International Review of Cytology: A Survey of Cell Biology, Volume 5

INTERNATIONAL

REVIEW OF CYTOLOGY

VOLUMEV

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INTERNATIONAL

Review of Cytology EDITED BY

G.H. BOURNE

J. F. DANIELLI

London Hospital

Zoology Deptutment King's CoUege London, England

Medical CoUege London, Englmul

VOLUME V

Prepared Under the Auspices of

The International Society for CeU Biology

ACADEMIC PRESS INC., PUBLISHERS NEW YORK

- 1956

Copyright @ 1956, by ACADEMIC PRESS INC. 125 East 23rd Street, New York 10, New York AN Rights Reserved NO PART BF THIS BOOK MAY BE REPRODUCED IN ANY FORM, BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS.

Library of Congress Catalog Card Number (52-5203)

PRINTED IN THE UNITED STATES OF AMERICA

Contributors to Volume V WARREN ANDREW, Wake Forest College, The Bowman Gray School of Medicine, Winston-Salem, North Carolina PETER C. CALDWELL, Biophysics Department, University College, London, England' ALBERT H. COONS, Department of Bacteriology and Immunology, Harvard Medical School, Boston, Massachusetts C. S. CUMMINS,Department of Bacteriology, The London Hospital Medical College, London, England JEAN C. DAN,Misaki Marine Biological Station, Miura-Shi, Japan JOHN W. HARMAN, Department of Pathology, University of Wisconsin, Madison, Wisconsina G. C. HIRSCH,Laboratory for Cell Physiology, Faculdade de Medicino, Univerdade de Stio Paulo, BraaiP L. C. U. JUNQUEIRA, Laboratory for Cell Physiology, Faculdade de Medicina, Univerdade de Stio Paulo, Brazil J. MANDELSTAEK, National Institute for Medical Research, Mill Hill, London, Enghnd DOUGLAS MARSLAND, Washington Square College, N e w York University, NewYork, and the Marine Biological Laboratory, Woods Hole, Massachusetts VISHWANATH,Department of Zoology, Panjab University, Hoshiarpur, Panjab, India T. A. J. PRANKERD, Medical Unit, University College Hospital Medical School, London, Englartd A. M. SCHECEITMAN, Department of Zoology, University of California, Los Angeles, California FRITIOF S. S J ~ S T R A N D ,Department of Anatomy, Karolinska Institutet, Stockholm, Sweden C. VENDRELY, Centre de Recherches sur les Macromole'cules, Strasbourg, France R. VENDRELY, Centre de Recherches sur les Macromole'cules, Strasbourg, France 1

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Present address : The Laboratory of the Marine Biological Association, Plymouth, England. Present address :Department of Pathology, St. Kevin's Hospital, Dublin, Eire Permanent address : Zoologisches Institut, Gottingen, Germany

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Foreword The aim of this series is to publish, over a period of years, articles in all branches of cytology (including cell physiology) in order to enable those interested in cell biology to form more easily a unified concept of the field. Research today is progressing so rapidly in a variety of distinct disciplines that it is difficult to correlate the findings in these many fields into a coordinated body of knowledge. This series strives to emphasize the unity of cytology. We have continued our general policy of publishing criticaal reviews of subjects which are included within the term cytology. Where it is possible to publish in the same volume two or three papers in the same field this has been done, but for obvious reasons it is not always feasible to do so. It is gratifying to note that the International Review of Cytology has become increasingly international. Included in this volume are five contributions from the United States, four from Great Britain, one from Brazil, one from Japan, one from India, one from France, and one from Sweden. The subjects dealt with also extend over a wide range-to name but a few, there are studies of bacterial structure, histochemistry with labeled antibodies, spermatogenesis, mitochondria of muscle and of the neuron, transfer of macromolecules, and intracellular pH.

G.H. BOURNE J. F. DANIELLI June, 1956

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CONTENTS

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v

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vii

Contributors to Volume V Foreword

Histochemistry with Labeled Antibody BY ALBERT H COONS.Depo7hnent of Bacteriology and Zmmmblogy. Howard Medicat School. Boston. MaPsachusetts

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I. I1 I11. IV V VI. VII VIII

Introduction

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. Technical Considerations .......................................... Fate of Injected Foreign Antigens ................................ . The Detection of Antigenic Substances Native to the Tissue ........ . Studies on Infectious Agents ...................................... Studies on Antibody Formation .................................... . Conclusion ........................................................ . References .......................................................

The Chemical Composition of the Bacterial Cell Wall BYC S CUMYINS. Depwtment of Bacteriology. The London Hospital Medical College. London. England I. Introduction ...................................................... I1. Preparation of Cell Wall Fractions ................................ I11 Properties of Cell Wall Fractions ................................. IV. Enzymatic Lysis of Bacterial Cell Walls .......................... V Quantitative Analyses and Structural Studies ....................... VI. Stability of Cell Wall Composition ................................ VII . Cell Wall Composition and the Gram Stain ......................... VIII. Cell Walls as Antigens .......................................... IX Cell Wall Composition and Bacterial Taxonomy .................... X . Conclusion ....................................................... XI . References .......................................................

1 2 10 14 17 19 20 21

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25 26 28 36 38 40 42 43 45 48 49

Theories of Enzyme Adaptation in Microorganism8 BY J MANDELSTAM. National Zwtitute for Medical Research. Mill Hill.

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I. I1 I11 IV V VI VII VIII IX X XI . XI1. XI11.

. . . . . . . . .

London. England

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Introduction Nomenclature .................................................... Some General Facts of Enzyme Adaptation ........................ The Mass Action Theory .......................................... The Plasmagene Theory .......................................... The Specific Precursor Theory .................................... The Organizer Theory (1) ........................................ The Organizer Theory (2) ........................................ The Organizer Theory (3) ........................................ The Kinetic Model ............................................... The Extended Mass Action Theory ................................ Conclusions References .......................................................

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

51 53 55 64

66 70 71 73 76 78 80 84 85

BY JOHN

The Cytochondria of Cardiac and Skeletal Huclcle W HARMAN. Department of Pathology. UniversiQ of W k c m . ~ . Madison. W i s c m h

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. Introduction ...................................................... 89 . History of Muscle Cytochondria .................................. 91 . Some General Properties of Muscle Mitochondria .................. 92 . Distribution and Morphology of Muscle Mitochondria .............. 93 . Chemical Composition of Muscle Cytochondria .................... 100 . Metabolic and Enzymatic Activities of Muscle Cytochondria ........ 105 Electron Transport system of Muscle Mitochondria ................ 112 . Oxidative Phosphorylation of Muscle Mitochondria ................ 116 . Distribution of Enzymes Acting on Phosphorylated Nucleotides .... 126 X . Morphology and Mitochondria1 Activity ............................ 132 XI. The Integration of Energy Metabolism in Muscle .................. 140

I I1 I11 IV V VI VII. VIII IX

XI1. References

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142

The Mitochondria of the Neuron BY WARRENANDREW. Wake Forest College. The Bowwm Gray School of Medicine. Winston-Salem* North Carolina

. Introduction ...................................................... . Early Observations ............................................... . Skepticism as to the Existence of Mitochondria in Nerve Cells .... . Extensive Studies of Cowdry ....................................... . The Problem of “Chromophil” Cells .............................. . Early Experimental Studies: Resistance of Mitochondria to Change . Recent Experimental Work ......................................

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

. The Ultrastructure of the Mitochondria of the Neuron ..............

.

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Mitochondria and the Aging Process ............................... Conclusion ....................................................... References .......................................................

147 148 151 152 154 156 160 161 164 167 169

The Results of Cytophotometry in the Study of the Deoxyribonucleic Acid (DNA) Content of the Nucleus BY R. VENDRELY AND C VENIIBELY. Centre de Recherches SUI Ips Mocromolicules. Strasbwg. Frame

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. Introduction ...................................................... 171 . Methods of Cytophotometry ....................................... 173 . The DNA Content of Interphase Nuclei of Normal Tissues ......... 177

I I1 I11 IV.

The DNA Content of Nuclei in Deep Physiological and Pathological Changes ....................................................... V. The DNA Content of the Nucleus during Cell Division .............. VI Conclusion VII References ........................................................

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182 187 193 194

Protoplasmic Contractility in Relatiom to Ged Structure: Tenaperatur&Prwure E.periments on Cytoldneeis and Amoeboid Movement BY DOUGLAS ~~ARSLAND Washington . S q w r e Colkge. NFWYork University. New York. ond the Marine Biological Laboratory. Woods Hole. Ma.rsachuJetts I Introduction ...................................................... 199 I1. Amoeboid Movement .............................................. 203 I11. lcyrdcinesis ...................................................... 210 IV Kinetics of Protoplasmic Contractility .............................. 219 V General Conclusions .............................................. 224 VI References ....................................................... 226

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Intracellular p H BY PET^^ C CALDWELL,Biophysics Department. University College. London. E n g M Introduction ...................................................... Theories about Intracellular pH .................................... Methods for the Determination of Intracellular pH .................. The Experimental Results which Have Been Obtained with the Different Methods for the Investigation of Intracellular pH ............ Discussion ........................................................ References .......................................................

..

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I I1 I11 IV

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V VI

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229 230 236 242

262 272

The Activity of Enzymes in Metabolism and Trarwport in the Red Cell BY T.A .J . PRANKEF~D. Medical Unit. University College Hospital Medical School. Lodon. England

. Introduction ...................................................... . Enzymes in Metabolism .......................................... . Enzymes in Transport ............................................

I 11 I11 IV

. References .......................................................

27!J 280 289

299

Uptake and Transfer ok Macromolscuh by C& with Special Refmmce to Growth and Development BY A M. SCHECHTMAN. Department of Zoology. U d v e r d y of California. Los Angeles. California I Introduction ...................................................... 303 I1 Transfer and Uptake of Macromolecules ............................ 304 I11 Macromolecular Uptake by Embryonic Tissues ..................... 310 IV Functions of Transferred Macromolecules in Embryos .............. 317 V References ........................................................ 320

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Cell Secretion: A Study of Pancreas and Salivary G b d s BY L. C W JUNQUEIRA AND 'G C HIRSCHLaboratory for Cell Physiology. FacuMade de Mediciw. Universidade de SZo Pcnclo. BraA I Introduction ...................................................... I1 Ingestion ......................................................... I11 Synthesis ........................................................ IV Extrusion ........................................................ V Kinetics ......................................................... V I References .......................................................

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323 326 328 353 355

360

The Acrorrome Reaction BY JEAN C DAN.Misaki Marine Biological Station. Miwa-Shi. J a w

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I I1 I11 IV V VI VII VIII IX

. Introduction ...................................................... . Structural Aspects of the Acrosome Reaction ...................... . Conditions Determining the Acrosome Reaction ...................... . Relation to the Agglutination Reaction ............................ . Acrosome Lysh .................................................. . Role of the A c r o m e Reaction in Sperm Entrance .................. . Possible Role of the Acrosome Filament ........................... . Acrosome Reaction in Relation to Specificity in Fertilization ........ . References .......................................................

365 366 381 385 386 387 389 391 393

Cytology of Spermatogenesis BY VIsHwh NATH. Departmest of zoology. Panjab university. Hoshiarpw. Punjab. India I Introduction and Scope ............................................ I1 Flagellate Sperm ................................................ I11 Non-Flagellate Sperm ............................................ IV Chromatoid Bodies in Spermatogenesis ............................ V Evolution and Functions of the Acrosame .......................... VI Evolution of the Mitochondria1 Nebenkern ......................... VII Origin of the Golgi Bodies in the Cell .............................. VIII Conclusion ....................................................... IX. References ........................................................

395 397 433 442 443 446 447 449 450

. . . . . . . .

The Ultrastructure of Cells as Revealed by the Electron Microscope S SJOSTRAND. Departmmf of Anatomy. KwolinSka Zndhrfet. BY FBITIOF Stockhob. Swedes I Introduction I1 The Development of Ultrathin Sectioning Techniques .............. I11 The Elestron Microscopy of Ultrathin Tissue Sections .............. IV The Problem of Fixation .......................................... V The Ultrastructural Organization of Mitochondria .................. VI The Golgi Apparatus ............................................ VII The Plasma Membrane ........................................... VIII The Basement Membrane ......................................... IX The Ground Substance of the Cytoplasm .......................... X The Nucleus ..................................................... XI Lipoprotein Structures ............................................ XI1 The Structural Organization of Whole Cells ........................ XI11 The Interpretation of Electron Microscope Observations ............ XIV Important Future Problems of General Interest .................... XV References ........................................................

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

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AUTHORINDEX........................................................... SUBJECT INDEX ............................................................

456 457 458 459 461 471 478 486 490 499 505 511 521 526 529 535 552

Histochemistry with Labeled Antibody

.

ALBERT H COONS Department of Bacteriology and Immunology. Howard Medical School. Boston. Massachusetts Page I. Introduction ........................................................ 1 I1 Technical Considerations ............................................. 2 1 The Preparation of Antisera ...................................... 2 2. Labeling Compounds .............................................. 2 3. The Preparation and Use of Fluorescein Isocyanate .................. 3 4. The Properties of Fluorescein-Carbamido-Proteins.................. 3 5 Preparation of Tissue Sections .................................... 4 6 Fixation ......................................................... 5 5 7 Use of Conjugates ................................................ 8. Fluorescence Microscopy .......................................... 5 9. Sensitivity of the Method .......................................... 6 10. Nonspecific Reactions ............................................. 7 11 Use of Layers .................................................... 8 I11 Fate of Injected Foreign Antigens .................................... 10 1 Bacterial Polysaccharides ......................................... 10 2. Animal Proteins .................................................. 11 I V The Detection of Antigenic Substances Native to the Tissue .............. 14 V . Studies on Infectious Agents ......................................... 17 1 Viruses .......................................................... 17 a Mumps ....................................................... 17 b. Influenza ...................................................... 17 c. Other Virus Infections ....................................... 18 2. Other Infectious Agents .......................................... 18 VI Studies on Antibody Formation ...................................... 19 VII Conclusion .......................................................... 20 VIII . References .......................................................... 21

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I . INTRODUCTION The use of specific antibody as a histochemical reagent depends on the fact. first clearly demonstrated by Marrack (1934). that dye molecules can be chemically linked to antibody molecules without impairing the capacity of the antibody to react specifically with the substance (antigen) which stimulated its synthesis. This immediately created a new approach to histochemical localization by allowing the histochemist to harness the remarkable specificity of immune reactions . The specific step in such a reaction is the deposit. from a solution. of labeled antibody molecules over those areas of tissues and cells where the antigen is present; unreacted antibody and inert labeled proteins remain in solution and can be washed away. leaving on the slide a labeled protein deposit which can be seen under the microscope. Like the use of an enzyme to identify a substance

1

2

ALBERT H. COONS

present in tissue, it is another way of utilizing the precision of biological specificity for cytochemical purposes. The use of labeled antibody requires a certain familiarity with the methods and tradition of immunology, as well as with the more strictly practicaI procedures used in the purification of antigens and the production of antisera. There are of course many antigenic substances which can theoretically be studied under a variety of circumstances by such means. The specificity of every reaction must be established by appropriate controls, the character of which will vary with the substance and the circumstance. Labeled antibodies so employed identify objects and localize them ; they are most effective when used to answer a morphological question.

11. TECHNICAL CONSIDERATIONS 1. The Preparation of Antisera Methods for the production of antisera are largely empirical. There are three considerations to be borne in mind: the animal must be stimulated repeatedly, the dose of antigen must be adequate but not overwhelming (cf. Kabat and Mayer, 1948 ; Dixon and Maurer, 1955) ,and, for nonliving antigens, the use of adjuvants will increase the titer considerably (eg., Freund and McDermott, 1942; Proom, 1943). Animals which fail to make a significant response within about one month will probably fail to do so. Those that do should be kept; for the production of high titers, the most important ingredient is time. After six months or more they may be cautiously reinjected. Antibodies produced in the rabbit, horse, monkey, chicken, dog, goat, and cow have so far been used successfully. 2. Labeling Compounds

The only compound used on any scale is fluorescein isocyanate (Coons, Creech, and Jones, 1941 ; Coons et al., 1942 ; Coons and Kaplan, 1950). The original choice was based on the brilliance of the fluorescence of fluorescein, and on the fact that yellow-green is a rare fluorescence in mammalian tissue (Hamperl, 1934). Two other labeling compounds have recently been reported by Clayton ( 1954) ; 1-dimethylamino-5-sulfonylchloridenaphthalene, producing a yellow fluorescence, and nuclear fast red (benzaldehyde-6-nitro-Z-sodium diazotate) , producing a red fluorescence, As Clayton points out, two fluorescence colors would be a great advantage in many studies.

HISTOCHEMISTRY WITH LABELED ANTIBODY

3

3. The Preparatiolz and Use of Fluorescein Isocyanate Fluorescein isocyanate is prepared through the intermediates nitrofluorescein and aminofluorescein. The nitrofluorescein itself is prepared by the fusion of Cnitrophthalic acid and resorcinol, two isomeric nitrofluoresceins resulting. The isocyanate was first prepared as a mixture of isomers by Berliner (Coons et al., 1942), who noted its instability. Later the isomers were separated (Coons and Kaplan, 1950). However, the isocyanate itself has not been crystallized or characterized, firm chemical ground stopping at the aminofluorescein step. Because of the instability of the isocyanate, it has been the usual practice to prepare the isocyanate as needed and to use it immediately. However, Marshall (1951) reported that a solution of fluorescein isocyanate in acetone retained its activity for as long as a year when protected from light, heat, and moisture. The author has found that such acetone solutions sealed in glass and stored in the dark at room temperature were satisfactory for at least as long as eight weeks. One remaining ampule shows appreciable darkening at the end of twelve weeks. DeRepentigny and James (1954) have recently analyzed the two aminofluorescein isomers chromatographically and found impurities amounting to between 5 and 10%. They could be removed by chromatographic separation. Coupling of the isocyanate to protein solutions is a simple procedure and has produced no comment in the literature since the original descriptions (Coons et d., 1942; Coons and Kaplan, 1950). One warning is perhaps pertinent : the attractive possibility of diazotizing aminofluorescein and coupling it to the protein through an azo-linkage, thus avoiding the isocyanate (and the necessity to use phosgene), fails; the resulting yellow conjugate is nonfluorescent. There is evidence that some antibodies are converted to the nonprecipitating type by the conjugation procedure ; this has been demonstrated for the case of horse diphtheria antitoxin (Scheibel and Coons, unpublished). This does not impair their activity on tissue sections. 4. The Properties of Fluorescein-Carbamido-Proteins The labeling of crude globulin solutions under the stated conditions produces fluorescent conjugates containing about two groups of fluorescein per globulin molecule (Coons et al., 1942; Coons and Kaplan, 1950). Schiller et d . (1953) labeled bovine serum albumin with progressively increasing amounts of fluorescein isocyanate up to six times the optimum for antibody solutions. They found that the number of fluorescein molecules introduced into the albumin molecule increased from 1.4 to 1.9 groups.

4

ALBERT H. COONS

They also made careful physicochemical and fluorimetric measurements of these conjugates. They reported that of their four preparations, three had a viscosity identical with that of the native protein, while one was 5% lower. Sedimentation patterns indicated that less than 10% of this latter preparation had a smaller frictional coefficient than the native protein. The only systematic change in properties as a result of labeling was a fall in the isoelectric point corresponding to the amount of fluorescein introduced. They concluded that their fluorescein-bovine albumin conjugates did not differ appreciably in size, shape, or homogeneity from bovine albumin. Schiller et aI. also compared the absorption spectra of bovine albumin, fluorescein, and fluorescein-carbamido-bovinealbumin over the range from 2200 to 5200 A, recording peaks for fluorescein at 2400 and 4900,and a minor peak at 2800. The maximum absorption of bovine albumin was at 2800;the conjugates had major peaks at 2800 and 4900. They also measured the emission intensity of their conjugates at various concentrations, finding an increase linear with the concentration. Spectrophotometric absorption measurements at increasing concentrations had the same slopes, but in only one of three conjugates were the absolute values obtained by the two methods of measurement similar. The discrepancies in the other cases were large, due to much lower fluorimetric values. These findings imply that such conjugates may contain colored side-products which do not fluoresce. 5. Preparation of Tissue Sections Since the specific activity of the antigen is vital for the success of the method, the procedures used in the preparation of tissue sections must guard it carefully. Although each antigenic substance has its own unique chemical properties to be considered, the two available general methods rely on the cutting of unfixed tissue: freeze-drying has been used for this purpose by Marshall (1951), and the Linderstr$m-Lang cryostat for the cutting of unfixed frozen blocks by Coons ef al. (1951). Preparing sections of frozen tissue with a chilled knife has also been employed (Mellors et al., 1955). The first method appears to have advantages over the others for cytological precision since 2 p sections may be cut, and since when used with care (cf. Bell, 1952) there would probably be fewer artifacts; the second method is very convenient for histological purposes. There appears to be no careful comparison of the two. The unresolved problem of the movement of substances during processing sections after cutting will be discussed below. Stable substances such as some bacterial polysaccharides can be studied in tissues after chemical fixation and conventional embedding in paraffin (Kaplan et al., 1950; Hill et al., 1950), although care must be taken not to

HISTOCHEMISTRY WITH LABELED ANTIBODY

5

leach them out of the sections during subsequent manipulations (Schmidt,

1952). 6. Fixation With either freeze-dried (Altman-Gersh) or frozen sections, the best fixative for the substance under investigation must be sought in experiments on single sections. For proteins, those so far employed are ethanol (95% v/v), and absolute methanol (Coons et al., 1951 ; Marshall, 1951 ; Gitlin et al., 1953). In the study of viruses, acetone has been used exclusively; ethanol destroys the antigenic activity of those on which it has been tried (mumps, influenza, canine hepatitis).

7. Use of Conjzcgates The use of purified conjugates (see nonspecific reactions below) is simple. A small drop is layered over the section, and the slide placed in a humid environment for 10-30 minutes at room temperature. Marshall (1951)carried out his reactions for as long as 48 hours in the cold, but in the author’s laboratory such long exposures have not thus far been found necessary. The preparation is then washed in buffered saline at neutral or slightly alkaline pH; ten minutes usually suffices for this. The section can then be mounted in buffered glycerol at p H 7.0.

8. Fluorescence Microscopy The amount of fluorescent protein deposited over antigen-containing sites in tissue sections is small, and the level of intensity of the fluorescence correspondingly low. Intense bombardment of the section with ultraviolet light is therefore necessary, rendering the light source a factor of great importance. The light sources successfully used so far are carbon arcs of 10-20 amperes (direct current), and high-pressure mercury vapor arcs of 250 and lo00 watts (Coons and Kaplan, 1950;Coffin et al., 1953; Marshall,1951 ; Hill and Cruickshank, 1953). The more intense the light source (up to the limits of present experience) the more is visible; the 20ampere carbon arc has revealed earlier stages in the formation of antibody, for example, than were detectable with an arc operating at 13 amperes. The details of condensing lenses and filters will be found in the papers cited above. The dark-field condenser was introduced into fluorescence microscopy by Barnard and Welch (1936),and fruitfully employed with fluorescent antibody by Marshall (1951). The increase in contrast and definition over that of the transmitting condenser (Coons and Kaplan, 1950) is considerable. Quartz is not necessary.

6

ALBERT H. COONS

The successful application of labeled antibody to antigen localization is due in large measure to the brilliance of the fluorescence of fluorescein. Measurements of the quantum efficiency of fluorescein by several investigatorssrange between 75% (Vavilov, 1924) and 85% (Umberger and LaMer, 1945). In alkaline solution it has absorption peaks at 3200 and 4900A (Vavilov, 1924;Ghosh and Sengupta, 1938;Umberger and LaMer, 1945). The values of the latter workers: peak absorption 495 mp, peak emission 521 mp. Pringsheim (1949) gives an absorption range from 4406 to 5200 A, with the maximum at 4940; emission wavelength 5100-5400A, maximum at 5180. Since any light absorbed is converted to emitted light with about 80% efficiency, it is evident that to achieve maximum bombardment with a given light source, ail light up to 4900 A should theoretically be allowed to beat on the object, and all light below 5000 A should be excluded from the observer's eye above the object. Ocular filters to achieve this, however, are green or yellow. Observations made through such filters can only be observations of the intensity of the green or yellow rays transmitted. There are luminous objects in tissues fluorescing with bright blue or whitish light containing green or yellow components visible through such filters. The specificity of the yellow-green fluorescein label, readily apparent to the eye, is lost. It is better therefore for visual observation to bombard heavily with less readily absorbed shorter wavelengths so as to achieve a black background and to use a colorless filter in the ocular, cutting off light below 4200 A. The dark-field condenser helps greatly in this connection, allowing the use of thinner filters in the optical path below the object. Mellors et ul. (1955) have used a yellow filter for photographic purposes and made relative measurements of the fluorescence intensity of deposited labeled antibody by photographic photometry,

9. Sercsitivity of the Method There is only one object so far studied from which the order of magnitude of the amount of antigenic material detectable by labeled antibody can be estimated, the bacterial cell. A single pneumowccus, for example, is readily detectable microscopically at low magnifications after reaction with specific fluorescent antibody. A single bacterium contains about 5 x 10" mgm. bacterial N. (This figure, 2 x lozoorganisms/mgm. bacterial N, was determined by Hershey (1938) for E. coli, but the order of magnitude would not be different for the pneumocwcus.) Heidelberger and Kabat (1937)have published data concerning the amount of antibody which reacts with pneumwoccal cells. The ef€ective antigen, the capsular polysaccharide, constitutes about 10% of the nitrogen, and the effective

HISTOCHEMISTRY WITH LABELED ANTIBODY

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combining ratio of antibody with this antigen is about 10: 1 (mgn. Ab N/mgm. antigen). In any case, the amount of antibody N reacting approximates the bacterial N present, as de!ermined by Heidelberger and Kabat. Hence, the amount of capsular antigen in a pneumococcal cell is about 5 x 1@la mgm. This is readily detected by the deposit on its surface of about 5 x 10” mgm. antibody N carrying about 1.5 x 1Olamgm. of fluorescein (Coons and Kaplan, 1950). However, such a cell contains a high concentration of antigen. Neglecting for this purpose the fact that the antigen is on the surface of the organism, if the diameter of cell and capsule is 2p. then the volume of the organism is 4$, and the antigen concentration is about 1 mgm./ml., a high biological concentration. This is not the limiting concentration detectable ; assuming that lJlOth this amount is at present directly detectable, and that the sensitivity could be increased by a factor of ten by the use of layers as described below, then the limit approaches perhaps 10 pgm./ml. as an ideal. The practical working limit at present with good antiserum is perhaps ten times this. 10. Nonspecific Reactions The nonspecific reactions referred to above are due to at least three elements : side reactions producing derivatives of fluorescein not readily removed by dialysis (these “stain” elastic tissue, blood vessel endothelium, and perhaps other elements), probably proteins in serum which react with tissue components (cf. Kidd and Friedewald, 1942), and finally antibodies against tissue components occurring as a result of immunization (e.g., Watson and Coons, 1954). The first two of these have been present in all conjugates of antiserum and fluorescein so far prepared, and must be removed by absorption with tissue powder on an empirical basis until better methods emerge. Reactions due to extraneous antigen-antibody systems can also be removed by the same method, a proper choice of tissue powder being the only requirement. However, this method may be selfdefeating when the antigenic components of normal tissue are under study. Even here, means may be devised to remove unwanted reactions and to establish the specificity of the reaction observed. For example, pituitary hormones can be obtained concentrated but not demonstrably antigenially pure. Antiserum prepared against one of them may contain other antibodies than those against the hormone under study. By quantitative absorption of aliquots of the conjugate with assayed pituitary fractions containing widely differing amounts of the hormone, it might readily be established that the smallest amount of these various fractions which just abolished the observed reaction on sections ran parallel with

8

U B E R T H. COONS

the amount of hormone known to be present in the fractions. This would go far toward establishing the identity of the objects stained by the labeled

antiserum. O r experimental endocrinological manipulation of the test animals before removal of the pituitary for study could be used to demonstrate the specificity of the staining reaction. Even in the simplest case, where an antigen foreign to the tissue under study is the object of investigation, controls of specificity are essential. Here, of course, normal tissue serves as an obvious control. Then, the staining reaction can be blocked, though reversibly, by prior treatment with unconjugated specific antibody. Or, again, aliquots of the conjugate can be treated with antigen to remove the specific antibody present and so abolish the staining reaction. Each case must be dealt with appropriately, but a vital component of any staining experiment with labeled antibody is a clear demonstration of specificity (Coons and Kaplan, 1950; Marshall, 1951; Hill and Cruickshank, 1953; Watson and Coons, 1954; Weller and Coons, 1954). 11. Use of Layers Physical studies of antigen-antibody reactions with the antigen fixed in space have been carried out by several groups of workers by the use of monolayers of antigen and optical measurement of the thickness of antibody deposited specifically on them. In some instances it was possible to build structures several layers thick composed of alternating layers of antigen and antibody (Langmuir and Schaefer, 1937; S M e r and Dingle, 1938; Porter and Pappenheimer, 1939; Harkins et al., 1940; Bateman et al., 1941; Rothen and Landsteiner, 1942). The physical state of immunologically active substances in cells is obviously markedly different from that in monolayers fixed to barium stearate films. Nevertheless, the findings of Batemen et al. are probably of significance for histochemistry with such reagents. Working with the M-protein of the streptococcus and its specific antibody, they found that the rate of deposition of the antibody on the antigen film varied with the concentration of antibody, as was to be expected. The time for one-half the surface to be covered varied from 5 minutes for undiluted serum to 30 minutes for serum diluted 1/3GQ. The striking finding was that the maximum thickness of the antibody layer attainable was 150 A for undiluted serum, but only 55 A for a 1/300 dilution. Assuming that the antibody molecules were prolate ellipsoids, they suggested that with high concentrations the antibody molecules were attached to the film by their ends and stood at an angle of 90"to the plane of the film,whereas when they reacted from a dilute solution and were deposited more slowly, they lay more nearly flat on the film, and so prevented closer packing. If similar conditions obtain in sections of tissue,

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prolongation of staining with dilute antibody solutions would not achieve the brightness produced by concentrated solutions. No systematic studies on this point have been carried out with fluorescent antibody. Two applications have been made of the use of layers with fluorescent antibody, one in the demonstration of antibody in cells, and the other in the use of fluorescent antiglobulin solutions to detect unlabeled specific antibody deposited over an antigen-containing site. In the demonstration of antibody in tissue cells, tissue sections were fixed in 95% ethanol (v/v) at 37" C. for 15 minutes, and then exposed to a solution of specific unlabeled antigen containing about 0.5 mgm. antigen/ml. After 30 minutes, this was rinsed off with buffered saline, washed for 10 minutes, and then exposed to specific fluorescent antibody. A control slide prepared by omitting the treatment with antigen allowed the increment of fluorescent antibody due to in vitro fixation of antigen to the cells to be recognized; the antibody in the cells could thus be localized (Coons et a!., 1955). Labeled antiglobulin sera have been employed successfully in a number of cases. The circumstances are somewhat different from the use of alternating layers of antigen and antibody in that the middle layer acts as an antibody on one side of the layer, and as an antigen on the other. This method is useful in cases of infectious disease where antigen is unavailable or difficult to produce in large amounts; convalescent serum as the intermediate layer has been successful in revealing tissue-culture cells infected with varicella and herpes zoster viruses, and in demonstrating that the two agents have marked cross-reactions (Weller and Coons, 1954). Liu and Eaton (1955) have been able to demonstrate strains of atypical pneumonia virus in the bronchial epithelium of the infected chick embryo by the same method; this virus causes no visible cytopathogenic effect in these cells, and would otherwise be undetectable except by neutralization tests in cotton rats (Eaton et al., 1944). Six different systems have been used :anti-human y-globulin rabbit serum (Watson ; Weller and Coons, 1954), anti-rabbit y-globulin chicken serum, anti-rabbit y-globulin goat serum, anti-chicken y-gfobulin rabbit serum (Watson), anti-rabbit y-globulin bovine serum, anti-human y-globulin horse serum (Gitlin). The use of two layers in this way increases the sensitivity appreciably, presumably because of the number of reactive sites on antigen molecules. If in such a layered structure half the sites on the intermediate layer were available for reaction with the labeled antiglobulin, the gain would be by a factor of about 5 (cf. Hooker and Boyd, 1942). It has been demonstrated that the specific sera used for the intermediate layer can be diluted considerably (sometimes to 1/100) before the reaction

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is too faint to be detectable. Indeed, Liu has used this as a method for the titration of such sera. Appropriate absorption of unlabeled sera is often necessary to remove nonspecific reactions. The general limitation on the use of such a two-step staining procedure is that the globulin-antiglobulin system employed will usually have to be derived from a species different from that of the tissue under study. 111. FATEOF INJECTED FOREIGN ANTIGENS The uptake by cells of a few subtances has been followed in the mouse by the use of labeled antibody.

I . Bacterial Polysaccharides Kaplan et d.,(1950) traced the fate of two pneumococcal capsular polysaccharides, and Hill et al., (1950) made a similar study of the capsular polysaccharide of the Friedander bacillus, Type B. [The pneumococcal polysaccharides have molecular weights when prepared under optimum conditions of 140,OOO (Type 11), and S00,OOO (Type 111) (Haworth and Stacey, 1948).] These acidic substances are immunologically similar and showed identical behavior in the mouse. After the intravenous injection of large doses, they were found in phagocytic cells both in the connective tissue and in the liver, spleen, lymph nodes, and bone marrow. They were also identified in epithelial cells: hepatic, adrenal cortical, and the macula densct of the renal tubule. Many fibroblasts throughout the body contained the material, and some lymphoid cells in the lymphoid follicles of the spleen and lymph nodes took up small quantities of them. In the joints, FriedIiinder polysaccharide was found in the joint cavity, in the cells of the synovial membrane, and in cartilage cells adjacent to the joint cavity. It was also present in osteoblasts and in an occasional osteocyte (Hill et al., 1950). After some days all three substances gradually disappeared from most of these locations, being slowly excreted in the urine and bile, but they showed a remarkable persistence in macrophages and Kupffer cells, still being present when the observations were discontinued after six months. Persistence of pneumococcal polysaccharide has been established by precipitin tests by Felton (1949), Stark (1955), and Felton et d. (1955). A quite different behavior was found for the case of the type-specific polysaccharide of Streptococcw pyogenes (Group A) by Schmidt ( 1952). This neutral substance, with a molecular werght of S,OOO, was excreted rapidly by the mouse into the urine, and could be found in the renal tubular epithelium for only 15 minutes after injection, and except for a rare phago-

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cytic cell, nowhere else. Its concentration fell rapidly in the blood and rose rapidly in the urine as determined by precipitin tests; the author found its behavior comparable to that reported for inulin.

2. Anha1 Proteiw Similar simple experiments with hens’ ovalbumin, bovine plasma albumin, and human y-globulin were conducted by Coons et d. (1951), who found an initial distribution very similar to the polysaccharides:phagocytic cells, fibroblasts, capillary endothelium, hepatic epithelium, renal tubular epithelium, lymphoid cells in lymphoid follicles, and traces in the epithelial cells of the adrenal cortex. Like the polysaccharides, the proteins were found in the perivascular connective tissue in all organs examined. They were also found in the Paneth cells of the gastrointestinal tract. Following intradermal injection, Waksman and Bocking (1953) found ovalbumin and bovine y-globulin in macrophages and extracellularly at the site of injection, and in cells lining the sinuses of the draining lymph node. Cells in the borders of the lymphoid follicles also contained antigen. In the cells which took them up, the proteins were demonstrated both in the nucleus and the cytoplasm. Macrophage ingestion was increased in the presence of antibody, but polymorphonuclear leukocytes were empty. Two principal differences between the acid polysaccharides and the bland proteins emerged: the period of their persistence in amounts detectable by this histochemical method, and their intracellular distribution. The proteins studied were detectable for only a few days at best, and whether by coincidence or not, disappeared at rates inverse to their molecular size. The persistence of protein antigens has been a subject of careful investigation because of its theoretical importance in developing theories of antibody formation. However, the results obtained by different methods applied to this problem are at great variance ; data derived from disappearance rates from the blood (see Coons, 1954), and tissues of proteins trace-labeled with I1al indicate persistence for only a few days, whereas data derived by reverse passive anaphylaxis indicate persistence of very small amounts for 3 months in mice and 2 months in rabbits (McMaster and Kruse, 1951; McMaster et al., 1954). Garvey and Campbell (1955) gave a series of injections of hemocyanin-azo-phenyl-Ss~-sulfonate to rabbits, and 6 weeks later found antigenically active material in the liver in amounts of 10-100 p p . or about 0.1% of that injected. This is probably within the range of immunohistochemical methods, particularly as the antigen was concentrated in the Kupff er cells. - Azo-proteins are, however, handled differently from native ones (Dixon et al., 1951), and the hemocyanin molecule is different from those studied above and has a somewhat higher molecular

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weight [unknown but assumed to be 200,000 (Garvey and Campbell, 1954) 1. A more complete summary of the available data concerning the persistence of protein antigens in laboratory animals has been given elsewhere (Coons, 1954). There is little doubt, however, that they persist longer in smaller amounts than can be determined by the use of labeled antibody, but for shorter periods than the acid polysaccharides mentioned above. The other striking difference in their behavior was the finding of ovalbumin, bovine globulin, and human y-globulin associated with the nuclei of those cells into which they penetrated (Coons et crt., 1951). These substances could be detected in the nuclei of hepatic cells, renal tubular cells, fibroblasts, Kupffer cells, adrenal cortical cells, lymphocytes, Paneth cells, and macrophages. Only a fraction of each of the cell types enumerated contained antigenic material, although in some organs it was a large one, for example, in the renal tubular epithelium. The authors considered the possibility that this finding was an artifact produced either by the spread of antigenic material during the thawing of the frozen section, or of movement during the fixing and staining procedures. There is sometimes clear evidence of the flow of fluid in a tissue section cut in the Linderstrplm-Lang cryostat, the method in use in the reviewer’s laboratory. Occasionally the contents of a blood vessel, for example, can be seen to have moved as a unit as much as 2-3 p, so that some of it overlaps the vessel wall. This is by no means always the case, however. There is therefore much to be said for Marshall’s use of the Altman-Gersh method of freeze-drying, and for the use of liquid nitrogen rather than solid carbon dioxide as the cooling agent during the initial freezing (Bell, 1952) for cytological studies with labeled antibody. Despite this difficulty, however, there was intrinsic evidence in the sections to support the validity of the observation. Perhaps the best was the differences found between the localization of ovalbumin and human 7-globulin in the kidney. In repeated observations, human y-globulin was invariably present in cells in the glomeruli, both in the cytoplasm and in the nuclei, as well as in the stroma of the cortex, and in the nuclei of the convoluted tubules. Ovalbumin was never found in the glomeruli, but was present in the other adjacent locations. Movement during thawing or diffusion during staining should affect both these antigens in a similar way. When antigen was found in hepatic cell nuclei, the negative shadows of nucleoli were frequently visible, another hint that the antigen had not drifted over the nucleus during processing. A similar intranuclear distribution was found by Gitlin et al. (1953), in their study of the distribution of various protein fractions of human plasma in young children. These homologous antigens, particularly albu-

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min and y-globulin, had a very similar distribution to that described for foreign proteins in the mouse. Exceptions were recorded for fibrinogen, which apparently is largely excluded from cells, and @-lipoprotein,which was found in many cell nuclei, but not in the cytoplasm. One puzzling finding was that of little albumin in hepatic cell cytoplasm, and only traces of fibrinogen, although there is excellent evidence for their synthesis by the liver (cf. Miller and Bale, 1954). The striking amounts of these different antigens, except fibrinogen, in cell nuclei, although they were visible in varying amounts in the near-by connective tissue and vessels, is further evidence against the existence of a systematic artifact ; however, they used the same methods as Coons et d. (1951). Schmidt (1954) found a limited distribution in mice of a protein fraction from the Group A streptococcus similar to the animal proteins ; these proteins disappeared (after 10 mgm. doses) within 60 hours. They had a granular distribution in the cytoplasm, but could not be found inside nuclei. Schiller et a2. ( 1953), injecting fluorescein-labeled bovine albumin intravenously into rats, were unable to find any material in the nuclei of cells which had high concentrations in the cytoplasm. The author has repeated this experiment in mice, and found that the distribution of labeled bovine albumin differed from that of native albumin detected by labeled antibody, and that nuclear penetration was rare, although it did occur. Other evidence bearing on the permeability of the nuclear membrane to protein molecules has since accumulated. Anderson (1953) and Dounce (1954) have reviewed the evidence derived from studies of the localization of enzymes in cell fractions isolated by differential centrifugation. They both concluded that the nuclear membrane is permeable to proteins. (However, see caution by Hogeboom and Schneider, 1953.) Dounce cited unpublished experiments by Holtfretter that the nuclei of frogs’ eggs were quite permeable to hemoglobin, and reported that isolated hepatic nuclei were permeable to hemoglobin. And M. L. Watson (1955) has summarized the findings to date concerning the structure of the nuclear membrane as revealed by electron microscopy, and added much new data of his own. He finds that all nuclear membranes studied (including liver and renal tubular epithelium) have pores through them ranging from 300-600 A in internal diameter. Each of these pieces of evidence, the finding of proteins in nuclei after intravenous injection, the diffusion of hemoglobin into isolated liver nuclei, and the universal finding of pores through the nuclear membrane, is subject to its own artifacts, but the possible artifacts seem to have little in common. It is likely that the nuclear membrane is permeable to proteins ; by the use of freeze-drying to eliminate movement during thawing, and of nonprecipi-

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tating antibody to diminish the possibility of diffusion of antigen during staining, it should be possible to establish this by immunohistochemical means in the intact cell.

IV. THEDETECTION OF ANTIGENIC SUBSTANCES NATIVE TO THE TISSUE The problems faced when attempts are made to employ labeled antiserum in the study of normal tissue components are somewhat different from the ones described above. In the first place, daring the production of an antiserum specific for the substance in question, it is almost inevitable that other antibodies will also be formed. Moreover, the antibody response to impurities is often disproportionatelyhigh (cf. Vaughan and Kabat, 1953, 1954). Some method must therefore be found to sort out such mixtures. Marshall (1951)was the first to apply labeled antibody to the study of a normal tissue component. He prepared antiserum in rabbits against adrenocorticotropic hormone (ACTH) prepared commercially from swine pituitaries. The resulting globulin fractions of the sera were prepared by ethanol fractionation, labeled, purified by repeated ethanol fractionation, and employed to study freeze-dried sections of hog, sheep and beef pituitary. and hog kidney. Plasma proteins were stained by the crude preparations in hog and sheep tissues, but this effect was removed by treatment of the conjugate with hog serum and hog kidney powder. Thereafter, there was left only the brilliant staining of certain cells in the hog pituitary, identified as basophil cells. The pituitary of the other species gave no reaction. Marshall showed that the conjugate would precipitate active hormone from a highly active hormone preparation. He concluded that, because the conjugate could react with the hormone, because the gland is known to contain hormone, and because only basophils reacted with the fluorescent conjugate, these cells must contain the hormone. The crux of the proof is the fact that the sought-for substance is biologically active and could be measured by an independent method. Fuller proof (but in this case perhaps not necessary) would have been to test the supernatant of an aliquot of conjugate after precipitation by hormone to see whether its staining ability had been removed-or by measured additions of hormone preparation to aliquots of conjugate, to determine whether the abilities of two different hormone preparations, one crude, to abolish the histochemical reaction were parallel to their hormone content. Of course, even here there is a theoretical pitfall, since some biologically active materials lose this activity while retaining activity as an antigen (e.g., bacterial exotoxins) . Hill and Cruickshank (1953) faced similar problems in their study of the kidney antigen responsible for an artificial disease, “nephrotoxic nephritis,” resulting from the injection of anti-kidney serum produced in another

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species (op. cit. for literature). Previous investigations, including the injection of Ifsl-labeled antiserum and its localization by radioautography (Pressman et al., 1949), had indicated that the antigen responsible was located in the glomerulus. Hill and Cruickshank found that labeled antirat kidney antibody stained the basement membrane of Bowman’s capsule, the tubular basement membrane, and tubular cytoplasm in the renal cortex. A fine green line beneath the endothelium outlined the capillary loops. Reticular fibers in the media of blood vessels reacted as well. Sera prepared against lung or isolated glomeruli also reacted in the same way with basement membrane but not with renal tubular epithelium. Absorption experiments with aliquots of these conjugates were consistent with the above findings, e.g., anti-kidney conjugate absorbed with lung or isolated glomeruli still reacted with the tubular cells but not with glomeruli. Inhibition tests followed a similar pattern, indicating the presence of a tubular antigen and an antigen common to lung, glomeruli, and basement membrane. The anti-glomerulus serum produced nephrotoxic nephritis. Immunological crossing with mouse tissue, previously described by Pressman (1949) , was confirmed; there was no crossing with guinea pig, rabbit, or human kidney. These workers then went on to a study of other organs (Cruickshank and Hill, 1953), demonstrating a common antigenic component in basement membrane, reticulum, sarcolemma, and neurolemma. There was no reaction with collagen. Absorption with rat spleen (containing reticulum but no basement membrane) abolished staining of both reticulum and basement membrane. Nonspecific staining reactions which could not be inhibited by pretreatment with unlabeled serum were found in scattered large cells (120 p )( 90 p) otherwise resembling human plasma cells, eosinophils, the cytoplasm of tracheal and bronchial epithelium, and the membrane lining the lacunae in which lay the cartilage cells of the tracheal rings. Mellors et ul. (1955) , extended the kidney studies by injecting anti-rat kidney serum, prepared in rabbits, into rats, and then treating frozen sections of the kidney with fluorescent anti-rabbit globulin prepared in chickens. By this means they were able to demonstrate the fixation of the rabbit globulin in the glomeruli. None of this intravenously injected anti-kidney globulin was found in the basement membrane of the tubules. Since it has been shown that such antibodies are rapidly removed from the circulation by the kidney, and probably also by the lungs (Sarre and Wirtz, 1939; Pressman et al., 1950), the absence of lesions in organs other than the kidney is perhaps not surprising, although Cruickshank and Hill’s findings suggest that antigen-antibody reactions might take place through-

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out the capillary bed. However, there is evidence (Pressman and Sherman, 1951a, b) of differing organ-specificity in the capillary beds, and Cruickshank and Hill’s findings do not exclude a mixture of antibodies reacting with the “single fluorescent reticular membrane” in the capillaries of all tissues in the rat. Indeed, Goodman et al. (1954), have presented evidence of at least two antigens in the glomerulus, one present also in kidney basement membrane, the other not. The only disagreement between Melbrs et d.and Cruickshank and Hill is the findings in the lung, an organ from which both groups had difficulty in preparihg frozen sections. Here, the latter workers found no basement membrane in the alveolar wall, only in the capillaries; this finding appears in the illustrations to be the more reliable. There is, of course, a basic difference in the two approaches. Cruickshank and Hill have described all the sites in the rat where antigen(s) is present and hence might react with injected antibody; Mellors et al. have determined where it does react, dependent presumably on the accessibility of the antigen to circulating antibody (Pressmen and Eisen, 1950). The only other example so far published of the study of an antigen native to the tissue is that of Marshall (1954) on the pancreas. He prepared and labeled antisera against bovine crystalline chymotrypsin, carboxypeptidase, deoxyribonuclease, and ribonuclease. Freeze-dried sections (2 p ) , with aqueous dioxane-formaldehyde fixation (details to come), were studied. Chymotrypsinogen and procarboxypeptidase were found in the apex of the acinar cells and in pancreatic juice in the ducts ;the zymogen granules were intensely stained, as well as the cytoplasmic matrix around them. The findings with the depolymerases were less definite, probably because the antigens were mixtures, although there was an indication that they occurred in different sets of cells. It is noteworthy that all four enzymes were of bovine origin and were studied in bovine pancreas (pancreas from other species were not examined). In these limited studies on naturally occurring antigens, each antigen used to stimulate the production of the necessary antiserum was as pure as possible; the serum itself was absorbed with appropriate material to remove contaminating antibodies, and the material studied was species specific. Although there are antigenic materials more or less widely distributed in many species (e.g., Forssman antigen), most antigenic molecules, even where they have identical biological activity, possess distinct species specificity. Studies of hormones, enzymes, or structural proteins, or other antigenic substances by means of labeled antibody must therefore be carried out with this point in mind. This is a misfortune, because these are the very substances of most interest to the histochemist; obtaining suitable antigens for the production of serum will often be difficult.

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V. STUDIES ON INFECTIOUS AGENTS 1. Viruses To the classical methods which have evolved in the study of viral infections have recently been added observations on the cytopathogenic effect of many viruses on cells in tissue culture (see Enders, 1954) and observations of ultrathin sections of infected tissue under the electron microscope (e.g., for literature, Harford et ul., 1955). In addition, studies on a few viruses have been carried out with specific labeled antibody. All three of these methods allow the identification of infected cells, and the latter two provide information concerning the locus of infection within the cell. Labeled antibody localizes antigenic material, of course, some of which consists of clumps of viral particles, and some is demonstrably not active virus but material derived from it (Liu, 1955b). a. Muwps. Mumps viral antigens have been localized by means of labeled anti-mumps monkey serum in the cytoplasm of acinar cells of the parotid, the cells lining the radicles of the parotid duct, and in scattered extracellular sites in the brain and spinal cord. (Coons et aZ., 1950; Chu et al., 1951). In infected Maitland-type tissue cultures of chick embryo brain and chorioallantois, it grew in the cells on the surface of the fragments (Watson, 1952a). In chick embryos infected with mumps virus by intra-amniotic injection, the virus multiplied in cells contiguous to the amniotic fluid: cells lining the amnion and covering the skin and pharynx and its extensions. Tiny fluorescent granules in the cytoplasm were the first manifestation of infection and were thought to represent small colonies or aggregates of virus. These increased in size and number until they filled the cytoplasm (Watson, 1952b). b. Inpuenza. Cells infected with influenza virus have been investigated in the chick embryo (Watson and Coons, 1954) and in the ferret (Liu, 1955a, b). In the chick embryo the same cells proved infected after intraamniotic inoculation as in the case of mumps, but the earliest visible stages were in close association with the nuclear membrane, whether just inside or just outside it (or conceivably within it) could not be determined. Occasional spots of antigenic material were visible within the nucleus. Later during the course of the infection the antigen came to lie entirely within the cytoplasm and the plasma membrane was brilliantly fluorescent. Under the conditions used in these experiments, the antigen was homogeneously distributed, no granularity being apparent. In the infected ciliated epithelium overlying the nasal turbinate in the ferret, Liu ( 1955a) demonstrated cytological localization of viral antigens in the cytoplasm and along the ciliated border of the infected cells, and

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irregular patches of antigenic material inside the nuclei. He noted marked cross-reactions among three strains of influenza A viruses, and was able to demonstrate (Liu, 1955b) that the intranuclear antigen was one common to all three and could be removed by absorption with supernatants of infected allantoic fluid from which infectious viral particles had been sedimented-the “S antigen” of Wiener et d. (1946). This antigen is itself sedimentable at 30,000 r.p.m. in 60 minutes. It is clear from his absorption experiments that this particle, or antigenic material derived from it, accounts for the intranuclear antigen. The antigens associated with the active virus particles remain in the cytoplasm. Harford et d. (1955) found aggregates of particles in the cytoplasm of the ciliated epithelium of the bronchi in infected mice, but none in the nuclei. These two morphological methods, then, supplement and support each other in this instance. c. Other Virus Infections. Infectious canine hepatitis, a disease of dogs and foxes, produces large intranuclear inclusion bodies in hepatic cells. These were found to contain viral antigen. Stages in the development of these inclusions from the first appearance of antigen on or within the nuclear membrane to the final inclusion readily visible in histological preparations were described. The only other cells demonstrably infected were the endothelial cells of small vessels, especially in the brain and lung (Coffin et d.,1953). Varicella (chicken pox) virus was first isolated in tissue cultures of human cells by Weller (1953). During the course of infection the infected cells develop intranuclear inclusions, swell, become rounded, and eventually degenerate. By means of convalescent human serum, and fluorescent anti-human y-globulin (see Section 11, 11 on Use of Layers), varicella antigen was found in and on the surface of these cells (Weller and Coons, 1954). The reactions were carried out on fixed whole cells grown on coverslips and for this reason the authors were cautious in their interpretation of cytologic details, although the photographs indicate antigen in both the nucleus and the cytoplasm. These cells (human skin-muscle cultures) were grown in a medium of bovine origin containing no human serum ; they contained no demonstrable human globulin.

2. Other Infectious Agents Kupffer cells and cells in the peritoneal exudate of cotton rats infected with the rickettsiae of epidemic typhus and Rocky Mountain spotted fever were specifically stained with labeled antibody by Coons et ul. (1950) ; individual rickettsiae were visible. Moulton and Brown (1954), studying smears of the bladder epithelium in infected dogs, found specific staining of the inclusion bodies of canine distemper. By means of layers using convalescent human serum Liu and Eaton (1955) have localized the Mac.

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strain of the virus of atypical pneumonia in the bronchial epithelial cells of infected chick embryos, where it causes an otherwise inapparent infection. Sheldon (1953) has demonstrated the antigens of Leptospira icterohemorrhagke in human muscle lesions. Goldman (1953) was able to differentiate between Entamoeba hktolytica and Errtamoeba coli by means of labeled antiserum. Bacteria have so far not been studied with fluorescent antibody, presumably because their investigation does not present quite the same difficulty as does that of viruses, and because current investigations have tended to neglect them. Nevertheless, it would probably be fruitful to investigate chronic infections by this means. VI. STUDIES ON ANTIBODY FORMATION By means of two layers, an examination of the antibody content of individual cells was carried out in rabbits under various conditions of antigenic stimulation. It wa,s determined that the clumps of plasma cells, found in hyperimmune animals in the medullary areas of spleen and lymph nodes and long associated with the antibody response, contained antibody in their cytoplasm, and occasionally in their nuclei as well (Coons et al., 1955). After one injection of antigen only a rare cell contained antibody four days later in the medullary area of the lymph node draining the site of injection. When two injections had been administered one month apart, hundreds of antibody-containing cells appeared in the same area. Cells with the morphology of hemocytoblasts contained detectable antibody on the second day after stimulation; these cells multiplied, differentiated through successively more mature stages, and ultimately became morphologically typical plasma cells. The antibody content in these cells increased, as judged by observation of the intensity of fluorescence, during this sequence. In some animals there was also antibody associated with the lymphoid follicles. (Leduc et al., 1955). Adsorption of the antigen to aluminum phosphate, long known to increase antibody titers, prolonged the response, and antigen was detectable 3 weeks later in the granuloma which formed at the injection site. Antibody-containing plasma cells were demonstrated in the regional lymph node and in the local granuloma (White et al., 1955a). Freund's adjuvant mixture (a water-in-oil emulsion containing killed tubercle bacilli and antigen) , which results in very high circulating antibody levels, was investigated by the same means. In this case, antibody production was widespread in the lymph nodes and spleen and in plasma cells in granulomata scattered in the lungs and liver. In some lymph nodes the whole extrafollicular area of the node was filled with plasma cells

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containing antibody, Oddly enough, the lymph node draining the injection site contained no demonstrable antibody, having been almost completely replaced by epithelioid cells (White et al., 195513). In a study of Russell bodies induced in mice and rabbits by the repeated injection of Profeus wlgaris, White (1954) demonstrated anti-proteus antibody in the cytoplasm of the plasma cells bearing them. In some of his preparations only the periphery of the Russell body itself was fluorescent, the cytoplasm of the cell being otherwise empty of demonstrable antibody. He concluded that there was antibody in the Russell body itself. Repeated attempts to demonstrate antigen as well were negative, although an effort was made to elute masking antibody. Witmer (1955), in an investigation of antibody formation in the rabbit eye, has found antibody in plasma cells infiltrating the iris, the ciliary body, and to a lesser extent the choroid. VII. CONCLUSION The use of antibody coupled with a visible label has made available a method allowing the microscopic study of cells for their content of complex biological substances, and potentially it has placed a large body of immunological knowledge at the service of the histochemist. Thus far, however, it has been exploited principally for immunological ends, by far the easiest problems to attack because a firm background of immunological information w a s at hand. Beginnings have been made in the investigation of antigens normally present in cells, but even in these cases there was previous immunological data to furnish a starting point. The principal problem facing the investigator who wishes to use these immunohistochemicalprocedures in the study of normal tissue components will be the purification of the chosen antigenic material, derived from the species to be explored, in order to stimulate the synthesis of the necessary antibody in some other convenient species. Unexpected reactions are inevitable as small amounts of active antigens contaminating the material stimulate a disproportionate amount of antibody. The antigenic complexity of the erythrocyte is a strong hint as to the complexity of other cell types.

ACKNOWLEDGMENTS That portion of the investigations reviewed here originating from the author’s laboratory was supported at one ar another time by grants from the Life Insurance Medical Research Fund, the Helen Hay Whitney Foundation, the American Heart Association, and the Eugene Higgins Trust. This review was written during tenure of a Career Investigatorship of the American Heart Association, The work on viruses w a s done under the

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sponsorship of the Commission on Immunization, Armed Forces Epidemiological Board, and was supported by the Office of the Surgeon General, Department of the Army. The author owes unpayable debts to those members of the university who first gave him encouragement and help : John F. Enders, the late J. Howard Mueller, Allan L. G r a i n , George B. Wislocki, and Louis F. Fieser. VIII. REFERENCES Anderson, N. G. (1953) Science 117, 517. Barnard, J. E.,and Welch, F. V. (1936) J . Roy. Microscop. SOC.S6, 361. Bateman, J. B., Calkins, H. E., and Chambers, L. A. (1941) J. I m m u t ~ l 41, . 321. Bell, L. G. E. (1952) Infem. Rev. Cytot. 1, 35. Chu, T. H., Cheever, F. S., Coons, A. H., and Daniels, J. B. (1951) Proc. SOC. Exptl. Biol. Med. 76, 571. Clayton, R. M. (1954) Nature 174, 1059. Coffin,D. L,Coons. A. H., and Cabasso, V. J. (1953) J . Exptl. Med. 98, 13. Coons, A. H. (1954) Ann. Rev. Microbiol. 8, 333. Coons, A. H.,Creech, H. J., and Jones, R. N. (1941) Proc. SOC.Exptl. Biol. Med.

47, 200. Coons, A. H., Creech, H. J., Jones, R. N., and Berliner, E. (1942) 1. Zmmu~o2. 45, 159. Coons, A. H., and Kaplan, M. H. (1950) J . Exptl. Med. 91, 1. Coons, A. H., Leduc, E. H., and Connolly, J. M. (1955) J . Exptl. Med. 102, 49. Coons, A. H., Leduc, E. H., and Kaplan, M. H. (1951) J . Exptl. Med. 98, 173. Coons, A. H., Snyder, J. C., Cheever, F. S., and Murray, E. S. (1950) 1. Exptl. Med. 91, 31. Cruickshank, B., and Hill, A. G. S. (1953) J . Pofhol. Bacterial. 66, 283. DeRepentigny, J., and James, A. T. (1954) Nature 174, 927. Dixon, F. J., Bukantz, S. C., and Dammin, G. J. (1951) Science lB, 274. Dixon, F. J., and Maurer, P. H. (1955) J . Exptl. Med. 101,245. Dounce, A. L (1954) btem. Rev. Cytol. 8, 199. Eaton, M. D., Meiklejohn, G., and van Herick, W. (1944) J. Exptl. Med. 79, 649. Enders, J. F. (1954) Ann. Rev. Microbiol. 8, 473. Felton, L. D. (1949) J . Zmmunol. 61, 107. Felton, L. D.,Prescott, B., Kauffmann, G., and Ottinger, B. (1955) J. Immzcnol. 7% 205.

Freund, J.. and McDermott, K. (1942) Proc. SOC.Exptl. B b l . Med. 49, 548. Garveyi J.. S., and Campbell, D. H. (1954) J. Immu~ol.72, 131. Garvey, J. S.,and Campbell, D. H. (1955) Federation Proc. 14,463 (Abstract). Ghosh, I. C., and Sengupta, S. B. (1938) Z . p h y d . Chem. BU,117. Gitlii, D. Unpublished data. Gitlin, D., Landing, B. H., and Whipple, A. (1953) J. Exptl. Med. 97, 163. Goldman, M. (1953) Am. I . Hyg. 68, 319. Goodman, M.,Greenspon, S., and Krakower, C. (1954) Federation Proc. 18, 495, (Abstract). Hamperl, H. (1934) Yirchozu's Arch. Pathol. Anat. 2c. Physiol., 292, I. Harford, C. G., H d m , A., and Parker, E. (1955) J. Ezptl. Med. 101, 577.

zz

ALBERT H. COOfS

Harkins, W. D., Fourt, L., and Fourt, P. C. (1940) J. Bioi. Chem. lS4, 111. Haworth, N., and Stacey, M. (1948) Ann. Rm. Biochm. 17,97. HeideIberger, M., and Kabat, E. A. (1937) I. Ezptl. Med. 65, 885. Hershey, A. D. (1938) Proc. Sac. Ezptl. Biol. Med. 38, 127. Hill, A. G. S., and Cruickshank, B. (1953) Brit. J. Exptl. Puthol. 84, 27. Hill, A. G. S., D m e , H. W., and Cootis, A. H. (1950) I . Exptf. Med. Sa, 35. Hogeboom, G. H., and Schneider, W. C. (1953) Scimce 118, 419. Hooker, S. B., and Boyd, W. C. (1942) J . Zmmuml. 46, 127. Kabat, E. A.,and Mayer, M. M. (1948) “Experimental Imrnunochemistry,” p. 543. Thomas, Springfield, Illinois. Kaplan, M. H., Coons, A. H., and Deane, H. W. (1950) J. Ezptl. Med. 91, 15. Kidd, J. G., and Friedewald, W. F. (1942) J. Ezpfl. Med. 78, 557. Langmuir, I., and Schaefer, V. J. (1937) J. Am. C h m . Sac. 69, 1406. Leduc, E. H., Coons, A. H., and Connolly, J. M. (1955) J. Ezptl. Med. la,61. Liu, .C. (1955a) I . Ezptl. Med. 101, 665. Liu, C. (1955b) J. Ezptl. Med. 101, 677. Liu, C., and Eaton, M. D. (1955) Bucterial. Proc. p. 61 (Abstract). Marrack, J. (1934) Nutwe #B, 292. Marshall, J. M., Jr. (1951) J . Exptl. Med. 94, 21. Marshall, J. M.,Jr. (1954) Ezptl. Cell Research 8, 240. McMaster, P. D., and Kruse, H. (1951) J. Ezpfl. M e d 84, 323. McMaster, P. D.,Kruse, H., Strum, E., and Edwards, J. (1954) Fedemtion Proc. 13, 505 (Abstract). Mellors, R. C., siegel, M., and Pressman, D. (1955) Lob. Invest. 4, 69. Miller, L. L., and Bale, W. F. (1954) J. Exjtl. Med. 99, 125. Moulton, J. E., and Brown, C. H. (1954) Proc. SOC.Ezptl. Biol. Med. 86, 99. Porter, E. F., and Pappenheimer, A. M., Jr. (1939) I. Ezptl. Med. 69, 755. Pressman, D. (1949) J. Immlueol. eS, 375. Pressman, D.,and Eisen, H. N. (1950) J. Immurcol. 64,273. Pressman, D., Eisen, H. N., and Fitzgerald, P. J. (1950) J. Zmmuml. 64, 281. Pressman, D.,Hill, R F., and Foote, F. W. (1949) Sciertce 108, 65. Pressman, D., and Sherman, B. (1951a) J. Zmmmal. 67, 15. Pressman, D.,and Sherman, B. (1951b) J. Zmmwnol. 67, 21. Pringsheim, P. (1949) “FIuorescence and Phosphorescence,” p. 423. Interscience, New York. Proom, H. (1943) I. Puthol. Bacterial. 66, 419. Rothen, A.,and Landsteiner, K. (1942) J . Ezptl. Med. 76, 437. Sarre, H., and Wirtz, H. (1939) Klin. Wockrchr. 18, 1548. Scheibel, I., and Coons, A. H. Unpublished observations. Schiller, A. A., Schayer, R. W.,and Hess, E. L (1953) I . Gm. Physiol. S8, 489. Schmidt, W. C. (1952) I. Exptl. Med. 86, 105. Schmidt, W. C. (1954) in “Streptoccocal Infections,” (McCarty, ed.). Columbia U. P., New York Shaffer, M. F., and Dingle, J. H. (1938) Proc. SOC.Exptl. Biol. Med. 38, 528. Sheldon, W. H. (1953) Proc. Sor. Exptl. Biol. Med. 84, 165. Stark, 0. K. (1955) I. Immunol. 74, 130. Umberger, J. Q.,and LaMer, V. K. (1945) J. Am. C h . Sac. 87, 1099. Vaughan, J. H.,and Kabat, E. A. (1953) J. Exjtl. M e d 87, 821. Vaughan, J. H.,and Kabat, E. A. (1954) J, Immuwl. 13, 205.

HISTOCHEMISTRY WITH LABELED ANTIBODY

23

Vavilov, S. J. (1924) Z . Physik. B, 266. Waksman, B. H., and Bocking, D. (1953) Proc. SOC.ExQtl. Biol. Med. 83, 738. Watson, B. K. (19%) Proc. SOC.Exptl. Biol. Med. IS, 222. Watson, B. K. (1952b) I . Erptt. Med. 96, 653. Watson, B. K. Unpublished data. Watson, B. K., and Coons, A. H. (1954) J . ExQtl. Med. SS, 419. Watson, M. L. (1955) I . Biophy~.aNd Biochem. Cytol. 1, 257. Weller, T. H. (1953) Proc. SOC.Exptl. Biol. Med. 83, 340. Weller, T. H., and Coons, A. H. (1954) Proc. SOC.Expfl. Biol. Med. MI 789. White, R. G. (1954) Brit. J . Exfitl. Pathol. 86, 365. White, R. G., Coons, A. H., and Connolly, J. M. (1955a) I . Exptl. Med. lOa, 73. White, R. G., Coons,A. H., and Connolly, J. M. (1955b) 1. ExQtl. Med. 10%83. Wiener, M.,Henle, W., and Henle, G. (1946) J . ExQtl. Med. 83, 259. Witmer, R. (1955) Schweia. med. Wockrchr. 86, 332.

This Page Intentionally Left Blank

The Chemical Composition of the Bacterial Cell Wall

.

C S. CUMMINS Department of Bacteriology. The London Hospitul Medical College. London. England

Pwe I Introduction ........................................................ 25 I1 Preparation of Cell Wall Fractions .................................. 26 1 Methods of Disintegration ...................................... 26 a. Mechanical .................................................... 26 b Heat Rupture of Bacteria ...................................... 26 2. Separation and Purification of Cell Wall Material .................. 27 111 Properties of Cell Wall Fractions .................................... 28 1 General Properties ............................ .................... 28 2 Components Liberated by Acid Hydrolysis ........................ 28 a Sugars ........................................................ 30 b. Amino sugars ................................................. 32 c. Amino acids ................................................... 32 d Diaminopimelic acid 33 e Lipid Components of the Cell Wall ............................ 35 I V Enzymatic Lysis of Bacterial Cell Walls .............................. 36 1 Effects of Proteolytic Enzymes .................................... 36 2. Enzymes Specifically Attacking Bacterial Cell Walls ................ 36 V Quantitative Analyses and Structural Studies .......................... 38 VI Stability of Cell Wall Composition .................................. 40 VII Cell Wall Composition and the Gram Stain ............................ 42 VIII Cell Walls as Antigens .............................................. 43 IX Cell Wall Composition and Bacterial Taxonmy ...................... 45 X Conclusion .......................................................... 48 XI References .......................................................... 49

.

.

.

.

.

. .

.

.

. .

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

.

. ... . . .

I

.

INTRODUCTION

Because of its physical properties. the cell wall is probably the only part of the bacterial cell which can easily be separated and purified. although the results of Weibull (1953a. b) suggest that it may also be possible to prepare a fraction from bacterial lysates which consists mainly of the cytoplasmic membrane Early work on the chemical nature of the bacterial cell wall was limited almost entirely to attempts to show that it was or was not cellulose or chitin (see reviews by Knaysi. 1938; Lewis. 1941) The techniques then available did not permit of the preparation of the material in a pure state. and even if this had been attained the subsequent analysis would have been extremely laborious. in view of what is now known about its composition. The fact that it is possible to write even a brief review of the chemistry of

.

.

26

C. S.

CUMMINS

the bacterial cell wall is due almost entirely to two technical advances: electron microscopy and paper partition chromatography. The use of the electron microscope has clearly demonstrated that the major part of the insoluble fraction obtained from mechanically disintegrated bacteria is composed of cell wall fragments, and the purification of such fractions by washing or enzyme digestion can be followed. This enables chemical analysis to be done on samples of known homogeneity, and paper chromatography allows of at least a qualitative examination on small samples of the material. The present review is not intended to be an exhaustive summary of the literature, but is an attempt to give a general picture of what is at present known about cell wall composition in bacteria, and how this may be related to other properties of the bacterial cell.

11. PREPARATION OF CELLWALL FRACTIONS The bacterial cell wall appears to be a tough, insoluble membrane, and the general method of preparation has been to rupture the cell mechanically or by heat, separate the insoluble cell wall fractions by centrifugation, and purify this material by washing, or in some cases by treatment with proteolytic enzymes.

1 . Methods of Disintegration a ) , Mechanical. These methods have been reviewed by Hugo ( 1954) in relation to the preparation of cell-free enzymes from bacteria, but are also applicable to the production of cell wall fractions. They include such procedures as grinding with abrasives (e.g., powdered glass, alumina), shaking with minute glass beads, forcing through a very small orifice at low temperature in a press, or using supersonic vibrations. One of the most generally useful methods, which has been employed by a number of workers (Hotchin et d., 1952; Mitchell and Moyle, 1951a, b; Salton, 1952b; Cummins and Harris, 1956) employs glass beads (ballotini, 0.150.20 mm.) in the tissue disintegrator described by Mickle (1948). b ) . Heat Rzrptztre of Bacteria. Salton and Horne (1951a) demonstrated that the walls of gram-negative species are ruptured if a small volume of a bacterial suspension is squirted into a large volume of water at 80-10°C. Gram-positive species do not rupture under such conditions, and the greater susceptibility of gram-negative organisms may be due to the differences between the cell walls of gram-positive and gram-negative species which Salton (1953) has described (see below, Section VII).

CHEMICAL COMPOSITION OF BACTERIAL CELL WALL

27

2. Separation and Purification of Cell Wall Material Whatever method of disintegration has been used the cell wall fraction

can be separated from the soluble constituents by centrifugation, after which a variety of methods have been employed to purify it further. Holdsworth (1952), working with a strain of Corynebwterium diphtheria, shook the material repeatedly in 2% sodium acetate until no more soluble constituents could be removed, and then further extracted the insoluble residue with W% phenol; but most investigators have used less drastic methods. Salton and Home (1951a, b) in particular have published electron micrographs of pdrified cell wall material from Streptococcus faecalis, Escherichia coli, and other organisms, which had been obtained by differential centrifugation and washing in distilled water. Cummins and Harris (1956), in a survey of cell wall composition in various genera, used digestion with trypsin and ribonuclease, followed by pepsin. McCarty (1952b) used a rather similar procedure in the preparation of material from Str. pyogenes. As is mentioned later, the use of proteolytic enzymes may remove not only cytoplasmic remnants but also various (originally surface) antigens from the cell walls themselves. Whatever method of preparation is used, it appears that electron microscopy is the only satisfactory way of establishing the purity of the final material, and shadowed preparations are essential for the demonstration of small amounts of impurities. Except in the walls of SpiriZlum (Houwink, 1953; Salton and Williams, 1954) and of an unidentified bacillus (Labaw and Mosley, 1954) in both of which a periodic structure has been observed, the preparations examined appear to consist of thin homogeneous membranes about 15-20 mp thick, and without structural differentiation. Estimates of what proportion of the cell is made up of cell walls have given very variable results. For example Mitchell and Moyle (1951b) estimated that the cell wall of Staphylococczcs aureus comprised about 20% of the cell dry weight, and Cummins (unpublished observations) in a similar estimation on C. di)&theriae, found a figure of about 16%. On the other hand Holdsworth (1952) has given the much higher figure of 40% in C. dipktheriae, and Fernell and King (1953) stated that the amount of insoluble material in organisms which had been disintegrated (and which they regarded as cell walls) varied from 40% in Staph. pyogenes to 76% in Pseudomonas aeruginosa. These authors did not examine their fractions in the electron microscope, and the figures seem unexpectedly high. Weibull (1953a) working with lysozyme and suspensions of Bacillus megaferiurn, whose cell wall is susceptible to the enzyme, found that about 20% of the dry weight of the cells was removed by its action.

28

C.

111.

S. CUMMINS

PROPERTIES OF CELL WALLFRACTIONS

1. General Properties One of the main features of cell walls of bacteria, as usually prepared, is their extreme insolubility and apparent inertness in a wide variety of reagents. This is convenient for their preparation, since they can easily be separated from other constituents by centrifugation, but makes the investigation of their chemical structure correspondingly more difficult. Holdsworth (1952), for example, working with cell walls of C. dipktheriae, found them resistant to formamide at 37", diethylene glycol, 50% aqueous pyridine, 90% phenol, and boiling 10% acetic acid, and Mitchell and Moyle (1951b) found that the cel1 wall of Staphylococcus aweus was unaffected by a similar range of reagents, including 50% aqueous guanidine hydrochloride. Salton (1952b) noted a similar resistance on the part of the cell walls of a strain of Str. faecdis. Apart from the use of specific enzymes, such as lysozyme, the only reagents in which the walls are easily soluble are strong mineral acids, and most of the information at present available about their structure has been obtained by chromatography of acid hydrolyzates. Investigations using these methods have given a general picture of the components present, at least in gram-positive species, and the next section deals with the results so far obtained. The effect of proteolytic and other enzymes will be considered later. 2. Components Liberated by Acid Hydrolysis At the time of writing, the only large series of results available are those of Salton (1953) and Cummins and Harris (1955,1956) and these are presented in detail in Tables I and 11. They refer almost exclusively to grampositive species, except for the strains of E. coli and Salmonella pullorum examined by Salton (Table I in this review). In both tables, the results refer to cell walls prepared by mechanical disintegration in a Mickle shaker. Salton also examined cell walls prepared by heat rupture (see Section 11, 1.b) in the case of the two gram-negative organisms, but could find no significant difference between cell wall fractions made in this way, and those obtained by mechanical disruption. Cummins and Harris treated the cell wall fractions with trypsin, ribonuclease, and pepsin before hydrolysis and this may account for the slightly simpler amino acid patterns reported by these authors. A similar series of results from gram-negative species is not yet available, so that general conclusions about the nature of cell walls in gram-negative bacteria must be more speculative. In addition to these two more extensive series of analyses, there are also the papers dealing with single strains by Holdsworth (1952) on C. dipktheke,

TABLE I* COMPARISON OF CELL WALL COMPOSITION IN GRAM-POSITIVE AND GRAM-NEGAT~ BACTEBIA Gram-Positive

Gram-Negative

StreptoMicro-

COCMdS

Substance

Streptococcus pyogenes

pyogms (trypsin

treated)

+++ +++ ++ ++ +++ ++ ++ + ++ + + + ++ ++ ++ + +++ + + -

Alanine Aspartic acid Glutamic acid Serine Glycine Threonine Lysine Valine Leucine/iso-Ieucine Proliie Arginiie Sulphur-containing amino acids Aromatic amino acids Diaminopimelic acid Hexosamine Galactose Glucose Mannose Rhamnose Ribose a Combined data from Salton (1953).

+ + +

++ -

++ I

++ -

Streptococcus faecalis

lysodkktincs

+++ +++ + ++ ++ +++ ++ ++ + + ++ -

-

-

++ -

++ + +++ -

-

c1

COCCUS

-

-

++ ++ -

SarcinO lutea

Bacillus dtilis

+++ +++ + + +++ +++ + ++ + + ++ ++ + -

-

-

-

++ -

+++ ++ ++ -

-

-

++ -

-

Eschm'chi Salmonella coli pallorum

+++ +++ ++ ++ +++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +++ ++ + ++ + ++ ++ + + ++ ++ + + + + + ++

++

!

! r +

0"

E

0

f!

A

8 0

w

I b&

3!a + r I 4

2!

E:

< $ r

N \o

30

C. S. CUMMINS

Mitchell and Moyle (1951b) on Staph. azcrew> Salton (1952b) on Str. faecdk, and Weidel (1951) on E. coli. The data on Str. faecdis given by Salton (1952b) are incorporated in his later paper (1953) and appear in Table I :they refer to strain N.C.T.C. 6782 which also was examined by Cummins and Harris (Table 11). The general nature of the cell walls of gram-positive bacteria is clear from the results detailed in these two tables. They are complexes containing sugar, amino sugar, and amino acid residues, and thus fall into the class of mucosubstances, although their extreme insolubility distinguishes them from most other substances of this kind. Whether they should be called mucopolysaccharide or mucoprotein would depend on the relative proportions of protein and plysaccharide fractions (see Kent and Whitehouse, 1955) and in most cases this is not known. The total number of different components is relatively small; in many cases the amino acid content, for example, is virtually represented by three substances. This small number of amino acids distinguishes the cell walls from mucoids such as blood group antigens. The poup A substance from ovarian cyst fluid, examined by Aminoff et al., ( 1950), contained eleven amino acids of which threonine was present in largest amount. a ) . Szcgars. In those species examined so far, the only unusual sugars found in hydrolyzates of the cell wall are the pentose arabinose and the methyl-pentose rhamnose: other sugars are represented by one or more of the three hexoses--glucose, galactose, and mannose. Neither ribose nor deoxyribose have been found in any species, and Salton reported that the cell walls of the species he examined (Table I) showed no ultraviolet absorption corresponding to nucleic acid. Unlike the three hexoses, arabinose and rhamnose seem to have a more restricted distribution among bacteria, being almost confined to the cell walls of certain genera, and the significance of this from a taxonomic point of view is discussed later (Section IX). These two sugars have already been reported from bacterial products, notably in polysaccharides from the tubercle bacillus. Haworth et d., ( 1948b) have described a lipo-polysaccharidecomplex from Mycobacterizcm tuberczclosis, in which the polysaccharide fraction contained arabinose, galactose, and mannose (see also Asselineau and Lederer, 1953). Another such polysaccharide, extracted by alkali, was found to contain arabinose, mannose, and rhamnose (Haworth et al., 1948a). In these polysaccharides the arabinose present was D-arabinose, and this was the variety found by Holdsworth (1952) in C. diphtheriae. In natural products such as gums L-arabinose normally occurs. In most cases the configuration of the sugars present in cell walls has not been determined, since only small amounts of material, sufficient for chromatography, have been prepared.

2

WAl.

E

PPV

-1

-

I

,

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

I I I I++I++ I I$++I++ I I I I l&+itl++++

I I I*+&

. . . . . . . . . .4-. . . . . . . . . . . . . . . . . . . . . . . . . .

,I

-

I --

+++e

-

++++L+*+H

+++ +++ +++ +++ +++ +++ +++ +++ -

++++ +

++++++ ++++++ ++++++

I II I1+1

-

++++++ ++++

+++ +++ --

++

-

I I I I+ I I It%

Streptococcus Group D N.C 1.6782 Strebtococcus Grouo D N.C Striptococcus Group E N.C.T.C. 5385 Streprococcus Group F N.C.T.C. 5389 Streptococcus Group G N.C.T.C. 4549 StaPhvlococcus aureus (3) Stabhylococcus albus ( 3 ) . Staphylococcus citreus (1) Swcina lutea N.C.T.C. 611 Micrococcus lutetu N.C.T.C. 8512 (A.T.C.C. 398) Micrococcirs conglomnatus N.C.T.C. 2677 Micrococcus ciniwbarcus N.C.T.C. 7502 Micrococciu r h o d o c h r m N.C.T.C. 7510 Anococcw viridans N.C.T.C. 8251 Anococcns viridans N.C.T.C. 7593 Acrococcus riridons N.C.T.C. 7592 A n o c o c c v ~M’ridaru N.C.T.C. 7761 Lactobacillus Y from Y Lactobacillus aci6ophilus r 6 r $ . C . 1899 Lactobacillus hrlvcticirg N.C.T.C. 76 Lactobacillus ferment; (bifidus) N.C.T.C. 2797 Lactobacillus brruis N.C.I.B. 8038 Lactobacillus casei N.C.I.B. 8019 rr Lactobacillus plantartrm N.C.I.B. 8030 NOTE: T r indicates trace. Combined results from Cummins and Harris (1956). slightly modified. In order to br!ng out the pattern of amino acids more c!early. only major components are recorded. except in the u s e of Sta/hy/ococcssdbtu a d SWhYlococcusmtrfllr. where the difference in the amount of serine appeared to be of significance. The numbers in parentheses represent the numbers of s t r u of the species concerned.

CHEMICAL COMPOSITION OF BACTERIAL CELL WALL

d

Corynebacteriiinr diphtheriae (4) Corynebacterium ukwans (3) Corynebacterium ovis N.C.T.C. 3460 Cor~nebacteriuirrxerosis (2) Corynebacterium hofmanni (2) Coryirebacteriiiin renale (1) Corynebacteriii?n equi N.C.T.C. 1651 Corynebacteriiriir mtiritim ( I ) Corynebactfriirm pyogeiies N.C.T.C. 5222 Strebtococcus Groun A I I )

I -

I

P-

------r CaL

32

C. 5. CUMMINS

There is no reason to suppose that arabinose is the only pentose likely to be found in bacterial cell walls. For example Hofmann (1953) noted the occurrence of xylose, together with rhamnose and galactose, in hydrolyzates of whole cells of Nitrosomonus, and it would be interesting to examine the cell walls of this group. b). Amino sugars. These have been found in all samples of cell walls examined by various investigators. Their presence has in most cases been recorded as “hexosamine,” without further differentiation, but Cummins and Harris (1956) using two-dimensional chromatography in phenol/NHs and lutidine, in which glucosamine and galactosamine can be separated, noted that while glucosamine and another, unknown, hexosamine-like substance were invariably present, galactosamine was not found in all species (Table 11). This unknown, hexosamine-like substance appeared to be identical with that reported by Strange and Powell (1954) in soluble peptides from germinating spores of B. cereus, B. subbizis, and B. meguterium. It was not fully identified by Strange and Powell, but gave the reactions of a sugar amine with ninhydrin, with the Elson and Morgan reagent (Partridge, 1948), and with ammoniacal silver nitrate. It seems probable that amino sugars occur in the cell wall as acetyl derivatives, since SaIton (1954) found that lysozyme digests of the cell walls of susceptible organisms gave a strong Morgan and Elson reaction for acetyl hexosamine. McCarty (1952b) also found acetyl hexosamine (N-acetyl glucosamine) in the carbohydrate obtained by enzymatic lysis of the cell walls of Str. pyogenes (see Section IV, 2). Such compounds would be deacetylated during the hydrolysis with strong mineral acids which is usually necessary to liberate cell wall components. c). Amino acids. These are relatively few in number in gram-positive species, which seem to contain three or four principal amino acids. Others are either absent, or present in very small amounts. Alanine and glutamic acid are principal amino acids in all species so far examined, and are accompanied by some combination of lysine, glycine, aspartic acid, and diaminopimelic acid. The last named, first identified in bacteria by Work (1951), is an unusual substance not previously found in biological material, and its occurrence and possible functions are described more fully below. It seems likely from Salton’s comparison of the cell walls of some grampositive and gram-negative species (Table I) that the latter contain a larger number of amino acids, including those which are aromatic and sulphur-containing. There also seems to be a suggestion from a comparison of his results and those of C u m i n s and Harris in Table I1 that gram-negative organisms contain both diaminopimelic acid and lysine in

CHEMICAL COMPOSITION OF BACTERIAL CELL WALL

33

the cell walls in about equal amounts, whereas in gram-positive species cell walls appear to contain one or the other in large amount, but not both. Apart from diaminopimelic acid, no other new or unusual amino acids have so far been identified in cell wall hydrolyzates, but several authors have reported ninhydrin-reacting spots which did not correspond to any known substance, suggesting that bacterial cell walls may contain other uncommon components. d ) . Diaminopimelic Acid. The presence of this substance in bacteria was first noted by Work (1949, 195Oa, b, 1951) in the ethanol-insoluble fraction of whole cells of C. diphtheriae, although it had been synthesized by S9rensen and Andersen in 1908 (see Gendre and Lederer, 1952). It is a,€-diaminopimelic acid (DAP) ,a straight chain diaminodicarboxylic acid whose formula is COOHCH (NH2) CH&HZCHZCH(NHz) COOH. and which can theoretically exist in three optically active forms since it has two asymmetric carbon atoms. All three isomers have been synthesized (Work et al., 1954), and so far two have been found in bacteria: the meso (DL-, internally compensated) isomer is that present in C. diphtheriue, DAP from E. coli appears to be a mixture of the meso- and LL-isomers, and in Clostridium welchii and Propionibacteria the LL-isomer occurs alone (Hoare and Work, 1955). The distribution of DAP among 118 species of bacteria, fungi, algae, protozoa, and plant viruses has been studied by Work and Dewey (1953) who examined hydrolyzates of dried intact cells. It was found in all the bacteria examined, with the important exception of gram-positive cocci where it was completely absent. In those species in which it was found, the amount varied considerably, from as much as 2% (dry weight) to 0.02%. In Myxophyceae DAP was present, but it was absent from other algae, from fungi and yeasts, and from two species of protozoa. It appeared to be absent from the strains of streptomyces and actinomyces examined, but it was later reported (Work, 1953) that these strains contained a very similar substance, which may be a methyl diaminopimelic acid. Work and Dewey also examined two virus preparations, tobacco mosaic and turnip yellow, but these did not contain DAP, and Knight (1954) did not find it in tobacco mosaic, cucumber virus 4, tomato bushy stunt, influenza A, influenza B, shope papilloma, or Ta bacteriophage. Present evidence suggests that in those gram-positive species in which it occurs the bulk of the DAP is in the cell wall. This is shown by a comparison of the results of Work and Dewey (1953) studying whole cell hydrolyzates, and those of C u m i n s and Harris (1956) who examined cell walls (Table 111). It can be seen that where DAP had been found in whole cell hydrolyzates, it was always present in the cell walls. Until

34

C.

S. CUMMINS

much more is known about cell wall structure in bacteria, the function of this unusual amino acid will be obscure, but Work and Dewey suggested that it “plays the part of an insolubilizing cross-linking agent, in a manner similar to that played by cystine in the keratinous group of proteins.” It cannot be essential for this, however, since there are no noticeable differences in physical properties between the cell walls of species containing DAP and those from which it is absent. Work and Dewey have also commented on the usefulness of the presence or absence of DAP as a point in classification, and this is discussed in a later section (Section IX). In addition to occurring in the cell wall, DAP is found in other situations in the bacterial cell. Strange and Powell (1954) for example, have found it in the soluble, hexosamine-containing peptide produced by germinating spores of Bacillus subtilis, B. cereus, and B. megateriwn. This interesting finding is discussed later (Section V) . The distribution of DAP in mycobacteria also is of considerable interest. In this genus, the lipopolysaccharide (wax D) fractions from virulent human strains of M. tuberculosis contain alanine, glutamic acid, and DAP, while these amino acids are absent from similar fractions of avirulent strains (Asselineau and Lederer, 1950). On the other hand Gendre and Lederer (1952) have shown that hydrolyzates of whole cells contained DAP whether the strain was virulent or not. These authors reported the presence of DAP in human, bovine, and avian strains of M.twberculosis, and in saprophytic acid-fast strains such as M. phlei. Work (1951) had also noted its presence in human, avian, and bovine strains, and in BCG. The simplest explanation of these findings would seem to be that DAP is present in the cell walls of all strains, but in the lipopolysaccharide of virulent strains only. However no analysis of purified cell wall preparations from mycobacteria are yet available, and the presence of DAP in their cell walls is not definitely established. Apart from its function in presumably stable cell constituents, diaminopimelic acid is also concerned in intermediary metabolism as a precursor for lysine, to which it gives rise by decarboxylation COOH

I CH.NH2 I \CHz)s CH-NHZ

I

COOH diaminopimelic acid

coz

+

CHzNHz

I

CH.NH2

I

COOH 4 lysine

+ COz

CHEMICAL COMPOSITION OF BACTERIAL CELL WALL

35

Dewey and Work (1952) described an enzyme (diaminopimelic acid decarboxylase) in E. coli which performed this reaction. Davis (1952) found mutants of E.coli with an absolute requirement for lysine, which accumulated DAP in large amounts in the growth medium, and Dewey and Work (1952) showed that these lysine-requiring mutants lacked the decarboxylase. I n a later paper, Dewey (1954) has investigated the distribution of this enzyme in various strains. Of 58 organisms of the coli-aerogenes group tested, 48 (83%) contained detectable diaminopimelic acid decarboxylase activity. There was, however, no correlation between the amount of enzyme and the amount of DAP in cell hydrolyzates, since the amino acid was found in several organisms in which no decarboxylase activity could be detected. Work (1955) has shown a similar lack of correlation in other species. For example two strains of Luctobucillus planturz4m (urabinoszcs), which contain DAP (see Table 11) had no decarboxylase activity, while Micrococcus lysodeikticus, Sarcina luteu, and Staphylococcus uure2cs (one strain each), although containing no detectable DAP, had decarboxylase activity of the same order as that found in many strains of E . coli. Work suggests that in these cocci, DAP is only a transient intermediate, since it is not found in hydrolyzates of any fraction of the cell. A recent note by Hoare (1955a) indicates that the situation is even more complex since he has found that the decarboxylase is specific for the meso (DL-) isomer of DAP, and will not attack the DD- or LL- isomers. Organisms such as A. aerogenes, which appear to decarboxylate the LL-isomer, are only able to do this because of the presence in them of another enzyme which converts LL- into meso-DAP. The distribution of this second enzyme in different organisms has not yet been studied. e ) . Lipid Components of the Cell Wdl. The nature of these is so far completely unknown, but they appear to be for the most part firmly bound. S&on (19526) for example could extract nothing from the cell walls of Str. faeculis with boiling ether, but 50 : 50 alcohol-ether containing 0.5% HC1 removed lipid material amounting to 2.3% of the dry weight of the cell wall preparation. In a later paper in which he examined the cell walls of gram-positive and gram-negative bacteria, Salton (1953) compared the amounts of lipid material he obtained by extracting cell walls with ether, by reflwring with 95% methanol, and by extracting with ether after hydrolysis in 6N HC1. In the case of E . coli the amounts obtained were 8.2%, 8.6%, and 22.5% (of dry weight of cell walls) while in the case of B. mbtilb the figures were 0.3%, 1.65, and 2.5%. Mitchell and Moyle (1951b) found total lipid of 4.4% in Stuph. pyogenes cell walls, by extracting with ether after hydrolysis.

36

C. S. CUMMINS

IV. ENZYMATIC LYSISOF BACTERIAL CELLWALLS 1. Effects of Proteolytic Enzymes Living bacteria appear to be resistant to proteolytic enzymes, and can grow and multiply in their presence, for example Str.pyogenes (Lancefield, 1943) and C. diphtheriae (Cummins, 1954). On the other hand suspensions of bacteria which have been killed by heating at 80-100" C. show a varying degree of lysis when incubated with trypsin. Salton (1953) estimated the amount of such lysis turbidimetrically and found about 12% in 1 hr. at 37" with B.megaterium, 2540% with M.lysodeikticus, and 85-9076 with E . coli and Ps. guorescens. Str. faecalis suspensions were quite unaffected by trypsin, even after autoclaving. Examination of the residual material (after maximum clearing) in the case of E.coZi and Ps.flzco~escensshowed it to be almost pure cell walls. Salton suggested that the effect was due to the heat rupture of gram-negative species at 80-100", thus allowing the trypsin to attack the cytoplasm. Heat rupture of gram-positive species does not occur, as judged by the electron microscope, so the lysis observed with heated gram-positive bacteria may be due to slow penetration by trypsin of heat-altered cell walls. In other experiments Salton examined purified cell wall preparations from B. megaterium prepared by mechanical disintegration and found that they were quite unaffected by trypsin. The resistance of bacterial cell walls to trypsin has also been noted by McCarty (1952b), and Cummins and Harris (1956) used both trypsin and pepsin in the purification of cell wall fractions prepared from more than 40 species. In all cases the cell walls were resistant to both enzymes, in that no visible decrease in opacity was caused by either. However, these authors considered that their routine use may simplify the amino acid pattern obtained on hydrolysis by removing superficially situated proteins, as Salton found to be the case in Str. pyogenes (see Table I). It would seem therefore that the basic material of the bacterial cell wall is resistant to proteolytic enzymes, although these may remove proteins which are probabIy superficial and not necessary for structural rigidity. 2. Enzymes Specifically Attacking Bacterial Cell Walls

The best example of these is lysozyme, a bacteriolytic enzyme present in body fluids, such as tears, and obtained in crystalline form from egg white. The organism most susceptible to its action is M~crococcusZysodeikticus (Meming, 1922) suspensions of which are rapidly cleared by lysozyme so that its substrate is obviously intimately connected with the maintenance of cell structure. This has been confirmed by SaIton (19%)

CHEMICAL COMPOSITION OF BACTERIAL CELL WALL

37

who found that purified cell wall suspensions from M. lysodeikticus were rapidly made soluble by lysozyme. Electron microscopy of the residue after 60 minutes digestion at pH 6.2 showed debris with a very occasional cell wall fragment which had apparently resisted lysis. There was no evidence for a lysozyme-resistant framework, and Salton suggested that the main action was a depolymerization of the whole cell wall structure. Later Salton (1954) , working with M. Zysodeikticus and two other lysozymesensitive bacteria, Sarcina lutea and B . megaterium, reported that when their cell walls were dissolved by lysozyme the major components found in the digests possessed molecular weights of the order of 10,000-20,000. It is probable that the action of lysozyme requires some combination of glucose and hexosamine in the cell wall, since these were the reducing substances present in all three species (see Salton, 1956a). Quite apart from actual depolymerization and lysis of the wall of completely susceptible species, lysozyme may have effects short of actual lysis in other cases because Webb (1948) has reported that heat-killed cells of Clostridium welchii and Stuph. albus can be made gram-negative by lysozyme, while Becker and Hartsell (1954) found that lysozyme and trypsin together produced a much greater degree of lysis of bacterial suspensions heated at 70°C.for 15 minutes, than did trypsin alone. Apart from lysozyme, the only other example which has been studied in detail of an enzyme specifically lysing bacterial cell walls is that produced by Streptomyces albus and described by Maxted (1948). This was originally used for preparing extracts from intact cells for antigenic grouping, but McCarty ( 1952a, b) has described the purification of the enzyme concerned, and demonstrated that it rapidly lyses cell wall preparations from Str. pyogenes (Lancefield Group A) , liberating a nondialyzable carbohydrate fraction. This was found to be serologically identical with the "c" carbohydrate of HCl or formamide extracts (Lancefield, 1928; Fuller, 1938) and was composed of rhamnose and glucosamine. These two substances had previously been reported in the group A antigen by Schmidt (1952) and are the same as those identified in cell wall hydrolyzates of Str. pyogenes by Salton (1953) and Cummins and Harris (1956). McCarty found that the streptomyces strain also produced proteolytic enzymes, but these were without action on the cell wall fraction. Maxted described his original crude enzyme preparations as being active against streptococci of groups A, B, C, D, F, and G, but there have so far been no reports of the effects of the purified enzyme on the cell walls of streptococci of groups other than A. Such an investigation might provide interesting information on the detailed chemical structure of the streptococcal cell wall. Since the cell walls of streptococci of groups A 4 all contain rhamnose

38

C.

S. CUMMINS

and glucosamine, it is probable that the presence of these substances is essential for the action of this streptolytic enzyme, in much the same way as glucose and glucosamine seem to be essential for the action of lysozyme. Salton (1955) has searched for other enzymes of this type among strains of Streptomyces, Micromonospora, and Nocardia, and in two strains of Myxococczcs fulvus. The test substrates were cell wall preparations dispersed in agar to give a solid, opaque medium on which the various organisms could be streaked, and those producing lytic enzymes could be identified by zones of clearing. He found that the strains of Streptomyces, Nocardia, and Micromonospora which he tested were all more or less active in lysing cell walls from Str. faecdis, B. megaterkm, M . Zysodeikticus, and the yeast Candida pulcherrimu, but were quite inactive in the case of cell walls of gram-negative species. This lack of activity with the latter may be another indication of broad differences in chemical structure between the walls of gram-positive and gram-negative bacteria. The Myxobacteria on the other hand, were quite unable to lyse the walls of any species of bacteria tested. Salton pointed out also that the use of heat-killed intact bacteria as test substrates in searching for enzymes active against the cell wall is likely to be misleading, because considerable clearing of such suspension is produced by the proteolytic enzymes of actinomyces (see Born, 1952 ; Muggleton and Webb, 1952; Tai and van Heyningen, 1951), although the cell walls themselves are unaltered. This is particularly noticeable with gramnegative bacteria.

V. QUANTITATIVE ANALYSES AND STRUCTURAL STUDIES One of the most detailed investigations so far reported on any one species is that of Holdsworth (1952), who examined the cell walls of C.diphtheriae (P.W.8 strain). The bacteria were distintegrated in 2% sodium acetate, shaken in the same solution until no more material was extracted, and the insoluble material finally purified with So% phenol. Holdsworth estimated that the cell walls (the phenol-insoluble fraction) comprised 45% of the weight of the cell, and contained 25% reducing sugar (as glucose), 60% protein, and 10% ash. The amino sugar content was 4%. The purity of the material examined was unfortunately not checked in the electron microscope. The cell wall complex could be dissociated into protein and polysaccharide components by boiling with saturated aqueous picric acid, and this treatment resulted in an insoluble protein picrate and a soluble oligosaccharide which could be precipitated with acetone, The sligosaccharide contained arabinose, galactose, and mannose in the proportions 3 :2 :1, but no amino sugars, which remained attached

CHEMICAL COMPOSII'ION OF BACTERIAL CELL WALL

39

to the protein. It was established that two of the sugars were D-arabinose and D-galactose ; insufficient material was available to determine the optical configuration in the case of mannose. By studying the action of periodate on this oligosaccharide Holdsworth concluded that it was a straight chain of six units with galactose in the end positions, since after periodate oxidation this sugar could no longer be demonstrated chromatographically. Cummins and Harris ( 1954) examined hydrolyzates of cell wall material from C. diplztheriae before and after it had been treated with periodate, and found that 0.01 molar HIOI at pH7 appeared to attack the mannose more readily than either arabinose or galactose. The difference between the effects of periodate on the oligosaccharide isolated by Holdsworth, and on the undissociated cell wall, may indicate that galactose is in some. way concerned with the attachment of the oligosaccharide in the cell wall complex. Holdsworth did not report on the amino acids present except to state that in his material diaminopimelic acid was present almost exclusively in the cell wall. The few quantitative results available in other species indicate that the proportion of sugars to amino acids in the cell wall of bacteria covers a wide range. Salton ( 1952b) found about 60% reducing sugar (as glucose), and 20% protein in the cell wall of Str. faecalis, while Mitchell and Moyle (1951b) found only about 0.5% of glucose and 1% of glucosamine in the cell walls of Staph. aureus, the rest of which was composed of amino acids. The finding of a very small amount of glucose in the cell wall of the latter species is confirmed by the qualitative results of Cummins and Harris (Table 11) who detected no glucose chromatographically in the cell walls of three strains of it. The extreme difficulty in dissociating cell wall material except by drastic chemical methods makes it likely that the use of specific enzymes such as lysozyme offers the best method of approach in structural studies. As already mentioned (Section IV.2) lysozyme appears to depolymerize the cell walls of susceptible species to soluble, nondialyzable components of a molecular weight of 10,000-20,000, which should be suitable for more detailed analysis. The results of McCarty (1952a, b) with the lysozymelike enzyme which attacks streptococcal cell walls suggest that this enzyme acts in the same way, and a search for similar enzymes, along the lines indicated by Salton (1955) might yield further results of interest. The hexosamine-containing peptides (Strange and Powell, 1954) liberated by germinating spores of various members of the Bacillus group are of interest in connection with cell wall composition in those species. These peptides are composed entirely of three amino acids-alanine, glutamic acid, and diaminopimelic acid, and two hexosamines-glucosamine and an

40

C. S. CUMMINS

unknown sugar amine. Although Strange and Powell were unable to attach any significance to them beyond suggesting that they may have been derived from the spore surface when its permeability was modified during germination, it seems much more likely that they represent a soluble fragment of the cell wall elaborated by the germinating protoplasm, because alanine, glutamic acid, and diaminopimelic acid are the amino acids found in the cell walls of these species (Salton, 1953 (see Table I) ; Cummins and Harris, unpublished observations). If this is so, the detailed analysis of these peptides would give a clearer picture of at least part of the structure of the cell wall in Bacillaceae. It is of interest also to note that in these peptides hexosamines are combined with amino acids, while in the fragments obtained from cell walls by the action of specific enzymes they seem to be combined with sugars : the polysaccharide fragment split off from the cell walls of Str. pyogenes by Maxted's enzyme, for example, was composed of acetyl glucosamine and rhamnose (McCarty, 1952b).

VI. STABILITY OF CELLWALLCOMPOSITION The constancy or otherwise of cell wall composition in any one strain when grown under a variety of cultural conditions does not seem to have been specifically investigated, but a number of workers (Stokes and Gunness, 1946; Work, 1949; Fernell and King, 1953) have examined the amino acids present in whole-cell hydrolyzates of several species grown on different media. In general their results indicate that while the composition of the free amino acid pool (extractable with ethanol) varied considerably with the medium, the composition of the ethanol insoluble residues was unaffected. For example Work (1949) observed that the amino acids of the insoluble cell residues of C.diplztkeriae were the same whether the organism was grown on a casein digest or a synthetic amino acid medium. More recently Hoare (1955b) has investigated the amino acid composition of cells of Sarcino lzctea grown on six different media. He found that while the amino acids in ethanol extracts of acetonedried organisms varied widely, the composition of the insoluble residues was remarkably constant. Since the amount of these residues varied from 52 to 79% of the dry weight of the cells they almost certainly contain material other than cell walls, a fact which is also suggested by their relatively high content of valine, leucine, proline, and aspartic acid, which are absent, or present only in traces, in the cell walls of this species (Tables I and 11). The results nevertheless suggest that the cell wall composition remained unaltered under different cultural conditions. It is noteworthy also that where different workers have investigated the same species there is general agreement in their results, although their media and conditions of growth were

CHEMICAL COMPOSITION OF BACTERIAL CELL WALL

41

necessarily different. All those who have examined Str. pyogenes, for example, (McCarty 1952b, Salton 1953, Cummins and Harris 1956) are agreed that the carbohydrate fraction of its cell walls is composed of rhamnose and hexosamine, and there is good correspondence between the results in the cases of Sarciw lutea and Str. faecalis 6782, recorded in Tables I and 11. A further indication of stability of composition is the remarkable “all-or-none” distribution of diarninopimelic acid, and the correspondence between its occurrence in hydrolyzates of whole cells and in cell wall fractions (see Table 111). TABLE I11 DISTRIBUTION OF a,e-DIAMINOPIMELIC ACIDIN GUN-POSITIVE BACTERIA

Bacterial Genus or Group Streptococci Corynebacteria Corynebacteriunz pyogenes Other species Staphylococci

sarcina Micrococci Bacillaceae Bacillus

}

Clostridum Lactobacilli Lactobaccilw plantamm Other species

Presence or absence of diaminopimelic acid in hydrolyzates of Whole Cells Cell Walls (Work and (Cummins and Dewey, 19.53) Harris 1956)

- (3)*

- (7).

Not examined

+ (1) - (3)

- (4)

+ (3)

Not examined

+ (4)

- (6)

The numbers in parentheses indicate numbers of species examined, or, in the case of a single species (e.g., C. pyogenes) the number of strains. b 1 strain of C1. tetani.

There is evidence, however, that quantitative differences may occur in the amounts of various components present in the cell walls of different strains of the same species. McCarty and Lancefield (1955) have shown that in the cell wall of Str.pyogenes the rhamnose/glucosamine ratio is normally between 1.5 and 2.0, but that in a variant (V) found after repeated mouse passage, the ratio was between 4.0 and 6.0. An intermediate type (I) was also found with a ratio of about 3.0. The alteration in ratio was accompanied by alteration in antigenic specificity, since antigenic ex-

42

C. S. CUMMINS

tracts of normal strains reacted scarcely at all to sera prepared to V-strains, and vice-versa, while extracts of I-strains reacted to sera prepared against either normal or V-strains. In short, present information suggests that the composition of the insoluble cell wall fraction of bacteria is constant in any species, in the sense that major qualitative differences do not occur, but strain differences in the amounts of the components present may be revealed by further quantitative work, which is at present almost entirely lacking. VII. CELL WALLCOMPOSITION AND THE GRAMSTAIN Gram-positive and grarn-negative organisms appear to differ fundamentally in physiological characteristics, and might be expected to differ also in cell wall composition. A number of the points of difference have already been mentioned, and are apparent from Salton’s findings in Table I. Apart from his results with the two species E. coli and S. pullorunz no systematic comparison has been made. The amino acid content of the cell wall in gram-negative species seems to be more complex, and includes aromatic and sulphurcontaining amino acids which are absent from the purified cell walls of gram-positive organisms. Salton also noted that the two gram-negative organisms contained about 20% of lipids in the cell walls, while in three gram-positive species the lipid content varied from 1.2-2.6%. The high proportion of lipid in the cell walls of the gram-negative strains may well be due to the O-antigens, which are known to be lipo-protein-polysaccharide complexes. In Shigella shigae for example, a phospholipin makes up about 10% of the antigen (Morgan and Partridge, 1940). Further facts suggesting fundamental differences in constitution between the cell walls of gram-positive and gram-negative species are the rupture by heat of gram-negative but not gram-positive organisms (see Section 11, 1) and the complete insusceptibility of the walls of gram-negative organisms to the lytic enzymes of the various actinomyces species tested by Salton (1955), although these were active against the walls of gram positive bacteria. It is possible that both of these properties are connected with the much higher proportion of lipid in the cell walls of gram-negative species. The relationship of cell wall composition to the actual staining of the cell by Gram’s method is at the moment quite obscure. It has been known for some time that crushing gram-positive cells renders them gram-negative (Benians, 1920; Burke and Barnes, 1928, 1929; Mittwer et aZ., 1950), and cell walls prepared by mechanical disintegration are uniformly gramnegative, even if prepared from strongly gram-positive species such as

CHEMICAL COMPOSITION OF BACTERIAL CELL WALL

43

C. hofmanni (Cummins, unpublished observations), which suggests that an intact cell wall is necessary for the dye complex to be retained in grampositive bacteria. Possibly, as Mitchell and Moyle (1954) suggest, there is a difference in permeability between the cell walls of gram-positive and gram-negative bacteria, the walls of the former being less permeable to the outward diffusion of some complex formed within the cell during the process of gram staining. As more information becomes available it may be possible to explain any such difference in terms of chemical composition. VIII. CELLWALLSAS ANTIGENS Except in the cases already mentioned (Section 11, 2), electron microscopy has revealed no evidence of structural differentiation in the cell walls of those species examined, but antigenic studies of purified cell wall material have shown that it is complex, despite its appearance of homogeneity in electron micrographs. McCarty (1952b) and Salton (1953) have both demonstrated that the cell wall of Str. pyogenes (Lancefield Group A) contains the polysaccharide group antigen, and Salton noted that the typespecific protein (M-antigen) was also present in cell walls of this species, and was therefore not removed in the process of mechanical disintegration. It could, however, be removed from the cell walls by tryptic digestion, in keeping with the findings of Lancefield (1943),who showed that trypsin would remove the M-antigen from living cells without affecting their viability. Salton further observed that after removal of the M-protein in this way the amino acid pattern in hydrolyzates of the cell wall was simplified, and that sulphur-containing and aromatic amino acids were no longer present (see Table I). A similar antigenic complexity has been demonstrated by Cummins (1954) in the cell wall of a strain of C. diphthedae mitis. Two antigenic components could be demonstrated in the wall of this species, a superficially situated type-specific protein antigen which corresponded to that giving agglutination in intact organisms, and a polysaccharide group antigen apparently shared by all strains of C. diphtheriae. The latter was the antigen responsible for the agglutination of suspensions of cell wall fragments, whether or not the specific antigen was still present, presumably because the group antigen formed the major part of the artificial surfaces produced by disintegration. The specific protein could be removed from the cell wall fragments by pepsin, though not by trypsin, and the situation was analogous to that found by Salton in Group A streptococci. In the case of C. diphtheriue, however, there was no clear-cut difference in amino acid pattern in cell walls hydrolyzed before and after peptic digestion (Cummins and Harris, unpublished observations). The finding that the polysaccharide part of the cell wall of group A

44

C. S. CUMMINS

streptococci represents the group antigen, or at least determines its specificity, makes it reasonable to suppose that the same situation exists in streptococci of other groups, and possibly in organisms in other genera such as Lactobacillzcs, which have recently been subdivided into groups by precipitation tests (Sharpe, 1955). There seems no reason to suppose that antigenic complexity of the cell wall is confined to streptococci and corynebacteria. Loss of surface antigens, normally accompanied by obvious change in surface properties such as smooth-ro.ugh variation in colony type and loss of suspension stability, is a well-known phenomenon in bacteria, especially in gram-negative bacilli such as Skigella. In Sh. skigue for example, Morgan (1936, 1937) has shown that the O-antigen possessed by smooth strains is a polymolecular aggregate of polysaccharide, protein, and phospholipin residues. This could be extracted with diethyleneglycol, and after purification represented about 5-776 of the dry weight of the organisms. From rough strains, on the other hand, similar extraction procedures produced only 0.2% of inactive material, showing that in these strains the O-antigen was absent. Antigens of similar type have been extracted from Sh. f i m e r i (Goebel et al., 1945), from Br. melitensis (Miles and Pirie, 1939),and from other gram-negative bacilli. Recently Davies (1955) has examined the polysaccharide fraction of such antigens extracted from various species of Sdmonella by the trichloracetic acid method of Boivin (Boivin et al., 1933 ; Boivin et al., 1934). The sugars identified chromatographically in hydrolyzates of the polysaccharides included pentoses (xylose and arabinose) , methyl pentoses (rhzunnose and fucose), hexoses (glucose, galactose, and mannose), and in some cases heptoses and other uncommon sugars. The number of sugars present in the polysaccharide varied from four to eight in different species. Glucosamine and chondrosamine were also present in some preparations. The protein part of the complex was not investigated. These results indicate the complexity likely to be found in the cell walls of Enterobacteriaceae. In rough strains, where the O-antigen is absent, the composition might be expected to be simpler, and a comparison of cell wall composition in rough and smooth strains of the same species should prove interesting. Salton (1953) has pointed out that his results with Sty. pyogenes (Group A) indicated quite clearly “the presence of molecular species which are not involved in the structural rigidity of the cell walls.” This is supported by the findings of Cummins (1954) in C. diphtheke, and by the loss of surface antigens by gram-negative bacilli, already referred to. In view of this, a tentative distinction may perhaps be drawn between essential and nonessential components in the cell wall, the latter

CHEMICAL COMPOSITION OF BACTERIAL CELL WALL

45

being those which may be lost without affecting the function of the wall and hence the viability of the cell. If this is found to be true, the composition of the essential component would be the more important, as representing a more fundamental unit in the cell, without which it could not survive. IX. CELLWALLCOMPOSITION AND BACTERIAL TAXONOMY One of the striking findings of Work and Dewey’s (1953) survey of the distribution of diaminopimelic acid among microorganisms was its complete absence from the gram-positive cocci. They suggested that its occurrence might qualify as a feature to be considered in bacterial classification, and the comparison of their results with the distribution of DAP in cell wall fractions recorded by Cummins and Harris (Table 111) strongly supports this suggestion, at least in gram-positive bacteria. Apart from the distribution of this unusual amino acid, which appears to be confined to bacteria, the results of the latter authors suggest that each bacterial genus may have a distinctive pattern of cell wall components, in particular among the amino acids present (Table 11). I t appears that the amino acids of the cell walls are represented by alanine and glutamic acid, together with some combination of aspartic acid, lysine, glycine, and diaminopimelic acid. An additional complication is that lysine and DAP appear to be mutually exclusive, so that one or the other is always present, but not both, a fact which is of some interest since lysine is produced by decarboxylation of DAP, and such a decarboxylase is known to occur in bacteria (see Section 111, 2d). These findings, however, refer only to five genera of gram-positive bacteria, and may well require modification as information on other species and genera becomes available. The type of result which is obtained by paper chromatography of cell wall hydrolyzates is illustrated in Figs. 1 and 2, which represent photographs of tracings of chromatograms from representative species of four different genera. The substance labeled “ ?Hexosamine” is that thought to be identical with the unknown hexosamine found by Strange and Powell ( 1954). Among the sugar residues identified, rhamnose and arabinose are the only ones whose distribution appears to have possible taxonomic importance, since the former seems to be a characteristic major component of streptococcal cell walls, and the latter of those of corynebacteria. The occasional occurrence of rhamnose in the cell walls of species in other genera (for example, C. murium and L. casei) does not seem to invalidate this when the amino acid components are also taken into consideration. The traces of arabinose found in L. casei can be disregarded for similar reasons.

46

C. S. CUYYINS

---a-

-

uu(NDII

SIaphybcOctlai C i t m a

1

phrnol-

FIG.1. Tracings of chromatograms showing the pattern of sugar components, hexoses and pentoses, in hydrolyzates of the cell walls of species representing four genera.* (From Cummins and Harris, 1956.)

* The strain called Lactobcacillus bifidus in Figures 1 and 2 is the same as that referred to as Lactobon'llus fermsnti (N.C. T.C. 2797) in Table 11.

CHEMICAL COMPOSITION OF BACTERIAL CELL WALL

Corymbockrium hofmanni

47

Streptocoma. groupF

-

LVSINE

FIG.2. Tracings of chromatograms showing the pattern of amino acids and amino sugars in hydrolyzates of the cell walls of species representing four genera.* (From Cummins and Harris, 1956.)

48

C. S. CUMMINS

On the basis of these results, Cummins and Harris have suggested that some species should be reclassified, and in particular that C.pyogenes should be included in the genus Streptococcus since it shows a typical streptocqal cell wall pattern both in sugars and amino acids. It also shares other properties with streptococci, such as lack of catalase activity. On the other hand, the two strains labeled Micrococcus rhodochrous and Micrococcus cinnabareus, which had been rejected from the group of catalase-positive cocci by Shaw et al., (1951) and Cowan (1955) on the grounds that they were diphtheroid in morphology, have the cell wall composition characteristic of Corynebacterium, and might more appropriately be included in the latter genus. The anomalous results of cell wall analyses in the case of LactobaciZlus plantarum are more difficult to interpret, since there are no obvious physiological differences between these strains and other lactobacilli, although their cell wall composition is sharply different. The results in three other strains were almost identical (see Cummins and Hams 1956) and the presence of diaminopimelic acid in this species was also noted by Work and Dewey (1953) (see Table 111). The results so far obtained seem sufficientlyinteresting to indicate the need for a wider survey of cell wall composition in bacteria, to enable a proper assessment to be made of the use of this character in bacterial classification. X. CONCLUSION The chemical structure of the bacterial cell wall raises a number of interesting questions in different fields. For example there is the purely chemical problem of detailed structural investigation, and if major cell antigens are located in the cell wall, as seems to be the case, there is the related immunochemical problem of correlating chemical constitution and antigenic specificity. Furthermore the enzyme-substrate systems afforded by the action of lysozyme and similar enzymes on cell walls could contribute both to a knowledge of cell wall composition and to an understanding of enzyme specificity. The cell walls of gram-positive species in particular seem well adapted to studies of this sort since they contain a relatively small number of basic units. The wall of Str. pyogenes, for example, is almost entirely made up of rhamnose, two hexosamines, and three amino acids. Apart from this, and probably of greater biological interest, there is the possibility of comparing cell wall composition not only in different bacterial genera, but between bacteria and other microorganisms such as yeasts, algae, fungi, and protozoa. Since this articIe was written, a review of the subject by Salton (1956b) has also appeared.

CHEMICAL COMPOSITION OF BACTERIAL CELL WALL

49

ACKNOWLEDGMENTS The author wishes to thank Professor C. F. Barwell, Professor F. L. Warren, Dr. F. C. 0. Valentine and Dr. H. Harris for help and advice during the writing of this review.

XI. REFERENCES Aminoff, D., Morgan, W. T. J., and Watkin, W. M. (1950) Biochem. J. 46,426. Asselineau, J., and Lederer, E. (1950) Compf. rewd. SOC. biol. aS0, 142. Asselineau, J., and Lederer, E. (1953) FortJchr. Ckem. org. Natwsfofle 10, 170. Becker, M. E.,and Hartsell, S. E. (1954) Arch. Biochem. and Biophys. 59, 402. Benians, T. H.C. (1920) J . Pathol. Bacteriol. aS, 401. Boivin, A., Mesrobeanu, I., and Mesrobeanu, L (1933) Compt. rend. SOC. biol. 114, 307. Boivin, A., Mesrobeanu, I., Mesrobeanu, L., and Nestorescu, B. (1934) Compt. rend. SOC. biol. llS, 306. Born, G. V. R. (1952) J. Gen. Microbiol. 6, 344. Burke, V., and Barnes, M. (1928) J. Bacteriol. 16, 12. Burke, V., and Barnes, M. (1929) J. Bacteriol. 18, 69. Cowan, S. T. (1955) personal communication. Cummins, C. S. (1954) Brit. J. Exptl. Pathol. S6, 166. Cummins, C. S., and Harris, H. (1954) Biochem. J . 57, xxxii. Cummins, C. S.,and Harris, H. (1955) J. Gen. Microbiol. IS, iii. Curmnins, C. S., and Harris, H. (1956) J. Gem Microbiol. 14, 583. Davies, D. A. L. (1955) Biochem. J . 69,696. Davis, B. D. (1952) Nuture 169, 534. Dewey, D. L. (1954) J. Gen Microbiol. 11, 307. Dewey, D. L., and Work, E. (1952) Natwre 169, 533. Fernell, W. R., and King, H. K. (1953) Biochem. J . 66, 758. Fleming, A. (1922) Proc. Roy. SOC.SSS, 306. Fuller, A. T. (1938) Brit. I . Exptl. Pathol. 19, 130. Gendre, T.,and Lederer, E. (1952) Biochim. et. Biophys. Acta 8, 49. Goebel, W. F., Binkley, F., and Perlman, E. (1945) J. Exptl. Med. 81, 315. Haworth, W. N., Kent, P. W., and Stacey, M. (1948a) J . C h m . Soc. p. 1211. Haworth W. N., Kent, P. W., and Stacey, M. (1948b) J . Chem. SOC.p. 1220. Hoare, D. S. (1955a) Biochem. J. 69, xxii. Hoare, D. S. (1955b) J. Gm. Microbiol. a,534. Hoare, D. S., and Work, E. (1955) Biochem. J . 60, ii. Hofmann, T. (1953) Biochem. J . 64, 293. Holdsworth, E. S. (1952) Biochim. et Biophys. Acta 9, 19. Hotchin, J. E., Dawson, I. M., and Elford, W. J. (1952) Brif. J. Expt?. Potkol. Sa, 177. Houwink, A. L. (1953) Biochim. et. Biophys. A c f a 10, 360. Hugo, W. B. (1954) Bacteriol. Revs. l8, 87. Kent, P. W., and Whitehouse, M. W. (1955) “Biochemistry of the Aminosugars.” Butterworths, London. Knaysi, G. (1938) Botan. Rev. 4, 83. Knight, C. A. (1954) Advances ilo Virus Reseurch, 2, 153.

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Labaw, L. W., and Mosley, V. M. (1954) Biochim. et. Biophys. Acta l6,325. Lancefield, R. C. (1928) J. Exptl. Med. Ca, 91. Lancefield, R. C. (1943) J. Exptl, Med. 78, 465. Lewis, I. M. (1941) Bacteriol. Revs. 6, 181. McCarty, M. (195%) J. Exptl. Med. 96, 555. McCarty, M. (195%) J. Exptl. Med. 96, 569. McCarty, M., and Lancefield R. C. (1955) I . Exptl. Med. la, 11. Mated, W. R. (1948) Lmcet ii, 255. Mickle, R. (1948) 1. Roy. Microscop. Soc. a,10. Miles, A. A., and Firie, N. W. (1939) Brit. J. Exptl. PathL 20, 83. Mitchell, P., and Moyle, J. (1951a) I . Gen; Microbiol. S, 966. Mitchell, P., and Moyle, J. (1951b) I . Gen. Microbiol. 6, 981. Mitchell, P., and Moyle, J. (1954) J. Gen. Microbiol. 10, 533. Mittwer, T., Bartholomew, J. W., and Kallman, B. J. (1950) Stain Technol. 26, 169. Morgan, W. T. J. (1936) Biochem. I. So, 909. Morgan, W. T. J. (1937) Biochem. I. ti, 2003. Morgan, W. T. J., and Partridge, S. M. (1940) Biochem. J. Sr, 169. Muggleton, P. W., and Webb, Id. (1952) Biochim. t t Biophys. Acta 2, 431. Partridge, S. M. (1948) Biochem. J. Ca, 238. Salton, M. R. J. (1952a) Notorre 170, 746. Salton, M. R. J. (195%) Bioclpim. et Biophys. Act& 8, 510. Salton, M. R. J. (1953) Biochim. et Biophys. Acta 10, 512. Salton, M. R. J. (1954) J. Gm. Microbiol. 11, ix. Salton, M. R. J. (1955) J. Gen. Microbiol. l2, 25. Salton, M. R. J. (19%) Proc. 3rd Intern. Congr. Biochem. Brussels 1955, p. 404. Salton, M. R. J. (1956b) i~ “Bacterial Anatomy” (Spooner and Stocker, eds.), p. 81. Cambridge U.P., London. Salton, M. R. J., and Home, R. W. (1951a) Biochim. et Biophys. Acta 7, 19. Salton, M. R. J., and Home, R. W. (1951b) Biochim. et Biophys. Acta 7, 177. Salton, M. R. J., and Williams, R C. (1954) Biochim. et Biophys. Acta 14, 455. Schmidt, W. C. (1952) I. Exptl. Med. SS, 105. Sharpe, M. E. (1955) J. Gen. Microbiol. Za, 107. Shaw, C., Stitt, J. M., and Cowan, S. T. (1951) J. Gm. Microbiol. 5, 1010. SBrensen, S. P. L,and Andersen, A. C. (1908) 2. physiol. Chem. 66, 250 (quoted by Gendre and Lederer, 1952). Stokes, J. L., and Gunness, M. (1946) J. Bacteriol. 52, 195. Strange, R. E., and Powell, J. F. (1954) Biochem. J . 68, 80. Tai,T. Y., and van Heyningen, W. E. (1951) I . Gpm Microbiol. 6, 110. Webb, M. (1948) J. Gm. Microbiol. a, 260. Weibull, C. (1953a) I . Bacterial. 86, 688. Weibull, C. (1953b) J. Bacteriol. 66, 6%. Weidel, W. (1951) 2. Nutturforsch. 8b, 251. Work, E. (1949) Biochim. et Biophys. Acts S, 400. Work, E. (195Oa) Biockim. ef Biophys. Acta 6, 204. Work, E. (195Ob) Biochem. J. 46, v. Work, E. (1951) B i o c h m J. 49, 17. Work, E. (1953) J. Gm. Microbiol. 9, ii. Work, E. (1955) in “Amino Acid Metabolism” (McElroy and Glass,eds.), p. 462. Johns Hopkins Press, Baltimore. Work, E., and Dewey, D. L. (1953) J. Gen. Microbiol. 9, 394. Work, E.. Birabaum, S. M., Winitz, M., and Greenstein, J. P. (1955) I . Am. Chem. SOC.77,1916.

Theories of Enzyme Adaptation in Microorganisms J. MANDELSTAM National Institute for Medical Research, Mill Hill, London, E n g l d Page

I. Introduction ........................................................ 11. Nomenclature ....................................................... 111. Some General Facts of Enzyme Adaptation .......................... 1. The Basic Phenomenon .......................................... 2. The Source of the Adaptive Enzyme .............................. 3. Protein Turnover in the Bacterial Cell ............................ 4. The Role of the Inducer ........................................ IV. The Mass Action Theory ........................................... V. The Plasmagene Theory ............................................ VI. The Specific Precursor Theory ...................................... VII. The Organizer Theory (1) .......................................... VIII. The Organizer Theory (2) .......................................... fX. The Organizer Theory (3) .......................................... X, The Kinetic Model ................................................. XI. The Extended Mass Action Theory .................................. XII. Conclusions ........................................................ XIII. References ..........................................................

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I. INTRODUCTION It is of interest that, in a subject which has existed for only fifty years and been studied seriously for less than twenty, there should be a sufficient number of theories to warrant the writing of a review article. Although the ratio of theories to facts is much higher than in other branches of biochemistry it is unlikely that the subject would have advanced so rapidly if these theories had not been formulated. The value of a theory in the early stages of development of a subject does not necessarily depend upon accuracy of prediction nor upon the correlation of a large number of facts into a unified system; it depends more simply upon providing a defined point of view and a set of postulates on the basis of which the investigator can decide what sort of observations he should make among the bewildering number that could be made in the absence of any guiding principle. Periodically, however, theories need to be reassessed in terms of the available facts to see to what extent they are still tenable. The present review is an attempt to do this. In the first three decades of this century a considerable number of scattered observations on enzyme adaptation had been made. An early, and much quoted, example is provided by the work of Dienert (1900) on the adaptation of yeast to galactose. The phenomenon itself was easily 51

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described. It had been found that an organism which was normally unable to ferment a particular sugar acquired the ability to do so if left in contact with the sugar for some hours; the new property was quickly lost if the organisms were grown in the original medium. The explanation, when any was given, was teleological, and little real progress was made. In the absence of any theory as to the nature of the process it was difficult to know what to look for, and examples of enzyme adaptation were collected in a random fashion more reminiscent of natural history than of biochemistry, The trend of research altered as a result of the theories put forward, first by Yudkin and then by Spiegelman and Monod. The consequence oi posing the problem in biochemical terms was that the experimental work acquired a biochemical orientation which it has since retained. At first, enzyme adaptation was studied for its own sake but more recently its relevance to other topics has been realized. Its d u e as a tool in the field of protein synthesis is obvious, for, as will be shown, it usually involves the specific synthesis of a protein that is easily identified and measured. Enzyme adaptation has also found application in the study of metabolic pathways for, occasionally, it is found that the addition of a single substrate leads to the adaptive formation of a sequence of enzymes. This happens because the metabolic product of each enzyme reaction causes adaptive formation of the next enzyme in the series. Compounds which are presumed to be intermediate metabolites can be tested and their position in the sequence established. A classic example of such an investigation can be found in the work of Stanier (1947). The literature on adaptation, which is by now considerable, has been discussed in a number of excellent reviews by Yudkin (1938), Monod ( 1947), Spiegelman (1950), Stanier (1951), Monod and Cohn (1952). It is not the object of this review to discuss recent advances as such, but rather to consider those that are relevant to the theories of enzyme adaptation. The following plan has been adopted for the presentation of the subject. A brief outline of the “main” facts will first be given and it will be readily appreciated that the selection of these facts is, by its nature, subjective, and that other writers might have chosen them quite differently. No other procedure was possible, because lack of space precludes a full discussion of all the facts in relation to all the theories. The theories chosen for discussion will then be described in turn and an attempt made to assess them in terms of the general facts already set forth and also in relation to more particular findings that are relevant in each case. There has been no attempt to provide a complete bibliography; when similar results have

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been reported by several different authors only one or two will be quoted in illustration. For convenience theories have been grouped into two categories : those that are specifically concerned with enzyme adaptation, and those that are based on more generalized cell models and can be used, in principle at any rate, to account for other phenomena such as cell growth.

11. NOMENCLATURE Enzyme adaptation is often not “adaptive” in the sense of fitting the organism to its environment. Thus, an enzyme may be formed in response to some compound which cannot be metabolized, or it may be formed under conditions where it cannot act. A case in point is the formation of tyrosine apo-decarboxylase in pyridoxin-deficient cells (Bellamy and Gunsalus, 1945). For these reasons, and also to distinguish true enzyme synthesis from changes in activity caused by permeability or other effects, it has been suggested that the term enzyme induction should be used instead of enzyme adaptation (see Cohn et al., 1953b). Further terms suggested are inducer for the specific substance inducing the enzyme synthesis ; inducible for the enzyme-forming system, and induced for the enzyme produced in this way. Though there is a strong case for adopting these terms, especially inducer, the older term enzyme adaptation has gained wide acceptance over a considerable period; it will therefore be retained in this review. Enzyme adaptation will be defined, in the way suggested by Stanier (1951), as an increase in the activity of a specific enzyme brought about by the presence of a specific substance, usually the substrate, occurring without any change in the genotype. The substance whose presence is specifically required for the adaptation will be called the inducer. The small amount of activity which is frequently encountered in cells before adaptation will be called the basaE enzyme. Those enzymes which are formed in large amounts in the absence of exogenous inducer will, as suggested by Karstrom (1938), be termed constitutive as opposed to adaptive. Two points must be emphasized here: the first is that any particular enzyme may be constitutive in one organism and adaptive in another; the second is that the distinction between adaptive and constitutive is an arbitrary one made on quantitative grounds. It might be said that when the basal activity is already so high that little or no further increase takes place in response to the inducer, the enzyme concerned is constitutive in that organism. It should also be remembered that an enzyme may appear to be constitutive because its inducer is a normal metabolite of the cell. Where this is the case, the adaptive nature of the enzyme will be masked because of the continuous endogenous production of the inducer. An

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example is N-acetylornithinase in Eschevichk coli, an enzyme which hydrolyzes N-acetylornithine to ornithine and is apparently constitutive. However, mutant strains were isolated which were unable to synthesize N-acetylornithine (Vogel and Davis, 1952 ;Vogel, 1953) and these mutants produced the enzyme only when the substrate was present in the growth medium, i.e., the enzyme behaves as a characteristic adaptive enzyme. This is the only instance in which an apparently constitutive enzyme has been shown to be adaptive and there is, at present, not enough evidence to make it possible to say whether all constitutive enzymes are actually adaptive. Even if this proposition were true it might be difficult, for technical reasons, to demonstrate. Nevertheless, as a working hypothesis in the study of protein synthesis it can be assumed that constitutive and adaptive enzymes are formed in the same way. The assumption is supported by investigations into the properties of constitutive and adaptive 8-galactosidase in E. coli (Monod and Cohn, 1952) and of basal (i.e., constitutive) and adaptive penicillinase in Bacillus subtilis and B. cereus (Manson et al., 1954; Kogut et al., 1956). In these studies, the properties examined included immunochemical specificity, the rate of thermal inactivation, Michaelis constants, etc. In no instance was any significant difference found. Since, as pointed out by Manson et d. (1954) it is unlikely that the same cells would synthesize the identical protein in two different ways, it is reasonable to accept what Cohn and Monod (1953) have called the “unitary hypothesis.” The original definition of enzyme adaptation requires some amplification. It has so far been assumed that the increased activity caused by the inducer is due to enzyme synthesis. In many instances this assumption has been shown to be correct, but increased activity could be due to the activation of existing apoenzyme by the synthesis of cofactors or otherwise; it could also be due to permeability changes. Examples of both of these processes can be found. Thus, Rodwell (1953) working with ornithine decarboxylase in Lactobacillus sp. showed that, among other factors, the presence of the specific substrate affected the rate of synthesis and the stability of the cofactor, pyridoxal phosphate. With regard to permeability, a case in point is the behavior of Pseudomoaus fEfcorescem (Kogut and Podoski, 1953). Cells grown in a citrate medium oxidized citrate immediately, while cells grown in a succinate medium oxidized citrate only after a lag period, and the curve of enzyme activity against time was of the type commonly observed in enzyme adaptation. Similar effects were obtained with other intermediates of the tri-carboxylic cycle. However, mechanically disintegrated cells possessed the appropriate enzymes whether they had been previously “adapted” or not. The increased activity

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caused by the substrate was therefore attributed to a specific permeability or transport factor. Analogous results, also with intermediates of the tricarboxylic cycle have been reported in Psezcdomonas by Barret et al., (1953) and in Azotobocter by Stone and Wilson ( 1952). These examples will suffice to show that caution is necessary in interpreting results obtained by the assay of enzyme activity in intact cells.

111. SOME GENERAL FACTS OF ENZYME ADAPTATION

I . The Basic Phenomenon Before proceeding to a consideration of theories of enzyme adaptation it is necessary to know what the main facts are that the theories are expected to cover. Some simple questions come to mind immediately. What are the precursors of the adaptive enzyme? What part does the inducer have in conferring specificity upon the new enzyme molecules? This section will be concerned with these questions and with others that arise from a closer consideration of them. It will become apparent that unequivocal answers are difficult to give, and that few generalizations can be made without contradiction. Many of the findings will be seen to hold only for a particular enzyme in a particular type of organism under particular experimental conditions. The basic phenomenon is quite clear. If organisms are incubated with the substrate, or some chemically similar compound, increased enzyme activity occurs quite rapidly, often within a few minutes, and this can generally be shown to be due to synthesis of the specific enzyme. A source of energy is invariably necessary, and if the inducer itself cannot provide energy, some other suitable donor must be provided. The rapidity of the reaction in most instances precludes the operation of mutation and selection, and it has been tacitly assumed for many years that the ability to form the enzyme is not restricted to a small proportion of the cells in a population but is a property common to all the cells. Earlier work had shown that this supposition was probably correct. For instance, yeast cells adapted to galactose in washed suspension had about half the activity of cells grown in galactose and therefore presumably fully adapted (Spiegelman and Dunn, 1947). It was therefore unlikely that only a small proportion of the population was involved in the process. However, a convincing demonstration of this point was given very recently by Benzer (1953) who showed that adaptive formation of 8-galactosidase in E. coli is a general property of the cells. In principle the method is easy to understand and is as follows. A population of cells is incubated with a nitrogen source but with no carbon source so as to cause carbon starvation. The cells are

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then infected with phage and lactose added. Those cells which have pgalactosidase can utilize the lactose, thereby allowing growth of the phage and consequent lysis of the cells. Bacteria having no enzyme are unable to produce energy and material for phage multiplication and they only lyse later when the enzyme liberated by the lysed cells becomes available to them. In a population containing both types of cells, the curve of lysis will therefore exhibit two phases. In the first phase those cells with enzyme lyse and discharge their enzyme into the medium ; in the second phase, the remaining cells lyse while the extracellular enzyme remains constant. On the other hand, in a population in which the enzyme is uniformly distributed there will be only one phase during which all the cells lyse and enzyme is continuously liberated during the process. In practice, Benzer found that artificial mixtures of adapted and unadapted cells gave a biphasic curve indicating that some of the cells had all of the enzyme and the rest none, while adapted cells gave a monophasic curve showing that the enzyme was distributed throughout the population. Although this is the only clear demonstration that virtually all the cells in a population can produce an adaptive enzyme there is no reason to believe that it is in any way atypical.

2. The Source of the Adaptive Enzyme An adaptive enzyme could theoretically be derived in one of two ways: it might be formed, by a small chemical change, from a relatively specific protein precursor which is already present; or it might be formed de novo, i.e., by complete synthesis from amino acids. A classic example of the first type of reaction is the conversion of trypsinogen to trypsin (Northrop, 1937) which can be initiated by traces of trypsin and then proceeds autocatalytically. Northrop (1949) was of the opinion that this mechanism was general and could also account for enzyme adaptation. There is however no evidence to support the idea that adaptive enzymes arise in this way. Proteins similar in structure to adaptive enzymes, and which might therefore be precursors, have not been found except in one instance. This is the protein Pz which is present in all coliform organisms capable of synthesizing p-galactosidase (= Gz). P z is antigenically related to Gz but has no enzyme activity. When the cells are made to produce Gz adaptively the rate of production of Pz is reduced by about 40% (Cohn and Torriani, 1952, 1953). This finding could mean that Pz is a precursor of Gz or that both proteins have a common precursor. The first of these possibilities was rendered improbable by experiments on the production of p-galactosidase in mutant strains of E. coli with a specific amino acid requirement. When inducer was added to such a culture, after growth had stopped because of exhaustion of the essential amino acid, no adaptation occurred.

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In other words, the cells contained Pz but could not synthesize Gz from it. Addition of the essential amino acid restored growth together with ability to form the adaptive enzyme (Monod et al., 1952). Later work has definitely ruled out the possibility that Pz, or any other preformed protein, could be a direct or indirect precursor of the enzyme (Hogness et al., 1955). It appears (see below) that the synthesis of /?-galactosidase in E. coli represents the formation of a new protein molecule from newly synthesized amino acids. The same appears to be true of the adaptation to maltose in yeast. A number of amino acid analogs were tested for their effect on adaptation to maltose (Halvorson and Spiegelman, 1952, 1953a, b ; Spiegelman and Halvorson, 1953). Of the compounds examined, the halogen substituted phenylalanines, particularly o-fluorophenylalanine (O.F.P.) , were most active. The analogs which inhibited growth also inhibited enzyme adaptation and the inhibitory effect on both processes could be reversed by adding the amino acid concerned. For example, the effect of O.F.P. was competitively reversed by phenylalanine. Furthermore O.F.P. interfered with the utilization of the free amino acid pool of the cells. When normal yeast cells metabolized glucose in the absence of an added source of nitrogen, the amino acid pool became depleted by incorporation into protein. If O.F.P. was present during this period it inhibited the incorporation not only of phenylalanine but of the amino acids in the pool generally. The effect of O.F.P. on growth and on enzyme adaptation can therefore be attributed to the fact that it blocks the free assimilation of amino acids in the pool. All these findings are consistent with the idea that adaptation involves the complete synthesis of a new protein from free amino acids.

3. Protein Turnover in the Bacterial Cell If it is accepted provisionally that the adaptive enzyme is synthesized from free amino acids a further question has to be considered. Can the amino acids needed for the new enzyme be derived from pre-existing cell protein or must there be a supply of nitrogen in the external environment? More generally, it may be asked whether there is a turnover of protein in microorganisms. If it is assumed that there is a turnover of protein, it follows that enzyme adaptation should be possible in the absence of any exogenous source of nitrogen, and it should be possible to show that the adaptive enzyme formed under conditions of nitrogen deprivation is synthesized at the expense of other cell proteins. With regard to the first point the evidence is conflicting and depends upon the enzyme and upon the organism. For example, adaptive enzymes which oxidize a variety of aromatic compounds are formed by Pseudomonas

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fluorescens in the absence of an external source of nitrogen (Stanier, 1947, 1951) . Similarly, yeast cells generally adapt to maltose and galactose in the same way. If, however, cells are starved of nitrogen by incubation with glucose for some time they may lose much of their ability to adapt (Spiegelman and Dunn, 1947). Addition of an ammonium salt to the medium restores the adaptive response. In E. coli and related organisms variable results have been reported. Thus, paratyphoid organisms can produce tetrathionase in the absence of an added nitrogen source and with no demonstrable increase in the weight of bacteria or the number of cells (Knox and Pollock, 1944). On the other hand, the synthesis of p-galactosidase in E. coli is closely bound up with general protein synthesis and in the presence of optimal amounts of inducer the adaptive enzyme is formed as a constant fraction of the total new protein synthesized during growth. Under conditions which precluded protein synthesis (e.g., exhaustion of an essential amino acid) no P-galactosidase was formed (Monod et. al., 1952). Similar results with the same enzyme have been reported by Rickenberg et al. (1953). Some interesting observations have been made by Wainwright and Nevi11 (1956). Using a strain of E . coli they confirmed the fact that P-galactosidase cannot be formed under conditions of nitrogen deprivation, but the same cells were able to synthesize appreciable amounts of tetrathionase and nitratase even after three hours of nitrogen starvation. A similar investigation has been carried out by Mandelstam (1955). A strain of Bacterium cadaveris was obtained which was able to synthesize adaptively both lysine decarboxylase and ornithine decarboxylase. The latter enzyme was similar to p-galactosidase in that it was formed only if the bacteria were able to grow, and it was produced as a constant fraction of the total new protein. In contrast, lysine decarboxylase was synthesized in nitrogen-starved bacteria and the rate of production remained linear for the duration of the experiments (2.5 hours). The rate was roughly half that obtained with non-starved bacteria. Amino acid analogs5-methyltryptophan and p-phenylserine-inhibited the synthesis, and the inhibition could be reversed by addition of the appropriate amino acid, i.e., tryptophan or phenylalanine. The effect of these compounds resembled the effect of amino acid analogs on adaptation to maltose in yeast and pointed to the existence of a pool of free amino acids in Bact. cadaveris. Accordingly the following experiments were carried out. Bacteria, grown in a medium consisting of glucose, lactate, and ammonium salts (Mandelstam, 1954), were harvested, washed, and heated in water at 100°C. for 20 minutes. The extract was poured through a column of acid-treated Dowex 50 resin and eluted with ammonia. When an amount of effluent

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corresponding to 25 mg. dry weight of bacteria was chromatographed on paper with butanol-acetic acid-water, thirteen distinct ninhydrin spots were found. The heaviest spots were due to glutamic acid, alanine, and glycine; leucine, valine, and aspartic acid were also present but in much smaller quantities. Several other strains of E. coli have been examined and all have been shown to possess similar pools. The failure of Taylor (1947) to detect any free amino acid pool in colifonn bacteria is probably due to the fact that the manometric techniques she employed were not sensitive enough to detect the small quantities present. The pool, which represents 0.5% to 1% of the total dry weight in B a d . cadaveris, was not significantly reduced when the bacteria were starved by incubation with glucose, in a nitrogen-free medium, for several hours. The adaptive formation of lysine decarboxylase also produced no significant depletion. However, the addition of phenylserine to a suspension of nitrogen-starved cells caused the pool to increase about 50% in the first hour and to remain at this level for the duration of the experiment (2.5 hours). The free amino acid pool in Bact. cadaveris is, in many respects, similar to that in yeast (Halvorson et al., 1955), and its behavior in both types of organism is consistent with the occurrence of protein breakdown and re-synthesis. It is difficult to prove this point unambiguously, and until this has been done the ultimate source of adaptive enzymes in nitrogenstarved bacteria, and the question of protein turnover, will remain in doubt. What emerges clearly from the evidence already available is that different enzyme-forming systems, even in the same cells, may differ greatly in their ability to utilize endogenous nitrogen sources. The subject of interconversion of cell proteins has been studied by only a few workers. Spiegelman and Dunn (1947) found that when yeast cells were adapted to galactose in the absence of a nitrogen source and then adapted to maltose, the second adaptation was severely limited in extent. The effect was overcome by adding a source of nitrogen to the adapting suspensions. Cells could also be made to adapt to galactose and maltose simultaneously by suspending them in a solution containing both sugars. The maltose fermenting system, having a shorter lag period, increased rapidly at first and then, as the galactose fermentation became established, reached a peak and began to decay. It thus appeared that the adaptation to galactose was carried out at the expense of the other adaptive system. Again the competitive effect was abolished when ammonium salts were present. The same two enzyme systems in a different strain of yeast, were examined by Mandelstam and Yudkin (1952) and the competitive interaction was confirmed. In assessing the significance of these experiments it must be remembered

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that only an over-all rate of fermentation is being measured and this does not necessarily reflect the quantity of the enzymes present (see Sheffner and McClary, 1954). Nevertheless, the fact that one enzyme system appears to be derived at the expense of another under conditions of nitrogen deprivation, but not otherwise, is prima facie evidence for the occurrence of interaction between the proteins concerned and certainly warrants a fuller investigation. Although there may thus prove to be a fairly rapid interconversion of protein between the galactose and maltose fermenting systems, this is probably not true of yeast proteins in general. This conclusion follows from the fact, mentioned earlier, that nitrogen starvation abolishes the capacity to adapt to galactose. Now it is unlikely that the two enzymes involved in the adaptation, galactokinase and phosphogalactoisomerase, constitute more than a small fraction of the total intracellular protein. If the cell proteins broke down at any significant rate, say 1% per hour, enough material should pass into the pool within an hour or two to provide ample material for the synthesis of the two adaptive enzymes. The fact that prolonged nitrogen starvation causes depletion of the pool could therefore mean that those proteins which are relatively unstable and which can revert rapidly to the amino acid pool have already done so during the period of nitrogen starvation, and the amino acids derived from them have been incorporated into more stable protein molecules (see Halvorson et uz., 1955). Interaction between two adaptive enzymes has not been demonstrated in bacteria. When Wainwright and Pollock ( 1949) examined tetrathionase (El) and nitratase ( G )from this point of view they found that there was no loss of El during subsequent development of Ea and the level reached by each enzyme was the same whether previous adaptation of the other system had occurred or not. More recently the problem has been investigated in an elegant series of experiments by Hogness sf d. (1955). The enzyme was /3-galactosidase which can be isolated from extracts of E. coli by the use of a specific antiserum. The bacteria were grown in the presence of SS6-labeledsulphate, without inducer, in a medium in which the sulphur was growth limiting. During this period all the sulphur in the medium became incorporated into the cell proteins which were consequently all labeled with radioactive sulphur. Non-labeled sulphur arid inducer were then added and, after a short period of adaptation, the j3-galactosidase was isolated and examined for radioactivity. If turnover of protein had taken place to any significant extent during adaptation, the enzyme should have contained radioactive sulphur derived from the other proteins of the cells. Instead, the radio-

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activity of the enzyme was negligibly low by comparison with p-galactosidase synthesized in the presence of labeled sulphur, indicating that virtually all the sulphur-containing amino acids in the adaptive enzyme were “new.” This experiment finally disposed of the possibility that the enzyme was formed from a pre-existing protein precursor. The stability of p-galactosidase was shown as follows. The enzyme was synthesized in nonlabeled medium, the inducer was removed by washing and the cells allowed to grow in the presence of radioactive sulphur. When the bacterial mass had increased tenfold it was found that (a) the total amount of enzyme had not altered although its concentration per cell had, of course, fallen to one-tenth; (b) the enzyme was not labeled with Sg6. This showed that the enzyme molecules were entirely stable during growth and that there was no exchange of material between them and the other proteins of the cell. The possibility remained that since the inducer catalyzed the synthesis of the enzyme it might also catalyze its breakdown. This possibility was eliminated by the following experiment : 8-galactosidase was synthesized in the presence of Sg6so that all the adaptive enzyme was fully labeled. The inducer was removed and the cells grown in nonlabeled medium until the bacterial mass had increased tenfold. During this period the specific radioactivity of the cell proteins generally, fell to one-tenth of the original value while the specific radioactivity of the p-galactosidase remained the same. Further adaptation, also in unlabeled medium, was then allowed to take place and the enzyme isolated and examined as before. Within the limits of experimental error it was shown that all the radioactive enzyme synthesized initially was still present although subsequently growth and further enzyme synthesis had occurred. The enzyme was therefore apparently just as stable in the presence of the inducer as in its absence. The authors drew the following conclusions from their findings: (a) pgalactosidase is a stable protein and its synthesis is irreversible; (b) all the other proteins of E . coli are also stable, i.e., there is no turnover of protein in the bacterial cell. The first conclusion is completely justified by the results presented and is supported independently by the work of Rotman and Spiegelman (1954) who used C14 instead of Sa6 and obtained very similar results with the same enzyme system. Rickenberg et al. (1953)also found that p-galactosidase was stable and during growth merely became diluted out in the increasing bacterial mass. A parallel observation had been made in the case of adaptive nitrqtase in E . coli (Wainwright and Pollock, 1949). The second conclusion regarding the stability of all the proteins in the cell is however an extrapolation which may not be true. In this context it is relevant to consider the work of Gale and Folkes (1954). When a

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cell-free extract of disintegrated staphylococci was incubated with adenosinetriphosphate, hexosediphosphate, and C14-labeled glutamic acid some of the glutamic acid became incorporated into the protein of the preparation. This took place through an exchange between glutamic acid in the medium and glutamic acid residues in the protein. The exchange reaction was not confined to glutamic acid but occurred with amino acids generally and it has also been demonstrated in intact staphylococci (Gale and Folkes, 1953). It is clear that if a pool exists, and if amino acids are exchanged between it and the proteins of the cell, the resultant state of dynamic interaction is not easily distinguished, experimentally, from protein turnover. There is not enough evidence to prove the occurrence of dynamic interaction of this type in coliform bacteria, but a free amino acid pool has now been demonstrated, and experiments with Sss-methionine suggest that E. coli may incorporate amino acids in much the same way as staphylococci (see Melchior et al., 1948 ; Melchior et al., 195 1). These observations are not necessarily in conflict with those of Hogness st d. (1955) for it is possible that the rate of amino acid exchange (or of protein turnover) in E. coli is negligibly low in comparison with the rate of growth. It is, however, also possible that considerable exchange or turnover occurs, but that the amino acid molecules involved are not used for the synthesis of 8-galactosidase. Reasons have already been given for believing that different enzyme-forming systems may differ in their ability to utilize the contents of the free amino acid pool. It is to be hoped that the experiments of Hogness et al. (1955) will be repeated on staphylococci. If these organisms were grown in labeled medium and then made to synthesize 8-galactosidase in non-labeled medium, the enzyme should contain some radioactivity. If it did not, it would mean that the 8-galactosidase system is unsuitable for detecting interaction between the proteins and the pool. O n the other hand, the formation of labeled enzyme would contradict the generalization from the experiments with E. coli and underline the differences between different types of microorganism. 4. The Role of the Indwer It is clear that the inducer must initiate adaptation by reacting with some receptor molecule which is probably specific. While the first and most obvious choice for the receptor is the enzyme molecule itself, in at least two instances-p-galactosidase and penicillinase-the evidence indicates that the receptor is something other than the enzyme. In an extensive and thorough investigation, Monod et al. (1951) used a series of substituted galactosides and compared affinity for ,3-galactosidase

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with ability to act as an inducer of the same enzyme. In general, substrates of the enzyme were also inducers but this correlation was not invariable. For example, o-nitrophenyl-a-L-arabinosidewas a substrate but not an inducer ; conversely some compounds like methyl-a-D-galactoside were inducers but not substrates. Furthermore there was no correlation between the potency of an inducer and its affinity for the enzyme. Thus, phenyl-bD-galactoside had a high affinity for the enzyme but was not an inducer, while some compounds like melibiose had little or no affinity for the enzyme but were nevertheless efficient inducers. The mode of action of the inducer in penicillinase production is also of great interest. Pollock (1950) found that cells of Bacillzcs cereus, treated with penicillin and then washed, could produce penicillinase apparently indefinitely and at an undiminished rate during subsequent growth in the absence of any free penicillin. The use of SWabeled penicillin (Pollock and Perret, 1951) showed that penicillin was fixed by the cells in two ways. There was a nonspecific fixation proportional to the concentration of penicillin in the medium, and a specific fixation having the form of an adsorption isotherm with a maximum value at 1.0 units penicillin per ml. The complex formed by the specific fixation appeared to be responsible for the subsequent penicillinase production, since the rate of enzyme synthesis was closely correlated with the level of SS5specifically fixed. A maximum rate of synthesis was obtained after the fixation of about 80 atoms of SS6 per bacterial cell, and appreciable synthesis was obtainable at even lower levels. Subsequently, when penicillinase was purified (Pollock and Torriani, 1953) and its turnover number estimated, it became clear that about twenty molecules of enzyme were produced per hour for every molecule of inducer initially fixed, that is, the inducer was acting as a catalyst for the enzyme synthesis. In this system, and in the case of 8-galactosidase, it is apparent that adaptation is not initiated by the enzyme-inducer complex and that the inducer must be combining with some other receptor molecule. The complex formed from penicillin and its receptor must be stable since enzyme synthesis continues at a steady rate for a long time in the absence of free penicillin and the labeled penicillin sulphur is retained in the cells. In the case of the galactosides the combination seems to be unstable, and removal of the inducer stops further synthesis of the enzyme. The chemical nature of the receptor is unknown but it must be presumed to be structurally similar to the enzyme molecule since it exhibits similar specific combining properties. Identification of the receptor, and a study of the complex formed between it and the inducer, might be of help in solving the problem of the specificity of enzyme synthesis. The receptor of B. cereus is a

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particularly suitable system for investigation because it forms a stable complex. Some work has been done on this problem using staphylococci which fix penicillin in the same sort of way (see Rowley et al., 1950; Cooper, 1955). The receptor appears to be associated with lipid particles and to be located at or near the cell surface. In the absence of more detailed information, it is impossible to know whether the inducer plays any part in conferring the specific configuration upon the enzyme molecule or whether specificity is determined by some cell constituent which is already present.

ACTION THEORY IV. THEMASS The mass action theory, which was proposed by Yudkin (1938), was the first attempt to present a scientific, as opposed to a teleological, explanation for enzyme adaptation. I t has considerably affected subsequent thinking in the subject and its influence on the theories of Spiegelman and Monod will become apparent. The basic assumption of the theory is that the adaptive enzyme E is formed from some precursor P with which it is in chemical equilibrium. Itl the case of most adaptive enzymes the equilibrium is considered to lie in the direction of the precursor so that the basal activity is very low. When the substrate is added, it combines with the enzyme; the equilibrium is upset and production of E takes place to restore the equilibrium. The adaptation thus represents the expansion of an existing system rather than the formation of an entirely new molecular species. This point is sometimes difficult to prove experimentally but, where sufficiently refined methods of assay have been used, some basal activity has usually been detected. It follows from the theory that adaptation could be caused by compounds other than the substrate, provided that they can combine with the free enzyme molecules. Yudkin therefore suggested that the end products of an enzyme reaction might, in some instances, be able to act as inducers, and the same property was postulated for other competitive or noncompetitive inhibitors of enzyme action. A further prediction made was that the rate of adaptation should be proportional to the degree of saturation of the enzyme by the inducer. These predictions will be considered in turn. When the theory was published some instances were already known where enzyme adaptation was apparently caused by the end product of the reaction. Thus lactase of yeast was increased by galactose as well as lactose (Dienert, 1900), an observation that was later confirmed in E. coti (Monod et al., 1951). Fructose and glucose were inducers of invertase in yeast (Euler and Cramer, 1913). More recent examples are the adaptive

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formation of benzaldehyde dehydrogenase in response to benzoic acid (Stanier, 1947) and of succinic dehydrogenase in response to fumaric acid (Karlsson and Barker, 1948). Although some of the earlier experiments may be invalid because of possible contamination of the reagents, particularly when sugars were used, this is much less likely in the more recent work. A more serious objection can be raised on the grounds that the end product of the reaction is not itself the inducer but that it is metabolized to the natural inducer. This would certainly happen when the enzyme reaction is reversible and could, in any case, occur in a less direct fashion. Since it is known that very low concentrations of inducer may be effective it is difficult to eliminate this possibility. However, if these findings are taken at their face value they are consistent with the mass action theory though they do not prove that the inducer acts by upsetting the equilibrium between E and P. The work on penicillinase and P-galactosidase shows that, in these systems, the inducer combines primarily with something other than the enzyme and its function in the former is catalytic and not stoichiometric as would be required by the mass action theory. The kinetic prediction, that the rate of adaptation should be proportional to the degree of saturation of the enzyme, obviously does not hold for the penicillinase and ,B-galactosidase systems, and very few attempts have been made to test it in other systems. The synthesis of lysine decarboxylase in Bact. cadaveris has been studied from this point of view (Mandelstam, 1954). The enzyme was produced equally well aerobically and anaerobically by washed suspensions of cells. Under anaerobic conditions the rate of synthesis of the enzyme was directly proportional to the degree of saturation of the enzyme by lysine, but aerobically a maximum rate of adaptation was obtained at such low concentrations of lysine that the enzyme was almost completely unsaturated. The anaerobic experiments suggest that the inducer causes adaptation by combining with the enzyme, while the aerobic experiments suggest that it combines with something else. A number of explanations for the apparent discrepancy were considered, including permeability of the cells to lysine under the two conditions, stability of the enzyme, etc. These were eliminated experimentally so that the apparent anomaly has not yet been resolved. A similar study of the kinetics of maltase adaptation in yeast has been made by Spiegelman and Halvorson (1954). The activity of a-methylglucoside as an inducer was found to be approximately 250 times greater than its affinity for the enzyme. This marked difference was quoted as evidence against the hypothesis that the inducer acts primarily by combining with the enzyme, and the existence of some other receptor substance

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was postulated. Now these kinetic experiments, and those of Mandelstam, are unequivocal only if the enzyme synthetic pathway is itself not rate limiting in the conditions of the experiment. If it is rate limiting, then the rate of synthesis of the enzyme cannot be used to measure the affinity of receptor for inducer because enzyme production may be maximal even when the receptor is only partially saturated. In the absence of information regarding the rate-limiting step in the enzyme synthesis, a close positive correlation between the rate of synthesis and the degree of saturation of the presumed receptor may be significant, or is at any rate a remarkable coincidence, a negative correlation by itself is inconclusive. This point does not seem to have been considered by Spiegelman and Halvorson who have merely shown a negative correlation without any evidence that the synthetic pathway is not itself rate limiting. While their conclusion may well prove to be correct it is not justified by the experimental evidence so far presented. A fuller assessment of the mass action theory will be given later. It will suffice at this stage to point out that it differs from most subsequent theories in that only one type of specific molecule is postulated, i.e., the enzyme itself; the precursor, as Yudkin stated, is not necessarily considered specific, As we have seen, this concept is insufficient to account for the adaptive formation of penicillinase and of 8-galactosidase, and another specific type of molecule is presumably present in both these systems. The inducer, in the mass action theory, is presumed to have the essential role of stabilizing the adaptive enzyme and so preventing its reversion to the precursor state. This idea is one of the main assumptions of the next two theories to be discussed.

V. THEPLASMAGENE THEORY The plasmagene theory (Spiegelman, 1946) was designed mainly to cover the followiqg points : 1. Competitive interaction between the galactose and maltose fermenting systems of yeast. 2. The sigmoid shape of the adaptation curve. 3. The finding that yeast cells, maintained in the presence of the inducer, could continue to synthesize the adaptive enzyme although they had in the meantime lost the requisite gene. The first of these phenomena has already been outlined; the other two require some description. Spiegelman had noted that the curve representing adaptation to galactose or maltose was sigmoid in form and resembled the growth curve of an autocatalytic system such as a culture of growing bacteria. Now, if it is

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assumed that the adaptive enzyme (E) is part of a self-duplicating molecule, then its production from precursor (P) is autocatalytic and is represented by the equation

-

E =

-

P 1

+ ea-kt

where P is the amount of enzyme finally formed, and a and k are constants. The experimental values were found to agree very closely with those calculated from the equation, and this agreement was adduced in support of the self-duplication hypothesis. The genetic experiments are somewhat more complicated. Lindegren et al. (1944) had obtained a hybrid strain of yeast by crossing Succhuromyces cerevisiae (unable to adapt to melibiose) with S. curlsbergensis (able to adapt to melibiose). The hybrid was phenotypically positive for adaptive melibiose fermentation due to the dominant nature of the fermentative capacity. When a second hybrid was obtained by mating a spore of the first hybrid with a negative haploid of S. cerevisk, the resulting diploids segregated to give a 1: 1 ratio of fermenters to nonfermenters. Spiegelman (1946) reported that this ratio was found if the segregation occurred in the absence of melibiose, whereas in the presence of melibiose all the spores could adapt to melibiose and this capacity was retained indefinitely provided that the organisms were always grown in the presence of this sugar. On removal of the melibiose two out of every four spores irreversibly lost the ability to form the adaptive enzyme. Similar behavior of the galactose adaptive system was exhibited in hybrids derived by mating S. cerevisiue with S. bayanzcs. These results pointed to the conclusion that although only two spores in every four had the essential gene, all four could produce the enzyme by virtue of a self-duplicating cytoplasmic enzyme-forming system which remained functional as long as the substrate was present. To account for these findings, the following theory, shown schematically in Fig. 1, was proposed. Each gene is assumed to produce plasmagenes which are more or less complete replicas of itself. These are formed continuously and enter the cytoplasm, where being potentially self-duplicating, they will, like all self-duplicating systems, compete for material and energy. The plasmagene, once formed, may decay to inactive protein ( I P ) or, if suitable material (M) is available, it may duplicate itself. Alternatively it may combine with precursor ( P r ) converting it to enzyme ( E l ) and SO give rise to the plasmagene-enzyme complex (PllE1). Since normally there is very little adaptive enzyme it is assumed that PllE1 breaks up into its component parts and that the El thus liberated decays to IP. If the sub-

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strate (S1)is added it combines with El. This stabilizes the enzyme and diminishes its rate of decay. Also S1 can combine with El while it is still part of the complex PIIEI; this stabilizes the complex. Spiegelman then argued that “inherent in the very definition of a self-duplicating entity is the concept that, should such a unit undergo a modification at any given moment, all subsequent replicas would bear this modification.” On this basis when PI1 exists alone it duplicates only Pll, but in the presence of

FIG.1. Plasmagene theory of enzyme adaptation (Spiegelman, 1946). G,, gene;

PI,, plasmagene; Pr, enzyme precursor; El,enzyme; S,, substrate; M, cytoplasmic material for duplicating plasmagenes; K is a constant measuring the rate of production of plasmagenes from genes. Broken arrows indicate decay to inactive protein (IP). The major tendency of a reversible reaction is shown by the longer of the two arrows. Double arrows indicate self-duplication.

the substrate the whole complex PllElSl is duplicated. The substrate therefore causes the formation of a new self-duplicating entity which reproduces the plasmagene as well as the enzyme. This model postulates no less than three self-duplicating entitiecthe gene, the plasmagene, and the plasmagene-enzyme-substratecomplex. Much of the complication of the theory is due to, and can only be justified by, the attempt to explain the genetic experiments. Unfortunately, the facts themselves are in some doubt. Lindegren and Lindegren (1946) carried out further experiments on continued exposure of yeast hybrids to the adaptive substrate. Their results were negative in that they were unable to show that continued exposure to the substrate produced any significant increase in the number of asci containing four fermenting spores. A further fact upon which the hypothesis of selfduplication rests is the sigmoid (autocatalytic) nature of the adaptation curve. In this connection it must be stressed that, while adaptation curves may often have this shape, they are not inherently autocatalytic. Thus Pollock (1950) has obtained a linear

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rate of penicillinase production ; Mandelstam and Yudkin (1952) found that the curve of galactose adaptation could be either linear or autocatalytic, and Ephrussi and Slonimski (1950) found a falling rate of production for cytochromes of yeast. In any case, the occurrence of an autocatalytic curve does not prove the existence of self-duplicating molecules. Monod (1947) pointed out that when the adaptive enzyme itself produces the energy for its own further formation its rate of appearance will necessarily be autocatalytic. Hinshelwood (1952, 1953a) and Mandelstam (1952) have both described in detail how the rate of formation of an enzyme may be autocatalytic although the enzyme molecules are not self-duplicating. Finally, with regard to competition between different enzyme-forming systems, it is apparent that this could be caused by any shortage of material or of energy and would then occur whether the enzyme-forming system were self-duplicating or not. More recently it has been claimed by Spiegelman and his colleagues that the behavior of “slow adapting” yeast cells (see Winge and Roberts, 1948) supports the plasmagene hypothesis. A strain of Succhuromyces chevulieri was used which adapted to galactose fermentation in about seven days. The cells retained their positive character, i.e., ability to ferment galactose rapidly, provided that they were grown in a galactose medium. With growth in the absence of galactose, positive cells were produced for about five generations when a “burst” of negative cells appeared in the culture (Spiegelman et ul., 1950). A closer examination of the reversion to the negative form was carried out by Spiegelman et at. (1951). A positive cell was grown in dextrose, and the buds were tested for the positive character. The first five generations from a positive cell were always positive. At about the sixth or seventh generation the buds became negative and the mother cell itself lost its positive character. The results were interpreted as evidence for the existence of cytoplasmic enzyme-forming particles which could increase in the presence of the substrate but not in its absence. The statistical data were consistent with the assumption that a positive cell contained roughly 100 such particles of which about half were transferred to the daughter cell at each cell division; the possession of one such particle might be sufficient to confer the positive property. The experiments are of considerable interest but they are still open to the criticism made by Monod and Cohn (1952). This was to the effect that, while it had been shown that positiveness is conveyed by discrete particles which can be “diluted out” during cell division, it had not been shown that these particles are not the actual enzyme molecules ; the possession of even a single molecule of enzyme might suffice to allow the cell to utilize galactose

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when placed in a galactose medium and would lead to the autocatalytic synthesis of more molecules of enzyme. VI. THE SPECIFIC PRECURSOR THEORY An attempt was made by Monod (1947) to formulate a theory of enzyme adaptation which would take account of the genetic control of the ability to produce any particular enzyme, and which would also explain the phenomenon of diauxie (see Monod, 1945, 1947). Diauxie was observed when coliform bacteria were grown in the presence of two sugars, one of which (A) could be utilized constitutively and the other (B) adaptively. The growth curve exhibited two distinct phases. In the first phase, substrate A was metabolized exclusively until it was exhausted. Growth ceased, and there followed a lag period during which the bacteria adapted to substrate B, which was then utilized for the second phase of growth. Apparently the presence of A prevented adaptation to B. With a suitable mixture of substrates such as glucose, sorbitol, and glycerol a growth curve exhibiting three phases could be obtained, each phase corresponding to the complete utilization of one substrate. Diauxie was shown to be a quantitative rather than a qualitative effect. With a mixture of glucose (A) and xylose (B) the form of the growth curve depended on the ratio A / B . With A / B = I, diauxie was marked ;with A / B = 1/10, the lag period separating the two phases was decreased ; and with A / B = l/lW, it was abolished so that a normal monophasic growth curve was obtained. It therefore appeared that, with a suitable ratio, adaptation to xylose could occur although glucose was still present. Diauxie was at that time attributed by Monod to a competitive interaction between enzyme-forming systems but is now believed to be due to a permeability effect in which glucose prevents the penetration of the adaptive substrate. Diauxie will be referred to again in connection with the organizer theory. With regard to the genetic control of enzyme formation Monod (1947) described experiments in which strains of E. coli were plated on media containing sugars not normally fermented by the strains. The occasional large colonies of mutants which appeared were isolated and found to differ from the parent strains only in their behavior towards the one sugar concerned. Apparently, therefore, a single mutation affected a single fermentative capacity and this one gene-one enzyme relationship was consistent with the facts of genetical inheritance of the ability to ferment sugars in yeast. Monod argued that acceptance of the hypothesis that the specific properties of an enzyme were determined by a single gene, was operationally equivalent to assuming that the gene conferred its specific pattern directly

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by forming some (cytoplasmic) replica or partial replica of itself. The inducer then acted by stabilizing the enzyme molecules that had been formed. Schematically the theory can be presented as follows :

Gi Bi Ei

GI is the gene and

+

Bi

+ 2i + iBi (=

+ Si + EiSi

El)

its cytoplasmic replica which is also the specific precursor of the enzyme. This combines with nonspecific precursor molecules, i, to give the active enzyme molecule El which is stabilized through combination with S1. Enzymes are assumed to be derived from a common pool of precursors and, unless they are stabilized by their substrates, they undergo inactivation and their material returns to the pool. Competition between enzymeforming systems arises when the material in the pool is limited. The similarities between this theory and those previously discussed are at once apparent. The specific precursor theory combines the stabilizing function of the substrate (derived from the mass action theory) with the genetic production of specific enzyme precursors (derived from the plasmagene theory). Since the precursor molecules are not endowed with selfreplicating properties the theory is not open to the same objecti’bns as the plasmagene theory, but it has the same essential limitations as the mass action theory and has since been abandoned by its author in favor of the organizer theory. B1

VII. THEORGANIZER THEORY (1) Reasons have already been given for believing that penicillin acts as an inducer of penicillinase by combining irreversibly with a specific receptor in the cell. The complex so formed appears to act catalytically because a number of molecules of enzyme are produced for every molecule of inducer originally fixed. Since the adaptation continues at an undiminished rate for a long period in the absence of exogenously supplied penicillin, and in the presence of large quantities of active enzyme, it is clear that the “real” inducer cannot be free penicillin. It was accordingly suggested by Pollock (1953a) that the active inducer is either the penicillin-receptor complex or some metabolic derivative of it. In a detailed consideration of the adaptive formation of penicillinase, Pollock distinguished three separate stages. The first stage consists of the specific fixation of penicillin by the receptor of the cell. The reaction is rapid, being half completed in one minute at 35°C. (Pollock, 1952). The next stage is a “latent” period of 14 minutes during which the free

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adaptive enzyme begins to appear. This is followed by the “linear” stage during which enzyme is synthesized at a constant rate. The initial stage, in which penicillin is fixed by the receptor, appears to be independent of the cell metabolism. During the second stage, the reactions which occur depend upon active metabolism and they can be stopped by the withdrawal of oxygen. If oxygen lack is imposed at the beginning of the latent period there is still a lag of 14 minutes after restoration of air before the linear stage is reached. The latent period could be the time needed to metabolize the inducer to its active form, or it could be the time needed to establish a steady state concentration of a specific enzyme precursor. The latter possibility is supported by the following piece of indirect evidence. If, during the linear phase, the cells are rendered anaerobic, growth and penicillinase production stop immediately. When oxygen is restored, there is a short period during which the rate of enzyme synthesis is markedly greater than it was initially; growth and oxygen consumption do not show this transient burst of activity after anaerobiosis (Pollock, 1953b). The experiments are consistent with the assumption that, during anaerobiosis, some more or less specific precursor accumulates. Inducer+ receptor (1 specific) ? Stages

‘Mbtabolisme des inducteun’

I

t

- - “Organizer’

Amino-

I

)

I

Precursor type 1

@

? Precursor type 2

Enzyme

FIG.2. Organizer theory of enzyme adaptation (Pollock, 1953a). For explanation see text.

These facts are summarized in the scheme shown in Fig. 2 (Pollock, 1953a). The “real” inducer is termed the organizer and is held to arise from the penicillin-receptor complex, possibly as a result of some metabolic process. The organizer is held to retain the specificity of the original inducer and to act as a catalyst for the formation of enzyme. Its nature is not further specified, but it could be regarded as a template which determines the specific properties of the enzyme molecule. The organizer theory differs from the mass action theory in two essential respects. Firstly, the action of the inducer is catalytic and not stoichiometric and, secondly, the existence of adaptive enzyme before addition of

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the inducer is not postulated. The gene would, by implication, be concerned in the production of specific receptor molecules. The theory schematized in Fig. 2 is a generalized statement of the known facts regarding adaptive penicillinase formation. While it may well be true of other adaptive systems, its general validity might be difficult to prove because of the technical difficulty of demonstrating the catalytic role of the inducer in any system where the continuous presence of the inducer is necessary for enzyme synthesis. In all such systems the possibility remains that the inducer is acting stoichiometrically. VIII.

THEORGANIZER THEORY (2)

A modification of the organizer theory was proposed by Cohn and Monod (1953) which is essentially similar to Pollock's theory, but which contains additional assumptions to cover the constitutive synthesis of enzymes and the inhibition of enzyme adaptation. The experiments on constitutive synthesis were based on the unitary hypothesis of enzyme formation, namely, that all enzymes are adaptive, but that some appear to be constitutive because the inducer is a normal cell metabolite and is consequently always present. Now, in cultures of E. coli which could produce p-galactosidase adaptively, mutants occurred which produced the enzyme constitutively. These were isolated by an enrichment technique in which the cells were grown alternatively in glucose and lactose (Cohen-Bazire and Jolit, 1953). I t follows from the unitary hypothesis that the constitutive mutants synthesize an endogenous inducer of p-galactosidase which is similar in structure to the compounds that act as inducers in the adaptive strain. Furthermore, it might be expected that structural analogs which inhibit adaptive formation of the enzyme would also inhibit constitutive formation. Accordingly suitable analogs were tested for their effect on the constitutive synthesis of p-galactosidase. Monod and Cohen-Bazire (1953a) reported some curious results which may be summarized as follows: 1. Thiophenol-p-galactoside, which is a competitive inhibitor of adaptive synthesis, had no effect on constitutive synthesis. 2. a-Galactosides, which are not substrates but some of which are inducers, had no effect on constitutive synthesis. 3. Galactose, the product of the enzyme action, and an inducer in the adaptive strain, inhibited constitutive synthesis. 4. p-Galactosides, which are substrates of the enzyme and also inducers, were inhibitors of constitutive synthesis. 5. Glucose, which causes a diauxic inhibition of adaptive synthesis, had no effect on constitutive synthesis.

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The extraordinary finding that the substrate of an enzyme, or the end product of enzyme action, may inhibit constitutive synthesis is not confined to @-galactosidase, for Monod and Cohen-Bazire (1953b) have demonstrated a similar reaction with tryptophan desmolase of Aerobacter aerogenes. This enzyme catalyzes the reaction indole

+ serine + tryptophan

The synthesis of the enzyme was markedly inhibited (65-70%) by the addition of either indole or tryptophan to the growth medium. The effect was fairly specific; other amino acids tested stimulated enzyme synthesis except glutamic acid and proline which were weakly inhibitory. Another example of the phenomenon was found in the methionine synthase of E. coli, an enzyke which catalyzes the synthesis of methionine. The production of this enzyme is virtually stopped altogether if the cells are grown in the presence of methionine. Although most amino acids had some stimulatory or inhibitory effect, none approached in magnitude that of methionine (Cohn et al., 1953a). The scheme represented in Fig. 3 is an attempt to account for the constitutive and adaptive synthesis of @-galactosidaseand for the various types

4*

AminWacids

Organizer

Enzyme

FIG.3. Scheme for adaptive and constitutive synthesis of @-galactosidasein Eschericlaia coli (Cohn and Monod, 1953). The various types of inhibition that may occur are as follows: (1) Diauxic inhibition of adaptive synthesis. (2) Inhibition of adaptive synthesis by thiophenol-&o-galactoside. (3) Metabolic inhibition, present in adaptive strains, absent in constitutive strains. (4) Inhibition of constitutive SWthesis by @-galactosides.

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75

of inhibition that may occur. Diauxic inhibition of adaptive synthesis is attributed to a permeability effect in which the penetration of the inducer is inhibited (e.g., by glucose). This is shown by a block (block 1) between the exogenous inducer and the cell wall. If the inducer penetrates, it is metabolized to the co-organizer. This is the stage at which thiophenol-pgalactoside is assumed to inhibit the adaptive process (block 2). In the constitutive strain, internal inducer is synthesized by the cell and metabolized to the same co-organizer; in the adaptive strain this reaction cannot occur (block 3). In either case, once the co-organizer has been formed it combines with a specific structure, the apo-organizer, thereby giving rise to the active catalyst for the synthesis of the enzyme. This stage in the formation of the organizer is inhibited in the constitutive strain by the substrates of the enzyme (block 4). The reaction which results in the formation of the organizer is represented as reversible to allow for the fact that the synthesis of adaptive 8-galactosidase stops on withdrawal of the external inducer. The general applicability of the organizer concept has already been discussed and the comments made in the preceding section apply equally to the present system. However, Cohn and Monod (1953) have attempted to include constitutive synthesis and its inhibition by the substrate of the enzyme. This scheme, which combines the organizer concept with the unitary hypothesis of enzyme formation, has genetical implications which could, in principle, be verified experimentally. Microorganisms may be classified as follows with reference to their ability to form any particular enzyme: (a) negative strains which cannot form the enzyme at all; (b) adaptive strains which form the enzyme when inducer is added; (c) constitutive strains which form the enzyme without added inducer. In terms of the organizer hypothesis it must be presumed that a negative strain is unable to form the specific receptor ; an adaptive strain possesses the receptor but is unable to form sufficient inducer ; a constitutive strain forms both. These capacities seem to be genetically determined, and a change from one type to another can occur as a result of a single mutation. An example, already quoted, is the formation of constitutive mutants by a strain of E. coli which synthesizes p-galactosidase adaptively. Similarly, constitutive penicillinase mutants have been isolated from an adaptive strain of B. cereus by Pollock (1955). If R+ and I+ denote the ability to form the receptor and the inducer, the following combinations are possible : (1) R- I- (negative) (2) R- I+ (negative) (4) R+ I+ (constitutive) (3) R+I- (adaptive)

76

J. MANDELSTAM .

The production of constitutive mutants (Rf I+) by an adaptive strain (R+ I-) would then measure the probability of the I- + I+ mutation. Similarly the production of adaptive mutants by a negative population (R- I-) would measure the probability of the R- + R+ mutation. The product of these values should determine the probability of obtaining R+ I+ from R- I-. Negative strains of the type R- I+ should, in principle, be obtainable and they should have the characteristic property of giving rise primarily to Constitutive mutants at a rate determined by the frequency of the R- + R + mutation. In addition, it should be possible to show that these organisms are capable of synthesizing the endogenous inducer. By contrast, the other type of negative strain R- I- should tend to produce adaptive mutants and should lack the endogenous inducer. The demonstration of mutant strains of the same organism with the postulated properties would provide supporting evidence for the theory of Cohn and Monod. The problem created by the fact that the same compound can cause adaptive synthesis and inhibit constitutive synthesis could obviously be resolved by abandoning the unitary hypothesis. Cohn and Monod, seeking to retain the hypothesis, suggested that the inhibitors of constitutive synthesis block the formation of organizer by virtue of their structural similarity to the co-organizer. This assumption does not really dispose of the difficulty, because if the co-organizer is identical in the adaptive and constitutive systems it is difficult to see why its combination with apoorganizer should be inhibited in the one case, but not in the other. Some additional properties would have to be postulated ad hoe to account satisfactorily for the differences between the constitutive and adaptive systems. In the absence of further information it is difficult to know what weight should be attached to this objection to the unitary hypothesis. Reference to the findings on the inhibition of the synthesis of constitutive /3-galactosidase will show that inhibition is produced by galactose and by the substrates of the enzyme which all give rise to galactose. The inhibition coufd therefore be due to the substrates themselves, or to the galactose which is formed from them, or to some metabolic derivative of galactose, A similar argument will apply to the inhibition of the synthesis of tryptophan desmolase and of methionine-synthase. The evidence so far available does not distinguish between the various possibilities and more detailed information is desirable. IX. THE ORGANIZER THEORY (3) Some highly interesting experiments have been carried out by Gale and Folkes (1954, 1955) on protein synthesis and enzyme adaptation in cellfree extracts obtained from supersonically disintegrated staphylococci. The extracts consisted of “broken cell envelopes” from which most of the nucleic

77

ENZYME ADAPTATION IN MICROORGANISMS

acids could be removed by extraction with NaCl solution followed by treatment with ribonuclease (RNase) and deoxyribonuclease (DNase) If such a preparation was incubated with C14-labeled glutamic acid, adenosinetriphosphate (ATP) , and hexosediphosphate ( H D P ) , the labeled glutamic acid became incorporated by exchange with glutamate residues in the proteins. The process was not accompanied by any net synthesis of protein. When a complete mixture of amino acids was added, the incorporation of glutamic acid was accompanied by a parallel increase in protein. The rate of incorporation by preparations depleted of nucleic acid could be considerably increased by addition of either RNA or DNA, the latter being somewhat more effective. The synthesis of the following enzymes by the cell-free preparations was also investigated : catalase, the enzymes which produce acid from glucose, and adaptive p-galactosidase. In contrast to the other enzymes, p-galactosidase was only formed in the presence of a suitable inducer, e.g., galactose. When preparations which were only partly resolved (i.e., still contained appreciable amounts of nucleic acid) were incubated with galactose, ATP, HDP, and amino acids, synthesis of p-galactosidase took place. The rate was increased by the further addition of a purine-pyrimidine mixture ( P P ) . In more highly resolved preparations, incubation with galactose, ATP, HDP, and amino acids did not produce much enzyme and PP had no stimulatory effect. However, addition of DNA restored enzyme synthesis and the response to PP. This effect of DNA depended upon the presence of RNA and was abolished by treatment with RNase. Experiments with C14-labeled uracil showed that the cell-free preparations synthesized nucleic acid and that the rate of synthesis was markedly increased in the presence of galactose. Penicillin inhibited the incorporation of C14-uracil and the synthesis of 8-galactosidase to about the same extent. In the light of these results the authors suggested the following scheme for enzyme synthesis. The initial organizer or template is DNA, possibly in combination with protein, which determines the formation of specific RNA. The RNA then catalyzes the synthesis of the enzyme. The rate a t which the enzyme is synthesized depends upon the amount of specific RNA in the system. The initiatory role of DNA is shown by the fact that preparations treated with DNase will not synthesize 8-galactosidase, and that the ability to do so is restored by adding DNA. The response to PP is accompanied by the synthesis of RNA whose essential role in the synthetic process is shown by the fact that RNase abolishes the response to DNA and PP. If these findings are shown to hold for enzyme-forming systems generally, the essential role of nucleic acids in enzyme formation, often suggested by

.

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J. MANDELSTAY

earlier workers, will be confirmed and a more precise chemical definition may be found for the terms “organizer” and “receptor.” In addition, the use of cell-free extracts should be of value in solving problems, like that of diauxic inhibition, where permeability factors arise.

X. THEKINETICMODEL Hinshelwood‘s kinetic theory of the bacterial cell has been developed in a series of publications (see Hinshelwood, 1946, 1952, 1953a, 1953b). The kinetic model is not specifically designed to account for enzyme adaptation but is a generalized model of the bacterial cell in which adaptation is one of the properties. In the simplest case it is assumed that there‘ are two substances (enzymes) X and Y each of which grows by incorporating a substance produced by the action of the other. Thus dX dY - = aY and - = PX dt dt These equations yield the following solutions:

+ %(XO - -ak Yo)e-kt

a

X = S(X0

+

Y = %(Yo

+Xo)@ + %(Yo - - X o ) r k t a a

k

(la)

k

(1b)

Where X Oand Yo are the initial values of X and Y and k is defined by the relation ap = ka. When t is large enough, the terms in e-kt become negligibly small and the ratio of the two enzymes is given by

X/Y

= (XO

+

a

Yo)/(Yo

+ -k XO) = a/k

(2)

a

If at this stage the bacteria are assumed to be subcultured, the X and Y of

&. (2) become the initial values (i.e., X o and Y o ) for the new system so that

Xo/Yo = a/k Equations ( la) and (lb) now reduce to the simple relationships X . = Xoent and Y = Yoekt (3) and each of the two enzymes increases as though it were autosynthetic. This argument has been developed and shown to hold for systems comprising more than two components, It has also been shown that the sequence need not be linear but can be of the following type

ENZYME ADAPTATION IN MICROORGANISMS

I Y1

JI

79

+ I Xi

c--------R

where X I and Y1 are enzymes whose formation is dependent on that of R( = DNA). Both X I and Y1 contribute material for the further synthesis of R. In all these systems it can be shown that the following will occur: (a) The proportion of the various constituents will become automatically adjusted in such a way that the rate of growth is maximal. (b) In the steady state thus attained each constituent will be apparently autosynthetic so that X = X O @ ~R, = Roekt,etc. (c) In the steady state all components of the cell will be in constant ratios to one another. If an inhibitor is added which reduces the rate of formation of a cell component essential for growth, it can be shown by a simple calculation that an adaptation will occur automatically to overcome the inhibition and a new steady state will arise in which the initial growth rate is restored. The basic premise of the theory is that the cell consists of a linked system in which enzymes provide substrates for one another and in which each enzyme contributes ultimately to its own formation and to that of all other components of the system. Although this picture may be considered oversimple, it nevertheless corresponds essentially to what is known about bacterial metabolism and is certainly justifiable as a first approximation. Hinshelwood's theoretical studies have been of value in that they have developed the logical consequences of this picture and shown the type of adjustments that might occur in an autosynthetic linked system of enzymes. Unfortunately, single enzyme systems have not been studied from this point of view, so that it is not possible to state whether the kinetic model has any practical application. Hinshelwood and his collaborators have generally used growth rate as a measure of adaptation, and they have often ascribed to enzyme adaptation changes that are more probably due to mutation and selection. Two examples are the development of drug resistance and the development of the ability to ferment sugars over a long period of time (e.g., in E. coZi mutabile) With regard to drug adaptation, Baskett (1952) reported that, if frequent additions of small amounts of proflavine were made to a growing culture of B. lactis uerogenes, the cells continued to grow and, in the space of a few hours, the concentration of the drug could be raised to a level many times higher than that required to inhibit the growth of the original popuIation. Because of the short duration of the experiments, mutation and selection could not have occurred and the resistance was therefore attributed to a metabolic adaptation. However, recent work does not support these

.

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J. MANDELSTAM

conclusions. Sinai and Yudkin (1955) have confirmed the effects described by Baskett, but showed that they were due to inhibition of the antibacteria1 effects of proflavine by the metabolic products of the growing culture. The organisms at the end of the experiment were no more resistant to proflavine than they had been at the beginning] and the medium in which they had grown now supported the growth of “unadapted” cells even though a very high concentration of drug was apparently present. In addition, Thornley and Yudkin (1955) have been able to demonstrate] by the replica plating technique (see Lederberg and Lederberg, 1952), the spontaneous occurrence of proflavine resistant mutants. The study of such complex phenomena as the development of drug resistance and the behavior of mutabile strains provides no basis for an adequate assessment of the kinetic model. Nevertheless, Hinshelwood’s premises are in conformity with what is known of cell metabolism, and adaptations of the type predicted should be observable in suitably chosen systems. One of the advantages of a theory that can be stated precisely and unambiguously is that the conditions in which it is applicable are clearly defined. It is apparent that the assumptions underlying the kinetic model restrict the situations in which it might be valid. Firstly, the adaptations are adjustments within a system that is growing autocatalytically, and the model cannot therefore apply to adaptation in nongrowing suspensions. Secondly, it is assumed that each of the enzymes in the system provides material for its own growth and that of the other members of the system. Consequently, application of the model is excluded in instances where the product of the enzyme action is not further utilized by the cells, or where the inducer is not a substrate. These restrictions will exclude a considerable proportion of the phenomena studied under the heading of enzyme adaptation. While the kinetic model will not therefore serve to explain enzyme adaptation in general, it might still be a valid description of the behavior of bacterial enzymes in certain conditions. It is possible that adjustive changes of the type predicted are more likely to be found in enzyme systems that are usually classed as constitutive. For example, if bacteria were grown on one sugar and then transferred to a medium containing another which they can also utilize constitutively, the ratio of the enzymes concerned might be found to alter in conformity with the predictions of the theory. XI. THEEXTENDED MASSACTIONTHEORY The extended mass action theory (Mandelstam, 1952) is a generalized statement of Yudkin’s equilibrium theory. The basic assumptions are that the enzymes and other proteins of the cell are in dynamic equilibrium with

ENZYME ADAPTATION I N MICROORGANISMS

81

one another through a common pool from which they are all synthesized] and that the end product of an enzyme reaction is partly catabolized and partly contributed to the pool. The pool is assumed to contain all materials needed for the synthesis of proteins-amino acids, energy source, vitamins] inorganic ions, etc. Constitutive and adaptive enzymes are assumed to be formed in the same way and to differ only quantitatively. The model, which is schematically shown in Fig. 4, represents a balanced metabolic system in which, for example, the proteins Prl and Pr2 will

I

Catabolism

FIG.4. Mass-action model of enzyme adaptation (Mandelstam, 1952). B, pool from which proteins are synthesized; Pr,, Pr,, Pr,, proteins; P, and P2,enzyme precursors; El and E,, enzymes; S, and S2, substrates; K,, K,, etc., are velocity constants. Broken arrows indicate that a number of reactions may be involved.

be in a fixed ratio to one another and to all other proteins of the cell. The ratios of the various proteins are determined by the equilibrium constants concerned. The model is not restricted to enzyme adaptation but can be applied to other processes. For instance, growth of microorganisms is explained as follows: Let S1 be the sole source of carbon and energy, and El the first of the enzymes concerned with its metabolism. (For purposes of this illustration Elmay be either constitutive or adaptive ; in the latter case its initial concentration will be very small.) A part of the products derived from Slthrough the action of El will pass directly, or after further metabolism, to the pool B. If S1 is present in excess, its concentration can

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J. MANDELSTAM

be regarded as constant and the rate of “feed back” of products to B is then directly proportional to E l . Since an increase in B leads to an increase in E l , and this in turn increases the flow of material to B, it follows that the rate of growth of Elis proportional to its own concentration, i.e., dEl/dt = kE1 and El = E1(O)eLt (4) where El,o, is the initial concentration of E l and k is a constant. Since the other proteins of the cell are also formed from B, the growth of each of them, and that of the culture as a whole, will be represented by similar autocatalytic equations. It should be noted that although the model differs from that of Hinshelwood, the same growth equations are obtained (cf. Eqs. (3) and (4)>. Enzyme adaptation can also occur in the absence of growth and, in terms of the model, this would mean that either the products of metabolism of S1 were not “fed back” to the pool, or that some other essential metabolites or growth factors were lacking. In such a system, when S1 is added it combines with El and thereby disturbs in turn the equilibria between PI and El and hence between P1 and B. The system tends to restore the equilibria by synthesizing further enzyme at the expense of B. If the nitrogen thus removed from B can be easily replenished, e.g., from an exogenous source, the pool can be regarded as a reservoir whose contents are always adjusted to a constant level. A simple calculation then shows that a steady state concentration of enzyme will be established whose value depends on the concentration of the substrate. The relationship is given by

= %(Sb -k

-I- KID)

(5) where e, and eb are the steady state concentrations of enzyme produced, in eb

Km)/(Sa

response to two different concentrations of substrate S,, and sb;KM is the Michaelis constant. The equation indicates that the adaptive enzyme will increase linearly with the concentration of the substrate. In starved cells the material in the pool is restricted in that it can only be replenished from the cell proteins already present. The relationship between enzyme and substrate now becomes

where K is a constant and the other terms are as defined above. A plot of enzyme produced against substrate concentration now gives a falling curve. The theory has also been applied to the problem-of interaction between

ENZYME ADAPTATION IN MICROORGANISMS

83

enzyme-forming systems. If starved cells are forced to synthesize two adaptive enzymes simultaneously there will be competition for the material in the pool. The situation, in terms of the model, is complex, and fluctuations in the levels of the two enzymes are predictable on theoretical grounds. However, calculation shows that the ratio of the enzymes should tend towards a value defined by the equation

edez = &iKmz(Si

+ Km1)/Ke&m1(Sz + Kmz)

(7)

where Sl and Szare the concentrations of the two substrates and el and ez are the steady state concentrations of the corresponding enzymes; Kml and Km2 are Michaelis constants; Ke1 and Ke2 are the equilibrium constants for the over-all reactions between the enzymes and the pool. A method was described by Mandelstam for the indirect estimation of Kel and Kez and the values for all other terms are obtainable by measurement. It follows from the equation that, if SZis kept constant, the ratio of the two enzymes will vary linearly with SI. It is also of interest to note that Eq. (7) provides an alternative explanation of diauxic inhibition. Thus, if El is adaptive and E2 constitutive, the ratio el/e2 will normally be very small, and assuming that the Michaelis constants are similar, will be determined by the ratio Kel/KeZ. It can be seen that the ratio of SJS2 would have to be made very large before El approached the normal levels of EP. Unless the two substrates were present in such a high ratio, SZwould act as an inhibitor of adaptation to S1, a prediction which is consistent with the facts of diauxic inhibition. Equations ( 5 ) and (6) , which express the relationship between the concentration of the inducer and the amount of enzyme formed, were applied to the galactose and maltose fermenting systems of yeast and a close agreement was found between the observed and calculated values (Mandelstam and Yudkin, 1952). Also, when cells were made to adapt to galactose (Sl) and maltose (Sz) simultaneously, it was shown that if Sz was kept constant, the ratio of the enzymes increased linearly with S1as required by Eq. (7). If suitable precautions were taken to keep the concentrations of the sugars constant during adaptation, the ratios of the enzymes approached the theoretical values calculated from the equation. Although the mass action model describes, qualitatively and quantitatively, the behavior of these two systems it was stressed by Mandelstam that this did not constitute “proof” of the theory to the exclusion of other possible explanations. Equations which are formally identical could, in theory, be developed from different premises, or alternatively, entirely different equations might describe the observed behavior equally well. Diauxic inhibition is a case in point; while it is directly predicted by the

84

J. MANDELSTAM

mass action model, and might even be found to agree in a strictly quantitative manner, an alternative explanation can be given in terms of permeability factors. The criticisms of the original mass action theory apply to the extended theory as well. For example, it is impossible, without the introduction of ad hoc assumptions, to explain the adaptive formation of penicillinase or /3-galactosidase. Nevertheless the theory does account for galactose and maltose adaptation in yeast, and the close agreement between the observed and calculated values still requires explanation for it is not likely to be entirely fortuitous. In general, if an adaptive enzyme is unstable in the absence of its substrate it will be difficult, in practice, to decide whether the inducer is acting catalytically or stoichiometrically. Thus, even if production of such an enzyme were caused by catalytically small amounts of substrate, the amount measured would depend on stabilization by the substrate, and would therefore reflect the degree of saturation of the enzyme by the substrate. The equations of the mass action theory would then give an accurate description of the behavior of the system even though the primary mechanism of adaptation differed fundamentally from that postulated in the theory. It can be concluded that the mass action theory, in its present form, is not applicable to any system in which the inducer can be shown to act in a catalytic fashion or in which the enzyme is stable in the absence of its substrate. The theory should, however, be usefuI in predicting the behavior of unstable adaptive enzymes, and its value will depend on how often such enzymes are encountered experimentally. So far, unstable adaptive enzymes have rarely been found in bacteria. An example is the adaptive system concerned with the anaerobic utilization of glucose in E. coli (Fowler, 1951). Instability of adaptive enzymes has been more frequently found in yeast where the evidence points to an exchange of material between the maltose and galactose fermenting systems, and where Spiegelman and Reiner ( 1947) have shown that galactozymase activity disappears rapidly in the absence of the substrate ; the loss of activity apparently depends on active metabolism since it does not occur under anaerobic conditions and is inhibited by sodium azide. XIT.

CONCLUSIONS

The theories that have been discussed have served the useful purpose of stimulating experimental work, but reasons have been given for considering that none of them is satisfactory as afi explanation of enzyme adaptation in general. Most of the theories cover only a very restricted number of the

ENZYME ADAPTATION I N MICROORGANISMS

85

available facts. This is unavoidable at the moment because the facts are themselves often apparently at variance and it follows that no theory which is internally consistent could explain them all. It will also be apparent that many of the basic theoretical assumptions depend largely upon observations made on single enzyme systems. For example, the concept of the catalytic action of the inducer-fundamental to the organizer theory-is based on the behavior of the penicillinase-forming system ; the conclusion that there is no turnover of protein in the bacterial cell rests upon the experiments with /3-galactosidase ; the mass action theory derives its support from the behavior of the sugar-fermenting systems in yeast. These examples, and others that might have been quoted, all indicate the necessity of studying a greater number of adaptive enzymes in a wider variety of organisms. Until this has been done, it will not be possible to formulate a satisfactory theory of enzyme adaptation because of the difficulty of deciding which facts are general and which facts are peculiar to specific enzyme-forming systems or to the organisms in which they occur. It has been asumed by most workers that all instances of enzyme adaptation will ultimately be explained in terms of a single theory, but the possibility cannot be excluded that there are such fundamental differences between enzyme-forming systems that no one theory can account for all the phenomena. XIII. REFERENCES Barrett, J., Larson, A. D., and Kallio, R. E. (1953) 1. Bacteriol. 65, 187. Baskett, A. C. (1952) Proc. Roy. SOC.BUD, 251. Bellamy, W.D.,and Gunsalus, I. C. (1945) 1. Bacteriol. 60, 95. Benzer, S. (1953) Biochim et Biophys. Acfa 11, 383. Cohen-Bazire, G., and Jolit, M. (1953) Ann. inst. Pmfeur 84, 937. Cohn, M., and Monod, J. (1953) L “Adaptation in Micro-Organisms” (Gale and Davies, eds.), p. 132. Cambridge U. P., London. Cohn, M., and Torriani, A. M. (1952) J. Immunol. a,471. Cohn, M.,and Torriani, A. M. (1953) Biochim. et Biophys. Acta 10, 280. Cohn, M.,Cohen, G., and Monod, J. (1953a) Comfit. rmd. 288, 746. Cohn, M., Monod, J., Pollock, M. R., Spiegelman, S., and Stanier, R. Y. (1953b) Nature 172, 1090. Cooper P. D. (1955) J . Gen. Microbial. 12, 100. Dienert, F. (1900) Ann. inst. Pastew 1% 139. Ephrussi, B., and Slonimski, P. P. (1950) Biochim. et Biophys. Actu 6, 256. Euler, H., and Cramer, H. (1913) 2. Physiol. Chem. 88, 430. Fowler, C. B. (1951) Biochim. et Biophys. Acfo 7, 563. Gale, E. F.,and Folkes, J. P. (1953) Biochem. J. 65, 721, 730. Gale, E. F., and Folkes, J. P. (1954) Nature 175, 1223. Gale, E. F., and Folkes, J. P. (1955) Biochem. J . 5B, 661, 675. Halvorson, H.O.,and Spiegelman, S. (1952) 1. Bacteriol. U, 207.

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585. Monod, J., and Cohn, M. (1952) Advances in Enxymol. l3, 67. Monod, J., Pappenheimer, A. M., and Cohen-Bazire, G. (1952) Biochim. et Biophys. Acta 0, 648.

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Yudkin, J. (1938)

Biol. Revs. 13, 93.

The Cytochondria of Cardiac and Skeletal Muscle JOHN

W. HARMAN

NOTEADDEDIN PROOF

The following recent publications on mitochondria and muscle have appeared in the literature or reached my attention subsequent to this paper’s completion for publication. A valuable phase and electron microscopic study of Dipteran fiight muscle has been presented by A. J. Hodge [ (1955) J . Biophys. and Biochem. Cytol. 1, 3611, in which the mitochondrial surface structure illustrated is essentially the same as depicted by Chapman (1954) and Weinreb and Harman (1955). In a recent review of muscle structure and function S. V. Perry [ (1956) Physiol. Revs. 36, 13 gives the muscle mitochondria considerable attention and discusses their significance in muscle contraction. Additional evidence that the oxidative and phosphorylative enzymes of muscle mitochondria have differential fragilities is adduced by B. Sacktor and R. Sanborn [ (1956) J . Biophys. urrd Biochem. Cytol. 2, 1051, who demonstrated that phosphorylating enzymes are more labile at higher temperatures. This is evident from both reduced P/O ratios and morphological deterioration in the mitochondrial population. An attempt to correlate ATPase activity with the E.M. morphology of mitochondria isolated in sucrose solutions of various molarities (Witter, R. F., Watson, M. L., and Cottone, M. (1955) J. Bioplzys. atzd Biochem. Cytol. 1, 127), permits the authors to define no clear-cut association. Although they did not characterize population trends the illustrations demonstrated a distinct type of mitochondrial morphology in each molarity. They also illustrate a mitochondrial matrix which shrinks independently of the finer gel. On the other hand in B1 avitaminosis there is shown to be a decrease of ATP-resynthesis and a concomitant decrease in optical density of mitochondrial suspensions, confirming a parallel derangement of structure and function [Frei, J., and Ryser, H. (1956) Expem’entia 12, 1051.

88

The Cytochondria of Cardiac and Skeletal Muscle JOHN W. HARMAN Department of Pathology, University of Wisconsin, Madison, Wisconsin I. 11. 111. IV. V. VI. VII. VIII. IX.

X. XI. XII.

Page 89

Introduction ....................................................... History of Muscle Cytochondria ................................... Some General Properties of Muscle Mitochondria ..................... Distribution and Morphology of Muscle Mitochondria ................. Chemical Composition of Muscle Cytochondria ....................... Metabolic and Enzymatic Aotivities of Muscle Cytochondria ..................................................... Electron Transport System of Muscle Mitochondria ................. Oxidative Phosphorylation of Muscle Mitochondria ................. Distribution of Enzymes Acting on Phosphorylated Nucleotides ...................................................... Morphology and Mitochondrial Activity ............................. The Integration of Energy Metabolism in Muscle ................... References .........................................................

91 92 93 100 105 112

116 126 132 140

142

I. INTRODUCTION In recent years there has emerged and been developed a type of cytological experimentation which affords considerable scope into the investigation of cellular physiology and biochemistry. The trend involves the application of an axiom of analytical chemistry to cytological studies, to separate separable things prior to proceeding with their analysis ( Bensley, 1942), because the analysis of the particulate constituents of cytoplasm is anly as perfect as the species purity of the preparation. The application of the principle to cellular investigation required the dispersion of the cells by gentle comminution procedures and the isolation, from the protoplasmic mass, of suspensions of relatively stable subcellular morphologic structures (Bensley and Hoerr, 1934; Claude, 1944, 1946a, b; Hogeboom et al., 1948). The particles thus isolated in a high state of purity from a variety of tissues and from both animals and plants (Bonner and Millerd, 1953; Laties, 1953, 1954) have well repaid the cytologist’s and biochemist’s efforts, with a rapid dividend of advance in our knowledge of the enzymatic and functional integration of the particles in fundamental cellular processes. The principles and expedients suitable for the extraction of particles from cell homogenates in pure suspensions have been exhaustively analyzed (de Duve and Berthet, 1955) with special regard for the technique of fractional centrifugation. A general survey of the chemistry and physiology of mitochondria and other cellular particles is also extant (Lindberg and Ernster, 1954). 89

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JOHN W. EABMAN

The singular discovery associated with utilization of the technique of cellular fractionation is the realization that mitochondria, basic constituents of the cytoplasm of all animal cells, are the predominant locale of the system of enzymes participating in cellular oxidation. It is especially through the work of Hogeboom and his colleagues (1948) that the mitochondria have been recognized as the special vehicles of the oxidative enzymes. Concurrently Green’s school (1951) reached the conclusion that the complex of enzymes which catalyze the reactions of the Krebs citric acid cycle are compounded in some subcellular unit and are arranged structurally as a system. Green and his associates defined this as the cyclophorase system. At about the same period it was observed independently (Schneider and Potter, 1949; Lehninger and Kennedy, 1948) that isolated mitochondria catalyze the gamut of enzymatic reactions peculiar to the citric acid cycle. Subsequently it was established that the cyclophorase of Green and the systems of the other workers are essentially identical, in so far as the activity of the cyclophorase depends upon the presence of intact mitochondria (Harman, 19%) and that there is a numerical relation between cyclophorase activity and mitochondria1 density (Harman, 195%; Paul and Sperling, 1952). In view of the role which the mitochondria play in the cellular metabolism of numerous tissues it is apposite to inquire into their participation in the metabolism and mechanisms of muscular systems. It is relevant that muscles may be distinguished from each other on the basis of functional criteria and also that they have been classified in accordance with the abundance or relative absence of minute granules located between the myofibrils (Knoll, 1891). There is indeed close correlation between the concentration of such particles in a muscle and its biological significance, as Knoll (1889) pointed out, “The abundance of interstitial granules in the active muscles of all animals points to a correlation between these interstitial granules and the function of the muscle.” Subsequent comparative investigations tend to substantiate this concept that particulate density and function are related. It is within our purview to survey here the recent knowledge of the cytological and enzymatical characteristics of the granules of muscle. Owing to the fact that muscle contains several types of granules the particles have been grouped together and designated as cytockondriu, as suggested (Opie, 1947a) for other tissues. The mitochondria are so called since they are similar to such particles in other tissues in all respects and the term sarcosome is reserved for a granule peculiar to muscle. This will cause no confusion, but will apply emphasis to the identity of the true mitochondrion and direct proper special interest to other particles which

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are found only in muscle. Since the identification of the granules has been made by several workers with the mitochondria (Regaud, 1909; Duesberg, 1910; Luna, 1913) cytologically, and more recently - biochemically (Watanabe and Williams, 1951; Harman, 195Oa), reversion to the nomenclature of Retzius (1890) is a stultification. On the other hand, the true sarcosome is not homologous with any structure in other cell types, so that restriction of this term to so distinctive a particle is certainly indicated in the interests of clarity. I t is realized that until the present the terms mitochondria and sarcosome have been used by some authors interchangeably, but with current characterization of the several muscle particles, this is no longer valid. 11. HISTORY OF MUSCLECYTOCHONDRIA Over a century ago Bowman (1840) contributed the fundamental observations which today remain the essence of our current concepts of myofibrillary and sarcomeric structure. During the same period for the first time attention was directed by Henle (1841) to the minute granule situated in the sarcoplasm between the myofibrils ; with this observation was initiated the study of the particles now known as mitochondria. Later reference to the granules was made by Lebert and Robin (1846) in regard to the fine granules in invertebrate muscle and by Aubert in 1853, who commented upon the considerable size and abundance of the particles in thoracic muscles of insects. But undoubtedly the most comprehensive study was made by Koelliker in a series of three papers (1857, 1866, 1888). H e referred to the particles as interstitial granules and regarded them as an essential component of the muscle cell. In his later study he separated the particles from insect muscle by teasing and analyzed the effect of various environmental factors on their morphology ; he was especially impressed by the considerable swelling which occurred when they were suspended in water. He was obliged to make the following comment on the phenomenon, “The granules swell considerably in water and are transformed into vesicles which have a distinct although very delicate mernbrane. During the process the components assume the configuration of a half-moon at one side and suffer partial solution.” Subsequently Retzius ( 1890) reexamined the interstitial granules during his investigations on sarcoplasm and at that time alluded to them as “sarcosomes” without specifying the connotation of the term. This designation was rendered superfluous by the identification of the interstitial granules with mitochondria (Regaud, 1909; Bell, 1911; Bullard, 1913, 1916; Duesberg, 1910; Luna, 1913), although Galeotti (1895) had previously shown that they were the fuchsinophile granules described by Altmann (1894). The biological range of the distribution of muscle mitochondria was especially

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JOHN W, HARMAN

studied in several surveys (Knoll, 1891; Holmgren, 1910; Bullard, 1913, 1916; Bell, 1911). Floegel (1872) found a second small, isodiametric particle situated between the myofibrils, which he distinguished from the granules of Koelliker because of difference in size, shape, and position. Retzius (1890) confirmed this observation. Jordon (1919) commented on the distribution of the interstitial granules of Koelliker and the small particles of Floegel and preferred to regard the latter as precursors of the larger. Holmgren (1909, 1910, 1913) classified them as I and Q granules in accordance with their predominant relation to the parts of the sarcomeres, but inferred that the smaller units may be waste products derived from disintegration of the larger interstitial granules. This smaller granule is now designated as the sarcosome (Kitiyakara and Harman, 1953). 111. SOME GENERALPROPERTIES OF MUSCLEMITOCHONDRIA Mitochondria occur in cells as either rods or spheres. The principal intracytoplasmic form of muscle mitochondria is rod shaped and irregular. This essential appearance in muscle has been established by numerous investigators employing the conventional Altman technique or some variant of it (Regaud, 1909; Holmgren, 1907; Ciaccio, 1940, 1941; Villmitjana, 1949; Harman and Feigelson, 1952a). Meyer (1926) critically examined this problem of in vivo configuration and demonstrated the important influence of the fixative and the tonicity of the medium on the shape of muscle mitochondria. In general it may be assumed in regard to all mitochondria that the spherical shape is a result of some stress or transforming agency and represents a deformity of the natural structural condition. That such stress may occur in vico is exemplified by the study by Holmgren of the effect of fatigue on the size and shape of the granules in the wing muscles of Libellzlla (Netzfluegler) which was allowed to beat its wings into a state of exhaustion prior to dissection of the musculature (Holmgren, 1910). Under such stress the granules lost the usual rod shape, swelled, and assumed a round, bloated appearance, The mitochondria of muscle are easily stained by the variety of techniques peculiar to mitochondria1 staining, such as the procedures of Regaud, Altman, and Benda, and by Baker’s method (Meyer, 1926; Kitiyakara and Harman, 1953). They are fixed with the reagents generally suitable for mitochondria and are only slightly impregnated with osmic acid, in contrast to the sarcosomes which are very osmiophilic. Although it has been indicated that Janus green B is specific for vital staining of the mitochondria (Cowdry, 1918>,it is not usually stated that this peculiarity is applicable only to the intracytoplasmic mitochondria, in muscle as among

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other cells. It has been observed that such staining is especially capricious for extracellular or isolated mitochondria, so that the use of this criterion for the identification of the mitochondria in homogenates is difficult and requires careful arrangement of the proper conditions for the test (Showacre, 1953; Lazarow and Cooperstein, 1953). By transmitted light mitochondria normally have a bluish to green tint which may be confused with Janus green staining. As Koelliker initially observed (1857) the manner in which the interstitial granules or the mitochondria swell and the shapes they assume in hyposmotic or dilute solutions are characteristic phenomena of the particles. This metamorphosis is not unique for muscle mitochondria but is uniformly observed in the mitochondria of all tissues (Bang and Sjovall, 1916; Knocke, 1909; Opie, 1948; Zollinger, 1948). It is studied easily by phase microscopy and is so characteristic that it has served in the identification of the particles during mitochondrial counts (Harman, 19SOa ; Allard et al., 1952; Shelton et al., 1953). The importance of this structural deformity in relation to mitochondrial metabolism has received considerable study and will be discussed later.

IV. DISTRIBUTION AND MORPHOLOGY OF MUSCLE MITOCHONDRIA An amazingly exhaustive survey of muscles has revealed (Rollet, 1889; Knoll, 1889, 1891) that the distribution of the granules among muscle types is diverse. Some muscles all but lack granules, whereas others are exceptionally granular. It is possible to distinguish two classes of muscle fiber, dark and light ; the dark fibers possess a large volume of sarcoplasm and numerous embedded granules, while the light fibers are deficient in both constituents. The gross character of any muscle depends on the relative proportion in it of the two fiber types. The majority of the mammalian muscles are made up of mixtures of dark granular and light granular fibers. On the other hand the flight muscles of soaring birds such as the pigeon, hawk, and seagull and of the bat, a soaring mammal, are composed chiefly of granular fibers. In the leg muscles of these animals light fibers predominate. In birds and all other animals (Knoll, 1889) the heart muscle is opaque and dark red owing to its heavy concentration of granules. The distinction is not made in these earlier studies between the different types of granule, but it may be presumed that the interstitial granules described by Koelliker are represented. The association between granule distribution and fiber type has recently been reinvestigated and confirmed (Kitiyakara and Harman, 1953). Jordon ( 1919) had previously observed a curious relationship in the mantis, where the leg muscles are poorly endowed with the granules and the powerful wing appendages are excep-

94

JOHN W. HARMAN

tionally rich ; this is correlated with the predatory habits of the insect. By the use of tetrazolium salts to indicate the presence of succinoxidase the distinction between granular and agranular fibers is beautifully demonstrated (Rutenberg et d., 1953). The intracellular mitochondria (Figs. 1 and 2) are perhaps best studied in freshly teased or homogenized muscles by phase microscopy (Harman,

FIG.1. Portion of a myofiber is depicted by phase microscopy, with Dark L contrast. The myofibrils are not conspicuous, whereas the linearly arranged mitochondria and the refractile sarcosomes are evident. The mitmhondria are elongated ( M )and the sarcosomes are in places (S) paired at the extremities of the mitochondria. Magnification 2990 X.

195Oa). In situ they are mainly arranged parallel to the long axis of the myofibrils, stretch along several segments of the myofibrils, and their total length may span two or more sarcomeres. There is no delimitation in relation to either the isotropic I segment or the anisotropic A segment of the sarcomere ; neither is the sarcomere related to the surface irregularities so common in the mitochondria. On the other hand, as Holmgren has depicted for insect muscle (1910) and as has also been observed in cardiac muscle (Harman and Feigelson, 1952b) some mitochondria lie transversely and under the circumstances are situated exactly across the A seg-

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95

ment of the sarcomere. These appear to bend and curve in conformity with the contour of the apposed myofibril. Exploration by electron microscopy of the structure of cardiac (Weinstein, 1954; Kisch and Bardet, 1951) and of skeletal muscle (Bennett and Porter, 1953 ; Chapman, 1954 ; Ruska, 1954 ; Weinreb and Harman, 1955; Edwards and Ruska, 1955) has substantiated the longitudinal loca-

FIG.2. A similar fiber from pigeon breast muscle examined by phase contrast, Light L, in which the mitochondria ( I ) are only slightly opaque and the sarcosomes stand forth owing to considerable refractility (2). The apparent spaces between the mitochondria1 rows are occupied by the myofibrils, which are not evident because of the refractive index of the sucrose suspending medium. Magnification 2990 X. tion of the interfibrillary mitochondria. Although the dimensions of the mitochondria vary considerably, the relation between the long axis of the particle and that of the adjacent myofibrillae is a constant feature. The spanning length of the mitochondria corresponds with the previous observations by phase microscopy. The electron micrographs have allowed elucidation especially of the finer structure and relationships of the mitochondria (Figs. 3 and 4). As indicated by Bennett and Porter ( 1953), the mitochondria constitute the largest and most conspicuous formed bodies situated in the sarcoplasm. This applied not only to the breast muscle of Gallus, but is also true for

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J O H N W. HARMAN

the breast of Colulnba Zivia, heart of Rana pipiens (Harman and Weinreb, unpublished observations), and the heart muscle of the cat, dog, guinea pig, human, snake, turtle, and chicken (Weinstein, 1954). In cardiac and breast muscle of the pigeon (less so in the domestic fowl) the mitochondria are nearly as conspicuous as the myofibrils (Fig. 1). It is of some importance that in none of these studies has the presence of a mitochondria1 double limiting membrane been depicted. Chapman

FIG.3. Electron micrograph of pigeon breast skeletal muscle. Between the myofibrils (F) are linear rodlet mitochondria ( M )with transverse laminations. The mitochondria and the myofibrils are closely abutting, and the mitochondria have no distinctive membranes. The dense electron opaque sarcosomes (S) are arranged at the sides and ends of the mitochondria to which they are connected by delicate strands (D),also communicating with the myofibrils. Close to the sarcosomes are finer granules, (G), especially near the 2 lines. Magnification 21,775 X.

97

CYTOCHONDRIA OF CARDIAC AND SKELETAL MUSCLE

(1954) evinces considerable doubt as to the evidence for a membrane in his preparations of insect muscle, and other investigators (Weinreb and Harman, 1955) have discarded the interpretation of the existence of limiting membranes in the mitochondria of pigeon breast muscle, which is in accord with the electron microscope analysis of that tissue (Fig. 5). This absence of a surface double membrane, as described by others (Sjostrand, 1953; Sjostrand and Rhodin, 1953; Rhodin, 1954 ; Palade, 1953) may be

FIG.4. A dense sarcosome lies between the extremities of two mitochondria. It connects with both mitochondria and the adjacent 2 line by the fine strands. On the strand to the 2 line are accumulations of minute granules ( G ) which are especially electron opaque. Fine transverse lamination is evident in the mitochondria. The arrow indicates the naked mitochondria1 surface. Magnification 61,750 X

.

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JOHN W. HARMAN

peculiar to skeletal muscle mitochondria. I n view, however, of the findings that the mitochondria in Paramecium lack membranes (Powers et al., 1955), and that there are many mitochondria in the neurons which fail to manifest membranes (Hartmann, 1953), it is likely that the nonmembranous mitochondria1 interface is the native condition (Frederic, 1954). These studies introduced the possibility that the procedures oi preparation and impregnation may be conducive to simulation of an interfacial barrier by piling up of methacrylate, and others have indicated that during washing the osmium silts up and may be deposited as in a membrane.

FIG.5. Electron micrograph of a mitochondrion from pigeon breast muscle. The longitudinal section demonstrates the absence of the effternal limiting membrane and the internal lamellar arrangement. Magnification 64,500 X .

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The absence of structural membranes1 would bring the matrix of the mitochondrion into contiguity with the sarcoplasm and the adjacent myofibrillae. In transverse sections of skeletal muscle an interpenetration of myofibrillary substance and mitochondrial matrix is demonstrated (Weinreb and Harman, 1955). In addition to the approximation by contact, the mitochondria are linked to both the myofibrillae and the sarcosomes by delicate reticular strands, as in Fig. 4 (Bennett and Porter, 1953; Weinreb and Harman, 1955), which are capable of rotating the cytochondria into transverse positions when shrinkage occurs. The fine inner structure of the muscle mitochondria, as revealed by electron microscopy of longitudinal sections in several species, does not correspond with that demonstrated hitherto for several other tissues. Even after closest examination the hollow center described and illustrated by Palade (1952) is not delineated, nor are cristae seen. O n the other hand the surface membranes of Sjostrand are absent, whereas the inner structure resembles that shown by him and Rhodin (1954). The inner laminated transverse doubled membrane structures are platelike in one plane and have terminal loops in others, so that this is believed to represent an inner folded fibrous gel which is embedded in a fine gel matrix (Weinreb and Harman, 1955). The surface of the inner gel is lined by osmium and may be interpreted as hollow, as in the mitochondria of Paranzeciuwa (Powers et al., 1955). The sarcosomes, the small particles of Floegel (Figs. 1 and 2) , are seen by phase microscopy as small, dense, highly refractile spherical bodies situated adjacent to the mitochondria (Kitiyakara and Harman, 1953 ; Harman and Osborne, 1953). They have a special tendency to location at the mitochondrial poles although some are situated between the sides of the mitochondria and the myofibrils. Electron microscopy demonstrates spherical structures in the same positions as the sarcosomes observed by phase microscopy. They are usually the most electron-opaque constituents in the section, are centered in cleared areas, and appear to be suspended from the adjacent mitochondria and myofibrils by fine strands which emanate from the substance of the bodies and fuse indistinguishably with the mitochondria and myofibrils. No ultrastructure has been demonstrated in sarcosomes with certainty, even after sectioning at 0.025~.The significance of the clear zone around the sarcosomes is open to speculation. It may indicate either severe shrinkage or alternatively the solvation of a peripheral layer by the organic fluids employed in the preparative procedures. The 1 Membrane implies a structural entity which surrounds a biological system, and which is different from the confined system in composition and function.

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latter view would suggest separate cortical and medullary layers. Features which distinguish the sarcosomes and mitochondria are enumerated in Table I. To the distinction should be added the observation that the mitochondria are complexly structured by electron microscopy, whereas the sarcosomes are uniformly electron opaque and have no inner differentiated structure. TABLE I COMPARISON OF MORPHOLOGIC CHARACTERISTICSOF MITOCHONDRIA AND SARCOSOMES OF PIGEON BREASTMUSCLE Property

Mitochondria

Size Shape Refractility Osmophiliaa Chromophilia (aniline dyes) Osmotic swelling Solubility in organic solvents Orientation

2 to 10 p Irregular rods Slightly refringent Slight Specific Characteris tic Slight Parallel to myofibrils

Sarcosomes 0.1 to 0.3 p

Round or elliptical Strongly refringent Strong Slight Absent Complete Transverse to myofibrils

* Osmophilia is the property of certain cellular structures of staining black with tetroxide of osmium. V. CHEMICAL COMPOSITION OF MUSCLE CYTOCHONDRIA In many laboratories extensive studies have been made of the chemical composition of mitochondria. There is agreement that the phospholipid and protein components are especially significant (Chargaff, 1942 ; Swanson and Artom, 1950). Perry (1952) has studied this in the lipoprotein granules isolated from the leg muscles of rats and rabbits and suggests the essential similarity of these granules to the mitochondria of other tissues. This has been verified for insect muscle mitochondria also (Watanabe and Williams, 1951) . The phospholipid, phosphoprotein, and PNA content has been reinvestigated (Harman, 1955) in a study of the granules of the rabbit psoas muscle and of the mitochondria and sarcosomes of the pigeon breast (Table 11). In some respects the small granules from the rabbit muscle resembled the mitochondria more closely than pigeon breast sarcosomes. In all three particles a considerable pentose nucleic acid fraction is detected, whereas the phosphoprotein component is negligible.2 Parallel analyses of rabbit liver mitochondria demonstrate that the phospholipid content of the muscle mitochondria is double that found in the liver particles. 2 The proportion of P N A and phosphoproteins are reciprocal and the ratios found depend. on whether the Schneider (1945) or Schmidt and Thannhauser (1945) technique is used.

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The protein composition of muscle mitochondria has received only cursory study. Although soluble fractions are obtained from the hepatic mitochondria by sonic vibration (Hogeboom and Schneider, 195Ob), the bulk of the mitochondrial constituents are not amenable to solubilization by this technique. The proteins of skeletal muscle have been examined by differential salt extraction (Kitiyakara, Harman, and Weinreb, unpublished observations). A minimal amount of protein is eluted by distilled water (Fig. TABLE I1 COMPAR~SON OF PHOSPHOLIPID, PHOSPHOPROTEIN, AND NUCLEICACID 'CONTENT OF MUSCLE MITOCHONDRIA,SARCOSOMES, AND LIVERM I ~ H O N D R I A

NUlllber Particle

Source

Mitochondria

Pigeon breast Pigeon breast Rabbit psoas major Rabbit liver

Sarcosomes Sarcosomes Mitochondria

of Experiments

Lipid P

AcidSoluble Proteins P -P DNA-P PNA-P

11

72.3

19.5

0.8

0.7

31.2

4

83.0

26.4

0.0

0.0

26.9

3

69.9

25.5

4.2

1.2

23.9

6

48.1

32.0

0.6

1.5

23.8

NOTE: The phosphorus values are expressed as P/mg. N., as determined by h e technique of Schmidt and Thannhauser (1945). Pigeon breast mitochondria and sarcosomes were prepared as described previously (Harman and Osborne, 1953). Sarcosomes from rabbit psoas major muscle were isolated in 0.24 M sucrose according to Perry's method (1952) with slight modification of the procedure. The mitochondria from rabbit liver were isolated in 0.25 M sucrose and freed from both microsomes and the particles contained in the fluffy layer. T,he averages listed were derived from individual estimations which differed by not more than 5 per cent.

6 ) but as the concentration of potassium chloride is increased it is possible to remove two components (Fig. 7). One is eluted by dilute salt, up to a concentration of 0.45 M KCl, the other is removed, with nearly complete solubilization of the particles at 0.8 M KCl. The results of this investigation show that the mitochondrial proteins are very slightly soluble in water, as determined elsewhere (Harman, 1950b). This substantiates other observations that the mitochondria are not susceptible to rupture and lysis in distilled water (Dianzani, 195313). The fraction of protein removed by the "high" salting-in method is fibrous and depicted as such by electron micrograph (Fig. 8) ; that eluted by lower ionic strength extraction is globular in behavior and appearance. Analyses of the globular component

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JOHN W. HARMAN

al

44

e c -

-ii 0.055

!i

0

*/

0

0

0

MgN/MI Suspension

FIG.6. Mitochondria are isolated from M. pectoralis major of the pigeon in 0.25 M sucrose by the fractionation procedure of Kitiyakara and Harman (1953). Varying concentrations of mitochondria are suspended in glass distilled water to a volume of 10 ml. The nitrogen concentrations were determined in the filtrates and suspensions after equilibration at 0°C. for 2 hours. Filtration was performed through No. 42 Whatman paper under suction in the cold. Centrifugation at 18,000 g for 30 minutes yields a similar extract. The plateau of 0.024 mg. N demonstrated in the figure is attained in either procedure. Second and third extractions of the residue release identical quantities of nitrogen. Prolongation of the extraction time for 24 hours obtains no additional release d nitrogen.

I

I

0.2

I

I

a4

0.6

m

1

I

0.8

1.0

KCI

FIG.7. Extraction by varying molarities of potassium chloride. A water suspension of skeletal muscle mitochondria was divided into aliquots containing 0.5 ml. aliquots containing 1.43 mg. of nitrogen. Each aliquot was made up to 10 ml. with potassium chloride, p H 7.0 of varying molarity. The nitrogen figure of the filtrate multiplied by 10 gives the result in mg. of nitrogen as a function of the original nitrogen concentration of the suspension. A t 1.0 M KCl this represents above 98.576 of the mitochondria1 nitrogen. Dialysis of the 1.0 M extract for 3 hours allows precipitation of most of the solubilized protein.

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reveal four peaks such as occur in proteins derived from liver mitochondria (Hogeboom and Schneider, 1950b). By variation in pH there is removal of considerable protein from the mitochondria on both the acid and alkaline sides of neutrality (Fig. 9 ) , with extraction of nearly 100% at p H 12. The analyses which demonstrate two proteins in skeletal muscle mitochondria, one fibrous and the other globular, are compatible with the elec-

FIG.8. Electron micrograph of the protein extracted from the muscle mitochondria by 1.0 M ICCI, pH 7.0. Prior to depositing proteins on the Formvar film they were dialyzed against distilled water for 3 hours a t 0°C. The fraction which precipitates out is fibrous, with a tendency to aggregate into long bundles and some forms clumps. The fraction remaining in the supernate after dialysis is nonfibrous, globular. Electron micrographs of the fibrous protein obtained by either dialysis or dilution are essentially similar, although the fibers from the dilution technique are shorter. Salt crystals were removed by washing, after thorough drying on the film. Preparations were shadowed, in vucuo, with uranium prior to electron microscopy in an RCA EMU-2. Magnification 17,779 X .

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J O H N W. HARMAN

tron microscope representations of the finer structure which also suggest the existence of two gels. The more easily removed gel may represent the fine matrix which surrounds a coarse folded ribbonlike gel that is extracted at high ionic strength, as are most fibrous gels. The capacity of salt solutions to obtain constituents of the mitochondria in solution is also demon-

1

100-

a

90

-

80

-

70

-

60

-

I

w

2

4

6

8

10

12

14

PH

FIG.9. Solubility of mitochondria1 proteins at varying pH values. Mitochondria were suspended in 0.03 M potassium phosphate405 M potassium chloride solution at different pH levels. Each aliquot contained 1.55 mg. nitrogen diluted to 10 ml. final volume. Equilibration in the Dubnoff shaker for three hours at 0°C. was allowed, before the suspension was filtered through Whatman No. 42 paper. The quantity of released nitrogen is expressed as a function of pH level, with point of minimal extraction at pH 7.0 and 100% extraction attained at p H 11. strated by the studies of de Duve’s group (Berthet st al., 1951; Berthet and de Duve, 1951; de Duve and Berthet, 1955) on the extraction of acid phosphatases. Some information may be obtained concerning the structural disposition of the protein and lipoprotein constituents in the mitochondrial architecture by the several studies which have employed the action of external enzymes.

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105

In liver mitochondria the claim has been presented (Nygaard et al., 1954) that lecithinase A induces surface alterations which are visualized by electron microscopy. This result is interpreted as an indication that the surface has a membrane susceptible to the enzyme. On the other hand in skeletal muscle mitochondria the use of Crotalus3 venom, lysophosphatides, and bacterial lecithinase evokes no alteration, whereas the submission of the mitochondria to minimal amounts of crystallized trypsin is rapidly followed by gigantic swelling of the particles and eventual solution ( H a r man and Osborne, unpublished observation). [Dianzani ( 1953a) digested liver mitochondria with trypsin and estimated the effect by mitochondrial count.] It is likely therefore that the surface of the mitochondrion is constituted by the matrix gel in which is buried the coiled lipoprotein fibrous gel seen by electron microscopy. This would also conform with the observations made by Green (1955) that tryptic digestion has the ability to convert the D P N H oxidase particle of pig heart into lipoprotein components of heavy and light lipid content. Among enzymes which are incapable of manifesting any detectable alteration are PNAase, DNAase, lipase, lysozyme, hyaluronidase, and collagenase. A N D ENZYMATIC ACTIVITIES OF MUSCLECYTOCHONDRIA VI. METABOLIC

Since the time of the pioneer physiological studies of Ranvier (1874) and Bonhoffer (1890) and the later metabolic work of Battelli and Stern (1912), it has been realized that differences in gross muscular structure are associated with differences in both function and oxidative capacity. The latter authors studied oxidation in white and red muscles; the red more vigorously oxidized succinate, pyruvate, and phenylenediamine. They proposed the concept that the oxidative enzymes are closely associated in the particulate structure and coined the name “oxydon” to express the idea (see the current concept by Green ( 1951) cyclophorase) . Another worker (Ahlgren, 1921) subsequently redefined the clear-cut oxidative capacities of the two muscle types. However, at the time, no exact correlation was made in these or other comparative tissue studies that suggested a connection between the granular content and oxidative capacity. More recently studies have been made which correlate the mitochondrial densities of several muscle types with the oxidative competency (Paul and Sperling, 1952). Stare and Baumann ( 1939) had cataloged the oxidative rates of muscles in several animals and established the high Qo2 of mammalian heart and pigeon breast muscles. Later investigations indicate that the scale of activity is a reflection of the density of the mitochondria1 popu3

Venom of Crotolus H . horridus

(R. Allen

snake farm).

106

JOHN W. HARMAN

lation. This result was reached either with the use of the endogenous repiration of whole homogenates or the employment of washed residues fortified with metabolites of the citric acid cycle and cofactors. TABLE I11 MIT~CHONDRIAL FOPULATION IN BREASTMUSCLESOF Experiment Muscle Major Minor Major Minor Major Minor

THE

PIGEON

Slide

p3 unita

Units Counted

Total Counts

Mt./ml

1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

988.1 1012.5 1406.5 956.25 1406.25 1575.0 1110.9 1420.0 993.0 1054.0 1012.0 1131.0 843.0 844.0 858.0 890.0 762.0 1037.0

765 850 850 850 850 850 850 850 850 850 850 850 850 850 850 1190 1700 1700

693 640 962 433 667 786 452 664 600 198 166 265 470 370 420 170 185 232

9.7 x 108 7.4 x 108 8.0 x 108 5.3 x 108 5.6 x 108 5.8 x 108 4.8 x 108 5.5 x 10s 6.0 x 108 2.3 x 108 1.9 x 108 2.6 x 108 6.6 x 108 5.1 x 108 5.3 x 108 2.3 x 108 2.8 x 108 2.6 x 1Oe

1 2 3

a “Unit” is the area of fluid between the cover slip and slide and bounded by the borders of a square in the Whipple disc. From the square dimensions of the areas in the disc obtained by the use of a stage micrometer and the depth of the liquid from the use of the micrometer fine adjustment of the microscope, the unit volume is derived. The muscles are M. pectoralis major and minor. Muscles homogenized in 0.25 M sucrose and squeezed through cheesecloth are used; 20 gm. of muscle are contained in a final volume of 300 ml. Suspensions are held at 0°C. until counts are made at 26°C. In experiments 2 and 3 the amount of muscle homogenized was reduced to half that in experiment 1. The measurements of the squares in the Whipple disc were made as previously described (Harman, 1950a). Depth of the fluid level is obtained by use of the micrometer caliper scale on the fine adjustment of the microscope using 100 as the point of reference.

The activity of the citric acid cycle in different muscles has also been assessed by the injecting of sodium fluoroacetate into the animals and estimating the accumulation of citrate in various muscles. The mitochondria are first counted by a reproducible method (Table 111). When the citrate pileup is compared with the mitochondria1 density of the muscles

107

CYTOCHONDRIA OF CARDIAC A N D SKELETAL MUSCLE

a relationship is apparent (Table IV) between the particulate density and the rate of citrate accumulation (Harman, 1955). There are exceptions, however, especially in the case of M. pectoralis major of the pigeon, which though exceedingly rich in mitochondria aggregates less citrate than the minor muscle which has fewer granules. The method of metabolic blockade TABLE IV OF CITRATEI N VARIOUSMUSCLESFOLLOWING INJECTION OF ACCUMULATION SODIUM MONOFLUORACETATE Species Rat

Rabbit

Pigeon

No. of Experiments 25

10

10

Tissue

pM'Citrate/lOO gm.

M. tibialis M. gastrocnemius Diaphragm Heart Kidney M. tibialis M. seminiembranosus Diaphragm Heart Kidney Flexor leg muscles M. pectoralis minor M. pectoralis major Heart

0.4

2.5 33.0 90.0 117.0 0.0 2.6 9.5 71.2 161.0 14.0 44.0 32.0 101.0

Mitochondrial Density 1 1 2 3

4 0 1 2

3 4 1 2 3 4

NOTE: Citrate is determined by the method of Potter and Busch (1950) on animals injected with m o u n t s of sodium fluoroacetate as indicated by them. An hour after injection the animals are anesthetized with Nembutal and the selected muscles dissected out and frozen instantly by crushing between blocks of solid carbon dioxide. Portions of the crushed muscle are deposited in tared flasks containing a measured volume of 10% trichloracetic acid and weighed. After further maceration and sufficient extraction of the tissue, aliquots of filtrate are used for citrate analysis in triplicate. Mitochondria1 density is determined on fresh homogenate in 0.25 M sucrose by phase microscopy.

may prove a general guide to the metabolic intensity of the Krebs cycle in various muscles, but the few exceptions observed limit its specificity. I t is possible that in some tissues and muscles an alternative pathway permits the citrate to escape accumulation by passage along another route. By use of a new mitochondrial counting method (described in the legend to Table 111) , a more precise relationship between the mitochondria1 population and the oxidative capacity of the muscles is available (Harman, 1955). When the oxygen uptake of the various skeletal muscles and cardiac muscle is estimated per unit number of mitochondria instead of on a basis of total

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JOHN W. HABMAN

mitochondrial population it has been found (Table V)* that in the muscles which are richest in mitochondria the average mitochondrial oxidation is lowest. Each mitochondrion in heart and M. pectoralis major oxidizes at a lower rate than it does in the leg muscles. From his observations on the finer mitochondrial structure of cardiac mitochondria Palade ( 1952) has suggested that the metabolic capacity of these units should be greater than in other tissues, because in cardiac mitochondria the inner folds containing TABLE V BETWEEN MITOCHONDIUAL COUNTAND OXIDATIVEACTMTYIN CORRELATION VARIOUSPIGEON MUSCLES Muscle

Mt./gram. (WW).

Heart M. pectoralis major M. pectoralis minor Flexors (leg)

1.2 x 1011 6.9 x 1010 3.3 x 1010 2.2 x 1010

p102/hour/gram (WW) p1O2/1010Mt. 10.0 103

6.5 103 4.4 103 3.3 x 103

833 942 1333 1500

a Mitochondria/gram wet weight of muscle. Mitochondria are counted as described in the text in preparations as indicated in Table IV. Oxygen uptake is measured by conventional Warburg procedure. The cups contain in a final volume of 2 ml. : 60 pM phosphate buffer, pH 7.20; 16 pM of MgCl; 1 mg. DPN (85% Schwartz) ; 7.5 pM A M P ; 40 pM alphaketoglutarate. To each flask 240 mg. of fresh homogenized muscle is added. Temperature is 37°C. Gas phase is O,,and CO, is absorbed by KOH in center well.

enzyme mosaics are more numerous. If the quantity of cytochrome c and the abundance of ridges is in “good correlation’’ in the order suggested it is difficult to reconcile this with the slower oxidation of the average cardiac mitochondrion and the more rapid oxidation of the particles from the other skeletal muscles. Levenbrook (in press) has applied the earlier counting method (Harman, 1950a) to enumeration of total mitochondrial populations of insect muscles and observes different oxidative capacities also. Analyses of the above type do not locate the exact site of the oxidative enzymes in the muscle cells, except by analogy with other tissues. It is possible, though the bulk of the oxidative enzymes are situated in the mitochondria elsewhere (Hogeboom and Schneider, 1951; Harman, 195Oa) that in muscle other components of the cell may contribute to the integrated system. The investigations in several laboratories provide clarification. It is demonstrated in cardiac muscle by fractionation studies that the mito4 This table, as originally published, had two figures in error, which have been corrected above (Harman, 1955).

CYTOCHONDRIA OF CARDIAC AND SKELETAL MUSCLE

109

chondria show the highest respiratory activity (Harman and Feigelson, 1952a). Other workers using blowfly muscle have established that the isolated mitochondria manifest a cytochrome oxidase activity, which is twice that of the whole muscle ; they assume that, since the particles represented about this proportion of the total muscle volume, the oxidative activity is therefore essentially mitochondrial (Watanabe and Williams, 1951). None of these investigations exclude participation of the myofibrils or other muscle constituents in oxidative processes. Subsequently two groups (Harman and Osborne, 1953; Chappell and Perry, 1953) independently have observed by fractionation analyses that the oxidative reactions consistent with the Krebs cycle are located exclusively in the mitochondria. It has also been determined (Harman and Osborne, 1953) that pure suspensions of myofibrils are totally without oxidative capacity (Fig. 10). On the other hand the sarcosomes, small particles of Floegel, possess a negligible oxidative capacity, but are unique in their ability to augment the oxidative rate of the mitochondrial suspensions (Fig. 11) . It is postulated that they may participate in muscle metabolism by regulative interaction with the mitochondria. This relationship may be similar to the influence (Pressman and Lardy, 1952) of the submicroscopic particles of liver on mitochondrial metabolism. The ability of the muscle cyclophorase from both cardiac and skeletal muscles to oxidize all the citric acid substrates catalytically is not precisely paralleled by the mitochondria in pure suspension. There is sluggish citrate oxidation in particular (Potter, 1945; Meduski, 1950; Harman and Feigelson, 1952a) in the cardiac mitochondria and some lag in skeletal muscle. This has been associated with diminution in the apparent isocitric acid dehydrogenase activity in mitochondria, since most of the activity of this enzyme observed in homogenates is recovered in the supernatant fraction (Hogeboom and Schneider, 19%). This observation is paradoxical, since the presence of the acetate activating, condensing (von Korff et al., 1954), and other enzyme moieties of the Krebs cycle are located in the mitochondria, and it has been shown that acetate can be oxidized by the muscle mitochondria. It is possible that, in contrast to the metabolically formed citrate which is situated where it is most available within the structure of the mitochondria, the externally added substrate penetrates with difficulty and has less access to the site of the enzyme. Alternatively it has been suggested (Plaut and Plaut, 1952) that a specific cofactor is washed out of the isolated mitochondria during preparation, for which contention these authors provide some evidence. In this regard it is significant that the manner of preparing the mitochondria has considerable influence on the results of assay procedures for specific enzymes in the isolated particles

110

JOHN W. HABMAN

(Dickman and Speyer, 1954; TSOU, 1951)P This renders the results and interpretations of the intracellular distribution of particulate associated enzymes more difficult, if the relative activities of the enzymes under study are in large part determined by the assay conditions. The sluggish metabolism of citrate in isolated mitochondria is also noticed in both the cyclophorase preparations and the separate mitochondria of chimpanzee heart 16001-

Time in minutes

FIG.10. The relative oxidative capacities of the three pure suspensions M, mitochondria; S,sarcosomes, and My,myofibrils are depicted. Substrate is 40,p M alphaketoglutarate, with 0,gas phase, and the addition of 20 &M Mg.; 60 p M phosphate buffer, p H 7.5; 1 mg. DPN (85% Schwartz) in a final volume of 2 ml. Temperature is 37°C. CO, is absorbed by KOH in the center well. Oxygen uptake is expressed as microliters/mg N.

(Harman, unpublished data), whereas the whole homogenate of this tissue oxidizes this substrate vigorously. Since this cyclophorase differs in this respect from other types it is suspected that some essential component or enzyme of the integrated complex is washed out during preparation. 6 Some indications have been obtained that when isocitric dehydrogenase activity is assayed in water-bloated mitochondria the distribution of the enzyme among cell constituents is reversed.

CYTOCHONDRIA OF CARDIAC AND SKELETAL MUSCLE

111

Analogous investigations have been conducted on several varieties of insect flight and thorax muscle. Most of the studies centered around three species of Diptera-the blowfly Phorrnica regina (Watanabe and Williams, 1951, 1953), the housefly Musca domestica (Sacktor, 1952, 1953a, 1954, 1955), and the fruit fly Drosophila (Watanabe and Williams, 1953). In their dimensions the mitochondria in these insects compare with the giant

2ooo

t

Time in minutes

FIG.11. The effects on oxygen uptake by the mitochondria (M)

by the addition of sarcosomes (S). System is the same as in Fig. 10. (From Harman and Osborne, 1953.)

units of pigeon breast muscle; the mitochondria varied in size somewhat with the age of the insect and in adult Drosophila are about 2 p and in Phormica about 3 p in diameter. On the other hand the particles in pigeon breast in some instances exceed 10 p and the average is about 5 p for both the major and minor pectoral muscles (Harman, unpublished data) as the result of the mensuration of 5000 representative rodlets in both muscles. There is thus a corresponding gigantism of these particles in several types of flight muscle. In insect muscle the oxidative metabolism is distributed comparably to that of mammalian and avian types. The separation of the flight muscle

112

J O H N W. HARMAN

constituents of Phormica into washed residues, pure mitochondria, and a nonparticulate supernatant isolates nearly the total oxidative metabolism in the mitochondria (Sacktor, 1955). Catalytic oxidation of the moieties of the Krebs cycle also identifies the mitochondria as the location of the citric acid cycle and establishes the validity of the extensive documentation of component enzymes which have been worked out previously (Sacktor, 1953a, b; Watanabe and Williams, 1953 ; Levenbrook, 1953). The glycolytic enzymes are absent from the mitochondria which are by themselves therefore incapable of oxidizing glycogen and glucose (Levenbrook, 1953; Sacktor, 1954). Both the washed residues and the mitochondria resuspended in the supernatant regain a capacity for oxidizing these substrates ; recombination of the particulate fractions with the supernatant causes several hundredfold augmentation in oxidation of glycogen and all the tested derivatives of the Meyerhof-Embden system, as well as acetate. Pyruvate and phosphophenolpyruvate are also oxidized, but lactate is not, which suggests that lactate is not a normal end product of glycolysis in insect flight muscle. Although preparation of pure myofibrils is technically difficult in insect muscle it appears that admixture of the contractile elements with mitochondria induces no significant alteration of the metabolism observed in isolated particles ( Sacktor, 1955). Some beneficial influence is detected in the oxidative phosphorylation of pigeon breast mitochondria, however (Berger and Harman, 1955), by the addition of myofibrils and nuclei to the mitochondrial suspensions, perhaps owing to nonspecific colloid protection by myofibrillar proteins. The implementation of the oxidative phosphorylation resembles the effect observed with liver nuclei and mitochondria (Johnson and Ackermann, 1953; Stern and Timonsen, 1955).

VII. ELECTRON TRANSPORT SYSTEM OF MUSCLEMITOCHONDRIA The occurrence of the citric acid-cycle-integrated system of enzymes in the mitochondria of several muscle types might presuppose the occurrence also of the Keilin-Hartree prototype electron transport system. Several lines of investigation have verified the accuracy of this assumption. Perhaps one of the most convincing pieces of evidence is the demonstration that a suspension of “colloidal particles” can be obtained by fragmentation of cardiac muscle mitochondria, which behaves enzymatically like the KeilinHartree preparation (Cleland and Slater, 1953a). Certain morphological features of the shattered mitochondria have led the observers to insist that the particles are mitochondrial “membranes,” and that consequently the succinoxidase system and the electron chains are situated in the “membranes” of the mitochondria. This view is supported by the opiniori that succinate does not penetrate into the mitochondrion because of a per-

CYTOCHONDRIA OF CARDIAC AND SKELETAL MUSCLE

113

meability barrier (Cleland, 1952). Since the metabolite is effectively oxidized, the enzymes may be concentrated at the surface. This concept is at variance with interpretation by others who isolated populations of small particles by high speed centrifugation (Hogeboom and Schneider, 1952) from disintegrated mitochondria. These particles also contain the bulk of the original succinoxidase, cytochrome c reductase combined in a single aggregate. The dimensions of the particles are related to the size of the intramitochondrial ridges, determined by centrifugal force, whence it appears that this system may be located within the mitochondrial framework. The responses of the mitochondrial morphology to the variations of molarity as correlated with alterations in cytochrome oxidase activity and in the succinic dehydrogenase activity are evidence against the concept that these enzymes are situated on the surface of muscle mitochondria (Figs. 12 and 13). Furthermore the process of fragmentation is attended by augmentation of activity, which is hardly conceivable for an enzyme complex already situated at the surface in position suitable for maximal access to substrate and release of split moieties. The number of enzymes relegated to the mitochondrial surface is almost tantamount to a problem of overpopulation. Several observations that mitochondria lack membranes would lend support to this opinion (Powers et al., 1955; Weinreb and Harman, 1955) whereas such electronmicrographs as Sjostrand’s permit distribution of the system both at the surface and in the inner fibrous gel (Sjostrand and Hanzon, 1954; quoted by Glimstedt, Lagerstedt, and Ludwig, 1954). Most individual components of the chain have been identified in the intact mitochondrial system of insect muscle (Watanabe and Williams, 1951) and some of them in skeletal muscle (Chappel1 and Perry, 1953). But the Keilin-Hartree heart preparation has subserved particularly the role of prototype preparation for studies on the composition of the chain. Use of BAL has encouraged serious consideration of a labile catalytic factor situated between cytochrome b and cytochrome c of cardiac muscle (Slater, 1949). The type of inhibition resulting from application of antimycin A is similar, suggesting that this factor exists in other mitochondrial succinoxidase systems. Whether it prevails in the main pathways of respiratory catalysis is not decided. More recently a DPNH oxidase has been isolated from beef heart mitochondria as a particulate entity (Green et al., 1954; Mackler, 1955). It catalyzes the oxidation of the DPNH by molecular oxygen, and not significantly by cytochrome c. It contains copper and a flavin. The brief evidence available indicates that the complex is a natural cellular structure; on alteration by degradation with deoxycholate it splits into two reaction links which resemble those of the conventional Keilin-Hartree

114

JOHN W. HARMAN

chain, although the cytochrome c is not present in the parent particulates. A DPNH oxidase is reported in protozoa, which may have similar properties to that from the heart (Eichel, 1955) since BAL and antimycin A inhibit each type of oxidase.

0

0.250 0.500 Molarity of sucrose

0.750

FIG.12. The influence of osmotic gradient on the mitochondrial crescent population and the concomitant cytochrome oxidase activity. Mitochondria isolated in 1.0 M sucrose, resuspended in the various levels of molar sucrose and the cytochrome oxidase activity determined by the method of Straus (1954). Samples concurrently were fixed at the end of the incubation period with formalin (as in Kaltenbach and Harman, 1955) and differential counts performed by phase microscopy. Cytochrome oxidase activity is determined on unit basis; one indophenol unit is that amount of indophenol blue formed by interaction between 100 mg. alphanaphthol and 100 mg. of diphenylene diamine (HCI), with 0.6 ml. of potassium dichromate as oxidant a t 30°C. in a final volume of 1.5 ml. Extraction of the indophenol blue is accomplished in the enzyme preparations by an actone-ethanol mixture, 2:1, which stops the reaction and elutes the dye from the protein coagulum. (In high sucrose concentrations the sucrose causes turbidity which is removed by centrifugation.) In the data presented the 100% cytochrome oxidase activity is taken as that found in distilled water. Cytochrome c is applied in concentration 0.1 ml. of 5 X 104 in a final volume of 1.5 ml. Crescent population is given as that actual concentration observed in the suspensions; if the water suspension is accepted as 100% the crescent curve and the activity curve overlie.

CYTOCXONDRIA OF CARDIAC AND SKELETAL MUSCLE

115

Verification of the importance of the DPNH oxidase is imperative, if it is regarded as a principal pathway for oxidation of several substrates. Although it relieves the usual electron system of some strain, it may also require revaluation of the recently proposed action of tocopherol. This vitamin has been alleged to affect the skeletal muscle electron chain reactions in several ways, some of which are incompatible with one another

l2O0r

-

\e

1.000 i c \

100

-

z

El

80 3 c

E

-

2a6

ul

9

60

a

2 E 0 al

200

t-

b

I

I 0

1

I

0.2

0.4

I

I

0.6 0.8 Molarity of sucroses

I

-

40 n.

-

20

I

0

ti

1.0

FIG.13. The activity of succinic dehydrogenase is analyzed in the mitochondria suspended in various osmolar levels of sucrose. The mitochondria of pigeon breast muscle are prepared in 1.0 M sucrose and aliquots dispensed into several molar levels of sucrose. The succinic dehydrogenase activity is determined by the method of Kun and Abood (1949) using neotetrazolium as the dye. Standard curve for this was obtained using 1% hydroxylamine BCl as the reductant in place of the capricious sodium hydrosdlfite. The E h of the hydroxylamine at alkaline pH is suitable for the reaction. Double extraction of the precipitated protein is required to secure complete removal of the reduced neotetrazolium into the solvent. (Houchin, 1942 ; Babinski and Hummel, 1947; Rabinovitch and Boyer, 1951). The most recent study demonstrates that the cytochrome c reductase of skeletal muscle is stimulated at least sixfold on addition of tocopherol to the test system (Nason and Lehman, 1955). Extraction of the enzyme fraction with isoijctane eliminates a component, for which only tocopherol can substitute as a means of restoring the activity of the enzyme to its initial rate. The system is also sensitive to antimycin A, which indicates the presence of an additional factor in the soluble system. How this may be related to the Slater factor is not yet apparent. The pro-

116

JORN W. HARMAN

duction of experimental muscular dystrophy (Mason, 1951) by vitamin deficiency may hinge on the disturbance of this particular enzymatic reaction, provided it represents a dominant pathway associated with structural maintenance and anabolism. If the DPNH oxidase shunts past this system in the normal muscle its significance in muscular dystrophy is considerable.

VIII. OXIDATIVEPHOSPHORYLATION OF MUSCLEMITOCHONDRIA The occurrence of phosphorylation as an obligatory concomitant of electron transfer was initially postulated by the Russian workers (Belitzer and Tsibakowa, 1939). The responsibility of the intermediate steps of electron transfer as the points for the conversion of the energy types has been appreciated by these workers in its broader implications. This has been subsequently substantiated by Ochoa ( 1941) who corrected the estimated efficiency of the process and also reviewed the evidence for substrate linked phosphorylation. Although the ratios of phosphorus esterified to oxygen consumed indicate that the connection occurs in the electron chain, some time elapsed before the experiments were devised which demonstrated this association (Friedkin and Lehninger, 1949). In cardiac muscle mitochondria it is found that both cytochrome c and oxygen allow the same P/O ratios (Slater, 1950), which would restrict the process to the region below the cytochrome c6 and impose a limit on the total phosphorylation capacity below that suggested by other evidence (Hunter, 1951). The Cambridge group prefers to believe that the higher ratios obtained in other laboratories may result from inadequate temperature equilibration during manometry (Slater and Holton, 1954). This is further averred by the similarity of the P: a-Kg ratios by both the Cambridge experimenters and others who establish higher ratios in their studies (Copenhaver and Lardy, 1952). However, more recently that argument has been considerably weakened by the counterevidence that skeletal muscle mitochondria, stabilized with albumin and without fluoride, attain a P :a-Kg of 3.77,in a malonate blocked system (Harman, 1955 ; Berger and Harman, 1955). Moreover, other direct experiments with ferrocytochrome c as hydrogen donor have established that phosphorylations may Occur in the cytochrome oxidase region (Neilson and Lehninger, 1954), and, although this evidence is obtained with a liver preparation, it has been indicated by the workers above that these particles and cardiac mitochondria have essentially the 6 The question of the participation of cytochrome b in oxidation of DPNH and succinate 'by mitochondria is not pertinent in intact mitochondria where the reaction is operative (Chance and Williams, 1954) although it may not so function in mitochondrial fragments (Slater, 1955a).

CYTOCRONDRIA OF CARDIAC AND SKELETAL MUSCLE

117

same role in oxidative phosphorylation mechanisms. It is likely therefore that the phosphorylations are distributed over the chain and not solely between the substrate and cytochrome c, although a reasonable reserve still restrains observers from unequivocal acceptance of this (Slater, 1955a). Moreover, evidence presented concerning “cross-over” points of the electron chain during enhanced oxidative phosphorylation in intact mitochondria (Chance and Williams, 1955) favors cytochrome c, DPNH, and cytochrome b as the sites of phosphorylations. There has also been considerable difference of opinion as to what constitutes the “specific nucleotide acceptor” for oxidative phosphorylation in the muscle mitochondria. Close scrutiny of the mechanism in several laboratories (Lindberg and Ernster, 1952; Slater and Holton, 1953) with cardiac mitochondria indicates that ADP is the primary nucleotide acceptor. With sufficient quantities of hexokinase imposed on the system, the action of ATPase is excluded from participation and the diversion of H P eliminated. This indicates that ATP is the compound formed and not succinylphosphate as intermediate. The entrance of AMP into the process is believed to be mediated by myokinase, which may vary in operative capacity from one animal heart to another (Slater and Holton, 1953). On the other hand it has been stated that AMP can also be directly phosphorylated even though the rate is only 5% of that at which ADP is employed as the acceptor (Lindberg and Ernster, 1952). However, in view of the work concerning the pyrophosphorylation of acetyl-Co-A (Lipmann et al., 1952) a mechanism for the pyrophosphorylation of AMP is extant. It is likely (Lindberg and Ernster, 1954) that there is opportunity for both paths, with the ADP normally responsible for the bulk of the phosphorylation capacity of the system. The considerable participation of myokinase in the regulation of mitochondria1 oxidative rates has been verified in other types of mitochondria (Siekevitz and Potter, 1953, 1954; Siekevitz, 1954). This may be similar in skeletal muscle mitochondria, where myokinase is plentiful (Kitiyakara and Harman, 1953;Perry, 1952; Sacktor, 1953a), although it has been claimed that myokinase activity is slight in cardiac mitochondria ( Slater and Holton, 1953). The segregation of oxidative enzymes and consequent phosphorylations in the mitochondria of cardiac muscle (Harman and Feigelson, 1952c; Cleland and Slater, 1953a), pigeon breast muscle (Harman and Osborne, 1953;Chappell and Perry, 1953), and insect flight muscle ( Sacktor, 1955), and their absence from the myofibrils indicates that the morphological and functional state of the myofibrils is ultimately dependent on the cytochondrial metabolism. The value of the oxidative metabolism in cell economy resides in the efficiency with which its utilizable energy is trapped

118

J O H N W. H A R Y A N

in the form of high-energy phosphate linkages. Some of this energy may be diverted to the perpetuation of the system’s own structure (Harman and Feigelson, 1952c), and part coupled to the mechanism of myofibrillar contractility (Harman and Osborne, 1953). Considerations for the critical importance of the high-energy reservoir in so many cellular processes has oriented attention toward examination of the efficiency of the process of oxidative phosphorylation in both isolated mitochondria and in muscle function. Much emphasis has been laid upon the environmental factors and preparative procedures essential for the stabilization of mitochondria with competent phosphorylating capacities, It is observed that the application of ions, e.g., fluoride ions, which avert the influence of deleterious phosphoryloclastic enzymes, enhances the yield of high-energy bonds even at the expense of limiting oxidation somewhat, and that morphology is simultaneously benefited (Harman and Feigelson, 1952~).Inclusion of citrate, which is only slowly oxidized, exerts, probably by its mildly chelating action, an effect comparable to the more tenacious agents. Employment of EDTA achieves a dramatic stabilization of oxidative phosphorylation in cardiac mitochondria (Slater and Cleland, 1952). This observation has led many workers to include a chelating agent routinely during the isolation steps and as an adjuvant in the determination of P/O ratios, on the contention that it neutralizes an injurious contaminant, bound to the mitochondria, which has been identified as calcium (Slater and Cleland, 1953). The ability of EDTA to insure both morphological and metabolic stability in the isolated mitochondria has been examined thoroughly (Slater and Cleland, 1953; Cleland and Slater, 1953a, b) and exploited as a most beneficial tool for the determination of phosphorylation mechanisms. The chelating agent permits the mitochondria to survive thermal transformation without morphological deformity, but is incapable of preventing osmotic reactivity (Cleland, 1952 ; Harman and Kitiyakara, 1955). This chelating effect, by which the influence of ambient temperature is excluded, obviates the necessity of refrigeration and lends maneuverability in exploration of permeability and metabolic phenomena (Cleland, 1952). The principal action of the agent is the chelation of calcium, which the mitochondria of cardiac and other muscles tend to accumulate in considerable concentration during isolation manipulations (Cleland and Slater, 1953a) and which incites rapid morphological and biochemical degeneration (Harman and Kitiyakara, 1955). This structural dissolution or unfolding is also seen to a lesser degree under the influence of magnesium in skeletal muscle mitochondria. From these observations it may be presumed that the inactiva-

CY’IQCHONDRIA OF CARDIAC AND SKELETAL MUSCLE

119

tion of the oxidase systems is caused by the structural disruption, as in distilled water. But recent evidence demonstrates (Harman and Miner, unpublished observations) that although the initial effect of low concentrations of calcium on the succinic dehydrogenase of muscle mitochondria is an enhanced reaction rate, as the level of calcium is increased the enzyme is suppressed (Fig. 14). (The possibility of a direct action on enzymes must be entertained.) The belief that the calcium alters the mitochondria by activating endogenous cathepsins or proteolytic enzymes can not be

n

n

0 Magnesium I Calcium

.003 M ,005 M ,007 M .OlO M Molarity of Ca++ 81 Mg+* FIG.14. The influence of calcium and magnesium ions at several levels was determined on muscle mitochondria isolated in 0.25 M sucrose by the method of Kitiyakara and Harman (1953). Succinic dehydrogenase activity was determined as in Fig. 13. Concentrations of ions mentioned represent the final concentrations in the reactant system. 0

HzO

confirmed by assay of either the amino or the SH-groups (Harman and Kitiyakara, 1955). The evidence for the various possibilities which explain the calcium transformation has led to the opinion that the ion interferes with endogenous stores of high energy P and facilitates its loss. This concept finds support in the data which show that added P and active phosphorylation provide protection to mitochondrial structure (Raaflaub, 1953a,b ; Brenner-Holzach and Raaflaub, 1954; Sacktor, 1954; Harman and Feigelson, 1952c; Cleland and Slater, 1953b; Perry, 1955). An examination of the mitochondrial morphology in the reconstructed phosphorylating systems of mitochondria, which have been preincubated CY

-

120

JOHN W. HARMAN

in the presence of calcium might advance our knowledge of ion effect on phosphorylation. It has been clearly shown (Ernster and Low, 1955; Hunter et al., 1955), that by optical density measurement the mitochondria alter in opacity, presumably because of condensation, as the return of phosphorylation occurs. The rarefaction of structure induced by the calcium stress is reversed by inclusion of A T P and manganese in the incubation medium. It has been indicated (Beyer, personal communication) that in the similar system studied by the Wenner-Gren group (Beyer et al., 1955) there is a restitution of mitochondrial morphology with the rise of phosphorylation and the percentage of active target mitochondria is appreciable. Some effect on phosphorylation has been also observed in cardiac mitochondria with the addition of manganese, although the expedient of calcium preincubation was not applied to the test system (Slater, 1955b). The latter study did not include either optical density or morphological investigations. It is possible that the manganese and A T P together supply the reorganization of mitochondrial structure which is imperative for the reintegration of the oxidative and phosphorylating systems. In this regard it may be said that the employment of the Cleland-Raaflaub technique of optical density for the determination of mitochondrial alteration is suitable for the detection of gross rarefaction and condensation of mitochondria1 mass. It would fail to register the subtle alterations in configuration, however, which have come to represent morphological states that reflect the oxidative and phosphorylating conditions of the mitochondria. This would also apply to the studies of Hollunger (1955) concerning quanidine in which it would be most important to determine whether his effects are coincident with disturbance of the mitochondrial structure. Among the environmental factors which require judicious control perhaps the most significant is the osmolarity and ionic composition of the medium (von Korff et al., 1954) in which the mitochondria are dispersed. Extensive analyses of molarity in relation to morphology, oxidation and phosphorylation in cardiac mitochondria reveal a paradoxical situation. T o preserve the native rodlet configuration it is essential to use hypertonic solutions of nonelectrolytes, such as sucrose and mannitol, in about 0.45 M concentration or higher. Yet this tonicity of osmolarity when maintained in the extracellular metabolizing system is injurious to the preservation of morphology, oxidation, and phosphorylation (Harman and Feigelson, 1952a, c ; Slater and Cleland, 1953). It is also apparent from these studies that the oxidative mechanism is considerably more sensitive to the variations in osmolarity than is the phosphorylating system. It is inferred accordingly that there is a great excess of the phosphorylating enzymes over oxidative, although there is no further evidence for this

CYTOCHONDRIA OF CARDIAC AND SKELETAL MUSCLE

121

and the conclusion that the two systems have differential fragilities is not excluded. Various oxidase systems, e.g., succinoxidase and a-ketoglutaric acid oxidase systems are very different in their susceptibility to osmotic stress. This differential fragility is always a problem in the assessment of whether the point of injury occurs in either the oxidative or the phosphorylation perimeter (Cross et al., 1948). It is certainly true, however, in regard to the a-ketoglutaric acid oxidase that these studies have established a suppression of oxidation by high osmolarity without disproportionate depression of phosphorylation, until a decreased margin of phosphorylation becomes incapable of sustaining the whole structure. On the other hand exposure of the mitochondria to hyosmolar media ultimately destroys integrated oxidation and eliminates phosphorylation concomitantly with dispersion of mitochondrial structure. There may be an initial acceleration of oxidation as the units swell and the substrate enters to saturate the enzyme, but further disruption disperses the compact inner mitochondrial gel (Weinreb and Harman, 1955) and leaches out the essential cofactors (Green, 1951). Cardiac and skeletal muscle mitochondria are resistant for a longer period to hyposmolar stress than are liver mitochondria (Lehninger, 1951), but eventually pass into typical degenerative forms (Harman, 19%). Whereas the manner in which hyposmosis injures the mitochondria is explicable, the cause for hyperosmotic stress damage is elusive, unless as Tyler (1954) recently suggested, the finer gels of the mitochondria are independently susceptible to osmotic reactions, as is the Keilin-Hartree preparation, where morphological osmotic barriers are not operative (Slater and Cleland, 1953). The investigations on morphology of insect muscle mitochondria in &tro has led to the conclusion that not only is the tonicity of the medium important but also the addition of certain proteins is imperative for preservation of form (Watanabe and Williams, 1953). This has induced other workers to employ bovine albumin in the studies of oxidative phosphorylation in insect muscle and the development of systems which assures a high reproducible P/O ratio (Sacktor, 1954). The level of albumin required is considerable and exceeds the normal proportion of this type of sarcoplasmic protein as determined by extraction of whole muscle (Bailey, 1944), although the regional concentration in the interfibrillary fluid may be high. On the other hand, in contrast with the benefit obtained in cardiac mitochondria by employing EDTA the insect particles are not only inhibited from phosphorylating but prevented from oxidizing ( Sacktor, 1954), which would substantiate the observation (Watanabe and Williams, 1953) that the mitochondria of Musca domestica withstand addition of EDTA poorly. Both calcium and manganese ions completely abolish the

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JOHN W. HARMAN

albumin stabilized phosphorylation of the insect particles and induce considerable depression of oxidation, which is augmented, on the contrary, by the presence of magnesium. This manganese effect is in contrast to the benefit the metal confers in the reconstruction of phosphorylation in preincubated liver mitochondria, where it is essential for the reestablishment of the phosphorylating mechanism after calcium transformation is initiated (Ernster and Low, 1955). These investigations on insect muscle mitochondria have been extended (Lewis and Slater, 1954) by scrupulous control of the osmolarity, restudy of the action of EDTA, and assessment of the significance of added albumin in the blowfly Calliphora erythroceplzaka. In this insect EDTA provides a stabilizing isolation medium which affords protection to the phosphorylating system, whereas further addition of albumin gives no suggestion of improvement in phosphorylation, although the oxidation is increased considerably. In general the P/O ratios of this insect are lower than those obtained with M . domestica (Sacktor, 1954). Further work is certainly needed in order to reconcile the discrepancy that is manifest in the effect of EDTA on the mitochondria of these two insects, which are in other respects similar. In view of the satisfactory results obtained with the EDTA chelation in oxidative phosphorylation of cardiac muscle and the conclusions (Watanabe and Williams, 1953) that proteinaceous media aid morphology, the conditions conducive to the stabilization of phosphorylation in pigeon skeletal muscle have been reinvestigated (Berger and Harman, 1954, 1955 ; Harman, 1955). Circumstances of homogenization, osmolarity, ion content, fluoride incorporation, and addition of EDTA (or sequestrene) and of bovine albumin are systematically analyzed. In contrast with the value placed on the Waring blendor for isolation of healthy mitochondria from disruption of viscera, such as liver and kidney, this instrument is the most suitable technique for dispersing pigeon breast muscle. The action of this procedure resides chiefly in the shearing force of the differentially rotating liquid layers, which slide the myofibrils apart and allow the exposed mitochondria to shake free into the medium without direct stress upon the units. On the other hand with the all glass homogenizer, the compactness of the pestle and tube distribute the force over the compressed muscle mass, twist and spread the myofibrils, and comminute the mitochondria in sitzc. Subsequent differential centrifugation is incapable of separating the fine myofibril fragments from the mitochondria, so that contamination is considerable. Comparative studies of the mitochondria obtained from muscles dispersed by both techniques and by Latapie mincing establish the superiority of the blendor method for isolation of mitochondria which actively phos-

123

CYTOCHONDRIA OF CARDIAC AND SKELETAL MUSCLE

phorylate (Berger, 1954, unpublished observations). This precludes acceptance of the dictum (except, perhaps, with reference to liver) that “absolute preference should be given to homogenizers of the type described by Potter and Elvehjem” (de Duve and Berthet, 1955). The components of the suspending medium also considerably alter P/O ratios. Both EDTA and albumin considerably enhance the P/O ratios with all the Krebs cycle substrates used, although albumin is preferable to the EDTA in certain respects and eliminates the capriciousness hitherto encountered in the ratios obtained (Tables VI and VII). It has also

TABLE VI OXIDATIVE PHOSPHORYLATION OF PIGEON BREASTMITOCHONDRIA SUSSTUTE : U-ICETOGLUTAXATE ~~~~~

~

Flasks without Albumin AP A 0 P:O ~

EXp2I-iment 1 2 3 4 5 5

+ .01 M

~

~

Average

P:o

~

~

~~

Flasks with Albumin

AP

A 0

P:o

18.8 30.4 23.8 26.4 45.9 32.4

7.1 13.8 8 10.8 18.4 10.1

2.65 2.20 2.98 2.45 2.48 3.24

~

Average

P:o

~

pM

Microatoms

6.4 17.4 5.6 9.1 18.0 23.0

6.6 12.6 6.9 8.7 8.5 9.0

.97 1.38 .81 1.12 2.10 2.55

1.58

2.53

NaF

NOTE:Average mitochondrial N 0.59 mg. per flask. The flasks contained 45 p M of substrate, 50 pM phosphate buffer, pH 7.4, 20 pM MgSO,, 6.5 pM ATP, 0.4 ml. of the mitochondrial suspension; 0.4 ml. purified yeast hexokinase and 60 pM glucose in the side arm. The final volume was made up to 2.0 ml. with 0.15 M KC1-Bovine serum albumin was added at a final concentration of 0.17%. Incubation at 30°C. for 20 minutes.

proven beneficial to include KCl, along with other components, in the suspending medium (Fig. 15). By using fluoride and malonate the theoretical maximum for a-ketoglutarate oxidation is approximated. The mitochondria in pigeon breast differ from those of insect muscle in the absence of EDTA effect on the system unless it is blocked by malonate. Albumin, however, is efficacious in both open and blocked systems. As in suggested for the insect preparations (Slater, 1955) it appears that albumin is a true activator. It may act in a manner similar to its reversal of antimycin inhibition on succinoxidase (Reif and Potter,1953) or it may be capable of fusion with the surface of fine mitochondrial matrix gel which it protects against denaturation. It is certain that albumin is specific, since other

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JOHN W. HAXMAN

TABLE VII EFFECT OF SEQUESTRENE AND BOVINEALBUMIN ON PHOSPHORYLATIONS ASSOCIATED WITH

MALONATB B ~ o ~ g g a-KET0GLUT-m n

OXIDATION

c

Experiment 1

Additions None 10-6 M sequestrene

2 3 4

5

None 10-6 M sequestrene None 0.17% allbumin None 0.17% albumin 0.17% dbbmin 0.17% albumin .01 M NaF

A€’ A 0 AKG ILM microA p.M

P:O

P:KG

7.1

1.01 3.19 1.83 2.25 1.79 3.10 1.54 2.83 3.03 3.48

1.66 2.70 3.77 3.45

7.2 33.5 15.6

20.6 8.4 29.9 8.3 18.4 22.3 24.6

10.4 8.5 9.2 4.7 9.7 5.4 6.5 7.4

7.1

5.0 6.8 5.9 7.3

0:KG

1.08

0.96 1.25 1.0

NOTE:Average mitochondrial N 0.71 mg. per flask. 45 pM of a-ketoglutarate, 20 pM malonate in all flasks. Other flask constituents as in Table I. Incubation at 30°C. for 20 minutes.

n

k

[o :C01U Y

!5 M :rose

HtO

0.15 M NaCl

0.15 ul KCI

FXG. 15. The effect of flask diluents on P/O ratios. The basic contents of the flasks per ml. are: 25 p M A T P ; 2.5 p M alphaketoglutarate; 25 p M glucose; 0.2 ml. of the mitochondria suspended in 0.25 sucrose, Hexokinase (Pabst) is tipped in from the side arm. As diluents appropriate amounts of the fluids indicated in the columns are added to bring the final volumes to 2 ml. The ratio found for 0.25 M sucrose is accepted as 100%. Each column represents the average of five experiments. In all experiments the trend was identical, the magnitude slightly different.

CYTOCHONDRIA OF CARDIAC AND SKELETAL MUSCLE

125

proteins applied are either inert or detrimental (e.g., protamine). The influence of the various constituents of the medium alone and in combination, illustrates the efficacy of the albumin, ATP, and sucrose on the preservation of morphological integrity (Fig. 15a) in nonmetabolizing RODS

90t 80 v)

SPHERICAL DENSE

-

0CRESCENTS

L:

a 70-

z

-I

a

60-

0

p

50-

0

g 40f

* 30eo

ALB.

ATP

KCI

PC .

FIG.15a. The mitochondria in all experiments were prepared in 0.25 M sucrose. Aliquots of 0.1 ml. of each suspension were dispersed in 2.5 ml. of fluids of various composition and allowed to incubate for 30 minutes at 20°C. before performance of differential counts. The control in sucrose was maintained at 0°C. until counted. Symbols under the different columns signify the fluids used as follows: S, 0.25 M sucrose at 0°C.; ALB, a mixture of equal parts of 0.25 M sucrose and 0.34% serum albumen; ATP, 0.125 M sucrose containing 4 pLM A T P ; KCl, 0.154 M potassium chloride; P04, potassium phosphate buffer, 0.1 M, p H 7.2; P04/ALB, the 0.1 M phosphate buffer contains 0.17% bovine serum a h m e n and finally H,O is doubly distilled water, not buffered. Each column represents the results of four experiments.

units. It is observed that the combination of sucrose, ATP, KCl, and albumin (0.7%) with phosphate buffer 7.4 pH, allows appearance of numerous target mitochondria (Fig. 19), at total osmolar concentration of 0.30 M.r 7 The osmolar concentration or the total molar concentration of all molecules and ions is defined as: osM = 8 (@f) where osM signifies the osmohr concentration; 11 is the number of particles per molecule and M is the wlarity of the substances. It is assumed that the osmotic coefficients of all the substances are unity under the conditions of study.

126

JOHN W. HARMAN

IX. DISTRIBUTION OF ENZYMES ACTINGON PHOSPHORYLATED NUCLEOTIDES The elaboration of the high-energy phosphate bonds has attained a critical role in energy conversions associated with the maintenance of biological systems. The transfer of this bound energy is mediated principally if not exclusively through the turnover of ATP, which can act as a primary source of energy. There are in tissues many enzymes or kinases which subserve the purpose of appropriately redistributing the WP from ATP to special acceptors. On the other hand there are also various enzymes concerned with the splitting off of the terminal WP from ATP with dissipation of energy and without recognizable synthetic reaction. Some of these are located in the mitochondria in proximity to the integrated system responsible for the elaboration of the ATP. This would imply that under optimal metabolic conditions the ATP-splitting enzymes, or ATPases, are inert or restrained, and evidence has been accumulated to verify this view (Keilley and Keilley, 1951;Potter and Recknagel, 1951) for liver mitochondria (see also Lardy and Wellman, 1953). Appreciation of the occurrence of this type of ATPase in muscle is due to Keilley and Meyerhof (1948,1950) who isolated the enzyme which is distinct from that found in the myofibrils, is characterized by magnesium stimulation, and is of lipoprotein nature. Association of the magnesium ATPase and myokinase with cellular, lipoprotein particles in rat and rabbit has followed (Perry, 1952), although the precise nature of the particles is not verified. That they are lipoprotein and consistent with the mitochondria is inferred. The mitochondria of pigeon breast (Kitiyakara and Harman, 1953) are found to contain a rich concentration of this type of ATPase and myokinase. This has been independently confirmed (Chappell and Perry, 1953). Selective isolation of the mitochondria in both studies establishes them as the locale of the enzyme. In the determination of the distribution of enzymes dephosphorylating ATP the expedient of separating cellular particles into pure suspension has obviated the necessity of summation studies, such as are employed in analysis of oxidative systems. With the ATPases this separation of particles is prerequisite for localization, since the application of the ions on the homogenate has diverse effects on the calcium- and magnesiumactivated enzymes in muscle and the isolated particles. By the fractionation technique a characteristic magnesium-stimulated ATPase, as indicated above, is identified in the mitochondria of pigeon breast (Kitiyakara and Harman, 1953 ; Chappell and Perry, 1953),insect flight muscle ( Sacktor, 1953a), and heart muscle (Harman, unpublished

CYTOCHONDRIA OF CARDIAC A N D SKELETAL MUSCLE

127

observations). In the presence of an optimal level of activating ion the rate of dephosphorylation in skeletal muscle mitochondria is considerable and approximates a QPN of 21,000, greatly exceeding the highest turnover of the myofibrillar calcium-activated ATPase at its optimal level. In all studies the ion effects are found comparable and the point of optimal stimulation is about the same level, although there is some difficulty in exact comparison because the criteria of activity used differ slightly. One worker expresses his results as mg. P/hr./mg. dry weight, another as Qp(N) which represents the P liberated as microliters of gas (at NTP) per hour by an enzyme preparation containing 1.0 mg. of nitrogen, and a third has converted the nitrogen value of the preparation to protein equivalent and calculates the result as mg. P/mg. protein. Irrespectively the relative areas of maximal stimulation operate in the same’short range. There is also agreement to the fact that calcium produces a slight and poorer response compared with the magnesium and that a much higher ionic concentration of calcium is required. The specificity of this enzyme has been assayed by testing a range of phosphorylated substrates, from which it is obvious that the only substrates used are ATP and, to a lesser extent, ADP. The dephosphorylation of ADP is not appreciable in the absence of magnesium ions in either insect or pigeon muscle mitochondria. This dephosphorylation of ADP is shown to arise from the conversion of the ADP first into A T P and AMP under the action of myokinase with subsequent splitting of orthophosphate from the ATP obtained by the dismutation. The early lag encountered during the dephosphorylation is explained as the zone of initial myokinase participation with buildup of substrate for the ATPase. The myokinase present in skeletal muscle, insect thoracic muscle, and heart muscle may be implicated in the mechanism of primary phosphorylation on occasion when the ADP level is depleted and acceptors are in demand. Although it is well established that the muscle mitochondria are rich in specific ATPase, the function of the enzyme has eluded explanation. The absence of ATPase activity or its insignificant rate in carefully prepared mitochondria from liver have induced some to interpret it as a consequence of mitochondria1 damage which activates a “latent” mechanism (Potter and Recknagel, 1951). An enzyme which is normally a transferase, under new environmental conditions is enabled to react with water and hydrolyze one of its substrates. At the osmolarity of 0.25 M sucrose conventionally used for such studies on liver mitochondria the enzyme activity is minimal, but as the molarity diverges away from this range toward either hyperosmolar or hyposmolar conditions the ATPase activity is much increased above the so-called “latent” level. Correlation of this variation of activity

128

JOHN W. HARMAN

attendant upon osmolar fluctuation with the morphology of the mitochondria indicates that the structural configuration is the determinant factor involved (Kaltenbach and Harman, 1955). Solubilization of the enzyme is excluded (Novikoff et d., 1952; Kaltenbach and Harman, 1955). This would imply that in the tissues, where the rodlet shape is observed in the osmolar zone above 0.4 M sucrose, the ATPase is capable of substantial activity for the reason that the cryptic enzyme is more accessible in the rod than in the sphere. Participation of the enzyme in cellular phenomena cannot be eliminated, since the b vivo rod forms are very active. The sluggishness of the ATPase of the mitochondria of liver isolated in the osmolar range of 0.125 M to 0.25 M sucrose is not the maintenance of a natural enzymatic inertia, but is a result of structural deformation which imposes a diffusion gradient previously absent. The mitochondria isolated from muscle are not latent at most osmolar levels (Kitiyakara and Harman, 1954) but manifest a zone of minimal activity identical with that seen in liver particles (Chappell and Perry, 1953). Cardiac mitochondria on the other hand more closely simulate the behavior of those from liver. In skeletal muscle the magnesium-activated ATPase is almost inextricably united to the hull of the mitochondria and defies extraction by drastic procedures such as thorough fragmentation and salt treatment which remove large amounts of protein (Harman and Kitiyakara, 1955). This strict attachment of the enzyme to the structure of the particles substantiates the observations that a similar state exists in liver mitochondria (Novikoff et al., 1952; Kaltenbach and Harman, 1955). On the other hand there have been claims of considerable solubilization of the liver enzyme by both simple and vigorous manipulations (Keilley and Keilley, 1953; Lardy and Wellman, 1953). The close conjunction of the ATPase with the mitochondria1 structure has permitted it to be employed as an index of the morphological condition of the units. In the process of application of uncouplers, such as dinitrophenol, usnic acid, gramicidin, and Janus green B, the morphology of the particles is altered, with formation of crescents and degenerate forms which represent a state associated with both the loss of phosphorylation and the increase in ATPase activity. It is not valid to imply that the uncoupling is a consequence of the ATPase intervention which vitiates the accumulation of C.IPfor several reasons. In the first instance the phosphorylations derived from anaerobic dismutation of alphaketoglutarate are resistant to the effect of uncouplers (Hunter, 1951). And it is unlikely that ATPase eschews the ATP derived from this source and from substrate-linked phosphorylation with a preference for that obtained from the

CYTOCHONDRIA OF CARDIAC AND SKELETAL MUSCLE

129

electron chain. Furthermore the rate of incorporation of inorganic phosphate in the course of oxidative phosphorylation is inhibited by DNP (Lehninger, 1949) which suggests that ATP formation is impaired, and it is also known that the DNP inhibits the turnover of the oxygen of the inorganic phosphate in the uncoupling action (Cohn, 1953; Boyer et ul.,

1955). A possible role of mitochondria1 ATPase in normal pathways of the cellular physiology is to some extent hinted at by the influence of phosphate acceptors and turnover on oxidative rates. The mitochondria under the conditions of proper molarity and fortified with cofactors and substrate (Lardy and Wellman, 1952) oxidize slowly and in some instances not at all, for the first five to ten minutes. The addition of hexokinase and glucose as a N P acceptor system greatly accelerates oxidation. As shown in Figs. 16 and 17 this may also be observed with cardiac mitochondria (Harman, unpublished data) when hexokinase or creatine transphosphorylase mechanisms are invoked to drain away the UP from the mitochondrial ATP and provide ample ADP-primary acceptor. It is obvious also that adenylic acid operates much in the manner of the hexokinase drain. Other work concerning the intramitochondrial control of oxidation by the hexokinase and citrulline synthesis (Siekevitz and Potter, 1953) shows how the lability of mitochondria1 ATP affects oxidative metabolism as well as mitochondrial form (Raaflaub, 1953a, b; Harman and Feigelson, 1952~). On the other hand recent studies (Swanson, 1955) also have demonstrated a nonoxidative incorporation of PS into ATP by resting, so-called “latent” mitochondria, at a rapid rate. Manifest ATPase is absent under the experimental conditions. This substantiates prior observations which employed PS and Ole analysis (Boyer et al., 1954) and demonstrates an exchange of phosphate with ATP, independent apparently of oxidation. In either scheme of silent phosphorylation it is difficult to visualize the ATP nucleotide as ready to accept the available phosphate unless some mechanism first converts it to ADP, and since there is maintenance of phosphate balance this is accomplished presumably by splitting of the terminal phosphate and not necessarily through a transferase. The participation of ATPase in the reaction is feasible, although the authors propose a$ an alternative explanation a reversal of the primary steps of esterification associated with electron transport. One reason for excluding ATPase from the reaction is that the phosphate turnover may be inhibited by agents without development of manifest ATPase activity. A suitably unifying hypothesis for phosphate transfer has many interwoven facets to be considered. Both in the silent phosphorylation exchange

130

JOHN W. HARMAN

and in the acceleration of oxidation by introduction of phosphate acceptors the incursion of a dephosphorylation enzyme may be occurring at all levels of mitochondrial activity. The importance of this in muscle is more likely, because there is no truly latent ATPase such as occurs in the liver mitochondria. This may follow some anomaly of preparation, but may also signify a peculiar metabolic requirement in muscle. The ATPase is 70 r

37O c.

f

MINUTES

FIG.16. The influence of hexddnase and ATP on the rate

of oxidation of cardiac mitochondria. Mitochondria isolated from rabbit heart by the method of Harman and Feigelson (1952a), in 0.5 M sucrose. The Warburg flasks contained 0.5 ml. m i t e chondrial suspension, 6 pM alphaketoglutarate; 3 p M of either AMP or ATP; 20 pM potassium phosphate buffer; pH 7.25; 1.0 pM succinate; 20 pM MgCl,; 25 pM NaF; 30 pM glucose and 3.0 mg. lyophilized hexolcinase. Final sucrose concentration in wash flask was adjusted to osmolarity equivalent to 0.25 M sucrose by the addition of solutions of proper concentration to give a final volume of 2 ml. Gas phase was oxygen. The flasks were thermally equilibrated for 8 minutes before the hexokinase was tipped in from the side arm. Reaction allowed to proceed at 37%. for 20 minutes.

firmly united to the fibrous coiled or looped inner gel, which is presumed to be compacted by ATP. Release of ATPase activity may unlatch the A T P links and be part of the mechanism of mitochondria1 motion, which occurs as the fibrous gel is compacted and unfurled with reformation and dephosphorylation of ATP. On the other hand the ATPase may serve the interests of metabolic turnover in total muscle activity. The close collusion of the glycolytic sys-

CYTOCHONDRIA OF CARDIAC AND SKELETAL MUSCLE

131

tem and citric acid cycle is nowhere more intimate than in muscle, where the products of the former, pyruvate and phosphoenolpyruvate, are natural substrates of the latter. It has been established that an inhibition of the glycolytic cycle ensues when all the adeninenucleotides are converted to ATP, when the ADP acceptor is unavailable. Under resting conditions,

MINUTES

FIG.17. The influence of hexokinase and creatinephosphokinaseon the oxygen UPtake of cardiac mitochondria. The conditions and type of preparation were similar to those in Fig. 16. In some flasks creatinephosphokinase replaced hexokinase as the transphosphorylation system in association with 20 mg. creatine. The pH in such flasks is adjusted to 8.2 to force the back transfer of phosphate from CP to ATP. Final molarity and volume are 0.25 and 2 ml. respectively.

where silent transfer may be effective this is satisfactory, but in active metabolism such an impediment to glycolysis may deprive the mitochondria of a principal metabolite. Since the ATP of the myofibrils is firmly bound to the contractile units the ATPase of this system is unlikely to assist in breaking such a deadlock. On the other hand the ATP and ADP of the mitochondria are able to diffuse across the mitochondria1 interface and ex-

132

JOHN W. HARMAN

change freely with those in the sarcoplasm. Under proper stimulation the mitochondrial and perhaps the soluble ATPase may shift the ATP:ADP ratios with the balance in favor of facilitating glycolysis and oxidation. Some observations similar to those on electrical stimulation of brain mitochondria (Abood and Gerard, 1953, 1954) are needed to permit evaluation of the influence of stimulation in muscle mitochondria. The trigger action of ATPase is feasible as an incitant to the onset of the restitutive phase of muscular contraction. Of course the mitochondria1 density of a muscle will largely determine how important it may be. In essentially glycolytic muscles this may be replaced by the creatine transphosphorylase, which interacts between the A T P reservoir in the sarcoplasm and the ADP in the fibrils derived from the action of ATPase during contraction. This enzyme is mainly in the sarcoplasm (Kitiyakara, 1955, unpublished observations).

X. MORPHOLOGY AND MITOCHONDRIAL ACTIVITY In several previous sections attention has been directed to the importance of mitochondrial structure. In view of the increasing regard for the significance of mitochondrial integrity on the biochemid and morphological levels it is appropriate to summarize our current knowledge of this relationship. The introduction of the phase microscope has provided a tool which has enabled the student of mitochondrial phenomena to visualize, under native and experimental conditions, the alterations in mitochondria which may be correlated with other levels of mitochondrial change simultaneously. Up to the moment most of such studies have been contributed with phase microscopy (Zollinger, 1948, 1950; Harman, 195Oa, b) The examinations of muscle mitochondria with such purpose in view have been limited, but the reactions generally of muscle mitochondria parallel those of other tissues faithfully. One of the most satisfying experiences in the examination of the mitochondria by phase microscopy has been the application of this technique to the study of contracting muscle by cinematography. The intention of this approach had been to analyze the alterations occurring in the various components of the sarcomere during the different periods of contraction and relaxation (Harman, 1954). It is clearly shown that the sequence as outlined by Jordon (1920) depicted the events in reverse. The exact series of changes follows the pattern described subsequently in isolated fibers suspended in salt solution after glycerol extraction (Huxley and Hanson, 1954), with cinematography. In active contraction the I bands shorten from the resting length until they entirely disappear, whereas the A bands are unaltered in length but they come to abut against the 2 lines

.

CYTOCHONDRIA OF CARDIAC AND SKELETAL MUSCLE

133

whereas the H zone vanishes. The diameter of the fibril is not changed, which is important in regard to compression of adjacent structures. This degree of contraction shortens the fibrils by about 20% to 25% of resting length. With relaxation, which is a rapid and obviously active process, the I bands reappear at the resting length, and the A bands split, when the H zone is especially conspicuous and may reveal the M line. There are some differences between the process seen in the preparation of homogenates spontaneously contracting and relaxing and those stretched artificially by the glycerol technique. The homogenates are closer to what Huxley and Hanson (1954) speak of as a plasticized muscle and this may mean that the A T P is no longer operating upon the contractile units but merely satisfying structural requirements. But the special interest of this spontaneous contraction lies in its occurrence only in metabolizing skeletal muscle (Harman and Osborne, 1953). A second type of muscle contraction, which corresponds with the delta state of Ramsey, and the onset of contraction bands is irreversible with shortening of the myofibrils to about 40% of the resting length. During this type of contraction there is a preliminary confused jumble of the bands with an elimination of the A band except for a fine faint line and a considerable accentuation of the Z lines as if some constituents of the A bands had aligned against the Z lines. This usually is observed as the system approaches exhaustion of metabolic life in vitro. It very closely resembles the picture most often observed in fixed postmortem tissue. The events of spontaneous myofibril contraction are not associated with deformity of the adjacent mitochondria by the shuttling fibrils. In the initial stages of the study most of these are rod shaped. With the full development of the oxidative process there is a change in the mitochondria1 configuration. The rods are enspherulated (Fig. IS), and this is best seen in the free floating forms unaffected by optical distortion caused by the contracting fibrils. There is a differentiation of the sphere into two concentric zones like a target (Figs. 19 and 20), after which this form is named. It has been observed in cardiac muscle that such a form is a criterion of a high rate of oxidative phosphorylation, and that the degree of phosphorylation may be assessed by enumerating them with differential counts (Harman and Feigelson, 1 9 5 2 ~ ) .Later studies on the relationship of the spontaneously contracting myofibrils and the population density of the target forms have established that they are both either present or absent simultaneously, that factors which perpetuate one support the other, e.g., fluoride, and that substances which eliminate one destroy the other, such as DNP and other uncouplers (Fig. 21). Since the myofibrils contain no systems for formation of ATP, which the mitochondria do, and the

134

JOHN W. HARMAN

targets are most active during oxidative phosphorylation it is apparent that they provide the myofibrils with the required W P for their function. On the other hand the glycolytic system is incapable of supporting contraction in vitro. When the target forms vanish and are replaced by a type called a crescent, which is also induced by hyposmolarity (Fig. Z Z ) , the spon-

FIG.18. A mixture of long and short rodlet mitochondria are shown in a suspension of mitochondria from pigeon breast muscles in 0.25 M sucrose. A few early crescents are present. Magnification 1400 X .

taneous contractions are no longer observed, but are followed by the irreversible contraction bands. Omission of cofactors, as DPN and magnesium, or low substrate levels permits early disappearance of targets and failure of the contractile system. In the muscle cell the mitochondria maintain a rod configuration. This has not only been clearly depicted by the classical studies of fixed prepara-

CYTOCHONDRIA OF CARDIAC AND SKELETAL MUSCLE

135

tions but has recently been verified by phase microscopy of teased and muscle homogenates (Fig. 18). By cinematography with phase microscopy it has been established that during the events of muscle contraction by the adjacent myofibrils, the mitochondria are inert and not affected by the movements of the sarcomeres ( Harman, 1954). When the mitochondria are expelled from between the fibrils by homogenization they maintain

FIG.19. A target type mitochondrion from a suspension of muscle mitochondria suspended in metabolic mixture containing alphaketoglutarate as substrate and the flask contents specified in Fig. 10. Magnification 1400 X.

the rod shape (Fig. 18) if the niolarity of the medium is satisfactory and refrigeration excludes the onset of autolysis. The spherical shape of muscle mitochondria obtained by several groups during isolation procedures (Watanabe and Williams, 1951, 1953 ; Cleland and Slater, 1953b) is a departure from the native appearance. The molarity of the media used by these workers is designedly rendered isosmotic with the serum of the species, being at the 0.25 to 0.35 M sucrose region. It has been shown however that the normal intracellular osmolarity in visceral and muscle cells (Opie, 1954) is considerably higher than that of the interstitial fluid and the serum. It is apparent that the manner of isolation initiates hyposmotic stress. And still this is not the entire story, because the preparation of pigeon skeletal muscle mitochondria in 0.25 M

136

JOHN W. HARMAN

sucrose permits the extrusion of mainly rod shaped mitochondria. It is perhaps that in one instance the comminution procedure (Cleland and Slater, 1953b) is abrasive, owing to grinding with sand, and in the other the interval of isolation (Watanabe and Williams, 1951, 1953) is not safeguarded by refrigeration. Earlier investigations with cardiac mitochondria (Harman and Feigelson, 1952a, b) have illustrated that cardiac mito-

FIG.20. A type of target sometimes seen when the ends of the mitochondria1 rodlet fold over, causing central groove. Metabolic conditions as in Fig. 19. Magnification 2700 X.

chondria freed from the ruptured cells and dispersed into 0.25 M sucrose may assume a spherical shape, whereas when they are released into 0.88 M and 0.4 M sucrose they persist as elongated rodlets, provided refrigeration is continued. It might be pointed out parenthetically that spherical mitochondria are rarities in carefully prepared electron micrographs of skeletal muscle (Palade, 1952; Chapman, 1954 ; Weinreb and Harman, 1955; Weinstein, 1954). Extracellular treatment with hyposmotic agents produces considerable distortion in the electron micrographs in muscle mitochondria isolated and sectioned after fixation with osmic acid (Weinreb and Harman, unpublished). This alteration begins to appear when the molarity falls to about 0.35 M sucrose, as Novikoff (1955) has also demonstrated in liver mitochondria.

CYTOCHONDRIA OF CARDIAC A N D SKELETAL MUSCLE

137

A series of phenomena called transforming agents have been analyzed in order to clarify the conditions peculiar to the crescent degeneration (Fig. 22) and the biochemical alterations which attend it (Harman, 1950a). These agents have been classified into categories according to their physical or chemical action on the particles. The measure of their activity is threefold, (1) crescent formation, (2) splitting out of pyridino0

Whole hornogenate at zero time

A

No substrate 2 micromoles a-ketoglutarate/ml.

8

20 micromoles a-ketoglutarate/ml.

A

0

40

80 120 Time in minutes

160

FIG.21. The behavior of targets in whole homogenates is demonstrated. The mitochondria1 counts are performed as described by Harman and Feigelson (1952a). Each point specifies the time of removal of the flask from the manometer for the performance of the count during the phases of oxidation. (From Harman and Osborne, 1953). proteins, and (3) leaking away of adenine nucleotides, with activation of enzymes which convert the nucleotides into residues. Side effects are influx of water, loss of electrolyte binding, and some release of enzymes such as acid phosphates in liver mitochondria and a slight amount of ATPase in muscle (Harman and Kitiyakara, 1955). These transformers are conveniently subdivided into five classes depending upon the mode of action: (1) osmotic, (2) thermal, (3) solvent, (4) specific ionic agents, and ( 5 ) mechanical. Expedients may be applied to obviate the action of the various transformers which combine to induce rapid degeneration in

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mitochondrial suspensions. Refrigeration delays thermal change. Hyperosmolarity diverts any osmotic effect, E D T A eliminates some ionic transformers, gentle treatment and use of mild dispersive procedure during isolation obviates the mechanical injury, and finally the suspension of the mitochondria in nonelectrolyte aqueous solutions prevents solvent transformation, an effect which electrolytes may have on proteins and liquid solvents may have on the lipoproteins. By the examination of the deleterious transformers it has been possible to establish conditions conducive

FIG.22. A mitochondria1 suspension derived from pigeon breast muscle was prepared in 0.25 M sucrose and after centrifuging the particles the pellett resuspended in distilled water for 30 minutes. Examined and photographed by phase microscopy. Dark L contrast used. Magnification 2700 X.

to the perpetuation of better morphology and biochemical activity in mitochondria. O n the other hand by judicious use of selected transformers the insight into the mechanisms of intramitochondrial energy transformations has been rewarding. I n the instance of calcium, which is a specific ionic transformer, the swelling caused by minute amounts of this ion is rapid and pervades the whole mitochondrial population (Harman and Kitiyab r a , 1955) and it has been shown that the ion is responsible for the failure of the a-ketoglutarate oxidase system and the cessation of phosphorylation (Slater and Cleland, 1953). EDTA is an efficient prophylactic for protection of both morphology and phosphorylation. The opinion has been expressed that the transformation phenomena are the consequence of changes in a mitochondrial membrane (Cleland and Slater, 1953b). However the reactivity of the mitochondria to fluctuations in the molarity of the medium even with rents in the interface indicates that the surface plays a limited part in the morphological alterations. On

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the other hand the swelling and shrinkage which occur with variation in the pH of the medium, in the absence of other linkage changes, suggest that hydrogen bonds within the inner gel may exert a considerable influence on the mitochondrial configuration ( Harman and Kitiyakara, 1955). The inability of the mitochondria to return to the primitive shape after transformation is not caused by loss of osmotically active constituents alone, but is a result of the rupture of inner linkages. The pathological phenomenon of cloudy swelling has been associated with the distention of the mitochondria of many tissues (Opie, 1947a, b ; Zollinger, 1950; Harman, 1950a). In muscle mitochondria the process is identical and is attended by the disassociation of oxidation from phosphorylation. The in vitro studies of the process have been reexamined with the use of mitochondrial preparations obtained from the muscles and hearts of animals intoxicated with diphtheria and tetanus toxins, which are capable of inducing cloudy swelling (Fonnescu and Severi, 1953, 1954 ; Michelazzi et al., 1955). These observations confirm the in vitro analyses and establish the concomitant depression of phosphorylation in the crescent or in swollen mitochondria from such animals. Another approach has been adopted (Billings and Harman, unpublished observations) by the injection of plasmocid into different animals and subsequent examination of various muscles to determine the relationship between the mitochondrial density and the incident necrosis. In view of the action of plasmocid on certain oxidative systems (Hicks, 1950) it might be assumed that the vulnerability of the muscle would be proportional to its dependence on the oxidative system and the mitochondria. As expected there is a liability on the part of the granular fibers although the muscles which were exceptionally rich tended to escape doses much below the lethal level. In this regard the heart is much less susceptible than the skeletal muscles (Puletti, unpublished observations) .E The order of damage coincided in general with the mitochondrial density or rather with the proportion of mitochondria-rich fibers in the muscle. It is apparent that the mitochondria not only provide a rich supply of energy to the contractile process, but that they are also able to alter its reaction to injury and toxins. It is likely that this difference may affect the variable response of muscle to induction of dystrophy. 8 On the other hand the dystrophy induced by cortisone in the rabbit (Ellis, 1955) has a predilection for some granular muscles but avoids others (Billings, Puletti, and Harman, unpublished observations).

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XI. THEINTEGRATION OF ENERGY METABOLISM IN MUSCLE It is feasible, with the relevant information derived from the studies of cardiac muscle, insect flight muscle, and avian flight muscle to reconstruct the correlation between metabolism and structure in muscle generally (Harman, 1952). There are according to our present knowledge two systems in muscle concerned with the conversion of the energy obtained from foodstuffs or substrates into utilizable constituents (Fig. 23). The first stage is the series of degradations by either the glycolytic steps of the

ATP

ADP

MY

FIG.23. The following abbreviations are used; Chd, glycogen, glucose and fructose ; PA, pyruvic acid ; AMP, adenosine monophosphate ; ADP adenosinediphosphate ; ATP, adenosinetriphosphate; C, creatine ; CP, creatinephosphate; Mt, mitochondrion ; Sc, sarcosome ; my, myofibril ; --P, high energy phosphate. The glycolytic breakdown of the carbohydrate molecule is brought anaerobically to the level of pyruvic acid (PA) by a partly reversible pathway. (1) The pyruvate is incorporated into the Krebs cycle of aerobic metabolism (2) under the influence of the activating and condensing enzymes contained in the mitowhondrion. Here the activated group is converted through cyclic oxidation into carbon dioxide and water with the simultaneous formation (3) of 24 high energy phosphate bonds, -P. The latter are transferred by proper transphosphorylase mechanisms on to the adenine nucleotide AMP and ADP to form ATP. The ATP thus formed can release -P to creatine ( 4 ) under the influence of creatine transphosphorylase to build up a reservoir of creatinephosphate. The same enzyme can again transfer w P from creatinephosphate to ADP made available in the muscle fibril during the process of contraction. ( 5 ) Compared with the 24-P units available from PA dnring aerobic metabolism {a total of 48 from the glucose molecule) there are formed only four -P during the glycolytic formation of PA (6). The sarcosomes lie adjacent to the mitochondria, held by intracellular reticulum and alter the rate of oxidation (7) either directly or by affecting phosphorylation.

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Embden-Meyerhof scheme (GS)followed by the piecemeal oxidations and decarboxylations of the Krebs cycle, which is principally housed in the mitochondria (MMS). The myofibrils contain no machinery for anabolic energy conversions and are incapable of providing -P from energy-rich sources by either of the above mechanisms. In all muscles they are therefore dependent on the other components, which furnish them with the energy needed for both structural maintenance and contractile capacity. In many muscles the basic metabolic machinery associated with contraction is conjoined with the GS of energy production, which is limited not only by restriction to use of carbohydrate substrates but also by adequacy of free inorganic phosphates and the necessity of sufficient induction levels of ATP for primary phosphorylation by kinase action on the carbohydrate prior to glycolysis. During the process of glycolysis the net yield of high-energy phosphate linkages amounts to two -P, equivalent to about 20,OOO calories, which are rendered accessible to the contractile units (CU) of the muscle. The remainder of the 500,ooO calories locked within the sugar molecule originally are not obtained, and if the residues of glycolysis are not further metabolized they are converted to lactic acid and diffuse away for the main part. For this reason the glycolytic system itself is primitive and necessitates that a very expensive purchase be made for the act of contraction, with rapid depletion of the energy sources. In muscles of this type the force of contraction may be considerable but the capacity for sustained activity is limited. The enzymes of this GS are moreover dispersed throughout the sarcoplasm ( Sacktor, 1955) and heterogeneously arranged, dependent entirely on diffusion gradients and random collision for dissemination of the energy-rich products. The intervention of the MMS, which has a more efficient mechanism of oxidative phosphorylation and the organization of its enzyme systems into compacted units with structural orientation, has obviated the dilemma of both substrate impoverishment and localization of high-energy products close to the work sites in suitable concentration (Harman and Feigelson, 1952b). This is especially necessary in cardiac and flight muscles, and muscles of respiration, e.g., the diaphragm, when irregular exhaustion of energy supply may be lethal. The introductions of the MMS guarantees a constant supply of NP to the contractile units (Fig. 23). Moreover, the mitochondria can use as substrate both pyruvic acid and lactic acid, both end products of the GS, and can salvage the enormous waste implied in this system, In some insect muscle this waste is further avoided by excluding the conversion of pyruvate to lactic acid and the absence of lactic acid oxidase (Sacktor, 1955). It has been demonstrated by Ed-

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wards and Ruska (1955) that in mitochondria1 rich insect flight muscles the tracheole system penetrates the fibers and establishes an intimate surface to surface apposition with the mitochondria; in such muscle the MMS is amply assured of its oxygen import by this arrangement. In insect white (coxal) muscle both mitochondria and tracheoles are infrequent. Muscles therefore which are replete with mitochondria have an advantage of paramount functional importance; their harvest of rapidly available N P prevents the seeping away of energy-rich compounds, e.g., lactic acid by the Cori cycle, and insures a constant supply of energy production. The purview of the MMS is, therefore, peculiar in that it provides not only h ample supply without storage of YY P immediately on demand, but through a stable composite system of multi-metabolic oxidative and phosphorylating enzymes extends vastly the range of substrates denied to the GS. ACKNOWLEDGMENTS These studies were in part supported by the Life Insurance Medical Research Fund and the Muscular Dystrophy Associations of America.

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The Mitochondria of the Neuron WARREN ANDREW Wake Forest College, The Bowman Gray School of Medicine, Winston-Salem, North Carolina

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

Introduction ........................................................ Early Observations ................................................. Skepticism as to the Existence of Mitochondria in Nerve Cells ......... Extensive Studies of Cowdry ........................................ The Problem of “Chromophil“ Cells ................................. Early Experimental Studies : Resistance of Mitochondria to Change ..... Recent Experimental Work ......................................... The Ultrastructure of the Mitochondria of the Neuron ............... Mitochondria and the Aging Process ................................. Conclusion ......................................................... References .........................................................

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I. INTRODUCTION With the rapid progress which has been made in the past few years in our knowledge of the cell organelles, it is now becoming possible to concentrate attention on particular types of cells and on the morphology, distribution, and alterations under various experimental and pathological conditions of the organelles in these cell types. The nerve cell occupies a unique position among the cell types in several respects. From the functional standpoint, the fact that these are cells which carry the impulses over long distances within the body and which integrate and coordinate the various body activities makes them of special interest. In the human nervous system they carry on that function, at least on the physical side, which we call “thinking,” perhaps the highest type of biological activity. On the morphological side, they are unusual cells in having processes, often of tremendous length, in which the cytoplasm would seem to be specially modified for their highly developed functions. Also on the morphological side, the junction of two neurons, the synapse, has been for many years a subject of great interest and the newer knowledge of the cell organelles and of the ultrastructure of the cell promises to aid in our understanding of that important area. There has been little opposition to a belief in the reality of the mitochondria since they are readily visible in a number of living cells, especially when contrast-increasing devices or vital staining procedures are used for their study. They can be seen in well-fixed material by means of a number of characteristic staining reactions. Their widespread occurrence in both 147

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animal and plant cells of the most varied types and their peculiar activity, consisting of both active and passive motions and of frequent divisions, have attracted the attention of many cytological workers. With the development of satisfactory techniques for the isolation of the mitochondria, a literature has developed covering their biochemical characteristics. A close relationship is seen between the enzymatic activity in the cell and the presence of these organelles (Hogeboom et al, 1948). Further development in the knowledge of the mitochondria has come in a striking manner in the studies of their ultrastructure, as revealed by the electron microscope (Palade, 1952, 1953). It has been found that the structural pattern of mitochondria is a complex one and one which is so general in its occurrence as to be considered as probably a basic, fundamental pattern in the organization at least of the animal cell. Each mitochondrion is found to possess a limiting membrane and a system of internal ridges (cristae mitochondriales) which protrude from the inner surface of the membrane into the interior of the organelle. In many mitochondria these cristae are perpendicular to the long axis and occur in series lying parallel to one another, at more or less regular intervals within any particular series. The interior of the mitochondrion appears to be filled with a matrix that appears structureless with the highest powers of resolution of the electron microscope. I n addition to these findings, the most favorable electron micrographs show the membrane about the mitochondrion to be double and the cristae to be folds of the inner layer of this membrane. 11. EARLY OBSERVATIONS The knowledge of the existence of mitochondria in the cytoplasm of nerve cells goes back to the very early days of study of these cell elements. Richard Altmann in 1890 published pictures showing the mitochondria in nerve cells including those in the Purkinje cells of the cerebellum. It will be recalled that Altmann (1852-1901), Professor at Leipzig, saw in the structures which we now call the mitochondria, elements of great significance, in fact, the true ‘(elementary organisms” of which all cells and tissues are composed. These he called the “bioblasts.” Such bioblasts, then, were present in the highest types of cells, the nerve cells, as well as in the lower types of cells, The concept of Max Verworn (1862-1921), Professor of Physiology at Gttingen, that the cell is the true unit of living organisms (1909) and that there is not any smaller fundamental unit was of course to become predominant over the concept of Altmann. The component parts of the cell, while still receiving great interest, were no longer thought of as being pos-

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sible elementary organisms in the sense in which Altmann had used the term. Following the earliest studies, descriptions of mitochondria in the nerve cells of a variety of vertebrates, representing in fact all of the various classes, were published by different authors. Thus Levi in 1896 and Held in 1897 described them in the nerve cells of Lepus ; Duesberg in the anuran tadpole in 1912; Busacca in 1912 described them in Testudo; Mawas (1910) in the cyclostome Petromyzon ; Duesberg in Gallus in 1910 and Cowdry in Cotulnba in 1912. They were also described in the guinea pig by Nageotte (1909a, b, 1910), in Felis by Altmann (1890) and by Lobenhoffer (1906), and in Canis by Lobenhoffer again in 1906. The variety of the form of the mitochondria in nerve cells was recognized by some of these early investigators. Thus Laignel-Lavastine and Jonnesco (1911) described the mitochondria in the Purkinje cells of the cerebellum of the guinea pig as appearing as “granules, rods and rows of granules.” Their technique consisted of fixation in 12% formalin followed by treatment with Weigert’s neuroglia mordant, staining with hematoxylin and by the methods of Altmann and Benda. They used also Regaud’s formolbichromate-hematoxylin technique and found the structures appearing essentially the same with these various methods. The variety of form of mitochondria is seen readily even when neurons of the same type are studied (Figs. 1* and 2). From the descriptions of some of the early observers, it is difficult to be certain whether they were dealing with mitochondria or with some other elements. Thus Furst (1902) described peculiar rings, threads, and knots in ganglion cells of embryos of salmon and also in several other species of fish. Such structures seemed to be absent from the cells in the brain and spinal cord. Because of this fact and also because the fixatives which he employed are not generally suitable for demonstration of mitochondria, it seems probable that the objects seen were not actually mitochondria in this case. It was recognized early that the mitochondria are distinct from some of the other elements found in the nerve cell. Thus Levi (1896) used Galleotti’s modification of Altmann’s method, which involves the use of methyl green as a differentiator in place of picric acid and which brings out both the mitochondria and Nissl substance. H e found the mitochondria as rodlike bodies which stained deeply with the anilin fuchsin and hence stood out as bright red bodies in contrast to the green-colored Nissl bodies.

* All photomicrographs are from specimens prepared in our laboratory, fixed in Regaud‘s fluid for 4 days, changing each day, post-chromated for 6 days, washed, and stained in acid anilin fuchsin.

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FIG.1. Purkinje cell of a young male mouse of the C57 Black strain, age 6-10 months. The cytoplasm shows long, filamentous mitochondria, together with some thicker rods. The molecular layer is above and to the left, the granular layer, with its mitochondria-rich glomeruli, below and to the right. X1215. FIG.2. Purkinje cell of an old female mouse of the C57 Black strain, age 19 months. The mitochondria appear chiefly as short rods and granules, although some filaments are present. Orientation of layers as in preceding figure. X1215.

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The Nissl bodies were lacking in the axon hillock, whereas the red rods were present there and indeed on out into the axon. Schirokogoroff ( 1913) found filamentous and rod-shaped mitochondria, independent of the Nissl bodies, in spinal ganglion cells, in cells from the medulla and other parts of the brain, from the spinal cord, and from the retina. He used fixation in Regaud’s fluid and stained the tissue according to the methods of Altmann, Heidenhain, and Benda. In fact, even the very early studies of Altmann (1890) would tend to show that the mitochondria are independent of the Nissl material, for he pictures them in the axon of the Purkinje cell as well as in the dendrites, and Nissl material is never found in the axon. Lobenhoffer (1906) showed the independence of the mitochondria from Nissl substance by counterstaining the Nissl substance with toluidin blue while the mitochondria were stained with the anilin fuchsin, thus presenting the contrast of red and blue. H e worked with the spinal cord, brain, and retina. The studies of Mawas (1910) on Petrowzyzon, while carried out on a form on which few cytological studies have been made, are of particular interest in showing the diversity of form of the mitochondria and even in suggesting one alteration of mitochondria which may be of considerable importance-namely, vesiculation. H e described the structure of the spinal ganglion cells in Petromyzon and in Ammocoetes branchialis studied by means of the Regaud method. I n the adult animal he found that the nerve cells may be divided into two groups, the large and the small. The small cells show granules and filaments distributed throughout the cytoplasm. I n the larger cells the cytoplasm is less intensely stained and there are present in addition to the granules and filaments a number of vesicles which are considerably larger than the granules and filaments. The vesicles seem to replace the granules and filaments in certain parts of the cells. Thus they are more numerous in the region of the nucleus although they also extend into the dendritic processes. H e felt that the vesicles are allied to the granules and filaments. There is no particular reason to think that his material was not from normal specimens. 111. SKEPTICISM AS TO IN

EXISTENCE OF MITOCHONDRIA NERVECELLS

THE

The theory of Meves (1908) concerning the potency of the mitochondria to transform into various differentiated elements of the cytoplasm had a special effect upon the concepts of the mitochondria in the nerve cells. Meves claimed that the neurofibrils arise by transformation of mitochondria. Since the neurofibrils are present in the adult nerve cell, it was not un-

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reasonable to feel that the mitochondria might be absent there. Thus, we find statements by Meves (1910), Duesberg (1910), and Hoven (1910) that there are no mitochondria in fully developed nerve cells. Probably partly as a result of this a number of investigators who looked for them there failed to observe them or gave up the search too readily. The convincing work of Cowdry (1914a) helped to refute this view of the change of mitochondria into neurofibrils and their consequent loss from the cytoplasm of the nerve cells.

IV. EXTENSIVE STUDIES OF COWDRY Cowdry proceeded to show the presence of mitochondria in a very definite way in the nerve cells of a variety of vertebrates and also to cite the observations of earlier workers who had felt that they had seen such elements in this type of cell. H e published (Cowdry, 1914b) the results of a comparative study on the spinal ganglion cells of the frog, Necturus, snake, turtle, pigeon, white rat, guinea pig, monkey, and man. H e found a remarkable degree of constancy in the morphology, distribution, and relative numbers of mitochondria in the spinal ganglion cells from these various animals. His description and picture of the mitochondria in a human spinal ganglion cell apparently are the first of their kind for a human nerve cell. This material was from a female child about two years of age and it is rather remarkable that although it is listed as having been fixed four hours after death the mitochondrial picture as presented is an excellent one. W e have the privilege of including in the present article what we believe to be the first photomicrograph of mitochondria in a human neuron (Fig. 3). In all of the specimens studied the morphology of the mitochondria was very constant. They vary from granules 0.25-0.75~ in diameter to rods 1-2 p and filaments 2-4 p in length. The granules are described as being sometimes arranged in rows. The rods are sometimes shaped like dumbbells or like pears and the filaments occasionally show enlargements or varicosities. Cowdry stated that he was unable to distinguish between the spinal ganglion cells of any of the animals on the basis of the mitochondrial content in spite of the variety of vertebrate material studied. H e found also that the microchemical reactions of the mitochondria are very constant. Microchemical reactions studied included the reaction of these elements to Janus green and diethylsafranin, although the author states that these reagents gave very disappointing results as compared with their action, for instance, on blood cells. Cowdry noted also a very definite relationship between the numbers of mitochondria in individual nerve cells and the number of lipoid globules.

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The larger the number of lipoid globules, in general, the smaller the number of mitochondria which were present. He states that both mitochondria and lipoid stained with nilblau B extract in the cells of all of the animals but that he could not see an actual transformation of one into the other. The lipoid globules themselves are all spherical and vary from 1 to 5 p in diameter.

FIG. 3. Large pyramidal cell of the motor cortex, left cerebral hemisphere, human. Specimen from fourteen-year-old female, fresh-fixed at hemispherectomy, carried out as a therapeutic measure for uncontrollable epileptic seizures. X 3800. The lipoid apparently is not to be confused with pigment, for Cowdry says that pigment was seen only in the spinal ganglion cells of Necturus and Rana. The pigment was bright orange in color. Cowdry says (1914b, p. 16) “no relationship was observed between the pigment and mitochondria, although I would not deny that such may exist.” Cowdry does state that there are differences in the mitochondria1 content of different individual spinal ganglion cells of the same animal, the exact degree of

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which cannot be determined on account of the difficulty in enumerating mitochondria. Cowdry postulated that the mitochondria are of importance in the metabolism of the nerve cell. Key (1916) had pointed out that in exocrine gland cells of the pancreas the mitochondria do not seem to be directly involved in the secretion process. I n this same article Cowdry mentions in a note that he had observed mitochondria in the nerve cells of a variety of invertebrate animals, including several species of crustaceans, Limulus, Nereh, and some molluscs. H e says that they are rather more variable in size, number, and staining reactions in the invertebrate nerve cells than in vertebrate nerve cells. Of course, we may note that this was in comparison probably with vertebrate nerve cells from a particular part of the nervous system, namely, the spinal ganglia. Cowdry (1914a) showed that the mitochondria are present in very early stages of embryonic development in the nerve cells in the chick, even before the differentiation of any somites. There are some interesting morphological differences between the mitochondria in developing nerve cells and those in later stages however. In the spinal ganglion cells of a thirty-five somite embryo, the average length of mitochondria is from 3 to 5 p while in the spinal ganglion cells of an adult fowl they seldom are more than 1 p in length. These measurements were made on fixed and stained tissue.

V. THEPROBLEM OF “CHROMOPHIL” CELLS Cowdry (1916) again has a good deal to say about the mitochondria of nerve cells in a paper concerning particularly the structure of chromophil cells, a peculiar but common “type” of nerve cell which shows a capacity for staining deeply and a slightly shrunken appearance, together with an obscuration of the nucleus. In this paper he points out that Busacca (1913) had noted that the mitochondria in certain cells in the brain of Testudo stain intensely with iron hematoxylin and that in some cells the mitochondria even seem to lose their definite outlines and to form homogeneous masses. Cowdry’s own studies on chromophilic cells were made on apparently normal white mice of known age. Fixation was by a type of perfusion method and modifications of the classical methods for a demonstration of mitochondria were used. The fixative was that of Regaud applied by injection through the blood vessels. Potassium permanganate and oxalic acid were used to facilitate the staining of the mitochondria with anilin fuchsin and the tissue was counterstained with methyl green according to the technique of Galleotti. I n describing the observations on the chromophil cells, Cowdry is willing to class with such cells those which show a slight increase in the amount

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and in the intensity of staining of the mitochondria. H e states specifically that in these cells there is apparently no corresponding change in the Nissl substance nor is the morphology of the mitochondria altered. Other chromophil cells show a remarkable increase in the number of mitochondria such that a particular cell may contain three or four times as many of these elements as does its neighbor. Such an increase in the number of mitochondria is associated with a slight but perceptible increase in the amount of the diffuse Nissl substance in the cytoplasm (stained with methyl green) and with a darker staining of the acidophilic and basophilic nucleoli and of the ground substance of the nucleus. Such cells, however, still may show no evidence of shrinkage. They may be recognized in Cajal preparations by the changes in the nucleus and the Nissl substance, although here of course the mitochondria are not evident. An apparent further stage is seen in a great increase in the Nissl substance, which now is present as a diffuse deposit. In this stage some of the mitochondria often lose their sharp outlines and seem to merge into the general cytoplasm. Mitochondria may not be very numerous in cells of this type. The nucleus stains intensely. The cell also has a few shrinkage spaces about it. Such cells again can be identified in sections stained with toluidin blue and in Cajal preparations. A further stage in the development of chromophilia shows an unusually large amount of Nissl substance and further evidences of disappearance of the formed mitochondria, especially in the processes of the cells. The outlines of the nucleus are very indistinct. In a still further stage only a few typical mitochondria persist near the origins of the cell processes. The Nissl substance appears to be “overshadowed” by a large amount of material which stains the same way as the formed mitochondria do in adjacent cells. Such cells usually show a considerable degree of shrinkage. Cowdry says that he does not know whether this shrinkage presents an actual diminution in size of the cell during life or whether it represents simply the result of the difference in the reaction of the chromophil cells to fixation and to the subsequent treatment. In relation to the mitochondria, Cowdry states that in chromophil cells in advanced stages they may disappear more or less completely in certain cells and their place be taken by a mass of amorphous material. The nucleus may be invisible, but if the same tissue is stained with hematoxylin and eosin one finds that these stains will not color the amorphous deposit or the Nissl substance and the nuclei are shown to have distinct and definite outlines and appear but little altered except that they contain rather more than the usual amount of stainable material. In this article Cowdry, in addition to going into considerable detail on

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the appearance of the mitochondria in the chromophil cells, raises the question as to whether or not some of the mitochondria in the nervous system may be intercellular in position. They are extremely abundant in the neuropile, but it is of course very difficult to be sure whether they are always within fibers or sometimes between them. W e shall see later that new evidence concerning this interesting problem has accumulated in the years intervening. Cowdry concludes that it seems highly probable that chromophil cells occur normally in the brain of the white mouse and that we have to recognize a partial solution of mitochondria just as investigators had recognized for many years a solution of the Nissl substance, or “chromatolysis.” Therefore, a process which may well be called “chondriolysis,” a term first employed by Romeis (1912) to describe the disintegration of certain mitochondria which escaped from cells into the uterine fluid of Ascaris, occurs also in the cells of the nervous system. Thurlow (1917) had found that the individual cell types in different parts of the nervous system are fairly uniform in the numbers of mitochondria present. Thus there are certain average numbers for the ventral horn cells, others for the Purkinje cells, and others for the spinal ganglion cells, a condition which might well be expected from the differing size and different morphological characteristics both of the cells and of the types of mitochondria in them. The great degree of difference which can be seen between the mitochondrial content of cells in different parts of the nervous system is well illustrated in a figure taken from Thurlow’s work and reprinted in Cowdry’s “General Cytology’’ (1924). This picture (Figure 13, p. 316 shows side by side a small cell of the locus coeruleus and a large cell of the mesencephalic nucleus of the fifth nerve of a white mouse. The mitochondria in the small cell are threadlike and few in number while those in the large cell from the mesencephalic nucleus are coarser, more granular in type, and very numerous, seeming to fill almost all of the cytoplasm. While the differences in numbers of mitochondria are marked in different parts of the nervous system, there appears not to be, according to Thurlow (1916), a constant difference in this respect between cells of motor and cells of sensory type.

VI. EARLY EXPERIMENTAL STUDIES : RESISTANCE OF MITOCHONDRIA TO CHANGE With the existence of the mitochondria in the nerve cells of adult animals firmly established, the way was open for experimental studies of the reaction of these cell elements in the nervous system. It seemed particularly perti-

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nent to undertake some such studies since it had already been asserted by some workers that the number, size, shape, and staining reaction of the mitochondria are altered with different degrees of cellular activity, especially in gland cells. Strongman (1917) attempted to find whether there were any results of muscular fatigue on the mitochondria in the nerve cells of white mice. H e found relatively little change. He described what appeared to be a tendency to clumping together of the mitochondria in the nerve cells of fatigued animals. This effect was particularly apparent at the base of the large dendrite in the Purkinje cells of the cerebellum. A still earlier study by Luna (1913) had indicated that shortly after the cutting of a large peripheral nerve tract the mitochondria of the corresponding ganglion cells loose their regularity of distribution, increase in size, and show an increased affinity for iron hematoxylin stain. In more advanced stages of the degenerative process the mitochondria disappeared entirely, according to Luna. This work was done on the toad. Some evidence had been obtained that mitochondria of nerve cells are peculiarly resistant to change even in marked pathological conditions. McCann (1918) stated that the mitochondria persist in normal number in cells where the Nissl substance has disappeared and even in late stages of phagocytosis of nerve cells in experimental poliomyelitis. Rasmussen (1919) felt that a good test of the resistance or lack of resistance to change of the mitochondria of the nerve cell would be to ascertain their behavior during natural hibernation, the state of profound dormancy in mammals such as is characteristic of woodchucks during the winter season. It was thought by this investigator that the enormous reduction in the vital processes of such animals during hibernation would help to give evidence concerning the connection between mitochondria and functional activity of the nervous system. The mitochondria of the nonhibernating animal are described as being granular, rod-shaped, and long-filamentous in the central nervous system and in the spinal ganglia as granules or very short rods. In the central nervous system the mitochondria near to the nuclei are usually granular. They tend to become short rods more peripherally and to be long-filamentous in the bases of the processes and also out in the dendrites and axons. In the spinal ganglia they are uniformly distributed as granular or short rodlike structures except in places where lipoid has accumulated. In such regions the mitochondria tend to be excluded. This had been noted by Cowdry in other species. In the preparation of the tissues of the control and experimental animals, blood was washed out by gradual perfusion with oxygenated Locke’s fluid and then Regaud’s fixative was allowed to perfuse

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the entire animal for one hour. Some animals were perfused only for short periods of time and it was found that there was no difference in the apparent manner of fixation of the mitochondria in those which had been perfused for the longer period. Staining was by means of acid anilin fuchsin and methyl green after various degrees of dechromation. Cells of the ventral horn of the spinal cord and of the lateral horn, spinal ganglion cells, the cerebellar cortex, and other regions of the nervous system were studied. Mitochondria were counted and their numbers expressed as millions per cubic millimeter of cytoplasm following the method devised by Thurlow (1917). In relation to the effects of hibernation the surprising result was obtained that there was no noticeable effect upon the mitochondria! Thus complete inanition for three months during the winter sleep and for three weeks after awaking from it did not modify the shape, the number, or the distribution of the mitochondria in the nerve cells. This rather remarkable result would tend to indicate that the mitochondria are necessary or fundamental elements of cellular life but probably not elements, in the case of the nerve cell at least, which are apt to change in accordance with the state of activity of the cell. The comments which Rasmussen has to make upon the matter of clumping of mitochondria are of some interest in relation to the vexed question of the chromophilic cells of the nervous system. He says that in “imperfectly fixed tissue” mitochondria frequently clump together into larger masses which are “clearly not representative of the normal condition.” H e also says (1919, p. 42) “to what extent this occurs in the case of the best fixation is difficult to say.” If imperfect fixation would explain the clumping of the mitochondria in the chromophilic cells there perhaps would be little point in further investigation of this phenomenon. However, one of the outstanding characteristics of the distribution of these cells is that they are found widely scattered and often in the closest relationship to apparently “well-fixed” cells in the sense that these neighboring cells show mitochondria of typical form and distribution. Cowdry (1926) attempted to summarize the results of studies on the reactions of mitochondria to cellular injury. H e points out how wide and varied the literature on mitochondria was, that important facts concerning them frequently are contained in papers which do not mention them in the title so that a great deal of collateral reading must be done, and much of the important work goes back (at that time) for almost forty years. H e mentions as the most complete summary of the earlier work that of Duesberg ( 1912) which covered all the available papers-about 500-from the very beginning of work on the subject up to October 15, 1911. The

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next review of the subject after that he cited as being his own survey in “General Cytology” which extended up to July, 1923, and also included a synopsis of about 500 papers. Whereas Duesberg had stressed the importance of mitochondria in development and inheritance, the latter synopsis by Cowdry stressed their importance in cellular physiology and pathology. The chief types of reaction of mitochondria to cellular injury which Cowdry lists, while applying to cells of the various tissues, are of interest to us in considering possible changes in injury of the nerve cell. Therefore, we will repeat them briefly here. They are : (1) the breaking up of filamentous mitochondria into granules-this apparently is the most delicate qualitative response of these elements ; (2) the passing into solution and the disappearance of granular mitochondria, the process which has been called “chondriolysis” ; (3) the enlargement of the granular mitochondria to form droplets followed by their disappearance or their change into fatty elements so that the condition may merge insensibly into one of fatty degeneration ; and (4) the agglutination of the granules, first into clumps, followed by a fusion of individual elements in the formation of large droplets which then are changed into lipoid, a process described by Scott (1916) in phosphorus poisoning. I n addition to the qualitative changes, quantitative changes in mitochondria had been recorded at that time. Cowdry, however, does not believe that the methods of estimating the quantitative changes have been in general very accurate and that many sources of error are present. H e states that a diminution in the amount of mitochondria is often encountered but that an increase above normal is comparatively rare. The following statement is of interest for our subject (Cowdry, 1926, p. 246) : “It is safe to assume that a decrease in mitochondria is indicative of a depression of functional activity and that an increase, in the rare cases in which it has been proved to occur, points to heightened activity, unless it is accompanied by fatty changes in the manner already referred to.” A third type of change is the topographical alteration, in which the distribution of the mitochondria within the cell is altered. The great mobility of the mitochondria as shown in the studies with the phase microscope and by cinematographic records would indicate that one must be careful in judging of such a change in distribution but at the same time in selected groups of cells where the topographical differences are constant they should be accepted as real phenomena. In discussing specifically the modification of the mitochondria in the nerve cell in relation to various factors, Cowdry points out that they appear not to be noticeably influenced by extreme fatigue (Strongman, 1917), nor in experimental poliomyelitis (McCann, 1918). In the latter condition

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the mitochondria retain the usual filamentous shape in very degenerate cells. Marinesco and Tupa (1922), as a result of experiments on tearing out of the axons, stated their opinion that the mitochondria are among the most resistant formations of the cell. However, work by Ma (1925) showed that they were not so unmodifiable in some nutritional deficiencies as Clark (1914) had thought. Ma found that the rods and filaments of the spinal ganglion cells break up into granules in beriberi, even in early stages. In later stages the number of mitochondria is much reduced while that of the lipoid globules in the cell is increased. Ma agrees with Scott (1916) in the opinion that mitochondria actually change into lipoid globules. In another part of this same paper, and without referring specifically to the nerve cells. Cowdry says (1926, p. 248) “Mitochondria are concerned in the formation of lipochrome pigments.” H e comments also that in regenerating cells and in some tumor cells they are greatly increased in number. VII. RECENT EXPERIMENTAL WORK Only within relatively recent times has an attempt been made to follow in a thorough manner the time course of mitochondria1 changes after section of axons. Hartmann (1948) studied the ventral horn cells of the rat following section of the sciatic nerve. H e has made a quantitative investigation of the mitochondria and has tried to correlate the mitochondria1 picture with the steps in chromatolysis. Animals were sacrificed at daily intervals from the first to the eleventh day after cutting of the nerve. Fixation was by perfusion with Regaud‘s fluid and removal to the same fixative. Alternate slides were stained for mitochondria and for Nissl substance, so that a fairly direct comparison could be made, Mitochondria were counted and calculations made to express them in terms of millions per cubic millimeter of cytoplasm, as had been done by Rasmussen ( 1919). Hartmann (1948) finds a definite and progressive change in the mitochondrial content postoperatively. The mitochondria on the operated side are increased in number, stain more intensely, and are somewhat increased in size. These changes are concomitant with the progress of chromatolysis. Thus the cells in the most advanced stages of chromatolysis are in all cases the same cells in which the increase in number of mitochondria is most pronounced. Hartmann mentions the finding by Homing (1929) of an enormous increase in numbers of mitochondria in the cells of regenerating land planarians. He suggests that the increased metabolic rate necessary to

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regeneration may be effective in the increase in number of mitochondria in the nerve cells which are growing new axons. Bodian and Mellors (1944) had found a significant increase in the amount of acid phosphatase in ventral horn cells of rhesus monkeys following section of the sciatic nerve. Their work fits into the concept of an increased metabolism in nerve cells after section of axons. Hartmann himself points out the problem involved in absolute identification of all of the elements counted as mitochondria but has followed up his earlier work with an electron microscope study of the cytoplasm of the nerve cell (Hartmann, 1953) and then with such a study on cells the axons of which had been severed (Hartmann, 1954). VIII. THEULTRASTRUCTURE OF THE MITOCHONDRIA OF

THE

NEURON

In the general study of nerve cells with the electron microscope Hartmann used cerebral cortex, cerebellar cortex, hypoglossal nucleus, ventral horn of spinal cord, and spinal ganglia of the rat. Hartmann describes the mitochondria as varying in appearance. Some appear to have a “granular” internal structure while others, even in thin sections, are opaque. Again, some show a limiting membrane while others do not, and he finds no correlation between the difference in this feature and the apparent difference in internal structure. The shapes of the mitochondria include spheres, short rods, and elongated filaments. For the electron microscope study of motor cells following section, Hartmann chose the hypoglossal nucleus of the rat and studied specimens taken on successive days through the 15th postoperative day. While no attempt was made to count the mitochondria, he says (1954, p. 21) : “The electron microscope study fully confirms the earlier observations made with the light microscope, inasmuch as the number of identifiable mitochondria is increased on the operated side as contrasted with the control side.” The mitochondria both in control and experimental material, he says, present a similar spectrum of variation, and one like that described in his 1953 paper. There are, however, more filamentous forms on the operated side and these are less dense to the electron beam. H e says (p. 22) : “It seems likely that these mitochondria are recently formed.” Hartmann states explicitly that the difference in mitochondria1 picture in control and experimental cells is not absolute, but in sorting 200 electron micrographs from the two sides, selection was 95% accurate. Of considerable interest is his statement that only a “small proportion” of mitochondria show transverse striations such as those described by Palade (1953), Sjostrand (1953), and Sjostrand and Rhodin (1953), as

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characteristic of mitochondria in various other types of cells. Thus a suggestion seems to be implied that mitochondria of nerve cells may present a rather marked difference from those in other types of cells. Geren and Schmitt (1954) describe the mitochondria in the axon of invertebrate nerve fibers, as observed with the electron microscope (Figs. 4 and 5). They say (p. 867) : “The mitochondria of the nerve fiber resemble those found in the perikaryon and other tissue cells.” The loca-

FIG.4. Mitochondria in a giant axon of the squid. Irregular inclusions are seen within the large mitochondria, which show clearly a double membrane at the periphery. (Geren and Schmitt, 1954.)

tion of the mitochondria is of interest in that in lobster fibers (large fibers) and in medium-sized fibers of squid they are clustered almost exclusively just below the surface of the Schwann cell. The axon surface of the Schwann cell presents outpocketings which are “highly suggestive” of a possible origin of mitochondria from the Schwann cell and their transmission to the cytoplasm of the axon. The authors, however, are commendably cautious and say that drawing of such a conclusion “would require reliance on highly circumstantial evidence.” Reading the description by these authors reminded us of a case where mitochondria definitely are furnished to certain cells by another cell. The apical cell of the testis in Diptera passes these elements and other cytoplasmic material into the spermatogonia (Carson, 1945). That the ex-

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tended cytoplasm of neurons might have “nurse-cells” in this respect is indeed an interesting possibility. I t should be pointed out here that not all investigators agree that some of the structures in invertebrate nervous tissue which have been called “mitochondria” should be so termed. Thus Robertson (1955) would not be willing to call the structures in our Fig. 4 mitochondria without further

FIG.5. Mitochondria in an axon from the leg of a lobster. Internal lamellar structure, so characteristic of mitochondria from cells of many kinds, is seen. (Geren and Schmitt, 1954.) evidence, particularly since he feels that the term now has such definite enzymatic and functional implications. Beams et al. (1952) say that in Regaud preparations mitochondria in the neuron are not to be confused with any other components present. They used both the light and the electron microscope in their studies of spinal ganglion cells. Palade (1954) has described the structure of synapses as seen with the

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electron microscope both in the central and in the peripheral nervous system. The portions of brain studied were cerebellar cortex, including the glomeruli in the granular layer and some synapses in the molecular layer, and neuropile of the medulla oblongata. In the ending of the axon, an accumulation of mitochondria, together with peculiar small vesicles, is found. The dendrite shows fewer mitochondria and fewer vesicles. In the peripheral, e.g., neuromuscular ending, the axon has the same appearance as in synapses within the central nervous system and is completely distinct from the sarcoplasmic “sole” of the muscle. No “extracellular” mitochondria were described in the nervous tissue in these studies on synapses. It is interesting to contemplate the very great numbers of mitochondria which must be present in an individual neuron when one considers the length of some of the axons in, for example, the nervous system of man. In a description of the morphology of the synapse in the neuropile of the earthworm, DeRobertis and Bennett ( 1955) speak of mitochondria within the nerve fibers which display the “characteristic cristae” of Palade. A comprehensive survey of the fine structure of neurons (Palay and Palade, 1955) recently has been published. In this survey only one paragraph of the text is devoted to the mitochondria as such. The conclusion in regard to the ultrastructure of these organelles, however, appears to be very clear cut. They do possess a double membrane at the periphery. The outer membrane is smooth, the inner one folded to form cristae. The cristae usually are of the “conventional, shelflike type” and are placed more or less at right angles to the long axis of the mitochondrion. In some of the mitochondria, however, they are parallel to the long axis, while in still others the projections from the inner membranes have the form of “villi,” appearing in some fields as circular sections within the matrix of the mitochondrion. These variations in pattern are seen in mitochondria of cell types other than the neuron and no distinctive features of ultrastructure for nerve cell mitochondria are noted. The technique of these workers presents pictures of mitochondria which are incomparably beautiful and detailed (Figs. 6 and 7). IX.

MITOCHONDRIA AND

THE

AGINGPROCESS

Our own interest in the mitochondria of the neuron has been stimulated by our desire to compare the structure of cells of particular types a t different periods in the life history and in particular to study the changes

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165

which occur in old age. We now are initiating investigation in this field. Figure 1 and 2 indicate the kind of difference which we have found between neurons in young and old mice. Hess and Lansing ( 1954) have made electron microscope observations

FIG. 6. Mitochondria in a cell of the superior cervical ganglion of a rat. The peripheral double membrane and the lamellae projecting into the interior are seen. X48,OOO. (Electron micrograph courtesy of Doctor George E. Palade.)

on mitochondria in the spinal ganglion cells of the guinea pig, using specimens 3 days, “adult,” and 6 years of age. They say that degenerative mitochondria can be found in young nerve cells and that normal mitochondria are seen in senile cells. Hence, it is difficult to use the condition

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WARREN ANDREW

of the mitochondria as an index of aging. They believe that the pigment of senile ganglion cells is formed by the mitochondria. If mitochondria are concerned more with fundamental metabolic processes of the cell, it seems to us logical to look for changes in senility in

FIG.7. Mitochondria in a cell from Auerbach’s plexus, intestine of a rat. X49,667. (Electron micrograph courtesy of Doctor George E. Palade.)

these elements even though they may be little affected by fatigue, hibernation, or other factors. Payne (1949) has shown profound changes in mitochondria in old age in the pituitary gland of the fowl. These consist chiefly in the formation of great vesicles from individual mitochondria, with eventual destruction of the cell.

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167

W e have noted a predominance of the filamentous type of mitochondria in neurons (Purkinje cells and motor horn cells) of younger mice and of short rods and granules, with a few vesicles, in neurons of mice in the early senile period (Andrew 1954). W e are looking forward to studying still older animals in the near future and have material from mice up to 800 days of age under preparation.

X. CONCLUSION We cannot help feeling that the story of possible relationship of mitochondria to the function of the neuron is still a very incomplete one. The problem of the chromophilic cells (Fig. 8) remains completely unsettled.

FIG.8. Juxtaposition of Purkinje cell with “normal” mitochondria1 picture with one of chromophilic type where no detail can be seen in the cell due to the very deep stain which it has taken. From a young male mouse of C57 Black strain. Such proximity of chromophilic to ordinary cells seems to argue against the concept of chromophilia as an artifact due to poor fixation. X1263.

In our own material we have been encouraged to further observations on such cells by the discovery of some areas of the brain where they occur in rather constant numbers and where there appear, at least, to be transitional stages between cells with a “normal” mitochondrial picture and those with the deeply-stained aspect of the “chromophilic” cells. Such a region is found in Ammon’s horn (Fig. 9).

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Future studies making use both of new modes of experimental approach and of the new tools available for the study of ultrastructure should be of much interest in the advance of our knowledge of the mitochondria of the neuron.

FIG.9. Cells of the horn of Ammon in a young male mouse of the C57 Black strain. In this region we have found what seem to be transitional stages between the “normal” mitochondria1 picture and the truly chromophilic cells, such as the one seen in the center of the figure. The transitional stages show many large spheroidal mitochondria which seem to merge together as chromophilia is attained. X1000.

ACKNOWLEDGMENTS The personal researches of the author described here, including the photomicrographs, are part of a project for study of the aging process in nerve cells, supported by a grant from the Josiah Macy Jr. Foundation. W e are much indebted to Mr. Benjamin Morton for aid with the photomicrographs.

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169

XI. REFERENCES Altmann, R. (1990) “Die Elementarorganismen und ihre Beziehungen zu den Zellen,” p. 145 Veit, Leipzig. Andrew, W. (1954) Proc. 3rd Congr. Intern. Assoc. Gerontol. London p. 11 (Abstract). Beams, H. W.,Van Breeman, V. L., Newfang, D. M., and Evans, T. C. (1952) J. Comp. Neurol. 96,249. Exptl. Biol. Mod. 66, 243. Bodian, D., and Mellors, R. C. (1944) Proc. SOC. Busacca, A. (1912) Anat. A n z 42, 620. Busacca, A. (1913) Arch. Zellforsch. 11, 327. Carson, H. L. (1945) J . Morphol. 77, 141. Clark, E. (1914) 1. Comp. Neurol. 24, 61. Cowdry, E. V. (1912) Intern. Monatsschr. Anat. Physiol. 29, 473. Cowdry, E. V. (1914a) Am. J . Anat. 15, 389. Cowdry, E. V. (1914b) Am. J. Anat. 17, 1. Cowdry, E. V. (1916) Carnegie Contribs. Embryol. 4, 27. Cowdry, E. V. (1924) “General Cytology.” Hoeber, New York. Cowdry, E. V. (1926) Arch.. Pathol. 1, 237. Duesberg, J. (1910) Arch. Zellforsch. 4, 602. Duesberg, J. (1912) Ergeb. Anat. 14. Eiztwicklirngsgesrhicltte 20, 567. DeRobertis, E. D. P., and Bennett, H. S. (1955) J. Biophys. and Biochem. Cytol. 1, 17. Furst, C. M. (1902) Anat. Japan., Anat. Hefte 19, 389. Geren, B. B., and Schmitt, F. 0. (1954) Proc. Natl. Acad. Scis. 40, 863. Hartmann, F. J. (1948) Anat. Record 100,49. Hartmann, F. J. (1953) J. Comp. Neurol. 99,201, Hartmann, F.J. (1954) Anat. Record 118, 1Y. Held, H. (1897a) Arch. Anut. u. Physiol. Anat. Abt. p. 204. Held, H. (1897b) Arch. Anat. u. Pitysiol. Szcppl. p. 273. Hess, A.,and Lansing, A. J. (1954) 1. Gerontol. 9, 361. Hogeboom, G. H., Schneider, W. C., and Palade, G. E. (1948) J . Biol. Chem. 172, 619. Horning, E. S. (1929) Australian J . Exptl. Biol. Med. Sci. 6, 11. Hoven, H. (1910) Arch. biol (Lihge) 25, 427. Key, J. A. (1916) Anat. Record 10, 215. Laignel-Lavastine, M.,and Jonnesco, V. (1911) Compt. rend. biol. 71, 699. Levi, G. (1896) Riv. patol. nervosa e mentale 1, 1. Lobenhoffer, W. (1906) Arch. mikroskop. A n d . IC. Entwicklungsmech. 68, 491. Luna, E. (1913) Anat. Anz. 44, 413. McCann, G. F. (1918) J . Exptl. Mcd. 27, 31. Ma, W. C. (1925) Am. J. Anat. 36, 215. Marinesco, G., and Tupa, A. ( 1 9 2 ) Compt. rend. SOC. biol. 87, 292. Mawas, J. (1910) Compt. rend. 160, 126. Meves, F. (1908) Arch. mikroskop. Anat. u. Entwicklungsmech. 73, 816. Meves, F. (1910) Arch. mikroskop. Anat. w. Entwicklungsmech. 75, 642. Nageotte, J. (1909a) Compt. rend. SOC. biol. 67, 472. Nageotte, J. (1909b) Compt. rend. soc. biol. 66,825. Nageotte, J. (1910) Compt. rend. SOC. biol. 68, 39.

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Palade, G. E. (1952) Anat. Record ll4, 427. Palade, G. E. (1953) I. Histochem. Cytochem. 1, 188. Palade, G. E. (1954) Anat. Record 118, 335 (Abstract). Palay, S. C., and Palade, G. E. (1955) I . Biophys. and Biochem. Cytol. 1, 69. Payne, F. (1949) I . Gerontol. 4, 193. Rasmussen, A. T. (1919) J . Cornp. Neurol. 91, 37. Robertson, J. D. (1955) Personal communication. Romeis, B. (1912) Arch. mikroskop. Alzat. u. Entwkklwgsmech. 80, 129. Schrikogoroff, J. J. (1913) Anat. A m . 43, 522. Scott, W. J. M. (1916) Am. J . Anat. 20, 237. Sjostrand, F. S. (1953) Nature 171, 30. Sjostrand, F. S., and Rhodin, J. (1953) Exptl. Cell Research 4, 426. Strongman, B. T. (1917) Anat. Record 12, 167. Thurlow, M. DeG. (1916) Arrat. Record 10, 253. Thurlow, M. DeG. (1917) Curwgie Contribs. Embryol. 6, 35. Verworn, M. (1909) “Allgemeine Physiologie,” 5th ed. Fischer, Jena.

The Results of Cytophotometry in the Study of the Deoxyribonucleic Acid (DNA)Content of the Nucleus R. VENDRELY

AND

C. VENDRELY

Coifre de Recherches sur les Macromolkcules, Strasbourg, France Page I. Introduction ....................................................... 171 11. Methods of Cytophotometry ....................................... 173 1. Cytophotometry in Visible Light ................................. 173 2. Cytophotometry in Ultraviolet Light ............................. 176 111. The DNA Content of Interphase Nuclei of Normal Tissues.. ........... 177 1. The Constancy of the DNA per Nucleus ......................... 177 2. Variations of the D N A per Nucleus Related to the Variations of Chromosome Number ........................................... 179 IV. The DNA Content of Nuclei in Deep Physiological and Pathological Changes ........................................................... 182 1. Physiological Changes ........................................... 182 2. The DNA of the Nucleus in Pathological Cases ................... 185 V. The D N A Content of the Nucleus during Cell Division.. ........... 187 1. The Moment of the Synthesis of DNA in the Process of Mitosis. 187 2. The D N A in Embryonic and Differentiating Tissues ............. 188 3. The D N A of the Nucleus during Maturation, Fertilization, and Cleavage ........................................................ 191 VI. Conclusion ......................................................... 193 VII. References ......................................................... 194

I. INTRODUCTION During the last few years, there has been considerable interest in the question of the DNA content of the cell nuclei. The main controversies have been concerned with the constancy, or nonconstancy, of the amount of DNA per nucleus related, or not, with the chromosome number. This problem was first attacked by chemical analysis, but, very rapidly cytophotometry in visible light took possession of the field and became the most efficient and convenient tool for the study of the DNA of the nucleus. It is worth pointing out that the extensive development of cytophotometry in visible light started from the discovery of a close relationship between the DNA content of the nucleus and the number of its chromosomes. The wide use of this technique yielded highly reproducible results which were checked by counts of chromosomes, biochemical data, and ultraviolet microspectrophotometry and it can now be considered as a sufficiently safe and precise means of investigation for the biologist. I n 1948, Boivin et al. reported chemical data on the DNA content in nuclei of various organs of cattle. They studied the amount of DNA contained in individual somatic nuclei (calf thymus, beef or calf liver, kidney, 171

172

R. VENDRELY AND C. VENDRELY

spleen, and pancreas) compared to the DNA of gametes (bull spermatozoa). Boivin and Vendrely (1947) had previously put forward the hypothesis that the gene could be imagined as a macromolecule of DNA, after the experiments of directed mutations on bacteria (Avery et al., 1944; Boivin et al., 1945a, b ; McCarty and Avery, 1946). If this postulate holds true, the amount of DNA per nucleus must be the same in all the cells of an animal and must represent double the DNA content of the nucleus of the gamete of the same animal, in the same way as the set of genes born by the chromosomes would be doubled. In fact, the results reported by Boivin et al. (1948) and Vendrely and Vendrely ( 1948, 1949) were quite consistent with the theory. They found about 6.4 ppgm. in the nucleus of all the organs of calf and beef studied and 3.3 ppgm. per sperm. These results were confirmed by other authors especially in fishes in which Mirsky and Ris (1949) and Vendrely and Vendrely (1952) found a very constant ratio of % between the DNA content of the sperm nucleus and that of the TABLE I D N A CONTENT(MICROMICROGRAMS PER NUCLEUS)OF THE NUCLEI OF CELLSOF DIFFERENT TISSUES OF CATTLE, RAT,AND FOWL FowlC

Organs Liver Thymus Kidney Pancreas Spleen Erythrocyte Leucocyte Lung Intestine Salivary gland Heart Bone marrow a

b e

6.4 6.4 6.4 6.6 6.8

9.40

2.44-2.68

6.72

2.31-2.48 2.43-2.73 2.52-2.66 2.55-2.61

6.52 6.60 6.71 7.60 7.55 6.46 6.90

2.57-2.65

Vendrely (1952). Thomson et al. (1953). Davidson et aE. (1950).

erythrocyte nucleus in a number of species (Tables I and 11). Davidson et d. (1950) found a constant individual amount of DNA in the nuclei of all the organs they studied in the chicken. All these data were obtained by gross chemical analysis performed upon nuclei isolated from one organ or the other ; an enumeration of nuclei in the suspension allowed a computation of the amount of DNA per nucleus. Of course, this method yields in fact a mean value for the amount of DNA of one nucleus and this value

CYTOPHOTOMETRIC STUDY OF NUCLEAR DNA

173

does not correspond to the reality if the suspension of nuclei so studied is heterogeneous, which is the case when embryonic, rapidly growing tissues are concerned, or when some of the nuclei are polyploid. The great superiority of the cytophotometric technique in such a field is that it makes possible a direct measurement of the amount of DNA in a single nucleus chosen among the others under the microscope. TABLE I1 DNA CONTENT(MICROMICROGF.AMS PER NUCLEUS) OF NUCLEI OF DIPLOID CELLS AND SFE ' RM IN SEVERAL SPECIES

Species

Erythrocyte

Cattle Fowl Toad Shad &P

Brown trout Rainbow trout Pii Tench

2.34 7.33 1.97 3.49 3.3 5.79 4.9 1.7 1.7

Liver

Sperm

6.4

3.3

2.39

1.26 3.70 0.91 1.64 1.6 2.67 2.45 0.85 0.85

2.01 3.33 3.0

Reference Boivin et al. (1948) ; Vendrely (1952) Mirsky and Ris (1949) Mirsky and Ris (1949) Mirsky and Ris (1949) Mirsky and Ris (1949) Vendrely and Vendrely (1952) Mirsky and Ris (1949) Vendrely and Vendrely (1952) VendreIy and Vendrely (1952) Vendrely and Vendrely (1952)

11. METHODSOF CYTOPHOTOMETRY 1. Cytophotometry in Visible Light The cytophotometry in visible light which was extensively used in the study of the DNA content of the nucleus was inspired by the famous tdchnique of ultraviolet microphotometry developed by Caspersson ( 1940), but the former does not require such an expensive set-up and it is quite easy and rapid to work with, so that it was used by a number of people for routine work. The method consists in first staining the nuclei (in tissue section or in a smear of isolated nuclei) by the Feulgen technique, then measuring the absorption of light by the Schiff-combined DNA of the whole nucleus. The first apparatus for such measurement was described by Pollister and Ris (1947), Pollister and Moses (1949), Pollister ( 1952). Another setup was described by Lison in 1950.l In the apparatus of Pollister, the nucleus or a part of the nucleus, observed through the microscope is isolated from the rest of the preparation by means of an iris diaphragm and the intensity of light transmitted through this surface is measured with a photo1 Two new models of histophotometers have been recently described by Vialli and Perugini (1954).

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R. VENDRELY AND C. VENDRELY

cell, which can be carried on the top of a bellows adapted to the ocular of the microscope. In Lison's apparatus, the enlarged image of the nucleus formed in the microscope is projected on a screen with a hole in its center ; behind this hole is fixed the photocell. One can choose an area of the nucleus and center it on the hole; the photocell gives the intensity of light passing through this part of the nucleus. In both techniques it is easy to compute, in arbitrary units, the amount of absorbing material in the nucleus, from two measurements with the photocell-( 1) the intensity of light transmitted through the nucleus, (2) the intensity of transmitted light in a "blank" (i.e., in a portion of the cytoplasm without any nucleus)-and from the determination of the surface of the nucleus, which is done by planimetry in Lison's apparatus or it is calculated from its dimensions measured with a micrometer (Pollister). The validity of the technique of cytophotometry in visible light has sometimes been questioned. It is conditioned by the specificity for the DNA of the Feulgen reaction and the important point is to know with certainty if this reaction stains specifically the DNA and, on the other hand, if the intensity of the color developed is proportional to the concentration of DNA in the nucleus. It is easy to answer the first point. The Feulgen technique is certainly one of the most reliable techniques of cytochemistry and its chemical significance is well known. It depends upon the production of aldehydes by acid hydrolysis of the deoxypentose of DNA and these aldehydes would restore the staining of the Schiff reagent (fuchsin decolorized by SO,). Bauer (1932, 1933) made a careful study of this reaction used in histology. He showed that the Schiff reagent is able to stain directly some cytoplasmic components, this "plasrnaYJ reaction is due to the presence of some polysaccharides and lipids. But if no color is developed when a control preparation without any hydrolysis is made one can be sure that all the purple color developed in the hydrolyzed preparation is due to the presence of DNA. Bauer (1932) determined also the optimal time of hydrolysis to obtain the maximum coloration. In spite of certain criticisms, the findings of Bauer were confirmed and it is now generally admitted that the Feulgen reaction performed under standardized conditions is highly specific for DNA, as more recently stated by Di Stephano, 1948a,b). The second point concerns the utilization of the Feulgen reaction for quantitative purposes. A priori, this did not seem possible after the findings of Widstrom (1928) and Caspersson (1932), who studied in vitro the intensity of the Feulgen reaction in connection with the concentration of DNA. They found that, in a certain range of concentration (between 0.02% and 1.5%) the intensity of dye is proportional to the

CYTOPHOTOMETRIC STUDY OF NUCLEAR DNA

175

concentration of DNA, but it is not so when proteins are present in the solution. More recently, Sibatani (1950) showed that the addition of histone to the nucleic acid increases markedly the intensity of the color reaction. In further studies, Sibatani (1953) showed that the presence of histone with DNA, in vitro, enhances the intensity of the Feulgen reaction in cases of overhydrolysis, lower pH, and absence of a large excess of sodium metabisulfite or Schiff reagent; the intensity of the dye is on the contrary depressed, in cases of optimal hydrolysis and in the presence of a large excess of sodium metabisulfite or Schiff reagent in the reaction mixture. But, on the other hand, it is possible that the behavior of the DNA toward the Feulgen reaction is not the same in sit% as in solution. Moreover, a number of data indicate that the intensity of dye of the Feulgen reaction performed in nuclei follows the Beer Lambert law in spite of the presence of variable amounts of proteins in the nuclei : Sibatani and Naora (1953), Sibatani (1954) made an extensive study of this problem. They extracted and measured the quantity of DNA and fuchsin combined in isolated nuclei stained by the Feulgen reaction and found no difference in the relative quantity of fuchsin and DNA thus combined in liver nuclei and in thymus nuclei, in spite of marked difference in the protein/DNA ratio in these two types of nuclei. On the other hand, Swift (1950b) cut Ambystoma liver nuclei at different thicknesses and he found a remarkably linear relationship between the extinctions and the various thicknesses of the absorbing layer, Schrader and Leuchtenberger (1950) showed that in the Pentatomid Arvelizcs there are three sorts of spermatocytes which differ considerably in volume (200, 400, 1600 cubic microns) and in their protein content, but the photometric Feulgen measurement of DNA per nucleus yielded exactly the same result in each case. So, a great dilution of DNA in a nucleus and the occurrence of more or less protein does not seem to affect the Feulgen reaction and quantitative photometric measurements are possible in all these conditions. The sources of errors inherent to the photometric technique have been carefully analyzed by Pollister and his group, Pollister and Swift ( 1950), and Swift (1950b) and also by Ris and Mirsky ( 1949), and the optimal conditions for the measurements were determined. Let us cite, however, Naora’s criticism (1952) of this method. This author suggested that all the cytophotometrical work which had been done so far was erroneous because the authors had not taken into account the possible errors due to the Schwarzschild-Villiger effect (1%). This effect consists of an increase of the light passing through a minute part of the preparation when a large area, including the part to be measured, is illuminated ; stray light is

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B. VENDmLY AND C. VENDRELY

then added to the image and may cause an error in the transmission measurement. Naora (1951, 1954), proposed an apparatus for cytophotometry where only a minute part of the preparation is illuminated, the illuminating optical system is made of another microscope reversed between the light source and the histological preparation, and an iris diaphragm of variable diameter ( 2 1 0 mm.) serves as a light source and is imaged with a reduction to 1 :2ooo by the system. This modified apparatus for cytophotometry does not seem to be indispensable. Pollister (Ornstein and Pollister, 1952) has shown that with his apparatus, there is no considerable error due to the Schwarzschild-Villiger effect within the ranges of concentration of DNA normally found in the nuclei studied. In most microspectrophotometric studies performed by his group small condenser apertures have been used; in these conditions glare is reduced. Naora has in fact overestimated the magnitude of the glare error. A few authors have sometimes stained the nuclei with purified methyl green instead of using the Feulgen reaction for the microspectrophotometry in visible light. Among these are Pollister and Leuchtenberger (1949), Kurnick (195Oa), and Frazer and Davidson (1953). The specificity of methyl green for DNA has been questioned. It seems that this reaction used under strictly standardized conditions can be used for a quantitative study of the DNA. Kurnick (19504 and Pollister and Leuchtenberger ( 1949) described respectively a personal procedure for staining with methyl green. Kurnick ( 195Oa, b, 1952) and Kurnick and Foster (1950) demonstrated that the methyl green stains selectively highly polymerized DNA and fails to stain, to any significant extent, depolymerized DNA and RNA. Thus methyl green could be used as a test for the degree of polymerization of DNA. But further researches (particularly by Taft, 1951) showed that other factors than the depolymerization of DNA must play a part in the variations of stainability with methyl green. This technique has certainly not the high specificity of the Feulgen reaction and its use is limited to a few studies in cytophotometry.

2. Cytophotometry in Ultraviolet Light Cytophotometry in ultraviolet light finally has yielded very interesting results in the study of DNA although it has not been used as extensively as cytophotometry in visible light (Leuchtenberger et al., 1952b), Frazer and Davidson (1953), Walker and Yates ( 1952a). It can be carried out in nuclei of living cells in tissue cultures as well as in isolated nuclei or nuclei in tissue sections. Microphotographs of these nuclei are performed in ultraviolet light at a wavelength of 2650 A. Densitometry is carried out on the negative and the areas of the nuclei are measured by planimetry.

CYTOPHOTOMETRIC STUDY OF NUCLEAR DNA

177

A simple calculation gives the amount of absorbing material in each nucleus in arbitrary units. Leuchtenberger et ul. (1952b) calculated the absolute amount of DNA per nucleus according to the following formula: Extinction x area DNA ’=

f

e

= extinction of a standard solution of DNA of 1 mg. per 1 cc. in a

1 cm. path. The absolute amounts of DNA calculated were in a very satisfactory agreement with the results obtained by chemical analysis. The results obtained with these techniques in various cases, will reveal some of the possibilities and the limitations of cytophotometry in visible and ultraviolet light.

111. THEDNA CONTENT OF INTERPHASE NUCLEIOF NORMAL TISSUES 1. The Constancy of the D N A per Nzccleus The first point which was studied by the cytophotometric technique was the amount of DNA per nucleus in different organs of the same animal. As the chemical work indicated a constancy of this amount (which was double the sperm content), throughout all the organs, it was quite important to settle this point. Two opposite opinions were then considered. On one hand American workers found a very good agreement between the DNA content of the nuclei and the number of chromosomes/the diploid cell nuclei in resting phase contain twice as much DNA as the haploid cell nuclei, and the polyploid cell nuclei, particularly in some organs of rodents (liver, pancreas), contain amounts of DNA 4 and 8 times higher than in the haploid cell nuclei. Ris and Mirsky (1949), for instance, showed in rat liver three classes of nuclei, the DNA content of which was in the ratio 2:1, 4:1,8:1, and this was proportional to the chromosome number. Swift (1950b) studied the DNA content of the nuclei of ten different tissues of mice and found in all the nuclei of kidney tubules, small intestine epithelium, spleen, neurons (spinal cord), interstitial cells of testis, what we shall call the “diploid value” of DNA ; the liver and pancreas were found to contain three classes of nuclei, the thymus lymphocytes and Sertoli cells two classes of polyploids (Table 111). On the contrary, Pasteels and Lison (19%) working with Lison’s apparatus for cytophotometry obtained quite different results in the rat. They found, for instance, in the liver and pancreas nuclei of the lower cIass, a DNA content which was only 1.3 to 1.5 times that of the sperm of the same animal and the two other classes of nuclei contained exact multiples of this DNA content. In other organs (kidney and adrenal gland, for in-

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R. VENDRELY A N D C. VENDRELY

stance) they found exactly the diploid theoretical value of twice that of the sperm. Pasteels and Lison concluded that there existed different amounts of DNA from one organ to the other, the amount being characteristic of the organ. However, their data were never confirmed. Leuchtenberger et al. (1951) made a cytochemical and chemical study of rat liver TABLE I11 AVERAGE AMOUNTS OF DNA PER NUCLEUS (ARBITRARY UNITS)IN FORMALIN-FIXED SOMATIC TISSUES OF YOUNG AND ADULT MICE,AS OBTAINED BY PHOTOMETRIC DETERMINATIONS ON FEULCEN PREPARATIONS" ~~

Cell types Liver

Pancreas

Thymus Lymphocytes Sertoli cells

Class I Class I1 Class I11 Class I Class I1 Class I11 Class I Class I1 Class I Class I1 Class I Class I1

Kidney tubule Small intestine epithelium Spleen Neurons (spinal cord) Interstitial cells (testis) Spermatids

~

-

~~

DNA in arbitrary units

Standard error

Number of nuclei measured

3.34 6.77 13.2

0.05 0.07 0.25

21 52 12

3.10 6.36 12.4

0.06 0.09

-

20 15 5

3.28 6.17

0.06 0.18

33 21

3.20 6.00

0.08 0.22

19 9

3.00 6.40

0.12 0.26

18

3.14

0.04

30

2.97 3.12 3.14

0.04 0.04 0.07

20 33 20

3.05 1.68

0.08 0.02

20 28

7

~

II

From Swift, 1950th

and kidney nuclei and their results were in very good agreement with the theory of the constancy of DNA ; the three classes of the liver nuclei contained respectively diploid, tetraploid, and octoploid amounts of DNA. Swift ( 195Ob) and Pollister et al. (1951) reported similar results, and also Fraser and Davidson (1953), Alfert and Swift (1953), Thomson and Frazer (1954). Even Pasteels and Lison (1953) were not able to confirm their first data. They published a series of measurements made in other rats and their results were then in complete agreement with the

CYTOPHOTOMETRIC STUDY OF NUCLEAR DNA

179

findings of the other authors. As their first experiment was performed in one rat, they claim that this exceptional case constitutes a valuable argument against the constancy of the DNA in the resting nuclei. As a matter of fact this stability of DNA is confirmed by a great many data and, as we shall see later, even in pathological cases with deep cytoplasmic and even nuclear changes, the DNA of the nucleus appears to be a remarkably constant amount and the theory of Pasteels and Lison seems now to be abandoned. 2. Variations of the D N A per Nucleus related to the Variations of Chromosome Number If the premises are admitted that the DNA content of the nucleus is related to the number of chromosomes the measurement of DNA per nucleus represents an easy way of studying polyploidy. We have seen how intratissular polyploidy can be demonstrated in certain organs of mammals, the significance of this polyploidy is not well known, it does not exist in very young animals and seems to be correlated somewhat with great functional activity of the cell being studied. Lecomte and De Smul (1952) have studied the effect of a hypoprotein diet upon the DNA classes of nuclei in young rat livers. They found by cytophotometric analysis (Lison’s apparatus) that in normal rats the nuclei of Class I1 (tetraploids) are predominant, while in animals with a hypoprotein diet the nuclei of the third class (octoploids) are completely absent and the diploid nuclei are the most numerous. So it seems that a lower activity in protein metabolism in the hepatic cell lessens markedly the degree of polyploidy. Let us quote also a very interesting study of Leuchtenberger et al. (1954b) showing the possible influence of hormones in the constitution of DNA classes in liver nuclei. Helweg-Larsen (1952) had previously demonstrated that dwarf mice showed a lack of, or a reduction in, nuclear size classes and when such dwarfs were injected with anterior pituitary growth hormone, the different nuclear size classes appeared as in normal mice. The cytophotometric investigations of Leuchtenberger et al. ( 1954b) showed that the dwarf mice had no DNA classes in their nuclei, all the nuclei contained the diploid amount. When these dwarfs are treated with anterior pituitary growth hormone the DNA classes are completely restored. Not only the chromosome number must be considered in correlation to the DNA content of the nuclei, but also the number of chromonemata in each chromosome. I n mammals, the chromosomes are the same in all the organs but in lower organisms, for instance in insects or in plants, the number of chromonemata in chromosomes, that is to say the degree of polyteny, may become considerable. The example of the chromosomes of

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the salivary glands of Drosophila or Chironomus is very characteristic and here the process of polyteny leads to giant chromosomes. It is evident that the process of multiplication of the chromonema involves the multiplication of its essential components and particularly of the DNA. Kurnick and Herskowitz ( 1952) determined photometrically (methyl green method) the DNA content of the nucleus of a salivary gland of Drosophila and found 712 x mg. for the biggest cells, while the anlage limb cells, probably diploid, contain 1.7 x mg. of DNA in their nucleus. This means that the nucleus of the salivary glands contains about lo00 times the haploid value of DNA. The chromosomes of salivary glands would thus be formed of about lo00 chromonemata. The degree of polyteny is more or less marked besides and the variations of the DNA content of the nuclei in the salivary gland ranged from 5.6 to 712 X mg. Meria and F4.k(1954) studied the DNA content of various tissues of the honeybee and they found a high degree of polyploidy in the different tissues with a rough positive correlation between the degree of ploidy and the secretory activity of the cell concerned. These authors found also a marked polysomaty in the tissues, the variability of the DNA per nucleus in this case is attributed to variations in chromosome numbers which are known to occur in certain cases or to an asynchronous multiplication of the chromonemata in the chromosomes. But, of course, this interpretation which appears quite satisfactory should not be accepted without consideration. One cannot prove cytologically that such a resting nucleus contains an abnormal number of chromosomes since chromosomes are invisible in resting nuclei. It is however possible to measure their size and it seems that there is, to a certain extent, a correlation between the size of a nucleus and the degree of its ploidy, but we cannot appreciate morphologically in resting nuclei a change in the degree of polyteny or of polysomaty. If we admit the strict Correlation between the DNA content of a nucleus and the number of chromosomes or more exactly of chromonemata, the measurement of this amount would be the easiest way of studying polyploidy, polyteny, and polysomaty in interphase nuclei. The possibility of physiological variations of the DNA, independent of the chromosomal set, will be discussed below, but, so far, it does not seem very likely. Plants, too, in many cases exhibit polyteny and polysomaty of nuclei in their tissues, and in fact Schrader and Leuchtenberger (1949) found that the nuclei of different tissues (root tip, leaf, bud tapetum, bud anther), in Tradescantia carry different amounts of DNA. Bryan (1951) on Tradescantia (bud-scale epidermis), Huskins and Steinitz (1948) on Rhoeo, found Similar results. Swift (1950a) studied also plant nuclei in various tissues of corn and of Tradescantiia and found different amounts of DNA in these nuclei, but

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these variations seem to be related to typical polyploidy or polyteny for the nuclei contained 2, 4, 8, 16, or 32 times the amount of DNA of the microgamete (haploid value). On the contrary, the results reported by Schrader and Leuchtenberger, Huskins and Steinitz are not always exact multiples of the DNA value of microgametes ; these intermediates could be interpreted as the result of a lack of synchronization in the reduplication process of the chromonemata, It is evident that further investigations are necessary in this field, for the behavior of the DNA per nucleus in plants seems to be quite different from that of animals and an extensive study of the DNA in plants should certainly lead to an interesting approach in problems concerned with polyploidy, polyteny, and endomitosis. Be that as it may, the deoxyribonucleic content of the nucleus in spermatid or in normal diploid nuclei in a given species appears to be characteristic of this species? and an evolutionary process involving a polyploidization from one species to another close species can be readily detected by means of photometric analysis. This is quite valuable inasmuch as sometimes chromosome counts are not possible or not clearly significant. The beautiful work of Hughes-Schrader (1951) on mantids is a good example of the utilization of the DNA constant in some problems of evolution. The author studied the DNA content of a number of close species of mantids whose karyotypes were not analyzable by the methods of comparative cytology. In three species with a number of chromosomes quite different, one having twice the number of chromosomes of the other, the DNA content of the spermatid was the same. This result was in good agreement with the cytological studies of the author, which lead to the conclusion that a redistribution of chromosomic material, not involving polyploidy, had been associated with the evolutionary divergence of these species, On the contrary, in two other close species a ratio very close to 1:2 was found between the DNA content of spermatids. So, polyploidy had probably played a role in the evolution of these karyotypes. Moses and Yerganian (1952) studied by cytophotometric analysis the DNA content of the nuclei in various tissues of two close species of Cricetinae which exhibit a great difference 2 The chemical analysis (Vendrely and Vendrely, 1949) of a number of different species show that the DNA content of the nucleus in mammals ranges from 5.0 to 6.4 ppgm. and in birds from 1.7 to 2.2 ppgm., that is to gay they are situated within narrow limits, but in fishes some high values would indicate the Occurrence of polyploidy in the process of evolution (Vendrely and Vendrely, 1950). Mirsky and Ris (1951) pointed out that the DNA content per cell in Iungfishes, amphibians, and reptiles decline from the first to the latter. O n the other hand, they noted that in invertebrates the DNA per cell is greater in higher forms than in more primitive forms. But of course, these are only indications and further studies are necessary for a clear view of the variations of the amount of DNA per cell throughout the animal kingdom.

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in their chromosome numbers (Mesocricetzcs azcratus, 2n = 38 (Koller, 1938; Muldal, 1948) or 2n = 44 (Matthey, 1951) and Cricetulus griseus, 2n = 22 (Matthey, 1951)). Their data suggest that the evolutionary process must involve chromosome fragmentation rather than polyploidy for the DNA content of the nucleus was the same in these species.

IV. THEDNA CONTENT OF NUCLEIIN DEEPPHYSIOLOGICAL AND PATHOLOGICAL CHANGES 1. Physiological Changes We have seen that accumulated evidence suggests that the DNA content of interphase nuclei in normal tissues can be considered as a constant characteristic of the species. But one may suppose that this amount can be affected by severe physiological or pathological disturbances which strongly affect the whole cell. As a matter of fact, some cytological studies suggested variations in the charge of chromatin in the nucleus correlated with the secretory activity of the cell for instance, but these studies were only qualitative and investigations with specific staining and quantitative measurements were necessary. At the present time, a number of cytophotometrical data are available concerning the DNA content of the nucleus in physiological and pathological conditions, but we must stress that, if the nucleus undergoes slight changes around the normal values, these variations cannot be detected by cytophotometric analysis, the errors of these techniques can reach 15 and even 30% when the distribution of chromatin becomes highly heterogeneous, and, at any rate, errors of 10% must be commonly expected. Nevertheless, as Leuchtenberger et al. (1951) have pointed out, in the liver of rodents, measurements of DNA in individual nuclei may exhibit a variation of -t-25% though the mean amount may be the same in all the diploid nuclei. So, these variations do not seem to be attributable exclusively to errors of the Feulgen photometric method. We shall see further that intermediate values of DNA can be due to DNA synthesis in preparation for a coming mitosis and the fact is particularly marked in developing or regenerating tissue. Even in a nondeveloping tissue, a few cells among the others must prepare for mitosis. But the question is to know whetherexcepting during the physiological preparation for mitosis or polyploidythe DNA content of the nucleus may undergo variations from the normal value under certain circumstances. A number of results argue against this possibility. Chemical measurements of a great number of authors have shown that prolonged fasting or a protein deficient diet, which cause a considerable decrease of RNA in rat liver, does not affect the DNA content of the nuclei (Mirsky and Kurnick, 1951; Campbell and Kosterlitz, 1952;

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Villela, 1952;Thomson et ul., 1953; Davidson, 1953; Fukuda and Sibatani, 1953). Measurements by cytophotometric techniques have not been performed in all these cases. We have seen that Lecomte and De Smul (1952),working on young rats, found that a hypoproteic diet caused a different repartition in the frequency of the nuclei within the different classes of polyploids. Moreover, the DNA content per nucleus within the classes is higher in animals submitted to a hypoproteic diet than in normal animals. The authors think that this variation is due to the fact that they are working with young animals. As Thomson et ul. (1953)point out “it is conceivable that protein deficiency might inhibit growth and mitosis to a greater extent than it affects the premitotic synthesis of DNA, and this could result in an apparent increase in the average DNA content per nucleus.” In rapidly regenerating liver (48hr. after hepatectomy) , Thomson and Frazer (1954)found an increased proportion of Class I11 (octoploid) nuclei; this fact seems to be related to the occurrence of mitosis. Laird (1953)reported that thioacetamide provokes a doubling of volume of the nuclei of rat liver cells, but the DNA content remained unchanged. The effect of some hormones has also been tried. For instance Alfert and Bern (1951) injected estrogens into ovariectomized rats. In such conditions, the nuclei of the uterine gland cells doubled their volume. I n these nuclei the protein content (measured photometrically with the Millon reaction) was doubled while the DNA content remained unchanged. On the other hand, Alfert et al. (1953) studied the DNA and proteins of thyroid gland nuclei of rat after hypophysectomy or treatment with propylthiouracil. The nuclear volumes in thyroids of normal, hypophysectomized, and propylthiouracil-treated rats formed a series of 1 :2 :3 and the total protein content was correlated with the volume. But the DNA content of the nuclei was that of typical diploid cells in the three series with occasionally a few polyploids. In thyroids of propylthiouracil-treated rats, where mitotic activity is visible, intermediates between diploid and tetraploid values are found. So, the functional activity of the thyroid gland, though it affects the volume and the protein content of the nuclei has no effect on the individual amount of DNA of these nuclei. But Roels (1954),working with the Lison’s apparatus, had reached quite different conclusions : he found that the amount of DNA in the thyroid nucleus increases with the cell activity (stimulated by the thiouracil) and decreases when the cell is inhibited by thyroxin. Perhaps these variations could be related to more or less mitotic activity in the gland under these conditions and not really to the cell secretory activity. The hypophysectomy, which produces a considerable decrease of the nuclear and cytoplasmic RNA in rat liver, does not affect the DNA content of the nucleus (Di Stephano, et al., 1952).

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These authors pointed out that the administration of growth hormone to hypophysectomized rats provokes an increase of the amount of both nuclear and cytoplasmic RNA, but no change of the DNA content of the nuclei. But, Bergerard and Tuchman-Duplessis (1953) (Lison’s apparatus) found that hypophysectomy in rats determines a slight decrease of the DNA content of the hepatic cell nuclei “B” (tetraploids). After the injection of growth hormone this content came back to the normal values. This discrepancy could perhaps be explained by the fact that Bergerard and Tuchman-Duplessis worked on young rats while Di Stephano worked on adult rats, where the tissues are no longer in development. Fautrez and Moerman (1954) (Lison’s apparatus) reported also possible variations from the diploid value in the DNA content of the hepatic cells of the fish Lebistes reticubtus; these could be related to the physiological activity of the organ. Govaert (1953) in Fmciola hepatica reported also some variations of DNA during physiological changes of the cells. Fautrez eb id. (1955) studied the compensatory hypertrophy of the kidney in the rat and they found in control nuclei an amount of DNA very markedly lower than the diploid value. This value would be increased in the kidney of the nephrectomized rat but was still lower than the diploid value. These results concerning the normal rat are in contradiction with the data of other authors (Leuchtenberger et al., 1951; Thomson and Frazer, 1954) who always found the diploid value in normal rat kidney. In summary, one can hardly come to a definite conclusion from these few results. It seems that Lison’s apparatus has shown irregularities in the DNA content of the nucleus ;the results obtained with Pollister’s apparatus, on the contrary, stress the constancy of this amount. A close comparison and a simultaneous study of the same material with the two apparatuses would be necessary to clear these discrepancies. The physiological changes in the DNA content of the nucleus that we reported are not important, and sometimes it is difficult to decide whether such differences are real or correspond to an error of measurement. Some of the changes noticed affect considerably the protein and RNA content of the nucleus ;this could change the conditions of measurements af the Feulgen intensity and atrect the results to a certain extent, Let us quote at least one very striking example of variation of the DNA content of the nucleus correlated with the secretory function. This remarkable case is reported by Leuchtenberger and Schrader (1952) in the salivary gland cells of Helix aspemu. These authors found that the DNA of the cells in this tissue varies in the order of magnitude of 30 :1 from nucleus to nucleus. This amount is higher in nonsecretory cells and decreases gradually as the amount of secretion increases in the cell. The authors suggest

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185

that the DNA would be utilized directly in the manufacture of cytoplasmic secretions of polysaccharide nature. A similar process has been described by Schrader and Leuchtenberger (1952) in the ovary of certain Hemiptera, where a part of the DNA of the nucleus of some cells seems to be transformed into nutritive material which is transferred to the growing eggs. Lison and Fautrez-Firlefyn (1950) found a similar process in certain cells of the ovary of a crustacean, Artemia salina, but interpret this fact as a process of degeneration consecutive to an excessive polyploidy. Such facts were never encountered in vertebrates and especially in mammals ; these particular cases speak in favor of the possibility of a direct participation of the DNA in the secretory act which seems to be a rare and even exceptional process. 2. The D N A of the Nucleus in Pathological Cases

Coming back to vertebrates and to mammals, we must now examine to what extent pathological changes affect the DNA of the nucleus in a tissue. Since 1950, Leuchtenberger, studying pycnotic nuclear degeneration, had shown that the DNA content of the nucleus remains the same until an advanced stage of pycnosis while the proteins of the nucleus (measured by the Millon reaction intensity) had already decreased considerably. More recently, Leuchtenberger et al. (1953, 1 9 5 4 ~ studying )~ the DNA content of spermatozoa of fertile and infertile human males, found a lowered value for this content (about 78% of the normal value) in some cases of infertility. A great deal of work had been done by chemical and cytochemical analysis on the mean amount of DNA per nucleus in various neoplastic tissues. Most of them indicate that this amount is the same in cancer as in normal tissue (Mark and Ris, 1949; Cunningham et al., 1950a,b; Price and Laird, 1950; Price et al., 1950; Metais and Mandel, 1950; Davidson et aZ., 1951a,b). In some cases, this amount of DNA per nucleus is higher than in normal tissues (McIndoe and Davidson, 1952; Klein and Klein, 1950; Klein 1951). Some investigations have been made with the cytophotometric technique. Leuchtenberger and Lund ( 1952) reported in senile keratoma of Freudenthal very high values of DNA per nucleus. Bader (1953) found in four out of five types of tumors values of DNA per nucleus higher than normal and attribute this fact to mitosis and polyploidy. Leuchtenberger et d. ( 1952a) estimated by ultraviolet microspectrophotometry combined with chemical analysis the DNA content of nuclei of ascites tumors. They found a tetraploid amount of DNA in Ehrlich ascites tumors, In the DBA ascites lymphoma nuclei, they found the typical diploid value of DNA. Mellors et al. (1952) using ultraviolet microspectrog-

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raphy found in the nuclei of squamous cancer eells a geometrical progression of the total nucleic acid content of the interphase nucleus, which is consistent with a process of polyploidy, endomitosis, or polyteny. Thomson and Frazer (1954) reported that the tumor-bearing liver, produced by a prolonged administration of p-dimethylaminoazobenzene to rats, contains a higher proportion of Class I nuclei than normal. A few Class IV nuclei were found in both regenerating liver and tumor-bearing liver. Recently, Leuchtenberger et al. (1954b) have reported a very important cytophotometrical work upon approximately 2500 individual cells of 49 normal and 27 malignant tissues of humans and they found that, while in the normal tissues the DNA content of the nuclei show a limited degree of variation from cell to cell, precancerous and malignant tissues have not the same uniformity in this respect. Malignant tissues show a much larger scatter from cell to cell than do the normal. These variations can easily be interpreted on the basis of mitosis occurring in malignant cells ; the synthesis of DNA in nuclei preparing mitosis may cause these deviations from the normal value. Leuchtenberger et al. (1954b) point out the fact that in some cases. which pathologists designate as borderline cases, where mitotic figures are scanty or absent, the cytophotometric analysis could be a very valuable help for the diagnosis of cancer; for it is able to detect in nuclei, which seem morphologically normal, a large scatter of DNA values per nucleus indicating a dividing process in preparation. At any rate, the neoplastic process has no specific effect on the DNA content of the nucleus and the variations of DNA found in such cancerous tissues are comparable to the variations occurring in rapidly growing tissues (embryo for instance) as we shall see further. We have seen that the DNA content of the nucleus can be studied by cytophotometric analysis. As for the reliability of this technique, Leuchtenberger ( 1954) has recently published an impressive number of cytophotometric results (upon 15,000 nuclei) accumulated for years, which are perfectly in agreement with chemical data and chromosome counts. Most of the time, the photometrical measurement for DNA did not differ very much from one nucleus to the other and this constant value represents double the amount of DNA measured in the sperm of the same animal; these data are in a very good agreement with the indications of chemical analysis. Cytophotometry is particularly valuable for the study of heterogeneous tissues where the occurrence of polyploid nuclei or mitosis make impossible the analysis with chemical technique of the DNA content of the nucleus. For that reason, the study of the behavior of DNA in the nuclei in developing tissues, particularly in the nuclei undergoing mitosis, is best carried out by cytophotometry.

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V. THEDNA CONTENTOF THE NUCLEUS DURING CELLDIVISION 1. The Moment of the Synthesis of D N A in the Process of Mitosis The very first cytophotometrical investigations on mitosis were made by Caspersson ( 1939). Using ultraviolet cytophotometry he studied the cell divisions in the spermatogenesis of a grasshopper and suggested that the synthesis of DNA necessary for the doubling of the chromosome might occur during the first phases of the division and reach a maximum at metaphase. Later on, Ris (1947) using the Feulgen cytophotometric technique, studied mitosis in meristem cells of onion root tips and in the spermatogenesis of Chortophaga and concluded also that there was a doubling of the DNA of the nucleus beginning at prophase and being completed at metaphase. But in the last years a great deal of work has been carried out on this subject and it seems that the synthesis of DNA is already complete when the first morphological changes of the prophase appear in the nucleus. First, spermatogenesis provided good material for such a study and a great number of authors have shown in testes of various animals that the first spermatocyte nuclei in prophase contain twice the amount of DNA of diploid cells, that is to say four times the amount of DNA in a sperm ; so all the DNA necessary for the two meiotic divisions is synthesized before the prophase in the first spermatocytes (Lison and Pasteels, 1949; Swift, 1950b ; Schrader and Leuchtenberger, 1950). Moses and Taylor (1953), studying the formation of pollen in Tradescantia concluded also that DNA synthesis occurs in predivisional stages. Taylor and McMaster (1954) studying the DNA content of the nuclei during microgametogenesis in Lilium longiftorum found also that the DNA is synthesized before mitosis. In Ciliates, Seshachar (1950) shows also that the synthesis of DNA must occur during interphase. As for mitosis in developing tissues, there were some controversies. Many authors studying embryos or developing tissues noted that in those materials the amount of DNA per nucleus showed values ranging between the diploid and twice this amount. For instance, Swift (1950b) in the developing liver tissue of an ll-day mouse embryo and in the erythrocytes of a recently hatched Ambystoma larva, found that the post-telophase nuclei showed diploid values whereas the early prophase nuclei contained twice this value. Alfert (1950), in the nuclei of the first stages of development of mouse embryo, found similar results. More recently, Patau (1953), working on mitosis of onion, showed that no significant DNA changes are found during mitosis (mean DNA value of 15 metaphases was 98.9% and that of 11 anaphases was 105.2%, of that of 19 prophases). But Pasteels and Lison (1950b), Lison and Pasteels (1951), and their school (Bul-

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lough and Pasteels, 1951; Marinone, 1951a) developed a quite different conception on the duplication of DNA in the course of mitosis. Working on erythroblasts of the rat embryo, Lieberkiihn glands of the adult rat and fibroblasts of the chick heart in tissue culture, they found that the synthesis of DNA occurred in telophase and was completed during the reconstitution of the two daughter cells. On the other hand, the value reached at telophase by these nuclei was higher than the theoretical diploid value (half of the amount of DNA of the primary spermatocytes), the ratio was 1.67:l in erythroblasts and 1.92:l for Lieberkiihn cells for instance, so that, this amount was by no means correlated with the chromosome number. The theory of Pasteels and Lison was not confirmed. On the contrary, Alfert and Swift (1953) showed that the discrepancies between the results of Pasteels and Lison and other authors’ results are probably due to errors of technique and interpretation. They reported for instance that, in the rat crypts of Lieberkiihn, anaphase and telophase nuclei are small and the density of stain is high. Errors due to stray light are important under those conditions, but Pasteels and Lison interpreted the increase of extinction as true DNA variation associated with synthesis. As Table I shows the measurements of Alfert and Swift (which have been made at a wavelength off the Feulgen absorption peak to minimize the stray light error) demonstrate a remarkable constancy of the amount of DNA in the chromosomes during the anatelophase (Table IV) Moreover, the studies of Walker and Yates (1952b) on living cells in tissue culture established beyond a doubt that the DNA of the nucleus is doubled during interphase before prophase. These authors have combined microcinematography in phase contrast of a living cell and the measurement of the ultraviolet absorption of its nucleus at different times. The examination of the film allows an exact knowledge of the developmental phase of the cell when the measurements are performed. So, the exact time of the synthesis of DNA for mitosis, is established as occurring during interphase. prior to the onset of mitosis (Fig. 1). When the rhythm of mitosis is very rapid, the interphase may be so short that the synthesis of DNA begins just after the telophase without any resting phase, as shown by Bergerard (1955) in neuroblasts and embryonic cells of the insect Clitzcmnus extradentatus, Br.

.

2. The D N A in Embryonic and Differentiating Tissues Another question, which was opened by the work of Pasteels and Lison concerns the amount of DNA in the nuclei of developing tissues. Most of the authors admitted that the increased mean values of DNA found by chemists for nuclei of dividing tissues compared to normal tissues are due

0

MEANAMOUNTS OF DNA

Stage

Pasteels and Lison (1950b)

Interphase

Continuous variation from 740 to 2620 1470, 1810, 1850, 1950 1377 f28

Prophase Metaphase Anaphase Telophase

IN

Continuous increase proportional to nuclear surface, 650-1300

3

TABLE IVa NUCLEIOF RATCRYPTS OF LIEBERKUHN Number of Measurements

38 4 21

23

Alfert and Swift Class I : 16.2 2 0.34 Class 11: 30.2 , 32.3 23.5, 32.0 32.0 34.7, 37.5 32.3 k 0.91 16.5 f0.46 Early 17.0 f0.42 Mid 17.5 f0.40 Late to early interphase 16.6 0.52

0

v 5:

0

Number of Measurements

8

3s 2 5 29 24 21 26 26 19

cl

g

mcf

E

m

3rc 0

w

Z

4Pr 4

k?E!

I-

a

From Alfert and Swift, 1953.

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R. VENDRELY AND C. VENDRELY

to the synthesis of DNA necessary for the mitoses, which are scanty or absent in normal tissues. And, indeed, these authors found in the nuclei of embryos (Swift, 1950b), of malignant tissues (Leuchtenberger, 1954b) values of DNA scattering from the normal value to double this value. But Lison and Pasteels (1951) and Pasteels and Bullough (1953) thought that in proliferating tissues, the amount of DNA of the nucleus is conP

M A T

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/$$ 1

I

,

I

I

I

I

l

4 N - ,..._..._ 4..........,,

I

I

,

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....,...

P

M A T

i

; I ; I

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I

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FIG.1. Development of the D N A content of the cell in the course of mitosis. a. Theory of Caspersson (1939) and Ris (1947). b. Theory generally accepted-Swift (1950b), Alfert (1950), Walker and Yates (1952b). c. Theory of Pasteels and Lison (195Ob) compared with the preceding theory.

siderably increased and that this increase cannot be explained merely by the preparation for mitosis. This opinion was based on results which are probably partially erroneous as Alfert and Swift (1953) demonstrated. In tissue culture, they demonstrated that the nuclei in the migration zone are greatly flattened, while in the dividing zone, the nuclei are spherical. When an appropriate correction is done, the results fall in an expected range of values and the discrepancies reported by Pasteels and Lison are no longer seen. The results of Lison and Pasteels (1951) in the developing sea urchin embryo suggested also a great increase of DNA during intense cellular multiplication and this increase was not correlated with the rhythm of nuclear division. But recently McMaster (1952) studying another sea urchin species found results in complete agreement with the constancy hypothesis. Nevertheless some other authors reported discrepancies from

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191

this constancy hypothesis in embryonic tissue or differentiating cells. Moore (1952), working on embryos of Rana pipiens, reported results which seem to indicate a correlation between differentiation and a wide range of values of DNA in the nuclei which could not be explained by the number of mitoses. Marinone (1951b) found a marked decrease of nuclear DNA during the maturation of erythroblasts and granuloblasts in man. Reisner and Korson (1951) reported similar results in erythroblasts. Moore (1952) suggested that the variations of DNA in differentiating cells could indicate a morphogenetic activity of the gene. But, of course, other data are necessary to settle this aspect of the problem of the DNA content of the nucleus.

3. The D N A of the Nucleus during Maturation, Fertilization, and Cleavage A number of authors have admitted for a long time that DNA is a very transitory substance which appears on the chromosomes and undergoes large fluctuations in the vital cycle of the cell. In fact, measurements of DNA in nuclei, as we have seen, suggested a quite different aspect of DNA as a biological substance: its remarkable constancy. But one fact is particularly puzzling: it is the failure of the Feulgen reaction in developing oijcyte nuclei of a great number of species. Many authors concluded that the DNA was absent from the nucleus at that stage of maturation. A very careful study by Alfert (1950) and Pollister et al. (1951) on oogenesis and cleavage in the mouse indicated very clearly that the amount of DNA per primary Gcyte nucleus is constant and that this amount becomes progressively diluted as the nucleus increases in time (Table V) so that the concentration of DNA is below the sensitivity of the Feulgen reaction. The decrease and disappearance of the Feulgen staining in oocytes, which have been described, is thus due to the progressive dilution of a constant amount of DNA in the increasing nucleus. After the two meiotic divisions, pronuclei contain one-fourth the DNA that was present in primary oocytes. Mulnard (1952) reaches the same conclusion in oogenesis of the insect Acanfhoscelides obtectus, and Swift and Kleinfeld (1953) in the grasshopper Melanoplus also obtained similar results (Table VI) . In the first cleavage stages, Alfert (1950) found no changes in the amount of DNA per nucleus : the diploid amount at early interphase doubled prior to the onset of mitosis. This observation was in contrast with the view of Lison and Pasteels, who found a high level for DNA content in the sea urchin embryo, this level decreasing gradually to the diploid amount in the course of development. Pasteels and Lison (1951) had also studied the fertilization and first cleavage stages in the annelid Sabellaria and found at

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these stages very high amounts of DNA. But Alfert and Swift (1953) studied the same material and found exactly the same pattern as in mouse oocyte cleavage. Swift and Kleinfeld (1953) in the grasshopper Melunoplus differentialis report aiso that the DNA “does not follow any comTABLE V AMOUNTS OF DNA (FEULGEN), IN ARBITRARY UNITS,IN PRIMARY O ~ C Y TNUCLEI E OF THE MOUSEa

Number measured

Description

Mean volume

Mean amount DNA (in arbitrary units)

Early peripheral oocytes with a follicle of a single layer of flattened cells (10-day-old mouse)

15

438p3

6.45 f0.14

Medium-sized oijcytes with a follicle of a single layer of cuboidal cells (10-day-old mouse)

15

1365p3

6.46 2 0.16

Large oiicytes with a follicle of 2-3 layers of cells (10-day-old mouse)

20

601Op3

6.68 f0.18

Oocytes of various sizes with follicles of 1-3 layers of cells, in a S-weekold mouse

10

a

6.02 f0.16

From Alfert, 1950. TABLE VI INDIVIDUAL NUCLEIDURING STAGESOF GRASSHOPPER EGG FORMATION^

O~GENESIS: AMOUNTSOF DNA

Stage Oogonia Primary oocytes Leptotene Zygotene Pachytene Diplotene Diakinesis Total Metaphase I Secondary oocytes Ova Second polar body 9

IN

D N A Class

Amount of DNA (in arbitrary units)

Standard error

NO Measured

2C+ 4C

7.1

0.45

23

4c 4c 4c 4c 4c 4C 4C

10.0 9.9 9.2 9.2 8.4 9.5 8.6 4.5

2c C C

From Swift and Kleinfeld, 1953.

9

5 4 8 0.12

2 28 1

2 7 4

CYTOPHOTOMETRIC STUDY OF NUCLEAR DNA

193

plex course of synthesis and segregation” but behaves just as in any normal dividing tissue. VI. CONCLUSION Summarizing we have seen that the first approach to the question of the DNA content of the nucleus was made by chemical analysis. The biochemists, very rapidly established the constancy of the DNA per nucleus as a general law comparable, for instance, to the law of the constancy of the number of chromosomes. In fact, considering the results of chemical analysis, this law of constancy appeared very likely, but, it must be borne in mind that the results established statistically upon a very great number of nuclei do not exclude the possibility of small variations to and from the mean value. Moreover, when dealing with developing tissues with a number of mitoses going on, the synthesis of DNA for new cells must occur very often and cause an increase in the DN A content of a number of nuclei. The work of Pollister and his school by measuring individual nuclei by cytophotometric methods confirmed very well the law of constancy, and the perfect agreement of these cytophotometrical datas and the previous biochemical results seem to reinforce the value of this law. But, at the same time, Belgian workers energetically withstood this theory and put forward personal opinions of the occurrence of various DNA contents of nuclei in the various tissues of animals. According to these authors, the DNA content of the nuclei gave a clue to the problem of tissue differentiation. This conception was based mainly on a number of results obtained in the different organs of one single rat. Later after studying a great number of tissues undergoing strong physiological changes, especially developing tissues, cells in tissue culture, and embryos, they reported a considerable range of variations of the DNA content of the nucleus, so that it was impossible to correlate this content with the chromosome number. A great confusion resulted from this controversy, and the whole question had to be reconsidered critically. First, some biochemists and cytologists, working together compared very carefully their results on the very same material especially on the rat. Parallel work was carried out with ultraviolet cytophotometry and the great number of results now available are in contradiction with the theory adopted by the Belgian authors. So in the light of recent work it seems that the general theory of Pasteels and Lison cannot be retained and the theory of the constancy of the DNA content of the nucleus is adopted by a great number of authors as a valuable working hypothesis.

194

R. VENDRELY AND C. VENDRELY

Deviations from this theory can be found in some cases, particularly in plant cells and in invertebrates. In such cases the occurrence of polyteny or aneuploidy (different number of chromosomes in the different cells of a tissue) can explain these discrepancies but sometimes direct participation of the DNA to the secretory process was suggested. In addition the possibility of variations of this DNA constant in some particular circumstances of cellular life cannot be excluded, for instance in differentiating cells, but we can hardly decide with the cytophotometric techniques whether these deviations are due to real variation of the DNA of the nucleus or to the synthesis of DNA for the future mitosis ; further work is necessary in this field. Nevertheless, the cytophotometric technique has proved highly valuable and made possible the study of some problems which it was impossible to solve otherwise, for instance the moment of synthesis of DNA for mitosis. And let us stress the progress in the field of cytology conferred by quantitative cytochemistry. The DNA which appeared to the cytologist as a variable element fixed upon the chromosomes during mitosis and of no genetic importance, appears, in fact, as a quantitatively constant element of the nucleus and an important component of chromosomes.

VII. REFERENCES Alfert, M. (1950) J . Cellular Comp. Physiol. 98, 381. Alfert, M., and Bern, H. A. (1951) Proc. Natl. Acad. Sci. (U.S.) 57, 202. Alfert, M., and Swift, H. (1953) Exptl. Cell Research 5, 455. Alfert, M., Bern, H. A., and Kahn, R. (1953) Anat. Record 117, 585. Avery, 0. T., MacLeod, C. M., and McCarty, M. (1944) J . Exptl. Med. 79, 137. Bader, S. (1953) Proc. SOC.Exptl. Biol. Med. 83, 312. Bauer, H. (1932) 2. Zellforsch. u. mikroskop. Anat. 15, 225. Bauer, H. (1933) 2. Mikroskop. anot. Forsch. SS, 143. Bergerard, J. (1955) Compt. r e d . M, 564. Bergerard, J., and Tuchman-Duplessis, H. (1953) Compt. rend. 236, 1080. Boivin, A,, Delaunay, A., Vendrely, R., and Lehoult, Y . (1945a) Comfit. rend. 2a1, 718.

Boivin, A., Delaunay, A., Vendrely, R., and Lehoult, Y. (1945b) Comfit. rend. soc. biol. 139, 1046. Boivin, A., and Vendrely, R. (1947) Experientia 8, 32. Boivin, A.,Vendrely, R., and Vendrely, C. (1948) Compt. rend. Z46,1061. Bryan, J. H.D. (1951) Chsomosoma 4, 369. Bullough, W. S.,and Pasteels, J. (1951) Nature 168, 608. Campbell, R. M., and Kosterlitz, H. W. (1952) Science 116, 84. Caspersson, T. (1932) Biochem. Z . 253, 97. Caspersson, T. (1939) Chrormsomo 1, 147. Caspersson, T. (1940) J . Roy. Microscop. SOC.60, 8. Cunningham, L., Griffin, A. C., and Luck, J. M. (1950a) Cancer Research 10, 211. Cunningham, L., Griffin, A. C., and Luck, J. M. (1950b) J. Gen. Physiol. 34, 59. Davidson, J. N. (1953) BuZZ. soc. chim. biol. 35, 49.

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Davidson, J. N., Leslie, I., Smellie, R. M. S., and Thomson, R. Y. (1950) Biochem. J. 46, Proc. XL. Davidson, J. N., Leslie, I., and White, J. C. (1951a) Lancet i, 1287. Davidson, J. N., Leslie, I., and White, J. C. (1951b) J. Pathol. Bacterial. 89, 471. DiStephano, H. S. (1948a) Proc. Natl. Acad. Sci. (U.S.) S4, 75. Di Stephano, H. S. (1948b) Chromosoma S, 282. Di Stephano, H. S., Bass, A. D., Diermeier, H. F., and Tepperman, j . (1952) Endocrinology 61, 386. Fautrez, J., and Moerman, J. (1954) Compt. rend. assoc. anat. 80, 554. Fautrez, J., Cavalli, G., and Pisi, E. (1955) Nature 176, 684. Frazer, S. C., and Davidson, J. N. (1953) Exptl. Cell. Research 4, 316. Fukuda, M., and Sibatani, A. (1953) Exptl. Cell. Research 4, 236. Govaert, J. (1953) Conipt. rend. soc. biol., 147, 1494. Helweg-Larsen, H. F. (1952) “Nuclear Class Series.” Munksgaard, Copenhagen. Hughes-Schrader, S. (1951) Biol. Bull. 100, 178. Huskins, G. L., and Steinitz, L. M. (1948) J. Heredity S9, 34. Klein, E., and Klein, G. (1950) N a t w e 186, 832. Klein, G. (1951) Exptl, Cell. Research 2, 518. Koller, P. C. (1938) J. Genet. S6, 177. Kurnick, N. B. (1950a) Ezptl. Cell. Research 1, 151. Kurnick, N. B. (195Ob) J. Gen. Physiol. 98, 243. Kurnick, N. B. (1952) Stuin Techml. 27, 233. Kurnick, N. B., and Foster, M. (1950) J. Gen. Physiol. S4, 147. Kurnick, N. B., and Herskowitz, I. H. (1952) J . Cellular Comp. Physiol. 39, 281. Laird, A. K. (1953) Arch. Biochem. and Biophys. 46, 119. Lecomte, C., and De Smul, A. (1952) Compt. rend. a94, 1400. Leuchtenberger, C. (1950) Clwomosoma 8, 449. Leuchtenberger, C. (1954) Science 120, 1022. Leuchtenberger, C., and Lund, H. (1952) Cancer Research l2, 278. Leuchtenberger, C., and Schrader, F. (1952) Proc. Natl. Acad. Sci. (U.S.) 98, 99. Leuchtenberger, C., Vendrely, R., and Vendrely, C. (1951) Proc. Natl. Acad. Sci. (US.)97, 33. Leuchtenberger, C., Schrader, F., Weir, D. R., and Gentile, D. P. (1953) Chomosorm 6, 61. Leuchtenberger, C., Klein, G., and Klein E. (1952a) Cancer Research 12, 480. Leuchtenberger, C., Leuchtenberger, R., Vendrely, C., and Vendrely, R. (1952b) Exptl. Cell. Research S, 240. Leuchtenberger, C., Helweg-Larsen, H. F., and Murmanis, L. (1954a) Lab. Invest. 8, 245. Leuchtenberger, C., Leuchtenberger, R., and Davis, A. M. (1954b) Am. J. Pathol. 90, 65. Leuchtenberger, C., Weir, D. R., Schrader, F., and Leuchtenberger, R. (1954~) Excerpta Med. I 8 , 418. Lison, L. (1950) Acta Anaf. 10, 330. Lison, L., and Fautrez-Firlefyn, N. (1950) Nature 166, 610. Lison, L.,and Pasteels, J. (1949) Comfit. rend. soc. biol. 143, 1607. Lison, L., and Pasteels, J. (1951) Arch. biol. (Likge) 62, 1. McCarty, M., and Avery, 0. T. (1946) J. Exptt. Med. 88, 89. McIndoe, W. M., and Davidson, J. N. (1952) Brit. J. Cancer 6, 200.

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McMaster, R. (1952) A w t . Record 118, 26. Marinone, G. (1951a) Experientia 7, 227. Marinone, G. (1951b) Le Sang 28, 89. Mark, D. D., and Ris, H. (1949) Proc. SOC.Exptl. Biol. Med. 71,727. Matthey, R. (1951) Experientia 7, 340. Mellors, R. c., Keane, J. F.,and Papanicolaou, G. N. (1952) Science 116,265. Meria, M. R. N., and Ris, H. (1954) Chromosoma 6, 522. Met% P., and Mandel, P. (1950) Compt. rend. soc. bid. 144, 277. Mirsky, A. E.,and Kurnick, N. B., quoted by Mirsky, A. E., and Ris, H. (1951) J. Gen. Physiol. S4, 451. Mirsky, A. E.,and Ris, H. (1949) Nature 163, 666. Mirsky, A. E.,and Ris, H. (1951) J. Gen. Physiol. 34, 451. Moore, B. C. (1952) Chromosonm 4, 563. Moses, M. J., and Taylor, J. H. (1953) Records Genet. SOC.Amer. 22, 88. Moses, M. J., and Yerganian, G. (1952) Records Genet. SOC. Amer. 21, 51. Muldal, S. (1948) Ann. Rept. J . Innes Hort. Imt. 88, 23. Mulnard, J. (1952) Compt. rend. assoc. anat. 69, 740. Naora, H. (1951) Science 114,279. Naora, H. (1952) Science 116,248. Naora, H. (1954) Exptl. Cell. Research 8,259. Ornstein, V . , and Pollister, A. W. (1952) Science 116,203. Pasteels, J., and Bullough, W. S. (1953) Arch. biol. (LiEge) 64, 271. Pasteels, J., and Lison, L. (1950a) Compt. rend. W , 780. Pasteels, J., and Lison, L. (1950b) Arch. biol. (Li?ge) 61, 445. Pasteels, J., and Lison, L. (1951) Nature 167, 948. Pasteels, J., and Lison, L. (1953) Compt. rend. 296, 236. Patau, K. (1953) Records Genet. SOC. Amer. B,90. Pollister, A. W. (1952) Lab. Invest. 1, 106. Pollister, A. W., and Leuchtenberger, C. (1949) Proc. Nutl. Aced. Sci. (US.)

36, 111. Pollister, A. W., and Moses, M. J. (1949) J. Gen. Physiol. S!2, 567. Pollister, A. W., and Ris, H. (1947) Cold Spring Harbor Symposia Quant. Biol. 12, 147. Pollister, A. W., and Swift, H. (1950) Science ill, 68. Pollister, A. W.,Swift, H., and Alfert, M. (1951) J . Cellular Comp. Physiol.

38, 101. Price, J. M., and Laird, A. K. (1950) Cancer Research 10, 650. Price, J. M., Miller, E. C., Miller, J. A., and Weber, G. M. (1950) Cancer Research

10, 18. Reisner, E. H.,and Korson, R. (1951) Blood 6, 344. Ris, H. (1947) Cold Spring Harbor Symposia Quant. Biol. 12, 158. Ris, H.,and Mirsky, A. E. (1949) J . Gen. Physiol. 33, 125. Roels, H. (1954) Nature 178, 1039. Schrader, F.,and Leuchtenberger, C. (1949) Proc. Natl. Aced. Sci. US.86, 464. Schrader, F., and Leuchtenberger, C. (1950) Exptl. Cell. Research 1, 421. Schrader, F., and Leuchtenberger, C. (1952) Ezgtl. Cell. Research 8, 136. Schwartzschild, K., and Villiger, (1906) Astrophys. J . 40, 317. Seshachar, B. R. (1950) Nature 166,848. Sibatmi, A. (1950) Nature 166, 355. Sibatani, A. (1953) J . Biochem. (Japan) 40, 119.

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CYTOPHOTOMETRIC STUDY OF NUCLEAR DNA

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Sibatani, A. (1954) Biochim. et. Biophys. Actu 13, 66. Sibatani, A,, and Naora, H. (1953) Biochim. et Biophys. Acta 12, 515. Swift, H. (195Oa) Proc. Natl. Acud. Sci. US.96, 643. Swift, H. (195Ob) Physiol. Zool. 23, 169. Swift, H., and Kleinfeld, R. (1953) Physiol. Zool. 26, 301. Taft, E. B. (1951) Expfl. CelZ Research 2, 312. Taylor, J. H., and McMaster, R. D. (1954) Chronzosoma 6, 489. Thomson, R. Y., and Frazer, S. C. (1954) Exptl. Cell. Research 6, 367. Thomson, R. Y., Heagy, F. C., Hutchinson, W. C., and Davidson, J. N. (1953) Biochm. J . 63, 460. Vendrely, C. (1952) Bzcll. B i d France et Belg. 86, 1. Vendrely, R., and Vendrely, C. (1948) Experientia 4, 434. Vendrely, R., and Vendrely, C. (1949) Experientia 6, 327. Vendrely, R., and Vendrely, C. (1950) Compt. rend. 230, 670. Vendrely, R., and Vendrely, C. (1952) Compt. rend. B36, 444. Vialli, M., and Perugini, S. (1954) Riv. Histochim. normole e patol. 1, 149. Villela, G. G. (1952) Rev. b r a d bioZ. 12, 321. Walker, P. M. B., and Yates, H. B. (1952a) Symposia SOC.Exgtl. Biol. 6, 265. Walker, P. M. B., and Yates, H. B. (1952b) Proc. Roy. SOC.BlM, 274. Widstrom, G. (1928) Biochem. 2. 199, 298.

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Protoplasmic Contractility in Relation t o Gel Structure: Temperature .Pressure Experiments on Cytokinesis and Amoeboid Movement DOUGLAS MARSLAND

Washington S q w r e College. N e w York University. N e w York. a d the Marine Biological Laboratory. Woods Hole. Massachwetts Page 199 I . Introduction ....................................................... 199 1. General Approach ............................................... 2. Gel Structure in Relation to Pressure and Temperature ............. 200 201 3. Pressure as a Physiological Tool ................................. 201 4. Special Techniques and Apparatus ............................... 203 I1. Amoeboid Movement ............................................... 1. Pressure Effects on Amoeboid Form and Movement ............... 203 206 2. Influence of Temperature ....................................... 207 3. Decompression Observations .................................... 210 I11. Cytokinesis ........................................................ 210 1. General Hypothesis ............................................. 211 2. Pressure Effects on Dividing Eggs .............................. 213 3. Temperature Effects ........................................... 4. General Interpretations .......................................... 216 I V. Kinetics of Protoplasmic Contractility ............................... 219 219 1. Earlier Evidence ................................................ 2. A T P Effects on Furrowing Strength .......................... 219 3. A T P Effects on Gel Structure ................................... 221 4. A T P Effects on Amoeboid Form ................................ 221 5. Experiments with Mersalyl Acid (Salyrgan) ................... 223 V . General Conclusions ................................................ 221 V I. References ......................................................... 225

I. INTRODUCTION 1 . General Approach Reversible sol-gel transformations have been observed in various cells for many years . It was not until 1926. however. starting with Mast's classical work on amoeboid movement. that the functional significance of these transformations began to be understood . Now. about thirty years later. there is considerable evidence which indicates that protoplasmic gel structures are potentially contractile. The gelational process thus seems to represent a mechanism which enables the cell to perform mechanical work . The purpose of this paper is to review the evidence. with special reference to amoeboid movement and the furrowing movements (cytokinesis) in animal cells. It seems probable that the development of mechanical energy by muscle tissues also represents a related phenomenon. but the present dis199

200

DOUGLAS MARSLAND

cussion will be concerned primarily with contractile processes in less highly specialized kinds of cells. A prior( the development of contractility in an essentially fluid system such as protoplasm would seem to presuppose the formation of some kind of gel structure. If, as seems likely, the contractile force originates from the folding of elongate protein molecules or molecular aggregates, it is difficult to see how such a folding could be effective in performing work unless the extended protein units were interlinked, forming a continuous and fairly extensive system throughout the cell. In fact it is generally agreed (see Kopac, 1950) that gelation represents the formation of a three-dimensional network from fibrillar units present in the system. Thus the contraction of a gel structure represents a rapid sort of syneresis, whereby the protein components of the colloidal network undergo forcible folding without relinquishing their intermolecular linkages (Goldacre and Lorch, 1950; Goldacre, 1952). Subsequent to folding, moreover, when the extended units have assumed a more globular form, the system reverts to the sol condition, merely by the loosening of the intermolecular bonds. The effective contraction of a gel structure necessarily seems to demand that the fluid component of the system, which initially occupies the interstices of the colloid network, be forced forth from the interstices, thus allowing for a fairly drastic shrinkage of the residual framework of the gel. The final dimensions, after syneretic shrinking, in some gels may represent a rather small fraction (about 10%) of the original volume. Thus the work performed during contraction may be considerable. Moreover, since the primary orientation of the elongate protein components of such a system need not be unidirectional, such gels need not display anisotropy, either in the expanded or contracted state.

2. Gel Structure in Relation to Pressure and Temperature The early work of Brown (1934) on Arbacia eggs and of Brown and Marsland (1936) on two species of Amoeba, showed that a drastic weakening and finally a complete solation of the plasmagel structures of these cells occurred when they were exposed to increasingly high hydrostatic pressures in the range up to 8000 lb./in.2. This result was difficult to understand until Freundlich ( 1937) published his analysis of gelational phenomena which forms the basis of the classification of gels shown in Table I. Then it was realized that protoplasmic gel systems, of which quite a number have now been studied (see Marsland, 1950), are uniquely different from such more familiar types as gelatin or agar. Instead of solating when warmed, protoplasmic gel structures become more firmly set ; and when higher pressures are applied, cellular gel structures are weakened

201

CYTOKINESIS AND AMOEBOID MOVEMENT

and eventually solated-which is just opposite to the behavior of the gelatin system (see Marsland and Brown, 1942). In short, since protoplasmic gelations are endothermic and since a volume increase (+AV) is involved, the sol & gel equilibrium is shifted toward the right by increasing temperature, and toward the left by increasing pressure. Thus both temTABLE I GEL TYPES(FREUNDLICH) , Type

Characteristics

Examples

~~~

I

Gelation involves volume decrease (- AV) Gelation liberates heat (exothermic)

Gelatin gels

I1

Gelation involves volume increase (4- AV) Gelation absorbs heat (endothermic)

Methyl cellulose Actomyosin Other protoplasmic gels

'I1

Gelation involves no volume changes Gelation (isothermic)

Sodium oleate Other soap gels

perature and pressure have provided useful tools not only for analyzing the functional significance of intracellular gelations, but also for studying the metabolic processes which supply energy to these reactions.

3. Pressure as a Physiological Tool I n thermodynamics, of course, pressure, like temperature, has always occupied a primary position. But in physiological work pressure has not received an adequate share of attention. Probably the specialized equipment involved is partly responsible for this neglect. But also pressure seems to have been forgotten merely because the pressure variations in the terrestrial environment are rather small and the direct effects of such small changes are almost negligible from a physiological viewpoint, But in the oceanic environment a rich variety of organisms live at graded levels down to and below a depth of six miles (Fig. 1). Here the pressure keeps mounting, up to more than 16,000 1b./im2, and with pressures of this magnitude the direct physiological effects are very significant. It is clear, therefore, that pressure has played an active role in the natural selection of many species and that it must be recognized as a normal and important physiological variable.

4 . Special Techniques and Apparatzcs Direct observation of the living specimens during exposure to high pressure is most essential. Characteristically the effects of pressure are rapidly and completely reversible and in several instances earlier workers

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DOUGLAS MARSLAND

obtained erroneously negative results simply because their observations were delayed until after decompression, following the removal of the material from the pressure chamber. Consequently progress in the field was greatly facilitated by the development of a microscope-pressure chamber in which small organisms and individual cells may be observed a t magnifications up to 600 diameters while they are subjected to pressures up

FIG.1. Deep-sea photograph of an unidentified sea cucumber; taken off coast of North Carolina at a depth of one and a half miles. Courtesy of the Woods Hole Oceanographic Institution. to 15,000 lb./in.2 (see Marsland and Brown, 1936; Marsland, 1950). Also the development of the pressure centrifuge (Brown, 1934) and of an adaptable temperature control housing (Marsland, 1950) were important. This equipment, in fact, has made it possible to obtain centrifugal measurements of the gelational state of the cytoplasmic components of different cells under systematically varied conditions of temperature and pressure throughout the physiological ranges, and to relate these gelational measurements to the observed changes in the activity and behavior of the cells. The centrifugal method of measuring the relative consistency (gel

CYTOKINESIS AND AMOEBOID MOVEMENT

203

strength) of gelated parts of the protoplasm at various temperatures and pressures is based on the usual assumption that Stokes’ law provides a useful approximation when applied to protoplasmic systems. Quite obviously the resistance to the displacement of visible granules through a gel cannot be regarded as an index of viscosity in a strictly physical sense. However, assuming that a uniform centrifugal force is used and that the density differential between the granules and the surrounding medium is not altered by the experimental treatment, it seems valid to take the centrifuge time required to produce a standard displacement of granules through the gelated cytoplasm as an index of the relative “consistency,” “stiffness,” or “strength” of this part of the protoplasm. Measurements of the relative gel strength have now been made on a number of different cells, including two species of Amoeba (Brown and Marsland, 1936), two species of Arbacia (Brown, 1934 ; Marsland, 1938, 193%) , and Elodea canadensis (Marsland, 1939b) ; and the consistent pattern of all these data tends to justify the basic assumptions involved. 11. AMOEBOID MOVEMENT 1 . Pressure Eflects on Amoeboid Form and Movement Anticipating the more detailed account which follows, it may be said that the pressure studies (Marsland and Brown, 1936; Landau et al., 1954b) generally provide strong support for the hypothesis of Mast (1926) in regard to the mechanism of amoeboid movement. Initially this hypothesis rested upon Mast’s beautifully accurate but purely visual observations of the form and movement of Amoeba protezcs. Recently, however, considerable experimental evidence has been obtained which tends to substantiate the main tenets of the Mast hypothesis, namely: (1) that the flowing of the plasmasol is caused by a contraction of the surrounding plasmagel, (2) that there is a gradient in the structural state of the plasmagel system such that the newly formed parts out near the extremities of an extending pseudopodium are less firmly gelled and less strongly contractile than the older parts back in the “body” of the amoeba, and (3) that the plasmagel layer in a typical pseudopodium constitutes a structural wall which guides the flow of plasmasol and sustains the pseudopodial form. The main results of the pressure experiments performed at 25” C., are summarized in Fig. 2. Her: it may be seen that the structural strength of the plasmagel of the amoeba falls off exponentially as the pressure of the environment is raised and that concomitantly drastic changes occur in the form and activity of the cells. If the pressure is raised rapidly, catching the pseudopodia in an extended elongate form, the first effect, which occurs within 2 or 3 seconds after the

204

DOUGLAS MARSLAND

pressure reaches 6ooo lb./in.2, is a collapsing of the pseudopodial walls. This collapse usually involves approximately the distal half of the pseudopodium, so that orit3 or more rounded masses, or balls, of cytoplasm are formed from each pseudopodium (Fig. 3 ) . Sometimes, indeed, especially at pressures higher than 600 Ib./in.2, the rounded masses into which an extended pseudopodium has been converted may become pinched off completely from the rest of the cell. In any event, during the 5-10 minutes following pseudopodial collapse, each of the specimens slowly rounds up into

AMOEBA PROTEUS

PRESSURE, Iba, / Ing FIG.2. Plasmagel strength and amoeboid form in relation to high pressure. Gel strength values relative to 100, the arbitrarily selected atmospheric value. (Data from Brown and Marsland, 1936.)

one large inert sphere, if pinching-off has not occurred, or into two or more smaller balls, if pinching-off did take place. The rounding of the specimens undoubtedly results from the drastic solation of the plasmagel system, which is demonstrated by the centrifugal measurements given in Fig. 2. But, in addition, the observations plainly indicate that the more recently formed plasmagel, out towards the ends of the pseudopodia, is relatively weaker, so that the solating effects of pressure are observed here sooner than in the more firmly set plasmagel in the “body” of the amoeba. This is in accord with Mast’s view that a gradient exists in the plasmagel system, the “older” parts being more firmly set and more strongly contractile than the “younger” parts. A different set of observations is obtained when lower pressures are applied in a step-wise fashion, allowing the amoebae to reach an equilibrium form by maintaining the pressure at each level for some 10 to 15 minutes.

CYTOKINESIS AND AMOEBOID MOVEMENT

205

At relatively low pressures (loo0 to ZOO0 Ib./in.2) the pseudopodia are definitely more slender and slightly longer, compared to normal specimens ; few of the amoebae display more than two pseudopodia, and a pseudopodium once formed rarely undergoes retraction. Also the slender pseudopodia tend to display a slightly zizzag form, since lateral ruptures of the walls, very near the tip, frequently produce small deflections in the direction of extension; and the plasma-gel sheet (Mast, 1926) is absent in most of the pseudopodial tips.

FIG.3. Collapsing and “pinching off” of pseudopodium, which occurs when the amoeba is suddenly exposed to pressures above a critical level. I n this case, the pressure was 7000 lbJin.2, at 25’ C. 1. One minute before compression; 2. Five seconds after Compression; 3. One minute later. 4-6. Successive photos taken at 1.5minute intervals thereafter, pressure maintained. These observations also tend to confirm the existence of a gradient in the structural state of the plasmagel system. Apparently the most labile o r weakly gelled parts, which are first to show signs of pressure solation, are the plasmagel sheet and the most distal part of the pseudopodial wall, out at the tip. Moreover, the observations indcate that the normal function of the plasmagel sheet may be to control and limit the pseudopodial length. It seems to provide a focus for the building of a plasmagel barrier at times when a pseudopodium stops extending and is about to undergo retraction. When somewhat higher pressures (20W5ooO Ib./in.2) are maintained, the “body” of the amoeba tends to become more and more rounded and the pseudopodia become smaller and smaller, both in diameter and length. At 4500 Ib./in.2, each specimen appears as an almost spherical mass from which protrudes a number of very small papilliform pseudopodia (see Fig. 2). Some very weak activity persists in such rounded specimens since

206

DOUGLAS MARSLAND

very slowly and occasionally a new tiny pseudopodium may appear and an old one disappear ; but the specimens display no apparent orientation and no capacity for locomotion. The progressive weakening of the plasmagel structure (Fig. 2) which occurs with increasing pressure in this range (2000-5000 lb./in.2) unquestionably must bear a causal relationship to these observed changes of form and activity. At the pseudopodial walls become weaker, stability seems to be maintained by compensatory reductions in the diameter of the pseudopodia1 tubes. Also as the plasmagel of the "body" of the amoeba loses structural strength, its contractility seems to weaken. The streaming of the plasmasol grows feebler, the pseudopodia become shorter and finally, at 6000 lb./in.2, the cell rounds up into a perfectly immobile sphere.

2. Influence of Temperature Similar experiments, performed at 20", 15", and 10" C., give an entirely parallel series of observations. Generally speaking the same changes occur, only more slowly; and what is even more significant-the pressure level required to induce each change becomes lower and lower as the temperature is reduced. This type of behavior is not surprising, of course, in view of the expected influence of temperature on the sol-gel equilibrium of the system, and in view of the centrifugal measurements given in Fig. 4. These data show that the gel strength/pressure curve at low temperature (15" C.) parallels the high temperature curve, but that all the values at 15" are approximately 40% slower. The minimum pressure required to induce a complete rounding of the amoebae into quiescent spheres falls off progressively-from 6OOO 1b./ins2 at 25"; to 5000 at 20"; to 4000 at 15"; and to 3000 at 10" C. At low temperature the rounding process is more sluggish, requiring some 15-20 minutes for completion at lo", as opposed to 5-10 minutes at 25" C. Moreover, the intermediate forms, in which reduced pseudopodia persist when the amoebae are maintained at moderate pressures, are similar to those described for 25". But the pressure level at which each equilibrium form is seen falls off by about loo0 lb./in.2 each time the temperature is lowered by 5" C. Another difference between the high and low temperature observations is provided by the pinching-off phenomenon. This becomes less frequent and distinct at lower temperatures. The pinched-off part frequently involves r/4 to % of the whole cytoplasm at 25" and 20" ; whereas at 15", the pinched-off spheres were very small; and at 10" pinching-off almost never occurs. Also, since the pseudopodia are less elongate at lower temperatures, the abrupt type of pseudopodial collapse seldom occurs below

CYTOKINESIS AND AMOEBOID MOVEMENT

207

15" C. Instead, the rounding of the specimens is very slow presumably because the viscosity of the plasmasol tends to increase at lower temperatures. 3. Deconzpression 0 bsemtations Evidence as to the intrinsic contractility of the plasmagel system is also provided by the striking reaction displayed by the specimens when they are released, especially if they have been maintained for some time at a

" W

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I

P

4

PRESSURE

-

6

8

1000 LB%/

10

so. IN.

FIG. 4. Plasmagel strength and pseudopodial stability in relation to pressure and temperature. Note that at 15 ." C. a pressure of 4000 1bJin.z is adequate to weaken the gel structure to the point where the pseudopodium becomes unstable, as compared to 6000 lb. at 25" C. (From Landau et al., 1954b).

relatively high pressure (Fig. 5 ) . Within 10 to 15 seconds of the decompression, each develops a broad hyaline zone between the cell membrane and the subjacent granular plasmagel layer, which previously was in contact with the membrane (see Fig. 5 ) . The plasmagel layer seems to detach itself from the membrane over the entire cell surface and to contract vigorously, squeezing forth a clear fluid which fills the broadening space between the membrane and the outer boundary of the granular plasmagel. Thus finally each of the still-rounded specimens displays three concentric zones : (1) a central granular mass, which accounts for about 50% of the total cell volume ; (2) a broad clear fluid zone around the granular mass ; and (3) the cell membrane, which remains in its original position. Such

208

DOUGLAS MARSLAND

a form is also seen in many of the pinched-off spheres, except when these are very small or very full of food masses. Following this sudden contraction, usually within 10-20 seconds, the amoebae commence to show signs of renewed activity. The deep-lying

FIG.5. Post-compression plasmagel contraction phenomenon in Amoeba proteus. (From Landau et al., 1954b.) A. Normal specimen, atmospheric pressure, 25 O C. B. Same specimen at 6000 lbJin.2, maintained 5 min. C. Fifteen minutes later, rounding complete ; "pinched-off" part has shifted around to left side of main mass. D. Fifteen seconds after decompression. Note the sudden and drastic contraction of the granular plasmagel, which has given rise to a broad hyaline zone between the cell membrane and the contracted gel. E. Ninety seconds after decompression. Amoeboid activity commencing. F. Five minutes later. Activity now quite vigorous.

CYTOKINESIS AND AMOEBOID MOVEMENT

209

granular cytoplasm begins to break out into the clear fluid zone in several places, bulging the membrane IocaUy in an irregular and disoriented fashion. Soon, however, well-developed pseudopodia begin to form, and four or five minutes later, normal locomotion reappears. The decompression contraction phenomenon is most plainly observed at higher temperatures-where the pressure required to cause a rounding of the specimens is likewise high. Also the reaction is more pronounced as the compression period is lengthened, reaching a maximum in about 20 minutes at 6OOO lb./in?, at 25" C. At lower temperatures, where the critical rounding pressures are lower, the reaction is weaker. In fact, at 10"/3000 lb./inP, no contraction was observed, although a small but unmistakable reaction did occur after these specimens had been raised to 7000 1b./h2 for 20 minutes. A complete interpretation of the decompression reaction is problematical. However, a tentative explanation may be given in terms of the gel contraction hypothesis. The inert spherical form of the amoeba observed at high pressures undoubtedly represents a more or less complete and generalized solation of the plasmagel layer, and the complete disappearance of any orientation or gelational gradient in the system. When suddenly released from the pressure constraint, apparently, a strong and uniformly distributed gelation reaction occurs in all of the peripheral part of the cytoplasm, fortified perhaps by the accumulation of one or more active metabolites during the pressure period, while amoeboid activity is suspended. Then there is a strong generalized contraction of the newly reconstituted plasmagel system, accompanied by a detachment of the cell membrane all over the surface of the cell. The contraction of the plasmagel, apparently, involves the squeezing forth of a fluid component which initially is enmeshed in the colloidal threedimensional network of the gel s t r u c t u r w r otherwise the network proper could not display such a drastic degree of shrinkage. Naturally this expressed fluid would be hyaline, since visible granules are too coarse to escape from the colloidal network. Ordinarily the escape of hyaline fluid from the contracting plasmagel cannot be seen, except locally in the hyaline cap of a pseudopodium or at the point where a new pseudopodium is about to form, since elsewhere a firm attachment of the cell membrane to the subjacent plasmagel prevents the fluid from escaping peripherally-and centrally the hyaline fluid imperceptibly intermingles with the granular flowing plasmasol. Probably not all of the fluid seen in the peripheral hyaline zone which develops after decompression is derived from the interstices of the gel structure. Probably during contraction the plasmagel layer also acts as a sieve which permits the egress of fluid, but not of granules, from the

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DOUGLAS MARSLAND

deeper-lying plasmasol, provided that there has been a detachment of the surface membrane. In fact, such a sievelike action seems to occur during normal locomotion, in the plasmagel sheet at the tip of an advancing pseudopodium, or at a locus more posteriorly, when the formation of a hyaline blister marks the place where a new pseudopodium is about to appear. In any event, the simplest interpretation of the decompression reaction seems to be that it represents a strong generalized contraction of the newly constituted plasmagel system. Temporarily this system appears to lack any kind of oriented organization or gradient, and normal locomotion is not resumed until such an organization has been re-established. The endothermic nature of protoplasmic gelations presupposes that energy from some metabolic source must be utilized in the building up of the plasmagel structure. The controlling factor in the present experiments, however, seems to be a direct action of pressure and temperature on the intermolecular linkages of the gel structure. In other words, the experiments give little information in regard to the pattern of metabolism which must determine the cyclic and oriented reactions of gelation and solation during normal locomotion. They suggest, however, that some phases of this metabolism may continue even at pressures which completely inhibit gelation, since the exceptionally strong contraction, observed as soon as the pressure is released seems to indicate that there has been an accumulation of one or more active metabolites during the compression period.

111. CYTORINESIS 1. General Hypothesis Normal cell division unquestionably represents a complex series of events and processes. Here, however, we shall deal mainly with the furrowing process, by which the animal type of cell divides itself into two daughter cells. The evidence to be reviewed represents a thorough testing of the cortical gel contraction theory of cytokinesis. This theory, as formulated by Marsland (1950), postulates that the furrowing potency in animal cells depends upon the structural state and hence the contractile capacity of a strongly gelated cortical layer of cytoplasm, especially in the furrow region. Thus the basic mechanisms for the development of mechanical energy in cytokinesis and in amoeboid movement are thought to be essentially the same. The existence of a cortical plasmagel layer in an egg cell (Arback pwnc&&fa) was first demonstrated clearly by the experiments of Brown in 1934. These experiments showed that the cortically embedded pigment bodies (red chromatophores) are considerably more resistant to centrifugal displacement than those in the deeper lying (medullary) cytoplasm

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CYTOKINESIS AND AMOEBOID MOVEMENT

of the egg. And in addition Brown showed that this well-defined plasmagel layer, like that of the amoeba, is susceptible to solation under pressure ;and that the plasmagel layer of the egg suddenly becomes very much more firmly gelled prior to and during the furrowing period.

2. Pressure Effects on Dividing Eggs The main effects of pressure on the gel structure and furrowing performance of Arbacia eggs during first cleavage are summarized in Fig. 6.

WNCTULATA 23O

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L

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2

3

4

5

6x103

PREssw1E, Ibr./ing

FIG.6. Plasmagel strength and rate of progress of furrows in relation to pressure. (Data from Marsland, 1938.) Here it may be seen : (1) that an exponential weakening of the cortical gel structure occurs as the pressure increases; (2) that the progress of the furrow as it impinges on the spindle axis becomes proportionally slower and slower as this weakening of the gel system progresses; and (3) that furrowing is aborted completely at SO00 lb./in.a (at 20" C.), when the weakening of the plasmagel system has reached a certain critical level. A furrowing egg in the pressure chamber can be observed continuously as the pressure is built up. When the pressure reaches 5000 Ib./in.a or more (at 20" C.), the advancing furrow halts; and then begins to recede. The recession is relatively more rapid at higher pressures. Thus within 4 minutes at 5000 lb. (or 2 minutes at 7000) the egg has resumed its original spherical form. This retreat of the furrow changes back to an advance, however, as soon as the pressure is released. In fact the furrow can be induced to retreat and advance alternately for several cycles by releasing the pressure after each successive compression. Finally, how-

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DOUGLAS MARSLAND

ever, if the furrow is held back for more than 15 minutes, it will fail to cut through the egg after the pressure has been released. Then no refurrowing occurs until it is time for the second cleavage, whereupon a double furrowing usually takes place, simultaneously dividing the egg into four (sometimes three) blastomeres. Although the pressure block to cleavage was first observed in the eggs of Arbacia pzcnctzclata, many other eggs, derived from animals in five different phyla, have now been studied (see Pease and Marsland, 1939).

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FIG.7. Plasmagel strength in relation to temperature. Time in seconds. (From Marsland, 195Or. To be sure, the minimum pressure required to block the furrowing reaction shows some variation, but in each case there is a critical blocking pressure and usually this falls in the range of -4 lb./in? (at 20"-25" C.). At pressures below 5000 Ib./in?, the furrow does not recede, but the rate at which it cuts through the egg is definitely retarded (Fig. 6). At the 4OOO lb. level, for example, it takes 9 minutes for the furrow to pass from the equator to the spindle center, instead of the normal atmospheric time

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CYTOKINESIS .AND AMOEBOID MOVEMENT

of 3 minutes (at 20" C.), In fact, the curve obtained by plotting the log of this retardation as a function of pressure gives an excellent fit when superimposed upon a similar plot of the cortical gel strength in relation to pressure, as measured by the pressure-centrifuge technique at the time when the furrows are about to form.

,.MO

GEL STRENGTH FOP0 * I FURROWING

-MINIMUM

-.'

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TEMPERATURE, O C FIG.8. Plasmagel strength in relation to temperature at various pressures. Measurements made on cleaving eggs of Arbocia lisula. (Data from Marsfand and Landau, 1954.)

3. Temperature Effects The data plotted in Figs. 7 and 8 show that the cortical plasmagel of the egg cell, like that of the amoeba, may be characterized as a type I1 system in which the structural strength of the gel network increases exponentially as the temperature is raised within the physiological range. This applies, apparently, not only to the relatively weak gel system of the unfertilized egg (Fig. 7) but also to the strongly fortified gel of the furrowing egg (Fig. 8), where much higher centrifugal forces were required to achieve a displacement of the embedded chromatophores even

214

DOUGLAS MARSLAND

though fairly high pressures were employed to bring the gel strength down into a measurable range. Correlated apparently with this action of temperature in fortifying the structure of the plasmagel system, the furrowing strength of the egg be-

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FIG.9. Temperature variations in the minimum pressure required to block the first cleavage furrow in several different eggs. (Data from Marsland and Landau, 1954.) comes progressively greater at higher temperatures-= may be seen in Fig. 9. With each increment of 5" C.,the magnitude of the pressure required to block the furrows becomes greater-by about lo00 lb./in? for the eggs of Echinurachnizcs and Arback-and by about 500 lb./in.a for the amphibian and annelid eggs. Moreover, the correlation between gel struc-

CYTOKINESIS AND AMOEBOID MOVEMENT

215

ture and furrowing strength becomes even plainer in light of the data shown in Fig. 10. This family of curves shows that a furrowing block occurs whenever the structural strength of the cortical gel system falls to a certain critical level. At higher temperatures the pressures required to produce this critical weakening are higher, and at lower temperatures they are lower; but in each case the cell is rendered incapable of performing the work of cleavage by any combination of temperature and pressure treatments that jointly weakens the gel structure to approximately the same degree. Pressunflemperature when division can occur

1

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FIG.10. Effects of temperature and pressure on the structural state of the phsmagel layer of the ArbacM egg in relation to cleavage capacity. Note that furrowing is aborted whenever the plasmagel strength falls below a critical level, as a result of the various temperaturepressure combinations. The centrifuge times are given directly, in seconds, and the extent of variation is indicated by the length of the markers. (Data from Marsland, 1950.) At first sight it might seem that the very ingenious measurements of Mitchison and Swann (1954) would indicate that the “cell surface” of the unfertilized sea urchin egg becomes stiffer and more resitant to stretching when the temperature is lowered (from 21.5” to 3.0”C.). However, these measurements of “stiffness” are complicated by the internal pressure factor. Also, and this is even more important] the measurements do not take into account the greatly increased Viscosity which occurs in the deeper

216

W UGL AS MARSLAND

cytoplasm when the temperature is lowered (Costello, 1934). Undoubtedly this factor would make it more difficult for the cell to be deformed by the elastimeter and would help to account for the downward drift of the measurements with time. Also it appears that these measurements deal primarily with the relatively thin (estimated by Mitchison and Swann at not more than 1,.5p thick) cell membrane rather than the cortical plasmagel layer of the cytoplasm proper, which is observed to be 5-6p thick (see Marsland and Landau, 1954). It is difficult to understand how Mitchison and Swann (1954) reach the conclusion that the pressuretentrifuge measurements (Figs. 7 and 8) deal with “cytoplasmic viscosityJ’ rather than the structural state of the cortical plasmagel layer. It is well known that many of the pigment bodies of the unfertilized Arbacia egg do lie in the deeper part of the cytoplasm. But some pigment bodies are also present in the cortical plasmagel and it is the displacement of the cortical pigment which forms the basis of our measurements. Moreover, some of the measurements are derived from fertilized eggs just prior to furrowing and in these eggs most of the pigment bodies have migrated into the cortex (Harvey, 1910). Therefore, it seems clear that the pressure-centrifuge experiments provide a valid measure of the structural state of the cortical plasmagel layer. Also it seems likely that the lower “yield point” observed by Mitchison and Swam in their low temperature elastimeter experiments actually represents a weakening of the structure of the cortical plasmagel layer.

4 . General Interpretations The foregoing temperature-pressure data clearly indicate that there is a quantitative reIationship between the gerational state of cortical plasmagel layer in the equatorial region of the dividing cell and the strength of the furrowing reaction. Recent workers, indeed, are generally agreed that a decisive role in cytokinesis is played by the cortical plasmagel layer of the cytoplasm. However, there is no agreement as to the precise mechanism of the furrowing process. In fact, two opposite viewpoints are currently held: first, that the cell membrane is pushed down into the developing furrow by a process of “growth” (Schechtman, 1937 ; Chambers, 1938) or of “expansion” (Swann, 1952; Mitchison, 1952) occurring in the cortical protoplasm; and second, that the cell membrane is pulled inwards into the furrow by a contraction of the subjacent plasmagel in the equatorial region (Lewis, 1938, 1942, 1951; Masland, 1938, 1942, 1950). Details of the cortical gel growth viewpoint cannot be considered, since Schechtman’s short preliminary report has not been followed by further

CYTOKINESIS AND AMOEBOID MOVEMENT

217

publication. However, it seems likely that this theory would be subject to criticisms similar to those sustained by the expansion theory of Mitchison, which will be discussed in some detail. Furrowing, according to the expansion theory (Mitchison, 1952), involves two successive processes : first, a preliminary contraction of the cell surface, in a broad equatorial region; and second, an active and sustained expansion, which enables the furrow to push inward to its completion. Apparently it is necessary to postulate the preliminary contraction phase to account for the initiation of furrowing, but the theory places main emphasis upon the expansion phase as the active process which develops the energy expended in furrowing. No difficulty is encountered in regard to the contraction phase of the Mitchison theory. In fact, this phase seems to represent a necessary postulate in all cortical gelation theories of cytokinesis. But serious difficulties, of a mechanical nature, are encountered in regard to the expansion phase. Provided that the dividing cell were prevented from undergoing elongation beyond a certain limit (as by the constricting influence of the fertilization membrane or other extraneous coats), it seems probable that an active expansion of the surface might lead to cleavage. But Mitchison’s experiments were carried out on cells from which the fertilization membranes and hyaline layers had been removed, and for such unconstrained cells it seems certain that a sustained expansion of the surface would merely lead to elongation. It is difficult to understand, therefore, how the surface expansion (or growth) mechanism can be applied to “naked” cells which, nevertheless, continue to cleave in normal fashion. Moreover, it is difficult to see how the expansion mechanism would operate in the case of fibroblasts and other amoeboid cells, which may display a highly irregular form even during the furrowing period. There is no need to question the birefringence measurements upon which the surface expansion theory is based. These observations merely indicate that the surface expands at the poles during early telophase, while simultaneously it contracts in the broad, bandlike area encircling the equator. This is in agreement with the observations of Dan (1943, 1948) of Dan and Dan (1940, 1947), and of Schechtman (1937) who focused attention upon the displacement of particles adhering to various parts of the cell surface. However, it is not necessary to assume that surface expansion is the active process which generates the energy expended in furrowing. In fact, a similar polar expansion (stretching) would be expected to occur passively on the basis of the cortical gel contraction theory. As a prelude to division, before the cell elongates or displays any defini-

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DOUGLAS MARSLAND

tive furrow, a structural gradient must develop in the plasmagel system, which initially seems to be strongly and uniformly set throughout. Cytokinesis is initiated, apparently, by a localized solation which weakens the cortical gel structure at each pole. Such a polar weakening is revealed by the experiments of Edward Chambers (unpublished personal communication, 1951) who found that early telophase eggs exposed to hypotonic swelling always burst at the poles. Also it is shown by the fact that the pigment bodies at the poles, compared to those in the equatorial zone, are more readily displaced when early telophase eggs are subjected to high centrifugal forces. This polar weakening, apparently, permits the more strongly gelled bandlike portion of the plasmagel system in the broad equatorial zone to contract forcibly, bulging the cell out at the poles as the cell undergoes elongation, which stretches the cell membrane in the polar regions. Such a polar stretching seems quite plain in the dividing neuroblasts depicted by Roberts (1954). In this case, however, one pole appears to display solation slightly before the other; and Roberts attributes the polar bulging to an active expansion rather than to a passive stretching. At present, however, there does not seem to be any decisive evidence which makes it necessary to assume that an active process of expansion provides energy for furrowing ;and in the absence of such evidence it seems best to retain the simpler theory-namely, that the furrowing cell derives all its energy from the contractility of the plasmagel system, as in the case of the moving amoeba. Following the contraction of the broad equatorial zone, which causes the cell to elongate, the contracting part of the plasmagel becomes more narrowly restricted to the equator, which leads to the appearance of a definitive furrow-as is likewise postulated in the surface expansion theory. Thus the area of passive surface stretching also becomes greater, so that the sides of the prospective blastomeres near and even in the developing furrow may become involved. Moreover, as the furrow deepens new plasmagel is drawn into an operative position along the walls of the furrow. Thus in a well-developed furrow there are two fairly broad “annuli” of newly mobilized pfasmagel constituting the walls of the deepening furrow. Now this newly mobilized plasmagel can contract and complete the work of cleavage. Meanwhile, the plasmagel at the very trough of the furrow, having expeiided its contractile energy, is free to undergo solation. This permits the cell membrane to fuse at the division axis, thus completely separating the daughter blastomeres (see Marsland and Landau, 1954).

CYTOKINESIS A N D AMOEBOID MOVEMENT

219

IV. KINETICSOF PROTOPLASMIC CONTRACTILITY 1. Earlier E d e n c e Since protoplasmic gelations are endothermic, we must look for a basic metabolic pattern by which the cell provides energy and determines when and where its essential gel structures shall be formed. Apparently the metabolic energy which the cell diverts into the building of its gel structures finally appears in the form of mechanical work during the contraction phase of the sol-gel cycle. The importance of adenosine triphosphate (ATP) as an energy source in muscular tissues naturally suggested that this important metabolite might contribute energy to the sol-gel cycle in cells generally. Indeed, considerable evidence in this regard has begun to come from several directions. Runnstrom (1949) showed that egg cells immersed in ATP solution became more resistant to hypotonic cytolysis, which seemed to be related to a gelling effect upon the cytoplasm. Kriszat (1949, 1950) found that A T P distinctly modifies the movements of Amoeba proteus. Loewy (1952) extracted an actomyosin type of protein from the amoeba Pelomyxa, and showed that this preparation displayed considerable changes in its gelational structure in the presence of A T P and related compounds. H. H. Weber demonstrated that glycerol-extracted fibroblasts underwent a quick and forceful contraction of their elongat: pseudopodia when treated with AT P solutions and that this remarkable contraction could be stopped quickly and reversibly when the cells were treated with mersalyl acid (salyrgan), a compound that inhibits the hydrolytic splitting of the high energy bonds of A T P (cf. Weber and Portzehl, 1954). And finally, Hoffman-Berling (1954) found that fiibroblasts, killed and glycerol-extracted just at the beginning of telophase when shallow cleavage furrows first appeared, showed a remarkable deepening of the furrows, virtually to the point of complete cleavage, when appropriate concentrations of ATP were added to the immersion medium. Accordingly, adenosine triphosphate was chosen as the first metabolite to be investigated in relation to the temperature-pressure parameters of the plasmagel system ;and mersalyl acid (salyrgan) was used as an inhibitor of the ATP system.

2. ATP Effects on Furrowing Strength Both Arbacia and Chaetopterus eggs displayed a distinct increase in the strength of the furrowing reaction as a result of adding A T P (O.OOO5 M ) to the sea water, starting approximately 25 minutes prior to the onset of first cleavage. This may be seen in Figs. 11 and 12 which show that the minimum pressures required to block the furrows are distinctly higher

220

DOUGLAS MARSLAND

i 4

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TEMPERATURE

95 C*

FIG 11. ATP-induced increase in the strength of the furrowing reaction. Each point indicates the pressure level which is just sufficient to block the furrows at each given temperature. (Data from Landau et al., 1955.)

CONTROL; No ATP

I

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25 TEMPERATURE

30 C.

FIG.12. Another example of the ATP-induced strengthening of the furrowing reaction. Points are same as in Fig. 11. (Data from Landau ef al., 1955.)

CYTOKINESIS AND AMOEBOID MOVEMENT

221

at each of the different temperatures. At atmospheric pressure also, the additional ATP enabled the eggs to complete their furrowing at temperatures which ordinarily are too low to allow for successful cleavage. Specifically for Arbacia, more than 90% of the ATP-treated eggs achieved successful furrowing at 9" C. (compared to 10% for the untreated specimens) ; and for Chaetopterus at 17" C., 89% of the treated eggs went through (compared to 4% in the untreated specimens). In fact, to achieve an equivalent low temperature inhibition of furrowing in the ATP-treated eggs, it was necessary to reduce the temperature by 2" C., to 7" in Arbacia and to 15" in Chaetopterus. In effect, therefore, it may be said that the A T P treatment permits a two-degree extension of the low temperature range of the species, at least as judged by the achievement of first cleavage.

3. ATP Effects on Gel Structure The data presented in Figs. 13 and 14 provide further support for the view that the improved furrowing performance of the ATP-treated eggs results from a fortification of the structure of the plasmagel layer. However, further experiments should be done. The present data were derived from the unfertilized eggs of Arbacia, because centrifugal forces high enough to measure the exceedingly great gelational strength of the furrowing eggs were not available. Moreover, the concentration of ATP required to give a measurable effect on the gel structure was higher than that used in demonstrating the increased strength of the furrowing reaction.

4. ATP Eflects on Amoeboid For% Further evidence that protoplasmic gelations may derive energy from the high potential phosphate bonds of A T P is provided by a study of the form assumed by Amoeba protezls when exposed to high pressures at various temperatures in the presence of A T P (0.0005M) added to the culture medium. In these experiments the minimum pressure required at each temperature to bring about a rounding of 75% of the ATP-treated specimens into completely quiescent spheres was found to be distinctly higher than for the controls-as may be seen in Fig. 15. These results, like the ATP effects upon the egg cells, seem to hinge upon the high potential phosphate bonds. Virtually no effects were obtained when adenosine monophosphate, adenosine, or inorganic phosphate were used in place of ATP (Landau et al., 1955). It is difficult, of course, to believe that ATP, a large highly polar molecule can penetrate into the cells in significant amounts, and it may not be necessary to postulate such penetration. Perhaps energy from ATP in the

222

DOUGLAS MARSLAND

I

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2

ATM

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PROSSURL

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1000 ~ o s . sa. 1 IN.

FIG.13. ATP-induced fortification of the plasmagel structure. Centrifugal measurements made at various pressures on the unfertilized eggs of Arbmia #nciulufu. (Data from Landau et al., 1955.)

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96

50

TEMPERATURE 'C.

FIG.14. ATP-induced strengthening of the plasmagel structure. Measurements at various temperatures on the unfertilized eggs of Arabaciu glcttcfdda. (Data from Landau et a[., 1955.)

CYTOKINESIS AND AMOEBOID MOVEMENT

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surrounding medium is transferred across the cell surface to the superficial layers of the cytoplasm, as is suggested by the work of Lindberg (1950).

5. Experiments with Mersalyl Acid (Sulyrgun) The experiments of Weber and Portzehl (1954) indicate that salyrgan strongly inhibits the hydrolytic splitting of the high-energy phosphate bonds of ATP and consequently studies have been started on the effects of this compound upon the furrowing strength and gel structure of dividing

L

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20

TEMPERATURE

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25

6.

FIG.15. ATP-induced increases in the minimum pressure required to abolish pseudopodium formation at different temperatures. Each paint indicates the minimum pressure which induced the complete rounding of 75% of 200 exposed specimens. (Data from Landau rt at., 1954b.)

eggs (Arbacia and Chaetopterw). The results so far likewise indicate that energy from the phosphate splitting can be utilized in building up the structure of the plasmagel system and finally serves to fortify the furrowing reaction. These results are shown in Figs. 16, 17, and 18. Here it may be seen that salyrgan added to the sea water (10 min. subsequent to fertilization) at a concentration (0.004 M ) which is not adequate to block the first cleavage, does nevertheless produce a distinct weakening of the furrowing reaction at each of the temperatures tested. Thus for both Arbuciu and Chaetopterus, a lower (by 500 1b./h2) minimum pressure is

224

DOUGLAS MARSLAND

required to block furrowing at each different temperature (Figs. 16 and 17). This weakening of the furrowing strength is related apparently to a simultaneous weakening of the cortical gel structure (Fig. 18). These measurements of the structural state of the gel system were made upon eggs which were about to divide ( 5 minutes prior to furrowing). At this time the gelational state of the cortical cytoplasm had already risen sharply to its high maximum, and a centrifugal force of 25,000 g was required to achieve a good displacement of the cortically embedded pigment bodies within a reasonable period of centrifugation.

I

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I 1 1

*woRLuL GLEAVAQE IN N R S U Y L ACID

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05

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TEMPERATURE .G.

FIG.16. Decreases in the strength of the furrowing reaction induced by mersalyl acid. Each point indicates the pressure level required to block the first cleavage furrow. (Data from Landau ct or., 1954a).

V. GENERALCONCLUSIONS All in all, the various experiments here reported seem to provide substantial support for the hypothesis that gel structures formed by the cell, particularly the cortical plasmagel layer of the cytoplasm, are intrinsically contractile and thus are instrumental in the performance of mechanical work. Metabolic energy, diverted into the endothermic gelation process, apparently, reappears in the form of mechanical work, such as amoeboid

CYTOKINESIS AND AMOEBOID MOVEMENT

225

movement and cytokinetic furrowing, when the fibrillar protein components of the gel structures undergo a forceful folding-thus reconverting the protein units into the more globular form which is characteristic of the sol condition. Apparently the strength of the intermolecular linkages of the gel structure is a critical factor in determining the strength of the contractile force

CHAETOPTERUS

I

3.0

f

54>

f

P.0

g e I

L

2

f

2o

15

I

I

I

20

25

30

TEMPERATURE .C.

FIG.17. Mersalyl acid-induced weakening of the furrowing reaction in another egg. Points as in Fig. 16.

that it can develop, In any event, under widely varying experimental conditions with several different kinds of cells, it has been found that whenever the tensile properties of the gel structure are weakened, the force of the mechanical movements of the cell is proportionately reduced. And conversely, all treatments that fortify the gel structure have been found to give a corresponding increase in the mechanical performance. Some evidence is now on hand which indicates that the high potential

226

WUGLAS MABSLAND

phosphate bonds of adenosine triphosphate may be utilized by the cell in deriving energy for the building of its gel structures. However, further studies are needed on the metabolic aspects of protoplasmic gelations.

MBMU PUNGWTA

i! "t

\

W'G

FIG.18. Mersalyl acid-induced weakening of the plasmagel strength. Measurements made exactly five minutes prior to furrowing. At this time the gel strength has risen to its very high cleavage maximum. ACKNOWLEDGMENTS This work was supported by grant series C807 from the National Cancer Institute, United States Public Health Service.

VI. REFERENCES Brown, D. E. S. (1934) J . Cellirlar Comp. Physiol. 6, 335. Brown, D. E. S.,and Marsland, D. A. (1936) J . Cellulw Comp. Physiol., 8, 159. Chambers, R. (1938) I . Cellular Comp. Physiol. U,149. Costello, D. P. (1934) J . Cellular Comp. Physiol. 4, 421. Dan, f. C. (1948) Physiol. 2061. 21, 191. Dan, K. (1943) I . Fac. Sci. Imp. Univ. Tokyo 6, 323. Dan, K., and Dan, J. C. (1940) Biol. Bull. 78, 486. Dan, K., and Dan,J. 6. (1947) Biol. Bwll. M, 139. Freundlich, H. (1937) I . Phys. Chem. U , 901. Goldacre, R. J. (1952) I ~ t e m Rev. . Cytol. 1, 135. Goldacre, R. J., and Lorch, I. J. (1950) Nature 166, 497.

CYTORINESIS AND AMOEBOID MOVEMENT

227

Harvey, E. N. (1910) I. Exptl. Zool. 8, 335. Hoffman-Berling, H. (1954) Biochim. et Biophys. Act5 15, 332. Kopac, M. J. (1950) Ann. Rev. Physiol. U, 7. Kriszat, G. (1949) Arkiv 2001.1, 81. Kriszat, G. (1950) Arkiv Zool. 2, 477. Landau, J. V., Marsland, D. A,, and Zimmerman, A. M. (1954a). Anat. Record, 220, 789.

Landau, J. V., Zimmerman, A. M., and Marsland, D. A. (1954b) J. Cellzdar Comp. Physiol. 44,211. Landau, J. V., Marsland, D. A , and Zimmerman, A. M. (1955) J. Celldw Comp. Physiol. 46, 340. Lewis,W. H. (1938) Arch. exptl. Zellforsch Gewebesiicht. 23, 270. Lewis,W.H. (1942) in “The Structure of Protoplasm” (Seifriz, ed.), p. 163. Iowa State College Press, h e s . Lewis,W. H. (1951) Ann. N. Y. Acad. Sci. 61, 1287. Lindberg, 0. (1950) Exptl. Cell Resewch 1, 105. h w y , A. G. (1952) I. Cellular Comp. Physiol. 40, 127. Marsland, D. A. (1938) J. Cellular Comp. Physiol. ia, 57. Marsland, D. A. (1939a) I.Cellular Comp. Physiol. 18, 15. Marsland, D. A. (1939b) I. Cellwlar Comp. Physiol. 18, 23. Marsland, D. A. (1942) in “The Structure of Protoplasm” (Seifriz, ed.), p. 127. Iowa State College Press, Ames. Marsland, D. A. (1950) I. Cellular Comp. Physiol. 36, 205. Marsland, D. A., and Brown, D. E. S. (1936) J. Cellular Comp. Physiol. 8, 167. Marsland, D. A., and Brown, D. E. S. (1942) I. Cellular Comp. Physiol. So, 295. Marsland, D. A., and Landau, J. V. (1954) J. Exptl. Zool. Isa, 507. Mast, S. 0. (1926) J. Morphol. and Physiol. U, 347. Mitchison, J. M. (1952) Symposia SOC.ELzptl. Biol. 6, 105. Mitchison, J. M.,and Swann, M. M. (1954) J. Exptl. Biol. Sl, 461. Pease, D. C., and Marsland, D. A. (1939) I. Cellular Comp. Physiol. 14 (suppl.) 1. Roberts, H. S. (1954) A d . Record ISO, 726. Runnstrom, J. (1949) Advances in Enzymol. 9, 241. Sehechtman, A. M. (1937) Science 86, 222. Swann, M. M. (1952) Symposia SOC.Exptl. Biol. 6, 89. Weber, H.H.,and Portzehl, H. (1954) Progr. Biophys. and Biophys. Chem. 4, 60.

This Page Intentionally Left Blank

Intracellular pH PETER C. CALDWELL Biophysics Department, University College, L d o n

Page I. Introduction .......................................................... 229 1. General ..........................................................229 2. Previous Reviews ................................................ 230 11. Theories about Intracellular pH ....................................... 230 1. Introduction ...................................................... 230 2. Theories Based on the Donnan Equilibrium ......................... 230 3. Theories which Account for Differences in Hydrogen Ion Activity which (Cannot Be Explained by the Donnan Theory ........................................................... 235 111. Methods for the Determination of Intracellular pH ................... 236 1. Measurement of the pH of a Tissue Brei, Tissue Extract, or of Expressed Cell Sap ....................................... 236 2. Measurement of the pH of a Sample of Tissue Fluid which Has Been Withdrawn from the Cells ....................... 237 3. Measurements in which the ,Colors of pH Indicators, which Are Either Present Naturally or Introduced Artificially, Are Studied ............................................. 237 4. Methods in which the Amounts in the Cells of a Weak Acid or Base and Its ,Corresponding Ion Are Measured.. ........... 238 5. Methods in which Micro-Electrode Systems Are Used ............. 241 IV. The Experimental Results which Have Been Obtained with the Different Methods for the Investigation of Intracellular 242 pH .................................................................. 1. Comparison of the Values Obtained for Intracellular pH 242 by Different Methods ............................................. 2. Changes of Intrarellular pH during Activity or after Alterations in the External Conditions ............................. 258 V. Discussion ........................................................... 262 1. Observed Values of the Intracellular pH and the Donnan Theory ...................................................... 262 2. Some Effects of Heterogeneity within the Cell on the Donnan Relations and on Certain Types-of pH Determination ......................................................... 265 3. Concluding Remarks ............................................. 271 VI. References ........................................................... 272

I. INTRODUCTION 1. General This review is divided into four main parts. In the first, certain theoretical aspects of the relations between the pH of the cell interior and its surroundings are considered. In the second, the various experimental ap229

230

PETER C. CALDWELL

proaches which have been used for measuring intracellular pH are briefly described together with their relative merits and disadvantages. In the third, the results which have been obtained are compared, in an attempt to assess the reliability of the methods, and some of the pH changes which have been observed in cells are described. The fourth part consists of a discussion of some of the material presented in the other three.

2. Previous Reviews The most recent reviews specifically devoted to intracellular pH appear to be those of Spek (1937,1938) and Lison (1941). The reviews of Fenn (1936),of Sendroy (1945),and of Albert (1952) although not primarily concerned with intracellular pH, refer to it. Certain aspects of the subject are reviewed in papers by VEs (1924),and Brandt (1945). The earlier literature includes a monograph by Reiss (1926). Aspects of pH in relation to plants have been discussed in three monographs by Small (1929,1946, 1954). A more recent monograph is that of Small and Wiercinski (1955). 11. THEORIES ABOUT INTRACELLULAR PH 1. Introduction The theories which have been advanced to explain differences in p H between the cell and its surroundings and between various regions of the cell itself can be divided into two groups. Theories of the first group, which are based on the Donnan membrane theory (Donnan, 1911), consider the variations in pH to be expected under conditions of thermodynamic equilibrium if the hydrogen ions are free to move to all parts of the cell and the surrounding medium, but certain other ions are not. Theories of the second group, which involve active transport of hydrogen ions at the expense of metabolic energy, are invoked when the first type of theory proves inadequate and the inadequacy cannot be explained by an inability of the hydrogen ions to move from one region to another.

2. Theories Based on the Donnan Equilibrium An equilibrium of the Donnan type is to be expected under conditions of thermodynamic equilibrium at any boundary between solutions containing ions, if one or more of the ionic species is unable to cross the boundary. The nonmobile ions may be unable to cross the boundary for a variety of reasons. In the classical case (Donnan, 1911) there is a membrane at the boundary through which the nonmobile ions are unable to pass. Alternatively the nonmobile ions may be attached to some macromolecu-

INTRACELLULAK

231

pH

lar structure such as a protein gel or ion exchange resin. A particular case of this type, which has been investigated by Danielli (1937, 1941), arises at interfaces between water and solutions of fatty acids in solvents immiscible with water and at protein surfaces. Ionized groups at the interface or protein surface, such as the anions of the fatty acids dissolved by the solvent immiscible with water or the acid and basic groups of the protein, are unable to escape into the bulk of the aqueous phase and give rise to a Donnan equilibrium between the latter and that part of the aqueous phase in the immediate vicinity of the interface or protein surface. The chief consequences of the Donnan type of theory, which are of importance for the purposes of this review, are those which relate to the distribution at equilibrium of those ions which are able to cross the boundary. Under these conditions the ratio of the activities in the two phases of all the ionic species of the same valency and sign is the same. The relation between the ratio r of the monovalent cations and that of the other ions can be expressed in general terms as follows: Activity of Cation (a) in phase ( 1)

r=(

Activity of Cation (a) in phase (2) Activity of Cation (b) in phase (1)

1

etc.,

Activity of Cation (b) in phase (2) Activity of Anion

(u)

in phase (2)

Activity of Anion

(a)

in phase (1)

Activity of Anion (19) in phase (2)

1

(1)

1

etc. Activity of Anion (a) in phase (1) In cases where the ionic strength is the same in the two phases, the ratio of the activities is equal to the ratio of the concentrations. A potential difference is also set up across the phase boundary, the magnitude of which is given by E (potential change on going from phase (1) to

RT

phase (2)) =

- log,r

F At 20°C.this can be written in the form E (in millivolts) = 58 loglor

232

PETER C. CALDWELL

If the extracellular and intracellular hydrogen ion activities are governed by the Donnan equilibrium theory, then from (1) and (3) (H+extraceuar)

(H+intraceunhr)

and

(4)

= r

-

Membrane potential (in millivolts) = 58 (intracellular pH extracellular pH) (5) where (H+extraceu*r) and (H+htraceunlar) are hydrogen ion activities and pH = - log10 (hydrogen ion activity). For hydrogen ions to obey these relations it is not essential for the membrane or phase boundary to be permeable to them as such since their concentration is linked to those of other ions. Thus if both the undissociated form and the corresponding ion of an acid (HA) or base (BOH) are free to penetrate, then (HAextraceuular) = (HAintraceumr) (BOHextmceuar) = (BOHintmce11nlar) and (A-intmce~uhr) - (B +extraceudr) = r (A-extrnceuuhr) (B +intrnceunhr) Since ( H + ) (A-) = K, (HA) and (B + ) (OH-) = Kb (BOH) (H+ex+xaceuar) or

(H +intracenvhr) Intracellular pH

-

(A-intraceunhr )

(B +extraceliuhr) -=r

(A-extraceunhr)

(B +intrnceunhr)

= extracellular p H

= extracellular pH

+ log10

+ loglo

(A-intmceunkr)

(A-extraceu&r) (B+extraceuuhr)

(6) (B +intraceuubr)

For the particular cases of water and OH-ions and COz and bicarbonate ions this becomes (H+extraceuar)

- (OH-intraceuar) - (HCOs-intmceutllar)

(7)

(HCOS-extraceuar) Thus a failure of hydrogen ions to obey the Donnan theory implies a similar failure on the part of the ions of weak acids and bases in the cell, provided the undissociated forms of each of these is present in the same concentration in the intracellular and extracellular phases. (H+intraceuar)

(OH-extraceunlar)

INTRACELLULAR p H

233

There is evidence in certain cases, notably muscle and nerve, that the intracellular and extracellular potassium ions may be in an equilibrium of the Donnan type. If both potassium ions and hydrogen ions obeyed the Donnan relationships, then the relationship

- (K+extraeenular)

(H+extraee~&r)

(H+intraceUn&r)

(K+intraeeUu&r)

(8)

should hold. That the hydrogen ions in cells should be in a Donnan equilibrium with those in their surroundings is implied in Donnan’s original paper (Donnan, 1911) and this possibility has also been considered by Reiss (1926). Netter (1928)and Mond and Netter (1930)have considered some of the consequences of (8) holding for cells, in particular for muscle, and some reasons against its being true for muscle have been advanced by Fenn and Cobb (1934),Fenn and Maurer (1935),and Hill (1955). That (7) and (8) do hold for muscle has been suggested by Conway and his collaborators (Boyle and Conway, 1941 ; Conway and Fearon, 1944). It is possible that uneven distributions of ions occur within cells as the result of the occurrence of Donnan equilibria between different regions. The existence of these equilibria could lead to differences in pH between various parts of the cell. The possibility of such differences between the fibrils and sarcoplasm of muscle has been considered by Fenn and Maurer (1935) and by Dubuisson (1942). The part local variations in pH may play in the functioning of cells is mentioned by Peters (1937). The existence of Donnan equilibria between different regions of the cell and the resulting local differences in pH would lead to limitations both in the applicability of the Donnan relationships to the mean intracellular activities of ions and in the use of certain types of pH determination. These limitations are dealt with in the last part of this review, in a discussion of some of the consequences of heterogeneity within the cell. Danielli has concluded that hydrogen ions are accumulated at certain interfaces and surfaces as the result of the existence of an equilibrium of the Donnan type. Measurements of the surface tension at the interface between solutions of fatty acids in bromobenzene and water (Danielli, 1937) suggested that the pH of the aqueous phase in the immediate vicinity of the interface is often as much as two units lower than that in the bulk of the aqueous phase. These results could be accounted for if it was assumed that negative fatty acid groups held at the interface give rise to a Donnan equilibrium, and hence a p H difference, between the two regions of the aqueous phase. Similar conclusions were reached, from electrokinetic measurements and studies with indicators, about the aqueous phases

234

PETER C. CALDWELL

in the vicinity and at a distance from the surface of ovalbumin molecules (Danielli, 1941). In this case the surface pH can be higher or lower than the pH of the bulk phase according to which side of the isoelectric point of ovalbumin the latter lies, being higher when the ovalbumin is positively charged and lower when it is negative. Danielli suggested that in cells these effects at interfaces and surfaces might give rise to quite large local variations of pH. Eddy and Hinshelwood (1950) have also considered the accumulation of hydrogen ions at protein surfaces and have discussed its relation to the accumulation of potassium. As a result of studies with bacteria they have suggested a competition of hydrogen ions with potassium ions for the negatively charged sites at the surfaces of certain enzymes which are activated by potassium. The mechanism which Eddy and Hinshelwood have proposed for this competition is based, however, not on the Donnan theory, but on the Langmuir adsorption isotherm. The equation used by Danielli for calculating the surface pH from electrokinetic measurements was that of Hartley and Roe ( 1940), namely that : 0

pH (surface

(at 25°C.)

(9)

(where g is the electrokinetic potential in millivolts and 60 the value of lo00 X 2.303 RT/F at 25°C.). Hartley and Roe applied this equation successfully in studies by electrokinetic measurements and with indicators of the pH at the surface of alkylamine and alkyl sulfonate micelles. The electrokinetic potential g is probably equivalent to the Donnan potential to be expected between the surface and the bulk phase on the basis of Danielli’s theory, since Eq. (9) can be obtained from Eq. (2) if the two potentials are identified with each other. Hartley (1934), in an investigation of the effects of alkylamine and alkyl sulfonate micelles on indicators, found that indicators, both forms of which were charged and of the same sign as the micelle, tended to be unaffected and to measure the pH of the bulk phase. Indicators consisting of a neutral form and a charged form of the same sign as the micelle and all those consisting of one or more forms of the opposite sign, were however affected. Most of these indicators gave pH values which were displaced toward the pH value to be expected at the surface of the micelles. It is likely, therefore, that they are held at the micelle surface and tend to measure the pH there. Certain indicators consisting of two charged forms of the opposite sign to the micelle gave pH values which were displaced from that of the bulk phase in a direction away from the pH to be expected at the micelle surface and Hartley concluded that in such cases, other more specific factors were at work. Effects similar to those found by Hartley

INTRACELLULAR

pH

235

were found by Danielli (1941) in his studies of the surface p H of ovalbumin. Danielli (1941) has pointed out that surface pH effects are probably the principal cause of the “protein” errors (Sorenson, 1909) which are found when the p H values of protein solutions are measured with indicators. It is also possible that they may be partly responsible for the “metachromatic” errors of pH indicators which have been found to occur in the presence of sulfate esters (Lison, 1935). The studies of Hartley (1934) suggest that the effects of surface p H phenomena on indicators, and therefore the errors introduced by them, can be largely eliminated if indicators consisting of two charged forms of the same sign as the surface are used. Similarly Danielli (1941) found that the errors introduced by the presence of ovalbumin were absent when the indicator was of the same sign as the ovalbumin. Indicators of the same sign as the surface should therefore be used for measurements of the p H of the bulk phase in the presence of molecules, such as proteins, at whose surfaces p H effects occur. Caldwell and Harris (1952) have pointed out a consequence of the hydrogen ion activities on the two sides of cell membranes being related to each other by Eq. (5). According to (5) depolarization of the membrane should cause the intracellular pH to increase to the extracellular value. In the case of frog muscle fibers, with resting potentials of -90 millivolts in Ringer of pH 7.3, the internal pH would be about 5.8 and depolarization with some agent, such as KCl isotonic with the Ringer and of pH 7.3, would bring the intracellular pH to about 7.3. It was suggested by Caldwell and Harris that such pH changes might influence enzyme activity and hence account for the changes observed in the rate of metabolism of tissues subjected to conditions which bring about depolarization. Experimental findings show, however, that these pH changes do not occur (Caldwell, 1955, 1956). The part that pH changes of this type may play in certain phenomena observed in heart muscle has been discussed by Hajdu (1953).

3. Theories which Account for Diferences in Hydrogen Ion Activity which Cannot Be Explained by the Donnan Theory The theories just outlined predict the hydrogen ion activities to be expected in the cell and its surroundings under conditions of thermodynamic equilibrium. If the activities are not in accordance with these theories, barriers may exist between regions of different pH through which hydrogen ions and those ions to which they are linked are unable to pass. Alternatively, processes may be at work which result in the maintenance of the nonequilibrium hydrogen activity differences at the expense of free

236

PETER C. CALDWELL

energy released by metabolism. In most cases the latter explanation seems to be preferred. The most striking example of the hydrogen ion activities in cells and their surroundings not being in accord with the Donnan theory is found in the gastric mucosa. Here the pH difference between the two outer cell boundaries is about 6 p H units while the potential difference, although of the right sign, is in the case of frog mucosa only about 30 millivolts (see for example Crane et al., 1948) instead of about 350 millivolts as required by Eq. ( 5 ) . The active extrusion of hydrogen ions at the expense of metabolic energy seems to be the only satisfactory way of explaining the secretion of hydrochloric acid by the mucosa and the maintenance of the nonequilibrium p H difference. Various schemes for the active transport of hydrogen ions have been discussed, notably by Crane et d. (1948), Patterson and Stetten (1949), Davies and Ogston (1950), Davies and Krebs (1952),and Conway (1952,1953). Active secretion of hydrogen ions by mechanisms similar to those functioning in gastric mucosa may also occur in yeast, pancreas, and kidney (Conway et al., 1950;Davies and Krebs, 1952). Fenn and Cobb (1934) and Fenn and Maurer (1935) have suggested that some form of active extrusion of hydrogen ions may take place in frog muscle, and Hill (1955) has recently discussed the evidence for this.

111. METHODSFOB

THE

DETERMINATION OF INTRACELLULAR PH

1. Measurement of the pH of a Tissue Brei, Tissue Extract, or of Expressed Cell Sap The most straightforward but least exact way in which an estimate of the intracellular pH of tissues and cells can be obtained is by measurement of the pH of a brei, an extract, or expressed cell sap. In most of the earlier work with this method, such as that of Michaelis and Davidoff (1912) on hemolyzed blood and Michaelis and Kramsztyk (1914) on various mammalian tissues, the platinumfiydrogen electrode was used for the pH measurement, although in a few cases indicators were used. Meyerhof and Lohmann (1926), in their studies on frog muscle, have used the quinhydrone electrode as well as the platinumfiydrogen electrode. In more recent work the glass electrode, first used for this purpose by Furusawa and Kerridge (1%7a, b) in work on muscle, has been used. The most serious objection to this method is that the destruction of the cells may lead to the occurrence of processes which bring about changes in pH. Certain workers have attempted to prevent such changes either by heat treatment (Michaelis and Kramsztyk, 1914) or by working at a

INTRACELLULAR

PH

237

temperature in the region of 0°C. during the measurement ( V l b et al., 1924a, b, c), or until just before the measurement (Furusawa and Kerridge, 1927a). If the platinumfiydrogen electrode is used for the p H measurement there is the possibility of interference by oxidation/reduction effectssuch as occur with the protoplasm of Nitella (Taylor and Whitaker, 1927), while measurements with indicators are subject to salt, protein, and metachromatic errors (Sorenson, 1909; Clark, 1922; Lison 1935). This method gives a pH value which lies somewhere between the p H value of the cell interior and the pH of the extracellular space and where the extracellular space is large and its pH different from the intracellular value the measured pH may differ appreciably from the latter. In spite of the errors to which it is subject, the method has given results which are in reasonable agreement with those obtained with other methods.

2. Measurement of the pH of a Sample of Tissue Fluid which Has Been Withdrawn from the Cells This method, which can normally be applied only to very large cells, has been used by a number of workers for the large plant cells Nitella, Valonia, and Halicystis, and by Bodine (1927) for Fundulus eggs. In the case of the large plant cells it is possible to withdraw from the cell a quantity of the cell sap large enough for a p H determination, with very little damage to the cell itself. The pH of the sap can then be determined either with a platinumfiydrogen electrode, an indicator, or a glass electrode. If the platinum/hydrogen electrode is used the determinations may be affected by reducing substances while if indicators are used they may be subject to salt, protein, and metachromatic errors.

3. Measurements in which the Colors of pH Indicators, which Are either Present Naturally or Introdwed Artificially, Are Stzcdied A number of workers have made use of observations on intracellular pigments which are also pH indicators in attempts to obtain information about the intracellular pH. Willstatter and Mallison ( 1915), Haas (1916) and Smith (1933) assumed that the natural color of the anthocyanins in flower petals depended on the acidity of the petal sap and Haas (1916) and Smith (1933) drew conclusions about the pH of the sap from the color changes of the anthocyanins with pH. Jacobs (1920b) and Smith (1923) used the color changes of the anthocyanin pigment to study the penetration of carbon dioxide into flower petals. Crozier (1918) drew conclusions about the pH in certain invertebrates from the color of their intracellular pigments. Spectrophotometric studies of intracellular p H sensitive pigments have been made by Vllts and Vellinger (1928) on sea urchin eggs

238

PETER C. CALDWELL

and by Drabkin and Singer (1939) on red cells whose hemoglobin had been converted to methemoglobin by treatment with nitrite. Chambers (1941) studied the accumulation in the cell nuclei of the pH sensitive pigment of tulip petals which occurs after crushing. From his observations he concluded that the pH of the nucleus was greater than 7.0. Most of the work with p H indicators has however been done with dyes which are not natural to the cells. Some workers have introduced these indicators by allowing them to permeate into the cells. Early examples of the use of this technique are the investigations of Atkins (1922a, b) and Pantin (1923) while a more recent one is the work of Bradford and Davies (1950) on gastric mucosa. Many indicators do not however penetrate easily into cells and various ways of introducing them have been devised. Staining with indicators of freshly cut sections has been used for certain plant tissues by various workers including Rohde (1917), Atkins ( 1 9 2 2 ~ ) ~ and Small (1929). A method described by V l b (1924) and by Reiss (1926) which involves a crushing of the cells in the presence of indicators, has been used by these two workers and their colleagues. Various techniques for the microinjection of indicators have also been used and these cause much less damage to the cells than the two methods which have just been mentioned. Workers with microinjection techniques include Schmidtmann (l!Z4), Needham and Needham (1925, 1926), Damboviceanu and Rapkine ( 1925), Rapkine and Wurmser (1926), and Chambers and his colleagues (Chambers and Pollack, 1927 ; Chambers et d.,1927 ; Chambers 1928, 1930, 1932; Chambers and Ludford, 1932; Chambers and Kerr, 1932; Pandit and Chambers 1932). The main advantage of the indicator method is that in some cases it is possible to locate in the cell regions of different pH. Its main disadvantage is that in many instances protein, metachromatic, and salt errors occur. Another disadvantage-is that in cells with little internal buffering the amount of indicator needed to produce an adequate color may be so large that it causes an alteration of the intracellular pH. Also, serious errors may arise if one of the forms of the indicator dissolves in the lipoids of the cell and the other form does not (FaurC-Fremiet, 1923).

4. Methods in which the Amounts in the Cells of a Weak Acid or Base and Its Corresponding Ion Are Measured In this type of measurement the cells are exposed to a weak acid or base and this is allowed to equilibriate between them and the surrounding medium. The intracellular concentration of the undissociated form of the acid or base is then assumed to be the same as that in the extracellular phase. Hence, if the combined concentration of the acid or base and its

INTRACELLULAB

pH

239

corresponding ion in the cells is measured, the concentration of the latter can be obtained from the difference. The p H of the cells can then be calculated from the pK of the acid or base and the appropriate form of the equation of L. J, Henderson; i.e., pH = pK, log10 (anion concentration)/ (undissociated acid concentration) or p H = p K , - pKb - loglo (cation concentration)/(undissociated base concentration) ( 10) Alternatively, the intracellular p H can be calculated from Eq. (6) if the pH and the concentration of the ion in the extracellular phase are also known and the activity coefficient of the ion is assumed to be the same in the two phases. The most widely used method of this type is that in which the amounts of carbonic acid and bicarbonate in the cell are measured. Such measurements were originally used to obtain the pH inside erythrocytes (Warburg, 1922; Van Slyke et d., 1923; Henderson et al., 1924) and were extended to other tissues by Fenn (1928, frog muscle and nerve) and Stella (1929, frog muscle). Normally, the total carbon dioxide content of the tissue is determined by treatment with acid. The intracellular partial pressure of carbon dioxide is assumed to be the same as that of the surroundings and the total amount of carbon dioxide present as H2COs and physically dissolved COZ is calculated from this partial pressure. The form of the L. J. Henderson equation used in this case is that known as the Henderson-Hasselbakh equation

+

where (C02) represents the combined concentrations of H&Os and physically dissolved CO2. Since concentrations, instead of activities, are used in the logarithmic term, a value of the pK appropriate to the ionic strength of the inside of the cells, must be used. Normally the pK found for serum is used and this seems to be justified by the work of Danielson et al. (1939) who determined the pK in breis of cat muscle and found it to be the same as that for serum. Sometimes, in measurements by this method, the intracellular pH is determined for a range of external carbon dioxide tensions. A possible source of error in this type of measurement is that part of the carbon dioxide released from the cells on treatment with acid arises not from carbonic acid and bicarbonate but from other substances which decompose under acidic conditions to form carbon dioxide. Such a state

240

PETER C. CALDWELL

of affairs exists in erythrocytes, particularly under reducing conditions, where some of the intracellular carbon dioxide combines with hemoglobin to form carbhemoglobin. In this case the difficulty can be overcome by the measurement of an empirical value of the p K of carbonic acid in hemolyzates of red cells under reducing conditions. The effect of the combination of some of the carbon dioxide with reduced hemoglobin is to give an apparent pK up to 0.2 lower than that for serum (Van Slyke et al., 1925; Stadie and Hawes, 1928; Dill et aZ., 1937). This apparent pK must be used for calculations of the intracellular pH of red cells under reducing conditions. Other substances which can form carbamino compounds, such as ammonia, glycine, and peptides (Meldrum and Roughton, 1933),may interfere with this method. The formation of such compounds in cells is discussed in some detail by Brandt (1945). There is also the possibility of interference from the carbon dioxide formed by acid labile /3 keto acids such as oxaloacetic acid and acetoacetic acid. Conway and Fearon (1944) have produced evidence to show that part of the carbon dioxide obtained from rat, rabbit, cat, and guinea pig muscle on treatment with acid arises, not from bicarbonate, but from some substance which does not form an insoluble barium salt and which does not appear to be a carbamino compound. This compound may not occur in sufficient quantities in cat muscle to affect the pH determinations since the values obtained by Danielson et al. (1938)for the pK of carbonic acid in the presence of cat muscle brei gave values close to those for other solutions of the same ionic strength. Since, however, these workers added a certain amount of sodium bicarbonate to their muscle breis, it is possible that the effects of Conway and Fearon’s compound may have been reduced. This method, like that based on the measurement of the pH of tissue breis and extracts, gives a pH value which lies somewhere between the intracellular p H value and the extracellular p H value. Provided that the extracellular space is small and its p H not too different from the cell interior, this source of error is not serious. In certain cases the volume of the extracellular space and its carbon dioxide content can be determined and reliable values for the intracellular bicarbonate and p H obtained. Such corrections have been applied in the case of frog muscle (Fenn and Maurer, 1935). Other acids which have been used for this type of measurement are acetic acid, glyceric acid, and hydrogen sulfide. Conway and Downey (1950) have used measurements of intracellular acetate and acetic acid to obtain information about the intracellular p H of yeast. They have also used measurements of the uptake of glyceric acid to obtain information

INTRACELLULAR

pH

241

about the pH of an outer region of the yeast cell. The amount of hydrogen sulfide taken up by Valonio and its relation to the pH of the cell sap has been studied by Osterhout (1925). The only basic substance which appears to have been used for this type of study is ammonia. The uptake of ammonia and its relation to the p H of the cell sap has been studied for Nitella by Irwin (1925)’for Valonia by Cooper and Osterhout (1930),and for Halicystis by Blinks (1933). The pH of frog muscle has been calculated from the uptake of ammonia by Netter (1934). Netter (1929) has also studied the uptake of ammonia by red cells and its relation to their internal pH. The uptake of ammonia by frog muscle has also been studied by Fenn et al. (1944) who showed that the ammonium ion ratio across the cell membrane corresponded to the hydrogen ion ratio obtained from carbon dioxide measurements rather than to the potassium ion ratio.

5. Methods in which Microelectrode Systems Are Used In this type of determination a suitable pH sensitive electrode system is inserted into the tissue, in some cases into individual cells, and the p H determined from the changes in potential. Three main types of p H sensitive electrode, usually in a micro form, have been used. Micro forms of the platinum/hydrogen electrode have been designed and used by Schade et al. (1921, subcutaneous p H measurements in man), Taylor and Whitaker (1927, intracellular measurements on the cell sap of Nitella) and by Dorfman (1936a,b, measurements on frogs’ eggs). Electrodes of certain metals whose electrode potential is sensitive to pH have also been used. Micro antimony electrodes have been used by Buytendijk and Woerdeman (1927, amphibian and hens’ eggs) and micro tungsten electrodes by Caldwell (1953, 1954b, intracellular measurements of p H in crab muscle fibers). A MnOZ/platinum electrode was used by Ritchie (1922) far <.measurementson whole frog gastrocnemius muscles, the values obtained probably representing the p H of the extracellular fluid. Small forms of the glass electrode for measuring the p H of tissues have been designed by Voegtlin ef al. (1935) and Sonnenschein et al. (1953, cat’s brain). A micro form of the glass electrode suitable for the direct determination of the intracellular p H of individual crab muscle fibers and squid giant axons has been designed and used by the author (Caldwell, 1954a,b; 1955; 1956). The advantage of the use of these microelectrode systems is that in certain cases it is possible to determine the pH inside single cells and single nerve and muscle fibers. Unfortunately the results obtained with the platinumfiydrogen electrode (Taylor and Whitaker, 1927) and with metal

242

PETER C. CALDWELL

electrodes such as the tungsten electrode (Caldwell, 1954b) are not always reliable, possibly on account of interference from reducing substances. On the other hand, the glass electrode appears to be affected only by hydrogen ions in aqueous solutions whose pH is less than 9.0 (Dole, 1941) and when used as an intracellular electrode it probably provides the most reliable and direct means of measuring intracellular pH.

IV. THEEXPERIMENTAL RESULTS WHICH HAVE BEENOBTAINED WITH DIFFERENT METHODS FOR THE INVESTIGATION OF INTRACELLULAR PH

THE

1. Comparison of the Values Obtained for Intracellzclar pH by Diferent Methods In Figs. 1 to 14 an attempt has been made to summarize and compare some of the values which have been obtained for the intracellular p H of a variety of tissues and cells by means of the different methods. The values for similar types of tissues or cells have been grouped together so that a comparison of the different methods can be made. The figures are not comprehensive but most of the available values for those tissues which have been the subject of a number of investigations are included. It will be seen in certain cases, notably muscle, liver, and marine eggs, that the results of Rous and those of Vl&s,Reiss, and their colleagues are appreciably lower than those obtained by other workers. The values given by Rous for muscle have been criticized by Fenn and Maurer (1935) on the grounds that the indicators which he used do not penetrate into cells and that the values are therefore for the extracellular space. Fenn and Maurer have also suggested that Rous’s experimental technique may have given rise to conditions which would lead to lactic acid formation. Vlks, Reiss, and their colleagues crushed their tissues to enable the indicators to penetrate into them. Other authors, who have used less brutal techniques such as microinjection for the introduction of the indicators, have obtained higher pH values which are in better agreement with those obtained with the other methods. Some of these authors (e.g., Needham and Needham, 1926; Chambers and Pollack, 1927; Chambers et al., 1927; Chambers and Ludford, 1932) and also Fenn and Maurer report that injured regions of cells show a more acid reaction to indicators than do the undamaged parts. It is possible, therefore, that the low pH vaIues obtained by Vlb, Reiss, and their colleagues are due to the injuries sustained by the cells when they are crushed. Injury of the cell does not, however, always result in a large decrease of pH, particularly if steps are taken to slow down enzyme reactions. Thus, the pH values obtained for certain tissue extracts and breis are in

INTRACELLULAR

pH

243

reasonable agreement with those obtained for the tissue by other methods, and the author, in work with intracellular glass electrodes on crab muscle and squid nerve (Caldwell, 1954b, 1956), found no indication of an appreciably lower pH in more damaged regions of the cell. Increased acidification in regions of injury has usually been observed in work with indicators and it is just possible that the color differences observed may in some cases be due to changes which lead to an alteration in the protein or metachromatic errors, rather than to an actual acidification. An alternative explanation of the discrepancy between the low pH values obtained by Vlb, Reiss, and their colleagues and the higher values of other authors has been discussed by Reiss (1926). This is that the higher values are due to an alkalization brought about by the loss of carbon dioxide from cells. While such an explanation may apply to values obtained from the measurement of the p H of tissue breis and extracts, it is not likely to apply to values obtained from the other types of measurement, in particular those involving the estimation of carbon dioxide under different carbon dioxide pressures or the use of microinjection and intracellular microelectrode techniques. It will be noted that the value of 6.0 given by Conway and Fearon (1944) for the intracellular p H obtained from measurements of the carbon dioxide in rabbit muscle is lower than most of the values for muscle, in particular from other carbon dioxide measurements. This low value was obtained by Conway and Fearon after allowance had been made for the presence in the muscle of compounds, other than bicarbonate, which give rise to carbon dioxide on treatment with acid. Such a correction has not been applied, in calculations by other workers, of intracellular pH from carbon dioxide measurements and failure to apply it may have given rise to errors in the values obtained. It is possible that the errors introduced by such compounds are not usually serious. Thus Hill (1955) has pointed KEYTO FIGURES 1 TO 14

Normal or resting d u e

0 Fatigue Rigor Cytolyzing or injured 8 In these determinations by Michaelis and Kramsztyk, the enzymes in the tissue were not inactivated by heating. 0 Nucleus @ Potassium deficient (D

8

NOTE:Normally the mean value given by the authors, or the mean of their range of values, is given. In a few cases the range of values is given and this is indicated by the sign

++.

PH

Method Measurement of the p H of breis or extracts

Indicatorsintro-

Animal

1. Frog 2. Frog 3. Frog 4. Cat 5. Cat 6. Cat 7. Cat (uterus) 8. Cat (uterus) 9. Cat (heart) 10. Cat (heart) 11. Rat 12 Dog (heart) 13. Mouse 14. Guinea pig IS. Guinea pig (heart) 16. Rabbit 17. Crab (Eupugurur) 18. Crab (Maia) 19. Crab (Cwcuucs) 20. Lobster (Homarus d g o r i s ) 21. Scyllium canicuh 22. Pecten operunrloris 2.3. octapus 24. Human

References Pechstein (1915) Meyerhof and Lohmann (1926) Reiss and Velliiger (1926) Micbaelis and Kramsztyk (1914) Furusawaand Kerridge (19na) Mackler et al. (1930) Furusawa and Kerridge (I527a) Kerridge and W i t o n (1929) Michaelis and Kramsztyk (1914) Furusawa and Kerridge (1927a) Woglom (1924) Michaelis and Kramsztyk (1914) Reiss and Vellinger (1926) Michaelis and ?Sramsztyk (1914) Michaclis and Kramsztyk (1914) Carlstr6met 01. (1928) Furusawa and Kerridge (1923%) FumsawaandKerridge (1927b) Caldwell (1956) Furusawaand Kerridge (1923%) Furasam and Kerridge (1927b) Furusawa and Kerridge (1927b) Quagliariello (1921) Griiff (quoted by Reiss, 1926)

FIG.la. MUSCLE

5

6

7

I

I

I

(D

a 0

8 1

0 .

1

0

2 3 4 5

0 0

6 7 8

0)

C B O

(Do

0 0

e o


0

0. @

9 10 11

‘d

m

Fj PI p

12

13

14

eo (Do 0 @

15

16

0

17 18

(D (D

0

0 0

0 (D

0 B)

19

20 21

0

22 23

0

24

E; 3

Method d u d by vital staining or by the staining of crushed cells.

25. 26.

27. 28. 29.

30. Microinjectim or miuoinsertion of indicators.

CO, measurements

31.

Animal Mouse Mouse Mouse Mouse (heart) Cat Patelk picta Vax'ious vertebrates and invertebrates Frog, N e c t w w

32. 33. Mouse 34. Frog 35. Frog 36. Frog 37. Frog 38. Frog (heart) 39. cat 40. Rat 41. Rabbit (leg) 42. Rabbit (abdominal) 43. Dog

44. Human 45. Crab (Maiu) NH, meaSWaentS Intracellular glass electrode

46. Frog

47. crab (CMcinUs) 48. Crab (Maia)

References 5 I Reiss (1924a) Rous (1925) Vlb and de Coulon (1924) Rous (1925) Reiss (1924a) Reiss (1924a) Schmidtmam (1925, quoted by Reiss,1926) Chambers et al. (1927)

Schmidtmann (1924) Fenn (1928) Stella (1929) Root (1933) Fenn and Maurer (1935) calculated from Brody (1930). Wallace and Hastings (1942) Gardner et d. (1952) Conway and Fearon (1944) calculated by Hill (1955) from Conway and Fearon (1944) Irving ef al. (1932) calculated from Gardner et al. (1952) Cowan (1933) Netter (1934) Caldwell (1954b) Caldwdl(1956)

hc. Ib. MUSCLE

..... .

PH

8

6

7

I

I

8 I

25 26

27 28 29

a

30 31

Method Microinjection of indicators

CO, measwements IntraCellUlW glass electrode

Animcrl 1. LoPhiur 2. SeM

RrferenceJ chambers (1930) Arvanitakiand Chaazonitis (1951,1954) Fenn (1928) Fenn et d.(1934) Cowan (1933) Caldwell (1956)

3. Frog ( R w ) 4. Frog (Ram pipiem) 5. Crab (Mmk) 6. Squid (Loligo forbeci)

Mctlrod Measurement of the pH of breis or extracts

Indicators introduced by vital staining or by the stainiig of crushed cells

Microinjection or microinsertion of indicators

FIG.2. NERVE Animal

1. Dog 2. Dog 3. Rabbit 4. Pig

5. Sheep 6. Beef 7. Cat 8. Mouse 9. Mouse 10. TrochococMealineafa (hepatopanueas) Rat, mouse, cat, rabbit, hamster 11. Parenchyma

12. Islets 13. Frog, Nec-

References Michaelis and Kramsztyk (1914) Carnot and Gruzewska (1925) Carnot and Gnuewska (1925) Long and Fenger (1915,1916) Long and Fenger (1915, 1916) Long and Fenger (1915, 1916) Reiss (1924a) Reiss (1924a) Rous (1925) Reiss (1924a)

Schmidtmann (1925, quoted by Reiss, 1926) Schmidtmann (1925, quoted by Reiss, 1926) Chambers et al. (1927)

FIG.3. PANCREAS

PH

6

5 I

8

7

I

1

I

a

a

0

I

1

2

a I

a

3 4

a

5

a

6 I

I

cd

PH I

a a a a

6

7

8

I

I

1

8.

1 2

a a

3

4 5

6

0

7 8 9

a 0

a

I

10

11 12 @ 13

a

8 I

x

I

I

3m

w F CI

6<

P r

Method Measurement of the pH of breis or extracts

Animal

1. 2. 3. 4. 5. 6.

Frog Frog cat Rat Rat Rat 7. Rat 8. Mouse 9. Mouse 10. Guineapig 11. Guinea pig 12. Guineapig 13. Rabbit 14. Rabbit 15. Rabbit 16. Dog 17. Dog 18. Pig 19. Sheep

References Pechstein (1915) Duval et al. (1925) Michaeb and Kramsztyk (1914) Michaelis and Kramsztyk (1914) Woglam (1924) Duval et al.( 1925) Leutllardt (1940) Michaelis and Kramsztyk (1914) Petow et d. (1925) Michaelis and Kramsztyk (1914) D u d et d. (1925) Petow et al. (1925) Michaelis and Kramsztyk (1914) Duval et d. ( 1 9 3 ) Carnot and Gruzewska (1925) Michaelis and Kramsztyk (1914) Catnot and Gruzewska (1925) Lung and Fenger (1916) Long and Fenger (1916)

FIG.4% Llvpg

. ... .. ... . ... PH

5 I

6

7

I

I

8.

0

a3

CB.

8

8

L

I

a 1

1

2 3 4 5 6

7

a

9

.

11 10 12 13

.

14 15 16

I

17 18 19 1

P Q c

c 'd

X

Method Measurement of the pH of breis or ex-

Animul 20. Pigeon 21. Duck

tracts

22, carp

Indicators introduced by vital staining or by the staining of crushed cells Microinjection or microinsertion of indicators

23. A s t u w fEuviotilis 24. Platycwcinw pugurur 25. CurcircusmaencM 26. Por&nus puber 27. OctoPuJ vrrlgwis 28. Sepio oficinalis 29. Anodonta cynea 30. Cat 31. Mouse 32. Mouse 33. Mouse 34. Mouse Cat, rabbit, guinea pig 35. Periphery of lobule

36. Center of lobule 37. Cells of Kupfer 38. Frog, Necturus

References Duval et al. (1925) D u d et ul. (1925) Dwal et ul. (1925) Duval et ul. (1925)

Duval ef ul. (1925) Duval et ol. (1925) D u d et el (1925) D u d et ul(l925) Duval et d(1925) Duval et d (1925) Reiss (1924a) Reiss (1924a) Reiss (1924b) Vl&sand de Coulon (1924) Rous (1925) Schmidbnann (1925, quoted by Reiss, 1926) Schmidtmann (1925, quoted by Reiss, 1926) Schmidtmam (1925, quoted by Reiss, 1926) Chambers et d. (1927) FIG.4b. LIVER

PH

5

6

1

I

a

7

1

I

0 0 0 0 0 0 0 0

0

0

0 0 0 0 0 0

0 L

1

0

e

20 21 22 23 24 25 26 27 28

1 m RI c1

29 30

c)

31 32 33 34 35

3

36

0

37 038

I

I

6

F

Method Measurement of the pH of breis or extracts Indicators introduced by vital staining or by the staining of crushed cells Microinjection or miaoinsertion of indica-

tors

Refemues Michaelisand Kramsztyk (1914) Michaelis and Kramsztyk (1914)

Adtd

1. Rat 2. Dog

PH

5 I

a a

Petow et ol. (1925) Chambers and Cameron (1932)

7

I

I

e

Reiss (1924a) Reiss (1924a) Rous (1925)

3. cat 4. Mouse 5. Mouse 6. Mouse] guinea pig 7. Chick (mesonephros) various mammals 8. Straight tubules 9. Convoluted tubules 10. Reticular cells

6

a

€3

schmidtmann (quoted by Lison, 1941)

8 1

1

a

2 3

a a a

Schmidtmann (quoted by Limn, 1941) schmidtmann (quoted by Lison, 1941)

FIG.5. K ~ N E Y Method Measurement of the pH of breis Indicators introduced by vital staining or by the staining of isolated mucosa Microinjection of indicators CO, measurements

References

Animal 2. Dog (pyloric mucosa)

Irving (1932) Irving (1932)

3. Frog, toad, cat, and polecat 4. Mouse, guinea pig

Bradford and Davies (1950) Petow et 01. (1925)

1. Dog (fundic mucosa)

5. Frog (epithelial cells) 6. Frog,Necturur 7. Dog (fundic and pyloric

Chambers (1930) Chambers et 01. (1927) Irvingand Wilson (1932)

mucosa)

FIG.6. GASTRIC MUCOSA

Cytoplasm of the oxyntic cells. sllghtly alkaline. lntracettular canalicuti. gastric ClyPtS and tubules; pH< 1.4

e

a a a a

3

0

0

4 5

6 7

Method Measurement of the pH of breis or extracts Indicators introduced by the staining of crushed cells Microinjection of indicators

Method pH of hemdyzates measured with a glass electrode Spectrophotometric studies of methemo-

Anid

References

1. Rat (grafted sarcoma) 2. Rat (spontaneous sarcoma) 3. Rat (Jensen sarcoma)

4. Mouse (grafted sarcoma) 5. Mouse (spontaneous mammary -r) Mouse, tissue cultures of 6. Normalrnammarygland 7. Mammarycarcinoma 8. Tarcarcinoma 9. Crockersarcoma

WogIom (1924) Woglom ( 1924) Leuthardt (1940)

5

6

r

I

V l h and de Coulon (1924) Vlbs and de Coulon (1924)

a

PH

8

I

i

a a a

a

8 8 8 0

7

3 4

5

0

6 7 8 9

a a

1. Dog 2. Human

RefereHces Drabkin and Singer (1939) Harris and Maizels (1952) (for an external pH of 7.4-7.6)

3. Dog

Drabkin and Singer (1939)

4. Horse 5. Human 6. Dog .7. Snapping turtle (Chelydra serpeutinu)

Van Slyke et al. (1923) Henderson et al. (1924) Rapoport and Guest (1939) Henderson (1928) p. 318

FIG.8. ERYTHR~CYTE~

w td

cj

r)

0 9

G

8

PH

5 r

6

7

1

I

!

a1 a 2 a 3

globin COz measurements

1

2

Chambers and Ludford (1932) 0 Chambers and Ludford (1932) Chambers and Ludford (1932) 1 I J Chambers and Ludford (1932) ' (Chambers and Ludford found the p H of the nuclei to be greater than 7.2 in all four cases) FIG.7. TUMORS

Animal

E

a

s

a a

5 6

a L

1

I

7 I

P

r

Method Measurement of the pH of a purb Measurement of the pH samples of intracellular fluid Spectrophotometric studies of an intracellular pigment Indicators introduced by vital staining or by the staining of crushed cells

Animal References 1. Sea urchin (P. lividru) Vlts et 01. (1924%b, c) 2. sea urchin (P.lividus) vellinger (1926,1927) 3. Sea ur&in (A. O~&WW- Vellinger (1926,1927) Crcloto) 4. Fundulus

Bodine (1927)

PH 4

5

I

I

0 culata)

6. Sea urchin (P. lividws) 7. sea urchin (P. lividus) 8. Seam& (E.cordaturn) 9. Subellark alveolata

Reiss (192% 1W) Reiss (1924% 1925) FaurC-Fremiet(1923) (initial value) Reiss (1%) Needham and Needham (1926) Reiss (1928) Rapkine and Wurmser (19%)

Needham and Needham (1926) Needham and Needbarn (1926) Needham and Needham (1926) FIG.9. MARINEEGGS

5 6 7

a

0 0

0 0

0

0 0 0 0 0

€3 0

0

Needham and Needham (1926) Pandit and Chambers (1932) chambers (1932) Chambers and Pollack (1927) Needham and Needham (1926) Rapkine and Wunnser (1926)

4

0

CY-1

14. Sea urchin (E.cordofurn) 15. Sea urchin (A. puncttllato) 16. Fzcnddus 17. Starfish (A. fwbesii) 18. Starfish (A. glocialis) 19. Starfish (A. rrrbm, ov+ eytes) 20. Ophiura laccrtosa 21. Ascidia menMa 22. Sabelluria alveobta

0

0 0 0 0

Vl&S(1924)

10. Sabellaria dwokatn Microinjection of in- 11. Seaurchin (P.lividw) dicators 12. Sea urchin (P. lirvidus) 13. Sea urchin (P.lividus, ovo-

1 2 3

a

0

5. Sea urchin (A. ~ q u i t u b ~ -Vl2s and Vellinger (1928)

8

7

6

0 0

0 0 I

I

I

9 10 11 12 13 14 15 16 17 18 19

20 21 22 I

1

Method pH of samples of sap measured with Pt/H, electrode

pH of samples of sap measured with indicators

pH of samples of sap measured with glass electrode

pH of sap measured with intracellular Pt/H, electrode

-

Species 1. Nitello (clumtu)

References Hoagland and Davis (1x3) PH

2. Nitetlu

Pearsall and Ewing (1924) 3. Nitelka (clavutu) Hoagland and Davis (1x3) 4. Nitello Irwin (1923) 5. Vubnio (nuccrophysu) Osterhout (1925) 6. VdorpiCr (macroplrjsa) Crozier (1919) 7. vd~(yenhicosa) Brooks (1930) 8. Hdicystit Blinks and Jaques (1930) 9. H&ystis (ovalis) Hollenberg (1932) 10. Valonio (wentricosu) Brooks (1930)

5

6

7

I

I

1

0

Taylor and mitaker (1927)

FIG.10. LARGEPLANT CELLS(CELLSAP)

1

1 2 3 4

JE

6

a

0 0

11. Nitello

a

10 11

g

Method Measurement of the p H of expressed cell sap or of extracts

References

Plunt 1. Beaqleaf 2. Bean,seed 3. Bryophylluon, leaf 4. Sunflower,leaf 5. Sunflower, stem 6. Sunflower,seed 7. Potato 8. Potato

9. Potato 10. Carrot 11. carrot 12. Beet 13. Beet 14. Beet

PH

Gustafsm (1924) 3.5 4 I # Nemec (Im) Gustafson (1925) Gustafson (1924) * Gustafson (1924) Nemec (1925) Pearsalland Ewing (1924) Stolclasa (1924) Wagner (1916) Pearsall and Ewing (1924) Rohde (1917) stdrlasa (1924) Harvey (1920) Fife and Frampton (1935) I

FIG.

Ila

PLANTS

'

5

6

7

I

I

I

*

0

i

1

2 t

0 0

6 7 8 9

F %

10

0

11.

0 0 I

3P

4

0

a0

5

3

e

0

1

7.5

I

12 13 14 1

i2

s cd

X

N

cn

-9.

Method Measurement of the pH of expressed cell sap or of extracts Indicators introduced by the staining of sections or of crushed tissues

Microinjection of indicators Not stated

Plan# 15. Corn,leaf 16. Corn, stem 17. Zeunrais, root 18. RiCirwM communis

References Gustafson (1924) Gustafson (1924) Stoklasa (1924) Harvey (1920)

19. Bean

Rea and Small (1927)

20. Sunflower 21. Potato 22. Carrot 23. Wheat,leaf 24. Wheat,root 25. RicinrUcmm~is Limnobiurn, root hair cells 26. Cytoplasm 27. Vacuole 28. Bryophyllnm, leaf

Martin (19n)

3.5

4

5

1

1

I

P”

6

7

I

I

.7.5 ‘i

Small (1929) Rohde (1917) Atkins (1922c) Atkins (1922c) Atkins (1922c)

Chambersand Kerr (1932) Chambers and Kerr (1932) Pucher et al. (1949)

The above list is by no means exhaustive. Small (1954, p. 62) states that the usual pH range for plants is 4.0-62 and that in a study of over 229 species of angiosperms, no remarkable exceptions have been found. Most of the pH values given in the Figure probably lie somewhere between the pH values of the cytoplasm and vacuole. FIG.llb. PLANTS

Method Indicators introduced 1. 2. 3. 4.

by vital staining

5. 6.

Species Marine IinULt. amoebae Ectoplasm, active Eetoplasm, resting Endoplasm, active Endoplasm, resting Pseudopodia, active Pseudopodia,moment of re-

References

traction

Microinjection of indicators

7. Amoeba pr0teu.s

8. Amoeba profeus 9. Amoebadubia 10. Amoebudubiu 11. Amoebudubia

PH

Pantin (1923) Pantin (1923) Paatin (1923) Pantin (1923) Pantin (1923) Pantin (1923)

Needham and Needham (1925) (no changes seen in pseudopodia) Chambers et nJ. (1927) Chambers ef nl. (1927) Chambers (1928) Pollack (1928)

FIG.12. AMOEBAE

5 t

7

6 I

8 1

I

0

1

0

2 0 3 0

0

4

5

0

0 0 0 0

6 0

7

a 9

10 11

F

i? ld (d

X

Method Measurement of the pH of an extract or of cells subjected to freezing and thawing Indicators introduced

References 1. Brandt (1945) (anaerobic cells) 2. Conway and O’Malley (1946) 3. Conway and Downey (1950) 4. Gutstein (1932)

by vital staining Microinjection of indicators CO, measurements

5. Mahdihassan (1930)

6. Conway and O’Malley (1946)

ments

7. Conway and Downey (1950) 8. Conway and Downey (1950)

Glyceric add measure-

9. Conway and Downey (1950)

Acetic acid measure-

ments (outer region of

the yeast cell)

FIG.13. YEAST

PH

7

6

5 I

I

I

0 0

0 0 0 0 0 0 0

8 t

1

2 3 4 5 6 7

a 9

INTRACELLULAX

PH

257

PH 5

7

6

8

-1

0 0

2

3 0 0

I

Method Indicators introduced by vital staining

I

I

Species Staphylococci Staphylococci Streptococci B. coli 5. B. friedliinder 1. 2. 3. 4.

4 5 J

References B a h t (1924) Gutstein (6932) Gutstein (1932) Gutstein (1932) Gutstein (1932, similar values were obtained for a number of other species)

FIG.14. BACTERIA OTHER ORGANISMS,

TISSUES, ETC.

Armstrong (1929) obtained values between 5.5 and 6.9 for the p H of the expressed sap of various fungi. Indicator studies on the tissues of these fungi gave pH values between 4.8 and 6.8. Atkins (1922a, b ) obtained values in the range 6.2-7.5 for the pH of certain coelenterates and marine algae, from studies involving vital staining with indicators. Damboviceanu and Rapkine (1925) found, from studies involving the microinjection of indicators, that the pH of the urns and enigmatic vesicles in the cavity fluid of SifilmclJW fiudw was normally about 7.4 while that of the plasma was 7.6. The pH of the secretion of the urns was about 6.0. They found a decrease in the pH of the urns, the enigmatic vesicles and the secretion of the u r n s in imunisation experiments. Rapkine and Damboviceanu (1925), in experiments involving the microinjection of indicators into certain elements of the blood and tunic of Ascidio mentula, found that the pH of the adipophorus cells and the vesicular elements of the blood and of the vacuolar elements of the tunic is less than 2.8, while that of the plasma is 7.0-7.4. These cells and elements therefore probably contain the sulfuric a d d which is liberated when the blood of certain Ascidians is plasmolyzed.

out that calculation of the intracellular pH of rabbit abdominal muscle from Conway and Fearon’s data gives a value nearer to the usual range of values, namely 6.75, even after allowance has been made for carbon dioxide from sources other than bicarbonate. Furthermore the results which have been obtained with the carbon dioxide method for other types of muscle and for other cells and tissues usually show reasonable agreement with

258

PETER C. CALDWELL

those obtained with other methods. Nevertheless, the occurrence of carbon dioxide-forming compounds other than bicarbonate introduces an uncertainty into this kind of p H determination and a further investigation of these compounds and of the errors introduced by them seems desirable. The agreement, which is found in certain instances between the results obtained with the different methods, is particularly striking in the case of Maia muscle. Here the p H values obtained by measurement of the p H of a muscle brei (Furusawa and Kerridge, 1927b), by carbon dioxide measurements (Cowan, 1933) and by the use of an intracellular glass electrode (Caldwell, 1956), are very similar. In the case of yeast, the intracellular p H values from different methods also show a reasonable agreement as do the values for nerve, gastric mucosa, and human erythrocytes. A direct comparison of the p H values, obtained for individual muscle fibers of Carcinus meanas with an intracellular glass electrode, with that of brei prepared subsequently from the same fibers has been made by Caldwell (1956) and here tcx, agreement was found. These comparisons suggest that measurements of the pH of a tissue brei or of the carbon dioxide content of the tissue can often give quite reliable values for the intracellular pH and that these methods can therefore be used with reasonable confidence for cells too small for measurements with intracellular glass electrodes. 2. Changes of Intracellular

PH

d u ~ n gActivity or after Alterations in the

External Conditions In this section some of the more important intracellular pH changes which have been observed during the activity of cells and tissues will be mentioned, together with some of the effects of alterations in the environment. A tissue whose intracellular pH during activity has been widely investigated is muscle. A number of workers (including Pechstein, 1915; Schade et d., 1921 ; Ritchie, 1922; Meyerhof and Lohmann, 1926; Furusawa and Kerridge, 1927a; Fenn, 1928) have reported an acidification in fatigued frog and mammalian muscle. This acidification is more pronounced when the muscle has gone into rigor. The changes in frog muscle in the earlier stages of activity have been studied by Lipmann and Meyerhof (1930); Meyerhof st al. (1932),and Hill (1940) who followed the uptake and release of carbon dioxide; by Margaria and Pulcher (1934), who followed changes in the color of indicators ; and by Dubuisson ( 1937, 1939, 1950) who followed the pH changes at the surface of frog muscle with glass electrodes. Changes in mammalian muscle have been followed with glass electrodes by Maison et al. (1938).The results indicate that in

INTRACELLULAR

pH

259

the earlier stages of activity there is an alkalization of the muscle, which probably arises from the breakdown of creatine phosphate, followed by an acidification which probably arises from the formation of lactic acid, Dubuisson (1950) also found an initial alkalization followed by an acidification which he suggested might represent changes in the muscle protein followed by the breakdown of adenosine triphosphate. Margaria (1934) and Dubuisson (1950) have also reported an alkalization when muscles are stretched. On the other hand Caldwell (1955, 1956) working with intracellular glass electrodes, was unable to detect any changes greater than 0.1 of a pH unit in crab muscle fibers during contractures initiated with 0.6 M KC1. The pH changes detected by Dubuisson and the probable pH changes corresponding to the carbon dioxide changes (Hill, 1955) are however of the order of 0.1 of a pH unit. The failure to detect any significant intracellular pH changes therefore was probably due to the insensitivity of the method, with which it was not possible to detect changes of less than 0.1 of a pH unit on account of interference from changes in the membrane potential. Another intracellular pH change which has been studied by a number of authors is that of cells which secrete either hydrogen or hydroxyl ions. An idea originally due to Briicke (1859) namely that the secretion of hydrogen ions might involve an alkalization of the secreting cell while the secretion of hydroxyl ions would involve an acidification, is discussed by Carnot et a2. (1925) and Reiss (1926). Such changes appear to occur in gastric mucosa, yeast, and pancreas. The gastric mucosa of dogs and rabbits was found by Carnot et a2. (1925) to become more alkaline during digestion. Bradford and Davies (1950) have investigated the secretion of a number of dyes, some of which were pH indicators, in isolated amphibian gastric mucosa and with pieces of mammalian gastric mucosa. They have concluded that the site of hydrochloric acid secretion is the wall of the canaliculi of the oxyntic cells. In a secreting mucosa the pH of the intracellular canaliculi, the gastric crypts, and the gastric tubules was found to be less than 1.4 while the cytoplasm of the oxyntic cells and the lower regions of the mucosa were slightly alkaline. The alkali formed during secretion, which is equivalent to the acid produced, is neutralized by carbon dioxide to form bicarbonate which passes into the blood or nutrient solution surrounding the mucosa (Davies, 1948). Davies (1948) and Crane et d. (1948) have suggested that the primary process in the secretion involves the splitting of water into hydrogen and hydroxyl ions at the expense of metabolic energy. The hydrogen ions are secreted while the hydroxyl ions form bicarbonate by interaction with carbon dioxide in the presence of

260

PETER C. CALDWELL

carbonic anhydrase. The secretion of hydrogen ions by yeast has been investigated by Conway et aZ. (1950) who have shown that during secretion the intracellular pH rises. Here also the alkali formed appears to be equivalent to the hydrogen ion secreted. The secretion of pancreatic juice, which is alkaline, has been studied by Carnot et al. (1925), who state that the pancreas becomes more acid during secretion. The intracellular pH of erythrocytes varies very slightly as the blood circulates, being lower in the venous blood than in the arterial blood, in response to the decreased pH and increased carbon dioxide tension of the serum of the former (Van Slyke et al., 1923; Henderson et al., 1924). Henderson et al. (1%4), and Henderson (1928) give nomograms from which the intracellular pH of the erythrocytes, which is normally less than that of the serum, can be calculated under various conditions. Henderson (1928) also gives figures for cases of nephritis and diabetic coma which show that the intracellular pH of the erythrocytes responds to the decreased pH of the serum, both values being up to 0.4 of a pH unit lower than usual. A similar change was found by Rapoport and Guest (1939) in dogs during pyloric obstruction. In many cells and tissues the intracellular pH usually appears to be affected only slowly, if at all, by quite large changes in the pH of their surroundings (Hoagland and Davis, 1923 ; Pantin, 1923 ; Schmidtmann, 1924; Reiss, 1926; Fern and Cobb, 1934; Wallace and Lowry, 1942; Amnitaki and Chalazonitis, 1954 ; Hill, 1955 ; Caldwell, 1955, 1956). Changes in intracellular pH can, however, be brought a b u t by particular acidic and basic substances, notably carbon dioxide (Jacobs, 1920b ; Smith, 1923 ; Fenn, 1928 ; Stella, 1929; Spek and Chambers, 1933 ; Cowan, 1933 ; Amnitaki and Chalazonitis, 1954 ; Caldwell, 1955, 1956) which probably permeates into the cell in an un-ionized form. In the presence of one atmosphere of carbon dioxide the intracellular pH of muscle and nerve is decreased by about one p H unit. Carbon dioxide enters cells very rapidly as is shown by pH measurements (Spek and Chambers, 1933; Arvanitaki and Chalazonitis, 1954; Caldwell, 1955, 1956), by measurements of carbon dioxide uptake (Fenn, 1928) and by heat production measurements (Hill, 1928; Stella, 1929). In the case of Sepia nerves, Arvanitaki and Chalazonitis (1954) have shown that the penetration of arbon dioxide is accompanied by a large but reversible decrease in the size of the action potential. That ammonia can penetrate into cells and increase the internal pH is shown by the effects of extracellular ammonium chloride on the pH of erythrocytes (Netter, 1929), on the pH of

INTRACELLULAR p H

261

the sap of Nitella (Irwin, 192S), Vulonia (Cooper and Osterhout, 1930), and Hulicystis (Blinks, 1933), and on the p H of rhododendron flowers and starfish eggs (Jacobs, 1922). Various authors have attempted to follow pH changes in various marine and amphibian eggs during development. Some of the work on the subject is discussed by Needham (1931,1942) and by Brachet (1947). Needham and Needham (1926)and Chambers and Pollack (1927)did not find any changes in starfish eggs and Bodine (1927)did not find any in FunduZus eggs. Reiss (1925)reported small changes in sea urchin eggs but Needham and Needham (1926)did not find any changes. Changes in amphibian and hens’ eggs have been reported by Buytendijk and Woerdeman (1927) and by Dorfman (quoted by Brachet, 1947). There is some evidence that hydrogen ion concentration gradients may occur during development, in particular that the animal pole is at a higher pH than the vegetal pole (Spek, 1930, 1933, 1934;Dorfman, 1936a). Differences in pH have been invoked by Willstatter and Mallison (1915)to account for the fact that the petals of different species of flower can be given widely different colors by the same or similar anthocyanin pigments. This view is supported to a certain extent by the work of Bwrton and Darbishire (1929) who found some correlation between the p H of petal extracts and the natural color of the anthocyanin. The color of anthocyanins is, however, modified not only by pH but also by the action of substances known as copigments. It is conceivable that some of these are polyelectrolytes and that they act on the anthocyanins by surface pH effects in the same way as proteins and sulfate esters act on indicator dyes to produce protein and metachromatic errors. Other types of interaction are probably involved, however, since Robinson and Robinson (1931) found that a variety of organic substances, which were probably un-ionized under the acid conditions used, exert a copigment-like action on anthocyanins. The diurhal variations of p H in certain plant tissues have been investigated by various authors. Gustafson ( lSZS), and Pucher et ul. ( 1949) have studied the changes in leaves of the succulent plant Bryophyllum calycinum and have found the p H to rise during the day and fall at night. Pucher et ul. showed that the changes are correlated mainly with alterations in the amounts of malic acid and citric acid in the leaves, the main changes being in the malic acid. Isocitric acid on the other hand showed little or no variation. Diurnal variations in the alga Spirogyru have been studied by Rohde (1917) and in this case the pH is greater at night than during the day.

262

1. Obsewed Y a k s of

PETER C. CALDWELL

V. DISCUSSION the Intracellwlar PH and the Donnan Theory

The results given in the previous section show that while for amoebae and certain vertebrate tissues, in particular muscle, nerve, liver, and the cells of gastric mucosa, most of the pH values which have been obtained lie in the region 6.5-7.5, for yeast and certain plant cells and tissues many of the pH d u e s lie below 6. In a number of cases the intracellular pH value is not that to be expected on the basis of the Donnan theory as the following discussion will show. The failure of the Donnan theory to account for the pH differences across gastric mucosa has already been mentioned. It seems likely that it also does not apply to muscle, nerve, and certain large plant cells. Mond and Netter (1930), Fenn and Cobb (1934), and Fenn and Maurer (1935) were among the first to suggest that Eq. (8) might not hold for frog muscle. The pH of the extracellular fluid in frog muscle is normally about 7.4 (Fenn and Maurer, 1935) and calculation of the intracellular pH, either from the intraceIlular and extracellular potassium concentrations and Eq. (8), or from the normal resting potential of about -W millivolts and Eq. ( 5 ) , shows that it should be below 6.0. Most of the experimental values which have been obtained, however, show that it is in the region of 7.0. Similar calculations for frog nerve also give intracellular pH values below 6.0 in contrast to the observed values which are in the region of 7.0. In the case of crab muscle the pH of the hemolymph and also of the usual bathing saline, is about T.5. For relatively undamaged fibers, whose resting potential is in the region of -50 to -65 millivolts, Eq. ( 5 ) predicts an intracellular pH of 6.7-6.4 in contrast to the observed values which are between 6.91 and 7.15 (Caldwell, 1954b, 1956). For squid nerve the state of affairs is less clear cut (Caldwell, 1956). For relatively undamaged regions of the axons, where the resting potentials lie between -50 and -60 millivolts, Eq. ( 5 ) predicts an intracellrtlar pH 0.85-1.0 lower than that of the surroundings. For axons immersed in sea water of the usual pH of about 8.1 this appears to be so, the calculated intracellular pH being 7.1-7.25. It is nut so, however, for axons in sea water whose pH has been lowered to about 7.5 since the calculated intracellular pH is 6.5-6.65 compared with the observed value of about 7.0. This second observation suggests that Eq. ( 5 ) may not hold for axons in the intact animal since it is likely that in this case also their surroundings are at a pH appreciably lower than 8.1 (Caldwell, 1956). Cells of Valonk and Halicystis immersed in sea water are found to have potential differences between the cell sap and the surroundings of about +lo millivolts (Osterhout et al., 1927; Blinks, 1930) and -60 to -80

INTRACELLULAB

pH

263

millivolts (Blinks, 1929, 1932) respectively. O n the basis of Eq. ( 5 ) the p H of the sap should be 8.27 for Valonicc and 6.7-7.1 for Halicystis if the sea water p H is 8.1. These calculated values are in rather sharp contrast to the experimental values of 5.8-6.3 for Valonk and 5.46.2 for Halicystis. The failure of the Donnan theory to account satisfactorily for the p H differences across cell membranes becomes more apparent when the extracellular pH, the intracellular pH, or the resting potential is varied. As has already been mentioned, there are many cases in which there is no change or only a slight change of the intracellular pH even when the extracellular p H is varied between quite wide limits. In most of these cases the Donnan relations do not appear to hold for hydrogen ions. The decrease produced by carbon dioxide in the intracellular p H of crab muscle and squid nerve has been studied (Caldwell, 1956) and it has been found that after an initial rapid fall to a value in the region of 6.0, the pH then remains constant over a period of hours, even though the value to be expected from the resting potential and the extracellular p H on the basis of Eq. ( 5 ) is considerably lower. The most likely interpretation of these results is that the membrane behaves as though it were relatively impermeable to bicarbonate ions but very permeable to carbon dioxide or undissociated carbonic acid. The intracellular p H in this case, therefore, instead of being governed by the Donnan equilibrium, seems to depend on the carbon dioxide tension to which the tissue is exposed, and on the bicarbonate content and buffering power of the cells. The effects of ammonia and ammonium ions on the intracellular pH and the potential between the cell sap and the surroundings have been studied in Halicystis osterlzoutii by Blinks (1933). Increases in the amount of ammonium chloride in the sea water with which the cells are surrounded lead to a corresponding increase in the pH of the sap. The potential between the sap and the surroundings is not however related to the difference in p H in the manner required by Eq. ( 5 ) , since initially it is not affected by the increasing intracellular p H and then it rapidly reverses in sign from about -68 millivolts to 30 or 40 millivolts when the external ammonium concentration reaches a critical value. The pH difference across the protoplasm of Halicystis and Valonia has been removed in experiments in which either the cells are immersed in a sample of the cell sap or in which their cell sap is replaced by sea water (Osterhout et al., 1927; Blinks 1929,1935). In no case is the potential difference across the protoplasm abolished as required by Eq. ( 5 ) . In certain cases there are practically no changes in the potential, while in others there are, the sign of the potential being reversed in one instance.

+

+

264

PETER C. CALDWELL

The intraceliular p H of muscle and nerve seems to be unaffected by depolarization. Thus in crab muscle and squid nerve exposed to a constant extracellular pH there appear to be only slow and often very minor changes in intracellular pH when the cells are depolarized either by injury or by treatment with potassium chloride (Caldwell, 1955, 1956). Equation (5) predicts that complete depolarization of the cells should result in the intracellular p H changing to that of the surroundings, but this occurs only slowly, if at all. The suggestion of Caldwell and Harris (1952) that the effects on metabolism often associated with depolarization may be due to changes in intracellular p H brought about by the operation of the Donnan equilibrium is therefore unlikely to be correct. In spite of the fact that there appear to be many cases where the p H differences across cell membranes cannot be adequately dealt with by the Donnan theory, there are some cases where the theory seems to be applicable, The best known of these is that of erythrocytes, where the hydrogen ions, bicarbonate ions, and chloride ions are distributed between the cells and the surroundings according to the equation (HCO~~-intraee~~u~ar)(a-intraceu&r)

-

- (H+extraceuubr)

(12)

(HCO~-ertrace~~nlar) (a-extraceu&r) (H+intraeeuur) (Warburg, 1922; Van Slyke, et csl, 1923 ; Henderson eb al, 1924; Henderson, 1928;Rapaport and Guest, 1939;Harris and Maizels, 1952). Also, it has been shown by Netter (1929)that

(H+intraeeuar) (NH4+intrace~ular) Equation (8) on the other hand does not appear to apply. It is not possible to decide whether Eq. ( 5 ) applies to erythrocytes since their resting potential is not known. The Donnan theory may apply to the pH differences across some of the cell membranes in frog skin, since Meyer and Bernfeld (1946)have shown that the potential difference across the skin is sensitive to the p H of the solution bathing the inner surface. When the pH of the solution in contact with the inner surface was varied, the potential varied with the p H in the manner required by Eq. ( 5 ) . The author, in some unpublished experiments in which unbuffered Ringer's solution, initially of the same pH, was placed on the two sides of frog skin, found that after a time a p H difference was set up which was similar to that calculated from the skin potential and Eq. (5). A similar state of affairs may exist for cells in the roots of wheat plants since if the roots are bathed in solutions of different pH, the

INTRACELLULAR

pH

265

potential difference between the root and the solution varies (Lundegirdh, 1938). The variations are of the right sign but are not, however, as large as those predicted by Eq. ( 5 ) .

2. Some Effects of Heterogeneity within the Cell on the Donnan Relations and on Certain Types of PH Determination The most likely explanation of the failure of the Donnan theory to account for the values of the hydrogen activities in cells in the cases just considered is that there is some form of active transport across the cell membranes. This active transport could operate either directly on the hydrogen ions themselves, or on one of the ions to which they are linked, such as bicarbonate. Another possible explanation, which was originally mentioned by Fenn and Maurer (1935) in connection with frog muscle and which may be valid in some instances, is that the failure is the result of a lack of homogeneity of the cell interior. The Donnan relationships hold rigidly only between phases which are homogeneous. If the cell interior is not homogeneous the applicability of the relationships to the cells and their surroundings is limited. Similarly, the law of mass action holds only for a homogeneous phase, and the applicability of pH determinations, such as indicator and carbon dioxide measurements, which depend on it is also limited if the cell interior is not homogeneous. These limitations will now be considered in some detail. The heterogeneity in the cell could be due either to the presence of discrete structures such as nuclei, mitochondria, or myofibrils, or it could arise from the presence of charged molecules such as proteins, between whose surfaces and the bulk aqueous phase of the cell, according to Danielli (1937, 1941), Donnan equilibria can exist. In a cell which is not homogeneous, ions which are free to permeate to all parts of the cell and which are not subject to active transport between the different parts, will be distributed between the different regions according to the Donnan theory. Donnan relationships of the type given in Eq. (2) will not hold for the mean intracellular activities of such ions and their activities in the extracellular fluid, but, as will now be shown Donnan relationships of the type given in Eq. (1) will apply to the mean intracellular activities and the extracellular activities, though to a limited extent. For the present purpose, only the limitations applying to monovalent ions will be dealt with. Suppose that the cell consists of regions of volume VI,VZ,. . . V,,, etc., between which Donnan equilibria exist, then the mean intracellular activity -. (afintraeeUular)of any cation a+ can be defined as being

- ss+v* 8 v*

266

PETER C. CALDWELL

the mean intracellularactivity (5+bheuar) of any cation b+ as being

and the mean intracellular activity ( z - b t a a a ) of any anion U- as being

-

&-Vn

E vn the mean intracellularactivity ( p - h h w n d r ) of any anion /3- as being

- EPn-v*

etc.

E Vn where a,,+, bn+, a,,-, region.

pn-, etc., are the activities of

the ions in the nth

Since for monovalent cations (from Eq. 1) &+

G+Vn

Therefore from (14) and (16)

= =

( a+ertracenuIIu)

be+, (b+8Xt?tddJ (a+ertrsceudr)

bn+ Vn (b +extcaceuulu)

INTRACELLULAR

PH

267

From (14) and (19)

-

(a+ intncennbr)

and since

(a+extraceunlar)

and

-

- ration #

(a-intracellar)

-- ranion

(20)

(a-extroceunlar)

(a+introceuliLr)

Equations (17), (18), and (20) show therefore that in cells whose interior is heterogenous the Donnan relationships can hold between the mean intracellular activities and the extracellular activities of monovalent ions of the same sign. The relationships will not hold, however, between the activities of ions of different sign. Therefore, for a cell whose interior is heterogeneous (K+extmceu*r)

-

(K+intnceuar)

-

(H+extna-r) (R+intraceuabr)

-

(NHlfextmcellulizr) (m4+intnceuUr)

- rmtion (21)

268

PETER C. CALDWELL

and

(HCO8-htnceUukr)

-

-

(OH-lntmcallalrr)

(OH-extmesunlar)

(HC08-eIhC0Uuk)

-

(a-htrwellular)

- ?anion

(C1-extraeeuuhr)

(22) but

Eation

# ;;nnion

and therefore (K+extnrceuhr)

#

(HCOa-iatrrceuular)

-

(a-intraceU*r)

(23) (Cl-extrace~ular) (It should be noted that the mean intracellular hydrogen and hydroxyl (K'htmeen-r)

(HCO8-extnce~n~ar)

-

and ( O H - h h d a r ) , in Eqs. (21), (22), and ion activities, ( H + h h = U * r ) (25) are not to be identified with the hydrogen and hydroxyl ion activities of a brei of the cells, since the latter are determined by the relative buffering capacities of the different cell regions as well as by the pH of these regions.) These considerations show that in cells in which Donnan equilibria exist between different intracellular regions, the monovalent cation ratio and the inverse of the monovalent anion ratio are not equal but can have widely different values. Such an inequality can exist in the complete absence of permeability barriers and active transport. Equation (23) shows that if the fibers are heterogeneous the different potassium ion and bicarbonate ion ratios found in muscle need not conflict with the Donnan theory. The fact that the ammonium ion ratio is not equal to the potassium ion ratio as required by Eq. (21) but is similar to the inverse of the bicarbonate ratio (Fenn et al., 1944) suggests however that heterogeneity of the muscle fiber does not provide an adequate explanation in this case. Equation (23) also shows that if red cells were heterogeneous, both the potassium ions and the chloride and bicarbonate ions in them could be in a Donnan equilibrium with the serum even though the potassium ratio and the inverse of the chloride and bicarbonate ratios are very different. Effects of the type under discussion probably occur in polyelectrolyte gels, Thus, Katchalsky and Michaeli (1955) have shown that the simple Donnan theory does not account for the uptake by polymethacrylic acid gels of lithium, sodium, potassium, and chloride from solutions of lithium, sodium, and potassium chlorides. They obtain an adequate theory, however, if activity factors calculated from the contractile free energy of the gel network and from the electrostatic free energy which arises from the interactions of the ions and the fured charges in the gel network are introduced. The second effect, which is the most important, might be expected on the basis of Danielli's (1937) hypothesis to give rise to Donnan

INTRACELLULAR

269

pH

equilibria between the vicinity of the fixed charges attached to the network and the bulk aqueous phase formed by the imbibed water. A consequence of heterogeneities within the cell which give rise to variations in pH between different regions is, as was mentioned earlier, that the applicability in certain types of pH measurement of the mass action formulae for the dissociation of acids and bases is curtailed. The variations in p H could be due either to the existence of intracellular Donnan equilibria of the type just discussed, but they could also be due to permeability barriers or to the local production of acid. Consider a weak acid, the concentration (HA) of the undissociated form of which is the same in all regions. The amount (An-) of the corresponding anion in the nth region will be given by

where

and (Hnf), (OHn-) are the H+ and OH- activities in the nth region. From (15) and (24) (hltraeellular)

= &(HA)

x&) -SVn

- K, (HA) E(OHn-)Vn K,

K, -(HA) (FH-intncenar) KO

SV,

(25

where (A-h-cellar) and ( T H - i n t m ~ ~ ~are r ) the mean intracellular activities of A- and OH-. Equation (25) shows that the mass action equation

KO

(A-) = -(HA) (OH-)

Kc0

can be used to calculate the mean intracellular OH- activity from the mean intracellular activity of A-, but the mass action equation

(A-) =

KO (HA)

(H+>

270

PETER C. CALDWELL

cannot be used to calculate the mean intracellular hydrogen ion activity since

v*

1 (~+iutraceunlar)

#

c,,sv, .

SimilarIy it can be shown that for a weak base BOH, the mass action

Ka

equation (B+)= -(BOH)(H+) can be used to calculate the mean

JGo

hydrogen ion activity from the mean activity of B+, but the mean OHactivity cannot be calculated from the equation

(B+)

(BOW

(OH- 1 This shows that in cells in which differences of p H occur between different regions measurements of pH based on observations of mean values of the extent of dissociation of a weak base do in fact give a p H value which corresponds to the mean hydrogen ion activity. Measurements based on the dissociation of a weak acid do not however give a p H value which corresponds to the mean hydrogen ion activity. They can, however, be used to obtain a value of pOH which corresponds to the mean OH- activity of the cells. Therefore if the pH of the cell interior is determined by measurements of the dissociation of a base such as ammonia or a p H indicator of the type BOH, the value obtained corresponds to the mean hydrogen ion activity in the cells. If on the other hand it is obtained from measurements of the dissociation of an acid such as carbonic acid or a p H indicator of the type HA, the pH value obtained will represent not the mean hydrogen ion activity but the pH which would be found if the cell interior contained the number of hydroxyl ions that it does, but was homogeneous. Hence, measurements of carbonic acid and bicarbonate can only give information about pH for cells whose interiors are of uniform pH. These considerations also show that in the presence of molecules such as proteins, at whose surface the p H is different from that in the bulk phase, p H measurements based on bicarbonate and ammonia determinations should be subject to the same anomalies as measurements with indicators. They emphasize therefore the desirability of carrying out intracellular pH determinations by a number .of methods. If the values obtained from bicarbonate, ammonia, and indicator measurements are in

INTRACELLULAR PH

271

agreement it is probably safe to conclude that the cell interior is reasonably uniform and that the value represents the actual hydrogen ion activity. Most of the values obtained for muscle by the different methods, in particular for frog and crab muscle are, as Fig. 1 shows, in reasonable agreement. It is unlikely therefore that in this case the failure of the Donnan relationships can be ascribed to heterogeneity of the cell interior and some form of active transport therefore seems likely. A similar conclusion can be reached in the case of the large plant cells Valonia, Nitella, and Hdicystis and also probably in nerve. There may however be other cases in which Donnan equilibria between different intracellular regions produce appreciable effects.

3. Concluding Remarks The main conclusion which can be drawn from the work presented in this review is that there are many cells in which the simple Donnan relationships do not hold when applied to the hydrogen ions. In some instances this may be due to permeability barriers or to heterogeneity in the cells. In cases where the intracellular pH fails to respond in the manner required by the Donnan theory to changes in the extracellular pH, the failure is probably due primarily to the relatively small numbers of hydrogen ions and bicarbonate ions passing across the membrane, the small number of hydrogen ions being due to their small intracellular and extracellular concentrations and the small number of bicarbonate ions to permeability barriers. It seems likely, however, that in many cases an active transport takes place across the cell membrane either of hydrogen ions or of ions such as bicarbonate to which they are linked. It is perhaps not surprising that the Donnan theory has such a limited applicability since it deals with systems which have reached a state of thermodynamic equilibrium,whereas one of the most important features of living cells is that they consist not of an equilibrium but a steady state. The Donnan theory and its modifications can only be expected to apply rigidly for dead cells in which the processes responsible for the maintenance of nonequilibrium ionic gradients are no longer active. One of the more interesting features of the intracellular p H is its sensitivity to the external carbon dioxide tension. This tension is probably one of the main factors which determines the intracellular pH of cells and tissues. The ease with which carbon dioxide, in contrast to many other acidic substances can penetrate into tissues and alter their intracellular pH is no doubt partly responsible for many of its effects. Among these effects are those on mitosis in amoebae (Voegtlin and Chalkley, 1935), on the division of marine eggs (Smith and Clowes, 1924), on the cells of the

272

PETER C. CALDWELL

respiratory center (Haldane and Priestley, 1905 ;Jacobs, 192Oa, b) on the carbohydrate changes in and the formation of lactic acid by muscle (Kerly and Ronzoni, 1933), on the energy and heat production of muscle (Hill, 1955) and on the action potential of nerve (Arvanitaki and Chalazonitis, 1954). It is also possible that the failure of certain bacteria to grow in the complete absence of carbon dioxide (Gladstone et id., 1935; Dagley and Hinshelwood, 1938; Kempner and Schlayer, 1942) may be due to a loss of carbon dioxide from the cells as a result of which they become too aIkaline. At present it seems likely that the further development of the study of intracellular pH is likely to be slow. The micro forms of the glass electrode which have been evolved so far can only be used for very large cells and there are major technical difficulties in making them any smaller. The extension and refinement of the other methods of measurement also present difficulties on account of the various errors to which they are subject. In the case of indicator measurements the application of the observations and theories of Hartley (1934), Hartley and Roe (1940) and of Danielli (1937, 1941), which have been mentioned in this review, may lead to information about the pH at surfaces and in the cell fluid. In particular] studies with indicators of different charge types and sign may lead to interesting results on this point. Here too there are difficulties] however, since both the experiments of Robinson and Robinson (1931), in which substances which were not polyvalent electrolytes modified the colors of anthocyanins, and the conclusion of Hartley (1934) that in certain cases specific factors at surfaces can affect the pH response of indicators show that the theory of indicator errors is by no means complete. Finally mention must be made of the extent to which the concept of pH is valid in dealing with the very small volumes which comprise the different regions of a cell. One cubic micron of a solution of pH 7.0 contains, as Netter (1934) has pointed out, about 69 hydrogen ions. In volumes of this order, therefore, pH ceases to have its usual meaning of the hydrogen activity at any given instant, but becomes the average hydrogen activity over a long period. VI.

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The Activity of Enzymes in Metabolism and Transport in the Red Cell

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T A. J PRANKERD MeaYcal Unit. University College Hospital Medical School. London Page I. Introduction ......................................................... 279 I1 Enzymes in Metabolism ............................................... 280 1 Metabolic Substrates ............................................ 280 a. Sugars ...................................................... 280 b Nucleosides .................................................. 283 c Other substrates ............................................. 283 2 Hexose Monophosphate Shunt ................................... 284 3 Phosphatases .................................................... 285 4 Acetylcholine-esterae (AChE) ................................... 285 5 Proteinases ..................................................... 286 6. Phosphopyridine Nucleotidase ................................... 286 7. Methemoglobin Reductase ....................................... 286 8 Glutathime ..................................................... 287 9. Stroma ......................................................... 287 10 Energy Utilization ............................................... 289 I11 Enzymes in Transport ............................................... 289 1 Mechanisms of Transport ....................................... 289 a. Active Transport ........................................... 289 b. Facilitated Diffusion ......................................... 290 c Passive Diffusion ............................................ 290 2 Phosphate Transport ............................................ 290 3 Sugar Transport ................................................ 293 4 Glycerol Transport ............................................. 295 5 Cation Transport ............................................... 296 IV References ........................................................... 299

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INTRODUCTION

The last twenty years have seen a remarkable change in ideas about the activity of the mature red blood cell No longer is it regarded as an inert sac, but rather as an active metabolic unit It is the aim of this article to review some of these activities Plenty of evidence exists to show that the metabolism of the red cell is directly related to the cell’s viability in vitro and probably also in Vivo. and it is likely therefore that a more intimate knowledge of this subject will shed light upon the normal process of aging of a cell whose life span has known limitations. as well as upon the mechanisms by which the cell is destroyed in disease. In addition a better understanding of the conditions for optimal storage of transfusable human blood can be derived from a thorough knowledge of this subject

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The discussion is divided into two parts, the first covering those metabolic reactions known to occur in red cells, and the second those systems of transport by which various substances enter and leave the cell and which are thought to be under enzymic control. Such a division is artificial as in reality both processes must be closely integrated, but it reflects two entirely different experimental approaches. During the course of the ensuing discussion gaps in our knowledge will become evident because much of the data is scanty and disconnected. To draw attention to these is an important purpose of this article. Except when stated otherwise the remarks apply to the human red cell which has been most extensively studied. 11. ENZYMES IN METABOLISM Structurally the red cell is divisible into a membrane, or stroma, and a semifluid interior which constitutes the hemolyzate after rupture of the cell. This subdivision is purely theoretical from a metabolic standpoint for many chemical reactions must be continuous between the two phases. It might be more appropriate to consider the surface phase separately as Rothstein (1954) has done since some of the specialized activities concerned with transport take place on the cell surface, but this division raises its own difficulties as it is not known how deeply these surface activities extend. In this discussion the cell will be considered as a whole except where certain specialized reactions are involved.

1. Metabolic Substrates a. Sugars. For many years the red cell has been known to utilize glucose (Macleod, 1913), and Lundsgaard (1933) showed that its disappearance was quantitatively related to lactic acid production. This observation implied that glucose was metabolized anaerobically, a conclusion borne out by the negligible oxygen consumption of the mature red cell, and the failure to isolate enzymes capable of the oxidation of pyruvate. All the intermediary compounds of glycolysis have been isolated from this cell and chromatography has permitted accurate estimation of the quantities of many of the intermediates present. In addition to these compounds the enzymes necessary for their continued degradation have been demonstrated. The quantities of a number of intermediates given below are those found by Bartlett et al. (1953). The red cell contains no glycogen so that for its continued metabolism it must have constant access to glucose. It is not known whether free glucose exists inside the cell, but certain considerations which will be dealt with in Section I11 make it likely that in certain species it does, particularly because glucose penetration of permeable cells is more rapid

ENZYMES I N RED CELL METABOLISM AND TRANSPORT

28 1

than its metabolism. It is therefore probable that hexokinase phosphorylates glucose after the latter has gained access to the cell interior, and not on the cell surface as Gourlay (1952) has suggested. Most of the esters in Table 1 are present in the cell in small amounts only, but 2,3-diphosphoglycerate (2,3-DPG) is particularly abundant. This ester is a coenzyme in the conversion of 3-phosphoglycerate to 2phosphoglycerate (Sutherland et al., 1949) and is therefore present in most cells only in catalytic amounts. One may fairly assume that in the red cell it has an additional role. TABLE I PHOSPHATE

DERIVATIVES OF GLYCOLYSISIN

Derivative Glucose monophosphate Fructose monophosphate 3-Phosphoglycerate Fructose diphosphate 2,3-Diphospbglycerate Adenosine triphosphate Adenosine diphosphate Adenosine monophosphate

THE

RED CELL

Micromoles per 100 ml. whole blood

2.0 1.o 3.0 10.5 157.0 45.2 11.6 1.1

From experiments with Ci4-labeled glucose (Bartlett and Marlow, 1953) and the uptake of Pm (Prankerd and Altman, 1954) it appears that 2,3DPG has a high turnover of the labeled atom and must be taking an active part in glycolysis. Prankerd and Altman found that it had the highest specific activity of the esters they separated, and since the specific activity of the extracellular orthophosphate bore a precursor relationship to that of the cellular 2,3-DPGJ they concluded that 2,3-DPG must be in equilibrium with the lJ3-ester. Rapoport and Luebering (1950) have produced evidence supporting this equilibrium by isolating a mutase from rabbit cells which activates the conversion of 1,3- to 2,3-diphosphoglycerate. Whether this reaction is reversible, or whether 2,3-DPG is dephosphorylated to a monophosphate ester is not known, but in either instance 2,3-DPG is ideally situated in the glycolytic sequence to act as a store of phosphate which could be utilized for the phosphorylation of ADP. Thus, if the cell were deprived of glucose it would still be able to synthesize ATP for a limited time at the expense of its stores of 2,3-DPG. This function would be a real advantage to the red cell which is faced with the problem of hemoconcentration and diminished supplies of metabolite in its passage through the spleen. In relation to this role of 2,3DPG it is interesting that those species having cells with a small diameterthickness ratio, and therefore less likelihood of stagnating in tissues, have

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amounts of 2,3-DPG much reduced in comparison to their reduction in size. It is probabie that the equilibrium of the conversion of 1,3-DPG to 2,3-DPG is much in favour of the latter as the reversal of this reaction would be thermodynamically difficult. It will be pointed out later that the energy stored in the red cell is utilized for a number of purposes essential for the maintenance of the cell’s integrity. If the cell is poisoned by such substances as fluoride or iodoacetate it lyses after a relatively short time as a result of rising intracellular osmotic pressure (Ponder, 1948), and one may presume that if it were metabolically defective its life span in vivo would be shortened. Thus Prankerd et d. (1954, 1955) have accounted for the anemia of hereditary spherocytosis on the basis of a metabolic defect in these cells, and one which has also been found by Motulsky et d . (1955). It appears that other sugars besides glucose can be used by the red cell. For instance Maizels (1951) found that fructose and mannose led to acid production when incubated with red cells and that these sugars would also support cation movements, whereas this activity was less evident with lactose and maltose, and absent with xylose, arabinose, and sucrose. Ribose is also not utilized. Similar observations have also been made by Spicer and Clark (1949), but the cells of all species do not utilize glucose at the same rate and some investigators have attempted to link this with the intracellular potassium concentrations, potassium being a coenzyme in the dephosphorylation of phosphopyruvate. Kerr (1937) lists an analysis of many different cells with respect to their potassium concentrations and rates of glycolysis, but no correlation is evident. The optimal potassium concentration for the dephosphorylation of phosphopyruvate is 0.05-0.015 M in &tro (Solvonuk and Collier, 1955), and human cells contain ten times more potassium than this and about ten times more potassium than the red cells of dogs, yet their glucose consumption is much the same. If, however, potassium is compartmentalized in the cell as Solomon and Gold (1955) have suggested, then only a small amount of potassium might be available to act as a catalyst, and some of these anomalies might be explicable. In support of this possibility Prankerd (1955a) has found that a reduction in cell potassium by cold storage slows the rate of synthesis of ATP on reincubation, and since there was about half the cell potassium still present he assumed that not all the cell potassium could have been available for catalytic action. Galactose is also utilized by human red cells, and galactose-l-phosphate normally converted to glucose-l-phosphate by an isomerase. In congenital galactosemia it appears that the red cells are deficient in this enzyme (Schwarz et d., 1955).

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b. Nucleosides. The first evidence that the red cell could break down these substances came from the work of Dische (1951) who showed that hemolyzates would esterify orthophosphate in the presence of nucleosides with the formation of ribose, triose, and hexose phosphates. Prankerd and Altman (1954), and Finch and Gabrio (1954), have recently shown that these substances may be important also in the metabolism of the intact red cell. Thus the former authors showed that in the absence of glucose adenosine would promote PSzuptake by red cells and also its incorporation into esters within the cell. They proposed that the nucleoside underwent phosphorylative cleavage on the cell surface and that the ribose phosphate formed was then incorporated into glycolytic reactions in the cell by way of the hexose-monophosphate shunt. Some of their work suggested that in certain circumstances adenosine would be a more satisfactory substrate than glucose. Finch and Gabrio approaching the matter from a different angle found that the storage of red cells in media to which adenosine had been added improved their post-transfusion survival time. Thus although it is doubtful if nucleosides play any physiological role in red cell metabolism they may prove of practical importance in blood storage. Prankerd and Altman also showed that even in the absence of glucose human red cells could utilise adenosine and guanosine for the resynthesis of phosphate esters including ATP. Gabrio et al. (1955) have since reported findings confirming this, whilst Sanberg et al. (1955) have recently investigated the nucleoside phosphorylase responsible for splitting these substances and shown that it is only active against purine nucleosides. c. Other Substrates. Although little is known about any other substrates the red cell may utilize, it appears from the work of Spicer and Clark (1949) that malic and fumaric acid can be converted to lactate by intact rabbit cells. This and the possible utilization of pyruvate are mysteries at present but these substances may be involved in the synthesis of phosphopyruvate from pyruvate, a step which involves thermodynamic difficulties along the reversed glycolytic route, but for which Krebs (1954) has proposed the following alternative pathway. Phosphopyruvate Oxaloacetate ATP ADP DPN

1 1

Pyruvate DmH

Lactate

1

TPNH

> Malate

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Red cells also contain the enzyme glyoxalase (Jones and McCance, 1949), which converts methyl glyoxal to lactic acid, and a large amount of its coenzyme glutathione (Schelling, 1932) ; methyl gIyoxal could be derived by dephosphorylation of 3-glyceraldehyde phosphate, but whether t h i s pathway is normally operative is not known. Nothing is known either of any part ascorbic acid may play in red cell metabolism.

2. Hexose Monophosphate Shunt This is an alternative pathway for the metabolism of glucose through which this sugar is oxidized with the formation of pentose and triose phosphates. The pathway occurs in many animal tissues and its composite reactions have recently been reviewed by Horecker and Mahler (1955). The following over-all reactions probably occur :

+ + +

+

1. 6 Glucose phosphate 3 0 2 3 6 COa 6 pentose phosphate 2. 4 Pentose phosphate + 2 hexose phosphate 2 tetrose phosphate 2 tetrose phosphate -+ 2 hexose phosphate 3. 2 Pentose phosphate 2 triose phosphate 4. 2 Triose phosphate + hexose phosphate inorganic phosphate

+

+

Only reaction 1 requires oxygen, triphosphopyridine nucleotide being the redox catalyst. The other reactions occur anaerobically. Dische (1938, 1951) first drew attention to the possibility of this pathway functioning in the red cell. He demonstrated in cell hemolyzates the esterification of orthophosphate in the presence of the nucleosides adenosine and guanosine. Ribose phosphates were formed and ultimately triose and hexose phosphates. The role of nucleosides in the metabolism of the red cell has already been described ;the pentose phosphate so derived could be utilized anaerobically by this shunt. Ling and Chow (1954) found that considerable ribose formation could be detected in red cells incubated aerobically in phosphate buffer with glucose. They suggested that ribose may therefore be an important pathway of glucose utilization in the mammalian erythrocyte. However if glucose were to be metabolized by this route there would have to be a reasonable consumption of oxygen, and in the mature red cell this is not found. Their suggestion would thus seem unlikely. The addition of methylene blue to incubating red cells is well known to increase their oxygen consumption to a range approaching that for nucleated cells (Harrop and Barron 1928). Further work by Warburg et d. (1930) later showed that the site of action of methylene blue was in the oxidation of glucose 6-phosphate to 6-phosphogluconate, the dye acting as a hydrogen acceptor from triphosphopyridine nucleotide. In this

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connection methylene blue may be substituting for cytochrome c lost from the cell during maturation. 3. Phosphatases SeveraI phosphatases occur in the red cell, and specific phosphatases such as ATPase are present in the stroma (Garzo et al., 1952; Clarkson and Maizels, 1952). The latter enzyme is activated by Mg++ and has a pH optimum at about 7.0. It occurs in the stroma in a concentration twice that of the cell interior, and both fractions are very sensitive to the presence. of copper. Whether this protein has contractile properties like its equivalent in muscle is not known, but its presence in greater concentration in the stroma raises the possibility that it might be concerned in maintaining the biconcave shape of the human red cell. A specific phosphatase splitting 2,3-DPG also appears to act at or near the cell surface. It is inhibited by Ca++, and, in contrast to other specific phosphatases, a nonstromal cofactor is required for its maximal activity. Contrary to the claims of Rapoport and Guest (1939), Clarkson and Maizels (1952)were unable to demonstrate 2,3-DPG splitting activity in hemolyzates. Although a number of phosphomonoesterases have been isolated from red cells with apparently different pH optima, Tsuboi and Hudson (1953) consider that there is probably only one ester with a p H optimum about 5.5. It is mentioned later how their activity is thought to account for ihe presence of inorganic phosphate within the cell, and how the stromal ATPase may lead to the escape of inorganic phosphate from the cell by hydrolysis of ATP at the cell surface.

4. Acetylcholine-esterase (AChE) This enzyme has received much attention recently as a result of the claims of Greig and Holland (1949)that it plays a significant part in the transport of ions across the cell membrane. The relevant historical details can be found in the reviews of Augustinsson (1948),and the demonstration of AChE in stroma by PalCus (1947). The enzyme has been purified by Zittle et al. (1954) who have reported an isoelectric point at p H 6.0, in contrast to that at 4.7 found by Augustinsson. AChE of the red cell differs in substrate requirements from cholineesterase (ChE) in the serum. This difference is related to the number of active centers on the enzyme molecule’s surface, the red cell enzyme having two and that of serum only one (Bergmann and Segal, 1954). Stroma AChE must therefore be considered an intrinsic stroma protein rather than a plasma protein adsorbed on the cell surface, and it is likely that it has been a part of the surface since the cell’s formation. The finding that certain liver diseases cause a decrease in serum without affecting red cell AChE supports this view (Fischer and Maier, 1953).

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Work bearing on the role of this enzyme in ion transport will be dealt with in Section 111, and it will be made clear that although there is no doubt about its existence in the stroma, the actual function of this enzyme is uncertain as its substrate is synthesized in too small a quantity to provide energy for transport of ions against concentration gradients (Mathias and Sheppard, 1954). 5. Proteinass The work of Morrison and Neurath (1953) shows that at least three different proteinases are present in red cell stroma. One of these extracted with KCNS had a p H optimum of 7.4, was activated by Zn++ and Fe+++ as well as by reducing agents like ascorbic acid, cysteine, and glutathione, and could utilize hemoglobin as a substrate. It was readily inhibited by Hg++ suggesting a requirement for -SH groups. The two other proteinases extracted with butanol had p H optima at 7.4 and 3.2. The former was found to be less stable, was unaffected by reducing agents and Hg++, but was also activated byZn++ and Fe+++. The latter was not activated by Zn++ and Fe+++. The presence of proteinases appears to be species dependent as Goetze and Rapoport (1954) failed to detect these enzymes in rabbit cells at neutral and alkaline pH, but found one active at p H 3.5. These authors have attempted to correlate cell maturation with proteolytic activity by showing that mature cells were less active in this respect than reticulocytes. Their possible role in cell destruction does not appear to have been investigated, and at present their function remains a mystery, although those with pH activity outside physiological ranges are presumably not active in vivo. 6. Phosphopyridine Nucleotidase Alivisatos and Denstedt (1951) found that the lactic dehydrogenase activity of red cells could be increased by the removal of stroma and concluded that stroma contains a DPNase which inactivates the coenzyme of lactic dehydrogenase. Upon addition of nicotinamide to the complete hemolyzate, DPN is protected from the action of DPNase, and the activity of the dehydrogenase equals that of a stroma-free hemolyzate. Red cells also contain triphosphopyridine nucleotide-requiring dehydrogenases and it is likely that the stroma contains TPNase activity.

7. Methemglobin Reductme The rate of auto-oxidation of hemoglobin in cells soon results in the formation of the stable form methemoglobin if cell metabolism is stopped (Cox and Wendel, 1942). Continuation of glycolysis enables the con-

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stantly formed methemoglobin to be reduced, so that the amount of this product in the living cell is very small. The mechanism of these reactions is now largely understood as a result of the work of Kiese (1944) and Gibson (1943, 1948). Reduction of methemoglobin is linked to the oxidation of reduced diphosphopyridine nucleotide (DPNH) which in its turn is linked to the reduction of DPN by the triose phosphate dehydrogenase system. The unsettled problem in these reactions is the activation of the first reaction since direct electron transfer from DPNH to methemoglobin proceeds very slowly. Kiese has suggested that a flavoprotein reductase activates this step, and evidence in support of this suggestion has been given recently by Altman (1954) who utilized an enzyme prepared from yeast in an artificial medium. In the rare familial disease of methemoglobinemia the methemoglobin reductase system is deficient presumably because of the absence of this enzyme. Its absence can be compensated for by methylene blue which bridges the gap between the reduction of methemoglobin and the triphosphopyridine nucleotide system (Gibson, 1948). 8. Glutathione This tripeptide occurs in large amounts in the red cell (Schelling, 1932) but its exact role in the metabolism of the cell is unknown. Its possible functions can be surmised from those known to occur in other tissues, of which the best described are : a. Coenzyme activity for glyoxalase in the conversion of methyl glyoxal to lactic acid (Racker, 1951). b. Formation of the prosthetic group of 3-phosphoglyceraldehyde dehydrogenase (Krimsky and Racker, 1952). c. Redox catalyst for DPN and ascorbic acid. A function described only in plants (Mapson and Gaddard, 1951). d. Provision of -SH groups at the cell surface, active in ion transport. As with most of the enzymes the problem is to decide whether a particular substance is still active in the metabolism of the mature erythrocyte, or merely a metabolic remnant from the cell’s nucleated life.

9. Stroma Many of the enzymes already discussed are probably situated near the surface of the cell and may be considered to form part of the protein matrix of the stroma. Certain metabolic activities are however peculiar to the stroma by virtue of its structure and these will be discussed next. They consist of exchange of materials with the external medium and of independent anabolic and catabolic changes, providing for the continuous renewal of the stromal architecture.

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It has already been pointed out that glycolysis proceeds close to the cell surface, and in this connection it is noteworthy that A T P synthesis occurs in stroma at a rate different from that inside the cell (Prankerd and Altman, 1954). This suggests independent activity of the stroma from that in the hemolyzate, and other evidence in its favor has been provided by Miinyai and SzCkely (1954) who found that the addition of KsFe(CN)e to NaF-poisoned cells caused a partial resynthesis of ATP, presumably by oxidizing reduced DPN which had accumulated as a result of enolase inhibition. Since the red cell is impermeable to KsFe(CN)e (Keilin and Hartree, 1946) the oxidation of DPNH and resynthesis of A T P must have taken place near the cell surface. One of the synthetic processes which has been demonstrated in stroma is the incorporation of C14-2-acetate into certain lipid fractions. This was demonstrated in phospholipid and sphingolipid fractions in vivo and in vitro (Altman et al., 1951; Altman, 1953). This incorporation is affected by environmental conditions such as storage at 4” or 37°C. At either temperature the cells lose their ability to take up labeled acetate before any change attributable to storage becomes apparent (Altman and Swisher, 1954). Certain diseases affecting the red cells, such as hemolytic anemias, were found to increase incorporation of labeled acetate, whereas in pernicious anemia there was decreased incorporation. The C14-activity was distributed between the phospholipid and springolipid fractions and to a much smaller extent the cholesterol fraction. Hagerman and Gould (1951) have presented evidence that free plasma cholesterol exchanges readily with the cholesterol of the cell. In an artificial medium of y-globulin and albumin with little cholesterol the exchange of red cell cholesterol does not take place, so that some factor in native plasma is required (Ruhenstroth-Bauer, 1953). London and Schwarz (1953) have pointed out that the exchange of cholesterol and other labile stroma components is probably of great importance in maintaining a viable cell. Lovelock (1954) suggests that this may be so as he found that cells from which phospholipid had been removed selectively hemolyzed within 30 seconds if cooled suddenly from 37” to 0°C. H e also demonstrated that cells stored in glycerol at -78°C. lose considerable amounts of lipid from their surface. The hemolytic action of digitonin appears to involve surface lipids and is dependent on a definite saturation threshold. Ruhenstroth-Bauer ( 1950) states that hemolysis does not begin until at least five adjacent cholesterol residues on the cell surface are covered by digitonin. This implies that a minimum number of cholesterol molecules must be rendered functionally “inactive” before critical changes in the stroma occur, and lead to hemol-

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ysis. Before hemolysis occurs cells lose potassium, gain sodium, and swell (Schatzmann, 1953). The means by which adenosine inhibits this exchange of cations is not known (Harris and Prankerd, 1955). I n plasma, where an active exchange of red cell and plasma cholesterol can continually occur, the probability of hemolysis from loss of surface lipids must be much smaller than in salt solutions, but in the presence of agents which block this exchange hemolysis may occur even in plasma; such a situation as this might develop in certain hemolytic diseases when the red cells are coated by antibody. Recent findings by Lovelock (1955) suggest that red cell stroma possesses intrinsic mechanisms for the destruction of its exogenous cholesterol since under conditions of stress cells lose cholesterol which cannot be found in the external medium. In contrast to the stroma lipids, the proteins are metabolically as stable as the intraerythrocytic hemoglobin (Muir et al., 1952).

10. Energy Utilization Having considered a number of the metabolic activities shown by the red cell, the question arises as to what use the energy derived from glycolysis is put. It will be seen in Section I11 that most of the transport systems are energy dependent and it is axiomatic that those maintaining the greatest concentration gradients will use the greatest amount of energy for their operation. Thus the transport of sodium and potassium has been calculated to use up to one-third of the available free energy of glycolysis by red cells (Solomon, 1952 ; Bernstein, 1953). What remains is then left for the operation of the many activities already described.

111. ENZYMES IN TRANSPORT 1. Mechanisms of Transport This subject was covered from a general point of view by Rosenberg and Wilbrandt (1952) in an earlier review of this series. These authors laid down certain rigid criteria for identifying enzymatic transport, but although these may be useful for this purpose they do not reveal the individual mechanisms involved. For the purpose of this article the following mechanisms will be considered : a. Active transport. This is a mechanism whereby a substance is moved from the lower end of an electrochemical gradient to the higher at the expense of energy derived from the metabolism of the cell. The mechanism is enzyme-dependent in so far as cell metabolism is enzymatically controlled, but enzymes do not necessarily play any intimate role in

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the actual movement of any substance. If the transported substance is an integral part of the chemical metabolism of the cell then it may be adsorbed at the cell surface, incorporated into the intermediary metabolism of the cell, and later released inside the cell. Phosphate might be regarded as an example of this form of transport in red cells, and pyruvate in yeast cells (Foulkes, 1955). Alternatively, energy may be utilized to perform osmotic work by means of a contractile protein (Goldacre, 1952) or it may be required to maintain the integrity of certain constituents of the cell membrane, as by the turnover of lipids, and the activation of possible carrier substances. b. Facilitated Diflw’on. In this case the movement across certain areas of the cell membrane is speeded up catalytically without energy expenditure by the cell, whereas diffusion across the rest of the membrane is slow (Davson and Danielli, 1952; Danielli, 1954). To do this there may be special surface groupings adjacent to membrane “pores” which lower the activation energy for membrane penetration, possibly by providing hydrogen-bonding groups. Alternatively a substance may be enzymatically changed after surface adsorption, and thus trapped, and a concentration gradient built up between this and another site inside the cell where the original substance is reconstituted. This process would not promote transport against a concentration gradient although there seems no reason why it should not be coupled with the previous mechanism. Facilitated diffusion is not energy dependent except in so far as the constitution of the membrane may be. c. Pwsive Diflw‘on. In this case the movement through a membrane is dependent simply on molecular agitation. It may occur alone or in conjunction with the first and second processes, but it is slower. It appears from recent work that the first two mechanisms often play the predominant role in transport through living membranes. It is these two which are enzymatically dependent and they will be considered here in their relationship to transport in the erythrocyte. In neither system is the movement of substances directly proportional to concentration.

2. Phosphate Transport The aqueous concentrations of this ion inside and outside the red cell are not equal, the preponderance being inside (Helpern, 1936; Prankerd and Altman, 1954). The gradient which results is only small but its presence and the failure of the two concentrations to be related by simple concentration laws (Helpern, 1936; Hahn and Hevesy, 1941) imply the presence of a specialized mode of transport. Furthermore, phosphate transport does not occur in the absence of glucose or in the presence of

ENZYMES IN RED CELL METABOLISM AND TRANSPORT

29 1

metabolic inhibitors, such as fluoride or iodoacetate, and must therefore be metabolically dependent. From analyses of the Relative Specific Activity (RSA) of intracellular phosphate esters and inorganic phosphate and their relationships to the extracellular orthophosphate, it would appear that very little free exchange between the two orthophosphate pools occurs and that labeled phosphate is first incorporated into esters inside the cell and later released into the intracellular pool by phosphatase action on these esters. This conclusion is drawn from the work of Gourlay (1952) and Prankerd and Altman (1954). It has already been mentioned that phosphate esters and some enzymes have been located in the red cell membrane and that it seems fair to conclude that glycolysis is proceeding close to the cell surface. This would be necessary if phosphate were to be taken up by the cell without first penetrating it. From an analysis of the RSA of intracellular phosphate compounds Prankerd and Altman have proposed that the uptake of phosphate is enzymatically linked to the conversion of 3-glyceraldehyde phosphate to 1,3-diphosphoglycerate, close to the cell surface. They found that the activity of extracellular orthophosphate bore a precursor relationship to the activity of cellular 2,3-DPG, and that this ester had a higher activity than ATP. Gourlay (1952) had previously proposed from the data of similar experiments that phosphate uptake was linked to the phosphorylation of ADP, also occurring at the cell surface. In each case the arguments have been based on the intracellular esters showing the highest RSA. Although Gourlay did not differentiate between the RSA of phosphorus atoms having different rates of turnover in the above compounds, Prankerd and Altman pointed out that taking this into consideration his results would be brought into line with theirs. From analogy with other tissues, kidney, etc., in which it is widely thought that phosphate transport is closely linked with glucose transport, the same might be supposed for the red cell. Below levels of 8C100 mg. per cent glucose the exchange of Ps2-orthophosphate appears to depend on the concentration. Jonas (1954) and Jonas and Gourlay (1954) suggest that the transport of inorganic phosphate ions across the red cell membrane is mediated by a complex involving ADP, ATP, hexokinase, Mg++, and K+. These authors concluded that phosphate is transferred to gIucose 6-phosphate at the cell surface with the aid of the above mechanism, and is in accord with similar conclusions reached by Schild and Maurer (1952) on the basis of Pa partitioning experiments. The latter workers also claimed that creatine phosphate also participated in the transphosphorylating process.

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In spite of these findings it seems unlikely that the hexokinase system plays a primary role in the transport of phosphate into the cell because inorganic phosphate can enter the glycolytic scheme only at the trios phosphate stage. Only after the action of glycerophosphokinase accompanied by phosphate transfer from lJ3-diphosphoglycerateto ADP can phosphate (as terminal P of ATP) be transferred to glucose. Thus the primary step in phosphate exchange most likely takes place at the glyceraldehyde 3-phosphate level. The works of all authors are, however, in agreement in proposing the phosphorylation of some intermediate compound maintaining the steady state between the intracellular and extracellular pools of orthophosphate. Further light has been cast on phosphate transport by observations on the use of nucleosides by the red cell. In this case the formation of a phosphorylated intermediate is clearer. I t has already been mentioned that when red cell hemolyzates are incubated with adenosine, ribose, triose, and hexose phosphates are formed, and that using intact cells Prankerd and Altman were able to show that adenosine or guanosine would promote P=-exchange in the absence of glucose and that this occurred at the same rate as in its presence. It was supposed that the mechanism of this was the splitting of the nucleoside by a phosphorylase at the cell surface and the metabolism of the phosphorylated pentose by way of the hexose monophosphate shunt. Data suggesting this mechanism were obtained by poisoning with arsenate. Since then a nucleoside phosphorylase has been demonstrated in red cell stroma (Prankerd and Altman, 1954; Gabrio and Huennekins, 1955) and Prankerd (1955b) has shown that adenosine is quantitatively converted to adenine by red cells and that the purine portion does not enter the cell. Although pentoses penetrate the red cell (see Section 111, 3) the phosphorylated sugar does not, and as pentoses will not serve as a substrate for cell metabolism it appears that they can do so only when phosphorylated by the cell. This may well be because the pentose phosphate does not penetrate the cell surface sufficiently to reach the necessary enzymes for its degradation. Although the transfer of phosphate across the red cell membrane is enzymatically controlled there remains the possibility that, like other ions, a passive diffusion may occur synchronously. This is suggested by some of the experiments of Gourlay and Matschiner (1953) who, by taking frequent samples of blood shortly after the addition of P32, found that two rate processes were present and suggested that the faster initial process was a passive flux; it might equally well have been an adsorption phenomenon. There has been little work on the mechanism by which phosphorus leaves the cell, but since it is enzymatically taken in it probably

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escapes by similar means, and a possible way would be by the hydrolysis of ATP at the surface by the stromal ATP-ase already described. Such a mechanism must be carefully geared to its uptake since cell P remains constant. The examination of the rate of phosphate exchange in the red cells of a number of other species was found to be closely related to the rate of glycolysis of those cells, and it is probable that the same mechanism of phosphate transfer is operative in all these cells (Prankerd and Altman, 1954). 3. Sugar Transport Since glucose is biologically the most important of sugars it will be the one chiefly considered. The red cells of certain mammals and adult primates, and some mammalian fetal cells are permeable to glucose and the distribution of glucose between plasma and cell water is therefore equal (Kozawa, 1914). This finding holds only between certain limits of extracellular glucose concentration (Klinghoffer, 1935). Concentrations greater than 2% in the extracellular fluid do not equalize and thus red cells in iso-osmotic glucose (5%) do not hemolyze. This anomaly, which implies that glucose does not penetrate the cells by a process of passive diffusion, has been studied in great detail by Wilbrandt, Le Fevre and Widdas. Kinetic studies of cell swelling in glucose-saline solutions have shown that a saturation point is reached above which no increase in the rate of glucose transfer can be produced by increasing its concentration outside, and above which presumably some specialized receptor site is full to its capacity. The absence of any concentration gradient between the outside and inside of the cell does not require any active transport. A number of assumptions have been made as to the nature of the receptor but no very definite findings have been made. Studies with iodoacetate have shown that this does not inhibit glucose penetration and that the process is not therefore linked to cell metabolism (Wilbrandt, Guensberg, and Lauener, 1947). Widdas (1955) has also pointed out the disproportion between the rate of glucose transfer and its rate of consumption by the cell, which would be a priori evidence for this. A number of other agents do block glucose transfer, Phloridzin does so and has been thought to interfere with phosphorylation of the sugar at the cell surface. Wilbrandt ( 1954) has reported that phosphorylated phloretin, which does not penetrate the cell surface, shows a greater inhibition of outwardly to inwardly transported glucose. He has suggested, since phloridzin possibly acts on phosphatases (although Shapiro (1947) does not think this so), that glucose is phosphorylated as the first step of transport and then finally

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dephosphorylated by phosphatases. This is speculative and since glucose does not accumulate against a concentration gradient it is probably not so. Also, since phloridzin is a glycoside it may well compete with glucose for certain receptor sites as other sugars do, especially since inhibition is to some extent proportional to concentration. Other inhibiting agents include mercury and silver, which Le Fevre (1948) thinks implicate sulfhydryl groups. Copper, however, which is active in inhibiting glycerol transport (Section 111, 4) and is a well known inactivator of sulfhydryl groups, has no effect on glucose transfer. Cysteine and glutathione both reverse the effect of mercury and silver, lending some support to the sulfhydryl group theory. The mere demonstration that inhibitors, known to act on particular chemical groups and thus inactivate enzymes, are effective is no proof that the particular enzymes being inactivated participate in transport. It may be that the same groups are common to both certain enzymes and certain parts of the cell surface, and that on being altered the membrane loses its selective properties because of the change in these surface groups. That certain areas of the membrane are selective in facilitating transport has been shown for glycerol by Danielli (1954), and Le Fevre (1954) has shown that the selectivity of tfie membrane for facilitating transport of sugars is specific for aldoses and that ketoses penetrate the cell in accordance with simple concentration laws. Le Fevre was able to show competitive inhibition between the aldoses but was surprised to find the same for mixtures of aldoses and ketoses. This apparent anomaly has been explained by Widdas (1955) by showing that the carrier equilibration constants of these sugars are different. That for aldoses is low as predicted from its approximation to saturation carrier kinetics whereas that for ketoses is high as predicted from simple diffusion laws. Studying the kinetics of glucose transfer Widdas (1954) has confirmed Le Fevre’s work (1952) and has also shown that the rate of penetration in human cells is proportional to the differences of the reciprocals of the intracellular and extracellular concentrations and that they fit a near saturation carrier equation. Most of the evidence in the case of the penetration of glucose into erythrocytes points away from any system of active transport and toward one of facilitated diffusion in which either some unknown carrier system exists, or chemical groups at certain sites of the cell surface are active in overcoming the restraint imposed by the polarity of the sugar in an aqueous medium. Although phosphorylation might be involved in this process there is no evidence for it and Le Fevre (1948) has shown that such a process does not involve inorganic phosphate.

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It is interesting to find that these active surface sites or carriers are not constant in a particular species throughout its life. Widdas (1955) has shown that the cells of the fetuses of some animals-sheep, pig, and rabbit-are permeable to glucose, whereas cells of their adult forms are not. This type of change is most likely to be associated with structural and chemical differences in the surface membrane, a difference which would not be surprising when one considers that hemoglobins differ between young and old, and so do the physical properties of the cell membrane (Sjolin, 1954). In those species in which glucose does not penetrate the cell its metabolism must begin close to the cell surface. The penetration of the cell by pentoses has been investigated by Le Fevre and found to show a behavior similar to the hexoses, particularly with regard to their subdivision into aldoses and ketoses. 4. Glycerol Transport

Glycerol penetrates the cells of many species and in contrast to glucose, glycerol penetration does not show the limiting effects of concentration, so that all cells permeable to glycerol eventually hemolyze in it whatever its concentration. It is not metabolized by the cell but its physical properties enable red cells to be stored for long periods at low temperatures (Lovelock, 1954). I t was first noticed by Jacobs and Corson (1934) that small amounts of copper, even in concentrations as low as lo-' M, retarded the rate of hemolysis of cells suspended in glycerol. Such small amounts of copper could only cover a small proportion of the cell surface and its effect would suggest that there are certain sites on the cell membrane at which glycerol absorption can be blocked. By an analysis of the kinetic studies of Jacobs, Danielli was able to show that glycerol penetrates the red cells of some species by two processes; a slow process of simple diffusion and a fast process which occurred only at certain membrane sites which involved less than 1% of the cell surface. He suggests that these active sites resemble enzymes and are similar to the findings of Davson (1942) in relation to the permeability of cats' erythrocytes to sodium. Le Fevre (1948), thinking that this effect would result from blocking of sulfhydryl groups, tried the effect of other agents known to show this affinity. He found that mercury, iodine, and chloromercuric benzoate had an effect similar to that of copper and that the effect could be inhibited by substances known to supply available sulfhydryl groups. Phloridzin, however, also inhibited the copper'effect. This inhibition and its reversal makes

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it unlikely that copper acts by structurally altering the cell hemoglobin as suggested by Parpart et al. (1947).

5. Cation Transport The permeability of the cell membrane to cations has now been firmly established where previously it was only implied by the high concentration gradients of these ions between cell and plasma water, and the inability to account for them in terms of the Donnan equilibrium. It has been shown that glucose is essential for the active transport and that it ceases with the interruption of glycolysis by poisoning with fluoride or iodoacetate. Respiratory inhibitors such as cyanide and dinitrophenol are ineffective in this respect (Maizels, 1951). Furthermore the active fluxes of these ions are independent of external concentrations over wide ranges (Harris and Maizels, 1951; Solomon, 1952). The following are main problems remaining to be solved now that so much has been unraveled in connection with cation movement.

1. Whether the transported cations are intimately incorporated into cell metabolism, analogous to the way in which phosphate is moved. 2. Whether the active movement of one ion will lead secondarily to the movement of the other in order to maintain electrochemical balances. 3. If both ions move actively, whether the same transport system operates for both ions or whether two separate systems exist. 4. What is the exact nature of the transporting agent? With regard to the first question, since glycolysis is proceeding close to the cell surface, one might suppose that some of the intermediary compounds of carbohydrate metabolism could act as transporting agents for sodium and potassium, depending on their affinities for these ions. The active fluxes of sodium and potassium are in the region of 4.3 and 2.45 p equiv. per ml. cell water per hour respectively (Harris, 1954), and these figures approximate closely the uptake of glucose and liberation of lactate by the cell under aerobic conditions; 2.5 and 4.7 p mols respectively per ml. of cell water per hour (Bird, 1947). Although this would be an attractive theory and does not seem to have been explored there is some evidence against it. Thus it is extremely probable that whatever transporting agents are active in the immature nucleated red cell would also be active in the mature red cell, as it is unlikely that a new transporting system arises with the changeover from aerobic to anaerobic metabolism. Maizels (1954) has examined cation transport in the nucleated erythrocytes of the chicken and found, by poisoning these cells with suitable substances, that cation transport is dependent on the respiratory cycle and not on

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glycolysis. As this cycle is only partially represented in the un-nucleated erythrocyte it is unlikely that any intermediary metabolite is the transporting agent. The second question has been examined by Harris and Maizels (1952) who have pointed out that if the distribution of potassium was secondary to the active extrusion of sodium and the electrical inequality arising from it, then (K) cell/(K) plasma should equal (H) cell/( H) plasma and (Cl) plasma/( Cl) cell They tested this problem experimentally and showed that the conditions are not satisfied ; thus ( K ) cell (Cl) plasma = 1.4 and = 30 (a)cells (H) plasma On the other hand, although active transport of sodium alone might lead to the characteristic cation distribution between the cell and plasma, the interior of the cell would perforce become too acid for normal functioning. It must therefore be concluded that one or two active transport systems operate for both sodium and potassium and there are several pieces of evidence bearing on the third question, the relationship between the two. Firstly there is a close relationship between the ability of the cell to move one ion and its ability to move the other if a restraint is imposed on the movement of one of them (Maizels, 1954b) or if lithium is substituted for sodium (Flynn and Maizels, 1949). Secondly Harris (1954) has examined in detail the linkage between the transport of these ions and drawn attention to the numerical association between the two active fluxes, that of sodium being twice that of potassium in most cells ; he concludes that a single mechanism brings about sodium extrusion and potassium accumulation. This conclusion is strengthened further by the effect of digitalis glycosides, which, without interfering with cell metabolism, cause a loss of K and gain of Na by the cell and must presumably affect the transport system itself (Schatzmann, 1953). Solomon (1952) however does not share this conclusion and his recent experiments (Solomon and Gold, 1955) have shown that the intracellular potassium is two compartmental, whereas he did not find this to be so for sodium. These results need confirmation, as the behavior of intracellular sodium and its exchangeability is variable especially after a few hours of storage (Harris and Prankerd, 1953). It has been mentioned earlier that Greig and her colleagues have suggested in a number of publications (Greig and Holland, 1949; Holland

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and Greig, 1950; Lindvig et d.,1951; Greig et al., 1953) that the hydrolysis of acetylcholine (ACh) by AChE at the cell surface provides energy for potassium transport across the cell membrane. Glycolysis is required for this system since it provides ATP for the activation of acetate in the choline acetylase system. Greig’s concept is based on a number of experimental findings : 1. Buffered suspensions of red cells lose less potassium and hemolyze to a lesser extent when ACh is metabolized. 2. In the presence of an AChE inhibitor red cell permeability is increased with loss of potassium and hemolysis. 3. The addition of ACh to cells which have previously been exposed to an AChE inhibitor does not decrease hemolysis and potassium loss. 4. Cells which have lost potassium are able to reaccumulate this ion against a concentration gradient when AChE is supplied. For such a system to operate adequate amounts of ACh, continuously synthesized, are required. The energetics of AChE hydrolysis reveal that for every mole of ACh hydrolyzed a maximum of two moles of potassium can be transferred (Mathias and Sheppard, 1954). Although red cells contain some choline acetylase activity, Mathias and Sheppard found that ACh synthesis amounted to, at best, 1/50th of the minimum ACh requirement. Other investigators (Taylor et aZ., 1952) also have been unable to find any correlation between the concentration of AChE necessary for complete enzyme inhibition and that concentration required to bring about potassium loss and hemolysis. The inhibitor concentration which will achieve hemolysis is 5&1000 times greater than the concentration for complete inhibition, Furthermore in vivo experiments with octamethylpyrophosphoramide reveal no direct correlation between potassium leakage and AChE activity (Goodman et UZ., 1955). It may be concluded that the substrate for AChE activity is synthesized in too small a quantity to provide energy for transport against a concentration gradient. If one mechanism operates for the transport of sodium and potassium its nature is quite unknown and the only clue to this is that transport probably occurs in the lipid phase of the membrane. This would have to be so be=use the hydrated sodium ion is larger than that of potassium yet it penetrates the cell more rapidly and could not therefore do so through aqueous channels. Further examination of the lipids of the cell membrane in this connection may well prove fruitful.

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ACKNOWLEDGMENTS The author is grateful to Dr. K. I. Altman for a number of suggestions in the preparation of this paper.

IV. REF-NCES Alivisatos, S. G. A., and Denstedt, 0. F. (1951) Science 114, 281. Altman, K. I. (1954) Biochim. et Biophys. Acta. 16, 155. 33, 168. Altman, K. I. (1953) Arch. Biochem. and Biophys. 42, 478. Altman, K. I., Warman, R. N., and Salomon, K. (1951) Arch. Biochem. und Biophys. 33, 168. Akman, K. I., and Swisher, S. (1954) Nafure 174, 459. Augustinsson, K. B. (1948) Acta Physiol. S c a d . 16, 1. Bartlett, G. R., and Marlowe, A. A. (1953) J. Lab. Clin. Med. a,178. Bartlett, G. R., Savage, E., Hughes, L., and Marlowe, A. A. (1953) J. Appl. Physiol. 6, 51. Bergmann, F., and Segal, R. (1954) Biochem. J. 68,692. Bernstein, R. E. (1953) Nature 172, 911. Bird, R. M. (1947) I . Biol. Chew. 16B, 493. Clarkson, E. M., and Maizels, M. (1952) J. Physiol. (Loltdon) 116, 112. Cox, W. W., and Wendel, B. (1940) I. Biol. C k m . 189, 33. Danielli, J. F. (1954) Symposia SOC.Exptl. Biol. 8, 502. Davson, H. (1942) J. C e l l d w Comp. Physiol. 20, 325. Davson, H., and Danielli, J. F. (1952) “The Permeability of Natural Membranes.” ‘Cambridge U. P., London. Dische, Z. (1938) N a t ~ & s m c h a f f m 26, 252. Dische, Z. (1951) in “A Symposium on Phosphorus Metabolism” (McElroy and Glass, eds.), Vol. 1, p. 171. Johns Hopkins Press, Baltimore. Finoh, C. A., and Gabrio, B. W. (1954) J. Clin. Invest. 33, 932. Fischer, R., and Maier, E. H. (1953) Klin. Wochschr. 32, 566. Flynn, F., and Maizels, M. (1949) J. Physiol. (London) 110, 301. Foulkes, E. C. (1955) J. Gen. Physiol. 38, 425. Gabrio, B. W., Hennessey, M., Thomasson, J., and Finch, C. A. (1955) I . Biol. Chem. 216, 357. Gabrio, B. W., and Huennekins, F. M. (1955) Fedwarion Proc. 14, 217. Garzo, T., Ullman, A., and Straub, F. B. (1952) Acta Physiol. Acud. Sci. Hung. S, 513. Gibson, Q. H. (1943) Biochem. I. 31, 615. Gibson, Q. H. (1948) Biochem. 1. a,13. Goetze, E., and Rapoport, S. J. (1954) Biochem. 2.326, 53. Goldacre, R J. (1952) Intern. Rev. Cytol. 1, 135. Goodman, J. R., Marrone, L. H., and Squire, M. C. (1955) Am. J . Physiol. 180, 118. Gourlay, D. R. H. (1952) Arch. Biochem. and Biophys. 40, 1. Gourlay, D. R. H., and Matschiner, J. T. (1953) I. Celldar Comp. Physiol. 41, 425. Greig, M. E., and Holland, W. C. (1949) Arch. Biochem. a d Biophys. 33, 370. Greig, M. E., Faulkener, J. S., and Mayberry, T. C. (1953) Arch. Biochem. and Biophys. 48, 39. Hagerman, J. S., and Gould, R. G. (1951) Proc. SOC. Ezjtl. Biol. Med. 78, 329.

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Hahn, L., and Hevesy, G. (1941) Acto Physiol. S c a d 3, 193. Harris, E. J. (1954) Symposio SOC.Exptl. Biol. 8, 228. Harris, E. J., and Maizels, M. (1951) J, Physiol. (London) 113, 506. Harris, E. J., and Maizels, M. (1952) I. Physiot. (London) ll8, 40. Harris, E. J., and Prankerd, T. A. J. (1953) J. Physiof. (London) 121, 470. Harris, E. J., and Prankerd, T. A. J. (1955) Biochem. 1. 81, i. Harrop, G. A., and Barron, E. S. G. (1928) I. Expbl. Med. 48, 207. Helpern, L (1936) I. Biol. C h . 114, 747. Holland, W.C., and Greig, M. E. (1950) Am. J . Physiol. 162, 610. Horecker, B. L., and Mahler, A. H. (1955) Ann. Rew. Biochem. !&, 207. Jacds, M. H., and Corson, S. A. (1934) Biol. Bull. 67, 325. Jonas, H. (1954) Biochim. et Biophys. Acto 18, 241. Jonas, H., and Gourlay, D. R. H. (1954) Biochim. et Bbphys. Acto 14, 335. Jones, P. H., and MoCance, R. A. (1949) Biochem. 1. 46, 464. Keilin, D., and Hartree, E. F. (1946) Nature 167, 210. Kerr, S. E. (1937) J. Biol. Chetn. 117, 227. Kiese, M. (1944) Biochem. 2. 316, 264. Klinghoffer, K. A. (1935) Am. J. Physiol. 111, 231. Kozawa, S. (1914) Biochem. 2. 60, 321. Krebs, H. A. (1954) Bull. Johns Hopkins H o d . B6, 19. Krimsky, I., and Racker, E. (1952) J . Biol. Chew. 198, 721. Le Fevre, P. G. (1948) 1. G m . Physiol. 31, 505. Le Fevre, P. G. (1954) Symposclr Sot. Exptl. Biol. 8, 118. Le Fevre, P. G., and Le Fevre, M. E. (1952) I. Gen. Physiol. Sa, 891. Lindvig, P. E., Greig, M. E., and Paterson, S. W . (1951) h c h . Biochm. and Biophys. SO, 241. Ling, C. T., and Chow, B. F. (1954) Federation Proc. 13, 253. London, I., and Schwarz, H. (1953) J. C l b . Itwest. a,1248. Lovelock, J. E. (1954) Nature 173, 659. Lovelock, J. E. (1955) Biochem. J. 60, 629. Lundsgaard, E. (1933) Ergeb. Enzymfmsch. 2, 179. Macleod, J. J. R. (1913) J. Biol. C h . 16, 497. Maizels, M. (1951) I. Physiol. (London) 112, 59. Maizels, M. (1954a) J. Physiol. (London) 186, 263. Maizels, M. (1954b) Symposia SOC.Exptl. Biol. 8, 202. Mbnyai, S,. and SzCkely, M. (1954) Acta. Physiol. Acad. Sci. Hung. 6, 7.. Mapson, L. W., and Goddard, D. R. (1951) Biochem. I. 49, 529. Mabhias, P. J., and Sheppard, C. W. (1954) Proc. SOC.Exptl. Bsol. Med. 86, 69. Morrison, W. L., and Neurath, H. (1953) I. BioE. Chem, !NO, 39. Motulsky, A. G., Giblett, E., Coleman, D., Gabrio, B. W., and Finch, C. A. (1955) 1. Clin. Invest. 34, 911. Muir, H. M., Neuberger, A., and Peronne, J. C. (1952) Biochem. J. 52, 87. Pal4us, S. (1947) Arch. Biochem. and Biophys. U,153. Parpart, A. K., Barron, E. $. G., and Day, T. (1947) Biol. Bull. @8,199. Ponder, E. (1948) “Hemolysis and Related Phenomena.” Grune, New York. Prankerd, T. A. J., and Altman, K. I. (1954) Biochem. I. 68, 622. Prankerd, T. A. J. (1955) Clin. Sci. 14, Prankerd, T. A. J., Altman, K. I., and Young, L. E. (1954) I: C&n. Invest. I, 957. Prankerd, T. A. J., Altman, K. I., and Young, L. E. (1955) I . Clin. Invest. M,1268.

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Racker, E. (1951) J. Biol. Chem. 190, 685. Rapoport, S. J., and Guest, G. M. (1939) J. Biol. Chew. 129, 781. Rapoport, S. J., and Luebering, J. (1950) J. Biol. Chem. W, 507. Rosmberg, T.,and Wilbrandt, W. (1952) Ilztern. Rev. Cytol. 1, 65. Rothstein, A. (1954) Protoplmtologia 2, E4. Ruhenstroth-Bauer, G. (1950) Noun~n-Schmiedeberg’sArch. ezptl. Pathol. Pharwakol. all, 32. Ruhemtroth-Bauer, G. (1953) 2. ges. Exptl. Med. 121, 475. Sanberg, A. A., Lee, G. R., Cartwright, G. E., and Wintrobe, M. M. (1955) J. Clin. Invest. 84, 1823. Schatzmann, H.J. (1953) Helv. Physiol. et Pharmacol. Acta 11, 346. Schelling, V. (1932) I. Biol. Chem. 96, 17. Schild, K. T.,and Maurer, W. (1952) Biochem. 2. SaS, 235. Schwarz, V., Goldberg, L., Komrower, G. M., and Holzel, L. (1955) B i o c h . J . 59, xxii. Shapiro, B . (1947) Biochem. J. U,151. Sjolin, S. (1954) Act4 Paediat. 43, Suppl. 98,5. Solomon, A. K. (1952) J. Gen. Physiol. 36, 57. Solomon, A. K., and Gold, G. L. (1955) J. Gm. Physiol. 98, 371. Solvonuk, P. F., and Collier, H. B. (1955) Can. J. Biochem. SS, 38. Spicer, S. S.,and Clark, A. M. (1949) I. Biol. Chem.179, 987. Sutherland, E.W., Postern&, T. Z., and Cori, C. F. (1949) I. Biol. Chem. 181, 153. Taylor, I. M.,Weller, J. M., and Hastings, A. B. (1952) Am. I . Ptcydol. 168, 658. Tsuboi, K. L.,and Hudson, P. B. (1953) Arch. Biochem. and Biophys. a,339. Wafiburg, O.,Kubowitz, F., and Christian, W. (1930) Biochem. 2. 227, 245. Widdas, W. F. (1954) I. Phydol. (Lodot&)m, 163. Widths, W. F. (1955) J. Physiol. (London) 127, 318. Wilbrandt, W. (1954) Proc. SOC.Exptl. Bio!. Med. 86, 136. Witbrandt, W., Guenskrg, E., and Lauener, H. (1947) Helv. Physiol. ef Pharmucol. Acta I, C20. Zittle, IC,A., Dellamonica, E. S., and Custer, J. H. (1954) Arch. Biochem. and Biophys. 48, 43.

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Uptake and Transfer of Macromolecules by Cells with Special Reference to Growth and Development A. M. SCHECHTMAN Department of Zoology, University of California, Los Aweles, Califor& Page I. Introduction ........................................................ 303 11. Transfer and Uptake of Macromolecules ............................. 304 1. Uptakes versus Transfer ......................................... 304 2. Uptakes by Nonphagocytic Cells ................................. 304 3. Uptake by the Avian Ovum ..................................... 307 4. Conditions Favorable for Demonstration of Uptakes ............... 309 111. Macromolecular Uptake by Embryonic Tissues ....................... 310 1. The Role of Phagocytosis in the Embryo ......................... 310 2. Criteria for Transfers to the Embryo ............................. 311 3. Transfers to Mammalian Embryos .............................. 312 4. Transfers to Avian Embryos ..................................... 313 5. Uptakes in Tissue Cultures and from Transplants.. ................. 315 6. Intracellular Sources of Protein ................................. 316 IV. Functions of Transferred Macromolecules in Embryos ................. 317 1. Trace vwsus Metabolic Transfers ................................. 317 2. Antibody and General Functions ................................. 317 3. Passive Plasma Formation ....................................... 318 4. Differentiation Functions ......................................... 319 V. References ......................................................... 320

I. INTRODUCTION The entrance of soluble heterologous proteins and other macromolecules into cells has been known for some time as a result of immunological studies. However, so long as soluble macromolecules were found mainly in such organs as liver, spleen, and bone marrow-centers of high phagocytic activity-it was reasonable to assume that some process similar to phagocytosis was involved. The uptake of soluble macromolecules might be visualized, in the words of Haurowitz (1954, p. 6 ) , as “a process akin to phagocytosis, an engulfing of the antigen molecules by pseudopodia or protuberances of the cytoplasm.” Such an assumption avoids the possibility that the proteolytic hypothesis of protein absorption may not be applicable universally. However, if the processes of phagocytosis and protein uptake are dissociable, if cells capable of the one are incapable of the other, then we may question whether the phagocytosis hypothesis is applicable to both types of phenomena. Whatever may be the mechanism by which soluble macromolecules are taken into cells, its dissociability from phagocytosis, and the association of foreign proteins with microsomes and mitochondria

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(Crampton and Haurowitz, 1950, 1951; Fields and Libby, 1952; Eichenberger, 1953), call the proteolytic hypothesis into question. We have perhaps gone too far in extrapolating this hypothesis from its original foundations-studies of digestion in the gut, of the specialized red blood cell, and of entire organisms such as invertebrate eggs and unicellular and relatively simple plants. In conformity with the generalized proteolytic concept of protein absorption, it has been assumed generally that the embryo synthesizes its proteins from small molecular metabolites transmitted directly from the maternal organism or indirectly through the yolk. A well-known exception is the antibody of embryonic plasma and tissues which is of maternal synthesis. Other macromolecules which escape proteolysis and enter the embryo include the natural isoagglutinins (better called lectins, Boyd and Shapleigh 1954, since both as to origin and physiological function their equivalence to antibodies is highly doubtful), heterologous serum proteins, and possibly enzymes. The passage of these macromolecules justifies the working hypothesis that macromolecular transfers other than antibodies may play a significant role in embryonic development.

11. TRANSFER AND UPTAKE OF MACROMOLECULES 1. Uptakes versw Transfers When a protein is injected into the circulation and later identified within a cell which is in direct contact with the circulation, it is logical and highly probable that the protein passed directly into the cell. This is cellular uptake, macromolecular transmission at its simplest. On the other hand, when the protein passes from one fluid compartment into another or into cells, and in the process traverses one or more layers of cells, we may, following the usage of Brambell and his associates (1952), apply the terms passage or transfer. Different as well as common mechanisms of transmission may be involved in the two processes. Uptake may involve phagocytosis at the submicroscopic level or actual passage into the cytoplasmic surface of the cell. Transfer may involve the same processes plus their converses, de-phagocytosis and secretion ; or interstitial passage between cells (the “seepage” of Brambell et al., 1952) ; or possibly a combination of both. Strict use of these terms is of minor importance; the possibility that different mechanisms may be involved is worthy of note.

2. Uptakes by Nonphagocytic Cells There can be little doubt that cells which phagocytize visible particles also take up soluble proteins and polysaccharides. The uptake of soluble antigens by organs rich in reticulo-endothelial elements was clear from the older

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literature (see Gay, 1935) and is not a controversial matter in the recent studies with radioisotope-labeled proteins, dye proteins, and native proteins detected by the use of fluorescein-labeled antibodies. As to uptake by nonphagocytic cells the results vary, and have perhaps been influenced by the types of macromolecules used. Autoradiographs of the spleen and liver of mice injected with S3s-sulfanilazoglobulinshowed most of the activity in the Kupffer cells and “comparatively little” in the hepatic cells ; in the spleen activity was found in the red pulp but not in the white pulp (Haurowitz, 1954). On the other hand, Coons (1953) and his associates, using fluorescein-labeled antibody, found that crystalline egg albumin, crystalline bovine albumin, human gamma globulin, and several bacterial polysaccharides are promptly recognizable in hepatic cells as well as in reticulo-endothelial cells. The fluorescein-antibody occurred in the connective tissue matrix of all organs studied, in lymphocytes, lymphoid follicles of the spleen and lymph nodes, and in the epithelial cells of the proximal convoluted tubules of the kidney. Pneumococcal polysaccharides were detected in the heart myocardial cells although the three proteins were not. All three proteins could be found in the nuclei of hepatic cells, renal tubule epithelium, lymphocytes, fibroblasts, and reticulo-endothelial cells. Stark ( 1955), using C’*-labeled Pneumococcus polysaccharide, concluded that spleen, liver, kidney, muscle, heart, stomach, intestines, and red cells contain about the same amounts of the polysaccharide eight months after injection. Gitlin et al. (1953) detected substances possessing the antigenicity of various human plasma proteins, in the cytoplasm and nuclei of human tissues. Using anti-beta lipoprotein, fluorescence could be observed in nuclei of a variety of cell types. Gamma globulin antigenicity was found in the nuclei of both hepatic and Kupffer cells. Tissues from a patient with almost complete absence of blood gamma globulin showed only faint fluorescence ; after injection of normal human serum the same tissues showed clear fluorescence. In a study of children with congenital deficiency of gamma globulin or fibrinogen this was confirmed (Gitlin, 1954). Crampton and Haurowitz (1950) injected ovalbumin and beef serum pseudoglobulin, both iodinated with SS1,into rabbits and fractionated the livers into nuclear, mitochondrial, microsomal, and supernatant fractions. Most of the activity was associated with the mitochondrial fraction, a small amount with the nuclear fraction. Fields and Libby (1952) injected I1slhuman serum albumin and gamma globulin and Psa-tobacco mosaic virus into mice and also obtained high activity in the mitochondrial fraction. Mice immunized with one of these antigens were shown to accumulate more of the same antigen on or in their liver mitochondria. These experi-

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ments indicate that foreign proteins, rather than being segregated into hypothetical vacuoles, become associated with normal cellular components. Unfortunately it is impossible to say if nonphagocytic cells were involved. However, Eichenberger ( 1953) found that ovalbumin becomes associated with the mitochondria of the kidney. Although it would appear from the above and from studies of avian egg considered below, that macromolecular transfers are probably not limited to phagocytic cells, the variant of phagocytosis called “cell drinking” or pinocytosis (Lewis, 1931) deserves special attention. The uptake of proteins in solution by engulfing of droplets of fluid surrounding cells has been described in tissue cultures of normal macrophages and fibroblasts (Lewis, 1931), in some but not all tissue cultures prepared from mouse and rat tumors (Lewis, 1937),and in several species of amoeba (Mast and Doyle, 1934; Holter and Marshall, 1954). In addition, it has been suggested that the vacuoles seen in chick embryo fibroblasts and in certain malignant cells may originate in this manner. Now since macrophages have been observed to transform into fibroblasts, and tumor cells are occasionally seen to engulf solid matter, it would appear that the observed instances of pinocytosis are limited to phagocytizing cells. However, we cannot conclude that pinocytosis is merely phagocytosis acting upon a purely liquid medium, since Lewis (1937)notes that malignant cells which are seen only occasionally to ingest dead cells may be very active in engulfing liquid media. Hence it is quite possible that cells not known to phagocytize may take up soluble proteins by the drinking method, especially since Holter and Marshall (1954) observed that pinmytizing amoebae contain vacuoles ranging in size from 10 p to the limits of resolution of the microscope. It thus becomes probable that amoebae take up droplets too small to be detected with the ordinary microscope, and this, in turn, suggests thatother cells may practice a form of pinocytosis which is difficult to detect. However, all this remains purely hypothetical and until we have definite evidence that such elements as hepatic cells, lymphocytes, heart myocardial cells, renal tubule epithelium, and ovarian eggs of birds carry on some form of pinocytosis, we can hardly accord this process a role of general significance in macromolecular uptakes. Moreover, pinocytosis cannot be considered a method of transfer as distinct from uptake, since all observers agree that the engulfed droplets are not extruded from the cells but shrink and disappear in the cytoplasm, their contents probably having been digested and absorbed. Chapman-Andresen and Holter (1955)have shown that C1*-labeled glucose passes rapidly through the surface of the pinocytized vacuoles into the cytoplasm, and it is doubtlessly true that amino

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acids derived from the digestion of engulfed proteins do the same. Finally, it may be noted that there is no necessity at present to assume that macromolecular uptakes and transfers are accomplished by any one kind of mechanism. Phagocytosis, pinocytosis, the fine channels of brush-border cells, facilitated diffusions, and active transports may all be involved. The discussions of Danielli (1954) and Brambell and Hemmings (1954) may be recommended to gain a broad perspective of the various possibilities.

3. Uptake by the Avian Ovum The literature apparently contains no evidence that the ovarian egg in its phase of rapid growth is capable of phagocytosis in the usual sense. Cytological evidence suggests that young wcytes take up cellular particulates from the follicle cells, but this ceases when the oijcytes reach a diameter of about 800 p (Brambell, 1926). Much later in the development of the ovum bovine serum crystalline albumin and gamma globulin are taken up by the cell after intravenous injection of the hen (Fig. 1). Each completed ovum may contain as much as 15-20 mg. of bovine protein per yolk according to estimates made on the basis of serological titers (Knight and .E 50e!

g

.-c C

c % 40-

R

l\

I t

1 % I I I I

t \ t \

FIG.1. Bovine protein concentrations in the “livetin” fraction of yolks from eggs laid by hens injected intravenously with 1.5 gm. of protein as a 5% solution in saline. Concentrations were estimated from serological titers obtained with antisera of tested sensitivity (Knight and Schechtman, 1954).

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Schechtman, 1954). Considering the weight of the ovum at ovulation and the total body weight of the hen, this is what one would expect if the proteins were evenly distributed through the body. Considering the declining increments of yolk deposited in the ovum during the last 2 or 3 days in the ovary (Romanoff, 1949) there is probably an elective accumulation of bovine protein in the several days following injection. Extracts of the yolk showed that the two bovine proteins exist in a free or loosely bound state since large amounts of yolk protein could be precipitated without altering the titers of the bovine proteins. In electrophoresis experiments the bovine proteins showed their normal respective electrophoretic mobilities (Fig. 2). There can be little doubt that the bovine macromolecules were transferred with relatively little or no hydrolysis into the ovum. The period of perceptible transfer into the ovum corresponds with the phase of maximal growth. This occupies approximately the last week of residence in the ovary when 8549% of the total yolk is laid down. This means that if a hen is injected on day 1, the eggs laid on days 243 contain yolks which were in rapid growth at the time of the injection. Since intravenously injected foreign proteins are detectable in the blood for at least 4-8 days, young ova will enter the growth phase during a corresponding period after the injection. The distribution of the injected bovine proteins conformed quite well to these conditions (Fig. 1). Maximal titers for both bovine proteins occurred in eggs laid 5-8 days after injection and the entire period of transfer extended over about 2 weeks. Riddle (1911) found that Sudan 111passes into ovarian eggs exclusively during the phase of rapid growth. Ramon (1928a, b) injected tetanus toxin subcutaneously into hens and found highest toxin concentration in yolks laid on the 5th day, and none after about 2 weeks. Maximum radiophosphorus activity occurs in eggs laid 5-8.6 days after injection of inorganic phosphate (Chargaff, 1942 ; Spinks et d.,1948; Smith et al., 1954). Maximal values of C1*activity added as labeled acetate to the feed of laying hens were reached in eggs laid on the 6th day (Kritschevsky et al., 1951). Living Newcastle disease virus is deposited in egg yolks at 2-9 days after injection (Zargar and Pomeroy, 1950). The deposition of such varied materials corresponds to the period of rapid growth and suggests that the physiological conditions of the ovum and perhaps of the associated follicle cells and blood vessels is more important in determination of uptake than chemical nature and molecular size. Ovarian eggs of diverse types of reptiles take up diphtheria and tetanus toxins after injection into various parts of the body (Grasset and Zoutendyk, 1929). This is evidently not a matter of toxic injury since reptiles

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susceptible to the toxins and those which show no apparent ill effect allow the toxin to pass. No toxin could be found in eggs present in the uterus at the time of injection.

4. Conditions Favorable for Demonstration of Uptake It appears, then, that there is no strict correspondence between phagocytic capacity and macromolecular uptake. Since some and possibly all

FIG.2. Electrophoretic patterns of yolk extract (“livetin” fraction) showing the localization of bovine gamma globulin and albumin after injection of the bovine proteins into the circulation of the hen. Above, typical patterns obtained with the Tiselius apparatus. Below, filter paper electrophoresis patterns. No, 1. Normal yolk extract without bovine protein activity. No. 2. Normal yolk extract with added bovine gamma globulin. No. 3. Normal yolk extract with added bovine crystalline albumin. No. 4. Livetin from an egg laid 6 days after injection of the hen with bovine gamma globulin. Localization of bovine protein, as shown by serological test of eluates, is indicated by solid arrowhead. No. 5. Livetin from an egg laid 5 days after injection of hen with bovine albumin. Localization and relative magnitudes of the bovine protein concentrations is indicated by hollow arrowheads (Knight and Schechtman, 1954).

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phagocytic elements are also capable of macromolecular uptake, the latter capacity must have a wider distribution. Why has it not been apparent in nonphagocytic cells ? The causes are no doubt partly methodological, since formerly only foreign antigenic substances could be traced. The wellknown proclivity of the reticub-endothelial elements for foreign particulates and soluble antigens resulted in foreign protein concentrations which were easy to detect. In addition the soluble antigens seem to be retained longer in the phagocytic cells. Coons (1953) noted that several proteins could be detected in reticulo-endothelial cells after all traces had disappeared from other cells. Bird and reptilian ova resemble reticulo-endothelial elements in these respects. They accumulate materials rapidly and conserve them ; quantitatively they far exceed the reticulo-endothelial cells in both respects. As an example of retention, bovine protein transferred from the hen’s circulation to the yolk can be detected in apparently undiminished concentration after 18 days incubation and at least 24 days after injection into the hen (Sounhein and Schechtman, 1955). 111. MACROMOLECULAR UPTAKE BY EMBRYONIC TISSUES 1. The Role of Phagocytosis in the Embryo

Phagocytosis in the adult vertebrate is largely concerned with the elimination of foreign materials and homologous necrotic tissues, and the establishment of immunity. In the embryo, however, phagocytosis serves totally different functions : it is largely concerned with uptake and transportation of nutriments. Phagocytosis is most pronounced in forms which possess a large amount of nutriment located outside the embryonic body-fish, reptiles, and birds. In some mammals the outermost layer of embryonic tissue (the trophoderm) engulfs maternal red cells and uterine cellular debris and is assumed to perform a similar nutritive function. The phagocytic capacities of the chick embryo were shown by Heine ( 1936), Steinmuller ( 1937) , and Canat and Opie (1943). All three germ layers of the early chick can phagocytize red cells and the carbon particles of India ink. The carbon particles can be transmitted by cell to cell transfer from the endoderm to other parts of the body. During development phagocytic capacity is restricted to specific types of cells but such differentiation is not associated with immunity functions. Thus the limitation of phagocytic capacity occurs progressively through the first half of development whereas the earliest evidence of an immunity reaction to bacteria is seen at about the 14th-15th days of incubation (Goodpasture and Anderson, 1937; Buddingh and Polk, 1939a, b, c). Up to this time phagocytization has little perceptible ill effect upon bacteria ; on the contrary, the intracellular en-

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vironment seems to be favorable for the multiplication of certain bacteria. Even when antibacterial action becomes apparent, phagocytosis per se is of doubtful significance, for it is at about the same time (14th-15th day) that antibodies originally deposited in the yolk become effective in humoral reactions. It is, then, quite apparent that phagocytic uptake during at least the first half of the developmental period of the chick does not have the same functional significance as in later life. It should be added that phagocytosis and cell-to-cell dissemination of yolk, although highly probable, is no more than a deduction based on the observation of yolk in various cells and the ability of such cells to take up and disseminate carbon particles. This problem should be investigated by the use of marked yolk.

2. Criteria for Transfers to the Embryo The passage of soluble macromolecules was first demonstrated in the embryo by the work of Ehrlich (1892) although it was not seen in this light until much later when the nature of antibodies became known. Curiously enough, the older theory of fetal and neonatal immunity, which survived well into the present century, postulated that a great variety of bacterial and viral antigens passed across the placental barrier. The discard of this hypothesis began with Ehrlich’s (1892) demonstration that immunity against the plant toxins abrin and ricin passes from the mother to the fetus and newborn by way of the placenta and mammary secretion. Ehrlich established one of the criteria for antigen transfer which has been used widely in subsequent investigations and has led to the erroneous impression that antigens are not passed to the young. Not knowing whether the fetus was capable of forming antibodies, Ehrlich considered the possibility that antigens might pass into the fetus and give rise to antibody in addition to that derived from the mother. His conclusion that abrin and ricin are not transferred was based on two observations. First, these substances although highly toxic for the adult caused no apparent injury to the fetus. This was not considered a decisive criterion since the fetus might have higher tolerance than the adult. Secondly, the immunity of the fetus and newborn was of short duration (passive immunity) in comparison with the durable (active) immunity of the adult. I have dwelt on Ehrlich’s classical experiment because many investigators have relied upon the duration of immunity, or the trend of antibody titers in the young, as a criterion for the passage of antigens to the fetus or newborn. Since it is usually true that the young organism lacks durable immunity, after inoculation of the mother, the impression gained ground

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that antigens, and by extrapolation other macromolecules, do not reach the fetus. The inadequacy of this criterion is apparent when we consider that the rate of clearance of antigens from the blood or tissues of the embryo is quite unknown. Thus, the antigen may be degraded or masked before the antibody-forming mechanism has matured. In preliminary experiments with bovine serum proteins taken up by the chick embryo from the yolk sac, we recently obtained evidence that the proteins disappear from the circulation within a few days and are not detectable in tissue extracts. The criterion is further inadequate since antigens may specifically inhibit subsequent antibody formation. This possibility is indicated by the work of Buxton (1954), Kerr and Robertson (1954), and Hanan and Oyama (1954). Experiments utilizing direct tests for transferred antigens have been very few in comparison with those concerned with transferred antibodies. It is understandable that from the practical viewpoint of those interested primarily in the immunity of the young, there seems to be little value in looking for antigens which are neither injurious nor capable of conferring immunity. This view must now be questioned since, as already mentioned, humoral antibody production may be inhibited in later life. Moreover, there are indications that tissue antigens induce some fundamental alterations in embryos (possibly through modification of antibody production) which affect interspecific tissue transplantation (Billingham et d., 1953).

3. Transfers to Mammalian Embryos We shall omit consideration of homologous antibody transfers since these are beyond doubt. Direct tests of embryonic tissues for other types of macromolecules have yielded fairly consistent data in favor of the transfer of plasma proteins but not of substances normally foreign to plasma. Nattan-Larrier et d. (1927a, b) injected tetanus toxin and anatoxin into pregnant rabbits during the last third of pregnancy and could not detect these materials in the fetal blood, although toxin remained in the maternal circulation for at least 48 hr. Negative results were also obtained with bacteriophage and diphtheria toxin injected into guinea pigs and rabbits during the last few days of pregnancy. In contrast, Nattan-Larrier ( 1935), using complement-fixation, could detect horse serum, egg white, and horse antitoxin in fetal guinea pigs after injecting the mothers during advanced pregnancy. Holford (1930) injected horse hemoglobin, crystalline egg albumin, horse and beef sera, and fractions of these sera into rabbits during the last week of pregnancy. Positive precipitin tests were obtained in the blood of a variable percentage of the newborn. Horse hemoglobin could

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not be detected. On the other hand, egg albumin which contains one or more proteins closely resembling serum proteins, was readily detected. Antibody from man, dog, guinea pig, cow, horse is transferred to the rabbit fetus after injection into the uterine lumen and there is clear selectivity of one kind of antibody over another (Batty et al., 1954). Bovine crystalline serum albumin injected into the circulation of the pregnant rabbit on the 19th day post coitum was detected by precipitin test in the fetal blood on the 20th day (Schechtman and Knight, 1955). McCance et al. (1949) described an apparent instance of enzyme transfer into the newborn puppy. Cholinesterase activity is relatively high in dog colostrum and in the blood of suckling puppies. It occurs at lower levels in puppies fed evaporated cow’s milk. For the early stages of mammals our knowledge of macromolecular transfer is limited to the work of Brambell and his associates on the rabbit (Brambell et al., 1948; Brambell et al., 1951). The large cavity of the rabbit blastocyst at 7-8 days Post coitum is essentially a yolk sac. Electrophoretic and ultracentrifugal examination of its fluid contents showed components like those in the plasma of the adult. Fibrinogen is present as shown by clotting reactions. Agglutinating antibodies for Brucelta, produced in rabbits or in cattle, were found in the yolk sac fluid after injection into the mother. The resemblance between the components of the yolk sac fluid and adult plasma, the proportions of the several components, and the demonstrated uptake of antibody injected into the mother, make it highly probable that the nonantibody macromolecules are transferred from the maternal circulation. Morris (1953) could not detect fibrinogen in the early yolk sac of the hedgehog and ferret ;in later development the yolk sacs fluid showed measurable quantities of protein at concentrations of about 0.1 mg./ml. in the ferret, and attaining 2.9 mg./ml. in advanced pregnancy of the hedgehog. The presence of proteins closely resembling or identical with those of the serum is not limited to the rabbit yolk sac. About one-fifth of the total protein of the bird yolk is composed of such proteins on the basis of amino acid composition, N :P ratios, etc. (Jukes and Kay, 1932). Some of the components have been shown to be antigenically and electrophoretically similar to or identical with adult serum proteins (Shepard and Hottle, 1949; Marshall and Deutsch, 1950,1951). However, fibrinogen has not been described in bird yolk. 4. Transfers to Avian Embryos The transfer of antibodies in birds, from hen to yolk to embryo, is well established. When we turn to transfers of other macromolecules there is little available evidence. Both bovine crystalline albumin and gamma globu-

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lin have been detected in the blood stream of the embryo after injection into the yolk sac (Schechtman and Knight, 1955). Gamma globulin appears within 8 hr. after injection and apparently disappears within 3 4 days. The embryo shows “dosed” and “open” periods of development and these correspond quite well with periods of homologous antibody uptake. Thus, gamma globulin was detected in only a few embryos at 8-9 days of incubation, in many at 10-15 days, and in every specimen at 16-20 days. The earliest uptake of antibody from the yolk into the blood is at about 10 days incubation (Jungherr and Terrell, 1948 ; Buxton, 1952) and antibody titers increase toward the later days of development, The concentration of bovine protein in the serum was 0.410.8mg./ml. in some instances, and higher values will doubtless be obtained with larger injection doses. According to Emanuelsson (1951) the yolk contains proteolytic enzymes ; obviously antibodies and the bovine proteins must escape these enzymes. It is possible that the enzymes are bound to yolk granules and have little effect on proteins existing in the soluble state, or perhaps they are attached to special types of granules. During development yolk shows an increasing free amino acid or peptide content (Emanuelsson, 1951; Smith et al., 1955). It may be suggested that these are derived largely from yolk phosphoproteins which are certainly hydrolyzed before they enter the embryonic blood (Marshall and Deutsch, 1950). A possible instance of uptake of antigenic substances by the chick embryo is recorded by Budon (1954). Killed Salnzonella pullorurn were injected into the yolk sacs of W a y embryos. In one group no antibodies were detectable at 27 days after hatching; in another group rising antibody titers were found in the second and third post-hatching months. This late formation of antibody indicates that some kind of Salmonella antigen, whether the entire bacteria or a soluble component, entered the chick‘s tissues but it is uncertain when the entry took place. In view of the known absorption of bacterial antigens from the digestive tract of the adult, it would not be surprising if small amounts were taken up by the lining of the yolk sac. A striking observation on uptake was made by Brandly et a1. (1946) who found that antibodies can exist in the tissues of embryos from immunized hens when none is detectable in the serum. Neutralizing activity for the virus of Newcastle disease occurred in the body extracts of 6-day embryos and in the fetal membranes, but could not be detected in the serum until some time between 12 and 15 days of incubation. The authors suggest that the apparent absence of antibodies in the blood may indicate some physiological immaturity, perhaps the lack of some substance required

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for this kind of antibody activity. It is also possible that the antibody entered the embryo much earlier by way of the circulation, or perhaps during the first few days of incubation when yolk granules appear to be phagocytized.

5. Uptakes in Tissue Cultures and from Transplants A promising approach to macromolecular transfers is the method of labeled tissue culture media used by Francis and Winnick (1953). One or two Cl4-1abeled amino acids were incorporated into embryonic chicks which were then converted into embryo extracts for the culture of heart fibroblasts. From the point of view of macromolecular transfers experiments of this kind are of little significance unless the role of split-off amino acids and their metabolites is given due consideration. To overcome this difficulty an excess of free and unlabeled amino acids was added to the culture medium, thus providing competitors for radioactivity which might be split off as amino acids. The partial effectiveness of this procedure in reducing specific radioactivity in the tissue proteins showed that labeled free amino acids were being utilized. However, the amount of activity and the proportions of the two labeled amino acids in the protein fraction of the fibroblasts indicated that peptides or whole proteins probably enter the cells. The conversion of C14 into small metabolites other than amino acids, and their utilization in protein synthesis, requires further investigation. It is interesting that the nonprotein of the medium seemed to be the main source of tissue protein during the first 4 days of culture, but from the 4th to the 6th days both protein and nonprotein contributed about equally. Winnick and Winnick (1953) obtained similar results. The experiments of Langman (1953),considered below, offer a striking parallel. There can be no doubt from these experiments that amino acids are utilized by tissue cultures and this, of course, is amply supported by the large body of work on the constitution of optimal culture media. It should, however, be noted that the necessity for macromolecules in the culture of animal tissues has been emphasized for a long time and that media lacking macromolecules are still inferior to complex “natural” media for active growth of animal tissues over long periods (Waymouth, 1954). Ebert (1954) restudied the enlargement of the embryonic spleen which follows upon implantation of pieces of adult chicken spleen on the chorioallantoic membrane. This is not a splenomegaly in the ordinary sense of the term, for heterologous (mouse) spleen is not effective, and other organs of the chicken (e.g., thymus, liver, kidney) are less effective than the spleen.

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After implantation of Saa-methionine-labeledtissues from the adult chicken and mouse on the chorioallantois, the protein fraction of the embryonic spleens showed more than twice as much specific activity as the proteins of the liver and kidney from the same embryos. Such selective transfer of Sari to the homologous host organ was also observed in the case of the kidney. In these experiments an attempt was made to rule out the effects of small Sa6-metabolites by injecting the Ss6-methionine into the yolk sac and implanting pieces of normal spleen (without the radioactive label) on the chorioallantois. In such embryos there was no selective accumulation of Sa6 in the spleen. These results are consistent with the hypothesis that units larger than amino acids, and possibly entire proteins, pass from the adult implant into the host spleen. Langman (1953) reported more direct evidence for macromolecular uptake in vitro. Pieces of fetal rabbit gonad were maintained in media, containing rabbit or cat serum plus spleen extract. Kept for a short time (e.g., 6 days) in cat medium, the rabbit gonadal fragments gave no reaction with anti-cat sera. After 18 days in cat medium the tissue extracts showed positive precipitin tests. It seems rather improbable that adsorbed cat antigens are involved, since 6 days of culture would seem to provide ample opportunity for adsorption.

6. ~ ~ ~ r a c e l l Sources u ~ a r of Protein The studies of Christensen and his associates (1948-1952) have emphasized the importance of intracellular amino acid concentrations in embryonic and other rapidly growing cells. Although free amino acids constitute a very small fraction of the total nitrogen of any tissue, there appears to be a tendency to maintain the intracellular level above that of the surrounding medium. This concentrative activity is more pronounced in earlier stages of development than in later stages, and in regenerating liver as compared with normal liver. In the rapidly growing tissues, including certain tumors, the differences between intracellular and extracellular amino acid concentrations may reach ratios as high as 12:l (Christensen and Streicher, 1948; Christensen and Henderson, 1952; Christensen and Riggs, 1952; Christensen et al., 1952). Relatively high amino acid concentrations in embryonic and tumor tissue were also reported by Winnick and Winnick (1953) and Babson and Winnick (1954). It should be emphasized that the concentrations of free amino acids within cells may be increased by enriching the medium with free amino acids. This does not necessarily mean that rapidly growing cells obtain their free amino acids exclusively from the unconjugated pool in the plasma, nor that amino acids absorbed

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as such are the main source available for protein synthesis. Yuile et al. (1951) found that lysine-C1* incorporated into plasma proteins is used more efficiently than the free amino acid made available from the digestive tract. Dogs fed ly~ine-C?~-proteineliminated much more C14 activity in the respiratory air and in the urine than dogs receiving the protein parenterally. Babson and Winnick (1954) have pointed out that free leucine-C14 seems to be utilized less efficiently than le~cine-C~~-protein in protein synthesis by Walker carcinoma. This tumor accumulated higher specific activity than liver or kidney when free 1 e ~ c i n e - Cwas ~ ~ used, but the difference was accentuated with radioplasma. Since the addition of an excess of free nonradioactive leucine did not greatly decrease incorporation of Cl4activity from radioplasma, it is probable that the activity was transferred in some nonamino acid form and possibly as protein.

IV. FUNCTIONS OF TRANSFERRED MACROMOLECULES IN EMBRYOS 1. Trace versus Metabolic Transfers In considering the passage of macromolecules a distinction must be made between trace and metabolic transfers. The latter refers to materials which enter in sufficient quantities to play a significant role as sources of energy or structural materials. It is well known that a great variety of foreign antigens can gain entrance in trace quantities into the adult body from the digestive tract, nasal cavity, or lungs (Drinker and Yoffey, 1941). These can have little significance as sources of energy or structure. The embryo uses such great quantities of protein that transferred serum proteins, if they play any appreciable part in growth, ought to amount to some significant fraction of the total nitrogen used. There is some evidence that this may be true. We have already mentioned the quantities of bovine protein which appear to be transferred from the blood of the hen to the yolk, and from the yolk sac to the circulation. Since the circulating bovine protein is being removed from the blood, it seems probable that metabolic quantities are taken up. The work of Winnick and his associates, points to the same conclusion. Macromolecular trace transfers may have significance for the embryo in other respects, as they have for resistance and allergies in the adult body.

2. Antibody and General Functions The functions of transferred antibodies in the circulation of the young organism seem obvious but it may be doubted whether immunity is their sole function, It should be recalled that the term antibody includes dissimilar

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proteins varying in molecular size and physicochemical properties. Furthermore, the transfer of the so-called isoantibodies (see for example, Zuelzer and Kaplan, 1954) which have no immunity value for the embryo, makes it improbable that immunity functions are unique in macromolecular transfers. We have no reason to assume that these various proteins forfeit all other possible roles because they participate in immune reactions or cause trouble in blood transfusions ! Whatever specific functions are involved, molecular properties and amino acid content of transferred proteins must be assumed to permit them to function as physicochemical factors (osmotic pressure, transport, etc.) and sources of metabolites. As pointed out previously, the importance of transferred macromolecules in this respect will depend upon evaluation of their respective quantitative contributions. Until further evidence is available such generalized functions are the most plausible that can be assigned to macromolecules transferred to avian or mammalian yolk sacs or to the embryonic circulation. The same can be said of proteins, peptides, or other macromolecules, which are apparently taken into cells and incorporated into, or associated with, protein fractions of tissues. Even assuming the reality of such transfers into cells, the concept of incorporation-assimilation into the structure of cytoplasm-has as yet no justification. Obvious foreign materials such as heterologous proteins and tobacco mosaic virus become closely associated with mitochondria (Crampton and Haurowitz, 1950; Fields and Libby, 1952) without justifying the assumption of “incorporation” in this sense. Survival time in the cytoplasm is no better as a criterion since sulfanilazo-proteins and certain bacterial polysaccharides apparently persist for months (Haurowitz, 1954; Coons, 1953).

3. Passive Plasma Formation It has been suggested that certain of the yolk proteins in the bird or mammalian yolk sacs are transferred into the circulation and, with perhaps slight modifications, become functional plasma proteins ( Schechtman, 1952, 1955). The only direct evidence for this hypothesis is the uptake of homologous antibodies and of heterologous albumin and gamma globulin into the blood of the embryonic chick and rabbit. Jukes and Kay (1932) first speculated that the blood phosphoprotein of the hen may be transferred without intermediate hydrolysis into the ovum. However, there is little probability that it reaches the embryonic circulation in macromolecular form since Marshall and Deutsch ( 1950) were not able to find any phosphoprotein in the blood of the 13-day embryo. Other components of the yolk, egg white, and serum are very similar in

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their electrophoretic and serological behavior. The serum albumin and gamma globulin fractions of the chick during the second half of development closely resemble the ovalbumin and conalbumin respectively of the egg yolk and white (Marshall and Deutsch, 1951). Several unusual electrophoretic components which appear in the serum of the hen at sexual maturity have been studied by Brandt et ul. (1950), Marshall and Deutsch ( 1950), and Heim and Schechtman ( 1954). These “prealbumins” also occur in the embryonic serum until about the 3rd day after hatching, at which time they disappear until sexual maturation of the hen. However suggestive of transfers such evidence may be, in the last analysis it simply indicates similar materials at different places.

4. Differentiation Fztnctions Weiss (1947, 1950, 1955) and Ebert (1954) have suggested that transferred macromolecules may act as templates in somewhat the manner postulated for antigens in relation to antibodies. Entry of a suitable macromolecule into an embryonic cell would thus establish a new order of specificity and perhaps initiate a discrete step in morphological or functional differentiation. Nace ( 1953) has postulated that transferred serum proteins may serve as templates or models on which the embryo patterns its own serum proteins. Weiss (1950) has explored the possibilities of the template hypothesis in relation to various developmental processes, regeneration, cell division, and growth regulation, and the theoretical consequences that might ensue when a macromolecule of specific pattern is lost from or added to oriented molecular populations. A possible role of transferred macromolecules in permitting or stimulating differentiation of the vertebrate gastrula has been postulated (Schechtman, 1955). I t is suggested that the vertebrate embryo has evolved to a state of dependence upon the mother for certain complex substances including proteins and that it utilizes these macromolecules until its own synthetic capacities have matured. This is an extrapolation of our knowledge of the origin and function of antibodies. Winnick (1952) and Ebert (1954) have suggested that entire protein molecules or fragments thereof may be incorporated into the structure of cells, serving as ready-made building blocks. Comment has already been made on the lack of suitable criteria for the incorporation of macromolecules into cellular structure.

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V. REFERENCES Babson, A. L., and winnick, T. (1954) Cancer Research 14, 606. Batty, I., Brambell, F. W. R, Hemmings, W. A,, and Oakley, C. L. (1954) Proc. Roy. SOC.Bl42, 452. Billingham, R. E., Brent, L., and Medawar, P. B. (1953) Nature 172, 603. Boyd, W.C., and Shagleigh, E. (1954) J. Zmrrmttol. 78, 226. Brambell, F. W. R. (1926) Trans. Roy. SOC.B214, 113. Brambell, F. W. R., and Hemmings, W. A. (1954) Symposia SOC.Expt2. Biol. 8, 476. Brambell, F. W. R., Hemmings, W. A., and Henderson, M. (1951) “Antibodies and Embryos.” Athlone Press, University of London. Brambell, F. W. R., Hemmings, W. A., Henderson, M., and Oakley, 6. L. (1952) Proc. Roy. SOC.BUS, 567. Brambell, F. W. R., Hemmings, W. A., and Rowlands, W. T. (1948) Proc. Roy. SOC.B W , 390. Brandly, C. A., Moses, H. E., and Jungherr, E. L. (1946) Am. J . Vet. Research 7, 333. Brandt, L. W., Clegg, R. E., and Andrews, C. A. (1950) J. Biol. Chew. 101, 105. Buddingh, G. J., and Polk, A. (1939a) J . Exptl. Med. 70, 485. Buddingh, G. J., and Polk, A. (1939b) J. Exptl. Med. 70, 499. Buddingh, G. J., and Polk, A. (1939~) J . Exgtl. Med. 70, 511. Buxton, A. (1952) J. Gen. Microbiol. 7, 268. Buxton, A. (1954) J. Gen. Microbiol. 10, 398. Canat, E.H., and Opie, E. L. (1943) Ant, J . Pafhol. 10, 371. Chapman-Andresen C., and Holter, H. (1955) Exptl. Cell Research Suppl. 3, 52. Chargaff, E. (1942) J. Biol. Chem. 143, 505. Christensen, H. N., and Streicher, J. A. (1948) I. Biol. C h m . 176, 95. Christensen, H. N., and Henderson, M. E. (1952) Cancer Research l2, 229. Christensen, H. N., and Riggs, T. R. (1952) J. Biol. Chem. 194, 57. Christensen, H. N., Riggs, T. R., Fischer, H., and Palatine, I. M. (1952) J. BioZ. Chem. 198, I. Coons, A. H. (1953) in “The Nature and Significance of the Antibody Response” (Pappenheirner, ed.), p. 200. Columbia U. P., New York. Crampton, C. F., and Haurowitz, F. (1950) Science lE2, 300. Crampton, C. F., and Haurowitz, F. (1951) Federation Proc. 10, 405. Danielli, J. F. (1954) SymposM SOC.Exptl. Biol. 8, 502. Drinker, C. K., and Yoffey, J. M. (1941) “Lymphatics, Lymph, and Lymphoid Tissue.” Harvard U. P., Cambridge. Ebert, J. D. (1954) Proc. Notl. A d . Sci. (U.S.) (0, 337. Ehrlich, P. (1892) 2. Hyg. Tnfektiomtskrankh.12, 183. Eichenberger, M. (1953) Exptl. Cell Researck 4, 275. Emanuelsson, H. (1951) Nafwe 188, 958. Fields, M.,and Libby, R. L. (1952) J. Zmauwl. 09, 581. Francis, M. D., and Winnick, T. (1953) J. Biol. C h m . aOa, 273. Gay, F. P. (1935) “Agents of Disease and Host Resistance,” Chapter 6. Thomas, Springfield, 111. Gitlin, D. (1954) in “Serological Approaches to Studies of Protein Structure and Metabolism” (Cole, ed.), p. 23. Rutgers, U. P., New Brunswick.

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Hanan, R., and Oyama, J. (1954) I . Immmol. 73, 49. Haurowitz, F. (1954) ia “Serological Approaches to Studies of Protein Structure and Metabolism” (Cole, ed.), Rutgers U. P., New Brunswick. Heim, W. G., and Schechtman, A. M. (1954) J . Biol. Chem. 209, 241. Heine, F. (1936) W i l h ~ l mRoud Arch. Entwicklungsmech, Organ. 184, 283. Holford, F.E. (1930) J. Zmmunol. 20, 177. Holter, H., and Marshall, J. M., Jr. (1954) Compt. rend. tvav. lab. Carlsberg S/r. Chem. aS, 7. Jukes, T. H., and Kay, H. B. (1932) J. Nutrition 6, 81. Jungherr, E. L., and Terrell, N. (1948) Am. J . Vet. Research 9, 201. Kerr, W. R., and Robertson, M. (1954) J. Hyg. 62, 253. Knight, P. F., and Schechtman, A. M. (1954) J. Exptl. Zool. 127, 271. Kritschevsky, D., Grau, C. R., Tolberf B. M., and Krueckel, B. J. (1951) Proc. SOC.Exptl. Biol. Med. 76, 741. Langman, J, (1953) Koninkl. Ned. Akad. Wetwrchap. Proc. C60, 219. Lewis, W. H. (1931) Bull. Johm Hopkins Hosp. 49, 17. Lewis, W. H. (1937) Am. 1. Cancer aS, 666. McCance, R. A., Hutchinson, A. O., Dean, R. F. A., and Jones, P. E. H. (1949) Biochem. 3. 46, 493. Marshall, M. E., and Deutsch, H.F. (1950) J . Biol. Chem. 186, 155. Marshall, M. E., and Deutsch, H. F. (1951) J . Biol. Chem. 189, 1. Mast, S. O., and Doyle, W. L. (1934) Protoplasma 20, 555 Morris, B. (1953) I . Embryol. Enptl. Morpbol. 1, 147. Nace, G. W. (1953) I . Enptl. 2001.Im, 423. Nattan-Larrier, L. (1935) Rev. immunol. 1, 455. Nattan-Larrier, L., Ramon, G., and Grasset, E. (1927a) Ann. inst. Pasteur 41, 848. Nattan-Larrier, L., Ramon, G., and Grasset, E. (192%) Ann. inst. Parteur 41, 862. Ramon, G. (1928a) Compt. rend. SOC. biol. 99, 1473. Ramon, G. (192813) Compt. rend. SOC.biol. 99, 1476. , Riddle, 0. (1911) J. Morphol. !d2, 455. Romanoff, A. L., and Romanoff, A. J. (1949) “The Avian Egg.” Wiley, New York. Schechtman, A. M. (1952) Ann. N . Y.Acad. Sci. 66, 98. Schechtman, A. M. (1955) in “Biological Specificity and Growth” (Butler, ed.), p. 1. Princeton U. P., Princeton, New Jersey. Schechtman, A. M., and Knight, P. F. (1955) Nature 176, 786. Shepard, C. C., and Hottle, G. A. (1949) J . Biol. Chem. 179, 349. Smith, A. S., Knight, P. F., and Schwhtman, A. M. (1955) Unpublished data. Smith, A. H., Winget, C. M., and Blackard, J. R. (1954) Poultry Sci. 98, 908. Sounhein, E., and Schechtman, A. M. (1955) Unpublished data. Spinks, J. W. T., O’Neil, J. B., Jowsey, J. R., Lee, C. C., and Reade, M. (1948) Can. J . Research D%, 163. Stark, 0. K. (1955) J. Zmmunol. 74, 130. Steinmuller, 0. (1937) Wilhelm Roux‘ Arch. Entwicklwgsmech. Organ. lS7, 13. Waymouth, C. (1954) Intern. Rev. Cytol. 3, 1.

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Weiss, P. (1947) Yale 3. Biol. Med. 19, 235. Weiss, P. (1950) Quart. Rev. Biol. a6, 177. Weiss, P. (1955) in “Biological Specificity and Growth” (Butler, ed.), p. 195. Princeton U. P., Princeton, New Jersey. Winnick, T. (1952) Texas Repts. Biol. and Med. 10, 243. Winnick, R. E., and Winnick, T. (1953) 1. Natl. Cancer Inst. 14, 519. Yuile, C. L. Lamson, B. G., Miller, L. L., and Whipple, G. H. (1951) 1. Exptl. Med. 98, 539. Zargar, S. L,and Pomeroy, B. S. (1950) Am. J. Vet. Research 11, 272. Zuelzer, W. W., and Kaplan, E. (1954) Am. J . Diseases Children 88, 158.

Cell Secretion: A Study of Pancreas and Salivary Glands L C. U.JUNQUEIRA and G. C. HIRSCH Laboratory for Cell Physiology, Faculdade de Medicine, Ut&w&hde de Sdo Paulo, Brazil

I. Introduction ......................................................... 11. Ingestion ............................................................ 111. Synthesis ............................................................ 1. Microsomes ....................................................... 2. Mitochondria ..................................................... 3. Golgi bodies ...................................................... 4. Nucleus .......................................................... IV. Extrusion ........................................................... V. Kinetics ............................................................. VI. References ...........................................................

Page 323 326 328 328 335 345 351 353 355 360

I. INTRODUCTION Through every living cell flows a stream of molecules. In a general way the activities of this stream may be divided into three-ingestion, intracellular metabolism, and extrusion. The protoplasm is separated from the surrounding matter by a cell membrane, through which the living cytoplasm obtains material necessary for the vital processes. This influx of material may be called ingestion. Inside the protoplasm two main processes occur, i.e., the building up of molecules (anabolism) and their breakdown (catabolism). Anabolism is a series of processes by which simple molecules are bound together to form more complicated ones such as carbohydrates, proteins, fats, enzymes, and hormones. A stream of molecules is also constantly flowing out of the cytoplasm. The material passing out may be divided into three groups-products of secretion, recrements, and excretions (Fig. 1). a. Secretions: Products of more or less complex molecules that are built up, accumulated in the cytoplasm, and later extruded to the outside of the cell. The best known examples are hormones, digestive enzymes, mucus, and poisons. b. Recrements: Inorganic substances such as water and ions flow into the cytoplasm during ingestion and are eliminated as such (from “Rekrete” according to Frey-Wyssling (1945). c. Excretions: This third group is of waste material derived from the breakdown of cellular components.

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.I C. U. JUNQUEIRA AND G. C. HIRSCH

The following scheme elucidates this :

I

Ingestion

* anabolism (synthesis)

--+

.1

Eliminated material

catabolism (analysis)

I

+ Excretions > Products of secretion

I

> Recrernents

The term secretion is very old and derives from the Latin secretzcs, i.e., severed, placed apart ; or from secretere, to part, to separate, to sift.

1. Secretions: enzymes, mucus hormones, etc.

METABOLISM Anabolism Syntheeis of higher molecules

2. Recrements: HaO, ions 4 3. Excretions: waste material e.g. urea, uric acid, e t c . k

acquirement at lone, micromolecules e.g. amindacids, glucose, fatty ?ids

-[

H40,ions Catabolism Breaking down and origin of excretion material

As in many other scientific terms ending in “tion” (e.g., mutation, adaptation) the term secretion has been used in two different ways: for the material that has been separated and for the process by which this “separation” is carried out. For clearness sake one should therefore call the material “the product of secretion” and the process by which the material is separated or built up “the process of secretion.” Several dictionaries (Oxford Dictionary, 1946; Webster’s New World Dictionary, 1952) give the following meaning for the word secretion: “1). Process by which special substances are separated from blood or sap for service in the organism or for injection; 2). any substances produced by

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such process, as saliva, urine.” Such a definition emphasizes the process as being a separation from blood or sap, and derives from the widespread conception that saliva, for example, would be present as such in the blood and later separated from it by a kind of “mechanical filtration” into the cells of the salivary gland. It was assumed that blood pressure played a great role in this process of separation or “filtration.” Since Ludwig ( 1851) made his studies showing that in the submaxillary gland of the dog the secretory pressure exceeds the blood pressure, this view has been modified and the process of secretion is considered to be much more than a simple filtration from the blood. As a matter of fact Kolliker (1852) expressed the view that “The true glands either separate certain constituents from the blood, or by means of it, elaborate peculiar substances or structural elements, . . .,, The usual dictionary definition may therefore be correct in saying that special substances are “separated,” but not from the blood. They should state that these substances are elaborated to higher substances within the cytoplasm as a result of cellular activity. Haldane and Huxley (1929) in their definition restricted the term secretion to the products formed by the activity of a gland, e.g., bile, gastric and pancreatic enzymes, saliva, They add “secretion may also be the formation of such a substance.” Certainly the term secretion was at first applied to both : the products of glands and the extrusion of these products from glands. It would not be advisable to extend the term secretion to the expulsion of different excretion substances or of typical recrements as water or ions. Two revised editions of medical dictionaries are also not correct. Gould‘s Medical Dictionary, 5th ed., 1947, says : “Secretion : 1) The art of secreting or forming from materials furnished by the blood a certain substance which is either eliminated from the body or used in carrying on special functions; 2) the substance secreted.” This definition is right in saying “forming.” And Stedman’s Medical Dictionary, 14th ed., 1939 : “Secretion: 1) The production by a cell . . . of some substance differing in chemical and physical properties from the body from which or by which it is produced; 2) the product, solid, liquid, or gaseous, of cellular or glandular activity. A secretion is stored up in or utilized by the animal or plant in which it is produced, thereby differing from an excretion which is intended to be expelled from the body.” This definition fails in the fact that not all products of cellular activity are secretion products. To s u m up we might define secretion as being a chain of processes in which the following three main steps may be observed:

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1). Ingestion, comprising the penetration of raw material into the cell such as amino acids, sugars, fatty acids, HzO,ions, etc. 2). Synthesis, in which more or less complex molecules are built up, concentrated, and stored in granules, vacuoles, crystals, etc. 3). Extrm‘on, the expulsion of these secretions from the cell. In this paper we will limit ourselves to the analysis of the activity of the protein-secreting cell of the type usually found in the pancreas and salivary glands not only in order to avoid an increase in complexity but mainly because we feel that a protein-secreting cell is an ideal material in which to study one of the most fascinating problems of biology, i.e., protein synthesis. This review therefore will deal mainly with studies in which these glands were used. 11. INGESTION The pancreatic exocrine cell has a single cell membrane of 6OA (Fig. 2) and a basement membrane of 15OA which is continuous in the basal region of all cells of one acinus (Sjostrand and Hanzon, 1954). Through these two membranes the ingestion of raw material takes place from the blood via the extracellular fluids. In the pancreatic cell, ingestion has been studied mainly in relation to ions. Thus it has been shown by Ball (1930) and Montgomery et d. (1940) that injected sodium and potassium appear promptly in the pancreatic juice. These last authors detected the appearance of radioactive Na in the pancreatic juice as soon as 3 minutes after an intravenous injection. Bivalent cations such as Ca and Mg pass much less freely (Ball, 1930). The pancreatic cells are however not completely permeable even to Na and K since the concentration of these ions is markedly smaller in the juice than in blood plasma (Solomon, 1952). The transport of COZ from the blood to the pancreatic juice is rapid and efficient in the dog (Ball et d., 1941). Very little has been published on the permeability to more complex substances. Reference should be made here to the work of Visscher (1942) who studied the “passage” of 85 dyes (cationic and anionic) and observed that only anionic and amphoteric dyes appeared in the juice while not one cationic dye was observed to pass to the juice in all the tests performed. It is interesting to remember that the same author described an inverse phenomenon (transport of cationic dyes) to occur in the gastric mucosa. A thorough review on ion and dye transport phenomena in the pancreas has been published recently (Solomon, 1952) including a calculation of the amount of energy spent to transport the inorganic components of 1 liter of pancreatic juice. This amount would be about 329 calories which is less

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than 8% of the 4300 calories available (calculated through the pancreatic Qoz and rate of juice secretion). The author concludes by pointing to the fact that no data exist as to the participation, or otherwise, of the excretory duct and centroacinar cells in the ion transport mechanism. The passage of more complex substances into the pancreatic cell has hardly been studied and this problem under different physiological conditions deserves further attention. It would be of interest to know whether this passage is or is not increased in stimulated glands. Unfortunately the known methods of stimulation promote a strong local vascular congestion of the pancreas, a serious cause of error in the interpretation of any result obtained. In vidro experiments should be attempted in order to study this problem. Reference should be made here to the observation of Hirsch (1931b) on the living pancreas where the penetration of neutral red was found to occur more intensely in cells previously stimulated by pilocarpine which suggests a change in cell permeability as a result of the stimulus. This does not occur with Janus green where permeability is independent of stimulation. The speed with which ions pass from the blood to the pancreatic juice, judging from the literature referred to in the beginning of this chapter, is quite great, and one has the impression that permeability is no limiting factor in their transport. As a result of studies in this laboratory on the rate of appearance of free C1*glycine into the rat pancreatic tissue after intravenous injection, a maximal radioactivity was found before the first 10 minutes (the shortest time observed) following the injection. These, as yet unpublished, results suggest that in the case of this amino acid absorption is rapid and represents no limiting factor. Rat blood plasma proteins tagged by a previous injection of C1’glycine when injected in another rat do not appear in the pancreatic juice suggesting that there is no passage of these proteins through the pancreatic cells (Junqueira et ul., 1955). A special reference should be made to the phenomenon described by Lewis (1937) occurring in macrophages and named “pinocytosis” by which extracellular fluid penetrates into the cells through invaginations of the plasma membrane. Recent electron microscopy observations suggest the existence of this phenomenon on a submicroscopical level in a variety of cells. Thus, infoldings of various dimensions in cell membranes leading deep into the cytoplasm have been observed in endothelial vascular cells, smodth and striated muscle, kidney epithelial cells, collecting tubules of salivary

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glands, parotid secretory cells, and parietal cells of the stomach. It appears that these infoldings contribute to the endoplasmic reticulum in macrophages and endothelial cells (Porter, 1955). This seems therefore to be a rather widespread phenomenon probably of great importance in cell physiology and which should be considered in any reference to ingestion.

111. SYNTHESIS We consider the process of synthesis to include the complex of phenomena occurring after the penetration of the raw material into the cell up to the complete formation of the product of secretion. In the glands we are considering (pancreas and salivary glands) the main process which occurs is the synthesis of proteins or proteins mixed or combined with variable amount of polysaccharides. Although the presence of polysaccharide substances (mucus) has been described since the beginning of this century in the secretory portions of the submaxillary and sublingual glands of mammalia, it seems to be a more general phenomenon than suspected. Thus the human pancreas presents a positive periodic-acid-Schiff (PAS) reaction after amylase treatment (Hartz, 1948) indicating the presence of polysaccharides. The parotid gland of 10 different species of mammals presents, also, a positive PAS reaction (Junqueira eb al., 1951) while unpublished results of this laboratory confirm this result for 18 different species of Brazilian mammals. The rabbit parotid gland reported to be the only one giving a negative PAS reaction (Junqueira et d.,1951) was found later on to be positive in three other specimens. It is of interest that no relation whatsoever could be found between the intensity of the PAS reaction and the morphological characteristics of the so-called %erous and mucous cells.” Thus one may find cells with all the morphological aspects of serous cells presenting a much stronger PAS reaction than typical mucous cells. We will review here the main topics related to the participation of the microsomes, mitochondria, Golgi bodies, and the nucleus in synthesis.

1. Microsomes The term “microsomes” was introduced in modern cytology by Claude (1943) to represent one of the submicroscopic cellular components isolated by centrifugation with a diameter of 50-200 mp. The components constitute 15-20% of the dry weight in protein-secreting glands (Claude, 194.8). The main characteristic of these particulates is their high content in ribose polynucleotides, a fact that led to the belief that they are the main

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329

elements of the “ergastoplasm” or chromidial substance. The ergastoplasm has been recently explored with the electron microscope (Figs. 2 and 3) ; for reviews (see Weiss, 1953; Sjostrand and Hanzon, 1954; Palade, 1955) and is considered to be constituted of vesicular and tubular elements sur-

FIG.2. Mouse pancreas cell under the electron microscope. Endoplasmic reticula and small granules. Intracellular cytoplasmic membranes arranged in pairs. On the outside of every pair opaque granules are attached: in the lower part of the picture the cell membranes of two adjacent cells are observed and close to the cell border a mitochondrion is seen. (Courtesy of Sjostrand and Hanzon, 1954.)

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L. C. U. JUNQUEIRA AND G. C. HIRSCH

rounded by a membrane containing a homogeneous substance and of a small granular component. Part of these granules are attached to the membranes on the outside of these “cytoplasmic membranes” while another part lies free in the cytoplasm. To the vesicular and tubular component Palade gave the name of endoplasmic reticula.

FIG.3. Electron micrograph of rat pancreas cell. Endoplasmic reticulum and small granules. 42,250 X. (Courtesy of Palade, 1955.)

A STUDY OF PANCREAS AND SALIVARY GLANDS

33 1

Whether microsomes are really derived from the ergastoplasm or not has been discussed recently by Bernhard et al. (1954) and Palade (1955). However, recent results of Palade and Siekevitz (1955a, b) obtained by studying sections of microsome pellets prepared by centrifugal fractionation showed that these elements are morphologically identical with the elements of the endoplasmic reticula of intact cells (Fig. 4). Treatment of microsomes with deoxycholate dissolves the vesicles and chemical analysis suggests that the R N A is localized in the small granular component and that the proteins, phospholipids, and DPNH-cytochrome c reductase activity are associated with the vesicles or tubules, i.e., the membrane portion or the substance to be found between the membranes (Fig. 5) * I n this article we will discuss the literature on the participation of ergastoplasm and RNA in cell secretion. An important role of the ergastoplasm in cell secretion had already been postulated in 1900 by Garnier and discussed since then (for recent literature see Rabinovitch et al., 1952b). The problem was a difficult one because it was impossible until recently to quantitate the amount of ergastoplasm during the secretory cycle. A new impetus to this problem was given by the introduction of cytochemical methods by Brachet (1950) and Caspersson (1950) who described a coincidence between cytoplasmic basophilia and activity related to protein synthesis in different types of cells. During the cell secretion cycle Caspersson et al. (1941), using microspectrophotometry, described changes in the amount of R N A in pancreas cells. Unfortunately the amount of R N A was apparently judged by the ultraviolet absorption of selected regions of the cells considered as representative. A satisfactory quantitation of the RNA with the spectrophotometer is possible only by integrating all areas of the cell. This type of study is now possible with the integrating microspectrophotometer described by Caspersson et al. (1953). No mention will be made here, because of the pitfalls present in the method, of the numerous papers in which the amount of RNA was surmised by the histological aspect without biochemical control. In the meantime several laboratories tackled the problem with chemical methods and changes of the R N A content have been described in several glands such as the pigeon crop gland (Jeener, 1948), seminal vesicles (Rabinovitch et al., 1951), and submaxillary glands under different secretory conditions (Rabinovitch et al., 1952a). As these organs however

332

L. C. U. J U N Q U E I R A A N D G. C. HIRSCH

present changes of cell size in the conditions studied, growth phenomena may interfere in the interpretation of the results. Experiments were therefore performed with pancreas during the secretory cycle promoted by pilocarpine for under these conditions no changes

FIG.4. Electron micrograph of pancreas microsome pellet embedded in methacrylate and sectioned. Observe vesicles and small granules. 33,000 X . (Courtesy of Palade, 1955.)

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333

of cell size and mitosis are observable. Contrary to the previous literature no change in the content of the R N A was observed (Rabinovitch et al.,

FIG.5. Electron micrograph of liver microsomes after deoxycholate treatment. Observe almost complete solubilization of the vesicles. The small granules remain apparently unchanged. 42,250 X. (Courtesy of Palade, 1955.)

334

L. C. U. J U N Q U E I R A A N D G . C. HIRSCH

1952b). These results were confirmed by Daly and Mirsky (1952), De Deken-Grenson (1952), and Langer and Grassi (1955). Hokin and Hokin (1954a) could observe no change in the RNA content of pigeon slices in vitro when stimulated to synthesize enzymes by the presence of amino acid mixtures. The idea that a more rapid turnover of RNA might be linked to cell secretion was then explored in different laboratories. The literature on the turnover of P32is conflicting and while Gubernieve and Il’Ina (1950) describe an increased uptake of Ps2 in the RNA of stimulated dog pancreas, De Deken-Grenson (1953) with mouse pancreas, and using better methods, could find no variation. Hokin and Hokin (1954b) working with pigeon pancreas in vitro also observed no increase of uptake. Fernandes and Junqueira (1955) studying the in vivo incorporation of glycine l-CI4 into the RNA and proteins of carbaminoylcholine-stimulated pigeon pancreas described a parallelism in its incorporation into these two substances, during the secretory cycle. These results by the way do not support Schucher and Hokin’s (1954) suggestion that “the so called cycle of secretion and resynthesis of pancreatic enzymes . . .” may merely reflect a constant rate of enzyme synthesis with superimposed variations in the secretory rate. Comparing results obtained in vivo with those in vitro Fernandes and Junqueira (1955) present data that suggest the influence of RNA in the laying down of the characteristic amino acid sequence required for the synthesis of a specific protein. It is interesting to observe that Koritz and Chantrenne (1954) came to a similar conclusion working with reticulocytes. The fact that injected radioactive amino acids concentrate first in the microsome fraction has been described by different authors (see Allfrey et al., 1953 for literature) and is considered suggestive of the participation of these particles in the phenomena of protein synthesis. A reference should be made here to the work of Gale and Folkes (1954) although their results were obtained with bacteria. These authors obtained evidence for the participation of DNA and RNA in the synthesis of proteins and incorporation of labeled amino acids. Furthermore the nature of the protein synthesized seems to be determined by the nucleic acids present. Electron microscopy has progressed rapidly these last years and several papers have appeared on the structure of basophilic substance in different secretory cells (Sjostrand and Hanzon, 1954; Dalton, 1951; Bernhard et al., 1952 ; Palade, 1955 ; Weiss, 1953). The images obtained are complex and might in the future furnish a morphological basis for the better understanding of the mechanism of protein synthesis. So far results of studies of glands under different physiological conditions, although of im-

A STUDY OF PANCREAS AND SALIVARY GLANDS

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portance, are scarce and should be the subject of more research. Gautier and Diomede-Fresa (1953) described changes of the ergastoplasm in parotid gland cells stimulated by pilocarpine, such changes however could not be observed by Sjostrand and Hanzon (1954) on similarly stimulated pancreatic cells. Weiss (1953) studied the ergastoplasm of normal and fasted mice pancreas acinar cells and described the secretory granules as arising from the “ergastoplasmic sacs” (endoplasmic reticula of Palade) . Reference should also be made to a paper recently published by Dempsey and Peterson (1955) on the electron microscopy of the normal, hypophysectomized, and stimulated thyroid. An interesting result obtained from electron microscopy on the pancreas is the study of the toxic action of neutral red; this drug promotes a loss of definition of the mitochondria, ergastoplasm, zymogen granules, and possibly Golgi bodies which become continuous with the cytoplasmic matrix (Weiss, 1955). If these results are confirmed, data obtained so far by vital staining methods should be assessed with great care. 2.

Mitochondria

Recently three general reviews on mitochondria have been published by Schneider and Hogeboom (1951), Lindberg and Ernster (1954), and Hirsch (1955a). The participation of mitochondria in cellular activity has been postulated since the beginning of this century owing to the fact that changes in the number and morphology of these elements could be observed in different conditions. A considerable amount of data was rapidly accumulated to such an extent that as early as 1918 Cowdry published a list of eighty substances in the formation of which mitochondria were said to be concerned. Many papers have since been published on the mitochondria of glandular cells, mainly on the changes in the number and form of mitochondria during the secretory cycle and the mitochondria1 contribution to secretory processes (Hirsch, 1931c, 1932a, b ; Enjo, 1947; Ries, 1935 ; see Huber, 194%, b for reviews). Even in pathological material such as tumors mitochondria have been linked to secretion (Ludford, 1948). The action of X-rays on the secretory processes of the pancreas in vivo were studied by Hirsch (1931a). Deformation of the mitochondria and retardation of the process of secretory granule synthesis was observed. Only after rebuilding of the mitochondria does the formation of zymogen granules continue. Apparently X-rays have a harmful effect on mitochondria (understandable through the biological action of radiation on

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L. C. U. JUNQUEIRA AND G. C. HIRSCH

enzymes, see Barron, 1953) hindering their participation in the processes of cell secretion. a. The Structure of the Mitochondria. A comprehensive analysis of the structure of the mitochondria of protein-producing cells is presented by Sjostrand and Hanzon (1954) on the pancreas (Fig. 2). The mitochondria are surrounded by an outer double-edged membrane. The total thickness of this membrane is 140A and the width of the less opaque space between the two lines is 70A. The inside of every mitochondrion is packed with plates oriented perpendicularly to the long axis. These plates also appear double edged. Every outside layer (lipoids?) has a diameter of 40A, the total thickness of every plate being 170A. The inner less opaque space between the outside layers (proteins ?) was calculated as 9OA wide. One end of these plates is in contact with the surface membrane while the other end is free. The distance between the plates generally varies from 70 to 4OOA. There are no indications that the plates represent folds of a single-edged surface membrane. b. The Physiological Action of Mitochondria. Following the historical results of Bensley and Hoerr (1934), mitochondria were isolated by differential centrifugation and subjected to chemical analysis. Many papers have been published since then, analyzing the chemical nature of mitochondria and studying their enzymology. The recent results are of such importance that a summary of these findings will be presented in order to provide a basis for a discussion of the functions of the mitochondria in secretory cells (for a detailed analysis of this aspect see the reviews cited at the beginning of this chapter). The idea first postulated by Kingsbury (1912) that mitochondria participate in cell respiration is now supported by solid experimental evidence and at present these cell organelles are considered to be the centers of biological oxidation. Enzymes for the oxidation of pyruvate (aerobic metabolism of carbohydrates), fatty acids, and amino acids are located in the mitochondria (see Schneider, 1953; Lehninger, 1951; Green, 1951 for reviews). Most of these enzymes have been studied (see Greenberg, 1954) and with the recent isolation of those related to fatty acid oxidation (Lynen and Ochoa, 1953; Green, 1954)) almost every one has been purified. Figure 6 shows the interrelationship of the pathways for the oxidation of several substrates by mitochondria. Glucose is metabolized, outside of the mitochondria and in the cytoplasm, to pyruvate (in a process called glycolysis) with the release of two pairs of hydrogen atoms. As a result of pyruvate, fatty acid, and amino acid oxidation, carbon dioxide and hydrogen are produced. Hydrogen is further oxidized to water. This exergonic reaction pro-

337

A STUDY OF PANCREAS A N D SALIVARY GLANDS

vides energy for life. Amino acid nitrogen is converted by mitochondria and cytoplasmic enzymes to urea (see Greenberg, 1954). The oxidation of glucose provides 686 kcal. per mol and can be represented as energy obtained by hydrogen oxidation:

CaHizOs 24 H + CaH12Os

+ +

+

6 0 2

- 6C02 - 12 H2O

6

- 6 C02

6 H2O 0 2

+

+

+

24H+ 686 kcal. 6 Ha0

+

686 kcal.

RCH.OHO~OO-OOA

FATS

b PYRWATE G A C f T Y L -

ORNITHINE

WA

-

ACETYLATIONS

x

I

QLUTAMATE

ASPARTATE

4

ACIDS A

AM~NO ACIDS

FIG.6. Diagram representing some oxidative pathways for the oxidation of lipids, aminoacids, and carbohydrates in the mitochondria.

338

L. C. U. JUNQUEIRA AND G. C. HIRSCH

The oxidation of a pair of hydrogen atoms provides 57 kcal., so that 12 pairs provide 684 kcal. The oxidation of two of these 12 pairs of atoms occurs during the degradation of a molecule of glucose to two pyruvate molecules (glycolysis) and the other ten pairs during the oxidation of the two pyruvate molecules (five for each molecule). Hydrogen is oxidized stepwise beginning with its removal from the

I

I

/

1

CYTO~HROMEc Bi-foc\ "dehydrogenaser"

FLAVOPROTEINREWCTASES

4

PYRIDINE NUCLEOTIDE

I

"flavin dehydrogem',

COENZYMES

CHOUNE SUCCINATE

SUBSTRATE

FIG.7. Some pathways of electron transport in mitochondria. substrate and continuing to its reaction with oxygen. These intermediate steps are performed by mitochondriat enzymes-the electron (or hydrogen) transport chain (see Westeimer et al., 1954; Anfinsen and Killey, 1954). Figure 7 shows some of the better known pathways for the electron (or hydrogen) transport observed in mitochondria1 preparations or in reconstructed solubilized enzyme preparations. Other electron carriers are known, but the real intramitochondrial pathway or the relative importance of each alternative pathway is still obscure. The electron transport chain has three main steps mediated by pyridine

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339

nucleotides ( D P N and T P N ) , flavoprotein reductases, and the cytochrome-cytochrome oxidase system. In this way a stepwise liberation of the 57 kcal. per hydrogen pair mol occurs. A definite amount of energy (14 kcal.) is liberated at each of the four levels represented in Fig. 8. An unknown enzymatic system transforms the energy liberated by the oxidation, into the chemical energy of the adenosine triphosphate (ATP) 57

42

I

I

OXYGEN

I \ CYTOCHROME c

0

k

9

28

FLAVO PROTEIN

W

m

zi t. l3

'6 z 14.

il"Y311 Ylrs

Succinyl

- coA

(

FIG.8. Diagram representing energy release and oxidative phosphorylation. pyrophosphate bond. This enzymatic process called oxidative phosphorylation occurs in mitochondria (Hunter, 1951). It is a very labile system and is inhibited by many compounds particularly 2,4-dinitrophenol and gramicidin. The nature of this unstable system is obscure, with the exception of the enzymes responsible for A T P formation, during the transformation of succinyl-coenzyme A to succinic acid (Lynen and Ochoa, 1953; Green, 1954). This reaction has guanosine diphosphate as coenzyme. The enzymes responsible for the other three phosphorylations are unknown and only recently a soluble system that couples oxidation of

340

L. C. U. JUNQUEIRA AND G. C. HIRSCH

reduced D P N to cytochrome c with A T P synthesis was isolated from mitochondria. This system requires inosine triphosphate or a contaminant of ITP preparations (Raw, 1955). A T P provides energy for different cell activities, such as protein synthesis and synthesis of other chemical compounds, cell contraction, light and electricity production, osmotic work (Kaplan, 1951), and cell secretion (Fernandes and Junqueira, 1953). Mitochondria are, therefore, at the present status of our knowledge, responsible for the aerobic metabolism represented mainly by the Krebs tricarboxyiic acid cycle and tissue oxygen consumption (Qo,) and the main energy-producing mechanism. I t is therefore logical that pancreas and salivary glands, organs with appreciable amounts of mitochondria, have their secretory processes inhibited by anoxia (Chardon and Gross, 1946) and that stimulation by secretin, pharmacological agents, or electricity increases the oxygen consumption (Davies et al., 1949 ; Hattingberg, 1932 ; Barcroft and Starling, 1%; Still et al., 1933; Deutsch and Raper, 1938). The participation of glycolysis in the processes of cell secretion in these glands has been discussed. Himwich and Adams (1930) and Deutsch and Raper (1938) could find no change in lactic acid production during secretion, concluding that this process does not serve as an important source of energy. Other authors however described an increased lactic acid production after stimulation (Ferrari and Hober, 1933 ; Bergonzi and Bolcato, 1930). Brock et al. (1939), however, believe that the increase of glycolysis in the presence of pharmacological agents is a nonspecific reaction resulting from toxicity. The participation of the cytochrome-cytochrome oxidase system present in mitochondria in cell secretion has so far not been studied ; data of Ferrari and Hober (1933) working with cyanide inhibition suggest the presence of this system in submaxillary glands. A study of this system in different physiological conditions would be of interest. The participation of phosphorus during secretion has been suggested by the fact that the inorganic phosphorus of venous blood increases after gIand stimulation (Camis, 1924 ; Ferrari and Hober, 1933). More direct evidence for the participation of energy-rich phosphorus compounds come from data of Northup (1936) who observed breakdown of creatine phosphate in stimulated salivary glands. Unfortunately no parallel studies on the aspect and number of mitochondria were performed in the experimental material referred to above, so that an integration of biochemical data with morphology is so far impossible.

A STUDY O F PANCREAS AND SALIVARY GLANDS

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The above mentioned experiments, although very suggestive as to the participation of mitochondria and their enzymes in secretory processes, are subject to criticism for nonstimulated glands are not resting glands (as several authors such as Himwich and Adams, 1930, state). It is a known fact that both pancreas and salivary glands are continuously secreting, and even if no juice or saliva is being expulsed, the cell is working on the synthesis of new material. The comparison of the cytological and biochemical phenomena related to cell secretion in a normally functioning gland with one producing no secretory material would be a much more valuable study. This comparison was made by Junqueira (1951) and Fernandes and Junqueira ( 1953) who studied the normal and excretory-duct-ligated submaxillary glands of mice and rats. It was observed that the ligature of the excretory duct of these glands leads to an abolition of secretion with disappearance of secretory granules, diminution of cell and gland size, and accentuated decrease in the activity of the digestive enzymes it contains (Junqueira, 1951). No degenerative phenomena were observed and the histological picture remains stable for at least one year ; it is furthermore a reversible process (Junqueira, 1951 ; Junqueira and Rabinovitch, 1954 ; Valeri, 1954b). A comparative morphological, histochemical, and biochemical study was performed in this material and the results are presented in Table I. From the analysis of the table one may see that glycolysis is similar both in secreting and in nonsecreting glands, suggesting the nonparticipation of glycolysis in processes of cell secretion. One is tempted to speculate that the energy derived from glycolysis is utilized by the cell mainly for its basic needs rather than for its specialized function. That the source of energy for cell secretion is derived mainly from the oxidative metabolism is suggested by the differences in Qoz and succinic dehydrogenase activities observed in both glands. In regard to succinic dehydrogenase activity it is interesting to observe that, using the histochemical tetrazolium method, different authors have described a much more intense reaction in the excretory ducts of pancreas and salivary glands than in the acini (Hill and Bourne, 1954; Neumann, 1952 ; Padykula, 1952 ; Stier, 1952). The impression is gained that these tubules have a more intense metabolism and one is tempted to relate it to a probable ion transport mechanism by these structures. A study of ion transport in these glands wifh suitable methods (radioautography and freezing-drying fixation) wovld be of great interest in clarifying which of the gland structures are responsible for the so-called “electrolyte secretion” recently reviewed by Solomon (1952) in the pan-

w

R TABLE I CHANGESIN RAT AND

Cell size and form (rat) Mitochondria (rat and mice)

Average gland weight

rats mice

Control gland

Ligated gland

Pyramidal

Cubical or flat

Abundant and rodlike in the secretory tubule cells Round or short rods in acinar cells

Greatly reduced

234.0 mg. 54.0 mg.

76.4mg. 30.0 mg.

Strong reaction

Proteins (mice) Ribonucleoproteins, histochemical (mice)

Strong basophilia RNAP ~DNAP

Ribonucleoproteins, biochemical (mice)

2.18

17.9 y tyrosinely D N A P

Cathepsin activity (mice) rats mice

2.8 mg. pheno1/100 mg. tissue 2.0 mg. pheno1/100 mg. tissue

Method used Helly fixative routine stains Regaud fixative Ferric and phosphotungstic hematoxylin

l

r ?

9

1

2 z

10

Very weak reaction

576.0 y pheno1/100 mg. tissue

Protease activity (rats)

Alkaline phosphatase

MICESUBMAXILLARY GLANDSPROMOTED BY DUCT LIGATION

Very weak basophilia RNAP -0.84 DNAP 266.0 y pheno1/100 mg. tissue

10.3 y tyrosinely D N A P

1.7 mg. pheno1/100 mg. tissue 2.4 mg. pheno1/100 mg. tissue

d

E ?-

Millon’s reagent histochemical Brachet toluidin blue ribonuclease test

*

1

3 9

l

?

Smith and Thanhauser’s method modified

2

Elx

Anson’s method

3

Anson’s method

6

Greenstein’s method

1,3

w

TABLE I (Continued) Control gland Acid phosphatase

Ligated gland

rats mice

1.8 mg. pheno1/100 mg. tissue 2.0 mg. pheno1/100 mg. tissue

mice

50% reduction of methylene blue color in 12 min.

rats

63% increase in Qo2 after addition of succinate

Succinic dehydrogenase

Qoz = 4.9

Oxygen consumption (rat) Glycolysis (rat)

N2 QG

=3.2

1.8 mg pheno1/100 mg. tissue 0.42mg. pheno1/100 mg. tissue 12% reduction of color in 12 min.

28%

increase in Qoz QoZ= 1.8

N2

QG = 3.3

Method used Greenstein’s method

3.

Thunberg’s methylene blue

l

Warburg apparatus

4

Warburg apparatus Warburg apparatus

4

4 1 : 1

C y t d r o m e oxidase (mice)

Dark blue color

Light blue color

Indophenol oxidase test

Adenosinetriphosphate plus diphosphate phosphorus ( A T P A D P ) (rat)

13.7 y/y D N A P

3.2 y/y D N A P

Le Page fractionation

Phosphocreatine phosphorus (rat) Inorganic phosphorus (rat)

8.5 y/y D N A P

2.7 y/y DNAP

44.8 y/y D N A P

18.6 y/y D N A P

Le Page fractionation

36.2y/y D N A P

method Pyruvate determination as described in 7

method

+

90.2 y/y D N A P

References

1. Junqueira (1951). 2. Rabinovitch et al. (1952a). 3. Junqueira and Rabinovitch (1954).

vl

H

2 % 3.

8 N $ 3.

4

5

E

Le Page fractionation method

Pyruvate utilization

1,3

4

%

rc 4

E

5 w

4. Fernandes and Junqueira (1953).

5. Fernandes and Junqueira (unpublished). 6. Rothschild and Junqueira (1951). 7. Friedeman and Haugen (1943).

G,

&

344

L. C. U. JUNQUEIRA AND G . C. HIRSCH

creas. The difference in cytochrome oxidase activity suggested by the histochemical method has not so far been checked by biochemical methods and should be further investigated. The marked decrease of the content of energy-rich phosphorus compounds (ATP, ADP, P C ) of the nonsecreting gland strongly suggests the participation of these compounds as immediate sources of energy for cell secretion. It is difficult at present to have even an approximate idea of the energy requirements of each of the main steps (ingestion, synthesis, and extrusion) of cell secretion. Protein synthesis is considered to be a process consuming relatively little energy, using, according to data based on the total protein turnover of the body (Borsook, 1950) about 1/300 of the basal metabolism. Although protein synthesis occurs with more intensity in the pancreas than in most of the tissues it is probably not the main energy consumer of the secreting cell. Calculations of Solomon (1952) suggest that the energy necessary for ion transport in the pancreas is about 8% of the energy utilized by the cell. If both of these calculations are approximately correct one arrives at the rather startling conclusion that the pancreas spends most of the energy available in activities not directly related to its main function, i.e., ion transport and protein synthesis. Besides the enzymes referred to above, others not present in mitochondria have been studied in working and resting submaxillary glands. Cathepsin activity is parallel to the gland secretory activity and has been shown to be controlled in vivo by androgenic sexual hormones also in other glands beside the submaxillaries (Rothschild and Junqueira, 1951 ; Junqueira and Rothschild, 1953). A parallelism was shown between catheptic activity and protein synthesis in different organs while no increase of catheptic activity could be detected in organs in which protein degradation was promoted. The behavior of the phosphatases is interesting for while the alkaline enzyme presents reduced activity owing to duct ligation in rats, the same reduction could be observed only for acid phosphatase in mice. It is an example of species differences observable also in some histochemical peculiarities and physiological behavior of these glands. Submaxillaries of mice for example are more sensitive to androgens than are those of rats. Summing up one may state that the role of mitochondria in the processes of cell secretion appears to be mainly that of energy suppliers. Integrated studies of morphological and biochemical aspects of glands under different conditions although scarce are of great interest and should furnish very interesting results. Electron microscopy under these conditions will prob-

A STUDY OF PANCREAS AND SALIVARY GLANDS

345

ably reveal new aspects and give rise to new ideas in the field of the physiology of secretion.

3. Golgi Bodies Since Cajal made his pioneer studies, from 1903 to 1915, the Golgi bodies have been related to cell function. No cell constituent has been the subject of so much research and discussion; suffice it to say that even until recently its existence has been doubted. The subject has been covered several times, one of the more comprehensive reviews being a monograph by Hirsch (1939),who collected at that time more than 2000 references. Since then the reviews of Bourne (1951a,b), Hiiber (1949a,b), and Hirsch ( 1955a) have appeared. The hypothesis that the Golgi bodies are artifacts is improbable owing to the following facts : a. In a number of cells these bodies have been studied in vivo or in vidro with phase contrast microscopy (Hirsch, 1948, Fig. 10; Schneider et al., 1953;Dustin, 1949;Inhuma and Yangiwasa, 1953; Sosa, 1953). b. The Golgi bodies have been separated by centrifugal fractionation of homogenized cells (Worley, 1951; Schneider et aZ., 1953; Dalton and Felix, 1954; Schneider and Kuff, 1954). c. Their ultrastructure has been recently studied with the electron microscope by Sjostrand and Hanzon ( 1954) and Dalton and Felix (1954) (Fig. 11). d. The fact that the Golgi bodies stain by vital dyes should not be considered a definite proof of the existence of these structures in view of the recent studies of Weiss (1955) demonstrating the toxic effect of neutral red promoting profound changes in the endoplasmic reticula, mitochondria, zymogen granules, “and possibly in the Golgi complex.” The so-called “Golgi apparatus” is considered to consist, in pancreas and salivary glands, not of a network but of more or less single Golgi systems lying together in a Golgi field (Hirsch, 1948) (Fig. 9). Every Golgi system consists, according to Hirsch (1939),of a homogeneous ground substance (the Golgi externum) in the interior of which vacuoles are found (the Golgi internum) that gradually grow and become denser (Fig. 10). The classical netlike structure described for the “Golgi apparatus” is considered by several authors to be an artifact (see Hirsch, 1939;Baker, 1944,1949;Palade and Claude, 1949). The nature of these vacuoles depends on the type of secretion product the cell elaborates. The ground substance is considered to be an elastic (Worley, 1946), sometimes birefringent, gel (MonnC, 1942). Its density is less than that

346

m

L. C. U. JUNQUEIRA AND G. C. HIRSCH

f"

3

LIVING CELL NEUTRALRED

10

9

A STUDY OF PANCREAS AND SALIVARY GLANDS

m i

CHAMf’Y FIXATION &Or IMPREGNATON

lnu)

HUNOER

I b. AFTER PILOCARPIN

2 h.AFTER PILOCARPIN

347

FIG.9. Mouse pancreas viewed by six different techniques; cells during hunger, 1 and 2 hr. after pilocarpin stimulation. (1) nucleus; (2) zyrnogen granule; (3) mitochondria; (4) little movable granules wandering from the surface of the mitochondria to the Golgi field ; ( 5 ) Golgi ground substance ; (6) vacuoles in the Golgi ground substance; (7) larger vacuoles; (8) denser vacuoles; (9) and (10) neutral red granules. (After Hirsch, 1918.)

348

L. C. U. JUNQUEIRA AND G. C. HIRSCH

of the cytoplasm (Beams and King, 1934; Simpson, 1941). Histochemical tests suggest the presence of lecithin and cephalin or sphingomyelin (Baker, 1944, 1949) while tests for cholesterol have been negative. Cain (1947, 1950) detected the presence of carotenoids. Granaglia (1950) and Berg ( 1951) confirmed these results. Corroborative evidence is presented by the fact that the treatment of pancreatic cells with lipoid solvents prevents the impregnation of the Golgi bodies by Aoyama’s method. Golgi ground substance Vacuoles = interna

0

1

3

2

Zymogen granule

4

6

5

7

FIG.10. The Golgi ground substance during the secretion of an enzyme or mucus producing cell. (After Hirsch, 1948.) Proteins are probably present but not in high concentration. Baker (1944, 1949) got negative tests for arginine and glutathione but a positive result with Millon’s, xanthoproteic, and tryptophane tests. The protein concentration is probably low (Baker, 1953; Bourne 1951a, b ) . A positive PAS test for polysaccharides has been described by Gersh (19491, Arzac and Flores (1952), and Schneider and Kuff ( 1954) in the Golgi fields of various tissues of different species. Sosa (1955) has produced evidence for the presence of lipids and polysaccharides in the Golgi apparatus. Alkaline phosphatase has been described by Emmel (1945) in the intestinal epithelium of mice and by Schneider et al. (1953) in Golgi bodies isolated from mouse epididymus cells. A histochemical analysis of

A STUDY OF PANCREAS AND SALWARY GLANDS

349

the Golgi bodies of the exocrine and endocrine pancreas cells was recently performed by Lacy (1954). The study of its composition has been initiated with chemical methods as a result of the application of centrifugal fractionation techniques (Dalton and Felix, 1954). In a recent paper Schneider and Kuff (1954) studying Golgi substance isolated by centrifugal fractionation from rat epididymus demonstrated a high content of pentosenucleic acid, phospholipid, and alkaline phosphatase activity. Cytochrome oxidase and deoxyribonuclease were absent from this fraction. The participation of Golgi substance in phenomena of secretion has been postulated mainly on the basis of two facts : a. Change in its size and morphology during the secretory cycle. b. The presence of vacuoles in the Golgi bodies and their gradual transformation into secretory gtanules (Fig. 10). This fact, observed by several authors, was described in the living pancreas by Hirsch (1932a, b) and in fixed material by Sluiter (1944) who studied the amount of Golgi vacuoles and zymogen granules during the secretory cycle. He could demonstrate that after pilocarpine stimulation the number of Golgi vacuoles increased in the first 7 hours, decreasing from then on ; the number of zymogen granules decreased during the first hour, increasing slowly to attain its maximum only by the 15th hour. This fact suggests that the Golgi vacuoles precede and are probably related to the formation of the zymogen granules. Hirsch in 1939 presented the theory that the Golgi bodies are the site of congregation of cytoplasmic products from which zymogenic granules arise. Biochemical data suggest that the Golgi bodies are probably not sites of protein synthesis or energy production. One is tempted therefore to speculate on the possibility that presubstances are transformed into ready-made products. A thorough biochemical and enzymatic analysis of the Golgi body is badly needed before further speculation is made. The data presented by Sjostrand and Hanzon (1954) on the ultrastructure of the Golgi bodies (Fig. 11) show an intimate relation between the Golgi vacuoles, considered to be precursors of zymogen granules, and the Golgi ground substance and membranes described by the authors. They state: “There are all transition stages observed between on the one hand, bodies with a pronounced elongated form and with the most direct topographic relations to the Golgi membranes, and Golgi granules embedded in the ground substance on the other. The impression when observing these pictures is that they show snapshots of a conversion of membranes into granules and vice-versa. The small granules seem to

350

L. C. U. JUNQUEIRA AND G. C. HIRSCH

FIG.11. Mouse pancreas cell under the electron microscope. Above, one mitochondrion. In the middle the Golgi membrane pairs partly bounding vacuolar spaces. Above them less dense Golgi vacuoles surrounded by the Golgi ground substance. Two large zymogen granules; the left one shows a marked dense surface layer, lower opacity, and coarser structure than the right zyrnogen granule. (Courtesy of Sjostrand and Hanzon, 1954.)

A STUDY OF PANCREAS AND SALIVARY GLANDS

351

coalesce to bigger granules which gradually gain the size, form and opacity of the zymogen granules.” These results on a submicroscopical level present evidence that supports Hirsch‘s theory (1939) in which GoIgi bodies are considered as a site of condensation of secretory products formed in other portions of the cell.

4. Nucleus A number of reviews are available of the classical literature on the behavior of the nucleus during cell secretion (Montgomery, 1898; Noll, 1905 ; Maziarski, 1910). In these, references will be found concerning variations in the position, shape, volume, and “chromaticity” of nuclei in different phases of cell secretion. Data pertaining to the pancreas will be found in Laguesse (1905, 1906) and Hiiber (1949a, b). No attempt will be made here to cover thoroughly this extensive literature, but only to point out some of the trends. a. Nuclear Volumes. Quantitative data on nuclear volume variations in pancreas and salivary glands under different conditions are scarce. A diurnal rhythm in the nuclear volumes of the exocrine pancreas has been reported, with modal nuclear volumes (%-hour periods) varying from 58 to 100 cu. p (Nicolaj, 1939). In a paper reporting on variations in nuclear volumes of salivary glands and pancreas after pilocarpine administration, the species of the animal used is not stated (Toni, 1948). Altmann ( 1952) , to refers to a drop in the dodal nuclear volumes from 400 and 800 cu. U 336 and 672 respectively soon after pilocarpine administration to mice. Later on an increase in the nuclear volumes over those of the resting phase was found. In adult male mice castration was followed by a significant decrease in the mean nuclear volumes in the cells of the secretory tubules of the submaxillary gland. Testosterone administered to either castrated or normal animals increased the mean nuclear volumes by about 20% (Valeri, 1954a). In untreated mice ligature of the excretory duct of the submaxillary gland was followed by a reduction in the mean volumes of the secretory tubule nuclei. Both in the control and in the duct-ligated glands testosterone administration promoted an increase in the mean nuclear volumes (Valeri, 1954b). This further connects secretory status with nuclear volumes in this material. It should be noted that in none of these cases could a “rhythmic” nuclear growth be found (see Schreiber, 1949). It is probable that these changes in nuclear volume are not coincident with changes in the DNA content but with changes in the protein (Altmann, 1952 ; see also Bern and AIfert, 1954). b. Nucleolar Volumes. Although the classical literature points to an

352

L. C. U. JUNQUEIRA AND G. C. HIRSCH

increase in the nuclear volumes in secreting cells, owing undoubtedly to technical factors, the problem has only occasionally been approached quantitatively. Yokoyama and Stowell ( 1951) measured nucleolar volumes in the exocrine cells of the mouse pancreas after pilocarpine administration. A definite increase was observed in the recovery period. Unfortunately the drug was administered in divided doses over a period of 6 hours, which makes it difficult to compare the results with previous data on the granule (Hirsch, 1932b) and enzyme (Daly and Mirsky, 1953) restoration of the acinar cells, Of interest are the recent observations by Schreiber and co-workers ( Schreiber et al., 1955) demonstrating quantitative variations in the nucleolus/nucleus ratio. They studied the size and number of nucleoli in actively secreting cells of the pancreas and salivary glands and compared them with other cells in the same organs with a lower secretory activity. These variations were observed to occur along fixed multiples of a basic unit (quantic variation). Similar phenomena were observed in the kidney (where the nuclei of convoluted tubules are compared with those of the ascending portion of Henle’s loop) and the liver (Pellegrino et al., 1955). The authors explain these results as due to a probable multiplication of the nucleolar organizer independently of quantitative changes in the nuclear size (Schreiber, 1955). c . Morphological evidence interpreted as indicating transfer of nuclear (and nucleolar) substances to the cytoplasm. 1n”the analysis of these data it is difficult to exclude artifacts arising during the histological procedure (see Laguesse, 1905), which prompted Wilson’s comment ( 1928) that “until the facts have been decisively demonstrated by the study of living cells, judgement on these cases should be suspended.” References are abundant in the classical literature and attention was recently drawn to these morphological phenomena by Hiiber (1949a, b) and especially by Altmann ( 1952). Altmann studies mouse pancreas after pilocarpine administration. A series of nucleolar and chromatin changes, interpreted as a cycle, are described. They ranged from extrusion of nucleolar substance into the cytoplasm (agreeing with Huber, 1949a, b) , with disappearance of the nucleoli, to reconstitution and enlargement of these organelles. After extrusion of the nucleolar material, nuclei enlarged and chromatin knots disappeared, giving an empty appearance to the nuclei (“dekondensationsphase”). This was followed by the reappearance and growth of the chromatin granules and reconstitution of the nucleoli. Unfortunately the author does not report the frequency with which these nuclear changes were found in a given section. The fact that the study of living cells by means of the phase contrast microscope was reported to agree with the results obtained in fixed material is important.

A STUDY O F PANCREAS AND SALIVARY GLANDS

353

Recently Pavan and Breuer (1955), and Breuer and Pavan (1955) described differences in the apparent RNA and DNA content of localized regions of larval Rhynchosciara salivary gland chromosomes in different phases of development. These authors suggest a possible relationship of these changes to secretory phenomena in the glands. This would be an example of localized chromosomal changes coincident with cell secretion. To what extent these data are related to Altrnann’s (1952) above-mentioned results is a matter of pure speculation. To summarize, although there is morphological evidence linking nuclear behavior to cell secretion in the pancreas and salivary glands, more work is needed on the subject, mainly a correlation of morphology with chemistry, in order to obtain a clear view of the whole problem. Pancreas seems still to be a promising material for such studies. Biochemical study of the behavior of nuclear components in different secretory levels has been pioneered by Mirsky and co-workers (see Allfrey et al., 195513). This work brought out the rather unexpected result that incorporation of N16 glycine is increased in DNA from mice pancreas when fasted animals are fed (Daly et al., 1952). On the other hand, increase in the incorporation of the isotope was found in the “histone” and “residual chromosome” fractions. These last two increases were roughly proportional to the ones found in the whole homogenate, in the “ribonucleoprotein pellett” and supernatant fractions (Allfrey et al., 1955a). I t would be of interest to determine whether the vascular factor to which attention was called elsewhere in this review is not playing a role in the different behavior of pancreas from fasted and fed mice.

IV. EXTRUSION Extrusion is the process by which cells eliminate the secretion products of their cytoplasm. Although cell secretion is a much studied process little is known of the morphology and mechanism of extrusion. Up to the present we have no definite idea whether extrusion is an active process promoted by cellular movements or contractions, or if it is a passive elimination of previously fluidified secretory granules, or if both processes may occur simultaneously. To our knowledge the first observations referring to extrusion were made on living rabbit pancreas by Kuhne and Lea (1882) who observed a dissolution of the secretory granules close to the cell apex. Results obtained with the classical methods of fixation are scarce in the literature and of little value because of the known artifacts they produce. I n vivo observations and those performed after freezing-drying fixation are therefore more valuable and will be discussed here.

354

L. C. U. JUNQUEIRA AND G. C. HIRSCH

Cove11 (1928) made a description of the extrusion occurring in pilocarpine-stimulated mouse pancreas. He observed A vivo the discharge of both fluid vacuoles and zymogen granules into the lumen of the acini. The intensity of the process was proportional to the amount of pilocarpine injected. According to Ries (1935) extrusion occurs by expulsion of granules and of vacuoles formed in the cell by coalescence of zymogen granules. After very intense stimulation fragments of the cytoplasm are also eliminated. The author is of the opinion that the cell membrane disappears at the site of extrusion, for no sharp limit between the cell apex and acinus lumen could be observed. Hirsch (1955b), from his experience with the living mouse pancreas, describes two types of extrusion. The first one occurs in nonstimulated pancreas and is characterized by a slow dissolution of the secretory granules and passage of this fluid material to the lumen of the acini. It is a synchronous process, for all the cells of one acinus extrude simultaneously. This type of extrusion is responsible for the “hunger secretion” described in several animals and recently reviewed and studied in the rat by Hirsch et al. ( 1 9 5 5 ~ ) . The other type occurs in stimulated glands and is characterized by the appearance of large vacuoles around the secretory granules followed by their dissolution. The contents of these vacuoles are then extruded through the cell membrane. According to Danielli (1951) this process might be of a mechanical nature because of differences in surface tensions present owing to the fact that the vacuole membrane has a higher curvature than the plasma membrane. When the stimulation is very intense the vacuoles are formed quicker and are larger. Some vacuoles are extruded surrounded by a cytoplasmic layer and later on burst in the secretory ducts. Recently a study of carbaminoylcholine-secretion stimulated rat pancreatic juice, obtained directly from the pancreatic duct through a polyethylene cannula, was performed under phase microscopy. No secretory granules as such could be observed, suggesting that these granules dissolve during extrusion or a short time after. Of interest are the observations of De Robertis (1942) on the secretory processes of thyrotropic hormone stimulated thyroid cells fixed by freezeing-drying. In this material the secretory granules were eliminated to the interior of the follicles, frequently surrounded by a thin layer of cytoplasm. To our knowledge no detailed study with freeze-drying fixation has been performed so far in salivary glands or pancreas after stimulus and this should be attempted.

A STUDY OF PANCREAS AND SALIVARY GLANDS

355

A better study of extrusion would be possible with the use of time lapse cinephotomicrography of living cells. Recently a method has been described for the cinematographic study of the living mouse pancreas (Haselmann et ul., 1953). A more detailed analysis of the intimate mechanism of extrusion is at the moment difficult owing mainly to the fact that simultaneously the cell is rapidly absorbing raw material through the opposite pole for the synthesis of new secretory granules. This probably explains why Daly and Mirsky (1952) could observe no change in the mouse pancreatic cell protein content during the secretory cycle promoted by pilocarpine. Apparently the entrance of amino acids, and their synthesis to compounds precipitable by trichloracetic acid is so rapid that it happens simultaneously while extrusion is occurring (see section on ingestion).

V. KINETICS Pancreas and salivary glands are favorable material for the study of the kinetics of secretion, for these glands can be easily stimulated to extrude the secretory product. This can be collected, measured, and assayed, and furthermore the rate of restitution can be judged through morphological data or digestive enzyme determinations. If the gland secretes proteins as in the case of the rat pancreas it is also an unusually favorable subject for the study of protein synthesis. The first attempt to study the rate of extrusion and synthesis was performed by Heidenhain (1875) who correlated in dog pancreas, the amount of secretory granules with enzymatic activity. Unfortunately few have followed Heidenhain’s example and the majority of the studies published so far deal with the morphology or biochemistry of these glands separately. Several papers have appeared studying the cell changes during the secretory cycle (Covell, 1928; Hirsch, 1931c, 1932a, b ; Duthie, 1933; Ries, 1935; Jarvi, 1939; Sluiter, 1944). It was however Hirsch (1915) who first applied biometrical techniques to the problem. In pancreas he divided (1932a) the cells into different types according to the amount of zymogen granules and followed the number of each cellular type during the secretory cycle promoted by pilocarpine (Fig. 12). Curves were obtained representing graphically the rate of extrusion and synthesis (Fig. 13). Ries ( 1935) arrived at similar results and described restitution taking under his conditions from 12 to 20 hours. These same techniques were applied to salivary glands by Jarvi (1939). The outcome of all these studies was that generally speaking for the pancreas, the maximum of degranulation (or extrusion) occurs between

356

L. C. U. JUNQUEIRA AND G. C. HIRSCH

the first 30 to 60 minutes after stimulation. Regranulation occurs approximately from 6 to 14 hours later. On the other hand, in the dog submaxillary gland, the rate of synthesis is very slow, and complete restoration of a gland which has undergone strong stimulation takes from 3 to 6 days (see Langstroth et al., 1938; Langstroth et al., 1939, for literature).

3 -Stage

-

4 Stow

FIG.12. Diagram of the four stages the pancreatic cell passes through during the restitution phase after pilocarpine stimulus. (Hirsch, 193Zb.) One of the first papers correlating morphology with chemistry is that of van Wee1 and Engel (1938) who studied the amount of secretory granules and compared this with the carboxypolypeptidase and dipeptidase activity of the mouse pancreas. Carboxypolypeptidase is a digestive enzyme that appears in the pancreatic juice while dipeptidase is present only in the pancreatic tissues. They observed that the carboxypeptidase activity in the pancreas reaches a minimum 3 hours after pilocarpine injection increasing from then on up to the 9th hour where a normal prestimulus level is reached. The aminopeptidase activity however presents changes in the opposite direction. With biochemical methods the rate of the resynthesis of amylase, protease, lipase, and carboxypeptidase has been recently studied by Daly and

357

A STUDY O F PANCREAS AND SALIVARY GLANDS

Mirsky (1952) in the pancreas of pilocarpine injected mice. They conclude that the minimum activity is reached about the first hour after the injection with a resynthesis usually passing the initial level at 6 hours. Data from Rabinovitch ( 1954) on mouse pancreatic ribonuclease suggest similar results. I n carbaminoylcholine-stimulatedpigeons results obtained with successive pancreatic biopsies showed a sharp decrease of the amylase activity after the first hour and a return to the initial level about the 6th hour after the injection (Fernandes and Junqueira, 1955). : 90 80

70 60

[/!b

Stage

.

L

\

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0 .

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50 40

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20

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0

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X

0

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5

6

7

8

9

10

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hours FIG.13. Graph showing the percentage of each cell type in the mouse pancreas during the restitution phase after pilocarpine stimulation. (From Hirsch, 1932b.)

Daly and Mirsky (1952) basing their views on the fact that the totaI protein content of the pancreas remains unchanged during the secretory cycle, conclude that during the expulsion of the enzymes, synthesis of new proteins takes place relatively rapidly. These proteins are then gradually transformed into the characteristic pancreatic enzymes. The shortest period studied was of 30 minutes after stimulation. Observations after shorter periods would be of importance and are being undertaken in this laboratory in order to attempt to isolate intermediates of protein synthesis. W e feel that these results of Daly and Mirslcy are of the greatest interest and they will be referred to later on. The introduction of radioactive isotopes in this field proved to be of interest for it was possible to study the rate of appearance of radioactive proteins in the pancreatic juice of rats previously injected with C14 glycine or S35 methionine and stimulated with carbaminoylcholine plus secretin

358

L. C. U. JUNQUEIRA AND G. C. HIRSCH

(Junqueira et al., 1955). The results obtained (Fig. 14) give an idea of the rate of protein synthesis occurring in the pancreas of rats under the conditions above mentioned. It can be seen that the maximum radioactivity is attained on an average of about 165 minutes after the glycine injection, suggesting that the secretory process occurs with maximal intensity between the second and fifth hours after the initial stimulation. 12000

1.75

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5000

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FIG.14. Appearance of radioactivity in the rat pancreatic juice proteins after injection of 3 . 3 ~C (1-14C) glycine and after secretin-carbamoylcholine stimulation A Rate of pancreatic juice flow 0 The asterisk indicates the time of the first stimulation and the arrow the time at which glycine was injected.

.

These results were recently confirmed with the use of phenylalanine C14, alanine CI4, and histidine C14 (unpublished results). With the conditions under which Junqueira and co-workers made their studies, a more detailed analysis of the time necessary for ingestion, synthesis, and extrusion was possible and this was performed in order to obtain an idea of the time participation of each of these components. The appearance of nonprotein radioactivity in the pancreatic tissue after its injection was studied (unpublished results from this laboratory) and the results so far obtained in nine rats suggest that the maximum level of free glycine in the pancreas occurs in less than 10 minutes. Penetration of this amino acid is therefore rapid and does not represent

A STUDY OF PANCREAS AND SALIVARY GLANDS

359

an important time factor in computing the rate of synthesis. Had we an idea of the time necessary for extrusion we might by subtraction estimate the average time for synthesis. Extrusion is considered to be a rather rapid process as can be inferred from the in vivo observations of Cove11 ( 1928) and Hirsch (1932a, b) and by the usual observation that an increased flow of pancreatic juice follows promptly (in the first 5 minutes) the stimulatory injection. The in vivo observations inform us about the time necessary for extrusion but give no idea, however, of the time necessary for the secreted substance to run through the whole length of the excretory ducts. An attempt was therefore made to obtain an approximate idea of this “time” by injecting Pa2-labeled phosphate intravenously in a secretin-carbaminoylcholine stimulated rat and observing the rate of appearance of radioactivity in the pancreatic juice. Three minutes after the Pa2 injection a strong radioactivity could be detected. This suggests that a short time is necessary for the juice to flow through the excretory ducts system. This result is, however, capable of a different interpretation, if the duct system in the rat actively transports ions, a supposition still to be proved but quite possible (see earlier discussion). A considerable portion of the duct system is however constituted of large ducts surrounded by a distinctive connective tissue sheath and lined by an epithelium with no cytological aspect suggesting active ion transport such as prismatic epithelium, orientated mitochondria, etc. The source of error suggested above would therefore be considerably decreased by this fact. The observation of Montgomery et al. (1940) that injected radioactive sodium and potassium appear promptly in the pancreatic juice corroborates this view. To summarize, it is quite probable that ingestion and extrusion are rapid processes and might be neglected in the computation of the time necessary for synthesis. We might conclude therefore that in the conditions worked under, synthesis occurs mainly from 2 to 5 hours with a maximum at 3 hours. The results presented by Daly and Mirsky (1952) which have already been discussed, suggest that the synthesis of protein molecules precipitable by trichloracetic acid is a rapid process requiring less than 30 minutes and occurring simultaneously with the process of extrusion. If this is so, one may postulate that a major part of the process of synthesis is spent in the transformation of these proteins into the characteristic pancreatic enzyme proteins. . I t is suggestive that these processes, i.e., protein synthesis and active enzyme synthesis can be separated, as shown by Fernandes and Junqueira ( 1955). The authors working with carbaminoyl-

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choline-stimulated pigeon pancreas in vivo and in%“to observed an active incorporation of C14 glycine into proteins in both conditions, while amylase synthesis could be detected only in the in vivo experiments. As the amount of RNA decreases in the in vitra slices they conclude that possibly the RNA might be somehow related to the second phase of synthesis, i.e., the transformation of proteins into enzymes. ACKNOWLEDGMENTS The authors are grateful to Dr. I. Raw for advice on mitochondria1 enzymology, Dr. M. Rabinovitch for the chapter on the nucleus, and Mrs. T. Schreiner for efficient aid in the manuscript. These studies were aided by grants from the Rockefeller Foundation, Deutsche Forschungsgemeinschaft, and Conselho Nacional de Pesquisas.

VI. REFERENCES Allfrey, V., Daly, M. M., and Mirsky, A. E. (1953) J . Gen. Physiol. 37, 157. Allfrey, V., Daly, M. M., and Mirsky, A. E. (1955a) J. Gelz. PhysioZ. 98, 415. Allfrey, V., Mirsky, A. E., and Stern, H. (1955b) Advances in Enzymol. 16, 411. Altmann, H. W. (1952) 2. Zellforsch. u. nzikroskop. Anat. 68, 632. Anfinsen, C. B., and Killey, W. W. (1954) Ann. Rev. Biochem. 29, 17. Arzac, J. P., and Flores, L. G. (1952) Stain Technol. 27, 9. Baker, J. R. (1944) Quart. J . Microscop. Sci. 86, 1. Baker, J. R. (1949) Quart. J . Microscop. Sci. 90, 293. Baker, J. R. (1953) Bull. microscop. appl. 121 9, 1. Ball, E. G. (1930) J. Biol. Chew. 86, 433. Ball, E. G., Tucker, H. F., Solomon, A. K., and Vennesland, B. (1941) J . Biol. Chew. 140, 119. Barcroft, J., and Starling, E. H. (1904) J. Physiol. ( L o n d o n ) 91, 491. Barron, E. S. G. (1953) T e x a s Repts. Biol. and Med. 11, 653. Beams, H. W., and King, R. L. (1934) Amt. Record 89, 363. Bensley, R. R., and Hoerr, N. (1934) Anat. Record 60, 449. Berg, N. 0. (1951) A c t a Pathol. Microbiol. Scund. Sappl. 90. Bergonzi, M., and Bolcato, B. (1930) Arch. sci. biol. ( I t a l y ) 14, 573. Bern, H. A., and Alfert, M. (1954) Rev. b r a d . biol. 14, 25. Bernhard, W., Gautier, A., and Rouiller, C. (1954) Arch. Anat. microscop. Morphol. exptl. 89, 236. Bernhard, W., Haguenau, F., Gautier, A., and Oberling, C. (1952) 2. Zellforsch 1c. mikroskop. Anat. 91, 281. Borsook, H. (1950) Physiol. Revs. 50, 2Q6. Bourne, G. H., ed. (1951a) ‘Cytology and Cell Physiology,” 2nd ed., p. 232. Oxford U. P., New York. Bourne, G. H. (1951b) J . Roy. Microscop. SOC.70, 367. Brachet, J. (1950) “Embryologie Chimique.” Masson, Paris. Breuer, M. E., and Pavan, C. (1955) Chrornosoma 7, 371. Brock, N., Druckrey, H., and Herken, H. (1939) Arch. exptl. Pathol. Phrrnakol. 191, 687.

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Cain, A. J. (1947) Quart. J. Microscop. Sci. 88, 151, 467. Cain, A. J. (1950) Biol. Revs. S,73. Camis, M. (1923-24) Arch. intern. physiol. 22, 343. Caspersson, T. (1950) “Cell Growth and Cell Function.” Norton, New York. Caspersson, T., Jacobson, F., Lomakka, G., Svensson, G., and Safstrom, R. (1953) Exptl. Cell Research 6, 560. Caspersson, T., Landstroem-Hydh, H., and Aquilonius, L. (1941) Chromosoma 2,111. Chardon, G., and Gross, A. (1946) Compt. rcnd. soc. biol. 140, 1004. Claude, A. (1943) Biol. Symposia 10, 111. Claude, A. (1947-48) Harvey Lectures Ser. IS, 121. Covell, W. P. (1928) Anut. Record 40, 213. Cowdry, E.V. (1918) Carnegie Comtribs. Embryol. 8, 39. Dalton, A. J. (1951) Am. J. Anat. 89, 109. Dalton, A. J., and Felix, M. D. (1954) Am. 1. Anat. 94, 171. Daly, M. M., and Mirsky, A. E. (1952) J. Gen. Physiol. 56, 243. Daly, M. M., Allfrey, V., and Mirsky, A. E. (1952) J. Gen.Physiol. 36, 173. Danielli, J. F. (1951) in “Cytology and Cell Physiology,” (Bourne, ed.), 2nd ed., p. 150. Oxford U. P., New York. Davies, R. E., Harper, A. A., and Mackay, I. F. S. (1949) Am. J. Physiol. 167, 278. De Deken-Grenson, M. (1952) Biochim. et Biophys. Acta 8, 481. De Deken-Grenson, M. (1953) Biochim. et Biophys. Acta 10, 480. De Robertis, E.D. P. (1942) Anat. Record 84,125. Dempsey, E. W., and Peterson, R. R. (1955) Endocrinology 66, 46. Deutsch, W., and Raper, H. S. (1938) J. Physiol. (London) a,439. Dustin, P. (1949) Acta. din. belg. 4, 9. Duthie, E. S. (1933) Proc. Roy. SOC.BllS, 459. Emmel, V. M. (1945) Anat. Record 91, 39. Enjo, K. (1947) Cytologia (Tokyo) 14, 70. Fernandes, J. F., and Junqueira, .L C. U. (1953) Exptl. Cell Research 6, 329. Fernandes, J. F., and Junqueira, L. C. U. (1955) Arch. Biochem. and Biophys. 66, 54. Ferrari, R., and Hober, R. (1933) PfEiigers Arch. ges. Physiot. 2SB, 299. Frey-Wyssling, A. (1945) “Erniihrung und Stoffwechsel der Pflanzen.” Gutenberg, Zurich. Friedeman, T. E., and Haugen, G. E. (1943) J. Biol. Chem. 147, 415. Gale, E. F., and Folkes, J. P. (1954) Nature 179, 1223. Gamier, C. (1900) I. Anut. Paris. 86, 22. Gautier, A.,and Diomede-Fresa, V. (1953) Mikroskopie 8, 23. Gersh, T. (1949) Arch. Pathol. 47, 99. Granaglia, G. (1950) Monit. zoo!. ital. 68, 100. Green, D. E. (1951) in “Enzyme and Enzyme Systems” (Edsall, ed.). Harvard U. P., Cambridge. Green, D. E. (1954) Biol. Revs. 2B, 330. (Greenberg, D. M., ed. (1954) “Chemical Pathways of Metabolism,” Vols. I and 11. Academic Press, New York. Guberniev, M. A., and Il’Ina, L, T. (1950) Doklady Akad. Nawk. S.S.S.R.71, 351. Gurwitch, A. (1904) “Morphologie und Biologie der Zelle.” Fiscber, Jena. Haldane, J. B. S., and Huxley, J. (1929) “Animal Biology.” Oxford U. P., New York. Hartz, H. (1948) Acta Brevia. Neerland. Physiol. Phurmacol. Microbiol. 14, 64.

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Haselmann, H., Junqueira, L. C. U., Michel, K., Menezes, J. R., Raia, S., and Sessq A. (1953) Mikroskopie 8, 400. Hattinger, I. (1932) Nawyn-Schmiedeberg’s Arch. exptl. Pathol. Pharmakol. 166, 333. Heidenhain, R. (1875) PfEiigers Arch. ges. Physiol. 10, 557. Hill, R., and Bourne, G. (1954) Acta Anat. 20, 116. Himwich, H. E., and Adams, M. A. (1930) An$. J. Physiol. B3, 568. Hirsch, G. C. (1915) 2002.Jahrb. Abt. Physiol. 56, 357. Hirsch, G. 4C. (1931a) Withelm Roux’ Arch. Entwicklungswwch. Organ. 123, 92. Hirsch, G. C. (1931b) Z . Zellforsch. u. mikroskop. Anat. 14, 517. Hirsch, G. C. (1931~)Biol. Rats. 6, 88. Hirsch, G. C. (1932a) 2. Zellforsch. u. mikroskop. Anat. 16, 290. Hirsch, G. C. (1932b) 2. Zellforsch. u. mikroskop. Anat. 16, 36. Hirsch, G. C. (1939) “Form und Stoffwechsel der Golgi-Koerper.” Protoplasma Monographien. Vol. 18. Borntrager, Berlin. Hirsch, G. C. (1948) Verhandl. deut. 2001. Ges. Kiel , 226. Hirsch, G. C. (1955a) in “Handbuch der Allgemeine Pathologie,” Vol. 2. Springer, Berlin. Hirsch, G. C. (1955b) Personal communication. Hirsch, G. C., Rothschild, H. A., Dohi, S. R., and Junqueira, L. C. U. (1955~) Pjliigers Arch. ges. Physiol. in press. Hokin, L. E. (1952) Biochim. et Bioghys. Acta 8, 225. Hokin, L E., and Hokin, M. R. (1954a) Biochim. et Biophys. Acta 13, 236. Hokin, L. E., and Hokin, M. R. (1954b) Biochim. et Biophys. Acta 13, 401. Hiiber, P. (1949a) 2. Zellforsch. u. mikroskop. Anat. Abt. B34, 428. Hiiber, P. (1949b) Vierteljahrsschr. naturforsch. Ges. Zurich 94, 73. Hunter, F. E., Jr. in “Phosphorus Metabolism.” (McElroy and Glass, eds.) Vol. 1, Johns Hopkins Press, Baltimore. Inhuma, M., and Yangiwasa, N. (1953) Folia Anat. Japon. 26, 202. Jarvi, 0. (1939) 2. Zellforsch. 21. mikroskop. Anat. Abt. B30, 156. Jeener, R. (1948) Biochint. et Biophys. Acta 2, 439. Junqueira, L. C. U. (1951) Exgtl. Cell Research 2, 327. Junqueira, L. C. U., and Rabinovitch, M. (1954) Texas Repts. Biol. and Med. 12, 94. Junqueira, L. C. U., and Rothschild, H. A. (1953) Acfn Physiol. Latinoemer. 3, 247. Junqueira, L. C. U.,Hirsch, G. C., and Rothschild, H. (1955) Biochem. J. 61, 275. Junqueira, L. C. U., Sesso, A., and Nahas, L. (1951) Microscopie 1, 133. Kaplan, N. 0. (1951) in “The Enzymes” (Sumner and Myrback, eds.), Vol. 11, Part 1, p. 55. Academic Press, New York. Kingsbury, B. F. (1912) Amt. Record 6, 39. Kolliker, A. (1852) “Manual of Human Histology,” Vol. 1, p. 101. Sydenham Society, London. Koritz, S. B., and Chantrenne, H. (1954) Acta Biochem. and Biophys. 13, 209. Kiihne and Lea. (1882) Untermch. fikysiol. I m f . Univ. Heidelberg 2, 448. (Quoted by Noll, 1905). Lacy, D. (1954) Nature 173, 1235. Langer, H., and Grassi, A. (1955) Z . physiol. Chem. 299, 139. Langstroth, G. O., McRae, D. R., and Komarov, S. A. (1939) Can. J. Research D17, 137.

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Langstroth, G. O., McRae, D. R., and Stavraky, G. W. (1938) Proc. Roy. SOC.Bl26, 335. Laguesse, E. (1905) Rev. gtn. Histol. 1, 543. Laguesse, E. (1906) Rev. gtn. Histol. 2, 1. Lehninger, A. L. (1951) in “Enzyme and Enzyme System” (Edsall, ed.), Harvard U. P., Cambridge. Lewis, W. H. (1937) Am. I. Cartcer 29, 666. Lindberg, O.,and Ernster, L. ( 1954) “Frotoplasmotologia,” Vol. 3. Springer, Vienna. Ludford, R. J. (1948) J . Roy. Microscop. SOC.68, 1. Ludwig, 0. (1851) 2. rat. Med. 1, 271. Lynen, F., and Ochoa, S. (1953) Biochim. et Biophys. Acta 12, 299. Maziarski, S. (1910) Arch. Zellforsch. 4, 443. MonnC, L. (1942) Arkiv. Zool. BM, 1. Montgomery, M. L., Sheline, G. E., and Chaikoff, I. L. (1940) Am. I. PhysioZ. 131, 578. Montgomery, T. H. (1898) J . Morphol. I,265. Neumann, K. H. (1952) Klin. Wochschr. SO, 605. Nicolaj, P. (1939) Boll. soc. itul. biol. sper. 14, 23. Noll, A. (1905) Ergeb. Physiol. 4,84. Northup, D. (1935-36) Am. J . Physiol. 114, 46. Padykula, H.A. (1952) Am. J . Anat. 91, 107. Palade, G.E. (1955) J . Biophys. and Biockem. Cytol. 1, 59. Palade, G.E., and Claude, A. (1949) J . Morphol. 86, 35, 71. Palade, G. E., and Siekevitz, P. (1955a) Federation Proc. 14, 262. Palade, G. E., and Siekevitz, P. (1955b) Anat. Record 121, 347. Pavan, C., and Breuer, M. E. (1955) Rev. Univ. Minas Geruis ( B r a d ) 11 (Suppl.) p. 90. Pellegrino, B., Kaiserman Abramof, I. R., and Schreiber, G. (1955) Rev. Univ. Minas Gerais ( B r a d ) 11 (Suppl.) p. 121. Porter, K. (1955) Personal communication. Rabinovitch, M. (1954) Proc. SOC.Exptl. Biol. Med. 86, 685. Rabinovitch, M., Junqueira, L. C. U., and Rothxhild, H. A. (1951) Science 114, 551. Rabinovitch, M., Rothschild, H. A., and Junqueira, L. C. U. (1952a) J . Biol. Chem. 194,835. Rabinovitch, M., Valeri, V., Rothschild, H. A., Camara, S., Sesso, A., and Junqueira, L. C. U. (1952b) I . Biol. Chem. 198, 815. Raw, I. (1955) J . Am. Chem. SOC.TI, 503. Ries, E. (1935) 2. Zellforsch. $4. mikroskop. Anat. 23, 523. Rothschild, H. A., and Junqueira, L. C. U. (1951) Arch. Biochm. and Biophys. w,453. Schneider, W. C. (1953) I . Histochem. Cytockem. 1, 212. Schncider, W.C., Dalton, A. J., Kuff, E. L., and Felix, M. D. (1953) Natztre 172, 161. Schneider, W. C., and Hogeboom, G. H. (1951) Cancer Research 11, 1. Schneider, W.C., and Kuff, E. L. (1954) Am. J . Anat. 94, 209. Schreiber, G. (1949) Biol. Bull. 97, 187. Schreiber, G. (1955) Publ. staz. sool. Najoli in press. Schreiber, G., Melucci, N., Kaiserman Abramof, I. R., and Pompeu Memoria, J. M. (1955) Rev. Univ. Minas Gerais (Brasit) 11 (Suppl.) p. 100.

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Schucher, P., and Hokin, L. E. (1954) J. Biol. Chem. 210, 551. Simpson, W. L. (1941) Anat. Record 80, 173. Sjostrand, F. S.,and Hanzon, V. (1954) Exptl. Cell Research 7, 393, 415. Sluiter, J. W. (1944) 2. Zellforsch. u.. mikroskop. Anat. s3, 187. Solomon, A. K. (1952) Federation Proc. 11, 722. Sosa, J. M. (1953) Anat. Record 116, 413 (Abstract). Sosa, J. M. (1955) Personal communication. Stier, A. (1952) Z . Anat. Entw’cklungsgeschichte 116, 399. Still, E. U.,Bennett, A. L., and Scott, V. B. (1933) Am. J. Physiol. 106, 509. Toni, G. (1948) Boll. soc. ital. biol. sper. !& 260. I, Valeri, V. (1954a) Compt. rend. 238, 1613. Valeri, V. (1954b) Science 120, 984. van Weel, P. B., and Engel, C. (1938) 2. vergleich. Physiol. 23, 214. Visscher, M. B. (1942) Federation Proc. 1, 246. Weiss, J. M. (1953) J. Exptl. Med. 88, 607. Weiss, J. M. (1955) J. Exptt. Med. 101, 213. Westeimer, F. H. (1954) in “A Symposium on the Mechanism of Enzyme Action” (McElroy and Glass, eds.), p. 321. Juhns Hopkins Press, Baltimore. Wilson, E. B. (1928) “The Cell in Development and Heredity,” 3rd ed., p. 95. Macmillan, New York. Worley, L. G. (1946) A w . N.Y. Acad. Sci. 47, 1. Worley, L. G. (1951) Exptl. Cell Research 2, 684. Yokoyama, H.O., and Stowell, R. E. (1951) I. Natl. Cancer Znszst. 11, 939.

The Acrosome Reaction JEAN C. DAN

Misaki Marine Biological Stntion, Mizlra-Shi, Japnn Page

I. Introduction ....................................................... 11. Structural Aspects of the Acrosome Reaction ....................... 1. Echinoidea ..................................................... 2. Asteroidea, Holothuroidea, Ophiuroidea ......................... 3. Mollusca: Pelecypoda ........................................... 4. Gastropoda ..................................................... 5. Annelida ....................................................... 111. Conditions Determining the Acrosome Reaction ....................... 1. “Physiological Maturity” of Spermatozoa ....................... 2. Presence of Calcium ............................................ 3. Presence of Self-species Eggs or Egg-Water ................... 4. Hyperalkalinity ................................................. 5. Contact ......................................................... 6. Conditions Special to Mytilus .................................... IV. Relation to the Agglutination Reaction ............................. V. Acrosome Lysin ................................................... VI. Role of the Acrosome Reaction in Sperm Entrance ................... VII. Possible Role of the Acrosome Filament ............................. VIII. Acrosome Reaction in Relation to Specificity in Fertilization . . . . . . . . IX. References .........................................................

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I. INTRODUCTION “The acrosome has long been treated with rather scanty respect,” according to Wilson in 1925. At that time the concept of its mechanical role as the “perforatorium,” of boring into the egg cell, had been rendered problematical by the general acceptance of Lillie’s chemical reaction theory of fertilization, and no substitute function had been established. In 1923, Bowen, studying the part played by the Golgi apparatus in the formation of the acrosome, had suggested that it might contain an egg-activating enzyme, and a claim that such an enzyme had been found in the acrosome of amphibian spermatozoa was later put forward (Einsele, 1930; Parat, 1933 ; Wintrebert, 1933)) although it was based on rather inadequate experimental data. Since then, no evidence has been produced to substantiate this suggestion of Bowen’s. Meves’ 1915 description of fertilization in Mytilus edulis includes the observation that the large acrosome is completely absent from spermatozoa which are just within the cytoplasm of eggs fixed soon after insemination; he comments that the acrosome substance must be very quickly dissolved. Popa, in 1927, was the first to report that in vitally stained sea urchin 365

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spermatozoa, a small amount of a sticky substance is extruded from the acrosomes of the spermatozoa in the presence of egg-water. Although subsequent observation with vastly improved optical apparatus has required the revision of many of Popa’s conclusions, his work stands as the pioneer effort to treat the spermatozoon as an individual, reactive cell. A series of studies of living spermatozoa by means of the phase contrast microscope and fixed material with the electron microscope, has supported Popa’s conclusion that the sea urchin acrosome undergoes a change in the presence of substances from the egg. This has, furthermore, been found to be true of the spermatozoa of other phyla, and it appears that the reaction of the acrosome holds a general significance in connection with the fertilization process. THE ACROSOME REACTION 1. Echinoidea

ASPECTSOF IT. STRUCTURAL

In living sea urchin spermatozoa (Hemicentrotus pulcherrimus, Pseudocentrotus depressus, Anthocidaris crassispina, Mespiliiz glo bulus) as well as in those of the sand dollars Clypeaster japonicus and Astriclypeus nzanni, with the oil immersion objective of the phase contrast microscope, the acrosome region is seen to consist of a dense apical granule and a more hyaline proximal substance between the granule and the anteriormost part of the nucleus. Afzelius (1955) has shown in electron micrographs of thin-sectioned sea urchin spermatozoa that the apical granule consists of homogeneously distributed, fine particles (Fig. 1) , while the hyaline region appears as a deep depression extending into the nucleus, filled with a less dense, homogeneously granular material. I n Strongylocentrotus droebachiensis this depression may or may not contain an elliptical vacuolelike structure ; in Psawnechinus miliaris, Echinus esculentzcs, and Echinocardium cordatuin no such vacuole is present. The acrosome reaction is most easily observed in sea urchin spermatozoa by adding egg-water to a fresh sperm suspension (Dan, 1952). With phase contrast, reacted spermatozoa are found attached at their tips to the slide and cover-glass surfaces by a flexible strand which allows rotational movement of the sperm. Careful observation shows that the apical granule is missing from such spermatozoa; the flexible strand is almost invisible and best detected by its restraining effect on the swimming activity of the spermatozoa. Electron micrographs of Hemicentrotus spermatozoa fixed after one minute in sBa water and two seconds and twenty seconds after the addition of egg-water to a similar sea water suspension are shown in Figs. 2, 3, and 4, respectively. It is evident that some sort of rapid change

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FIG.1. Strongylocentrotw droebackiensis, spermatozoon longitudinally sectioned. In the lower right part of the picture there is a transverse section through a spermatozoon showing a vacuole in the acrosomal region. Magnification xx 000 x. Inset a longitudinal section through a spermatozoon acrosme with a vacuole. Magnification xx 000 x. (Photographs by Afzelius (1955), reproduced by permission.)

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FIG.2. Spermatozoa of Hemicentrotus pulcherrimw in sea water fixation).

(formalin

FIG.3. Hemketzfrotw spermatozoa, formalin-fixed 2 seconds after exposure to egg-water. Material of acrosome granule has been pushed away from sperm head by extrusion of filament. (Breakage in filament, usually found in preparations of sperm fixed immediately after acrosome reaction, is believed to be due to effect of desiccation on recently formed filament.) FIG.4. Hemicentrotus spermatozoa fixed 20 seconds after acrosome reaction. Material of acrosome granule and cytoplasmic membrane has largely dissipated, leaving only slender filament.

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has occurred in the more hyaline region of the acrosome underlying the apical granule, producing a filament (Fig. 3) which carries the granular material on its distal end. Judging from the way in which this material disperses after a few seconds in sea water (Fig. 4), and from its appearance in Fig. 5, it is suggested that the investing cell membrane has broken at the tip of the acrosome, exposing the material of the granule directly to the sea water. The fact that the filament usually breaks on drying in preparations fixed 2-5 seconds after the addition of egg-water but much less often in spermatozoa fixed at 20-30 seconds, indicates that it gains in tensile strength on exposure to sea water, or perhaps simply with time. No further change in the appearance of the filament can be detected in either living spermatozoa or those fixed 10-20 minutes after exposure to egg-water ; it also apparently retains its tensile strength, since spermatozoa once attached to a glass surface by it do not swim free in spite of their very vigorous flagellar activity. Among eleven species of Japanese and American sea urchins directly observed by the writer, only one, Diadem setoswm, has spermatozoa in which the form of the acrosome departs from the usual type. In this species, the acrosome region consists of a large, spherical, refringent knob (Fig. 6) ; in the presence of egg-water the sphere disappears, leaving a stubby clump of apparently fibrous material projecting from the tip of the spermatozoon (Fig. 7).

2. Asteroidea, Holothuroidea, Ophiwoideal The spermatozoa of three species of starfish, two brittle stars, and one species of sea cucumber (Colwin and Colwin, 1956) are similar in shape and quite different from those of the Echinoidea, having almost spherical heads with the acrosomes in the form of blunt cones embedded in the nucleus so that the base of the cone forms the anterior surface of the sperm head (Fig. 8A). With phase contrast the acrosome appears hyaline and more refringent than the nuclear material. In order to induce the acrosome reaction with egg-water in these forms, it is necessary to treat the sperm suspension with an “adjuvant,” such as a solution of egg-albumin (Metz, 1945). If egg-water is added to spermatozoa suspended in 0.5% crystalline egg-albumin-sea water, an irreversible agglutination takes place, the spermatozoa uniting head to head in small and large clumps (Dan, 1954a). Projecting from the center of the acrosome surface there can be seen a very slender filament (Fig. 9), between 1 The following species were investigated. Asteroidea : Asterina pectinifera, Asterias amuren&, Astropecten scoparius; Holothuroidea: Holothuria atra; Ophiuroidea: Ophiarachnelta gorgonia, Ophioplocus japonicus.

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FIG.5. Hemicentrotus spermatozoa which were fixed in sihb after reacting on contact with collodion membrane. Note that substance of acrosome granule appears to be exposed in the medium. FIG.6. Spermatozoa of Diadem setosum, in sea water. FIG.7. Diadema spermatozoa fixed after addition of egg-water.

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22 and 28 p in length in Asterias amurensis and Asterina pectinifera, about 2 0 p in Holothuria atra, and up to 3 0 p in the much larger Ophioplocus japonicus spermatozoon (Figs. 10, 11) . These filaments are rather rigid and are stable in sea water, although they could not be fixed with neutralized formalin. Electron micrographs show that they have a fibrous

FIG.8. Sketches of starfish spermatozoa. A. Before reaction; middle-piece is closely pressed against nucleus, flagellum extends directly back from head ; acrosome appears as refringent body embedded in nucleus. B. After reaction; middle-piece has become rounder, flagellum extends sideways from between nucleus and middle-piece, long slender filament has been projected anteriorly from center of acrosome.

FIG.9. Electron micrograph of Asterina spermatozoon, fixed with osmium vapor on cofldon membrane. Note that acrosome filament consists of central fiber surrounded by membrane (swellings in membrane are fixation artifacts). (From Dan, 1954a)

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FIG.10. Sketches of ophiuroid spermatozoa, drawn to same scale as that of Fig. 8. A. Unreacted. B. Reacted.

FIG.11. Spermatozoa of Ophioplocus jupotticus, osmium vapor-fixed on collodion membrane after treatment with ammoniacal sea water of pH 9. Filament of center spermatozoon broken in handling ; filament of sperm at right incompletely extruded, stouter than normal.

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core covered by a very delicate membrane which may form blisterlike vesicles, presumably as an effect of the fixative (Os04 vapor). Concurrent with the extrusion of the acrosome filament, a change takes place in the arrangement of the parts of the sperm head. The middle-piece becomes more nearly spherical and is less closely pressed against the nucleus ; and the tail, which originally extends directly backwards, in the reacted spermatozoon projects laterally from between the head and middlepiece (Figs. 8B, IOB). The acrosome loses its high refringency, appearing grayer than the nucleus with phase dark contrast, but no change in the shape of this region can be detected.

3. Mollusca: Pelecypoda The bivalve molluscs form particularly appropriate material for a study of acrosome behavior (see Dan and Wada, 1955) because several species of this group have strikingly large spermatozoa in which some of the structural details are observable even in the living state. This is especially true of Mytilus edulis, which will be described as representative of the group. The MytiZus sperm head, together with its circle of five small spheres composing the middle-piece, is about 7 p in length, with a long, tapering acrosome making up more than half the total length (Fig. 12). Figure 13

FIG.12. Mytilw spermatozoon, formalin-fixed in chilled sea water suspension. (From Dan and Wada, 1955)

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is a composite sketch of the fine structure of such a spermatozoon, particularly of its acrosome, as disclosed by a thin-section study now in progress. Electron micrographs of longitudinal sections through the spermatozoon show that the acrosome consists of an outer sheath enclosing a central

FIG.13. Sketch of fine structure of Mytiltis spermatozoon acrosome region; details taken from a number of electron micrographs of thin-sectioned cells. Left side of acrosome sheath is shown in wrinkled condition found in sections; right side is drawn smoothed as it appears (with phase contrast oil immersion) in osmium-fixed whole spermatozoa before methacrylate embedding. See text for description of structures.

tube, both composed of strongly osmiophilic granular material, although the structure of the sheath appears to be less stable, since it is invariably found thrown into folds (as in the left side of the sketch), presumably as the result of partial breakdown caused by the action of the fixative (about 0.5% OsOlin sea water). The central tube ends just beneath the rounded

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end of the sheath in a knoblike granule, the diameter of which is slightly greater than that of the tube immediately proximal to it. Extremely tenuous connecting fibrils extend across the narrow space between the two structures. The walls of the two concentric tubes unite to form the wall of the widened basal part of the acrosome, and are continuous with a thick ring of the same substance which is in contact with the nucleus. Through the central cavity of this basal part there runs an extremely slender tube which

FIG. 14. Mytilus spermatozoon which has reacted in presence of egg-water. (From Dan and Wada, 1955)

widens into a funnel at each of its ends-distally making contact with the inner wall of the central tube, and proximally opening into the end of a cylindrical cavity which extends into the nuclear material, ending near the insertion of the flagellum. Most of this complex structure disappears almost instantaneously when the acrosome reaction occurs, as on the addition of egg-water, leaving a slender filament about l o p in length (Fig. 14). It is also possible, by exposing sperm suspensions to low temperature or to sea water containing excess calcium, to cause the breakdown of the sheath, leaving the inner tube exposed (Fig. 15). I n such cases there is very little increase in its length, and it has a perceptibly greater diameter than that of the filament

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produced in the presence of egg-water. Moreover, when Mytilus spermatozoa are exposed to between 0.5 and 5% neutralized formalin in sea water at room temperature, in most of them the acrosomes elongate and are fixed in curved and even spiral shapes. This effect is less marked if the suspension is chilled. The oyster spermatozoon, on the other hand, is among the smallest that have been found in any of the groups studied, and its acrosome

FIG.15. Mytilus spermatozoon, showing breakdown of acrosome sheath (partial reaction) on addition of excess calcium to sea water suspension. consists of a very low, broad-based cone, in which, however, some differentiation can be detected in the living state (see Dan and Wada, 1955). On reaction, the cone disappears and a filament about 8 p in length is extruded (Fig. 16). This filament also, like that of the starfish, shows evidence of a central fiber surrounded by an extremely delicate membrane.

4. Gastropoda In several genera among the Archeogastropoda (Turbo, Scutus, Umbonium, Tegula, Monodonta, Cdcar, Lunella, Clypidina) in which fertilization takes place externally, it has been found that the large spermatozoa possess a prominent acrosome, which undergoes a reaction of a different

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sort from that observed among the bivalves. Both before and after the acrosome reaction, the spermatozoa of these genera resemble each other closely, differing chiefly in over-all size of the head and middle-piece, which varies between 5 and 7 p . In Scutus scaplza, a limpet, the nuclear part of the sperm head is elongated-cylindrical in shape, with a bluntly tapering acrosome making up 30% of the total length of 10 p (Fig. 17). In the living spermatozoon,

FIG.16. Spermatozoon of Crassostrea eckinata, reacted in presence of egg-water on collodion membrane (osmium vapor fixation). The sharply pointed tip and the expanded condition of the surrounding membrane are generally characteristic of these filaments. Note the flattened anterior surface of the sperm head and the remnants of the former acrosome membrane. (Reacted spermatozoa are often found with this region stuck to glass or collodion surfaces.) with dark contrast, the acrosome appears brightly refringent, and no internal structure can be seen in it. In reacted spermatozoa (Fig. 18) the material filling the acrosome has been ejected, leaving only a membrane delicate enough to appear transparent even with the electron microscope. To what seems to be the original acrosome membrane has been added an even more delicate extension, and a rather sturdy filament, attached at its base to the nuclear surface, extends through the empty acrosome membrane and for some distance beyond it.

THE ACROSOME REACTION

FIG.17. Spermatozoon of

FIG.18. Reacted

Scutus scapha in sea water.

Scutw spermatozoon ( formalin fixation in suspension).

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5 . Annelida A few observations made on the spermatozoa of annelids have shown that the often conspicuous acrosome reacts at the egg surface, and sometimes in egg-water. Of three nereids, Perinereis cultrifera and Platynereis dumerilii produce a short (about 3 p ) , stout filament on reaction (Figs. 19 and 20 A, B, D, E) ; Nereis mirabilis has a very small acrosome and

FIG.19. Reacted spermatozoon of Perinereis (formalin fixation in suspension containing egg-water) .

no filament could be found on reacted spermatozoa (Fig. 20 F, G). Hydroides norvegicus has a cap-shaped hyaline acrosome apparently without internal structure, which breaks down at the egg surface or in sea water at p H 8.8, but apparently without the extrusion of a filament (Fig. 20 H, I). The acrosome of a terebellid species is nearly hemispherical (Fig. 20 J, K), completing the curve of the nuclear part of the head; this acrosome breaks down on contact with the vitelline membrane, but it has not yet been established that a filament is formed in this species. In both this form and Perinereis, a transparent spherical membrane is often found in the place of the acrosome on sperm exposed to egg-water (Fig. 20 C, L) ; this is believed to represent an intermediate stage in the acrosome reaction.

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THE ACROSOME REACTION

111. CONDITIONS DETERMINING THE ACROSOME REACTION

1. “Physiological Maturity” of S p e r m a t o z o a The first requisite for obtaining a reaction of the acrosome in the spermatozoa of any of the species studied is that the cells must be fully mature in a physiological sense. Admittedly there are unknown factors involved in attaining this condition. While all the sea urchin spermatozoa obtained

n

FIG.20. Sketches of various annelid spermatozoa: A,B,C : Perinereis cultrifera; D,E : Platynereis dumerilii; F,G : Nereis mirabilis; H,I : Hydroides norvegicus; J,K,L : an unidentified terebellid worm. A,D,F,H,J : before reaction ; B,E,G,K : reacted spermatozoa ; C,L : “partial” or atypical reaction.

by a forced shedding are capable of reacting, molluscan gametes require some sort of final “processing” just before or at the time of spawning in order to achieve an optimally reactive condition. For this reason it is highly desirable to induce a quasi-natural shedding by one of the effective methods (see Dan and Wada, 1955). In some cases the spermatozoa from excised molluscan testes, which are at first quite inert, become reactive after being suspended for some time in sea water. This characteristic is not entirely peculiar to the molluscs. As mentioned above, starfish sperm shed from excised testes are usually immobile,

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and must be treated with an “adjuvant” before they will become actively motile, agglutinate, or undergo the acrosome reaction in response to eggwater. However, like those of many molluscan species under this condition, such starfish spermatozoa become immediately active on being mixed with unfertilized eggs and are quite capable of fertilizing them. In the sea urchin D i a d e m setosum spawning occurs only at the full of the moon (Fox, 1924). Spermatozoa obtained three or four days before the spawning date can be made to agglutinate by egg-water, but most of them do not undergo the acrosome reaction, although all the spermatozoa react when they are tested on the day of spawning.

2. Presence of Calcizlm In all but one of the species which have been investigated, the removal of calcium from the medium (see Dan, 1954b) is the only method which has so far been found to suppress the acrosome reaction without causing any appreciable morphological changes in the spermatozoa or impairing their motility. The one exception is the sand dollar Clypeaster japonica; in this species, the mere suspension of “dry” sperm in Ca-free sea water causes the acrosomes of all the cells to react, and fertilization takes place readily in Ca-free sea water. The addition of up to 2% of 0.36 M sodium citrate to the Ca-free sea water does not change the result, but no reaction occurs in Ca-Mg-free sea water. However, the spermatozoa lose their motility and show morphological changes after 2-5 minutes in this medium. If Clypeaster spermatozoa were exceptional only in that their acrosomes could react in the absence of calcium, it would be reasonable to assume, and probably simple to show, that they are able to use magnesium as a substitute for calcium. The fact that lack of calcium induces the acrosomes to react complicates the problem, for which so far no explanation is in sight. The general phenomenon, that most spermatozoa do not react to the usually effective stimuli when the calcium concentration is below certain thresholds, is also without explanation; it can only be added to the list of facts indicating that calcium is of the greatest importance in cellular irritability. 3. Presence of Self-Species Eggs or Egg- Water

With no exception that can be detected, the most effective agent for inducing the acrosome reaction is the presence of self-species eggs (in calcium-containing sea water) . Since echinoid spermatozoa react very readily to several kinds of stimuli, including even dilute solutions of egg jelly, they are not good material for testing the relative effectiveness of

T H E ACROSOME REACTION

383

various conditions. On the contrary, the acrosomes of gastropod spermatozoa are usually unaffected by egg-water, but can be seen to react as the spermatozoa force their way through the wide jelly layers surrounding the egg membrane. Whether the spermatozoa react as readily in the presence of fertilized as of unfertilized eggs is difficult to determine, because the jelly layers found around most eggs must be saturated with any specific substance characteristic of the unfertilized egg, and the complete removal of such layers involves a rather drastic change in the conditions of the experiment. The oyster egg, however, is quite lacking in a jelly layer; when fertilized oyster eggs are washed and reinseminated, spermatozoa react at their surfaces apparently as readily as they do with unfertilized eggs.

4 , Hyperalkalinity Increase in the alkalinity of the medium to about p H 9 has been shown to result in a breakdown of the acrosome in gastropods (Tyler, 1949), sea urchins (Dan, 1952), and bivalves (Dan and Wada, 1955). In both the molluscan groups, raising the pH causes an increase in the motility of sluggishly moving spermatozoa, and is a necessary procedure if fertilization is to be obtained under laboratory conditions in some species (Wada, 1941). It is therefore believed that this treatment in the molluscs tends to favor the occurrence of a typical acrosome reaction, rather than to cause an atypical breaking down of the acrosome region. Sea urchin spermatozoa show a somewhat less typical reaction (Fig. 21), and in dense suspensions they agglutinate irreversibly ; on the other hand, raising the p H has long been the favorite method for increasing the rate of fertilization in hybridization experiments with echinoderms (Loeb, 1916).

5. Contact Reaction of the acrosome on contact with glass surfaces is observable any time a suspension of sea urchin spermatozoa in sea water is placed under the microscope, as indicated by the characteristic rotation around the acrosome filament which is stuck to the glass. Figure 5 is an electron micrograph of )Hemicentrotus spermatozoa which were placed in a living suspension in plain sea water on a collodion film and formalin-fixed in situ. The spermatozoa of other groups besides the sea urchins also respond to contact with a reaction of the acrosome, at rates which parallel their readiness to react to egg-water. It has been observed that oyster spermatozoa show very few reacted acrosomes in the bulk of a suspension when egg-water is added, but a high percentage of these same spermatozoa

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react if they are brought into contact with a glass surface or if the suspension is stirred vigorously with a glass rod in the presence of eggwater. It is tempting to relate this dependence on contact of the oyster acrosome reaction to the fact that, since oyster eggs have no jelly, contact with the egg surface must constitute the normal stimulus to reaction of these spermatozoa.

FIG.21. Hemicentrotzcs spermatozoon, showing somewhat atypical acrosome reaction at pH 9.

6. Conditions Special to Mytilus As was mentioned above, Mytalus spermatozoa have been found to undergo a partial acrosome reaction if they are left for several hours at 2-4" C., or if the calcium content of the sea water is increased by the addition of about 10% of 0.36 M CaC12. Since these conditions are ineffective in extremely dense suspensions in which the activity of the spermatozoa is reduced, it is believed that they can be regarded as acting by stimulating, although inadequately, the acrosome to react, rather than by in some way directly causing a breakdown of the acrosome sheath. These agents have not been found to be equally effective with the spermatozoa of other species, in the few cases in which they have been tested. A 0.05% solution of merthiolate in sea water also causes the Mytilus acrosomes to undergo a partial reaction, but in this case the spermatozoa are immobilized before the acrosome breaks down, and cytolize soon afterward.

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385

TO THE AGGLUTINATION REACTION IV. RELATION Popa (1927) believed that the extrusion from the acrosome of a small amount of sticky substance could explain agglutination of Arbacia spermatozoa, the gradual dissolution of the substance setting the sperm free and leading to the “reversal” of agglutination. However, several considerations indicate that such a mechanism is inadequate to account for all the aspects of the phenomenon. In the first place, the dissolution of substance which does take place is complete within 10 to 20 seconds, while agglutination usually lasts for 2 to 3 minutes. Moreover, the small, regular, head-to-head clusters appear almost instantaneously on the addition of egg-water, precluding the possibility that they are formed by random acrosome-to-acrosome collisions. I n the third place, microscopic observation of an agglutinated suspension shows that the spermatozoa are not held immobile in the clusters and that whole clusters merge to form larger ones. Furthermore, the same sort of acrosome reaction occurs in the spermatozoa of several genera which show no agglutination at all (Clypeaster), or a faint, irreversible reaction (MespiZkz, Astriclypeus) . Agglutination is extremely variable in Diad e m , sometimes being slight and irreversible, occasionally strong and reversible, both with and without the breakdown of the large hyaline knob covering the acrosome (Fig. 6). Finally, in the three Misaki species of regular sea urchins which undergo typical reversible agglutination in the presence of egg-water ( H . pulcherrimus, A . crassispinu, P . depressus), the acrosome reaction is completely suppressed in a Ca-low medium, although the spermatozoa are strongly and reversibly agglutinated in this medium (Dan, 1954b). Such suspensions, moreover, retain their full fertilizing capacity after the reversal of agglutination, when they are used to fertilize eggs in sea water. A reduction in the fertilizing capacity of sperm suspensions which have been reversibly agglutinated in sea water was first reported by Lillie (1913) ; the fertilizin theory explains this as the result of a chemical binding, by the jelly substance (fertilizin) dissolved in the egg-water, of another substance (anti-fertilizin) borne on the sperm head, which must be available if fertilization is to take place (see Tyler, 1948). However, the finding reported above, that spermatozoa do not lose their fertilizing capacity after agglutination if the acrosome reaction is suppressed by lack of calcium, indicates that reversible agglutination is a matter of less than primary importance so far as fertilization is concerned. The problem of specificity in agglutination has been thoroughly examined by Elster (1935), who found such lack of correlation between crossagglutination and cross-fertilization among the Neapolitan sea urchin

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species that he was led to question the significance of fertilizin in fertilization (see also Runnstrom, 1952, for further discussion of this problem). The relation between interspecies agglutination and the corresponding acrosome reaction is similarly confusing, so far as it has been investigated. For example, in the cross, Clypeaster sperm X Anthocidaris egg-water, there is no agglutination but all the sperm react. In the reverse cross, there is strong, irreversible agglutination, but only about one-third of the spermatozoon acrosomes react. In both crosses of Hemicentrotus x Pseudocentrotus, there is strong reversible agglutination, but about half of the sperm show no acrosome reaction. It is probably significant that there is no combination in which some acrosome reaction does not occur ; this agrees with the experimental finding that increase in the sperm concentration usually results in a certain amount of cross-fertilization even in “difficult” crosses.

V. ACROSOME LYSIN The attempt to interpret the difference between the condition of the sea urchin acrosome immediately following reaction (Fig. 3) and after 20 seconds (Fig. 4) led to the assumption (Dan, 1952) that a sea-watersoluble substance is released by the breakdown of the cytoplasmic membrane at the anterior tip of the spermatozoon, and that this substance is a lysin specific for the vitelline membrane of the unfertilized egg. That such lysins are contained somewhere in the sperm cell had been established by Tyler (1939) and his students (Berg, 1949, 1950; Krauss, 1950a) in molluscan material ; this was confirmed by von Medem ( 1942). Krauss (1950b) also extracted from sea urchin sperm a lysin which dissolved preserved molluscan membranes, but he was unable to test its effect on the sea urchin membranes because of the side effects caused by other substances present in the extract. Taking advantage of the fact that the extraordinarily large acrosome of the Mytilus spermatozoon can be made to react by various means (see above) releasing its substance into the suspending medium and leaving the rest of the cell undamaged, Wada et al. (1956) have shown that this substance contains a lysin which dissolves the vitelline membrane of the Mytilus egg. Extraction of suspensions in which !%-950/0 of the sperm have reacted indicates that there is no lytic activity left in cells which have released their acrosome substances, while similar extracts of intact cells have lytic activity comparable to that of the acrosome-substance-containing supernatant of reacted suspensions. On comparing Fig. 15 with Fig. 13, it is evident that the major portions of the substance included in the intact acrosome which have disappeared on reaction are that making up the sheath and the material which fills

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the enlarged basal cavity. Until more definite information is obtained concerning the chemical characteristics of the lysin, however, it is premature to speculate concerning its state in the intact acrosome. Tyler (1939) has demonstrated with Megathura, the giant keyhole limpet, that when eggs are placed in a 1% sperm suspension, the egg membrane, which is separate from the egg surface, is rapidly dissolved by a lysin carried by the spermatozoa. This observation has been repeated with the Japanese limpet, Scutus scapha, a member of the Fissurellidae, the family to which Megathura belongs. If the eggs are inseminated with a dilute sperm suspension and single spermatozoa are followed, under phase contrast (40 X objective), as they swim through the dense jelly layer, it is easy to see that the acrosome reacts (Figs. 17, 18) as the spermatozoon approaches the egg membrane. The spermatozoon is stopped at the membrane surface for about 30 seconds, although its flagellar activity continues without change ; then it abruptly penetrates the membrane and swims rapidly in the perivitelline fluid. The first spermatozoon to penetrate the membrane usually goes directly to the egg surface and is taken into the cytoplasm ; later arriving spermatozoa swim about within the perivitelline space. If the focus is raised so that the surface of the membrane can be observed obliquely, and attention is directed to the point where a spermatozoon has just arrived, a bright spot can be seen to appear and spread to a diameter of about 5 p before the spermatozoon suddenly disappears through it. Even after the sperm has penetrated, the appearance is the same, as though the material were softened but not dissolved completely, so that a small dent is formed in the smooth curvature of the membrane.

VI. ROLEOF THE ACROSOME REACTION I N SPERM ENTRANCE I n the discussion of the factors which determine the occurrence of the acrosome reaction, it was stated that lack of calcium in the medium is the only general condition which prevents reaction without causing any other detectable changes in the structure or activity of the spermatozoon. It can hardly be a coincidence that this condition also prevents fertilization, so far as the writer is aware, in all cases which have been tested with the sole exception of Clypeaster japonica, the only species which has been found to show a reaction of the acrosome as well as fertilization in a Cafree meduim. Further support for the thesis that the acrosome reaction is a prerequisite for sperm entrance comes from the “classical” method of increasing the alkalinity of the medium, in order to obtain fertilization in experiments involving species crosses, self-fertilization in self-sterile forms (Morgan, 1927), and Ca-Mg-deficiency of the medium (Loeb, 1914, 1915), since

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such increase in p H has been found to be the most generally effective means of inducing the acrosome reaction in a wide variety of species, Elster (1936), studying the role of fertilizin in sea urchin fertilization, obtained a result in crossing experiments for which he was unable to find a satisfactory explanation within the limits of the fertilizin theory. He found (Table I) that in the cross, Arbacia pustulosa sperm x Paracentrotus Svidus eggs, there is a marked increase, from 1 to 40%, in the TABLE I EFFECT OF ADDED Arbacia EGG-WATER IN CROSS: Arbacia SPERMX Paracentrotus EGGS(From Elster, 1936) Time after suspension of sperm in (a) (b) (c)

0 min. 5 min. 30 min. 1 hr. 3 hr. 6 hr

% Fertilized Eggs A. Eggs in pure sea water B. Eggs in egg-water (a>

(b)

(a)

(b)

(c)

1 0 1 1 0 1

40 2 2 0 1 1

27 42 38 27 29 23

31 14

33 5 1 1 2 1

7 15 13 13

Note : (a) sea water (untreated sperm) (b) weak egg-water (c) strong egg-water

cross-fertilization rate if Arbacia egg-water is added to the sperm suspension (A ( a ) , ( b ) ) , so long as the egg-water-treated sperm are used immediately, but this improvement in the fertilizing capacity is lost within five minutes. H e found the same increased fertilization rate when Arbacia egg-water was added to the Paracentrotus egg suspensions (B ( a ) ) , but this enhancing effect was also effaced after the first five minutes by treatment of the fertilizing sperm suspensions with Arbacia egg-water ( B ( b ) ), especially B (c)). If these results are interpreted in terms of the acrosome reaction, it would appear that the presence of the self-species eggwater increased the rate of acrosome reaction, and thereby the crossfertilization, in the first case (A ( b ) ) ; this effect would be expected to disappear in a short time, as the acrosome lysin was dissipated in the medium. When the Arbacia egg-water was added to the Paracentrotus egg suspension, the enhancing effect would appear each time untreated sperm were added to the eggs (B (a) ), but pretreatment with egg-water ( B (c) ) , as in the first case, would reduce the fertilizing capacity of the sperm after the first few minutes.

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Since the acrosome reaction in sea urchin spermatozoa does not cause any change in the shape of the rest of the cell, it is impossible to determine whether or not the reaction has occurred in a fertilizing spermatozoon in the process of penetration. In the other forms which have been studied, however, there are unmistakable evidences that the reaction has occurred in the fertilizing spermatozoon. In starfish, not only is the filament visible, extending through the jelly layer, but the changed relation of the tail and middle-piece is readily apparent. In all the bivalve species in which the process has been observed, the fertilizing spermatozoon remains closely attached to the vitelline membrane for some time; in every case it is clearly in the reacted form, with the acrosome absent, the nuclear part of the head rounded, and the four or five units making up the middlepiece more nearly spherical than before reaction (see Dan and Wada, 1955). In the gastropods Scutus, Calcnr, and Turbo, the acrosome can be seen to react as the spermatozoon forces its way through the wide layer of jelly, and only those in which the reaction has occurred close to the egg membrane are able to penetrate it. TABLE I1 FERTILIZING CAPACITY OF Mytilus SPERM EXPOSED TO EGG-WATER BEFORE INSEMINATION (% FERTILIZATION) Relative Sperm Concentration Time after suspension

15 min. 30 scc.

2 4 8 Spermatozoa suspended in : Egg Egg Egg Sea Sea Sea Sea Egg Water Water Water Water Water Water Water Water 1

44

10.5

0 83

85 20

1

90

77

50

1.5

94

2.5

85

Experiments with Mytiius gametes (Wada, 1955) have shown that (a) pretreatment with Mytilus egg-water greatly reduces the fertilizing capacity of the suspension when it is tested 15 minutes after the addition of the egg-water; and (b) exposure to egg-water immediately (30 seconds) before insemination “sensitizes” the spermatozoa so that the fertilization rate is greater than that of the controls. The results of two representative experiments are given in Table 11. VII. POSSIBLE ROLEOF THE ACROSOME FILAMENT The first evidence suggestive of something like a spermatozoon acrosome filament appears in the results of Lillie’s ( 1912) “partial fertilization” experiment, in which he centrifuged off the fertilizing spermatozoon from Nereis eggs, showing that the spermatozoon initiates the cortical changes

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long before it penetrates the egg. Later Loeb (1916) showed in a hybridization experiment that Strongylocentrotus franciscanus eggs could be made to elevate a fertilization membrane by starfish sperm, although the spermatozoa themselves did not enter the eggs. Reverberi (1935) found the same effect in a cross between two ascidian species; eggs were activated by spermatozoa which did not penetrate the eggs, but were detached from the surface. Such eggs showed monasters but did not segment. A consideration of the normal fertilization process in the starfish brings up several points which bear on the function of the acrosome filament in these animals, some of which are supported by certain aspects of the fertilization process in the bivalves. For example, the block to polyspermy is established by the filament; although several sperm filaments may be in contact with the egg surface at first, only one of these spermatozoa is eventually drawn through the jelly. Moreover, the other processes in the activation of the egg-i.e. cortical granule breakdown, separation of the fertilization membrane and formation of the fertilization cone-are all set off in the egg by the filament alone, while the sperm head is still outside the membrane, and even separated from it by a distance of several microns. Finally, there is at present no way to account for the passage of the sperm through the jelly layer, since the tail is practically immobile, except by assuming that the egg cytoplasm is in some manner able to draw in the filament to which it is attached (see Dan, 1954a). Since the sperm head is about 2Op away from the egg surface at the beginning of this process and separated from the active cytoplasm of the fertilization cone, when this develops, by a length of extremely tenuous filament, it seems improbable that surface tension or any sort of phagocytic activity is involved in the shortening of the filament. Unless an alternative mechanism is found, starfish fertilization thus provides reason to think that the acrosome filament not only establishes contact between the spermatozoon and the egg cytoplasm but also enables the egg cytoplasm to exert some degree of pulling force on the sperm head. In addition to such circumstantial evidence, there are observations (see Runnstrom, 1952 p. 78, for a full discussion of the submicroscopic factors involved in activation) which indicate that changes occur in the cytoplasm around the penetrating spermatozoon. Pending the securing of actual data on the subject, it may be permissible to advance the suggestion that a filament, attached to the sperm head and extending into the deeper layers of the cytoplasm ahead of the spermatozoon, offers a concrete mechanism for converting forces arising from condensation and reorientation of sub-

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microscopic fibers into traction forces acting to pull the spermatozoon into the egg cytoplasm. In Mytilus and in three species of Crassostrea, relatively long acrosomal filaments are also found (Dan and Wada, 1955), but in contrast to the starfish the acrosome reaction takes place at the surface of the vitelline membrane and the filaments are presumably discharged directly into the egg for their whole length, since not only the fertilizing spermatozoon but all supernumerary sperm are found closely attached to the membrane surface in the reacted condition when eggs are fixed 2 seconds after insemination. Within 15 to 20 seconds, all but one of these spermatozoa lose their close contact and become detached from the vitelline membrane ; the filaments are still present but are usually much shorter than those found on spermatozoa which have reacted in egg-water (Wada, unpublished observations). The eggs of these species have a thin vitelline membrane separated from the cytoplasmic surface by a rather thick (0.5-1 p ) layer of hyaline substance. No fertilization cone is formed in normal sperm entrance; after remaining outside the vitelline membrane for some time ( 1 % 4 minutes) , the spermatozoon appears simply to sink through the covering layers into the cytoplasm. It seems not unreasonable to imagine that in these forms, as in the starfish, a traction force is being applied to the filament to draw the sperm head into the cytoplasm. Moreover, since the polyspermy-preventing mechanism resulting in the ejection of all but one of the spermatozoa which first attach to the egg takes effect while the head of the fertilizing spermatozoon is still outside the vitelline membrane, it seems again, as in the starfish, to be the acrosome filament which is directly responsible for setting off this mechanism. VIII. ACROSOME REACTION I N RELATION TO SPECIFICITY IN

FERTILIZATION One of the most impressive aspects of the fertilization reaction is its specificity under natural conditions ; experimentally, however, this specificity has been found to be far from absolute (see Runnstrom, 1952). Loeb ( 1916), discussing the results of his hybridization experiments, states, “We reach the conclusion, therefore, that the specificity which allows the sperm to enter an egg is a surface effect which can be increased or diminished by an increase or decrease in the concentration of OH as well as of Ca. The writer has shown that an increase in the concentration of both substances may cause an agglutination of the spermatozoa of starfish to the jelly which surrounds the egg of purpuratus. It is thus not impossible that the specificity which favours the entrance of a spermatozoon into an

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egg of its own species may consist in an agglutination between sperniatozoon and egg protoplasm (or its fertilization cone) ; and that the agglutination is favoured if the COHor Coa or both are increased within certain limits.” (pp. 77-78.) If it is permissible, on the basis of the evidence presented in this paper, to substitute for Loeb’s idea of “agglutination” the concept of an “acrosome reaction,” his conclusion introduces the possibility that the factors responsible for the specificity of fertilization should be looked for in some aspect of the acrosome reaction. A t present three main aspects are distinguishable in the process of sperm entry as it is conceived on the sole basis of the acrosome reaction: the “trigger” process which sets off the reaction, the action of the released lysin in dissolving the metaplasmic coverings of the egg, and the function of the filament in activating the egg (as found in starfish and molluscs). If the experimental data are accepted as they stand, there are results for one species or another which negate the absolute specificity of each of these processes. For example, sea urchin acrosomes react rather readily to such naturally occurring, nonspecific influences as contact and the jelly substance of other species. The egg-membrane lysins are generally specific, but Krauss (1950b)has found that a lysin extracted from sea urchin sperm dissolves preserved Megathztra membranes. Finally, the fact that interspecies cross-fertilization occurs in some cases with no more artificial assistance than a moderate increase in the sperm concentration indicates that eggs can be activated by the spermatozoa (acrosomal filaments) of other than their own species. Continued study of this problem may well disclose that no one broad generalization will cover all cases ; that among different groups of animals, one or another of these processes in turn may prove to be the determiner of specificity in fertilization within that group. On the other hand, the probability that the activity of the acrosome lysins depends on specific enzymes opens the interesting possibility that further investigation of such lysins, isolated from acrosomes with a minimum of contamination from substances contained in other parts of the cell, might fix on them the responsibility for the specificity of the fertilization reaction.

ACKNOWLEDGMENTS The writer is deeply indebted to Mr. Seiji Wada, of the Department of Fisheries, Kagoshima University, for his invaluable cooperation in the preparation of this manuscript, especially with respect to the molluscan material, as well as for many other observations not individually attributed to him.

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She wishes also to express her gratitude to the several institutions which have been most generous in sharing their facilities during the course of this work: the Misaki Marine Biological Station, the Tokyo Institute of Technology, The Keio University Hospital Electron Microscopical Research Laboratory ;gratitude is also expressed to the Japanese Ministry of Education, for grants-in-aid.

IX. REFERENCES Afzelius, B. A. (1955) 2. Zellforsch. u. mikroskop. Anat. 411, 134. Berg, W. E. (1949) Am. Naturalist 83, 221. Berg, W. E. (1950) Biol. Bull. 98, 128. Bowen, R. H. (1923) (cited from Wilson, 1925). Colwin, A. L., and Colwin, L. H. (1955) J . Morphol. 97, 543. Dan, J. C. (1952) Biol. Bull. 103, 54. Dan, J. C. (1954a) Biol. Bull. 107, 203. Dan, J. C. (1954b) Biol. Bull. 107, 335. Dan, J. C., and Wada, S. K. (1955) Biol. BuU. 109, 40. Einsele, W. (1930) Wilhelm Roux’ Arch. Entwicklungsmech. Organ. 123, 279. Elster, H.-J. (1935) Wilhelm Roud Arch. Entwkklungsmech. Organ, 133, 1. Elster, W.-J. (1936) Zool. Anc. Supjl. 9, 168. Fox, H. M. (1924) Proc. Roy. SOC.B96, 523. Krauss, M. (1950a) J. Exptl. 2001.114, 239. Krauss, M. (I950b) J. Exptl. Zool. 114, 279. Lillie, F. R. (1912) J. Exptl. Zool. l2, 427. Loeb, J. (1914) Science [N.SI 40, 316. Loeb, J. (1915). Am. Naturalist 49, 257. Loeb, J. (1916) “The Organism as a Whole.” Putnam, New York. Metz, C. B. (1945) Biol. Bull. 89, 84. Meves, F. (1915) Arch. mikroskop. Anat. u. Etztwkklungsmech. 78, 47. Morgan, T. H. (1927) “Experimental Embryology.” Columbia U.P., New York. Parat, M. (1933) Compt. rend. SOC. biol. ll2, 1134. Popa, G. T. (1927) Biol. Bull. 62, 238. Reverberi, G. (1935) Pubbl. stac. cool. Napoli 16, 175. Runnstrom, J. (1952) Symposia SOC.Exptl. Biol. 6, 39. Tyler, A. (1939) Proc. Natl. Acnd. Sci. U.S. 26, 317. Tyler, A. (1948) Physiol. Revs. 18, 180. Tyler, A. (1949) Am. Naturalist 83, 195. von Medem, F. G. (1942) Biol. Zetttr. 62, 431. Wada, S. K. (1941) Kagaku Nadyo 4 (3), 202 (in Japanese). Wada, S. K. (1955) Mem. Fat. Fisheries. Kagoshima Univ. 4, 105. Wada, S. K., Collier, J. R., and Dan, J. C. (1956) ExptE. Cell Research in press. Wilson, E. B. (1925) “The Cell in Development and Heredity.’’ MacmPllan, New York. Wintrebert, P. (1933) Compt. rend. SOC. biol. lU, 1636.

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Cytology of Spermatogenesis VISHWA N A T H

Department of Zoology. Panjab University. Hoshiarpur. Panjab. India Page I Introduction and Scope ............................................. 395 I1. Flagellate Sperm ................................................... 397 1. Typical Flagellate Sperm ...................................... 397 2. Atypical Flagellate Sperm with the Centriole at the Anterior Tip of the Nucleus ............................................ 397 3. Atypical Flagellate Sperms without Mitochondrial Formations .... 398 4. Acrosome Formation in the Flagellate Sperm .................. 398 a . Direct Method of Acrosome Formation ...................... 398 b. Indirect Method of Acrosome Formation .................... 411 5 . Perforatorium and Periodic Acid-Schiff Technique .............. 429 6. The Fate of Mitochondria in the Flagellate Sperm .............. 429 a . Flagellate Sperms with Mitochondria in the Neck Region .... 430 b. Flagellate Sperms with a Distinct Mitochondrial Middle431 Piece ...................................................... c. Flagellate Sperms of Insects and Scorpions .................. 432 I11. Non-Flagellate Sperm .............................................. 433 1. Vesiculiform Sperms .......................................... 433 a . Decapod Crustacea ......................................... 433 b . Millipedes .................................................. 438 2. Tubuliform Sperm of Ticks .................................... 439 3. Amoeboid Sperm of Nematodes ................................ 439 I V . Chromatoid Bodies in Spermatogenesis .............................. 442 V . Evolution and Functions of the Acrosome ............................ 443 1. Occurrence .................................................... 443 2. Position of the Acrosome in the Sperm ........................ 443 3. Pro-acrosome ................................................. 443 4. Degeneration of the Acrosome in Certain Animal Sperms ........ 444 5 . Functions of the Acrosome .................................... 445 V I Evolution of the Mitochondrial Nebenkern .......................... 446 VII . Origin of the Golgi Bodies in the Cell .............................. 447 VIII . Conclusion ......................................................... 449 I X. References ......................................................... 450

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INTRODUCTION AND SCOPE

In the present review the author has attempted to discuss the recent work on spermatogenesis with particular reference to extranuclear components. such as the mitochondria. the Golgi bodies. the centrioles and their products. the axial filaments. the mitochondria1 nebenkern. the chromatoid bodies. and the acrosome. These cell components in the spermforming cells have been treated from their morphological and functional 395

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aspects; and an attempt has also been made to examine some of them (acrosome and mitochondrial nebenkern) from their evolutionary aspects. Broadly speaking, two types of spermatogenesis have been reviewedflagellate sperm and non-flagellate sperm--each with its subdivisions. These have been discussed from the point of view of the evolution of the acrosome and the mitochondrial nebenkern. The morphology of the Golgi bodies in spermatogenesis, as studied in the living cells examined under the ordinary or phase contrast microscopes, has also been considered particularly from the point of view of the personal observations of the present author and his colleagues. Technique. As stated by Baker (1933), M. Flesch was the first person to recommend Flemming-without-acetic in 1879. But priority apart, Gatenby, while working at Oxford more than thirty years ago, recommended Flemming-without-acetic (F.W.A.) followed by 0.5% iron-hematoxylin for the study of cytoplasmic inchions in spermatogenesis. Since then the author and his students have worked out the spermatogenesis of a large number of species of animals-mammals, birds, reptiles, amphibia, fishes, scorpions, ticks, insects, millipedes, decapod crustacea, and others-exclusively with this technique. This technique does not introduce any artifacts. It stains the Golgi bodies black, and clearly brings out their chromophilic and chromophobic parts, when such parts are present. It stains the mitochondria gray to black, depending on their metabolic state. It has the added advantage of fixing and staining the chromosomes and the centrosomes brilliantly, despite the absence of acetic acid. Many years ago Strangeways and Canti (1927) showed that the cell after a short period of fixation with 2 per cent osmic acid is almost like the living cell in vitro with regard to all the inclusions. The writer has been successfully employing this technique on eggs since 1928 for the demonstration of the Golgi bodies in several examples of oogenesis. Recently this method has also been employed by Shafiq (1953) for the demonstration of the Golgi spheroids (his lipochondria) . Nevertheless, this technique is not suitable for section-cutting, as osmic acid, used alone, does not make the tissue sufficiently hard to be cut into sections with the microtome. For this reason, Flemming-without-acetic acid is an ideal fixative, as the chromic acid in it makes the tissue sufficiently hard without introducing any artifacts. In the opinion of the writer, therefore, much of the controversy with regard to the Golgi apparatus could have been avoided if F.W.A. followed by 0.5% iron-hematoxylin had been used exclusively by various investigators working on spermatogenesis instead of the silver (Da Fano, Ao-

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yama) or long osmication ( Mann-Kopsch, Kolatchev) techniques, which admittedly produce serious artifacts. For this reason I have laid stress in this review on conclusions drawn by me, my colleagues and research scholars from the study of F.W.A.hematoxylin preparations and of the living cells under the phase contrast microscope. F.W.A. stained and unstained preparations appear to be identical with whole mounts of living cells in saline and since they are permanent they are in that sense superior to them. 11. FLAGELLATE SPERM 1. Typical Flagellate Sperm A typical flagellate sperm may be defined as a sperm with a vibratile tail, an acrosome always in front of the nucleus, and one or more centrioles behind the nucleus. The tail invariably consists of the axial filament, which is a backward fibrillar extension of the centriole. The greater part of the axial filament is ensheathed by a very thin cytoplasmic envelope; and the spermatid mitochondria, or their products, are always relegated to a part, greater or smaller, of the tail. Such a typical flagellate sperm is met with in mammals, birds, tortoises and turtles, frogs and toads, fishes, insects (except Cicindela, Lepisma, and the dragonfly, Sympetrum hypomelas), molluscs and the scorpions. Wilson (1925) gives an excellent review of the extensive work of Ballowitz and Retzius on flagellate sperm, “whose indefatigable labours have made known the sperms of a great number of animals from the coelenterates up to the mammals.”

2. Atypical Flagellate Sperm with the Centriole at the Anterior Tip of the Nucleus I n the tiger beetle, Cicindela (Goldsmith, 1919), the sperm is flagellate, but it has become atypical on account of the migration of the centriole to the anterior tip of the nucleus. The axial filament springs up from the centriole, and, running backward by the side of the nucleus, it enters the tail. The acrosome retains its typical position at the anterior tip of the nucleus. But in Lepisma (Bowen, 1924a; Nath and Bhatia, 1953), the centriole and the acrosome have exchanged places, the former lying at the anterior tip of the nucleus and the latter in the neck region of the sperm ! Reviewing the work of Koltzoff (1909) on the sperm of barnacles (Lepas, Bdanzls) and schizopods, and of Broman (1900a, b) on the sperm of the toad, Bombinator, Wilson (1925) states that in these forms also the centriole shifts forward to the anterior tip of the nucleus, as in Ci-

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cindela and Lepisma. The axial filament thus runs backward from the centriole parallel to the elongated nucleus.

3. Atypical Flagellate Sperms without Mitochondria1 Fornuttions Flagellate sperms may become atypical for an entirely different reason. In some sperms examined in this laboratory the entire mitochondria1 contents may be cast off during spermateleosis, so that the ripe sperm is without any mitochondria1 formations. Examples of such atypical flagellate sperms are met with in Rhabdocoeles and Acoeles (Plagiostoma, Amphanostoma, Darwinia, Macrorhynchus) ; in Polyclads (Leptoplana) ; and in Triclads (Procerodes), as reviewed by Wilson (1925). Other examples are found in digenetic trematodes (Issoparorchis, Phyllodistomum, Cyclocoelum, Cotylophoron, CastrothyEax, Asymphylodora; Dhingra, 1954a, b, c ; 1955a, b, c) ; in spiders (Plexippus paykulli, Pardosa sp.; Sharma, 1950; Sharma and Gupta, 1956) ; and in snakes (Natrix p. piscator; Sud, 1955). In the dragonfly, Sympetrune hypomelas (Nath and Rishi, 1953), the mitochondria are conspicuous by their absence throughout spermatogenesis. Dhingra has also shown that the platyhelminth sperm is devoid of an acrosome.

4. Acrosome Formation i n the Flagellate Sperm Recent research on cytoplasmic inclusions in spermatogenesis has clearly shown that the acrosome of the sperm is always formed by the Golgi bodies of the spermatid. The details of this process vary widely in different animal groups ; but, broadly speaking, there are only two methods by which the acrosome is formed from the Golgi bodies. One is the direct method of acrosome formation, in which the Golgi bodies are directly transformed into the acrosome. The other is the indirect method, in which the Golgi bodies secrete the acrosome, and after doing so are themselves sloughed off along with the residual cytoplasm. a. Direct Method of Acrosome Formution. In this section the direct method will be considered in various forms-scorpions, insects, birds, amphibia, reptiles, fishes, spiders, and ticks. Scorpions. A very convincing case of the direct origin of the acrosome from the Golgi bodies is met with in the scorpion, Buthus acute-carinatus (Nath and Gill, 1950). These authors describe the Golgi apparatus of the primary spermatocyte as consisting of two pieces, which in early spermateleosis fuse to form a single compact body. This places itself at the anterior end of the elongating nucleus and grows directly into the acrosome (Figs. la, lb). Recently Nath has examined the living sperm-forming cells of this

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scorpion under the phase contrast microscope. It seems that the Golgi apparatus in the primary spermatocyte consists of three granules, one of which is very small; and in the preparations of Nath and Gill this seems to have fused with one of the two bigger granules. In fact their text figure 111, 2 (reproduced in Fig la), gives a clear indication of the small

F

a

b

FIG.1. a, Primary spermatocyte and b, spermatid of Buthus (Nath and Gill, 1950). NOTE:K e y to Notation on Figzcres. A, acrosome; Ac, acroblast; AB, accessory body ; AF, axial filament; AG, acrosomal granule ; AP, amoeboid process ; AT, axial tube ; AV, acrosomal vesicle ; C, centrosome ; C,, proximal centrosome ; C,, distal centrosome ; CB, chromatoid body ; CG, chromatin granules ; CV, cytoplasmic vesicle ; EP, end-piece; G, Golgi body; W, sperm head; IG, idiosomic Golgi complex; M, mitochondria ; MN, mitochondria1 nebenkern ; MP, middle-piece ; MP‘, main piece ; MR, mitochondrial ring; N, nucleus; Ne, neck; P, pro-acrosome; PC, post-nuclear cap; R, ray; RC, residual cytopIasm; RF, ray figure; T, tail; V, vacuoles.

granule having fused with one of the two bigger ones (Fig. 2a). Figure 2c is a photograph of a living spermatid in which the Golgi body (proacrosome) formed by the fusion of Golgi granules has placed itself in front of the nucleus; and behind the nucleus is the bipartite mitochondrial nebenkern (Fig. 2b). Figure 2d is a microphotograph of the nearly ripe sperm in which the acrosome, the elongated nucleus, and the sperm tail with its cytoplasmic blebs all stand out very clearly. Insects. As early as 1911, Montgomery, working on the Hemipteran Euschistus, described the origin of the acrosome directly from a body called the “sphere” or the “idiozome,” which we now know to be the Golgi apparatus. Gatenby (1917a), in his pioneer work on Smerinthus, stated that “the acrosome is finally formed by the running together of several acroblasts.” According to Gatenby, “the acroblasts are quite spherical, and their wall is of equal thickness, not more bulging or thicker on one side than the other.” Each “acrosomal vesicle” differentiates an “acrosomal granule” within it ; and ultimately all the acroblasts unite together to form the acrosome. Later Bowen ( 1922a), working on Pygcera bzlcephulu, interprets the acrosome as the secretory product of the Gofgi bodies. He agrees with

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Gatenby inasmuch as there is one acrosomal granule to each acrosoinal vesicle. He also agrees with Gatenby that the acrosomal granules fuse to form one large granule, which forms the acrosome, but he differs from Gatenby inasmuch as the acrosomal vesicles are sloughed off after secreting the acrosomal granules.

FIG.2. Microphotographs of living testicular material of Bwthus under positive phase contrast; a, primary spermatocyte; b, c, spermatids; d, ripe sperm. (See key on p- 399.) Bawa (1954) , working in this laboratory on Prodenia litacra and Anaphaeis sp., has seen Golgi vesicles in the living spermatids of the former species under the phase contrast miscroscope. According to this author the Golgi bodies in both the Lepidopteran species collect near the spermatid nucleus, and soon a deeply staining granule, the acrosomal granule, appears amongst the Golgi bodies. During the growth of the acrosomal granule the outer multiple structure formed by the fusion of the Golgi vesicles gets converted into a single envelope, the acrosomal vesicle. The acrosomal vesicle subsequently disappears and the acrosomal granule, which has much increased in size by this time, prepares to form the acrosome of the mature sperm (Figs. 3a-3e).

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Amongst the insects there are three very clear cases of the direct origin of acrosome from the Golgi bodies, namely in the louse, the dragonfly, and the silver-fish insect, examined in this laboratory. Doncaster and Cannon (1920), and Cannon (1922), worked out the spermatogenesis of two species of the louse, Pediculus corporis and P . capitis. They showed clearly that the acrosome in the louse was formed

a

C

b

e

FIG.3. a, b, Spermatids of Prodenia; c-e, spermatids of AmfJhcaeis(Bawa, 1954). (See key on p. 399.)

directly from a rounded body, which was considered by them to be Golgi apparatus. Sarkaria (1944), one of my students, working on the cattle louse, Haemutopinus tuberculatus, in the Zoological Laboratories of the Panjab University at Lahore (now in Pakistan), confirmed the findings of Doncaster and Cannon. It may be noted that these authors avoided the silver and long osmication methods and used Flemming-without-acetic acid exclusively for the study of the Golgi apparatus. For this reason, as pointed out by Nath (1944), every line of their account bears the imprint of careful observation and interpretation. Subsequently Sharma and Malik (1953) worked on both the species of Pediculus at Hoshiarpur and confirmed in every detail the findings of Doncaster and Cannon (Figs. 4a4c).

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In the Hoshiarpur dragonfly, Sympetrum hypomelar also, which is remarkable for the complete absence of the mitochondria throughout spermatogenesis, the acrosome arises directly from the Golgi bodies (Nath and Rishi, 1953). In the earliest spermatid there are a few prominent,

m

FIG.4. a, Spermatocyte; b, spermatid ; c, nearly ripe sperm of louse (Sharma and Malik, 1953) ; d-f, spermatids; g, ripe sperm of Sympetrum (Nath and Rishi, 1953) ; h-m, spermatids of Lepisma (Nath and Bhatia, 1953) ; n-t, spermatids, u, ripe sperm of domestic duck (Gupta, 1955). (See key on p. 399.)

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darkly staining Golgi bodies. Later these fuse together to form a single mass, the pro-acrosome. This is found at the anterior end of the nucleus and grows directly into the acrosome (Figs. 4 d 4 g ) . Bowen (1924a) reported an important and unique discovery that in the sperm of Lepisma domestica, the silver-fish insect, the acrosome remains in the neck region, from where it sends backwards into the tail region a filamentary process, which becomes indistinguishable from the axial filament. Bowen also reported that in Lepismu sperm, as in Cicindela (Goldsmith, 1919), the centriole shifts to the anterior tip of the nucleus. Later Gatenby and Mukerji (1929) published a paper on the spermatogenesis of Lepisma and strongly contradicted Bowen’s conclusions. According to these authors, (1) the acrosome in the Lepisma sperm lies as usual at the anterior tip of the nucleus, and (2) the “acrosome” of Bowen or the “middle-piece” of Charlton (1921) is the “post-nuclear body.” This controversy seems to have been laid to rest with the publication of a paper by Nath and Bhatia (1953) on the same subject. These authors are in general agreement with the broad conclusions of Bowen, although there are some important differences of detail. One of these differences of detail is with regard to the manner in which the Golgi bodies form the acrosome. According to Bowen the acrosome in the Lepisma sperm is secreted by the Golgi bodies, whereas, according to Nath and Bhatia, it is formed by the direct fusion of the Golgi vesicles (Figs. 4 h 4 m ) . Indeed Bowen is himself doubtful about the acrosome being a secretory product of the Golgi elements (his acroblasts). In his figures 93 to 95 he correctly shows the Golgi elements coming together, but “this multiple construction of the acroblast makes it difficult to observe the actual deposition of the acrosome, which is perhaps deposited a little at a time as in the Lepidoptera.” Bowen admits that in the scanty material at his command, “the steps in the deposition of the acrosome could not be followed satisfactorily. . . ” The material studied by Bowen, however, had been admittedly prepared with fixatives containing fat solvents. Birds. In the birds a very clear example of the direct origin of the acrosome from the Golgi bodies has been recently discovered in the domestic duck by Gupta ( 1955). Gupta’s conclusions are based not only on the study of fixed but also of the living material under the phase contrast microscope. According to this author, the Golgi elements in the earliest spermatid of the duck are in the form of discrete granules and spheroids, some of which show a chromophilic cortex and a chromophobic medulla. Gradually, in the course of spermateleosis, all the Golgi elements, granules and spheroids alike, come together to form a single prominent spherical or ovoid body,

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Later this body also shows a duplex structure and attaches itself to the anterior aspect of the nucleus. This is the pro-acrosome, which is directly differentiated into the acrosome. The pro-acrosome, which in its early stages of formation shows a duplex structure, gradually becomes entirely chromophobic, and assumes the appearance of a vacuole, standing prominently under the phase contrast microscope. In late stages of spermateleosis the pro-acrosome becomes more or less triangular and a deeply staining granule appears at its apex. Gradually this granule grows in size, extending through the whole pro-acrosomal triangle till finally the acrosome is formed (Figs. 4 n 4 u ; 5a). Zlotnik (1947), working on the spermatogenesis of the domestic fowl with the silver nitrate techniques of Da Fano and Aoyama, also describes an archoplasmic vacuole, which develops in close association with the Golgi

FIG.5. Microphotographs of living spermatids under positive phase contrast ; a, domestic duck (Gupta, 1955) ; b, domestic fowl (Sharma et al., 1956). (See key on p. 399.) material. Later the pro-acrosome arises in this vacuole in the form of a deeply staining granule. This together with the vacuole is differentiated into the acrosome, and the Golgi material itself is ultimately sloughed off. Sharma et al. (1956) have also worked out the spermatogenesis of the domestic fowl, but they have employed Flemming-without-acetic and .5 % hematoxylin exclusively, avoiding both the silver and long osmication techniques. They have examined along with the present writer the living sperm-forming cells of this material under the phase contrast microscope. In this material as in the duck, a prominent vacuole, corresponding to the archoplasmic vacuole of Zlotnik, can be seen sticking to the nucleus of the spermatid (Fig. 5b), and in the fixed preparations a deeply staining pro-acrosomal granule can be detected in its interior. It appears that in the fowl, contrary to the situation in the duck, all the Golgi elements do not participate in the formation of the archoplasmic vacuole, as a few of them can always be seen being sloughed off (Figs. 6a-6d). Amphibia. In the amphibia Sharma and Sekhri (1955) and Sharma and Dhindsa (1955) have clearly shown that in the frog (Rana tigrina)

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and the toad (Bufo stomaticus) respectively the acrosonie is directly formed by the fusion of the Golgi elements. Sharma and Dhindsa’s conclusions in the case of the toad are also supported by the study of the living material under the phase contrast.

N

a

b

FIG.6. 0-d, Spermatids of domestic fowl (Sharrna

et d.,1956). (See key on p.

399.)

In the earliest spermatid of the frog the Golgi elements are in the form of granules. Soon most of the Golgi granules, but by no means all, collect together to form a big deeply staining granule, the pro-acrosome. This is situated at the anterior end of the spermatid nucleus and is gradually differentiated into the acrosome. First the pro-acrosome, which hitherto stained uniformly, shows a duplex structure with a chromophilic cortex and a chromophobic medulla. Soon the chromophilic cortex disappears and the pro-acrosome assumes the form of a vacuole with gluelike contents, reminding one of a similar structure in the duck and the fowl (Gupta, 1955; Sharma et al., 1956), the snake, Nutrix, and the turtle (Sud, 1955, 1956). The chromophobic vacuole forms directly the acrosome of the ripe sperm (Figs. 7 ; 8a-8e).

FIG.7. Microphotograph of the living ripe sperm of frog under positive phase contrast. (See key on p. 399.)

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The formation of the acrosome in the toad proceeds on more or less the same lines as in the frog, except that the acrosome is a pointed needlelike structure. The authors have also seen the chromophobic vacuolar proacrosome in the living material studied under the phase contrast (Figs. 8f-8j). Reptiles. I n Reptiles the checkered snake, Natrix p . piscutor, and the turtle, Lissemys pzcncbatu, have unexpectedly turned out to be excellent

h'

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FIG.8. a-e, Spermatids of frog (Sharma and Sekhri, 1955) ; f-i, spermatids ;j , ripe sperm of toad (Sharma and Dhindsa, 1955) ; R-q, spermatids ; r, ripe sperm of Natrix (Sud, 1955) ; s-u, spermatids of turtle (Sud, 1956). (See key on p. 399:)

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materials for the demonstration of the acrosome arising directly from the Golgi substance (Sud, 1955, 1956). In the spermatogonia of the turtle the Golgi elements exist as prominent chromophilic granules, much bigger than the mitochondria. But in the primary spermatocytes (of the turtle as well as the snake) very distinct chromophobic vacuoles with gluelike contents appear, one in close association with a Golgi granule. Some, but by no means all, of the Golgi granules develop these vacuoles. In the spermatids there are no Golgi granules, but the chromophobic vacuoles unite to form one large vacuole, the contents of which seem to become denser. This is the pro-acrosome, which, placing itself at the anterior end of the spermatid nucleus, depresses the nuclear membrane, and is later on differentiated into the acrosome (Figs. 8k-8u). The pro-acrosome in the snake Natrix and in the krait, sticking to the anterior face of the nuclear membrane of the spermatid, has been seen by Sud and the present author in the living material under the phase contrast and photographed (Figs. 9a-9e).

FIG.9. Microphotographs of living testicular material of Natrix under positive phase contrast; a-d, spermatids; e, ripe sperm (Sud, 1955). (See key on p. 399.) Mr. Sud showed to the writer living spermatids of Uromastix hardwikii under the phase contrast, each spermatid showing a prominent and firm vacuole sticking to its nucleus. Fishes. In fishes also, both cartilaginous and bony, Vasisht (1953, 1954a, b) , in his remarkable publications, has shown very clearly that the acrosome in nine species of Hypotremata (elasmobranchs) , in three species

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of Pleurotremata (elasmobranchs) , and in three species again of teleosts (Actinopterygii, Cyprinodontidae) the acrosome is formed directly by the fusion of the Golgi elements. In the Hypotremata (Vasisht, 1953), either a single Golgi body advances toward the nucelus to form the acrosome as in AetomyZeus nichofii, Dasyatis zugei, Dasyatis kuhlii, and Torpedo marmorata, or a few Golgi bodies come close together and ultimately fuse to form the forerunner of the acrosome (pro-acrosome) as in Aetomyleus maculatus, Rhynobatw obtusus, Rhynobatus granulatus, and Dasyatis sephen (Figs. 1Oa-1Od). Vasisht stresses the point that all the Golgi bodies present in the cell in all these species do not take part in the formation of the pro-acrosome. A few, which are left unused, remain as the Golgi remnant and are sloughed off along with the residual cytoplasm. Finally Vasisht states that in all these species there is a short acrosome. In his second publication, Vasisht (1954a) has worked out the spermatogenesis of three species of elasmobranchs (P1eurotremata)-Chiloscy Zlium griseum, Carcharinus limbatus, and Sphyrna blochii. In Chiloscyllium griseum a few Golgi bodies come together to form the pro-acrosome. This, sooner or later, settles down on the nucleus and is transformed into an acrosome. The acrosome is at first pearshaped, but ultimately it becomes wavy. In Carcharinus limbatus most of the Golgi bodies come close together, and ultimately fuse to form the pro-acrosome. This forms the acrosome, which is generally pear shaped, but sometimes it may assume the form of a filament. The process of acrosome formation in Sphyrna blochii is more or less similar, except that the acrosome is conical (Figs. 1Oe-log). Vasisht (1953) has given an excellent review of the previous work on Elasmobranch sperm. All the previous workers mentioned by him, with the single exception of Ratnavathy (1941), employed exclusiveIy fixatives containing fat solvents and carried out their investigations at a time when the Golgi bodies and the mitochondria had not even been discovered. Nevertheless, these workers gave a very faithful account of what they saw in their preparations. Hermann (1882), the earliest worker on the subject, describes a body, “nodule cephalique,” which is later on applied to the surface of the nucleus of the spermatid. The nodule cephalique forms, according to Hermann, the “cephalic point,” which Vasisht correctly interprets as the acrosome in modern terminology. Similarly Vasisht has correctly interpreted the greasy granules of Hermann, which unite to form the nodule cephalique, as the Golgi bodies, although these bodies are very much distorted in Hermann’s preparations. Jensen ( 1883), who studied some fresh elasmobranch material also, describes two refringent bodies in fresh spermatids slightly osmicated.

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These bodies always take a very deep coloring in osmic acid. They fuse together to form one voluminous body, which ultimately takes a position in a depression at the anterior aspect of the nucleus. Here the voluminous body becomes cylindrical. Again Vasisht has correctly interpreted Jensen’s refringent bodies and the voluminous body as the Golgi bodies and the acrosome respectively.

M P -

R F-

d FIG.10. a-c, Spermatids; d, ripe sperm of Hypotremata (Vasisht, 1953) ; e-f, spermatids; g, ripe sperm of Pleurotremata (Vasisht, 1954a) ; h, ripe sperm of Gambusia (Vasisht, 1954b). (See key on p. 399.) Swaen and Masquelin (1883), while working on some elasmobranch sperms, describe a problematic body, which gradually comes near the anterior aspect of the nucleus and ultimately comes in contact with it. Here again Vasisht concludes that the problematic body of these authors corresponds to his pro-acrosome. Moore ( 1895) describes an “archoplasmic vesicle,” which becomes first flattened and then elongates out, together with the nuclear chromatin, forming a definite “cephalic point to the spermatozoon head.” The archoplasmic

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vesicle of Moore and his cephalic point have again been correctly interpreted by Vasisht as his pro-acrosome and acrosome respectively. In his second paper on the spermatogenesis of three species of sharks, Vasisht (1954a) shows very clearly that the acrosome in sharks also is directly formed by the fusion of Golgi bodies as in rays and skates (Vasisht, 1953). He does not agree with Ratnavathy (1941) that the acrosome in the shark Chikoscyllium griseum is secreted by the Golgi bodies. Vasisht did not see in all the twelve species of elasmobranchs studied by him either the acroblast or the acrosomal vesicle of Ratnavathy. I t may be noted that Ratnavathy used the silver and long osmication techniques, which admittedly introduce serious artifacts. In his third paper, Vasisht (1954b), working on three species of teleost fish, likewise concludes that the acrosome is formed by the direct fusion of the Golgi elements, as in the rays, skates, and sharks (Fig. 10h). In all the three teleost species studied by Vasisht he has observed a small acrosome in the living sperm even under an ordinary microscope, and in the case of Gambusia wrayi this has been further confirmed by the study of fresh testicular material under the phase contrast microscope. Under this microscope the acrosome has been seen by the present author and also by Vasisht as a very conspicuous conical structure in front of the nucleus

FIG.11. Microphotograph of the heads of living sperm of

Gambusia.

of the Gambusia sperm (Fig. 11). Vasisht stresses the point that in fixed preparations the tiny acrosome is liable to collapse. H e further states that the acrosome stains very poorly ; hence it has completely escaped the notice of Vaupel (1929). For this reason and also on account of having used fat-solvents she could not give a clear picture of the Golgi bodies, which form the acrosome. Spiders. In the spider Plexippus also (Sharma, 1950), the acrosome is directly formed by the Golgi granules. In the spermatid the Golgi granules have a distinct tendency to come together to form bigger granules, One of these big Golgi granules directly forms the acrosome, the rest of the Golgi granules being sloughed off along with the entire mitochondria1 mass, Sharma and Gupta (1956) have confirmed this account in the case of the spider, Pardosa (Figs. 12a-12f). Ticks. In the tick sperm also Sharma (1944) has clearly shown that

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the acrosome in all the three species that he studied is formed by the direct fusion of all the Golgi elements (Figs. 13a, 13b ; 14a-14d). b. Indirect Method of Acrosome Formation. I did not believe in the secretory hypothesis of the acrosome by the Golgi bodies until about four years ago when I examined some excellent preparations of the coleopteran forms Coccinella septumpunctata and Aulocophora foveicollis, prepared by Bhardwaj and Gupta respectively (Nath et aE., 1951), and more recently N

FIG.12. a-e, Spermatids; f, ripe sperm of Plexippus (Sharma, 1950). ( S e e key on p. 399.)

FIG.13. Microphotographs of the living spermatids of cattle-tick under positive phase contrast. (See key on p. 399.)

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of the orthopteran form Acheta domesticus Linn. (Gryllus domesticus), prepared by Bhimber (Nath and Bhimber, 1953), and still more recently of the slug, Anadenys altivagus, prepared by Chopra (Nath and Chopra, 1955).

d h

i

FIG.14.

a-c, Spermatids; d, ripe sperm of ticks (Sharma, 1944) ; e-h, spermatids of Coccinella and i,j , spermatids of Aulocophora (Nath et al., 1951). (See key on p. 399. )

The mammalian sperm has also been included in this section, as the majority of the workers on this material have favored the view that the acrosome of the mammalian sperm is a secretory product of the Golgi bodies. This view is supported by the recent work of Sharma et al. (1953) on Caviu and that of Dhillon (1955) on the white rat which was carried

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out on living material examined under the phase contrast microscope in this laboratory, although these workers differ widely from the previous workers, with regard to (1) the details of the formation of acrosome, (2) the morphology of the Golgi bodies, and ( 3 ) some other structures in the mammalian sperm. Coleoptera. Bowen (1924a) in his remarkable paper on the “Formation of Sperm in Coleoptera and Aptera” admits that the testicular material of Chelymorpha is unsuitable both for the study of the condensation of the mitochondria1 nebenkern and for the development of the acrosome from the Golgi apparatus. But the coleopteran form Coccinella septumpunctata (Nath et al., 1951) , is an excellent example for the demonstration of the acrosome being secreted by the acroblast, presumably formed by the fusion of the Golgi bodies. Having secreted the acrosome, the acroblast is sloughed off along with the residual cytoplasm. In this species the acroblast is a large structure, elongated in form with one end broad and the other narrower. It stains homogeneously with hematoxylin and never reveals any internal structure. Suddenly a vacuole, the acrosomal vesicle, appears in the substance of the acroblast, appearing rather unexpectedly but invariably toward its broad end. For a long time the acrosomal vesicle remains closely attached to the acroblast. Later, the staining capacity of the acrosomal vesicle increases and a deeply staining granule soon appears within its interior. This is the acrosomal granule, which grows in size. At the same time the acrosomal vesicle seems to shrink, the granule thus filling up the whole space in the vesicle. The enlarged acrosomal granule migrates to the anterior aspect of the nucleus, where it forms the acrosome. The elongated acroblast shifts backwards into the tail, where it begins to degenerate and is ultimately sloughed off (Figs. 14e-14h). In Aulocophora foveicollis (Nath et al., 1951), the form of the acroblast is perfectly spherical. An acrosomal granule, deeply staining, puts in its appearance in the close neighborhood of the acroblast, but never in the interior of the acroblast’s substance. The acrosome is derived directly from the acrosomal granule, after it has shifted to the anterior aspect of the sperm nucleus, whereas the acroblast shifts backwards in the tail and disappears (Figs. 14i, 14j). Orthoptera. Bowen makes occasional references to his work on the Orthoptera in his papers on the Lepidoptera (1922a) and Hemiptera (1922b). Chang-Chun Wu (1946) worked out the spermatogenesis of an orthopteran form, Diestrammenu sp. with special reference to Golgi bodies and mitochondria. Earlier Payne (1916), published a paper on Gryllotalpa borealis and G. vulgaris.

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Nath and Bhimber (1953) and Nath et al. (1954) have studied the sperm-forming cells of Achetu domesticus Linn. ( Gryllus domesticus) in the living condition under the phase contrast microscope. In an early spermatid, as studied in the living condition under the phase contrast, the Golgi bodies appear in the form of a few very dark granules, lying close to the mitochondrial nebenkern, but in the sectioned and stained preparations most of the Golgi bodies appear in the form of crescents, although a few Golgi granules may also be present. A little later the Golgi bodies come together and most probably fuse to form the acroblast, although the actual fusion was not observed. A few of the Golgi bodies do not cooperate in the formation of the acroblast, and they are ultimately sloughed off. The acroblast appears first in the form of a vesicle and soon an acrosomal granule is differentiated within it. The acroblast with the acrosomal granule has been seen by the authors in the living cell at this stage. The acroblast now moves toward the posterior aspect of the nucleus and appears horseshoe shaped. The acrosomal granule is pushed out of the acroblast, and is deposited on the nuclear membrane at its posterior aspect. After this the acroblast starts moving backwards, and is finally sloughed off along with other Golgi bodies. Simultaneously, the acrosomal granule itself starts moving forward along the periphery of the nucleus, and finally deposits itself at its anterior aspect. It then grows directly into the acrosome through an interesting process of differentiation (Figs. 15a-15). In Chrotogonus trachypterus (Nath et al., 1954; Bawa, 1955) the acrosome is also secreted by the Golgi elements. The vacuolelike proacrosome can be very clearly seen sticking to the nuclear membrane in the living material (Fig. 16a). Hemiptera-Heteroptera. Bawa (1953) has worked out the spermatogenesis of two species of this group of insects, Laccotrephes muculatus and Sphaerodem rusticurn. In both these species there is a single acroblast formed by the fusion of the Golgi spheres. The acrosome is secreted by the acroblast, which is finally sloughed off. This process of acrosome formation is exactly like that of orthopteran species described above. In Hulys dentatus (unpublished) the whole process of acrosome formation in the same cell has been very clearly examined and microphotographed in the living condition under the phase contrast (Figs. 16b-16d). Pulmonate Gastropods. The common slug of Simla (Punjab, India), Anadenus altivagus, has unexpectedly turned out to be the very best material for the demonstration of (1) the origin of Golgi dictyosomes from the mitochondrial granules by alignment, and (2) the secretion of the acrosome by the Golgi dictyosomes, which are subsequently cast off.

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CYTOLOGY OF SPERMATOGENESIS

Nath and Chopra (1955) have worked out the spermatogenesis of Antadenus exclusively with F.W.A. and iron-hematoxylin, and they have also examined the living material under the phase contrast. According to these authors the early spermatids have two to four Golgi dictyosomes each. When there are three dictyosomes they are arranged in the form of a characteristic triangle, and when there are four these are arranged

A

a

TT

C

M

e

b

-h M.N

f

FIG.15. a-i, Spermatids of Achefu (Grylttls) ; g-i from living material under phase contrast (Nath and Bhimber, 1953). (See key on p. 399.)

again in the form of a characteristic rhomboid. To begin with, the dictyosomes are devoid of any chromophobic material, but in late spermatids a very clear chromophobic sphere appears in close association with each dictyosome. Subsequently the Golgi complex, triangular or rhomboidal in form, shifts in front of the nucleus of the elongating spermatid, and in that situation, more often than not, it is in the form of an open U, with the concavity of the U facing the nuclear membrane and holding in it the chromophobic spheres fused in one large vesicle. This last structure bears a very close resemblance to the acrosomal vesicle of insects, e.g., rlcheta and Coccinella.

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But, whereas in Acheta and Coccinella and in insects in general only one acrosomal granule is secreted in the acrosomal vesicle, in Anadenus a very large number of tiny deeply staining granules make their appearance. These are soon released by the Golgi complex, and they are seen deposited in’the form of a cap just in front of the nuclear membrane. Soon the granules fuse together and the cap fits tightly over the nucleus, reminding

FIG.16. Microphotographs of the living spermatids under positive phase contrast ; on p. 399.)

a, Chrotogonus; b-d, Halys. (See key

one of the conditions prevailing in the mammalian sperm. The acrosomal cap stains deeply and uniformly. But this is only a temporary phase; the cap is gradually condensed into the small conelike acrosome of the ripe sperm. The Golgi complex, having apparently performed its function of secreting the acrosomal granules, shifts back to the tail region of the elongated spermatid and is subsequently sloughed off. In its early periods of shifting backwards, each Golgi dictyosome has its chromophobic vesicle intact, and this last is Ioaded with acrosomal granules, which were not released by the complex. Sooner or later, the dictyosomes of the Golgi remnant begin to stain poorly, and along with this the chromophobic vesicles completely disappear from view (Figs. 17a-17k). The figures of Watts (1952), who also studied the living material of the slug Arion under the phase contrast microscope, bear a very close re-

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semblance to those of Nath and Chopra (1955). She has described the Golgi element as a dictyosome, and she also states that each dictyosome develops a chromophobic vesicle in close association with it in the course of spermateleosis. She has also produced some excellent photographs of the living material studied under the phase contrast, but she has missed

F M

N

N

a

FIG.17. a, b, Primary spermatocytes; c, late telophase I ; d, secondary spermatoe-K, spermatids of Anadems (Nath and Chopra, 1955).. (See key on p. 399.)

cyte;

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the details of the process of the deposition of the acrosome. Nevertheless she states clearly that the acrosome is secreted by the Golgi complex. Soon after the manuscript of this review had been sent to the editors on April 1, 1955, the author was delighted to read the excellent paper of Roque (1954) on the morphology of the cytoplasmic inclusions of the living spermatocytes and spermatids of Helix as revealed by the phase contrast and interference microscopy and supra-vital dyes. According to Roque, the following structures are found in the paranuclear region of the late primary spermatocytes of HeZix: (1) composite bodies which are spheres, each sphere being partially or completely surrounded by a sheath of dark contrast ; some of these spheres are clear, vacuolelike, but others look like solid bodies; (2) rods which are not associated with spheres; ( 3 ) granules; and (4) pale gray spheres of homogeneous structure. The composite bodies are the more numerous of these structures: and the granules and homogeneous bodies are not observed in every spermatocyte and their number varies from one cell to another. Finally, according to Roque, there are in many spermatocytes small granules in the cytoplasm adjacent to the paranuclear region. Nath and Gupta (1956) thoroughly examined under the phase contrast microscope the living testicular material of the Simla slug, Anadenus altivagus, on the spot in August, 1955, and obtained some very instructive photomicrographs. They entirely agree with Roque ; but, according to their studies, most of the composite bodies have only a partial sheath of dark contrast round them, as most of them appear in the form of crescents with attached spheres (Fig. Ma). Rarely the composite bodies come up in the form of rings in their photographs, which means that the sheath of dark contrast is complete in only a few cases (Fig. 1%). Rods of dark contrast not associated with spheres are very common (Fig. 1&), as are the gray spheres of homogeneous structure of pale contrast (Fig. 18d). As the result of his studies with interference microscopy, Roque comes to the conclusion that the sheaths of the composite bodies and the rods have a higher refractive index than the spheres and the granules. Nath and Gupta (1956) in Anadenus agree with Roque that the sheaths of the composite bodies and the rods have a very dark contrast as compared with the pale contrast of the spheres; but they are convinced that the granules in the paranuclear region have also a contrast as dark as the sheaths of the composite bodies and the rods (Fig. 18e). Nath and Gupta (1956) have shown that the granules of dark contrast, in the paranuclear region (as well as the “small granules” of Roque adjacent to the paranuclear region), are some of the mitochondria1 granules, which are being transformed into the Golgi bodies.

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FIG.18. Microphotographs of the living primary spermatocytes of Anadems (Nath and Gupta, 1956) ; a-e under positive phase contrast; f, stained with neutral red chloride and examined under bright field illumination.

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Nath and Gupta (1956) have also stained with neutral red the “pale grey spheres of homogeneous structure” of Roque or the “chromophobic spheres” of Nath and Chopra (Fig. l8f). Roque (1954) did not find any evidence “of a direct participation of the paranuclear bodies in the formation of the acrosome” in Helix ; but Nath and Gupta have fully confirmed with phase contrast microscopy the account of acrosome formation in Anadenus given by Nath and Chopra (Figs. 19a-19d).

FIG.19. Microphotographs of the living spermatids of Anadems under positive phase contrast (Nath and Gupta, 1956). Srivastava (1953) states that in the pulmonate gastropod Vuginula maculata, “there is no connection between the Golgi body and the acrosome,” as he never saw the Golgi body moving in front of the spermatid nucleus. Srivastava describes a single Golgi body in the “spermatogonium, spermatocyte, or spermatid,” which, according to him, is easily observable even under an ordinary microscope. It is difficult to believe that the body, which Srivastava takes pains to prove to be the Golgi body, is in fact the

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42 1

Golgi body, as all the other previous workers on the spermatogenesis of the pulmonate gastropods have described the Golgi body as a dictyosome (Gatenby 1917b, 1918; Hickman, 1931; Baker, 1944, 1949; Watts, 19523. T o the best of my knowledge, Srivastava is the only worker who has denied any connection between the acrosome and the Golgi complex. Experience shows that the process of the deposition of the acrosome by the Golgi complex is very short-lived; and it is possible that Srivastava has missed the crucial stage, if the body described by him as the Golgi body is in fact the Golgi body. Nath and Chopra (1955) have suggested that the “Golgi body” of Vuginula may well be a chromatoid body as described by Gupta (1955) in the domestic duck and by Sud (1955) in the snake Natrix p . piscator. Mammals. Gresson (1951) has given an excellent * review of the structure and formation of the mammalian spermatozoon. It is not intended to go over the same ground in this review in the stereotyped manner, but rather to interpret the observations of previous workers in the light of what I have seen under the phase contrast microscope in the living sperm-forming cells of the guinea pig, the white rat, the mouse, and the squirrel in this laboratory. Sharma et al. (1953) published a paper on the spermatogenesis of Cuviu, employing Flemming-without-acetic and iron-hematoxylin. Sharma has recently examined (along with the author) the living cells of Cuvia under the phase contrast microscope and has photographed them. Similarly Dhillon (1955) has examined (along with the author) the living spermforming cells of the white rat, the mouse, and the squirrel, and has also photographed them. Figure 20a has been drawn after figure 1 of Gresson. It will be profitable to quote him verbatim with regard to the general make-up of the mammalian sperm. “In broad outline the mammalian spermatozoon (Fig. 1) is composed of the following regions : head, neck, middle-piece, and flagellum. The head is made up of the nucleus and the acrosome. The latter is a cap-like structure covering the anterior region of the nucleus; it varies in size and in shape in different animals. In certain mammals, at least, the posterior region of the nucleus fits into a cup-shaped body, the post-nuclear cap. It is probable that the whole head is enclosed by a thin protoplasmic sheath or membrane which is continued over the neck, middlepiece and part of the flagellum. According to Green (1940) this membrane contains an ‘albuminoid.’ The neck contains the proximal centriole and other structures to be described later. The axial filament begins in the neck and extends to the end of the flagellum; in the middle-piece it is surrounded by the mitochondria1 sheath. At the extreme distal end of the

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middle-piece the ring centriole surrounds the axial filament. The flagellum consists of two parts. The anterior part is called the main-piece and is composed of the axial filament surrounded by the protoplasmic sheath. The terminal part of the flagellum, or end-piece, is shorter than the mainpiece and consists of the axial filament which in this region is probably enclosed by the protoplasmic sheath.” The process of spermateleosis in mammals is admittedly complicated, especially the formation of the acrosome by the Golgi bodies. There are

n

Ne

d

G

a

C

e

FIG.20. a, Typical mammalian sperm (Gresson, 1951) ; b-e, spermatids of Caz& (Sharma et aL, 1953). (See key on p. 399.) numerous papers on the subject with widely differing interpretations, particularly with regard to the morphology of the Golgi bodies ; but the writer believes that the processes of spermateleosis in different species of mammals follow a uniform pattern with minor variations, and the differences are more due to differences in technique than to any other factor. The formation of the acrosome by the Golgi bodies is closely bound up with the morphology of these bodies. It is proposed, therefore, to consider first the morphology of the Golgi bodies and acrosome formation. Golgi Bodies and Acrosome Formation. Well before the discovery of the Golgi bodies Meves (1899) describes and figures small granules (kornchen) embedded in a rounded “idiozom” (idiosome of modern authors) in the spermatocytes, both primary and secondary, and in the spermatids. Later these granules fuse together to form the acrosome

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(spitzenkopf) . In the early stages of its differentiation the acrosome shows two regions, the inner intensely black and the outer pale yellow in color. In the course of acrosome formation the idiosome moves away and enters the tail region of the sperm, where it disappears. It is interesting to note that Meves employed sublimate and osmium mixture as a fixative (which has no fat solvent in it) amongst others. As quoted by Sharma et al. (1953), Papanicolaou and Stockard (1918) have also described granules (idiogranulomes) embedded in the idiosomic region in Cavia, the cortex of the idiosome being known as idioectosome. Each idiogranulome is surrounded in the spermatid by a clear vacuole (idiogranulotheca). By rapid fusion the idiogranulomes form a single large idiosphaerosome enclosed in a large vacuole, the idiosphaerotheca, which in turn is formed by the fusion of the idiogranulothecae. The outer shell of the idiosome, the idioectosome, which now begins to move away is renamed as the idiophthartosome. In the meantime the idiosphaerosome secretes a crescentic idiocalyptosome, and is itself known from now onwards as the idiocryptosome. In the ripe sperm the idiophthartosome disappears along with the cytoplasm. The idiocryptosome and the idiocalyptosome together form a double cap, spermiocalyptra, for the sperm head. The idiosphaerotheca persists through all the later stages and develops into a membranous cover for the cap and the head of the sperm and it is then known as the “spermiocalyptrotheca”. Bell (1929) worked out the spermatogenesis of the dog by employing a special technique of his own and avoiding all the silver nitrate techniques. His special technique consisted of fixation in a special mixture of formalin, mercuric chloride, and picric acid in certain proportions. After the initial fixation the testicular material was osmicated in 2 per cent osmic acid from three to nine days. With this special technique Bell showed t h t the Golgi bodies embedded in the idiosome and the general cytoplasm were granular in form. He never observed Golgi crescerzts, rods or networks. Sharma et al. (1953) also describe the Golgi elements embedded in the idiosorne and the general cytoplasm as granules. According to these authors, the process of spermateleosis is heralded by a distinct tendency of the Golgi elements to come together. ‘‘ This results in the formation of fewer and bigger Golgi granules not only in the idiosome but also in the cytoplasm. The fusion of the Golgi granules in the idiosome, however, continues till only a single big Golgi granule is produced. This is situated almost in the middle of the idiosome. Soon after its formation it becomes surrounded by a clear vacuole which gradually increases in size as the idiosome moves towards one side. The single Golgi granule now moves through the vacuole and comes in intimate contact with the nuclear mem-

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brane.” This Golgi granule, according to the authors, grows in size and develops into the acrosome. The spermatid nucleus “grows under the vacuole in the form of a conical projection with the acrosome situated at its tip.” The acrosome now gets differentiated into two regions, an inner darkly-staining and an outer lightly-staining (Figs. 20b-2Oe ; 21a). The living cells of Cavia studied under the phase contrast show Gale granules embedded in the idiosome, their fusion to form the acrosome, and the differentiation of the acrosome into two regions. But Dhillon (1955) has excellent photographs of the living material of the white rat studied under the phase contrast. Although the process of spermateleosis is more difficult to elucidate in the white rat on account of the smaller size of its cells as compared with the guinea pig, it has been followed clearly by Dhillon, both in fixed and in living material. In the early spermatid the Golgi granules are embedded in the idiosome in much the same way as in Cavia, except that in the white rat the idiosome is smaller (Fig. 21b). From then on the details of the formation of the acrosome by the Golgi bodies differ in the two species. Whereas in Cawia the pro-acrosome is formed by the union of a number of Golgi granules, in the white rat only one Golgi granule forms this structure. The rest of the idiosomic Golgi granules in the white rat shift to the periphery of the idiosome and there form a thick highly chromophilic shell round the idiosome, in the center of which the pro-acrosomic granule is embedded. At this stage the idiosome is a very compact structure with a thick deeply staining cortex, and a chromophobic medulla containing the pro-acrosomal granule. After the idiosome has become horseshoe shaped (Fig. 21c), the pro-acrosomal granule comes out of the idiosome, and, as it does so, it drags with it a part of the chromophobic material. Later the discarded idiosomic shell shifts behind the nucleus into the tail region and is ultimately sloughed off. But the pro-acrosomal granule surrounded by the chromophobic vesicle continues to develop into the acrosome. The chromophobic vesicle is soon converted into a transparent vacuole, with the proacrosomal granule suspended in its interior. Gradually the vacuole grows in size and expands over the anterior aspect of the spermatid nucleus. As it does so, the nucleus puts out a beak anteriorly underneath the vacuole, with the acrosomal granule seated at its tip. The pro-acrosomal granule never grows in size and is gradually absorbed in the surrounding vacuole. At this time the contents of the vacuole seem to have condensed, and the vacuole has spread itself out in the form of a cap fitting the elongating nucleus (Figs. 21e-21g). In the mouse (Fig. 21d) and the squirrel also the Golgi bodies are in the form of granules both in the idiosome and the general cytoplasm.

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Gatenby and Woodger (1921), working on guinea pig with the Golgi apparatus techniques of Cajal and Mann-Kopsch, described the Golgi apparatus in the spermatid as “consisting of an inner core of archoplasm, and a cortical region formed of curved plates and rods-the dictyosomes.” With formalin-silver nitrate techniques, however, “the Golgi apparatus

FIG.21. Microphotographs of the living testicular material of rodents under positive phase contrast. a, ripe sperm of Caziu; b, c, spermatids of white rat (Dhillon, 1955) ; d, e, spermatids of mouse (Dhillon, unpublished) ; j, g. sperm of white rat (Dhillon, 1955). (See key on p. 399.)

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either appears as a reticulum, or the whole cortex of the apparatus reduces the silver, and then appears homogeneous : with Mann-Kopsch techniques, the individual dictyosomes are often very clearly marked.” In conformity with all authors on mammalian spermatogenesis, Gatenby and Woodger derive the acrosome from pro-acrosomic granules, which appear in the archoplasm during the later stages of the spermatocyte. But they regard these granules as secretions of the Golgi bodies and not as Golgi bodies themselves. Walker (quoted by Rau and Brambell, 1925) takes exception to the description of the formation of the acrosome given by Gatenby and Woodger, and he considers the technique used by them as “drastic.” Likewise Nath (1944) and Baker (1953) point out that the silver nitrate and long osmication techniques introduce serious artifacts. Rau and Brambell (1925), working on the sperm-forming cells of Cavia and mouse, do not describe a reticulum, but figure the Golgi apparatus as consisting of banana-shaped pieces. Gresson (1951), while reviewing his previous work, also states that the localized Golgi material of spermatocytes and spermatids of mammals is not a network but is composed of rods, filaments, and perhaps also of granules, which closely surround the archoplasm (Gresson, 1942 ; Gresson and Zlotnik, 1945, 1948). As previously stated, the writer has examined the idiosome in the living sperm-forming cells of the guinea pig, the white rat, the mouse and the squirrel, but he has never seen the Golgi elements in a form other than granular. With regard to the formation of the acrosome in mammals, the writer believes that this structure is formed directly from the pro-acrosome and the vacuole surrounding it, the pro-acrosome having been formed itself by the fusion of some of the Golgi elements embedded in the idiosome. The Golgi granules, which do not take part in the formation of the acrosorne, are sloughed off along with the whole of the idiosomic mass. Strictly speaking, this method of acrosome formation is mid-way between the direct and indirect methods of formation, inasmuch as the whole of the Golgi substance in the idiosome is not used up in the formation of the acrosome. The Protoplasmic Bead. Meves (1899) figures a prominent protoplasmic bead without any granules in it in the region of the middle-piece of the sperm of CUT&. Gradually this bead shifts downwards along the axial filament and is ultimately lost. As quoted by Gatenby and Woodger (1921), Retzius has also figured a small bead of protoplasm on some part of the middle-piece in many mammalian spermatozoa. Gatenby and Woodger (1921) were first to give a detailed account of

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this bead in the spermatids of Cavia. According to these authors the Golgi apparatus of the elongating spermatid buds off a small part of itself. This part ultimately comes to lie in the protoplasmic bead of the middle-piece. “With formalin and silver nitrate techniques the protoplasmic bead is found to contain a number of argentophil platelets or rods, which impregnate exactly like the Golgi apparatus of younger sperm cells.” Later Gatenby and Wigoder (1929) figured the Golgi apparatus of the protoplasmic bead of the ripe sperm of Cavia as a reticulum. Rau and Brambell (1925) in Cavk, Gresson (1942) in mouse, and Gresson and Zlotnik (1945) in pig, sheep, dog, cat, rat, and golden hamster have all described a protoplasmic bead on the middle-piece. Gresson (1951) states that “a number of granules and irregularly shaped bodies” (italics by author) are seen in the protoplasmic bead. According to Sharma et al. (1953), it is very difficult to show with certainty that the Golgi elements of the protoplasmic bead are derived from the main mass, and they believe that the Golgi granules of the bead may well be some of the Golgi granules which do not participate in the formation of the pro-acrosome, and are subsequently sloughed off. This observation of Sharma is strongly supported by the statement of Gresson (1951) that “separation of the Golgi material into two parts does not occur in the spermatids of the rabbit (Gresson and Zlotnik, 1945) nor in those of the bull (Gresson and Zlotnik, 1948). . . .” The most important point, however, may be noted that Sharma et al. (1953) like Bell (1929) , have described and figured Golgi granules in the protoplasmic bead. As stated by Gresson (1951) “there is considerable difference of opinion as to the fate of the protoplasmic bead,” but he adds that “according to Gresson and Zlotnik, after the sperms have entered the epididymis the protoplasmic bead moves down the middle-piece and is ultimately lost. . .” Sharma et al. (1953), and Mukherjee and Bhattacharya (1949) also agree that the protoplasmic bead is ultimately sloughed off. There is hardly any doubt that the protoplasmic bead found on the middle-piece of the mammalian sperm is simply the residual protoplasm containing Golgi granules (Figs. 21a, 21e, 2lf). The Post-nuclear Cap. Gatenby and Wigoder (1929) described postnuclear granules in the sperm-forming cells of the guinea pig. These granules were first observed by the authors in the spermatocytes in the neighborhood of the Golgi apparatus. In the spermatid these granules take up a position behind the nucleus: hence they are described as postnuclear granules. They are “elongate, hollow structures,” which expand till they “unite to form the solid covering behind .the nucleus.J’ This gives

.

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rise to the argentophilic post-nuclear cap on the posterior part of the nucleus. It is significant that the authors stress the point that the postnuclear cap cannot be demonstrated except by the Da Fano method. Gatenby and Beams (1935) did not observe any post-nuclear granules in the human sperm-forming cell ; nevertheless they describe a post-nuclear cap in the human sperm. But they admit that “both the origin and the subsequent history of these sharply impregnated structures are somewhat difficult to make out. . . .” They are not certain whether the post-nuclear cap is “ever a separate rudiment in the cytoplasm of the spermatid, or merely a thickening on the nuclear membrane.” Zlotnik (1943) describes an argentophilic post-nuclear cap in the spermatid of the dog and the cat, and he derives it from the argentophilic granules deposited along the nuclear ring (anterior). Gresson and Zlotnik (1945) describe in addition a (posterior) nuclear ring at the distal end of the spermatid nucleus of the dog, cat, and rabbit, and they derive the post-nuclear cap mostly from the argentophilic material of the (anterior) nuclear ring, the (posterior) nuclear ring forming only the extreme distal end of the cap. Randall and Friendlaender (1950), as quoted by Gresson (1951), also identified a nuclear ring and a post-nuclear cap in electron micrographs of the spermatozoon of the ram. Nath (1944), while stressing that no reliance could be placed on silver and long osmication techniques, quoted the remarkable work of Friend (1936), who worked on the sperm nuclei of a large number of species of British Muridae. Friend discovered that there was an asymmetrical deeply staining area in the posterior part of the nucleus in all the sperms he had seen, which gave the characteristic stain with Feulgen. He named this area the “dense posterior region,” and he pointed out that in position it agreed with Gatenby’s “post-nuclear body.” From this Nath (1944) concluded that silver and osmium granules could settle down on structures totally unrelated to the Golgi apparatus. Most recently Sharma et al. (1953) also concluded “that the post-nuclear body is nothing but a part of the nucleus, probably containing most of the dense chromatin material,” as it gives the “characteristic Feulgen reaction” (Fig. 21a). Chromatoid Body or the Accessory Body. Gatenby and Beams (1935) described an accessory body in the human spermatocyte. They conclude that the accessory bodies are those Golgi elements which do not collect in the idiosome. Gresson (1942) describes an accessory body in the neck region of very late spermatids and sperms of the mouse. He aIso concludes that this

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granule is derived from the Golgi material, which had moved away from the pro-acrosome. Gresson and Zlotnik (1945) describe a number of accessory bodies in the male germ cells of some mammals, which originate from the localized Golgi material of the spermatocytes and spermatids. Finally an accessory body is embedded in the neck region. Commenting on these observations, Sharma et al. (1953) conclude “that some of those Golgi elements also which do not come together to be in the localized condition] and remain dispersed throughout the cytoplasm of the spermatocytes and the spermatids, may have been labelled as chromatoid bodies.”

5. Perforatoriurrs and Periodic Acid-Schif Technique Clermont et d. (1955), working on the rat sperm with ordinary, phase contrast, and electron microscopy, have described in great detail a structure in front of the sperm nucleus, which they have named the “perforatorium.” This structure is absolutely independent of the acrosomic system, consisting of an acrosome and a head cap. The perforatorium itself is an elongated, three-sided pyramid, and is a modification of the nuclear membrane in this region. The authors treated the sperms with IN-NaOH and discovered that the pyramid gives rise posteriorly to three prongs, of which one is dorsal and the remaining two ventro-lateral. In this laboratory Dhillon (unpublished) has produced excellent photomicrographs of the rat sperm (Figs. 22a-22d) and has confirmed the account of Clermont et al. Clermont and Leblond (1955), in man, monkey, ram, and other mammals have demonstrated at the Golgi phase “PA-Schiff reactive particles” in the idiosome, which “fuse into the acrosomic granule-a structure composed of an inner and an outer zone.” This account of acrosome formation in mammals supports in general the account given by Sharma et al. (1953) in Cavia.

6. The Fate of Mitochondria in the Flagellate Sperm The mitochondria of the spermatid are always relegated to some region of the tail of the maturing sperm, unless they are cast off as in Peripatus (Montgomery, 1912), the spiders Plexippus and Pardosa (Sharma, 1950; Sharma and Gupta, 1956), and the snake Natrix (Sud, 1955). As has already been indicated the mitochondria are not cast off in the vast majority of animals. In this connection three types of flagellate sperms may be recognized: (1) sperms in which the mitochondria are closely grouped together in the neck region, whether the centriole remains single or divides into two; (2) sperms with a mitochondria1 middle-piece of varying length,

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whose anterior and posterior limits are determined by the anterior and posterior centrioles respectively ; and (3) sperms in which the centriole remains single in the neck region (or even if the centriole divides, its products remain in the neck region), and the mitochondria form a sheath of the axial filament in by far the greater length of the tail, except a small end-piece which appears to be naked (as in insects and scorpions).

FIG.22. Microphotographs of the fresh rat sperm treated with IN-NaOH and examined under positive phase contrast (Dhillon, unpublished). a. Flagellate Sperms with Mitochondria in the Neck Region. The sperms of the frog Rana tigrina (Sharma and Sekhri, 1955) and the toad Bufo stomaticus (Sharma and Dhindsa, 1955) conform to this type. In both these species the centriole divides into two, but the proximal and the distal centrioles lie immediately one behind the other in the neck region. The mitochondria remain inactive, and do not fuse to form a nebenkern. They remain very closely grouped together in both the species, so that a very inconspicuous middle-piece is formed which, in the living sperm under the phase contrast, can be seen as a small nodule behind the nucleus (Figs.

h,@I.

Wilson (1925) has given an excellent review of such primitive flagellate sperms in which the mitochondria remain grouped together in the neck region and do not descend down the axial filament, as described by Retzius, Ballowitz, and Duesberg. Such a primitive sperm is conspicuously found in some Hydromedusae, Scyphomedusae, and Anthozoa and also in several

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43 1

of the higher animal groups. Reference may be made to Wilson for details ; but Wilson makes a very reasonable suggestion that “this simple type of sperm resembles an embryonic type of sperm which in many higher animals only appears as a transitory stage in the formation of a more highly elaborated type.” b. Flagellate Sperms with a Distinct Mitochondria1 Middle-Piece. From this primitive type of sperm can be derived the sperms of mammals, birds, tortoises, and turtles amongst the reptiles, fishes, and some molluscs, in which there is a distinct mitochondrial middle-piece of varying length. The anterior and posterior limits of this middle-piece are marked off by the proximal and distal centrioles respectively. Behind the middle-piece there is the main-piece in which region the axial filament is ensheathed by a thin cytoplasmic envelope, and behind the main-piece there is a short end-piece in which the axial filament is naked (Fig. 4u). Sometimes, as in the slug Anadenus (Nath and Chopra, 1955), there is no main-piece, the very long mitochondrial middte-piece being followed directly by a short end-piece (Fig. 17k). For details of this type of sperm also reference may be made to the review of Wilson (1925). Reference may also be made to Sharma et al. (1953) for mammals, Gupta (1955) for birds, Sud (1956) for turtles, and Vasisht (1953, 1954a, b) for fishes, for full bibliographies (Figs. 23a, 23b).

FIG.23. Microphotographs of the living sperm. a, sperm of fowl with dark field illumination (Sharma et al., 1956) ; b, sperm of turtle under positive phase contrast (Sud, 1956). (See key on p. 399.)

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c. Flagellate Sperms of Insects and Scorpions. In insect sperm the centriole remains single and is usually lodged in the neck region, except in the tiger beetle Cicindela (Goldsmith, 1919), and the silver-fish insect Lepisma (Bowen, 1924a; Nath and Bhatia, 1953), in which the centriole is situated at the extreme tip of the elongated nucleus (Fig. 4m). Early in insect spermateleosis the mitochondria usually become bubblelike and fuse together to form a rounded highly characteristic nebenkern, which is generally differentiated into a cortical chromophobic and a medullary chromophilic area. It was shown by Bowen (1922a) that the nebenkern has a plate-work structure and not a spireme structure as described by Gatenby (1917a). As the nebenkern is pulled down the axial filament its chromophilic medullary part shrinks progressively till it ultimately disappears. In Prodenia litura, however, Bawa ( 1954) states that the chromophilic part of the nebenkern is sloughed off. Bawa has confirmed this observation under the phase contrast in the living maturing spermatid. The plate-work structure also gradually disappears. Simultaneously with these changes a new substance, the “central substance” of Bowen ( 1922a) or the “sheath substance” of Nath (1925) appears in the chromophobic part : this substance ultimately forms the sheath of the axial filament. Very recently Beams et al. (1954) have studied the mitochondria1 nebenkern of the spermatids of the grasshopper under the phase contrast and electron microscopes, and have confirmed the “plate-work” theory of Bowen. These authors state that “the mature nebenkern is composed of concentric lamellae about 400 A in thickness.’’ Some of these lamellae appear to be composed of double membranes. “The lamellae are arranged like the layers in an onion and they alternate with layers of matrix.” Later in spermateleosis “the nebenkern loses its lamellar structure, divides, and its two halves elongate” down the axial filament. Nath et al. (1951) state that the “central substance,” which generally appears in the insect nebenkern, is completely absent in Aulocophora foveicollis and Coccinelh septumpunctata. Bawa (1955) also reports the complete absence of the central substance in Chrotogonus trachypterus. In the scorpion sperm also, as is generally the case in insects, the centriole does not divide and remains in the neck region ; and the mitochondria form the sheath of the axial filament for its greater part. In the scorpions Opbthacanthus, Hadrurus, and Vejovis (Wilson, 1916), each spermatid receives a few large spherical mitochondria, whose number varies from 5 to 7 in the first two species and 4 to 6 in Vejovis. These form a ring immediately behind the spermatid nucleus surrounding the axial filament and are then drawn out to form its sheath but in this case

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without twisting. In Palamnaeus bengalensis (Gatenby and Bhattacharya, 1925) the number of mitochondria in the spermatid varies from 4 to 11. They become grouped together at the base of the nucleus and later form the tail-sheath as in Opistkanthus, Hadrurus, and Vejovis. I n the scorpions Centrurus and Centruroides (Wilson 1916, 1931 ; WiIson and Pollister, 1937), there are two spheroidal mitochondria1 mitosomes in the spermatid. These become closely associated to form a bipartite nebenkern, which is spun out spirally round the axial filament as the spermatid elongates. In both these genera the mitochondria of the spermatogonia are small, numerous, separate bodies. These unite in the primary spermatocyte to form a single homogeneous, open ring. I n meiosis I the ring places itself parallel to the spindle, then elongates, and in late anaphase I is cut across at each polar extremity, thus producing two rods lying parallel to each other and to the long axis of the spindle. I n the final telophase I each rod is cut transversely at its middle point, each secondary spermatocyte thus receiving two short rods-two-fourths of the original ring. In meiosis I1 each of these two rods is again equally divided transversely, producing two still shorter rods. Consequently each spermatid receives two short rods-two-eighths of the original ring-which becoming spheroidal form the bipartite nebenkern of the spermatid. Nath and Gill (1950) have confirmed the above account in the scorpion Buthus acute-carinatus, which, like Centrurus and Centruroides, belongs to the family Buthidae.

III. NON-FLAGELLATE SPERM The non-flagellate sperms can be conveniently divided into three types : ( 1 ) Vesiculiform (decapod Crustacea ; millipedes), (2) Tubuliform (Ticks), and (3) Amoeboid (Nematodes). In all these types the axial filament, if present, is not vibratile in the usual way.

1. Vesiculiform Sperms a. Decapod Crustacea. Nath (1932) published for the first time an account of the bizarre sperm of a decapod crustacean (fresh-water crab, Paratelphusa spkigera) , employing fixatives free from fat solvents. The previous workers, without exception, had employed fixatives containing strong fat solvents, which completely destroyed or distorted the mitochondria and the Golgi bodies. Consequently they had described several fantastic structures in the decapod sperm, unheard of in the flagellate sperm. Nath showed that the crab sperm, in spite of its weird form, is in regard to its components exactly like a typical flagellate sperm. It possesses a cup-shaped nucleus with the margin of which the ringlike acro-

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some is fused. The mitochondria-like mass of Koltzoff (1906), Binford (1913), and Fasten (1918) consists of the fused Golgi elements destined to form the acrosome. The cavity of the cup is completely filled by the mitochondrial vesicle, corresponding to the “primary vesicle” of Fasten. The mouth of the cup is very efficiently plugged by the ringlike, distal centrosome, which is identical with Fasten’s %hromatin ring.” At the bottom of the mitochondrial vesicle lies the vesicular, proximal centrosome, answering to the description of the “secondary vesicle” of Fasten. Between the two centrosomes runs a thick axial filament, corresponding to Fasten’s “central body.” Nath also produced evidence to show that at the time of fertilization, when the spermatozoa explode, the unusual form of the sperm changes into that of a typical one. The so-called “chromatoid bodies” of Fasten were also shown by Nath to be the mutilated Golgiidiosome complex, which is directly transformed into the ringlike acrosome, fusing with the lips of the nuclear cup of the sperm (Figs. 24a-24c). In 1937 Nath likened the spermatozoon of the macruran form, Palaemon lamarrei, to a cricket ball having a small hole at the top. The outer leather covering of the ball represents the nucleus, and the internal stuffing the cytoplasmic vesicle, in the formation of which almost the whole of the cytoplasm of the spermatid along with its entire mitochondrial and Golgi material is sacrificed. The hole is very efficiently plugged with a ringlike rentrosome, which gives off inwardly a large number of axial filaments radiating through the vesicle toward the nucleus and outwardly a long, stout spine ending in a fine point. The axial filaments and the spine were both homologized by Nath with the axial filament of a typical flagellate sperm, inasmuch as both the structures are outgrowths from the centrosome. Nath further interpreted the so-called “chromatoid bodies,” described by Fasten (1914) in a crayfish, as the Golgi masses. The acrosome is never formed as the Golgi material is completely merged along with the entire mitochondrial material into the cytoplasmic vesicle. Finally Nath suggested that, as in Paratelphusa spinigera, the cytoplasmic vesicle forms the mechanism, which is responsible for the explosion of the sperm at the time of fertilization (Figs. 24d, e). In 1942 Nath published a comprehensive monograph on the spermatogenesis of three Macruran (Penaeus indicus, Panulirus polyphagus, Palaemon lamarrei) , four Anomuran (Clibanarius longitarsis, C. nathi, Coenobita rugosus, Pagurus punctulatus) , and 27 Brachyuran species. The sperm of Penaeus indicus and Panulirus polyphagus (Figs. 24f, g ) is built up on the same plan as that of Palaemon lamerrei, except that there is neither a spine nor axial filaments, which are so characteristic of Palaemon. But the sperms of all these three macruran species resemble

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CYTOLOGY OF SPERMATOGENESIS

each other in having a cuplike nucleus, a large ringlike centrosome, a cytoplasmic vesicle in the formation of which the entire mitochondria1 and Golgi material is sacrificed, and in the complete absence of an acrosome. In all the four Anomuran species studied by Nath the nucleus of the sperm is also cup shaped, but the nuclear cup is lined by an attenuated layer of residual cytoplasm, forming a nucleocytoplasmic cup. In Clibanarius the nucleocytoplasmic cup is deep, whereas it is shallow in Coeno-

LR

C

N

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c

e

g

h

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i

FIG.24. a, Ripe sperm; b, c, spermatids of Paratelpkusa (Nath, 1932) ; d, e, sperm of Palaemon (Nath, 1937) ; f, sperm of Penaeus; g, sperm of Panulirus; h-j, sperm of Clibanarius (Nath, 1942). (See key on p. 399.)

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bita and Pagurus. In both species of Clibanarius the hollow of the deep nucleocytoplasmic cup is filled by the mitochondria1 nebenkern or capsule, which is very large and chitinous in C. longitarsis. In both an axial tube with chitinous walls develops in the axis of the nebenkern, in which a very large tri-partite centrosome is safely ensconced. Proximally the axial tube is closed and distally it is plugged by a mass of capsular material. Between the cytoplasmic and nuclear parts of the nucleocytoplasmic cup of Clibanarius longitarsis there lies a prominent triangular, quadrangular, or pentangular figure, from the angles of which the rays arise. Both the rays and the figure were shown to be formed of fine filaments, which arise directly from the proximal part of the centrosome. Nath, therefore, homologized the rays with the axial filament of the flagellate sperm. In C. nathi the ray figure is triangular, so that there are invariably three rays in the sperm of this species. In both the species of Clibanurius the acrosome is absent, the single Golgi granule of the earliest spermatid quickly disappearing in spermateleosis. Nath further showed that the opening out of the sperm of Clibanarius at the time of fertilization is probably due to the lengthening out of the spinelike distal part of the tri-partite centrosome (Figs. 24h-24j). In contrast with Clibattarius, the nucleocytoplasmic cup of Coenobita and Pagurus is shallow. Into this shallow cup is inserted one end of an elongate mitochondrial nebenkern. In Pagurus, as in Clibanarius nathi, there is a triangular ray-figure from the angles of which the rays arise. But in Coenobita the rays (of which there are three) originate directly from the proximal part of the tri-partite centrosome. Nath (1942) argues that a more convincing evidence in favor of the view that the rays are homologous with the axial filament of the flagellate sperm could not be wished for. In both Pagurus and Coenobita a chitinous axial tube develops in the median axis of the elongate mitochondrial nebenkern. Again, as in Clibanarius, in both Coenobita and Pagurus the acrosome is conspicuous by its absence, the single Golgi granule of the earliest spermatid of Coenobita quickly disappearing in spermateleosis. In Pagurus the Golgi material degenerates even earlier during the meiotic stages (Figs. 25a-25f). Nath (1942) worked out the spermatogenesis of 19 brachyuran genera. These genera readily fall into two well-defined groups. In one group represented by Paratelphusa (also Nath, 1932), Potamon, Ocypoda, Macrophthalmu, and Matuta the sperms are bi-centrosomal and the acroblast forms an acrosomal ring which, after fusing completely with the lips of the nuclear cup, cannot be recognized as a separate structure (Figs. 24a-24c ; 25g). In the second group, which comprises 14 genera represented by Leptodius and its allies, the sperms are tri-centrosomal and the acrosomal

*

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CYTOLOGY OF SPERMATOGENESIS

a

C

8

n

m

1

C.G

k

P

4

0

R,

r s t FIG.25. a-c, Sperm of Pagurus; d-f, sperm of Coewobita; g , spermatid of Ocypoda; h, i, sperm of Eriphin ;j , sperm of Leptodius ; k, sperm of Scylla ; 1, sperm of Menippe ; m, sperm of Mefopograpszls; w, sperm of Carcitroplux (Nath, 1942) ; 0-q, sperm of Thyroglufw (Nath and Sharma, 1952) ; r, s, spermatocytes of Porrocaecum (Vadehra, unpublished) ; t, sperm of Ascaris (Collier, 1936). (See key on page 399.)

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ring is never formed, the acroblast degenerating completely. Of the two centrosomes in the first group, the distaf one plugs the mouth of the nuclear cup which contains the mitochondria1 nebenkern, and the proximal, by lengthening out, causes the sperm to explode. Between the two centrosomes runs an axial filament. Of the three centrosomes in the second group the distal has a plugging function, the proximal merely serves as a shelf on which the axial filament rests, and the middle centrosome, by lengthening out, causes the sperm to explode. Nath further showed that the processes of the Brachyuran sperm are pseudopodial in nature: they are extensions of the fine film of cytoplasm investing the nuclear cup and have no connection whatsoever with the centrosomal apparatus (Figs. 25h254. Using the number of centrosomes as a test, Nath (1942) evolved a scheme of evolution of the different groups of the order Decapoda. This coincides with the natural scheme based on morphological and other characters. Nath further showed that the force, which by exploding the mitochondrial vesicle drives the nucleus of the sperm into the egg, is supplied by the lengthening out of the centrosomal apparatus. It is, therefore, comparable with the rotary force, which is believed to be supplied by the axial filament of the normal flagellate sperm, driving the sperm head into the egg. b. Millipedes. The earliest work on the sperm of chilognaths is by Gilson (1886), who gave a very brief account of the ripe sperms of a few species. In Julus GiIson described a cup-shaped sperm reminding one of the Decapod sperm. H e also described a cup-shaped sperm in Polydesmus complanatus, although the cup in this form seems to be very shallow as compared with that of the Julus sperm. Amongst the older literature other papers on the subject are by Oettinger (1909), Sokoloff (1914), and Warren ( 1934). The latest paper on the subject is by Nath and Sharma (1952) on the sperm of the millipede, Thyroglutus malayus. In this paper the authors have reviewed the whole of the earlier literature on the subject, more particularly the conclusions of Warren, which they showed to be untenable. According to Nath and Sharma the ripe sperm of Thyroglutus malayus is a simple structure consisting of three cell-elements only-the nuclear cup, the centrosome, and the cytoplasmic vesicle. The acrosome, the middle-piece, and the axial filament of the typical flagellate sperm are conspicuous by their absence. During spermateleosis the, vesicular nucleus of the early spermatid is converted into a wide and shallow cup. Sirnultaneously vacuoles appear in the cytoplasm, and both the mitochondria and the Golgi elements merge into these. By a twofold process of growth and

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439

coalescence these vacuoles form a large cytoplasmic vesicle, which is pushed inside the nuclear cup just before the ripe sperm is formed. Nath and Sharma studied the detailed structure of the centrosomal apparatus and supposed that its large size was in conformity with the belief that it is in some way connected with the opening out of the sperm (Figs. 250-

w.

2. Tubuliform Sperm of Ticks Sharma (1944) worked out in detail the spermatogenesis of three species of ticks-the cattle tick (Hyalomma aegyptium), the dog tick (Rhipicephdus sanguineus), and fowl tick (Argus persicus) , with special reference to the Golgi elements, mitochondria, and centrosome, and the unique form of locomotion in this bizarre and aberrant sperm. For earlier literature on the subject reference may be made to Sharma. Sharma has recorded a rare phenomenon-that the final stages of spermateleosis in ticks are completed in the genital tract of the female (cf. Collier, 1936 in Ascaris). The ripe sperm in all the three species of ticks described by the author is in the form of a long, nonvibratile tube with a single, large, ringlike centrosome placed at one end of the tube, which must be considered as the anterior end since Sharma and the present writer have both seen the centrosome pulling the long tube behind it. What would be the head (consisting of the nucleus and the acrosome) in other sperms becomes enclosed in the posterior end of the tube. This very unusual position of the sperm head and the centrosome is brought about by a very complicated process of invagination and subsequent evagination of the sperm tube (Fig. 14d). 3. Amoeboid Sperm of Nematodes Nath and Raina (1929) examined the living sperm of Ascaris Iumbricoides stained with neutral red. The visibility of the mitochondria and the Golgi elements improves after treatment with this dye, although they do not stain with it. The mitochondria (first described by Meves, 1911) appear as large spheres and the Golgi elements as small granules on their margins. In fixed preparations Nath and Raina described the Gold elements as vesicles, each vesicle showing an osmiophilic rim and an osmiophobic central substance. Vadehra (unpublished) ,working on the nematode Porrocaecum angusticolle of the vulture, confirmed these findings and showed that the GoIgi vesicles are strongly argentophilic also (Figs. 25r, s) . But Collier (1936), working on the cytoplasmic components in the fertilization of Ascaris suilh, has shown very clearly that the fully matured sperms are found only in the uterus of the female. The fully ripe sperm

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has a large prominent acrosome, which occupies almost as much as onethird of the total volume of the sperm. The acrosome is formed by the Golgi bodies of the spermatid. After the amoeboid sperm has entered the egg at the time of fertilization, the acrosome breaks up into a number of refringent bodies, which gather at the periphery of ,the fertilized egg. Subsequently the refringent bodies disappear after becoming vacuolated. Collier gives reasons to believe that the thick cell wall of the egg is formed at the expense of the material derived from the fragments of the acrosome, the refringent bodies (Fig. 25t). Collier (1936) does not make any reference to the paper of Sturdivant (1934) on the spermatogenesis of Ascaris nzegalocephala. Nor does she give any account of spermatogenesis in A . suilla. After the manuscript of this review had been sent to the editors, the author’s attention was drawn to Sturdivant’s paper in the course of his investigations in collaboration with Sant Singh on the spermatogenesis of Ascaris suilla and the allied form, Porrocaecum angusticolle from the vulture. Sturdivant has described the secretion of refringent granules by the Golgi bodies in their attached chromophobic spheres in the primary spermatocytes. The author used 20 per cent acid fuchsin in anilin water on Weigl or Kolatchev preparations for 2 minutes-a rather drastic procedure -to demonstrate this secretion. Consequently Sturdivant’s account and figures of secretion of the refringent granules by the Golgi bodies are not at all convincing. After secreting the refringent granules all the Golgi bodies are said to be cast off with the cytophore and the plasma lobe formed in the earliest spermatids. Hence the ripe sperm is stated to be without any Golgi bodies, contrary to the findings of Collier (1936) in A . suilla. The refringent granules, according to Sturdivant, later develop rods inside them (which are homologous with the acrosomal granules), and unite together progressively to form one large refringent body, the acrosome of the ripe sperm, which is found only in the uterus of the female. Nath and Singh (1956) in A . suilla and Porrocaecum have generally confirmed the account of Sturdivant. But, according to them, the refringent granules are not secreted by the Golgi bodies ; on the other hand the Golgi bodies are directly metamorphosed into the refringent bodies. That is the reason why Sturdivant could not find any Golgi bodies in the ripe sperm. Nor do Nath and Singh find any casting off of the Golgi bodies with the cytophore and the plasma lobes. Again these workers find that in Porrocaecum granules and not rods appear in the refringent bodies. Nath and Singh’s work will be published in due course, but the following photomicrographs of the living material of Porrocaecum are very instructive. Figure 26a is a photomicrograph of a living sperm of Porrocaecum

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from the uterus of the female seen under the phase contrast. In this figure there are two Golgi granules lying very close together on the large conelike acrosome just below its tip ; the nucleus is very small and faint behind ;and the granular mitochondria do not show at all. In the photomicrograph

FIG.26. Microphotographsof the living material of nematodes under positive phase contrast; u, sperm; b, c, spermatids of Porrocaecunz; (I, e, sperm of Ascaris milla (Nath and Singh, 1956). (Fig. 26b) an early spermatid is seen choked with refringent bodies, and one of these bodies, containing the acrosomal granule, has accidently wandered into the plasma lobe. In the photomicrograph (Fig. 26c) the plasma lobes are absolutely devoid of any material, Figures 26d and 26e are photographs of the living ripe sperms of A . szcilla. In Fig. 26d the nucleus and acrosome are very clear ; and in Fig.

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26e the mitochondria are masking the nucleus, and the acrosome is also very clear. Lastly in Figs. 25r and 25s of this review showing spermatocytes of Porrocaecum (after Vedehra, unpublished), the structures labeled mitochondria must be interpreted as refringent bodies in the light of the work of Sturdivant, and Nath and Singh.

IV. CHROMATOID BODIESIN SPERMATOGENESIS It has already been stated that the chromatoid bodies or the accessory bodies in mammalian spermatogenesis take their origin from the spermatid Golgi apparatus. Gatenby and Beams ( 1935), Gresson (1942), Gresson and ZIotnik (1945), and Sharma et ul. (1953) all agree on this point. Indeed Sharma et al. do not recognize any chromatoid bodies as such in the spermatogenesis of Cawia, and they quite rightly conclude “that some of those Golgi elements also which do not come together to be in the localised condition and remain dispersed throughout the cytoplasm of the spermatocytes and the spermatids may have been labelled as chromatoid bodies.” Similarly Nath ( 1932) interpreted the chromatoid bodies described earlier by Fasten (1918) in the primary spermatocytes of Cancer as the typical Golgi-idiosome complex. Likewise Nath ( 1937) interpreted the chromatoid bodies described by Fasten (1914) in the spermatogonia and spermatocytes of the crayfish, Cambarus virilis, as the Golgi masses. Again Nath and Bhatia (1953) in Lepisma interpreted the “chromatoid body” of Bowen (1924a), Gatenby and Mukerji (1929), and Mukerji (1929) as that part of the Golgi material, which does not merge into the acrosome. The “spermatid remnant” of Bowen was likewise interpreted as that part of the Golgi material, which does not participate in the formation of the acrosome. A review of the old literature shows that the earliest workers were indeed describing the Golgi apparatus (which had not yet even been discovered) under fantastic names other than the chromatoid bodies (Nath, 1942). Gilson (1&86),Hermann (ISSO), La Valette St. George (1892), and Grobben (1878), all working on the spermatogenesis of Astacus, have undoubtedly described the Golgi apparatus under the names of nebenkern or corpuscule albuminoide, corpuscle paranucleare, knauelformigen nebenkern (ball-like nebenkern), and nebenkorper respectively. Again the mitochondria-like mass of Koltzoff (1906), Binford ( 1913), and Fasten (1918) described in the spermatids of certain decapod sperms (Brachyura) are the fused Golgi elements destined to form the acrosome (Nath, 1932). But there is no doubt that there are present in some cases of spermatogenesis enigmatic chromatoid bodies, which do not answer to the descrip-

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tion of the typical Golgi-idiosome complex. Recently Sud (1955) in the checkered water snake, Natrix p. piscator, and Gupta (1955) in the domestic duck have described such an enigmatic chromatoid body. In the duck this body is present in all the stages of spermatogenesis from the spermatogonia onward, but it is sloughed off in late spermateleosis. In the water snake the behavior of the chromatoid body is similar except that it appears for the first time in the primary spermatocyte. In both the duck and the snake it can be seen in the living cells under an ordinary microscope; it does not contain any vacuoles; it is not blackened in silver and long osmication techniques ; it is not destroyed or even slightly corroded in Bouin’s fluid; it is not blackened by osmic acid; and lastly it is not stained by sudan black, which is supposed to be a specific stain for the lipoids (Golgi bodies). It may be that the single “Golgi body” of the gastropod pulmonate, VaginuZa maculata, described by Srivastava (1953), is such an enigmatic chromatoid body.

V. EVOLUTION AND FUNCTIONS OF THE ACROSOME 1. Occurrence Except in certain well-understood cases, an acrosome seems to be present in all the phyla of the animal kingdom from the sponges upward. 2. Position of the Acrosome in the Sperm The position of the acrosome is almost always in front of the nucleus except in the silver-fish insect, Lepisma domestica (Bowen, 1924a ; Nath and Bhatia, 1953), ticks (Sharma, 1944), and five genera of Brachyura which have bi-centrosomal sperms (Nath, 1932, 1942). In Lepismu domestics the acrosome is formed in the neck region of the sperm from the Golgi bodies and remains there, sending back into the tail region a long filamentary process. I n the same paper Bowen also states that in the beetles Chelymorpha and Lixus the acrosome lies along the whole length of the sperm head, whereas in Hemiptera the bulk of the acrosome is spread out along one side of the head. In ticks the acrosome, along with the nucleus, is present at the posterior end of the tubelike sperm, the centrosome being right at the anterior end of the tube. In the bi-centrosomal sperms of Brachyura (Paraklphusa and its allies) the ringlike acrosome fuses with the lips of the nuclear cup, which are posterior in position.

3. Pro-acrosome It may be stated without fear of contradiction that the acrosome is invariably formed from the Golgi bodies. The details of this process are complicated ; and the pro-acrosome may be a secretory product of the

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Golgi bodies or may be formed by their direct fusion. But the writer has no doubt that the pro-acrosome, when it emerges for the first time, is frequently in the form of a chomophobic vacuole, variously described as the archoplasmic vacuole, acrosomal vesicle, or simply the idiosome. This chromophobic vacuole has been seen in this laboratory sticking closely to the anterior aspect of the nuclear membrane of the spermatid in the living cells of many species under the phase contrast microscope. The proacrosomal vacuole, gradually ripens into the acrosome. Its contents become more and more gluelike. An acrosomal granule may appear in its interior, as for example, in the domestic fowl ; or many minute acrosomal granules may appear in the vacuole as in Anadenus.

4 . Degeneration of the Acrosome in Certain Animal Sperms Nath (1932) described a prominent ringlike acrosome in the vesiculiform sperm of Paratelphusa spinigera. This completely fuses with the lips of the nuclear cup, and can no longer be distinguished as a separate structure. Nath (1942) showed that the acrosomal ring in Potamon, Ocypoda, Macrophthalmus, and Matuta becomes progressively more and more slender, until it completely disappears in the Brachyura having tri-centrosoma1 sperms (e.g., Leptodius). In these crabs the Golgi elements of the spermatid fuse together to form a pro-acrosome, which, to begin with, grows in size, and becomes quite a prominent structure in the cell. But it is not destined to form an acrosome. It never becomes ringlike. After becoming granular or vacuolated, it completely disappears from the cell. In the Macrura (Nath, 1937, 1942), the pro-acrosome is never' formed, the Golgi elements disappearing in spermateleosis. In the Anomura, Nath (1942) showed that in both species of Clibanurius and Coenobita also the acrosome is conspicuous by its absence, the single Golgi granule of the spermatid disappearing in spermateleosis. In the Anomura the climax is reached in Pugurus in which the Golgi material degenerates even earlier, during the meiotic stages. The vesiculiform sperm of the millipedes (Nath and Sharma, 1952) is also completely devoid of an acrosome, the Golgi material disappearing in the course of sperrnateleosis. In Isoparorchis eurytremum (Dhingra, 1954a) there is a prominent Golgi granule in each of the spermatogonia, primary and secondary spermatocytes, and spermatids, which has been microphotographed by the author in the living material. Under the phase contrast the Golgi granule appears as a dark, very refractile body in sharp contrast with the dull appearance of the mitochondria. But the Golgi granule completely dis-

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appears in the course of spermateleosis, with the result that the ripe sperm has no acrosome whatsoever. The climax is reached in Cotylophoron elongatum and Gastrothylax crumenifer (Dhingra, 1955a, b), in which the Golgi material has completely disappeared in all categories of sperm-forming cells. Nath (1942) showed that the degeneration of the acrosome in the decapod crustacea does not follow the generally accepted line of evolution in this group of animals or in the animal kingdom in general. Why the acrosome should disappear completely from certain sperms must still remain unexplained.

5. Functions of the Acrosome Waldeyer (1901) considered the acrosome to act as a boring or cutting apparatus for the penetration of the sperm into the egg. Hence the acrosome has been known as a perforatorium since the time of Waldeyer. But it has now been known for a long time for various reasons set forth by Cowdry (1924), Bowen (1924a), and Nath ( 1 x 6 ) that Waldeyer’s theory is totally untenable. Bowen (1922b) has suggested that the acrosome may represent the “sperm receptors” postulated in Lillie’s theory of fertilization. That the acrosome is the seat of certain chemical substances, which may be of the nature of catalytic agents bringing about the changes concomitant with fertilization, is a tempting hypothesis. But it is irreconcilable with the complete absence of the acrosome or even of the Golgi material in certain well-understood cases of spermatogenesis, e.g., certain species of platyhelminths (Dhingra, 1955a,b) There does not seem to be any doubt that the acrosome, if present, enters the egg cytoplasm along with the nucleus. Lillie (1912) in Nereis has traced it into the egg where it breaks up into a number of deeply staining granules, which persist for sometime in close contact with the sperm nucleus and later disappear. Collier (1936) in Ascuris suilla states that the thick cell wall of the fertilized egg is formed from material derived from the acrosome: this is in the form of a large “refringent body” formed by the Golgi granules. The acrosome breaks up into a number of large refringent pieces in the cytoplasm of the egg :these grow in size and are used up as material for the formation of the thick cell wall. Dan (1950), working with phase contrast microscope on five species of sea urchins and one of starfish, has shown that the entire spermatozoon, including the whole of the tail, is engulfed by the egg in fertilization. This author has made an important observation that “in every case the fertiliza-

.

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tion cone makes its first appearance after contact has been established between the sperm head and the living egg cytoplasm, and persists in normal cases for from 5 to 15 minutes after the beginning of sperm penetration.” A little later Dan (1952) published another paper on the reaction of the acrosome to egg water and other stimuli in two sea urchin species. In this paper the author shows very clearly by photomicrographs that a gelatinous substance is extruded from the tip of the acrosome in response to the stimulus of egg water, alkaline sea water and contact with solid surfaces. “However, careful study of all the electron photographs seems to indicate rather that the anterior part of the acrosome undergoes a drastic change within one or two seconds after such stimulation.” Dan proposes “that the condition of the acrosomal region in sperm which have so reacted (Fig. 5 ) can best be explained by imagining that the plasma membrane investing the tip of the acrosome undergoes local autolysis, leaving the underlying substance exposed.” Dan conjectures that this substance may be, or may include, an egg-membrane lysin such as that isolated by Tyler and Krauss, or an egg-surface lysin like Androgamone I11 of Runnstrom et al. ; if so “the postulated breakdown of the acrosome membrane would leave the lysin free to attack the vitelline membrane of the egg as soon as the spermatozoon carried it into contact with the egg surface.” Still later Dan (1954) showed very clearly in three starfish species that the reaction of the acrosome to homologous sea water is the putting out of a “very slender, straight filament which possesses considerable rigidity” -contrary to the assumption of Fol that this well-known filament originates from the “attraction cone.” Dan believes that this filament, extending through the jelly, stimulates the egg cortex, and following this stimulation, the egg cytoplasm draws in the filament with the attached spermatozoon and simultaneously forms a fertilization cone beneath the vitelline membrane, which separates as the fertilization membrane. VI. EVOLUTION OF THE MITOCHONDRIAL NEBENKERN It has been shown that amongst the flagellate sperms it is in the insects that the mitochondria1 nebenkern reaches its highest state of development. It has also been shown that amongst the non-flagellate sperms the mitochondrial nebenkern is most complicated in structure in the Anomura, particularly in the species Clibanarius and Coenobita. I n the silver-fish insect, Lepisma domestica, Nath and Bhatia (1953) have described a typical insect mitochondrial nebenkern in early spermatids in conformity with Bowen (1924a), but later it occupies an unusual position

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in this atypical flagellate sperm. Its anterior boundary is marked by the centriole, which has shifted to the anterior tip of the nucleus, and its posterior by the acrosome, which remains in the neck region. But Nath and Bhatia have shown that the mitochondrial nebenkern in Lepism pales off and disappears completely without forming the sheath of the axial filament in the typical insect manner. Why the mitochondrial nebenkern, having been evolved in the insects as a characteristic complicated structure, should completely disappear in Lepima is still an unexplained puzzle. But its disappearance is obviously on a par with the disappearance of the acrosome in many animal species. In the checkered water snake Natrix p . piscator (Sud, 1955) in the spiders Plexippus paykulli and Pardosu sp. (Sharma, 1950; Sharma and Gupta, 1956), and in the sperms of digenetic trematodes (Dhingra 1954a, 1955a, b), the entire mitochondrial contents of the spermatid are cast off. This has been checked in the case of the snake and the digenetic trematodes in the living cells studied under the phase contrast. Finally the climax is reached in the Hoshiarpur dragonfly, Sympetrum hyponzelus (Nath and Rishi, 1953) , in which the mitochondrial material is conspicuous by its absence in all stages of spermatogenesis.

VII. ORIGINOF THE GOLGIBODIESIN THE CELL In the first flush of extensive work carried out on a variety of cells, cytologists were generally inclined to the view that the Golgi bodies (and also the mitochondria) conform to the principle of genetic continuity like the chromosomes and the centrioles. Despite the fact that it can be demonstrated very clearly in a variety of dividing cells that the Golgi bodies (and also the mitochondria) are sorted out roughly into two equal parts and distributed to the daughter cells, the hypothesis of genetic continuity is now totally untenable. Hirsch (1939), as quoted by Bourne (1951), derives the Golgi “presubstance,” i.e., the Golgi granules, from the mitochondria. The granule is later differentiated into two parts, an “externum” and an “internum.” Thus is formed, according to Hirsch, a “Golgi system,” the secretory product appearing in the internum. Working on the spermatogenesis of the slug Anadenus, Nath and Chopra (1955) have recently collected very strong evidence in favor of Hirsch’s view. These authors state that in the earliest spermatogonia of Anadenus there are no granulations in the cytoplasm at all, the mitochondria appearing for the first time in the form of granules in late spermatogonia. The Golgi material appears for the first time in the earliest primary spermatocytes, in the form of prominent dictyosomes, which are formed from the

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mitochondrial granules by alignment. At first the dictyosomes have a crenated appearance, but soon they acquire a uniform contour. I n the fully grown primary spermatocyte the dictyosomes all collect together. At this stage there is no chromophobic vesicle (internum) attached to the dictyosomes. I n the following diakinesis the dictyosomes can be clearly seen breaking up into granules, which merge into the mitochondrial granules, so that in metaphase I not a trace of them is left. During late telophase I the dictyosomes are again formed by the alignment of mitochondrial granules; and during metaphase I1 and anaphase 11, the dictyosomes again break up to merge into the mitochondrial granules, to re-appear again during late telophase 11. I t is in the course of spermateleosis that each dictyosome develops a chromophobic vesicle, in which fine acrosomal granules appear in large numbers. In the opinion of the writer stronger evidence in favor of Hirsch's view could not be wished for, as the Golgi dictyosomes in Anadenus not only arise from the mitochondria but also merge into them during metaphases I and 11. This material also provides strong evidence in favor of Hirsch's view that the secretion arises in the internum of the Golgi complex. Roque (1954) is doubtful about the origin in Helix of Golgi dictyosomes (his paranuclear bodies) ; but Nath and Gupta (1956) in their phase contrast studies of Anadenus have fully confirmed the account of Nath and Chopra (1955) of the origin of the dictyosomes from the mitochondria. Figure 27a is a photomicrograph of a late spermatogonium of Anadenus. The mitochondria exist in the form of a juxtanuclear mass, but there are no traces of the Golgi dictyosomes. In early prophases I, however, when the mitochondria have spread out uniformly throughout the cytoplasm, some mitochondrial granules appear to have become more refractile than the rest as they have a darker contrast. These mitochondria of darker contrast gradually align themselves to form Golgi dictyosomes (Fig. 27b). Stages of meiosis I were not available ; but Nath and Gupta ( 1956) successfully photomicrographed early and late telophase I1 stages. Figure 27c is a photomicrograph of early telophase 11. It will be seen that in this figure there are no traces of Golgi dictyosomes. But in late telophase I1 (Fig. 27d), the mitochondria can be very clearly seen aligning themselves to form the Golgi dictyosomes-third time in spermatogenesis. Nath (1942) observed in the decapod spermatogenesis and Sharma (1944) observed in ticks that in many species the earliest spermatogonia are devoid of any Golgi material, which appears in later spermatogonia in the form of a very few sharply staining granules, embedded in the juxtaor circumnuclear mass of pale mitochondria.

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VIII. CONCLUSION The apparent complete absence of the Golgi material (or its product, the acrosome) or of the mitochondria, or of both, in the spermatozoa of many species of animals leads us to the irresistible conclusion that neither of these cytoplasmic inclusions is essential in the process of fertilization.

FIG.27. Microphotographs of the living ovo-testicular material of Anadaus examined under positive phase contrast ; a, late spermatogonium; b, early prophase I ; c, early telophase 11; d, late telophase I1 (Nath and Gupta, 1956). This conclusion is strongly supported by the remarkable work of Hughes-Schrader (1946) on iceryine coccids in which “the sperm is formed by the invasion of the cytoplasmic anlage of the spermatid tail by a nuclear diverticulum containing the chromosomes,’’ which “are arranged in linear order and are attached end to end.” The spermatid “tail develops directly into the mature sperm,” and “the rest of the cell, with nucleus and all formed cytoplasmic constituents, is discarded.” It is clear, therefore, that chromatin is not only the physical basis of heredity par excellence but also of enzymatic activity.

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ACKNOWLEDGMENT It is a great pleasure to me to thank all my colleagues for the help that they have given me in the preparation of this review, particularly Mr. B. L. Gupta who has taken almost all the microphotographs and has drawn all the figures after the original authors. I wish also to thank the Vice-Chancellor of the Panjab University, for allocating the necessary amount of funds for the purchase of two phase contrast microscopes out of the grants made to the Panjab University by the Universities Grants Commission. IX. REFERENCES Baker, J. R. ( 1933) “Cytological Technique.” Methuen, London. Baker, J. R. (1944) Quart. J. Microscop. Sci. 85, 1. Baker, J. R. (1949) Quart. J. Microscop. Sci. 90, 293. Baker, J. R. (1953) Nature 171, 617. Bawa, S. R. (1953) Research Bull. East Panjab Univ. 38, 181. Bawa, S. R. (1954) Research Bull. Panjab Univ. 45, 39. Bawa, S. R. (1955) Research Bull. Panjab Univ. 89, 47. Beams, H. W., Tahmisian, T. N., Devine, R. L., and Roth, L. E. (1951) Biol. Bull. 107, 47. Bell, A. W. (1929) I. Morphol. and Physiol. 48, 611. Binford, R. (1913) J . Morphol. 24, 147. Bourne, G. H. (1951) “Cytology and Cell Physiology.” Oxford U. P., New York. Bowen, R. H. (1922a) Quart. 1. Microscop. Sci. 66, 595. Bowen, R. H. (192%) J. Morphol. 37, 79. Bowen, R. H. (1924a) J. MOrphOl. and Physiol. 39, 351. Bowen, R. H. (192413) Am. J . Anat. 33, 197. (Quoted by Collier, 1936.) Broman, I. (1900a) Anat. Anz. (Jenu) 17, 20. (Quoted by Wilson, 1925.) Broman, I. (1900b) Anat. Anz. ( J e n a ) 17, 129. (Quoted by Wilson, 1925.) Cannon, H. G. (1922) Quart. J. Microscop. Sci. 66,657. Chang-Chun Wu (1946) Quart. J. Microscop. Sci. 87, 31. Charlton, H. H. (1921) J . Morphol. 35, 381. Clermont, Y., Einberg, E., Leblond, C. P., and Wagneg, S. (1955) Ataat. Record 121, 1. Clermont, Y., and Leblond, C. P. (1955) Am. J . h a t . 96, 229. Collier, V., Jr. (1936) Quart. J. Microscop. Sci. 78, 397. Cowdry, E. V. (1924) “General Cytology.” Hoeber, New York. Dan, J. C. (1950) Biol. Bull. 99, 399. Dan,J. C. (1952) Biol. Bull. 103, 54. Dan, J. C. (1954) Biol. Bull. 107, 203. Dhillon, B. K. (1955) Research B J l . Panjab Univ. 76, 119. Dhillon, B. K. Unpublished. Dhingra, 0. P. (1954a) Research Bull. Panjab Univ. 44, 21. Dhingra, 0. P. (1954b) Research Bull. Panjab Univ. 61, 101. Dhingra, 0. P. (1954~)Research Bull. Panjab Univ. 61, 159. Dhingra, 0. P. (195Sa) Research Bull. Panjab Univ. 64, 1.

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Nath, V. (1944) Presidential Address to the 31st Indian Sci. Congr. Sect. 2001. Entomol. Indian Sci. Congr. ASSOC.,Gouranga Press, Calcutta. Nath, V., Bawa, S. R., and Bhimber, B. S. (1954) Nature 173, 312. Nath, V., Bawa, S. R., Bhardwaj, R., and Gupta, M. L. (1951) Research Bull. East Panjab Univ. 16, 39. Nath, V., and Bhatia, C. L. (1953) Research Bull. East Panjab Univ. 27, 33. Nath, V., and Bhimber, B. S. (1953) Research Bull. East Panjab Univ. 37, 145. Nath, V,. and Chopra, H. C. (1955) Research Bull. Panjab Univ. 74, 91. Nath, V., and Gill, G. K. (1950) Research BuEI. East Panjab Univ. 1, 1. Nath, V., and Gupta, B. L. (1956) Quart. J. Microscop. Sci. 97, in press. Nath, V., and Raina, J, L. (1929) Proc. Indian Sci. Congr. 16, 197. Nath, V., and Rishi, R. (1953) Research Bull. East Panjab Univ. 31, 67. Nath, V., and Sharma, G. P. (1952) Research 82611. East Panjab Univ. 22, 99. Nath, V., and Singh, S. (1956) Research Bull. Panjab Univ. 91, in press. Oettinger, R. (1909) Arch. Zellforsch 3, 563. Papanicolaou, G. N.,and Stockard, C. (1918) Am. J. Anat. 24, 37. (Quoted by Sharma et al., 1953.) Payne, F. (1916) J. Morphol. 28, 287. Randall, J. T.,and Friedlaender, M. H. G. (1950) Ezptl. Cell Research 1, 1. (Quoted by Gresson, 1951.) Ratnavathy, C. K. (1941) J. Madras Univ. 19, 227. Rau, A. S., and Brambell, F. W. R. (1925) J. Roy. Microscop. SOC. 46, 438. Roque, A. L. (1954) J. Roy. Microscop. SOC.74, 188. Sarkaria, D. S. (1944) Proc. 31st Indian Scd. Congr. p. 81. Shafiq, S. A. (1953) Quart. J. Microscop. Sci. 94, 319. Sharma, G. P. (1944) Proc. Natl. Inst. Sci. Zndia 10, 305. Sharma, G. P. (1950) Research Bull. East Panjab Univ. 6,67. Sharma, G. P., Chaudhuri, G. C., and Sattee, V. S. (1953) Research Bull. East Panjab Univ. 38, 157. Sharma, G. P., and Dhindsa, K. S. (1955) Research Bull. Panjab Univ. 82, 175. Sharma, G. P., and Gupta, B. L. (1956) Research Bull. Panjab Univ.84, 5. Sharma, G. P., Gupta, B. L., and Nayyar, K. K. (1956) Research Bull. Panjab Univ. 93, in press. Sharma, G. P., and Malik, A. P. (1953) Research Bull. East Panjab Univ.32, 73. Sharma, G. P., and Sekhri, K. K. (1955) Research- Bull. Panjab Univ. 79, 145. Sokoloff, J. (1914) Zool. Ane. 44, 558. Srivastava, M.D. (1953) Nature 172, 689. Strangeways, T. S. P., and Canti, R. G. (1927) Quart. J. Microscop. Sci. 71, 1. Sturdivant, H. P. (1934) J. Morphol. 66, 435. Sud, B. N. (1955) Research Bull. Panjab Univ. 71, 101. Sud, B. N. (1956) Research Bull. Panjab Univ. 86, in press. Swaen, A., and Masquelin, H. (1883) Arch. biol. (LGge) 4, 749. (Quoted by Vasisht, 1953.) Vadehra, N. P. Unpublished. Vasisht, H. S. (1953) Research Bull. East Panjab Univ. 40, 193. Vasisht, H. S. (1954a) Research Bull. Panjab Univ. 46, 49. Vasisht, H. S. (1954b) Research Bull. Panjab Univ. 62, 169. Vaupel, J. (1929) J. Morphol. and Physiol. 47, 555.

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Waldeyer, W. (1901) in “Handbuch der vergleichenden und experimenteilen Entwicklungslehre der Wirbeltiere” (Hertwig, ed.), Vol. 1, 86, Fischer, Jena. (Quoted by Bowen, 1924.) Warren, E. (1934) Ann. Natal Museum 7, 351. Watts, A. H. G. (1952) J . Morphol. 91, 53. Wilson, E. B. (1916) Proc. Natl. Acad. Sci. (US.)2, 321. Wilson, E. B. (1925) “The Cell in Development and Heredity.” Macmillan, New

York. Wilson, E. B. (1931) J. Morphol. 62, 429. Wilson, E. B., and Pollister, A. W. (1937) J . Morphol. So, 407. Zlotnik, I. (1943) Nature 161, 670. Zlotnik, I. (1947) Quart. J . Microscop. Sci. 88, 353.

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The Ultrastructure of Cells as Revealed by the Electron Microscope F R I T I O F S. SJOSTRAND Department of Anatomy. Karolinska Znstitutet. Stockholm. Sweden I. I1 I11. IV. V. V I. VII

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Introduction ....................................................... The Development of Ultrathin Sectioning Techniques ................. The Electron Microscopy of Ultrathin Tissue Sections ............... The Problem of Fixation ........................................... The Ultrastructural Organization of Mitochondria ................... The Golgi Apparatus ............................................... The Plasma Membrane ............................................. 1. The Brush Border of the Intestinal Epithelium ................. 2. Tubular Invaginations of the Cell Membrane ................. 3. Intracellular Folds of the Cell Membrane ..................... 4. Specialized Regions of the Cell Membrane ..................... 5 . The Intercalated Discs of Heart Muscle ..................... 6. The Free Surface of Ciliated Cells ........................... The Basement Membrane ........................................... The Ground Substance of the Cytoplasm ............................. 1. Cytoplasmic Membranes ....................................... 2. The Endqlasmic Reticulum ................................... 3. Vesicles ...................................................... 4. Opaque Particles ............................................. 5. The Basophilic Component of the Cytoplasm ................... 6. The Synaptic Granules ....................................... 7. Osmiophilic Granules ......................................... 8. Fibrillar Structures ........................................... The Nucleus ....................................................... 1. The Nuclear Membrane ..................................... 2. The Nucleoplasm ............................................. Lipoprotein Structures ............................................. 1. General ...................................................... 2. The Myelin Sheath ........................................... 3. Retinal Rods ................................................. 4. Chloroplasts .................................................. 5 . The Osmium Staining ......................................... The Structural Organization of Whole Cells ....................... 1. The Retinal Rods ............................................. 2. The Tubular Cells of the Kidney ............................... 3. The Exocrine Cells of the Mouse Pancreas ..................... 4. Cross-Striated Mammalian Muscle Fibers ..................... The Interpretation of Electron Microscope Observations ............... Important Future Problems of General Interest ....................... References .........................................................

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I. INTRODUCTION The development of ultrathin sectioning techniques has made possible a systematic study of the ultrastructural organization of cells. As a result a great number of electron microscope studies on various cellular components have been published and it seems quite justifiable to review the results obtained as they have drastically extended our knowledge of the organization of cells. The number of papers published' and the great variety of material studied make it impossible to produce a complete review and to discuss all the basic problems of electron microscopy as applied to the study of tissue cells. This review therefore may be found to be incomplete in some respects. Only a fairly limited number of morphologists have any appreciable experience in electron microscopy that would enable them to evaluate the various contributions which have been made on this subject. In a new field such as this it is very tempting to overestimate the significance of the observations made. Therefore, this review is intended to be critical and to give as balanced and moderate a presentation of the collected facts as is possible. Different attitudes are represented among those who have applied the electron microscope in the study of cell structure. I n this review a certain emphasis will be put on the type of work that has aimed at as precise a description of the ultrastructural components of the cells as present techniques permit. Between these studies, in which the resolving power of the electron microscope has been used to its limit, and the old light microscopic studies, there are all kinds of intermediate levels. It is striking how few really new structural components have been revealed by the electron microscope as compared with the careful classical light microscope studies. In most cases the electron microscope's contribution has been a detailed analysis of the geometrical morphology of already well-known components of the cells. It is to be hoped that this will help to establish a better understanding of the functional significance of these various components and of the basic processes taking place in them. For the moment it is reasonable to state that the electron microscope studies have not yet contributed any new, really basic concepts in cell research. The reason might be that the technique is still too imperfect to give the most essential information or that new concepts have to be founded on data collected not only in electron microscopy but from several fields of biological sciences. 1

The earlier papers have been reviewed by Selby (1953) and by Swerdlow (1954).

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TECHNIQUES

The perfecting of the technique for cutting ultrathin tissue sections has been made possible through contributions from several researchers. Embedding in plastic, as described by Newman et al. (1949), represents a very important contribution. Really ultrathin sections cannot be obtained from tissues embedded by any other known technique. The most important problem in the development of microtomes has been to provide a useful cutting edge and to study the conditions under which such an edge will give the best results. The designing of a reliable “specimen-advance” mechanism is a second problem which especially affects the reproducibility of the sectioning procedure. Ultrathin sections have, however, been obtained in a less reproducible way with rather crude solutions of this problem as for instance the modification of the Spencer microtome No. 810 (Sjostrand, 1953a). This modification represented a continuation of the work of Hillier and Gettner (195Oa, b) who in their turn based their work on the pioneer work of Pease and Baker (1948). It seems statistically possible to obtain some ultrathin sections from almost any sectioning mechanism. Useful knife edges can be obtained in two different ways : by sharpening steel edges to a quality superior to what theoretically seemed almost impossible and the breaking of glass plates to obtain a sharp edge (Latta and Hartmann, 1950). The method for sharpening microtome knives was greatly improved by Hillier (1951) and the knife edges he obtained allowed the cutting of 0.1-0.2 p-thick sections. The method worked out by Sjostrand (1951a, 1953a) for polishing steel edges and the application of this technique on razor blades made it possible to obtain ultrathin sections, that is sections about 200 A thick. The controlled conditions under which the sectioning could be done when using these knives made it possible to establish the importance of the rake angle and to show that this should be about 30°,that is a rake angle within the range used in the cutting of metals (Sjostrand, 1955a, 1956a). The technique of Hillier and Gettner allowed the cutting of sections 0.1-0.2 p thick routinely. The combination of methacrylate embedding and glass knives as cutting edge made it possible for Palade (1952a, b) to cut sections 500-1000 A thick with a microtome designed by Claude and Blum (Claude, 1948). The resolution obtained by Hillier and Gettner l20A. The was about 500 A and that demonstrated by Palade was o@ step down to high resolution, which here refers to a resolution of 30 A or better, was taken in 1952 with the production of sections about 203 A thick with a modified Spencer microtome (Sjostrand, 1953a, b, d ) . The

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cutting of such sections was made a routine business through the construction of a new microtome in 1952 (Sjostrand, 1953e). Since then several microtome constructions have been improved (Porter and Blum, 1953) or designed (Hodge ek al., 1954) with which excellent results have been obtained. Since the conditions for ultrathin sectioning are now well known the construction of new types of microtomes is a fairly simple task. MICROSCOPY OF ULTRATHIN TISSUESECTIONS 111. THEELECTRON To reveal in a more exact way the morphology of ultrastructural components, high resolution electron microscopy is indispensable. Tissue sections measuring 200 A or less in thickness allow a specimen resolution of better than 30 A and, therefore, a more exact study of these components. With the conventional method of mounting the sections on Formvar or collodion film covered grids the limit for specimen resolution is 25-30 A even when the resolving power of the instrument is better than 20 A (Sjostrand, 1955b, 1956b). The reason for this limit is the low contrast of the specimen which makes it impossible to make real use of in-focus pictures. This situation may be improved through mounting the sections on metallized Formvar nets (Sjostrand, 1955b, 1956b). I n this case the absence of a more or less diffusely scattering Formvar or collodion film improves the contrast drastically. The use of carbon films (Bradley, 1954; Watson, 1955a) may mean a certain improvement as compared with Fornivar films. However, no pictures at the limit of resolution have been presented of specimens mounted on such films which would make a comparison possible. The elimination of a supporting film through the mounting on nets seems the radical solution of the problem. The term “high resolution” is used to characterize very different standards of resolution by various electron microscopists. I n most cases it just means a better resolution than the average and that varies very much in different laboratories. The present author has accepted the definition of high resolution proposed by Williams (1952) as a basis of discussion, namely one of 30 A or better. In order to report on the resolution, actual measurements should be made of the minimum distance of image points that are resolved in the pictures. T o avoid mistakes through a misinterpretation of groups of film grains or occasional defects in the emulsion these measurements should be performed on at least two independent exposures of the same specimen area (Ruska, 1954). There are so many uncritical statements regarding resolution appearing in papers on electron microscopy (e.g., claiming a resolution of 20 A when taking pictures at an electron optical magnification of 3000-6000 times), that such statements should be considered very cautiously. The pre-

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requisite for a resolution of 20 A is that the picture be taken at an electron optical magnification of at least 25,000 times, because of the grain factor of the film which is far more disadvantageous for low contrast than for high contrast pictures. The limit for the resolving power of the emulsion generally used seems to be 50-60 p according to an investigation by Ruska and collaborators (personal communication) and according to our own experience with Kodak lantern slide plates. According to Ruska this figure is almost independent of the type of emulsion within a fair range of fine grain emulsions. It is to be hoped that, in the future, the electron microscopists will be more cautious in this respect. OF FIXATION IV. THEPROBLEM

Before ultrathin sectioning had been developed to any degree of perfection many electron microscopists and other morphologists took a rather pessimistic view of the possibilities of preserving the structure of cells well enough to make an ultrastructural analysis worth while. We owe a good deal to Palade (1952a) who modified the osmium fixation to give a decent preservation of the fine structure of cells. His introduction of the veronalacetate buffer as a medium for the osmium tetroxide has improved the quality of fixation for several tissues. However, the p H of the osmium tetroxide solution seems not to be so important as he originally claimed. Variation of p H within a wide range from pH 4,and in some cases even from p H 2, to p H 8 does not substantially affect the result in many tissues, e.g., the kidney (Rhodin, 1954), the intestinal epithelium (Zetterqvist, 1956), and the sensory epithelium of the inner ear (Wersall, 1956). Most of the great differences of the quality of fixation described by Palade (1952a) in his study at a low resolution seem to represent various degrees of post-mortem changes. The osmium tetroxide solution of Palade is strongly hypotonic and in several cases, as for instance the retina of the eye (Sjostrand, 1953a), the kidney tubular cells (Rhodin, 1954), and the intestinal epithelium (Zetterqvist, 1956), hypotonic solutions of osmium tetroxide produce a more or less marked swelling of the cells. The importance of using isotonic solutions has therefore been stressed (Sjostrand, 1953a). It has even been possible to obtain as good a preservation of the cell structure by using isotonic non-buffered solutions of osmium tetroxide as for instance a solution of isotonic sodium chloride (Sjostrand and Rhodin, 1956). The most important factor for good preservation is the time elapsing between the shutting off of the blood supply to a tissue and the establishing of a sufficient concentration of osmium tetroxide inside the cell to fix the structure. This time must not exceed 5 to 15 minutes (Rhodin, 1954;

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Sjcstrand and Hanzon, 1954a). The slow penetration of osmium tetroxide through the tissue therefore makes it necessary to restrict the study of the cell structure to an approximately 40 p-thick surface layer of the tissue blocks, that is the first few layers of cells (Rhodin, 1954). The systematic study of post-mortem changes by preserving tissues at various intervals after the killing of the experimental animal helps an investigator to become acquainted with the most obvious artifacts that may appear. The time of fixation is important since a too long stay in the osmium tetroxide solution may result in an extraction of material from the cells. Fixation at a low temperature (0-+2"C.) increases the chances for a good preservation. How can we accept the structural patterns observed in osmium-fixed material as representing the preformed structure of the living cells? A Comparison of the appearance of tissue cultured cells by means of light microscopy before and after fixation with osmium tetroxide is often sufficient for the light microscopists but is certainly not sufficient to convince the electron microscopist. It has therefore been important to compare the structural patterns observed in frozen dried material with those appearing in the osmium-fixed tissue. In these two cases the tissue has been subjected to such different treatments that it seems unlikely that the same artifacts would appear. The only comparison of this sort that has been published deals with the cytoplasmic membrane structures of the exocrine pancreas cells ( Sjostrand, 1953f, g) . The same characteristic pattern could be observed in both cases. The most convincing evidences that make it justifiable to accept osmium fixation as reasonably reliable are to be found in those cases where the results of optical polarization analysis and those of electron microscopy are in harmony. In all cases studied until now where electron microscopic data have been compared with data from optical polarization analysis they have been found to be in complete agreement. However, it seems justifiable to point out that the osmium fixation may give a rather crude and incomplete picture of the ultrastructure qf cells and it cannot be stressed too much how important it is to find new techniques of fixation. The chances of obtaining reIiable preservation increase with the strength of the structure .which itself is dependent on the chemical binding forces within it. A highly organized structure with strong bonds between protein molecules, as for instance collagen and various membrane structures, will be more resistant than the presumably less organized and labile ground substance of the cytoplasm.

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V. THEULTRASTRUCTURAL ORGANIZATION OF MITOCHONDRIA Indications of an internal structure in mitochondria were reported by several electron microscopists at an early stage of the development of the ultrathin sectioning technique. These investigations, however, did not make it possible to define any fundamental structural pattern. Palade (1952b) published a description of the fine structure of mitochondria according to which the mitochondria were bounded by a singlelayered surface membrane which was folded with the folds extending some distance towards the center of the mitochondrion. These folds were called by Palade cristae mitochondrules. In the interior of the mitochondrion he observed a central space free of cristae and extending along the whole length of the mitochondrion. The thickness of the outer surface membrane was estimated at 70-120 A and that of the folds at 180-200 A. Through studies independent and parallel to those of Palade, Sjostrand (1953b, h),l and Sjostrand and Rhodin (1953a, b ) l arrived at a more detailed and different description of the ultrastructure of mitochondria in the inner segments of retinal rods (Fig. 1), the intestinal epithelium, and tubular cells of the mouse kidney (Fig. 2). The mitochondria were described as bounded by a double membrane representing the surface or outer mitochondrial membrane. I n the interior, double membranes, classified as inner mitochondrial membranes, were arranged in parallel and mainly oriented perpendicularly to the long axis of the mitochondrion. These inner membranes may extend across the whole diameter of the mitochondria of the kidney tubular cells “or be separated by a distance of 100-200 A from the outer membrane.” They therefore constitute almost complete septa. In the mitochondria of the inner segments of retinal rods the inner membranes showed very irregular outlines, like pieces of a jigsaw puzzle. No direct continuity between inner and outer membranes was observed although the inner membranes were in close contact with the outer surface membrane along at least part of their rims. No continuity between the space bounded by the two dark layers of the inner mitochondria membranes and the surrounding cytoplasmic milieu could be seen as the surface membrane formed a complete sheath around the mitochondrion. The dimensions of the mitochondrial membranes of the tubular cells of the kidney, and of the inner and outer mitochondrial membranes of the inner segments of retinal rods, were 1%-160 A. The membranes consisted of two opaque layers measuring 40 A in thickness in the kidney 1 These results were presented at the meeting of the Electron Microscope Society of America in Cleveland, November, 1952, and at the meeting of the Scandinavian Electron Microscope Society, June 6, 1952.

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FIG.1. Mitochondrion in the inner segment of a retinal rod of the guinea pig eye. A triple-layered surface membrane bounds the mitochondrion. In the interior triple-layered inner mitochondria membranes or lamellae are observed. I n the surrounding ground substance of the cytoplasm some groups of opaque particles are present. Magnification : X 120,000. (Sjostrand, 19531.)

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tubular cells and separated by a 70 A-wide, less opaque, interspace. The corresponding values for retinal rod mitochondria were 55 and 40 A. Some figures for these mitochondrial membrane dimensions have been put together in Table 1. These dimensions and those obtained from a similar structure in the outer segments of retinal rod (Sjostrand, 1953a, b) made it justifiable to assume that the layered structure corresponded to two protein layers separated by a double layer of lipid molecules. I n the case of the outer segments of the retinal rods some indications of the mechanical stability of the opaque layers were available. Furthermore optical polarization data were useful when trying to interpret the latter structure. The proposed arrangement would correspond to the one proposed by Danielli and Dawson ( 1934) and by Danielli (1936, 1951) for the plasma membrane. This interpretation is supported by the optical polarization data obtained by Rollhauser (1954) from a study of the kidney mitochondria. Figure 3 shows in a schematic way the main data collected in the ultrastructural organization of mitochondria. The inner mitochondrial membranes are more or less densely packed in mitochondria from different types of cells. In the distal convoluted tubule of the mouse kidney (Rhodin, unpublished ; see Sjostrand, 1954a) and in the skeletal muscle mitochondria they are especially densely packed with a distance between the membranes comparable to that between the two opaque layers of each individual membrane. A similar dense packing also occurs in muscle mitochondria (Chapman, 1954 ; Andersson, 1956). In a later paper Palade (1953a) accepted the double-layered structure of the outer mitochondrial membrane, but interpreted tentatively the inner mitochondria1 membranes as folds of the inner opaque layer of the outer membrane. This interpretation which is based on the intimate contact between inner and outer mitochondrial membranes has not been supported by the detailed morphology at the site of contact as analyzed on high resolution pictures. Studies of post-mortem changes taking place within 15-30 to 45 minutes after death have shown that the mitochondria are changed very soon after death (Khodin, 1954; Sjostrand and Hanzon, 1954a). They swell after the shutting off of the blood supply, the inner niitochondrial membranes appear fragmented and pulled apart leaving a more or less extensive central space free from inner membranes. It might be that such postmortem changes are responsible for the central space described by Palade. In order to see the inner and outer mitochondrial membranes it is necessary for the plane of the section to be oriented perpendicularly to the plane of the membranes, if the sections are not less than 200 A thick. This depends on the contrast conditions which are favorable when the opaque

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FIG.2. Mitochondrion in a proximal convoluted tubule cell of the mouse kidney. The inner mitochondria membranes extend across the whole diameter of the mitochondrion. They make contact with the outer surface membrane of the mitochondrion but do not appear as folds of the latter membrane. Magnification: X 184,000. ( Rhodin, 1954.)

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TABLE I DIMENSION s OF MITOCHONDRIA MEMBRANES Outer membranes Total thickness in A 150

Thickness of osmiophilic layers in A 55

Thickness of osmiophobic interspace inA 40

Total thickness in A 160

Prox. convol. tubule cell of mouse kidney Exocrine cells of mouse pancreas

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Skeletal muscle of mouse Heart muscle guinea pig Intestinal epithelium of mouse Ciliated cellsa Goblet cellsa

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Thickness of osmiophobic interspace in A References 40

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Andersson, in preparation Andersson, in preparation Zetterqvist, 1956 Rhodin and Dalhamn, 1956 Rhodin and Dalhamn, 1956 Rhodin and Dalhamn, 1956 Rhodin and Dalhamn. 1956

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FIG.3. Schematic three-dimensional presentation of the ultrastructural organization of two types of mitochondria: A . kidney mitochondria, B . retinal rod mitochondria. C and D show the appearance of these two types of mitochondria in thin sections. E gives some dimensions of the mitochondria membranes (see further Table I). F illustrates the tentative interpretation of the observed patterns in terms of molecular organization with two protein layers separated by a double layer of lipid molecules.

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electron-scattering layers are lined up parallel to the electron beam so that the scattering power at various levels of the section is added up to produce contrast in the image plane. For an exact study of the arrangement of the mitochondrial membranes and for estimating the number of membranes it is therefore necessary to work with extremely thin sections. The studies of the behavior of the mitochondrial membranes under various conditions of fixation have shown that the inner ones are more susceptible to swelling than is the outer one. Through this swelling the space in between the two opaque layers is increased. Furthermore, in well preserved mitochondria, for instance, in muscle tissue ( Sjostrand and Anderson, 1956) and the exocrine cell of the pancreas (Sjostrand and Hanzon, 1954a)) shown in Fig. 4 the dimensions of the outer and inner mitochondria1 membranes are quite different. This might well indicate that they have a different chemical composition and molecular organization. These facts, in addition to the actual morphology of the place of contact between inner and outer membranes make it justifiable to classify them in two different categories. In the ground substance of the mitochondria opaque areas are frequently observed which sometimes may be sharply outlined and may show a complicated inner structure. Such opaque areas have been observed especially in kidney and pancreas mitochondria (Sjostrand and Rhodin, 1953a, b ; Sjostrand and Hanzon, 1954a). The description of the ultrastructure of the mitochondria of the tubular cells of the kidney given by Sjostrand and Rhodin (1953a, b) and by Rhodin (1954) has been confirmed by Pease (1955) and by Bargmann et al. (1955). The general patterns described by Sjostrand (1953h) have furthermore been observed in the mitochondria of the exocrine pancreas cells (Sjostrand and Hanzon, 1954a), of intestinal epithelium (Zetterqvist, 1956), of heart and skeletal muscle (Sjostrand and Anderson, 1956), in the goblet cells of the respiratory epithelium of the trachea of rat (Rhodin and Dalhamn, 1954, 1956), and in insect flight muscle (Chapman, 1954). Some of the fundamental data regarding the ultrastructural organization of mitochondria have been surveyed by Sjostrand ( 1954a). A completely different interpretation of the organization of mitochondria in muscle tissue has been presented recently by Harman (1955) and by Weinreb and Harman (1955). According to Harman there is no outer surface membrane bounding the mitochondrion but a folded inner membrane is responsible for the structural pattern observed in the mitochondria. The resolution in the published pictures is low and a tremendous drift parallel to the inner membranes makes it impossible to draw conclusions about any structures oriented perpendicularly to the drift. Biochemists

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FIG.4. Mitochondrion in an exocrine cell of the mouse pancreas. The inner mitochondria1 membranes form incomplete septa extending across the mitochondria1 body. Some opaque regions are observed in the mitochondrion. Magnification: X 70,000. (Sjostrand and Hanzon, 1954a.)

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who want to make use of niorphologic data in their research must be cautious in accepting such conclusions as Green and Beinert have done in their recent review article in the Annual Review of Biochemistry (1955). The results obtained on mitochondrial fractions isolated through fragmentation and differential centrifugation appear now in a new light. The earlier electron microscope studies of isolated mitochondria have been accepted by many as demonstrating the existence of a mitochondrial membrane. However, the more detailed study of such fractions that has been possible on ultrathin sections through the fractions has made it obvious that membranes always appear in every cell fraction. They might perhaps be formed artificially in the lipid-protein mixtures obtained from tissues or emanate from the numerous membranes of various kinds observed in intact cells. A recent study of fractionated mitochondria (Glimstedt and Lagerstedt, 1953a, b, 1954; Glimstedt et ai., 1954a, b) has led the authors to assume the existence of a granular component in the ground substance of the mitochondria measuring 300 A in diameter and arranged in rows. The published pictures illustrate the danger of making drop preparation of such material where enormous surface tension forces may produce rather misleading drying patterns. No sufficiently well defined particles which do not represent contaminating material have been demonstrated to allow the concIusions of the authors. Mitochondria showing structural patterns slightly different from the one just described have been observed by Rhodin and Dalhamn (1956) in the ciliated cells of the tracheal epithelium in rat. All the inner mitochondrial membranes are connected through segments that extend between adjacent membranes. The interior of the mitochondrion therefore appears to be divided into a great number of compartments. The functional significance of the ultrastructural organization of mitochondria has been the object of obvious speculations. The membranes appear to be a useful means o f arranging enzyme molecules in certain patterns. These patterns would be important for the sequences of coordinated enzymatically controlled reactions. The enzyme molecules could also be oriented in such a way that the chances for an enzyme-substrate interaction will increase. W e have, however, no real information regarding the localization of enzymes within the mitochondria. The arguments presented by Palade (195213) would not stand any serious criticism.

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47 1

VI. THEGOLCIAPPARATUS Discussion of the Golgi apparatus among light microscopists is characterized by great confusion. Which components observed by various techniques in light microscopy represent the Golgi apparatus? T o what degree are the impregnation pictures that have been observed artifacts? T o what extent does a preformed structure exist in living cells that corresponds to the Golgi apparatus? What should the Golgi apparatus be called? These are some of the problems that have been discussed at a recent symposuim (Lacy, 1955). The uncertainty regarding the structural significance of the various pictures of the Golgi apparatus was not changed by the first electron microscopic studies of this structure since the resolution was not good enough to add very much that was new to the light microscope observations. It has, however, been possible with improved resolution to define in a clear-cut way the structural organization of the Golgi apparatus (Dalton and Felix, 1954; Sjostrand and Hanzon, 1954b, c, d ) . This new picture of the Golgi apparatus reveals it as a highly organized constantly present region of the cytoplasm. The most characteristic component of the Golgi apparatus is a system of 60 A-thick membranes arranged in pairs and with the membranes of a pair fused along their rims, thus bounding a narrow interspace (Fig. 6 ) . Three to five or more such membrane pairs are fairly densely packed within the Golgi zone. In many cases the space bounded by a membrane pair is rather wide, appearing as a large vacuole. The width of the space between the membrane pairs, on the other hand, may be very constant, 60 A. In Table I1 some published data regarding the dimensions of the Golgi membranes have been collected. These membrane pairs are embedded in a ground substance which is homogeneous or finely granulated or even reticulated. In the exocrine pancreas cells of the mouse (Sjostrand and Hanzon, 1954b, c, cl) this ground substance is clearly differentiated from the rest of the cytoplasm due to the existence of the cytoplasmic membranes in the latter regions. In other cell types there is no very marked difference between the ground substance of the cytoplasm inside and outside the Golgi zone. In Some types of cells, for instance the exocrine pancreas cells, there are a great number of granules differing in form, size, and opacity embedded in the Golgi ground substance. They show a very intimate topographic relationship to the Golgi membranes. All intermediate stages from minute granules of low opacity to the large zymogen granules of high opacity may be Seen in the exocrine pancreas cells (Sjostrand and Hanzon, 1954b, c, d ) .

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FIG.5. Golgi apparatus in an exocrine cell of the mouse pancreas. The Golgi membranes or y-cytomembranes bound large vacuolar spaces. Their smooth appearance contrasts with the dotted appearance of the a-cytomembranes seen in the lower part of the picture. Above the y-cytomembranes a region containing a great number of granules varying in size, opacity, and form is seen. The well-defined fairly opaque granules are characteristic zymogen granules. Magnification : X 44,000. (Sjostrand and Hanzon, 1954c.)

ULTRASTRUCTURE OF CELLS

473

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FIG,6. Higher magnification of Golgi membranes, y-cytomembranes. These membranes are arranged in pairs. The two membranes or layers are fused at their rims and bound a narrow space which frequently exhibits a fairly high opacity or is enlarged to a vacuolar space. The height of the space in between the membrane pairs, which shows a rather low opacity, is very constant. The vacuoles are in most cases bounded by a pair of membranes. In the lower left corner a part of a mitochondrion is seen. Magnification : X 150,000. (Sjostrand and Hanzon, 1954c.)

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475

TABLE I1 DIMENSIONS OF THE GOLCIMEMBRANES

Organ Pancreas mouse

Number of membrane pairs

2-5

Thickness of single membranes in A

60

Width of interspace between membrane pairs in A

60

Width of interspace between the two constituent single membranes of a pair in A

w

T!

-

Sjostrand and Hanzon, 1954b, c Dalton and Felix, 1954

w P

~

cn 0:

7

Varies with function

70

70

140

Kidney mouse

4-6

60

90

50-200

Rhodin, 1954

Intestinal epithelium mouse

2-6

60

60

50-200

Zetterqvist, 1956

Epididymis mouse

2

References

0

g z

ULTRASTRUCTURE OF CELLS

477

In other cell types such as the tubular cells of the kidney few or no granules may be observed (Rhodin, 1954). The large vacuolar spaces bounded by the Golgi membranes do not appear in the tubular cells of the kidney (Rhodin, 1954), in ganglion cells (Palay and Palade, 1955), in the corneal epithelium (Sheldon, 1956), or in the skeletal muscle fibers (Anderson, 1956). In the intestinal epithelium of the mouse, on the other hand, they are frequently very large and may contain rounded, very opaque, osmiophilic granules (Zetterqvist, 1956). A study comparing the Golgi apparatus of normal and cancerous cells has been published recently by Haguenau and Bernhard ( 1955). In the auditory hair cells of the organ of Corti a similar system of membranes has been observed constituting the Hensen’s body (Engstrom and Sjostrand, 1954). In these cells similar systems of membranes also form a layer along the cell membrane with the constituent membrane pairs oriented parallel to the cell surface. No observations have been made until now which could throw some light on the functional significance of this structure. Experiments using pilocarpine to stimulate the exocrine pancreas cells (Sjostrand and Hanzon, 195413, c, d ) have not elucidated what is really going on. That the Golgi apparatus of secretory cells seems to play a role in the process of secretion has been assumed from light microscope observations. This assumption is based mainly on topographic relationship between the area in which secretory granules appear and the Golgi zone. The electron microscope observations present the same topographic relationship. However, carefully controlled experiments have to be devised which will make it possible to obtain a real, objectively selected time sequence of morphologic stages before a more precise and detailed interpretation of the functional significance will be possible. Electron microscopy has revealed a rather well defined ultrastructural organization of the Golgi apparatus. This high degree of organization, and its astonishing stability, for instance to post-mortem influences ( Sjostrand and Hanzon, 1954c, d ) , seem to justify the acceptance of the Golgi apparatus as a preformed structure of living cells. The Golgi membranes appear to be structurally fairly uniform in a great variety of cell types. The granules and vacuoles vary as to occurrence, number, and size. This might well explain the diff erent results obtained with impregnation techniques. It is also obvious that a Golgi apparatus appears in cells that are not secretory but it still shows the same basic component, i.e., the Golgi membranes. The morphologically similar systems of membranes in the auditory hair cells might not be homologous structures. If they represent Golgi elements it would be justifiable to replace the term Golgi apparatus with a

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more practical classification based on the morphological characteristics of its components.

VII. THE PLASMA MEMBRANE High-resolution electron microscopy has revealed the dimensions of the cell membrane and some of its components (Sjostrand and Rhodin, 19531, ; Sjostrand and Hanzon, 1954a). Earlier observations on the cell membrane were made on specimens which were too thick to allow any well defined picture. A general increase of the opacity toward the cell surface has frequently been classified as the cell membrane. The electron microscope has revealed one osmiophilic component or layer of the plasma membrane measuring 60 A in thickness (Figs. 7, 8). When two cells are in close contact, for instance in the acinar components of the pancreas or in the tubules of the kidney, the osmiophilic layers of the two plasma membranes are separated by a less osmiophilic interspace. The thickness of this space is strikingly uniform along the whole surface of contact and measures 110-130 A. Because of the uniform thickness of this interspace it has been assumed to correspond to an organized layer of material, for instance, lipids. The total thickness of the plasma membrane in the exocrine pancreas cells and the tubular cells of the kidney, therefore, would be about 120 A. The less opaque interspace may also correspond to a layer of cementing material. 1. The Brush Border of the Intestiml Ebitheliuwt

This consists of cylindrical processes (Granger and Baker, 1949, 1950 ; Dalton et nl., 1950; Zetterqvist, 1956). High-resolution electron microscopy (Fig. 9 ) has shown that they are bounded by a complete and continuous cell membrane (Zetterqvist, 1956). The thickness of this part of the cell membrane is 105 A and it consists of three layers, two osmiophilic layers 40 A thick separated by a less osmiophilic 25 A-thick interspace (Zetterqvist, 1956). At the bases of the brush border processes the cell membrane extends continuously between the processes. There are no indications of any pores of a size over 50-100 A in diameter. The present material does not exclude the existence of pores smaller than 50-100 A. When examining the brush border of cells during resorption of carbohydrates, fats, and proteins no pores have been observed. Preliminary observations seen1 to show that the structure of the cell membrane is radically changed during absorption. Thus, only one osmiophilic layer may be observed in absorbing cells (Sjostrand and Zetterqvist, unpublished). If confirmed this observation would indicate that the cell membrane in this zone is a labile, dynamic structure.

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FIG.7. Survey picture of the a-cytomembranes in the cytoplasm of exocrine pancreas cells. In the lower part of the picture a mitochondrion extends along the cell boundary separating two cells. Opaque particles are seen attached to the 30-40 A thick basic membrane of the a-cytomembranes. These are arranged in pairs bounding a space of low opacity. Magnification : X 97,000. (Sjostrand and Hanzon, 1954a.)

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FIG.8. Higher magnification of the cell boundary seen in Fig. 7.. The picture shows in a distinct way the osmiophilic components separated by a less osmiophilic component of the plasma membranes. From such pictures it is possible to obtain quite good data regarding the dimensions of the different components. Magnification : X 310,000. (Sjostrand and Hanzon, 1954a.)

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481

FIG.9. Cross section through the brush border of the intestinal epithelium (jejunum) of mouse. The plasma membrane covering the free surface of these cells is a triple layered structure. The total thickness of the plasma membrane is 105 A, the thickness of the two opaque, osmiophilic layers is only 40 A and the width of the separating space 25 A. Magnification : X 130,000. (Zetterqvist, 1956.)

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The brush border of the kidney proximal tubule cells is rather difficult to analyze because the tubules collapse with fixation so that the brush border becomes extremely compressed. In a paper by Sjostrand and Rhodin (1953b) this structure was falsely interpreted but the mistake was corrected by Rhodin (1954) when studying the tubule cells during various functional conditions, under some of which the tissue could be fixed and a free lumen retained. Also in this case we are dealing with cylindrical processes nieasuring only 500-700 A in diameter and 1.4 p in length. The thickness of the osmiophilic component of the cell membrane is here 60-80 A.

2. Tubular Invaqinations of the Cell Membrane These have been observed in the proximal convoluted tubule by Rhodin (1954) who presented the correct interpretation of the tubular structure observed earlier (Sjostrand and Rhodin, 1953a, b) in the apical part of the tubule cells.

3. Intracellular Folds

of

the Cell Membrane

These were first observed in the tubule cells of the kidney (Sjostrand and Rhodin, 1953a, b ; Rhodin, 1954) forming more or less extensive compartments in which the mitochondria were lined up (Fig. 10). The thickness of these folds, 250 A, corresponds to the thickness of two cell membranes in close contact. In these cells, therefore, these folds do not seem to mean an increase of the free surface area between the cytoplasm and the outer surrounding milieu. This milieu is, furthermore, separated off by the basement membrane which covers the surface of the cell from which these folds extend. I t seems reasonable to assume that the functional significance of these folds is to increase the amount of active cell membrane and to extend the functional potentialities of this membrane to the interior of the cell body. A similar but more elaborate arrangement of the cell membrane has been observed in the ciliary epithelium of the eye (Holmberg, 1955). In these two types of cells, the tubule cells of the kidney and the ciliary cells, there is considerable active transport of ions and water. It might be that the folding of the plasma membrane represents an adaptation to this transport. There is a certain difference between the plasma membrane folds in these two cases. I n the tubule cells of the kidney the spacing between the two osmiophilic layers in a fold is very constant, indicating a close contact between the surfaces of a fold. In the ciliary epithelium, on the other hand, this spacing is more variable perhaps depending on a capillary space filled with liquid separating the two plasma membrane sur-

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483

FIG.10. p-cytomembranes representing folds of the plasma membrane extending into the cytoplasm of a proximal convoluted tubule cell of the mouse kidney. Compare the morphology of these membranes and the a-cytomembranes in Fig. 7 and the y-cytomembranes in Figs. 5 and 6. Magnification: X 46,000. (Rhodin, 1954.)

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faces of the fold. If such a space exists it is separated from the aqueous humor in the posterior chamber by a basement membrane.

4. Specialized Regions of the Cell Membrane These occur in several cases as for instance at the base of the inner segments of the receptor cells in the retina of the eye. These cells are at this level surrounded by the most peripheral parts of Muller’s cells. A close contact exists between the receptor cells and the Muller’s cells from this region all the way down to the synaptic body of the rods and cones. At the base of the inner segments a zone of the cytoplasm adjacent to the cell membrane is very opaque owing to the osmiophilia and the density of the cytoplasm (Sjostrand, 1953i). A narrow less opaque space separates the osmiophilic layers of the two cell membranes. No connecting structures are seen extending between the cells. They seem to be packed without any noticeable interstitial space between them. This zone corresponds to the external limiting membrane, which is a misleading term as no special membrane exists. When staining for light microscopy the regions of dense cytoplasm are easily stained and may give the impression of a perforated membrane. This region of the cell membrane may represent a spot of especially firm contact between the cells. The cytoplasm is here considerably denser than the rest of the cytoplasm and the whole arrangement might constitute a rigid ring structure, which stabilizes the cells mechanically as an intracellular skeleton. Similar arrangements have been observed in the excretory cells of the pancreas (Sjostrand and Hanzon, 1954a), in the tubule cells of the kidney (Rhodin, 1954), in the liver cells (Fawcett, 1955), and in the intestinal epithelium (Zetterqvist, 1956). I n the latter case the osmiophilic layer of the cell membrane in this region is divided into three layers, two osmiophilic layers separated by a less osmiophilic interspace. That the osmiophilic layer of the cell membrane is split into two osmiophilic layers may perhaps indicate that this is a region of the cell membrane with a specialized function other than a purely mechanical one. These regions correspond to the “terminal bars” described in light microscopy. It frequently occurs that the cell membranes of two adjacent epithelial cells are folded with the folds projecting more or less far into the adjacent cell (Fawcett, 1955 ; Sjostrand and Hanzon, 1954a). These folds increase the surface area of contact and may increase the firmness of contact between cells.

ULTRASTRUCTURE OF CELLS

485

5. The Intercalated Discs of Heart Muscle These have been studied by van Breemen (1953) at a too low resolution to clarify the structural organization. High-resolution electron microscopy has revealed that the intercalated discs represent cell boundaries (Sjostrand and Anderson, 1954). At these boundaries the cell membrane is in contact with a very opaque, osmiophilic region of the cytoplasm. The myofibrils do not pass across the boundary. A bright zone 150-200 A wide extends in between the osmiophilic layers of the two plasma membranes which are facing each other. This boundary frequently shows a wavy course which may explain the rodlike structure observed in the light microscope. According to Bourne ( 1953) histochemical studies would indicate high metabolic activity in this region. H e assumed that the discs might act as boosters of the contraction wave spreading through the cardiac tissue. The electron microscopic observations should be of interest to the physiologists.

6. The Free Surface of Ciliated Cells These have been carefully studied by Fawcett and Porter (1954) in molluscs, amphibia, mouse, and man. The internal structure of cilia was found similar in all the animals studied. The cylindrical processes are bounded by the cell membrane and contain nine double filaments arranged in a ring around the periphery of the cilium and at a distance of 5 0 0 A from a pair of single filaments placed in the center of the cilium. The diameter of the filaments was found to be 300 A and they showed a strikingly regular spacing. Between the peripheral filaments the spacing was found to be 5W A from center to center. A more or less well developed basal corpuscle is associated with the cilium. The structure of this corpuscle is different in various species. It consists of “a dense osmiophilic shell surrounding a protoplasmic core of lesser density” and shows an asymmetrical form with the asymmetry related to the direction of the ciliary beat. I n molluscs and amphibians cross-striated rootlets extend from the basal body a varying distance through the cytoplasm. The periodicity of these rootlets was estimated at 550-700 A. In a high-resolution study of the cilia of the respiratory epithelium of the rat trachea (Rhodin and Dalhamn, 1954, 1956; Dalhamn, 1956) the principal observations of Fawcett and Porter have been confirmed and more detailed data added. The peripheral filaments are described as 300 A thick in a chordal direction and 180 A thick in a radial direction. A 50 .&-thickosmiophilic surface layer surrounds a less opaque center. Across

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this center a radially oriented, 50 A-thick osmiophilic septum is seen which makes the filaments appear as double (Fig. 11) . The dimensions indicate, however, that we might prefer to consider these filaments as single units of a fairly complicated structure rather than to describe them as pairs of filaments in close contact. The basal body is described as consisting of the basal ends of the peripheral filaments. Below the cell surface the peripheral filaments become thicker and divided by several intrafilamentous septa. I n this way a continuous ring structure is formed which surrounds a less opaque center, in which sometimes a spherical osmiophilic granule may be observed. Further down below the cell surface the thickened filaments coalesce to form a body which in cross sections appears as a reticulated structure owing to the dense packing of the osmiophilic surface layers and septa of the filaments (Figs. 12, 13). The central filaments divide at their ends just below the cell surface and are here in mutual contact. In a study of the effect’of certain poisonous gases, e.g., SiOz, on the ciliary apparatus Dalhamn (1956) could observe no structural change in the cilia. A very marked reduction of the speed with which the mucus was transported was recorded under these conditions. This effect could, however, be explained by the changed topography of the surface of the ciliated epithelium. This surface, which is normally smooth, showed extensive crypts and irregularities in the animals that had been exposed to poisonous gases. This would result in the beat of the cilia becoming inefficient. The frequency and the speed of the various components of the ciliary beat were quite normal after exposure to poisonous gases which is in agreement with the fact the structure of the cilia is unaffected. For further .information regarding the very careful technique for recording ciliary activity the reader is referred to the monograph of Dalhamn (1956). A functional interpretation of the structures observed in the cilia seems difficult to present. The complex structure of the basal body and the characteristic aggregation of mitochondria in this cell region might well indicate that it has an important functional significance. Studies of the cilia at different stages of the beat cycle might help in unveiling the mechanism of ciliary motion. Such stages will probably need to be fixed by freezedrying. VIII. THEBASEMENT MEMBRANE Electron microscopy of the basement membrane has not contributed any conclusive data regarding its structural organization. The occurrence of a true basement membrane may, however, be easily demonstrated through electron microscopy as in the case of the epidermis (Ottoson et al., 1953).

ULTRASTRUCTURE OF CELLS

487

FIG. 11. Cross section through cilia of the ciliated epithelium of the rat trachea. Magnification: X 130,000. (Rhodin and Dalhamn, 1956.)

488

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FIG.12. Cross section through cilia of the ciliated epithelium of the rat trachea. These cross sections pass through the basal part of the free segment of the cilia and through the basal bodies at various levels. Magnification: X 96,000. (Rhodin and Dalhamn, 1956.)

ULTRASTRUCTURE OF CELLS

489

FIG.13. Longitudinal section through the basal part of two cilia of the ciliated epithelium of the rat trachea. Magnification : X 68,000. (Rhodin and Dalhamn, 1956.)

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It appears as a continuous easily observed membrane, the outlines of which do not appear sharply defined. In most cases it is quite homogeneous even at a high resolution. This might well be due to a lack of contrast of the structural components involved. An indication of an irregular fibrillar structure oriented parallel to the surface has been observed by Rhodin (1955). Special techniques for electron staining are needed for a detailed study. Between the basement membrane and the adjacent cell membrane there is a certain space the width of which varies considerably ; it is sometimes as wide as 200-300 A (Ottoson et aZ., 1953). The thickness of the basement membrane also varies from a minimum of 150 A measured in the exocrine part of the pancreas (SjGstrand and Hanzon, 1954a), to 200-300 A in the epidermis of the frog (Ottoson et al., 1953), 500-1O00 A in the tubule cells of the kidney (Sjostrand and Rhodin, 195313; Rhodin, 1954), and 650 A in the glomerulus (Rhodin, 1955). I t might be that the less opaque interspace between the osmiophilic layer of the cell membrane and the basement membrane corresponds to a layer of less osmiophilic material, for instance, oriented lipid molecules. The existence of such interspaces is especially obvious in the capillary loaps of the glomeruli of the kidney (Rhodin, 1955). IX. THEGROUNDSUBSTANCE OF THE CYTOPLASM 1. Cytoplasmic Membranes The term, ground substance, refers to that part of the cytoplasm which is not organized as mitochondria, Golgi apparatus, or specialized cell inclusions (e.g., zymogen granules) or structures such as myofibrils. The light microscopists have already revealed certain regions in the ground substance which exhibit special staining properties or structural characteristics. Basophilic regions have been described corresponding to regions of high RNA concentration according to the studies of Caspersson and collaborators and of Brachet. In the exocrine pancreas cells, for instance, Heidenhain (1880) had observed a lamellar structure in the basal parts of the cells. These lamellar structures have been called "Basallamellen." The term ergastoplasm was introduced by Garnier ( 1899). The electron microscope has revealed a very varied organization of the ground substance when comparing various types of cells and different regions within the cytoplasm of a cell (Sjostrand, 1954b). A very striking component of the ground substance is a system of membranes, which are associated with small opaque particles. This structure has been studied at various levels of resolution. The lowresolution studies of Hillier ( 1950), Dalton et al. ( 1950), Bernhard et al.

ULTRASTRUCTURE OF CELLS

491

(1951, 1952), Dalton (1951), and Palade and Porter (1952) resulted in incorrect descriptions of it. Palade and Porter (1952) described a system which “appears with the same features as in cultured units and shows similar morphological variations ranging from isolated vesicles to complicated networks of canaliculi. The diameters of its elements vary from 200 mp (vesicles) to 70 my. (canaliculi). These elements have a dense membrane 8 mp in thickness and a less dense content.” The canalicular elements were considered particularly numerous and definitely predominant in glandular cells as the pancreas exocrine cells. At the Electron Microscope Society of America Meeting in 1952 a different description was presented (Sjostrand, 1953f, g ) for this cytoplasmic component in the exocrine cells of the pancreas. According to this study the ground substance of the cytoplasm consisted of membranes which extended over large distances and were arranged in pairs (“double membranes”). They were oriented approximately concentrically around the nucleus. This system of membranes had also been studied in frozen-dried material and an optical polarization analysis of living pancreas tissue examined in situ had shown a birefringence of the cytoplasm. The analysis of this birefringence showed that its properties were in harmony with the assumed existence of membranes arranged parallel to the cell surface. These results made it probable that the membrane structures observed in the electron micrographs were present in the living cells. This description was so different from that presented at that time by Palade and Porter for the mainly canalicular “endoplasmic reticulum” of cells in tissue cultures that it was not justifiable to homologize them. Furthermore, it was pointed out that the cytoplasmic membranes observed by Sjostrand and Rhodin (1953a, b) in the tubular cells of the kidney and described at the same meeting were morphologically so different from the membranes of the pancreas that there was no justification in considering these membranes as homologous structures as Palade and Porter (1952) had done. The first detailed high-resolution study of this type of membrane was performed on exocrine pancreas cells (Sjostrand, 19533 ; Sjostrand and Hanzon, 1954a). According to this study the cytoplasmic membranes consist of an approximately 40 A-thick basic membrane on one side of which opaque particles are attached (Fig. 7). These particles are angularly shaped and are of a rather uniform size the largest diameter measuring 150 A and the smallest diameter 130 A (these diameters were measured at two directions oriented mutually perpendicularly). The membranes are arranged in pairs with the two membranes of a pair fused along their rims. They therefore,

492

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bound a narrow space filled with homogeneous or finely granular material which in most cases exhibits a low opacity. The opaque particles are attached to the outer surface of the membranes, the surface facing the closed narrow space between the membranes being smooth. The width of this space, at right angles to the membranes, may vary considerably with the type of cell. I t is very narrow in the exocrine pancreas cells, 70-700 A (Sjostrand, 1953f, g, Sjostrand and Hanzon, 1954a), and very wide, for instance, in the thyroid epithelium (Ekholm and Sjostrand, unpublished). These membranes may be very large, e.g., in the exocrine pancreas cells, where only a few narrow passages pass through the membrane pairs. Nothing is known about the material present in the narrow space bounded by the membrane pairs. It might be that this space represents an organized layer of, for instance, lipid molecules or some other material that has been extracted during fixation and embedding. In such a case each membrane pair should be considered as a layered membrane structure or as a layered lamella. Another possibility is that the two membranes of a pair bound a liquid milieu and that they separate this milieu from the surrounding cytoplasm. The cytoplasm would then consist of two different milieus separated by a membrane. This interppetation has been presented by Porter (1953). The classification of the cytoplasmic membrane structures as flat “sacs” (Weiss, 1953 ; Robertson, 1954) reflects an interpretation similar to the second alternative. For the moment we cannot give a definite interpretation. The swelling of the spaces bounded by the cytoplasmic membranes that may occur post mortem may distort the picture considerably and shows that we are dealing with a very labile structure. I n frozen-dried material (Fig. 14) the spacing between the two opaque layers of a membrane pair is far more uniform than after osmium fixation. The space in between the membrane pairs appears as continuous all through the cytoplasm. It exists in zones about 150-1000 A wide in between the membrane pairs and these zones communicate by means of narrow passages through the membrane pairs. In the exocrine pancreas cells this system of membranes fills the whole ground substance of the cytoplasm. Mitochondria, Golgi apparatus, and zymogen granules are located in the continuous space in which the membrane pairs are embedded. This space is also in direct contact with the cell membrane. The nuclear membrane, on the other hand, is associated with a single membrane of the same type as the membranes of the cytoplasm. The opaque particles of this membrane are attached to the peripheral surface of the membrane. The nucleus is therefore screened off from the continuous space of the cytoplasm.

ULTRASTRUCTURE OF CELLS

493

I n other types of cells the number of such membrane pairs is much less and they appear separated by large spaces. They may be concentrated in a certain region of the cytoplasm as for instance in the liver cells (Bernhard et al., 1952; Fawcett, 1954, 1955) and the nerve cells (Palay and Palade, 1953, 1955; Palay, 1954), where these regions correspond to the Nissl substance. In many types of cells they are completely absent or are observed rarely as, for instance, in the receptor cells of the eye, the tubular

FIG.14. a-cytomembranes in an exocrine cell of the mouse pancreas, fixed through freeze-drying and embedded without any electron staining in methacrylate. Notice the rather constant spacing of the membrane pairs. Magnification: X 19,000. (Sjostrand, 1953h.)

cells of the kidney, skeletal muscle and heart muscle tissue. They are therefore not a constant component of cells as are, for instance, mitochondria and the Golgi apparatus. As to the functional significance of these cytoplasmic membranes their extensive occurrence in the exocrine pancreas cells might well indicate that they are of importance for the synthesis of the secretory products of these cells. The membranes may divide the cytoplasm into two separate systems of compartments or intracellular milieus. At least ont

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of these milieus is continuous. The narrow compartments bounded by the membranes which are embedded in this milieu might well communicate all through the cell but these relations have not been studied in detail. However, some observations by Rhodin (unpublished) on serial sections through plasma cells would indicate that this might be the case in that particular type of cell. Membranes certainly seem to represent a useful mechanism of organization for enzymatically controlled synthetic activities. That the regions of the cytoplasm where this type of membrane occurs correspond, in many cases, to basophilic areas was assumed by Dalton et al. (1950) and by Palade and Porter (1952) and was demonstrated by Bernhard ot al. (1952) who recognized this structure as the ergastoplasm. Basophilia is, however, not always associated with this type of organization of the cytoplasm (see further discussion on page 497). It seems, however, quite obvious that this type of membrane represents the structural background for the structures described by Heidenhain (1880) as filaments and by Zimmermann (1927) as “Basallamellen”. What was observed must have been rather crude aggregations of the partially destroyed membranes. The electron microscope has made it possible to analyze and describe the geometry of the elementary components of a structure already known in light microscopy, and to demonstrate its very widespread Occurrence and its frequently very exhaustive nature. The knowledge of the ultrastructural organization of these cytoplasmic regions will certainly become the starting point for a more detailed analysis of its biochemical and physiological significance, which no doubt represents a very fundamental problem in cell physiology.

2. The Endophmic Rebic.ulum In American publications the term “endoplasmic reticulum” has been introduced by Porter and Kallmann ( 1952). This term originally referred to a structure, presumably vesicular and tubular, which was observed in whole cells in tissue cultures. The low resolution of the pictures that could be obtained with that kind of specimen did not allow any detailed structural characterization of these vesicles. The early electron microscopic studies of tissue sections revealed a structure in the cytoplasm which could not be analyzed in detail owing to the low resolution that could be obtained with the technique of that time. This structure was therefore misinterpreted as representing fibrils but was identified as the ergastoplasm described in light microscopy (Bernhard et al., 1951, 1952). In their first studies using thin sectioning Palade and Porter (1952) reported the occurrence of tubules and vesicles in the cytoplasm of various

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495

types of cells and they claimed that these structures represented the “endoplasmic reticulum” already observed in tissue culture cells. The tubular appearance they described, for instance, in the exocrine cells of the pancreas was certainly partly due to a fragmentation of the membranes which take place post mortem (Sjostrand and Hanzon, 1954a). At the low resolution used in Palade and Porter’s first work it was impossible to differentiate membranous or vesicular structures of different kinds and therefore everything occurring in the cytoplasm that appeared as tubules or vesicles was described as the “endoplasmic reticulum.” The very frequent double-lined pattern was interpreted as representing a longitudinal section through a tubule. So they described the “endoplasmic reticulum” of the tubular cells of the kidney, and in that case they included membranes that were demonstrated by Sjostrand and Rhodin (1953a, b) and by Rhodin (1954) to be folds of the cell membrane. Palay and Palade (1955) have described the membrane structures in the Golgi apparatus as the “agranular reticulum” which they consider related to the “endoplasmic reticulum” as “most of these distinguishing characteristics are only quantitative in nature.” Recently Palade ( 195513) has claimed the formation of “endoplasmic reticulum” components from folds of the cell membrane. The tubular structures observed in muscle fibers (see Section XII, 4) have also been included in the “endoplasmic reticulum” (Bennett and Porter, 1953; Bennett, 1955). With improved technique Palade (1953b, 1955a), and Palade and Porter (1954) have observed the granular component of the cytoplasmic membranes, and were therefore able to differentiate between granular and agranular components. The vesicular and tubular structures observed in whole cells in tissue cultures then appear bounded by a granulated membrane. This means that the endoplasmic reticulum as described in tissue cultured cells seem to correspond to the “Basallamellen” in for instance the pancreas and salivary gland cells as described in light microscopy. Fawcett (1955) seems to use the term “endoplasmic reticulum” in a more strict sense. According to him this term refers to the granulated membranes, which seems quite reasonable. In a recent paper (Burgos and Fawcett, 1955), however, he includes membranes lacking any attached opaque particles as components of the “endoplasmic reticulum.” This mixing of various structural elements under one and the same term “endoplasmic reticulum” would be justified if it had been demonstrated that we are dealing with homologous components from the point of view of function or cytogenesis, or if we were not able to differentiate between them structurally. It would also be more logical to stick to any term already existing in light microscopy as for instance “ergastoplasm.” How-

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ever, it seems justifiable to create a new terminology when dealing with ultrastructural elements if analyzed on an ultrastructural level. Such a terminology should be based on the structural characteristics that have been observed. Furthermore, if this analysis has clearly demonstrated structural elements of rather different morphological characteristics these elements should be given individual names. These names have to be based on morphology as long as we lack any knowledge regarding the functional significance of the elements. And as long as we know nothing about the homology, structural differences should be considered good reasons for differentiating the structures through different names. The term “endoplasmic reticulum” seems unsuitable as the structures it refers to is not reticular and there is little reason to talk about endoplasm in tissue cells. The membrane structures observed in the cytoplasm of various types of cells were called intracellular cytoplasmic membranes ( Sjostrand, 1953f, g) which name referred to membranes as a unit of structural organization of the cytoplasm. At that time it was already obvious that these cytoplasmic membranes were of different types. For them a shorter term has been proposed (Sjostrand, 1955a, 1956a) namely cytomembranes with the differentiation of the various types of membranes through letters of the Greek alphabet. The membranes of the type observed] for instance, in the exocrine pancreas cells will be called a-cytomembranes and they will be defined as membranes measuring about 40 A in thickness, arranged in pairs or as single membranes associated with the nuclear membrane, and with angularly shaped particles measuring 150 A in diameter attached to the outer surface of the former membranes. The size and form of the spaces bounded by these membranes may vary considerably in different types of cells. The folds of the cell membrane which extend more or less far into the cytoplasm will be called p-cytomembranes. The membrane pairs observed in the Golgi apparatus and also in some structures not recognized earlier as Golgi apparatus will be called y-cytomembranes. They are defined as 60 A thick membranes arranged in pairs with the membranes of a pair fused at their rims and with 2 to 6 pairs arranged in parallel. Frequently vacuolar spaces separate the membranes of a pair. This way of describing the observed structures would be objective and as free from preconceived ideas as possible. It seems quite reasonable to assume that the very different structural appearance of these three kinds of membranes corresponds to different functional potentialities.

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3. Vesicles These have been described as constituents of the cytoplasm (Sjostrand, 1953i; Zetterqvist, 1956). They may be defined as rounded or spherical components bounded by a 50 A-thick membrane and appearing to be empty in the fixed and embedded specimens. The term “vesicles” refers to their appearance in such specimens and does not exclude the fact that they may represent granules that have been dissolved during fixation and embedding. In the tubule cells of the kidney such an extraction of material resulting in the formation of vesicles has been demonstrated (Sjostrand, 1944; Sjostrand and Rhodin, 1953b). Vesicles have been suggested by Palade ( 1 9 5 3 ~ )to represent a transportation mechanism for water through endothelial cells. The surface of these cells is very irregular and shows deep crypts. The possibility has to be excluded that the vesicles observed are not such crypts. If there are vesicles that move, their movements have to be demonstrated through experiments, showing different stages in the process of transportation. The idea of transportation through movements of vesicles seems quite plausible but it needs to be proved.

4. Opaque Particles Opaque particles were described by Sjostrand (19539 and Sjostrand and Rhodin (1953b) as occurring in groups in the cytoplasm. The diameter of the smallest particles was found to be 50 A and the diameter of the smallest groups about 100 A. These groups showed angular outlines. The general Occurrence of similar opaque particles in a variety of cell types was pointed out by Palade (1953b, 1955a) who described them as spherical particles measuring 80-300 A in diameter. According to careful measurements of the diameter of such particles in the intestinal epithelium of the mouse their diameter is 150 A (Zetterqvist, 1956). It may be questioned whether all these particles despite their variation in size are of identical chemical composition. They are similar to the particles observed as components of the a-cytomembranes and according to Palade all such particles are identical.

5. The Basophilic Component of the Cytoplasw This has as yet not been identified in electron microscopy with any degree of certainty. Almost every structural component of the ground substance that has been observed has been interpreted as being responsible for the basophilia. When Porter and Thompson (1947) observed the socalled “growth granules” they interpreted them as the basophilic component. When the “fibrils” of the ergastoplasm and the vesicles and

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canaliculi of the “endoplasmic reticulum” were observed this component was made responsible (Bernhard et a/., 1951, 1952; Porter, 1953) and when it was obvious that the strongly basophilic regions, for instance, of embryonic cells did not contain many vesicles but did contain a great number of opaque particles, the interest was directed toward these particles as representing the basophilic component (Palade, 1955a). In a study using differential centrifugation combined with electron microscopic control Palade and Siekevitz (1955) claimed that the opaque particles occurred in the RNA-containing microsome fraction. This result has not been confirmed by Kuff, Hogeboom, and Dalton (1956) in a study with a very efficient technique for separating the various microsome fractions. The least convincing evidence for a correlation of basophilia with the cytoplasmic membranes and the opaque particles that has been presented is that of Porter (1954) who demonstrated that the basophilic regions of the pancreas cells varied with the amount of zymogen granules occupying the apical zone of the cell body. Such evidence shows only that the zymogen granules certainly cannot be made responsible for the basophilia. For the moment it seems reasonable to assume that basophilic regions of the cytoplasm may show various degrees of ultrastructural organization. Frequently a-cytomembranes are present in these regions and frequently only free opaque particles are present. None of these components have been demonstrated without doubt to be responsible for the basophilia. At this point it seems justifiable to point out that the pictures of osmiumimpregnated cytoplasm may not show all components present and that we are not in a position to present a complete description of the ultrastructural organization of the cells.

The Synaptic Granules Granules measuring 200 A in diameter and characterized by an osniiophilic peripheral region surrounding a less osmiophilic center are found associated with synapses (Figs. 15 16). They represent the most striking component of the cytoplasm of a variety of synapses studied (Sjostrand, 1953k, 1954c, d ; Palade, 1954; De Robertis and Bennett, 1954). They are sometimes referred to as vesicles (De Robertis and Bennett, 1954). The osmiophilic periphery, however, does not appear as a marked membrane structure. The opacity decreases gradually from the periphery toward the center. In some cases, as for instance the receptor cell synapses of the perch eye the particles are osmiophilic in their entirety. These granules have been observed in the receptor cell synapses of the eye (Sjostrand, 1953k, 1954c), in the synapses of the cochlea (Engstrom and Sjostrand, 1954), in the synapses of the sensory epithelium of ampulla 6.

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cristae and maculae (Wersall, 1956), in the interneuronal synapses of the central nervous system (De Robertis and Bennett, 1954). It may be that these granules represent a characteristic component of the synapse cytoplasm. Their functional significance is unknown.

7. 0smiop hilic Granules Various kinds of osmiophilic particles have been described in the cytoplasm. In the tubular cells of the mouse kidney large, strongly osmiophilic granules appear in the intermediate cell zone (Sjostrand and Rhodin, 1953a, b) . These granules sometimes show a layered structure (Rhodin, 1954). Rhodin and Dalhamn (1954) observed osmiophilic granules with a regular crystalline packing of smaller units in a new type of cell, the socalled brush cells, in the tracheal epithelium of the mouse. Weiss (1955) described the occurrence of granules in the exocrine pancreas cells and in tubular cells of the kidney after injections of neutral red. H e claimed that they had been formed from mitochondria. A layered structure in some of the observed granules was interpreted as evidence in favor of such an interpretation. This structure is, however, a frequent occurrence in granules in normal tubular cells and no relationship between these granules and the mitochondria has as yet been demonstrated. The morphology is so different in these granules and in the mitochondria that such a relationship is morphologically improbable. A very specific type of granule has been observed in graniilocytes (Sheldon and Zetterqvist, 1955). These granules consist of ovoid bodies containing a strongly osmiophilic structure in the center. This structure was shown to consist of densely packed lamellae.

8. Fibrillar Structures These may be observed in the ground substance as for instance in the tubular cells of the kidney (Sjostrand and Rhodin, 1953b; Rhodin, 1954) and in the ciliary epithelium of the eye (Holmberg, 1955).

X. THE NUCLEUS 1. The Nuclear Mevwbrane The double character of the nuclear membrane was demonstrated by Callan and Tomlin (1950) with fragmentation technique and by Hartmann (1953) on thin sections. The dimensions of the membrane were measured at high resolution to 230 hi for the total thickness 60-70 d for the two osmiophilic layers and 100 A for the less osmiophilic interspace (Sjostrand and Rhodin, 1953b).

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FIG.15. Synaptic bodies of retinal rods in the guinea pig eye. In the upper part of the picture are two small rod spherules and below them a large conically shaped rod synapse. The rod cells extend to the left side. Along the right side of the picture some branches of the dendrites from the bipolar nerve cells are seen. Notice the characteristic appearance of the cytoplasm of the synapses. Magnification : X 25,000. (Sjostrand, 1953k, 1954c.)

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FIG.16. Higher magnification of parts of two adjacent synaptic bodies of retinal rods in the guinea pig eye. The four lines running roughly parallel through the picture represent the plasma membranes of the rod synapses and of the interposed Muller’s cell. The triple-layered structure formed through the combination of rod cell and Muller’s cell plasma membranes is characteristic for all presumably conducting structures of the retinal rods and the dendrites of the retinal neurones. Magnification : X 110,000. (Sjostrand, 1953k, 1954c.)

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In the exocrine pancreas cells the nuclear membrane appears as a single osmiophilic layer but a single a-cytomembrane is associated with this nuclear membrane (Sjostrand and Hanzon, 1954a). Fawcett (1955) has published some figures for the nuclear membrane of liver cells. This nuclear membrane consists of two membranes, about 90 A thick, pursuing a parallel course 100-120 A apart. Fawcett writes that “it is not entirely clear whether both membranes should be considered part of the karyotheca or whether only the inner layer is to be identified with the nucleus and the outer one regarded as the limiting membrane of the cytosome.” From a functional point of view it seems justifiable to consider the different layers of the nuclear membrane as components of one structural element, This seems even more so when considering the possibility that there might exist a continuous layer of oriented molecules in between the two osmiophilic layers observed in the electron micrograph and that the apparent individuality of these two layers may result from an artificial swelling of the intermediate layer during fixation and embedding. The existence of pores in the nuclear membrane was demonstrated by Callan and Tomlin, who located the pores in the outer sheath of the nuclear membrane. The inner sheath, on the other hand, was continuous. In thin sections through the nuclear membrane of the sea urchin oocyte, Afzelius ( 1955) observed a rather complicated structure. In tangential sections a characteristic ring structure is seen. At the edges of the less opaque central region of these rings the two opaque layers of the nuclear membrane are fused and a single layer extends across the inner diameter of the rings. The pronounced opacity of the rings is due to diffusely outlined electron-scattering material which forms an annular structure protruding 600 A toward the interior of the nucleus and 150-250 A toward the surrounding cytoplasm and arranged around the edge of the singlelayered zone of the nuclear membrane. The number of “holes” per square micron was estimated at 40-80. The total thickness of the double-layered nuclear membrane was 300400 A and the thickness of the constituent osmiophilic layers was 90 A each. Kautz and de Marsh (1955) also found evidence that no holes exist in the nuclear membrane of chick embryo cells. This conclusion was based on photometer tracings along the nuclear membrane clearly showing high opacity across the “holes.” In a paper by Dawson et al. (1955) the characteristic ring structure observed in tangential sections through the nuclear membrane of spinal ganglion cells has been interpreted as representing holes in spite of the fact that these regions appear elevated in sections shadowed after extraction of the methacrylate. This fact the authors explain as caused by

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material passing through the “holes” so that they become filled. With such assumptions it is possible to satisfy a desire to see holes. Their pictures of cross sections through the nuclear membrane are not clear enough to demonstrate the detailed structure. A ring structure was observed by Rhodin (1954) in the nuclear membrane of mammalian cells. Some papers have been published by Watson (1954, 1955b) in which the existence of holes in the nuclear membrane of various types of rat cells is claimed. The evidence in favor of real holes is, however, not convincing. The irregular spacing of the nuclear membrane layers in Watson’s material has led him to assume that the nuclear membrane is a sac or flattened vesicle comparable to the “vesicles” occurring in the cytoplasm. The observations made on the structure of the nuclear membrane point to a complicated structural organization of this membrane, presumably corresponding to regions of different permeability properties. It is also obvious that the structure of the nuclear membrane varies considerably in different types of cells. The existence of holes cannot be considered to have been convincingly demonstrated.

2. The Nucleoplasm This does not show up as a highly organized component in the electron micrographs. It appears in the exocrine pancreas cells to consist of rather irregularly distributed anisodiametric particles with an average diameter of 170 A. The maximum diameter measures 190 A and the minimum diameter 150 A (Sjostrand and Hanzon, 1954a). In the tubular cells of the mouse kidney the nuclear particles measure 250 A in length and 150 A in width (Rhodin, 1954). Fawett (1955) gives the figure 150-200 A for the granular material of the liver cell nucleoplasm. A coiled or lamellar structure of the nucleolus has been reported by Porter (1954) and Bernhard, et al. (1955). The poor structural pattern of the nuclei might well be the result of improper preservation and it seems too early to discuss further the structure of the nucleus on the basis of its appearance in the electron microscope.

XI. LIPOPROTEIN STRUCTURES 1. General Lipoprotein structures of the myelin sheath of the myelinated nerve fibers, the outer segments of the rods and cones of the retina, and the grana of the chloroplasts have been studied at high resolution. Since it is possible to compare these results with those of in Vivo optical polariza-

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tion analysis these structures might be the subject of a discussion on the preformation or otherwise of objects observed with the electron microscope. 2. The Myelin Sheath The myelin sheath (Fig. 17) shows a very regularly layered structure with a periodicity of 120 A in the sciatic nerve of the mouse (Sjostrand, 1953c, d) . The opaque, osmiophilic layers measure only 25 A in thickness. In between these layers a faint interperiod is seen located at the center of the interspace (Sjostrand, 1953c, d, 1 9 5 4 ~ ) .This structural pattern varies considerably with focusing. It is therefore important to analyze close-tofocus pictures (Sjostrand, 1953c, d, 1 9 5 4 ~ ) .Fernhdez-Morin, who first observed indications of this layered structure (1950, 1952), has recently (1954) presented excellent material on this problem. The interpretation of the observed pattern is difficult as long as we do not know the chemical composition of opaque and less opaque regions in the electron micrographs. The pattern reflects the concentric layering of protein and lipid molecules and it may tentatively be assumed that the opaque layers correspond to the protein component. The formation of the myelin sheath during embryological development has been studied by Geren (1954). She has shown a spiral structure of the myelin sheath at the early stages of development. The spiral appears as an invagination of the cell membrane of the Schwann cells. Her observations have been confirmed by Robertson (1955) studying adult nerve fibers.

3. Retinal Rods The outer segments of retinal rods consist of a pile of rounded discs with a diameter corresponding to the diameter of the outer segment (Fig. 18). In each disc two membranes about 30 A thick, which are fused along their edges, enclose a space 70-80 A wide (Sjostrand, 1953a, b). In studies of fragmented outer segments (Sjostrand, 1949) these thin membranes could be isolated. This means that they are mechanically rather stable, which may indicate that they are composed of proteins. According to optical polarization data (Schmidt, 1928, 1934, 1937) the lipid molecules are oriented parallel to the long axis of the outer segment. Some indirect evidence points to their location within the narrow space bounded by the membranes of the double membrane discs. The structural pattern of the rod outer segments is somewhat similar to that of most mitochondria. This fact has assisted in the interpretation of the structural pattern of mitochondria ( Sjostrand, 1953h) described above. Two different interpretations of the functional significance of the disc structure have been proposed by Wald (1954) and by Ingelstam (1955).

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FIG.17. High-resolution pictures of myelin sheath of a 4-p thick nerve fiber in the sciatic nerve of mouse. A-D from a through-focus-series with A and B underfocused, C in focus, and D overfocused. The dependence of resolution on focussing is illustrated as well as the change of the image pattern with variations in focussing. E shows a slightly underfocused picture of another through-focus-series of the same nerve fiber and the identical region as shown in A-D. The layered structure of the myelin sheath appears as a regular periodicity with a tnain period and a fainter interperiod frequently accentuated by the existence of opaque dots. Magnifications : A-D X 160,000, E X 300,000. (Sjostrand, 1953d, 1954c.)

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FIG.18. Longitudinal section through the outer and inner segments of retinal rods of the guinea pig eye at the junction between outer and inner segments. The connecting structure extending between these two segments is seen as well as the layered structure of the outer segments and some mitochondria in the inner segments. Magnification : X 38,000. (Sjostrand, 1953a.)

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The latter author has treated the structure of cone and rod outer segments as representing electromagnetic wave receptor antennae using the spacings of the periodic structure for his calculations. Wald, on the other hand, has discussed the problem from a purely photochemical point of view. The structure of the outer segments of the cones differs somewhat from that of the rod outer segments. In the first study they were found to consist of a pile of single-layered discs (Sjostrand, 1953a, b) but with improved technique for preservation it has been possible to observe a similar double membrane structure of the individual discs as described for the rod outer segments (Sjostrand, unpublished). The spacings of the periodic structure of rod and cone outer segments have been confirmed by X-ray diffraction analysis of fixed material (Finean et al., 1953). This result is of interest in demonstrating the reliability of the figures obtained from measurements on electron micrographs in spite of the restricted number of observations that can be made with this technique. 4. Chloroplasts These have been studied in Aspidistra elatior at a high resolution by Steinmann and Sjostrand (1954, 1955; see also Sjostrand, 1954c, e, f ; Finean et aZ., 1953). They consist of membranes extending right across the chloroplast. The thickness and the regularity of the spacing of these membranes are different in different regions. In some parts the thickness of the membranes is 65 A and the spacing is very regular. These regions represent the grana and have the shape of irregularly formed columnae. In between these regions in the stroma the membranes measure only about 30 A in thickness. The parts of the membranes located within the grana are fused in pairs. This structure is very similar to that of retinal rods and cones. The organization in membrane pairs is also obvious from X-ray diffraction data (Finean et al., 1953). The periodicity as measured on the electron micrographs was 2S270 A and the repeating unit as calculated from X-ray diffraction patterns was 250 A. This is a further striking confirmation of data collected from electron micrographs. The difference in thickness between the stroma membranes and the grana membranes corresponds to the thickness of two layers of chlorophyll molecules. The arrangement of the chlorophyll molecules as monalayers has been proposed by Wolken and Schwertz (1953) studying chloroplasts of lower plants. A layered structure of the chloroplasts was observed by Wolken and Palade (1952), Leyon ( 1953a,b, 1954a, b) , Cohen and Bowler ( 1953),

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and by Sager and Palade (1954). In a recent study by Mercer et at. (1955) chloroplasts of Nitella have been studied in a swollen state. The published pictures of swollen chloroplasts may be interpreted as showing the chloroplast membranes arranged in pairs. Such an assumption would easily explain the transformation of the membrane into vesicles when swelling in hypotonic media. 5. The Osmium Stizining The studies of lipoprotein structures have shown a structural organization of great regularity. The interpretation of the patterns observed has to be considered as presumptive. However, the observations made, for instance, on the retinal rod outer segments and the chloroplast stroma certainly make it reasonable to assume that the opaque layers appearing in cross sections through the different layered structures represent the protein component. Another possibility would be that the opaque layers represent the boundary between lipids and proteins or a lipid layer bound to a protein layer. The staining properties, for instance of muscle myofibrils and of collagen, clearly demonstrate osmium tetroxide as a powerful electron stain for proteins. A combination of osmium staining and staining with phosphotungstic acid does not change the various patterns observed in any respect other than that of increasing the contrast by staining the same components as the osmium tetroxide does. Among the lipids there are a number of compounds that react only weakly with osmium tetroxide (Bahr, 1954) and which might well be extracted during the embedding procedure. Bahr (1955) has shown that a high percentage of the lipids are still soluble in alcohol after treatment with osmium tetroxide. The staining of proteins might well be responsible for the general blackening of tissues when fixed in this substance. The blackening does not help very much in differentiating structures in light microscopy. Here the heavy reduction of osmium tetroxide by certain lipids dominates the picture. But this general background blackening has been shown to be a very valuable contrast-enhancing factor in electron microscopy. A better understanding of the chemical background for the electron staining with osmium tetroxide is badly needed. The studies of simple systems as individual chemical components in solutions (Bahr, 1954) and of oriented components such as can be obtained with pure lipids (Geren and Schmitt, 1953) are of great importance. If, however, a periodic pattern can be obtained in the latter case, as has been demonstrated by Schmitt and Ceren, this does not exclude the possibility that organized structures of many different materials may give similar patterns. The lipoprotein structures are of special interest because their ultra-

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structural organization may be studied to some extent with optical polarization methods in the fresh, living state. It is of fundamental interest for a critical evaluation of the data collected in electron microscopy that all components that have been accessible for an optical polarization analysis have shown in electron microscope photographs a structural pattern that is in harmony with the optical polarization data. In fact the polarized light microscope should be the standard equipment of the electron microscopists. But it has to be used on living cells and it has to be equipped for analyzing very weak birefringence as in the microscope developed by InouC. XII. THESTRUCTURAL ORGANIZATION OF WHOLE CELLS In a few cases whole cells have been subjected to a complete ultrastructural analysis. As the topographic relationship between the different structural components is of some interest some examples will be described.

1. The Retinal Rods In the guinea pig eyes retinal rods have been studied in detail by Sjostrand (1953a, b, i, k, 19%). These cells are characterized by being organized in discrete segments with drastically different organization (see Fig. 19). The structure of the outer segments has already been briefly described. It may be added b t the pile of double membrane discs is enveloped in a cell membrane, which also covers most of the basal surface of the outer segment. Only at a rather narrow region is there a connection between the outer and the inner segments (Fig. 18). This connection consists of a thin stalk in which two concentrically arranged rings of fibrils are seen. The nine fibrils of the outer ring are in close contact with the cell membrane of this constricted part of the cell. The nine fibrils of the inner ring in this connecting structure penetrate into the apical part of the inner segment and end abruptly 0.4 p under the surface. They are firmly attached to a cross-striated, diffusely outlined, fibrous structure running through part of the inner segment. The connecting structure and the cross-striated fibers are to some extent similar to the cilia described by Fawcett and Porter (1954). They have, however, two sets of fibrils arranged in rings instead of one set in the cilia and they lack the two central fibrils present in cilia. I n the inner segment a dense aggregation of mitochondria is seen. In cases where a discrete ellipsoid is present, this consists of closely packed mitochondria. The rod fiber extending from the inner segment to the synaptic body shows a structural organization similar to that of an unmyelinated nerve fiber. In the latter case the cell membrane of the nerve cell and that of

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FIG.19. Schematic survey p i c b e of the retinal receptor cells of the guinea pig eye.

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the Schwann cell are in close contact and form a layered surface structure with two opaque osmiophilic layers separated by a less opaque interspace. The same structure is seen in the rod fiber owing to the combination of the cell membrane of the rod cell and that of Miiller’s cells. The rod synapse (Figs. 15, 16) is a highly specialized structure with the typical granular structure of synapse cytoplasm. Two extensions from the bipolar nerve cells penetrate into the synaptic body and are located in invaginations of the rod cell membrane. Inside the rode spherule these extensions make contact with some tubular structures of the rod cell. This structural organization has allowed a hypothetical interpretation of the functional significance of the various segments of the rod cell (Fig. 20). The dense aggregation of mitochondria in the inner segment might indicate that this segment is responsible for generating energy for the excitatory process. The outer segment is adapted as an antenna for picking up light energy. The minute amount of energy needed for stimulating the outer segment might, however, be considered insufficient to produce a full response of the receptor cell without a mechanism of amplification. This mechanism might be represented by the inner segment. The outer segment would then act by means of a kind of trigger mechanism on the inner segment. From the inner segment to the synaptic body the excitatory state might be spread through a mechanism of conduction similar to that of the unmyelinated nerve fibers. The large contact surface between the rod synapse and the extensions of the bipolar nerve cells might represent a structural adaptation for a direct electrotonic spreading of the excitation to the nerve cell. The absence of mitochondria in this region is striking. An analysis of the distribution of certain enzyme activities in the retina (Sjostrand and Gierer, unpublished) has shown that the enzymatic activity of the outer segments is low as compared to that of the inner segments as far as aldolase, cytochrome oxidase, cholinesterase, and ATPase activity is concerned. These results would point to the inner segment as the metabolically most active component of the two.

2. T ~ Tubular s Cells of the Kidney The tubular cells of the kidney were analyzed at a high resolution by Sjostrand and Rhodin (1953a, b) and by Rhodin (1954). In the cells of the proximal convoluted tubules three different zones may be distinguished. The apical zone is characterized by the brush border which represents a large surface area facing the tubular lumen. Below this zone there is an intermediate zone characterized by the

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tubular invaginations of the cell membrane and of granules containing a fluorescent material soluble in water (Sjostrand, 1944) and therefore dissolved during the osmium fixation. This granular component seems to have been overlooked by Pease (1955) who interpreted the persisting membranous wall of the granules as widened parts of the tubular invaginations. However, these structures are not continuous and the dimensions

FIG.20. Three-dimensional reconstruction of the synaptic body of the retinal rods of the guinea pig eye. The dendritic branch of the bipolar nerve cell which makes contact with the receptor cell penetrates into the rod spherule with two terminal processes. These processes are in contact with a tubular or vesicular structure inside the rod spherule. (Sjostrand, 1953k, 1954c.)

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of the bounding membranes of the fluorescent granules and of the invaginations are different. The large basal part of the cells contains a great number of mitochondria and is divided by folds of,the cell membrane at the basal cell surface, the fl-cytomembranes, into compartments. The amount and extension of these folds vary considerably. However, there is an intimate topographic relationship between the p-cytomembranes and the mitochondria. No a-cytomembranes are observed. Golgi membranes but no Golgi granules are seen along the sides of the cell nucleus. The basal surface of the cells is covered with a basement membrane. Figure 21 shows in a schematic way the structural organization of the proximal convoluted tubule cells.

FIG.21. Schematic drawing of a proximal convoluted tubule cell of the mouse kidney summarizing the observations made by Sjostrand and Rhodin (1953%b) and by Rhodin (1954).

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The description presented by Sjostrand and Rhodin and by Rhodin has been confirmed by Pease (1955) and by Bargmann et al. (1955). Pease added some observations on the distal convoluted tubule and the loop of Henle. No structural changes could be observed when varying the functional state within a physiological range (Rhodin, 1954). After injection of egg albumin according to the experimental conditions of Oliver ( 1945, 1948), Rhodin found that the mitochondria were extensively damaged with the formation of large granules. This effect is reversible and the formation of new mitochondria from the large granules can be followed with a restoration of the internal organization and of the outer surface membrane of the mitochondria. It was possible to follow the excretion of urine at various morphological stages and to correlate those changes with the resorptive activity of the tubular cells. The resorption was decreased when there was destruction of the internal organization of the mitochondria and reached a normal level when this organization was restored. Therefore, it seems reasonable to assume that the activity of the tubular cells depends on an intact ultrastructural organization of the mitochondria. No other ultrastructural changes could be observed within the cells under these experiments. 3. The Exocrine Cells of the M o w e Pancreas These have been studied at a high resolution (Sjostrand and Hanzon, 1954a, b, c) and the most characteristic structural feature is the great number of a-cytomembranes. They are distributed all over the cytoplasm except for the region of the Golgi apparatus (Fig. 22). The abundance of such membranes makes it justifiable to assume that they have an important functional significance in connection with the synthesis of the secretion products. According to the experiments by Weiss (1953) these membranes disappear during prolonged fasting and reappear after feeding proteins to the experimental animals. The experimental conditions in this case, were, however, so drastic that we cannot exclude the fact that there was severe cell damage and that the changes were not just a physiological adaptation. The same argument seems valid in connection with the study of comparable changes in the a-cytomembranes of liver cells as a result of fasting (Bernhard et d., 1952; Fawcett, 1955). Such experimental conditions are, however, useful when searching for a system to correlate basophilia and structural organization (Bernhard et ul., 1952). It seems very important when designing experiments for morphologic studies to have a certain guarantee that the effects are within the physiological range and are not pathological adaptations with risks of secondary

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FIG.22. Schematic survey picture of exocrine cells of the mouse pancreas showing the topographic relationships between a-cytomembranes, mitochondria, zymogen granules, Golgi apparatus, and nucleus. (Sjastrand and Hanzon, 1954%)

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ef€ects. Furthermore, it should be required that the activity of the cells be quantitatively measured to make possible a correlation of a particular change of structural organization with a known state of activity. Extensive trials have been made to produce a clear-cut morphological change in the exocrine pancreas cells by repeated injections of pilocarpine hydrochloride in doses varying to far above what can be considered a physiological range (Sjostrand and Hanzon, 1954a, c). The result was negative. The difficulties in judging the average picture of the cells were obviously because of the limited number of ceIls that can be analyzed in the electron microscope in a reasonable time and the asynchronous activity of the cells. The problem of obtaining a statistically representative sampling with the small fields of view of the electron microscope affects the fundamental designing of animal experiments in electron microscopy. We have to satisfy a fundamental requirement that the activity of the cells should be synchronized. 4. Cross-Striated Mamnzalim Muscle Fibers High-resolution studies have been made by Huxley (1953), Huxley and Hanson (1954), and Hanson and Huxley ( 1955), who observed two types of elementary filaments measuring 50 and 120 A in diameter. In the I bands only the thin filaments were present but in the A bands both types were mixed in a regular array. The thick filaments terminate at the A-I boundary. The two types of filaments were assumed to correspond to actin and myosin respectively. Recent studies (Sjostrand and Anderson, 1956) have revealed a more complicated structure. There are elementary filaments running all along the A and I bands but the diameter of these filaments varies from a rather thick (160 A) diameter within the H band to a somewhat smaller diameter (130 A) in the A band. Close to the A-I boundary the diameter gradually decreases to the small (50 A) diameter characteristic for the I-band filaments. Within the A bands the filaments are mutually connected through cross-bridges as described by Bennett and Porter (1953) and by Hodge (1954). In longitudinal sections at a high resolution the filaments show a very complicated form within the A bands. They appear folded or winded like a cork screw. The presumed helical structure has a pitch of 50-100 A. The cross bridges appear more as due to a direct mutual contact of these folded or helical filaments than as due to discrete bridges connecting well defined cylindrical filaments. Due to this irregular form of the filaments and the small pitch of the presumed helix the measurements of the diameter of the filaments within the A bands will be inexact. The thin filaments observed in the A bands by Huxley are no continuous

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FIG.23. Cross section through cross striated skeletal muscle fiber of mouse showing the thick myofilaments within the A band characterized by their irregularly angular shape and numerous cross connections. In the lower left corner the section passes through the I band with thin myofilaments, which are continuous with the thick A band filaments. Magnification: X 300,000. (Sjostrand and Anderson, in press.)

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filaments but appear as an artificial representation in cross sections of the cross-bridges. When comparing sarcomeres of different lengths the length of the I band as well as the A bands is reduced. As the reduction of the I band length does not result in any new structural pattern the I band pattern seems to have changed into an A band pattern. Therefore, the contraction seems to take place both in A and I bands. A simple coiling of the filaments is quite possibly an explanation for these changes. Further data with more precise measurements at improved specimen resolution are, however, indispensable for a more precise interpretation. The conclusions regarding the distribution of actin and myosin in the thin and thick filaments (Hanson and Huxley, 1953) have been criticized by Szent-Gyorgyi et al. (1955). In a paper that appeared during the preparing of this survey Hodge ( 1955) has presented some excellent data regarding the ultrastructural organization of the flight muscle of the blowfly. I n cross sections the elementary filaments in the A bands appear in these muscles as ring structures with a regularly arranged transverse connection between the filaments. These run continuously along the whole length of the sarcomere. In the A bands they consist of a less dense core about 4 0 A in diameter surrounded by a thin dense cortex, the over-all diameter of the myofilanient being about 100-120 A. The less dense core of the myofilament appears to be structurally continuous within the filaments of the H , I , and Z bands. Hodge assumes that the less dense core consists of F actin filaments. The myosin may be located in the A band or be uniformly distributed throughout the sarcomere. The greater thickness of the myofilaments within the A bands may be explained by the presence of the unidentified protein of Szent-Gyorgyi et al. (1955). The transverse connections between the myofilaments are supposed to consist of actotroponiyosin. Hodge declares that "it seems premature to attempt a description of the structure observed in flight muscle in terms of actin, myosin, etc." What is lacking are inter alia reliable methods for extraction of the various components in the flight muscle which could be combined with electron microscopy for a detailed localization of the extracted components. Neither these observations of Hedge nor those of Sjostrand and Andersson agree with the interpretation of Huxley and Hanson. It is not too obvious from the pictures and data presented by Hodge that the interpretation that the thin filaments within the I band are continuous with the less opaque core of the A-band filaments and therefore would represent the same material as the core substance is correct. There are Some obvious structural differences between the myofibrils of the blow-

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fly flight muscle and those of the vertebrate skeletal muscle. No lessopaque core to the A-band filaments has been observed in the latter case (Sjostrand and Anderson, 1956). The mitochondria of the vertebrate skeletal muscle fibers are very numerous and may be very regularly arranged within the I bands at the A-I-band boundary (Fig. 24). They are organized structurally in a way similar to that of most other mitochondria (Anderson, 1956). They appear similar to the sarcosomes of insect flight muscle (Chapman, 1954; Hodge, 1955). A network of tubes with a rather uniform diameter and wall thickness has been observed corresponding to the network observed in light microscopy by Retzius (1881, 18%). I t is striking that this three-dimensional continuous network cmsists of meshes oriented longitudinally within the A bands and to a great extent transversally in the I bands with the transverse branches located within that part of the I band which is adjacent to the A band. This network, for which the name siircotubes has been proposed, shows intimate relationships with the sarcolemma and the mitochondria. No continuity with the mitochondria has, however, been observed. It has not as yet been possible to decide whether a continuity exists between the sarcotubes and the sarcolemina. The sarcotubes have been observed by Bennett and Porter (1953) but were described as consisting of vesicles and were identified with the “endoplasmic reticulum.” The fopographic arrangement of the mitochondria and the sarcotubes points to the I bands as structurally complex regions of the myofibrils. I t has been suggested that the functional significance of the sarcotubes is that they function as a conducting system which would be of importance for the spreading of the excitation through the mass of myofibrils within the muscle fiber. XIII.

THE INTERPRETATION OF ELECTRON MICROSCOPE OBSERVATIONS

Many new observations have been made possible through the recent development of ultrathin sectioning techniques for electron microscopy. These observations have freyuently been presented with far reaching conclusions about the functional significance of what has been seen. A more conservative attitude is recommended. It would be better to make use of the imagination in finding out about the pitfalls and the weakness in argumentation than to use it in building precarious hypotheses. The organization of the cytoplasm appears very complicated when studied on this new level. It seems justifiable to warn against too extensive generalizations. The complex structural differentiation of cells should not be underestimated or oversimplified. Careful quantitative de-

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FIG.24. Longitudinal section through cross striated skeletal muscle fiber showing the topographic relationships between myofibrils, mitochondria, and sarcotubes. Magnification : X 60,000. (Sjostrand and Anderson, in press.)

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scription of observed structures instead of the frequent superficial deScriptions so often presented would help to prevent many of the too facile generalizations which appear at present. An obvious conclusion that can be drawn from the present experience in electron microscopy of tissue cells is that membrane structures occur frequently in the cytoplasm. The presence of membranes seems to represent a very fundamental principle of organization of living matter. We must, however, consider that the conditions under which our observations are made may very well be especially favorable for observing membranes. Such structures are for instance easy to observe under the low contrast conditions of electron microscopy because they may be oriented perpendicularly to the plane of the section. The electron-scattering taking place in the membrane will then produce good contrast. The osmium tetroxide might also be especially favorable in preserving structures such as a membrane with a firm binding of the constituent molecules, but may destroy more labile structures. It seems reasonable to propose caution in reconstructing movements and changes of form from electron micrographs. They are to be considered as still pictures and nobody would try to reconstruct a football match from a still picture even if we know what game is going on. In the cells the situation is worse because we do not even know the rules of the game. We should also be a little cautious when accepting the morphological appearance of various components as presented by the electron microscope after the necessarily drastic treatment of the tissue required for this technique. It would be justifiable to recommend taking into consideration all possible modifications of the structure that may take place as a result of the changed conditions of metabolism, when cutting out pieces of tissue. One should also consider the effect of the fixing agent before the concentration in the cell has reached the limit where preservation takes place, the disorientation caused by the reactions between the fixing agent and the structural elements, the extraction of water soluble and lipoid soluble substances during fixation and dehydration in alcohol and embedding in methacrylate, and the effects in connection with the polymerization that frequently give rise to a complete destruction of the structural organization. When for instance electron microscopists talk about more or less flat vesicles, this description refers to the appearance in the electron micrograph but not necessarily to the real morphology of the component described. The vesicular appearance may be the result of swelling of a triplelayered membrane structure owing to a disorganization of an intermediate layer. The or-cytomembranes observed in the exocrine pancreas cells preserved through freeze-drying do not appear as vesicles (Sjijstrand,

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1953f, g). On the other hand, the vesicular appearance may be more obvious when post-mortem changes have been produced either through delayed fixation or because of slow penetration into the center of a tissue block (Sjostrand and Hanzon, unpublished). The tonicity of the fixing agent represents another factor that greatly influences the morphology of “double membrane” structures. When the spacing between membranous components is very constant it seems reasonable to assume a structural continuity. The lack of such a constancy in the spacing, however, cannot be considered as indicating a free space. It is, for instance, very easy to separate the two opaque layers of the triple-layered discs in the outer segments of the retinal rods through exposure to hypotonic media. The sensitivity of this structure to the osmolar concentration of the osmium solution was in fact the reason for adjusting the osmolar concentration of the osmium mixture carefully (Sjktrand, 1953a). In this respect our experience differs from that of Palade (1953a). The vesicular appearance of discs, which have been provoked in this way should be considered an artifact when compared with the very regular spacing observed when fixation under conditions introducing less osmotic stress has been used. The great difficulties in separating the two membranes of a disc in fragmentation experiments should also be considered (Sjostrand, 1949) and so should the indications of material in between the opaque layers of nonswollen discs. When reconstructing a three-dimensional picture from the twodimensional electron micrograph image, statistical considerations seem very useful. Such considerations were part of the background for interpreting the pattern observed in the cytoplasm of pancreas exocrine cells as membranes (Sjostrand, 1953f,g) and not as longitudinal sections of tubes (Palade and Porter, 1952). In thin sections these membranes could be observed in various degrees of tilt with respect to the direction of the beam, which further supported the first mentioned interpretation. It has been obvious that the dimensions of various ultrastructural components are very constant. The rather limited dispersion of the values that has been reported in various measurements made in published studies from our laboratory (Sjostrand, 1953a, b, d, h, i ; Sjostrand and Rhodin, 1953a, b ; Rhodin, 1954) may seem a little surprising. However, we have to realize that the measurements have been made on randomly chosen points along, for instance, a mitochondria1 membrane. The restriction of randomness here is that the structure to be measured should appear sufficiently well defined to allow a precise choice of points between which the dimensions should be measured. A very simple geometric consideration makes it obvious that such a definition of the contours is possible only when

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the membranes are oriented parallel to the beam. Rather small deviations from this orientation means a considerable reduction of contrast. The distance through which the fraction of the beam which contributes to an image point passes through the more opaque layers of a membrane, will correspond to the thickness of the section when the membrane is oriented parallel to the direction of the beam. The maximum scattering influence will be obtained under this condition. With increasing tilting of the membrane the same fraction of the beam will pass through a decreasing length of the opaque layers and a reduction of the contrast will follow. A tilt of 30" will mean that the distance along which the beam passes through the opaque layers will be only twice the thickness of these layers. Assuming a thickness of the section of 200 A and a thickness of the opaque layer of 40 A a tilt of 30" will reduce the contrast to less than 50% of that at a parallel orientation. At a smaller angle the two opaque layers of a mitochondrial membrane will overlap a fact which would exclude any measurements of the individual components. Such a consideration also indicates that the contrast will be insufficient to observe the membrane structures at a fairly minute tilt if the sections are not extremely thin. This condition we have accepted as an explanation of why the amount, for instance, of mitochondrial membranes observed has increased with the reduction of the thickness of the sections following improvements of the sectioning technique. When estimating quantitatively the number of membranes in the mitochondria of various cell types, this factor has to be taken into consideration. As we ate dealing with contrast conditions the density of the ground substance will also influence such estimates. The low density of the ground substance of certain mitochondria will make it possible to detect the mitochondrial membranes even when tilted at a large angle (Sjostrand, 1953i). The justification for these assumptions has been clearly demonstrated by Williams and Kallman (1955) by taking electron micrographs of sections at various angles, using a stereo-holder, and comparing the influence of the angle of tilt on the image for instance, of mitochondria. The fact that adjacent sections in a series do not match too well has been discussed by Williams and Kallman (1955). One explanation is that during an ordinary exposure of the sections to the beam they will be reduced to about half the original thickness owing to sublimation of methacrylate. This means that great surface tension forces will act on the structure and distort the various components. Williams and Kallman also draw the conclusion that material is removed from the tissue in between the sections because, for instance, membranous structures appear sharply indented in shadowed sections that have

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not been exposed to the beam. This effect, however, might be explained as resulting from a “compression” of the sections, that is, an increase of the thickness of the methacrylate part of the section through flowing combined with a decrease of the dimension parallel to the direction of the cutting. Such effects are easily observed on a macroscopical as well as microscopical scale, for instance when trimming the tissue blocks. The anomalies observed when trying to match adjacent sections in a series seem to be well explained through the effect of surface tension forces when the methacrylate evaporates, by the dependence of the contrast of the tilt of the membranous structures with respect to the beam, and perhaps to losses of material from the cell structures through a tearing off of minute particles. These factors will represent sources of error when interpreting electron micrographs qualitatively and quantitatively.

XIV. IMPORTANT FUTURE PROBLEMS OF GENERALINTEREST The first problem which appears to require investigation is that of fixation. The present technique of osmium fixation appears to preserve only certain types of structures and makes it difficult to combine fixation with methods for electron staining of specific chemical components. As to the artifact problem, the fact that the same structural patterns may be observed after fixation in some other chemical fixative should not be considered sufficiently strong evidence in favor of the preformed character of the pattern. The similarity of results when using two different chemical fixations might very well depend upon one effect in common for both fixatives, for instance upon a similar precipitation of the proteins. The ideal method to use as a check, and as a starting point for cytochemical work, would be freeze-drying preservation. Freeze-drying has been demonstrated as a valuable technique for electron microscopy in connection with the study of the a-cytomembranes in the pancreas (SjGstrand, 1953f,g) . Bad results were, however, obtained by Bretschneider and Elbers ( 1952). It is true that freeze-drying may give excellent results for light microscopy if no ice crystals detectable by this method of microscopy could be detected (see for instance Sjostrand, 1944, 1951b). Only a very narrow zone at the periphery of the piece of tissue shows this good preservation. When applying this technique in electron microscopy it has been obvious that the zone of good preservation may be only a fraction of a cell diameter broad. A very good control of the sectioning procedure is necessary to be able to select the right zone. Qndom choice of the region to be sectioned would give little chance to hit a useful part of the tissue. This fact in combination with the extraction of the crude embedding medium (carnuba

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wax and paraffin) might explain the complete lack of success of Bretschneider and Elbers. The main artifact of freeze-drying is ice crystal formation during freezing. The tendency for crystal formation and the size of the crystals depends on the water content of the tissue. High water content increases the risks of this artifact. When applying this technique to electron microscopy we have to consider the water content of various cell components, down to the elementary supramolecuIar components, more than that of the tissue. It’seems likely that even if ice crystals appear in the ground substance of the cytoplasm there will be a fair chance that no ice crystals will be formed within elementary components of low water content. With a certain change of topographic relationships owing to the ice crystals formed in the ground substance it will, in such a case, still be possible to preserve the ultrastructural organization of the elementary components. The results of Williams ( 1953) when applying freeze-drying to preserve viruses support such an assumption. The main problem when making use of freeze-drying in electron microscopy and studying ultrathin tissue sections, is that of electron optical contrast. The a-cytomembranes are sufficiently dense to show up in the electron micrograph for finer details,, for instance, of mitochondria, are less favorable. To improve the contrast, attempts have been made to combine freeze-drying with subsequent electron staining using osmium tetroxide or phosphotungstic acid (Sjostrand, unpublished). The structural patterns observed in such specimens are identical with those occurring after the osmium solution has acted directly on the more labile, living, tissue cells. However, it seems possible that the preservation is improved since many structural components appear rather fragmentary after direct osmium fixation as compared with the appearance after preservation with freeze-drying combined with subsequent osmium staining. This result may be interpreted as indicating that the ultrastructural organization is well preserved after freeze-drying and that the difficulties in applying this technique without staining are due to low contrast. It may also be considered as supporting the idea that the structural patterns observed are preformed and not caused by artifacts formed by the action of osmium tetroxide. .It may be assumed that osmium tetroxide acting on living cells and on dead dried cells (that is under very different conditions) would not produce identical artifacts. The application of cytochemical methods in electron microscopy is a still more delicate problem than the working out of such techniques for light microscopy. Some preliminary trials have been made but little new information as compared- to that of light microscopy has been presented.

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The histochemical reaction for acid and alkaline phosphatase has been tried by Sheldon et al. (1955) and Brendes et al. (1956). Even a method that would differentiate between lipids and proteins would be very valuable for our interpretations of the structural patterns. At last it seems justifiable to stress the importance of high-resolution electron microscopy in the study of ultrastructural problems. The most fundamental and primary events in the cell that could be presented in images take place at a molecular and supramolecular level. The assembly of secretory products starts with a synthesis of molecules and continues with an aggregation of these molecules into secretory granules. T o be able to follow this mechanism we have to follow the process from the very beginning and not be satisfied only by establishing topographic relationships between secretory granules and, for instance, the Golgi membranes. To correlate on the basis of topographic relationships is to start from the assumption that the functional capacity of each cell region is restricted to only one function. When a structural component has been clearly shown to represent the structural background for one specific function the important problem will be to analyze the molecular organization of this structure. When doing so we are aiming at collecting information such that, when combined with biochemical and physical-chemical data, it will aid in gaining an understanding of the basic processes in living matter. In the case of skeletal muscle tissue, where we have reached furthest in such an analysis, the need for such a detailed description is also most imperative. Most of the work presented today represents morphology at another level, and many old and new problems of morphology may be solved at lower resolution in establishing, for instance, approximately where a certain component is formed or a certain reaction takes place. The ambition of ultrastructure research is, however, to go further and to contribute to an understanding of how a function may proceed. The hope that such contributions will be possible represents the really new and fascinating prospect of modern morphology. By emphasizing the aim of ultrastructure research the importance of high-resolution work is made clear. To improve the effective resolution and to work with a quantitative approach seems then of fundamental importance. This survey has not presented all the kinds of speculation that have appeared in electron microscopy of tissue cells. Only a limited number have been examined. Experimental data that have not been presented in a quantitative way have not always been considered too seriously. Science has to be based on objective quantitative estimations. In electron micro-

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scopy such a demand may easily be satisfied, as the electron microscope provides us with clear-cut pictures of well defined structural components and we only have to use our measure and count to get the figures. A reaction against too loose interpretations and a demand for a quantitative approach seem to be important to prevent electron microscopy, as applied to tissue cell studies, from becoming the Science of Guesses.

XV. REFERENCES Afzelius, B. A. (1955) Exptl. Cell Research 8, 147. Andersson, E. (1956) I n preparation. Bahr, G. F. (1954) Exptl. Cell Research 7, 457. Bahr, G. F. (1955) Exptl. Cell Research 9,277. Bargmann, W., Knoop, A., and Schiebler, T. H. (1955) Z . Zellforsch. u. nzikroskap. Anat. 42, 386. Bennett, H. S. (1955) Am. J. Phys. Med. 34, 46. Bennett, H. S., and Porter, K. R. (1953) Am. J. Anat. 93, 61. Bernhard, .W., Bauer, A., Gropp, A., Haguenau, F., and Oberling, C. (1955) Exptl. Cell Research 9, 88. Bernhard, W., Gautier, A., and Oberling, C. (1951) Compt. r e d . soc. biol. 146, 566. Bernhard, W.,Haguenau, F., Gautier, A., and Oberling, C. (1952) Z . Zellforsch. u. mikraskop. Anat. 37, 281. Bourne, G. H. (1953) Nature 172, 588. Bradley, D. E. (1954) Brit. I . A p p l . Phys. 61, 65. Brandes, D., Zetterqvist, H., and Sheldon, H. (1956) Nature 177, 382. Bretschneider, L. H., and Elbers, P. F. (1952) Koninkl. Ned. Akad. Wetenschap. Proc. Ser. C 66, No. 5, 675. Burgos, M. H., and Fawcett, D. W. (1955) J. Biophys. and Biochem. Cytol. 1, 287. Callan, H. G., and Tomlin, S. G. (1950) Proc. R o y . SOC.B137, 367. Chapman, G. B. (1954) J. Morphol. 96, 237. Claude, A. (1948) Harvey Lectures Ser. 49, 121. Cohen, M., and Bowler, E. (1953) Protoplasma 42, 414. Dalhamn, T. (1956) Acta PhysioE. S c a d . 36, Suppl. 123. Dalton, A. J. (1951) A n . 1. Anat. 89, 109. Dalton, A. J., and Felix, M. D. (1954) Am. J. Anat. 94, 171. Dalton, A. J., Kahler, H., Striebich, M. J., and Lloyd, B. (1950) 1. Natl. Cancer Inst. 11, 439. Danielli, J. F. (1936) 1. Cellular Comp. Physiol. 7, 393. Danielli, J, F. (1951) in “Cytology and Cell Physiology” (Bourne, ed.), p. 150. Oxford, New York. Danielli, J. F., and Dawson, I. M. (1934) J. Cellular Comp. Physiol. 6, 495. Dawson, I. M., Hossack, J., and Wyburn, G. M. (1955) Proc. R o y . SOC.144, 132. De Robertis, E. D. P., and Bennett, H. S. (1954) Federation Proc. 13, 35. Engstrom, H., and Sjostrand, F. S. (1954) Acta Oto-Laryngol. 44, 490. Fawcett, D. W. (1954) Anat. Record 118,422. Fawcett, D. W. (1955) I. Natl. Cancer Zitst. 16, Suppl. 1475. Fawcett, D. W., and Porter, K. R. (1954) I . Morphol. 94, 221. FernLndez-Moran, H.(1950) Exptl. Cell Research 1, 309. Fernindez-Morin, H. (1952) Exptl. Cell Research 3, 1.

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Fernhdez-Morin, H. (1954) 8th Cell. Biol. Congr. Leyden, unpublished. Finean, J . B., Sjostrand, F. S., and Steinmann, E. (1953) Exptl. Cell Research 6, 557. Garnier, C. (1899) ThBse mCd., Nancy. Geren, B. B. (1954) Exptl. Cell Research 7, 558. Geren, B. B., and Schmitt, F. 0. (1953) J. Appl. Phys. 24, 1421. Glimstedt, G., and Lagerstedt, S. (1953a) Kgl. Fysiograf. Sallskap. Lund Forh. [N.F.] 25, 1. Glimstedt, G., and Lagerstedt, S. (1953b) Kgl. Fysiograf. Sallskap. Lund Forh. LN.F.1 84, 3. Glimstedt, G., and Lagerstedt, S. (1954) Anat. Anz. 100, 97. Glimstedt, G., Lagerstedt, S., and Ludwig, K. S. (1954a) Exptl. Cell Research 7, 575. Glimstedt, G., Lagerstedt, S., and Ludwig, K. S. (195413) Experientia 10, 462. Granger, B., and Baker, R. F. (1949) Amt. Record 109, 459. Granger, B., and Baker, R. F. (1950) Anat. Record 107, 423. Green, D. E., and Beinert, H. (1955) Ann. Rev. Biochewa. 24, 1. Haguenau, F., and Bernhard, W. (1955) Arch. Anat. microscop. Morphol. Exptl. 27, 44. Hanson, J., and Huxley, H. E. (1953) Nature 172, 530. Hanson, J., and Huxley, H. E. (1955) Symposia Sac. Exptl. Biol. 9, 228. Harman, J. W. (1955) Am. J. Phys. Med. 34, 68. Hartmann, F. J. (1953) J . Comp. Neurol. 99, 201. Heidenhain, M. (1880) in “Herrmann’s Handbuch der Physiol,” Band 5, p. 174. Hillier, J. (1951) Rev. Sci. Instr. 22, 185. Hillier, J. (1950) Compt. rend. Congr. Intern. Microscopie Electronique Paris 1953, p. 592. Hillier, J., and Gettner, M. E. (1950a) J . Appl. Phys. 22, 135. Hillier, J., and Gettner, M. E. (1950b) Science 112, 520. Hodge, A. J. (1954) Proc. Intern. Conf. Electron Microscopy, London, in press. Hodge, A. J. (1955) J . Biophys. and Biochm. Cytol. 1, 361. Hodge, A. J., Huxley, H. E., and Spiro, D. (1954) J. Histocheni. Cytochem. 2, 54. Holmberg, A. (1955) Acta Ophthalwaol. 33, 377. Huxley, H. E. (1953) Biochim. et Biophys. Acta 12, 387. Huxley, H. E., and Hanson, J. (1954) Nature 173, 973. Ingelstam, E. (1955) Proc. Meeting ProBIem in Contemporary Optics, Florence 1954, in press. Kautz, J., and de Marsh, Q. B. (1955) Exptl. Cell Research 8, 394. K u f f , C. L., Hogeboom, G. H., and Dalton, A. J. (1956) J. Biophys. Biochem. Cytol. 2, 33. Lacy, D. (1955) Nature 175, 1235. Latta, H., and Hartmann, J. F. (1950) Proc. Soc. Exptl. Biol. Med. 74, 436. Leyon, H. (1953a) Exptl. Cell Research 4, 371. Leyon, H. (1953b) Exptl. Cell Research ti, 520. Leyon, H. (1954a) Exptl. Cell Research 6, 497. Leyon, H. (1954b) Exptl. Cell Research 7, 265. Mercer, F. V., Hodge, A. J., Hope, A. B., and McLean, J. D. (1955) Australian Commonwealth Sci. and Ind. Research Organisation Bull. Tech. Paper 8, 1. Newman, S. B., Borysko, E., and Swerdlow, M. (1949) J. Research Natl. Bur. Standards 43, 183. Oliver, J . (1945) Harvey Lectures Ser. 40, 102.

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531

Oliver, J. (1948) I. Mt. Sinai Hosp. N.Y. 15, 175. Otto-, D., Sjiistrand, F., Stenstrh, S., and Svaetichin, G. (1953) Acta Physiol. S c u d . 29, Suppl. 106, 611. Palade, G. E. (1952a) J . Exptl. Med. 95, 285. Palade, G. E. (195%) Anat. Record 114, 427. Pdade, G. E. (1953a) I. Histochew. Cyfochem. 1, 188. Palade, G. E. (1953b) J. ApPl. Phys. 24, 1419. Palade, G. E. (1953~)I . Appl. Phys. 24, 1424. Palade, G. E. (1954) Amt. Record ll8, 335. Palade, G. E. (1955a) J. BCophys. und Biochem. Cytol. 1, 59. Palade, G. E. (1955b) Anat. Record l21, 445. Palade, G. E., and Porter, K. R (1952) Atcat. Record 112, 370. Palade, G. E., and Porter, K. R. (1954) 1. Exfitl. Med. 100, 641. Palade, G. E., and Siekevitz, P. (1955) Federatior, Proc. 14, 262. Palay, S. L (1954) Amt. Record 118, 336. Palay, S. L.,and Palade, G. E. (1953) I . Appl. Phys. MI1419. Palay, S. L., and Palade, G. E. (1955) I. Biophys. and Biochem. Cytol. 1, 69. Pease, D.C (1955) Anat. Record l9l, 723. Pease, D. C.,and Baker, R. F. (1948) Proc. SOC.Exptl. Biol. Med. 67, 470. Porter, K. R. (1953) I . Exptl. Med. 97, 727. Porter, K. R. (1954) I. Hisfochem. Cytochetn. 3, 346. Porter, K. R., and Blum, J, (1953) Amt. Record 117, 685. Porter, K. R., and Kallman, F. L. (1952) Ann. N.Y. Acarb. Sci. 64, 882. Porter, K. R., and Thompson, H. P. (1947) Cancer Research 1, 431. Retzius, G. (1881) Biol. Untetmch. 1, Ser. 1, 1. Retzius, 6. (1890) Biol. Unterwch. IN.F.1 1, 51. Rhodin, J. (1954) “Correlation of Ultrastructural Organization and Function in Normal and Experimentally Changed Froximal Convoluted Tubule Cells of the Mouse Kidney.” Stockholm. Rhodin, J. (1955) Exptl. Cell Reseurch 8, 572. Rhodin, J., and Dalhamn, T. (1954) J . Appl. Phys. 25, 1463. Rhodin, J., and Dalhamn, T. (1956) 2. Zellforsch. u. mkoskop. Anat. in press. Robertson, J. S. (1954) A u s t r d b J. Exptl. Biol. Med. Sci. 82, 229. Robertson, D. J. (1955) J. Biophys. md Biochem. Cytol. 1, 271. Rollhiher, H. (1954) Ber. Oberhess. Ges. Natur. w. H d l k . Giessen Natwrw. Abt. [N.F.] 27, 177. Ruska, E. (1954) Cowegtw di Elettronica e Telm%orte Milan April 1954. Sager, R., and Palade, G. E. (1954) Exptl. Cell Research 7 , 584. Schmidt, W. J. (1928) Arch. exptl. Zellforsch. Gezvebeeiicht. 6, 350. Schmidt, W.J. (1934) 2. Zelljorsch. w. mikroskop. Anat. 22, 485. Schmidt, W. J. (1937) “Die Doppelbrechung von Karyoplasma, Zytoplasma und Metaplasma” Borntraeger, Berlin. Selby, C. C. (1953) Cancer Research lS, 753. Sheldon, H. (1956) I. Bwchrm. Biophys. Cyfol. in press. Sheldon, H., and Zetterqvist, H. (1955) Bwll. Johns Hojkins Hasp. 96, 135. Sheldon, H.,Zetterqvist, H., and Brandes, D. (1955) Exptl. Cell Reseorch 9, 592. SjGstrand, F. S. (1944) Acta Am& Suppl. 1, 177. Sjiistrand, F. S. (1949) I . Cellukar Comb. PhySiol. a,383. Sjsstrand, F. S. (1951a) Nature 168, 646.

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SjGstrand, F. S. (1951b) SymPosillnr Free&ng Drying, London, p. 177. Sjastrand, F. S. (1953a) J. Cellular Comp. Physiol. 4 15. Sjijstrand, F. S. (1953b) J. Appl. Phys. 24, 117. Proc. Electron Microscope Sac. Amer. Clevekmd 1952. SjBstrand, F. 5. (1953~)J. Appl. Phys. 24, 117. Proc. Electron Microscope SOC. Am#. CZeveW 1952. Sjostrand, F. S. (1953d) Experkntia 9, 68. Sjijstrand, F. S. (1953e) Experientia 9, 114. Sjostrand, F. S. (1953f) J. Appl. Phys. 24, 116. Proc. Electron Microscope SOC. Amer. Cleveland 1952. Sjostrand, F. S. (1953~)Nature 171, 31. Sjostrand, F. S. (1953h) Nature 171, 30. Sjostrand, F. S. (19.531) J. Cellular Camp. Physiol. 42, 45. Sjostrand, F. S. (19533) J. Rppl. Phys. 24, 1423. Sjostrand, F. S. (1953k) J. A$#. Phys. 24, 1422. Sjostrand, F. S. (1954a) Symposia 8th Congr. Cell Biot. Leyden, p. 16. Noordhoff, London, 1955. Sjostrand, F. S. (1954b) Sym@osia8th Congr. Cell Biol. Ley&*, p. 222. Noordhoff, London, 1955. Sjijstrand, F. S. (1954~)2. wks. MikrosRop. 62, 65. Sjostrmd, F. S. (1954d) Proc. Intern. Conf. Electron Microscopy London, in press. Sjostrand, F. S. (1954e) Rapport Elrropean Congr. Electron Microscopy Ghent April 1954, p. 69. Centrum Electronenmicroscopie. Sjostrand, F. S. (1954f) Proc. Intern. CoHf. Electron Microscopy London, in press. Sjostrand, F. S. (1955a) Compt. rend. Coltoque Intern. Tech. Rec. Microscopie Electroniqne Corpurc. Toulouse, 151. Sjostrand, F. S. (1955b) Sci. Tools 8, 25. Sjostrand, F. S. (1956) in “Physical Techniques in Biological Research.” (Oster and Pollister, eds.), Vol. 111, p. 241. Academic Press, New York Sjijstrand, F. S. (1956b) Exptl. Cell Research in press. Sjostrand, F. S., and Andersson, E. (1954) Experientia 10, 369. Sjostrand, F. S.,and Andersson, E. (1956) ExpN. Cell Research, in press. Sjostrand, F. S., and Hanzon, V. (1954a) Esptl. Cell Research 7, 393. Sjastrand, F. S., and Hanzon, V. (1954b) Experientia 10, 367. Sjostrand, F. S., and Hanzon, V. (1954C) Exptl. Cell Research 7, 415. Sjastrand, F. S., and Hanzon, V. (1954d) Proc. Intern. Conf. Electron Microscopy London, in press. Sjgstrand, F. S., and Rhodin, J, (1953a) 3. Appl. Phys. %, 116. Proc. Electron Microscope SOC.Amer. Cleveland 1952. Sjbstrand, F. S., and Rhodin, J. (1953b) Exptl. Cell Research 4, 426. Sjdstrand, F. S., and Rhodin, J. (1956) In preparation. Sjostrand, F. S., and Zetterqvist, H., in preparation. Steinmann, E., and Sjostrand, F. S. (1954) Proc. Intern. C m f . Electron Microscopy Lodon in press. Steinrnann, E., and Sjastrand, F. S. (1955) Exptl. Cell Research 8, 15. Swerdlow, M. (1954) Anal. Chew 26, 34. Szent-Gybrgyi, A. G., Mazia, D., and Szent-Gyiirgyi, A (1955) Biochim. et Biophys. Acta 16, 339. van Breemen, V. L. (1953) A M . Record 117, 49. Wald,G. (1954) Science llS, 887. Watson, M. L. (1954) Biochh. et Biophys. Acta l6, 475. Watson, M. L. (1955a) J. Biophys. and Biochem. Cyfol. 1, 183.

ULTRASTRUCTURE OF CELLS

533

Watson, M. L. (1955b) I. Biophys. and Biochem. Cytol. 1, 257. Weinreb, S., and Harman, J. W. (1955) J . Exptl. Med. 101, 529. Weiss, J. M. (1953) J . Expfl. Med. 98, 607. Weiss, J. M. (1955) J. Exptl. Med. 101, 213. WerGll, J. (1956) Actu Oto-Laryrtgol. Suppl. 128. Williams, R. C. (1952) Biochdm et Biophys. Actu 8, 227. Williams, R. C. (1953) Exptl. Cell Reseurch 4, 188. Williams, R, C., and Kallman, F. (1955) J . Biophys. et Biochem. Cytol. 1, 301. Wolken, J. J., and Palade, G. E, (1952) Notwe 170, 114. Woken, J. J., and Schwertz, F. A. (1953) I. Gen. Physiol. 37, 111. Zetterqvist, H. (1956) “?.be Ultrastructural Organization of the Columnar Epithelial Cells of the Mouse Intestine.’, Stockholm. ’Zhmermann, K. W. (1927) in “Handbuch der mikroskopischen Anatomie des Menschen” (von Mollendorff, ed.), Vol. 5, Part 1, p. 86. Springer, Berlin.

This Page Intentionally Left Blank

Author Index Numbers in italic type indicate the pages on which references are listed.

A

Baker, R F., 457,478,530,531 Bale, W. F., 13, 22 Balint, M., 257, 273 Ball, E. G., 326, 360 Bang, I., 93, 142 Barcroft, J., 340, 360 Bardet, J. M., 95,144 Bargmann, W., 468,516, 529 Barker, H. A., 65,86 Barnard, J. E,,5,21 190,191, 192,194,196,351,360 Barnes, M., 42, 49 Allard, C., 93, 142 Barrett, J., 55, 85 Allfrey, V.,334, 353,360, 361 Aftman, K. I., 281, 282, 283, 287, 288, Barron, E. S. G., 2%4,296,300,336,360 Bartholomew, J. W., 42, 50 290,291, 292, 293, 299,300 Bartlett, G. R., 280, 281, 299 Altmann, H. W., 351,352, 353,360 Altmann, R., 91, 142, 148, 149, 151, 169, Baskett, A. C., 79, SO, 85 Bass, A. D., 183,195 Alwisatos, S. G. A., 286,299 Batelli, F.,105, 142 Aminoff, D., 30, 49 Bateman, J. B., 8, 21 Andexsen, A. C., 33, 50 Batty, I., 313, 320 Anderson, K,,310, 321 Bauer, A., 505,529 Anderson, N. G., 13, 21 Bauer, H., 174,194 Andersson, E., 463, 466, 468, 477, 484, Baurnann, C. A., 105,146 518, 520, 521, 522, 529, 532 Bawa, S. R., 400, 401, 411, 412, 413, 414, Andrew, W., 167,169 432, 450, 452 Andrews, C.A., 319, 320 Beams, H. W., 163, 169, 348, 360, 428, Anfinsen, C.B., 338,360 432,442,450,451 Appelmans, F., 104, 142 Becker, M. E., 37,49 Aquilonius, L., 331, 361 Beinert, H., 470, 530 Armstrong, J. I., 257, 272 Bell, A. W., 423, 427, 450 Artom, C., 100,146 Bell, E. T., 91, 92, 142 Arvanitaki, A., 246,260, 272, 272 Bell, L. G. E., 4, 12, 21 Arzac, J. P., 348,360 Belitzer, V. A., 116, 142 Assilineau, J., 30,34, 49 Bellamy, W. D., 53, 85 Atkins, W. R. G., 238,254, 257,272 Benians, T. H. C., 42,49 Aubert, H., 91, 142 Bennett, A. L., 340, 364 Augustinsson, K.B.,285, 299 Bennett, H. S., 95, 99, 142, 164, 169, Avery, 0. T., 172,194,195 Abood, L G., 115, 132, 142,144 Abrahams, M.D., 254, 261,276 Ackermann, W. W., 112,144 Adams, M. A., 340,341, 362 Afzelius, B. A., 366,367,393,504, 529 Ahlgren, G., 105,142 Albert, A., 230,272 Alfert, M, 178, 183, 187, 188, 189, 190,

495,498,499, 518, 521,529

B Babinski, D. H., 115,142 Babson, A. L,316,317,320 Bader, S., 185,194 Bahr, G. F., 105,145, 510,529 Bailey, K., 121, 142 Baker, J. R., 345, 348, 360, 396, 421, 426, 450

535

Bensley, R. R.,89, 142, 336, 360 Benzer, S., 55, 85 Berg, N. O., 348,360 Berg, W. E., 386,393 Berger, M., 112, 116, 122, 123,142 Bergerard, J., 184, 188,194 Bergmann, F., 285, 299 Bergonzi, M., 340,360

536

AUTHOR INDEX

Berliner, E., 2, 3,21 Berman, H.,245, 274 Bern, H.A., 183,194,351,360 Bernfeld, P.,264, 275 Bernhard, W.,331, 334, 360, 477, 490,

Bradford, N. M., 238,249,259,273 Bradley, D.E.,458,529 Brady, T.G.,233,236,260,273 Brambell, F. W. R., 304, 307, 313, 320,

493, 494, 498, 505, 516, 529, 530 Bernstein, R. E., 289,299 Berthet, J., 89, 104,123,142,143 Berthet, L.,104,142 Beyer, R.E.,120,142 Beyer, T.,120,142 Bhardwaj, R., 411,412,413,432,452 Bhatia, C. L.,397, 402, 403, 432, 442, 443 446, 452 Bhattacharya, D. R., 433,451 Bhattacharya, P.,427, 451 Bhimber, B. S.,412,414,415,452 Billingham, R.E.,312,320 Binford, R.,434,442,450 Binkley, F.,44, 49 Bird, R. M.,296,299 Birnbaum, S. M.,50, 50 Black, S., 117,145 Blackard, J. R.,308,321 Blinks, L . R.,241, 252,261,262,263,273 Blum, J., 458,531 Bock, A.V.,239,250,260,264,275 Bocking, D.,11, 23 Bodian, D.,161,169 Bodine, J, H., 237, 251,261, 273 Boivin, A., 44, 49 Bolcato, B., 340, 360 Bonhoffer, K., 105,142 Bonner, D.M.,61,86 Bonner, J., 89, 142 Born, G.V.R., 38,454 Borsook, H.,344, 360 Borysko, E.,457,530 Bourne, G. H.,341, 345, 348, 360, 362, 447,450,485,529 Bowen, R. H.,365, 393, 397, 399, 403, 413,432,442, 443,445,446,450 Bowin A., 171,172, 173,194 Bowler, E.,509,529 Bowman, W., 91,142 Boyd, W.C.,9,22,304,320 Boyer, P.D.,115, 129,142,143,145 Boyle, P.J., 233,273 Brachet, J., 261,273,331,360

Brandes, D., 528,529,531 Brandly, C. A., 314, 320 Brandt, K.M.,230,240,256,273 Brandt, L. W.,319,320 Brenner-Holzach, O.,119, 143 Brent, L., 312,320 Bretschneider, L. H.,526, 527,529 Breuer, M.E.,353,360,363 Brock, N.,340,360 Brody, H.,245, 273 Broman, I., 397, 450 Brooks, M.M.,252, 273 Brown, C. H.,18,22 Brown, D. E. S., 200, 201, 202, 203, 204,

426, 427, 452

210, 211, 226,227 Briicke, E.,259,273 Bryan, J. H.D., 180,194 Buddingh, G. J., 310, 320 Bukantz, S. C., 11,21 Bullard, H. H.,91,92,143 Bullough, W.S., 188, 190,194,196 Burgos, M. H.,495,529 Burke, V.,42, 49 Busacca, A.,149, 154,169 Busch, H., 107,145 Buxton, A.,312, 314,320 Buxton, B. H.,261,273 Buytendijk, F.J. J., 241, 261, 273 C Cabasso, V. J., 5, 18,21 Cain, A. J., 348,361 Caldwell, P. C., 235, 241, 245 243, 244, 245, 246, 258, 259, 260, 262, 263, 264,

273 Calkins, H. E., 8, 21 Callan, H.G.,499,504,529 Camara, S.,331,333,363 Cameron, G.,238, 249,251,273 Camis, M.,340,361 Campbell, A. M.,69,87 Campbell, D.H., 11, 12,21 Campbell, R. M.,182,194 Canat, E.H.,310,320

537

AUTHOR INDEX

Cannon,H. G., 401,450,451 Cantero, A., 93, 142 Canti, R. G., 3%, 452 Carlstrom, A. B., 244,273 Carnot, P., 246,247,250, 259,260,273 Carson, H. L,162,169 Carton, E., 233, 236,260,273 Cartwright, G. E., 283,301 Caspersson, T., 173, 174, 187, 190, 194, 331, 361 Cavalli, G., 184, 195 Chaikoff, I. L., 326,359,363 Chalazonitis, N., 246, 260, 272,272 Chalkley, H. W., 241,271,277 Chambers, L.A,, 8,21 Chambers, R., 216, 218, 226, 238, 245, 246, 248, 249, 250, 251, 254, 261, 273, 276, 277 Chance, B., 116, 117,143 Chang-Chun Wu, 413, 450 Chantreme, H., 334, 362 Chapman, G. B., 88, 95, 96, 136,143, 521, 529 Chapman-Andresen, C., 306,320 Chappell, J. B., 109, 113, 117, 126, 143 Chardon, G., 340, 361 Chargaff, E., 100,143,308,320 Charlton, H. H., 403, 450 Chaudhuri, G. C., 412, 421, 422, 423, 428, 429, 431, 452 Cheever, F. S., 17, 18,21 Chopra, H. C., 412, 415, 417, 420, 431,447, 448, 452 Chow, B. F.,284,300 Christensen, H. N., 316, 320 Christian, W., 284, 301 Chu, H. I., 239, 240, 274 dhu, T. H., 17,21 Ciaccio, G ,92, 143 Clark, A. M., 282, 283,301 Clark, E., 160,169 Clark, W. M., 237, 273 Clarkson, E. M., 285,299 Claude, A,, 89, 143, 328, 345, 361, 457, 529 Clayton, R. M., 2, 21 Clegg, R. E., 319,320 Cleland, K. W., 112, 113, 117, 118, 120, 121, 135, 136, 138, 143, 146

Clermont, Y.,429, 450 Clowes, G. H. A., 271, 276 Cobb, D. M., 233,236, 246,262,274 Coffin, D. L., 5, 18, 21 Cohen, G., 74, 85 Cohen, M., 509, 529 Cohen-Bazire, G., 56, 58, 62, 64, 73, 74, 85, 86

Cohn, M., 52, 53, 54, 56, 57, 60, 62, 64, 69, 73, 74, 75, 76, 85, 86, l29, 143 Coleman, D., 282, 300 Collier, H. B., 282, 301 Collier, J. R., 386, 393 Collier, V., Jr., 437, 439, 440,445, 450 Colwin, A. L., 370,393 32, Colwin, L H., 370,393 260, Connolly, J. M., 9, 19, 20, 21,22, 23 Conway, E. J., 233, 236, 240, 243, 245, 256, 260, 273 Coons, A. H., 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 17, 18, 19, 20, 21, 22, 23, 305, 463, 310, 318, 320 Cooper, P. D., 64,85, 87 128, Cooper, W. C., 241, 261,273 Cooperstein, S. J., 93,145 Copenhauer, J. H., 116,143 Cori, C. F., 281,301 Corson, S. A., 295,300 427, Costello, D. P., 216, 226 Cottone, M., 88 Covell, P.,354, 355, 359, 361 Covo, GA., 121,143 421, Cowan, S. L., 245, 246,260, 274 Cowan, S. T., 48,49,50 Cowdry, E. V., 92, 143, 149, 152, 153, 154, 155, 156, 157, 158, 159, 160, 169, 335, 361, 445, 450 Cox, W. W., 286,299 Cramer, H., 63, 85 Crampton, 'C. F., 304,305, 318, 320 Crane, E. E., 236, 259,274 Creech, H. J., 2, 3, 21 Cross, R. J., 121, 143 363, Crozier, W. J., 237, 252, 274 Cruickshank, B., 5, 8, 14, 15, 21, 22 Cummins, C. S., 27, 28, 30, 31, 32, 36, 37, 39, 40, 41, 43, 44, 45, 46, 47, 48, 49 119, Cunningham, L., 185, 194 Custer, J. H., 285, 301

w.

538

AUTHOR INDEX

D Dagley, S., 272, 274 Dalhamn, T., 466,468,470, 485, 486, 487, 488, 489, 499, 529, 531 Dalton, A. J., 334, 345, 349, 361, 363, 476, 478, 490, 494, 498, 529, 530 Daly, ,C., 240, 274 Daly, M. M., 334, 352, 353, 355, 356, 357, 359, 360, 361 Damboviceanu, A,, 238, 257, 262, 274, 276 Dammin, G. J., 11, 21 Damon, E. B., 262, 263,276 Dan, J. (C.,217, 226, 366, 370, 372, 374, 376, 377, 381, 382, 383, 385, 386, 389, 390, 391, 393, 445, 446, 450 Dan, K.,217,226 Danielli, J. F., 231, 233, 234, 235, 265, 268, 272, 274, 290, 294, 299, 307, 320, 354, 361, 463, 529 Daniels, J. B., 17, 21 Danielson, I. S., 239, 240, 274 Darbishire, F. V., 261, 273 Davidoff, W., 236, 275 Davidson, J. N., 172, 176, 178, 183, 185, 194, 195, 197 Davies, D. A. L., 44, 49 Davies, R. E., 236, 238, 249, 259, 273, 274, 340, 361 Davis, A. M,,179, 186,195 Davis, A. R., 252, 260,275 Davis, B. D., 35, 49,54, 87 Davson, H., 290, 295, 299 Dawson, I. M., 26,49,463, 504, 529 Day, T., 296, 300 Dean, R. F. A., 313, 321 Deane, H. W., 4, 10,22 Deutsch, H. F., 313,314, 318, 319,321 Deutsch, W., 340, 361 de Coulon, A,, 245, 248, 250, 277 De Deken-Grenson, M., 334,361 de Duve, C., 89,104,123,142,143 de Lamirande, G., 93,142 Delaunay, A., 172,194 Dellamonica, E. S., 285, 301 De Lorenzo, W. F., 69,87 de Marsh, Q. B., 504,530 Dempsey, E. W., 335,361 Denstedt, 0. F., 286, 299

De Repentigny, J., 3,21 De Rabertis, E. D. P., 164,169, 354, 361, 498, 499, 529 De Smul, A., 179,183,195 Devine, R. L, 432,450 Dewey, D. L, 33, 34, 35, 41, 45, 48, 49, 50 Dhillon, B. K., 412, 421, 424, 425, 429, 450 Dhingra, 0. P., 398, 444, 445, 447, 450, 451 Dhindsa, K. S., 404,406,430,452 Dianzani, M. U., 101,105,139,143, 145 Dickman, S. R., 110,143 Dienert, F., 51, 64,85 Diermeier, H. F., 183, 195 Dill, D. B., 240, 274 Dingle, J. H., 8, 22 Diomede-Fresa, V., 335, 361 Dische, Z., 283,284,299 Di Stephano, H. S., 174,183,195 Dixon, F. J., 2, 11, 21 Dohi, S. R., 354, 362 Dole, M., 230, 242, 274 Doncaster, L, 401, 451 Donnan, F. G., 231, 233, 260, 263, 264, 265, 267, 268, 269, 271, 274 Dorfman, W. A,, 241,261,274 Dounce, A. L, 13, 21 Downey, M., 233,240, 245,256,273 Doyle, W. L,306, 321 Drabkin, D. L, 238,250,274 Drinker, C. K., 317,320 Druckrey, H., 340,360 Dubuisson, M., 233, 258, 259,274 Duesberg, J., 91, 143, 149, 152, 158, 159, 169 Dunn, R., 55, 58, 59, 87 Dustin, P., 345,361 Duthie, E. S., 355, 361 Duval, M., 247,248,274

E Eaton, M. D., 9, 18, 21, 22 Ebert, J. D., 315, 319, 320 Eddy, A. A., 234,274 Edwards, G. A., 95,142,143 Edwards, J., 11, 22

AUTHOR INDEX

Ege, R., 244, 273 Ehrlich, P.,311, 320 Ehret, C.F., 98, 99, 113,145 Eichel, H. J., 114, 143 Eichenberger, M.,304, 306,320 Einberg, E., 429,450 Einsele, W., 365,393 Eisen, H. N.,15,16,22 Elbers, P.F., 526, 527,529 Elford, W.J., 26, 49 Ellis, J. T.,139, 143 Elster, H.-J., 385, 388,393 Emanuelsson, H., 314, 320 Emmel, V. M.,348,361 Enders, J. F.,17, 21 Engel, C.,356, 364 Engstriim, H.,498,529 Enjo, K.,335, 361 Ephrussi, B., 69,85 Ernster, L,89, 117, 120, 122, 142, 143, 145, 335, 363 Euler, H., 63,85 Evans, T. C., 163,169 Ewing, J., 252,253,258, 276

539

Ferrari, R., 340, 361 Field, H.,Jr., 239,250, 260,264, 275 Fields, M.,304,305,318,320 Fife, J. M.,253,274 Fildes, P.,272, 274 Finch, C. A.,282,283,299,300 Finean, 3. B., 509,53U Fischer, H.,316,320 Fischer, R.,285, 299 Fitch, R. H.,241,277 Fitzgerald, P. J., 15,22 Fleming, A., 36,49 Flesch, M.,451 Flick, J. B.,241, 268, 274 Floegel, J. H. L, 92,143 FIores, L. G.,348,360 Mynn, F.,297, 299 Flynn, R. M.,117,145 Folkes, J. P.,61,62,76,85,334, 361 Fonnescu, A., 139,143 Foote, F.W.,15, 22 Forbes, W.H., 240,274 Ford, L,120,144 Foster, H.C.,245, 275 Foster, M.,176,195 F Foulkes, E.C.,290,299 Fdcone, A. B., 129,142,143 Fourt, L.,8,22 Fasten, N.,434, 442, 451 Fourt, P.C.,8, 22 Faulkener, J. S.,298, 299 Fowler, C.B.,84,85 FaurOFremiet, E., 238, 251,274 Fox, H.M.,382,393 Fautrez, J., 184, 195 Frampton, V. L,253,274 Fautrez-Ferlefyn, N.,185,195 Francis, M. D.,315,320 Fawcett, D. W.,484, 485, 493, 495, 504, Frazer, S. C., 176, 178, 183, 184, 186,

505, 511, 516, 529

195, 197

Fearon, P. J., 233,240,243,245,273 Frederic, J., 98,143 Feigefson, M., 92, 94, 109, 117, 118, 119, Frei, J., 88 120, 129, 130, 133, 136, 137, 141, 144 Freund, J., 2, 21 Felix, hd. D, 345, 348, 349, 361, 363, 476, Freundlich, H., 200, 226 529 Frey-Wyssling, A.,323, 361 Felton, L D., 10,21 Friedewald, W.F., 7,22 Fenger, F., 246,247,250,275 Friedkin, M.,116, 143 Fenn, W. O., 230, 233, 236, 239, 240, Friedlander, M.H.G., 428,452 241, 242, 245, 246, 258, 260, 262, 265, Friedeman, T. E., 343, 36l 274 Friend, G. F., 428, 451 F e r g u m , J. K. W., 245,275 Fry, W., 59,60,86 Fernandes, J. F., 334, 340, 341, 343, 357, Fukuda, M.,183,195 Fuller, A. T.,37, 49 359, 361 Fernandez-Morh, H.,!%6, 529,530 Furst, C. M.,149,169 Furusawa, K.,236,237, 244,258,274 Fernell, W. R.,27,40,49

540

AUTHOR INDEX

G

Green, D. E., 90, 105, 113, 121, 143, 336,

Gabrio, B. W., 282,283,292,299,300 Gale, E.F.,61,62,76,85,334,361 Galeotti, G., 91, 143 Gander, J. G., 129,142 Gardner, L I., 245,274 Garnier, C.,331,361,490,530 Garvey, J. S., 11, 12,21 Garzo, T.,285, 299 Gatenby, J. B., 399, 403, 421, 425, 426,

Green, W.W., 421, 451 Greenberg, D.M.,336,337,361 Greenspon, S., 16,21 Greenstein, J. P.,50, 50 Greig, M.E.,285,297,298,299,300 Gresson, R. A. R., 421, 422, 426, 427,

339, 361, 470, 530

428, 429, 442, 451

Griffin, A. C.,185,194 Grobben, C.,442, 451 427, 428, 432, 433, 442, 451 Gropp, A.,505,529 Gautier, A., 331, 334, 335, 360, 361, 490, Gross, A., 340,361 493,494,498, 516,529 Gruzewska, Z.,246, 247,259,260,273 Gay, F. P., 305,320 Guberniev, M. A.,334,361 Gendre, T.,33, 34,49 Guensberg, E.,293,301 Gentile, D.P., 185,195 Guest, G. M.,250, 260,264,276,285,301 Gerard, R. W., 132,142 Gueylard, F.,247, 248, 274 Geren, B. B., 162, 163,169, 506,510, 530 Gunness, M.,40,50 Gersh, T.,348,361 Gunsalus, I. C.,53, 85 Gettner, M.E.,457,530 Gupta, B. L., 398, 402, 403, 404, 405, 410, Ghosh, I. C.,6,21 418, 419, 420, 421, 429, 431, 443, 447, Giblett, E.,282, 300 451, 452 Gibson, Q.H., 287,299 Gupta, M.L.,411, 412, 413, 432, 448,452 Gill, G. K.,398, 399,432,452 Gurwitch, A., 361 Gilson, G., 438,442,451 Gustafson, F.G., 253,254,261,274 Gitlin, D.,5, 12,21, 305,320,321 Gutstein, M.,256,257,262,274 Gladstone, G. P.,272,274 H Glaman, G. V., 109, 120,146 Glhard, R.,259,260,273 Haas, R., 237, 27# Haege, L.F,, 241, 268, 274 Glimstedt, G., 113, 143,470,530 Hagerman, J. S.,288,299 Goddard, D.R.,287,300 Haguenau, F., 334, 360, 477, 490, 493, Goebel, W.F.,44,49 Goetze, E.,286, 299 494, 498, 505, 516, 529, 530 Hahn, L., 290,300 Gold, G.L.,282, 297,301 Goldacre, R. J., 200, 226,290,299 Hajdu, S.,235, 274 Haldane, J. B. S., 325,361 Goldberg, L,282,301 Haldane, J. S.,272,274 Goldman, M.,19,21 Goldsmith, W.M.,397,403,432, 451 Halpert, A.,241,258,276 Halvorson, H. O., 57, 59, 60, 65, 66, 85. Goodman, J. R.,298,299 86, 87 Goodman, M.,16,21 Hamlin, A,, 17, 18, 21 Goodpasture, E.W.,310,321 Hamperl, H.,2, 21 Gould, R.G., 288,299 Gouriay, D. R. H., 281,291,292,299,300 Hanan, R.,312,321 Hanson, J., 132, 133,144,518, 520,530 Govaert, J., 184,195 Hanzon, V., 113, 146, 326, 329, 334, 335, Granaglia, G., 348,361 336, 345, 349, 350, 364, 460, 463, 466, Granger, B., 478,529 Grasset, E.,308, 312,321 468, 469, 471, 472, 474, 476, 477, 478, 479, 480, 484, 490, 491, 492, 495, 504, Grassi, A., 334,362 505, 516, 517, 524, 532 Grau, C. R.,308,322

AUTHOR INDEX

Harford, C. G., 17, 18, 21 Harkins, W.D., 8,22 Harman, J. W.,88, 90,91, 92,93, 94, 95, 96, 97, 99, 100, 101, 102, 105, 106, 107, 108, 109, 110, 111, 113, 114, 116, 117, 118, 119, 120, 121, 122, 126, 128, 129, 130, 132, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 146, 468, 530, 533 Harper, A. A., 340,361 Harris, E. J., 235, 250, 264, 273, 274, 289, 296, 297, 300 Harris, H., 27, 28, 30, 31, 32, 36, 37, 39, 40, 41, 43, 44, 45, 46, 47, 48, 49 Harrison, W. H., 129,142 Harrop, G. A., 284,300 Hartley, G. S., 234,235,272,274 Hartmann, F. J., 98, 144, 160, 161, 169, 457, 499, 530 Hartree, E. F., 288,300 Hartsell, S. E., 37, 49 Hark, H., 328,361 Harvey, E.N., 216,227 Harvey, R. B, 253,254, 275 Haselmann, H., 355,362 Hastings, A. B., 239, 240, 245, 274, 277, 298, 301 Hattingberg, I., 340,362 Haugen, G. E., 343, 361 Haurowitz, F., 303, 304, 305, 318, 320, 321 Hawes, E. R., 240,277 Haworth, N., 10, 22 Haworth, W. N., 30, 49 Heagy, F. C., 172, 183,197 Hocht, E., 128,145 Hegnauer, A. H., 246,274 Heidelberger, M., 6, 22 Heidenhain, M., 494, 530 Heidenhain, R., 355, 362 Heim, W. G., 319,321 Heine, F., 310, 321 Held, H., 149,169 Helpern, L,290,300 Hel~eg-Larse~~, H. F., 179,195 Hemmings, W. A., 304,307,313,320 Henderson, L J., 239,250,260, 264,275 Henderson, M., 304,313,320 Henderson, M. E., 316, 320

541

Henle, G., 18, 23 Henle, J., 91,144 Henle, W., 18, 23 Hennessey, M., 283, 299 Henriques, V., 244, 273 Herken, H., 340,360 Hermann, G., 408,442,451 Hershey, A, D., 6, 22 Herskowitz, I. H., 180,195 Hess, A., 165,169 Hew, E. L., 3, 13, 22 Hevesy, G., 290,300 Hickman, ,C.P., 421,451 Hicks, S. P., 139,144 Hill, A. G. S., 4, 5, 8, 10, 14, 15, 21, 22 Hill, A. V., 233, 236, 243, 247, 259, 260, 275 Hill, D. K., 258, 272, 275 Kill, R., 341, 362 Hill, R. F., 15, 22 Hiller, S., 238, 246, 248, 249, 250, 255, 273 Hillier, J., 457, 490, 530 Himwich, H. E., 340,341,362 Hinshelwood, C. N., 69, 78, 80, 86, 234, 272, 274 Hirsch, G. C., 327, 335, 345, 347, 348, 349, 351, 352, 354, 355, 358, 359, 362, 447, 451 Hoagland, D. R., 252,260,275 Hoare, D. S., 33,35,40,49 Hodge, A. J., 88, 4.58, 510, 518, 520, 521, 530 Hober, R., 340,361 Hoerr, N. L., 89,142,336,360 Hoffman-Berleng, H., 219,227 Hofmann, T., 32, 49 Hogeboam, G. H., 13, 22, 89, 90, 101, 103, 108, 109, 113, 144, 148, 169, 335, 363, 498, 530 Hogness, D. S., 57, 60,62, 86 Hokin, L. I?., 334,362,364 Hokin, M. R., 334,362 Holdsworth, E. S., 27,28,30,38, 39,49 Holford, F. E., 312, 321 Holland, W. C., 285,297, 299,300 Hollenberg, G. J., 252, 275 Holluager, G., 120,144 Holmberg, A., 482,499,530 Holmgren, E., 92,144

542

AUTHOR INDEX

Holter H., 306, 320,321 Holton, F. A., 116, 117, 146 Holzel, L., 282, 301 Hooker, S. B., 9,22 Hope, A. B., 510,530 Horecker, B. L., 284,300 Horne, R. W., 26, 27, 50 Homing, E. S., 160,169 Hossack, J., 504, 529 Hotchin, J. E., 26, 49 Hottle, G. A,, 313, 321 Houchin, 0. B., 115,144 Houwink, A. L,27,49 Hoven, H., 152,169 Hudson, P. B., 285,301 Hiiber, P., 335, 345,351, 352, 362 Huennekins, F. M., 292,299 Hughes, L., 280, 299 Hughes-Schroder, S., 181,195, 449, 451 Hugo, W. B., 26, 49 Hummel, J. P., 115, 142 Hunter, F. E., Jr., 116, 120, 128, 144, 339, 362 Huskins, G. L., 180,195 Hutchinson, A. O., 313,321 Hutchinson, W. C., 172, 183, 197 Huxley, H. E., 132, 133, 144, 458, 518, 520, 530 Huxley, J., 325, 361 I Il’Ina, L. T., 334, 361 Ingelstam, E., 506, 530 Inhuma, M., 345,362 Irving, L,245, 249,275 Irwin, M., 241,252,275

J Jacobs, M. H., 237, 260, 261, 272, 275, 295, 300 Jacobson, F., 331, 361 Jacques, A. G., 252, 262,263,273,276 J a d , O., 355, 362 James, A. T., 3,21 Jeener, R., 331,362 Jensen, 0. S., 408, 451 Johnson, R., 112,144 Jolit, M., 73, 85 Jonas, H., 291, 300

Jones, M. E., 117,145 Jones, P. E. H., 313,321 Jones, P. H., 284,300 Jones, R. N., 2, 3, 21 Jonnesco, V., 149,169 Jordon, H. E., 92,93, 132,144 Jowsey, J. R., 308,321 Jukes, T. H., 313, 318,321 Jungherr, E. L, 314,320,321 Junqueira, L. C. U., 327, 328, 331, 333, 334, 340, 341, 343, 344, 354, 355, 357, 358, 359, 361, 362, 363

K Kabat, E. A., 2, 6, 14, 22 Kahler, H., 241, 277,478, 490, 494, 529 Kahn, R., 183,194 Kaiserman Abramof, I. R., 352,363 Kallio, R. E., 55, 85 Kallman, B. J., 42, 50 Kallman, F. L., 494, 525,531,533 Kaltenbach, J. C., 114, 128,144 Kaplan, E., 318, 322 Kaplan, M. H., 2, 3, 4, 5, 7, 8, 10, 11, 12, 13, 21, 22 Kaplan, N. O., 340,362 Karlsson, J. L, 65, 86 Karstrom, H., 53, 86 Katchalsky, A., 268, 275 Kauffmann, G., 10, 21 Kautz, J., 504, 530 Kay, H. B., 313, 318,321 Keane, J. F., 185, 296 Keilin, D., 288, 300 Keilley, R. K., 126, 128, 144 Keilley, W. W., 126, 128, 144 Kempner, W., 272, 275 Kennedy, E. P., 90,145 Kent, P. W., 30,49 Kerly, M., 272, 275 Kerr, T., 238,254,273 Kerr, S. E., 282,300 Kerr, W. R., 312, 321 Kerridge, P. M. T., 236, 237, 244, 258, 274, 275 Key, J. A., 154, 169 Kidd, J, G., 7,22 Kiese, M., 287, 300 Killey, W. W., 338, 36U

543

AUTHOR INDEX

King, H. K., 27,40,49 King, R. L., 348,360 Kingsbury, B. F., 336,362 Kisch, B., 95,144 Kitiyakara, A., 92, 93, 99, 101, 102, 117, 118, 119, 126, 128, 132, 137, 138, 139, 144 Klein, E., 185, 195 Klein, G., 185, 195 Kleinfeld, R., 191, 192,197 Klinghoffer, K. A., 293,300 Klioze, O., 62, 86 Klotz, I. M., 62, 86 Knaysi, G., 25, 49 Knight, C. A., 33, 49 Knight, P. F., 307, 309,313,314,321 Knocke, V., 93,144 Knoll, P., 90,92, 93, 144 Knoop, A., 468,516, 529 Knox, R., 58, 86 Koelliker, A,, 91, 93, 144, 325, 362 Koeppe, A. J., 129,143 Kogut, M., 54, 86 Koller, P. C., 182,195 Koltzoff, N. K., 397, 434, 442, 451 Komarov, S. A., 356,362 Komrower, G. M., 282,301 Kopac, M. J., 200, 227 Koritz, S. B., 334, 362 Korson, R., 191,196 Kosterlitz, H. W., 182, 194 Kozawa, S., 293, 300 Krakower, C., 16, 21 Kramsztyk, A., 236, 243, 246, 247, 249, 250, 275 Krauss, M., 386,392, 393 Krebs, H. A., 236, 259,274,283,300 Krimsky, I., 287, 300 Kriszat, G., 219, 227 Kritschevsky, D., 308, 321 Krueckel, B. J., 308, 321 Kruse, H., 11, 22 Kubowitz, F., 284, 301 Kiihne, 353, 362 Kuff, C. L, 498,530 Kuff, E. L., 345, 348, 349,363 Kun, E., 115,144 Kumick, N. B., 176,180, 182,195,196

L Labaw, L. W., 27, 50 Lacy, D., 349, 362,471,530 Lagerstedt, S., 113,143,470, 530 Laguesse, E., 352, 363 Laignel-Lavastine, M., 149, 169 Laird, A. K., 183, 185,195,197 La Mer, V. K., 6, 22 Lamson, B. G., 317,322 Lancefield, R. C., 36, 37,41,43,50 Landau, J. V., 203, 207, 208, 213, 214, 216, 218, 220, 221, 222, 223, 224, 227 Landing, B. H., 5, 12, 21, 305, 321 Landsteiner, K., 8, 22 Landstroem-Hyd6nJH. 331,361 Langer, H., 334,362 Langman, J., 315, 316, 321 Langmuir, I., 8, 22 Langstroth, G. O., 356, 362, 363 Lansing, A. J., 165,169 Lardy, H. A., 109, 116, 126, 128, 129, 143, 144, 145 Larson, A. D., 55,85 Laties, G. C., 89, 144 Latta, H., 457, 530 Lauener, H., 293, 301 La Valette St. George, 442, 451 Lazarow, A., 93, 145 Lea, -, 353, 362 Leavenworth, C. S., 254, 261, 276 Lebert, H., 91, 145 Leblond, C. P., 429, 450 Lecomte, C., 179, 183,195 Lederberg, E. M., 80,86 Lederberg, J., 80,86 Lederer, E., 30,33, 34,49 Leduc, E. H., 4, 5, 9, 11, 12, 13, 19, 21,22 Lee, C. C., 308, 321 Lee, G. R., 283,301 Lehman, J, R., 115,145 Lehninger, A. L,90, 116, 121, 129, 143, 145, 336, 363 Le Fevre, M. E., 294,300 Le Fevre, P. G., 294,295,300 LehouIt, Y., 172,194 Lemmer, K. E., 258,275 Leslie, I., 172, 185,195

544

AUTHOR INDEX

Leuchhberger, C., 175, 176, 177, 178, 179, 180, 182, 184, 185, 186, 187, 190, 195, 196 Leuchtenberger, R., 176, 177, 179, 186, 195 Leuthardt, F., 247,250+275 Levenbrook, L,108, 112,145 Levi, G., 149, 169 Levy, J. F., 120,144 Lewis, I. M., 25,50 Lewis, S. E., 122,145 Lewis, W. H., 216,227,306, 321,327,363 Leyon, H., 509,530 Libby, R. L, 304,305,318, 320 Lillie, F. R., 365,385,389,393,445,451 Lindberg, O., 89, 117, 145, 223, 227, 335, 363 Lindegren, C. C., 67,68,86 Lindegren, G., 67,68,86 Lindvig, P. E., 298,300 Ling, C. T., 284,300 Lipmann, F.,117,145,258,275 Lison, L, 173, 177, 178, 185, 187, 189, 190, 191, 195, 196, 230, 235, 237, 251, 275 Liu, C., 9, 17, 18, 22 Lloyd, B., 478,490,494,529 Lobenhoffer, W., 149, 151,169 Loeb, J., 383, 387,390,391,393 E w , H., 120,122,242,143 Loewy, A. G.,219,227 Lohmann, K., 236,244,258,275 Lwnaacka, G., 331,361 London, I., 288,300 Long, J. H., 246,247,250, 275 Longmuir, N. M., 236,259,274 Lorch, I. J., 200, 226 Lovelock, J. E.,288,289,295,300 Lowry, 0. H., 260,277 Luchsinger, W. W., 129, 143 Luck, J. M., 185,194 Ludford, R. J., 238, 242, 250, 253, 273, 335, 363 Ludwig, K. S.,113,143,470,530 Ludwig, O., 325,363 Luebering, J., 281, 301 Luna, E., 91,145,157,169 Lund, H., 185,195 Lundegardh, H., 265,275

Lundsgaard, E., 280,300 Lynen, F.,336,339,363

M Ma, W. C., 160,169 McCance, R. A., 284,300,313,321 McCann, G. F., 157,159,169 McCarty, M., 27, 32, 36, 37, 39, 40, 41, 43, 50, 172, 194, 195

McClary, D. O., 60,87 McDermott, K., 2, 21 Machlachlan, E. A., 245,274 McIndoe, W. M., 185,195 Mackay, I. F,S., 340,361 Mackler, B., 113,143,145 Mackler, H., 244, 275 McLean, F. C., 239, 240, 250, 260, 264, 277

McLean, J. D., 510, 530 M a c h d , C. M., 172,194 Macleod, J. J. R., 280,300 McMaster, P. D., 11,22 McMaster, R., 190,196 McMaster, R. D., 187,197 MacPherson, E. H.,109,120,146 McRae, D. R., 356,362,363 Mahdihassan, S., 256,275 Mahler, A. H., 284,300 Mahler, H. R., 113,143 Maier, E. H., 285. 299 Maison, G. L,258,275 Maizels, M., 250, 264, 274, 282, 285, 296, 297, 299, 300 Malik, A. P.,401, 402,452 Mallison, H., 237, 261,277 Mandel, P., 185, 196 Mandlestam, J., 58, 59, 65, 66, 69, 80, 81, 83, 86

Manson, E., 54, 86 Mhyai, S.,288,300 Mapson, L. W., 287,300 Margaria, R., 258, 275 Marinesco, G., 160,169 Marinone, G., 188, 191,196 Mark, D. D., 185,196 Marlowe, A. A., 280, 281, 299 Marrack, J., 1, 22 Marrone, L. H., 298,299 Marsh, B. S., 246, 274

545

AUTHOR INDEX

Marshall, J. M., Jr., 3, 4, 5, 8, 14, 16, 22, 306, 321 Marshall, M. E., 313, 314,318,319, 321 Marsland, D. A., 200, 201, 202, 203, 204, 208, 210, 211, 212, 213, 214, 215, 216, 218, 220, 221, 222, 223, 224, 226, 227 Martin, S. H., 254,275 Mason, K. E., 116,145 Masquelin, H., 409, 452 Mast, S. O., 203, 205, 227, 306,321 Mathias, P. J., 286,298, 300 Mathieu, R., 93, 142 Matschiner, J. T., 292, 299 Matthey, R., 182, 196 Maurer, F. W., 233, 236, 240, 242, 245, 262, 265, 274 Maurer, P. H., 2, 21 Maurer, W., 291,301 Maws, J., 149, 151, 169 Maxted, W. R., 37,40,50 Mayberry, T. C., 298,299 Mayer, M. M., 2'22 Mazia, D., 520,532 Maziarski, S., 351, 363 Medawar, P. B., 312,320 Meduski, J. W., 109,145 Meiklejohn, G., 9, 21 Melchior, J. B., 62, 86 Meldrum, N. V., 240, 275 Mellors, R. C., 4, 6, 15, 22, 161, 169, 185, 196 Melucci, N., 352, 363 Menezes, J. R.,355, 362 Mercer, F. V., 510, 530 Meria, M. R. N., 180,196 Mesrobeanu, I., 44,49 Mesrobeanu, L., 44, 49 Metais, P., 185, 196 Metz, C. B., 370, 393 Meves, F., 151, 152, 169, 365, 393, 422, 426, 439, 451 Meyer, A,, 92, 145 Meyer, K. H., 264,275 Meyerhof, O., 126,144,236,244,258,275 Michaeli, I., 268, 275 Michaelis, L., 236, 243, 246,247, 249, 250, 275 Michel, K., 355, 362

Michelazzi, L,139, 145 Mickle, H., 26, 28,50 Miller, E. C., 185, 196 Miller, J. A., 185, 196 Miller, L..L., 13, 22, 317, 322 Millerd, A., 89, 142 Miles, A. A., 44,50 Mirsky, A. E., 172, 173, 175, 177, 181, 182, 196, 334, 352, 353, 355, 357, 359, 360, 361 Mitchell, P., 26, 27, 28, 30, 35, 39, 43, 50 Mitchison, J. M., 215, 216, 217, 227 Mittwer, T., 42, 50 Mohle, W., 2.58, 275 Moerman, J., 184,195 Mond, R., 233,262, 275 Monn6, L., 345,363 Monod, J., 52, 53, 54, 56, 57, 58, 60, 62, 64,69, 70, 74, 75, 76, 8.5, 86 Montgomery, M. L., 326, 359,363 Montgomery, T. H., 351, 363, 399, 429, 451 Moore, B. C., 191,196 Moore, J. E. S., 409, 451 Mor, M. A,, 139,145 Morgan, T. H., 387,393 Morgan, W. T. J., 30,32,42, 44,49,50 Morris, B., 313, 321 Morrison, W. L,286,300 Moses, H. E., 314,320 Moses, M. J., 173, 181, 187,196 Mosley, Q. M., 27, 50 Motulsky, A. G., 282,300 Moulton, J. E., 18, 22 Moyle, J., 26, 27, 28, 30, 35, 39, 43, 50 Muggleton, P. W., 38, 50 Muir, H. M., 289, 300 Mukerji, R. N., 403,442, 451 Mukherjee, D. P., 427, 451 Muldal, S., 182, 196 Mulnard, J., 191,196 Murray, E. S.,17, 18, 21

N Nace, G. W., 319,321 Nageatte, J., 149, 169 Nahas, L., 328,362 Naora, H., 175, 176,196, f97 Nason, A., 115,145

546

AUTHOR INDEX

Nath, V., 397, 398, 399, 401, 402, 403, 411, 420, 435, 443,

412, 413, 414, 415, 417, 418, 419, 421, 426, 428, 431, 432, 433, 434, 436, 437, 438, 439, 440, 441, 442, 444, 445, 446, 447, 448, 451, 452 Nattan-Larrier, L., 312,321 Nayyar, K. K., 404,405,431,452 Needham, D. M., 238, 242, 251, 255, 261, 275 Needham, J., 238, 242, 251, 255, 261, 275 Nemec, A., 253, 275 Nestorescu, B., 44, 49 Netter, H., 233, 241, 245, 260, 262, 264, 272, 275, 276 Neuberger, A., 289, 300 Neukirch, P., 241, 258, 276 Neumann, K. H., 341,363 Neurath, H., 286, 300 Nevill, A,, 58, 87 Newfang, D. M., 163,169 Newman, S. B., 457, 530 Nicolaj, P., 351, 363 Nielsen, S. O., 116, 145 Noll, A., 351, 363 Northrop, J. H., 56, 86 Northup, D., 340, 363 Novikoff, A. B., 128, 136,145 Nygaard, A. P., 105,145

0 Oakley, C. L,304,313,320 Oberling, C., 334, 360, 490, 493, 494, 498, 505, 516, 529

Ochoa, S., 116,145,336,339,363 Oettinger, R., 438, 452 Ogston, A. G., 236, 274 Oliver, J., 516, 530, 531 Olmstead, J. M. D., 244, 275 O'Malley, E., 233, 256, 273 O'Neil, J. B., 308, 321 Opie, E. L., 90, 93, 135, 139,145,310, 320 Omstein, V., 176,196 Orth, 0. S., 258,275 Osborne, U. H., 99, 101, 105, 109, 111, 117, 118, 133, 137,144

Osterhout, W. J. V., 241, 252, 261, 262, 263, 273, 276

Ottinger, B., 10, 21

Ottoson, D., 486, 490, 531 Oyama, J., 312, 321

P Padykula, H. A., 341,363 Palade, G. E., 89, 90, 97, 99, 108, 136, 144, 169, 335, 491, 524,

145, 148, 161, 163, 164, 165, 170, 329, 330, 331, 332, 333, 363, 457, 459, 461, 463, 470, 493, 494, 495, 497, 498, 509, 531 Palatine, I. M., 316, 320 Palay, S. C., 164,170 Palay, S. L., 477, 493, 531 PalCs, S.,285, 300 Pandit, C. G., 238,251,276 Pantin, C. F. A., 255,260,276 Papanicolaou, G. N., 185,196,423,452 Pappenheimer, A. M., 56, 58,86 Pappenheimer, A. M., Jr., 8, 22 Farat, M., 365, 393 Parker, E., 17, 18,21 Parpart, A. K., 296,300 Partridge, S. M., 42, 50 Pasteels, J., 177, 178, 187, 188, 189, 191,194,195,196 Patau, K., 187,196 Paterson, S. W., 298,300 Patterson, W. B., 236, 276 Paul, M. H., 90, 105,145 Pavan, C., 353,360,363 Payne, F., 166,170,413, 452 Pearsall, W. H., 252, 253,258,276 Pease, D. C., 212, 227, 457, 468, 514, 531 Pechstein, H., 244, 247, 258, 276 Pellegrino, B., 352, 363 Perlman, E., 44, 49 Peronne, J. C., 289,300 Perret, C. J , 63, 86 Perry, S. V., 100, 101, 109, 113, 117, 126, 128,143,145 Perugini, S.,173, 197 Peters, R. A., 233, 276 Peterson, R. R., 335, 361 Petow, H., 247, 249, 252, 276 Pietrkowski, D., 247, 249, 252, 276 Pinska, E., 69, 87 Pirie, N. W., 44, 50

166, 334 477, 510,

190,

516,

119,

AUTHOR INDEX

Pisi, E., 184, 195 Plaut, G. W. E., 109,145 Plaut, K. A., 109,145 Podoski, E. P., 54,86 Podber, E., 128,145 Pok, A., 310,320 Pollister, A. W., 173, 175, 176, 178, 191, 196, 433, 453 Pollack, H., 238, 242, 245, 246, 248, 249, 251, 255, 261, 273, 276 Pollock, M. R., 53, 54, 58, 60, 61, 63, 68, 71, 72, 73, 75, 85, 86, 87 Pmeroy, B. S., 308,322 Pompeu Memoria, J. M., 352, 363 Ponder, E., 282,300 Popa, G. T., 365, 366, 385,393 Porter, E. F.,8, 22 Porter, K., 95,99,142,328,363 Porter, K. R., 458, 485, 491, 492, 494, 495, 497, 498, 505, 511, 518, 521, 524, 529, 531 Portier, P., 247, 248, 274 Portzehl, H., 219,223, 227 Postern&, T. Z., 281, 301 Potter, V. R., 90, 107, 109, 117, 123, 126, 127, 129,145,146 Powell, J. F.,32, 34, 39, 45,50 Powers, E. L, 98,99, 113,145 Prankerd, T. A. J., 281, 282, 283, 288, 289, 290, 291, 292, 293, 297, 300 Presmtt, B.;10, 21 Pressman, B. C., 109,145 Pressman, D., 4,6, 15, 16, 22 Price, J. M.,185,196 Priestly, J. G., 272, 274 Pringsheim, P., 6, 22 Proam, H., 2, 22 &her, G. W., 254,261,276 Pulcher.. C... 258.275 .

Q Quagliariello, G., 244, 276

R Raaflaub, J., 119, 129, 143,145 Rabinovitch, M., 331, 333, 341, 343, 357, 362, 363 Rabinovitz, M., 115,145 W e r , E., 287, 300,301

547

Raia, S., 355, 362 Raina, J. L., 439, 452 Ramon, G., 308, 312, 321 Randall, J. T., 428, 452 Ranvier, L., 105, 245 Rapaske, R., 113,143 Raper, H. S., 340,361 Rapkine, L,238, 251, 257, 262,274,276 Rapoport, S., 250, 260,264, 276 Rapoport, S. J., 281, 285, 286,299,301 Rasmussen, A. T., 157, 160,170 Ratnavathy, C.K., 408,410,452 Rau, A. S., 426,427* 452 Raw, I., 340,363 Rea, M. W., 254,276 Reade, M.,368, 321 Recknagel, R. 0, 126, 127,145 Regaud, C., 91,92,145 Reif, A. E., 123, 146 Reiner, J. M., 84, 87 Reisner, E. H., 191, 196 Reiss, P., 230, 237, 238, 242, 243, 244, 245, 246, 248, 249, 251, 259, 260, 261, 276, 277 Retzius, G., 91, 92, 146, 521, 531 Reverberi, G., 390, 393 Rhodin, J., 97, 98, 146, 161, 170, 459, 460, 461, 463, 464, 466, 468, 470, 476, 477, 478, 482, 483, 484, 485, 487, 488, 489, 490, 491, 494, 495, 499, 505, 513, 515, 516, 518, 524,531,532 Richardson, G. M., 272,274 Rickenberg, H. V., 58,61,86 Riddle, O., 308, 321 Ries, E., 335, 354, 355, 363 Riggs, T. R., 316,320 Ris, H., 172, 173, 175, 177, 180, 181, 185, 187, 190, 196 Rishi, R., 398, 402, 447, 452 Ritchie, A. D., 241, 258, 276 Roberts, C. C., 69, 87 Roberts, H. S., 218, 227 Roberts, P. W., 64,87 Robertson, D. J., 506, 531 Robertson, J. D., 163, 170 Robertson, J. S., 492, 531 Robertson, M., 312, 321 Robin, C. H., 91,145

548

AUTHOR INDEX

Robinson, G. M.,261,272,276 Robinson, R,261, 82,276 Rodwell, A. W., 54, 86 Roe, J. W., 234, 272,274 Roels, H., 183, 196 Rohde, K., 238,253,254,261,276 Rollett, A., 93,146 Rollhfiuser, H., 463, 531 Romanoff, A. J., 308,321 Romanoff, A. L., 308,321 Romeis, B., 156, 170 Ronzoni, E., 272,275 Root, W. S., 245, 276 Roque, A. L.,418, 420,448,452 Rosenberg, T., 289,301 Roth, L. E., 98, 99,113,145,432,450 Rothen, A., 8, 22 Rothschild, H. A., 327, 331, 333, 343, 344, 354, 358, 362, 363 Rothstein, A., 280,301 Rotman, B., 61, 86 Roughton, F. J. W., 240,275 Rouiller, C., 331, 360 Rous, P., 242,243,245, 246, 2-48] 249,276 Rowlands, W. T., 313,320 Rowley, D., 64,87 Ruhenstroth-Bauer, G., 288,301 Runnstrom, J., 219,227,386,390, 391,393 Ruska, E., 458, 531 Ruska, H.,95, 142,143,146 Rutenberg, A. M., 94,146 Ryan, J., 128, 145 Ryser, H., 88

S Sacktor, B., 88, 111, 112, 117, 119, 121, 122, 126, 141, 146 Safstrom, R., 331, 361 Sager, R., 510, 531 Salomon, K., 288,299 Salton, M. R. J., 26, 27, 28, 29, 30, 32, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 50 Sanberg, A. A., 283,301 Sanborn,R., 88 Sarkaria, D. S., 401, 452 Sarre, H., 15.22 Sattee, V. S., 412, 421, 422, 423. 427, 428, 429, 431, 452 Savage, E., 280,299 Schade, H., 241,258,276

Schaefer, V. J., 8, 22 Schatnnann, H. J., 289,297, 301 Schayer, R. W., 3, 13, 22 Schechtman, A. M., 216, 217, 227, 307, 308, 309, 310, 313, 314, 318, 319, 321 Scheibel, I., 3., 22 Schelling, V., 284,301 Schiebler, T. H., 468, 516, 529 Schild, K. T., 291,301 Schiller, A. A., 3, 13, 22 Schlayer, C., 272, 275 Schmidt, G., 100, 101,146 Schmidt, W. C.,5, 10, 13, 22,50 Schmidt, W. J., 506,531 Schmidtmann, M.,238, 245, 246, 248, 249, 260, 276 Schmitt, F. O., 162, 163,169,510,530 Schneider, W. C., 13, 22, 89, 90, 93, 100, 101, 103, 108, 109, 113, 144, 146, 148, 169,335,336, 345,348,349,363 Schrader, F., 175, 180, 184, 185, 187, 195, 196 Schreiber, G., 351,352,363 Schrikogoroff, J. J., 151,170 Schwartzschild, R., 175,196 Schwarz, V., 282,288, 300,301 Schwenmin, D., 59,60,86 Schwertz, F. A., 509, 533 Schucher, P., 334,364 Schulz, W., 247, 258, 275 Scott, V. B., 340, 364 Scott, W. J. M.,159, 160,170 Segal, R., 285, 299 Sekhri, K. K., 404,406,430,452 Selby, C. C., 456,531 Seligman, A. M.,94, 146 Sendroy, J., 230, 276 Sengupta, S. B., 6, 21 Sesachar, B. R., 187,196 Sesso, A., 328,331, 333, 355,362,363 Severi, C., 139,143 Shaffer, M. F., 8, 22 Shafig, S. A,, 396, 452 Shapiro, B., 293, 301 Shapleigh, E., 304, 320 Skarma, 6. P., 398, 401, 402, 404, 405, 406, 410, 411, 412, 421, 422, 423, 427, 428, 429, 430, 431, 437, 438, 439, 443, 444,447,448,452 Sharpe, M.E.,44,50

AUTHOR INDEX

shaw, c., 48,50 Sheffner, A. L,60,87 Sheldon, H., 477,499, 528,529,531 Sheldon, W. H., 19,22 Sheline, G. E., 326, 359,363 Shelton, E., 93, 146 Shepard, C.C.,313,321 Sheppard, C.W., 286,298,300 Sheridan, E, 241,268,274 Sherman, B., 16,22 Showacre, J. L,93,146 Sibatani, A., 175, 183,195,196,197 Siegel, M.,4, 6, 15, 22 Siekevitz, P., 117, 129, 146, 331, 363,

549

Sonnenschein, R. R., 241, 276 Sorenson, S. P. L,33, SO, 235,237,276 Sosa, J. M., 345,348,364 Sounhein, E., 310,321 Spek, J., 230, 260,261,276, 277 Sperling, E., 90, 105,145 Speyer, J. F., 110,143 Spicer, S. S., 282, 283, 301 Spiegelman, S., 52, 53, 55, 57, 58, 59, 61, 65, 66, 67, 68, 69, 84, 85, 86, 87 Spiro, D., 458, 530 Spinks, J. W. T., 308, 321 Squire, M. C., 298, 299 Srivastava, M. D., 420, 443,452 Stacey, M., 10, 22, 30, 49 498, 531 Stadie, W. C., 240, 277 Simpson, W. L., 348, 364 Stanier, R. Y.,52, 53, 58, 65, 85, 87 Simpson, W. W., 244,275 Sinai, J., 80,87 Stare, F. J., 105,146 Stark, 0. K., 10, 22,305,321 Singer, R. B., 238,250, 274 Starling, E. H., 340,360 Singh, S.,440, 441,452 Sjolin, S., 295, 301 Stavraky, G. W., 356,363 Sjiistrand, F. S., 97, 113, 146, 161, 170, Stein, S. N., 241, 276 326, 329, 334, 335, 336, 345, 349, 350, Steinitz, L. M.,180,195 364, 457, 458, 459, 460, 461, 462, 463, Steinmann, E., 509, 530, 532 466, 468, 469, 471, 472, 474, 476, 477, Steinmuller, O., 310, 321 478, 479, 480, 482, 483, 484, 485, 486, Stella, G., 239, 245, 260, 277 490, 491, 492, 493, 495, 496, 497, 498, Stenstriim, S., 486,490,531 499, 500, 501, 504, 505, 506, 507, 508, Stern, H., 112,146, 353, 360 509, 511, 513, 514, 515, 516, 517, 518, Stern, L., 105, 142 519, 520, 521, 522, 523, 524, 525, 526, Stetten, D., Jr., 236, 276 527, 529, 531, 532 Stier, A., 341, 364 Still, E. U., 340, 364 Sj3val1, E., 93,142 Slater, E. C., 112, 113, 116, 117, 118, 119, Stitt, J. M., 48, 50 120, 121, 122, 123, 135, 136, 138, 143, Stockard, C., 423,452 Stoddard, J. L., 239, 250,260, 264,275 145, 146 Stokes, J. L., 40, 50 Slonimski, P. P., 69, 85 Stoklasa, J., 253, 254,277 Sliuter, J. W., 349, 355, 364 Stone, R. W., 55, 87 Small, J., 230, 238, 254, 276 Stowell, R. E., 352, 364 Smellie, R. M. S.,172,195 Strange, R. E., 32, 34, 39, 45, 50 Smith, A. H., 308,321 Strangeways, T. S. P., 396, 452 Smith, A. S.,314,321 Straub, F. B., 285, 299 Smith, E. L,64,87 Straus, W., 114,146 Smith, E. P., 237,260, 276 Streicher, J. A., 316, 320 Smith, H. W., 271,276 Striebich, M. J., 93, 146, 478, 490, 494, Snyder, J. C., 17, 18, 21 529 Sokoloff, J., 438, 452 Strongman, €3. T., 157, 159,170 Solomon, A. K,282, 289,2%, 297, 301, Strum, E., 11, 22 326,341,344,360,364 Sturdivant, H. P., 440,452 Solvonuk, P. F., 282, 301

550

AUTHOR INDEX

Sud, B. N., 398, 405, 406, 407, 421, 429,

431, 443, 447, 452 Sussman, R. A., 69,87 Sutherland, E. W., 281, 302 Svaetichin, G.,486,490, 532 Svensson, G.,331,362 Swaen, A., 409, 452 Swann, M.M.,215,216,227 Swanson, M. A., 100, 129,146 Swerdlow, M.,456, 457,530,532 Swift, H., 175, 177, 178, 180, 187, 188, 189, 190, 191, 192,294,296, 297 Swisher, S., 288,299 Sz&ely, M.,288, 300 Szent-Gyorgyi, A., 520,532 SzentaGyorgyi, A. G.,520,532

U Ullman, A., 285,299 Umberger, J. Q.,6,22

V Vadehra, N. P., 437,439,442,452 Valeri, V., 331,333, 341,351, 363, 364 Van Breeman, V. L., 163,269,485,532 van Herick, W., 9, 22 van Heyningen, W.E., 38,50 Van SIyke, D.D., 239, 240,250,260,264,

277 van Weel, P. B., 356, 364 Vasisht, H. S.,407, 408,409, 410, 431, 452 Vaughan, J. H., 14,22 Vaupel, J., 410,452 Vavilov, S.J., 6, 23 T Veflinger, E.,237,244,251,276, 277 Vendrely, C.,171, 172, 173, 176, 177, 178, Taft, E. B., 176,297 181, 182,184,294,295,297 Taggart, J. V.,121, 243 Vendrely, R., 171, 172, 173, 176, 177, 178, Tahmisian, T. N., 432,450 181, 182,184,294,295,297 Tai, T. Y.,38,50 Vennesland, B., 326,360 Taylor, C. V., 237,241,252,277 Verworn, M.,148,170 Taylor, E.S.,59, 87 Vialli, M.,173,197 Taylor, I. M.,298,302 Vickery, H.B., 254,261,276 Taylor, J. H., 187, 296, 297 Villela, G. G., 183,297 Tepperman, J., 183,295 Villiger, W.,175,296 Terrell, N., 314,322 Villmifjana, L.,92,246 Thannhauser, S. J., 100, 101,146 Visscher, M.B.,326,364 Thomasson, J., 283,299 Vl&, F.,230, 237, 238, 242, 245, 248, 250, Thompson, H. P.,497,531 251, 277 Thomson, R. Y.,172, 178, 183, 184, 186, V~egtlin,,C., 241, 271, 277 295, 297 Vogel, H. J., 54,87 Thornley, M. J., 80, 87 von Korff, R. W., 109, 120,246 Thurlow, M.DeG.156, 158,270 von Medem, F. G., 386,393 Timonsen, S., 112,246 Tolbert, B. M.,308, 322 W Tomlin, S. G., 499, 504,529 Wada, S. K., 374, 376, 377, 381, 383, 386, Toni, G., 351,364 389,391,392.393 -_Torriani, A. M.,56,63,85,86 Wagneg, S., 429,450 Tridgell, E, J., 54,86 Wagner, R. J., 253, 277 Tsibakowa, E.T., 116,242 Wainwright, S. D.,58,60,61, 87 Tsou, C. L,110,246 Waksman, B. H.,11,23 Tsuboi, K.L.,285,301 Wald, G.,506,532 Tuchman-Duplessis, H., 184,294 Waldeyer, W., 445, 453 Tucker, H.F., 326,360 Wdker, P.M.B., 176,188,190,197 Tupa, A.,160,269 Walker, R. M.,241,276 Tyler, A., 383,385, 386, 387,393 Wallace, W.M.,245, 260, 277 Tyler, D.B., 121, 246 Warburg, E.J., 239, 264,277 -I

AUTHOR INDEX

Warburg, O., 284,301 Warman, R. N.,288,299 Warren, E., 438, 453 Watanabe, M. I., 91, 100, 109, 111, 112,

551

Wilson, P. W., 55, 87 Winge, O.,69,87 Winget, C. M.,308,321 Winitz, M.,50, 50 113, 121, 122, 135, 136,146 Winnick, R.E.,315,316,322 Watkin, W. M.,30,49 Winnick, T.,315, 316,317, 319,320,322 Watson, B. K.,7, 8, 17,23 Winton, F.R.,244,275 Watson, M. L,13, 23, 88, 458, 505, 532, Wintrebert, P.,365,393 533 Wintrobe, M. M.,283,301 Watts, A. H. G., 416, 421,453 Wirtz, H.,15, 22 Waymouth, C.,315, 321 Witmer, R.,20, 23 Webb, M.,37, 38,50 Witter, R. F., 88 Weber, G.M.,185,196 Wittkower, E., 247,249, 252,276 Weber, H.H.,219,223,227 Woerdeman, M.W., 241,261,273 Weibull, C.,25, 27, 50 W o g l a , H., 244,247, 250,277 Weidel, W., 50 Wolken, J. J., 509,533 Weinreb, S., 88, 95, 96, 97, 99, 101, 113, Wolman, M.,94,146 121, 136,146,468, 533 Woodger, J. H.,425,426,451 Work, E.,32, 33, 34, 35, 40, 41, 45, 48, Weinstein, H. J., 95,96, 136, 146 Weir, D. R., 185,195 49,50 Weiss, J. M., 329, 334, 335, 345, 364, Worley, L. G., 345,364 Wu, H.,239,240,250,260,264,277 492,499,516,533 Wurmser, R.,238, 251, 276 Weiss, P.,319, 322 Wyburn, G.M.,504,529 Welch, F. V.,5, 21 Weller, J. M.,298,301 Y Weller, T.H.,8,9,18,23 Yangiwasa, N., 345,362 Wellman, H.,126, 128, 129,144 Yanoksky, C, 61,86 Wendel, B., 286,299 Yates, H.B.,176, 188,190,197 Wersall, J., 459, 499, 533 Yerganian, G.,181,196 Westeimer, F. H.,338, 364 Yoffey, J. M.,317, 320 Whipple, A,, 5, 12, 21, 305,321 Yokoyama, H.O.,352,364 Whipple, G. H.,317, 322 Young, L.E.,282,300 Whitaker, D.M.,237,241,252,277 Yudkin, J., 52, 59, 64, 66, 69, 80, 83, 86, White, J. C.,185,195 87 White, R. G.,19, 20,23 Yuile, C. L., 317, 322 Whitehouse, M.W., 30,49 z Widdas, W. F.,293,294,295,301 Zargar, S. L., 308,322 Widstrom, G.,174,197 Zetterqvist, H., 459, 466, 468, 476, 477, Wiener, M.,18, 23 478, 481, 484, 497, 499, 528, 529, 531, Wiercinski, F.,230, 276 532, 533 Wigoder, S. B., 427, 451 Zimmerman, A. M.,203, 208, 220, 221, Wilbrandt, W., 289, 293,301 222,223, 227 Williams, C. M., 91, 100, 109, 111, 112, Zimmermann, K.W., 494,533 113, 121, 122, 135, 136,146 Zittle, C. A., 285, 301 Williams, C.R.,116, 117, 143 Zlotnik, I., 404, 426, 427, 428, 429, 442, Williams, R. C.,27,50, 458, 525, 527, 533 451, 453 Willstatter, R.,237, 261, 277 Wilson, E.B., 352,364,365,393,397, 398, Zollinger, H. U., 93, 132, 139,146 Zoutendyk, A.,308,321 430,431,432,433,453 Zuelzer, W. W., 318,322 Wilson, M.J., 245, 249,275

Subject Index A ATP, see Adenosinetriphosphate ATPase, see Adenosinetriphosphatase Accessory body, of human spermatocyte, 428-429 Acetylcholinesterase, cation transport in cells and, 286, 298 in erythrocytes, 285 difference between serum AChE and,

285 N-Acetylornithase, 54 Acids, measurement of intracellular p~ with, 238-241,270 Acrosome, see also Acrosome reaction behavior in annelids, 380 in asteroids, 446 in echinoids, 381, 382 in gastropods, 377-379,383,389 in holothuroids, 370 ff. in molluscs, 374-377, 381, 382, 383,

hyperalkalinity, 383 presence of calcium, 382 presence of self-species eggs or eggwater, 382-383,388 history, 365-366 role in sperm entrance, 387-389 specificity in fertilization and, 391-392 structural aspects of, 366380 Actin, distribution in muscle, 520 Actinomyces, proteolytic enzymes of, 38 Actom~osin~ distribution in muscle, 520 Adenosinetriphosphate, effect on cytokinesis, 219-221 on oxidation of cardiac mitochondria,

130 as energy source, in muscular tissues, 219 in sol-gel cycle in egg cells, 219, 221, 222, 226 389 of, 219, 223 a f l g m as in sea-urdn, 366-370,371, 381, 382, synthesis in erythrocytes, 281,283 383, 384, 386-387 in stroma, 288 degeneration of, 444-445 effect of egg substance on, 365-366, Ad~SinetriPhosPhatase, magnesium-activiated, in muscle mito368, 370,446 chondria, 126 ff. egg-activating enzyme in, 365 nwscular contraction an4 132 filament of, 368,370,372,373,377,446 possible role of, 389-391 Adrenocorticotropic hormone, formation of, 443 preparation of antiserum to, 14 in flagellate sperm, 398-429 Amobacter aerogenes, from Golgi bodies, 398 ff. decarboxylation of LL-diaminopimelic direct method, 398-411 acid by, 35 indirect method, 411-429 Aerwmus, in mammals, 422 ff. cell wall, composition, 31 function of, 365,445-446 lysin of, 386-387 Agents, infectious, labeled antibodies in studies occurrence, 443 on, 17-19 position in sperm, 443 Acrosome reaction, 365-393, see also A g d u t h t i o n , of spermatozoa, 385 Acrosome acrosome reaction and, 385-386 fertilizing capacity and, 385 agglutination of spermatozoa and, 385386 Aging, factors affecting, 381-384 effect on neuronal mitochondria, 164contact, 383-384 167

552

SUBJECT INDEX

Albumin, as activator of oxidative phospborylation of mitochondria, 123 ff. fluorescein-labeled, fate of injected, 13 serum, labeling with fluorescein isothiocyanate, 3-4 properties of conjugate, 4 synthesis by liver, 13 Algae, diaminopimelic acid in, 33 Amino acids, in bacterial cell walls, 32-35 proportion of sugars and, 39 htracellular, role in growing cells, 316317 uptake by tissue cultures, 315 Amino sugars, in bacterial cell wall, 32, 47 Amoebae form, effect of A T P on, 221,223 of pressure and temperature on, 203206 intracellular pH, 255, 262 determination, 260 movement, 200 effect of A T P on, 219 of pressure and temperature on form and, movement of, 203-206 mechanism of, 203 Amphibia, acrosome formation in, 404-406 eggs of, changes of p H in, 261 Anabolism, 324 definition, 323 Androgamone 111, 446 Annelids, acrosome behavior in, 380 spermatozoa of, 381 Anomura, non-flagellate sperm of, 434,435-436 absence of acrosome from, 444 structure of mitochondria1 nebenkern in, 446 Anthocyanins, color of, pH of petals and, 261 Antibodies, association with lymphoid foIlieles, 19 formation of, 19-20

553

inhibition of, 312 in the rabbit eye, XI labeled, in estimation of pituitary hormones, 7-8, 14 with fluorescein isocyanate, 5 ff. fixation, 5 fluorescence microscopy of, 5-6 nonspecific reactions, 7-8 preparation of tissue sections for use with, 4-5 use of, 5 histochemical studies with, 1-23 sensitivity of the method, 6-7 technical considerations, 2 in study of kidney antigens, 15-16 of normal tissue components, 14 of pancreas antigens, 16 of viruses, 17-18 use of layers in studies with, 8-10 labeling compounds for, 2 linking of dye molecules to, 1 transfer to arian embryos, 313-315 to mammalian embryo, 313 transferred, functions of, 317-318 uptake into embryonic blood, 318 Anti-fertilizin, 385 Antigen-antibody reactions, use of layers in study of, 8 ff. Antigens, 18, 43, 44 bacterial cell walls as, 43-45 foreign, entrance into body, 317 fate of injected, 10-14 localization of antibody following injection of, 19-20 of viral, 17-18 native, detection of, 14-16 protein, persistence in laboratory animals, 12 soluble, uptake by phagmytic cells, 304305 transfer to embryo, 311-312 Antiglobulin sera, labeled, 9-10 systems of, 9 use of layers in studies with, 9-10 Anti-glomerulus serum, nephrotoxic nephritis produced by, 141s

554

SUBJECT INDEX

Anti-kidney globulin, fate of injected, 15 Antimycin A, effect on mitochondria1 enzymes, 113, 114, 115 Antiserum, to AGTH, preparation of, 14 labeled, differentiation between Brfamoeba histolytica and Entamoeba coli with 19 preparation, 2 Arbacia, eggs, effect of pressure on dividing,

BAL (British antilewisite), effect on mitochondrial enzymes, 113,

114, 115 Basallamellen, 490, 494, see also Ergastoplasm endoplasmic reticulum and, 495 Basement membrane, 486, 490 dimensions of, 490 Bases, measurement of intracellular pH with,

241,270 Basophilic components, cytoplasmic, 497-

498 Benzaidehyde-6-nitro-2-sodiumdiazotate of temperature on, 213-216 (nuclear fast red), Ascidians, labeling of antibodies with, 2 intracellular pH, 257 Beriberi, Ascorbic acid, effect on mitochondria, 160 red cell metabolism and, 284 Bioblasts, 148 Birds, Aspidistra elatior, nuclear DNA content, 172,181, 194 chloroplast of, 509 sperm of, structure, 431 Asteroids, spermatogenesis in, 403-404 acrosome behavior in 370 ff., 381, 382, Blood group substances, 389 amino acids in, 30 fertilization process in, 390 role of acrosomal filament in, 390, 391 Brachyura, non-flagellate sperm of, 434,436,438 Axons, absence of acrosome from, 444 cutting of, effect on mitochondria, 160- Brush border, 161 of intestinal epithelium, structure of, B 478-482 Bacillus group, of tubular kidney cells, 513 hexosamine-containing peptide in spores of, 39-40 C Bocillws cereus, "C' carbohydrate, 37 constitutive penicillinase mutants, 75 composition, 37 Bacteria, see also Microorganisms and Calcium, individual bacteria effect on acrosome reaction, 382 cell wall of, chemical composition, 25m muscle mitochondria, 118, 119 ff. 50 role in fertilization reaction, 391, 392 in Gram-positive and Gram-negative, Cancer, 29 cytophotometry in diagnosis of, 186 diaminopimelic acid in, 41 Carbamino compounds, effect of proteolytic enzymes on,36 formation in cells, 240 heat rupture of, 26 intracellular pH, determination, 257 Carbon dioxide, lysozyme-sensitive, 37 penetration into tissues, effects of, 271Bactm'w cadaver&, 272 formation of adaptive enzymes in, 58-59 uptake by cells, 260 211-213

SUBJECT INDEX

Carbonic acid, measurement in cells, 239-240 Carboxypolypeptidase, pancreatic, 356-357 Catabolism, 323, 324 Cations, transport in red cells, 296-298 acetylcholinesterase and, 298 dependency on respiratory cycle, 296 possible mechanisms, 291-298 Cuvia, see Guinea pig Cell division, see Mitosis Cell membrane, see also Cytomembrane, Cytoplasmic membrane folds of, in tubular kidney cells, 515 tubular, invaginaticms of, 482 Cell sap, of large plants, intracelluar pH, 252, 271 Cell walls, bacterial, amino acids in, 32-35 proportion of sugars to, 39 antigenic complexity of, 44 as antigens, 43-45 chemical composition of, 25-50 classification and, 45-48 constancy of, 40-42 Gram stain and 3 0 , 4 2 4 strain differences m, 41-42 enzymatic lysis of, 36-38, 39 fractions of, 26 ff. general properties, 28-35 preparation of, 26-27 separation and purification, 27 lipid components, 35 methods of disintegration, 26 quantitative analysis of, 38-40 structure of, 27 sugars and amino sugars in, 30, 32, 39, 46 taxonomic importance, 45 Cells, active surface sites, 294, 295 active transport across membranes in, 265 activity of mitochondria and, 158, 159 animal, furrowing process, see Cytokinesis antibody content of, 19-20 bacterial, kinetic model of, 78-80 turnover in, 57-62

555

chromophil, 154-156, 158, 167, 168 distribution of, 158 mitochondria in, 155-156 heterogeneity of interior of, 265 effect on intracellular pH, 265-271 maturation, proteolytic activity and, 286 membranes of, see Cell membrane, Cytomembranes nerve, see Neurons pH within, 229-277 applicability of Donnan theory to, 262 ff. effect of intracellular heterogeneity on, 265-271 methods for determination, 236-242, 246 ff. theories concerning, 230-236 pancreatic, 466-477, 478 ff., 490 ff., 496, 504, 516-518 electron microscopy of, 329, 330 permeability to ions, 326 secretory activity, 326-360, 516 phagocytic, uptake of soluble antigens by, 304-306 protein-secreting of pancreas and salivary glands, 326 ff. red, see Erythrocytes regeneration, effect on mitochondria, 160-161 reversible sol-gel transformations in, 199 role of intracellular amino acid concentrations in growing, 316-317 secretion, 323-364 secretory, structure of basophilic substance in, 334-335 structural organization of whole, 511521 ultrastructure, derived from electron microscopy, 455-533 high-resolution technique, 528 interpretation of observations, 521, 523-526 dimension of components, 524-525 uptake and transfer of macromolecules by, 303-322 phagocytic capacity and, 309-310 viability, exchange of labile stroma components and, 288

556

SUBJECT INDEX

wall of, see Cell wall diaminopimelic acid in, 33 Cerebral cortex, motor, sugars in, 30,39 pyramidal cell of human, 153 cmytId??7acterircm pyogmes, Chlorophyll, streptococcal cell wall pattern of, 48 arrangement in chloroplasts, 509 Cowdry's studies on neurond mitochonChloroplasts, dria, 152-154 structure of, 509-510 Crarsostre5, Cholesterol, role of acrosomal filament in fertilizaexchange between plasma and eryjhrotion process, 391 cytic, 288, 289 Creatine transphosphorylase, 132 of stroma, 288-289 Cricetinae, Cholinesterase, nuclear DNA content in, 182 transfer to mammalian offspring, 313 crustaceans, Chromatin, decapod, vesiculiform sperm of, 433-438 enzyme activity and, 449 absence of acrosome from, 444-445 as physical basis of heredity, 449 Cyclophorase, Chromatoid body, citrate oxidation by, 109-110 in spermatogenesis, 428-429, 442-443 mitochondria and, 90 Chromatolysis, 160 Cytochondria, see Mitochondria Chromophil cells, see Cells, chromophil Cytochrome c reductase, Chromosomes, of skeletal muscle, effect of tocopherol giant, of salivary glands of Drosophila, on, 115 180 Cytochrome oxidase, DNA content, 180 activity in muscle mitochondria, 109, number of, nuclear DNA content and, 113 171, 177, 179-182, 193 effect of osmotic gradient on, 114 chymetrypsin, localization in pancreas, 16 Cytokinesis, 199, 200, 210-218 Cilia, cortical gel contraction theory, 210 internal structure of, 485 cortical plasmagel layer of cytoplasm functional significance, 486 and, 216 Clostrhm welchii, effect of ATP on, 219-221 diaminopimelicxcid in, 33 mechanism of, 216-218 Coccids, iceryne, Cytomembranes, 4%, see also Cell memspermatogenesis in, 449 brane, Cytoplasmic membranes CoeIenterates, a-Cytomembranes, 4% intracellular pH, 257 of exocrine pancreas cells, 496, 504, 516 Coleoptera, electron microscopy of, 524, 526 acrosome formation in, 413 functional significance, 516 Corti's organ, auditory hair cells of, membranes in, @-Cytomembranes.496 of tubular kidney cells, 515 477 7-Cytomembranes, 4% Corynebactm*w d i p h t h h e , cell wall of, 27 ff. Cy tophotometry, antigenic complexity of, 43 in cancer diagnosis, 186 composition of, 31 methods of, 173-177 fractions of,general properties, 28 in ultraviolet light, 176-177 preparation of, 27 in visible light, 173-176 oligosaccharide in, 38-39 sources of errors, 175-176

557

SUBJECTINDEX

in study of nuclear deoxyribonucleic acid, 171-197 reliability of, 186 Cytoplasm, basophilic component of, 497-498 cortical plasmagel layer of, 216 in Rrbach egg, 210-211 elimination of molecules from, 323 ff. excretions, 323, 324 ground substance of, 490-499 membrane structures of, 523 ff. metabolism, 323,324 opaque particles of,497 recrements, 323 secretions, 323, 324 survival of bacterial polysaecharides in,

of gametes, 172 in liver, 173 during maturation, fertilization and cleavage, 191-193 number of chromonemata and, 179-

182 of chromosomes and, 171, 172, 179-

182, 193

in neoplastic tissues, 185-186 in oocyte, 191, 192 in plants, 180, 194 secretory activity of cells and, 185 in somatic tissue of mice, 178 in sperm, 173,181, 194 variations in, 179-182,184, 193-195 in tumor tissues, 186 Diaminopimelic acid, 318 in bacterial cell walls, 32,33-35 of sulfanilazoproteins in, 318 transfer of nuclear and nucleolar subfunction of, 34 stances to, 352-353 distribution in microorganisms, 45 vesicles of, 497 Diaminopimelic acid decarboxylase, Cytoplasmic membranes, 490-494,see also distribution of, 35 Cell membrane, Cytomembranes in Escketfchia coli, 35 specificity of, 35 dimensions, 491 terminology, 4% Diauxie, enzyme adaptation and, 70,75 D Digitonine, DNA,see Deoxyribonucleic acid hemolytic action, mechanism of, 288-289 DPNH oxidase, 113-114 I-Dimethylamino-5*ulfonyl c h 1 o r i d e isolation from cardiac mitochondria, 113 naphthalene, muscular dystrophy and, 116 labeling of antibodies with, 2 of protozoa, 114 Dipeptidase, pancreatic, 356 role of, 115 2,3-Diphosphoglycerate, 281 Deoxyribonucleic acid, in erythrocytes, 281 gene as macromolecule of, 172 Diphtheria antitoxin, nuclear, cell division and, 187-193 horse, effect of labeling on, 3 constancy of, 171,172, 177, 193 Distemper, cytophotometry in study of, 17 localization of inclusion bodies of of diploid cell, species differences in, canine, 18 173 Dog, effect of hormones on, 183-184 spermatogenesis of, 423 of oogenesis on, 191-192 Donnan theory, of pathological conditions on, 185applicability to intracellular pH, 230-

186 of physiological changes on, 182-

185 in embryonic and differentiating tissue, 188,190, 191 in erythrocytes, 173

235, 262-265 Drug adaptation, in microorganisms, 79-80 Dyes, linking to antibodies, 1 as intracellular pH indicators, 238

558

SUBJECT INDEX

permeability of pancreatic cells to, 326327 Dystrophy, muscular, DPNH oxidase and, 116 experimental, 116

E EDTA, as mitochondrial stabilizer, 118, 121, 122,123, 138 Echinoids, acrosome behavior in, 381,382 Eggs, avian, uptake of macromolecules by, 307-308 Electrolyte secretion, by pancreas, 341 Electron microscopy, of bacterial cell wall, 26 of basement membrane, 486,490 cellular ultrastructure revealed by, 455533 interpretation of observations 521, 523-526 of ciliated cells, 485-486, 488,489 of cytoplasmic ground substance, 490499 of Golgi apparatus, 471-478 of mitochondria, 95-98, 148, f61 ff. of muscle components, 95-98, 518 of nuclear membrane, 499 of ultrathin tissue sections, 458-459 Electron transfer, phosphorylation as concomitant of, 116 Electron transport, in mitochondria, 112-116, 338-339 enzymes participating, 338-339 Embryo, antibody transfer to, 312 role of phagocytosis in, 310-311 transfer of antigens to, 311-312 of macromolecules to, 310, 311-315 to avian, 313-315 criteria for, 311-312 functional significance, 317-319 to mammalian, 312-313 uptake of bovine serum proteins from yolk sac by chick, 312 Entamoeba hktolytica, differentiation between Entamoeba coli, and, 19

Enzyme adaptation, 52 see also Enzymes, adaptive adaptation curves, 67 ff., 70 basic phenomenon, 55-56 definition, 53 diauxie and, 70, 75 inducer of, 62-64, 65,71 ff. action mechanism, 85 mass-action model of, 81 ff. mass action theory of, 85 in microorganisms, 51-87 in cell-free extracts, 76-78 mass action theory, 64-66, 80-84 organizer theory of, 71-78 plasmagene theory, 66-70 specific precursor theory, 70-71 theories of, 51-87 nomenclature, 53-55 protein synthesis and, 52, 54 source of adaptive enzyme, 56-57 unitary hypothesis, 54, 76 Enzyme (s) , acting on phosphorylated nucleotides, distribution of, 126-132 action on mitochondria, 105 activity, chromatin and, 449 adaptive, see also Enzyme adaptation and individual enzymes interaction, 60 precursors, 56 cellular, mitochondria and, 148 distribution in mitochondria, 113, 336 ff. in retina, 513 effect of x-rays on, 336 egg-activating, in acrosome, 365 in erythrocytes, role in metabolism, 279 289 in transport, 289-298 oxidative, in mitochondria, 90,105-112 potassium-activated, surface pH of, 234 proteolytic, effect on bacterial cell walls, 36 streptolytic, carbohydrates essential for action of, 38 synthesis, constitutive inhibitors of, 73, 74 mechanism of, 77-78 in pancreas, 355 ff.

SUBJECT INDEX

559

Epithelium ciliary of eye, arrangement of cell membrane in, 482 intestinal, osmophilic granules in, 477 ultrastructure of brush border of,

constitutive @-galactosidase mutants,

Erythrocytes, acetylcholinesterase in, 285-286 antigenic complexity, 20 glucose metabolism in, 280-281 pathway of, 284 glutathione in, 287 intracellular pH of, changes in, 260 determination of, 239-240,250 Donnan theory and, 264 metabolism in, 279 defective, results of, 282 role of enzymes, 280-289 of nucleosides, 283,284 substrates for, 280-284 nuclear DNA content, 173 phosphate derivatives of glycolysis in,

Fertilization, Lillie’s theory of, 365, 445 Fertilization reaction, specificity of, 391 acrosome reaction and, 391-392 factors affecting, 391 Fertilizin, 385 role in sea urchin fertilization, 388 Feulgen reaction, 174-175 specificity for DNA, 174 Fibrillar structures, cytoplasmic, 499 Fibrinogen, fate of injected, 13 synthesis by liver, 13 Fibroblasts, transformation of macrophages to, 306 Fishes, acrosome formation in, 407-410 sperm of, 431 Fixation, of tissue sections for ultrastructure studies, 459-460 Fluorescein-carbamido proteins, properties, 3-4 Fluorescein isocyanate, labeling of antibodies with, 2, 3 preparation, 3 Fluorescence microscopy, in study of labeled antibody, 5-6 0-F1uorophenylalanine, effect on adaptation of yeast to maltose, 57 Freeze-drying, of tissue sections, 526-527

73-75 diaminopimelic acid in, 33 diaminopimelic acid decarboxylase in,

35 478-482 preparation of cell wall fractions from, of mitochondria in, 461, 466,468 27 respiratory of rat trachea, cilia of, 485- Extrusion, 353-355 486,487,488,489 mechanism of, 353-354 Ergastoplasm, 329-330,335 F of exocrine pancreas cells, 490 ff.,495 functional significance, 331, 493 Fatigue, muscular, microsomes and, 331 effect on mitochondria of nerve cells, in pilocarpine stimulated glands, 335 157, 159

281 phosphatases in, 285 phosphopyridine nucleotidase in, 286 poisoned, lysis of, 284 potassium in, 282 proteinases in, 286 species dependency of, 286 sttucture of, 280 transport in, 289 ff. active, 289-290 cations, 296-298 enzymes and, 289-298 facilitated diffusion, 290 of glycerol, 295-296 passive diffusion, 290 of phosphate, 290-293 of sugar, 293-295 Escherichia COG, composition of cell wall, 29,30

560

SUBJECT INDEX

Friedlilnder bacillus, Type B, capsular polysaccharide of, fate of injected, 10 Fungi, diaminopimelic acid in, 33 intracellular pH, 257 G Galactokinase, 60 Galactose, adaptation of yeast to, 51, 55 Galactosemia, congenital, erythrocytic isomerase and, 282 p-Galactosidase, 54 formation, adaptive, 60-61, 62, 66, 74, 84 in cell-free extracts, 77 diauxic inhibition, 75 in E. co$ 55-56 constitutive, 74 effect of analogs on, 73 role of inducer in, 62-63, 65 isolation, 60 protein Pz and, 56-57 stability, 61 synthesis in E. co& 58 Gastric mucosa, hydrochloric acid secretion by, site of, 259 intracellular pH, 249 changes in, 259 determination, 249 secretion of p H ions in, 236, 259 Gastropods, acrosome behavior in, 377-379, 383, 389 acrosome formation in pulmonate, 414421 Gels, classification, 200, 201 Gene, as macromolecule of DNA, 172 Glands, protein-secreting, mkrosomes in, 328 ribonucleic acid content of, 331 Globulin, labeling with fluorescein isocyanate, 3 y -Globulin, fate of injected, 11, 12

Glucosamine, in bacterial cell walls, 37 Glucose, transport in red cells, 293-295 inhibitors of, 293-294 utilization by erythrocytes, 280-281, 284 Glutathione, in erythrocytes, 284,287 Glycerol, transport in red cells, 295-296 inhibitors of, 295 Glycine, absorption by pancreatic tissue, 327 Glycolysis, in cells, secretion and, 340, 341 site of, 2% in erythrocytes, 289, 291 in mitochondria, 338, 340 role in muscle metabolism, 141 Glyoxalase, in erythrocytes, 284 glutathione and, 287 Goblet cells, ultrastructure of mitochondria in tracheal, 466,468 Golgi apparatus, 345-351 acrosome formation from, 365, 398 ff., 443 composition of, 348-349 ground substance of, 345, 349,471 membranes of, 471, 474, 475 dimensions, 476 morphology of mammalian, 422-429 nature of, 345 of normal and cancerous cells, 477 origin in cell, 447-449 phase contrast microscopy, 345 as preformed structure of living cells, 471, 477 role in cell secretion, 349, 351 of scorpion spermatocyte, 398-399 of secretory cells, functional significance, 477 structure, 345-346, 471-478 Golgi bodies, see Golgi apparatus Granules, muscular, nomenclature, 90-91, 92 Granulocytes, osmiophilic granules in, 499

561

SUBJECT INDEX

Ground substance, of cytoplasm, 490-4559 of Gold bodies, 345,349,471 Guinea pig, spermatogenesis, cytology of, 421-427

H H d y s dentatus, acrosome formation in, 414,416 Heart muscle, intercalated discs of, 485 Helix ~ Q W C I , variations in nuclear D N A content in, 184 Hemiptera, acrosome formation in, 414 variations in nuclear DNA content in, 185

Hemocyanin-a2ophenyl-Sw-sullonate, fate of injected, 11 Hemoglobin, erythrocytic, stability, 289 permeability of nuclear membrane to, 13 Hepatitis, infectious canine, localization of vital antigens in, 18 Heteroptera, sacrosome formation in, 414 Hexokinase, effect on oxidation of cardiac mitochondria, 130, 131 Hexosamine, in peptide of Bacillus group, 39-40 Hexosamine-like substance, in bacterial cell walls, 32 Hexose monophosphate shunt, in erythrocytes, 284-285 Hibernation, effect on neuronal mitochondria, 157158 Holothuroids, acrosome behavior in 370 ff. Hormones, effect on nuclear DNA content, 183-184 Hydrogen-ion concentration, intracellular, 229-277 application of Donnan theory to, 262 ff.

changes in, 258-260 determination of, 236-242 effect of intracellular heterogeneity on, 269-271 indicators for, 272 microelectrodes for, 272 results obtained, 242-258 extracellular and, 233 ff ., 235 sensitivity to external CO, tension, 271-272 theories concerning, 230-236 active extrusion theory, 236 based on Donnan equilibrium, 230235 I Indicators, pH, effect of surface pH phenomena on, 234-235 intracellular pigments as, 237-238 metachromatic errors and, 235 microinjection techniques, 238 Influenza, localization of viral antigens in, 17-18 Insects, flagellate sperm of, 432 mitochondria1 nebenkern in, 432,446 flight muscle, ultrastructure of, 468, 520 spermatogenesis, cytology of, 399-403 Intestines, epithelium ultrastructure of brush border of, 478-482 Invertebrates, nuclear DNA of, 181, 194 Ions, permeability of pancreatic cells to, 326327 transport across cell membrane role of acetylcholinesterase in, 285, 286 Isomerase, erythrocytic, galactosemia and, 282

K KeiIin-Hartree chain, 112 components, 113 Kidney, antigens of, labeled antisera in study of, 15-16

562

SUBJECT INDEX

intracellular pH, determination of, 249 tubular cells of, intracellular fold of cell membrane in, 482, 483 plasma membrane of, 478, 482, 483 specialized regions of cell membrane,

484 structure, 511-516 functional significance of, 516 of mitochondria in, 461, 463, 464,

465,466,467, 468 Krebs cycle, mitochondria and, 109 ff., 112, 141,337,

340

L Lactobacilli, cell wall, composition of, 31 Lactobacilk p l a n t a m , cell wall pattern, 48 Leptospira icterohmorrhagiae localization of antigens of, 19 Lillie's theory of fertilization, 365, 445 Lipids, in cell wall, 35 cation transport and, 298 stroma, incorporation of labeled acetate into, 288 @-Lipoprotein, fate of injected, 13 Lipoprotein structures, cellular, electron microscopy of, 50.5-511 Liver, intracellular pH, 247,248 determination of, 247,248 nuclear DNA content, 173, 182 effect of hormones on, 183-184 subparticles, effect on mitochondria1 metabolism, 109 Liver cells, nuclear membrane of, 504 Liver disease, effect on serum acetylcholinesterase,

285 Lysin, acrosomal, 386-387,446 Lysine, diaminopimelic acid as precursor of, 34 Lysine decarboxylase, formation, role of inducer in, 65 synthesis by Bacterium cadoveris, 58,

59

Lysozyme, carbohydrates essential for action of, 38 effect on bacterial cell walls, 39

M M-antigen, streptococcal, 43 Macromolecules, uptake and transfer by cells, 303-322 differentiation functions, 319 functions of transferred, in embryos,

317-319 mechanisms involved, 304 phagocytic capacity and, 309-310 passive plasma-formation, 318-319 trace versus metabolic transfers, 317 transfer to avian embryo, 317 uptakes by nunphagocytic cells, 304-

307 uptake in witru, 315-316 uptake versus transfers, 304 Macrophages, pinocytosis in, 327 transformation into fibroblasts, 306 Macrura, non-flagellate sperm of, 434,435 Maltose, adaptation of yeast to, 57 kinetics of, 65 Mammals, nuclear DNA content of, 172,181 spermatogenesis, cytology of, 422-429,

431 Manganese effect on phosphorylation in cardiac mitchondria, 120, 121, 122 Mantids, karyotypes of, polyploidy and evolution of, 181 Mersalyl acid, effect on cytokinesis and plasmagel structure of dividing eggs, 223-224,

225, 226 as inhibitor of ATP, 219,223 Microphotometry, ultraviolet, 173 Marine algae, intracellular pH, 257 Marine eggs, intracellular pH, changes in, 261 determination of, 257

SUBJECT INDEX

Membranes, cellular, 523, see also [Cell wall, Cytomembranes plasmocytic, see Plasma membrane Metabolism, cellular, mitochondria and, 90 of erythrocytes, enzymes and, 280-289 Methemoglobin, fate in cells, 286-287 Methemoglobin reductase in erythrocytes, 286-287 Methemoglobinemia, 287 Methionine synthase, action of, 74 constitutive synthesis, inhibition of, 74, 76 Micrococcus, cell wall, composition, 31 pattern of, taxonomic importance, 48 Micrococcus lysodeikticus, effect of lysozyme on, 36-37 Microelectrodes, measurements of intracellular pH with, 241-242 Microorganisms, see also Bacteria and individual microorganisms ability to form enzymes, classification, 75 distribution of diaminopimelic acid in, 33-35 drug adaptation in, 79-80 enzyme adaptation in, theories of, 5187 Microsomes, 328-335 electron micrographs of, 332 protein synthesis and, 334 ribose polynucleotides in, 328 structure, 328 Millipedes, vesiculiform sperm of, 438-439 absence of acrosome from, 444 Mitochondria, 147-148, 335-345 activity, 148 structural requirements, 132-139 assmiation of foreign proteins with, 318 of tobacco virus with, 318 biological oxidations in, 336 fi. pathways of, 336-338 sources of energy for, 336 ff.

563

cellular activity and, 148, 157, 158, 159 chemistry and physiology of, 89, 100105, 336 chondriolysis of, 156, 159 of chromophil cells, 154-156 effect of enzymes on, 105 of X-rays on, 335 electron transport in, 338-339 enzymology of, 336-344 fate in flagellate sperm, 429-433 function of, 159, 160 of glandular cells, 335 Golgi bodies and, 447-448 ground substance of, granular component in, 470 insect, oxidative metabolism in, 111-112 oxidative phosphorylation in, 121-122 isolation, 89 Krebs cycle and, 109, 112, 141, 337, 340 membranes of, dimensions, 461, 466 metabolism, myofibrils and, 117-118 muscle, 89-146, 521 chemical composition, 100-105 distribution of, 93 electron transport system of, 112-116 enzyme distribution in, 113 history of, 91-92 magnesium activated ATPase in, 126 ff. role of, 128, 129 morphology of, 93-100 oxidative phosphorylation, 116-125 phase contrast microscopy of 94 ff., 132 properties of, 92-93 respiratory activity in cardiac, 109 shape of, 92, 134-136 alterations of, 128, 136-139 effect of stress on, 92 staining of, 92-93 nebenkern of, see Nebenkern, mitochondrial of neurons, 147-170 aging process and, 164-167 clumping of, 157, 158 Cowdry’s studies on, 152-154 early observations on, 148-151 function of, 154 in invertebrates, 154, 162, 163

564

SUBJECT INDEX

lipoid globules and, 152-153 morphology of, 152 neurofibrils and, 151 number of, difference in, 156 possible origin of, 162 resistance to change, 156-161 Nissl substance and, 149? 151, 155 occurrence, 148 as vehicles of oxidative enzymes, 90, 105-112 physiological action of, 336-345 post-mortem changes in, 463 reaction to cellular injury, 158, 159 of retinal rods, 461, 462, 463, 466,467 role in cell respiration, 336 role in fertilization, 449 in muscle metabolism, 141-142 shape of, 157 effect of dilute solutions on, 93 of hibernation on, 157 variety of, 148, 151 ultrastructure, 148, 161-164, 336, 461470 functional significance, 470 vesiculation of, 151 Mitosis, D N A and, 187-191 Molluscs, acrosome behavior in, 374377,381, 382, 383, 389 Mucombstances, in bacterial cell walls,30 Mumps, localization of viral antigens in, 17 Muscle, contraction, 132 6. ATPase and, 132 mitochondria1 configuration and, 132133 energy metabolism in, integration of, 140-142 frog, active extrusion of hydrogen ions in, 236 heart, intercalated discs of, 485 intracellular pH, 258 changes in, 258-259 depolarization and, 264 determination of, 239, 244-245 Donnan theory and, 262,263

mammalian, structure of cross-striated fibers, 518-521 mitochondria of, 89-146, 521 ultrastrucure of, 466,468,470 oxidative capacities, 105 ff., 110 type differences in, 305 sarcotubes of, 521 Mycobacteria, distribution of diaminopimelic acid in, 34 Mycobactmim tl4bmcs&lo~s, diaminopimelic acid in, 34 lipopolysaccharide complex in, 30 Myelin sheath, formation of, 506 structure of, 506, 507 Myofibrils, cytochondrial metabolism and, 117-118 M yokinase, role in phosphorylation, 117, 127 Mytilus, acrosomal filaments of, 391 Mytilss eduI&, acrosome behavior in, 374-377, 384, 386-387, 389 spermatozoa, structure of, 374376

N Nebenkern, mitochondrial, evolution of, 432-433, 446-447 in insects, 432 in scorpions, 432-433 structure of, 432 Nematodes, amoeboid sperm of, 439-442 Nephritis, “nephrotoxic”, 14 kidney antigen responsible for, 14-15 Nerves, cells of, see Neurons intracellular pH, 260 depolarization and, 264 determination of, 246 Donnan theory and, 262,263 uptake of CO, by, 260 Neurofibrils, mitochondria and, 151 Neurons, function of, 147

565

SUBJECT INDEX

mitochondria of, 147-170 function of, 154 ultrastructure of, 164 Neutral red, toxic action on pancreas, 335 Nissl bodies, chromatolysis of, 156 mitochondria and, 149, 151 Nitratase, 60 Nitrosomar, pentoses in cell wall of, 32 Nucleic acids, protein synthesis and, 334 role in enzyme synthesis, 77-78 Nucleoplasm, structure of, 505 Nucleoside phosphorylase, in erythrocytic stroma, 292 Nucleosides, erythrocytic, role in phosphate transfer, 292 as metabolic substrates for erythrocytes, 283 Nucleotides, phosphorylated, distribution of enzymes acting on, 126132 Nucleus, 351-353 behavior during cell secretion, 351 ff. classes, 177, 178 DNA content of, derived from cytophotometric studies, 171-197 electronmicroscopy of, 499-505 membrane of, permeability to protein molecules, 12, 13 structure of, 13, 504-505

0 0-antigen, of Shigella shigae, 44 OH concentration, role in fertilization reaction, 391, 392 Oligosacchar ide, in cell walls of Corylrebocterium diphtherioe, 38-39 Oocyte, nuclear D N A content, 191 Ophiuroids, acrosome behavior in, 370 ff. Ornithine decarboxylase, synthesis by Bacterium CQ&V~&, 58

Orthoptera, acrosome formation in, 413-414 Osmophilic granules, cytoplasmic, 499 Osmium tetroxide, as electron stain for proteins, 510411 as fixative, 523, 526 for tissue sections, 459-460 Ovalbumin, fate of injected, 11, 12 surface pH of, 234,235 Oxydon, 105

P Pancreas, antigens of, labelled antisera in study of, 16 behavior of nuclear components at different secretory levels, 353 cells, electronmicrographs of, 329, 330 exocrine, a-cytomembranes, 478, 479, 493, 496, 504 dimension, 326 ergastoplasm of, 490-492 Golgi bodies of, 471, 472, 473, 476, 477 mitochondria of, structure, 466,468, 469 specialized regions of cell membrane, 484 structure of, 516-518 protein-secreting, activities, 326-353 structure, 346, 347 extrusion in, 353-354 types of, 354 of fasting animals, effect of feeding on, 353 Golgi apparatus of, 345, 350, 471, 472, 473, 476, 477 intracellular pH, changes in, 251, 253 determination of, 246 localization of chymotrypsinogen and procarboxypeptidase in, 16 method for cinematographic study of, 355 nuclear and nucleolar volumes of, variations in, 351, 352 protein synthesis in, 344

566

SUBJECT INDEX

rate of protein extrusion and synthesis in, 355357,358-360 secretory activity, 326-360 cellular changes during, 355, 356-357 electrolyte secretion, 341 toxic action of neutral red on, 335 Paper chromatography, in study of bacterial cell wall, 26 Penicillinase, 54 formation, adaptive, 66, 72, 73, 84 inducer for, mode of action, 63, 71, 75 Pentoses, penetration of cells by, 295 Peptide, hexosamine-containing, in spores of Bacillus group, 39-40 Perforatorium, 429 role of acrosome as, 365 structure, 429 Phagocytosis, in adults, 310 cellular uptake of macromolecules and, 309-310 development and, 310-311 role in embryo, 310-311 Phase contrast microscopy, of Golgi bodies, 345 of mitochondria, 94 ff., 132 Phosphatases, in red cell, 285 Phosphate, transport in red cells, 290-293 enzymatic control of, 292 glucose transport and, 291 mechanism of, 292-293 Phosphogalactoisomerase, 60 Phospholipids, in mitochondria, 100, 101 2. Phosphoprotein, blood, transfer into avian ovum, 318 Phosphopyridine nucleotidase, in erythrocytes, 286 Phosphorus compounds, energy-rich, as energy sources for cell secretion, 344 Phosphor ylation, oxidative, in mitochondria, 116-125, 339-340 environmental influence, 120

inhibitors of, 339 mechanism of, 117 role of ATPase in, 129 ff. specific nucleotide acceptor for, 117 stabilizers for, 118-119, 122, 123 Pigments, intracellular, as p H indicators, 237 lipochrome, formation, mitochondria and, 160 Pinocytosis, 306-307, 327-328 definition, 327 Pituitary, hog, basophil cells as hormone source, 14 Pituitary hormones, labeled antibodies and determination of, 7-8, 14 Plant viruses, diaminopimelic acid in, 33 Plants, cell sap of large, intracellular p H of,252, 262-263 applicability of Donnan theory to, 271 determination of, 237, 256 intracellular pH, changes in, 254, 261 determination of, 254, 258, 259 Donnan theory and, 260 nuclear D N A in, 180, 194 Plasma, human, intranuclear distribution of protein fractions of, 12, 13 Plasma membrane, folds of, 495 endoplasmic reticulum and, 495 intracellular folds of, 482-484 functional significance, 482 osmophilic component of, 478 specialized regions of, 484 ultrastructure of, 478-486 Plasmagel, contractility, 199-227 kinetics of, 219-224 mechanism of, 200, 209-210 relation to mechanical work, 224 ff. cortical layer, of Arbacio eggs, 210-211 classification, 213 effect of pressure and temperature on, 211-216

567

SUBJECT INDEX

thickness of, 216 decompression reactions, 207 effect of pressure on, 200, 201, 206, 207, 211-213 Pneumonia, atypical, localization of virus of, 19 Poliomyelitis, experimental, effect on mitochondria, 159 Polyploidy, in hepatic cells, protein metabolism and, 179, 182 measurement of nuclear DNA in study Of 179-182 Polysaccharides, bacterial, antigenic, 44 fate of injected, 10-11 molecular weights of pneumococcal, 10

survival of cytoplasm, 318 in pancreas and salivary glands, 328 Potassium, in erythrocytes, 282 intracellular, behavior of, 297 Prealbumins, 319 Pressure, effect on protoplavmic gel structures, zoo, 201-206 as physiological tool, 201 Pro-acrosome, 399, 404, 426, 443-444 Procarboxypeptidase, localization in pancreas, 16 Proflavine adaptation, in microorganisms, 79-80 Protein Pz, B-galactosidase and, 56-57 Protein metabolism, nuclear DNA content and, 182, 183 Protein synthesis, energy requirement for, 344 enzyme adaptation and, 52, 54 microsomes and, 334 nucleic acids and, 334 in pancreas, 344, 355 ff. Proteinases, in erythrocytes, 286 Proteins, absorption, proteolytic concept of, 303304

antigenic, animal, fate of injected, 1114 cellular uptake, 306306 cellular, interconversion, 59-60 stability of, 61-62 heterologous, penetration into cells, 303 incorporation into cellular structure, 319 intracellular sources, 316-317 mitochondrial, 100, 101-104 osmium staining of, 510-511 pH indicators suitable for, 235 permeability of nuclear membrane to, 12, 13 plasma, absorption by pancreatic cell, 327 formation, 318-319 transfer to mammalian embryos, 312313 serum, template function of transferred, 319 streptococcal, fate of injected, 13 turnover in bacterial cell, 57-62 Prateus vulgaris, formation of antibody to, 20 Protoplasmic head, of mammalian sperm, 426-427 fate of, 427 Protoplasmic gel structures, see Plasmagel Protozoa, diaminopimelic acid in, 33 Pseudomow fluorescm, formation of adaptive enzymes by, 58 Pycnosis, effect on cellular components, 185 Purkinje cells, 150

R Rat, nuclear DNA content of Lieberkiihn crypts, 189 spermatogenesis in, 424 Reptiles, acrosome formation in, 406-407 Reticulum, agranular, 495 endoplasmic, 494-496, 498 “basallamellen” and, 495 origin of, 495 sarcotubes and, 521

568

SUBJECT INDEX

Retina, Secretion, cellular, 323-364 enzyme distribution in, 513 behavior of nucleus during, 351 Retinal rods, definition, 324-326 structure of, 461, 462, 463, 466, 467, kinetics of, 355-360 506, 508, 509, 511-513 nuclear and nucleolar volumes in, 352 functional significance, 509, 513 in pancreas and salivary glands, 326synaptic bodies of, 500-503 360 Rhanmose, role of ergastoplasm in, 331 in bacterial cell wall, 37 of Golgi bodies in, 349, 351 taxonomic importance, 45 of RNA in, 331-334 Ribonucleic acid, source of energy for, 341, 344-345 localization in microsomes, 331 cytoplasmic, 323, 324 pancreatic, role in transformation of Shigella, proteins to enzymes, 359-360 0-antigen of, 44 role in cell secretion, 331-334 SiPunCUluJ *, Ribose polynucleotides, intracellular pH, 257 in microsomes, 328 Snakes, Rocky Mountain spotted fever, fate of mitochondria in sperm of, 429 localization of individual rickettsiae of, 18 sodium, s intracellular, behavior of, 297 “S antigen,” 18 Spherocytosis, hereditary, Salivary glands, anemia of, 282 cell secretion and chromosomal changes, Spectrophotometry, 353 in studies of intracellular pH, 237-238 Golgi apparatus of, 345 Sperm, see also Acrosome, Spermatozoa nuclear volume variations in, 351 acrosome degeneration in, 444-445 synthetic activity in, 328 flagellate, 397-433 rate of protein synthesis, 356 acrosome formation in, 398-429 Salmonella @llorum, atypical, 397-398 composition of cell wall, 29 fate of mitochondria in, 429-433 Salyrgan, see Mersalyl acid types of, 429-433 Sarcosomes, 90, 91 typical, occurrence, 397 chemical composition, 101 structure of, 397 morphologic characteristics, 100 mammalian, formation, 421 ff. oxidative capacity, 109, 110 photomicrographs of, 429,430,431 role in muscle metabolism, 109 structure, 421 Sarcotubes, 521 non-flagellate, 433-442 Sea urchin, amoeboid, 439-442 acrosome behavior in, 366-370,371, 381, tubuliform, 439 382,383,384,3~-3a7,389 types of, 433-442 sperm agglutination and, 385-386 vesiculiform, 433-439 fertilization process in, 390 nuclear DNA content, 173 role of fertilizin in, 388-389 position of acrosme in, 4 3 spermatozoa, structure of, 366,377 Scorpion, Spermatogenesis, flagellate sperm of, 432-433 cytology of,395-453 nebenkern in, 432-433 techniques used in study, 306-307 spermatogenesis of, 398-399 nuclear DNA content and, 187

569

SUBJECT INDEX

Spermatozoa, see also Acrosome Sperm annelid, 381 asteroid, 370-374 D N A content, fertility and, 185 echinoid, acrosome behavior in, 366-370 electron micrographs of, 367 ff. fertilizing capacity of agglutinated, 385 Spiders, spermatogenesis in, 410 fate of mitochondria in sperm of, 429 Spirogyra, diurnal variations of pH in, 261 Staphylococcus a u r w , cell wall fractions, general properties, 28 Sfre@tococnrJfaccalis, cell wall, cornposition, 29, 30 fractions of, general properties, 28 preparation of, 27 Streptococcus pyogaes, cell wall, composition, 29, 48 preparation of cell wall fractions from,

27 Streptococcus #yogenes (Group A), polysaccharide of, fate of injected, 1011 Streptococci, cell wall, composition, 31 group A, antigen of, carbohydrate fraction, 37 antigenic complexity of cell wall, 43, 44

strom labile components, cellular viability and exchange of, 288 lipids of, 288-289 incorporation of labeled acetate into, 288 metabolic activities of, 287-289 nucleoside phosphorylase in erythrocytic, 292 Submaxillary glands, cytochrome-cytochrome oxidase system in, 334 effect of duct ligation on, 341, 342, 343 stimulated, cell changes in, 355,356 Succinoxidase, in mitochondria, 113, 115 Sugars,

in bacterial cell walls, 30, 32, 39, 46 as metabolic substrates for erythrocytes, 280-284 penetration of cells by, 294-295 Synapses, 147 cytoplasmic granules of, 498-499 ultrastructure, 164 Synaptic body, of the retinal rod, 513, 514

T Tetrathionase, 60 Thyroid, electron microscopy of, 335 Ticks, acrosome formation in, 410-411,412 tubuliform sperm of, 439 Tissue cultures, uptake of macromolecules in, 315 Tissue sections, preparation for use with labeled antibodies, 4-5 ultrathin, electron microscopy of, 458459 techniques for obtaining, 457-458 Tissue (s), components, use of labeled antibody in study of, 14 DNA content of interphase nuclei of normal, 177-182 detection of antigens native to, 14-16 determination of htracellular pH in brei, extract, or sap, 236-237 embryonic, macromolecular uptake by, 310-317 neoplastic, nuclear DNA in, 185-186 Tocopherol, effect on cytochrome c reductase of skeletal muscle, 115 Trypsin, effect on bacterial cell walls, 36 Tumors, intracellular pH of, determination of, 250 nuclear DNA content and, 185-186 Turtles, sperm of, 431 Typhus, epidemic localization of individual rickettsiae of, 18

570

SUBJECT INDEX

Thyroid, secretory processes in freeze-dried stimulated cells of, 354 T r ypsinogen, conversion to trypsin, 56 Tryptophan desmolase, action, 74 constitutive synthesis, inhibition of, 74, 76

V Varicella (chicken pox), isolation of virus in, 18 localization of viral antigens with labeled antisera, 18 Viruses, labeled antibodies in studies on, 17-18

X X-rays, effect on mitochondria, 335-336 on pancreas, 335

Y Yeast, adaptation to galactose, 51, 55, 59 enzymes involved in, 59,60 nitrogen supply and, 59, 60 to maltose, 57, 59 kinetics of, 65 to melibiose, genetic aspects, 67 formation of adaptive enzymes in, plasmagene theory of, 67-70, 71 intracellular pH, changes in, 251, 253 determination, 256 secretion of hydrogen ions by, 260 Yolk proteins, conversion to plasma proteins, 318 Z

Zymogen granules, of pancreas cells, 498