International Review of Cytology, Volume 59

INTERNATIONAL

REVIEW OF CYTOLOGY VOLUME 59

ADVISORY EDITORS H. W. BEAMS HOWARD A. BERN GARY G. BORISY ROBERT W. BIUGGS STANLEY COHEN RENE COUTEAUX MARIE A. DIBERARDINO CHARLES J. FLICKINGER M. NELLY GOLARZ DE BOURNE K. KUROSUMI MARIAN0 LA VIA GIUSEPPE MILLONIG ARNOLD MITTELMAN DONALD G. MURPHY

ROBERT G. E. MURRAY ANDREAS OKSCHE VLADIMIR R. PANTIC W. J . PEACOCK DARRYL C. REANNEY LIONEL I. REBHUN JEAN-PAUL REVEL WILFRED STEIN ELTON STUBBLEFIELD HEWSON SWIFT DENNIS L. TAYLOR TADASHI UTAKOJI ROY WIDDUS ALEXANDER L. YUDIN

INTERNATIONAL

Review of Cytology EDITED BY

G . H. BOURNE St.

J. F. DANIELLI

George's University School of Medicine St. George's, Grenada West lndies

Worcester Polytechnic Institute Worcester, Massachusetts

ASSISTANT EDITOR K . W. JEON Depurtment of Zoology University of Tennessee Knoxville, Tennessee

VOLUME59

ACADEMIC PRESS New York San Francisco London A Subsidiary of Harcourt Brace Jovanovich. Publishers

1979

COPYRIGHT @ 1979, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRlEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

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LIBRARY OF CONGRESS CATALOG CARD NUMBER: 52-5203 ISBN 0-12-364359-7 PRINTED IN THE UNITED STATES OF AMERICA

79 80 81 82

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Contents LIST OF CONTRIBUTORS . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii

The Control of Microtubule Assembly in Vivo EIJZABETH C . RAFF I . The Secret: Observations of Microtubule Assembly in Vivo. The Importance of Microtubule-Organizing Centers . . . . . . . . . . . . . . . . . . . 11. The Dance: Experimental Dissections of Microtubule Assembly in Vivo . . . Ill . The Suppositions: Conclusions about the Control of Microtubule Assembly in Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 36 81 83

Membrane-Coating Granules A . F . HAVWARD

I . Histology of Stratified Squamous I1. Membrane-Coating Granules . 111. Summary and Future Prospects References . . . . . . . . .

Epithelia . . . . . . . . . . . . . . .

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97 99 123 124

Innervation of the Gastrointestinal Tract GIORGIOGABELLA

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I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . Vagus Nerve . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Nerves from the Abdominal Plexus . . . . . . . . . . . . . . . . . . IV . Pelvic Nerves . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Distribution of Intramural Plexuses . . . . . . . . . . . . . . . . . . VI . Shape of Intramural Plexuses . . . . . . . . . . . . . . . . . . . . VII . Number of Neurons . . . . . . . . . . . . . . . . . . . . . . . . VIII . Size of Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . IX . Types of Neurons . . . . . . . . . . . . . . . . . . . . . . . . . X . Histochemistry for Acetylcholinesterase . . . . . . . . . . . . . . . . XI . Catecholamine Histochemistry . . . . . . . . . . . . . . . . . . . . XI1 . Serotoninergic Neurons . . . . . . . . . . . . . . . . . . . . . . . XI11. Immunofluorescence Histochemistry . . . . . . . . . . . . . . . . . XIV . Ultrastructure of the Myenteric Plexus . . . . . . . . . . . . . . . . XV . Nerve Endings and Synapses in the Myenteric Plexus . . . . . . . . . . XVI . Glial Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . XVII . Surface of Ganglia and Vascularization . . . . . . . . . . . . . . . . XVIII . Ultrastructure of the Submucosal Plexus . . . . . . . . . . . . . . . . XIX . Innervation of the Muscularis Extema . . . . . . . . . . . . . . . . . XX . Innervation of the Mucosa . . . . . . . . . . . . . . . . . . . . . XXl . Innervation of the Blood Vessels . . . . . . . . . . . . . . . . . . . XXII . Extrinsic Nerve Fibers . . . . . . . . . . . . . . . . . . . . . . . V

130 130 131 131 132 135 137

140 140 143 144 151 152 154 159 165 166 167 167 170 171 171

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CONTENTS

XXIII . Exogenous Adrenergic Transmitters and “False XXIV . Adrenergic Innervation . . . . . . . . . . xxv . Cholinergic Innervation . . . . . . . . . . XXVI . Innervation by Other Types of Nerves . . . . XXVII . Afferent Fibers . . . . . . . . . . . . . XXVIII . Development . . . . . . . . . . . . . . XXIX . Coupling between Muscle Cells . . . . . . xxx . Interstitial Cells . . . . . . . . . . . . . References . . . . . . . . . . . . . . .

Transmitters” . . . . . . .

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172 174 175 176 177 178 181 183 187

Effects of Irradiation on Germ Cells and Embryonic Development in Teleosts NOBUO EGAMIA N D KEN-ICHIIJIRI 1. Introduction

I1 . I11 . IV . V. VI .

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Radiation Effects on Ovaries . . . . . . . . . . Radiation Effects on Testes . . . . . . . . . . . Genetic Effects on Radiation . . . . . . . . . . Effects on Embryonic Development . . . . . . . Concluding Comments . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . .

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195 196 206 217 218 243 245

Recent Advances in the Morphology. Cytochemistry. and Function of Balbiani’s Vitelline Body in Animal Oocytes SARDUL S. GURAYA 1. Introduction

I1. Ill . 1V. V.

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Balbiani’s Vitelline Body in Invertebrates . . . Balbiani’s Vitelline Body in Protochordates . . Balbiani’s Vitelline Body in Vertebrates . . . . General Discussion and Conclusions . . . . . References . . . . . . . . . . . . . . . . .

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24.9 25. I 267 251 314

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Cultivation of Isolated Protoplastsand Hybridization of Somatic Plant Celhs RAISAG . BUTENKO I. I1 . 111. IV . V. V1. VII .

Introduction . . . . . . . . . . . . Historical Outline . . . . . . . . . . Isolation and Properties . . . . . . . . Cultivation . . . . . . . . . . . . . Fusion and Somatic Hybridization . . . Incorporation of Foreign Genetic Material Conclusion . . . . . . . . . . . . . References . . . . . . . . . . . . .

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SUWECTINDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONTeNTS OF PREVIOUS VOLUMES . . . . . . . . . . . . . . . . . . . . . .

323 325 325 340 351 363 36.5 36’6

375 379

List of Contributors Numbers in parentheses indicate the pages on which the authors' contributions begin.

RAISAG . BUTENKO (323), Timiryazev Institute of Plant Physiology, Academy of Sciences of the USSR, Moscow 127273, USSR NOBUOEGAMI(199, Zoological Institute, Faculty of Science, University of Tokyo, Tokyo 113, Japan GIORGIO GABELLA (129), Depurtment of Anatomy, University College, London WClE 6BT, England SARDULS . GURAYA (249), Department of Zoology, College of Basic Sciences and Humanities, Punjab Agricultural University, Ludhiana, Punjab, India A. F . HAYWARD (97), Royal Dental Hospital School of Dental Surgery, London

W.C.2, England KEN-ICHIIJIRI(195), Zoological Institute, Faculty of Science, University of Tokyo, Tokyo 113, Japan ELIZABETH C. RAFF( l ) , Program in Molecular, Cellular, and Developmental Biology and Department of Biology, Indiana University, Bloomington, Indiana 47401

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INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 59

The Control of Microtubule Assembly in Vivo ELIZABETH C. RAFF Program in Molecular, Cellular, and Developmental Biology and Department of Biology, Indiana University. Bloomington. Indiana

1. The Secret: Observations of Microtubule Assembly in Vivo.

The Importance of Microtubule-Organizing Centers . . . . . A. The Morphogenesis of Basal Bodies and Centrioles . . . . B. The Assembly of the Axoneme of Cilia and Flagella . . . . C. The Morphogenesis of Microtubule-Containing Organelles in Protozoa . . . . . . . . . . . . . . . . . . . . D. The Assembly and Disassembly of Labile Microtubule Arrays: The Mitotic Apparatus . . . . . . . . . . . . . . . E. The Assembly and Disassembly of Labile Microtubule Arrays: Cytoplasmic Microtubules . . . . . . . . . . . . . 11. The Dance: Experimental Dissections of Microtubule Assembly in Vivo . . . . . . . . . . . . . . . . . . . . . . . A. The Biochemistry of Tubulin, the Structural Microtubule Protein B. Microtubule Assembly in Vitro: Possible Regulatory Factors . C. The Growth of Microtubules in Vitro onto Isolated Microtubule-Organizing Centers . . . . . . . . . . . D. Experiments in Which Microtubule Assembly Is Elicited in Vivo by the Injection of Microtubule-Organizing Centers into Eggs E. Calcium and Other Small Molecules as Possible Regulators of Microtubule Assembly in Vivo . . . . . . . . . . . . F. Time-Dependent Properties of Tubulin and Microtubules . . G . Spatial Localization of Microtubules: Association with Membranes . . . . . . . . . . . . . . . . . . . H. Microtubule Regulation: Genetic Studies . . . . . . . . 111. The Suppositions: Conclusions about the Control of Microtubule Assembly in Vivo . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

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We dance round in a ring and suppose, But the secret sits in the middle and knows. Robert Frost ( 1 949)

With the adv nt of electron microscopy, it was recognized that microtubules were ubiquitous components of eukaryotic cell organelles and were in fact participants in many of the most basic cellular processes, most notably as the spindle Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-364359-7

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fibers of the mitotic apparatus and as the 9 + 2 axoneme tubules in cilia and flagella. Somewhat later they were found to be one of the main components of neural tissue, and it is now known that microtubule networks exist in the cytoplasm of most eukaryotic cells. The exquisite and delicate control over the timing of appearance and positioning of microtubules and microtubule-containing organelles is spectacularly obvious in many cellular events, and the question of the nature of their regulation is thus a fascinating problem. But it is also a particularly frustrating problem because of the abundance of tantalizing data which approach the question but fall short of answering it. The secret, in this case, is all too elusive. The vastness of the microtubule literature and the frequency with which it is reviewed are often noted [generally, as here, at the beginning of the very reviews in question: for example, see Porter (1966) for the first review of microtubule function, and Burnside (1975) for a more recent historical overview; reviews by Hepler and Palevitz (1974), Jacobs and Cavalier-Smith (1977), Newcomb (1969), Olmsted and Borisy (1973), Pickett-Heaps (1975b), Roberts (1974), Snyder and McIntosh (1976), and Stephens and Edds (1976) provide several viewpoints on microtubule structure and function in animals and plants.] What follows therefore is in no way a comprehensive review of the microtubule literature or even of that part of it which might be supposed to be included under the title. The representation of papers discussed is just that, a selective representation. In 1974 Roberts summed up rather nicely what was then known about the control of microtubule assembly in vivo by noting that it was “all rather hazy at the moment.” Unfortunately, it is still rather hazy, but in the last several years much more information has been accumulated which both gives direct clues about the molecular mechanisms underlying the temporal and spatial regulation of microtubule assembly and makes the interpretations of some earlier observations (particularly on the role of microtubule-organizing centers, for example) more sound. This article, then, addresses the following questions. First, what exactly do microscopists see when they look at a cell in which microtubules are in the process of coming or going? Second, what kind of experiments are possible to examine microtubule assembly events in living cells? And third, what molecules in addition to the primary constituent of microtubules, tubulin, are involved in controlling these events?

I. The Secret: Observations of Microtubule Assembly in V i v a The Importance of Microtubule-Organizing Centers One of the surprises of the close look at cells allowed by electron microscopy was not only the ubiquity of microtubules but also the fact that very disparate structures were formed from them. For instance, the two most prevalent

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microtubule-containing organelles, mitotic spindles and cilia and flagella, are very different in that the first is a labile structure repeatedly and rapidly assembled and disassembled at each cell division, while the others are stable structures with diverse accessory structural components, persistent under treatments such as cold, pressure, and various mitotic poisons (e.g, colchicine) which cause the mitotic spindle to disappear. The question of how the precise regulation of both the temporal appearance and spatial organization of microtubules is achieved immediately became the subject of an enormous amount of research. Crucial to this question is the point of origin of microtubules, and investigation of this revealed yet another microtubule-containing organelle. Light microscopy showed dense structures at the poles of many mitotic spindles and at the base of cilia and flagella-the centriole and the basal body, respectively. Electron microscope studies showed these two structures to be morphologically equivalent, consisting of a cylindrical structure with a basic pattern of nine triplet tubules arranged around a central “cartwheel” structure [see Fulton (1971) and Pitelka (1974) for historical and morphological reviews of these structures]. However, although the outer doublet tubules of the axoneme are direct extensions of the basal body triplet tubules, the microtubules in the mitotic apparatus are not continuous with centriole tubules, and many mitotic spindles, notably those in higher plants, have no centrioles at the poles at all. The polar mitotic microtubules in fact appear to arise out of amorphous electron-dense material which surrounds the centriole (or, particularly in anastral mitotic figures, the polar tubules appear, like the earth in Genesis, to arise out of nothing at all). Furthermore, in addition to the highly structured basal body and the seemingly structureless pericentriolar material there are diverse other structures from which microtubules arise. The useful term “microtubule-organizing center” was coined by Pickett-Heaps (1969) to denote the structures or material from which microtubules initiate. A. THEMORPHOGENESIS OF BASAL BODIES A N D CENTRIOLES

Some of the most elegant descriptions of the process of microtubule assembly in vivo are the original observations of the formation of centrioles and basal bodies which were made as soon as the technology of electron microscopy permitted. These observations have stood in the literature awaiting complete interpretation from biochemical data (but the biochemical technology in this area has not quite caught up yet). Early on, centrioles were postulated to be selfreplicating organelles; the fact that new centrioles often arose close to mature ones, the difficulty of discerning intermediate forms, and the complexity of the organelle all seemed to indicate that they must be autonomous-‘ ‘reproducing” by dividing-r at least that the foramtion of a new centriole required the presence of a mature one and was directed by it. Even after it was realized that they

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could in fact arise de novo (Dirksen, 1961), the idea persisted. This problem has been discussed at length by Pickett-Heaps (1969, 1971, 1975a). In fact, centrioles and basal bldies in vivo arise out of electron-dense material of uncertain structure and composition, which may or may not be associated with a mature organelle. Studies on basal body and centriole morphogenesis in various vertebrate tissues have yielded similar reconstructions of the development of the mature organelle from precursor structures which are in turn derived from amorphous or fibrous electron-dense masses in the cell interior. Dirksen and Crocker (1966) examined the formation of centrioles in differentiating ciliated cells of fetal rat tracheal epithelium. Subsequently, Dirksen (197 I ) described centriole morphogenesis in the ciliated epithelium of mouse oviduct, which took place in a brief period after birth but was not synchronized, even within a single cell. Centriole morphogenesis was similar in both tissues, proceeding through four sequential stages. First, in the center of the cell clusters of electron-dense fibrillar masses 60-80 nm in diameter appeared, which later became organized into larger aggregates 100-700 nm in diameter in cleared areas of the cytoplasm. The structure of these aggregates was difficult to ascertain because of their electron opacity; they were usually amorphous, but occasionally microtubules or a mature centriole was present. As shown in Fig. 1 , these electron-dense masses then gave rise to procentrioles, becoming surrounded by as many as nine immature centrioles in various stages of development. The first stage in centriole development was the appearance of an annulus or disk of indistinct structure which later developed the typical centriolar cross section. The microtubules of the procentrioles were often connected to the central mass by fine strands. Concomitantly with centriole maturation the central mass of material either disappeared or became clearly hollow, suggesting that the centrioles had in fact been formed from this material. Occasionally procentrioles were found surrounding mature centrioles. In neither of these studies was Dirksen able to define clearly the relationship between mature centrioles and the amorphous material which appeared to be the earliest procentriole precursor. Steinman (1968) followed the differentiation of ciliated cells in epidermis and trachea of Xenopus faevis. His electron micrographs show dense, amorphous masses around which are clustered smaller electron-dense masses which apparently become procentrioles, cylinders 150 nm in diameter with nine single tubules. These structures appeared deep in the cytoplasm of the cell near the nucleus; correlated with their disappearance multiple mature centrioles 200 nm in diameter with a typical cross-sectional structure appeared in the apical cytoplasm. The inference was that the procentrioles rapidly matured and migrated to the cell surface, although no intermediate forms were found. The centrioles then aligned at the apical surface, ciliary shafts grew out of them, and basal body accessory structures such as rootlets appeared. Small, electron-dense bodies close to the base of the basal body and the growing cilium were interpreted as

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Flc. I . Centriole morphogenesis in developing mouse oviduct epithelium. Three centriole generative complexes, each consisting of the central electron-dense precursor structure (clearly “hollow” at this stage of development) surrounded by nine developing procentrioles. Two procentrioles in the upper right complex were not in the plane of section. X47.450. Reprinted from Dirksen (1971), with permission.

axoneme precursors; similar but less electron-dense material was interpreted as the source of the basal body rootlets. The various morphological events described did not occur in synchrony; a single cell often contained both basal bodies with growing axonemes and unaligned centrioles still in the supranuclear cytoplasm. Attachment of the centriole at the cell surface appeared to be the signal for assembly of the axoneme; Steinman saw only two cases in which an unaligned centriole bore an axoneme. A similar pattern of centriole formation was observed by Kalnins and Porter (1969)during ciliagenesis in chick tracheal epithelium. Basal bodies formed in a

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region of fibrous material associated with the cell centrioles; procentrioles arose in a cluster around a core of dense material sometimes containing a cylindrical structure resembling a centriole. The earliest procentrioles were short cylindrical structures without microtubules; tubules appeared first as singlets, progressing to doublets and triplets. The basal bodies then elongated, matured, and migrated to the cell surface, where the cilia were assembled. In this tissue basal body development appeared to be synchronous. Sorokin (1968) studied the formation of centrioles and subsequent ciliagenesis in fetal rat lungs. He observed two patterns of morphogenesis. The first occurred early in development in interphase pulmonary cells of all types and involved the centrioles subsequently associated with the mitotic spindles. Usually one, but sometimes as many as eight, new centrioles arose directly adjacent (at right angles) to the wall of a preexisting centriole, first as procentrioles; during development these annular structures lengthened into cylinders. The triplet tubules started as singlet tubules to which the second and third tubule walls were added. These daughter centrioles were released into the cyoplasm when they had matured about halfway. An interesting and somewhat puzzling aspect of the regulation exerted in this system is Sorokin’s observation of the occasional growth of transitory rudimentary cilia from one of the pair of centrioles in differentiating fetal pulmonary cells. These are transitory embryonic organelles and are rare in adult tissues; they are distinguishable from adult cilia because they are incompletely formed, especially at the tips, and the central pair of tubules is lacking. It is tempting to interpret this observation as a lapse in control, that is, a centriole growing a cilium. The second pattern of centriole morphogenesis Sorokin observed was the acentriolar pathway, which occurred late in the fetal period and involved formation of the basal bodies of ciliated epithelial cells. Masses of fibrous, granular material accumulated in close proximity to Golgi elements in the apical cytoplasm. The fibrogranular areas increased in size and apparently condensed into spherical masses, around the periphery of which procentrioles arose. Ultimately the mature centrioles aligned in rows underneath the apical end of the cell membrane. The signal for ciliagenesis appeared to be placement of the basal bodies at the cell membrane; accessory structures such as satellites and roots appeared, and then the ciliary shaft grew out. The ciliated border cilia grew faster than the rudimentary cilia and had the complete 9 + 2 cross-sectional pattern. The mature basal bodies did not ususally have procentrioles associated with them, but Sorokin observed this in a few cases and concluded that the capacity for formation of an associated organelle is retained in basal bodies produced by the acentriolar pathway. A similar dual set of pathways for centriole morphogenesis was observed by Anderson and Brenner (1971) in rhesus monkey oviduct. After ovariectomy, deciliation and loss of basal bodies occurred in ciliated epithelial cells of the

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oviduct; estrogen treatment caused redifferentiation of these cells. The major pathway was acentriolar, in which basal bodies were formed from procentrioles generated from aggregates of fibers with no structural resemblance to centrioles. First, fibrous granules of 40-60 nm in diameter appeared in the apex of future ciliated cells. These granules usually were aggregated into sheets or spheres but sometimes were dispersed in the cytoplasm and sometimes associated with the nuclear membrane; occasionally a few microtubules were present among them. The fibrous material appeared to fuse to form the procentriolar ring. These investigators found the stages in procentriole development difficult to interpret. The central cartwheel structure formed before the triplet tubules; the A tubules then formed in sequence around the ring, but tubule growth after that was not synchronous. The transition from procentriole to basal body involved lengthening of the procentriolar cylinder and migration to the cell surface, followed by the addition of accessory structures and changes in the internal cartwheel arrangement. The ciliary shaft was assembled after the basal body reached the cell surface. The second, minor, pathway Anderson and Brenner observed was the centriolar pattern, in which 1 to 10 procentrioles formed at right angles to walls of the preexisting pair of centrioles. As in the acentriolar pathway, these procentrioles also appeared to form from amorphous electron-dense material which in this case surrounded the walls of the mature centrioles and in which their bases were embedded. The maturation pattern of these centrioles was the same as the acentriolar pathway; mature centrioles were released into the cytoplasm and apparently migrated to the surface along with those formed through the acentriolar pathway. Since all procentrioles associated with any one mature centriole were always at the same stage of development, it was inferred that morphogenesis at any one site was synchronous. These workers also observed occasional formation of transitory rudimentary cilia, often with abnormal and incomplete microtubule patterns in cross section. These appeared several days before the main ciliature formed. Dippell (1968) detailed the sequential assembly of basal body structure in Paramecium, as shown in Fig. 2. Basal body assembly in this ciliate is under very tight spaital and temporal control. New basal bodies form immediately anterior to an existing adult basal body but separated by a few hundred angstroms; the majority form in a 20-minute period, 50 minutes before the completion of cell division. Dippell did not find the diverse electron-dense aggregates reported in the vertebrate studies. The first structure she observed was the direct precursor of the basal body cylinder, a flat disk of dense, fibrous material with no discernible substructure. Microtubule assembly always started at a specific point and proceeded around the disk, forming a ring of nine singlet tubules. B-tubule assembly then started, often before completion of all the A tubules and not always sequentially. Dippell observed short fibers between adjacent tubules in the beginning stages, which later disappeared; she speculated that these fibers functioned in

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FIG. 2. Morphogenesis of basal bodies in Parumetlium. (a) Formation of the first tubule. 140.000. (b) A ring of singlet tubules partially completed. X 138,000. (c) Formation of the B tubules; doublets are partially completed. X 140,000. (d) Adult basal body (in cross section) with a new basal body anterior and at right angles. X140,ooO. (e) Four generations of basal bodies. X

~68,000. Reprinted from Dippell (1968), with permission.

development of the ninefold symmetry. After the triplet tubules formed, the other internal structures of the central cartwheel appeared. New basal bodies moved to the cell surface, where the cilia ultimately formed. Dippell often observed cytoplasmic microtubules associated with both growing and mature basal bodies; these were never continuous with basal body microtubules but inserted or originated in dense material around the basal body. The position of an existing basal body determined the position of the new one; Dippell occasionally ob-

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served new basal bodies forming near immature ones that had not yet moved toward the cell surface. A sequence of events similar to that reported by Dippell was observed by Johnson and Porter (1968) during formation of basal bodies at cell division in the unicellular biflagellate alga Chlamydomonas reinhardii. The earliest stage in basal body development they observed was a ring of nine singlet tubules. Cavalier-Smith (1974) reported that basal body morphogenesis in this organism, as in the vertebrate studies, could proceed through two alternative pathways. In vegetative cells flagella are resorbed prior to cell division, but the basal bodies remain attached to the plasma membrane.. A new basal body arises close to the old one, physically attached to the wall of the old one by amorphous material. In zygotes the basal bodies and associated structures as well as the flagella disappear, and there is no trace of the flagellar apparatus throughout zygospore maturation; basal bodies are then assembled de n o w close to the plasma membrane during zygospore germination. Basal body assembly is apparently the same in both the vegetative and sexual cell cycles, the first recognizable intermediate being the ring of nine singlet tubules around a central cartwheel structure. Cavalier-Smith also occasionally observed disklike structures which may have been earlier stages. The B and C tubules were apparently added on together, followed by elongation of the cylinder and finally the appearance of accessory structures such as roots and striated connections. As in other organisms, flagellar outgrowth began only after complete assembly of the basal body and its attachment to the plasma membrane. The cellular control over the number of flagella and accessory structures appears to be more stringent than control over the number of basal bodies; daughter cells may have four basal bodies but rarely more than one pair of flagella and associated structures. During mitosis, when the basal bodies are free of flagella, their identification as such or as centrioles is ambiguous. However, although they may sometimes be physically close to the mitotic apparatus, they do not function as mitotic centers (Cavalier-Smith, 1974; Coss, 1974; Johnson and Porter, 1968). Johnson and Porter (1968), however, suggested that their position may be directly involved in determination of the plane of cell division. Gould (1975) examined intermediate structures in preparations of basal bodies isolated from Chlamydomonas, as shown in Fig. 3. He also confirmed that the primary component of basal bodies is in fact the structural microtubule protein tubulin. Gould suggested that his results “revive” the possibility of the generation of new basal bodies by direct nucleation from an existing basal body. His electron micrographs showed that isolated basal body pairs have two probasal bodies attached to them through a complex of associated structures; just after cell division the probasal body consists of a ring or annulus of nine “dots” (rudimentary microtubules) connected to the mature basal body by fibers. This annulus

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FIG.3. Isolated basal bodies from C. reinhardii. Stages in morphogenesis. (a) Basal body pair isolated after cell division with two probasal bodies each consisting of an annulus of nine components, two of which are connected by long fibers to the proximal end of one of the mature basal . c) Basal body pairs isolated before cell division, showing elongation of the bodies. ~ 3 3 , 0 0 0 (band . Basal bodies isolated at the onset of mitosis, showing completion of probasal bodies. ~ 3 3 , 0 0 0 (d) probasal body maturation. X33.000. Reprinted from Gould (1975), with permission.

was not visible in thin sections of cells, either because it is too thin or possibly because it is unstable. Just before the next cell division the probasal body elongates and forms a new mature basal body; at this time Gould was able to distinguish that each dot is in fact a triplet microtubule, several of which are connected to the proximal end of the mature basal body by fibers. Gould interpreted his data to mean that development of the probasal bodies proceeds through simultaneous assembly of the A, B, and C tubules, but that the A tubules elongate some-

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what faster than the others so that the growing end of the probasal body might appear in cross section to represent a nine-singlet tubule structure. Basal body morphogenesis has been observed in a variety of other organisms; in many cases it appears to occur very rapidly without obvious intermediate forms. Millecchia and Rudzinska (1970) observed that basal body morphogenesis in the suctorian Tokophyryu infusionum proceeded similarly to that in Purumecium; new probasal bodies arose adjacent to mature ones, the A tubules being formed first. Allen (1969) reported that the earliest stage he observed in basal body morphogenesis in Tetruhymenu was a structure containing nine singlet tubules. Fulton and Dingle (1971) reported that no structure resembling a centriole or basal body could be found in the ameboid form of the ameba-flagellate Nuegleriu but that during transformation to the flagellated morphology basal bodies appeared about 10 minutes before the flagella themselves. These investigators found no structural precursors, although it appeared that basal bodies arose within the cytoplasm and then moved to the cell membrane. Outka and Kluss (1967) similarly did not observe basal body precursors in amebas of the ameba-flagellate Tetrumitus rostrutus, but during the ameba-to-flagellate transformation they found basal body-like structures in association with the nuclear membrane or with dense bodies in the cytoplasm. More recently, Ash and Stephens (1975) found that, during ciliagenesis in the gill of the bay scallop, Aequipecten irradiuns, the formation of basal bodies combined two aspects of morphogenesis seen separately in other studies. First, as in vertebrate studies, they observed the appearance of a complex of dense granules but thereafter, as in Naegleriu, mature basal bodies appeared very rapidly with no obvious organized intermediate stages. Basal body formation was not synchronous within a cell. As in other systems, the elaboration of accessory basal body structures and ciliagenesis was initiated after movement of the mature basal body to the cell surface. The above-mentioned studies, although differing in detail, give a fairly unified idea of the sequential assembly of basal bodies and centrioles. There are, however, several examples of different-I am tempted to say stranger-modes of formation of basal bodies, particularly in the development of multiple flagellated plant sperm (see reviews by Hepler and Palevitz, 1974; Paolillo, 1975). For example, Mizukami and Gall (1966), and more recently Hepler (1976) and Myles and Hepler (l977), have described spermiogenesis in the fern Marsilea. Marsilea sperm have over 100 flagella, the basal bodies of which arise from the blepharoplast, a spherical structure 0.8 pm in diameter formed before the last cell division in developing spermatids from a solid sphere of material of moderate electron density and complex substructure. This structure separates into two blepharoplasts which appear to serve as microtubule-organizing centers during assembly of the mitotic spindle, although they do not remain as the focal points of the mitotic tutubles after prophase. During metaphase the blepharoplast becomes hollow, and at this time the walls can be seen to consist of radially

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arranged, closely packed procentrioles. After cell division, the blepharoplast fragments and the procentrioles are released into the cytoplasm; they elongate and migrate to the cell surface, and the flagella are assembled. Similar events occur during spermiogenesis in the cycad Zamia, in which the sperm have upward of 20,000 flagella and the blepharoplast is huge, 10 p m in diameter (Mizukami and Gall, 1966), and in Equisetum (Duckett, 1973). B. THEASSEMBLYOF

THE

AXONEME OF CILIAA N D FLAGELLA

The assembly of the cilia or flagella axoneme may be thought of as a continuation of the process initiated by the formation or activation of the microtubuleorganizing center, in which the formation and placement in the membrane of the basal body are the next steps. The location and orientation of the axonemal shaft are specified by the location and orientation of the basal body; furthermore, although transitional structures are formed at the junction of the axoneme and the distal end of the basal body, the outer nine doublet tubules of the axoneme are specified by the A and B tubules of the basal body triplet tubules. The positioning of the basal body at the cell surface appears to be achieved by fitting either the basal body itself or some of the accessory structures into a specific site at or near the membrane [for example, Cavalier-Smith (1974) showed that in Chlamydomonas the basal body is attached to the cell membrane by the transitional fibers]. In some cases new basal bodies arise next to old ones, which places them close to the site they will occupy when mature; in others, the basal body arises in the cytoplasm and migrates to the cell surface. A mechanism may be envisioned in which the basal body travels along the cell surface until it “locks” into the specific site, as has been shown for another organelle, the trychocyst, in Paramecium (Pollack, 1974; Sonnebom, 1974). Thus control over the outgrowth of the axoneme is primarily temporal. The control over timing may be complex. For example, Nanney (1975) observed that in Tetrahymena new basal bodies were assembled adjacent to existing ones a complete cell cycle before they become ciliated. As discussed above, Sorokin (1968) and Anderson and Brenner (1971) observed that, when ciliagenesis took place too early, the resulting axonemes were transitory and defective. The regulation of ciliagenesis involves not only the initiation of axoneme assembly, but also determination of the final length of the axonemal shaft and in many cases disassembly of the axoneme as well, as many cells resorb their axonemes at some point in their life cycle (e.g., Chlarnydornonas before division). Electron microscope studies of basal body morphogenesis and subsequent ciliagenesis suggest that amorphous or fibrous material, similar to that identified as the precursor material for centriole formation, is the precursor material from which the axoneme is assembled (Ash and Stephens, 1975; Cavalier-Smith, 1974; Steinman, 1968). Outka and Kluss (1967) observed that during flagellar

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growth in Tetrumitus rostrutus a membrane-bounded vesicle which became filled with fibrillar material formed at the tip of the shaft; this vesicle disappeared as the flagella grew, suggesting incorporation of the material into the axoneme. The regeneration of amputated cilia and flagella has provided an ideal model system. Initial studies by Rosenbaum and Child (1967) and Rosenbaum and Carlson (1969) showed that, after amputation of the flagella of Ochromonas, Euglenu, and Astusiu or the cilia of Tetruhymena, there was a lag during which protein synthesis appeared to be required; thereafter, elongation proceeded at a rate which constantly decelerated as the final length was approached. Rosenbaum et ul. (1969) and Coyne and Rosenbaum (1970) extended the observations to Chlumydomonus. They reported that, in a single cell, assembly of one flagellum could occur at the same time as disassembly of the second. If only one of the two flagella were removed, the remaining one shortened at the same time as the amputated one began to elongate. When protein synthesis was inhibited with cycloheximide, the regenerating flagella reached only about one-third of the normal length; in cells with only one amputated flagellum, the length of the two flagella finally regenerated in the presence of cycloheximide depended on how long the stump of the remaining one had been. Tamm (1967) also studied control of flagellum length. He found that the initial rate of elongation of regenerating flagella of Puranema depended on the length of the stump of the amputated flagellum; that is, the longer the stump of a partially amputated flagellum, the slower the rate of regeneration. The final flagellum length was constant. Thus the cell appeared to “know” how long the flagellum was. Another interesting example was reported by Kerr (1972), who studied elongation of the flagella of the slime mold Didymium nigripes. This organism has two flagella of unequal length; these not only elongated at different rates but reached the maximum rate of elongation at different times. Cycloheximide caused flagellar growth to cease. She concluded from the complexity of the growth kinetics that the mechanism of regulation of elongation did not simply depend on the diffusion of subunits to the growing end of the axoneme nor was it a simple function of the total length. Bums (1973) observed that the regeneration of cilia in gastrulae of the sea urchin Tripneustes grutillu also took place after a lag and proceeded at a decreasing rate so that the final length was reached asymptotically. Interestingly, “animalized” cilia, which were up to three times the length of normal cilia, elongated at the same initial rate but had an extended period of elongation. Thompson et ul. (1974) observed the regeneration of Tetrahymena cilia in a scanning electron microscope study. They reported that ciliagenesis proceeded synchronously in recently divided cells but asynchronously in others and that, although oral and somatic cilia elongated at the same time, the rate of elongation of oral cilia was faster. Rannestad (1974) also investigated the regeneration of

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cilia in Tetrahymena. He found that, similar to the situation in Chlamydomonas, if only 75% of the cilia were removed, the remainder were resorbed before regeneration of the whole ciliature; again, however, some cilia began to regenerate even before all the remaining old cilia were completely resorbed. Regeneration required protein synthesis or resorption of old cilia; partially but not completely deciliated cells regenerated cilia in the presence of cycloheximide. However, the volume of cilia regenerated (about 90% of the cilia) in the presence of cycloheximide was greater than the volume resorbed (about 25%). Rannestad concluded that material contributed by the resorbed cilia potentiated the assembly of cilia from pools of already existing precursors. Certainly his results and those of Rosenbaum and co-workers indicate both the need for synthesis of some but not all axoneme components and the possibility of reutilization of resorbed components. All these studies illustrate the complexity of control and the possibility of separate regulation of similar organelles in a single cell. An obvious possibility for part of the control of assembly of the axoneme is the synthesis of a certain cornponenet in limiting amounts. This may occur during ciliagenesis in the sea urchin embryo. Regulation is not, however, achieved through the modulation of levels of tubulin. In an early study Auclair and Siege1 (1966) showed that even repeated regeneration of the cilia of gastrula stage embryos could take place in the presence of actinomycin D and puromycin (which decreased total protein synthesis by 90%), thus suggesting that cilia were assembled from a preexisting pool of subunits. Work in several laboratories has shown that a tubulin pool is made during oogenesis and is subsequently maintained throughout early development (Bibring and Baxandall, 1977; Borisy and Taylor, 1967b; Cognetti et a/., 1977; Raff, 1975; Raff and Kaumeyer, 1973; Raff et al., 1971, 1972, 1975). Work by Stephens (1972b, 1977b) has shown that, while pools of tubulin and several other axoneme components are maintained and synthesized at constant rates, the initiation of ciliagenesis is accompanied both originally and during regeneration by the synthesis of limiting amounts of two minor components. Several studies have examined the synthesis of tubulin (but not of other axoneme proteins) during the growth of cilia or flagella in other organisms. Nelson (1975) showed that, as in the sea urchin, ciliagenesis in Tetrahymena utilized preexisting stores of tubulin. In some cases, however, the onset of ciliagenesis is marked by tubulin synthesis. Dirksen and Staprans (1975) found that, although there were tubulin pools present prior to ciliagenesis in the mouse oviduct, 90% of the tubulin in 3-day-old and 75% in 5-day-old mouse oviducts was newly synthesized. Weeks and Collis (1976) found that tubulin synthesis was induced when flagella were removed from Chlamydomonas cells. However, Weeks et al. (1977) observed that the induction of tubulin was not dependent on the actual utilization of tubulin stores during flagella regeneration, both because

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of the timing of the induction-it took place before the tubulin pools were depleted-and because inhibition of regeneration with colchicine did not inhibit the induction. These investigators suggested that a feedback-type mechanism might operate once tubulin levels reached a certain point since, when flagella regeneration was blocked with colchicine, the induced synthesis decreased earlier than in the controls. Finally, Fulton and co-workers found that tubulin utilized in the formation of flagella was specifically synthesized during transformation from the ameboid to the flagellate form in Naegleria gruberi (Fulton and Kowit, 1975; Fulton and Simpson, 1976; Kowit and Fulton, 1974a,b). It is clear that overall assembly of the flagella or cilia shaft proceeds from the basal body outward. Oddly enough, however, it is still unclear whether microtubule assembly takes place distally (by the addition of subunits at the growing tip), as has been implicitly assumed in many discussions, or proximally (by the insertion of subunits at the basal body end). Early studies of flagellar growth in Ochromonas and Chlamydomonus by light microscope autoradiography indicated that, while most of the radioactivity from radioactively labeled amino acids incorporated into regenerating flagella appeared in the distal half of the organelles, some label always appeared in the proximal half of the shaft (Rosenbaum and Child, 1967; Rosenbaum et al., 1969). The possibility existed that some or all of the label seen in the proximal part of the flagella was due to proximal growth of the membrane or other nonmicrotubule parts of the axoneme. Witman (1975) repeated these experiments, pulse-labeling regenerating flagella of Chlamydomonas; he then isolated the flagella and purified the outer nine doublet fibers and examined the incorporation by electron microscope autoradiography . He found that 65% of the grains appeared over the distal half of the flagella and 35% over the proximal half; he concluded that the outer doublet tubules are primarily assembled at the tip, but the results were still ambiguous. More recently, Dentler and Rosenbaum (1977) reexamined flagellar growth and shortening in Chlamydomonas by electron microscopy and concluded that, while the outer doublet tubules appeared to be assembled by distal addition of subunits, the central pair of singlet microtubules appeared to be assembled by proximal insertion of subunits. They reached this conclusion partly through in vitro experiments. A central “cap” attaches the distal end of the central microtubules to the tip of the flagellar membrane; this structure stays in place throughout both elongation and resorption of the flagellum. There are also distal filaments present on the ends of the A tubules of the outer doublets. When isolated axonemes were attached to grids and incubated with purified brain tubulin, assembly occurred at the distal ends of the A tubules (in spite of the distal filaments), but the cap on the central pair prevented assembly. However, if the cap was lost during the isolation procedure, assembly of tubulin subunits took place at the distal ends of the central pair. Dentler and Rosenbaum pointed out that assembly of the central microtubules might take place distally in vivo even in the presence of the cap, but

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that attachment to the grids might in some way prevent the process in vitro. Nevertheless, their results reopen the old question concerning the mechanism of assembly of cilia and flagella in vivo. Control of the disassembly of flagella is a problem that has been treated as the obverse of assembly, although the regulatory mechanisms may in fact be different. In most of the studies cited above, flagellar regression, like growth, appeared to take place gradually and sequentially at the tip, in the reverse order of assembly (see, for example, Cavalier-Smith, 1974). A variation on this was observed by light and electron microscopy by Ishigami (1977) in the myxomycete Sternoniris pallida during transformation from the flagellate to the ameboid form. Within 60 seconds the entire axoneme was resorbed into the cell body by fusion of the flagellar and cell membranes. At first the entire axoneme was visible in the cytoplasm (sometimes still beating); then it disintegrated over about 90 minutes-B tubules first, and then A tubules, spokes, and central tubules in that order. Cellular control over the disassembly process was indicated by the fact that cytoplasmic microtubules and the basal bodies apparently persisted much longer. This organism has two flagella, a long one which beats and a short one which does not; the short one is always retracted first.

c. THEMORPHOGENESIS OF MICROTUBULE-CONTAINING ORGANELLES IN PROTOZOA In addition to simple cilia and flagella, many specialized protozoan organelles are constructed from specialized cilia or other patterned arrays of microtubules. The oral apparatus of Tetruhyrnena consists of closely arrayed cilia forming four “membranes” which are the basis of the organism’s generic name, plus additional microtubules. These specialized cilia are under different control than the regular somatic ciliature. Williams and Frankel (1973), Williams and Nelson (1973), and Williams (1975) have described the assembly of this organelle. A new oral apparatus is formed at cell division and also under conditions of amino acid starvation, when it is resorbed and then redifferentiated or replaced. At cell division, a new oral apparatus, which will become the oral apparatus in the posterior daughter cell, forms behind the future site of cell cleavage; in oral replacement the new apparatus forms just anterior to the site of the old one, which is resorbed. During resorption of the old apparatus the cilia are apparently withdrawn into the cytoplasm and disassembled; meanwhile basal bodies proliferate rapidly, new basal bodies being formed adjacent to mature ones. Very little protein synthesis is necessary for oral replacement; these investigators speculate that basal body proliferation may be controlled by the synthesis of a small number of regulatory proteins. The complexity of microtubule regulation is vividly obvious in this organism. During the final quarter of the cell cycle, a new oral apparatus begins to form in the posterior of the cell. The old apparatus

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meanwhile migrates to the cell surface (i.e.. the “mouth” flattens out) and undergoes regressive changes; the undulating membrane cilia are lost, and the buccal membranelle cilia shorten. Thus for a time two identical microtubulecontaining structures are present in a single cell, one in the process of assembly and the other in the process of disassembly. Ultimately the regressing old apparatus and the forming new apparatus reach the same stage, and then during cell cleavage both develop synchronously to the final completely differentiated structure. Tucker (1970, 1971) described the morphogenesis of the cytopharyngeal basket, the mouth structure of another ciliate, Nussula. This organelle is constructed of a cylinder of rods (the basket units) each of which is composed of a bundle of microtubules aligned in parallel along their long axes. Similarly to the situation in the oral apparatus of Tetruhymena, disassembly of the old basket occurs simultaneously with assembly of two new ones. During interphase the cytopharyngeal basket is located immediately anterior to a pore. Before cell division the old basket detaches and “floats” into the cytoplasm, where it breaks down. Meanwhile, at the anterior of the dividing cell a new basket forms at the site of the old basket, and a new pore appears just behind it; at the posterior, a second new basket forms just in front of the old pore. Thus in the two resulting daughter cells, the relative arrangement of basket and pore is maintained, their positions being determined by membrane sites. Morphogenesis of the basket is extremely complicated. At the proximal end of the basal bodies (which bear cilia at their distal ends) a structure called the laminated cap appears. It is an electron-dense, layered plate from which microtubules in a highly ordered parallel array emerge; Tucker has observed that the microtubules appear to arise from the middle layer of the cap and pass through the bottom layer. Each array ultimately forms one basket unit, as shown in Fig. 4. The units detach from the basal bodies but retain the cap structure; they then align in a row which curls in from both ends, forming the cylindrical basket. During the rolling up of the basket a reticulum of microtubules forms just beneath the pellicle in the region of the basket. Most of these tubules are caught inside the basket and resorbed; they possibly represent a “scaffold” around which the basket is formed. Occasionally Tucker observed mistakes; sometimes one of the units moved too fast or too slow and was “caught” in the middle of the forming basket or else left behind in the cytoplasm. The units left behind persisted in the cytoplasm near the basket until the next cell division, when they were disassembled along with the old basket. The arrangement of the units in mature baskets does not reflect the arrangement of the basal bodies near which the laminated cap and associated microtubules arose. The basal bodies themselves remain in place, the cilia they bear becoming the paroral ciliary row. The control over ciliagenesis also is complex in this organism; different classes of cilia persist andor are generated at different times in the cell cycle. Tucker pointed out that a mechanism of microtubule-nucleating

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FIG.4. Morphogenesis of the cytopharyngeal basket of Nussula. Longitudinal section through the top of one basket unit showing the microtubules, laminated cap, basal body, and cilium. x I13,OOO. Reprinted from Tucker (1970). with permission.

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sites plus diffusible subunits could not generate the events he observed. Rather, this complex control must also involve localization of the structural components or of the signaling or regulatory factors, or both. This interesting system has been further described more recently by Tucker et al. (1975) and Pearson and Tucker (1977), who observed in detail the bundles of microtubules arising from the laminated cap. The tubules in the units are packed in a hexagonal pattern; the pattern is disrupted if the organelle develops in the presence of colchicine. By comparing the development in the absence and in the presence of colchicine, they concluded that the pattern is determined by the specification of the site of origin of each tubule; that is, the cap comprises a microtubule-nucleating template. The microtubules in the bundle are subsequently connected by cross-linking structures which maintain the hexagonal cross-sectional pattern permanently. However, it is the nucleating sites in the laminated cap and not the links which establish the pattern, and the pattern is at first simply maintained by the close packing of the growing microtubules. Thus the dense sheets which arise at the proximal end of the basal bodies are microtubule-organizing centers in the classic sense. There are no data as yet on the biochemical nature of this material or how it is regulated. The function of the adjacent basal bodies remains obscure; it seems possible that they may serve as a means for localizing formation of the microtubule bundles rather than participating in generating them. Tucker (1977) reviewed the general problem of how the shape and pattern of microtubule arrays can be specified and concluded that, while the patterns themselves may be specified in several ways (e.g., self-linking of tubules, simple close-packing arrays, or template-type nucleation), additional regulatory mechanisms including compartmentalization of components must also exist. A third interesting protozoan model system is the formation and distribution of microtubules in Ochromonus, described in an elegant study by Bouck and Brown (1973a,b; Brown and Bouck, 1974). This organism is a biflagellate which maintains a teardrop shape although it lacks either a pellicle or cell wall; at the anterior of the cell is a “beak” from which the flagella emerge, and at the posterior is a narrow extension of the cytoplasm, the “tail.” Bouck and Brown have observed that the shape of the cell is maintained by two sets of cytoplasmic microtubules which appear to be under separate cellular control even though the microtubules in the two sets are physically very close to each other. The axoneme microtubules are the only microtubules directly connected to the two basal bodies, but both sets of cytoplasmic microtubules are associated with several fibrous structures which are themselves associated with the basal bodies. There are two striated fibers; one connects the two basal bodies and the second, the rhizoplast, shown in Fig. 5, extends from the region of the basal bodies toward the nuclear membrane. The rhizoplast consists of amorphous electron-dense material arranged in cross-

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FIG. 5 . Rhizoplast microtubules in Ochrornonas. (a, During interphase. The rhizoplast extends over the nuclear surface and is associated with a Golgi complex on the upper surface, while the microtubules extend from the lower surface. X 56,000. (h) During mitosis. The basal body, to which the mitotic rhizoplast remains attached, can be seen in the upper left. x20.800. Reprinted from Bouck and Brown (1973a). with permission.

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bands; the upper surface is associated with the Golgi complex, and from the lower surface extends the set of microtubules which determine the shape of the tail. There are also several unstriated fibers associated with the basal bodies. One set of these, the kinetobeak fibers, consists of amorphous material plus arrays of microtubules which begin in parallel and then curve and splay outward; these microtubules determine the beak shape. The independent regulation of these two sets of microtubules was determined from the fact that they were differentially sensitive to pressure and colchicine and that both disassembly and assembly of the two sets occurred separately in a specific sequence. In cells exposed to colchicine or put under increased pressure, the cytoplasmic microtubules disassembled with a concomitant loss of the typical cell shape, the process proceeding from posterior to anterior. When colchicine was removed or the pressure released, the microtubules reassembled and the cell shape was restored. Reassembly of the kinetobeak tubules occurred first, within 10 minutes, and after 15-30 minutes the rhizoplast microtubules reassembled. The rhizoplast itself was not altered by microtubule-depolymerizing treatments nor were the relative locations of the rhizoplast and the kinetobeak nucleation regions. A similar sequence of disassembly occurs before cell division. During mitosis the spindle microtubules are attached to a structure identical to the rhizoplast. After division the two sets of microtubules reappear, forming the beak first and then the tail. Bouck and Brown concluded that in Ochrornonas the two nucleation sites control the timing, orientation, and pattern, as well as position, of the microtubule arrays. Finally, the axopodia of heliozoan protozoa have provided a model system for study of the regulation of labile microtubules. These organelles are rods resembling miniature sea urchin spines. Tilney and co-workers showed that microtubules were responsible for both the production and maintenance of the form of the axopodia of Echitiosphaerium (formerly called Actirzosphaerium), which consist of microtubules packed in parallel longitudinally with a cross-sectional pattern of an interlocking double spiral (Tilney, 1968a,b, 1971a; Tilney and Byers, 1969; Tilney and Porter, 1965, 1967; Tilney et al., 1966). While the axopodia are permanent features of the protists that bear them, they are dynamic organelles in that the cell constantly changes their length during feeding or as a consequence of changes in the environment, by assembly and disassembly of the component microtubules. Experimentally they could be induced to disassemble by the application of cold, pressure, or colchicine, or to reassemble after release from these treatments, and were stabilized by deuterium oxide, as is typical of labile microtubule systems. Tilney and co-workers found that assembly and disassembly of the microtubules takes place sequentially at the tip of the organelle and that the subunits are reused during reassembly of the organelles. They concluded that in this organism the cross-sectional pattern is determined by specific cross-bridging of the growing microtubules.

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A mechanism explaining how the cross-sectional microtubule pattern in Echinosphaeriurn axopodia is generated by self-linkage was proposed by Roth

and co-workers; they formulated the “gradion” hypothesis, according to which the binding of the first linker to a particular tubulin subunit allows the binding of the second, and so on, in an allosterically induced conformational gradient (Roth and Pihlaja, 1977; Roth et al., 1970). Ockleford and Tucker (1973) observed contraction and regrowth of the axopodia of a related organism, Actinophrys sol. They reported that as in the assembly of flagella, the rate of elongation of the axopodia decreased as the final length was approached. Regrowth of a single axopodium contracted during feeding proceeded at the same rate as regrowth of the entire set of axopodia after cold treatment. Echinosphaeriurn and Acrinophrys belong to the Actinophryida, heliozoa in which the axopodia arise independently either at the nuclear membrane or in the cytoplasm. In the Centrohelida, the axopodia arise from the centroplast, a dense structure in the center of the cell, and the microtubules composing them are in hexagonal or triangular cross-sectional patterns (see Bardele, 1977, for a brief review of the heliozoan groups). Tihey (1971b) studied reformation of the axopodia of Ruphidiophrys after disassembly of the microtubules by cold treatment; the microtubules indeed initiated from the centroplast (which in this organism is highly electron-dense and without discernible substructure), but pattern formation did not begin until the microtubules had extended some distance from the centroplast. The pattern, as in Echinosphaeriurn, was generated by specific bridging of the microtubules; each microtubule could be bridged to four others. Tilney concluded that, once one bridge was attached, the position of the others was determined, similar to the mechanism proposed by Roth et al. (1970). Bardele (1977) concluded that, while in some Centrohelidan heliozoa the axopodial pattern was specified by self-linkage, as observed by Roth and Tilney, in others it appeared that a template-driven nucleation followed by bridging occurred, similar to the case in Nussula. Bardele’s electron micrographs of the centroplast of species of Heterophrys show that it is a “hollow” sphere containing an electron-dense striated disk(s) in the center; the axopodia microtubules radiate from the electron-dense surface of the sphere, but their mode of attachment is unclear because their proximal bases are surrounded by amorphous electron-dense material. A N D DISASSEMBLY O F LABILE MICROTUBULE ARRAYS: D. THEASSEMBLY THEMITOTICAPPARATUS

The literature on mitosis is at least as extensive as the literature on microtubules, part of which it includes. The classic review by Mazia (1961) gives a

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comprehensive historical view of observations of mitosis. Consideration of the mitotic apparatus has focused on two problems. One is the mechanism by which chromosome separation is achieved. The two basic theories of chromosome movement are, first, the dynamic equilibrium theory proposed by Inoue (1964) and Inoue and Sat0 (1967), whose elegant studies using birefringence microscopy established the labile nature of the mitotic fibers and who suggested that chromosome movement results directly from equilibrium assembly and disassembly of microtubules from a pool of subunits, and second, the sliding-filament model proposed by McIntosh et al. (19691, who suggested that movement occurs as a result of microtubules sliding relative to each other, similar to the mechanism accepted for the bending of cilia and flagella. As yet, however, no single hypothesis as to how microtubules function in chromosome movement explains all the data (for recent summaries and discussions of this problem, see Bajer and MolC-Bajer, 1975; Inoue, 1976; Inoue and Ritter, 1975; McIntosh et al.. 1975b, 1976; Nicklas, 1975; Salmon, 1975c, 1976). Recent evidence has suggested the possibility that actin may alsci be involved in the production of chromosome movement (Cande et al., 1977; Forer, 1976; Sanger and Sanger, 1976). The second problem of course is how the mitotic apparatus is formed and how the cell regulates its appearance, an important solo part in the overall orchestration of cell division. It may well be that how the mitotic appararatus functions and how it is regulated are not independent questions but, since the data are not complete, it seems useful and possible to consider the latter problem separately. Formation of the mitotic apparatus depends on the formation and/or functioning of microtubule-organizing centers. In a typical mitotic apparatus microtubules arise at two different kinds of sites, at the mitotic poles and at the kinetochores of the chromosomes. The formation, morphology, and mode of function of various astral and anastral mitotic spindles were reviewed by Bajer and Mole-Bajer (1971) and Nicklas (1971). As these workers pointed out, the point of origin of polar mitotic tubules may he unclear, since a variety of structures (or no structure) exists at mitotic poles in different organisms, but kinetochore microtubules clearly arise at this differentiated area of the chromosome. Recent electron micrographs by Roos (1977) show the kinetochore of mammalian chromosomes in cross section to consist of outer and inner electrondense layers separated by a less dense middle region; it is biochemically as well as structurally differentiated, since cytological staining shows no chromatin at least in the outer and middle layers. The kinetochore microtubules arise directly from the dense material of the outer layer and do not extend into the other layers. This structure is reminiscent of other microtubule-organizing centers such as the rhizoplast of Ochromonas and mitotic centers in certain other organisms discussed below.

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Since a centriole appears at each pole of an astral mitotic figure, it was assumed for a long time that the centriole was the actual focal point for the polar mitotic tubules and in some way produced them or caused their appearance. It is now abundantly clear that this is not so. For one thing, this proposal left unexplained anastral acentriolar mitotic figures, of which there are innumerable examples and which function perfectly well. Pickett-Heaps (1969, 1971, 1975a) has suggested that, while this structure in its role as a basal body is an example of a highly structured microtubule-organizing center, its association with the mitotic apparatus is in fact a mechanism to ensure its correct partitioning to the daughter cells during cell division. However, this rationalization is not entirely satisfactory either, since it is clearly possible for many cells to elaborate centrioles de n o w , and since even in a single organism centrioles may be present in some but not other mitotic figures. The centriole retains its position as the Mona Lisa smile of mitosis. Szollosi et crl. (1972) reported that, while centrioles are present at spindle poles of oogonia and oocytes of mice and other mammals, they are absent in meiotic figures subsequent to the pachytene stage. Their electron micrographs showed that at the time of germinal vesicle breakdown in mouse oocytes, aggregates of electron-dense fibrous material appeared, from which microtubules radiated forming acentriolar asterlike arrays; groups of these subsequently served as spindle poles. Recent studies by Berns and co-workers of centrioles and mitosis in rat kangaroo cells in culture have demonstrated that pericentriolar material but not the centriole is required for both assembly and function of the mitotic apparatus. The mitotic pole in these cells is of the typical astral type, consisting of a centriole surrounded by amorphous material from which the polar tubules extend. At prophase Berns et a / . (1977) irradiated with an argon laser microbeam one of the centriolar areas of cells sensitized to radiation by acridine orange treatment; the primary damage was dispersal of the pericentriolar material, possibly suggesting a nucleic acid component. These cells underwent nuclear breakdown, chromosome condensation, formation of the metaphase plate, and cytokinesis, but the chromosomes did not separate nor did any anaphase movements occur. Electron micrographs showed kinetochore microtubules on both sides of the chromosome mass, but the polar microtubules were not formed normally and microtubules were absent from the cleavage constriction. In a second series of laser microbeam experiments, Berns and Richardson (1977) directly irradiated the centriolar region during early prophase and observed mitotis in cells in which the centrioles were physically removed from the spindle, severely structurally damaged, or destroyed. The mitotic tubules remained focused on the pericentriolar material, and mitosis proceeded normally. This group also observed the dispensability of the centriole in tetraploid cells which underwent spontaneous reduction divisions;

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electron micrographs showed centrioles at only two of the four spindle poles formed, but the microtubules of all poles tenninated in pericentriolar-like material (Brenner et al., 1977). Newcomb (1969), Hepler and Palevitz (1974), and Pickett-Heaps (1975b) have reviewed the situation in plants; centrioles or basal bodies appear in algae and other lower forms, in ginkgos and cycads, but not in other gymnosperms and not at all in angiosperms. Higher plants have anastral mitotic figures, often with no discernible polar organizing site. In lower plants and other lower eukaryotes, a variety of specialized microtubule-organizing centers and mitotic spindle morphologies and modes of function occurs. Many of these organizing centers resemble some of those already mentioned and consist of layers or masses of electron-dense material from which microtubules initiate. In a review concerned with evolution of the mitotic apparatus, Kubai (1975) gives an exhaustive tabulation of spindle morphologies. Within the scope of this article this topic cannot be completely covered, but some particularly striking examples can perhaps serve to illustrate the point. Manton et a / . (1969) followed the development of the unusual mitotic spindle in spermatogonia of the marine diatom Lirhadesmiurn undulurum. No centriole was involved. Near the nucleus of nondividing cells was a spindle precursor consisting of a rectangular body composed of a series of parallel, electron-dense plates or layers. At prophase a spindle appeared between the precursor and nuclear envelope; microtubules arose out of extensions of the end plates of the precursor, which served as mitotic poles. During enlargement of the spindle the rest of the precursor structure disappeared, implying incorporation of precursor material into the developing spindle microtubules. Mitosis proceeded after the nuclear envelope disappeared, and the mitotic array “sank down” into the chromosomes. Zickler (1970) described the microtubule-organizing centers in four species of ascomycete fungi; again, centrioles were not involved. Mitosis in these cells occurred with the nuclear envelope intact. The microtubules of the mitotic or meiotic spindle assembled within the nucleus, arising at the poles from centrosomal plaques, sandwichlike structures with portions on both sides of the nuclear membrane. In some species, this structure is L-shaped, and part of it is not attached to the nuclear membrane but extends into the cytoplasm. Sometimes microtubules extend from the extranuclear side of the structure, giving the appearance of astral fibers. Similar spindle plaques consisting of layered, electron-dense bodies lying on the nuclear envelope function as spindle poles in the intranuclear mitotic apparatus of the yeast Succhuromyces cerevisiae (Moens and Rapport, 1971). After cell division, in each daughter cell a second spindle plaque arises adjacent to the old one; they then move apart and become the poles of the mitotic apparatus at the next division. Other organisms with layered, electron-dense microtubule-

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organizing structures are the mushroom Boletus rubinellus (McLaughlin, 1971), the slime mold Physarum polycephalum (Tanaka, 1973), and the cowpea rust fungus, Uromyces phaseoli (Heath and Heath, 1976). Spherical microtubule-organizing structures resembling the blepharoplast of Marsilea have been observed in marine protozoa. Perkins (1970) described mitosis in a species of Lahrynthub; in vegetative cells the spindle microtubules directly attached to and presumably arose from two electron-dense spherical masses 200-300 nm in diameter. These aggregates had a central cartwheel structure similar to that in centrioles but lacked the nine triplet tubules. Tippit and Pickett-Heaps ( I 977) described a large, extremely complex spherical microtubule center in the pennate diatom Surivella ovulis; this organelle was present during interphase as a dense mass uniformly granular in substructure, from which extended a few microtubules. In early prophase a spindle formed beneath it, the microtubules of which extended between two dense end plates. As the spindle elongated, the spherical microtubule center dwindled and disappeared; a new one later formed de novo in each daughter cell. Other large, atypical microtubule-organizing structures have been observed in flagellates inhabiting the gut of wood roaches and termites. Grimstone and Gibbons (1966) described the ultrastructure of the centriolar apparatuses in Trychonympha and Psuedotrychonympha, which consist of long fibrillar rods lacking any centriolelike substructure but which serve as mitotic centers during cell division. Tamm and Tamm (1973a,b) described a similar apparatus in Deltotrychonympha and Koroga. Interphase cells contain two club-shaped bodies of fibrillar or granular material, with no discernible substructure or association with microtubules, connected to each other and arranged at right angles, reminiscent of a typical centriole arrangement; at division the two structures separate and serve as mitotic poles, spindle microtubules being assembled from the ends and radiating toward the nucleus. These organisms have the remarkable feature of containing hundreds of thousands of basal bodies of unknown function free in the cytoplasm, 500,000 to 700,000 immature basal bodies being arranged in chains and about a fifth as many mature organelles scattered singly (Tamm, 1972). Unstructured sites similar to the pericentriolar material have also been reported. Pickett-Heaps and Fowke (1970) described the events of microtubule assembly in the alga Closteriutn littorale, in which the mitotic spindle components apparently are altered after cell division and reutilized to reestablish cell organization. During mitosis, microtubules emerged from the mitotic centers, which consist of regions of granular material. At telophase, the spindle microtubules disassembled, but the mitotic centers persisted and in each daughter cell moved to the side and migrated along the chloroplast. Microtubules then extended in parallel from the organizing center toward the nucleus, forming a cylinder through which the nucleus moved, repositioning itself in its original place in the cytoplasm.

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A series of experiments indicates that in some organisms the two classes of mitotic microtubules have somewhat different properties and thus may be subject to differential control. That is, kinetochore microtubules appear considerably more stable than polar microtubules. Brinkley et al. (1967) found that, if Chinese hamster cells in culture were treated with low levels of colcemid, monopolar spindles formed in which polar microtubules were lacking but the chromosomes were attached by kinetochore microtubules radially around the pair of unseparated centrioles. Apparently the position of this aberrant mitotic apparatus was determined by the position of the centrioles. When the colcemid was removed, a normal spindle formed as interpolar microtubules formed and the two poles separated. Brinkley and Cartwright (1975) observed that in cultured rat kangaroo fibroblasts not only were the kinetochore tubules more stable than the polar tubules but that the lability of the polar tubules changed during the course of mitosis. Salmon (1975a,b,c) and Salmon et a / . (1976) observed that kinetochore tubules of several species were less sensitive to depolymerization by increased pressure than were polar tubules; they speculated that the greater stability may simply reflect the number of "attached" ends (kinetochore tubules extend to the poles and do not have "free" ends in the cytoplasm as do polar tubules). Essentially all observations on the assembly of the mitotic apparatus are consistent with what has become dogma, that is, that the mitotic apparatus is assembled out of a preexisting pool of tubulin subunits and, as originally proposed by Inoue (1976), the tubulin subunits are in equilibrium with the assembled microtubules. Thus the control of its appearance involves mobilization rather than synthesis at least of the primary molecular components. Some experiments by Stephens (1972a, 1973) dealt directly with this question; he showed that not only the size but also the morphology of the mitotic apparatus of sea urchin embryos depend on the temperature at which they are grown. Eggs of the cold-water sea urchin Stronylocetitrotus droebachietzsis develop to normal plutei even at 0°C. Stephens found that in embryos grown at 8°C the mitotic spindle was of normal morphology with large polar asters, but that in embryos grown at 0°C the mitotic spindle was of the anastral type and in addition was much smaller. He maintained fertilized eggs at 0" or 8°C and then raised the temperature at division; in both sets of eggs the spindle size and birefringence increased. In 8°C eggs large asters formed, but only a few astral fibers formed in 0°C eggs. Both sets showed maximum spindle birefringence at 12"C, the upper temperature limit for viability of this species. The differences in mitotic apparatus size indicated the portion of the total tubulin available for utilization rather than the total size of the pool, since after slow perfusion with deuterium oxide the resultant increased spindle size was the same for eggs at both temperatures. McIntosh et a / . (1975a) studied the mitotic spindle in mammalian cells using rat kangaroo cells in culture. Their electron micrographs showed that in these cells the microtubules at the mitotic poles end (or, presumably, originate) in

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masses of amorphous electron-dense material; there were no asters such as observed in marine eggs. The metaphase spindle formed over a period of about $5 hour. Since treating the cells with puromycin an hour before the start had no effect on its formation, they concluded that there were probably no translational controls on microtubule assembly; however, the level of inhibition of protein synthesis attained (80%) did not preclude the synthesis of some components. They noted that determination of the spatial location of the mitotic apparatus persisted through premature disassembly of the mitotic microtubules by cold, colchicine, or increased pressure; when the cells were released from these treatments, the mitotic apparatus reformed in the same orientation it had had previously. This observation is similar to that reported earlier by Goode (1973) in a study of the kinetics of reformation after cold-induced disassembly of the mitotic apparatus in the ameba Chuos carolinensis; he concluded that this process involved only the final steps in mitotic apparatus formation, that is, microtubule elongation rather than initiation. Goode also concluded that the rate of reassembly was consistent with the diffusion of subunits to a single growing point per microtubule. How the mitotic apparatus is positioned in the correct orientation is an important question about which unfortunately little is known. The position of the mitotic apparatus is crucial to proper determination of the daughter cells. From observations of mitosis and the subsequent planes of cell cleavage it has been inferred that in many cells the orientation of the mitotic apparatus determines the plane of cleavage. This has been directly demonstrated in eggs of marine invertebrates, in which physically shifting the position of the mitotic apparatus has been shown to cause an accompanying shift in the subsequent position of cleavage (see the review by Rappaport, 1971). The classic example illustrative of this point is the embryonic development of the snail Lirnnaea peregru (Morgan, 1927). The symmetry of the adult snail, including the direction of coiling of the shell, is determined by the direction of spiral cleavage, which is in turn determined by the orientation of the mitotic apparatus at the second cleavage. This symmetry determination is controlled by a pair of alleles of a single gene; the majority of individuals are coiled dextrally, the allele for which is dominant over the allele for sinistral coiling. This gene is a maternal effect gene; that is, the genotype of the mother entirely controls the direction of coiling of the offspring. Recently Freeman (1977) has found that injection of cytoplasm from eggs or early embryos of dextral individuals into eggs produced by females homozygous for the recessive sinistral coiling pattern causes them to develop with dextral coiling. The reciprocal injection (from sinistral into dextral eggs) had no effect. These results imply the action of a gene product present in the dextral but not the sinistral form. A mechanism in which orientation is not determined until after formation of the mitotic apparatus has been described in the onion Allium by Palevitz and Hepler (1974a,b). During guard cell division the cell plate forms along the

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longitudinal axis of the cells (whereas in other cell types division is transverse). In these cells, however, the position taken by the mitotic apparatus is apparently simply that in which it has the most “room” to function-obliquely or longitudinally across the cell. Mitosis proceeds until late anaphase or early telophase, at which point the entire spindle with the daughter chromosome sets reorients, the poles going to specified points on the cell periphery. Bands of microtubules appear at these points on the membrane, but how they participate in the reorientation is not clear. The reorienting movement is slow, requiring 15-20 minutes, but is specific; these investigators did not observe any cases in which the final position was “overshot. The movement was reversibly blocked by metabolic poisons and prevented by treatment with colchicine and vinblastine, in which case aberrant and misplaced cell plates formed. Several workers have concluded that in viva formation of microtubules involves the closure of sheets of filaments at the tips (or opening of the tubule during disassembly). Such intermediates would be seen in cross section as C-shaped. C-shaped microtubules have indeed been observed in sections of both isolated spindles (Cohen and Gottlieb, 1971) and mitotic cells (Jensen and Bajer, 1973); such profiles are most common near the equatorial region in metaphase, which presumably represents a region of active microtubule assembly andor disassembly. C-shaped cross sections interpreted as intermediates have also been observed during the rapid assembly of microtubules in mammalian blood platelets (Behnke, 1967) and during the extremely rapid or “cataclysmic” shortening of Echinosphaeriurn axopodia caused by treatment with Cu2+ or Ni2+ (Roth and Shigenaka, 1970). ”

E. THEASSEMBLYA N D DISASSEMBLY OF LABILE MICROTUBULE ARRAYS: CYTOPLASMIC MICROTUBULES Many cells contain microtubule arrays during interphase as well as during mitosis. Like mitotic tubules, cytoplasmic tubules are labile and subject to disassembly by cold, high pressure, and treatment with antimitotic drugs such as colchicine and vinblastine. Cytoplasmic microtubules play diverse roles in different cells, including formation and/or maintenance of cell shape and involvement in secretion, transport, and other intracellular movement (see Roberts, 1974; Stephens and Edds, 1976). A special widely studied class of cytoplasmic microtubules are neurotubules; because of its quantify and ease of isolation neurotubulin has been the basis of most biochemical studies of tubulin (see Sections I1 A,B). An early study by Gibbins er al. (1969) on the function and regulation of cytoplasmic microtubules concerned formation of the primary mesenchyme in embryos of the sea urchin Arbacia puncrularu. They suggested that control over

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the distribution of microtubules (hence over the developmental sequence of cell shape changes) might consist of sequential “activation and repression of nucleating sites. In cells of early blastulae, the cytoplasmic microtubules originated from two or three “satellites,” electron-dense masses arranged around the basal body at the apical part of the cell, from which point they diverged outward and extended downward parallel to the cell periphery. In later-stage blastulae, microtubules in presumptive mesenchyme cells (which round up and migrate into the blastocoel) appeared randomly oriented. However, at this point is illustrated one of the difficulties of electron microscope studies, namely, that they give static pictures of dynamic events with concomitant difficulties in interpretation. The basal bodies in the early blastula cells observed bore cilia, but later studies have shown that the micromeres which arise at the 16-cell stage and ultimately become the primary mesenchyme do not become ciliated (Okazaki, 1975; Raff et a l . , 1975). As Gibbins and co-workers pointed out, it was difficult to locate these cells in thin sections in early blastulae; thus it was not certain that the two kinds of ectodermal cell microtubule arrangements in fact represented sequential alterations in the presumptive mesenchyme cells. In later stages, the sequence could be clearly followed, since the differentiating cells could be more easily identified. In newly formed mesenchyme cells microtubules radiated from the cell center; in some cases a few microtubules appeared to contact the wall of the centriole. During migration and formation of the cable syncytium the microtubules in both the pseudopodia and stalks appeared in arrays parallel to the direction of extension, while in the cell body the microtubules appeared to extend from dense masses, or satellites, near the nuclear envelope (but did not contact the centrioles). Tihey and Gibbins (1969) further studied these events by treating ernbryos with colchicine, deuterium oxide, or increased hydrostatic pressure; they concluded that the changes in microtubule orientation were important in developing but not in maintaining the changes in cell shape. Tilney and Goddard (1970) confirmed that the electron-dense satellites in ectodermal cells of early blastulae in fact represented sites of initiation of microtubule assembly. When they decreased the temperature to 0”C, the microtubules disassembled but the satellite structures remained; on rewarming, microtubules arose at the satellites. Warren (1974) followed the involvement of microtubules in the morphogenesis of developing muscle cells in the tail of Rana pipiens tadpoles by electron microscope observation of serial sections through portions of developing myoblasts. He observed that during development of these cells there was a shift in microtubule organization from radial arrays early in development to parallel arrays in differentiated cells. In mesenchyme cells and in some premyoblast cells the microtubules radiated from amorphous electron-dense centriolar satellites, whereas in more mature myoblasts and myotubes the microtubules were arranged along the length of the cell and were no longer focused on the cell center. The site ”

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of origin of microtubules in mature cells was unclear. Warren observed many apparently abrupt terminations of microtubules in serial sections; some ends of microtubules were free in the cytoplasm, but frequently these ends were associated with various cell membranes and in a few cases with amorphous, electron-dense material. Recently, the ability to observe the process of formation and disappearance of both mitotic and cytoplasmic microtubules has been greatly enhanced by the use of indirect immunofluorescence. Briefly, specific antibodies are prepared to purified tubulin. Cells to be examined are then fixed on slides and reacted with the antitubulin; the areas andor structures in the cell which have bound the antitubulin molecules are subsequently amplified as well as visualized by treatment with fluorescence-labeled antibodies from a second species (often sheep or goat) raised against immunoglobulins of the species (usually rabbit) in which the antitubulin antibodies were raised. A common feature of these experiments is that the antitubulin antibodies bind to microtubules from a wide variety of heterologous species. Antibodies prepared against mammalian brain tubulin have been shown to decorate microtubules and tubulin-containing structures in several mammalian and vertebrate cell types (Brinkley et a / . , 1975a,b,, 1976; Edelman and Yahara, 1976; Frankel, 1976; Fujiwara and Pollard, 1978; Fuller and Brinkley, 1976; Fuller et al., 1975a; Osborn and Weber, 1976a,b, 1977; Schliwa et al., 1978; Weber, 1976) and in invertebrates, plants, and protozoa (Franke et al., 1977; Weber et al., 1977b). Similar results have been obtained with antisera against sea urchin sperm tail tubulin (Weber, 1975, 1976; Weber et a/., 1975a,b), sea urchin egg tubulin (Sato et al., 1976), and tissue culture cell tubulin (Isenberg et a/., 1977). The structures stained were shown to be authentic microtubules by the use of known microtubule-depolymerizing agents such as cold, colchcine, and vinblastine; the binding appears to be specific for tubulin with the possible exception of some nuclear fluorescence, the nature of which is unclear, observed in only a few cell lines. As shown in Fig. 6, the most striking result of these studies is that not only is the mitotic apparatus stained, giving an appearance at each stage of mitosis virtually identical to that obtained by birefringence studies, but that an elaborate network or cytoskeleton of microtubules exists in nearly all the interphase cells examined. This cytoplasmic microtubule network disappears during prophase and is completely absent during mitosis, when most of the fluorescence appears in the mitotic apparatus, although in some cell lines background fluorescence increases slightly over the interphase level. The cytoplasmic microtubule complex then reappears after disassembly of the mitotic apparatus. Whether the same pool of tubulin subunits is utilized in both cytoplasmic and mitotic tubules is an interesting but unresolved question. The reciprocal appearance and disappearance of the two sets of microtubules have been taken as implicit evidence that this may be so, but there is no direct unequivocal biochemical proof. Certainly the

Fic;. 6. Microtubules in mouse 3T3 cells in culture visualized by indirect immunofluorescence with a monospecific antibody against tubulin. (a) Interphase cell, showing the cytoplasmic microtubule network. ~ 7 6 5 (b) . Interphase cell after treatment with colchicine ( I pLp/nil) for I hour. x765. In both (a) and (b) the microtubule-organizing structure in the cell center is clearly visible. (c) Mitotic cell. showing astral and spindle tubules. X765. Reprinted from Osborn and Weber (1976b). with permission.

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organizational events determining the pattern and orientation of assembly appear to be separate. The dismantling of cytoplasniic microtubules often involves the loss of asymmetry (i.e., the rounding up of cells before division); this may simply ensure proper formation of daughter cells. The cytoplasmic microtubule arrays revealed by immunofluorescence staining appear to emanate from a small number of sites in the cell center. Osborn and Weber (1976a,b) monitored elaboration of the cytoplasmic microtubule network in mouse 3T3 cells in culture recovering from treatment with cold or colchicine and during the process of spreading and attaching to glass substrates. They obtained clear evidence that microtubules arose from one or two discrete sites in the center of the cytoplasm and grew in a polar manner toward the cell periphery (Fig. 6a,b). When the microtubules first approached the plasma membrane, they often appeared to contact or at least approach it closely and in some cases appeared to “stretch” it. The microtubule-organizing structure close to the nucleus was visualized as a fluorescence-staining cylinder about 3 pm long, itself exhibiting polarity in that one end was above the plane of the microtubules, which arose directly from the bottom end of the cylinder. This structure persisted in cells in which the cyotplasmic microtubules were depolymerized by cold or drugs. Its relation, if any, to centrioles is unclear as yet. Osborn and Weber suggested that two types of regulation must be involved in the assembly of cytoplasmic microtubules: (1) positive regulation, in which assembly takes place with specific timing and orientation from the organizing site, and (2) negative regulation, in which assembly outside the specific pathway is inhibited, preventing the formation of random or unoriented microtubules. Frankel (1976) observed the outgrowth of microtubules in cultured mouse macrophages and fibroblasts. Macrophages showed a single microtubule-organizing center which appeared as a fluorescent ring with a dark center from which microtubules extended radially in an asterlike pattern; fibroblasts showed a typical complex network of microtubules. At initial stages of regrowth after microtubule depolymerization caused by cold or drugs, microtubules in fibroblasts grew out toward the cell membrane from one to three foci near the cell center. The cytoplasmic microtubule network has also been visualized at the electron microscope level. Two groups have employed the immunoperoxidase method, in which fixed cells are treated sequentially with antitubulins, then antiimmunoglobulins, and then a complex of peroxidase and antiperoxidase; after incubation with a peroxidase substrate, the reaction sites are ultimately made visible with electron microscopy by the deposition of osmium. Using this method DeMey et al. (1976) and DeBrabander et al., (1977ii,b) observed a typical network of cytoplasmic microtubules radiating from the cell center toward the cell periphery in mouse embryo cells in culture. These investigators interpreted the diffuse staining observed in the cytoplasm to indicate the presence of tubulin subunits;

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this background staining increased when cytoplasmic tubules were disassembled by colchicine treatment and decreased when vinblastine crystals, which stained heavily, were formed. Because the staining was evenly distributed, they suggested that no gross compartmentalization of tubulin subunits occurs in these cells. This group also reported that this method could be used at the light microscope level (DeBrabander et al., 1977b). In rat kangaroo cells in culture, Pepper and Brinkley (1977) observed staining in kinetochores, electron-dense amorphous pericentriolar material, and small viruslike particles associated with centrioles, in addition to that in centrioles and the mitotic spindle. Thus tubulin may be a component of microtubule-organizing centers. The peroxidase staining was uniform throughout the kinetochore, even though microtubules appeared to be associated only with the outer layer. Eckert and Snyder (1978) combined the indirect immunofluorescence technique with high-voltage electron microscopy by employing antibodies raised against glutaraldehyde-treated tubulin, confirming the identity of the networks observed at the light microscope level with microtubules. Immunofluorescence studies of the cytoplasmic microtubule network have raised the possibility that it may be altered in transformed cells. Brinkley et al. (1975a,b; 1976) reported that, whereas in several lines of normal fibroblastlike mammalian cells in culture they observed a typical complex network of cytoplasmic microtubules radiating from one or two densely staining areas near the nucleus, cells transformed by viruses, chemically or spontaneously, contained either randomly oriented or very few microtubules. However, during mitosis there was no detectable difference in the microtubule patterns between transformed or nontransformed cells. This group recently repeated these observations using hybrids of transformed mouse cells and normal human fibroblasts; only cells showing normal growth patterns exhibited the complete microtubule network, while cells showing intermediate or transformed growth patterns all showed diminished or absent microtubule networks (Miller et al., 1977). Similarly, Edelman and Yahara (1976) observed distinct microtubule networks in normal cells, whereas antitubulin staining in transformed cells was diffuse and unstructured. Both patterns could be observed in the same cells: chicken fibroblasts infected with a temperature-sensitive Rous sarcoma virus and grown at permissive temperatures showed the typical transformed morphology and diffuse pattern of antitubulin staining; at restrictive temperatures the typical cytoplasmic microtubule network, and normal cell shape, reappeared within 1 or 2 hours, However, Osborn and Weber (1977) demonstrated cytoplasmic microtubule networks in several lines of transformed mammalian cells in culture and suggested that the rounded morphology of transformed cells makes the observation of microtubules by indirect immunofluorescence technically difficult. DeMey et al. (1978) also observed microtubule networks in transformed cells at

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both the light and electron microscope levels using the immunoperoxidase technique; in some tumor cell lines their electron microscope observations revealed microtubule networks which were poorly visualized at the light microscope level.

11. The Dance: Experimental Dissections of Microtubule Assembly in Vivo

It is difficult to separate the observation of a biological system from experimentation; the very procedure of microscopic observation itself, after all, involves meddling with the system. Also, many of the observations discussed in Section I were made on systems in which microtubule assembly or disassembly had been induced by altering normal physiological conditions (i.e., treatment with cold, pressure, drugs, and so on). Nevertheless, the basic rationale behind these studies was to describe the processes of microtubule assembly as they actually normally occur in cells. The work to be discussed now is more clearly experimental in motive; that is, it is aimed at defining the mechanisms which govern microtubule assembly in vivo by altering andor isolating the components of the microtubule assembly process. A real difficulty with experiments on highly purified preparations is that they may not accurately reflect physiological conditions. However, experiments performed in sifu or on partly purified preparations have the reciprocal drawback, always present in examining complex systems, of the difficulty of sorting out specific from nonspecific effects. A. THEBIOCHEMISTRY OF TUBULIN, THE STRUCTURAL MICROTUBULE PROTEIN

Tubulin was first identified as the major component of microtubules by observation of its binding of the antimitotic drug colchicine (Borisy and Taylor, 1967a,b; Shelanski and Taylor, 1967, 1968; Weisenberg e f al., 1968; L. Wilson, 1970; Wilson and Meza, 1973). The recent reviews by Snyder and McIntosh (1976) and Stephens and Edds (1976) contain good summaries of the chemistry of tubulin and the structure of microtubules. The functional subunit in the assembly of microtubules is a 110,000-molecular-weight heterodimer of the two tubulin monomers (Luduena et a l . , 1975, 1977); a and /3 tubulin are related peptides with similar molecular weights and electrophoretic properties, but different primary structures (Luduena and Woodward, 1973, 1975) coded for by separate genes (Bryan et ui., 1978). Tubulin synthesis may accompany the formation of a microtubule-containing organelle but, as discussed above in connection with studies of ciliagenesis, the synthesis of tubulin only rarely appears to be the controlling point for microtubule assembly. Rather, in general it appears that most cells maintain substantial tubulin pools. For example, many developing embryos maintain a relatively constant level of tubulin throughout early development: the axolotl (Raff, 1977;

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Raff and Raff, 1978); Drosophila (Green et al., 1975); the sea urchin (reviewed by Raff et al., 1975; see also the discussion of ciliagenesis, above); and Spisula (Bumside et al., 1973). Modulation of tubulin levels occurs, as indicated by the changes in relative rates of tubulin synthesis observed during the cell cycle in mammalian cells in culture (Klevecz and Forrest, 1975; Lawrence and Wheatley, 1975), in sea urchin embryos (Rubin et al., 1976), and in Chlamydomonas (Pipemo and Luck, 1977). Such changes have not been found to be involved in the regulation of cell division, however. Given that cells contain reserves of tubulin which may be assembled into microtubules, to date it has been difficult to define which properties of the tubulin monomers serve regulatory functions in vivo. First, binding sites are present on tubulin molecules for several small molecules of which the most important appear to be: 1. Drugs such as colchicine, vinblastine, and podophyllotoxin, and so on, which act as mitotic poisons (see, for example, Bhattacharyya and Wolff, 1977; Bryan, 1972; Cortese et al., 1977; Garland and Teller, 1975; Harrisson et al., 1976; Kelleher, 1977; Lee et al., 1975; McClure and Paulson, 1977; Owellen et al., 1972; Pfeffer el nl., 1976a,b; Schmitt and Atlas, 1976; L. Wilson, 1970, 1975; Wilson et al., 1974, 1975a,b). 2. Guanine nucleotides (Berry and Shelanski, 1972; Bryan, 1972; Shelanski and Taylor, 1968; Stephens et a l . , 1967; Weisenberg et al., 1968). 3. Metal ions, including magnesium (Lee and Timasheff, 1975, 1978; Olmsted and Borisy, 1975), calcium (Hayashi and Matsumura, 1975; Rosenfeld et al., 1976; Solomon, 1976, 1977), and lithium (Bhattacharyya and Wolfe, 1976a). The binding of antimitotic drugs is the most widely used biochemical tool for tubulin characterization. How the drug-binding sites participate in normal function is unknown. The binding of nucleotides and magnesium is required for microtubule assembly in v i m , but it is not clear if they serve a regulatory function. Calcium is an inhibitor of microtubule assembly in vitro and is a good candidate for an in vivo regulator. These are discussed more fully in following sections. Several posttranslational modifications of tubulin monomers have been observed. Unfortunately, evidence on their possible functions in vivo is incomplete and sometimes conflicting. First, tubulin has been reported to be a glycoprotein (Margolis er al., 1972). Feit and Shelanski (1975) observed that tubulin from particulate fractions but not from soluble fractions was glycosylated after incorporation of gly~osamine-'~C into mouse brain in vivo. Second, Eipper (1969, 1974, 1975) showed that tubulin from rat brain is phosphorylated at a single serine residue in the p chain; she also observed that the labeling patterns of 32P

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incorporation into tubulin in brain slices from newborn rats were different from the patterns in adult brain. Phosphorylation of tubulin has also been reported in chick brain (Lagnado and Kirazov, 1975). Piras and Piras (1975) reported that the extent of phosphorylation of tubulin from HeLa cells varied during the cell cycle, but phosphorylation of tubulin could not be detected at all in Chinese hamster ovary (CHO) cells in culture (Rubin and Weiss, 1975) or in normal or differentiating neuroblastoma cells in culture (Solomon et al., 1976). Tubulin was earlier thought to possess intrinsic protein kinase activity, but it has been shown that this activity, although sometimes copurifying with brain tubulin assembled in virro, is separable (Rappaport et al., 1976; Sandoval and Cuatrecasas, 1976a). Finally, it has been shown that tyrosine is covalently linked to the carboxyl terminal residue of the a subunit of brain tubulin by a specific posttranslational enzymic reaction (Arce et al., 1975; Argarana et al., 1977; Barra et al., 1974; Raybin and Flavin, 1975, 1977a). Halleck et al. (1977) reported that the tyrosine residue was released under conditions which promoted assembly in vitro of microtubules from rat brain fractions. However, Raybin and Flavin (1977b) found that only some brain tubulin purified by assembly is tyrosylated and that tubulin assembled just as well in vitro with or without carboxyl terminal tyrosine. Furthermore, while they found tubulin-tyrosylating activity in other rat tissues besides brain, tyrosylation of tubulin appeared to occur primarily on tubulin found in insoluble fractions in cultured neuroblastoma cells and did not occur at all in various invertebrates they examined. Tubulin is evolutionarily a highly conserved protein. Fulton et al. (1971) originally showed by cross-reactivity of antibodies against tubulin from sperm tails of the sea urchin A . punctulata that flagellar and mitotic microtubules from several species of sea urchins and a sand dollar were extremely similar, although not identical. The immunofluorescence studies discussed in Section I, exploiting the cross-reactivity of antitubulin antibodies with diverse heterologous microtubules, also well illustrate this point. This evolutionary constancy of both the morphology of microtubules and the structure of the tubulin subunits has led many workers to place a quite reasonable implicit emphasis on the similarities of tubulins from different organisms. It has only recently been appreciated that a major means of regulation of tubulin function may lie in small differences in the tubulin molecules even of a single organism. That is, what has been thought of as the “tubulin pool” in a cell may actually consist of multiple subpools in which the tubulin subunits are heterogeneous either because of actual differences in primary structure or secondary posttranslational modifications (or both). Of course differences in the behavior of microtubules have been known almost as long as microtubules have been recognized. Behnke and Forer (1967) originally classified microtubules into four groups based on stability to fixation and solubilization, the most labile being those in the mitotic apparatus and the most stable those in the axoneme of flagella or cilia. It is still not clear whether these

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differences reflect differences in the biochemistry of the composite tubulins or whether they depend on the cellular environment of the various tubules. It is very obvious that the most stable microtubules are those which are part of more complex structures; associated structures such as arms, doublet or triplet tubules, spokes, linkers, and so on, may indicate the presence of binding sites not present on other microtubules. How such sites are specified has not been determined. Stephens (1975) has suggested that there may be differences in the tubulins comprising the A and B tubules in the outer doublet tubules of sea urchin sperm flagella. He obtained in v i m reassembly of tubulin isolated from A tubules into single tubules, but tubulin from B tubules formed only sheets or ribbons, possibly because the preferred mode of self-association of the tubulin which in vivo forms the doublet portion of the tubule precludes formation of a single tubule; however, no doublets formed in mixed solutions of tubulin from A and B tubules. The biochemical evidence was not definitive. Large cyanogen bromide cleavage peptides were similar for both Q and /3 tubulins from A and B tubules. There were some differences in the amino acid profiles of the different subunits, but total amino acid analysis of such large peptides is not a sensitive technique and the differences observed were slight. More recently Stephens (1977b) obtained evidence from radioactive labeling experiments that the tubulin pools for the A and B subfibers of the outer nine doublet tubules of sea urchin embryo cilia are separate. Wilson et al. (1975a) suggested that several populations of microtubules exist in brain because of the differential sensitivities they observed in preparations of brain tubulin to the antimitotic agent griseofulvin. Feit er al. (1977) found that two-dimensional gel electrophoresis resolved both a- and Ptubulin subunits from chick, adult mouse, and adult bovine brain, as well as from mouse neuroblastoma cells in culture, into multiple bands. Berkowitz et al. (1977) and Bryan er af. (1978) found that this method resolved the a- but not the P-tubulin subunit from calf and chick brain, respectively, into two bands. Similarly, Raybin and Flavin ( 1 977b) and Lu and Elzinga ( 1 977) found that the a- but not the P-tubulin subunit from rat and calf brain, respectively, separated into two peaks on hydroxyapatite chromatography; the biochemical basis for the separation was not clear but apparently did not involve carboxy terminal tyrosylation. Kobayashi and Mohri (1977) obtained a single peak on hydroxyapatite columns for both aand Ptubulin subunits from starfish sperm flagella, but both subunits subsequently yielded multiple bands on isoelectrofocusing gels. In the absence of further biochemical characterization, resolution into multiple bands, particularly on isoelectrofocusing gels, must be interpreted with caution, but there are certainly strong indications that microheterogeneity exists in tubulins from a single tissue or cell. There is good evidence for heterogeneity among tubulins from different tissues or structures in the same organism. Kowit and Fulton (1974a,b) and Fulton and

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Kowit (1975) showed that, during transformation from the ameba to the flagellate state in N. gruberi, essentially all the tubulin utilized in assembly of the flagella axoneme is synthesized de novo. This is not because the amebas lack sufficient tubulin; tubulin represents nearly 12% of the total cell protein, whereas the flagellar outer doublets comprise only 0.15% of the total protein. Rather, the tubulin synthesized appears to be a unique molecule; antibodies prepared against flagella tubulin did not cross-react with tubulin present in the amebas, or indeed with soluble tubulin present in the flagellates. Fulton and Simpson (1976) therefore speculated that in general, separate genes code for tubulins designed for specific functions. Pfeffer et al. (1976a,b) found that tubulin from sea urchin eggs had a binding constant for colchicine 10-fold lower than that from solubilized sea urchin sperm tail outer doublets (which had a binding constant similar to that observed for brain tubulin). Bibring et al. (1976) also examined tubulins from different sea urchin tissues. In electrophoresis on polyacrylamide gels containing both sodium dodecyl sulfate (SDS) and urea the a subunits of tubulin purified from isolated mitotic apparatus and from the A tubule of cilia, but not from outer doublet tubules of sperm flagella, separated into two bands, whereas in gel systems containing only SDS or only urea, the a subunits from all three migrated similarly, as did the fl subunits in all the gel systems examined. Isoelectrofocusing gels revealed several distinct differences in cyanogen bromide peptides obtained from a tubulin from mitotic apparatus and flagella outer doublets, although the majority of bands were the same. Finally, we have recently observed that tubulins from adult tissues (brain and testis) of the Mexican axolotl and the salamander Necturus differed in electrophoretic properties, although not in colchicine-binding properties, from the tubulin present in eggs and embryos (Raff and Raff, 1978). Furthermore, specific limited proteolysis with both chymotrypsin and a Staphlococcus protease yielded distinctly different peptide patterns from axolotl egg and testis tubulins; the microheterogeneity was more marked in the a than in the fl subunits.

B. MICROTUBULE ASSEMBLY in Vitro: POSSIBLE REGULATORY FACTORS Because microtubule assembly in vivo is complex and difficult to deal with directly, much effort has been devoted to delineation of the process as it occurs in vitro. Weisenberg (1972b) first reported that microtubules could be reconstituted in vitro from supernatants of brain homogenates. Microtubule assembly occurs under essentially physiological conditions at 37"C, slightly acid or neutral pH, and moderate ionic strength, and requires the presence of Mg2+ and GTP and the absence of Ca'+. The microtubules that form in vitro have the same appearance as cytoplasmic microtubules in vivo and in most cases are subject to disassembly by the same agents, namely, cold, colchicine and other antimitotic drugs, and increased pressure. The assembly process consists of two separable

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steps, nucleation or initiation, and elongation. Microtubule assembly in vitro has been extensively reviewed (see, for example, Borisy et al., 1975, 1976; Olmsted et al., 1974; Penningroth et al., 1976; Stephens and Edds, 1976). The majority of the studies of microtubule assembly in vitro have been performed on tubulin isolated from brain; this system is ideal because tubulin constitutes up to 20% of total brain protein. However, assembly of microtubules in vitro has also been demonstrated with tubulin isolated from developing embryos of Drosophila melanogaster (Green et al., 1975, 1979), marine invertebrate eggs (Kuriyama, 1977), and a variety of nonneural mammalian cell types (Barnes et al., 1975; Castle and Crawford, 1975; Doenges et al., 1977; Nagle et al., 1977; Weber et al., 1977a; Wiche and Cole, 1976; Wiche et al., 1977a,b). Several studies have been aimed at defining which amino acid residues of the tubulin subunits participate in the assembly process. Modification of tyrosine (Gaskin and Gethner, 1976) and histidine residues (Lee et al., 1976) prevents assembly. Several laboratories have proposed that sulfhydryl groups are involved in the control of tubulin function. The number of free sulfhydryl groups measured varies somewhat depending on the conditions used in preparing the tubulin but is usually reported to be seven per 55,000-molecular-weight monomer, of which two are required for assembly (Kuriyama and Sakai, 1974; Mellon and Rebhun, 1976a,b; Nishida and Kobayashi, 1977; Wallin et al., 1977). The details of the ionic requirements reported for the assembly of microtubules in vitro vary considerably, depending on the source and method of preparation of the tubulin and on other buffer components. Thus the standard assembly conditions require 0.5 mM magnesium (Shelanski ef a/., 1973; Weisenberg, 1972b); concentrations of 10 mM or above may also stimulate assembly (see below), but under other conditions inhibit assembly (Olmsted and Borisy, 1975) or cause aggregation and precipitation of tubulin (Weisenberg and Timasheff, 1970). Calcium is a potent inhibitor of microtubule assembly in vitro, but whether inhibition occurs at millimolar or micromolar concentrations depends on the magnesium concentration (Olmsted, 1976; Rosenfeld et al., 1976). Thus it has been difficult to determine which buffer conditions used in vitro represent conditions in vivo. The second requirement for microtubule assembly in vitro, that for guanine nucleotides, is also complex. Tubulin-nucleotide interactions have been reviewed by Jacobs (1975), Olmsted (1976), Weisenberg (1975), and Weisenberg et al. (1976b). Briefly, tubulin contains at least two binding sites for guanine nucleotides which differ in the extent of exchange with exogenous nucleotides. The role and function of nucleotides at these two sites is incompletely understood as yet, and the data thus give a somewhat conflicting picture. As Weisenberg et al. (1976a,b) have pointed out, the state of bound guanine nucleotide is highly sensitive to the conditions under which tubulin and microtubules are observed. The more readily exchangeable site, which Geahlen and Haley (1977) have shown to be on the /3 subunit, appears to be most important in microtubule

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polymerization. GTP or GDP (which is subsequently transphosphorylated to GTP) is bound at this site and is hydrolyzed under conditions which also allow polymerization (David-Pfeuty et al., 1977), but it is not clear if hydrolysis is a requirement for, or merely concomitant with, polymerization; in some studies it was found that hydrolyzable GTP was necessary to obtain polymerization (Maccioni and Seeds, 1977; Olmsted and Borisy, 1975), while in other studies nonhydrolyzable analogs of GTP supported polymerization (Arai and Kaziro, 1977; Penningroth and Kirschner, 1977; Penningroth et al., 1976; Weisenberg and Deery, 1976). Assembly was also reported in the absence of added nucleotides when glycerol or sucrose, which stabilize microtubules, was present in the assembly buffer (Shelanski et al., 1973). These results may be reconciled by the finding of Penningroth and Kirschner (1978) that the nucleotide specificity at this site is broad and that only substoichiometric amounts of bound trinucleotide are required for polymerization to occur. Sandoval et al. (1977) found that purified tubulin not only asembled more efficiently in the presence of a hydrolyzable GTP analog (guanylyl 5'-methylene diphosphonate, GMP-CH,-P-P), but that the resultant tubules were also more stable than those formed in the presence of GTP. This is consistent with the data of Arai and Kaziro (1977) and Penningroth and Kirschner (1977), who both concluded that binding of GTP at this site induces conformational changes favoring assembly. Jacobs and Caplow (1976), however, concluded that the role of GTP at this site is not as an allosteric effector. Zeeburg et al. (1977) reported that the GDP resulting at this site may also be cleaved, forming a covalent GMP-protein bond, which they suggested as a possible regulatory mechanism. GTP is also bound at the less readily exchangeable site; hydrolysis of this moiety has generally not been observed (Penningroth and Kirschner, 1977; Spiegelman et al., 1977; Weisenberg et al., 1976b) but has been reported to occur (MacNeal and Punch, 1977). Spiegelman et al. (1977) have suggested that the nucleotide bound at this site, unlike that at the exchangeable site, may be a stable structural component or cofactor and is not involved in the control of assembly or disassembly. In the initial studies of microtubule assembly in vitro, several laboratories observed that both crude preparations of brain tubulin and tubulin resolubilized from microtubules assembled in vitru could be separated by differential centrifugation or molecular seive chromatography into two fractions, both composed primarily of tubulin, one of which was competent to initiate self-assembly and one of which was not (Borisy and Olmsted, 1972; Borisy et al., 1974, 1975, 1976; Bryan, 1976; Erickson, 1974b; Kirschner and Williams, 1974; Kirschner et al., 1974, 1975a,b; Penningroth et al., 1976; Scheele and Borisy, 1976; Shelanski et al., 1973; Weisenberg, 1974; Weisenberg and Rosenfeld, 1975a). One fraction corresponded to the 1 10,000-molecular-weight 6 s tubulin dimer, but the other was composed of high-molecular-weight oligomers of tubulin with sedimentation constants from 18 to 36s. Electron microscopy of the latter frac-

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tions revealed highly ordered ring-or disklike structures, the exact structure of which depended on the source and conditions of preparation of the tubulin. Although the oligomeric fractions could reversibly break down into 6 s dimeric tubulin and were competent to reassemble into microtubules, the 6s fractions neither formed rings or disks nor reassembled into microtubules unless mixed with the 36s fraction. Many laboratories postulated that these disklike structures represented the initiation step of microtubule formation in vitro. However, this does not appear to be the case. Weisenberg (1974) showed that, although rings were present initially and disappeared during the subsequent course of polymerization, they were not incorporated directly into microtubules but were first broken down into smaller subunits. More recently, in detailed kinetic and electron microscope analyses, Bryan ( I 976) showed that the rings are not required either for nucleation or for elongation of microtubules. The function of these structures remains unclear; they appear to be purely in vitro structures resulting from the experimental manipulation of molecules which have the potentiality for several modes of self-association. Rings and disks are frequently observed to form under conditions (such as in the cold) under which microtubule assembly is inhibited. Certainly no structures resembling them have been observed in electron microscope studies of microtubule systems in vivo. Experiments designed to determine why some brain tubulin preparations were competent to initiate self-assembly into microtubules while others were not, and to define the nature of the rings and disks observed in assembly-competent fractions, brought up another point crucial to the understanding of both the process and the control of microtubule assembly: the possibility of a requirement for factors in addition to tubulin, which might act either stoichiometrically or catalytically to allow initiation andor elongation of microtubules. It was observed that microtubules formed in vitro frequently contained proteins in addition to tubulin, particularly a series of proteins in the molecular weight range from 200,000 to 350,000 which copurified with tubulin through several cycles of assembly and disassembly (for example, see Borisy et al., 1974; Bums and Pollard, 1974; Dentler et ul., 1975; Erickson, 1974b; Gaskin et al., 1974b; Haga and Kurokawa, 1975; Keates and Hall, 1975; Kirschner e t a / . , 1974; Kuriyama, 1975; Sloboda et a/., 1975, 1976a). These proteins together with a more recently identified group of proteins of lower molecular weight, collectively termed tau factor (Weingarten et ul., 1975), have been identified as such microtubule assembly-promoting factors. Amos (1977b) has presented an excellent short summary of this work. Tubulin fractions competent to self-assemble appear to be those also containing these proteins, which collectively have been termed microtubule-associated proteins (MAPs). In recent literature, tubulin, together with MAPs, has been referred to as microtubule protein(s). [A word of caution is in order here, since in earlier literature the latter term, “microtubule protein(s), was used synony”

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ELIZABETH C. RAFF

mously with “tubulin. ”1 Both the number and nature of the MAPs and the details of how they affect microtubule assembly vary in different experiments. Most studies of MAPs indicate that they act stoichiometrically to promote the assembly of microtubules in vitro and are more important in the initiation of assembly than in microtubule elongation. Whether they are regulatory proteins for microtubule assembly in vivo is currently a much-debated question. Recent work has emphasized the separation of pure 6s tubulin from MAPs by ion-exchange chromatography: either by anion exchange, in which case tubulin is retained on the column and the MAPs are eluted under low salt conditions, or by cation exchange, in which case the MAPs are retained and tubulin is eluted first. Under standard assembly conditions, tubulin so purified does not initiate extensive self-assembly into microtubules, but does so if the MAP fraction is added back. Murphy and Borisy (1975) and Murphy ef af. (1977a,b) examined the assembly of porcine brain tubulin separated from MAPs by DEAE-Sephadex chromatography. Of about 35 nontubulin proteins observable after two cycles of assembly and disassembly only a few bound to microtubules with high affinity and remained at constant stoichiometry through further cycles of assembly. In their preparations the main component which stimulated assembly was the high-molecular-weight proteins, but other stimulating proteins, including the tau group, also appeared to be present. At 5°C the addition of MAPs to the purified tubulin promoted the formation of ring structures; recent work by this group has shown that the type of oligomeric structure formed in the cold is a function not only of solution conditions such as pH and ionic strength but also of which species of MAP is associated with tubulin (Marcum and Borisy, 1978a,b; Scheele and Borisy, 1978; Vallee and Borisy, 1978). At 37”C, rapid assembly into microtubules occurred; while MAPs stimulated both initiation and elongation, they were required only for the former. Murphy et al. (1977a) observed that the stimulation of microtubule assembly by MAPs proceeded with sigmoidal kinetics, which they interpreted to indicate that stimulation of elongation occurred by reduction of the rate of depolymerization, thus shifting the equilibrium to favor assembly. In contrast to models involving the incorporation of MAPs into tubules during assembly they suggested that MAPs bind to sites which become available only after polymerization, thereby stabilizing the microtubule. Some evidence consistent with this idea has been reported by Meier and Jgrgensen (1977), who found that antibodies prepared against intact microtubules assembled from rat brain tubulin in vitro bound to intact microtubules and to MAPs (which in their preparations included tau factor and a 135,000molecular-weight component) but not to soluble 6s tubulin dimers. Sloboda et af. (1976b) reported similar results with bovine brain tubulin purified by chromatography on phosphocellulose; they found that, as the ratio between the amount of high-molecular-weight MAPs and tubulin was increased, a greater number of tubules were initiated and at a faster rate, and in addition the

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total mass of microtubules assembled increased; thus in this system MAPs were required for both nucleation and elongation. In contrast to these results, Weingarten et al. (1975) did not observe significant amounts of high-molecular-weight MAPs in their preparations of microtubules polymerized in virro from porcine brain, but found that tau factor, a group of proteins with molecular weights similar to that of tubulin, could be separated from tubulin by phosphocellulose chromatography. These proteins appeared to be incorporated stoichiometrically into microtubules and were required not only for the initiation of self-assembly of purified tubulin but also, in lower amounts, for its addition to preformed microtubule seeds (Witman er ul., 1976). Penningroth et a / . (1976) found that crude preparations of tau factor could be separated into two active fractions, and Cleveland et al. (1977a,b) recently reported that purified tau factor accounted for two-thirds of the total assemblystimulating activity in their MAP preparations and consisted of four closely related heat-stable proteins with apparent molecular weights between 55,000 and 62,000. The remaining microtubule assembly-stimulating activity in their MAP preparations was of much lower specific activity and was found in a fraction containing a spectrum of high- and low-molecular-weight proteins. These investigators suggested that tau proteins function by binding several tubulin molecules per molecule, thus facilitating the formation of longitudinal filaments, hence microtubule assembly. Fellous et ul. ( 1977) investigated the microtubule assembly-stimulating activity in MAP fractions from rat brain and found that in their preparations it resided entirely in the low-molecular-weight material. The association of nontubulin proteins with microtubules in vivo was inferred by Kirkpatrick et ul. (1970), who observed high-molecular-weight components in preparations of intact microtubules isolated from brain. Burton and Femandez (1973) observed “fuzzy” projections evenly spaced along the length of the tubule walls of axonal neurotubules in situ. Several workers subsequently observed similar projections on the surfaces of microtubules isolated intact from blood platelets and brain (Behnke, 1975), and on tubules assembled in vitro from combined purified tubulin plus MAP fractions (Amos, 1977a; Dentler et al., 1975; Murphy and Borisy, 1975; Sloboda et al., 1976a). These projections were interpreted as evidence that the high-molecular-weight MAPs were incorporated as structural components in the microtubule walls. Some microtubules formed in vitro are clearly smooth-walled, depending on the conditions under which they are assembled (Bloodgood and Rosenbaum, 1976; Sloboda et u / . , 1976a), and such projections are absent in most electron micrographs of microtubules in vivo. However, Amos (1977a) has recently reported that whether these projections are seen or not may depend on the methods used for fixation and staining. The similarities between the high-molecular-weight MAPs and dynein, the high-molecular-weight ATPase which constitutes the arms, or projections, on the outer doublet tubules of cilia and flagella (see Gibbons et a / . , 1976) have not

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ELIZABETH C. RAFF

escaped notice, but a relationship between these proteins, if there is one, has not been established. High-molecular-weight MAPs do not have ATPase activity (Bums and Pollard, 1974; Gaskin et al., 1974b) and appear to differ slightly from dynein in electrophoretic mobility (Murphy and Borisy, 1975). However, Bloodgood and Rosenbaum (1976) observed that the rate of assembly but not the final mass of tubules assembled was enhanced by heterologous high-molecularweight fractions, including dynein, isolated from invertebrate sperm flagella. Such activity of MAPs in heterologous systems is not unique. Bovine brain MAPs have been found to support the assembly of tubulin purified from marine invertebrate eggs by DEAE-Sephadex chromatography (Kuriyama, 1977), and tubulin purified from Drosophilu embryos by phosphocellulose chromatography (Green e f al., 1979). Wiche et al. (1977a) observed that porcine brain tau factor not only stimulated the assembly of rat glial tissue culture cell tubulin but also stabilized the polymerizing ability of the tubulin preparations, which in the absence of added tau factor was lost rapidly after preparation. One possibility which has been considered is that the tau proteins may represent fragments of the high-molecular-weight MAPs. Sloboda et al. (1976b) noted that the high-molecular-weight MAPs were degraded into smaller fragments rather rapidly during storage, with a concomitant loss of their ability to facilitate the initiation of microtubule assembly but retention of their effect on the total amount of assembly. Vallee and Borisy (1977) found that limited trypsin treatment of microtubules assembled in vitro selectively destroyed the highmolecular-weight MAPs and correspondingly removed the lateral projections observed on the tubules. However, the typsin-treated tubulin preparation retained its capacity for self-assembly. These investigators therefore proposed that the assembly-stimulating activity of the high-molecular-weight MAPs actually resided in a small fragment (putatively on the order of 30,000 to 35,000 molecular weight). Against this circumstantial evidence are the recent observations of Cleveland et al. (1977a,b) that peptide maps obtained from tau factor and the high-molecular-weight MAPs are different and of Connolly ef ul. (1978) that antisera against the two species of MAPs do not cross-react. As Amos (1977b) has pointed out, however, since the tau proteins would constitute only 20% of the high-molecular-weight MAP molecules, this matter cannot yet be considered definitively resolved. There is no direct evidence that MAPs are microtubule assembly factors which function in vivo. The strongest indication that this may be so is provided by recent indirect immunofluorescence studies showing that antibodies prepared against purified tau or high-molecular-weight MAP fractions decorate the same cytoplasmic network of fibers decorated by antibodies against tubulin. These studies do not answer the question of how MAPs function but do demonstrate that these proteins are at least localized along with microtubules in cells in vivo. As shown in Fig. 7, Connolly et al. (1977) found that, in mouse embryo fibro-

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FIG. 7. Microtubules in mammalian cells in culture visualized by indirect immunofluorescence with antibodies against tuhulin and against MAPs. (a-d) Interphase cells. (e-g) Mitotic cells. (a and e ) Mouse embryo fibroblasts stained with antibody against tubulin. (b and f) Pig embryo fibroblasts stained with antibody against tau factor. (c and g) Rat Ch glial cells stained with antibody against total high-molecular-weight MAPs. (d) Rat C6 glial cells stained with antibody against a-highmolecular-weight MAP. (a) ~ 8 5 0 (; h ) X 6 0 0 ; (c) X500; (d) X850; (e) X775; (f) x 775; (g) x 9 2 5 . Micrographs a, b, d, e and f were kindly provided by J . A. Connolly, Dept. of Anatomy, University of Toronto, Toronto, Ontario. Micrographs c and g reprinted from Connolly c/ ol. (1978) with permission.

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ELIZABETH C. RAFF

blasts, staining with antitau antibodies was identical in pattern although slightly more diffuse than staining with antitubulin antibodies in both interphase and mitotic cells; in addition, both were colchicine-sensitive; more recently this group demonstrated that in rat glial tissue culture cells, the staining pattern with antibodies prepared against high-molecular-weight MAPs is also similar to that obtained with antitubulin antibodies (Connolly et a l . , 1978). Tubulin, tau factor, and the high-molecular-weight MAPs were antigenically distinct; no crossreactivity was observed among the three antibody preparations. Similar experiments by Sherline and Schiavone (1977, 1978) showed that antibodies prepared against rabbit brain high-molecular-weight MAPs decorated the cytoplasmic microtubule network and the mitotic apparatus in mouse 3T3 cells and cultured neuroblastoma cells. The strongest evidence against the specificity of MAPs in microtubule assembly is the finding reported by several laboratories that ion exchange-purified tubulin can be assembled in their absence if the original assembly conditions described by Weisenberg (1972b) are altered. First, Shelanski et al. (1973) discovered that the inclusion of 4 M glycerol in the assembly buffer both facilitated the rate of assembly and stabilized the resultant microtubules. This modification has been widely employed, and many properties of tubulin assembly in vitro may reflect this stabilization even when glycerol-containing buffers are used only in the initial preparative steps, since glycerol appears to associate tightly with tubulin (Detrich et af., 1976). The minimum (or critical) tubulin concentration necessary for assembly originally observed in brain tubulin preparations also containing MAPs was as low as 0.2 mg/ml (Gaskin et af., 1974a; Olmsted et al., 1974). In the absence of MAPs, purified brain tubulin can be assembled in the presence of glycerol or other stabilizers if the tubulin concentration is on the order of 1 mg/ml (Himes et a l . , 1976, 1977; Lee and Timasheff, 1975; Wehland et af., 1977). Second, since MAPs may be separated from tubulin by virtue of the fact that they bind to anionic resins whereas tubulin does not, several laboratories investigated the effects on microtubule assembly of other polycations such as DEAE dextrans, histones, polylysine, protamine, and RNase A. Such molecules were indeed found to stimulate the polymerization of purified brain tubulin in vitro into either morphologically normal microtubules (Murphy et a l . , 1977b) or double-walled tubules consisting of a normal microtubule with the normal subunit arrangement but with an additional layer of tubulin subunits forming the second outside wall (Erickson, 1976; Erickson and Voter, 1976; Jacobs er a l . , 1975a,b). These observations led Erickson and Voter (1976) to raise the possibility that MAPs have been fortuitously and not specifically copurified with tubulin because of nonspecific electrostatic interactions of these proteins with tubulin. Conversely to the stimulation of polymerization by polycationic substances, Bryan et al. (1975a.b) and Nagle and Bryan (1976) observed that RNA and other

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polyanions inhibited microtubule assembly in vitro. The inhibition could be overcome by adding back fractions containing MAPs, suggesting that the polyanions inhibited assembly by binding to basic proteins necessary for assembly. These workers proposed that a similar mechanism might prevent spontaneous assembly of tubulin subunits in vivo. Finally, Herzog and Weber (1977) demonstrated that “nonphysiological” buffer components could be avoided altogether. Ion exchange-purified brain tubulin assembled in the absence of MAPs or other factors into morphologically normal microtubules sensitive to cold and colchicine when the magnesium concentration was raised to 10 mM and the tubulin concentration kept above 2.5 mg/ml; whether these conditions reflect those possible in vivo is at the moment controversial. In a detailed thermodynamic study of the effects of solution variables on the assembly of purified brain tubulin in vitro, Lee and Timasheff (1977) found that, depending on the buffer, the critical tubulin concentration ranged from 0.5 to 2 mg/ml; their data were consistent with a simple model of self-nucleated polymerization requiring the binding of magnesium but not requiring additional nontubulin proteins or other factors. Much less information is available on the properties of assembly in vitro of tubulin from nonneural sources, but interesting differences from, as well as similarities to, the properties of brain tubulin have been revealed, demonstrating points at which models for tubulin behavior based on data from brain may not be generally applicable. Doenges et a/. (1977) and Nagle et a / . (1977) found that tubulin from several lines of mammalian cells in culture could be polymerized in vitro in the presence of glycerol. Their preparations did not contain highmolecular-weight MAPs but did have protein components of a lower molecular weight which were removed concomitantly with a loss of polymerizing activity by ion-exchange chromatography. Perhaps most interesting is the fact that preparations of cultured cell tubulin did not normally form ring or disk structures, but did so in the presence of MAPs isolated from brain. We have investigated assembly in vitro from Drosophilu embryos (Green et ul., 1975, 1979). It is worth noting that Drosophilu embryos exhibit one of the highest rates of mitosis known in eukaryotes (as frequently as every 10 minutes) and are thus quite different from brain tissue in their biological demands for microtubules. In the presence of glycerol Drosophilu tubulin polymerizes into normal microtubules assiciated with a small number of nontubulin proteins from 46,000 to 200,000 in molecular weight, similar but not identical to brain MAPs. After purification by phosphacellulose chromatography this tubulin does not spontaneously reassemble alone but is capable of several modes of selfassociation: (1) forming morphologically normal microtubules with normal assembly kinetics in the presence of heterologous MAPS isolated from bovine brain or in the presence of low concentrations of RNase A, (2) very rapidly forming double- and triple-walled microtubules in the presence of 3.3 mg/ml or more

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RNase A, and (3) forming macrotubules, single-walled tubules with diameters two to three times that of normal microtubules, in the presence of magnesium concentrations of 16 mM or greater. These are similar to forms seen after vinblastine treatment of rat renal cells in vivo (Tyson and Bulger, 1973) and brain tubulin in vitro (Warfield and Bouck (1974, 1975). Kuriyama (1977) purified echinoderm egg tubulin by DEAE-Sephadex chromatography followed by polymerization in vitro in the presence of glycerol; this tubulin initiated spontaneous self-assembly at concentrations above 2.7 mg/ml; at lower concentrations MAPs isolated from porcine brain or microtubule fragments from cilia, which served as nucleation centers, were necessary in order to obtain polymerization. Maximal assembly occurred at salt concentrations much higher than those at which brain tubulin assembles, but which approach the physiological concentrations in marine eggs. All these experiments illustrate the general difficulty of extrapolating between in vitro and in vivo conditions and reemphasize that the details of tubulin assembly in vitro vary greatly, depending on both the source and also on experimental procedures; furthermore, a variety of tubulin structures may be stabilized in vitro which do not occur in vivo. Erickson and Voter (1976) hypothesized that microtubule assembly in vitro is faciliated by agents (including both MAPs and “nonphysiological polycations) which bind to and cause aggregation of tubulin molecules, thus producing areas of localized elevated tubulin concentrations, at which self-assembly occurs. Such a mechanism may also explain the stimulation of assembly by elevated magnesium concentrations, and of course by elevated tubulin concentrations. They suggested that functional tubulin concentrations in vivo might be on the order of 10-50 mg/ml. It is interesting to note, in light of this hypothesis, that the minimum concentration of soluble tubulin in Drosophila eggs is about 14 mg/ml (Green et al., 1979). Still unexplained is the function of MAPs in vivo; at this point the suggestion by Murphy et al. (1977a) that MAPs in vivo serve to stabilize microtubules seems most attractive. Although the mechanism of initiation of self-assembly of tubulin in vitro remains unknown, a substantial amount of evidence indicates that tubule elongation occurs by the addition of subunits to the ends of the protofilaments composing the tubule wall; the final step in tubule formation is then the folding of the sheet of laterally associated protofilaments into a complete tubule-that is, as has been deduced to occur in vivo, from a C-shaped to a closed, circular cross section (Bryan, 1976; Burton and Himes, 1978; Doenges et al., 1977; Erickson, 1974a; Johnson and Borisy, 1977; Kirschner et al., 1975a; Nagle et al., 1977). An interesting point is the directionality of microtubule elongation, discussed in the following section. A second question is the number of protofilaments which make up the tubules. Single microtubules both in vivo and assembled in v i m have generally been found to consist of 13 protofilaments (Fujiwara and Tilney, 1975; Jones, 1975; LaFountain and Thomas, 1975; Tilney er al., 1973). In ”

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doublet tubules of flagella and cilia the A tubule consists of 13 protofilaments and the B tubule consists of 10 additional protofilaments attached so that the common wall contains three of the A-tubule protofilaments (for a review of axoneme structure, see Warner, 1974). However, microtubules composed of 12 to 15 protofilaments have been observed in vivo (Burton et al., 1975; Nagano and Suzuki, 1975). Recently Burton and Himes (1978) and Pierson et al. (1978) showed that, although the majority of tubules assembled from bovine brain in vitro contained 13 protofilaments, the number of protofilaments incorporated could vary from 12 to 16 depending upon solution variables such as pH and on the number of cycles of polymerization.

c. THEGROWTHOF MlCRO'lUHULES in

VitrO O N T O

ISOLATED M I C R O T U R U L E -

ORGANIZING C E N T E R S

As is clear from the examples in Section I, microtubules in vivo generally do not appear to arise spontaneously but to grow from distinct, specific sites. This aspect of microtubule polymerization has been reproduced in vitro by several laboratories. A set of experiments on aster formation in homogenates of developing eggs of the surf clam, Spisulu solidissirnu, by Weisenberg (1973) and later by Weisenberg and Rosenfeld (1975a,b) demonstrated that the microtubule-organizing center is a real structure which can be isolated, and that its functionality develops with time. These investigators found that asters formed when homogenates of activated but not of unactivated eggs were warmed after preparation in the cold. Asters did not form in either the pellets or supernatants from low-speed centrifugation of homogenates of activated eggs; however, when a pellet from an activated egg homogenate was mixed with the supernatant from homogenates of either activated or unactivated eggs, asters formed. Thus the pellet was interpreted to contain the organizing center and the supernatant, soluble tubulin. The morphology of the aster formed depended upon the developmental age of the eggs. No asters and few microtubules formed in homogenates of unactivated eggs. Immediately after activation small asters formed in which microtubules arose from a dense central cylindrical structure. At later times large asters formed in which the microtubules arose from electron-dense material surrounding a discernible centriole; someimtes in the latest, largest asters a few microtubules appeared to originate directly from the centriole microtubules. Interestingly, pretreatment with colchicine did not inhibit the formation of a functional microtubule-organizing center, even though no asters formed in homogenates of treated eggs; when the pellet from a treated egg was mixed with a supernatant from an untreated egg, asters formed. Since aster-forming ability was present in activated egg cytoplasm before the appearance of centrioles, it was concluded that centrioles were not directly involved in aster formation.

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Experiments in several laboratories have shown that isolated structures such as flagella microtubules and basal bodies will serve as nucleating sites for the polymerization of purified tubulin in vitro. These experiments show that even heterologous tubulin assembles onto these structures, that the soluble tubulin need not be competent to initiate self-assembly, and that the assembly process is polar. Burns and Starling (1974) showed that tubulin in high-speed supernatants of rat brain homogenates, which did not spontaneously reassemble, reassembled when heterologous “seeds” consisting of sea urchin sperm tail fragments were added; conversely, soluble sea urchin tubulin assembled on fragments of rat brain microtubules. Similar results were obtained with solubilized flagella outer doublet tubulin (Kuriyama, 1976). The directionality of microtubule growth was demonstrated in a series of experiments showing that, as illustrated in Fig. 8, assembly of brain tubulin in vitro onto fragments of flagella isolated from Chlamydomonus and sea urchin sperm (Allen and Borisy, 1974; Binder et al., 1975) and basal bodies isolated from Chlumydomonus (Snell et al., 1974; Steams et a l . , 1976), retains the orientation of assembly which occurs in vivo. That is, soluble brain tubulin from high-speed supernatants (which did not initiate self-assembly) assembled primarily at the distal end of both basal bodies and flagella fragments, the rate of elongation depending on the brain tubulin concentration. Some growth of tubules at the proximal ends and at basal body accessory structures such as connecting fibers or rootlets occurred at high tubulin concentrations, but distal assembly was always favored. Microtubules added to both tubules of the central pair but almost entirely to the A tubules of the doublet or triplet microtubules. The formation of doublet or triplet tubules has not yet been observed in v i m . That directionality of growth is an intrinsic property of brain tubulin itself was shown by Dentler et al. (1974). These workers injected chicks intracerebrally with tritiated leucine, isolated labeled brain tubulin, and assembled it in vitro into microtubules; pieces of these microtubules were then incubated with unlabeled tubulin under assembly conditions, and the resulting tubules were exarnined by electron microscope autoradiography . They found that essentially all the radioactivity in the elongated tubules appeared at one end. Recent evidence on the intrinsic polarity of microtubules was provided by Margolis and Wilson (1977, 1978), from experiments on substoichiometric colchicine inhibition of microtubule assembly in vitro and on the rate of exchange of soluble tubulin with microtubules assembled in vitro in the presence and absence of podophyllotoxin, using 3H-GTP as a marker. Their results strongly suggested that, at least in vitro, assembly and disassembly of brain tubulin occur at opposite ends of the microtubule during the steady state. These investigators suggested that possible consequences of this in vivo are (1) an “intrinsic flow” of tubulin subunits through the microtubule, resulting in a specific directionality

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of microtubule function (e.g., during mitosis, see Margolis e t a / . , 1978), and ( 2 ) that MAPS may function by preventing disassembly. The interpretation of this model for microtubules which have attached ends (i.e., originate at a discrete structural site such as the kinetochore or the distal end of the basal body) seems less clear than for microtubules with two free ends (i.e., self-nucleated tubules assembled in vitro);the model requires that microtubule growth at a site proceed through continued insertion of subunits at the site rather than at the tip, which would then be the disassembly end. This question may relate to the ambiguity of results of experiments discussed in Section I which were designed to determine the point of insertion of tubulin subunits into the growing axoneme of cilia and flagella. At any rate these data are an important consideration as a reminder that the assumption implicit in many studies of microtubule polymerization, that disassembly is the reverse of assembly, may not be entirely valid. Another series of experiments has dealt with the assembly of purified tubulin subunits onto partially purified components of the mitotic apparatus. Cande e t a / . (1974) and lnoue rt ul. (1974) treated lysed mitotic cells and Rebhun et a / . (1974) treated mitotic spindles isolated from Spisulu with solutions containing soluble vertebrate brain tubulin and found that reversible addition of the tubulin to mitotic tubules, accompanied by typical changes in spindle birefringence, occurred. McGill and Brinkley (1975) reported that when HeLa cells arrested in metaphase by colcemid were lysed into solutions containing soluble brain tubulin, microtubules assembled both at the kinetochores and in association with centriole pairs. Milsted et a / . (1977) later exploited the use of brain tubulin solutions to stabilize the mitotic apparatus from blastema stage embryos of D . rnelunoguster so that it could be isolated and examined by scanning electron microscopy. Snyder and Mclntosh (1975) showed that the initiation of new microtubules in mitotic rat kangaroo cells lysed into tubulin solutions took place, as well as elongation of microtubules already present. Microtubules grew directly from the kinetochore but not from the centriole, although they emanated from the surrounding region. These workers suggested the possible involvement of the centriole in microtubule orientation or placement rather than nucleation. Similarly to the Spisulci experiments, this study also demonstrated that the potential for microtubule initiation by the centriolar region is a time-dependent process. “Ripening” occurred between early prophase and late prometaphase, as demonstrated by the increasing number and length of microtubules in asters formed during this period. The assembly of microtubules at the kinetochore was also time-dependent and did not take place before prometaphase. Differential control over the two kinds of microtubules was indicated by the fact that microtubules assembled more readily into asters but kinetochore microtubules were more stable. Pretreatment with colchicine prevented spindle formation but not ripening of the microtubule-organizing center.

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FIG. 8 . The directionality of microtubule assembly i n vifro on isolated organizing structures. Basal bodies (a) and fragments of intact flagella axonemes (b) isolated from Chlomydomonas were incubated with soluble chick brain tubulin. In both cases assembly was clearly preferential at the . from Snell ef al. (1974), with permission. distal end of the isolated structure. (a) ~ 2 8 , 4 6 0Reprinted Copyright 1974 by the American Association for the Advancement of Science. (b) X 13,600. Micrograph kindly provided by L. I. Binder from his Ph.1). Dissertation, Yale University, New Haven, Connecticut.

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Telzer e t d . (1975) found that, as shown in Fig. 9, when chromosomes isolated from HeLa cells were attached to grids and incubated with soluble chick brain tubulin before fixation, microtubules assembled only at the kinetochore region, starting in nearly parallel array close to the kinetochore (perpendicular to the long axis of the chromosome) and then radiating outward. No microtubules were visible on isolated chromosomes alone; it seemed likely that the microtubules were initiated at the kinetochore, but the possibility that the elongation of very short stubs of microtubules occurred could not be eliminated.

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ELIZABETH C . RAFF

FIG.9. Microtubule Assembly irr virru on the kinetochore of isolated chromosomes. Chromosomes isolated from HeLa cells were incubated with soluble chick brain tubulin; microtubule assembly took place only at the kinetochore. Marker: I p m . Reprinted from Telzer ef ( I / . (1975). with permission.

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Finally, as shown in Fig. 10, Gould and Borisy (1977) recently directly demonstrated that the amorphous electron-dense material observed in ultrastructure studies functioned as a microtubule-organizing center in v i m . They studied the formation of microtubules from the centrosomes of CHO cells in culture. This structure consists of a pair of centrioles surrounded by fibrous electron-dense material from which the cytoplasmic microtubules arise in interphase; during mitosis, there is one centrosome at each pole of the mitotic apparatus. These investigators presented electron micrographs of negatively stained material clearly showing that both cytoplasmic and mitotic microtubules arose directly from the amorphous pericentriolar material in normal cells, in cells recovering from colcemid pretreatment, and in lysates of colcernid-blocked cells incubated with soluble pork brain tubulin. Furthermore, microtubules were observed to grow out from isolated preparations of amorphous material (identified as the pericentriolar material by the presence of dense viruslike particles associated with the centrosome in vivo) when incubated with soluble brain tubulin. In the

FIG. 10. The nucleation of microtubule assembly iir rirro by isolated electron-dense pericentriolar material. (a and b) Patches of electron-dense fibrous material isolated from CHO cells in culture; inferred to he pericentriolar material by the presence of viruslike particles typically associated with the material iti v i ~ w~ 4 0 . 8 1 0 .(c and d ) Similar patches of isolated pericentriolar material after incuhation with soluble porcine brain tubulin. ~ 4 0 . 8 1 0 .Reprinted from Could and Broisy (1977). with permission.

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ELIZABETH c‘. RAFF

absence of pericentriolar material, isolated centrioles incubated with brain tubulin showed growth of tubules by direct elongation of centriole tubules. Could and Borisy have concluded that the pericentriolar material in these cells both initiates and anchors microtubules in vivo and in v i m but, as they have also pointed out, their results bring up a very interesting question about the function of the centriole in asters and in cytoplasmic microtubule arrays, and about the origin of microtubule arrays of very different shape and function (i.e., cytoplasmic and mitotic) from the apparently structureless pericentriolar material.

D. EXPERIMENTS IN W H I C H MICROTUBULE ASSEMBLY IS ELICITED in Vivo THE

INJECTION

OF

BY

MICROTUBULE-~R.GANIZING CENTERS I N T O EGGS

Several experiments have been reported in which the formation of asters or mitotic spindles, accompanied by the initiation of cleavage and some degree of embryonic development, is elicited by the injection into eggs of fractions containing microtubules. Such experiments may be considered the in vivo equivalent of the experiments described in the preceding section. Certain observations are common to all these injection experiments. First, at least p a t of the normal temporal sequence of postfertilization events must be preserved; that is, the egg must be capable of being activated, and activation of the egg must occur before cleavage can be initiated. Second, only particulate microtubule-containing structures elicit aster or spindle formation and cleavage initiation. It appears that the injected structures serve as organizing centers for polymerization of the tubulin already present in the egg. As in the in vitro experiments discussed above, microtubule-organizing structures from heterologous species function. Aspects of the results of‘ injection experiments which differ considerably are the type of microtubule-containing structure observed to cause cleavage initiation and the specific nature of the egg response to the injected structures, which varies from formation of multiple asters accompanied by abnormal cleavage to successful completion of parthenogenetic development. It is an old observation in embryology that frog eggs can be induced to undergo parthenogenetic development by pricking with a “dirty” needle. This process consists of two steps: (1) activation of the egg (pricking with a clean needle causes activation without subsequent development), and (2) initiation of cleavage and subsequent development triggered by an agent present in the blood or lymph into which the needle has been dipped. Fraser (1971) examined the extent of parthenogenesis induced in R. pipiens eggs by the injection of various fractions of homogenates of different tissues. Particulate fractions from low-speed centrifugation of homogenates from many tissues from several species elicited cleavage; the highest activity was found in the fraction from brain, which resulted in normal cleavage in 20% of the injected eggs, many of which developed to the hatched tadpole stage. From the effects of colchicine, trypsin, mercap-

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toethanol, deuterium oxide, and detergents, Fraser concluded that the agent responsible for initiating cleavage was microtubules. She then injected fragments of cilia and flagella axonemes from Tetrahymenu and Chlamydomonas but observed no cleavage. Fraser’s experiments were suggestive but inconclusive because the work was done with uncharacterized fractions. Similar experiments using a fish, the medaka (Oryzias latipes), showed that cleavage could be induced in unfertilized eggs by injecting flagella, purified fragments of sperm tail axonemes, or precipitated aggregates of soluble tubulin, but not by injecting solubilized sperm tail tubulin (lwamatsu and Ohta, 1974; Iwamatsu et al., 1976; Ohta and Iwamatsu, 1974). Heterologous sperm tail fractions elicited cleavage initiation, but the most normal-appearing cleavage resulted from the injection of homologous fractions. In many injected eggs the cleavage patterns were abnormal, resembling polyspermy, but a small percentage developed to gastrulation. Eggs which failed to activate during the injection process failed to cleave at all. The question of timing was recently studied in Ranu eggs by Elinson (1977). He found that cleavage occurred only if both an organizing center was supplied (in this study, by sperm) and activation of the egg had occurred, but that the normal order of these events could be reversed. Oocytes which had been induced to mature by hormone treatment but which were still between metaphase I and metaphase I1 (at which time they become activatable and are normally fertilized) could be inseminated, but no cleavage or development occurred. A small spindlelike structure formed around the sperm chromosomes. Elinson maintained inseminated immature eggs until they became mature and then activated them by electric shock; the eggs then began to cleave and progressed to blastula or blastulalike stages. The cleavage patterns were often irregular, but a small percentage of the eggs completed development to tadpoles. Heidemann and Kirschner (1975) injected basal bodies isolated from Chlumydotnonas and Tetruhymena into unfertilized eggs of X . laevis. They observed the formation of multiple asters and irregular cleavage furrows 20-60 minutes after injection (normally cleavage begins about 90 minutes after fertilization); since both the timing and cleavage pattern were abnormal, the events in the injected eggs probably did not reflect normal events of embryonic development. However, the capacity of the egg to assemble asters depended on the stage of development. Fully grown but immature oocytes were found to contain the same amount of tubulin, measured by colchicine-binding activity, as unfertilized eggs. However, these oocytes could not be fertilized or induced to form asters by the injection of materials which induced aster formation in eggs. Heidemann and Kirschner speculated that tubulin in oocytes is unable to polymerize. More recent work by these investigators has shown that after hormonal stimulation, germinal vesicle breakdown, and mixing of nucleoplasm and cytoplasm have taken place, Xenopus oocytes induced to mature in vitro form asters in response to injected

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basal bodies in the absence of the cortical events of activation (Heidemann and Kirschner, 1978). Aster formation was specific to basal bodies; the injection of soluble tubulin, microtubules prepared from porcine brain, fragments of sperm flagella, buffer alone, or fractions from E. coli had no effect, whereas crude fractions containing centrioles or basal bodies (such as crude nuclei preparations or Tetrahymena pellicle fragments) induced aster formation. No asters formed after the injection of basal bodies plus colchicine. Heidemann and Kirschner suggested that these results support the idea that centrioles play an active role in aster formation, and that whether the basal body or centriole organizes a flagellum or an aster depends on the intracellular environment. Heidemann et a l . (1977) attempted to define some of the components that control the functionality of centrioles in aster formation. They found that aster formation did not occur when basal bodies injected into Xenopus eggs were first treated with proteolytic enzymes or with RNase; treatment with DNase and various other enzymes had no effect. Treatment with proteases was correlated with gross structural damage to the basal bodies, but RNase treatment did not cause any structural damage which they could observe. The effects of RNase appeared to be specific, since treatment of basal bodies with RNase A, a basic protein, or S1 nuclease, an acidic protein, gave the same results. Earlier, however, Dippell (1976) showed that a structure consisting of a twisted fiber and dense granules seen in longitudinal sections of the lumen of the central part of Paramecium basal bodies contained both RNA and protein and that both were removed after either RNase or protease treatment. We have observed similar structures in basal bodies isolated from Chlamydomonas (E. Raff, R. Raff, and F. Turner, unpublished data). Thus it is possible that RNase causes subtle structural changes. However, since Heidemann et al. found that RNase-treated basal bodies served as templates for in vitro microtubule assembly when incubated with soluble brain tubulin, they concluded that the RNA present in basal bodies is required for aster formation and that the two functions of this structure, centriolelike activity (formation of mitotic asters) and basal body-like activity (serving as a direct template for microtubule elongation), are separable. Zackroff et ul. (1976) also investigated the possible functional role of RNA in aster formation using the partly in vitro system of aster formation in homogenates of meiotically dividing Spisula oocytes. However, while RNase treatment in this system also caused changes in aster morphology, the effects were mimicked by other basic proteins such as histones. Contrary to results reported for microtubule assembly in vitro, they observed enhancement of aster formation in the presence of certain polynucleotides and suggested the presence in vivo of an inhibitor of microtubule assembly bound by and thereby inactivated by specific RNA. Maller et al. (1976) reported that the formation of asters and spindlelike structures followed by apparently normal cleavage and development to the blas-

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tula stage was induced by the injection of sea urchin sperm head fractions into Xenopus eggs. Aster formation and cleavage initiation was correlated with the presence of centrioles in the preparations. Optimal results were obtained with the equivalent of 10 to 15 sperm heads per egg; 90% of injected eggs initiated cleavage at the correct time (90 minutes) and, of these, 16% progressed to blastula stages. Some association of condensed chromosomes with the induced spindles was observed. Thus the events in these eggs resembled normal postfertilization events. With higher doses of injected material, the normal pattern and timing of cleavage was suppressed and precocious multiple cleavage became predominant; after an injected dose of the equivalent of 350 sperm heads per egg, multiple asters formed in all the eggs, followed by rapid irregular fragmentary cleavage such as Heidemann and co-workers had observed. Maller and coworkers suggested that their results indicated a “titration” of the anticleavage threshold of the egg-that the introduction of a gross excess of potential microtubule-initiating sites overwhelms the control system in the eggs. These workers also injected sperm tail fractions, but it required the equivalent of 400 tails per egg to induce parthenogenetic development and only 30% of the eggs initiated cleavage. Because of the large dose needed, they concluded that the source of the activity of tail fractions was likely to be centrioles present from contaminating heads, rather than the flagella microtubules. Forer er nl. (1977) injected mitotic spindles isolated from sea urchin embryos into Rnnu eggs. In half of the eggs injected with glycerol-dimethyl sulfoxide (DMS0)-isolated spindles, or spindles isolated in hexylene glycol and immediately transferred to glycerol-DMSO, normal-appearing cleavage was initiated. The results were the same in enucleated eggs. No cleavage resulted after the injection of spindles isolated and subsequently incubated in hexylene glycol, These investigators preferred the hypothesis that cleavage initiation resulted from function (and chromosome movement) of the injected mitotic figures, but it seems more likely that the injected structures served as microtubule-organizing centers as in other injection experiments. We have examined a maternal effect mutation, nc, in the Mexican axolotl, A . mexicanum (Raff and Raff, 1978; Raff et al., 1976). Sperm enter mutant eggs, which then undergo many of the initial steps of activation such as the cortical response and swelling and migration of the pronucleus, but fail to initiate cleavage or subsequent development. When highly purified fragments of doublet tubules isolated from sea urchin sperm tails were injected into fertilized nc eggs, the eggs initiated cleavage and developed until a blastulalike stage was reached, at which time they arrested. No microtubule assembly or cleavage occurred after the injection of buffer, soluble tubulin, nontubulin particles, or normal nucleoplasm, nor did cleavage take place after the injection of tubule fragments if the eggs were incubated in colchicine. In preliminary experiments we injected basal bodies isolated from Chlamydomonas into activated nc eggs. However, although

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very few of the eggs initiated normal-appearing cleavage, most did not cleave at all and a few underwent multiple premature fragmentary cleavage similar to that routinely observed in Xenopus by Heidemann and Kirschner (1975) and by Maller et ul. (1976) with high levels of injected material. Normal timing was retained in mutant eggs corrected by the injection of tubule fragments. Normal fertile axolotl eggs begin cleavage at 18°C about 7 hours after spawning (and fertilization); fertile nc eggs injected with tubules within 1-2 hours after spawning initiated cleavage 5-6 hours later. Fertilization in axolotl eggs is normally polyspermic; only one sperm nucleus becomes the male pronucleus, but large asters form around each sperm. Sperm enter nc eggs, but no asters form. It is possible that the subsequent initiation of microtubule assembly induced by the injection of relatively large numbers of tubule fragments represents a ‘titration” of the cleavage potential of the egg, as suggested by Maller et al. (1976) for Xenopus. Mutant eggs contain a pool of tubulin essentially similar in size and properties to be found in normal eggs; the lesion in the mutant appears not to reside in the tubulin molecules per se but rather in an activation step preceding the formation or functioning of microtubule-organizing centers. The nc mutant condition could be phenocopied with either normal or nc unfertilized eggs. Unfertilized axolotl eggs can be artificially activated by means of electric shock, and the injection of microtubules into such artificially activated eggs gave the same results as in fertilized nc eggs, that is, initiation of cleavage and development to a partial blastula. In the other injection experiments discussed, the formation of asters was observed by light microscopy. Likewise, examination of sections of fertilized nc eggs or artificially activated axolotl eggs into which microtubule fragments had been injected showed asters and mitotic spindlelike structures in the cleaving blastomeres. As shown in Fig. 11, we also examined these structures by electron microscopy, which revealed that they represented large, parallel arrays of microtubules somewhat similar to those seen in the mitotic spindle of normal cleaving eggs. We occasionally observed doublet microtubules in cross sections of corrected eggs, indicating that the injected doublet tubules retained their morphological integrity. Locating such structures in sections of large, yolky amphibian eggs is technically difficult, and we were not able to examine the site(s) of initiation of the induced microtubules. The various injection experiments confirm that microtubule arrays can be induced to form in vivo by other microtubule-containing structures. The asters formed most likely do not arise through direct elongation of the tubules of the injected basal bodies and flagella axonemes as occurs when such structures are incubated with tubulin in vitro. A possible function of the injected structures might be to serve as a focal point for the amorphous material from which mitotic tubules appear to arise. Similarities in results appear directly related to the general nature of microtubule assembly systems; reasons for differences are less

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FIG. I I . Microtubules elicited in nr mutant axolotl eggs by the injection of microtubule fragments. Cleavage was induced in fertilized ric eggs by the injection of fragments of purified outer doublet tubules isolated from sea urchin sperm tails; after about 24 hours the resultant blastula-like embryos were fixed and sections were examined by electron microscopy. (a) Longitudinal section of a bundle of microtubules. This array of microtubules is similar to the mitotic microtubules observed in normal embryos. x 15.870. Reprinted from Raff and Raff (1978), with permission. (b) Highermagnification view of another longitudinal section of induced microtubules showing a cross section of an injected doublet tubule (arrow); the inset shows a cross-sectional view of another group of induced microtubules. ~ 5 7 . 2 7 0 .Both electron micrographs were taken by F. R. Turner, Dept. of Biology, Indiana University, Bloomington, Indiana.

clear. The most puzzling difference is that either basal bodies (or centrioles) or microtubule fragments, but not both, elicited aster formation and cleavage initiation in the various species studied. The tempting, if rather unsatisfying, rationalization is to ascribe this to differences in preparative or experimental procedures, or to species differences. For example, of the species studied in the injection experiments, eggs of Runa, the medaka, and Xenopus are all easily activated by pricking, whereas axolotl eggs can be activated only with difficulty by fairly severe electric shock. In other species, notably sea urchins, the formation of asters and even complete parthenogenetic development can be induced without the introduction of microtubule-organizing centers (Brandriff ei al., 1975; Dirksen, 1961; Kato and Sugiyama, 1971; Moy et al., 1977). Microtubule assembly

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ELIZABETH C:. RAFF

is only one of a complex battery of interlocking events which take place in the egg after fertilization (see Epel, 1977; Epel et af., 1969, 1974); the nature of the coupling between events of activation, microtubule assembly, and cell division may well differ in various species. A N D OTHERSMALL MOLECULES AS POSSIBLE REGULATORS OF E. CALCIUM MICROTUBULE ASSEMBLY in Vivo

Calcium appears to be important in the regulation of microtubule assembly in vivo, although much of the evidence on this point is indirect (reviewed by Bryan, 1975). Of primary consideration is the fact that calcium inhibits microtubule assembly in vitro. Fluctuations in local cellular calcium levels are thus envisioned to participate in microtubule regulation, with relatively high cellular calcium preventing assembly, or alternatively, promoting disassembly, and lower calcium levels allowing assembly. However, how far the in vitro effect can be extrapolated to the conditions in vivo is unclear. As discussed above, the level of calcium which inhibits microtubule assembly in vitro varies considerably, depending on the concentration of magnesium and other ions, but at any rate it has not been reported to be significantly inhibitory at concentrations much M . According to Rasmussen and Goodman (1975) this is one or even below two orders of magnitude greater than the calcium concentrations which exist in mammalian cell cytoplasm. However, as they point out, microsomes and mitochondria have much higher calcium levels (potentially lop2M in mitochondria) although in bound form. Some experiments by Nishida and Sakai (1977) indicated that microtubles may be more calcium-sensitive in vivo than has been observed in vitro. They found that microtubule assembly in crude extracts of M calcium, whereas the same extent of porcine brain was 50% inhibited by inhibition of assembly of microtubule proteins purified by two or more cycles of assembly was achieved at only lo-:$ M calcium. They suggested the presence of a factor controlling calcium effects, since they were able to restore calcium sensitivity to the purified microtubule proteins by adding back approximately equal amounts (measured by protein content) of partially purified supernatant; the relatively large amount of material needed, however, makes this result somewhat difficult to interpret. Circumstantial evidence for calcium control of microtubules is provided by the observations of Gallin and Rosenthal(1974), who found that, during chemotactic movements of human granulocytes, microtubules proliferated in the cell processes concomitantly with changes in the state of cellular calcium levels, including both the release of calcium and a shift of' cytoplasmic calcium to a particulate fraction. Several laboratories have reported experiments aimed at directly assessing calcium effects on microtubules in vivo. Schlaepfer and Bunge (1973) simply

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exposed axonal microtubules of cultured rat sensory ganglia to the external medium by transecting them; rapid disruption of microtubules occurred in calcium-containing medium but not in the presence of EGTA. Many recent studies of calcium effects have employed the ionophore A23 187 which promotes passive transport of calcium and other divalent cations through lipid barriers, including biological membranes, thus releasing intracellular calcium and bringing it into equilibrium with the surrounding medium (Reed and Lardy, 1972; Wong et d., 1973). Schliwa (1976) studied the effect of the ionophore on the retraction and reformation of heliozoan axopodia; he observed that in the presence of A23 187, disassembly of axopodia microtubules could be controlled by the amount of calcium in the external medium. At lo-” M calcium, slow, orderly disassembly of the axoneme microtubules occurred, and cross sections showed the pattern of remaining microtubules to be intact. At lo-” M calcium, disassembly occurred rapidly, and cross sections showed considerable disarray both in the pattern of remaining microtubules and in individual microtubules (many C and S shapes were present). The axopodia reformed in calcium-free medium containing EGTA. These experiments did not show whether or not calcium is the normal in vivo regulator of axopodiuni length, but Schliwa felt that the levels of calcium which in the presence of ionophore caused retraction of axopodia were low enough to be physiologically significant. Fuller and Brinkley ( I 976) observed that the cytoplasmic microtubule network in cultured 3T3 mouse fibroblasts demonstrated by immunofluorescence staining with antitubulin antibodies disappeared after a 100-minute exposure to medium M calcium. The microtubule network reapcontaining A23187 and 4.9 X peared after the cells were returned to normal medium. As a model for the regulation of assembly by sequestration of calcium, this group added preparations of mitochondria to microtubule assembly mixtures in vitro; mitochondria1 uptake of calcium relieved the inhibition of assembly caused by 5 x M calcium in the presence of equimolar magnesium (Fuller and Brinkley, 1976; Fuller et al., 1975b). Osbom and Weber (1977) tested the effect of calcium on cytoplasmic microtubules directly; they observed that the treatment of cultured mammalian cells with nonionic detergents in buffers containing GTP and EGTA produced a “cytoskeleton” in which the microtubule network remained intact. The inclusion M calcium, however, caused disruption and fragmentation of the of 4 x microtubules. In both these studies the level of calcium observed to cause microtubule disassembly was higher than probable physiological levels. Fulton (1977) observed that, while the transformation of Naegleriu from the ameboid to the flagellated state (during which a cytoplasmic microtubule system develops as well as the flagella) took an hour and required both transcription and translation; the opposite change, back to the ameboid state, required less than a minute. He found that a fraction isolated from amebae caused transient shape changes in flagellates resembling those that took place during actual transforma-

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tion. The treatment of flagellates with A23187 caused similar shape changes. Fulton postulated that the microtubule cytoskeleton in the flagellate is rapidly dismantled in the presence of the increased levels of free calcium, which in turn are required for the actin-based motile system in the amebae and that cyclic changes in free calcium levels in vivo are controlled by a factor present in the isolated fraction. Complex changes in both divalent and monovalent ions take place at fertilization and in general in cells changing from a nonproliferating to a mitotic state. Calcium fluctuations and pH changes are involved in the step-up from the quiescent metabolism of the unfertilized egg to initiation of cell division and early embryonic development (reviewed by Epel, 1977). There is some difficulty in sorting out the specific postfertilization events which control assembly of the mitotic apparatus. Treatment with the ionophore A23187 has been found to cause activation of eggs of diverse vertebrate and invertebrate species, apparently through release of intracellular calcium and/or transport of calcium across the cell membrane (Belanger and Schuetz, 1975; Chambers et d . , 1974; Lallier, 1974; Schuetz, 1975; Steinhardt and Epel, 1974; Steinhardt et d.,1974). Ionophore-treated eggs undergo the cortical changes typical of activation and complete meiosis but in most cases do not subsequently begin mitosis. Recent studies with the calciumsensitive photoprotein aequorin have demonstrated that during the activation of both vertebrate and invertebrate eggs a rapid, transient rise in calcium due to the release of intracellular calcium occurs at the surface of the egg; the calcium concentrations reached are on the order of 10-"-10-" M (Gilkey et d.,1978; Ridgway et d.,1977; Steinhardt et [ I / . , 1977). At least in sea urchin eggs, the wave of calcium release is followed by a rise in pH caused by exchange of extracellular sodium ions for intracellular hydrogen ions (Johnson et d.,1976; Lop0 and Vacquier, 1977). Fluctuations in calcium and magnesium ion concentrations appear to regulate the activity of factors which control metaphase arrest and germinal vesicle breakdown in amphibian oocytes (Masui et d . , 1977; Meyerhof and Masui, 1977; Wasserman and Masui, 1976). In other cell types, Jensen et a / . (1977) reported that A23187 had a mitogenic effect on human peripheral lymphocytes, supporting their hypothesis that an increase in the calcium level in some intracellular compartment is an important event in the change to a proliferating state, while Cone and Cone (1976) found that mitosis could be induced in vitro in fully differentiated chick embryo neurons by agents which cause depolarization by increasing intracellular sodium ions and decreasing potassium ions. The complexity of such systems was pointed out by Baltus et d . (l977), who examined ionophore-induced changes in calcium levels in a study of oocyte maturation in Xenopus (which includes formation of a meiotic spindle); these workers concluded that it is an oversimplification to attribute the regulatory role to calcium alone, and that what must be considered is the total balance

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between mono- and divalent ion levels. Furthermore, they concluded that strong compartmentalization of ions must occur in vivo. Rasmussen and Goodman (1975) also emphasized that changes in calcium levels are accompanied by changes in the total ion balance of the cell. Certainly regulatory mechanisms involving changes in calcium or other ion concentrations must be either temporally transient or spatially localized, or both. For example, while assembly of the mitotic apparatus presumably requires low calcium concentrations, calcium is required for subsequent formation of the cleavage furrow and completion of cytokinesis (Baker and Warner, 1972; Hollinger and Schuetz, 1976; Rebhun, 1977). A calcium-activated ATPase associated with the mitotic apparatus isolated from sea urchin eggs (Mazia et a / . , 1972; Petzelt, 1972; Petzelt and Ledebur-Villiger, 1973) has frequently been postulated to play a role in controlling calcium levels, but there is no direct evidence for this. Implicit in many of the studies mentioned above is the assumption that in analogy to its effects in virro, calcium also acts directly on microtubule assembly in vivo. Another possibility is that it acts indirectly, possibly in coordination with adenosine cyclic 3' 3'-monophosphate (CAMP) or other cyclic nucleotides. Rasmussen and Goodman (1975) have reviewed the relationships between calcium and CAMP, mutually regulatory molecules involved in the translation of cell surface events into metabolic responses within the cell. Many of the known effects of cAMP are mediated through the stimulation of protein kinases with resultant phosphorylation (and a change to the active state) of the target protein(s); in some cases calcium also functions directly in this manner (reviewed by Greengard, 1978). Interest has also focused on a second cyclic nucleotide, cGMP, as a possible microtubule regulator, because of the involvement of guanine nucleotides demonstrated in microtubule assembly in virro and because of the reciprocity of the action of cAMP and cGMP observed in some other cellular processes (see Goldberg er NI., 1974). An interesting possibility exists that some aspect of microtubule assembly may be controlled by cyclic nucleotide-stimulated protein kinase activity through modification not of tubulin but of MAPs. Preferential phosphorylation both of high-molecular-weight MAPs and tau factor by a CAMP-stimulated protein kinase which copurifies with brain tubulin during cycles of polymerization in vitro has been observed in several laboratories (Cleveland et a / . , 1977a,b; Lagnado and Kirazov, 1975; Rappaport er d.,1976; Sheterline, 1977; Sloboda e t a / . 1975, 1976b). Sandoval and Cuatrecasas (1976b) reported that CAMP-stimulated phosphorylation of proteins in brain tubulin preparations was antagonized by cGMP. Unlike calcium, cyclic nucleotides have not been demonstrated to affect microtubule assembly in vitro. However, workers in several laboratories have observed changes in microtubule patterns in various cell types associated with changes in cyclic nucleotide levels (reviewed by Willingham, 1976). The most

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extensive studies have been with CHO cells in culture. When treated with dibutyryl cAMP or other agents which cause elevation of cellular cAMP levels, these cells undergo a marked change in morphology from a knobbed epithelial cell shape to an elongated fibroblast shape; the cell shape change is accompanied by a proliferation of arrays of microtubules in the cell processes, parallel to the cell axis, and appears to be mediated by the stimulation of protein kinase activity (Borman et al., 1975; Hsie et al., 1976; Li et al., 1977; O’Neill et al., 1977; Porteret al., 1974). Rubin and Weiss (1975) determined the amount of assembled microtubules in CHO cells (measured as a percentage of the total colchicinebinding activity pelleted in the presence of a microtubule-stabilizing buffer) and found that the increase in amount of microtubules after dibutyryl cAMP treatment ranged from 30 to 300% depending on culture variables including cell density. Hennebeny et al. (1975) observed that in calcium-containing medium the ionophore A23 I87 prevented the elongation of CHO cells induced by dibutyryl cAMP treatment, suggesting reciprocal effects of calcium and cyclic nucleotides on microtubule assembly in these cells. Microtubule proliferation with concomitant cell elongation has also been observed to accompany increased cellular cAMP levels in several other cell types (Brinkley et al., 1975a,b; Nath et ul., 1978; Willingham and Pastan, 1975). DiPasquale et al. (1976) found that, although the proporition of tubulin in assembled microtubules increased after the treatment of cultured melanoma cells with dibutyryl CAMP, the total amount of tubulin remained unchanged. Several observations, mostly indirect evidence based on colchicine and other drug studies, indicate cGMP-related changes in microtubule patterns in certain cell types. In a study on the capping of binding sites for the lectin concanavalin A (Con A) on peripheral blood polymorphonuclear leukocytes, Oliver (1975) and Oliver et al. (1975) observed that the effects of colchicine and cGMP or agents which stimulated its production were mutually antagonistic, suggesting cGMP promotion of microtubule assembly. Similarly, lysosomal enzyme release in this cell type is inhibited both by colchicine and under conditions where cAMP levels are increased, but is stimulated by cGMP (Goldstein et ul., 1973; Smith and Ignaro, 1975; Weismann et ul., 1975). Kaliner (1977) reported similar observations for immunologic secretion in human lung tissue. Generalizing from the consideration of CHO cells, Puck (1977) has suggested that cAMP mediates the maintenance of the cytoskeleton of microtubules and microfilaments and that this network is involved in the coordination of growthregulating information exchanged between the cell surface and the nucleus; one of the consequences of the disorganization of this network may then be malignant transformation. Observations on CHO and other cells had earlier led to the suggestion that in transformed cells the levels of cAMP are lower than in normal cells; however, not all transformed lines revert to fibroblastlike shape after

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cAMP treatment, nor do all transformed lines have decreased cAMP levels (see reviews by Pastan et al., 1975; Rebhun, 1977). The question of calcium, cyclic nucleotides, and their interrelated involvement in the events of cell division has recently been extensively reviewed by Rebhun (1977), who pointed out that, while coordination of the events of mitosis appears universally to involve changes in the distribution of calcium ions, no generalizations can be drawn concerning the actions of cyclic nucleotides since cell division, and apparently also microtubule assembly, can be stimulated, inhibited, or unaffected by cyclic nucleotides, depending on the cell type in question. Finally, another class of small molecules which may be involved in the regulation of microtubules are sulfydryl-containing compounds; fluctuations in cellular levels of free sulfydryl groups have long been implicated in control of the formation and maintenance of the mitotic apparatus (see Mazia, 1961). In studies on the effects of various metabolic inhibitors on the mitotic apparatus of eggs of sea urchins and other marine invertebrates Rebhun (1976) and Rebhun et al., (1975, 1976) observed that the disassembly of mitotic microtubules was correlated with rises in oxidized forms of glutathione and other sulfhydryl-containing compounds in the cytoplasm; this group suggested that calcium levels might be mediated through the oxidation of sulfhydryls. Mellon and Rebhun (1976a,b) found that, at concentrations of calcium which inhibited polymerization of tubulin in vitro, the titer of free sulfhydryls was also decreased. In studies with human peripheral polymorphonuclear leukocytes, Oliver et al. (1976) and Burchill et ul. (1978) observed the disassembly of cytoplasmic microtubules under conditions in which gluathione oxidation and formation of protein-S-S-glutathione occurred; these workers emphasize that no evidence exists for direct control of microtubules by the interaction of tubulin with glutathione but rather that control of microtubule assembly is part of the general physiological balance of cell metabolism and the cellular response to changes in oxidative conditions. F. TIME-DEPENDENT PROPERTIES OF TUBULIN A N D MICROTUBULES There are numerous reports in the literature that biochemical properties of tubulin may vary depending on the developmental age or stage of differentiation of the tissue from which it is isolated. Such changes in properties may reflect mechanisms which govern the temporal regulation of microtubule assembly and are also part of the general question of tubulin heterogeneity discussed earlier. Some observations are consistent with a single population of tubulin molecules acted upon by changing batteries of regulatory factors, while other observations indicate that different tubulin molecules may be present at different times in development. Both may occur. The timed synthesis of tubulin specific to flagella

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in the ameba-flagellate protozoan Naegleria described by Fulton and co-workers (see discussion above) appears to be a good developmental model system; in higher organisms, gene switches during development are well known. There are recent indications that a general eukaryotic regulatory mechanism may involve the presence of multiple forms of key proteins, perhaps differing only subtly. For example, several of the histones have been found to consist of two or more very closely related proteins present at specific times in development. Relative comparisons of the details of the biochemical properties of tubulin must be made with some care, because the method of preparing tubulin fractions, the method used to measure drug binding, and so on, influence the results (Bamburg etal., 1973a,b; Hains et ul., 1978; Owellen et al., 1972; Raff, 1977; Sherline et al., 1975). Bamburg et al. (1973a,b) first observed that as development progressed the lability of the colchicine-binding activity of tubulin in supernatants from homogenates of chick brain and sensory tissue increased and also that increasing amounts of tubulin occurred in particulate fractions. They suggested that these changes in properties reflected differences in tubulin molecules present at different times during development. Gonzales and Gee1 (1976) observed similar changes in the properties of brain tubulin during the postnatal development of rats. Hains et al. (1978) found that the affinity of tubulin from newborn rat brain for both colchicine and vinblastine differed from that of tubulin from adult rat brain. These workers suggested that in general tubulin in young brain tissue is different from that in adult brain and in light of this offered an intriguing speculation that such differences might explain the fact that the neurotoxicity of vincristine is much less for young children than for adults. The development of brain tissue involves the formation and maintenance of large numbers of neurotubules. Work from two laboratories suggests that at least partial control over this process lies in regulatory factors, possibly the MAPs studied in vitro. Fellous et al. (1975) and Nunez et al. (1975) investigated the extent of microtubule assembly which could be attained in virro in supernatants from homogenates of rat brains of different developmental ages. They found that both the rate of polymerization and the final amount of polymerized microtubules were much lower in preparations from fetal or newborn rats than in those from older rats. The amount of tubulin present, measured by colchicine binding, was about the same and thus did not appear to be the limiting factor in the massive postnatal neurotubule proliferation. Nor was capability for elongation of microtubules the limiting factor; fetal tubulin elongated microtubules when sonicated fragments of adult brain microtubules were added. Polymerization was also stimulated by the addition of adult supernatant (at a concentration which did not support initiation of self-assembly). These workers concluded that an initiation factor(s) was missing in brains at early developmental stages. Fellous et al. (1976) found that MAPs prepared from adult brain as described by Weingarten et

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al. (1975) stimulated the rate of assembly in vitro of brain tubulin from fetal and newborn rats but did not increase the final amount polymerized, which was about half that in supernatants from adult brains. Rat brain maturation is believed to be under thyroid hormone control and in recent work Francon et al. (1977) suggested that thyroxine might be one regulatory signal involved in microtubule assembly. They found that, although brains from hypothyroid and normal rats contained about the same amount of tubulin, the rate of microtubule assembly in virro was much slower in preparations from brains of hypothyroid rats. However, the final extent of polymerization was about the same. The rate of assembly of tubulin from brains of hypothyroid rats was considerably stimulated by the addition of MAPs isolated from normal brain but did not reach the normal rate. However, tubulin made from brains of thyroxine-treated hypothyroid rats assembled at a nearly normal rate. Schmitt er al. (1977) reported similar experiments confirming that tubulin in supernatants from brains of 5-day-old rats polymerized in virro at a much slower rate and to a much lesser extent than tubulin from adult brains. However, in the presence of 4 M glycerol, both the rate and extent of assembly of tubulin from newborn rat brain were similar to those for tubulin from adult brain (which polymerized at the same rate and to the same extent in the presence or absence of glycerol). These workers were unable to correlate the presence of highmolecular-weight MAPs either with the age of the brain or with the rate of in vitro polymerization; they tentatively identified an 82,000-molecular-weight component as being present in tubulin preparations from brains of rats 10 days old or older but not in younger brains. They postulated a membrane-bound initiation factor to explain both the temporal onset and the spatial localization of microtubules in growing neurons. A series of studies related to the question of brain development concern the differentiation of mouse neuroblastoma cells in culture. Schubert et al. (1971) showed that, when these cells were cultured in serum-free medium, they rapidly and reversibly differentiated into a neuronlike morphology, extending long neurites containing numerous microtubules. Protein synthesis was required for the differentiative process. Morgan and Seeds (1975) and Schmitt (1976) showed that the synthesis of tubulin was not a controlling factor, however, since the total tubulin levels remained constant during proliferation of the microtubule network. Recently, Seeds and Maccicmi (1978) suggested that the appearance of microtubules in differentiating neuroblastoma cells might be controlled through MAPs. They examined the ability of high-speed supernatants from cell extracts to promote microtubule assembly in vitro of purified lamb brain tubulin, under conditions in which the brain tubulin did not initiate self-assembly unless brain MAPs were added back. Polymerization was also stimulated by supernatants from differentiated but not from undifferentiated neuroblastoma cells. The stimulation of assembly by differentiated cell supernatant plus brain MAPs was additive; con-

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versely the presence of undifferentiated cell supernatant did not inhibit MAPstimulated assembly. The ability of cell supernatants to stimulate assembly correlated with morphological differentiation; supernatants from cells that were serum-deprived under conditions in which differentiation did not occur, for example, did not stimulate assembly. The differentiated cell supernatants did not contain microtubule fragments nor initiate self-assembly at the concentration used (0.1-0.2 mg/ml); the factor(s) responsible for the brain tubulin assemblystimulating activity was not identified. Mizel and Bamburg (1975) showed that nerve growth factor-induced extension of neurites by chick embryo dorsal root and sympathetic ganglia took place under constant total tubulin levels. Levi et al. (1975) reported that nerve growth factor bound to mouse brain tubulin in vitro and under some conditions stimulated polymerization, but there is no evidence showing that this occurs in vivo. The brain studies involve stages very late in development. Fewer data are available on tubulin properties during early embryonic development. We observed that several changes in the properties of tubulin in supernatants of axolotl egg homogenates, including a marked increase in the lability of colchicinebinding activity, took place after the initiation of cleavage; however, the results of mixing experiments indicated that at least some of the differences in properties were not due to differences in tubulin per se (Raff, 1977). Kuriyama (1977) found no differences in polymerizability in vitro in tubulin from unfertilized and fertilized sea urchin eggs. A question closely related to development and differentiation concerns microtubule regulation during different physiological states. Perhaps the most striking such change is transformation, when both cell morphology and mode of growth alter drastically. As previously discussed, it has been suggested that cytoplasmic microtubule networks are modified during transformation. Biochemical evidence on this point is somwhat unclear. Several groups have compared mouse 3T3 cells with SV40-transformed 3T3 cells; both the polymerization of t u b u h in vitro and the amount of tubulin present were found to be the same in both cell types (Fuller et al., 1975b; Weber et al., 1977a; Wiche et al., 1977b). Fine and Taylor (1976), however, reported that, while the amount of soluble tubulin was the same in both cell lines, the transformed cells had only about two-thirds the normal amount of total tubulin because of a decrease in tubulin appearing in particulate fractions. Ostlund and Pastan (1975) reported that virally transformed rat kidney fibroblasts had only half as much tubulin, measured by colchicine binding, as the parent cells. Pipeleers et al. (1977a,b) examined total tubulin and polymerized microtubule levels in several mammalian cell types under different physiological conditions and concluded that the total tubulin level and the degree of polymerization were independently regulated and could be modulated by many factors. For example, during fasting the total tubulin level in rat and mouse liver fell only slightly,

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whereas the percentage of tubulin in polymerized microtubules was drastically decreased relative to that in liver from normally fed animals. After glucose feeding, the percentage of polymerized microtubules increased, but the total tubulin level remained constant. G. SPATIAL LOCALIZATION OF MICROTUBULES: ASSOCIATION WITH MEMBRANES A good (teleologically speaking) mechanism for cellular control of the localization andor orientation of microtubule arrays would be to anchor either the initiation sites or the tubules themselves onto some other subcellular structure. It appears that many cells do just that; microtubule-organizing centers, microtubules, and tubulin itself have all been observed in intimate association with membranes. There is both morphological and biochemical evidence on this point. First, in many (though by no means all) cases microtubules arise near membranes or approach them closely. Where the initiation site is associated with membrane, this may imply a way of determining the spatial arrangement of the microtubule array, whereas association with membranes of microtubules which originate elsewhere may imply a funtional rather than a regulatory involvement. In many lower organisms with specialized forms of mitosis, the mitotic microtubule-organizing structures are situated directly on the nuclear membrane (reviewed by Hepler and Palevitz, 1974; Kubai, 1975); several examples of this type were discussed in Section I. In some primitive organisms chromosomal attachment at mitosis is directly to the nuclear membrane; Pickett-Heaps (1969, 1975a) postulated that mitosis arose through such chromosome-membrane connections and that the kinetochore and other microtubule-organizing centers were relics or derivatives of primitive membrane specializations. This hypothesis carries with it the idea that, evolutionarily, microtubules first arose for the purpose of cell division (i.e., mitotic before axoneme microtubules, for instance). The evolutionary shift of chromosome movement from membranes to microtubules has been traced by Kubai (1975). Many other observations discussed in Section I also illustrate membrane involvement in the initiation or placement of microtubules, for example, in the location of protozoan organelles and basal bodies at sites on the cell membrane. The location of centrioles within the cytoplasm may also be membrane-related. Bornens (1977) has recently reported electron micrographs showing that preparations of rat liver nuclear envelopes contain numerous centrioles some of which appear to be physically connected to the membranes by dotlike strands of material of rather low electron density. Ultrastructural examination has revealed the close association of cytoplasmic microtubules in both animal and plant cells (Franke, 1971; Schliwa, 1977; H. J. Wilson, 1970). Such associations are particularly prevalent in neural tissue, and clear associations of microtubules with both pre- and postsynaptic membrane

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specializations have been shown (Bird, 1976; Gray, 1975; Westrum and Gray, 1976, 1977). Observing that the electron-dense aggregates from which tubules arose in the cell center of fish melanophores are in close physical association with elements of the internal cell membrane system, Schliwa (1978) postulated that this represented positioning of the microtubule-organizing structures at sites from which calcium levels could be controlled. Sherline et al. (1977) found that microtubules assembled in vitro bound to isolated pituitary secretory granule membranes. In an ultrastructural study of diploid human fibroblasts in culture using highvoltage electron microscopy Wolosewick and Porter (1976) observed a continuous three-dimensional internal cellular lattice in which microtubules, microfilaments, internal cell membranes, and other cell components were interconnected by 3- to 6-nm filaments. Byers and Porter (1977) observed a similar lattice in cultured fish erythropores; the filaments forniing the lattice were absent during pigment aggregation and reformed when the pigment granules dispersed. These investigators postulated that MAPS might be a component of the filaments. Workers at several laboratories have concluded that microtubules are also closely associated with the plasma membrane. Much of the evidence is derived from studies of the binding of Con A and other lectins to cell surfaces (see reviews by Edelman et a l . , 1976; Nicolson, 1976a,b). In many cases Con A binding is accompanied by clustering or capping of the binding sites; concomitant proliferation andor rearrangement of microtubule arrays immediately adjacent to the cell membrane has been observed (Albertini and Anderson, 1977; Albertini and Clark, 1975; Clark and Albertini, 1976; Hoffstein er al., 1976). In other cell types rearrangements of cell surface components occur only after treatment with agents that disrupt microtubules, including colchicine (Berlin et a l . , 1974; Edelman er al., 1973; Oliver, 1975; Oliver et al., 1975; Yahara and Edelman, 1973, 1975) and calcium ionophores in calcium-containing medium (Poste and Nicolson, 1976). Finally, there is considerable evidence that tubulin is a component of membranes from a variety of sources (reviewed by Stephens and Edds, 1976). Tubulin has been shown to be present in synaptic and postsynaptic membrane fractions (Blitz and Fine, 1974; Walters and Matus, 1975); Estridge (1977) found that some of the tubulin in neural membranes may be exposed on the cell surface. Bhattacharyya and Wolff (1975) showed that tubulin with properties similar but not identical to those of soluble tubulin was a bona fide component of membrane fractions from mammalian brain and thyroid. In subsequent work, Bhattacharyya and Wolff (1976b) found that tubulin solubilized from brain membranes could be copolymerized in vitro with soluble tubulin; these investigators speculated that membrane-bound tubulin functioned in nucleating microtubules but had no direct evidence for this. Stephens (1977a) recently demonstrated that tubulin is the main protein component in the membrane fraction from scallop gill cilia but is

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not present in that from sperm tail flagella; furthermore, the ciliary membrane tubulin was glycosylated to a small extent, whereas ciliary axoneme tubulin was not. As Stephens points out, it is not clear what functional role tubulin plays in the ciliary membrane, nor is it clear why the ciliary and flagellar membranes differ. Related to the question of the association of tubulin with membranes is the existence of other particulate forms of tubulin. Weisenberg (1972a, 1973) examined the organizational state of tubulin before and during meiosis in eggs of the surf clam, S. sofidissima. He made homogenates of eggs by gentle methods in a buffer known to stabilize microtubules, separated them into soluble and particulate fractions, and then determined the distribution of tubulin by colchicine binding. As expected, metaphase eggs contained a significant amount of particulate tubulin in low-speed pellets, presumably representing the mitotic apparatus. The more striking result was that interphase eggs contained the same amount of particulate tubulin (about 10-15% of the total). Unlike that from metaphase, the interphase tubulin-containing particle was very fragile. In the low-speed pellet from gently handled homogenates, Weisenberg isolated granular spheres 10-20 p n in diameter with a membranous structure on one side. The structures contained no microtubules, but microtubules were frequently observed radiating from them. Rougher handling caused this structure to disperse, and the particulate tubulin then appeared in higher-speed pellets. During prophase the amount of tubulin in the particulate fractions declined to a minimum; Weisenberg therefore postulated that the interphase structure was broken down and the component tubulin utilized in assembly of the mitotic apparatus. Weisenberg and Rosenfeld (1975a) speculated that possible functions of the interphase structure could relate to determination of the correct spatial orientation of the mitotic apparatus or to sequestering the specific pool of tubulin from which it would be elaborated. The interphase structure is reminiscent of the basal body and mitotic precursor structures observed in the electron microscope studies discussed in Section I. In fact, Staprans and Dirksen (1974) performed similar experiments to determine the organizational state of tubulin during ciliagenesis in mouse oviduct, which Dirksen (1971) had previously followed in ultrastructure studies (see above). The appearance of increasing amounts of tubulin in the particulate fractions of homogenized newborn mouse oviducts correlated in timing with the presence of the centriole precursors seen in ultrastructure studies. Dirksen and Staprans (1975) found that ciliagenesis was accompanied by a burst of de novo tubulin synthesis and observed by electron microscope autoradiography that at this stage of development more label was present in centriole precursors and basal bodies than in other organelles. However, in a detailed study of labeling patterns during this period of rapid tubulin synthesis Dirksen and Staprans (1977) concluded that the labeling kinetics were too complex to fit a simple model of sequential transfer of tubulin to centriole precursors and then on to the axoneme.

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H. MICROTUBULE REGULATION: GENETIC STUDIES Genetic analysis is a potentially powerful tool for the study of microtubule regulation which has been relatively little exploited. The maternal effect mutation nc in the Mexican axolotl discussed above is an example of the kind of mutation which may help to delineate the points of control over microtubule assembly. An ideal system for examining the developmental control over tubulin and microtubule function is spermiogenesis in Drosophila. Many mutations affecting spermiogenesis have been described, among which are several resulting in altered microtubule assembly or function including disruption of the normal organization of the axoneme (reviewed by Kiefer, 1973; Romrell, 1975). Wilkinson er al. (1974) described a male sterile mutant of D . rnelanogasfer in which the sheath of cytoplasmic microtubules normally attached to the nuclear envelope of developing sperm cells failed to be assembled, with concomitant failure of nuclear shape change; nevertheless, axonemal microtubules assembled. The mutant could be phenocopied by treating wild-type flies with colcemid. Studies of this mutant combined with observations of the effect on spermiogenesis of vinblastine, which prevents assembly of the axoneme but to some extent allows elaboration of the cross-linked nuclear sheath microtubules (Wilkinson et al., 1975), led this group to conclude that the two classes of microtubules necessary for sperm differentiation are separately regulated. Lifschytz and Harevan (1977) and Lifschytz and Meyer (1977) described a series of male sterile sperm dysfunction mutants of D . rnelanogaster including several with defects in the morphology of the meiotic spindle. One of these produced a monastral but dipolar spindle at the second meiotic division, demonstrating independence of the spindle pole from the astral array; two centrioles were present at the single astral pole. These workers concluded that the defects in spindle morphologies observed more likely resulted from defects in the control of microtubule assembly rather than lesions in the structural tubulin gene. The abnormalities observed were confined to meiosis, indicating independent control over other microtubule assembly events. Finally, Rungger-Brandle (1977a,b) reported a mutant of Drosophila hydei in which spermatid differentiation was blocked at an early elongation step. As shown in Fig. 12, electron micrographs of mutant testes cultured in vivo show several microtubule-related abnormalities in morphology which, taken together with the similar changes caused by antimitotic drugs in developing spermatids from normal flies, suggest that the lesion is in microtubule assembly. For example, nuclear and cell elongation failed to take place, apparently because of failure to assemble normal cytoplasmic microtubule arrays; cytoplasmic microtubules were fewer in number than normal, and the majority observed had grossly abnormal cross sections. In addition, the central pair was missing from the flagella axoneme. Interestingly, some of the abnormal microtubule cross sections ob-

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FIG. 12 Abnonnal cytoplasmic microtubules in spermatids of the mutant f(3)pl (lethal polyploid) of L ) . hytlri. Cross sections o f sperinatid microtuhule arrays showing the abundance of various ahnornial forms. The arrows in (d) denote tuhules connected by long linkers. (a) X61.600; (h) ~ 6 1 , 6 0 0(;c ) ~ 8 4 , 7 0 0 ;( d ) ~ 8 3 , 1 6 0 .Reprinted from Rungger-Brindle (1977b). with permission.

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FIG.13 Cytoplasmic microtubules in variant lines of CHO cells in culture after treatment with dibutyryl CAMP. (a) Epithelium-like CHO cell variant showing randomly oriented cytoplasmic microtubules. These cells (with or without dibutyryl cAMP treatment) are similar to untreated normal . Fibroblastlike CHO cells. x 12.325. Inset: Phase-contrast micrograph of a whole cell. ~ 6 8 0 (b) CHO cell variant, showing a long, parallel array of microtubules in the elongated cell processes. Normal CHO cells respond similarly to dibutyryl cAMP treatment, but the fibroblastlike variant cells . Phase-contrast micrograph of a whole cell. ~ 6 8 0 . become even more elongated. ~ 2 8 , 4 7 5 Inset: Reprinted from Borman er a / . (1975), with permission.

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served by Rungger-Brandle in the mutant spermatids resembled those seen by Burton and Himes (1978) after the assembly of brain tubulin in v i m at slightly suboptimal pH levels. Chlaniydomonas is another organism in which mutations involving defects in microtubule assembly have been observed, particularly in formation of the flagella axoneme. Nonmotile mutants with elongated or stumpy flagella or axonemes lacking one or both central pair microtubules or with other defects in microtubule arrangement have all been reported (McVittie, 1972; Pipemo et al., 1977; Warr, 1974; Witman e t a / . , 1978). Goodenough and St. Clair (1975) have described a particularly interesting Chlamydomonas mutant in the basal bodies of which a ring of singlet microtubules is formed instead of the typical triplet tubules. This strain lacks flagella, but occasionally the flagellar transition region, a short axoneme, and the tunnel in the cell wall through which flagella usually emerge do form; the axoneme formed, however, is defective in the same sense as the basal body; that is, it contains only singlet tubules. Cytoplasmic microtubules in this mutant are normal. Control over the number of flagella was illustrated in studies by Warr (1968) of a mutant with a defect in cell division such that multinucleate cells formed; the number of pairs of flagella was the same as the number of nuclei. In addition to mutations there are numerous examples of species in which the axonemal arrangements of the sperm flagella deviate from the typical 9 + 2

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pattern but in which the morphology of the microtubules is normal (for example, see Baccetti et al., 1973, 1974; Phillips, 1974; Schrevel and Besse, 1975; Thomas, 1975; Tulloch and Hershenov, 1967; Van Deurs, 1974). There are also examples in which the microtubules themselves are of variant morphology. Turner (1972) described the ultrastructure of the testis of the water strider, Gerris remigis; the cytoplasm of the sheath surrounding the spermatid bundles is filled with modified microtubules resembling a normal microtubule of typical circular cross section and diameter with one to three curved or hooklike projections. The projections gave some of the modified microtubules the appearance of opened doublet or triplet tubules. Some of the cross sections resembled those discussed above in the D. hydei mutant and in brain tubulin assembled at suboptimal pH. Longitudinal sections showed that the projections were continuous with the tubule wall. The projections disappeared alfter colchicine treatment, leaving microtubules of normal cross section. Baccetti and Dallai (1978) have recently described the feebly motile multiflagellated sperm of the termite Mastotermes durwiniensis in which the centrioles have doublet instead of triplet tubules; also, the flagella axoneme is of an atypical 9 + 0 pattern, and the doublets have only single arms. Two studies have demonstrated abnormal microtubule regulation in mammalian cells in culture. As shown in Fig. 13, Borman et al. (1975) reported a variant line of CHO cells which maintained an epithelium-like shape even after cAMP levels were increased; microtubules appeared in response to cAMP but were randomly distributed and did not give rise to the marked shape changes seen in normal cells; these workers also reported a fibroblastlike variant which normally maintained extensive microtubule arrays. Oliver (1975) and Oliver et a / . (1975) found that some of the deficiencies observed in cells from mutant beige or Chediak-Higashi mice were consistent with abnormalities of microtubule regulation; whereas leukocytes from normal mice required colchicine treatment in order for Con A capping to occur, leukocytes from the mutant mice capped spontaneously. Dooker and Bennett (1974) reported that, in sterile male mice homozygous for the tit" allele marked morphological abnormalities occurred during spermiogenesis, apparently caused by premature disassembly or disorganization of cytoplasmic microtubules. These investigators suggested that disruption of the microtubule arrays was in turn related to abnormalities in the membranes with which these arrays were normally associated. Finally, there have been several reports of various cell types resistant to antimitotic drugs, particularly colchicine. However, there has to date been no demonstration of the existence of the putative modified tubulins. For example, Ling and Thompson (1974) demonstrated that a line of CHO cells resistant to colchicine and vinblastine were less permeable to these drugs than normal CHO cells; mechanisms unrelated to tubulin such as reduced permeability to the drugs,

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increased drug efflux, and the like, have not been unequivocally eliminated in other such studies.

111. The Suppositions: Conclusions about the Control of Microtubule Assembly in Vivo At this point a summary of what is known about the control of microtubule assembly in vivo very nearly constitutes a restatement of the entire problem, but a restatement of the problem may be helpful in assimilating the mass of extraordinarily diverse observations on the topic. Microtubule assembly depends on the formation or activation of microtubule-organizing centers or structures, the primary cellular signals for which are largely unknown. A variety of microtubuleorganizing centers has been observed; the most ubiquitous consists of amorphous electron-dense material, sometimes exhibiting a fibrous or granular substructure, of uncertain biochemical composition. There are some indications that one component of these centers may be tubulin itself, but it is not clear if this material has a general or typical composition. In different cell types or at different times this material may give rise either directly to microtubules or to more elaborate microtubule-containing organelles, particularly centrioles. Centrioles (after some morphological modifications and in some cases after physical relocation to the cell surface) may function as basal bodies and give rise directly to the microtubules of the flagella or cilia axoneme, thus themselves constituting a microtubule-organizing structure. In other cases centrioles may subsequently be associated with the electron-dense material from which either cytoplasmic and mitotic microtubules or other centrioles may arise. Numerous specialized microtubuleorganizing centers of more complex structure exist; most common are spherical or laminated structures composed of electron-dense material resembling that observed in a more amorphous form. The nature of the information which specifies the temporal occurrence and spatial orientation of microtubules is beginning to be defined. Some of the answers are apparent if not explicit; calcium ion fluctuations, membrane involvement, and heterogeneity of tubulin subunits are clearly among the most important regulatory mechanisms. The former two have been appreciated for quite a long time, although the biochemical details are only just now being worked out, while the last point has only relatively recently been understood. One of the most studied current problems concerns the nature and function of nontubulin MAPs. These proteins clearly exist and almost certainly are real and not fortuitously coisolated components of microtubules. The confusing and sometimes contradictory nature of the data concerning their number, identity, and function in vivo may well stem from genuine cellular diversity. There are likely several different kinds of MAPs with perhaps subtly differing and/or interrelated

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functions; MAPS may participate both in the structural scaffolding of the cell a5 well as in the regulation of microtubule assembly or function. A third point which cannot be overemphasized is the difficulty of correlating in vivo observations with in vitro data. The biology of microtubules and the chemistry of tubulin have frequently been considered separate problems, and this may explain apparent anomalies in the two sets of data. A cell may function like a bureaucracy (what is not explicitly permitted is forbidden), while in the more “democratic” in vitro environment of the test tube many tubulin selfassociations are permitted including those which do not occur in the cell. While one may speak in general of the regulation of microtubule assembly, it must be recognized that control must actually occur, perhaps separately, at various levels: that is, control at the cellular level to determine when and where microtubules will be assembled, and control at the molecular level governing the actual process of assembly of tubulin subunits. The latter is the level at which most in vitro data apply. Some regulatory signals may operate exclusively at one level or another. Thus hormonal stimulation of microtubule assembly (such as proliferation of basal bodies in mammalian oviduct in response to estrogen o r neurotubule proliferation in response to thyroxine) clearly are cellular signals removed at least one step from the actual fitting together of microtubule subunits, while guanine nucleotide availability most likely operates at the subunit level. Other signals-such as calcium ions or MAPS-may operate in different ways at both levels. Similarly, there are two aspects of the control over the timing of microtubule appearance: the actual or developmental time at which microtubules must be assembled (or disassembled), and the rate at which tubulin subunits assemble, also an important component of microtubule regulation. Genetic studies may be one of the most helpful approaches in differentiating between different levels of control. It may be a mistake to look for a single mechanism in which all the data can be made to fit into a general statement of how microtubule regulation is achieved. It is at least my own distinct impression that a lot of the data will be left hanging out of the suitcase no matter how hard we sit on it. Just as the obvious long-standing evolutionary constancy of microtubule morphology and tubulin structure for a while obscured the fact of microheterogeneity of tubulin subunits, the large amount of data from studies of assembly of neurotubulin in virro-while of enormous value-may have tended somewhat to obscure the diversity of control mechanisms in other tissues. For example, the mechanisms by which individual microtubules are organized into patterned arrays have clearly been shown to be diverse even in organisms as closely related as various heliozoan protozoa, in some of which an axopodium pattern is established by specific linkage and in others of which it is established by template nucleation. Even the assembly of morphologically indistinguishable organelles-basal bodies and centrioles-has been shown to take place through different morphological steps in different

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organisms (thus the modes of formation in the most extensively studied groups-vertebrates and protists-are quite different and both are radically different from the mode of formation in motile plant sperm). At the conclusion of biochemical reviews it is customary to comment on the necessity for more work on the structure and function of the protein in question; in view of the staggering amount of data already available on tubulin and microtubules perhaps in this case it is more suitable to remark on the need for new analytical insight. Finally, one must remember that control of microtubule assembly is not an isolated problem, but is just one aspect-a central and still puzzling o n e 4 f the general problem of modem biology, initiated in the studies of eighteenth- and nineteenth-century embryologists, of how the morphology of cells and organisms is controlled.

ACKNOWLEDGMENTS I acknowledge with thanks the many people who very kindly sent me the photomicrographs which so elegantly illustrate the topic considered here, R. A. Raff for critical reading of the manuscript, the staff of the Indiana University Department of Biology Office for patient and rapid typing and retyping, and the National Science Foundation for support through Grant PCM 73 02130 A01.

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IN'IhRNAIIONAL REVIEW OF C'Y I'OI.OGY. VOI. S Y

Membrane-Coating Granules A. F. HAYWARD Royiil Den/til Hospitul School oJ Dcnttrl Surgery, London, England

I . Histology of Stratified Squamous Epithelia . . . . . . . . . 11. Membrane-Coating Granules . . . . . . . A. Identification, Distribution, and Morphology

B . Origin . . . . . . . . . . . C . Fate . . . . . . . . . . . D. Composition . . . . . . . E. Functions . . . . . . . . I l l . Summary and Future Prospects . . References . . . . . . . . . .

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I. Histology of Stratified Squamous Epithelia The histology of stratified squamous epithelia has been reviewed many times, and reference may be made to articles such as those by Squier (1971) and Squier et al. (1975) based on the oral mucosa, and those by Breathnach (1971) and by various distinguished authors in Zelickson (1967) referring to the skin. Stratified squamous epithelia, keratinized or nonkeratinized, may be of ectodermal or endodermal origin and in mammals is typically represented by the epidermis of the skin and by the epithelia lining the oral cavity, vagina, urethra, anal canal, esophagus, and forestomach. For descriptive purposes, stratified epithelium can be divided into a series of cellular layers, four or five in number in keratinized epithelium (Fig. 1) and two or three in nonkeratinized epithelium. These layers are convenient for description and also provide a series of stages of development through which differentiating postmitotic cells eventually pass. The events of differentiation are therefore more-or-less easily followed in sequence in one section. The basal layer of the epithelium consists of a coherent germinative zone in which, for a given species and body region, the cells possess fairly fixed kinetic characteristics such as generation times and mitotic indexes. Half of the daughter cells produced by the mitoses of the basal layer migrate into the parabasal zone and then into the stratum spinosum, so called because of the numerous prominent intercellular bridges and desmosomes visible with light microscopy. In general, the cells of the stratum spinosum are amitotic, but in some tissues mitoses persist into the deeper layers. 97 Copyright @ 1979 by Academic Press. Inc All nghb of reproduction in any form rexrved ISBN 0-12-364359-7

FIG. I . Histological section of typical keratinized stratified epithelium (rat hard palate) showing the conventional layers. A , Level at which MCGs appear in the cytoplasm of epithelial cells; B , level at which discharge into intercellular spaces occurs. ~ $ 5 0 .

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Ultrastructural changes occumng in the stratum spinosum include an increase in the number of desmosomes and in bundles of tonofilaments which represent the accumulation of fibrous keratin in the cytoplasm. There is a decrease in the nucleocytoplasmic ratio and an increase in cell volume (Alvarez and Meyer, 1971). Cells of the more superficial layers of the stratum spinosum are flattened and, in many keratinized epithelia though not in all, large basophilic granules known as keratohyalin granules develop. Much has been written about keratohyalin granules. The cells in which they are found constitute the stratum granulosum. At the upper limit of the stratum granulosum, keratinization or cornification occurs. The formed organelles of the cell become lost in an acidophilic mass of keratin. The nuclei degenerate or disappear, and ultimately desquamation of the cell occurs. Perhaps the most important single morphological event in the sequence of cell differentiation is membrane thickening. This is believed to be due to apposition on the cytoplasmic surface of the cell membrane of a layer of material hitherto unrecognizable within the cytoplasm (Farbman, 1966). The earlier confusion between this process and the fate or function of membrane-coating granules (MCGs) is dealt with in Section II,E,I. Suffice it to say here that membrane thickening may well be the event which terminates any interaction between the cell’s contents and its surroundings, including the exocytosis of MCGs. Thereafter it remains only for the cell to be shed as a keratinized squama. The differentiation of cells in nonkeratinized epithelia differs from that in the keratinized type in that keratohyalin granules are seldom formed and the nucleus and cytoplasmic components do not lose their integrity in the surface layers. The plasma membrane of the cells undergoes an apparently similar thickening from the cytoplasmic surface (Hashimoto et d., 1966b; Hackemann et a / . , 1968). In very general terms nonkeratinized epithelia are thicker than keratinized epithelia and have a higher mitotic index, but these features are of course subject to great variation. For descriptive convenience nonkeratinized epithelia are divided into a stratum basale and deep and superficial stratum spinosum.

11. Membrane-Coating Granules

In order to make the following descriptions intelligible a summary of the place of MCGs in this pattern of cell differentiation is given as an introduction but without supporting references. MCGs appear first in the cells of the stratum spinosum of keratinized epithelia and at about the same distance from the basal cells in nonkeratinized epithelia. At about 0.2 jm in diameter they are below the resolution of the light microscope. A large number of them appear along the upper or superficial border of each cell

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FIG. 2 . Distribution of MCGs in cells of the deeper layers of the stratum granulosum (rat soft palate). KH, Keratohyalin granules; M , mitochondria. X8925.

(Fig. 2), and as differentiation proceeds they are discharged into the intercellular spaces by exocytosis. Thus they decrease in number in the cytoplasm. Practically all the intracellular MCGs disappear prior to membrane thickening. A.

IDENTIFICATION, DISTRIBUTION, AND

MORPHOLOGY

1. Crireria for Identification of Membrane-Coating Granules

The criteria for identification of granules as MCGs are ( 1 ) size (Section II,A,4,a), (2) distribution (Section II,A,3,b), and (3) internal structure (Section II,A,4,b). The internal structure consists of alternating thick and thin electron-dense lamellations. In sections such a structure is seen only in a proportion of granules and only in the granules of keratinized epithelia. However, there are many nonlamellated bodies in these epithelia which have the same distribution and size and which are generally believed to be those cut in such a plane that lamellations are not visible. Bodies with the same distribution but without internal lamellations occur in nonkeratinized epithelia (Grubb et al., 1968).They share some other characteristics of MCGs such as a glycoprotein reaction (Hayward and Hackemann, 1973)

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and an acid phosphatase reaction (Silverman and Keams, 1970) and are now believed to be MCGs (Squier, 1977). 2 . Nomenclature None of the names so far given’to MCGs is entirely satisfactory, nor has any achieved unanimous acceptance: Initially purely descriptive epithets were applied. Into this category fall the “small spherically shaped granules” of Selby (1957) and “corpuscula” of Frei and Sheldon (1961). “Small dense granules” (Schroeder and Theilade, 19661, “small lamellated bodies’’ (Frithioff and Wersail, 1965; Squier, 1968), and “lamellated dense bodies’’ (Farbman, 1964) also represent no more than a description. Attempts to name these bodies based on functional attributes have had little more success. Thus “transitory dense bodies’’ (Osmanski and Meyer, 1967) merely implies a short life, and “cementsome” (Hashimoto, 1971b) implies a function of intercellular adhesion which has not received support from other observations. The early description of the bodies by Odland (1960) has been recognized eponymously by some authors (Wolff and Holubar, 1967). The two most widely used terms are “membrane-coating granule” (Matoltsy and Parakkal, 1965; Matoltsy, 1966) and “keratinosome” (Wilgram, 1965). The name “membrane-coating granule” was based on the view, no longer accepted, that the disappearance of the granules from the cytoplasm was followed by and functionally related to the thickening of the cell plasma membrane. The process of membrane thickening is better understood now and is clearly due to the accretion of material, as yet unidentified, on the cytoplasmic surface of the membrane (Farbman, 1966). The contents of the granules are discharged into the intercellular spaces, so that the two phenomena appear to be unconnected, depriving the name of its original significance. However, the concept of coating the outer surface of the cell membrane has been revived in more recent views of the granules (Martinez and Peters, 1971), and the name still appeals to many authors. It is used in this article. The name “keratinosome” has also been used, particularly by Wilgram and his colleagues (Wilgram and Weinstock, 1966; Weinstock and Wilgram, 1970; Wilgram et al., 1970). The only objection seems to be that the bodies are not restricted to keratinized epithelium, and it is not conventional to refer to the cells of nonkeratinized epithelium as keratinocytes. 3 . The Distribution of Membrane-Coating Granules a. In Epithelial Tissues. MCGs are found in almost all stratified squamous epithelia. Their presence does not appear to depend on the existence or degree of keratinization, although most studies have been carried out on keratinized epithelia. MCGs appear to be absent from the special nonkeratinized junctional epithelium of the gingiva (Section II,E,4), but granules with several features

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similar to those of MCGs are found in the adjacent and contiguous sulcular epithelium (Innes, 1973). The number of granules in cells appears Lo depend on the body region. It is said to differ from site to site in the skin (Wilgram and Weinstock, 1966; Wilgram, 1965) and between the skin and the oral mucosa (Wilgram and Caulfield, 1966) in such a way as to suggest that epithelia with more rapid production of cells display more MCGs. This suggestion has been supported by a study of skin diseases, especially the rare congenital keratoses (Wilgram and Weinstock, 1966). The number of MCGs was observed to be greater where the mitotic index was known to be high and to be less where desquamation was reduced in the presence of a low or normal mitotic index. The objection to these observations is the subjective nature of the assessment of numbers of MCGs in electron micrographs. Only the more objective stereological methods such as those used by Schroeder’s group (Schroeder and MunzelPedrazzoli, 1970; Bernimoullin and Schroeder, 1977) can eliminate the doubts left by such an assessment. Granules similar to MCGs are seen in reptiles, for example, in the epidermis of turtles where they have been referred to as lamellar bodies (Matoltsy and Bednarz, 1975). In the epidermis of newborn chicks a larger body has been described within which several discrete lamellated structures are present, each of which resembles one MCG. They have been called multigranular bodies (Matoltsy , 1969). b. In Cells. Within the epithelial cells it is now generally agreed that MCGs are found near the upper, distal, or superficial border of the cells, and a few occur near the opposite border (Frithioff and Wersall, 1965; Farbman, 1964) (Fig. 2). They increase progressively in number toward the upper part of the stratum spinosum and stratum granulosum but decrease thereafter and virtually disappear from the cell before membrane thickening occurs. 4. Morphology

a. Size. There is universal agreement on the size of MCGs. The smallest reported size is 0.1 pm in diameter (Frithioff and Wersall, 1965; Parakkal, 1967), and the largest is 0.3 p n in diameter (Farbman, 1964). There is no evidence that the dimensions vary from site to site, from tissue to tissue, or with the pathological state. b. Volume Density. The fraction of the total volume of the cell occupied by MCGs has been measured by stereological methods. The most convincing estimates have come from publications arising from a program of quantitative studies on the structure of oral epithelium and skin (Schroeder and Munzel-Pedrazmli, 1970; Meyer and Schroeder, 1975; Landay and Schroeder, 1977; Bernimoullin and Schroeder, 1977). The methodology has recently been computerized (Hammer and Schroeder, 1977).

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Values for volume density of MCGs in keratinized epithelium have been given by Klein-Szanto et al. (1976), Klein-Szanto (1977), and Meyer and Schroeder (1975). Typical figures obtained for the volume density ( V , ) of MCGs in epidermal cells of normal human skin from the iliac crest are (Klein-Szanto et al., 1976): stratum spinosum, middle, 1.4 k 0.13 mm3/cm3 or 0.14%; stratum spinosum, upper, 7.96 2 1.57 mm3/cm" or0.79%; stratum granulosum, 11.44 k 0.75 mm3/cm3of 1.14%. In nonkeratinized epithelium of the oral mucosa where no stratum granulosum is present much smaller values have been obtained. Those of Landay and Schroeder (1977) are given here, but comparable figures were obtained by Bernimoullin and Schroeder (1977): stratum spinosum, deep, 1.5 2 0.7 mm3/cm3or 0.15%; stratum spinosum, upper, 2.2 -+ 1.1 mm3/cm3 or 0.22%; stratum spinosum, surface, 0.7 2 0.7 mm3/cm3or 0.07%. These figures confirm the generally observed progressive rise in the number of MCGs as cells differentiate, and the latter set shows the fall in MCGs at the surface of nonkeratinized epithelium. So far no numerical evidence has been produced for a fall in the number of MCGs in the upper layers of the stratum granulosum in keratinized epithelium accompanying exocytosis. Clearly any future claims of an increase or reduction in the relative volume of MCGs under any condition will need to be supported by comparable stereologically derived data. The figure for percentage of the cell volume occupied by MCGs of 2-15% obtained by Elias and Friend (1975) in neonatal mouse epidermis appears to be too high. A minor difference could be explained in terms of tissue differences, but this figure appears unrealistic. It is perfectly possible of course for MCGs to occupy 15% of limited volumes of cytoplasm because of their nonrandom distribution. An alternative stereological method, which may be preferable in studying a structure of such small relative volume and uniform size, is to count the number of profiles of MCGs in a given area of section. This method has been used to show a reduction in the number of MCGs in experimental comedo formation (Woo-Sam, 1978). Substantial errors can be encountered because of the similarity of the dimensions of MCGs and the section thickness. c. Internal Structure. MCGs have a single outer limiting membrane 10 nm in thickness with a triple-layered structure (Fig. 3). In the MCGs of keratinized epithelia, there is a rather complex internal structure of parallel lamellations (Farbman, 1964; Frithioff and Wersall, 1965) which consists of alternating electron-dense and electron-translucent bands (Fig. 4). The principal electrondense lamellae 3 nm in width are separated by an electron-translucent band of 5 . 5 nm. This is further subdivided into two equal lamellae by a thin (intermediate) electron-dense band approximately 1 nm thick. The dimensions are shown in Fig. 5 (Hayward, 1978). As Frithioff and Wersall (1965) have commented, this structure bears a strong resemblance to artificially prepared phospholipids. An

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FIG.3. Groupof MCGs, showing the lamellated internal structure and trilaminar outer membrane . Hayward (1973). in granules cut in a suitable plane (rat hard palate). ~ 6 8 , 0 0 0 From

FIG.4. MCGs, showing alternating broad and n a m w electron-dense bands and the presence of two different sets of lamellae. X380.000. From Hayhard (1973).

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MEMBRANE-COATING GRANULES

extracellular spaces

granules major dense band

A

3nm

7

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

1-

translucent major 5.5nm band

intermediate dense band

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

A

3.3nm

’I

I

7.6nm

............ ............. ............ ..rm

............ ............. ............... .......... ............ ............. ............ FIG. 5 . Diagrammatic representation of the pattern and mean spacing of lamellations in MCGs and intercellular spaces in the epithelium of a hamster cheek pouch (not to scale). From Hayward (1978).

individual granule may have more than one set of lamellations arranged at angles to each other (Frithioff and Wersall, 1965, Hayward el al., 1973) (Fig. 4). Successive thick, dense lamellations may appear to be continuous at their outer edges so that they “enclose” a narrow lamella as if folded back on themselves (Farbman, 1964). Evidence of another structure superimposed on the lamellations has been observed but not confirmed. Farbman (1964) saw evidence of tubules penetrating the lamellations, and Hayward and Hackemann (1973) reported peripheral arcades just beneath the outer membrane as viewed in oblique sections. There is a remote possibility, which no investigator has seriously considered, that the lamellations represent an artifact of fixation, that is, that no lamallae are present during life at body temperature. In nonkeratinized epithelia MCGs do not usually possess a lamellated internal structure (Grubb et al., 1968; Silverman, 1967; Silverman and Kearns, 1970; Hayward and Hackemann, 1973). The granules are enclosed in a trilaminar

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FIG.6. MCGs of nonkeratinized epithelium (human buccal mucosa) showing the trilaminar touter membrane and central electron-dense core from which lamellae are absent. ~ 7 6 , 5 0 0 .

membrane, but the contents are finely granular and aggregated centrally so iis to leave a clear zone beneath the limiting membrane (Fig. 6). Hashimoto ei al. (1966b) observed lamellated MCGs in the otherwise nonkeratinized human posterior buccal mucosa. Nonlamellated MCGs have been observed in the hyperkeratinized epidermis in Kyrle-Flegel disease (Squire et af., 1978). Under pathological conditions, for example, of the oral mucosa, where normally nonkeratinized epithelium is replaced by the keratinized form in leukoplakia, the MCGs, not unexpectedly, take on the lamellated keratinized form (Hackemann and Hayward, 1972). Furthermore, the relative volume of the cells occupied by MCGs, which is low in nonkeratinized epithelium, rises to more than 1% of the total cell volume in the stratum spinosum in leukoplakia (KleinSzanto et af., 1976). This figure is more typical of human epidermis.

B. ORIGIN Early studies, lacking the detailed information now available, led investigators to interpret MCGs as degenerate mitochondria (Selby, 1957; Odland, 1960). Others believed them to have originated from the cell membrane by the pracess of pinocytosis (Rupec and Braun-Falco, 1965; Olson et al., 1969). Among the first

MEMBRANE-COATING GRANULES

I07

descriptions was one in which MCGs were reported to be viruslike particles (Zech et al., 1961). Several general statements have been made about the relationship of MCGs to the Golgi apparatus (Weinstock and Wilgram, 1970; Hashimoto et al., 1966a; Suzuki and Kurosumi, 1972). The only evidence cited that this is other than a fortuitous relationship is the observation of rudimentary lamellated material in Golgi cisternae (Wolff-Schreiner, 1977). It is likely from general principles of protein synthesis and secretion that substances found in MCGs such as glycoproteins (Section II,D,l) and lysosomal enzymes (Section II,D,3) probably have passed through the Golgi apparatus. Takaki (1 974) challenges this assumption and, on the basis of enzyme histochemical observations, believes that MCGs originate directly from the rough endoplasmic reticulum (ER). Observations on a pathological condition of the human oral mucosa known as white spongy nevus (A. F. Hayward, unpublished) has produced evidence of granules intermediate in structure between those typically found in keratinized epithelium and those of nonkeratinized epithelium. There appeared to be lamellated and nonlamellated granules present in the same cell, but the natural incidence of granules without lamellae in randomly cut sections renders this a difficult feature to confirm. Granules were observed with a central dense core surrounded by a translucent zone beneath the limiting membrane with lamellations within the central dense core. This appeared to be intermediate in form between granules from keratinized and nonkeratinized epithelia. Some MCGs contain endocytosed material (Section II.D,3), so they are secondary lysosomes and may not be produced directly from the Golgi apparatus but may form by fusion of a cytoplasmic vesicle produced as a sequel of endocytosis with a primary lysosome. As in many other tissues, primary lysosomes remain unidentified in epidermis, but by analogy with tissues where they have been recognized they are probably produced in the Golgi-Endoplasmic ReticulumLysosome (GERL) system of the smooth ER (Novikoff el a l ., 1971; Novikoff, 1976). Bonneville et af., (1968) put forward a hypothetical scheme associating the formation of MCGs with large coated vesicles found near the Golgi apparatus. These workers were unable to see any direct spatial relationship between MCGs and the enlarged Golgi apparatus present in epidermal cells in psoriasis. Data are available which indicate a possible time scale for the elaboration of MCGs. Colloidal thorium dioxide, injected subepithelially into oral mucosa, was found in MCGs 3 hours after injection (Hayward, 1976), but this was the shortest period at which specimens were taken and was not necessarily the minimum period required for synthesis of MCGs. When radioactively labeled precursors of glycoprotein synthesis were administered to mice, the label was incorporated into the cytoplasm of the cells of the oral epithelium and then localized at the cell surface 4 hours later (Susi, 1973). MCGs were not mentioned by Susi but are one mechanism by which such a process could have occurred. The evidence from

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these two studies is that the elaboration and release of MCGs is a rapid process taking 3-4 hours. The origin of the MCGs may also be studied during the differentiation of epithelia in embryological development or during regeneration in wound healing. The study of developing epithelia has not led to any conclusions about the origin of MCGs. Papers on the subject by Bonneville (1968) and DuBml (1972) failed to mention MCGs at all. Krawczyk and Wilgram (1975) looked specifically at MCGs in healing wounds of the epidermis and observed that they were present before the stratum corneum formed. Baratz and Farbman (1975) made the contrary observation. In the development of the soft keratin-bearing areas of the rat tongue the stratum comeum was present before the MCGs appeared in the cdls. These investigators concluded that MCGs were not essential for keratinization to proceed. Stereological measurements merely show a progressive increase of volume density of MCGs with time in the epithelium of a healed wound (Andersen, 1978). C. FATE MCGs discharge their contents into the intercellular spaces by fusing their outer membrane with the cell plasma membrane (Fig. 7) (see, for example,

FIG.7. Exocytosis of the contents of a MCG of keratinized epithelium into the intercellular space between the most superficial cells of the stratum granulosum (SG) and stratum corneum (SC). Human From Hayward and Hackemann (1973). gingival epithelium. ~76,500.

MEMBRANE-COATING GRANULES

109

Farbman, 1964; Frithioff and Wersall, 1965; Matoltsy and Parakkal, 1965; Hashimoto et d.,1965). Many investigators have reported the presence of lamellated material in the intercellular spaces, which persists through the cornified layer (Figs. 8 and 9). Martinez and Peters (1971) described a process of dissociation of the granule contents along the thin (intermediate), dense lamellae into pentalaminar disks of which the central layer comprised the original thick, electron-dense lamella and the two outermost comprised half the original thin, electron-dense lamellae. Before this occurred the discharged lamellae were orientated at right angles to the cell membrane, but after splitting apart they spread along the cell surface to modify the outer leaflet of the plasma membranes which were then separated by the pentalaminar disks so as to produce a multilayered “sandwich” which might itself then split along the line of the central thick leaflet of the original pentalaminar disk. One curious feature is that the original thick central lamella of the disk increases in thickness as it passes up to the stratum comeurn. Lavker rejected the view that the pentalaminar disks of Martinez and Peters were the form into which the MCG contents dispersed. He first described (Lavker, 1974) dispersion into an amorphous mass from which lamellae reformed. Subsequently (Lavker, 1976) the amorphous material was ascribed to

Fic. 8. Intercellular spaces between cells of the stratum granulosum (SG) and stratum comeum (SC), showing lamellated material resembling contents of MCGs in all respects. The cell membrane of the stratum comeum is thickened by the apposition of electron-dense material to the cytoplasmic From Hayward (1973). surface. Rat soft palate. ~46,750.

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oblique sectioning, but the lamellar structure of the MCGs was found to coalesce into broad sheets so that membranelike figures or bilayers of indeterminate length were produced parallel to and often in close contact with the cell membranes. Similar, very extensive stacks of intercellular lamellations far greater in length than would be possible within a membrane-coating granule have been shown intercellularly by Elias and Friend (1975) using both conventional sectioning and freeze-fracture. Observations on the epithelium of the hamster cheek pouch provide support for Lavker’s view (Hayward, 1978). The intercellular material is organized into stacks of membranes of considerable length, far greater than could be accounted for by the dimensions of individual granules (Fig. 9). After primary fixation with osmium tetroxide solution myelinlike figures can also be found. It is interesting to note that the dimensions of the lamellae differ substantially from those of the contents of the granules. They also differ in the stacks of membranes and in the myelin figures. The reason for these differences is not known but has been tentatively ascribed to a change in hydration (Hayward, 1978). Under normal conditions exocytosis of the contents of MCGs is completed prior to the onset of membrane thickening (Fig. 8). In dyskeratosis some lcells keratinize prematurely with thickening of the cell membrane while the cell is still

FIG.9. Lamellated material of the intercellular spaces in the stratum corneum of a hamster c heek pouch, showing the rearrangement into long stacks of lamellae with dimensions a5 shown in Fig. 5 . ~76,500.From Hayward (1978).

MEMBRANE-COATING GRANULES

111

in the stratum spinosum. In some instances this leads to the retention of MCGs after keratinization has occurred (Sato et al., 1977), suggesting that they are unable to leave the cells after membrane thickening. D. COMFQSITION

1. Glycoproteins Most knowledge of the cytochemistry of glycoproteins is based on the periodic acid-Schiff (PAS) method for 1 :2 glycol linkages. Periodic acid oxidation is effective under a wide range of conditions including those prevailing in thin sections prepared for electron microscopy. Several alternative methods have been designed to demonstrate the aldehyde groups in such a way that an electron-dense material is produced. Among such methods are the silver methenamine technique (Seligman et al., 1965) and the thiocarbohydrazide (TCH)-silver proteinate technique (Thiery, 1967). For these methods, adequate controls are essential (Hayward and Hackemann, 1973; Courtoy and Simar, 1974). It is advisable to include phospholipid extraction (Hayward and Hackemann, 1973), since some phospholipids give a positive PAS reaction (Pearse, 1968), and the possibility that the reaction may be due to these or related compounds such as glycolipids had not received serious attention until recently (Elias et al., 1977a). Both the silver methenamine and TCH-silver proteinate techniques have been applied to MCGs. The former gave positive results in MCGs in human epidermis (Hashimoto et al., 1966a; Olson ct al., 1969) but not in the hands of all investigators (Mercer et al., 1968). TCH-silver proteinate reacts with MCGs in several but not all oral epithelia (Hayward and Hackemann, 1973; Hayward, 1973) (Fig. 10). Controls show that the reaction is almost certainly due to a glycoprotein. A positive reaction is also found in the extracellular spaces after MCGs are discharged from the cells (Fig. 11). The silver deposit from this stain is closely applied to the distal plasma membrane of the cells and only to a lesser extent to the proximal or deep surface. It is notable that this distribution corresponds to the surface at which most of the granules are released. However, it is equally important that, in keratinized epithelium where the intercellular spaces are known to be filled with lamellae presumably derived from the granules, no lamellae are seen after TCH staining. The lamellae are not preserved without postfixation with osmium tetroxide, and this may introduce problems with the specificity of staining. Certainly primary fixation with osmium tetroxide can result in a positive TCH reaction with proteins and lipids (Rambourg, 1974). It seems unlikely then that the distribution of TCH staining accurately reflects the distribution of the carbohydrate-containing material in vivo.

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FIG. 10. MCGs stained with the periodic acid-TCH method for complex carbohydrates The internal lamellar structure is not preserved. Human gingiva. ~ 4 5 , 9 0 0 .

In an attempt to provide a simple staining method for demonstration of MCGs, perhaps with a view to easier quantitation, Ashrafi et af. (1977) used the periodic acid-alkaline bismuth method of Ainsworth et al. (1972). This was thought to be specific for glycoproteins, and controls showed it to be mediated by the production of aldehyde groups. In discussing glycoprotein staining it is relevant to note that MCGs contain lysosomal enzymes (Section II,D,3) of which many if not all are glycosylated proteins. Lysosomes can be stained with both the PAS stain and a hexarrrinesilver stain (Capanna et ul., 1966). 2 . Phospholipids and Lipoproteins The lamellated appearance of the contents of MCGs inevitably leads to the suspicion that polar lipids or phospholipids are present. Frithioff and Wersall have already pointed out the similarity between the images of the lamellations and those of prepared phospholipids. Such lamellae are characteristic of phospholipids in contact with water (Mercer, 1961; Revel et al., 1958; Stoeckenius, 1959; Stoeckenius et al., 1960). The alternating thin and thick bands in NLCGs are visually reminiscent of the appearance of the lipoprotein complex myelin. However, the latter is formed in a special way by alternate apposition ad the

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113

FIG. 1 1 . Periodic acid-TCH-positive material in intercellular spaces after the discharge of MCG contents. Although probably not representing the distribution of carbohydrate-containing material in vivo the stain adheres to the distal cell membrane, that is, that from which discharge of the granules occurred. x 17,000.

cytoplasmic faces and external faces of cell plasma membranes. Much work (see, for example, Finean, 1961) has been done on the ultrastructure of myelin, and the dimensions vary considerably according to the method of preparation. Unfortunately it appears unlikely that any useful comparison between MCG contents and myelin is possible in terms of dimensions of lamellae at the present time. Histochemical tests for phospholipids have been applied to MCGs with positive results. They stain with acid hematein (Olah and Rohlich, 1966), and the reaction is abolished by extraction with pyridine which also destroys the lamellated internal structure. Digestion with phospholipase C also abolishes the lamellar structure (Hashimoto, 1971b), but it is worthy of note that the lamellae are in any event fairly sensitive to the preparatory method. They are not, for example, preserved by aldehyde fixation unless postfixation in osmium is also used (Hayward and Hackemann, 1973). MCGs stain with a block impregnation method using osmium-zinc iodide which is reputedly specific for lipids other than phospholipids, that is, nonpolar lipids (Niebauer ef ul., 1969). Additional support for the specificity of the method comes from the observation that the staining is abolished by mild extrac-

1 I4

A . F. HAYWARD

tion with hexane which reputedly extracts only nonpolar lipids (Wilgram et al., 1973). The behavior of the intercellular lamellae in freeze-fracture preparations also suggests a neutral lipid composition (EJias et al., 1977b). More recently the problem of lipid composition was approached in a different way by Elias et af. (1977a). Lipid histochemistry suggests that nonpolar or neutral lipids become progressively more prominent in squamous epithelia as cells pass from the stratum granulosum to the stratum corneum. Lipid extracted from the whole epidermis of newborn mice in which hair and sebaceous glands are absent shows an equal proportion of neutral and polar lipids. Twenty-five percent of this lipid is present as glycolipid, and such compounds are PASpositive. Two conclusions may be tentatively drawn from these observations. The PAS-positive component of MCGs often regarded as a glycoprotein may be a glycolipid, and the polar lipids of the contents may be converted to neutral lipids after extrusion from the cells. 3. Lysosomal Enzymes Two cardinal features of a lysosome are that it is enclosed by a single trilaminar lipoprotein membrane and contains a battery of hydrolytic enzymes with a more-or-less acid pH optimum (de Duve and Wattiaux, 1966). Primary lysosomes are those whose enzyme contents have not been involved in cellular activity other than their own synthesis, whereas secondary lysosomes are those which have been involved in cellular digestive activity. They may therefore contain material of extracellular origin or material derived from autophagic activities. The picture is complicated by the recently developed ideas about the GBRL system (Novikoff et al., 1971) which may give rise to both lysosomes and secretory droplets such as those of exocrine cells. There have been many otlservations of acid phosphatase in secretory granules not otherwise regarded as lysosomes (Osinchak, 1964; Sobel and Arvin, 1965; Hand and Oliver, 1977). MCGs are enclosed by a single trilaminar membrane (Martinez and Pelers, 1971) which by analogy with other such membranes is probably lipoprotein in nature. They also contain lysosomal enzymes as demonstrated by electroncytochemical techniques (Figs. 12-14). In epidermis, Wolff and Holubar (l967), Wolff and Schreiner (1970), Squier and Waterhouse (1970), and Gonzalez e! a f . (1976) demonstrated acid phosphatase using a lead-containing medium. Not all the MCGs in these studies were positively stained, and there was a suggestion that the enzyme could more easily be shown in the guinea pig than in humans (Wolff and Schreiner, 1970). There appears to be an increase in the proportion of MCGs in which acid phosphatase can be stained as the cells move from the spinous to the granular layer (Wolff and Schreiner, 1970; de Rey et al., 197’6). Results with other lysosomal enzymes at a histochemical level have not k e n uniform. Arylsulfatase has been demonstrated by some investigators (Olson et a l . , 1969; Gonzalez et af., 1976; Takaki, 1974) but not by others (Rowden,

FIG. 12. Part of a cell of the stratum spinosum after the application of an acid phosphatase reaction. The end product appears black and is localized to MCGs. There is none in the intercellular spaces. Human keratinized oral mucosa. X23.800.

Fic. 13. Acid phosphatase reaction in nonkeratinized epithelium with the reaction localized in the MCGs and absent from the intercellular spaces. Human buccal mucosa. x 10,625.

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A . F. HAYWARD

FIG. 14. Same tissue as in Fig. 13 but at a more superficial level, showing the acid phosphatase end product localized to the intercellular spaces as a result of the exocytosis of MCGs. x 10,625.

1968). Nonspecific esterase was found in only a proportion of granules (Squier and Waterhouse, 1970; Gonzalez et al., 1976), even though it was consistently shown in extracellular lamellated material. The occasional presence of endocytosed material in MCGs categorizes them as secondary lysosomes (Hayward, 1976) (Fig. 15), and it is of interest that the complex bodies found in epidermal cells which ingest colloidal material also contain identifiable MCGs (Fig. 16). The presence of lysosomal enzymes in MCGs may not mean that they function as lysosomes within the cell. They could be a vehicle for the extracellular release of a hydrolytic enzyme (Wolff and Schreiner, 1970). Alternatively, since the presence of acid phosphatase in secretory granules for no known reason is well established, the same situation may exist in MCGs. The increase in the proportion of granules containing enzyme with progressive differentiation referred to earlier contrasts with the picture in known secretory granules where the am'ount of enzyme decreases as the granules mature (Hand and Oliver, 1977). In the epidermis of skin, irradiation with ultraviolet light produces changes in acid phosphatase and phospholipid content which suggest that lysosomes, are damaged (Johnson, 1968; Johnson and Daniels, 1969). Moderate exposure to ultraviolet light has been found rapidly to reduce the number of MCGs in human epidermis (Wilgram et al., 1970). X-irradiation also destroys MCGs, at the s,ame

Fic;. 15. A group of MCGs near the cell membrane of rabbit buccal epithelium 3 hours after colloidal thorium dioxide was injected into the subepithelial tissues. Some of the material is seen in one MCG (arrow). xY3,SOO.

FIG. 16. . Complex body present in epithelium 24 hours after the injection of thorium dioxide subepithelially. It represents a phagosome or a secondary lysosome into which both thorium dioxide and a membrane-coating granule have been incorporated. x 9 5 . 2 0 0 . From Hayward (1976).

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time inducing an overall increase in the acid phosphatase level in the epidermis (de Rey et a l . , 1976). Secondary lysosomes in epidermal cells can be labeled with an electron-dense marker such as thorium dioxide (Wolff and Honigsmann, 1971; Hayward, 19’76). Subsequent exposure to ultraviolet light causes a release of this label into the cytoplasm, presumably indicating a breakdown of the lysosomes (Honigsmann et al., 1974). Other cytoplasmic components exhibit damage before this breakdown, so it is not assumed to be the primary effect of ultraviolet irradiation. MCGs also take up endocytosed material (Hayward, 1976), so their response to ultraviolet light may be the same as that of other secondary lysosomes. Acid phosphatase can readily be demonstrated in the intercellular spaces (Fig. 15) and in keratinized epithelium is often associated with lamellated material derived from MCGs. Most of the investigators already referred to in this section have achieved similar results.

E. FUNCTIONS The functions of MCGs have not been conclusively established, but several plausible hypotheses have been advanced. 1. Membrane Thickeriitig The first realistic suggestion concerning the part played by these granules in the epithelium was put forward by Matoltsy and Parakkal (1965). They investigated various aspects of the chemical resistance of the thickened cell membrane of the stratum corneum, at the same time drawing attention to the granules, previously largely ignored. They then proposed that the granules provided the material to create the thickened membrane and as a result coined the expression “membrane-coating granules. It was subsequently shown by careful analysis of electron micrographs that membrane thickening was accompanied by other changes such as disappearance of the outer leaflet of the triple membrane :,trueture (Farbman, 1966). The thickness of the inner leaflet is augmented by apposition on the cytoplasmic face of the membrane of a thick, electron-dense layer of unknown origin. Membrane thickening occurs from the cytoplasm, whereas the MCGs are discharged prior to membrane thickening and are on the outside where they play no part in the process (Hashimoto, 1971a,b; Grubb er ul., 1968). ”

2 . Cell Adhesion

The factors involved in the adherence of epidermal cells to each other include desmosomes, tight or gap junctions, and possibly an intercellular cement. In cells of mesodermal origin such as cultured fibroblasts, cell surface glycoproteirns are responsible for vital cell properties such as intercellular adhesion and adhesion to substrates. There is a glycoprotein layer on the surface of epidermal cells

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throughout the depth of the epithelium (Wislocki et al., 1951; Cohen, 1968). Extracellular material stainable by the PAS method and presumably glycoprotein in nature becomes thicker and more intensely stained in the more superficial layers (Meyer and Gerson, 1964). In psoriatic epidermis there are greater quantities of extracellular glycoprotein than in normal skin (Braun-Falco, 1958). Psoriasis is characterized by increased stickiness of epidermal cells. It may well be that this results from a glycoprotein-cementing substance, distinct from the cell surface protein, whose production in abnormal quantities is the primary lesion in psoriatic epidermal cells (Bonneville et al., 1968). Hashimoto (1971a,b) takes the view that MCGs with their glycoprotein content provide this cementing substance and that this is their function. He prefers to call them cementsomes. Hashimoto and Lever (1966) claim to have observed abnormally large numbers of MCGs in psoriasis, and there is evidence for this in earlier publications on the same topic (Zech et af., 1961; von Wettstein et al., 1961; Van der Staak et af., 1969). There is no statistically tested objective support for such a finding. On the contrary Bonneville and her colleagues ( 1968) observed no qualitative differences from normal in MCGs in psoriasis but found an enlarged Golgi apparatus with discrete vacuoles possibly of secretory material. From their work it appears that MCGs provide an extracellular material other than a cementing glycoprotein. The structural appearance of lamellated intercellular material suggests that the lipid content of a lipid complex predominates over the glycoproteins. 3 . Production of a Cell Surface Coat Surface coats are an established feature of metazoan cells (Martinez-Palomo, 1970; Parsons and Subjeck, 1972). They are generally demonstrable by cationic stains such as lanthanum hydroxide, colloidal iron, and ruthenium red. There is little doubt that the reaction is due to anions on the surface provided by either sulfate groups or sialic acid which are both often present in glycoproteins. It is possible that glycosaminoglycans provide the anions responsible for surface coat staining. They are included in the vague designation “acid mucopolysaccharides,” for which ruthenium red staining was believed to be specific (Luft, 1971). It seems likely that ruthenium red most often stains glycoproteins (Blanquet, 1976). The origin of cells stained by such compounds must be taken into account when assessing results, as there is little evidence for the occurrence of glycosaminoglycans in epidermal cells. In epidermis there is a surface coat stainable with ruthenium red (Wolff and Schreiner, 1968), which is almost completely removed from cultured cells by neuraminidase (Fritsch et a l ., 1975). It is reconstituted within 24 hours of removal. Since MCGs may contain glycoproteins, it is apparent that they might contribute to the surface coat. The presence of stainable material throughout the depth of the epidermis (Wislocki et al., 1951; Rambourg et al., 1966), with the possible

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exception of the basal cells (Mercer et al., 1968), militates against such a proposal because of the level at which MCGs are released. 4. Cell Desquamation MCGs contain the lysosomal enzyme acid phosphatase demonstrable by electron microscopy and, as at other sites, it is assumed that other lysosomal enzymes, not as readily demonstrated, are also present. Given the right pH, at least two processes occur in the intercellular spaces in which such enzymes could participate. One is the progressive breakdown of desmosomes which occurs throughout the stratum corneum (Odland and Reed, 1967), and the other is the breakdown of the outer leaflet of the plasma membrane which occurs during and after membrane thickening (Farbman, 1966). These two phenomena may be related. Further, if the intercellular adhesive material already referred to in relation to psoriasis (Section II,E,2) has to be broken down so that cells can be desquamated, this breakdown is another potential function of hydrolytic enzymes at this site. Lysosomes may contain enzymes capable of breaking down lipoproteins as well as glycoproteins, but there is no evidence that the intercellular pH could be as low as the optimum for these acid hydrolases. Allen and Plotten (1975) assumed, from the fact that release of MCG contents preceded bireakdown, that the two events were consequential. However, there was no experimental evidence to support such a conclusion. Circumstantial evidence linking MCGs with cell desquamation has been obtained from the study of rare congenital hyperkeratoses. In recessive ichthyosis, thickening of the stratum corneum is associated with an apparently reduced number of MCGs (Anton-Lamprecht, 1974). In Kyrle-Flegel disease MCGs were believed to be absent in the hyperkeratotic papules (Frenk and Tapenoux, 1976) but they have since been shown to be present and abnormal in structure for a keratinized epithelium (Squier el al., 1978). Comedo formation in acne vulgaris is the result of cohesive hyperkeratinization in hair follicles and sebaceous ducts and a reduced number of MCGs has been reported in those sites in acne (Knutson, 1974). There is evidence, both qualitative (Woo-Sam, 1977) and quantitative (Woo-Sam, 1978) of a reduction in the number of MCGs during experimental comedo formation producing an acne-like condition.

5 . Permeability Probably the most attractive hypothesis concerning the function of MCCis has come from observations on transepidermal movement of various substances. That skin is permeable to some extent has been known for many years. It is preferentially permeable to lipid-soluble materials. Water and other solutes can penetrate whole skin to a measurable degree which is limited by the intact epidermis (Winsor and Burch, 1944). Galey et al. (1976) confirmed that the

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barrier to permeability lay in the epidermis and showed that permeability was due solely to diffusion and depended on the molecular weight of the material studied. The epithelium of oral mucosa has traditionally been regarded as more permeable than skin. There is evidence that this is so for water (Adams, 1974b; Galey et d.,1976) and some evidence that nonkeratinized oral epithelium is more permeable than the keratinized form (Adams, 1974a). A detailed consideration of the many aspects of permeability of epidermis to low-molecular-weight materials is not relevant here, but a description of the work which has been done has been reported by Scheuplein (1 976). The epidermis of both skin and oral mucosa is remarkably impermeable to substances of higher molecular weight. The passage of large molecules across squamous epithelia can be monitored by various means such as radioactive labeling (Tolo, 1971), fluorescence (Brill and Krasse, 1958), or enzymic properties of the substance (Squier, 1973; Squier and Hopps, 1976). The results have been more or less uniform. Whether keratinized or nonkeratinized epithelium is studied, substances of moderate or high molecular weight applied to the surface penetrate only two or three cell layers between the cells. There appear to be two likely mechanisms by which the diffusion of materials through the intercellular spaces could be prevented. One is the presence of tight (gap) junctions between cells, which mechanically close the intercellular spaces. The other is the presence of impervious material filling the spaces, perhaps contributed by the extruded contents of the MCGs. Specialized intercellular junctions originally described as tight junctions (Farquhar and Palade, 1964) are a common mechanism for sealing intercellular spaces. Many of these junctions are permeable to such molecules as lanthanum salts and are now referred to as gap junctions. In epidermis Friend and Gilula (1972) precisely defined the different types and showed that it may not be possible to categorize a given example by transmission electron microscopy alone. An extensive network of gap junctions throughout the more superficial layers of an epithelium could provide a permeability barrier. In the epithelia which have been examined with this in mind no such network was present in the stratum corneum, and only a very restricted one was found in the stratum granulosum (Shimono and Clementi, 1976; Caputo and Peluchetti, 1977). A comprehensive study using a variety of techniques leads Elias et a/., (1977b) to conclude that gap junctions do not significantly contribute to the permeability bamer of squamous epithelia. The view that MCGs provide the permeability barrier when their contents are extruded into the intercellular spaces has been put forward several times (Wolff and Honigsmann, 1971; Elias and Friend, 1975; Squier and Johnson, 1975). Certainly the presence of lipids of one sort or another in the contents of MCGs correlates well with the observed increase in epidermal permeability produced by a variety of fat solvents (Sweeney and Downing, 1969).

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Circumstantial evidence of the part played by MCGs can be obtained by injecting a traceable substance beneath an epithelium and observing permeaion toward the surface. With the use of thorium dioxide (Wolff and Honigsmann, 1971; Hayward, 1976), horseradish peroxidase (Squier, 1973; Elias and Friend, 1975; Squier and Hopps, 1976; Squier and Rooney, 1976; Wolff and WolffSchreiner, 1976), lanthanum nitrate (Schreiner and Wolff, 1971), and feiritin (Elias and Friend, 1975; Nordqvist et al., 1966) it has been found that injected material permeates through the epithelium only as far as the stratum granulowm. The limit to which penetration occurs corresponds with the level at which the contents of MCGs are extruded into the intercellular spaces. In an experiment using thorium dioxide, 3 hours after injection, none of the foreign material was observed in spaces occupied by lamellated material derived from MCGs. After 24 hours injected material was seen together with MCG contents, but this could be explained by passive movement toward the surface during cell migration when MCG contents were expelled into spaces already containing the injected substances (Hayward, 1976). Another test of the hypothesis that MCGs play a role in the permeability barrier is to investigate epithelia where no MCGs are present. There appear lo be two in which this occurs. Oral epithelium can be grown in tissue culture, and in one particular system (Jepson, 1974) differentiation and keratinization 0 1 the constituent cells occurs without the production of MCGs (Fejerskov et a l . , 1974). In this system the epithelium becomes permeable to compounds which do not ordinarily penetrate oral epithelium (Squier et ul., 1978). Permeability is enhanced in the absence of MCGs but not necessarily because of their absence. The second site from which evidence may be obtained is the specialized oral epithelium known as the junctional epithelium. A detailed review of this region, especially of its clinical aspects, has recently been published by Schroeder (1977). In humans, gingival epithelium is of three types (Fig. 17). Exposed to the oral environment and masticatory forces is a keratinized epithelium in which MCGs with lamellated contents are found (Hayward and Hackemann, 1973). In contact with tooth enamel but not attached to it is crevicular or sulcular epithelium which is not keratinized. A similar epithelium in cats contains granules resembling MCGs (Innes, 1973). Continuous with the sulcular epithelium, but firmly attached to the enamel and thus closing the sulcus apically, is the junctional epithelium (Schroeder and Listgarten, 1977). 'This epithelium is not keratinized, and descriptions that have been published have not shown the presence of MCGs (Schroeder and Listgarten, 1977; Frank and Cimasoni, 1970). Permeability studies in this area are bedeviled by problems of identification of the precise epithelium being studied. Fluorescein injected into the bloodstream passes into the gingival sulcus very rapidly (Brill and Krasse, 1958). Horseradish peroxidase applied to the rat gingiva penetrates to the apical limit of the junctional epithelium in 10 minutes, even though it does not penetrate gingival

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Junctional Epithelium

\ 1/

123

d

FIG. 17. Diagram of the arrangement of three types of epithelial tissues around the cervical margin of a tooth as seen in humans and most mammals. The junctional epithelium is physically attached to the enamel surface. The gingival sulcus, lined with sulcular epithelium, is shown as a wide trough, but this is not normally so. The sulcular epithelium is normally in contact with, but not attached to. [he enamel. E, Enamel; D, dentine.

epithelium at all (McDougall, 1971). The same protein appears to penetrate the junctional epithelium of dogs as judged by cellular infiltration after sensitization (Kahnberg et a / ., 1978), but there is evidence that horseradish peroxidase itself is chemotactic so that the white cells migrate toward the applied protein. It may also be true that the permeability of this region is not limited to the truly junctional epithelium but involves the sulcular epithelium as well (Tolo, 1971; McDougall, 1972; Caffese and Najleti, 1976). More work is needed on the gingival sulcus, preferably in a species where the sulcular epithelium is a distinct entity and is nonkeratinized, to establish (1) whether MCGs are present or absent in the junctional epithelium and (2) whether there is a significant difference in the permeability of sulcular and junctional epithelia to moderate- or high-molecular-weight materials.

Ill. Summary and Future Prospects MCGs represent a specific secretory product of almost all stratified squamous epithelia. In keratinized epithelia they contain lysosomal enzymes, but this probably

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reflects their origin rather than their function. Their contents are extruded from the cells so as to fill the intercellular spaces where it probably provides the efficient permeability barrier found in most such epithelia. The work on these granules has been almost entirely structural. Definitive information on the composition of MCGs awaits their isolation, presumably by differential centrifugation. Epithelia have always been difficult subjects for this procedure because of the massive quantity of insoluble fibrous protein present in the cells. The much larger, more prominent keratohyalin granules have only seldom been isolated (Matoltsy and Matoltsy, 1970). MCGs would probably prove even more labile. The outstanding ultrastructural problem is the origin of MCGs. A spatial association with the Golgi apparatus is not convincing evidence of their origin from this structure. Cytochemical or radioactive labeling has not so far led to a more definite conclusion and awaits the investigation of an experimental situation where the production of MCGs is halted or where they are reduced in number so that a surge in production rate shows their origin more clearly. The association of a change in MCGs with diseases or an experimental change in squamous epithelia can only be substantiated by adequate stereological or other morphometric data.

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INTEKNATIONAL REVIEW OF CYTOLOGY VOL . 59

Innervation of the Gastrointestinal Tract GIORGIO GABELLA

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Depurtnrent of Anutomy. University College London. England

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . Vagus Nerve 111. Nerves from the Abdominal Plexus . . . . . . . . . . . IV . Pelvic Nerves . . . . . . . . . . . . . . . . . . . V . Distribution of Intramural Plexuses . . . . . . . . . . . VI . Shape of Intramural Plexuses . . . . . . . . . . . . . . VII . Number of Neurons . . . . . . . . . . . . . . . . . v111 . Size of Neurons . . . . . . . . . . . . . . . . . . IX . Types of Neurons . . . . . . . . . . . . . . . . . . X . Histochemistry for Acetylcholinesterase . . . . . . . . . . XI . Catecholamine Histochemistry . . . . . . . . . . . . . A . Myenteric Plexus . . . . . . . . . . . . . . . . . B . Suhmucosal Plexus . . . . . . . . . . . . . . . . C . MuscleCoat . . . . . . . . . . . . . . . . . . D . Mucosa . . . . . . . . . . . . . . . . . . . . E . Blood Vessels . . . . . . . . . . . . . . . . . . XI1 . Serotoninergic Neurons . . . . . . . . . . . . . . . . XI11. Immunofluorescence Histochemistry . . . . . . . . . . . XIV . Ultrastructure of the Myenteric Plexus . . . . . . . . . . xv . Nerve Endings and Synapses in the Myenteric Plexus . . . . . XVI . Glial Cells . . . . . . . . . . . . . . . . . . . . XVII . Surface of Ganglia and Vascularization . . . . . . . . . . XVIII . Ultrastructure of the Suhmucosal Plexus . . . . . . . . . . XIX . Innervation of the Muscularis Externa . . . . . . . . . . xx . Innervation of the Mucosa . . . . . . . . . . . . . . . XXI . Innervation of the Blood Vessels . . . . . . . . . . . . XXII . Extrinsic Nerve Fibers . . . . . . . . . . . . . . . . . XXIII . Exogenous Adrenergic Transmitters and "False Transmitters" XXIV . Adrenergic Innervation . . . . . . . . . . . . . . . . xxv . Cholinergic Innervation . . . . . . . . . . . . . . . . XXVI . Innervation by Other Types of Nerves . . . . . . . . . . XXVII . Afferent Fibers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XXVIII . Development XXIX . Coupling between Muscle Cells . . . . . . . . . . . . . xxx . Interstitial Cells . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

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Copyright @ 1Y7Y by Academic Press Inc . All nghlr of reproduction in any forin reberved. ISBN 0-12-361359-7

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I. Introduction The structures involved in nervous control of the activities of the gastrointestinal tract are examined in this article. They can be classified as (1) nerve pathways reaching the gastrointestinal tract, including afferent and efferent pathways; (2) ganglia and plexuses located within the wall of the stomach and the intestine; and (3) terminal parts of nerve fibers making contact with the effectors (muscular and glandular) or forming sensory endings. The effectors include secretory cells imd smooth muscle cells of the intramural blood vessels and of the muscle layers proper. The presence of highly developed intramural plexuses is a characteristic feature of the gastrointestinal tract. It is probably related to the complexity of functions of the vertebrate gut, notably regulated propulsive activity. An analysis of the structure of the intramural plexuses forms the bulk of this article. The efferent pathways leading to the gastrointestinal tract can be divided into three major groups: (1) the vagus nerves; (2) the splanchnic nerves, with the abdominal plexus and its perivascular nerves reaching the stomach and intestine; and (3) the pelvic plexus and its nerves. The afferent pathways are less well understood. 'The pathways within the central nervous system that are related to gastrointestinal functions are not considered here.

11. Vagus Nerve

Nerve fibers originating from neurons in the medulla oblongata and in the jugular and nodose ganglia travel in the vagus nerve along the neck and thorax and enter the abdomen after passing through the diaphragm in the same orifice as the esophagus. This part of the nerve is the abdominal tract of the vagus; it is usually in the form of two plexuses adhering to the ventral (arising mainly ]from the left thoracic vagus) and to the dorsal (arising mainly from the right thoracic vagus) aspects of the abdominal esophagus (Mitchell, 1941). There are many quantitative studies on the composition of the abdominal vagus, which as: reviewed elsewhere (Gabella, 1976a). In all the mammalian species investigated the abdominal vagus is made up of several thousand axons of which the great majority are unmyelinated. Apart from differences in size and the presence or absence of a myelin sheath, the axons of the abdominal vagus show no structural features which allow types of fibers to be recognized. It is well known that the nerve contains afferent and efferent fibers. The latter are cholinergic (preganglionic), but there are also noncholinergic inhibitory fibers (Langley, 1898) which originate from the brain stem. Moreover, the abdominal vagus contains a small number of adrenergic fibers, demonstrable by fluorescence microscopy usually after experimental ligature of the nerve (Muryobayashi et al., 1968;

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Nielsen et a / . , 1969; Lundberg et a/., 1976), which originate in the superior cervical ganglion and reach the vagus through an anastomosis. There are also anastomoses between the thoracic vagus nerve and intercostal nerves, and afferent fibers from the abdomen reach the thoracic spinal cord via these nerves [demonstrated in physiological experiments by Harper et a/. (1 9391. In the rat there are a few chromaffin cells scattered along the vagus nerve (Gabella and Pease, 1973), and in the dog this nerve contains many neurons, either isolated or clustered in small groups (Dolgo-Saburov, 1935). Several small paraganglia accompany the abdominal vagus of rodents (Goormaghtich, 1936; Hollinshead, 1941; Mascorro and Yates, 1974). The majority of the fibers of the abdominal vagus penetrate into the wall of the stomach and terminate in this organ. Some vagal fibers reach parts of the intestine, descending along its wall from the stomach or passing through the coeliac ganglion.

111. Nerves from the Abdominal Plexus

The sympathetic fibers for the stomach and the intestine originate from neurons in the prevertebral ganglia (coeliac ganglion, superior mesenteric ganglion, and inferior mesenteric ganglion, together forming the abdominal plexus). These ganglion neurons are adrenergic, and their axons (postganglionic fibers) reach the wall of the gut (Sections XI and XXIV). The preganglionic fibers originate from the thoracolumbar spinal cord and reach the prevertebral ganglia through the thoracic and the lumbar splanchnic nerves (see a review in Gabella, 1976a). The prevertebral ganglia receive, in addition to input from the spinal cord, input from neurons situated in the alimentary tract (Kuntz, 1938, 1940); afferent and efferent fibers seem therefore to converge on the same ganglion neurons (Crowcroft and Szurszewski, 1971; Crowcroft e t a / . , 1971). The mesenteric nerves emerge from the abdominal plexus directed toward the intestine and run in the mesentery parallel to the large vessels. They are small nerves, usually two or three in number, in the proximity of each artery. They are composed of unmyelinated fibers; myelinated fibers are rare, representing less than 1% of all the fibers.

IV. Pelvic Nerves The intestine receives nerve fibers from preganglionic neurons in the sacral spinal cord (sacral parasympathetic fibers), which pass through the pelvic plexus and emerge as colonic nerves and rectal nerves which reach the intestinal wall

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running parallel to large blood vessels. The pelvic plexus contains also adrenergic and cholinergic ganglion neurons, fibers of the hypogastric nerve which have an apparent origin in the prevertebral ganglia, and small nerves with an apparent origin in the sacral sympathetic chain (sacral splanchnic nerves). The colonic and rectal nerves contain therefore, in addition to the sacral parasympathetic preganglionic fibers (cholinergic), adrenergic postganglionic fibers from the pelvic plexus and probably other types of fibers including afferent fibers.

V. Distribution of Intramural Plexuses Intramural plexuses are present throughout the entire length of the gastroinltestinal tract. In some species, for example, the rat, a clear-cut plexus (myentericus) is present in the esophagus between the two layers of striated musculature (Gruber, 1968). Starting from the esophagus, the plexuses can be visualized as networks which extend without interruption-but with great changes in shapedown to the upper part of the anal canal; they continue through the gastroesophageal junction, the pyloric junction, and the ileocolonic junction. Two major plexuses, or ganglionated plexuses, are found in the gut. The myenteric plexus (plexus myentericus or Auerbach’s plexus) is situated within the muscularis extema, between the two muscle layers. The submucosal plexus (plexus submucosus) is situated in the submucosa. Some investigators distinguish in the latter plexus a plexus entericus extemus (plexus submucosus eKternus, Schabadasch plexus or Henle’s plexus) situated close to the inner aspect of the circular musculature, and a plexus submucosus intemus (plexus submucosus or Meissner’s plexus) which lies closer to the mucosa (Schabadasch, 1930; Gunn, 1968; Stach, 1977). Two submucosal plexuses are best recognized in the pig intestine, where they can be dissected out separately into two layers of the submucosa (Gunn, 1968; Stach, 1977). A clear distinction of two plexuses has been made in the cat and sheep intestine, but applies less well to small laboratory animals. Some investigators have described in the stomach (and in the small intestine near the attachment of the mesentery) a subserosal plexus, with few ganglia, mainly associated with branches of the vagus nerve (Schabadasch, 1930; Ferri and Ottaviani, 1966), but in the opinion of other workers (e.g., Stohr. 1957) this plexus is not separable from the myenteric plexus.

FIG. 1. Myenteric plexus of the guinea pig, stained with a histochemical method for DPN diaphorase (described in Gabella, 1971). Stretch preparation of the longitudinal musculature with the plexus attached. (A) Colon. Note the wide range of cell sizes. A branching blood vessel is visible at top center. Marker: 200 /.un. (B) Rectum. To the right are neumns lying in a connecting strand. Marker: 100 p.(C) Colon. Note the wide range of cell sizes and shapes. Marker: 50 p n

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VI. Shape of Intramural Plexuses When the wall of the small intestine is distended, the intramural ganglia are virtually one cell thick and spread on a plane parallel to the intestinal outer surface. The plexuses are close to being two-dimensional neuronal circuits. The arrangement of ganglia and connecting strands can be studied in properly stained in toto preparations of the wall or of its layers, peeled off and mounted, and observed enfuce (Fig. I ) . Silver impregnation, methylene blue, and histochemical methods have been used to study the general appearance of the plexuses. They are netlike structures with ganglia (the nodes of the mesh, formed by clusters of neurons) and strands of nerve fibers connecting them. The size and shape of the meshes vary greatly between the myenteric and submucosal plexuses, within a plexus in different parts of the alimentary tract, and within the same part of the gut in different animal species. In all the examples studied the size of the ganglia and the pattern of the plexus are highly consistent and are characteristic of a given part of the alimentary tract in a given species (Schabadasch, 1930; Stohr, 1930; Irwin, 1931; Matsuo, 1934; Ohkubo, 1936a,b,c; Taxi, 1965; Stach, 1971). For example, in the guinea pig one can at first glance distinguish the myenteric plexus of the ileum from that of the duodenum, stomach, colon, or rectum (Fig. 2); similarly, one can easily tell the myenteric plexus of the rabbit from that of the rat, guinea pig, sheep, or monkey. Nothing is known of the morphogenetic processes which lead to the formation of a given type of mesh. It is also unknown whether the type of pattern has any functional significance or bears any relation to the activity of the part of the intestine where it occurs. The ganglia are more closely packed and relatively larger in young and immature animals (Section XXVIII) (Fig. 17). In the adult most of the neurons are situated within the ganglia, but some can be found in the connecting strands or in tertiary meshes of the plexus (Fig. IB). In the stomach of the rat and guinea pig the ganglia of the myenteric plexus are very numerous near the lesser curvature, and the density of neurons is high, but they become progressively less frequent when areas nearer the greater curvature are examined; here large areas of the stomach musculature have few ganglia or none at all. In the parts of the intestine which have taeniae the myenteric plexus is more tightly meshed and the neurons are more numerous in the regions beneath the taeniae than in the rest of the wall (Section VII). In the colon of the guinea pig the myenteric plexus has small ganglia and long strands forming large meshes in FIG.2. Drawings of the myenteric plexus from various portions of the guinea pig intestine, obtained by tracing ganglia and connecting meshes from photographic montages of stretch preparations stained as in Fig. I . (A) Duodenum. ( B ) Ileum. (C) Colon. (D) Rectum. In each preparation the left side is near the peritoneal attachment, and the longitudinal axis of the intestine runs vertically. Marker: 1 mm.

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the region beneath the attachment of the peritoneum (which constitutes up to one-eighth of the circumference of the canal) (Fig. 3A); on the other hand, in the region adjacent to the peritoneal attachment the ganglia are the largest of the gut and are so close to each other that they often merge with one another (Fig. 2C). The meshes become wider, and the ganglia more rarefied as the plexus spreads toward the antiperitoneal side of the canal (Figs. 1C and 3A). Compared with the myenteric plexus, the submucosal plexus has finer meshes and more numerous and smaller ganglia and shows a regular pattern (Schabadasch, 1930; Stohr, 1930; Ohkubo, 1936a; Gunn, 1968) (Fig. 3B). In some species the submucosal plexus is distributed at various levels in the submucosa, and a Meissner’s and a Henle’s plexus can be distinguished (Section V). The plexus of Henle resembles the myenteric plexus except that the ganglia are smaller and the strands thinner. This plexus is usually poorly developed; exceptions are the colon of the sheep and, in this and other species, the portions of the intestine near the ileocolonic junction and the anal canal (Gunn, 1968). In the submucosa of the stomach there is a nerve plexus, but it has been reported to have few ganglion cells or none at all (Kyosola et al., 1975). However, nerve ganglia in the submucosa of the stomach are described by several investigators (Stohr, 1930; Ohkubo, 1936a); it is possible that their distribution is not uniform in various parts of the stomach, as is the case with the myenteric plexus.

VII. Number of Neurons Counts of neurons in the myenteric plexus have been made in several mammalian species (Table I). All the figures obtained refer to the number of neurons per square centimeter of external surface of stomach or intestine. The values are thus only approximate, since the area of the external surface varies with the degree of distension of the organ, but they give a good indication of relative differences in neuronal density in various parts of the gut. In most experiments the duodenum has a higher neuronal density than the ileum, and the colon a higher density that the small intestine. It would be interesting to have more data and to determine

FIG. 3. (A) Stretch preparation of part of the circular musculature with the myenteric plexus attached, from the guinea pig colon, stained for AChE. The left half of the preparation corresponds to the part of the wall which is under the attachment of the peritoneum and has a characteristically wide-meshed plexus. (B) Stretch preparation of the suhmucosa from the guinea pig ileum, stained for AChE. Intense activity is seen in the ganglia of the submucosal plexus (individual neurons are not recognizable). (C) Neuron of the myenteric plexus of the guinea pig injected intracellularly with Procion yellow (courtesy of Dr. G. M. Lees, Department of Pharmacology, University of Aberdeen). Note the variety of processes arising from the perikaryon. The long, thin process oriented vertically runs in an aboral direction and could be followed for about 390 p n . Marker: 50 prn.

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N U M B t R OF NEURONS I N T H t

TABLE 1 MYENTRK PLEXUS OF VARIOUS PORTIONS O F THF ALIMENTARY ' I R A < T IN

SI W R A L

SPECIES

Species

Region

Neurons"

Method

Referenceh

Guinea pig

Esqphagus Stomach Cardia Pylorus Duodenum Ileum Cecum Beneath taeniae Between taeniae Colon

I .370

Methylene blue (spread preparations)

(1)

Sigmoid Rectum Guinea pig

Monkey

Guinea pig

Esophagus Stomach, PYIOIUS Duodenum Upper Middle Ileum Cecum Colon Sigmoid Rectum

-

3.500 20,000 10,000 7,500 12,000 4,500 15.000 19,000 17,000 18,000

1,300 16.500

Methylene blue (spread preparations)

9,300 9,800 7.200 4,100 14,800 14,800 16,000

Stomach Cardia Fundus Pylorus Duodenum Jejunum Ileum Cecum Colon Rectum

2,500 1,100 3,500 1,700 2.700 2,400 1,300 1,400 3,500

Stomach Cardia Body Pylorus Duodenum Ileum Colon Rectum

2.200 9,500 16,250 6,700 5,300 12,500 15,600

Methylene blue (spread preparations)

Methylene blue (spread preparations)

(4)

(rontinued)

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TABLE I (continued) Species

Region

Neurons

Method

49.08 I 15,41 I

Thionine (sections)"

Ileum

7,786

Thionine (sections)"

Guinea pig

Ileum Cecum Colon

14,210 9.290 35,570

Toluidine blue (spread preparations)"

Mouse

Ileum Cecum Large intestine

29,600 50,200 90,800

Toluidine blue (spread preparations)'

Cat

Duodenum

12.170

Bielschowski (tangential sections)'

Rabbit

Duodenum Mesentery Antimesentery Jejunum Mesentery Antimesentery Ileum Mesentery Antimesentery Cecum Rectum Mesentery Antimesentery Anus

2,940 2,840 2,640

Small intestine

9.405

Cat

Duodenum Ileum

Dog

Rat

Referenceh

Methylene blue 3,500 2,280

2.900 2,088 2,500 2 ,000 1.760

Tetrazolium stain (spread preparations)"

"Number of neurons per square centimeter of outer surface of the organ. "About 45,000 neurons were counted (from five cats). 'About 23.000 neurons were counted (from three dogs). "In this paper both the figures of 1421/mm' (and 929 and 3557) and 14,210/cmZ(and 9300 and 35600) are given. The counts were carried out on a total surface of 0.01 I cm2. "The actual figures given in this paper are 2960, 5012, and 9080/mm2.The counts were carried out on a total surface of 0.067 cm2. T h e counts were carried out on a total surface of 0.067 cm2. "The counts were canied out on a total surface of I 1 .60cm2 (from three rats). The standard deviation was 2677. hReferences: ( I ) Irwin (1931); (2) Matsuo (1934); (3) Ohkubo (1936b); (4) Ohkubo (1936~); (5) Sauer and Rumble (1946); (6) Filogamo and Vigliani (1954); (7) Tafuri (1957); (8) Tafuri and De Almeida Campos ( I 958); (9) Leaming and Cauna ( I 96 I); (10) Maslennikova (1962); ( I 1 ) Gabella ( 197 I ) .

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whether there is a true correlation between neuronal density and the thickness of the muscle coats (volume of the tissue innervated) as some of these figures suggest. In the guinea pig cecum the packing density of neurons is nearly three times greater in the myenteric plexus beneath the taeniae than in the rest of the organ (which is almost devoid of longitudinal musculature) (Irwin, 1931). In the case of the rat (9400 neurons/cd) histochemical staining has k e n carried out on the entire length of the small intestine, and it has therefore been possible to calculate that the total number of neurons in the myenteric plexus of the small intestine is about 1.85 million. The total number of enteric neurons in the whole gut is probably over three times as great. Sauer and Rumble (1946) calculated that in the intramural plexuses of the cat small intestine there could be as many as 6 million neurons. Fewer counts have been carried out on the submucosal plexus. This plexus has about 3600 and 4500 neuronskm', respectively, in the ileum and colon of' the guinea pig (Ohkubo, 1936a). The submucosal neurons are two or three times more numerous than the myenteric neurons in the cat intestine (Sauer and Rumble, 1946) and about half as numerous in the rat ileum (Gabella, 1976a).

VIII. Size of Neurons A feature of the neurons of the myenteric plexus is that their sizes span an extremely wide range (Fig. 1). A range of cell sizes is found in all autonatmic ganglia but is minute compared to that observed in myenteric ganglia (Fig. 4) (Gabella, 1971). In the rat cecum the cell surface of myenteric neurons ranges from approximately 100 pnz to well over 2000 pn'. The range is smaller in other portions of the alimentary tract but always covers one order of magnitude. Characteristically, in the ileum small neurons predominate over large ones, and they are relatively more numerous than in other parts of the gut. The significance of these wide ranges and regional differences is unknown, but they are in agreement with other indications that enteric ganglia contain neurons with different functional specializations. In the submucosal plexus ganglion cells are usually smaller and less heterogeneous in size than in the myenteric plexus (Ohkubo, 1936a; Gunn, 1968).

IX. Types of Neurons Attempts to classify enteric neurons morphologically, mainly on the basis of silver impregnation methods, have produced a vast literature (see review.s in Schofield, 1968; Gabella, 1976a). The best known classification is that proposed

141

INNERVATION OF THE GASTROINTESTINAL TRACT

0

100 200 300 400 500 600 700 800 Area of cell maximal profile (p?)

100 200 300 400 500 600 700 Area of cell maximal profile (,urn’)

D

100 200 300 400 500~x)7008009001000 Area of cell maximal profile (!ma)

loo

200 300 400 500 600 700 800 Area of cell maximal profile (itmy)

FIG. 4. Histograms of nerve cell sizes in the myenteric plexus of different segments of the alimentaly canal in the adult rat. The measurements of cell profiles were carried out on preparations stained as in Fig. 1. The total cell surface area is about twice these values. (A) Stomach (N = 1590). (B) Small intestine (N = 3100). (C) Cecum ( N = 2400). (D) Rectum ( N = 1930). The figures to the right of the histograms indicate the percentage of cells whose size exceeded the highest value on the abscissa. (From Gabella, 1971.)

by Dogie1 (1895,1899): type I neurons (motor neurons with one axon and many short dendrites), type I1 neurons (sensory neurons with long processes only), and type 111 neurons (with dendrites branching around other ganglion neurons). Dogiel’s classification has been confirmed and elaborated by some investigators (e.g., Stohr, 1930; Lawrentjew, 1931; Gunn, 1959, 1968), modified by some (e.g.. Hill, 1927), and denied by others (e.g., Kuntz, 1922; Schofield, 1968). Classifications have also been suggested based on the affinity and the lack of affinity of neurons for silver impregnation (Honjin et al., 1959), on cell size (Gunn, 1959, 1968; Feher and Vajda, 1972), or on the number of processes (unipolar, bipolar, and multipolar neurons) (Schofield, 1968). Unfortunately, silver impregnation methods are selective for a small number of neurons in the enteric ganglia (Fig. 5 ) ; they appear to be successful only in some species, often do not allow the axon to be distinguished from the dendrites, and can hardly allow a neuron to be identified as motor or sensory or as an intemeuron; only

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rarely do the impregnated neurons fit into clear-cut morphological categories (although undoubtedly some neurons conform to one of the three categories of Dogiel). Age and pathological conditions can affect the morphology as well as the number of enteric neurons (Cavazzana and Borsetto, 1948; Rintoul and Kirkman, 1961; Friese and Pearse, 1963; Smith, 1967; Gabella, 1976a; Eckardt and LeCompte, 1978). The problem of identifying types of neurons in the enteric plexuses remains obscure and obviously awaits clarification from a systematic electron microscope study. Very refined experiments with intracellular electrical recording have been carried out in order to classify enteric neurons on the basis of their electrical properties (Nishi and North, 1973; Hirst et al., 1974; Hirst and McKirdy, 1975). Since the same neurons used for recording can be injected with a dye (Nishi and North, 1973) (Fig. 3C), these experiments open up an interesting field in the study of intramural plexuses, but the results so far have shown little correlation between electrophysiological and morphological characteristics (Hodgkiss and Lees, 1978).

X. Histochemistry for Acetylcholinesterase

In the cat intestine the acetylcholinesterase activity (AChE) is intense in many neurons of the submucosal plexus and weak in those of the myenteric plexus (Taxi, 1965). Similarly, in the mouse 50% of the neurons in the submucosal plexus have intense activity, but only 5-10% in the myenteric plexus (Taxi, 1965). In the myenteric plexus of the rat ileum the vast majority (possibly all) of the neurons show AChE activity (Gunn, 1968), but the intensity varies considerably among neurons (Koelle, 1951; Learning and Cauna, 1961; Jacobowitz, 1965; Taxi, 1965; Van Driel and Drukker, 1973); in the colon activity is intense in about a quarter of the neurons (Donhoffer, 1958; Gunn, 1968). Similar continuous variations in AChE activity from intense to very low (or negative) occur in intramural ganglia of the pig (Gunn, 1968), ram (Ruckebusch and Santini, 1968), and cat (Kyosola ef ul., 1975). With this histochemical method a submucosal plexus cannot be identified in the stomach of the cat (Gunn, 1968) or the rat (Kyosola ef ul., 1975).

FIG. 5. Myenteric plexus of the cat intestine silver-impregnated with the Bielschowki-Gros method. ( A ) An elongated bipolar neuron. (B) A large bipolar neuron with two large bulbous projections from the perikaryon. At top center is a lightly inpregnated bipolar neuron. In this and in the other micrographs of this set the numerous dark, oval-shaped profiles are nuclei of glial cells. (C) Several multipolar neurons. (D)A multipolar neuron; the arrow indicates a process with spines. (E) A connecting strand of the plexus showing the vast number of glial cells present. Markers: (A) 50 p ; (B) 20 F .

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XI. Catecholamine Histochemistry A. MYENTERIC PLEXUS In the intestinal wall, freeze-dried and exposed to formaldehyde vapors [the method of Falck and Hillarp (Falck, 1962)], the myenteric plexus shows many fluorescent fibers with the histochemical characteristics of noradrenergic fibers (Norberg, 1964; Jacobowitz, 1965). Stretch preparations (longitudinal muscle and myenteric plexus stretched on glass slides and air-dried) display the plexus in foto, and many fluorescent varicosities can be clearly visualized in the ganglia and the connecting strands of the plexus (Gabella and Costa, 1967; Read and Bumstock, 1968a; Costa and Gabella, 1971; Furness and Costa, 1974) (Fig. 6). The stretch preparation technique [which is a modification of the method used by Ambache (1954) for pharmacological studies in v i m ] is rapid and beautifully displays the pattern of fluorescent fibers over wide surfaces (1 cm2 or more); however, whereas it can be easily applied to some organs, such as the rabbit and guinea pig intestine, it is difficult to use with others, such as the intestine of the chicken, rat, and cat. It seems that the longitudinal musculature of the guinea pig ileum has the ideal thickness and strength, and that a natural cleavage plane occurs between the circular and longitudinal muscle layers. Apart from a few exceptions (see below) the neurons of the myenteric plexus do not display formol-induced fluorescence, all the adrenergic fibers being therefore of extrinsic (or extramural) origin. The neuronal cell bodies of the plexus are identified as large, nonfluorescent areas among the numerous fluorescent varicosities and can be visualized by a counterstain. (It should be mentioned that diffusion of the fluorophore around a cluster of varicosities produces a floorescent patch, and that this artifact may be interpreted as a fluorescent cell body). Fluorescent fibers are varicose, with faint or no fluorescence in the intervaricose portions (Fig. 6B and C). The beaded appearance of individual fibers is clear in the connecting strands of the plexus, where some fibers can be followed for a considerable length across ganglia and along several strands; most varicosities are uniform in size and spacing, but some very large varicosities are found (Fig. 6B). Inside the ganglia the varicosities are arranged in such 81 way that the course and branching of individual fibers can hardly be recognized (Fig. 6A). Varicosities appear over the entire ganglion, but occasionally there are denser clusters associated with some ganglion cells. The fluorescent varicosities

FIG. 6. Fluorescence microscopy for catecholamines. Stretch preparations of longitudinal muscle of the guinea pig ileum with the myenteric plexus attached. (A) A ganglion of the plexus. Marker: 100 pin. (B) A connecting strand showing a fluorescent axon with unusually large varicosities. Marker: 20 p n . (C) Varicose fiber5 of tertiary meshes of the plexus. Marker: 20 p .

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in the myenteric ganglia are not of uniform size, and many of them are larger than those found, for example, within the muscle coat. The emission speclra of these structures, studied cytofluorometrically, are entirely due to norepinephrine, and 5-hydroxytryptamine (5-HT) is absent (Ahlman and Enerback, 1974). The distribution of adrenergic fibers, as seen in fluorescence microscopy, appears rather uniform in the myenteric ganglia of various parts of the alimentary tract (Costa and Gabella, 1971). Norepinephrine has been detected by quantitative fluorometric studies in various parts of the gut (for a review, see Holzbauer and Sharman, 1972). BiochLemical studies on the myenteric plexus of the guinea pig have been carried out after separating the longitudinal musculature (with the plexus attached) from the: rest of the wall (Fig. 7). The concentration of norepinephrine ranges between 0.56 (ileum) and 1.3 (colon) p d g m fresh tissue (Juorio and Gabella, 1974). It is lowest in the middle part of the gut (ileum) and increases toward both ends. In the ileum the norepinephrine concentration in the myenteric plexus-longitudinal muscle is similar to that of the rest of the wall (0.53 pg/gm); since the latter component represents 83% by weight of the intestinal wall, it follows that only about one-sixth of the total norepinephrine content is in the myenteric plexus. If the absence of intramural adrenergic (fluorescent) neurons is the rule, few exceptions are known. The myenteric plexus of the guinea pig colon contains

0

bngitudinol muscl.

fl

cirrulor musclo

- mymntwic

pl*xus

- submucous pl*xus - submucosa - murosa

stomach duodenum

ileum

cecum (taenia)

colon

rectum

FIG. 7. Distribution of norepinephrine in the longitudinal muscle-myenteric plexus and circular muscle-subrnucosal plexus-submucosa-mucosa of different regions of the guinea pig a1irnc:ntary canal. Values are in micrograms per gram of fresh tissue k S E M . (Data from Juorio and Gabella, 1974.)

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some adrenergic ganglion cells (Costa et al., 1971). They represent about 1% of the total neuronal population of the plexus, show a wide range of fluorescence intensities, and have one to four processes which are also fluorescent and can be followed for several microns until they become lost among the great number of fluorescent fibers in the ganglia (Furness and Costa, 1971). These cell processes are initially smooth but become varicose some distance from the cell body; some fibers penetrate into the circular muscle layer (Furness and Costa, 1971). After extrinsic denervation only about 40% of the fluorescent endings disappear; the remaining ones therefore originate from the intrinsic adrenergic neurons. In agreement with these histochemical results, the norepinephrine content of the myenteric plexus of the colon is more than twice that of the ileum, and about a quarter of it is not depleted by extrinsic denervation (Gabella and Juorio, 1975). The intrinsic adrenergic neurons of the colon have not been identified in the electron microscope; their pharmacological properties have been investigated by Costa and Furness (1971). An exceedingly small number of fluorescent neurons is present in the myenteric plexus of the rectum (Furness and Costa, 1973). Intramural fluorescent neurons have been described in the myenteric plexus of the rat ileum (Oosaki and Sugai, 1974) but, as in the case of some of the earliest reports (Tafuri and Raick, 1964; Hollands and Vanov, 1965), the evidence is not completely convincing. The fluorescent cells associated with intramural plexuses at the choledochoduodenal junction (Kyosola and Rechardt, 1973) are probably nonneuronal cells (Kyosola, 1976). Fluorescent neurons in the myenteric plexus have been observed in the gizzard of the chick (Bennett et a / . , 1973) and in the large intestine of the lizard (Read and Burnstock, 1968a). B. SUBMUCOSAL PLEXUS

Numerous fluorescent varicose fibers are distributed around the ganglion cells and in the connecting strands of the submucosal plexus (Norberg, 1964; Jacobowitz, 1965; Gabella and Costa, 1967; Read and Burnstock, 1968a). The varicosities are usually finer and more uniform than those of the myenteric plexus (Costa and Gabella, 1971). When two plexuses can be distinguished in the submucosa (Section VI), they are both supplied by fluorescent fibers (Fumess and Costa, 1973). In the stomach of the rat (Muryobayashi et a / . , 1968), cat, guinea pig, and rat (Furness and Costa, 1974) the adrenergic fibers of the submucosa do not appear to be associated with a submucosal plexus. There are no fluorescent ganglion cells in the submucosal plexus in all the organs studied (including the colon of the guinea pig; see Section XI,A), but Krokhina (1973) observed a few fluorescent (adrenergic) submucosal neurons in the pyloric region of the cat stomach. [It has been reported that after extrinsic denervation not all the fluorescent fibers of the submucosal plexus in the guinea pig ileum disappear (Boyle et a / . , 1977).]

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C. MUSCLECOAT In addition to the fibers within the myenteric plexus, in the muscle coat (muscularis externa) of the intestinal wall there is a small number of adrenergic fibers directly supplying the muscle. They have been seen in fluorescence microscopy in the circular muscle layer of the gastrointestinal tract of all the species investigated (Gabella and Costa, 1967; Fumess, 1970; Silva et al., 1971; Costa and Gabella, 1971). The fluorescent fibers run approximately parallel to the muscle cells and have a fairly regular varicose shape; some are presumably perivascular, but many are associated directly with the muscle. There are few quantitative data on the density of adrenergic innervation in different muscles, organs, and animal species. Gillespie and Maxwell (1971) counted fewer adrenergic fibers (across the entire wall) in the rat jejunum than in the duodenum, and fewer still in the ileum and cecum; the colon and rectum have fewer fibers than the cecum, and the stomach has about the same number as the jejunum. In the small intestine there are fewer fluorescent fibers in the longituldinal muscle than in the circular muscle (cat, Silva et al., 1971; rabbit, Costal and Gabella, 1971); they are usually absent in the thin longitudinal musculature of the ileum, colon, and stomach in the rat and guinea pig, but some are found in the duodenum (Costa and Gabella, 1971). Adrenergic fibers are present in the guinea pig taenia coli (Aberg and Eranko, 1967; Bennett and Rogers, 1967); characteristically, these varicose fibers, when visualized in stretch preparations, do not lie strictly parallel to the muscle cells (Costa and Gabella, 1971). A high density of intramuscular adrenergic fibers occurs at certain junctions between segments of the alimentary tract (which are regarded as sphincters by some investigators). At the gastroesophageal junction (the cardia or lower esophageal sphincter) of the guinea pig, the muscle layer contains far more adrenergic fibers than the adjacent regions of the stomach and esophagus (Gabella and Costa, 1968a) (Fig. 8A); however, in the rat the adrenergic innervation of this junction does not differ from that of the adjacent areas of the stomach (but it is twice as dense as in the adjacent parts of the esophagus) (Gillespii: and Maxwell, 1971), and in the cat and monkey the circular muscle of the lower part of the esophagus has only sparse adrenergic innervation (Baumgarten and Lange, 1969). At the junction between the pylorus and duodenum in the rat the circular

FIG.8. (A and B) Fluorescence microscopy for catecholamines. (A) Musculature of the cardia of the guinea pig (section). Rich adrenergic innervation. (B) Mucosa of the stomach of the rabbit (section) (from Costa and Gabella, 1971). To the left is the muscularis mucosae, and to the right are the gastric glands with many intensely fluorescent chromaffin cells. (C) Autoradiography of the guinea pig ileum after incubation in norepinephrine-3HH. Silver grains outline the myenteric ganglia. There are also some silver grains in a submucosal ganglion (arrows), around blood vessels of the submucosa, and in the innermost part of the circular musculature. Marker: 100 pin.

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musculature is thickened, and within a band about 1 mm wide it shows dense uniform adrenergic innervation; the band has clear-cut boundaries, and the iidjacent areas of the pylorus and duodenum have only few adrenergic fibers (Gillespie and Maxwell, 1971). There is a similar arrangement in the guinea pig (Costa and Gabella, 1971). On the other hand, the same groups of investigators found no special adrenergic innervation at the ileocolonic junction in rats and guinea pigs, but Furness and Costa (1974) report that in rats and rabbits the ileocolonic sphincter is more densely innervated than the adjacent nonsphincter muscle. In the cat there are a few fluorescent (adrenergic) fibers in the longitudinal muscle of most of the colon, and along the rectum there is an increasing number of adrenergic fibers; in the circular muscle the increase occurs only from the middle part of the rectum, but in the internal anal sphincter the intramuscular adrenergic innervation is particularly rich (Howard and Garrett, 1973). A similar arrangement exists in the guinea pig (Costa and Gabella, 1971) and probably in humans (Baumgarten, 1967). In the guinea pig adrenergic fibers are more abundant in the circular muscle of the rectum than in the colon, and they increase in number toward the anal sphincter (Gabella and Costa, 1968a; Costa and Gabella, 1971; Furness and Costa, 1973). In the anal sphincter they form an extremely dense network with myriad fluorescent fibers running in several directions (whereas in the rectum and in most other parts of the alimentary tract they mainly run parallel to the muscle cells). A similar but less remarkable increase occurs in the longitudinal musculature of the rectum. Most of the adrenergic fibers to the rectal musculature and plexuses (in the guinea pig) originate from the inferior mesenteric ganglion; fibers for the blood vessels of the rectum arise from the posterior division of the pelvic plexus; some of the adrenergic fibers to the internal anal sphincter originate from the sacral sympathetic chain (Costa and Furness, 1973).

D. MUCOSA Fluorescent varicose fibers from the submucosa pierce the muscularis mucosae and branch in the basal part of the tonaca propria of the mucosa (Norberg, 1'364, 1967; Jacobowitz, 1965; Baumgarten, 1967; Gabella and Costa, 1968b; Read and Burnstock, 1968b; Silva et al., 1971). A few fluorescent fibers run parallel to the muscle cells of the muscularis mucosae, particularly where this muscle is thicker, as at the gastroesophageal junction and the anal canal (Baumgarten and Lange, 1969; Costa and Gabella, 1971). Most of the fluorescent fibers foim a network in the proximity of the basal parts of the glands and the blood vessels of this region of the wall of the stomach and intestine (Fig. 8B). Few fibers are directed toward the lumenal surface of the wall, running parallel to the glands of the stomach and rectum and within the axis of the villi of the small intestine.

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E. BLOODVESSELS The superior and inferior mesenteric arteries and the coeliac artery are richly supplied with fluorescent fibers running in the adventitia and in the outer third of the media (Mohri et al., 1969; Fumess, 1971). The density of innervation increases toward the intestine. All the small mesenteric veins are accompanied by adrenergic fibers, but these are less abundant than near the corresponding arteries. The density of innervation is greater in large veins but remains much less than that of arteries (Fumes, 1971). Arteries within the wall of the stomach and intestine are richly innervated by fluorescent fibers; these follow the arteries, forming a tightly meshed network in their adventitia, and can be traced down to the small arterioles (Fumess, 1971). Only near the smaller vessels do the fluorescent fibers acquire the characteristic varicose appearance. The veins of the wall of the gut are more sparsely innervated than the arteries; adrenergic fibers are generally confined to veins over 100 pm in diameter (Fumess, 1971). Blood capillaries and intramural lymphatic vessels do not have adrenergic innervation.

XII. Serotoninergic Neurons The interesting hypothesis that in the myenteric plexus there are serotoninergic neurons (transmitting by means of 5-HT) has been proposed and investigated by Gershon and collaborators. They confirm that in normal preparations all fluorescence is due to norepinephrine and not to 5-HT; however, by pooling myenteric plexus and longitudinal musculature from the entire small intestine of six guinea pigs a minute amount of 5-HT (81 ng/gm of wet weight) is detected (Robinson and Gershon, 1971). This result is confirmed by Juorio and Gabella (1974), whereas in the rest of the wall, on account of the large number of enterochromaffin cells, the concentration of 5-HT is almost 100 times higher (Benditt and Wong, 1957). A yellow fluorescence (presumably representing 5-HT) appears in neurons of the guinea pig myenteric plexus after treatment with reserpine, amethyl-p-tyrosine and a monoamine oxidase inhibitor, a result suggesting synthesis of 5-HT in the plexus (Robinson and Gershon, 1971). It is known that exogenous 5-HT and 5-hydroxytryptophan can be taken up in vitro also by adrenergic axons (Owman, 1964; Snipes et al., 1968); however, in the myenteric plexus of the rat and guinea pig intestine it is possible that other cellular elements (the hypothetical serotoninergic neurons) account for this uptake (Gershon et al., 1965, 1976; Robinson and Gershon, 1971). [However, by electron microscope. autoradiography, Taxi and Droz (1966) found a large uptake of norepinephrine but no uptake of 5-hydroxytryptophan in the rat intestine; negative results with the uptake of tryptophan are reported also by Dubois and Jacobowitz (1974).]

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Gershon and collaborators conclude that the myenteric plexus (of the mouse and guinea pig) contains a unique type of neuron, not found elsewhere in the peripheral nervous system, which uses 5-HT as a transmitter (Robinson and Gershon, 1971). These neurons (which, it is suggested, contain 5-HT in ii histochemically nondetectable amount) are intramural, since uptake of 5-HT (but not of norepinephrine) persists in organotypic cultures of mouse fetal intestine (Dreyfus et al., 1977a); and the serotoninergic axons are confined to the plexus, since no uptake is found in the longitudinal muscle. Other indirect evidence supporting this hypothesis is provided by developmental and organ culture studies (Rothman et al., 1976: Dreyfus et af., 1977b). Moreover, some intramural neurons can be immunochemically stained for tryptophan hydroxylase (the enzyme catalyzing the first step in the synthesis of 5-HT from tryptophan, which is used as a marker for serotoninergic neurons) (Gershon et uf., 1077). Wood and Mayer (1978), by recording intracellularly from myenteric neurons of the guinea pig small intestine, showed that 5-HT may be the transmitter involved in the production of the excitatory postsynaptic potential which can be evoked by electrical stimulation of an interganglionic strand. Finally, a considerable number of neurons in the submucosal plexus contain the enzyme aromatic 1-amino-acid decarboxylase (Costa et uf., 1976; Furness and Costa, 1978), surgical or chemical denervation abolishes all adrenergic fibers and then admin istration (in vitro) of a monoamine oxidase inhibitor and of a 5-HT precursor ( 1 dopa, which is not fluorescent) produces intense fluorescence in several ganglion cells of the submucosal plexus (and in a very few of the myenteric plexus), a result which indicates the presence of a decarboxylating enzyme. Fluorescence, however, also appears in some glial cells of the submucoal plexus and in some interstitial cells.

XIII. Immunofluorescence Histochemistry Antibodies raised against peptides and labeled with a fluorescent compound have provided a highly specific histochemical tool for studying enterochromaffin cells. Various “intestinal hormones” have been localized in these cells in addition to amines, and over 10 different types of cells have been recognized. More recently it has been found that some of the peptides isolated from the mammalian gut are localized in autonomic nervous structures and in the central nervous system. The problem is particularly important, since there is physiological evidence that other transmitters in addition to acetylcholine and norepinephrine are present in the enteric plexuses (Section XXVI). Substance P (von Euler and Gaddum, 1931) is an undecapeptide isolated from the hypothalamus (Chang et al., 1971). Substance P immunoreactivity (or substance P-like immunoreactivity) is localized not only in endocrine cells of the

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intestinal mucosa identified as enterochromaffin cells (Heitz et al., 1976), but also in intramural nerves [the myenteric plexus and the circular muscle layer of the mouse proximal colon, but not in the duodenum (Nilsson et al., 1975)] and in neurons and processes of the myenteric and submucosal plexuses in mammals (Pearse and Polak, 1975). The amount of substance P in aganglionic parts of the large intestine (in Hirschprung’s disease) is lower than in the controls, suggesting a correlation between substance P and ganglion cells (Ehrenpreis and Pernow, 1952; Pernow, 1953); based on an electron microscope study on similar material it has been suggested that there is a correlation between substance P and the granular vesicles of the intramural nerve endings (Tafuri et al., 1974). It has been proposed that in the spinal cord and dorsal root ganglia substance P is a transmitter in sensory pathways (Lembeck, 1953), a hypothesis which is supported by the high concentration of the peptide in the dorsal roots (Otsuka et al., 1972) and by its immunofluorescence localization (Hokfelt et al., 1975a). Another polypeptide isolated in large amounts from the intestine and brain is vasoactive intestinal peptide (VIP); it is composed of 28 amino acids (Said and Mutt, 1970). VIP immunoreactivity is localized in varicosities of the myenteric plexus and in some nerve fibers in the mucosa of humans (Bryant ef al., 1976). Immunoreactivity is found also in certain intramural neuronal cell bodies and the VIP-fibers are therefore considered to be entirely or mainly of intrinsic origin (Bryant et al., 1976; Larsson et al., 1976; Fuxe et al., 1977). Enkephalins are recently discovered peptides which have a high affinity for opiate receptors (Hughes, 1975; Hughes et al., 1975) and are present in large amounts in the gastrointestinal tract (Hughes et al., 1977). In the rat myenteric plexus (especially in the colon) there are varicose fibers labeled with antibodies to the peptide leucine-enkephalin (Elde et al., 1976), an interesting observations in view of the presence of specific opiate receptors in guinea pig myenteric neurons (Kosterlitz et al., 1972). Tritiated morphine is specifically taken up by glial cells of the guinea pig myenteric plexus (Diab et al., 1976). Somatostatin (a 1Camino acid peptide isolated from the hypothalamus; Brazeau et al., 1973) is abundant in the gut (Arimura et ul., 1975) and has been localized in varicose fibers of the myenteric and submucosal plexuses (Hokfelt et al., 1975b; Costa et al., 1977). Enkephalin-like immunoreactivity has also been found in the myenteric plexus of the human gut (Polak et al., 1977). Organ bath experiments suggest that somatostatin inhibits neuronal release of cholinergic and adrenergic transmitter substances in smooth muscles (Cohen et al., 1978). In organotypic tissue culture of small intestine from mouse embryos numerous VIP- , enkephalin- , substance P- and some somatostatin-immunoreactive fibers are found (Schultzberg et al., 1978). Such fibers, since they occur in the absence of the extrinsic innervation to the gut, must originate from neurons intrinsic to the intestinal wall. In ganglia of the myenteric plexus (from the taenia coli of newborn guinea pigs) grown in vitro there is a population of neurons with high

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specific uptake activity for GABA (y-amino-butyric acid) (Iessen, 1978, personal communication). Although it is not a immunohistochemical method, the technique of Olson et al. (1976) is mentioned in this section because it involves localization of another putative transmitter in the intestine. The fluorescent compound quinacrine, an acridine derivative with antimalarial activity, binds to some neurons of the m,yenteric plexus and to some varicose fibers. It also stains adrenomedullary cells, but not the nerves of the iris (which are all cholinergic or adrenergic). It is possible that quinacrine stains a specific population of gastrointestinal fibers which are neither cholinergic nor adrenergic (Olson et ul., 1976). Since quinacrine has an affinity for ATP and for several structures rich in ATP, this histochemical method may detect neurons and fibers rich in ATP or a purine compound (“purinergic fibers”) (Bumstock, 1975).

XIV. Ultrastructure of the Myenteric Plexus Fine-structural studies of the myenteric plexus are relatively few when the complexity and variability of the plexus is considered. Few species have been investigated (Richardson, 1958; Taxi, 1958, 1959, 1961, 1965; Baumgarten et al., 1970; Gabella, 1972a; Cook and Burnstock, 1976a; Yamamoto, 1977). A striking feature of the plexus in the electron microscope is that its components (nerve cells, glial cells, and glial and neuronal processes) are tightly packed together, in a manner similar to that found in the central nervous system (Figs. 9- 13). In other autonomic ganglia, individual neurons with their satellite cells and incoming nerve fibers are sheathed by collagen, and fibroblasts, mast cells, and capillaries are present among neurons. On the other hand, in the intramural ganglia of the intestine there is connective tissue around the ganglia and strands (Section XVII), but this (in adult animals) does not penetrate within the ganglia or within bundles of nerve fibers. The gap between adjacent structures in intramural ganglia is a space only 15-20 nm wide (Fig. 9A and C). Tracers such as peroxidase readily penetrate into and completely fill this space (Jacobs, 1977). During mechanical activity of the intestinal wall the shape of ganglia is grosdy altered, and there are therefore extensive changes in the shape of cell bodies iind

FIG. 9. Myenteric plexus of the guinea pig ileum. (A) A neuron with a large dendrite lying immediately beneath the basal lamina. Marker: 1 pm. ( 8 ) A process of a glial cell which form!;an expansion at the surface of the ganglion. Note the gliofilaments and the patches of electron-dense material beneath the surface membrane. Marker: I pm. (C) The arrows indicate vesicle-containing axons with clusters of vesicles and membrane specializations directly apposed to processes of a glial cell. Marker: 0 . 5 p m . c , Collagen fibrils; g, glial cell; n, neuron; i , interstitial cell.

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processes. Whether there is also some sliding of the various structures past each other is not known. The perikarya appear very heterogeneous in size, and also their fine structure varies greatly. There is, however, not enough evidence to attempt a nerve cell classification on this basis. In the myenteric plexus of the stomach, ileum, cecum, and colon of the guinea pig, Cook and Bumstock (1976a) describe eight types of neurons: neurons with processes accompanied by satellite cells, small (10-12 pm) granular neurons, elongate (10 x 20 pm) electron-dense neurons, medium-sized (20-25 pm) neurons with rough endoplasmic reticulum, medium-sized (20-25 p m ) neurons with smooth endoplasmic reticulum, medium to large (30 p m ) neurons with distinctive mitochondria, large (35 pm) neurons with many small mitochondria, and neurons containing numerous granular vesicles. Other investigators have noted the remarkable variability of the appearance of the cytoplasm of myenteric neurons; mitochondria, microtubules, neurofilaments, lysosomes, stacks of rough endoplasmic reticulum, and large granular vesicles (LGVs) can be highly developed or sparsely distributed. These data, together with the cell size (however difficult it may be to estimate cell size on sectioned material) and data on the cell’s processes, should provide the basis for subdividing this highly heterogeneous population of neurons. Fine-structural information on cell processes is meager (Gabella, 1972a). In the guinea pig ileum some myenteric ganglion cells have large dendrites which appear as a continuation in shape and structure of the cell body (Fig. 9A); other dendrites, which originate abruptly from the cell body, mainly contain microtubules and neurofilaments, together with rough endoplasmic reticulum, ribosomes, and granular vesicles. Some dendrites contain clusters of LGVs along their length (Fig. 12A). Cell bodies and large dendrites can be perfectly smoothsurfaced, but some have small fingerlike evaginations of their surface, with an amorphous content and no organelles (somatic and dendritic spines), usually bearing a synapse (Gabella, 1972a) (dendritic spines are occasionally seen also in silver-impregnated neurons; Fig. 5D). Undoubtedly, intramural neurons possess axons, but their origin from the cell body has not been documented with electron microscopy; this is partly because of sampling problems and partly because some axons may not originate from the soma but rather from dendrites, at some distance from the soma (but even the latter possibility has not been documented). In the neuropil (the dense network of

FIG. 10. Myenteric plexus of the guinea pig ileum. (A) Nerve ending with small AGVs synapsing on a dendrite. Note the prominent dense projections. Marker: 0.5 p n . (B) Nerve ending with LGVs synapsing on a dendrite. Note that in the immediately prejunctional region the ending contains mainly small AGVs. Marker: 0.5 gm. (C) Nerve ending with small AGVs and many granular vesicles of heterogeneous appearance (HGVs) synapsing on a perikaryon. Marker: 0.5 pm.

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Fic. 1 1 . (A) Nerve endings on the myenteric plexus of the guinea pig ileum, containing a large number of SGVs synapsing on an intramural neuron. Marker: 0.2 p n . (B)Nerve endings of the submucosal plexus of the guinea pig ileum after incubation with 5-hydroxydopamine. The endings contain mainly SGVs synapsing on a small dendrite. Marker: 0.5 p m . (C) A nerve ending of different morphology synapsing on a submucosal neuron of the guinea pig ileum. Marker: 0.5 pm.

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various cellular processes originating from neurons and glial cells) there are many profiles of typical axonal appearance (round in shape, containing mainly microtubules and a few neurofilaments and vesicles), but what the axons look like in their initial segments is not known. A remarkable characteristic of the myenteric plexus (in the guinea pig, rat, rabbit, and sheep) is that many ganglion cell somata and many large dendrites have parts of their surface not covered by glial cells or neuronal processes but directly exposed to the ganglion surface and lying immediately underneath the basal lamina (Gabella, 1972a) (Figs. 9A and 13). At this level the neuronal membrane is smooth and shows no structural specialization, but in the cytoplasm immediately underneath there is often a band of microfilamentous material. Some neurons have parts of their surface exposed to both surfaces of the plexus, which face the two muscle layers.

XV. Nerve Endings and Synapses in the Myenteric Plexus Nerve profiles containing large numbers of vesicles are abundant in the myenteric plexus (Taxi, 1958, 1959, 1965; Tafuri, 1964), but classification and identification of their tramsmitters has just begun. Naturally, this kind of analysis presupposes that there is a correlation between axonal structure and type of transmitter; but it should be borne in mind that to some extent this remains just an assumption and that such a correlation may turn out to be less good than expected. Baumgarten el u / . (1970) describe three types of nerve profiles in the myenteric plexus of the large intestine of the human, rhesus monkey, and guinea pig. One type, characterized by numerous electron-lucent vesicles (35-60 nm in diameter) and a few large (80-110 nm) vesicles with a granule of medium electron density, is identified as preterminal and terminal varicosities of cholinergic fibers; in the latter case the nerve endings can be in synaptic contact with ganglion neurons. A second type of profile, far more numerous in humans and monkeys than in guinea pigs, is characterized by vesicles (50-90 nm in diameter and spherical, elongated, or dumbbell-shaped) with a strongly osmiophilic granule accompanied by an equal number of agranular vesicles (AGVs) (40-60 nm) and a few large vesicles (90-130 nm) with a granule of medium electron density. These endings are identified as adrenergic (they show increased electron density after 5-hydroxydopamine treatment and degenerate after 6-hydroxydopamine treatment; see Section XXIII) and are never found to form synaptic contacts. A third type of nerve profile, which is the most often encountered, contain in addition to electron-lucent vesicles (40-60 nm in diameter) vesicles 85- 160 nm in diameter with a large granule of medium, nonuniform electron density sometimes not clearly separated from the vesicle membrane. In

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the terminal varicosities (but not in the pretenninal ones) the membrane of these large vesicles is often incomplete. These profiles are called p-type varicosities (“p” for “polypeptide,” because of the similarity of the granular vesicles to those found in neurosecretory neurons, as originally suggested by Taxi, 1965). In the myenteric plexus of the guinea pig ileum many vesicle-containing nerve profiles synapse on the soma and dendrites of ganglion neurons and, on the basis of vesicle morphology, several types have been tentatively identified (Gabella, 1972a). Some nerve profiles contain exclusively or predominantly electron-clear AGVs 40-60 nm in diameter; a few LGVs are often present in these endings which are presumably cholinergic. The synaptic specializations vary from a moderate thickening of pre- and post-synaptic membranes to prominent dense projections with a clustering of vesicles (Fig. 10A; see also Fig. 12A from the sheep ileum). Other nerve profiles mainly contain small granular vesicles (SGVs) 40-60 nm in diameter and having a highly electron-dense granule. Often the inner leaflet of the vesicular membrane is more electron-dense than the membrane of nongranular vesicles. With the technique used preservation of the dense cores is somewhat inconsistent. This type of ending is presumably adrenergic, and it has the characteristic pre- and postsynaptic specializations (50-60% of them are said to synapse on the soma or on somatic spines) (Fig. 11A). A third type of synapsing nerve profile is characterized by granular vesicles 90-140 nm in diameter having a dense core of variable appearance (they are called heterogeneous granular vesicles, HGVs) (Fig. 1OC). The cores have medium electron density, somewhat lighter in the larger vesicles, which also often display an incomplete vesicular membrane [similar vesicles are described by Baumgarten et al. (1970) in their p-type nerve profiles]. Mitochondria, microtubules, and many small AGVs are also present. Characteristically, in the region of the ending near the presynaptic membrane only SGVs are found, and granular vesicles are generally not observed closer than 200 nm to the presynaptic membrane. These nerve endings are also present in the duodenum, cecum, and colon; at least in the small intestine, they are of intrinsic origin, since they are not affected by extrinsic denervation. A fourth type of nerve profile (rare in the small intestine) synapsing on intramural neurons contains LGVs about 90- 150 nm in diameter having intensely electron-dense cores and a clear halo. Here again the region immediately adjacent to the presynaptic membrane contains only small AGVs (Fig. 10B). There are also a few nerve endings containing mainly flat AGVs FIG. 12. ( A ) Myenteric plexus of the sheep ileum. Nerve endings with small AGVs synapsing on a large dendrite which contains a few LGVs. Marker: 0.5 pn. (B) Myenteric plexus o f the sheep ileum. A large nerve profile at the surface o f the plexus has synaptic contacts with a dendrite. The ending is packed with small AGVs, but it also shows an area rich in LGVs. c , Collagen fibrils. Marker: I pm. (C) Myenteric plexus of the guinea pig colon. A nerve profile synapsing on a ganglion neuron contains vesicles which appear different from those in Figs. 10 and 1 1 . Marker: 0.5 pn.

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FIG. 13. Myenteric ganglion of the guinea pig ileum with circular musculature in transverse section. A smooth muscle cell of the longitudinal layer has a conspicuous process which reaches the surface of the ganglion. The arrow points to a vesiclecontaining nerve ending lying immediately beneath the basal lamina of the ganglion. c, Collagen fibrils; n, neuron; g, glial cell; m, smooth muscle cell. Marker: 2 p n . (reproduced from Gabella, 1972b).

(measuring 50-70 nm across) and synapsing on somatic spines. Whether these disk-shaped vesicles originate from the flattening of round vesicles during fixation with glutaraldehyde (as has been shown to occur in other tissues; Walberg, 1966; Valdivia, 1970) is not known, but it has been argued that even as artifacts these vesicles can still permit identification of a separate type of ending. Neuirons have been found which receive over a limited area of their surface synapses from three different types of endings. All the junctions described above (synapses; for

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junctions with glial cells see Section XVI) are areas of morphological specialization; this of course is not the same as evidence of functional transmission. Many vesicle-containing profiles do not show synaptic specializations or do not even contact a neuronal structure; most of them are due to an unfavorable plane of section, but there are some varicosities which do not form specialized contacts. Analysis of serial sections should clarify this problem and provide a quantitative answer. All the nerve profiles described are occasionally found connected to two thin axonal portions containing mainly microtubules and are therefore varicosities (although they may have a rather irregular shape); but how many of the synaptic endings are varicosities and form synapses en passage and how many are true endings has not been ascertained. Different vesicle-containing varicosities can lie side by side, and they sometimes show symmetric membrane thickenings along the region of apposition, but there is no good morphological evidence of axo-axonic synapses or of synapses on presynaptic endings. Cook and Burnstock (1976a), studying the myenteric plexus of stomach, ileum, cecum, and colon of the guinea pig found eight different types of nerve profiles, based on the appearance of the vesicle present. Some of the nerve types illustrated form a synaptic contact with ganglion cell bodies or dendrites. In the myenteric plexus of the human intestine a “presynaptic axon” of a presumptive adrenergic fiber (Pick, 1967) and presumptive cholinergic axo-somatic and axoaxonic synapses have been seen (Van der Zypen, 1967). Yamamoto (1977) describes four types of nerve profiles in the myenteric plexus (and in the muscle coat) of the small intestine of the bat and mouse: (1) profiles with larger dense-cored vesicles (140-160 nm in diameter) having a core of medium electron density and often without a clear-cut halo between the core and membrane, (2) profiles with large dense-cored vesicles (80-130 nm in diameter) with a core slightly denser than that in the previous type and a clear halo between the core and membrane, (3) profiles with small AGVs (40-60 nm in diameter) which are regarded as cholinergic endings (they are the only type seen to make axo-somatic and axo-dendritic synaptic contacts), and (4) profiles with AGVs 50-70 nm in diameter. The last type of profile is regarded as adrenergic, in spite of the absence of dense cores in the small vesicles (which is attributed to a fixation artifact); the identification is based on the size of the vesicles, since it has sometimes been observed that SGVs (adrenergic) are slightly larger than AGVs (cholinergic) (Honjin et a l . , 1965; Baumgarten et al., 1970; Kyosola and Rechardt, 1975). After fixation with permangante [a procedure which is elective for the detection of adrenergic vesicles (Richardson, 1966)] profiles containing SGVs are seen in the myenteric plexus of the cat small intestine (Feher et a l . , 1974). Some nerve profiles in the myenteric plexus of the guinea pig ileum lie at the surface of the ganglia or of strands, immediately underneath the basal lamina (Gabella, 1972a). Most of these profiles contain mainly small AGVs (Figs. 13

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and 14A); the rest mainly contain flat AGVs (Fig. 14C), and occasionally the two types occur side by side. At the free surface of these profiles the membrane often has dense projections and clusters of vesicles, an arrangement suggesting that they may be points of release of transmitters. In conclusion, correlating vesicle morphology and type of nerve ending in intramural plexuses is a difficult problem, and the available classifications should be regarded as tentative. In some animal species other than those mentioned the characterization of nerve ending types on the basis of vesicle morphology may turn out to be even more difficult than in the guinea pig. A striking feature of nerve endings in the enteric ganglia is the abundance of LGVs varying in size, electron density of the granule, and extent of the electron-lucent halo (structural aspects which are closely related to the preparative procedure) (Fig. 12B and C). As mentioned, LGVs can also be found in dendrites (Fig. 12A), and to a lesser extent in glial and interstitial cells.

XVI. Glial Cells The myenteric plexus contains many glial cells, and in the laboratory animals investigated they greatly outnumber the nerve cells (Gabella, 1972a; Yamamoto, 1977). They are readily indentified in the electron microscope by their elongated nuclei, rich in chromatin, and by the appearance of their cytoplasm (Figs. 9 and 13). Cook and Burnstock (1976b) argue that these cells should not be called glial cells, and they distinguish two cell types within this group: Schwann cells, enveloping many unmyelinated axons of the ganglia, and satellite cells, in close association with neuronal perikarya. Other investigators have observed structural variations in the glial cells of the plexus, but they have not distinguished different cell types. Some glial cells form an extensive coat surrounding a ganglion cell and also have processes wrapping around bundles of axons (Gabella, 1972a). Glial cells have an extremely irregular shape and many laminar processes which may extend a considerable distance from the cell body. They often have a centriole, and cilia are not uncommon (cilia have only rarely been seen in myenteric neurons). Filaments 10 nm in diameter (gliofilaments) are abundant in many glial cells and their processes (Fig. 9B). Some of the many processes of

FIG. 14. (A) A myenteric ganglion of the guinea pig ileum. A nerve ending situated at the surface of the ganglion is packed with small AGVs. Marker: 0.5 pn. ( B ) Longitudinal muscle of the guinea pig duodenum. A small nerve bundle with an AGV-containing varicosity lying close. to two muscle cells. Marker: 0.5 p n . (C) Myenteric ganglion of the guinea pig ileum. A nerve ending situated at the surface of the ganglion contains disk-shaped vesicles. Marker: 0.5 pn. (D) Guinea pig taenia coli incubated with 5-hydroxydopamine. A varicosity of this small nerve contains small AGVs and lies very close to a smooth muscle cell. Marker: 0.2 pn. m, Smooth muscle cell; s, Schwann cell.

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glial cells reach the surface of the ganglion and are in contact with the basal lamina. At these sites conspicuous patches of microfilaments underlie the cell membrane (Fig. 9B). A characteristic of intramural ganglia of the rat, guinea pig, and sheep is that numerous vesicle-containing nerve profiles abut on the surface of glial cell bodies and processes; at the area of contact some of the varicosities show a clustering of vesicles, thickening of the membrane, and occasionally dense projections, whereas there is no structural specialization on the side of the glial cell (Gabella, 1972a) (Fig. 9C). The significance of these neuroglial contacts is completely obscure. Some of them appear as half of a synapse, but it is not clear how stable these structures are. They may be structures through which glial cells are controlled by neurons, or they may be nonsynaptic sites of release of neurotransmitters.

XVII. Surface of Ganglia and Vascularization The surface of ganglia of the myenteric plexus is formed by the surface membrane of nerve cell bodies and large dendrites, of glial processes, and of vesicle-containing varicosities. In the connecting strands glial processes and varicosities appear at the surface. The entire surface of the plexus is covered b y a basal lamina, outside which is the extracellular space with microfibrils, collagen fibrils, and occasional elastic fibers. Flattened interstitial cells (Section XX) or their laminar processes lie parallel to the surface of the ganglion and may occasionally form an incomplete sheath around it, but often the surface of the ganglion or of a connecting strand is directly apposed to smooth muscle cells of the circular or longitudinal layer. Collagen fibrils and interstitial cells do not form anything resembling a capsule around the ganglion. When an electron-opaque tracer (horseradish peroxidase) is injected intravenously into guinea pigs, within minutes it penetrates the entire network of narrow gaps between adjacent stnctures of myenteric ganglia (Jacobs, 1977); similarly, colloidal lanthanum (applied together with the fixative) seems 10 diffuse freely into the ganglia (Bursztajn and Gershon, 1977). Occasionally a smooth muscle cell has a cytoplasmic process directed toward a ganglion or a nerve strand, which closely approaches its surface or even pierces the basal lamina and penetrates among nerve processes (Yamamoto, 197’7). Tapering ends of muscle cells reaching the surface of the ganglion, leaving a gap of only 20 nm (and no basal lamina in it), are also observed (Fig. 13) (Gabellla, 1972b). Blood vessels do not penetrate inside ganglia of the myenteric plexus, hut capillaries are often seen in their proximity. In the intestine of the cat and pig a

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periganglionic capillary network has been clearly shown (Iwanow and Radostina, 1937; Stach et a / . , 1977; Stach, 1978): it has a characteristic architecture and is denser than in any other part of the muscle coat.

XVIII. Ultrastructure of the Submucosal Plexus Little is known of the ultrastructure of the submucosal plexus. It is formed by tightly packed ganglion cells, glial cells, and processes, and it shares many basic characteristics with the myenteric plexus. In the submucositl neurons of the guinea pig ileum, large areas of the neuronal membrane are exposed to the surface of the ganglion and are covered by the basal lamina which surrounds the entire ganglion. On the other hand, vesicle-containing varicosities are rarely found at the surface of the ganglion. Nerve endings of various types synapse on dendrites and ganglion cell bodies (Figs. 11B and C). Among them endings containing SGVs (adrenergic) are clearly recognized; their granularity is readily enhanced by incubation with 5-hydroxydopamine (Fig. 11B). Nerve endings with a clustering of vesicles and membrane specialization can be found contacting glial cells.

XIX. Innervation of the Muscularis Externa In this section and in Sections XX and XXI the microscopic distribution of nerve fibers in the layers of the gastrointestinal wall is described. The term “innervation” is used in a loose way simply to indicate thal nerve fibers are present in a particular tissue. We do not attempt to determine whether these encounters between nerve fibers and other structures are silent ones or involve communication and of what type. A11 the axons in the wall of the stomach and intestine are unmyelinated, with very few exceptions (e.g., Schofield, 1968). By electron microscopy single axons are not normally found in the intestinal musculature (Richardson, 1958, 1960; Taxi, 1959, 1965; Gander, 1961; Lane and Rhodin, 1964a; Bennett and Rogers, 1967; Nagasawa and Mito, 1967; Gabella, 1972b), but rather nerves containing from three to seceral dozen axons (Fig. 15). (Rare exceptions are reported by Yamauchi, 1964; Bennett and Rogers, 1967.) These axons have expansions containing vesicles and mitochondria, which alternate with narrow portions (0.1-0.2 p m in diameter) containing microtubules (microtubules are usually continuous throughout the varicosities); probably the majority of axons (and not only the adrenergic ones) are varicose, but whether each varicosity is a point of transmitter release remains to be established. Schwann cells are associated with all nerve bundles; the smallest nerve

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bundles usually contain only small processes of Schwann cells. All nerve bundles have a single basal lamina around them, and along their length they become associated with processes of interstitial cells (Section XXX). Vesicle-containing varicosities can be found well inside a nerve bundle; but more often they lie at the surface of the bundle and can be apposed to a process of an interstitial cell, to an endothelial cell of a capillary, or to a smooth muscle cell (Fig. 15). The distance between a varicosity and the nearest smooth muscle cell is usually 80 nm or more and is therefore greater than that found at other autonomic neuromuscular junctions (e.g., vas deferens, sphincter pupillae). However, some axons are closely apposed to a smooth muscle cell (or to an interstitial cell) with a gap of only 15 nm (Fig. 14A): these junctions are relatively few compared with the number of intramural axons, but they may be the more important ones in the nerve control of muscle activity. The transverse diameter of varicosities ranges from 0.4 to over 1.O pm; some are almost fully packed with vesicles, and others contain only few vesicles. Although there have been only a few attempts to classify intramuscular nerve endings on the basis of vesicle morphology, it appears that axonal types similar to those found in the myenteric plexus can be identified in the muscle. Profiles containing mainly electron-lucent vesicles predominate over the other types. Vesicles can cluster beneath the axonal membrane where dense projections are sometimes found, but there are no junctional specializations in the corresponding areas of the smooth muscle cell. In the ileum nerve bundles are mainly found in the circular muscle; those of the longitudinal muscle tend to lie in the inner part of the layer, near the myenteric plexus, and are usually of the presumably cholinergic type (Fig. 14B). In the circular musculature of the guinea pig ileum there can be as many as 6500 axons (one-sixth of which are varicosities) per 10,000 transversely sectioned muscle cells, but presumably many of them are axons in transit between the myenteric plexus and submucosa. One-sixth of the axons counted are at the boundary with the longitudinal muscle (they could be considered tertiary meshes of the myenteric plexus); and one-half are situated at the innermost part of the circular muscle coat, that is, at the boundary between the bulk of the circular layer and a special layer of small, dark muscle cells (Gabella, 1972b). The occurrence of a denser innervation in the innermost part of the circular layer of the small intestine is well documented (Ramon y Cajal, 1911; Li, 1937, 1940; Taxi, 1965; Silva et a l . , 1971). The significance of these characteristic smooth muscle cells and their rich FIG. 15. (A) Circular muscle layer of the guinea pig ileum in transverse section. A small nerve bundle lies among smooth muscle cells, capillaries, and interstitial cells. Marker: 2 p n . (B) Guinea pig taenia coli in transverse section. An interstitial cell has several slender processes which approach nerve bundles and smooth muscle cells. Marker: 2 p n . m, Smooth muscle cell; s. Schwann cell; v, vessel; i, interstitial cell.

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innervation is unknown, but it has been suggested that they may be involved in detecting radial distension of the intestine (Gabella, 1974). In the taenia coli it has been reported that at intervals of about 1 mm along its length there is a marked decrease in the number of small intramuscular nerve bundles (Bennett and Rogers, 1967).

XX. Innervation of the Mucosa The mucous membrane (or mucosa) of the various parts of the gut is richly supplied with nerve fibers (see a review in Schofield, 1968). The fibers are unmyelinated and grouped in small bundles (Palay and Karlin, 1959); they penetrate into the mucosa from the submucosa, piercing the muscularis mucosae. Schofield (1960), using a silver impregnation method on the small intestine of various mammals, observed in the mucous membrane axons of intrinsic and of extrinsic origin, the latter being of larger diameter and disappearing after extrinsic denervation. The intrinsic fibers can be traced to submucosal ganglia (Kuntz, 1922); they are regarded as afferent fibers mediating the peristaltic reflex (Bulbring et al., 1958). The number of nerve fibers is probably very high; 180 alxons have been counted in the connective tissue near a single gland of the mouse colon (Silva, 1966; Silva et al., 1968), and histochemical methods show dense networks of fibers (Section XI,D). Some small nerves run parallel to muscle cells of the muscularis mucosae. Within these bundles of axons there are vesiclecontaining varicosites, some of which face the membrane of a smooth muscle cell; the gap between the two is usually 0.1 pm or more (Lane and Rhodin, 1964b; Honjin et al., 1965). Varicosities filled with either AGVs or SGVs (and regarded as cholinergic or adrenergic, respectively) are seen in the mucosa of the human intestine. Both types of varicosities lie at a distance of no less than 0.2-0.3 pm from smooth muscle cells and gland cells, but they are closer (12-20 nm) to interstitial cells, with which they are described as forming synaptic junctions (Honjin er al., 1965).

Many of the fibers in the mucosa are perivascular and assumed to be vasomotor; presumably many fibers are afferent, but the sensory endings are not identified with certainty in the electron niicroscope. Some fibers run in the proximity of secretory gland cells or enterochromaffin cells (Masson, 1928; Gasbarrini and Freyrie, 1967; HAkanson e? al., 1969), and possibly influence their secretory activity. In the cat and guinea pig vagal nerve stimulation produces a depletion of 5-HTin the enterochromaffin cells of the jejunum (Hohenleitner et al., 1971; Ahlman et al., 1976). Close neuroepithelial junctions and intraepithelial nerve fibers are only very rarely found (Dermietzel, 1971). 11 has been noted that varicosities are apposed to interstitial cells or fibroblasts more often than to any other cell type (Honjin et al., 1965; Dermietzel, 1971; Giildner

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et ul., 1972). Very few nerve fibers are found inside the lymphatic tissue of

Peyer’s patches (Pfoch and Unsicker, 1972). In the stomach, fluorescence microscopy reveals adrenergic fibers winding around the bottoms of the glands or running parallel to their bodies up to beneath the surface epithelium (Section X1,D). Adrenergic fibers are known to influence acid secretion (and therefore also peptic ulcer production); this occurs mainly through changes in the gastric mucosal blood flow (Reed et ul., 1971; Grund et ul., 1975), but it has also been suggested that adrenergic fibers modulate the stimulatory effect of vagal fibers (Moraes et ul., 1978). In an electron microscope study it has been reported that in the gastric mucosa of rats arterioles receive only adrenergic innervation, whereas capillaries receive only cholinergic innervation (Kalahanis et ul., 1976). Adrenergic innervation of the blood vessels of the gastric mucosa has been documented with fluorescence microscopy (Baumgarten, 1967; Norberg, 1967). Furness (1971) reports that in the cat gastric mucosa adrenergic innvervation is restricted to the arterioles near the bottoms of the glands.

XXI. Innervation of the Blood Vessels Fluorescence microscopy has shown the abundant supply of adrenergic fibers to intramural blood vessels, in particular the arteries (Section XI,E). By electron microscopy varicosities with SGVs are seen in the advertitia of blood vessels, 100 nm or more away from smooth muscle cells of the media. Small nerves,

formed by three to eight axons, run parallel to the arterioles of the submucosa (Devine and Simpson, 1967). The problem of other types of nerves innervating intramural blood vessels has not been investigated morphologically (but see Section XX for a discussion of the innervation of the vessels of the gastric mucosa). Physiologically, vasodilator fibers originating from intramural neurons have been found in the cat small intestine (Biber et al., 1973), prompting the suggestion that a nervous structure of local origin is involved in the control of intestinal blood flow in addition to the extrinsic fibers. Vesicle-containing varicosities are often found in the proximity of blood capillaries, both in the muscle coat and the mucosa (with a few tens of nanometers between the apposed membranes), but whether such encounters have any functional significance has not been established.

XXII. Extrinsic Nerve Fibers Extrinsic nerve fibers reach the gut through the vagus nerve, the nerve from the abdominal plexus, and the pelvic nerves. The only extrinsic fibers clearly identified histologically are the adrenergic ones (Section XI). They are post-

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ganglionic fibers from prevertebral ganglion cells, and they completely disappear from the gut after section of the mesenteric nerves or excision of the prevertebral ganglia. Vagal nerve endings (and those from the pelvic nerves) have proved difficult to recognize in the electron microscope, either under control conditions or after section of the nerves. With silver impregnation methods (degenerating) vagus nerve endings have been seen in the stomach (Schofield, 1962; Morrison and Habel, 1964) and, less convincingly, in the small intestine (see review in Schofield, 1968). In view of the slight structural disruption extrinsic denenation causes in the intramural plexuses, the extrinsic fibers are quantitatively a very minor component of the plexus.

XXIII. Exogenous Adrenergic Transmitters and “False Transmitters” Exogenous norepinephrine is taken up in vitro by the intestinal wall. The uptake is almost completely blocked by imipramine, and after extrinsic denervation it falls to less than 10% in the longitudinal muscle-myenteric plexus of the guinea pig ileum and to about one-third in the rest of the wall (within about 5 days) (Gabella and Juorio, 1973) (Fig. 16). Autoradiographically, the exogenous norepinephrine taken up in vifro appears localized at sites at the surface of the myenteric plexus and to a lesser extent at sites deeper in the ganglia. The “hot spots’’ at the surface of the ganglia are more numerous (as judged by light microscopy) than those situated in close proximity to ganglion cells (Fig. 8C), and there is therefore not a good correspondence with the distribution of nerve profiles containing small, dense-cored vesicles (Section XV). In the submucosal plexus, however, the hot spots deep in the ganglia far outnumber those at its surface. By electron microscopy, Taxi and Droz (1970) found labeled norepinephrine localized in nerve endings synapsing on dendrites in the: two intramural plexuses (rat intestine). The labeling of perivascular fibers ad the submucosa is more intense than that of intraganglionic nerve endings. In the guinea pig ileum exogenous norepinephrine is also taken up by some fibers in the circular muscle layer, particularly near its Imder with the submucosa (Fig. 8C). Fluorescent varicose fibers of the myenteric plexus of the guinea pig are greatly reduced in number after the injection of 6-hydroxydopamine (an adrenergic false transmitter which is specifically taken up by adrenergic endings and produces their degeneration; see Thoenen, 1972), and the few which remain have an abnormal appearance and no varicosities (Ross and Gershon, 1970; Qayyum, 1976). The norepinephrine content of the entire wall falls to less than lo%, and the uptake of exogenous norepinephrine to 20% (Ross and Gershon, 1970). In other similar experiments, the norepinephrine content of the myenteric plexus and longitudinal muscle was reduced to about 40%, but that of the rest of the wall to less than 25% (Juorio and Gabella, 1974).

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FIG. 16. Changes in norepinephrine concentration and in norepinephrine uptake (01 vitro) in the longitudinal muscle-myentenc plexus of the guinea pig ileum at various times after extrinsic denervation (data partly from Gabella and Juorio. 1973).

In the electron microscope, axons of the longitudinal and circular muscle layers and the muscularis mucosae of the rat duodenum show enhanced granulation of small vesicles after a short exposure (about 1 hour), either in vivo or in vitro, to 6-hydroxydopamine (Wong, 1975, 1977) (after a longer exposure to the drug the axons degenerate and dissappear). Also, nerve endings of the submucosal plexus, including those which synapse on cell bodies (Wong et al., 1974), are labeled following exposure to 6-hydroxydopamine (Wong, 1977). However, the effect of this drug on the myenteric plexus is less clear at the ultrastructural level; in spite of conspicuous changes observed with fluorescence microscopy (see above), only occasional degenerating endings and very little disruption of the plexus is observed by electron microscopy (Ross and Gershon, 1970). [Feher and Vajda (1976) have reported that the drug produces degeneration of endings in the cat myenteric plexus.] After the administration of 5-hydroxydopamine (another adrenergic false transmitter which is specifically taken up by adrenergic endings but does not cause their degeneration) there is enhanced granulation of small vesicles in intramuscular adrenergic axons (Fig. 14D), in the perivascular adrenergic axons particularly in the submucosa, and in the adrenergic varicosities of the submucosal plexus (including those synapsing on ganglion neurons) (Fig. 1 IB). In the myenteric plexus it appears that the exogenous transmitter is taken up mainly by endings

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situated at the surface of the plexus (cf. the similar distribution of exogenous norepinephrine as seen by autoradiography; Fig. 8C): this is a puzzling result, since under normal conditions the varicosities at the surface of the plexus do not show small, dense-cored vesicles.

XXIV. Adrenergic Innervation Noradrenergic fibers originating from neurons of the prevertebral ganglia reach the stomach and intestine. It is possible but not yet proven that some fibers (e.g . , those innervating the intramural blood vessels) originate from paravertebra1 ganglia. The (thoracic) splanchnic nerves contain some adrenergic postganglionic fibers (Dahlstrijm and Fuxe, 1964), although the majority are either afferent or preganglionic (Foley, 1948). Whether the same is true for the abdominal splanchnic nerves is not known. An indication of the degree of divergemce of impulses from the prevertebral ganglia to the enteric ganglia is given by the ratio of the number of neurons; there are about 50,000 neurons in the abdominal plexus of the rat (Klingman and Klingman, 1967), but there are a few inillion intramural neurons (Section VII). Of the adrenergic fibers which penetrate into the gastrointestinal wall some reach the myenteric and submucosal plexuses, some run within the muscle coats, some reach the mucosa, and some are associated with blood vessels. It is not known whether these are different su'bpopulations of adrenergic fibers or not, or whether some fibers can supply ganglia of the intramural plexuses and directly innervate some muscle cells or blood vessels. The occurrence of individual fluorescent fibers innervating blood vessels and muscle cells has been shown in the pupillary muscles of the iris, under experimental conditions (Malmfors and Sachs, 1965). The fluorescent fibers distributed in the myenteric plexus are an important component showing prominently in fluorescence microscopy. The importance of the observations of Norberg (1964) and Jacobowitz (1965) was enhancedl by the fact that up to that time the predominant view, based on silver impregnation and methylene blue studies, was that adrenergic fibers ended within the muscle layers (indeed, this is where they had to be found according to the classic notion that all autonomic pathways were two-neuron pathways consisting of a pre- and a postganglionic nerve). It should be remembered, however, that only about one-sixth of the norepinephrine content of the intestinal wall (in the guinea pig ileum) is accounted for by the endings in the myenteric plexus, the other five-sixths being present in the rest of the wall (Section XI). At the ultrastructural level one type of nerve profile has been identified with some confidence as adrenergic, since it contains SGVs (Section XV). At least in the guinea pig ileum many of these nerve profiles are found in synaptic contact with a perikaryon or a dendrite (or a spine from either). The problem has not been

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properly investigated, but it seems clear that some profiles do not form a synapse or have any junctional specialization. How often this happens is not known, but this type of ending is common in the connecting strands of the plexus and less so in the ganglia. In other parts of the gut (e.g., the colon of the guinea pig) there are probably adrenergic profiles which, instead of SGVs contain granular vesicles with are of medium size and less electron-dense. If the interpretation of the electron micrographs is correct, there is a discrepancy between the finding of adrenergic synapses on myenteric ganglion neurons and the electrophysiological results of Nishi and North (1973) and Hirst and McKirdy (1974, 1975). Since during intracellular recording it is found that adrenergic nerve stimulation or administration of norepinephrine has no direct effect on the membrane of the cell body, these investigators suggest that the effects of norepinephrine are not due to a synaptic effect but to “presynaptic inhibition”; that is, the norepinephrine released by adrenergic endings inhibits the release of acetylcholine from cholinergic endings or blocks the invasion of terminals by nerve impulses (Hirst and McKirdy, 1974, 1975), depressing or abolishing the excitatory junction potentials. Another discrepancy is that exogenous (tritiated) norepinephrine and 5-hydroxydopamine appear to be taken up mainly by processes situated at or near the ganglion surface (Sections XI1 and XXIII); but under control conditions varicosities at the surface of the ganglia do not show dense-cored vesicles. On the other hand, in the submucosal ganglia the uptake of exogenous adrenergic transmitters is confined to varicosities synapsing on or close to ganglion cells. A small number of adrenergic fibers lie close to smooth muscle cells of the muscularis externa, particularly near the innermost part of the circular muscle (Silva et af., 1971). In general adrenergic innervation of the longitudinal muscle is less abundant than that of the circular layer, but adrenergic fibers are readily detectable in the taenia coli (Fig. 14D) and in the longitudinal muscle coat of the rectum (Section X1,C). Particularly rich adrenergic innervation occurs in the musculature of the gastroesophageal junction and the anal canal, and probably of other so-called sphincters. Unfortunately, very little is known about the ultrastructure of these parts of the alimentary tract and about the distribution of cholinergic fibers and other types of nerves.

XXV. Cholinergic Innervation The intestinal plexuses are known to be able to produce acetylcholine in large amounts. The quantities released are greater in the small intestine than in the stomach or the large intestine and are greater in the guinea pig than in the dog, cat, or rabbit (Dikshit, 1938; Welsh and Hyde, 1944). Upon electrical field stimulation large amounts of acetylcholine are released from the myenteric

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plexus (Feldberg and Lin, 1950; Paton and Zar, 1968; Kosterlitz et al., 1970), and this occurs also after degeneration of the extrinsic nerves (Feldberg and Lin, 1950). The histochemical studies on acetylcholinesterase are inconclusive. Most of the intramural neurons show enzymic activity, but it is not certain that they are all cholinergic neurons. Cholinergic nerve endings have not been clearly identified with this method (see Gunn, 1968), although by electron microscopy it should be possible. Relatively few synaptic endings contain an almost pure population of mall AGVs and can be identified with some confidence as cholinergic. Whether some of the endings containing many AGVs together with a substantial number of LGVs are cholinergic cannot be excluded, and there is little reason to expect: that the cholinergic endings of the gut should all be of uniform appearance. In some species, such as the guinea pig and the sheep, the myenteric ganglia show at their surface many, often large, varicosities containing electron-lucent vesicles (Section XV) (Fig. 12B). This observation may account for the large amounts of acetylcholine released from the plexus upon electrical stimulation. Presumably some cholinergic endings are of extrinsic origin (e.g., vagal fibers). In spite of many laborious studies the distribution of vagal fibers in the stomach and small intestine remains poorly known. Very little has been contributed to this question by electron microscopy. Extrinsic cholinergic fibers and endings cannot be inore than a very minor component of intramural plexuses, since the concentration of choline acetyltransferase is unchanged after extrinsic denervation of the rabbit ileum (Filogamo and Marchisio, 19701, and the great majority of synaptic endings do not degenerate after extrinsic denervation of the guinea pig ileum (Gabella and Juorio, 1973).

XXVI. Innervation by Other Types of Nerves In addition to adrenergic and cholinergic efferent fibers and to sensory fibers, the intestine probably contains other types of nerves. There is good evidence for intrinsic inhibitory fibers which do not operate with either of the classic transmitters (Bennett et al., 1966). Inhibitory mechanisms play a dominent role in the control of intestinal motility (Wood, 1975), and it has been suggested that the circular musculature is mainly under the control of these inhibitory fibers (Anouras et al., 1977), whereas the longitudinal musculature is mainly controlled by cholinergic excitatory fibers (Paton and Zar, 1968). It has been proposed that the intrinsic inhibitory fibers use ATP or a related purine compound as a transmitter (see a detailed review in Bumstock, 1975). The supporting evidience for this hypothesis is mainly pharmacological, but histochemical methods such as that of Olson et a / . (1976) (Section XIII) may help to clarify this problem. The morphological correlate of ATP stores is described as LGVs (80-200 nm in diameter) with an ill-defined halo (large, opaque vesicles) (Bumstock, 19-75).

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Other putative transmitters are amino acids, peptides, and polypeptides. Uptake studies (Section XII) suggest that 5-HT and possibly some amino acids are stored as transmitters in some enteric neurons. As regards peptides, the field of peptidergic neurotransmission in the enteric plexuses is developing fast since the advent of immunofluorescence and radioimmunochemical techniques for these substances. Although only a few pharmacological studies have been made on the effects of these substances, biochemical evidence clearly indicates their presence in the intestine, and some of the immunochemical localizations are very convincing. This line of work provides also an interesting link between nerve cells and hormone-secreting cells. It is possible that some peptides are both gastrointestinal hormones and neurotransmitters (Bryant et a f . , 1976), but their role as transmitters is far from proved. It remains also to be seen whether peptides are stored in certain nerve endings as principal neurotransmitters or whether they are stored and released together with another transmitter. In some enterochromaffin cells polypeptides and amines coexist in the same cells (Erspamer, 1954; Pearse, 1969; Owman et al., 1973; Heitz et a f . , 1976) and possibly in the same vesicles (Alumets et a f . , 1977). Somatostatin and noradrenaline are both present in some neurons of the prevertebral ganglia (Hokfelt et a f . , 1977); (incidentally, the postganglionic fibers originating from these neurons reach the intestine). Moreover, in several types of nerve endings in somatic and autonomic nerves it has been shown that the neurotransmitter (e.g., acetylcholine, norepinephrine) is released together with other substances which include peptides and proteins, ATP, and so on. Peptide-releasing nerve endings have been tentatively identified as endings containing granular vesicles 85-160 nm in diameter with medium electron density (p-type varicosities; Baumgarten et a f . , 1970; see also Fig. 1OC) (Section XV). It should also be borne in mind that in the intramural plexuses most of the nerve endings contain a significant number of LGVs. Even in endings tentatively identified as cholinergic (since they mainly contain AGVs) there are more LGVs than in somatic cholinergic endings, and often the population of LGVs is heterogeneous in appearance. It is possible that some of the active substances identified with immunofluorescence are localized in cholinergic endings. In this context it is worth stressing that where the ending forms a synaptic junction only agranular vesicles are seen in the immediate proximity of the presynaptic membrane-even in endings which contain mainly LGVs (Gabella, 1972a).

XXVII. Afferent Fibers The distribution of afferent fibers in the gastrointestinal wall has been studied by many investigators by silver impregnation methods (see Acta Neuroveg. Vol. 28, 1966). Some fibers form complex arborizations parallel to the muscularis mucosae, between this structure and the bottoms of the glands, which are re-

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garded as mechanoreceptors (Stach, 1976). However, encapsulated nerve endings and sensory receptors are not seen with electron microscopy, and the mechanoreceptors and chemoreceptors, of which the gastrointestinal tract i s undoubtedly supplied (Iggo, 1957; Leek, 1972), have not been identified. If the sensory endings are free endings, they are not identified in the electron microscope; they probably contain some vesicles, since this seems to be the case for almost all the nerve endings of the gut. Nerve endings packed with mitochondria are not usually found. Some afferent fibers originate from intramural neurons, but the neurons of origin have not been identified with certainty (for attempts to identify electrophysiologically intramural afferent neurons, see Nishi and North, 1973; Hirst et a / . , 1974). Afferent fibers of extrinsic origin reach the gastrointestinal wall through the vagus nerve and the splanchnic nerves and have their cell bodies in craniospinal ganglia. Moreover, there are (unidentified) afferent neurons situated in the enteric ganglia which send out processes which synapse onto neurcms of the prevertebral ganglia (Kuntz, 1938, 1940). These fibers are activatcd by distension of the intestinal wall, and their activity can be detected by intrace:llular recording in the prevertebral ganglion cells (Crowcroft and Szurszewski, 1971). Degenerating nerve endings are described in the coeliac ganglion of the cat after partial resection of the small intestine (Ungvary and Leranth, 1970); and in the mesenteric nerves of the rabbit as many as 30% of the axons remain intact distal to a surgical division of the nerves (and therefore originate from intramural neurons) (Ross, 1958).

XXVIII. Development In a thorough review on the origin of enteric ganglia (Andrew, 1971) the various and numerous hypotheses on the source of intramural neurons are presented and critically discussed. The range of these hypotheses, which are inainly based on experiments on chick embryos, is remarkable; among the sug,gested sources of intramural ganglia are the local gut mesoderm, the endoderm, the neural crest, and/or the neural tube of the trunk, the vagal neural crest More recent experiments have shown that the precursors in intramural neurons do not originate within the wall of the gut but migrate into it at early embryonic stages (when they are structurally undifferentiated and therefore almost impossible to recognize). After extirpation of the neural crest, enteric ganglia fail to appear in the gut, provided that the extirpation is carried out at a very early stage (i.e., before migration occurs). In chick embryos precursors of enteric ganglion neurons may be present in the gut as early as the 10-somite stage (36 hours of incubation) (Andrew, 1964). Some experiments suggest that enteric neurons originate entirely from the vagal neural crest (Hammond and Yntema, 1947;

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Yntema and Hammond, 1954), whereas others indicate that in addition to the vagal neural crest the trunk neural crest is also involved (Andrew, 1969, 1970) or at least has the capacity of giving rise to enteric neurons (Andrew, 1971). An important step forward in this field was made with the use of a cell-marking technique based on structural differences in the interphase nucleus of two closely related species of birds, the chick and the quail (Le Douarin, 1973). By transplanting segments of quail neural tube and neural crest into chick embryos it has been shown that the neurons of the intramural ganglia of the whole gut, including the large intestine, originate from the vagal portion of the neural crest (corresponding to somites I to 7). with a small contribution from the lumbosacral portion (below the level of somite 18) (Le Douarin and Teillet, 1971a,b, 1973). There is no contribution from the neural crest at the level of somites 8 to 28; this portion of the neural crest (the cervicothoracic neural crest) contributes cells to the paraand prevertebral sympathetic ganglia and to the adrenal medulla (the latter from the neural crest at the level of somites 18 to 24). These sympathetic neurons are almost all adrenergic, whereas the enteric neurons which originate from the vagal and thoracolumbar neural crest are at least in part cholinergic. Using the same chick-quail transplant technique Le Douarin and Teillet (1974) showed that the “adrenomedullary neural crest,” when grafted in place of the vagal neural crest, gave origin to cholinergic enteric neurons, as the vagal neural crest does; the vagal neural crest, when grafted in place of the adrenomedullary neural crest, gave origin to adrenergic adrenomedullary cells. These and other experiments from the same group have lead to the conclusion that “the phenotype expressed by the autonomic ganglion cells depends on tissular interactions occurring at the site into which they are located at the end of their migration from the neural crest” (Le Douarin, 1977). The differentiation of intramural neurons has been studied with the histochemical method for acetylcholinesterase (AChE) (Cantino, 1970; Keller, 1976). In the chick AChE-positive neurons appear first in the gizzard and then in progressively more caudal regions of the gut. In rat and rabbit embryos AChE activity appears first in the myenteric neurons of the duodenum and rectum (at about 15-18 days of gestation), and then spreads to those in the intervening regions (in submucosal neurons it appears only a few days after birth) (Cantino, 1970), an observation which suggests a dual origin of enteric neurons, as shown in the chick. However, Webster (1973) found no evidence of a sacral source of enteric neurons and concluded that in the mouse intestine all the neurons were of vagal origin; they are said to migrate into and along the alimentary tract in a craniocaudal direction, a process occurring between days 10 and 15% of gestation. A similar developmental process has been suggested for the human intestine by Okamoto and Ueda (1967), who also report migration of neurons from the myenteric plexus into the submucosa to form the submucosal plexus between the twelfth and sixteenth weeks of gestation.

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Fluorescent (adrenergic) fibers are detectable in the myenteric plexus of the human intestine at 9-10 weeks of gestation, but 1 or 2 weeks earlier if the organ is incubated in the presence of norepinephrine; in the submucosal plexus fluorescent fibers are visible starting at the thirteenth week (Read and Burnstock, 1970). Adrenergic fibers appear earlier in the small intestine than in other organs (iris, vas deferens) and toward the end of embryonic life in the rat (De Champlain et d.,1970; Owman et a / . , 1971). A network of adrenergic fibers in the intramural plexuses and in the circular muscle layer is well developed in newborn guinea pigs. In rabbit embryos the intrinsic cholinergic excitatory innervation and the intrinsic inhibitory innervation of the intestine develop several days earlier than the adrenergic innervation (Gershon and Thompson, 1973). In newborn rats the packing density of myenteric neurons is over 60,000/cm2 (Fig. 17A and B), that is, over six times greater than in the adult, but the total number of neurons is only about one-quarter that found in the adult (Gabella, 1971), an observation suggesting that in this species a substantial number of enteric neurons develops after birth. In the guinea pig the plexuses are already well developed at birth, but the ganglia are much closer to each other than in the adult (Fig. 1 7 0 . The fine-structural development of nerve endings and their vesicles has been studied in the myenteric plexus of human and rabbit fetuses and has been correlated with the various steps of development of intestinal mobility (Kubozoe et al., 1969; Daikoku et d., 1975). In myenteric neurons of guinea pig fetuses synapses show prominent membrane densities (Fig. 18A) which become more attenuated after birth. Membrane densities are often present on the nerve cell membrane at sites where there are no nerve endings (Fig. 18B). Characteristically, embryonic smooth muscle cells have extensive contacts with the ganglia of the myenteric plexus (Fig. 18C).

XXIX. Coupling between Muscle Cells An important mechanism for spreading excitation in smooth muscle is based on the presence of electrotonic junctions between smooth muscle cells. These junctions were identified in the electron microscope by Dewey and Barr (1962) and are called nexuses or gap junctions. By freeze-fracture technique gap junctions are readily identified by the presence of tightly packed intramembrane particles (about 9 nm in diameter) on the P face of the junctional membranes and

FIG. 17. ( A and B ) Myenteric neurons of the small intestine of a newborn rat. Stretch preparation obtained as in Fig. I . ( C ) Myenteric plexus from the ileum o f a 3-day-old guinea pig. Stain as in Fig. I .

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of corresponding pits on the E face (see reviews in McNutt and Weinstein, 1973; Larsen, 1977) (Fig. 19A and B). In sectioned material nexuses have been observed in several smooth muscles of the alimentary tract (Dewey and Barr, 1962, 1964; Lane and Rhodin, 1964a; Oosaki and Ishi, 1964; Bennett and Rogers, 1967). It seems, however, that the distribution of nexuses is far from uniform in different muscles. The circular muscle of the small intestine in all the species examined is richly supplied with nexuses (Figs. 19A and B), whereas the longitudinal muscle has nexuses in the cat (Taylor er a / ., 1977) but not in the dog or the guinea pig (Henderson et a / . , 1971; Gabella, 1972b). The taenia coli has a few small nexuses according to some investigators (Fry e t a / . , 1977) or virtually none according to others (Gabella and Blundell, 1978). These differences between various muscles have been confirmed by the freeze-fracture technique, but many parts of the gut remain to be investigated. How coupling between muscle cells can occur in the absence of gap junctions is not known.

XXX. Interstitial Cells Interstitial cells were described by Ramon y Cajal (1893) as “interstitial sympathetic neurons” and have been the object of much controversy ever since. An excellent historical account of this problem has been presented by Taxi (1963, together with a thorough description of the morphology of interstitial cells of the intestine as studied by silver impregnation and methylene blue methods. In the electron microscope (Richardson, 1958, 1960; Hager and Tafuri, 1959; Taxi, 1959, 1965; Yamauchi, 1964; Rogers and Bumstock, 1966; Imaizumi and Hama, 1969; Gabella, 1972b; Cook and Bumstock, 1976b) interstitial cells are seen close to ganglia and strands of the myenteric plexus and to intramural nerves; some may be unrelated to nerves. Interstitial cells are also present in the mucosa, but their structure is less well known (Honjin et a / . , 1965) and some investigators describe them as fibroblasts (Guldner et al., 1972). Interstistial cells have and elongated nucleus and several cytoplasmic processes originating from an elongated or polygonal cell body. Conspicuous components of the cytoplasm are vesicles and distended cisternae of smooth and rough endoplasmic reticulum; their content is usually of medium electron density. Interstitial cells do not have a basal lamina, although there may be occasional exceptions (in the

FIG. IS. ( A ) Myenteric ganglion of the ileum of an 85-mm guinea pig fetus. Note endings containing AGVs and prominent synaptic junctions. Marker: 0.5 yn.(B) Same material as in (A). Three neurons adjacent to each other. Note dense areas in the neuronal membranes. Marker: 0.5 p m , (C) Myenteric ganglion of the ileum of a 45-mni guinea pig fetus. A smooth muscle cell forms an extensive contact with processes of the ganglion. m, Smooth muscle cell. Marker: I p m .

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lovebird, Imaizumi and Hama, 1969; in the mouse small intestine, Yamamoto, 1977). The cells are intensely positive for nonspecific cholinesterase (Koelle, 1951; Gunn, 1971). Morphologically there is nothing (except perhaps their stainability with methods such as silver nitrate or methylene blue, which are elective, although not specific, for nervous tissue) to suggest that these cells are neurons or neuronlike; they are also structurally different from Schwann cells, thus ruling out the suggestion of Lawrentjew (1926) that the two are one and the same type of cell. On the other hand, interstitial cells have many features in common with fibroblasts, in particular the highly developed endoplasmic reticulum and the absence of a basal lamina. On the basis of the evidence at hand it can be concluded, as already suggested by Dogie1 (1 8 9 9 , that interstitial cells are modified (or specialized‘?)fibroblasts (Richardson, 1958, 1960; Yamauchi, 1964; Rogers and Burnstock, 1966). Their structure is, however, variable, and they are not always identifiable with certainty; it may be that they do not represent a single cell type. Typical fibroblasts are absent from the muscle coat and the areas around the myenteric plexus (Richardson, 1958; Yamamoto, 1977) or only occasionally found (Nagasawa and Mito, 1967; Gabella, 1972b). In addition to the cells described above and to blood vessels, other cell types can be found in this part of the interstitial wall; they include histiocytes or macrophages (recognized by their colloidopessic or phagocytotic activity (Taxi, 1965), mast cells (Cook and Bumstock, 1976b), and myoblastlike cells (Yamamoto, 1977). Some interstitial cells lie close to the surface of ganglia of the myenteric plexus (Fig. 9B), but the basal lamina of the plexus and collagen fibrils intervene between the two structures. Rarely an interstitial cell is seen partly or entirely inside a ganglion of the plexus. Other interstitial cells and their slender processes are in contact with nerve strands of the plexus and intramuscular nerves, or form a sheath around them (Hager and Tafuri, 1959; Taxi, 1965; Rogers and Burnstock, 1966); these cells probably correspond to the “endothelial” sheath described by Ranvier ( 1 880) around the major trunks of the niyenteric plexus of the rabbit and considered equivalent to the Henle’s sheath of somatic nerves. At these sites sometimes

FIG. 19. ( A ) Freeze-fracture preparation of the circular muscle of the guinea pig ileum. Two muscle cells separated by a narrow intercellular space (white horizontal streak) come in contact with each other at the level of a nexus. This shows particles on the P face of one cell membrane and pits on the E face of the other cell membrane. Marker: 0.5 F . (B) Freeze-fracture preparation of the circular muscle of the guinea pig duodenum. A large nexus seen on the P face of the cell membrane and some caveolae fractured at the level of their necks. Nexuses are larger and more numerous in the duodenum than in other parts of the small intestine. Marker: 0.5 p n . (C) Circular muscle of the guinea pig ileum near the suhmucosa. showing, in addition to the ordinary smooth muscle cells (m), a layer of small and electron-dense smooth muscle cells (arrows). Between the two muscular components are several vesicle-containing varicosities. s, Schwann cell; m, smooth muscle cell. (From Gabella, 1974.) Marker: I p n .

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a vesicle-containing varicosity can be as close as 80 nm to an interstitial cell (Imaizumi and Hama, 1969). In the mucosa some nerve endings and interstitial cells form contacts with a gap of only 20 nm, described as synaptic (Honjin et ul., 1965) or synapsislike (Guldner e t a / . , 1972). Especially with their very long laminar processes, interstitial cells of the muscularis externa come in contact with smooth muscle cells (Fig. 15A); some electron micrographs show one interstitial cell contacting several smooth muscle cells (Fig. 15B). Laminar processes of interstitial cells often intervene between muscle cells and nerve bundles (which may have vesicle-containing varicosities at their surface) (Fig. 15A). Nexuses between interstitial cells and smooth muscle cells are clearly seen in the lovebird gizzard (Iniaizumi and Hama, 19691, in the mouse small intestine (Yamamoto, 1977), and (infrequently) in the cat small intestine (Taylor ei al., 1977); few small nexuses of this mixed type have been seen in the taenia coli (Gabella, 1976b). Nexuses between two cell types are known to occur in cells grown in vitro (e.g., cells of the ovarian granulosa and myocardial cells, 1Lawrence, rt ul., 1978), and they provide the pathway for metabolic and electrical coupling. The significance of the mixed type of nexus in smooth muscle is unknown, and these junctions have not yet been identified in freeze-fracture preparations. Ramon y Cajal’s idea that interstitial cells are the terminal neurons of the autonomic pathways is not supported by electron microscope evidence, but it still lives, in a sense, in the suggestion of Imaizumi and Hama (1969) that interstitial cells transmit stimuli from axons to smooth muscle cells. (If transmission takes place through interstitial cells, it can be argued that it may occur in the opljosite direction, that is, from muscle cells to nerves, providing the origin of an afierent pathway-but there is no evidence to this effect.) Taylor et ul. (1977) have suggested that interstitial cells may electrically couple longitudinal and circular muscle layers of the cat intestine, in view of the presence of nexuses between the two cell types. Yamamoto (1977), on the other hand, has proposed that inlerstitial cells are undifferentiated or immature smooth muscle cells and originate from embryonic myoblasts. Finally, it is possible that an important role of some interstitial cells is a purely mechanical one: helping to reshape muscle cell groups and allowing sliding and a change in the shape and position of intramuscular nerves during isotonic contraction.

ACKNOWLEDGMENTS 1 thank Eva Franke and Hilary Samson for help in printing the micrographs. Permission to reproduce previously published material has been ohtained from the following publishers: Springer Verlag (Figs. 8A and B , and 19C). Chapman & Hall (Fig. 13), and Cambridge University Press (Fig. 4).

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INNERVATION OF THE GASTROINTESTINAL TRACT Webster, W. (1973). J . Embryol. Exp. Morphol. 30, 573-585. Welsh, J . H . , and Hyde, J . E. (1944). Proc. SOC. Exp. Eiol. Med. 55, 256-257. Wong, W. C. (1975). Experientia 31, 1080-1082. Wong, W. C. (1977). J . Anat. 124, 637-642. Wong, W. C., Helme, R . D., and Smith, G . C. (1974). Experientia 30, 282-284. Wood, J . D. (1975). Physiol. Rev. 55, 307-324. Wood, J . D., and Mayer, C. J . (1978). Narure (London) 276, 836-837. Yamamoto, M. (1977). Arch. Histol. Jpn. 40, 171-201. Yamauchi, A. (1964). Acta Anat. Nippon. 39, 22-38. Yntema, C. L., and Hammond, W. S. (1954). J . Comp. Neurol. 101, 515-541.

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INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 59

Effects of Irradiation on Germ Cells and Embryonic Development in Teleosts NOBUOEGAMIA N D KEN-ICHIIJIRI Zoological Institute, Fuculty of Science, University of Tokyo, Tokyo, Japan

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. . . . . . . . . . . . . . . . . . . . . . . . . . 111. . . . . . . . . B. Fertility and Histological Studies on Irradiated Fish . . . . C. Autoradiographic Studies in Breeding Season . . . . . . D. Autoradiographic Studies in Sexually Inactive Seasons . . . E. Effects of Various Radiations . . . . . . . . . . . . 1V. Genetic Effects of Radiation . . . . . . . . . . . . . . V . Effects on Embryonic Development . . . . . . . . . . . A. Lethal Effects and Hatchability Experiments . . . . . . . B. Activity of Irradiated Nuclei during Development . . . . . C. Hertwig Effect . . . . . . . . . . . . . . . . . . D. Low-Dose and Low-Dose-Rate Irradiation . . . . . . . . E. Effects on Development of Germ Cells . . . . . . . . . F. Effects on Development of Various Tissues . . . . . . . VI. Concluding Comments . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Whole-Body Irradiation . . Local Irradiation . . . . . Fine Structures . . . . . Gonadectomy by Radiation .

. . . . . . A. . . . B. . . . C. . . . D. . . . E. Effects of Incorporated Radionuclides . . Radiation Effects on Testes . . . . . . . A. Castration Experiments . . . . . . .

I. Introduction

11. Radiation Effects on Ovaries

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. . . . . .

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

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195 196 196 200 20 1 203 205 206 206 206 207 209 214 217 218 218 224 226 221 229 24 I 243 245

I. Introduction Many investigators have reported that mammalian embryos and germ cells are highly sensitive to ionizing radiation, and these observations have been well considered in formulating the recommendations for radiation protection issued by the International Commission on Radiological Protection (1966). From the point of view of basic radiation biology, comparative studies with various animals on the radiosensitivity of embryos and germ cells may provide many suggestions useful in analyzing the mechanisms involved in the differences in the nature of radiation-induced injuries in different types of cells of various animals. Furthermore, in recent years ecologists and environmentalists have pointed out the I95

Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved ISBN 0-12-364359.7

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importance of the study of radiation effects on fish and fish populations in order to protect aquatic ecosystems from nuclear radioactive wastes. For these reasons several biologists have started quantitative evaluation of the effect of low doses of ionizing radiation on the fecundity and fertility of aquatic organisms. Against this background, our knowledge of radiation effects on teleostean germ cells and embryonic development is now increasing. Since several reviews have been published from the radioecological point of view (Chipman, 1972; Egami, 1973, 1979; Templeton et ul., 1971, IAEA, 1976, 1979; Blaylock and Traballka, 1978), in this article the problem is dealt with from the viewpoint of more basic cell biology. Roentgen (r) is the unit of exposure due to x or y rays. The rad is the unit of absorbed dose applicable to any ionizing radiation. The latter is a relatively recent term, so that dosage has been expressed in roentgens in much of the radiobiology literature. In this article, dosages are as given in the original papers, either in rads or roentgens without an attempt at conversion.

11. Radiation Effects on Ovaries

A. WHOLE-BODY IRRADIATION

The effects of radiation on fish ovaries have been investigated by sevseral workers. Solberg (1938a) reported in detail the effects of x rays on the fertilit:y of Oryzicrs lutipes. During the breeding season, extending from April to September, a pair of 0. lcitipes may lay a cluster of eggs almost every morning. The number of eggs laid by an Oryzicis female is markedly reduced for a few days after irradiation with 1980 r of x rays, but it later returns to normal (Solberg, 19313a). Similar results were obtained in fish given 2 kr of x rays, while in experiments with larger doses of x rays (4-32 kr) oviposition was irreversibly inhibited within 9 days (Egami and Hyodo, 1965b) (Fig. I ) . The ovary of normal laying Oryzias contains oogonia and many oocytes at various stages of growth. When the ovaries were dissected in an isotonic sodium chloride solution, large oocytes were easily isolated and became observable, while the oogonia and small oocytes were difficult to count (Egami, 1959a,b; Egami and Hyodo, 1965b). Histological and dissection studies on the ovaries of the irradiated fish indicated the following. In intact fish, at least eight stages in the development of oocytes are easily distinguishable, as reported by Yamamloto and Yoshida (1964): chromatin-nucleolus stage (A), perinucleolus stage (B), late yolk vesicle stage ([I2), early yolk vesicle stage (C), yolk vesicle stage (D,), primary yolk granule stage (E,), secondary yolk granule stage (Ep). and migratory nucleus stage (F). The frequency distribution of the mean numbers of intact and atrophic oocytes at each stage per section in irradiated and nonir-

.-.

197

EFFECTS OF IRRADIATION IN TELEOSTS

X----X

4, v)

A----I

8 kr

2 kr

m-rn

16 kr

4 kr

a - - ~ - a2 kr

0 kr

20.

E! 15

W

x-

15 DAYS AFTER IRRADIATION

~

-X

20

Fic;. I . Effects of whole-body irradiation on oviposition. (From Egami and Hyodo, 1965b. by permission of the Zoological Society of Japan.)

radiated fish is presented in Fig. 2 . This figure does not indicate the total number of oocytes, but it does show the relative differences in ovarian conditions among different groups of fish. During the experiment, the relative number of oocytes at different stages was approximately the same in the nonirradiated group, oocytes at the B stage being the most numerous.

DAYS AFTER IRRADIATION

FIG. 2. Changes in the frequency distribution ofoocytes at different developmental stages (A-F) after irradiation with 0, 2, or 16 kr of x rays, on the basis of histological studies of ovaries. Solid column indicates number of damaged oocytes. Columns or parts of columns represent different stages of oocyte development, as shown in the diagram in the lower right corner. (From Egami and Hyodo, 1965b, by permission of the Zoological Society of Japan.)

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NOBUO ECAMl A N D KEN-ICHI IJIRI

In the ovaries of fish irradiated with 2 kr of x rays, some large oocytes at the D2. El. and E2 stages underwent atrophy at S days, each exhibiting a pycnotic nucleus (Figs. 2 and 3C). In oocytes at the 112 and E, stages, the formation of yolk granules was strongly inhibited. At 7-9 days, the majority of the large oocytes disappeared (Figs. 2 and 3D), however, the small oocytes remained unaffected. In ovaries examined 14- 19 days after irradiation, large oocytes (stages E and F) were seldom encountered, although the small ones appeared to be normal (Fig. 3E). At 23 days after irradiation the ovaries were approximately normal in histological structure, damaged oocytes having been replaced by newly grown ones, probably derived from those which had been in the C and D stage!; at the time of irradiation (Figs. 2 and 3F). These findings seemed to show that oocytes at the D, and El stages were the most radiosensitive and were killed by 2 kr of x rays, while larger ones, at the E2 and F stages, which were not injured by the dose, continued to grow and were laid within 5-7 days after irradiation. Smaller oocytes at the A to D, stages were also resistant to radiation, being capable of undergoing maturation after irradiation. Consequently, damaged ovaries resumed their normal organization and egg-producing function within about 3 weeks. In the ovaries of 16-kr-irradiated fish, the first histological indication of damage appeared within 24 hours after irradiation (Figs. 2 and 3F). Following irradiation with this dose of x-rays, oocytes at all stages were affected, and no recovery followed (Figs. 2 and 3G and H) (Egami and Hyodo, 1965b). A detailed study of the radiation effects of a massive dose of x rays on the ovary of 0 . lufipes was carried out by Srivastava (1966) and Srivastava and Rathi (196’7). During sexually inactive seasons, the atrophic ovaries of 0 . latipes contain only small oocytes and oogonia. The ovaries develop within a few weeks when fish are transferred to a high temperature (Egami, 1954). However, if they are irradiated with 2 kr of x rays immediately beiore the transfer, development of the ovaries is strongly inhibited. Some of the inhibitory effects on ovarian development seem to be ascribable to depression of the release of gonadotropins from the pituitary gland, since implantation of desiccated pituitary glands attenuates the inhibitory effects of x rays on the ovaries. Irradiation with 8 kr or more destroys ovarian oocytes completely, and no gonadotropin effects are observed during the winter season (Egami and Hyodo, 1965a; Yamamoto and Egami, 1974). The histological effects of x-irradiation on the ovary of the common marine goby (Chasrnichthys gulosus) were also studied (Hyodo-Taguchi et al., 1970). Females were given 0.5, 1, 2, 3, or 4 kr of x rays and examined 2-24 days later. The effects were similar to those in Oryzias; however, the oocytes of the g;oby were more sensitive to x rays than those of the medaka. Some of the mediumsized oocytes of the goby were destroyed even when the dose was 0.5 kr, while the majority were destroyed at 1 kr (Fig. 4) (Egami and Hyodo-Taguchi, 1971).

FIG. 3. Histological changes in ovaries of irradiated fish. ( A ) Normal ovary of a nonirradiated fish. (B) Ovary of a fish killed 3 days after irradiation with 2 kr. (C) Ovary of a fish killed 5 days after irradiation with 2 kr. (D) Ovary of a fish killed 9 days after irradiation with 2 kr, showing damaged large oocytes and healthy-looking small oocytes. (E)Ovary of a fish killed 23 days after irradiation with 2 kr, showing complete recovery of ovarian structure. (F) Ovary of a fish killed 1 day after irradiation with 16 kr. (G) Ovary of a fish killed 5 days after irradiation with 16 kr. (H) Totally damaged ovary of a fish killed 6 days after irradiation with 16 kr. (From Egami and Hyodo, 1965b, by permission of the Zoological Society of Japan.)

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NOBUO EGAMI AND KEN-ICHI IJlRl

FIG.4. Histology ot ovaries of irradiated gobies. (A) Ovary, at the most advanced stage of development, of a nonirrddiated fish. (B)Ovary of a fish fixed 4 days after irradiation with 2 kr. (C) Ovary of a fish fixed 12 days after irradiation with 4 kr. (D)Ovary of a fish fixed 12 days after 1970, by permission of the Faculty of Science, irradiation with 0.5 kr. (From Hyodo-Taguchi 01 d., the University of Tokyo.)

B. LOCALIRRADIATION In order to study the abscopal effects of radiation, local irradiation experiments were carried out (Egami and Hyodo, 196Sb) (Fig. 5 ) . Even when the ovarian region of the fish was shielded, 2 kr of x rays was somewhat effective, and the temporary sterility induced by whole-body irradiation seemed to be ascribable both to the local effects on the ovary and to abscopal effects from somewhere outside the ovarian region. The complete damage to oocytes produced by 16 kr of x rays was induced only when the ovarian region was irradiated. In Oryzias, the effects of local irradiation of part of the ovary have not yet been studied in detail, chiefly because of the practical difficulty of conducting experiments in this species, which has small gonads. Therefore experiments along the same lines were attempted with the loach (Misgurnus anguillicaudatus) which has elongated ovaries (Egami and Aoki, 1966). Following whole-body irradiation with 2 kr, some oocytes at the yolk vesicle and yolk granule stages underwent vacuolation and were damaged. Even when only the anterior half of the body or heajd was

EFFECTS OF IRRADIATION IN TEJBOSTS

201

FIG.5. Diagram of plutial irradiation of 0. latipes. (A) Irradiation of ovarian region only. (B) Irradiation of whole body except ovarian region. (From Egami and Hyodo. 1%5b, by permission of the Zoological Society of Japan.)

irradiated with a 2-kr dose, the posterior half being shielded from radiation, ,changes similar to, but less marked than, those induced by whole-body irradiation took place in both irradiated and shielded parts of the ovaries. Damage in the anterior (irradiated) part and that in the posterior (shielded) part of the same ovary were essentially similar in degree. In contrast, when only the posterior half of the body was exposed to a 2-krdose, the ovarian portions of both irradiated and shielded parts of the body were almost unaffected. These results seem to show that the destructive changes were due to irradiation effects on organs located somewhere in the head region rather than to injuries directly inflicted on the ovaries. Oocytes at all stages were killed by whole-body irradiation with 16 kr. When either the anterior or the posterior half of the body, or any small part of the body, was irradiated with 16 kr, the majority of oocytes at all stages in the irradiated region of the ovaries underwent degeneration within 5 days. Although damage to the nonirradiated portion of the ovaries was slight, large oocytes were affected. In these cases, the irradiated portion was sharply defined from the shielded portion of the same ovary. It was concluded that oocytes at all stages were directly affected by 16 kr of x rays. C. FINESTRUCTURES

After whole-body irradiation of laying females with 0, 2, or 16 kr of x rays, ultrastructural changes in the oocytes and follicular cells of 0.latips were observed (Yamamoto, 1972). Within 10 hours after irradiation with 2 or 16 kr of x rays, the oocytes were ultrastructurally indistinguishable from those of intact ovaries, while some cells in the follicular epithelium were severely damaged. Such cells had diminished in size, and the electron opacity of their cytoplasmic matrix had increased. The nuclei of these cells exhibited varying degrees of

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NOBUO EGAMI AND KEN-ICHI IJlRl

EFFECTS OF IRRADIATION IN TELEOSTS

203

abnormality (Fig. 6). The first sign of abnormality in vitellogenic oocytes was observable 48 hours after irradiation. In the cytoplasm of these oocytes, numerous myelin-like figures, which are infrequently encountered in intact oocytes, appeared (Fig. 7). They arose mostly in contact with mitochondria (Fig. 8), but were sometimes observed within yolk vacuoles (Fig. 9) or near Golgi complexes (Fig. 10) or were contiguous with the membranes of the endoplasmic reticulum (Fig. 11). The disorganization of the overall structure of the vitellogenic oocytes was recognizable 72 hours after irradiation with 2 kr of x rays. In the early phase, large, empty vacuoles appeared in the peripheral cytoplasm. The vitellogenic oocytes were remarkable for their wealth of cytoplasmic organelles, such as mitochondria, rough-surfaced endoplasmic reticulum, and Golgi complex, in the peripheral area of the cytoplasm. This fact may indicate a change in synthetic activity in this area. It seems likely that the susceptibility to radiation of the medium-sized oocytes depends on the active state of the cytoplasmic organelles at this stage. The damage to the oocytes is a kind of interphase cell death by radiation. On the basis of electron microscope observations, several investigators have postulated that one of the critical structures in the interphase death produced by radiation is the cytoplasmic membrane system, since there is a close relationship between cellular radiosensitivity and the integrity of the membrane structure of the cells. The finding of the Oryzius oocytes mentioned above may support this idea (Yamamoto, 1972). It was also suggested based on electron microscopy that the mechanism of cell death induced by 16-kr irradiation was different from that induced by 2 kr. This series of experiments also suggests that the cause of radiosensitivity changes during oogenesis in fish may not be the same as that in mammals. In the case of fish ovaries, deposition of the yolk may modify the radiosensitivity (Yamamoto, 1972). D. GONADECTOMY BY RADIATION Since gonads are more radiosensitive than somatic tissues, irradiation was used as a method of gonadectomy in various organisms. In Xiphophorus hefferi and Poecifiu rericufatu several investigators have reported a relationship between radiation effects on the gonads and secondary sexual characteristics. Following FIG.6. Parts of damaged oocyte. (A) Necrotic follicle cell (NFC) surrounded by cytoplasmic feet of adjoining follicle cells with a normal appearance (FC), fixed 10 hours after 2-kR irradiation. (6) Previtellogenetic oocyte (OC) and surviving follicle cell (FC) containing a part of a necrotic follicle cell (arrow). A, Attaching filament; 6 . basement membrane of follicularepithelium; CH, chorion; N, nucleus of follicle cell; OC, oocyte. (From M . Yamamoto, 1972, by permission of the Faculty of Science, The University of Tokyo.)

FIG. 7 . Peripheral portion of vitellogenic oocyte fixed 48 hours after 2-kr irradiation. The many electron-opaque bodies (MF) are myelinlike figures. (M) Mitochondria; ( Y ) Yolk vacuoles. Bar, I p n . (From M . Yamamoto, 1972, by permission of the Faculty of Science, the University of Tokyo.)

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205

ovarian damage by x rays, the shape of the anal fin of females changed to some extent to that of the male type (Vivien, 1950, 1953; Follenius, 1952, 1953). However, the effects of gonadectomy by radiation on secondary sex characteristics are more evident in the case of male fish. OF INCORPORATED RADIONUCLIDES E. EFFECTS

A series of experiments designed to study the histological effects of p rays from incorporated 9"Sr-yoYon the ovaries of freshwater and marine fishes was carried out. The medaka and the common marine goby (C. gulosus) were used (Hyodo-Taguchi et a / . , 1970). In studies on the medaka, fish were kept in water containing 0, 0.1, 10, or 100 pCi/liter of YSr for a period of 10 days and then transferred to tap water. The histological structure of the ovary, the number of eggs laid, and the hatchability of the eggs were examined for 300 days after transfer. However, no significant effects of !'"Sf were observable in the 0.1 and 10 pCi/liter groups within this period (Kator and Egami, 1978; N. Egami and K . Kator, unpublished data). Two series of experiments were done with the goby in different seasons (HyodoTaguchi et al., 1971). In both seasons, in the ovaries of fish kept in ""Sr-water, unmistakable histological changes were recognizable, even at the lowest concentration (1-50 pCi/liter), within 10 days. In general, small oocytes were intact, at least in histological appearance, irregular vacuoles of various sizes were found in the cytoplasm of larger oocytes, and the effects became clear in fish exposed to !"'Sr for a longer period. The hisological changes in the ovary observed were similar to those induced by x-irradiation (Hyodo-Taguchi et a / . , 1970). The uptake of ""Sr by marine teleosts has already been examined by many investigators, and it is likely that the effects are produced by /3 rays from 9oSr-"oY concentrated in the fish body. The difference in radiosensitivity between the two species was found in both x-ray and P-ray experiments. Since the rate of incorporation of 90Sr into the body is different in freshwater and in marine fish, a quantitative comparison is difficult; however, at any rate, the species difference is of considerable importance in estimating the maximum permissive concentra-

FIG.8. Myelin-like figure arising in contact with a mitochondrion. Bar, 1 Fin. (From M . Yamamoto. 1972, by pemiission of the Faculty of Science. the University o f Tokyo.) FIG.9 . Myelin-likc figure arising within a yolk vacuole. Bar, 1 p m . (From M . Yamanioto, 1972, by perniission of the Faculty of Science, the University of Tokyo.) FIG. 10. Myelin-like figures arising near a Golgi complex. Bar, I p n i . (From M . Yamamoto. 1972, by permission o f the Faculty of Science, the University of Tokyo.) FIG. I I . Myelin-like figure contiguous with the membrane of endoplasniic reticulum. Bar, I p m , (From M . Yamamoto, 1972, by permission of the Faculty of Science, the University of Tokyo.)

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NOBUO EGAMI A N D KEN-ICHI IJIRI

tion of radioative substances in the environment with reference to the conservation of fish resources and from the readioecological point of view.

111. Radiation Effects on Testes

A. CASTRATION EXPERIMENTS Formerly, in order to examine the role of the testis in the maintenaince of secondary sex characteristics, x rays were used as a tool of castration. In teleost fish, Samokhvalova (1935) reported that x rays destroyed the testis of P . reticulata and inhibited development of the male-type characteristics. Aft’er this report, similar facts were confirmed by the same investigator in X. helleri and Carassius vulgaris. More detailed work along the same line was carried out by Follenius (1952, 1953, 1963), Mohsen (1956), Natali (1940), Vivien (1953), and Kessler and Luther (1957). According to Kessler and Luther (1957), the most radiosensitive stage in spermatogenesis in P . reticulata is the secondary spermatogonia, while the spermatocytes are also sensitive. They observed radiation effects in adult and young fish, and a close relationship between testis histology and the appearance of male sex characteristics (body coloration arid the gonopodium) was reported in fish irradiated with 0.5, 1,2, 3, and 4 kr of x rays.

B. FERTILITY A N D HISTOLOGICAL STUDIES ON IRRADIATED FISH In 0. latipes, Solberg (1938a) studied histologically radiation effects on spermatogenic cells. He irradiated adult males with 495, 990, 1485, and 19801 r of x rays. Histological effects were found 24 hours after irradiation, and these Ieffects became marked. Recently Egami and co-workers carried out a series 01’ more extensive investigations (Egami, 1979; Konno and Egami, 1966; Hyodo-Taguchi and Egami, 1971). The breeding female of 0. latipes, when kept together with a healthy male, lays fertilized eggs almost every morning. If the male fish is irradiated with 2 kr of x rays, however, the percentage of fertilized eggs laid by the partner decreases during a period ranging from 2 to 6 weeks. A temporary reduction in testicular weight occurs in males irradiated with 0.1-2 kr of x rays. When males are exposed to 8 kr of x rays, fertility completely fails about 1 week after the exposure. Histological observations have revealed that varying numbers of spermatogonia and spematocytes are affected by x rays. Within 24 hours, the multiplication of spematogonia is, for instance, slightly affected by 0.25-0.1 Ikr of x rays. It is clear that the effects on the testis are induced by a lower dose of x rays than in the case of the ovary. It was also observed that the youngesi: spermatogonia, spermatids, and mature sperm were not damaged, and that the

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fertility of the fish was not interfered with shortly after irradiation. Since repopulation of the youngest spermatogonia had started, when the irradiation dose was 2 kr or less, the fertility of the fish was restored. Oocytelike ceiis were frequently encountered 30-60 days after irradiation in testes recovering from damage (Egami, 1955; Konno and Egami, 1966). The results of local irradiation of the testes are different from those of irradiation of the ovaries. Radiosensitive spermatogonia suffer damage directly even when exposed to only 0.1- I kr of x rays, the decrease in the secretion of pituitary gonadotropins being of minor importance in bringing about sterilization. It is probable that, in the ovary, growing oocytes neither multiply nor synthesize DNA in their nuclei, and so visible effects of radiation do not appear upon irradiation of I kr or less. The basic mechanisms of fertility effects are thus different in the two sexes. Histological effects were observed in the x-irradiated male goby by Hyodo-Taguchi and Egami (1971). I N BREEDING SEASON C. AUTORADIOGRAPHIC STUDIES

Cell population change following x-irradiation in the testes of fish during the breeding season was examined by labeling spermatogenic cells with thymidineZ'H. 1. Rute of Spermutogenesis in Intact Oryzius

The duration of spermatogenic events in several species of mammals has been autoradiographically studied, and the definite duration of the later phases of spermatogenesis has been reported. Based on these reports, the time course of temporary sterility after irradiation has been explained in terms of the rate of repopulation of spermatogenic cells in some rodents. During the breeding season, each male of 0. f a t i p s was given a single intraperitoneal injection of 2.5 pCi of th~midine-~H dissolved in 0.025 ml of Ringer's solution. The injected males were then kept together with laying females at 25" or 15°C. The fish were killed at various times after the injection. In the peripheral part of the testis several spermatogonia were encountered. The nuclei of spermatocytes at the leptotene stage were characterized by strongly stained chromonemata. In the inner part of the testis there were many acini, each containing a cluster of spermatocytes at the zygotene and postsynaptic stages. Groups of spermatocytes at first and second meiotic metaphase occurred frequently, but secondary spermatocytes in interkinesis were seldom found. At metaphase, the first meiotic division was readily distinguishable from the second by its larger contour of nucleus and by a lower frequency in each acinus. The spermatids were small, round cells with densely distributed chromatin material. On the assumption that the relative length of a certain stage of spermatogenesis is reflected in the relative number of cells at that stage, it seems likely that the

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meiotic prophase stage is longer than meiotic metaphase 11. Moreover, spermatids seem to require a fairly long time for transformation into mature sperm. In autoradiographs, dense clusters of silver grains were found only over the nuclei of cells at a definite stage of spermatogenesis. At 1 hour after the injection a small number of nuclei of spermatogonia and spermatocytes at the preleptotene stage were labeled, but no other cells were labeled. In fish fixed at 2 or 3 hours, the labeled cells at the most advanced stage were at the leptotene stage. Some results of observations are summarized in Table I. From such observations, it has been found that the intervals from spermatocyte DNA synthesis to the early spermatid stage are at least 5 and 12 days at 25" and 15"C, respectively, arid that it takes about 7 days for a spermatid to differentiate into a spermatozoon at 25°C and about 8 days at 15°C (Egami and Hyodo-Taguchi, 1967). Such observations have also been reported by several investigators for several fish species under nonirradiated conditions. 2. Radiation Effects During the breeding season irradiated males were given a single injection of thymidine-w, after which autoradiographic observations were carried out. The incorporation results of 2-kr irradiation showed that ( 1 ) the rate of th~midine-~H into spermatogonia was decreased; (2) the active division of spermatogonia was temporarily inhibited, and consequently the supply of spermatocytes was iinterefered with for a period of about 2 weeks; (3) some dormant unlabeled spermatogonia escaped destruction by x rays and eventually began to proliferate about 2 weeks after irradiation; (4) a majority of labeled spermatocytes at the leptotene stage underwent degeneration within a few days after irradiation; (5) a small number of young spermatocytes in irradiated fish were capable of becoming mature spermatozoa (however, the rate of meiosis of these cells was slower

TABLE! I

MOSTADVANCED LABELED SPtRMATOGENETlC

CELLS FOLLOWLNG INTRAPEIUTONEAL INJECTION OF

T H Y M I D I N E - : ' H I N On~zziasTESTES"

-

Hours after the injection" Temperature ("C)

1

2

25

Pre

Lep

15

3

9

24

48

72

MI

Lep

Pac

Pac

Pac

Lep

Lep

Pac

Pac

120

288

480

tid

zoa

zoa

Pac

tid

zoa

702

zoa -

"From Egami and Hyodo-Taguchi, 1967, by permission of Academic Press, New York. "Pre, Preleptotene; Lep, leptotene; Pac, pachytene; M I , first meiotic metaphase; tid, spermatids; zoa, spermatozoa. In each group three males were examined.

EFFECTS OF IRRADIATION IN TELEOSTS

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than in the nonirradiated controls because of delay of meiosis); (6) nonlabeled spermatocytes at later stages of meiosis in irradiated fish continued to develop and finally became fertile spermatozoa within a few week; and (7) irradiated spermatids and spermatozoa were apparently left undamaged (Egami et a l . , 1967). D. AUTORADIOGRAPHIC STUDIES I N SEXUALLY INACTIVE SEASONS 1 . Initiution of'Spermutogenesis following Exposure to High Temperatures In winter the testis of 0. latipes is inactive, the number of spermatocytes is very small, and spermatogonia cease to proliferate. When males are transferred to a warm environment, however, the spermatogonia multiply within a short period after the transfer. It is found that this system is very useful in analyzing radiation effects on cell population change. With the use of this system, a series of experiments was therefore carried out by Hyodo-Taguchi and co-workers (Hyodo-Taguchi and Egami, 1975, 1976a,b,c; Hyodo-Taguchi and Maruyama, 1977). In the atrophic testis, spermatogonia can be classified into at least three stages on the basis of topographic features and nuclear morphology: spermatogonia Ia, Ib, and I1 (Hyodo-Taguchi and Egami, 1976a,b). Primary spermatogonia Ia have a small, oval, darkly stained nucleus. Spermatogonia Ib have a large, pale, round nucleus with a well-defined nucleolus. In the inner part of the testis there are secondary spermatogonia 11; the nucleus of the cells is smaller than that of the primary spermatogonia Ib, and it is characterized by heterogeneously stained chromatin. The number of these cells per cross section of the testis is easily counted. In winter, the testis contains residual sperm and acini of spermatogenic cells in earlier stages. Mitotic and meiotic figures of spermatogenic cells are seldom encountered. No spermatogonia are labeled with thymidine-"H, and the number of spermatocytes is very small. If these fish are transferred to a warm environment (25°C) from cold surroundings, a considerable increase in the mitotic figures of primary spermatogonia Ib and progress in meiotic prophase are found 2 days after the transfer. Three days after incubation, the number of primary spermatocytes increases by means of a rapid transformation of spermatogonia I1 to spermatocytes (Figs. 12 and 13). When the fish are maintained for 5-6 days at 25"C, the germinal zone consists mainly of acini of primary and secondary spermatocytes, but no spermatids are found. On the fifteenth to eighteenth day, the testes are well developed, the relative number of spermatogonia decreases, and the spermatogonial zone becomes thin, since spermatogenic cells after spermatocytes increase in number (Hyodo-Taguchi and Egami, 1976). From an autoradiographic examination it was found that, in winter, the transformation of spermatogonia I1 to primary

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FIG. 12. Spermatogenic cells of 0.larips at various stages. ( A ) Atrophic testis in winter. (B) Testis of fish 3 days after transfer to 25°C. (C and D)Testis of fish kept at 25°C for 6 and I8 days, respectively. gla, First type of primary spermatogonia la; glb, second type of primary spermatogonia Ib; gII, secondary spermatogonia 11; plcl, primary spmnatocytes at the preleptotene stage; m, mitoses of primary spermatogonia Ib; mel, primary spermatocytes at the meiotic metaphase of first civision; cII, secondary spermatocytes; t, spermatids; z, spermatozoa. (From Hyodo-Taguchi and Egami, 1976b, by permission of the Zoological Society of Japan.)

spermatocytes stopped and that primary spermatocytes ceased to develop at the pre-DNA synthetic stage. After the transfer to 25"C, DNA synthesis is stimulated, and later proliferation of spermatogonia Ib and progress of the maturation division of early speirmatocytes take place synchronously. The results show that, before establishment of a steady state of continuous spermatogenesis at a warm temperature, a temporary decrease in the number of spermatogonia Ib is observed. Changes in DNA synthetic activity and the mitotic rate o f spermatogonia Ia are not clear even when the fish are transferred to 25°C. It is likely that spermatogonia Ia are stem cells and that these cells do not react to the demand rapidly (Hyodo-Taguchi and Egami, 1976b). 2. Effects of Acute X-Irradiation When sexually inactive fish were irradiated with 100-1000 r and then transferred to a warm temperature, spermatogonia Ib, spermatogonia 11, and primary

EFFECTS OF IRRADIATION IN TELEOSTS

21 1

FIG. 13. Autoradiographs of testis of fish kept at 25°C for various lengths of time in winter. (A) Testis of a fish in winter. ( B ) Testis of a fish fixed I day after transfer to a high temperature. (C and D) Testis of a fish maintained at 25°C for 3 and 18 days, respectively. lab glb, Labeled primary spermatogonia Ib; I ah cl, labeled primary spermatocytes. (From Hyodo-Taguchi and Egami, 1976b. hy permission of the Zoological Society of Japan.)

spermatocytes were damaged within 3 days, and the number of cells per cross section of the testis decreased (Figs. 14 and 15), but spermatogonia la were unaffected. Characteristic pycnotic cells were observed in acini containing spermatogonia Ib and spermatogonia 11. In spermatogonia la no radiation effects were observable in the cell number, mitotic activity, or number of pycnotic nuclei. The data on the percentages of surviving spermatogonia Ib can be approximated by a single straight line on the logarithmic scale, but extrapolations to the low dose do not fit the original point. These observations suggest that a fraction of the cell population is more radiosensitive than the majority of spermatogonia Ib. When the irradiated fish were kept under high-temperature conditions for 30 days, the cell population changed as is shown in Fig. 16. The results indicate that, during the cell number recovery process, new spermatogonia Ib arise from spermatogonia la that have escaped destruction by x rays. This finding suggests that spermatogonia Ia are slow-cycling, being stem cells, in normal fish (Hyodo-Taguchi and Egami, 1974b, 1975, 1976b,c). These results were confirmed by Michibata (1975).

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NOBUO EGAMl AND KEN-ICHI IJIRI

FIG. 14. Histological changes in the testes of 0.laripes after x-irradiation. X 670. (A) Normal testis in winter (at time of irradiation). (B) Testis of a fish 3 days after x-irradiation with loo0 rads. cyte, Spermatocytes; gla, primary spermatogonia Ib: gII, secondary spermatogonia 11; s, sperm; pygIb, pycnotic primary spermatogonia la; gIb, primary spermatogonia Ib; pygII, pycnotic secondary spermatogonia 11. (From Hyodo-Taguchi and Egami, 1976c, by permission of Academic Press, New York.)

FIG. 15. Autoradiograph of the testes of 0.kuripes. (A) Testis of a nonirradiated fish. (B) Testis of a fish I day after 1000-rad x-irradiation. (C) Testis of a fish 3 days after 1000-rad x-irradiation. (Y. Hyodo-Taguchi, unpublished photograph.)

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NOBUO EGAMl AND KEN-ICHI IJIRl

r I ~0 U

I

I

a

I

10

20

30

I

0

10 DAYS

20 AFTER

IRRAl7lATION

FIG. 16. Change in the number of primary spermatogonia la (A) and Ib (B) for 30 days after x-irradiation. (From Hyodo-Taguchi and Egami, 1976c, by permission of Academic Press, New York.)

E. EFFECTSOF

VARIOUS

RADIATIONS

1 . Neutron Effects The effects of 2-MeV fast neutrons produced by a Van de Graaff accelerator on spermatogenic cells of 0. latipes were compared to those of 200-kVp x rays (Hyodo-Taguchi and Maruyama, 1977). In general, a weight loss of the testis and damage to the spermatogonia and spermatocytes the same as those produced by x-irradiation occurred after whole-body irradiation with neutrons. However, the 50% effective absorbed doses (ED,,) of neutrons for these effects were much less than those of x rays. For instance, for primary spermatogonia Ib 3 days after exposure the ED,,, value was 330 rads for neutrons and 810 rads for x rays. From these comparisons, the relative biological effectiveness (RBE) was examined at different times after irradiation for various biological indicators (damage to each stage of the spermatogenic cells, testicular weight, and thymidine-JH incorpora-

EFFECTS OF IRRADIATION IN TELEOSTS

215

tion into the testis as measured by the biochemical method). The RBE values for each criterion were from 2.5 to 3.8, smaller than those for the inhibition of hatchability in embryos of the same fish irradiated at developmental stages (Hyodo-Taguchi et al., 1973).

2. Effects of Low-Dose-Rate y Rays It is strongly recommended by the Committee on Nuclear Science, National Academy of Science, USA that biological hazards from low doses of ionizing radiation be estimated (National Academy of Science, 1974). It is suggested by various biological systems that the chronic effects of low doses or low-dose-rate radiation are quantitatively quite different from the effects of acute irradiation (Egami and Etoh, 1966). Blaylock (1969) studied the fecundity of the tap minnow, Gumbusia affnis, exposed to chronic radiation. Woodhead (1977) reported the effects of chronic irradiation on reproduction in the guppy. We have started a large-scale series of experiments designed to estimate lowdose effects on teleost fish. As part of the series, the effects of chronic y-irradiation at low dose rates on spermatogenesis were compared to those of acute irradiation (Hyodo-Taguchi and Egami, 1974a, 1976a). Males were irradiated under outdoor condition at the y-field in Ibaragi Prefecture in summer and in winter at dose rates of 160-3.6 r/day. In the winter experiment the fish were transferred to laboratory aquaria kept at 25°C. In another group of experiments the irradiation was carried out in the y-room of Tokyo University under controlled conditions. The results may be summarized as follows. When the dose rate was 100 r/day or higher, the radiation effects were cumulative, spermatogonia Ib were destroyed 10 days after the commencement of irradiation, and no indications of recovery were found. When the dose rates were 30-40 rlday, the number of spermatogonia Ib decreased to about 25-40% of those of the controls. Even when the total dose of irradiation increased, no further decrease in spermatogonia number took place, and another steady-state condition in the cell population kinetics was established under continuous irradiation. When the dose rate was 10-20 r/day or less, and almost normal number of spermatogonia was observed after a slight temporary decrease in the number of spermatogonia Ib. No detectable effects of low-dose-rate radiation were observed in the number of spermatogenic cells irradiated at 10 r/day or less. 3 . EfSects of SUSr-”Y p Rays In order to measure strontium uptake by fish and the testes, male fish were kept in tap water containing 1 and 10 pCi of 85SrC12per liter, respectively. Fish were killed at various times after the beginning of treatment, and the radioactivity of the whole body and of the gonad was measured (Fig. 17). Next, males were kept in water containing 1 or 10 pCi of in nitrate form for 1-30 days. Histological observations showed that dividing spermatogonia and spermatocytes

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NOBUO EGAMI AND KEN-ICHI IJlRI

FIG.17. Uptake of “Sr by the fish body and the gonads. (From Yoshimura et a / . . 1969. by permission of the Zoological Society of Japan.)

were severely damaged within 10 days, even in the 1-pCi group, while mature spermatogonia were left intact. In fish maintained in water with the radioisotope for a period of 30 days, the testes became more severely damaged and the spermatogenic cells disappeared almost completely, although supporting cells were left unaffected in the 10-pCi group. No signs of recovery were observed in this series (Yoshimura el al., 1969). Effects on the testis for a longer period (from a few months to a year) were analyzed by Kator and Egami (1978). 4. Effects of Tririated Water

Damage to spermatogenic cells in fish kept in tritiated water was compared to that in fish irradiated continuously with a comparable dose rate of y rays. Besides being of theoretical interest, this study is of practical importance because significant amounts of tritium are or will be produced by the nuclear power industry, including future nuclear fusion plants. The weight change in the testes and the behavior of primary spermatogonia Ia and Ib of 0. latipes continuously exposed to tritiated water at concentrations of 5 x 5 X and lo-’ CUliter were investigated. A marked weight loss of the testes was observed. No change in the number of spermatogoriia Ia was demonstrated, but the number of spermatogonia Ib decreased in fish of all groups during the first 5 days. Ten days after the exposure the reduction in the

EFFECTS OF IRRADIATION IN TELEOSTS

217

number of spermatogonia Ib was marked at 5 X lo-' CVliter and higher concentration groups. In fish kept in 5 x CVliter, the number of spermatogonia Ib recovered (Hyodo-Taguchi and Egami, 1974a, 1975, 1977). The uptake of tritium by a whole fish or a gonad was measured by liquid scintillation counting after the fish had been oxidized by means of a sample oxidizer. The estimated dose from the /3 rays was calculated, and the P-irradiation dose at the concentration of lo-' CVliter was estimated to give a whole-body dose rate of about 2 rads/day. From the results, it was concluded that the effects of tritium on spermatogenic cells were similar in quality to those of continuously irradiated y rays from outside the body, but quantitatively the tritium effects were slightly greater than those of y rays (RBE = 1.1-1.5) if the tritium was uniformly distributed.

IV. Genetic Effects of Radiation Genetic effects of radiation have been analyzed mainly with microorganisms, insects such as Drosophilu, and mice; however, in recent years quantitative studies on radiation-induced mutations in teleosts have been carried out by several investigators. The present situation has been well reviewed by Schroder (1973, 1978) and in a scientific report by the United Nations (1972). In most experiments, germ cells at various stages were irradiated, and the frequency of the induction of mutations was examined in subsequent generations. The dominant lethal mutation rate was ascertained in terms of the hatchability of fertilized eggs, the dominant visible mutation was examined by observing the offspring, the recessive visible mutation was demonstrated by the so-called specific loci method in Oryzius, and the frequency of chromosome aberration was observed in squash preparations of fish cells. As to visible mutations, the most frequently induced characteristics were spinal curvature (lordorsis, kyphosis, or scoliosis), malformations of the eyes and head, and hereditary changes in body color. In addition, swim bladder defects and quantitative meristic characteristics were induced. Recently the importance of behavioral and biochemical mutants was pointed out (Schroder, 1978). In X. helleri (Anders et al., 1971) and P. reticulata (Schroder, 1968, 1969a,b,c; Spieser and Schroder, 1978; Purdom, 1966; Purdom and Woodhead, 1973; Samokhvalova, 1935), oocytes and spermatogenic cells at various stages were irradiated in the gonads of the parents, and then fry laid at different periods after irradiation were examined. In Sultno gairrlnerii (McGregor and Newcornbe, 1968, 1972; Newconibe and McGregor, 1967) eggs or milt were irradiated outside the fish, and the effects were examined in the embryos. In order to estimate the induction rate of the dominant lethal mutations or the mutations influencing viability, the hatchability of fertilized eggs was examined in 0.

218

NOBUO EGAMI AND KEN-ICHI IJIRl

latipes and S . gairdnerii, and the brood size, postnatal death rate, or sex ratio was examined in P. reticulata. From the results, a linear relationship between the irradiation dose and the mutation rate was established in general under definite conditions. One of the most important problems in studying the genetic effects of radiation in teleosts is the change in radiosensitivity during gametogenesis, temperature effects on the induction of mutation (Egami and Hyodo-Taguchi, 1973), and species differences in radiosensitivity among vertebrates. Dose rate effects on the induction of mutation were observed at the spermatogonial and spermatocyte stages, but not in mature sperm. Based on these observations it is probable that the ability to repair radiation-induced damage in genetic materials in germ cells changes during the development of lhese cells. Besides gene mutation, the induction of chromosome aberration by radiation was examined under both in vivo (Hama trt a l . , 1976) and in vitro conditions (cultured fish cells). The relationship between the killing effects of radiaticln on germ cells and the induction of mutations in germ cells by radiation under various conditions is an important problem awaiting solution in the future.

V. Effects on Embryonic Development A. LETHAL EFFECTSA N D HATCHABILITY EXPERIMENTS 1. External Ionizing Radiations

a. Stage Sensitivity. The effects of acute x- and y-irradiation on developing fish embryos are well documented. Most of the reports agree that, for the end points of hatchability and lethality, fish embryos exhibit decreasing radiosensitivity with advancing development (Solberg, 1938b; Welander, 1954; Rugh and Clugston, 1955; Neyfakh, 1959; Allen and Mulkay, 1960; Bonham and Wdander, 1963). The decrease in sensitivity is steady except for an increase during the late cleavage and gastrula stages. Bonham and Welander (1963) showed the most radiation-sensitive period of development of eggs of the silver salmon (Oncorhynchus kisurch) to be a particular period (10 minutes after fertilization) of the one-cell stage. They calculated the lethal dose killing 50% of the organisms within 150 days (LD50,,5,Jof irradiation at that stage to be approximately 16 r. Kulikov (1970b) studied the radiosensitivity of embryos of the pike (Esox lucius) during fertilization and early cleavage after irradiation with 200 r of y rays. The first period of increased sensitivity was found to be the stage of convergence of the male and female pronuclei. Later periods of increase in radiosensitivity were the intermediate phases of the mitotic cycles of early cleavage. Belyaeva and Pokrovskaya (1958, 1959) determined changes in radiosensitivity of loach eggs from the time when

219

EFFECTS OF IRRADIATION IN TELEOSTS

the furrow of the first cleavage was formed up to the four-blastomere stage. The greatest sensitivity was observed when irradiation was applied at anaphasetelophase of the first cleavage, at a time when furrows appear in the developing eggs. This mitotic phase is 20 times as sensitive as interphase. Even 50 r administered at this time causes 30% lethality and produces a considerable number of deformed embryos at hatching. When eggs of the carp (Cyprinus carpio) are exposed to acute y-irradiation (Frank, 1973), sensitivity decreases as development increases, except for an increase in sensitivity during late cleavage. Stages in order of decreasing sensitivity are: one-cell, late cleavage, early'cleavage, and postorganogenesis. b. Hatchability and Lethality. The dose-hatchability relationships of 0. latipes embryos irradiated with x rays at different developmental stages are shown in Fig. 18 (Hyodo and Egami, 1962; Hyodo-Taguchi et al., 1973).

"-a

I

50

3 2 - c e l l stage

0 (3

zI 0

II Germ-ring stage

GI IL

0

t-

z

Ill B e g i n n i n g of blood c i r c u l a t i o n

W 0

[r:

W

I

a

Im 50[

7

E

:

0 1 - a . 0 IWO

l

y

g

Zoo0

LWO

DOSE

IN

e

EM0

;

16000

n

t

of t h e t a i l

32ax) 6LWO

RAE

FIG. 18. Effect of various doses of x rays on the hatchability of 0.lafipes embryos irradiated at different developmental stages. (From Hyodo-Taguchi e/ ul.. 1973, by permission of Academic Press, New York.)

220

NOBUO EGAMI AND KEN-ICHI IJlRl

Neutron irradiation was also carried out at the same stages, and it was found that neutrons were 5.6 and 6.0 times as effective at stages I and 11, respectively, as the same dose of x rays, but only 1.7 times as effective at stage IV. The delay in hatching time was also evident in embryos irradiated at stages I and I1 for both x rays and neutrons, but there was no apparent delay in those irradiated at stage IV. These results indicate that the mechanism controlling the inhibition of hatching by irradiation at stage IV is different from those at stages I and 11. Hatchini; of fish embryos involves complicated mechanisms. It has been demonstrated in 0. latipes embryos by electron microscope studies that the unicellular hatching gland, distributed in the epithelium of the endoderm, is differentiated at stage I11 (Yamamoto, 1963). The hatching enzyme from the cells of this gland acts on the chorion of the eggs to dissolve it at the time of hatching, the enzyme resembling a tryptic proteinase (Ishida, 1944). It is probable that irradiation at stages I and I1 affects the multiplication and differentiation of the cells of the hatching gland, and at stage IV the synthesis or secretion of the enzyme for hatching. Hyodo-Taguchi and Egami (1969a) examined the survival time of x-irradiated embryos of 0. latipes. They suggest that, as a result of embryonic differentiation, the critical tissues in embryonic death following irradiation may chiange during early development, since the shapes of the dose-survival time relationship curves are clearly different for embryos irradiated at different developmental stages (see also Hyodo-Taguchi and Egami, 1969b; Ijiri et al., 1978). Long-term experiments were carried out on the life-span of 0. latipes after x-irradiation at the embryonic stage. The dose range employed was 0-lo00 r. It was found that, when 100 r or more was given, the mortality rate increased even 450 days or later and the mean life span of the irradiated fish became shorter (Egami, 1971; Egami and Etoh, 1973). c. Malformations. Frequency of malformations of the eyes and body following the irradiation of trout (S. gairdnerii) embryos shows a peak value at the late cleavage stage (McGregor and Newcombe, 1968). In mammalian embryos, it is reported that malformations are induced at the greatest frequencies during active organogenesis. Prior to this period (i.e., from fertilization to implantation) irradiation causes a high mortality rate but a surprisingly low incidence of grossly deformed survivors (Russel and Russel, 1954). The failure of mammalian studies to demonstrate high frequencies of malformations as a result of irradiation prior to active organogenesis must be due to the selective loss of potentially malformed embryos as a result of failure to implant or postimplantation death (McCkegor and Newcombe, 1968). In this sense, mammals are not always the most suitable material for quantitative studies on the teratogenic effect of radiation during the very early period of development. Organisms better suited for this purpose are found among the egg-laying vertebrates, and transparent fish embryos seem to be especially suitable for studies of this kind.

EFFECTS OF IRRADIATION IN TELEOSTS

221

2 . Ultraviolet Light Irradiation Ultraviolet light irradiation at early stages of fish embryos also modifies their subsequent development. Hinrichs (1 925) reported experiments with Fundulus heteroclitus in which different embryo stages were exposed to ultraviolet light. A higher percentage of abnormalities is obtained by irradiating eggs immediately after fertilization than at any later period during development. Exposure to ultraviolet light immediately after fertilization is most apt to produce abnormalities in circulation in later development, resulting in the formation of a swollen pericardium and an elongated, weakly pulsating heart which often contains no circulating blood corpuscles. At and beyond the early cleavage stage, the developing circulation is less likely to be impaired, while eye defects and concomitant abnormalities of the anterior part of the brain are most frequent. The percentage of total abnormalities decreases as the time after fertilization increases, reaching its lowest point at the late cleavage stage. As the embryonic axis continues to develop, embryos again become highly susceptible to ultraviolet light, particularly exhibiting defects in eyes and tail regions. Such a pattern of change in sensitivity of fish embryos to ultraviolet light is somewhat different from that for ionizing radiations (see Section V,A, 1). This probably is due to the change in transparency of the embryonic body during development and the limited ability of ultraviolet light to penetrate into the embryos. Irradiation at early stages (e.g., early cleavage) caused a delay in hatching time, and the length of the delay depended on the dose. However, irradiation at later stages, for example, after the embryonic axis was formed, showed an apparent transitory “stimulation” with regard to time of hatching; more eggs hatched in the early part of the hatching period in the exposed groups than in the controls (Hinrichs, 1925). Recently, ultraviolet irradiation of 0. latipes eggs was performed before and after fertilization (Fig. 19). It was found that the stage up to 30 minutes after fertilization (at 25°C) was the most sensitive to ultraviolet light (254 nm) of all the stages examined. Moreover, visible light treatment of embryos after ultraviolet irradiation showed the photoreactivation phenomenon, indicating that dimerization of pyrimidine bases in the nucleus was responsible for such embryonic deaths (K. Ijiri, unpublished). Unlike the ultraviolet-caused sterility observed in amphibian anurans (Ijiri, 1976a,b, 1978; Ijiri and Egami, 1976), no differences were observed in germ cell numbers among 0 . latipes embryos or fry which developed from ultraviolet-irradiated eggs, eggs photoreactivated after ultraviolet irradiation, and unirradiated controls. 3 . Radionuclides

Radionuclides in the water are not the only radiation source by which fish embryos are irradiated externally. A high degree of accumulation of radionu-

222

NOBUO EGAMl AND KEN-ICHI IJIRl U.V. d o s e = 4 6 0 0 erg/rnm)

p.

1.0

N

1

0,

0

rd c

ul

2 0.5

-m .-> > L

3

m

0

i

10

20

30

40

50

60

80

FIG. 19. Stage-sensitivity curve of 0.luripes eggs for the effects of ultraviolet irradiation (4600 ergs/mm') on their subsequent development. Eggs were irradiated before fertilization or at vxious stages after fertilization up to 80 minutes (at 25"C), just before the beginning of the first cleavage. The survival rate was expressed as the ratio of the number of embryos which survived to stage 19 (stage of formation of optic buds) to the number of eggs initially irradiated. (K. ljiri, unpublished data.)

clides in or on the eggs can give rise to a radiation dose within or in the immediate vicinity of the developing embryos that is greater than that arising from the water alone (Polikarpov, 1966; Templeton er al., 1971; IAEA, 1976). Polikarpov and Ivanov (1961, 1962), Ivanov (1966), and Polikarpov (1966) reported on the effects of yoSr-yoY(/3 rays) at low concentrations in seawater on the development of eggs of Black Sea fish. Extensive studies were carried1 out with eggs of a large number of species over the goSr-sOYconcentration range 10-'4-10-4 Ci/liter. Reduced hatchability of larvae and early mortality were seen at concentrations of lop7Ci/liter and above, and the incidence of abnormalities increased significantly and with remarkable consistency at concentrations of lo-'"Ci/liter and above. The value of lo-'" Ci/liter corresponds to the irradiation of a dose rate of radday (Polikarpov and Ivanov, 1962), surprisingly a value smaller than that of the natural background radiation dose rate which fish in radday, Woodhead, 1971). Ivanov (1967) reported water receive (ca. 2 x on the effects of yUSr-yoYin seawater on the mitotic activity and production of chromosome aberrations in the dividing cells of eggs of the Black Sea scorpion fish Scorpaena poruns. As the concentration increased from to Ci/ liter, the mitotic activity of the cells decreased. At the same time, the percentage of chromosome aberrations increased with statistical significance when the concentration exceeded Cdliter. The types of aberrations were varied, with chromosomal and chromatid bridges and fragments observed most frequenltly.

EFFECTS OF IRRADIATION IN TELEOSTS

223

Fedorov et al. (1 964) also carried out experiments using plaice eggs to determine the effects on embryos of 9oSr-"0Y at low specific activities in seawater. Evidence of damage to developing embryos was found at activity levels of lo-" M i t e r , and the dose rate they estimated was 1.9 X rad/day in yoSr-yoY contaminated water. The above data thus suggest that sensitivity of fish eggs to radiation is very high for radionuclides in water, but other scientists failed to detect such a high sensitivity. Brown (1962), Brown and Templeton (1964), and Templeton (1966) conducted experiments similar to those of Polikarpov and Ivanov (1961, 1962), using eggs of the brown trout (Salmo trurta) and of plaice (Pleuronectes plaressa) maintained from immediately after fertilization until hatching in water contaminated with 9oSr-yoYover the range 10-'"-10-4 Cdliter. The accumulation factor for yttrium was found to be 10.5, and the integrated dose received by the embryos during the course of development was estimated to be 230 rads at a water specific activity of Cdliter, the highest level used. Parallel expetiments in which developing eggs were continuously irradiated by sealed y-ray sources of I3?Csat dose rates between background and 1 radhour were also carried out. It was concluded from these experiments that radiation doses of up to 500 rads received during embryonic development did not cause significant differences in either the number of eggs hatching or the number of abnormal larvae produced. In experiments with the tench (Tinca tinca) Kulikov et al. (1968) found that Cdliter of incubation of fertilized eggs in aquatic solutions containing lO-IoyoSr-90Ydid not have a noticeable effect on the rate of embryonic development or on the numerical yield of normal and deformed prelarvae. With eggs of pike (E. lucius) again no marked effect was established in the concentration range Cdliter from the moment of fertilization up to hatching. Only a concentration of lo-¶ Ci/liter induced a slight rise in the incidence of morphological deformities upon hatching (Kulikov, 1973). No decrease in mitotic activity was observed in the embryos of the Atlantic salmon (Salmo salar) under conditions of chronic irradiation with lop7Cdliter of yoSr-90Y (Migalovskaya, 1973). When the eggs of 0. lutipes were incubated in a yOSr-yoYsolution of lop6Cilliter, no significant difference in hatchability was observed (HyodoTaguchi and Egami, 1970; Kator and Egami, 1978). However, when the experiment was continued up to 70 days after hatching, there was some interference with growth, and abnormalities in gonads. Especially in the testes a marked suppression of proliferation of spermatogonia was observed (Hyodo-Taguchi and Egami, 1970). The effects of other radionuclides (e.g., 3H, I4C, "Na, "Cr, 6sZn, 13Ts, and I4¶Ce)on the development of fish eggs have also been studied (see Templeton et al., 1971; Ichikawa and Suyama, 1974; IAEA, 1976).

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NOBUO EGAMI AND KEN-ICHI IJIRI

In conclusion, it may be noted that available data on the harmful effect of a low level of radioactive contamination of water on the embryonic stages of fish are rather contradictory. The disparity in the results indicates, on the one hand, lack of knowledge concerning the radiosensitivity of the early stages of ontogenesis in fish and, on the other, the possibility of appreciable differences in the radiosensitivity of different species to irradiation from radioactive contamination of water and the accumulation of radionuclides by developing embryos. B . ACTIVITY OF IRRADIATED NUCLEIDURING DEVELOPMENT In general, the nucleus is far more susceptible to radiation than cytoplasm. A dose range therefore exists within which radiation can almost completely inactivate the genetic function of nuclei without damaging cytoplasmic structure to a significant extent. After such treatment, development proceeds only to the stage up to which the cytoplasm is capable of developing without receiving genetic information from the nucleus. X-irradiation experiments (20-60 kr) on embryos of the loach, Misgurnusfossilis, produced the results shown in Fig. 20 (Neyfakh, 1959, 1964). When nuclei are inactivated during period I (fertilization-early blastula), the arrested stage does not depend on the moment of irradiation. They all arrest at the late blastula stage, before gastrulation. This implies that during

2

4

Cleavage

6

8

10

12

14

Blaatula g ~ t t r u b t l o ~ Dovelopniental stager (h)

16

18

20

22

Organogenesla

Fic. 20. Dependence of developmental arrest on x-irradiated stages (40 kr. loach eggr,). Each arrow shows the development of an irradiated embryo from the moment of irradiation to the ,arrest of development. [From Neyfakh, 1964, by permission of Nurure (London).]

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EFFECTS OF IRRADIATION IN TELEOSTS

period I the nuclei do not perform a developmental function. The picture is quite different during period 11, suggesting active morphogenetic function of nuclei during this period. Thus irradiation at different stages of the embryo serves as a useful method for determining the active periods of the nucleus during embryonic development (Neyfakh, 1959). Generalized results on the time and duration of periods of nuclear function and the stages determined by such nuclear activity are presented in Fig. 21. In the early development of the loach, the nuclei function periodically, each period of activity being responsible for a subsequent episode of development. Nuclear activity during oogenesis ensures development of the egg up to the late blastula stage; nuclear activity during the early and midblastula stages ensures gastrulation. Development of axial organs and further organogenesis are determined by nuclear function beginning at the midgastrula stage. A further application of radiation inactivation of nuclei to the investigation of nucleocytoplasmic interactions during embryonic development is summarized elsewhere (Neyfakh, 1964). The cytoplasmic sensitivity of loach eggs becomes noticeable after irradiation with doses of 80 kr or more. As mentioned above, Neyfakh found the range of 10-60 kr to be optimal for complete inactivation of nuclei without damaging the cytoplasm extensively. In this dose range, karyokinesis is depressed (though cytokinesis is retained in early cleavage), and atypical and pycnotic forms of nuclei appear. A Feulgen-positive substance is found outside the nuclei and even outside the cells (Belyaeva and Pokrovskaya, 1958). Radiation-induced damage in the nucleus, manifest in decreased embryo survival, becomes evident after treatment with a dose as small as about 50 r. Chromosome aberrations can be observed even in a smaller dose range (Neyfakh, 1959).

During oogenesis

During e a r l y blastula Beginning f r o m g a s t r u l a m

Function of n u c l e u s 25

30

Organogenesis

FIG. 21. Scheme of the periods of nuclear functioning in the early development of the loach. The lower scale gives the hours and stages of normal development. The lines connecting the upper and lower horizontal lines indicate which particular stage of embryonic development is influenced by nuclear activity occurring at a given stage. Above the upper line the period at which the nucleus determines the given developmental stages is shown. (From Neyfakh, 1959, by permission of Company of Biologists Ltd.. London.)

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NOBUO EGAMl AND KEN-ICHI IJIRl

After x-irradiation of 1000 r at the four-cell stage of loach ( M . fossilir;) embryos, Pankova (1965) has studied the changes in chromosomes occurring in cells in the mid- and late blastula and gastrula stages. The proportion of cells containing damaged chromosomes increases as embryos advance froin the midblastula to gastrula stages. Most of the chromosome aberrations arise directly after irradiation and may be transmitted from cell to cell during the subsequent cleavage. Such types of aberrations exhibit the shape of fragments resulting, from the breakdown of chromosome bridges, which are known as “bridge fragments. However, together with these fragments of bridge origin, true chi-omosome fragments, which are clearly distinguishable in cytological preparations, arise during the midblastula to gastrula stages. This indicates the existence of latent damage not detected immediately after irradiation but leading to visible new chromosome breaks only after more than 10 cell generations. This appearance of new chromosome breaks may be explained by a disturbance in nuclear metabolism. The functioning of a nucleus from which parts of chromosomes have been lost during the series of divisions is greatly impaired. Such a nucleus sends distorted information to the cytoplasm, as a result of which definite changes occur in it also, which in turn have a detrimental effect on the structures of the nucleus, and new chromosome breaks occur (Pankova, 1965). Similar experiments are underway in 0. futipes embryos (Suyama and Etoh, 1978). ”

C. HERTWIG EFFECT In 1911, 0. Hertwig found that, when frog sperm were exposed to various doses of radiation (radium irradiation) and subsequently allowed to fertilize normal eggs, a paradoxical situation emerged in which embryos survived small exposures extremely poorly but displayed better survival characteristics at higher doses. This phenomenon is now commonly referred to in the literature as the Hertwig effect. The probable explanation of the Hertwig effect lies in the partial inactivation of sperm chromatin at low doses, with the result that embryos develop with an aneuploid chromosomal condition, which is detrimental to survival. At higher doses, however, all the sperm chromatin is inactivated or destroyed, leaving only the maternal haploid set of chromosomes to participate in further development. In the latter case, the (gynogenic) haploid condition %:ems to be far less deleterious to development in general than aneuploidy. The sperm essentially only gives an impetus to development, bringing into the ovum the centrosome necessary for subsequent divisions. The same phenomenon was also demonstrated in fish (Oppermann, 1913). Using F. hereroclitus, Lasher and Rugh (1962) reported that exposun: of sperm to 500 r (I3‘Cs y rays) had no apparent effect on the development of eggs, while a 5000-r exposure caused high mortality and abnormality, and above a 5000-r dose (to the sperm) the effect of ionizing radiation appeared to decrease so

EFFECTS OF IRRADIATION IN TELEOSTS

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that a greater percentage of near-normal embryos resulted from cleaving eggs. In cytological studies it was found that a maximum degree of damage to the developing embryos of the loach ( M . fossilis) and the carp (C. carpio), which occurs just below the dose range of the Hertwig effect, was matched by a maximum number of breaks in the sperm chromosomes (Romashov et al., 1960). Chromosome aberrations (mainly anaphase bridges) are observed also in sea urchin embryos inseminated with ultraviolet-irradiated sperm (Ejima and Shiroya, 1977; Ejima et al., 1978). Although in the dose range of the Hertwig effect no male nucleus participates in development, complete recovery of diploidy (with only a maternal set of genes) sometimes occurs after fertilization (Lasher and Rugh, 1962; Romashov et al., 1963). Such diploid gynogenesis can also be observed in several cases of distant hybridization, chiefly in fish and amphibians. The studies on radiationinduced diploid gynogenesis in fish open up interesting possibilities both in the theoretical and in the practical sense. The possibility of sex regulation in fish arises, since it is expected that such gynogenic individuals in species with female homogametry will be only females. In some cases, for example, in the breeding of sturgeon, it is very important to obtain a high percentage of females in the progeny because of the high commercial value of the caviar they yield. The homozygous fish produced can serve as interesting material in studies of function and regulation of gene expression in developmental biology and genetics and, if this technique is applied to the next generation, a “cloned” fish population will be obtained, which is also quite valuable in fisheries for the retention of properties a certain mutant fish displays. Recently, the Hertwig effect caused by ultraviolet light (254 nm) irradiation of fish sperm was studied in 0. latipes. Advantage was taken of the transparency of fertilized eggs of this species, and visible light treatment (ca. 8 klux during the fertilized but uncleaved stage was performed. The results clearly showed the existence of photoreactivation phenomenon, thus demonstrating the involvement of pyrimidine dimers in the ultraviolet-caused damage to sperm chromatin (Fig. 22; K. Ijiri, unpublished). D. LOW-DOSEA N D LOW-DOSE-RATE IRRADIATION

At dose rates of a very low level, continuous irradiation sometimes produces no observable effects on embryonic development. It is likely that responses to continuous irradiation may be masked because of recovery processes. At very low dose rates, there is competition between injury and repair and, if repair processes keep pace with injuries, and if all types of injuries are reparable, the two processes may be in equilibrium, hence irradiation will produce no obvious ill effects. Moreover, at low doses of irradiation, some interesting effects have been reported, effects which are not yet fully understood.

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NOBUO EGAMl AND KEN-ICHI IJlRl

-

m

U.V.

t

dirk

U.V.

t

visible light

--U---u

.-

D D

L

a

0

1 0

500

1000

1500

U.V. d o r e ( e r g / m + )

Fic. 22. Hertwig effect caused by ultraviolet irradiation of sperm of 0. Iutipes. and its photoreactivation phenomenon. Eggs inseminated with ultraviolet-irradiated sperm were kept in the dark (circles) or were treated by visible light (squares) for 10-70 minutes after fertilization. Later all the eggs were kept in the dark for their subsequent development (at 25°C). The survival rate was expressed as the ratio of the number of embryos which survived to stage 19 (stage of formation of optic buds) to the number of eggs initially inseminated. (K. Ijiri, unpublished data.)

Donaldson and Bonham (1964, 1970) and Bonham and Donaldson (1966, 1972) subjected Chinook (Oncorhynchus rshawysrcha) and Coho (0.kislvrch) salmon eggs to about 0.5 r/day of y radiation from shortly after fertilization until the alevins swam, resulting in a total dose of about 40 r. Irradiated fish were compared to similarly treated controls with respect to survival, growth, vertebral numbers and anomalies, other defects, and sex ratios. Juveniles, marked for identification, migrated to the ocean, whence some returned to the place of their liberation to spawn 2, 3, 4, or 5 years after irradiation. Various crosses were made, and the offspring were reared, marked, and liberated. These series of long-term experiments indicated that survival and growth of the young of the first generation tended not to be markedly affected by this level of radiation, but certain abnormalities were usually more numerous in the irradiated than in the control stock. Surprisingly, the total number of returning adults and egg production per female from the irradiated groups usually exceeded those of the controls. Using acute x-irradiation of 15-1530 r, Wadley and Welander (1971) treated Chinook salmon embryos at four different embryonic stages in duplicate exlperiments, one at ambient temperature (11.3"C) and the other at 2°C above the ambient. The effects of temperature and irradiation were evaluated in ternis of mortality and growth (length and weight). Growth stimulation attributable to low-dose irradiation was observed at both temperatures but was more apparent at

EFFECTS OF IRRADIATION IN TELEOSTS

229

the ambient temperature. When rainbow trout (S. gairdnerii) were reared for 2 years after irradiation with doses of 200 r or less during the eyed stages, the irradiation had no demonstrable effect on survival, growth, or fecundity. However, for the eggs obtained from such irradiated parents, there was a slight dose-related increase in the number surviving to the eyed stage (Welander et al., 1971). Egami and Hama (1975a,b, 1977) examined dose rate effects on hatchability and delay in the hatching time of 0. latipes embryos. Embryos were irradiated with different dose rates at various developmental stages and incubated at 25°C. With a total y-ray dose of 2 kr, the dose rate effect is clearly demonstrated if exposure is started at early stages (e.g., 0, 1, and 2 days after fertilization). The smaller the dose rate, the shorter the delay in hatching with higher hatchability. During prolonged exposure at lower dose rates, the embryonic stage advances more and sensitivity to radiation also changes (sensitivity decreases in general) during the course of development, which probably is one of the reasons for obtaining such clear dose rate effects in embryos beside the actual participation of repair processes. Temperature plays an important role in this respect. When the embryos were kept at 4°C only during the irradiation period and then incubated at 25”C, no dose rate effects were observed. During the cooling period at 4”C, the embryos ceased development almost completely, and no advance in developmental stage took place, the sensitivity remaining the same for both acute and prolonged irradiation. There is a report that irradiation of fertilized eggs of the tench (T.tinca) with y-irradiation doses of 25-100 r increases the survival rate of the prelarvae that hatch (in comparison with the controls) and also increases their resistance to large supplementary radiation doses (4000 r) given at a later stage of development (Kulikov et al., 1969). Other experiments (Kulikov, 1970a) show that irradiation of prelarvae with 250 r causes increased mortality but produces a radioprotective effect on the survivors with respect to subsequent irradiation at a dose of 1500 r. These experiments offer evidence of a “triggered” repair system, and it has been proposed that the effects of radiostimulation and an increase in radioresistance, as a result of preliminary irradiation with small doses, are regulated by the same mechanism (Kulikov et al., 1969). At any rate, the effects of low radiation doses on fish development seem to exhibit very complex but interesting phenomena and require further investigations to identify the mechanism.

E. EFFECTSO N DEVELOPMENT OF GERMCELLS Germ cells in embryos and fry are also known to be susceptible to radiation, as are most of the organs during such stages. Since germ cells are easy to distinguish histologically from somatic cells, and their number per animal is somewhat

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NOBUO EGAMl AND KEN-ICHI IJlRl

on order of 100 to loo0 at these stages, counting them permits a quantitdive assessment of radiation effects on such cells. Extensive studies have been carried out on 0. latipes. It is therefore worthwhile first to outline the normal devdopment of the germ cells in embryos and fry of this species (Gamo, 1961; Tsuzuki et al., 1966; Satoh and Egami, 1972, 1973). The stages of embryonic development are named according to Matsui's normal table for this species (Maltsui, 1949; Yamamoto, 1975). 1. Normal Development of Germ Cells a. Origin of Primordial Germ Cells (I): No presumptive primordial germ cells can be histologically recognized before gastrulation. Their appearance is first observed in the early gastrula stage (stage 14), when large-sized cells are noticeable in the blastoderm. Some are found close to the periblast and often in contact with periblast nuclei. Their main features are a size larger than that of surrclunding cells, a round and distinct outline, and larger nuclei. At the neurula stage, their distribution is almost limited to within the posterior half of the body. A s the myotomes differentiate (stage 20), the embryonic body elevates on the yolk sac and the future lateral plate is pushed peripherally, including the primordial germ cells within. At this stage they are found only in the peripheral endoderm. They are polymorphic in shape, increase in number, aggregate together, and, in most cases, are accompanied by either periblast nuclei or yolk protrusions or by both. Up to this stage, it is difficult to determine their number exactly, because of the existence of ambiguous cells. The approximate number, however, is about 20. As the elevation of the embryonic body proceeds, such cells are found more medially as the result of a medial shift of the periblast skirt and are also accompanied by periblast nuclei and yolk protrusions under the gut when the blood circulation begins. At this stage 26, about 3 days after fertilization at 25"C, when granules of melanin pigment appear in the eyes, primordial germ cells are clearly distinguishable from somatic cells. b. Germ Cell Migration Process (11). The primordial germ cells gathered under the gut at stage 26 start to ascend into the mesenchyme of the body proper, leaving the primitive endoderm, the periblast, and the yolk underneath. Primordial germ cells reach the somatopleura of the lateral plate, along which they migrate dorsally up to the dorsal end of the plate, the presumptive dorsal mesentery (future gonadal area), where they form a nest. The cells characteristically have large cells and a large nucleus, a distinct round outline, a less well-stained cytoplasm, and a single clear nucleolus (Fig. 23). c. Mitotically Dormant State (Ill). For a certain period (stages 26 to 31), the primordial germ cells remain in a mitotically dormant state. When a cell count is performed on germ cells situated in the gonadal area of embryos (Fig. 24), the apparent increase in number during this period is due to migration of the cells into the gonadal area. The average number of germ cells is about 60 to 70.

EFFECTS OF IRRADIATION IN TELEOSTS

23 1

FIG. 23. The primordial germ cells in 0. luiipes embryos at stage 27-. Here three distinct primordial germ cells, each at a different location, are shown: a germ cell under the gut, that is, before migration (a); a germ cell in the process of migration (b); and a germ cell which has reached the future gonadal area ( c ) . Bar: 20 p m . (K.Ijiri, unpublished observations.)

d. Onset of Mitosis (IV). At about stage 31 and on, mitotic figures are encountered in primordial germ cells. Concomitantly with mitotic division, the number of primordial germ cells increases, reaching about 90 immediately before hatching (stage 33). No sign of meiosis in primordial germ cells is, however, observed in any of the embryos. The number of somatic cells in the gonadal

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rudiment is very small. No indications of sex differences in the gonad primordia are seen by histological observation before hatching. e. Sex Differentiation of Germ Cell ( V ) . Hatched fry can be classified into two types according to the number and the nuclear activity of the germ cells in their gonadal area. One type consists of fry having about 100 germ cells and no cells in meiotic prophase. In other types of fry, the number of germ cells is increased through mitotic division. Furthermore, some of the germ cells begin to enter into meiotic prophase (leptotene). During subsequent development, the fry of the former type differentiate into males, while the latter differentiate into females. In other words, the morphological sex differentiation of germ cells occurs at the time of hatching. At this time, however, no sex differences in the morphology of somatic cells in the gonad were observable (Tsuzuki et al., 1966; Satoh and Egami, 1972).

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Germ cells in male fry, without mitosis or meiosis, keep the number of germ cells at about 100 up to the time when their proliferation finally takes place, about 15-20 days after hatching. Germ cells in female fry continue to increase in number and go through the stages of meiotic prophase: leptotene, 0 day after hatching; zygotene, 1 or 2 days after hatching; pachytene, 4 or 5 days after hatching; and diplotene, about 10 days after hatching (Figs. 25-28). The process is not synchronized among all the cells in an ovary, so that developing gonads contain germ cells at various stages. The total number of germ cells continues to increase, and the oocytes at the diplotene stage accumulate during formation of the ovary in young fry. 2. Effects of Rudiution on Germ Cell Development a. Effects on Primordiul Germ Cells. Ijiri and Egami (1977, 1978, 1979) and Ijiri (1977a) studied the effects of acute y-irradiation on primordial germ cells (i.e., before sex differentiation) in embryos of 0. lafipes. Their results are presented here. i. Stuge sensitiviry. Embryos were irradiated with 2000 rads of y rays at various developmental stages, and the total number of germ cells was counted in the fry at hatching (Fig. 29). It was found that y-irradiation at the early embryonic stages [0 days (stage 9), 1 day, and 2 days after fertilization] was more effective in reducing germ cell numbers than irradiation at later stages. Germ cells during the stages at 0, 1, and 2 days after fertilization are believed to be highly active in mitosis. This probably explains the high radiosensitivity at such stages. Stage 9 (0 days), which is the late blastula stage and is believed to occur before the germ cell determination process, exhibits the greatest sensitivity. The radiosensitivity of germ cells decreases sharply after 3 days. The primordial germ cells in the 3-day embryo (stage 26') are mitotically dormant and are also in the midst of migration toward the genital ridges. The low sensitivity at this stage indicates that irradiation probably has little effect on such germ cells undergoing migratory movement. It appears that the germ cell numbers in fry irradiated 5 and 6 days after fertilization are somewhat smaller than those in fry irradiated at 3 and 4 days, implying that a change takes place in germ cells during these stages, even though their number at the time of irradiation is the same as that at 3 and 4 days (Fig. 24). Probably, a change in nuclear structure during the stages of 5 and 6 days after fertilization, for germ cells to enter into mitotic phase (IV), is responsible for such results. ii. Effects of irrudiation before gertn cell determination. Exposure of 0day embryos (stage 9) to 500-2000 rads of y-irradiation produced an exponential dose-effect relationship for the end point of germ cell number at hatching. After a 2000-rad irradiation at stage 9 germ cells were counted to follow the time course changes in germ cell number in the irradiated embryos. The results are shown in Fig. 30.

FIGS.25-28. ( A ) Germ cells in male fry of 0. I d p e s . (B) Germ cells in female fry. FIG.25. Newly hatched fry within 24 hours after hatching. ( A ) Germ cells (arrow) show ng no sign of meiosis. G , gut; P, pronephric duct. (B)Germ cells in leptotene of meiotic prophase (anow). FIG.26. Fry 3 days after hatching. (A) No sign of meiotic prophase. (B) Germ cells in zygotene of meiotic prophase (arrow). Fic;. 27. Fry 5 days after hatching. ( A ) No sign of meiotic prophase. (B) Germ cells in pachytene of meiotic prophase (arrow). FIG. 2 8 . Fry I0 days after hatching. (A) No sign of meiotic prophase. (B) Germ cells in diplotene of meiotic prophase (arrow). (From Satoh and Egami. 1972, by permission of Company of Biologists Ltd., London, Courtesy of Dr. N . Satoh.)

235

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At stage 30-, the number of primordial germ cells in embryos irradiated at stage 9 was 13, compared with about 65 in unirradiated controls, as shown in Fig. 24. Subsequent changes in the pattern of total ypulation of germ cell and the onset of meiosis showed a close similarity between normal development and that of the irradiated cases. Similar observations were reported in amphibians (Ijiri and Egami, 1975; Ijiri, 1976b) and mammals (Beaumont, 1966). Despite a marked reduction in germ cell number, the fry that developed from 2000-radtreated embryos had already shown complete signs of sex differentiation of germ cells at 2 days after hatching (Fig. 30). During the normal development of 0. lutipes fry, there is a difference in germ cell number in future male and female fry at the onset of sex differentiation of germ cells (Section V , E , l ) . The above results suggest that such a difference in germ cell number is not the cause of sex differentiation, showing that the onset of sex differentiation of germ cells is

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independent of the size of the primordial germ cell population at the genital ridges. iii. Effects on primordial germ cells at the genital ridges. After a dose of 2000 rad to embryos at 4 days of development (stage 28), time course changes in g e m cell numbers have been examined (Fig. 31). Germ cell numbers remain essentially unchanged throughout the subsequent developmental stages examined up to 2 days after hatching. Parallel histological observations fail to reveal any degenerative or pycnotic cells within a short period after irradiation (e.g., within 36 hours), corresponding to the absence of an immediate reduction in germ (cells on the time course curve. These observations suggest that, under these exlperimental conditions, primordial germ cells at mitotic arrest stage (111) do not exhibit radiolesions in the form of immediate cell death. However, g e m cells with large nuclei (presumably undergoing a degenerative process) are observed from 48 hours after irradiation. They are characterized by a large diameter and vacuolization of their nuclei (for histology, see Ijiri and Egami, 1977; Ijiri, 1977a). These cells seem to remain at the gonadal area until about

237

EFFECTS OF IRRADIATION IN TELEOSTS

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hatching time, when nuclear swelling reaches its maximum. This swelling of nuclei appears to be correlated with the onset of mitosis (IV),which occurs a little before hatching in unirradiated control fry (Fig. 24). Quantitative studies on the changes in nuclear diameter have been performed employing statistical methods, and the results show that the appearance of enlarged nuclei reaches a

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NOBUO EGAMI AND KEN-ICHI IJIRI

maximum percentage at hatching time ( I 12 hours after irradiation), and that such swelling of nuclei has already started in primordial germ cells at 48 hours after irradiation (Fig. 31). At 2 days after hatching, irradiated fry contain fewer germ cells of this type, which suggests that some of the cells with an extremely enlarged nucleus have degenerated away from their populations. The !Same number of germ cells at this stage as at earlier stages may be explained by compensation for the lost germ cells through mitosis, which occurs just before the hatching time and thereafter. Unlike the time course observations on embryos and fry irradiated at 4 days after fertilization, in time course studies of 0-day-irradiated embryos and fry such abnormal type of cells are rarely encountered. Elimination through mitosis of radiation-damaged cells during early embryonic development is possible. [Similar observations were reported in amphibian (Ijiri, 1979).] Hatching fry which have survived irradiation administered at 0 days seem to contain fewer damaged cells than those irradiated at 4 days after fertilization. This may be one of the reasons why germ cells which have survived 0-day irradiation (Fig. 30) exhibit less delay in the onset of meiosis than those irradiated at 4 days after fertilization (Fig. 31). b. Effects on Sexually Differentiated Germ Cells. The response to x-irradiation of germ cells at gonial and gonocyte stages in embryos and fry of 0. lafipes has been studied through the observation of changes in the germ cell population. The results described here are based on the data published (Hamaguchi and Egami, 1975; Hamaguchi, 1976) and unpublished data. i. Effects on germ cells in meiotic prophase. Effects of acute x-irradiation on female germ cells at various stages of oogenesis were studied by irradiating fry at 2, 10, or 15 days after hatching with 1000 r. Postirradiation changes in the oocyte population were examined. The results show that there is an obvious difference between oocytes after diplotene and those before pachytene in the response to x-irradiation. When germ cells are irradiated at diplotene, the cells are killed within 24 hours after irradiation. And when oocytes in leptotene, zygotene, and pachytene are exposed to x rays, they can pass through the scages of meiotic prophase at a normal rate but fail to enter into diplotene and degenerate. Some oogonia are killed immediately after exposure, but others undergo the normal developmental process and degenerate at the critical stage between pachytene and diplotene. This implies that the apparently homogeneous oogonial population is heterogeneous in its response to x rays. More details on this problem are discussed in Section V,E,2,b,ii. Changes in the population of germ cells irradiated 15 days after hatching are shown in Fig. 32. The total number of germ cells decreased in two steps; first, by immediate cell death within 24 hours after exposure, and second, by delayed cell death which occurred between 3 and 5 days after irradiation. The former resulted

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DAYS AFTER IRRADIATION

FIG 32. Change in the number of various gem1 cells per fish in 0.lofipes fry irradiated 15 days after hatching. Solid circles. total number of genn cells per fish; open circles, number of oogonia per fish; triangles, number of oocytes in pachytene per fish; squares, number of oocytes in diplotene per fish. (From Hamaguchi and Eganii, 1975, by pennission of Taylor & Francis Ltd., London.)

mainly from the death of oogonia and of oocytes in diplotene, and the latter mainly from the death of oocytes in pachytene. When male fry were irradiated under the same conditions, the number of gonocytes continued to decrease throughout the observation period (until 20 days after irradiation). In fry irradiated 2 days after hatching, almost all the gonocytes were killed by 18 days after exposure. ii. Effects on gonial stages. Embryos and/or fry were exposed to 1000 r of x rays 1 day before hatching and at 0, 1, 2 , and 3 days after hatching. The change in the total number of germ cells in females is shown in Fig. 33. Delayed cell death at the critical stage between pachytene and diplotene is generally manifested as a decrease in the total number of germ cells between 5 and 12 days after hatching. Figure 33 shows that cell death at the critical stage begins to appear in fry irradiated 1 day after hatching and is very remarkable in fry irradiated 2 and 3

240

NOBUO EGAMI AND KEN-ICHI IJlRl

O'

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12 DAYS A F T E R HATCHING

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FIG. 33. Change in the radiation response of oogonia in 0.lutipes fry according to their stage of development. Open squares, irradiated 1 day before hatching; solid squares, irradiated within i~ day after hatching; triangles, irradiated I day after hatching; open circles, irradiated 2 days after hatching; open circles, irradiated 3 days after hatching. (From Hamaguchi, 1976. by permission of Taylor & Francis Ltd., London.)

days after hatching. However, a regenerative capacity is shown in the increase in the number of cells between 12 and 20 days after hatching. In fry irradiated 1 day before hatching, the total number of germ cells increases about 4 times during the period. However, when they are irradiated 3 days after hatching, the germ cell population is hardly able to regenerate. The earlier the stage of development at the time of exposure, the more remarkable the repopulation. These results show that the change in the response of oogonia to x rays begins to occur 1 day after hatching, when the number of germ cells in meiosis begins to increase rapidly. This suggests that the change is correlated with the initiation of meiosis. If the oogonia in the mitotic cycle are exposed to x rays, inhibition of proliferation occurs but, when oogonia which have started the meiotic cycle are irradiated, they degenerate at the critical stage between pachytene and diplotene. The failure of oogonia to repopulate in fry irradiated 2 and 3 days after hatching is probably due to the smaller number of oogonia in the mitotic cycle. When male embryos and fry were irradiated under the same conditions., the

EFFECTS OF IRRADIATION IN TELEOSTS

24 1

capacity for repopulation after exposure was abruptly lost at hatching time. Such a change may be correlated with cessation of the proliferation of germ cells in male embryos. iii. Hrrmuphroditic gonads produced by x rays. Egami and Hyodo-Taguchi ( 1 969) examined the gonadal development of 0. lutipes fry irradiated at various embryonic stages before hatching. When embryos were irradiated with 500-2000 r of x rays at 7-8 days after fertilization (just before hatching), testes in the developed fry transformed into ovotestes in about one-quarter of the males. The frequency of appearance of sterile gonads increased with the dose administered. When embryos were irradiated at 1-6 days after fertilization, the production of ovotestes was rare. Retardation of gonadal development due to x-irradiation at early embryonic stages has also been reported by several workers (Foster er al., 1949; Kobayashi and Mogami, 1958). 3. Chronic lrrudiation Chronic y-irradiation during germ cell development in 0.furipes embryos and fry is now being investigated by Egami and Hama. Preliminary data show that mitosis and differentiation of primordial germ cells are inhibited, depending on the exposure rate and/or accumulated doses. The total germ cell number in the fry decreases with increasing irradiation period, and entry into meiotic prophase is strongly inhibited at a dose rate of 130 r/day or more. At 50 days after fertilization a decrease in germ cell number is observed even in fry irradiated continuously at the rate of 27.7 r/day. In fry irradiated at a dose rate of 240 r/day the number of germ cells decreases to less than 10 (Hama and Egami, 1978). Dose rate effects are different under different temperature conditions, and details will be published elsewhere by Egami and Hama. Migalovskiy (1973) incubated embryos of the Atlantic salmon (S. salur) in water radioactively contaminated with !"'Sr-"Y and examined the primordial germ cells formed. The formation of primordial germ cells was depressed at a Wliter. In cases where the development of embryos concentration of 2 x occurred at activity levels of 0.7 x lop6 and 0.6 x lo-' Wliter, the number of germ cells, initially smaller than in the controls, increased and approached the number of cells in the controls when examined at the stage of 1-day larva. At 0.6 x Ci/liter, the number of germ cells even exceeded that of the controls somewhat (although this excess is not statistically reliable). F. EFFECTSON DEVELOPMENT OF VARIOUS TISSUES Allen and Mulkay (1960) made an excellent contribution to this problem, and their work still serves as a guideline for studies on radiation damage to various

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tissues in fish embryos. A brief outline of their results, obtained by irradiation of paradise fish embryos with 1000 r of x rays, is presented here. Developmental stages are described mostly in hours after fertilization at 27°C. 1. Blood and hemaropoietic organs: Of all embryonic tissues the hematapietic tissues are the most sensitive to irradiation. Irradiation at and before the gastrula stage destroys almost all blood cells. Irradiation between 7 hours (the beginning of gastrulation) and 16 hours (12 to 16 somites) reduces the number of erythrocytes that subsequently appear; the earlier the exposure, the greater the reduction. These blood cells disappear froin the circulation in 7-8 days, ,after which few or none can be found circulating. 2 . Eye: Irradiation at almost any stage from fertilization to hatching results in anomalous growth of thc eye. Variable responses of different portions of the eye are noted. 3. Central nervous system: This system is highly sensitive at all stages of development. Development of the keel is suppressed by irradiation during 0-7 hours prior to its formation. The posterior end of the neural tube is very sensitive from 4 to 9 hours, as evidenced by cellular necrosis. Gross contortions of various types, such as lateral displacement of the dorsal half of the brain, are produced by exposure at 4-6 hours. Development of the infundibulum is suppressed between 10 hours (3 to 4 somites) and 16 hours (12 to 16 somites). During 10-19 hours the optic lobes of the brain are more sensitive than any other region of the nervous system. Irradiation between 24 and 33 hours delays the narrowing of the brain cavities for several hours. 4. Muscle: Muscle tissue is susceptible to damage at all stages between fertilization and hatching, the period of greatest sensitivity being prior to the formation of the first somite at 8 hours after fertilization. 5. Gut: Irradiation between 2 and 7 hours produces necrosis and prevents differentiation into gill arches. After this time, up to 14 hours, differentiation is suppressed but not exactly prevented. Between 4 and 20 hours coiling of the gut is suppressed. Exposure any time after 7 hours causes a separation of the lalyers of the gut, between the mucosa and the muscularis. 6. Heart: The heart displays six types of specialized sensitivity depending on the time of irradiation. A generalized result of irradiation at all stages is a breakdown of the endocardium layer. No effect on the heartbeat can be detected. 7. Pronephros: The Wolffian ducts tend not to differentiate after irradiation between 7 and 9 hours after fertilization. Bowman’s capsule is sensitive at two periods; prior to 10 hours (three to four somites), irradiation produces a capsule of normal size but made up of abnormal, distorted cells; exposure between 18 and 21 hours causes a small but histologically normal capsule. The hematopctietic elements of the pronephros are completely destroyed in the early stages.

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8. Notochord: The notochord is sensitive to irradiation only during 4-7 hours. Exposure during this period reduces the subsequent size or completely suppresses the formation of the notochord. In many irradiated embryos the dorsal surface of the notochord is wrinkled, but this is attributable to shrinkage of the more sensitive adjacent neural tube. As a general result, during the first 4 days of development of paradise fish embryos (fertilization to the yolk-absorbed stage) the embryonic tissues may be listed approximately in the following order of decreasing radiosensitivity: hematopoietic tissue, eye, optic lobe of the brain, posterior nerve cord, central nervous system (other than above), pharynx, immature germ cells, muscle, olfactory organ, gut, heart, excretory system, notochord, chromatoblasts, cartilage, differentiated periblast, mature erythrocytes (Allen and Mulkay, 1960). Recently, effects of y-irradiation during embryonic stage on thymus have been studied quantitatively in Oryzius latipes, measuring thymus volume from the histological sections (Ghoneum et al., 1979).

VI. Concluding Comments In this article, the effects of radiation on gametogenesis in adults and on the developmental process in embryos of teleost fish have been discussed. The most significant conclusion to be drawn here is that the radiosensitivity of gametes and embryonic cells undergoes a considerable change during these processes. Sterility and weight reduction of testes after irradiation are due mainly to actual cell loss, and the order of decreasing sensitivity of spermatogenic cells is from the most sensitive spermatogonia, through primary and secondary spermatocytes and spermatids, to sperm, which are quite radioresistant. Even in morphologically homogeneous populations of spermatogonia, a heterogeneity of sensitivity exists. As shown in an autoradiographic experiment, th~midine-~Hlabeled spermatagonia are radiosensitive, while the unlabeled dormant and youngest spermatogonia are not. The same heterogeneity is also observed among the gonia population present in newly hatched fry. Although at present there are still limitations to the quantitative interpretation of these results, such radiosensitivity surely reflects the internal state and structure of the cell (mainly the nucleus) and will provide valuable information about the process during gametogenesis. The radiosensitivity of gametogenesis in the ovary and testis cannot be directly compared, except probably from the viewpoint of genetic effects of radiation. As pointed out in Section II,C, in fish oocytes yolk deposition, that is, a cytoplasmic component, may play an important role in modifying their radiosensitivity. The

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NOBUO EGAMl AND KEN-ICHI IJIRI

possibility of such damage to cytoplasmic components should also be kept in mind in interpreting the results obtained from irradiated embryos, a typical example of which is shown in local ultraviolet irradiation experiments on amphibian embryos (Ijiri, 1977b). The classic law of Bergonie and Tribondeau (1906) states that the sensitivity of cells varies directly with their mitotic activity and inversely with their degree of differentiation. Embryonic cells are no exception. Being made up largely of differentiating and mitotic cells, the embryo is considered the most radiosensitive stage in the entire life cycle of an organism (Rugh, 1960). Most studies concerning radiosensitivity of fish embryos have so far been performed for the end points of hatchability and lethality during development. This is mainly because of the simplicity of the technique. No doubt many irradiated larvae that appear normal at hatching cany radiation damage that becomes evident at some future stage. But the immediate effects of radiation on the development and hatchability of eggs at least show how radiosensitivity changes during embryonic development. However, this does not deny the importance of long-term studies on these irradiated animals. For instance, as in the case of primordial germ cells which have survived irradiation, although they are noimal in appearance, a slight change not immediately detectable in such cells has a greater effect later simply because they serve as stem cells for thousands of germ cells in adult gonads. Such a long-term experiment has various obstacles to overcome, but it is certainly essential for evaluation of the ecological effecls of radiations and radionuclides on the population of aquatic organisms. The degree of accumulation in and on the egg varies with the radionuclide, chemical composition of the water, epiphytes present on the egg, and the species of fish involved. Consequently, comparisons of the effect of radionuclide (:ontamination in fish eggs cannot necessarily be made solely in terms of the concentration of radioactivity in the water (Brown and Templeton, 1964). This may be one of the reasons for the disparity in the results obtained by different scientists with various species of fish eggs concerning the effects of radionuclides in w;ater. A methodology with definite conditions and criteria is now needed in this Field, and attempts have now been made (IAEA, 1979). An application of the Hertwig effect to diploid gynogenesis was discussed. Further investigations remain to be made into this problem, but it certainly has a promising future both in a theoretical and in a practical sense.

ACKNOWLEDGMENT We wish to thank Dr. Yasuko Hyodo-Taguchi for her collaboration and support

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INTERNATIONAL REVIEW OF CYTOLOGY. VOL. 59

Recent Advances in the Morphology, Cytochemistry, and Function of Balbiani's Vitelline Body in Animal Oocytes' SARDUL S. GURAYA Drpurttireiri of Zoology, Collegr .f basic^ Srietrcrs und Huitiutiities. Putijtrh Agric.ul/urtrl Utiivrrsiry, Luclhiutiu, Putrjah, Indin

I. Introduction . . . . . . . . . . . . . . .

. . . A . Spiders . . , . . . , . , . , . . . B. Scorpions and Myriapods , . , . , . . . C. Insects , . . , . , , , . . . . . . D. Crustaceans . . . . . . . . . . . . E. Molluscs . , . . . . . . . . . . . . F. Echinoderms . . . . . . . . . . . . G. Platyhelminthes . . . . . . . . . . . H. Acanthocephala . . . . . . . . . . . I l l . Balbiani's Vitelline Body in Protochordates . . A. Tunicates . . . . . . . . . . . . . B. Atnphio.rus . . . . . . . . . . . . . IV. Balbiani's Vitelline Body in Vertebrates . . . . . . . . . . . . . . . A. Elasmobranchs B. Teleosts . . . . . . . . . . . . . . C . Amphibians . , . . . . . . . . . . . D. Reptiles . . . . . . . . . . . . . . E.Birds . . . . . . . . . . . . . . . F. Mammals . . . . . . . . . . . . . V. General Discussion and Conclusions . . . . . References . . . , . , . , . , . . , . 11. Balbiani's Vitelline Body in Invertebrates

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I. Introduction The conspicuous juxta- or paranuclear complex of organelles surrounding the centrosome of vertebrate and invertebrate oocytes has been the subject of studies for many years (see Munson, 1912; Wilson, 1937; Chaudhry, 1952; Raven, 1961). According to Henneguy (1887) it was reported in the spider oocyte by von 'This article is dedicated to the memory of Professor Vishwa Nath who made outstanding contributions to the cytology of animal gametes. 249 Copyrighl @ 1Y7Y by Acaddemtc Press. Inc. All nghts of reproduction in any form reserved ISBN 0-12-364359-7

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Wittich in 1845 and was called the “Dotterkern” by Carus in 1850. Balbiani (1 864a,b) investigated this ooplasmic structure in detail in spiders and myriapods and summarized his extensive observations in 1893. Balbiani’s student, Henneguy, called this juxtanuclear ooplasmic structure “Balbiani’s vesicle” in 1887 and the “yolk body of Balbiani” in 1893. Van der Stricht (1923), in his classic monograph on the developing mammalian oocyte, made a clear distinction between the central vitelline or Balbiani’s body and the surrounding vitellogenic or mitochondria1 bed. Aykroyd (1938) designated these areas the “pallial layer” and “couche vitellogtne, respectively, while Chaudhry (1952) described them as the yolk nucleus and pallial substance, respectively, in the fish oocyte. Beams and Sheehan (1941) designated the whole juxtanuclear complex of organelles the “yolk nucleus complex” in human oocytes, as Guraya (1962, 1963a) also did in the oocytes of birds and reptiles where it showed a relatively more complex structure and histochemical composition. Raven (1961), however, believed that the whole juxtanuclear complex of organelles should be called the “Balbiiini’s vitelline body”-a term also used by Hertig and Adams (1967) and Hertig (1968) in their studies on human oocytes. Thus the term “Balbiani’s vitelline body” is used here for the whole juxtanuclear complex of organelles in young oocytes, which usually includes the cytocentrum with its archoplasm (or yolk nucleus), Golgi bodies, mitochondria, lipid bodies, and other componenl s of diverse morphology and nature. In this article the use of the term “yolk nucleus” is confined to more-or-less well-defined aggregations or bodies of a basophilic (or RNA-containing) substance usually preserved by the classic techniques of cytology (Munson, 1912; Wilson, 1937; Chaudhry, 1952; Raven, 1961). Such basophilic yolk nuclei may be formed in the region of Balbiani’s vitelline body. Because of their dense, organized nature, they may stand out in sharp contrast to the general ooplasm and form a substrate for the multiplication and accumulation of other cytoplasmic components of the Balbiani’s vitelline body. The surrounding Golgi bodies and mitochondria have often been called the pallial substance of the yolk nucleus (see Raven, 1961). Ultrastructurally, the yolk nucleus usually has been found to consist of endoplasmic reticulum or ribosomes [or ribonucleoprotein (RNP) particles] or both, which show many variations in development and organization in different animal species (Rebhun, 1956a,b; Reverberi, 1966a). In earlier studies, camed out with classic techniques of cytology, it was mostly the yolk nucleus component of the Balbiani’s vitelline body that was apparently preserved because of its protein, lipoprotein, and RNA composition, whereas the other cell components consisting of lipids and lipoproteins were wholly or partially removed because of inadequate fixation. This led to considerable controversy about the correct identification of organelles and inclusions in the Balbiani’s vitelline body in different animal species (see Munson, 1912; Wilson, 1937; Chaudhry, 1952; Raven, 1961). Divergent opinions also con”

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tinued to be expressed about the behavior and functions of the Balbiani’s vitelline body in the developmental processes of growing oocytes. The purpose of this article is to summarize and discuss the origin, development, morphology (including ultrastructure), cytochemistry, relationships, and functions of various components of the Balbiani’s vitelline body in the oocytes of invertebrates, protochordates, and vertebrates, which have been recently revealed with the use of modem cytochemical and electron microscope techniques. The correlation and discussion of recent data obtained with these techniques are considered necessary for a complete understanding of its origin, composition, and functions. The views with regard to its origin, morphology, composition, and function as reported by classic cytologists were conflicting (see Munson, 1912; Wilson, 1937; Chaudhry, 1952; Raven, 1961), but much of the more recent research has not previously been critically examined and assessed. Therefore full attention is given to the mounting volume of recent histochemical and electron microscope work. For a better understanding of the diversity of the structure, behavior, composition, and cytochemistry of the Balbiani’s vitelline body in the oocytes of different groups of invertebrates, protochordates, and vertebrates, these various aspects are first described in relation to the Balbiani’s vitelline body in the spider oocyte, which is relatively better developed and was also the first ooplasmic structure to attract the attention of cytologists.

11. Balbiani’s Vitelline Body in Invertebrates A. SPIDERS The Balbiani’s vitelline body forms a very conspicuous cytoplasmic structure in the oocytes of some species of spiders and has been investigated with various techniques of cytochemistry and electron microscopy (Jacquiert, 1936; Andre and Rouiller, 1957; Sotelo and Trujillo-Cenoz, 1957; Andre, 1958; Krishna, 1958; Nath et al., 1959; Sareen, 1963, 1964; Costanzo, 1964; S. S. Guraya, unpublished, 1978). S. S. Guraya (unpublished, 1978) recently examined the details of the origin, development, differentiation, morphology, and behavior of the Balbiani’s vitelline body during the successive stages of oocyte growth in the spider Araneus (Fig. IA). The smallest oocytes (or oogonia) seen in the ovary do not show the Balbiani’s vitelline body (Fig. 1A and B). With the initiation of oocyte growth the basophilic substance, designated here as the yolk nucleus substance, begins to differentiate close to the nuclear envelope, apparently around the centriole (Fig. 1C). At first it is of more or less homogeneous appearance and is associated with deeply sudanophilic phospholipid granules and a few

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FIG. I . The origin, development, and morphology of the yolk nucleus and other cell components of the Balbiani’s vitelline body in successive stages (A-I) of oocyte growth in the spider Ar.9neuu. CZ, central zone of yolk nucleus; L, lamellae; LB, lipid body; LY, lipid yolk; LZ, lamellar 2.one of yolk nucleus; M, mitochondria; N , nucleus of oocyte showing nucleoli; PZ,peripheral zone of yolk nucleus; TZ, transitional zone of yolk nucleus; V, vacuole; Y N , yolk nucleus.

granular or rodlike mitochondria; the latter are moderately sudanophilic and have the usual phospholipid-protein composition. Some clear vacuoles of small size, which react negatively to histochemical tests, are also seen in the yolk nucleus substance which itself stains faintly with Sudan black B; its Sudan black-

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positive reaction becomes negative in material extracted with hot pyridine. This indicates that the yolk nucleus substance of the Balbiani’s vitelline body in the spider oocyte contains diffuse lipids due to lipoproteins. The peripheral portions of the yolk nucleus are basophilic and contain RNA, as judged from the positive reaction with methyl green-Pyronine and the negative reaction in control sections treated with RNase. With further growth of the oocyte, the yolk nucleus, still lying adjacent to the nuclear envelope, differentiates first into two zones-the inner and the outer (Fig. ID). The inner or central zone is smaller and appears homogeneous in appearance, except for the presence of vacuoles. It stains very faintly with Sudan black B and reacts negatively with tests used for the detection of phospholipids and triglycerides. This indicates the presence of lipoproteins in the central zone. Phospholipid granules are either absent or relatively few in number. The outer zone of the yolk nucleus is larger and is associated with concentric lamellae consisting of a lipoprotein (or phospholipid-protein) complex, as they stain more deeply with Sudan black B than the matrix in which they are embedded. Besides the concentric lamellae, phospholipid granules and a few small mitochondrial granules are also associated with the outer zone of the yolk nucleus. The phospholipid granules stain more deeply with Sudan black B and acid hematein than the lipoprotein lamellae and mitochondria1 granules. A few small vacuoles are also seen in the yolk nucleus substance. These observations showed that the Balbiani’s vitelline body in young oocytes of the spider consisted of a wellorganized yolk nucleus, mitochondria, lipid bodies, and possibly Golgi bodies; the last-mentioned could not be distinguished from the yolk nucleus proper in the histochemical preparations used, but the yolk nucleus is differentiated into zones. With further development of the oocyte, the Balbiani’s vitelline body separates from the nuclear envelope and occupies the central ooplasm (Fig. 1E) where it attains maximum size and differentiation (Fig. 1F and G). The fully developed and differentiated Balbiani’s vitelline body is a well-organized spherical structure having four well-demarcated zones: an inner or central zone, a lamellar zone, a transitional zone, and a peripheral zone; the last two zones develop after the Balbiani’s vitelline body has moved away from the nuclear envelope (Fig. 1E-G). Actually these zones are formed mainly as a result of complex developmental processes in the yolk nucleus proper. The factors which govern their formation are still not known. The structure and histochemical composition of the central and lamellar zones remain more or less the same as those described for the previous stages, except that the number of lamellae and phospholipid bodies increases further. Very small mitochondrial granules also increase in number in the lamellar zone. Sometimes a few lamellae, lipid granules, and vacuoles are also seen in the central zone. The transitional zone surrounding the lamellar zone has a relatively small width and is more or less devoid of lamellae and phos-

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pholipid granules. Outside the transitional zone lies the broad peripheral zone of very loose consistency, which consists of a granular basophilic substance, granular mitochondria, and phospholipid granules of variable size. The basophilic substance stains mainly for RNA and protein, and some lipoprotein is present in this zone. This material, consisting of RNA, protein, and lipoprotein, appears to move away from the peripheral zone along with the mitochondria and phospholipid bodies of variable size, which simultaneously accumulate in the outer ooplasm. Finally all the lipid bodies are shifted to the peripheral zone of the Balbiani’s vitelline body where they form dense aggregations (Fig. 1H) and simultaneously become rich in triglycerides. At this stage the outer ooplasm shows a rapid accumulation of lipid (or triglyceride) and protein yolk bodies (Fig. IH and I). Except for some lipid bodies, which originate from the Balbiani’s vitelline body, most of the fatty yolk droplets and all the protein yolk bodies are formed without any morphological association with the Balbiani’s vitelline body (Fig. 1H and I). Jacquiert (1936) studied isolated Balbiani’s vitelline bodies (yolk nuclei) of spiders. He reported the lamellar structure of the Balbiani’s vitelline body based on polarized microscopy. He also found that the yolk nucleus, which constituted the major component of the Balbiani’s vitelline body, was soluble in acids and alkalies. It gave numerous protein reactions, particularly those of tyrosine and cysteine (sulfhydryl groups), and contains phospholipids associated with proteins. Jacquiert believed that the yolk nucleus of spider oocytes, which was associated with many mitochondria, acted as a site of intense secretion and a center of activity of the oocyte. Sareen (1963, 1964), using various histochemical tests, investigated the Balbiani’s vitelline body of the spider Lycosu chaperi. As in Araneus, the yolk nucleus substance also originates around the centriole in the juxtanuclear cytoplasm of the newly differentiated oocyte. With the growth of the oocyte it develops concentric lamellae. Under the phase-contrast microscope, the yolk. nucleus of Lycosa also shows concentric lamellae near the central zone, which are more closely packed close to this zone. Sareen also distinguished the same four zones in the fully differentiated Balbiani’s vitelline body of Lycosa as those described above for Araneus (Fig. 1G). These zones are formed by the complex differentiation of the yolk nucleus proper. ‘The yolk nucleus of Lycosa contains lipoproteins, unsaturated lipids, carbohydrates (acid and neutral mucopolysaccharides, and glycogen), proteins (which contain tyrosine, tryptophan, histitline, arginine, and sulfhydryl and disulfide groups), RNA, traces of DNA, iron., and vitamin C. The amount of these substances varies greatly in different zones of the yolk nucleus. The fourth zone, corresponding to the peripheral zone of Balbiani’s vitelline body in Aruneus, is also rich in RNA and mitochondria. Sareen could not distinguish granular mitochondria in the lamellar zone of the yolk nucleus as described for Araneus; however, he reported the presence of phosphcllipid

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granules. The yolk nucleus of Balbiani’s vitelline body in the oocytes of the spider Pholcus is rich in RNA (Fautrez, 1950; Urbani, 1955). Nath er ul. (1959) applied histochemical techniques for lipids to the study of Balbiani’s vitelline body in Plexippus and described four zones, namely, an innermost zone of diffuse lipids, a clear zone, a zone of circularly arranged and closely packed mitochondrial fibers, and an outermost zone of diffuse lipids, which were again due to complex differentiation of the yolk nucleus. It should be mentioned here that their clear zone, resembling the transitional zone of the Balbiani’s vitelline body in Aruneus (Fig. lG), is between the central and lamellar zones rather than between the lamellar and peripheral zones. The circularly arranged, closely packed mitochondria1 fibers described by Nath et ul. (1959) clearly correspond to the lamellae of S. S. Guraya (unpublished, 1978) and Sareen (1963, 1964). According to S. S. Guraya (unpublished 1978), the lamellae consist of lipoproteins and phospholipids. Krishna (1958) mentioned that the yolk nucleus of the Balbiani’s vitelline body in Lycosa birmanica was free of triglycerides, and that its concentric lamellae showed a thick coating of phospholipids covering lipoproteins of varying consistency, thus supporting the observations of S. S. Guraya (unpublished, 1978) on the histochemical nature of lamellae in Araneus. S. S. Guraya (unpublished, 1978) observed that phospholipid bodies mostly developed first in the lamellar zone of the yolk nucleus; however, a few lipid granules were also formed in its central zone (Fig. 1D-F).But according to Nath et uf. (1959) and Sareen (1963, 1964), the phospholipid bodies are restricted to the innermost or central zone of the yolk nucleus where they originate and finally pass toward the outermost zone as in Aruneus (Fig. IG-I). Meanwhile, the lipid bodies grow in size and become rich in triglycerides. The lipid droplets consisting mainly of triglycerides, which aggregate in the outermost zone of the yolk nucleus, are finally released into the outer ooplasm, as also demonstrated for Aruneus (Fig. 11). As in Araneus, most of the fatty yolk and all the protein yolk globules have no relation to the Balbiani’s vitelline body of Lycosa (Sareen, 1964). They arise independently in the ooplasm. Koch (1928) observed the origin of mitochondria and lipid bodies in the yolk nucleus of the Balbiani’s vitelline body in Tegenaria domestica. The fat droplets were formed in the clefts separating the lamellae and were also dispersed throughout the cytoplasm. This is also in agreement with the observations of Guraya (unpublished, 1978) in Araneus.

The fine structure of the Balbiani’s vitelline body in the oocytes of spiders has been studied (Sotelo and Trujillo-Cenoz, 1957; Andre and Rouiller, 1957; Andre, 1958; Costanzo, 1964). According to Sotelo and Trujillo-Cenoz (1957), light microscope preparations show the yolk nucleus to be composed of a central core of granular or vesicular elements and a cortex of concentrically arranged lamellae which clearly correspond to the central and lamellar zones of the yolk

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nucleus in the early oocytes of Aruneus (Fig. 1D-F). This also shows that Sotelo and Trujillo-Cenoz (1957) might have investigated only the yolk nucleus of young oocytes, as it is differentiated into four zones in the relatively large previtellogenic oocytes of Araneus (Fig. 1G). The electron microscope studies of Sotelo and Trujillo-Cenoz show that the cortex or lamellar zone of the Balbiani’s vitelline body is composed of concentrically arranged granular membranes of endoplasmic reticulum among which mitochondria and Golgi elements are intermingled. The core or central zone is made up of a so-called capsulated body which consists of vesicles and multivesicular bodies. The lipoproteins demonstrated histochemically by s. s. Guraya (unpublished, 1978) seem to constitute the ultrastructural elements of vesicles, endoplasmic reticulum, multivesicular bodies, Golgi bodies, and so on, of the Balbiani’s vitelline body (Fig. 2). However, Sotelo and Trujillo-Cenoz (1957) did not demonstrate phospholipid bsdies of histochemical techniques (Fig. ID-G). Andk and Rouiller (1957) have described the Balbiani’s vitelline body of the spider Tegenaria parientina as differing somewhat in fine structure from that found by Sotelo and Trujillo-Clenoz (1957). In Tegenaria the Balbiani’s vitelline body consists of four zones (Andre, 1958): (1) a central zone containing granules and vesicles, (2) a lamellar zone consisting of many concentric lamellae, (3) a zone of transition composed of structures ranging from granules to lamellae, and (4) a vesicular zone containing ergastoplasm, ribosomes, mitochondria, and Golgi bodies (Fig. 2). These various zones are clearly identical to the corresponding four zones described for the closely related genus Aruneus (Fig. 1G). Costanzo (1964) investigated :jtmctural, ultrastructural, and cytochemical aspects of the yolk nucleus in the Balbiani’s vitelline body of T. domestics oocytes, which at full development consists of three zones (Fig. 3) which clearly correspond to the first (or central), second (or lamellar), and third (or transitional) zones in Araneus. However, according to Costanzo, the peripheral lamellar zone has basophilic regions and the whole: yolk nucleus shows strong positive reactions for polysaccharides and proteins and weak reactions for proteins coupled with sulfhydryl groups; the external zone of the yolk nucleus gives positive reactions for lipids, however, the central and lamellar zones do not. But the histochemical investigations of various workers, including Guraya, have demonstrated the presence of lipoproteins and phospholipids in both the central and lamellar zones of the yolk nucleus. According to Urbani (1955) the lamellar part of the yolk nucleus consists of proteins but contains no RNA. The pallial layer, identical to the peripheral zone of the yolk nucleus in Araneus, is rich in RNA. Urbani (1955) did not describe lipoproteins in the lamellar zone, which includes the lamellae (or ultrastructural membranes of endoplasmic reticulum). To summarize the observations at the ultrastructural and cytochemical levels, it is evident that the Balbiani’s vitelline body of the spider oocyte is very complex both morphologically and cytochemically . The morphological complexity is mainly due to the differentiation of its yolk niicleus

FIG. 2. The ultrastructure of various components of the Balbiani’s vitelline body in previtellogenic oocyte of the spider T. parienfina. z.c., central zone; 2.1.. lamellar zone; z.t., transition zone; z.v., vesicular zone of yolk nucleus; b.b., plasma membrane with microvilli; ch., chorion; e.d., ergastoplasmic vesicles; e.e., stack of flattened ergastoplasmic pouches; e . n . , nucleolar buds in the nucleoplasm; ep., spheres of concentric ergastoplasmic lamellae; G, Golgi vesicles; g.i., granules in ergastoplasmic vesicles; m, mitochondria; m.n., nuclear membrane with pores; np.. nucleoplasm; nu., nucleolus. (From Raven. 1961, after Andre, 1958.)

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F I G 3 . Ultrastructure of the Balbiani's vitelline body from an oocyte of 7'. dowe.viim, showing the various zones of its yolk nucleus. zc, Central zone; zl, lamellar zone; I. lipid bodies. (From Costanzo. 1964.)

component into zones of variable width and ultrastructural organization, At the ultrastructural level the development and arrangement of membranous components vary greatly in these zones. B . SCORPIONS AND

MYRIAFQDS

In the yellow scorpion (Buthus hendersoni), the yolk nucleus of the Balbiani's vitelline body originates close to the nucleus of the young oocyte and forms a

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conspicuous juxtanuclear mass of hemispherical shape, with which are associated filamentous mitochondria (Sareen, 1970). In the dense substance of the yolk nucleus proper phospholipid bodies are also present; the large lipid bodies lying at the periphery of the yolk nucleus also contain triglycerides. The yolk nucleus gives positive reactions for proteins (tyrosine, arginine, and sulfhydryl groups), RNA, and carbohydrates. These observations have shown that the Balbiani’s vitelline body in the young oocytes of scorpions consists of a spherical yolk nucleus, mitochondria, and lipid bodies. It does not develop the morphological complexity discussed above for the Balbiani’s vitelline body of spider oocytes. But, as in spiders, in the scorpion it continues to persist in large oocytes in which enough yolk has already formed, although it does not participate directly in the formation of yolk. The fully formed yolk nucleus of the Balbiani’s vitelline body in the oocytes of the myriapod Geophilus consists of a central and peripheral zone (Koch, 1925); the latter usually has a concentric lamellar structure. Fat droplets pass from the yolk nucleus into the outer ooplasm; especially at later stages huge masses of fat arise from the yolk nucleus, as already discussed for the spider. Finally it moves to the periphery, flattens, and degenerates. In the early oocytes of Glomeris an acidophil perinuclear cap develops, which thickens and then breaks apart into fragments in the cytoplasm (Faure Fremiet et a l . , 1950). The paranuclear acidophil bodies consist of proteins and nucleoproteins. Mitochondria accumulate at their surface. Finally, the Balbiani’s bodies consisting of a yolk nucleus and mitochondria break down completely, and their components are distributed throughout the ooplasm before yolk deposition starts.

C. INSECTS Sareen and Kapal (1970), using histochemical techniques, have demonstrated the presence of a yolk nucleus of loose consistency in the oocytes of the bedbug, Cimex lecrularius. It originates around the centriole adjacent to the nuclear envelope in the young oocyte. With the growth of the oocyte, granular and filamentous mitochondria accumulate around the yolk nucleus which lies in the juxtanuclear ooplasm. With the deposition of yolk, the whole paranuclear complex of organelles forming the Balbiani’s vitelline body moves away from the germinal vesicle (nucleus) and attains a rounded form. Three zones can be distinguished: (1) The innermost zone stains with azure A, methyl greenPyronine G , and the Feulgen stain; (2) the intermediate zone shows yolk precursors (composed of carbohydrate and protein) and small phospholipid granules; and (3) the outermost zone is rich in lipid bodies (composed of triglycerides) and mitochondria. In more advanced oocytes, the Balbiani’s vitelline body migrates toward the posterior end of oocyte where it disintegrates and its various components are dispersed in the outer ooplasm. The fine structure of young and developing oocytes of various species of

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insects has been studied by several workers (see references in Beams, 1964; Norrevang, 1968). The presence of a well-organized Balbiani’s vitelline body (or bodies) has not been reported in their oocytes. In other words, the yolk nucleus, which develops great morphological complexity in spider oocytes, does not show appreciable development in the oocytes of insects. However, the perinuclear ooplasm of young oocytes in insects shows aggregations of ribosomes, and sparsely scattered elements of endoplasmic reticulum and mitochondria, which are not organized into a Balbiani’s vitelline body comparable to that of spidlers or scorpions. The ribosomes (or electron-dense granules) lying in the perinuclear ooplasm are presumed to be transferred from the nucleus to the ooplasm through the nuclear pores (see Beams, 1964; Norrevang, 1968).

D. CRUSTACEANS In Arremia, the Balbiani’s vitelline body consists of multivesicular bodies, dense bodies, and small vesicles (Anteunis et a f . , 1964). Later Anteunis et a f . (1966) described a peculiar assemblage of mitochondria, ergastoplasmic vesicles, and ribosomes in previtellogenic oocytes of Arternia. These constiluents were arranged in a paracrystalline pattern. It is difficult to interpret these structures which seem to appear only after glutaraldehyde fixation.

E. MOLLUSCS The oocytes of different molluscan species have a well-organized Balbiani ’s vitelline body as revealed by histochemical techniques and electron microscopy. Guraya (1969b), using histochemical techniques, has reported the presence of a dense yolk nucleus in the Balbiani’s vitelline body of young oocytes in the slug (Ariofimaxcofumbianus). It gradually develops adjacent to the nuclear envelope in the juxtanuclear ooplasm and finally forms a dense spherical or hemispherical mass. Histochemical tests have revealed the presence of protein, lipoprotein, and RNA in the yolk nucleus; phospholipid bodies and dictyosomes also arise in close morphological association with the yolk nucleus, forming a conspicuous Balbiani’s vitelline body in the juxtanuclear ooplasm. With the growth of the oocyte, the Balbiani’s vitelline body disintegrates, and its various components are distributed into the central ooplasm where they further multiply by growth and differentiation. The Balbiani’s vitelline body is also well developed in the oocytes of the slug (Laevicaufisalre). Its yolk nucleus attains a larger size while still in the juxtanuclear ooplasm (Fig. 4). However, it also stains for RNA, protein, and lipoprotein (S. S. Guraya, unpublished, 1978). Phospholipid bodies, dictyosomes, and mitochondria are also closely associated with the yolk nucleus and form ;I conspicuous Balbiani’s vitelline body. It has not been determined whether they

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Fic;. 4. The yolk nucleus (YN), dictyosome (D), mitochondria (M), lipid bodies (LB), and basophilic substances (BS) of the Balbiani’s vitelline body in a young oocyte of the slug L . a h .

originate in association with the Balbiani’s vitelline body (Fig. 4). Several phospholipid spheres aggregate on one side of the Balbiani’s vitelline body or in the outer ooplasm. The highly sudanophilic lipid droplets also form conspicuous aggregations of variable size in growing oocytes in which the slightly sudanophilic yolk nucleus substance and other components are distributed throughout the ooplasm (Figs. 5 and 6). In Spisula (Rebhun, 1956a,b) the basophilic yolk nuclei of the Balbiani’s vitelline body are found near the nuclear envelope in two forms: (1) platelike, consisting of double lamellae perforated by annuli, like the nuclear membrane, or (2) cylindrical to spherical or ellipsoidal. The latter are believed to derive from the former by the transformation of the lamellae into flat vesicles and the disappearance of annuli. In fine structure, the yolk nuclei of the Balbiani’s vitelline body in the oocytes of different species of molluscs have been related to the ergastoplasm or granular endoplasmic reticulum (Rebhun, 1956a,b, 1961; Bedford, 1966; Reverberi, 1966a, 1967; Ubbels, 1968; Bottke, 1973). They often have a concentric lamellar structure and may be associated with mitochondria and other components (Fig. 7). In the gastropod Bembicius, the Balbiani’s vitelline body consists of ergastoplasmic whorls and mitochondria (Bedford, 1966). Guraya (1969b) has suggested that the lipoprotein component demonstrated with histochemical techniques in the yolk nucleus substance of the Balbiani’s vitelline body may be derived from its ultrastructural membranous system

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FIG.5 . Histochemical preparation of an oocyte of the slug L. o h . showing accumulations of highly sudanophilic lipid droplets (LD) and slightly sutlanophilic elements of yolk nucleus subslance, which are not seen in the nucleus ( N ) . Frozen section stained with Sudan black B. ~ 4 4 0 .

FIG.6. Higher-power view of a portion of the oocyte in Fig. 5 showing the details of distribution of highly sudanophilic lipid droplets (LD) and slightly sudanophilic yolk nucleus substance, which are not seen in the nucleus (N). X 1000.

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which forms whorls; the R N A and protein component may be due to the amorphous or granular substance or ribosomes associated with the membranous system of the yolk nucleus (Fig. 7). The organization and amount of both membranes and granular or amorphous substance greatly vary in the yolk nuclei of different molluscan species (Rebhun, 1956a,b, 1961; Bedford, 1966; Reverberi, 1966a, 1967; Ubbels, 1968; Bottke, 1973). Their density is apparently dependent upon the development and compact organization of the membranous system described with electron microscopy. Guraya (1969b) has suggested that the main function of the yolk nucleus and its highly organized derivatives in the slug oocyte is to act as a site of the synthesis and distribution of their slightly sudanophilic material in the ooplasm of rapidly developing oocyte (Figs. 5 and 6), which at the ultrastructural level consists of elements of endoplasmic reticulum or ergastoplasm or vesicles of various sizes (Rebhun, 1956a,b, 1961; Reverberi, 1966a, 1967; Ubbels, 1968). Schmekel and Fioroni (1974) studied the ultrastructure of Balbiani’s vitelline bodies (yolk nuclei) during early cleavage of the gastropod Nassarius reticulatus L., which occur in all blastomeres from the 4- to the 16-cell stage. Most of the Balbiani’s vitelline bodies are spherical in shape, lie in the juxtanuclear cytoplasm, and contain many different organelles such as endoplasmic reticulum vesicles, mitochondria, Golgi stacks, ribosomes, and glycoprotein granules (Fig. 8). Although they are ultrastructurally heterogeneous, they fall into two categories or types (Fig. 8). Type I is a massive spherical accumulation of mitochondria embedded in an intermitochondrial substance which consists of both granules and filaments (Fig. 8A). Type I1 is a ball of radially arranged small Golgi stacks clustered around a center of Golgi vesicles and other organelles embedded in ground cytoplasm (Fig. 8B) structurally similar to the intermitochondria1 substance of type I. The functions of both types are unknown, but they are not involved in the synthesis or breakdown of yolk. F. ECHINODERMS

In the oocytes of A m d o n (Harvey, 1931; Cotronei and Urbani, 1957), the yolk nucleus arises in somewhat older oocytes through the concentration of basophilic substances-first in the form of granules which then unite to form a homogeneous, transparent, crescent-shaped band that surrounds the nucleus on one side. Cotronei and Urabni (1957) have compared the yolk nucleus of Antedorz with the ergastoplasm of other cells. According to Urbani (1955) the yolk nucleus contains much RNA, besides proteins and lipids; the last-mentioned probably consist of lipoprotein with which RNPs are associated. In the oocytes of sea urchins, the ergastoplasm or yolk nucleus (consisting of granular endoplasmic reticulum) is reported to be the sole, or by far the most prominent, component in the Balbiani’s vitelline body (Afzelius, 1957). It consists of concentric

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Fic. 7. Electron micrograph showing the yolk nucleus of a Spisulu vitellogenic oocytc: in the form of whorls of granular endoplasmic reticulum. Note also the yolk bodies and mitochondria inside the total structure. (From Rebhun, 1961.)

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0 0

0

A FIG.8 . Spherical yolk nuclei of types I ( A ) and 11 ( B ) from early cleavage of N . rdculnius L. (From Schmekel and Fioroni. 1974.)

double membranes with granules 150 8, in diameter arranged along the outer side. The membranes do not possess annuli. In the center of the yolk nuclei of sea urchins, yolk platelets or mitochondria may be found. In conclusion, it can be stated that the Balbiani’s vitelline body in the oocytes of echinoderms consists mainly of a yolk nucleus (or ergastoplasm) and mitochondria. G. PLATYHELMINTHES Koulish (1963, in an electron microscope study on oocytes in the trematode has described bodies in the cytoplasm close to the nucleus which have a density, a structure, and staining qualities in common with the nucleolus. They thus consist of densely packed ribosomelike granules with a finely granular substance between the granules.

Gorgoderina,

H . ACANTHOCEPHALA Guraya ( 1969a), using histochemical techniques, described the Balbiani ’s vitelline body in the oocytes of Acanthocephala belonging to the genus Prosrhenorchis. In the ooplasm of young oocytes, a dense, homogeneous substance accumulates gradually in the form of a crescent or cap adjacent to the nuclear envelope (Fig. 9A). Ultimately it forms a juxtanuclear mass with a spherical shape, which is called the yolk nucleus (Fig. 9B and C). It is reactive for protein, lipoprotein, and RNA. Some mitochondria and phospholipid spheres develop in

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FIG.9. Successive stages of acanthocephalan (Proshenorchis sp.) oocyte growth. (A-C) The development and morphology of various components of the Balbiani’s vitelline body. ( D ) Fraginentation of the yolk nucleus and distribution of other com]mnents of the Balbiani’s vitelline body. (From Guraya, 1969a.)

close association with the basophilic yolk nucleus which forms a substrate for them. The Balbiani’s vitelline body in the growing oocyte of Centrorhyrrchus corvi also consists of yolk nucleus, mitochondria, and lipid inclusions (Parshad and Guraya, 1977). A homogeneous subspherical or spherical mass known ;is the yolk nucleus is gradually formed in the juxtanuclearcytoplasm (Figs. 10 and 11). It consists of RNA, proteins (Fig. 1 l ) , lipoproteins, and phospholipids (Fig 10). Small, dense, irregular bodies which histochemically resemble the yolk nucleus

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FIG. 10. Histochemical preparation showing the sudanophilic yolk nucleus (YN) of the Bal~ . Cortical region of supporting syncytium; Id, lipid hiani’s vitelline body in an oocyte of C . U J ~ V css, droplets; n, nucleus, og, young oogonium; 0s. oogonial syncytium; yn, yolk nucleus. (From Parshad and Guraya, 1977.) FIG. 1 1 . Histochemical preparation of an acanthocephalan (C. corvi) oocyte showing proteins in the yolk nucleus (yn) and nucleus (n). Mercuric broniphenol blue. (From Parshad and Guraya, 1977.)

are also seen in the outer ooplasm. After the Balbiani’s vitelline body is fully developed in the juxtanuclear ooplasm, its various components are distributed in the outer ooplasm (Guraya, 1969a; Parshad and Guraya, 1977). The yolk nucleus fragments, and its derivatives, which take the form of spherical masses of variable size, shape, and density, are gradually distributed throughout the ooplasm where they form conspicuous structures (Figs. 9D and 12A). As the oocyte grows, these yolk nucleus masses multiply, presumably by growth and further fragmentation; their number increases without any direct morphological association with the nuclear envelope. In the fully developed oocyte, the yolk nucleus derivatives, mitochondria, and lipid inclusion bodies finally form a cortical layer (Fig. 12B). After fertilization the yolk nucleus substance is segregated into conspicuous masses of various sizes (Fig. 12C) which are distributed to different blastomeres .

111. Ralbiani’s Vitelline Body in Protochordates

A. TUNICATES Guraya ( 1 968b) has reported the presence of organized, dense, spherical bodies among the mitochondria and granular basophilic substance of the perinuclear ooplasm in young oocytes of the tunicate Molgula (Fig. 13A). With growth

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FIG. 12. Successive stages of growing acanthocephalan (Prosrhenorchis sp.) oocyte. (A and B) Morphology and distribution of yolk nucleus derivatives, lipid bodies, and mitochondria. (C) Distribution of yolk nucleus derivatives, lipid bodies and mitochondria. (From Guraya, 1969a.)

of the oocyte, their number and size increase (Fig. 13B and C). According to Guraya (1968b), the resulting large, dense inasses of spherical shape are comparable to the yolk nuclei of other animal species, as they consist of RNA, protein, and lipoprotein. They do not show any morphological association witlh the mitochondria which are, however, dispersed in the perinuclear ooplasm ,along with the uniformly distributed finely granular basophilic substance consisting of

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FIG. 13. Stages (A-E) in the growth and di\tribution of ooplasmic organelles in the developing oocyte of the tunicate Molgula (From Guraya, 1968b.)

RNA and protein. As the development of the oocyte proceeds, the basophilic substance of the yolk nuclei is distributed throughout the central ooplasm, along with the other cell components, where the yolk bodies consisting of protein, carbohydrate, and lipids first originate (Fig. 13D and E). In young oocytes of Herdmania (Guraya, 1972a) one basophilic spherical yolk nucleus is seen in the juxtanuclear ooplasm instead of the several yolk nuclei which develop in the perinuclear ooplasm of young oocytes of Molgula (Guraya, 1968b). However, its histochemical reactions are the same, and it is reactive for protein, lipoprotein, and RNA. The yolk nucleus in the oocytes of Herdmania stands out from the surrounding cytoplasm because of its greater density. Surrounding the dense yolk nucleus, there are also present mitochondria, a granular basophilic substance (rich in RNA), and a lipid inclusion body in the juxtanuclear

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ooplasm; some mitochondria, however, form a close morphological association with the yolk nucleus. After the yolk nucleus has fully developed in the juxtanuclear ooplasm, it moves away from the nucleus (or germinal vesicle) and eventually fragments. Its derivatives, along with the mitochondria and finely granular basophilic substance, are distributed throughout the central ooplasm, where the yolk bodies first originate, suggesting a role in yolk formation. The yolk nuclei and their derivatives correspond to the ultrastructural membranous system which occurs in a variety of forms in growing oocytes of different tunicates (Kessel and Kemp, 1962; Mancuso, 1964; Kessel, 1966); their membranous system is closely associated with variable amounts of a granular or amorphous substance which may be composed of RNA and protein demonsmated with histochemical techniques. Based on the composition of yolk nuclei, Guraya (1972a) has suggested that they may be important sites for the formation of cytoplasmic membranes and RNPs during the growth of tunicate oocytes. Similar functions for the yolk nucleus in tunicates have also been proposed by Mancuso (1964). In summary, it can be stated that the basophilic yolk nuclei of the tunicate oocyte consist of endoplasmic reticulum and RNP particles, as discussed above for echinoderms, molluscs, and so on. But they do not develop any appreciable morphological association with other cell components to form a complex Balbiani’s vitelline body.

B. Amphioxus In young oocytes of Brunchiostoma (Amphioxus) treated with histochemical techniques a homogeneous substance accumulates gradually in the form of a crescent or cap adjacent to the nuclear envelope (Guraya, 1968a). Ultimately, it forms a juxtanuclear mass called a yolk nucleus which also associates with granular mitochondria to form a Balbiani’s vitelline body. The yolk nucleus of the Amphioxus oocyte is reactive for RNA, protein, and lipoprotein. After the Balbiani’s vitelline body has fully differentiated, it moves away from the germinal vesicle and eventually breaks up into masses of variable size. As the growth of the oocyte proceeds, these masses consisting of yolk nucleus substance and mitochondria multiply, probably by growth and further differentiation. Ultimately, yolk nucleus substance and mitochondria are distributed throughout the central ooplasm. The protein yolk bodies first originate in association with the masses consisting of the yolk nucleus substance and mitochondria. From the electron micrographs of Amphioxus oocytes presented by Reverberi (1966b), it appears that the yolk nucleus substance described by Guraya (1968a) includes elements of endoplasmic reticulum associated with numerous ribosomes 01 RNP particles. Thus the yolk elements in the Amphioxus egg apparently arise directly under the influence of and within a well-developed system of granular endoplasmic reticulum or yolk nucleus substance (or ergastoplasm); the mitochondria

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27 1

closely associated with the yolk nucleus substance may be the source of energy required in the formation of yolk material. In conclusion, young oocytes of Amphioxus develop a Balbiani’s vitelline body which consists of a yolk nucleus and mitochondria. The yolk nucleus, containing RNA, protein, and lipoprotein, is comparable to ergastoplasm as it consists of endoplasmic reticulum and ribosomes.

IV. Balbiani’s Vitelline Body in Vertebrates A. ELASMOBRANCHS Applying histochemical techniques, S. S. Guraya (unpublished, 1978) recently examined the details of the origin, differentiation, structure, and histochemical composition of Balbiani’s vitelline body in the oocytes of the dogfish (Scoliodon sorrukowuh), which consists of a yolk nucleus, mitochondria, and lipid bodies. With the growth of the oocyte, the yolk nucleus develops gradually adjacent to the nuclear envelope in the juxtanuclear ooplasm (Fig. 14A-C). Ultimately, it forms a large juxtanuclear mass of subspherical shape (Fig. 14D). From the beginning of its origin and differentiation, two zones can be distinguished in the yolk nucleus of the dogfish oocyte (Fig. 14B and C): (1) a central spherical or hemispherical zone of a more dense, organized nature, and (2) a peripheral zone of loose consistency; both zones may be separated by a transitional zone of clear cytoplasm (Fig. 14C). Sometimes several dense, organized bodies resembling the central zone of the yolk nucleus are seen in the oukr intensely basophilic substance which has a granular appearance (Fig. 14C and D). The various components of the yolk nucleus are reactive for lipoprotein, protein, and RNA; the peripheral zone is relatively richer in RNA than the central dense zone and the other dense, organized bodies of the juxtanuclear ooplasm. Mitochondria are associated with the outer basophilic substance. The phospholipid bodies are present in both zones of the yolk nucleus. After the Balbiani’s vitelline body has attained full development and differentiation, its various components such as the yolk nucleus derivatives, mitochondria, and phospholipid bodies are distributed throughout the central ooplasm where they multiply further (Fig. 14E and F). Then the protein yolk bodies begin to develop in the regions of the ooplasm where the various organelles of Balbiani’s vitelline body are distributed (Fig. 14G). No electron microscope studies have been carried out on the Balbiani’s vitelline body of cartilaginous fish. But in light of the various studies summarized above, it can be stated that the yolk nucleus of the dogfish oocyte is differentiated into zones, apparently as a result of the complex development and arrangement of its ultrastructural components (possibly endoplasmic reticulum and ribosomes), as already discussed for spiders, molluscs and so on.

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FIG. 14. The origin, development, morphology, and behavior of the yolk nucleus and otkler cell components of the Balbiani’s vitelline body in successive stages (A-G) of oocyte growth in the dogfish (S. sorrakowah). BS, Basophilic substance; CZ, central zone of yolk nucleus; M , mitochondria; N, nucleus of oocyte showing nucleoli; PY, protein yolk; PZ, peripheral zone of yolk nucleus; TZ, transitional zone of yolk nucleus; YN, yolk nucleus; YND, yolk nucleus derivatives.

273

BALBIANI’S VITELLINE BODY

B. TELEOSTS Guraya (1963b, 1965a) has described the origin, morphology, and histochemical composition of the Balbiani’s vitelline body in oocytes of teleosts (Channa murulius and Heteropneustes fossilis). A granular substance begins to arise in the form of a crescent or cap adjacent to the nuclear envelope. It is strongly basophilic and contains RNA and protein (Fig. 15A-D). The amount of this basophilic substance increases further, and it forms a large juxtanuclear dense mass of hemispherical shape, which is called the yolk nucleus (Fig. 15E-G) (see also Kraft and Peters, 1963). Besides the RNA and protein, the yolk nucleus of the Balbiani’s vitelline body in the teleost oocyte also contains a faintly sudanophilic lipoprotein substance (Figs. 16 and 17) (Guraya, 1965a). The development of the yolk nucleus in the oocytes of teleosts is accompanied by a proliferation of mitochondria and phospholipid bodies, for which the former forms the substrate (Figs. 16 and 17). Thus the Balbiani’s vitelline body of the

@ @ @ C NUCLEUS DENSE BODY

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YOLK-NUCLEUS SUBSTANCE

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NUCLEUS

I FIG. 15. Oocytes of fish, drawn from material fixed in Zenker’s fluid and stained with methyl green pyronine. (A) Very young oocyte of Chtrrlrw. showing a dense body. (B) Very young oocyte of Chtrrincr. showing hasophitic yolk nucleus substance and a dense body adjacent to the nuclear envelope. (C) Very young oocyte of Hrrerc~p,irirsrrs,showing yolk nucleus substance in the perinuclear cytoplasm. (D-G) Young oocytes of Chtrriritr. showing the growth of a yolk nucleus and a dense body. ( H ) Growing oocyte of Chtr~rrici.showing the proliferation of basophilic masses from the yolk nucleus. ( I ) Large previtellogenic oocyte of Chtrnrrcl, showing a homogeneous yolk nucleus and uniformly distributed basophilic substance proliferated from the yolk nucleus. (From Guraya, 1963b.)

274

SARDUL S . GURAYA

FIG. 16. Histochemical preparation of very young oocyte of Chutmu. showing slightly sudanophilic yolk nucleus substance, mitochondria, and deeply sudanophilic lipid granules of the Balbiani’s vitelline body (BVB) which lies in the juxtanuclear cytoplasm. The nucleus (N) is sudanophobic. Frozen section stained with Sudan black B . x 1000.

teleost oocyte consists of a yolk nucleus, mitochondria, and phospholipid bodies, as also reported for the dogfish oocyte. Nayyar (1964) found the Balbiani’s vitelline body of Heteropneustes to be composed of lipids and mitochondria. and reported that its function was to initiate the synthesis of lipids. He appears to have overlooked the yolk nucleus proper, which forms the substrate for other components such as the mitochondria and phospholipid bodies (Guraya, 1963b, 1965a). The Balbiani’s vitelline body in the oocytes of Channa may have one or two dense bodies composed of RNA and protein (Fig. 15D-G) (Guraya, 1963b), which appear to be the extruded nucleoli (Kraft and Peters, 1963; Guraya, 1965a). The dense body is separated from the granular substance of the yolk nucleus by a clear zone. Gopal Dutt (1964) described a similar dense structure in the yolk nucleus of Anabas scandens oocytes, which originated adjacent to the nuclear envelope and was reactive for RNA. He did not pay much attention to the mitochondria and lipid bodies closely associated with the yolk nucleus to form a Balbiani’s vitelline body. Abraham et al. (1966) found the yolk nucleus of teleost oocyte extremely basophilic and rich in RNA. Livni (1971) reported the following enzymes to be concentrated in the Balbiani’s vitelline body of three

BALBIANI’S VITELLINE BODY

275

F I G 17. Histochemical preparation o f a growing oocyte o f Chunnrr, showing a slightly sudanophilic yolk nucleus and mitochondria1 elements of the Balbiani’s vitelline body ( B V B ) which has moved away from the nuclear envelope. The nucleus ( N ) i s sudanophobic. Frozen section stained with Sudan black B. x 1000.

species of teleosts: glucose-6-phosphate dehydrogenase, a-glycerophosphate dehydrogenase, and succinate dehydrogenase. Yamamoto ( 1 964) found that the yolk nucleus of the Balbiani’s vitelline body in the oocytes of Oryzias was composed of a network of small granules which, according to him, were probably RNP and of nuclear origin. This is supported by the fact that Ulrich (1969), in the teleost Panio, described the migration of nuclear ribosomes through the pores of the envelope into the perinuclear cytoplasm where they aggregated in small groups and often became closely associated with mitochondria. Wegmann and Gotting (1971) have also reported a network of granules associated with development of the Balbiani’s vitelline body in the teleost Xiphophorus. According to Riehl (1976, 1978), the Balbiani’s vitelline -body (yolk nucleus) in young oocytes of the freshwater teleosts Normacheilus barbatulus (L.) and Phoxirius phoxirzus (L.) consists of a nucleolar substance and mitochondria, and partly of dictyosomes (Figs. 18 and 19). His nucleolar material, which corresponds to the yolk nucleus substance of light microscopy (Fig. 15) (Guraya, 1963b, 1965a), leaves the nucleus through the pores of the nuclear envelope and accumulates in the juxtanuclear cytoplasm (Figs. 18 and 19); it mainly consists of RNA as

276

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FIG. 18. Electron micrograph of a portion of a very young oocyte of the fish N. barbarulu.! (L.), showing the nucleolar substance (6), mitochondria (7), dictyosome (8), and ribosomes of a developing Balbiani’s vitelline body adjacent to the nuclear envelope. Nucleoplasm ( I ) , nuclear envelope (2), and nuclear pores ( 3 ) are also seen. (From Riehl, 1976.)

confirmed with autoradiographic studies (Riehl, 1978). Clerot (1976) has also investigated the ultrastructural aspects of the juxtanuclear complex of organelles in young oocytes and spermatocytes of several species of cyprinid fish. ‘They consist of intermitochondrial “cement, annulate lamellae, mitochondria, and so on. Annulate lamellae appear to be part of the intermitochondrial cement which is identical to the nucleolar or yolk nucleus substance of other workers and also derives from the nucleus. Intermitochondrial cement is believed to be involved in active mitochondria1 biogenesis. As the growth of the oocyte in teleosts proceeds, the Balbiani’s vitelline body or juxtanuclear complex of organelles separates from the nuclear envelope and becomes spherical in shape (Figs. 15H and I and 17) (Chaudhry, 1952; Guraya, 1963b, 1965a; Kraft and Peters, 1963; Gopal Dutt, 1964; Nayyar, 1964; Wegmann and Gotting, 1971; Guraya et al., 1975, 1976). According to Gmaya (1963b, 1965a), the yolk nucleus of the Balbiani’s vitelline body produces from its peripheral portions a basophilic substance that is rich in RNA. This subs1:ance moves into the outer cytoplasm as irregular masses (Fig. 15H). The mitochondria also increase in number and form association with the masses of basophilic ”

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F i c . 19. Electron micrograph of a portion o f a young oocyte o f the fish N . hrrrhritrtlus, showing the yolk nucleus substance (9) associated with mitochondria, which originates from the nucleolar substance (6); the latter resembles the nucleoli ( 5 ) very closely. (From Riehl, 1976.)

material during previtellogenesis. Finally, the Balbiani’s vitelline body shifts to the cortical ooplasm (Figs. 151 and 17) where it finally disintegrates (Kraft and Peters, 1963). The protein yolk bodies first originate in regions of the ooplasm rich in RNA-containing yolk nucleus substance and mitochondria derived from the Balbiani’s vitelline body (Guraya, 1965a). Beams and Kessel (1973) made correlative light and electron microscope studies on young oocytes of the trout, that is, during previtellogenesis and early stages of vitellogenesis. They described the behavior of the yolk nucleus in the Balbiani’s vitelline body from its origin in the youngest oocyte to its disappearance at the beginning of vitellogenesis. It first originates adjacent to the juxtanuclear ooplasm, where it takes the form of a duplex body composed of a nonbasophilic component (an idiosome and a highly basophilic portion, the pallial substance, as already described) in the yolk nucleus of dogfish oocytes (S. S. Guraya, unpublished, 1978). The electron micrographs of Beams and Kessel (1973) reveal the yolk nucleus of trout oocyte to be a dense, finely granular substance apparently unrelated to the Golgi apparatus or mitochondria. Beams and Kessel (1973) note that its exact role in oogenesis is still not clear but that it probably constitutes an essential precursor substance (perhaps RNA) necessary

278

SARDUL S. GlJRAYA

for oocyte growth and vitellogenesis, which corresponds to the RNA-rich yolk nucleus substance of Guraya (1963b, 1965a). Riehl (1976) believes thar the Balbiani’s vitelline body (yolk nucleus) has no relationship to the formation of yolk in the oocytes of N . barbatulus. C. AMPHIBIANS Employing histochemical techniques, Guraya (1965a, 1968c, 1970a) has investigated the origin, development, structure, and histochemical composition of para- or juxtanuclear complexes of organelles and inclusions in the oocytes of different species of amphibians (toads and frogs). The yolk nucleus substance of loose consistency, which is reactive for RNA, protein, and some lipoprotein, first accumulates adjacent to the nuclear envelope in young oocytes (Fig. 20A and B). With the growth of the oocyte the yolk nucleus substance, along with the mitochondria, continues to accumulate in the perinuclear or juxtanuclear ooplasm but remains of loose consistency and stains faintly with Sudan black B (Fig. 21). Its developmentand differentiation vary greatly among different species of amphibians, and it does not form conspicuous aggregations in young oocyles of the toad Sufo americanus (Fig. 22A and B). Slightly sudanophilic components consisting of the yolk nucleus substance and mitochondria are sparsely distributed in the perinuclear ooplasm. Extremely sudanophilic lipid granules are also seen. The basophilic yolk nucieus substance of amphibian oocytes, which is closely associated with mitochondria, does not consist of the compact, organized structures described as yolk nuclei in the oocytes of other animal species (Guraya, 1965a, 1968~). The yolk nucleus substance of amphibian oocytes is clearly identical with the perinuclear concentration of tiny granules and short rods, intermitochorrdrial cement, annulate lamellae and membrane systems arranged in a circular pattern, and large masses of juxtanuclear vesicles seen in electron microscope studies (Figs. 23 and 24) (Kemp, 1956; Lanzavecchia, 1961; Balinsky and Devis, 1963; Kessel, 1963; Anderson and Choymyn, 1964; other references in Wischnitzer, 1966; Clerot, 1968). These electron microscope studies also indicate that the yolk nucleus substance of histochemical studies (Guraya, 1965a, 1968c, 1970a) shows several variations in subcellular organization in the oocytes of different amphibians but is not organized into dense structures. The vesicles and annulate lamellae of the yolk nucleus substance have been found to arise from the outer lamella of the nuclear envelope (Fig. 24). Besides the membranous components and mitochondria, the Balbiani’s vitelline body of amphibian oocytes also shows an amorphous granular substance at the ultrastructural level (Fig. 23), which may consist of RNA and protein demonstrated with histochemical techniques; the lipoprotein component of the yolk nucleus substance may be due to its membranous elements (Guraya, 1968~).

BALBIANI’S VlTELLlNE BODY

279

FIG.20. Successive stages (A-F) of a growing oocyte of the toad Bufo woodhousii, showing the development, differentiation, morphology, and distribution of various components of Balbiani ‘s vitelline bodies which form conspicuous ooplasmic structures. (From Guraya, 1968c .)

The aggregations of mitochondria which appear during oocyte development have been generally confused with the yolk nuclei (Wartenberg, 1962; Balinsky and Devis, 1963; Wischnitzer, 1966), and the sparsely distributed basophilic yolk nucleus substance which forms the substrate for the mitochondria in Balbiani’s vitelline bodies has been overlooked (Figs. 20C-F and 25) (Guraya, 1965a, 1968c, 1970a). The yolk nucleus substance and mitochondria form slightly sudanophilic masses which show many variations in form, size, density,

280

SARDUL S . GURAYA

FIG.21. Histochemical preparation of a portion of a bullfrog (R.caresbeiana) ovary, showing the localization of sudanophilic Balbiani’s vitelline bodies (arrows) in the perinuclear ooplasrn, which are finally shifted to the peripheral ooplasrn o f the growing oocyte. The nuclei (N) are sudanopliobic. Frozen section stained with Sudan black B. X400.

and distribution in the growing oocytes of different amphibian species (Figs. 20C-E, 21, 22, 25, and 26-28). Some workers have identified the yolk nucleus substance as the vesicular cytoplasm or clusters of vesicles or intermitochondrial cement forming part of the “yolk nucleus” or “aggregation of mitochondlria” (Wischnitzer, 1962, 1964, 1966; Balinsky and Devis, 1963; Clerot, 1968). Similarly, the vesicles and granular substance of the yolk nucleus substance are also seen in close association with the aggregations of mitochondria described in other studies (see references in Wischnitzer, 1966). Kessel (1963) has clearly dktinguished the yolk nucleus substance from the mitochondria in Balbiani’s vitelline bodies and has investigated their origin and behavior in greater detail. Guraya

BALBlANl’S VITELLINE BODY

28 1

FIG.22. Histochemical preparation of portions of a toad ( B . americanus) ovary showing the relatively poor development of Balbiani’s vitelline bodies (arrows) in successive stages (A and B) of oocyte growth. Deeply sudanophilic lipid bodies and slightly sudanophilic organelles (yolk nucleus substance, mitochondria, Golgi bodies, and so on) are sparsely distributed throughout the ooplasm, which can be distinguished in contrast to the sudanophobic nuclei (N). Frozen sections stained with Sudan black B. ~ 8 3 0 .

282

SARDUL S . GURAYA

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FIG. 23. Balbiani's vitelline body formation in the amphibian oocyte. (A) Nucleolar materiiil (n) which passes through a discontinuity in the nuclear envelope (ne) and comes in contact with several adjacent mitochondria (m). (B) The nucleolar or yolk nucleus substance binds the mitochondria into a mass, the Balbiani's vitelline body. (C) The Balbiani's vitelline body consisting of yolk nucleus substance (yn) and mitochondria migrates toward the periphery of the oocyte (arrow). See also Figs. 20 and 21. (From Wischnitzer, 1966.)

BALBIANI'S VlTELLlNE BODY

283

FIG. 24. Formation of the annulate lamellae in Neclurus. The sequence of events appears to involve blebbing of the outer membrane of the nuclear envelope (A), simultaneous release of the blebs (B), movement of the row of blebs toward the periphely (C), formation of several rows of released blebs (D), fusion of some of the vesicles (E), and continued fusion resulting in the formation of cisternae (F). (From Wischnitzer, 1966.)

(1965a, 1968c, 1970a) has suggested that the conspicuous cytoplasmic masses or Balbiani's vitelline bodies which differentiate in developing amphibian oocytes (Figs. 20C-E, 21, 22, and 25-28), are not only sites of the accumulation of mitochondria but also of accumulation of the yolk nucleus substance which

284

SARDUL S . GURAYA

BALBIANI’S VITELLINE BODY

285

consists of elements of endoplasmic reticulum and a granular substance composed of RNA and protein. This close anatomical relationship indicates a close functional relationship between the mitochondria and yolk nucleus substance in Balbiani’s vitelline bodies of developing amphibian oocytes. Clerot (1968) believes that juxtanuclear mitochondrial groups associated with intermitochondrial cement form important centers for mitochondria1 multiplication in the amphibian oocyte. Some ooplasmic masses or Balbiani’s vitelline bodies consisting of a yolk nucleus substance and mitochondria are transformed into lipid droplets (or lysosomes) in the ooplasm of growing oocytes in Rana caresbeiana (Guraya, 1968~). Such fatty metamorphosis of cell organelles is a conspicuous feature of previtellogenic oocytes in Rana pipiens (Fig. 27B) (Guraya, 1970a). These ooplasmic masses of cell organelles undergoing fatty metamorphosis are clearly identical with the “yolk nuclei” of Kemp (1956) and the “fatty yolk centers” of Ward (1962) in the oocytes of R . pipiens. The latter investigator called the yolk nucleus substance the “fatty yolk” and the mitochondria the “fatty yolk mitochondria’ ’; both cell components are considerably altered during their fatty metamorphosis. As the fatty metamorphosis is advanced, both the yolk nucleus substance and mitochondria of Balbiani ’s vitelline bodies are gradually transformed into lipid droplets (or possibly lysosomes) containing first phospholipid and then phospholipid and triglyceride (Guraya, 1970a). Kemp (1956) has also stated: “In the peripheral cytoplasm a yolk nucleus appears to consist of an aggregate of mitochondria together with lipochondria of various sizes. It is believed that the larger lipochondria are formed by the coalescence of smaller ones, which seem to come from the ground cytoplasm.” From this statement it appears that the amorphous yolk nucleus substance could not be distinguished from the ground cytoplasm. Results of cytochemical studies have clearly demonstrated that the precocious aggregations of lipid droplets or lipochondria (or lysosomes) which develop in the oocytes of R . pipiens are formed by the fatty metamorphosis in the yolk nucleus substance and mitochondria of the Balbiani’s vitelline body (Guraya, 1970a). The biological significance of this phenomenon could not be determined in the oocytes of frog (R. pipiens and R . catesbeiana), as no such transformation of cell organelles (yolk nucleus substance and mitochondria) into fatty droplets was observed in developing oocytes of various species of toad (Guraya, 1965a, 1968~).

FIG.25. Histochemical preparation of two growing previtellogenic oocytes (A and B) of the toad B . woodhousii, showing the accumulation and distribution of Balbiani’s vitelline bodies (arrows) which consist of slightly sudanophilic yolk nucleus substance and many sudanophilic granular mitochondria. See details in Fig. 21. Nuclei (N) are also seen. Frozen sections stained with Sudan black B . x840.

286

SARDUL S . GUKAYA

FIG.26. Histochemical preparation of growing previtellogenic oocytes ( A and B) of the toild B . umcriccurus, showing the development and differentiation of Balbiani's vitelline bodies (arrows) The slightly sudanophilic yolk nucleus substance, granular mitochondria, and so on, are more-or-less uniformly distributed throughout the ooplasm. The nuclei (N) are sudanophobic. Frozen sections stained with Sudan black B . ~ 8 3 0 .

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287

Guraya (l965a, 1968c, 1970a), using histochemical techniques, has demonstrated local differentiations and variations in the same system of the yolk nucleus substance in Balbiani’s vitelline bodies of developing amphibian oocytes. With electron microscopy very little work has been carried out on the origin of, differentiation of, and various changes in the cytoplasmic organelles in Balbiani’s vitelline bodies of growing oocytes in different amphibians (Wartenberg, 1962; Wischnitzer, 1962, 1964, 1966; Balinsky and Devis, 1963; Kessel, 1963). Almost all the electron microscope studies have described the Golgi elements or dictyosomes in amphibian oocytes (Wischnitzer, 1966), but none has examined their origin and the morphogenetic changes in the yolk nucleus substance and other cellular organelles of Balbiani’s vitelline bodies at the subcellular level; it is essential to study them extensively throughout the oocyte growth of amphibians (Wartenberg, 1962; Balinsky and Devis, 1963). since isolated observations always lead to confusion. It will be very interesting to know whether the entire membranous system of the yolk nucleus substance in the amphibian oocyte is derived directly or indirectly from the nuclear envelope, or if it is also formed by the transformation of a granular amorphous substance abundantly present in the ooplasm. Kessel (1963) directed his attention to this important problem of morphogenetic changes in organelles of amphibian oocytes. In conclusion, it can be stated that the yolk nucleus substance of Balbiani’s vitelline bodies in amphibian oocytes is not organized into dense bodies comparable to the yolk nucleus of spider oocytes. D. REPTILES Guraya (1963a, 1968d), using histochemical techniques, investigated the origin, differentiation, development, structure, and histochemical nature of Balbiani’s vitelline body in the oocytes of different species of reptiles including snakes, lizards, and turtles, which consists of a yolk nucleus, mitochondria, Golgi bodies, and diverse lipid bodies. The yolk nucleus of the Balbiani’s vitelline body differentiates adjacent to the nuclear envelope and finally forms a juxtanuclear mass (Fig. 29A-P). It is a homogeneous, spherical or subspherical mass, and histochemical tests indicate that it is rich in RNA and protein; some lipoproteins are also present, as it stains faintly with Sudan black B (Fig. 30) (Guraya, 1965b, 1976a). The Balbiani’s vitelline body in oocytes of reptiles is a center of high synthetic or metabolic activity, as different types of lipid inclusions (L,. L,,and L,) and mitochondria appear in abundance in association with a yolk nucleus which forms a substrate for them (Fig. 30). L, bodies consist of phospholipids and triglycerides, L2bodies are made up of phospholipids, and spheres consist of triglycerides. In winter oocytes of the snake Bungarus cueruleus, most of the highly sudanophilic lipid droplets which obscure the yolk nucleus and mitochondria of the Balbiani’s vitelline body (Fig. 30A) disappear

288

SARDUL S . GURAYA

BALBIANI’S VlTELLlNE BODY

289

FIG.28. Histochemical preparation of a growing oocyte of the frog ( R . catesbeiunu), showing the localization of sudanophilic Balbiani’s vitelline bodies (arrows) in the cortical ooplasm where they form conspicuous structures. N , Nucleus. Frozen section stained with Sudan black B. ~ 9 5 0 .

(Fig. 30B). Eventually, the Balbiani’s vitelline body disintegrates, and its RNA-containing yolk nucleus substance, as well as the other inclusions and organelles, are distributed throughout the cytoplasm. However, in oocytes of the wall lizard (Hemidactylus), fixed in March through April, the yolk nucleus continues to persist as an organized structure through various stages of previtellogenesis (Fig. 3 1A) (Guraya, 1968d). This persisting yolk nucleus of spherical shape is a dense, organized structure which stains for protein, lipoprotein, and RNA. It does not form a close morphological association with other cell components such as the mitochondria, lipid bodies (L), spaces or canals, diffuse sudanophilic substance, and dense bodies, which are arranged in three zones

FIG. 27.

Histochemical preparations of growing previtellogenic oocytes of a toad (Bufo

sromuticus) and a frog (Rana pipiens). (A) The localization of some Balbiani’s vitelline bodies

(arrows) in the cortical ooplasm of toad, which consist of a slightly sudanophilic yolk nucleus substance, mitochondria, and a few deeply sudanophilic lipid bodies. (B) The distribution of Balbiani’s vitelline bodies of variable size throughout the ooplasm (arrows) of frog oocyte some of which are in the peripheral ooplasm and increase in their sudanophilia. Frozen sections stained with Sudan ; ~420. black B. (A) ~ 9 8 0(B)

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0 A

FIG. 29. Young oocytes of reptiles, drawn from material fixed in 2enker's fluid and stained with methyl green-pyronine, showing the development and differentiation of the yolk nucleus. (A) From a snake (Naja) ovary. (B) From a snake (Boiga)ovary. (C) From a house lizard (Hemidactylus) ovary. (D)From a garden lizard (Calotes)ovary. (E) From a snake (Bungarus) ovary. (F) From a snake (Naja) ovary. ( G ) From a snake (Boiga) ovary. (H) From a house lizard (Hemidactylus) ovaiy. (I) From a garden lizard (Culotes) ovary. (J) From a turtle (Lissemys) ovary. (K and L) 0ocyt:es of Boiga. showing dense bodies and engulfed follicle cell nuclei in association with the yolk nucleus. (M) From a Naja ovary. (N) From a Hemidactylus ovary. (0)From a Lissernys ovary. (P) From a spiny-tailed lizard (Uromastix) ovary. (From Guraya, 1963a.)

FIG. 30. Histochemical preparations of oocytes from the ovary of a snake (Bungarus cueruleus). (A) Origin and development of the yolk nucleus (lightly sudanophilic), mitochondria, and deeply sudanophilic lipid droplets (LD) of the Balbiani's vitelline body (BVB) in the juxtanuclear ooplasm; the nucleus (N) is sudanophobic. (B) Winter oocyte showing a large Balbiani's vitelline body (I3VB); sudanophilic lipid droplets of large size are not seen. The outer ooplasm ( 0 )has relatively few cell components. Follicular epithelium (FE) containing lipid bodies (L)is also seen. (From Giiraya, 1976a.)

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SARDUL S. GURAYA

FIG.31. Histochemical preparations of portions of growing previtellogenic oocytes from the ovary of a house lizard (Hemiductyhs). (A) The dense spherical yolk nucleus (YN) is surrounded by three zones ( I , 2, and 3) of cell components, including filamentous mitochondria (M).lipid bodies (L2),canals or spaces (C), a diffuse sudanophilic substance (DSS), and its dense bodies (DB).(B) The nucleus or germinal vesicle (GV),cortical layer of cell components (CLC), and stntified follicular epithelium (FE). (From Guraya, 1968d.)

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293

around the yolk nucleus (Figs. 31A and 32). The physiological significance of this dissociation of various cell components from the yolk nucleus has not been determined. The mitochondria stain for lipoprotein, the LL bodies consist of phospholipid, the spaces do not contain material demonstrable with histochemical techniques, and the ooplasm containing the diffuse sudanophilic substance and dense bodies (Figs. 31A and 32) shows lipoproteins, protein, and RNA. Eventually, the persisting yolk nucleus disintegrates, and its contents as well as the other surrounding cell components gradually accumulate in the cortical ooplasm of oocytes (Fig. 318) which are ready to form yolk bodies. In the oocytes of the lizard Anofis (S. S. Guraya, unpublished observations), the yolk nucleus of the Balbiani's vitelline body also moves away from the nuclear envelope and continues to persist as a large, spherical, homogeneous structure in the center of the ooplasm, which is reactive for protein, lipoprotein, and R N A . Mitochondria and lipid bodies (composed of phospholipids), how-

F I G 32. 1)iagrainmatic presentation of yolk nucleus of dense organization and the three zones ( I . 2 , and 3) of various cell coinponents shown in Fig. 31A. (From Guraya. I9hXd.)

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SARDUL S. GLJRAYA

ever, still continue to be associated with this persisting yolk nucleus, whereas most of the other components, including L,, L2,and L bodies, and mitochondria, disassociate and move away from the yolk nucleus to occupy the cortical ooplasm where they constitute a cortical layer. Meanwhile, the yolk nucleus releases its substance from the peripheral portions into the outer ooplasm. To the best of our knowledge, no electron microscope studies have been carried out so far on the Balbiani’s vitelline body of reptilian oocytes. Electron microscope studies on this paranuclear complex of organelles, which becomes large and complex, will be of interest. E. BIRDS The Balbiani’s vitelline body forms the characteristic feature of young oocytes of birds (Fig. 33A-C) (Guraya, 1962, 1975a, 1976b; Chalana and Guraya, 1979). It differentiates adjacent to the nuclear envelope and contains morphologically and histochemically diverse cell components, including a yolk nucleus, mitochondria, Golgi bodies, and lipid bodies of variable size and composition. Electron microscope studies have also revealed the complex nature of the Balbiani’s vitelline body in young oocytes of fowl (Greenfield, 1966; Bellairs, 1967). The initial aggregation of the various organelles and inclu:srons . adjacent to the nuclear envelope clearly suggests that the nucleus (or gerrninal vesicle) plays an important role in their development in the juxtanuclear area. The Balbiani’s vitelline body is formed in the oocytes of both immature and adult birds (Greenfield, 1966; Bellairs, 1967; Guraya, 1975a, 1976b). The paranuclear complex of organelles and inclusions has also been described in various ways, for example, as the yolk body of Balbiani (de’ Hollander, 1904), a yolk body (Loyez, 1906), a yolk-forming layer or yolk bed (Van Durme, 1914), a mitochondria1 yolk body (Brambell, 1926), and early yolk (Konopacka, 1933). All these terms imply that this region is involved in the formation of yolk, but it now appears unlikely that it manufactures yolk directly. The accumulation of various cell components in the Balbiani’s vitelline body clearly suggests that this region is the site of high metabolic activity concerned with the initial multiplication and accumulation of various organelles and inclusions (Guraya, 1962, 1975a, 1976b). The basophilic hemispherical or spherical yolk nucleus gradually develops adjacent to the nuclear envelope and forms a substrate for the multiplication and aggregdtion of other ooplasmic structures of the Balbiani’s vitelline body (Fig. 34 and 35A-D) (Guraya, 1962, 1975a, 1976b). It is not homogeneous, sirice it contains various reticular and granular substructures (Figs. 34 and 35A -D). Histochemical tests indicate that it consists mainly of RNA, protein, and lipoprotein (Guraya, 1962, 1975a, 1977; Chalana and Guraya, 1979). The proteins of the Balbiani’s vitelline body are acidic and basic (Chalana and Guraya, 1979).

BALBIANI’S VITELLINE BODY

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Fm. 33. Histochemical preparations of oocytes. (A) Portion of a common myna (Acriclo/heres) ovary, showing the accumulation of highly sudanophilic lipid bodies in the Balbiani’s vitelline body (BVB); the nuclei ( N ) are sudanophobic. (B) Oocyte of a pigeon (Columba livicr), showing sudanophilic lipid droplets (LD) and diffusely distributed lipids of a Balbiani’s vitelline body (BVB). (C) Oocyte of a dove (Strepmpelitr ~lectrocto),showing heavy accumulation of highly sudanophilic lipid droplets (LD) aruund and in the Balbiani’s vitelline body (BVB) which also shows diffuse sudanophilic lipids. (D) Oocyte from the pigeon ovary, showing fragmentation of a Balbiani’s vitelline body. Sudanophilic lipid droplets are accumulating in the cortical ooplasm. Frozen section stained with Sudan black B. ( B and C) X 7 6 5 ; ( D ) ~ 3 4 0 .

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FIG.34. Young oocytes (A-D) from the ovary of a sexually immature fowl, illustrating :iuccessive stages in the development and differentiation of the yolk nucleus (YN) and its associated mitochondria (M)and lipid inclusions (L) adjacent to the nucleus (N). Similar organelles and lipid bodies are also sparsely distributed in the follicular epithelium (FE). The basement rneinhrane ( B M ) is intensely stained. A darkly stained follicle cell (DF'I is also seen. (E) An oocyte from the oviiry of a sexually immature fowl, illustrating dispersion of the yolk nucleus (YN), mitochondria, lipid bodies, and other components. Champy-iron hematoxylin. (From Guraya, 1976h.)

FIG.35. Oocytes ( A - E ) from the ovary of a ring dove, illustrating successive stages in the development, differentiation. and dispersion of the yolk nucleus (YN) and its associated mitochondria ( M ) , Golgi hodies, and lipid hodies ( L ) , which may form secondary yolk nuclei (SYN) in the cortical ooplasm of large oocytea. The nucleus ( N ) shows nucleolus-like bodies (NB). Single-layered follicular epithelium ( F E ) is associated with a deeply stained basement membrane (BM). Champyiron hematoxylin. (From Guraya. 1976b.)

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Electron microscope studies of this structure in the oocytes of fowl have: not identified the yolk nucleus proper (Greenfield, 1966; Bellairs, 1967). However, these investigations have shown a large paranuclear aggregate of vesicles or saccules interpreted as a Golgi complex or vesicles (Fig. 36). Actually the relationship of the yolk nucleus to the Golgi bodies was not clearly revealed in the ultrastructural studies of Greenfield (lc)66), who appears to have described both components as Golgi vesicles. Bellairs (1967) seems to have made a distinction between them, as she stated that the most conspicuous feature of this region (or yolk nucleus of Guraya, 1962, 1975a, 1976b) of the oocyte was the large number of vesicles (Fig. 36). The largest of these are patches of Golgi apparatus, which are scattered throughout the Balbiani’s vitelline body. The individual Golgi vesicles are irregular in shape, may measure as much as 1 p.m in maximum length, and have an “empty” appearance. These patches of Golgi apparatus or vesicles also correspond to the Golgi bodies of light microscopy, which form an anatomical association with the yolk nucleus as observt:d in Kolatchev and Aoyama preparations (Fig. 37) (Guraya, 1976b). According to Bellairs (1967) large empty vesicles are scattered apparently as separate units in this region of the oocyte, though it is possible that they may be in communication with other parts of the Golgi system. Besides these large vesicles, vesicles of variable size and density are also present, as well as RNP particles (Fig. 36) which, on close examination, appear to be embedded in an electron-dense matrix; the granular contents are less electron-dense in the large vesicles than in the small ones. All these ultrastructural components represent the yolk nucleus of light microscopy (Figs. 34, 35A-D, and 37) (Guraya, 1962, 1975a, 15176b; Chalana and Guraya, 1979), which consists of RNPs and lipoproteins (Guraya, 1962). The lipoproteins of the yolk nucleus, which stain faintly with Sudan black B (Fig. 33B and C), appear to constitute the various ultrastructural elements of endoplasmic reticulum (or vesicles of variable size), and its RNP apparently consists of RNP particles and possibly the finely granular matrix. These coi-relations between the findings of light and of electron microscopy cIearly suggest that the Golgi complex and yolk nucleus are two separate cytoplasmic structures in the avian young oocyte, which are, however, closely associated with each other (Fig. 37) (Guraya, 1976b). The fully grown yolk nucleus in some oocytes of birds, for example, quail and fowl, is sometimes differentiated into zones of variable width and density: (1) an inner zone, (2) a middle zone, and (3) an outer zone. The yolk nucleus in the young oocytes of quail also has highly chromophilic bodies of variable size (Fig. 38A), which consist mainly of protein and RNA (see also Guraya, 1962). They seem to have originated from the nucleus. Mitochondria having the usual phospholipid-protein composition are i n the form of granules and filaments which accumulate in close association with the yolk nucleus (or ultrastructural vesicles of variable size and density) in the

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FIG. 36. ( A ) Electron micrograph of a section through the Balbiani’s body. (B) Electron micrograph of part of the same section, showing the nuclear membrane and edge of the Balbiani’s body. Note the small vesicles lying in clusters between the vesicles. Inset: Enlargement of part of a Balbiani’s body to show twn sizes of granule-filled vesicles. (Froni Bellairs, 1967.)

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FIG.37. Young oocytes (A and B) from the ovary of the brown dove Srrepropelia senegalensis, showing the development and differentiation of the yolk nucleus (YN) and Golgi bodies (G)adjacent to the nucleus (N). Single-layered follicular epithelium also shows darkly stained Golgi complexes. Kolatchev preparation. (From Guraya, 1976b.)

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FIG. 38. (A) Young oocytes of quail (Coturnir coturnix), showing highly chromophilic dense bodies (arrows) in the yolk nucleus (YN) forming a mass in the juxtanuclear cytoplasm. A nucleus (N) is seen. Champy-iron hematoxylin preparation. x IOOO. (B) Oocyte of quail, showing fragmentation of yolk nucleus (YN). N , Nucleus. Aoyama preparation. x 1000.

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juxtanuclear area (Fig. 34A-D). This is in agreement with the electron microscope studies of Greenfield (1966) and Bellairs (1967) who showed the presence of granular and filamentous mitochondria in the Balbiani’s vitelline body of the fowl oocyte (Fig. 36). Mitochondria lying in the outer ooplasm have apparmtly moved away from the region of the Balbiani’s vitelline body. The highly sudanophilic lipid bodies which accumulate in and around the Balbiani’s vitelline body are in the form of granules and spheres (Fig. 33A-C) (Guraya, 1957, 1959, 1962, 1975a, 1976b: Chalana and Guraya, 1978). L’ltrastructural studies have also revealed the presence of lipid drops in the vicinity of the Balbiani body of the fowl oocyte (Fig. 36B) (Greenfield, 1966; Bellairs, 1967). The various types of lipid bodies have been studied in frozen sec1:ions fixed in formaldehyde-calcium, postfixed in dichromate calcium, and stained with Sudan black B (Fig. 33A-C). The lipid granules are composed of phospholipids and triglycerides, whereas the spheres consist of triglycerides and cholesterol and/or its esters (see Guraya, 1957, 1959, 1975a, 1976b; Chalana and Guraya, 1979). The presence of lipid bodies intermediate between granules and spheres suggests that the latter are derived from the former by growth and chemical change. The differences in their chemical composition are also supported by their fixation behavior with classic techniques of cytology; lipid granules containing phospholipids are partially preserved, whereas lipid spheres composed of triglycerides and cholesterol andor its esters are completely removed (Fig. 34A-D and 35A-C) (Guraya, 1957, 1959, 1962, 1975a, 1976b; Chalana and Guraya, 1979). Ultrastructural osmiophilic structures and lamellated bodies (Greenfield, 1966), and multivesicular bodies (Fig. 36) (Bellairs, 1967), apparently correspond to some of the partially fixed lipid granules embedded in the yolk nucleus. Most of the highly sudanophilic lipid bodies that tend to hide the yolk nucleus (Fig. 33A-C) are usually lost in the paraffin sections (Figs. 34, 35A-C, and 37). Besides the various cell components discussed above, the Balbiani ’s vitelline body of the avian oocyte also has annulate lamellae and a crystalline lattice (Fig. 39) (Bellairs, 1967). According to Bellairs (1967), the annulate lamellae may be sites of vesicle formation. The possible origin and function of annulate lamellae, which occur in the oocytes of some animal species, have been discussed in previous reviews (Kessel, 1964; Norrevang, 1968; Wischnitzer, 1970). After the various components of the Balbiani’s vitelline body have attained maximum development in the juxtanuclear ooplasm, they are dispersed in the cytoplasm of the growing oocyte. The yolk nucleus itself breaks up first into irregular masses (Fig. 38B) and then into small elements (Fig. 33D). In the vitellogenic ovaries of some birds, the yolk nucleus still persists, and as a result of continued growth becomes almost as large as the nucleus of the oocyte (Figs. 35C and 40A and B). Owing to its dense, organized nature, the yolk nucleus stands out in sharp contrast to the general ooplasm. Sometimes, two yolk nuclei

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FIG. 39. Electron micrograph of two serial sections ( A and B ) through the annulate lamellae lying in a Balbiani's body. The arrows in (B) indicate that the annulate lamellae may be the site of vesicle formation. (From Bellairs, 1967.)

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develop simultaneously in the oocytes of the vitellogenic hen ovary and continue to persist in oocytes which measure 236 pm in diameter; the large one is in the juxtanuclear ooplasm and the small one at the animal pole. Part of the fully developed yolk nucleus in the oocytes of the: vitellogenic chicken takes the form of radiating projections which extend into the outer ooplasm (Fig. 40A). The yolk nucleus and its long extensions sometimes develop into a complicated structure (Fig. 40A-C). Its extension may take the form of an arch with its concavity toward the nuclear envelope. The arch consisting of yolk nucleus substance is of variable width (Fig. 40A and C). Secondary projections may also arise from the outer surface of the arch, which reach the cytoplasmic membrane of the oocyte. The yolk nucleus and its various extensions appear to contribute their contents to the outer ooplasm where it is distributed. The fully developed yolk nucleus in the oocytes of the nonvitellogenic chicken does not develop such complex extensions (Fig. 34D). With further growth of the oocyte in the vitellogenic hen ovary, the persisting yolk nucleus and its extensions finally disappear as their contents consisting of RNA, protein, and lipoprotein are distributed into the cortical ooplasm where they further multiply and accumulate (Guraya, 1975a, 1976b). The mitochondria, Golgi bodies, and sudanophilic lipid bodies are also dispersed into the outer ooplasm (Fig. 33D) and continue to multiply. In some oocytes of vitellogenic birds, the yolk nucleus or nuclei and associated components move away from the juxtanuclear ooplasm and continue to persist as organized structures in the outer ooplasm (Figs. 35E, 40D, and 41) where they appear to proliferate further. The cortical ooplasm of some growing oocytes in vitellogenic hens has two to seven compact spherical bodies which in morphology and histochemical reactions closely resemble the juxtanuclear yolk nucleus (Fig. 40D). They are also associated with the mitochondria, Golgi elements, and lipid granules consisting of phospholipids and triglycerides. These cortical ooplasmic structures or secondary yolk nuclei (Fig. 35E) are apparently formed from fragments of original yolk nucleus, which attain a large size by further growth and differentiation. They finally develop into a compact, orFIG.40. (A) Oocyte from the ovary of a sexually mature fowl, illustrating the morphological complexity of the yolk nucleus ( Y N ) and its extensions (arrows) lying close to the nucleus (TO. A portion of follicular epithelium (FE) is also seen. Ixwitsky-iron hematoxylin preparation. (B) Higher-power view of the yolk nucleus (YN) shown in (A), illustrating its three zones. Most of the mitochondria (M) are present in the peripheral zone. A portion of the nucleus ( N ) is also seen. Lesitsky-iron hematoxylin preparation. (C) A higher-power view of the yolk nucleus extension shown in (A), illustrating several mitochondria (M)and sparsely distributed yolk nucleus substance in its vicinity. A portion of the nucleus ( N ) is also seen. Lewitsky-iron hematoxylin preparation. (D) Portion of a oocyte from the ovary of a sexually mature fowl, showing two yolk nuclei ( Y N ) and associated organelles such as mitochondria (M). Portions of the follicular epithelium (FE) and nucleus (N), are seen. Lewitsky-iron hematoxylin preparation. (From Guraya, 1976b.)

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FIG.41. Portion of a fowl oocyte showing a large yolk nucleus (YN) in the cortical ooplasm. Follicular epithelium (FE) is also seen. Lewitsky-iron hematoxylin preparation. ~ 4 0 0 .

ganized structure in the cortical ooplasm, similarly to the juxtanuclear yolk nucleus (Figs. 35E, 40A and B, and 41). These secondary Balbiani’s vitelline bodies of the cortical ooplasm seem to be additional sites of multiplication of the yolk nucleus substance, mitochondria, Golgi bodies, and possibly lipid granules. Finally, they also disappear before the yolk vesicles form in the central ooplasm. The cortical yolk nuclei are not developed in the corresponding oocytes of the nonvitellogenic chicken (Guraya, 1975a, 1976b). The amount of yolk nucleus substance and other cytoplasmic components seems to increase during their dispersal (Guraya, 1962, 1975a, 1976b). It becomes relatively greater in the cortical ooplasm where the various ooplasmic components derived from the Balbiani’s vitelline body form a conspicuous cortical layer (Fig. 42). In the final

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FIG.42. Histochemical preparation of a growing oocyte of a sparrow (Passer dowesricus), showing the dispersal and localization of various cell components including lipid droplets of the Balbiani’s vitelline hody in the cortical ooplasni (arrows). The nucleus is sudanophobic. Frozen section stained with Sudan Black B . x400.

stages of previtellogenic oocyte growth, the lipid spheres consisting of triglycerides and cholesterol and/or its esters appear to be absorbed by the cortical cytoplasm. In summary, it can be stated that the Balbiani’s vitelline body of the avian oocyte is very complex morphologically and chemically. It is the site of high metabolic activity as evidenced by the presence of various enzymes, namely, acid phosphatase, 5’-nucleotidase, and NAD and NADP diaphorase (Chalana

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and Guraya, 1979). These enzymes may be responsible for the production of NAD and NADP coenzymes which are well known to play a significant role in different metabolic reactions. F. MAMMALS Primordial oocytes in mammals remain in arrested meiotic prophase for a long time in the ovarian cortex (Mauleon, 1969). The various ooplasmic structures such as the yolk nucleus, granular basophilic substance, mitochondria, and phospholipid bodies form a juxta- or paranuclear complex which is usually designated the Balbiani’s vitelline body (Figs. 43 and 44) (Guraya, 1964, 1965c, 1967a,b, 1968e,f, 1970b,c,d, 1974). Electron microscope studies have revealed a rather simple, large paranuclear aggregate of vesicles or saccules interpreted as a large Golgi complex or cytocentrum (Figs. 45 and 46) (Hadek, 1965; Hertig and Adams, 1967; Stegner, 1967; Hertig, 1968; Zomboni, 1972), which is identical to the yolk nucleus of light microscopy and histochemical studies (Figs. 43 and 44) (Guraya, 1964). The various components of the Balbiani’s vitelline body accumulate in the juxtanuclear cytoplasm of differentiating oocytes during development of the mammalian ovary (Figs. 44D, 45, and 46). The relationship of

FIG.43. Diagrams of human primordial oocytes showing different organelles of the paranuclear complex (or Balbiani’s vitelline body). (A) Primordial oocyte from the ovary of a woman 18 ,years old. (B) Primordial oocyte from the ovary of a woman 25 years old. (C) Primordial oocyte frorn the ovary of a woman of 35 years old. Changes in the spherical bodies (SB) are shown. BS, Basophilic substance; F. slightly sudanophilic filaments; FE, follicular epithelium; G, bodies probably corresponding to the Golgi bodies of ultrastructural studies; L, lipid bodies; M , mitochondria; N, nucleus or germinal vesicle; NI, nucleolus; SB, spherical bodies; V, vacuole; YN, yolk nucleus. (From Guraya, 1970b.)

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Flci. 44. Histochemical preparation of Primordial oocytes of different mammalian species, showing the yolk nucleus (YN)(or Golgi complex), mitochondria (M),and lipid bodies (LB) of the Balbiani’s vitelline body (BVB). The yolk nucleus is slightly sudanophilic. Such diffuse lipids are not seen in the nucleus ( N ) . A follicular wall (FW) and stronia ( S ) are also seen. (A) Primordial oocyte from a cow ovary. ( B ) Primordial oocyte from a buffalo ovary. (C) Primordial oocyte from an American opossum ovary. (D)Primordial oocyte from 40-day fetal guinea pig ovary. V, vacuole; ST, stroma. Frozen sections stained with Sudan black B. x700.

the yolk nucleus, consisting of protein, lipoprotein, and very little RNA, to the Golgi complex is still unknown in the mammalian oocytes, and Hadek (1965) has stated that “the yolk nucleus of the eutherian mammal could be mistaken for the Golgi complex, but for the absence of parallel running lamellae, and also for the smooth endoplasmic reticulum, but for the uneveness and irregularity of the channels.” In young oocytes of mammals the Golgi bodies are apparently associated intimately with the yolk nucleus or cytocentrum (Fig. 46) (Hertig and Adams, 1967; Hertig, 1968). Zamboni (1972) has reported structural differences in the organization of the Golgi apparatus in different mammalian species. Ac-

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FIG.45. Electron micrograph of germ cell nest from a 40-day fetal guinea pig ovary showing the origin and differentiation (arrows) of the Balbiani’s vitelline body in the juxtanuclear cytoplasm. ~ 3 8 5 0 (.S . S . Guraya, H.-E. Stegner, and C. Pape, unpublished.)

cording to Anderson (1972), acid phosphatase activity appears in the Golgi complex of mammalian oocytes. A granular basophilic substance consisting mainly of RNA, protein, and lipoprotein, along with mitochondria, is unifoimly distributed around the yolk nucleus or Golgi complex (Fig. 43) and corresponds to the ultrastructural RNP particles or ribosomes and various profiles of endoplasmic reticulum (Fig. 46) (Adams and Hertig, 1964; Hadek, 1965; Stegner, 1967; Guraya, 1973, 1974). Its slightly sudanophilic lipoprotein component (Fig. 44) may derive from the ultrastructural vesicles of the endoplasmic reticulum, which are sparsely distributed in the cytoplasm (Figs. 45 and 46). Sparsely distributed mitochondria having an usual phospholipid-protein coniposition (Fig. 43) show great diversity in ultrastructure (Figs. 45 and 46) (see Guraya, 1974). In the oocytes of some mammals, the granular basophilic substance forms dense aggregations in which are embedded the mitochondria

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(Adams and Hertig, 1964; Odor, 1965; Weakley, 1966; Guraya, 1970d); the physiological significance of their morphological association has been discussed by Odor (1965). Phospholipid bodies of variable size and morphology are usually associated with the yolk nucleus and other cytoplasmic components of the Balbiani’s vitelline body and are also present in the peripheral ooplasm where they may be associated with pinocytotic vacuoles (Figs. 43 and 44) (see Guraya, 1973, 1974). In electron microscope studies they appear to have been overlooked. However, in the young oocytes of some mammalian species, they have been noted and described as dense aggregates, dense bodies, or electron-dense inclusions (Blanchette, 1961; Hope, 1965; Odor, 1965; Weakley, 1966; Hertig and Adams, 1967; Hertig, 1968). Hertig and Adams (1967) and Hertig (1968), in their electron microscope studies of human primordial oocytes, identified lipid

FIG.46. Portion of a primordial oocyte from a fetal guinea pig ovary showing the Golgi complex (G), mitochondria (M). and ribosomes (R),of the Balbiani’s vitelline body. Elements of the endoplasmic reticulum (ER) and dense bodies are also seen. The nucleus (N) shows a nuclear envelope (NE). x 110.230. (S.S.Guraya, H.-E. Stegner, and C . P a p , unpublished.)

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bodies as small compound aggregates which were not membrane-enclosed ((see Guraya, 1974). The Balbiani’s vitelline body of primordial oocytes in the human and chimpanzee also shows masses of stacked or concentric annulate lamellae, and heterogeneous spherical bodies (or ultrastructural large compound aggregates) (Hertig and Adams, 1967; Hertig, 1968; Guraya, 1970b; Barton and Hertig, 1972). They consist of RNA and lipoprotein. Spherical bodies, which conskt of RNA and lipoprotein in young women, develop free phospholipid and triglyceride in women in their middle reproductive years (Fig. 43), suggesting that human primordial oocytes become aged in terms of somatic cell life (Guraya, 1967a, 1970b, 1974). The spherical bodies or ballooned compound aggregates of the Balbiani’s vitelline body in human oocytes have not been found in the oocytes of other mammals (Guraya, 1970~).However, the oocytes of pregnant cats have spberical inclusion bodies which stain moderately for phospholipids. The same bodies in the oocytes of estrous cats appear nearly sudanophobic, having a few highly sudanophilic phospholipid granules embedded in their matrix. These histoche:mical changes in the inclusion bodies of cat oocytes apparently occur in response to alterations in the nature and amount of hormones, as suggested by development of histochemically demonstrable lipids in them during pregnancy when high levels of progesterone are secreted by the corpora lutea. This observation should be confirmed by other techniques. Such studies will increase our knowledge of the effects of hormones on the morphology and biochemistry of ooplasmic organelles in the Balbiani’s vitelline body, about which nothing is known. He:rtig and Adams (1967) have described the effects of environmental hormones, on certain orgnalles of the Balbiani’s vitelline body in human primordial oocy tes. The inclusion bodies of the cat primordial oocyte apparently correspond to the membrane-bound bodies illustrated by Weakley (1968, Fig. 14 and 16). In his paper, it is not mentioned whether the ovaries of pregnant or estrous cats were used. The similarities between the inclusion bodies of Guraya (1970~)and the membrane-bound bodies illustrated by Weakley (1968, Figs. 14 and 16) is ,also supported by his statement that the most frequent and consistently occurring type of membrane-bound bodies contain fragments of membrane associated with an amorphous substance and occasionally a small inclusion which may be lipid; his small inclusion is clearly identical to the phospholipid granules embedded in the matrix of inclusion bodies described by Guraya (1970~).Inclusion bodies similar to those of estrous cats also develop in large, growing oocytes of the rhesus rrionkey (Guraya, 1970~).The inclusion bodies of these oocytes may serve as notrients during oocyte growth, as they are not seen in the fully mature egg. The growth of oocytes in mammals is accompanied by the dispersion, proliferation, and accumulation of various components of the Balbiani’s vitelline body such as the yolk nucleus derivatives, granular basophilic substance, mitochon-

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dria, phospholipid bodies, and inclusion bodies of cat oocytes. They show greater concentration in the peripheral ooplasm of rapidly growing oocytes (Guraya, 1970c, 1973, 1974). The yolk nucleus derivatives appear in a variety of forms having different sizes and densities. The large, spherical masses derived from the original yolk nucleus, which lie in the cortical ooplasm, seem to constitute secondary sites for the growth and multiplication of the yolk nucleus substance. They are clearly identical to the ultrastructural Golgi areas, Golgi cornplexes, or large aggregates of vesicles in the cortical ooplasm of growing oocytes (Adams and Hertig, 1964; Hope, 1965; Baca and Zamboni, 1967; Zamboni, 1972; Guraya, 1974). The other derivatives of the yolk nucleus presumably constitute the ultrastructural cytoplasmic lamellae and endoplasmic reticulum; these systems of cytoplasmic membranes show great diversity in development, amount, and form in developing oocytes of different mammalian species (see Adams and Hertig, 1964; Hadek, 1965, 1966; Weakley, 1967a, 1968). Weakley (1968) has stated that these variations, which occur in rapidly expanding cytoplasm, are probably simply different methods of coping with the problem of how to manufacture enough membranous components to serve the growing oocyte. Based on histochemical observations, correlated with electron microscope studies (Guraya, 1970c, 1973, 1974), it can be stated that the yolk nucleus and its large derivatives apparently are sites of the synthesis and proliferation of mernbranous elements which are gradually distributed into the outer ooplasm; their RNA content may be of physiological significance for this purpose. Weakley (1968) has also stated that the various membranous structures may function as site for the poliferation of smooth membranes. During oocyte growth mitochondria form a close morphological association with the granular basophilic substance and loosely organized derivatives of the yolk nucleus (Guraya, 1970c, 1973, 1974). This indicates a physiological relationship between these organelles. The hamster ovarian oocyte, surrounded by two or three layers of granulosa cells, develops a large paranuclear complex of organelles, consisting of a basophilic substance, mitochondria, and phospholipid bodies (Guraya, 1975b); the basophilic substance, staining for RNA, protein, and lipoprotein, forms the basic material with which the mitochondria apparently multiply. This paranuclear complex of organelles has also been designated “Balbiani’s vitelline body” by Weakely (1967b, 1971), who used electron microscope and autoradiography techniques. The basophilic substance of histochemical studies corresponds to the ultrastructural granular-fibrillar cytoplasmic bodies and intermitochondrial substance which are highly electron-dense and granular, have filamentous components, and consist of basic protein and RNA; the lipoprotein component demonstrated in the basophilic substance may derive from the elements of endoplasrnic reticulum and other filamentous structures described in the juxtanuclear complex (Weakely, 9167b, 1971). Weakley (1971) has suggested that the intermitochond-

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rial substance (or granular basophilic substance) of the Balbiani ’s vitelline body represents machinery for the manufacture of mitochondrial proteins for which. the mitochondrial DNA is insufficient to code. Guraya (1975b) believes that the phospholipid inclusion bodies of the Balbiani’s vitelline body act as a storage depot for membrane-forming phospholipids which apparently combine with protein to form the lipoprotein component of organelles including mitochondria. The results of histochemical and electron microscope studies on the hamster oocyte, as discussed by Guraya (1975b), have shown that two juxtanuclear entities (or Balbiani’s vitelline bodies) are developed at different stages OF its growth; they differ in structure and to some extent in chemical nature. The organelles accumulated in the Balbiani ’s vitelline body of primordial oocytes are finally distributed in the cortical ooplasm, whereas those of the second paranuclear complex (or Balbiani’s vitelline body), which develops later in oocytes surrounded by two to three layers of granulosa cells, are mostly distributed into the inner regions of ooplasm (Guraya, 1975b). The initial development and accumulation of RNA-containing organelles of both complexes adjacent to the nuclear envelope suggest that the nucleus is probably stimulated twice by factors of cytoplasmic origin in order to contribute specific substances (RNAs) to the ooplasm, which may influence the formation of organelles to be finally disitributed in two specific regions of the growing oocyte.

V. General Discussion and Conclusions The correlation of recent cytological, histochemical, and electron microscope data has revealed that the Balbiani’s vitelline body in oocytes of different animal species represents the aggregation of different organelles and inclusions of diverse morphology and nature. Therefore it shows extreme variability in development, organization, and composition among different animal species. The nature of the factors causing these variations have not been determined, but genetic, physiological, and environmental factors perhaps influence its formation and differentiation. Depending upon the animal species or group, the Balbiani’s vitelline body shows many combinations of various organelles and inclusions such as a yolk nucleus (or ultrastructural endoplasmic reticulum or RNP particles or both), mitochondria, Golgi bodies, annulate lamellae, multivesicular bodies, lipid inclusions, and so on, which aggregate to a variable degree in the juxtimuclear cytoplasm. Of these organelles and inclusions, the yolk nucleus shows great diversity in development and morphological organization. In the oocytes of different groups of invertebrates, protochordates, and vertebrates, the yolk nucleus always develops adjacent to the nuclear envelope, suggesting an important influence of the nucleus on its initial formation. Several workers using electron microscopy have shown the origin and transfer of IWP

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particles and elements of endoplasmic reticulum from the nucleus and its nuclear envelope into the paranuclear ooplasm of young oocytes in some animal species, where they constitute the yolk nucleus of the Balbiani’s vitelline body (see Beams, 1964; Wischnitzer, 1966; Scharrer and Wiirzelmann, 1968a,b; Baker and Franchi, 1969; Riehl, 1976). The initial formation and development of the yolk nucleus adjacent to the nuclear envelope also suggests that its components (especially RNA) may carry genetic information from the nucleus during this period of close contact. The size, structure, and density of the fully developed yolk nucleus vary greatly in animal oocytes. It is sometimes differentiated into zones of variable width in the oocytes of some animals, such as spiders, fish, reptiles, and birds. Thus the yolk nuclei in the oocytes of certain animals are highly organized, dense structures having an intricate internal structure resulting from the various arrangements of its components, especially the endoplasmic reticulum. The details of the developmental processes involved in the formation of dense, organized yolk nuclei are still to be worked out. The yolk nuclei in the oocytes of invertebrates, protochordates, and vertebrates are related to the ergastoplasm or endoplasmic reticulum. They show considerable diversity in development and organization at the subcellular level; often they have a concentric lamellar structure. This is evidently the result of variable development, complex differentiation, and the arrangement of their membranous system, controlled either by extracellular factors or by genetic factors, about which very little is known. The relative amounts and arrangements of both the membranes and the amorphous or granular substance constituting the yolk nuclei in the animal oocyte vary greatly not only among different species but also within different regions of the yolk nucleus in the same species (e.g., spiders and molluscs). The amorphous or granular substance seems to be the predominant feature of yolk nuclei in some animals (e.g., some insects and amphibians), in comparison to the membranous system which is poorly developed, as it consists of masses of vesicles, annulate lamellae, or sparsely scattered vesicles (Wischnitzer, 1966). The poor development of the membranous system appears to be responsible for the loose, flocculent consistency of amphibian yolk nuclei. In the yolk nuclei of other animal species such as spiders, molluscs, and sea urchins, the membranous system is highly developed in comparison to the granular substance. These yolk nuclei in fine structure have therefore been related to the ergastoplasm or granular endoplasmic reticulum. The yolk nucleus in the oocytes of mammals has been identified as a Golgi complex or Golgi apparatus owing to the specific nature and arrangement of its membranous system. In spite of the diverse morphology of the yolk nucleus in different groups of animals, all workers using histochemical techniques have demonstrated variable amounts of RNA, protein, and lipoprotein in it; a sudanophilic lipoprotein component apparently derives from the ultrastructural elements of the endoplasmic

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reticulum, which show many variations in development in different animal species. In agreement with Fautrez (1958), Raven (1961) concluded that variously modified yolk nuclei are complexes of RNPs, originating in close contact with the nucleus, which often later disperse before the formation of yolk. Recent cytochemical studies have also revealed the presence of lipoproteins in yolk nuclei. All these studies have shown that the yolk nucleus has the same fundamental chemical composition and ultrastructure in most oocytes, the few exceptions involving variations in detail rather than basic deviations from the general composition of endoplasmic reticulum, ribosomes, and so on. It was the basophilic yolk nucleus of the Balbiani’s vitelline body that was identified in most earlier studies carried out with classic techniques of cytology (see Raven, 1961). The other cell components were not properly preserved, probably becmse of their lipid content, as discussed in detail for reptiles, birds, and so on. Different cell components such as the mitochondria, Golgi bodies, annulate lamellae, multivesicular bodies, and lipid bodies may accumulate to a variable degree in close association with the yolk niicleus of young oocytes to fomi the Balbiani’s vitelline body which is a site of their initial formation, multiplicalion, and accumulation. The number and nature of these cell inclusions and organelles, as already stated, vary greatly in the oocytes of different species of animals (see also Weakley, 1967b). The yolk nucleus of mammals designated the Golgi complex does not develop morphological association with the mitochondria. Previous workers have given the name “yolk nucleus” to the accumulation of mitochondria, Golgi bodies, and lipid bodies in the juxtanuclear area or in the outer cytoplasm, overlooking the yolk nucleus proper of the Balbiani’s vitelline body (see Chaudhry, 1952; Kemp, 1956; Balinsky and Devis, 1963; Nayyar, 1964; more references in Wischnitzer, 1966). They did not mention the basophilic yolk nucleus substance around which the various other separate inclusions and organelles of the Balbiani’s vitelline body usually aggregate and multiply. The Balbiani’s vitelline body is very complex both morphologically and histochemically in the oocytes of reptiles and birds. This complexity may be related to the large size of their eggs. After the yolk nucleus and other cell components of the Balbiani’s vitelline body have attained maximum development in the juxtanuclear cytoplasm of young oocytes, their subsequent behavior differs from one group of animals to another. Generally, the Balbiani’s vitelline body, especially its yolk nucleus, breaks up into irregular masses of various sizes while still in the juxtanuclear cytoplasm, indicating that it is a transitory ooplasmic structure concerned with the multiplication and accumulation of organelles and inclusions. In the oocytes of other animals the Balbiani’s vitelline body moves away from the nuclear envelope and persists as an organized structure throughout various stages of previtellogenesis; meanwhile it contributes its components to the outer ooplasm. Its persistence may be related to the rapid growth of previtellogenic oocytes.

BALBIANI’S VITELLINE BODY

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Sometimes several secondary Balbiani ’s vitelline bodies also develop in the growing oocytes of animals, which appear to constitute the site for the multiplication of organelles and inclusions. The persisting and secondary Balbiani’s vitelline bodies finally disintegrate and disappear before yolk deposition starts in the ooplasm of large oocytes. However, in some spiders and molluscs they can be seen even in yolky eggs as well as in embryogenesis. The persisting Balbiani’s vitelline body consists of a yolk nucleus, mitochondria, Golgi bodies, and lipid bodies in the oocytes of fish and spiders. It is interesting that the mitochondria, lipid bodies, and other cell components dissociate completely from the persisting yolk nucleus of the lizard (Hemidactylus) oocyte (Guraya, 1968d). The mitochondria, Golgi bodies, lipid bodies, and nucleolar extrusions to a variable degree are seen to be at times associated with the yolk nucleus proper of the Balbiani’s vitelline body. But it is not known whether this structural relationship has some physiological significance or is to be considered fortuitous. The different cell components in the growing oocytes of Hemiclucfylus form three zones around the persisting yolk nucleus (Guraya, 1968d). Their exact physiological significance has not been determined. Before yolk deposition starts, various organelles including the yolk nucleus substance, mitochondria, lipid inclusions, and other components are generally distributed in the outer ooplasm, except in some spiders and molluscs where the yolk nucleus associated with some cell components continues to persist during vitellogenesis and early embryogenesis. Divergent views have been expressed about the functions of the Balbiani’s vitelline body in animal oocytes. From the results of various studies discussed here, it can be concluded that these juxtanuclear entities act as initial centers for the formation, multiplication, and accumulation of organelles (e.g., endoplasmic reticulum, ribosomes, Golgi bodies, mitochondria, and annulate lamellae) and inclusions (lipid bodies) which are finally distributed in the ooplasm before yolk deposition starts. The Balbiani’s vitelline body is an important site for metabolic processes, as evidenced by the presence of intense enzyme activity. The various organelles and lipid bodies are subsequently distributed in the outer ooplasm. The lipid bodies are either used for oocyte growth processes or accumulate for future use in embryogenesis. These observations suggest that the Balbiani’s vitelline body is not only a very significant ooplasmic structural entity, but that it may be morphologically and biochemically very complex and diverse. Use of the electron microscope and histochemical techniques has confirmed this and has allowed a good description to be obtained of the detailed organization of the Balbiani’s vitelline body in the oocytes of various animal species. As a result, we now have a deeper understanding of both the morphology and functional importance of Balbiani’s vitelline body in oocyte development. Its various organelles and components are apparently elaborated in response to the especially intensive metabolic needs of the growing oocyte. The RNA contents of its yolk nucleus

318

SARDUL S. GURAYA

substance must be involved in the synthesis of raw materials (especially proteins) needed for the construction of various organelles, which are required in abundance because of the large size of the egg cell. Further autoradiographic and histoenzymological studies should be carried out on the Balbiani’s vitelline body to determine more precisely the origin and metabolic functions of its various components, as very little work has been carried out previously along these lines. In general, regulation of the biogenesis of cell organelles during cell growth, reproduction, or differentiation forms an important area of biology about which we have the least knowledge. Guraya (1975a), after studying the conspicuous differences in the Balbiani’s vitelline body of oocytes in juvenile and sexually mature fowls and wall lizards, has suggested that the metabolic status of the female apparently has a great influence on the developmental processes and composition of ooplasmic structures during previtellogenesis, as the Balbiami ’s vitelline body shows a great morphological and histochemical complexity in the oocytes of sexually mature females. Most ultrastructural studies have shown that yolk bodies do not originate directly in the Balbiani’s vitelline body. But it is well-established that they bzgin to arise after its basophilic yolk nucleus substance, mitochondria, Golgi bodies, RNP particles, and other Components are distributed throughout the ooplasm, suggesting that its organelles have a role in early yolk formation. Yolk has k e n described as arising within various animal oocytes either freely in the cytoplasm or within ergastoplasmic cistemae, within preexisting vacuoles, inside mitochondria, or by a combination of two or more of these methods (see Beams, 1964; Wischnitzer, 1966; Norrevang, 1968). It is therefore clear that not all organisms deposit their yolk in the same way. After the ooplasmic organelles and RNPs needed to sustain protein synthesis during early embryogenesis have accumulated during oocyte growth, they are usually rearranged to form conspicuous gradients in the ooplasm of the fully mature egg (Guraya, 1965a, 1969b, 1970c, 1972b, 1974). There is thus clear-cut evidence of histochemical (or biochemical) and morphological differences between the animal and vegetal halves of the fully grown egg. The gradients of ooplasmic components orginally derived from the Balbiani ’s vitelline body may also be of great significance in embryogenesis (Guraya, 1975b). In this regard, its RNAs must be of special significance in embryogenesis. It will be usefill to work out their perspective importance during fertilization and early embryogenesis. Davidson (1977) believes that the RNA stored during oogenesis may act as an inducer of early development.

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Afzelius, B. A. (1957). Z. Zellforsch. Mikrosk. Anar. 45, 660. Anderson, E. (1972). In “Oogenesis” (J. D. Biggers and A. W. Schuetz, eds.), pp. 87-117. Univ. Park Press, Baltimore, Maryland. Anderson, E., and Beams, H. W. (1960). J . Ulfrastrucr. Res. 3, 432. Anderson, E., and Chomyn, E. (1964). Anar. Rec. 148, 254. And& J. (1958). Bull. Microsc. Appl. [2] 8, 93. And& J., and Rouiller, C. (1957). J. Biophys. Biochem. Cyrol. 3, 977. Anteunis, A., Fautrez-Firlefyn, N., Fautrez, J., and Lagasse, A. (1964). Exp. Cell Res. 35, 239. Anteunis, A., Fautrez-Firlefyn, N., Fautrez, J., (1966). J . Ulfrasrrucr. Res. 15, 122. Aykroyd, 0. E. (1938). Z. Zellforsch. Mikrosk. Anat. 27, 691. Baca, M., and Zamboni, L. (1967). J . Ulfrasrrucr.Res. 19, 354. Baker, T. G., and Franchi, L. L. (1969). Z. Zellforsch. Mikrosk. Anat. 93, 45. Balbiani, E. G. (1864a). C . R. Hebd. Seances Acad. Sci. 58, 584. Balbiani, E. G. (1864b). C. R. Hebd. Seances Acad. Sci. 58, 621. Balbiani, E. G. (1893). J . Anal. Physiol. Norm. Parhol. Homrne Anim. 29, 145. Balinsky, B. I., and Devis, R. J. (1963). Acta Embryol. Morphol. Exp. 6, 55. Barton, B. R., and Hertig, A. T. (1972). Biol. Reprod. 6, 98. Beams, H. W. (1964). In “Cellular Membranes in Development” (M. Locke, ed.), pp. 175-219. Academic Press, New York. Beams, H. W., and Kessel, R. G. (1973). Am. J . Anar. 136, 105. Beams, H. W., and Sheehan, J. F. (1941). Anat. Rec. 81, 545. Bedford, L. (1966). J . Embryol. Exp. Morphol. 15, 15. Bellairs, R. (1967). J . Embryol. Exp. Morphol. 13, 215. Blanchette, E. J. (1961). J . Ulrrasrrucr. Res. 5, 349. Bottke, W. (1973). Z. Zellforsch. Mikrosk. Anar.. 138, 239. Brambell, F. W. R. (1926). Philos. Trans. R. SOC. London, Ser. B 214, 113. Cams, J. V. (1850). Z . Wiss. Zool. 2, 97. Chalana, R. K., and Guraya, S. S. (1979). Poulrry Sci. 58, 1. Chaudhry, H. S. (1952). Z . Zellforsch. Mikrosk. Anar. 37, 455. Clerot, J.-C. (1968). J. Microsc. (Paris) 7, 973. CICrot, J.-C. (1976). J. Ulrrasrrucr. Res. 54, 461. Costanzo, D. G. (1964). Acta Hisrochem. 17, 377. Cotronei, G . , and Urbani, E. (1957). Pubbl. Sfn. Zool. Napoli 29, 15. Davidson, E. H. (1977). “Gene Activity in Early Development,” 2nd ed. Academic Press, New York. de’ Hollander, F. (1904). Arch Anat. Microsc. 7, 117. Fauk Fremiet, E., Courtines, H., and Mugard, H. (1950). Exp. CellRes. 1, 253. Fautrez, J. (1950). C. R. Seances SOC.Biol. Ses Fil. 144, 1129. Fautrez, J. (1958). C. R. Assoc. Anat. 45, 1. Gopal Dutt, N. H.(1964). Q. J . Microsc. Sci. 105, 349. Greenfield, M. L. (1966). J . Embryol. Exp. Morphol. 15, 297. Guraya, S. S. (1957). Q . J . Microsc. Sci. 98, 407. Guraya, S. S. (1959). Res. Bull. Panjab Univ. 10, 119. Guraya, S. S. (1962). Q . J . Microsc. Sci.103, 411. Guraya, S. S. (1963a). Anar. Rec. 146, 17. Guraya, S. S. (1963b). Z. Zellforsch. Mikrosk. Anar. 60, 659. Guraya, S. S. (1964). Am. J . Anat. 114, 283. Guraya, S. S. (1965a). Z. Zelyorsch. Mikrosk. Anat. 65, 662. Guraya, S. S . (1965b). J . Morphol. 117, 151. Guraya, S. S. (1965~).J . Exp. 2001. 160, 123. Guraya, S. S. (1967a). Res. Bull. Panjab Univ. 18, 203.

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Nath, V., Gupta, B. L., and Manocha, S. L. (1959). Cellule 59, 387. Nayyar, R. P. (1964). Q. J . Microsc. Sci. 105, 353. Norrevang, A. (1968). Int. Rev. Cytol. 22, 113. Odor, D. L. (1965). Am. J . Anal. 116, 493. Parshad, V. R., and Guraya, S . S. (1977). Parasilology 74, 243. Raven, C. P. (1961). “Oogenesis.” Pergamon, Oxford. Rebhun, L. 1. (1956a). J . Eiophys. Eiochem. Cyrol. 2, 93. Rebhun, L. I . (1956b). J . Eiophys. Eiochem. Cyrol. 2, 159. Rebhun, L. I. (1961). J . Ultraslruct. Res. 5, 208. Reverberi, G. (1966a). Exp. Cell Res. 42, 392. Reverberi, G. (1966b). Arch. Zool. Ital. 51, 903. Reverben, G. (1967). Acta Embryo/. Morphol. Exp. 10, 1. Riehl, R. (1976). Z . Naturforsch., Teil C. 31, 761. Riehl, R. (1978). Cytobiologie 17, 137. Sareen, M . L. (1963). Curr. Sci. 32, 24. Sareen. M. L. (1964). Res. Bull. Panjab Univ. 15, 269. Sareen, M. L. (1970). Zool. Pol. 20, 373. Sareen, M. L., and Kapal, V. K. (1970). Res. Bull Panjab Univ. 21, 525. Scharrer, B., and Wiirzelmann, S . (1969a). Z . Zellforsch. Mikrosk. Anat. 96, 325. Scharrer, B., and Wiirzelmann, S. (1969b). Z . Zellforsch. Mikrosk. Anat. 101, I . Schmekel, L., and Fioroni, P. (1974). Cell Tissue Res. 153, 79. Sotelo, J . R., and Trujillo-Cenoz, 0. (1957). J . Eiophys. Eiochem. Cylol. 3, 301. Stegner, H. E . (1967). Ergeb. Anat. Entwicklungsgesch. 39, 7. Ubells, G . A. (1968). “A Cytochemical Study of Oogenesis in the Pond Snail Limnea stagnalis. ” Bronder-Offset, Rotterdam. Ulrich, E. (1969). J . Microsc. (Paris) 8, 447. Urbani, E. (1955). Puhhl. Sfn. Zool. Nupoli 26, 63. Van der Stricht, 0. (1923). Arch. Eiol. 33, 229. Van Durme, M. (1914). Arch. Eiol. 29, 71. von Wittich, W . H. (1845). “Dissertatio sistens observations quaedam de sistens Aranearum e x ovo evolutione. Halis, Saxonum (Halle, Germany). Ward, R. T. (1962). J . Cell Eiol. 14, 309. Wartenberg, H. (1962). Z . Zellforsch. Mikrosk. Anat. 58, 427. Weakley, B. S. (1966). J . Anal. 100, 503. Weakley, B. S. (1967a). Z . Zellforsch. Mikrosk. Anat. 81, 91. Weakley, B. S. (1967b). Z . Zellforsch. Mikrosk. Anat. 83, 582. Weakley, B. S. (1968). Z . Zellforsch. Mikrosk. Anal. 85, 109. Weakley, B. S. (1971). Z. Zellforsch. Mikrosk. Anat. 112, 69. Wegmann, I., and Gotting, K. J. (1971). 2. Zellforsch. Mikrosk. Anur. 119, 405. Wilson, E . B. (1937). “The Cell in Development and Heredity,” 3rd ed. Macmillan, New York. Wischnitzer, S. (1962). Z . Zellforsch. Mikrosk. Anat. 57, 202. Wischnitzer, S. (1964). J . Ultrastruct. Res. 10, 14. Wischnitzer, S. (1966). A h . Morphog. 5, 131. Wischnitzer, S. (1970). Int. Rev. Cyrol. 27, 65. Yamamoto, M. (1964). J . Fac. Sci. Univ. Tokyo, 10, 335. Zamboni, L. (1972). In “Oogenesis” ( J . D. Biggers and A. W. Schuetz, eds.), pp. 5-46. Univ. Park Press, Baltimore, Maryland. ”

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INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 59

Cultivation of Isolated Protoplasts and Hybridization of Somatic Plant Cells RAISAG. BUTENKO Tiinirycizev Institute of Plant Physiology, Accidemy of Sciences of the USSR, Moscow. USSR

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Methods and Conditions of Isolation . . . . . . . . . . C. Properties . . . . . . . . . . . . . . . . . . . D. Plasmalernrna . . . . . . . . . . . . . . . Cultivation . . . . . . . . . . . . . . . . . . . . A. Techniques . . . . . . . , . . . . . . . . . . B. Regeneration of the Cell Wall . . . . . . . . . . . . C. Division and Formation of Cell Colonies . . . . . . . . Fusion and Somatic Hybridization . . . . . . . . . . . . A. Technique . . . . . . . . . . . . . . . . . . . B. Selection of Hybrid Cells . . . . . . . . . . . . . . C. Intraspecific Somatic Hybridization . . . . . . . . . . D. Intergeneric Somatic Hybridization . . . . . . . . . . Incorporation of Foreign Genetic Material . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

I . Introduction . . . . 11. Historical Outline . . 111. Isolation and Properties A. Sources . . . .

. .

.

,

. . . . . . . . .

,

IV.

,

. . .

,

,

V.

VI. VII.

,

323 325 325 325 328 330 336 340 340 341 348 351 351 353 359 361 363 365 366

I. Introduction Technological advances in plant protoplast research, along with progress in the use of cell cultures, have opened up new perspectives for studying the biology of plant cells and practical application of the knowledge obtained. They have facilitated study of the composition and structure of the primary cell wall and the mechanism of action of the enzymes capable of degrading it (Rodionova et al., 1969, 1971; Albersheim, 1974). It is now possible to look into the molecular, metabolic, and structural aspects of cell wall regeneration and the effect of external factors on the synthesis and orientation of microfibrils of cellulose during the formation and growth of the cell wall (Pojnar et al., 1967; Cocking, 1970; Burgess and Linstead, 1976b, 1977b; Robenek and Peveling, 1975, 1977). Isolated protoplasts are invaluable in studying the properties of the surface cell membrane-plasmalemma (Hartman et al., 1973; Glimelius et al., 1974; 323 Copyright @ 1979 by Academic Press, Inc. All rights of reproduction In any form reserved. ISBN 0-12-364359-7

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Williamson ef a / . , 1976; Burgess and Linstead, 1976a). The absence of a cell wall facilitates investigation of the transport and cotransport of low-molecularweight compounds across the membrane. Absorption (endocytosis) of larger particles can also be investigated, as well as the effect of hormonal and inhibitory factors on their uptake. Investigation of the interaction of bacteria and fungi with sensitive and resistant cells and study of the effect of their toxins are other possible areas in which plant protoplasts may be used in fundamental and applied research (Strobel, 1973; Strobe1 and Hess, 1974; Pelcher et ~ z l . , 1975). Isolation of cell organelles with the use of isolated protoplasts allows oine to obtain more active organelle fractions than are obtained by means of mechanical degradation; this has been demonstrated with respect to nuclei (Blaschek ei' al., 1974; Ohyama et al., 1977b) and chloroplasts (Rathnam and Edwards, 1976). Fractions of individual vacuoles (Wagner and Siegelman, 1975; Lorz et al., 1976) and surface membranes (Galbraith and Northcote, 1977) have been isolated from protoplasts. This allows one to compare the composition and properties of the plasmalemma and tonoplast. Isolated plant protoplasts represent an ideal population of individual cells which is very infrequent in suspension cell cultures. Suspension of single cells from protoplasts is a prerequisite for quantitative studies of mutagenesis and cell selection (Aviv and Galun, 1977; Gleba and Gleba, 1978). Plant protoplasts are of great interest mostly as a very promising technique in genetics. Isolated protoplasts have the following useful properties: (1) fusability, including interspecies fusion (Cocking, 1971; Keller and Melchers, 1973; Kao and Michayluk, 1974); (2) the ability to take up from the external solution macromolecules (Ohyama et al., 1972; Gleba et al., 1974; Suzuki and Takebe, 1976), virus particles (Takebe, 1975), cell organelles (Potrykus and Lbrz, 1976), and microorganisms (Davey and Cocking, 1972); (3) the ability to divide after regeneration of the cell wall and to form colonies of callus cells, which can sometimes produce a whole plant (Nagata and Takebe, 1971; Takebe et al., 1971). It is the last-mentioned property that, when combined with the totipotency of plant cells in in vitro cultures, makes isolated protoplast techniques important in practical genetics and selection. In addition, the fusion of isolated plant protoplasts facilitates study of the genetics of somatic cells, which is essential for fundamental plant genetics. Hybridization of somatic cells opens up unique possibilities in the study of cytoplasmic heredity (Burgutin ef al., 1977; Belliard and Pelletier, 1978; Gleba ef al., 1978). Somatic cell hybridization is also a good tool for investigating the action of genes and the interaction between the nucleus and cytoplasm in cytodifferentiation. The genetic aspects of the utilizaticin of plant protoplasts have been analyzed in several reviews (Cocking, 1975, 1976a, 1976b; Carlson, 1973; Gamborg, 1975a,b, 1977; Gigot and Hirth, 1975; Vasil, 1976; Butenko, 1979). This article deals largely with the cytological aspects of

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325

the isolation, cultivation, and fusion of plant protoplasts and also with the cytophysiological problems pertaining to isolated protoplasts and those problems that can be resolved with the use of isolated protoplasts.

11. Historical Outline

The first steps in isolating plant protoplasts were made by Klercker (1892); protoplasts from thin sections of plasmolyzed tissue of sugar beet root, cucumber mesocarp, and Strutiotes aloides stem were mechanically isolated in a medium containing an osmotic stabilizer. The quantity of isolated protoplasts was small, and only tissues consisting of large, vacuolized, parenchymatous cells could be used. As a early as 1897 Townsend described the ability of plant protoplasts to regenerate the cell wall. According to him, subprotoplasts (cytoplasts) lacking a nucleus did not form a cell wall. The fusion of isolated protoplasts was later reported (Kiister, 1909). In 1919 yeast protoplasts were isolated with the use of gut juice of Helix pornatia. The true history of plant protoplast isolation techniques began in 1960, when Cocking applied hydrolytic enzymes from a culture medium of molds in the large-scale production of protoplasts from tomato root tips. Then observations followed on the absorption of latex and ferritin by protoplasts from an external solution (Cocking, 1966), the absorption of virus particles (Takebe et al., 1968), the sensitivity of protoplasts to the action of auxin (Ruesink and Thimann, 1965; Power and Cocking, 1970), spontaneous fusion and the methods inducing fusion (Cocking, 1971, 1972; Eriksson, 1971; Kao and Michayluk, 1974), the induction of division in cells arising from isolated protoplasts (Nagata and Takebe, 1970), and the regeneration of plants from newly formed callus tissue (Takebe e f al., 1971). In 1972 in Versailles, France, the First International Colloquium on isolated protoplasts was convened, which discussed problems pertaining to the isolation of protoplasts and the hybridization of somatic plants cells.

111. Isolation and Properties

The properties of plant protoplasts and their ability to regenerate the cell wall and to start division depend on the source from which they have been obtained, its state, and the methods of isolation and cultivation. A. SOURCES

Protoplasts are prepared from tissues of various plant organs-leaves, roots, tubers, petals, fruits, leguminous plants root nodules-and from tumors of different origin and tissues and cells cultivated in vifro. Protoplasts can be isolated

326

RAlSA G . BUTENKO TABLE I PLANT SPECIES IN WHICH PROTOPLASTS HAVEBEENSUCCESSFULLY CULTURED Origin of protoplasts

Species

Growth response

Asparagus oflcinalis

Cladode’s

Cell lines, shoots

A tropa belladonna

Tissue culture of stem Mesophyll cells

Cell lines, plants

Tissue culture of ovule Mesophyll cells Tissue culture of root Cell suspension Mesophyll cells Cell culture Mesophyll cells Placenta

Cell lines, embryos Cell lines, plants Cell lines, plants

Brassica napus Citrus sinensis Datura innoxia Daucus carota Daucus carota x D . capillifolius Hordeum vulgare Hyoscyamus niger Lycopersicon pimpinellifolium Nicotiana tabacum Nicotiana silvestris Nicotiana knightiana Nicotiana glauca X N . langsdorfli Nicotiana tabacum X N . tabacum carrying N . debneyi cytoplasm Oryza saliva Petunia hybrida Petunia parodii Petunia hybrida P . parodii

X

Pisum sativum Phaseolus vulgaris Ranunculus sceleratus Saccharum officimrum Solanum chacoense

Cell lines, plants

Reference Bui-Dang-Ha and Mackenzie ( 1973) Gosch et al. (1975) Kartha et al. ( 1974); Thomas et al. ( 1976) Vardi ef al. (1975) Schieder (1975) Grambow et al. (1972)

Somatic hybrids

Dudits et al. (1977)

Cell lines Cell lines Cell lines

Koblitz (1974) Kohlenbach and Bohnke (1975) Sharma (1974)

Mesophyll cells Mesophyll cells

Cell lines, plants Cell lines, plants

Mesophyll cells Mesophyll cells

Cell lines. plants Somatic hybrids

Mesophyll cells

Somatic hybrids

Nagata and Takebe (1971) Nagy and Maliga (1976); Banks and Evans ( I 976) Butenko et al. (1979) Carlson et al. (1972) Smith et al. (1976) Gleba et al. (1977) Belliard et al. (1977)

Mesophyll cells, tissue culture Mesophyll cells

Cell lines, roots

Deka and Sen (1976)

Cell lines, plants

Mesophyll cells Mesophyll cells

Mesophyll cells Mesophyll cells Meosphyll cells

Cell lines, plants Cell lines, somatic hybrids Cell lines Cell lines Cell lines, plants

Durand et al. (1973); Frearson et al. (1973) Hayward and Power (1975) Power Y I al. (1977)

Tissue culture

Cell lines

Maretzki and Nickell (1973)

Mesophyll cells

Cell lines, plants

Butenko et al. (1977b)

Constabel et al. (1973) Pelcher ef al. (1974) Dorion et al. (1975)

(continued)

ISOLATED PROTOPLASTS AND SOMATIC PLANT CELLS

327

TABLE I (coririrrued)

Species Soltrriurri dtd(~urtiunr

Solurrurn rubc,rosurri

Viris viriijiera Z m muvs

Origin of protopla5ts

Growth response

Mesophyll cells Mesophyll cells

Cell lines. plants Cell lines, plants

Tissue culture of pericarp Stem

Cell lines

Binding and Nehls ( 1977) Kuchko and Butenko ( 1976); Butenko e t d . (l977b); Shepard and Totten (1977) Skene (1974)

Cell lines

Potrykus

Reference

CI

a / . (1977b)

from microspores at the tetrad stage or from unicellular microspores (Bajaj, 1974; Deka et d., 1977). Algal protoplasts have also been isolated successfully (Braun and Aach, 1975; Schlosser rt d . , 1976; Gresshoff, 1976; Berliner, 1977). Protoplasts were isolated from mosses (Binding, 1966; Stumm et a / . , 1975) and liverworts (Schieder, 1975). The number of plants species from which isolated protoplasts have been obtained is close to 200 and is increasing. Table I lists the species from which protoplasts capable of sustained division and plant regeneration have been obtained. Leaf mesophyll is commonly used to obtain large amounts of homogeneous and homoploidal protoplasts. From 1 gm of potato leaf mesophyll 5-8 X 10" protoplasts can be isolated, of which 70-80% are viable (Butenko e t a / . , 1977b). The age of the plant, the age of the leaf, and the growth conditions affect the quantity of viable protoplasts and their behavior in cultivation. Reproducible results are as a rule obtained with plants grown in greenhouses under artificial illumination or in test tubes. Binding (1975) and Butenko et al. (l977b) demonstrated that the best results were obtained with test tube cultivation of plants that originated from meristematic cultures and were maintained by cloning under sterile conditions. Leaf mesophyll protoplasts can be readily separated and purified from tissue and cell debris, but sometimes they fail to divide. This is especially true of protoplasts obtained from leaves of cereals and leguminous plants (Potrykus rf ml., 1976; Avetisov rt 1978). When peas or beans were placed in the dark o r weakly (about 300 lux) illuminated for 20- 162 hours prior to the isolation of protoplasts, the number of viable protoplasts capable of division increased (Constabel r t a / . , 1973; Pelcher et nl., 1974). A short day (6 hours) and weak illumination produced the same effect on potato leaf protoplasts (Shepard and Totten, 1977). The number and the activity (estimated from the incorporation of leucine-:'H, uridine-:'H, and thymine-:'H) of oat protoplasts increased after the leaves were pretreated with cycloheximide ( 1 pg/ml) and kinetin (5 pg/ml) (Kaur-Sawhney et NI., 1976).

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RAISA G . BUTENKO

Protoplasts isolated from callus or suspension cultures are better adapted to division and growth on nutritive media. But their disadvantages are morphophysiological heterogeneity and heteroploidy. The separation of protoplasts and cells in suspension is sometimes easy, but in other cases it is very difficult. Cell suspensions growing at a high rate maintained by frequent subculturing (every 3 days) into fresh media (Gamborg, 1976) are preferable. The yield of isolated protoplasts from a suspension of Haplopappus gracilis cells was reported to increase when the cells were preincubated (1) with a high concentration of auxins, ( 2 ) with a low concentration of sucrose, and (3) with additional methionine, cysteine, and mercaptoethanol. The amount of isolated protoplasts increased from very little to 40-50%. Nethertheless, combining of all these conditions failed to give positive results (Wallin et a / . , 1977). B. METHODS A N D CONDITIONS O F ISOLATION The conditions for isolating protoplasts, that is, the type and concentration of enzymes and osmotic stabilizer, pH, temperature, time, and the use of protectors, determine the quantity and quality of the resulting protoplasts. The goal is to degrade the cell wall without impairing the living protoplast. This is achiev'ed by creating a water stress resulting in plasmolysis. The plasmolysis depends on the type of plasmolytic agent used and its concentration; 0.45-0.8 M manniitol or sorbitol is routinely used, as is a mixture of 0.3 M sorbitol and 0.3 M mannitol (Kao et al., 1971). The application of a metabolizing osmotic agent, sucrose or glucose, can be more advantageous, as a decrease in its concentration during the cultivation of protoplasts can be beneficial for division and growth of the cells (Kao et al., 1974). On the contrary, when the aim is to decrease the influx of an osmotic stabilizer in the cell, a high-molecular-weight substance, for example, polyethylene glycol (PEG) (MW >3000) (Pavlenko et al., 1978) is used. Mineral salts are not frequently employed for this purpose, because in the presence of certain salts some of the enzymes used for degradation of the cell decrease their act.ivity; besides, salts readily penetrate into the cell. However, the isolation of protoplasts from leaf mesophyll of clover, alfalfa, and ryegrass gave better results in the presence of ionic osmotic stabilizers than with mannitol and sucrose (Mesentzev ef uf., 1976). But tobacco mesophyll protoplasts isolated and cultivated in a medium where the osmotic potential was maintained by a mixture of salt:$produced a weak pseudowall and were not capable of sustained division. Placing such protoplasts in a sucrose-containing medium resulted in the formation of a rigid wall and regular cell division (Meyer and Abel, 1975). The effect of osmotic stabilizers is estimated mostly by the quantity of viable cells capable of division, although there is no doubt that the properties of the plasmalemma and the osmotic properties of protoplasts can to a great extent depend on the type of

ISOLATED PROTOPLASTS AND SOMATIC PLANT CELLS

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plasmolytic agent used. Lysis of protoplasts induced by addition of water to the osmotic stabilizer solution proceeds in different ways. When mannitol is diluted with water, the membrane disintegrates into small pieces; the addition of sucrose leads to the formation of comparatively long strands; a mixture of calcium chloride and potasium chloride lyses the plasmalemma but does not disintegrate it into fragments (Ruesink, 1973). Protoplasts were successfully isolated under comparatively low osmotic pressure provided by 0.3 M sucrose with 2% polyvinylpyrrolidone that served as a stabilizer (Shepard and Totten, 1977). Enzyme Preprrrcitions

The primary cell wall can be degraded by hydrolytic enzymes, that is, cellulase, hemicellulase, and pectinase. The composition of the enzyme mixture, pH, and duration of treatment of a tissue are derived empirically with due regard to the species, age, and physiological state of the plant. The age of the plant o r tissue determines the composition of the primary cell wall; the older the plant o r tissue, the higher the cellulose content and the lower the hemicellulose content. Thus the degree of polymerization of cellulose was reported to increase 14-fold as pea cells grew (Spencer and Maclachlan, 1972). When the aromatic polymer lignin appears in the cell wall, the isolation of plant protoplasts by enzymic techniques becomes impossible. For large-scale production of protoplasts crude commercial preparations are used; the most common are cellulase Onozuka R-10 (Kinki Yakult, Nishinomiya), cellulysin 219466 (Calbiochem, Switzerland), Driselase (Kyowa Kogyo, Tokyo), Macerozyme R-10 (Kinki Yakult, Nishinomiya), pectinase 31660 (Serva, Heidelberg), glusulase (Endo Laboratories, Inc., New York), and Xylanase (Moscow Enzyme Factory). For the isolation of protoplasts from the majority of plant tissues and cell cultures to be successful, the presence of preparations with pectolytic and cellulytic activities is, as a rule, required. Xylanase is active even without pectinases and has been successfully used for isolating protoplasts from leaf mesophylls of clover, alfalfa, potato, blue grass, tobacco, and different species of asters, and also from cell cultures of carrot and tobacco (Butenko and Ivantsov, 1973; Mesentzev et ~ 1 1 . . 1976; Butenko ct N I . , 1977b; Landova and Landa, 1975). Protoplasts from leaf mesophylls of succulent plants have been isolated with a single enzyme, pectinase (Salema and Brandao, 1976); the same was reported for protoplasts from tomato leaf mesophyll (Wit, 1976). There is no doubt that a crude enzyme preparation containing not only cellulases and pectinases but also proteases, nucleases, lipases, and phosphorylases has a damaging effect on protoplasts. This is why some investigators (Prat, 1972; Pilet, 1972; Gerasimov, 1976) still favor mechanical means-they construct a special apparatus that facilitates the production of tissue sections for obtaining protoplasts. The damaging effect of enzymes can be lessened by reducing the time of their action; to

330

RAISA G. BUTENKO

attain this the temperature must be raised, or alternatively it may be lowered to 10°C but then the time of incubation with the enzymes must be prolonged. Potrykus et al. (1977b) recommend that protoplasts be isolated from corn internodes by placing the sections in a medium with enzymes first for 16 hours at 12°C and then for 6 hours at 32°C. The effect of light on the isolation of protoplasts is not well known; usually they are isolated in the dark or under weak illumination. The pH for the isolation of protoplasts from various sources )using different enzyme mixtures is from 5.4 to 6.2. For leguminous plants the 6.0-7.0 pH range is the most favorable (Pelcher el al., 1974). Consecutive and snmultaneous enzyme treatments of tissues are also employed. With the former method (Takebe et al., 1968) a tissue is first treated with pectinase, to induce maceration into individual cells, and then placed in a solution containing cellulase to obtain isolated protoplasts. With the latter method (Cocking, 1972) the tissue is treated with pectinase and cellulase at the same time, and hemicellulase is somelimes added. In this case, maceration of the tissue into cells and isolation of the protoplasts occur simultaneously. The advantage of the consecutive treatment is the reduced possibility of spontaneous fusion of protoplasts and a more homogeneous population from leaf tissue, since the cells of spongy parenchyma are destroyed (Pisetskaya er' al., 1975). The disadvantage of the method is that the protoplasts inside the cell may undergo degradation during maceration of the tissue. The disadvantage of the simultaneous treatment is greater spontaneous fusion of protoplasts, resulting in a large number of multinucleate cells. Isolated protoplasts are thoroughly washed from the enzyme solution nith a medium containing an osmotic stabilizer and calcium chloride. The optimal conditions for isolation, washing, and purification (centrifugation in the washing medium) are determined by calculating the number of viable protoplasts. Viable protoplasts are detected with the use of Evans blue, methylene blue, or fluorescein diacetate (Larkin, 1976). C. PROPERTIES The cytomorphological properties of plant protoplasts isolated from various plant tissue or from in vitro cultures usually correspond to those of the initial cell with a correction for the plasmolyzed state iFig. 1). The size of plant protoplasts varies from 15 to 60 pm in diameter. Small protoplasts can result from meristematic cells or appear as a result of plasmolysis leading to fragmentation of the cell contents into several subprotoplasts (Binding and Kollmann, 1976); this can also occur after budding. All plant protoplasts are as a rule spherical in shape, the only exception being cylindrical protoplasts from leguminous plants root nodules filled with Risobium sp. bacteroids. Plant protoplasts membranes, unlike certain

ISOLATED PROTOPLASTS AND SOMATIC PLANT CELLS

33 1

FIG. I . Plant protoplasts isolated from N . r t r h u c ~ t m leaf mesophyll (A). N . knighriroitr leaf mesophyll ( B ) . V . firhtr leaf mesophyll ( C ) , S. thcrc~ooiwleaf mesophyll (D), S. rrthrmsurrr cell culture (E), and D . ('tirortr cell culture (F). ~ 3 9 0 .

bacerial protoplasts (Marquis and Comer, 1976) and surface membranes of micoplasmas (Maniloff and Morowitz, 1972), cannot retain the form of the cell. The morphological markers for mesophyll protoplasts are chloroplasts, which uniformly spread in the thin layer of cytoplasm on the protoplast periphery. Chloroplasts that are clustered on one side and form a compact agglutinated mass indicate that the mesophyll protoplasts is not viable. Leukoplasts and numerous proplastids are plastid markers for protoplasts isolated from cell cultures. In protoplast plastids there is a large amount of starch. This is also observed when the initial cells contain no starch (Fowke et ul., 1973; Sidorov et al., 1977). A . L. Kursanov and N. A. Ryabushkina (personal communication) demonstrated that protoplasts isolated from tobacco leaf mesophyll incubated with bicarbonate-I4Cconverted the labeled products into chloroplast starch via photosynthesis. Within 15 minutes 45% of the assimilated label was found to be present in the alcohol-insoluble fraction, mostly i n starch. These results point to a decrease in the metabolic activity of protoplasts as a result of osmotic stress. However, the accumulation of transitory starch can be associated with preparation for cell wall regeneration synthesis (Hanke and Northcote, 1974). In isolated protoplasts, compared to normal cells, the number of profiles of rough endoplasmic reticulum localized in the vicinity of the nucleus, and also in the periphery of the protoplasts parallel to the plasmalemma, first decreases and then increases (Fowke et al., 1973; Robenek and Peveling, 1977; Belitser el af., 1977). This is also true of mitochondria and ribosomes-the increase in the

332

RAlSA G. BUTENKO

number of these organelles is preparation for regeneration of the cell wall and cell division. Plant protoplasts are normally uninucleate. Polynucleate protoplasts that are sometimes observed are either the result of spontaneous fusion of protoplasts during the course of isolation (Fowke et al., 1975) or arise later during culltivation when, for different reasons, karyokinesis is not followed by cytokinesis (Eriksson and Jonasson, 1969; Bawa and ‘Torrey, 1971; Withers and Cocking, 1972; Reinert and Hellmann, 1973). In isolated protoplasts nuclei often acquire an irregular lobe form which iri also characteristic of callus cells (Yakovleva, 1970). After removal of the cell wall, a plant protoplast promptly reaches osmotic equilibrium with the external medium. Osmotic lability is a property inherent in virtually all plant protoplasts, and isolated plant protoplasts can act as osmometen. A constant increase in the osmotic pressure of the medium leads to the dehydration and shrinkage of protoplasts and then to their degradation (Kuchko and Butenko, 1971). Gradual dilution of a medium containing an osrnotic stabilizer with water, or dialysis of a protoplast suspension in a medium coritaining an osmotic stabilizer against water, causes swelling of protoplasts, a considerable increase in their volume, and then bursting. A sharp decrease in the osrnotic pressure of the external solution induces instantaneous lysis of protoplasts. The rate of swelling and the time between the beginning of dilution and the onset of lysis depend on the molecular weight of the osmotic stabilizer. The study of the osmotic properties of plant protoplasts, and of the effect on its stability af the type of osmotic stabilizer, hormonal factors and ionic conditions are still in the initial phase. Isolated protoplasts represent a convenient system for studying water metabolism in a plant cell (Pavlenko et al., 1978). Studies in which the physiological activity of isolated plant protoplasts have been investigated are rather few. Attention has so far been focused on regeneration of the cell wall and cell division. This is only natural, because interest in the production of callus tissues and plants regenerated from them has been great. Now greater attention is being paid to the physiological study of protoplasts. The preparation of isolated protoplasts involves a shock that is expressed in vac uolization, ribosome impoverishment, and nucleus and chloroplast condensaltion. Then, under favorable cultivation conditions, dedifferentiation and preparation for division occur. Thus, on the one hand, there is degradation of initial cell structures (Figs. 2 and 3) and, on the other, formation of new profiles of endoplasmatic reticulum, mitochondria, and ribosomes (Gigot et al., 1975; Belitser et al., 1977; Sidorov et al., 1977). A study on the functional activity of isolated protoplasts that have passed the shock stage but have not yet started dediffenmtiation would be of great interest. In the majority of articles there is no indication as to the stage of the studied protoplasts. Therefore we cite only works in which the protoplasts were in the first 24 hours of cultivation, although under conditions

ISOLATED PROTOPLASTS AND SOMATIC PLANT CELLS

333

FIG.2 . ( A ) Mesophyll protoplast of N . rtrhtrc,um immediately after isolation. Note localization of the catalase activity in the peroxisome associated with a chloroplast (arrow) [3,3-diaminobenzidine (DAB) reaction]. (B) After 2 days of cultivation. DAB reaction shows the peroxidase activity of a tonoplast (arrow). (Courtesy of Dr. N. B. Belitser.)

leading to regeneration of the cell wall, dedifferentiation, and division. Plant protoplasts are capable of absorbing from the external solution and metabolizing amino acids, sugars, precursors of nucleic acids, and terpenoids. Incorporation into the total protein of labeled amino acids by protoplasts isolated from Avena coleoptiles hardly ever differed from incorporation by the coleoptiles themselves

3 34

RAlSA G . BUTENKO

FIG. 3 . Dedifferentiation of a chloroplast (degradation of the lamellar structure and accumulation of starch) during the cultivation of an isolated mesophyll protoplast of N . /cih[icum (Belitser PI d , , 1977).

(Yasmine and Esnault, 1976). Synthesis and degradation of protein in isolated pea leaf protoplasts were similar to protein metabolism in the initial plant material (Watts and King, 1973; Watts et af., 1974). Le~cine-~H, ~ridine-~H, and thymidinesH were incorporated in an acid-insoluble fraction of protoplasts from oat leaf mesophyll. Incorporation started immediately after isolation and lasted for 6 hours for Ie~cine-~H and ~ridine-~H, and for 21 hours for thymidin~e-~H (Fuchs and Galston, 1976). Autoradiographs showed that the incorporation of labeled thymidine-gH into nuclei DNA of protoplasts isolated from tobacco leaf mesophyll began 24 hours after isolation and reached a maximum at 48-72 hours (Viktorova et al., 1977). Protoplasts freshly isolated from Calendula officirzafis leaves weakly incorporated Na2'4CO:,and acetate- 1-I4C; the incorporation intensified 15-20 hours later, especially under illumination. Up to 40% of latxled precursors were found in the fraction containing fatty acids and unsaponificated lipids (Kasprzyk er al., 1976). This evidence shows that the metabolic activity of protoplasts is comparable to that of the initial cells. However, there are obsixvations indicating that the incorporation of amino acids and sugars into isolated

ISOLATED PROTOPLASTS AND SOMATIC PLANT CELLS

335

protoplasts is four times as low as in the plasmolyzed cells (Ruesink, 1973). Absorption of precursors by plant protoplasts undoubtedly depends on the type of osmotic stabilizer and the pH of the external solution, as well as on the species and tissue specificity of the protoplasts and their density. Rubin and Zaitlin (1976) pointed out that the density of protoplast suspensions markedly affected the absorption of le~cine-~H and ~ r i d i n e - ~precursors. H A 2-order decrease in the density of a protoplast suspension caused a 125-fold increase in the absorption of precursors per protoplast. These workers believed that in concentrated suspension labeled precursors were diluted by nonlabeled ones of cellular origin. Respiration of protoplasts decreases during isolation and during the shock phase (Maretzki and Nickell, 1973). The intensity of respiration of Petunia leaf protoplasts considerably increases (by one order) after 3 hours of cultivation (Hoffmann et al., 1975; Kull and Hoffmann, 1975). Zeatin at first increases the intensity of respiration compared to that of the controls and then sharply decreases it. The respiration of mesophyll Petunia protoplasts rapidly increases with age (Kull and Hoffmann, 1975). The respiration of isolated protoplasts is sensitive to the action of respiratory inhibitors, and 1 x 10-4M 2,4-dinitrophenol (DNP) induces an uncoupling effect (Pavlenko et a l . , 1978). Protoplasts isolated from the leaf cells of photosynthesizing plants retain the ability to photosynthesize (Wegmann and Miihlbach, 1973). The photosynthetic activity of protoplasts from spinach leaves was the same as that of leaf tissue (Nishimura and Takashi, 1975). The photosynthesizing activity decreased with the age of the protoplast suspension (Kull and Hoffmann, 1975). Possible reasons for this are the excessive saturation of chloroplasts with starch (Hanke and Northcote, 1974; Ryabushkina and Kursanov, 1978) and gradual degradation of chloroplasts associated with the forthcoming division (Belitser er al., 1977). Protoplasts isolated from leaf mesophyll of C3 and C4 plants are similar to the initial tissue cells with respect to all parameters of photosynthetic carbon metabolism. Mesophyll protoplasts of C, plants are characterized by a high activity of phosphoenolpyruvate (PEP) carboxylase and a low activity of ribulose diphosphate (RuDP)-carboxylase; they are capable of fixing carbon dioxide into C4 dicarboxylic acids if the medium is supplemented with C, pathway precursors. Mesophyll protoplasts of various C3 plants have a complete set of enzymes for the accumulation of carbon via the Calvin-Benson cycle. Under favorable conditions their photosynthetic ability can be maintained for about 20 hours after isolation, and they are potentially capable of creating their own energy sources for metabolism and growth in a photosynthetic fashion. Photorespiration is inherent in C 3 but not in C4 protoplasts (Gutierrez et d.,1975; Edwards er al., 1976). The enzyme activity in isolated protoplasts has been studied by several investigators. Protoplast cytoplasm from Hippeastrum petals was shown to contain active esterases, proteases, fl-galactosidase, a-glucosidase, and &lucosidase;

336

RAlSA G . BUTENKO

in the fraction of vacuoles obtained from these protoplasts these enzymes were not present. In the cytoplasm and vacuoles of these protoplasts acid phosphatase, RNase, and DNase were also found (Butcher et al., 1977). The change in the isozyme spectrum of peroxidase was ascribed by Mader and co-workers (1976) to the preparation for dedifferentiation. The appearance of new isozymes in this case can be inhibited by actinomycin and cycloheximide. Premecz et al. (1977) reported an increase in the activity of RNase in isolated tobacco mesophyll protoplasts which they believe to be the consequence of osmotic shock. In protoplasts isolated from soybean root nodules, nitrogenase activity has been studied (Schelter and Hess, 1977). Protoplasts from nodules of another leguminous plant, Vignu, contained much leghemoglobin and were capable of reducing acetylene. Reduction of acetylene started 24 hours after isolation. The nitrogenase activity was preserved for 11 days; ATP, PEP, and succinate increased this activity, whereas leghemoglobin inhibited it. The maximal rate of acetylene reduction was 2-5% of that for the nodules (Broughton et al., 1976). There are grounds for believing that the physiology of isolated protoplasts, will in the near future attract the attention of more and more scientists. Plant protoplasts are especially useful in the study of photosynthesis and photorespiration, because they represent a system intermediate between chloroplasts and :plant tissues. Plant protoplasts have an advantage over chloroplasts in that they allow one to consider the effect of cytoplasmic factors on photorespiration and also to study the photosynthetic metabolism of carbon supported by functionally active cytoplasm. D. PLASMALEMMA Plant protoplasts represent a unique biological system for studying the properties of a surface membrane of a plant cell, the plasmalemma, and regulation of its activity. Moreover, comparison of the response of the plasmalemma to a certain action in the intact cell and in the isolated protoplast allows one to study the structural and fuctional character of the interaction of the plasmalemma and cell wall. Like other features of plant protoplasts, the properties of the plasmalemma are determined by genetic factors, by the epigenetic and physiological state of the initial cells, and by the medium (osmotic stabilizer, pH, and ion composition) in which the protoplasts existed. The structure of the protoplast membrane is investigated by several electron microscope techniques, namely, ultrathin sections, complementary replicas, freeze-etching and deep etching, and scanning electron microscopy (SEM). Lectin microprobes are just coming into use. The application of physiological methods allows one to investigate transmembrane transport of metabolites, physicochemical parameters of protoplast membranes, and the response of the membrane to the action of hormonal factors, toxins, and inhibitors.

ISOLATED PROTOPLASTS AND SOMATIC PLANT CELLS

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The population of plant protoplasts is hetemgepeous with respect to the properties of the membrane. This heterogeneity is less noticeable in protoplasts isolated from leaf mesophyll and is more pronounced in protoplasts isolated from other plant organs or cell cultures. The degree of folding is one of the signs of the heterogeneity of the surface membrane (Fowke et al., 1973); in addition, some protoplasts invariably lyse even with an optimal concentration of osmotic stabilizer (Gleba et al., 1974; Avetisov et a / . , 1978). The surface membrane of protoplasts is elastic and can stretch significantly. The maximal increase in the surface of protoplasts is about four times. The same data were reported for tobacco leaf mesophyll protoplasts treated with auxin in a concentration of 1 x lop4to 1 x 10-:’M (Pavlenko et al., 1978). The surface area of the membrane can increase at the expense of unfolding, but this alone cannot account for the 4-fold increase. The volume of protoplasts achieves a maximum within 30-40 minutes after the addition of auxin. This rate renders improbable an increase in the membrane material at the expense of new formation. The elasticity of plant protoplast membranes observed experimentally does not fit the theory of a liquid mosaic membrane structure (Singer and Nicholson, 1972). This is also the opinion of Marquis and Comer (1976) regarding bacterial protoplasts and Evans and La Celle (1975) regarding erythrocytes. The plasmalemma of plant protoplasts behaves like a bacterial membrane devoid of sterols, rather than like the sterol-containing membrane of erythrocytes. Ruesink (1971, 1973) showed that the surface membrane of protoplasts from oat coleoptiles was resistant to polyene antibiotics, such as nystatin and amphotericin, which destabilize sterol-rich membranes. That plant plasmalemmas are poor in sterols is demonstrated by the response of the membranes to cationic and anionic detergents. There is much controversy about the surface charge of the plasmalemma of isolated plant protoplasts. Ruesink (1973) believes that on the surface of protoplasts isolated from oat coleoptiles there is a strong negative charge. Grout and Coutts (1974) and Coutts and Grout (1975), on the basis of electrophoretic evidence, support the idea that isolated protoplasts are negatively charged. According ot them, plycations can recharge membranes without degrading them. Galston et al. (1977) studied the change in the surface charge on the membrane of tobacco mesophyll protoplasts that are capable of regenerating the cell wall and dividing, and mesophyll protoplasts of oat and corn that do not form the cell wall and could not divide. According to these investigators, the charge on the plasmalemma of the initial cells was always negative. Right after isolation the charge on the plasmalemma of all the protoplasts became weakly positive. On the surface membranes of protoplasts from oat and corn the positive charge remained throughout formation of the cell wall, whereas the charge of tobacco protoplast plasmalemmas again became weakly negative and, during the formation of a rigid cell wall, still more negative. Protoplasts of oat and corn, even after foma-

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RAlSA G . BUTENKO

tion of the cell wall, collapsed when the osmotic pressure was raised, but tobacco protoplasts plasmolyzed normally. These workers attributed the osmotic instability of the former to the irreversibility of the sign of the charge on the plasmalemma of the protoplast. It is possible that there is a direct relationship among the charge on the plasmalemma, the formation of the cell wall, and the ability of protoplasts to undergo sustained cell division. The adhesion of particles to the surface of the plasmalemma of plant protoplasts and the phenomenon of endocytosis are related to the charge on the plasmalemma. Endocytosis of latex globules, femtin, and virus particles was reported (Cocking, 1965, 1966; Mayo and Cocking, 1969; Suzuki et al., 1977). In the last-mentioned work it was shown that the uptake of polysterol spheres by tobacco mesophyll protoplasts proceeded at a fast rate. Both individual sphleres and groups of spheres were absorbed, and great amounts of endocytotic vesicles containing the spheres were found to be present in the cytoplasm. The upmke decreases, as does the temperature, in the presence of DNP and azide, which shows that the process is energy-dependent. Poly-L-omithine and other polycations stimulate the uptake. The mechanism of the uptake of virus particles is under discussion. Suzuki and co-workers (1977) favor the uptake of viruses via endocytosis. Burgess et al. (1973a,b) believe that viruses penetrate into the protoplast through lesions and breaks in the plasmalemma, and that poly-L-omithine induces these breaks. Zhuravlev et al. (1975) believe that the absorption of viruses involves several mechanisms, the operation of which can be regarded as a single electroosmotic mechanism. The use of lectins as microprobes can be useful in studying the properties of the plasmalemma. Lectins bind to the plasmalemma of plant protoplasts (Burgess and Linstead, 1976a, 1977b; Williamson et al., 1976) and cause them to agglutinate (Glimelius et al., 1974; Larkin, 1977). In animal cells it is known that lectin-concanavalin A (Con A) binds with a carbon receptor which is mobile in the membrane. This mobility leads to the formation of clusters as a result of the association of Con A molecules. In the work of Williamson et al. (1976) (Con A labeled with hemocyanin was shown to cluster in the plasmalemma of soytean protoplasts. Burgess and Linstead (1976a) demonstrated by thin-sectioning and SEM techniques that a calloid gold-Con A complex clustered on the surface of tobacco protoplasts. It was demonstrated that a protoplast could bind 108 molecules of Con A , which was commensurable with the number of sites, 3 x lo7,per cell for fibroblasts. When the temperature was raised from 5" to 30°C the mobility of the complexes increased but was much lower than in animal tumor cells. The formation of clusters in the plasnialemma of tobacco mesophyll protoplasts required 30-60 minutes. Studies involving protoplasts have also provided evidence that the binding sites of specific toxins produced by the pathogens Helminthosporium sacchari

ISOLATED PROTOPLASTS AND SOMATIC PLANT CELLS

339

and H. mciydis are located on the plasmalemma (Strobel and Hess, 1974; Pelcher et al., 1975).

Phytohormone auxin was shown to induce a rapid effect on the membrane and on the osmotic properties of isolated plant protoplasts of different origin (Ruesink and Thiniann, 1965; Power and Cocking, 1970; Bayer, 1973; Hall and Cocking, 1974; Bayer and Sonka, 1976; Pavlenko et al., 1978). Auxin ( I x M ) added to a ptotoplast suspension in osmotic equilibrium with the medium induces the absorption of water and swelling and lysis of protoplasts. The rate of the process depends on the type of osmotic stabilizer; it is higher for low-molecular-weight stabilizers and lower for those of high molecular weight. The curves in Fig. 4 show the dependence of the diameter of the protoplasts and the time of lysis after the addition of auxin on the type of stabilizer. These results indicate the importance of plasmalemma stretching and the auxin-induced increase in the size of water channels for an increase in cell volume. The cascade character of the process results in such an increase in channel size that transport of the osmotic stabilizer into the protoplast becomes

(A I

5

5 L

yz

10

0

15 20 25 T (minutes)

30

w

,

35

40

I 2 3

40

4

a

(B)

5

10

15 20 25 T (minutes)

30

35

40

FIG. 4. ( A ) Same as in ( B ) hut in the presence of auxin ( 1 x M iodoacetic acid). Arrows show the beginning of lysi\ of protoplahts. ( B ) The increase in the size of isolated mesophyll protoplasts of N . itrhtrcrtrti o n incuhation with different osmotic stabilizers. The osmotic potential of the incubation mcdium was X.7 a1m in all cases. I. Sucrose; 2. mannitol; 3, PEG (MW 400); 4. PEG (MW 3000)

340

RAISA G. BU'I'ENKO

possible. It is most important here that the size of a channel correspond to that of the stabilizer molecule. The primary mechanism of auxin action on the plasmalemma of mesophyll protoplasts from Nicotiana and Vicia was studied by Bayer (1973) and Bayer and Sonka (1976). Within less than 1 second after the addition of 1 x 10 M auxin to a protoplast suspension from Vicia, the pH dropped by 0.2. DNP (lo-'' M ) decreased the auxin-induced pH effect; 50-60 minutes later the protoplasts lost their ability to respond to the action of auxin by acidification of the medium. It was restored by the addition of potassium chloride. The results were interpreted as indicating a H+ efflux and an influx of K+. The bacterial toxin fusicoccin produces a similar effect. Rollo et a f . (1'977) compared the effect of fusicoccin on cell cultures and protoplasts of Boston ivy, platan, tobacco, and other plants. In all cases acidification of the medium was observed resulting from a H+ efflux and cotransport of K+ and 3O-methylglucose into the cells. The relationship between the events in the membrane and the metabolism and growth of protoplasts is still obscure and requires more comprehensive study.

IV. Cultivation A. TECHNIQUES

Plant protoplasts washed from enzyme preparations and purifed from cell debris and chloroplasts are placed in culture media similar to those used for cell suspensions (Gamborg et al., 1976) but modified in accordance with the species to be studied. Optimal combinations of conditions for different species are sought empirically. Potrykus et af. (1976) suggested a microtechnique for large-scale screening of culture conditions for protoplast suspensions. His group cultivated protoplasts in 4O-pl hanging drops, to each of which about 400 protoplasts were added. Under such conditions protoplasts formed a monolayer. A more common method is to place protoplasts in 0.1-ml drops (Kao et al., 1971). Also, prcltoplasts can be suspended in liquid media and grown in petri dishes in a thin layer of liquid; they are sometimes plated onto agar media or onto liquid over agar. :Petri dishes are sealed with Parafilm and placed under constant conditions, at 25°C in the dark or under 200-400 lux illumination. With the drop technique the periodic addition of fresh medium is recommended. Sometimes success in cultivation is achieved by a stepwise decrease in the concentration of an osmotic stabilizer in solution (Kameya, 1975). However, cells from protoplasts can be placed in a medium without an osmotic stabilizer only after large clusters of cells have k e n formed.

ISOLATED PROTOPLASTS AND SOMATIC PLANT CELLS

34 1

The ultimate goal of the cultivation of protoplasts is to obtain cell colonies which can then be grown by routine methods.

B. REGENERATION OF THE CELLWALL At the first stages of cultivation under the most favorable conditions, from 5 to 20% of plant protoplasts undergo degradation. This is a result of the heterogeneity of the population in relation to osmotic properties. Viable protoplasts rapidly (within 24 hours) regenerate the cell wall. The formation of the cell wall is indicated by a change in protoplast shape, budding, and the ability to repeated plasmolysis. Formation of the cell wall can be detected even earlier with the help of fluorescent brighteners (e.g., Calcofluor White), specific staining for polysaccharides, all kinds of electron microscope techniques (thin sections, deep etching, surface replication, and scanning (Pojnar et al., 1967; Nagata and Takebe, 1970; Nagata and Yamaki, 1973; Burgess and Fleming, 1974; Willison and Cocking, 1972; Fowke et a/., 1973; Williamson et al., 1977; Asamizu et a / . , 1977). Formation of the cell wall is not only the decisive point in the development of plant protoplasts but is also a useful model for studying the synthesis of polysaccharides and the self-assembly of polymers on the surface of the plasmalemma. The majority of investigators who have studied regeneration of the cell wall by plant protoplasts believe that at the beginning of cultivation the surface of the protoplast plasmalemma has no remnants of the old wall. They are contradicted by Prat and Roland (1971), who think that random localization of microfibrils at the beginning of deposition and great uniformity at the later stages point to the necessity for a template for self-assembly. The rate and regularity of cell wall regeneration depend on the species of plant and the differentiation of the initial cell. Protoplasts from leaf mesophyll of the genera Nicotiana, Petunia, Datura, and Brussica quickly form the cell wall. In 24 hours spherical protoplasts became oval, and the cell wall can be detected with the help of Calcofluor White (Hess et al., 1973; Frearson et al., 1973; Gleba et al., 1974). Leaf mesophyll protoplasts of cereals and some leguminous plants require about 4 days for cell wall regeneration. Cell wall regeneration is extremely difficult in protoplasts from oat coleoptiles and cell cultures of rose (Pearce and Cocking, 1973). The same is true of protoplasts isolated from cell cultures of H. gracilis (Eriksson and Jonasson, 1969). Giles (1972) described protoplasts from mesophyll cells of Digilaria sanguinalis grass that could not form the cell wall. They acquired it only after fusion with soybean protoplasts that formed the cell wall normally. Ultraviolet-irradiation inhibits regeneration of the cell wall and simultaneously the synthesis of protein and RNA (Sakai and Takebe, 1974). The formation of cellulose microfibrils and regeneration of the cell wall can also be reversibly

342

RAISA G . BUTENKO

inhibited by certain concentrations of cumarin. The same concentration can induce characteristic changes on the surface of the plasmalemma (Burgess and Linstead, 1977a). Concentrations of sucrose exceeding 0.3 M and of sorhitol above 0.5 M can inhibit cell wall formation (Shepard and Totten, 1975). It is possible that different factors that inhibit cell wall regeneration come into play at different stages of this multistep metabolic process. Most often protoplasts from various plant tissues and cell cultures can form the cell wall within 4 days. The wall has an almost normal structure, but the microfibril network of cellulose is looser at the first step. Asamizu er d.(1977) studied cell wall regeneration by protoplasts from a suspension of carrot cells; they showed that the synthesis of cellulose started without a lag phase immediately after the protoplasts were placed in the medium. The deposition of cellulose microfibrils on the surface of the plasmalemma was at first very slow and became more intensive after 8 days of cultivation. At first glucan chains of low molecular weight were formed, and then their molecular weight increased. The degree of polymerization of cellulose was at first very low but baciime normal by the time of cell division; however, its quantity was only I / I O that in the initial cell. These workers showed that short microfibrils were at first randomly localized on the surface of the plasmalemma and then became oriented parallel to it. Williamson et al. (1977) used the platinum-palladium replica technique to reveal the time course of formation of the cell wall in a suspension of protoplasts from Vicia hujastuna leaf mesophyll. The deposition of microfibrils on the surface of the plasmalemma was observed as early as 10-20 minutes later, and after 20 hours they formed a rather dense network on the surface of the pmtoplasts. After 4 hours 50%, and after 16 hours 90%, of all protoplasts had fonned the wall. Robenek and Peveling (1975, 1977) studied cell wall regeneration by leaf protoplasts and cell cultures of Skimmia japonica stem. They used freezeetching, deep etching, scanning, and thin-sectioning to show the absence of any remnants of the cell wall on the surface of the protoplasts. At the onset of cultivation typical changes in the cytomorphology of the protoplast were observed. The number of profiles of rough endoplasmic reticulum increased. The majority of them were seen close to the plasmalemma, which formed invaginations toward them. In addition to stretched endoplasmic reticulum profiles, there were many wider tubular structures that had no definite localization. The number of polysomes and mitochondria increased. The changes on the external and internal surfaces of the plasmalemma were specific. Particles that had tieen localized uniformly on both surfaces of the plasmalemma increased in numlber, and their arrangement in rows started in 4 hours; in 48-72 hours all the parti'cles were arranged in dense rows and formed hexagonal structures. The particles were projected onto the external surface as pores. The first microfibrils appeared in 24 hours; they were oriented as rows of particle-pores. It has been suggested that the

ISOLATED PROTOPLASTS AND SOMATIC PLANT CELLS

343

sequence of events is as follows. Transfer of protoplasts into the culture medium induces transcription activity of the nucleus; a protein synthesis apparatus (polysomes) then forms, and the number of endoplasmic reticulum profiles (the site of cellulose precursors synthesis) increases. In the case of Skimmia protoplasts, the dictyosomes and vesicles of the Golgi body do not play an important role in the synthesis of the products responsible for cell wall regeneration. Cellulose synthetase forms on the profiles of rough endoplasmic reticulum and is then transferred to the plasmalemma to be activated. The rows of particle-pores on the surfaces of plasmalemma consist of proteins that stabilize the channels through which polymer molecules penetrate to the outer surface of the plasmalemma, where the self-assembly of fibrils is controlled by the specific arrangement of the particlepores (a template of 14 particles arranged in a row) begins. The results obtained with isolated protoplasts have aroused controversy with respect to the participation of cell organelles in the synthesis of substances for cell wall regeneration. Vesicles of the Golgi body (Prat, 1972; Roland, 1973), specific cell wall bodies (Pearce ef al., 1974), vesicles formed by invagination and folding of the plasmalemma during plasmolysis (Cocking, 1970; Burgess and Fleming, 1974), and different forms of endoplasmic reticulum (Robenek and Peveling, 1977) are suggested sites of synthesis. It is possible that there is no unique scheme for diverse species, and that the observed differences only reflect genetic peculiarities of the plants or epigenetic specificities of the tissues from which the protoplasts have been obtained. There is abundant evidence that cell wall regeneration by plant protoplasts may have anomalies that are expressed in budding, in the absence of cytokinesis or an abnormal position of the middle lamella and cell plate, and also in incomplete formation of cell plates between divided cells. A deficiency or loss of pectins in the medium may be one of the reasons for malformation of the cell wall. Out of four pectin substances that should be present in the cell wall, Hanke and Northcote (1974) detected only three, and the fourth, major pectin fraction was not form in the protoplasts. That there is a relationship between the loss of pectins in the medium and cell wall malformation is proved by the fact that much better regeneration is achieved by plating protoplasts on agar rather than on a liquid medium (Frearson et al., 1973; Avetisov et a / . , 1978), and also by better and faster formation of the cell wall on the surface of a plasmolyzed cell compared to the surface of isolated protoplasts (Prat and Roland, 1971; Roland and Prat, 1973). The loss of pectin substances or their insufficient synthesis can result in anomalies in the stretching of the cell wall as the volume of the protoplast increases. Cellulose microfibrils do not display the typical slipping over one another; this leads to uneven stretching of the wall and expansion (budding) of the protoplast at its weakest site (Willison, 1976). The budding of protoplasts

344

RAlSA G. BUTENKO

FIG.5 . Different forms of budding during the cultivation of mesophyll protoplasts o f V.faba in drops of liquid medium.

ISOLATED PROTOPLASTS AND SOMATIC PLANT CELLS

FIG. 5 .

(continued)

345

346

RAISA G . BUTENKO

during cultivation has been described by many investigators (Pojnar et al., 1!)67; Bawa and Torrey, 1971; Horine and Ruesink, 1973; Pearce and Cocking, 1!)73; Hanke and Northcote, 1974; Fowke et al., 1974). The budding observed in protoplasts from Vicia faba mesophyll has different forms (Fig. 5 ) . The bud can lose or retain a connection with the bulk of the protoplasts; it can contain almost all of the vacuole or part of the cytoplasm with the organelles and the nucleus. If the connection with the protoplast is retained, the bud may have cytoplasmic strands that are common for the whole cell, and on the surface a cell wall of the normal thickness if formed (Avetisov et al., 1978). Bawa and Torrey (1971) showed that malformation of the cell wall by Convolvulus protoplasts resulted in the appearance of multinucleate protoplasts, since karyokinesis was not followed by cytokinesis. The same observation was made by Reinert and Hellmann (1973) on protoplasts from carrot and by Sharma (1974) on protoplasts from Lycopersicon pimpinellifolium. In the latter case the protoplasts did not form the cell wall at all, and specific coenocytic structures arose. These structures were also described by Gosch and co-workers (1975) for protoplasts obtained from cell suspensions of Arropa belladonna. Anomalies in cell plate formation in protoplast cultures have been reported in the literature (Fowke et al., 1973, 1974, 1975; Prat and Poirier-Hamon, 1975). The common reason for anomalous formation of the cell wall and cell plate by plant protoplasts is insufficient synthesis of pectins (Hanke and Northcote, 1974). Even in protoplast cultures from tobacco leaf mesophyll Sidorov el al. (1 977) observed an incomplete first cytokinesis (Fig. SA), although tobacco mesophyll protoplasts are known normally to form the cell wall and divide. Belitser et al. (1977) has reported that in protoplasts grown in liquid medium,the first cytokinesis is characterized by the formation of a wide cell plate made of loosely packed microfibrils. The cell wall at this moment is far from being complete. On the surface of the plasmalemma there is a thin layer of very loosely packed microfibrils. The periplasmic space is not very well formed (Fig. 6B). The studies of Belitser et al. (1977) and Sidorov et al. (1977) on tobacco protoplasts suggest that the formation of a normal cell wall and division of the nucleus may be independent processes. However, the results of SchildeRentschler (1977) are at variance with this idea. Having added cellulase to the culture medium in which tobacco mesophyll protoplasts had been grown, she observed the inhibition of cell wall regeneration and nucleus and cell division. Thermal denaturation of the enzyme decreased and removed the inhibition. The addition of cellobiose, which inhibits the activity of cellulase, caused noimal formation of the cell wall and cell division, as a result of which colonies were formed. It seems that there is a direct relationship between the ability of protoplasits to regenerate a normal cell wall and divide to form cell colonies.

ISOLATED PROTOPLASTS AND SOMATIC PLANT CELLS

347

FIG.6 . The first mitosis of a mesophyll protoplast of N . rabacum. (A) Incomplete cytokinesis. (B) Loosely packed cellulose fibrils of a regenerated cell plate. (Courtesy of Dr. N. V. Belitser and Dr. V . A. Sidorov.)

348

RAISA G. BUTENKO

C. DIVISION A N D FORMATION OF CELLCOLONIES Induction of division and sustained division in plant protoplasts cultures is less frequent than successful isolation of protoplasts. The list of species of plants for which the conditions for the formation of cell colonies from protoplasts have been determined is increasing. It is beyond doubt that the ability of a cell that has originated from a protoplast to begin dividing depends on differentiation of the initial cell. Sometimes, however, regardless of the differentiation of the tissue cell, the protoplasts obtained from it can be either capable or incapable: of dedifferentiation and division. For example, the photosynthesizing cells from leaves of Nicotiana, Petunia, Datura, and Solanum (Fig. 7) easily form protoplasts capable of dividing whereas in cereals division is rare (Potrykus et al., 1976; Deka and Sen, 1976). Koblitz (1974) used cereal cells as the initial material for obtaining protoplasts, that is, barley cells grown in a suspension culture; the protoplasts gave colonies of callus cells. Potrykus et al. (1977b) obtained a callus culture as a result of sustained division of protoplasts isolated from stem tissue of corn plants, although they had unsuccessfully tried to obtain dividing protoplasts from corn leaf tissue (Potrykus er al., 1977a). The ability of the cells formed from isolated protoplasts to undergo sustained division apparently also

FIG. 7.

Induction of division and formation of cell colonies in a culture of isolated protoplasts of

S, chacoerise.

ISOLATED PROTOPLASTS AND SOMATIC PLANT CELLS

349

depends on the genetic and epigenetic specificities of the initial material. Such features are the presence of heterochromatic nuclei or a special state of the membrane which cannot accept phytohormones to activate the nucleus and also the inability of the protoplast to form a normal cell wall. It is difficult to say whether these blocks can be removed by altering the external conditions. Attempts to achieve this empirically have shown that it is difficult (Potrykus et al., 1976, 1977a). Potrykus and co-workers (1976, 1977b) studied the conditions required for sustained division in a culture of protoplasts isolated from leaves of 75 species and varieties of wheat, barley, rye, oat, and corn. To overcome the block in nuclei division, they tested 40,000 combinations of 144 phytohormones and growth stimulators, but without success. However, the block could be removed by the fusion of protoplasts from barley leaves with protoplasts isolated from the cells of an actively proliferating suspension of carrot cells (Constabel et al., 1975b; Dudits et al., 1976a). These were the first results for plant material showing that it was possible to overcome the block and induce the division of interphase nuclei using fusion with a premitotic partner. The results of the study on the change in the membrane potential of cereal protoplasts during isolation and in the course of cultivation, compared to tobacco mesophyll protoplasts capable of dividing, demonstrate the special state of the membrane of the former (Galston et al., 1977). It is an established fact that the conditions of cultivation affect the frequency and rate of formation of cell colonies by protoplasts capable of dedifferentiation and division induction. Of great importance is the plating density of the protoplasts. The optimal density is 1 X l o 4 to 5 X los protoplasts/ml. Such a density gives confluent growth in a petri dish. In isolating a cell line from an individual protoplast the density should be decreased. Protoplasts isolated from pea root tip were capable of dividing at a lower plating density, 6 x lo3 protoplasts/ml (Landgren, 1976). Kao and Michayluk (1975) used a much more complicated culture medium and thus succeeded in obtaining callus tissues with a plating density of 3-5 protoplasts/ml. The feeder layer technique also gives good results with a low (1 x 102) plating density (Raveh et al., 1973; Raveh and Galun, 1975; Kuchko and Butenko, 1977; Butenko et al., 1977a). Raveh et al. (1973) and Raveh and Galun (1975) used isolated tobacco protoplasts x-irradiated with a dose that inactivated but did not kill the cells, which they mixed with agar to form a feeder layer on top of which isolated citrus protoplasts mixed with agar were plated. In the studies of Kuchko and Butenko (1976a) protoplasts isolated from the leaves of two species of potato Solanurn tuberosum and S. chacoense were plated on a feeder layer formed of tobacco or potato cells inactivated with 6oCo rays and mixed with agar. The use of a feeder layer allows the plating density of the protoplasts to be deckased by two orders of magnitude; it also permits one to

350

RAlSA G. BUTENKO

improve the plating efficiency and facilitates the development of colonies capable of further growth as a cell culture (Table 11). The plating efficiency also depends on the composition of the medium and the concentration of the osmotic stabilizer. Kameya (1975) showed with Pelargonium protoplasts that the induction of division required one set of conditions and sustained division another. According to this investigator the optimum for induction of division is Murashiqe-Skoog (MS) medium diluted 10-fold with 10% mannitol. For colony formation 5% mannitol and a normal MS medium is better. The concentration and composition of mineral salts in the medium was of great importance in the division of protoplasts isolated from potato and tobitcco leaf mesophyll (Upadhya, 1975; Kuchko and Butenko, 1977; Uchimiya and Murashige, 1976; Shepard and Totten, 1977). Carbohydrates are also very important in some cases. Protoplasts isolated from pea root tip divided in a medium with sucrose but not with glucose and proved extremely sensitive to the composition and concentration of phytohormones (Landgren, 1976). The conditions of cultivation, that is, light, temperature, and humidity, also play an important role in the successful formation of colonies by isolated protoplasts. The most favorable is high humidity, 25"-28"C, and darkness or very low-intensity illumination (100-400 lux). Sometimes good results are achieved if protoplasts are first cultivated in complete darkness and then placed under lowintensity light (Cocking, 1976b; Banks and Evans, 1976). Division induction and colony formation by protoplasts of even related species may differ in their requirements for temperature, light, nutrition, and regulatory factors (Banks and Evans, 1976; Power et al., 1976b; Zapata et al., 1977). The optimal conditions for division induction and their maintenance for the formation of colonies can also differ (Kuchko and Butenko, 1976). In a culture of protoplasts that are readily induced to divide, the first division has been reported to occur in 48-72 hours, and the second and the following ones

EFFPCTOF

A

FIXDERLAYER ON

TABLE I1 THE CULTIVATION

OF

PROTOPLASTS

FROM

-

Solririurn tuhemsurn I'

Effect on Cell wall formation (%) First division ('70) Number of colonies after 2 weeks (a) Size of colonies (mm) After 4 weeks After 6 weeks "y-Ray-inactivated cell suspensions of N .

With feeder layer

Without feeder layer

75-80 50-55 40-45

75-80 15-20 3-5

2.4-3. I 3.9-4.8

0.8- I .3

I .2-2.0

o r S. tuhemsurn were used as a feeder layer.

~ ~ J U C U , ~

ISOLATED PROTOPLASTS A N D SOMATIC PLANT CELLS

35 1

at intervals of 48 hours (Gleba et al., 1974; Gosch et a l . , 1975; Kuchko and Butenko, 1977; Schieder, 1975). Sometimes the lag phase before the first division lasts as long as 7-25 days. Such a long lag phase has been reported for cotton protoplasts (Bhojwani et al., 1977; Khasanov and Butenko, 1979). Mitotic division in protoplasts cultures does not differ from that in cell cultures. The divisions are asynchronous. The first mitoses are observed in 2-3 days; in a week 10-20% of the cells divide (Schieder, 1975; von Arnold and Eriksson, 1976, 1977). The data on plating efficiency are contradictory. Different investigators give figures from 1-2% to 50-60%. For protoplasts isolated from leaf mesophyll of Nicotiana silvestris, Banks and Evans (1976) reported the frequency of divisions to be not more than 1%, whereas Nagy and Maliga (1976) reported a plating efficiency of 60-90% for the same species. The differences in the values for the plating efficiency may be due to the quality of the material and cultivation conditions, as well as different methods of calculation. Cell colonies as large as 1.5-2 mm can be placed under conditions that are common for the cultivation of cells and tissues.

V. Fusion and Somatic Hybridization Isolated plant protoplasts are useful in somatic hybridization because until a short time before the cell wall is formed they can fuse together. A. TECHNIQUE

The spontaneous fusion of protoplasts can be due to the plasmodesmata which expand rather than break. It is more frequently observed in protoplasts isolated from in vitro cultures than in those obtained from plant tissues. In the case of plant tissues fusion occurs more often between protoplasts isolated from meristematic cells than between leaf mesophyll cells (Cocking, 1971, 1972). Several methods have been suggested for the stimulation of protoplast fusion. Cocking (1971, 1972) used nitrates, mostly sodium nitrate, as a stimulant. The percentage of fusion was not very high, and protoplasts were not infrequently damaged. Nitrates proved inadequate for parenchymatous, vacuolized cells. The use of Ca2+ at a high (9.5-10.5) pH gave better results for such cells (Keller and Melchers, 1973). The action of nitrates and alkaline solutions is based on alteration of the surface charge of protoplast membranes, which causes their adhesion and then fusion. The adhesion of plant protoplasts does not always result in fusion. Agglutination of protoplasts can be induced by specific antibodies (Hartman et al., 1973) and lectins (Glimelius et al., 1974), but no fusion is observed. A solution of PEG (20% w/v) with a molecular weight of 1500-6000 proved to be advantageous for the induction of fusion in plant protoplasts (Kao

352

RAISA G . BUTENKO

FIG.8 . Fusion of mesophyll protoplasts of N . tcrhucurri with a protoplast isolated from a cell culture of Arthitlopsis rhdirrricr. Three hours after fusion the cytoplasm has not mixed. (Sidoi:ov d . . 1978.)

and Michayluk, 1974; Wallin e t a / . , 1974). PEG, first suggested for inducing the fusion of plant protoplasts, found application in the hybridization of animal cells (Pontecorvo, 1975) and for inducting the fusion of plant protoplasts and animal cells (Ahkong et u / . , 1975). Protoplasts agglutinate after PEG is added to the medium and after 20-40 minutes incubation the suspension is diluted with a solution containing no PEG, and fusion of protoplasts occurs (Fig. 8). PEG is necessary for both agglutination and fusion of protoplasts, but the concentrations required are different. The mechanism of action of PEG is obscure. Then: are several hypotheses, but none of them have been substantiated by facts. Fusion, especially induced fusion, always produces deterioration. PEG induces osrnotic stress and a rapid shrinkage of protoplasts at the first stage; just as rapid deplasmolysis occurs at the dilution stage. Protoplasts of various plant species isolated from differentiated tissues display differences in resistance to the damaging action of PEG. The concentration and time of treatment are important for the viability of the products of fusion. The protective action of Ca'+ ions in the fusion of plant protoplasts was reported by Cocking (1976b). In animal cell fusion induced by PEG, 5 1 0 % dimethyl sulfoxide was used as a protector (Norwood et ul., 1976).

ISOLATED PROTOPLASTS AND SOMATIC PLANT CELLS

353

Two methods of plant protoplast fusion induction by PEG have been described (Kao and Michayluk, 1974; Glimelius et al., 1974). The more popular is the fusion of protoplasts in a drop on a coverslip as described by Kao and Michayluk (1974). The protoplasts obtained in this way are damaged to a lesser degree than during the centrifugation required in the method of Glimelius and co-workers. The first method also enables one to carry out microscopic monitoring of the agglutination and fusion of protoplasts. After treatment with PEG, a suspension of protoplasts (both fused and nonfused) is washed with medium containing an osmotic stabilizer and calcium chloride and cultivated by standard methods. B. SELECTION O F HYBRID CELLS

1. Cytology of Fusion The cytology of fusion is studied by light and electron microscopy. SEM and transmission electron microscopy (TEM) are used, and the adhesion of surface membranes of protoplasts and cytological specificities of the products of fusion and daughter cells are investigated. For sufficient PEG-induced fusion of protoplasts there should be no cellulose microfibrils on the surface of the protoplasts (Ito, 1973; Weber et ul., 1976; Williamson et al., 1977). A TEM study of fusion of the protoplasts of pea and Vicia (Fowke et al., 1975), and pea and soybean (Fowke et al., 1977), showed that the membrane contact was discontinuous. Wallin et ul. (1974) observed continuous contact in the fusion of carrot protoplasts, and Burgess and Flemming (1974) reported that membrane contacts of both types were possible. The fusion of membranes takes place at the external edges of the contact, and internal zones degrade and form several vesicles which later gradually disappear (Fig. 8). A mosaic structure for the common membrane of fusion products of plant protoplasts has not been proved. PEG-induced fusion may involve two or more protoplasts. With 30% of the protoplasts being fused, 14% were due to 1 + 1 fusions (Constabel et al., 1975b). Usually the number of fused protoplasts is less than 10% (Cocking, 1976b). The fusion of cytoplasms is a rather slow process, requiring 3- 12 hours. The fusion of protoplasts of different plants leads to the formation of homokaryons (of each of the lines of the partner species) and heterokaryons. The formation of large multinucleate products of fusion was observed that, although capable of forming a cell wall, were not viable (Fowke et al., 1977). The regeneration of a cell wall hybrid with respect to chemical composition and structure has never been reported. If the two protoplasts to be fused are obtained from vacuolized cells, the nuclei in the fused protoplasts are often separated in space, which makes their fusion difficult. Premitotic nuclear fusion has been reported for soybean protoplasts (Miller et al., 1971), soybean and pea (Constabel et al.. 1975a), soybean and tobacco (Kao, 1976, 1977), and oat and carrot (Dudits et al., 1976a). The fusion of

354

RAISA G. BUTENKO

closely located interphase nuclei is facilitated by the formation of nuclear ml:mbrane bridges (Fowke et a / . , 1975, 1977). The formation of synkaryons was observed 24-48 hours after the beginning of cultivation. The hybrid nucleus was larger in volume and had common chromatin which mixed slowly. The daughter cells also had a hybrid nucleus (Constabel er a/., 1975a, 1977b; Fowke et a / . , 1976, 1977). Nuclei fusion is more often observed during mitosis when the movement of the cytoplasm is very active and the nuclei can come very close to each other. Synchronous division of nuclei provides a better chance for the formation of a common spindle and the fusion of nuclei (Gosch and Reinert, 1976). In heterokaryons resulting from the fusion of protoplasts of soybean and corn, about 50% of the first and 30% of the second mitoses were synchronlous (Kao et al., 1974; Kao, 1976), whereas divisions in the Viciu-soybean combination were not synchronous (Kao, 1976). The fusion of protoplasts from soybean and Nicorinima glaucu involved anomalies in chromosome behavior (chromatid bridges, ring chromosomes, fragments, and so on). After 6 months this line of the hybrid cells continued to reproduce, retaining the soybean chromosomes after having lost many of the N . glaucci chromosomes. The loss of N . gluucu chromosomes was accidental (Kao, 1977). One of the anomalies in the division of the product of protoplast fusion is multipolar mitoses leading to chimerical colonies. The behavior of nuclei in the products of fusion is difficult to predict. Nuclei can fuse to form a synkaryon and then a hybrid cell line. In a heterokaryon, the nuclei can divide independently. In this case the products of division form a cluster of cells after the cell wall is regenerated and, as a result, chimerical callus tissue arises. The nuclei can segregate and give rise to cells with hybrid cytoplasm and a nucleus of one of the partners. Such cells have been given the name “cybrids. They are of interest in studying the mechanisms of cytoplasmic heredity. Power et al. (1975) were the first to suggest the possibility of obtaining cybrid cell lines. The fusion of protoplasts of leaf mesophyll of Perunia with those from a cell culture of crown gall of Parrhenocissus resulted in the selection of a cell line with a Parthenocissus nuclei but with a peroxidase isoenz:yme spectrum that included proteins specific for both partners. The isoenzymes of peroxidase specific for Petunia persisted in the cells for a year and were later lost. These investigators believed this to be proof of the cybrid nature of the selected line. Cybrid cells and cybrid plants were obtained by Gleba er a / . (1!)74) and Burgutin er a / . (1977). In their studies normal and chlorophyll-deficient chloroplasts served as markers indicating a mixed cytoplasm. The karyological and morphological characteristics of the plant demonstrated that the nucleus of only one of the partners was retained. During multiplication of the cybrid cell in the dark or at low light intensity, and after multiplication during stem apex formation under the same conditions, mainly selection of chlorophyll-deficient plastids was observed. In contrast, in the development of the plant under normal illumination, normal green plastids were mostly selected (Fig. 9). The selection of plastids of a definite type after sexual hybridization was described by Schotz ”

ISOLATED PROTOPLASTS A N D SOMATIC PLANT CELLS

355

FIG. 9 . Preferential selection of normal plastids in the ontogenesis of a cybrid plant obtained by fusion of nuclear and plastoin chlorophyll-deficient mutants of N . rtrhtrcurn.

(1954) and Sager (1972). The cybrid character of the plants obtained as a result of fusion of mesophyll protoplasts isolated from a plastom chlorophyll-deficient mutant of Nicoriuna tabucum and a cytoplasmic male sterile analog of N . tubacum carrying cytoplasm of N . debneyi was proved by Gleba et ul. (1978) by the appearance of variegated plants and the presence of large subunit polypeptides of both N . tabucum and N . debneyi types in fraction I protein of these plants. Preferential selection of cytoplasm structures was demonstrated by Belliard et ul. (1977). These investigators fused protoplasts isolated from leaves of fertile plants of Samsum tobacco with leaf protoplasts of Techne tobacco having a characteristic male sterility (the genome of N . tabucum and the cytoplasm of N . debneyi). Somatic hybridization produced plants that differed in flower morphology and degree of fertility. Nicotiana tabacum X N . debneyi mixed cytoplasm facilitated the emergence of plants that were morphologically close to Techne, but were-unlike it-fertile. This can be indicative of the existence of conditions favorable for the selection of cytoplasmic structures of N . tabacum. Microscopic evidence obtained during the cultivation of cell lines with a hybrid cytoplasm also suggest that it is the organelles of one of the partners that are preferentially retained (Binding, 1976). Fowke et al. (1976, 1977) reported a decrease in the number and degradation of chloroplasts of sweet clover and pea in the fusion of their protoplasts with those of a soybean cell culture; the number of soybean leukoplasts did not decrease considerably. It should be noted that this process may be only the result of degradation of chloroplasts in dedifferentiation and proliferation of callus cells (Gigot et al., 1975; Belitser et al., 1977). 2. Conditions for Selection of the Products of Fusion and Hybrid Cells Identification of products can be based on the cytomorphological differences in the parent cells, for example, different pigmentation and the localization of pigment in various organelles of the cell, and a difference in the structure of cell

356

RAlSA G . RUTIiNKO

organelles (the presence of mature normal chloroplasts or proplastids and amyloplasts). The type of vacuolization of the cytoplasm, the size and structure of the nucleus, and the number of nucleoli can serve as morphological markers. Identified heterokaryons can be observed during further cultivation. Monitoring is possible at a low plating density in agar media. This can be achieved by using media of complex composition (Kao and Michayluk, 1975) or the feeder layer technique (Raveh and Galun, 1975; Kuchko and Butenko, 1977). The low percentage of fusion, especially in the required combinations, the infrequent formation of synkaryons, difficulties in inducing the division of the hybrid cell, and formation of colonies all account for a very low frequency of emergence of a hybrid line (1 x lo-”). The alternative is to provide conditions for selective reproduction of heterokaryons and hybrid cells. Selective media are widely used in the hybridization of mammalian cells. Work with animal cells was facilitated by the use of auxotrophic and resistant mutant lines (Kao and Puck, 1968; Shapiro et al., 1972; Thompson and Baker, 1973). The preparation and isolation of mutants of somatic cells of plants has just begun (Kovaleva et al., 1971; Lescure, 1973; Widholm, 1974, 1977; Carlson, 1973; Maliga et al., 1973, 1975, 1976; Karanova et al., 1973; Karanova and Shamina, 1978; Miiller and Grafe, 1976; Mendel and Miiller, 1976; Aviv and Galun, 1977). The high synthesizing activity of plant cells makes the production of auxotrophic mutants almost impossible (Widholm, 1974, 1977). Risistance mutants arise more frequently (Widholm, 1974, 1977; Maliga, 1976). The use of mutant cell lines obtained purposely for the isolation of plant protoplasts, their fusion, and the selection of hybrid cells has so far been described only in the works of Grafe et al. (1977) and Glimelius et al. (1978). A s a rule attempts are made to find essential differences between the fusion partners (Cocking, 1973). When hybrid somatic cells are selected, the aim is either to create selective conditions resulting in the cells of both parent lines being eliminated, or semiselective conditions under which the cells of one parent are eliminated and both the heterokaryons and the cells of the other parent reproduce. In the hybridization of plant somatic cells, in addition to selection at the cellular level, selection at the level of the resulting plants is possible. Selection at the hybrid cell level enables one to study the nucleus-plasma relationships and the regulation of gene operation in the hybrid product and in reproduction; besides, new possibilities are provided for determining the conditions required for regeneration of the whole plant from the callus tissue. At the same time, subculturing of the cells results in the accumulation of hereditary changes not directly associated with somatic hybridization. Polyploidization, segregation of chromosomes, and chromosomal aberrations are often involved in the emergence and reproduction of callus cells (Shamina, 1965; Tomy, 1967; D’Amato, 1975, 1977). It is important to reduce the time from the onset of division in the hybrid cell until an embryoid or stem structure is formed. Pavlova and Butenko (1!)69)

ISOLATED PROTOPLASTS AND SOMATIC PLANT CELLS

357

observed stem organogenesis when individual cells of tobacco callus tissue were plated onto an induction medium. Three to four cell generations were necessary for the meristematic regions to form and the stem apex to be differentiated; this time is sufficient for anomalies to appear (Gleba and Gleba, 1978). Liirz et al. (1977) suggested a medium for the induction of somatic embryogenesis in tobacco cells originating from mesophyll protoplasts. The distance from the protoplast to the plant is thereby considerably reduced compared to organogenesis, and the possibility of emergence of karyological anomalies is lessened. At present several approaches to the selection of products of plant protoplast fusion and hybrid cells have been described. The following are the most promising. a. Hormone Prototrophy and Resistance to High Hormone Concentrations. Carlson et al. (1972) and Smith et al. (1976) used hereditary phytohormone prototrophy in combining Nicotiana glauca and N . 1angsdorjE. Selection after the fusion of protoplasts was carried out in a medium containing no auxin or cytokinin. In the first experiment a plant identical to a sexual amphyploid was obtained (Carlson et al., 1972). In the second experiment (Smith et al., 1976) the hybrid nature of 23 independently obtained plants was proved. Smith (1976) believes that this principle of hybrid selection can also be applied to somatic hybridization in the Nicotiana genus in cases when sexual hybrids are unknown but indirect data point to their prototrophy with respect to phytohormones. Differences in the requirements for hormonal factors in protoplasts from Petunia hybrida and Parthenocisus tricupidata (Power et a l . , 1975) and P. hybrida and P . parodii (Power et al., 1977) were used in establishing selective conditions in the somatic hybridization of these species. As a result of complementation, the hybrid P . hybrida x P . parodii cells that resulted from the products of fusion of mesophyll protoplasts of these species were tolerant to high concentrations of phytohormones; this made it possible to obtain hybrid callus tissue and then hybrid plants. b. Temperature Selection. This method is doubtlessly very promising, especially in interspecific hybridization when the protoplasts and the cells of the partner species differ in resistance to extreme temperatures. The work of Dudits (1976) is a good example of the use of high temperature for the selection of heterokaryons in the fusion of protoplasts isolated from cell cultures of soybean and rice. At 37°C cells of rice divided, and those of soybean did not. However, in a medium of a certain composition only soybean cells could divide (Kao et a l . , 1974). After fusion, the protoplasts were first plated onto a semiselective medium and then exposed to 37°C. Th~midine-~H was incorporated only into the nuclear DNA of the heterokaryons; but no thymidine label was observed in the parent cel Is. c. Sensitivity to Unusual Metabolites and Xenobiotics. Canavanine, an unusual amino acid, did not inhibit the division of cells originating from protoplasts

358

RAISA G . BUTENKO

of Caragana, alfalfa, and sweet clover. However, protoplasts of soybean and pea did not divide in its presence; neither did the heterokaryons. In this case the sensitivity dominated (Constabel er al., 1975b). The resistance of rice to the toxic effect of propanyl was used in the work of Dudits and Nemet (1976) to prepare a semiselective medium for fusion with wheat protoplasts (Trirr'cum monococcum). When plated after fusion onto a medium with 40 mg/ml of propanyl, only cells that originated from the rice protoplasts and heterokaryons divided. Thus the resistance to propanyl associated with the activity of arylacylamidase dominated in the hybrid product. d. Cotnplementation between Recessive Genes. Protoplasts obtained ~ k o m liverwort (Sphaerocarpos doneflii) mutants that are auxotrophic with respect to nicotinic acid and glucose were selected after fusion on a simple mineral medium. As a result of complementation, hydrids were selected and karyologically identified (Schieder, 1974). Photosynthesis-deficient mutants of tobacco were also used for selecting the hybrid products. In the work of Melchers and Labib (1974) mesophyll protoplasts of two different mutant haploid plants of N. rahacum were fused. In both cases the mutation involved sensitivity to highintensity light but was determined by nonallelic recessive genes (S and V ) . As a result of complementation and selection under high-intensity light, co1onit:s of hybrid cells were obtained having a bright-green pigmentation, unlike the yellowgreen coloration of the parent lines. From the hybrid cells plants were obtained that were identical to sexual hybrids between these mutants. Gleba er al. (1974) fused mesophyll protoplasts of a varigated plastoni mutant of Samsun tobacco with protoplasts of seedlings of the genome mutant which was homozygous for the defective gene Su. The selected trait was restoration of the photosynthesizing ability as a result of nucleus-plastom complementai.ion. The principle of selection is shown in Fig. 10. As the nucleus mutation was

A

B

C

FIG. 10. Scheme o f an experiment on the hybridization o f somatic cells of nuclear ( - 1 and plastid ( - ) chlorophyll-deficient mutants of N . ttrhocunr. ( A ) Product of fusion of protoplasts--;i true hybrid. (B) Product of fusion of two protoplasts after nuclei segregation. (C) Product of fubiori of a protoplast and cytoplast. ( B ) and (C) are cybrids (containing a nucleus o f one of the partners :and a mixed cytoplasm).

ISOLATED PROTOPLASTS AND SOMATIC PLANT CELLS

359

semidominant with respect to the defective gene, two classes of events could be marked: ( I ) the emergence of true hybrids and (2) the production of cybrids. In the work of Burgutin et ul. (1977) the somatic cybrids obtained were analyzed. The method suggested by these workers postpones selection of the hybrid form until the stage of plant regeneration. This obviously decreases the possibility of selection of the desired event (the hybrid product); in addition, there are doubts as to the independent origin of regenerated plants. But if the principle of cornplementation of the nucleus and plastids is used, cybrids can be obtained to investigate nucleus-plastom and plastom-plastom relationships.

c. INTRATPECIFIC SOMATIC HYBRIDIZATION It is also appropriate to analyze here cases in which somatic hybridization occurred not only within a species but between different species of one genus, when sexual hybrids from such hybridization are known. The study of plants obtained by somatic hybridization techniques and their comparison with sexual hybrids is of interest in elucidating the effect of the mixed cytoplasm and higher ploidy (Burgutin rt ul., 1977; Belliard etul., 1977, 1978). It is necessary then to rule out properties that could have arisen on reproduction of the hybrid cell during cultivation. Somatic hybrids have been studied both at the level of plants regenerated from hybrid callus tissue and, in some cases, at the level of their progeny (Smith, 1976; Melchers, 1977; Burgutin et al., 1977). Tobacco plants obtained as a result of intraspecific somatic hybridization have been studied. The parents used for somatic hybridization were haploid in the case of Melchers’ experiments (Melchers and Labib, 1974; Melchers, 1977) and diploid in the work of Gleba et ul. (1974) and Burgutin et (11. (1977). Somatic hybrids at the plant level were obtained by intrageneric hybridization via fusion of protoplasts of N . glaucu and N . langsdorffii (Carlson et al., 1972; Smith et d . , 1976), P . purodii and P . hybrida (Power et ul., 1976a,b; Cocking et ul., 1977), Daucus carotu and D . capillifolius (Dudits et al., 1977) and Solunum tuherosutn and S . chucoense (Butenko and Kuchko, 1979). When diploid protoplasts were used in fusion, the resulting plants morphologically resembled amphiploid sexual hybrids; when haploid protoplasts were fused, they resembled diploid forms of sexual hybrids. Dudits et al. (1977) reported the intermediate character of the type of leaf inherited by somatic hybrids originating from the fusion of protoplasts from a cell culture of D. carota (an albino mutant) and D . capillifolius (normal green). The dominant traits were the type of leaf hairs of D . carotu and the form and color of the root of D. capillijolius. In the peroxidase isozyme spectrum of the hybrids, in addition to the common bands, there was a band characteristic of D . carota only (Dudits et al., 1977). Somatic hybrids between P . parodii and P . hybridu (Power et al., 1975) were morphologically identical to the sexual amphiploid but had new components in the peroxidase isozyme spectrum. Based on morphological traits

360

RAISA G . BUTENKO

(height, rate of growth, flowers, and tendency toward tumor formation) somatic hybrids of N. glauca and N . langsdo@i were similar to sexual amphipboids (Smith, 1976). The zymograms of aminopeptidase, lactate, and alcohol dehydrogenase of the somatic hybrid and amphiploid differed from those of the parents (Wetter and Kao, 1976; Wetter, 1977). The leaves and tubers of somatic hytirids between Solanum tuberosum and S . chacoense were intermediate with respect to the parents. On the other hand, the height of the hybrid plants exceed that of the parent plants (Butenko and Kuchko, 1979). Cytological ana1ysi:s of chromosome numbers in the somatic hybrids cells showed that they often differed from the expected ones (Table 111). As a result of the fusion of protoplasts isolated from the cells of diploid plants, a 6 n range of ploidy was observed instead of a 4n range. Smith (1976) and Dudits et al. (1977) interpreted this as resulting from the fusion of three protoplasts. In the experiments of Smith et al. (1976), the chromosome numbers of the somatic hybrids were 66 and 60, which in the opinion of these workers can reflect the fusion of two proto24 18), or vice plasts of N. glauca with one of N. langsdor-i (24 18 24). Some triploid plants originating from fused haploid versa (18 protoplasts of N. tabacum were described by Melchers (1977). Mixoploidy with 3n predominant was observed in the cells of cybrid tobacco plants described by Burgutin et al. (1977). It can be suggested that, as is often the case in plant cell cultures (Kovaleva er al., 1971; Karanova et al., 1973; Karanova and Shamina, 1978), the 3n cells are selected on reproduction. Analysis of the available data, mostly on the Solanaceae, does not allow one to estimate the possibility of obtaining karyologically stable hybrid lines by cell culture techniques. In addition to genome and chromosome polymorphism, arising as a result of dedifferentiation of the initial hybrid cell and its futher reproduc-

+

+

+

+

TABLE 111 PLOIDYLEVELOF PLANTSOBTAINED FROM FUSEDPROTOPLASTS

Partners in somatic hybridization

Number of chromosoms

-

Reference

56-64 (average 60)

Smith (1976)

Nicotiana rabacum (2n = 48) and N . tabacum (2n = 48)

144 (hybrid) 72 (cybrid)

Gleba et al. (1974) Burgutin ef al. (1977)

Nicotiana tabacum ( n ( n = 24)

=

48, 72

Melchers ( 1977)

Daucus carom (2n (2n = 18)

18) and D . capillifolius

34-54

Dudits et a / . (1977)

Nicotiana glauca (2n (2n = 18)

=

=

24) and N. langsdorfii

24) and N . tabacum

ISOLATED PROTOPLASTS AND SOMATIC PLANT CELLS

36 1

tion, the source of karyological variation may be the release of some of the nuclear material during protoplast budding and also segregation of chromosomes during the first divisions (Burgutin et al., 1977). The fertility of somatic hybrids was very high in the cases described by Melchers (1977) and Smith (1976), and low in those reported by Burgutin et al. (1977).

D. INTERGENERIC SOMATIC HYBRIDIZATION Regardless of the taxonomic position of plants, their protoplasts can fuse without any incompatibility being revealed at this stage. Table IV shows the results of successful fusion of evolutionarily rather distant species. In most cases, the hybrid products have been studied microscopically at the stage of fusion of protoplast membranes, cytoplasm, and nuclei. These results were analyzed in the previous section. It is appropriate to review here the results pertaining to the characteristics of the cell lines obtained by intergeneric somatic hybridization and the attempts to estimate the validity of hybrid cells in studying the manifestations of gene activity. This is often difficult to do, because few hybrid cell lines have been obtained as yet, and few divisions of heterokaryocytes and hybrid cells have TABLE IV INTERSPECIFIC SOMATIC HYBRIDIZATION VIA PLANTPROTOPLAST FUSION Pattners and source Nicotiana glauca (mesophyll) and N . langsdorfii (mesophyll) Daucus carota (cell culture) and D. capillifolius (cell culture) Petunia hybrida (mesophyll) and P . parodii

(mesophyll) Nicotiana rabacum (mesophyll) and N . tabacum on N . debneyi cytoplasm (mesophyll) Nicotiana glauca (mesophyll) and Glycine m u (cell culture) Colchicum autumnale (mesophyll) and G . m u (cell culture) Oryza sativa (seedlings) and T . monococrum (cell culture) Solanum tubermum and Lycopersicon esculentum Solanum tuherosum and S . chacoense Arabidopsis thaliana and Brassica campestris

Fusion products Hybrid cell plants Hybrid cell plants Hybrid cell plants Hybrid cell plants

Reference

lines,

Smith et al. (1976)

lines,

Dudits er al. (1977)

lines,

Power et al. (1976b)

lines,

Gleba et a / . (1977)

Hybrid cell lines

Constabel er al. (1976)

Division of hybrid cell Heterokaryons, hybrid cells Hybrid cell lines, plants Hybrid cell lines, plants Hybrid cell lines, plants

Constabel et a / . (1976) Dudits (1976) Melchers er al. (1978) Butenko and Kuchko (1979) Gleba and Hoffmann (1979)

362

RAISA G. BU’TENKO

been observed. Only a few lines originating from the fusion of mesophyll protoplasts of N. glauca with protoplasts from a soybean cell culture and mesophyll protoplasts of Brassica campestris with protoplast from callus tissue of Arabidopsis thaliana kept reproducing for several months (Kao, 1976; Gleba and Hoffmann, 1978). In all the known cases of intergeneric fusion, one of the partners was a leaf mesophyll protoplast and the other a protoplast from a1 cell culture. This resulted not only in an additional cytological marker for studying fusion products (chloroplasts of mesophyll protoplasts and leukoplasts of protoplasts of cultivated cells), but also stimulated nuclei division in mesophyll protoplasts (Constabel et a l . , 1977a). However, there is no detailed descripticm of activation of the interphase nucleus by a premitotic division partner. In a hybrid line originating from the fusion of protoplasts of soybean and N. glauca, almost all the chromosomes of N. glauca were eliminated during several months of cultivation (Kao, 1976), whereas in the case of a hybrid line obtained from B. cumpestris and A . thuliana protoplasts fusion of all metaphases examined contained chromosomes of both species (Gleba and Hoffmann, 1978). Data on the regulation of gene activity in hybrid cells are few. An idea albout gene expression can only be gained from a few observations on the faie of physiological and biochemical markers in hybrid cells and heterokaryons. The canavanine sensitivity of soybean appeared to be dominant in heterokaryocytes obtained as a result of the fusion of canavanine-resistant protoplasts of Cura,pana urborescens with protoplasts of soybean (Constabel et al., 1975b). The temperature stability of rice was dominant in the products of fusion of protoplasts of unstable soybean with rice protoplasts (Dudits, 1976). Stability toward propanyl is also dominant in the fusion products of protoplasts from stable rice and unstable wheat (Dudits and Nemet, 1976). N o direct confirmation has so far been obtained that the activity of both genomes is retained in the synthesis of RNP,and specific proteins. Shoots and flowering plants have been regenerated by using the hybrid cell lines obtained by intergeneric protoplasts fusion (Gleba and Hoffmann, 1979; Melchers et al., 1978). Several cases of fusion of plant protoplasts and animal cells induced by PEG have been described. In 1975 Ahkong et 1x1. fused protoplasts isolated from a yeast cell and chicken erythrocytes. Dudits et al. (1976b) described the fusion of protoplasts from carrot cells with HeLa cells. The nuclei in the heterokaryons differed in ability to be stained and in that the HeLa nuclei were labeled with thymidine-:’H. Seventy-two hours after fusion the cells formed a cell wall, and 0.3-0.6% of fused interphase nuclei were observed. Jones et al. (1976) induced fusions and observed their consequences when tobacco mesophyll protoplasts were combined with HeLa cells. Light microscope examination revealed agglutination of the protoplasts and the animal cells and the presence of HeLa nuclei (labeled with thymidine-:’H) in the plant protoplasts. In the work of Willis et al. (1977) SEM and TEM were employed to

ISOLATED PROTOPLASTS AND SOMATIC PLANT CELLS

363

follow the fusion of protoplasts from tobacco leaf with chicken erythrocytes. SEM allows one to monitor the process and to show that the fusion of plant and animal membranes takes place. This was substantiated by observation of formation of the products of degradation of the fused membranes. Fusion took place only when 1% protease was added to the medium. Sidorov et al. (1978) performed an electron microscope study on the fusion products of Arabidopsis protoplasts and human lymphocytes; they observed agglutination followed by fusion of membranes. Protoplasts of a leaf of a variegated plastom mutant of tobacco fused with cells from a tissue culture of Drosophila melanogaster (Nikiforova, 1979). In all described cases there is no evidence on the fate of the fusion products. Moreover, the viability of the partners is questionable, since they have different requirements for osmolarity, nutrition, and temperature and differ in sensitivity to the action of PEG.

VI. Incorporation of Foreign Genetic Material Somatic hybridization of taxonomically remote species can result in genetic incompatibility when the morphological structures were formed. Translocation of the genetic material could be expected, as was the case in the reproduction of hybrid animal cells (Miller et al. , 1971; Schwartz et a l . , 1971), but no such data for hybrid plant cells have ever been reported. Cultivation of isolated protoplasts has given impetus to studies on the possibility of genetic transformation of plant cells with the aid of foreign genetic material. The data pertaining to genetic transformation of plant cells have been obtained with different species and by different methods, hence defy analysis. There are formidable difficulties in choosing a system that would be equally adequate for proving the possibility of transformation by biological means and for biochemical study of the fate of the DNA introduced into the cell. The fate of homologous DNA would be especially hard to follow. The possibility of incorporation of foreign DNA by plant protoplasts was studied by several investigators (Ohyama et al., 1972, 1977a; Gleba et a l . , 1975; Suzuki and Takebe, 1976). In these studies use was made of heterologous labeled DNA of Escherichia coli, Bacillus subtilis, and bacteriophage fd. Ohyama et al. (1977a) investigated the uptake of homologous DNA from soybean cells. In all these studies, DNA absorption and incorporation into the trichloroacetic acid (TCA)-insoluble fraction was estimated by the activity of the preparation that had incorporated the DNA label. And in all the studies following the washing of the protoplasts that had been incubated with foreign DNA by DNase there was an activity in the cytoplasm fraction of the protoplast homogenates and TCA precipitates obtained from these protoplasts. The binding in-

364

RAlSA G. BUTENKO

creased with the time of incubation; the maximum being achieved between 20 minutes and 2 hours. The addition of DEAE dextran, poly-L-lysine, and poly-~ornithine (Ohyama et a / . , 1972; Gleba e t a / ., 1975), as well as zinc, magnesium, or calcium (Suzuki and Takebe, 1976), to the incubation medium of the protoplasts enhanced the uptake of the label. Suzuki and Takebe (1976) showed that for 1 hour 30% of the labeled single-stranded DNA of bacteriophage fd was takein up by protoplasts and 60-80% of this was in an acid-insoluble form. Analysis by centrifugation on a sucrose gradient showed that about 30% of DNA in the cytoplasm retained the size of intact bacteriophage fd DNA. Ohyama et a / . (1977a) point out that EDTA and pronase inhibit the uptake of homologous DNA by soybean protoplasts. In none of the studies on uptake and binding of DNA is there unambiguous proof of its being incorporated into plant protoplasts. The enhanced effect of protectors can also be observed when DNA has been absorbed on the outer membrane of protoplasts. Kool (1977) used autoradiography to demonstrate that, on incubation of soybean protoplasts with homologous labeled DNA, the label was detected in the remnants of the cells walls contaminating the protoplast suspension. In the cells the label appeared much later, and at that time the low-molecular-weight products of degradation of DNA by nucleases could be detected. The addition of an excess of unlabeled thymidine totally inhibited the appearance of activity in the cells. DEAE dextran increased the binding of the labeled material and its resistance to nucleases, but the material bound to the remnants of the cell walls rather than to protoplasts. The same difficulties concerning determination of the binding site of exogerlous DNA are encountered on incubation with the fraction of nuclei isolated from protoplasts (Hess, 1976). Attempts have been made to study the possibility of expression of foreign genetic material (Hess, 1976). Nuclei were isolated from protoplasts of Petunia and incubated with E. coli DNA. It was determined with the help of DNA-RNA hybridization that the nuclei transcribed not only DNA of Petunia but a certain amount of that of E . coli. But when the nuclei were washed after incubation and placed in a transcription medium, no transcription of E. coli DNA was observed, although the DNA of Petunia was transcribed as before. It may be that in this case E . coli DNA was observed on the nuclear membrane or on the membranes that contaminated the nuclear fraction. The possibility of utilization of vehicles, that is, phages, phytoviruses, and plasmids, for the transfer of foreign DNA into plant protoplasts was investigated (Doy e t a l . , 1973; Gresshoff, 1975; Carlson, 1973; Johnson et al., 1973; Kapitsa et al., 1977). Results showing the possible transfer of a lactose operon of E. ,co/i in cultivated Arahidopsis, tomato cells, and Acer with the help of bacteriophages A plac+ and 480 plac (Doy et a / . , 1973; Johnson et a / . , 1973) were not confirmed by Kapitsa et a / . (1977).

ISOLATED PROTOPLASTS AND SOMATIC PLANT CELLS

365

Plasmids attract much attention as vehicles for transferring foreign information into plant cells. The cloning technique can be used for designing plasmids with replicator genes for both prokaryotic and eukaryotic cells. It has been suggested that plasmids may be used for cloning genes to study the possibility of transformation of specific mutant plant cells (Cannon, 1976). However, verification of this possibility by Hess (1976) failed to confirm it. A mutant of Chlarnydomonas reinhardii that forms no cell wall was employed to study the possibility of transformation, using the factor of resistance to kanamycin from a kanamycin-resistant E . coli. Colonies of C. reinhardii resistant to the antibiotic were selected, but after 40 generations they lost their resistance, hence the foreign genetic material. Materials that have natural replicators, for example, chloroplast DNA and caulimoviruses, can be useful in transformation experiments with plant cells. However, systematic studies are required on the possibility and mechanism of their introduction into plant protoplasts, their integration, and their replication. Auxotrophic mutants in which the absence of a given metabolite is lethal are ideal receptor systems for proving such biological transformations. Foreign genetic material can be introduced into plant protoplasts in the form of cell organelles (nuclei, plastids, chromosomes, and cells of microorganisms). Such an approach is interesting not only in genetic modification of the cell but also in studying nucleus-cytoplasm and plastom-plastom relationships. Evidence has been reported on the possibility of introducing nuclei (Potrykus and Hoffmann, 1973; Binding, 1976; Lorz, 1976), chloroplasts (Potrykus, 1973; Carlson, 1973; Bonnet and Eriksson, 1974), and bacterial cells (Davey and Cocking, 1972; Davey and Power, 1975) into isolated protoplasts; but it has not been reported whether they retain their intactness and what happens to them later. On the contrary, Davey et af. (1973, 1976) showed that chloroplasts and cells of Rhizohiurn and Anacysris nidulans were considerably damaged in the PEG solution used for fusion. The transfer of organelles using the fusion of protoplasts of one species with subprotoplasts of the other would have been preferable (Binding and Kollmann, 1976) in this case.

VII. Conclusion The isolation and cultivation of plant protoplasts is a new high-resolution technique for studying the biology of plant cells. There are many problems to be solved that are associated with obtaining protoplasts active in division and morphogenesis from all species of theoretical or practical interest. There are few studies devoted to the properties of the surface membrane of protoplasts, its behavior during the uptake of substances and particles, and its response to hor-

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Subject Index

A Abdominal plexus, nerves, gastrointestinal tract and, 131 Acanthocephala, Balbiani’s vitelline body in, 265-267 Acetylcholinesterase, gastrointestinal histochemistry of, 143 Amphibians, Balbiani’s vitelline body in, 278287 Amphioxus, Balbiani’s vitelline body in, 27027 1 Axoneme, assembly of, 12-16

B Balbiani’s vitelline body in invertebrates Acanthocephala, 265-267 crustaceans, 260 echinoderms, 263-265 insects, 259-260 molluscs, 260-263 platyhelminthes, 265 scorpions and myriapods, 258-259 spiders, ,251-258 in protochordates Amphioxus. 270-271 tunicates, 267-270 in vertebrates amphibians, 278-287 birds, 294-308 elasmobranchs, 271 -272

mammals, 308-314 reptiles, 287-294 teleosts, 273-278 Basal bodies, morphogenesis of, 3-12 Birds, Balbiani’s vitelline body in, 294-308

C Calcium, microtubule assembly and, 64-69 Catecholamines, gastrointestinal histochemistry blood vessels, 151 mucosa, 150 muscle coat, 148-150 myenteric plexus, 144-147 submucosal plexus, 147 Cell wall, regeneration of, 341-347 Centrioles, morphogenesis of, 3-12 Cilia, axoneme, assembly of, 12-16 Crustaceans, Balbiani’s vitelline body in, 260 Cytoplasm, assembly and disassembly of microtubule arrays, 30-36

E Echinoderms, Balbiani’s vitelline body in, 263-265 Eggs, injection of microtubule-organizing centers into, 58-64 Elasmobranchs, Balbiani’s vitelline body in, 27 1-272 Embryonic development, radiation and activity of irradiated nuclei, 224-226 effects on germ cells, 229-241 315

376

SUBJECT INDEX

effects on tissue development, 241 -243 Hertwig effect, 226-227 lethal effects and hatchability experiments, 218-224 low-dose and low-dose rate irradiation, 227229 F Flagella, axoneme, assembly of, 12- 16

0

vagus nerve and, 130-131 Genetic material, foreign, incorporation into plant cell protoplasts, 363-365 Glial cells, gastrointestinal, 165-166

I Insects, Balbiani's vitelline body in, 259-260 Intramural plexuses, gastrointestinal distribution of, 132-134 shape of, 135-137

M Gastrointestinal tract abdominal plexus nerves and, 131 Mammals, Balbiani's vitelline body in, 308-314 adrenergic innervation, 174- I75 Membrane(s), microtubules and, 73-75 afferent fibers, 177-178 Membrane-coating granules, 99-100 composition, 1 1 1 - 1 18 blood vessels, innervation of, 171 fate, 108-1 1 1 catecholamine histochemistry functions, 118-123 blood vessels, 151 identification, distribution and morpholog:y, mucosa, 150 muscle coat, 148-150 100-106 myenteric plexus, 144-147 origin, 106-108 summary and future prospects, 123-124 submucosal plexus, 147 cholinergic innervation, 175-176 Microtubule assembly experimental dissections in v i m coupling between muscle cells, 181-183 biochemistry of tubulin, 36-40 development, 178-181 calcium as possible regulator, 64-69 exogenous adrenergic transmitters and false transmitters, 172-174 growth of microtubules, 51-58 injection of organizing centers into eggs, extrinsic nerve fibers, 171-172 58-64 glial cells of, 165-166 histochemistry of acetylcholinesterase, 143 possible regulatory factors, 40-51 immunofluorescence histochemistry , 152- 154 regulation: genetic studies, 76-8 1 spatial localization, 73-75 innervation by other types of nerves, 176-177 interstitial cells, 183-1 86 time dependent properties, 69-73 intramural plexuses control, conclusions regarding, 81 -83 distribution of, 132-134 observations in vivo, 2-3 shape of, 135-137 assembly and disassembly of labile arrays cytoplasmic, 30-36 mucosa, innervation of, 170-171 muscularis externa, innervation of, 167-170 mitotic, 23-30 assembly of axoneme of cilia and flagella, myenteric plexus catecholamine histochemistry, 144-147 12-16 morphogenesis of basal bodies and cennerve endings and synapses in, 159-165 ultrastructure, 154-159 trioles, 3-12 morphogenesis of organelles in protozoa, neurons number of, 137-140 16-23 Mitotic apparatus, assembly and disassembly of size of, 140 microtubule mays, 23-30 types of, 140-143 pelvic nerves and, 131-132 Molluscs, Balbiani's vitelline body in. 260-2153 serotoninergic neurons, 151-152 Muscle cells, gastrointestinal, coupling between, 181-183 submucosal plexus, ultrastructure of, 167 Myenteric plexus surface of ganglia and vascularization, 166catecholamine histochemistry , 144- 147 167

377

SUBJECT INDEX nerve endings and synapses in, 159-165 ultrastructure, 154-159 Myriapods, Balbiani’s vitelline body in, 258259

N Neurons, gastrointestinal number of, 137-140 serotoninergic, 151 -152 size of, I 4 0 types of, 140-143

0 Ovaries, radiation and effects of incorporated radionuclides, 205-206 fine structures, 201-203 gonadectomy by radiation, 203-205 local irradiation, 200-201 whole-body irradiation, 196-200 P Pelvic nerves, gastrointestinal tract and, 131132 Plant cell protoplasts cultivation division and formation of cell colonies, 348-351 regeneration of cell wall, 341-347 techniques, 340-341 fusion and somatic hybridization intergeneric, 361 -363 intraspecific, 359-361 selection of hybrid cells, 353-359 technique, 351-353 historical outline, 325 incorporation of foreign genetic material, 363-365 isolation and properties methods and conditions of isolation, 328330

plasmalemma, 336-340 properties, 330-336 sources, 325-328 Platyhelminthes, Balbiani’s vitelline body in, 265 Protozoa, morphogenesis of rnicrotubulecontaining organelles in, 16-23 R Radiation embryonic development and, 218-243 genetic effects of, 217-218 of ovaries, 196-206 of testes, 206-217 Reptiles, Balbiani’s vitelline body in, 287-294

S Scorpions, Balbiani’s vitelline body in, 258-259 Spiders, Balbiani’s vitelline body in, 251-258 Stratified squamous epithelium, histology of, 97-99 T Teleosts, Balbiani’s vitelline body in, 273-278 Testes, radiation and autoradiographic studies in breeding season, 207-209 autoradiographic studies in sexually inactive seasons, 209-214 castration experiments, 206 effects of various radiations, 214-217 fertility and histological studies on fish, 206207 Tubulin biochemistry of, 36-40 time-dependent properties of, 69-73 Tunicates, Balbiani’s vitelline body in, 267-270 V Vagus nerve, gastrointestinal tract and, 130-1 31

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Contents of Previous Volumes Volume I Some Historical Features in Cell BiologyARTHURHUGHES HUSKINS Nuclear Reproduction-C. LEONARD Enzymic Capacities and Their Relation to Cell Nutrition in AnimakaEORGE w . KIDDER The Application of Freezing and Drying Techniques in Cytology-L. G. E. BELL Enzymatic Processes in Cell Membrane Penetration-TH. ROSENBERG A N D W. WiLBRANDT

Bacterial Cytology-K. A. BlssET Protoplast Surface Enzymes and Absorption of Suger-R. BROWN Reproduction of Bacteriophage-A. D. HERSHEY

The Folding and Unfolding of Protein Molecules as a Basis of Osmotic Work-R. J. GOLDACRE

Nucleo-Cytoplasmic Relations in Amphibian Deve1opment-G. FRANK-HAUSER Structural Agents in Mitosis-M. M. SWANN Factors Which Control the Staining of Tissue Section with Acid and Basic Dyes-MARCUS SINGER The Behavior of Spermatozoa in the Neighborhood Of Eggs-LORD ROTHSCHILD The Cytology of Mammalian Epidermis and Sebaceous GhIdS-WILLlAM MONTAGNA The Electron-Microscopic Investigation of Tissue Sections-L. H. BRETSCHNEIDER The Histochemistry of Esterases-G. GOMORI

The Nature and Specificity of the Feulgen Nucleal Reaction-M. A. LESSLER Quantitative Histochemistry of Phosphatases-WILLIAM L. DOYLE Alkaline Phosphatase of the Nucleus-M. CHLVREMONT A N D H. FIRKET Gustatory and Olfactory Epithelia-A. F. BARADI A N D G. H. BOURNE Growth and Differentiation of Explanted Tissues-P. J. GAILLARD Electron Microscopy of Tissue Sections-A. J. DALTON A Redox Pump for the Biological Performance of Osmotic Work, and Its Relation to the Kinetics of Free Ion Diffusion across Membranes-E. J. CONWAY A Critical Survey of Current Approaches in Quantitative Histo- and CytochemistqDAVIDCLICK Nucleo-cytoplastmic Relationships in the Development of Acetabularia-J. HAMMERLING Report of Conference of Tissue Culture Workers Held at Cooperstown, New York-D. J. HETHERINGTON AUTHOR INDEX-SUBJECT

INDEX

Volume 3

The Nutrition of Animal Ct?k---cHARITY WAYMOUTH Caryometric Studies of Tissue CUltUreS"TT0 BUCHER AUTHOR INDEX-SUEJECT INDEX The Properties of Urethan Considered in Relation to Its Action on Mitosis-IvoR CORNMAN Volume 2 Composition and Structure of Giant Chromosomes-MAx ALFERT Quantitative Aspects of Nuclear NucleoproHow Many Chromosomes in Mammalian Soteins-HEWSON SWIFT matic Cells?-R. A. BEATTY Ascorbic Acid and Its Intracellular Localization, with Special Reference to Plants-J. CHAYEN The Significance of Enzyme Studies on Isolated Cell Nuclei-ALEXANDER L. DOUNCE Aspects of Bacteria as Cells and as OrThe Use of Differential Centrifugation in the ganiSmdTUART MUDDA N D EDWARDD. Study of Tissue E n z y m e d H R . DE DUVE DELAMATER A N D J. BERTHET Ion Secretion in Plants-J. F. SuTcLiwE Enzymatic Aspects of Embryonic Multienzyme Sequences in Soluble ExtractsDifferentiation-TRuccvE GUSTAFSON HENRYR. MAHLER

319

3 80

CONTENTS O F PREVIOUS VOLUMES

Azo Dye Methods in Enzyme HistochemistryPEARSE A. G. EVERSON Microscopic Studies in Living Mammals with Transparent Chamber Methods-Roy G. WILLIAMS The Mast C e l l - G . ASBOE-HANSEN Elastic Tissue-EDWARDS w. DEMPSEYAND ALBERTI. LANSING The Composition of the Nerve Cell Studied with New Methods-svEN-OLoE B R A T T ~ R D AND HOLGERHYDEN AUTHOR INDEX-SUBJECT

INDEX

The Results of Cytophotometry in the Study of the Deoxyribonucleic Acid (DNA) Cont(mt of AND C. VENthe Nucleus-R. VENDRELY DRELY

Protoplasmic Contractility in Relation to Gel Structure: Temperature-Pressure Experiments on Cytokinesis and Amoeboid MovementDOUGLASMARSLAND Intracellular pH-PETER C. CALDWELL The Activity of Enzymes in Metabolism and Transport in the Red Cell-T. A. J. PRANKERD

Uptake and Transfer of Macromolecules by Cells with Special Reference to Growth and Development-A. M. SCHECHTMAN Cell Secretion: A Study of Pancreas and Salivary Glands-L. C. J. JUNQUEIRAAND C;. C. HIRSCH The Acrosome Reaction-JEAN c . DAN Cytology of Spermatogenesis-VrsHwa N ~ T H The Ultrastructure of Cells, as Revealed by the Electron Microscope-FRITloF s . SJOSTIUND

Volume 4

Cytochemical Micrurgy-M. J. KOPAC Amoebocytes-L. E. WACGE Problems of Fixation in Cytology, Histology, and Histochemistry-M. WOLMAN Bacterial CytO1Ogy-ALFRED MARSHAK Histochemistry of Bacteria-R. VENDRELY Recent Studies on Plant Mitochondria-DAVID P. HACKETT AUTHOR The Structure of Chloroplasts-K. M~HLETHALER

Histochemistry of Nucleic Acids-N. 9. KURNICK

S. Structure and Chemistry of Nucleoli-W. VINCENT On Goblet Cells, Especially of the Intestine of Some Mammalian Species-HARALD MOE Localization of Cholinesterases at Neuromuscular Junctions-R. COUTEAUX Evidence for a Redox Pump in the Active Transport of Cations-E. J. CONWAY AUTHOR INDEX-SUBJECT

INDEX

Volume 5 Histochemistry with Labeled AntibodyALBERTH. COONS The Chemical Composition of the Bacterial Cell Wall-C. S . CUMMINS Theories of Enzyme Adaptation in Microorganisms-J. MANDELSTAM The Cytochondria of Cardiac and Skeletal Muscle-JOHN W. HARMON The Mitochondria of the Neuron-WARREN ANDREW

INDEX-SUBJECT

INDEX

Volume 6 The Antigen System of Paramecium aureliaG. H. BEALE The Chromosome Cytology of the Ascites Tumors of Rats, wtih Special Reference to the Concept of the Stemline C e l l - S m R o MAKINO The Structure of the Golgi Apparatus-ARTHUR AND PRISCHIAF. POLLISTER W. POLLISTER An Analysis of the Process of Fertilization and Activation of the Egg-A. MONROY The Role of the Electron Microscope in Virus Research-RoBLEY C. WILLIAMS The Histochemistry of PolysaccharidesARTHURJ. HALE The Dynamic Cytology of the Thyroid GlandJ. GROSS Recent Histochemical Results of Studies on Embryos of Some Birds and Mammals-ELIO BORGHESE Carbohydrate Metabolism and Embryonic Determination-R. J. O’CONNOR Enzymatic and Metabolic Studies on Isolated AND R. M. S . SWELLIE Nuclei-G. SIEBERT

38 1

CONTENTS OF PREVIOUS VOLUMES

The Cell Surface of Paramecium<. F. EHRET AND E. L. POWERS The Mammalian Reticulocyte-LEAH MIRIAM LOWENSTEIN The Physiology of Chromatophores-MILTON FINGERMAN The Fibrous Components of Connective Tissue with Special Reference to the Elastic AUTHOR INDEX-SUBJECT INDEX Fiber-DAVID A. HALL CUMULATIVE SUBJECT INDEX Experimental Heterotopic Ossification-J. B. (VOLUMES 1-5) BRIDGES A Survey of Metabolic Studies on Isolated Volume 7 Mammalian Nuclei-D. B. ROODYN Trace Elements in Cellular Function-BERT L. Some Biological Aspects of Experimental VALLEEAND FREDERICL. HOCH Radiology: A Historical Review-F. G. Osmotic Properties of Living Cells-D. A. T. SPEAR DICK The Effect of Carcinogens, Hormones, and Vitamins on Organ Cu~tures-ILSE LASNITZKI Sodium and Potassium Movements in Nerve, Muscle, and Red Cells-I. M. GLYNN Recent Advances in the Study of the Pinocytosis-H. HOLTER Kinetochore-A. LIMA-DE-FARIA AUTHOR INDEX-SUBJECT INDEX Autoradiographic Studies with S35-Sulfate-D. D. DZIEWIATKOWSKI The Structure of the Mammalian Volume 9 SpermatOZQOn-DON W. FAWCETT The Lymphocyte-0. A. TROWELL The Influence of Cultural Conditions on BacteThe Structure and Innervation of Lamellibranch rial Cytology-J. F. WILKINSONAND J. P. Muscle-L. BOWDEN DUGUID Hypothalamo-neurohypphysial NeurosecreOrganizational Patterns within Chromosomestion-J. C. SLOPER BERWIND P. KAUFMANN, HELENGAY,A N D Cell Contact-PAUL WEISS MARGARET R. MCDONALD The Ergastoplasm: Its History, Ultrastructure, Enzymic Processes in Cells-JAY BOYDBEST and Biochemistry-FRANGOISE HAGUENAU The Adhesion of Cells-LEONARD WEN Anatomy of Kidney Tubules-JOHANNES RHO- Physiological and Pathological Changes in

Recent Approaches of the Cytochemical Study of Mammalian TissuesaEORGE H. HOGEBOOM, EDWARD L. KUFF, AND WALTER C. SCHNEIDER The Kinetics of the Penetration of Nonelectrolytes into the Mammalian ErythnxyteFREDABOWYER

DIN

Structure and Innervation of the Inner Ear Sensory Epithelia-HANS ENGSTR~M A N D JAN WERSALL The Isolation of Living Cells from Animal Tissues-L. M. RINALDINI AUTHOR INDEX-SUBJECT

INDEX

Volume 8 The Structure of CytOphSeHARLES OBER-

Mitochondria1 Morphology-CH. ROUILLER The Study of Drug Effects at the Cytological L e v e l - G . B. WILSON Histochemistry of Lipids in OogenesisVISHWANATH Cyto-Embryology of Echinoderms and Amphibia-KUTSUMA DAN The Cytochemistry of Nonenzyme ProteinsRONALD R. COWDEN AUTHOR INDEX-SUBJECT

INDEX

LING

Wall Organization in Plant Cells-R.

D. PRE-

Volume 10

STON

Submicroscopic Morphology of the apse-EDUARDO DE ROBERTS

Syn-

The Chemistry of Shiff s Reagent-FREDERICK H. KASTEN

3 82

CONTENTS OF PREVIOUS VOLUMES

Spontaneous and Chemically Induced Chromo- In Vivo Implantation as a Technique in Skeletal Biology-WILLIAM J. L. FELTS some BR~~S-ARUN KUMARSHARMA A N D The N a m and Stabilityof Nerve Myelin-J. B. ARCHANA SHARMA RNEAN The Ultrastructure of the Nucleus and Nucleocytoplasmic RelationdAuL WISCH- Fertilization of Mammalian Eggs in Vitro-C. R. AUSTIN NITZER Physiology of Fertilization in Fish E g g s The Mechanics and Mechanism of CleavagTOKI-oYAMAMOTO LEWSWOLPERT The Growth of the Liver with Special Reference AUTHOR MDEX---SUBJECT INDEX to Mammals-F. DOUANSKI Cytology Studies on the Affinity of the CarVolume 13 cinogenic Azo Dyes for Cytoplasmic The Coding Hypothesis-MARTYNAs YEAS Components-YosHrMA NAGATANI HERBERT Epidermal Cells in Culture-A. GEDEON Chromosome Reproduction-J. MATOLTSY TAYLOR Sequential Gene Action, Protein Synthesis, and AUTHOR INDEX--SUBJECT INDEX Cellular Differentiation-REED A. FLICCUMULATIVE SUBJECT INDEX KMGER (VOLUMES 1-9) The Composition of the Mitochondria1 Membrane in Relation to Its Structure and Volume 11 FunctioeEruc G . BALL AND CLIFFl3 D. JOEL Electron Microscopic Analysis of the Secretion Pathways of Metabolism in Nucleate and AnuMechanism-K. KUROSUMI cleate Erythrocytes-H. A. SCHWEIGER The Fine Structure of Insect Sense OrgansSome Recent Developments in the Field of AlELEANORH. SLIFER kali Cation Transport-W. WILBRANDT cytology of the Developing Eye-ALFRED J. Chromosome Aberrations Induced by Ionizing COULOMBRE Radiations-H. J. EVANS The Photoreceptor Structures-J. J. WOLKEN Use of Inhibiting Agents in Studies on Fertiliza- Cytochemistry of Protozoa, with Particular Reference to the Golgi Apparatus and the tion MechanismdHARLEs B. METZ MitochondrieVIsHwA NATH AND G. P. The Growth-Duplication Cycle of the Cell-D. DUTTA M. PRESCOTT A N D (JHOHistochemistry of Ossification-RoMULo L. Cell Renewal-FELIX BERTALANFFY SEN LAU CABRINI Cinematography, Indispensable Tool for AUTHOR I N D E X 4 U B J E C T INDEX Cytology-C. M. Pomerat AUTHOR INDEX-SUBJECT

INDEX

Volume 14

Inhibition of Cell Division: A Critical and Experimental AndysidEYMoUR GELFANT Sex Chromatin and Human Chromosomes Electron Microscopy of Plant Protoplasm-R. BUVAT -JOHN L. HAMERTON Chromosomal Evolution in Cell Populations Cytophysiology and Cytochemistry of the Organ of Corti: A Cytochemical Theory of T. C. Hsu Hearing-J. A. VINNIKOV AND L.K. TITOVA Chromosome Structure with Special Reference to the Role of Metal Ions-DALE M. STEF- Connective Tissue and Serum Proteins-R. E. MANCINI FENSEN Electron Microscopy of Human White Blood The Biology and Chemistry of the Cell Walls of Higher Plants, Algae, and Fungi-D. H. cells and Their Stem Cek-MARCEL BESSIS NORTHCOTE AND JEAN-PAUL THIERY

Volume 12

3 83

CONTENTS OF PREVIOUS VOLUMES Development of Drug Resistance by Staphylococci in Vizro and in Vivo-MARY BARBER Cytological and Cytochemical Effects of Agents Implicated in Various Pathological Conditions: The Effect of Viruses and of Cigarette Smoke on the Cell and Its Nucleic Acid ~ E C I L I ELEUCHTENBERGER AND RUDOLF LEUCHTENBERGER The Tissue Mast Wall-DOUGLAS E. SMITH

In Vivo Studies of Myelinated Nerve Fibers-

CARLCASKEY SPEIDEL Respiratory Tissue: Structure, Histophysiology, Cytodynamics. Part I: Review and Basic Cytomorphology-FELIX D. BERTALANFFY AUTHOR INDEX-SUBJECT

INDEX

Volume 17

The Growth of Plant Cell Walls-K. WILSON Reproduction and Heredity in 1. .nosomes: A AUTHOR INDEX-SUBJECT INDEX Critical Review Dealing Mainly with the African Species in the Mammalian Host-P. J. WALKER Volume 15 The Blood Platelet: Electron Microscopic Studies-J. F. DAVID-FERREIRA The Nature of Lampbrush Chromosomes-H. The Histochemistry of MucopolysaccharidesG.CALLAN ROBERTc. CURRAN The Intracellular Transfer of Genetic Respiratory Tissue Structure, Histophysiology, Information-J. L. SIRLIN Cytodynamics. Part 11. New Approaches and Mechanisms of Gametic Approach in PlantsInterpretations-FELIX D. BERTALANFFY LEONARD MACHLIS A N D ERIKA The Cells of the Adenohypophysis and Their RAWITSCHER-KUNKEL Functional Significance-MARC HERLANT The Cellular Basis of Morphogenesis and Sea AUTHOR INDEX-SUBJECT INDEX A N D L. Urchin Development-T. GUSTAFSON WOLPERT Plant Tissue Culture in Relation to Development Volume 18 CYtOlOgY~ARLR. PARTANEN The Cell of Langerhans-A. S. BREATHNACH Regeneration of Mammalian Liver-NANCY L. The Structure of the Mammalian Egg-ROBERT R. BUCHER HADEK Collagen Formation and Fibrogenesis with Spe- Cytoplasmic Inclusions in Oogenesis-M. D. L. cial Reference to the Role of Ascorbic SRIVASTAVA Acid-BERNARD s. COULD The Classification and Partial Tabulation of EnThe Behavior of Mast Cells in Anaphylaxiszyme Studies on Subcellular Fractions IsoIVANMOTA lated by Differential Centrifuging-D. B. Lipid Absorption-ROBERT M. WOTTON ROODYN AUTHOR INDEX-SUBJECT INDEX Histochemical Localization of Enzyme Activities by Substrate Film Methods: Ribonucleases, Deoxyribonucleases, Proteases, Volume 16 Amylase, and Hyaluronidase-R. DAOUST B. Ribosomal Functions Related to Protein Cytoplasmic Deoxyribonucleic Acid-P. GAHANAND J. CHAYEN Synthesis-Tow HULTIN Physiology and Cytology of Chloroplast Forma- Malignant Transformation of Cells in V i m KATHERINE K. SANFORD tion and “Loss” in Euglena-M. GRENSON Cell Structures and Their Significance for Deuterium Isotope Effects in Cytology-E. s. BOSE, H. I. CRESPI, AND Ameboid Movement-K. E. WOHLFARTH- FLAUMENHAR, J. J. KATZ BOTTERMAN Microbeam and Partial Cell 1rradiation-C. L. The Use of Heavy Metal Salts as Electron S t a i n s . RICHARDZOBELAND MICHAEL SMITH BEER Nuclear-Cytoplasmic Interaction with Ionizing AUTHOR INDEX-SUBJECT INDEX Radiation-M. A. LESSLER

3 84

CONTENTS O F PREVIOUS VOLUMES

Electromyography: Its Structural and Neural Basis-JOHN V. BASMAJIAN “Metabolic” DNA: A Cytochemical Study-H. Cytochemical Studies with Acridine Orange and ROELS the Influence of Dye Contaminants in the The Significance of the Sex ChromatinStaining of Nucleic Acids-FREDERICK H. MURRAY L. BARR KASTEN Some Functions of the Nucleus-J. M. MITCHI- Experimental Cytology of the Shoot Apical Cells SON during Vegetative Growth and Flowering-A. Synaptic Morphology on the Normal and DeNOUGAREDE generating Nervous System-E. G. GRAY Nature and Origin of Perisynaptic Cells of the A N D R. W. GUILLERY Motor End Plate-T. R. SHANTHAVEEICAPPA Neurosecretion-W. BARGMANN AND G. H. BOURNE Some Aspects of Muscle Regeneration-E. H. AUTHOR INDEX-SUBJECT INDEX BETZ,H. FIRKET,AND M. REZNIK The Gibberellins as Hormones-P. W. BRIAN Volume 22 Phototaxis in Plants-WOLFGANG HAUPT S. Phosphorus Metabolism in Plants-K. Current Techniques in Biomedical Electron ROWAN MicroscopySAuL WISCHNITZER

Volume 19

AUTHOR INDEX-SUBJECT

INDEX

The Cellular Morphology of Tissue Repair-R. M. H. MCMINN Structural Organization and Embryonic Volume 20 DifferentiatiOn--GAJANAN V. SHERBET AND The Chemical Organization of the Plasma MemM. S. LAKSHMI brane of Animal Cells-A. H. MADDY The Dynamism of Cell Division during Early Subunits of Chloroplast Structure and Quantum Cleavage Stages of the Egg-N. FAUrREZConversion in Photosynthesis-RoDERIc €3. FIRLEFYN A N D 1. FAUTREZ PARK Lymphopoiesis in the Thymus and Other TisControl of Chloroplast Structure by Lightsues: Functional Implications-N. B. LESTERPACKERAND PAUL-ANDRE. SIEGENCC EVERETT A N D RUTHW. TYLER(CAFFREY) THALER Structure and Organization of the Myorieural The Role of Potassium and Sodium Ions as Junction-C. COeRs Studied in Mammalian Brain-H. HILLMAN The Ecdysial Glands of Arthropods-WILLIAM Triggering of Ovulation by Coitus inthe RatS . HERMAN CLAUDE ARON.GITTAASCH,AND JAQUELINE Cytokinins in Plants-B. I. SAHAI SRIVASTAVA Roos AUTHOR INDEX-SUBJECT INDEX Cytology and Cytophysiology of Non- CUMULATIVE SUBJECT INDEX Melanophore Pigment Ceh-JOsEPH T. (VOLUMES 1-21) BAGNARA The Fine Structure and Histochemistry of ProstaVolume 23 tic Glands in Relation to Sex HormonesDAVIDBRANDES Transformationlike Phenomena in Somatic Cerebellar Enzymology-LUCIE ARVY Cells-J. M.OLENOV AUTHOR INDEX-SUBJECT

INDEX

Recent Developments in the Theory of Control and Regulation of Cellular ProceswROBERTROSEN Volume 21 Contractile Properties of Protein Threads from Sea Urchin Eggs in Relation to Cell DiHistochemistry of Lysosomes-P. B. GAHAN Physiological Clocks-R. L. BRAHMACHARY vision-HIKOICHI S A K A I Microscopic Morphology of Ciliary Movement and Coordination in Electron Ciliates-BELA PARDUCA Oogenesis-ARNE N ~ R R E V A N G

385

CONTENTS OF PREVIOUS VOLUMES Dynamic Aspects of Phospholipids during Protein Secretion-LowELi. E. HOKIN The Golgi Apparatus: Structure and FunctionH. W. BEAMSA N D R. G. KESSEL The Chromosomal Basis of Sex DetenninationKENNETHR. LEWISA N D BERNARD JOHN AUTHOR INDEX-SUBJECT

INDEX

Isozymes: Classification, Frequency, and Significance-CmRLEs R. SHAW The Enzymes of the Embryonic NephronLUClE ARVY Protein Metabolism in Nerve Cells-B. DROZ Freeze-Etching-HANS MOOR AUTHOR INDEX-SUBJECT

INDEX

Volume 24

Volume 26

Synchronous Cell Differentiation4EoRGE M. PADILLA A N D IVAN L. CAMERON Mast Cells in the Nervous SyStem-YNGVE OLSSON Development Phases in Intermitosis and the Preparation for Mitosis of Mammalian Cells in VifrO-BLAGOJE A. NESKOVIC. Antimitotic S u b s t a n c e s a u u DEYSSON The form and Function of the Sieve Tube: A Problem in Reconciliation-P. E. WEATHERE LEY A N D R. P. C. JOHNSON Analysis of Antibody Staining Patterns Obtained with Striated Myofibrils in Fluorescence Microscopy and Electron Microscopy-FRANK A. PEPE Cytology of Intestinal Epithelial Cek-PETER G.TONER Liquid Junction Potentials and Their Effects on in Biology Potential Measurements Systems-P. C. CALDWELL

A New Model for the Living Cell: A Summary of the Theory and Recent Experimental Evidence in Its S U P ~ O I ~ ~ I L N. B ELING RT The Cell Periphery-LEONARD WEISS Mitochondria1 DNA: Physicochemical Properties, Replication, and Genetic Function-P. BORSTA N D A. M. KROON Metabolism and Enucleated Cek-KONRAD KECK Stereological Principles for Morphometry in Electron Microscopic CytOlOgy-EWALD R. WEIBEL Some Possible Roles for Isozymic Substitutions during Cold Hardening in Plants-D. W. A. ROBERTS

AUTHOR INDEX-SUBJECT

INDEX

Volume 25 Cytoplasmic Control over the Nuclear Events of Cell Reproduction-NOEL DE TERRA Coordination of the Rhythm of Beat in Some Ciliary Systems-M. A. SLEIGH The Significance of the Structural and Functional Similarities of Bacteria and Mitochondria-SYLvAN NASS The Effects of Steroid Hormones on Macrophage Activity-B. VERNON-ROBERTS The Fine Structure of Malaria ParasiteS-MARIA A. RUDZINSKA The Growth of Liver Parenchymal Nuclei and Its Endocrine Regulation-RITA CARRIERE Strandedness of Chromosomes-sHELDoN WOLFF

AUTHOR INDEX-SUBJECT

INDEX

Volume 27 Wound-Healing in Higher Phts-JACQUES LIPETZ Chloroplasts as Symbiotic OrganekS-DENNIS L. TAYLOR The Annulate L a m e l l a e S a u L WISCHNITZER Gametogenesis and Egg Fertilization in Planarians-C. BENAZZILENTATI Ultrastructure of the Mammalian Adrenal COrteX~IMONIDELMAN The Fine Structure of the Mammalian Lymphoreticular system-1 AN CARR Immunoenzyme Technique: Enzymes as Markers for the Localization of Antigens and AntibodiedTRATIS AVRAMEAS AUTHOR INDEX-SUBJECT

INDEX

Volume 28 The Cortical and Subcortical Cytoplasm of Lymnaea EggxHRISTIAAN P. RAVEN

386

CONTENTS O F PREVIOUS VOLUMES

The Environment and Function of Invertebrate Nerve Cells-J. E. TREHERNE A N D R. B. MORETON Virus Uptake, Cell Wall Regeneration, and Virus Multiplication in Isolated Plant Protoplasts-E. C. COCKING The Meiotic Behavior of the Drosophila OOCyte-ROBERT c . KING The Nucleus: Action of Chemical and Physical Agents-RENB SIMARD The Origin of Bone Celk-MAUREEN OWEN Regeneration and Differentiation of Sieve Tube Elements-WILLIAM P. JACOBS Cells, Solutes, and Growth: Salt Accumulation AND in Plants Reexamined-F. C. STEWARD R. L. MOTT AUTHOR INDEX-SUBJECT

INDEX

Volume 29 Gram Staining and Its Molecular M e c h a n i s m s B. B. BISWAS,P. S . BASU,A N D M .K. PAL The Surface Coats of Animal Cells-A. MARTfNEZ-PALOMO Carbohydrates in Cell Surfaces-RICHARD J. WINZLER Differential Gene Activation in Isolated Chromosomes-MARKUS LEZZI lntraribosomal Environment in the Nascent P e p tide Chain-HIDEKO KAJI Location and Measurement of Enzymes in Single Cells by Isotopic Methods Part I-E. A. BARNARD Location and Measurement of Enzymes in Single Cells by Isotopic Methods Part 1 1 4 . C. BUDD Neuronal and Glial Perikarya Preparations: An Appraisal of Present Methods-PATRICIA v . JOHNSTONA N D BETTYI. ROOTS Functional Electron Microscopy of the Hypothalamic Median Eminence-HIDESHI KOBAYASHI, TOKUU,MATSUI,A N D SUSUMI ISHII Early Development in Callus Cultures-MICHAEL M. YEOMAN AUTHOR INDEX-SUBJECT

INDEX

Volume 30 High-pressure Studies in Cell Biology--ARTHUR M. ZIMMERMAN Micrurgical Studies with Large Free-Living Amebas-K. W. JEON AND J. F. DANIELLI The Practice and Application of Electron Microscope Autoradiography-J. JACOB Applications of Scanning Electron Microscopy in Biology-K. E. CARR Acid Mucopolysaccharides in Calcified TiSSUeS-sHlNJlRO KOBAYASHI AUTHOR INDEX-SUBJECT

INDEX

CUMULATIVE SUBJECT INDEX (VOLUMES

1-29)

Volume 31 Studies on Freeze-Etching of Cell Membranes-KURT M ~ H L E T H A L E R Recent Developments in Light and Electron Microscope Radioautography-C. C. BUDD Morphological and Histochemical Aspec~s of Glycoproteins at the Surface of Animal Cells-A. RAMBOURG DNA Biosynthesis-H. S. JANSZ,D. VAN DER MEI, A N D G. M. ZANDVLIET Cytokinesis in Animal Cells-R. RAPPAFORT The Control of Cell Division in Ocular Lens-<. N. J. UNAIKAR, V. HARDING,J. R. REDDAN, A N D M. BAGCHI The Cytokinins-HANS KENDE Cytophysiology of the Teleost PituitaryMARTINSAGE AND HOWARDA. BERN AUTHOR INDEX-SUBJECT

INDEX

Volume 32 Highly Repetitive Sequences of DNA in Chromosomes-W. G. FLAMM The Origin of the Wide Species Variation in Nuclear DNA Content-H. REES A N D R. N. JONES Polarized Intracellular Particle Transport: Saltatory Movements and Cytoplasmic Streaming-LIONEL I. REBHUN The Kinetoplast of the Hemoflagellates-LP,RRY SIMPKIN

387

CONTENTS OF PREVIOUS VOLUMES Transport across the Intestinal Mucosal Cell: Hierarchies of Function-D. S. PARSONA N D C. A. R. BOYD Wound Healing and Regeneration in the Crab Pararelphusa hydrodromous-RITA G. ADIYODI The Use of Ferritin-Conjugated Antibodies in Electron MicrOSCopy~OUNCILMANMORGAN Metabolic DNA in Ciliated Protozoa, Salivary Gland Chromosomes, and Mammalian C e l l s S . R. PELC AUTHOR INDEX-SUBJECT

Mechanisms of Ion Transport through Plan Cell Membranes-EMANuEL ERSTEIN Cell Motility: Mechanisms in Protoplasmic Streaming and Ameboid Movement-H. KOMNICK, w. STOCKEM, A N D K. E. WOHLEFARTH-BOTTERMANN The Gliointerstitial System of MollusesCHISLAIN NICAISE Colchicine-Sensitive Microtubles-LYNN MARGULlS AUTHOR INDEX-SUBJECT

INDEX

INDEX

Volume 35 Volume 33 Visualization of RNA Synthesis on ChromosOmes-0. L. MILLER. JR. A N D BARBARA A. HAMKALO Cell Disjunction (“Mitosis”) in Somatic Cell Reproduction-ELAINE G . DIACUMAKOS, SCOTT HOLLAND, A N D PAULINE PECORA Neuronal Microtubles, Neurotilaments, and Microfilaments-RAYMOND B. WUERKER A N D JOEL B. KIRKPATRICK in Antibody Lymphocyte Interactions Responses-J. F. A. P. MILLER Laser Microbeams for Partial Cell Irradiation-MICHAEL w . BERNSA N D CHRISTIAN SALET Mechanisms of Virus-Induced Cell FusionGEORGEPOSTE Freeze-Etching of Bactena-cHARLES C. RE MSEN A N D STANLEY W. WATSON The Cytophysiology of Mammalian Adipose Celb-BERNARD G . SLAVIN

The Structure of Mammalian ChromosomesELTONSTUBBLEFIELD Synthetic Activity of Polytene ChromosomesHANSD. BERENDES Mechanisms of Chromosome Synapsis at Meiotic PrOphase-PETER B. MOENS Structural Aspects of Ribosomes-N. NANNlNGA

Comparative Ultrastructure of the Cerebrospinal Fluid-Contacting Neurons-B. VlGH A N D I. VIGH-TEICHMANN Maturation-Inducing Substance in StarfishesHARUOK A N A T A N I The Limonium Salt Gland: A Biophysical and Structural Study-A. E. HILL A N D B. S HILL Toxic Oxygen Effects-HAROLD M. SWARTZ AUTHOR INDEX-SUBJECT

INDEX

Volume 36

Molecular Hybridization of DNA and RNA in SitU-WOLFGANG HENNIG The Relationship between the Plasmalemma and Plant Cell Wd-JEAN-CLAUDE ROLAND Volume 34 Recent Advances in the Cytochemistry and UItrastructure of Cytoplasmic Inclusions in MasThe Submicroscopic Morphology of the Interphtigophora and Opalinata (Protozoa-. P. ase Nucleus-SAUL WlSCHNlTZER DUTTA The Energy State and Structure of Isolated Chloroplasts: The Oxidative Reactions Involv- Chloroplasts and Algae as Symbionts in ing the Water-Splitting Step of MOIIUSCS-LEONARD MUSCATINE AND Photosynthesis-ROBERT L. HEATH RICHARD W. GREENE Transport in N e u r o s p o r a ~ E N E A. SCAR- The Macrophage--hlMoN GORDON A N D ZANviL A. COHN BOROUGH AUTHOR INDEX-SUBJECT

INDEX

388

CONTENTS O F PREVIOUS VOLUMES

Degeneration and Regeneration of Neurosecretory Systems-HORST-DIETER DELLMANN

Cell Separation by Gradiant Centrifugation-R. HARWOOD

AUTHOR INDEX-SUBJECT

SUBJECT INDEX

INDEX

Volume 37

Volume 39

Units of DNA Replication in Chromosomes of Eukaroytes-J. HERBERT TAYLOR Viruses and Evolution-D. C. REANNEY Electron Microscope Studies on Spenniogenesis in Various Animal SpeCieS"ONPACHIR0 Y ASUZUMI Morphology, Histochemistry, and Biochemistry of Human Oogenesis and OVUhtiOn-sARDUL S. GURAYA Functional Morphology of the Distal LungKAYEH. KILBURN Comparative Studies of the Juxtaglomerular Apparatus-HIRoFuMI SOKABE AND MITZUHO OGAWA The Ultrastructure of the Local Cellular Reaction to Neoplasia-IAN CARR A N D J . c . E. UNDERWOOD Scanning Electron Microscopy in the UltraStNCtural Analysis of the Mammalian Cerebral Ventricular System-D. E. SCOTT, G . P. AND M. N. SHERIDAN KOZLOWSKI,

Androgen Receptors in the Nonhistone Protein Fractions of Prostatic Chromatin-TuNc YUE WANGA N D LEROYM. NYBERG Nucleocytoplasmic Interactions in Development of Amphibian Hybrids-STEPHEN SUBTIELNY The Interactions of Lectins with Animal Cell SUffaCeSAARTH L. NICOLSON Structure and Function of Intercellular Junctions-L. ANDREW STAEHELIN Recent Advances in Cytochemistry and Illtrastructure of Cytoplasmic Inclusions in Ciliophora (Protozoa)--G. P. DUTTA Structure and Development of the Renal Glomerulus as Revealed by Scanning Electron Microscopy-FRANC0 SPINEI.L.1 Recent Progress with Laser MicrnbeamsMICHAEL W. BERNS The Problem of G e m Cell Determinants--H. W. BEAMSA N D R. G. KESSEL

AUTHOR INDEX-SUBJECT

INDEX

SUBJECT I N D E X

Volume 40

B-Chromosome Systems in Flowering Plants and Animal Species-R. N. JONES Genetic Engineering and Life Synthesis: An In- The lntracellular Neutral SH-Dependent Protease Associated with Inflammatory troduction to the Review by R. Widdus and C. Reactions-HIDE0 HAYASHI Auk-JAMES F. DANIELLI Progress in Research Related to Genetic En- The Specificity of Pituitary Cells and Regulittion of Their Activities-VLADIMIR R. PANTIC gineering and Life Synthesis-Roy WIDDUS Fine Structure of the Thyroid Gland-HIsAo A N D CHARLES R. AULT FUJITA The Genetics of C-Type RNA Tumor VirusesPostnatal Gliogenesis in the Mammlian J. A. WYKE Brain-A. PRIVAT Three-Dimensional Reconstruction from Projections: A Review of AlgorithmsRICHARD Three-Dimensional Reconstruction from Serial Sections-RANDLE w . WARE A N D VINCENT GORDAN AND GABOR T. HERMAN LOPRESTI The Cytophysiology of Thyroid CellsSUBJECT INDEX VLADIMARR. PANTIC The Mechanisms of Neural Tube FonnationPERRYKARFUNKEL Volume 41 The Behavior of the XY Pair in MammalsThe Attachment of the Bacterial Chromosome to ALBERTO J. SOLARI the Cell Membrane-PAUL J. LEIBOWITZ AND Fine-Structural Aspects of Morphogenesis in MOSELIOSCHAECHTER Acetabularia". WERZ

Volume 38

CONTENTS OF PREVIOUS VOLUMES

389

Regulation of the Lactose Operon in Escherichiu Biochemical Studies of Mitochondria1 Transcription and Translation<. SACCONEA N D E. coli by c A M P - C . CARPENTER A N D B. H. QUAGLIARIELLO SELLS The Evolution of the Mitotic Spindle-DONNA Regulation of Microtubules in TerrahymenuNORMANE. WILLIAMS F. KUBAI Cellular Receptors and Mechanisms of Action of Germ Plasma and the Differentiation of the Germ Cell Line-E. M. EDDY Steroid HOrmOneS-sHUTSUNG LIAO A Cell Culture Approach to the Study of An- Gene Expression in Cultured Mammalian Cells-RoDY P. COX A N D JAMESc. KING terior Pituitary Cells-A. TIXIER-VIDAL, D. GOURDJI, A N D C. TOUGARD Morphology and Cytology of the Accessory Sex AND Glands in Invertebrates-K. G. ADIYODI lmmunohistochemical Demonstration of R. G. ADIYODI Neurophysin in the Hypothalamoneurohypophysial System-W. B. WAT- SUBJECT INDEX KINS

The Visual System of the Horseshoe Crab Limulus polyphemus--WOLF H. FAHREN-

Volume 44

The Nucleolar Structure-SIBDAs GHOSH The Function of the Nucleolus in the Expression of Genetic Information: Studies with Hybrid Animal Cells-E. SIDEBOTTOM A N D I. I. DEAK Volume 42 Phylogenetic Diversity of the Proteins RegulatRegulators of Cell Division: Endogenous Mitotic ing Muscular Contraction-WILLIAM Inhibitors of Mammalian Celk-BISMARCK LEHMAN B. Lozzio, CARMEN B. Lozz~o,ELENAG. Cell Size and Nuclear DNA Content in BAMBERGER, A N D STEPHEN V. LAIR VertebrakS-HENRYK SZARSKI Ultrastructure of Mammalian Chromosome Ultrastructural Localization of DNA in Ultrathin A N D WALTER Aberrations-B. R. BRINKLEY Tissue Sections-ALAIN GAUTIER N. HITTELMAN Cytological Basis for Permanent Vaginal Computer Processing of Electron Micrographs: Changes in Mice Treated Neonatally with A Nonmathematical Account-P. W. Steroid Hormones-NoeoRu TAKASUGI HAWKES On the Morphogenesis of the Cell Wall of Cyclic Changes in the Fine Structure of the Staphybcocci-PETER GIESBRECHT, JBRG Epithelial Cells of Human EndomewiumWECKE,A N D BERNHARD REINICKE MILDRED GORDON Cyclic AMP and Cell Behavior in Cultured The Ultrastructure of the Organ of CortiCeb-MARK c . WILLINGHAM ROBERTS. KIMURA Recent Advances in the Morphology, HisEndocrine Cells of the Gastric Mucosa-ENRICO tochemistry, and Biochemistry of SteroidSOLCIA.CARLOCAPELLA,GABRIELEVASSynthesizing Cellular Sites in the NonmamSALLO, A N D ROBERTO BUFFA malian Vertebrate O V ~ ~ ~ ~ A RS.D U L Membrane Transport of Purine and Pyrimidine GURAYA Bases and Nucleosides in Animal CellsSUBJECT INDEX RICHARD D. BERLINA N D JANET M. OLIVER EACH

SUBJECT I N D E X

SUBJECT INDEX

Volume 45 Approaches to the Analysis of Fidelity of DNA Repair in Mammalian Ceh-MICHAEL W. LIEBERMAN The Evolutionary Origin of the Mitochondrian: A Nonsymbiotic Model-HENRY R. MAHLER The Variable Condition of Euchromatin and Heterochromatin-FRIEDRICH BACK A N D RUCOLFA. RAFF

Volume 43

390

CONTENTS O F PREVIOUS VOLUMES

Effects of 5-Bromodeoxyuridine on Tumorigenicity, Immunogenicity, Virus Production, Plasminogen Activator, and Melanogenesis of Mouse Melanoma CellsSELMA SILAGI Mitosis in Fungi-MELVIN S. FULLER Small Lymphocyte and Transitional Cell Populations of the Bone Marrow; Their Role in the Mediation of Immune and Hemopoietic Progenitor Cell Functions-CoRNELrus ROSSE The Structure and Pmperties of the Cell Surface Coat-J. H. LUFT Uptake and Transport Activity of the Median Eminence of the Hypothalamus-K. M. KNIGGE,S. A. JOSEPH,J. R. SLADEK, M. F. NOTTER,M. MORRIS,D. K. SUNDBERG, M. A. HOLZWARTH, G. E. HOFFMAN,A N D L. O'BRIEN

Chemical Nature and Systematization of Substances Regulating Animal Tissue GrowthVICTORA. KONYSHEV Structure and Function of the Choroid Plexus and Other Sites of Cerebrospinal Fluid Formation-THOMAS H.MILHORAT The Control of Gene Expression in Somatic Cell Hybrids-H. P. BERNHARD Precursor Cells Of Mechanocytes-ALEXANDER J. FRIEDENSTEIN SUBJECT INDEX

Volume 48

Mechanisms of Chromatin Activation and Repression-NORMAN MACLEAN AND 'VAUGHAN A. HILDER Origin and Ultrastructure of Cells in Vitrc-L. M. FRANKS A N D PATRICIA D. WILSON SUBJECT INDEX Electrophysiology of the Neurosecretory Cell-KINJI YAGl AND SHIZUKO IWASAKI Reparative Processes in Mammalian Wound Volume 46 Healing: The Role of Contractile A N D DENYS Neurosecretion by Exocytosis-TOM CHRISTIAN PhenomenaSruLro GABBIANI MONTAND~N NORMANN Genetic and Morphogenetic Factors in Hemo- Smooth Endoplasmic Reticulum in Rat Hepatocytes during Glycogen Deposition and globin Synthesis during Higher Vertebrate Depletion-ROBERT R. CARDELL,JR. Development: An Approach to Cell Differentiation Mechanisms-VICTOR NIGON Potential and Limitations of Enzyme Cytochemistry: Studies of the Intracellular A N D JACQUELINE GODET Digestive Apparatus of Cells in Tissue Cytophysiology of Corpuscles of Stannius-V. Culture-M. HUNWEN G. KRISHNAMURTHY Ultrastructure of Human Bone Marrow Cell Uptake of Foreign Genetic Material by Plant Protoplasts-E. C. COCKING Maturation-J. BRETON-GORIUSA N D F. The Bursa of Fabricius and lmmunoglobulin REYES Synthesis-BRUCE CLICK Evolution and Function of Calcium-Binding SUBJECT INDEX Proteins-R. H. KRETSINGER SUBJECT INDEX

Volume 49 Cyclic Nucleotides, Calcium, and Cell Volume 47 Division-LIONEL 1. REBHUN Spontaneous and Induced Sister Chromatid ExResponses of Mammary Cells to Hormones-M. changes as Revealed by the BUdR-Labeling R. BANERJEE Method-HATAo KATO Recent Advances in the Morphology, Histochemistry, and Biochemistry of Steroid- Structural, Electrophysiological, Biochemical, and Pharmacological Properties of Synthesizing Cellular Sites in the Testes of Neuroblastoma-Glioma Cell Hybrids in Cell Nonmammalian VertebratesAARDuL S. Culture-B. HAMPRECHT GURAKA Epithelial-Stromal Interactions in Development Cellular Dynamics in Invertebrate Neurosecretory Systems-ALLAN BERLIND of the Urogenital Tract-GERALD R. CUNHA

CONTENTS OF PREVIOUS VOLUMES Cytophysiology of the Avian Adrenal Medulla-AsoK GHOSH Chloride Cells and Chloride Epithelia of Aquatic Lnsects-H. KOMNICK Cytosomes (Yellow Pigment Granules) of Molluscs of Cell Organelles of Anoxic Energy PI'odUCtiOn-IMRE ZS.-NAGY SUBJECT INDEX

Volume 50

391

Volume 52 Cytophysiology of Thyroid Parafollicular Celk-ELADlo A. NUNEZAND MICHAELD. GERSHON Cytophysiology of the Amphibian Thyroid Gland through Larval Development and Metamorphosis-ELIANE REGARD The Macrophage as a Secretory Cell-Roy C. PAGE,PHILIPDAVIES,A N D A. C. ALLISQN Biogenesis of the Photochemical Apparatus-TIMOTHY TREFFRY Extrusive Organelles in Protists-KLAUS HAUS-

Cell Surface Enzymes: Effects on Mitotic Activity and Cell Adhesion-H. BRUCEBOSMANN MANN New Aspects of the Ultrastructure of Frog Rod Lectins-JAY c. BROWN A N D RICHARDc . Outer Segments-JORGEN ROSENKRANZ HUNT Mechanisms of Morphogenesis in Cell SUBJECT INDEX Cultures-J. M. V A S I I I E V AND I. M. GELFAND

Cell Polyploidy: Its Relation to Tissue Growth and Functions-W. YA. BRODSKY A N D I. V. URYVAEVA Action of Testosterone on the Differentiation and Secretory Activity of a Target Organ: The Submaxillary Gland of the Mouse-MoNrQuE CHRBTIEN SUBJECT INDEX

Volume 51 Circulating Nucleic Acids in Higher STROUN, PHILIPPE Organisms-MAURICE ANKER,PIERRE MAURICE,AND PETER B. GAHAN Recent Advances in the Morphology, Histochemistry, and Biochemistry of the Developing Mammalian O V ~ ~ ~ - S A R D S. UL GURAYA Morphological Modulations in Helical Muscles (Aschelminthes and Annelida)-GluLlo LANZAVECCHIA Interrelations of the Proliferation and Differentiation Processes during Cardiac Myogenesis and Regeneration-PAVEL P. RUMYANTSEV The Kurloff C~II-PETER A. REVELL Circadian Rhythms in Unicellular Organisms: An Endeavor to Explain the Molecular Mechanism-HANS-GEoRG SCHWEIGER A N D MANFRED SCHWEIGER SUBJECT INDEX

Volume 53 Regular Arrays of Macromolecules on Bacterial Cell Walls: Structure, Chemistry, Assembly, and Function-UwE B. SLEYTR Cellular Adhesiveness and Extracellular Substrata-FREDERICK GRINNELL Chemosensory Responses of Swimming Algae A N D D. C. and Protozoa-M. LEVANWWSKY R. HAUSER Morphology, Biochemistry, and Genetics of Plastid Development in Euglena grucilis-V. NIGONA N D P. HEIZMANN Plant Embryological Investigations and Fluorescence Microscopy: An Assessment of Integration-R. N. KAPILA N D S. C. TIWARI The Cytochemical Approach to Hormone Assay-J. CHAYEN SUBJECT INDEX

Volume 54 Microtubule Assembly and Nucleation-MARC W. KIRSCHNER The Mammalian Sperm Surface: Studies with Specific Labeling Techniques-JAMES K. KOEHLER The Glutathione Status of Cek-NECHAMA s. KOSOWER A N D EDWARD M. KOSOWER Cells and Senescence-ROBERT ROSEN lmmunocytology of Pituitary Cells from Teleost Fishes-E. F O L L ~ N I UJ.S , DOERR-SCHOTT, A N D M. P. DUBOIS

392

CONTENTS OF PREVIOUS VOLUMES

Follicular Atresia in the Ovaries of Nonmamma- Methods of Measuring lntracellular Calciumlian VertebrateS-SRINIVAS K. SAIDAPUR ANTHONY H. CASWELL Hypothalamic Neuroanatomy: Steroid Hormone Electron Microscope Autoradiography of CalBinding and Patterns of Axonal Projectionscified Tissues-ROBERT M. FRANK DONALD W. PFAFFA N D LILYC. A. CONRAD Some Aspects of Double-Stranded Hairpin StrucAncient Locomotion: Prokaryotic Motility tures in Heterogeneous Nuclear RNASyStemS-LELENG P. TO A N D LYNNMARHIROTONAORA GULIS Microchemistry of Microdissected Hypothalamic An Enzyme Profile of the Nuclear Envelope-1. Nuclear Areas-M. PALKOVITS B. ZBARSKY SUBJECT INDEX SUBJECT INDEX

Volume 57 Volume 55

The Corpora Allata Of InseCtS-PIERRE CASSIER Kinetic Analysis of Cellular Populatjons by Means of the Quantitative Radioautography-J.-C. BISCONTE Cellular Mechanisms of Insect Photorcception-F. G. GRIBAKIN Oocyte Maturation-YosHIo MASUIA N D HUGH J. CLARKE The Chromaffin and Chromaffin-like Cells in the Autonomic Nervous SyStem-JACQUES TAXI The Synapses of the Nervous System-A. A. MANINA SUBJECT INDEX

Chromatin Structure and Gene Transcription: Nucleosomes Permit a New SynthesisTHORUPEDERSON The Isolated Mitotic Apparatus and Chromosome Motion-H. SAKAI Contact Inhibition of Locomotion: A Reappraisal-JOAN E. M. HEAYSMAN Morphological Correlates of Electrical and Other Interactions through Low-Resistance Pathways between Neurons of the Vertebrate CenSOTELO A N D tral Nervous System<. H. KORN Biological and Biochemical Effects of PhenylaVolume 58 lanine Analogs-D. N. WHEATLEY Recent Advances in the Morphology, HistoFunctional Aspects of Satellite DNA and chemistry, Biochemistry, and Physiology of Heterochromatin-BERNARD JOHN A V D Interstitial Gland Cells of Mammalian OvaryGEORGE L. GABORMIKLOS SARDUL S. GURAYA Determination of Subcellular Elemental ConcenCorrelation of Morphometry and Stereology tration through Ultrahigh Resolution Electron with Biochemical Analysis of Cell FractionsMicroprobe Analysis-THOMAS E. HUTCHINR. P. BOLENDER SON Cytophysiologyof the Adrenal 2ona FasciculataGASTONE G. NUSSDORFER,GIUSEPPINA The Chromaffin Granule and Possible Mechanisms of Exocytosis-HARVEY El. MAZZOCCHI, A N D VIRGILIO MENEGHELLI POLLARD, CHRISTOPHER J. PAZOLES, C A R LE. SUBJECT INDEX CREUTZ,A N D ORENZINDER The Golgi Apparatus, the Plasma Membrane, and Functional Integration-W. G . WHALEY Volume 56 A N D MARIANNE DAUWALDER Control of Meiosis-I. N Synapses Of Cepha~opodS-cOLETTE DUCROS Genetic GOLUBOVSKAY A Scanning Electron Microscope Studies on the Development of the Nervous System in Vivo Hypothalamic Neurons in Cell Culture-A. A N D F. DE VITRY TIXIER-VIDAL and in Vitro-K. MELLER Cytoplasmic Structure and Contractility in The Subfornical Organ-H. DIETERDELLMANN A N D JOHNB. SIMPSON AND Amoeboid Cells-D. LANSING TAYLOR SUBJECT INDEX JOHN S. CONDEELIS