility decreases, the narcotic potency increases. This is typical of any series of indifferent narcotics, just as it is with the carbamate series. Accordingly, it leads one to suspect that in the benzene derivatives one is dealing purely with narcosis, and not an effect like that induced by colchicine (which doesn’t fit any series, being very potent despite its water solubility).

URETHAN I N RELATION TO MITOSIS

123

IV. BIOCHEMISTRY

Can we look deeper and find in biochemical mechanisms the explanation of these cytologic events? We should acknowledge first the early contributions of Warburg (1911), showing that a series of carbamates forms a series of varying narcotic effectiveness when tested on sea urchin eggs, and that cell division is disrupted at concentrations that barely affect oxygen consumption. Brock et al. (1939) verified this for phenylurethan. Bodine and Fitzgerald (1949) showed that a series of carbamates reduced the oxygen consumption of dividing cells and non-dividing cells in the grasshopper embryo to the same extent. Tyler and Horowitz (1938) found the typical urethan effect in Urechis, nuclear division in the absence of cytoplasmic division, at concentrations of phenylurethan that did not affect respiration. Now this would seem to minimize the importance of respiration in the mitotic process, but this brings us up against Fisher and his co-workers (Fisher and Stern, 1940; Fisher and Henry, 1944) who find from the relationships of concentration to inhibition that there are two parallel respiratory systems affected by urethan, and that when one of these is inactivated, cell division is blocked. This was demonstrated in Arbmk, yeast, Te.trahymmna, and Colpoda (Fisher and Stern, 1940; Fisher and Henry, 1944; Burt, 1945; Ormsbee and Fisher, 194.4). This whole question of the dependence of mitosis on respiration is a touchy one. Scholander et al. (1952) have demonstrated waves of increased respiration coincident with cytoplasmic division in some species, and we have emphasized that cytoplasmic division is the first visible process inhibited by carbamates. In the frog egg, Zeuthen (1946) demonstrated cycling of respiration in time with cell division, but it is disconcerting to find that the cycling continued in one egg where there was nuclear division in the absence of cleavage. A typical effect of carbamates is to permit nuclear division in the absence of cytokinesis. One would be more hopeful of establishing a connection between cleavage and the effect of carbamates on respiration if non-cleaving mitotic eggs lost the undulations of respiratory activity. In Arbacia the evidence is that energy for cell division is eventually made available through the cyanide-sensitive system (Krahl and Clowes, 1940; Krahl, 1950). If we look at particular enzymes we find that the carbamates and other narcotics affect dehydrogenases. It is when urethan acts as a narcotic that it affects cell division, so if it does this by depriving the division process of oxidative energy, the point of interference is the dehydrogenase end of the cytochrome chain (Keilin and Hartree, 1939; Kreke and Suter, 1945; Cadoni and Imperati, 1930; Sen, 1931). Inasmuch as Meyerhoff and Wilson (1948) have demonstrated an

124

IVOR CORNMAN

inhibition of hexokinase and phosphohexokinase with phenylurethan, this appears to be another narcotic function of carbamates. From this Boyland ( 1950) derives encouragement for his hypothesis that some carcinogenic and carcinoclastic substances interfere with the phosphorylations necessary for maintaining the normal chromosome cycle. Perhaps both respiratory and glycolytic pathways supply energy for cell division, but one must conclude along with Brachet (1950) that the link between mitotic processes and respiration is remote in some species. Caution is to be observed in ascribing importance primarily to respiration in the effects of carbamates on cell division. Decreased and even increased activity of a number of other enzymes in vitro have been reported (cf. Heilmeyer et al., 1948). This involves one in the usual difficulty of assigning the isolated enzyme to its role in mitosis. Cell fractions give an answer in terms of the morphologic components we can see. Bodine and Lu (1950) have shown that urethan not only decreases the oxygen uptake of mitochondria and microsomes, but also affects the isolated nuclei in the same way. This takes on added interest in terms of a detail mentioned earlier: the relative decrease in prophases in the mitotic counts of urethan-treated material. Von Miillendorff ( 1937a, b) maintained that his time-lapse photographic method demonstrated a genuine shortening of the prophase. Might there be some enzyme at the cell surface which maintains the membrane until the end of prophase, and which is inactivated by urethan? This in turn harks us back to Darlington’s precocity theory of meiosis. Perhaps a single urethan-sensitive enzyme determines whether the karyokinetic process is to be mitotic or meiotic. With phenyl urethan Straub changed a mitotic pattern of division in Actinophrys into meiotic division, Moment used narcotics in an attempt to shift meiosis in the direction of mitosis. Another contribution from the biochemists is the observation that carbamates at low concentrations increase the oxygen uptake (Bodine and Fitzgerald, 1948; Runnstrijm, 1928; Brock et al., 1939; Cadoni and Imperati, 1930; Fellinger and Schmid, 1948; Williams et al., 1952; Huisman, 1951). Perhaps it is this stimulatory amount which shortens prophase. And there is other cytologic evidence of stimulation. Bucher (1949a, b) reports a genuine increase in the mitotic rate. This occurs at a low concentration where there is no prolongation of mitosis and therefore accumulation of mitosis can not be blamed for a spurious increase. Lushbaugh et d. (1948) produced increased proliferation in skin wounds of the rat with doses of urethan well below those which produced abnormal division. Lasnitzki ( 1949) obtained increased proliferation of carcinoma and sarcoma cultures with urethan concentrations that inhibited non-

URETHAN IN RELATION TO MlTOSIS

125

malignant growth. This latter may be a clue to the chemistry of malignant cells. As to the place of stimulation in the narcosis picture, it is hard to say in the face of results that are not in agreement. Enzymatic increases have been obtained with ethylphenyl, propyl, and butyl carbamates, which is as it should be if the stimulation is part of the narcotic process. The amount of increased dehydrogenation in guinea pig liver obtained by equal doses of a series of carbamate esters correlates inversely with the narcotic seriation (Cadoni and Imperati, 1930). One suspects that adjusting dose to obtain equal stimulation would give a reverse order. In grasshopper embryos, however, Bodine and Fitzgerald ( 1949) obtained stimulation of oxygen uptake only with urethan. This is another question we must leave open. A look at how urethan attacks cellular processes should give us a clue as to what it works on, and where. As a first question, is one part of the molecule more important than the other? To answer this we labeled urethan with C14 in the carboxyl group and in the methylene carbon of the ethyl group (Fig. 3). With carboxyl-labeled urethan, labeled bi-

f NaHC140~

~~r;c'400 CH~CH,

H&COO C'4H&Ha

liOC'*H?CHa

.

FIG.3. Isotope- labeled urethan and comparison moieties.

carbonate was run as a control, and with the ethyl-labeled molecule, labeled ethanol was the control. All were in amounts too small to affect cleavage. After 30 minutes or an hour the eggs were centrifuged. any unfixed urethan was washed or sublimated away, and the amount of fixed carbon was counted. The amount was infinitely small, and no more than was fixed from alcohol or bicarbonate. Eggs accumulate urethan rapidly and then lose it readily to resume physiologic function. Eggs completely blocked with a heavy dose of urethan will resume cleavage when placed in sea water free of the drug. It appears, then, that urethan exerts its narcotic effect while held only lmsely in the cell. \Veil, then, does it combine chemically, or does it simply dissolve in

126

IVOR CORNMAN

the protoplasm, or is it adsorbed on a surface? If it attaches, neither end would seem to be a logical site, for we have seen that substitution at the ester or the amino end has only one effect, and that is to increase the effectiveness as more carbon atoms are added. Again, any argument based on a series of carbamates must ask whether the higher congeners are really doing the same thing as urethan. For the cytologist, the morphologic evidence presented earlier should be fairly convincing. For the biochemist the change in response to temperature might carry more weight. The amount of urethan needed to produce a unit delay in the rate of cell division is greatest for the sea urchin at about 21", and decreases as one raises or lowers the temperature (Fig. 1). The doubly substituted nitrogen, ethyl-N,N-diethyl carbamate, too, is least effective at 21", and more effective at higher or lower temperatures. No claim is made for the precision of the slopes-they are roughly drawn from exploratory data (Cornman, 195Ob). The point to be made is that this similarity of temperature effect is not to be expected if alteration of the molecule alters its mechanism of action. Considerable alteration of the ends of the molecule in the chemical sense can be made without removing its narcotic activity. Accordingly, it seems improbable that urethan combines at either end. These temperature effects should tell us something about the way in which the carbamates act, and that indeed was the main reason for undertaking the experiments. The experiment was intended to show that urethan does two things to the cell proteins : at low temperatures it adsorbs and thereby interferes. As temperature increases, the urethan is driven off, and hence is less effective. But then a temperature is reached where the cell proteins start to denature, and urethan accelerates this process : hence, again more efficient action. The inspiration and argument were drawn from the work of Johnson et aE. ( 1948). Evidence that urethan accelerates enzyme denaturation is derived from the simultaneous use of pressure, which prevents that type of denaturation which presumably requires unfolding and expansion of the protein molecule. Johnson et al. (1948) in their figure 4 show that beyond a critical temperature the enzyme (in this case invertase) rapidly becomes inactive. If urethan is added, this inactivation sets in at a lower temperature, but pressure nullifies the urethan. At the lowest temperatures tested, pressure no longer offsets the urethan. Even in this range urethan still inhibits invertase, and Johnson is of the opinion that here it keeps the substrate from reaching the enzyme. It is doubtful whether this interference is competition in the sense of an antimetabolite, considering the variety of molecular patterns which are found among narcotics.

URETHAN IN RELATION TO MITOSIS

127

How does a carbamate attack a protein it is going to block or denature? Adsorption is a strong possibility, but Taylor offers evidence against it. Solution in cell lipids remains a consideration in view of the efficiency of the higher congeners. At least this efficiency of the more fat-soluble members of the series can not be ascribed to faster penetration, for we have seen that urethan enters the egg within seconds. This, then, sketches our knowledge of urethan in terms of its narcotic action on dividing cells. We might touch briefly-and finally-on another highly important effect on the chromosomes which can not be definitely assigned to the narcotic syndrome. It was mentioned earlier that chromosome fragmentation has been repeatedly observed. These involve not only breaks of the entire chromosome, but also the more delicate breaks (of which X-rays are also capable) : nipping off a chromatid, or breaking it twice so it can invert and reattach. Oehlkers (1943, 1946; Oehlkers and Linnert, 1949) has obtained as high as 38% translocations in Oenothera, and 31% with only 5 mM. urethan (1943). Mitotic block in the onion requires 200mM. Chromosome fragmentation has been produced in buds of other species by Oehlkers and Marquardt (1950) and by Gottschalk (1951). Vogt (1950) has obtained mutations in Drosophila by means of urethan. Harking back to our tracer experiments, by using sperm we found a slightly greater fixation of radioactivity than resulted from carbonate or ethanol. Urethan so combined was a very small fraction of the total, but it did combine with this compact genetic material which is sperm, whereas it did not combine detectably with egg cytoplasm. Boyland (1952) mentions experiments by Koller in which chromosome fragmentation induced in tumor cells by urethan was prevented with thymine. We have tested this combination with sea urchin eggs and could get no antagonism of cleavage inhibition. Here, then, seems to be a separate mutagenic action of urethan, operating perhaps via an attack on purine metabolism. V. SUMMARY With so little known about the action of carbamates there is little that can be presented as solid conclusions. Rather, the ideas discussed are summarized here uncritically as ideas to be more mercilessly attacked in the future. Urethan rapidly penetrates a cell, and the whole molecule attaches where it alters the action of one or more enzymes, and whence it can easily be dislodged. As mitosis begins, low concentrations stimulate enzymes, increase the number of dividing cells, and shorten prophase. In metaphase, the ever-sensitive spindle is disturbed, but not completely

128

IVOR C O R N M A N

inactivated, resulting in some blocked metaphases and all intergrades of chromosome maldistribution. At telophase, furrow constriction proceeds well until the final pinching off, at which point urethan interferes. Incomplete, the furrow regresses. In the binucleate egg, twin mitotic figures form and divide the cell into four blastomeres. Finally, in the interphase, urethan in small amounts attacks the chromosome, perhaps by chemical combination causing breaks and mutations.

VI. REFERENCES Badger, G. M. (1946) Nature, 168, 585. Balduini, M. (1949) Haemafologicu, 33, 55. Bastrup-Madsen, P. (1949) Acta Pathol. Microbiol. S c a d , 26, 93. Bastrup-Madsen, P. (1950) Le Sang, 21, 345. Bastrup-Madsen, P. (1951) Acfa Radiol., S6, 452. Battaglia, E. (1949) CaryoZogb, 1, 229. Battle, H. I., and Laing, M. A. (1949) Anat. Record, 106, 527. Bodine, J. H., and Fitzgerald, L. R. (1948) Physiol. ZoBI., 21, 303. Bodine, J. H., and Fitzgerald, L. R. (1949) Phpiol. Zoal., a,117. Bodine, J. H., and Lu, K-H. (1950) Anat. Record, 108, 536. Boyland, E. (1950) Riock. cf Biophys. Acta, 4, 293. Boyland, E. (1952) Cancer Research, l!2, 77. Boyland, E., and Williams-Ashman, H. G. (1951) Acta Unio Intern. Contra Cancrum 7, 432. Brachet, J. (1934) Arch. Biol, 46, 611. Brachet, J. (1950) Chemical Embryology, Chapter 5. Interscience Publishers, New York. vanBreemen, V. (1949) Anof. Record, 105, 588. Brink, F., and Posternak, J. M. (1948) I. Cellular Comp. Physiol., 32, 211. Brock, N., Druckrey, H., and Herken, H. (1939) Naunyn-Schmiedeberg’s Arch. exptl. Pathol. Phurmukol., 193, 679. Bucher, 0.(1947) Schweia. wed. Wockrchr..,77, 1zz9. Bucher, 0. (194%) Schwria. med. Wochschr., 79, 483. Bucher, 0. (1949b) Helv. Physiol. et PhurwcoI. Actu, 7 , 37. Burt, R. L (1945) Biol. BJI., 88, 12. Cadoni, G., and Imperati, L. (1930) Riv. patol. sper., 6, 65. Cornman, I. (1950a) I. Natl. Cancer Inst., 10, 1123. Cornman, I. (1950b) BWl. Bull., 99, 338. Cornman, I. (195oC) And. Record, 108, 535. Cornman, I., Skipper, H. E., and Mitchell, J. H., Jr. (1951) Cancer Research, 11, 195. Deufel, J. (1951) Chrotnosoma, 4, 239. Deysson, G. (1944) Compt. rend. Acod. Sci., 219, 366. Deysson, G. (1945) Compt. rend. Acod. Sci., 230, 367. Doxey, D. (1949) Ann. Bot., 13, 329. Dustin, P., Jr. (1947) BKf. I. Cancer, 1, 48. Ennis, W. B., Jr. (1948) Am. J. Botany, 36, 15. Ennis, W. B., Jr. (1949) Am. I. Botany, S6, 823. Fellinger, K., and Schmid, J. (1948) Wien. 2.iw. Med., 29, 245.

URETHAN IN RELATION TO MITOSIS

129

Ferguson, J. (1939) Proc. Roy. SOC.(London), Bl97, 387. Fisher, K. C., and Henry, R. J. ( 1 9 4 ) J. Gen. Physiol., 27, 469. Fisher, K. C., and Stern, J. (1940) Biol. Bull., 79,342. Fuhner, H. ( 1903) Naz~nyn-SclvmiedcDerg'sArch. exptl. Paihol. Pharmakol., Sl, 1. Fuhrmann, K. (1950) Virchods Arch. pathl. Anat. u. Physiol., 319, 1. Gavaudan, P., Dodi, M., and Poussel, H. (1946) Trav. sci. Sfa Essais Bowhe#, 2, 1. Gavaudan, P., and Poussel, H. (1944) Compt. r e d . SOC. biol., 188, 246. Geiersbach, U. (1939) Arch. exptl. Zellforsch. Gewebeziicht., 23, 210. Gottschalk, W. (1951) Chromosoma, 4, 342. Guyer, M. F., and Claus, P. E. (1947) Proc. SOC.Exptl. Biol. Med., 64, 3. Haddow, A., and Sexton, W. A. (1946) Nature, 167, 500. Heilmeyer, L.,Merk, R., and Pirwitz, J, (1948) Beiheffe Med. Monafsschr., No. 4, 120 pp. Hindmarsh, 11. M. (1951) Proc. Linncan Soc, New South Wales, 76, 158. Hohl, K. (1947) Experientia, 8, 109. Hughes, A. F. W. (1950) Quart. J . Microscop. Sci., (3) 91, 251. Huisman, T. H.J. (1951) Acta Physiol. ef Pharmacol. Neerl., 2, 88. Ivens, G. W., and Blackman, G. E. (1949) SymposiO SOC. Exftl. Bbl., 3,266. Johnson, F. H., Kauzmann, W. J., and Gender, R. L. (1948) Arch. Biochem., 19,229. Keilin, D., and Hartree, E. F. (1939) Pror. Roy. SOC.(London), B127, 167. Krahl, M. E. (1950) Bwl. Bull., 98,175. Krahl, M.E.,and Clowes, G. H. A. (1940) J. Gen. PhycysiOl., aS, 413. Kreke, C. W.,and Suter, Sister M. St. A. (1945) J . Biol. C k m . , 160, 105. Iarsen, C. D. (1946) J . Natl. Cancer Insf ., 7, 5. Larsen, C. D. (1947a) J. Natl. Cancer Inst., 8, 99. Larsen, C. D. (194%) Cancer Research, 7, 726. Larsen, C. D. (1948) J. Natl. Cancer Inst., 9,35. Lasnitzki, I. (1949) Brit. J. Cancer, 9, 501. Lefhre, J. (1939) Compf. vend. Acad. ScC, 208, 301. Lillie, R. S. (1914) J. B b l . Ckem., 17, 121. Ludford, R. J. (1936) Arc& exptl. Zellforsch. Gewebesiicht., 18, 411. Lushbaugh, C.C., Green, J. W., and Storer, J. B. (1948) J. NaB Cmcer Inst., 8,201. Luther, W.,and Lorenz, W. (1947) Strahlmthmpie, 77, 27. Meyerhof, O., and Wilson, J. R. (1948) Arch. Biochem., 17, 153. Moeschlin, S. (1947) Helv. Med. Actq Ser. A., 14, 279. yon Mollendorff, W. (1937a) Arch. exptl. Zellforsch. Gewebeziicht., 19, 263. von Mdlendorff, W. (1937b) 2. Zellforsch. u. mikroskop. Anaf., 27, 301. von Mlillendorff, W. (1938) Arch. exptl. Zellforsch. Gmbeziicht., 21, 1. Moment, G. (1938) Bull. Mt. Dtsert Is. Biol, Lab. 40th semon, p. 19. Nettleship, A.,and Henshaw, P. S. (1943) J. Natl. Cancer Inst., 4, 309. Oehlkers, F. (1943) Z. indukt. Abstamm-u. Yererblchre, 81, 313. Oehlkers, F. (1946) Biol. Zentr., 66, 176. Oehlkers, F.,and Linnert, G . (1949) 2. i m h k t . Abstamm.-zr. Vererblehrc, 83, 136. Oehlkers, F.,and Marquardt, H. (1950) 2. indukt. Abstahm-u. Vererblekre, 83, 299. Ormsbee, R. A., and Fisher, K. C. ( 1 Y 4 ) J. Gen. Physiol., 27, 461. Ustergren, G. (1944) Heredifas, SO, 429. Ustergren, G. (1951) Colloq. intern. centre natl. recherche sci. (Paris). 26, 77. Painter, T. S. (1915) J . Exptl. Zoo[., 18,299. Painter, T. S. (1918) 1. Exptl. Zool., 24, 445.

130

IVOR CORNMAN

Paterson, E., and Thompson, M. V. (1949) Nutwe, 163, 563. Reirner, E. E. (1948) Wkn. 2. inn. M e d , 29, 318. Rosin, A. (1951) Blood, 6, 652. Runnstriim, J. (1928) Actu Zool., 9, 475. Scholander, P. F., CIaff, C. L., Sveinsson, S. L, and Scholander, S. I. (1952) Biol. Bzcll., 102, 185. Schulze, E.,Fritze, E., and hliiller, H. H. (1947) Dm:.med. Wochsck., 72, 371 Sen, K. C. (1931) Bbchem. I., 26, 849. Sentien, P. (1949) Compt. rend. Assoc. And., 96,613. Sentien, P. (1950) I. SUisJe twd, So, 1218. Sentien, P. (1951/52) Arch. aiutt. hktol. et embryol., S4, 377. Sirnonet, M.,and Guinochet, M. (1939) Compt. rend. Acad. Sci., '208, 1667. Skipper, H.E.,and Bryan, C. E. (1949) J. Natl. Cancer Inst., 9,391. Skipper, H. E.,Bryan, C. E., Riser, W. H., Jr., Welty, M., and Stelzenmuller, A. (1948) I . Nutl. Cancer hut., 9,77, Straub, J. (1951) Biol. Zetttr., 70, 24. Taylor, G. W. (1935) J. Celluluv Comb. Phy.601., 7, 409. Tyler, A., and Horowitz, N. H. (1938) Biol. Bult., 74, 99. Vogt, M. (1950) 2. id&. A b s t m m u . Vererblehre, 88, 324. Warburg, 0. (1910) 2. physiol. Chpm., 66, 305. Warburg, 0.(1911) 2. physiol. Chon., 70, 413. Williams, W. L., Aronsohn, R. B., and Meyer, R. H. (1952) Federatim Proc., 11, 402. Zeuthen, E. (1946) Compt. rend. Wav. lab. Curlsberg. Sir. chim., 26, 191.

Composition and Structure of Giant Chromosomes MAX ALFERT Department of Zoology, University of California at Berkeley Page I. Introduction ........................................................ 131 11. Recent Advances in Chromosome Chemistry and Structure ............. 132 1. Constancy of Nuclear DNA ...................................... 132 2. Isolated Chroinosomes ........................ i ................... 134 3. The Extended State of Chromatin ................................ 134 4. Chemical Composition of Chromatin ............................... 135 5. Phase Contrast and Electron Microscopy .......................... 136 111. The “Salivary” Chromosome ......................................... 136 1. The Euchromatic Regions ........................................ 138 2. The Heterochromatic Regions ..................................... 143 3. The Theories of Salivary Chromosome Structure ................... 146 151 IV. The Lampbrush Chromosome ........................................ 1. The Lateral Projections .......................................... 157 2. The Chromosome Axis ........................................... 157 3. Development of Lampbrush Structure .............................. 159 4. Discussion and Conclusions ....................................... 159 V. The Functional Significance of Giant Chromosomes ; General Discussion 161 VI. References .......................................................... 164

I. INTRODUCTION I n only a few cases is it possible to see and study chromosomes in the interphase nucleus. The outstanding objects of such investigations have been the giant chromosomes’that occur in the cells of dipteran larvae and those which are found in telolecithal oocytes of vertebrates. I n recent years much work has been concentrated on the composition and structure of “interphase chromosomes” and, with the aid of new techniques, significant progress has been made. In the light of these results it seems justified to review critically the available information on giant chromosomes, especially the relation which these particular chromosomal types have to each other and to interphase chromosomes in general. As far as vertebrate oocyte chromosomes are concerned, they were first mentioned by Flemming (1882) and described in great detail by Riickert (1892) , who labeled them as “lampbrush” chromosomes. In the following half-century little progress was made in the study of these structures except for the firm establishment of their chromosomal nature and continuity as postulated by Riickert, but challenged previously by Schultze (1887) and subsequently in a series of papers by Carnoy and Lebrun (cf. 1900). Following Koltzoff’s (1938) stimulating essay, lampbrush chromosomes were the object of several investigations which 131

132

M A X ALFERT

have led to two essentially contradictory and seemingly irreconcilable views of their structure, sponsored by Duryee (1941, 1950) and Ris ( 1945) respectively. Aside from several electron microscope studies which will be discussed later, there has been surprisingly little recent information on these chromosomes until publication of GuyCnot and Danon’s (1953) monograph. The giant chromosomes in larval dipteran tissues such as the salivary glands, where they were first described by Balbiani (1881), have been the object of innumerable investigations ever since their chromosomal nature became established by Heitz and Bauer and independently by Painter in 1933, and their unique usefulness for genetic research was recognized. A large number of articles reviewing previous work on this subject have been published, beginning with Alverdes (1912) and including Painter ( 1934, 1939), Bauer ( 1936a), Metz and Lawrence (1937), Geitler (1938), Muller (1941), Caspersson (1950), and Bauer and Beermann (1952), aniong others. The reader is referred to these for detailed consideration of the numerous steps by which knowledge in this field has progressed. The present review will be limited to structural aspects of “salivary” and similar chromosomes and not deal with cytogenetic problems as such. The evidence for the major theories of giant chromosome structure will be discussed and supported by a representative sample of references. In addition, the reviewer will attempt to evaluate the available information according to his lights. In the following sections, recent advances in chromosome chemistry, the salivary gland type chromosome, the lampbrush chromosome, and a comparison of both types and their relation to nuclear function will be taken up consecutively.

11. RECENTADVANCES I N CHROMOSOME CHEMISTRY AND STRUCTURES The nucleoprotein composition of the interphase nucleus in a variety of cell types has been investigated by a combination of biochemical techniques and by microspectrophotometric studies in situ and on isolated cell components. For references and a critical discussion of the methods and findings, see Swift (1953). In the following paragraphs several points relevant to the further discussions are singled out for special consideration : 1. Constancy of Nuckar D N A An outstanding discovery, resulting from the independent work of several groups of investigators using a variety of techniques (chemical

COMPOSITION AND STRUCTURE OF GIANT CHROMOSOMES

133

analysis, cytophotometry and autoradiography) , is the finding that deoxyribonucleic acid (DNA) appears to be quantitatively correlated with the chromosome complement present in a nucleus and is by far the least variable of the known nuclear fractions. A characteristic quantity of DNA is associated with the haploid chromosome set of each species, and depending on their degree of ploidy, nuclei in non-dividing tissues contain multiples of the basic amount. Correlated with chromosome reproduction, nuclear DNA doubles prior to division in mitotically active cells and increases in geometric steps when nuclei become polyploid. In the latter case DNA measurements on individual nuclei cannot distinguish between different types of chromatid aggregation (i.e., polyploid vs. polytene condition) in the multivalent nuclei. In meiosis a single chromosomal reproduction in primary gonocytes is followed by two division cycles, and one finds that DNA is reduced to the basic (haploid) quantity in two equal steps, corresponding to the two meiotic divisions (see Caspersson as early as 1939, for nucleic acid content of meiotic nuclei). Functional changes in cells can bring about considerable differences in protein and ribonucleic acid content of nuclei as well as changes in size and appearance of nucleoli and chromocenters, but seem to affect the total DNA content of the nucleus little, if at all. While quantitative measurements were usually confined to interphase nuclei, an ingenious photometric method developed independently by Ornstein (1952a) and Patau (1952) has made it possible to follow the DNA content of whole chromosome sets through mitosis; by this method it was found that the total DNA of the chromosomes does not change during the course of mitosis (Patau and Swift, 1953), a conclusion confirming the evidence obtained by autoradiographic techniques (Pelc and Howard, 1952 ; Taylor, 1953). This subject was reviewed in great detail by Vendrely .( 1952) and the above-mentioned observations have generally confirmed the hypothesis of nuclear DNA constancy, originally advanced by Boivin ct al. (1948) and also maintained by Mirsky and Ris (1949). In its restricted form, taking into account polyploidy, chromosomal aberrations and DNA synthesis prior to mitosis, the hypothesis is well supported by numerous data. The exceptions to this rule which have so far been reported in the literature require further investigation. In some cases they seem to be based on faulty technique or fallacious interpretation of results (see discussion by Alfert and Swift, 1953). Since giant salivary as well as oocyte nuclei have been interpreted as resulting from internal chromosome reproductions, a knowledge of their DNA content is a valuable criterion of their possible niultivalence.

134

MAX ALFERT

2. Isolated Chromosomes The attempt to isolate and analyze L‘chromosomes” from interphase nuclei has led to considerable controversy in the recent literature. Chromatin threads were obtained from blood cells by Mirsky and Pollister (1943), and from leukemic tissues by Claude and Potter (1943). In a series of papers Mirsky and Ris (cf. 1951) have described the isolation of chromosomes from different vertebrate tissues. Such chromosomes were reported to be morphologically recognizable and consist mainly of DNA, histone (s), “residual protein,” and ribonucleic acid (RNA) . While the DNA-histone fraction is quantitatively constant in chromosomes from different tissues of one animal, the residual proteinRNA fraction varies considerably in amount, being most abundant in physiologically active cells with a large amount of cytoplasm, such as liver and kidney. Lamb (1950) has questioned the chromosomal nature of these strands and regards them as mechanical artifacts of the isolation procedure, an opinion shared by Hughes ( 1952). Recently, however, Polli (1952) and Denues (1952, 1953) have published evidence in favor of Mirsky and Ris’ view. This particular controversy is not easy to resolve, since chromosomes are not visible in the intact nuclei from which they are claimed to have been isolated and one consequently lacks a reliable criterion of their structure. If they have no similarity to mitotic chromosomes there is no reason for calling them chromosomes; and if they look too much like chromosomes (e.g., Yasuzumi, Yamanaka, et al., 1952) they may turn out to be contaminant bacteria (Houwink, 1952). Leaving aside the exact morphologic significance of “isolated chromosomes” it would still appear safe to accept the essential chromatin nature of the isolated material, as well as the significance of the quantitative differences in “residual protein” between physiologically active and inactive cells. Changes in mitotic chromosome size are known to occur during development, and chromosome volumes may differ in various tissues (eg., Biesele 1946). Such differences have been attributed ‘to variations in residual protein by Biesele (1947).

3. The Extended State of Chromatk The absence of visible chromosomes in most interphase nuclei has puzzled cytologists for a long time, and various theories were proposed to account for it. These have been summarized and elaborated by Ris and Mirsky (1949), who describe the DNA-histone fraction of the chromosomes as existing in an “extended,” swollen state, filling the whole interphase nucleus evenly. Phase contrast and electron microscopy following adequate fixation (Ornstein and Pollister, 1952) confirm this view : only

COMPOSITION A N D STRUCTURE OF GIANT CHROMOSOMES

135

nucleoli and a few dense but otherwise structureless regions appear on a homogeneous background in many types of nuclei. The extended nucleohistone fraction is able to condense reversibly under various conditions resulting in the appearance of visible chromosomal elements in the nucleus (Borysko, 1953). The importance of DNA in the swelling of chromosomal elements and whole interphase nuclei under the influence of certain ions has recently been confirmed by Anderson and Wilbur (1952) and by Kaufmann (1952).

4 . Chemical Composition of Chromatin Analysis of extracted nucleoproteins and differential chemical and enzymatic digestion of cells, combined with cytochemical procedures in situ, have been used to determine the various chromosomal fractions and how they are put together. With the development of rigorously controlled digestion procedures by Kaufmann, McDonald, and Gay (cf. 1951) and especially mild extraction methods (Bernstein and Mazia, 1953) the picture of the interrelation of nuclear components and of chromosomal architecture has become increasingly complex. There are diverse chemical indications far the occurrence of different DNA fractions (Zamenhof and Chargaff, 1949; Barton, 1952 ; Bendich, 1952), and Hamer (1951) reviewed the evidence, brought forth by several independent groups of workers, for the existence of four different general categories of proteins in nuclei. This does not take into account the presence and localization of possibly many nuclear enzymes (e.g., Stern et al., 1952; Lang et al., 1953). While histones appear to be quantitatively correlated with nuclear DNA (Mirsky and Ris, 1951; supported also by correlation of data by Stedman and Stedman, 1951, and Vendrely and Vendrely, 1952), the digestion and staining experiments of the Kaufmann group and of Mirsky and Ris indicate that DNA is combined with both histone and nonhistone protein in the chromosome; the same may be true for RNA. Kaufmann, et aE. ( 1951) have concluded that the chromosome represents an intricate structural framework in which no single component is responsible for the morphologic integrity of the whole structure. On the chemical level this may be paralleled by the existence of complex particles containing DNA, histone and non-histone protein in intimate association (Bernstein and Mazia, 1953). Lacking precise information on the nature of the bonds holding the various components together as well as on the native configurations of the components themselves, it appears hopeless to this reviewer to attempt any definition of a molecular chromosome model at present. However,

136

M A X ALFERT

chemical methods provide information on the gross composition of chromatin, and $tochemical techniques allow localization of certain substances in the chromosome. In combination with cytophotometric techniques introduced by Caspersson (1936), it is now possible to perform quantitative or semiquantitative analysis in sit% of substances which have either a strong natural absorption or for which specific and quantitative color reactions are available. This is notably true for nucleic acids and certain protein and carbohydrate groups (cf. Pollister ef al., 1951). In this connection it is important to remember that the failure to obtain a cytochemical reaction does not provide conclusive evidence for the absence of the substance under investigation: in relatively thin histologic sections a colored compdund must reach a fairly high concentration before it becomes visible to the eye or measurable by a photoelectric cell. This restriction does not apply to microphotometric work on solutions where the thickness of the absorbing layer can be extended at will with the aid of special cuvettes.

5. Phase Contrast and Electron Microscopy Phase contrast and electron microscopy both depend on differences in density for visualization of structure. A structure surrounded by a medium of very similar density may consequently not be detected. Recently attempts have been made to increase contrast by use of specific electron stains (Lamb et al., 1953). Ornstein (1952b) discussed the reason why the degree of contrast that can be achieved in this way is limited and cannot approach the contrast possible in visual microscopy by means of ordinary stains and color reactions. While application of the electron microscope has been highly successful in the case of some cytoplasmic elements and viruses, this technique has done very little for nuclear cytology so far. Pictures of chromosomes obtained by different workers often show a great deal of variation and do not permit unequivocal interpretation. Within the cell, chromosome structure does not produce much contrast, and isolated chromosomes appear to be very labile and are easily distorted beyond recognition.

111. THE“SALIVARY” CHROMOSOME The giant chromosomes found in dipteran salivary gland nuclei will be used here as the prototype of a category of chromosomes present in many dipteran larval tissues, secretory as well as non-secretory, but all growing by increase in cell size rather than cell number (cf. Trager, 1935). Makino (1938) and Cooper (1938), among others, have mentioned the tissues in which such chromosomes occur in Drosophila : they include

COMPOSITION AND STRUCTURE O F GIANT CHROMOSOMES

137

different regions of the gut, muscle, trachea, adipose and certain nervous tissues. M i n x (1949) lists different families of Diptera in which such chromosomes were observed. Aside from numerous Drosophila species on which intensive cytogenetic studies were concentrated, Chironoww, Sckra and Simulium were the genera most frequently used for cytologic investigations. A number of authors (eg., Buck, 1937; Painter and Griffen, 1937; Melland, 1942; Mainx, 1949; Beermann, 1952) have described the different types of chromosomal structures that occur, and their ontogeny, in dipteran larval tissues. A summary and further references on the development of the salivary glands in Drosophila can be found in Bodenstein’s (1950) review. The true nature of the peculiarly segmented “nuclear filaments” remained unexplained for over fifty years after their discovery in Chironomus plzlmosus by Balbiani-as Iong as observations were made on stained sections and whole mounts of glands, or on living or accidentally damaged and broken cells. In 1933, Heitz and Bauer applied the simple acid squash technique, which permitted them to understand the chromosomal configuration in salivary glands of Bibio hortulanus: in the same year, Painter published similar conclusions derived from acetocarmine squashes of Drosophila melanogaster glands and presented the first evidence for a close correspondence between genetic crossover maps and the cytologic configuration of the X chromosome. The general appearance of salivary chromosomes in smear preparations can be summarized as follows : Enormously enlarged homologous chromosomes are more or less closely paired and loosely twisted around each other so that the chromosomal elements appear to occur in haploid number ; these elements are usually present as discrete units, although end-to-end associations occur in some instances (e.g., Bauer, 1936a). Each unit exhibits a characteristic longitudinal differentiation, an individual pattern of alternating dense- and light-staining regions, which correspond point by point in the homologous chromosomes unless certain structural alterations have taken place. The units thus described correspond to what used to be called the “genetically active regions” (Muller and Painter, 1932), or the euchromatic regions (Heitz, 1933a) of the ordinary mitotic chromosomes. Kostoff (1930) seems to have been the first to comment on the possible genetic significance of the longitudinal differentiation exhibited by the salivary gland chromosomes. ‘ Although the reviewer considers the distinction between euchromatin and heterochromatin to be arbitrary, the structure of the chromosome regions conventionally thus defined will be discussed separately, and the terms “band” and “interband” will be used in a purely descriptive sense.

138

MAX ALFERT

1. The Euchronzatic Regions

The distinctly cross-banded chromosome parts correspond presumably to Heitz’ mitotic “euchromatin” ( 1928). They have an approximately cylindric shape, but in some instances (e.g., Chironomus) exhibit, along their axis, a series of markedly constricted or expanded regions. They also show a variable number (sixteen to several hundred, depending on the age and type of material) of faint, longitudinal or helical striations when in moderately stretched condition. The presence of these striations, which give the chromosome the appearance of a cable of loosely twisted threads has been taken as prima facie evidence of their polytene structure. Electron micrographs of unstretched chromosomes (Herskowitz, 1952) or formvar replicas thereof (Palay and Claude, 1949) as well as of sections of salivary gland nuclei fixed in neutral OsOl or formalin (Borysko, 1953) show no longitudinal striations ; these appear only when the chromosomes are stretched or the nuclei are fixed in acid fluids. The situation here is somewhat analogous to that of the mitotic spindle, which may appear structureless in living cells and in electron micrographs (Rozsa and Wyckoff, 1950), although clear evidence of an oriented structure can be obtained by use of polarized light ( e g . , InouP, 1952). In both cases the presence of submicroscopic fibrous structure is further indicated by anisotropic swelling and shrinkage, which occurs mainly at a right angle to the long axis (BElaf, 1929; Ambrose and Gopal-Ayengar, 1952). Thus the obliquely longitudinal microscopic striations which appear under certain conditions may be regarded as significant artifacts to which a submicroscopically oriented structure may predispose. However, if such striations were simply “stress lines” in stretched chromosomes, as interpreted by Metz (1936) and Buck (1942), one would expect them to run only parallel to axes of stress rather than take the helical course particularly well illustrated in Beermann’s (1952) photomicrographs (his figure 17). The characteristic banding pattern along the chromosome axis visualized routinely by acetocarmine or orcein staining was demonstrated by Caspersson (1936) to be due to alternating regions of high and low nucleic acid concentration. The bands had been interpreted as disks (Balbiani, 1881), supeTficia1 folds or coils (Korschelt, 1884), and rings (Carnoy, 1884) by early investigations, and the pros and cons of their chromatin nature were discussed. Except for Metz and co-workers (e.g., Metz and Lawrence, 1937) they were commonly considered, during the late thirties, to represent the chromomeres of packed chromonemal bundles. Following the interpretation of Koltzoff (1934), the chromonemes and associated chromomeres were at first believed to be peripherally located, coiling loosely around an achromatic chromosome axis. Heitz (1934)

COMPOSITION AND STRUCTURE OF GIANT CHROMOSOMES

139

also described the stainable bands as peripheral ringlike structures, and considered this to provide evidence for the spiral nature of the chromosome. His critical observations were made on upturned chromosome ends which show the chromatic ring structure particularly well. Bauer (1935a), however, clearly demonstrated the disk structure of the chromatic regions and later (Bauer, 1936b) provided an explanation for the ringlike structures seen at chromosome ends : The unsaturated valences of terminal chromomeres cause them to approach lengthwise and bring about a funnel-shaped deformation of the disk, which then appears ring-shaped in surface view. The telomeres described by Warters and Griffen (1950) may be due to the same effect. Heitz and Bauer (1933) demonstrated the presence of DNA in the bands by means of the Feulgen reaction, but also reported a weak reaction in regions between the bands. This would agree with Palay and Claude’s ( 1949) observation that the longitudinal striations which appear in electron micrographs of stretched chromosomes are susceptible to deoxyribonuclease action. The latter observation was not confirmed, however, by Yasuzumi, Odate, and Ota (1951). The variations in microscopic structure exhibited by different bands have been described in minute detail since the bands came to be regarded as the visible representatives of genetic loci by a majority of geneticists (e.g., Bridges, 1935 ; Muller and Prokofjeva, 1935), although not by all (Koltzoff, 1934; Marshak, 1936). Metz (1937), in particular, discussed the possible relationship of genes to chromatic bands and closely adjacent or included achromatic material. H e had also noted (Metz, 1935) that the structure of any one band may vary considerably, depending on the treatment to which the preparation had been subjected. The bands may appear as rows of variable numbers of granules or vesicles, as wavy lines, or single and double lines of different thickness. The heavier bands can often be separated into several thinner ones when the chromosome is stretched. The compound nature, in the direction of the chromosome axis, of certain bands has also been demonstrated by cytogenetic techniques (Muller and Prokofjeva, 1935) , as well as optically, by use of the increased resolution provided by ultraviolet light (Ellenhorn ~t el., 1935). Electron micrographs (Palay and Claude, 1949; Pease and Baker, 1949; Schultz et al., 1949; Yasuzumi, Odate, and Ota, 1951; Herskowitz, 1952) show the bands, as well as in some instances the interband regions, to be composed of numerous submicroscopic granules. Various data on microscopic and submicroscopic structure are summarized in Table I. On the basis of ultraviolet absorption studies, Caspersson (1940a) described the occurrence of tyrosine-containing, globulin-type proteins

TABLE I MICROSCOPIC APPEARANCE OF SALIVARY CHRohlOSOMES ~~~~~~

Organism Chironumus-

Clrironomus thtlnrmi Cltirmrnus defectus Chironomru tentans Drosophila meIamgarter Drosophila melanogaster Drosophila melanogarter Llrosophita pseudoobscilra Simulium zirgatum SimdiuSciara Sciura

~

Number of Number of Longitudjnal Chromomeres Threads Observed in Bands

16

ca. loo

350-400 several 100 16 16 4

-

16 ca. 100 350-400

16 16

8-30. av.16 64-128

96

20-30

8

I

~

Dimensions

of Salivary Chromosomes

Corresponding Dimensions of Mitotic Chromosomes

-

-

Largest: 275 X 20-25 p Largest: 270 X 20 p Total length of set: 1180 p X chrom.: 200 p long

7.5p

X chrom.: 240-260 p long

2.8 p

-

-

-

Authors Koltzoff, 1934 Bauer, 1935a Bauer, 1936a Beermann, 1952 Bridges, 1935 Muller, 1935 Kodani, 1942 Koller, 1935 Painter and Griffen, 1937 Geitler, 1938 Buck, 1937 Ris and Crouse, 1945

SUBMI~XOSC~PIC DIMENSIONS OF SALIVARY CHROMOSOME PARTS Or nanism Drosophila wlanogader Drosophila pseluioobscura Drosoph2a melanogarter Drosophila .m'dk

Fibrils

Particles

-

210-330 mp diameter (mostly 250-290 m p )

5-8 rnp thick SO mp thick (10 mv after extraction of nucleic acids)

Authors

Palay and Claude, 1949

50-150 X 25 X 8 mp Pease and Baker, 1949 100-150 mp diameter (20 X 140 mp after extraction Yasuzumi, Odate and Ota, 1951 of nucleic acids)

? F7 X

z1

COMPOSITION A N D STRUCTURE O F GIANT CHROMOSOMES

141

throughout the chromosome, and in addition to these, more basic, histonelike proteins in the bands. The presence of the latter is inferred from a shift of the typical tyrosine absorption peak toward a longer wavelength. Since this histone shift was not present in absorption spectra of pure nucleohistones investigated by other workers (e.g., Mirsky and Pollister, 1946), its exact significance is not fully established. Serra and QueirozLopes (1943) applied a histochemical arginine reaction and found that the bands react more strongly than interband regions. From this they concluded that, assuming the over-all protein concentration to be similar in bands and interbands, the bands have a much higher arginine concentration, which would be expected if they contained histones. The foregoing assumption is not justified, however, unless independently proved. Since most proteins contain arginine, a strongly positive arginine reaction may simply indicate a higher local concentration of almost any protein. Mazia and Jaeger (1939) noted that the chromosomes give a positive ninhydrin reaction and are completely digested by buffered trypsin solutions ; peptic digestion, believed not to attack histones, produces great shrinkage of the chromosomes without destroying their basic structure. The authors concluded that histones are responsible for the linear integrity of the chromosomes, since enzymatic removal of nucleic acids also leaves the basic chromosome structure intact. The latter point was confirmed by Frolova (1944), who observed, however, that pepsin digests chromosomes from which the nucleic acid had previously been removed. Kaufmann et al. (1951) later found that histones are susceptible to the action of pepsin, and Daly et ad. (1951) extended this observation to isolated mammalian chromosomes : while pure histones are completely digested by pepsin, combined histones are only partially liberated from chromosomes by the enzyme. It appears likely that the susceptibility of histones to pepsin action depends on their association with other proteins and with nucleic acids, as well as on the physical state of the histone itself (Mazia, 1950). Since histones are known constituents of animal nuclei and chromosomes, one might expect them to be present in salivary chromosomes also, and to be generally correlated with the nucleic acid distribution. However, the data available at present are inconclusive with respect to their exact location in the chromosome, and their presence, like that of nucleic acid, in low concentration throughout the chromosome cannot be excluded. A series of new cytochemical reactions for protein components were developed and applied to salivary chromosomes by Danielli (1947, 1950), who concluded that the bands contain tryptophan, tyrosine, and histidine, and that interband regions appear to be deficient in these but do contain amino groups. The fast green-stainable components of interband regions

142

MAX ALFERT

reported by Schultz (1941) may also well be amino groups. Microincineration (Barigozzi, 1937) has demonstrated the high ash content of the bands, which are also regions of higher concentration of mass than interband spaces, according to Engstrom and Ruch (1951). A differential distribution of phosphates and potassium salts, in bands and throughout the chromosome, respectively, was claimed by Yasuzumi and Sawada ( 1950). Microdissection, polarized light, and studies of natural and induced dichroism have been used by several groups of investigators. In some respects the results obtained by these different methods are in basic agreement with one another and demonstrate fundamental differences between band and interband regions : Buck (1942) and d'Angelo (1946) manipulated individual chromosomes from glands treated with osmic vapors and unfixed glands, respectively. The chromosomes are highly elastic and, when stretched, elongate mainly in the interband regions ; the bands are more resistant to deformation, an observation already made by Balbiani (1881). Fixed and unfixed chromosomes differ in some respects, as might be expected: the unfixed ones are much more extensible and can be shredded into longitudinal fibrils. D'Angelo also presented evidence for a chromosome pellicle, a structure not visible in electron micrographs. Buck noted that chromosomes can, under certain conditions, be pulled out into smooth and homogeneous strands which stretch indefinitely. The artifact produced in this way may have some bearing on the single axial filament which some authors described in lampbrush chromosomes (see section IV) . The negative birefringence of the bands, discovered by Ullrich (1936), is attributable to their nucleic acid content. This optical effect is small in fresh nuclei and increases somewhat upon standing and during dehydration. Schmidt ( 1941) discussed the significance of birefringence with respect to chromosome structure and concluded that the bands contain nucleic acid chains parallel to the chromosome axis. However, Caspersson (1940b, 1941) emphasized the advantage of making use of ultraviolet dichroism for the study of nucleic acid orientation and maintained that the very small observable optical effects indicate a low degree of nucleic acid orientation. Frey-Wyssling ( 1943) concurred with Caspersson on the latter point, but considered birefringence and dichroism measurements to be of equal sensitivity. Pfeiffer (1941) observed that the over-all negative birefringence of salivary chromosomes decreases with stretching. By means of a refined technique he proved this to be due to separate properties of bands and interbands respectively (Pfeiffer, 1952; see also Schmitt, 1938). In the unstretched chromosome the net optical effect is

COMPOSITION A N D STRUCTURE OF GIANT CHROMOSOMES

143

due to the nucleic acid in the numerous bands ; upon stretching the isotropic protein material in the interband regions becomes oriented and at the same time positively birefringent. In the over-all effect, this positive birefringence compensates for the unchanged negative birefringence of the bands. Ambrose and Gopal-Ayengar (1952) found that neutral red becomes attached in an orderly fashion to the protein chains in interband regions, as indicated by dichroism in polarized light. In summary, there appears to be considerable evidence for a submicroscopic fibrillar organization of the chromosome, based on extensible protein chains. The banding pattern is due to high local concentrations of nucleic acids whose relative lack of orientation might indicate the presence of coiled nucleoprotein fibers, as proposed by Ris and Crouse (1945). Such postulated coils, however, would have to be of a much smaller magnitude than those figured by the last-named authors if the electron microscopic evidence is taken into account. Subject to the limitations previously mentioned, localization and quantitative estimation of nucleic acids is possible with considerable accuracy. This is especially true for DNA and less so for RNA, for which no simple, direct, and specific test comparable to the Feulgen reaction exists. The .specificity and accuracy of histochemical methods for other substances are in many cases not so well established. Recalling the observations by Schultz (1941) and Swift (1953) of Feulgen-negative but fast green-stainable “bands,” particular caution is warranted when the results obtained by various group reagents are referred to as indicating the composition of bands and interbands in the sense in which these terms are used by cytogeneticists. This particular point is ilfustrated in Figure 3 (an unpublished contribution by Swift and Rasch), showing how markedly the distribution of. Feulgen- and fast green-stainable material may vary. Only Krugelis (1946) has previously made a similar comparison of alkaline phosphatase distribution to the Feulgen pattern of salivary chromosomes. Finally, it appears to this reviewer that many of the arguments about the significance of variations in the fine structure of bands are rather futile: Although the acid smear technique made it possible to demonstrate the chromosome nature and linear differentiation of the “nuclear filament (s) ,” it also undoubtedly introduces considerable artifact. Slight differences in the material and the technical skill of the preparator are intangible variables which may influence the final appearance of any preparation.

2. The Heterochromatic Regions In his first communication on salivary chromosomes, Painter (1933) noted that the “genetically inert” region of the X chromosome as well as

144

M A X ALFEBT

most of Y do not show up in the preparations and assumed that this material had either been eliminated or existed in some unrecognized form in the nuclei. The situation was further investigated by Heitz (1933a, b) who determined the distribution of heterochromatin in two Drosophila species and correlated it, in Drosophila melanogaster, with the known distribution of genes ; heterochromatin was found to contain fewer genes than euchromatic regions of similar length. The heterochromatic chromocenter in various types of nuclei was homologized in the salivary gland nuclei with a dense, more or less vacuolated mass from which the chromosome arms radiate out. In other forms, e.g., Bibio (Heitz and Bauer, 1933) and Chironornus (Bauer, 1935b), the paired chromosomes are not connected to a common chroniocenter but may exhibit a variable degree of end-to-end association (Bauer, 1936a) also sometimes attributed to heterochromatin. Later work proved the cytologic appearance of heterochromatin to be as manifold and confusing as its genetic manifestations (see reviews by Schultz, 1947; Barigozzi, 1949; and Hannah, 1951). Heitz (1934) observed that the distribution of heterochromatin in mitotic cells does not correspond to the degree to which heterochromatic material spreads from the chromocenter into the arms of the salivary chromosomes ; he therefore postulated two types of heterochromatin: a, the compact middle portion of the chromocenter, which corresponds to the heterochromatin of the mitotic chromosomes ; and p, which is unrecognizable in mitotic chromosomes but grows in size together with the euchromatic chromosome parts and has a more diffuse structure. Heitz did not recognize the banded structure in either type. PGnter (1935) also defined heterochromatin as an “amorphous mass of chromatin material” and Koller (1935) called it an “undifferentiated magma.” On the other hand, Bauer (1936a) and Frolova . ( 1936) described a fundamentally similar banded structure in euchromatin and heterochromatin, although Bauer emphasized structural differences in the chromomeres composing the heterochromatic bands. Pavan (1946) reviewed several instances in which the distribution of heterochromatin in salivary chromosomes was found to differ from that of mitotic chromosomes in extent, and also interpreted his own observations in terms of two different kinds of heterochromatin. Poulson and Metz (1938) studied the structure of different types of nucleolus-forming and related, presumably heterochromatic regions (“puffs” and “bulbs”) in salivary chromosomes. They suggested that the differences between heterochromatin and euchromatin may be “of degree rather than of kind”; stainability is modified .not by changes in the amount of chromatic material, but by differential accumulation of achromatic

COMPOSITION AND STRUCTURE O F GIANT CHROMOSOMES

145

material which may lead to a more or less pronounced disorganization of the normal structure. [This interpretation is in accord’with recent studies on mammalian liver (e.g., Campbell and Kosterlitz, 1952; Thomson et al., 1953) which show that the total amount of DNA per nucleus remains unchanged in situations, such as starvation, which lead to great variations in the appearance of heterochromatin (Lagerstedt, 1949) .] Albuquerque and Serra (1951) also attributed the condensed or loose appearance of heterochromatin in salivary chromosomes to different degrees of dispersion of chromatic material; this, in turn, they thought to be due to the differential effect of acetic acid treatment on the proteins in the respective chromosome regions. In the past the terms heterochromatin and euchromatin have often been interpreted to indicate that the difference in chromaticity is due to a difference in nucleic acid, particularly DNA, content. It would seem possible that the more specific claims of this nature (e.g., Caspersson and Schultz, 1938) could be reinterpreted, without violation of the observed facts, in terms of IocaI changes in concentration of DNA. The terms “heteropycnosis” or “heterochromasy,” which emphasize changes in appearance of chromatin, seem more justified than “heterochromatin” and “euchromatin,” which suggest the existence of two different substances. According to Ris (1945) heteropycnosis is generally a matter of differential coiling or condensation of chromosome parts. This view seems to account in the simplest way for the observable facts, and does not exclude th.e possibility that the degree of chromosome reproduction may be modified in some instances in condensed regions (see below). Differential condensation appears to be obscurely related to a host of genetic phenomena and different manifestations of chromosome behavior, but it is by no means certain that there is any necessary connection between the phenomena exhibited at such different levels of chromosome activity. It would seem desirable to this reviewer if the term heterochromatin were at least restricted, as originally intended, to situations where demonstrable differences in stainability exist. This is not the case in the so-called intercalary heterochromatin identified by criteria such as non-homologous association, or increased breakability of chromosomes (eg., Kaufmann, 1946). This latter property was attributed by Schultz (1947) to the lack of an elastic protein component in interband spaces of heterochromatic regions. Quantitative analysis of heterochromatic regions is especially difficult since the issue may be confused by structural aspects, namely the relation of chromosome parts to accessory materials. In salivary gland nuclei, Caspersson (194Oa) described the presence of histonelike proteins in heterochromatic regions as well as in nucleoli, and emphasized the

146

MAX ALFERT

similarity of their ultraviolet absorption spectra. However, the observed general resemblance of these absorption spectra may be due to the dispersal of nucleolar material among the chromosome fibrils, rather than to any similarity in composition of chromosome material and nucleolar material per se. Indeed, the nucleic acid component of the nucleolus is RNA while that of the chromosome is (at least predominantly) DNA, but these cannot be directly distinguished by ultraviolet absorption spectroscopy. On basis of staining in combination with differential enzymatic digestion, Kaufmann et al. (194s) and Lesher (1951a) have detected the presence of RNA in heterochromatic regions of salivary chromosomes. In this respect the heterochromatic regions would again be similar to nucleoli, as well as to condensed mitotic chromosomes, which some authors (e.g., Semmens and Bhaduri, 1939; Jacobson and Webb, 1950) believe to be coated with nucleolar material. The latter assumption is supported by the fact that condensed chromosomes sometimes eliminate sizeable chunks of ribonucleoprotein (Cooper, 1939 ; Ris and Kleinfeld, 1952).

3. The Theories of Salivary Clzromosome Structure

All theories developed to account for the appearance of these chromosomes fall into one of two categories ( I and 111) or a combination of both (11) : I. Salivary chromosomes are the result of several cycles of intranuclear chromosomal reproduction and consist of bundles of unfolded ordinary chromonemes (the polytene theory sponsored by Bauer, 1935a ; Hertwig, 1935; Cooper, 1938; Painter, 1939; and Beermann, 1952). 11. Salivary chromosomes consist of bundles of chromonemes ; their size is due at least in part to the accumulation of extra, material in the center of the chromosomes, and/or to an actual growth in length of the chromonemes (Koltzoff, 1934; Heitz, 1934; Painter, 1934; Calvin et d., 1940; Ris and Crouse, 1945; White, 1945). 111. Salivary chromosomes are paired chromosomes which have grown enormously in length and width by addition or incorporation of extraneous material not present in ordinary chromosomes (the early alveolar concept of Metz, 1935 ; theories proposed by Kodani, 1942, Kosswig and Sengun, cf. 1947a, and by Darlington, 1949). The historical development of these theories will be discussed below. In 1934 Koltzoff advanced the theory that salivary chromosomes are bundles of paired genonemes resulting from internal chromosomal divisions. H e figured a total of 16 strands in Drosophih and referred to the then meager evidence for polyploidy and its relation to nuclear size in support of his view, which soon became widely quoted and accepted. Hertwig

COMPOSITION A N D STRUCTURE OF GIANT CHROMOSOMES

147

(19351, applying Jacobj’s (cf. 1935) principle of the “rhythmic growth of nuclei,” attempted to estimate the valence of salivary chromosomes by measuring volumes of nuclei in different larval organs of DrosophiZa. He found a geometric series of nuclear size classes and considered the largest salivary gland nuclei to be 256 to 512 ploid, an estimate far surpassing any of those based on counts of the longitudinal striations, or of the chromomeres composing the disks of the chromosomes. The same discrepancy between chromosome size and number of visible strands, as well as the great variability of the appearance of bands in Sciara under different conditions of treatment and fixation, led Metz and co-workers (e.g., Metz and Lawrence, 1937) to reje‘ct the “polytene” theory; they substituted the “alveolar concept,’’ regarding salivary chromosomes as huge cylinders composed of chromatic material whose apparent structural pattern is determined by the distribution of numerous achromatic vesicles suspended within the chromatic continuum. Berger’s ( 1938) and Geitler’s (cf. 1938) classic descriptions of endopolyploidy in insect tissues were soon followed by the discovery of numerous similar cases (e.g., Painter and Reindorp, 1939) which demonstrated the widespread occurrence of intranuclear chromosolsie reproduction and resulting polyploidy or polyteny in many animal and plant tissues. Consequently the polytene concept of salivary chromosomes became more firmly established. In 1941 Metz summarized his position by stating (1) that individual chromonemes may be present, but can not be microscopically resolved, and (2) that no evidence for a longitudinally fibrillar organization of the chromosome exists. It must be noted however that the first of these assertions had already been taken into account by the adherents of the polytene theory, since Bauer (1938) had described how giant chromosomes in nurse cells of muscids fall apart into small chromosomes more numerous than the previously visible longitudinal striations, and since Painter ( 1939) had of fered a revised concept on the compound structure of the visible chiornomeres. The second of Metz’s assertions is based at least in part on Buck’s ( 1942) micromanipulation studies ; however, these have not furnished reliable evidence against fibrillar structure, since the treatment of chromosomes with osmic vapors is likely to introduce cross-linkages between neighboring protein chains, thereby producing an artificial three-dimensional fabric. During the forties three new theories of salivary chromosome structure were proposed; one is based on an unorthodox concept of chromomere structure, and the other two approach the problem from exactly opposite sides : an analysis of the developing salivary chromosome, on the one hand, and a chemical dissection of the mature chromosome into its constituent parts, on the other.

148

MAX ALFERT

The conclusions based on the earlier digestion experiments with NaOH h,y Calvin et d. (1940) were revised by Kodani (1942) on the basis of new observations resulting from the application of alkali-urea mixtures. Such treatments produce a series of constant and reproducible changes and lead eventually to complete breakdown of the chromosome structure. Interpreting these events, Kodani postulated the existence of four huge chromatids forming flattened loops to which nucleic acid “bulbs” are attached in the banded regions. During certain phases of their breakdown the chromosomes exhibit a fuzzy appearance, also described in a similar connection by Painter (1941), and compared by both authors to “lampbrush” images. (This turns out not to have been a happy choice of expression, since the radiating chromatic fibrils thus described have no relation or structural similarity to the lampbrush loops of vertebrate oocytes.) Although it would be interesting to know why chrpmosomes disintegrate under certain conditions in such a characteristic fashion, the method employed seems to be too harsh to permit precise conclusions regarding the fine structure of the intact chromosome. Even the much gentler and more refined enzymatic digestion procedures of the Kaufmann group have so far only given some hints on how various chromosome components are put together; however, they point to a multiple-fiber structure of the salivary chromosomes (Kaufmann, 1952). Ris and Crouse ( 1945), unsatisfied with the available interpretations, advanced the view that salivary chromosomes consist of a cable of uniformly staining chromonemes which remain coiled in certain regions, thus producing the denser bands where the recurrent fibers overlap. The increase in length of the chromosomes is attributed to a true growth in length of the chromonemes. This view is not well supported by the electron micrographic evidence showing the bands to be composed of numerous very small particles, as well as contrary to Bauer and Beermann’s (1952) explanation of the structure of Balbiani rings (see below). It has also been criticized by Hinton (1946), who observed that the disks can be pulled out as units from broken chromosomes. Hinton regarded the interband material as accessory nucleoplasm since he failed to find continuous fiber structure throughout the length of the chromosome. Beginning in 1946, Kosswig and Sengun have published a series of observations in which they relate the banded structure of giant chromosomes in different tissues of Chironomus to a “spiral stage” during the development of these chromosomes. Such a spiral stage had previously been described by Alverdes (1912). Kosswig and Sengin claimed that the early paired chromosomes exhibit a chromomeric structure which later disappears when the homologues become relationally coiled. The

COMPOSITION AICD STRUCTURE OF GIANT CHROMOSOMES

149

double coil then fuses into a single one which grows in length and width and increases in number of gyres. Finally, multiple breaks occur in the heavily staining coil, transforming its gyres into chromatic blocks that become separated by accumulation of weakly staining interband material. This transformation proceeds at different rates and to different extents in various tissues, so that the final banding pattern of the same chromosome differs in midgut, rectum, malpighian tubule and salivary gland (Kosswig and Sengiin, 1947a and b). Differences in the banding patterns in different tissues of Chironowms were also reported by Pennypacker (1950), but could not be detected in Sciara by Berger (1940), or in Drosophila by Slizynski (1950a). Pavan and Breuer (1952) have published photomicrographs demonstrating the essential similarity in banding patterns of the same chromosome from several larval and one adult tissue of Rhynchosciara. This case appears to be the most convincing since the reader is able to compare a large number of excellent photomicrographs and need not depend on the judgment of the authors in the execution of drawings. A sample of Pavan and Breuer’s comparisons is presented in Figure 8. The claims of Kosswig and Sengiin led Beermann (1952) to undertake, in Chirunomus, what constitutes to date the most extensive and detailed investigation of giant chromosome development in different larval tissues : Pairing of two-stranded homologues takes place at a very early stage; the chromosomes later exhibit a spiralized condition and grow by lateral addition and longitudinal unfolding of fiber bundles. During their development the chromosomes pass through a “meander stage” in which they become deformed by a longitudinal series of flattenings, alternatingly offset at right angles; this condition simulates the existence of two relationally coiled strands and forms the probable basis of Kosswig and Sengun’s misinterpretation (see Figs. 6 and 7). The characteristic banding pattern can be followed from a fairly early stage until the time when the largest salivary chromosome reaches a size of about 270 by 20 p and contains about 500 bands. Beermann estimated the degree of polyteny to be of the order of 16,000. No despiralization of the twisted chromonemal cable occurs during its development to maximal size. Chromosomes in different tissues show a constant banding pattern and go through the same developmental stages, although they may stop at different points and at various degrees of polyteny; they also exhibit different extents of chromonemal unfolding so that the ratio of length to width may vary considerably in the fully developed giant chromosomes of different tissues. Although Beermann’s view is in full agreement with Pavan and Breuer’s demonstration of the constancy of banding patterns in different tissues, the

150

M A X ALFERT

former author also carefully analyzes and describes specific modifications in the expression of the banding pattern resulting from accumulation of various types of achromatic materials. (On a limited scale, these observations on the modifying effect of achromatic material would therefore seem to this reviewer to justify some of Metz’s views which were discussed before.) The changes in appearance and stainability thus produced in certain bands are regarded as significant functional modifications, giving direct evidence for differential activity of certain chromosome regions in different tissues, or in the same tissue at different stages of development and under different experimental conditions (see Fig. 9). Beermann pays special attention to the variable expression of “Balbiani rings” present only on the fourth chromosome in salivary gland nuclei, but sometimes on other giant chromosomes in other tissues. These structures appear superficially as diffuse, weltlike protuberances surrounding definite chromosome regions and consist of intermingled chromatic and achromatic material. Their fine structure was successfully analyzed by Bauer and Beermann (1952) in especially favorable inversion heterozygotes of Chirononzus. The Balbiani ring is actually a region where the chromonemal cable opens up and branches out into repeatedly subdividing bundles of decreasing numbers of strands which form a collar of loops projecting laterally around the chromosome before they collect again on the other side into the typical chromosome structure. The banding pattern can be followed into the branches of these bundles as long as they still contain a sufficient number of fibrils to remain microscopically visible (see Figs. 4 and 5). Droplets of achromatic material are dispersed among the loose fibrils and their accumulation was observed in living chromosomes after larvae had been exposed to cold treatment (Beermann, 1952). Bauer and Beermann (1952) regarded the Balbiani ring as a natural experiment revealing the polytene. condition of the chromosome and discussed the available evidence in favor of this view. In addition to the observations already mentioned, this evidence includes White’s ( 1948) demonstration of an intermediary condition between typical banded giant chromosomes and numerous dissociated threads in different regions of the salivary gland of the Cecidomyid Dasyneura a f i n k An experiment by Slizynski (1950b), supposed to have demonstrated partial breakage and rearrangement of chromosomes following X-ray treatment of embryos, presents, in this reviewer’s opinion, no conclusive evidence for the polytene structure : in his diagrammatic representation of the rearrangements, the author figured involvement of one-fourth of the total salivary chromosome ; since four chromatids are probably present when the chromosomes pair and before they become polytene, this adds no new evidence for the later-

COMPOSITION AND STRUCTURE OF GIANT CHROMOSOMES

151

arising polyteny. A clear demonstration that only a few fibrils out of a large bundle are involved in a structural rearrangement would add the formidable problem of explaining how the large bundle can become deformed to the typical inversion or deficiency loop structures at such a late stage. According to Koller (1935)’ the formation of these configurations is facilitated by relational coiling of the early paired chromosomes, although the stages pictured by him may in part be based on a misinterpretation of a condition similar to Beermann’s “meander stage.” Indication for the occurrence of endomitotic cycles has also been claimed by Painter (1941), who pointed out that chromosomes from different nuclei of a single preparation may exhibit characteristically different reactions to chemical digestion procedures, probably depending on their respective stage of endomitosis. One further important point of evidence may be added in favor of the polytene theory. This concerns the DNA content of salivary gland nuclei and is complementary to Hertwig’s (1935) demonstration of nuclear size classes. Kurnick and Herskowitz (1952) have estimated, from photometric measurements of individual nuclei stained with methyl green, that the large salivary gland nuclei in Drosophila nzelanogcwter have undergone a 420-fold increase in DNA content over that of diploid nuclei. Correcting for possible error in the determination of the lowest values, the authors assume that 8 cycles of internal duplication have resulted in chromosomes consisting of about 1,000 strands. On their published graph they draw a straight line to represent the direct relationship between nuclear size and DNA content, but the experimental values are widely scattered around this line. The data reported-a total of 15 nuclei measured-are too few to reveal the discontinuous nature of nuclear DNA synthesis, and the methyl green method employed is not the best available for such a purpose (cf. Alfert, 1952). Swift and Rasch (1954) have obtained much more numerous data by photometric Feulgen dye determinations on nuclei of Drosophila salivary gland cells and closely associated cell types. This work is presented in Figures 1 and 2. The authors found a geometric series of 10 DNA classes, ranging from presumably diploid (2C) anlage cells to 1,024-ploid (1,024C) salivary gland cells. The DNA values fall into generally non-overlapping groups (see also Swift, 1950), indicating that they resulted from a series of successive synchronized duplications. The corresponding nuclear sizes seem to vary in a much more continuous fashion and exhibit considerable overlap among nuclei belonging to neighboring DNA classes. Although there are also aberrations in some details from a direct proportionality between nuclear size and amount of DNA, the general trend toward an over-all linear relationship is obvious. When

152

MAX ALFERT

Swift and Rasch’s data are considered in conjunction with Hertwig’s (1935) more extensive nuclear volume measurements, it seems clear that the growth of salivary gland nuclei results from an endomitotic process and is not a gradual and continuous hypertrophy due to the chromosomes’ becoming bloated with their own products, as postulated by Darlington (1949). The results obtained by the above-mentioned authors would also appear to resolve the problem (see Geitler, 1938) whether growth of salivary gland nuclei is entirely due to increasing polyteny of unfolding chromonemal elements or whether polyteny is accompanied by a gradual growth of the chromosomes: in the latter case one would expect the nuclear volumes to increase more rapidly than their DNA content, or-an improbable assumption-the nuclear mass to increase in density as the nuclei grow in size. This latter alternative could be excluded by application of the X-ray mass absorption technique developed by Engstrom (1950) o r by photometric measurements of nuclear protein content. Some deviations from a linear relationship between nuclear size and DNA content may be expected to arise from possible variations of certain chromosome components in localized regions such as Balbiani rings (see discussion in section V), as well as from variations in the amount of accessory nucleolar material, like those demonstrated by Beermann (1952). The main nucleolus was found to increase more or less proportionally with nuclear size during long periods of larval development in Drosophila robztsta but then to stop growing before nuclear volumes reach their peak (Lesher, 1951b). An excellent direct proportionality between average nuclear volume and DNA content in cells known to belong to different polyploid classes can sometimes be observed in situations where it appears safe to assume that all the cells are in similar physiologic condition, an assumption which might be less justified in case of dipteran salivary glands. Unpublished data of this nature, obtained by Dr. Wm. Carnes, are given in Table 11. The same point is illustrated by Frazer and Davidson (1953, their fig. 4). Before leaving this topic it should also be mentioned that measurements of nuclear size d o n e do not always constitute a good criterion of polyploidy or polyteny and may lead to very misleadiiig conclusions, especially when different cell types or cells in different physiologic conditions are compared. Rhythmic changes in nuclear size are known in some cases not to reflect changes in ploidy (Alfert, 1950; Schrader and Leuchtenberger, 1950; Alfert and Bern, 1951). The “super giant” salivary gland nuclei in Cecidomyia sp. may belong in this category, judging by their appearance as described by White (1948). Although the present reviewer considers the polytene theory to be best supported by the available evidence, several interesting problems remain

t

TABLE I1 Nuclear Class

Number of Nuclei Measured

Amount of DNA (Extinction X Area) Mean S.D.

3 Volume MEUl

S.D.

Amounts of DNA in arbitrary units of Feulgen dye, and corresponding volumes iu p’ of mouse liver nuclei. Microphotometric measurements on individual nuclei isolated in 2% chilled sucrose, fixed in formalin, stained in bulk with the Feulgen reaction, dehydrated and suspended in mounting medium on a slide. (Contributed by Wm. Carnes.)

154

MAX ALFEBT

unresolved. One of these is concerned with the question to what extent different chromosomes in one nucleus, or even different regions within a chromosome, can become polytene to different degrees. The “sex ratio” phenomenon analyzed by Sturtevant and Dobzhansky (1936) represents a well substantiated case of this type. Other cases which suggest this possibility have been reported by Schultz (1941), Melland (1942), and White (1948), who discovered that certain chromosomes appear to be out of step with the rest of the set, when judged by their size and stainability. I t will eventually be determined by means of photometric DNA measurements whether these cases are due to unequal polyteny or merely to abnormal states of contraction of certain chromosomes. This latter possibility raises another point of interest, concerning the increase in chromosome length during their development : the mechanism of differential chromonemal “unfolding” in different tissues and possibly in different regions of the same chromosome is still completely obscure. The causes of the breakdown of certain polytene chromosomes into their constituent fibrils (Bauer, 1938; White, 1948) are equally unknown. A curious occurrence of simultaneous polyploidy and polyteny in Lestodiplosis (White, 1946) is perhaps most easily explained by assuming fusion of nuclei containing polytene chromosomes. Although such a process has not been directly observed in this case, Alverdes (1912) clearly pictured binucleate cells in the salivary gland of Chironomus, and described as amitosis what seems to represent various stages of nucIear fusion.

IV. THELAMPBRUSH CHROMOSOME A short historical review of work on the specialized chromosomes in certain vertebrate oocytes is included in Dodson’s paper (1948). These chromosomes can be found in some fishes, amphibians, reptiles, and birds but have been most frequently studied in amphibians, where they reach their most extreme development in oocytes whose growth period may extend over a number of years. Lampbrush chromosomes develop from typical diplotene chromosomes (homologues exhibiting chiasmata in characteristic number and distribution) and maintain the general diplotene configuration throughout their development. They increase in size together with the nucleus and cytoplasm and reach their peak of development when yolk formation in the cytoplasm is well advanced; after this they become smaller again while cell and nucleus continue to increase in size. The largest of these chromosomes, in Triturus pyrrhogaster, are considerably over a millimeter in length in the unfixed condition (Duryee, 1941) and they have a fuzzy and irregular outline due to numerous lateral projections from their surface. Great numbers of nucleoli arise in contact

COMPOSITION A N D STRUCTURE OF GIANT CHROMOSOMES

155

with these enlarged chromosomes and disperse through the nuclear volume, which is at no time homogeneously filled by the chromosomal structures. Since these chromosomes finally contract to the condensed metaphase condition, the tremendous size changes which they undergo have repeatedly given rise to speculations concerned with possible relationships between chromosome size and function : the pronounced growth of the oocyte resulting from synthesis of various materials has been correlated with quantitative variations in functional chromosome components, distinguished from the genetically important fractions which alone make up the condensed metaphase chromosomes that become distributed during the meiotic divisions. Riickert’s concepts of Somatoplusma and Reinzplasma are in this sense homologous to Goldschmidt’s ( 1904) “trophochromatin” and “idiochromatin,” as well as to Mirsky and Ris (1949) “variable and constant” chromosome components, although the chemical entities included in these concepts have not always been the same. The study of lampbrush chromosomes is complicated by the fact that their component threads are of small dimensions and do not stain well with most cytochemical staining procedures. Dodson ( 1948) reported failure to obtain positive ninhydrin, Millon, and biuret tests. Hematoxylin, whiGh stains a number of compounds (Vendrely, 1950), has usually given the best results, and a recently described protein stain (Mazia et al., 1953) is also useful for their study (see Fig. 11). The question of the nucleic acid content of these chromosomes is of special interest. Several workers (e.g., Ris, 1945; Dodson, 1948) have been able to stain lampbrush chromosomes with pyronin at least in some stages, a fact interpreted by many to indicate the presence of RNA. However, the pyronins used in recent years by American workers are of doubtful specificity (Pollister and Leuchtenberger, 1949; Kurnick, 1952) and allow no conclusions unless used in conjunction with carefully controlled enzymatic digestion (cf. Brachet, 1953). Brachet (1929) and Koltzoff (1938) reported that nucIei temporarily become totally Feulgen-negative during the growth period of amphibian and other oocytes containing lampbrush chromosomes. From this Koltzoff concluded that DNA was completely absent and consequently could not be a gene component. The fallacy of such a conclusion was mentioned in the introduction and has been discussed in greater detail by Alfert (1950). Painter ( 1940), arguing from theory, considered lampbrush chromosomes to be polytene, but later (Painter and Taylor, 1942) retracted this proposition in consideration of the weak Feulgen stainability of the oocyte nuclei. Brachet (1940) also revised his earlier conclusions, for he was able, by use of appropriate fixation procedures, to demonstrate DNA by

156

MAX ALFERT

the Feulgen reaction at all stages. To increase the visibility of the greatly dispersed chromosomes, he also fixed and stained oocytes after centrifugation and noted that the thereby packed Feulgen-positive material did not seem to change in quantity during oogenesis. This point should be tested by means of photometric techniques in order to determine how amphibian material compares with the conditions found in mouse (Alfert, 1950) and grasshopper oocytes (Swift and Kleinfeld, 1953), where an apparent decrease in Feulgen intensity is attributable to dilution of the reactive material. (This dilution is not meant to indicate dispersion into the nuclear sap, but a dilution within certain regions of the enlarged chromosomes themselves. Brown et al. (1950) have found that nucleic acids are absent from the nuclear sap of amphibian oocytes.) Both Brachet (1940) and Painter and Taylor (1942) mention the presence of small Feulgen-positive granules, free from chromosomal threads and possibly in connection with nucleoli, dispersed in the nuclear space. This observation could not be confirmed by Callan (1952) but may find an explanation in the discussion of a similar finding by Guyknot and Danon (1953) : these authors observed that Feulgen-positive granules in nucleoli of Triton give a Schiff reaction even without preceding hydrolysis ; consequently, in this case, the reaction cannot be attributed to DNA. Except for Duryee (1950), there is at present general agreement that granules or blocks of Feulgen-positive material are visible at least along the axial regions of lampbrush chromosomes at all stages. Dodson (1948) also described the small lateral projections of early lampbrush chromosomes of Amphiuwzza as Feulgen-positive. This description has been confirmed on Triturm and Necturus by observations of the reviewer (see Fig. 12c) and is considered to be of prime importance for the interpretation of these structures. Experimental work on isolated amphibian oocyte nuclei was recently reviewed (Callan, 1952). The principal workers in this field (Callan and co-workers, and Duryee, 1941) agree on the adverse effect of Ca” in isolation media. Duryee ( 1941) also manipulated isolated chromosomes with glass needles and noted their physical properties. Whether such isolated chromosomes are “alive” is questionable, especially since Briggs and King (1953) found that Duryee’s isolation medium does not provide an environment able to sustain whole living nuclei. There has been considerable controversy about the structure of the chromosome axis and the lateral projections. According to the views of Ruckert (1892) and Ris (1945), these two chromosome parts are fundamentally the same, the lateral projections being extensions of chromonemal fibers. All other workers regard the axis alone as represent-

COMPOSITION AND STRUCTURE OF GIANT CHROMOSOMES

157

ing the chromosome and the lateral projections as accessory material arising in connection with definite chromosome regions. The evidence for these contentions will be discussed separately in the following paragraphs dealing with fully developed lampbrush chromosomes.

1. The Lateral Projections Hemming (1882) pictured a cross section of a lampbrush chromosome in Siredon as a starlike structure composed of radially projecting fibrils, while Riickert ( 1892) maintained that the lateral projections actually represent loops. He described the arduous task of tracing a chromosome thread through several loops by writing “when one has followed a fibril through several gyrations one gladly stops its further pursuit.”* Clark, Barnes, and Baylor (1942) indicated that the chromosomes have a branched, fernlike appearance in electron micrographs, but Clark, Quaife, and Baylor (1943) retracted the implications arising from the previous description. Neither Tomlin and Callan (1951), nor Gall (1952) using Tritzwus were able to demonstrate the loop structures or any of their finer morphologic details in electron micrographs. Duryee (1941), Ris ( 1945), and GuyPnot and Danon (1953), working with isolated chromosomes from varied amphibian material, could clearly determine the loop structure of the lateral projections, although the last-named authors also describe the occurrence of straight radial fibrils in addition to the loop5 (see Fig. 10). Using different methods of isolation in different media, Ris (1952) and GuyCnot and Danon ( 1953) obtained electron micrographs of loops which show a similar submicroscopic structure. However, the two groups do not interpret their results in the same way: Ris describes coiled bundles of tightly twisted threads, while GuyPnot and Danon speak of chains of particles lined up in a complex zig-zag pattern. In spite of these different interpretations, the factual agreement is evident in Figures 15 and 16. According to Ris, twisted microfibrils of similar dimensions (about 500 A. thick) are present in variable number in the chromosomes of three different genera of salamanders. 2. The Chromosome Axis Stained preparations are often described as showing a discontinuous linear arrangement of dense particles from which the loops appear to project. However, Riickert (1892) considered these dense regions as being “simulated by optical cross sections of crossing threads,” a view also *This and the following quotations from Ruckert’s article are given here in literal translation by the reviewer. They represent findings and opinions of an early observer, unbiased by preconceived ideas about chromosome structure.

158

MAX ALFERT

adopted by Ris (1945). Duryee (1950) was able to demonstrate that the chromosome axis is highly elastic and can be stretched several hundred per cent, whereby successive bundles of loops become separated but not straightened out. He also noted that the loops themselves were less elastic and reacted differently to chemical treatments than the chromosome axis. Although the axis is described as an optically single, very thin, and uniform thread by both Duryee (1950) and Gall (1952), the former had previously claimed that X-radiation demonstrates the existence of a latent longitudinal fissure which one would expect to find in a diplotene chromosome (Duryee, 1939). The electron micrographic evidence presented by various authors is extremely controversial : Tomlin and Callan (195 1) describe a single homogeneous strand of 200 A. diameter, Gall (1952) claims the diameter of the axial strand to be 1,OaO A. and GuyPnot and Danon (1953) maintain that there are two strands of about 150 A. diameter each. The structure described by Guyenot and Danon is illustrated in Figure 13. Other pictures by the same authors (see Fig. 14), however, show the chromosome as a much thicker (1,600 A.) multistranded cable, and this condition is interpreted as being due to a fibrous envelope of possibly acidic proteins, surrounding the two chromatids. The distinction thus postulated between chromonemal and accessory fibers appears to this reviewer to be somewhat arbitrary. The electron micrograph obtained by Boche and Anderson (Morton, 1941), referred to by Duryee (1950) as evidence for the loop structure of the side projections, is at the same time not favorable for Duryee’s concept of the chromosome axis because the latter appears as a relatively thick structure; it might well represent a cable of smaller units which seem to be pulled out into the loops in an irregular fashion and do not appear to be inserted at one level into paired granules (see below). According to Ris’ view there is no axis distinct in structure from the side loops. Feulgen-stained sections of various amphibian oocytes prepared by this reviewer (Alfert, unpublished) have consistently given a picture of the chromosome axis which is at variance with the description of the chromosome as a single strand to which granules of Feulgen-positive material are attached. In some chromosome regions the axis appears as a uniform, double-stranded, Feulgen-positive coil (Fig. 12a), in others it has a less regular structure (Fig. 12b), and at times it seems to disperse into numerous fine fibrils which collect again after a short distance to reconstitute a dense axial cable. These observations are in accord with those of Riickert and Ris.

COMPOSITION AND STRUCTURE OF GIANT CHROMOSOMES

159

3. Development of Lampbrush Structure The different opinions cited above are reflected in the accounts of the formation and regression of the lampbrush structure given by different workers: Ruckert (1892) described how the side loops appear to grow out as rodlets, then divide into small particles which move apart to form beaded threads that are least stainable at the periphery; he ascribed the later chromosomal condensation to a reversal of the same stages. Riickert’s observations were made on stained sections. Duryec (1941) described the process of loop formation in fresh material: The loops begin as short connecting rods between pairs of dense “chromioles” (granules) which occur in a definite longitudinal pattern embedded in the chromosome axis; their subsequent growth, attributed to “lateral synthesis” by chromioles, causes these rods to buckle and project out from the chromosomes. The loops thus formed have a beaded structure and not all of them are morphologically alike. At a later stage the loop material is sloughed off, a view in general accord with Koltzoff’s (1938) description of the events. GuyCnot and Danon (1953) devote much of their account to the appearance and behavior of nucleolar substances in the oocytes. They regard nucleoli as synthetic centers for “nucleoplasmic filaments” which fill the nuclei during the early growth stages of the chromosomes. At that time the lateral chromosome projections are solid bristles of the type previously described by Dodson ( 1948). GuyCnot and Danon attribute loop formation not to synthetic activity of the chromosomes, but to an addition of material from the “nucleoplasmic filaments” to the lateral rod-shaped projections. At a later stage the loops are resorbed in an unspecified manner. Ris (1945) denies the existence of corpuscular chromomeres and regards them as optical artifacts produced by narrowly pitched coils in the chromonemes. He described the genesis of the lampbrush structure in relation to other meiotic chromosomes showing at times a similar fuzzy outline: the diffuse appearance of diplotene chromosomes in grasshopper testes, and even to a greater extent in amphibian oocytes, is ascribed to laterally projecting gyres of chromonemes which have grown enormously in length ; they remain condensed and markedly Feulgen-positive only in the axial region. The later chromosome condensation is presumed to involve simple retraction of the side loops.

4 . Discussion and Conclw’om The present reviewer considers Ris’ theory to furnish, in a general way, the most probable explanation of lampbrush chromosome structure, although several important details are in need of clarification. In an attempt to refute Ris, Duryee (1950) condemned the “obsolete techniques of fixation

160

M A X ALFERT

and staining” and affirmed his confidence in observations made on fresh material. His clear demonstration of a chromosomal axis which exhibits different properties than the loops seem to constitute the most serious, but not necessarily unsurmountable objection to Ris’ theory. However Duryee’s distrust in orthodox cytologic procedures is unfounded. Riickert’s description of the lampbrush chromosome as a “whorl of densely tangled threads, winding in a complicated fashion” can be easily verified in early lampbrush stages when the chromosomal loops are quite thick and readily stainable. Moreover at this time they are also, at least at their bases, clearly Feulgen-positive. This latter fact probably represents the best criterion of the chromonemal nature of the loops. Individual loops are not necessarily formed by whole chromatids (which would restrict the number of loops possible at any one point), but by variable bundles of microfibrils of which the chromatids are composed. In later stages a differential elongation of the loops might lead to a dilution of the Feulgenpositive material below the visual threshold and also cause a sufficient differentiation in chemical composition and/or physical state of the extended loop material to account for its differential behavior in comparison with the more condensed central regions which remain Feulgen-positive. It is also possible that the presence of matrix material (Duryee, 1950; Guyknot and Danon, 1953) or a pellicle, restricted to the chromosome axis, contributes to the latter’s special properties. At the peak of their development the chromosomes exhibit thousands of loops and, on basis of Ris’ interpretation, would seem to contain a much greater mass than the ordinary mitotic chromosomes. Using Dodson’s (1948) measurements of the average number and dimensions of loops of a chromosome in Awtphiuwzu, one arrives at an average chromosome volume of about 6900 p3 as against 72 p3 for the average mitotic chromosome. I t is not clear how simple loop “retraction” could result in the necessary reduction in volume unless one assumes a hundredfold difference in density of chromosomal material between lampbrush and metaphase condition. If the size differences indicate true differences in mass and not in density then the chromosomal threads must be able to grow at one stage and might later condense by eliminating some of their substance in a manner similar to that postulated by Koltzoff (1938) or Duryee (1950). A possible alternative, avoiding this problem, might consist in the assumption that no true growth but only an unfolding of chromonemal elements occurs. In this case it would be necessary to postulate that most of the thickness of the elongated loops is due to the addition of extra-chromosomal substance perhaps as described by Guyknot and Danon (1.c.). At present it seems impossible to decide between these two possibilities. It can only

C03IPOSITION AND STRUCTURE OF GIANT CHROMOSOMES

161

be said, in favor of the first hypothesis, that it is not incompatibIe with radioautographic evidence by Pelc and Howard (1952), showing that certain chromosomes may continue to synthesize sulfur-containing protein at a time when their DNA content is not increasing. As far as microscopic and submicroscopic structure is concerned, one might take issue with Ris’ use of the terms “major and minor coils” in connection with lampbrush chromosomes. The coil-within-a-coil structure described in plant chromosomes (see review by Manton, 1950) was put in doubt by Ruch (1949) in Tradescantk and appears to be best established on the microscopic level in certain protozoans (Cleveland, 1949). There is no demonstrated continuity between the loose and irregular lampbrush loops and the coils of the mitotic chromosome. With respect to the submicroscopic fibrils, their segmented appearance (see Figs. 15 and 16) may equally well be due to the presence of linear arrays of particles as to tight twists in a homogeneous thread. According to the concept of Ris, lampbrush chromosomes may appear to be microscopically multistranded since the chromatids split up into many constituent fibrils. They could consequently be regarded as being “polytene,” in the literal sense of the word. However, in so regarding them, one would render the term polyteny meaningless with respect to its intended implication of genetic multivalence of certain chromosomes. Although it might at times be difficult to distinguish microscopically between the two kinds of multistrandedness exhibited by lampbrush and salivary chromosomes respectively, the two conditions should easily be distinguishable by the criterion of DNA content. According to R k (1952), the bundles of microfibrils composing a haploid chromosome set of any one species carry a characteristic amount of DNA. Polyteny can therefore be detected, since it results in a multiplication of the basic DNA content; this is to a high degree the case in salivary gland nuclei, but so far there is no evidence that it occurs in oocyte nuclei beyond the extent of a single duplication in preparation for the ensuing meiotic divisions.

SIGNIFICANCE OF GIANTCHROMOSOMES ; GENERAL V. THEFUNCTIONAL

DISCUSSION As far as lampbrush chromosomes are concerned, their functional interpretation by different workers has not given rise to much controversy, since they are universally connected with synthetic activity in the growing oocyte. To Duryee they are perfect examples of chromosomes at work in a non-mitotic cell, and allow distinction between two different types of chromosomal activity : (1) the production of lateral loops (==growth of chromosomal fibrils, according to Ris), and ( 2 ) the production of

162

MAX ALFERT

swarms of nucleoli which arise at definite loci along the chromosomes. Both of the materials thus produced become incorporated into the cytopIasm of the egg: some nucleoli evert their substance through the nuclear membrane, and the loop material and remaining nucleoli mix with the cytoplasm after breakdown of the germinal vesicle. Dodson (1948) stressed surfacechemical considerations and believes that the enormous surface exposed by the lanipbrush chromosomes is of importance in their catalytic activity. Aside from the cytologic demonstrations that in lampbrush as well as salivary chromosomes substances can be seen to arise and accumulate in conjunction with definite chromosome regions, the exact relations between chromosomes and cellular synthesis are still obscure. The most comprehensive hypothesis in this field, recently summarized (Caspersson, 1950), is due to the work of Caspersson and collaborators. The results of modern biochemical work, involving radioactive tracers, are not incompatible with their view that the nucleus is a center of RNA synthesis (cg., Marshak, 1948; Smellie et al., 1953), which may have its visual expression in production of nucleoli. However, a t the same time such studies (e.g., Hultin, 1950) have given no decisive indication that the nucleus is a center of cellular protein synthesis. The problems involved in such considerations have been critically reviewed by Mazia (1952) and Danielli ( 1953). The functional interpretation of salivary chromosomes has been attempted from several viewpoints, in relation to growth, synthetic activity, and cellular differentiation : Caspersson (1940a) has used the salivary chromosome as a model of an intermitotic chromosome in a synthetically active cell. He described its activities in terms of genetically specific syntheses in euchromatic regions, and a correlation between heterochromatin, nucleolus, and cytoplasmic protein synthesis mediated by RNA and basic proteins. Lesher (1951a) extended previous observations by Painter (1945) and Hsu (1948) on the cytoplasmic aspects of dipteran salivary gland function, and reapplied the Caspersson viewpoint on the basis of the picture obtained by use of basic stains. Kurnick and Herskowitz (1952), on the other hand, have used Mirsky and Ris’ (1949) criterion of a nucleus with a relatively low DNA concentration in their discussion of nuclear function in synthetically active cells. It must be remembered, however, that such considerations about nuclear function need have no relation at all to the polytene structure of the salivary chromosome, since similar polytene chromosomes are present in many different larval tissues and may just represent a growth pattern peculiar to these organisms (Cooper, 1938). Caspersson himself has chosen the salivary chromosome simply as a convenient model system whose huge dimensions facilitate the

COMPOSITION AND STRUCTURE OF GIANT CHROMOSOMES

163

use of microabsorption techniques. I t would be interesting to extend such studies to polytene chromosomes in organs that are not particularly active, as well as to different stages in the development of these chromosomes. Several authors have considered the possible relationship between chromosome reduplication and cellular differentiation (cf. Huskins, 1947 ; Geitler, 1948 and before ; D’Amato, 1952). Geitler in particular pointed to the instances in which characteristic degrees of polyploidy or polyteny are reached in different insect tissues, and Schultz (1952) discussed the possible significance of this fact in terms of threshold reactions. There is as yet no direct evidence that polyploidy per se is related to cell differentiation but such a possibility cannot be excluded. Polyploid cells occur in many mammalian tissues and in this case are certainly not related to cell differentiation, since they arise in already differentiated tissues and increase in frequency with the age of the animal (Swift, 1950) as well as under intense irritation (Bader, 1953). In at least some cases, however, this polyploidy results from very different processes, e.g. nuclear fusion, from those that occur in larval insect and plant tissues. Comparing salivary and lampbrush chromosomes in toto, there appears to be no over-all structural similarity between these two chromosome types, as had sometimes been postulated in the past (Painter, 1940; Calvin et al., 1940). However a striking structural resemblance in certain features becomes obvious when one compares Bauer and Beermann’s (1952) explanation of the Balbiani ring with Ris’ interpretation of Iampbrush loop structure: in both cases a lateral dispersion and radial projection of chromosome fibers takes place in regions where nucleolar material is formed. One might speculate that the whole lampbrush chromosome is organized in this fashion to supply the oocyte with numerous materials necessary for the growth of the embryo. In polytene chromosomes this activity is restricted to definite regions which differ in nuclei of various tissues according to specific cell requirements. Differential chromosomal activity in dipteran tissues may also be expressed in less extreme structural modifications than represented by the loop formations. Finally, one might discuss the conditions responsible for the visibility of these two chromosomal types in the interphase nucleus: many polytene nuclei appear not to differ from other nuclear types in that very little if any intra-nuclear structure is visible in perfectly undisturbed cells. This corresponds to the “extended state’’ of chromatin. Somatic pairing of chromosomes is a phenomenon most strikingly expressed in Diptera and may be responsible for keeping the multiple sets of submicroscopic chromosome fibrils aligned and insure appearance of orderly structure when the extended chromatin condenses. I n nuclei of other organisms this condensa-

164

MAX ALFERT

tion, unless it occurs in preparation for mitosis, results only in irregular clumping and appearance of fibrous networks. The lampbrush clmmosome, on the other hand, is regarded by Ris and Mirsky (1949) as a natural example of a ‘5-esidual chromosome’’ visible in situ simply because of its enormous size. These two types of chromosomes could thus be considered (Ris, 1951) as representing extreme examples of two possible patterns of chromosome growth : ( 1) duplication of chromonemal elements, which leads to a more or less proportional increase of all chromosomal and nuclear components, and (2) differential growth of a chromosome fraction specifically, although perhaps indirectly, concerned with cellular synthesis. These two patterns may be of very general significance but can only be visualized under exceptional conditions (i.e. somatic pairing in dipteran tissues), or when they reach extreme degrees (high ploidy in insect tissues, or hypertrophy of the “residual chromosome”). ACKNOWLEDGMENTS The author is greatly indebted to all who have contributed to this review or permitted use of published material. The constructive criticism of Professor Franz Schrader, Dr. Sally Hughes-Schrader and Professor Kenneth W. Cooper are gratefully acknowledged, and Dr. Aloha Hannah is cordially thanked for her critical reading of the manuscript.

VI. REFERENCES Albuquerque, R. M., and Serra, J. A. (1951) Portugaliae Acta Biol., AS, 187. Alfert, M. (1950) J. Cellular Camp. Physiol., 88, 381. Alfert, M. (1952) Biol. Bull., 103, 145. Alfert, M., and Bern, H. A. (1951) Proc. Natl. Acad. Sci. US.,37, 202. Alfert, M., and Swift, H. (1953) Exptl. Cell Research, 5, 455. AIverdes, F. (1912) Arch. Zellforsch., 9, 168. Ambrose, E. J., and Gopal-Ayengar, A. R. (1952) Nature, 169, 652. Anderson, N. G., and Wilbur, K. M. (1952) J. Gar. Physiol., SS, 781. Bader, S. (1953) Proc. SOC.Exptl. Biol. Med., 82, 312. Balbiani, E. G. (1881) A n d . Ans., 4, 637, 662. Barigozzi, C. (1937) 2. Zellforsch. (L.nrikruskofi. Anot., 26, 462. Barigozzi, C. (1949) Porfugaliae Acfa Biol., A, R. B. Goldschmidt Vol., 593. Barton, J. (1952) Biol. Bull., 103, 319. Bauer, H. (1935a) Z . Zellforsch. u. mikroskop. Anat., aS, 280. Bauer, H. (1935b) Nafurwiss., El,475. Bauer, H. (1936a) 2001.lahrb., 66, 239. Bauer, H. (1936b) Proc. Nutl. A d . Sci. U. S., 22, 216. Bauer, H. (1938) Nafurwt’ss., 26, 77. Bauer, H., and Beermann, W. (1952) Chronrosoma, 4, 630. Beermann, W. (1952) Chromosoma, 6, 139. BClai, K. (1929) Wilhelm Roux, Arch. Entwicklungsmech. Organ., 118, 374. Bendjch, A. (1952) Exbil. Cell Research, Supfil., 2, 182. Berger, C. A. (1938) Carnegie Inst. Wash. Publ,, Confribs. Embryol., 496, 167.

COMPOSITION AND STRUCTURE OF GIANT CHROMOSOMES

165

Berger, C. A. (1940) J. Heredity, 81, 2. Bernstein, M. H., and Mazia, D. (1953) Biochim. et Biophys. Acto, 10, 600. Biesele, J. J. (1946) J. Gerontol., 1, 433. Biesele, J. J. (1947) Cancer Research, 7, 70. Bodenstein, D. (1950) Biology of Drosophila, Chapter 4. John Wiley & Sons, New York. Boivin, A., Vendrely, R., and Vendrely, C. (1948) Conzpt. rend., 226, 1061. Borysko, E. (1953) Bull. Johm Hopkins Hosp., 92, 151. Brachet, J. ( 1 W ) Arch. biol. (Liige), S9, 677. Brachet, J. (1940) Arch. biol. (Lihge), 61, 151. Brachet, J. (1953) Quart. J. Microscop. Sci., 94, 1. Bridges, C. B. (1935) J . Heredity, 26, 60. Briggs, R., and King, T. J. (1953) J. Exptl. Zoot., 122, 485. Brown, G. L., Callan, H. G., and Leaf, G. (1950) Ndture, 165, 600. Buck, J. B. (1937) Proc. Natl. Acad. Sci. U.S., 23, 423. Buck, J. B. (1942) J. Heredity, 93, 3. Callan, H. G. (1952) Symposia Soc. Exptl. Biol.,6, 243. Calvin, M., Kodani, M., and Goldschmidt, R. (1940) Proc. NatE. Acad. Sci. U.S., 26, 340. Campbell, R. M., and Kosterlitz, H. W. (1952) Science, 116, 84. Carnoy, J. B. (1884) La Biologie cellulaire. J. Van In & Cie., Lierre. Carnoy, J. B., and Lebrun, H. (1900) Cellule, 17, 199. Caspersson, T. (19336) Skuid. Arch. Physiol., S~ppl.,73, 8. Caspersson, T. (1939) Chromosoma, 1, 147. Caspersson, T. (1940a) Chroinosomca, 1, 562. ’ Caspersson, T. (1940b) Chromosoma, 1, 605. Caspersson, T. (1941) Chromosoma, 2, 247. Caspersson, T. (1950) Cell Growth and Cell Function. W. W. Norton & Co. New York. Caspersson, T., and Schultz, J. (1938) Nature, 142, 294. Clark, G. L., Barnes, H. R., and Baylor, E. R. (1942) Science, 95, 250. Clark, G. L., Quaife, M. L., and Baylor, M. R. B. (1943) Biodynamica, 4, 153. Claude, A., and Potter, J. S. (1943) J. Exptt. Med., 77, 345. Cleveland, L. R. (1949) Trans. A m . Phil. Soc., S9, 1. Cooper, K. W. (1938) Proc. Natl. Acad. Sci. US.,24, 452. Cooper, K. W. (1939) Chromosoma, 1, 51. Daly, M. M., Mirsky, A. E., and Ris, H. (1951) J. Gen. Pltysiol., 34, 439. D’Arnato, F. (1952) Caryologia, 4, 311. D’Angelo, E. G. (1946) Biol. Bull., 90, 71. Expfl. Biol., 1, 101. Danielli, J. F. (1947) Synzposiu SOC. Danielli, J. F. (1950) Cold Spring Harbor Symposia Quant. Biol., 14, 32. Danielli, J. F. (1953) Cytochemistry. John Wiley & Sons, New York. Darlington, C. D. (1949) Proc. 8th 1qtterr.t. Congr. Genet., Hereditas Suppl., p. 189. Denues, A. R. T. (1952) Exptl. Cell Research, 3, 540. Denues, A. R. T. (1953) Exptl. Cell Research, 4, 333. Dodson, E. 0. (1948) Utziv. Calif. (Berkeley) Publs. Zual., 63, 281. Duryee, W. R. (1939) Anat. Record, 76, Suppl. 1, 144. Duryee, W. R. (1941) Univ. Penn. Bicentennial Conf. on CytoE., Genet. Evolution. p. 139.

166

MAX ALFERT

Duryee, W. R. (1950) A m . N . Y.Acad. Sci., 60, 920. Ellenhorn, J., Prokofjeva, A., and Muller, H. J. (1935) Compt. rmd. acad. sci. U.R.S.S., 1, 234. Engstrom, A. (1950) Prop-. Biophys. and Biophys. Chew., 1, 164. Engstrom, A., and Ruch, F. (1951) Proc. Natl. Acad. Sci. US.,37, 459. Flemming, W.(1882) Zellsubstanz, Kern und Zelltheilung. F. C. W. Vogel, Leipzig. Frazer, S. C., arid Davidson, 3. N. (1953) Exptl. Cell Research, 4, 316. Frey-Wyssling, A. (1943) Chronzosotna, 2, 473. Frolova, S. L. (1936) Biol. Zhiir., 6, 271. Frolova, S. L. (1944) J . Heredify, 36, 235. Gall, J. G. (1952) Exptl. Cell Rcsearclt, Suppl., 2, 95. Geitler, L. (1938) Biol. Zentv., 68, 152. Geitler, L (1948) listerr. Bofan. 2., 96, 277. Goldschmidt, R. (1904) 2002.Jahrb., 21, 41. Guyknot, E.,and Danon, ht. (1953) Reu. suisse zool., 60, 1. Hamer, D. (1951) Brit. J . Carrcer, 6, 130. Hannah, A. (1951) Adznnces in Genet., 4, 87. Heitz, E. (1928) Jahrb. &ss. Botan., 69, 762. Heitz, E. (1933a) 2. Zellforsclt. w. tnikroskop. Anat., u), 237. Heitz, E. (1933b) Z . Zrllforsch. 16. mikroskop. Anat., 19, 720. Heitz, E. (1934) Biol. Zcnfr., 64, 588. Heitz, E.,and Bauer, H. (1933) 2. Zdlforsch. u. mikroskop. Anat., 17, 67. Herskowitz, I. H. (1952) J. Heredity, 43, 227. Hertwig, G. (1935) 2. indukt. Abstantm.-u. Vererblehre, 70, 4%. Hinton, T. (1946) 3. Hcredity, 37, 99. Houwink, A. L. (1952) Expericntia, 8, 385. Hsu, W. S. (1948) Quart. J . Microscop. Sci., 89, 401. Hughes, A. (1952) The Mitotic Cycle. Academic Press, New York. Hultin, T. (1950) Exptl. Cell Research, 1, 376. Huskins, C. L. (1947) Am. Nutziralist, 81, 401. Inouk, S. (1952) Exptl. Cell Kcsearch, Sicppl., 2, 305. Jacobj, W. (1935) 2. mikroskop.-artat. Forsch., 38, 161. Jacobson, W.,and Webb, M. (1950) J. Physiol., 1U, PI-oc. 1. Kaufmann, B. P. (1946) J . E x j f l Zool., 102, 293. Kaufnmn, B. P. (1952) Proc. Natl. Acad. Sci. US., 38, 464. Kaufmann, B. P., hkDonald, If. R., Gay, H., Wilson, K., Wyman, R., and Okuda, N (1948) Carnegie Inst. Waslt. J’corbook, 47, 144. Kaufmann, B. P., McDonald, M. R. and Gay, H. (1951) 1. Crlldar Co~rtp.Physiol. Szcppl., 1, 71. Kodani, M. (1942) J . H t ~ r d i t y ,SS, 115. Koller, P. C. (1935) Proc. Roy. Sac. (London), B118, 371. Koltzoff, N. K. (1934) Srknce, 80, 312. Koltzoff, N. K. (1938) BioZ. Zhrtr., 7, 3. Korschelt, E. (1884) 2001.Anz., 7, 189. Kosswig, C.,and Sengiin, A. (1947a) Rev. fac. sci. rmiv. Istanbul, B12, 107. Kosswig, C.,and Sengiin, A, (1947b) J . Heredity, 38, 235. Kostoff, D. (1930) J. Zieredity, 21, 323. Krugelis, E. J. (1946) Biol. BuIL, 90, 220.

COMPOSITION AND STRUCTURE OF GIANT CHROMOSOMES

167

Kurnick, N. B. (1952) J. Natl. Cancer Inst., 13, 262. Kurnick, N. B., and Herskowitz, I. H. (1952) J. Cellular Cump. Physiol., 39, 281. Lagerstedt, S. (1949) Acta Anat., Suppl., 9. Lamb, W. G. P. (1950) Exptl. Cell Research, 1, 571. Lamb, W. G. P., Stuart-Webb, J., Bell, L. G., Bovey, R., and Danielli, J. F. (1953) Exptl. Cell Research, 4, 159. Lang, K., Siebert, G., and Fischer, F. (1953) Biochem. Z., 324, 1. Lesher, S. (1951a) Exptl Cell Research, 2, 577. Lesher, S. (1951b) Exptl. Cell Research, 2, 586. Mainx, F. (1949) Proc. 8th I n t m . Congr. Genet., Hereditas Suppl., p. 622. Makino, S. (1938) Cytologia, 272. Manton, I . (1950) Biol. Revs., 25, 486. Marshak, A. (1936) Am. Naturalist, 70, 181. Marshak, A. (1948) J. Celhlaa Comp. Physiol., 32, 281. Mazia, D. (1950) Ann. N.Y. Acad. Sci., 60, 954. Mazia, D. (1952) I n Modern Trends in Physiology and Biochemistry. Academic Press, New York. Mazia, D., Brewer, P. A., and Alfert, M. (1953) Biol. Bull., 104, 57. Mazia, D., and Jaeger, L (1939) Proc. Natl. Acad. Sci., 26, 456. Melland, A. M. (1942) Proc. Roy. SOC.Edinburgh, B61, 316. Metz, C. W. (1935) I. Heredity, 26, 491. Metz, C. W. (1936) Biol. Bull., 71, 238. Metz, C. W. (1937) Genetics, 22, 543. Metz, C. W. (1941) Cold Spring Harbor Symposia Quunt. Biol., 9, 23. Metz, C. W., and Lawrence, E. G. (1937) Quart. Rev. Biol., 12, 135. Mirsky, A. E., and Pollister, A. W. (1943) Biol. Syinposia, 10, 247. Mirsky, A. E., and Pollister, A. W. (1946) J. Gen. Physiol., SO, 117. Mirsky, A. E., and Ris, H. (1949) Nature, 163, 666. Mirsky, A. E. and Ris, H. (1951) J. Gen. PhyySiol., 34, 475. Morton, G. A. (1941) R C A Rev., 6, 144. Muller, H. J. (1941) Cold Spring Harbor Synp. Quant. Biol., 9, 290. Muller, H. J. and Painter, T. S. (1932) 2. Ldukt. Abstamm.-u. Yererb.-lehre, SZ, 316. Muller, H. J., and Prokofjeva, A. (1935) Proc. Natl. Acad. Sci. U.S.,21, 16. Ornstein, L. (1952a) Lab. Invest., 1, 250. Ornstein, L. (1952b) J. Natl. C a w w Illst., 13, 242 (in discussion following S. Mudd, c t al., p. 241). Ornstein, L., and Pollister, A. W. (1952) Trans. N.Y. Acad. Sci., 14, 194. Painter, T. S. (1933) Science, 78, 585. Painter, T. S. (1934) J. Heredity, 26, 465. Painter, T. S. (1935) Genetics, 20, 301. Painter, T. S. (1939) Am. Natzrralist, 73, 315. Painter, T. S. (1940) Proc. N ~ t l Acad. . Sci. US.,26, 95. Painter, T. S. (1941) Cold Spring Harbor Symposia Quant. Biol., 9, 47. Painter, T. S. (1945) I. Exptl. Zoo!., 100, 523. Painter, T. S., and Griffen, A. B. (1937) Genetics, 22, 612. Painter, T. S., and Reindorp, E. C. (1939) Chromosoma, 1, 276. Painter, T. S., and Taylor, A. N. (1942) Proc. Natl. A d . Sci. US.,28, 311. Palay, S. L, and Claude, A. (1949) J. Exptl. Med., 89, 431. Patau, C. (1952) Chromosoma, 6, 341.

168

MAX ALFERT

Patau, C., and Swift, H. (1953) Chromosoma, 6, 149. Pavan, C. (1946) Proc. Natl. Acad. Sci. US.,52, 137. Pavan, C., and Breuer, M. E. (1952) 1. Heredity, 49, 151. Pease, D.C., and Baker, R. F. (1949) Science, 109, 8. Pelc, S. R., and Howard, A. (1952) Exptl. Cell Research, Suppl., 2, 269. Pennypacker, M. (1950) J. Heredity, 41, 155. Pfeiffer, H. H. (1941) Chromosoma, 2, 77. Pfeiffer, H. (1952) Cellicle, 6S, 341. Polli, E. E. (1952) Chromosoma, 4, 621. Pollister, A. W.,Himes, M., and Ornstein, L. (1951) Federatiotc Proc., 10, 629. Pollister, A. W., and Leuchtenberger, C. (1949) Proc. Natl. Acad. Sci. US.,96, 111. Poulson, D. F., and Metz, C. W. (1938) 3. Morphol., 63, 363. Ris, H. (1945) Biol. Bull., 89, 242. Ris, H. (1951) In Symposium on Cytology, p. 21. Michigan State College Press, East Lansing, Mich. Ris, H. (1952) Records Genet. SOC.Amer., 21, 63. Ris, H.,and Crouse, H. (1945) Proc. Natl. Acad. Sci. US.,31, 321. Ris, H.,and Kleinfeld, R. (1952) Chromosome, 6, 363. Ris, H.,and Mirsky, A. E. (1949) J. Gen. Physiol., 32, 489. Rozsa, G., and Wyckoff, R. W. G. (1950) Biochim. ef Bioplrys. Acta, 6, 334. Ruch, F. (1949) Chromosomo, S, 357. Riickert, J. (1892) Anat. AM., 7, 107. Schmidt, W.J. (1941) Cltromosom, 2, 86. Schmitt, F. 0. (1938) J. Appl. Phys., 9, 109. Schrader, F.,and Leuchtenberger, C. (1950) Exptl. Cell Research, 1, 421. Schultz, J. (1941) Cold Spviitg Harbor Symposia Quunt. Biol., 9, 55. Schultz, J. (1947) Cold Spring Harbor Symposia Quunt. Biol., 12, 179. Schultz, J. (1952) Ezptl. CeZl Research, Suppl., 2, 17. Schultz, J., MacDuffee, R. C., and Anderson, T. F. (1949) Science, 110, 5. Schultze, 0.(1887) 2. w'ss. Zool., 46, 177. Semmens, C. S. and Bhaduri, P. N. (1939) Stain Technol., 14, 1. Serra, J. A., and Queiroz-Lopes, A. (1943) Ckromosoma, 2, 576. Slizynski, B. M. (1950a) 1. Genet., 60, 77. Slizynski, B. M. (1950b) Genetics, 35, 279. Smellie, R. M. S., Mclndoe, W. M., Logan, R., Davidson, J. N. and Dawson, I. hl. (1953) Biochem. J., 64, 280. Stedman, E. and Stedman, E. (1951) Phil. Trans. Roy. Sac. (London) B236, 565. Stern, H.,Allfrey, V., Mirsky, A. E., and Saetren, H. (1952) J. G m Physiol., 56, 559. Sturtevant, A. H., and Dobzhansky, T. (1936) Genetics, 21, 473. Swift, H.F. (1950) Physiol. Zool., a3, 169. Swift, H. (1953) frttem. Rev. Cytol., 2, 1. Swift, H.,and Kleinfeld, R. (1953) PhySiol. Zool., 26, 301. Swift, H.,and Rasch, E. M. (1953) Unpublished data and observations. Swift, H., and Rasch, E. M. (1954) J. Hisfocltem. Cytockm., 2 (Abstr., in press). Taylor, J. H. (1953) Erptl. Cell Research, 4, 164. Thomson, R. Y.,Heagy, F. C., Hutchison, W. C., and Davidson, J. N. (1953) Biochem. J., 65, 460. Tomlin, S. G., and Callan, H. G. (1951) Qiraat. J. Microscop. Sci., 92, 221.

COMPOSITION AND STRUCTURE O F GIANT CHROMOSOMES

169

Trager, W. (1935) 1. Exptl. Zoul., 71, 489. Ullrich, H. (1936) Planta, 26, 311. Vendrely, C. (1950) Arch. mat. histul. entbryol., 53, 81. Vendrely, C. (1952) Bull. biol. France et Belg., 86, 1. Vendrely, R., and Vendrely, C. (1952) Cumpt. rend., 236, 395. Warters, M.,and Griffen, A. B. (1950) J. Heredity, 41, 183. White, M. J. D. (1945) Animal Cytology and Evolution. Cambridge University Press, Cambridge, England. White, M. J. D. (1946) J. MorpAoZ., 78, 201. White, M. J. D. (1948) J. Murphol., 82, 53. Yasuzumi, G., Odate, Z., and Ota, Y. (1951) Cyfulugia, 16, 233. Yasuzumi, G., and Sawada, H. (1950) Cytologiu, 16, 295. Yasuzumi, G., Yamanaka, T., Morita, S., Yamamoto, Y., and Yokoyama, J. (1952) EzpcrientiQ, 8, 218. Zamenhof, S., and Chargaff, E. (1949) 1. Bid. Cheal., 178, 531.

170

MAX ALFERT

FIG. I

o gland 8

1.251

-..;

4

fat

duct antage

<*-

Nuclear

Volume

FIG.1. The relation between amounts of DNA, in arbitrary units, and nuclear volume in p3 in salivary gland tissues of Drosophilu melanogastm. Glands fixed in 10% neutral formalin, mounted whole, and stained with the Feulgen reaction. Adage, duct, and some anterior fat body nuclei were measured with light of 560 mp; gland and fat body nuclei were measured a t 600 mp. Ten fat body nuclei were measured at both wave lengths to obtain a correction factor, so that data could be compared. (Contributed by Swift and Rasch, unpublished).

FIG,2. Photographs of Feulgen stained D. melanogacter salivary glands. The location of the various DNA classes, as graphed in Figure 1, are shown. (Contributed by Swift and Rasch, unpublished.) FIG.3. Photographs of a n Xxhromosome from D. nwhogmter salivary gland nuclei smeared in 45% acetic acid, followed by 70% alcohol, stained with the Feulgen reaction and counterstained with 0.2% fast green in 95% alcohol. 3a photographed with green light to show distribution of Feulgen dye; 3b photographed with red-purple light to show fast green distribution. The two stains reveal different banding patterns especially in regions 9-7 (a,b,c). (Contributed by Swift and Rasch, unpublished.)

COMPOSITION AND S T R U C T U R E OF GIANT C H R O M O S O M E S

171

172

MAX ALFERT

FIG.4. Drawing of a Balbiani ring from the fourth salivary chromosome of Ch2ononzzts tenfans. X 2,750. (From Beermann, 1952 ; courtesy of Chronzosonza.) FIG.5. Photomicrograph of a Balbiani ring from the fourth salivary chromosome of an inversion heterozygous 6.dentairs. About X 1,800. (From Bauer and Beermann, 1952; courtesy of Chronzosowa.) FIG. 6. Photomicrograph of a salivary chromosome region of C. t e n t a w exhibiting the meander structure simulating two relationally coiled strands. X 2,000. (From Beermann, 1952; courtesy of Chromosome.)

FIG. 7. Plasticin model illustrating the actual structure of the meander stage. (From Bccrniann, 1952 ; courtesy of Clzro~~oso+t~n.) FIG. 8. Comparison of the banding pattern in sections 16-18 of chromosome A in two different tissues of Rkrynchosciara larvae. Drawing of standard map on top, photographs of corresponding regions from aceto-orcein smears of salivary glands ( 5 ) and Malpighian tubules ( M ) below. About X 1,300. (From Pavan and Breuer, 1952; courtesy of J . Heredity.) FIG.9. Drawings of corresponding regions of the first chromosome in four different tissues of one C. tentails larva. From left to right : salivary gland, hlalpighian tubule, rectum, midgut. About X 2,200. (From Beermann, 1952; courtesy of Chro~>zosontn.)

COMPOSITION AND STRUCTURE O F GIANT CHROMOSOMES

173

174

3 I A X ALFERT

FIG. 10. Phase contrast photograph of laiiipbrusli-chrotiiosome of T i i t n t L rristaftis isolated in acidified water. About X 750. Numerous loops arid two straight lateral fibrils a r e shown. (From Guyknot and Danon, 1953; courtesy of Rr-,~.Strissc Z o o / . )

FIG.11. Photograph of a section from a young oocyte of Uutraclioscps attmcatirs. Fixed in Bouin’s fluid, stained with bromphenol blue. X 470. (Preparatioo and photo~i~icrograpliy by P . A. Brewer ; courtesy of Bid. Brt/[.) FIG.12. Photographs of various larnpbrush chromosomes in sections from young oocytes of Mt-ctinirs rrzc~cidosiis. Fixed in acetic alcohol, stained with the Feulgeii reaction. X 2,000. 12a and b show different aspects of the appearance of thc clironiosomc axis ; 12c shows a Feulgen-positive loop base. (Preparation by M, Alfcrt, photomicrography by 0. P. Pearson.)

FIG.13. Electron micrograph of lampbrush chromosome of T . cvistnfiis, isolatrti in 45% acetic acid. About X 7,600. Loops and most of matrix presumed to be lysctl: arrows point to axis composed of two filaments. (From Guytnot and Danon, 1953; courtesy of Rw. Srrissc Zool.) FIG.14. Material and preparation as in Figure 13. About X 16,500. Presurnctl tcl show the presence of a fibrous matrix hiding tlic two chromatids from vie\\-. From Guytnot and Danon, 1953; courtesy of Rev. S’riLse Zool.)

FIG.15. Electron micrograph of lampbrush chromosome of T . rr-isfafirs, isohtcd in 4% saline containing 570 acetic acid. Ahnut X 17,000. The chromosome axis (-4) exhibits a swollen region, described as chroinoinere, at wliicli sidehranchrs ( L ) are inserted. Compare structure of (L) to that shoun in Figure 16. (From Guytnot and Danon, 1953; courtesy of RFZI.S~risscZool.) FIG.16. Electron micrograph of lampbrush loop structure from

R

chromosome of

Ncctitviis. Isolated in 10% sucrose, fixed in 1% 0 ~ 0 ,About . X 23,000. (Contributed

by H. Ris.)

COMPOSITION AND STRUCTU5.E .OF GIANT CHROMOSOMES

17.5

This Page Intentionally Left Blank

How Many Chromosomes in Mammalian Somatic Cells? R . A. BEATTY* Institute of Awinial Genetics. Ediiiburglz. Scotletid Paye I. Introduction ....................................................... 177 178 I1. Chromosome Number iii Germ Cells ................................ 1 . Spermatocytes and Oocytes of Man ............................ 178 2. Spermatogonia and Oogonia of Man ............................ 178 3. Germ Cells of Mammals Other Than Man ...................... 179 4. Claim that the Haploid Number in Man is 8 .................... 179 5. Reconciliation of Discrepancies in Chromosome Counts of Human Germ Cells .......................................... 150 6. Isolated Polyploid Cells in the Testis ........................... 181 7. Conclusion ..................................................... 181 111. Chromosome Number in Somatic Cells iiz Sitz~...................... 152 1. Somatic Cells i iz. Sitit in Man .................................. 182 2. Somatic Cells irs Sitti in M a m n d s Other Than Man ............ 183 IV . Chroniosonie Number iti Somatic Cells of Man in Tissue Culture ...... 183 V. Discussion ........................................................ 186 1. Assessment of the Reality of Somatic Inconstancy . . . . . . . . . . . . . . . . 156 2. Interpretation of Chromosome Counts from Tissue Cultures ...... 187 3. D N A Content of Nuclei and Somatic Inconstancy . . . . . . . . . . . . . . 188 4. Use of the Term Hetcroploidy .................................. 188 5 . Origin of Inconstancy in the Life of the Individual .............. 189 6. Are Cells with Abnormal Chronisome Numbers at the End of a Cell Lineage? ........................................ 190 7. Dying and Transitory Tissues .................................. 190 8. Some Consequences of Assumins. That Non-diploid Cells Do Form Cell Lineages ......................................... 191 9. Research Outlook .............................................. 193 VI . Conclusions and Summary ......................................... 191 V I I. Addendum ........................................................ 195 V I I I . References ........................................................ 195

I. INTROIXJCTION The generalization that there is a characteristic haploid chromosome number for each species of animal. and that each somatic cell of the body contains two such haploid sets. is subject to well-known exceptions. such as polyploidy. polysomaty. and heteroploidy in general . Of rather different nature are reports that in the somatic cells of mammals there is a wide cell-to-cell variation in chromosome number. termed somatic inconstancy. or chromosomal inconstancy in somatic cells. or somatic aneuploidy. The purpose of this review is to present the data with special reference to man and to outline some of the consequenct:s for embryology and genetics.

* Member

of Scientific Staff. Agriculturd Research Council.

177

178

R. A. BEATTY

The reviewer’s credentials are that he has had experience in counting chromosomes in somatic tissues of enibryu mammals, and the subject of chromosonial inconstancy, thougli not a direct research interest, has arisen in h i s own work and is of importance for it. As a matter nf terniinology, the custoin of grouping spermatogonia with other body cells as “somatic cells” obscures the present issue. Tissues on which cliroiiiosome counts are niade will therefore be classified as geriniiinl tisstrcs (-gonia and -cytesj on the one hand, arid soittcitic tissires (all other tissues except spermatids and gametes j on tlic other. The term “somatic tissues irr sitii” is used to contrast with “somatic cells in tissue culture,’’ and is not intended to mean that cells are exaruined while still attached to the organism. 11. CHROMOWME NUMBERI N GER?rI CELLS

1. Spcriiiatocgtes and Oocytes of Man With the recent exception of Lams (1950), which will be discussed later, there is now general agreetnent that the haploid chromosome nuniher in human sperniatocytes is 21. This number was first reported in 1912 by de Winiwarter. From 1900 to 1924 a number of 12 or near 12 was commonly reported. It niay be seen from Fig. l a how the “24 school” waxed as the “ I 2 school” waned. \Ve niay coticlude that an early period of hunian cytology lasted to about 1911, was succeeded by a transitional period up to about 1924, and gave way to a recent period thereafter. The successive reports from 1892 onward narrow down to a figure oF 24. The data for oocytes (Fig. l c j are scanty, but generally consistent with 24 rather than 12 or 48. The recent period coincides almost exactly with the “era of cytogenetics” which Matthey (1949) dates froiii 1925. Sonie cytologists have, of course, been well in advance of their period, though detailed attention to this point has been impracticable in the construction of Figs. 1 and 2. 2. Spcrwtatogoiiia mid Oogonin of Man

It is now generally agreed that the cliroiiiosome number in human sperniatogonia is 45, tlie first report near this number being again due to yon U’iniwarter (1912). Exactly tlie same tendencies seen1 to esist as with spermatocyte counts (see Fig. l b ) , probably not unconnected with the fact that most authors examined both spermatogonia and spermatocytes. There is an early period in which numbers of about 24 were reported, until 1910; a transitional period until 1924 in which numbers of 24 and 48 were recorded, and thereafter a recent period in which the number was determined as 48. In the recent period there are also figures

CHROMOSOMES I N MAMMALIAN SOMATIC CELLS

179

of 47. But this small discrepancy is not important for present purposes, depending as it does on a long-standing controversy as to whether the sex chromosomes are of the X-0 or X-Y types, with the X-Y type now generally accepted on both cytologic and genetic grounds. Again, as for spermatocytes, it may be seen how the “24 school” waned as the “48 school” waxed. Of the six records for oogonia (Fig. Id) five are at or near 48,and the fifth has an undetermined upper limit not exclusive of 48.

3. Germ Cells of Mammals Other Than Man Man has served as the main example. But a glance at, for instance, Makino’s atlas of chromosome number.; (1951) shows much the same historical tendencies-a narrowing down to a haploid number in spermatocytes and oocytes which is half the diploid number in spermatogonia and oogonia, with an early period up to a b u t 1910, a transitional period to about 1924, and a more recent period of better agreement. As in man, a tendency for early erroneous counts to be multiples of 2 or 6 can be detected. For the sake of accuracy, the special case of an XXY mechanism requires mention (Matthey, 1949), involving an odd number in the diploid count, but the general conclusions above are only slightly affected.

4. Claim That the Haploid Number in. Man is 8 We have assumed so far that there is “a” chromosome number to count in germ cells of all human beings, and that polyploid individuals, if found, would be a self-evident exception, presumably very rare. In a startling paper by Lams (1950) the literature of i.he germinal and somatic chromosome number of man is grouped into three reports of diploids, twelve of triploids, six of tetraploids, three of pmtaploids and fourteen of hexaploids, with a basic haploid number of S instead of 24. If the literature is representative, then it would seem .:hat the human race consists of diploid and polyploid individuals in proportions of this order. Thirty-eight years before, in what we have called the early period of cytology, Lams had reported 8 as the spermatocytal haploid number for man, and on reexamination of his original preparations he confirmed this figure. H e reconciled differences in chromosome number reported by different cytologists by saying that all reports were red and exact, and not simply reflections of difficulties in technique and interpretation. He claimed that these reports were grouped around a lowest common multiple of 8; thus, investigators reporting a diploid numbei- of 16 were examining diploids, and so on up to hexaploids with 48 chromosomes. Although the possibility of polyploid eipolution in mammals is not wholly excluded (Darlington, 1953; Flschberg and Beatty, 1952b; Gates, 1942 ;

180

R. A. BEATTY

Sachs, 1952), there are various a pra’ori objections to the origin of polyploid races in bisexually-reproducing animals (Muller, 1925), and all the more so for a mixed polyploid race such as Lams envisages. These objections, in short, are based on evidence from plants and animals for upset of the normal sex ratio in the progeny of polyploids; breakdown of the mechanics of meiosis in polyploids, leading to inviable gametes or to no gametes, and including a breakdown of the polyploid state itself during gametogenesis ; intersexuality of individuals bearing certain polyploid combinations of sex chromosomes. The result is that we should expect an enormous amount of infertility in human breeding, much greater than is actually observed. We should also expect other peculiarities. If the pattern of some plant and invertebrate polyploids is followed, we should expect the same number of cells in polyploids as in diploids, but cell size and body size would be larger than normal. Yet cytologists do not report giants and dwarfs in their human material. Alternatively, if the pattern of amphibian polyploids is followed, we should expect the body to remain relatively constant in size, but be composed of fewer and larger cells ; yet we do not find reports of human beings with the volume of blood cells and other cells falling into one of the five relative size classes of 2 :3 :4 :5 :6, according to the individual examined. Lams’ interpretation of the data can also be questioned; it would seem too much of a coincidence that the data quoted by him show some indication of a rise from diploidy to hexaploidy, suspiciously in correspondence with the technical advance of cytology during the last half century. And finally, where are the trivalents, tetravalents, and so on expected in polyploid meioses ; and the complex segregation of Mendelian characters typical of a polyploid? It does not seem possible to avoid concluding that Lams’ theory is untenable, though the chance of an occasional polyploid individual remains open.

5. Reconciliation of Discrepancies in Chromosome Counts of Human Germ Cells Although rejecting Lams’ theory, we still have to account for the discrepancies between the reports of different cytologists. Many cytologists of the early period were exceptionally keen observers. Yet, some subjective tendencies seem to have been at work. The first is that even numbers are reported more often than odd, in all classes of animals. Hut the early reports were so wide of the mark that there was no observational basis at the time for knowing whether the true count was odd or even, and no a priori reason why spermatocytes should have an even number. Presumably we are conkerned with the human bias to-

CHROMOSOMES I N M A M M A L I A N SOMATIC CELLS

151

ward estimating in even numbers, combined with the idea that, in spermatogonia, the presence of two haploid :jets must give an even number. Another fairly obvious tendency may be due to the influence of multiples of half-a-dozen. In the ,early erroneoils counts for spermatocytes and spermatogonia (i.e. all plots below 20 an81 40 respectively in Fig. l a and b, up to 1924), it will be seen that 16 c a t of the 33 relevant points, or 48%, are divisible by 6. On a random 'mis, only 1 in 6, or 17%, would be expected; or, if superimposed on the tendency to estimate even numbers only, 1 in 3, or 33%. The tendency to report multiples of 6 could have a possible basis in human chromo:;omes; 48 is itself divisible by 6, and if, in a not very good preparation, pairs of chromosomes were mistaken for single chromosomes, we should obtain a figure of 24; if 4 were mistaken for 1, a figure of 12, and so on. But if we select an animal in Makino's 1951 list which fulfils the requirements of being well documented and of having a diploid number not a multiple of 6-e.g., the mouse, with a diploid number of &there is still the same tendency, and to about the same extent as in man. Finally, it is evident that individual authors rarely fail to confirm their earlier papers, even when new material is examined, perhaps from a different race of man. To conclude : discrepancies from 48 in spermatogonia and 24 in spermatocytes may be attributed in general to technical difficulties in the early and transitional periods of human cyto"ogy, combined with a subjective tendency to report multiples of 2 or 6.

6. Isolated Polyploid Cells in the Testis Although, in general, counts differing from 48 in spermatogonia and 24 in spermatocytes have been rejected, there is one special case in which there seems to be a real divergence, in the giant cells of the testis (Andres, 1933 ; Koller, 1937 ; Montgomery, 1912 ; Painter, 1923a). These cells are tetraploid as spermatogonia, diploid as spermatocytes, and give rise to diploid spermatids and giant diploid sperm. It would be of great interest to know if these diploid sperm can fertilize an egg, since a triploid embryo might result. Venge's report of inconstancy in the chromosome number of rabbit spermatogonia (1953), including numbers at or near polyploid numbers, may perhaps mark the beginning of a reorientation of our ideas of germinal constancy, but its implications will not be considered in this review.

7.

Conclurion

We have been at some pains to elablate with special reference to man the generally accepted fact that each speaes of mammal has a characteristic haploid chromosome number in -cytes, and twice that number in -gonia

182

R. A. BEATTY

(with a special case of an XXY sex chromosome complement). Against this background, the numbers in somatic tissues may now be considered.

111. CHROMOSOME NUMBER IN SOMATIC CELLSIN SITU 1. Somatic Cells in Situ in Man The historical tendency of counts in germ cells-that is, a narrowing down to a haploid number of 24 in -cytes and a diploid number of 48 in gonia-is completely reversed with somatic cells (see Fig. 2a). Instead, from 1897 onward, numbers are reported which deviate further and further from 48, and culminate with a range of from 4 to 104. The recording of a range of numbers is especially marked in the recent period of cytology, from 1924 onward. I t can scarcely be maintained that cytologists, as they increase the accuracy of their counts of germ cell chromosomes, are simultaneously becoming less expert in counting the chromosomes of somatic cells. Rather, the data of the last hdf-century point to a real cell-to-cell variation in the chromosome number of at least some somatic cells. Two reports approaching recent times are those of Rappeport (1922) who, with human embryos, reported a range of 38 to 48 in the amnion and 32 to 53 in the pleura; and of Rotter (1922), with a range of 19 to 24 in the human fetus. Schachow (1927), with sections of chorion and decidua of 2 to 3 month human embryos, found numbers of 8, 18 to 20, 23, 24, and 48, with 24 as the commonest count. A brief note that chromosome number in an 8.5 mm. human embryo was about 24 rather than 48 was given by Adamstone (1929). Evans and Swezy (1929) reported only the number 48 in somatic cells of 23 to 25 mm. human embryos, but an earlier paper (Evans and Swezy, 1928) seems to imply that selection of cells containing 48 chromosomes may have been practiced. I n the subserous connective tissue of a 123 mm. human embryo, Karplus (1929) found numbers of about 30 to 64, and in the amnion of other embryos 38. Andres and Shiw (1935), with total preparations of amnion and sections of other tissues, found a variation of from 32 to 73: the tissues were amnion ; mesenchyme cells of chorionic villi ;nerve cells of the brain ; mesenchyme and epithelial cells of the skin, lung, and small intestine. Similar results were reported in 1936 by Andres and Jiv (= Shiw), who found that embryonic and extra-embryonic tissues, and the separate tissues of the embryo, all exhibited about the same range of inconstancy. The figures on bone-marrow cell from adult man by Slizynski (1945) were 48 chromosomes (personal communication). In squash preparations of normal uterine mucosa, Barigozzi (1947) reported only counts of 48, but gave no details. The subject of inconstancy was brought to a head

CHROMOSOMES IN MAMMALIAN SOMATIC CELLS

183

by Timonen (1950) , who counted chromosomes in 1,000 cells from normal human uterine endometrium in the proliferative stage, and found a range from 4 to 104, with the highest peak in the frequency distribution near the haploid number, and a smaller peak near the diploid number. I t is of importance to note that there was confirmation from a different kind of evidence; the frequency distribution of volumes of 2,OOO nuclei gave a curve of similar appearance. Timonen and Therman (195Oa), again with normal endometrium in the proliferative stage, noted hypo- and polyploid cells, but instanced only counts of 18 and 48. In this paper it is important to note that both sections and smears u w e used. Timonen and Therman (195Ob) also examined tissues of 6 to 24 week human embryos, and found a similar range of cell-to-cell variation in chromosome number in skin, brain, liver, intestine, cartilage and bone marrow. Thus ten reports of somatic inconstancy, from nine workers, apply to at least eleven major organs or tissue systems in man: amnion, bone marrow, cartilage, chorion, connective tissue, endometrium, intestine, liver, lung, nerve, and skin; to embryonic and adult tissues; and to results from both sections and smears.

2. Sonzatic CelEs in Situ of Mammals Other Than Man Our main example has been man, but inconstancy in chromosome number of somatic cells is reported from other mammals also. In the omentum of the cat, Pletnev (1941) found a range of 32 to 64. Wodsedalek (1941), in a paper entitled “Fetal membranes as unreliable sources for accurate studies of chromosonies in mammals,” found a range from the haploid to over the tetraploid number in amnion and chorion of many ungulates, carnivora and rodents iiicluding rat and mouse. It will be noticed that the title of his paper begs the question now at issue; a worker with a different point of view might perhaps have called it “Fetal membranes as reliable sources for accurate studies of somatic chromosomes in mammals.” Tetraploid mammalian blood cells were described by La Cour (1944). Sorokina (1950) found a range of 15 to 69 in pig amnion (and 20 to 56 in tissue cultures of embryo pig gut). Fischberg and Beatty (1951) reported briefly the observation of abnormal chromosome numbers in blood cells of the embryo mouse. The details of a preliminary note of somatic constancy in the mouse (Boothroyd and Walker, 1952) are awaited with interest.

IV. CHROMOSOME NUMBER IN SOMATK CELLSOF MAN IN TISSUE CULTURE In tissue cultures, the historical tendencies are less clear (Fig. 2%). A range of numbers, or the possibility of a range, is usually reported.

184

R. A. BEATTY

Kemp (1928; 19%, b ; 1930), with in toto preparations of tissue cultures of liver, heart and spleen from human embryos, found a fairly con-

r

40 -

(b] Spermatogonia

i

'1

20 -

! I

FIG.1. Historical

summary

I

t I

I

of chromosome numbers reported for the germ cefls

of man. Each dot represents a n observation. A range is shown by two dots joined by a line. An arrow indicates an uncertainty in an upper and/or lower direction. No selection of data has been made, in order to avoid bias. All papers which could be consulted in the original have been included. The main reference sources are the bibliograrhies in Harvey (1920), Painter (1923a), Oguma and Kihara (lPW), Hcberer (19351, McClung (1939) and Makino (1951); only a few of these references have proved inaccessible, and have been excluded. The detailed references are: oogonia-Andres and Navaschin (19361, Andres and Viigel (1936), Molas (1926). Swezy and Evans (1930). von Winiwarter (1912, 19ao); oocytes-Alten er al. (1930), Hoadley and Simons (1928), Pincus and Saunders (1939) ; spermatogonia-von Bardeleben (1892, 1897), Branca (1924), Duesberg (I%), Evans and Swezy (1928, lag), Friedenthal (1921), Guyer (1910, 1927), King and Beams (1936), Roller (1937), Minouchi and Ohta (1934), Moore and Walker (I%), Oguma (1930, 1937, 1939), Oguma and Kihara (1923), Painter (1923a, b, 1924), Shiwago and Andres (1932a, b), Wieman (1917), von Winiwarter (1912, 19ZJ); van Winiwarter and Oguma (1926); spermatocytes-van Bardeleben (1692, IW), Branca (1910, 1911, 19241, Duesberg (1%), Evans and Swezy (1929), Friedenthal ( l a l ) , GuCherz (19121, Guyer (1910, 1914), Jordan (1914), King and Beams (1936), Lams (1950), Minouchi and Ohta (19341, Montgomery (1912), Moore and Arnold (I%), Oguma and Kihara (1923), Fainter (1!423a, b, 1924), Wieman (1917), Wilcox (1900) van Winiwarter (1912, 1920, 1921), van Winiwarter and O g u m a (1926).

CHROMOSOMES I N MAMMALCAN SOMATIC CELLS

185

stant chromosome number of either 48, or, owing to uncertainty over 1 or 2 chromosomes, perhaps slightly more or less. He specifically (1930) does not exclude the possibility of small departures from 48 in normal

1

(a) Somatic cells in situ.

~~

80 70 60 50

1 .;'1 '

40

1

30

I

.20 . 10 '

8o 70

t

Ib)

Somatic cells in tissue culture

60 . 50 . 40 .

30 . - 1

1890

1900

1910 Year

1920 1930 of pub ication

1940

1950

FIG.2. Historical summary of chromosome numbers reported for the somatic cells of man. The table is constructed on the same principles as Fig. 1. Reference sources are: somatic cells in tissue culture-Andres and Navaschin (1933, Caffier (1932), Chrustschoff and Berlin (1935), Hsu (1952), Kemp (1% 19Z!?a, h, 1930), Shiw (1938); somatic cells in situ-Adamstone (lag). Andres and Shiw (1935), Andrer and Jiv (193611, Barigozzi (1947), Evans and Swezy (1929). Fleming (18%'). Karplus (1929), Rappcport (1922), Rotter (1972). Schachow (1927), Slizynski (1945), Timonen (1950), Timonen and merman (1950a), Wieman (1913).

tissues. CaiKer (1932) with total preparations of tissue cultures of human embryonic lung, reported twelve counts of 45 to 50; three of 33 to 40; one of under 30; and two of over 80: observational uncertainty was 3 to 4 chromosomes or less. 33% of his counts were therefore outside the diploid range. Chrustschoff and Berlin (1935), using total mounts of tissue cul-

186

R. A. BEATTY

trres of human leukocytes, listed a single count of 52. Andres and Navaschin (1936), in sections of tissue cultures of human embryonic tissue, found only the numbers 47 and 48; the tissues were lung mesenchyme, mesenchyme, and skin epithelium. Shiw (1938), in tissue cultures of embryonic human ovary, found 43 to 53 in connective tissue cells, and 48 in epithelial cells. Special mention must be made of the work of Hsu (1952) with tissue cultures of embryonic skin and spleen of a 4-month human fetus, Skin cultures were difficult to examine; the only four counts possible were of 48. In the spleen cultures, the results were one count of 44, five of 45, eleven of 46, eleven of 47, ninety-one of 48, four of 49 and one of 91. Thus 27% of the spleen counts were not of the diploid number, a figure which is close to Caffier’s 33% for embryonic lung, and certainly not significantly different from it. The particular value of HSU’S work is that his preparations were exceptionally clear. This was due in part to a fortunate accident, in which the tissues were exposed for a time to a hypotonic solution, thus giving a c-mitotic effect of shortened and easily counted chromosomes. It is rarely that such exact counts can be made with mammalian somatic cells. Being total mounts, the preparations could be assumed to be relatively free of mechanical disturbance of mitoses during preparation. The use of hypotonic swelling as a general method for simplifying the counting of chromosomes had already been forecast independently by Hughes ( 1952), who had studied the phenomenon in detail in living chick tissue cultures. Thus reports of somatic inconstancy in cultures apply to at least four major organs or tissues : connective tissue, leukocytes, lung, and spleen. In general, the range of variation is less than for somatic cells in situ.

V. DISCUSSION 1. Assessment of the Reality of Somatic Inconstancy The existence of somatic inconstancy, as a phenomenon, is beyond question; it is well known in cancers. The present paper is limited, however, wholly to non-pathologic cells. The data seem to constitute a strong case for the existence of a cell-to-cell inconstancy in the chromosome number of at least some somatic tissues of normal mammals. In embryonic membranes, which are particularly suitable for observation, there would Seem little doubt of the reality of chromosomal inconstancy. In other tissues the evidence is strong. It is perhaps too early to conclude that inconstancy is characteristic of all mammalian somatic tissues, but the data certainly point in that direction. Detailed assessment of individual reports has not been made here, though some general possibilities of

CHROMOSOMES I N MAMMALIAN SOMATIC CELLS

187

error have been outlined. As maintained in the last section of this review, the future of the subject demands improred techniques, whose results may place us in a better position to measure the observational uncertainties of existing reports, and clarify the actual range of the variation in a given tissue. Now that the subject has come into prominence, it is important that it should soon be settled to everyone’s satisfaction. Recognition of the reality of somatic inconstancy has met with resistance in some quarters and with overenthusiastic acceptance in others. The acceptance or rejection of somewhat novel observations is often influenced by factors other than the existing data themselves. For instance, recognition is often withheld until a result has been confirmed. It should be pointed out that Timonen and Therman’s results are themselves a confirmation of a body of earlier work. When in doubt, one can only exercise one’s judgment, but here judgments vary considerably. Eastern genetics, which visualizes only a minor role for the chromosomes in heredity and development, is predisposed to accept somatic inconstancy. Western genetics, which believes in a more detailed control of development and heredity by chromosomal determinants, is inclined to suspect reports of somatic inconstancy. In fact, somatic inconstancy is quite reconcilable with Western genetics. Western genetics is already committed, with certain obvious reservations, to belief in a Weismannian isolation bebwen soma and germ cells, and the chromosomal constitution of the somatic cells need not necessarily affect the process of passing unit characters from one generation to the next. As to the problem of how an individual grows under the influence of its own genes, several solutions in the presence of somatic inconstancy can be envisaged, and are discussed below. One r.efreshing point is that ideologic differences do not seem to be affecting the observations themselves. It is noteworthy, for instance, thai Timonen and Therman, who do not appear to belong to the Eastern school of genetics, report a greater range of inconstancy than Sorokina, who clearly subscribes to the Eastern school. Further, Andres and co-workers, who report somatic inconstancy as already reviewed (Andres and Shiw, 1935; 1936), report only constancy or near-constancy in germ cells (Andres and Navaschin, 1936: Andres and Vogel, 1936; Shiwago and Andres, 1932a, b ) ; we do not therefore have any general suspicion that their techniques and/or interpretations must always lead to a report of inconstancy. 2. Interpretation of Chromosome C a n t s from Tissue Cultures

With tissue cultures, there is an upfortunate paradox. They are ideal for observation and accurate chromosome counts, but, as against

188

R. A. BEATTY

this, they are not normal tissues. The somatic cell in situ is the real object of study, but is technically less favorable material. If an author reports wide chromosomal inconstancy in tissue cultures, then either this reflects pre-existing inconstancy in the piece of tissue used to start the culture, or it may have been caused by the abnormal environment of the cells in the culture. If an author reports extreme constancy in cells of a tissue culture, then either inconstancy in the initial piece of tissue is disproved, or, alternatively, it may be that cells with abnormal chromosome numbers are viable only in the setting of a surrounding soma, and perish in a culture, with the result that the culture displays only the diploid number. Thus, whatever the findings, it does not seem that existing results from tissue cultures can prove or disprove inconstancy in tissues in situ.

3. D N A Content of Nuclei and Somatic Inconstancy Demonstration of inconstancy rests on two main lines of evidence-the counting of chromosomes in cell divisions, and (in Timonen’s work) confirmatory observations of relative nuclear size in a tissue. If the deoxyribonucleic acid content of nuclei (see Frazer and Davidson, 1953, and Swift, 1950, for data and references) is a measure of the chromosomal content, we should expect DNA determinations in individual nuclei to provide a third line of evidence. I n mammalian somatic tissues, DNA measurements do in fact show a scatter around the diploid peak in the frequency distribution curve ; in certain tissues with a proportion of polyploid cells, there are also other peaks corresponding to cells with extra chromosome sets. It is not clear, however, how much of this scatter is due to technical difficulties. It would be of great interest to have available DNA measurements on proliferating endometrium nuclei, for the expectation from Timonen’s and Therman’s work is that the major peak in this tissue should be nearer the haploid than the diploid value. Use of the Term Heteroploidy The term heteroploid denotes cells, tissues, organs, or organisms in which there are chromosomal counts differing from the haploid and diploid numbers characteristic of the species. I n amphibia, for instance, a diploid animal contains only diploid cells. If numbers of non-diploid cells are found, the amphibian can safely be called a heteroploid. But in view of somatic inconstancy (and even on the extreme view that all somatic inconstancy is an artifact) it seems that the examination of all normal mammals except early embryos rnust result in classifying them as hetero ploid. There is a breakdown of the nomenclature. In studying mammalian polyploidy, as pointed out by Thernian and Timonen (1951 1, it is neces4.

CHROMOSOMES I N MAMMALIAN SOMATIC CELLS

189

sary to be careful that interpretation of abnormal chromosome counts should take account of a normal inconstancy. Thibault (1952), referring to post-implantation mammalian polyploids, goes so far as to imply that in the presence of somatic inconstancy, conclusive evidence of polyploidy needs analysis of spermatogenesis and oogenesis. 5.

Origin of Inconstancy in the Life of the Indiwidzcal

An attractive supposition to explain the cytology of origin of sotnatic inconstancy is that multipolar divisions occur. With tetrapolar divisions, we should expect the products of division to contain approximately the haploid number of chromosomes, but cells with numbers below and above this figure would also occur. A certain proportion of tetrapolar divisions could give exactly the same kind of distribution of chromosome numbers as reported by Timonen-Le., a peak about the haploid number, a smaller peak about the diploid number, a scatter around both peaks, and on the whole more hypo- than hyperploid cells. Unfortunately, the evidence is that multipolar divisions are rare in normal human embryos and endometrium (Andrcs and Jiv, 1936; Therman and Timonen, 1951 ; Timonen, 1950). The time of origin of somatic inconstancy during embryonic life is of interest. In the early embryo (blastocyst) or the mouse, the reviewer, originally in collaboration with Dr. h4. Fischberg, has examined large numbers of squash preparations of 3% -day embryos, and found that clear-cut identification of haploids, diploids, triploids, tetraploids, and hexaploids could be made. Other evidence confirmed that our observations reflected a real phenomenon ; changing the mating system altered the proportion of lieteroploids (Beatty and Fischberg, 1951a; Fischberg and Beatty, 1952b) ; triploidy (Fischberg and Beatty, 1952a) or tetraploidy (Beatty and Fischberg, 1952) could be induced experimentally; cell number in polyploids was in inverse proportion to the number of chromosome sets present (Beatty and Fischberg, 1951b). In these embryos, the outstanding types were diploids and polyploids, with whole niultiples of the haploid number in each mitosis of an embryo ; aneuploids with incomplete chromosome sets were relatively rare. In the last-mentioned paper we followed heteroploid development forward and identified triploids at 4% and 5% days of development ; at these times, embryos we re either diploid or polyploid, with no aneuploids and no cell-to-cell variation in chromosome number. Finally, at 9% days, we reported triploids, but here chromosome counts were more difficult, and we reported some variation. Whether this was a real variation, or due to difficulties in preparation and observation could not be determined. In an allegedly triploid adult rabbit, and to a minor

190

R. A. BEATTY

extent in a control, variation was reported (Melander, 1950). The evidence from rodents, then, may be summarized as follows: no somatic inconstancy up to about gastrulation time ; possibility of inconstancy just after the neural groove was formed and before the anterior limb buds form; somatic inconstancy reported after birth. In 6- to 24-week human embryos, Timonen and Therman (195Ob) found inconstancy; limb buds are already formed in &week embryos. If we generalize between rodent and man, the evidence, scanty as it is, suggests three conclusions : ( 1) in the non-diff erentiated embryo up to about gastrulation there is little or no inconstancy ; (2) some time between gastrulation and limb-bud formation inconstancy may begin ; and (3) after limb-bud formation, when much organogenesis and differentiation has taken place, inconstancy is reported. Gastrulation and primary organogenesis may therefore quite possibly be free to proceed in the absence of inconstancy. The relation between inconstancy and differentiation just outlined has been expressed in terms of mammals only, in order to keep the argument self-contained, but Oksala (1939), with non-mammalian material, has already suggested that somatic heteroploidy is related in some way to differentiation, and Huskins (1949) has taken up the subject in interesting detail ; both authors are, however, concerned with plyploidy in particular, rather than with the large aneuploid element of somatic inconstancy reported in mammals.

6. Are Cells with Abnormal Chromosome Nwmbers at the End of a Cell Lineage? Perhaps somatic cells with abnormal chromosome numbers are each at the end of their life history, and are not destined to divide further. But we cannot be looking at the very end of a cell lineage. Most of the counts of abnormal numbers were made on prophases and metaphases, which must have resolved themselves from a nucleus containing an abnormal number, which in turn must have come from a preceding abnormal anaphase. Thus, if we accept somatic inconstancy at all, we are committed to accepting at least two divisions of cells with abnormal numbers of chromosomes. And if the cells have divided twice, they may have divided more than twice.

7. Dying and Transitmy Tissues An intermediate attitude between complete belief in somatic inconstancy and complete rejection of it is that it does occur, but "merely" (useful word!) in dying and transitory tissues, It is reported (Roosen-Runge, 1953) that cell division in rodents can continue after death of the animal. As the cells themselves become moribund, the abnormal conditions might

CHROMOSOMES I N MAMMALIAN SOMATIC CELLS

191

lead to abnormal divisions. In the living animal, the outer cells of the epidermis are progressively dying and open to the possibility of abnormal divisions. The same might apply in a less obvious case, such as the death of individual cells of internal organs. Further, particularly clear somatic inconstancy is to be observed in the transitory embryonic membranes and in the monthly-renewed endometrium. The more we believe in somatic inconstancy applying only to dying and transitory tissues, the more we are committed to what may be called somatic Weismannism. In this situation, we have to visualize the mammalian body as containing a basic network or patchwork of cells with 48 chromosomes, which is responsible for the continuity of genetic factors in the somatic tissues, and which gives rise to the other cells with abnormal numbers. The evidence of such a system may already be before our eyes ;the specialized cells of an organ often originate from a restricted zone of so-called “germinal epithelium” (Weiss, 1949). 8. Some Consequences of Assuming That Non-diploid Cells Do Form Cell Lineages a. Gene Action. Large and Small Orgmu. Let us consider the alternative hypothesis that cells with. abnormal chromosome numbers are not at the end of their developmental history, but divide many times and constitute an appreciable part of the mammalian body. How then could the genes be imagined to exert their action when whole blocks of hereditary determinants, as represented by whole chromosomes, must often be entirely missing, and there is, in effect, a chromosomal anarchy? If we take a normal cell with 48 chromosomes, and consider an abnormal division in which the two daughter sets of chromosomes are distributed unequally among the two daughter cells, we might end with chromosome numbers of say 56 and 30, respectively. The average of these numbers is still 48. If each daughter cell undergoes a similar kind of division, we might get such numbers as 57, 55; 45, 35; but the average of the four is still 48. Now in most tissues these daughter cells are close to one another. Thus we can imagine the somatic tissues to contain little groups of cells of common descent in which balanced chromosome sets are present, though spread unequally between the different cells of the group. This explanation was originated in more extended form by Timonen and Therman (195Ob). They argued that if the 48 chromosomes in the fertilized human egg all divide at the same rate, we shall end up with an organism containing equal numbers of each type of chromosome. The human organism, considered as a whole, would contain a very large number of complete chromosome sets, and the fact that they were parceled out unequally among the different

192

R. A. BEATTY

cells need not affect their functioning as complete sets. They suggest “that the whole human organism acts as a giant polyploid cell.” As a corollary to this theory, it has to be assumed that gene action takes place across cell walls (Timonen, 1950). This theory of Therman and Timonen implies that with enormous numbers of cells under consideration, the merage number of each type of chromosome in any particular region will be equal. But if we take the particular case of organs composed of small numbers of cells containing self-differentiating hereditary factors, we should, by chance, occasionally find that one of these organs happens to have one particular single chromosome lacking in all its constituent cells, and we might expect in some cases a visible expression of this event. Consider, for example, coat color in mammals. It is known from skin transplantation work that small patches of transplanted skin can grow hairs of their own type, and not that of the surrounding host skin, Now consider a rabbit heterozygous for the albino factor. It may rarely happen that all the cells generating a hair have lost the chromosome carrying the factor for color, leaving only the albino factor, and such a hair would be expected to be white. In more general terms, we should expect in some cases that a mammal heterozygous for a self-differentiating color factor should show an expression of the homozygous factor in a few of its hairs. For this there is possibly some observational basis, which is presented more as a subject for study than as clear-cut evidence. On a non-agouti background (aa), the hornozygous light chinchilla rabbit (cChkchL) is blackishbrown; the heterozygote with albino (ccchL) is sandy; the homozygous albino (cc) is white. Close examination of the heterozygote reveals a number of scattered white hairs which are rare in the homozygous light chinchilla. Are these white hairs perhaps due to loss of the chromosome carrying the color factor, thus leaving the albino factor uncovered? b. Determination and Somatic Inconstancy. An alternative level of explanation of how a mammal could develop under the influence of its own genes in the presence of somatic inconstancy involves the concept of cytoplasmic determination. I t may be that the cytoplasm in many mammalian tissues has been determined in embryonic development, before somatic inconstancy began; that gene action took place many generations of cells previously, and the chromosomal content of the later cell generations was less important for their development than cytoplasmic components such as plasmagenes. In correspondence with this, we have already seen that there is some evidence of absence of inconstancy in early embryos. c. Nature of the Relation between Somatic Hetevoptoidy and Differentiation. The observation of a relation between heteroploidy and differ-

C H R O M O S O M E S IN MAMMALIAN SOMATIC CELLS

193

entiatiori is a datum in its own right. The nature of the relation is a different subject. One of the central problems of embryology and genetics is how an organism, all of whose cells are customarily thought to contain the same chromosome and gene complements, yet differentiates its cell types, At first sight, the connection between inconstancy and differentiation might suggest that here at last we have the possibility of a chromosomal differentiation which might be a direct cause of cellular differentiation. We could imagine, for instance, that skin is skin because of certain chromosomal combinations in its cells ; cartilage is cartilage because of other combinations. But this is not true for amphibia, in which chromosome number is highly uniform from cell to cell, and the main problem remains unsolved. If the main problem is unsolved in amphibia, it seems unlikely that a problem of such a general nature has been solved in a particular way for mammals only. Rather, the somatic inconstancy in mammals is to be thought of as random. 9. Research Outlook It is highly desirable that the extent of somatic inconstancy in mammals should be confirmed beyond all doubt, and that certain details should be available. In particular, the exact time of origin during ontogeny is of interest, and also the question of whether or not cells with abnormal chromosome numbers divide to give cell lineages. Separate consideration should be given to results from germ cells; from somatic cells in situ; fmm somatic cells in tissue culture; and from pathologic cells. More suitable material is desirable. The reviewer at one time attempted to obtain specimens of Cricetulus p'seus, for which a haploid number of only 7 had been reported (Pontecorvo, 1943). This figure has now been amended to 11 (Matthey, 1951; Sachs, 1952), the same number as in Cricetus cricefzw ( Matthey, 1951; Sachs, 1952). Nevertheless, these two hamsters seem to offer the best-known material in higher mammals, in comparison with the haploid numbers of 20 or more in mouse, rat, rabbit, guinea pig, man and others. There are also marsupial species with low chromosome numbers. Improved technique is also needed. Are we to continue indefinitely with the suspicion that smear and squash techniques damage somatic mitoses (though not those of germ cells) and give an untrue appearance of inconstancy ; that sections also give untrue pictures of somatic chromosomes (but not those of germ cells) by loss of parts of mitoses and difficulty in observation? In the work of Hsu (1952) the ease with which the chromosomes could be counted was remarkable. This was due in part to an accidental c-mitotic treatment with hypotonic solution. Deliberate use of a c-mitotic agent such as hypotonic solution or colchicine is now becoming a

194

R. A. BEATTY

general method of increasing the accuracy of chromosome counts in mammals. Another possibility for improved technique would be the observed squash ; it should be possible to select an undamaged and undisturbed mitosis in a fragment of tissue and then, under continuous observation with the microscope, squash it in order to separate the chromosomes for counting, while at the same time ensuring that no chromosomes were being driven away from or entering the field of observation. Finally, it should be noted that there is a situation in which the attitude toward a technique is affected by the nature of the results. If, under repeatable conditions, the diploid chromosome number and no other could be clearly and unambiguously demonstrated in all or nearly all the cells examined in samples of a tissue, then we should cease to question technique, and know that for that tissue the subject of chromosomal inconstancy was to be dismissed.

VI. CONCLUSIONS AND SUMMARY During the last half-century, reports of the chromosome number in germ cells of man and other mammals have narrowed down in general to a definite haploid and diploid number characteristic for each species. Discrepancies among the early reports can be attributed to technical and observational difficulties, combined with certain subjective influences. Over the same half-century, with the same technical background, an increasingly greater cell-to-cell variation in chromosome number of mammalian somatic cells has been reported. The trend is particularly marked in the recent period of cytology. This variation, which may be called somatic inconstancy, or somatic aneuploidy, is reported from at least eleven organs or tissues in man; from embryonic and extra-embryonic tissues, and from results of both smear and section techniques. Inconstancy is reported aIso from other mammals. A smaller but definite inconstancy has been reported from tissue cultures of somatic cells, Inconstancy in mammalian embryonic membranes seems well established. In other tissues also there is strong evidence. Reaction to these reports has been marked, and perhaps not unconnected with prevailing ideologies. Results from tissue cultures are not thought to solve the main problem of inconstancy in dtu, though these studies have an interest of their own. Somatic inconstancy as judged by chromosome counts is not contradicted by deoxyribonucleic acid determinations on single nuclei. Somatic inconstancy has repercussions on the study of mammalian polyploidy. The cytology of origin of somatic inconstancy is not clear; the time of origin would possibly be between gastrulation and limb-bud formation. Inconstancy may be typical only of dying and transitory tissues, and

CHROMOSOMES I N MAMMALIAN SOMATIC CELLS

195

cells with abnormal chromosome numbers may be near the end of a cell lineage. But there is some evidence for division of these cells ; this seems an important point for examination. On the hypothesis that cell lineages are formed, some problems are outlined: the mode of gene action in large and small organs, and the connection of inconstancy with determination and differentiation. The research outlook is thought to demand improved material and technique, for which a few suggestions are made.

VII. ADDENDUM Some important papers have been published since this review was written. Hsu and Pomerat (1953a, b) have confirmed in detail the existence of somatic inconstancy in tissue cultures from mouse, guinea pig, dog and cotton-rat. Their second paper summarizes an even greater inconstancy in various rat tissues in sittr reported by Tanaka (1951 : in Japanese). Sachs (1953), in normal uterine endometrium of man, rat and a vole, does not find the extensive sub-diploid variation in chromosome number reported by Timonen and Therman for this tissue in its proliferative stage. Manna (1953), however, confirms Timonen and Therman, though the range and characteristics of the variation were different ; he also observed inconstancy in human uterine cervix tissue. VIII. REFERENCES Adamstone, F. B. (1929) Anat. Record, 44, 232. Allen, E., Pratt, J. P., Newell, Q. U., and Bland, L. J . (1930) A m J. Anat., 46, 1. Andres, A. H. (1933) 2. Zrllfursch. t4. mikroskop. Anat., 18, 411. Andres, A. H.,and Shiw, B. W. (1935) Biol. Zhrcr., 4, 505 (German summary). Andres, A. H., and Jiv, B. V. (Shiw, B. W.) (1936) Cyfologia (Tokyo), 7, 371. Andres, A. H.,and Navaschin. M. S. (1936) Z . Zellforsch. u. mikroskop. Anaf., 24, 411. hndres, A. H., and Viigel, I. I. (1936) 2. Zellforsch. u. mikroskog. And., 24, 552. Bardeleben, K. von (1892) Verhandl. Amt. Ges., Wdpn, Jena, p, 202. Bardeleben, K. von (1897) Arch. A n d . u. PhySiol., Suppl., p. 193. Barigozzi, C. (1947) Arch. Julius Klaus-Stift. Vererbungsforsch. Soaialantlrropol. u. Rassenhyg. 22, 342. Beatty, R. A., and Fischberg, M. (1951a) I. Genet., 60, 345. Beatty, R. A., and Fischberg, M. (1951b) I. Exptl. Biol., 26, 541. Beatty, R. A.,and Fischberg, M. (1952) J . Genet., W ,471. Boothroyd, E. R.,and Walker, B. E. (1952) Genetics, 87, 567. Branca, A. (1910) Comfit. rend. asssac. anot., 12, 5. 3ranca, A. (1911) Biblwg. anat. 21, 233. Branca, A. (1924) Arch. zool. exfitl. et g h . , 62, 53. Caflier, P. (1932) 2. Geburtshiilfe u. GynZkul., 101, 262. Chrustschoff, G, K., and Berlin, E. A. (1935) I. Genet. 31, 243. Darlington, C. D. (1953) Nature, 171, 191. Duesbcrg, J. (1906) Anat. An&, 28, 475.

1%

R. A. BEATTY

Evans, H. M., arid Swezy, 0. (1928) Genctics, 13, 532. Evans, H. M.. and Swezy. 0. (1929) Mmn. Uniz-. Calif., 9 ( l ) , 40 pp. Fischberg, AT., and Reatty, R. A. (1951) 1. Exptl. Zool., 118, 3-71. Fischberg, M., and Beatty, R. A. (1952a) 1. Genet., 60, 455. Fischberg, M., and Beatty, R. A. (1952b) Evoliction, 6, 316. Fleming, W. (1897) Anat. Ana., 14, 171. Frazer, S. C., and Davidson, J. N. (1953) Exbtl. Cell Research, 4, 316. Friedenthal, H. (1921) '4rch. Rasseit- 14. Gesellschuftsbiol. 13, 257. Gates, R. R. (1942) Science, 96, 336. Gutherz, S. (1912) Arch. niikroscop. Anat. u. Entm'cklungsmech., 79 (II), 79. Guyer, M. F. (1910) Biol. Bull., 19, 219. Guyer, M. F. (1914) Science, 39, 721. Guyer, M. F. (1927) Being Well Born, 2nd ed. Bobbs-Merrill Co., Indianapolis. Harvey, E. B. (1920) J. Morpltol., 84, 1. Heberer, G. (1935) 2. menschl. Yererbungs- u. Konstifutiodehre, 19, %. Hoadley, L., and Simons, D. (1928) Ant. J. Anat., 41, 497. Hsu, T. C. (1952) J. Heredity, 43, 167. Hsu, T. C., and Pomerat, C. M. (19%) J. Hered., 44, 23. HSU,T. C., and Pomerat, C. M. (1953b) I. Morph., SS, 301. Hughes, A. (1952) Quurt. I. Micro. Sci., 93, 207. Huskins, C. L. (1949) Proc. 8th Intern. Congr. Genetics, p. 274. Jordan, H. E. (1914) Camegie Inst. Wash. Publ. No. 182, 163. Karplus, H. (1929) 2. Zellforsch. u. mikroskop. And., 10, 38. Kemp, T. (1928) Compt. rend. SOC. biol., 99, 1601. Kemp, T. (1929a) 2. mikroskop.-anat. Forsch. 16, 20 pp. Kemp, T. (1929b) Biol. Meddel., 7, 1. Kemp, T. (1930) 2. Zellforsch. u. mikroskop. Atlat. 11, 429. King, R. L., and Beams, H. W. (1936) Anat. Record, 66, 165. Koller, p. B. (1937) Proc. Roy. SOC.(Edinburgh) Sect. B, 67, 194. La Cour, L. F. (1944) Proc. Roy. SOC.(Edinburgh) Sect. B, 62, 73. Lams, H. (1950) La Cellrrle, 64, 67. Makino, S. (1951) An Atlas of the Chromosome Numbers in Animals. Iowa State College Press. Manna, G. K. (1953) Nature, 175, 271. McClung, C. E. (1939) Tabdue Biol., 18 ( l ) , 1. Matthey, R. (1949) Les chromosomes des vertibres. F. Rouge, Lausanne. Matthey, R. (1951) Exjtmkntio, 7, 340. Melander, Y. (1950) Heredifus, 36, 335. Minouchi, O., and Ohta. T. (1934) Cytologia, 6, 472. hfolas, L. G. Guilera (1926) Trav. Lab. reclrerches biol. Univ. Madrid, 24, 333. Montgomery, T. H. (1912) 1. A d . Nat. Sci. Phil., 16, 1. Moore, J. E. S., and Arnold, G. (1906) Proc. Roy. SOC.(London) Ser. B., TI, 563. Moore, J. E. S., and Walker, C. E. (1906) Liverpool Univ. Lob. R e p . , (N.S.) 7 ( l ) , 75. Muller, H. J. (1925) Am. Nuturdst, 69, 346. Oguma, K. (1930) Arch. Biol., 40, 205. Oguma, K. (1937) J. Mmfhol., 61, 59. Oguma, K. (1939) Botany 15 zoology (Tokyo), 7, 179 (in Japanese; extract in Makino, 1951).

CHROMOSOMES I N MAMMALIAN SOMATIC CELLS

197

Oguma, K., and Kihara, H. (1923) Arch. Biol., 59, 493. OksaIa, T. (1939) Hereditas, !46, 132. Painter, T. S. (1923a) J. Enpfl. Zool., 37, 291. Fainter, T. S. (1923b) Anat. Record, 23, 129 (Abstr.). Painter, T. S. (1924) Am. Naturalist, 68, 506. Pincus, G., and Saunders, B. (1939) Amt. Record, 76, 537. Pletnev, S. A. (1911) Compt. rend. w a d . sci. U.R.S.S., (N.S.) 31, 491. Pontecorvo, G. (1943) Proc. Roy. SOC.(Edinburgh) Sect. B, 62, 32. Rappeport, T. (1922) Arch. Zrllforsch., 16, 371. Roosen-Runge, E. C. (1953) Exptl. Cell Research, 4, 52. Rotter, H. (1922) Z. Krebsforsch., 18, 171. Sachs, L. (1952) Heredity, 6, 357. Sachs, L (1953) Nature, 172, 205. Schachow, S. D. (1927) Anat. Anz., 62, 122. Shiw, B. W. (Jir, B. V.) (1938) Biol. Zhzw., 7, 545 (German summary). Shiwago, P. I., and Andres, A. H. (1932a) Biol. Zhur., 1, 82 (French summary). Shiwago, P. I., and Andres, A. H. (1932h) 2. Zellforsch. u. mikroskop. Atuat., 16, 413. Slizynski, B. M. (1945) Nature, 166, 427; and personal communication. Sorokina, N. I. (1950) Imest. Akad. NaMk. (S.S.S.R.), (6), 97. Swezy, O., and Evans, H. M. (1930) J . Morphol., 49, 543. Swift, H. F. (1950) Phy&ol. Zoiil., as, 169. Therman, E., and Timonen, S. (1951) Hereditas, 37, 266. Thibault, C. (1952) Refit. 2nd Internat. Coltgr. Phyhsdol. Pathol. Animal Reproduction and Artificial Insemination, 1, 7. Timonen, S. (1950) Acta Obsfet. Gywnrcol. Scand., 31, Suppl. 2, 1. Timonen, S., and Therman, E. (1950a) Cancer Research, 10, 431. Timonen, S.,and Therman, E. (1950b) Nature, 166, 995. Venge, 0. (1953) Kgl. Lantbruks-Hiigskol. Ann., 19, 233. Weiss, P. (1949) The Chemistry and Physiology of Growth, p. 135. Princeton University Press. Wieman, H. L. (1913) J. Anat., 14, 461. Wieman, H. L. (1917) Am. 1. Anat., 21, 1. Wilcox, E. V. (1900) Antat. AM., 17, 316. Winiwarter, H. von (1912) Arch. Biol.. 27, 91. Winiwarter, H. von (1920) Rev. anthropol., 30, xxvi. Winiwarter, H. von (1921) Conipt. rcnd. SOC. bid., 85, 266. Winiwarter, H. von, and Oguma, K. (1926) Arch. Bid., 36, 99. Wodsedalek, J. E. (1941) Anat. Record, 81, Suppl., 79.

This Page Intentionally Left Blank

The Significance of Enzyme Studies on Isolated Cell Nuclei ALEXANDER L. DOUNCE* Biochernistry Departmcnt, University of Rochester School of Medicine and Dentisfry, Strong Memorial Hospital, Rochester, New York

I. Introduction ....................................................... 11. Work of Mirsky and Collaborators on Cell Nuclei Isolated by a Modification of the Technique of Behrens .............................. 111. Work of Lang and Collaborators .................................. IV. Recent Work of Hogeboom and Schneider on Synthesis of DPN by Nuclear Preparations ............................................ v. The Problem of Oxidative Enzymes in Cell Nuclei .................. VI. General Discussion ................................................ VII. Summary and Conclusions ......................................... VIII. References ........................................................

Page 199

202 208 212 213 218 221 221

I. INTRODUCTI~N Within recent years work on cell nuclei has advanced to the point where, instead of intermittent and scattered efforts by a few individuals, we now find a number of laboratories publishing the results of extensive research devoted exclusively to the chemistry and enzymology of cell nuclei. This situation is probably the result of general advances in cytochemical technique as well as the growing realization that the cell can never be understood completely from the physiologic or biochemicaI standpoint until the functions of the major cell components are elucidated. The need to understand the causes for and the mechanism of cell division as the basis for a comprehension of cancer also has stimulated research on the chemistry of cell nuclei. Much of the newer work is concerned with investigations of isolated nuclei and nuclear components, although some of it deals with histochemistry, microspectrography, and microdissection. This article does not cover all recent work on cell nuclei but is instead restricted to an attempt to analyze the recent work of major importance dealing with the enzymes of cell nuclei isolated by biochemical techniques. It is quite certain from a brief consideration of some well-established facts in the realm of biology and biochemistry that some enzymes at least must be present within the cell nucleus. Many of these facts have been presented previously (Dounce, 1952a). An additional consideration is the synthesis of deoxyribonucleic acid (DNA). It seems out of the question, *The support of the National Cancer Institute of the National Institutes ef Health, U.S. Public Health Service, is gratefully acknowledged.

199

200

ALEXANDER L. DOUNCE

for example, that a high molecular weight material such as deoxyribonucleic acid ( D N A ) could be synthesized itt toto in the cytoplasm and then be transferred to the nucleus, subsequently to become localized in the chromosomes in an ordered manner. Moreover, DNA is not found in detectable amounts in the cytoplasm of mammalian somatic cells. If we admit that at least the late stages in the synthesis of deoxyribonucleic acid must therefore take place within the nucleus of the non-mitotic cell, it follows at once that enzymes involved in this synthesis must also be located within the cell nucleus, since enzymes do not act over any considerable distance. To consider the late stages in the synthesis of DNA as nonenzymatic would be as unwarranted as to assume the migration of finished DNA molecules from the cytoplasm to the nucleus. Of course it might be argued that DNA synthesis occurs only during mitosis, after the disappearance of the nuclear membrane and loss in distinction between nucleus and cytoplasm. However, the best evidence available at present is against such an idea, since DNA appears to be synthesized just prior to prophase, as a step necessary to prepare the cell for subsequent mitotic division (Vendrely, 1952; Thomson et al., 1953). The problem of determining exactly what enzymes do occur within the cell nucleus has been under investigation for a number of years by various workers who have relied mainly on the technique of cytochemistry on a “macroscopic” scale or on histochemical procedures. It might be thought that, given a mild method for isolating cell nuclei in test tube quantities, reasonably free from cytoplasmic components such as microsomes and mitochondria, the determination of what enzymes are present in the cell nucleus would be a routine matter. However, two considerations arise which complicate the problem immensely and which in general have not been given sufficient study in past investigations. One of these considerations is the possibility of adsorption of enzymes by the nuclei, and the other is the problem of whether the nuclear membrane is permeable to protein, at least under the conditions obtaining during isolation of the nuclei. I t has recently become evident (Dounce, Kay, and Pate, 1953) that adsorption can complicate the picture with insoluble mitochondria1 enzymes, although previous studies have argued against adsorption by nuclei playing a very significant role in the cases of certain soluble enzymes (arginase, aldolase, and catalase) (Dounce, 1952a ; Dounce and Beyer, 1948). It has been thought by some investigators that the nuclear membrane is impermeable to soluble enzymes, provided it has not been “damaged.” Relatively recent work indicating a permeability of isolated nuclei to

ENZYME STUDIES ON ISOLATED CELL NUCLEI

201

protein (Dounce, 1952a) might be criticized on the grounds that the device used for rupturing the cells (the Waring Blendor) also injured the nuclear membranes, and a similar argument might be brought against the finding (previously unpublished) that histone can be extracted from liver cell nuclei isolated at pH 4.0, without changing the microscopic appearance of the nuclei. However, still more recent and also previously unpublished experiments carried out by Dr. J. Holtfreter of the University of Rochester with frog egg nuclei isolated by microdissection have indicated that these nuclei are quite permeable to hemoglobin in a variety of solutions of different types commonly used as media in large-scale isolations of cell nuclei. Since electron microscope studies (Callan and Tomlin, 1950; Bairati and Lehmann, 1952; Harris and James, 1952; Palade, personal communication) have demonstrated considerable similarity among the nuclear membranes of amphibian egg cell nuclei, nuclei of amoebas, and nuclei of liver cells, it is quite possible that liver cell nuclei will behave more or less in the same manner as those of the two other forms mentioned, as far as permeability to protein is concerned. Further evidence supporting the idea that cell nuclei can be permeable to hemoglobin is the observation that the nuclei of chicken erythrocytes isolated at p H 6.8 under very mild conditions do not contain hemoglobin (Dounce and Lan, 1943), whereas nuclei of chicken erythrocytes isolated by Stern et al. (1952) with a modified Behrens’ technique do contain very appreciable quantities of hemoglobin. It might be argued that the saponin used in the isolation procedure damages the nuclear membranes, but this is only conjecture. Moreover, saponin does not as a rule destroy cell membranes other than those of the erythrocyte. In addition to this, it has been observed (previously unpublished) in this laboratory that nuclei of liver cells, isolated in distilled water at a pH of about 7.0 by means of a new homogenizer that permits a very complete cell breakage without apparent damage to nuclei or mitochrondia, apparently take up cow hemoglobin readily, judging from the color, and also lose a major fraction to the solution on washing with water. Hemoglobin is, of course, a protein of moderately low molecular weight, but certain enzymes have even lower molecular weight and hence may be expected to pass through the nuclear membrane quite readily. When the experiment involving hemoglobin can be repeated with a high molecular weight chromoprotein such as horse liver catalase (molecular weight about 250,000) it will be possible to say still more about permeability of the nuclear membrane. Recent papers by Anderson (1952, 1953a, b) also give evidence that the nuclear membrane can be permeable to large molecules, such as hemoglobin

202

ALEXANDER L. DOUNCE

and other proteins. The argument is advanced by Anderson (1953a) that it is hard to imagine genetic information being passed to the cytoplasm in the form of small molecules, and this argument is difficult to dispute. If it is accepted, either the nuclear membrane must be permeable to large molecules or the nucleus must exert its genetic effects only during mitosis. The latter possibility is very improbable. The concept of a nuclear membrane permeable to relatively large molecules is by no means universally accepted, however. For example, Callan and Tomlin (1950), working with the nuclear membrane of amphibian egg cells (Xenopus), have apparently concluded that this membrane is not permeable by diffusion to protein, although clear-cut evidence for this statement seems to be lacking. Recently, Hogeboom and Schneider (1952) have concluded that the nuclear membrane of liver cell nuclei may not be permeable at least to one soluble enzyme, and hence the question of nuclear membrane permeability, which is vital in our attempt to understand nucleuscytoplasm relationships, remains unsettled. It seems pertinent, therefore, to review critically some of the arguments which have been advanced in connection with nuclear-membrane permeability. Since this paper is concerned primarily with enzyme studies, and since most of the recent work on enzymes bears directly or indirectly on nuclearmembrane permeability, the remainder of the paper will deal with what appear to be the most important recent studies of enzymes in isolated cell nuclei. 11. WORKOF MIRSKYA N D BY A

CELL NUCLEIISOLATED TECHNIQUE OF BEHRENS

COLLABORATORS O N

MODIFICATION OF

THE

Mirsky and collaborators have isolated cell nuclei from a number of animal and plant tissues (Stern et al., 1952, Allfrey et al., 1952; Daly et al., 1952; Stem and Mirsky, 1952), with a modification of the technique originally developed by Behrens (1939). The lyophilization was carried out by a modern technique, and cyclohexane was substituted for benzene in the specific gravity flotations. Photographs of the isolated nuclei (Stem et el., 1952) indicate a high degree of purity in the case of bird erythrocyte nuclei, but do not dearly indicate that nuclei prepared from mammalian tissue are free from surrounding pieces of cytoplasm, although the latter nuclei do appear to be entirely free from fine material. It is not at all clear to this author that the nuclei isolated from liver tissues by Mirsky ot QZ. are, as claimed by them, greatly superior to those obtained in the author’s laboratory by a very similar procedure (Dounce, Tishkoff, et al., 1950). In any case it is of the utmost importance to estimate how much, if any, bound cytoplasm is present in the nuclei, since the concentrations

ENZYME STUDIES ON ISOLATED CELL NUCLEI

203

of some of the nuclear enzymes are reported by Mirsky ef aE. as being rather low compared with corresponding concentrations in cytoplasm. The significance of enzymes in this class must remain in doubt until the degree of cytoplasmic contamination can be estimated. (The word concentration as applied to enzymes is used only for convenience throughout this paper, since what is really measured is, of course, activity of enzyme per unit dry weight of material.) It was found by Mirsky et al. that many enzymes survived the lyophilization and sedimentations in organic solvents which are used in the isolation procedure of Behrens, and generally, when enzymes were destroyed, it was the lyophilization rather than the solvents which caused the trouble. However, no data are given showing concentrations of the enzymes in a fresh homogenate, and this is unfortunate, since it is impossible to know just how much destruction of a given enzyme may have occurred. If appreciable destruction does occur, it is possible that the degree of destruction of a given enzyme might be different in the nuclei and the cytoplasm. The results obtained by Mirsky and collaborators concerning enzyme distribution cannot easily be generalized or even summarized, and therefore only enough of the results will be given here to serve as a basis for discussing the arguments presented. Allfrey et d. (1952) have classified enzymes for the purposes of their work as follows: (1) special enzymes, more or less characteristic of a given tissue ; (2) enzymes of more general distribution among tissues. I n addition, hemoglobin and myoglobin have been studied. According to Mirsky, the nuclei of different tissues differ as much as the tissues themselves, among the special enzymes. Thus arginase is present in high concentrations in the cytoplasm and nuclei of mammalian liver cells. Arginase was also found, although in much lower concentrations, in the cytopIasm of calf kidney nuclei. (The reader is referred to a paper by Dounce, 195Oa, and to work by Lang and Siebert, 1951, for a comparison of these results with the results obtained using nuclei prepared by means of aqueous procedures.) In the same class of enzymes, catalase is also found in high concentrations in the cytoplasm and nuclei of horse and calf liver cells, but it is stated to be absent from the nuclei of fowl erythrocyte and kidney tissue, although it is present in high concentrations in the cytoplasm of cells from these organs. (In nuclei prepared by an improved aqueous procedure, Dounce, 195Oa, found catalase in the nuclei of both liver and kidney cells.) In considering enzymes of more general distribution, Allfrey et d. (1952) found that the nuclear concentration of a given enzyme cannot

204

ALEXANDER L. DOUNCE

be predicted from the corresponding cytoplasmic concentration of the same enzyme. For instance, it is stated that alkaline phosphatase is low or absent from the nuclei of most tissues, with the exception of horse liver. ( I n spite of this the data show that the concentration of the alkaline phosphatase in the nuclei of calf liver cells is 12% of the concentration in cytoplasm, and results where the nuclear concentration is above 7% of the cytoplasmic concentration are stated to be of significance.) The enzyme ATP-ase was low or absent from all nuclei studied. (This is in contrast to nuclei prepared in aqueous media, which generally have high ATP-ase activity.) Esterase was present in significant concentrations in the nuclei from a number of tissues, whereas P-glucuronidase was not present in significant amounts in the nuclei studied except in mucosal tissues. An enzyme of particular interest was nurleoside phosphorylase, which was found in particularly high concentrations in all nuclei studied. It was thought that this enzyme might make a particularly significant contribution to the metabolism of the cell nucleus. A study of fetal tissue was also made. Here one of the principal findings of interest was that a number of enzymes present in the adult, such as Iipase and amylase of pancreas, were only barely detectable in the fetus. In addition, it was found that the ratio of nuclear concentration to cytoplasmic concentration for a given enzyme was greater in embryonic than in adult tissues. The effect of starvation on nuclear enzymes also was noted. Whereas starvation usually caused an increase in the concentration of a given enzyme in cytoplasm, just the reverse was true of the concentration of nuclear enzymes. An exception occurred with catalase, since this enzyme decreased sharply in concentration in both nuclei and cytoplasm during starvation. Stern and Mirsky (1952) have also studied cell nuclei obtained from wheat germ by the modified Behrens’ technique. These nuclei were thought to be about 85% pure, and, interestingly enough, contained ribonucleic acid in concentration nearly equal to the ribonucleic acid concentration of cytoplasm. (Cf. nuclei of rat liver cells studied by Dounce et d. 1950.) The following glycolytic enzymes were found in the nuclei in rather high concentrations : aldolase, 3-phosphoglyceraldehyde dehydrogenase, enolase, and pyruvate kinase. Lactic dehydrogenase was not tested for. Except for enolase, the concentrations of the glycolytic enzymes were slightly higher in nuclei than in whole tissue ; with enolase, the nuclear concentration was 1.6 times its concentration in whole tissue. Dounce et al. (1950) found that aldolase was damaged by the Behrens’ procedure when the latter was applied to liver nuclei, but this would not be necessarily true for plant cell nuclei.

ENZYME STUDIES ON ISOLATED CELL NUCLEI

205

The enzyme p-amylase was also studied, and it was found to be almost entirely confined to cytoplasm. In fact, the amount of 8-amylase found in nuclei was used to estimate the amount of cytoplasmic contamination of the nuclei, on the assumption that the true concentration of this enzyme in the nuclei studied is zero. From this work it was concluded that nuclei of plant cells, at least, can derive energy from glycolysis. From a study of the diphosphopyridine nucleotide content of nuclei prepared from calf pancreas, liver, and heart, it was concluded in addition that glycolytic reactions may well occur in mammalian nuclei also, since the DPN concentrations in all cases were slightly higher in the nuclei than in the whole tissues. It should be noted that total glycolysis was in no case studied. Hexose diphosphate was the earliest-occurring substrate of the glycolysis cycle which was used, and it was even surmised that reoxidation of DPN probably occurred as the result of nucleocytoplasmic interaction rather than through the reduction of pyruvate to lactate in the nuclei. In the discussion of this work on glycolytic enzymes, it is not stated that Dounce and collaborators found a number of glycolytic enzymes in reasonably high concentrations in cell nuclei prepared at p H 6.0 by an aqueous procedure (Dounce, 1950a, 1952a), and emphasized the possible importance of glycolysis in cell nuclei, although the work of Lang et al. (Lang and Siebert, 1951) on glycolysis with isolated cell nuclei is discussed. Hemoglobin and myoglobin were studied using nuclei of chicken erythrocytes and muscle cell nuclei, respectively ; hemoglobin was present in significant amounts in the erythrocyte nuclei, but no myoglobin was found in the muscle cell nuclei. However, the structure of the muscle cell is so specialized that it would be very hazardous to try to derive general conclusions from work on muscle nuclei, especially in regard to permeability of the nuclear membrane (see discussion below), since other membranes may intervene between the myoglobin and the nuclei. Mirsky has concluded from his studies that passive permeability of the nuclear membrane to enzymes is very unlikely. H e makes the following statement in regard to this point: “That this ‘passive diffusion’ is unlikely from a general biological standpoint should be apparent, but it is worth emphasizing that in the entire pattern of distribution observed, the physical factors likeliest to be associated with such a diffusion processsolubility and molecular size-bear no relation to the actual presence or absence in the nucleus of the various components. . . Furthermore, it is clear that passive diffusion tends to an equalization of concentrations ; no such tendency has been observed. In fact, where enzymes are more

.

206

ALEXANDER L. DOUNCE

concentrated in the nucleus than in the cytoplasm, or where, in the case of starvation, changes in concentration run oppositely in cytoplasm and nucleus, the occurrence of a mechanism of passive diffusion cannot be postulated. If so, the presence of special cytoplasmic components within the nucleus must be considered as an active and hence necessary part of nuclear metabolism.” This reasoning, however, appears somewhat superficial to the writer, since a number of pertinent considerations seem to have been neglected. In the first place, the statement concerning equalization of concentrations (in nuclei and cytoplasm) by passive diffusion across the nuclear membrane can refer correctly only to concentration based on the soluble aqateour phase, since it is obvious that large particles, such as secretory granules or mitochondria, cannot diffuse across the nuclear membrane at all. It is quite likely that digestive enzymes, such as are present in the pancreas, for example, exist essentially as zymogen granules within the cytoplasm, and that such enzymes become soluble only after breakdown of the zymogen granules in the pancreatic ducts. It is thus futile to speak of solubility or molecular weight of an enzyme which may never exist in solution until it leaves the cell, or until it has been extracted from such particulates as secretory granules or mitochondria during isolation procedures. Moreover, an enzyme present within the cell nucleus might be bound to a greater or lesser extent by nucleic acid, so that, again, total concentration of even an intranuclear enzyme should be based only on its conceiztration in the soluble aqueous phaxe, and such concentration cannot be estimated at the present time. The argument concerning irreconcilability of the effects of starvation on intracellular enzyme distribution with the postulate of free diffusion of enzymes across the nuclear membrane must also remain inconclusive until the concentrations of enzymes in the soluble phase of nucleus and cytoplasm can be measured. It is very possible, for example, that insoluble phases of cytoplasm, including material such as glycogen, will be lowered much more rapidly than the insoluble phases of nuclei, which include the chromosomes. Therefore, if there is only a slight decline in total amount of a given soluble enzyme, this enzyme may be lowered in concentration in nuclei and raised in concentration in cytoplasm (when concentration is based on total dry weight). The findings of Stern and Mirsky (1952) in regard to glycolytic enzymes in plant cell nuclei are of interest. As previously noted, the presence of glycolytic enzymes has been reported by three other workers (Dounce, 1950a, 1952a; Lang and Siebert, 1951) in isolated cell nuclei, although in one case (Lang and Siebert, 1951) the amount of glycolysis was thought

ENZYME STUDIES ON ISOLATED CELL NUCLEI

207

too slight to be of metabolic significance. Thus far, hexokinase has not been found in cell nuclei, and if it is really absent, the glycolytic system of nuclei must be considered as incomplete. Stern and Mirsky have concluded that the final step in glycolysis may also be lacking in nuclei and believe that D P N reduced in the early stages of glycolysis is probably transferred to the cytoplasm before it is reoxidized, since it was felt that otherwise large amounts of lactate would accumulate. However, these authors did not investigate lactic dehydrogenase, an enzyme which Dounce has found in reasonably high concentration in rat liver cell nuclei isolated at p H 6.0 (Dounce, 1950a, b). If D P N which has been reduced in nuclei is reoxidized in cytoplasm, it would seem most likely that such reoxidation should occur within the mitochondria ; but it is doubtful whether mitochondria would be permeable to DPN. I t seems more likely that the DPN-dependent mitochondria1 enzymes act in conjunction with intramitochondrial DPN which cannot escape to the outside. I t would seem preferable to assume that glycolysis in nuclei proceeds to the lactate stage, and that lactate then diffuses out to the cytoplasm, where it is oxidized. We know that in the case of liver, for example, lactate is readily utilized, undoubtedly by cytoplasmic enzyme systems, and there seems therefore to be no particular reason to advance the argument that if glycolysis were complete in nuclei, large quantities of lactate should accumulate. Furthermore, there seems to be no more reason for assuming the accumulation of pyruvate from nuclear metabolism than of lactate from the same source. If we accept the hypothesis of intranuclear glycolysis, in favor of which there is now some experimental evidence, the question arises as to the possible biologic function of such glycolysis. Is glycolysis within nuclei necessary to furnish energy for intranuclear protein and nucleic acid synthesis, or is it necessary to furnish certain intermediate metabolites needed for special intranuclear metabolism ? Or, finally, is the occurrence of intranuclear glycolytic enzymes without functional significance relative to nuclear metabolism? This problem has already been discussed by the author, and only one additional thought will be added here. Recent work by Racker ( 1952) has apparently demonstrated that deoxyribose can be synthesized by the action of an enzyme called DR aldolase on the substrates 3-phosphoglyceraldehyde and acetaldehyde. The suggestion has been made by Dr. Stotz of this department in a private conversation that intranuclear glycolysis might, in addition to furnishing a source of intranuclear energy, be necessary to furnish the 3-phosphoglyceraldehyde apparently needed for the synthesis of 2-deoxyribose. The validity of this suggestion will depend upon future localization of the site of synthesis of 2-deoxyribose.

208

ALEXANDER L. DOUNCE

As concluding remarks concerning the work of Mirsky et al. with the Behrens-type nuclei, it can be stated that the results are challenging from the standpoint of experimentation and represent a definite advance in the struggle to determine the metabolic role of the nucleus in the resting cell. The task of determining the amount and kind of extranuclear contamination in various instances no doubt will gradually be accomplished so that the fullest possible significance can be ascribed to the results obtained. The disagreements in interpretations between Mirsky et al. and the author are only of transitory significance, since they will ultimately be resolved by further experimentation. 111. WORKOF LANGAND COLLABORATORS Another approach to the problem of metabolic function of the cell nucleus has been made by Lang and his colleagues (Lang, Siebert, et d., 1950; Lang and Siebert, 1950; Lang, Siebert, et al., 1951; Siebert, Lang, and h n g , I951; Ling and Siebert, 1951; 1952 ; Lang, Siebert, and Lucius, 1952; Siebert, Lang, et al., 1953; Lang, Siebert, and Fischer, 1953), who also have used isolated cell nuclei for enzyme studies as well as for some other work, such as a study of the distribution of metals between nucleus and cytoplasm. I n this work, the nuclei were isolated by differential centrifugation in strong sucrose solution. The main innovation was the use of a special mechanically-operated homogenizer of a new design which was said to break the cells without damaging the nuclei. The tissue was subjected to preliminary homogenization in a ground-glass homogenizer which was an elaboration of the device first described by Hagan (1922) and later reinvented in a slightly modified form and applied to tissue homogenization by Potter and Elvehjem (1936). The mechanical homogenizer through which the suspension subsequently passed has an outside member, consisting of a slightly tapered steel cylinder, and an inside member, consisting of a solid polished steel cylinder of the same taper as the outside piece. The inside piece was rotated by means of a heavy-duty motor. The machining of outside and inside members of the homogenizer was very accurate, and the spacing was adjustable. The homogenizer was jacketed so that it could be kept at 0"C. by an ice-water bath. The previously homogenized material was run in to the top of the homogenizer and was fed through by gravity alone. Forty per cent sucrose was used as suspending fluid. The rate of rotation of the inner member of the homogenizer was slow enough (1,400 r.p.m.) so that laminar flow was always maintained. In photographs the isolated nuclei look very clean, but too few nuclei are included to enable one

ENZYME STUDIES ON ISOLATED CELL NUCLEI

209

to judge of the quality of the preparations from inspection of the photograph. Some of the results obtained by Lang and co-workers from a study of nuclei obtained by their procedure will now be stated briefly. (1) Enzymes of oxidation (with the exception of certain dehydrogenases) were said to be entirely absent from the nuclei. For example xanthine oxidase, L-amino oxidase, L-proline oxidase, D-amino oxidase, and succinoxidase could not be found in nuclei isolated from rat liver or pig kidney. Flavin adenine dinucleotide was added when testing D- and L-amino oxidase, but without effect. (Cf. results of T. H. Lan, who found high concentrations of uricase in liver cell nuclei isolated in aqueous media at pH 6.0. This finding, which is probably the result of. adsorption, will be discussed later.) (2) Certain hydrolytic enzymes were found in the nuclei in high concentrations. For example, pig and calf kidney nuclei contained nearly all of the DNA-ase of the tissue (nothing was said, however, of the possibility that inhibitors might be present in cytoplasm), and these nuclei were also said to be rich in cathepsins. Phosphatase was mentioned as being present. Arginase was found to be present in rat liver nuclei in about the same concentration as in the cytoplasm; but, contrary to the results obtained by Dounce and Beyer (1948), the nuclear arginase could be activated by the addition of manganese. ATP-ase was found in high concentrations in nuclei isolated from the livers and kidneys of rats and pigs. (3) Glycolysis was studied using nuclei obtained from pig kidney. It was possible to detect an anaerobic “glycolysis” starting from fructose diphosphate, but the observed rate was rather small. No aerobic glycolysis at all could be measured. In this work the presumed formation of lactate was followed gasometrically through COZ evolution. It was necessary to remove sugar by dialysis in order to obtain measurable glycolysis. The apoenzymes of 3-phosphoglyceraldehyde dehydrogenase and lactic dehydrogenase were found in the nuclei, and the activity of these apoenzymes was not increased by the addition of kochsaft. However, in spite of the fact that Thunberg technique with methylene blue was used, no diaphorase seems to have been added, so that the latter findings appear to have little quantitative significance. Other workers, including the author in an early investigation, have also failed to add diaphorase in testing for the dehydrogenases of isolated cell nuclei by means of methylene blue decolorization. Lang and co-workers believe that the apoenzymes of glycolysis (with the exception of one or more enzymes above the fructose diphosphate level) ’

210

ALEXANDER L. DOUNCE

are all present in cell nuclei, because of the finding of anaerobic glycolysis, but they believe that, quantitatively, glycolysis is probably insufficient to fulfill the energy requirements of the nuclei. They therefore conclude that the most likely source of energy in the cell nucleus is the splitting of ATP. The latter conclusion might seem inconsistent with the failure of Lang et al. to find phosphate esters or high energy phosphates within the cell nuclei, since these authors previously expressed the belief that many types of organic molecules, as well as protein, are mainly retained by the nuclei during the isolation procedure. This belief, however, is not founded on a single shred of real evidence, in the opinion of the writer, but is arrived at by unjustified reasoning based on failure to extract material from the already isolated nuclei. But in spite of the fact that failure to find phosphate esters in the isolated nuclei does not constitute valid objection to the hypothesis of energy production by A T P hydrolysis, there still does not seem to be any justification whatsoever for such an hypothesis. ATP generally transfers energy through reactions in which high energy phosphate is first transferred from the A T P to other molecules, and then inorganic phosphate is split off during the process of molecular condensations. The latter process seldom, if ever, has anything to do with ATP-aso activity, with the possible exception of the case of muscle contraction. It should be noticed that Lang et al. have really not proved that complete glycolysis can occur with their isolated nuclei, but only that acid can be produced by the action of the nuclei on fructose diphosphate. Nevertheless, in a general way their work is in agreement with that of the writer (Dounce, 195Oa) and with that of Stern and Mirsky (1952), since at least some of the apoenzymes of glycolysis have been found in isolated nuclei. The quantitative aspects of the work appear to be so weak, however, that little basis is furnished for deciding whether glycolysis is or is not an important energy-yielding mechanism in the cell nucleus, largely because severe losses of soluble enzymes may have occurred during isolation of the nuclei. In two very recent papers (Siebert, Lang, at al., 1953.; Lang, Siebert, and Fischer, 1953), Lang and collaborators have made an extensive study of proteolytic enzymes and enzymes concerned with amino acid metabolism. Cell nuclei (generally kidney or liver) were found to contain transaminase, glutamic acid dehydrogenase, glutathionase, and peptidases for L-leucylglycine and glycylglycine. I t was noticed that in kidney, a D-peptidase for D-leucylglycine was low in concentration in nuclei compared with the concentration in cytoplasm. Histidase, transpeptidases, and folic acid conjugase were not found in appreciable quantities in any nuclei studied.

ENZYME STUDIES ON ISOLATED CELL NUCLEI

211

No peptide synthesis was observed as the result of incubating nuclei with amino acids and ATP. I t should be noted that, in testing for glutamic dehydrogenase, it was necessary to add large quantities of DPN. This is not surprising, in view of the fact that no diaphorase appears to have been added. The reaction between methylene blue and DPN is very slow indeed in the absence of diaphorase, and cell nuclei prepared in our laboratory, at least, have always been very deficient in flavoprotein. In a study of pancreas nuclei, Lang et al. find that the concentration of trypsin in the nuclei is higher than in the cytoplasm. The nuclear trypsin was enhanced in activity by enterokinase. Catheptic activity was also very easily measurable in the isolated nuclei. The activity of the nuclear cathepsin was enhanced by cyanide. (Cf. work of Miller, Dounce, et al. on nuclear cathepsin. See Dounce, 1952a.) The statement is made that the trypsin of nuclei isolated from pancreas could not have arisen from adsorption of soluble cytoplasmic trypsin, since in this event the activity of fivefold-washed nuclei would have been less, although less than what is not clear. This statement is similar to previous statements made by Lang and collaborators regarding loss of material by the nuclei and seems to ignore the possibility that such loss or gain might occur in the earliest stages of the isolation procedure. The argument is also made that the nuclear trypsin could not be the result of an admixture of cytoplasmic particles, since subjecting the nuclei to quite slow centrifugation, which would remove such cytoplasmic particles, did not cause a lowering of tryptic activity, and since microscopic observation did not show contaminating particles in the nuclei. This argument is good as far as it goes, but the possibility of adsorption of microscopically invisible fine pieces of broken cytoplasmic particles seems to have been overlooked. This sort of adsorption evidently occurs when liver ceIl nuclei are prepared at pH 6.0 in very dilute citric acid, and causes contamination of the nuclei with cytochrome oxidase. The phenomenon will be discussed in more detail shortly. As concluding remarks concerning the work of Lang et d., it should be stated that these workers have recognized certain difficulties in the experimental techniques commonly used for isolating cell nuclei and have apparently succeeded in overcoming certain of these, mainly by the use of an improved technique for homogenization. They have made a serious effort to attack experinientafly certain fundamental problems concerned with the metabolism of the cell nucleus, and the writer wishes to emphasize that only in matters of interpretation is there disagreement between h n g et al. and himself. Such disagreements are inevitable, especially in the

212

ALEXANDER L. DOUNCE

early stages of scientific investigations. Work on cell nuclei is certainly in the early stages as far as basic understanding is concerned, in spite of the length of time that research of this sort has been going on.

IV. RECENTWORKOF HOGEBOOM A N D SCHNEIDER ON SYNTHESIS OF DPN BY NUCLEAR PREPARATIONS It was suggested by Brachet (1951a) that some of the effects of the nucleus on the cytoplasm might be mediated by control of coenzyme synthesis by the nucleus. Later, Hogeboom and Schneider (1952) claimed to have shown that practically all of the enzyme involved in synthesis of DPN according to the following reaction: NMN* -4- ATP DPN pyrophosphate

+

was recoverable in the nuclear fraction. The concentration of this enzyme in the nuclei was five to six times greater than its concentration in the whole homogenate, and it was found that at least 85% of the enzyme could be brought into solution from the nuclei by means of the sonic oscillator. The latter finding was taken to indicate that the nuclear membrane must be impermeable to the enzyme in question. This work was claimed by the authors as the first clear-cut demonstration of a nuclear enzyme, in spite of the amount of work on nuclear enzymes previously reported in the literature. This statement seems somewhat extravagant. Without going into a detailed comparison of this work with previous studies, one possible theoretical difficulty will be mentioned which would follow from the restriction of DPN synthesis to the cell nucleus. If the nucleus is the sole site of DPN synthesis in the cell, it is hard to explain the presence of DPN in mitochondria, considering the apparent impermeability of mitochondria to rather small molecules, such as sucrose, for instance. Nevertheless, mitochondria must depend on DPN to carry out certain reactions, such as the dehydrogenation of P-hydroxybutyric acid, for example, and one might therefore expect synthesis of DPN within the mitochondria. A possible experimental difficulty with this work lies in failure of the authors to show whether they could obtain recovery of DPN added to the various cytoplasmic fractions under the conditions of the assay method which was used. Moreover, the absorption spectrum of reduced DPN was not shown as a proof that DPN was actually synthesized by'the action of the nuclear enzyme, but instead the absorption was reported only at one point in the spectrum. The last objection may be a trifle *Nicotinamide mononucleotide.

E N Z Y M E STUDIES O N ISOLATED CELL NUCLEI

213

weak, but the former at least must be satisfied before the claim can be substantiated that an insignificant amount of the DPN-synthesizing enzyme occurs in the cytoplasmic fractions. The liberation of the DPN-synthesizing enzyme from nuclei by the action of the sonic oscillator would seem to point very definitely to the occurrence of the enzyme zerithin the nuclei. But from this evidence alone it could not be safely concluded that the nuclear membrane in vivo is not permeable to this enzyme, in view of the possibility that permeability of the membrane may be lessened by the addition of calcium chloride to the homogenizing medium, or that the calcium might act in such a manner as to bind the enzyme firmly to the nuclear material. Thus the evidence of Hogeboom and Schneider that the nuclear membrane is not permeable to at least one soluble enzyme is not yet conclusive.

V. THEPROBLEM OF OXIDATIVE ENZYMES IN CELL NUCLEI One of the problems concerned with enzyme systems of cell nuclei is whether the nuclei contain any oxidative enzymes. This problem does not have a direct bearing on nuclear membrane permeability but is of importance in connection with adsorption of cytoplasmic material by the nuclei. The author (Dounce, 1943) originally found cytochrome oxidase in apparently significant concentrations in liver cell nuclei isolated with citric acid at p H 6.0, and this observation was subsequently confirmed (Dounce, 195Oa, b ) . In spite of this finding, little or no succinic dehydrogenase activity could be measured, using suspensions of the same nuclei. The absence of succinic dehydrogenase correlated with the observed absence of contaminating whole mitochondria, and hence the absence of succinic dehydrogenase was taken as a possible criterion for the absence of contaminating cytoplasmic particles in the nuclei. Schneider (1946a, b) claimed that cytochrome oxidase, as well as succinic dehydrogenase, was not a true nuclear constituent, on the basis of works with nuclear fractions isolated from sucrose homogenates. However, their nuclear fractions contained both cytochrome oxidase and succinic dehydrogenase in concentrations approaching the corresponding concentrations in whole tissue. The basis for the conclusion that these enzymes did not occur in nuclei was the argument that enzymes should not be considered as belonging to a given type of intracellular particulate if only a relatively small fraction of the totaE enzyme of the cell were found in the particulate in question (Schneider and Hogeboom, 1951). This point of view was attacked by the writer (Dounce, 1951) who pointed out that the use of such a criterion would preclude the possibility of finding a given biochemical constituent in nucleoli if this constituent

214

ALEXANDER L. WUNCE

occurred anywhere else in the cell, since the nucleoli constitute an extremely small fraction of the total cell volume, and therefore could not contain a very appreciable fraction of the total amount of any constituent that occurred in similar concentrations in other parts of the cell. The conclusions of Hogeboom et al. were also attacked on the grounds that their nuclear fractions were so grossly contaminated with microscopically visible impurities that conclusions concerning the enzymatic composition of the nuclei could not safely be drawn. Recently Hogeboom, Schneider, and Striebich ( 1952) have developed an improved small-scale method for isolating nuclei of far better quality than that of their original nuclear fractions. Even these nuclei could stand some improvement, however, according to our experience with the method. The chief basis of the new method was the use of calcium chloride as an agent to prevent agglutination of the nuclei, as recommended by Schneider and Petermann (1950). Trouble from contamination by erythrocytes was avoided by perfusing the liver, first with physiologic saline and then with calcium chloride-sucrose solutions. When nuclei prepared as just described were analyzed by Hogeboom and Schneider for cytochrome oxidase, it was found that the concentration of this enzyme was only 10% that of its concentration in the homogenate, and, moreover, in a set of experiments the cytochrome oxidase concentration in the nuclei was found to correlate almost perfectly with the concentration of residual mitochondria which could not be removed from the nuclei. Similar results were obtained with the enzyme uricase, which Lan had found in very high concentration in nuclei of liver cells prepared at pH 6.0 by the use of the Waring Blendor and very dilute citric acid. Following the publication of the work just described, wet have spent a considerable length of time in an attempt to confirm or refute the results and to settle once and for all in our own minds at least the question of whether any cytochrome oxidase belongs to the cell nucleus. W e have finally come to the conclusion that Hogeboom et al. are probably correct in their assertion that cytochrome oxidase is not a nuclear constituent. We have also been able to discover the probable reason for finding cytochrome oxidase in considerable concentration in nuclei isolated at pH 6.0 with very dilute citric acid. We first isolated nuclei according to the procedure of Hogeboom et al. Using the Potter and Elvehjem modification of the Hagan homogenizer, considerable trouble was encountered with residual whole cells. Further?This work was carried out by Mrs. published previously.

s.

G. M. Pate and the writer, and has not

ENZYME STUDIES ON ISOLATED CELL NUCLEI

215

more, as the purification proceeded, small particles became increasingly difficult to remove. Nevertheless reasonably satisfactory nuclei were obtained, but in such small quantities that analysis for enzymes was not at the time attempted. Next, many large-scale experiments were carried out in which liver was homogenized by means of the colloid mill or the Waring Blendor, the nuclei being subsequently isolated at pH 5.9 by differential centrifugation in water or in l or 2% gum arabic solution (Dounce and Litt, 1952). I t was immediately noticed that the use of gum arabic resulted in a marked decrease in the color of the nuclei compared with the color of those isolated with water as the suspending medium. This loss in color was particularly marked when 2% gum arabic was used. It was found that homogenization in gum was desirable as far as obtaining a product of little color was concerned, but undesirable in regard to yield. Homogenization was most easily accomplished using water with the p H adjusted to 5.9. Gum arabic solution was then added for all subsequent centrifugation. The decrease in the reddish brown color of nuclei brought about by the use of gum arabic in the suspending medium suggested to us that the gum might be displacing colored material adsorbed on the nuclear surface, and that insoluble enzymes adsorbed in the form of very finely divided particulate matter might also be similarly displaced. I t was found that to a certain extent the latter supposition was true, since the concentration of cytochrome oxidase in nuclei prepared with gum arabic as suspending medium occasionally was found to be only 10% of the corresponding concentration in the whole homogenate. Using distilled water alone as suspending medium in the large-scale isolations just described, the nuclear concentration of this enzyme is usually greater than 60% that of the concentration in whole tissue, and sometimes equal to the latter concentration. I t was not always possible, however, to achieve a very marked lowering of cytochrome oxidase concentration in nuclei isolated with gum arabic, but the results obtained tended definitely in this direction. The use of the Waring Blendor for obtaining homogenates from which nuclei are to be isolated has been severely criticized on the grounds that the violent action of the blades tends to disrupt the nuclei. The same sort of criticism could be applied to the colloid mill, to a somewhat lesser degree. However, the fact remains that both the Waring Blendor and the colloid mill, if run at the proper speeds and with the p H of the suspension adjusted to the proper value, are capable of producing suspensions of nuclei containing so few whole cells that subsequent isolation of the nuclei by differential centrifugation is very satisfactory,

216

ALEXANDER L. DOUNCE

On the other hand, use of the Potter-Elvehjem modification of the Hagan homogenizer invariably leaves so many unbroken whole cells that subsequent isolation of the nuclei by differential centrifugation is not satisfactory, since it is very difficult or impossible to separate nuclei from appreciable quantities of whole cells by differential centrifugation procedures. This situation led Lang and co-workers to produce the homogenizer described earlier in this paper, but this apparatus requires precision machining and therefore is expensive. We have lately devised a ground glass homogenizer which seems to fulfill the requirements for breaking virtually all of the whole cells in a liver homogenate without subjecting the nuclei to violent conditions. Moreover this homogenizer permits the isolation of mitochondria in at least as good a condition as has been possible to obtain previously, provided of course that the proper suspending medium is used, The homogenizer, which consists of a glass ball on the end of a heavy glass rod that is worked up and down by hand in a ground cylinder, will be described elsewhere. With this new type of homogenizer it is possible to obtain nuclei in a variety of aqueous suspending media, since no special precautions such as lowering of the pH need be taken to avoid breaking the nuclei themselves. Plain water, water with sufficient citric acid to adjust the pH to values between 6 and 7, sucrose, sucrose-calcium chloride, gum arabic solution adjusted to various p H values, and strong ethylene glycol (final concentration about 70%) all have been used to produce homogenates containing large numbers of free nuclei with few or no whole cells. As a rule, if agglutination of the nuclei is to be avoided the p H must be lowered at least to 6.2, or else very dilute calcium chloride must be introduced into the medium. However if ethylene glycol is used in a final concentration of 70%, no agglutination occurs, although the nuclei swell considerably. In regard to the condition of the mitochondria, it can be stated that in all cases where the medium is hypotonic the mitochondria are damaged, or more generally they disintegrate completely. Complete disintegration of the mitochondria makes it easy to obtain nuclei that are microscopically almost free from small particulate matter, but it must be admitted that removal of most or all of the mitochondria without damaging them would be highly desirable from the standpoint of obtaining nuclei uncontaminated with mitochondria1 enzymes. This statement applies both to the insoluble enzymes of mitochondria, such as cytochrome oxidase, succinic dehydrogenase, and uricase, and to the enzymes which may be brought into solution

ENZYME STUDIES ON ISOLATED CELL NUCLEI

217

by disrupting the mitochondria, such as acid phosphatase and malic dehydrogenase. We are at the present time working with the new homogenizer in an attempt to obtain nuclei which have been freed from mitochondria without disintegrating appreciable numbers of the latter. The problem of wholecell contamination no longer is troublesome, but instead of whole cells, the cell membranes described by Hogeboom et al. (1952) may cause a certain amount of trouble. The general procedure followed by us has been to homogenize in isotonic sucrose containing about 0.005 M CaClz (Schneider and Petermann, 1950; Hogeboom et al., 1952). The nuclei are then centrifuged after being underlaid with stronger sucrose solution (0.34 M ) which also contains a small concentration of CaCla (ca. 0.0002 M ). The nuclei are next washed twice in isotonic sucrose which must be free from calcium chloride, the pH having been adjusted to 6.2 after adding the sucrose, and finally they are stirred up and centrifuged twice in 1% gum arabic solution adjusted to p H 6.2. The latter solution removes red cells (by laking) and any residual mitochondria (by causing them to disintegrate). A refinement would be to perfuse the livers with saline and then sucrose (Hogeboom et d.,1952) before homogenizing. The method just described produces nuclei of excellent microscopic appearance, Nuclei obtained by procedures of the sort just described have been obtained with a cytochrome oxidase concentration as low as 10% that of the whole homogenate. I t is felt that eventually we may succeed in reducing the cytochrome oxidase content to a still lower value. The concept of surface adsorption of insoluble enzymes from broken mitochondria has been supported qualitatively by experiments, carried out by Mr. E. R. M. Kay (1953) of this laboratory, in which nuclei isolated in various ways were allowed to act upon 2,3,5-triphenyl tetrazolium chloride in the presence of buffered sodium succinate. In cases where succinic dehydrogenase activity was detectable, microscopic examination showed that the dye appeared on the surface of the nuclei but never in the interior. In working with succinic dehydrogenase, we have found that this enzyme is subject to a more rapid decay than is cytochrome oxidase, so that our early failure to find any succinic dehydrogenase in nuclei isolated with very dilute citric acid at p H 6.0 was undoubtedly attributable to this decay phenomenon, and did not, as originally thought, indicate that there are no mitochondria1 fragments in the preparation. Thus far we have not carried out new experiments with uricase, but it is likely that the same general type of results would occur as with succinic dehydrogenase or cytochrome oxidase.

218

ALEXANDER L. DOUNCE

VI. GENERALDISCUSSION Evidence is accumulating which indicates that the nuclear membrane may be quite permeable to ordinary organic molecules, and even to protein of at least as high molecular weight as hemoglobin. Certain evidence against this point of view, which has been discussed in this paper, is by no means conclusive. Some of the results of electron microscopic studies of the nuclear membrane are compatible with a highly permeable membrane but are, of course, not conclusive, owing to the possibility of fixation artifacts. If the nuclear membrane is really a relatively coarse structure, permeable to enzymes, then the question of intranuclear and extranuclear metabolism has to be viewed in a somewhat different light from what has been customary in the past. Intranuclear enzymes would include all of the enzymes present in soluble form in the cytoplasm, and the concentrations of such enzymes would be the same in the non-particulate soluble aqueous phase common to the nucleus and cytoplasm. Intramitochondrial enzymes could be, and as we now know are, quite different, in general, since mitochondria are known to be quite impermeable. Of course the possibility that some bound intranuclear enzymes might be found which would not occur in any part of the cytoplasm would have to be admitted (Cf. Hogeboom and Schneider, 1952). If intranuclear metabolism should be in any way different from metabolism going on in the soluble aqueous phase of cytoplasm, it would be different according to the hypothesis just presented mainly because of differences in bound or insoluble substrates (such as DNA or glycogen) or because of the unavailability of diffusible cytoplasmic substrates in the nucleus, If the hypothesis of cytoplasmic screening presented elsewhere by the author (Dounce, 1950a, b) is of any validity, it follows that a given soluble substrate present in cytoplasm may be present in nuclei only in much lower concentration. It can be pointed out here, in regard to the cytoplasmic screening hypothesis, that the one metabolite thus far found not to be synthesized at a relatively rapid rate in resting nuclei is DNA, and hence the point of application of screening must presumably be restricted to a substrate or substrates involved in the synthesis of DNA. This makes the problem of investigating cytoplasmic screening of substrate from the nucleus more susceptible to experimental investigation than previously, although there is still not enough known about DNA synthesis to make such investigation easy at the present time. What direction should future research follow to make possible a more rapid clarification of the biochemical role of the nucleus in the resting cell? The answer to this question will of cmrse depend upon the point

ENZYME STUDIES ON ISOLATED CELL NUCLEI

219

of view of the investigator. One obvious thing to do is to extend to other chromoproteins of higher molecular weight the work done with hemoglobin in connection with studies of permeability of the nuclear membrane. Work with single cells, such as the nuclear transplantations carried out by Danielli and others (Commandon and dc Fonbrune, 1939; Lorch and Danielli, 1950; Briggs and King, 1952) and the recent microdissection experiments of Brachet and collaborators (Brachet, 1950, 1951a, 1952; Brachet and Chantrenne, 1951 ; Chantrenne-van Halteren and Brachet, 1952 ; Urbani, 1952), Mazia and Hirshfield (1950), and Danielli and collaborators (Lorch, Danielli, and Horstadius, 1953 ; and Hijrstadius, Lorch, and Danielli, 1953), no doubt will be of paramount importance in elucidating nucleus-cytoplasm interactions and investigating general questions of importance such as the permeability of the nuclear membrane. f t is very doubtful whether isolation procedures could safely be relied upon to furnish unequivocal information without the checks that can be made through microdissection studies of single-celled organisms, and to some extent through histochemical studies. One of the most interesting recent findings concerned with nuclear metabolism is that the rate of turnover of nuclear RNA is appreciably higher than that of cytoplasmic RNA (Marshak and Calvert, 1949; Barnum and Huseby, 1950; Jeener and Szafarz, 1950; Hurlbert and Potter, 1952; Smellie and McIndoe, 1952; Smellie et al., 1953). This finding is particularly interesting when considered together with the drop in RNA synthesis that occurs in amoebas following denucleation (Brachet, 1950; Mazia and Hirshfield, 1950). The follow-up of this work should certainly throw considerable light on the metabolic role of the nucleus. It is the opinion of the writer that in the field of nuclear enzymes, the most useful information can be obtained from comparative studies on nuclei isolated by different methods and obtained from different tissues, as already has been emphasized (Dounce, 1952a, b). Correlation of such work with histochemical studies, whenever possible, is also highly desirable. This point of view has also been expressed by Behrens and Taubert (1953) in an article on a recent modification of Behrens’ technique for isolating nuclei. There are now enough different procedures available for isolating nuclei to make comparative investigations really worth while. These methods include isolations with very dilute citric acid at p H 6 or 4, isolations with isotonic sucrose containing calcium chloride, isolations with strongly hypertonic sucrose including the procedure of Lang and a recently described procedure of Krakaur, Graff, and Graff (1952), isolations with 70% ethylene glycol or glycerol, and various modifications of the Behrens’ technique.

220

ALEXANDER L. DOUNCE

We do not believe, as Mirsky apparently does, that all aqueous methods should be abandoned in favor of the Behrens’ type of procedure. Neither do we believe that the latest method of Hogeboom and Schneider produces such excellent nuclei that other methods are superfluous. In regard to the latter method, we wish to make the claim that these nuclei are not unaltered, since they show no gel formation with alkali or molar chloride. In this respect they are similar to most other nuclei prepared in aqueous media at pH 6 to 7, although chicken erythrocyte nuclei and nuclei made in glycerol or ethylene glycol at a final concentration of 70% constitute exceptions, since the latter do form gels. This gel formation has been discussed previously (Dounce, 1949, 1950b). Failure to obtain gel formation is undoubtedly caused by autolysis, either proteolytic or nucleolytic in nature, or both. If the hypothesis of a highly permeable nuclear membrane is correct, one might wonder why any soluble enzymes at all should be found in cell nuclei isolated in aqueous media. A reasonable answer is that more or less of the soluble enzyme protein of the nuclei is held as a complex with nucleic acid, chiefly with DNA. I t has previously been mentioned (Dounce, 1952a) that the DNA of cell nuclei probably has many or all of its phosphate groups free, and that the concept of nucleo-histone salt within living cell nuclei is very likely an artifact. It is probable that the phosphate groups of DNA are neutralized with alkali metals such as potassium, and that during isolation of the nuclei the alkali metal ions diffuse out of the nuclei faster than the protein, with the result than an interchange occurs between the alkali metal bound to the DNA by ionic forces and positively ionized groups on the protein, forming a DNAprotein complex of a salt type. Such an interchange should decrease in amount as the p H increases up to the point where the bulk of the soluble intranuclear protein becomes strongly anionic. However, since the isoelectric points of proteins are not all the same, certain proteins should be lost from the nuclei before others as the pH increases. Factors of molecular size and charge distribution would doubtless also be of importance in determining the relative binding of various proteins to DNA. In view of what has just been said, it would appear to the writer that much more time should be spent in designing experiments in an attempt to evaluate the significance of finding a given enzyme in cell nuclei, and that less time should be spent at present in assaying a given type of isolated nucleus for a large number of enzymes. The concept of a nuclear membrane permeable to protein is advantageous from the standpoint of explaining the mechanism of gene action. The one gene-one enzyme hypothesis of Beadle (1946 ; 194Sa, b, c) might be

E N Z Y h l E STUDIES ON ISOLATED CELL NUCLEI

22 1

subject to criticism on the grounds that an ayoenzyme may operate with a prosthetic group or coenzyme that would require the participation of a number of genes for its synthesis. But if the hypothesis is restated as the one gene-one protein hypothesis, it constitutes a very plausible and convincing general mechanism by which genes could act. If the hypothesis as restated is correct, then the nuclear membrane should be permeable either to protein or to large molecules, such as polypeptides or ribonucleic acid, which might be involved in the synthesis of protein. It is conceivable that the nuclear DNA might act by causing synthesis of RNA which then wouId pass out to the cytoplasm and function there in the synthesis of protein (Dounce, 1952~). The reported differences in composition between nuclear and cytoplasmic RNA (Crosbie, Smellie, and Davidson, 1953) do not necessarily rule out transfer of nuclear RNA to the cytoplasm, if independent RNA synthesis also occurs in cytoplasm. But if the nuclear membrane were permeable to RNA it would in all probability also be permeable to protein. The idea of simple diffusion is certainly more attractive to the writer than the idea of an “active” transport of these molecules through the membrane by the aid of some chemical process. It is hard to see, for instance, how phosphorylation could be helpful in transporting through the nuclear membrane a molecule as large as that of an average protein.

VII. SUMMARY AND CONCLUSIONS Certain problems concerned with the enzyme content of cell nuclei isolated by chemical procedures have been presented and discussed, and recent findings bearing on these problems have been analyzed. The point of view taken in this paper is in favor of a highly permeable nuclear membrane with probable diffusion through the nuclear membrane of molecules at least as large as those of certain proteins. Some evidence for and against such an hypothesis has been discussed and correlated with recent work on the enzymes found in isolated cell nuclei. The validity of recent conclusions of various authors concerning nuclear enzymes has been discussed, and two possible energy sources for nuclear metabolism have been considered in detail. Probable future trends in research concerned with cell nuclei have been discussed.

VIII. REFERENCES Allfrey, V., Stern, H., Mirsky, A. E., and Saetren, H. (1952) J. Physiol., 36, 559 Anderson, N. G. (1952) J. Tettrt. Acad. Sci., 27, 198. Anderson, N. G. (1953a) Science, 117, 517. Anderson, N. G. (1953b) Exptl. Cell Remmh, 4, 306. Bairati, A., and Lehmann, F. E. (1952) Experientia, 8, 60.

222

ALEXANDER L. DOUNCE

Barnum. C. P., and Huseby, R. A. (1950) Arch. Biochern., 29, 7. Beadle, G. 1%'. (1946) Am. Scientist, 34 31. Beadle, G. W. (1948a) Aw. Scientist, 36, 69. Beadle, G. W. (1948b) Physiol. RPJS., 26, 643. Beadle, G. 1%'. (1948c) Ann. Rev. Eiocknn., 17, 727. Behrens, If. (1939) 2. Physiol. Chem., 268, 27. Behrens, hf., and Taubert, M. (1953) 2. Physiol. Chem., 291, 213. Brachet, J. (1950) Expericwtia, 6, 294. Brachet, J. (1951a) Natnre, 168, 205. Brachet, J. (1951b) Ex&rierlrfiu, 7, 34-1. Brachet, J. (1952) Experientia, 8, 347. Brachet, J., and Chantrenne, H. (1951) Natrwe, 168, 950. Briggs, R., and King, T. J. (1952) Proc. NafL Acad. Sci. U.S., 38, 455. Callan, H.G., and Tomlin, S. G. (1950) Proc. Roy. SOC.(London), B,137, 367. Chantrenne-van Halteren, M. B., and Brachet, J. (1952) Arch. intern. Php-iol., 60. 187. Commandon, J., and deFonbrune, P. (1939) Compf. rend. SOC. b i d , 130, 740. Crosbie, G. W., Smellie, R. M. S., and Davidson, J. N. (1953) Biochent. J., 63, 287. Daly, M. &I., Allfrey, V. G., and Mirsky, A. E. (1952) J . Grn. Physiol., 36, 173. Dounce, A. L. (1943) J. Bliot. Ckem., 147, 685. Dounce, A. L. (1949) Science, 110, 442. Dounce, A. L. (195Oa) A m . N . Y . Acad. Sci., 60, 982. Dounce, A. L. (1950b) I n The Enzymes, Vol. 1, p. 187. Academic Press, New York. Dounce, A. L. (1951) Comer Research, 11, 562. Dounce, A. L. (1952a) The Enzymes of Isolated Nuclei in Chemistry atid Physiology of the Nucleus. Exptl. Cell. Rescarch, Suppl., 2, 103. Dounce, A. L. (195%) 1. Cell. Comp. PhySiol., 39, SzippL 2, 43. Dounce, A. L. (1952~)Exytnologiu, 16, 251. Dounce, A. L., and Beyer, G. T. (1948) J . Biot. Chent., 174, 859. Dounce, A. L., Kay, E. R. M., and Pate, S. G. h,l. (1953) Fcderation Proc., 12, 198. Dounce, A. L., and Lan, T. H. (1943) Scifitce, 97, 584. Dounce, A. L., and Litt, M. (1952) Federation Proc., 11, M3. Dounce, A. L., Tishkoff, G. H., Barnett, S. R., and Freer, R. M. (1950) J . Gen. Physiol., 33, 629. Hagan, W.A. (1922), J . Exptl. Itled., 36, 711. Harris, P.,and James, T. W. (1952) Expcricrrtia, 8, 384. Hogeboom, G. H., Schneider, W. C., and Striebich, M. J. (1952) J . Biol. Chem., 196, 111. Iiogeboom, G. H., and Schneider, W. C. (1952) 1.Biol. Chent., 197, 611. Horstadius, S.,Lorch, I. J., and Danielli, J. F. (1953) Exptl. Celt. Research, 4, 263. Hurlbert, R. B., and Potter, V. R. (1952) J . Biol. Che?~.,196, 257. Jeener, R.,and Szafarz, D. (1950) Arch. Riochcm., 26, 54. Kay, E. R. 11. (1953) Doctorate Thesis, University of Rochester School of Medicine and Dentistry. Krakaur, R., Graff, A. M., and Graff, S. (1952) Camer Research, 12, Proc., 276. Lang, K., and Siebert, G . (1950) Biochrm. Z., S O , 402. Lang, K., and Siebert, G. (1951) Biocherrr. Z., S22, 1%. Lang, K.,and Siebert, G. (19552) Biochem. Z., 322, 360. Lang, K.,Siebert, G., Baldus, I., and Corbet, A. (1950) E x p m P n f k 6, 59.

ENZYME STUDIES ON ISOLATED CELL NUCLEI

223

Lang, K., Siebert, G., and Fischer, F. (1953) Biochent. Z.,324, 1. Lang, K.,Siebert, G., and Lucius, S. (1952) Experientio, 8, 228. Lang, K., Siebert, G., Lucius, S., and Lang, H. (1951) Biochem. Z.,321, 538. Lorch, I. J., and Danielli, J. F. (1950) Nature, 166, 329. Lorch, I. J., Danielli, J. F., and Hiirstadius, S. (1953) Exgtl. Cell Research, 4, 253. Marshak, A., and Calvert, F. (1949) J. Cell. Cornp. Physiol., S4, 451. hfazia, D., and Hirshfield, H. I. (1950) Sciewce, 112, 297. Palade, G. Personal cQmmunication. Dr. Palade has found that the nuclear membrane of the mammalian liver cell has a double layer structure and in this respect is therefore similar to the nuclear membranes of amphibian eggs and amebas. Potter, V. R., and Elvehjem, C. A. (1936) J . Uiol. Chem., 114, 495. Racker, E. (1952) J . Bwl. Clzem., 196, 347. Schneider, R. M., and Petermann, M. L. (1950) Cancer Research, 10, 751. Schneider, W. C. (1946a) 1. Biol. Chpm,, 166, 585. Schneider, W. C. (1946b) Cancer Research, 6 , 685. Schneider, W. C., and Hogeboom, G. H. (1951) Cancer Research, 11, 1. Siebert, G.,Lang, H., and Lang, K. (1951) Biochmn. Z., 321, 543. Siebert, G., Lang, K., Miiller, L., Lucius, E., Miiller, E., and Kiihle, E. (1953) Biochem Z., 323, 532. Smellie, R. M. S., and McIndoe, W. M. (1952) Biochem. J., 62, Proc. XXII. Smellie, R. M. S., McIndoe, W. M., Logan, R., Davidson, J. N., and Dawson, I. M (1953) Biochem. J., 65, 280. Stern, H., and Mirsky, A. E. (1952) J . GPIZ.Physiol., 36, 181. Stern, H., Allfrey, V. G., Mirslry, A. E., and Saetren, H. (1952) J. Gen. Physiol., 36, 559. Thornson, R. Y., Heagy, F. C., Hutchinson, W. C., and Davidson, J. N. (1953) Biochem. J., 65, 460. Urbani, E. (1952) Arch. intern. Phy&Z., 60, 189. Vendrely, C . (1952) Bull. biol. Frame ef Belg., 86, 1. 0

This Page Intentionally Left Blank

The Use of Differential Centrifugation in the Study of Tissue Enzymes CHR. D E DUVE AND J. BERTHET* Labomfwy of Physiological Chemistry, University of Lowmin, Belgium

......................................................... The Technique of Differential Centrifugation ..........................

1. Introduction

11.

111.

1. Theoretical Considerations . ., .. .. . .. . . ... . . .. . .. , ... . . . . .. . . . . . . . . 2. Practical Procedures ............................................ . 3. Special Techniques ................. . ...... ......... . , ....... ..... Scope and Limitations of Differential Centrifugation as Revealed by Enzyme Distribution Studies . ...... .. . . .... ... . . .. .. . . .. ... ..... . . .. . . . 1. Summary of Factual Knowledge .... ................ .. ..... ....... 2. Validity of Fractionation Scheme ... ........... ........... .. . ... . 3. Efficiency of Separation of Particulate Fractions . ........... ....... 4. Artificial Redistribution of Enzymes .............................. 5. Multiplicity and Heterogeneity of Cytoplasmic Particles .. . ... . .. . 6. Artifacts Associated with Enzyme Assays . ..... . .................. 7. Truly Heterogeneous Enzyme Distributions .. . .. . ... .. .... ... ... . . . Biological Evaluation of the Results of Tissue Fractionation Studies . .. . 1. Hydrolytic Enzymes ... , . .. . . .. . . . . . . . . . . . . . . . . . . . .. . , . .. , . .. ... . 2. Cellular Oxidations ...... ....... , .. .. . . . . . .. .. . . . . . .... .. . . . ~.~. 3. Oxidative Phosphorylations . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .............. . . . ... . . . . . . .. . . . . . . . . . . . . . . . 4. Synthetic Processes 5. Permeability of Intracellular Bodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary and Conclusions .................... .... .. .. . . ..... . . .. ... References ...................... ............. . ... ... . . . . . . .. . . .. . .. . d

..

IV.

...

V. VI.

Page 225 226 227

231 238 239 240 240 241 245 250 253 258 259 260 261 262 265 267 269

no

I. INTRODUCTION+ Centrifugation has often been used in the past for the isolation of cellular constituents, particularly of nuclei, but it is only in recent years that techniques of complete fractionation have been introduced, thanks to the pioneer investigations of Bensley and Hoerr (1934), Claude (1938, 1941, 1943, 1946a, b), Brachet and Jeener (1944), and Hogeboom, Schneider, and Palade ( 1948). These techniques have been applied extensively during the last five years, and already several reviews have been devoted to the results which they have yielded (Bradfield, 1950; Claude, 1948, 1949; Dounce, 1950; deDuve, 1952; Hogeboom, 1951; Hogeboom, Schneider and Striebich, 1953 ; Holter, 1952 ; Lang, 1952 ; Potter, Recknagel, and Hurlbert, 1951 ; Schneider and Hogeboom, 1951).

* Chercheur

agrG de 1’Institut Interuniversitaire des Sciences Nucliaires. The following abbreviations have &en used in this review :-DNA : deoxyribonucleic acid : RNA : ribonucleic acid ; A T P : adenosine triphosphate ; ADP : adenosine diphosphate ; D P N : diphosphopyridine nucleotide (coenzyme I) ; T P N : triphosphopyridme nucleotide (coenzyme 11).

t

225

226

CPIR. DE DUKE AND J. BERTHET

In considering the amount of factual knowledge acquired so far by means of differential centrifugation, one is impressed both by the fundamental importance of the results achieved and by the remarkable possibilities offered by this new method. Some difficulties are becoming evident, however, and these must not be overlooked. The first are purely technical. Differential centrifugation is a delicate method, and small modifications in the procedures applied may in many cases alter quite significantly the manner in which a preparation is finally fractionated. Other difficulties concern the interpretation of the results obtained. Here again, technical details may be of paramount importance. In writing the present review, the authors have deemed it of particular interest to call attention to those facts which, in the experience of other workers and in their own, are likely to affect the successful performance of a centrifugal fractionation as well as the significance of the collected data. They have chosen to deal in a fairly detailed manner with the theoretical and practical aspects of the technique and to put greater emphasis on the problems that the use of differential centrifugation has raised than on those that it has helped to solve. The references are mostly illustrative and do not represent a complete survey of the present literature. They include unpublished data obtained recently in this laboratory.* OF DIFFERENTIAL CENTRIFUGATION 11. THETECHNIQUE

Since the publication in 1948 by Hogeboom, Schneider, and Palade of a complete scheme of fractionation in 0.58 M sucrose, and its adaptation by Schneider (1948) to 0.25 M sucrose, most authors have followed one of these two schemes, usually the latter. Both are similar in design to the original method of Claude (1946a, b) who established the principle of separating the cytoplasmic constituents into three fractions : 1. The krqe granules, sedimentable at low speed, which have been shown by Hogeboom, Schneider, and Palade (1948) to contain essentially mitochondria; 2. The small granules or microsomes, distinctly different from mitochondria and requiring high centrifugal fields to sediment completely ; 3. The unsedimentable constituents representing the final mpernatant or soluble fraction. In the new schemes, a fourth fraction containing the w Z e i is isolated

* A number of the results referred to in the text under Appelmans and de Dwc (unpublished), Appelrnans et at. (unpublished), and Gianetto and de Duve (unpublished) have been recently summarized in a note by de Buve, Gianetto, Appelmans, and Wattiaux (1953).

DIFFERENTIAL CENTRIFUGATION A N D ENZYMES

227

in a first step, and a complete honrogenate, representative of the whole tissue, is prepared initially and used as a standard of comparison. Great care is taken to work throughout on a quantitative basis, and a final check is introduced in the form of a recomzsfituted hoimgerzate, made up by recombining the four fractions in suitable amounts. As will be shown later, a rigid adherence to this basic pattern cannot be expected to answer all the problems that can theoretically be tackled by differential centrifugation. On the other hand, there are firm grounds for the belief that the scheme is essentially sound, from a cytologic point of view. In addition, whatever the advisability of introducing new methods at a later stage, the necessity remains that some uniform procedure should be followed as closely as possible in the initial steps of an investigation. Only in this manner can the important requisites be met of reproducibility and comparability between different laboratories. It is fortunate, in this respect, that most workers have followed similar schemes. However, the actual performance of a complete fractionation meets with a number of technical difficulties familiar to all workers in the field but not always solved in the same manner in individual laboratories. In addition, the theoretical basis of the technique is sometimes overlooked, and the description of the methods used is not always sufficient to ensure easy reproducibility in other laboratories. Both aspects will be examined here, in relation to the various steps of a complete fractionation according to Schneider (1948). 1. Theoretical Considerations

Fractionation by means of centrifugation is based on the fact that particles of different size or density sediment at different speeds in a field of centrifugal force. The conditions for centrifugation are chosen each time so as to cause complete sedimentation of the heavier particles with as little contaniination as possible by the lighter ones. a. General Forwiulas. The rate of sedimentation of a spherical particle in a gravitational field is given by the formula dx

-=

G 91 distance traveled along the direction of the field (in cm.), sec.), r the radius of the particle (in cm.),dp its density d , the density of the suspension medium (in g./cu.cm.), of the medium (in poises), and G the gravitational field dt

where z is the b the time (in (in g./cu.cm.), '1 the viscosity (in crn./sxz).

29 ( d p - - d m )

228

CHR. DE D W E A N D J. BERTRET

When the field is one of centrifugal force, it is directed outwards and radially with respect to the rotation axis and equals 4 2 (r.p.m.) G = W'X = x (2) 3,600 where w is the angular velocity (in radians/sec.), r.p.m. the number of revolutions per minute, and x the radial distance between the axis of rotation and the point at which the field is measured (in cm.). By combining equations 1 and 2 and integrating, one finds f 2-

2ra (d,--dm)

In- = -r O

(3)

ddt

-

91 0

where xo and x represent the positions of the particle measured radially from the axis of rotation, at times 0 and t respectively. Since one usually aims at complete sedimentation, the most interesting form of equation 3 is written: ,t Rmax. 2r2 (dp-dm) -ln~ dt (4) Rmin. 91

/ 0

in which R m a X . is the maximal radius (radial distance between the axis and the bottom of the tube) and Rmfn.the minimal radius (radial distance between the axis and the upper layer of fluid during centrifugation). This equation describes the relation between the variables which affect sedimentation for a particle which is present at the top of the tube when the centrifugation is started and which has just reached the bottom of the tube when the centrifugation is stopped. Obviously, all particles of the same type must then have reached the bottom of the tube and the formula therefore applies to complete sedimentation. A useful form of formula 4 is the following:

T

0

in which the time T is expressed in minutes and g,,. represents the average gravitational field, expressed in terms of the gravity constant, prevailing in the middle of the centrifuge tube, i.e., at a point measured by the average radius Rav.= 0.5 ( R m a r . Rmin.) :

+

DIFFERENTIAL CENTRIFUGATION AND ENZYMES

229

Rnv. 4 2 (r.p.m.)2 RaT. - = 11.1787 X 10° (r.p.m.)a lLaV. 981 3,W X 981 (6 )

o2

gnv.

=

_ I _

These problems have been discussed in a recent paper (de Duve and Berthet, 1953), in which it has been suggested, as an alternative, that the conditions of centrifugation be described by the sedimentation constant s of the lightest spherical particles which are sedimented completely :

b. Types of Centrifuges. The preceding formulas are applicable only if sedimentation occurs along the direction of the field, as it does, for instance, in a centrifuge equipped with a horizontal yoke allowing the tubes to swing out radially during operation. Such centrifuges are rarely used in fractionation, which requires high rotational speeds, obtainable only with conical heads in which the tubes are placed radially but are inclined at a fixed angle with respect to the horizontal plane.* In such heads, sedimentation starts out horizontally but proceeds further along the walls of the tubes. Fortunately, it has been shown by Pickels (1943) that an inclined tube may be treated for most practical purposes as a horizontal tube with R,,,. and R,,,, equal to the radial distances which separate the top and bottom of the fluid layer from the axis. Hence the formulas given above may be taken to apply to all types of centrifuges, and it follows that the most important factors dependent on the type of instrument used are the values R,,,. and &in., the latter being also dependent on the degree of filling of the tubes. c. Particles and Medium. The factors affecting the rate of sedimentation in a given field are the size and density of the particle and the density and viscosity of the suspension medium. In addition, one has to take into account the shape of the particle, the formulas given being applicable only to spherical particles. However, only pronounced asymmetry will modify the rate of sedimentation appreciably, and in most cases the assumption of sphericity may be made without undue error.

* A horizontal rotor of tne “swing out” type has recently been developed for high-speed centrifugation by the Spinco Company (Belmont, California).

230

CHR. DE DUVE A N D J. BERTHET

In a given medium, the governing factor is represented by the product (dp-dm). Hence, it is theoretically possible for smaller particles with higher density to have the same Sedimentation rate as larger particles with a lower density. In fact, the smaller microsomes have k e n shown by Holter et al. (1953) to have a density range significantly higher than that of the mitochondria (1.25 to 1.30 against 1.10 to 1.20). This difference does not seem to be large enough to compensate for the large difference in size but could render separation more difficult in hypertonic than in isotonic solutions. However, the hypertonicity may cause the removal of more water from the larger granules than from the smaller ones, thus reducing the density difference. There are, at least, no indications from the data published in the literature that changes in the tonicity of the medium affect the efficiency of separation. d . Field and Time. In all the above equations, the total force applied has been purposely represented by the time integral of the field or of the squared angular velocity. With relatively slow centrifuges, the time taken for acceleration and deceleration is generally small with respect to the time during which a constant field is applied, and it is justifiable to describe the centrifugation in terms of the latter two variables. Such is not the case with fast centrifuges. With these, adequate sedimentation can often be obtained by mere up-and-down runs lasting a few minutes, with very short periods at plateau speed. The field developed during acceleration and deceleration then represents the dominant factor, and complete integration becomes necessary. If the instrument is equipped with a revolution meter, a chart giving the value of the integral for different speeds can easily be constructed. The total force applied is then best expressed in terms of a composite unit g-min., or, alternatively, as the equivalent of x minutes at y g . It must be further noted that field and time are not the only factors affecting sedimentation but that the final result is also influenced by the values of R,,,. and Rmin.. Hence the latter must be mentioned, in addition to the time integral of the average field or of the squared angular velocity, to ensure complete reproducibility of the experiment. e. Eficicncy of Scparatiori. Formulas 4, 5, and 6 give directly the total force just necessary to ensure complete sedimentation of a given popidation of particles. Obviously, if centrifugation is stopped at this moment, some of the lighter particles will also have reached the bottom of the tube and will contaminate the precipitate. The importance of this contamination is worth emphasizing. For instance, it can be calculated that, with a centrifuge having R,,,.= 10 and Rm10.=5, the contamination of the precipitate by particles having a value of t.2(dp--dm) equal to oney2

DIFFERENT IAI. CENTRIFUGATION A N D ENZYMES

231

third that of the heavier particles is already 40% at the moment when complete sedimentation of the heavier particles occurs. Even with particles for which this value is one-tenth that of the heavier ones, the contamination would still be 13%. Such values are of course minimal, since field-time integrals slightly larger than the theoretical values are generally chosen in order to allow for a certain packing of the sediment. It may be concluded from this that, unless the sedimentation properties of the populations to be separated are very different, gross coutatnination of the first sediments is unavoidable. In other words, careful washing of the precipitates is essential. This necessity greatly limits the number of fractions which can be isolated under good conditions, for each washing requires time and causes increased dilution of the subsequent fractions. Another point worth mentioning is that even the lighter particles which have not yet reached the bottom of the tube have nevertheless started moving toward the lower fluid layers, Therefore, the manner in which decantation is performed may affect the composition of the fractions in a decisive way, especially with poorly packed precipitates such as are obtained when very low centrifugal forces are used to separate the larger cytoplasmic granules. Finally, it must be remembered that, although both size and density determine the rate at which particles sediment, density remains the only factor affecting the position of the completely sedimented particles within the precipitate. Therefore the upper layer of the sediment tends to become enriched in less dense particles, whatever their size, as the time-field integral increases with respect to its minimum value.

2. Practical Procedures a. General Precautions. Differential centrifugation has been applied successfully to a number of different organs and, although some tissues are less suitable than others owing to their heterogeneous cellular content or tough connective framework, the indications are that approximately the same techniques can be used with most types of biologic material. However, the main experience so far has been gained on the liver of small rodents, and this discussion will be limited to the technique as applied to this particular tissue. Unless otherwise indicated, it is preferable to work with animals subjected to a short fast of approximately 12 hours. A low glycogen content is thereby ensured, without too great a rise of the fat content. In addition, the main precautions which should be observed are to work throughout at a temperature as near 0" C. as possible (without, however, going below this limit) and as rapidly as possible, in order to minimize

232

CHR. DE DUVE A N D J . BERTHET

the artifacts resulting from autolysis, agglutination, and other changes occurring in the homogenate. b. The Honzogmate. To be suitable for centrifugal fractionation, the homogenate should (a) be free of extraneous elements such as blood and connective tissue; (b) contain all the particulate components of the tissue’s cells in a state of morphologic and ch,emical integrity, as well as in one of perfect division from each other and from the soluble components. Contamination by extraneous elements can be minimized by a preliminary perfusion of the organ and by pressing the organ through a 1-mm. screen mesh to separate the parenchymatous pulp from the connective and vascular framework (Claude, 194Ga). Many workers omit these steps. The measure in which the second set of conditions is met depends essentially on the type of device used for grinding the tissue and on the composition of the suspension medium. (1) GRINDINGDEVICE. Absolute preference should be given to homogenizers of the type described by Potter and Elvehjem (1936). Simple rubbing in a mortar, as recommended by Claude (1946a), disrupts only a fraction of the cells, and mechanical choppers such as the Waring Blendor cause excessive damage to the particulate components of the cells (Berthet et d.,1951 ; Gianetto and de Duve, unpublished ; Hogeboom, 1951;Potter, Recknagel, and Hurlbert, 1951; Schneider and Potter, 1949; Walker, 1952 ; Wilbur and Anderson, 1951). The Potter-Elvehjem instrument is, of course, not entirely free of these drawbacks and should be used with discrimination. In recent years, there has been a tendency to replace the original all-glass model with smooth-walled glass tubes fitted with. resistant plastic pestles. The latter do not lose their fit as quickly as the former and do not release powdered glass during operation. Lang and Siebert (1952) have recently described two new types of pestle-homogenizers, allowing a constant flow of material. The first operates in closed circuit and is used for coarse pregrinding. The latter is a precision mill made of stainless steel. Both instruments were specially designed for the isolation of nuclei. A good technique, whereby complete disruption of the cells can be ensured with a minimum of damage to the intracellular bodies, is to combine the grinding with the isolation of the nuclei. The tissue is first homogenized with a small quantity of fluid during a short time, insufficient to cause complete breakage of the cellular structures. The resulting suspension is centrifuged at a speed a little higher than is necessary to cause complete sedimentation of the nuclei, and the supernatant is decanted. The precipitate is then rehomogenized in a new quantity of

DIFFERENTIAL CENTRIFUGATION AND ENZYMES

233

fluid and recentrifuged, this time at the exact speed required for the nuclei, and the same procedure is repeated once more. When these operations are properly conducted, the final precipitate is practically free of unbroken cells and contains all the nuclei, with rather less than their usual inevitable contamination by cytoplasmic material. The three supernatants, recombined, contain most of the cytoplasmic material and are ready for further fractionation. The advantage of this procedure is that the fragile cytoplasmic particles are removed from the homogenizer almost as soon as they are released from the cells and are therefore not subjected to excessive mechanical strain or to prolonged contact with the nuclei, with which they tend to agglutinate. At the same time, complete homogenization of the tissue is ensured and time is gained. The procedure has one drawback, namely that no really complete homogenate has been prepared. The homogenate can, of course, be reconstituted by mixing suitable quantities of the two fractions.

(2) SUSPENSION MEDIUM. With homogenization, the intracellular components are released into an unnatural environment, and it seemed at first logical to choose as a suspension medium a “physiologic” isotonic salt solution, buffered at an approximately neutral pH. Further work, although confirming the requirement for isotonicity and approximate neutrality, has shown that salt solutions are entirely unsuited for the purpose at hand. Such solutions strongly favor the agglutination of particles of all sizes (Dalton et,aE., 1949; Hers et al., 1951 ; Hogeboom and Schneider, 1950b; Hogeboom, Schneider and Palade, 1948 ; Kennedy and Lehninger, 1949; Pressman and Lardy, 1952), do not preserve the morphologic integrity of the large granules (Dalton et al., 1949; Hogeboom, Schneider, and Palade, 1948), and are unable to protect them osmotically (Berthet et al., 1951; CleIand, 1952). Sucrose solutions, first introduced by Hogeboom, Schneider, and Palade (1948), show little of these effects and must be given strong preference. They are not, however, entirely free of drawbacks. In the first place, they are unable to oppose the progressive fall in p H which occurs in tissue homogenates. This acidification may be extremely harmful, since p H values lower than 6 have been found to cause extensive agglutination of particulate material (Claude, 1946b; Hers et d.,1951) and also to increase the permeability of the larger granules to sucrose, thereby exposing them to osmotic disruption (Cleland, 1952). Fortunately, liver preparations, when kept properly cooled, rarely reach critical pH values, but with tissues such as kidnev, which acidify much more strongly, one

234

CHR. DE DUVE AND J. BERTHET

must choose between two equally harmful procedures: either to let the acidification proceed or to oppose it by the addition of a suitable base, thereby increasing the ionic strength. For this reason, the results with kidney are never as satisfactory as with liver (Hers et al., 1951). Another drawback of sucrose solutions is that they facilitate the adsorption of soluble proteins on the surface of the granules (Beinert, 1951; Berthet et al., 1951). According to Anderson and Wilbur (1952) and IVilbur and Anderson (1951), salt-free sucrose solutions are unable to preserve the morphologic integrity of isolated nuclei. I n their original publication, Hogeboom, Schneider, and Palade ( 1948) advocated the use of hypertonic sucrose solutions (0.88 M ) on the basis that only in such solutions did the mitochondria maintain their elongated shape. There are, however, many objections of a practical nature to the use of such a dense and viscous medium in centrifugation, and the results obtained so far indicate that in most cases hypertonic sucrose may be replaced by the isotonic, 0.25 M solution. The influence of calcium ions deserves a special comment. By addition of 0.00018 M CaClz to the suspension medium, Schneider and Petermann (1950) and Hogeboom, Schneider, and Striebich (1952) have succeeded in isolating nuclei practically uncontaminated by cytoplasmic material. The subsequent steps of the fractionation, however, were adversely affected by the presence of calcium ions, which have been shown to cause aggregation of cytoplasmic material (Hogeboom, Schneider, and Striebich, 1952). On the other hand, Slater and Cleland (1952) have shown that the addition of versene (ethylenediamine-tetraaceticacid), to the suspension medium greatly increases the stability of the oxidative and phosphorylating systems in mitochondria (heart sarcosomes) , and their data support the conclusion that this effect is due to the removal of calcium ions. It thus appears that conditions suitable for the successful isolation of one type of cellular component may influence unfavorably the separation of another, and vice versa. At the present time, salt-free solutions would appear to be the best medium for a complete fractionation. c. Cerzfrifugation. As was shown in the theoretical section, the conditions of centrifugation are best described by mentioning the following data : ( a ) The type of centrifuge and rotor used, and especially, the values of R,,,, and Rmfn..I t will be remembered that the latter value depends on the amount of fluid present in the tubes. (b) The time integral of the average gravitational field applied. (1)

NUCLEI.Using the horizontal yoke No. 269 of the International

DIFFERENTIAL CENTRIFUGATION AND ENZYMES

235

centrifuge PR-1, Schneider (1948) separates the nuclei from 1 :10 homogenates (100 mg. of wet tissue per milliliter) in 0.25 M sucrose by a centrifugation of 10 minutes at W g (6,000 g-min.). With 0.88 M sucrose, Schneider and Hogeboom (1950a) use 16,000 g-min. When the isolation of the nuclei is combined with the initial homogenization, as described above, it is advisable to homogenize with small quantities of fluid (approximately 2.5 ml. for each gram of tissue) to make the final cytoplasmic extract no more diluted than 1 :lo. The first homogenate is then fairly viscous and slightly higher fields are required to effect adequate sedimentation. Using 0.25 M sucrose as the suspension medium and a Corda centrifuge, type 2,047, having a horizontal yoke with tangential tubes, of Rm,,.=15cm. and Rmh,=%cm., we separate the first nuclear sediment at lO,O00 g-min., and wash twice at 6,000 g-min. The nuclei form a well-packed grayish sediment above a thin red layer of erythrocytes. The cloudy supernatant is easy to decant and there is little difficulty in removing most of the large granules which tend to accumulate above the nuclear sediment, since their brown color is easily distinguishable against the gray background of the nuclei. Nevertheless, the contamination of the nuclear fraction by cytoplasmic material remains fairly important, owing to the large volume of the sediment and to the tendency of the cytoplasmic particles to adhere to the nuclei. The nuclear fraction also contains most of the erythrocytes and of the connective and vascular debris, the cells which have escaped disruption, bile canaliculi (Novikoff ef al., 1953) and cell membranes (Hogeboom, Schneider, and Striebich, 1952 ; Palade, 1951). (2) LARGEGRAKULES.The separation of the larger cytoplasmic granules represents the most delicate step of a complete fractionation, because hepatic cells contain at least two types of particles sedimentable at fairly low speeds : the classic “respiring” mitochondria, to be referred to in this paper as mitochondria A, and other granules, free of cytochrome oxidase but containing several hydrolytic enzymes, which will be designated as mitochondria B. This will be discussed in greater detail later in the paper. I n the original procedure of Schneider (1948), the “mitochondria” are separated from 0.25 M sucrose by centrifuging 10 minutes at 8,500g in the high-speed conical head of the International Centrifuge. With 0.88 M sucrose, Hogeboom, Schneider, and Palade ( 1948) centrifuge 20 niinutes at 24,OOOg. In a more recent paper, Schneider and Hogeboom (195Oa) report slightly different values: 10 minutes at 5,ooOg in 0.25 M sucrose and 20 minutes at 29,OCOg in 0.88 M sucrose.

236

CHR. DE DUVE AND J. BERTHET

According to the experience of the reviewers, these procedures are adequate for the complete sedimentation of mitochondria A but do not allow a complete recovery of the B type. In addition, the sediment always contains some poorly packed material as well as the pinkish “fluffy layer” which will be referred to later, and the results may be very different according to the manner in which the supernatant fluid is decanted. There is every reason to believe that these steps are not performed in the same way in different laboratories and that the fractions referred to in the literature as “mitochondrial” and “microsomal” do not always have the same composition. It has been found in this laboratory (Appelmans, et al., unpublished) that the large granules are best isolated from a 1 :10 cytoplasmic extract in 0.25 M sucrose by spinning down at 160,OOO g-min. in the Spinco preparative ultracentrifuge model L, using rotor n‘40, with R,,,.=8.1 and R,,,.=4.8 cm. The two subsequent washings are performed at 130,000 g-min. Under these conditions, the sediment contains most of the large granules of both types and is contaminated by only small amounts (4 to 6%) of microsomes, as measured by their glucose-6-phosphatase activity. I t is also found, when this procedure is followed, that the microsomal sediment contains no granules visible in the ordinary microscope, nor any buff-colored material at all, as it often does when lower centrifugal fields are applied and the supernatant of the large granules is decanted thoroughly. This procedure does not, of course, distinguish between the two types of large granules. In order to do that, a heavier fraction must be isolated at 50,000 g-min., with two washings at 30,000 g-min., following which the remaining larger granules are sedimented from the combined supernatants as described above. Only a partial separation between the A and B granules is obtained in this manner, and their respective proportions in the two fractions must be estimated by adequate enzymatic assays, since they vary considerably with minute changes in technique. ( 3 ) SMALL GRANULES. By definition, the microsome fraction should contain all the particulate material which has not sedimented with the large granules. It follows that (a) the composition of this fraction is to a certain extent dependent on the technique used for the large granules; (b) the field and duration of the centrifugation used to separate the fraction are not critical but should be high enough to ensure complete sedimentation of the smaller microsomes. Originally, the small granules were isolated at l,OOO,OOO to 1,800,OOO g-min. in small conical rotors,

DIFFERENTIAL CENTRIFUGATION AND ENZYMES

237

such as the high-speed head of the International or MSE Major Centrifuges. Under such conditions the smallest microsomes do not sediment completely and are found in the final supernatant, from which they can be separated either by centrifugation at higher speed (Barnum and Huseby, 1948; Beaufays and de Duve, unpublished) or by irreversible agglutination at pH 5 (Hers ct al,, 1951). Practically complete sedimentation of the microsomes (estimated by their glucose-6-phosphatase activity) is obtained in 0.25 M sucrose at 3,000,000 g-min. When 0.88 M sucrose is used, it is customary to dilute the mitochondria1 supernatant to half its original concentration of sucrose and to use higher field-time values (5,000,000 to 8,000,000 g-min.) . d . Decanfafionand Rcsuspension. The difficulties of decantation have already been mentioned. A convenient method is to use a pipet with a narrow drawn-out tip bent to an angle of 90 degrees, and a specid rubber bulb fitted with valves, allowing controlled sucking. Another problem is the resuspension of the packed sediments. Perfect emulsification must be produced ; otherwise, the subsequent sedimentation will be grossly irregular and the final fractions will contain clumps. This step is best performed by means of a thick tube blown out into a spherical bulb fitting closely into the bottom of the plastic centrifuge tubes. The rod is rotated by means of a motor controlled by a rheostat, and the instrument is used as a microhomogenizer of the Potter Elvehjem type, the tubes being kept in ice during operation. The fluid must be added in small portions at first, but once complete emulsification has been effected in a volume of 1 to 1.5 ml., further dilution requires no special precaution. In this way, a sediment is completely resuspended in less than one minute. Finally, every care must be taken to ensure quantitative recoveries. It is convenient to use the wet weight of processed tissue as reference standard and to transfer each fraction quantitatively into graduated vessels, where the final volume is then adjusted to make a definite multiple of the original weight. Using the techniques described, and washing each particulate fraction twice, it is easy to isolate final fractions of the following dilution : nuclei, 1:4;large granules, 1 : l ; small granules, 1 :2 ; final supernatant, 1 9 . In this procedure, the washings are each time combined with the first supernatant. Hence, three times more fluid must be centrifuged to recover the microsomes or the yield of these is proportionately smaller, and the final supernatant is diluted nine times with respect to the cytoplasmic extract. If three particulate fractions are isolated instead of two, these difficulties become even greater. They can be obciated by keeping the washings separate, but then the number of fractions to analyze becomes prohibitive.

238

CHR. DE DUVE AND J. BERTHET

3. Special Techniques In addition to the standard routine procedure described above, many variants have been evolved for special purposes. Several authors have attempted to increase the number of fractions. The scheme used in this laboratory for the separation of two fractions of large granules has already been described. Somewhat similar procedures have been used by McShan and Meyer (1952) and McShan et al. (1953) in their studies on pituitary gonadotropin, and by Laird, ef aI. (1952, 1953). The latter authors have concentrated on the isolation of the ‘‘fluffy layer,” a fraction which does not seem to be identical with our B mitochondria (see below). Several microsome fractions have been isolated by Chantrenne (1947), Barnum and Huseby (1948) and Hazhn et al. (1953). The most involved scheme of fractionation has been applied by Novikoff, ct aE. (1953) who have isolated as many as ten unwashed fractions and six washed fractions in 0.88 M sucrose. Many special techniques have also been worked out with the aim of isolating a given class of intracellular components in as great a state of purity and integrity as possible, whatever the quantitative yield of the procedure. Such components include particulate glycogen (Claude, 1946b ; Lazarow, 1942), chromatin threads (Claude, 1942 ; Claude and Potter, 1943; Mirsky and Ris, 1947), melanin granules from amphibian liver (Claude, 1942), ferritin granules (Stern, 1939), and myofibrils (Perry and Horne, 1952; Schick and Hass, 1949). However, the most extensive investigations have dealt with the isolation of nuclei, for which a large number of methods have been described. As has been pointed out above, the nuclear fraction which is isolated in the usual type of fractionation is always contaminated by a number of extraneous elements and by a fair amount of cytoplasmic material. In addition, it is generally agreed that the sucrose solutions used in fractionation work do not preserve the integrity of the nuclei, probably owing to the high degree of permeability of the nuclear membrane (Anderson, I953 ; Anderson and Wilbur, 1952). It bsignificant in this respect that most of the techniques which have been worked out for the isolation of l~uclei depend on the use of special suspension media believed to reduce the exchanges of matter across the nuclear membrane. Among these may he listed : anhydrous organic solvents (Allfrey et al., 1952; Behrens, 1932 ; Dounce, et d . 1950), dilute citric acid (Dounce, 1943; Dounce and Beyer, 1948; Marshak, 1941 ; Mirsky and Pollister, 1946; Stoneburg. 1939), highly hypertonic (40%) salt-free sucrose (Lang and Siebert, 1 9 5 4 , isotonic sucrose containing 0.00018 M calcium chloride (Hogeboom,

DIFFERENTIAL CENTRIFUGATION A N D ENZYMES

239

Schneider, and Striebich, 1952 ; Schneider and Petermann, 1950), or other electrolytes (Wilbur and Anderson, 1951), solutions of macromolecular substances such as gum arabic (Dounce and Litt, 1952). Various grinding devices and special centrifugation schemes have been described in connection with the use of each of these media (for a survey of the earlier methods, see Dounce, 1950). It is too early to assess the relative merits of these procedures. Their very multiplicity, together with the numerous discrepancies which exist between the reported data one the chemical and enzymatic composition of isolated nuclei, are sufficient to illustrate the great complexity of this problem. Finally, reference should be made to two variant procedures intended to increase the selectivity of the separations. One is the layering technique in which a small quantity of suspension is layered over several volumes of a slightly denser medium and centrifuged (Hogeboom, Schneider, and Palade, 1948; Hogeboom, Schneider, and Striebich, 1952; Wilbur and Anderson, 1951). Obviously, contamination of the sediment by lighter particles should be practically avoided in this manner, and very efficient washing must be achieved, since the particles continuously encounter fresh medium in the course of sedimentation. Unfortunately this procedure causes too great dilution of the subsequent fractions to be used in a complete fractionation. The other technique, recently described by Holter et al. (1953), combines the layering method with the use of suitable density gradients. The centrifugation is performed in a horizontal swingingtube rotor and allows the fractions to separate according to their respective densities and independently of their size. It will be of great interest to compare the results obtained by means of this method with those furnished by the classic procedures based on sedimentation rates.

LIMITATIONS OF DIFFERENTIAL CENTRIFUGATION AS REVEALED BY ENZYMEDISTRIBUTION STUDIES The use of differential centrifugation and, especially, its application to the study of enzymes are complicated by a large number of errors and artifacts, and it is of primary importance that these should be identified as accurately as possible. Morphologic and chemical controls, useful as they are, can hardly be expected to answer all the problems which are raised. More selective tests are needed, and it is the object of this section to show how the objects of the studies, the enzymes, can furnish a wealth of important information, provided the initial assumption is made thut a given enzyme belongs to a .n'nqk intracellular component itz the living cell. If the assumption can be substantiated in a reasonable manner by additional 111.

SCOPE A N D

240

CHR. DE DUVE A N D J. BERTHET

experiments, the enzyme then becomes a sensitive indicator of the fate suffered in the course of fractionation by the component to which it is attached. It need not be verified in every case to be useful, since a few selected enzymes suffice to help considerably in the interpretation of the results of other experiments of the same kind.

1. Sutnmary of Factual Knowledge Table I summarizes the main data obtained on the liver of rodents fractionated in 0.25 or 0.88 M sucrose. They have been arranged in four groups, depending on whether the largest proportion of enzyme was recovered in the final supernatant (I), the microsomal fraction (11), the mitochondria1 fraction (111) or the nuclear fraction (IV). Each group has been divided in a number of subgroups, corresponding to fairly similar patterns of distribution. This arrangement has been made solely for the purpose of facilitating the following discussion and is not meant to imply a closer relationship between the enzymes of the same subgroup. 2. Validity of Fracfiionation Scheme Doubts have been repeatedly raised about the validity of the fourfractions principle on which the method of differential centrifugation is based. Several authors have expressed the belief that the division between large and small granules is arbitrary; they consider either that the cell contains a continuous spectrum of bodies of all sizes with gradually changing properties ( Chantrenne, 1947 ; Jeener, 1948) or that the microsomes represent artificial degradation products of larger intracellular particles (Green, 1951). At first sight, the results summarized in Table I would appear to support this contention, offering, as they do, a bewildering variety of patterns, few of which conform to the simple picture required by the four-fractions principle. A closer examination of these results, however, reveaIs th.at a number of enzymatic distributions do in fact afford strong support to this principle. In the first place, it is clear that some enzymes occur in such overwhelming proportion in the final supernatant that this fraction may safely be taken as representing a cytologically distinct part of the cell’s content. Amongst such enzymes may be listed those of group Ia and presumably also of group Ib-the latter could be microsomal, but the low extent at which they occur in the other particulate fractions argues against this possibility-as well as a number of those of group Ic, although here the lack of a complete balance renders a definite conclusion impossible. Glucose-6-phosphatase (IIa) has been recovered to a very large extent in the microsomes, and there is good experimental evidence that its presence

DIFFERENTIAL CENTRIFUGATION AND ENZYMES

241

in other fractions is an artifact. These facts argue strongly in favor of the genuine character of the microsomes. As to the mitochondria1 fraction, it is obvious that it contains particles which are quite distinct from the microsomes, since many enzymes (IIIa), of which cytochrome oxidase and the related succinoxidase are the most typical ones, sediment completely or almost completely with this fraction. If any such demonstration were necessary for the nuclei, one could mention the fact that the nuclear fraction contains most of the DNA, and most of at least one enzyme, namely the DPN-synthesizing enzyme of Kornberg (1950) (IVa). One may therefore conclude from these results that at least one population of distinct cytologic entities is effectively concentrated in each of the fractions isolated by differential centrifugation. This conclusion is further strengthened by a number of other observations, such as morphologic examinations, chemical analyses, and turnover experiments, which will not be considered in detail here (for a discussion of these points see de Duve, 1952; Smellie et al. 1953), and by the recent density-gradient analyses of Holter et d.(1953).

3. Eficiency of Separation of Particdote Fractions Once the four-fractions principle is established, it becomes of great theoretical and practical importance to ascertain whether the particulate enzymes mentioned in the previous section do in fact belong exclusively to the cytologic population corresponding to each fraction. Two enzymes may be mentioned for which this demonstration has been made in what appears to be a satisfactory manner, namely glucose-6-phosphatase and cytochrome oxidase. In the case of glucose-6-phosphatase, it has been found that the proportion of enzyme recovered in the nuclear and mitochondria1 fractions is greatly influenced by the ionic strength of the suspension medium and by the time taken to separate the microsomes (Hers et d . 1951). It is about twice as great in saline or buffered sucrose solutions as in salt-free sucrose and is always larger when the nuclei and mitochondria are isolated successively than when they are centrifuged off together. Under optimal conditions, as much as 95% of the enzyme can be separated from the nuclei and mitochondria, and it can be safely concluded from the ease with which this yield is lowered under less favorable conditions that the remaining 5% belong to contaminating microsomes. As to the small proportion which is found in the final supernatant when the microsomes are separated at l,&Oo,OOO g-min., it can be irreversibly agglutinated at pII 5 (Hers ~t al., 1951) and comes down quantitatively at 3,000,000

CHR. DE DUVE AND J. BERTHET

242

TABLE I Distribution of Enzymes in Rodent Liver Fractions Isolated in 0.25 M or 0.88 M Sucrose Percentage of total recovered in

subGroup group

I

I1

a

Phosphoglucomutase Hexosediphosphatase b Glutathione reductase Aldolase Glycolysis c Adenosinedeaminase Glucose-6-phosphate dehydrogenase Nucleoside phosphorylase Phosphorylase d Isocitric dehydrogenase Leucine peptidase Alkaline phosphatase Glycolysis Catalase . Myokinase a

b

I11

Enzyme

a

Nuclear Large Small Final Molarifrac- gran- gran- super- tyof ReferAnimal tion Utes ules natant sucrose ences Rat + < 1 + Rat + 1+ Rat 0.3 0.4 Hat 3 1 Rat 5 3 Mouse Rat

-

-

Mouse Rat

-

-

Mouse

3

Rat Mouse Rabbit Rat Rat

3.5

10

13 4.5 5

Glucose-6-phosphatase

Rat 6 Rat +5 Guineapig + 5 Cytochrome c reductase Rat (DPN-specific) Mouse 9 Esterase Rat 6.5 D PN-nucleosidase Rat 37 Succinoxidase

Cytochromeoxidase Rhodanese Myokinase Choline oxidase

Rat Rat Rat Rat Mouse Rat Mouse Rat Rat Mouse Rat Rat

13 8 18 14 20 19 20 15 17

15 16 9

<1

100

0.25

2 96 t903

0.25

0.25

-

0.25

5

0.25 0.25

4 1

025 0.25 0.88 0.25 0.25 0.25

6

12 16 9 0

11 6 3

45 29

7 1

13

75 88

--$ --$

27 28

17 4 63 56 72 70 56

77 79 62 77 72 65 78

0.25 0.88 0.88

t96+ t82+ - 90

1

82 48

>8S

82 74 81

53 49 31 6 11 8

58

4 3 20

53

42

59

4+ t123 4-

0 4 t

0

0.5 4+

2 4

0 0 5

1.5 1.5 t

3 6+

9 3 c13+

0.25 0.25 0.25 0.88 0.25 0.25

0.25 0.25 0.88 0.88 0.88

0.25 0.25 0.25 0.25 0.25 0.25 0.25

0.25

7 8 9 10 11

1 1 1 12 13 10 14

15 15 16

17 18 19 18 10 19 20 21 22

243

DIFFERENTIAL CENTRIFUGATION AND ENZYMES

TABLE I (confinwed) Percentage of total recovered in

subGroup group

I11

Enzyme

Oxalacetic oxidase Octanoic acid oxidase Glutamic dehydrogenase P-Aminohippuric acid synthesis

b

c

IV

a b

Uricase ATP-ase (hlg" act.)

Nuclear Large Small Final Molarifrac- gran- gran- super- tyof ReferAnimal tion ules ules natant sucrose ences Rat Rat Rat

10 5 3

45 95 81

0

Mouse

28

70

Mouse

2

90

Rat Rat Mouse Rat Mouse Mouse Rat

7

73 64 50 41 58 73 46

20 31 24 10 10 31

ATP-ase (Ca*+act.) Ribonuclease Deoxyribonuclease Cathepsin Cytochrome c reductase Mouse 12 (TPN-specific) Uricase Rat 6 Mouse 3 Acid phosphatase Rat 5 Rat 6 MOU5C 7 p-Glucuronidase Mouse 14 Betaine aldehyde osidase Rat 12 Cytochromc c Rat 10 'Rat 6 Rat < 5 Ribonuclease Rat 14

49 65 55 38 64 19 31 50 51 51 60 35

DPN-synthesizing enzyme Arginase Alkaline phosphatase

3 15 13

Mouse Rat Rat

71 34 40

5

0

23

0.25 0.88 0.25

24

c 6+

0.25

25

c 03

0.25

25

17 11 27 11

c

o+

0

3

8

2

0.88

14

7

0.58

15 14 12

5 5

0.25 0.88 0.25 0.25 0.25

28 7

0.25 0.25 0.25

6 4 4

0.88

29 19,30 8 31 32 33 33 34 35

5

c21+ 16

7.5

36 7 32 2 4 8+ 21 34 18 11 18 29 40 13 8 22 2 40 34

0.25 0.88 0.25 0.25 0.25 0.88 0.25 0.88

+30+ 27 8 26 21

0.25 0.25 0.25

7

<5

17

-

28

36 10

10

References l o Table I : 1. Hers e l 01. (1951) ; 2. Rall and Lehninger (1952) ; 3. Kennedy and Lehninger (1959) ; 4. Schneider and Hogeboom (1952a) ; 5. Mueller and Miller (1919) ; 6. Hogeboom and Schneider ( 1 9 5 0 ~ ) ;3. Mnvcr and Greco (1951); 8. Tauboi (1952); 9. LePage and Schnsidcr (1948); 10. Ludewig and Chanutin (1950); 11. Novikoff er al. (1952); 12. Hogeboam (1949); 13. Hogehoom and Sehneidcr (1950b): 14. Sung and Williams (1952); 15. Sehneidcr and Hogeboom (195ba); 16. Hogeboom ct d . (1948); 13. Srhcin er a[. (1951): 18. Schneider and Hogehoom (1950b); 19. A p p e h a n i cr sf. (unpublished). 20. Kielley and Kiellep. (1951) ; 21. Williams (1952.4 : Z2. Kensler and Langemnnn (1951) ; 23. Schneider and Potter (1959); 24. Schneider (1918); 25. Hoaebwm and Schneider (1953s); 26. Kieiley sud Schneider (1950) ; 23. Scbneidt.r ef 01. (1950) ; 28. Schneider m d Hogeboom (1952b) : 29. Palad. (1951) ; 30. Berrhet and da Duve (1951); 31. Walker (1958); 32. Williams (1952b); 33. Schncider et d. (1948); 34. Beinert (1951) ; 35. Pirotto and Desrour (1952) ; 36. Hogeboom and Sebneider (1952b).

244

CHR. DE DUVE AND J. BERTHET

g-min (Appelmans et al., unpublished ; Beaufays and de Duve, unpublished). Glucose-6-phosphatase may therefore be considered to belong exclusively to true microsomes, and it thereby becomes a good indicator of microsomal contaminations in other fractions. A comparison of these results with the data obtained on the distribution of esterase (IIb) in sucrose (Ludewig and Chanutin, 1950; Novikoff, et ul., 1953) and saline media (Omachi et al., 1948) suggests that this enzyme is also essentially microsomal, except that the amount of enzyme found in the final supernatant by some authors (Ludewig and Chanutin, 1950; Omachi et al., 1948)-but not all (Novikoff ef al., 1953)-appears to be too large to be accounted for entirely by unsedimented microsomes. The alkaline phosphatases of kidney (Kers et aE., 1951 ; Kabat, 1941) and intestine (Hers et nl., 1951), a lactonase present in kidney (Meister, 1952) and the renal glucose-6-phosphatase (Hers et d.,1951) have similar distributions. The vitamin A esterase of liver has been reported recently to be recovered entirely in the microsomes (Ganguli and Deuel, 1953). Cytochrome oxidase and the related succinoxidase are found mainly in the mitochondria, but also to a significant extent in the nuclear iraction. It has long been known that the latter fraction is always contaminated by a fairly large amount of mitochondria. The observation of Kennedy and Lehninger (1949), showing that the mitochondrial octanoic oxidase system sediments quantitatively with the nuclear precipitate in saline homogenates, has served to emphasize the tendency of the large granules to aggregate together and with the nuclei. More recently, Hogeboom, Schneider, and Striebich (1952) have succeeded in isolating 70 to 90% of the total nuclei with less than 1% of the total cytochrome oxidase activity. Even this small amount could be accounted for by the number of mitochondria counted in the nuclear fraction. The absence of cytochrome oxidase in isolated nuclei has recently been confirmed by Dounce, Kay, and Pate (1953). In the experiments of Hogeboom, Schneider, and Striebich (1952), 93% of the cytochrome oxidase activity was present in the mitochondrial fraction and 1% in the combined supernatants. As can be seen in Table I (IIIa) higher amounts of enzyme sometimes escape sedimentation with the large granules, in which case they come down with the microsomes, but one can safely attribute this fact to contamination either by small mitochondria or by “mitochondrial ghosts” (see below). It may be concluded from these experiments that cytochroine oxidase is an essentially mitochondrial enzyme, which again can be used to ascertain the extent of overlapping between particulate fractictns. For instance, an examination of Table I will show that several other enzymes have distributions very similar to that of cytochrome oxidase (IIIa),

DIFFERENTIAL CENTRIFUGATION A N D ENZYMES

245

from which it would seem to follow that they are also essentially mitochondrial. I n the case of rhodanese, this parallelism has been verified experimentally (Appelmans et af., unpublished ; de Duve, Appelmaas, and Wattiaux, 1952). Reference should be made here to the artifacts resulting from incomplete homogenization of the tissue. I n this case, the nuclear fraction contains an abnormal proportion of essentially cytoplasmic enzymes. The high content of cathepsin found in the nuclei by Maver and Greco (1951) (IIIb) can probably be explained in this manner, for experiments in this laboratory have shown this enzyme to be present in the nuclear fraction only to the extent of 3 to 55% (Gianetto and de Duve, unpublished).

4 . Artificial Redistribaitioia of Enzymes Besides incomplete separation, several other factors can complicate the results of enzyme distribution studies. The most important are: (a) the retention of soluble enzymes by particulate fractions through surface adsorption; (b) the release of soluble enzymes from particles, either through mechanical injuries or autolysis, eventually complicated further by secondary adsorption; (c) the formation from damaged particles of insoluble debris of differing sedimentation rate. a. Adsmption of Soluble Enzymes. When an enzyme is recovered in a practically quantitative manner in the final supernatant, or when being particulate, it turns out to be essentially insoluble, there can usually be little doubt as to its intracellular location. But when a soluble enzyme is recovered partly in the final supernatant, partly in one or more particulate fractions, the significance of the results is more questionable. It then becomes of crucial importance to determine to what extent adsorption is involved and to what extent the enzyme should be considered as truly belonging to the particles. Only a few investigators have tried to settle this point by direct experiments. The most straightforward studies were made by Beinert (1951) on the distribution of cytochrome c (IIIc). By adding radioactive cytochrome c to the homogenate, this author was able to demonstrate that adsorption accounts quantitatively for the amount of catalyst present in the nuclear and microsomal fractions, but only for a small percentage of the mitochondria1 content. In their studies on acid phosphatase, Berthet et al. (1951) were able to show that part of the enzyme present in the particulate fractions is retained by adsorption. I n this case, the distinction between adsorbed and truly particulate enzyme could be made, thanks to the fact that the former is fully active under all conditions while the latter is essentially inactive as long as the particles maintain their structural

246

CHR. DE DUVE A N D J. BERTHET

integrity, owing to the existence of a permeability barrier to the substrate. Both Beinert (1951) and Berthet et al. (1951) have observed that the adsorption phenomena are much more important in salt-free sucrose solutions than in media containing electrolytes. More recently, Schneider and Hogeboom (1952b) have obtained evidence for the adsorption of deoxyribonuclease and ribonuclease by particulate fractions, especially by the microsomes. The fact that the mitochondria were much richer in these enzymes than the niicrosonics, despite their smaller adsorptive capacity, together with the existence of somewhat reduced activities in intact mitochondria, compared with granules disrupted by sonic vibrations, appeared to refute the possibility of an artificial concentration of the enzymes in mitochondria. A survey of Table I indicates that there are inany other cases in which studies of this kind would be of considerable help in the interpretation of the experimental data. b. Artificial Solubilization of Particulate Enzymes. In order to clarify this point a distinction should be made between particulate and insoluble enzymes. Insoluble enzymes may be provisionally defined as those firmly linked with structural components, presumably of lipoprotein nature, from which they can be detached only with loss of activity or by fairly severe lytic conditions, such as treatment with organic solvents, strong salt solutions, surface-active agents or enzymes. Particulate enzymes, if defined as those associated with intra-cellular bodies, may be so either because they belong to the insoluble class and form part of the structural framework of the particle or because, although freely soluble, they are retained within the granule by a membranelike barrier. It has been known since the studies of Claude (1%6b) that the osmotic disruption of mitochondria releases into the medium a fair amount of soluble proteins. A similar result obtains after treatment of the granules by means of sonic vibrations (Hogeboom and Schneider, 1950a), exposure to the Waring Blendor (Gianetto and de Duve, unpublished), etc. Although questioned by some authors (Green, 1951, 1952 ; Harman, 1950a, b; Huennekens and Green, 1950), the existence of a mitochondrial membrane has been demonstrated by the early electron microscope studies of Claude and Fullam (1945), Dalton et aE. (1949) and Miihlethaler et d. (1950), as well as by permeability studies (Appelmans and deDuve, unpublished; Berthet et al., 1951 ; Cleland, 1952). More recently, investigations of thin tissue sections in the electron microscope by Palade (1952, 1953), Watson (1952), Sjostrand (1953), and Sjostrand and Rhodin ( 1953) have revealed the existence of a complex mitochondrial framework, composed of a peripheral double membrane and of a system of internal ridges forming a series of incomplete septa in the interior of the mitochon-

DIFFERENTIAL CENTRIFUGATION AND ENZYMES

247

drial body. Within this framework is enclosed an almost structureless matrix, which presumably contains the soluble proteins referred to above. Several enzymes have been identified among the soluble protein fraction which is released from large granules subjected to niechanical or osmotic disruption, namely cytochroine c (Beinert, 1951 ; Schneider, Claude, and Hogeboom, 1938), acid phosphatase (Berthet et al., 1951 ; Berthet and de Duve, 1951), p-glucuronidase (Gianetto and de Duve, unpublished ; Walker, 1952), cathepsin (Gianetto and de Duve, unpublished), glutamic dehydrogenase (Hogeboom and Schneider, 1953a), and myokinase (Barkulis and Lehninger, 1951) . This enzyme (Kielley and Kielley, 1951), as well as ribonuclease and deoxyribonuclease ( Schneider and Hogeboom, 1952b), has also been solubilized by exposing the mitochondria to sonic vibrations. However, this treatment seems to cause a more complete disintegration of the granules, since it even releases part of the cytochrome oxidase (Hogeboom and Schneider, 1952a). All these enzymes show a complex distribution which has not yet been entirely clarified in all cases, but which, in the case of acid phosphatase, can be described as follows (Berthet and de Duve, 1951) : Approximately 70 to 80% are recovered in what may be described as the native bound form, i.e., retained within intact granules. These are concentrated in the mitochondria1 fraction, but also occur in small quantities in the nuclear and microsonial fractions. Of the remaining 20 to 30%, about half is found in the final supernatant, the other half being adsorbed on the surface of the various particles which presumably have taken it up from the soluble fraction. Parallel distributions are found for P-glucuronidase and cathepsin (Gianetto and de Duve, unpublished). Such results raise the interesting question of whether the “free enzyme” found in the supernatant and, under adsorbed form, in the particulate fractions, truly exists in soluble form in the intact cell or has been released accidentally from damaged granules in the course of the homogenization and fractionation procedures. Such a question cannot be answered unequivocally, but experiments on the fragility of the granules indicate that at least part of the enzyme, and possibly all of it, must be considered to originate from intact granules (Berthet and de Duve, 1951 ; Gianetto and de Duve, unpublished). If the latter view is correct, the implication o€ these findings is that some 20 to 30% of these granules are injured in a complete fractionation, releasing their soluble proteins in the suspension medium, where they redistribute themselves amongst the various fractions according to their affinities for their respective components. Whether this type of reasoning can be extended to the other enzymes listed in groups IIIb and IIIc remains to be investigated. The question

248

CHR. DE DUVE AND J. BERTHET

becomes particularly delicate in the case of cytochrome c (IIIc), catalase (Id), possibly the myokinase of rat liver (Id)-here, however, the recovery is unsatisfactory and the results disagree with those obtained 011 mouse liver (1IIa)-as well as of vitamin BIZ and folic acid (Swendseid, Bethel1 and Ackermann, 1951) coenzyme A and pantothenic acid (Higgins et al., 1950) and thiamine pyrophosphate (Goethart, 1952). In all these cases, an approximately 50% partition between large granules and final supernatant has been found, with only small amounts in the nuclear and microsomal fractions. Are these cases similar to those discussed above, but with a higher proportion of injured granules, or do they illustrate a true state of affairs? The point is at present unsettled but is of considerable importance. Nuclei are also known to possess well-defined membranes and to contain soluble proteins. The extent to which the latter are lost in the course of fractionation has given rise to long debates. Of considerable interest in this respect is the recent discovery of Hogeboom and Schneider (1952b) that the DPN-synthesizing enzyme of Kornberg (1950)-a protein which is released by sonic disruption-is almost entirely, if not exclusively confined to the cell nuclei. Unless special precautions were taken, approximately 30% of this enzyme was recovered in the mitochondria1 supernatant,* a strong indication that up to one-third of the nuclei may be damaged in a complete fractionation. Similar phenomena have not been described for true microsomes. Indeed these bodies have not been found to release significant amounts of protein nor to have typical saclike structures ; they are denser than mitochondria (Holter et d.,1953), and there are some indications thatithey may be components of reticular or membranelike structures which have been described in the cytoplasm by a number of investigators (Bernhard et al., 1951; Bernhard et al., 1952; Dalton et d.,1950; Oberling et al., 1953; Palade and Porter, 1952; Porter, 1953; Porter and Thompson, 1947; Sjostrand, 1953; Sjostrand and Rhodin, 1953). In the case of the microsomes, however, as in that of the structural framework of the other particulate components, another possible artifact must be considered, namely the release of enzymes from insoluble structures by autolysis. So far there are few clear-cut experimental data concerning the importance of autolysis in homogenates and isolated cell fractions. This process has been shown to cause the disintegration of mitochondria (Appelmans and

* Unfortunately the authors have not subfractionated this supernatant and therefore offer no proof that they are dealing with a soluble enzyme. Sonic vibrations have been shown to solubilize “insoluble” enzymes (Hogeboom and Schneider, 1952a).

DIFFERENTIAL CENTRIFUGATION AND ENZYMES

249

de-Duve, unpublished; Berthet et al., 1951) and the solubilization of part of the microsome-bound alkaline phosphatase of kidney (Hers et al., 1951; Kabat, 1941). c. Artificial Redistribution of Insoluble Enzymes. As was seen in the preceding section, there are strong indications that a significant percentage (perhaps 20 to 30%) of both the nuclei and the large granules are injured and release soluble proteins in the course of a complete fractionation. This process is probably an all-or-none effect, in that the membrane of the particles, once it is damaged, will let through all the soluble proteins of the particle. At least, such seems to be the case for the acidphosphatase-bearing granules, for it has been found in a variety of experiments in which graded damage was caused that, in each individual case, identical proportions of three different proteins, acid phosphatase, P-glucuronidase and cathepsin, were released (Gianetto and de Duve, unpublished). A corollary of these findings is that a fairly large amount oi corresponding “ghosts” containing insoluble enzymes must be included in the fractionation, and it is quite possible that these do not exhibit the same sedimentation properties as the intact particles from which they derive. Besides nuclear and mitochondrial debris, the cell membranes themselves must also be considered. According to the observations of Palade (1951) and Hogeboom, Schneider, and Striebich (1952), the cell membranes probably come down with the nuclei. The fate of the damaged nuclei can be roughly estimated from the recorded data on the distribution of DNA. In most investigations, a varying proportion of the total DNA, ranging from only a few per cent to as much as 20, escapes sedimentation with the nuclei and is recovered in the mitochondrial and microsomal fractions (Hogeboom and Schneider, 1952b; Hogeboom, Schneider, and Striebich, 1952 ; Schneider and Peterman, 1950 ; Schneider and Hogeboom, 1952b ; Schneider and Potter, 1949). In addition, microscopic examinations indicate that a number of damaged nuclei must be present in the nuclear fraction itself (Hogeboom et al., 1952), a conclusion which is also suggested by the fact that the amount of DNA lost to the cytoplasmic fraction is smaller, in the experiments of Hogeboom and Schneider (1952b), than the amount of DPN-synthesizing enzyme released. The sedimentation behavior of damaged mitochondria can be estimated from the observed distributions of insoluble mitochondria1 enzymes, such as cytochrome oxidase and succinoxidase. Presumably, the small amounts of these enzymes usually recovered in the microsomal fraction belong to the mitochondrial debris. In addition, in view of the extent of damage estimated previously, it would seem that the mitochondrial fraction itself

250

CHR. DE DUVE AND J. BEBTHET

contains a fair amount of injured granules. Of special interest in this connection is the so-called “fluffy layer” described by several authors, a pinkish, poorly sedimented layer which is found on top of the wellpacked, buff -colored mitochondrial precipitate. According to Potter, Recknagel, and Hurlbert (1951) and Laird et d . (1952, 1953), this material resembles microsomes in that it is much richer in RNA than the mitochondria and is unable to reduce Janus B green to diethylsafranin at 37” C., a reaction characteristic of the mitochondrial precipitate (Potter, Recknagel, and Hurlbert, 1951). On the other hand, its average density is smaller than that of the mitochondria, which themselves are less dense than the microsomes (Holter et al., 1953), and it contains the typically mitochondrial succinoxidase system at a concentration, expressed in activity per milligram of nitrogen, 0.76 times that of the mitochondria (Laird et al., 1952, 1953). Laird et al. (1952, 1953) have examined this fraction in the electron microscope and believe it to be formed mainly of small mitochondria. This opinion, however, is not shared by Novikoff et al. (1953), who, while confirming the analytic data of Laird et d. (1952, 1953), state their preparation to be a “gross mixture of mitochondria (mostly small) and microsomes (including most of the larger dense microsomes of the homogenate and a great many less dense and dense varieties).” Results obtained in this laboratory indicate that the fluffy layer is richer in glucose6-phosphatase (microsomes) than in acid phosphatase (mitochondria B ). Such data are still difficult to reconcile with one another and the results of more complete examinations should be awaited. In the opinion of the reviewers, the possibility that this fraction might contain ‘a fair amount of mitochondrial ghosts would bear consideration, for a reasonable explanation would then be found for its peculiar sedimentation behavior, for its high specific succinoxidase activity (despite the microsomal contamination), and perhaps, also, for at least part of its high RNA content, if, as certain experiments suggest (Claude, 1946b), the latter is associated in the mitochondria with their insoluble framework.

5. Multiplicity and Heterogeneity of Cytoplusrnk Particles As was pointed out in a previous section, there are strong grounds for the belief that each of the four fractions isolated by differential centrifugation does indeed concentrate to a large extent a definite population of intracellular components. This fact, however, does not preclude the possibility that living cells may consist of more than four distinct components, in other words, that there may be more than two types of cytoplasmic particles. Although difficult to interpret, the data of classical morphology would

DIFFERENTIAL CENTRIFUGATION AND ENZYMES

25 1

seem to argue in favor of a greater multiplicity of cytoplasmic granules. It may be recalled that the fate in differential centrifugation of such formations as secretory granules, centrosomes, and the Golgi body* is entirely unsettled at the present time, and that some indications have been obtained of the existence of two distinct types of mitochondria (KandavelVendrely, 1949). Electron microscope studies also support the concept of multiplicity, and reference may be made here to the so-called “growthgranules” described by Porter ( 1953) and Porter and Thompson (1947). A number of differential centrifugation studies also point toward a greater complexity of the cytopIasmic content. Of particular interest in this connection are the recent studies of Novikoff et al. (1953), who have investigated the distribution of DNA, RNA, and nitrogen, and of several enzymes in ten unwashed fractions isolated at increasing gravitational forces from a single homogenate prepared in 0.88 M sucrose, as well as in a complete fractionation furnishing six washed fractions. The results obtained reveal marked differences in sedimentation behavior between two pairs of enzymes, all previously believed to be essentially mitochondrial, namely succinoxidase and adenosinetriphosphatase, which are concentrated in the earliest fractions, and uricase and ’the sedimentable acid phosphatase, of which the greatest part comes down at centrifugal forces intermediate between those used for the isolation of the mitochondria and the microsomes. Esterase and RNA appear in a third group, concentrated mostly in the final particulate fractions. Alkaline phosphatase is present in small quantities in all the sediments but appears mostly in the final supernatant, which also contains the soluble portion of acid phvsphatase (35%). Finally, a complex distribution was found for adenosine-5phosphatase, showing two peaks, one in the nuclear fraction, the other coinciding roughly with the acid phosphatase-uricase peak. The authors have also examined the various sediments by phase contrast microscopy. They maintain the distinction between mitochondria and microsomes but describe large and small mitochondria, as well as three types of microsomes. I n discussing these results, however, the authors seem to favor an explanation based on heterogeneity rather than on multiplicity. They stress the fact that the cytoplasmic granules differ enzymatically only in extent of activity (italics theirs) and do not seem to consider the possibility of contamination or overlapping. As was shown above, there are many reasons, both theoretical and experimental, to believe that these processes are inevitable. The results mentioned could therefore also be interpreted

* Schneider, Dalton, et al. (1953) have recently described the isolation of Golgi particles from mouse epididymis. This material was very rich in alkaline phosphatase.

252

CHR. DE DUVE AND J. BERTHET

as signifying that abnormal distributions, such as those of acid phosphatase and uricase, reflect the sedimentation pattern of special populations of granules, containing the totality of these enzymes and different both from the succinoxidase-containing mitochondria and from the esterase-bearing microsomes. To mention an extreme case, these special granules may even be entirely undetectable by the usual morphologic tests, since they could be very scarce and still contain the total enzyme activities, which correspond to only minute amounts of protein. Investigations performed in this laboratory support the concept of multiplicity. In confirmation of the results of Novikoff et al. (1953) it has been found that the acid phosphatase-containing granules require higher centrifugal forces for sedimentation than the cytochrome oxidasebearing mitochondria, but in addition a fair amount of evidence has been obtained indicating that two entirely distinct types of granules are involved. For instance, although the extent of overlapping is too great to permit complete separation, it has been possible to isolate a head fraction containing almost no bound acid phosphatase and approximately one third of the respiratory activity, as well as a tail fraction rich in acid phosphatase and practically devoid of cytochrome oxidase. Both fractions contain less than 5 % microsomes, as judged by the presence of glucose-6-phosphatase. Also, much of the bound phosphatase (intact granules) can be washed off a mitochondrial fraction without loss of cytochrome oxidase, and the amount of phosphatase associated with a given proportion of the oxidase has been found to differ greatly with very small changes in the experimental procedures. In contrast to these findings, the content of rhodanese was found to parallel closely that of cytochrome oxidase, whatever the fraction examined (Appelmans et al., unpublished; de Duve, et al., 1952), while more recent experiments have revealed a similar relationship between acid phosphatase, /3-glucuronidase, and cathepsin (Gianetto and de Duve, unpublished). In short, the indications are that the mitochondria1 fraction concentrates, besides the totality of the mitochondria containing cytochrome oxidase and rhodanese, a variable proportion of a second class of granules differing from the first and from true microsomes and containing the three hydrolytic enzymes mentioned. These two groups correspond to the mitochondria A and B, referred to in the section on techniques. In addition, it must be pointed out that the data not only support the multiplicity concept, but also argue against heterogeneity within a given class, since different subfractions are found to contain associated enzymes in the same proportions. Other studies dealing with the problem are the investigations of the “fluffy layer” referred to above and those of McShan and Meyer (1952)

DIFFERENTIAL CENTRlFUCATION A N D E N Z Y M E S

253

and McShan ct al. (1953), who found the pituitary gonadotropin to be concentrated in a fraction intermediate between the mitochondria and microsomes. Holter et al. (1953) also conclude from their densitygradient studies that the mitochondria fraction is much more heterogeneous than the microsome fraction. These findings have an important implication, namely that two enzymes may be entirely unrelated cytologically, even though they are concentrated in the same fraction by differential centrifugation. Such a possibility should at least be suspected if small but significant differences in distribution are observed between the two enzymes. It has been verified experimentally in the cases mentioned and could be so in others of a similar kind. The concept of heterogeneity within a given class of granules, which the reviewers feel has not yet been verified with certainty in the case of the larger particles, could, however, apply to the smaller ones, for several investigators (Barnum and Huseby, 1948; Chantrenne, 1947; H a d n et aE., 1953) have shown that the composition of microsomes changes progressively with their size. The possible existence of several distinct classes of microsomes should not, however, be overlooked (see for instance : Porter, 1953 ; Porter and Thompson, 1947).

6. Artifacts Associated with Enzyme Assays When differential centrifugation is used for enzyme distribution studies, additional difficulties are encountered, arising from the necessity of estimating enzymes quantitatively in an extremely complex environment. There are numerous examples in the literature of the various types of errors and artifacts associated with this part of the experimental procedure. u. Monoenzymatic Systems. With monoenzymatic reactions, the main factors which have been found to affect the observed distribution pattern are : a. The lability of the enzyme studied toward enzymatic or nonenzymatic inactivation. b. The existence of structural barriers restricting the availability of enzyme and substrate. c. The presence of inhibitors or activators. d. The lack of specificity of the substrate used for the assay. e. The presence of competing systems likely to affect the kinetics of the reaction studied. A particularly striking example of the first type of artifact is provided by the difficulties which were experienced in the study of liver phosphorylase, an enzyme which is known to suffer a very rapid enzymatic

254

C H R . DE DUVE A N D J. BERTHET

inactivation in liver extracts (Sutherland and Cori, 1951). Only by sedimenting all the particulate components of the homogenate in a single highspeed centrifugation was it possible to demonstrate the essentially soluble nature of the enzyme (Hers ct d . , 1951). Similar complications have been encountered in studies on glucose-6-phosphatase (Hers et al., 1951) myokinase, and adenosinetriphosphatase (Novikoff et al., 1952). Several instances of the second type of artifact have also been described. In this laboratory (Appelmans and de Duve, unpublished; Berthet et al., 19.51 ; Berthet and de Duve, 1951; de Duve, Berthet, et al., 1951), it has been found that bound acid phosphatase is unable to split added pglycerophosphate unless the granules have been subjected to a treatment, either prior to or concomitant with the enzymatic test, which disrupts their native structure and releases the enzyme in soluble form. Osmotic protection experiments showed that the granules are impermeable to glycerophosphate and that the results observed could be explained by the existence of a structural barrier preventing an effective contact between the internal enzyme and the external substrate. Similar observations have been made by Walker (1952) in the case of P-glucuronidase, using phenolphthalein glucuronide as substrate. His findings have been confirmed in this laboratory and a third system of the same kind has been added to the list, namely cathepsin, with henioglobin as substrate (Gianetto and de Duve, unpublished). Interestingly enough, as has already been pointed out, all three enzymes belong to the same granules and are released simultaneously in identical proportions when the granules are subjected to a graded disruption. Another interesting case of structural hindrance has been described by Lehninger (1951a, b), who found that saline-washed mitochondria, although capable of oxidizing p-hydroxybutyrate via their own internal DPN and cytochrome c, were unable to oxidize externally added DPNH unless cytochrome c was also added. Even in the latter case, the reaction occurred at a reduced rate and was considerably accelerated when the particles had been pretreated with distilled water during a short time. More recently, Hogeboom and Schneider (1953a) have described a similar phenomenon of structural hindrance in the assay of the mitochondria1 glutamic dehydrogenase. The presence of inhibitors or activators has been frequently encountered in one or the other form and may cause very troublesome complications, especially when the active agent is unequally distributed with respect to the enzyme. In many cases, this fact results in incomplete or excessive balances, but not necessarily so. For instance, the final supernatant contains a thermostable inhibitor of glucose-6-phosphatase (Beaufays and de Duve, unpuhlished) . However, the increase of activity which should be observed

DIFFERENTIAL CENTRIFUGATION A N D ENZYMES

255

in washed microsomes is offset by the inactivation of this labile enzyme, and the recoveries often appear unusually good. Only when fractions are recombined in various ways does the true picture of what has happened emerge. This process of recombining fractions to test for interactions has been used by many authors and has often provided important additional information. A conspicuous example of its failure to afford a satisfactory explanation is shown in the experiments of Sung and Williams (1952) on the distribution of DPN-nucleosidase (IIb). The sum of the four fractions totaled 136% of the activity of the homogenate, whereas recombined they had essentially the same activity as the homogenate. From partial recombinations it could only be concluded that all fractions appeared to be mutually inhibitory. Studies on P-glucuronidase have shown another type of complexity. Walker and Levvy (1953) have found that liver homogenates contain an inhibitor of this enzyme and that the addition of a surface active agent suppresses this inhibition. Recent experiments have shown that this type of inhibition is due essentially to insoluble components, microsomes and mitochrondial debris, that it is considerably increased when this material is aged and, further, that the degree of inhibition is strongly pH-dependent, resulting in considerable alterations in the pH activity curve of the enzyme (Gianetto and de Duve, unpublished). Particularly striking are the experiments of Pirotte and Desreux (1952), who found the ribonuclease of sucrose homogenates to be inhibited up to 95%. The activity could be released by treatment with 0.25 M sulfuric acid or with strong salt solutions. The final supernatant was particularly rich in inhibitor, being not only entirely inactive, unless pretreated with sulfuric acid, but even able to suppress completely the activity of added crystalline ribonuclease. This inhibition may not have been as great in the experiments of Schneider and Hogeboom (1952b) who, using other assay conditions, found a small amount of activity in the final supernatant. However, this proportion is much smaller than that reported by Pirotte and Desreux (1952) for the fully activated enzyme (see Table I, groups IIIb and IIIc). Atypical distributions can also be encountered in cases where the substrate used for the assay is acted upon by more than one enzyme. This sort of artifact is a’common complication in studies on phosphatases, of which many different types of varying specificity are known, both. in the acid and alkaline classes. It is readily recognized in a study on mouse liver by Tsriboi (1952) with phenyl phosphate as substrate, His “acid phosphatase” ( TIIc) is obviously heterogeneous, since it contains a labile component, revealed by a low recovery and direct inactivation experiments.

256

CHR. DE DUVE AND J. BERTHET

This component is none other than glucose-6-phosphatase, which is fairly active on phenyl phosphate, but not on /l-glycerophosphate (Beaufays and de Duve, unpublished). Finally, the last type of artifact, namely the presence of competing systems, is responsible for the failure of Hers et al. (1951) to make an adequate study of the distribution of hexokinases. These enzymes are fairly abundant in the final supernatant and are easily assayed in this fraction, but they cannot be determined accurately in the homogenate and in the particulate fractions, owing to the antagonising effects of adenosinetriphosphatase and glucose-6-phosphatase. b. PoEyenzytatic Systems. When the reactions involve more than one enzyme, all the complications mentioned above are of course multiplied, and in addition there arises the difficult problem 0; ascertaining which step is rate-limiting in the over-all process. For instance, it is often claimed that the mitochondria are the main sites of cellular oxidations, and that anaerobic glycolysis occurs essentially in the extramitochondrial spaces, corresponding to the final supernatant. Indeed, the ability of homogenates to metabolize appropriate substrates such as fatty acids, amino acids and various Krebs cycle intermediates aerobically ( IIIa) is concentrated mainly in the mitochondria1 fraction (Harman, 195Oa ; Hogeboom and Schneider, 195Oc ; Hogeboom, Schneider, and Palade, 1948; Kennedy and Lehninger, 1948, 1949; Leuthardt and Mauron, 1950; Novikoff et ul., 1953; Schein et aE., 1951; Schneider, 1948; Schneider, Claude, and Hogeboom, 1948; Schneider and Hogeboom, 195Oa, b ; Schneider and Potter, 1949). On the other hand, their ability to glycolyse carbohydrates anaerobically ( I d ) is recovered essentially in the final supernatant (Kennedy and Lehninger, 1949; LePage and Schneider, 1948). However, as was pointed out by Hogeboom and Schneider (Hogeboom, 1951 ; Hogeboom and Schneider, 1 9 % ~; Schneider and Hogeboom, 1951), all the oxidative reactions studied in the first class require cytochrome oxidase for their oxygen consumption and could therefore only occur in the fractions containing this enzyme, whereas at least one enzyme necessary for glycolysis, aldolase (Ib), is known to be almost entirely recovered in the final supernatant. When a quantitative balance obtains, as in the case of the fatty acid-oxidizing system in the experiments of Kennedy and Lehninger (1949), the results are compatible with the hypothesis that all the enzymes and cofactors involved are concentrated in one fraction, but they only demonstrate that this fraction contains most of the rate-limiting factor and enough of the others to prevent them from becoming rate-limiting. When, on the other hand, the recovery is low and can be corrected by recombining fractions, as was the case in studies on glycolysis (LePage and Schneider, 1948)

DIFFERENTIAL CENTRIFUGATION AND ENZYMES

257

and on the oxidation of succinate (Schneider and Hogeboom, 1950a, b), octanoate (Schneider, 1948), oxalacetate (Schneider and Potter, 1949), isocitrate (Hogeboom and Schneider, 195oC), and a-ketoglutarate (Siekevitz, 1952), the probabilities are that at least two factors are so unequally distributed that each becomes rate-limiting in the fraction where its concentration is low. This fact has of course been clearly recognized and has been pointed out repeatedly ( Hogeboom and Schneider, 1950c; Kennedy and Lehninger, 1949). I t has been evidenced in a particularly striking fashion by the studies of Hogeboom and Schneider ( 1 9 5 0 ~ )on the distribution of isocitric dehydrogenase (Id), This enzyme is recovered mostly in the final supernatant, and its concentration in the mitochondria fraction is sufficiently low to raise some doubt about its significance. When studying the oxidation of isocitrate, the authors found that only the mitochondria and to a certain extent the nuclei were able to carry out the reaction, but at one third of the rate of the homogenate. Addition of the supernatant to the active fractions raised their activity significantly, an indication that isocitric dehydrogenase probably was rate-limiting. Hawever, the combined activities obtained in this manner totalled only 50% and were then restored to the original level by the addition of the microsomes. Presumably, another enzyme had now become rate-limiting, namely the TPN-specific cytochrome c reductase, of which an important part is present in the microsame fraction (IIIb). Another clear case, in which two interdependent enzymes were artificially separated by the fractionation, is given by the experiments of Mueller and Miller (1949). Perhaps the most impressive example of the difficulties associated with enzymatic assays is furnished by the studies on the ability of the liver to dephosphorylate ATP. The substrate contains three phosphate groups, one or more of which can be detached by an astonishing variety of enzymes, acting either singly or together and subject to different types of activations and inhibitions (Kielley and Kielley, 1953; Lardy and Wellman, 1953; Swanson, 1951). A determined effort has been made by Novikoff et al. (1952) to resolve this highly complex system into its individual components. But their results, while confirming the presence of a large proportion of the magnesium-activated adenosinetriphosphatase activity of liver in the mitochondria (IIIb), have failed to clarify completely the nature and distribution of the various enzymes involved. The problem is further complicated by the fact that adenosinetriphosphatase has been shown to be essentially latent in intact mitochondria and to exhibit full activity only when the granules have been preincubated during a certain time at 28" C. without oxidizable substrate (Kielley and Kielley, 1951).

258

CHR. DE DUVJZ AND J. BERTHET

This activation is inhibited by adenylic compounds (Kielley and Kielley, 1951), but much remains to be learned about this phenomenon, which can be evoked also by exposure to hypotonic media (Potter and Recknagel, 1951), by calcium ions (Lardy and Wellman, 1953; Potter and Sinionson, 1952) and by dinitrophenol (Hunter, 1951 ; Lardy and Wellman, 1953; Potter and Recknagel, 1951; Siekevitz and Potter, 1953a).

7. Truly Heterogeneous Enzyme Distributions Although the evidence available so far appears to argue in favor of a monistic concept of the intraceliular localization of enzymes, it is much too early to decide to what extent this concept can be considered valid or how far it will stand generalization. In the first place, it is fairly probable that enzymes exist which are truly present in two or more distinct loci. Some may even be ubiquitous, for instance if they belong to some basic structural or functional constant which is repeated in all the particulate formations of the cell. Allcaliiie phosphatase ar,d, perhaps, adenosinetriphosphatase, could belong to this class. However, the examples of apparent heterogeneity are already so numerous that such a conclusion should only be reached after all monistic explanations-including those based on the existence of discreet differences such as might exist between two enzymes of the same class-have been excluded by adequate investigations. Another point which must be kept in mind is that the limitations of the technique are such that a monistic interpretation can only be established with a certain degree of probability but never demonstrated in a clear-cut fashion. Perhaps the greatest interpretive difficulties will be encountered in the assessment of the true enzymatic composition of the non-particulate fraction of the cell and of the nuclei. In the former case, the uncertainty arises from the fact that a homogenate cannot be prepared without causing a fairly large proportion of soluble enzymes to leak out of injured particles. One can estimate roughly the extent of this damage by separate experiments and thereby guess at the proportion of enzyme present in free form in the intact cell, but the conclusion reached will always be the result of a somewhat arbitrary extrapolation. In the case of the nuclei, one is met with the problem of whether their enzymatic machinery is entirely distinct from that which exists in the cytoplasm, or whether a number of processes occur simultaneously in both sites. The latter point of view has been vigorously defended by Dounce (1950) and, more recently, by Stern ef al. (1952) and by Lang and Siebert (1951), who have found a number of enzymes to be present in significant quantities ia samples of nuclei isolated by special techniques

DIFFERENTIAL CENTRIFUGATION A N D ENZYMES

259

from various tissues. However, most of the enzymes thus identified occur also in the cytoplasm in large and sometimes overwhelming quantities, and too little is known about the possible artifacts associated with the techniques used to permit a definite conclusion at the present time. On the other hand, the results obtained so far by the sucrose technique seem to argue in favor of the former concept. Most of the enzymes studied are essentially cytoplasmic, and their ccintent in the nuclear fraction can be satisfactorily accounted for by contaminations, while one enzyme at least is now known to be associated mainly, if not exclusively, with the nuclei (Hogeboom and Schneider, 1952b). Three exceptions have, however, been found : alkaline phosphatase (Ludewig and Chanutin, 19SO), arginase (Ludewig and Chanutin, 1950; Rosenthal, 1953), and adenosine5-phosphatase (Novikoff et &., 1953). The nuclear content of these enzymes is high enough to appear significant. T o conclude this discussion, reference must be made to the greatest limitation of differential centrifugation experiments, namely their essentially statistical nature. At their best, they only provide a global picture, in which all the disparities existing between different cell types, as well as between different functional states of the same cell type, are merged and averaged. Further discrimination can only be achieved by other methods, such as microdissection associated with microanalysis and the various techniques of cytochemistry. Another fruitful approach, which has already been applied in a limited number of cases, is to vary the state of the tissue investigated and to look for changes in distribution patterns. If correlated with cytologic modifications, such observations may provide additional information concerning the physiologic significance of the results obtained.

EVALUATION OF THE RESULTSOF TISSUE IV. BIOLOGICAL FRACTIONATION STUDIES

Differential centrifugation can serve two purposes. Either it can provide a starting point for more advanced enzymologic work by furnishing new types of preparations particularly suited to the study of certain metabolic pathways or to the further isolation of enzymes, or it can be used as a tool in cytologic research and serve to study the higher levels of organization of the cell. In the first type of application, artifacts are of secondary importance. The enzyme chemist takes advantage in a straightforward manner of the separation procedure offered by differential centrifugation and from then on proceeds to analyze the system further by means of the usual techniques of biochemistry. I t would be beyond the scope of this review to survey the manifold applications of this kind. As an example, one may mention

260

CHR. DE DUVE AND J. BERTHET

the progress which has been made recently in the study of fatty acid oxidation. First demonstrated in cell-free extracts by Leloir and Mufioz (1939), this process was then localized in the insoluble residue of homogenates (Lang, 1939; Lehninger, 1945 ; Mufioz and Leloir, 1943), and later in the mitochondria fraction by Kennedy and Lehninger (1948 and 1949) and Schneider (1948). A further advance was made when Drysdale and Lardy (1953) succeeded in extracting from isolated mitochondria a soluble system capable of oxidizing fatty acids, culminating in the recent identification and purification by a number of workers of the main enzymes involved in this process (see Lynen and Ochoa, 1953). When differential centrifugation is used as a cytochemical tool, far greater caution has to be exercised in the evaluation of the results, since in this case every type of artifact must prove misleading or confusing, unless properly recognized. As was shown in the preceding chapter, the limitations of the technique are rather formidable and it is possible that some of the conclusions arrived at in earlier work will need reconsidering in the light of the newly gained knowledge. It is, however, of some interest to examine the available data as they are today and to attempt an evaluation of their biologic significance.

1. Hydrolytic Enzymes The most striking aspect of the distribution of hydrolases is that most of them appear to be associated with particulate entities. The significance of this fact remains somewhat obscure, since the true function of many of these enzymes is still unknown. It can, however, be pointed out that if hydrolytic enzymes were free to act within the living cell, as they do for instance in homogenates, they would lower to a very great extent the efficiency of the synthetic processes and might even interfere with the structural integrity of the cell as a whole. I t therefore seems reasonable to assume that the intracellular segregation of hydrolases constitutes one of the means whereby hydrolytic activity is held in check or kept localized at specific sites of the cell. Of particular interest in this respect is the finding, briefly recorded in this paper, that the cell contains a special type of granule, within which three different soluble hydrolytic enzymes, all with an acid pH optimum, are confined, surrounded by a membrane which appears to be impermeable to their substrates. The nature and function of these granules raise intriguing problems. Exceptions to the above generalization are furnished by hexose diphosphatase (Hers et d., 1951) and leucine peptidase (Maver and Greco, 1951), which are recovered to a large extent in the final supernatant. However, these enzymes have narrow specificities and show absolute

DIFFERENTIAL CENTRIFUGATION AND ENZYMES

26 1

requirements for metallic ions, and they could be controlled in a different manner. 2. Cetlzclar Oxidations Contrary to a statement which is often found in the literature, the oxidizing enzymes are far from being confined exclusively to the mitochondria. As was pointed out in a previous section, this distribution is almost unique for cytochrome oxidase. Cytochrome c is about equally distributed between the mitochondria and the soluble fraction, and, although one cannot exclude the possibility of artificial solubilization, this explanation seems unlikely in view of the fact that osmotically disrupted mitochondria do not release their cytochrome c in non-ionic media (Beinert, 1951 ; Schneider, Claude, and Hogeboom, 1948; TSOU,1952). The cytochrome c reductases are both recovered partly in the mitochondria1 fraction and partly in the microsomes, and the latter ,fraction is the richer for the DPN-specific enzyme. In view of previous discussions, such findings are strong indications that the enzymes do not belong exclusively to the mitochondria and could even be compatible with their association with a distinct type of particle. However, results such as those of Lehninger (1951a, b) on P-hydroxybutyrate oxidation suggest the presence of active cytochrome c reductase within the mitochondria themselves. Turning now to the D P N and TPN-linked dehydrogenases, it is found that several of them, namely those acting on glucose-6-phosphate (Mueller and Miller, 1949), isocitrate (Hogeboom and Schneider, 1 9 5 0 ~ )and glutathione (Rall and Lehninger, 1952) are largely concentrated in the final supernatant. This fraction must also contain a good part of the triose phosphate and lactic dehydrogenases, in view of the results on glycolysis (Kennedy and Lehninger, 1949; LePage and Schneider, 1948) ; and probably several others (Dianzani, 1951a, b, c). On the other hand, it is likely that the dehydrogenases acting on fatty acids (see beginning of this chapter) and on glutamic acid (Hogeboom and Schneider, 1953a), as well as some of the transaminases which are required for the deamination of other amino acids by means of the glutaniate-a-ketoglutarate system (Hird and Rowsell, 1950; Miiller and Leuthardt, 1950b), are present mainly in the mitochondria, which also contain most of the cytochrome-linked succinic dehydrogenase. However, in these cases, quantitative data are only exceptionally available. In considering these results, even with due regard for the incompleteness of the information and for the various errors and artifacts which are likely to distort the picture, it is difficult to escape the conclusion that there nlust exist two distinct pathways of electron transfer within the living cell. One is entirely mitochondrial and is exemplified by the

262

CHR. DE DUVE A N D J. BERTHET

oxidation of /3-hydroxybutyrate, which, in the experiments of Lehninger (1951a, b), proceeded via internal DPN and cytochrome c. The other is mainly extramitcchondrial and operates, for instance, in the oxidation of isocitrate (Hogeboom and Schneider, 1 9 5 0 ~ )which ~ could involve the soluble isocitric dehydrogenase and soluble TPN, the microsomal T P N specific cytochrome c reductase and soluble cytochrome c, and finally the mitochondria1 cytochrome oxidase, which in this case should be situated on the surface of these granules. As for the distinguishing features between the two systems, they could be determined essentially by the nature of their respective substrates, As far as can be ascertained from the scanty data available, carbohydrate intermediates seem to be oxidized mainly by the extramitochondrial system, fatty acids and perhaps amino acids by the intramitochondrial one; and both systems may be involved in the oxidation of the common Krees cycle intermediates.* Interestingly enough, the two pathways have a common meeting ground in the cytochrome oxidase reaction, although even here the existence of two distinctly located enzymes may have to be considered ; one internal, acting on internal cytochrome c, and the other superficial, acting on external cytochrome c. I n any case, whether identical or different enzymes are involved, their close proximity would render the two systems mutually competitive for the available oxygen. One would then expect the external pathway to be favored in this competition, at least if equally supplied with oxidizable substrates. Such a possibility would go far in explaining the well-known balance obtaining in the liver cell between the metabolism of carbohydrate and that of fat and protein. Incidentally, the oxidases which act independently of the cytochrome system are not concentrated exclusively in the mitochondria, as is shown by the results of Novikoff et al. (1953) on uricase. The fact that catalase appears to have a heterogeneous distribution (Ludewig and Chanutin, 1950) may also be significant.

3. 0xida tizpe Plz 0splz oryla ti0n s If the dual picture of cellular oxidations presented in the preceding section is correct, the possibility exists that it may also apply to the associated phosphorylations. The ability of the mitochondria to catalyze these processes in the presence of a variety of oxidizable substrates has k e n well established by a number of authors and requires no further comment. On the other hand, the possible existence of an extra* Contrary to the “cyclophorase” concept (Green, 1951, 1952; Green, Loomis, and Auerbach, 1948), at least two enzymes operating in the Krebs cycle are present in significant quantities in the mitochondria1 supernatant, namely isocitric dehydrogenase (Hogeboom and Schneider, 19.50~) and fumarase (Appelmans et at., unpublished).

263

DIFFERENTIAL CENTRII’UGATION AND ENZYMES

mitochondriat pathway of oxidative phosphorylations has not yet been sufficiently investigated to warrant definite conclusions. In discussing this problem, a distinction has to be made between the phosphorylations coupled with the dehydrogenation of the substrate, with the electron transfer between coenzyme and cytochrome c and, finally, with the oxidation of cytochrome c by molecular oxygen. At least one reaction of the first type is known to occur outside the mitochondria: the phosphorylation associated with the triose phosphate-dehydrogenase reaction, which operates in glycolysis. As to the third type, assuming that it can occur-a point which is still disputed by some authors (Slater, 1950) -it must necessarily be mitochondria-linked. As in the case of cytochrome oxidase, one may have to consider in addition the possibility that two distinct mitochondrial sites are involved, one internal, the other superficial, but this distinction would be hard to demonstrate unequivocally. In the case of the intermediate step, the question can be put more clearly: are the electron transfers catalyzed by the extramitochondrial cytochrome c reductases phosphorylative or non-phosphorylative ? This problem has not yet been subjected to a direct experimental test. The nearest approach to it has been made by Lehninger (1951a, b), who was able to demonstrate phosphorylation coupled with the oxidation of DPNH by “native” mitochondria, whose internal systems were unavailable to the added DPNH. This evidence suggests that the external oxidative pathway was involved, but the fact that a mitochondria1 preparation was used renders the interpretation uncertain. Lehninger ( 1951a) also found evidence that the system operating in his experiments could catalyze a nonphosphorylative type of oxidation when the level of DPNH was kept very low compared with that of DPN by the addition of alcohol dehydrogenase and alcohol. Of considerable interest in this respect are the recent observations showing that intact mitochondria seem to be unable to carry out a nonphosphorylative type of oxidation. Their rate of respiration is very low in the absence of a suitable phosphate acceptor, and it is stepped up considerably by the addition of ADP (Kielley and Kielley, 1951) or of any system capable of regenerating ADP continuously from the ATP formed by oxidative phosphorylation, such as hexokinase glucose (Hunter, 1951; Kielley and Kielley, 1951; Lardy and Wellman, 1952; Niemeyer ef al., 1951 ; Potter and Recknagel, 1951 ; Rabinovitz et ai., 1951 ; Siekevitz and Potter, 1953b), creatine phosphokinase creatine (Lardy and Wellman, 1952), or simply the substrates necessary to induce the ATP-dependent synthesis of citrulline, which is carried out by the mitocliondria themselves (Siekevitz and Potter; 1953b). These experiments

+

+

264

CHR. DE DUVE A N D J. BERTHET

are of fundamental importance to our understanding of the mechanisms by which catabolic reactions are adjusted to the energy requirements of the cell. The respiration of mitochondria is also stimulated by a decrease in the tonicity of the medium (Potter and Recknagel, 1951 ; Slater and Cleland, 1953) and by the addition of 2,4-dinitrophenol (Hunter, 1951; Hunter and Spector, 1951 ; Judah and Williams-Ashman, 1951 ; Lardy and Wellman, 1952, and 1953; Potter and Recknagel, 1951; Siekevitz and Potter, 1953b; Witter, et d.,1953), of calcium ions (Siekevitz and Potter, 1953b) or of the nuclear fraction (Johnson and Ackerman, 1953; Potter, Lyle, and Schneider, 1951), of microsomes and of an acetone extract of microsomes, the latter effect being enhanced by potassium ions (Pressman, 1952; Pressman and Lardy, 1952). All these agents have been found to increase the adenosinetriphosphatase activity in the preparation (Hunter, 1951; Lardy and Wellman, 1953; Potter and Recknagel, 1951; Potter and Simonson, 1952; Pressman, 1952; Pressman and Lardy, 1952), and it has been suggested that, like those mentioned above, they act essentially by accelerating the rate of renewal of ADP from A T P (Potter, Lyle and Schneider, 1951; Potter and Recknagel, 1951 ; Siekevitz and Potter, 1953b). However, this cannot be the sole explanation, since it does not account for the observations that hypotonicity (Lehninger, 1951a, b ; Slater and Cleland, 1953) and the addition of nuclei (Johnson and Ackerman, 1953) stimulate the uptake of phosphate as well as the rate of respiration, when hexokinase and glucose are present. In the former case, it is probable that increased accessibility between the intramitochondrial enzymes and the external substrates and cofactors plays an important part in the observed stimulations. As to dinitrophenol, in addition to its effect on adenosinetriphosphatase, it must exert a specific action on one or more of the unknown steps involved in the phosphorylations associated witri the electron transfers between the reduced coenzymes and oxygen (Hunter, 1951; Lardy and Wellman, 1953). To conclude this discussion, some experiments on aging should also be mentioned. Preincubation of mitochondria without oxidizable substrates causes a progressive loss of oxidizing and phosphorylating capacity. This phenomenon can be reversed at an early stage by the addition of substrate (Kielley and Kielley, 1951), but later becomes irreversible; it is retarded by adenine nucleotides (Kielley and Kielley, 1951; Raaflaub, 1953) and is accompanied by swelling of the mitochondria (Harman and Fiegelson, 1952; Raaflaub, 1953) and by a considerable increase of the adenosinetriphosphatase activity of the preparation (Kielley and Kielley, 1951). These

DIFFERENTIAL CENTRIFUGATION AND ENZYMES

265

phenomena indicate that oxidative phosphorylations may be necessary to maintain the functional and structural integrity of the mitochondria. 4. Syitthetic Processes

Most of the present information concerning the intracellular localization of synthetic processes is derived from indirect studies in which tracer methods were combined with the technique of differential centrifugation. Only in exceptional cases has it been possible to determine directly the distribution of the enzymes systems involved. A number of authors have studied the turnover of RNA. The most striking fact revealed by these investigations is the very high rate of turnover of nuclear R N A which has been uniformly demonstrated, both with P32(Barnum and Huseby, 1950; Jeener and Szafarz, 1950; McIndoe and Davidson, 1952; Marshak, 1948; Marshak and Calvet, 1949; Payne, Kelly and Jones, 1952 ; Smellie and McIndoe, 1952 ; Smellie et al., 1953 ; Tyner, Heidelberger, and LePage, 1953) and with a number of organic precursors labeled with C1' or N16 (Eliasson et aE., 1951; Hurlbert and Potter, 1952 ;Payne, et al., 1952 ; Smellie and McIndoe, 1952 ; Tyner et al., 1953). Cytoplasmic R N A is renewed much more slowly, but there is still considerable disagreement about its relative rate of turnover in the various fractions. As pointed out by Smellie et al. (1953), differences in the techniques used for fractionation, as well as a varying degree of contamination of the cytoplasmic extract by nuclear RNA, are probably responsible for these discrepancies. On the basis of their results, Jeener and Szafarz (1950) have put forward the attractive hypothesis that the nucleus may be the main source of R N A in the cell, and that the freshly secreted RNA may serve as a seedling around which rnicrosomes and later mitochondria are built up (Chantrenne, 1947; Jeener, 1948). However, a closer examination of the time curves (Barnum and Huseby, 1950; Smellie et al., 1953), as well as the demonstration of profound chemical differences between nuclear and cytoplasmic R N R (Crosbie et al., 1953; Elson and Chargaff, 1951 ; McIndoe and Davidson, 1952), appear to argue against this possibility. I n addition to being active centers of RNA and, of course, of DNA synthesis, the nuclei may also be involved in the formation of some coenzymes, as shown by the distribution of the DPN-synthesizing enzyme (Hogeboom and Schneider, 1952b). On the other hand, recent investigations have failed to support the long-accepted theory that the nucleus may play an essential part in the synthesis of proteins. As shown by Brachet, Chantrenne, and their co-workers (Brachet and Chantrenne, 1951, 1952; Vanderhaeghe, 1952), enucleated halves of giant monocellular organisms

266

CHR. DE DUVE AND J. BERTHET

go on growing and synthesizing proteins at an almost undiminished rate several weeks after having been sectioned, and also retain the ability of producing adaptive catalase (Brachet and Chantrenne, 1953; Chantrenne, Brachet and Brygier, 1953). Moreover, it has been found by a number of investigators (Borsook, 1950 ; Borsook et al., 1950b; H u h , 1950; Lee et al., 1951 ; Tyner et al., 1953) that by far the greatest turnover of protein occurs not in the nucleus but in the microsomes. The latter also have the highest phospholipid turnover (Ada, 1949 ; Smellie et d.,1953). Several synthetic processes have been shown to occur in isolated mitochondria, namely the formation of p-aminohippuric acid (Kielley and Schneider, 1950), hippuric acid (Leuthardt and Nielsen, 1951 ; Nielsen and Leuthardt, 1949), citrulline (Grisolia and Cohen, 1953 ; Leuthardt and Miiller, 1948; Leuthardt, Miiller and Nielsen, 1949; Miiller and Leuthardt, 1950a : Siekevitz and Potter, 1953b), phosphatides (Kennedy, 1953) and fatty acids (Brady and Gurin, 1952). In the latter case, supplementation by the soluble fraction was essential. Owing to their high content in glutamic dehydrogenase and in transaminases (see above), mitochondria are probably important loci of amino acid formation. The incorporation in vitro of labeled amino acids into protein molecules has been studied in various isolated fractions (Borsook, 1950; Borsook et al., 195Oa; Peterson and Greenberg, 1952; Siekevitz, 1952). Of particular interest are the experiments of Siekevitz (1952), who showed that isolated microsomes were able to build radioactive alanine into their proteins, provided they were incubated with respiring mitochondria or with a soluble factor which appears to be formed by mitochondria by a process involving oxidative phosphorylation. The mitochondria alone did not take up radioalanine. As to the soluble fraction, it would appear from the distribution of the enzymes involved to be the main site of glycogen synthesis. These findings raise the interesting problem of how the synthetic processes are supplied with the necessary energy. If, as was surmised above, there are two more or less independent catabolic pathways in the cytoplasm, one may first wonder to what extent extramitochondrial syntheses can be supported by intramitochondrial reactions, and vice versa. As far as phosphate-bound energy is concerned, the recent investigations of Siekevitz and Potter (195313) indicate that the transfer of A T P can occur across the mitochondria1 membrane, but that when two differently located synthetic processes are in competition with each other, the one occurring on the site of A T P generation will probably be favored over the other. This discrimination may be much stricter when an exchange of reduced coenzymes becomes necessary, if the impermeability of intact mitochondria to D P N H reported by Lehninger (1951a, b) is a general phenomenon.

DIFFERENTIAL CENTRIFUGATION AND ENZYMES

267

The problem of energy supply becomes of particular importance in the case of nuclear syntheses, since it now appears very probable that the nucleus is unable to carry out oxidative phosphorylations. According to Lang and Siebert (1951), isolated nuclei are able to glycolyze hexose diphosphate and could derive most of their energy from this process. However, a comparison of their data with those of Kennedy and Lehninger (1949) suggests that the glycolytic activity of isolated nuclei is probably no more than a small percentage of the activity of the whole liver cell. In view of the low energy yield of glycolysis, it seems doubtful that enough energy could be supplied in this manner to cover the needs of nuclear syntheses. Another possibility is suggested by the observations made by FrPdCric (1951) and FrCdCric and Chkvremont (1952) on growing tissue cultures by means of phase contrast microcinematography. These workers have found that mitochondria frequently move up toward the nucleus and form close temporary attachments with the nuclear membrane. The images suggest that exchange of matter is taking place during these contacts. I t is not impossible that high-energy compounds may be supplied to the nucleus in this manner. Conversely, the mitochondria might derive some compounds from the nucleus, for instance DPN. The stimulating effect of nuclei on the oxidative and phosphorylative capacity of mitochondria, referred to above (Johnson and Ackerman, 1953), could be related to a process of this sort.

5. Permeability of Intracellular Bodies Several studies have dealt directly or indirectly with the permeability properties of mitochondria. Reference has been made in preceding sections to the various observations showing the existence of structural barriers between intramitochondrial enzymes and external substrates. Such observations may be of great significance but are not always easy to interpret. A more direct approach has been made possible, thanks to the fact that mitochondria behave as fairly typical osmotic systems, swelling in hypotonic media and also in media which are made isotonic by means of substances which penetrate through their membrane. In the latter case, the rate of swelling depends on the rate of diffusion of the solute and can serve to measure this rate. Studies of this kind on heart-muscle sarcosomes have been made by Cleland (1952)) using an optical method based on the observation, also made by Raaflaub (1953), that the swelling of mitochondria is accompanied by a decrease in the turbidity of their suspensions. In this laboratory, the osmotic swelling or disruption of mitochondria has been followed by the increased accessibility of the internal acid phosphatase to external glycerophosphate (Appelmans and de Duve, unpublished ;

268

CHR. DE DUVE AND J. BERTHET

Berthet et al., 1951). Although two different types of particles were studied (sarcosomes and B mitochondria, see p. 235), the results of both investigations have yielded comparable results showing that the granules are much more permeable to sodium and potassium chloride than they are to sucrose. Glucose and several divalent salts were found to enter the particles at a reduced rate, while glycerol penetrated almost instantaneously. In addition, Cleland (1952) found the permeability of sarcosomes to be strongly pH-dependent, with a pronounced minimum at p H 7.0. H e also made the interesting observation that one species of phosphate ions, the divalent form, enters the sarcosomes very rapidly. These results are in agreement with earlier morphologic observations showing that mitochondria do not retain their structural integrity in saline solutions as they do in sucrose (Dalton et al., 1949; Hogeboom et al., 1948). Their interpretation is complicated, however, by the recent findings of Bartley and Davies (1952), MacFarlane and Spencer (1953), and Spector ( 1953), that mitochondria are capable of maintaining a concentration of sodium and potassium ions above that of the surrounding medium, provided they are adequately supplied with oxidizable substrates. This behavior seemed to be associated with the capacity for oxidative phosphorylation and required the presence of adenine nucleotides, but was not inhibited by dinitrophenol. With an optical method, Raaflaub ( 1952) found that ATP, which protects the mitochondria against the inactivating effects of aging, also prevents their swelling in the absence of oxidizable substrates. The protection of heart-muscle sarcosomes by the removal of calcium demonstrated by Slater and Cleland (1952) may also be relevant to this phenomenon. A possible clue to these effects may be provided by the observation of Macfarlane and Spencer (1953) that mitochondria can actually decrease their water content under good conditions. This fact suggests the existence of a contractile system within or around the mitochondria-a distinct possibility in view of the striking motility of these granules in the living cell (Bourne, 195I ; Chbremont and Frkdkric, 1952 ; FrCdCric, 1951; FrCdCric and Chcvremont, 1952). The permeability of the nuclear membrane has given rise to many debates, but its direct study has been thwarted by the difficulty of isolating nuclei in good condition. Recent investigations by Anderson (1953), Anderson and Wilbur (1952), and Wilbur and Anderson (1951) indicate that isolated nuclei do not behave as osmotic systems, a fact in agreement with the generally accepted opinion that the nuclear membrane is very permeable. Characteristic morphologic changes could be induced by a number of electrolytes but seemed to be associated with physical modifications of the nuclear DNA rather than with osmotic phenomena.

DIFFERENTIAL CENTRIFUGATION AND ENZYMES

269

Hogeboom and Schneider (1953b) have recently commented on the nature of the nuclear envelope.

V. SUMMARYAND CONCLUSIONS The most impressive fact emerging from the preceding survey is the fundamental and far-reaching importance of the new technique of differential centrifugation in the study of cellular organization. As a bridge between the fields of cytology and biochemistry, it offers tremendous possibilities, which even the most carefully worked out techniques of cytochemistry could never have been expected to fulfill. I t must be remembered, however, that the application of differential centrifugation is fraught with many technical difficulties and open to a large number of errors. The methods that have been worked out today represent significant improvements over the earlier ones, but much still remains to be done to augment their accuracy and selectivity. For this purpose, it is important to have in mind the theoretical basis of the technique as well as the various factors of practical nature which have been found to affect the results. These have been surveyed in detailed manner in the first part of this review. The limitations of differential centrifugation become particularly severe when the technique is applied to the study of tissue enzymes. It is now quite clear that the observed partitions provide only the roughest sort of information concerning the true intracellular distribution of enzymes. They can only be considered as clues which have to be followed by many additional experiments in order to arrive at their real significance. As has been shown in the second part of this review, the a prwn’ assumption that specific enzymes are entirely concentrated in a given cellular site has proved extremely profitable in guiding these additional experiments, even in those cases in which it is found not to hold true. The use of enzyme determinations to ascertain the composition of isolated fractions is also of great interest and deserves more frequent application. As to the final interpretation of the results obtained in these studies, it should be emphasized not only that it is important to know what reactions do and can occur in isolated systems, but that the main object of cellular physiology is to ascertain which of the many processes discovered by biochemists actually do take place within the cell. Fundamental as they are, experiments on complex systems, planned with a more or less preconceived idea of the reactions likely to occur, are apt to be tnisleading and to confuse the picture. Such studies must be supplemented with accurate determinations of the distribution of the individual enzymes concerned and of the permeability properties of the structural barriers

270

CHR. DE DUVE AND J. BERTHET

interposed between them, before an attempt can be made to reconstruct the true organization of the cell.

ACKNOWLEDGEMENTS The following organizations have provided financial support to the personal investigations referred to in this review : The Fonds National de la Recherche Scientifique; the Centre National de Recherches sur la Croissance Normale et Pathologique ; the Institut Interuniversitaire des Sciences Nucleaires ; the Lilly Research Laboratories ; the Rockefeller Foundation. VI. REFERENCES Ada, G. L. (1949) Biochem. J., 46, 422. Allfrey, V., Stern, H., Mirsky, A. E., and Saetren, H. (1952) J . Gen. Phynol., 96, 529. Anderson, N. G. (1953) Enptl. Cell Resemch, 4, 307. Anderson, N. G., and Wifbur, K. M. (1952) J . Gen. Physiol., 36, 781. Appelmans, F., and de Duve, C. Unpublished results. Appelmans, F., Wattiaux, R., and de Duve, C. Unpublished results. Barkulis, S. S., and Lehninger, A. L (1951) 3. BWZ. ChPm., 190, 339. Barnum, C. P.,and Huseby, R. A. (1948) Arch. Biochm., 19, 17. Barnum, C. P.,and Huseby, R. A. (1950) Arch. Biochem., 29, 7. Bartley, W., and Davies, R. E. (1952) B i o c h . J., 62,xx. Beaufays, H., and de Duve, C. Unpublished results. Behrens, M. (1932) Hoppe-Scyler's 2. physiol. C h m . , 209, 59. Beinert, H. (1951) 3. Biol. Chem., 190, 287. Bensley, R. R.,and Hoerr, N. L. (1934) Anat. Record, 60,449. Bernhard, W.,Gautier, A., and Oberling, C. (1951) Compt. rend. SOC. hiof., 145, 566. Bernhard, W.,Haguenau, F. Gautier, A., and Oberling, C. (1952) Z . Zellforsch. 97, 281. Berthet, J., Berthet, L., Appelmans, F., and deDuve, C. (1951) Biochem. J . , 60, 182. Berthet, J., and deDuve, C. (1951) B i o c h . J., 60, 174. Borsook, H. (1950) Physiol. Revs., SO, 206. Borsook, H., Deasy, C. L., Haagen-Smit, A. J., Keighley, G., and Lowy, P. H. (1950a) 3. Biol. Chzm., 184, 529. Borsook, H., Deasy, C. L., Haagen-Smit, A. J., Keighley, G., and Low, P. H. (1950b) J . Biool. Chem., 187, 839. Bourne, G. H.(1951) Cytology and Cell Physiology, 2nd ed., p. 232. Oxford University Press, London. Rrachet, J., and Chantrenne, H. (1951) Nature, 166, 950. Brachet, J., and Chantrenne, H. (1952) Arch. irtterrz. physiol., 60, 547. Brachet, J., and Chantrenne, H. (1953) Arch. ititem. physiol., 61, 248. Brachet, J., and Jeener, R. (1944) EwyvzoZogia, 11, 1%. Bradfield, J. R. G. (1950) BioZ. Revs. Cambridge Phil. Soc., 26, 113. Rrady, R. O.,and Gurin, S. (1952) J. Bid. Chem., 199, 421. Chantrenne, H. (1947) Biochim. et Biophys. Acfa, 1, 437. Chantrenne, H., Brachet, J., and Brygier, J. (1953) Arch. intern. physiol., 61, 41Y. ChCvremont, M.,and Frkderic, J. (1952) Arch. biol. (Likge), gS, 259.

DIFFERENTIAL CENTRIFUGATION A N D ENZYMES

27 1

Claude, A. (1938) Proc. SOC.Exptl. Biol. Med. 39, 398. Claude, A. (1941) Cold Spring Harbor SymposM Quant. Biol., 9, 263. Claude, A. (1942) Tram. N.Y. Acad. Sci., (2) 4, 79. Claude, .4. (1943) B i d . Symposia, 10, 111. Claude, A. (1946a) I . Exptl. Med., 84, 51. Claude, A. (1946b) J . Exptf. Med., 84, 61. Claude, A. (1948) Harvey Lectures, 45, 121. Claude, A, (1949) In Advances in Protein Chemistry, Vol. 5, p. 423. Academic Press. New York. Claude, A., and Fullam, E. F. (1945) J . Exptl. Med., 81, 51. Claude, A., and Potter, J. S. (1943) J . Exptl. Mrd., 77, 345. Cleland, K. W. (1952) Natwre, 170, 497. Crosbie, G. W., Smellie, R. M. S., and Davidson, J. N. (1953) Biochem. 1.. 64, 287. Dalton, A. J., Kahler, H., Kelly, M. G., Lloyd, B. J., and Striebich, M. J. (1949) J . Natl. Cancer Iitst., 9, 439. Dalton, A. J., Kahler, H., Striebich, M. J., and Lloyd, B. J. (1950) J . Natl. Canscr m.,11, 439. deDuve, C. (1952) In Expos& Annuels de Biochimie Midicale, Vol. 14, p. 46. Masson, Paris. de Duve, C., Appelmans, F., and Wattiaux, R. (1952) 2nd Intern. Congr. Oiochem. Abstr. of Commun., p. D8. de Duve, C. and Berthet, J. (1953) Nahre, 174, 1142. deDuve, C., Berthet, J., Berthet, L., and Appelmans, F. (1951). Nafure, 167, 389. de Duve, C., Gianetto, R., Appelmans, F., and Wattiaux, R. (1953) Nature, 172, 1143. Dianzani, M. U. (1951a) Arch. f;sioI., 60, 175. Dianzani, XI. U. (1951b) Arch fisio!., 60, 181. Dianzani, Id.U. (19%) Arch. fisiol., 60, 187. Dounce, A. L. (1943) 1. Biol. Chena., 147, 685. Dounce, A. L. (1950) In The Enzymes, Vol. 1, p. 187. Academic Press, New York. Dounce, A. L., and Beyer, G . T. (1948) J . Biol. Chem., 174, 859. Dounce, A. L., Kay, E. R. M., and Pate, S. G . M. (1953) Federation Proc., l2, 198. Dounce, A. L., and Litt, M. (1952) Federation Proc., 11, 203. Dounce, A. L., Tishkoff, G. H., Barnett, S. R., and Freer, R. M. (1950) J. Gen. Physiol., SS, 629. Drysdale, G. R., and Lardy, H. A. (1953) J . Biof. Chem., 203, 119. Eliasson, N. A., Hammarsten, E., Reichard, P., Aquist, S., Thorell, B.. and Ehrensvlrd, G. (1951) Acta Chem. Scand., 6, 431. Elson, D., and Chargaff, E . (1951) Fedpratioiz Proc., 10, 180. Frkdiric, J. (1951) Compt. rend. SOC. biol., 146, 1913. FrbdCric, J., and Chivremont, M. (1952) Arch. Dial. (Lihge), 63. 109. Ganguly, J., and Deuel, H. J., Jr. (1953) Nutiire, 172, 120. Gianetto, R., and de Duve, C., Unpublished results. Goethart, G. (1952) Biochim. et Biophyys. Acta, 8, 479. Green, D. E. (1951) In Enzymes and Enzyme Systems, p. 15. Harvard University Press, Cambridge. Green, D. E. (1952) 2nd Intern. Corigr. Biochm., Symposium sur le cycle tricarboxylique, p. 5. Sedes, Paris. Green, D. E., Loomis, W. F., and Auerbach, V. H. (1948) J. Biol. Chtm., 172, 389. Grisolia, S., and Cohen, P. P. (1953) J . Biol. Chew, 753.

272

C H R . DE DUVE A N D J. BERTHET

Harman, J . W. (1950a) Exbtl. Cell Research, 1,382. Harman, J. W. (195Ob) Enptl. Cell Research, 1, 394. and Fiegelson, M. (1952) Exbtl. Cell Research, 3, 509. Harman, J. W., Hazin, S. J., Singal, S. A., Sydenstricker, V. P., and Littlejohn, J. M. (1953) Federation Proc., 12, 216. Hers, H. G., Berthet, J., Berthet, L.,and de Duve, C. (1951) Bull. soc. c h b . biol., s9, 21. Higgins, H., Miller, J. A., Price, J. M., and Strong, F. M. (1950) Proc. SOC.Erfitl. Biol. Med., 76, 462. Hird, F. J. R., and Rowsell, E. V. (1950) Naticre, 166, 517. Hogeboom, G. H. (1949) J . B i d . Cherri., 177, 847. Hogeboom, G. H. (1951) Federntion Proc., 10, 640. Hogeboom, G.H., and Schneider, W.C. (1950a) Nature, 166, 302. Hogeboom, G. H., and Schneider, W. C. (1950b) I. Natl. Cattcer Znsf. 10, 983. Hogeboom, G. H., and Schneider, W. C . (1950~)J . Biol. Chem., 186, 417. Hogeboom, G. H., and Schneider, W. C. (1952a) J. Biol. Chern., 194, 513. Hogeboom, G. H., and Schneider, W. C. (1952b) 1. BioI. Chem., 197, 611. Hogeboom, H.,and Schneider, W. C . (1953a) J. Biol. Chcm., 204, 233. Hogeboom, H., and Schneider, W. C. (1953b) Science, 118, 419. Hogeboom, G. H., Schneider, W. C., and Palade, G. E. (1948) I . Biol. Cltem., 172, 619. Hogeboom, G. H., Schneider, W. C., and Striebich, M. J. (1952) J. Biol. Chem., 196, 111. Hogeboom, H., Schneider, W. C., and Striebich, Id.J. (1953) Cancer Research, lS, 617. Holter, H. (1952) Adzances in Emymol., 13, 1. Holter, H., Ottesen, M., and Weber, R. (1953) Enperientka, 9, 346. Huennekens, F. M., and Green, D. E. (1950) Arch. Biochem., 27, 418. Hultin, T. (1950) Exptl. Cell Research, 1, 376. Hunter, F. E., Jr. (1951) In Phosphorus Metabolism, Vol. 1, p. 297. The Johns Hopkins Press, Baltimore. Hunter, F. E., Jr., and Spector, S. (1951) Federation Proc., 10, 201. Hurlbert, R. B., and Potter, V. R. (1952) J . Biol. Chem., 196, 257. Jeener, R. (1948) Biochim. et Biophys. Acta, 2, 633. Jeener, R., and Szafarz, D. (1950) Arch. Biochem., 26, 54. Johnson, R. B., and Ackermann, W. W. (1953) J. Biol. Chem., 240, 263. Judah, J. D., and Williams-Ashman, H. G. (1951) Biochem. /., 48, 33. Kabat, E. A. (1911) Sciarce, 93, 43. Kennedy, E. P. (1953) J. BioZ. Chem., 201, 399. Kennedy, E. P., and Lehninger, A. L. (1948) J . Biol. Chem., 172, 847. Kennedy, E. P., and Lehninger, A. L. (1949) J . Biol. Chem., 179, 957. Kensler, C. J., and Langemann, H. (1951) J . Biol. Chem., 192, 551. Kielley, W. W., and Kielley, R. K. (1951) J . Biol. Chent., 191, 485. Kielley, W.W., and Kielley, R. K. (1953) J . Biol. Chem., 200, 213. Kielley, R. K., and Schneider, W. C. (1950) J . Biol. Chem., 186, 869. Kornberg, A. (1950) /. B i d . Chem., 182, 779. Laird, A. K., Nygaard, O., and Ris, H. (1952) Canccr Research, 12, 276. Laird, A. K., Nygaard, O., Ris, H., and Barton, A. D. (1953) Exptl. Cell Researck, 6,

147.

DIFFERENTIAL CENTRIFUGATION AND ENZYMES

2?3

Lang, K. (1939) Hoppe-Seyler‘s 2. physiol. Chem., 261, 240. Lang, K. (1952) In Mikroskopische und Chemische Organisation der Zelle, p. 24. Springer Verlag, Berlin. Lang, K., and Siebert, G. (1951) Biochem. Z.,322, 196. h g , K., and Siebert, G. (1952) Biochenz. Z., 322, 360. Lardy, H. A., and Wellrnan, H. (1952) I. Biol. Chem., lQ6, 215. Lardy, H. A., and Wellrnan, H. (1953) J. Biol. Chem., 201, 357. Lazarow, A. (1942) Anat. Record, 84, 31. Lee, N. D., Anderson, J. T., Miller, R., and Williams, R. H. (1951) I. B i d . Chem., 192, 733. Lehninger, A. L. (1945) J. Biol. Chem., 161, 437. Lehninger, A. L. (1951a) In Phosphorus Metabolism, Vol. 1, p. 344. The Johns Hopkins Press, Baltimore. Lehninger, A. L. (1951b) J. Biol. Chem., 190, 345. Leloir, L. F., and Muiioz, J. M. (1939) Biochetn. I., 33, 734. LePage, G. A., and Schneider, W. C. (1948) J. Biol. Chem., 176, 1021. Leuthardt, F., and Mauron, J. (1950) Helv. Physiol. et Phrnaacol. Actu, 8, 386. Leuthardt, F., and Miiller, A. F. (1948) Enperientiu, 4, 478. Leuthardt, F., Miiller, A. F., and Nielsen, H. (1949) H e h . Chint. Acta, 32, 744. Leuthardt, F., and Nielsen, H. (1951) Helv. Chim. Acta, 34, 1618. Ludewig, S., and Chanutin, A. (1950) Arch. Biochem., 29, 441. Lynen, F., and Ochoa, S. (1953) Biochiw. et Biophys. Acta, 12, 299. Macfarlane, M. G., and Spencer, A. G. (1953) Biochem. I., 64, 569. McIndoe, W. M., and Davidson, J. N. (1952) Brit. I. Cancer, 6, 200. McShan, W. H., and Meyer, R. K. (1952) Etrdocriitology, 60, 294. McShan, W. H., Rozich, R., and Meyer, R. K. (1953) Endocrinology, 62, 215. Marshak, A. (1941) J . Gen. Physiol., 26, 275. Marshak, A. (1948) J . Cellular Covip. Physiol., S2, 381. Marshak, A., and Calvet, F. (1949) J. Cellular Comp. Physiol., 34, 451. Maver, M. E., and Greco, A. E. (1951) J. Natl. Cancer Inst., U,37. Meister, A. (1952) Scieme, 116, 521. Mirsky, A. E., and Pollister, A. W. (1946) I . Gen. PhytySiol., SO, 117. Mirsky, A. E., and Ris, H. (1947) I . Gob. Physiol., 31, 1. Mueller, G. C., and Miller, J. A. (1949) 1. Biol. Chem., 180, 1125. Miihlethaler, K., Muller, A. F., and Zollinger, H. U. (1950) Exjen’entia, 6, 16. Miiller, A. F., and Leuthardt, F. (1950a) Helv. Chim. A c f q SS, 262. Miiller, A. F., and Leuthardt, F. (1950b) Helv. Chint. Acta, S3, 268. Muiioz, J. M., and Leloir, L. F. (1943) J. Biol. Chem., 147, 355. Nielsen, H., and Leuthardt, F. (1949) Helv. Physiol. Acfu, 7, C, 53. Niemeyer, H., Crane, R. I<., Kennedy, E. P., and Lipmann, F. (1951) Federation Proc., 10, 229. Novikoff, A. B., Hecht, L., Podber, E., and Ryan, J. (1952) I. Biol. Chem., 194, 153. Novikoff, A. B., Podber, E., Ryan, J., and Noe, E. (1953) J . Histochern. Cytochem., 1, 27. Oberling, C., Bernhard, W., Gautier, A., and Haguenau, F. (1953) Presse mid., 61, 719. Ornachi, A., Barnum, C. P., and Glick, D. (1948) Proc. SOC.Enpfl. Biol. Med., 67, 133.

Palade, G. E. (1951) Arch. Biochem., SO, 144.

274

CHR. DE DUVE AND J. BERTHET

Palade, G. E. (1952) Anut. Record, 114, 427. Palade, G. E. (1953) J . Histochem. Cytochem., 1, 188. Palade, G. E.,and Porter, K. R. (1952) Anat. Record, 112, 370. Payne, A. H.,Kelly, L. S., and Jones, H. B. (1952) Cancer Research, 12, 666. Perry, S. V.,and Horne, R. W. (1952) Biochim. et Biophys. Actq 8, 483. Peterson, E. A.,and Greenberg, D. M. (1952) 1. B i d . Chem., 194, 359. Pickels, E. G. (1943) J . Gen. Physiol., 26, 341. Pirotte, M.,and Desreux, V. (1952) Bull. SOC. chim. Belges, 61, 167. Porter, K.R. (1953) J. Exptl. Med., 97, 727. Porter, K. R., and Thompson, H. P. (1947) Cancer Research, 7, 431. Potter, V. R., and Elvehjem, C. A. (1936) J. Biol. Chem., 114, 495. Potter, V. R.,Lyle, G . G., and Schneider, W. C. (1951) J . BioI. Chem., 190, 293. Potter, V. R., and Recknagel, R. 0. (1951) In Phosphorus Metabolism, Vol. 1, p. 377. The Johns Hopkins Press, Baltimore. Potter, V. R., Recknagel, R. O., and Hurlbert, R. B. (1951). Federation Proc., 10, 646. Potter, V. R., and Simonson, H. C. (1952) Federation Proc., 11, 270. Pressman, B. C. (1952) Federation Proc., 11, 271. Pressman, B. C., and Lardy, H. A. (1952) J . Biol. Chem., 197, 547. Raaflaub, J. (1953) Helv. Physiol. Acta, 11, 142. Rabinovitz, M.,Stulberg, M. P., and Boyer, P. D. (1951) Science, 114, 641. and Lehninger, A. L. (1952) I. Biol. C k . , lB4, 119. Rall, T.W., Randavel-Vendrely, C. (1949) Acta Atuzt., 7, 2 5 . Rosenthal, 0. (1953) Federation Proc., 12, 260. Schein, A. H., Podber, E., and Novikoff, A. B. (1951) J . Biol. Chem., 190. 331. Schick, A. F.,and Hass, G. M. (1949) Science, 109, 486. Schneider, R. M.,and Petermann, M. L. (1950) Cancer Research, 10, 751. Schneider. W. C. (1948) 1. Biol. Chrm., 176, 259. Schneider, W. C.,Claude, A., and Hogeboom, G. H. (1948) I. Biol. C k m . , 172, 451. Schneider, W.C., Dalton, A. J., Kuff, E. L., and Felix, M. (1953) Nafure, 172, 161. Schneider, W.C., and Hogeboom, G. H. (195Oa) J. Biol. Chm., 18S, 123. Schneider, W.C., and Hogeboom, G. H. (1950b) 1. Natl. Cancer Inst., 10, %9. Schneider, W.C., and Hogeboom, G. H. (1951) Cancer Research, 11, 1. Schneider, W.C., and Hogeboom, G. H. (1952a) J. Biol. Chem., US, 161. Schneider, W. C., and Hogeboom, G. H. (1952b) J . Biol. C h , 198, 155. Schneider, W.C., Hogeboom, G. H., and Ross, H. E. (1950) I . Natl. Cancer Irrrt., 10, 977. Schneider, W. C., and Potter, V. R. (1949) J. Biol. Chem., 177, 893. Siekevitz, P. (1952) 1. Biol. Chem., 196, 549. Siekevitz, P., and Potter, V. R. (1953a) J . Biol. Chem., 200, 187. Siekevitz, P., and Potter, V. R. (1953b) J. Biol. Chem., aOl, 1. Sjostrand, F. S. (1953) Nature, 171, 30. Sjostrand, F. S.,and Rhodin, J. (1953) Exptl. Cell Research, 4, 426. Slater, E. C. (1950) Nature, 166, 982. Slater, E. C., and Cleland, K. W. (1952) Nature, 170, 118. Slater, E. C., and Cleland, K. W. (1953) Biochem. I., M, 557. Smellie, R. M. S., and McIndoe, W. M. (1952) B i o c h I., 62, xxii. Smellie, R. hi. S., McTndoe, W. M., Logan, R., Davidson, J. N., and Dawson, I. M. (1953) Biochm. J., 64, 280.

DIFFERENTIAL CENTRIFUGATION AND ENZYMES

275

Spector, W. G. (1953) Proc. Roy. SOC.(London), B141, 269. Stern, H.,Allfrey, V., Mirsky, A. E., and Saetren, H. (1952) J. Gefi. PhySiol., 36, 559.

Stern, K. G. (1939) Cold Spring Harbor Symposia Quant. Biol., 7, 312. Stoneburg, C. A. (1939) J. Biol. Chm., 129, 189. Sung, S. C.,and Williams, J. N., Jr- (1952) J. Biol. Chew, 197, 175. Sutherland, E.W., and Cori, C. F. (1951) J. Biol. Chem., 188, 531. Swanson, M.A. (1951) J . Biol. C h m . , 191, 577. Swendseid, M. E., Bethell, F. H., and Ackermann, W. W. (1951) J. Biol. C h . , 190, 791. Tsou, C. L. (1952) Biochem. J., 60, 493. Tsuboi, K. K. (1952) Biochim. ef Biophys. Acta, 8, 173. Tyner, E. P.,Heidelberger, C., and LePage, G. A. (1953) Cancer Research, 13, 186. Vanderhaeghe, F. (1952) Arch. idem. physiol., 60, 190. Walker, P. G. (1952) B i o c h m ~J., 61, 223. Walker, P. G.,and-Levvy, G. A. (1953) Bwchem. J., 64, 56. Watson, M. L. (1952) University of Rochester Atomic Energy Project, unclassified report, U.R. 185. Wilbur, K. M., and Anderson, N. G. (1951) Exptl. Cell Research 2, 47. Williams, J. N., Jr. (1952a) J. Biol. Chem., 194, 139. Williams, J. N.,Jr. (1952b) J. Bhl. Chem., 196, 37. Witter, R. F.,Newcomb, E. H., and Stotz, E. (1953) J . Biol. Chem., 202, 291.

This Page Intentionally Left Blank

Enzymatic Aspects of Embryonic Differentiation TRYGGVE GUSTAFSON The Wenner-Gren Zttsfifute for Experimental Biology, University of Stockholm, Sweden

I. 11. 111. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII.

Page Introduction ....................................................... 277 Analytic Data from the Developing Sea Urchin Egg ................ 277 Intracellular Localization of Enzymes in Eggs and Embryas .......... 279 Factors Modifying the Enzyme Activity in Homogenates and ifi dvo .. 284 Cofactors and the Control of Their Formation ...................... 291 On the Genesis of New Mitochondria in Embryonic Differentiation .. 295 The Primary Pattern of Mitochondrial Distribution .................. 301 The Metabolic Background of the Mitochondria1 Distribution ........ 305 The Gradual Complication of the Mitochondria1 Pattern ............ 309 On Qualitative Biochemical Differentiation ........................ 311 The Mode of Operation of Mitochondria in Morphogenesis .......... 314 On the Mode of Action of Li Ions in the Developing Egg ............ 316 References ........................................................ 320

INTRODUCTION Julius Sachs, in a paper Uber Stoff und Form der Pflansenorgam, postulated that the morphologic development of a plant is determined by its chemical constitution. On the basis of this concept, Herbst (1892) tried to modify morphogenesis in animals by changing the chemical composition of the embryos. Marine invertebrate eggs are suitable for this purpose, since chemical agents may be administered via the surrounding medium. In the course of this investigation, Herbst observed a remarkable effect of lithium ions, Li, on sea urchin development. Li in fact brought about a change designated as vegetalization, i.e., enormous hypertrophy of the endoderm at the expense of the ectoderm, which thus became rudimentary (Fig. 1). Maximal morphogenetic effects are obtained if the Li treatment coincides with the period of exponential respiratory rise during the cleavage stages of the egg (Lindahl, 193913, 1940) (Fig. 2). Li added at any time during this sensitive period reversibly checks the further respiratory rise, but not the actual respiratory activity. It was suggested that this Li sensitive respiratory rise was connected with the elaboration of particulate units which control the synthetic activity in later stages of development and thereby also control the morphogensis (Gustafson 1950).

11. ANALYTICAL DATAFROM

THE

DEVELOPING SEAURCHINEGG

A series of biochemical studies were undertaken in order to investigate whether the action of Li could be correlated with changes in synthetic

277

278

TRYGGVE GUSTAFSON

activity, and if such changes could be referred to the development of particulate units. An essential part of these studies was devoted to enzymatic development. The enzyme studies were supplemented by an investigation of the amino acid composition of the developing egg, and studies of its serologic and cytologic properties.

A

66 8

C

FIG.1. Vegetalized sea urchin larvae; vegedization increasing in strength from the left to the right. Redrawn after Lindahl and ohman (1938).

501

I

4 8 I2 16 20 24 28 FIG.2. Relative respiratory intensity of the sea urchin egg in different stages of

0

development. Hatching indicated by H ; the invasion of primary mesenchyme cells in the blastocoele indicated by I. The respiratory rise during the “cleavage stages” before H is reversibly inhibited by lithium ions. Redrawn after Lindahl (1939b).

ENZYMATIC ASPECTS OF EMBRYONIC DIFFERENTIATION

279

The total changes in the amino acid composition of the developing egg were found to be rather insignificant. As a rule, no changes appeared before the hatching blastula stage or around the onset of visible differentiation in the advanced blastula. More pronounced changes were noted in the fractions of free amino acids, peptides, and certain peptidic growthpromoting compounds (Fig. 6). Transient or permanent depletions in these fractions signalize the onset of new and intense biochemical processes in the advanced blastula (Fig. 6) (Gustafson and Hjelte, 1951; Kavanau, 1953, 1954 ; Gustafson, HjeIte and Hasselberg, 1952 ; Kavanau, Gustafson, and Binhidi, 1954). Studies on antigenic and enzymatic development gave support to the impression that the cleavage stages are a “silent phase” in many respects. New antigens thus could not be demonstrated in sea urchin development before the early gastrula stage (Perlmann and Gustafson, 1948). Between this stage and the pluteus stage a new serologic component turned up. Furthermore, the activity of a series of enzymes was low and comparatively constant during the cleavage stages, but increased in the advanced blastula or during gastrulation (Fig. 3a, b). This series of enzymes includes alkaline phosphatase (Mazia et aE., 1948 ; Gustafson and Hasselberg, 1950), dehydrogenases, glutaminase I, cathepsin 11, apyrase (Gustafson and Hasselberg, 1951), and cholinesterase (Augustinsson and Gustafson, 1949). In another series of enzymes, however, the activity was constant throughout the early development up to the pluteus stage (Fig. 3c). This series comprises aldolase, adenosindeaminase, phenylsulfatase, acid phosphatase, enzymes splitting inorganic pyro- and hexametaphosphate, a proteolytic enzyme (Gustafson and Hasselberg, 1951), deoxyribonuclease (Mazia, 1949), and a peptidase attacking alanylglycine (Holter and Lindahl 1940). It is evidently possible to classify the enzymes in two main groups, those showing constant activity up to the pluteus stage, and those increasing in activity at the onset of visible differentiation. I t may now be asked to what extent this classification reflects the intracellular localization of the enzymes.

LOCALIZATION O F ENZYMES I N E G G S AND EMBRYOS In 1913 Warburg demonstrated the respiratory activity of minute particles extracted from liver. This observation initiated a new branch of biochemistry which emphasized the importance of the particulate localization of certain enzymes (cf. Holter, 1952; Green, 1952; Lindberg and Ernster, 1954). The larger particles have often been identified as mitochondria, long known to the cytologists. These particles were shown to be the site of numerous enzymes, e.g., those active in the intracellular 111.

INTRACELLULAR

280

TRYGGVE GUSTAFSON

oxidation and associated processes of phosphorylation. The smaller granules, the microsonies, are generally considered to be poorer in enzymes than the mitochondria. The intracellular localization of enzymes may be severely altered during the process of homogenization (cf. Holter, 1952). This source of error can be circumvented by studying the enzyme distribution in eggs stratified or even fragmented by centrifugal force according to the method of Harvey (1931). Using this technique, Holter (1936) showed that no formed inclusions could be the sole carriers of peptidase in marine eggs such as Arbucia and Chuetopterw. The activity showed mainly the same distribution as the ground cytoplasm in eggs stratified by means of centrifugation. Catalase ought to have a similar localization in the unfertilized egg (Holter 1937). Using the same technique, Mazia (1949) concluded that deoxyribonuclease occurs in the soluble state in the unfertilized egg. Glycolytic enzymes are further localized in the supernatant of centrifuged homogenates of Echinus according to Cleland and Rothschild (1952a).

/oo

'

/O-

'

10 HOUfS

-

20

0

30

60

50

FIG.3(a)

FIG.3. Relative enzyme activity in normal and Li-treated sea urchin eggs in different stages of development; time for Li treatment indicated; a : apyrase; b : glutaminase ; c : aldolase. From Gustafson and Hasselberg (1951).

ENZYMATIC ASPECTS OF EMBRYONIC DIFFERENTIATION

Hours

-

Hours

FIG.3 ( b ) .

-

FIG.3 ( c ) .

281

282

TRYGGVE GUSTAFSON

On the basis of studies by Shapiro (1935) and Navez and Harvey (1935), HoIter (1936) pointed out that respiratory enzymes and peptidases have an essentially different localization. Studying the respiratory activity of the light and heavy fragments of the centrifuged egg, Shapiro thus showed that the sedimentable granules play a most important role in the process of oxidation. Similar results were obtained by Navez and Harvey for the indophenoloxidase reaction in centrifuged eggs. Ballentine (1939) furthermore demonstrated that a great fraction of the dehydrogenases is bound to such granules, and Keltch et al. (1950) state that the mitochondria contain the enzymes necessary for cellular oxidation and the associated reactions of phosphorylation. I t may be postulated that the whole complex of the tricarboxylic acid cycle, as demonstrated in'the sea urchin egg by Cleland and Rothschild (195Zb), resides in the mitochondria. Acid phosphatase is also bound to granules of mitochondria1 or vitelline origin, but not to the nuclei (Mazia, 1949). Mitochondria may also contain proteolytic enzymes. Woodward ( 1949) thus demonstrated proteolytic activity in cytoplasmic granules obtained from unfertilized Arbacia eggs by means of fractional centrifugation, and Lundblad (1952) reports a similar localization in Pararentrotus. Studies on enzymatic localization in other invertebrate eggs show a similar intracellular distribution as described for the sea urchin. Different criteria indicate that the tricarboxylic acid cycle enzymes of Ostrea are bound to granules and behave as a complex in a centrifugal field (Cleland, 1951; cf. 1950). Cleland fractionated the intracellular granules into four groups : protein yolk, mitochondria, microsomes, and lipid or Golgi material. The enzymes of the tricarboxylic cycle were found only in the mitochondria fraction and in the protein yolk. In contrast, lipase, acid phosphatase, and apyrase were distributed applroximately uniformly between ground cytoplasm and granules, whereas amylase was present only in the ground cytoplasm. The glycolytic system was most active in homogenate supernatants where it was qualitatively similar to the system in whole homogenates. In Mactru a proteolytic enzyme with p H optimum 7.5-7.6 is present in the small granules and in the clear supernatant, whereas another proteolytic activity with a p H optimum 3.5-4.0is associated with the granule-free supernatant (Woodward, 1950). Respiratory enzymes are not always localized in mitochondria. In Chaetopterus the enzymes glucosed-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, which are essential to the oxidative formation of pentose phosphate, reside in the supernatant of homogenized embryos. The enzymes show a constant activity up to a 100 cell swimming stage at least (Cohen, 1951). These enzymes are probably important in the carbo-

ENZYMATIC ASPECTS OF EMBRYONIC DIFFERENTIATION

283

hydrate metabolism in the sea urchin egg (Lindberg, 1946; Lindberg and Ernster, 1948 ; Hultin, 1953 ; Cleland and Rothschild, 1952b), where they may behave in a similar way as in Chaetopterus. Studies on vertebrate material reveal an intracellular localization of enzymes which is in many details similar to that in the sea urchin egg and adult mammalian tissues. Steinbach and Moog (1945) already showed that adenylpyrophosphatase as well as acid and alkaline phosphatase in the chick embryo is bound to intracellular granules. They suggest that these granules are components of a cytoskeleton on which the enzymatic activities of the intact cell are orientated. Spratt (1952), reviewing the chemical embryology of the chick, suggests an association between the dehydrogenase activity, the cytochrome system, and the mitochondria. In the ovarian frog egg, Recknagel (1950) found that a major fraction of the cytochrome oxidase is localized in large granules. Brachet (1952) stresses, furthermore, that alkaline phosphatase and the respiratory activity show the same animal-vegetal gradient in the frog germ as do the sulfydryland nucleic acid-rich granules to which these enzymes are generally bound. The data above indicates that the enzymes in the sea urchin egg with a developmental rise in activity may be mitochondrial, whereas those with a constant activity are, as a rule, non-mitochondrial. The percentage rise in activity and the time for the rise of the different enzymes were not uniform. Yet it may be fair to assume that the rise in activity mainly reflects an increase in both the amount of enzyme and the mitochondrial number at the onset of visible differentiation. Li treatment during cleavage stages retarded the subsequent rise in activity of a series of enzymes, as well as the changes in amino acid composition (Fig. 3a, b) . This Li treatment should thus check the subsequent rise in mitochondrial number, which is in conformity with the assumptions of Gustafson (1950). The hypothesis above was supported by aid of mitochondrial counts (Gustafson and Lenicque, 1952). Particles stainable with the mitochondrial vital stain Janus green were observed in the pluteus. These bodies were also stainable with Nile blue sulfate and Dahlia violet. They were also recognized in the phase microscope as spheres or dumbbell-shaped structures. After staining with Nile blue sulfate, Dahlia, or similar stains, the particles resembled granules or rods, as a rule with thickened ends. The use of basic stains for staining of the chondriome has been applied by Harvey in the sea urchin (1931), by Ries and Gersch (1936) in invertebrates, and by Holtfreter in amphibians (1946a, b). The stainability may be due partly to a rim of basophilic material which often surrounds mitochondria and which may show quantitative variations (cf. Opie, 1947).

284

TRYGGVE GUSTAFSON

The stainable elements were counted in sit%in larvae flattened beneath the coverslip. Curves on the relative mitochondria1 number in different early stages were obtained, using the above method. These curves (Fig. 4) resemble the enzyme curves, even with regard to the retarding effect of Li.

Hours

FIG.4. The relative mitochondria1 number in normal and Li-treated developmental stages of the sea urchin egg; Li treatment between 3 and 6 hours after fertilization; dotted curve : Li-treated larvae. From Gustafson and Lenicque (1952).

IV. FACTORS MODIFYINGTHE ENZYME ACTIVITY IN HOMOCENATES AND In Vivo The cytologic and the enzymologic studies on sea urchin eggs are evidently in good agreement, since both indicate formation of mitochondria at the start of visible differentiation, but certain enzyme curves show an unexpected course which apparently does not fit into the picture. This may depend upon the interference of different factors blocking the enzyme a'ctivity in vivo, in homogenates, or in both. Some examples of such factors in the sea urchin egg and elsewhere will be reported. The curve for catalase in sea urchin development is different from that of most of the other enzymes studied. After a slight, somewhat uncertain drop upon fertilization (Doyle, 1938), the activity is constant up to the late blastula, but a second drop then occurs (Deutsch and Gustafson,

ENZYMATIC ASPECTS O F EMBRYONIC DIFFERENTIATION

285

1952) (Fig. 5a). This drop is about synchronous with the rise in activity of the mitochondria1 enzymes. The marked decrease in actiV;ty of catalase suggests a decrease in the amoutif of this enzyme. The marked increase in

20

t

"0 4 Hours

Hours

8

I2

16

20

24

28

32

FIG.5a.

FIG.5b.

FIG.5. Relative enzyme activity in the sea urchin egg in different stages of development ; a : catalase. b : cytochrome oxidase. From Deutsch and Gustafson (1952).

286

TRYGGVE GUSTAFSON

activity noted upon aging of a dilute Iysate and the inhibitory effects of an embryo lysate kocksuft suggests, however, that the decrease merely concerns the activity and reflects the formation of an inhibitor. The actual concentration of the enzyme may remain constant or even increase. The general importance of the catalase inhibitor is suggested by a series of papers on catalase activity in relation to neoplastic growth. Greenstein, et d.,(1941) demonstrated that liver catalase of rats and mice bearing extrahepatic tumors is markedly lowered, as has been repeatedly verified. Hargreaves and Deutsch ( 1952) recently demonstrated the inhibition in vitro of both crystalline catalase and liver homogenate enzyme by kocksuft from Jensen rat sarcoma. The inhibitor was a low molecular weight substance, rather stable to acid and alkali. Spectroscopic studies suggest that its inhibition is effected through the iron porphyrin group of the enzyme. The reaction is a function of the inhibitor concentration and time of incubation. The inhibitor was evidently present in a bound and inactive state in the tumors, but was released upon heating to 100" C. Reversal of the inhibition occurred upon dilution as in the sea urchin lysates. The inhibitor appeared to be transported via the blood stream from the tumor to the liver. In homogenates of this organ it was probably destroyed by some factor. The inhibitor was more or less characteristic for tumor tissues, and probably for embryonic cells, since adult material only showed slight or no activity. The identity of the inhibitor with glutathione has been suggested (Rybak and Boeri, personal communication). An inverse variation of the SH content and catalase activity has, in fact, been described from germinating seedlings (Goksiiyr, et al., 1953). The catalase inhibitor may also explain the low figures for the catalase activity of sea urchin spermatozoa given by Rothschild (1950). Rybak and Gustafson (1952) found that the catalase activity of this material very much depends upon the state of the spermatozoa. Fresh spermatozoa had a very low activity, which increased upon aging of the cells. One explanation for this is that an inhibitor, initially present in high concentration, was gradually destroyed or washed away from the spermatozoa. The catalase activity may also be influenced, however, by the intracellular structure, which changes during aging of the spermatozoa in sea water. This possibility is in conformity with the observations by Kaplan (1952), who showed that intracellular catalase of erythrocytes has a much lower activity, but also a higher stability than the extracted enzyme. In this respect the erythrocyte catalase shares the properties of the interfacially unfolded catalase in vitro compared with soluble catalase. Kaplan suggests that erythrocyte catalase occurs reversibly absorbed at some intracellular

ENZYMATIC ASPECTS OF EMBRYONIC DIFFERENTIATION

287

interface of sufficiently low interfacial tension to permit desorption into the rolled-up, soluble form upon Iysis of the cell. The above-mentioned studies on living cells by aid of centrifugation indicate that catalase in the unfertilized sea urchin egg is mainly localized in the ground cytoplasm (+ microsomes?) (Holter, 1952). The enzyme in the developing larva may, however, adhere progressively to intracellular surfaces, since part of the catalase in liver cytoplasm is firmly bound to mitochondria (Euler and Heller, 1949). Kaplan’s observations suggest that an adsorption of catalase to an increasing intracellular surface might depress the catalase activity at the onset of the visible differentiation, and thus act in the same direction as the heat stable inhibitor. The curve for cytochrome oxidase in the sea urchin shows a strong decrease in activity somewhat prior to the rise in the mitochondrial number (Fig. 5b) (Deutsch and Gustafson, 1952). This result closely resembles the curve for catalase and the changes in the indophenoloxidase and “benzidinperoxydase” reactions (Ries, 1937 ; Ranzi and Falkenheim, 1937; Pitotti, 1939). A similar curve has also been reported for cytochrome oxidase in amphibian development (Spiegelman and Steinbach, 1945). Cytochrome oxidase may be expected, however, to show the same rise exhibited by mitochondrial enzymes in general. Lindahl (193%) in fact postulated a synthesis of a CO-sensitive, 02-transferring enzyme in the sea urchin, basing his conclusions on the CO effect on respiration. A synthesis of cytochrome oxidase has also been demonstrated in the grasshopper and chick embryos, and even in amphibians, at least in more advanced stages of development (cf. Boell, 1947). The enzyme synthesis in sea urchin and early frog development is thus probably masked, perhaps by an inhibitor, e.g., the catalase inhibitor. This is, in fact, not restricted to catalase, but is active against other iron porphyrin enzymes as well (Hargreaves and Deutsch, 1952). Albaum and Potter (1943) also dealt with an inhibitor of succinoxidase present in autolytic or necrotic, but not in healthy tumors. The drop in activity in the embryo may also be due, however, to structural changes. Spiegelman and Steinbach (1945) thus discussed the drop in activity in Raria in terms of changes in structural relations. A gradual aggregation of enzyme-active elements into mitochondria may be an important factor. Acid phosphatase is probably localized in mitochondria to a great extent (cf. Mazia, 1949). Its activity could thus be expected to rise during the sea urchin development. The activity in fresh homogenates is rather constant, however, in Avbmia (Mazia et al. 1948) as well as in Paracmtmtus. (Gustafson and Hasselberg, 1950). But Lindvall (1952) demonstrated a strong rise in activity, in aged lyophilized material of

288

TRYGGVE GUSTAFSON

Paracentrotm. The manifestation of this rise may be due to the structural disintegration of the mitochondria. Berthet and de Duve (1952) in fact demonstrated the existence of a mitochondria-linked, enzymatically inactive form of acid phosphatase in rat liver tissue. Different treatments, such as aging of the preparations or repeated freezing and thawing, all lead to solubilization of the enzyme. In contrast to its bound form, the free enzyme exhibits high activity. When considering the manifold factors which influence, for example, catalase activity, all enzyme data from complicated systems must be interpreted with great care. On different occasions one factor or another may contribute to a deflection of the curves of activity. It may be possible, however, to resolve the curves into their components. Developmental changes in an inhibitor for tissue hemolysins have been demonstrated by Tyler (1951). Whole cell extracts of fetal preparations are inactive due to the presence of an inhibitor. The data support the hypothesis that there exists in the liver a dissociable lysin-inhibitor complex which can be broken by means of ultracentrifugation. The inhibitor is fixed to the cellular structures that sediment after 25,000 g for 1 hour. Ponder (1951) suggests that the lysin inhibitor complexes in the supernatant of, say, mouse liver homogenates are lipoproteins. According to Tyler, natural inhibitors may play an important role in determining differences in activity between various tissues. The formation of enzymes in the amphibian embryo has been interpreted by Boell (1947) as a mere reflection of the utilization of yolk. This does not exclude that the enzyme formation reflects the mitochondria1 development as well. Enzymes attacking yolk, glycogen, and starch, and especially the inhibitors and activators of these enzymes, probably hold a key position in the developing organism. Plant physiology has supplied the literature with interesting examples (Kneen and Sandstedt, 1946; Militzer et d., 1946; Miller and Kneen, 1947; Myrback and Urtenblad, 1938). Certain water-soluble natural substances have been reported as inhibitory to various amylases, e.g., in Leoti sorghum. The sorghum inhibitor is mainly localized in the germ and the bran and seems to be an organic acid of relatively high molecular weight. The indole group appears to be a structural integer of importance in the inhibition of amylases. The inhibition is reversed upon germination, when the inhibitor is evidently destroyed or used up. The activation of &amylase which occurs upon germination in barley may, on the contrary, depend upon its liberation from a high molecular weight, insoluble protein caused by the action of proteolytic enzymes (Myrback and Ortenblad, 1938). Similar conditions may regulate the utilization of

ENZYMATIC ASPECTS O F EMBRYONIC DIFFERENTIATION

289

the carbohydrate-rich yolk and glycogen which occur in the sea urchin egg (cf. Mom4 and Slautterback, 1950; Immers, 1952). Studies on hatching enzymes present many examples of inactivation and activation of enzymes and enzyme precursors. A membrane-dissolving agent with proteolytic properties ( Sugawara, 1943) was demonstrated in the blastules of the sea urchin at hatching (Ishida, 1936). An activator of the enzyme occurs in the unfertilized egg and in early cleavage stages. An inhibitor appears, in contrast, in recently hatched blastulas. A similar situation prevails in amphibian development (Esposito, 1950). W u and Wang (1948) also found an anti-proteolytic agent in hatching fish and amphibian embryos. This agent was present in the mucus on the surface of the embryo, which thus might be protected from attack by the hatching enzymes. The problem of regulation of histolysis and histogenesis in insects should be discussed in this connection. The U-shaped metabolic curve of Lichtenstein (1946) in holometabolic insects is caused, according to Agrell (1949, 1951c), by a corresponding drop and rise in the apodehydrogenase activity. There is an obvious parallelism between decreasing metabolism and histolysis and also between increasing metabolism and histogenesis. Agrell ( 1 9 5 1 ~ )raises the question of an interdependence between oxidative metabolism and histolysis-histogenesis. He assumes that the histolysis is partly brought about by digestive proteolytic enzymes and shows the presence of a “peptic” enzyme with a very low p H optimum. There is no change in amount of this enzyme as inferred from homogenate studies. The enzyme might be activated, however, by a transient decrease in the intracellular p H in the sites of histolysis. A remarkably large drop in the pH has been demonstrated during the critical period, both in the hemolymph and in homogenates, and this over-all change may imply still greater changes in small localized areas. The pH decrease which activates the proteolytic enzymes may in turn be caused by an altered metabolism, possibly a disturbed oxidative chain and accumulation of acid metabolites. Agrell traces the ultimate cause of the changed metabolism to the wellestablished hormonal control of insect metamorphosis. Histolysis might thus be a final link of a long chain :hormonal control, oxidative metabolism, intracellular pH, proteolytic activity, histolysis. H e suggests, however, that a mutual interdependence occurs between oxidative metabolism and proteolysis, i.e., the activated proteolytic enzymes decompose the oxidative enzymes, which further alter the oxidative metabolism and lower the pH. In other words, there is a ckctclw Vitiosus. The sea urchin egg contains a zymogen or blocked proteolytic enzyme which is activated upon fertilization (Lundblad, 1949, 1950, 1952). The

290

TRYCCVE GUSTAFSON

activity is abolished, however, about 30 minutes after fertilization by some process in zivo. The enzyme preparation can often be activated by thiol compounds. Lundblad correlates the high transient proteolytic activity with a temporary high sensitivity to thiol reagents (Kriszat and Runnstrom, 1952b), and with a transient high content of glutathione (Shearer, 1922; fnfantellina and L a Grutta, 1947). He concludes that the natural peak in proteolytic activity is due to the appearance of SH-groups, and he emphasizes that a similar correlation prevails in germinating seeds. It is possible that this inverse relation between the glutathione content and the enzyme activity also holds for later stages of development. The formation of glutathione may thus contribute to the increase in activity of certain enzymes in the blastula, as well as to the decrease in activity of the iron porphyrin enzymes (Rybak, personal suggestion). Heilbrunn and co-workers (see Heilbrunn, 1945) assume that the release of calcium plays a decisive role in the process of activation of the unfertiiized egg. Thus, upon addition of calcium to crushed eggs, a breakdown of cytoplasmic granules was observed (Heilbrunn, 1927 ; Gross, 1951). This breakdown is accompanied by a sudden oxygen uptake and the formation of an acid. Thiol groups are probably involved in this process, since iodoacetate is inhibitory (Hultin, 1949 ; 1950a, b). Many proteolytic enzymes, e.g., in the sea urchin egg homogenates, are activated by calcium. Lundblad suggests that calcium liberates a proteolytic sulfydryl enzyme similar to that demonstrated by Woodward (1950) in the cytoplasmic granules, as well as in the non-granular fraction of Mactru eggs. He stresses that when it was possible to activate the enzyme with cysteine, this could generally be done also with calcium. On the breakdown of granules, Lundblad points out that thiols are liberated which further activate the process. Runnstrom (1949) underlines the hypothesis that the initiation of development of the egg may be due to the activation of various enzymes. Studies on the chemical nature of the jelly coat substance (cf. Vasseur, 1952) reveal its great similarities with heparin and suggest that the jelly coat might function as enzyme inhibitor. Various experiments support this hypothesis and suggest that a heparin like inhibitor of proteolytic enzymes is present in the vitelline membrane and the cortical layer. The inhibitor is assumed to be removed as it reacts to substances in the sperm. Periodate treatment removing the jelly substance may facilitate the fertilization and further development of underripe eggs. In fact, this treatment activates the eggs or at least changes the state of an unfertilized egg into a transition state (Runnstrom and Kriszat, 1950; Kriszat and Runnstrom, 1952a).

EXZYNATIC ASPECTS O F EMBRYONIC DIFFERENTIATION

291

The proteolytic enzyme activated upon fertilization may bring about certain changes in the proteins which result in unmasking reactive groups. Certain of these unmasked groups may exert an enzymatic activity. As was stressed by Runnstrom (1949), it is not yet possible to indicate the sequence of the different processes released in the activation of the eggs. “Interactions between later and earlier links in the concatenated initiating reaction probably occur. This may contribute to give the over-all process a certain autocatalytic character. If for example the process A causes the process B, and this in turn C, the last process-let it be for example release of calcium-may accelerate process A, which again will accelerate B and C. Parthenogenetic agents may attack different links in the chain of events, but in view of the interaction the whole chain might come into operation irrespective of which link was primarily affected.” This consideration may be generally applied and may concern the control of the exponential respiratory rise during the cleavage stages, and evocation in the vertebrate egg, as well as histolysis in insects, etc. (cf. above). AND V. COFACTORS

THE

CONTROL OF THEIR FORMATION

Mitochondria contain a relatively high amount of cozymase, triphosphopyridine dinucleotide, coenzyme A, flavin adenine dinucleotide, diphosphothiamine, a-lipoic acid, pyridoxal phosphate, folic acid, biotin, and vitamin BIZ (Green, 1952). A multitude of cellular reactions depends upon the presence in the mitochondria of such factors. The choline oxidase activity, for example, is lost upon freezing and thawing, which cause a release of the coenzyme, but the activity is restored upon addition of extracts from boiled mitochondria (Williams, 1952). Adenylic acid, as a second example, has to be added to mitochondria in order to make them perform oxidative phosphorylation. The development of chick embryos presents many examples of changes in vitamins and coenzymes, although little is known about their intracellular localization. The need for vitamins is illustrated by the various abnormalities caused by maternal deficiencies and the application of antimetabolites. Vitaniin injections in the yolk at an early stage may reverse these changes ; cf. the review of Cravens (1952). A synthesis of nicotinic acid and its gradual incorporation into DPN has been demonstrated (Levy and Young, 1948. Coenzyme synthesis is, however, often fed with vitamins accumulated in the yolk or egg white. There is, for example, a transfer of riboflavin, mainly from the egg white to the embryo, where it is gradually transformed into flavin adenine mono- and dinucleotides. On the whole, this transformation fails in the extraembryonic tissues (Matsuura, 1951). Thiamine shows a similar pattern of transfer, conjugation, and distribu-

292

TRYGGVE GUSTAFSON

tion but is mainly derived from the yolk (Okamoto, 1951). In the grasshopper egg the decrease in riboflavin in late development (Bodine and Fitzgerald, 1948) can be correlated with the appearance of a pterin compound which stimulates the growth of certain lactic acid bacteria (Burgess et d.,1952). Further examples on the developmental role of and changes in vitamins may be obtained from the sea urchin egg. In this material drastic changes in the citrovorum factor (leucovorin) , niacin, and other vitamins have been demonstrated (Bhhidi, Kavanau, and Gustafson, 1%4), and the niacin analogue acetylpyridine induces changes in animal egg halves, reminding of vegetalization (Horstadius and Gustafson 1954). The cofactors are sometimes firmly bound to the mitochondria (Scheid and Schweigert, 1951), but the binding capacity may change. The cozymase content of tumor mitochondria has been reported to be low (Euler, st at., 1938; Bernheim and Felsovanyi, 1940), and the addition of cozymase causes a strong response in this material compared with normal mitochondria (Wenner and Weinhouse, 1953). It was suggested that mitochondria of neoplastic tissue do not bind cozymase as strongly as mitochondria from certain normal tissues, and that this may result in a corresponding higher level of this coenzyme in the soluble portion of the cytoplasm. The high level of cozymase might in turn determine the high rate of glycolysis in neoplastic material, cf. amphibian cleavage stages. Lindahl and Lennerstrand (1942) studied the content of cozymase in the development of Ram and Bufo. From their methods one may infer that the authors examined mainly the variations in “free” or weakly bound cozymase. The changes were unimportant up to the tailbud stage, when a drastic, though transitory drop occurred. The present author thinks that this drop reflects the rapid binding of accumulated soluble cozymase in mitochondrialike enzyme vacuoles or granules which develop at this time (cf. Holtfreter, 1946b). A marked depletion in alcohol-extractable growth factors (75 per cent alcohol) appears in the advanced sea urchin blastula. It is less pronounced in Li-treated material. The depletion is thus inversely proportional to the number of mitochondria and may reflect the binding of soluble factors to these organella. If water or tungstic acid is used for the extraction, the depletion is only transitory and an increasing amount of the factors can be extracted in subsequent stages of development. (Fig. 6) In a very synchronous material the increase in the gastrula can be resolved into a series of peaks (Kavanau, Gustafson, and Bhnhidi, 1954), each probably corresponding to one wave of mitochondria1 development. The abundant occurrence of ascorbic acid in differentiating areas underlines its importance to embryonic development. An increase in ascorbic

293

ENZYMATIC ASPECTS OF EMBRYONIC DIFFERENTIATION

acid content occurs in the amphibian tail bud stage. This is followed, however, by a sudden drop (Urbani-Mistruzzi and Carducci-Pitoni, 1948) ; cf. S a h o (Daniel, 1949). Part of this drop might be due to the binding of ascorbic acid in the argentophilic material of granules which often characterize differentiating areas. The existence of tissue-bound ascorbic acid which mostly escapes determination has in fact been demonstrated (Summerwell and Sealock, 1952). o=normal Water exfracfs. 28 Cows.

u

.s f lu

1'

4

8

;2

I6

20

24

20

32

36

40

64

&

Hours of egg development

FIG.6. Relative growth-promoting activity of water extracts of sea urchin eggs in different stages of development ; test organism Lactobacillus case;; turbidimetric estimation of the growth. From Gustafson Hjelte, and Hasselberg (1952). The synthesis of enzyme protein and the prosthetic group may have a different course. Either of these might thus limit the rise in enzyme activity. Such a dissociation is applicable to the synthesis of hemoglobin. Here two consecutive stages are discernible, and the prosthetic hemin is formed during the second stage (Thorell, 1947). A complete dissociation between the synthesis of apoenzyme and coenzyme is known from Chlamydowonas (Moevus, see Sonneborn, 1951), where the apoenzyme for formation of rutin reproduces cytoplasmatically as part of the chloroplasts even in mutants which do not form the corresponding prosthetic group. Synthesis of rutin may be induced in those mutants by extracts from normal cells. Diapauses or resting stages in the embryonic or postembryonic development of insects may partly reflect discordances in the availability of apoand coenzymes. The hibernation diapause in the butterfly Phalaera bucephda may thus be explained as vitamin deficiency (AgrelI, 1951a, b). The low respiratory quotient, in fact, indicates that the decarboxylating

294

TRYGGVE GUSTAFSON

part of the Krebs cycle is blocked, e.g., by cocarboxylase or coenzyme A deficiencies. Injection of thiamine and pantothenic acid, precursors of these coenzymes, brought about a transient increase in the respiratory quotient and the oxygen consumption. The production of coenzymes evidently plays an outstanding role for the realization of the potential enzyme activities of the mitochondria, and these coenzymes or their precursors may be provided by the nuclei (Brachet, 1952). Biotin, p-aminobenzoic acid, and adenosine may thus be produced in the nuclei, since the genes controlling the formation of each substance are closely adjacent (Pontecorvo, 1952). This view is supported by the relatively high content of various coenzyme precursors in the nucleus, e.g., riboflavin, thiamine, pantothenic acid, and nicotinic acid (Isbell ef ai., 1942). The high amount of adenosinedeaminase and nucleosidephosphorylase in the nuclei suggests that adenylic acid is of nuclear origin, as pointed out by Mirsky (1951), and Brachet presents a similar concept (1952). The role of the nucleus as a regulator of the mitochondria1 activity appears from enucleation experiments, from the biochemistry of lethal bastards (Brachet, 1952), and from the effects of nuclei added to mitochondrial suspensions (Potter et ul., 1951). The nucleated half of a sea urchin egg, divided by means of ultracentrifugation, has a higher P32incorporation than the heavy p.art, in spite of the richness of the latter in mitochondria and respiratory enzymes. Lethal hybrids, i.e., animals with disturbed nuclear-cytoplasmic relations, may further have a decreased capability of maintaining A T P in the phosphorylated state (Barth and Jaeger, 1936). It is also notable that the mitochondria in the spermatogonia of certain bastards of Drosophila behave abnormally (Dobzhansky, 1934). Most of these observations support the idea that the nucleus supplies the adenylic acid necessary for the coupling between oxidation and phosphorylation. The basic role of this reaction in development is elucidated by the fact that usnic acid, which uncouples these two processes, stops P3*penetration as well as the development of the sea urchin without inhibiting the oxidative processes (Marshak and Hartiiig, 1948). The metabolism during the cleavage stages in the sea urchin may depend upon compounds released from the oocyte nucleolus dissolving upon maturation. The oocyte nucleolus of the starfish is, in fact, rich in acidsoluble nucleotides similar to adenylic acid (Vincent, 1952). When certain of these compounds are consumed or incorporated in mitochondria1 precursors, a respiratory plateau or diapause may be reached, lasting until the nuclei have attained full control of the cytoplasmic activity. The rapid rise in the activity of many enzymes after the respiratory

ENZYM.4TIC

ASPECTS O F EMBRYONIC DIFFERENTIATION

295

diapause (Gustafson and Hasselberg, 1951), the strongly increasing mitochondria1 number (Gustafson and Lenicque, 1952), and the intensified ATP metabolism (Lindberg, 1950) are very likely influenced by an increased production of “coenzymes” in the nuclei. An increased nuclear control in this stage is in fact indicated by the reappearance of nucleoli (MonnC, 1951) . The problem now appears of how nuclear control is regulated, a subject discussed by Zeuthen (1951). He showed that the 0 2 consumption changes rhythmically and presents arguments that this rhythm concerns the rate of energy output (Zeuthen 1946, 1949, 195Oa, b). The period of increasing respiration is correlated with periods of reappearance and persistence of new nuclei, whereas the respiration remains constant or decreases at the dissolution of the nuclear membrane. The nuclear stimulation of the cytoplasmic metabolism in the early cleavage stages is thus restricted to short periods. When the interval between the waves of mitoses begin to lengthen, synchronously with the exponential respiratory rise (cf. Lindahl 1939b), the respiratory waves progressively become larger. This suggests that the nuclei now begin to control syntheses in the cytoplasm. The nucleus seems thus to have the alternative choice of dit.iding and reorganizing itself o r of controlling the growth processes in the whole cell (Zeuthen, 1951). The question, however, remains how the nuclear activity is switched in the latter of these two directions, During the period of intense cleavage, the rapid growth in the total nuclear volume takes place at the expense of material which may be stored in the cytoplasm of the mature egg, and which cannot be synthesized as yet, at least not to a sufficient rate (Zeuthen, 1951). Studies on the DNA content of developing frog eggs (Hoff-Jorgensen and Zeuthen, 1952) plus calculations on the total nuclear DNA content in the sea urchin egg indicate that this stored material includes the DNA precursor postulated by Abrams (1951) (Zeuthen, 1951). The gradual depletion in this DNA precursor very likely prolongs the interphases and may thus increase the nuclear stimulation of the cytoplasmic activity. The duration of the diapause may depend upon the relative rate of depletion of the DNA precursor and of coenzymes from the oocyte nucleolus. If the relative rate of consumption of the nucleolar material is high, the diapause will be long.

VI. ON THE GENESIS

OF NEW MITOCHONDRIA IN EMBRYONIC DIFFERENTIATION

The respiratory curve of the sea urchin egg shows an exponential rise during the early cleavage stages, a plateau, and a second rise (Lindahl, 1939b). The second rise is abrupt and coincident with the development of

2%

TRYGGVE GUSTAFSON

new mitochondria. It presumably reff ects an increased nuclear stimulation of the cytoplasmic activities. The first exponential respiratory rise may also be linked to an elaboration of particulate units (Gustafson, 1950). This elaboration and the accompanying respiratory rise may be limited to structural rearrangements, since the enzyme synthesis is weak in this phase of development (Runnstrom, 1952). The first respiratory rise is reversibly inhibited by Li (Lindahl, 1939b), which may indicate that this agent also disturbs the development of the particles concerned (Gustafson, 1950; Lallier, 1951, 1952a, b). The particles may be considered as precursors of mitochondria, since the number of ripe mitochondria becomes retarded subsequent to Li treatment. The gradual development of mitochondria from non-mitochondria1 precursors has been studied by Chantrenne (1947), who found that intracellular granules of adult mouse liver could not be classified in two main groups only, big granules and microsomes, as suggested by Claude ( 1946). There was, rather, a continuous variation of the particles, both in size and chemical composition. The greater the particles the greater their content of enzymes and phospholipids and the smaller their content of nucleic acid. Chantrenne suggested that granules of different sizes represent different developmental stages of lipoprotein complexes which grow and produce enzymes around centers rich in ribonucleic acid. Jeener (1948) was able to fragment these granules. The smallest granules gave rise to a high proportion of small nucleic acid-rich but enzyme-poor fragments, and a low proportion of large enzyme-rich but ribonucleic acid-poor fragments, Upon fragmentation of the larger liver granules the result was reversed, the large enzyme-rich fragments being most abundant. Jeener suggested that the small nucleic acid-rich particles gradually aggregate and that the interior of these aggregates is the site of enzyme production. This enzyme synthesis should be paralleled by the loss of ribonucleic acid in the interior of each aggregate, while new nucleic acid-rich particles are continuously added to the periphery. Mitochondria should be a final stage of this development. The development of mitochondria has been studied by aid of electron microscopy (Eichenberger, 1953). Upon injecting an animal with egg albumin, the kidney mitochondria accumulating the foreign protein disintegrate, but gradually regenerate. The first stages of regeneration are characterized by membrane-free minute granules which grow and finally reach the size and shape of mitochondria. In this connection one should mention that injected ovalbumin is first incorporated in the microsomes and is then found in the mitochondria (Crampton and Haurowitz, 1951). The development of intracellular granules has also been studied in the

ENZYMATIC ASPECTS OF EMBRYONIC DIFFERENTIATION

297

egg of Twbifex (Lehmann, 1950). Here the organ-forming polar plasms exhibit a strong Nadi reaction and thus contain indophenoloxidase (Lehmann, 1948). Electron microscopy revealed that the Nadi-positive regions contain particles of 30 to 100 mp in diameter. From this uniform population two different groups develop, one in the ectodermal somatoblast Zd, with small globules, and the other in the mesodermal somatoblast 4d, with greater globules of the size 600 x 300 mp. The strong electron absorption of the particles indicates a high phosphorus content. The particles are very labile to many fixatives. These properties together with the Nadi reaction of the somatoblasts suggest that they represent different developmental stages of enzyme-bearing particles. Holtfreter ( 1946a, b) studied the development of intracellular inclusions in the amphibian egg. In the unfertilized egg of Rana he recognized a great number of “lipochondria” stainable with Nile blue sulfate. In the later development the lipochondrial substance fused with the yolk granules and covered their surfaces. The yolk granules then disintegrated and dissolved, presumably due to the lipochondrial enzymes. The yolk liquefaction in an epithelium cell was accompanied by the formation of vacuoles. When these vacuoles were stained with Nile blue sulfate they exhibited a shrinkage. Such a shrinkage also occurred in normal development, and the large vacuoles were thus transformed into granules. A strong staining caused the death of the animal, which may indicate that the vacuoles contain respiratory enzymes. It is quite probable that the development of vacuoles and their transformation into granules represents the development of new mitochondria by a process of coacervation. In the sea urchin egg, the development of mitochondria from precursors is supported by experiments of Harvey (1946), who divided the egg into light and heavy fragments by means of centrifugal force. The fertilized clear quarter of the Arbucia egg is free from large granular inclusions such as mitochondria, yolk, and pigment. These fragments can still develop into small plutei of a fairly normal shape. The mitochondria are not replaced during early development, at least not until the blastula stage. In the plutei, however, mitochondria are present. The big granules must consequently be formed de novo and not by division of preformed mitochondria. Upon fertilization a small amount of mitochondria is introduced into the egg with the sperm; these do not infect the whole cytoplasm, but lie close together in a single blastomere in the 32-cell stage (cf. Bourne, 1951). The eggs may, however, contain distinct mitochondria1 precursors which are not removed by means of ordinary centrifugation. Certain small particles can in fact be removed from the clear cytoplasm by aid of still stronger centrifugation (Harvey, 1946). In an electron microscopic

298

TRYGGVE GUSTAFSON

study of high-speed centrifuged eggs, Lansing et al. (1952) also demonstrated that the eggs contain two layers of what appears to be mitochondria, one in the centripetal lipid layer and the other just above the yolk granule layer. Also, the clear middle zone contained numerous small doublemembraned lenslike bodies. The mitochondria1 number is low and rather constant during the cleavage stages (Gustafson and Lenicque, 1952). A certain rise in the number of stainable particles could, however, be observed (Lenicque and Gustafson, 1954). This rise was of a much smaller magnitude than the rise in the late blastula, but followed the course of the respiratory curve. I t was suggested that these stainable elements correspond to the biggest granules in a particulate population which develops as long as certain essential factors accumulated in the egg cell are available. After a certain lag, when the nuclei begin to produce more of these compounds, the development continues. A closer examination of data available on the intracellular localization of enzymes gives support to the idea of a gradual development of mitochondria from smaller units. Enzymes in embryos are, in fact, often localized unconventionally. Bodine and Lu (1950, 1951a, b ) , who studied the respiratory activity of cytoplasmic fractions of grasshopper embryo homogenates did not find any functional difference between mitochondria and microsomes. Hutchens, Kopac, and Krahl (1942) report that the cytochrome oxidase of Arbacia eggs is associated with granules smaller than and with a different stainability from mitochondria. Enzymes which are soluble in early development may, however, change their localization as the development proceeds. Mazia (1949) showed that deoxyribonuclease, soluble in the unfertilized egg of Arbacia, is gradually fixed on sedimentable elements as the embryonic development proceeds. Krahl (1950) suggests that the total reducing activity, the cytochrome oxidase activity, and the deoxyribonuclease which are in solution or fixed on minute corpuscles in the unfertilized egg progressively incorporate in granules which stain like mitochondria. The binding of enzymes to specific granules would parallel the development of specific functions. I n the chick embryo the apyrase activity, to a great extent localized in granules, becomes more and more concentrated in these granules as development proceeds (Steinbach and Moog, 1945). In an earlier section of this review, a developtnental drop in the cytochrome oxidase and catalase activity was discussed, as well as a masked rise in activity of acid phosphatase. It was also emphasized that the extractability of various low molecular weight factors decreases in the course of development. These changes appear to be synchronous with

ENZYMATIC ASPECTS OF EMBRYONIC DIFFERENTIATI0,N

299

the development of mitochondria, and one may tentatively suggest that they reflect the process of coacervation and fixation of soluble coenzymes as well as enzymes in granules. The elaboration of big, complex granules by a process of coacervation may be elucidated by the studies of Brachet and Chantrenne (1944), who observed a progressive combination of “free” nucleic acid with large granules in the embryos. The carotinoid-containing granules in the eggs of Paracentrotus are evidently subjected to a similar combination with nucleic acid during development (Monroy et al., 1951; Monroy and De Nicola, 1952). These granules are originally complex and contain proteins, lipids, and carbohydrate, but no nucleic acid. The same results were obtained with granules derived from early stages of development and up to the early blastula. In the advanced blastula, however, the preparations appeared to contain some ribonucleic acid, which probably is not contaminative, but constitutes an integral part of the granules, The carotinoid containing nucleic acid-free granules may be considered as old mitochondria of the ovocyte to which nucleic acid-rich elements aggregate at the formation of new mitochondria. The formation of ribonucleic acid-rich particles from material of almost molecular dimensions has been studied in the development of Ascaris (Panijel and Pasteels, 1951). In this egg, the basophilia due to ribonucleic acid decreases during oogenesis, but rises abruptly upon fertilization. This “primary basophilia” appears around the male pronucleus in the fertilized egg, and it is evidently dependent upon the Gram-positive material which is introduced with the sperm and gradually disappears. The minute elements of the primary basophilia are progressively transformed into larger and heavier particles where the nucleic acid is relatively loosely bound. The particles are probably rich in enzymes, since their elaboration can be correlated with an increase in the respiratory activity of the egg. The topographic relation between the chondriome and the granules of the secondary basophilia indicates that the preformed mitochondria of the zygote is probably necessary for the transformation of the primary elements into secondary elements, at least in the uncleaved egg. Sedimentation of the mitochondria of the egg and sperm by means of centrifugation may, in fact, cause serious abnormalities, at least if it is effected at a sufficiently early stage after fertilization. The mitochondria of the egg of Ascaris may consequently be considered as “nurses” of the new generation of mitochondria. This is evidently not necessarily the case in the sea urchin, where development may proceed in a mitochondrial-free fragment. The relation between soluble ribonucleic acid and ribonucleic acid bound

300

TRYGGVE GUSTAFSON

to granules has also been studied in the testicles of Ascaris by Panijel (1951), who stresses that two stages are discernible in the particulate development. In the first stage soluble ribonucleoproteins are accumulated ; during the second, granules are elaborated from this material. Jeener (1948) and Panijel (1951) emphasize the similarities between the development of viral particles and ribonucleoprotein-containingparticles which normally occur in the cell. Gustafson (1953) also suggests that the intracellular development of a virus particle may serve as a model for the mitochondrial development ; cf. the reviews on virus development by Sanders (1952) and Hershey (1952). Upon entering a susceptible cell the virus particles break down into smaller units. The dissociated units act as organizing centers for the formation of replicas and of further, biologically inert, constituents. The actual synthesis of virus constituents probably occurs early in the constant phase of the growth cycle and may be traced by an increasing rate of isotope incorporation (P32)in deoxyribonucleic acid. This early “silent phase” is followed by a phase of gathering already synthesized subordinate units into the complete infective virus. The reappearance of antigenicity and the hemagglutinative capacity of virus may precede the return of infectivity. Thus the phase of gathering may take place in a series of stages, which are to a certain degree discernible in the electron microscope (Levinthal and Fisher, 1952). The attainment of infectivity in some phage can be correlated with the appearance of a protein membrane and a tail. The analogy between viral and mitochondrial development is disturbed by the fact that the mitochondria in an embryo develop during a period of varying nuclear control. The course of the growth cycle is in both cases dependent upon the criteria used for the measurements, i.e., whether one chooses serologic properties, enzymatic activities, infectivity, cytologic manifestations, metabolic rates, etc. Most criteria give essentially the same picture for the mitochondrial development in the sea urchin egg, indicating an increased number in the advanced blastula. Studies by aid of isotopes reveal important activities already in the cleavage stages, as well as in the “silent phase” of the viral development. The development of mitochondrial precursors during the cleavage stages of the sea urchin egg is elucidated by the isotope studies of Hultin. The incorporation of NIG m-alanine during the cleavage stages is thus concentrated in the microsome fraction (Hultin, 1 9 5 3 ~ ) .The increased rate of rebuilding of this fraction during the cleavage stages is supported by the continuous increase in the rate of incorporation of N16 ammonia, glycine, and nL-alanine, and ClC carbonate into the non-soluble proteins which may represent the intracellular particles (Hultin, 195Oc, 1952,

ENZYMATIC ASPECTS O F EMBRYONIC DIFFERENTIATION

301

1953e, Hultin and Wessel, 1952). The purine and pyrimidine metabolism, which increase synchronously with the exponential respiratory rise, may also indicate a progressive multiplication or reconstitution of small nucleic acid-rich microsomelike particles during the cleavage stages (Hultin, 1953). From the mesenchyme-blastula stage onwards the rate of incorporation of N16 DL-alanine into the mitochondria fraction equaled that for the submicroscopic fraction, which indicates that new mitochondria are formed (Hultin 1953c), possibly by a gathering of precursor elements. The qualitative difference between the particulate population in the cleavage stages and those in the differentiating larva is manifested in metabolic studies. The metabolism of amino acids during the cleavage stages is thus largely restricted to exchange reactions such as transpeptidation and transamidation. On the other hand, ammonia incorporation into amino acids as well as transamination remains low up to the advanced blastula and thereafter increases (Hultin, 1953b). Glutamic acid shows the highest metabolic activity. The increased amino acid metabolism very likely reflects the development of new mitochondria, since glutamic acid is closely connected with the Krebs cycle (Hultin, 1953e) and holds a central position in the amino acid metabolism. The increased amino acid metabolism agrees well with the amino acid analyses (Gustafson and Hjelte, 1951), indicating that variations in the total amounts of different amino acids are initiated when new mitochondria appear around the onset of differentiation. The rate of incorporation of CI4 acetate into free hypoxanthine, into ribonucleic acid nucleotides, fatty acids, and amino acids showed a minute rise during the cleavage stages of the sea urchin egg. The rate of incorporation was, however, drastically intensified in the advanced blastula (Hultin, 1953). This indicates a qualitative change of the intracellular granules from precursors, unable to metabolize acetate, into ripe mitochondria with a high capacity to convert acetate into various metabolites. The rate of incorporation of NIB ammonia, alanine, and glycine in the soluble proteins increases distinctly in the advanced blastula. This indicates that the particulate population then becomes capable of synthesizing proteins according to the actual demands of the differentiating organism. Labeled proteins are thus emitted in the cellular fluid (Hultin 1952, 1 9 5 3 ~ ) ~ and some of these proteins may even exhibit a new serologic specificity (Perlmann and Gustaf son, 1948).

VII. THEPRIMARY PATTERN OF MITOCHONDRIAL DISTRIBUTION The over-all biochemical changes of a germ reveal little about morphogenesis if not supplemented with data on the different embryonic regions,

302

TRYGGVE GUSTAFSON

such as different Mastomeres or the germ layers. Microrneres from the dividing sea urchin egg have been isolated in considerable numbers by making use of the countercurrent centrifuge constructed by Lindahl and Kiessling (1950). It is difficult, however, to collect pure entodermal or ectodermal tissues from sea urchin larvae, but a comparison between normal and Li-,egetalized material may give some idea about the biochemistry of the primary germ layers (Gustafson and Hasselberg, 1950, 1951 ; Augustinsson and Gustafson, 1949; Gustafson and Hjelte, 1951 ; Gustafson, Hjelete and Hasselberg, 1952). This indirect approach has not revealed any permanent, qualitative differences between the primary germ layers. The rise in enzyme activity was, however, distinctly retarded in a series of cases in vegetalized material as compared with the normal material (Fig. 3a, b). These observations suggest that there is a gradient with regard to the rate of enzyme synthesis in the egg, the animal or ectodermal

U

b

C

FIG.7. Mitochondria1 distribution in the sea urchin egg in different stages of development The gradient curves show the relative density of the mitochondrial population (R.M.D.) in different levels along the animal-vegetal axis ; animal pole to the left; vegetal pole to the right; age in hours indicated in each single figure; Li and IBA denote lithium and iodosobenzoic acid treatment; a to f : larvae developing from whole eggs. From Gustafson and Lenicque (1952). g: isolated animal (an.), and vegetal (veg.) halves, and animal halves with four (an. 4- 4), or eight (an. 8) micromeres implanted. From Lenicque, Horstadius, and Gustafson (1953).

+

EKZYMATIC ASPECTS OF EMBRYONIC DIFFERENTIATION

s

b

26 I B A

f

e

80

60

40

20

veg.

d

s

Qi An. pole

Veg. pole 9

FIG.7. (contiwed)

303

304

TRYGGVE CUSTAFSON

parts being more active than the vegetal or endodermal parts. This holds at least for the early stages of differentiation (Gustafson and Hasselberg, 1951). This conclusion is supported by cytologic studies which give information about the mitochondria1 distribution (Fig. 7). This is uniform up to the mesenchyme blastula stage (Fig. 7a), but an animal-vegetal gradient then appears (Fig. a),growing steeper as the development proceeds (Fig. 7d). After Li treatment, the gradient is concave, or the mitochondria-rich region is at least smaller than in normal larvae (Fig. 7c). For larvae animalized with iodosobenzoic acid the phenomenon is reversed (Fig. 7f) (Gustafson and Lenicque, 1952; Gustafson, 1952a). The mitochondrial curves for isolated animal and vegetal halves resemble those of the animalized and vegetalized whole eggs. Implantation of micromeres in an animal half changes the curve to that of a normal vegetalized larva, depending upon the number of micromeres implanted (Fig. 7g) (Lenicque, et al., 1953). The distribution of mitochondria, as studied by cytologic methods, gives a preliminary orientation about the distribution of certain enzymes in the embryo, Such studies, however, reveal nothing about the soluble enzymes or about enzymologic differences between mitochondria in different regions. Direct determinations of enzymatic activities in different parts of the embryo thus remains a task of primary importance; cf. the work of Holter and Lindahl (1940). The application of quantitative micromethods to amphibian material has given results relevant to different vertebrate eggs as well, and consistent with the mitochondrial studies on sea urchins. Alkaline phosphatase (Krugelis, 1947) , detectable SH-groups in trichloroacetic acid-fixed material (Brachet, 1940), reducing properties (Child, 1948, and others), and respiratory activity (various authors, cf. Brachet, 1952) thus mainly show the same distribution as the ribonucleic acid-rich granules, which are abundant in areas of high morphogenetic potential or intense differentiation (Brachet, 1941 ; Steinert, 1951a). These granules probably represent sulfydryl- enzyme- nucleic acid-rich complexes such as mitochondria and microsomes (Brachet, 1952). Their distribution evidently determines the relative metabolic intensities in various developmental stages or regions of the embryo. The C14-incorporation from glycine in proteins, for example, show an acceleration from the beginning of the gastrula through the early larval stages (Friedberg and Eakin, 1949) and therefore are similar to the curve for the synthesis of ribonucleic acid (Steinert, 1951a), an important constituent of the granules. The C1* of glycine and Ss5 methionine is more rapidly incorporated in proteins or intermediate compounds by the dorsal halves of a gastrula or neurula

ENZYMATIC ASPECTS OF EMBRYONIC DIFFERENTIATION

305

than by ventral material (Friedberg and Eakin, 1949; Eakin et d.,1951), and the synthesis of nucleic acid and A T P is likewise concentrated in the dorsal halves (Brachet and Chantrenne 1946). The gradients in the gastrula have been subjected to further studies in Amblystoma by Gregg and L#vtrup (1950), who have established gradients in yolk, fat, carbohydrate, alanylglycine, and p-glycerophosphatase by using non-yolk N and total N as reference components.

VIII. THEMETABOLIC BACKGROUND OF THE MITOCHONDRIAL DISTRIBUTION The ribonucleic acid gradient in the amphibian germ appears to be established already in the young oocyte (Brachet, 1942). In the sea urchin egg, on the other hand, no gradient in nucleic acid content has been established in early developmental stages, but other kinds of gradients have been postulated. Boveri emphasized that the morphogenetic properties showed a graded change along the egg axis. This gradient might correspond to an aninial-vegetal gradient in metabolic intensity, as postulated by Child (1941). Runnstrom (1928, 1929, 1931, 1933) considered that this decrease in intensity of an “animal” metabolism is accompanied by a corresponding increase in intensity of an antagonistic, “vegetal” type of metabolism (Fig. 8a). According to this double-gradient concept the

U

b

C

FIG.8.

Diagrams symbolizing the double gradient system of a developing sea urchin egg ; a : normal egg; b : vegetalized egg ; c : animalized egg.

relative intensity of the competitive metabolic types determines the extension of the primary germ layers. Li treatment favors the vegetal gradient at the expense of the animal gradient (Fig 8b) (Runnstrom, 1933; Lindahl, 1936), and so does the implantation of micromeres (Horstadius, 1935). This results in a hyperdevelopment of the endoderm at the expense of the ectoderm. Thiocyanate and iodosobenzoic acid and the removal of vegetal material has the opposite effect (Fig. 8c) (Lindahl, 1936, Runnstrom and Kriszat, 1952). The animal metabolism evidently favors mitochondrial development, whereas the vegetal metabolism has an inhibitory action. A primary mitochondrial gradient is thus established. Li depresses the mitochondria1 gradient by inhibiting the animal process. Iodosobenzoic acid changes the

306

TRYGGVE GUSTAFSON

mitochondrial gradient in an opposite way, e.g. by removing the vegetal inhibition (Gustaf son 1952a). The mitochondrial gradient is opposed by a mitotic gradient which obviously appears at the formation of the mesenchyme blastula (Agrell, 1953). Agrell concludes that the mitotic acivity and mitochondrial formation are to a certain extent antagonistic (cf. Zeuthen, 1951). An antagonism between the mitotic and the mitochondrial activity is also indicated by the work of FredCric and Ch6vremont (1952) ; cf. Daneel and Gutters (1951), who showed how mitochondria disintegrate upon mitosis. The localization of reducing enzymes in the “centriole” in embryos and various adult tissues (Spratt, 1951) may support the view that various constituents may be transiently dissociated from the mitochondria and associated with the organella of cell division. According to this view, the animal metabolism favors mitochondrial development, whereas the vegetal, at least indirectly, favors the mitotic activity in the advanced blastula. The animal type of metabolism is probably not bound to mitochondria. Normal larvae may thus be formed from mitochondria-free fragments in which mitochondria do not reappear in the cleavage stages (Harvey, 1946). Li, furthermore, acts on whole stratified eggs irrespective of the direction of the centrifugal force to the egg axis, and both light and heavy fragments are open to the action of Li (Horstadius, 1953). Much effort has been made to get direct information on the animal and vegetal metabolic types. Child (1941) found an animal vegetal gradient in the reduction of vital stains in the blastula of Strongylocentrotus. In the advanced blastula, a second, reversed reduction gradient appeared, coexistent with and stronger than the first gradient. These gradients may be changed by the action of Li or thiocyanate (Ranzi and Falkenheim, 1937; Ranzi, 1939), or operatively (Horstadius, 1952), like the mitochondrial gradients (Gustafson and Lenicque, 1952), of which they are forerunners and with which they are partly coexistent. An isolated vegetal half thus shows a vegetal, but in most cases no animal reduction gradient. An isolated animal half, on the contrary, has a strong animal, but no vegetal gradient. Micromeres implanted in the animal material depress the animal gradient and induce a vegetal, so that the reduction gradients resemble those of a normal egg. The reduction gradients do not reveal much about the nature of the animal and vegetal metabolic processes. Lindahl and Holter (1940) made an attempt to elucidate the metabolic gradients by respiratory measurements with the Cartesian diver technique of LinderstrZm-Lang. They found the oxygen consumption to be the same in isolated animal and vegetal halves of the egg. The sum of respiration of the half larvae was

ENZYMATIC ASPECTS OF EMBRYONIC DIFFERENTIATION

307

equal to the respiration of corresponding whole larvae. Furthermore, addition of Li, KCN, glyceraldehyde, and pyocyanine had the same influence upon the. respiration in animal and vegetal halves and whole eggs. I t may be noted in this connection that the Cartesian diver technique was unable to demonstrate any respiratory gradient in Planark (Qvtrup, 1953). The respiratory studies evidently did not reveal any gradient in respiration along the animal-vegetal axis in the sea urchin. Such a gradient, however, appears in the egg of Mytilzar. Thus Berg and Kutsky (1951) found that the respiratory intensity of the vegetal polar lobe is lower than that in the remainder of the egg. The polar lobe cytoplasm is relatively free from granules of the large visible size, and the authors suggest that it might be lacking to some extent in smaller granules with attached oxidative enzymes. It is interesting to note that the respiratory rise in animal material exhibits a diapause, which is absent in the vegetal material. The results of Holter and Lindahl might have turned out otherwise if the respiratory activity of animal and vegetal halves could have been measured in the intact egg, since metabolic regulation may precede a morphogenetic regulation. Horstadius ( 1952) showed in fact that the pattern of differential reduction of animal and vegetal material in situ is not identical with that of isolated animal and vegetal halves. In this connection he emphasizes the work of Barth and Sze (1951) showing that the respiratory activity of the combined pieces of organizer and ectoderm of Rana is higher than that of the separated pieces of these regions, when isolated at the gastrula stage and measured at the neurula stage. This is similar to the findings of Minganti (1951) that isolated animal and vegetal halves of ascidian embryos are devoid of melanizing enzyme activity, whereas the activity is present in the animal part when in contact with vegetal material. Studies on the respiratory quotient of the Li-sensitive, exponentially growing respiratory fraction suggest that the animal metabolism involves the oxidation of carbohydrates (Lindahl and Uhman, 1938). A carbohydrate breakdown may, however, proceed through different pathways, e.g., via trioses or by a stepwise oxidative decarboxylation of glucose-6phophate, both of which pathways occur in the sea urchin (Lindberg, 1945 ; Gustafson and Hasselberg, 1951 ; Cleland and Rothschild, 1952b). The sea urchin egg has the capacity of incorporating CI4 carbonate. This capacity increases during the Li-sensitive respiratory rise. The carbonate fixation depends upon the degree of reduction of triphosphopyridine dinucleotide ( T P N ) which in turn may depend upon the conversion of glucose6-phosphate to ribose phosphate via phosphogluconic acid (cf. Hultin

308

TRYGGVE GUSTAFSON

1953e). Hultin finds it plausible that the ribose phosphate-generating, TPN-reducing carbohydrate metabolism is accelerated during the Lisensitive respiratory rise before the tricarboxylic acid cycle becomes more important (Hultin and Wessel, 1952). One may thus suggest that the oxidative decarboxylation of glucose-&phosphate, not bound to mitochondria (Glock and McLean, 1952), is a characteristic feature of the animal, non-mitochondria1 metabolism (Hultin, 1953e). Phosphogluconic acid and propanediol phosphate, which both appear to play a role in the special type of carbohydrate metabolism discussed (Lindberg, 1946), actually have an animalizing effect (Horstadius and Gustafson, 1947). The importance of a TPN-reducing, ribose phosphate-generating carbohydrate metabolism in the animal trend of development is supported by the fact that animalization (Runnstrom and Kriszat, 1952) as well as carbonate fixation ( H u h and Wessel, 1952) are favored by the same agent, iodosobenzoic acid (Hultin, 1953e). The period of the Li-sensitive respiratory rise, furthermore, coincides with the period when iodosobenzoic acid brings about animalization (B’ackstrom, 1953a). The exponential respiratory rise which may involve an increased ribose phosphate generation, coincides with an accelerated purine and pyrimidine metabolism (Hultin, 1953a; Hultin and Wessel, 1952). The central position of ribose phosphate in purine metabolism is well known (Greenberg, 1951). Hultin thus suggests that the metabolism of ribose phosphate and purines may form a link between the exponential respiratory rise and the processes of determination (Hultin, 1953e), notably the animal processes. Part of the nucleic acid metabolites formed through the exponential increasing metabolism during the cleavage stages may accumulate as deoxyribonucleic acid. This nucleic acid shows a rapid synthesis (Abrams, 1951), accelerating after an initial lag (Villee st al., 1949; Zeuthen, 1951). Another part of the nucleic acid metabolites may be incorporated in ribonucleic acid, which is immediately consumed by the growth of microsomelike precursors of mitochondria (cf. Gustafson, 1950; Hultin, 1953e). Hultin underlines ( 1953e) that the “hexose-monophosphate shunt” (Horecker, 1951) becomes more important if the triose phosphate dehydrogenation step of the glycolytic cycle is blocked by appropriate concentrations of SH reagents (Crane and Ball, 1951; Lindberg and Ernster, 1948). One may consequently assume that there is a choice between the two types of carbohydrate metabolism in the egg. Iodosobenzoic acid, which oxidizes SH-groups, may favor an oxidative, TPN-reducing, ribose phosphate-generating animal type of carbohydrate metabolism (Hultin,

ENZYMATIC ASPECTS OF EMBRYONIC DIFFERENTIATION

309

1953e). The micromeres, on the other hand, probably emit a principle which favors an antagonistic type of metabolism. Thiocyanate treatment before fertilization may bring about animalization if the treatment is performed with good oxygen supply. C O - 0 2 mixtures in the dark, low oxygen pressure, and KCN check the animalizing processes (Lindahl, 1933). Addition of the redox dye pyocyanine, on the other hand, strongly promotes animalization (Runnstrom and Thornbloom, 1938). Lindahl thinks that by promoting swelling of the cytoplasm thiocyanate ions allow certain oxidative reaction to proceed, e.g., the oxidation of SH-groups (Lindahl, Swedmark and Lundin, 1951). The mode of action of thiocyanate may thus be related to that of iodosobenzoic acid already discussed. The above conclusion appears to be in conflict with the observation that animalization can be induced by thiomalic acid, a thiol (Lallier, 1 9 5 2 ~ ) . This and other reducing compounds also induce the formation of axial structures in ventral explants of young amphibian gastrulae, whereas oxidizing agents are inhibitory (Rapkine and Brachet, 1951; Lallier, 1951). The conflict referred to is not unexpected if one realizes the multiple points of attack of thiol compounds in the cell, e.g., on a wide variety of enzymes, reversibly denaturated proteins, and low-molecular weight substances (Barron, 1951;Rapkine and Brachet, 1951). Thiol compounds may for instance induce precytolytic changes favoring neuralization and parthenogenesis (Holtfreter, 1945, 1947; Rapkine and Brachet, 1951). They also bring about localized shrinkage of the Echinocmdium egg, probably corresponding to the presumptive oral side of the larva (Gustafson, 1952b). They may also control the breakdown of granules in the egg, since a thiol reagent inhibits this process in egg homogenates (Hultin, 195Ob). The effects of the application of thiol-reagents and thiols may consequently be complicated and involve processes synergistic or antagonistic to each other.

IX. THE GRADUALCOMPLICATION OF

THE

MITOCHONDRIAL PATTERN

The simple primary mitochondrial gradient is not permanent but grows complicated as development proceeds. This can be studied in a Li-vegetalized larva (Gustafson and Lenicque, 1952) (Fig. 9a, b). Secondary and tertiary peaks gradually appear, but there is a regression of the primary peak. This development of mitochondria in the vegetal area may indicate a gradual defeat of the vegetal inhibitory activity. The emancipation of the mitochondria1 activity does not, however, proceed immediately in the animal-vegetal direction, but a “mitochondrial vacuum,” the presumptive proctodaeum, persists long after mitochondria have appeared in more

310

TRYCGVE GUSTAFSON

vegetal parts, such as the stomach. Finally, mitochondria develop in the proctodaeum rudiment as welt. It thus appears as if the ectodennal part or any other part with a rapid mitochondria1 development suppresses a similar process in the adjacent areas. After iodosobenzoic acid treatment, the primary vegetal inhibition is abolished and thereby the incitement of a 2000

0

I50

loo

50

\ro

b

\)p

43Li

0

4

r ’

P

34 Li

a b FIG.9. The complicated mitochondria1 distribution in advanced stages of development of Li treated ( a and b) a n d iodosobenzoic acid treated larvae (c) ; cf. legend to Fig. 7. From Gustafson and Lenicque (1952).

gradually complicating pattern (Fig, 9c). These changes in mitochondria1 pattern in Li- or iodosobenzoic acid-treated larvae are paralleled by similar changes in the development of isolated vegetal and animal halves (Lenicque, Horstadius, and Gustafson, 1953). The double-gradient system becomes fractionated into a series of separate centers, probably as a result of interaction between different levels. Each of these new centers then creates conditions for a further complication by means of interaction with its surroundings. This may be an application of Cohen’s (1942) principle of self-increasing complexity as cited in Lwoff’s (1950) book on morphogenesis in ciliates. “Once attained, a certain degree of complexity will increase under its own momentum, physically, chemically or both” . . . “Given just the number of asymmetrical conditions in the

ENZYMATIC ASPECTS OF EMBRYONIC DIFFERENTIATION

31 1

environment, the chemical complexity of protoplasm is sufficient to evoke further spacial patterning. This, in turn, increases the complexity of the gradients and phases and so evokes a still more complex patterning, the whole cycle repeating itself until the system has reached a condition of dynamic stability, or of rigidity as in fructification. . . .’’ The interaction between the different areas in an embryo, discussed in a theoretical paper by Spiegelman (1945), may be mediated by waste products secreted from cells of high metabolic activity, or by a depletion of adjacent, less active cells in diffusible agents. In a paper on the differentiation of Rhodnius, Wigglesworth (1948) also discusses “the uptake from the substrate by an active centre of the material necessary for a particular determination and the consequent inhibition in the surrounding zone of centres with the same potential activities.” The importance of this depletion is suggested by the studies on peptides, growth-promoting compounds, cozymase, etc., quoted above, showing drastic drop at the onset of differentiation, presumably due to the binding of these compounds (cf. Steinert, 1951b, 1952). Such interactions may also determine the suppression or the development of a secondary oral side (Lindahl, 1936; Horstadius, 1938), or a secondary ciliary tuft in the embryo (Lindahl, 1936). They are not confined to early stages of development, since vegetal material from an early pluteus, if transplanted to an animal egg, retains the property of checking the development of the ciliary tuft (Horstadius, 1950). The separation of the bristle-bearing plaques on the abdomen of the insect Rhoditizrs also leads to the emergence of new bristles { Wigglesworth, 1948).

X. ON QUALITATIVE BIOCHEMICAL DIFFERENTIATION The development of the sea urchin egg has been discussed in terms of interaction between two antagonistic types of metabolism. One of these types was supposed to favor mitochondrial development, whereas the other type was inhibitory and gradually disappeared. A sequential development of mitochondrial populations was brought about under the influence of these metabolic types and according to Cohen’s principle of selfincreasing complexity. In this discussion little attention was, however, paid to the qualitative biochemical differences between various tissues of the differentiated larva. The biochemical differentiation in the sea urchin development may be illustrated by the pigment granules, which show a continuous change in staining properties along the animal-vegetal axis of the vegetalized exogastrula (Gustafson and Lenicque, 1952). This gradual change presupposes a heterogeneous metabolic pattern in the larva. Specific localized metabolic processes have in fact been demonstrated in this material. Sulfate thus

312

TRYGGVE GUSTAFSON

becomes indispensable in the advanced blastula (Herbst) , and this sulfate requirement is confined to cells with a vegetal trend in differentiation (Lindahl and Stordahl, 1937). Lindahl suggested that sulfate may be required for the detoxication of waste products of aromatic amino acids locally produced in areas of high vegetal activity. H e furthermore demonstrated the presence of a phenolsulfatase, which might catalyze this reaction. The sulfate metabolism may be related to the formation of pigment associated with the vegetal trend in differentiation (Lindahl, 1936). It may, however, even be involved in the early processes of determination, since the animalization induced by iodosobenzoic acid becomes fortified in the absence of sulfate (Backstrom, 1953a). Serologic technique may reveal the formation of new specific components in the course of development. Such components have been demonstrated in the sea urchin gastrula (Perlmann and Gustafson, 1948) and in the amphibian tail bud stage (Flickinger and Nace, 1952). Chick and amphibian lens antigens were furthermore detected shortly before the morphologic differentiation of the lens occurred, but a refined technique might demonstrate these antigens in still earlier stages (Ten Cate and Van Dooerenmaalen, 1950). Certain adult organ antisera are in fact detectable as early as in the primitive streak stage of the chick (cf. Ebert, 1950, 1952). In the hybrid sea urchin larvae, paternal antigens were detected as early as in the mesenchyme blastula, which indicates that the nucleus has begun to take part in embryonic development at least by that time (Harding, Harding, and Perlmann, 1954). The new components in the early development demonstrated by Flickinger and Nace could not be sedimented by high speed centrifugation and are thus either water soluble or residing in the microsome fraction. Antisera to this fraction inhibit the development most effectively (Brachet; see Flickinger and Nace, 1952). The authors thus suggest that the new antigens reside in the microsomes where they may be synthesized. The origin of specific molecules and the regulation of their spatial distribution is the basic problem of ontogenetic enzymology. Morgan (1934) considered that the nuclei, equipotential in early developmental stages, change under the influence of the different cytoplasmic properties in various parts of the germ, the genes being activated or suppressed. The nuclear changes in turn retroact on the cytoplasm. Differences in enzymatic properties of nuclei isolated from various organs have in fact been demonstrated by Mirsky (1951), and in certain' observations of Brachet (1940). Krugelis (1947) and Lison and Pasteels (1951) indicate that such a differentiation may start in early developmental phases. A study of the conditions of activation of genes in different cytoplasms may thus

ENZYMATIC ASPECTS OF EMBRYONIC DIFFERENTIATION

313

be considered a fundamental problem in developmental physiology (Waddington, 1950). According to another concept, differentiation is rather a cytoplasmic question. Poulson (1945) suggested that differentiation is controled by plasmagenes. The serologic specificity of a differentiated tissue may be determined by the competition between populations of serologically different plasmagenes (Sonneborn, 1950). Spiegelman ( 1948) suggested that the plasmagenes are involved in the synthesis of various enzymes. According to him, differentiation is the result of the multiplication of different kinds of plasmagenes in different regions, directed, for example, by the amount of substrates in various parts of the embryo. These hypotheses have been further commented by Brachet ( 1952), who considers that the plasmagenes are microsomes of different specificity. A recent contribution to the theory of differentiation has been given by Rose (1952), who suggests that differentiation consists of the successive inhibition of one gene-initiated reaction after another. A major assumption in this hypothesis is that a center with high metabolism and rapid development inhibits less active and slower developing regions to attain the same level of differentiation. The inhibitors should be specific metabolites which diffuse from one cell group to another and inhibit metabolic reactions in the mass-action rdationship. An initial test seems to support this concept according to Rose. Nerveless tadpoles have thus been produced by culturing embryos with pieces of brain in the medium, and various other deficiencies have been induced in an analogous way. The bearing of enzyme adaptation on problems of genetic and cellular differentiation was discussed by Monod ( 1947). Direct evidence for adaptive enzyme formation in embryonic material was given by Gordon (1952), who studied the adenosine deaminase activity in the chick liver. In normal development, this enzyme is not demonstrable in the liver until some 24 to 48 hours after hatching. Following injection of adenosine into the yolk sac, however, considerable adenosine deaminase activity is present in the liver two to five days prior to hatching. Addition of succinate, furthermore, accelerates the developmental rise in succinic dehydrogenase in Amblystoma (Boell, 1949). The close correlation between the rise in cholinesterase activity and the development of coordinated functions in the sea urchin larva (Augustinsson and Gustafson, 1949) indicate that the enzyme is not synthesized and accumulated before it is needed. I t seems, rather, that the synthesis begins when the enzyme is required, i.e., at the onset of coordinated ciliary activity somewhat prior to hatching. The enzyme is probably located in fibrillar structures serving as a conducting mechanism and connecting the base of

314

TRYGGVE GUSTAFSON

the individual cilia (cf. Seaman and Houlikan, 1951 ; Seaman, 1951). The rise in cholinesterase activity may reflect an enzyme adaptation in these structures as a response to a local accumulation of acetylcholine. This view is in accordance with Sawyer’s (1943) interpretation of the cholinesterase curve in the development of Amblystoma. According to this, an increasing amount of acetylcholine would be liberated in the early stages of muscular response, but the enzyme content would be insufficient to hydrolyze the acetylcholine within the refractory period of the system, and consequently the muscular response becomes tetanic. A non-tetanic response is not possible until the enzyme content has increased considerably, i.e., the enzyme seems to be synthesized as a response of the substrate. The apparent differences in time of the increase in activity of the respiratory enzymes and the cholinesterase in amphibian and sea urchin embryos (Boell, 1947; Gustafson and Hasselberg, 1951) do not necessarily imply that the mechanisms of enzyme synthesis differ. The differences between the enzymes may rather be due to the different availability of the specific substrates as suggested by Augustinsson and Gustafson ( 1949). Monod, Stainer, and others (cf. Monod and Cohn, 1952) also pointed out that the formation of the so-called constitutive enzymes as well as of adaptive enzymes may involve the participation of their substrates.

XI. THEMODEOF OPERATION OF MITOCHONDRIA I N MORPHOGENESIS The role of mitochondria in the synthesis of proteins is suggested by their rich assortment of various enzymes including cathepsins. Protein synthesis may result, however, from a cooperation between nitochondriathe sites of energy generation, and microsomes-the supposed sites of formation of new proteins. The mitochondria appear to give off certain rather stable low molecular weight substances which initiate the incorporation of amino acids in the microsomes (Siekievitz, 1952). The proteinsynthesizing microsomes may, however, be more or less closely attached to the surface of the mitochondria. Opie (1947) and others in fact observed that ribonucleic acid-containing basophilic material is concentrated on the surface of the large cytoplasmic granules. The stainability of the mitochondria in the sea urchin larva may be due to such basophilic material. The appearance of mitochondria in part of the blastula wall in the sea urchin is followed by a stretching of the cells from a cylindric to a more flattened shape. This process is presumably mediated by fibrous structural proteins elaborated under the influence of the mitochondria. Each mitochondrial population will thus correspond to and contribute to a deformation of the thick blastula wall, which consequently becomes fractionated into a series of vesicles (Fig. 10). The extent of the ectoderm thus appears to

ENZYMATIC ASPECTS OF EMBRYONIC DIFFERENTIATION

315

be determined by the spatial extension of a mitochondrial population of a certain minimal density at a certain critical time of development. The localized development of mitochondrial populations in the remaining rnaterial is followed by the gradual elaboration of the entodermal vesicles. In

*

FIG.10. Optical median sections of Li-treated larvae in different stages of cell stretching. Abbe’s drawing apparatus. Age in development: no. 1 : 26, no. 2: 29, nos. 3-8: 33 hours; no. 3 with abnormal cell stretching in a small part of the presumptive entoderm. From Gustafson and Lenicque (1952).

a normal larva the first critical border in the mitochondrial frequency lies within the vegetal hemisphere of the blastula, In a strongly Li-treated larva it is, however, forced in the animal direction, and the first wave of stretching only brings about a rudimentary ectodermal protuberance (Fig. 10, no. 5 ) . If the mitochondrial frequency is about uniform in the egg subsequent to iodosobenzoic acid treatment, the entire thick blastula wall is stretched simultaneously, and a single ectodermic vesicle is formed (Fig. 9c). I n larvae, finally, which develop without plasma cleavage into vacuolized swimming syncytia, little stretching occurs and the structure protein fibers accumulate in bundles and clusters close to the vacuoles (Gustafson, 1954). Mitochondria appear to be consumed during intense processes of differentiation. The mitochondria in the mammalian epidermis thus fragment progressively in the stratum granulosum where keratohyalin granules ap-

316

TRYGGVE GUSTAPSO’N

pear (cf. Montagna, 1952), and the newly formed mitochondria in the sea urchin blastula appear to be consumed during the process of cell stretching. Raven (1946) also states that the number of mitochondria in Limnuea, abundant in the ectoderm of the gastrula, decreases with the onset of differentiation except in certain specialized cells. This also holds for the iron-containing P-granules (Arendsen de Wolff-Exalto, 1947). Mitochondria stuffed with material for morphogenetic purpose, however, may persist for a long time and only discharge their content upon certain stimuli. The cortical granules thus break down upon fertilization and supply material for the fertilization membrane (Runnstrom, 1949). The process of gastrulation in the sea urchin also starts parallel to the breakdown of mitochondria1 granules in the vegetal part of the gastrula (Gustafson and Lenicque, 1952). The carotinoids of the sea urchin egg (Paracentrotus) are bound to granules (Monroy et al., 1951). During early development there is a strong continuous drop in the carotinoid content of the germ, which may initiate a breakdown of the whole granules. The pigment probably disappears from the micromere material at the time preceding invagination which agrees with the vegetal breakdown of granules observed by Gustafson and Lenicque (1952). The drop in carotinoid content during the cleavage stages only fails to occur when gastrulation is abnormal. Monroy’s observation may thus illustrate how old mitochondria, stuffed with morphogenetic material, are consumed for morphogenetic purposes, e g , preliminary cell-stretching during stages when a new active mitochondrial generation has not as yet appeared. Granules displaceable by centrifugal force may in fact be active in the cell-stretching processes, the blastula cells rich in these granules showing a precocious flattening (Lindahl, 1932). XII. ON THE MODEOF ACTIONOF LI IONSIN

THE

DEVELOPING EGG

Sensitivity to Li ions is not restricted to the sea urchin egg but is also demonstrated in a wide variety of invertebrates as well as vertebrates. It has thus been possible to use Li as a tool in studies of morphogenetic fields and of the course of embryonic determination (Lindahl, 1936), Horstadius (1936), Gustafson, (1950, 1952a, b), Child (1941), Raven (1952), Lehmann (1945), Pasteels ( 1945), Lombard (1952), Backstrom (1953b), Ranzi (1952), Lallier (195Za, b) and others. In the sea urchin, Li treatment brings about vegetalization as described above. I n Limnaea it induces a series of head malformations (cf. Raven, 1952). In Xenopus, finally, Li treatment may bring about “ventralization,” i.e., the larvae are reduced in their axial parts to a little hump attached to the great entoderm

ENZYMATIC ASPECTS OF EMBRYONIC DIFFERENTIATION

317

and ventrolateral ectoderm (Backstrom, 1953; cf. Pasteels, 1945). In all these cases the effects indicate that Li weakens polar gradient fields. The abnormalities mentioned above can only be induced during certain developmental periods (Lindahl, 1940; Horstadius, 1936 ; Raven, 1952 ; Pasteels, 1945 ; Backstriim, 1953 ; Backstrom and Gustafson, 1953). Periods of high Li sensitivity coincide with the early cleavage stages. If the Li treatment is restricted to the period before or after the “sensitive” period, the effects are weak or qualitatively different and, as a rule, spatially more restricted. In the sea urchin egg, the period of vegetalization is thus followed by a period of low sensitivity. A second period of high sensitivity appears in the gastrula, but treatment in this stage only suppresses the further elaboration of the germ layers, eg., the formation of arms (Backstrom and Gustafson, 1953). Li treatment of the amphibian gastrula may also bring about rather restricted spatial effects. For example, it may act rather selectively on the presumptive chordamesoderm in Triton, bringing about a SoMtitisierung of the presumptive chorda material (cf. Lehmann, 1945). If the treatment is confined to short time periods, the chorda defects are restricted to short segments which are about to lose their power of regulation at the time of treatment (Lehmann, 1945; cf. Backstrom, 1953). Treatment of gastrulae may also bring about cephalocaudal and dorsoventral hypomorphoses (Pasteels, 1945), which may be regarded in part, however, as the integral effect of a series of spatially restricted traumata. Embryonic differentiation includes the successive elaboration of a series of particulate populations, vast or restricted, isolated from each other or partly superimposed in time and space (cf. Gustafson and Lenicque, 1952). Li treatment may interfere with the development of any of these. The developmental effects of Li may thus depend upon the spatial extension and time course of development of the various particulate populations, the Li concentration, the stage and time of exposure, the rate of penetration of the agent in the various parts of the germ, as well as upon the amount of Li retained by the various blastema after the treatment. The phenomenon of revitalization known from the heat-shock studies by Brachet (1952) may finally reverse the primary effects, i.e., the primary trauma in a restricted area may heal under the influence of certain substances diffusing from adjacent non-affected tissues. Such a revitalization after Li treatment has been demonstrated by Lombard (1952) in amphibians ; cf., however, Lallier (1952b). Li interferes with the formation of enzymes and mitochondria in the sea urchin egg and in amphibians and probably in any kind of embryonic material which is sensitive to Li (Lallier, 1951, 1952a, b). This does not,

318

TRYGGVE tiUSTAFSON

however, settle the primary point of attack of Li, either its nature or its intracellular localization. Various approaches have been made to this problem. There is much evidence that Li interferes with the potassium distribution in the cells. K thus counteracts the vegetalizing effect of Li (Runnstrom, 1928) and also antagonizes the Li inhibition of the respiratory rise (Lindahl, 1936). Li also enhances the tubular excretion of K in the kidneys, although simultaneous infusions of the two ions failed to reveal evidence for competitive excretion ( Foulks et al., 1950). Various organic acids fortified the morphologic effect of Li (Lindahl, 1940), possibly due to their power of interfering with the K distribution as does Li (Gustafson, 1950; Malm, 1950; cf., however, Raven, 1952). A competition between K and hydrogen ions for secretion by the renal tubules has in fact been suggested to explain certain relationships between the K metabolism and urine acidification (Orloff and Kennedy, 1952). The fact that CO (Runnstrom, 1933), KCN, and partial anaerobiosis (Lindahl, 1936, 1940) fortify the effects of Li in the sea urchin egg might be due to acids appearing in the egg (Lindahl, 1940). Lindahl (1936) suggested that Li displaces K from certain groups in some enzyme, the action of which requires K. Such enzymes have been described, for example, by Kachmar and Boyer (1951), who found that K is necessary for the phosphate transfer from 2-phosphopyruvate to ADP is a partially purified rabbit muscle system. This observation indicates that Li, competing with K, may cause an abnormality in the phosphate metabolism. This has actually been demonstrated in yeasts, by Lindahl and Lindberg j1946), as well as in developing sea urchin eggs, where the addition of Li causes an accumulation of inorganic pyrophosphate (Lindahl and Kiessling, 1951). Lindahl and Kiessling suggest that the draining of phosphate from the first phosphorylation product is retarded in the presence of Li. Li also inhibits the anaerobic glycolysis of the amphibian gastrula to a certain extent (Lallier, 1952a). It also inhibits the aerobic and anaerobic glycolysis of human spermatozoa and destroys their motile activity. On the other hand, it stimulates the aerobic lactic acid production of rat testicular tissues without affecting the anaerobic glycolysis or the respiration (McLeod, 1949). Li added to sea urchin egg homogenates inhibits niethylene blue reduction in the presence of hexose monophosphate (Lindahl, 1936) but has no significant effect on the alkaline phosphatase (Gustafson and Hasselberg, 1950). Isolated triosephosphate dehydrogenase is inhibited to a certain extent, whereas isolated aldolase and hexokinase are unaffected (Lallier 1952a). On the whole, one gets the impression that the enzymatic effect of Li on isolated enzymes or enzyme

ENZYMATIC ASPECTS OF EMBRYONIC DIFFERENTIATION

319

extracts is weaker than on whole cells or cellular structures. The accumulation of inorganic pyrophosphate is, in fact, a rather unspecific phenomenon indicating cell damage (Lindberg and Ernster, 1953). When Li acts upon the sea urchin germ, it brings about a distinct coarsening of the cytoplasm ( Kunnstrom, 1928 ; Lindahl, 1936), whose elastic properties are considerably changed ( Runnstroni, 1935). Ranzi and collaborators (1952) underline the morphogenetic role of these changes in the physical structure of the cytoplasm and emphasize that the morphogenetic effects of Li may be induced by compounds which have a similar precipitating effect on the cytoplasmic colloids, such as Mg, sulfate, tartrate, citrate, and ethyl alcohol (Ranzi, 1952; Raven, 1952). Neither of these compounds, however, reaches the efficiency of Li. Analogously, the morphogenetic effects of thiocyanate should also, to a certain extent, distinguish other hydrating ions (Ranzi, 1952). Ranzi and collaborators (1952) studied the influence of vegetalizing and animalizing agents on the viscosity of proteins, e.g., extracted from amphibian and sea urchin embryos. Solutions of fibrous proteins, or proteins with a tendency to be converted into the fibrous state, showed a decrease in viscosity induced by animalizing substances, and an increase induced by vegetalizing substances. The viscosity changes are also brought about in vivo, as appeared from measurements on proteins extracted from animalized and vegetalized sea urchin embryos. The changes are proportional to the morphogenetic effects. The globular proteins, as opposed to the fibrous, showed an increased viscosity both when treated with vegetalizing and with animalking substances. Electron microscopic studies on actomyosin and thymonucleohistone solutions reveal a loss of the fibrillar structure upon addition of thiocyanate, whereas the fibrillar structure is very pronounced when Li is added. These observations are in good agreement with the decrease in viscosity and flow birefringence upon addition of thiocyanate. Ranzi concludes that fibrillar proteins and proteins with a fibrous trend are intimately involved in the processes of embryonic determination. These proteins are supposed to constitute an important fraction of the network of the cytoplasmic ground substance, as studied by MonnP (1946) and Lehniann (1947) and which can be studied with the electron microscope (Lehmann and Biss, 1949). Agents which bring about animalization and chorda hypertrophy should cause a fibrillar protein denaturation, expressed by a depolymerization of the network of fibrous proteins. This should agree with the observation that it is easier to extract proteins ( Lindahl, Swedmark, and Lundin, 1951) from readily animalized sea urchin eggs than from less sensitive material. Yegetalizing agents such as

320

TRYGGVE GUSTAFSON

Li should inhibit the depolymerization and thus bring about greater cytoplasmic stability, which may influence the formation of mitochondria. Raven and co-workers revealed new features of the Li problem by studying the direct effect on the Limnaea egg. They emphasize (Raven, 1952) the difficulties of piecing together all observations into a coherent picture. Li, however, obviously influences the density of the cytoplasm, especially in the cortex. It may hereby inhibit the cortical attraction of material which is to constitute the animal pole plasm (Raven, 1952). The period of maximum sensitivity to Li concides with the formation of the animal pole plasm by cytoplasmic segregation. If the formation of the polar plasms is disturbed by centrifugation, abnormalities may be brought about showing a certain resemblance to the Li malformations. When centrifugation and Li treatment are combined, the effect is found to be additive, which indicates that these two treatments have the same mechanism of action. There seem to be great differences in the mode of action of Li in the sea urchin egg and in L h m e a , but these differences should not be overemphasized. There is thus evidence that Li in both these cases operates by the modification of mitochondria1 gradients. The establishment of the mitochondria1 gradient in the sea urchin larva is determined by syntheses in the individual cells. An ooplasmic segregation appears to be of little importance for the process of determination (cf. Gustafson, 1946), although the formation of the equatorial pigment band in Pwucentrotus reveals important displacement in the cortex (cf., however, CostelIo, 1947). In Linznuea on the other hand the mitochondrial gradient appears to be established in early cleavage stages by a Li-sensitive process of ooplasmic redistribution of a mitochondria-rich ectoplasm, and this explains why centrifugation may have the same effect as Li treatment. The primary mitochondrial gradient is then perhaps consolidated by Li-sensitive development of new mitochondria similar to that in the sea urchin egg. It is tempting to suggest that the mitochondria of the primary gradient act as “nurses” for these new mitochondria; cf. Ascaris above. The cell surface, which may be regarded as a cell organ comparable with mitochondria (Runnstram, 1952), may have a similar “nurse” function in the sea urchin egg, where the development .of the new generation of mitochondria appears to be independent of the preformed mitochondria of the unfertilized egg. XIII. REFERENCES Abrams, R. (1951) Exptl. Cell. Research, 2, 235. Agrell, I. (1949) Acta Physiol. Scund., 16, 247, 355. Agrell, I. (1951a) Nuhcre, 167, 283. Agrell, I. (1951b) Ann. bid. (Pa&), 27, 287. Agrell, I. (1951~)Acta Physbl. Scand., 29, 179.

ENZYMATIC ASPECTS OF EMBRYONIC DIFFERENTIATION

.

32 1

Agrell, I. (1953) ArRw. 2001. (In press). Albaum, H. G., and Potter, V. R. (1943) Cancer Research, 3, 303. Arendsen De Wolff-Exalto, E. (1947) Proc. Koninkl. Akad. Wetemchap. A m f e r dam., 60,315. Augustinsson, K. B., and Gustafson, T. (1949) J. Cellular Comb. Physiol., S4, 311. Backstrijm, S. (1953a) Arkiv. Zool., 4, 485. Backstrom, S. (1953b) Arkiv. Zool. (In press). Backstrijm, S., and Gustafson, T. (1953) Ark&. Zool., 6, 185. Ballentine, R. (1939) Biol. Bull., 77, 328. BLnhidi, Z., Kavanau, J. L, and Gustafson, T. (1954) (In press). Barron, E.S.G. (1951) Advances in Enzymol., 11, 201. Barth, L. G., and Jaeger, L (1946) Physiol. ZoBI., 20, 133. Barth, L. G., and Sse, L C. (1951) Exptl. Cell. Research, 2, 608. Berg, W. E.,and Kutsky, P. B. (1951) Biol. Bull., 101,47. Bernheim, F.,and Felsovanyi, A. von (1940) Science, 91, 76. Berthet, J., and De Duve, C. (1952) Biochem. I., 60, 174. Bodine, J. H.,and Fitzgerald, L. R. (1948) Physiol. Zoi61., 2l, 93. Bodine, J. H.,and Lu, K. H. (1950) Phy.sio1. 2051, 28, 301. Bodine, J. H.,and Lu, K. H. (1951a) Physiol. Zool., !M, 120. Bodine, J. H.,and Lu, K. H. (1951b) B i d . Bull., 100, 153. Boell, E. J. (1947) Ann. N. Y. Acad. Sci., 49, 773. Boell, E. J. (1949) Anat. Record, 106, 600. Bourne, G. H. (1951) Cytology and Cell Physiology, p. 232. Oxford University Press, New York. Brachet, J. (1940) Arch. biol. (Liige), 61, 167. Brachet, J. (1941) Enaymlogia, 10, 87. Brachet, J. (1942) Arch. biol. (Liige), 63, 207. Brachet, J. (1952) Actualifis biochim., 16. Brachet, J., and Chantrenne, H. (1944) Acta Biol. Belgico, 2, 451. Brachet, J., and Chantrenne, H. (1946) Compt, rmd. soc. biol., 140, 892. Burgess, L. E., McNeil, E. H., and Rolfe, D. T. (1952) Federdion Proc., 11, 20. Castiglia-Pitotti, M (1948) Boll. sot. itul. biol. sper., !U, 117. Chantrenne, H. (1N7)Biochim. et Biophys. Acto., 1, 437. Child, C. M. (1941) Patterns and Problems of Development. University Chicago Press, Chicago. Child, C. M. (1948) J. Ezptl. Zool., 109, 79. Claude, A. (1946) I. Esjtl. Med., 84, 51, 61. Cleland, K. W. (1950) Proc. Linneas Sot. New South Wales., 76, 2%. Cleland, K. W. (1951) Alufraiian I. Exptl. Bwl. Med. Sci., 29, 35. Cleland, K. W.,and Rothschild, Lord (1952a) I. Expfl. Biol., 29, 285. Cleland, K. W.,and Rothschild, Lord (195%) I. Expfl. Bid., 29, 416. Cohen, A. L (1942) Growth, 6,259. Cohen, S. S. (1951) Biol. Bull., 101, 237. Costello, D. P. (1947) A m N. Y. Acad. Sci., 49, 663. Crampton, C.,and Haurowitz, F. (1951) Federation Proc., 10, 405. Crane, R. K.,and Ball, E. G. (1951) I. Biol. Ckem., 189, 269. Cravens, W. W. (1952) Ann. N. Y. Acad. Sci., 66, 188. Daneel, R., and GGtters, E. (1951) Natunuiss., 38, 117. Daniel, R. J. (1949) Biochem. I., 46, 435.

322

TRYCCVE GUSTAFSON

Deutsch, H. F., and Gustafson. T. (1952) Arkiv. Kemi, 4, 221. Dobzhansky, T. (1934) 2. Zellforsch. u. Mikroskop. Anaf., 21, 169. Doyle, W. L. (1938) Compt. rend. frm.lab. Curlsberg., Sir. chim., 21, 291. Eakin, R. If., Kutsky, P. B., and Berg, W. E. (1951) Proc. SOC.Expfl. Biol. Med., 78, 502. Ebert, J. D. (1950) J. Exptl. Zool., 116, 351. Ebert, J. D. (1952) Aim. N. Y. Acad. Sci., 66, 67. Eichenberger, M. (1953) Exptl. Cell. Research, 4, 275. Esposifo, S. M. (1950) Boll. Zool. Turin, 17, 105. Euler, H. von, and Heller, L. (1949) 2. Krebsforsch., 66, 393. Euler, H. von, Schlenk, F., Heiwinkel, H., and Hijgberg, B. (1938) 2. Physiol. Chem., 266, 208. Flickinger, R. A., and Nace, G. W. (1952) Exptl. Cel!. Research, 3, 393. Foulks, J. G., Mudge, G. H.. and Gilman, A. (1950) Federation Proc., 9, 41. FredCric, J., and Chbremont, M. (1952) Arch. biol. (Liige), 63, 109. Friedberg, F., and Eakin, R. M. (1949) 1. Expfl. Zool., 110, 33. Glock, G. E., and McLean, P. (1952) Nafure, 170, 119. Gordon, 11. W. (1952) Fe&ration Proc., 11, 220. Green, D. E. (1952) Symposium sur le cycle trkarboxylique, 2nd Intern. Congr. Biochem., Paris., p. 5. Greenberg, G. R. (1951) I. Biol. C h . ,190, 611. Greenstein. J. P., Jenrette, W. V., and White, J. (1941) 1.B i d . Chent., 141, 327. Gregg, J. R., and L~vtrup,S. (1950) C'ompt. rend. traz'. lab. Carlsbrrg, Sfr. chim., 27, 307. Goksoyr, Y., Boeri, E., and Bonnichsen, R. K. (1953) Acta Chem. Scand. 7, 657. Gross, P. R. (1951) Biol. Bull., 101, 209. Gustafson, T. (1946) Arkiv. Zool., S8A, No. 4. Gustafson, T. (1950) Rai. suisse zool., 67, 77. Gustafson, T. (1952a) Nitrogen metabolism, enzymic activity and mitochondria1 distribution in relation to differentiation in the sea urchin egg. Thesis, University of Stockholm. Gustafson, T. (1952b) Arkiv. Zool., 3, 273. Gustafson, T. (1953) J. Embryol. a d Exptl. Morphol., 1,251. Gustafson, T. (1954) (In press). Gustafson, T., and Hasselberg, I. (1950) Exptl. Cell. Research, 1, 371. Gustafson, T., and Hasselberg, I. (1951) Expfl. Cell. Research, 2, 642. Gustafson, T.,and Hjelte, M.-B. (1951) Exptl. Cell. Research, 2, 474. Gustafson, T., Hjelte, M.-B., and Hasselberg, I. (1952) Exgtl. Cell. Research, 5, 275. Gustafson, T., and Lenicque, P. (1952) Exptl. Cell. Research, 3, 251. Harding, C. V., Harding, D., and Perlmann, P. (1954) Exptl. Cell Research, 6, 202. Hargreaves, A. B., and Deutsch, H. F. (1952) Cancer Research, 12, 720. Harvey, E. B. (1931) Biol. Bull., 61, 273. Harvey, E. B. (1932) Biol. Ball., 62, 155. Harvey, E. B. (1941) Biol. Bull., 81, 114. Harvey, E. B. (1946) J. Expfl. Zool., 102, 253. Heilbrunn, L. V. ( 1 9 n ) Arch. exptl. Zellforsch. Gewebeziicht., 4, 246. Heilbmnn, L. V. (1945) An Outline of General Physiology. W. B. Saunders Co. Philadelphia Herbst, C. (1892) 2. wks Zoo'l., 66, 446.

ENZYMATIC ASPECTS OF EMBRYONIC DIFFERENTIATION

323

Hershey, A. D. (1952) Intern. Rev. Cyfol., 1, 119. Hoff-JZrgensen, E.,and Zeuthen, E. (1952) Nature, 169, 245. Holter, H. (1936) J. Cellular Comb. Physiol., 8, 179. Holter, H. (1937) Arch. enptl. Zellforsch. Gewebezuchf., 19, 232. Holter, H. (1952) Advances in Enaymol., 13, 1. Holter, H., and Lindahl, P. E. (1940) Compt. rend. trav. lab. Carkberg, Sir. chim., 23, 249. Holtfreter, J. (1945) J. Expzptl. Zool., 98, 161. Holtfreter, J. (1946a) I. Exptl. Zool., 101, 355. Holtfreter, J. (1946b) I. Exptl. ZooL, lOS, 81. Holtfreter, J. (1947) J. Exptl. Zool., 106, 197. Holtfreter, J. (1948) SymposiO SOL.Exptl. Biol., 2, 17. Horecker, B. L. (1951) Phosphoroils Metabolism, 1, 117. HBrstadius, S. (1935) Pubbl. stae. zool. Napoli., 14, 251. Horstadius, S. (1936) Wilhelm Rotrx' Arch. Enh*cklzcngnnech. Orgatr., 196, 1, 40. Horstadius, S. (1938) Wilhelm Row2 Arch. Enizvicklungsmech. Organ., 138, 197. Horstadius, S. (1950) J . Exptl. Zool., 119, 245. Horstadius, S. (1952) .J. Expfl. Zool., 120, 421. Horstadius, S. (1953) Pubbl. staz. 8001. Napoli., %, 45. Horstadius, S., and Gustafson, T. (1947) Zoo?. Bidr. Uppsala., 26, 571. Horstadius, S., and Gustafson, T. (1954) 1. Emhyol. and Exptl. Morphl. (In press). Horstadius, S., and S t r h b e r g , S. (1940) Wilhelm R o d Arch. Entm'cklungsmech. Organ., 140, 409. Hultin, T. (1949) Arkiv Kemi, Mineral. Geol.. =A, No. 27. Hultin, T. (1950a) Exptl. Cell Research, 1, 159. Hultin, T. (1950b) Expfl. Cell Research, 1, 272. Hultin, T. (1950~)Exptl. Cell Research, 1, 599. Hultin, T. (1952) Exptl. Cell Research, 3, 494. Hultin, T. (1953a) Arkiv Kemi, 6, 267. H u b , T. (1953b) Arkiv Kemi, 6, 543. Hultin, T. (1953~)Arkiv Kemi,6, 559. Hultin, T. (1953d) Arkiv Kemi, 6, 195. Hultin, T. (1953e) Studies on the structural and metabolic background of fertilization and development. Thesis, University of Stockholm. Hultin, T., and Wessel, G. (1952) Exptl. Cell Research, 3, 613. Hutchens, J. O., Kopac, M. J., and Krahl, M. E. (1942) J. Cellulur Comp. Physiol., 20, 113. Immers, J. (1952) Arkiv. Zool., 3, 367. Infantellina, F., and La Grutta, G. (1947) Boll. soc. ifal. biol. sper., 23, 492. Isbell, E. R., Mitchell, H. K., Taylor, A., and Williams, R. J. (1942) Univ. Texas Publ. No. 4357, 81. Ishida, J. (1936) Annotat. Zool. Japonenses., 16, 453. Jeener, R. (1948) Biochinr. et Biophys. Acta, 2, 633. Kachmar, J. F., and Boyer, P. D. (1951) Fe&ration Proc., 10, 204. Kaplan, J. G. (1952) PhysioZ. Zool, 26, 123. Kavanau, J. L. (1953) I. Exjtl. Zool., 124, 285. Kavanau, J. L. (1954) Exptl. Cell Research, 6, 563. Kavanau, J. L., Gustafson, T., and BAnhidi, Z. (1954) Exptl. Cell. Rescarch, 6, 567. Keltch, A. K., Strittmayer, C. F., Walters, C. P., and Clowes, G. H. A. (1950) I. Gen. Physiol., 33, 547.

324

TRYGGVE GUSTAFSON

E., and Sandstedt, R. M. (1946) Arch. Biochan. 9,235. Krahl, M. E. (1950) Biol. Bull., 98, 175. Kriszat, G., and Runnstrom, J. (1952a) Exptl. Cell. Research, 3. 597. Kriszat, G., and Runnstrlim, J. (1952b) Exptl. CelE Research, S, 500. Krugelis, E. J. (1947) B i d . Bull., 9S, 215. Lallier, R. (1951) Bull. SOC. chim. biol., 33, 439. Lallier, R. (1952a) Comptes rend., 236, 98. Lallier, R. (195%) Comptes remi., 236, 260. Lallier, R. (1952~)Experientia, 8, 271. Lansing, A. I., Hillier, J., and Rosenthal, T. B. (1952) Biol, Bull., 103, 294. Lehmann, F. E. (1945) Einfiihrung in die physiologische Embryologie. Birkhauser, Basel. Lehmann, F. E. (1947) Rev. suisse zool., 64, 246. Lehmann, F. E. (1948) Rev. misse zool., 66, 1. Lehmann, F. E. (1950) Experientia, 6, 382. Lehmann,F. E.,and Biss, R. (1949) Rev. slcisse zool., 66, 264. Lenicque, P., and Gustafson, T. (1954) Exptl. Cell Research (In press). Lenicque, P., Horstadius, S., and Gustafson, T. (1953) Exptl. Cell Research, 6, 400. Levinthal, C., and Fisher, H. (1952) Biochim. et Biophys. Acta, 9,419. Levy, M., and Young, N. F. (1948) I. Biol. Chem., 176, 185. Lichtenstein, N. (1946) Enzymologia, 12, 156. Liidahl, P.-E. (1932) Wilhelm Roux’ Arch. EntzPrickZungsntech. Organ,, 127, 323. Lindahl, P.-E. (1933) Wilhelm Ruux’ Arch. Entm’cklungsmech. Organ., 128, 661. Lindahl, P.-E. (1936) Acta eool. (Stockholm), 17, 179. Lindahl, P.-E. (1939a) 2. vergleich. Physiol., 27, 136. Lindahl, P.-E. (1939b) 2. vergleich. Physiol., a?, 233. Lindahl, P.-E. (1940) Wilhelm Row’ Arch. Entwicklzmgmch. Organ., 140, 168. Lindahl, P.-E., and Holter, H. (1940) Comfit. rend. truv. iab. Curlsberg, Sir. chim., 23, 257. Lindahl, P.-E., and Kiessling K.-H. (1950) Expevientia, 6, 425. Lindahl, P.-E., and Kiessling, K-H. (1951) Arkiv. Kemi, 8, 97. Lindahl, P.-E., and Lennerstrand, A. (1942) Arkiv. Kemi, Mineral. Geol., lSB., No. 13. Lindahl, P.-E., and Lindberg, 0. (1946) Nature, 167, 335. Lindahl, P.-E., and Bhman, L. 0. (1938) Biol. Zentr., 68, 179. Lindahl, P.-E., and Stordahl, A. (1937) WGheEm R o d Arch. Entm*ckEungsmech. Organ, 136, 44. Lindahl, P.-E., Swedmark, B., and Lundin, J. (1951) Exptl. Cell Research 2, 39. Lindberg, 0.(1946) Arkiv. Kewi, Milzeral. Geol., =A, No. 2. Lindberg, 0.(1950) Erptl. Cell Research, 1, 105. Lindberg, O.,and Ernster, L. (1948) Biochim et Biophys. Acta., 2, 471. Lindberg, O., and Ernster, L. (1954) Chemistry and Physiology of Mitochondria and Microsomes, Springer Verlag, Wien. Lindvall, S. (19.52) Personal communication. Lison, L., and Pasteels, J. (1951) Arch. biol. (Likge), 62, 1. Lombard, G. L (1952) An Experimental Investigation of the Action of Lithium on the Amphibian Development. Thesis, University of Utrecht. Wvtrup, E. (1953) J. Exptl. Zool., l24, 427. Lundblad, G. (1949) Nature, 163, 643. Kneen,

ENZBhfATIC ASPECTS OF EMBRYONIC DIFFERENTIATION

32 5

Lundblad, G. (1950) Exptl. Cell Research, 1, 264. Lundblad, G. (1952) Arkiv. Kemi, 4, 537. Lwoff, A. (1950) Problems of Morphogenesis in Ciliates. John Wiley and Sons, New York. McLeod, J., Swan, R. C., and Aitken, G. A. (1940) Am. 1. PhyysiOl., 167, 177. Malm, M. (1950) Physiol. Plantarum., 3, 376. Marshak, A., and Harting, J. (1948) J . Cellular Comp. PhysioE., S1, 321. Matsuura, K. (1951) Igaku to Seibutsugaku, 18, 96. Mazia, D. (1949) J. Cellular Comp. Physiol., 34, 17. Mazia, D., Blumenthal, G., and Benson, E. (1948) Biol. Bdl., 96, 250. Militzer, R., Ikeda, C., and Kneen, E. (1946) Arch. Biochm., 9, 309, 321. Miller, B. S., and Kneen, E. (1947) Arch. Bioclzem., 16, 251. Minganti, A. (1951) Pubbl. staz. zool. Napoli, 2S, 52. Mirsky, A. E. (1951) Cold Spring Harbor Symposia Quant. Biol.,16, 481. Monnh, L. (1946) Experieatia, 4 153. Monn6, L. (1951) Personal communication. Monni, L., and Slautterback, D. B. (1950) Exptl. Cell. Resmclz, 1, 477. Monod, J. (1947) Growth, 11, 223. Monod, J., and Cohn, M. (1952) Admnces in Emymot., 13, 67. Monroy, A. Monroy-Oddo, A., and De Nicola, M. (1951) Expfl. Cell Research, 2, 700. Monroy, A., and De Nicola, M. (1952) Experientia, 8, 29. Montagna, W. (1952) Intern. Rev. Cytol., 1, 265. Morgan, T. H. (1934) Embryology and Genetics, New York. Myrback, K., and tjrtenblad, B. (1938) Enzymologia, 2, 305. Navez, A. E., and Harvey, E. B. (1935) Biol. Bull., 69, 342. Okamoto, B. (1951) Igaku to Seibutsugaku, 18, 214. Opie, E. L. (1947) J . Exptl. Med., 86, 339. Orloff, J., and Kennedy, T. J. (1952) Federation Proc., 11, 115. Panijel, J. (1951) Experientia, 7, 100. Patiijel, J., and Pasteels, J. (1951) Arch. b i d (Lihge), 62, 353. Parrish, D. B., Williams, R. N., Hughes, J. S., and Payne, L. F. (1950) Arch. Biochem., 29, 1. Pastels, J. (1945) Arch. biol. (Libge), 66, 105. Perlmann, P., and Gustafson, T. (1948) Experientia, 4, 481. Pitotti, M. (1939) Pubbl. staz. zool. Napoli, 17, 193. Ponder, E. (1951) J. Gen. Physiol., 34, 551. Pontecorvo, G. (1952) Symposia soc. exptl. biol., 6, 218. Potter, V. R., Lyle, G. G., and Schneider, W. C. (1951) J .Biol. Chem., 190, 293. Poulson, D. F. (1945) Am. Natwalist, 79, 340. Ranzi, S. (1939) Arch. zool. ital., 26, 427. Ranzi, S. (1952) Arch. leierl. BOO!., 10, 92. Ranzi, S., and Falkenheim, M. (1937) Pubbl. staz. 2001. Napoli, 16, 436. Rapkine, L., and Brachet, J. (1951) Bull. SOC. chim. biol., 53, 427. Raven, C. P. (1944) Arch. nkerl. zool., 7, 353. Raven, C. P. (1952) Experientia, 8, 252. Recknagel, R. 0. (1950) J. Cellzdar Comp. Ph3doZ., SS, 111. Ries, E. (1937) Pubbl. staz. zool. Napoli, 16, 363. . 16, 223. Ries, E., and Gersch, M. (1936) Pubbl. staz. ~ 0 0 1 Napoli,

326

TRYGGVE GUSTAFSON

Rose, S. M. (1952) Am. Naturalist, 86, 337. Rothschild, Lord. (1950) J . Exptl. Biol., 26, 306. Runnstrom, J. (1928) Wilhelm Roux‘ Arch. Enkwicklungsmech. Organ., 113, 556. Runnstrom, J. (1929) Wilhelm Roux’ Arch. Entwicklungsmeck. Organ., 117, 123. Runnstrom, J. (1931) Wilhelm R o d Arch. Entzticklungsmech. Organ., 124, 127,a3. Runnstrom, J. (1933) Wilhelm R o d Arch. Enh‘cklungsmech. Organ., l29, 442. Runnstrom, J. (1935) Bwl. Bull., 68, 378. Runnstrom, J. (1949) Advances in Enzymol., 9, 241. Runnstrom, J. (1952) Modern Trends in Physiology and Biochemistry, p. 47. Academio Press, New York. Runnstrom, J., and Kriszat, G. (1950) Exptl. Cell. Research, 1, 355. Runnstrom, J., and Kriszat, G. (1952) Exptl. Cell. Research, 3, 497. Runnstrom, J., and Thornblom, D. (1938) Acta Biol. Lat, 8, 97. Rybak, B., and Gustafson, T. (1952) Arkiw. 2002.3, 189. Sanders, F. K(1952) Symposia SOC.Exptl. Biol., 6, 201. Sawyer, C. H. (1943) 1. Exptl. Zool., 92, 1. Scheid, H.,and Schweigert, B. S. (1951) J. Biol. Chem, 193, 299. Seaman, G. R. (1951) Proc. SOC.Exptl. Biol. Med., 76, 169. Seaman, G. R.,and Houlikan, R. K. (1951) J. Cellular Comp. Physiol., 37, 309. Shapiro, H. (1935) J. Cellular. Comp. Physiol., 6, 101. Shearer, C. (1922) Proc. Roy. SOC.(London), BSS, 213. Siekevitz, P. (1952) J. Bwl. Chem., 196, 549. Sonneborn, T. M. (1950) Heredity, 4, 11. Sonneborn, T. M. (1951) Cold Spring Harbor Symfiosia Quant. Biol., 16, 483. Spiegelman, S. (1945) Qacart. Rev. Biol., 20, 121. Spiegelman, S. (1948) Symposia SOC.Exptl. Biol., 2, 286. Spiegelman, S., and Steinbach, H. B. (1945) Biol. Bull., 88, 254. Spratt, N.,Jr. (1951) Anat. Record, 111, No. 3. Spratt, N., Jr. (1952) Anw. N. Y . Acad. Sci., 66, 40. Steinbach, H.B., and Moog, F. (1945) 1. Cellzdar Comp. Physiol., aS, 175. Steinert, M. (1951a) Bull. SOC. chim. biol., 33, 549. Steinert, M. (1951b) Expm*entiu,7,342. Steinert, M. (1952) Arch. intern. phytysiol., 60, 192. Sugawara, H. (1943) J . Faciclty Sci. Imp. Univ. Tokyo, Sect. IV, 6, 109. Sumerwell, W. N.,and Sealock, R. R. (1952) I. Biol. Chem.,196, 753. Ten Cate, G., and Van Doorenmaalen, W. J. (1950) Proc. Koninkl. Ned. Akad. Wetenschap., Ser. C, 63, 3. Thorell, B. (1947) Acta. Med S c a d . S@pl., 200. Tyler, D. B. (1951) Am. J. Physa’ol., 164, 467. Urbani-Mistruzzi, L.,and Carducci-Pitoni, A. (1948) Atti accad. naa. Lincei, Rend. Classe sci. fis., mat. e nat. Ser. XA, 4, 228. Vasseur, E. (1952) The Chemistry and Physiology of the Jelly Coat of the SeaUrchin Egg, Thesis, University of Stockholm. Villee, C. A., Lowens, M., Gordon, M., Leonard, E., and Rich, A. (1949) 1. Cellular Comp. Physiol., 33, 93. Vincent, W . S. (1952) Proc. Natl. Acad. Sci. U.S., S8, 139. Vogel, H. J., and Davis, B. D. (1952) Federation Proc., 11, 485. Waddington, C. H. (1950) Rev. misse aool., 57, Suppl. 1, 153. Wenner, C. E.,and Weinhouse, S. (1953) Cancer Resmrch, 13, 21.

ENZYMATIC ASPECTS OF EMBRYONIC DIFFERENTIATION

Wigglesworth, V. B. (1948) Symposia SOC. Exptl. Biol.. 2, 1. Williams, J. N., Jr. (1952) I. B i d . Chm., 197, 709. Woodward, A. A., Jr. (1949) Bid. Bdl., 97, 264. Woodward, A, A., Jr. (1950) B i d . Bull., 99, 367. Wu, H. W., and Wang, T. H. (1948) Sirzensia, 19, 6. Zeuthen, E. (1946) Compt. red. truv. lab. Carlsberg, Sir. chk., 26, 191. Zeuthen, E. (1949) Am. Naturalist, 89, 303. Zeuthen, E. (19%) B i d . Bull., 98, 144. Zwthen, E. (195Ob) B i d . Bull., 98, 152. Zeuthen, E. (1951) Pubbl. stm. zool. Napoli, Swppl., 23, 47.

327

This Page Intentionally Left Blank

Azo Dye Methods in Enzyme Histochemistry

. .

A G EVERSON PEARSE Post-graditate Medical School. Hammersmith. London. E n g l a d

Page I . Introductiori ....................................................... 329 TI. Criteria for Azo Dye Methods ...................................... 330 1 The Substrate ................................................. 330 2 The Primary Reaction Product (Simultaneous Methods) ........ 331 3. The Final Reaction Product (All Methods) ..................... 331 4. The Diazonium Salt (Simultaneous Methods) .................. 331 5 The Primary Reaction Product (Post-coupling Methods) ........ 331 111. The Non-coupling Azo Dye Methods .............................. 334 I V . .The Simultaneous Coupling Azo Dye Methods ...................... 335 1. Effects Due to the Nature of the Substrate ...................... 335 2. Diffusion of the Primary Reaction Product ...................... 336 3 The Rate of Coupling .......................................... 336 4. Modifications of Substrate ...................................... 338 5. A Modified Naphthol A S Method for Esterase .................. 344 6. Effects of Increasing Complexity of the Substrates .............. 347 7. Conclusions ................................................... 349 349 V . Effects Due to the Nature of the Diazotate ........................ 1. Non-specific Coupling in the Tissues ............................ 349' 2. Staining of the Tissues by the Diazotate or its Breakdown Products 350 3 Inhibition of Enzymes .......................................... 350 4. Conclusions .................................................... 351 5. Criticisms of Particular Simultaneous Coupling Methods .......... 351 VI . The Postxoupling Azo Dye Methods .............................. 353 VII Final Conclusions .................................................. 355 \TI11 References ........................................................ 357

. .

.

.

.

. .

I . INTRODUCTION The production by Menten. Junge. and Green in 1944 of the first azo dye method for the demonstration of alkaline phosphatase in tissue sections marked the introduction of a new histochemical principle . The nine years following their discovery have seen the application of this coupling azo dye principle not only to the demonstration of the phosphatases but to many other enzymes as well. It may therefore be appropriate to take stock of the situation. at a time when there is more than a suggestion that the azo dye methods may be eclipsed by methods depending on the production of substituted indigos (Holt. 1952; Holt and Withers. 1952) . The azo dye methods can be divided into three groups. non.coupling. simultaneous coupling. and post.coup1ing. depending on whether or not a diazonium salt is used to produce the final colored reaction product and. if so. on whether it is used in the incubating medium (simultaneous

330

A. G. EVERSON PEARSE

coupling) or in a separate bath after stopping the enzymatic reaction (post-coupling) The original method of Menten et al. (1944) depended on the hydrolysis of calcium P-naphthylphosphate and on the reaction in situ of the liberated @-naphtholwith freshly diazotized a-naphthylamine at pH 9.4. It was thus the forerunner of all the simultaneous coupling methods. The first recorded non-coupling method was that of Friedenwald and Becker ( 1948), who used a highly colored soluble substrate (1-0hydroxyphenylazo-2-naphthyl glucuronide) which was hydrolyzed to an insoluble colored reaction product. The post-coupling methods have been developed mainly by Seligman and his associates, although Danielli (1946) produced a method for alkaline phosphatase (see Table I ) which falls into this category. The method of Seligman et d.(1949), using 6-bromo2-naphthyl glucuronide as substrate for P-glucuronidase, was the first of a series of very similar post-coupling methods for different enzymes. In this type of method, incubation takes place at a p H generally considered to be too low for efficient coupling with most diazotates, and the colorless, water-insoluble reaction product which is deposited in the tissues is therefore subsequently coupled with a diazonium salt, at a more suitable (alkaline) pH, to produce a highly colored azo dye. A list of the azo dye methods employed in enzyme histochemistry appears in Table I. In this chapter I propose to consider the theoretical and practical advantages and disadvantages of the three a m dye methods, and to report a small amount of experimental work which has bearing on the problems of enzyme specificity and accuracy of localization which are involved. Methods for demonstrating enzymes which depend on other than azo dye substrates will be discussed only indirectly, hut some attempt will be made to decide whether the azo dye methods have a future in applied histochemistry, or whether they are doomed to extinction. Tnevitahly, the substance of this chapter must be considered as an interim report. 11. CRITERIA FOR Azo DYEMETHODS

.

For all the azo dye methods certain general characteristics are desirable in the substrate, and for the simultaneous coupling reactions certain additional qualities should reside in the chosen coupling agent. A list of the most important criteria appears below.

1. The Substrate should be (1) stable in the solvents employed, over a wide range of pH ; (2) free from strong ionic charges and (3) able to penetrate cell membranes;

A 2 0 DYE METHODS I N ENZYME HISTOCHEMISTRY

33 1

(4) strongly attracted to enzyme receptor groups and rapidly hydrolyzed ;

( 5 ) insoluble in, and unbound by, other tissue components; ( 6 ) soluble up to its optimal concentration for enzyme activity.

2. The Primary Reaction Product (Simultaneous Methods) should be ( 7 ) insoluble in water, buffer, and other solvents employed; (8) insoluble in, and unbound by, all tissue components.

3. The Final Reaction Product (A11 Methods) should be ( 9 ) insoluble in water, bufier, and other solvents employed; (10) insoluble in, and unbound by, all tissue components; ( 1 1) highly colored; i.e., highly absorbing in the red or blue regions of the visible spectrum; (12) (usually) non-particulate under high powers of the light microscope.

4. The Diazoniirm Solt (Simultuneous Methods) should be ( 13) sufficiently soluble at the pH employed ; (14) stable at the pH employed ; (15) non-inhibitory to the enzyme concerned; ( 16) lipid insoluble ; (17) capable of rapid coupling with the primary reaction product.

5. The Priiimry Reactioiz Product (Post-cozhpling Methods) must be (18) insoluble in water, buffer, and other solvents employed; (19) insoluble in, and unbound by, all tissue components. It must be observed, at once, that not one of the methods so far described is able to satisfy all these relatively simple criteria, and that this failure provides a series of Achilles heels which may render the whole principle of the azo dye methods vulnerable if not mortal. Having said this, it is possible to qualify most of the criteria to allow the practical utility of many of the methods while admitting their theoretical imperfections. Between the theoretical data, based on chemical and physical measurements of the various reactions in zitvo, and the results of their application to tissue sections, there is a great gulf. Absolutely critical localization of an enzyme on the microscopic intracellular level, that is to say, localization to mitochondria, rnicrosomes, or other intracellular structures is not entirely necessary for work in applied histocheniistry. Biochemical evidence may suggest, for instance, that a given enzyme is contained entirely within the mitochondria. Granted this knowledge, it may then be important to determine the presence or absence of the enzyme in a certain type of cell,

TABLE I Azo DYE METHODSmn ENZYMES Type of method'

Enzyme

Substrate

Precipitant

Demonstrator

Author (I)

NC

p-glucuronidase

Nil

Nil

NC

alkaline phosphatase alkaline phosphatase alkaline phosphatase alkaline phosphatase acid phosphatase acid phosphatase esterase

l-o-hydroxyphenylazo2-naphthyl glucuronide p-nitrobenzene-azonaphthol-1-phosphate calcium 8-naphthyl phosphate calcium p-naphthyl phosphate sodium a-naphthyl phosphate calcium a-naphthyl phosphate sodium a-naphthyl phosphate p-naphthyl acetate

Nil

Nil

a-naphthylamine (diazo) a-naphthylamine (stable diazo) o-dianisidine (stable diazo) anthraquinone (stable diazo) o-dianisidine (stable diazo) a-naphthylamine (stable diazo)

Nil

Friedenwald and Becker (1948) Loveless and Danielli (1949) Menten et (11. (1944)

sc

sc sc sc

sc sc

Nil Nil Nil Nil

Nil

Manheimer and Seligman (1949) Gomori (1951) Seligman and Manhrimer (1949) Grogg and Pearse (1952b) Nachlas and Seligman (1949)

? 0

TABLE 1-( Continued) Type of method.

Enzyme

Substrate

sc

esterase

a-naphthyl acetate

sc

esterase

sc

cholinesterase

2-hydroxy-3naphthoic anilide 6-bromocarbonaphthoxy choline sodium phenolphthalein phosphate 6-bromo-2-naph thy1 glucuronide 6-benzoyl-2-naphthyl sulfate 6-bromo-2-naphthyl p-galactoside 6-bromo-2-naphthyl p-galactoside

PC PC

alkaline phosphatase p-glucuronidase

PC

aryl sulfatase p-galactosidase

PC

p-glucosidase

PC

NC, non.soupling: SC. rimultancous coupling; PC. pcat-coupling.

Precipitant o-dianisidine (stable diazo) o-aminoazotoluene (stable diazo) a-dianisidine (stable diazo) Nil Nil Nil Nil Nil

z Demonstrator Nil

Author (s) Gomori (1950)

Nil

Gomori (1952a, b)

Nil

Ravin et a[. (1951)

NH,OH o-dianisidine (stable diazo) o-dianisidine (stable diazo) o-dianisidine (stable diazo) o-dianisidine (stable diazo)

Danielli (1946)

Seligman and Nachlas (1950) Seligman et al. (1949)

0 U

;$m

E4

z

: M

2z

m

Ecn c1

Cohen t t al. (195Za)

Eg

Cohen ~t 01. (1952b)

1

I4

v1

0

.c

334

A. G. EVERSON PEARSE

the amount that is present at a given time, and the changes in amount that occur in various physiologic or pathologic conditions. These processes may be more easily followed (and even measured) if the reaction product, though confined by the cell membrane, stains all the cytoplasmic coniponents indifferently, dissolves in them, or fills the whole cell. The theoretical requirements of 10, above, may therefore be undesirable in some instances. Notwithstanding the above, the method which gives the most critical intracellular localization should always be employed in applied histochemical research unless there are good reasons to the contrary. In the discussions that follow, the azo dye methods will be considered largely as experimental tools for the applied histo-chemist and not primarily as methods for critical intracytoplasmic localization of enzymes. Nevertheless, it will be shown that some, at least, of the simultaneous coupling azo dye methods are capable of a degree of intracellular localization approaching that given by the substituted indoxyl methods at their best.

111. THE NON-COUPLING Azo DYE METHODS It will be observed from Table I that only two methods of this type have been evolved, and both can be dealt with fairly briefly. The substrate used by Friedenwald and Becker (1948) has been shown by Campbell (1949) and by Burton and Pearse (1952) to give a comparable result with active and inactivated sections. The method of Loveless and Danielli ( 1949) for alkaline phosphatase, though theoretically excellent, produced results that are difficult to interpret. In kidney sections, for instance, provided that some free phenol was present, the nuclei alone were stained. In the presence of free phosphate as well, the final dye reaction product was also found in the brush borders. These results were regarded by the authors as indicating a differential activation of nuclear and brush border phosphatases. I consider that before this ittterpretation of the facts can be accepted alternative explanations (diffusion of partly soluble reaction product, non-specific modifiable absorption of phenol) must be overruled. The failure of kidney alkaline phosphatase to hydrolyze the pure substrate (p-nitrobenzeneazonaphthol-l-phosphate) must also be explained. A wide variety of naphthol and substituted naphthol phosphates have proved to be readily hydrolyzed, in the pure state, by mammalian alkaline phosphatase. Whether a non-coupling azo dye method free from such objections as these can be produced remains to be seen. It is probable, with the shifting of emphasis toward the post-coupling and simultaneous coupling methods,

AZO DYE METHODS I N ENZYME

HISTOCHEMISTRY

335

and even away from the azo dye methods altogether, that further attempts to synthesize soluble colored esters of insoluble colored am dye bases will be few and far between. In the case of other dyes it is possible that non-coupling methods may be evolved. The soluble colored esters of leucobases of the benzanthrone series of dyes, for instance, yield highly colored and highly insoluble bases on hydrolysis and oxidation. If these large molecules are hydrolyzed sufficiently rapidly by the protected enzymes of tissue sections, the localization of the precipitate should be closely related to the sites of enzyme activity. IV. THE SIMULTANEOUS COUPLING Azo DYEMETHODS All the simultaneous coupling azo dye methods listed in Table I can be criticized (a) on account of the use of unnatural substrates and (b) on account of the employment in the incubating medium of strong bases (diazonium hydroxides) which have affinity for acid groups in the tissues and which are inhibitory toward the various enzymes. The fact remains, however, that esterases are capable of hydrolyzing the unnatural naphthyl and other substrates at very high rates (in some cases the rates of hydrolysis in z4tro exceed those of the natural substrate), and attempts to modify the substrate in order to make it resemble the natural one (Wolf ef ad., 1950) have usually resulted in a deterioration in respect to criteria 2 and 5. The necessary implications of the second objection will be considered, below, in a section on the effects due to the nature of the diazotate. 1. Efects Date to the Nature of the Substrate Many naphthyl substrates (the acetates for instance) are subject to slow non-enzymatic hydrolysis in buffer solutions. This feature is not of much importance, perhaps, with small incubation times, but it must be considered increasingly important as the length of incubation increases. All the older simultaneous coupling azo dye methods fail to satisfy criterion 7 and are subject to diffusion of the primary reaction product. The process of diffusion can be shown, when it is not obvious, by reducing the strength of the diazonium salt below its minimum value for effective coupling or by interference with its availability. Attempts to measure the m o u n t of diffusion by the face-to-face method produce a diffusion effect by preventing coupling in this way. ( I n the face-to-face method active and inactivated slides are incubated with a small space between them, maintained by a pair of coverslips or pieces of cellophane at either end of the slides. Coupling is markedly slowed when the distance between the sections is reduced to about 100 p , so that Grogg and Pearse (1952b), for instance, were unable to measure the diffusion of a-naphthol at pH 5.0

336

A. C. EVERSON PEARSE

below this relatively enormous figure. It is probable that the maintenance of a forced flow of incubating medium between the two slides would enable diffusion to be measured at lower levels but unlikely that diffusion of the order of 1 to 5 p could be measured by this means. 2. Difusion of the Prhmry Reaction Product

Provided that the final reaction product ( F R P ) is absolutely insoluble, and discounting for the moment those usually insignificant errors of localization which are due to enzyme diffusion, all displacement of the F R P from the site of active enzyme hydrolysis will be due to diffusion of the primary reaction product (PRP). This in its turn is governed by several factors, notably by the rite of production of the P R P (dependent on the turnover number of the enzyme for the chosen substrate), by the diffusion constant of the PRP, and by the rate of its coupling with the diazonium salt. All three can be subjected to some measure of control, first by chemical modifications of the substrate, affecting both diffusion constant (solubility) and coupling rate, and secondly by modifications of the diazonium salt, affecting the rate of production of the PRP (by enzyme inhibition) and also the rate of coupling.

3. The Rate of Coupling The naphthols, which form the PRP of most current simultaneous coupling methods, couple more rapidly with diazonium salts than do most phenols or amines. The substitution of hydrogen atoms of the coupling agent (i.e., the phenol or naphthol) by electronegative groups (eg., nitro, sulfo, cyano, carboxyl, carbonyl, halogen) has the effect of slowing the reaction rate, while the presence of these groups in the diazonium salt increases the rate. Conversely, substitution of the coupler by electropositive groups (e.g., hydroxyl, amino, methoxy, ethoxy, methyl) increases the rate, while similar substitution of the diazonium salt retards it (Fierz-David and Blangey, 1949). The rate of coupling may be increased by lowering the acidity, by raising the concentration of the salt, or by raising the temperature, and also by the addition of water-binding agents. Undesirable side effects are introduced, however, both by warming and by increasing the alkalinity, in that breakdown of the diazonium salts becomes rapid. These effects are considered below in the section on diazonium salts. In order to determine just how much the rate of coupling affects the final histochemical result, some experiments were carried out with a simple type of flowmeter, designed to measure the coupling rate of the various combinations of PRP and diazonium salt used in enzymatic histo-

337

A20 DYE METHODS IN ENZYME HISTOCHEMISTRY

chemistry (Defendi and Pearse, 1954). The apparatus, suggested by the experiments of Ilartridge and Roughton (1923) on the dissociation of oxygen and hemoglobin, consisted simply of two large bottles connected, with the minimum dead space, to a long capillary tube, and finally to a collecting bottle. After mixing of P R P and diazonium salt solutions their rate of flow through the capillary tube was controlled by the application of negative pressure. The amount of FRP production (azo dye) was determined photometrically at various points along the capillary tube, and curves were constructed in which the percentage of F R P formed the ordinate and the time of reaction the abscissa. The times listed in Table I1 for various combinations of PRP and diazonium salt were determined by this method; in each case the time taken to reach the point of 50% conversion to the FRP is listed. TABLE I1 COUPLING I N VITF.0 (15-17" c.) (50% final dye concentration)

RATES OF

a

Diazonium salt of

Coupling agent

o-dianisidine o-dianisidine o-dianisidme o-dianisidine o-dianisidine a-dimisidhe a-dianisidine o-dianisidine a-dianisidine o-dianisidine

a-naphthol a-naphthol a-naphthol a-naphthol a-naphthol p-naphthol p-naphthol p-naphthol p-naphthol p-naphthol

o-dimisidhe I-arninoanthraquinone 1-aminoanthraquinone o-dianisidine 1-aminoanthraquinone o-dianisidine o-dianisidine o-dianisidine o-dianisidine o-dianisidine a-dianisidine o-dianisidine o-dianisidine o-dianisidine

thymol thymol a-naphthol guaiacol guaiacol Naphthol AS Naphthol AS-BG Naphthol AS-FR Naphthol AS-OT 6-bromo-%naphthol 1-chloro-2-naphthol 1,6-dibromo-?-naphthol 2,4-dichloro-l-naphthol 2,4-dibromo-l -naphthol

In 50% ethanol. o.syO pyriaine.

b IU

Buffer pH f0.2 5.0 6.2 7.4 8.2 9.2 9.2 8.2 7.4

6.2 5.0 7.4

7.4 7.4 7.4 9.2

7.4' 7.4' 7.4"

7.4" 7.4' 7.4' 7.4 7.4b 7.4

Time (secs.) 2.75 0.298 0.165 0.130

0.092 0.080 0.155 0.185 20.0 Not recorded 1.57 1.65 0.30 8.0 1.o 47.0 92.0 41.0 82.0 0.328 1.36 Not recorded 0.240 Not recorded

338

A. G. EVERSON PEARSE

Although it is not claimed that the figures recorded in the Table are necessarily completely accurate they are considered sufficiently accurate to form a basis of comparison. Without entering into mathematical calculations of the extent of possible diffusion in each case, it is clear from the figures in the top half of Table I1 that with a- and P-naphtholic substrates the coupling interval under these optimal in vitro conditions is in nearly all cases sufficiently large to allow some diffusion of the soluble PRP. Direct comparison between U- and p-naphthol, coupling with Fast Blue B salt at p H 7.4, shows that there is little difference between the two. If their respective diffusion constants are similar, any observable difference in diffusion under histochemical conditions is likely to be due to a different affinity for reactive groups in the tissue, that is to a different degree of non-specific absorption. A series of experiments was carried out with modified substrates, designed to establish the precise conditions most favorable to accurate enzyme localization. Where the rates of coupling of the P R P of these modified substrates were also measured, they appear in the lower half of the table.

4. Modifications of Sbbstrate The two obvious lines of attack were, first, the use of substrates '(and diazotates) which might be capable of more rapid coupling than the conventional ones and, secondly, the use of substrates whose phenolic P R P would diffuse less before the occurrence of coupling, whether that were slow or fast, than the relatively highly soluble naphthols. The possibilities of these two lines of attack were explored by using the substrates listed in Table 111, in all cases in a standard simultaneous coupling method applied usually to cold formalin-fixed frozen sections either attached to slides or free floating. The possibilities of the second line of attack have already been explored, by other workers in the field, in two particular directions. First Seligman and his associates (Ravin, Zacks, and Seligman, 1953; Ravin, TSOU, and Seligman, 1951; Rutenburg, Cohen, and Seligman, 1952) have carried out experiments with halogen- and aroyl-substituted naphthol esters and, secondly, Gomori ( 1952a) has used 2-acetoxy-3-naphthoic anilide (Napthol AS acetate) in place of U- and 0-naphthyl acetates. Most of the work of Seligman was carried out with the idea of increasing the insolubility of the P R P to a point at which simultaneous-coupling could be replaced by a post-coupling technique. Where his substrates can be used in simultaneous coupling techniques, however, they may be expected to yield better results than substrates having more soluble P R P (such as the unsubstituted naphthols). Naphthol AS acetate was used by Gomori primarily with

TABLE 111

DYE SU BSTA N C ES (All phosphates used a s disodium salts)

PERFORhIAKC'E OF ~ , l O U I F I E DA20

Substrate

Quality

Thymol acctate Thyrnol phosphate Guaiacol acetate 6-bromo-2-naphthyl phosphate

Poor

Good

1-chloro-2-naphthyl phosphate

Moderate

1,6-dibro11~0-2-naphthylphosphate

Moderate

2,4-dichloro-l-naphthyl phosphate

Good

2,4-dibromo-l-naphthyl phosphate

Moderate

2-acetoxy-3-naphthoic anilide ( A S acetate) 2-acetoxy-3-naphthoic-2,5' dimethoxy anilide (AS-BG acetate) 2-acetoxy-3-naphthoic utoluidide (AS-OT acetate) 2-acetoxy-3-naphthoic o anisidide (AS-FR acetate) 2-acetoxy-6-bromonaphthalene-3carboxylic acid

tiood

Poor

Poor

Moderate Excellent

blistochcmical Kesult Kemarks Flocculent FRP with diffusion Flocculent F R P with ditiusion Flocculent F R P with diffusion Equivalent to a-naphthyl phosphate at p H 9.2 arid at pH 5.0 Diffusion of P R P with naphthyl phosphates Equivalent to naphthyl phosphates at p H 9.2 and a t p H 5.0 Better than naphthyl phosphates a t p H 5.0 Better than naphthyl phosphates a t p H 5.0, worse at p H Y.0 Some diffusion of P K P a t p H 6.8 Large crystalline FRP, some diffusion of P R P Better than AS acetate

Good

Equivalcnt results to AS acetate

Bad

Not hydrolyzed by tissue esterases

z

0

&

&

\o

340

A. G. EVERSON PEARSE

the idea of reducing diffusion of the PKP and thus increasing the accuracy of localization of esterase. Applying the method to his standard cold acetone-fixed paraffin sections, however, Gotnori ( 1952a, 1952b) described a series of localizations of esterase, which led him to postulate the existeixe of a separate AS-esterase. In my view the importance of the Kaphthol AS acetate method is not that it allows demonstration of a separate enzyme, the existence of which is open to question, but that with suitable tissue preparations it permits far more accurate localization within the cell of the biochemically established non-specific esterases than any previously recorded azo dye method. The use of cold acetone-fixed paraffin sections for the localization of enzymes in the tissues, long sanctioned by custom, is completely indefensible. This is not only because of the admittedly large destruction of most enzymes, which the process involves, but because of relatively enormous movements of enzyme which occur when the intracellular structures of which they formed an integral part are altered or, if they are of lipid nature, totally removed. I have hitherto recommended (Grogg and Pearse, 1952a, b ; Pearse, 1953a) that cold formalin-fixed frozen sections be employed for all enzymes not sensitive to this fixative, whatever technique is used, but especially with the azo dye methods. I must add thereto the proviso that, if the most accurate intracellular localization is desired, free-floating and not mounted sections should be employed. No prejudice to the position of freeze-dried sections is intended by these remarks, although I have not found such material entirely satisfactory for use with the coupling azo dye methods (see Figs. 1, 2, 3 and 4). a. Substituted Phenylphosfhafes. The possibilities of the first line of attack referred to above were explored by using esters of thymol and guaiacol as substrates. The rates of hydrolysis were found to be rapid, but it can be seen from Table I1 that the rates of coupling with diazonium salts were far slower than those obtained with U- or p-naphthol. Effective localization of esterase and alkaline or acid phosphatase was not obtained, therefore, with either thymol or guaiacol esters, and in both cases a flocculent FRP was produced which diffused widely in the incubating medium above the section, rising as a cloud from areas of active enzymatic hydrolysis. The results obtained with twenty different diazonium salts were in no case as good as those produced by using the naphthyl acetates. b. Halogen-Substilufed Maphtlzyl Plzosphates. Two mono-substituted and three di-substituted naphthols were investigated as substrates in simultaneous coupling methods for alkaline (pH 9.2) and acid (pH 5.0) phosphatases. One of the mono-substituted pair (6-bronio) was found to be as good as a-naphthyl phosphate, but the other was less good at both

AZO DYE METHODS I N E N Z Y M E HISTOCHEMISTRY

34 1

FIG.1. Rat kidney, freeze-dried and acetone-fixed 5 p section. AIkaIine phosphatase using sodium a-naphthyl phosphate at pH 9.2. (Incubation 10 minutes.) Note apparent presence of cells within the so-called brush border. Hemalum, X 490.

FIG.2. Rat kidney, as Figure 1. Esterase using a-naphthyl acetate/Fast Blue B salt at p H 7.4. (Incubation 5 minutes.) X 520. FIG.3. Rat kidney, as Figures 1 and 2. Esterase using AS-BG acetate a ~ ~ d diazotized 4-benzoylaniino-2,5-dimethoxy aniline at pH 6.8. (Incubation 25 minutes.) Note suggestion of localized perinuclear concentrations of enzyme. X 430. FIG.4. Rat kidney, freeze-dried, 5 p, free-floating section. Esterase using Naphthol A S acetate/Fast Red RC salt. (Incubation 20 minutes.) There is no nuclear counterstain; the enzyme is present solely in spherical bodies of various sizes. X 520.

342

A. G. EVERSON PEARSE

alkaline and acid p€I levels. One of the di-substituted naphthyl phosphates was inferior to a-naphtliyl phosphate, but the other two (2,4-dibromo and 2,4-dichloro) were sometimes considerably better, especially at p H 5.0, where the visible diffusion of PRP from a strong enzyme locus (prostatic acini) was very considerably diminished. In the case of rat kidney, using the 2,4-dibronio salt, the apparent localization of acid phosphatase was observed closely to resemble that afforded by some of the newer esterase techniques, in that the FRP was concenerated in the subnuclear region of the

FIG.5. Rat kidney, cold formaiin-fixed 10 p frozen section. Acid phosphatase using 2,4-dibromonaphthyl-l-phosphate/Purplesalt at pH 5.0. (Incubation 3 hours.) The enzyme appears predominantly in the perinuclear “GoIgi region”. X 240. FIG.6.

Rat liver, as Figure 5. Distribution of esterase using 5-bromoacetylindoxyl.

x 550. FIG.7. Rat liver, as Figures 5 and 6 . Distribution of esterase using Naphthol AS acetate/Fast Blue B salt. Note essential similarity to Figure 6. X 500.

AZO DYE METHODS I N E NZ YM E HISTOCHEMISTRY

343

cell. This is illustrated in Figure 5. It is considered that the improvement produced by these two substrates warrants their extended trial in place of a-naphthyl phosphate in a coupling azo dye method for acid phosphatase such as that described by Grogg and Pearse (1952a). According to Fierz-Ilavid and 13langey (19493 coupling does not take place with a-naphthol derivatives which are substituted in the 2 and 4 positions. I n vi t ro, in aqueous solutions of buffer, coupling did not take place with either of the 2,4-substituted naphthols, though it did so under histochemical conditions and also in the presence of alcohol (50%) or pyridine (0.5%). Because of the slow rate of coupling and because of the great increase of the rate observed in the presence of pyridine, it may be surmised that the diazonium salt displaces one of the substituent bromine groups to form the azo dye final product. A c i d . The original c. Esters of the A ' d i d e s of 2-l~ydron-y-3-na~lithoic ester used by Gomori (1952a), Naphthol AS acetate, and three derivatives of this compound, were used as substrates for esterases in cold formalinfixed, or freeze-dried acetone-fixed, free-floating and mounted sections. With this type of material the intracellular localization of non-specific esterase was very good with the acetate of the plain anilide (Naphthol AS), fairly good with the acetates of the anisidide (AS-FR) and the dimethoxyanilide (AS-BG), and best with the acetate of the o-toluidide (A S - O T ) . The equation on p. 314 illustrates the reaction that takes place when this last substrate is used. The addition of one ( n S - F R j or two (AS-BG) methoxy groups to the original molecule might have been expected, on simple theoretical grounds, to increase the rate of coupling but, in practice, the results were not so satisfactory. Table I1 shows that the coupling rate of AS-FR is indeed faster than that of Naphthol AS, but for AS-BG it is considerably slower. Substitution by a methyl group (AS-OT), having the theoretical and actual effect of slowing the coupling rate, in practice produced superior results. An attempt was made to determine the effect on the solubility of the PKP of the introduction of a carboxyl group as in 2-acetoxy-6-bromonaphthalene-3-carboxylic acid. Although 6-bromo-2-naphthyl acetate is hydrolyzed by rat liver at one-quarter the rate of 2-naphthyl acetate (Ravin et al., 1953), no hydrolysis of the 3-carboxylic acid substituent could be obtained under histochemical conditions. Experiments with monoand di-halogen-substituted naphthoic anilides (e.g. 6-bromo-2-acetoxy-3naphthoic anilide) are obviously necessary. Details of the esterase method which, as a result of these experiments, I regard as the best at present available when fine localisation is required, are given below.

344

A. G. EVERSON PEARSE

-

6 El'

Q

5. A Modified Naphthol A S .Ilcthod

~ O YEstemses

1. Fix thin slices ( 2 to 3 mm.) of tissues in cold (0 to 4" C ) , neutrai but not buffered, 15% formalin for 10 to 16 hours. 2. Cut frozen sections at 7 to 15 p, into 1% neutral formalin. 3. Incubate the free-floating sections for 30 seconds to 15 niiiiutes at 17 to 20" in the substrate 4. Wash briefly in distilled water. 5. Counterstain the nuclei, if required, in half-strength Ehrlich's hematoxylin ( 1 minute, nuclei blue) or in aqueous methyl green, 10 drops of a 1% solution in 5 ml. distilled water (1 to 16 hours, nuclei green).

* T o 10 ml. of 1% solution of propylene glycol (Goniori, 195213) iii 0.2 M phosphate buffer a t pH 6.8, add 0.1 ml. of a 1% solution in acetone of Naphthol AS acetate (2-acetoxy-3-naphthoic anilide, M.P. 160-161") or, better still, Naphthol AS-OT acetate (2-acetoxy-3-naphthoic-o-toluidide,M.P. 93-96"), Add 10 to 20 mg. of the stable diazotate of o-dianisidine (Fast Blue B salt) or of 4-amino-2,5-dimethoxy-4nitroazobenzene (called Purple salt). Shake well and filter through a Whatman No. 40 paper. Use as soon as possible, preferably at once.

AZO DYE METHODS I N ENZYME HISTOCHEMISTRY

345

6. hlount in glycerine jelIy. (For the preservation of methyl green stained nuclei add one drop of 0.5% methyl green to 10 drops of the warm jelly.) The final azo dye is either a deep bluish purple (AS acetate, Fast Blue U ) , blackish purple (AS acetate, Purple salt), deep reddish brown (AS-OT acetate, Fast Blue H ) , or deep purple (AS-OT acetate, Purple salt). The results of the applicatioii of the above method, using the recommended substrates, are illustrated in Figures 7 and 9, and 10 to 14. I n the primary tubules of rat kidney it will be observed that the localization of esterase in certain round bodies, first demonstrated by Holt and Withers (1952) and illustrated in Figure 5, is almost exactly reproduced. This is best shown in Figure 9 (AS-OT, Fast Blue B ) and less well in Figure 10 (AS, Purple salt). The nature of the bodies thus demonstrated is uncertain. It appears that they were first observed by Sjostrand (1945-46) in freeze-dried sections examined by ultraviolet light. They had not apparently been demonstrated by any other technique until they were shown by Holt and Withers (1952) to be the site of non-specific esterase. I t has been observed by Holt (1953) that no esterase can be shown in the round bodies in freeze-dried sections which have been subjected to the influence of lipid solvents, suggesting that they are of lipid or lipoprotein nature. Histochemical examination by a variety of procedures ( Pearse, 1953b) failed to disclose any trace of lipid in the form of a membrane and suggested that the bodies consist of a condensed, or polymerized, basic protein possessing strong reducing groups in its molecule. Figure 4 shows the bodies in a freeze-dried parafin-embedded section of rat kidney, dewaxed in light petroleum and fixed for one minute in absolute alcohol. It should be compared with Figure 2 showing the application of the standard a-naphthyl acetate method, and Figure 3, showing the use of * G - B G acetate as substrate, in each case on similar material. In both the last two figurcs the FRP is concentrated, to some extent, in the subnuclear region. I t is unlikely that the staining of the round bodies in kidney is due to diffusion of the YRP and its non-specific absorption since they cannot be demonstrated by treatment with Naphthol AS or AS-OT in 50% alcohol, followed by a diazonium salt as in the post-coupling methods. The fact, moreover, that they are revealed by the very different indoxyl method of Holt and Withers argues strongly against non-specific absorption of the reaction product. I n sections of rat liver the observed distribution of esterase (Figs. 6 and 7, 12 to 14) is unlike that produced by the older azo dye methods, and it most closely resembles the picture produced by the indoxyl method

346

A. G. EVERSON PEARSE

FIG.8. Rat kidney, cold formalin-fixed 10 p frozen section. Distribution of esterase in convoluted tubules using 5-bronioacetylindoxyl. X 650. FIG.9. Rat kidney, as Figure 8. Distribution of esterase using Naphthol AS-OT acetate/Fast Blue B salt. (Incubation 30 miuutes.) X 550.

FIG.10. Rat kidney as Figures 8 and 9. Distribution of esterase using Naphthol 4 5 acetateipurple salt. (Incubation 2 hours.) Note that in spite of long incubation the enzyme is still localized in perinuclear spherical bodies. X 550. FIG.11. Rat kidney exactly as Figure 10. In the collecting tubules the predominant localization of esterase is in spherical bodies lying on the side of the nucleus away from the lumen. X 550.

A20 DYE METHODS IN ENZYME HISTOCHEMISTRY

347

(Fig. 6 ) ,especially when Naphthol AS acetate is used as substrate (Fig. 7). 1Vhen AS-OT acetate is used esterase is seen to be concentrated in round or rectangular bodies which are sometimes located in the subnuclear or perinuclear “Golgi region” of the cell but more often as a lining to the cell along its borders adjacent to the bile canaliculi (Figs. 12, 13, and 14). This picture is predominantly periportal in the rat liver. In many other types of cell in which non-specific esterase has hitherto been demonstrated to occur diffusely throughout the cytoplasm, the AS-OT method reveals the enzyme in small round bodies usually, but not always, perinuclear in location and with a definite bias in favor of the secretory pole of the cell.

6. Efects of Increasing Complexity of the Substmte The effect on the apparent localization of enzyme produced by Gomori’s (1952a, b ) Naphthol AS modification has been referred to already. In cold acetone-fixed paraffin sections it was sufficiently great to cause the author to postulate the existance of separate enzymes (a-esterase and AS-esterase) in such sections. If the two methods are compared by using cold formalin-fixed free-floating frozen sections the differences of localization, organ for organ, are far less marked. I n many cases they disappear altogether. With muscular and neuromuscular structures, however, differences remain. In particular, the motor end plates of rat striated muscle, which stain rapidly and diffusely by the naphthol acetate methods (a- and p-naphthyl acetate, 6-bromo-2-naphthyl acetate) cannot be demonstrated with the acetates of naphthoic anilides as substrates. Only an occasional faint deposition of dye is observed in the region of the sole plate. This lack of hydrolysis of the more complex substrates might be considered to be due to enzyme specificity, but the specific acetylcholinesterase (AChE) of the nerve cells in rat brain was observed to hydrolyze all four AS acetates rapidly. I n appears more likely that the latter are unable to penetrate some kind of semipermeable membrane in the motor end plates which is permeable to the naphthyl acetates but not to more complex compounds. Such a hypothesis is supported by observations on unfixed freeze-dried material, where there is often interference with the hydrolysis of AS acetates by tissues that attack these substrates rapidly when prepared by the cold formalin or cold acetone methods in frozen or paraffin sections. From the theoretical point of view, interference with the ability of the substrate to penetrate various membranes is not the only important result of increasing the complexity of the substrate molecule. If coupling is insufficiently rapid (as will usually be the case) the PRP may be attracted to tissue components other than those that have affinity for unsubstituted naphthols. A different localization of the final azo dye product will thus

348

A. G. EVERSON PEARSE

FIG.12. Rat liver, cold formalin-fixed 10 p frozen section. Distribution of esterase using Naphthol AS-OT acetate/Fast Blue B salt. (Incubation 45 minutes.) The cnzyme is either in perinuclear spheres or in irregular bodies lining the bile canaliculi. Methyl green, X 470.

FIG.13. As Figure 12, using Naphthol AS-OT acetate/Purplc salt. The relationship of the esterase-containing material to the bile canaliculi is wcll shown. X 550.

FIG.14. A high-power view of the same section as Figure 13. Shows the shape and character of the esterase-coiitaining material. X 1150.

AZO DYE METHODS I N ENZYME HISTOCHEMISTRY

349

be produced. Diffusion of the order of 10 p and over is usually quite obvious under the niicroscope, and with most of the simultaneous coupling methods it is produced easily by prolonged incubation. It should be noted that with the O-acetyl-5-bronioindoxyl method of Holt and Withers (1952), prolonging the incubation period up to 1000 times more than that necessary to produce a satisfactory reaction has no effect on localization of the indigo deposit in the tissues. This may be due to blocking of the enzyme receptor groups by the indigo FRP. JVith the AS acetate methods diffusion is always much less than with the riaphthyl acetates but, even with the best diazotates that have so far been used, prolonging the incubation period produces some apparent widening of the loci that are stained, probably attributable to diffusion of PRP.

7 . Conclusions From the experimental work recorded above it is concluded that the factor of greatest importance in providing clear and accurate localization of enzyme, in the simultaneous coupling azo dye methods, is not the rate of coupling but the solubility (and thus the diffusion constant) of the PRP. If the PRP is sufficiently insoluble, the rate of coupling becomes relatively unimportant, and it is then more important to choose a diazonium salt giving a highly colored non-particulate FRP, preferably relatively insoluble in lipids as well as in aqueous solutions. Tf, on the other hand, the PRP is relatively soluble (e.g., U - or P-naphthol), then the rate of coupling remains a critical factor. At present the greatest drawback to the production of new azo dye methods is the fact that trial and error remains the only method by which the histochemical result of such inetliods can be determined. It is possible to predict only very approximately the effect of any given alteration of substrate, diazoniuni salt, buffer or solvent, on the final histochemical result. This is in marked contrast to the state of affairs existing with the indoxyl substrates where a given effect may be postulated on chemical grounds and observed to occur when the substrate is used on tissue sections.

v.

DUE TO T H E NATUREO F TIZE DIAZOTATE 1. Nowspecific Coirpling iit the Tissues

EFFECTS

Diazoniuin salts will couple, at least at alkaline pH levels, with reactive groups present in tissue sections. Among these are the phenol group of tyrosine, the indole group of tryptophan and the itnidazole group of histidine. 'IYhen mono-diazonium salts are used, the resulting tissue compounds, which are usually pale yellow in color, will not subsequently react with phenols or arylamines. If bis-diazonium salts are used, coupling

350

A. G. EVERSON PEARSE

with the reactive tissue groups may take place at one end of the molecule only, leaving the other end free to couple with a suitable phenol or arylamine. From these observations one would expect that the employment of mono-diazonium salts in simultaneous coupling reactions at any p H level would not result in the production of false localizations. Bisdiazonium salts, on the other hand, after reaction with the tissues, might be capable of leaving free diazo groups to be attached to the phenolic PIIP. Tests with the mono-diazotates of 5-chloro-o-toluidine and 4-chloro-oanisidine have shown that no non-specific azo dye production occurs after 1 hour's incubation of tissues at p H 5.0, 7.4, or 9.2, and subsequent treatment with saturated aqueous a-naphthol at p H 7.4. With the bisdiazotate of o-dianisidine (Fast Blue B salt), however, at pH 7.4 and 9.2 a red azo dye is formed in the tissues after treatment with a-naphthol. No dye is produced at p H 5.0. These results indicate the need for caution when using bis-diazotates at alkaline pH levels, although with Fast Blue B salt the F R P of enzymatic hydrolysis is usually a blackish or bluish purple dye, easily distinguishable from the red false reaction product. 2. Stainkg of the Tissues by the Diazotate or Its Breakdown €'roducts

Some of the more highly colored diazotates are in themselves dyestufis and they stain the tissues deeply from the moment of first contact. The majority do not do this, however. Much more common is the non-specific staining of tissue components by the colored breakdown products of the diazotates. These are produced far more rapidly at alkaline than at acid pH levels. From cold acetone-fixed paraffin sections the (brown) staining due to breakdown products can be removed almost entirely, as shown by Gomori (1951), with dilute HCl or with acid alcohol. It is usually resistant to removal from frozen sections, however. If the FRP in a given method differs sharply in color from the breakdown product of the diazotate employed, only esthetic objections need be raised. If, on the other hand, as is so often the case, the two colors are distinguishable with difficulty, then it is obviously impossible to use the method for localization of small amounts of enzyme. If the absorption spectra of the two are similar, even spectrophotonietry will be impossible.

3. Inhibition of Enzymes Diazonium salts in aqueous solutions are strong bases, and they inhibit enzymes in their own right. No quantitative or even semiquantitative data are available, however, €or the inhibition of enzymes by diazonium bases, and only roughly quantitative data (Pearse, 1953) have been given for a

AZO DYE METHODS I N ENZYME HISTOCHEMISTRY

35 1

variety of stable diazotates. These have shown wide differences in the degree of inhibition, and a close dependence on the p H of the medium, as a property of the individual bases. Perhaps equally important when stable diazotates are used is the inhibitory effect of the various stabilizing agents used in their preparation. The diazotates most commonly employed in the dye industry and in histochemistry are zinc-diazo double salts, sulfonic acid derivatives, naphthalene sulfonates or borofluorides. These complexes are often further stabilized by the addition of a relatively large proportion of aluminum or magnesium salts. Removal of aluminum salts (as their purpurin lakes) has been shown to have an appreciable effect on the speed of enzymatic breakdown of the naphthyl esters (Pearse, 1953). Attempts to remove the zinc completely by means of chelating agents (such as dithizone) were uniformly unsuccessful. I n view of the natural reluctance of the manufacturers to allow publication of details of stabilization methods for particular salts precise details cannot be given. It can be stated, however, that the presence of zinc is important in the development of phosphatase methods at acid and alkaline p H levels (since it is an inhibitor at the concentrations used) and that aluminum and magnesium salts are also inhibitory when present in high concentrations. An additional effect of the common diazotates, which has been overlooked or ignored, is a lowering of the p H of the incubating medium. Under certain conditions this effect may itself be responsible for enzyme inhibitions. A further side effect attributable to the diazotates, caused by high concentrations of aluminum and other salts, is an alteration in the permeability of cell membranes. This means that in the presence of certain stable diazotates the substrate is unable to penetrate such membranes (the red cell envelope, for instance), and the recorded localization of enzyme is thus inaccurate. 4. Conclusioias It is concluded that the majority of effects due to diazotates are inseparable from the simultaneous coupling methods but that, provided one is aware of their existence, and provided that in every given case a variety of conditions is employed to determine the optimum, there need be no lack of confidence in the apparent localization of enzymes demonstrated by such methods.

5. Criticism of Particular Simultaneous-Coupling Methods It is necessary to comment only briefly on the individual methods recorded in Table I. Of those for alkaline phosphatase only the methods of Gomori (1951), using paraffin sections, or methods resembIing that of

352

A. G . EVERSON PEARSE

Grogg and Pearse (19524, using frozen sections, may be expected to be in current use. The latter method, especially, suffers from failure to conform with criterion 12 in that the F R P is particulate. With the use of halogen-substituted naphthyl phosphates in place of a-naphthyl phosphate it may be possible to use a more slowly coupling diazotate which will provide a non-particulate F R P at p H 9.0. Allowing these strictures, the coupling azo dye methods for alkaline phosphatase are sometimes capable of results greatly superior to those produced by the older techniques. Figure 14 illustrates the application of the method to growing bone (guinea-pig, femoral epiphysis) where the zoning of the enzyme is particularly striking. The acid phosphatase method of Grogg and Pearse (1952b) suffers very severely from failure in respect to criteria 7 and 10; at pH 5.0 diffusion from the majority of sites is considerable (unless a barrier relatively impermeable to the PKP is present) and the FRP is lipid-soluble. As would be expected from inspection of Table 11, where the rate of coupling between a-naphthol and Fast Blue B salt is shown to rise drainatically between pH 5.0 and 6.2, raising the p H of the incubation medium from pH 5.0 to pH 6.0 does diminish the amount of observed diffusion. Considerable improvement can also be recorded by the use of 2,4 dibronio( o r dichloro-)-l-naphthyl phosphate as substrate, but the ultimate object of conformity with all the criteria listed on p. 330 has not yet been reached in this instance. Figure 16 shows that in certain sites, such as human fetal femoral epiphysis, localization of acid phosphatase by the method of G r o g and Pearse is not unsatisfactory. In this tissue no improvement is recorded by using the halogen-substituted naphthol phosphates as substrates, and in others (Fig. 5) the localization of enzyme differs greatly from that shown by the older methods. In this case it will be observed that acid phosphatase is apparently concentrated, in a proportion of the primary convoluted tubules, in the Golgi region of the cell. In other tubules it is more diffusely situated in the cytoplasm, a localization which agrees with that shown by the older azo dye techniques. These older methods for esterases (Nachlas and Seligman, 1949 ; Goiori, 1950) fail to satisfy criteria 7, 8, 9, and 10 and they are useful only as broad histochemical rather than cytochemical indicators for esterase. Because of the diffuse nature of the staining which results from their use, in certain cases where even staining of a particular cell is produced, there may be advantages for spectrophotometry. The newer methods for esterases (Gomori, 1952a, b ; and the method described in this article, p. 344) are chiefly objectionable for failure to conform with criterion 3. This lack of penetration causes a negative result to occur in sites known to contain an

A20 DYE METHODS I N ENZYME HISTOCHEMISTRY

353

esterase capable of hydrolyzing the substrate. It is a heavy price to pay for satisfactory perfornlance in respect to criteria 7, 8, 9, and 10, and no way around the difficulty is at present in sight. Successful simultaneous coupling azo dye methods are pre-eminently niethods of coinpromise.

FIG.15. Guinea-pig, newborn, lower femoral epiphysis. Cold formalin-fixed 25 p frozen section. Alkaline phosphatase using sodium a-naphthyl phosphate diazotized 5-chloro-o-toluidine. (Incubation 30 minutes.) Upper left, the epiphysis ; lower right, the metaphysis ; between zones of ossifying cartilage showing different enzyme activity. Methyl green, X 145.

Azo DYEMETHODS VI. THEPOST-COUPLING There are four current post-coupling methods used in enzyme histochemistry (see Table I ) , three for glycosidases (P-glucuronidase, P-galactosidase and p-glucosidase) and one for an esterase (aryl sulfatase). The pH optimum for each of these enzymes is on the acid side of neutrality, and the methods which have been developed for their demonstration depend on the hydrolysis of suitable naphthyl substrates, at the individual pH optimum for the enzyme, in a suitable buffer, and in the presence of activators if necessary, but in the absence of diazonium salts. All objections to the use of a diazonium salt in the incubating medium are thus removed, and the enzyme is enabled to act on the substrate without inhibition.

354

A. G. EVERSON PEARSE

The substrates in common use for the simultaneous coupling methods cannot be used in post-coupling routines since their P R P are quite unsuitable in respect to criteria 18 and 19 (solubility in water, non-specific binding). The unsubstituted naphthols ‘‘stain’’ a wide variety of tissue components and this staining is easily revealed by the subsequent addition of a diazonium salt. No histologic use has yet been found for such a method of staining. The substrates evolved by Seligman and his associates for the four enzymes listed in Table I were designed to yield insoluble P R P which would be deposited at the precise loci of enzyme activity. Unfortunately, 6-bromo-2-naphthol, the P R P of the three glycosidase methods, fails completely to satisfy criteria 18 and 19. It is still sufficiently soluble in water to diffuse from its site of production, but it appears to have a strong affinity for protein, and it is readily soluble in lipids. I attribute to these latter properties the fact that if incubation is carried out in the presence of 1.0 M NaCl, 6-bromo-2-naphthol cannot be found free in the incubating medium. Similarly, 6-benzoyl-2-naphtho1,the P R P of Rutenburg E t d ’ s (1952) method for aryl sulfatase, has a high affinity for proteins and is lipid-soluble. In these circumstances it is not surprising that organs

FIG.16. Human fetus, 23 weeks gestation, lower femoral epiphysis. Cold formalinfixcd 35 CL frozen section. Acid phosphatase using sodium a-naphthyl phosphate/Fast Blue B salt a t p H 5.0. (Incubation 3 minutes.) Strong deposits in the periosteum, above, and in the ossifying cartilage, below (right), on the side of the shaft. Methyl green, X 130.

FIG.17. Rat adrenal gland, cold formaliii-fixed 10 p frozen section. Shows disposition of aryl sulfatase using potassium 6-benzoyl-2-naphthyl sulfate and post. coupling with Fast Blue B salt at pH 7.4. Hemalum, X 520.

AZO DYE METHODS IN ENZYME HISTOCHEMISTRY

355

having high aryl sulfatase activity are diffusely stained and that fat globules are colored electively. Figure 16 shows a section of rat adrenal incubated for 3 hours with potassium 6-benzoyl-2-naphthyl sulfate at p H 6.1, and subsequently coupled at p H 7.4 with Fast Blue B salt. The cells of the cortex are uniformly stained red, except where demonstrable fat globules occur. These are more intensely stained. I t is possible to use the sulfatase substrate in a simultaneous coupling procedure and if this is done, in rat kidney for instance, the more intense activity is noted in those tubules which contain the strongest non-specific esterase. This is in contrast to the uniform picture obtained in the postcoupling method. The latter can, in fact, be considerably modified by postcoupling at pH 6.1 instead of at 7.4 as in the original method. This observation provides an additional reason for the use of caution in accepting the apparent localization of enzyme afforded by the original method. I have severely criticized the post-coupling azo dye methods in the past (Burton and Pearse, 1952; Pearse, 1953) and I can only reiterate here my belief that those methods so far described afford a relatively gross histologic localization of enzyme. No post-coupling method should be employed when there is any possibility of operating a simuItaneous coupling variant. VII. FINALCONCLUSIONS It will by now be apparent that I believe the simultaneous and postcoupling methods can alone be considered to have any future in applied histochemistry. Should satisfactory non-coupling methods be evolved this opinion might, of course, have to be altered. In the case of the acid and alkaline phosphatases, and of non-specific esterase, the available azo dye methods can be considered complementary to the substituted indoxyl methods. In the majority of cases they afford a strikingly similar localization of enzyme. I n the case of the cholinesterases of the motor end plate, however, the degree of fine localization afforded compares unfavorably not only with that of the indoxyl methods (Holt and Withers, 1952) but also with that of the acetyl thiocholine methods (Koelle, 1951 ; Couteaux and Taxi, 1952) and with that of the myristoyl choline method (Gomori, 1948; Denz, 1953). Although it is not true, as recently stated by Barnett (1952), that oxidation of indoxyl to indigo takes place only in an alkaline medium, there are at present some inherent deficiencies in the application of the substituted indoxyl methods to enzymes having pH optima on the acid side of neutrality. These deficiencies are due to the crystalline nature of the substituted indigos which are produced and to consequent interference

356

A. G. EVERSON PEARSE

with precise intracellular localization. The azo dye methods do not suffer from these deficiencies and effective coupling can be obtained at quite low p H levels on the acid side by a suitable choice of diazotate. The indoxyl methods suffer also from the fact, first emphasized by Holt and Withers (1952), that there is always a tendency for their indigo FRP to be further oxidized to colorless dehydroindigos. If, at a given site, this process is taking place at a rate equivalent to or greater than the rate of production of indigo no permanent deposition of dye will occur and the site will be recorded as negative. Provided that there is no barrier to the substrate and the diazonium salt, such a site will be recorded accurately and quantitatively by an azo dye method. A further possible example in which the FRP of the azo dye methods may prove to be more suitable than the highly colored aggregates of the indigo FRP is in the application of spectrophotometry. The qualities of the azo dyes produced in the simultaneous coupling methods are almost infinitely variable, and for spectrophotometry, which calls for much less color per unit area than does standard visual microscopy, suitable modifications of the azo dye methods may be more easily made than in the case of the indoxyl methods. TABLE IV DYE METHODS (Formalin-fixed, preferably free-floating sections) COUPLING AZO

pH and

Enzyme

Substrate

Colorant

buffer

Non-specific esterases

Naphthol A S or AS-OT acetates

Fast Blue B salt or Purple Salt'

Phosphate

a-naphthyl 6-bromo-2-naphthyl 2,4-dichloro- or 2,4-dibromoI-naphthyl phosphates

Fast Red TL saltb or Fast Blue B salt

9.2 Verond acetate

Acid phosphatases

2,4-dichloro- or 2,4-dibromoI-naphthyl phosphates

Fast Blue B salt or Purple salt

5.0 Acetate

Acid phosphatases

a-naphthyl phosphate

Fast Blue B salt

Alkaline phosphatases

A d sulfatases 8

b c

6.8

6.1 Acetate

6-ben~~ylS naphthyl sulfate

Tbe diazolale of 4-amino.2,5.dimethory-4'~nitroa~obelume. The diarolats of 5-chlom.o-tolddina The diazotstc of 4-nim-o-anidin0.

Fast Scarlet Rc

6.1 Acetate

AZO DYE METHODS I N ENZYME HISTOCHEMISTRY

357

In the above section only those instances in which the azo dye methods may be superior to the indoxyl methods have been mentioned. It is not necessary to enumerate, in this context, all those instances in which the indoxyl methods are superior to the azo dye methods. A list of simultaneous coupling methods recommended for present use appears in Table IV. It will probably have to t x modified extensively in the light of investigations now proceeding in various localities, but it seems safe to affirm that the rich vein which was struck by Menten, Junge, and Green in 1944 is at present by no means exhausted.

A c K N O w LEDGN ENTS I am indebted to I.C.I. Ltd., Blackley, blanchester, for a generous supply of stable diazbtates, and to Ciba Ltd., Basle (Dr. Kd. Meier), for the potassium-6-benzoyl-2naphthyl sulfate. For the newer phosphates and acetates used as substrates I have to thank Professor E. J. King and Dr. W. Klyne respectively. Figures 6 and 8 appear through the kindness of Dr. S. J. Holt. The process of freeze-drying was made possible by grants from the Central Research Fund of the University of London.

vrrr.

REFERENCES

Barnett, R. J. (1952) Anaf Record, 114, 577. Burton, J. F., and Pearse, A. G. E. (1952) Brit. J . Exptl. Pathol., 33, 1. Campbell, J. G. (1949) Brit. J . ExptE. Pathal., SO, 548. Cohen, R. B., Rutenburg, S. H., Tsou, K-C., Woodbury, M. A., and Seligman, A. hl. (1952a) J. Biol. Chem., 196, 607. Cohen, R. B., Tsou, K-C., Rutenburg, S. H., and Seligman, A. M. (1952b) J. Biol. Chem., 196, 239. Couteaux, R., and Taxi, J. (1952) Arch. onat. microscop. morphol. expt!., 41, 352. Danielli, J. F. (1946) J. Exbtl. Biol., 22, 110. Defendi, V., and Pearse, A. G. E. (1954) To be published. Dew, F. (1953) Brit. J. Exptl. Pathol., 54, 329. Fierz-David, H. E., and Blangey, L. (1949) Fundamental Processes of Dye Chemistry. Interscience Publishers, New York. Friedenwald, J. S., and Becker, B. (1948) 1. Celldar Contp. Physiol., 31, 303. Gomori, G. (1948) Proc. Soc. E r p t l . Bid. Med., 66, 354. Gomori, G. (1950) In Menstruation and Its Disorders. Charles C Thomas, Springfield, Ill. Gomori, G. (1951) J. Lab. Clin. &fed.. 37, 520. Gomori, G. (1952a) Intern. Rev. Cytol., 1, 323. Gomori, G. (195%) Microscopic Histochernistry. Chicago University Press, Chicago. Grogg, E., and Pearse, A. G. E. (1952a) Natuve, 170,578. Grogg, E.,and Pearse, '4. G. E. (1952b) J . Pathol. Bacterial., 64, 627. Hartridge, H., and Roughton, F. J. W. (1923) Proc. Roy. Soc. ( L o d o n ) A104, S76. Holt, S. J. (1952) Nature, 169, 271. Holt, S. J. (1953) Personal com~nunication. Holt, S. J., and Withers, R.F.D. (1952) N a t w e , 170, 1012. Koelle, G. B. (1951) J . Pharwiacul. E.rptl. Therap., 103, 153.

358

A. G. EVERSON PEARSE

Loveless, A., and Danielli, J. F. (1949) Quart. 1. Microscop. Sci., 88, 159. Manheimer, L. H.,and Seligman, A. M. (1949) J. 1Vutl. Cancer Inst., 9, 181. Menten, M. L.,Junge, J., and Green, M. H. (1944) J. BbE. Ckem., 163, 471. Nachlas, M. M.,and Seligman, A. M. (1949) I . Natl. Cancer Inst., 9,415. Pearse, A. G. E. (1953a) Histochemistry. J. & A. Churchill, London. Pearse, A. G. E. (1953b) 1. Patkol. Bucteriol., 66, 331. Ravin, H. A., Tsou, K-C., and Seligman, A. M. (1951) I . Biol. Chem., 191, 843. Ravin, H. A., Zacks, S. I., and Seligman, A. M. (1953) I. Pharmu.coZ. Exptl. Therap., 107, 37. Rutenburg, A. M., Cohen, R. B., and Seligman, A, M. (1952) Science, 116,539. Seligman, A. M., and Manheimer, L. H. (1949) J . NutZ. Cancer Inst., 9, 427. Seligman, A. M.,and Nachlas, M. M. (1950) J . Clin. Invest., 29, 31. Seligman, A. M.,Nachlas, M. M., Manheimer, L. H., Friedman, 0. M., and Wolf, G. (1949) Ann. Surg., S O , 333. Sjiistrand, F. (1945-46) Actu Anat., Suppl. 1. Wolf, G., Friedman, 0. M., Dickinson, S. J., and Seligrnan, A. M. (1950) I . Am. claem. SOC., 74 390.

Microscopic Studies in Living Mammals with Transparent Chamber Methods* ROY G . WILLIAMS Departmen.t of Anatomy, University of Pennsylwnia. Philadelphia. Pamsylmtiia I . Introduction ................................................ I1. Construction and Installation of Chamhers .......................... 111. Blood Vessels ..................................................... 1. Growth ........................................................ 2. Caliber Changes ................................................ 3 . Anastomoses ................................................... 4. Effects of Various Agents ...................................... I V Lymphatic Vessels and White Cells ................................ 1. Growth of Vessels and Development of Function ................ 2. White Cells ................................................... V Other Tissues of the Ear .......................................... 1 . Nerves ........................................................ 2 Connective Tissue .............................................. 3 . Fat ............................................................ 4. Epidermis ..................................................... 5. Cartilage and Bone ............................................. VI. Grafts ............................................................ 1 . Autogenous and the Technique of Making ........................ 2. Homologous .................................................... 3 Tumors ........................................................ 4. Parasites and Eggs ............................................. VII . Tuberculosis ....................................................... VIII. Conclusion ........................................................ I X . References ........................................................

.

.

.

.

Page

364 364 364

367 367 368 371 371 372 373 373 374 373 375 375 376 376 386 387 392 393 394 394

I. INTRODUCTION The possibility of creating in mammals a thin region suitable for prolonged study with transmitted light and high magnification was a concept of Dr . E . R . Clark. Naturally occurring thin regions such as the tails of frog larvae have been used for histologic study since the beginning of microscopy. For many years, Dr . Clark himself used the tadpole tail for his investigations of blood vascular and lymphatic growth . H e has stated (1931) that he conceived the transparent chamber method as a means for extending his observations to mammals. Such a method depends upon the capacity of blood vessels. connective tissue. and other cells to grow into and fill actual spaces in the body if the spaces are *The work of the author is being supported by a grant from the National Institutes of Health. United States Public Health Service.

359

360

ROY G. WILLIAMS

sufficiently small. Clark (1931) further stated, “In 1875, Ziegler made studies on the new vessels and tissues which had grown into a space between two coverslips inserted under the skin of mammals, and in 1!2002 Maximow gave a beautiful description of the new tissue present in celloidin chambers which were inserted and removed at different intervals and then fixed and stained. Although both of these investigators made studies on fixed material, their results showed conclusively that new tissue, including blood vessels, will invade thin artificial spaces.” Although Dr. Clark had speculated about the feasibility of the method as long ago as 1910, actual work did not begin until 1922 when the problem was suggested to one of his students, J. C. Sandison. Sandison (1924) made a preliminary report of his studies and in 1928b published a more detailed account of a chamber together with a general survey of the growth and behavior of living cells and tissues as they appeared in it. According to Clark (1931), Sandison then decided to carry out his original intention to complete his surgical training and did nothing further about perfecting the method. However, since then, Clark and others working in his laboratory and elsewhere have perfected and adapted the technique until it may now be applied to all the common laboratory mammals and, depending on the type of chamber used, histologic and histophysiologic studies can be made in the same animal for a period of years if necessary. The transparent chamber technique might in its broadest sense include all transparent devices for exteriorizing internal organs so that they may be studied microscopically for more than a few hours, e.g., intestine (Zintel, 1936) ; pancreas (Flory and Thal, 1947) ; ovary and tube (Estable, 1948), or for replacing part of the body surface with a window so that underlying parts may be visualized, e.g., the skull chamber (Wentsler, 1936) or the transparent calvarium of Shelden et al. (1944). But, in this review consideration will be given only to those methods concerned with establishing a thin area of living tissue suitable for microscopic work at the surface of mammals and the uses to which such preparations have been put.

11. CONSTRUCTION AND INSTALLATION OF CHAMBERS The design and construction of most chambers for use in rabbits’ ears stem from one or another of the four types described by Clark, KirbySmith, et d. (1930). Of these four types, two, the “preformed tissue” and “round table,” are still in general use without fundamental changes ; one, the “bay chamber,’’ is obsolete, and the other, the “combined chamber,” is useful only for the limited purpose of comparing the appearance and behavior of newly formed vessels with original ear vessels.

STUDIES WITH TRANSPARENT CHAMBER METHODS

361

The preformed tissue chamber is used chiefly for visualization of the original ear vessels with their nerve supply intact. The principle of this chamber is the retention of a layer of skin and subcutaneous tissue on one side and the substitution of a thin mica covering for the cartilage and skin of the other. The parts of the chamber and their relationship to the tissues of the ear are shown in Fig. 1B. The thinness of the retained tissue is maintained by pressure exerted from both sides. Details of construction are given by Clark, Kirby-Smith, et al. (1930). This chamber has been adapted to the mouse by Algire (1943a, 1946b, 1947). The round table chamber is a device for studying the growth and behavior of blood and lymphatic vessels, nerves, connective tissue, and other tissues and cells. It was originally described by Clark, Kirby-Smith, et al. (1930) and has been modified in various ways by Williams (1934a), Ebert, Florey, and Pullinger (1939), Ebert, Ahern, and Bloch (1948), Essex (1918), Ahern et d . (1949), and Robertson (1951, 1952). It has been adapted to a surgically made skin flap by Williams (1934b), to the dog’s ear by Moore (1936), and to a dorsal skin flap on the mouse by Algire (1943a) and Algire and Legallais (1949). J o s h (1952) has modified Algire’s procedure in the mouse. The parts of a round table chamber and their relationships to the tissue of an ear are shown diagrammatically and not to scale in Fig. 1C. In installing this chamber, the skin but not the subcutaneous tissue is removed from both sides of the ear over an area the size of the chamber. A hole is cut through the center of the denuded area to take the table of the chamber. Holes are punched through the ear for the three bolts that hold the base and cover together against projections from the table, the projections being the feature that insures an observation space of uniform thickness, 40 to 75 p , which growing tissues can invade. When the chamber is installed in an ear, access may be had in two ways to the tissue filling the observation space : (1) through a hole in the table, closed with a plug when not in use (Clark, Kirby-Smith, et al. 1930; Ebert, Florey, and Pullinger, 1939), or (2) incorporating in the chamber devices that permit removal and replacement of the cover (Williams, 1934a: Williams and Roberts, 1950). The useful life of preformed tissue and round table chambers as originally described or in any of their modifications is something less than one year although now and then a round table chamber will stay in place longer, but that cannot be counted on. Neither of the chambers described nor any of their modifications can be used satisfactorily without external protection. There is no firm contact union of chamber and tissue, and the mechanical interlocking of the

362

ROY G. WILLIAMS

A Outer skin and subcutaneous tissue Cartilage

Inner skin

8.

C. PI

Mica

Mica

Plastic

Tantalum

--_ ___Tantalum

gauze

Paper

Lucite

Other metals

FIG.I. Diagrams showing the parts of three commonly used chambers and theii relationships to each other and to the tissues of an ear. A, section through a portion of an ear. B, cross section of a “preformed tissue” chamber. C, cross section of a “round table” chamber. D, cross section through the long axis of a tantalum chamber. Diagrams are not to scale. The bolts are made of tantalum and the nuts of brass.

STUDIES WITH TRANSPARENT C H A M B E R METHODS

363

two is inadequate to prevent some movement of the device in the ear or wherever it is installed. By using plastic splints and removable shields so placed that they nowhere come into contact with the chamber, a satisfactory degree of chamber stability can be obtained (Clark and Clark, 193Zb). Unanesthetized rabbits are immobilized for study by placing them on their backs on an animal board equipped with a head stock (Clark, Sandison, and Hou, 1931). Essex (1948) immobilizes them in their natural sitting position, and Algire (1943a) places mice in a brass cartridge containing a slitlike opening through which the flap containing the chamber protrudes. To expose tissues in chambers to fluids of known composition or to withdraw fluid for analysis, two means have been devised. A glass chamber (Abell and Clark, 1932b) may be made so that it contains a well or “moat” accessible to the outside through silver tubes and internally in communication with the space into which tissues grow, or a mica cell with inlet and outlet holes can be used to replace the cover on a removabletop chamber (Williams and Roberts, 1950). Injection ducts and “lacunae” may be built into a round table chamber (Ebert, Sanders, and Florey, 1940). Of the chambers so far devised, only one consistently permits studies for longer than one year and provides free access to the contents (Williams and Roberts, 1950). It is made of tantalum and mica and, when properly installed between perichondrium and cartilage, cannot be extruded. It may be used for at least four years and probably for the animal’s remaining life. When equipped with an internal reflecting surface, it may be installed in the skin of any laboratory mammal as large or larger than a rat without the necessity for making a skin flap. Light-reflecting chambers must be studied with vertical illumination, the Leitz Ultrapak being most suitable. The principles of constructing and installing tantalum and mica chambers are entirely different from those previously described. The parts and their relationships to each other and to the tissue of an ear are illustrated diagrammatically in Fig. 1D. The body of the chamber is composed of a flat tantalum ring enclosed in a double layer of tantalum gauze which projects well beyond the ring. Four bolts carry devices that maintain the observation space and permit removal of the mica cover. I n installing this chamber, immediate stability is achieved by the killing compression of skin and cartilage under the upper tantalum ring. This holds the chamber firmly until connective tissue and vessels have time to grow through the metal gauze from side to side and effectively anchor it in place. External protection is at no time necessary.

364

ROY G. WILLIAMS

Because this chamber is placed in a naturally occurring cleavage plane, its installation in an ear is easily and quickly done, Through a short incision in the skin and cartilage on the inner ear surface, the perichondrium and overlying tissue are elevated away from the cartilage by means of a semisharp instrument designed for the purpose, over an area slightly larger than the chamber. Incisions at right angles to each other are then made through inside skin and cartilage and the partly assembled chamber laid between perichondrium and cartilage. Inside skin and cartilage cover the chamber. The remaining rings are installed and forced firmly against the included tissue by tightening the nuts on four bolts. Tissue over the observation space is trimmed away and the cover and retaining devices applied. The chamber then requires no further attention. Vessels appear in the observation area somewhat later than they do in round table chamber because of the greater distance they have to grow, and nerves do not grow into them, but events transpiring as vascularization develops are otherwise the same. Epidermis which occasionally ruins round table chambers cannot grow into the tantalum variety. The tissue that grows into tantalum chambers, or probably any chamber, is, after some weeks, laminated, consisting of three discrete layers, the outer ones composed of avascular connective tissue, 5 to 10 p thick, and between them the vascular layer, the total thickness being uniform in any single chamber. It is the inner avascular connective tissue layer that permits opening the chamber with no extravasation of blood or other obvious damage to the contents. Minimal thickness of tissue that can be achieved in the observation space is 18 p . Vessels will not grow into a thinner space. Optimal thickness for most purposes is from 40 to 75 p.

111. BLOODVESSELS 1. Growth The presence of a fluid-filled space in the body is not of itself an adequate stimulus to vascular growth (Clark, 1936aa). There must be present in the environment growth-promoting conditions which involve both physical and chemical properties. Some of the physical factors that stimulate or influence vascular growth are : space relationships in the region being vascularized, temperature conditions, consistency of the surrounding medium, rate of blood flow, and amount of interchange through the capillary wall. It is unlikely that there is in the body a chemical which under any circumstances is specifically responsible for vascular growth. Embryonic tissue extracts and inflammatory exudates contain substances that favor growth of lymphatics, nerves, and connective tissue as well as blood vessels and other tissues (Clark and Clark, 1936a, 1939). It is therefore reason-

STUDIES WITH TRANSPARENT CHAMBER METHODS

365

ably certain that there are chemical substances which can stimulate local growth in general and that these substances are potent components of embryonic extracts and inflammatory exudates, but just what they are is by no means clear at present. Capillaries form from endothelial sprouts arising from pre-existing capillaries. Solid, pointed endothelial projections extend out from the side of a circulating capillary or venule. In the base of these projections, a liimen develops which is continuous with that of the parent vessel. Endothelial nuclei migrate into the process by ameboid activity of their surrounding endoplasm and also increase in number by mitosis. No outside cells contribute to these endothelial extensions. The growing sprout eventually comes in contact with another sprout or capillary whereupon it forms a connection through which the lumen gradually extends. Capillaries appear in chambers after about seven days and advance at rates varying from 0.2 to 0.6 mm. per day (Clark, 1936a; Clark, Clark, and Abell, 1933). Newly formed capillaries are generally larger with walls softer and more fragile than older ones. These differences last for only a day or so, although they may return in older capillaries following chemical or mechanical stimuli not necessarily involving inflammation. In early stages of growth, the endothelial cells are arranged as a syncytium within which nuclei move around, sometimes passing one another in the wall or migrating across the lumen. There is evidence that the endothelium consists of an exoplasm and an endoplasm, the former as a continuous homogeneous layer and the latter containing the nuclei and surrounding cytoplasm (Clark and Clark, 1939). Capillaries beget capillaries in the initial vascularization of a region and are produced in great excess of those found in the more stable vasculature that develops as the stimulus to growth subsides. Within the indiscriminate capillary plexus resulting from the growth stimulus, arterioles and venules develop. Arteries conveying blood to the plexus influence the development of new arteries within it. Whether a capillary becomes an arteriole or not depends on the blood pressure and volume of flow and the times over which those factors are applied. Pressure regulates the thickness of wall, and volume of flow determines the size of lumen-these are parts of the histomechanical principles of Thoma (Clark, 1936b). The various features of capillary growth and circulatory changes previously mentioned are not limited to conditions in which a whole area is being newly vascularized. The normal vasculature of the adult mammal is, except for larger vessels, in a labile state and subject to change in form and endothelial consistency in response to minute chemical, mechanical,

366

ROY G. WILLIAMS

and thermal stimuli (Clark and Clark, 1932a; Clark, Hitschler, et al., 1931). Gradually, over long periods, vessels functioning as venules may become arterioles, or the reverse may occur. Both arterioles and venules may revert to capillaries, and, in response to mild inflammations, these vessels may produce a new plexus in which new arterioles and venules differentiate (Clark and Clark, 194Oa). Loss of capillaries occurs at the same time new ones are forming. In some instances the process is the reverse of sprouting, and in others it appears to result from dissohtion of endothelium. If a capillary does not have flow in it for a sufficiently long period, occasionally no longer than 24 hours, it will disappear. Loss of capillaries is greater in the neighborhood of arterioles than near venules (Clark and Clark, 1935, 1939). Capillary sprouting is, perhaps, not the only mechanism concerned in the vascularization of a region. Chalkley, Algire, and Morris (1946), in studying wound repair in mice, observed that as much as 49% of the vascular bed was restored before vascular sprouts appeared. This suggested three things to the authors, one of which was that there could have been an increase in length of formed vessels and an extension of them into the wound area without the vascular sprouting process playing a part. This was an interesting and astute explanation of a finding that could have been made in a living animal only by the use of an accurate method for determining the level of vascularity (such a method was devised by Chalkley, 1943). How the increase in length could have been produced was explained hypothetically. Although the authors did not mention it, the process may well have been similar to that observed in tissue culture by Lewis (1931), who with time-lapse motion pictures demonstrated conclusively that, in Vitro, a formed capillary plexus can become exteEded in the absence of hemodynamic factors and without endothelial sprouting. The mechanism by which this was achieved was not explainable from the motion picture film. In typical first vascularization of parts, embryologically, the first veins are at a distance from the arteries or, as in the brain, at the opposite side of the organ or alternating, as in the lung. In the vascularization of chambers, arteries and veins tend to alternate and venae comites develop only as a secondary formation. Some of the conditions leading to the formation of companion veins are thought to be: the development of a growth-promoting medium in which a secondary growth of capiIlaries takes place in the interstices of the original set; the existence next to the artery of an unimpeded growth space; the thick layer of muscle cells on the artery making it impervious to lateral growth of its own endothelium or invasion from outside endothelium; the splinting effect of the artery which lessens outside pressure on adjacent veins (Clark and Clark, 1943b).

STUDIES WITH TRANSPARENT CHAMBER METHODS

367

2. Caliber ChaPiges Adventitial (Rouget) cells on regenerating capillaries form from connective tissue cells. Sparsely distributed inert cells resembling fibroblasts assume a flattened, longitudinal position with their processes parallel with the vessel wall. In capillaries that regress, these cells remain behind. On vessels becoming venules, the number may be slightly increased. Vessels differentiating into arterioles tend to straighten, lose side branches, reduce their caliber, and increase the strength and thickness of endothelium. During these changes, the number of adventitial cells rapidly increases. At the same time their axes change from longitudinal to transverse, and they become converted to smooth muscle cells. None of the vessels having only longitudinally arranged adventitial cells show active contractility. Vessels having transversely arranged cells develop active contractility if they are reached by a regenerating vasomotor nerve (Clark and Clark, 1937a, 194Oa; Clark, Clark, and Williams, 1934; Sandison, 192&, d, 1929, 1931, 1932). Beecher (1936a) stated that capillary contractility is present and common in the rabbit and that it is produced by the Rouget cells and by swelling of endothelial nuclei, both of those elements being controlled by the sympathetic nervous system (Beecher, 1936b). Clark and Clark (1940a), who, after many years of study, could find no evidence for active contractility of capillaries in the mammal, state that “the real factors responsible for control of peripheral circulation in the mammal have been shown to be the smooth muscle cells on arteries, arterioles, arterio-venous anastomoses and a few of the larger veins mediated through the sympathetic nervous system.” Sanders et al. (1940) concluded that capillary contractility does occur in rabbits’ ear chambers and that it is under control of sympathetic nerves. They believe it is produced by endothelial swelling but could find no evidence that the Rouget cells assist in the contraction. While Clark and Clark (1933b, 194Oa, 1943a) nowhere deny that caliber changes occur in capillaries of mammals, they feel that such changes are so “slight, infrequent and passive” as to be completely outweighed in importance by the “positive, violent, spontaneous and rhythmic” contractility of vessels with muscle cells on them.

3. Anastomoses The governing factor for the formation and maintenance of arteries and arterial anastomoses at the periphery appears to be Thoma’s histomechanical principle that size of lumen and thickness of wall are determined by amount of blood flow and pressure respectively. Tn absence of flow for

368

ROY G. WILLIAMS

sufficiently long, an artery becomes obliterated, For maintenance of small arterial anastomoses, frequent reversal of flow is essential. This is produced by unsynchronized contractions of vessels or parts of vessels and by varying external pressures. Between vessels protected from external pressure or in which unsynchronized contractions do not occur, there are few anastomoses (Clark and Clark, 1946, 1947). Arteriovenous anastomoses occur normally in many regions of which the rabbit’s ear is one. These operate as mechanisms for partially shunting blood around the capillary bed. They increase rapidly in number in response to sudden and sustained increase in blood flow. They may be retained, or they may disappear completely when the flow returns to normal. These connections have well-developed cellular walls that contract vigorously, being able to close the lumen completely. The diameter of lumen in any single contracting arteriovenous anastomosis may vary from 0 to 60p Anastomoses may have their own nerve supply and contract independently of the arteries or in conjunction with them, or they may have no nerves and contract only on local stimulus (Clark, 1938; Clark and Clark, 1934a, b). 4. Effects of Varioiis Agents The transparent chamber method affords an excellent means for studying changes in the peripheral circulation and the effect of drugs and other agents on blood vessels. Hou (1932) studied the action of ephedrine and related substances, as did Levinson and Essex (1943a) and Vigran and Essex (1950). Wilson (1936) used Adrenalin, ephedrine, ergotoxine, histamine, nitroglycerin, and tyramine, and Solis and Essex (1951) studied the action of protarnine sulfate. Abell and Page (1941) investigated the effect of angiotonin in hypertensive rabbits and in 1942a, b reported that angiotonin and rennin elevated arterial pressure with little reduction in blood flow by constricting the arteries and augmenting the force of the heart beat. Tyramine and methylguanidine sulfate acted similarly. Angiotonin, unlike epinephrine or pitressin, acted on ear vessels in a manner suggesting that it may be capable of producing chronic hypertension. Seldon et al. (1942) studied the effect of Pentothal sodium, cyclopropane, nitrous oxide and oxygen, ethylene, and ether on minute peripheral vessels, All these anesthetics produced a sustained increase in systolic blood pressure except ethylene, with which the increase was slight. Pentothal sodium produced dilatation of both arteries and capillaries, and all others caused arteriole and capillary constriction except cyclopropane, with which the capillary bed only was dilated. They concluded that Pentothal sodium because of its dilating effect on both arteries and capillaries may be

STUDIES WITH TRANSPARENT C H A M B E R METHODS

369

responsible for the increased oozing of blood observed in operations with that anesthetic. Ergonovine maleate, a drug used to prevent postpartum hemorrhage and in the relief of migraine, caused no obvious injury to blood vessels with forty times the usual dose (Abell, 1946a). Massive doses of the drug, unlike ergot, ergotoxine, and ergotamine, did not cause gangrene, although small thrombi and endothelial stickiness occurred (Abell, 1946b). Cortisone (Ebert and Barclay, 1952) tended to protect the vasculature against the effects of inflammation, improving arteriolar tone, diminishing damage to endothelium, and decreasing diapedesis and exudation. I t was concluded that if the results of inflammation are useful, as they would be in combating a bacterial invader, then the use of cortisone is harmful. But in a useless inflammation, such as rheumatoid arthritis, cortisone would be beneficial. Abell and Clark (1932a, b, 1933, 1937a, b) and Abell (1934, 1935, 1937a, b, 1939, 1940) used a glass chamber so constructed that blood vessels and related tissues could be exposed to the action of various fluids and the fluid withdrawn for analysis. When zinc-free methylene blue was placed in the well of the chamber, it was rapidly toxic to vessels if the concentration was 0.5% or greater. The dye diffused into the tissue for only about 0.2 mm. By variations in staining intensity, it was determined that a gradient of oxygen tension was present at the periphery of the vascular tissue. Abell (1934) placed buffered phosphate solutions at p H 7.4 and 6.2 in the moats of chambers and found that the solutions injured the vessels and were precipitated at the injury sites. These solutions entered the capillaries in concentrations diminishing as the distance from the source increased. Precipitate from pH 6.2 solution was gradually absorbed, but that from pH 7.4 solution was much less soluble. Urea (Abell, 1937a, b) passed by diffusion from the chamber moat into blood vessels. Amount of diffusion was proportional to the concentration. The amount of decrease of urea concentration was directly proportional to the concentration and to the capillary area but inversely proportional to the volume of solution. Phenol red in 0.4% isotonic solution diffused into tissue a visible distance of 1 mm. I t was not concentrated and not toxic. With the circulation free, the indicator pointed to a pH of 7.2 for the intercellular substance. With the circulation stopped, the p H dropped to 6.8 within 10 to 15 minutes, indicating the accumulation of acid metabolites. Following restoration of the circulation, the pH returned to 7.2 within 1 or 2 minutes (Abell and Clark, 1937b).

370

ROY G. WILLIAMS

Carbon dioxide perfused through a chamber in the presence of phenol red caused the pH of the intercellular substance to drop from 7.2 to 6.6, without damage to the endothelium. If the endothelium was sticky to leukocytes before perfusion, the gas eliminated the stickiness. It had no effect on the caliber of denervated vessels. When the circulation was poor, the acid change was more rapid and produced thickening and vacuolation of endothelium in arteries. If the COz perfusion was then stopped, the vacuoles disappeared rapidly, but if continued, plasma hemorrhage occurred in all vessels with stoppage of flow in some. The changes were reversible if COZ perfusion was not too prolonged (Abell and Clark, 1937a). Abell (1939, 1940, 1 9 4 6 ~ )found all capillaries permeable to protein, mature ones being much less so than those newly formed. Metaphen, a mercurial antiseptic, in concentrations of 1 :2500 produced no serious damage to blood vessels after 12 hours of contact. Four days of continuous contact destroyed the vessels but only for a distance of 1.5 mm. from the source. In contrast, 70% alcohol produced extensive hemorrhage and other damage within 15 minutes (Abell, 1941). Burns (Abell and Page, 1943) produced vasoconstriction of all arteries and larger veins. This resulted in reduced blood flow to the tissues and inadequate return of blood to the heart. It was thought likely that the burned tissue produced a substance that entered the blood plasma and produced the vasoconstriction. When rabbits were sensitized to horse serum, injection of that antigen or its introduction into the moat of an ear chamber resulted in arteriolar contraction, injury to endothelium, emigration of leukocytes, and emboli formation in capillaries and venules. Repeated introduction of serum into the moat exaggerated the changes and resulted in areas of complete endothelial destruction. However, reparative processes began immediately, eveti in the continued presence of serum (Abeil and Schenck, 1938). Ebert and Wissler (1950, 1951a, b) observed similar changes. They determined that cortisone tended to protect against the damage. Levinson and Essex (1943b) reported that shock resulting from intestinal manipulation produced vasoconstriction which was altered in denervated parts. Algire ( 1946a, c ) produced tourniquet shock in mice and compared the result with that following injection of bacteria polysaccharide. The results were similar in that both produced decrease in capillary circulation hut were dissimilar to the extent that edema was produced by the bacterial derivative but not by tourniquet shock. These experiments did not support the concept that in tourniquet shock there is increase in systemic capillary permeability. Algire, Legallais, and Park (1947) also found that a bacterial polysaccharide caused progressive decrease in rate of blood flow

STUDIES WITH TRANSPARENT CHAMBER METHODS

371

and decrease in percentage of functional capillaries in striated muscle. Occlusion of vessels reached a maximum within 3 to 4 hours and recovery occurred within 18 hours. No hemorrhage or necrosis was observed in muscle, but as circulation decreased, contraction was retarded and finally ceased. Algire and Chalkley (1945) and Chalkley et al. (1946) studied vascular reactions to traumatic wounds and the effect of dietary protein on vascuiar repair of wounds in mice. The growth sequence in wound repair was similar in most ways to that already described as chambers became vascularized. Both low and high protein diets retarded vascular growth, but the latter did so only if fed for long periods before wounding. A high protein diet for only fifteen days before injury did not delay vascularization and might have been beneficial. When thioflavine S in 4% aqueous solution is injected into the blood stream during ultraviolet irradiation, the circulating blood becomes yellow fluorescent, large emboli develop, and arteries are constricted. The emboli are secondary to dye aggregation, but the arterial constriction is a photodynamic reaction associated with radiation between 3200 and 38QO A., the spectral range strongly absorbed by the dye. The reaction is blocked by reduction of tissue oxygen tension or by filtering out the spectral band (Algire and Schlegel, 1950). Silica granules implanted in chambers are taken up by macrophages where they remain for months. The silica-laden macrophages produce no visible effect on connective tissue, lymphatics, or blood capillaries, and there is no tendency toward the formation of silicotic nodules (Gark and Haagensen, 1939, 1940).

VESSELS AND WHITECELLS IV. LYMPHATIC Grvwth of Lymphatic P’essels and Development of Function 1. All new lymphatic endothelium comes from pre-existing lymphatic endothelium. The growth process is similar to that in blood capillaries. Endothelial sprouts are sent out into the bases of which a lumen gradually extends. The lymphatic system is normally closed during growth and subsequently (Clark, 1 9 3 6 ; Clark and Clark, 1931, 1932b, 1933a, C, 1937b, c ) . Ingrowth of new lymphatics is always later and more sporadic and the number of vessels less than is the case for blood vessels. Rate of growth may equal that of blood vessels, but anastomoses are less frequent and the vessels are less labile. “The adult mammalian lymphatic system, like the blood-vascular system, retains the same growth properties present in its

372

ROY G . WILLIAMS

embryonic development, after the stage of primary differentiation" (Clark and Clark, 1932b). The growth of lymphatic vessels is influenced by the consistency of the medium into which they grow. When growth of blood vessels precedes that of lymphatics, the latter tend to occupy the clear space surrounding veins. Since there is little pressure in lymphatics, they are easily compressed and portions of them may be cut off and isolated. Isolated lymphatics persist for months and retain their growth properties. They may eventually reunite with others or gradually disintegrate (Clark and Clark, 1933a, c, 1937b, c). Because of the close proximity of peripheral lymphatics to blood vessels and the fragility of the endothelial walls, it is common to have extravasation of blood into lymphatics. Holes in the walls of vessels heal quickly. Blood cells in lymphatic vessels may pass along and disappear within 24 hours, but if trapped in the system they can exist unchanged for weeks. Fluid passes into newly formed lymphatics but only slightly, and the flow of lymph is extremely slow or absent for long periods. The lymphatics play no significant part in the removal of extravasated erythrocytes or of other extraendothelial debris. During edema, fluid in lymphatics increases, but flow may be diminished. Regions without lymphatics show no disturbed physiology. In the absence of external pressures, lymph flow is extremely sluggish (Clark and Clark, 1937b, c). Henry (1933) found that the total area of lymphatics of the ear about equals that of the blood vessels. Lymph flow was variable but extremely small even with massage. Maximum flow was 0.0845 cu. mm. per square millimeter of lymphatic surface per hour. Following injuries, lymphatic vessels may develop holes in the endothelium that may persist for as long as thirteen days, if there is fluid outside, and if pressure and suction alternate (Clark and Clark, 1933a, c).

2. White Cells Monocytes migrate from the circulating blood into surrounding tissue and become macrophages or tissue histiocytes. They increase in size and are phagocytic. Their ability to change their position is limited; hence they tend to stay in the neighborhood where they first appear. Many of them become oriented along blood vessels with the long axes parallel to the vessels (Ebert and Florey, 1939). Clark and Clark (1948) made similar observations and also saw macrophages divide, although the circulating blood seemed to be the main source of macrophages for combating infection. A single mocrophage may ingest as many as twenty

STUDIES WITH TRANSPARENT CHAMBER METHODS

373

erythrocytes. After an active period of phagocytosis, the cells diminish in size and remain as tissue components for many months. In certain locations, macrophages may fuse and form multinucleate giant cells (Sandison, 193i ) Macrophages shed portions of their peripheral cytoplasm 4.5 hours after taking up antigenic azoprotein ( Evans blue linked to horse serum), and the process may continue for many days (Robertson, 1952). Lymphoid cells that ingest the antigen are not seen after the first day. The shed cytoplasm is ingested by neighboring macrophages, and it is possible that this may be a process by which the antigen is shared by macrophages in a region. The dye-protein complex is not taken up by endothelial cells. Small lymphocytes are very actively motile cells. Ebert, Sanders, and Florey (1940) obtained no evidence that these cells may be converted to cells of some other type. Clark and Clark (1936b) and Clark, Clark, and Rex ( 1936), under circumstances that were particularly favorable for studying the fate of polymorphonuclear leukocytes, determined that such cells may change to small clear cells with round nuclei that might easily be mistaken for lymphocytes. However, they stated that the cells were probably undergoing degeneration and not transformation to a different type. They suggested that the small round cell infiltration frequently seen in pathologic conditions may be made up of these altered polymorphonuclear leukocytes. The injection of various substances into the blood stream, e.g., hydatid cyst fluid, acacia, glycogen, and dextran (Essex and Grana, 1949), causes leukocytes to cohere and form large clumps that adhere to the enduthelium. This condition is temporary, rarely exceeding 90 minutes in duration. Since leukopenia frequently precedes leukocytosis after the injection of such substances as glycogen, this observation on the behavior of leukocytes in the peripheral circulation may provide a clue to the reason for the letikopenia. I

OF T HE EAR V. OTHERTISSUES 1. Nerves

There is a marked variation in the extent of new formation of nerves in chanibers, depending on mechanical conditions, position of cut nerves with respect to the observation space, and whether or not growth-promoting conditions exist (Clark and Clark, 1938). I t appears that nerves regenerate where the growing ends are in a favorable growth-promoting environment and not because there is a need for them. The growing ends of regenerating ear nerves cannot he seen in chambers because of tissue density. However, the growth cones of fibers growing from autografts of sympathetic ganglia

374

ROY G. WILLIAMS

can be seen and the process of fiber formation observed. Once a medullated fiber has formed, it can be seen as a pair of highly refractile lines. Medullation begins at a Schwann cell and progresses in both directions. Isolated stretches of medullation may start at several Schwann cells simultaneously. Nodes of Ranvier are usually absent at first but may develop later. Myelin degeneration occurs rapidly over the entire length of nerve beyond a point of injurv. The Schwann cells may persist after injury and remedullation occur, at times centripetally (Clark and Clark, 1938). When nonmyelinated nerves are present but not visible in a chamber, they may be visualized by vital staining with methylene blue (Clark, Clark and Williams, 1934). By staining at various time intervals in the same animal, it was demonstrated that development of contractility in an artery progressed along the vessel at the same rate that accompanying nerves grew in length, whereas other vessels without accompanying nerves did not develop active controlled Contractility. As a preliminary to nerve repair after injury, Essex and deliezende ( 1943) emphasized the importance of vascularization. Repair does not rest solely with the nerve cell body and proximal part of the axone and distal sheath but also in the vascularity of the regenerating area. Thus, in manipulating nerves, special effort must be expended in maintaining their blood supply. 2. Connective Tissue Fibroblasts invade the observation space in round table chambers about six days after operation. Their time of appearance in the field parallels that of the blood vessels. Fibroblasts are essential for the development of connective tissue fibers (Stearns, 194Oa, b), and the cells are intimately associated with the actual formation of fibers. Orientation of fibroblasts appears to influence the orientation of fibers. There is no evidence in chambers indicating that connective tissue fibers are continuations of preexisting fibers or formed by transformation of a fibrin net or from any type of cell other than the fibroblast. Connective tissue fibers develop extracellularly as a result of fibroblast activity. The rate, amount, and direction of fiber formation is influenced by tensions. The presence of epidermis alters the tempo and pattern of fiber formation (Stearns, 1939). Intercellular substance is of a gelatinous rather than a free-fluid nature (Clark and Clark, 1933c) and, when blood circulation is free, has a pH of 7.2 (Abell and Clark, 1939).

3. Fa# Fat is normally present in rabbits’ ears and may form in newly grown tissue. Its presence is not related to thickness of tissue or season of the

STUDIES W I T H TRANSPARENT C H A M B E R METHODS

375

year or to the age, sex, or state oi nutrition oi the animal. It frequently, but not invariably, appears first in cells close to blood vessels. There appears to be no relationship between fat and the presence of lymphatic vessels. Fat cells develop from cells indistinguishable from connective tissue cells. Minute refractile droplets develop in the cytoplasm, cell processes retract, the droplets increase in size, and the cell becomes rounded and larger. Loss of fat is the reverse-globules become smaller and break up into smaller and smaller droplets that finally disappear, leaving a granulated cytoplasm. Fat appears to enter cells in a soluble form and not by phagocytosis of visible fat droplets (Clark and Clark, 1940b).

4. Epidermis Epidermis sometimes grows into round table chambers from the surrounding normal skin. Clark and Clark (1944) have summarized the main points of its behavior and properties as follows : Migrating epidermal cells resemble fibroblasts in shape. The extension of a line of these cells is slightly more rapid than that of a growing line of fibroblasts and capillaries. During growth, small islands of epidermal cells may become completely surrounded by vessels. The cells become vacuolated and die, blood vessels then invade the islands, and the remains are disposed ot by macrophages. Fibrin dissolves in the neighborhood of growing epidermis, suggesting that the cells produce a fibrinolytic enzyme. Blood vessels do not invade living epidermis, and those near it are always wide, sinusoidal channels. Connective tissue that forms next to epidermis is coarse and arrayed in regular parallel rows in contrast with the finer irregularly arranged connective tissue fibers present elsewhere.

5. Cartilage and Bone Both cartilage and bone arise in chambers from time to time and for no certain reason. Cartilage arises from elongated motile cells resembling fibroblasts and containing a characteristically granulated cytoplasm. The cells finally lose their motility, enlarge, and become rounded. They lie in lacunae and contain commonly, in a mature cell, only one large fat droplet which more or less indents the nucleus. The cytoplasmic granules are uniform in size and in constant motion as is the cell itself, although it does not change position. The cell outline varies from minute to minute as short, blunt processes are sent out and withdrawn. Once the cells are fixed in position, a homogeneous ground substance appears between them (Clark and Clark, 1942). When the ground substance is well developed, the tissue then undergoes no change for many months unless bone develops in it. No mitoses were seen in chondroidal cells.

376

ROY G . WILLIAMS

Cartilage formation occurs in regions in which cessation of growth and retraction of capillaries is a conspicuous feature, although laying down oi the cartilagenous ground substance appears to precede the vascular change. Clark and Clark (1942) state, “it seems evident that some special localized chemical condition of the tissues must serve as stimulus for the metaplasia of connective tissue cells, or the differentiation of specific precartilage cells, into cartilage in the chamber areas, in view of the sporadic and restricted formation of cartilage.” Blood vessels appear to have only a secondary importance in the formation of new cartilage and bone. Within the chondral areas, dark, amorphous masses occasionally develop. These are composed of many minute granules thickly distributed in the hyaline intercellular substance. High magnification demonstrates the presence of typical bone lacunae and canaliculi in the granular areas. Cartilage itself may never be invaded by vessels, but if bone is laid down in it, capillaries then grow and invade the bone. However, vascularization of newly formed bone is not a very vigorous process. Bone formation and resorption occur simultaneously, but the process of resorption is obscure (Sandison, 1928a ; Kirby-Smith, 1933).

VI. GRAFTS 1. Autogemus and the Technique of Making There are three ways in which chamber methods have been used for the study of grafted tissue. (1) The grafts may be included in the chamber when it is installed. This is the method that was first used. It is now obsolete since better methods are available. ( 2 ) Tissue in the observation space of round table chambers may be approached from below through a small hole in the table, kept closed by a plug when not in use. This was first described by Clark, Kirby-Smith, et al. (1930) and improved by Ebert, Florey, and Pullinger (1939) and by Robertson (1951, 1952). ( 3 ) The chamber may be so built that the mica cover can be removed and replaced under fluid (Williams, 1934a; Williams and Roberts, 1950). This method provides easy access from above, requires no special apparatus, does not injure the exposed tissue, and is the method that has been most extensively used in the study of transplants. Methods 1 and 3 have been adapted to mice by Algire and Legallais (1949) and their adaptation has been used with changes by Conway, Joslin, and Stark (1951a) and by Joslin (1952). The method as used in mice permits studies of about two months’ duration. With tantalum chambers in rabbits, observations may be made in the same chamber for a period of years. In the following consideration of grafts, any statements or other data

STUDIES WITH TRANSPARENT CHAMBER METHODS

377

for which no literature references are given will be unpublished data from the reviewer's records. The technique of grafting is of primary importance in the study of transplants, for therein lies the greatest opportunity for introducing artifacts in the results. The literature is replete with contradictory statements concerning the survival, vascularization, and behavior of autografts. Any graft in any region not connected to the host by sewed anastomoses of fairly large blood vessels must be sufficiently small so that growing vessels may reach the center before cells die from lack of oxygen or, if blood vessels are slow in growing, so that the cells may be supplied by diffusion from host vessels. This requirement limits the size of grafts to fractions of a millimeter if they are not to have necrotic centers. For chamber work, there is an additional reason for small grafts, namely, the semirigid limited space in which they must lie. In rabbits, using the third method described above, optimal diameter of grafts after compression with the cover has been found to be from 50 to 300 p on host regions from 50 to 75 p thick. Thickness of grafts after compression with the cover is from 20 to 30 p. Grafts with those dimensions do not appreciably increase the thickness of tissue in chambers, since they rapidly become incorporated in it instead of existing as excrescences. Maximum diameter of grafts, except for thin tissue such as choroid plexus or omentum, is about 700 p. Compression by the cover of such small grafts does not occlude the underlying vessels because when the donor piece is removed from its circulating blood supply the cells undergo immediate softening. Microdissection of cells in vivo with the blood circulating and the nerves intact show the cells of thyroid, for example, to be much more viscous and turgid than they are when blood flow is stopped or than they are in excised cells surviving in Tyrode solution (Williams, 1944). For reproducible results, it is necessary to follow a standardized procedure in making giafts in chambers. A satisfactory sequence has been found to be as follows: (1) Exposure of the donor part, which is then covered to prevent drying. (2) Sterilization of the chamber cover and its removal under Tyrode solution. ( 3 ) Excision of 1 cu.mm. of tissue from the donor part and immediate immersion of the piece in Tyrode solution at 4"C. (4) Shaping of the grafts under a dissecting microscope, using instruments sharpened and pointed as for operating on embryos. ( 5 ) Pressure injury of the site at which the graft is to lie, using the point of a forceps. (6) Floating the graft on the point of a forceps and placing it, under a second microscope, on the injured site. (7) Replacement of the cover and retaining devices. (8) Closure of the wound through

378

ROY G. WILLIAMS

which the donor piece was obtained. Three or four grafts may be placed and covered by this means in less than 20 minutes from the time of excision of the donor piece. Injury of the host region at the graft site is not an important factor in vascularization of autotransplants. Stimulus to vascular growth comes from the graft itself and affects both the host vessels and endothelium carried over in the graft. The stimulus does not extend beyond the lateral graft margins and is uniform throughout the area of contact between host and graft. Table I shows a list of tissues that have been studied in grafts and TABLE 1 TISSUES STUDIED AS AUTOCRAFTS AND AS HOMCGRAITS Those marked S survived, those labeled N S did not survive, and those followed by P S survived but were altered in some way. Autografts Thyroid Adrenal Glomerulosa Fasciculata Medulla Ovary Follicles Interstitial cells Lymph node Bone Sympathetic ganglion Brain Brown fat Parathyroid Fat, abdominal Spleen Red bone marrow Epidermis Pancreas Omentum

-

~

N S

S

N S P S N S

N S N S

S

N S N S

S

S

PS N

S1

S S

N Sa N S N S

S

S N S

PS N S1 S'

N S N S

S

Leydig cells Tubules Pineal Choroid plexus Ciliary body V, carcinoma ~~

S

PS

Testis

~~

Homografts

PS

N S1 S2 S1 S

~

~

and 2 studied in one or two animals respectively. All other tissues studied in not less than 3. Total number of animals used was 110. 1

STUDIES WITH TRANSPARENT CHAMBER METHODS

379

whether or not each survived. Survival was arbitrarily defined as occurring in any tissue which, at the end of four months, gave no signs of regression such as diminution in external dimensions, abrupt decrease in number of countable cells, decrease in blood vessels, and no leukocyte infiltration. The period of four months was selected because experience indicated that if a tissue was not going to survive it would disappear well within that time. Minimum time in which any surviving graft was studied was eight months and the maximum fifteen months. When autografts are made in chambers in the standard manner described above, there is great constancy in behavior and vascular response of each tissue. Tissues survive every time, or they do not survive at all, or they survive but are modified in some way, always the same way. Certain generalizations about surviving autografts can be made. Endotheliuni of the host will begin to invade them, and their own endothelium begin to grow within 24 hours. Endothelium of the graft survives and is an important factor in revascularization. Hemodynamic factors are not important in the first growth of vessels but are in determining the final pattern of vascularity. By 48 hours, a fully formed and blood-filled capillary plexus will be present within the graft, but it will contain no flowing blood. By three days, circulation will be free in all parts of the plexus. By four days, the circulation will reach its final form for each tissue with arterioles and venules well developed. A stable vascular condition will be reached by the eighth day. Further generalizations about surviving autografts can be made. Because of space limitations, the data on which they are based may not here be stated. The graft determines the nature of the vasculature in it, not the host vessels on which it is placed. The final vasculature resembles that of the whole part from which the graft was derived. The vascularization of surviving autografts tends to be a repetition of the same process as that by which the whole part was vascularized embryologically. Growth potentialities of endothelium characteristic of the embryo are retained in the adult and are similar in a wide range of species. The internal structure and organization of a surviving autograft tends to duplicate that of the whole part from which it was taken; e.g., grafts taken at random from red pulp of spleen reconstitute themselves to complete splenic lobules with central Malpighian bodies, penicillus arteries, ampullae of Thoma, sinusoids, etc.; thyroid grafts become oriented with small follicles a t the center and larger ones at the periphery; and adrenal glomerulosa grafts, under appropriate stimulation, reproduce the typical zonation of the gland. Agents that affect the donor tissue also affect the graft and in the same way. In other words, in tissues where nerves are

380

ROY G. WILLIAMS

not functionally essential, if a graft survives without alteration of structure, it functions. Halstead‘s “law” which states that grafts of glands survive in proportion to the need for them does not apply for surviving autografts of glands given in Table I, since no deficiencies were created by the grafting operations. If a graft survives and is structurally unaltered with chamber methods, then it is available for histologic and histophysiologic experimentation. But if it does not survive, or if it is altered in some way, then it is up to the investigator to determine the extent to which the results were produced by technical difficulties or limitations of method. Among those autografts listed in Table I, the method was responsible for failure of survival of ovarian follicles and probably red bone marrow. I t was also responsible for the altered behavior of epidermis. But it was not responsible for results with adrenal medulla, brain, and pancreas, for those tissues did not survive, irrespective of the graft site. The changes in seniiniferous tubules resulted from excision of tissue for grafting and not from other limitations of the method. Routine magnifications used in the study of grafts are from 14 X to 600 x. For some cells in favorable locations, magnifications of lo00 x may be used, e.g., in zona glomerulosa of the adrenal cortex or testicular interstitial cells. But in other cells, e.g., parathyroid gland, where contrast between cells is poor in the living state, high magnification is generally unsatisfactory. In many cases, the same cells can be located day after day and the changes they undergo recorded by camera lucida tracings, photomicrographs, or cinephotomicrography. The tissues listed in Table I were not all studied in the same detail. Those not mentioned hereafter have been examined chiefly from the standpoint of survival only. Prolonged study of living grafts and microdissections in vivo have provided the information about thyroid that follows. Lateral limits of individual cells in follicles cannot be seen and nuclei are not often visible. The inner and outer cell boundaries are at most times very sharp in optical section, the inner more so than the outer. The colloid is clear, amorphous, and homogeneous and never presents the peripheral serrations so conspicuous in some fixed sections. I t has the same osmotic pressure as blood serum. Colloid pressure is about the same in all follicles irrespective of size and is less than capillary pressure. The viscosity of colloid varies in different follicles. Evans blue injected into small follicles disappears within 10 minutes, whereas in larger peripheral follicles it does not disappear in 12 hours, suggesting that the colloid in different follicles varies in other ways than in viscosity, probably in protein content. Methylene blue

STUDIES WITH TRANSPARENT CHAMBER METHODS

381

injected into colloid with no extravasation disappears within 20 minutes from all follicles, and extrafollicular cells become blue, indicating that diffusions from colloid can take place through the cell wall. The greatest visible activity of which a follicle is capable is a cyclic change that has been divided into four stages, the last and shortest stage being one of rapid collapse associated with great hut not complete loss of colloid (Williams, 1937). Just before collapse, some cells in the wall manifest pinocytosis. The coniplete four-stage cycle has been seen within 19 hours, but it may require many days, or follicles may never complete a cycle, activity being limited to oscillation between stages 2 and 3. Nothing leaves the follicles in a visible form. Normally, there are no holes or defects in the follicle wall. No evidence has been obtained from living follicles indicating that basal secretion occurs. When the gland is stimulated, the cells secrete toward the lumen in the apocrine manner. In unstimulated glands, secretion is very slow and is associated with the formation of droplets in the apical ends of cells and their extrusion into the colloid. When colloid volume changes occur, they are generally, but not always, accompanied by reverse volume changes in the cells. Colloid volume may increase without cordesponding decrease in cell volume. The evidence from living grafts is that the follicle is the functional unit of the gland, not individual cells. The cells secrete only into the lumen, where various changes in the secreted material can occur. The colloid cannot be looked upon as exclusively a stored and reserve secretion. Substances can diffuse out of the cqlloid without changes in colloid volume and with no visible changes in the cell wall (Williams, 1944). When colloid is reduced in volume, the reduction is probahly accomplished by diffusion through the cell wall. By the same token, it is conceivable that substances .could diffuse into the colloid without active participation of the cells. Iodine operates to reduce cell volume, increase colloid, and decrease its viscosity, but the response to iodine is not uniform in all follicles. Thyrotropic hormone increases cell volume and decreases colloid, but in some follicles it may cause increase in colloid without cell changes. in 50microcurie doses in rabbits produces no change in structure of grafts, It is concentrated in the colloid, and autographs of living grafts can be made with 12-day exposures. The autographs so made demonstrate nothing except that iodine is concentrated by grafts of thyroid. The film must of necessity be too far removed from the radioactive source to permit the useful kind csf autographs that can be made from sections. Fifty microcuries of I I 3 l , given 24 hours before grafting, completely

382

ROY G. WILLIAMS

eliminates the initial stimulus to vascular growth that is otherwise so prominent a feature with thyroid grafts. All follicular structure is quickly lost. However, many thyroid cells remain alive and new follicles form after about five weeks. Thereafter, their appearance and behavior is the same as in any untreated thyroid autografts. Thiouracil in doses of 100 mg. daily causes loss of colloid, increase in cell volume, and increase in vascularity. With prolonged use, small follicles are completely eliminated, the drug apparently acting chronically as a specific poison. The number of follicles per unit of volume is thereby reduced by as much as 35%. With thiouracil, large follicles at the periphery of grafts may lose most of their colloid and regain it within two weeks, and then repeat the loss and gain in the following two weeks, in each filled stage retaining their large size. These large follicles are generally thought to be less active than the smaller ones, but they are certainly not inactive. New follicles form in grafts, but the process is not active in untreated animals. No instance of new follicle formation by budding from prcexisting follicles have been seen in many hundreds of follicles. New follicles form from interfollicular cells, some of which are cells remaining after dissolution of follicles. Anything interfering with the integrity of the connective tissue capsule of a follicle results in its dissolution, but some of the cells remain alive and capable of forming new follicles if they gain the proper relationship to each other and to the surrounding connective tissue (Williams, 1937, 1939a, b, 1941, 1944). Autografts of adrenal cortex were first made in chambers by Hou (1929). These did not survive long, and no studies of the cells were made. With improved methods, grafts of zona glomerulosa and capsule survive indefinitely in the animal’s lifetime. The cells are clearly visible with high magnification. Their cytoplasm is filled with granules that appear and disappear according to a sequence that has been determined (Williams, 1945). Survival of zona glomerulosa is not regulated by body need for adrenal secretion. With the main glands in place, zona fasciculata generally does not form from zona glomerulosa grafts, although it may if grafts are old ones. When the main glands are removed, new zona fasciculata forms rapidly from glomerulosa. If two glomerulosa grafts are so located that one is separated from the only available artery by another graft, the former will be supplied with blood by a portal circulation. This has nothing to do with adrenal as such but with the location of grafts with respect to arteries. The first vessels in any graft are always capillaries. Within the capillary plexus, Some vessels ordinarily develop into arteries and others into venules, but arteries

STUDIES WITH TRANSPARENT CHAMBER METHODS

383

will not develop unless at some point or points an already formed artery sends blood into the plexus. If only veins carry blood to a plexus, no arteries develop and the circulation is portal. This finding would indicate, among other things, that it is possible to alter experimentally the vascular gradient in a tissue surviving as an autograft. The vascular gradient normally in adrenal cortex has been adduced to explain the differences in cells in the various zones. I n the case of the zona glomerulosa grafts mentioned, one graft occupied a position in a vascular gradient comparable to the zona fasciculata in whole glands. This altered position had no effect on the cells as far as could be determined. Zona fasciculata transplanted alone behaves quite differently from zona glomerulosa. The transplants have no power to stimulate growth of endothelium, neither that grafted nor that of the host region. The cells gradually become aligned along the host vessels, chiefly along veins, and slowly disappear over many months by a process which in some exes may be holocrine secretion. The cytoplasm of fasciculata cells is filled with minute granules that obscure the nucleus. They coalesce and become larger and highly refractile. These droplets then change in consistency, take a position in contact with the cell membrane, and rather rapidly decrease in size until they disappear, presumably by diffusion through the cell wall. The results from studies of grafts of adrenal cortex support the concept that cells arise at the periphery and are moved through the gIand toward the medulla where their existence ends. In grafts, the zona fasciculata has no regenerative capacity. The cells are largely post mitotics (Williams,

1945, 1947). Pieces of seminiferous tubule and islands of interstitial cells survive readily as autografts in chambers. Testicular interstitial cells arise from cells indistinguishable from connective tissue. They acquire cytoplasmic granules, reach a certain size, at which time they have a characteristic appearance, and then undergo regression. The process of granulation and d e g r ad a t i o n is not cyclic. They are fixed post mitotics with a life span of about nine months. This is the first instance in which information concerning the length of life of a highly differentiated cell has been obtained in a living animal, and it illustrates an important potentiality of the method, since such data can be obtained, at present, by no other means. Pieces of seminiferous tubule become closed vesicles. The spermatogenic cells rapidly disappear or never progress beyond the stage of secondary spermatocytes. They tend to separate from the wall and lie free in the lumen. Sertoli cells persist. They do not constitute a syncytium. They produce droplets that are discharged into the lumen and slowly disappear.

384

ROY G . WILLIAMS

Luteinizing hormone stimulates excessive droplet production by Sertoli cells, and the process is repeatable at fairly frequent intervals. Droplets appearing in the lumen arise as the result of apocrine secretion by Sertoli cells. The inferences from the foregoing are that the pituitary controls discharge of sperm cells into the tubules and that secretion pressure of Sertoli cells provides the means for moving material along the tubule (Williams, 195Oa). Leydig cells do not arise at random from the connective tissue of grafts. They arise only near tubules or other interstitial cells. A piece of tubule without interstitial cells near it becomes fibrotic with great increase in the collagenous fibers of the tunica propria and shrinkage of Sertoli cells. Interstitial cells are largest when located near tubules. These and other findings suggest that there is a reciprocal relationship between interstitial cells and tubules. With interstitial cells missing, the tubules resemble those seen in the aged. Whatever relationships there may be between the two types of cells are of a local nature and not produced through the blood stream, since the animals studied were young and had no testicular deficiency. The inferences from these findings are several, one of which is that testicular age changes may not be primary results of age as such but secondary to closure of tubule outlet or failure of interstitial cells or both ( Willianis, 195%). The vasculature in and around islands of interstitial cells resembles one form of simple hemangioma. Similar vascular arrangements were not found in whole testis by injection methods, Interstitial cell grafts produced a spreading factor and did so without themselves degenerating. These findings give a lead toward the experimental production of some hemangiomas, a condition about which knowledge has not progressed much since the time of Ribbert (Williams, 1949). The spleen in autografts duplicates the structure of the organ as described by Mollier (1910). With the spleen removed, grafts may increase threefold in size, but with the spleen in place, they do not increase. Malpighian body transplanted alone does not survive. Red pulp taken at random reconstitutes itself to a splenic lobule complete, except for nerves, smooth muscle, and sheathed arteries, with a new Malpighian body, penicillus arteries, ampullae of Thoma, and an intermediate sinusoidal zone demonstrating the structural characteristics as described by Mollier ( 1910). Various experiments concerning functions of spleen grafts substantiate many of the prevailing ideas obtained by other methods. Intravascular phagocytosis occurs and also intravascular fragmentation of protoplasmic masses which may be platelet formation. Information has been obtained

STUDIES W I T H TRANSPARENT CHAMBER METHODS

385

about spleen as a source of cellular additions to the blood stream (Williams, 195Ob). Autografts of omentum, in which the original omental vessels were clearly visible before and after transplanting, demonstrated conclusively that, in that tissue, the original vessels survive and constitute a major factor in revascularization. The original vessels are subject to realignment in response to hemodynamics in the same way that vessels respond in intact parts. Conway ef al. (1951a, b, c, 1952a, b) studied autografts and homografts of skin in mice. They found that, shortly after placing of grafts, a plasmatic circulation enabled them to survive during the first week. Blood vessels then developed by capillary budding, and the vascularization was complete by about fifteen days. While capillary budding (sprouting is a more descriptive term) as a process of vascularization of grafts undoubtedly occurs, nevertheless some of their data are open to question. I n none of the fields as shown in the photographs is it possible to see capillary sprouts, because the magnifications are too low. Capillary sprouting cannot be seen without using the compound microscope at magnifications of about 200 >(: or more. The process of vascularization as given in their diagrams explaining the photographs is not the full story as it purports to be but only the end stage ; the stage of venule development and all vessels shown have the arrangement characteristic of venules. The parallelism of external vessels related to grafts as shown in the diagrams is a common occurrence and has no special or important significance. In one diagram, it is said that a large vessel is growing into the graft. I t can be stated categorically that large vessels do not grow into grafts but form from pre-existing capillaries. In incised wounds, there is some suggestive evidence that adjacent formed vessels are concerned with the repair, in addition to vascular sprouting, but that possibility does not apply in the vascularization of grafts. The times of vascularization as given in the articles quoted have no significance because the authors could determine nothing about the first stages of the process, since the magnifications used were inadequate to see the extremely delicate sprouts with which vascularization begins. While capillary sprouts do form from venules, it is doubtful if those shown in the diagrams were capillary buds, and certainly no sprouts show in the photographs. It can be stated with assurance that in normal, uninfected tissue, endotlielial sprouting occurs only within the graft and from the area on which it is placed and that the stimulus to such formation does not extend beyond its lateral margins. Vessels do not grow toward a normal graft, from outside its lateral limits, but they do receive blood drainage from it. Conway, J o s h , ef al. (1952b) could find no evidence that corticotropin regulated

386

ROY G. WILLIAMS

the survival time of skin homografts and with this finding there is no disagreement. 2. Honaologozts Grafts

.

The results with homografts hereinafter discussed summarizes the studies made with chamber methods, and no attempt will be made to quote or reconcile the contradictory statements with which the extensive general literature abounds. As can be seen from Table I, survival of homografts is not the rule. Except for the V p carcinoma, the only homografts that survived were from choroid plexus and ciliary body. It is generally agreed that homografts survive longest in the anterior chamber of the eye or in the brain. Therefore, it was thought that the cells probably producing the fluid in those regions should themselves survive as homografts. Choroid plexus grafts develop a series of temporary fluid-filled vesicles of various sizes. The fluid slowly disappears and reappears in other places. Ciliary process survives as a mass of deeply pigmented cells. No vesicles formed in these grafts. Tissue taken from iris alone does not survive. Corticotropin, cortisone, and deoxycorticosterone have no effect in prolonging survival of thyroid, adrenal fasciculata, or spleen, and thiouracil fed to the host for many weeks before homografting of spleen has no effect in prolonging survival of that tissue. A striking characteristic of most autografts is their ability to stimulate the growth of endothelium, both their own and that of the vessels with which they are in contact. Homografts generally have no such ability, or if they do, the vessels cannot be maintained. I n the same tissue in different animals, transplanted endothelium may grow well for a time in homografts, or it may not grow at all, or its growth may be limited. The same variety of response applies to the host endothelium. In spleen, for example, the transplanted endothelium may grow vigorously for the first 24 to 48 hours, but no connections are made with host vessels and the blood never circulates in the newly formed plexus. The blood becomes laked and the grafts rapidly disappear. In thyroid, a fully circulating plexus may develop and the follicles appear perfectly normal. This may continue for several weeks or even months, and then the blood flow stops, the blood becomes laked, and the grafts rapidly disappear. In other thyroid grafts, no vessels form and the grafts disappear within a few days despite the fact that they were sufficiently small so that they could have been supplied by diffusions from the host vessels with which they were in contact. Unlike autografts, one cannot predict what the vascular response in a homograft will be or for how many days the graft will survive. The only thing about

STUDIES WITH TRANSPARENT CHAMBER METHODS

387

which one can be certain is that for the tissues studied (Table I), with the exceptions noted, survival time will not compare favorably with autografts. On first thought it might be supposed that the vascular differences between autografts and homografts are important factors in the failure of homograft survival. However, they may be effects of something else rather than causes. Autografts of adrenal fasciculata have no ability to stimulate growth of endothelium, but many of them survive for months. II3' temporarily suppresses the growth of endothelium in autografts of thyroid, which then resemble homografts of the gland. But some cells survive and after a time reconstitute follicular structure and become vascularized so that one cannot tell that vascularization had been delayed. It would seem likely, therefore, that whether or not a graft can stimulate formation of a new vascular plexus is not the deciding factor in its survival. The failure of homo- and heterografts to survive consistently or at all has been ascribed to anaphylaxis, genetic factors, and other things. The body undoubtedly has defenses against foreign invasions of any sort. A piece of glass, for example, becomes etched by the corrosive action of tissue fluids. But there is an additional factor that may play a part and which has not heretofore been emphasized. Each surviving graft of whatever sort creates its own internal environment. Failure to survive may therefore be related in the first instance to inability of transplanted cells to function adequately or at all in a foreign region or another animal, thus preventing maintenance or renewal of their protective intercellular environment. It is doubtful if host lymphocytes or other white cells are important in the survival of homografts, at least insofar as their presence locally is concerned. As homografts disappear, the region is sometimes infiltrated with white cells, but not in excessive numbers, such as would be produced by an infection. In many instances, no more white cells are present than would be there normally. There may or may not be sticking of leukocytes to the walls of host vessels in the neighborhood, a delicate sign of vascular injury that might be expected near dying cells. These variations in white cell response indicate that the circumstances surrounding disappearance of a graft are not always the same. Many homografts appear to dissolve and fade away rather than die and have the remains removed by phagocytes. The differences noted apply to the same tissue in successive hosts as well as to different tissues. 3. Tuntors Tumor grafts are generally homografts. Tumor might survive as autografts, but none of .the accounts encountered in chamber literature were concerned with them. A striking difference between a tumor homograft

388

ROY G. WILLIAMS

and a homograft of normal cells in which the tumor was primary is that the former commonly survives but the latter does not. The tumor graft behaves in that respect like an autograft. The tissue differentials (an expression used by Loeb, 1945), or whatever it is that ordinarily prevents survival of most homografts in most regions, seems to be missing or modified in cancer cells, although even tumors tend to be species specific. However, by serial transplantation in the brains of rats and other animals (Greene, 1951), the species factor can be overcome and heterografts be made to survive. Vascular reaction to transplanted tumor has attracted investigators and was the first phase of the cancer problem to be studied with chamber methods. Ide et al. (193%) studied the Brown-Pearce rabbit epithelioma. They used the first method of transplanting as described above, wherein the transplant was made when the chamber was installed and before it was vascularized. Vessels appeared from three to eight days later. Tumor growth, as seen grossly, coincided with the onset of vascular growth and progressed steadily until the chamber was completely filled. The tumors then suffered complete or partial resorption. The authors stated that, since the rapidly growing tumor was able to initiate in an unprepared site an adequate blood supply which was characteristic and not observed in the controls or in injury repair sites, it was probable that the tumor elaborated a vessel-growth-stimulating substance. While it cannot be denied that the tumor may have produced such a substance, that possibility does not automatically follow from the fact that its vascularization was different from the controls or from that in injured sites, for if it did, one would have to suppose that there was something common about the vascularization of any tissue and with the process of injury repair. But, aside from the general mechanism of endothelial sprouting, that is not necessarily the case. The final form of the vasculature is a function of each tissue and varies from tissue to tissue, depending upon the internal environment and other factors that each provides. Any surviving graft acquires a vasculature characteristic for that tissue, and the fact that it may be different from that produced by another type of tissue does not prove the presence or absence of a vessel-growth-stimulating substance, for there are many other factors involved. It should be pointed out that the authors only expressed a probability and did not claim to have proved the matter. The vascularization of a malignant tumor was also studied by Algire (1943b), who used a melanoma implanted in transparent chambers installed in dorsal skin flaps on mice. In this case, vascularization did not begin for twenty days, and during that time there was little tumor growth. After vascularization began, migration and proliferation of tumor cells

STUDIES WI TH TRANSPARENT CHAMBER METHODS

389

accelerated. Algire and Chalkley (1945) carried the study further, using sarcomas and mammary gland carcinomas, and also investigated the repair of wounds. They found that capillaries arose from the host and that endothelial proliferation appeared as early as three days after implantation, whereas in wounds it did not begin for six days. Despite the fact that the tumors became well vascularized, differentiation of vessels into arterioles and venules was not evident. The authors believe that an outstanding characteristic of the tumor cell is its capacity to elicit continued growth of new capillary endothelium from the host. “This characteristic of the tumor cell, rather than some hypothetical capacity for autonomous growth inherent within the cell, is, from the standpoint of the host, an important expression of neoplastic change.” This generalization implies that growth potentialities in the tumor cells themselves may be secondary in importance to the endothelial proliferation which the tumor can induce, or, stated otherwise, that the tumor grows because it can cause proliferation of blood vessels. There are those, of course, who think just the opposite, namely, that vessels proliferate because the tumor itself has the capacity for continued growth. This is the sort of thing that can lead to endless argument and is similar in its futility to the question, which came first, the chicken or the egg. However, it is clear that the suggested generalization of Algire and Chalkley does not apply to all malignancy, although it cannot be denied that it may apply to some. The Vz carcinoma, an epidermoid carcinoma of rabbits, has no ability to stimulate the growth of vessels (Williams, 1951). It infiltrates among the host vessels, causing changes in them, and finally eliminates all vessels centrally located by external pressure upon them, thus guaranteeing that the tumor center will be necrotic. This tumor is invasive and rapidly fatal, although it does not metastasize freely. The only viable part of it, after the first few days following transplantation, is a narrow peripheral rim where host vessels are available. The vascular response to the Vz carcinoma is very similar to that in normal growing epidermis, which suggests that a malignant tumor may retain, to some degree at least, the ability to influence its vasculature in the same manner as do the normal cells in which it was primary. Vessels near growing normal epidermis are wide, thin-walled, sinusoidal channels, and this is characteristic of those among the growing cells in V t carcinoma. Algire and Chalkley (1945) and Ide et al. (1939b) found large, thin-walled vessels consisting of endothelium only in sarcomas, niarnmary gland Carcinomas, and the Brown-Pearce epithelioma. They noted that arterioles and venules did not develop in the plexus. In growing mouse melanomas, capillaries were smaller and there was a greater tendency

390

ROY C. WILLIAMS

for arterioles and venules to differentiate (Algire and Legallais, 1948j . Since arterioles and venules normally develop within a capillary plexus in response to hemodynamic factors, it is curious that they sometimes do not develop in tumors, since if there is blood flow, there must be pressure and other differences in vessels. However, to have the formation of arterioles in a capillary plexus, there must be already formed arterioles sending blood into the plexus, and vessels in the plexus must not be of too large caliber. For example, an ampulla of Thoma in the spleen is an abruptly enlarged cone-shaped venous extension of an arteriole. As the spleen becomes vascularized, arteriolar formation does not extend beyond the ampulla. In some forms of hemangiomas, the vasculature is sinusoidal and arterioles do not develop in them. The size of capillaries and venules in a part may be an expression of the nature of the ground substance. If it affords little support for the walls, those composed chiefly of endothelium could be expected to dilate even under normal capillary pressure (Williams, 1949). The walls of vessels in Brown-Pearce tumors are so fragile that they are easily ruptured, and it would seem to be an easy matter for small tumor fragments to be dislodged and swept into the ruptured vessels and hence into the general circulation { Ide et al., 1939). But the authors found no metastases in over 105 cases. They believe that a study of the local defenses of body organs, particularly the lungs, against tumor fragments should be of considerable interest, as indeed it would. But the sweeping into the general circulation of tumor fragments through traumatic openings in vessels may not be so common. When a vessel ruptures, blood is extravasated until the pressure outside the vessel is locally equal to that inside or until the rupture heals or the hemorrhage stops for some other reason. To have tumor cells enter the blood stream, the rent in the vessel would have to stay open and a loose tumor fragment be forced in by external pressure. I t would, presumably, not often be sucked or swept in by the moving blood stream, since if there were any cell movement in the extravascular regions, it would tend to be away from the vessels because of the pressure of extravasation. There is no clear-cut evidence in chamber literature, or if there is it hasn’t been located, as to how metastases begin. Since the vessels in tumors are frequently so thin-walled and large and the tumor cells so actively growing and invasive, it is easily conceivable that tumor could grow into a vessel as a tuft and then be dislodged by the moving blood. Tumor cell masses in vessels have been described in microscopic sections, but they appear not to have been seen by direct observation in the living. The effect of roentgen irradiation on transplants of Brown-Pearce rabbit

STUDIES WITH TRANSPARENT CHAMBER METHODS

391

epithelioma was investigated by Downing cf al. (1940). Within one hour of the 10,000-r. dose, a red haze developed microscopically around the tumor and in control regions, but a gross erythema did not appear until five days later. The tissues were then suffused with extravasated red cells to an extent that made microscopic study impossible. Before that stage, all vessels in the irradiated sites became beaded and irregular in contour, and blood flow was reduced, erratic, or absent. However, normal responses to paling and flushing were almost intact. After five days vascular dissolution began, with slow recovery during the following week. The authors observed that endothelial response to irradiation was the same in both tumor and control areas which, they state in effect, indicates that a tumor does not change endothelium in it as far as the response to irradiation is concerned. The effects of irradiation on mammary tumor implants in mice were studied by Merwin et al. (1950). Dosage of 2000 to 3000 r. produced marked regression followed by regrowth. Tumor vessels narrowed progressively for about one week, but when tumor growth was resumed the vessels enlarged. The growth rate of the tumor after the first effects of irradiation subsided was initially about the same as it had been without treatment hut was later gradually reduced, apparently because after irradiation the growth potentialities of the endothelium were less. Vessels in regrowing tumor foci finally broke down, and the now nonvascularized opaque tissue stimulated the growth of adjacent vessels that had not been irradiated. When this occurred, such tumor as was still viable began to grow at its usual rate. Grafts not themselves irradiated were also made on vessels that had been. As in the previous cases, it was evident that irradiated vessels could not regenerate. I t appeared that anything short of complete tissue destruction by irradiation affected the growth properties of the tumor secondarily by damage to its blood supply. In this connection, it is pertinent to note again that 1131 suppressed the growth of endothelium in autografts of normal thyroid. Peripheral hypotension induced by histamine and in other ways reduces the blood supply in transplanted tumors (Algire and Legallais, 19.51). Reduction of tumor circulation is directly correlated with the duration and degree of peripheral hypotension and does not require toxic or lethal doses of histamine. Tumor necrosis results only after large, but not lethal, doses. Blood pressure in tumor vessels approximates normal venous pressure. “If a hypotensive state were maintained for a sufficiently long period one would expect to find histologic or gross evidence of damage to the tumor tissue from the resultant ischemia.” Bacterial potysaccharide affects vessels in mice, both normal and those

392

ROY

G. WILLIAMS

in tumors, in the following manner. The blood flow and percentage of functional capillaries is progressively decreased. Stasis and occlusion of capillaries develop, reaching a maximum in 3 to 4 hours with recovery in normal tissues within 18 hours. Hemorrhage and necrosis do not occur in normal tissues. The stasis and occlusion in all vessels seem to be associated with edema, suggesting increase in capillary permeability. In tumor vessels, after various periods, vascular occlusion is followed by the sudden appearance of petechiae throughout the tumor, followed by extensive necrosis. However, some tumor cells survive and retain their ability to stimulate the growth of vessels, and tumor growth is then resumed. There appears to be no primary action of the polysaccharide on the tumor cells. The tumor-necrotizing effect of this agent seems to be brought about by ischemia and circulatory stasis induced by hypotension (Algire, 1 9 6 ; Algire, Legallais, and Park, 1947; Algire and Legallais, 194s; Algire, Legallais, and Anderson, 1952). Mechanical obstruction can duplicate the action of bacterial p l y saccharide in producing hemorrhage and tissue damage in some tumors but not in others (Youngner and Algire, 1949a, b). Vascular reactions of inice to mouse fibroblasts cultivated in vitro and therein treated with methylcholanthrene were investigated by Algire, Chalkley, and Earle ( 1950). Various cell strains were created, depending on the length of time each was exposed to the carcinogen. These cultures were then implanted in mice by means of a chamber method. The vascular reaction to these transplants from tissue cultures was parallel with the capacity of the culture strains to give rise to sarcomas in other animals. Plasma clots used as controls produced only a mild foreign body reaction, indicating that cells must be present for a more vigorous vascular proliferation. With increased time in vitro after removal of the carcinogen, the ability of each cell strain to form sarcomas was reduced. This, the authors suggested, may indicate that growth of cultures in an entirely heterologous media for extended periods may alter cell characteristics so that they are less able to live when transplanted to an animal and less able to give rise to sarcomas. 4 . Parasites and Eggs This heading is included mainly because of the promise of the method in such investigations and not because it has been used extensively in the study of parasites. Only a single reference concerning such work has been found. Hoeppli and Hou (1931) placed various parasite eggs in chambers. The devices they used were not well adapted for transplanting, since the effective techniques now available had not then been developed. They found that Ascaris lumbricoides and FasciolopSis eggs produced little leukocyte infiltration, but it was much increased when the egg shells were

STUDIES WITH TRANSPARENT CHAMBER METHODS

393

broken. The shell of Fa.~ciolopSis buski eggs was less resistant to dissolution by tissue Auid than was the shell of Asccw-is tumbricoides. They also found that worm capsules of Spirocerca sanguinolenta larvae were very resistant to white cells. Physaloptera clausa from hedgehogs and Enterobius znermicduris from man survived only about two days. Physalojtera produced tissue liquefaction around its anterior end, but no similar changes were observed with Enterobius.

VII. TUBERCULOSIS

The early reaction, nine to twenty-three days, to lung bovine tubercle bacilli in chambers is minimal. However, the late reaction, ten to twentyfour days, is an explosive necrotizing response. As determined in one animal by systematic skin testing with old tuberculin, the beginning of late reaction coincides with the development of skin sensitivity. Tissue destruction followed progressive vascular damage resulting .in venous thrombosis (Ebert, Ahern, and Bloch, 1948). The thrombosis occurred only in small vessels, 30 to 40 p in diameter. It was impossible to tell whether the vascular changes were primary or secondary, but there was little doubt that they played an important part in tissue destruction. Ebert and Barclay (1950) studied the effect of chemotherapy in tuberculous infections. Animals were generally sensitized before inoculation of the chamber with 0.004 mg. of virulent bovine tubercle bacilli. They were then treated for six to eight weeks, some with streptomycin (100 mg. daily), some with p-aminosalicylic acid (0.900 mg. daily), and others with the two combined. Thrombosis of small vessels and infarction occurred as it did in untreated animals. In general, healing and extension of the disease process occurred simultaneously in different parts of tubercles as they do without treatment, but in treated animals there was more extensive healing. Healing and extension were commonly cyclic in that there were times when healing was predominant and others when it was not. When treatment was begun before inoculation, the initial necrotizing response was not inhibited. Cortisone reduces the inflammatory response to tuberculous infection of a hypersensitive animal (Ebert, 1951, 1952). In the presence of cortisone, vascular tone is better maintained, damage to endothelium of arterioles and venules is reduced, and there is less diapedesis of leukocytes and less exudate. The reduction in inflammation causes poorer localization of infection. This recalls studies previously quoted (Ebert and Barclay, 1952) in which it was stated that if an inflammation is useful to the body cortisone may be harmful, but it would not be if the inflammation were useless, as in rheumatoid arthritis.

394

ROY G. WILLIAMS

As a preliminary to a study of its effect in tuberculous infection, Markham et al. (1951) investigated the behavior in Zrivo of an almost insoluble antibiotic, micrococcin. A fine suspension of the material was forced into chambers through an access hole and also, in other animals, by removing the chamber cover. The micrococcin was taken up by macrophages and retained for many months without apparent damage to them. Vascular endothelium was unaffected. The antibiotic behaved in the body like a bland foreign body, When carbon and micrococcin are injected intravenously, the particles are agglutinated by the platelets and the masses adhere to the walls of vessels in the neighborhood of tuberculous lesions and tend to stay in the vessels. The micrococcin produces no effect on the growth of tubercles even when in the center of necrotic tubercles (Sanders et al. 1951). Micrococcin in Triton WR-1339, a detergent of low toxicity, was tested in tuberculous infection by Heatley et al. (1952). The course of experimental tuberculosis and the nature of the lesions in guinea pigs were not influenced by intravenous injections of the solution.

VIII. CONCLUSION The transparent chamber method is a means for creating a microscopic section, as it were, without the disadvantages of killing the cells, subjecting them to potent chemicals, and converting three dimensions to two. With such preparations, studies need not be limited to descriptive histology. They also reduce the need for inference in determining a vital sequence. The method will continue to be used as it has been because none of the subjects studied has been exhausted. The most likely extensions of its use, with suitable, easily made modifications, would seem to be with polarized light, ultraviolet light, reflecting microscopes, fluorescent microscopy, microdissection, and precise chemical methods adapted to living cells. ACKNOWLEDGMENTS The author is deeply indebted to Miss Patricia Rochford for her invaluable assistance in the preparation of this review, and to Dr. E. R. Clark, who kindly read the manuscript.

REFERENCES Abell, R. G. (1934) Anat. Record, 60, 161. Abell, R. G. (1935) Amat. Record, 64, 51. Abell, R. G., (1937a) Anat. Record, 69, 11. Abell, R. G. (1937b) Anat. Rrcord, 67, Sztppl. 3, 1. Abell, R. G. (1939) Biol. Bull. 77, 320. Abell, R. G. (1940) Anat. Record, 78, 215.

STUDIES WITH TRANSPARENT CHAMRER METHODS

395

Abell, R. G. (1941) Anat. Recmd, 81, 477. Abell, R. G. (1946a) Biol. Bull., 91, 219. Abell, R. G. (1946b) Biol. Bull., 91, 230. Abell, R. G. (1%) Am. I. Physiol., 147, 237. plbell, R. G., and Clark, E. R. (193Za) Ailat. Record, 6'2, S%ppE., 45. Abell, R. G., and Clark, E. R. (1932b) Anat. Record, I , 121. .&bell, R. G., and Clark, E. R. (1933) Ailat. Rccord, 66, Suppl., 1. Abell, R. G., and Clark, E. R. (1937a) Biol. Bull., 73, 371. Abell, R. G., and Clark, E. R. (1937b) Biol. Bull., 73, 370. Abell, R. G., and Clark, E. R. (1939) Ancrt. Kccord. 73, SuppI. 2, 1. Abell, R. G., and Page, I. H. (1941) Biol. Bull., 81, 293. Abell, R. G., and Page, I. H. (194Za) J. Exptl. Med., 76, 305. Abell, R. G., and Page, I. H. (194Zb) I. Exptl. Med., 76, 673. Abell, R. G., and Page, I. H. (1943) Surg. Gynccol. Obstef., 77, 348. Abell, R. G., and Schenck, H. P. (1938) J . Imrmrnol., 34, 195. Ahern, J. J., Barclay, W. R., and Ebert, R. H. (1949) Science, 110, 665. Algire, G. H. (1943a) J. Natl. Cuncer Inst., 4, 1. Algire, G. H. (1943b) J. Natl. Cancer Insf., 4, 13. Algire, G. H. (1946a) Federation Proc., 6, 217. Algire, G. H. (1946b) Anat. Record, 97, 410. Algire, G. H. (1%) Cancer Research, 6, 491. Algire, G. H. (1947) Approaches to Tumor Chemotherapy, Sec. 11, p. 13, F. R. Moulton, ed., Science Press, Lancaster, Pa. Algire, G. H., and Chalkley, H. W. (1945) J. Nutl. C m e r Inst., 6, 73. Algire, G. H., Chalkley, H. W., and Earle, W. R. (1950) I. N d . Cancer Inst., 11, 555. Algire, G. H., and Legallais, F. Y. (1948) Spec. Publ. N. Y . Acad. Sci., 4, 159. Algire, G. H., and Legallais, F. Y. (1949) 1. Notl. Cuncer Inst., 10, 225. Algire, G. H., and Legallais, F. Y. (1951) J. Natl. Cancer Iwf.,1%399. Algire, G. H., Legallais, F. Y., and Park, H. D. (1947) J. Nutl. Cancer Inst., 8, 53. Algire, G. H., Legallais, F. Y., and Anderson, B. F. (1952) J. Nutl. Cancer Insf., 12. 1279. Algire, G. H., and Schlegel, J. U. (1950) J. Celldur Comp. PhySiol., S6, 95. Beecher, H. K. (1936a) Skand. Arch. Physiol., 73, 1. Beecher, H. K. (1936b) Skand. Arch. Physiol., 73, 123. Chalkley, H. W. (1943) J. Natl. Cottcpr. Inst., 4, 47. Chalkley, H. W., Algire, G. H., and Morris, H. P. (1946) 1. Natl. Cancer Inst., 6, 363. Clark, E. R. (1931) ColZectirqp Net, 6, 279. Clark, E. R. (1936~)Ann. Znternal Med., 9, 1043. Clark, E. R. (1936b) Trans. Coll. PhySiciaJts Phila., 6, 1. Clark, E. R. (1938) PhySioE. Revs. 18, 229. Clark, E. R., and Clark, E. L. (1931) Anut. Record, 48, 13. Clark, E. R., and Clark, E. L. (1932a) Am. J. Amt., 49, 441. Clark, E. R., and Clark, E. L (1932%) Am. J . Anat., 61, 49. Clark, E. R., and Clark, E. L. (1933a) Am. 1. Phykol., 106, 19. Clark, E. R., and Clark, E. L. (1933b) Biol. Bull., 66, 363, Clark, E. R., and Clark, E. L. (1933~)Am. J . Aliet., U2, 273. Clark, E. R., and Clark, E. L. (1934a) Am. 1. Anut., 64, 229. Clark, E. R., and Clark, E. L. (1934b) Ant. J . Anat., 66, 407. Clark, E. R., and Clark, E. L. (1935) Am. I. Amt., 67, 385.

3%

ROY G. WILLIAMS

Clark, E. R., and Clark, E. L. (1936a) Biol. Bull., 71, 405. Clark, E. R., and Clark, E. L. (1936b) Am J. Md. Sci., 191, 302. Clark, E. R., and Clark, E. L. (1937a) Bid. BUN, 73, 371. Clark, E. R., and Clark, E. L. (1937b) Am. J. Anat., 62, 59. Clark, E. R., and Clark, E. L. (1937~)Am. I. Anat., 60, 253. Clark, E. R., and Clark, E. I-. (1938) Anat. Record, 70, Swppl. 3, 14. Clark, E. R., and Clark, E. 2.. (1939) Am. J . Anof., 64, 251. Clark, E. R., and Clark. E. 1.. ( 1940a) AVW.J . Anot., 66, 1. Clark, E. R., and Clark, E. L. (194Ob) Am. I. Anat., 67, 255. Clark, E. R., and Clark, E. L. (1942) Am. J. Anat., 70, 167. Clark, E. R., and Clark, E. L. (1943a) Am. J. Anat., 73, 215. Clark, E. R., and Clark, E. L. (1943b) Anat. Record, 86, 303. Clark, E. R., and Clark, E. L. (1944) Anat. Record, 88, 426. Clark, E. R., and Clark, I?. L. (1946) Biol. Bit/[., 91, 221. Clark, E. R., and Clark, E. L. (1947) Anat. Record, 97, 326. Clark, E. R., and Clark, E. L. (1948) Anat. Record, 100, 650. Clark, E. R.,Clark, E. L., and Abell, R. G. (1933) Anet. Record, 66, 50. Clark, E. R., Clark, E. L., and Rex, R. 0. (1936) Am. J. And., 69, 123. Clark, E. R., Clark, E. L., and Williams, R. G. (1934) Am. J. Anat. 66, 47. Clark, E. R., and Haagensen, D. E. (1939) Proc. Air Hygiene Foundation Amer., 4th Fall meeting, p. 49. Clark, E. R., and Haagensen, D. E. (1940) Proc. Air Hygiene Fozlndation Amer., 5th Fall meeting, p. 36. Clark, E. R., Hitschler, W. J., Kirby-Smith, H. T., Rex, R. O., and Smith, J. H. (1931) Anat. Record, W, 129. Clark, E. R.,Kirby-Smith, H. T., Rex, R. O., and Williams, R. G. (1930) Anat. Record, 47, 187. Clark, E. R., Sandison, J. C., and Hou, H. C. (1931) Anat. Record, SO, 169. Conway, H., Joslin, D., and Stark, R. B. (1951a) Plastic Reconstructive Surg., 8, 194. Conway, H., and Stark, R. B. (1951b) Plastic ReconstructizJe Surg., 8, 354. Conway, H., Stark, R. B., and Joslin, D. (1951~)Plastic Reconstwfive Surg., 8, 312. Conway, H., Stark, R. B., and J o s h , D. (1952a) Plastic Reconstmctive Surg., 10, 67. Conway, H., Joslin, D., Rees, T. D., and Stark, R. B. (1952b) Plastic Reconstructive Swg., 9, 557. Downing, V., Bishop, F. W., and Warren, S. L. (1940) Am. J. Roentgenol. Radium Therapy, 43, 249. Ebert, R. H. (1951) Trans. Natl. Tubcrc. Assoc., 47, 140. Ebert, R. H. (1952) Am. Rev. Tuberc., 66, 64. Ebert, R. H., Ahern, J. J., and Bloch, R. G. (1948) Proc. SOC.Exptl. BioL Med., 68, 625. Ebert, R. H., and Barclay, W. R. (1950) J. CZin. Invest., 29, 810. Ebert, R. H., and Barclay, W. R. (1952) Ann. Internu2 Med., 57, 506. Ebert, R. H., and Florey, H. W. (1939) Brit. I . Exfill. Pathol., 20, 342. Ebert, R. H., Florey, H. W., and Pullinger, B. D. (1939) J. PathaZ. Bacteriol. 48, 79. Ebert, R. H., Sanders, A. G . , a i d Florey, 11. W. (1940) Brit. J. Exptl. Pathol., 21, 212. Ebert, R. H., and Wissler, R. W. (1950) J. Lob. Clin. Med., 36, 818.

STUDIES WITH TRANSPARENT CHAMBER METHODS

397

Elm$ R. H., arid Wissler, R. W. (195la) 1. Lab. Clin. Mrd., 98, 497. Ebert, R. H., and Wissler, I<. W. (1951b) J. Lab. Clitt. bled., 38, 511. Essex, H. E. (1948) Methods in Medical Research, Yo]. 1, pp. 139-145. Year Book Publishers, Chicago. Essex, H. E., and de Rezende, N. (1943) Am. J . Physiol., 140, 107. Essex, H. E., and Grana, A. (1949) Am. I . PbysioE., 168, 3%. Estable, C. (1948) Proc. SOC.Exptl. Uiol. Ned., 67, 445. Flory, C. M., and Thal. A. (1947) Anat. Record, 97, 33. Greene, H. S. N. (1951 ) Cumer Research, 11, 529. Heatley, N. G., Gowans, J. L., Florey, H. W., and Sanders, A. G. (1952) Brit. J . EXptl. !'lltkOl., 39, 105. Henry, C. G. (1933) Ailof. Rccord, 67, 263. Hoeppli, R. J. C., and Hou, K. C. (1931) Chinese 1. Phytysiol., 6, 19. Hou, H. C. (1929) Proc. SOC.Expfl. Biol. ilfed., 26, 745. Hou, H. C. (1932) Chiinfse J . P h y ~ o l . 6, , 243. Ide, A. G., Baker, N. H., and Warren, S. L. (1939a) Am. J. Roewtyenol. Radiiu~i Therapy, 42, 891. Tde, A. G., Harvey, R. A., and Warren, S. L. (1939b) Arch. Patkol., 28, 851. Joslin, D. (1952) Sciertcf, 116, 601. Kirby-Smith, H. T. (1933) Am. J . Anat., 63, 377. Levinson, J. P., and Essex, H. E. (1943a) . 4 m J. Physiol., 139, 423. Levinson, J. P., and Essex, H. E (1943b) Proc. Soc. ExptI. Biol. M e d . , 62, 301. Ixwis, W. H. (1931) h'zili., lohiis Hopkbts Hasp., 48, 242. J.oeb, L. (1945) Biological Basis of Individuality, Charles C Thomas, Springfield, Ill. hiarkham, N. P., Heatley, N. G., Sanders, A. G., and Florey, H. W. (1951) Brit. J . Exptl. Pathol., 32, 136. Merwin, R., Algire, G. H., and Kaplan, H. S. (1950) J . N a t l . Cancer Inst., 11, 593. Riollier, S. (1910) Arch. mikroskop. Allat. 14. Entwirkhiigsmech., 76, 608. Moore, R. L. (1936) Attut. Record, 64, 387. Robertson, J. S. (1951) Aristraliniz J . Exptl. Biol. M e d . Sci., 29, 393. Robertson, J. S. (1952) Australian J . Exptl. Biol. Med. Sci., 50, 59. Sanders, A. G., Ebert, R. H., and Florey, H. W. (1940) Quart. J . Exptf. Physiol.,

30, 281.

Sanders, A. G., Florey, 3.W., and Wells, A. Q. (1951) Brit. J , Exptl. Pathol., 32, 452. Sandison, J. C. (1924) Anat. Record, 28, 281. Sandison, J. C. (1928a) Anat. Record, 40, 41, Sandison, J. C. (1928b) Am. J . Anat., 41, 447. Sandison, J. C. (1928~)Am. J . Anat., 41, 475. Sandison, J. C. (1928d) Am. J . Mcd. Sci., 9, 21. Sandison, J. C. (1929) Anat. Record, 42, 62. Sandison. J. C. (1931) Anat. Record, 60, 355. Sandison, J. C. (1932) .4nat. Record, 54, 105. Seldon, T. H., Lundy, J. S., and Essex, H. E. (1942) Anesthesiology, 3, 146. Shelden, C. H., Pudenz, R. H., Restarski, J. S., and Craig, W. M. (1944) J . N t w o s u r g . , 1, 67. Solis, J. T., and Essex, H. E. (1951) Am. J. Physiol, 167, 1. Stearns, M. L. (1939) Anat. Record, 73 SuppE., 49. Stearns, M. L. (1940a) Am. J . Anat., 66, 133.

398

ROY G. WILLIAMS

Stearns, M . L. (1940b) Am. J . ‘4nat., 67, 55. Vigran, M., and Essex, H. E. (1950) Ant. J . Physiol., 162, 230. Wentsler, N. E. (1936) 4nat. Rccord, 66, 423. Williams, R. G. (1934a) Anat. Record, 60, 487. Williams, R. G. (1934b) Anat. Record, 60, 493. Williams, R. G. (1937) Am. J . Anat., 62, 1. Williams, R. G. (1939a) J . Morshol., 65, 17. Williams, R. G. (1939b) ,412at. Record, 73, 307. Williams, R. G. (1941) Arrat. Record, 79, 263. Williams, R. G. (1944) Am. J . Anat., 76, 95. Williams, R. G. (1945) Am. J . Amt., 77, 53. Williams, R. G. (1947) Am. J . Alwt., 81, 199. Williams, R. G. (1949) Anat. Record, 104, 147. Williams, R. G. (1950a) Ant. J. Anat., 86, 343. Williams, R. G. (1950b) Am. J . Anat., 87, 459. Williams, R. G. (1951) Cancer Research, 11, 139. Williams, R. G., and Roberts, B. (1950) Anat. Record, 107, 359. Wilson, H. C. (1936) J . Pharmacol. Exptl. Therap. 66, 97. Youngner, J. S., and Algire, G. H. (1949a) J . Natl. Canccr Inst., 10, 565. Youngner, J. S., and Algire, G. H. (1949b) Cancer Research, 9, 559. Zintel, H. A. (1936) Anat. Record, 66, 437.

The Mast Cell G. ASBOE-HANSEN Laboratory for Connective Tisme Research. University Institute of Medical Amtomy. Copenhagen. Denmark

I. Introduction ....................................................... I1. Origin ............................................................ 1. Homoplastic Regeneration ...................................... 2. Heteroplastic Regeneration ..................................... I11. Morphology ....................................................... 1. Nucleus ....................................................... 2. Granules ....................................................... 3. Mitochondria .............................. ................ 4. Golgi Apparatus ................................................ 5 . Cytoplasm ..................................................... 6. Cellular Membrane ............................................. IV . Distribution ....................................................... 1. Normal Connective Tissue ...................................... 2. Pathologic Connective Tissue ................................... V. Cytochemistry ..................................................... VI . Function .......................................................... 1. “Nutrition” .................................................... 2 . Elastin Formation .............................................. 3: Pigmentation .................................................. 4. Phagocytosis .................................................. 5 . Defense ........................................................ 6. Relation to Fat ................................................ 7. Histamine Production .......................................... 8. Mucopdysaccharide Secretion ................................... VII . Physiologic Variability ............................................ VIII . References ........................................................

Page 399 400

400 400 402 402 402 405 405 405 405 405 406 410 415 422 422 422 423 423 423 424 424 425 426

431

I. INTRODUCTION Mast cells are present wherever there is connective tissue . They are large mesenchymal cells containing in their cytoplasm granules and a substance with mucopolysaccharide characters . They are-like the fibroblasts and histiocytes-integral individuals of the cell population in normal connective tissue . The term 11.zart #ell was coined by Ehrlich. and means an overnourished cell. The connective tissue has recently commanded increased interest . It has been discovered that certain hormones-primarily those of the adrenal cortex-influence the mesenchymal tissue and that the clinical action of cortisone is due essentially to the connective tissue response . These findings have made the mast cell an object of particular interest . And indeed. it

399

400

G . ASBOE-HANSEN

deserves special attention, not least because in most pathologic laboratories it is neglected as it is not demonstrable in histologic sections stained by current methods. 11. ORIGIN The origin of the mast cells is not definitely known, although various theories have been advanced in the course of time. Most recent workers support the histogenous hypothesis, although the h m t o g e n o u s theory still has its advocates (Turk, 1904; Brack 1925; and others). Owing to common tinctorial properties (metachromatic staining of the granules with toluidine blue and other basic dyes), the basophile leukocytes of the blood have been interpreted as “blood mast cells.” Furthermore, the basophile leukocytes may migrate into the tissues. The nucleus of the blood cells, however, is usually segmented, unlike that of the tissue mast cells. Apart from the metachromatic staining of the granules, the two cells have nothing in common and a large number of workers repudiate the view that the two should represent the same type of cell (Maximow, 1913; Michels, 1938; Doan and Reinhart, 1941).

1. Homoplastic Regeneration Mitotic figures are rarely seen in the mast cells of an adult organism (Maximow, 1906; Downey, 1913). I n young fetuses they are far more common (Maximow, 1907; Alfejew, 1924; Lehner, 1924; Nagayo, 1928: Michels, 1938). Pappenheim (1904) regarded the mast cell as a degenerating cell. “Definitely to disestablish this notion,” Michels ( 1922) demonstrated normal chromosomal configuration during mitosis in mast cells from the spleen of the lizard Gongylils. Maximow (1906) and others have observed definite mitoses in an adult cat, and Deringer and Dunn (1947) explain the mast cell increase in fetal as well as in neoplastic tissue by mitotic division. Various findings indicate, however, that amitotic divisions occur in mast cells (Sabraz6s and Lafon, 1908; Lehner, 1924 ; Stockinger, 1927 ; Bloom, 1942). Lehner observed that mast cells are liable to form groups, and that neither mitoses nor heteroplastic regeneration are found in such groups. Moreover, mast cells with bipartite nuclei have been observed, and these cells are sometimes grouped like cartilage cells. Finally, Lehner observed central constrictions of the nuclei, phenomena interpreted as an indication of amitotic division. 2. Hetwoplastic Regeneration Numerous authors believe that heteroplastic regeneration, i.e. development of mast cells from other types of cells, takes place. Lymphocytes

T H E MAST CELL

401

have been reported to be transformed into mast cells in some way or other ( Audry, 18% ; Downey, 1911 ; Michels, 1922 ; and others). Sabrazis and Lafon ( 1 W ) mention pro-mastcells, meaning large mononuclear cells with basophile granules, not staining metachromatically. In their opinion, these cells are transitional stages from lymphocytes or other mononuclear cells. Plasma cells have also been interpreted as the precursors of mast cells (Downey, 1911 ; Sabrazk and Lafon, 1908; Michels, 1935;and others). According to several authors, histiocytes may develop granules and are transformed into mast cells (Downey, 1911; Herzog, 1916;and others). Finally, it is believed that fibroblasts, the mesenchymal prototype, may be transformed into mast cells. At present, this is the most popular theory. Bates (1935), calling attention to the marked similarity between the nuclei of fibroblasts and of mast cells, claimed to have observed signs that fibroblasts may become mast cells. Studying the tissues of rats and mice, Alfejew (1E4) found signs oi heteroplastic regeneration from mesenchymal cells, but he observed mitoses as well. On the basis of the examination of cat, rabbit, rat, and human fetuses, Holmgren (1946) maintained that mast cells develop from mesenchymal cells. They form a special type of cell with their own line of development. He makes a distinction between two types of mast cells which appear, however, to represent merely two stages in the development of the mast cell proper. Bensley (1952) studied the histogenesis of mast cells in skin after subcutaneous injection of histamine. Mast cells were seen to develop from small round cells in the connective tissue, and granules appeared in fibroblasts as well as in endothelial cells. The continued discussion regarding heteroplastic regeneration is presumably due to the fact that faintly granulated mast cells often escape detection with ordinary histologic technique. Recent technical advancesprimarily freeze vacuum dehydration, electron microscopy, and autoradiography-have, however, afforded important and in fact indispensable means of solving these problems. Mast cells without demonstrable granules look like other mononuclear connective-tissue cells ; their granulation may also vary according to the functional condition of the tissue. The reported instances of heteroplastic regeneration may, therefore, represent merely the granulation of previously degranulated mast cells which have escaped histologic detection.

402

G. ASBOE-HANSEN

111. MORPHOLOGY The mast cells may vary within wide limits in shape and size depending on various factors, among others on the structure, water content, etc. of the surrounding tissue. According to Bates ( 1935) , their size increases with advancing age. They may be flat, spherical, spindle-shaped, or stellate and-when situated between coarse fibrils-almost filiform. The same cell may assume varying shapes under the influence of different physiologic actions (p. 426). According to Lehner (1924), the plump mast cells have a diameter ranging from 8 to 15 p, while the elongated ones may attain twice this size. The size of mast cells depends largely on the fixative used. When fixed in alcohol, they are relatively small, at least constantly smaller than in tissues fixed in 4% lead subacetate (the method of Holmgren) , Freezevacuum fixation conserves the mast cells in their largest and most regular varieties of form. 1. Nucleus The nucleus is round or oval, in some cells with a small notch. Its situation is usually central though sometimes excentric. In proportion to the volume of the cell, it is relatively small, about 4 to 6 p . According to nuclear appearance, Holmgren has classified mast cells into two types : Type A, with a large nucleus poor in chromatin, resembling the nucleus of a fibroblast; and Type B, with a small nucleus rich in chromatin, suggesting the nucleus of a lymphocyte. There seem to be all transitions, however, and probably the functional state of the cells, their age, movement, etc., combined with the media of preparation, give a varying histologic appearance of the mast-cell nucleus. In man, binuclear mast cells are occasionally seen, whereas the segmented nucleus as in basophilic leukocytes does not occur. The pattern of the nuclear chromatin is regular and without characteristics. The apparent presence of a nucleolus in rare cases no doubt represents a localized fusion of the chromatin structure and is probably an incidental occurrence of no real significance.

2. Granules Mast cells contain granules of globular or-rarely-oval shape (Harris, 1900). A few investigators have described elliptic or rod-shaped granules (Ranvier, 1893; Sylvkn, 1950), and this shape has been attributed to pressure exerted by adjacent granules. Harris (1900) found the granules to measure 0.2 to 0.4 p . Other workers have arrived. at similar results (Maximow, 1906; Lehner, 1924; Michels, 1938; Julkn et al., 1950).

THE MAST CELL

403

Studying freeze-dried mast cells from the rat mesentery in the phase contrast microscope, SylvCn ( 1950) found granules ranging in diameter from 0.3 to about 1 /I. I n recent studies of the dermal connective tissue by electron microscopy, the writer (1954b) found the granules to be almost invariably globular in shape and of constant size (0.3 p ) . In tissues fixed in a buffered solution of osmic acid (after Palade, 195Z), alcohol, basic lead acetate, or formalin, ;is well as in freeze-dried specimens, intergranular cytoplasmic connections were observed (Fig. 1). Their biconcave shape might indicate that these structures consist of dried Cytoplasm. No granular structures were observed in the cytoplasm. Nakajima (1928) and Nagayo (1928) reported that in young individuals the mast cells had fewer and smaller granules. According to these authors, the topographic factors play a role in this connection, mast cells

FIG.1. Electron microscopy of a mast cell. Magnification: approx. 1.5000 X . Sector with the nucleus in the top right-hand corner and granules which are spherical and of uniform size. Note the intergranular bridges of cytoplasm.

404

G. ASBOE-HANSEN

from the alimentary tract and lymphoid organs, for instance, presenting particularly small granules. Bates ( 1935j also found fewer-but not smaller-granules in young individuals. The globular shape oE the granules has repeatedly given rise to the presumption that the granules actually are droplets of fluid suspended in the cytoplasm. Julkn ct al. (1950) used differential centrifugation of suspensions of ox liver capsule, rich in mast cells and fibroblasts, and in this way they separated a fraction consisting of large mast granules (0.2 to 0.3 p ) and another fraction consisting of submicroscopic particles in the cytoplasm (p. 416). Lehner ( 1924) found canal-like non-granulated stripes in the niast-cell cytoplasm which he interpreted as a kind of secretory ducts through whicli dissolved granule substance might reach the cell surface. A jzixtaiizrclear u”oiie, devoid of granules, is sometimes observed. Presumably, this is a phenomenon depending on the methods of preparation. So far, the writer has never seen it in freeze-dried tissue. Maximow ( 1906), Lehner ( 1924j and other workers have interpreted nncuolkafiow in mast cells as a sign of advanced age and degeneration. In the phase contrast microscope SylvCti ( 1950) saw clear spaces or pseudovacuoles. Others, particularly recent workers, believe this phenomenon to be related to secretion, perhaps a sign of secretory exhaustion. The present writer ( 1950g; 1952b) found vacuolization of mast cells derived froin individuals subjected to the action of cortisone or corticotropin. Granules are sometimes found outside the cells, surrounding the perimeter of the mast cells and situated in the free connective-tissue ground substance. A t times they may be so far away from the cells that they are not likely to be situated witliin invisible cell boundaries. Maxiniow’s interpretation (1904) that the mast cells are secreting cells, emitting their granules to the connective tissue, cannot he rejected. In that case, however, it is surprising that the phenomenon of extracellular granules is not inore cominon. It is striking also that this scattering of granules is widespread in some preparations and absent in others. The phenomenon ia now widely attributed to artifacts (shrinkage during preparation, damage due to the-microtome knife, etc.). Plt times, the body of the mast cell is surrounded by a halo of cloudy amorphous nietachromasia, no granular structure being distinguishable. This phenomenon is also generally interpreted as an artifact ( e g . dissolution of granules). It is important to note that SylvCn and Larsson (1948) by chemical and S y l v h (1940) by actinic action upon the skin, induced degranulation of the mast cells and observed extracellular occurrence of nietachromatic substance. The same

THE MAST CELL

405

effect was obtained by Brodersen (1928) in the rat by mechanical action upon the skin, and by the present writer (1950f) in human skin by producing urticaria1 derniographism.

3. Mitochondria These structures are occasionally mentioned as coniponents of mast cells, but their demonstration is still rather doubtful. I n the hamster, Compton ( 1952) found no or exceedingly few mitochondria. Zollinger ( 1950) interprets mast granules as mitochondria altered by saturation with heparin. H e could not, however, demonstrate significant amounts of ribonucleoprotein or deoxyribonucleoprotein in the granules.

4. Golgi Apparatus A Golgi apparatus (or Golgi bodies) has been observed in a few mast cells but is far from constant (Compton, 1952).

5. Cytoplasm On microscopic examination the intergranular cytoplasm invariably presents itself as a homogeneous mass devoid of any structure. It is oxyphilou\ (Michels, 1938 ; i\l aximow and Hloom, 1938) as well as metachromatic (JulPn et al., 1950). Upon ultracentrifugation and electron microscopy the latter authors found a microsome structure in the cytoplasm made up of minute submicroscopic particles with a diameter of less than 10 mp and a marked tendency to form aggregates. The substance is freely soluble in water. 6. Cellular Membrane No distinct membrane is visible in the ordinary microscope, nor could Sylven (1950) make out such a structure by phase contrast microscopy. I n the electron microscope the writer (195413) found a distinct cell membrane.

IV. DISTRIBUTION 1. Normal Connective Tissue The reported distribution of mast cells in the body differs according to the experimental animal employed. There is a marked difference between the mast-cell content in a given organ of different animals. A feature cornmon to all animals, however, is that the cells are present in the connective tissue (Staemmler, 1921, and others) and that they appear to vary in number with the quantity of hyaluronic acid in the tissues ( Asboe-Hansen, 1950d, e). Their number does not, on the other hand, vary with the

406

G. ASBOE-HANSEN

quantity of metachromatic substance (metachromasia is given by other acid mucopolysaccharides too) in the connective tissue. It is characteristic also that mast cells are especially numerous in the adventitia of the Blood vessels and their immediate surroundings (Jorpes, Holrngren, and Wilander, 1937, and many others). The mast cells have been studied in detail in the skin. The corium is connective tissue of a rather dense fibrillar structure, containing connectivetissue ground substance in moderate quantities and comparatively few cells. In addition to hyaluronic acid, the dermal ground substance also contains chondroitinsulfuric acid (Meyer and Chaffee, 1941 ; Rleyer and Rapport, 1951) but according to Meyer and Rapport this chondroitinsulfuric acid (type B) is not sensitive to hyaluronidase. In normal skin, the mast cells are relatively few in number, usually small and spindleshaped, as a rule rather faintly granulated, but showing unmistakable metachromasia. They are preferably of a perivascular habitat, but a considerable number of mast cells will invariably be encountered in relation to the hair follicles too. Those mast cells which are situated free in the tissue without relation to the vessels are most plentiful in the superficial layer of the corium, the papillary layer. Hellstrom and Holmgren (1947) tried to count the mast cells per square millimeter of skin. The number proved to decrease with advancing age, and granulation as well as metachromasia were less pronounced in old than young subjects. These alterations of course entail difficulties in identifying the mast cells. Women under 40 years of age had higher mastcell counts than men. Mast cells may in rare cases be found among the epithelial cells of normal epidermis. This has been taken as a proof that the mast cells are capable of moving in the tissues (Williams, 1900; Unna and Schumacher, 1925). I n the synozial membrane-where the synovial fluid is forined-mast cells are present in large iiumbers, varying somewhat according to the site (Hamtnar, 1894; Petersen, 1930; Davies, 1942-43 ; Janes and Rlcr?onald, 1948 ; Asboe-Hansen, 1950d ; and others). The “synovioblasts” described by Vaubel (1933) may have been mast cells. The cells, which he studied in tissue culture, he characterized as “round and spindle-shaped, polygonal and epithelioid cells which form in their cytoplasm large, highly refractive granules that stain deeply red with neutral red.” In this as in other tissues the number evidently depends in some measure on the age of the subject, being on the whole most abundant in youth. The mast cells may occur in all synovial strata, but are most plentiful-in fact accumulated -along the lining facing the joint cavity. In the deeper layers they are mainly situated perivascularly. Davies’ ( 1942-43) contention that mast

T E E MAST CELL

407

cells never occur in the synovial lining among the surface cells does not accord with the present writer’s findings (1950d). Preparations fixed in lead subacetate 457 or freeze-dried, and stained in an aqueous or alcoholic solution of toluidine blue show numerous mast cells right among the surface cells (Figs. 2 and 3 ) . The most superficial ones are, however, usually faintly granulated and therefore hard to identify unless the technique is good, especially if the fixative is less favorable. Deeper in the synovial connective tissue there are numerous mast cells, often so densely granulated that the nucleus is hardly visible. While the granulation of the mast cells decreases toward the articular cavity, the quantity of hyaluronidase-sensitive metachromatic substance increases. The writer interprets the varying granulation of the mast cells as different phases of secretion and concludes from the above-mentioned findings that evidently the mucopolysaccharide in the synovia-hyaluronic acid--is produced by the mast cells in the synovial connective tissue (p. 426). Mast cells are present in the oczrlar ~fiemtbranes,primarily along the vessels, but in appreciable numbers only in the ciliary body, iris, corneal limbus, and in the subconjunctival loose connective tissue (Jorpes, Holmgren, and Wilander, 1937 ; Undritz, 1946 ; Asboe-Hansen, 1950e). The sclera, which gives a rather intense metachroniatic stain with toluidine blue owing to its content of chondroitinsulfuric acid, holds only a few mast cells. I n the umbilical cord, Wharton’s jelly contains large quantities of hyaluronic acid as well as chondroitinsulfuric acid. I n 1950d the writer showed that the embryonic cells of the cord have a metachromatic cytoplasm and stain like polysaccharides with the method of Hotchkiss and of Hale. Granulated mast cells may be demonstrated in sections stained with an alcoholic solution of toluidine blue. Prakken and Woerdemann (1952) found numerous well-granulated mast cells in the umbilical cord, after having eliminated the metachromatic ground substance by prolonged action of hyaluronidase. In an untreated preparation stained with an aqueous solution of toluidine blue the mucinous ground substance is so abundant and shows such intense metachromatic staining as to mask the characteristics of the mast cells. Tn addition to the sites mentioned, mast cells are present in varying quantities wherever there is connective tissue. On the whole, they appear to be abundant around organs which vary in volume or which move (vessels, ureters, urinary bladder, etc.) ; relatively high counts may also be found where there is ample fatty tissue. Parenchymal organs, such as the liver and kidneys, are rather sparsely supplied with mast cells. The digestive tract, mainly its subniucous and

408

G . ASBOE-HANSEN

FIG.2. Spnovial membrane of knee joint. Fixation: basic lead acetate 4%. Staining : Toluidine blue 1 O/oo (aqueous solution). Numerous mast cells along the surface. Figure 3 represents the framed field. ( A m . Rltmwzatic Diseases, 1950, 9, 154). FIG.3. T h e same preparation as figure 2 (framed field). Sgnoviab rlewzents including ten mast cells, four of which are marked : (1) densely granulated deep mast cell; ( 2 ) a more superficial mast cell, less saturated with granules; ( 3 ) in the syiiovial lining, a poorly granu!ated mast cell showing an even fainter staining; (4) mast cell containing no granules free in the joint cavity, identified by a faint rnetachromasia of its cytoplasm ( A m . Khezmafic Diseases, 1950, 9, 155).

T H E MAST CELL

409

410

G. ASBOE-HANSEN

subserous layers, is well supplied. In the tongue, as well as the gingiva, there is constantly quite a striking number. I n the urogenital tract, the highest counts are found in the bladder, ureters, and glans penis. The lungs also Iiold ample quantities. In the nervous system, mast cells are present wherever connective tissue is in evidence, eg., in the perineurium. The posterior lobe of the pituitary has been found to contain numerous mast cells (Gray, 1935). The thymus is generally claimed to be particularly rich in mast cells, but here again the mast-cell count is in proportion to the amount of connective tissue, the number increasing when involution sets in (Staemmler, 1921; Lehner, 1924). There are no mast cells in cartilage or bone, but in the perichondriutn and periosteuni they are numerous.

2. Pa tli olog ic Conii ect k e Tisst1e In certain pathologic conditions the number of mast cells in the connective tissue is increased. Real mast-cell tuinors have not been observed in man, but in dogs, mice, and horses ( SabrazGs and Lafon, 1908; Bloom, 1942, 1952; Deringer and Dunn, 1947; Oliver c t al., 1947). Man, on the other hand, may develop a skin disease, uvticarin piginentosa, cliaracterized by patchy accumulations of mast cells of all sizes and degrees of granulation in the connective tissue (Fig. 4 ) . At the corresponding sites melanotic pigment is seen in the basal epithelial layers and certain cells in the corium.

FIG.4. Urticaria pignientosa. Myriads of mast cells in the connective tissue. hfagnificatiotl: approu. 300 X . (G. Asboe-Hansen (1951) On the hlucinous Substances of the Connective Tissue. Rosenkilde 8: Bagger, Copenhagen).

THE hlAST CELL

411

This disease is characterized by, among other things, the formation of an urticaria1 wheal upon rubbing or scratching the surface of the elements. In rare cases it has been accompanied by skeletal changes such as cystic osteoporosis (Sagher et al., 1952), indicating that the disorder is a systemic disease. Rarely the disorder is accompained by cutaneous hemorrhages (Asboe-Hansen, 1950b). It is not known with certainty whether the mast cells play some role in inducing the symptom of itching. In patients with urticaria pigmentosa, neuroderniatitis, and urticarial dermographism, the writer ( 1950f) observed emission of the mast-cell granules. The formation of an urticarial wheal may be accompanied by local itching. In Cazal’s (1942) opinion scattered mast-cell granules act as antigens to which eosinophilic cells respond by multiplying. Cazal and-in 1953-Riley and West discuss the possibility that mast cells may produce histamine. This throws new light on the urticarial wheal as well as itching. An increase in the mast-cell count has been observed in a number of itching skin diseases, such as neurodermatitis, lichen planus, urticaria, Besnier’s prurigo, pityriasis rosea, acute as well as more chronic eczema. In fresh elements of Zupzts erythevtatosus and sderoderwza the writer ( 1951) observed considerable numbers where there is accumulation of metachromatic ground substance in the connective tissue ; the same applies to pempkigirs vidgaris, zdiligo (Marc, 1594), exfoliutive dennutitis (Bender, 1900) and scarlet fever (Sniirnowa-Zanikowa, 1926). In Bzisclzke’s scleroderma addtorum, Keining and Braun-Falco ( 1952) have reported accumulation of mast cells. In periarteritis iiodosu the vessets are surrounded by granulocytes, lymphocytes-and numerous mast cells (Altsliuler and Angevine, 1949), In p o i k i l o d w m atropllicans wsczdlure the latter cells are large and numerous (Asboe-Hansen, 1950f; Burkl and Leonhartsberger, 1951). Other diseases of the skin are accompanied by an accumulation of mast cells in the dermal connective tissue. This is true of wyxedetlza and quite particularly of circumscribed tmyxsdewia, a condition believed to be due to an increased activity of the thyrotropic hormone of the pituitary. In both conditions, the skin shows an accumulation of metachromatic mucinous connective-tissue ground substance, consisting mainly of hyaluronic acid, but also of chondroitinsulfuric acid (Watson, 1946 ; Watson and Pearce, 1947). There is an abundance of mast cells in the perivascular areas as well as free in the connective tissue. Although all degrees of granulation may be observed, most of the cells are well granulated, large, round, oval, or polyhedric (Ashoe-Hansen, 1950~). In many cases of Izyperthyroidisnz, small, spindle-shaped mast cells

412

G . ASBOE-HANSEN

abound in the perivascular areas, whereas only a few are found free in the connective tissue. There is little or no metachromatic substance in the connective tissue. Presumably the mast cells pray an iinportant role in the process of wound healing, because they seem to supply important substances to the matrix, the mother substance, of the connective tissue and because the formation of connective tissue is the first and most important process in wound healing (SylvCn, 1941 ; and others). In abnormal wound healing with kcloid formation, preparations stained with toluidine blue show extremely pronounced metachromasia. Part of this metachromatic substance is sensitive to hyaluronidase. There are numerous mast cells of varying but predominantly marked granulation (SylvPn, 1945 ; Asboe-Hansen, 195Of; Keining and Braun-Falco, 1952). The subepithelial zone of the connective tissue is excepted (Fig. 5 ) . These changes are most pronounced in keloids of fairly short standing. It is worth mentioning in this connection that keloids-spontaneous as well as trau-

FIG.5. Iieloid. Numerous mast cells and intense metachromasia, primarily in the deep layers. Rlagnification: approx. 150 X.

THE hfAST CELL

413

niatic-are often observed in patients with increased activity of the thyrotropic hormone-eg., in acromegaly. In graiiuloina pyogetiicit+n or t~lcangicctatirit~it the mast cells are outstanding cell elements in addition to fibroblasts, lymphocytes, and granulocytes. In a number of skin h ~ ~ i o rbenign . ~ , as well as malignant, mast cells may occur in great quantities. In skin carcinoma, the connective tissue exhibits, at some distance from the epithelial masses, strands, and islets, tnassive inetachroniasia with numerous mast cells, f orrriing at times a mantle around the tumor at the transition to normal tissue (Fromme, 19%; Staemmler, 1921; Bierich, 1922 ; Higuchi, 1930; Cranier and Simpson, 1944; Asboe-Hansen, 19501; and others). Cramer and Simpson (1934) and others have also found high mast-cell counts in the connective tissue in experimental skin cancer in mice (Fig. 6). The mast cells are particularly

FIG.6. Precancerous papillonla in the skin oi a mouse painted with carcinogenic hydrocarbon (9, lO-dimethyl-l,2-benzanthracene). Myriads of mast cells in the connective tissue.

414

G . ASBOE-HANSEN

plentiful at the stages preceding the development of carcinoma and in animals which proved relatively resistant to carcinoma. Other tumors may also be accompanied by an abundance of mast cells in the connective tissue. This applies, for example, to sarcoma, uterine fibroniyoma, fibroma, Recklinghausen’s neuro-fibroma, condyloma and lipoma. Wherever there is new formation of connective tissue, there are mast cells (Staemmler, 1921; Brack, 1925; Sylvkn, 1941; and others). This presumably explains their abundance in hepatic cirrhosis and chronic subcutaneous edema. Prakkeii and Woerdeniann, in 1952, ascertained that a high degree nf tissue eosinophilia is always accompanied by a marked increase in the nuniber of mast cells, but high mast-cell counts were also found in some conditions without tissue eosinophilia. I t is a fairly general experience that mast-cell counts are low in acute iil.flamiaations, whereas in chronic infections they are high (tuberculosis, syphilis). Besides other pathologic changes, the synovial membrane in patients with rheumatic fever and rheumatoid arthritis shows markedly increased mast-cell counts (Altshuler and Angevine, 1949 ; and others). According to Ragan and Meyer (1949) the content of hyaluronic acid in the articular fluid is increased in chronic rheumatoid arthritis, but it is depolymerized. Accumulations of mast cells have been reported in the eye-especially in the ciliary body and iris-in glaucomu, quite particularly glaucomatous iridocyclitis (Holingren and Stenbeck, 1940 ; Zollinger, 1949 ; AsboeHansen, 1950ej. Since the ocular fluids are rich in hyaluronic acid which binds water, the content of hyaluronic acid and its degree of polymerization might influence the intraocular pressure (Meyer, 1947). The number End functional state of the mast cells may perhaps be interpreted as the morpliologic evidence of the amount of water-binding plysaccharide in the eye. This view is supported by the depressing effect of cortisone on the intraocular pressure in glaucoma (Bardram) and its effect on mast cells (p. 427 j. Hamilton and Syverton ( 1950) found highly increased mast-cell counts in the heart in cases of rheumatic fever; at the same time intense metachroniasia of the ground substance was observed. Saphir (1950) reported accumulation of mast cells in non-rheumatic varieties of myocarditis. Normally, the bone fizmrow contains very few mast cells. They may, however, be present in large numbers in cases of aplastic and heinolytic aregenerative anemia of a chronic course, resistant to therapy and with a poor prognosis (Bremy, 1950). E’rimary amyZoidosis is often accompanied by an accumulation of mast

THE MAST CELL

415

cells in the affected tissues (Cazal, 1942). The mucopolysaccharide in amyloid is known to be an ester of monosulfuric acid containing equimolar parts of acetyl glucosamine and a uronic acid. It is not sensitive to hyaluronidase (Meyer, 1947).

V. CYTOCH EM ISTRY As early as 1877 Ehrlich tried to form an idea of the chemistry of the mast cells, and other workers after him have made great efforts in this respect in order to throw some light on the histophysiology of mast cells. At an early date some knowledge was reaped about the solubilitv properties of the mast granules. It is agreed that differences exist in the different species. In the rabbit, the granules are freely soluble-which may explain why many histologists have seen but a few mast cells in the rabbit connective tissue-and in the rat they are much less soluble in water. The solubility of the human mast-cell granules is somewhere between these two extremes. Nagayo (1928) found no marked solubility in water, ether, acids or alkaline solvents. Finding mast-cell granules sensitive to pepsin and trypsin, Nagayo could confirm Ehrlich’s observations and support numerotis subsequent workers’ assumptions that mast cells contain albumin, Werme1 and Sassuchin (1927) found that the granules of rabbit mast cells gave positive Millon’s xanthoproteic and biuret reactions and, in addition, they were stained with carmine and iodine. These authors concluded that the cells must contain a glucoprotein. Nakajima (1928) also believed that they must contain a glucoprotein; this view is supported, moreover, by dietetic experiments conducted by de GiorgiFerrari (1933). Having observed a positive Rest reaction, Arnold (19M) thought that mast cells contained glycogen. This gave rise to energetic protests, i n f o nlitr. frcm Lehner (1924)’ Nakajima (1928), Kirkman, (19SO), and Wislocki and Dempsey (1946). RTeJ-er (1W4), van HervITerden ( 1919), and others advanced histologic evidence that the granules contain nucleic acid. Zollinger ( 1950) was unable to demonstrate appreciaMe quantities of ribonucleoprotein and deosyribonucleoprotein in the granules. Ribonuclease or deoxyribonuclease does not affect mast granules (l$X&cki and Dempsey, 1946 ; Zollinger, 1950). Tn Pappenheim’s opinion ( 1908) the granules contain a niucdecitide. Many workers, including Hoper (1890), Harris (1900), and Staemmler (1921), ascertained that the granular substance must be related to mucin owing to the similarities in staining reactions. Mast cells stain positively with miicicarmine and niucihematin and metachromatically with various basic dyes, such as toluidine blue and thionine. Moreover, the mast granules stain metachromatically

416

G . ASBOE-HANSEN

with a large number of dyes, such as methylene azure, methyl violet, cresyl violet, neutral ethyl violet, brilliant cresyl blue, amethyst, acridine red, neutral red, pyronine, safranine, and azure (Michels, 1938). The blue dyes give a red metachromasia, the red ones a yellowish. So far, the cause of the metachromatic staining- reaction has not been definitely elucidated. Lison (1935, 1936) found that metachromasia with toluidine blue wab produced solely by high molecular esters of sulfuric acid of the type R-OS03H, counting chondroitinsulfuric acid, inucoitinsulfuric acid, and heparin-in other words sulfuric niucopolysaccharides. On the basis of Lison’s results and after Jorpes and associates had characterized heparin as a poly-sulfuric acid ester, Holmgren and Wilander ( 1937), Jorpes, Holmgren, and Wilander ( 1937), and Wilander ( 1938) claimed that the mast-cell granules contained heparin. Studying tissues rich in mast cells, such as the hepatic capsule of the cow, Holmgren and coworkers compared the content of poly-sulfuric acid ester with tissues poorer in mast cells, e.g. the pig or sheep liver capsule. These morphologic and chemical studies led to the theory that mast cells produce heparin (p. 425). The Swedish workers fixed their specimens in basic lead acetate, as it is known to precipitate heparin (Howell, 1928). By this means Holmgren (1938) believed that he could prevent the dissolution of the mast granules in aqueous media. The theory that the granules should contain heparin was, however, opposed by J u l h et al. (1950). On the basis of studies including differential centrifugation and electron microscopy, they maintained that the granules did not contain heparin, at least not in the form of an active anticoagulant and metachromatic substance. On the other hand, there is heparin in the intergranular cytoplasm, which contains the above-mentioned subinicroscopic microsomes. The intergranular substance is characterized as metachromatic and oxyphilous, water soluble, and consisthg mainly of a heparin-protein complex, of higher molecular weight and of greater anticoagulant power than commercial heparin. In other words, the substance differs from heparin isolated by the method of Charles and Scott. By heparin JulQ et al. evidently mean sulfuric mucopolysaccharide, i.e., esters of sulfuric acid in different degrees of esterification. The granules are believed to contain protein and lipoids as well as enzymes (vide i w f m ) . In a subsequent study-also including differential centrifugation-Snellman et al. (195 1) maintain that the heparin complex is made up of the polysaccharide heparin loosely bound to a low-molecular polypeptide component (containing only six amino acids) and a fatty component. Zollinger (1950) opposed the view that the metachromatic substance is contained in the intergranular cytoplasm, not in the granules, his contention

THE MAST CELL

417

being founded upon studies by the phase contrast microscope. The granules are claimed to contain “heparin” and to release this substance ilz zlitro if suspended in distilled water. Metachroniasia is no longer considered specific of poly-sulfuric acid esters as originally maintained by Lison. It is known that this staining reaction inay be observed in other substances as well. I n tissues, there is primarily a question of the closely related but nonsulfuric mucopolysaccharide hyaluronic acid (Blix, 1941 ; Wislocki e t al., 1947; Altshuler and Angevine, 1949; Bunting, 1950; Asboe-Hansen, 1950d, e ; SylvCn and Malmgren, 1952 ; Persson, 1953). Preparations of pure hyaluronic acid stain nietachromatically and the inucinous substances of the vitreous body and the synovial fluid stain rnetacliromatically without containing sulfur. Jorpes, \17erner, and dberg (1948) and Friberg et al. (1951) observed staining of mast cells with fuchsin-sulfite iollowing oxidation with periodic acid (by the method of McManus-Hotchkiss) . By this procedure it is possible to stain hpaluronic acid and mucoitinmonosulfuric acid, but not chondroitinsulfuric acid and heparin. From this the authors deduce that the substance contained in the mast cells is not heparin, but possibly its precursor. Friberg ef al. conclude that the metachromatic substance is in the granules and state that “the assumption that the metachromatic substance exists intergranularly is not well-founded.” The acidophily of the cytoplasm is taken as an indication against it. Like Jorpes et al., these authors refer to heparin as a mixture of di- and trisulfuric acid esters of a polymer, forming upon hydrolysis glucosamine and glucuronic acid. There is some difference of opinion about whether all mast cells are stainable with the periodic acid-leucof uchsin method. At any rate, some differences appear to exist between the individual species (Lillie, 1948, 1950; Kirkman, 1952 ; Compton, 1952). The periodic acid oxidizes polysaccharides to polyaldehydes. This enables one to disclose polysaccharides containing 1,Z-glycol groups or a similar structure in which the hydroxyl groups are replaced by amino or alkyl amino groups. I n tissues, these polpsaccharides are, among others, hyaluronic acid, glucuronic acid, glucosamine, and glycogen (Hotchkiss, 1948). Glegg et al. ( 1952) could not, however, stain a hpaluronic acid preparation in vitvo. As far as the mast cells are concerned, it has been shown that diastase digestion does not affect positive staining with the McManus-Hotchkiss method (Lillie, 1948 ; Leblond, 1950). No recent author has been able to demonstrate glycogen in mast cells. By autoradiography Asboe-Hansen (1953) demonstrated radioactive sulfur in tissue mast cells of experimental skin tumors in mice after injection of sodium sulfate labeled with S35. Yet some mast cells-granu-

418

G . ASBOE-HANSEN

lated and not differing histologically in any respect from the othersdid not contain radiosulfur (Figs. 7, 8, 9).

FIG. 7. Autoradiograph of an experimental skin tumor (mouse painted with 9,10-dimethyl-1,2-benzanthracene). Unstained preparation. Dark-field microscopy. Magnification : approx. 400 X . The hypcrplastic epithelium a t the top with deep extensions into the coriutn. In the connective tissue the mast cells present themselves as shining spots. The small dots indicate the diffuse blackening of the film caused by the radiosulfur contained in the connective-tissue ground substance. (Cancer Research, 1953, 13, 587).

Hyaluronic acid and the heparinlike anticoagulants are closely related. Both are polymers, built up of glucosamine and glucuronic acid (Jorpes and Bergstriim, 1936; Palmer et al., 1937; Stacey, 1943), and it seems likely that both are derived from the same precursor. Experimental sulfonation of hyaluronic acid and related polysaccharides (Balazs et al., 1951; Walton, 1952 ; and others) results in substances with highly anticoagulative proper ties.

T H E MAST CELL

419

From histochemical studies, chemical analyses, and autoradiographs it may be deduced that the mast cells contain a sulfuric mucopolysaccharide closely related to heparin and hyaluronic acid, but not identical with either. It is not known which chemical changes (desulfonation, sulfonation, or the like) take place before, during or after the passage from cell to connectivetissue ground substance. The metachromasia of the mast cells is not influenced by hyaluronidase treatment of fixed tissue (Davies, 1942-43 ; Wislocki et al., 1947; AsboeHansen, 195Od ; Compton, 1952 ; Prakken and Woerdemann, 1952). On the other hand, human mast cells react by degranulation when the hyaluronic acid in the surrounding connective-tissue ground substance is broken down by local injection of hyaluronidase in Vivo (Asboe-Hansen, 1950-51). This finding could net, however, be confirmed by Compton (1952) in experiments with the hamster. Compton used a different preparation of hyaluronidase and a different technique. I t is generally agreed that the mast granules are basophilic, i.e, stain with basic dyes. Wislocki e t al. (1947), staining with methylene blue and eosin following fixation in Zenker’s solution, pointed out that the basopliilia bears no relation to the degree of metachromasia. Thus, for instance, the jelly of Wharton, vitreous body, and synovial fluid, which stain metachromatically, do not possess basophile staining properties, unlike cartilage, certain mucous glands, and mast granules, which exhibit a marked affinity for methylene blue. This difference is believed to be due to the p H of the different substances, the sulfuric type which make up the latter group having an appreciably lower pH than the mucinous substances of the former group, which partly have a lower content of sulfuric acid (umbilical cord) and partly are nonsulfuric (vitreous body and synovial fluid). As mentioned above, Snellman p t al. (1951) noted the presence of lipids in the mast cells. The granules were found to contain lecithin, cholesterol, and neutral fats. Fat has been demonstrated histochemically (by Sudan black B) in the granules of occasional human mast cells (Wislocki and Dempsey, 1946; Wislocki e t al., 1947; Lillie, 1948; Montagna and Noback, 1948). According to Ciaccio (1913), mast cells do not normally hold lipoid granules, although the latter may be present in certain pathologic conditions. In tissue samples from the hamster, Compton (1952) found a negative reaction except in the granules of a few mast cells lying in the vicinity of fatty tissue. Montagna and Noback, using the Baker formol-calcium-cadmium technique, believed that the lipids present in the mast-cell granules consisted of phospholipids. Several enzymes have been found in the mast granules. A lipase content in the granules has been demonstrated by Montagna and Parks

420

G. ASBOE-HANSEN, M.D.

FIG.S. Autoradiograph. Sector of the connective tissue from the prrparatioii shown in figure 7. Light-field microscopy. T h e mast cells stand out as black patches ; the ground substaiicc gives diffuse blackening of the film. Magnification : approx. 1600 X. (Cancer Research, 1953, 13, 587). FIG. 9. Autoradiograph of a vessel with two mast cells in the immediate surrouridings. At the sites of the mast cells there is intense hlackening of the film. Staining : Toluidiiie blue, $/2kaqueous solution. Magnification : approx. 1800 X. (Cancer Research, 1953, 13, 587).

THE MAST CELL

42 1

422

G. ASBOE-HANSEN

(1948) and Montagna and Noback (1948) using the Gomori technique. Alkaline phosphatases are also claimed to have been demonstrated histochemically in mast granules (Noback and Montagna, 1946, Wislocki and Dempsey, 1946; Dalgaard and Dalgaard, 1948), and acid phosphatases too (Nohack and Montagna, 1946; Wislocki et al., 1947 ; Montagna and Noback, 1948). By means of the M-Nadi reagent, Montagna and Noback demonstrated stable cytochrome oxidase in mast-cell granules. Kirkman (1350) and Compton (1952) were unable to confirm this finding. Both the last-mentioned authors also found negative reactions with the G-Nadi reagent. Riley and West (1953) found a notable content of histamine in tissue mast cells.

VI. FUXCTION The function of the mast cells has for a long time been an object of intensive research. Numerous theories have been advanced. Those dating from the era before any knowledge of the chemistry o€ the mast cell may partly be disregarded. Still, they are worth mentioning, for historical reasons and because they have contributed to further research into an understanding of this important problem. The different notions regarding function are based on the distribution of the mast cells, their number and chemistry. 1. “Nzttrition” Ehrlich, in 1877, interpreted these cells as storing connective-tissue cells ; thus the term : Mastzelleiz. Many subsequent workers have supported the theory that the mast cells were important factors in nutrition. Their localization at the bases of the intestinal villi made Hardy and IVesbrook, in 1895, suggest that the “basophilic cells” play a synthesizing role during the absorption of the food through the intestinal niucosa. Many authors, including Michels (1923) have interpreted the mast cells as having something to do with food-storage and nutritional conditions. Bolton (1933), however, did not find sufficiently convincing evidence of this. I n this connection particular interest attaches to Nakajinia’s ( 1928) and Nagayo’s ( 1928) inanition and nutritional experiments. They found the mast-cell count in the tissues to vary according to the nutritional state.

2. Elastin Formation The assumption that mast cells form elastin (Loewenthal, 1904) is founded upon the observation that common staining reactions with toluidine blue and orcein exist. This does not, however, apply to all staining reactions (Bujard, 1930, h’akajima, 1928) - Bierich (1922) and Higuchi

THE MAST CELL

423

(1930) found a parallelism between the mast-cell count and elastic fibers in carcinomatous tissue. 3. Piqittentatioit The theory that mast cells form pigment was advanced by Rheindorf (NOS), Meirowsky (1908) and others. Meirowsky interpreted the effect of Fitisen rays upon the skin as follows: The nuclear substance of th? mast cells is appreciably increased, migrates to the cytoplasm, d i e r e it breaks into granules which become metachromatic, eventually to be transformed into pigment grains. I n Staffel’s (1907) opinion the mast cells contributed to the accuniulation of pigment in urticaria pigmentosa. According to Lehner ( 192-t), pigmented grains are never found in zii:po in the unstained mast cells Michels (1938) believes that the theory has arisen from technical errors, such as the transport o€ pigmented grains by the microtonie knife acros5 the inast cells of the histologic tissue slices.

4. Phugocytosis At intervals, it has been stated that mast cells possess macrophagic properties (Arnold, 1906 ; Weidenreich, 1908; Weil, 1919 ; Broclersen, 1928 ; and others), and many have interpreted the granules as phagocytized material. This theory has been opposed by Fahr (1905), Naka jima ( 192S), Michels ( 1938), and many others. 5. Defense The role of the mast cells in the defense system of the organism was aired as early as 1896 by Cajal and has later been discussed by a host of other workers. Fahr, in 1905, clainied that the mast cells showed negative and positive chemotaxis upon injection of virulent and now virulent bacteria and toxins respectively. In 195Og the writer suggested that iiiast cells might play a role in the defense against infection by the production of hyaluronic acid. The latter is an important component of the connective-tissue ground substance and is of decisive significance to the degree of permeability of the connective tissue and its resistance to bacterial and viral infection. Breakdown prodticts of the niast-cell polysaccliaride-such as acetyl groups, glucuronic acid, and sttlfuric acid-tnay also be iiiiagined to be factors in the local defense of the organism. AS mentioned ahove, mast cells are abundant in the connective tissue surrounding certain malignant tilntovs (spontaneous as well as induced). We cannot yet explain the purpose of their presence in these cases. Conflicting opinions have been expressed. The mast cells may destroy or

424

G. ASBOE-HANSEN

neutralize the presumed toxic substances of cancer (Fronime, 1906, and others) ; their proximity may be due to cheinotaxis (Higuchi, 1930) ; in producing mucopolysaccharide they may be imagined to counteract the spread of the tumor in the tissues by reducing the permeability of the connective tissue (Cramer and Simpson, 1944 ; Asboe-Hansen, 1950f). It may also be assumed that the sulfuric inucopolysaccharide of the mast cells acts as a hyaluronidase-inhibitor and thus counteracts the spread of hyaluronidase-producing growths (Boyland and McClean, 1935 ; Coman, 1946, McCutcheon and Coman, 1947). Heparin and similar polysaccharides possess an antihyaluronidase effect (McClean, 1942). Heparin inhibits the growths of tissue ceIls in vitro (Fischer, 1936). Stuart (1952) suggests that the mast cells act in the defense mechanism against alterations induced by anaphylaxis. 6. Relatiotz to Fat T o the mast cells have also been attributed the role of participating in some way or other in the new formation or metabolism of fat. They are present in abundant quantities in fatty tissue, among fat cells, of all shapes from globular to semilunar, wedged between the individual fat cells. Some authors (Lombardo, 1908, and others) have believed that mast cells could be transformed into fat cells. Lombardo also found the granules stainable with Sudan TI1 and osniic acid. Tuma (1928) observed that an otherwise distinct fat staining was lost if rats were avitaminosed. Studies on the effect of the thyrotropic hormone of the pituitary on connective tissue by Asboe-Hansen and Iversen (1951) and Iversen and Asboe-Hansen (1952) showed that at the same time as fat is mobilized from the normal depots there is an accumulation of mast cells and nen formation of hyaluronidase-sensitive mucopolysaccharide, evidently hyaluronic acid. The mast cells seem to lie in the fatty tissue in a state of preparedness under hormonal control and to be the origin of the hyaluronic acid. There seems to be an equilibrium-regulated by hormonal factorsbetween fatty tissue and the mucinous substance. This is probably also the explanation of Harma and Suomalainen’s (1951) finding of a marked increase in the mast-cell count in the hedgehog during hibernation.

7. Histamine Productioit Cazal (1942) suggested that mast cells might be the source of histamine. According to Graham’s finding, the basophile leucocytes of the blood contain large amounts of histamine; on this basis Dempsey (1952) discussed the possibility that tissue mast cells also contain and maybe produce histamine. Stuart (1952) observed rupture and disappearance of mast cells and

THE MAST CELL

425

extracellular dispersion of granules during anaphylaxis, changes which could be prevented by antihistaminics. In 1953 Riley and West found a relation between the number of tissue mast cells and the content of histamine and advanced the theory that mast cells produce histamine.

8. Mucopolysaccharide Secretion Harris ( 1900), observing conformity between the stainability with mucicarmine and niucihematin of mast cells and mucin, concluded that mast granules contained mucin and that the mast cells formed the mucinous substance of the connective tissue. Staemmler (1921) also believed that the mast cells-which he interpreted as unicellular glands-formed the mucin of the interfibrillar cement substance. Brack (1925) was in doubt about this theory, but was of the opinion that the mast cells participate in the growth of connective tissue, because they are present in larger numbers in young connective tissue than in older and because there are no mast cells in scar tissue. Because of common staining reactions with toluidine blue, Schaff er (1907) formed the theory that mast cells produce the chondroitinsulfuric acid of cartilage. In 1937-38 the Swedish workers Holmgren, Jorpes, and Wilander published investigations into the chemistry and function of the mast cells which surpassed all previous studies and theories. Holmgren and Wilander (1937) found that a tissue rich in mast cells has more pronounced anticoagulative properties than other tissues more sparsely supplied with mast cells. They referred to Lison, who had reported that metachromasia with toluidine blue is due to the presence of high molecular poly-sulfuric acid esters, and to papers by Jorpes and Bergstrom elucidating the chemistry of heparin (Jorpes, 1935; Jorpes and Bergstrom, 1936) and they stated that the “mast granules” contain heparin and that probably they give off an anticoagulative substance to the tissues and the blood. Sylv6n (1941) believed that the mast granules contain poly-sulfuric acid esters which they can release to the growing tissues. The investigations mentioned on p. 417, which showed that staining with toluidine blue is not specific of sulfur-esterified mucopolysaccharide but may be obtained with nonsulfuric kindred substances as well, and in part that human mast cells do not contain heparin but perhaps its precursor, have led us further on the road toward the elucidation of the function of the mast cells. Asboe-Hansen (1950-51) did not find a definite relation between the mast-cell count in tissues and the tissue content of sulfomucspolysac-

426

G . ASBOE-HANSEN

charides. O n the other hand, he found the mast-cell count to accord well with the amount of hyaluronic acid in tissue as determined by chemical and histochemical methods. The author studied normal skin, skin from patients with skin diseases and with internal disorders related to the endocrine system, umbilical cords , synoliial membranes, and eyes. Mast cells proved to be present wherever there is hyaluronic acid. In the presence of permanent increase in hyaluronic acid, the mast cells are increased in number and size. Conformity was observed between the stainability of mast-cell granules and the hyaluronidase-sensitive connective-tissue ground substance, the jelly of Wharton, the ocular fluids, and the synovia. There were signs of (compensatory?) concourse of mast cells from the vessels to connective tissue the hyaluronic acid of which had previously been broken down enzymatically. The mast cells appeared to emit granules iii this presumed secretory phase. A new formation of metachromatic substance seemed to take place in the areas abounding in mast cells. In the synovial connective tissue, mast cells showed various degrees of saturation with granules (different functional phases ?). The author concluded that the mast-cell granules contain mucopolysaccharides of heparin type and he advanced the following theory : Under hornzonal influence the mast cells secrete tlic wcesenchyvtal wzucopolysaccharide hyaluronic acid, perhaps by w a y of n heparinlike precursor. It is by no means impossible and indeed quite probable-that the mast cells are the origin of an anticoagulative sulfuric polysaccharide of heparin type as well as of a non-anticoagulative, sulfurfree polysaccharide (hyaluronic acid). This opinion has also been expressed by Prakken and J4‘oerdeniann ( 1952). Battezzatti (1951) and Hissard et al. (1951) also consider that the iliast cells supply the precursor of both, inclining to the view that hyaluronic acid is convertible into “heparin.” Campani (1951) finds it equally probable that the mast cells convert hyaluronic acid and other components of the ground substance into heparinlike substances. VII. PHYSIOLOGIC VARIABILITY Recent investigators have observed variations in the number, localization, distribution, shape, and granulation of the mast cells in various physiologic conditions and as a response to various experimental conditions. Similar changes are found in the connective tissue in cases of various endocrine disorders. In myxedematous tissue the number of mast cells is increased in the perivascular areas as well as free in the connective tissue. The cells are on the whole large and all more or less saturated with granules. Follow-

T H E MAST CELL

427

ing treatment with thyroid hormone the mast cells become small, often spindle-shaped and filiform, with sparse granulation, and occurring mainly in the perivascular areas (Asboe-Hansen, 195oC, e). The same occurs in thyrotoxicosis. They may at times be present in quite large numbers. No doubt many mast cells escape detection because they are degranulated and consequently no longer nietachromatic. The writer (195Oe) suggested that in fact it is the thyrotropic pituitary hormone which acts upon the connective tissue, and that the thyroid hormone acts by inhibiting this action. In animal experiments (guinea pig), Asboe-Hansen and Iversen (1951) found increased quantities of tnast cells and hyaluronidase-sensitive mucopolysaccharide in the connective tissue following injection of thyrotropic hormone. This action is observed after and simultaneously with mobilization of fat from the normal depots. The mucinous substance replaces fat transported by the ldood stream to the muscles, liver, and kidneys, where it accumulates. The development of exophthalmos during the administration of thyrotropic hormone is explained by an increase in volume of the retrobulbar tissue due to the capacity of hyaluronic acid to bind water. The thyroid-stimulating, exophthalmogenic, and fat-mobilizing power of the pituitary extract is inhibited by thyroxin (Iversen and Asboe-Hansen, 1952). Corticotropin and cortisone also act upon the mast cells. The adrenal cortical hormone cortisone inhibits the new formation of connective tissue and alters the fibroblasts in healing wounds (Ragan et al., 1949; Plotz et al., 1950). Since new formation of connective-tissue ground substance appears to be essential to the new formation of the connective tissue as a whole, and since several findings ( Asboe-Hansen, 1950g ; Kulonen, 1951) indicate that the quantity of hyaluronic acid in connective tissue decreases during adniinistration of cortisone and corticotropin, attention has been centered on the mast cells. In experiments with human subjects, rabbits, mice, and guinea pigs, the writer (19Xg, 1952b) found a decrease in the mast-cell count and certain niorphologic changes (cf. also Videbaek, et 01. 1950). True, some cells in the Connective tissue of individuals treated with corticotropin and cortisone seem to be quite normal, but most show signs of degranulation especially in the human material ; the granules are collected in aggregates of varying size, not as normally evenly distributed in the cytoplas~n. Some cells are truly vactiolized; the walls separating the vacuoles are made up of a homogeneous or partly granular substance which stains in part orthochroniatically and in part inetachromatically with toluidine blue. The cells have irregular outlines, a more or less ragged shape, and vary in size, although most are small. The tissue specimens used were fixed in 470 lead subacetate or freeze-dried. (Figs. 10, 11, 12, 13. Caval-

428

G . ASBOE-HANSEN

FIG.10. Normal mast cell from human skin before administration of cortisone. The fine granules are uniformly distributed throughout thc cytoplasm. FIG. 11. After admiiiistratiori of cortisone, 1800 mg. Mast cell of dermal connective tissue showing vacuolization.

T H E MAST CELL

429

FIG. 12. After administration of cortisone, 1800 mg. Another mast cell with granules distributed in major and minor clusters and lumps, staining more or less intensely. FIG.13. After administration of cortisone, 1800 mg. Mast cell with granules of irregular distribution, partly in the periphery, partly in lumps around the central nucleus. Vacuolization of the cytoplasm. Figs. 10, 11, 12 and 13: Staining %% toluidine blue solution, aqueous magnification approx. 3000. X. (Proc. SOC. Exp. Biol. Med., 1952, 80, 677).

430

G. ASBOE-HANSEN

lero and Braccini (1951) observed a reduction in the mast-cell counts in skin, muscles and myocardial tissue of the rat. Bloom (1952) found similar morpliologic changes and a reduction in the mast-cell count in malignant niastocytomas in dogs. Several workers have confirmed the effect of cortisone on mast cells (Stuart, 1951; Fulton and Maynard, 1953; Baker, 1953). Schoch and Glick (1953) could not confirm these observations. These authors used alcoholic fluids for fixing and staining, and the experimental animals were rats. So the different results may be due to the different methods. In tissue cultures of embryonic skin and in spleen cultures the activity of the mast cells is inhibited by cortisone (free alcohol) while the fibroblastic activity remains undisturbed (Paff and Stewart, 1953). Studying the effect of cortisone on experimental skin tumors in mice, Engelbreth-Holm and Asboe-Hansen ( 1953) observed a marked inhibition of tumor growth associated with alterations in the number and morphology of the mast cells. In those cases where tumors developed despite administration of cortisone, numerous mast cells were found in the connective tissue. Thus in some cases cortisone in moderate doses did not appear to be able to prevent the activity of the mast cells. Autoradiographic studies on experimental skin tumors in mice under the influence of cortisone showed a reduced uptake of radiosulfur in the mast cells after intraperitoneal injection of S"-labeled sulfate ( Asboe-Hansen, 1954a). Painting of the skin with coal tar results in a marked increase in the number of histologically demonstrable mast cells in the connective tissue. It is known that the connective tissue, primarily the ground substance, is affected in scurvy (Bunting and White, 1950; Persson, 1953; and others) and that ascorbic acid and dehydroascorbic acid influence these changes. No reports are known to the writer concerning the condition of the mast cells during avitaminosis, but this important problem is now the object of research. Hyaluronidase is presumably present in the skin in active or inactive form. Its activity in the tissues depends to a marked extent on endocrinologic factors (Opsahl, 1949a, b, c ; Asboe-Hansen, 1952a). Hyaluronidase exerts a certain effect on the granulation of mast cells. As mentioned on p. 419, it has been shown in experimental work that if hyaluronidase is injected into living tissue, the mast cells are degranulated, perhaps in an endeavor to re-establish stntzts quo, as hyaluronidase breaks down the hyaluronic acid in the connective-tissue ground substance. Other chemical alterations in the tissues also appear to produce degranulation. A single painting with phenol, benzene, and naphthalene is followed by degranulation of the mast cells in the dermal connective tissue (SylvCn and Larsson,

THE MAST CELL

43 1

1948). These authors mention the possibility that the granular substance takes part in the organism’s efforts of detoxification, X-irradiation is primarily followed by degranulation of the mast cells ( SylvCn, 1945), and some time later by a marked increase in their number. Degranulation may also be induced by a mechanical action upon the skin (Asboe-Hansen, 1951). W e do not know which factors are mobilized in the tissues by these actions. The morphologic changes induced by various physiologic and pathophysiologic factors indicate that the cell responds promptly-like a physiologic entity in the connective tissue-to endogenous and exogenous stimuli. VIII. REFERENCES Alfejew, S. (1924) F o ! k Harnaatol., 30, 111. Altshuler, C. H., and 24ngevine, D. M. (1949) Ant. I . Pathol., 25, 1061. Arnold, J. (1906) Miinclt. wed. LVochschr., 63, 585. Asboe-Hansen, G. (1950a) Bull. histol. ajpl. ct tech. microscop., 27, 5. Asboc-Hansel], G. ( 1950b) Acta Ue,.,iiato-T’c?aercoZ., SO, 159. Asboe-Hansen, G. (19.k) J . Iwesf. Dcmcafol, 15, 25. Asboe-Hansen, G. (1950d) AWI.Khetmatic Discascs, 9, 149. Asboe-Hansen, G. (1950e) A c f a Dcrniato-Yeno-eol., 30, 221. Asboe-Hansen, G. (1950f) Acta Derniato-VcBereo/., 30, 338. 2, 271. Asboe-Hansen, G. (19509) Srand. J . C h . G Lab. Ir~z~cst., Bindewvets Mucinfise Substanser. ( 0 1 1 the Mucinous Asboe-Hansen, G. (1951) 0111 Substances of Connective Tissue. English Summary) Rosenkilde i% Bagger, Copenhagen. Asboe-Hansen, G., and Iversen, Kurt (1951) Acfa Endocrhol., 8, 90. Asboe-Hansen, G. (1952a) Acfa Emfocritiol., 9. 29. Asboe-Hansen, G. (1952b) Proc. Sac. Esptl. Biol. N e d . 80, 677. Asboe-Hansen, G. (1953) Caiicer Research, 13, 587. Asboe-Hansen, G. (1954a) Cancer Researclt. 14. 94. Asboe-Hansen, G. (19S4b) Trans. 5th Conf. on Connective Tissues,Macy Foundation, New York. Audry, C. (1896) Mowtsch. prakt. Dmatol., 22, 393. Baker, B. L. (1953) t h i . N . Y . Acad. Sci., 56, 684. Balazs, E. A., Hljgberg, B., and Laurent, T. C. (1951) Acfa Physiol. Sramf., 23, 168, Bardram, hf., Unpublished. Bates, E. (1935) Anaf. Record, 61, 231. Battezzatti, M. (1951) Press€ me‘d., 59, 1628. Render, 0. (1900) Virckow’s Arch., pntltol. rlriol. 11. Pltysiol., 159, 86. Bensley, S. (1952) Anat. Record, 112, 310. Bierich, R. (1922) F ~ ~ Y C ~ W W ’ S Arch., jaflznl. And. If. Physiol., 239, 1. Blix, G. (1941) Cited by SylvPn and hIalingren (1952). Bloom, F. (1942) Arch. Pathol., 33, 661. Bloom, F. (1952) Prcac. Soc. Exptl. Biol. ~Mcd.,79, 651. Bolton, L. (1933) J . Mar-jhal., 64, 549. Boyland, E., and hlcClean, D. A. (1935) I . Pafhol. Bactcuiol., 41, 553. Brack, E. (1925) FoZia Hacmatol., 31, 202.

432

G. ASBOE-HANSEN

Brcmy, P. (1950) Die Gewebsniastzellen iin nienschlichen Knochenmark. G. Thieme, Stuttgart. Brodersen, J. (1928) 2. vzikroskop.-anaf. Forsch., 14, 60. Bujard, E. (1930) B i d . histol. appl. ct tech. microscop., 7, 264. Bunting, H. (1950) Ann. N.Y. Acad. Sci., 62, 977. Bunting, H., and White, R. F. (1950) Arch. Pathol., 49, 590. Burkl, W., and Leonhartsberger, F. (1951) Wicii klin. Wochschr. 63, 647. Cajal, R. Y . (1895) Rev. trimestr. n k r o g r . , 1, 83. Campani, M. (1951) Lancet, 260, 802. Cavallero, C., and Braccitii, C . (1951) Proc. Snc. Exptl. Biol. Med., 78, 141. Cazal, P. (1942) Un nouvel aspect de la nikdicine tissulaire: Ie reticulopathie. Vigot Frhes, Paris. Charles, A. F., and Scott, D. A. (1933) J . Biol. CIzenr, 102, 431. Ciaccio, C. (1913) Zcntr. allpnz. Pathnl. 11. pafhol. Anat., 24, 49. Coman, D. R. (1946) Am. J. Med. Sci., 211, 257. Compton, A. (1952) Ain. J . Anat., 91, 301. Cramer, W., and Simpson, W. L. (1944) Caircrr Research, 4, 601. Dalgaard, E., and Dalgaard, J. (1948) Ugeshrift Lreger, 110, 513. Davies, D. V. (1942-43) J . Anat., 77, 160. Dempsey, E. W. (1952) Trans. 3rd Conf. on Conncctive Tisszm, hlacy Foundation, New York. Deringer, M. K., and Dunn, T. B. (1947) J . Natl. Cancer Inst., 7, 289. Doan, C. A., and Reinhart, H. L. (1941) Am. J. Clin. Pathol., 11, 1. Downey, H. (1911) Anat. An:. Erg. Heft, 38, 74. Downey, H. (1913) Folia Haenzatol., 16, 49. Ehrlich, P. (1877) Arch. nzikroshob. Anat. 24. Eirtze.icBh~iigsmccIt., 13, 263. Engelbreth-Holm, J., and Asboe-Hansen, G. (1953j A c f a Patltol. Microbiol. Scatid., 32, 560. Fahr (1905) Virchozw's Arch. pathol. A l i a f . ZL. Pltysiol., 179, 450. Fischer, A. (1936) Protoplasma, 26, 344. Friberg, H., Graf, W., and Aberg, B. (1951) A c f a Patlzol. MicrobioL S c a d . , 29, 198. Frommc, F. (1906) Z e ~ t r Gyniikol., . 30, 1146. Fulton, G. P., and Maynard, F. L. (1953) Proc. Soc. Exptl. Bid. Med. s4, 259. de Giorgi-Ferrari (1933) Moilit. 2001. ital., 44, 260. Glegg, R. E., Clermont, Y., and Leblond, C. P. (1952) Stain Tcchrtol. 27, 277. Graham, H., cited by Dempsey (1952) Tratrs. 3rd Conf. o n Conncctiz~eTissztes, Macy Foundation, New York. Gray, J. (1935) J. Anat., 69, 153. Hale, C. W. (1946) Nature, 167, 802. Hamilton, T. R., and Syverton, J. T. (1950) A n t . J . Patltol., 26, 705. Hammar, J. A. (1894) Arch. nzikroskop. Ailat. 11. Eittwickbtiigsnleclz., 43, 266. Hardy, W., and Wesbrook, F. (1895) 1. Gcn. Phy.c!siol., 18, 490. Harma, R.,and Suomalainen, P. (1951) A c f a Physiol. S c a d . , 24. 90. Harris, H. (1900) Phila. Med. J., 6, 757. HellstrGm, B., and Holmgren, H. (1947) Svrnska LuBartidn., 11, 1. vanHcrwerden, hl. A. (1919) Folia Microbiol. (Delft), 6, 19. Herzog, G. (1916) Beitr. pathol. Anat. i ~ allgcnz. . Puthol., 61, 377. Higuchi, K. (1930) Folia Haematol., 41, 401. Hissard, R.,Moncourier, I.., and Jacquet, J. (1951) Ann. nzed. (Paris), 62, ,583. Holnigren, H. (1938) 2. wiss. Mikroskop, 65, 419.

T H E MAST CELL

433

Holmgren, H. (1946) A c t e Atfat., 2, 40. Holmgren, H., and Stenbeck, A. (1940) Acta Ophthalniol., 18, 271. Holmgren, H., and Wilaiider, 0. ( 1937) 2'. wiikroskop.-niiat. Forsch., 42, 242. Hotchkiss, R. D. (1948) Arch. Bioclzcwr., 16, 131. Howell, W.H. (1928) Bzrll. Joltits Hopkitis I l o s p . , 42, 199. Hoyer, H. (1890) Arch. wzikroskop. Anat. 16. Eittzcick1i1ragsniech., 36, 310. Iversen, Kurt, and Asboe-Hanseri, G. (1952) A c f a Eitdocriml., 11, 111. Janes, J., and McDonald, J. R. (1948) Arr-lz. Pafhol., 45, 622. Jorpes, E. (1935) Biochenc. J., 29, 1817. Jorpes, E., and Bergstrrjm, S. (1936) Iluppe-Sryler's 2'. ph>m'ol. Chenz., 244, 253. Jorpes, E., Holmgren, H , and Wilander, 0. (1937) 2. mikroskop.-anat. Forsrh., 42, 279. Jorpes, E., Werner, B., and Aberg, B. (1948) J . Biol. Chena., 176, 277. J u l h , C., Snellman, O., and Sylven, B. (1950) A c t a Physiol. S c a d . , 19, 289. Keining, E., and Braun-Falco, 0. (1952) Dcrtnatol. Wocksclz~.,126, 633. Kirkman, H. (1950) An$. J . Attat., 86, 91. Kirkman, H. (1952) Cited by Compton (1952). Kulonen, E. (1951) A c t a Physiol. S C ~ J24, L ~Suppl. . , 88. I.eblond, C. P. (1950) .4nz. J . And., 86, 1. Lehner, J. (1924) Ergeb. Ailat. Y . En~"cklzlllysgeschichte,25, 67. Lillie, R. D. (1948) Histopathologic Technic. Blakiston Co., Philadelphia. Lillie, R. D. (1950) Aitat. Record, 108, 233. Lison, L. (1935) Arch. biol. (Park), 46, 5%. Lison, L. (1936) Histochimie animale. MCthodes et probl6mes. Gauthier-Villars, Paris. Loewenthal, N. (1W4) Arch. mikroskop. Anat. 21. Entwicklztngsmcch., 63, 389. Loinbardo, E. (1908) Foliu Haenmtol., 6, 42. Marc, S. (1894) Virchoza's Arch. putlzol. Aiiat. u. Pltysiol., 136, 21. Rlaximow, A. (1904) Aeitr. patkol. Anat. 21. allgem. Pathol. 35, 53. Maximow, A. (1906) Arch. mikroskop. Anat. u. Entwkklungsnzech., 67, 680. Rfaximow, A. (190f) Folin Haematol., 4, 611. Maximow, A. (1913) Arch. mikroskop. A w t . u. Entm*cklungsmech., 83, 247. Maximow, A.,and Bloom, F. (1938) A Textbook of Histology. W. B. Saunders Co., Philadelphia. McClean, D. (1942) J . Patlzol. Bacterial., 54, 284. McCutcheon, M.,and Conian, D. R. (1947) Cafrcer Research, 7, 379. McManus, J . F. A. (1948) Stain Tcchttul., 23, 99. Meirowsky, E. (1908) Uber den Ursprung des melanotischen Pigmentes der Haut und des Auges. Klinkhardt, Leipzig. Meyer, A. (1904) Cited by Werniel and Sassuchin (1927). Meyer, K. (1947) Physiol. Revs., 27, 335. Meyer, K., and Chaffee, E. (1941) J. Bid. Chenz., 138, 491. Meyer, K., and Rapport, M. M. (1951) Science, 113, 596. Michels, N. A. (1922) Comfit. r e d . SOC. hiol., 86, 111. Michels, N. A. (1923) Cellule, 33, 339. Michels, N. A. (1935) Am. J . Almt., 67, 439. Michels, N. A. (1938) The mast cells. In Handbook of Hematology, Hoebei, New York. Montagna, W., and Noback, C. R. (1948) Anat. Record, 100, 535.

434

G. ASBOE-EIANSEN

Montagna, W., and Parks, H.F. (1948) A m t . Record, 100, 297. Nagayo, M. (1928) Zciztr. allgcwz. Pathol. zi. pathol. Anat., 43, 289. Nakajima, Y. (1928) Trans. J a j a n Pathol. Soc., 18, 150. Noback, C. R., and Montagna, W. (1946) A m t . Record, 96, 279. Oliver, J., Bloom, F., and Mangieri, C . (1947) J . E.rptl. Mcd., 86, 107. Opsahl, J. C. (1949a) Yale J . DioZ. Med., 21, 255. Opsahl, J. C. (194%) Yalc J . Biol. Med., 21, 487. Opsahl, J. C. (1949~) Yalc 1. Biol. Med., 22, 116. and Stewart, R. (1953) Proc. SOC.Exptl. Biol. hied., 83, 591. Paff, G. H., Pafade, G. E. (1952) J. Exjtl. Med., 95, 285. Palmer, J. W., Smyth, E. M., and Meyer, K. (1937) J . Biol. Chcnt., 119, 491. Pappenheim, A. (1904) Folia Hacmutol., 1, 165. Pappenheim, A. (1908) Folia Hnentafol., 5, 156. Persson, B. H. (1953) Dissertation, Studies on Connective Tissue Ground Substance. Uppsala. Petersen, H. (1930) I n Handb. niikroskop. Axat. M m c h . , 2, 664, 666. Plotz, C., Howes, E., Blunt, J., Meyer, K., and Ragan, C. (1950) Arch. Dermatol. u. Syphilis, 61, 919. Prakken, J. R., and Woerdemann, M. J. (1952) D m m t o l o g i c a , 106, 116. Ragan, C.,Howes, E., Plotz, C., Meyer, K., and Blunt, J. (1949) Proc. SOC.Expfl. Biol. Mcd., 72, 718. Kagan, C., and Meyer, K. (1949) J. Clin. Invest., 28, 56. Xanvier, L. (1893) Compt. r e d , 116, 234. Nheindorf, A. (1905) Folia H ~ ~ ? J z2, u 821. ~o~., Riley, J. F., and West, G. B. (1953) J. Plzysiol., 120, 528. Sabrazss, J., and Lafon, C. (1908) Folia Hatwatol., 6, 3. Sagher, F.,Cohen, C., and Schorr, S. (1952) J . Inzvst. Dcrinafol., 18, 425. Saphir, 0. (1950) Anz. J . Pathol., 26, 706. Schaffer, J. (1907) Zattr. Physiol., 21, 258. Schoch, E.,and Glick, D. 1953) J . Itwest. Dcrmatol., 20, 119. Smirnowa-Zamkowa, A. (1926) Virchow’s Arch. pathol. Anat. ti. Physiol., 261, 191. Snellman, O.,SylvCn, B., and Julkn, C. (1951) Biochitn. et Biophys. Acta., 7, 98. Stacey, M. (1943) Chemistry & Industry (Rev.), 62, 110. Staemmler, M. (1921) Fratztfiwt. 2.Pathol., 26, 391. Staffel (1907) Folia Hammtol., 3, 576. Stockinger, W. (1927) Z.Zellforsch, 6, 27. Stuart, E. G. (1951) Anat. Record, 109, 351. Stuart, E. G. (1952) Anat. Record, 112, 394. SylvCn, B. (1940) Acta Radiol., 21, 206. Sylven, B. (1941) Acta Chir. S c a d . , 86, SnppI. 66. Sylvh, B. (1945) Acta Radiol., 26, Suppl. 69, 5. Sylvkn, B. (1950) Exptl. Crll Kcscarch, 1, 492. SylvCn, B., and Larsson, L. G. (1948) Caizcer Research, 8, 449. SylvPn, B., and Malmgren, H. (1952) Lab. Invest., 1, 413. Tuma, V. (1928) Compt. rcizd. Assoc. An&., 23, 455. Turk, W. (1904) Vorlesungen iiber klinische Haematologie. Braunnidller, IVien. Undritz, E. (1946) Schw&. nted. Wockschr., 76, 88. Unna, P. G., and Schumacher, I. (1925) Lebensvorgange in der Haut der Menscheti und der Tiere, Leipzig.

T H E MAST CELL

435

Vaubel, E. (1933) 1. Enptl Med., 68, 63. Videbaek, Aa., Ash-Hansen, G., Astrup, P., Faber, V.,Hamburger, C., Schmith, K.. Sprechler, M.,and BrBchner-Mortensen, K. (1950) Acta Endocrinol., 4, 245. Walton, K. W. (1952) Brit. 1. Pharnucol.. 7 , 370. Watson, E. M. (1946) Carl. ,%fed. Assoc. J., 64, 260. Watson, E. M., and Pearce, R. H. (1947) Brit. J. Dermatol., 69, 327. Weidenreich, F. (1908) Folia Haematol., 5, 135. Weil, P. (1919) Arch. mikroskop. Am:. u. Entwicklicngsmech. 93, 1. Wermel, E. M.,and Sassuchin, D. (1927) 2. Zellforsch., 6, 424. Wilander, 0. (1938) S&and. Arch. Physiol., 81, Suppl., 15. Williams, H. (1900) Anr. J . Med. Sci., 119, 705. Wislocki, G. B., and Dempsey, E. 1%'. (1946) Anat. Record, 96, 249. Wislocki, G. B., Bunting, H., and Dempsey, E.W. (1947) Am. J. Anat., 81, 1. Zollinger, H. U. (1949) Ophfhalmologica, 117, 249. Zollinger, H. U. (1950) Experientiu, 6, 384.

This Page Intentionally Left Blank

Elastic Tissue EDWARD W. DEMPSEY AND ALBERT I. LANSING Department of Anatomy, Woshington University School of Medicine, St. Louis, Missouri I. 11. 111. IV. V. VI.

Introduction ......................................................... Chemistry of Elastic Tissue .......................................... Staining Reactions and Histochemistry of Elastic Tissue ................ Intrafibrillar Architecture of Elastic Tissue .......................... Age Changes and Pathology of Elastic Fibers ........................ References ..........................................................

Payr

437 438 442 445 451 452

I. IXTRODUCTIBX Although Haller appears to have been the first to distinguish the elastic recoil of tissues, it remained for microscopists to recognize the specific elements now called elastic fibers. Henle (1841) first described the elastic lamellae of blood vessels and recognized both that they were different from white fibrous or collagenous tissue and that they were elastic in nature. Kolliker (1850), describing his own experiments, reviewed also the accumulated observations to that date. Von Ebner (1870) seems to have been the first to use dyes to achieve selective staining of elastic tissue. The staining characteristics, after the introduction of Weigert’s ( 1898) resorcin-fuchsin method and Tanzer’s ( 1891) orcein stain, have become, for practical purposes, the means of defining elastic fibers. In the extensive literature pertaining to elastic tissue, its formation, distribution, physical and, chemical characteristics, and pathologic changes, two contributions stand out as landmarks. The first of these is Mall’s extensive study of the properties characterizing elastic, collagenous, and reticulated fibers (Mall, 1W) and his later description of the formation of these fibrous elements (Mall, 1 x 2 ) . The second is the comprehensive review by Hass (1939a, b) followed by a series of papers on the physical and chemical changes in pathologic states (Hass, 1942a, b ; 1943). In these papers the distribution, ontogenetic and phylogenetic development, and degeneration of elastic tissue are presented so well that today only minor footnotes can be added. Except for occasional comments, therefore, these subjects will not be reviewed here; the reader is referred to the treatises cited above. For the purpose of definition, elastic tissue may be described as a system of fibers or fibrils, freely anastornosing with one another into a reticular network, occurring always in association with collagenous or reticulum fibers. The elastic fibers are nearly colorless early in the life of the 437

438

EDWARD W. DEMPSEY AND ALBERT 1. LANSING

individual; later they assume a yellowish tint deepening to brown in old age, from which fact the term yellow elastic tissue is derived. The elastic fibers stain selectively with resorcin-fuchsin and orcein, are heavily stained by hematoxylin in the Harris and Verhoeff procedures, but have normally only slight affinity for acid or basic dyes. With minor exceptions, such as the slight staining of cartilage matrix, mucus, eleidin, and mast cell granules with resorcin-fuchsin and the moderate coloration of nuclei with orcein, these staining procedures are operationally specific for elastic tissue ; that is, any fibrous material stained by them is regarded as elastic tissue, whereas any fiber failing to stain is believed to be non-elastic. It should be added, however, that newly forming fibers, recognizable by their refractility and their netlike pattern, put in their appearance in embryos before they acquire their charicteristic selective affinity for elastic tissue stains. Morphologists have recognized for many years that not all elastic tissues are alike, but that variations in a basic pattern exist. Thus, the elastic fibers of loose connective tissues such as the dermis or the connective tissue of mesenteries consist of fine fibers, 0.5 to 1.511 in diameter, which frequently branch in an anastomosing pattern so that no loose ends are ever visible. The individual fibers pursue relatively straight courses from one branch to the next; consequently the entire structure appears to be in a stretched condition. In elastic tendons such as the ligamentum nuchae this reticular pattern is modified. Large fibers, up to 4 or 5 p in diameter, pursue parallel courses. Careful observation reveals, however, that these parallel fibers branch and anastomose, so that the ligament can be regarded as a reticular net stretched between its two ends, in which the individual fibers are greatly hypertrophied. A third morpholhgic situation also exists as exemplified in the lamellae of the elastic tunics of large blood vessels. Here the tissue exists as fibrils dispersed within flattened plates or lamellae, in which frequent holes or fenestrations appear. Such a structure can be regarded as derived from a stretched reticular net in which the parallel fibers have hypertrophied along their lateral borders, or in which a platelike matrix has filled in the spaces of the net (cf. Dees, 1923. Other minor variants of this basic three-dimensional network also occur, as for example in the cufflike junction between elastic fiber and muscle cell which is common in all smooth muscles, and in the striated muscles of the face which attach to soft tissues. 11. CHEMISTRY OF ELASTIC TISSUE

The early chemical studies on the composition of elastic tissue were greatly hampered by three major difficulties. Elastic fibers everywhere

ELASTIC TISSUE

439

are associated with collagenous and reticulum fibers. These latter fibers must be separated from the elastic fibers before meaningful analyses can be performed. This separation has turned out to be quite difficult. Conheim’s original data on aniino-acid composition are certainly derived from material contaminated with collagen. Astbury’s ( 1940) attempt to determine the X-ray diffraction spectrum of elastin was defeated by the collagen pattern present in his best specimens. Recently, Gross (1950) reported a preparation of elastin from aortas by a method which, when repeated in this laboratory, yielded as an apparent elastin content 93% of the original dry weight of the whole organ. Obviously, such preparations are contaminated by other components. A second difficulty hampering chemical investigation of elastin concerns its insolubility and lack of reactivity. Except for its affinity for certain selective stains, to be discussed later, elastin appears to have singularly few reactive groups. As a consequence, analytic methods have been employed which are so drastic as to involve total hydrolysis of the protein into its constituent amino acids. Lastly, a third difficulty concerns the apparent fact that elastin, from different sources and even from the same source at different ages, has different compositions. Later sections of this review will discuss the altered calcium and amino-acid contents of elastin which occur upon aging. The insoluble albuminoid scleroprotein, elastin, can be prepared from fresh tissues by procedures which destroy other components (Lansing, Kosenthal et al., 1952). Although no criteria for chemical purity are yet available, it is possible to prepare reproducible elastin fractions. Fresh or frozen aorta or ligamentum nuchae may be defatted by refluxing in methanol or ethanol for 1 hour, followed by a second hour in acetone. Collagen and other elements may then be removed by a modification of the procedure of Lowry et al. (1941) involving hydrolysis in hot tenth normal NaOH for at least 45 minutes, by which time a constant dry weight is achieved. During this hydrolytic procedure, aliquots removed at various times and analyzed by paper chromatography for their amino acids demonstrate that tryptophan decreases progressively until only a trace remains at 45 minutes. This amino acid, a significant component of most proteins but absent in elastin, can therefore be used as a rough index of contamination in elastin. Likewise, hydroxyproline can also be used as an index of purity, since collagen is rich and elastin poor in this amino acid (Table I). Dried elastin, prepared as outlined above, preserves many if not all of its normal properties. Microscopically it remains as a fibrous network, with no detectable collagen or muscle when stained with Mallory’s tri-acid

440

EDWARD W. DEMPSEY A N D ALBERT I. LA N SI N G

procedure. The elastin fibers are optically homogeneous and highly refractile, and are not digested in solutions of crystalline trypsin. The fibers have a refractive index of 1.534. When stretched or dried, the fibers become strongly birefringent, but in their hydrated and relaxed state they are isotropic. They stain readily by the usual elastic tissue stains, orcein, and resorcin-fuchsin and by Verhoeff’s procedure. Inorganic constituents, such as calcium and phosphorus, vary with the age of the individual and the anatomic source of the sample. These constituents will be discussed separately. After removal of these elements by extraction overnight in tenth normal formic acid, the residual purified elastin contains no detectable sulfur. The nitrogen content has been found to be 15.6 to 16.0% in ligamentum nuchae and averaged about the same in elastin from blood vessels (Lansing, 1951). These figures are comparable to those obtained from other proteins and indicate that elastin, as prepared in this manner, contains only small amounts of other substances. Complete hydrolytic procedures carried out on elastin prepared from various sources indicate a varying amino acid composition. Table I summarizes the relative amounts of amino acids, as determined with microbiologic assay procedures by Lansing, Roberts, et al. (1951) and amplified by unpublished data from this laboratory. Qualitatively similar but quantitatively less accurate observations have been made using twodimensional paper chromatography. The results cited above are somewhat at variance with several reports on the analytic composition of elastin. For example, Hall et al. (1952) present data indicating that elastin has a carbohydrate content of about 2%. Banga and Schuler (1953) also report the presence of carbohydrate in their preparations, in quantities comprising 10 to 12% of their elastin preparation. They suggest that elastin is a glycoprotein and that its fibrous structure is due to the carbohydrate moiety. I t is difficult to see how a glycoprotein, having 12% of its weight represented by carbohydrate, could have a nitrogen content as high as the 16% reported by Lansing (1951). Using the anthrone reagent of Loewus (1952), only traces of carbohydrate were detected in elastin derived from human aortas and none in material prepared from the ligamenturn nuchae of the horse (Lansing, Rosenthal, et al., 1952). Such conflicting data suggest strongly that the elastin preparations which contain large amounts of carbohydrate are contaminated by collagen and its associated connective-tissue ground substance. Moreover, our results indicate that the fibrous structure of elastin is independent of its carbohydrate content, since after NaOH hydrolysis extensive enough to reduce carbohydrate to zero, the elastin was still fibrous, possessed its usual staining characteristics, and was elastic when stretched,

441

ELASTIC TISSUE

A new tool for investigating the chemical and morphologic structure of elastin has come from the discovery of an elastolytic enzyme by Ba16 and Banga (1949, 1950) which has recently been prepared in crystalline form (Banga, 1952). This enzyme is prepared from crude pancreatin by TABLE I AMINOACID COMPOSITION OF COLLAGEN AND (gN per 100 g. protein N )

ELASTIN

'

Amino Acid

Total N Glycine Alanine Leucine Isoleucine Valine Phenylalanine Tyrosine Tryptophan Serine Threonine Cystine Methionine Prolinc Hydroxyproliiie Lysine Hydroxylysine Arginine Histidine Aspartic acid Glutamic acid Amide N Total found

Collagenl Elastinh (Cowhide) (Ligamentum nuchae) 18.6 26.2 9.5 3.22 2.12 3.4 4.2 1.4 0.0 3.4 2.4 0.0 0.8 15.1 14.0 4.5 1.3 8.8 0.8 6.3 11.3

0.66 119.00

Elastine (Young) (aorta)

17.1 29.9 18.9 8.7 4.0 17.4 5.0 1.61 0.01 .82 .96 0.15 0.03 17.0 2.0 0.39

15.71 26.10 23.18

0.89 0.07 0.63 2.1 0.04 127.70

1.78 0.15 0.38 1.83 2.90 90.83

-

4.52 2.10 13.00 1.73 1.45 0.06 0.29 0.65

0.06 0.06 10.10

0.49

-

Elastin@ Elastine (Old) (Pulmonary) (aorta) (aorta)

-

15.56 21.30 21.58 4.76 2.33 11.50 1.97 1.76 0.24 0.70 1.13 0.10 0.35

16.00 32.00

9.20

11.0

1.17 4.35 0.75 1.11 3.01 2.86 90.17

-

4.78 2.28 12.9 _ .

-

__ __ --_ .

__

- ._ __

0.62 1.75

1.51 82.03

Bowes, J. H., and Kenton, R. H. (1948) Biochem. I. (London), 43, 358.365. Neuman, R. E. (1949) Arch. Blochem., 24, 289-298. C Lansing, A. I., Roberta, E., Ramasarma, G. B., Rosentbal, T. B., and Alex, M. (1951) Proc. SOC. E x p t l . BioI. Med.. 76, 714-717. a

b

successive salting-out procedures. Elastic tissue, or purified elastin, is rapidly rendered soluble by the enzyme. The soluble material derived from the action of elastase does not contain free amino acids, The tryptic activity of the elastase is very low or absent, depending upon the degree of its purification. Banga (1951) believes the enzyme converts elastin from a fibrous to a globular and therefore soluble form; however, more recent information indicates that some degradation does occur. During solubiliza-

442

EDWARD W. DEMPSEY AND ALBERT I. LANSIHG

tion of elastin, small droplets form from the elastin fibers and, upon standing, rise to the top of the solution to form a creamlike layer. The separation of this layer can be hastened by centrifugation, after which it may be collected as an oily layer floating at the top of the centrifuge tubes. Analyses of this layer indicate it comprises from 0.5 to 1% by weight of the dry, defatted elastin, and that it apparently is a sphingomyelinlike substance. Banga (1951) has also noted this oily substance in solubilized elastin. In his experiments, the addition of 15% trichloracetic acid to solubilized elastin caused a yellow, oily precipitate to form. Elastin has also been solubilized, and the properties of the soluble material have been studied, after treatment with oxalic acid (Adair et al., 1951) and strong solutions of urea (Hall et al., 1952).

111. STAINING REACTIONS AND HISTOCHEMISTRY OF ELASTIC TISSUE The identification of elastic fibers by histologic procedures does not present special problems. The fibers are generally accepted to be yellow, optically homogeneous, and refractiie, to form anastomosing networks, and, unlike collagenous fibers, to be unaffected by dilute acids. They stain selectively with orcein and resorcin-fuchsin, and may also be colored by a number of other procedures which are less selective, namely, Verhoefi's hematoxylin, Nile blue sulfate, basic fuchsin, osmic acid, Sudan black and the McManus periodic acid-Schiff reagents. They are ordinarily very lightly stained by acid or basic dyes. I n recent years, staining has been considered as an example of the physical-chemical binding of dye to substrate. Attention has been given to the conditions under which staining will or will not occur, with the aim of characterizing the staining reaction so that the bonding force between dye and structure may be identified. Thus, the Sudan dyes are thought to color lipids by solution forces; acid and basic dyes react with tissue structures under conditions of pH and ionic strength which indicate that salt-linkages or ionic forces are involved, whereas certain chromophobic substances such as elastic fibers are stained only under conditions in which ionic forces are absent or at a minimum. The analysis of acid-base reactions with dyes began with Ehrlich (1885) and has recently been summarized with respect to histologic staining by Singer (1952). The conditions under which orcein reacts with elastic fibers have been described by Dempsey, Vial, et al. (1952) ; Engle and Dempsey (1954), and Weiss (1954). The common staining reactions of elastic fibers are summarized in Table 11. The processes of fixation, embedding, and sectioning have relatively little effect on elastic tissue staining. Elastic fibers exhibit extraordinarily

443

ELASTIC TISSUE

little affinity for acid or basic dyes, regardless of the pH at which staining is carried out. Moreover, unlike most other tissues, oxidation with reagents such as periodic acid does not markedly increase the slight basophilia of elastic fibers. This observation is in accord with the low sulfur content of elastin, since the tissues exhibiting strong basophilia after oxidation are those with a high sulfur content, in which presumably the sulfide and TABLE I1 STAINING CHARACTERISTICS OF ELASTIC TISSUE Horse ligamentum nuchae

Stain

+ + +

Verhoeff Resorcin fuchsin Orcein

Human arterial elastic tissue

Rat loose connective tissue

H W ~ dermal elastic tissue

++ ++ + or ++ +

++

++

red

mixed, red and yellow

+

+

Nile blue sulfate

++

Congo red

red

young is red old is yellow

Schiff reagent

-

-

Periodic acid and Schiff reagent Mineral content after rnicroincineration

+

weak

+in periphery of lamellae

-

- Young

+

Osmic acid

weak

Sudan IV

+

weak young is dense blue, old is faint blue

Victoria blue

Sudan black

++ old

+

+

weak

weak

-

-

+ = positive ++ = strong positive

- = negative

-1-

+

+'+ + or ++ +

weak

+

variable

+ +

weak

+ +

444

EDWARD W. DEMPSEY AND ALBERT I. LANSING

sulfhydryl groups have been oxidized to sulfonic acids (Dempsey, Singer, and Wislocki, 1950). The affinity of elastic fibers for orcein is unaffected by pH over a wide range. Above p H 8.5 the orcein solution changes to a blue color and staining fails, but below this point the uptake of dye is almost unaffected by pH. In addition, with acid-base dyes, the best staining occurs in aqueous solutions and dye uptake is depressed by the addition of alcohol to the staining bath, whereas with orcein the reverse is true. Elastic fibers stain best from acid-alcohol solutions. These observations indicate that ionic forces play very little part in the uptake of orcein by elastin. Other forces must therefore be invoked to explain this type of selective staining. Elastic fibers stain, albeit rather variably, with various modifications of Schiff’s leukofuchsin procedure. Fresh elastic fibers from most rodents stain lightly with Schiffs reagent; the intensity of staining is greatly increased by prior oxidation in periodic acid (Hotchkiss reaction). The simplest explanation of these phenomena is that elastin contains free and potential aldehyde groups which react with leukofuchsin. Since orcein is a phenolic compound, and since phenols and aldehydes condense in reactions such as that in which bakelite and other artificial resins are formed, the thought occurred that these aldehyde groups might be the site of linkage with orcein. A further point supporting this thought is that oxidants have long been known to enhance the selective staining of elastic tissue. The function of the oxidants might thus be explained by their converting potential to free aldehyde groups which could then bind greater quantities of dye. Nevertheless, attractive though this theory may be, it is wrong. The aldehyde groups, with or without prior oxidation, may be bound irreversibly with phenylhydrazine or semicarbazide without interfering in any way with the capacity of the fiber to bind orcein (Deinpsey, Vial, et aE., 1952). Commercial preparations of orcein, whether derived from natural sources or by synthetic processes, contain a mixture of dyes which may be separated by paper or column chroniotography. Four colored dyes and one colorless but fluorescent substance have been isolated (Engle and Dempsey, 1954). When tested in a paper electrophoresis apparatus for their net charge, one fraction, having a blue color, migrated toward the negative pole, whereas the other fractions moved toward the anode or did not migrate at all. In staining tests, elastic tissue was stained almost equally well by the fractions carrying either the negative or the positive charge. This observation is in good accord with those mentioned above which indicate that orcein staining is relatively independent of p H and that the best staining occurs under conditions of minimal net charge of the protein.

ELASTIC TISSUE

445

Altogether, these observations suggest strongly that hydrogen bonding, rather than ionic forces, determines the uptake of orcein by elastin. Further support of this hypothesis has come from experiments by Weiss (1954) in which the energy of the orcein-elastin complex has proved to be about 6,000 calories per mol, a figure which agrees well with the energy of hydrogen bonds but is too low for ionic forces. As mentioned above, elastic tissue and elastin exhibit a marked aflinity for phenolic compounds. Since orcein and resorcin-fuchsin both are phenolic derivatives, the selective staining capacities of these dyes could be explained by the bonding of phenolic to elastic complexes. This concept was tested by Dempsey, Vial, et al. (1952). Sections of ligamentum nuchae were exposed to solutions containing phenol, naphthol, and naphthoic derivatives. After this exposure the slides were washed and stained with orcein. Phenol depressed the uptake of orcein perceptibly, a-naphthol moderately, and naphtholsulfonic acid and naphthylamine strongly. Thus it would seem that these substances compete with orcein for reactive sites upon the elastin molecule. Furthermore, these blocking experiments demonstrate that ionic reactions are inconsequential in elastin. This statement is derived from the fact that naphtholsulfonic acid, a negatively charged compound, and naphthylamine, which carries a positive charge, are both effective blocking agents. Indeed, it can be shoan that the negative charge of naphtholsulfonic acid is not involved in its reaction with orcein, since elastic fibers which normally are not stained with basic dyes become strongly basophilic after treatment with the sulfonic naphthoic substance. ARCHITECTURE OF ELASTIC TISSUE IV. INTRAFIBRILLAR In recent years, much attention has been given to the macromolecular organization of fibrous structures. Silk, keratin, collagen, cellulose, and other fibers have been extensively investigated with the electron microscope, the polarizing microscope, by their X-ray diffraction spectra, and by other methods which permit observations or deductions concerning their “ultrastructure” or “fine structure.” Elastin and elastic fibers have not been SO extensively studied as have the other fibers, perhaps because elastic tissue has not been of any particular commercial importance. The observations to be reported in this section, therefore, are scanty and incomplete, and point toward the need for much more extensive study. Elastic fibers as encountered in spreads of fresh tissue or in histologic sections are highly refractile and have sharply defined margins. When examined with a polarizing microscope they are isotropic in their re1a:xed condition, but if stretched they become markedly birefringent. Elastin,

446

EDWARD W. DEMPSEY AND ALBERT I. LANSING

prepared from defatted elastic tissue and freed from collagen and other components by extraction with hot sodium hydroxide, assumes, when dry, an irregular granular form which also is birefringent. Dried granules of elastin may be hydrated by prolonged soaking in water, or more rapidly by exposure to concentrated solutions of organic acids such as tartaric acid. The rehydrated elastin reveals fibrous structure when teased ; these fibers are isotropic unless stretched, when they become strongly anisotropic. The Wiener (1909) theory of anisotropism states that asymmetric micelles, one dimension of which is small in relation to the wave length of the incident light, are isotropic when randomly oriented but become anisotropic when oriented with their long axes parallel to one another. Frey-Wyssling ( 1948) has discussed the optical behavior of reticular or randomly-oriented asymmetric fibrils when placed under tension, and has shown how stretching of such a system leads to an increased orientation, much as the strands of a fish-net become parallel to one another if the net is stretched from two diagonally opposite corners. Such an explanation suffices to describe the behavior of elastic fibers. Submicroscopic fibrils, randomly oriented in the relaxed state, could be arranged in parallel arrays by stretching or by drying. This explanation postulates, therefore, that the intrafibrillar architecture of elastic fibers consists of submicroscopic elements, oriented at random to one another. This postulate will be examined further in a following section devoted to electron microscopy. The homogeneous, refractile elastic fibers seen in normal fresh tissues may be altered in certain pathologic states and experimental situations. Such altaations may be explained most conveniently by assuming a submicroscopic architecture of a particular kind. In aged skin and in the elastic tunics of old blood vessels, fractured or split fibers are frequently encountered. Such pictures are best explained by assuming cleavage planes in the axis of fracture. The presence of such planes would imply either a prevailing crystalline pattern in the plane of cleavage, or a region of attenuated elastic material in the same plane. Similar observations have been made on elastic fibers during digestion in elastase solutions (Lansing, Rosenthal, et al., 1952). Early in the digestion process, the glassy refractility of the fibers is lost, and small indentations begin to occur along the external cylindrical surfaces. Somewhat later, the smooth cylindrical structure changed to appear as a pair of loosely twisted fibrils. Each of these two fibrils in turn split into two more; beyond this point the process of digestion proceeds very rapidly by fragmentation and dissolution of the small fibrils. Elastic fibers from formalin-fixed tissues are digested in a similar manner, except that frequently transverse breaks appear in the center of the fiber, producing

ELASTIC TISSUE

447

an appearance similar to that of a string of beads encased in a transparent cylinder. These results, together with the observations that the circumference of elastic fibers may stain differently from the center (Mall, 1896), lead to two general statements. First, preferential planes for digestion with elastase imply at least some degree of longitudinal and transverse orientation, probably of a somewhat less dense material than that composing the fibrils, which are digested only after longer times. Secondly, a circumferentially arranged material resists digestion longer, particularly after fixation in formalin, and exhibits affinities for stains to a degree differing from that of the centrally located substance. With the electron microscope, the process of digestion by elastase can be followed down to smaller dimensions. Samples of a suspension of elastin can be removed from the digesting enzyme solution at different times, placed on the grid, and examined with the electron microscope. In such preparations, large fibers, completely opaque to the electron beam, are the only elements detectable after short periods of digestion. After longer times smaller fibers are present, and occasional fibers are seen, the ends of which are frayed into smaller fibrils. This process of fraying or splitting of the larger fibers continues until small, rather short fibrils about 180A. in diameter are present. Still longer times of digestion lead to dissolution of even these small fibrils. In all of the preparations examined after partial digestion in elastase solutions,. occasional collagen fibrils have been encountered on the electron microscope grids. These fibrils are easily identifiable by the characteristic cross-banded structure, the major periodicity of which is 640 A. No large bundles of collagenous fibers have ever been encountered ; those fibrils present have been solitary. Their frequency is greatest in the preparations digested longest in the enzyme. In one specimen, a collagenous fibril was observed emerging from a partially digested, large elastic fiber. These observations indicate that even “purified” elastin preparations are contaminated by collagen, probably because the collagenous fibrils are embedded in the elastic substance and are thus protected from the solubilizing effects of the hot NaOH solutions designed to destroy them. This interpretation is somewhat different from that of Schwartz and Dettmer (1953) and Dettmer (1952). These authors observed transverse periodic bands in fibers prepared from elastic tissues. In some cases, the periodicity was so regular that the fibers could be identified as collagenous, with their characteristic 640 A. spacing ; in others, the transverse bands had different spacings, and these yere regarded as the elastic fibrils. However, collagenous fibrils have been described in which spacings other than the 640 A. periodicity are present (Porter, 1951). Moreover, electron

448

EDWARD W. DEMPSEY A N D ALBERT I. LANSING

staining with heavy metals, particularly silver, as employed by Dettmer, is notably capricious, especially in small collagenous fibrils. Our own observations that collagenous fibrils actually penetrate into the substance of elastic fibers, taken together with the criticisms enumerated above, all combine to cast doubt upon the transverse banding of elastic fibrils. Another concept of the macromolecular structure of elastic fibers was published by Gross (1949). Using trypsin, Gross obtained partial digestion of elastic fibers and observed the appearance of tightly twisted helices as digestion proceeded. He postulated, therefore, that such helically coiled filaments formed a repeating unit of structure upon which elastic fibers were built. However, other investigators found that although crude trypsin preparations will destroy elastic fibers, recrystallized trypsin will not ; consequently, the digestion observed by Gross must be due to some contaminant, probably elastase (Lansing, 1951). Franchi and De Robertis ( 1951), using crude trypsin sterilized by ultrafiltration, were unable to obtain the twisted filaments observed by Gross, but found such filaments present in profusion in the unsterilized trypsin solutions themselves. They pointed out the similarity between the filamentous helices and the structure of bacterial flagella, Gross ( 195l), after restudying the problem, also found the twisted filaments in the enzyme solutions. In consequence of all this, the contention that helically coiled threads form the unit structure of elastic fibers no longer has a factual basis. In recent years, fixation and sectioning procedures have been developed which permit good preservation of cellular structure in sections of 0.1 p thickness or less. By now, a goodly number of papers have appeared in which cytologic descriptions of various tissues are presented. It is rather remarkable that no mention of elastic fibers has appeared in this literature, despite the fact that regions known to contain elastic fibers have been examined. The reason for this remarkable omission quickly became apparent upon examining sections of ligamentum nuchae and aorta, tissues rich in elastic fibers. In thin sections of tissues fixed in Palade’s buffered osmic acid solutions, elastic fibers have extremely little density in electron micrographs. By contrast, the collagenous fibrils which surround elastic fibers and occasionally penetrate into their substance are osmiophilic and stand out as dark objects in photographs. Thus, the elastic tissue appears only as a negative image in electron micrographs, and since elastic fibers always are closely associated with collagenous reticulum, the elastic fiber appears merely as a region in which the reticulum pattern seems distorted. Consequently, up to the present, these important fibers have been overlooked. Careful examination of micrographs made from thin sections of elastic

ELASTIC TISSUE

449

tissue reveals a feltwork of dense reticulum fibrils occupying the space between, and surrounding the periphery of, the elastic fibers. Occasional fibrils penetrate the elastic substance, dividing the elastic fiber into two or more segments. Within the elastic substance, occasional small, dense dots or strands are present. Whether these represent wisps of collagenous fibrils embedded within the substance of the elastic fiber or whether they are composed of some other dense or osmiophilic substance is not clear at present. I n most sections, except for the dense dots or fibers mentioned above, the elastic substance itself has a homogeneous appearance and remarkably little density. In occasional, very thin sections, micrographs have been obtained in which the elastic substance is resolved into a faint fibrillar appearance. However, the density of these micrographs is so slight that the arrangement of these fibrils with respect to one another has not been adequately visualized in such preparations. Specimens from which the methacrylate embedding mass has been removed and the sections shadowed with chromium have proved more illuminating. In these, the elastic substance is resolved as an anastomosing, three-dimensionaI network of fibrils, the diam7ters of which are approximately 200 A. The closeness with which these fibrils approach one another varies from place to place in the same elastic fiber. Some areas are dense, with many fibrils per unit area ; others occur in which the fibrils exist as loose meshwork (Dempsey,

1952). The X-ray diffraction spectrum of elastic fibers and elastin has received only scanty attention. Kolpak (1935) described an amorphous, powder pattern only as derived from relaxed elastin preparations. Upon stretching, a pattern similar to that of collagen was obtained. However, Astbury (1940) in reviewing the question concluded that the faint collagen pattern was caused by contamination of the elastin preparation. This conclusion is in accord with the finding, cited above, that collagenous fibrils may be seen in electron micrographs derived from even highly “purified” elastin. Although more investigation is clearly necessary, it is possible to construct, from the present knowledge, a hypothetical architecture for the normal elastic fiber. Preparations studied with the optical microscope reveal that elastic fibers occur as a completely branched, anastomosing system with no loose ends. Digestion of these fibers with elastase and pathologic changes, particularly those associated with aging, reveal that these fibers have a proclivity for splitting along their long axes. Transverse fractures, although possible, are less frequent than are longitudinal splits. This predisposition toward splitting indicates a submicroscopic organization oriented along the long axis of the fiber. Likewise, the anisotropy developed during

450

EDWARD W. DEMPSEY AND ALBERT I. LANSING

stretching, being positive with respect to the long axis of the fiber, is best interpreted as indicating an array of asymmetric aggregates that become oriented with their long axes parallel to the long axis of the fiber. The proclivity to splitting presumably represents merely a cleavage between these parallel aggregates. Several facts indicate that large elastic fibers are not completely uniform throughout their substance. The proclivity toward cleaving longitudinally bespeaks a longitudinally oriented structure, whether of different composition or of less dense concentration compared with other regions of the fiber. Upon partial digestion of a large fiber, it splits into two smaller fibers, which are loosely twisted around one another. Such an arrangement can be conceived to occur by postulating the twisting fusion of two sinall fibrils which are cemented together along their length by an adhesive of the same composition as that of the fibers themselves, but of somewhat less concentration. Such an arrangement would account for the dense and rarefied areas described in electron micrographs. The ultimate fibrillar structure of the elastic substance, in this hypothetical construct, is an anastomosing network of fibrils approximately 200 A. in diameter. Such networks are common in other elastic substances such as rubber, and could account for the stretchability of the fiber as well as for its elastic recoil. Collagenous fibers, wrapped closely around the surface of the elastic fiber and penetrating into its substance, would serve to anchor the elastic net to adjacent structures. The concept outlined above has obvious implications concerning the iormation of elastic fibers, For years a controversy has existed concerning the intra- vs. the extra-cellular origin of collagenous fibrils. Only our almost complete ignorance about elastogenesis has prevented the extension of this controversy to include this other unit of the connective tissue fiber system. However, it would appear from many of the facts cited above that the elastic fiber must have an extracellular origin. It is impossible to conceive of a fiber, 5 p in cross sectional diameter, cleavable into smaller fibers twisted around one another and cemented together along their lengths, and into which collagenous fibrils are inserted, as being built up in toto inside a cell. Such a fiber is easily visible in microscopic preparations ; therefore, if it were formed within the 'cell its presence there should be easily visible, and it is not. In embryos, moreover, small fibers appear first and gradually enlarge with time. Much of the elastic substance must be added, therefore, after the fiber is in its extracellular position. Since its fibrillar organization is the same, except for density, in both the peripheral and central portions of the fiber, it follows that elastin may be laid down extracellularly in its characteristic architectural pattern, and

ELASTIC TISSUE

45 1

it is therefore unnecessary to postulate an intracellular origin of the formed fiber. It would appear, rather, that the fiber forms by condensation out of precursors located in the intercellular substances.

V, AGE CHANGESAND PATHOLOGY OF ELASTIC FIBERS Just as the chemical composition of elastin has been difficult to determine because of its unreactivity, so has elastic tissue exhibited little change in altered physiologic states of the organism. Tumors or overgrowths of elastic tissue occur rarely if at all, and pathologic destruction of elastic fibers occurs so infrequently that some authors believe an elastic fiber, once formed, persists throughout the lifetime of the individual. However, accumulating evidence indicates that some degree of turnover does occur, particularly in certain pathologic situations, and that alterations in the amount and characteristics of elastic fibers are associated with aging. Perhaps the most clear-cut evidence for the destruction of elastic fibers occurs in tuberculous processes in the lung. During the inflammatory reaction, in the alveolar walls surrounding the lesion, the fine elastic fibers exhibit a frayed appearance and become separated from their characteristic network so that single fibers can be traced to an unattached end. Such an end is never encountered in a normal elastic reticulum. Moreover, at such a fiber-ending, the fiber is frayed and unraveled into two or more component fibrils loosely twisted around one another in a fashion reminding one of the appearances of fibrils digested with elastase. At further stages the elastic fibers fragment into an amorphous, granular substance still retaining the selective staining characteristics of elastic tissue. Still later, this substance disappears. Thus, complete destruction and disappearance of the elastic fiber occur during the development and consolidation of the tuberculous lesion. As individuals age, changes overtake elastic fibers in various portions of the body. Those in the skin and in the larger blood vessels have been studied more carefully than in those other locations. There is great variation in the aging processes of elastic fibers. In some individuals they occur early ; in others, late in life. They may be marked in some locations and absent in others. Although such senile changes have been studied most in man, with insufficient attention being paid to other animals, it also appears that marked species variations occur. In large arteries, and especially in the aorta, localized areas occur in which destruction of elastic tissue begins to appear at about 40 years of age in the human. After its beginning, the change is more or less progressive, so that in the eighth and ninth decades nearly all of the elastic larnellae are involved. The elastic tunics first become somewhat

452

EDWARD W. DEMPSEY AND ALBERT I. LANSING

frayed in appearance, and fibers apparently delaminating off from the elastica externa and interna appear scattered throughout the media. Breaks, with unravelled ends, and transverse fractures frequently appear in the individual fibers. The aging and altered fibers exhibit an increased basophilia and stain more intensely with resorcin-fuchsin and the other elastic stains. In incinerated preparations, the locations of the fibers are distinguished by a heavy deposit of white ash which gives the microchemical reactions of calcium, whereas the elastic fibers from young individuals are lacking in ash. Upon chemical examination, calcium contents as high as 14% have been found in elastin from old aortas. Associated with this increase in calcium content is a change in the relative proportions of the constituent amino acids. The dicarboxylic amino acids, glutamic and aspartic, are considerably increased (cf. Table I ) , and other amino acids also exhibit altered proportions (Lansing, Roberts, Ramasarma, Rosenthal and Alex, 1951). I n the skin, areas of senile degeneration known as elastosis frequently occur, particularly on the exposed portions of the body such as the hands, face and neck. The pathologic changes are similar to those seen in arteries, and involve fragmentation, fracture, and granular degeneration of the individual fibers. Such areas of elastosis have been studied by Weidman (1931), Cooper (1952), and Lansing, Cooper, and Rosenthal (1953), who have described the characteristic appearance and histochemical reactions of the lesions. Characteristically the altered fibers are pigmented and, according to Weidman, contain sudanophil lipid. Lansing, Cooper, and Rosenthal were unable to confirm the sudanophilia of elastotic lesions, but reported that the degenerating areas of the skin, unlike those in arteries, were poor in minerals when microincinerated specimens were examined.

VI. REFERENCES Adair, G. S., Davis, H. F., and Partridge, S. M. (1951) Nature, 167, 605. Astbury, W. T. (1940) J . Inter%. SOC.Leather Trades' Chemists, 24, 69. Ba16, J., and Banga, I. (1949) Schweiz. 2.allgem. Pathol. u. BaRtera'oZ., 12, 350. Ba16, J., and Banga, I. (1950) Uiochem. J., 46, 384. Banga, I. (1951) 2. Vitamin-Hormolz- u. Fermentforsch., 4, 49. Banga, I. (1952) Acta PhySiol. Acad. Sci. Hung., 3, 317. Banga, I., and Schuler, J. (1953) Acta Physiol. Acad. Sci. Hung., 4, 13. Cooper, Z. (1952) Problems of Ageing, 3rd ed. Williams & Wilkins Co., Baltimore. Dees, M. B. (1923) Anat. Record, 26, 169. Dempsey, E. W. (1952) Science, 116, 520. Dempsey, E.W., Singer, M., and Wislocki, G. B. (1950) Staim Technol., 26, 73. Dempsey, E. W., Vial, J. I)., Lucas, R. V., and Lansing, A. I. (1952) Anat. Record, 113, 197. Dettnier, N. (1952) 2. Zellforsclz. u. mikroskop. Anat., 97, 89.

ELASTIC TISSUE

45.3

Ehrlich, P. ( 1885) Das Sauerstoff-Bedurfniss des Organismus. Hirschwald, Berlin. Engle, R. L., Jr., and Dempsey, E. W. (1954) J . Hisfo- and Cytochem., 2, 9. Franchi, C. hl., and De Robertis, E. (1951) Proc. Snc. Exptl. Biol. Med., 76, 515. Frey-Wyssling, A. ( 1948) Submicroscopic Morphology of Protoplasm and its Derivatives. Elsevier Press, New York. Gross, J. (1949) J . EnptE. MPd., 89, 699. Gross, J. (1950) J. Gerontol., 6, 343. Gross, J. (1951) PI-oc. SOC.Exptl. BioE. Med., 78, 241. Hall, D. .4.,Reed, R., and Tunbridge, R. E., 11952) Mature, 170, 264. Hass. G. M. (1939a) Arch. Palhol., 27, 335. Hass, G. M. (1939b) Arch. Pathol., 27, 583. Hass, G. M. (1942a) drck. Pathol., 34, 807. Hass, G. M. (194%) Arch. Pathol., 34,971. Hass, G. M. (1943) Arch. Pathol., 36, 29. Henle, J. (1841) Allegemeine Anatomie, Leipzig. Iidliker, A. von (1850) Cited by Rothig, Mikroskopische Anatomie, oder Gewebelehre des Menschen. Engelmann, Leipzig. Kolpak, H. (1935) Kolloid-Z., 73, 129. Lansing, A. I. (1951) Trans. 2nd Conf. on Comective Tissues, pp. 45-85. Macy Foundation, New York. Lansing, A. I., Cooper, Z., and Rosenthal, T. B. (1953) Proc. A m . Assoc. d n a f . , .4nat. Record, 115, Sit.ppl., 340. Lansing, A. I., Roberts, E., Ramasarma, G. B., Rosenthal, T. B.. and Alex, M. (1951) Proc. Soc. Exptl. Bid. Med., 76, 714. Lansing, A. I., Rosenthal, T. B., Alex, M., and Deriipsey, E. W. (1952) Anat. Record, 114, 555. Loewus, F. A. (1952) Anal. Chent., 24, 219. Lowry, 0. H., Gilligan, D. R., and Katersky, E. hl. (1941) J . BioI. Chem., 139, 795. Mall, F. P. (18%). /ohm Hopkins Hosp. Reports, 1, 171. Mall, F. P. (1902) Am. J . Anat., 1, 329. Petry. G. (1952) 2. Zenss. Mikroskop., 61, 66. Porter, K. R. (1951) Trans. 2nd Conf. on Connective Tismcs, p. 126. Macy Foundation, New York. Schwartz, W., and Dettmer, N. (1953) Virchows Arch. patkol. Anat. u. Physiol., 323, 243-268. Singer, M. (1952) Ititern. Rat. Cytol., 1,211. Tamer, P. (1891) Mowtschv. prakt. Dermatol., 12, 394. von Ebner (1870) Rollet’s Unters. an der Inst. fur Physiol. u. Histol. in Gvaz. ( Leipzig). Weidman, Fred D. (1931) Arch. Devmatol. and Syphilol., 24, 954. Weigert, C. (1898) Zentr. allgcm. Pathol. u. pathol. Anat., 9, 287. Weiss, J. (1954) J. Hisfochcm. and Cytoclrem., 2, 21. Wiener, 0. (1909) Math-Physik. Kl., 32, 379.

This Page Intentionally Left Blank

The Composition of the Nerve Cell Studied with New Methods WEN-OLOF BRATTGARD

AND

HOLGER HYDBN

Department of Histology, The University of Giiteborg. Swedtn PWf 455 455

I. Introduction .................... ....................... 11. Mass Determination . . . . . . . . . . . . ....................... 1. X-ray Microradiography .................................... 2. Gravimetric Determination . . . 3. Comparison between the Method 111. Quantitative Determination of Ribmucleic Acid in Individual Nerve Cells 1. Microchemical Method .... 2. Ultraviolet Microspectrograp 3. Comparison between the Methods for R N A Determination . . . . . . . . IV. Changes in the Neurons with Increasing Age and Intracellular Differentiation ....................................... V. Chemical Changes Induced by Adequate Stimulation VI. Summary .......................................................... VII. References .........................................................

467 468 469 472 474 474

I. INTRODUCTION Both chemical processes of high velocity and slow chemical changes take place in the structural units of the central nervous system. It now seems possible to approach the problem of whether these changes are correlated with the function of the unit and the age of the individual. Owing to the complicated three-dimensional construction of the nervous tissue it is, however, scarcely possible to use biochemical methods involving a separation of structural units and analysis of the various fractions. The present article is an account of methods recently developed and adapted for the anaIysis of single nerve cells with respect to mass, lipids, proteins, and nucleic acids. The value of mass obtained from lipid-extracted sections of nerve cells is an approximate measure of the bulk of the proteins. Some examples are given of data obtained on the problem of age and function. 11. MASSDETERMINATION 1. X-Ray Micrmadiography The first pictures of microscopic objects containing elements of high atomic weight were taken by Goby (1913a, b, c ; 1925). The pictures were taken in the scale 1 : l and subsequently enlarged. Goby proposed the name microradiography. Dauvillier (1930) used soft X-rays for taking microradiograms. He was the first to use the Lippmann film. In a series

455

456

SVEN-OLOF BRATTGARD AND HOLGER HYDEN

of studies, Lamarque (1936, 1937, 1938) Lamarque and Turchini (1936) and Turchini (1937) made considerable improvements in the quality of the pictures obtained. They used an open X-ray tube provided with a filter for the radiation and with a chamber containing the film emulsion and the object. They were the first to use soft X-radiation filtered through an A1 foil. They also used a Li filter and radiation generated at 1 to 5 kv. These authors also applied the method as a histochemical tool for the localization of iodine. Independently of Lamarque and Turchini, Sievert ( 1936) published ;I paper describing two methods for taking microradiograms. In fact, in this paper the author sketches the principles and applicability of modern microradiography using X-radiation. In the first method described, the microscopic section is placed in close contact with the film emulsion and the picture is taken in the scale 1 :l. Jn the second method, a small diaphragm is used to reduce the effective focal surface. A magnified picture is obtained by placing the object between the diaphragm and the film. Sievert pointed out the possibility of using X-ray microradiography for qualitative chemical analyses of microscopic objects. H e also stated that, in using microphotometry and in introducing elements of high atomic weight in the cell, these methods could be applied for the determination of physiologically interesting elements in microbiologic work. In 1938, Gretchishkin published a paper applying the methods described by Sievert to study small, mineral-containing organisms and histologic sections. Prives and Gretchishkin (1935) are the first who have taken X-ray microradiograms of nervous tissue. They found that the grey substance retained the X-radiation less intensely than the white substance. In 1944, von Hamos and Engstrom described a procedure for determining small quantities of an element in a biologic specimen by a comparison between the secondary radiation from a sample containing the element in question and the same radiation from a known amount of. the element. Bohatyrschuk (1942, 1944) discussed some errors of importance in microradiography and described a method for preparing a suitable photoemulsion for this type of work. Engstrom (1946) worked out the theoretical basis and apparatus for quantitative histochemical analyses by measuring the absorption of a microradiogram in monochromatic X-radiation on both sides of an absorption edge characteristic of the element sought for. Engstrijm gave an account of the theoretical possibility of determining biologically interesting elements in microscopic sections and also made analyses of Ca and P. Clark and Eyler (1944) and Clark (1947) used monochromatic X-radiation to study objects of biologic interest.

THE COMPOSITION OF THE NERVE CELL

457

Engstrom and Lindstrom (1949, 1950) investigated the contribution of various elements to the X-ray absorption of biologic material. They found that, in the 4 to 12 A. region, the effect of the absorption edges of the elements in tissue of average composition can be neglected. Carbon, nitrogen, and oxygen in the tissue make the main contribution to the absorption of soft X-radiation in this wave-length region. These elements, together with hydrogen, account for the main part of the cell mass. A correction is made for the hydrogen. The biologic section is exposed, together with a step-wedge of known composition as a reference system, by means of which the amount of dry substance in the sample can be determined. The microradiograms are taken on Lippmann film in the scale 1 :1, according to the principle given by Goby, Lamarque, and Sievert. Engstrom and Lindstrom constructed an open X-ray tube provided with a copper anode. The maximum intensity of the radiation which is filtered through an A1 foil lies at 7 to 10 A. Some equipment for microradiography has been described by Engstrom and Wegstedt ( 1951). At the Histological Department of the University in Gteborg a modified type of X-ray tube has been constructed together with some auxiliary instruments (Brattgtd and HydCn, 1952). The apparatus and the method have been especially adapted for investigations of nervous tissue and of its individual components. a. Principle and Performance. The theoretical calculation of X-ray microradiography using soft X-radiation for the determination of the cell mass has been given by Engstrom and Lindstrom (1950). In the 6 to 12 A. wave-length region, the absorption of the X-radiation is proportional to the mass of the elements carbon, nitrogen, and oxygen. There may be a considerable variation in the ratio of these substances without any appreciable change in the absorption coefficient for their common mass. Engstrom and Lindstrom have also shown that other substances with an atomic number below 30 may be present in considerably higher concentrations than those in biologic samples, without affecting the results of the absorption measurements. One exception in biologic material is calcium in bone tissue. A correction must be made, however. for the hydrogen present in biologic samples. The absorption of X-rays is calculated from the formula P -(-)-

m

P

I, = lo: e or from I.

log-=

I*

m

cr

(-)

P

458

SVEK-OLOF BRATTCARD A N D HOLCER HYDfiN

where I1 is the intensity of the transmitted X-rays, I0 the intensity of the incident X-rays, tn the mass of the absorbing substance in g./cm.2 and p/p the mass absorption of the sample in question. The X-radiation was generated at 3,000 volts and filtered through a 9-p thick aluminum foil. The greatest intensity lay between 7 and 10 A . ; the mass absorption coefficient was calculated for 8.32 A. The difficulty of measuring II and I0 necessitates the use of a reference system. This must have the same dependence on the wave-length as that of C, N, and 0 in the biologic sample, i.e., in the protein substance which is the main component of the cell mass. Nitrocellulose (Klint and Bernhardt, Stockholm, Sweden) with a composition of 49.5% C, 6.63% N, 38.25% 0 and 5.62% H was used for the preparation of this reference system. Thin foils were prepared from the nitrocellulose and placed beside the preparation as a step wedge. Microradiograms were made simultaneously of both the preparation and the reference system, and the X-ray absorption determined by measurements of the density on a photographic film. That of the biologic sample was determined by a comparison with the density of the reference system, using the formula given by Engstrom and Lindstrom (1950)

where ?&/kHP is the weight of the biologic sample in question, kRref,and kHP the correction factors for the hydrogen in the reference system and in the sample, respectively, and mref, is the mass of the reference system. (p/p)CNOref. and ( p / p ) CNO, are the mass absorption coefficients for the reference system and the sample, respectively. Equation 3 may also be written m =k (4)

-

where m is the mass of the biologic sample in question and k is a constant determined by the correction factors for the hydrogen and the mass absorption coefficients. As these can be calculated, the formula, under the experimental conditions used, will be m 1.106 mre,, where mreI is the mass of the reference system with the same absorption as that of the sample. This absorption is determined experimentally. A 5- to 15-p thick section of the tissue is mounted on the supporting cellulose membrane of a special holder (Fig. 1) (Brattgzrd and Hydbn, 1952). On the same holder is placed the step wedge made of nitrocellulose

-

THE COMPOSITION OF THE NERVE CELL

459

films. This unit fits into a preparation holder of the X-ray tube. The section and the reference system are exposed on a Lippmann film in the

Sample Holder

FIG.1. Arrangement of the sample and the reference system above the slits of the sample-holder.

scale 1:l. The close juxtaposition of the film to the section is of great importance. b. The Apparatus. The apparatus for taking microradiograms of biologic specimens is seen in Figures 2 and 3 (Brattgard and H y d h ,

1952). The head of the X-ray tube (G), is made of forged brass of high purity and is water-cooled (E). The water-cooled anode is adjustable axially by means of a fitting (S) sealed by an annular rubber gasket (J). The target is exchangeable. The cathode is placed at an angle of 45" to the surface of the anode. The latter is placed horizontally. The filament, which is spiralized, is surrounded by a static shield concentrating the electron beam on the anode. The arrangement thus permits focused radiation and the selection of a radiation cone of high and even intensity. The radiation is filtered through a 9-p thick A1 foil shown at A in Figure 2. The inside walls of the X-ray tube are gilded. All vacuum connections are made by vacuum rubber gaskets. In the preparation holder, B in Figure 2, the sample lies in close contact with the film. This holder with fittings for sample

P

8

FIG.2. The construction of the X-ray tube and preparation holder. A. 9-p thick A1 filter B. Preparation holder C. Lippmann film D. Sample holder E. Cooling system for the head

F. Anode G. Head H. Porcelain isolator J. Annular vacuum gasket K. Cooling system for the gasket L. Window for the filament control

M. Filament holder N. Filament P. Pyranimeter intake R. Vacuum gasket S. Fitting for anode adjustment

THIS COMPOSITION OF T H E NERVE CELL

461

and film can be handled in daylight and provides for a rapid exchange of sample. The light trap consists of the 9 - p thick A1 foil, serving at the same time as an X-ray filter. The evacuation is made with a Siegbahn

FIG.3. The X-ray tube ( X ) mounted on the top of the Siegbahn molecular pump

(V). B is the preparation holder. At T the 2-step pre-vacuum pump. At U the high-vacuum valve.

molecular pump, ( V ) in Figure 3, provided with a two-step rotating pump (T) . The high-vacuum valve ( U) permits of breaking the vacuum for changing the specimen. The high voltage d.c. is obtained from the main a.c. by a transformer followed by an Se full-wave rectifier. The ripple is reduced by a chokeinput filter. Using a generating voltage of 3,000 v. and 15 to 30 ma., the

462

SVEN-OLOF BRATTGARD A N D HOLGER HYDBN

exposure time with this apparatus is 1 to 2 minutes. The maximum intensity of the X-radiation used lies at 8 to 10 A. Additional apparatus must be used, however, in this technique. For cytochemical purposes it is of primary importance to have microscopic sections in which the structures and the chemical properties are well preserved. All embedding procedures cause great changes in the chemical composition and in the structure of the cell material. A cryostat has therefore been constructed (Brattgird and Hydin, 1954), in which the material is sectioned at a low temperature. The frozen sections obtained are directly transmitted to a vacuum chamber, placed within the cryostat. In this chamber the sections can be dried at -4Q* C. and at lo3mm.Hg. Dried sections of uniform thickness are obtained in this way. Since the sections are never treated by chemical agents, they are especially appropriate for mass determinations as well as for cytochemical studies. A suitable apparatus is used for the preparation of the nitrocellulose films from which the supporting membrane for the section and the reference system are made (Brattgird and HallPn, 1952; HallCn 1953a). Since the X-ray absorption of the biologic structure is compared with that of the reference system, the mass of the latter must be known in every case. The thickness of the nitrocellulose films is determined with a double-slit interferometer with great accuracy ( H a l I k and Ingelstam. 1952; Djurle and HallCn, 1953). The mass of the films is calculated from these values. The absorption in the microradiogram taken is recorded directly with a special microphotometer (Bourghardt, Brattgird, et al., 1953). The photometer permits an automatic recording under visual control of the density of a surface corresponding to 2 to 3 p2 in the radiogram. The electrical stabilization and construction permits the use of an almost linear amplification. The main parts (Fig. 4) are a microscope, a system of prisms and mirrors, a phototube, an amplifier with a stabilization unit, and a recording writer, the whole set-up constituting a stable unit. With the optical system, the X-ray picture is enlarged 500 to tB3 times. The light intensity is recorded by means of an electron-multiplier provided with an iris diaphragm, of which the aperture corresponds to an area of 3 p2 in the picture. The microscope stage with the centered microradiograms is moved by means of a synchronous motor across the measuring beam. The logarithmic shape of the intensity curve due to the nature of the light absorption is compensated for by the characteristic of a special tube in the amplifier. c. The Errors and Their Elim&ation. There are several sources of

THE COMPOSITION OF THE NERVE CELL

463

error which must be eliminated or reduced when using the microradiographic method for mass determination.

FIG. 4. The microphotometer. H is the handle of the frictional gear, SS synchronous motor, S stable substructure, P phototube, MS micro-screws.

The errors of the method can be grouped in systematic and random errors dependent on the properties of the biologic material. ERRORS. The X-ray method is based on the fact (1) SYSTEMATIC that the carbon, nitrogen, and oxygen in the tissue account for the main part of the absorption of soft X-radiation. These elements taken in the same proportions as in proteins are assumed to be responsible for the total absorption. A correction is made for the presence of hydrogen. However, other elements than C, N, 0, and H contribute to the total absorption. The presence of these other elements is permitted to give a positive systematic error of 5%It is evident that the distribution of these other elements in a cell may vary and so give rise to a random error, but this error is included in the total random error dependent on the biologic material.

<

464

SVEN-OLOF BRATTGARD A N D HOLGER HYD6.N

(2) R A N D ~ M ERRORS. ( a ) Errors in the reference system. The reference system is made of thin nitrocellulose films arranged in the form of a step-wedge. These films can be prepared in various ways. According to Lindstrom (1953) the films used by him and measured with a gravimetric method have an error of t 1 6 % . With a special apparatus in preparing the films, this error can be reduced to k97. (Brattgsrd and HallPn, 1952). The gravinietric method has some obvious drawbacks however (Brattg%d, Hallkn, and HydCn, 1953a, b ; Djurle and HallCn, 1953), which render it unsuitable. For the quantitative study, the same part of the reference system that is used for the absorption measurements must also be measured with respect to mass. For the X-ray absorption measurements we use a 0.2 rnm2 surface of the film constituting each step of the reference system. The mass of the reference system in the same area is then determined interferometrically according to Hallkn and Ingelstam ( 1952) and Djurle and Hallkn (1953). This method has an accuracy of &l%. ( b ) Errors in the sidpporting film. The section is mounted on a supporting cellulose film covering the slit of the sample holder. However carefully this film is prepared, irregularities in its thickness are unavoidable. This error is determined concurrently with : ( c ) The t w o r introduced by variation in the uniformity of the X radiation intensity. The cathode filament in our apparatus is surrounded by a static shield; focused radiation, which provides the best conditions for radiation of high and uniform intensity, is thus obtained. This detail must nevertheless be checked from time to time. ( d ) Error in the photographic-photometric procedure. One possibility consists of enlarging the primary radiogram by photomicrography (Engstram and Lindstrom, 1950). Lindstriim (1952, 1953) determined the error by measurement of 21 duplicates, using sections of biologic material, and found it to be *23%. This error also includes certain biologic variations. The present authors have also tested this procedure (Brattgsrd, HallPn, and H y d h , 1953b) and found a standard deviation of *3770. It is evident from these figures that such a procedure cannot be used. A specially designed microphotometer was therefore constructed, with linear amplification, to measure the density directly in the primary radiogram (Bourghardt, Brattgird, ~t al., 1953). Using this photographic-photometric procedure, the random error in a determination was determined to *l.5% (Brattgird, Hydkn, and Hallin, 1953b). ( e ) Tkp X-ray method gives information regarding the w m s per Rirfuce unit. If the thickness of the section is known, the mass per unit

THE COMPOSITION OF T H E KERVE CELL

465

of volume can be determined. A large systematic error appears as soon as a piece of tissue is treated with a fixation solution and embedded (Stowell, 1941; SylvCn, 1951). If optical methods are used to measure the thickness of the section, cytologically prepared specimens are required. The outcome of this measurement, however, will never give the actual thickness of the fresh preparation. This is no doubt the most pertinent problem within cytochemistry. W e have therefore chosen whenever possible to use fresh and frozen sections. The thickness of a histologic section must be regarded as a mean value. If the tissue contains structures of varying hardness there will be considerable variations about this mean value, owing to physical factors in sectioning. These variations may be so great that it is scarcely feasible to speak of "the thickness of the section" but only of thickness within certain areas (Brattgird, 1954). Since absorption measurements are usually confined to small areas of single structures, e.g. the cytoplasm or the karyoplasm, and these structures are relatively homogeneous with respect to mass, the variation can be reduced. A hardness suitable for individual structures at sectioning is obtained by freezing the specimen. Various structures demand a special temperature ( Schultz-Btauns, 1931) . To preserve the structure, treatment according to the freezing-drying method can be used. Three prerequisites, i.e. fresh tissue, sectioning at suitable temperature, and preservation of the structures, can be fulfilled in using the following procedure. The nerve tissue is frozen down in liquid air and placed in a cryostat (Brattgird and Hydkn, 1954) in which the temperature is kept at -8 to -18" C. A motor-driven microtome of the Minot type is used for sectioning. The sections are immediately transferred to a vacuum chamber inside the cryostat. In this chamber the sections are dried at -4.0 to -60"' C. and at 10" mm.Hg. Furthermore, systematic errors due to biologic variations must be taken into account. The error inherent in the biologic material is mainly caused by treatment in the histologic procedure. Formaldehyde solution, for example, causes a 40% shrinkage of the fresh tissue (Stowell, 1941). As a rule, however, the aim of the investigation is to obtain information regarding differences between cells uniformly treated ;changes induced by fixation can therefore be disregarded. Another systematic error is due to the dissolving effect of the chemicals used in the preparation. Some of these errors are dealt with by B'rattgird and H y d h (1952), Brattgird (1952), H y d h (1952), and Edstrom (1953b). X-ray microradiography has also been used for chemical fractionation of nerve cells. The lipids are determined quantitatively by extractiox

466

SVEN-OLOF BRATTGARD AND HOLGER

HYDPN

with chloroform, and the nucleic acids together with the proteins can be released by enzyme digestion. Using these procedures, additional errors must be taken into account. Examples are given in the paper by Brattg5rd and HydPn (1952). W e wish to emphasize that only b y reducing the errors of the apparatus rn outlined in the aforegoing does it become possible t o perform m a s determinations zetith an accuracy of -+lo to 15%.

2. Gravimetric Determination The total amount of dry weight of nerve cells can also be determined by means of a gravimetric procedure, according to Lowry (1941, 1944, 1953). Small pieces of the tissue or large single nerve cells may be used. The limit of the method lies at 1Wmg. The procedure is the following. The specimen is frozen down, sectioned, and dried in vacuo. Single nerve cells are collected by dissecting the section with needles, or a small piece is cut out with a small scalpel. The specimen is placed on the scale of the quartz balance described by Lowry (1953). The balance is made from a thin quartz rod. The rod will be bowed by the weight of the specimen and the deviation is recorded with a microscope system. Since the balance is calibrated, the weight can be calculated from the value of the deviation.

3. Conzpwisoia betzueen the Methods for Mass Determination Table I shows the data from a mass determination in various parts of Ammon’s horn, obtained by means of X-ray microradiography and TABLE I TOTAL DRYWEIGHT OF DIFFERENT PARTS OF AMMON’S HORN Weight in 10-9 rng./p3

rllvez4.r Stratzlm oriens Strabm Pyramidale

Stratum radicatum Stratma locunosum

X-ray Microradiography

Gravimetric Method According to Lowry

0.38 0.23

0.37 0.B

0.19 0.21 0.25

0.16

0.22 0.26

according to Lowry’s method, respectively. It may be noted that the material was treated in the same way in both cases, i.e., frozen down and sectioned in a cryostat and dried in vacuo. In both cases the microtome had the same setting (lop). No statistically significant differences are found between the results of the two methods.

THE COMPOSITION O F THE NERVE CELL

467

111. QUANTITATIVE DETERMINATION OF RIBONUCLEIC ACID I N INDIVIDUAL NERVECELLS

1. Microchemical Method This method has been worked out by Edstrom (1953a, b). The principle is the following. A single nerve cell is obtained by means of micromanipulation. The specimen is digested with ribonuclease and the extract obtained is placed on a thin strip of cellophane. The amount of RNA can be computed from the absorption within the ultraviolet region. The practical procedure is as follows. Carnoy-fixed nervous tissue, embedded in paraffin, is cut into 70-p thick sections. Nerve cells entirely situated within the section are dissected out with a micromanipulator. The Carnoy solution ensures the precipitation of the nucleic acids. The paraffin is removed and the cell is passed through alcohol to water. The cells to be investigated are dissected free and separated from surrounding tissue, and their volume is then determined. A buffered water solution of ribonuclease digests the crushed cell during 20 minutes at 20" C. The extraction is repeated three times. The extracts are placed on a 30 by 40 p cellophane strip. This strip is photographed in the ultraviolet region at 2570 A. together with a rotating sector serving as a reference system. After the strip is developed, the density of the strip image is recorded photoelectrically along the strip. The amount of nucleic acid is calculated from the extinction values and the size of the absorbing surface. With the aid of the figures for the cell volume, the average RNA concentration is calculated. A minor correction must be made for contaminating protein. The systematic errors are caused by absorption from substances other than RNA, e.g. proteins, DNA, and ribonuclease. These positive systematic 1%. A possible negative systematic errors have been found to be about error could be anticipated on the grounds that the ribonuclease might not render all R N A soluble. This was tested by repeated digestions and successive treatment with perchloric acid. Since no RNA could be obtained after digestion by ribonuclease, this error may be considered as insignificant. The error introduced by an incomplete precipitation extraction of nucleotide material by the Carnoy solution has been discussed by Edstrom (1953b) ; it was found to be negligible. The accuracy of the method is about +lo .lO-**g. The amount of RNA contained in large and medium-sized nerve cells is of the order of magnitude of 100 to 1,000~1012g.The method consequently permits good accuracy in analyses of such cells. As has been emphasized in the aforegoing, all cytochemical methods will be affected by the variations in the biological material. This factor has been dealt with on page 465.

+

468

SVEN-OLOF BRA’ITGARJJ AND HOLGER

HYDBN

2. Ultromiolet Microspectrography This method was elaborated by Caspersson (1936; 1940a, b ; 1941). A microscopic section is placed in a microscope and a complete absorption spectrum from 2,400 to 3,000 A. is recorded from a small area in the preparation. The prerequisite is that the amount of ncm-specific light losses at 2,600 A. can be computed and that the object has sufficient optical homogeneity. The amount of nucleic acid in the specimen is computed from the extinction value at 2,600 A. Photography at 2,600 A., which gives a high resolution, also shows the relative distribution of the absorbing substances within the cell section. The method is described in detail in several papers (Caspersson, 1936 ; 1940a, b ; 1941) and has also been used in a series of investigations of nerve cells (HydCn, 1943; Hydin and Hartelius, 1948; HydCn and Lindstrijm, 1950; Hamberger and H y d h , 1945, 1949a, c). One of the present authors has used this method for the study of nerve cell material for more than ten years. The sources of error in ultraviolet microspectrography are, however, great. Regarding nerve cells and the specificity of the ultraviolet absorption, certain precautions must be taken because there are substances other than RN.4 which have a selective and high absorption at 2,600 A. (HydPn and Lindstrom, 1950). In studying nerve cells this source of error is, however, easy to avoid. With respect to other types of tissue, the .question whether a high absorption at 2,600 A. indicates the presence of nucIeic acid does not seem to be totally clarified. There are several papers discussing the validity of the results obtained by ultraviolet microspectrography, as well as the errors of this method (Glick, Engstrom, and Malmstrom, 1951 ; Wilkins, 1950; Davis and Walker, 1953). Among the main errors discussed may be mentioned inhomogeneities of the biologic material, non-specific light losses due to light scattering and reflection, and the effect of the radiation itself. As already discussed, another group of. errors is due to histochemical treatment of the specimen. The introduction of the ultraviolet procedure according to Caspersson nevertheless provided a great impetus in the field of cytochemistry and has given many valuable results. With due regard to these errors which impair the ultraviolet microspectrography as a quantitative method, we are now instead using the microchemical method according to Edstrom for the determination of RNA.

3. Comparison Between the Methods fm R N A Determination The RNA content of motor nerve cells from the ventrolateral group in the spinal cord of the rabbit has been investigated with ultraviolet microspectrography (HydPn and Hartelius, 1948 ; Niirnberger, Engstrom, and

469

THE COMPOSITION OF THE NERVE CELL

Lindstrom, 1952). Determination of the RNA content according to Edstrom in the same type of nerve cells has now been performed by Edstrom (1953a) and HydPn, Edstrom, and Brattggrd (1954). The results are shown in Table 11. The difference is considerable. The highest values of RNA are obtained by the method which has the smallest error. TABLE I1 RNA CONTENT I N MOTORROOTC ~ L FROM S Carnoy-Fixed Cells Weight in per cent

UV Microspectrography ~~

THE

RA~BIT

Microchemical Determination of RNA

~

NiirnbergerEngs tromLindstrom

HydCnHartelius

Edstrom

EdstriimBrattgHrd

1.7+0.30

1.7+0.09

2.420.10

2.6k0.12

HydCn-

IV. CHANGES IN THE NEURONS WITH INCREASING AGE A N D INTRACELLULAR DIFFERENTIATION Embryonal nerve cells have been studied with the methods described in the aforegoing. I t was found that, judging by the absorption spectra, the protein substances in the nerve cell cytoplasm increase more than 2,ooO times during the development of the nerve cell from neuroblast to adult cell with completed growth ( HydPn, 1943). The ultraviolet analysis showed the presence of a high RNA content in the cytoplasm at all stages of this development. Special attention was paid to the increase of RNA in the nucleus, localized both in the nucleolus and in the karyoplasm. I t was also found that the amount of RNA in the karyoplasm of the adult nerve cell could vary according to the type of cell. In connection with studies on a rabies strain, Sourander ( 1953) investigated the mass of chick embryo neuroblasts during development. He observed an increase in the amount of proteins of fourteen times in the cell body of embryonal motor anterior horn cells during the last two pre-natal weeks (Fig. 5). If the nerve cells were excluded from adequate stimulation or deprived of contact with the peripheral field, a disturbance took place in their development. If the differentiation of the neurons was inhibited by removing the peripheral field according to the technique of Barron (1948) and Mottet (1951), it was found that the increase in the mass of the cell, expressed both per cell and per volume unit, was inhibited (HydCn, 1953). (See Table 111.)

470

SVEN-OLOF BRATTGARD AND HOLGER

HMSN

TABLE Irr TOTALDRY WEIGHT OF CELLS F R O M THE VENTKO-LATERAL CELL GROUPOF SPINAL Cow OF THE CHICKEMBRYO

THE

Total Development : 6 Days Development after Operation : 3 Days

Mass mg.

*

10-9/@

Mass per Cell Cytoplasm mg. 10-B

-

Cells from Control Side

Cells Deprived of Peripheral Field

0.13f0.01

0.07 +O. 008

123

10

The post-natal development of the neurons with respect to mass has been studied by BrattG5rd (1952) on retinal ganglion cells. Provided that the anitiial received normal light stimulation, the mass of the cell body expressed per volume unit increased 100% during early post-natal development (Table IV) . If retinal ganglion cells are deprived of adequate light stimulation Cells from control embryos

Cells from infected embryos

"t

Days of incubation FIG.5. Total dry substance per cell cytoplasm in motor anterior horn cells of noninfected chick embryos and embryos infected with the Flury strain of rabies.

47 1

THE COMPOSITION O F T H E NERVE CELL

during the early post-natal period, there is no further increase of mass in the cells, and the chemical differentiation stops (Table V) (Brattghrd, 1952). There is, however, another type of chemical differentiation of the nerve cell during the post-natal period. As early as seven years of age, a yellow pigment appears in many motor cells. The mass of the pigment is greater than that in the remainder of the cell body (Table VI) (HydCn and TABLE IV TOTALDRYWEIGHTOF CARNOY-FIXED RETINALGANGLTON CELLSAT DIFFERENT AGES Weight in 10-9 mg./w2 ll-Day-Old Rabbits

10-Week-OId Rabbits

8-Month-Old Rabbits

0.4Zf0.016

0.78&0.013

0.98&0.038

TABLE V ADEQUATESTIMULATION ON THE DEVELOPMENT OF RETINAL GANGLION CELLS FROM BIRTHTO THE ADULT STAGE Weight in 10-9 mg./pz

T H E INFLUENCE OF

Rabbits Living in Ordinary Daylight for 11 Days

Rabbits Living in Ordinary Daylight for 10 Weeks

Rabbits Born in Darkness and Then Living in Complete Darkness for 10 Weeks

Rabbits Born in Darkness, Living in Darkness for 10 Weeks, Then Living in Ordinary Daylight for 3 Weeks

0.42-tO.016

0.78+-0.013

0.16rt0.012

0.58*0.021

TABLE VI TOTAL DRYWEIGHTOF MOTORROOTC ~ L S Weight in 10-8 mg./$ Cytoplasm Containing RNA and Protein

0.37

Yellow pigment

0.56

Lindstrom, 1950; HydCn, 1950). It also contains more lipids than the remainder of the cell body but does not contain any RNA. The results of absorption measurements throughout the ultraviolet and visible range of the spectrum, together with fluorescent data, indicate that this pigment belongs to the pterin group. Thus, during the growth and development of the neuron there is a steady increase of mass in lipid-extracted cells, i.e., the main bulk of proteins. During the same period, the RNA content of the cell is high.

472

SVEN-OLOF BRATTGARD A N D HOLGER H Y D k N

A disturbance in development is reflected, as could be expected, in the structural as well as the chemical differentiation of the neuron. The occurrence and increase of the yellow pigment, concurrent with a decrease in the RNA content of the celI, represent a post-natal chemical differentiation of the neuron which presumably continues during the life span. These changes may be the expression of the chemical renewal of the nerve cell, a chemical differentiation compensating for the missing mitoses. CHANCES INDUCED BY ADEQUATE STIMULATION V. CHEMICAL It has been shown in earlier studies using ultraviolet microspectrography that adequate stimulation of motor and sensory neurons is followed by a change in the purine and pyrimidine absorption. Thus, strong motor activity is reflected in the motor root cells by a decrease in the RNA content and its subsequent restoration (HydPn, 1943). Similar chemical changes were found to occur after adequate stimulation of sensory neurons, such as vestibular and cochlear ganglion cells and the retina1,cells (Hamberger and HydPn, 1945; 1949a, b, c ; Brattg&d, 1952). With X-ray microradiography, it has also been possible to study the lipids of the nerve cells which have been extracted by chloroform. W e found a considerable lipid content both in the nerve cell cytoplasm and in the nucleus. The values for the nucleus amounted to about 25% in nerve cells belonging to Deiters’ nucleus (Fig. 6, Table VII). These TABLE VII

TOTALDRYWEIGHT AND LIPIDS OF NERVECELLSFROM ADULT RABBITS Weight in 109

Nucleus

Cytoplasm

-

Total Dry Weight Deiters’ cells Purkinje cells Motor root cells

mg./~3

0.65 0.41 0.54

Lipids

0.32 0.09 0.14

Proteins

0.33 0.32 0.40

Total Dry Weight 0.47 0.33 0.33

Lipids Proteins

0.17 0.08 0.09

0.30

0.25 0.24

~~

values are in fairly good agreement with those given by Tyrell and Richter (1951). Another contribution to the understanding of the chemical organization of the nerve cell is the finding that the lipids can also vary following neuronal stimulation. Thus, in experiments on rabbits, it was found that after rotation the amount of lipids in the cytoplasm of the Purkinje cells in the flocculus of the cerebellum increased 40 to 50%. The following observation was made which may be of interest to

THE COMPOSITION O F T H E NERVE CELL

473

FIG.6. Nerve cells from Deiters’ nucleus of a rabbit photographed at 8 to 10 a. ; a. fresh, frozen section; b. the same section after treatment with chloroform; c. after digestion with ribonuclease.

474

SVEN-OLOF BRATTGARD AND HOLGER H M ~ N

biochemists in the preparation of cell material for enzyme or other chemical studies. The question has arisen whether solutions used in the preparation dissolve substances from isolated nuclei or cells. W e found that the solvents used in making microscopic sections from Carnoy-fixed nerve cells extracted the main part of the lipids situated in the cytoplasm and in the karyoplasm (Table VIII). Moreover, it may be stressed that substances other . than chloroform-soluble lipids are dissolved from the nucleus by the treatment. TABLE VIII THEEFFECT OF CARNOY'S SOLUTION O N THE CYTOPLASM A N D NUCLEUS OF MOTOR NERVECELLS Weight in 10-9 mg./p3 Fresh Frozen Cells

Cytoplasm Nucleus

Total Mass

After Lipid Extraction

Carnoy-Fixed Cells

0.71+0.056 0.48k0.016

0.35k0.035 0.30-+0.037

0.34%0.017 0.13f0.008

Summarizing the results obtained with the methods described in this article with regard to chemical changes following adequate stimulation, the following tentative picture can be given of the nerve cell. It has been found that adequate stimulation of motor and sensory neurons is followed by changes in the lipids, the KNA content, and the cell proteins. From the data obtained these changes seem to be correlated. The presence of a liponucleoprotein complex in the nerve cell may therefore be assumed. Furthermore, it seems likely that these liponucleoproteins constitute a nerve cell complex that is closely linked up with function.

VI. SUMMARY Methods for the quantitative determination of the mass, lipids, and KNA of single nerve cells are described. The capacity and errors of the methods are discussed. X-ray microradiography using soft X-radiation and a new microchemical procedure for R N A determination within the cell dimensions are discussed in detail. Some data on nerve cells during development and differentiation are given. Quantitative changes in the nerve cells with increasing age and following motor and sensory stimulation are reported.

VII. REFERENCES Barron, D. H. (1948) J . Comb. N e w o l . , 88, 93. Bohatyrschuk, F. (1942) Fortschr. Gebiete Rb'nfgens&ahle?L, 66,253. Bohatyrschuk, F. (1944) Acta Rudiol., 26, 351.

THE COXPOSITION OF T H E NERVE CELL

47s

Bourghardt, S., Brattgard, SO., HydCn, H., Jiewertz, B., and Larsson, S. (1953) J. Sci. Znstr., 80, 464. Brattgard, S-0. (1952) Acfo Radiol. S~ppl.,96, Brattglrd, S-0. (1954) I. Roy. Microscop. SOC.(In press). Brattgard, S-O., and HallCn, 0. (1952) Biochim. et Biophys. Acta, 9, 488. Brattgard, S-O., Hall&, O., and Hydin, H. (1953a) Biocltim. et Biophys. Acta, 10, 486.

BrattgHrd, S-O., HallCn, O., and H y d h , H. (1953b) Acta Radhl., SQ,494. Brattgilrd, S-O., and H y d h , H. (1952) Acta Radiol. Suppl., 94. Brattgird, S-O., and Hyd6n, H. (1954) Unpublished material. Caspersson, T. (1936) Scund. Arch. Physiol. Suppl., 8. Caspersson, T. (1940a) Chromosoma, 6, 562. Caspersson, T. (194Ob) J. Roy. Microscop. SOC.,80, 8. Caspersson, T. (1941) Nafunviss.,29, 33. Clark, G. L. (1947) Radiology, 49, 483. Clark, G. L,and Eyler, R. W. (1944) I d . Rdiography, 8, 13. Dauvillier, M. A. (1930) Compt. rend., 190, 1287. Davis, H. G., and Walker, P. M. B. (1953) Progr. in Biophys., S, 195. Djurle, E. and Hallin, 0. (1953) Erptl. Cell. Research, 6, 301. E d s t r h , J. (1953a) Biochim.. et Biophys. Acta, 11, 300. Edstrijm, J. (1935b) Biochim. et Biophys. Acta, l2,361. Engstrlim, A. (1946) Acta Radiol. SupPl., 6s. EngstrZim, A., and Lindstriim, B. (1949) Nahre, Iss,563. . Engstrom, A., and LindstrBm, B. (1950) Biochitn. et Biophys. Acta, 4, 351. Engstrom, A., and Wegstedt, L. (1951) Acta Radiol., 56, 345. Glick, D., Engstriim, A., and Malmstrom, B. G. (1951) Science, 114, 253. Goby, P. (1913a) Cutnpt. rend., 166, 686. Goby, P. (1913b) J. Roy. Mkroscop. SOC.,61, 373. Goby, P. (1913~) Arch Romfgen Ray, 18, 247. Goby, P. (1925) Compt. rend., lao, 735. Gretchishkin, S. V. (1937) Vestnik Reiitgenol. i Radiol., 17, 549. Gretchishkin, S. V. (1938) Vestnik Rmtgenol. i Radiol. a0, 397. Hallen, 0. (1953a) Expfl. Cell. Research, 4, 494. HallCn, 0. (1953b) Unpublished material. HallCn, O., and Ingelstam, E. (1952) Exptl. Cell. Research, S, 248. Hamberger, C-A., and Hyden, H. (1945) Acta Oto-Iaryngol. Suffil., 61. Hamberger, C-A., and HydCn, H. (194%) Acta Oto-luryngol. Sup& 76, 36. Hamberger, C-A., and Hyden, H. (1949b) Acla Oto-laryngol. SUppZ., 75, 53. Hamberger, C-A., and HydPn, H. (194%) Acfa Oto-larpgol. SZcppl., 76, 82. v. Hamos, L. and EngstrBm. A. (1944) Acfu Radiol., 26, 325. HydCn, H. (1943) Acta Physiol. Scad. Suppl., 17. HydCn, H. (1950) In Genetic Neurology, p. 177. Univ. of Chicago Press, Chicago. HydCn, H. (1952) I n Die Chemie und der Stoffwechsel des Nervengewebes. p. 1. Springer VerlaE, Heidelberg. HydCn, H. (1953) J . Embryol. w d Exptl. dforpho!., 1, 315. H y d h , H., Edstriim, J., and Brattghd, S-0. (1954) Unpublished material. HydCn, H., and Hartelius, H. (1948) Actu Psychfat. et Neurol. Suppf., 48. Hyden, H., and LindstrBm, R. (1950) DkczhFsions Faraday soc., 9, 436. Lamarque, P. (1936) I. radio!. et ilectrol., a0, 325.

476

SVEN-OLOF BRATTGARD AND HOLGER

HMBN

Lamarque, P. (1937) Bull. hisfol., 14, 5. Lamarque, P. (1938) J. brlge radio!., 27, 109. Lamarque, P., and Turchini, J. (1936) Cow@#.rend. sac. biof., 122, 294. Lindstrom, B. (1952) Acto Radio]., S8, 355. Lindstrijm, B. (1953) Biochim. ef Biophys. Acta, 10, 186. Lowry, 0. H. (1941) J . Biol. Chew., 140, 183. Lowry, 0. H. ( 1 M ) J . Biol. Chew., 162, 293. Lowry, 0. H. (1953) J . Hktochern. and Cytockm., 1, 420. Mottet, N. K. (1951) Yule J . BioI. and AIed., a3, 390. Niirnberger, J., Engstrom, A., and Lindstrom, B. (1952) J , Cellular Comp. Physiol., 39, 215. Prives, M., and Gretchishkin, S. V. (1935) Vcstlaik RPntgen. i Rudiol., 14, 201. Schultz-Brauns, 0. (1931) Klin. Wochschr., 10, 113. Sievert, R. (1936) Acta Radiol., 17, 299. Sourander, P. (1953) Acta P a t h l . Microbial. S c a d Suppl., 9s. Stowell, R. E. (1941) Stain Technol., l6, 67. SylvCn, B. (1951) Acta union intern. contrr cancer, 7, 708. Turchini, J. (1937) Bull Histol. app!. et tech. microscop., 14, 17. Tyrell, L. W, and Richter, B. (1951) Biochem. J., 49, Proc. LI. Wilkins, M. H. F. (1950) DkcusSions Faruifay Sac., 9, 363.

Author Index Numbers in italics indicate pages on which the reference is listed.

A

Aquist, S., 265, 271 Arendsen De Wolff-Exalto, E., 316, 321 Arnold, G., 184, 196 Arnold, J., 415, 423, 431 Aronsohn, R. B., 124, 130 von Am, A., 96, 97, 110 Asboe-Hansen, G., 403, 404, 405, 406, 407, 410, 411, 412, 413, 414, 417, 419, 423, 424, 425, 427, 430, 431, 431, 432, 433,

Abell, R. G., 363, 365, 368, 369, 370, 374,

394, 395, 396 Aberg, B., 417, 432, 433 Abrams, R., 43, 58, 295, 320 Ackermatin, W .W.,248, 264, 267, 272,

275 Ada, G. L., 266, 270 Adair, G. S.,442, 452 Pldamstone, F. B., 182, 185, 195 Agrell, I., 289, 293, 306, 320, 322 Ahern, J. J., 361, 393, 395, 396 Albaum, H. G.,287, 321 Albrecht, M., 53, 55, 64 Albrink, W. S., 8, 58 Albuquerque, R.M., 145, I64 Alex, M.. 439, 440, 446, 452, 453 Alfejew, S., 400, 401, 431 Alfert, M., 133, 151, 152, 155, 156, 164, 167, 174 AIgire, G. H., 361, 363, 366, 370, 371, 376, 388, 389, 390, 391, 392, 395,

397, 398

Allen, A., 3, 6, 63 Allen, E., 184, 195 Allfrey, V. G., 135, 168, 201, 202, 203, 221, 222, 223, 238, 258, 270, 275 Altshuler, C. H.,411, 414, 417, 431 Alrcrdes, F., 132, 148, 154, 164 Ambrocie, E. J., 138, 143, 161 Ames, A. M.,48, 65 Anderson, B. F., 392, 395 Anderson, J. T., 266, 273 Anderson, N. G.,135, 164, 201, 202, 221, 232, 234, 238, 239, 268, 270, 275 Anderson, T. F., 139, 168 Andres, A. H.,181, 182, 184, 185, 186, 187, 189, 195, 197 Andrus, W. de W., 7, 13, 63 Anfinsen, C. E., 23, 24, SZ, 58, 65 Angwinc, D. M., 411, 414, 417, 431 Appelmans, F., 226, 232, 2.33, 234, 236, 243, 245, 246, 247, 248, 249, 255 254, 262, 267, 268, 270,271

435 Astbury, W. T., 439, 449, 452 Astrup, P., 427, 435 Astrup, T., 10, 11, 26, 29, 30, 41, 45, 58,

62 Aub, J. C., 25, 40, 51, 52, 59 Audry, C., 401, 431 Auerbach, V. H.,262, 271

Augustinsson, K. B., 279, 302, 313, 314,

321

B

Baeh, S. J., 36, 39, 58 BBckstrCm, S., 308, 312, 316, 317, 321 Bacon, J. S. D., 7, 58 Bacq, 2. M., 53, 58 Bader, S., 163, 164 Badger, G. M., 122, 128 Bairati, A., 201, 221 Baitsell, G. A., 8, 58 Baker, B. L,430, 431 Baker, L. E., 4, 6, 9, 15, 26, 28, 29, 35, 36, 41, 42, 46, 47, 49, SO, 54, 55, 58 Baker, N. H., 388, 390, 397 Raker, R. F., 139, 168 Bakker, J. H., 70, 110 Balazs, E. A., 418, 431 Balbiani, E. G.,132, 138, 142, 164 Baldridge, G. D., 56, 58 Balduini, M., 120, 128 Baldus, I., 208, 222 Ball, E. G.,23, 24, 52, 58, 65, 308, 321 Ballentine, R.,282, 321 Ba16, J.. 441, 452 Banga, I., 440, 441, 442, 452 Binhidi, Z., i79, 292, 321, 323 Barclay, R. K., 39, 6 4

477

478

AUTHOR INDEX

Barclay, W. R., 361, 369, 393, 395, 396 Bardeleben, K . von, 184, 195 Bardram, M., 414, 431 Barigozzi, C., 142, 144, 164, 182, 185, 195 Barklay, H., 7, 59 Barkulis, S. S., 247, 270 Barnes, H. R., 157, 165 Barnett, R. J., 355, 358 Barnett, S. R., 202, 204, 222, 238, 271 Barnum, C. P., 219, 222, 237, 238, 244, 253, 265, 270, 273. Barron, D. H., 469, 474 Barron, E. S. G., 50, 59, 309, 321 Barry, E., 13, 46, 64 Barski, G., 11, 13, 56, 59 Barta, E., 34, 59 Barth, L. G., 294, 307, 321 Bartley, W., 268, 270 Barton, A. D., 238, 250, 272 Barton, J., 135, 164 Bastrup-Madsen, P., 120, 128 Bates, E., 401, 402, 404, 431 Battaglia, E., 122, 128 Battezzatti, M., 426, 431 Battle, H. I., 120, 128 Bauer, H., 132, 137, 139, 144, 146, 147, 148, 150, 154, 163, 164, 166, 172 Baumann, C. A., 49, 64 Baylor, E. R., 157, 165 Raylor, M. R. B., 157, 165 Beadle, G. W., 220, 222 Beams, H . W., 184, 196 Beaton, J. R., 7, 68 Beatty, R. A., 179, 183, 189, 195, 196 Beaufays, H., 237, 254, 256, 270 Becker, B., 330, 332, 334, 357 Beecher, H. K., 367, 395 Beeckmans, M. L., 45, 66 Beermann, W., 132, 137, 138, 146, 148, 149, 150, 152, 163, 164, 172 Behrens, M., 202, 219, 222, 238, 270 Beinert, H., 234, 243, 245, 246, 247, 261, 270 BPIIr, K., 138, 164 Bell, D. J., 7, 58 Bell, L. G., 136, 167 Bender, O., 411, 431 Bendich, A., 135, 164 Bennett, L. I-., Jr., 43, 67

Bensley, K. K.,225, 270 Bensley, S., 401, 431 Benson, E., 279, 287, 325 Berg, W. E., 305, 307, 321, 322 Berger, C. A., 147, 149, 164, 165 Bergrnann, M., 36, 59 Bergman, R. A. M., 8, 59 Bergstrom, S., 43, 66, 418, 425, 433 Berlin, E. A., 185, 195 Bern, H. A., 152, 164 Bernhard, W., 248, 270, 273 Bernheim, F., 292, 321 Bernstein, M . H., 135, 165 Berthet, J., 229, 232, 233, 234, 237, 241, 243, 244, 245, 246, 247, 249, 254, 256, 260, 268, 270, 271, 272, 288, 321 Berthet, L., 232, 233, 234, 237, 241, 243, 244, 245, 246, 247, 249, 254, 256, 260, 268, 270, 272 Bethell, F. H., 248, 275 Betker, N., 36, 59 Beyer, G. T., 200, 209, 222, 238, 271 Bhaduri, P. N., 146, 168 Bieri, J. G., 8, 61 Bierich, R., 413, 422, 431 Biesele, J. J., 134, 165 Birkenmaier, O., 69, 98, I10 Risceglie, V., 46, 59 Bishop, F. W., 391, 396 Biss, R., 319, 324 Bizzozero, G., 96, 110 Blackman, G. E., 122, 129 Bland, L. J., 184, 195 Blangey, L., 336, 343, 357 Rligh, J., 52, 59 Blix, G., 417, 431 Bloch, K., 45, 59 Bloch, R. G., 361, 393, 396 Block, R. J., 40, 59 Blocker, T. C., 8, 67 Eloom, F., 400, 405, 410, 430, 431, 433, 434

Blumenthal, G., 279, 287, 325 Blunt, J., 427, 434 Bodenstein, D., 137, 165 Bodine, J. H., 123, 124, 125, 128, 292, 298, 321 Boell, E. J., 287, 288, 313, 314, 321 Boeri, E., 286, 322

479

AUTHOR INDEX

Bohatyrschuk, F., 456, 474 Bahm, 3.. 70, 110 Boivin, A., 133, 165 Rolling, D., 40, 59 Bolton, L., 422, 431 Bonnichsen, R. K., 286, 322 Boothroyd, E. R., 183, 195 Borderioux, I., 31, 67 Borsook, H., 266, 270 Borysko, E., 135, 138, 165 Boscia, H., 12, 63 Bourghardt, S., 462, 464, 475 Bourne, G. H., 268, 297, 270, 321 Bovey, R., 136, 167 Boyer, H. K., 28, 43, 59 Boyer, P. D., 263, 274, 318, 323 Boyland, E., 124, 127, 128, 424, 431 Braccini, C., 430, 432 Brachet, J., 120, 124, 128, 155, 156, 165, 212, 219, 222, 225, 265, 266, 270, 283, 294, 299, 304, 305, 309, 312, 313, 317, 321, 325 Brack, E., 400, 414, 425, 431 Bradfield, J. R. G., 225, 270 Brady, R. O., 266, 270 Branca, A., 184, 195 Brattgard, S-O.,457, 458, 459, 462, 464. 465, 466, 469, 470, 471, 472, 475 Braun-Falco, O., 411, 412, 433 Braunstein, A. E., 50, 59 vanBreemen, V., 122, 128 Bremy, P., 414, 432 Breuer, M . E., 149, 168 Brewer, P. A., 155. 167, 174 Bridges, C. B., 139, 165 Briggs, R., 156, 165, 219, 222 Brin, M., 34, 59 Brink, F., 122, 128 de Brion, G., 56, 59 Br6chner-Mortensen, K., 427? 435 Brock, N., 123, 124, 128 Brodersen, J., 405, 423, 432 Brown, G. B.,43, 62 Brown, G. L., 156, 165 Brues, A. M., 15, 25, 27, 28, 31, 32, 33, 40, 41, 51, 52, 59, 68 Bryan, C. E., 115, 116, 130 Bryant, J. C.,21, 6i Brygier, J., 266, 270

Bucher, O., 69,70, 71, 72, 73, 74, 75, 76, 78, 79, 82, 83, 84, 87, 88, 89, 91, 93, 94, 96,!?8,99, loo, 101, 102, 103, 105, 106, 107, 108, 110, 120, 124, 128 Bucholz, D. J., 28, 54, 64 Buck, J. B., 137, 138, 142, 146, 165 Bujard, E., 422, 432 Bullough, W. S., 28, 33, 40, 55, 59 Bunting, H., 417, 419, 422, 430, 432, 435 Burgess, L. E., 292, 321 Burkl, W., 411, 432 Burrows, M. T., 5, 25, 33, 34, 35, 59, 60 Burt, A. S., 28, 50, 51, 59 Burt, R. L., 123, 128 Burton, J. F., 334, 355, 357 C

Cadoni, G., 123, 124, 125, 128 Caffier, P., 185, 195 Cajal, R. Y., 423, 432 Calcagno, O., 26, 66 Callan, H. G., 156, 157, 158, 165, 168, 201, 202, 2222 Calved, F., 219, 223 Calvet, F., 265, 273 Calvin, M., 146, 148, 163, 165 Cameron, G., 5, 24, 27, 48, 49, 56, 59, 60 Campani, M., 426, 432 Campbell, J. G., 334, 357 Campbell, R. M., 145, 165 Cappel, L., 54, 55, 63 Carducci-Pitoni, A., 293, 326 Carleton, If. M., 5, 59 Carminati, V., 44, 59 Carnoy, J. B., 131, 138, 165 Carrel, A., 2, 3, 5, 6, 7. 8. 9, 15, 25, 35, 36, 42, 58, 59, 60 Caspersson, T.. 132, 133, 136, 138, 139, 142. 145, 162, 165, 468, 475 Cavallero, C., 427, 432 Cazal, P., 411, 415, 424, 432 Chaffee, E., 406, 433 Chain, E., 37, 61 Chalkley, H. W., 366, 371, 389, 392, 395 Chambers, R., 48, 49, 56, 60 Champy, C., 6, 60 Chantrenne, H., 219, 222, 238, 240, 253, 265, 266, 270, 296, 299, 305, 321

480

AUTHOR INDEX

Chantrenne-van Halteren, M. B., 219, Costello, D. P., 320, 321 Couteaux, R., 355, 357 222 Coyne, B. A., 39, 66 Chanutin, A., 243, 244, 259, 262, 273 Craig, W. M., 360, 397 Chargaff, E., 135, 169, 265, 271 Cramer, W., 413, 424, 432 Charles, A. F., 416, 417 Crampton, C., 296, 321 Chevillard, L,33, 61 ChPvremont, M., 53, 60, 267, 268, 270, Crane, R. K., 263, 273, 308, 321 Cravens, W. W., 291, 321 271 Crosbie, G. W., 221, 222, 265, 271 Child, C. M., 304, 305, 306, 316, 321 Crouse, H., 143, 146, 148, 168 Chlopin, N. C., 6, 60 Christensen, H. N., 7, 37, 38, 39, 40, 48, Crumpler, H . R., 7, 60 Cunningham, B., 3, 61, 67 60 Chrustschoff, G. K., 185, 195 D Ciaccio, C., 419, 432 Dalgaard, E., 422, 432 Claff, C. L., 123, 130 Dalgaard, J., 422, 432 Clara, M., 103, 110 Clark, E. L., 363, 364, 366, 367, 368, 371, Dalton, A. J., 233, 246, 248, 251, 268, 271, 274 372, 373, 374, 375, 376, 395, 396 Clark, E. R., 359, 360, 361, 363, 364, 365, Daly, M. M., 141, 165, 20.2, 222 366, 367, 368, 369, 370, 371, 372, 373, D’Amato, F., 163, 165 Daneel, R., 306, 321 374, 375, 376, 395, 396 Danes, B., 6, 33, 61 Clark, G. L., 157, 165, 456, 475 Claude. A., 134, 138, 139, 165, 167, 225, D’Angelo, E. G., 142, 165 226, 232, 233, 238, 243, 246, 247, 250, Daniel, R. J., 293, 321 Danielli, J. F., 136, 141, 162, 165, 167, 256, 261, 274, 2%, 321 219, 222, 223, 330, 332, 333, 334, 357, Claus, P. E., 120, 129 358 Cleland, K. W., 233, 234, 246, 264, 267, 268, 271, 274, ZSO, 282, 283, 307, 321 Danon, M., 132, 156, 157, 158, 159, 160, 166, 174 Clermont, Y., 417, 432 Darke, S. J., 20, 64 Cleveland, L. R., 161, 165 Clowes, G. H. A., 123, 129, 282, 323 Darlington, C. D., 146, 152, 165, 179, 195 Dauvillier, M. A., 455, 475 Coca, F., 6, 60 Cohen, C., 411, 434 Davidson, J. N., 23, 43, 44, 45, 54, 55, Cohen, P. P., 51, 60, 266, 271 61, 64, 145, 152, 162, 166, 168, 188, 196, 200, 219, 221, 222, 223, 241, 265, Cohen, R. B., 333, 338, 354, 357. 358 266, 271, 273, 274 Cohen, S. S., 282, 310, 321 Cohn, M., 314, 325 Davies, D. V., 406, 419, 432 Davies, R. E., 268, 270 Comati, D. R., 424, 432, 433 Cornmandon, J., 219, 222 Davis, H. F., 442, 452 Compton, A., 2.2, 23, 25, 27, 66, 405, 417, Davis, H. G., 468, 475 419, 422, 432 Dawson, I. M., 162, 168, 219, 223, 241, 265, 266, 274 Conway, H., 376, 385, 396 Cooper, K. W., 136, 146, 162, 165 Deasy, C. L., 266, 270 Cooper, Z., 452, 452, 453 de Duve, C., 225, 226, 229, 232, 233, 234, 236, 237, 241, 243, 244, 245, 246, 247, Corbet, A., 208, 222 Cori, C. F., 254, 275 248, 249, 252, 254, 255, 256, 260, 262, Corkill, A. B., 27, 63 267, 268, 270, 271, 272, 288, 321 Cornman, I., 56, 60, 114, 115, 116, 117, Dees, M. B., 438, 452 126, 128 Defendi, V., 337, 358

481

AUTHOR INDEX

Delaunay, A., 34, 56, 61 Delaunay, M., 56, 61 Dempsey, E. W., 415, 417, 419, 422, 424, 432 435 439, 440, 442, 444, 445, 446, 449, 452, 453 Demuth, F., 15, 28, 35, 61, 62 DeNicola, M., 299, 316, 325 Dent, C. D., 7, 60 Denues, A. R. T., 134, 165 Denz, F., 355, 357 Deringer, M. K., 400, 410, 432 De Robertis, E., 448, 453 Desreux, V., 243, 255, 274 Dettmer, N., 447, 452, 453 Deuel, H. J., Jr., 244, 271 Deufel, J., 12, 128 Deutsch, H. F., 284, 285, 286, 287, 322 Deysson, G., 1.22, 128 Dianzani, M. U., 261, 271 Dickinson, S. J., 335, 358 Dingwall, J- A., 7, 13, 63 Djurle, E., 462, 464, 475 Doan, C. A., 400, 432 Dobzhansky, T., 154, 168, 294, 322 DodC, M., 122, 129 Ddson, E. O., 154, 155, 156, 159, 160, 142, 165 Dogliotti, G. C., 70, 73, 110 Doljanski, L., 4, 7, 10, 61, 63, 65 Dounce, A. L., 199, 200, 201, 202, m3, 204, 205, 206, 207, 209, 210, 211, 213, 218, 219, 220, 221, 222, 225, 238, 239, 244, 258, 271 Downey, H., 400, 401, 432 Downing, V., 391, 396 Doxey, D., 122, 128 Doyle, W . L., 284, 322 Drew, A. H., 22, 27, 61 Druckrey, H., 123, 124, 128 Drysdale, G. R., 43, 61, 260, 271 Dubin, I. N., 24, 61 Duerst, M. L,22, 65 Duesberg, J., 184, I95 Dunn, T. B., 400, 410, 432 Duryee, W. R., 132, 154, 156, 157, 158, 159, 160, 165, 166 Dustin, P., Jr., 116, 120, 121, 128 Duthie, E. S., 37, 61

E Eakin, R. M., 304, 305, 322 Earle, W. R., 2, 6, 13, 21, 22, 24, 27, 57, 61, 65, 66, 67, 392, 395 Ebeling, A. H., 3, 6, 8, 9, 15, 25, 28, 29, 35, 36, 41, 42, 49, 50, 54, 55, 58, 60, 61

Ebert, J. D., 312, 322 Ebert, R. H., 361, 363, 367, 369, 370, 372, 373, 376, 393, 395, 396, 397 Edstrom, J., 445, 467, 469, 475 Eggleston, L. V., 39, 64 Ehrensvard, G., 10, 11, 26, 29, 30, 32, 37, 39, 41, 42, 43, 44, 45, 58, 61, 62, 64, 265, 271 Ehrich, W., 69, 110 Ehrlich, P., 415, 422, 432, 442, 453 Ehrmann, R. L., 14, 26, 61 Eichenberger, M., 296, 322 Eliasson, N. A., 265, 271 Ellenhorn, J., 139, 166 Elliott, K. A. C., 56, 61 Ellis, J. P., 8, 61 Elman, R., 31, 61 Elson, D., 265, 271 Elvehjem, C. A., 208, 223, 232, 274 Enders, J. F., 8, 27, 61, 68 Engebreth-Holm, J., 430, 432 Engle, R. L., Jr., 442, 444, 453 Engstrom, A., 142, 152, 166, 456, 457, 458, 464, 448, 475, 476 Ennis, W. B., Jr., 122, 128 Ephrussi, B., 33, 61 Erlenbach, F., 27, 61 Erlichman, E., 5, 15, 26, B, 36, 41, 47, 48, 53, 54, 61, 68 Emster, L., 279, 283, 308, 319, 324 Esposito, S. M., 289, 322 Essex, H. E., 361, 363, 368, 370, 373, 374, 397, 398 Estable, C., 360, 397 Estey, K. C., 28, 64 Euier, H. von, 287, 292, 322 Evans, H. M., 182, 184, 185, 196, 197 Evans, V . J., 13, 21, 57, 61, 66, 67 Eyler, R. W., 456, 475 Ezell, D., 51, 52, 65

482

AUTHOR INDEX

F Faber. V., 427, 435 Fahr, 4173, 432 Falkenheirn, hf., 287, 306, 325 Fawns, H. T., 13, 66 Felix, M., 251, 274 Fell, H. B., 46, 47, 61 Fellinger, K., 124, 128 Felsovanyi, .4. von, 292, 321 Ferguson. J., 122, 1-39 Ferraniola, R., 53, 66 Fiegelson, hl., 264, 272 Fierz-David, H. E., 336, 343, 357 Fischberg, M., 179, 183, 189, 195, 196 Fischer, A,, 1, 3, 5, 6, 8, 9, 10, 11, 15, 26, 29, 30, 32, 35, 36, 37, 38, 39, 41, 42, 44, 45, 49, 50, 51, 52, 54, 61, 62, 67, 424, 432 Fischer, F., 135, 167, 208, 210, 223 Fischer, G., 32, 39, 62 Fischer, H., 39, 60 Fisher, H., 300, 324 Fisher, K. C., 123, 129 Fisher, R. A.. 87, 110 Fitzgerald, L. R., 123, 124, 125, 128, 292, 321

Flemming, W., 131, 157, 166, 185, 196 Flickinger, R. A., 312, 322 Florey, H. W., 361, 363, 367, 372, 373, 376, 394, 396, 397 Flory, C. M., 360, 397 Foa, P. P., 54, 62 de Fonbrune, P., 219, 222 Foulks, J. G., 318, 322 Franchi, C. M., 448, 453 Francis, M. D., 37, 62 Frazer, A. C., 45, 62 Frazer, S. C., 152, 166, 188, 196 FrCdiric, J., 53, 62, 267, 268, 270, 271, 306, 322 Freer, R. M., 202, 204, 222, 238, 271 Freerksen, E., 70, 110 Friedberg, F., 304, 305, 322 Frey-Wyssling, A., 142, 166, 446, 453 Friberg, H., 417, 432 Friedenthal, H., 184, 196 Friedenwald, J. S., 330, 332, 334, 357 Friedheim, A. H., 28, 62 Friedman, 0. M., 330, 335, 358

Fritze, E., 120, 130 Frolova, S. L., 141, 144, 166 Fromme, F., 413, 424, 432 Fuhner, H., 120, 129 Fuhrmatin, K., 120, 129 Fullam, E. F., 24-5, 271 Fulton, G. P., 430, 432 G

Gaillard, P. J.. 7, 48, 55, 62, TO, 110 Gall, J. G., 157, 158, 166 Ganguly, J., 244, 271 Gates, R. R., 179, 196 Gattiker, R., 70, 73, 75, 78, 79, 84, 88, 91, 93, 94, 96, 98, 99, 102, 103, 105, 106, 107, 108, 110 Gautier, A., 248, 270, 273 Gavaudan, P., 122, 129 Gay, H., 135, 141, 146, 166 Gebelein, H., 88, 110 Gebler, H., 43, 62 Geiersbach, U., 120, 129 Geiman, Q. M., 23, 24, 52, 58, 65 Geitler, L,132, 147, 152, 163, 166, 174 Gender, R. L., 126, 129 Gerarde, H. W., 35, 39, 43, 62 Gersch, M., 283, 326 Gey, G. O., 6, 7, 13, 14, 22, 23, 24, 25, 26, 27, 53, 61, 62, 67 Gey, hl. K., 6, 7, 13, 22, 2.3, 24, 27, 62 Gianetto. R., 226, 232, 245, 246, 247, 249, 252, 254, 255, 271 Gilligan, D. R., 439, 453 Gilman, A., 318, 322 de Giorgi-Ferrari, 415, 432 Glegg. R. E., 417, 432 (;lick, D., 244, 273, 430, 434, 468, 475 Glock, G. E., 308, 322 Gohy, P., 455, 475 Goethart, G., 248, 271 GoksGyr, Y., 286, 322 Goldhaber, G., 4, 61 Goldinger, J. M., 43, 50, 58, 59 Goldschmidt, J., 7, 63 Goldschmidt, R., 146, 148, 155, 163, 165, 166

Comes da Costa, S. F., 53, 62 Gomori, G., 332, 333, 338, 340, 343, 344, 347, 350, 351, 352, 355, 357

483

AUTHOR INDEX

Goodwin, R. F. W., 7, 62 Gopal-Ayengar, A. R., 138, 143, 164 Gordon, M., 308, 326 Gordon, M. W., 313, 322 Gordonoff, T., 46, 47, 49, 50, 62 Gottschalk, W., 127, 129 Gowans, J. L., 394, 397 Graf, W., 417, 432 Graff, A. M., 219, 222 Graff, S., 219, 222 Graham, H., 424, 432 Grana, A., 373, 397 Gray, J., 410, 432 Greco, A. E., 243, 245, 260, 273 Green, D. E., 240, 246, 262, 271, 272, 279, 291, 322 Green, J. W., 120, 124, 129 Green, M. H., 329, 330, 332, 357, 358 Greenberg, D. M., 266, 274 Greenberg, G. R., 43, 62, 308, 322 Greenberg, M.,54, 62 Greene, H. S . N., 388, 397 Greenstein, J. P., 286, 322 Gregg, J. R., 305, 322 Gretchishkh, S. V., 456, 47.5, 476 Grisolia, S., 266, 271 Grogg, E., 332, 335, 340, 343, 352, 357 Gross, J., 439, 448, 453 Gross, P. R., 290, 322 Grossfeld, H., 8, 62 Griffen, A. B., 137, 139, 167, 169 Guercio, F., 55, 67 Guillery, H., 35, 62 Guinochet, hl., 122, 130 Gunsalus, I. C., 50, 62 Gurin, S., 43, 67, 266, 270 Gustafson, T., 277, 279, 280, 283, 284, 285, 286,287, 292, 293, 295, 296, 298, 300, 301, 302, 304, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 321, 322, 323, 324, 325, 326 Gutherz, S., 184, 196 Gutters, E., 306, 321 GuyCnot, E., 132, 156, 157, 158, 159, 160, 166 Guyer, M. F., 120, 129, 184, 196

H Haagensen, D. E., 371, 396 Haagen-Smit, A. J., 266, 270 von Haam, E., 54, 55, 63 Haas, P., 7, 59 Haddow, A., 2, 63, 116, 120, 129 Hagan. W. A,, 208, 222 Haguenau, F., 248, 270, 273 Hale, C. W., 407, 432 Hall, D. A., 440, 442, 453 HallCn, O., 462, 464, 475 Hamberger, C.-A., 468, 472, 475 Hamburger, C., 427, 435 Hamer, D., 135, 166 Hamilton, H. L., 51, 63 Hamilton, P. B., 42, 63 Hamilton, T. R., 414, 432 Hammar, J. A., 406, 432 Hammarsten, E., 265, 271 v. Hamos. L., 456, 475 Hanes. F. M., 6, 64 Hanks. J. H., 22, 27, 63 Hannah, A., 144, 166 Harding. C. V., 312. 322 Harding, D., 312, 322 Hardy, W., 422, 432 Hargreaves, A. B., 286, 287, 322 Harma, R., 424, 432 Harman, J. W., 246, 256, 2h4, 272 Harris, H., 402, 415, 425, 432 Harris, M., 11, 12, 13, 14, 23, 24, 32, 44, 63. 64 Harris, P., 201, 222 Harrison, R. G., 5,' 63 Hartelius, H., 468, 475 Harting, J., 294, 325 Hartman, J. D., 34, 63 Hartree, E. F., 123, 129 Hartridge, H., 337, 3.57 Harvey, E. B., 184. 196, 280, 282, 283. 297, 306, 322, 325 Harvey, R. A., 389, 397 FTass, C;. M . , 12, 63, 65, 238. 274, 437, 453 Hasselberg. T., 279. 280, 287, 293, 295, 30-3, 304, 307. 313, 318, 322 llaurowitz. F., 296, 321 HazLn, S. J., 2.38, 253, 272

484

AUTHOR INDEX

Heagy, F. C., 145, 168, 200, 223 Heale, J., 437, 453 Healy, G. M., 26, 27, 63 Heaton, T. B., 6, 63 Heatley, N. G., 394, 397 Heberer, G., 184, 196 Hecht, L., 243, 254, 257, 273 Heiberg, K. A., 69, 110 Heidelberger, C., 265, 266, 275 Heilbrunn, L. V., 290, 322 Heilman, D. H., 56, 63 Heilmeyer, L., 124, 129 Heinrich, M. R., 43, 63 Heitc, H.-J., 88, 110 Heitz, E., 137, 138, 139, 144, 146, 166 Heiwinkel, H., 292, 322 Heller, L., 287, 322 HellstrGm, B., 406, 432 Henderson, M. E., 38, 60 Hengstmann, H., 48, 49, 50, 63 Henry, C. G., 372, 397 Henry, R. J., 123, 129 Henshaw, P. S., 113, 129 Herbst, C., 97, 110, 277, 322 Herken, H., 123, 124, 128 Hers, H. G., 233, 234, 237, 241, 243, 244, 249, 254, 256, 260, 272 Hershey, A. D., 300, 323 Herskowitz, I. H., 138, 139, 151, 162, 166, 167 Hertwig, G., 97, 110, 146, 147, 151, 152, 166 van Herwerden, M. A., 415, 432 Herzog, G., 401, 432 Hetherington, D. C., 26, 50, 51, 63 Higgins, H., 248, 272 Higuchi, K., 413, 422, 424, 432 Hill, R. T., 27, 63 Hillier, J., 298, 324 Himes, M., 136, 168 Hindmarsh, M. M., 122, 129 Hinton, T., 148, 166 Hintzsche, E., 69, 70, 74, 110, 111 Hird, F. J. R., 261, 272 Hirshfield, H. I., 219, 223 Hissard, R., 426, 432 Hitchcock, M. W . S., 7, 63 Hitschler, W . J., 366, 396 Hjelte, M.-B., 279, 293, 301, 302, 322

Hoadley, L,184, 196 Hoeppli, R. J . C.,392, 397 Hoerr, N . L., 225, 270 Hoff -Jdrgensen, E., 295, 323 Hoffman, R. S., 7, 13, 61, 63 Hogberg, B., 292, 322, 418, 431 Hogeboom, G. H., 202, 212, 213, 214, 217, 218, 222, 223, 225, 226, 232, 233, 234, 235, 238, 239, 243, 244, 246, 247, 248, 249, 254, 255, 256 257, 259, 261, 262, 265, 268, 269 272, 274 Hohl, K., 122, 129 Holmgren, H., 401, 406, 407, 414, 416 425, 432, 433 Holt, S. J., 329, 345, 349, 355, 356, 357 Holter, H., 225, 230, 239, 241, 248, 250, 253, 272, 279, 280, 282, 287, 304, 306, 323 Holtfreter, J., 283, 292, 297, 309, 323 Hopkins, F. G., 43, 63 Horecker, B. L., 308, 323 Horisberger, B., 70, 71, 72, 74, 76, 93, 110 Horne, R. W., 238, 274 Horowitz, N . H., 120, 123, 130 Horstadius, S., 219, 222, 223, 292, 302, 304, 305, 306, 307, 308, 310, 311, 316, 317, 323, 324 Hosono, S., 47, 63 Hotchkiss, R. D., 407, 417, 433 Hou, H. C., 363, 368, 382, 392, 396, 397 Houlikan, R. K., 314, 326 Houwink, A. L., 134, 166 Howard, A., 133, 161, 168 Howell, W. H., 416, 433 Howes, E., 427, 434 Hoyer, H., 415, 433 Hsu, T. C., 185, 186, 193, 195, 196 Hsu, W. S., 162, 166 Hudspeth, E. R., 33, 34, 63 Huennekens, F. M., 246, 272 Hueper, W. C., 3, 6, 36, 41, 63 Hrrggett, A. St. G., 7, 59 Hughes, A., 44, 63, 134, 166, 186, 196 Hughes, A. F. W., 6, 63, 120, 129 Huisman, T. H. J., 124, 129 Hull, W., 28, 43, 51, 63, 67

AUTHOR INDEX

485

Hultin, T., 162, 166, 266, 272, 283, 290, 300, 301, 307, 308, 309, 323 Hunter, F. E., Jr., 258, 263, 264, 272 Hurlbert, R. B., 219, 222, 225, 232, 250, 265, 274 Huseby, R. A., 219, 222, 237, 238, 253, 265, 270 Huskins, C. L., 163, 166, 190, 196 Hutchens, J. O., 298, 323 Hutchison, W. C., 145, 168, 200, 223 Hydkn, H., 457, 458, 459, 462, 464, 465, 466, 468, 469, 471, 472, 475

Johnson, R. B., 264, 267, 272 Jones, H. B., 265, 274 Jones, M., 35, 39, 43, 62 Jordan, H. E., 184, 196 Jorpes, E., 406, 407, 416, 417, 418, 425, 433 J o s h , D., 361, 376, 385, 396, 397 Judah, J. D., 264, 272 Juhisz-Schfiffer, A., 47, 64 Julen, C., 402, 404, 416, 419, 433, 434 Junge, J., 329, 330, 332, 357, 358

I

Kabat, E. A., 244, 249, 272 Kachmar, J. F., 318, 323 Kahler, H., 233, 246, 248, 268, 271 Kandutsch, A. A., 49, 64 Kaplan, H. S., 391, 397 Kaplan, J. G., 286, 323 Karplus, H., 182, 185, 196 Katersky, E. M., 439, 453 Katzenstein, M., 4, 64 Kaufmann, B. P., 135, 141, 145, 146, 148, 166 Kauzmann, W. J., 126, 129 Kavanau, J. L., 279, 292, 321, 323 Kay, E. R. M., 200, 217, 222, 244, 271 Keighley, G., 266, 270 Keilin, D., 123, 129 Keining, E., 411, 412, 433 Kelly, L. S., 265, 274 Kelly, M. G., 233, 246, 268, 271 Keltch, A. I<., 282, 323 Kemp, T., 184, 185, 196 Kendal, L. P., 4, 22, 24, 36, 68 Kennedy, E. P., 233, 243, 244, 256, 257, 260, 261, 263, 266, 267, 272, 273 Kennedy, T. J., 318, 325 Kensler, C . J., 243, 272 Kiaer, S., 6, 64 Kidder, G. W., 2, 32, 64 Kielley, R. K., 243, 247, 257, 258, 263, 264, 266, 272 Kielley, W. W., 243, 247, 257, 258, 263, 264, 272 Kiessling, K.-H., 302, 318, 324 Kihara, H., 37, 64, 1&I, 197 Kimura, T., 10, 64 King, G., 7, 59

Ide, A. G., 388, 389, 390, 397 Ignatjewa, 2. P., 70, 93, 111 Ikeda, C., 288, 325 Immers, J., 289, 323 Imperati, L., 123, 124, 125, 128 Infantellina, F., 290, 323 Ingebrigtsen, R., 6, 63, 64 Ingelstam, E., 462, 464, 475 InouC, S., 138, 166 Isbell, E. R., 294, 323 Ishida, J., 289, 323 Ivens, G. W., 122, 129 Tvers, J. B., 8, 55, 64 Iversen, Kurt, 424, 427, 431, 433

J Jackson, E. B., 15, 25, 27, 28, 31, 32, 33, 40, 41, 51, 52, 59, 68 Jacobj, W., 69, 70, 73, 98, 103, 111, 147, 166 Jacbbson, W., 146, 166 Jacoby, F., 3, 4, 9, 20, 44, 64, 68 Jacquet, J., 426, 432 Jacquez, J. A., 13, 39, 46, 64 Jaegar, L., 141, 167, 294, 321 James, T. W., 201, 222 Janes, J., 406, 433 Jazimirska-Krontowska, C., 25, 64 Jeener, R., 219, 222, 2 5 , 240, 265, 270, 272, 296, 300, 323 Jenrette, W. V., 286, 322 Jiewertz, B., 462, 464, 475 Jiv, B. V., 182, 185, 189, 195 Johnson, F. H., 126, 129 Johnson, M., 28, 33, 40, 59

K

486

AUTHOR INDEX

King, R. L., 184, 196 King, T. J., 156, 165, 219, 222 Kirby, D. B., 28, 64 Kirby-Smith, H. T., 360, 361, 366, 376, 396, 397 Kirk, P. L., 3, 14, 28, 43, 51, 59, 61, 63, 66, 67 Kirkman, H., 415, 417, 422, 433 Klatt, 0. A,, 37, 64 Kleinfeld, R., 146, 156, 168 Kligman, A. M., 56, 58 Knake, E., 4, 64 Kneen, E., 288, 324, 325 Knight, B. C. J. G., 3, 64 Kodani, M., 146, 148, 163, 165, 166 Koelle, G. B., 355, 357 Koller, P. B., 181, 184, 196 Koller, P. C., 144, 151, 166 Kijlliker, A. von, 437, 453 Kolpak, H., 449, 453 Koltzoff, N. K., 131, 138, 139, 146, 155, 159, 160, 166 Kopac, M. J., 298, 323 Kornberg, A., 241, 248, 272 Korner, F., 73, 111 Korschelt, E., 138, 166 Kosswig, C., 146, 149, 166 Kosterlitz, H. W., 145, 165 Kostoff, D., 137, 166 Krahl, M. E., 123, 129, 298, 323, 324 Krakaur, R., 219, 222 Kranta, H., 97, 111 Krebs, H. A., 23, 24, 39, 40, 47, 48, 50, 51, 52, 64 . Kreke, C. W., 123, 129 Krimsky, I., 41, 66 Kriszat, G., 290, 305, 308, 324, 326 Krugelis, E. J., 143, 166, 304, 312, 324 Kuczinski, M. H., 35, 53, 64 Kuff, E. L,251, 274 Kiihle, E., 208, 210, 223 Kulonen, E., 427, 433 Kurnick, N. B., 151, 155, 162, 167 Kutsky, P. R., 305, 307, 321, 322 Kutsky, R., 13, 64

L La Cour, L. F., 183, 196 La Grutta, G., 290, 323

Lafon, C., 400, 401, 410, 434 Lagerkvist, U., 43, 64 Lagerstedt, S., 145, 167 Laing, M. A., 120, I28 Laird, A. K., 238, 250, 272 Lallier, R., 296, 309, 316, 317, 318, 324 Lamarque, P., 456, 475, 476 Lamb, W . G. P., 134, 136, 167 Lambert, R. A., 6, 25, 64 Lams, H., 178, 179, 184, 196 Lan, T. H., 201, 222 Landschiitz, C., 11, 12, 32, 39, 62, 64 Landsteiner, K., 6, 64 Lang, H., 208, 223 Lang, K., 135, 167, 203, 205, 206, 208, 210, 222, 223, 225, 232, 238, 258, 260, 267, 273 Langemann, H., 243, 272 Lansing, A. I., 298, 324, 439, 440, 442, 444, 445, 446, 448, 452, 452, 453 Lardy, H . A., 43, 61, 65, 233, 257, 258, 260, 263, 264, 271, 273, 274 Larsen, C. D., 114, 115, 129 Larsson, L G., 404, 430, 434 1-arsson, S., 462, 464, 475 Lasfargues, E., 11, 68 Lasnitski, I., 36, 39, 58, 120, 124, 129 Latta, J. S., 28, 54, 64 Laurent, T. C., 418, 431 Lawler, T. G., 30, 64 Lawrence, E. G., 132, 138, 147, 167 Lazarow, A., 238, 273 Leaf, G., 156, 165 Leblond, C. P., 417, 433 Lebrun, H., 131, 165 Lebrun, J., 56, 61 Lee, N. D., 266, 273 Lefhvre, J., 122, 129 T.egallais, F. Y., 361, 370, 376, 390, 391, 392, 395 Lehmann, F. E., 201, 221, 297, 316, 317, 319, 324 Lehner, J., 400, 402, 404, 410, 41.5, 423, 433 Lehninger, A. I,., 233, 243, 244, 247, 254, 256, 257, 260, 261, 262, 263, 264, 266, 267, 270, 272, 273, 274 Leinfelder, P. J., 33, 61 Leloir, L. F., 260, 273

487

AUTHOR INDEX

Lenicquc, P., 283, 284, 295. 298, 302, 30.1, 306, 309, 310, 311, 315, 316, 317, 322, 324 Letinerstrand, A., 292, 324 Leonard, E., 308, 326 I,eonhartsberger, F., 411, 433 LePage, G. A., 243, 256, 261, 265, 266, 273, 275 Lipine, P., 11, 13, 59 Lesher, S., 146, 152, 162, 167 I ~ s l i e ,I., 43, 45, 54, 61, 64 Lettri, H., 53, 55, 64 Leuchtenberger, C., 152, 155, 168 Txuthardt, F., 256, 261, 266, 273 Levi, G., 105, 111 Levinson, J. P., 368, 370, 397 1-evinthal, C., 300, 324 Levy, M., 291. 324 Levvy, G. A., 255, 275 Lewis, M. R., 4, 5, 22, 24, 26, 8,28. 29, 30, 34, 64, 65 1-ewis, 1%'.H., 4, 5, 6, 22, 23, 24, 26, 30, 34. 64, 65* 70, 111, 366, 397 Ti. .I.G., 22, 6.5 Lichenstein, N., 289, 324 Liebow, A. A., 42, 67 des Ligneris, hl. J. A., 8, 65 Likely, G. D., 2, 6, 57, 65, 67 Lillie, R. D., 417, 419, 433 Lillie, R. S., 120, 129 I h , I., 43, 67 Tindahl, P.-E., 277, 278, 279, 287, 292, 295. 296, 302, 304, 305, 306, 307, 309, 311, 312, 316, 317, 318, 319, 323, 324 Lindan, O., 7, 60 Lindberg, O., 8 9 , 283. 295, 307, 308, 318, 319. 324 Lindstrom, B., 457, 458, 464, 4643, 471, 475, 476 Lindvall, S., 287, 324 Linnert, G., 127, 129 Lipmann, F., 263, 273 Lipnik, M. J., 56, 58 Lipton, M. A., 50, 59 Lison, L., 312, 324, 416, 433 I.itt, M., 215, 222, 239, 271 Littlejohn, J. M., 238, 272 Lloyd, B. J., 233, 246, 248, 268, 271 Locke, F. S., 22, 27, 65

Loeb, L., 6, 6.5, 388, 397 Loewenthal, N., 422, 433 h e w u s , F. A,, 440, 453 Logan, R., 162, 168, 219, 223, 241, 265, 266, 274 Lombardo, E., 424, 433 Lombard, G. L., 316, 317, 324 1-onberg-Holm, K. K., 34, 65 Loomis, W. F., 2G2, 271 Lorch, I. J., 219, 222, 223 Lorenz, W., 120, 129 Loveless, A., 332, 334, 358 Lbvtrup, S., 305, 322 Lowens, M., 308, 326 Lowry, 0. H., 439, 4.53, 466, 476 Lowy, P. H., 266, 270 Lu, K.-H., 124, 128, 298, 321 Lucius, E., 208, 210, 223 Lucius, S., 208, 223 Lucas, R. V., 442, 444, 445, 452 Ludewig, S., 243, 244, 259, 262, 273 Ludford, R. J., 120, 129 Ludwig, F., 46, 47, 49, 50, 62 Lundblad, G., 282, 289, 325 Lundin, J., 309, 319, 324 Lundy, J. S., 368, 397 Lushbaugh, C. C., 120, 124, 129 de Lustig, E. S., 8, 65 Luther, W., 120, 129 Lwoff, A., 310, 325 Lyle, G. G., 264, 274, 294, 325 Lyman, C . M., 50, 59 Lynch, E. L., 48, 60 Lynen, F.. 260, 273

M McClean, D. A., 424, 431, 433 McClung, C. E., 184, 196 McCutcheon, M., 424, 433 McDonald, J. R., 406, 433 McDonald, M. R., 135, 141, 146, 166 MacDuffee, R. C., 139, 168 Macfarlane. M. G., 268, 273 VcHenry, E. W., 7, 68 McIndoe, W. M., 162, 168, 219, 223, 241, 265, 266, 273, 274 McKee, R. W., 23, 24, 34, 52, 58, 65 Macklin, Ch. C., 102, 111 McLean, P., 308, 322

488

AUTHOR INDEX

Macleod, J., 34, 65 MacLeod, P. R., 43, 65 McManus, J . F. A., 417, 433 McNeil, E. H., 292, 321 McShan, W. H., 238, 252, 253, 273 Maganini, H., 12, 65 Mainx, F., 137, 167 Makino, S., 136, 167, 179, 181, 184, 196 Mall, F. P., 437, 447, 453 Malm, M., 318, 325 Malmgren, H., 417, 434 Malmstrom, B. G., 468, 475 Mangieri, C., 410, 434 Manheimer, L. H., 330, 332, 358 Mann, G. V., 50, 67 Manna, G. I<., 195, 196 Manners, D. J., 32, 65 Manton, I., 161, 167 Marc, S., 411, 433 Margoliash, E., 7, 10, 65 Markham, N . P., 394, 397 Marquardt, H., 127, 129 Marshak, A., 139, 162, 167, 219, 223, 238, 265, 273, 294, 325 Matsuura, K., 291, 325 Matthey, R., 178, 179, 193, 196 Maurin, J., 11, 13, 59 Mauron, J., 256, 273 Maver, M. E., 243, 245, 260, 273 Maximow, A., 400, 402, 404, 405, 433 Mayer, A., 33, 61 Maynard, F. L., 430, 432 Mazia, D., 135, 141, 155, 162, 165, 167, 219, 223, 279, 280, 282, 287, 298, 325 Medawar, P. B., 28, 33, 65 Meirowsky, E., 423, 433 Meister, A., 51, 65, 244, 273 Melander, Y., 190, 196 Melass, V . H., 52, 66 Militzer, R., 288, 325 Miller, B. S., 288, 325 Miller, J . A,, 243, 248, 257, 261, 272, 273 Miller, R., 266, 273 Minganti, A., 307, 325 Minouchi, O., 184, 196 Mirsky, A. E., 133, 134, 135, 141, 155, 162, 164, 165, 167, 168, 201, 202, 203, 204, 206, 210, 221, 222, 223, 238, 258, 270, 273, 275, 294, 312, 325

Mellanby, E., 46, 47, 61 Melland, A. M., 137, 154, 167 Menten, M. L., 329, 330, 332, 357, 358 Merk, R., 124, 129 Merwin, R., 391, 397 Messina, L., 48, 65 Metz, C. W., 132, 138, 139, 144, 146, 147, 167, 168 Meyer, K., 406, 414, 415, 418, 427, 433, 434 Meyer, R., 70, 111 Meyer, R. H., 124, 130 Meyer, R. K., 238, 252, 253, 273 Meyerhof, O., 123, 129 Michels, N . A., 400, 401, 402, 405, 416, 422, 423, 433 Miszurski, B., 7, 65 Mitchell, H . K., 294, 323 Mitchell, J. H., 43, 67 Mitchell, J . H., Jr., 116, 128 Moeschlin, S., 120, 129 Molas, L. G. Guilera, 184, 196 vonMBllendorff, M., 70, 111 von Miillendorff, W., 55, 65, 70, 111, 120, 124, 129 Mollier, S., 384, 397 Moment, G., 120, 129 Moncourier, L., 426, 432 MonnC, L., 289, 295, 319, 325 Monod, J., 313, 314, 325 Monroy, A., 299, 316, 325 Monroy-Oddo, A., 299, 316, 325 Montagna, W., 316, 325, 419, 433,434 Montgomery, T. H., 181, 184, 196 . Moog, F., 283, 298, 326 Moore, J . E. S., 184, 196 Moore, R. L., 361, 397 Moppett, W . A., 8, 65 Morgan, J. F., 5 , 15, 21, 26, 27, 40, 41, 42, 44, 45, 46, 47, 48, 49, 50, 51, 52, 63, 65 Morgan, T. H., 312, 325 Morris, H . P., 366, 371, 395 Morita, S., 134, 169 Morse, A., 7, 65 Morton, G. A., 158, 167 Morton, H. J., 15, 21, 26, 40, 41, 42, 44, 45, 46, 47, 48, 49, 50, 51, 52, 65 Mottet, N . K., 469, 476

489

AUTHOR INDEX

hlottram, J. C., 6, 65 hfudge, G. H., 318, 322 hlueller, G. C., 243, 257, 261, 273 Miihlethaler, K., 246, 273 hfuir, H.. 45, 66 hfiiller, A. F.. 246, 261, 266, 273 hfiiller, E., 208, 210, 223 hliiller, H. G., 73, 111 Sluller, H. J., 132, 137, 139, 167, 180. 196 Miiller, H. H., 120, 130 Nliiller, L., 208, 210, 223 Muiioz, J. M., 260, 273 hliinzer, F. Th., 102, 103, 105, 111 Myrhack, K., 288, 325

N Navez, A. E., 282, 325 Needham, J., 33, 65 Negelein, E., 12, 30, 68 Neidhardt, H. W., 8, 55, 64 Nettleship, A.. 113, 129 Nettleship, W. A., 28, 64 Newcomh, E. H., 264, 275 Neweli, Q. U.,184, 195 Neymann, C. A., 34, 35, 59 Nielsen, H., 266, 273 Niemann, C., 36, 59 Niemeyer, H., 263, 273 Nixon, D. A., 7, 52, 65 Nohack, C. R., 419, 422, 433, 434 Noc, E., 235, 238, 214, 250, 251, 252, 256, 259, 262. 273 Novikoff, A. B., 235, 238. 243, 244, 250, 251, 252, 254, 256, 257, 259, 262, 273, 274

Nowinski, W. W., 8, 33, 61, 65, 66, 67 Nungcster, W . J., 48, 65 Niirtiberger, J., 468, 476 Nygaard, O., 238, 250, 272 Nace, G. W., 312, 322 Nnchlas, hf. M., 330, 332, 333, 352, 358 Nagayo, If.,400, 403, 415, 422, 434 Nakajima, Y., 403, 415, 419, 423, 434 Narisawa, S., 47, 63 Navaschin. M. S., 184, 185, 186, 187, 195

0 Oberling, C., 248, 270, 273 Ochoa, S., 260, 273

Odate, Z., 139, 169 Oehlkers, F., 127, 129 Oesterling, M. J., 40, 66 Oguma, K., 184, 196, 197 @hlenschlager, V., 10, 11, 26, 29, 30, 41, 45, 58, 62 6hman, L. O., 278, 307, 324 Ohta, T., 184, 196 Okamoto, B., 292, 325 Oksala, T., 190, 197 Okuda, N., 146, 166 Oliver, J., 410, 434 Olivo, 0. M., 8, 65 Omachi, A., 244, 273 Opie, E. L,283, 314, 325 Opsahl, J. C., 430, 434 Orloff, J., 318, 325 Ormsbee, R. A., 23, 24, 52, 58, 65, 123, 129 Omstein, L., 133, 134, 136, 167, 168 brtenblad, B., 288, 325 Osgood, E. E., 22, 65 Bstergren. G., 122, 129 Ota, Y., 139, 169 Ottesen, M., 230, 239, 241, 248. 250, 253, 272

P

Paff, G. H., 430, 434 Page, I. H., 368, 370, 395 Pa&, J., 34, 61 Painter, J. T., 51, 52, 54, 65 Painter, T. S.. 120, 129, 132, 137, 143, 144, 146, 147, 148, 151, 155, 156, 162, 163, 167, 181, 184, 197 Palade, G., 201, 223 Palade, G. E., 225, 226, 233, 234, 235, 239, 243, 246, 248, 249, 256, 268, 272, 273, 274, 403, 434 PaIatine, I. M., 39, 60 Palay, S. L., 138, 139. 167 Palmer, J. W., 418, 434 Pannett, C., 22, 23, 25, 27, 66 Pappenheim, A., 400, 415, 434 Park, H. D., 370, 392, 395 Parker, R. C., 3, 6, 8, 15, 21, 22, 24, 26, 27, 33, 34, 40, 41, 42, 44, 45, 46, 47, 48, 49, 50, 51, 52, 55, 62, 63, 64, 65, 66

490

AUTHOR INDEX

Pukes, A. S., 27, 63 Parks, H. F., 419, 434 Parshley, M. S., 23, 25, 40, 66 Partridge, S.M., 442, 452 Panijel, J., 299, 300, 325 Pasteels, J., 299, 312, 316, 317, 324, 325 Patau, C., 133, 167 Pate, S. G. M., 200, 222, 244, 271 Paterson, E.. 49, 50, 66, 120, 130 Paulmann, F. K., 33, 66 Pavan, C., 144, 149, 168, 172 Payne, A. H., 265, 274 Pearce, R. H., 411, 435 Pearse, A. G. E., 332, 334, 335, 337, 340, 343, 345, 350, 351, 352, 355, 357, 358 Pearson, P. B., 52, 66 Pease, D. C., 139, 168 Pelc, S. R., 133, 161, 168 Pennypacker, M., 149, 168 Perlmann, P., 279, 301, 312, 322, 325 Perrone, J. C., 51, 63 Perry, S. V., 238, 274 Persson, B. H., 417, 430, 434 Petermann, M. L., 214, 217, 223, 234, 239, 249, 274 Petersen, H., 406, 434 Peterson, E. A., 51, 65, 266, 274 Ffeiffer, H., 142, 168 Pfeiffer, H. H., 142, 168 Pfuhl, W., 70, 103, 111 Pickels, E. G., Z29, 274 Pillsbury, D. M., 56, 58 Pincus, G., 184, 197 Pires Soares, J . M., 41, 66 Pirie, N. W.. 9, 66 Pirotte, hl., 243, 255, 274 Pirwitz, J., 124, 129 Pitotti, M., 287, 325 Plantefol, L., 33, 61 Platt, M.,3, 6, 63 Plaut, G. W . E., 43, 61 Pletnev, S. -4.,183, 197 Plotz, C., 427, 434 Plotz, H., 51, 63 Podber, E., 235, 238, 243, 244, 250, 251, 252, 254, 256, 257, 259, 262, 273, 274 Polli, E. E., 134, 168 Pollister, A. W., 134, 136, 141, 155, 167, 168, 238, 273

Pomerat, C. M., 8, 31, 33, 34, 51, 52, 54, 55, 56, 63, 64,65, 66, 67, 195, 196 Ponder, E., 288, 325 Pontecorvo, G., 193, 197, 294, 325 Popjik, G., 45, 66 Porter, K. R., 248, 251, 253, 274, 447, 453 Portugalow, W. W., 70, 97, 111 Posener, K., 12, 30, 68 Posternak, J. M., 122, 228 Potter, J. S., 134, 165, 238, 271 Potter, V. R., 208, 219, 222, 223, 2-25, 232, 243, 249, 250, 256, 257, 258, 263, 264, 265, 266, 272, 274, 287, 294, 321, 325 Poulson, D. F., 144, 168,313, 325 Poussel, H., 122, 129 Prakken, J. R., 407, 414, 419, 426, 434 Pratt, J. P., 184, 195 Pressman, B. C., 233, 264, 274 Preston, M. M’E., 6, 63 Price, J. M., 248, 272 Prives, M., 456, 476 Prokofjeva, A., 139, 166, 167 Pudenz, R. H., 360, 397 Pullinger, B. D., 361, 376, 396 Pybus, F. C., 13, 66

0 Quaife, M. L., 157, 165 Quarles, E., 52, 67 Queiroz-Lopes, A., 141, 168

R Raaflaub, J., 264, 267, 268, 274 Rabinovitz, M., 263, 274 Rachmilewitz, M., 34, 66 Rafelson, M., 32. 39. 62 Rafn, M. L., 37, 60 Ragan, C., 414, 427, 434 Rall, T. W., 243, 261, 274 Ramasarma, G. B., 440, 452, 453 Randavel-Vendrely, C., 251, 274 Ranvier, L., 402, 434 Ranzi, S.,287, 306, 316, 319, 325 Rapkine, L., 309, 325 Rappeport, T., 182, 185, 197 Rapport, M. M., 406, 433 Rasch, E. M., 143, 151, 167, 168, 170

AUTHOR INDEX

Raven, C. P., 316, 317, 318, 319, 320, 325 Ravin, H. A,, 333, 338, 343, 358 Recknagel, R. O., 2 5 , 232, 250, 258, 263, 264, 274, 283, 325 Reed, R., 440, 442, 453 Rees, T. D., 385, 396 Reichard, P., 43, 66, 265, 271 Reimer, E. E., 120, 130 Reindorp, E. C., 147, 167 Reinhart, H. L., 400, 432 Rerabek, E., 44, 66 Rerabek, J., 44, 66 Restarski, J . S., 360, 397 Rex, R. O., 360, 361, 366, 373, 376, 396 deRezende, N., 374, 397 Rheindorf, A., 423, 434 Rhoads, C. P., 34, 65 Rhodin, J., 246, 248, 274 Rich, A., 308, 326 Richter, B., 472, 476 Ries, E., 283, 287, 325, 326 Riggs, T. R., 38, 39, 60 Riley, J. F., 411, 422, 425, 434 Rolfe. D. T., 292, 321 Ringer, S., 22, 27, 66 Ris, H., 132, 133, 134, 135, 141, 143, 145, 146, 148, 155, 156, 157, 158, 159, 161, 162, 164, 165, 167, 168, 174, 238, 250, 272, 273 Riser, W. H., Jr., 115, 116, 130 Roberts, B., 361, 363, 376, 398 Roberts, E., 440, 452, 453 Robertson, J. S., 361, 373, 376, 397 Roffo, A. H., 22, 24, 25, 26, 53, 66 Roll, P. M., 43, 62 Roosen-Runge, E. C., 190, 197 Rose, G. G., 8, 55, 66 Rose, S. M.. 313. 326 Rose, W. C., 20, 36, 39, 40, 66 Rosenberg, S., 14, 66 Rosenthal, H. L,53, 66 Rosenthal, O., 259, 274 Rosenthal, T. B., 298, 324, 439, 440,446, 452, 453 Rosin, A., 34, 66, 120, 130 Ross, E. J., 54, 66 Ross, H. E., 243, 274 Rossi, F., 47, 49, 66

49 1

Rothschild, Lord, 280, 282, 283, 286, 307, 321, 326 Rotter, H., 182, 185, 197 Roughton, F. J . W., 337, 357 Roukhelman, N., 28, 62 Rowley, D., 7, 59 Rowseil, E. V., 261, 272 Rozich, R., 238, 253, 273 Rozsa, G., 138, 168 Ruch, F., 142, 161, 756, 168 Riickert, J., 131, 156, 157, 159, 168 Runnstriim, J., 124, 130, 290, 291, 296, 305, 308, 309, 316, 318, 319, 320, 324, 326 Ruskin, A., 56, 66 Ruskin, B., 56, 66 Russell, M. A., 3, 6, 36, 41, 63 Rutenburg, A. M., 333, 338, 354, 357, 358 Ryan, I., 235, 238, 243, 244, 250, 251, 252, 254, 256, 257, 259, 262, 273 Rybak, B., 286, 326 Ryley, J . F., 32, 65 S Sabrazes, J., 400, 401, 410, 434 Sachs, L., 180, 193, 195, 197 Saetren, H., 135, 168, 201, 202, 203, 221, 223, 238, 258, 270, 275 Sagher, F., 411, 434 Salisbury, P. F., 32, 66 Salle, A. J., 55, 66 Sanders, A. G., 363, 367, 373, 394, 396, 397 Sanders, F. K., 300, 326 Sanders, M., 22, 24, 27, 67 Sandison, J. C., 360, 363, 367, 373, 376, 396,397 Sandstedt, R. M., 288, 324 Sanford, K. K., 2, 6, 13, 21, 57, 61, 65, 66, 67 Saphir, O., 414, 434 Sarett, H. P., 53, 66 Sassuchin, D., 415, 435 Saunders, B., 184, 197 Sauser, G., 69, 98, 111 Sawada, H., 142, 169 Sawyer, C. H., 314, 326 Schachow, S. D., 182, 185, 197

492

AUTHOR INDEX

Schaffer, J., 425, 434 Schairer, E., 69, 111 Scheid, H., 292, 326 Schein, A. H., 243, 256, 274 Schenck, H . P., 370, 395 Schick, A. F., 238, 274 Schlegel, J . U., 371, 395 Schlenk, F., 51, 67, 292, 322 Schmid, J., 124, 128 Schmidt, W . J., 142, 168 Schmith, K., 427, 435 Schmitt, F. O., 142, 168 Schneider, R. M., 234, 274 Schneider, W . C., 202, 212, 213, 214, 218, 222, 223, 225, 226, 227, 232, 234, 235, 238, 239, 243, 244, 246, 248, 249, 251, 254, 255, 256, 257, 260, 261, 262, 264, 265, 266, 268, 272, 273, 274, 294, 325 Schoch, E., 430, 434 Scholander, P. F., 123, 130 Scholander, S. I., 123, 130 Schorr, S., 411, 434 Schrader, F., 152, 168 Schreier, K., 7, 67 Schuler, J., 440, 452 Schultz, J., 139, 142, 143, 144, 145, 163, 165, 168 Schultz-Brauns, O.,465, 476 Schulze, E., 120, 130 Schumacher, J., 406, 434 Schutze, O., 131, 168 Schwartz, W., 447, 453 Schweigert, B. S., 292, 326 Schweitzer, A. W., 12, 63, 65 Scott, D. A., 416, 432 Sealock, R. R., 293, 326 Seaman, A. J., 22, 65 Seaman, G. R., 314, 326 Seldon, T . H., 368, 397 Seligman, A. M., 330, 332, 333, 335, 343, 352, 354, 357, 356 Semmens, C. S., 146, 168 Semura, S., 54, 55, 67 Sen, K. C., 123, 130 Sengiin, A., 146, 149, 166 Sentien, P., 120, 130 Serra, J. A., 141, 145, 144, 168 Sexton, W. A., 116, 120, 129

217, 233, 247, 259, 269,

154,

338,

Shannon, J. E., 13, 21, 57, 61, 66, 67 Shapiro, H., 282, 326 Shearer, C., 290, 326 Shechmeister, I . L., 55, 66 Shelden, C. H., 360, 397 Sherwood, M . B., 8, 58 Sherwood, R. M., 52, 66 Shipp, M. E., 26, 63 Shiw, B. W., 182, 185, 186, 187, 195, 197 Shiwago, P. I., 184, 187, 197 Shooter, R. A., 25, 26, 67 Siebert, G., 135, 167, 203, 205, 206, 208, 210, 222, 223, 232, 238, 258, 267, 273 Siekevitz, P., 257, 258, 263, 264, 266, 274, 314, 326 Sievert, R., 456, 476 Signorotti, B., 28, 67 Simms, H . S., 8, 12, 22, 23, 24, 25, 27, 40, 66. 67 Simon-Reuss, I., 43, 63 Simonet, M., 122, 130 Simons, D., 184, 196 Sirnonson, H . C., 258, 264, 274 Simpson, W . L., 413, 424, 432 Sinclair, H . M., 51, 67 Singal, S. A., 238, 253, 272 Singer, M., 442, 444, 452, 453 Sjostrand, F. S., 246, 248, 274 Sjostrand, F., 345, 358 Skipper, H . E., 43, 67, 115, 116, 130 Slater, E. C., 234, 263, 264, 268, 274 Slautterback, D. B., 289, 325 Slizynski, B. M., 149, 150, 168, 182, 185, 197 Smellie, R. M. S., 162, 168, 219, 221, 222, 223, 241, 265, 266, 271, 274 Smirnowa-Zamkowa, A., 411, 434 Smith, J . A., 54, 62 Smyth, E. M., 418, 434 Smyth, H . F., 34, 67 Snedecor, G. W., 87, 111 Snell, E. E., 37, 52, 64, 67 Snellman, B., 31, 67 Snellman, O., 402, 404, 405, 416, 419, 433, 434 Sober, H. A., 51, 65 Solis, J. T., 368, 397 Sonne, J. C., 43, 67 Sonneborn, T. M., 293, 313, 326

493

AUTHOR INDEX

Sorokina, N. I., 183, 197 Sourander, P., 469, 476 Spector, S., 264, 272 Spector, W. G., 37, 67, 268, 275 Spencer, A. G., 268, 273 Spiegelman, S., 287, 311, 313, 326 Spratt, N., Jr., 283, 306, 326 Spratt, N. T., 2, 30, 31, 67 Sprechler, M., 427, 435 Stacey, M., 418, 434 Staemmler, M., 405, 410, 413, 414, 415, 425, 434 Staffel, 423, 434 Stare, F. J., 50, 67 Stark, R. B., 376, 385, 396 Steams, M. L., 374, 397, 398 Stedman, E., 135, 168 Stedman, E., 135, 168 Stein, R. J., 20, 67 Steinbach, H. B., 283, 287, 298, 326 Steinert, M., 304, 311, 326 Stelzenmuller, A., 115, 116, 130 Stenbeck, A., 414, 433 Stern, H., 135, 168, 201, 202, 203, 204, 206, 210, 221, 233, 238, 258, 270, 275 Stern, J., 123, 129 Stern, K. G., 42, 67, 238, 275 Stewart, D. C., 28, 67 Stewart, R., 430, 434 Stieg, H., 7, 67 Stillman, N. P., 8, 12, 67 Stjernholm, R., 32, 37, 39, 42, 44, 61, 62 Stock, C. C., 39, 64 Stockinger, W., 400, 434 Stoneburg, C. A., 238, 275 Stordahl, A., 312, 324 Storer, J. B., 120, 124, 129 Stotz, E., 264, 275 Stowell, R. E., 465, 476 Straub, J., 124, 130 Streicher, J. A., 7, 38, 40, 6U Striebich, M. J., 214, 217, 222, 225, 233, 234, 235, 238, 239, 244, 246, 248, 249, 268, 271, 272 Strittmayer, C. F., 282, 323 Strong, F. M., 248, 272 Stuart, E. G., 424, 430, 434 Stuart-Webb, J., 136, 167 Stuermer, V. M., 20, 67

Stulberg, M. P., 263, 274 Sturtevant, A. H., 154, 168 SubbaRow, Y., 25, 40, 51, 52, 59 Sugawara, H., 289, 326 Sumerwell, W. N., 293, 326 Sung, S. C., 243, 255, 275 Suomalainen, P., 424, 432 Suter, Sister M. St. A., 123, 129 Sutherland, E. W., 254, 275 Suvarnakich, K., 50, 67 Sveinsson, S. L., 123, 130 Swann, H. G., 33, 34, 63 Swanson, M. A., 257, 275 Swedmark, B., 309, 319, 324 Swendseid, M. E., 248, 275 Swezy, O., 182, 184, 185, 196, 197 Swift, H., 132, 133, 143, 151, 156, 163, 164, 168, 188, 197 Sydenstricker, V. P., 238, 253, 272 SylvCn, B., 402, 403, 404, 405, 412, 414, 416, 417, 419, 425, 430, 431, 433, 434, 465, 476 Syverton, J. T., 414, 432 Szafarz, D., 219, 222, 265, 272 Szarski, H., 8, 67 Sze, L. C., 307, 321 Szejnam, M., 102, 111

T Tanzer, P., 437, 453 Tarver, H., 51, 67 Taubert, M., 219, 222 Taxi, J., 355, 357 Taylor, A., 294, 323 Tazima, M., 9, 10, 67 Ten Cate, G., 312, 326 Tenenbaum, E., 7, 10, 35, 53, 61, 63, 64, 65 Tennant, R., 42, 67 Thal, A., 360, 397 Thalhimer, W., 53, 62 Therman, E., 183, 185, 188, 189, 190, 191, 197 Thibault, C., 189, 197 Thomas, J. A., 31, 52, 67 Thompson, H. P., 248, 251, 253, 274 Thompson, M. V., 49, 50, 66, 120, 130 Thompson, R. B., 51, 53, 67 Thomson, R. Y., 145, 168, ZOO, 223

494

AUTHOR INDEX

Thorell, B., 265, 271, 293, 326 Vial, J. D., 442, 444, 445, 452 Thornblom, D., 309, 326 Videbaek, Aa., 427, 435 Timonen, S., 183, 185, 188, 189, 190, 191, Vigran, M.,368, 398 192, 197 Vikbladh, I., 27, 67 Tinker, J. F., 43, 62 Villee, C. A., 308, 326 Tishkoff, G. H., 202, 204, 222, 238, 271 Vincent, W. S., 294, 326 Tiuey, H., 22, 65 Vogel, I. I., 184, 187, 195 Tomlin, S. G., 157, 158, 168, 201, 202, VogeIaar, J. P. M., 5, 15, 26, 27, 36, 41, 222 47, 48, 52, 53, 54, 68 Tompkins, E. R., 3, 67 Vogt, M., 127, 130 Townsend, F. M.,55, 66 Volkert, M., 10, 11, 58 Trager, W., 136, 169 Vollmar, H., 46, 47, 48, 49, 68 Trowell, 0. A., 7, 12, 25, 34, 44, 55, 56, Volpino, G., 6, 68 67 VonEbner, 437, 453 Tsou, C. L., 261, 275 Voss, H., 73, 111 sou, K-c.,333,338,357,358 W Tsuboi, K. K., 243, 255, 275 Tuma, V., 424, 434 Waddington, C. H., 313, 326 Tunbridge, R. E., 440, 442, 453 Walker, B. E., 183, 195 Turchini, J., 456, 476 Walker, C. E., 184, 196 Turk, W., 400, 434 Walker, P. G., 232, 243, 247, 254, 255, Tyler, A., 120, 123, 130 275 Tyler, D. B., 288, 326 Walker, P. M. B., 468, 475 Tyner, E. P., 265, 266, 275 Wallace, A. C., 8, 58 Tyrell, L. W., 472, 476 Wallbach, G., 54, 68 Tyrode, Ex. V.;22, 24, 27, 67 Walters, C. P., 28.2, 323 Walton, K. W., 418, 435 U Waltz, H. K., 13, 21, 57, 61. 66, 67 Uei, K., 26, 67 Wang, T. H., 289, 327 Ulloa-Gregori, O., 8, 67 Warburg, O., 12, 30, 68, 114, 120, 123, Ullrich, H., 142, 169 130 Undritz, E., 407, 434 S. L., 388, 389, 390, 391, 396, Warren, Unna, P. G., 406, 134 397 Urbani, E., 219, 223 Waters, M., 139, 169 Urbani-Mistruzzi, L., 293, 326 Wassermann, F., 103, 111 V Waterman, N., 8, 59 Van Doorenmaalen, W. J., 312, 326 Watson, E. M., 411, 435 Valentine, J. D., 43, 67 Watson, M. L., 246, 275 Vanderhaeghe, F., 265, 275 Wattiaux, R., 226, 236, 243, 245, 252, 262, Varossieau, W. W., 7, 62 no,271 Vasseur, E., 290, 326 Waymouth, C., 3, 5. 6. 8, 23, 43, 44, 55, Vaubel, E., 406, 435 61, 68 Veer, .W. L. C., 55, 62 Webb, M., 146, 166 Vendrely, C., 133, 135, 155, 165, 169, 200, Weber, R., 230, 239, 241, 248, 250, 253, 223 272 Vendrely, R., 133, 135, 165 Wegstedt, L., 457, 475 Venge, O., 181, 197 Weichselbaum, T. E., 31, 61 Vercesi, C., 55, 67 Weidenreich, F., 423, 435 Verga, G., 48, 65 Weidman, Fred D., 452, 453

495

AUTHOR INDEX

Wilson, H.,15, 27, 28, 31, 32, 33, 41, 68 Weigert, C., 437, 453 Wilson, H.C., 368, 398 Weil, P.,423, 435 Wilson, J. L., 26, 68 Weiner, R. von E.,28, 64 Wilson, J. R., 123, 129 Weinhouse, S.,292, 327 Wilson, K.,146, 166 Weinstein, H. R., 54, 62 Winiwarter, H.yon, 178, 184, 197 Weiss, J., 442, 445, 453 Winnick, T.,5, 35, 37, 39, 43, 62, 68 Weiss, P.,191, 197 Weller, T. H., 27, 68 Wirth, J., 23, 68 Wislocki, G. B., 415, 417, 419, 422, 435, Wellman, H.,257, 258, 263, 264, 273 Wells, A. Q., 394, 397 444, 452 Wissler, R. W., 370, 396, 397 Welty, M.,115, 116, 130 Wenner, C. E., 292, 327 Withers, R. F. D., 329, 345, 349, 355, Wentsler, N. E., 360, 398 356, 357 Wermel, E. M., 70, 93, 97, 111, 415, 435 Witter, R. F., 264, 275 Werner, B., 417, 433 Wodsedalek, J. E., 183, 197 Werthemann, A., 35, 53, 64 Woerdemann, M. J., 407, 414, 419, 426, Wesbrook, F., 422, 432 434 Wessel, G.,301, 308, 323 Wolf, G., 330, 335, 358 West, G. B., 411, 422, 425, 434 Wolken, J. J., 13, 68 White, J., 286, 322 Womack, M., 40, 66 White, J. M., Jr., 7, 68 Woodbury, M. A., 333, 357 White, M. J. D.. 146, 150, 152, 154, 169 Woodward, A. A., Jr., 282, 290, 327 White, R. F., 430, 432 Woodward, G., 3, 6, 63 White, P. R., 5, 11, 20, 21, 22, 26, 27, Worzniak, J. J., 31, 36, 59, 68 32, 39, 40, 41, 47, 49, 50, 51, 52, 68 Wright, G. P., 9, 27, 33. 68 Wielgosz, G.,11, 13, 59 Wu, H.W., 289, 327 Wieman, H.L., 184, 185, 197 Wyckoff, R. W. G., 138, 168 Wiener, O.,446, 453 Wyman, R., 146, 166 Wigglesworth, V. B., 311, 327 Y Wilander, O.,406,407, 416, 425, 433, 435 Wilbur, K. M.,135, 164, 232, 234, 238, Yamamoto, Y.,134, 169 239, 268, 270, 275 Yamanaka, T.,134, 169 Wilcox, E. V., 184, 197 Yasuzumi, G.,134, 139, 142, 169 Wilflingseder, P.,69, 74, 111 Yen, C. K.,24, 61 Wilkins, M. H. F., 468, 476 Yokoyama, J., 134, 169 Williams, H., 406, 435 Young, I. M., 7, 68 Williams, J. N., Jr., 243, 255, 275, 291, Young, N. F., 291, 324 327 Youngner, J. S . , 392, 398 Williams, R. H., 266, 273 Yrarrazaval, S., 56, 61 Williams, R. G., 360, 361, 363, 367, 374,

376, 377, 381, 382, 383, 384, 385, 389, 390, 396, 398

Williams, R. J., 294, 323 Williams, W. L., 124, 130 Williams-Ashman, H. G., 264, 272 Willigens, C., 81, 111 Willmer, E. N.,4, 5, 7, 22, 24, 27, 28, 31, 32, 36, 43, 44, 45, 55, 67, 68 Wilson, D. W., 43, 63, 67

2 Zacks, S. I., 338, 343, 358 Zeuthen, E.,123, 130, 295, 306, 323, 327 Zamenhof, S., 135, 169 Zintel, H. A,, 360, 398 Zollinger, H.U., 246, 273, 405, 414, 415. 416, 435 Zweibaum, J., 102, I l l

Subject Index A A T P (see Adenosine triphosphate) ATP-ase (see Adenosine triphosphatase) Acacia, effect on leukocytes, 373 Acetate, sodium, in tissue culture media,

18 Acetylcholine, accumulation of, and enzyme adaptation, 314 Acetyl glucosamine, 415 Achromatic figure (see also : Spindle, Mitosis) in cleavage of Echinarachnius and Lytechinzu eggs, 118, 119 in eggs treated with urethan, 119 Actinophrys, effect of phenylurethan on division pattern, 124 Activators, of enzymes, 253, 289 Actomyosin, electron microscope studies on, solutions of, 319 Adenine, 18, 44 Adenocarcinoma, effect of ascorbic acid in zttro on, 48, 49 Adenosine, 44, 294 Adenosine deaminase, 279, 294, 313 Adenosine diphosphate, 263, 264 Adenosine triphosphatase, 204, 209, 243,

251, 254, 256, 257, 258 in centrifugal fractions, 243 in nuclei, 204, 209 Adenosine triphosphate, 210, 257, 263, 264 cell nucleus, and splitting of, 210 pre-prophase inhibition in Vitro by, 44 in tissue culture media, 18, 29 Adenylic acid, 18, 44, 294 Adrenal, auto- and homografts of, 378 Adrenal cortex, autografts of, 382 Adrenaline (see Epinephrine) Adrenochrome, reduction in mitotic activity in vitro and, 55 Adrenocortical hormone, in tissue culture media, 55 Adsorption spectrum, of reduced DPN,

212 Agar, and trypsinized peptone medium.

34 Agglutination, in homogenate, during differential centrifugation, 232, 233 of isolated nuclei, prevention, 214

4%

Aging, of animals, and polyploid cells, I63 changes in mast cells with, 406 effect on elastic fibers, 437, 438, 446 species variations in, 451 of elastin, changes in composition, 439 of mitochondria, oxidative phosphorylations and, 264, 265 of neurons, chemical changes with, 469 of spermatozoa, and catatase activity, 286 DL-Alanine, in tissue culture media, 16 8-Alanine, in tissue culture media, 17, 52 Alanylglycine, gradients in, of Apnblystonio embryo, 305 Albumin, effect of, on mitochondria, 296 serum, in tissue culture media, 13, 35 Alcohol, effect on blood vessels, 370 Aldolase, 200, 204, 242, 279, 318 and deoxyribose synthesis, 207 Allantojc &id, use for tissue culture, 8 Amblystoma, cholinesterase in development of, 314 effect of succinate on succinic dehydrogenase in, 313 metabolic gradients in gastrula stage of, 385 Amines, 336 Amino acids, 256 collagen composition, 441 in developing egg, 279 effect on survival of fibroblasts, 10 in elastin, 439, 440, 441, 452 in elastic fibers, 439 formation of, mitochondria and, 266 growth stimulatim in eitro by, 40 infused, and carbohydrate source, 31 metabolism of, during cleavage, 210, 301 in tissue culture nutrient solutions, 16,

17, 34-42 P-Atninobenzoic acid, effect of genes on formation of, 294 and nerve fibers ifi zd'tro, 52 in tissue culture media, 18, 29 in medium for malarial parasites, 52 p-Aminohippuric acid, enzyme synthesis, in centrifugal fractions, and, 243

SUBJECT INDEX

Amitosis, in mast cells, 400 origin of binucleate cells and, 106, 107 in salivary gland of Clziroriomus, 154 and stages of nuclear fusion, 154 Amnion, human, chromosome numbers in, 182, 183 Amoeba, R N A synthesis following denucleation, 219 Amphibia, lampbrush chromosomes in,

154 pattern of polyploidy in, 180 Aqnplzhma, lampbrush chromosomes in,

154 Feulgen-positive material in, 156 volume of, compared with mitotic chromosomes, 160 Amylase, pancreatic, 204 p-Amylase, nuclear, and cytoplasmic, 205 Amylases, 288 Amyloidosis, mast cells in, 414-415 Anaphase, effect of urethan on, 120 Anaphylaxis, mast cells and, 424, 425 and survival of grafts, 387 Androgens, and mitosis, 55 Anesthetics, effects on circulation, 368,

369 Aneuploidy (see also : Somatic Inconstancy) in embryos, 189 somatic, 177 Angiotonin, effect of, on peripheral circulation, 368 Animal pole, enzyme activity of, in sea urchin egg, 304 Anions, in balanced salt solutions for tissue culture, 27 Anisotropism, theory of, 4l6 Anisotropy, of elastic fibers during stretching, 450 Anticoagulants, heparin-like, 418 Antigens, 312, 370 Antihistamines, 425 Antimetabolites, chick embryo development and, 291 ,and urethan, 126 Antisera, in primitive streak stage of chick, 312 Antuitrin, in tissue culture media, 19, 55

497

-4podehydrogenase, in holometabolic insects, 289 Apoenzymes, in isolated nuclei, 209 synthesis of, in Cklamydot~o~zas, 293 Apyrase, 279, 298 Aqueous humor, use of, for tissue cultures, 8 i\rabjnose, in tissue culture media, 30 .4vbaciu (see also: Sea urchin) eggs of, acid phosphatase in homogenates of, 287 cell division in, 123, 297 granular inclusions in, 297 peptidase in, 280 unfertilized, proteolytic enzymes in,

282 urethan-produced r e t a r d a t i o n of cleavage of, 117 Arginase, in centrifugal fractions, 243 cytoplasmic, 203, 209 nuclear, 200, 203, 209, 259 -4rginine, 17, 29, 30, 141 Artifacts, in differential centrifugation,

259 electron microscopy of salivary chromosomes, 138 in enzymatic systems, 253-258 incompIete tissue homogenization and,

in

245 isolation procedure and, 134 in mast cell preparations, 404 Ascarks, basophilia during oogenesis and in fertilized egg of, 299 formation of ribonucleic acid-rich par. ticle during development of, 299 mitochondria in, 320 ribonucleoproteins in, 300 Ascaris hmbricoides, eggs of, in transparent chamber studies, 392, 393 Ascites, 37, 55 Ascitic fluid, use for tissue culture, 8 Ascorbic acid, 430 in differentiating regions, 292, 293 influence on cell surface and cellular exchanges, 48 and phagocytic activity of leucocytes,

48 in synthetic media, 15, 17, 29, 48, 49 tissue-bound, 293

498

SUBJECT INDEX

Aspartic acid, in tissue culture media, 17, 29, 38 Assay, of enzymes, artifacts in, 253 microbiologic, of amino acids in elastin, 440, 441 Asters, in Echinarachnius and Lyfechim eggs, 119 Autografts, 373, 377, 385, 386, 387 tissues studied as, 378 vascularization of 377, 379, 383 Autographs, of grafts of thyroid gland, 381 Autolysis, 35, 232, 248 Autoradiography, 417, 418, 420, 421, 430 of nucleus, 133 Avitaminosis, and mast cell granules in rats, 424 Axone, of nerve cell, repair in, 374 Azo dye methods, 329, 330, 334, 336, 338, 349, 351-356 for acid phosphatases, 352, 356 for alkaline phosphatase 332, 333, 334, 341, 351, 352, 356 for aryl sulfatase, 333, 356 for cholinesterase, 333 diffusion in, 336, 338 for esterase, 332, 333, 341, 342, 352, 353 for p-galactosidase, 333 for p-glucosidase, 333 in histhemistry, 332-333 and indoxyl methods, 357 localization of precipitates and enzyme activity with, 335 modifications of substrates, for, 338343 non-coupling, 329, 330, 334-335, 355 for non-specific esterase, 356 Azoprotein, effect of, on macrophages, 373 B Bacilli, bovine tubercle, rekction to, in transparent chambers, 393, 394 Bacteria, contamination of isolated chromatin by, 134 effect on mast cells, 423 growth of, peptides and, 37 “Balbiani ring”, 163 analysis, 150, 152 in Chkononaus fentam, 172, 173

revealing polytene condition of salivary chromosomes, 150 Basophilia, of elastic fibers with aging, 452 metachromasia of mast cells and, 419 during wgenesis in Ascaris, 299 oxidation and, 443 Barley, enzymes in germination of, 288 Benzene, effect on mast cells, 430 Benzene derivatives, narcotic action on cells, 122 Benzidine peroxidase, 287 Bicarbonate, in synthetic tissue culture media, 24 Bibio, salivary chromosomes, and heterochromatin, 144 Bihio hovtularaw, salivary gland chromosomes, 137 Binucleation, 120, 122 Biotin, 17, 29, 43, 45, 51, 294 Birds, lampbrush chromosomes in, 154 Birefringence, of elastin fibers, 4-40 in salivary chromosomes, 142, 143 Blastocele, invasion of, by mesenchymal cells in sea urchin, 278 Blastocyst, of mouse, heteroploidy in, 189 Blastoderm, of trout, effect of urethan on, 120 Blastmeres, in urethan treatment, 128 Blastula, granules in, of Pamcmtrotw, 299 mitotic gradient in, 306 N15-labeled proteins in cellular fluid of, 301 of sea urchin, biochemical changes in, 279, 284, 285, 292, 311, 312 Blastula stage, enzymes in, 279 Blood, amount of pantothenate in chicken, 52 chromatin threads from cells, 134 contamination in homogenate of, 232 fetal, 7 glucose in, birds and mammals, mammalian, tetraploid cells in, 183 white cells of, urethan effects on, 116 volume of, 180 Blood flow,effect of burns on, 370 Blood vessels, see Vessels, blood Bodies, intracellular, permeability of, 267

n

SUBJECT INDEX

Bone, auto- and homographs, 378 growth of, in transparent chambers,

375, 376 resorption of, in transparent chambers,

376

vascularization of newly formed, 376 Bone marrow, chromosome number of in human. 182, 183 mast cells in, 414 and oxygen tension in Otitro, 34 of rabbit, hypoxanthine and nucleic acids in, 43 red, auto- and homografts of, 378 Brain, auto- and homografts of, 378 Buds (see: Limb buds and Tail buds) Buffers, for coupling azo dye methods,

356

Bufo, cozymase, in development of, 292 Burns, effect of, on blood flow,370

C C14, 39, 300, 301, 304 nuclear RNA turnover and, 265 urethan labeled with, 125 Calciferol (see also Vitamin D), in tissue culture media, 17 Calcium, activation by, 290 in elastin, 439, er0, 452 ions, and ATP-ase, 258 influence on isolation of nuclei, 234 stimulation of respiration in mitochondria by, 264 Calcium gluconate, for urcdele organ culture, 31 Calcium 6-naphthyl phosphate, 330 Calcium pantothenate, in tissue culture media, 17 Cancer, see Carcinoma Capillaries, 390 contractility of, and Rouget Celts, 367 effect of bacterial polysaccharide on occlusion of, 392 in grafts, 382 growth of, 365, 367, 371 morphology of, 365 percentage of, in striated muscle, and bacterial polysaccharide, 371 permeability, leukotaxin-like peptides and, 37

499

regenerating, adventitial cells in, 367 in transparent chambers, 365 Carbamates, 120 (see also: Urethan, Phenylurethan) as cawinoclasts and as carcinogens, 115 and cell division, 114, 115, 116, 117, 123 effect of temperature, 117 effect on enzyme activity in &fro, 124 esters of, 125 narcotic action of, 124 and oxygen consumption, 123 penetration into cells of higher animals,

117 and protein denaturation, 127 pycnosis in animal cells and, 120 solubility of, in cell lipids, 127 toxicity of, 116 Carbohydrates, anaerobic glycolysis, 256 gradients in Amblystoma emhyro, 305 oxidation of, in sea urchin, 307, 308 and protein synthesis, 31 in tissue culture media, 15, 27-34 Carbon, effect of injection of, in transparent chambers, 394 Carbon dioxide, effect on pH of intercellular substance in transparent chamber, 370 Carcinoclastic activity, of urethan, 114,

116, 124

Carcinoclasts, 115, 124 Carcinogens, carbamates as, 115 and phosphoryfation, 124 urethan as, 113, 114 Carcinoma, and cell division, 199 effect of urethan on cultures of, 124 experimental, mast cell counts in, 413 of mammary gland, vascularization in transparent chambers, 389 of mouse, effect of thiamine on, 49 of vitamin A, on, 46 of rat, growth in, 25 somatic inconstancy, in cells of, 186 V,, auto- and homografts of, 378 survival of, 386 vascular response, 389 Carnivora, chromosome number in chorion and amnion of, 183 Carotene, 17, 47 Carotinoids, in egg of Paracenhohw, 316

500

SUBJECT INDEX

Cartilage, 364, 425, 438 formation of, 48, 49, 375, 376 Caryometry, 69-109 Casein, effect of digests of, on tissue culture, 35 Catalase, activity of, during development, 298 distribution of, 200, 203, 204, 242, 243, 242, 266, 280, 284, 286, 287 effect of starvation on nuclear and cytoplasmic, 204 inhibitor of, in neoplastic growth, 286 permeability of nuclear membrane and, 201 Cathepsin, 252, 254 in nuclei, 209, 211, 245 Cathepsin 11, in sea urchin development, 279 Cations, in tissue culture media, 25 Crcidomyia sp., “super giant” salivary gland nuclei of, 152 Cecidomyid, see Dacynewa afittis, 150 Cell fractions, effect of carbamates on, 124 Cell lineage, 177, 191, 192, 193 Cells, adventitid (Rouget), in regenerating capillaries, 367 binucleate, 102-109 of blood, chromatin threads in, 134 of chick heart, 45, 49 chondroidal, mitosis in transparent chambers, 375 components of, isolation by centrifugal fractionation, 2.25, 238 of connective tissue, development of fat from, 375 death of, and abnormal divisions, in organs and skin, 191 degeneration of, in transparent chambers, 373 Deiters’, composition of, 472 differentiation of, and chromosomes, 162, 163 disruption of, for centrifugal fractionation, 232 division of, and abnormal number of chromosomes, 190, 191 and cancer, 199 after death, in rodents, 190

effect of carbamates on, 118, 123 of peptides on, 37 of radiation on, 121 of urethan on, 120, 121, 126, 127 and glycolysis, 124 and heterochromatin, 144 and respiration, 124 embryonal nerve, composition of, 469 embryonic, and oxygen requirements, 33 endothelial, 373 enzymatic digestion of, and chromosomal fractions, 135 enzymes of, isolated by biochemical techniques, 199-221 epidermal, in transparent chambers, 375 epithelial, 48 extrafollicular, of thyroid, 381 fixed tissue, effect of choline on, 52, 53 function of, and DNA concentration,

162 germ, chromosome numbers in, 177, 178, 179, 180, 181, 184, 185, 193, 194 somatic inconstancy in, 193 giant, formation of, and oxygen deficiency, 34 granules of, soluble hydrolytic enzymes in, 266 hyperploid, 189 hypoploid, 189 hypopolyploid, in endometrium, 183 in interphase, effect of adenosine and adenylic acid on, 44 interstitial, in seminiferous tubules, 383, 384 isolated, in tissue culture, 120 of Leydig, in connective tissue grafts, 378, 384 lineage, and abnormal chromosome numbers, 193, 195 of liver, bi- and multinucleate, in zz‘vo, 102 living, behavior, in transparent chambers, 261, 360 lymphoid, ingestion of antigen by, 373 lysis of, and erythrocytic catalase, 287 mass of, 457, 449 mast, 399-413 amitosis in, 400

SUBJECT INDEX

and anaphylaxis, 424, 425 autoradiography of, 420, 421 basophilia of, 419 cellular membrane of, 405 changes in, 406, 414, 423, 424, 427, 430, 431 chemotaxis of, 423, 424 chondroitinsulfuric acid production of, by, 425 chromosomes in, of lizard GongyZw, 400 conversion of hyaluronic acid into heparin-like substances by, 426 cytochemistry of, 415 cytoplasm of, 405 degranulation of, 405 distribution of, 399, 400, 405, 406, 407, 410, 411, 412, 413, 414 elastic fibers and, in carcinomatous tissue, 423 elastin formation by, 422 fat metabolism and, 424 food absorption and, 422 freeze-dried, 403 function of, 422-426 glucoprotein in, 415 glycogen in, 415 Golgi apparatus in, 405 granules of, 401, 403, 404, 405, 407, 415, 416, 419, 422, 424, 425, 438 enzymes and, 415, 422 nucleic acid in, 415 ribonucleoprotein in, 415 solubility of, 415 staining of, 438 histamine production by, 411, 424 juxtanuclear zone in, 404 lipids in, 419 of lizard, 400 metachromasia of, 407, 415, 419 mitochondria in, 405 mitosis in, 400 morphology, 402-405 mucopolysaccharides, 419, 425, 426 nucleus of, 402 origin of, 400-401 and permeability of connective tissue, 424 phagocytosis by, 423

501

physiologic variability of, 426-431 pigment formation by, 423 radioactive sulfur in, of experimental tumors, 417, 418 release of poly-sulfuric acid esters by, 425 tumors of, 410 in tumors, 423 vacuolization in, 404 membrane of, 56, 201, 202 mesenchyrnal, 278, 399, 401 metazoan, nutritional needs of, 57 migration of, 32, 56 mononuclear, as pro-mast cells, 401 moribund, 190 motor root, 468, 469,471, 472 multinuclear, iw vitro, 102, 373 nerve (see also Cells, motor root, of retinal ganglion) composition of, 455-474 effect of aging on, 474 ribonucleic acid in, 467-469 non-diploid, and cell lineage, 177, 191, 192, 193 non-particulate fraction of, 258 nucleus of, chemistry and enzymology of, 199

metabolic function of, 208 pathologic, somatic inconstancy in, 193 photoelectric measurements with, 136 plasma, as precursors of mast cells, 401 polyploid, 163, 177, 181, 183, 189 population of, growth determination in, 57 post-mitotic, 383 of Purkinje, composition of, 472 resting, and chromosomal activity, 161 metabolic role of nucleus in, 208, 218 of retinal ganglion, 470, 471 Rouget (see also : adventitial), contractility of capillaries and, 367 of Schwann, in transparent chambers, 374 of Sertoli, 383, 384 somatic, chromosome numbers in, 177, 180, 182, 183 DNA in cytoplasm of, 200 inconstancy in, 186, 193 strains of, 57, 3%

502

SUBJECT INDEX

surface of, and development of mitochondria, 320 synthesis in, and differential growth of chromosome, 162, 164 of thyroid, 380, 381 volume, in human blood, 180 white, in transparent chambers, 372, 373 whole, residual, in nuclear isolation procedures, 214 Centrifugation, differential (see also : Fractionation, complete) applications of, 239-259, 260, 269 conditions for, 234237, 259 of eggs and enzyme distribution, 280,

297,298 and

Feulgen-positive material in oocytes, 156 in isolation of nuclei, 215 homogenates used for, 227, 232 isolation by, of cytoplasmic contents, 225, 251 of mast cell granules, 404 of nuclei, 208, 215 of particulate fractions, 241, 244, 245 245 liver of rodents and, 231 technique of, 224-239 temperature requirements for, 231 tissues suitable for, 2‘25-269 and tonicity of medium, 2-30 Centrifuges, 229, 230, 302 Centrosomes, and differential centrifugation, 251 Cervix, uterine, somatic inconstancy in, 195 Chactopterus, 280, 282, 283 Chambers, transparent, 360-3154, 394 Chiasmata, 154 Chick, embryo, enzymes in, 283, 287 Chironomus, banding patterns in chromosomes, 149 binucleate cells in salivary gland, 154 giant chromosomes in various tissues, 137, 138, 140, 148, 149 and hetercchromatin, 144 Chirommus defectw, salivary chromosomes, 140

Chimmomw plumasus, “nuclear filaments” in, 137 Chironomus tentans, “Balbiani ring” in salivary glands of, 140, 172, 173 Chironomw thummi, salivary chromosomes, 140 Chlamydomanas, synthesis of apo-enzyme, coenzyme and of rutin in, 293 Chloroform, effect on nerve cells, 473 Chloroplasts, of ChZanydomonas, 293 Cholesterol, 15, 18, 419 Choline, 17, 29, 45, 52, 53 Cholinesterase, azo dye method for, 333 distribution of, 3 9 , 313, 314, 355 Chondriome, 283, 299 Chondroitinsulfuric acid, 406, 407, 41 1, 417, 425 production by mast cells, 425 Chordamesoderm, presumptive, “Somitisierung” of, in Triton, 317 Chorion, chromosome number in, of human embryo, 182, 183 Choroid plexus, auto- and homografts of, 378, 386 Chromatids, 127, 133, 148, 160 Chromatin, chemical composition of, 131 extended state of, 131, 134, 163 structure of, 135 Chromatography, paper, determination of amino acids in elastin by, 440 Chromium, shadowing with, for electron microscopy, 449 Chromocentws, and cell function, 133 heterochromatic, in salivary cells, 144 Chromomeres, 138, 139, 147 corpuscular, in lampbrush chromosomes, 159 number of, in salivary chromosomes, 140 in Triton cristatus, electron micrograph of, 174, 175 Chromonemes, bundles 5f, 138 duplication of, and chromosome growth, 164 in salivary chromosomes, 146, 148 Chromoprotein, and permeability, 201 Chromosomes, activity of, in non-mitotic cells, 161 axis of, 131, 156, 158, 160, 167

SUBJECT INDEX

banding of, constancy of patterns, 149, 150 breaks in, 127 (see also fragmentation) bridges, produced with carbamates, la0 and cellular synthesis, 162 chemistry of, 131, 132 clumping of, produced by carbamates, 120 coiling of, 151 and heteropycnosis, 145 condensation of, 159 contraction, abnormal, 154 counts of (see also number of), 177, 187, 193, 194 deoxyribonuclease, and, 139 DNA content of, during mitosis, 133 and differentiation, 164, 193 diploid number (see: number of) in diplotene, 154, 159 effect of carbamates on, 120, 127 of colchicine on, 186, 193 of urethan on, 119, 127 fibrils in, and chromatin condensation, 163 fractions of, determination of, 135 fragmentation of, 121, 127 giant, in Bibio hortzdanw, 137 in Chironomus, 137 composition of, 131 in Diptera, 137 in Drosophila, 137 functional significance of, 161-164 “meander stage” of, 151 in nurse cells of muscids, 147 in Sciara, 137 in Simuliitm, 137 structure of, 131, 132, 136 theories of, 132 growth of, 160, 164 haploid, and DNA, 133 number of, 177, 178, 181 and tetrapolar division, 189 histones, DNA arid RNA in, 135 human, causes for discrepancies in counts of, 181 in fertilized egg, 191 itisoluble phase of nuclei and, 206 it1 intermitotic, and salivary chromsomes, 162

503

and interphase nucleus, 131 irregularities in, 119, 120, 128 isolated, 14, 131, 134, 174, 175 “lampbrush”, 131, 132 in Aniphizlmu, 154 axis of, 131, 156, 160, 167 chromomeres of, 159 Feulgen reaction in, 155, 160 fixed and fresh, observations on, 160 isolated, electron micrograph of, 174, 175 lateral projections of, 155, 156, 157 loop structure in, 163, 174, 175 matrix of, 160 of hTecturms, 156, 174, 175 polytene nature of, 155 radioactive tracers and, 162 as “residual chromosomes”, 164 and salivary, artifacts of, 142, 163 of Siredon, 157 staining of, 155 Feulgen reaction, 155, 160 structure of, 159 surface of, and catalytic activity, 162 of Triton, 156 of Triton cristatiw, phase contrast, microscopy of, 174, 175 of vertebrates, 154 mammalian, isolated, 14 in metaphase, 155 mitotic, 160 condensed, and heterochromatin, 146 dimensions of, in Diptera, 140 effect of colchicine on, 186, 193 normal cycle of, and phosphorylations, 124 and ninhydrin reaction, 141 number of (see also Somatic inconstancy), 177, 182-190, 193, 195 mucosa, 182, 183 paired, 151 salivary chromosomes interpreted as, 146 pairing of, in Diptera, 163 pellicle of, 142 peptic digestion, 141 of plants, coils in, 161 polytene, 154, 162

504

SUBJECT INDEX

protein chains, and submicroscopic structure of, 143 reproduction of, 133, 145, 163 “residual chromosomes”, 164 salivary, 131, 132, 136 amino groups in, 141, 142 arginine in, 141 “Balbiani ring” in, 150, 172, 173 “bands” and “interbands” of, 137, 139 and genetic loci, 139 banding, pattern in, 143 interpretation of, 138 in Bibio, and heterochromatin, 144 birefringence of, 142, 143 and cellular differentiation, 162 in Chivonomu.r, and heterochromatin, 144 coiling, 148, 149 cytochernistry of, 141 in Dmpeura afinLr, 150 development of, in Chironomus, 149 dichroism, and nucleic acid orientation, 142, 143 dimensions of, 140 elasticity of, 142 and enzymatic digestion, 146 Feulgen reaction in, 139 histidine in, 141 and histones, 141 lampbrush and, 163 longitudinal differentiation ‘n, 137, 138 . and micro-absorption, 163 microdissection of, 142 microincineration of, 142 micromanipulation of, 147 microscopic and sub-microscopic appearance, 140 nucleic acids in, 138, 143 and nucleoplasm, 148 number of longitudinal threads in, 140 phosphates and potassium salts in, 142 polytene structure of, 138 and radioactive tracers, 162 in smears, 137, 138 striations in, and electron microscopy, 138

structure of, 146-154 submicroscopic fibrils in, 140 tryptophane in, 141 tyrosine in, 141 ultraviolet light studies on, 139, 141 sets of, and cell number in polyploids, 189 complete, in human dividing egg. 191 sex, and polyploidy, 180 size and function of, 155 somatic, 177, 182, 183, 185, 193 spiral nature of, evidence for, 139 stretched, and deoxyribonuclease, 139 structure of, in ontogeny, of Diptera, 137 and trypsin digestion, 141 in vertebrate m y t e s , 131 volume of, 160 X - 0 and X-Y types, and chromosomal counts, 179 X, in Drosophila melanogasfer, 137, 144, 170 Y,and the “genetically inert” region, 144 Ciliary body, 378, 407 Ciliary process, survival in grafts, 386 Ciliary tuft, suppression of, in embryo, 311 Circulation, in transparent chambers, 368-371, 379, 383 Citric acid, 213, 238 Citrovorum factor, 292 Citrulline, synthesis of, by mitochondria, 263 Cleavage, 117, 279, 291 effect of carbamates on, 123 of lithium on respiration during, 277 Coacervation, of soluble enzymes and coenzymes, 299 Coenzymes, in Chlamydomonas, 293 in chick embryo development, 291 precursors of, in cell nucleus, 294 nuclear control of, 212 Coenzyme A, distribution of, in fractions, 248 Colchicine, 28 effect of, I22 on binucleate cell production, 106 on chromosomes, 193, I94

505

SUBJECT INDEX

on mitosis, 121 on nuclear size, 98, 100 on spindle fibers, 113, 117 on thymus cells, 121 on tissue culture, 99 Collagen, 439 Colloid, in thyroid, 380 Colloid mill, use for isolation of nuclei, 215 Conjugase, of folk acid, in isolated nuclei,

21 0 Corium, 406, 410 Cornea, mitosis with phenylurethan, 120 Corticotropin, 386, 427 Cortisone, effect on diapedesis of leukocytes, 393 on endothelium, 370 on experimental skin tumors, 430 on fibroblastic activity, 430 on grafts, 386 on inflammation, 369, 393 on lymphocytes, 57 on malignant mastccytomas, 430 on mast cells, 427, 428, 429 on migration of cells, in zitro, 56 on permeability of cell membrane, 56 on secretory activity of renal tubules in vitro, 56 on tissue culture, 54, 56 Cotton-rat, somatic inconstancy in tissue culture of, 195 Cozymase, 29, 292 Creatine, 29, 263 Cricetulus griseits, chromosome number in, 193 Cricefus cricetus, chromosome number in, 193 Crocker mouse sarcoma (see Sarcoma 180) Crossover maps, and cytologic configuration of X chromosome, 137 Cryostat, 462 Cyanide, 57, 211 Cyclohexane, use in nuclear isolation, 202 Cysteine, 15, 17, 290 Cystine, 17, 29, 30, 54 Cytochemistry, of mast cells, 415 microradiography and, 462 of nuclear enzymes, 200

Cytochrome, effect of carbamate on, 123 Cytochrome oxidase (see Oxidase) Cytochrome c. 243, 345, 254, 261, 262 Cytokinesis, effect of carbamates on, 122, 123 Cytology, differential centrifugation, and, 269 Cytophotometry, of nucleus, 133 Cytoplasm, acidophily of, 417 alkaline phosphatase in, 204 amount of, and residual protein-RNA fraction, 134 @-amylasein, 205 arginase in, of rat liver, 209 bound, in nucleus, 202 catalase in, during starvation, 203 constituents of, in fractionation, 226. 251, 252 DNA synthesis and, 200, 207 droplets of fat, in transparent chambers, 375 effects of lithium on, of sea urchin egg, 319, 320 electron microscopy of, 136 enzymes in, 203, 206 granules in, multipljcity of, 251 of mast cells, 403, 404, 405 metachromatic, 407 of nerve cell, effect of fixation on, 474 particles of, types, 250 RNA synthesis by, 265 shedding of, by macrophages, 373 Cytoplasmic determination, 192 Cytoskeleton, 283

D DNA (see Deoxyribonucleic acid) DNA-ase (see Dmxyribonuclease) D P N (see Diphosphopyridine nucleotide) Dasynezwa .finis, salivary chromosomes in, 150 Deaminase, adenosine, in centrifugal fractions, 242 Deamination, 261 Death, in rodents, and cell division, 190 Decidua, chromosome number in, of human embryo, 182 Dehydroascorbic acid, 430 Dehydrogenase, effect of carhamates on. 123

506

SUBJECT INDEX

fatty acid, 261 glucose-6-phosphate, 242 glutamic, 243, 254 glutamic acid, 210,211,261 isocitric, 242,257 lactic, 204, 207, 261 malic, 217 succinic, 213,216, 217,313 3-phosphoglyceraldehyde, 204 triosephosphate. 279, 318 Dehydrogenation, with carbamate esters,

125 Denucleation. in amoeba, and RNA synthesis, 219 Deoxycorticosterone, effect on grafts, 386 Deoxyribonuclease, adsorption by microsomes, 246 in centrifugal fractions, 243 effect on mast cell granules, 415 in nuclei of pig and calf kidney, 209 in sea urchin development, 279, 298 and stretched chromosomes, 139 in unfertilized eggs, 280 Deoxyribonucleic acid, 146, 199, 200 amounts of, and nuclear volume, 153,

170

and cellular function, 162 in chromatin threads, 134 chromocenters and total nuclear, 133 in chromosomes, 133, 143, 155 during cleavage, 308 determination of, 133,467 distribution of, 251 in nuclear fraction, 241 in salivary gland nuclei, 151 in single nuclei, with somatic inconstancy, 194 in tissues, 133, 145, 188 fractions in nuclei, 135 histone fraction, 134 nuclear, constancy of, 131,132,133 and histones, 135 and internal chromosome duplication,

151

and nucleolus, 133 and somatic inconstancy, 177, 188 ploidy and, 133 precursor of, in eggs, 295

role in protein synthesis, 221 in RNA synthesis, 21 synthesis of, 43,133,199,200,218,26.5 Deoxyribonucleoprotein, in mast cell granules, 415 Deoxyribose, 18,207 2-Deoxyribse, synthesis of, and glycolysis, 207 Dermis, fibers of, morphology and pattern, 438 Despiralization. of salivary chromosomes,

149

Dextran, effect on leukocytes, 373 Dialysis, of biological tissue culture media, 9, 10, 11 Diapause, 293,295 Diapedesis, 369, 393 Diaphorase, 209, 211 Diazotates, in azo dye methods, 330,

350, 351

Dichroism, natural and induced, in salivary chromosomes, 142, 143 Dicot plants, effect of urethan on. I21 Diethylstilheetrol, and mitotic disturbances in iitru, 55 Differentiation, cellular, and enzyme adaptation, 313 intracellular, changes in neurons during, 469 biochemical, in sea urchin development,

311-314

and somatic heteroploidy, 190, 192-193 Diffusion, in incubating medium, for azo dye methods, 335, 336, 338, 340,

349

of lactate, during glycolysis, 207 passive, and nuclear enzymes, 205-206 Dihydroxy-3-napthoic acid, 343 Dinitrophenol, ATP-ase and, 258 Diphosphatase, hexose, 242 Diphosphopyridine nucleosidase, distr ibution of, 255 Diphosphopyridine nucleotide, 261, 262,

267

concentration of, i i i whole tissues and nuclei, 305 in mammalian nuclei, 205 in mitochondria, 212 reduced, adsorption spectrum of, 212

SUBJECT INDEX

507

reoxidation of, in cytoplasm, 207 Eggs, activation of, 290, 291 synthesis of, 212 of amphibians, intracellular inclusions transfer of, to cytoplasm in glycolysis, in, 297 207 of Arbacia, deoxyribonuclease in, 298 Diploids, in embryos, 189 of Ascaris luntbricoides, effect on leukocyte infiltration, 392, 393 Uiploidy, rise to hexaploidy from, 180 binucleate, and urethan treatment, 128 Diplotene, chromosomes, 154, 159 centrifuged, 280, 282, 297 Diptera, chromosomes of, 137, 140 DNA in, 295 salivary gland function, 162 developing, 278-284, 290, 316320 somatic pairing in, 163 of Echitzocardizcm, effects of thiol comDivision, meiotic, 124, 133, 161 (see also pounds on, 309 Meiosis) enzymes in, 278-284, 287, 290, 298, 302 mitotic (see Cell, division) of Farcialopsis buski, effect on leukomultipolar, 189 cyte infiltration, 392, 393 tetrapolar, and haploid number of of frog, cell division and respiration in, chromosomes, 189 123 Dog, somatic inconstancy in tissue culhuman, chromosome division in, 191 ture of, 195 of invertebrates, 277, 282 Drosophila, banding patterns in chromomarine, 118, 280 somes of, 149 ovarian, cytochrome oxidase in, 283 cytogenetics of, 137 of Paracentrotus, carotinoid-containing genonemes in salivary chromosomes of, granules in, 299 146 of parasites, in transparent chambers, location of giant chromosomes in, 136, 392, 393 137 penetration of, by urethan, 116, 127 mitochondria in spermatogonia of, 294 of sea urchin, 116, 120, 278, 283, 294, mutations, with urethan in, 127 304 nuclear volume in larval organs of, 147 stratified by centrifugal force and enDrosophila melanogaster, 137, 140, 151, zyme distribution, 280 170, 171 unfertilized, 280, 287, 290 Drosophila psefddoobscura, salivary o f Xenopus, permeability of nucleus in, chromosomes in, 140 202 Drosophila robusta, nuclear and nucleolar Egg yolk, 47, 282, 288, 297, 305 growth in, 152 Elastase, 441, 446, 447 Drosophila virilis, salivary chromosomes Elastin (see also: Fibers, elastic; Subin, 140 stance, elastic ; Anisotropy) Drugs, radiomimetic, 121 cornpasition of, 439, 440, 441, 443, 452 Dyes, azo, in enzyme histochemistry, 329formation by mast cells, 422 357 fractions, preparation of, 439 benzathrone series of, 335 properties of, 439, 440, 441, 445, 446 submicroscopic elements in, 446 E X-ray diffraction spectrum of, 439, 449 Echinarachnius, cytology of, 118, 119 Elastogenesis, 450 Echb~ocavdi~m, egg of, 309 Elastosis, degeneration of fibers in, 452 Ecltinus, glycolytic enzymes in egg homo- Electrolytes, in suspension media, 239 genates of, 280 Electron(s), transfer of, in living cell, Ectoderm, 277, 305 261

508

SUBJECT INDEX

Electron microscopy, of actomyosin, 319 of chromatin, 134 of chromosomes, 131, 132, 136, 138, 139, 157, 158, 174, 175 of collagen fibrils, 447, 448 of cytoplasmic elements, 136, 251 of elastic tissue, 447, 448, 449 of fibers, 445 of “fluffy layer”, 250 of mast cell, 403 of membranes, 201, 246 methacrylate embedding for, 449 of mitochondria, 296 of mitotic spindle, 138 and nuclear cytology, 136 of nucleolus, 135 specific stains for, 136 of Tubifex eggs, 297 of thymonucleohistone, 319 and viruses, 136, 300 Electrophoresis, paper, fraction’s of commercial orcein in, 444 Eleidin, staining of, 438 Embryology, ehromosmne and gene complements and, 193 Embryos, of amphibians, enzymes and yolk utilization in, 288 of ascidians, melanizing enzymes in, 307 ascorbic acid in, 292 of chick, 24, 4749, 287, 291, 298, 470 development of blood- and lymphatic systems in, 190, 1p2, 372, 379 differentiation in, 277-320, 295-301 effect of vitamin A, 47 enzymes in, 279-284, 287, 298 fibers of, 438, 450 of grasshopper, 123, 125, 287, 298 growth of, and organization of lampbrush chromosomes, 163 homogenized and respiratory enzymes, 282, 298 human, 49, 182, 183, 186, 189, 190 limb bud rudiments of, and vitamin A, 47 origin of somatic inconstancy in, 189 vascularization in, 366 of mouse, 189

Embryo extract, in biological media, 6, 9, 8-14,23, 41 “Embryonin”, 10, 14, 20 Endoderm, 277, 305 Endometrium, 183, 189, 191, 195 Endomitosis, 151, 152 Endopolyploidy, in insect tissues, 147 Endothelium, 365, 366, 370, 371, 379, 383, 386 Enolase, in wheat germ nuclei, a 4 EnterobiuF vermicdaris, survival in transparent chamber, 393 Enterokinase, and trypsin activity, 211 Enucleation, and role of the nucleus, 294 Environment, and cell nutrition, 2 Enzymes, activation of thiol compounds, 290 activity, 203, 252, 283, 284-291 adaptation of, 313, 314 adsorption of, by nuclei, 200 antagonistic effects of, in fractions, 256 artificial redistribution of, 245-250 assays for, artifacts associated with, 253 azo dyes in histochemistry of, 329-357 cellular, 331 classification of, 203 coacervation of, 299 concentration of, 203, 204, 206 cytoplasmic and nuclear, 258, 259 and DNA synthesis, 200 DPN-synthesizing, 212, 213, 241, 243 destruction in nucleus and cytoplasm, during isolation, 203 digestive, and zymogen granules, 206 distribution of, in adult mammalian digestion of chromosomes and, 146, 155 tissues, 2M atypical, and artifacts, 255 in developing eggs, 278-284 and differential centrifugation, 239259 in embryos, 288, 298 in fresh homogenate, 203 in granules, of liver cells, 296 and difforences in sedimentation, 252 heterogeneous, 258-259 intracellular, and starvation, 206, 258 in larvae of sea urchins, 279

509

SUBJECT INDEX

in nucleus, 199-221, 259 in Ram, during development, 287 effect of carbamates, on activity, 123, 124, 126, 127 elastolytic, and solubility of elastin, 441 of general distribution, 203 glycolytic, 204, 206, 242 hatching, of amphibia, fish and sea urchins, 289 hydrolytic, 209, 260 inhibition by diazoniuan salts, 350, 351 insoluble, Zoo, 216, 249 interdependent, separation of, by fractionation, 257 intracellular components, and, 239 iron porphyrin, 287 localization .by azo dye methods, 335, 349, 355 melanizing, in ascidians, 307 microsomal, sedimentation of, 251 mitochondrial, 207, 251, 283, 287 molecular weight of, 206 non-particulate, and nuclear fraction, 258 nuclear, 135, 200, 204, 205, 206, 210 oxidative, 209, 213 particulate, 246-249, 279-284 proportion of, in homogenate fractions, 240 pmteolytic, 210, 279, 282, 288, 289 respiratory, 282, 283 soluble, 200, 206, 217, 245 of soluble protein fraction, 247 special, 203 specificity of, with azo dye methods, 330 synthesis of, and respiration, in sea urchin, 287 of tissues, and differential centrifugation, 225-269 and yolk utilization, 288 Enzymology, ontogenetic, 312 Eosinophilia, mast cells and, 414 Ephedrine, 368 Epidermis, 33, 40, 374, 375, 378, 389 Epididymis, of mouse, Colgi particles in, 251 Epinephrine, 55, 368 Epithelioma, of rabbit, 388, 390, 391

Epitheilm, 11, 23, 28, 191 Ergonovine maleate, 369 Ergot, 369 Ergotamine, 369 Ergotoxine, 368, 369 Erythrocytes, of birds, purity of isolated nuclei from, 202 catalase in, 286 of chicken, effect of saponin on, 201 contamination of fractions by, 214 extravasated, removal by lymphatics, 372 gel formation by isolated nuclei of, 220 hemogloblin in nucleus of, 201, 205 ingestion of, by macrophages, 373 Eschatin, in tissue culture media, 19 Esterases, azo dye method for, 332, 333, 352, 353, 356 in centrifugal fractions, 242, 251 distribution of, 204, 332, 333, 341-348 Esters, 210, 343 of carbamates (see Carbamates) Estradiol, effect of, 55, 57 Estrone, effect of, in vitro, 55, 56 Ethylphenylcarbamate, effect on plants, 122 Ethylurethan (see Urethan) Euchromatin, 144 euchromatic regions, 131, 138 in salivary chromosomes, 137 and genetically specific syntheses, 162 Evolution, and polyploidy, 179, 180 Extracts, cell-free, and fatty acid oxidation, 260

F

Factors, genetic, and survival of grafts, 387 hemdynamic, and vascularization in transparent chambers, 390 Fasciolopsis bzwki, eggs of, in transparent chamber gtudies, 392, 393 Fat (s), abdominal, auto- and homografts of, 378 and blood vessels, in transparent chambers, 374, 375 droplets of, in cytoplasm, 375 gradients in, of Amblystoma embryo, 305 and mast cells, 419, 424

510

SUBJECT INDEX

Fatty acids, 45, 256, 260 Femur, epiphysis of, alkaline phosphatase in, 353 Ferritin, granules of, isolation by fractionation, 238 Fertilization, in sea urchin, 119, 284, 285, 289, 290, 291 Fetus, chromosome numbers in, 182 enzymes in, 204, 354 Feulgen reaction, in salivary chromosomes, 139, 155 in centrifugation, 156 Fibers, collagenous, 439, 450 of connective tissue, and fibroblasts, 374 elastic, 437-452 aging, 451-452 basophilia of, and oxidation, 443 density of, in electron micrographs, 448 description of, 437 effects of elastase on morphology and refractility, 446, 447, 449 histologic identification of, 442 isotropism of, in relaxed condition, 445 origin of, 450, 451 pathologic destruction of, 446, 451, 452 patterns of, 438 in polarizing microscopy, 445 properties of, in spreads of fresh tissue, and in sections, 445 selective staining of, 437, 438, 442, 443 separation of, for chemical analysis, 439 submicroscopic structure, 448, 450 trypsin digestion of, 448 X-ray diffraction spectrum of, 449 electron microscopy of, 445 fine and large, occurrence of, 438 formation of, 374 macromolecular organization of, 445 periodic bands in elastic tissue, 447 pigmentation of, in elastosis, 452 polarizing microscopy of, 445 reticulum, 439 stretchability of, 450 X-ray diffraction spectra of, 445

Fibrils, in chromosomes, 140, 154, 161 collagen, in electron microscopy, 447, 448, 450 reticular, optical behavior of, 446 Fibrin, proteose from, and growth of tissue culture, 35 Fibrin net, in transparent chambers, 374 Fibroblasts, 399, 404, 413 binucleatc, in culture, 102, 103, 106, 109 caryometric studies on, in culture, 70, 71 effect of amino acids and peptides on. 10, 35-37, 41, 43, 52 of glucose on, 28, 29 of hemoglobin on, 26 of hormones on, 52, 54, 55 of lipids on, 44 ff. of vitamins on, 46, 47, 51, 52, 54 growth measurements in cultures of, 3, 4, 8, 25 media for, 22, 23, 24, 26, 32, 55 and migrating epidermal cells, 375 migration of, 23, 33, 42, 55 nuclear size, 77, 82, 85, 86, 90, 91, 93, 94, 95, 109 orientation of, in transparent chambers, 374 pre-prophase inhibition in, 44 transformation into mast cells, 401 vascular reactions to mcthylcholanthrene-treated, 392 Fibroma, mast cells in, 414 Fibromyoma, uterine, mast cells in, 414 Filaments, “nucleoplasmic”, and nucleoli, 159 Fishes, lampbrush chromosomes in, 154 Flavoprotein, isolated nuclei and, 211 Flowmeter, measurements of coupling rate in histochemistry by, 336, 337 “Fluffy layer”, 250, 252 Fluids, amniotic, use in tissue cultures 8 ascitic, glycine concentration in, 38 biological, in tissue culture, 8 body, and nutrients for tissue culture, 1 of hydatid cyst, effect on leukocytes, 373 synovial, 406 Folic acid, distribution of, 17, 51, 248 and megaloblast maturation b t vitro, 51 and metabolism, in zitro, 43, 45

SUBJECT INDEX

Follicles, ovarian, survival in grafts, 378, 380 Fractions, centrifugal enzymes in, 243-244 final particulate, 251 microsomal, 236, 238, 240, 241, 244, 245, 25 1 mitochondrial, 240, 241, 244, 250-254, 257, 261 nuclear, 238, 240, 241, 244, 245, 264 particulate, 241 soluble, or final supernatant, 226, 266 Fractionation (see also : Centrifugation, differential) centrifugal, 227-241 complete, of cellular constituents, 225, 249 Fragmentation, intravascular, in spleen grafts, 384 nucleolar, in chick fibroblasts, 44 Frog, egg of, cell division and respiration, 123 DNA in, 295 Fructose, 7, 30 Fructose diphosphate, in tissue culture media, 29, 30 ’

G

Galactose, in tissue culture media, 29, 30, 31 p-Galactosidase, azo dye method for, 333 Gametogenesis, and polyploidy, 180 Ganglia, sympathetic, auto- and homografts of, 373, 378 Gastrula, 292 of amphibians, protein synthesis in, 304 enzymes in, 279 of sea urchin, 312, 316 Gastrulation, and somatic inconstancy in rodents, 190 Gel, formation of, by isolated chicken erythrocyte nuclei, 220 Genes, action of, 191, 192, 220,221 and differentiation, 193 and formation of biotin, p-aminobenzoic acid and adenosine, 294 and somatic inconstancy, 187 Genonemes, in salivary chromosomes, 146 “Ghosts”, in fractionation, 249, 250

51 1

Glands, adrenal, aryl sulfatase in, 354, 355 salivary, 132 Glucoprotein, in mast cells, 415 Glucosamine, 30, 418 Glucose, in blood of birds and mammals,

27 C14 labeled, incorporation into aspartic acid and alanine, in vitro, 39 in synthetic tissue culture media, 15, 18, 21, 27, 30, 31, 32, 33, 54 Glucose-6-phosphatase, 25, 241, 244 8-Glucosidase, azo dye method for, 333 Glucuronic acid, 418 B-Glucuronidase, 204, 243, 252, 255 azo dye method for, 332, 333 Glucuronides, in azo dye methods, 330 Glutamic acid, 7, 17, 29, 40, 301 Glutaminase I, in sea urchin development, 279 Glutamine, 17, 29, 30, 36, 42 Glutathionase, in isolated nuclei, 210 Glutathione, 15, 17, 30, 41, 261, 290 Glycerol, 18 Glycerophasphate, permeability of granules and, 254 in tissue culture media, 18, 29, 30 Glycine, 15, 16, 35, 38, 39 Glycogen, 30, 55, 206, 231, 238, 266, 288, 373, 415 Glycolysis, 30, 53, 205, 207 (see also: Enzymes, glycolytic) anaerobic, 33, 124, 209, 210, 256, 318 effect of lithium on, 318 nuclear, 207, 210 nucleic acid synthesis, and, 207 Glycoprotein, elastin and, 440 Glycosidases, azo dye m e t h d s for, 353 Glycyltyrosine, effect on Streptococcrw faecalis, 37 Golgi body, 251, 282, 405 Golgi particles, isolation from mouse epididymis, 251 “Golgi region”, 347 Gonadotropin, 238, 253 Congylus, chromosomes in mast cell from spleen of, 400 Gonocyte, primary, and DNA during meiosis, 133

512

SUBJECT INDEX

Gradients, density of, and layering technique, for centrifugal fractionation, 239 “double-gradient concept”, 305 metabolic, in gastrula of Amblystoma, 305 mitcchondrial, 305, 306, 309, 310 mitotic, in blastula, 306 reduction, 306 respiratory, 307 ribonucleic acid, in amphibian germ, 305 vascular, in autografts, 383 vegetal, effect of lithium and micrw mere implantation on, 305 Grafts in transparent chambers, 377-378 autogenous, 376-386 effect of drugs on, 386 homologous, 386-387 of spleen, functions of, 384 survival of, 380 of tumors, 387-392 Granules, argentophilic, and ascorbic acid in differentiating cells, 293 carotinoid-containing, in Pwacenfrofxs, 299 cellular, and hydrolytic enzymes, 260 cytoplasmic, enzymatic differences in, 251 disintegration with sonic vibrations, 247 of frog germ, and respiratory enzymes, 283 “growth granules”, 251 intracellular, classification of, 2% keratohyalin, 315 large, method of distinguishing, 226, 235, 236, 240 in mast cell, 438 secretory, 206, 251 sedimentation of, 252, 282 small, isolation of, 226, 236-237 special, enzyme activity in, 252 undetectable morphologically, and enzyme activities, 252 of vitelline origin, in invertebrate eggs, 282 zymogen, 206 8-Granules, 316

Granulomas, mast cells in, 413 Granulocytes, 411, 413 Grasshopper, embryos of, 123, 125, 287 Growth, of cells, differential, of chromosome fraction, 164 in zitro, 2, 3, 4 of lymphatic vessels, in vifro, 372 malignant, 125 neoplastic, 114, 125 “rhythmic”, of nuclei, 147 and salivary chromosomes, 162 Growth factors, 11, 292 Guanine, 18, 44 Gum arabic, 215, 239

H Hamsters, chromosome numbers in, 193 Hatching, enzymes concerned with, 289 Heart, of chick, in tissue culture, 25 Hemangiomas, 384 Hematogenous theory, of mast cell origin, 400 Hemin, 18, 293 Hemoglobin, 26, 201, 203, 205, 218, 219, 254, 293 Hemolysins, 288 Hernomhage, 370, 371 Heparin, 290, 416, 419, 424 Heterochromasy, 145 Heterochromatin, 131, 137, 143, 144, 145, 162 Heterochromatin a, 144 Heterochromatin p, 144 Heterogeneity, of cytoplasmic content, 251, 252, 258 Heteroploidy, 188, 189, 192, 193 Heteropycnosis, 145 Hexoploidy, in embryos, 180. 189 Hexokinase, 124, 207, 256, 318 Hexose diphosphate, 18, 205, 260 Hibernation, effect on mast cells in the hedgehog, 424 Histamine, 368, 401, 411, 424, 425 Histidase, in isolated nuclei, 210 Histidine, 17, 26, 29, 30, 141, 349 Histiocytes, 399 in cortisone-treated kidney cultures, 56 transformation into mast cells, 401 in transparent chambers, 372

SUBJECT INDEX

Histochemistry, of enzymes, azo dyes in. 329-357 of isolated nuclei, 199 Histogenesis, in insects, regulation of, 289 of mast cells, after histamine, 401 Histogenous hypothesis, of mast cell origin, 400 Histolysis, in insects, 289, 291 Histomechanical principle of Thoma, and vascularization in transparent chambers, 365, 367 Histones, and bands in salivary chromosomes, 141 and DNA, 135 in isolated chromatin threads, 134 in isolated nuclei, 201 Homogenates, 227, 232, 233, 254 enzymes in, 203, 234-291 oxidation of fatty acids in, 260 properties of, 232 Homogenization, 208, 211, 245, 280 Homogenizer, for isolation of nuclei, 208, 216 Homografts, 378, 386, 387, 388 Hormones, lactogenic, in vitro, 55 luteinizing, 384 thymtropic, 381, 424, 427 in tissue culture media, 53-57 Hyaluronic acid, 407, 411, 417, 418, 419, 426 depolymerized, in rheumatoid arthritis, 414 in dermal ground substance, 406 and mast cells, 405, 423 Hyaluronidase, 406, 407, 415, 419, 430 Hybrids, lethal, phosphorylated A T P in, 294 Hydrocarbons, carcinogenic, 113, 413 Hydrogen ion concentration, critical, for isolated nuclei, 216 for tissue culture, 11, 12, 57 for tissue homogenates, 233 effect of, on azo dye methods, 356 on enzyme activity, 255 on injury to blood vessels, 369 during histolysis in insects, 289 of intercellular substance, 374

513

Hydrolases (see also : Enzymes, hydrolytic), distribution of, 260 Hydrolysis, non-enzymatic, of naphthyl substrates, 335 Hydroxyproline, 17, 439 Hyperthyroidism, mast cells and, 412 Hypertrophy, and growth of salivary gland nuclei, 152 and “residual chromosome”, 164 Hypomorphoses, cephalocaudal and dorsoventral, in gastrulae, 317 Hypoxanthine, 18, 29, 43, 301

I 1131 (see also Iodine), 381, 387, 391 “Idiochromatin”, 155 Inclusions, granular, in the egg of Arback, 297 Indophenol oxidase, embryogenesis and, 287 Infecfion, macrophages and, in transparent chambers, 372 mast cells in, 423 tukrculous, 393 Inflammation, 37, 366, 414 Inhibitors, of enzymes, in hatching, 289 formation of, in sea urchin, 286 in monoenzymatic systems, 253 Inosinic acid, 29, 30 Inositol, 7, 17, 29, 45, 52 Insects, endopolyploidy in, 147 apodehydrogenase in holometabolic, 289 regulation of histolysis and histogenesis in, 289 Insulin, 19, 45, 53, 54 “Interband”, in salivary ’chromosomes, 137 Intercellular substance, nature and pH of, 374 Interphase, carbamates and, 119, 120, 128 chromosomes in, 131 effect of adenosine and adenylic acid on cells in, 44 of DNA precursor on, 295 of urethan on, of sea urchin eggs, 119, 128 Intersexuality, and polyploidy, 180 Inversion, loop, and structural rearrangement in salivary chromosomes, 151

514

SUBJECT INDEX

Invertase, inhibition of, by urethan, 126 Invertebrates, pattern of polyploidy in,

180 Iodine, effect on thyroid follicle, 381 localization of, by microradiography,

438 Larvae, dipteran, chromosomes of, 131,

456 Iodosobenzoic acid, 304, 305, 306, 309,

310, 315 Ionic environment, and tissue culture, 57 Iris, 386, 407 Irradiation, effect on tumors, 390, 391 Isocitrate, 261 Isolation, of chromatin strands, 134 of nuclei, 199-221 Isoleucine, 17, 29, 30 Isopropylphenylcanbamate, 116, 122 Isotropism, in relaxed elastic fibers, 445

J Jelly coat substance, 290 Juxtanuclear zone, in mast cells, 404

K Karyokinesis, 121 Karyomere, formation of, with urethane,

119 Karyoplasrn, of adult nerve cell, 469 “Keimplasma”, 155 . Keloids, mast cells and metachromasia in, 412 Keratinization, prevention by vitamin A, in vitro, 47 a-Ketoglutarate, oxidation of, 257 Kidney, 134, 203, 233, 345, 346, 407 Kinase, in wheat germ nuclei, 204 “Kochsaft”, 209, 286 Krebs cycle, intermediates of, and action of homogenates, 256 mitochondria1 and extramitochondrial systems and, 262 and rate of mitosis, 33 Kurtosis, use in caryometry, 79, 81, 83,

108

L Lactate, 30, 205, 207 Lactic acid, 31, 32

Lactobarillus casei, effect of sea urchin extracts on, 293 Lactoglobulin, and tissue culture growth,

36

Lactonase, in kidney, distribution in fractionation, 244 Lactose, in tissue culture media, 30 Lamellae, in elastic tunic of blood vessels,

132 of sea urchin, 278, 287 Layering technique, in centrifugal fractionation, 239 Lecithin, 44, 419 Lecithinase A, effect on chick heart, 45 Lesions, elastotic, histochemistry of 452 tuberculous, fate of elastic fibers in, 451 Lestodip[osis, polyploidy, and polyteny in,

154 Leucine, 7, 16, 17, 29, 30 Leuco- (see also: Leuko- ) Leucovorin, z92 Leucyltyrosine, effect on Streptococcus

faecalis, 37 Leukemia, effect of urethan on, of mice, 115, 116 Leuko- (see also: Leuco- ) Leukocytes, basophilic, and mast cells, 400 chromosome count from tissue culture of, 186 effect of acacia on, 373 of CO, perfusion on endothelium, 370 of dextran on, 373 of glycogen on, 373 of hydatid cyst fluid on, 373 of insulin in cultures of, 54 of thiamine on growth of, 49 of thyroxin on, in zitro, 55 and eggs and larvae of parasites in transparent chamber, 392, 393 fragility of, and ascorbic acid content,

48 migration of, in transparent chamber,

370 oxygen consumption in Zritro by, 34 phagocytosis by, and ascorbic acid, 48 polymorphonuclear, 373 somatic inconstancy of, in vitro, 186 “trephone” secretion by, 8 Leukocytosis, glycogen and, 373 Leukopenia, glycogen and, 373 Limb bud development, and somatic inconstancy, in rodents, 190

515

SUBJECT INDEX Limtiaea, 316, 320 Lipase, pancreatic, 204 Lipids, effect of carbamate on, 127 in invertebrate eggs, 282 in mast cells, 419 in nerve cells of adult rabbits, 472 extraction hy fixative, 473, 474 in tissue culture media, 44-46 Lipid synthesis, and vitamin B, 45 “Lipochondria”, in unfertilized egg of Rarm, 297 Lipogenesis, and carbohydrate metabolism, 45 Lipoma, mast cells in, 414 Liponucleoproteins, nerve cell function and, 474 Lithium, 310, 315 effect on cytoplasm, 319 on development of sea urchin, 277, 278, 280, 281, 304, 318 on gradients, 305 on respiration, 307, 308 Lithium ions, action on developing egg, 316-320 cells, bi- and multinuclear, 102, 134 Liver, amphibian, isolation of melanin granules from, 238 caryometric studies on, 70 centrifugal fractionation of, 231, 233, 240, 242, 243 dehydrogenation in, 125 dephosphorylation of ATP by, 257 effect of vitamin A on cultures of, 46 enzymes in, 203, 209, 287, 345, 347, 348 mast cells in, 407 nuclei of, 98, 145, 153, 201, 204, 207, 209, 213 of rodents, differential centrifugation of, 231 Loops, chromosomal (see lampbrush chromosomes), 161 Lung, 186, 410 Lymph, in tissue culture, 5 Lymph flow, in rabbit’s ear, 372 Lymph node, auto- and homografts of, 378 Lymphatic vessels (see Vessels, lymphatic)

Lymphocytes, 25, 44,56, 57 small. in transparent chambers, 373 survival of homografts and, 387 transformation of, into mast cells, 401 viability of, with cortisone, 56 Lyophilization, and enzymes, 203 isolation of nuclei and, 202 Lysine, 7, 29, 30 Lysolecithin, effect on chick heart, 45 Lytechinur, egg cleavage, 119

M Macrophages, choline and transformation of fixed tissue cells into, 52, 53 divisions of, in transparent chamber, 372 fusion of, to form multinucleate giant cells, 373 intake of erythrocytes by, 372-373 of micrococcin by, 394 of silica by, 371 nutritional requirements of, 3, 20 Mactro, eggs of, proteolytic enzyme in, 282, 290 Malpighian body, in spleen grafts, 384 Maltose, in tissue culture media, 30, 31 Mammals, chromosome number in, 193 polyploid evolution of, 179 transparent chamber methods in studies on living, 359-394 Mannose, in tissue culture media, 29, 30 Marrow, effect of carbamates on, 120 Marsupials, chromosome number in, 193 Mass, determination of, and microradiography, 463, 466 Mastmytomas, effect of cortisone on, 430 “hlastzellen”, 422 Matrix, in cartilage, staining of, 438 of connective tissue, and mast cells, 412 of lampbrush chromosomes, 160 of mitochondria, soluble proteins and, 247 “Meander stage”, and giant chromosomes, 151 Media, hypotonic, 216, 258 suspension, for centrifugal fractionation, 229, 230, 233, 231, 238, 239 for isolated nuclei, 216, 234, 238 tissue culture, biological, 5-23

516

SUBJECT INDEX

Medullation of nerve fibers, in transparent chambers, 374 Megaloblasts, maturation in vitro, 51, 53 Meiosis, DNA in primary gonocytes, during, 133 effect of narcotics on, 124 in polyploids, 180 Melanin, granules isolated by frac"rionation, 238 Melanomas, vascular response in, 389, 390 Membranes, cellular, of mast cells, 405 embryonic, 185, 191, 194 fetal, and chromosome counts in, 183 nuclear, morphology of, 268 nature of, 269 permeaibility of, 200, 205, 212, 213, ocular, mast cells in, 407 218, 268 penetration of, by substrate, in azo dye methods, 347 of phage, 300 synovial, mast cells in, 406, 408, 409 vitelline, proteolytic enzymes in, 290 Menadione, in tissue culture media, 17 Mercury, effect of, on lymphocytes, 57 Mesenteries, fibers in, 438 Metabolism, antagonistic types of, in sea urchin, 311 carbohydrate, in eggs, 283,308 of fats, 45, 419, 424 and mitochondria, 306 nuclear, and cytoplasmic components, 206 and RNA, 219 of nuclear acids, 43, 99 oxidative, in insects, 289 of purines, and urethan action, 127 vegetal, and mitotic activity, 306 Metabolites, intermediate, and intranuclear metabolism, 207 Metachromaaia, 412, 416, 419 Metachromatic substance, 407 Metals, in tissue culture media, 26 Metamorphosis, in insects, hormonal control of, 289 Metaphase, abnormal chromosome counts from, 190 effect of adrenorhome on, 55 of carbamates on, 120

of phenylurethan on, 120 of urethan on, 119, 120, 127 Metaphen, effect on blood vessels, 370 Metastases, 114, 390

Methacrylate, embedding medium, 449 Methionine, in tissue culture media, 17, 29 Methylcholanthrene, 37, 392 Methyl guanidine sulfate, 368 Methyl naphthohydroguinone sulfate, 29 Methyl testosterone, and mitotic disturb ances in vitro, 55 Microabsorption, and salivary chromosomes, 163 Microcinematography, movement of mitcchondria in phase contrast, 267 Micrmmin, intake by macrophages, in transparent chambers, 394 Microdissection, 394 of isolated nuclei, lW, 201 and nucleusqytoplasm interactions, 219 of salivary chromosomes, 142 of thyroid, in Vim, 380 Microincineration, of elastotic lesions and aging arteries, 452 of salivary chromosomes, 142 Micromanipulation, of salivary chromcsomes, 147 Micromeres, 302, 304, 305, 309 Microorganisms, nutrition of, 2 Microphotometer, in microradiography, 462, 463 Microradiograms, 455, 456, 457, 458, 459 Microradiography, 455, 456, 457, 459-465 chemical fractionation of nerve cells and, 465 cgtochemical purposes of, 462 and mass determinations, 455, 463 nerve cells and, 465, 4M, 472, 474 nucleic acids in nerve cells and, 466 qualitative chemical analysis by, 456 Microscopy, electron (see Electron mi-0scopY) fluorescent, 394 phase contrast (see Phase contrast microscopy) polarizing, of commercial fibers, 445 reflecting, 394

SUBJECT INDEX

Microsomes, 241, 246, 250, 255, 308 antigens in, 312 artificial degradation products of, 240 composition of, 241, 244, 246, 260, 280 effect of urethan on oxygen uptake by, 124 in eggs of invertebrates, 282 in fractions, 200, 226, 252 origin of, 265 properties of, 248 and respiration in mitochondria, 264 Sedimentation of, 230, 251 Microspectrography, of isolated nuclei, 199 ultraviolet, and RNA determination, 468 Microspectrophotometry, of interphase nucleus, 132 Millon test, and lampbrush chromosomes, 155 Mitochondria, 308, 309-311, 331 adsorptive capacity of, 246 aging, ATP and, 268 amino acid formation, and, 266 in amphibian embryos, 288 and basophilic material in sea urchin, 283 chemical factors in, 291 citrulline synthesis by, 263 contractile system and, 268 conversion of acetates by, 301 counts and enzymatic activity in embryogeny, 283, 284 D P N in, 212 during development of Arbacia, 297 development of, cell surface and, 320 disintegration of, 216, 246, 248 distribution of, 301309 effect of urethan on oxygen uptake in, 124 in eggs of Ascaris, 299 electron microscopy of, 296 electron transfer and, 261 enzymes and, 216, 217, 257, 261, 279, 280, 287 fragmentation of, in stratum granule sum, 315 and granules, in differential centrifugation, 226

5 17

introduction into the egg with the sperm, 297 of invertebrates, 282 in isolation of nuclei, 200 in kidney, effect of egg albumin on, 2% in mast cells, 405 in morphogenesis, 314-316 nuclear membrane and, 206 origin of, 265, 295-301 oxidative phosphorylations, 234, 262265 oxidations and, 254, 256 permeability of, 266, 267 in phase contrast, 251, 267 precipitate of, in “fluffy layer”, 250 and protein synthesis, 314 residual, and cytochrome oxidase in isolated nuclei, 214 and respiration during cleavage, 2% respiration of, 264 sedimentation behavior in, 249 in spermatogonia of Drosopkila, 294 tricarboxylic acid cycle in sea urchin egg and, 282 “Mitochondria1 ghosts”, contamination with, and cytochrome oxidase, 244 Mitosis, (see also: Cell, division of; Division, mitotic), in chondroidal cells in transparent chambers, 375 and chromatin condensation, 163-164 DNA, in chromosomes during, 133, 200 duration of, 55, 56 effect of adrenochrome on, 55 of amino acids on, 41 of carbamates on, 115, 124 of colchicine on, 121 of glycogen on, 55 of hormones on, 54, 55 of oxygen tension on, 33 of purines on, 44 of trypoflavine on, 107 of urethan on, 113-128 of x-rays, 4 in endothelial nuclei, and formation of capillaries, 365 in “ohserved squash” technique, 1% stimulation of, 5, 6 tripolar, in urethan treated material, 119

518

SUBJECT INDEX

Mitotic coefficient, in tissue culture, % Monocot plants, effects of urethan on, 121 Monocytes, 15, 41, 47, 55, 372 Monosulfuric acid, in amyloid, 415 Morphogenesis, 277, 301 mitochondria and, 314-316 Mouse, chromosome number, diploid, 181, Mucin, 415 Mucoitin monosulfuric acid, 417 Mucolecitide, in mast cell granules, 415 Mucopolysaccharides, 406, 415, 419, 424, 425 Mucosa, uterine, chromosome number, 182, 183 Muscids, giant chromosomes in nurse cells of, 147 Mutations, with urethan, 127, 128 Myoblasts, peptic digests and growth of, 38 Myocarditis, mast cells in, 414 Myelin, degeneration of, in transparent chambers, 374 Myofibrils, isolation of, by fractionation, 238 Myoglobin, 203, 205 Myokinase, 242, 254

N NIB, incorporation into microsome fraction, 300, 301 nuclear RNA turnover, and, 265 Naphthalene, 430 p-Naphthol, 330 Naphthols, 336 a-Naphthylamine, 330 Naph.thy1 phosphates, halogen substituted, in azo dye methods, 340 Narcosis, 116, 125 Necrosis, 371 :Vcchwus, lampbrush chromosomes in, 156 loop, electron micrograph of, 174, 175 Neqdasia, nutrition in, 2 Nerve fibers, growth of, and oxygen tension, 34 Nerves, in transparent chambers, 373-374, 379 Nervous system, mast cells in, 410

Neural groove stage, and somatic inconstancy in rodents, 190 Neuralization, induced by thiol compounds, 309 Neuroblasts, 469 Neurons (see also: Cells, nerve; Neuroblast), 470, 471, 472-474 Neurula, protrin synthesis in, 304 Nicotinamide, 17, 51 Nicotinamide mononucleotide, D P N synthesis, and, 212 Nicotinic acid, 17, 29, 45, 51, 294 Ninhydrin, reaction of chromosomes to, 141, 155 Nitrogen, distribution of, in fractions, 15, 25 1 Nitroglycerine, 368 Nitrophenols, metaphase block with, 122 Nodes of Ranvier, in transparent chambers, 374 Nodules, silicotic, 371 Nucleates, effect on migration of mouse fibroblasts, 42 Nucleic acids (see also : Deoxyribonucleic acid, Rihonucleic acid) effect of poisons on metabolism of, 99 in growing tissues, 42, 43 combination with carotinoid-containing granules in eggs, 299 cytophotometric determinations of, 136 in lampbrush chromosomes, 155 in mast cell granules, 415 metabolites of, during cleavage, 308 nuclear enzymes and, 206 in salivary chromosomes, 138, 142, 143 synthesis, and glycolysis, 207 in tissue culture media, 15, 35, 42-44 Nucleolus, 145, 146 biochemical constituents, determination of, 213-214 in cellular synthesis, 162 and Feulgen-positive granules, 156 and “nucleoplasmic filaments”, 159 in oocytes, coenzymes of, 154, 155, 294, 295 phase contrast and electron microscopy of, 135 production of, in noii-mitotic cells, 162

SUBJECT INDEX

regions forming, 144 size of, 71, 107, 133, 152 Nucleoplasm, accessory, and interband material of, 148 Nucleoproteins, 10, 42, 132, 135 Nucleosidase, in centrifugal fractions, 242 Nucleosidephosphorylase, in nuclei, 294 Nucleosides, 43 Nucleotides, 43 Nucleus, ATP-ase in, 204 adsorption by, and insoluble mitochondrial enzymes, Mo alkaline phosphatase in, 204 arginase, in rat liver, 209 biochemical role in resting cell, 218 of bird erythrocyte, 202 bound cytoplasm in, 202 catalase in, during starvation, 204 and cellular protein synthesis, 162 chemistry of, 199 of chicken erythrocyte, hemoglobin in, 205 classes of nuclear sizes, 69, 73, 75, 76, 108, 151 coenzyme synthesis by, 212, 294 DNA and RNA, 151, 265 enzymes of, 199, 203, 204, 206, 221, 248, 258, 259 of non-particulate fraction of, 258 esterase in, 204 of fat body, in Drosophila wlanogasfey, 170 of frog egg, isolated by microdissection, 201 fusion of, polytene chromosomes and, 154 p-glucuronidase in, 201 hexokinase, 206 incomplete glycolysis in, 207 injury to membrane during isolation procedures of, 201 insoluble phase of, 206 interphase, 131, 133, 163 isolated, 199-221, 238, 244, 258 enzymes in 199-21, 226, 258 microdissection of, 199 and microspectrography of, 199 morphologic integrity, and cornposition of suspension medium, 234

519

and peptide synthesis by, 21 1 and phosphate esters in, 210 mammalian, diphosphopyridine nucleotide in, 205 membrane of, 200, 205, 206, 213, 218, 220, 221, 248, 268, 269 metabolism of, 206, 208, 219 morphology, following histone extraction, 201 multivalent, and DNA content, 133 of muscle cell, and myoglobin, 205 of nerve cell, effect of fixation on, 474 oxidative phosphorylations, and, 267 oxygen uptake, with urethan, 124 in plant, and glycolysis, 205, 206 polyploid, and DNA, 133 protein of, and cell function, 133, 265 pycnotic, due to carbamates, 120, 121 regulator of mitochondria1 activity, 294 resting, DNA synthesis in, 218 "rhythmic growth" principle, 147 ribonucleic acid in, and cell function, 133 role of, during rhythmical changes in respiration, 295 of salivary gland, and DNA content, 161 in sea urchin eggs, urethane-treated, 119 sedimentation, during centriugal fractionation, 232 single, DNA determinations, and somatic inconstancy, 194 size of, 69, 95, 97,99, 102 frequency curves, 77, 80, 82, 85, 87, 88, 91, 92, 95, 96, W, lW, 101, 104,106 statistical analysis of, 71-78 soluble phase, and enzyme concentration, 206 splitting of, by ATP, 210 stimulation of cytoplasmic activity by, 295 synthesis by, 212, 218, 265, 294 transplantation of, 219 trypsin in, 211 voIume of, 69-72, 97, 147, 152, 153, 170, 183 of wheat germ, enzymes in, 204

520

SUBJECr INDEX

Nucleus-cytoplasm relationship, and permeability of nualear membrane, 202 Nutrients, essential, for tissue culture, 3 Nutrition, cellular, mechanism of, 1 Nutritional requirements, of animal cells, 1-56 0

Oxidation-reduction potential, and tissue culture, 57 Oxides, in tissue culture media, 15 Oxygen, 27, 33, 38, 123-125, 294, 304, 307 Oxygen tension, gradient of, in peripheral tissue in transparent chambers, 369 and mitotic rate in tissue culture, 33 production of emboli and, 371

Octanoate, oxidation of, 257 Ocular membranes, mast cells in, 407 Oetrothera, chromosome translocations with urethan in, 127 Oleic acid, effect of, on rat fibroblasts, 46 Omentuxn, auto- and homografts of, 378, 385 chromosome counts in, 183 Onion, mitotic block with urethan in, 127 Ontogeny, and chromosomal structures in Diptera, 137 and somatic inconstancy, 193 Oocytes, 131, 148, 154-156, 177, 178, 294 Oogenesis, and polyploidy, 189 Oogonia, human, chromosome numbers in, 177, 178, 179 Orcein, 444, 445 Organogenesis, primary, and somatic inconstancy in, 190 Oscillator, sonic, and DPN-synthesizing enzyme, 212, 213 Osmic acid, electron microscopy and, 448 Osteoblasts, 11, 32, 33, 70, 82, 90, 102 Ostrea, enzymes of tricarboxylic acid cycle in, 282 Ovalbumin, in microsomes and mltochondria, 2% Ovary, 120, 378, 380 Oxalacetate, oxidation of, 257 Oxidase, cytochrome, in development, 213-216, 244, 247, 252, 285, 287, 298,422 in fractions, 252 in mast cell granules, 422 and mitochondria, 214, 216, 244 in nuclei, 213-215 release of, by sonic vibrations, 247 in whole tissues, 213 Oxidases, 209, 243 Oxidations, cellular, 256, 260, 261-262

P 294, 300 and nuclear RNA, 265 pH (see Hydrogen ion concentration) Pancreas, auto- and homografts of, 378 Pantothenate, 29, 45, 52 Pantothenic acid, 2.18, 294 Papilloma, precancerous, mast cells in connective tissue of, 413 Paracentro tus, acid phosphatase in homogenates of, 287, 288 carotinoids in egg of, 316 formation of equatorial pigment band in, 320 proteofytic enzymes in unfertilized eggs of, 282 Parasites, in transparent chambers, 392, 393 Parathyroid, auto- and homografts of, 378, 380 Parenchymal organs, mast cells in, 407 Parthenogenesis, induced by thiol compounds, 309 Particles, cytoplasmic, 229, 250-253 Pellicle, in salivary chromosomes, 142 Pepsin, effect on mast cell granules, 415 Peptic digests, of normal and sarcoma cells, 36 Peptidase, 210, 242, 260, 279, 280, 282 Peptides, 10, 34-42, 211 Peptones, 15; 34, 35 Perichondrium, 364, 410 Perineurium, mast cells in, 410 Periodate, and fertilization, 290 Periosteum, mast cells in, 410 Peritoneal exudate, in tissue culture media, 8 Permeability, of cell membrane, 56 of nuclear membrane, 200, 201, 202, 205 P32,

52 1

SUBJECT INDEX

of intracellular bodies, 267 of mitochondria, 267 Phage, infectivity of, and appearance, 300 Phagocytes, 387 Phagocytosis, 48, 372, 375, 384 Phalaera Buccbphela, hibernation diapause, 293, 294 Phase contrast microscopy, of chromatin, 134 of chromosomes, 131, 136, 174, 175 of connective tissue, 403 of mast cells, 404, 417 of microsomes, 251 of mitochondria, 251 of nucleolus, 135 Phenol(s), 336, 430 Phenol red, in tissue culture media, 19 Phenolphthalein glucuronide, as substrate for p-glucuronidase, 254 Phenylalanine, in tissue culture media, 17, 29, 30 Phenylphosphates, substituted, in azo dye methods, 340 Phenylsulfatase, in sea urchin, 279, 312 Phenylurethan, cytological effects, 116, 118, 120, 122, 124 Phosphatase, acid, 256 activity of, 287, 298 and adsorption, 245 azo dye methods for, 332, 35.2, 356 bound, in fractions, 252 in centrifugal fractions, 243 distribution, 252, 279, 354 p-glucuronidase and cathepsin relationship to, 252 of mitochondria, 217 sedimentation, 25 1 adenosine-5-, 251, 259 alkaline, azo dye method for, 329, 332, 333, 334, 351, 352, 356 in centrifugal fractions, 242, 243 distribution, 201, 2.1-1, 258. 259, 279, 304, 334, 341, 353, 422 solubilization of, 249 gIucose-6-, 242, 252, 254, 256 in isolated nuclei, 209 Phosphate, 23, 142 Phosphate-, glucose-6, 261

Phosphoglucomutase, in centrifugal fractions, 242 Phosphogluconic acid, animalizing effeots on sea urchin, 308 Phosphohexokinase, inhibition with phenylurethan, 124 Phosphokinase, creatine, 263 Phospholipids, in mast cell granules, 419 Phosphoprotein, as active factor in “embryonin”, 10 Phosphorus, 23, 440 Phosphorylase, in centrifugal fractions, 204, 242, 253-254 Phosphorylation, 124, 221, 262-265, 267, 291 Photoemulsion, for microradiography, 456 PhyJaloptera claicsa, in transparent chamber, 393 Pigment, mast cells and, 410, 423 yellow, in motor cells, 471 Pigmentation, of fibers in elastosis, 452 Pineal body, auto- and homografts of,

378 Pitressin, 19, 55, 368 Pituitary, mast cells in, 410 Pituitary gonadotropin, in intermediate fraction, 253 Plants, (see also Monocot and Dicot plants) deb-elopment, and chemical constitution, 277 nucleus, glycolytic enzymes in, 206 pattern of polyploidy, 180 Plasma, 5-7, 19, 23, 37, 42, 46, 47, 50-52 Plasmagenes, 313 Platelets, 384, 394 Pleura, human, chromosome numbers in, 182,183 Ploidy, DNA quantity and degree of, 133 insect tissues, 164 salivary gland, estimation of, 147 Pluteus stage, 279, 283 Polynucleotides, free, in tissue culture, 43 Polyploidization, and amitosis, 106 Pulyploidy, amphibian, pattern of, 180 in animals, 147, 163 caryometric demonstration of, 101 cell number, and chromosome sets in, 180, 189

.

522

SUBJECT INDEX

and chromosome counts in human cells, 179 and differentiation, 163, 190 and evolution of mammals, 179 and gametogenesis, 180 and intense irritation, 163 and intersexuality, 180 invertebrates, pattern of, 180 larval insects, 163 mammalian, 188-189 meiosis and, 180 and nuclear DNA, 133 and nuclear size, 146, 156 in plants, 122, 147, 163, 180 and polyteny in Lestodiplosis, 154 and sex chromosomes, 180 spermatogenesis and oogenesis as evidence of, 189 in tissue culture, with colchicine, 101 Polysaccharides, 370, 391, 392, 418, 424 Polyteny, 149 and basic DNA content, 161 degrees of, in insect tissues, 163 and nuclear size, 152 and polyploidy, in Lestodiplosis, 154 and sabivary chromosome structure, 13S, 146-152 Potassium, 142, 264, 318 Pre-prophase, susceptibility to urethan, 121 Primary germ layers, 302 Primitive streak stage, antisera in, 312 Proctodaeum, mitochondria and formation of, 300, 310 Progesterone, effect on lymphocytes, 57 Prolan, effect on fibroblasts, 55 Proline, in tissue culture media, 17, 29 Propanediol phosphate, effect on sea urchin, 308 Prophase, abnormal chromosome counts from, 190 and DNA synthesis, 200 effect of urethan on, 120, 121, 124, 127 Protamine sulfate, 368 Proteins, 15, 143, 320, 371 cellular, 162 cytophotometric determinations, 136 denaturation of, and carbamates, 127

labeled with N15, in cellular fluid of advanced blastula, 301 and microsomes, 266 nuclear, 133, 135 permeability to, 200, 370 release of, during fractionation, 246, 249 “residual”, 134 solwble, fractionation of, 247, 248 in special granules, 252 stain for, in lampbrush chromosomes, 155 synthesis of, 161, 162, 207, 221. 265, 266, 304, 314 in thyroid, 380 viscosity changes in, 319 Proteose, and cell proliferation, 35 Protoplasm, 126, 311 Protozoa, enucleated, catalase production by, 266 chromosome coils in, 161 nutrition of, 2 Purines, 26, 38, 42-44, 127, 301, 472 Pycnosis, 120, 121 Pyridine, effect on rate of coupling in azo dye methods, 343 Pyrimidine, absorption of, and nerve cells, 38, 42-44, 301, 472 Pyridoxal, 17 Pyridoxine, 17, 29, 45, 50, 51 Pyronins, specificity of, and RNA in lampbrush chromosomes, 155 Pyruvate, 30, 31, 205

R RNA

also: Ribonucleic acid, Ribosenucleic acid) Rabbit, motor root cells of, RNA content in, 469 triploid, adult, 189 Radiation, effect of, on proliferating cells, 121 Radioactivity, fixation of, in sperm, 127 Radiogram (see : Microradiography) Kana, 287, 292, 297, 307 Rat, chromosome number in fetal metnbranes, I83 Reductase, cytochrome, DPN-specific, 242 (see

523

SUBJECT INDEX

cytochrome c, in mitochondria1 fraction, 243, 257, 261 glutathione, in centrifugal fractions,

242 Regeneration, heteroplastic, 400, 401 homoplastic, 400 Refractility, of young fibers, 438 Rennin, effect on circulation, 368 Reptiles, lampbrush chromosomes in, 154 “Residual chromosome”, hypertrophy and, 164 Resorption, of bone, i n transparent chambers, 376 Respiration, in ectoderm and organizer of RaFia, 307 and glycolysis, in rifro, with insulin, 53 of mitochondria, 264 nucleus and, 29.5 of sea urchin, 277, 287, 295, 2% Resuspension, of sediments, in centrifugal fractionation, 237 Revitalization, in amphibians, after lithium treatment, 317 Riboflavin, 17, 29, 45, 50, 291, 294 Ribonuclease, adsorption of by microsomes, 246 in centrifugal fractions, 243 digestion of nerve cells by, 473 effect on mast cell granules, 415 inhibition of, 255 Ribonucleic acid, 43, 135, 146 (see also : Ribose nucleic acid, RNA) cytoplasmic, 219, 265 determinations of, 467-469 differentiation of nerve cells and, 469 distribution of, 251 in chromosmnes, 143, 155 in final particulate fraction, 251 in “fluffy layer”, 250 in granules of liver cells, 296 in isolated chromatin, threads, 134 in motor root cells and motor activity, 472 in testes of Ascaris, 300 in wheat germ nuclei, 204 fraction of, in kidney cells, 134 gradient of, in amphibian germ, 305 and histones, 135 nuclear, 133, 219, 265

-

and purines, in rat, 43 and pyrimidines, 43 secretion of, and origin of microsomes, and mitochondria, 265 synthesis of, 162, 219, 221 Ribonuclmprotein (s) , in Ascaris, 300 elimination by condensed chromosomes,

146 in mast cell granules, 415 Ribose, I8 Rhodanese, 245, 252 Rhodnius, differentiation in, 311 Rhynchosciara, banding patterns in chromosomes of, 149, 172, 173 Rodents, chromosome number in, aannion and chorion of, 183, 190 cornea of, effect of phenylurethan, on mitosis in, 120 liver of, data obtained from fractionation, 231, 240, 3 2 - 2 6 Roots, effect of urethan on, 121 Rutin, synthesis in Ch[a,nydnnionas, 293

S S35,

304, 417

Salamander, effect of phenylurethan on mitosis in, 120 microfibrils in lampbrush chroinos m e s of, 157 Sulmo, 293 Salts, diazonium, 329, 331, 336, 349, 353 mono-diazonium, in azo dye methods,

349 in tissue culture media, 15, 16, 22, 23,

24, 27 Sand-dollar, eggs of, effect of urethan on, 118 Saponin, cell membranes and, 201 Sarcoma 180, ascorbic acid and growth of, it^ ziiro, 48 Sarcoma, cultures, proliferation with urethan, 124 of fowl, methylcholanthrene-induced, 38 fusocellular, growth of, 25 and lactic acid production, 31 mast cells in, 414 vascularization of, in transparent chambers, 389

524

SUBJECT INDEX

Sarcosomes, of heart-muscle, mitochondrid permeability, 2&7, 268 niitochondria, and versene, 234 Sciara, giant chromosomes in, 137 salivary chromosome bands, 140, 147, 149 Scleroprotein, allmninoid, 439 Sea urchin, biochemical differentiation in, . 311 cholinesterase activity in larva of, 313 DNA in eggs of, 115 effect of, carbamates on, 115 of lithium on, 277, 316 of phenyl urethan, 120 of usnic acid on, 294 metabolism during cleavage, 294 paternal antigens in hybrids of, 312 vegetalization, caused by lithium, 316 Secretion, effect of cortisone on, in renal tubules, 56 in thyroid follicles, 381 Sedimentation, and contamination, in differential centrifugation, 230 of enzymes of mitochondria1 and microsoma1 fractions, 251 Sedimentation constant, expression of, ZB Sedimentation rate, centrifugal fractionation, and, 2 8 , 228, 229, 230, 239 Sediments, of centrifugal fractionation, in phase contrast, 251 Seedlings, effect of urethan on, 121 Serine, 16 Serum, 370 of cortisone-treated rabbits, and cell migration in vifro, 56 in tissue culture media, 7, 8, 12, 13, 19, 20, 53 Shock, in mice, 370 Silica, granules of, implantation in transparent chambers of, 371 Sirnuliicmn, chromosomes in, 137, 140 S i d h n virgatwt, salivary c h r o m somes in, 140 Siredon, lampbrush chromosomes, in, electron micrographs of, 157 Skin, auto- and homografts of, 385 diseases of, and mast cells, 411 effect of amino acids on growth of, in vifro, 40

normal, in transparent chambers, 375 Sodium rhloride, and fibroblast migration, 23 Sodium salts, organic, in tissue culture media, 29 Solubilization, artificial, of particulate enzymes, 246-219 of elastin, 442 Solutions for centrifugal fractionation, 234 for chromosome counts, 186, 193, 1% for tissue culture, 16, 17, 18, 19, 21, 22, 23, 24, 27 Solvents, organic anhydrous, in suspension media, 238 Soma, Weismannian, 187 Somatic aneuploidy, 194 (see Somatic inconstancy) Somatic constancy, in mouse, 183 Somatic heteroploidy (see also : Aneuploidy, Somatic inconstancy) Somatic inconstancy, 177, 194, 195 and chromosomes in heredity and development, 187 and cytoplasmic determination in mammals, 192 and differentiation, 190, 193 distribution of, in cancerous and nonpathologic cells, 177, 186, 187 in embryos, 190 in edometrium, 195 in germinal epithelium, 191 in organs in sifu and in zz’tro, 186 in uterine cervix, 195 in rodents, after birth, 190 and embryo development in rodents, 190 origin of, in ontogeny, 189, 193, 194 and RNA determinations in nuclei, 1W and “somatic Weismannism”, 191 and tissue culture, 194, 195 Somatic pairing, in Diptera, 163 “Somatic Weismannism”, and somatic inconstancy, 191 Somatoblasts, of Tubifex, size of glob ules in, 297 “Somatoplama”, 155 Sorghum inhibitor, 288

SUBJECT INDEX

Specific gravity, and flotations with cyclohexane in isolation of nuclei, 202 Spectrophotometry, modifications of azo dye methods for, 356 Spermatocytes, number of chromosmes in, 177, 178, 180, 181 Spermatogenesis, and polyploidy, 189 Spermatogwia, chromosome number, 177, 178, 179, 181 mitochondria in, of Drosophilu, 294 Spermatozoa, catalase activity in, 286 fixation of radioactive urethan by, 127 introduction of mitochondria into the egg by, 297 Spinal cord, RNA content in cells of, 468, 469 Spinal ganglia, growth in tissue culture, and glucose, 28 Spindle, mitotic, effect of colchicine on, 113, 117, 122 of urethan on, 119, 121, 127 electron micrographs of, 138 !$leen, auto- and homografts of, 378, 384, 385 cultures of, 34, 46 somatic inconstancy in, 186 Sphingomyelin-like substance, in oily layer, from elastin solubilization, 442 Spiroccrca sanguinolenta, larvae of, resistance to white cells in, 393 Starch, 30, 288 Starfish, nucleotides in omyte nucleolus of, 294 Starvation, and enzyme concentration, 204, 206 Stimulation, chemical changes, in neurons, induced by, 472-474 Stratum granulosum, fragmentation of mitochondria in, 315 lacunosum, mass determinations by microradiography in, 466 oriens, mass determination by micrcradiography in, 466 pyramidale, mass determination by microradiography in, 466

525

radiatufn, mass determinations by microradiography in, 466 Strepogenin, as bacterial growth factor, 37 Streptococcw faecal&, effect of peptides on growth of, 37 Strongylocentrofw, gradients in blastula of, 306 Substance, elastic, 450 ground, of connective tissue, in elastin preparations, 440 intercellular, hyaline, within chondral areas, 374, 376 osmiophilic, in elastic tissue, 449 Substrates, availability of, in monoenzymatic systems, 253 modifications of, for am dye methods, 330, 331, 338-343, 347 for simultaneous coupling methods, 354 Succinate, effect on succinic dehydrogenase in Amblystomu, 313 oxidation of, 257 Succinoxidase, 242, 244, 251, 287 Sucrose, 30, 32, 208, 233, 238 Sudanophilia, in elastotic lesions, 452 Sulfatase, aryl, in adrenal gland of rat, 354 azo dye method for, 333, 356 Sulfomucopolysaccharides,in tissues, and mast cell count, 425-426 Sulfuric acid, enzyme inhibitor in supernatant and, 255 Supernatant, final, or soluble fraction, in differential centrifugation, 226 enzymes in, 240, 280 extramitochondrial spaces and, 256 Surface, nuclear, 73 Syncytium, 383 Syndrome, radiomimetic, 121 “Synovioblasts”, description of, 406 System, blood-vascular, in adult mammals, 371 central nervous, chemical processes in, 455 enzyme, and hexokinase, 256 lymphatic, in adult mammals, 371 monoenzymatic, artifacts of, 253, 254, 255, 256 polyemymatic, artifacts of, 256-258

526

SUBJECT INDEX

T T P N (see Triphosphopyridine dinucleotide) Tadpole, tail of, blood vascular and lymphatic growth, 359 Tail bud stage, of amphibians, ascorbic acid in, 293 serologic demonstration of specific components in, 312 Tantalum, in transparent chambers, 376 Telolecithal oocytes, of vertebrates, 131 Telomeres, 139 Telophase, effect of cysteine on, 41 of urethan on, 120, 128 Temperature, and amino-acid uptake in culture, 39 and effect of urethan on cell division, 126 Tendons, elastic, reticular pattern in, 438 Testis, auto- and homografts of, 378 of guinea pig, growth in vitro, 41 Testosterone, 55, 57 Trtrahymena, 2, 32 Tetraploidy, 189 Tetravalents, 180 Thiamine, 17, 29, 45, 49, 50, 292, 294 Thiamine pyrophosphate, distribution in fractions, 248 Thiocyanate, effect on animal gradient, 305, 309 Thioflavine S, effect on circulating blood, with ultraviolet radiation, 371 Thiomalic acid, animalization induced by, 309 Thiouracil, 382, 3% Threonine, 16, 29, 30 Thrombosis, and tissue destruction in reaction to tubercle bacilli, 393 Thymine, 18, 127 Thyrnonucleohistone, electron microscopy of solutions of, 319 Thymus, effect of colchicine on, 121 mast cells in, 410 Thymus nucleic acid, in tissue culture media, 18, 35 Thyroid, auto- and homografts of, 378 endothelium of, effect of 1131 on, 387, 391 follicles of, 381, 382

grafts of, and microdissection in vi'uo, 380 growth stimulation in vitro by aminoacids, 40 vascularization of, in grafts, 386 Thyroxine, 18, 54, 55 Tissue cultures, caryometric studies on, 69-109 cells in, adaptation to media, 21, 26, 32 chromosome number in, 183, 186, 188, 194, 195 effect of colchicine on, 99 of X-rays on, 4 growth, measurements of, 3, 4 of homeotherm tissues, 22 media, 3-21, 23, 25 mitotic activity in, 4, 28 nuclear size in, 69-109 Tissues, absorption of soft X-radiation by, 457 adult, ratio of nuclear to cytoplasmic enzyme concentration in, 204 aging, binucleate and multinucleate cells in, 106 animal, isolated nuclei of, 202 variations in residual protein-RNA fraction of, 134 autografts and homografts, list of, 378, 379 carcinomatous, elastic fibers and mast cells in, 423 of Chiyonomus tenfans, 172, 173 conjunctive, 96 connective, 371 avascular and vascular layers of, in transparent chambers, 364 contamination in homogenate, 232 dermal, in phase contrast microscopy, 403 effect of glucose on, 27 of oxygen tension on, 34 of trace metals on, 26 of thyrotropic pituitary hormone on, 427 in grafts of thyroid, 382 mast cells in, 399, 405, 407, 410, 424, 426 nuclei of, caryometric studies on, 70 somatic inconstancy in, 186

SUBJECT INDEX

in transparent chambers, 363, 374, 375 for differential centrifugation, 231 differentiated, serologic specificity of,

313 diphosphopyridine nucleotide concentration in, 205 dying, 177, 190, 191 of ear, in transparent chambers, 359-

394 ectodermal, in sea urchin, 302 elastic, 437-452 chemistry of, 438-442 description of, 437 electron microscopy of, 449 histochemistry of, 442-445 intrafibrillar architecture of, 445-451 osmiophilic substance in, 449 overgrowths and tumors of, 451 staining properties of, 437, 442, 443,

444,445 yellow, 438 embryonic, chromosome numbers in, 182, 183 ratio of nuclear to cytoplasmic enzyme concentration, 204 entodermal in sea urchin, 302 enzymes of, and differential centrifugation, 225-269 fatty, mast cells in, 407 fetal, amino acids in, 38 mast cells in, 400 fragments of, for mitotic counts, 194 germinal, chromosome counts in, 178 glucosamine in, 417 glucuronic acid in, 417 glycogen in, 417 growing, release of polysulfuric acid esters to, by mast cells, 425 in transparent chambers, 364 homoeotherm, in culture, 22 hyaluronic acid in, 417 of insects, polyploidy and polyteny in,

163

527

living, hohavior in transparent chamber, 360 malignant, effect of estrone on, 55 mammalian, polyploidy in, 163 mesenchymal, influence of hormones on,

399 mucosal, p-glucuronidase in nuclei of, 204 neoplastic, effects of insulin on, 53 mast cells in, 400 non-dividing, ploidy in, 133 of plants, 163, 202 poikilotherm, in culture, 22 of Rkyiuhosciaru, banding pattern in,

172, 173 of sea urchin, 302 secretory, in Diptera, 136 somatic, chromosome counts in, 178 stable elements in, 96 staining of, by diazotates, 350 subcutaneous, of rabbit, nuclear sizes in, 95 transitory, 177, 190, 191 whole, cytochrome oxidase and suecinic dehydrogenase in, 213 a-Tocopherol phosphate, in tissue culture media, 17 Toxins, effect on mast cells, 423 Tracers, radioactive, and RNA synthesis,

162 Transaminases, 39, 210, 261 Transaminations, 39, 50, 301 Translocations, effect of urethan on, in Oenothera, 127 Transpeptidases, in isolated nuclei, 210 Transplantations, nuclear, 219 Transplants, in transparent chamhers, 376 “Trephones”, 8, 20 Tricarboxylic acid cycle, in sea urchin egg, mitochondria and, 282 Triphosphopyridine dinucleotide, 261, 290,

307, 308

Triploidy, 189 invasiou of artificial spaces in transTribnewtes, effect of urethan on eggs parent chambers by, 360 of, 115, 116, 119 labile elements in, % Triton, effect of lithium on presumptive leukemic, chromatin threads in, 134 chordamesoderrn in, 317 liquefaction of, by Physaloptera c l a z r . ~ , Feulgen-positive granules in nucleolus in transparent chamber, 393 of, 156

528

SUBJECT INDEX

Triton crisfatits, chromomeres of, electron micrograph, 174, 175 lampbrush chromosomes of, phase contrast microscopy, 174, 175 Trihcrus, lampbrush chromosomes of, in electron micrographs, 157 Triticm pyrrhogoster, l a m p b r u s h chromosomes and nucleoli in, 154, 155 Trivalents, 180 “Trophochromatin”, 155 Trout, blastoderm of, effect of urethan on, 120 Trypaflavine, 102, 106, 107 Trypsin, 211, 415, 410, 448 Tryptophan, 17, 29, 30, 141, 349 Tuberculin, 393 Tuberculosis, transparent chamber method in study of, 393, 394 Tiibifex, eggs of, development of granules in, 297 Tumors, animal, effect of urethan and carbamates on, 116 ascites, and peptides, 37 b l d pressure in vessels of, 391 caryometric studies on, 69 catalase inhihitor in animals with, 286 cell division in, 114 in cultures, and vitamin D, 47 effect of bacterial polysaccharide on, 3% of urethan and thymine on chrome s m e s of, 127 experimental, autoradiography of cortisone treated, 430 in mice anesthetized with urethan, 113, 115 radioactive sulfur in, 417, 418 extrahepatic liver catalase and, 286 glycolysis in, 30 growth in tissue culture, 25 malignant, mast cells in, 423 mast-zell. 410 mitosis and oxygen tension in, 33 of skin, mast cells in, 413 succinoxidase inhibitor in, 287 transplanted, vascularization in, 388 Tunics, elastic, aging* 451, 452 Tungstic acid, 292

Tween 80, 18. 45 Tgramine, effect on peripheral circula. tion, 368 Tyrosine, 17, 37, 139, 141, 349 Tyrode solution, 9, 10, 22

U Ultraviolet absorption, and chemistry of salivary chromosomes, 139, 141 Ultraviolet light, effect of, on salivary chromosomes, 139 Ultraviolet microspectrography, ribonucleic acid and, 468 Ultraviolet radiation, effect of thioflavine S on circulating blood and, 371 Ungulates, chromosome number in chorion and amnion of, 183 Uracil, 18 Urea, 18, 369 Urcchis, effects of urethan on. 123 Urethan, biochemical changes with, 123-127 concentration of, in treated eggs, 116 effects on achromatic figure. 119 on avian cells in vitro, 120 carcinogenic, 113 on chromatid breakage, 127 on chromasomes, 119, 127 Cytologic, 115, 118-122, 125 on enzymes, 127 on invertase, 126 lethal to mice, 116 on mammalian cells, 120 on mitosis, 113-128, 211 on mutations, in Drosophilu, 127 narcotic, 125 on nuclear size in vitro, 98, 99, 100, 102 on oxygen uptake by mitochondria, 124 on proliferation of carcinoma and sarcoma in vitra, 124 on purine metablism, 127 on survival of eggs, 119 on translocations in OenothPra, 127 lung tumors in mice produced with, 113 mode of action, 114, 117, 118, 126, 127 penetration into cells, 127

529

SUBJECT INDEX

radioactive, 127 toxicity of, 116 Uricase, 209, 214, 216, 217, 243, 251, 252, 262 Urodeles, medium for organ culture of, 31 Uronic acid, 415 Usnic acid, 294

V Valine, 16, 29, 30 Vascularization, capillaries and, in transparent chambers, 345 of grafts, 379, 385, 387 nerve repair and, 374 of transplanted tumor in chamber, 388. 389 Vasoconstriction, 370 Vegetal pole, enzyme activity of, in sea urchin egg, 304 Vegetalization, 277, 2% Venules, 389, 390 Versene, in suspension medium for centrifugal fractionation, 2.34 Vertebrates, oocytes of, 131 Vessels, blood, 363, 364-367, 370, 375, 390, 406 lymphatic, 371-372 Vibrations, sonic, treatment of granules with, in differential centrifugation, 246, 247 Viruses, antigenicity and hemagghtinative capacity of, 300 culture of, medium composition for, 23 electron microscopy of, 136 incorporation of P32 into, 300 Virus particle, development of, and mitochondria, 300 Vitamin A, 15, 17, 46, 47 Vitamin A esterase, in liver microsomes, 244 Vitamin B in tissue culture media, 15, 35 Vitamin B,,, distribution of, 53, 248 Vitamin C (see also Ascorbic acid), 48, 49 Vitamin D (see also Calciferol), 47 Vitamin E, 47, 48 Vitamin K, in tissue culture media, 48

Vitamins, effect on development, 291, 293 in tissue culture media, 17, 18, 46-53 Vole, somatic inconstancy in endometrium of, 195

W Wharton’s jeliy, mast cells in, 407 Wheat germ, ribonucleic acid in nuclei of, 204 Wounds, effect of phenyl urethan on, 120 af urethan on, 124 repair of, 371, 389, 412

X X - 0 chromosome type, 179 XXY mechanism, 179 XXY, sex chromosome complement, 182 X-Y, chromosome type, 179 X-rays, and chromosome axis, 158 effect on mast cells, 431 on embryos and polytene structure of salivary chromosomes, 150 and fibroblast cultures, 4 maximum toxic effect on lymphocytes,

57 mitotic inhibition by, 4 monochromatic, in microradiography, 456 soft, in X-ray microradiography, 455474 X-ray absorption, by biologic materials, 456, 457 formula for calculation of, 457-459 X-ray diffraction spectra, ultrastructure of commercial fibs, 445 of elastin and elastic fibers, 449 X-ray microradiography (see hlicroradiography) Xanthine, 18 Xenopus, effect of lithium on, 316 permeability of nuclear membrane of, 202 Xylose, 30

Y Yeasts, lithium-potassium competition in, 318

530

SUBJECT INDEX

Yolk, 288, 297 calciferol in, 47 gradients in, of Amblystom embryo, 305 of invertebrate eggs, fractionated protein, 282

Z Zotla f w i c u l a t h grafts Of, 382, 383 glomm~losa,grafts of, 382, 383 Z ) m u ~ l in . sea urchin egg, 289 granules of. atid digestive enzymes, 206