INTERNATIONAL REVIEW OF CYTOLOGY V55, Volume 55 (v. 55)

INTERNATIONAL

REVIEW OF CYTOLOGY VOLUME55

ADVISORY EDITORS

H. W. BEAMS

ARNOLD MITTELMAN

HOWARD A. BERN

DONALD G. MURPHY

W. BERNHARD

ROBERT G. E. MURRAY

GARY G. BORISY

ANDREAS OKSCHE

ROBERT W. BRIGGS STANLEY COHEN

VLADIMIR R. PANTIC DARRYL C. REANNEY

RENE COUTEAUX

LIONEL I. REBHUN

MARIE A. DI BERARDINO

JEAN-PAUL REVEL

N. B. EVERETT

WILFRED STEIN

CHARLES J. FLICKINGER

ELTON STUBBLEFIELD

M. NELLY GOLARZ DE BOURNE

HEWSON SWIFT

K. KUROSUMI

DENNIS L. TAYLOR

MARIAN0 LA VIA

TADASHI UTAKOJI

GIUSEPPE MILLONIG ROY WIDDUS ALEXANDER L. YUDIN

INTERNATIONAL

Review of Cytology EDITED B Y

G . H. BOURNE Yerkes Regional Primate Research Center Emory University Atlanta, Georgia

J. F. DANIELLI Worcester Polytechnic Institute Worcester, Massachusetts

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

VOLUME 55

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

1978

COPYRIGHT @ 1978, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION M A Y BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

ACADEMIC PRESS, INC.

111 Fifth Avenue, New York, New York 10003

United Kingdom Edition published by ACADEMIC PRESS, INC. ( L O N D O N ) LTD. 24/28 Oval Road, London NW1 7DX

LIBRARY OF CONGRESS CATALOG CARD NUMBER:52-5203 ISBN 0-12-364355-4 PRINTED IN THE UNITED STATES OF AMERICA

Contents LISTOFCONTRIBUTORS .......................................................

ix

Chromatin Structure and Gene Transcription: Nucleosomes Permit a New Synthesis THORUPEDERSON I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . .. . . . . . . . . . . . . ... . . . . . . . . 111. Euchromatin Content and the Sequence Complexity of Nuclear RNA . . IV. Chromatin Fractionation: Its Logic and Logistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. The Euchromatin versus Active Chromatin Problem

V. VI. VII. VIII. IX. X. XI.

Histone Content of Active Chromatin .................. The Nucleosome Paradigm . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nucleosomes and Transcription . . .. .. Morphology of Active Transcripti Evidence for an Alteration of Nucleosome Structure during Transcription . . . . . . . . . Contemporary Research on the Structure of Active Chromatin Conclusion ............... .................... ....... . . ........... References

1 2 3 5 9 10 11 14 15 18 19 20

The Isolated Mitotic Apparatus and Chromosome Motion H. SAKAI I. Introduction. . . . . . . . . . . . . . . . . . . . . . . .

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

11. Egg Tubulin

111. The Isolated IV. Mitosis and t References . . . . .

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

23 24 31 39 44

Contact Inhibition of Locomotion: A Reappraisal JOAN

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

E. M. HEAYSMAN

................ Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . ................................... Contact Inhibition Type 1 ................................ Contact Inhibition Type 2 . . Failure of Contact Inh Tumor Cells and Contact Inhibition .............................. Mechanism of Action of Contact Inhib .............................. ............ .............. * ......... ............................ V

49 51 53 54 56 58 63

'64

vi

CONTENTS

Morphological Correlates of Electrical and Other Interactions through Low-Resistance Pathways between Neurons of the Vertebrate Central Nervous System C. SOTELO AND H. KORN

I. General Considerations

..

...

.....

IV. Electrical Synapses in the Mammalian C N S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

67 70 77 90

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

104

11. Ultrastructure of Electrical s ........................................ 111. Electrical Synapses in the CNS of Nonmammalian Vertebrates .....

Biological and Biochemical Effects of Phenylalanine Analogs D. N. WHEATLEY

... ................................................... 111. Incorporation into Protein ............ I. Introduction

IV. Analog Proteins: Molecula V. Analog Proteins: Cellular Effects.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Use of Analogs in Other Areas of Biological Research. . ............ V11. Degradation of Analo .............. VIII. Concluding Remarks .............. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

114

134 I48 159

162 I64

Recent Advances in the Morphology, Histochemistry, Biochemistry, and Physiology of Interstitial Gland Cells of Mammalian Ovary SARDULS. GURAYA 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Interstitial Gland Cells of the Developing and Maturing Ovary . . . . . . . . . . . . . . . . . .

III. Interstitial Gland Cells of the Prepubertal and Adult Ovary ..................... IV. General Discussion and Conclusions.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

171 173 180 227 240

Correlation of Morphometry and Stereology with Biochemical Analysis of Cell Fractions R. P. BOLENDER I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. The Technique of Stereological Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. The Technique of Differential Centrifugation and Marker Enzyme Analysis. . . . . . . .

247 248 271

CONTENTS IV . The Integration of Stereological and Biochemical.Data ......................... V . Summary and Conclusions ................................................ References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii 275 287 287

Cytophysiology of the Adrenal Zona Fasciculata GASTONE G . NUSSWRFER. GIUSEPPINA MAZZOCCHI.AND VIRGIL10 MENEGHELLI I . Introduction . . . I1. Fine Structure of Normally Functioning Adrenocortical Cells . . . . . . . . . . . . . . . . . . . I11. Fine Structure of Hyperfunctioning and Hypofunctioning Adrenocortical Cells ..... IV . Morphological-Functional Correlations in Adrenocortical Cells . . . . . . . . . . . . . . . . . . V . The Mechanism of Hormone Release by Adrenocortical Cells . . . . . . . . . . . . . . . . . . . VI . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

SUBJECT INDEX .............................................................. CONTENTSOFPREVIOUSVOLUMES ..............................................

291 292 316 321 347 354 355 367 37 1

This Page Intentionally Left Blank

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

R. P. BOLENDER (247), Department of Biological Structure, School of Medicine, University of Washington, Seattle, Washington 98195 SARDULS . GURAYA(171), Department of Zoology, College of Basic Sciences and Humanities, Punjab Agricultural University, Ludhiana, Punjab, India JOAN E. M. HEAYSMAN (49), Department of Zoology, University College, London, England H. KORN(67) , l Laboratoire de Neuromorphologie (U-I06 INSERM), Centre Midico-Chirurgical Foch, 92150 Suresnes, France GIUSEPPINA MAZZOCCHI (29 1 ) , Department of Anatomy, Laboratory of Electron Microscopy, University of Padua, 35100 Padua, Italy VIRGILIO MENEGHELLI (29 1) , Department of Anatomy, Laboratory of Electron Microscopy, University of Padua, 35100 Padua, Italy GASTONEG. NUSSDORFER (291), Department of Anatomy, Laboratory of Electron Microscopy, University of Padua, 35100 Padua, Italy THORUPEDERSON ( 1) , Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545 H. SAKAI(23), Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo,Japan C . SOTELO (67), Laboratoire de Neuromorphologie (U-I06 INSERM), Centre Midico-Chirurgical Foch, 92150 Suresnes, France

D . N . WHEATLEY (log), Department of Pathology, University Medical Buildings, Foresterhill, Aberdeen AB9 2ZD, Scotland

'Maitre de Recherches (INSERM, U 3).

ix

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INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 55

Chromatin Structure and Gene Transcription: Nucleosomes Permit a New Synthesis THORUPEDERSON Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts

I. Introduction

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

1

11. The Euchromatin versus Active Chromatin Problem . . . . . 111. Euchromatin Content and the Sequence Complexity of Nuclear RNA

IV . V. VI. VII. VIII. IX.

Chromatin Fractionation: Its Logic and Logistics . . . . . Histone Content of Active Chromatin . . . . . . . . . . The Nucleosome Paradigm . . . . . . . . . . . . . Nucleosomes and Transcription . . . . . . . . . . . . Morphology of Active Transcription Units . . . . . . . . Evidence for an Alteration of Nucleosome Structure during Transcription . . . . . . . . . . . . . . . . . . X . Contemporary Research on the Structure of Active Chromatin . XI. Conclusion . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

. . . . . .

.

2 3 5 9 10 11

14 15 18 19

20

I. Introduction A long-standing problem in the field of eukaryotic genome biology is the question of how chromatin structure is related to transcription. Besides its inherent appeal, an important heuristic reason for studying this issue is to identify the particular aspect of chromatin structure that might be most profitably exploited for fine-scale analysis of the mechanics of transcriptional control. Assuming at the outset that the transcribed loci of eukaryotic chromosomes must be specialized at the molecular level, this article attempts to analyze critically the issues of whether and how such specialization extends to supramolecular and higher orders of chromatin structure. This axis of modem chromosome research has had two partially overlapping phases. The first, which received particular emphasis in the 1960s, deals with the relationship of transcriptional activity to the degree of chromatin fiber packing within the interphase nucleus (euchromatin and heterochromatin). This is still a popular aspect of the problem, but one that may now require some clarification in light of more recent developments. The second phase has focused on the extent to which transcriptional activity is related to local changes in the nucleoprotein structure of chromatin fibers themselves, and this contemporary and highly active area of investigation is taken up in the 1

Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-364355-4

2

THORU PEDERSON

latter portion of this article. As will be seen, a recent revolution in the field of chromatin structure has made it possible to view the problem in a much more precise way than ever before.

11. The Euchromatin versus Active Chromatin Problem

Our point of departure here is the cytology of the interphase cell nucleus. In almost all eukaryotic cells, interphase chromatin exists as two distinct morphotypes: euchromatin and heterochromatin. Although these terms are sometimes also applied to biochemical fractions, they properly belong only in the vocabulary of cytology and, by extension, cytogenetics. The existence of two forms of interphase chromatin has been known for at least half a century (Heitz, 1928). Several studies have demonstrated that heterochromatin is genetically inert, the two classic examples being position effects in Drosophilu and mammalian X-chromosome mosaicism. In the biochemical context, it was Mirsky’s group that first attempted to relate interphase chromatin dimorphism to nuclear metabolism, specifically transcription. Autoradiographic analyses of uridine3H-labeled thymus lymphocyte nuclei served to localize sites of RNA synthesis (and accumulation) over the diffuse, euchromatin regions (Littau et al., 1964). This observation formed an apparent analogy with the earlier work of Gall and Callan (1962), and of the Rockefeller group (Izawa et al., 1963), demonstrating RNA synthesis on the loops of amphibian oocyte meiotic bivalents (lampbrush chromosomes). Another development that provided momentum for the view of a major degree of structural specialization of active chromatin was the discovery that “puffed” regions of dipteran polytene chromosomes represent sites of intense RNA synthesis. However, the dialectic problem with puffs is that it is very difficult to know whether they represent a structural motif causally related to transcription, or instead one that owes its structure to an accumulation of newly synthesized ribonucleoprotein (RNP). One does not visualize simply chromatin templates at these loci, but rather a template-transcript complex. The more contemporary view is that puffs simply represent the most intense sites of transcription, or the particular regions of the chromosome where completed RNP transcripts tend to accumulate, or both. Thus early autoradiographic studies by Rudkin (1962) suggested that, in addition to puffs, nonpuffed regions are also active in RNA synthesis, and it has been recently confirmed that the majority of interband regions synthesize RNA, although at lower levels than puffs (Zhimulev and Belyaeva, 1975; Pages and Alonso, 1976). Moreover, interband regions, as well as puffs, appear to be the major sites of RNA polymerase-B localization in Drosophila polytene chromosomes (Jamrich et al., 1977). While the concept that heterochromatin is transcriptionally inactive is reliably documented, at least at standard levels of autoradiographic or biochemical sen-

CHROMATIN STRUCTURE AND GENE TRANSCRIPTION

3

sitivity, the still prevalent view that euchromatin represents, in its entirety, sites of gene transcription warrants particular scrutiny. In the case of the thymus lymphocyte, which was the prototype for this thesis (Littau et al., 1964), the amount of euchromatin DNA is rather small, about 20%, with the remainder residing in facultative heterochromatin. In this particular case, it is perfectly possible that all the euchromatin is transcriptionally active. However, the issue that needs to be critically explored is the validity of the popular generalization of this view from the specific case of the thymus lymphocyte to a dogma. The concept of a stringent correlation between gross chromatin structure (i.e., euchromatin versus heterochromatin) and gene transcription has had a pronounced impact on the course of chromatin research over the past decade. In some quarters it continues to prevail as an implicit, and often explicit, component of contemporary thinking in the field of eukaryotic gene regulation. However, it now seems quite likely that this view is incorrect. 111. Euchromatin Content and the Sequence Complexity of Nuclear RNA A major reason for questioning the view that all euchromatin DNA is transcriptionally active comes from a consideration of the complexity of nuclear RNA.’ The fact is that the amounts of euchromatin in most mammalian cell nuclei exceed by an order of magnitude, or more, the fraction of the genome that is transcribed, measured in the same cell type. For example, in mouse liver approximately 90% of the nuclear DNA is in euchromatin (Yasmineh and Yunis, 1970), while RNA-driven hybridization reactions with purified nonrepetitive DNA indicate that not more than 8 -9% of the nonrepetitive fraction of mouse liver DNA is transcribed, or 6-7% of the total genome (Hahn and Laird, 1971; Grouse et al., 1972). While it is a formal possibility that the transcribed fraction of eukaryotic genomes has been greatly underestimated (to be discussed), these kinds of results suggest that the majority of euchromatin is, like heterochromatin, transcriptionally inert. As shown in Table I, the proportion of total DNA represented by the nucleotide sequences in nuclear or total cell RNA falls into a rather narrow set of low ‘As used here, and throughout this article, the “complexity” of a nucleic acid sample refers to the total number ofkinds of nucleotide sequences present, of an arbitrarily selected length, without regard to their respective concentrations. The use of this term closely parallels the meaning of “diversity” in the discipline of population biology. Just as diversity has nothing to do with biomass, complexity is a rather undemocratic notion that ignores the abundance of each component sequence. A quick analogy is to consider an unorthodox library whose collection consists of 1 copy of book A, lo00 copies of book B, and 10 million copies of book C. This library’s unfortunate patronage would find its selection of reading material to have the disappointing complexity of 3, despite the truly extraordinary size of the library’s total holdings.

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TABLE 1 ESTIMATES OF THE TRANSCRIBED FRACTION OF SINGLE-COPY DNA SEQUENCESI N MAMMALIAN CELLS"

Organism Mouse

Organ or cell Liver Liver Kidney Spleen

Hybridized single-copy DNA (% of total duplex)* 8.2 9.4 9.6

10.0

Liver Kidney Spleen Thymus Ascites cells

21.8

cow

Liver

10.8

Human

HeLa cells

17.8

Rat

10.6 9.6 9.2 8.4

Transcribed single-copy sequences expressed (% of total DNA in genome)" 5.74" 6.58' 6.72" 7.00' 16.35' 7.95' 7.20' 6.90' 6.3W 6.4Eh 1 I .O'

Excluding values for brain. Assuming asymmetric transcription. "Obtained by correcting values in first column for the repeated sequence content (mass) of the genome. dHahn and Laird (1971). "Grouse et al. (1972). 'Chikaraishi et at. (1978). UHolmesand Bonner (1974). hKohne and Byers (1973). 'Holland and Pederson, unpublished data. 'I

values (6-16%) for the mammalian organs and species that have been reliably studied, with the exception of brain (not shown). However, these values must be taken as minimum estimates for two reasons. First, in these measurements is it not always certain that the rarest RNA sequences have reacted. The reaction is driven by nuclear or cellular RNA as it exists in the steady state, and therefore extremely rare transcripts may be underrepresented and not a kinetically significant component of the observed reaction. Sequences can be rare because of low transcriptional production, low metabolic stability or, in the case of nuclear RNA, short nuclear residence time. However, the values in Table I for rat tissues and for HeLa cells probably include nuclear RNA sequences as rare as 0.1-1 .O copies per cell (Chikaraishi et al., 1978; Holland and Pederson, in preparation). A second consideration is that these experiments deal only with transcripts of single-copy DNA sequences. If all the repeated sequences (as mass) were transcribed in these cells, which is most unlikely, the values in the last column of Table I would range from 30 to 40% of the total DNA, which is still far below the

CHROMATIN STRUCTURE AND GENE TRANSCRIPTION

5

amount of euchromatin DNA. The view that only relatively small fractions of most mammalian genomes are transcribed also receives support from ultrastructural studies. Electron microscopy of detergent-lysed nuclei from growing mammalian cells reveals that, in nonnucleolar chromatin, transcription is an infrequent event (Hamkalo et al., 1974). Finally, additional evidence that the transcribed fraction of eukaryotic genomes is small comes from chromatin fractionation experiments.

IV. Chromatin Fractionation: Its Logic and Logistics Although it is not always recognized, the approaches employed to date for fractionating chromatin into its transcriptionally active and inactive components have had two distinct rationales. The Rockefeller group separated thymus lymphocyte chromatin by differential sedimentation into fractions that morphologically resembled the euchromatin and heterochromatin components of intact interphase nuclei (Frenster et al., 1963). Not unexpectedly, pulse-labeled RNA was largely confined to the euchromatin fraction. While these procedures may fractionate the two major chromatin morphotypes, euchromatin and heterochromatin, they do not necessarily separate transcriptionally active and inactive chromatin. It was Bonner’s group that first recognized the possibility that bona fide active chromatin might possess intensive physical properties different from those of bulk chromatin, that is, that the transcribed portion of eukaryotic genomes is both structurally unique and small. In a limited reaction, DNase I1 (spleen DNase) was found to attack rat liver or ascites cell chromatin so as to render a small portion (10-20%) soluble in 0.15 M NaCl, with the bulk of the chromatin retaining its characteristic insolubility in 0.15 M NaCl (Marushige and Bonner, 1971; Billing and Bonner, 1972), presumably because it remains unsheared by the DNase (see also Gottesfeld et al., 1974a; Pederson and Bhorjee, 1975). Other approaches, based upon the same rationale of a structurally specialized but small transcribed fraction of eukaryotic genomes, have involved mechanical shearing of chromatin followed by fractionation on the basis of sedimentation, solubility, or chromatographic properties (Chalkley and Jensen, 1968; McConaughy and McCarthy, 1972; Janowski et al., 1972; Murphy et al., 1973; Simpson and Reeck, 1973; Simpson, 1974; Turner and Hancock, 1974; Berkowitz and Doty, 1975). Based upon nascent RNA content or endogenous RNA polymerase activity, these latter approaches yield “template-active” fractions that are enriched 5- to 20-fold relative to the “template-inactive” fractions. Up until very recently, the most definitive criterion, enrichment of active gene sequences, had not yet been generally applied. The following discussion therefore relates specifically to nascent RNA content as an index of the purity of active chromatin and also considers the validity of this criterion.

6

THORU PEDERSON

Figure 1 shows the theoretical relationship between nascent RNA content and purity of active chromatin fractions, computed assuming either that 20% of the genome is transcribed (solid curve) or that 80% of the genome is transcribed (dashed curve). The numbered arrows refer to the observed values of nascent RNA content published in the articles listed in the legend. On the solid curve (20%of the genome transcribed), these observed values can be seen to correspond to purities of 76-93%. More importantly, it can be seen that the observed values of nascent RNA content (2.2 to 3.9 on the ordinate) do not intersect the theoretical curve based on 80% of the genome being transcribed. It follows that, if 80 % of the genome were transcribed, the observed values for nascent RNA per unit of DNA would signify purities of greater than 100% and, since this is impossible, the hypothesis that 80% of the genome is active is ruled out. This approach can, by extension, be used to set an upper limit on the transcribed fraction of a given genome. One first generates a family of curves such as shown in Fig. 1 for different values of percent of genome transcribed. Then, by taking a given reported value for nascent RNA content per unit of DNA (y), one simply determines by inspection the value of percent of genome transcribed whose curve intersects y at a value of x just greater than 100%. Since purity cannot of course exceed loo%, this gives the maximum value of percentage of genome transcribed that is compatible with a given value of nascent RNA content of the active fractions. For an observed nascent RNA per unit DNA value of 3.9, as reported for HeLa cell active chromatin fractions (Pederson and Bhorjee, 1975), it can easily be shown that this upper limit is 25.5% of the genome. It is emphasized

20 I genome transcribed

0.05

o'o'O 0

. 20

400

60 Purity (%)

2 80

10L 0

FIG.1. Relationship between observed enrichment of nascent RNA in template-active chromatin and calculated purity, based upon assuming that either 20%of the genome is transcribed (solid curve) or 80%is transcribed (dashed curve). The numbered arrows refer to data from the following articles: 1: Pederson and Bhorjee (1975); 2: Billing and Bonner (1972); 3: Murphyet al. (1973); 4: Frensteret al. (1963); 5: Turner and Hancock (1974).

CHROMATIN STRUCTURE AND GENE TRANSCRIPTION

7

that this is a limit, not the true value. This kind of analysis cannot give an actual value for the transcribed fraction of a genome unless one has an independent measure of purity for the active chromatin fraction. It simply indicates that, in this particular case (HeLa cells), a value of 3.9 for the nascent RNA content per unit DNA could not be observed unless the active fraction of the genome were 25.5% or less. It is intuitively obvious that, when the purity of an active fraction is less than loo%, which must be the case, and its observed nascent RNNDNA value is 3.9 (as in Pederson and Bhorjee, 1975), then the fraction of the genome actually transcribed must be less than 25.5%. As discussed later, it is of interest to note that a very different kind of data decomposition has led Bonner’s group (Gottesfeld et al., 1976) to a nearly identical estimate (20%) for the transcribed fraction of the rat liver genome, based upon the measured kinetic complexities of DNA from the template-active fraction versus total chromatin. According to the foregoing discussion, the results of chromatin fractionation experiments are compatible with the view that the transcribed fraction of the genome in most mammalian cells is small, on the order of 25% at most. In turn, this supports the view that the cytological entity termed euchromatin, comprising the majority of interphase DNA in these cells, cannot be synonymous with active chromatin. However, for this argument to hold, it is essential to show that the nascent RNA criterion of active chromatin enrichment is not spurious. One could argue that nascent RNA is released from its active DNA templates by endogenous or exogenous nucleases and subsequently fractionates spuriously with a small amount of special chromatin that is physically or chemically distinct for reasons other than its transcriptional activity, giving the false impression of a high enrichment of active chromatin. For the spleen DNase fractionation procedure (Gottesfeld et al., 1974a) this trivial explanation has been convincingly ruled out in two independent studies (Billing and Bonner, 1972; Pederson and Bhorjee, 1975). However, it is to be noted that reconstruction experiments have indicated that the nascent RNA criterion may not be as valid for other chromatin fractionation procedures, including those that involve extensive mechanical shear (Seidman and Cole, 1977). Additional evidence against a trivial basis for at least the spleen DNase chromatin fractionation procedure comes from an analysis of the reassociation kinetics of DNA from the active chromatin fraction (Gottesfeld et al., 1976). The template-active fraction of rat liver chromatin prepared by this method contains 11% of the total DNA and possesses a nonrepeated sequence complexity equivalent to approximately 1/10 of that of the total rat genome. Moreover, repeated DNA sequences in the active chromatin fraction represent a subset equivalent to 1/20 of the complexity of the intermediate kinetic component of total rat liver DNA. If certain not unreasonable assumptions are made, the lengths of DNA fragments in the active fraction and their sequence complexity can be used to

8

THORU PEDERSON

show, by straightforward algebra and curve-fitting, that the template-active fraction of the rat genome is 20 -+ 3% (Gottesfeld et al., 1976). These results demonstrate that both the repeated and nonrepeated DNA sequences present in the template-active chromatin fraction generated by the DNase I1 procedure represent a particular subset of the total genome. However, this fact does not demonstrate that these are actually the specific sequences that direct transcription in vivo. The coenrichment of nascent RNA with this fraction (Billing and Bonner, 1972; Pederson and Bhorjee, 1975) suggests that such a transcriptional correlation exists, but rigorous confirmation of this point requires a demonstration that the “active” chromatin DNA is actually enriched for sequences coding for cellular RNA, preferably a specific mRNA. In the initial study by Gottesfeld et al. (1974a) it was concluded that the DNase II-generated active chromatin fraction from rat liver was enriched approximately six- to sevenfold in nonrepeated DNA sequences complementary to total liver RNA. Using DMSO-induced Friend leukemia cells and specific globin cDNA probes, it has recently been shown that globin sequences are enriched 2.4- -7.0-fold (relative to total cell DNA) in the template-active fraction (Gottesfeld and Partington, 1977; Wallace et al., 1977). Gottesfeld and Partington also found a similar enrichment (2.4-fold relative to total cell DNA) for poly(A)-containing mRNAcoding sequences and for rDNA in the template-active fraction, while the inactive fraction displayed a quantitatively similar enrichment for highly repeated DNA sequences that may contain a large fraction of nontranscribed components, such as satellite DNA. However, these two groups obtained different results for the uninduced Friend cell. Gottesfeld and Partington found no enrichment of globin sequences in the template-active fraction of chromatin from uninduced cells, whereas Wallace et al. found an enrichment similar to that seen with the DMSO-induced cells. The basis for these differences is not yet resolved, but it is useful to recall that globin gene sequences retain their DNase I-sensitive configuration in the transcriptionally inactive mature chicken erythrocyte (Weintraub and Groudine., 1976). To summarize, it can be said that chromatin fractionation data support, but do not by themselves formally prove, the view that the proportion of DNA transcribed in somatic mammalian cells is small, on the order of 20% or less. As discussed earlier, this same conclusion emerges from a consideration of the sequence complexity of nuclear RNA. Since more than 85% of the interphase DNA in these cell types resides in euchromatin, it is most unlikely that all euchromatin is transcriptionally active. The results of chromatin fractionation experiments indicate that bona fide active chromatin is in some way structurally different from bulk inactive chromatin, but this anatomical specialization is apparently based upon an order of nucleoprotein structure lower than that which can be visualized and defined cytologically as euchromatin.

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V. Histone Content of Active Chromatin The chromatin fractionation studies discussed above and shown in Fig. 1 (Frenster et al., 1963; Billing and Bonner, 1972; Pederson and Bhorjee, 1975; Murphy et al., 1973; Turner and Hancock, 1974) are ones in which nascent RNA content was used as the criterion of active chromatin enrichment. In the absence of the ideal criterion-enrichment of specific expressed gene sequences-this is the best alternative, especially when the proper kinds of controls are employed (Billing and Bonner, 1972; Pederson and Bhorjee, 1975). Several other studies purporting to have used the nascent RNA index were excluded from Fig. 1 because the duration of labeling was too long to be reliably considered diagnostic of nascent transcripts, as opposed to other chromatin-associated RNAs that are possibly present in both active and inactive regions. And of course in many other studies, criteria less rigorous than nascent RNA content have been employed. Taken together, the compositional data for all these so-called template-active fractions are quite variable and, in the absence of a satisfactory measure of purity in each instance, it is not possible to know whether this variation in chemical composition is due merely to differences in purity or to intrinsic variations in the makeup of template-active chromatin among the various cell types used. One result that has been reported with reasonable consistency is an absence or greatly reduced complement of histone 1 in the active fraction (Simpson and Reeck, 1973; Pederson and Bhorjee, 1975; Berkowitz and Doty, 1975). However, since this histone can under certain conditions be particularly sensitive to endogenous chromatin proteases (Chong et al., 1974), and also because it is the most loosely bound of the five histones (Ohlenbusch et al., 1967), its reported absence from active chromatin warrants scrutiny. In one study, the possibility of a protease artefact was rather convincingly ruled out (Simpson and Reeck, 1973). Although the low ionic strengths used in these chromatin fractionation experiments are not normally capable of dissociating histone 1 from chromatin, the interesting possibility remains that histone 1 in active chromatin is somehow special (e.g., highly phosphorylated) and more easily dislodged than its counterparts in the inactive portions of the chromatin. The possible absence of histone 1 from transcribed chromatin regions clearly requires further investigation. One reason for drawing attention to the need for further study of the possible absence of histone 1 from active chromatin is that this is a potential key to understanding the elevated susceptibility of these regions of native chromatin to nuclease attack-an effect on which the more successful chromatin fractionation schemes are based, as discussed in Section IX. In addition, a lack or reduced content of histone 1 could go a long way toward explaining the relatively “open” structure of active chromatin as revealed by physicochemical properties such as circular dichroism (Polacow and Simpson, 1973; Gottesfeld et al., 1974b), melt-

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ing temperatures (McConaughy and McCarthy, 1972; Reeck et al., 1972; Berkowitz and Doty, 1975; Gottesfeld et a l . , 1975b), and DNase sensitivity of the isolated active fractions (Berkowitz and Doty, 1975; Gottesfeld ef a l . , 1975a). Histone 1 is also regarded as a major determinant of chromatin solubility (Ohlenbusch ef al., 1967), and an absence of histone 1 would not be incompatible with the solubility of active chromatin in 0.15 M NaCl, a salt concentration at which bulk chromatin is insoluble (Marushige and Bonner, 1971; Billing and Bonner, 1972; Gottesfeld et a l . , 1974a; Pederson and Bhorjee, 1975). A related issue is the possible role of histone 1 in determining chromatin fiber folding patterns. In particular, histone 1 has been implicated in the folding of the elementary 80- to 100-A chromatin fiber into 200- to 250-A fibers (Renz et al., 1977). It follows that a local removal of histone 1 could trigger a 200-250 A + 80-100 A fiber unfolding, and this could be an important early event in activating transcription. While the foregoing discussion has emphasized the possible absence of histone 1 from active chromatin fractions, it is not to be ignored that the same literature demonstrates the presence of the other four histones in active chromatin, thus eliminating the trivial possibility that these regions are merely naked DNA. Active chromatin is nucleoprotein. Much more importantly, the presence of histones 2a, 2b, 3, and 4 in active chromatin serves to connect the chromatin fractionation field with the recently emerged nucleosome model of chromatin organization.

VI. The Nucleosome Paradigm The emergence of the repeating subunit (nucleosome) model of eukaryotic chromosome structure has provided an exciting new perspective for examining the relationship between transcription and chromatin anatomy on a finer scale than was possible before. Our present understanding of the nucleosome structure of chromatin can be briefly summarized as follows. Each chromatin subunit is a histone octamer consisting of a (H3-H4), tetramer and two molecules each of H2a and H2b (Kornberg, 1974; Thomas and Kornberg, 1975), with the arginine-rich tetramer apparently constituting an inner "core" (Camerini-Otero et al., 1976). Each nucleosome is associated with approximately 190 base pairs of DNA, 140 of which are apparently associated with the H3-H4 tetramer core in a particularly nuclease-resistant form (Sollner-Webb and Felsenfeld, 1975; Axel, 1975). The total length of nucleosome-associated DNA varies phylogenically , but the length of the core-associated DNA appears to be a constant 140-base-pair unit (Noll, 1976; Morris, 1976; Lohret a l . , 1977). Limited digestion of nuclei or chromatin by endogenous nucleases (Hewish and Burgoyne, 1973; Burgoyne et a l . , 1974), micrococcal nuclease (Noll, 1974a; Sollner-Webb and Felsenfeld, 1975; Axel, 1975), or pancreatic DNase (Noll, 1974b) results in preferential

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attack at internucleosome bridges, generating a now-familiar series of DNA fragments that are integral multiples of the basic 190-base-pair repeat. More extensive digestion destroys the noncore (ca. 50 base pairs) nucleosome DNA and cuts the 140-base-pair core DNA into fragments that, when denatured, are found to comprise multiples of a 10-nucleotide repeat. This was first demonstrated by No11 (1974b) using pancreatic DNase and has recently been confirmed with this enzyme by four other groups (Camerini-Otero et al., 1976; Weintraub and Groudine, 1976; Garel and Axel, 1976; Simpson and Whitlock, 1976a). A similar pattern of fragments was also seen with micrococcal nuclease (Axel et al., 1974; Camerini-Otero et al., 1976), DNase I1 (Sollner-Webb et al., 1976), and endogenous nuclease (Simpson and Whitlock, 1976b). The 10-nucleotide spacing of nuclease cuts has been recently analyzed in some detail by SollnerWebb and Felsenfeld (1977). Reconstruction experiments with purified histones demonstrate that H3 and H4 are sufficient to generate a chromatin-like limit digest pattern in DNA (Camerini-Otero et al., 1976). Complexes reconstructed from all four histones are even more similar to native chromatin, as judged both by electron microscopy (Oudet et al., 1975) and by nuclease and protease digest patterns (Sollner-Webb et al., 1976). Several lines of evidence suggest that histone 1 is not part of the core (140 base pairs) nucleosome structure (Baldwin et al., 1975; Chalkley and Hunter, 1975; Bonner and Pollard, 1975; Whitlock and Simpson, 1976; No11 and Kornberg, 1977). This histone may be associated with the 50-base-pair noncore DNA, since its selective removal renders this DNA particularly nuclease-sensitive (Whitlock and Simpson, 1976; No11 and Kornberg, 1977). Finally, recent immunosedimentation experiments suggest that nucleosomes are homogeneous with respect to histone content (Simpson and Bustin, 1976). The localization of nonhistone chromatin proteins in relation to nucleosome structure has not yet been rigorously established. It has been claimed that nonhistone proteins are associated with monomer nucleosomes (Gottesfeld et al., 1975a; Liew and Chan, 1976), but these reports did not unequivocally establish an actual physical linkage as opposed to cosedimentation of nonhistone protein-containing structures with mononucleosomes. A somewhat more convincing case for the existence of nonhistone proteins in nucleosome-like, histone-containing particles has been made by Paul and Malcolm (1976). Some of these nonhistones may be RNA-packaging proteins complexed with nascent heterogeneous nuclear RNA transcripts (Pederson, 1974a,b).

VII. Nucleosomes and Transcription With the foregoing as a backdrop, we now take up the issue of whether the nucleosome motif characteristic of bulk chromatin applies also to the organization of transcribed chromatin regions. As discussed earlier, all histones with the

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possible exception of H1 are observed in biochemical fractions of active chromatin. The possibility that these histones are confined to small amounts of contaminating inactive chromatin is ruled out by the observed histone/DNA ratio of these fractions and also by physical measurements (thermal denaturation, circular dichroism) which show that the DNA of template-active fractions behaves like nucleohistone, not naked DNA. Since active chromatin contains the four histone building blocks of the nucleosome, it follows that these regions must contain either nucleosomes per se or, alternatively, a unique, nonnucleosome type of histone structure. Several recent biochemical and cytological studies demonstrate rather convincingly that the former possibility is the case. One of the first fine-scale probes of the nucleoprotein organization of active chromatin regions was made by Felsenfeld’s group (Axel er al., 1975). These investigators used poly-D-lysine to titrate accessible DNA in reticulocyte chromatin, followed by Pronase digestion to generate protein-free, poly-D-lysine-covered DNA (Pronase attacks only L-stereoisomer peptide linkages). Subsequent digestion with micrococcal nuclease and phenol-chloroform deproteinization yielded an “open” DNA fraction (i.e., representing DNA sufficiently accessible in native chromatin to bind poly-D-lysine). In parallel, a “covered” DNA fraction was prepared by digesting chromatin with nuclease directly, without prior poly-D-lysine titration. After deproteinization, this latter fraction presumably represented DNA that was nuclease-resistant in native chromatin by virtue of its association with (natural) chromatin protein. However, since the actual state of these DNA fractions in native chromatin is not certain, the terms “open” and “covered” DNA are used here merely for descriptive clarity. Annealing experiments with globin cDNA revealed that, while globin sequences were present in both the open and covered DNA fractions, the plateau levels of cDNA annealing for the open DNA fraction were consistently lower, by about 20%. This was not due to a lower total concentration of globin sequences in the open DNA, and further controls demonstrated that the portion of globin sequences missing from open DNA is covalently adjacent to the cDNA-annealing sequences. This means that the sequences missing from open DNA are not complementary to a minor, nonglobin sequence component of the cDNA probe which in chromatin is restricted to covered DNA. The most straightforward interpretations of these results are that (1) there is no region of the globin gene that, at some time in the steady state, is not complexed with chromatin protein and that (2) some specific region of the globin gene (about 20% of the cDNA length) is always complexed with protein. Thus all regions of the globin gene sequences are represented in the covered DNA fraction (but presumably not all of each globin gene), while a constant subset of the globin sequences is absent from the open DNA. These important experiments, published just as the nucleosome model was beginning to crystallize, constituted the first direct evidence that

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regions of a specific active gene contain chromatin proteins in direct contact with DNA. With the nucleosome model at hand, it became possible to ask directly whether or not nucleosome-associated DNA contains actively transcribed sequences. This was first investigated by Lacy and Axel (1975), who used sucrose gradients to purify monomer nucleosomes from rat liver nuclei that had been subjected to a limited digestion with micrococcal nuclease (4% total DNA hydrolysis). It was found that the kinetic complexity of the nucleosome-associated DNA was approximately the same as, and possibly equal to, that of total rat liver nuclear DNA. Moreover, the annealing kinetics of poly (A) -mRNA-derived cDNA were similar when driven by total nuclear DNA or by purified mononucleosome DNA. Finally, globin gene sequences were detected in DNA from purified mononucleosomes of duck reticulocyte chromatin. Very similar results were reported soon thereafter for human lymphocyte chromatin by Saunders’ group (Kuo ef al., 1976) and for Xenopus embryo chromatin by Reeves (1977). It is to be noted that these results imply a nucleosome structure for active chromatin only if it is assumed that all genomic copies of the hybridizable sequences are present in the nucleosome DNA fraction used as driver. Although the DNA sequences analyzed in these experiments (Lacy and Axel, 1975; Kuo et al., 1976; Reeves, 1977) are called “nonrepeated,” this is a purely operational term and the hybridization methodology upon which this nomenclature is based does not, as usually employed, distinguish between sequences present literally once per haploid genome, as opposed to a few times. Moreover, even for a sequence present only once per haploid genome, in diploid cells the copy number is obviously 2 to 4,depending on the cell cycle stage. Therefore the formal possibility still exists that the DNA sequences under analysis in these experiments simply represent the inactive copies of gene sequences that are represented several times (two to four) per cell. According to this view, the gene copies that are actually active in vivo reside in the micrococcal nuclease-hydrolyzed DNA fraction and are therefore not part of the analysis. Nevertheless, their inactive counterparts are present in the nuclease-resistant DNA and can therefore still react with the cDNA probe. Although intuitively this seems unlikely, it has not been formally ruled out. Of course, this problem is far more serious in the case of DNA sequences known to be repeated in the genome, that is, genes that can be reliably demonstrated to reside in the moderately repeated kinetic family of total DNA. It has been shown that at least some of the ribosomal cistrons in Tetrahymena and of the ribosomal and 5s rRNA genes in Xenopus have a nucleosome structure (Mathis and Gorovsky, 1976; Reeves and Jones, 1976). The issue of whether or not these nucleosome-associated ribosomal genes are actually part of the transcriptionaIIy active machinery in viva was addressed further by Reeves (1976). He showed that rDNA is organized in nucleosomes inXenopus tadpoles heterozygous for the +

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anucleolar mutation. Since there is evidence that these animals may rely on the transcription of their entire complement of ribosomal genes to support embryonic development (Knowland and Miller, 1970), these results tend to reduce considerably the possibility that the apparent nucleosome-associated form of rDNA merely represents an inactive subset of the repeated ribosomal genes.

VIII. Morphology of Active Transcription Units Recent ultrastructural studies of dispersed chromatin fibers and their nascent RNP have begun to form a cytological perspective for the biochemical investigations discussed above. To begin with, there is now general agreement that active ribosomal genes do not contain beaded structures analogous to nucleosomes (Woodcock et al., 1976; Laird and Chooi, 1976; Laird et al., 1976; Foe et al., 1976), at least under the conditions used to disperse, fix, and stain the nucleolar chromatin for these studies. This result contrasts sharply with the biochemical evidence (Mathis and Gorovsky, 1976; Reeves and Jones, 1976), indicating that the ribosomal genes are organized into the familiar pattern of alternating nuclease-sensitive and nuclease-resistant DNA that is the hallmark of nucleosome structure. A related finding is that the DNA in active ribosomal chromatin does not appear to be appreciably foreshortened, in contrast to the higher chromatin packing ratio of DNA in inactive regions. It has been estimated that there are only 1.2 p m of B-conformation DNA helix per micron of active ribosomal chromatin in embryos of the milkweed bug Oncopeltus fasciatus, as compared to 2.3 p m of DNA per micron of inactive chromatin in the same organism (Foe et al., 1976). Similar packing values have been estimated for active ribosomal gene transcription units in Drosophila embryos (Laird et al., 1976) and Xenopus oocyte chromatin (Scheer et al., 1977). It is most unlikely that a nonshortened B-conformation DNA structure in active ribosomal chromatin could coexist with nucleosome organization as we generally know it. However, it seems perfectly possible that histones may be present on transcribed rDNA but in a sufficiently modified form (e.g., by acetylation) that their mutual protein-protein associations are no longer recognizable ultrastructurally as nucleosomes. This interpretation receives support from the nucleohistone-like appearance of DNA underlying nascent ribosomal RNP matrixes in negatively stained spreads of newt chromatin (Woodcock et al., 1976), as well as from the biochemical observation that histones are present in purified nucleolar chromatin fractions (Higashenakagawa et al., 1977). If these histones were appropriately disposed along the active D N A , they could still conceivably generate the characteristic nuclease digestion pattern. A precedent for this is the case of adenovims DNA-histone complexes which, like active ribosomal chromatin, show a nucleosome-like nuclease digestion pattern (Cordon et al., 1976) and yet have a nonbeaded ultrastructural

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appearance (Miller and Hodge, 1975). Taken together, these observations suggest that the histone-histone interactions which constitute the cornerstones of normal nucleosome structure are in fact rather dynamic. In some cases (e.g., active ribosomal genes) it seems that nucleosome structure can be so significantly perturbed during transcription as to eliminate the typical beaded morphology detected by electron microscopy, but without disrupting the specific DNAhistone contacts that generate the characteristic repeating series of nuclease digestion products. Recently developed methods for mass isolation of nucleolar chromatin (Higashenakagawa et al., 1977) should facilitate the analysis of histone-DNA associations in and around active ribosomal genes. In contrast to reports of a nonbeaded ultrastructural appearance of active ribosomal chromatin, studies by Laird and co-workers reveal that nonribosomal transcription units in both Oncopeltus and Drosophila embryo chromatin have a definite beaded morphology (Laird and Chooi, 1976; Laird et al., 1976; Foe et al., 1976). Both the size and staining texture of these beads is the same as those seen throughout the nascent RNP-free (i.e., inactive) regions of chromatin, reinforcing their interpretation as nucleosomes (Laird et al., 1976). The DNA/ chromatin packing ratio of beaded nonribosomal transcription units in Oncopeltus is estimated to be 1.6-1.9 p m of B-conformation DNA per micron of chromatin, as compared to a compaction value of 2.3 for beaded inactive regions (Foe et al., 1976). It therefore seems probable that some longitudinal expansion of the usual (i.e., inactive) DNA folding pattern occurs during the transcription of nonribosomal genes. The nearly complete unfolding of active rDNA (Laird and Chooi, 1976; Foe et al., 1976) may simply represent an extreme case, possibly due to the intense rate of RNA polymerase initiation at these particular loci (McKnight and Miller, 1976).

IX. Evidence for an Alteration of Nucleosome Structure during Transcription Now we have come full circle and can logically take up the issue of whether or not the demonstrated nucleosome organization of (nonribosomal) active chromatin loci is in any way altered in comparison to that of the remainder of the genome. For purposes of historical perspective, it is instructive to develop this theme from the standpoint of the use of three rather different enzyme probes, micrococcal nuclease, spleen DNase, and pancreatic DNase. Clark and Felsenfeld (197 1) reported that micrococcal nuclease digests chromatin to a limit of 50% of the total DNA, measured by acid solubility. Mirsky (1971) criticized these experiments on the grounds that the limit digest value of 50% might only be apparent because of insufficient enzyme concentration or reaction time. However, his experiments employed pancreatic DNase, not

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micrococcal nuclease. Pederson ( 1972) showed that the digestion of chromatin DNA by pancreatic DNase proceeds to essentially 100% acid-soluble nucleotides in a reaction dependent upon the product of enzyme concentration and time. However, unlike Mirsky, Pederson (1972) emphasized that the pancreatic DNase results were not necessarily at variance with the micrococcal nuclease data of Clark and Felsenfeld and further suggested that specific regions of the DNA may be attacked at different stages in the pancreatic DNase digestion process. Subsequent experiments with micrococcal nuclease (Clark and Felsenfeld, 1974; Schmidt et al., 1975) have demonstrated that Clark and Felsenfeld's original conclusion was correct; the 50% digestion of chromatin DNA by this nuclease represents a true limit. At this point it became apparent that, of the two enzymes, micrococcal nuclease was the more interesting and also presumably the more pragmatic as a probe of chromatin structure. As is evident from the previous section, micrococcal nuclease has indeed been the key to identifying and to some extent dissecting the nucleosome organization of chromatin. However, this nuclease, as it is presently employed, does not display a preference for active chromatin regions (Axel et al., 1973). Pancreatic DNase and spleen DNase are different. As discussed earlier, spleen DNase (DNase 11), when used under suboptimal conditions (pH 6.5), generates double-strand cuts preferentially in or near active chromatin loci (Gottesfeld et al., 1974a; Pederson and Bhorjee, 1975). This reaction is most properly envisioned as site-selective shearing, since only 2% total DNA hydrolysis (measured as acid-soluble nucleotides) is required to shear out the template-active chromatin fragments (Pederson and Bhorjee, 1975). The fact that the inactive chromatin retains the solubility and sedimentation properties of intact chromatin implies that these regions are cut much less frequently, or not at all, during the limited DNase I1 digestion. DNase I1 apparently recognizes some specific aspect of active chromatin structure, even though these sites can be shown to have a nucleosome configuration (Gottesfeld et al., 1975a). More recently, a third pattern of chromatin attack, different from that of either micrococcal nuclease or DNase 11, has been observed with pancreatic DNase. Although this enzyme hydrolytically attacks total chromatin DNA progressively to a 95-100% limit digest (Mirsky, 1971; Pederson, 1972), Weintraub and Groudine (1976) have recently shown that a very brief digestion of chick erythrocyte nuclei with pancreatic DNase (10-20% of the total DNA digested) selectively attacks globin gene sequences,as measured by a reduction in the concentration of nuclease-resistant DNA sequences hybridizing with a cDNA constructed from globin mRNA. Essentially similar results have been reported by Garel and Axel (1976) for ovalbumin gene sequences in the hen oviduct. However, since the cDNA probes used in both of these studies were complementary to abundant mRNAs coding for specialized cell products, the possibility exists that the demonstrated pancreatic DNase sensitivities of globin and ovalbumin

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gene sequences represent special cases and may not be a general feature of active chromatin. This is unlikely in light of the similar pancreatic DNase sensitivity of chicken red blood cell chromatin sequences complementary to heterogeneous nuclear RNA, which obviously represents a highly complex population of gene transcripts (Weintraub and Groudine, 1976). This result with nuclear RNA also shows that the pancreatic DNase sensitivity of active chromatin includes both the coding as well as noncoding (cDNA-hybridizing) strand of transcribed DNA sequences. Moreover, Weintraub and Groudine also examined chromosomally integrated copies of avian myeloblastosis viral DNA sequences, which are transcribed only at very low rates in these cells. Like the chromatin DNA sequences complementary to globin cDNA or to nuclear RNA, these endogenous viral sequences were also found to be selectively destroyed by pancreatic DNase. A definitive demonstration that DNase I-sensitivity is a general feature of active chromatin and not one restricted to genes coding for high abundance mRNAs has been made recently by Garel et al. (1977). They have shown that the chromatin DNA sequences coding for the rarest mRNAs in the chicken oviduct are pancreatic DNase sensitive and, in fact, their results imply that this chromatin configuration is retained by gene sequences that are copied by RNA polymerase at rates as low as a few times per day. Another demonstration that pancreatic DNase sensitivity is a general feature of active chromatin is the work of Levy W. and Dixon (1977). Using a kinetically fractionated cDNA complementary primarily to mRNAs present in low intracellular concentration (i.e., the high-complexity mRNA class), they showed that trout testis chromatin sequences complementary to this cDNA were selectively destroyed by pancreatic DNase. In contrast, using a fractionated cDNA preparation complementary to the more abundant polyadenylated testis mRNAs, they found that the chromatin DNA sequences complementary to this cDNA were not preferentially attacked. This appears to disagree with the results for globin and ovalbumin sequences (Weintraub and Groudine, 1976; Garel and Axel, 1976). It is possible that in the trout testis system the more abundant mRNAs are not being actively transcribed at the stage of spermatogenesis at which the chromatin is probed with DNase, although the results of Weintraub and Groudine show that globin DNA sequences retain their pancreatic DNase sensitivity even in the mature erythrocyte, after globin mRNA transcription has demonstrably stopped. This is a highly significant issue, and further work is needed to clarify the basis of these differences. It seems likely that the selectivity of DNase I for active versus inactive chromatin will turn out to be somewhat less straightforward than first thought. Nevertheless, the data published so far, particularly those of Garel et al. (1977), permit the conclusion that, if not all, at least a large number of different active gene sequences in a given genome are organized in a chromatin structure that is particularly sensitive to pancreatic DNase attack. Taken together with the above-summarized biochemical and ultrastructural evidence that at least nonribosomal transcription units

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retain a nucleosome structure, the pancreatic DNase results strongly suggest that the nucleosome organization of active chromatin is in some way altered relative to that of inactive regions.

X. Contemporary Research on the Structure of Active Chromatin Two conclusions can be drawn with reasonable certainty. First, many transcribed chromatin loci, or at least substantial portions of each of them, are organized into nucleosome-like structures. Second, it now seems clear that the nucleosomes in these active regions have a significantly altered structure, and that their associated DNA is more susceptible to attack by either spleen DNase (shearing) or pancreatic DNase (hydrolysis) than in nontranscribed chromatin. Any view of the possible differences between nucleosome structure at active and inactive chromatin loci is limited by our presently incomplete understanding of nucleosome structure per se. The current view of the (inactive) nucleosome is based primarily on cross-linking and enzymatic digestion studies of native versus reconstituted chromatin, and on the thermodynamics of homotypic and heterotypic histone-histone interactions in solution (Kelley, 1973; D’Anna and Isenberg, 1973, 1974a,b; Kornberg and Thomas, 1974; Roark et al., 1974; Rubin and Moudrianakis, 1975; Weintraub et al., 1975; Thomas and Komberg, 1975; Baldwin et al., 1975; Chalkley and Hunter, 1975; Camerini-Otero et al., 1976; Sollner-Webb et al., 1976; Martinson and McCarthy, 1976). Similar studies with template-active chromatin fractions and their extracted histones would now be of great value in defining more precisely the ways in which nucleosome structure is modified during transcription. It could be that nucleosome structure is altered to varying extents at different active loci depending, for example, on the rate of RNA polymerase initiation. In some cases nucleosomes may merely “breathe” transiently about their dyad symmetry axis to accommodate local DNA strand separation and polymerase read-through, as suggested in two recent speculations (Weintraub et al., 1976; Lilley et al., 1977). In other instances, where transcription is more intense and polymerase loading is correspondingly high (e.g., in the ribosomal genes), the steady-state nucleosome structure might be more substantially disrupted. This consideration suggests that the analysis of how nucleosome structure is altered during transcription using total template-active chromatin fractions might be compromised by a high degree of heterogeneity. Another particularly important question is whether or not the electrostatic bonds between the DNA phosphate-sugar backbone and the relatively polar N-terminal regions of the histones are appreciably disrupted during transcription. Intuitively, this seems not to be necessary, and perhaps a more realistic possibility is that during transcription there is a transient relaxation of the histone-histone associations in the core of the nucleosome itself, which presum-

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ably involve the more nonpolar C-terminal domains of the component histones. Transient changes in the levels of posttranslational histone modifications such as acetylation and phosphorylation might play important roles here. These aspects of histone metabolism have been circumstantially linked to transcriptional activity, and one initial study using template-active chromatin suggests that histone acetylation and phosphorylation may indeed be slightly elevated during transcription (Levy-Wilson et al., 1977). However, it should be emphasized that, in addition to possibly modifying the structure of the nucleosome itself, histone acetylation and phosphorylation may actually have more to do with higher orders of chromatin folding and packing, as exemplified by chromosome coiling during mitosis (Gurley et al., 1974; Bradbury et al., 1974a,b) or the progressive condensation of chromatin that occurs during avian erythroid cell maturation (Ruiz-Canillo et al., 1975). An important related issue is the apparent absence of histone 1 from active chromatin regions, which was discussed earlier. If this is confirmed, it would then be of great interest to determine whether or not the structure of the nucleosome core itself is relaxed when histone 1 is deliberately removed from the flanking DNA. The localization in active versus inactive chromatin of special proteins such as the so-called high-mobility group of nonhistones (Goodwin et al., 1973) also appears to be a potentially incisive avenue of investigation. The presence in active chromatin of proteins that influence DNA structure (e.g., supercoiling) is another area of escalating interest at the present time, and initial results suggest that this approach may be profitable (Higashenakagawa et al., 1977). The analytical methodology needed to explore all these issues is currently available. The limiting factor is simply the availability of active gene chromatin of high purity. It can be anticipated that a prominent component of future research will be efforts to isolate chromatin fractions containing the active nucleoprotein forms of pure genes (such as those coding for histones, P-globin, ovalbumin, or rRNA). This will obviously permit the structure of active chromatin to be analyzed more cleanly than with currently available template-active fractions of total chromatin.

XI. Conclusion It is a remarkable fact that, despite three decades of research on chromatin composition and metabolism, starting with Mirsky and Pollister (1946), its underlying unit structure has been resolved only in the past 3 years. This is a striking example of the often saltatory nature of scientific progress, which has also been stressed by several historians of science, most notably Kuhn (1962). The recentness of the nucleosome’s emergence notwithstanding, it has already proven to be a highly catalytic perspective from which to investigate how

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chromatin structural changes determine or accompany transcription. After many years of sluggish progress, it is probable that our understanding of the relationship between chromatin structure and transcription will now advance quite rapidly, in the postnucleosome era of chromosome research.

ACKNOWLEDGMENTS Research cited from this laboratory was supported by NIH grants GM 21595 and CA 13400. 1 thank Jim Calvet, Bill Crain, and Jay Greenberg for critically reading the manuscript. Important suggestions were also made by Hans Ris and Pat Williamson. The advice of Gary Felsenfeld was especially helpful and is gratefully acknowledged.

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Gottesfeld, J. M., Garrard, W. T., Bagi, G., Wilson, R. F., and Bonner, J. (1974a). Proc. Natl. Acad. Sci. U.S.A. 71, 2193. Gottesfeld, J. M., Bonner, J., Radda, G. K., and Walker, I. 0. (1974b). Biochemistry 13, 2937. Gottesfeld, J. M., Murphy, R. F., and Bonner, J. (1975a). Proc. Natl. Acad. Sci. U.S.A. 72, 4404. Gottesfeld, J. M., Kent, D., Ross, M., and Bonner, J. (1975b). In “Florida Colloquium on Molecular Biology” (G. Stein and J. Stein, eds.), p. 227. Academic Press, New York. Gottesfeld, J. M., Bagi, G., Berg, B., and Bonner, J. (1976). Biochemistry 15, 2472. Gottesfeld, J. M., and Partington, G. A. (1977). Cell 12, 953. Grouse, L., Chilton, M. D., and McCarthy, B. J. (1972). Biochemistry 11, 798. Gurley, L. R., Walters, R. A,, and Tobey, R. A. (1974). J. Cell Biol. 60, 356. Hahn, W. E., and Laird, C. D. (1971). Science 173, 158. Hamkalo, B. A,, Miller, 0. L., Jr., and Bakken, A. H. (1974). Cold Spring Harbor Symp. Quunt. Biol. 38, 915. Heitz. E. (1928). Juhrb. W i n . Bot. 69, 762. Hewish, D. R., and Burgoyne, L. A. (1973). Biochem. Biophys. Res. Commun. 52, 504. Higashenakagawa, T., Wahn, H., and Reeder, R. H. (1977). Dev. Biol. 55, 374. Holmes, D. S., and Bonner, J. (1974). Biochemistry 13, 841. Izawa, M., Allfrey, V. G., and Mirsky, A. E. (1963). Proc. Natl. Acad. Sci. U.S.A. 49, 544. Jamrich, M., Greenleaf, A. L., and Bautz, E. K. F. (1977). Proc. Natl. Acad. Sci. U.S.A. 74, 2079. Janowski, M., Nasser, D. S . , and McCarthy, B. J. (1972). Gene Transcription Reprod. Tissue, Trans. Karolinska Symp. Res. Methods Reprod. Endocrinol., 5th, 1972, p. 112. Kelley, R. I. (1973). Biochem. Biophys. Res. Commun. 54, 1588. Knowland, J., and Miller, L. (1970). J. Mol. Biol. 53, 321. Kohne, D. E., and Byers, M. J. (1973). Biochemistry 12, 2373. Kornberg, R. D. (1974). Science 184, 868. Kornberg, R. D., and Thomas, J. 0. (1974). Science 184, 865. Kuhn, T. A. (1962). “The Structure of Scientific Revolutions.” Univ. of Chicago Press, Chicago, Illinois. Kuo, M. T., Sahasrabuddhe, C. G., and Saunders, G. F. (1976). Proc. Natl. Acad. Sci. U.S.A. 73, 1572. Lacy, E., and Axel, R. (1975). Proc. Natl. Acad. Sci. U.S.A. 72, 3978. Laird, C. D., and Chooi, W. Y. (1976). Chromosoma 58, 193. Laird, C. D., Wilkinson, L. E., Foe, V. E., and Chooi, W. Y. (1976). Chromosomu 58, 169. Levy W., B., and Dixon, G. H. (1977). Nucleic Acids Res. 4, 883. Levy-Wilson, B., Gjerset, R. A., and McCarthy, B. J. (1977). Biochim. Biophys. Acta 475, 168. Liew, C. C., and Chan, P. K. (1976). Proc. Natl. Acad. Sci. U.S.A. 73, 3458. Lilley, D. M. J., Pardon, J. F., and Richards, B. M. (1977). Biochemistry 16, 2853. Littau, V. C., Allfrey, V. G., Frenster, J . H., and Mirsky, A. E. (1964). Proc. Nurl. Acad. Sci. U.S.A. 52, 93. Lohr, D., Corden, J., Tatchell, K., Kovacic, R. T., and Van Holde, K. E. (1977).Proc. Nurl. Acud. Sci. U.S.A. 74, 79. McConaughy, B. L., and McCarthy, B. J. (1972). Biochemistry 11, 998. McKnight, S. L., and Miller, 0. L., Jr. (1976). Celf 8, 305. Martinson, H. G., and McCarthy, B. J . (1976). Biochemistry 15, 4126. Marushige, K., and Bonner, J. (1971). Proc. Nail. Acad. Sci. U.S.A. 68, 2941. Mathis, D. J., and Gorovsky, M. A. (1976). Biochemistry 15, 750. Miller, 0. L., Jr., and Hodge, L. D. (1975). J. Cell B i d . 67, 284a. Mirsky, A. E. (1971). Proc. Null. Acad. Sci. U.S.A. 68, 2945. Mirsky, A. E., and Pollister, A. W. (1946). J. Gen. Physiol. 30, 117.

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Moms, N. R. (1976). Cell 8, 357. Murphy, E. C., Hall, S. H., Shepherd, J . H., and Weiser, R. S. (1973). Biochemistry 12, 3843. Noll, M. (1974a). Nature (London) 251, 249. NOH, M. (1974b). Nucleic Acids Res. 1 , 1573. Noll, M. (1976). Cell 8, 349. Noll, M., and Kornberg, R. D. (1977). J . Mol. Biol. 109, 393. Ohlenbusch, H. H.,Olivera, B. M., Tuan, D., and Davidson, N. (1967). J . Mol. Biol. 25, 299. Oudet, P., Gross-Bellard, M., and Chambon, P. (1975). Cell 4, 281. Pages, M., and Alonso, C. (1976). Exp. Cell Res. 98, 120. Paul, J . , and Malcolm, S. (1976). Biochemistry 15, 3510. Pederson, T. (1972). Proc. Narl. Acad. Sci. U.S.A. 69, 2224. Pederson, T. (1974a). J . Mol. Biol. 83, 163. Pederson, T. (1974b). Proc. Natl. Acad. Sci. U . S . A . 71, 617. Pederson, T., and Bhorjee, J . S. (1975). Biochemisfry 14, 3238. Polacow, I., and Simpson, R. T. (1973). Biochem. Biophys. Res. Commun. 52, 202. Reeck, G . R., Simpson, R. T., and Sober, H. A. (1972). Proc. Nurl. Acad. Sci. U . S . A . 69, 2317. Reeves, R. (1976). Science 194, 529. Reeves, R. (1977). Eur. J. Biochem. 75, 545. Reeves, R., and Jones A. (1976). Nature (London) 260, 495. Renz, M., Nehls, P., and Hozier, J. (1977). Proc. Nafl. Acad. Sci. U . S . A . 74, 1879. Roark, D. E., Geoghegan, T. E., and Keller, G. H. (1974). Biochem. Biophys. Res. Commun. 59, 542. Rubin, R. L., and Moudrianakis, E. N. (1975). Biochemistry 14, 1718. Rudkin, G . T. (1962). Ann. Hisrochim., Suppl. 2 , 77. Ruiz-Canillo, A., Wangh, L. J., and Allfrey, V. G. (1975). Science 190, 117. Scheer, V., Trendelberg, M. F., Krohne, G., and Franke, W. W. (1977). Chromosomu 60, 147. Schmidt, G., Cohen, M. P., and DeMarco, P. (1975). Mol. Cell. Biochem. 6, 185. Seidman, M. J., and Cole, R. D. (1977). J. Biol. Chem. 252, 2630. Simpson, R. T. (1974). Proc. Narl. Acad. Sci. U . S . A . 71, 2740. Simpson, R. T., and Bustin, M. (1976). Biochemisfry 15, 4305. Simpson, R. T., and Reeck, G. R. (1973). Biochemistry 12, 3853. Simpson, R. T., and Whitlock, J. P., Jr. (1976a). Cell 9, 347. Simpson, R. T., and Whitlock, J . P., Jr. (1976b). Nucleic Acids Res. 3, 117. Sollner-Webb, B., and Felsenfeld, G. (1975). Biochemisfry 14, 2915. Sollner-Webb, B., and Felsenfeld, G. (1977). Cell 10, 537. Sollner-Webb, B., Camerini-Otero, R. D., and Felsenfeld, G. (1976). Cell 9, 179. Thomas, J . O., and Kornberg, R. D. (1975). Proc. Nutl. Acad. Sci. U . S . A . 72, 2626. Turner, G., and Hancock, R. (1974). Biochem. Biophys. Res. Commun. 58, 437. Wallace, R. B., Dube, S. K., and Bonner, J . (1977). Science 198, 1166. Weintraub, H.,and Groudine, M. (1976). Science 193, 848. Weintraub, H., Potter, K., and Van Lente, F. (1975). Cell 6, 85. Weintraub, H., Worcel, A., and Alberts, B. (1976). Cell 9, 409. Whitlock, J. P., J r . , and Simpson, R. T. (1976). Biochemisfry 15, 3307. Woodcock, C. L. F., Frado, L.-L.Y., Hatch, C. L., and Ricciardiello, L. (1976) Chromosoma 58, 33. Yasmineh, W. G., and Yunis, J . J . (1970). Exp. CellRes. 59, 69. Zhimulev, I. F., and Belyaeva, E. S . (1975). Chromosoma 49, 219.

INTERNATIONAL REVIEW OF CYTOLOGY,VOL. 55

The Isolated Mitotic Apparatus and Chromosome Motion H.SAKAI Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo. Hongo, Bunkyo-ku, Tokyo, /upcin

I. Introduction . . . . . . . . . . . . . . . . . . . 11. Egg Tubulin . . . . . . . . . . . . . . . . . . . . A. Tubulin Pool in the Egg . . . . . . . . . . . . . B. Assembly of Egg Tubulin into MTs . . . . . . . . . C. Regulation of MT Assembly in Yivo . . . . . . . . . 111. The Isolated MA . . . . . . . . . . . . . . . . . A. Isolation . . . . . . . . . . . . . . . . . . . . B. MTsandTubulin . . . . . . . . . . . . . . . . C.ATPases. . . . . . . . . . . . . . . . . . . D. Response to Heterologous Tubulin . . . . . . . . . IV. Mitosis and the Roles of MTs . . . . . . . . . . . . A. Anaphase Chromosome Movement . . . . . . . . . B. Induction of Chromosome Motion in the Isolated MA . . C. Actin and Myosin in Mitosis . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

. .

.

. .

. .

. .

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23 24 24 26 29 31 31 33 36 38 39 39 40 43 44

I. Introduction Formation of the mitotic apparatus (MA) and of the contractile ring are two important events in cell division in the sea urchin egg. The MA consists of a framework of microtubules (MTs) functioning in the mitotic movement of daughter chromosomes, and the contractile ring may be a device for cleavage. The latter process is not independent of the former, but a sequentially coordinated event to accomplish cell division. Recent advances in studies on the structure and function of MTs and contractile systems in nonmuscle cells have provided insights into molecular events taking place in the mitotic process and cleavage of the sea urchin egg. New knowledge based on electron microscopy, biochemistry, and biophysical studies on MTs has resulted in additional concepts of cell motilitflhis article discusses the possible involvement of MTs, tubulin, and dynein in mitosis. \

23 Copyright 0 1978 by Academic Press, Inc. All nghts of reproduction in any form reserved ISBN 0-12-364355-4

24

H.SAKAI

11. Egg Tubulin

A. TUBULINPOOL IN THE EGG The sea urchin egg contains tubulin as 0.37% of the total protein (Arbacia punctulata, Raff and Kaumeyer, 1973). If we assume that a tubulin pool of the same size is found in the Hemicentrotus egg, 0.25 ng of tubulin is present, calculating from the amount of total protein. In our measurements, theHernicentrotus egg contained 68 ng of protein. Since the average egg diameter is 94 p m and the volume of the egg is 4.35 X lo5pm3, the concentration of protein is 157 mg/ml. Based on an average diameter of 74pm (Harvey, 1932), the total protein of an Arbacia egg (33 ng/egg) obtained by Raff and Kaumeyer (1973) gives a total protein concentration of 156 mg/ml. This value agrees precisely with that obtained for the Hemicentrotus egg. Since 26.4 pg of tubulin is contained in the spindle at first-division metaphase (see Section III,B), the amount of tubulin incorporated into the spindle at metaphase accounts for 10.6%of the total egg tubulin. There is evidence that an unfertilized sea urchin egg contains at least three to five times the amount of tubulin polymerized into MTs of the first-division metaphase spindle (Raff and Kaumeyer, 1973). By counting the MTs in sections of the MA of A . punctulata, Cohen and Rebhun (1970) calculated that the MTs constitute 0.067%of the total egg protein (0.1% if astral rays are included in the calculation). Since the volume of the metaphase spindle in the first cell division is 2.1 X lo3p m 3 , if we assume that it is an ellipsoid of revolution and the cell volume is 4.35 X lo5 p m 3 , the volume ratio of spindle to egg is 0.5%.That 10.6%of the total egg tubulin is localized in the spindle means that tubulin is concentrated in the MA at 20 times the concentration in the cytoplasm. Quantitation of the tubulin pool during development was reported by several investigators, all of whom demonstrated that the pool remained relatively constant through gastrulation. The total pool size ranges from 0.4% (Drosophila, Green et al., 1975) to 0.4-1.7% (sea urchin, Raff and Kaumeyer, 1973; Raff et al., 1975; Pfeffer et al., 1976) of the total protein. In Spisula embryos, a higher value, 3.3%, was reported using vinblastine-induced precipitation of tubulin (Burnside et al., 1973), and in Urechis mature eggs values of 0.8 and 5% were given as minimum and maximum estimates, respectively (Miller, 1973). Raff (1977) reported that axolotl egg soluble tubulin, 0.27% of the total protein, remained constant through early development; this a little less than the concentration in sea urchin embryos. A large value for the tubulin pool (2.7 mg/ml), determined by Scatchard plots of the colchicine-binding reactions, was reported by Pfeffer et al. ( 1 976) for Strongylocentrotus purpuratus eggs. The glycerol-magnesium-Triton method can be used to isolate spindles at any early developmental stage (Fig. 1). The size of the spindles decreases, accom-

25

ISOLATED MITOTIC APPARATUS AND CHROMOSOME MOTION 1

2

3

4

7

8

FIG.1. Spindles isolated from sea urchin embryos at cleavage stages. The figure on each photograph indicates the number of cell divisions. Spindles from micromere-derived blastomeres are not shown.

Log N FIG.2. Change in spindle volume during the cleavage stage as a function of the number of

blastomeres. The ratio of the long axis to the short axis of isolated spindles ranged from 1.4 to 1.8 for Hemicentrotus and 1.8 to 2.2 forhthocidaris, independent of the spindle size. The spindle was assumed to be an ellipsoid of revolution. The straight line represents log (IIN”’) as a function of log N (number of blastorneres). V,, Volume of blastomere spindle; VO,volume of spindle at the first cell division.

26

H. SAKAI

panied by a decrease'in the diameter of the blastomeres. Although proliferating cells are clearly shown to synthesize tubulin periodically during cell cycles (Pipermo and Luck, 1977), the sea urchin egg or embryo synthesizes only a very small amount during the cleavage stage, and the tubulin pool remains constant. Figure 2 shows that the change in the spindle volume V , is proportional to the reciprocal of the square root of the total number of blastomeres N ; V , = Vo/(N)112, where V , is the spindle volume in the first cell division. This relationship holds tme at least until the blastula stage. This also indicates that the sum of the spindle volumes Vt of all the blastomeres in an embryo is proportional to the square root of the number of blastomeres; Vt = V , ( N ) l M . This equation gives a simple estimate of when the tubulin reservoir will fully take part in MT assembly for spindle formation, assuming that the concentration of tubulin in the spindle is constant no matter how small the spindle becomes after repeated cleavage. As described above, the metaphase spindle in the first cell division contains 10.6% of the total tubulin. Therefore the embryo exhausts the tubulin pool after the sixth division. This may account for the fact that the asters are no longer clearly visible after the fifth division of the sea urchin embryo. However, if we take the tubulin pool size of 2.7 mg/ml determined by Pfeffer et al. (1976), the tubulin pool will be sufficient even after 10 divisions. Whether or not the tubulin content of the spindle changes during development remains to be determined.

B. ASSEMBLY OF EGG TUBULIN INTO MTs Sea urchin egg tubulin has a relatively low affinity for colchicine. Bryan ( 1972) determined the maximum equilibrium constant for the colchicine-binding

reaction in vinblastine crystals isolated from S. purpurutus eggs. The constant was about 10 times lower than that obtained for brain tubulin from chick embryos. Similar binding constants were obtained for vinblastine crystals and soluble tubulin from the same species of sea urchin by Pfeffer et d.(1976.) Attempts to polymerize tubulin in egg extracts has not succeeded because of the low amount of tubulin and the presence of inhibitory material (Bryan et al., 1975) in marine eggs. However, Bums and Starling (1974) presented electron microscope evidence that sea urchin egg MTs were formed when short pieces of rat brain MTs were added as polymerization seeds to the soluble supernatant of egg homogenates. Weisenberg and Rosenfeld (1975) also showed an aster-like assembly of MTs using homogenates of surf clam eggs. Recently, Kuriyama (1977) purified egg tubulin from the sea urchin and starfish. The tubulin preparation was shown to consist of 6.3 S components having a molecular weight of 110,000 as determined by gel filtration. The egg tubulin dimer possesses activity to bind 0.8 mole of colchicine and 0.8-0.97 mole of exogenous GTP at the exchangeable site. Egg tubulin consists of a and /3 subunits with mobilities identical to those of porcine brain tubulin and outer fiber

27

ISOLATED MITOTIC APPARATUS AND CHROMOSOME MOTION

tubulin of sea urchin sperm flagella. The egg tubulin fraction obtained by DEAE-Sephadex A-50 column chromatography (0.2% of the total soluble protein of the egg) polymerizes into MTs upon warming at 35°C. The MTs pelleted by centrifugation are depolymerized by cooling. After centrifugation at 100,000 g for 45 minutes, the supernatant contains purified tubulin, but no highmolecular-weight component (MT-associated proteins) or tau protein. The viscosity of this fraction increases upon warming, showing the formation of numerous MTs without MT-associated proteins. Furthermore, the presence of 0.4 M potassium chloride still supports MT assembly at half the level attained under standard conditions. The assembled MTs are susceptible to low temperature, calcium ions, sulfhydryl-blocking reagents, and colchicine (Fig. 3). Although egg tubulin at a low concentration polymerizes only to a certain extent, the addition of nuclei fractions for polymerization (outer fiber fragments, brain MT fragments, high-molecular-weight components, or tau protein) greatly favors an increase in the rate of MT assembly (Fig. 4).

0.3 0

0.2

6?

0.1

Incubation time (min) 3. Sensitivity of starfish egg MTs to colchicine, cold, p-chloromercuriphenyl sulfonate (PCMPS) and calcium ions. (A) Depolymerization by colchicine. The first and second arrows indicate the addition of 1mM colchicine to final concentrations of 30 and 60 pM, respectively. Protein concentration was 16 pM as tubulin dimer. The dotted area represents the time of cold treatment. (B) Depolymerization by cold treatment and by PCMPS. The first arrow indicates the addition of potassium chloride to a final concentration of 0.05 M. The second and third arrows indicate the addition of PCMPS, causing a blockage of 1.6 moles of sulfhydryl groups per 55,000 gm of protein. (C) Depolymerization by calcium ions. The first arrow indicates the addition of potassium chloride. Calcium ions were added at the second arrow to a final concentration of 1.6 mM (Kuriyama, 1977). FIG.

28

H.SAKAI

0.6

0.4

0.2

P 0.6

04

0.2

Incubation time (min) FIG.4. Stimulation of egg MT assembly by nucleating fractions. (A) Flow-through fraction of porcine brain tubulin on Sephadex G-200. (B)0.35 M potassium chloride batch eluate (containing HMW)of porcine brain tubulin on DEAE-Sephadex A-50. (C) Outer fiber fraction from Tetruhyrnena cilia. (D) Partially purified brain tubulin fraction. (E) Tau protein. (F)Short pieces of porcine brain MTs. a, Hemicentrotus egg tubulin ( I .6 mg/ml) alone; b, egg tubulin plus each nucleating fraction; c, nucleating fraction alone. Dotted areas represent time of cold treatment (Kuriyama, 1977).

More recently, evidence was presented that tau protein might be contained in and associated with spindles of mouse fibroblasts. This was shown using anti-tau protein serum prepared in rabbits by the indirect immunofluorescence technique. The staining image was identical to that observed when cells were treated with antitubulin serum (Connolly et al., 1977). The significance of this taulike protein is not clear, because egg tubulin is highly competent to assemble into MTs without MT-associated proteins. Studies on the fine structure of the mitotic spindle have revealed that the fibrous element of the spindle is the microtubule. Polarization optical studies on living cells have demonstrated that the MTs of mitotic spindles are in dynamic equilibrium with tubulin dimers (InouC and Ritter, 1975; InouC et al., 1975). Furthermore, the application of Wiener’s equation to spindle birefringence demonstrated the entirety of the birefringence to be derived from spindle MTs (Sato et al., 1975). The Sluder’s experiment (1976) explains how the tubulin pool determines the size of the spindle formed in the sea urchin egg. A decrease in spindle length can be induced by pulse treatment of the sea urchin egg with Colcemid. Ultraviolet light irradiation causes the recovery of tubulin competent

ISOLATED MITOTIC APPARATUS AND CHROMOSOME MOTION

29

to polymerize from the tubulin-Colcemid complex, and the small spindle elongates quickly to the original length. Similar to their in vivo behavior, isolated MAS increase in size on the addition of heterologous tubulin (Rebhun et al., 1974; Inout et al., 1974; Sakai et al., 1975). C. REGULATION OF MT ASSEMBLY in Vivo One of the important problems in mitosis concerns the mechanism by which cyclic formation and breakdown of mitotic MTs are regulated, which is poorly understood. After syngamy is completed in the fertilized sea urchin egg, a monaster develops around the nucleus through the growth of MTs. The centrioles separate to locate on opposite sides of the nucleus. The breakdown of the nuclear envelope is followed by formation of the spindle. Since even an unfertilized sea urchin egg contains tubulin competent to self-assemble into MTs, some form of regulation must suppress polymerization until the MA begins to form. The tubulin pool is 0.6 mg/ml, as shown by Raff and Kaumeyer (1973). If we take this value to be applicable to the Hemicentrotus egg, this concentration of tubulin initiates selfassembly to only a small extent, as judged from the observation that viscosity increases only slightly at an egg tubulin concentration of 1 mg/ml, the critical concentration of egg tubulin (0.15 mg/ml) for polymerization being lower than that of brain tubulin (0.2-0.3 mdml). However, after the appearance of pericentriolar satellites (Brinkley and Stabblefield, 1970) or pericentriolar material (Gould and Borisy, 1977), rapid tubulin polymerization starts, mediated by the nucleating activity of these structures, since egg tubulin at a concentration lower than 1 mg/ml is polymerized quickly by the addition of a nuclei fraction. In in vitro experiments, MTs radiated from granular material surrounding the centriole, as observed in a homogenate of an artificially activated Spisulu egg (Weisenberg and Rosenfeld, 1975). Using Pt K, cells pretreated with Colcemid and lysed into tubulin-containing medium, Snyder and McIntosh (1975) observed an increase in the ability of the centriolar region to initiate MT assembly from early prophase through late prometaphase. Furthermore, Gould and Borisy (1977) demonstrated that the pericentriolar material isolated from Chinese hamster ovary cells nucleated the assembly of MTs. Only a few MTs were observed to associate with the centriole when incubated with porcine brain tubulin. In the sea urchin egg, clusters of granular material were seen around the centriole from prophase through anaphase (Endo, 1978), and this structure seemed to be a nucleating center for MT assembly at the poles. In in vitro assembly experiments, R. Kuriyama and G. G. Borisy (personal communication) observed the radial growth of heterologous MTs from centrosphere regions from isolated MAS. However, isolated metaphase chromosomes from HeLa cells initiated MT assembly only at the kinetochores when incubated with a

30

H.SAKAI

high-speed supernatant of chick brain extract that did not initiate MT assembly (Sloboda et al., 1976). McIntosh et al. (1969, 1975) proposed that MTs radiating from both poles form the framework of the spindle mediated by bridges interconnecting spindle MTs. Therefore the MTs growing from the kinetochores face the pole opposite the spindle MTs. At present, the organizing center of the MA seems to consist of the material assembled surrounding the centriole. We can consider that the framework of the MA is formed by the assembly of MTs if a sufficient amount of tubulin is supplied around the pericentriolar material. It might be easier to explain how MTs are formed in the cell if the nucleating structures were known. However, why the breakdown of MTs in the spindle, as indicated by birefringence decay near the pole (Inoue, 1976), occurs after anaphase is difficult to determine. We now have some information on the control of MT assembly in v i m . Binding of GTP to both the exchangeable and nonexchangeable sites of tubulin and hydrolysis of the GTP bound to the exchangeable sites are requisites for the assembly of MTs (Kobayashi, 1975; Kobayashi and Shimizu, 1976; Weisenberg et al., 1976; Arai and Kaziro, 1977). Ionic strength and pH are also important factors for regulating MT assembly (Olmsted and Borisy, 1973, 1975; Lee et al., 1974; Kuriyama, 1977). Furthermore, the polymerization of tubulin requires tubulin sulfhydryl groups (Kuriyama and Sakai, 1974; Mellon and Rebhun, 1976; Wallin et al., 1977). Two moles of cysteine residues in tubulin are required for MT assembly (Kuriyama and Sakai, 1974), although colchicine-binding activity still remains intact after chemical modification of the 2 moles of sulfhydryl groups. It is not conceivable that changes in ionic strength and nucleotide metabolism regulate MT assembly in living cells, and it still remains to be determined whether or not the reversible dehydrogenation of tubulin sulfhydryl groups (Kimura, 1973; Kuriyama and Sakai, 1974; Sakai, 1975; Mellon and Rebhun, 1976) corltrols the cyclic assembly of MTs during the cleavage cycle, as extensively discussed by Rebhun et al. (1976). Furthermore, a change in pH within the cell (D. Epel, personal communication) may possibly control the assembly. However, since the first report on MT assembly in vitro (Weisenberg, 1972), calcium ions have been presumed to be a regulator of the assembly and disassembly of MTs (Weisenberg, 1972; Haga et al., 1974). However, purified MT protein has been shown to be insensitive to calcium ions, inhibition of MT assembly being induced only by millimolar levels of free calcium ions (Olmsted and Borisy, 1973, 1975), although Rosenfeld et al. (1976) argued that the calcium concentration necessary for the inhibition of MT assembly depends on the coexistence of magnesium ions. We recently presented evidence that the sensitivity of a tubulin preparation to calcium ions depended on the extent of purification (Nishida and Sakai, 1977). The more tubulin purified, the more free calcium ions required for the inhibition of tubulin polymerization. Formation of MTs in a

'I

31

ISOLATED MITOTIC APPARATUS AND CHROMOSOME MOTION

100

-

x

ae

'\.

A

P

I

to-*

CO'+ Concentration (MI

FIG. 5. Calcium sensitivity of crude and purified MT protein fractions in polymerization. Solid symbols, Crude fraction; open symbols, purified fraction. Different symbols represent results from different preparations (Nishida and Sakai, 1977).

crude MT protein fraction was inhibited by micromolar levels of calcium ions. The concentration of calcium ions required for half-maximal inhibition was 10 pM. However, after purification by two cycles of temperature-dependent assembly-disassembly , tubulin became insensitive to calcium ions, halfmaximal inhibition occurring at a 0.7 mM concentration of free calcium ions (Fig. 5). This calcium sensitivity was due to a protein factor, calcium-sensitizing factor, contained in the crude extract of porcine brain. In the presence of the factor, purified MT protein developed high sensitivity to micromolar levels of free calcium ions. Although we do not know whether a similar factor is present in mitotic cells, a local change in free calcium concentration may control MT assembly-disassembly. There have been reports on many vesicles found in the matrix of the spindle and the centrosphere regions (Harris, 1962, 1975; Harris and Mazia, 1962; Friedlander and Wahrman, 1970; Robbins and Jentzsch, 1970). These vesicles may be responsible for the release of calcium ions when the spindle disintegrates after anaphase.

111. The Isolated MA

A . ISOLATION

MAS were first isolated by Mazia and Dan in 1952 from sea urchin eggs by fixing mitotic cells in cold ethanol and dispersing the cytoplasm with Duponol C. This work, together with the observation of birefringence in the spindle of living cells (briefly reviewed by Rebhun and Sander, 1967), has given substantial

32

H. SAKAI

support to the existence of a fibrous entity in the MA. Later, Mazia (1955) applied digitonin in the dispersion of cytoplasm to obtain a more fibrous specimen of MA. In a further improvement of the isolation technique, Mazia and his co-workers (1961b) isolated MAS directly from living eggs using dithiodiglycol, sucrose, and EDTA. This greatly contributed to the further improvement of isolation procedures. Kane’s earlier isolation method employing 1,6- or 2,5hexanediol was also used to isolate MAS from living cells (Kane, 1962a,b). Later, he introduced hexylene glycol (2-methyl-2,4-pentanediol) for use in MA isolation (Kane, 1965), and this compound is now widely employed. In Mazia’s laboratory, Sakai (1966) substituted dithiodipropanol for dithiodiglycol in isolation procedures. Isolation requires stabilization of the MA, as extensively studied by Kane (1962a,b, 1965). The MA was stabilized by long-chain glycols without causing sulfhydryl groups in the spindle to form disulfide bonds. Hexanediol or hexylene glycol at a concentration of 1 M was sufficient to stabilize MAS. Kane’s studies on the mechanisms of stabilization showed that many organic solvents can be substituted for the glycol (Kane, 1965). With the use of these isolation methods, many studies have been done on the chemical and biophysical properties of isolated MAS (Zimmerman, 1960; Miki, 1963; Miki-Noumura, 1965, 1968; Rebhun and Sharpless, 1964; Rebhun and Sander, 1967; Borisy and Taylor, 1967; Bibring and Baxandall, 1968, 1969, 1971; Cohen, 1968; Goldman and Rebhun, 1969; Forer and Goldman, 1969; Bryan and Sato, 1970; Wray and Stubblefield, 1970; Nevo et al., 1970; Milsted and Cohen, 1973). The properties of the isolated MA, including its solubility, have been well reviewed by Forer (1969). Mazia e? al. (1972) found that replacing sodium in seawater with lithium caused the formation of a large MA that was stabilized in vivo and did not break down even at the end of mitosis. Stabilized MAS were isolated from such eggs by the use of Triton X-100 after fixation in 30% ethanol at - 10°C and shown to be visibly stable in water. More recently, a strong stabilization of isolated MAS was achieved (Forer and Zimmerman, 1974, 1976a,b; Foreret al., 1976) upon isolation in a medium containing glycerol, dimethyl sulfoxide, magnesium, and phosphate buffer; the medium also stabilized brain MTs (Filner and Behnke, 1973). Such MAS have stable MTs, stable birefringence, and stable solubility properties. Stable MAS were also obtained in an isolation medium containing glycerol, magnesium, and Triton X-100 (Sakai et al., 1977). These MAS, however, differed from MAS in living cells in that their birefringence did not disappear at low temperatures. However, different approaches to MA isolation were carried out by several investigators. Based upon the findings by Weisenberg (1972), who demonstrated in vitro polymerization of tubulin by lowering the calcium concentration, Rebhun et al. (1974) reported that a modification of MT assembly medium allowed MAS

ISOLATED MITOTIC APPARATUS AND CHROMOSOME MOTION

33

to be isolated from eight species of eggs and that the MAS were cold-labile, calcium-sensitive, stable at room temperature, and possessed a birefringence approximately equal to that of in vivo MAS. Sakai and Kuriyama (1974) reported a different isolation medium that contained glycerol, EGTA, and 2(N-morpho1ino)ethanesulfonic acid (MES). The birefringence of the isolated MAS was quickly lost after exposure to cold, calcium, or sulfhydryl-blocking reagents (Sakai et al., 1975). These treatments released tubulin from the glycerol-isolated MAS. Further revised medium also contained tubulin, GTP, glutathione, ascorbic acid, and CAMP in experiments inducing in vitro chromosome motion (Sakai et al., 1975, 1976). B. MTS AND TUBULIN Schmidt (1937, 1939) reported that mitotic spindles in the living cell showed positive birefringence relative to the long axis. He concluded that oriented protein units were responsible for the birefringence. After other workers had confirmed this observation (Hughes and Swann, 1948; Swann, 1951a,b; Inout and Dan, 1951), Inout presented the first definitive evidence that spindle fibers existed in living cells using an improved polarized light microscope (InouC, 1951, 1953). Furthermore, bundles of spindle fibers in living plant cells were observed by Bajer and Allen (1966) with the Nomarski differential interference microscope. Electron microscope observation of mitotic cells revealed many MTs localized in the MA, and it was suggested that MTs seen with electron microscopy corresponded to the birefringent fibers seen with the polarization microscope. Isolated MAS were first shown to be birefringent by Mazia and Dan (1952), and several studies followed (Kane and Forer, 1965; Rebhun and Sawada, 1969; Rebhun and Sander, 1967; Goldman and Rebhun, 1969; Bryan and Sato, 1970; Stephens, 1972). The presence of 20-nm filaments (MTs) in the isolated MA was described by Kane (1962b), and many MTs were found in MAS isolated by different methods (Kane and Forer, 1965; Kiefer et al., 1966; Bibring and Baxandall, 1968, 1971; Cohen and Rebhun, 1970; Cohen and Gottlieb, 1971). Kane and Forer (1965) found a correlation between birefringence and tubular filaments (MTs); birefringence decay in spindles kept in the hexylene glycol isolation medium was accompanied by simultaneous degeneration of the filaments. They attributed the birefringence to the presence of the filaments. This was confirmed later by Rebhun and Sander (1967), although they observed a slower decay of birefringence. Rebhun and Sander (1967) established a correlation between the image of the MA in the electron microscope and that in the polarization microscope. Especially in the case of isolated MAS from Spisula eggs, the results suggested that the MTs alone contributed the MA birefringence. However, Behnke and Forer (1966) argued that MA birefringence was not due to MTs; in

34

H . SAKAI

crane fly spermatocytes the MTs could not have given rise to birefringence because the distribution of MTs seen with electron microscopy did not match the distribution of birefringent material; many MTs were found in the interzonal region where no birefringence was observed. It is not likely that MTs are totally responsible for all the spindle birefringence. For example, Goldman and Rebhun ( 1 969) suggested that chains of ribosome-like particles associated with MTs contribute to form birefringence. However, three reasons presented by Rebhun and Sander (1967) gave strong support to the idea that MTs give rise to the majority of spindle birefringence. (1) MTs are the most prominent oriented elements seen in electron micrographs of the MA in an intact cell as well as of the isolated MA. (2) Loss of birefringence of the MA with time is accompanied by a breakup of MTs, both events occurring in parallel. (3) The increase in birefringence of the MA of marine eggs in the presence of heavy water is accompanied by a corresponding increase in the number of MTs. In addition, an increase in the tubulin pool size causes a corresponding increase in spindle birefringence (Sluder, 1976; InouC et al., 1974). Incorporation of tubulin into the interzonal region of isolated anaphase MAS gives rise to strong birefringence otherwise only faintly observed (Sakai et al., 1975). Finally, the birefringence of the isolated spindle can be precisely fit to a theoretical birefringence curve over a wide range of imbibing medium refractive indexes (Sato et al., 1975). In 1974, Rebhun et al. reported important findings on the cold sensitivity of isolated MAS. The birefringence of MAS isolated in a reassembly buffer of MTs completely disappeared when the MAS were cooled to 0"-4"C within 1 minute of isolation. MAS were not visible in the polarization microscope. However, those cooled 5-6 minutes after isolation required 10-15 minutes to reduce birefringence to one-quarter to one-half that of normal. Furthermore, when more than 15 minutes elapsed between isolation and cooling, the reduction in birefringence was delayed for several hours. Residual birefringence was observed to persist even after the depletion of MTs from cold-treated MAS, which was confirmed by electron microscope observations. These results clearly indicate that even isolated MAS lose birefringence totally after cold treatment, and storage after isolation produces alterations in spindle structures associated with MTs. MAS isolated by the glycerol method quickly lose birefringence when the temperature is lowered (Sakai et al., 1975). However, MAS isolated by the glycerol-magnesium-Triton method (Sakai et al., 1977) are resistant to low temperature, more than half the spindle retardation remaining at 0°C. Since porcine brain MTs assembled in vitro are stable in the same isolation medium but are totally depolymerized by chilling, it is likely that magnesium ions at more than 2 mM cause structures of the spindle matrix to be altered, possibly producing artifacts affecting the evaluation of elements which give rise to birefringence. The stabilizing effect of magnesium ions has been reported by several investigators (Goidman and Rebhun, 1969; Cohen, 1968; Forer and Zimmerman, 1974, 1976a,b).

ISOLATED MITOTIC APPARATUS AND CHROMOSOME MOTION

35

In 1967, Borisy and Taylor suggested that a 6 s protein which binds colchicine was a subunit of MTs. In the analysis of colchicine binding in a variety of cells and tissues, they found that significant binding was obtained with proteins of isolated MA. Subsequently, Shelansky and Taylor (1967) reported that a major protein of the central pair of MTs from sea urchin sperm tails showed a sedimentation constant of 6 s and the protein bound 0.5-1 .O mole of colchicine per 105 gm of protein as determined by sucrose density gradient centrifugation. They showed that 1 mole of guanine nucleotide was bound per 60,000 gm of outer doublet MTs (Shelansky and Taylor, 1968). Furthermore, Weisenberg et al. (1 968) purified colchicine-binding 6 s protein from the brain. They demonstrated that the protein had two binding sites for GTP; one was exchangeable and the other nonexchangeable with exogenous free GTP. These facts present the strong possibility that the binding receptor of colchicine in the arrest of cell division is MT protein. Many reports on proteins in the MA have appeared since isolation of the MA was first described by Mazia and Dan (1952). However, only in recent years has one of the major proteins of the MA been identified as tubulin (Mohri, 1968). From hexylene glycol-isolated MAS, Bibring and Baxandall (1971) obtained a protein fraction by extracting MAS with a solution containing an organic mercurial, meralluride sodium. The MAS remained intact, but observations with a polarization microscope showed that birefringence was almost totally lost. The fraction contained about 10% of the protein of isolated MAS, and 90% of the extracted protein was precipitated by the addition of a final concentration of 4 mM vinblastine sulfate. The meralluride extract gave a major doublet band in electrophoresis on polyacrylamide gel containing 8 M urea identical to that of sperm tail outer doublet tubulin. However, their sodium dodecyl sulfate (SDS) polyacrylamide gel system failed to resolve a-and P-tubulin subunits in either MA or sperm tail tubulin. The sedimentation analysis gave a value of 8.9 S at pH 6.5 and 4.9 S at pH 9.0. These values seem to derive from the tubulin denatured by the organic mercurial. A few years later, Sakai and Kuriyama (1974) reported that glycerol-isolated MAS contained tubulin possessing a colchicine-binding activity higher than that ever reported. Upon partial purification by DEAE-Sephadex A-50 column chromatography, they showed that tubulin contained in the MA had a molecular weight of 105,000 as determined by gel filtration on Sephadex G-200. Amidomethylation of the tubulin fraction caused separation of the a and P subunits, which had an apparent molecular weight of 55,000 and 51,000, respectively, as determined by SDS polyacrylamide gel electrophoresis. Our measurements indicate that tubulin in the MA of the Hemicentrotus pulchirrimus egg occupies about 8% of the total MA protein (Sakai and Sakai, in preparation), as measured by scanning SDS polyacrylamide gels stained with fast green according to the method of Gorovsky et al. (1970). The isolated spindle contains 330 pg of proteins. Therefore 26.4 pg of tubulin is contained in the

36

H. SAKAI

spindle. The number of tubulin dimer molecules is estimated to be 1.44 x lo* Since the length of the metaphase spindle is 22 p m and this length of MT contains 2.86 X lo4 molecules of tubulin dimer, the number of MTs estimated for a whole spindle is 5000 (1.44 X 108/2.86 X I@). With the assumption that 60% of egg tubulin in the spindle is in the form of MTs in the tubulin-MT equilibrium at 20"C'[assessed value from Lee ef al. (1974) and Olmsted and Borisy (1973)], 3000 MTs exist in the mitotic spindle of the first division. This value is close to that measured with electron microscopy for A. punctulafa eggs at metaphase (2000 MTs, Cohen and Rebhun, 1970), assuming that the diameter of theHernicentrotus egg (94 p m ) is larger than that of the Arbacia egg (74 p m , Harvey, 1932). After the first cleavage of theHernicentrotus egg, the diameter of the divided blastomeres becomes 75.5 p m , very close to the diameter of the Arbacia egg. The volume of the second-division spindle gives an estimate of 2400 for the number of MTs. Recently, MAS were isolated from HeLa cells in a hexylene glycol-Tritonsucrose medium (Chu and Sisken, 1977). These mammalian MAS contained a 55,000-molecular-weight component which made up as much as 20-30% of the total soluble protein. Among the possibilities discussed, it is probable that a large proportion of nontubulin material in the spindle is lost during isolation and purification of MAS in the Triton-containing isolation medium. C. ATPASES An important step in understanding the mechanism of chromosome motion is analysis of ATPase activity in the isolated MA, since it may resemble that of other motile systems such as cilia or flagella that contain MTs and dynein. It is also important to find structures with which ATPases are associated. The first description of ATPase activity in the isolated MA was reported by Mazia et al. (1961a) for dithiodiglycol-isolated MAS and included nucleotide specificity and activation by divalent cations. Two pH optima were shown at 7.8 and' 8.6. Magnesium ions were the most effective in activating the ATPase. Calcium-activation was one-third the activation produced by magnesium ions. Subsequently, Miki (1963) reported that the ATPase activity of MAS isolated from ethanol-fixed eggs was three times higher than that of the cytoplasmic fraction. Magnesium ions were also found to be necessary for enzyme activity. In hexanediol-isolated MAS, no significant ATPase activity was found by Stephens (1967). Later, Weisenberg and Taylor (1968) obtained a soluble 13 S ATPase by sucrose density gradient centrifugation from sea urchin egg cytoplasm and from hexylene glycol-isolated MAS. The enzyme was activated by both magnesium and calcium ions. The ratio of magnesium-activity to calcium-activity was 1.1. The pH dependence of the activity resembled that of ciliary dynein (Gibbons, 1966), with two pH optima at about 7.2 and 9.3. ATP was specific for the enzyme, but the rates of hydrolysis of GTP and ITP relative to that of ATP were

ISOLATED MITOTIC APPARATUS AND CHROMOSOME MOTION

37

slightly higher than those reported for ciliary dynein. After the isolation of MAS using hexylene glycol, 0.5 M potassium chloride solubilized 50% of the total ATPase activity in the MAS, most of which migrated on a 5-20% sucrose gradient with a sedimentation constant of 13 S. These investigators suggested that the 13 S enzyme most likely had a functional role in mitosis, although the possibility that the 13 S enzyme found in isolated MAS is a contaminant from the cytoplasm cannot be excluded. However, recent observations by Mohri et al. (I 976), using antidynein (fragment-A) serum for indirect immunofluorescence staining, demonstrated that dyneinlike material became concentrated in the MA. The staining was also detected in isolated MAS (Fig. 6A and B), and in anaphase MAS the interzonal region stained as well. The 13 S enzyme found by Weisenberg and Taylor (1968) was recently confirmed to be localized in MAS isolated by the glycerol-magnesium-Triton method (S. Maekawa and E. Kuramoto, unpublished data).

FIG.6. (A) Localization of antidynein (fragment-A) fluorescence in the sea urchin egg at late anaphase by the indirect immunofluorescence technique. (B) Staining of isolated metaphase MAS using antidynein serum. (C) Staining of isolated metaphase MAS from Hemicentrotus egg with anti-starfish egg myosin serum. Very faint staining was obtained when the antiserum was previously treated with a small excess of antigen or replaced with nonimmune serum (Mohri et al., 1976). By permission of The Japanese Society of Developmental Biologists. Photographs kindly given by H. Mohri of The University of Tokyo.

38

H. SAKAl

A series of studies on a calcium-activated ATPase was performed by Mazia et al. (1972) and Petzelt (1972; Petzelt and Auel, 1977). Significantly higher activity of the ATPase in MAS was found only as the calcium-activated ATPase. This enzyme was inhibited by magnesium ions, and monovalent cations (sodium and potassium) did not have an activating effect on it but instead caused inhibitionquite different from the properties of dynein ATPase. The enzyme had two pH optima at about 8.5 and 5.0. It is not clear why this MA preparation did not show magnesium-activated ATPase as demonstrated by Weisenberg and Taylor (1968) and Mazia et a f . (1961a). For enzyme identification in MAS the purest MA specimens should be used, because of the presence of dynein-like ATPase (Mabuchi, 1973) and myosin (Mabuchi, 1973, 1974, 1976) in the egg cortex, fragments of which are usual contaminants in MA preparations. To minimize this the localization of ATPases can be determined by the fluorescence-labeled antibody technique. Recent observations show that myosin is actually localized in the spindle, especially in the region between the chromosomes and the poles (Fujiwara and Pollard, 1976), or in the central region of the centrospheres (Mohri et al., 1976) (Fig. 6C).

D. RESFQNSETO HETEROLOGOUS TUBULIN The isolation of cold-labile MAS creates interesting problems involving the reactivity of isolated MAS toward various agents in comparison with that of in vivo MAS. Rebhun et al. (1974) isolated MAS from eight species of eggs in a reassembly buffer of MTs including a proteinase inhibitor. The MA isolated from Spisula sofidissima was heat-labile and calcium-sensitive, showing birefringence approximately equal to that of in vivo MAS. The addition of brain tubulin from chick embryo increased the birefringence up to 37%. Lowering the temperature to 4°C caused the birefringence to decrease by 41%, and rewarming to 30°C restored it to 90%. These workers found a large increase in the size of isolated MAS in the presence of heterologous tubulin during a 2-hour incubation which induced widening of the spindle equator and elongation of the asters. Inout et al. (1974) measured the birefringence of the meiotic spindle of a Chaetopterus oocyte in the presence of 10 mg/ml porcine brain tubulin. After lysis of the oocyte, the spindle responded to heterologous tubulin, showing an increase in spindle retardation. The same was true with glycerol-isolated MAS (Sakai et al., 1975). Porcine brain tubulin was incorporated into the interzonal region of an isolated anaphase spindle, resulting in the appearance of strong birefringence in that region for the first time. These results are well explained by the observation that heterologous MTs were formed from brain tubulin and fragments of outer fibers from flagella (Allen and Borisy, 1974; Olmsted et a l . , 1974) or from egg tubulin and brain MT fragments (Kuriyama, 1977).

ISOLATED MITOTIC APPARATUS AND CHROMOSOME MOTION

39

IV. Mitosis and the Roles of MTs A. ANAPHASE CHROMOSOME MOVEMENT Anaphase separation of chromosomes involves two motile events, chromosomal spindle fiber shortening and central spindle elongation. Analyses of the anaphase movement using living cells show that shortening of the chromosomal fibers (kinetochore MTs) begins first, and then elongation of the central spindle ensues, causing a widening of the interzonal region and further separating the daughter chromosome sets. The shortening of the chromosome fibers is called anaphase A and the elongation of the central spindle anaphase B (Inout and Ritter, 1975). In some animal cells, only the chromosomal fibers shorten, the pole-to-pole length remaining unchanged; in others, both anaphase A and B occur in parallel. In most plant cells, however, only anaphase A is responsible for chromosome separation, and in extreme cases chromosomal fibers do not shorten appreciably, chromosomes separating principally through an increase in the pole-to-pole distance (reviewed by Mazia, 1961). The velocity of the anaphase movement ranges from 0.2 to 5 p m per minute. In the sea urchin egg, the chromosomes complete anaphase A within 4 minutes at 20°C (2.5 k m per minute). Ultraviolet microbeam irradiation of mitotic spermatocytes was performed by Takeda and Izutsu (1960) to clarify the interrelationship between anaphase chromosome motion and the kinetochore or kinetochore fibers. When one of the paired kinetochores of a bivalent of an Oxya spermatocyte was irradiated at the end of metaphase (10 minutes before the onset of anaphase), the half-bivalent with the irradiated kinetochore did not migrate to the pole but remained at the spindle equator. The irradiation of both bivalent kinetochores resulted in a failure of anaphase movement in both daughter chromosomes. When the anaphase chromosome was irradiated at the kinetochore site, the path of movement of the half-bivalent was disturbed, but it eventually migrated to the pole. Similar results were also described by Forer (1966). Anaphase chromosome movement stopped only when chromosomal fibers were irradiated by an ultraviolet microbeam. Before anaphase the spindle fiber was necessary for movement, but during anaphase irradiation no longer stopped the movement. Although chromosomes can move normally after irradiation of the chromosomal MTs adjacent to the kinetochore causes the birefringence to be greatly reduced or to disappear, this does not prove that chromosomal MTs do not take part in movement because there is no evidence that such a dose of ultraviolet irradiation depolymerizes the kinetochore MTs completely. It is also possible that some of the partly depolymerized MTs are repaired in a short period of time by the supply of tubulin dimers (cf. Nicklas and Staehly, 1967; Begg and Ellis, 1974; Sluder, 1976). Since MTs are the major fibrous constituent of the spindle, accounting for the

40

H. SAKAI

majority if not all of the spindle birefringence, and MTs are the only fibers attached to chromosomes at the kinetochore, there is general agreement that chromosomes are moved by forces acting on them in the kinetochore region, supported by experiments with colchicine, lowered temperature, or increased pressure; these treatments depolymerize MTs and prevent in vivo chromosome movement (InouC, 1964; InouC and Sato, 1967; Fuseler, 1975; Salmon, 1975a,b). Movement resumes only when chromosomal fiber birefringence reappears. Therefore kinetochore MTs are indispensable for anaphase movement of chromosomes. The mechanism of anaphase chromosome movement has been considered in close relation to the functions of MTs in the MA. MTs are in dynamic equilibrium with tubulin dimers, depending on the temperature and the tubulin pool size. The assembly-disassembly properties of spindle MTs were deduced principally through quantitative studies of birefringence in living cells long before MTs were studied in vitro. The reversible self-assembly of tubulin into MTs in vitro closely follows the behavior of spindle fibers in vivo (InouC, 1976). The assembly reaction is endothermic, entropy-driven, and presumably mediated through hydrophobic interactions. Based on retardation analyses using polarization optics, the motive force of anaphase movement is believed to derive from the depolymerization of kinetochore MTs near the spindle poles (InouC et al., 1975; InouC and Ritter, 1975; InouC, 1976). Fuseler’s experiments showed that the increase in anaphase chromosome velocity caused by raising the temperature closely paralleled the decay rate of anaphase half-spindle birefringence; the faster the daughter chromosomes moved to the pole, the faster the retardation decreased. These results suggest that the depolymerization of MTs in the halfspindle creates empty sites on the MTs, causing rearrangement of the MT surface lattice to fill these sites. This hypothesis is based on the assumption that, at anaphase onset, the pool of tubulin dimers is inactivated and, as the free tubulin concentration decreases, more subunits are drawn into the pool from the MTs to maintain the MT-tubulin equilibrium.

B. INDUCTION OF CHROMOSOME MOTIONIN

THE ISOLATED

MA

Almost all our information about the motility of chromosomes has been obtained from living mitotic cells. Moreover, establishment of an in vitro MA motile system is required in investigating biochemical characteristics of chromosome movement. Over the past 15 years, unsuccessful attempts have been made to induce chromosome motion in isolated MAS (Mazia, 1961; Forer and Zimmerman, 1974). This is mainly because of alterations in spindle MTs or the spindle matrix during isolation. Since MTs in the spindle and asters are as labile as cytoplasmic MTs, the stabilization of MTs has been a requisite for the isolation of MAS retaining structural integrity. However, excess stabilization results

ISOLATED MITOTIC APPARATUS AND CHROMOSOME MOTION

41

in irreversible changes in MTs as well as in the surrounding spindle matrix. Since the MA is originally labile, it is important to isolate MAS which are as labile as those in the living cell in investigating their functions in vitro. For the purpose of studying spindle functions in vitro, Cande et al. (1974) introduced a cell model prepared by lysing mitotic Pt K1cells into a solution of Triton X- 100 containing Carbowax 20M, EGTA, and piperazine-N,N'- bis(2ethanesulfonic acid) (PIPES) buffer at pH 6.9. This solution maintained the spindle length for hours and stopped the chromosome motion within a few seconds of apparent cell lysis. When 0.1-2.5 mM ATP or GTP was added to this solution, followed by lysis of an early-anaphase cell, the chromosomes kept on moving in a remarkably normal fashion. There is a possibility that this Triton model still contained endogenous ATP and tubulin, preventing experimental analysis to determine whether some proteins were active in chromosome motion. In a different approach to understanding spindle functions, cold-labile anaphase MAS were isolated in MT assembly buffer (Rebhun et al., 1974). In some cases, anaphase MAS cold-treated and incubated in the presence of tubulin for about 1 hour attained a configuration resembling that of early telophase with the chromosomes at the astral centers. Since the telophase-like configuration was achieved after the regrowth of anaphase MAS once depleted of MTs, it is not clear how the chromosomes moved to the astral centers. More direct experiments were performed using glycerol-isolated MAS (Sakai et al., 1975, 1976). MAS isolated in the glycerol medium without tubulin, GTP, and magnesium ions were unstable at 20°C (Sakai and Kuriyama, 1974). The chromosomes became disjoined from the spindle within 1 hour. The equator of the spindle became faint, and half-spindles could eventually be separated from each other by gently shaking. Upon isolation at temperatures lower than 20"C, the spindles were frequently separated from the asters, indicating that their association was temperature-sensitive. These MAS were too unstable to examine the induction of chromosome motion. After the addition of tubulin, GTP, and other MT-stabilizing agents, however, the isolated MAS retained chromosomes and asters over a period of 2 hours at 20°C. Furthermore, the addition of a certain amount of tubulin (1.3-1.4 mg/ml) caused as much as a twofold enlargement of the spindle and asters. As noted previously, the sea urchin egg contains tubulin at a concentration of 0.6 mg/ml, and the size of the spindle is controlled by the amount of tubulin surrounding the MA both in vivo and in vitro. We determined the in vitro concentration at which the spindle size was maintained constant or increased slightly. It was about 0.4-0.5 mg/ml, depending upon the preparation of porcine brain tubulin or outer fiber tubulin from sea urchin sperm. Under these conditions, chromosome motion of isolated anaphase MAS was induced by the addition of ATP and magnesium ions both at a final concentration of 0.7 mM. Although the chromosome-to-pole distance shortened to some extent, elongation

H.SAKAl

42

of the spindle due to the incorporation of brain tubulin seemed to contribute in part to the apparent separation of the half-bivalents. When the tubulin concentration was higher, its incorporation into the interzonal region probably pushed the half-bivalents apart, increasing the chromosome-to-chromosome distance. Shortening of the chromosome-to-pole distance was further demonstrated when MAS were isolated in the presence of a low concentration of magnesium ions together with tubulin, GTP, and other MT-stabilizing agents. Isolation often caused groups of chromosomes to adhere to each other, possibly disturbing chromosome motion in a normal fashion. There was a definite tendency for independent daughter chromosomes to move faster than clustered ones. In such MAS, ATP was the specific nucleotide inducing the motion, while GTP, ITP, UTP, and CTP had little effect. Colchicine strongly inhibited chromosome motion at a concentration of 5 p M , and the absence of tubulin also resulted in failure to induce motion. However, anti-starfish egg myosin serum did not significantly inhibit chromosome motion in isolated MAS from starfish eggs (Fig. 7). These results suggest that spindle MTs are involved in the in vitro motion of chromosomes. The sliding of a group of doublet MTs from a sperm tail was demonstrated by Summers and Gibbons in 197 1. This motion is specific for ATP and magnesium ions and its observation gives substantial support to the idea that dynein-outer fiber doublet interaction induces flagellar movement. Ogawa and Mohri (1975) 30 -

A

-

8

SL

20 40 60 80 100

t

20 40 60 00 (00

-I

20

SL

Ft 40

60

20

40

60

Time after addition of reaction medium ( m i d

FIG. 7. Effect of y-globulin fraction of anti-starfish egg myosin serum and antidynein serum on in v i m chromosome motion. (A) y-Globulin fraction of nonimmune serum. (8) y-Globulin fraction of anti-egg myosin serum. (C) Nonimmune serum. (D) Antidynein serum. Isolated early anaphase MAS were treated with the above fractions at a final concentration of 1 mg/ml each, followed by the addition of reaction medium containing ATP and magnesium ions. SL, Spindle length; CPD, chromosome-to-pole distance; ID, interzonal distance (Sakai ef al., 1976).

ISOLATED MITOTIC APPARATUS AND CHROMOSOME MOTION

43

treated flagellar dynein with trypsin to obtain a fragmented polypeptide (fragment A, Ogawa, 1973), and anti-fragment-A serum was made in rabbits. The antibody was shown to inhibit movement in the Triton model of sea urchin spermatozoa (Okuno et al., 1976), as well as the ATPase activity of fragment A (Ogawa and Mohri, 1975). When anti-fragment-A serum was applied to isolated MAS, chromosome motion was completely suppressed (Fig. 7). In some of the isolated MAS, early-anaphase half-bivalents moved back to the equator of the spindle, forming aggregates. If we assume that in vitro chromosome motion is caused merely by the growth of heterologous MTs within an interzonal region, which push the daughter chromosomes to both poles, the same motion should be observed in anaphase MAS treated with anti-fragment-A serum. However, this is not the case. These observations favor the idea that MT-dynein interaction produces the motive force for the shortening of kinetochore MTs. However, these results cannot explain the decay of birefringence accompanied by anaphase chromosome movement in vivo. Moreover, one can argue that the in vitro motion is quite slow (Forer et al., 1977). This may be attributed to the isolation medium and procedure which are still unable to isolate MAS retaining a fully operative motile system. Isolation was performed at pH 6.2, which would shift spindle MTs toward depolymerization even in the presence of heterologous or homologous tubulin. Isolation at pH 6.7 increased the velocity to more than fivefold that in MAS isolated at pH 6.2, though the yield of MAS was reduced, Further improvements in the isolation method would provide more precise information on in vitro motion. C. ACTIN AND MYOSININ MITOSIS In contrast to the detection of tubulin by the indirect immunofluorescence technique (Nagayama and Dales, 1970; Dales, 1972; Fuller et al., 1975; Weber et al., 1975; Cande et al., 1977; Connolly et al., 1977), actin filaments have been observed in meiotic and mitotic spindles by electron microscopy after the addition of heavy meromyosin (HMM) from rabbit skeletal muscle (Behnke et a!., 1971; Forer and Behnke, 1972; Gawadi, 1971; Hinkley and Telser, 1974). Furthermore, actin has been identified in the spindle and in isolated MAS using fluorescence-labeled HMM (Sanger, 1975; Cande er al., 1977). However, there are arguments about the presence of actin in MAS. Glycerination disrupts the organization of a cell, and HMM induces G actin to polymerize (Cooke and Morales, 1971; Kikuchi et al., 1969). The other objection is that many actin filaments are observed in animal cells, especially in the cell cortex (Ishikawa et al., 1969; Goldman and Knipe, 1973), and actin observed in the spindle may originate from outside the spindle during glycerination. Fluorescent image of actin was also observed by K. Izutsu (personal communication) in the mitotic spindle of cultured tissue cells by the indirect im-

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munofluorescence technique; similar images were seen after treatment with nonimmune serum and fluorescence-labeled goat antiserum to rabbit y-globulin. Furthermore, glycerination clearly induced an artifact in actin localization. However, it is conceivable that some actin filaments in the cell are nonspecifically incorporated into the MA during its organization. Microfilaments are observed in glutaraldehyde-fixed spindles in plant cells not treated with glycerol (Bajer and Molh-Bajer, 1969, 1971) and in crane fly spermatocytes (Muller, 1972). To avoid possible artifacts caused by glycerination, as well as by contamination of the spindle with cytoplasmic actin, Cande et al. (1977) fixed Pt K1 cells with Formalin. The fixed metaphase spindle showed antiactin fluorescence near the spindle poles and in discrete fibers running from the chromosomes to the poles. During anaphase, the fibers shortened as the chromosomes approached the poles, leaving the interzonal region devoid of fluorescence. These investigators suggested that the presence of actin in fibers running from the chromosomes to the poles but not in the interzonal region supports involvement of an actomyosin-like system in mitotic movement of the chromosomes. It is natural to suppose that myosin is also contained in the spindle if actin is a genuine spindle constituent. A careful study was done by Fujiwara and Pollard (1976) on the distribution of myosin in dividing HeLa cells. They found a high concentration of myosin-specific staining in the mitotic spindle, especially in the region between the chromosomes and the poles. Moreover, Mohri et al. (1976) presented evidence that fluorescence-labeled anti-starfish egg myosin serum specifically stains the spindle pole, staining of the regions between the chromosomes and the poles being not detectable. These results suggest the possible involvement of an actin-myosin motile system in chromosome movement. However, the presence of actin and myosin in the spindle does not prove that they play a role in mitosis, and present results do not support this possibility. Furthermore, recent studies by Mabuchi and Okuno (1977) clearly indicated that anti-starfish egg myosin serum did not inhibit mitosis when it was injected into the starfish egg, while it did inhibit cytokinesis. These facts make it evident that the MT4ynein system works on chromosomes and, after a cleavage signal is transmitted to the cell periphery (Hiramoto, 1956; Mazia, 1961; Rappaport, 1975), the actin-myosin system is activated in the form of a contractile ring (Schroeder, 1973), leading to constriction of the cell.

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INTERNATIONAL

REVIEW OF CYTOUXiY, VOL. 55

Contact Inhibition of Locomotion: A Reappraisal JOAN E. M. HEAYSMAN Department of Zoology, University College, London, England

I. Introduction

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11. Contact Inhibition Type 1 111. Contact Inhibition Type 2

IV. V. VI. VII.

Failure of Contact Inhibition between Normal Cells Tumor Cells and Contact Inhibition . . . . . . Mechanism of Action of Contact Inhibition . . . Conclusion . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . .

. . . . .

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

49 51 53 54 56 58 63 64

I. Introduction When cells come into contact with each other, their behavior is often considerably altered. An example of this is the phenomenon of contact inhibition of locomotion which, although it had been known for many years, particularly in epithelial wound healing, was first described in detail by Abercrombie and Heaysman (1954), working with chick heart fibroblasts moving on a plane surface in tissue culture. They defined contact inhibition as the stopping of the continued locomotion of a cell in the direction which has produced a collision with another cell or, alternatively, the prohibition, when contact between cells has occurred, of continued movement such as would carry one cell across the surface of another. They showed that this reaction was a direct response to cell-cell contact. Similar reactions have been described in several widely different types of fibroblasts (Abercrombie et al., 1968), pigmented retina epithelial cells (Middleton, 1972; Di Pasquale, 1975), polymorphonuclear leukocytes (Oldfield, 1963), and nerve fibers (Dunn, 1971). Some confusion arose in the literature when the term “contact inhibition” was also used for the inhibition of cell division found in dense cultures of many types of cultured cells. As Martz and Steinberg (1972) rightly point out, it is undesirable for one term to denote both the inhibition of movement and the inhibition of division, since they are operationally distinct and may well be mechanistically independent. While it is possible that cell contact plays a part in the inhibition of cell division, it is, according to present evidence, unlikely, and the two 49

Copyright @ 1978 by Academic Press, Inc. ifreproduction in any form reserved. ISBN 0-12-362355-4

All ri&s

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phenomena are probably due to entirely different reactions. An apparent correlation has been found (Castor, 1971) between the average rate of mitosis and the average rate of cell translocation at a given level of population density, but Martz (1973), using 3T3 cells (a mouse fibroblast cell line), found no correlation between the average speeds of individual cells and their generation times. The term “density-dependent inhibition of mitosis” (Stoker and Rubin, 1967) is now more generally used for the inhibition of mitosis, and this phenomenon is not considered further in this article. The original data on contact inhibition of locomotion (Abercrombie and Heaysman, 1954) were obtained from studies on the displacement of the whole cell. Explants were placed so that the cellular outgrowths met between them. It was found that, when these outgrowths met, little further movement of the cells in the direction which had produced collision occurred and the cells tended not to overlap one another. As a result, a monolayer (Abercrombie and Heaysman, 1954) was produced. The formation of a more-or-less single layer of cells over the substrate and the virtual absence of nuclear overlaps rapidly came to be used as criteria for the presence of contact inhibition, while a high incidence of nuclear overlap, multilayering, or piling up, was considered evidence of its absence. The amount of overlap in a particular situation was expressed as an overlap index. This value was derived by estimating the number of cell nuclei which would be expected to overlap if the cells were distributed at random and comparing this number with the number of overlaps actually occurring, the ratio of the observed number to the expected number being the index, It was soon evident that the situation was far from simple. Cells did not exhibit either complete contact inhibition or a complete failure of contact inhibition. Populations of fibroblasts from different sources can have significantly different overlap indexes under standard culture conditions (Abercrombie et al., 1968) and, when dissociated cells of two heterologous fibroblast populations are cultured together, the presence of cells of one kind can influence the homologous overlapping behavior of the other. Cells also have different overlap indexes in different culture situations (Hams, 1973a). Several theories evolved to explain the phenomenon of contact inhibition. They have been reviewed in detail by Abercrombie (1970) and Harris (1 974) and can reasonably be described as falling into two main groups. According to the first group, a cell might stop upon collision with another cell either because it is more firmly attached to the substrate than it would be to the other cell, or because the upper surface of the opposing cell is nonadhesive. The phenomenon would therefore be a question of differential adhesion. The second group consider it to involve direct inhibition of the cellular machinery which produces cell locomotion, brought about upon collision, either as a result of the transmission of a signal or by the interaction of certain properties of the two contacting surfaces. More detailed study of individual cells has added to our understanding of what happens to individual cells upon contact with each other. In the case of chick

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heart fibroblasts, contact inhibition has now been more specifically described as a process involving adhesion, paralysis, and contraction (Abercrombie, 1970). If this more detailed definition is applied to the reactions of some other types of cells, certain behavior which has been described as contact inhibition under the original broad definition (i.e., the directional restriction of displacement) no longer falls into this category, as Martz and Steinberg (1973) have emphasized. Vesely and Weiss (1973), as a result of their work on normal and neoplastic rat cells, have suggested the terms “contact inhibition type 1” and “contact inhibition type 2.” Type 1 is typified by the original reaction described between chick heart fibroblasts as it has now been redefined, while in type 2 extensive overlapping does not occur but there is no inhibition of lamellipodial activity, that is, no paralysis, no contraction, and probably no adhesion. This nomenclature has been used in several recent articles such as those by Armstrong and Lackie (1975) and Harris (1974). When considering a given case of monolayering or low nuclear overlap, it therefore appears to be advisable to determine whether it results from contact inhibition of the first or second type. Clearly these are two distinct phenomena with probably entirely different underlying mechanisms and should be identified as such.

11. Contact Inhibition Type 1 Chick heart fibroblasts moving on a plane surface in tissue culture show contact inhibition type 1. When the collision is between the leading lamella of one fibroblast and that of another, further movement in the direction of contact stops almost immediately in both fibroblasts, any ruffling of the leading lamellae stops, and eventually a retraction occurs and the leading edges separate. A further collision may occur, with further resultant inhibition; or, if free space is available, the fibroblasts may move away into it. When the collision is between the leading lamella of one fibroblast and the side of another, the movement forward of the first fibroblast may continue for a while, the lamella passing under the other fibroblast (Boyde et al., 1969), but then contact inhibition usually becomes effective, the cell’s forward movement ceasing and contraction of the leading edge occurring. The movement of the second fibroblast is unaffected, though it may be distorted as a result of retraction of the first fibroblast. Where detailed examination has taken place, a similar sequence of events has been seen to occur in fibroblasts from many different organs and species. Di Pasquale and Bell (1974) have suggested the alternative term “inhibition of ruffling” for this type of contact inhibition, the most obvious immediate effect of two cells coming into contact usually being the stopping of the ruffling of the leading lamellae. Ruffling of the leading lamella is frequently associated with cell movement but, as shown by Abercrombie et al. (1970a), a cell does not need to ruffle in order to move, indeed ruffling may indicate a failure of complete efficiency of move-

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ment. Cells moving under agar or methylcellulose show no ruffles, but upon collision exhibit the paralysis and contraction typical of type- 1 contact inhibition. Also, as Trinkaus ef al. (1971) have pointed out, the inhibition of ruffling is a local phenomenon, ruffling being inhibited at the point of contact but continuing at points a short distance away. New lamellae may also be initiated elsewhere on the cell’s surface. Ruffling may also cease before contact inhibition becomes effective, for example, when a leading lamella passes between the substrate and the ventral surface of the opposing cell. It would probably be more satisfactory to keep the original term for this type of inhibition, since it is after all the phenomenon we were describing, albeit in a rather imprecise manner. Although contact inhibition of locomotion of this type would be expected to result in a failure of overlapping and the production of a monolayer, a true statistical evaluation of it can be obtained only by the direct monitoring of many cellular collisions, a very time-consuming task but one which was undertaken by Vesely and Weiss (1973) and more recently by Stephenson and Stephenson (1978). Fixed material, however, has some advantages, mainly in regard to the much larger number of separate cultures that can be conveniently studied and the ease with which regularities in behavior can be detected. The two methods complement each other, but all cells assumed to exhibit contact inhibition of this type should be monitored by filming. Most collisions between normal tissue cells in culture that have been studied in detail in this way appear to exhibit contact inhibition type 1 . Where the collision is between two leading lamellae as described above, both cells are inhibited-both the cell that would move ventrally to the other and the cell that would move dorsally. This is not just an inhibition of overlapping, but of underlapping as well. Thus the idea that contact inhibition is due to differential adhesion is unlikely to be correct in this situation; the underlapping cell is not in fact changing its substrate. If a cell’s locomotion is inhibited upon contact because it cannot exchange its adhesion to the substrate for an adhesion to the colliding cell, only the overlapping cell should show this response. While contact inhibition is usually reciprocal, instances have been found in which only one of the cells is inhibited, the other apparently being more or less unaffected by the collision. Such a nonreciprocal situation has been described for methylcholanthrene-induced sarcoma cells of the mouse and normal chick fibroblasts (Heaysman, 1970) and for neoplastic and normal rat fibroblasts (Vesely and Weiss, 1973). In both cases the normal cell exhibited a typical contact inhibition response upon collision with the neoplastic cell, while the activity and locomotion of the neoplastic cell was unaffected. As these reports point out, estimates of contact inhibition based on nuclear overlap from fixed cultures of such cells would be thoroughly misleading. Attempts to reproduce the phenomenon of contact inhibition type 1 by causing a cell to collide with nonliving material, whether in the form of a fixed cell or of

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substrates covered with varying organic substances, have until recently been unsuccessful. While cells have been shown to be unable to move onto less adhesive substrates (Carter, 1967; ivanov and Margolis, 1973), they have not, when confronted with the boundaries of these substrates, been observed to undergo the characteristic immediate paralysis of ruffling activity and subsequent retraction described above. The forward movement of the cell is stopped, but the cell remains at the edge of the opposing strip, its leading lamella ruffling actively. Few data appear to be available on the reaction of cells upon collision with substrates assumed to be more, rather than less, adhesive, than their normal substrate. Against fixed cells, frequently no inhibition at all occurs. R. A. Weiss and S. A. Cairns (unpublished observations) found that chick heart fibroblasts moved readily onto and over the surfaces of glutaraldehyde-fixed fibroblasts as do embryonic rat fibroblasts (Vesely and Weiss, 1973). The latter investigators noted, however, that glutaraldehyde-fixed neoplastic rat fibroblasts did inhibit the movement of live neoplastic and normal rat fibroblasts, but here, as with the nonadhesive substrates described above, the leading lamellae continued their ruffling activity and there was no paralysis or retraction. Heaysman and Turin (1976), however, using zinc fixation of chick heart fibroblasts, described what they considered a typical contact reaction by a living cell upon collision with a fixed one. Zinc fixation had been used previously in the preparation of cell membranes (Warren et al., 1966), and it has been suggested that the surface structure is strengthened or stabilized so that it can withstand separation and isolation from the other cellular components, though the precise changes taking place in the membranes leading to this stabilization are apparently not known. There appears to be a difference in properties in zinc-fixed cells and glutaraldehydefixed cells, and further analysis of different fixation methods may lead to a better understanding of the surface structures or proteins involved in contact inhibition.

111. Contact Inhibition Type 2 As described above, when cells come into contact with a substrate which is less adhesive than the one on which they are moving, though their forward locomotion is inhibited, the ruffling of the leading lamella is not, and no signs of the adhesion and retraction characteristic of the classic contact inhibition response are seen. The ruffling of the leading lamella, far from being inhibited, is frequently very active indeed, suggesting possibly an inability to make a cell substrate adhesion to the new substrate (Abercrombie et al., 197Oa). Such adhesions are probably an important component of the cellular motile apparatus, and failure to make them against a less adhesive substrate prevents the cell from

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moving onto such a substrate. A layer of agar placed as a barrier to normal fibroblasts moving on glass elicits such a response but, if the agar is dried down or annealed to the glass, the fibroblasts will continue their forward movement onto and over it. Maroudas (1975) has suggested that this is due to the formation of microfibril aggregates to which the cells’ leading lamellae can attach. In most cases in which this type of contact inhibition has been described the collision was not between cell and cell but between a cell and the boundary of a nonliving substrate, but Vesely and Weiss (1973) have described it as occurring between Rous sarcoma virus (RSV)-transformed rat fibroblasts as well as between living normal rat fibroblasts and glutaraldehyde-fixed neoplastic rat fibroblasts. Armstrong and Lackie (1975) also describe it between rabbit peritoneal neutrophil granulocytes and rabbit fibroblasts, while recent work by Bellairs (1978) suggests that it may also occur between hypoblast cells in culture. In all these cases, inhibition of forward locomotion occurs, but not inhibition of lamellipodial activity. Such a reaction results in a failure to overlap and, when it occurs between cells, in the formation of a monolayer. But there is no evidence to suggest that an actual inhibition of the locomotory mechanism of the cell is involved; indeed Harris (1973a) reports that cells crowded at the boundary of a less adhesive substrate move onto it in preference to piling up and overlapping one another. Carter (1967) has suggested that, if cell surfaces were less adhesive to one another than to the substrate, they would tend not to overlap one another for the same reason that they tend not to move onto less adhesive substrates or to move up an adhesive gradient. Contact inhibition of locomotion would thus be a special case of haptotaxis. A similar explanation of monolayering has been suggested by Martz et al. (1974), who consider it a special case of Steinberg’s “differential adhesion” hypothesis (Steinberg, 1970). Monolayering would thus occur when cell-substrate adherence exceeds cell-cell adherence. Such theories may well explain the reactions described here, and this type of contact inhibition, in which inhibition is not in the lamellipodial activity but is a failure of forward translocation, probably due to a failure to adhere to the opposing substrate, whether it be the surface of another cell or an inert substrate, should more aptly be described as substrate-dependent inhibition of locomotion.

IV. Failure of Contact Inhibition between Normal Cells With the use of the overlap index as a measure of contact inhibition it has been shown that not only do few, if any, cells show a complete absence of nuclear overlap, but that the overlap index of a particular cell type can be altered in various ways, for example, by the presence of other cells of differing types, by

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alteration in the substrate, and by the addition of a substance such as urea to the medium. We are not referring here to the effect of such carcinogenic agents as viruses, and known chemical carcinogens. When overlaps or underlaps are studied in detail in cells cultured in situations in which contact inhibition is normally effective, they are seen to occur in the main as a result of the leading lamella of the underlapping cell passing under the side of the overlapping cell. While such a collision may still result in contact inhibition even after the lamella of the underlapping cell has passed some distance under the overlapping cell, in some instances this does not occur and a nuclear overlap is produced. Areas of cell substrate adhesion have been described as being more frequent at the leading edge of the cell (Harris, 1973b), and it is possible that the overlap described here may occur without the cell membranes coming into close enough contact for contact inhibition to become effective. The main volume of this work has been carried out on fibroblasts, but there is no reason to suppose that the same situation does not occur in other types of cells which exhibit the classic type of contact inhibition of locomotion. Cells plated on “dried” agar and other less adhesive substrates show increased overlap (Harris, 1973a), but it is probable that this is due not to a decrease in contact inhibition but to some other factor. Cells on dried agar certainly exhibit a normal contact inhibition response when the collision is between leading lamellae, but the cells tend to be less well spread on the substrate than they would be on glass. This lack of spreading may be correlated with a bigger gap between the cell and the substrate over large parts of the cell. Elongated cells probably adhere to the substrate at either end but not at the sides, and such a situation may well increase the likelihood of sideways collisions producing overlaps. In the presence of urea the overlap index of chick heaft fibroblasts and of 3T3 cells is also increased. When returned to normal medium, the cells rapidly return to their normal overlap value. It has been suggested that urea causes the release from the cell surface of a protein required for cells to contact-inhibit one another. However, preliminary results in our laboratory suggest that cells in a ureacontaining medium, while less notably elongated than those on dried agar, still have a significantly smaller spread area than cells in normal medium and that the increased overlap index may again be due to increased underlapping. Whether or not the gap between such cells and the substrate is actually of sufficient size to allow the passage of another cell without close contact occurring is yet to be shown, and a correlated light and electron microscope study is in progress. An apparent increase in the overlap index of a particular cell type may also be brought about by retraction clumping. This is particularly likely to take place on less adhesive substrates and occurs as a result of cells being pulled together when the adhesiveness of the substrate is reduced relative to the intercellular adhesiveness, not as a result of one cell moving onto or under another.

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V. Tumor Cells and Contact Inhibition Tumor cells apparently escape many of the controls and restraints that influence the behavior of normal cells in vivo, particularly those which maintain cell position and prevent the invasion of surrounding tissues. Consequently the most extensive interest in the concept of contact inhibition has probably arisen with regard to the possible difference in behavior, in this context, between normal and tumor cells, whether the tumor cells are derived from in vivo tumors or are produced in culture by the transformation of normal cells. Cells from mouse sarcomas S37 and S180 were shown not to exhibit contact inhibition when they came into collision with normal chick heart fibroblasts or mouse skeletal muscle fibroblasts (Abercrombie et al., 1957), neither the sarcoma cells nor the fibroblasts being inhibited; it was at first assumed that a behavioral characteristic showing a marked difference between normal and malignant cells had been identified. Tumor cells were already known to pile up in culture, and many workers initially assumed that tumor cells, besides possibly showing no contact inhibition with normal cells, that is, no heterologous contact inhibition, would also show no contact inhibition with each other (homologous contact inhibition). No such suggestion had been made by the original investigators, and indeed such a situation was not really anticipated. The action of contact inhibition has been suggested as the reason for the occurrence of oriented migration from explants in culture (Abercrombie, 1970), and such a migration is characteristic of tumor cells as well as normal cells. Decreased homologous contact inhibition could therefore be expected to depress the invasive performance of a particular cell type. Malignant cells in fact show considerable variation in their contact inhibition properties. Projan and Tanneberger (1973), using the overlap index as a measure of the effectiveness of homologous contact inhibition in a range of human malignant cells, showed a much greater variation in values than was found in normal fibroblasts, many malignant cells having a lower intensity of contact inhibition but some higher. Stephenson el al. (1974) found the nuclear overlap index of malignant mouse ascites carcinoma cells to be characteristically higher than that of embryonic normal mouse cells, while that of malignant mouse melanoma cells was typically lower. Guelstein et al. (1973), working with normal mouse fibroblasts and cells transformed by Moloney mouse sarcoma virus, found no deficiency in contact inhibition between transformed cells, or between transformed and normal cells, while Vesely and Weiss (1973), in a detailed description of individual cell collisions, showed that neoplastic rat fibroblast cells had a high degree of homologous contact inhibition. Since in the animal body malignant cells invade populations of normal cells, it is probable that their heterologous contact inhibition with normal cells has more relevance to their invasive capacity than their contact inhibition response to each other. Heterologous contact inhibition between tumor cells and normal cells in

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culture has frequently been found to be defective, but this is not always the case; and even where it is defective, it still shows considerable variation between one tumor type and another. Abercrombie and Heaysman (1 976), using the same experimental situation as that used by Abercrombie et al. in 1957, that is, placing two explants a set distance from each other and observing the changes in behavior of the cells in the outgrowths when they collide, found that there was considerable variation in the amount of mutual invasion achieved by different sarcoma cells and the standard fibroblasts. Sometimes invasion occurred as if the confronting population were not there, but frequently invasion was reduced, suggesting some degree of obstruction by the confronting population. Possibly there are varying degrees of defective heterologous contact inhibition, and it is the relative values of the heterologous and both the homologous contact inhibitions that are of prime importance. These workers have suggested that this variability may be associated with variable tumor invasivity in vivo. In this context not only is the heterologous contact inhibition between the tumor cells and the normal cells of interest, but so also is the reciprocal contact inhibition between the normal cell and the tumor cell. Abercrombie (1975) defined cellular invasion as the movement of a cell population into an area already occupied by another population and pointed out that this occurs in the junctional zone between the explants once a high, homogeneous density has developed, unless the intensity of the heterologous contact inhibition approximates that of the homologous contact inhibition for each population. While the usual method of identifying the intensity of contact inhibition (i.e., the overlap index) clearly indicates the amount of interaction when both types of cells exhibit a similar response to collision, it will give spurious values if one cell type exhibits contact inhibition and the other does not. In such a nonreciprocal situation only the monitoring of individual cell collisions can give a true estimate of the situation. The heterologous overlap index for methylcholanthrene induced sarcoma (MCIM) and normal fibroblasts (Abercrombie and Heaysman, 1976), for example, is higher (but only marginally so) than the comparable homologous indexes, and yet MCIM cells show considerable invasive power. Analysis of individual collisions shows that, when the leading edge of an MCIM cell collides with the leading edge of a chick heart fibroblast, the MCIM cell continues to move as before, but the chick heart fibroblast ceases to move in the direction of the contact and its leading edge contracts just as in ordinary contact inhibition. The speed of movement of the sarcoma cell, under our conditions, is considerably less than that of the fibroblast; and so, if there is free space around the fibroblast, this nonreciprocal inhibition causes the fibroblast to retreat from the sarcoma cell. If the fibroblast cannot move away because it is surrounded by other cells, the MCIM cell moves under or over the fibroblast. If the leading edge of an MCIM cell collides with the side of a chick heart fibroblast, it is not inhibited and, as in fibroblast-fibroblast side collisions, nei-

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ther is the fibroblast. The MCIM cell passes under or over the fibroblast, more often under than over, probably because its leading edge is more closely attached to the substrate than is the side of the fibroblast. A similar situation has been described by Vesely and Weiss (1973) for RSV-transformed rat fibroblasts in collisions with embryonic rat fibroblasts, and by Stephenson and Stephenson (1978) for human melanoma cells in collisions with human skin fibroblasts. Barski and Belehradek (1965) have suggested that the intermingling of cancer cells with normal cells in v i m is not necessarily due to reduced contact inhibition. Cancer cells may invade normal cells not by moving over them but by working their way through gaps in the outgrowth. They found that the cells they used, Moloney sarcoma virus (MSV)-transformed BALB/3T3 and sarcoma 180, did not move over a coherent sheet of normal fibroblasts, though they did invade less coherent outgrowths. Stephenson and Stephenson (1978) have described a similar situation between human melanoma cells and chick heart fibroblasts; no movement of human melanoma cells onto chick cells was seen, and invasion appeared to be mainly by the progress of radially oriented melanoma cells between radial rows of fibroblasts. They also described chick heart fibroblasts and human skin fibroblasts as being contact-inhibited by head-on collisions with human melanoma cells but able to pass beneath orthogonally oriented, bipolar melanocytes. Possibly this resembles the apparent loss of contact inhibition of normal cells on a poorly adhesive substrate described above. Indeed, Erickson (1 976) has suggested that the increased overlapping seen in polyoma-transformed baby hamster kidney (BHK) cells, as compared to that found in normal BHK cells, is due not to their being released from contact inhibition, but to active underlapping, resulting from the cells being less well attached to the substrate. When the ruffled membranes of two of these cells collide, a normal contact inhibition-type response results. Bell (1972) also suggests this as the explanation for the radical differences in overlap index of transformed 3T3 cells and normal 3T3 cells. Polyoma virus-transformed 3T3 cells have long, narrow lamellae with wide gaps between points of adhesion to the substrate, while 3T3 cells have broad lamellae and numerous closely placed substrate adhesions. In all these situations only the individual monitoring of head-on collisions between cells really shows whether contact inhibition is effective or not. There is obviously much more investigation to be done before we have anything resembling a complete understanding of the part played by contact inhibition in the behavior of tumor cells.

VI. Mechanism of Action of Contact Inhibition Little is known for certain about the mechanism by which the contact interactions involved in contact inhibition of locomotion exert their influence on cell behavior.

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The inhibition of locomotion produced as a result of collision with a nonadhesive or less adhesive substrate, whether it be the surface of another cell or a nonliving substrate, is probably due to failure to produce an adhesion plaque against such a substrate. A cell, in order to move, must not only extend over the substrate but must also attach itself to the substrate. Electron micrographs of the leading edge of fibroblasts (Abercrombie et al., 1971) show localized plaques where the unit membrane comes within 30 nm of the substrate. Here there is an electron-dense fibrous zone beneath the unit membrane, and in several cases an oblique tract of microfibrils extends backward from it to join the cortical microfibrils of the upper surface. Similar structures have been identified in other types of cells, and it has been suggested that they are attachment sites. If a cell does not make such plaques against the substrate, forward locomotion will not occur. Such a mechanism does not appear to underlie the phenomenon of contact inhibition as initially described in normal fibroblasts. Here close examination has shown that the process involves adhesion, paralysis, and retraction. Far from there being a failure of adhesion, recent evidence has suggested that the formation of a specialized contact between the colliding cells is a necessary part of the reaction. Using a method whereby changes in cell behavior, as observed in living cultures using phase-contrast optics, can be correlated with changes in the ultrastructure of the cells involved, Heaysman and Pegrum (1973a) have described areas of specialization of the cortical cytoplasm developing at points of close apposition of the unit membranes of colliding cells and have suggested a correlation between the changes occurring at these points of contact and the sequence of events characteristic of contact inhibition. When the collision is between leading lamellae, these specializations appear within 20 seconds of the cells coming into contact. Within 60 seconds microfilaments appear associated with these areas, “lined up” in a direction parallel to the longitudinal axis of the cells, and the cell surfaces become distorted in the area of close apposition. More than 2 minutes after collision the microfilaments are very well marked and apparently connect up with the layers of longitudinal cortical filaments beneath the dorsal surface of the lower and upper cells. Retraction probably begins at this stage, culminating in separation of the two cells. In the regions of separation there is a rapid pulling apart of the two cell surfaces, and electron micrographs of this stage show a rapid disappearance of the electron-dense areas. These areas of specialization resemble closely those described above as occumng between the cell and the substrate, and yet here apparently the lamellae do not continue to extend forward as they do when a cell-substrate plaque has been formed. It has been suggested by Hams (1973b) that the old adhesions of cells to the substrate break down when they come into contact and, if the new intercellular adhesions formed are weak, they may give way under the tension transferred to them and retraction then follows. Abercrombie and Dunn (1975), however, using the interference-reflection mi-

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croscope invented by Curtis (1964) and subsequently developed by Lochner and Izzard (1973), showed that adhesions of a fibroblast to a glass substrate usually persist in the neighborhood of a contact with another fibroblast during the process of contact inhibition. Adhesion points or plaques were seen to disappear elsewhere. If the formation of these contact specializations is fundamental to the process of contact inhibition, cells showing a lack of contact inhibition would not be expected to produce them upon collision. Such appears to be the case for the only situation so far studied in detail; no such areas occur between chick heart fibroblasts and S180 tumor cells (Heaysman and Pegrum, 1973b). Many other cells exhibiting or failing to exhibit contact inhibition must be studied in this way before a definite correlation can be assumed. Chick heart fibroblasts have been shown to exhibit contact inhibition with zinc-fixed cells (Heaysman and Turin, 1976), and preliminary results suggest that contact areas and associated fibrous tracts similar to those described above are formed by the chick heart fibroblasts against the fixed cells. Again the question arises as to how these areas differ from those produced against the substrate. Possibly the cell surface is least stable at the leading edge, and contact between normal cells somehow stabilizes it at the point of collision, preventing further surface flow, hence locomotion. Albrecht-Buehler and Chen (1977) suggest that surface flow may be locally inhibited by surface networks predominantly built of LETS protein and have demonstrated that such networks appear to originate from points of cell-cell contact. A flow of surface material on the upper surface of fibroblasts in culture is suggested by the backward movement of particles frequently described (Abercrombie et al., 1970b; Harris and Dunn, 1972). Bray (1970) observed the same phenomenon on advancing nerve fiber tips. These observations have given rise to a theory of tissue cell locomotion according to which the dorsal surface of the Iocomoting cell moves back and new surface appears along the leading edge (Abercrombie et al.. 1972). If the assembly of membrane at the leading edge were to be inhibited by contact with another cell, the movement of particles on the surfaces of colliding cells should also stop. Do these particles in fact stop moving on contact? Differences of opinion exist, at least as far as confluent 3T3 ceils are concerned; Hams (1973~)says the movement of particles is strongly ihhibited, but Albrecht-Buehler (1973) says the movement, at least of gold particles, is not. If the local nature of the contact inhibition response is borne in mind, such differing results are understandable. Our experience with particles on chick heart fibroblasts is that, when they are in the immediate vicinity of the contact, they stop moving. Is a surface difference between normal and malignant cells at the root of their difference in contact inhibition behavior? Barnett et al. (1975) say that normal 3T3 cells have ordered membrane lipids and aggregated intramembranous parti-

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cles, whereas virally transformed cells have fluid membrane lipids and randomly distributed intramembranous particles; and Poste (1973) describes the rate of synthesis of the surface as being significantly faster in transformed than in normal cells. Fine and Taylor (1976) suggest that the change in the polymerizability or membrane association of actin, which accounts for the decreased particulate/ soluble ratio they found to occur after SV40 transformation, may result in the marked change in plasma membrane-associatedproperties such as contact inhibition of locomotion. If, upon the stimulus of contact, adhesive molecules move within the fluid lipid bilayer to the site of contact, the microfilaments seen in relation to the contacts may perform the work of moving them, and such a change as described by Fine and Taylor might prevent the appearance of such contact specializations. However, homologous contact inhibition has been described between transformed cells and, where they have been studied in detail, they have been shown (Heaysman and Pegrum, 1973~)to make adhesion plaques against the substrate. Such a change in the surface activity of the transformed cell also does not explain the apparent lack of contact specialization in a normal fibroblast when it is in contact with a tumor cell, unless it is the complementary nature of the specializations that makes the cell-cell contact so different from the cellsubstrate contact. Are these adhesion areas regions of intercellular communication? Flaxman et al. (1969) have suggested that tight junctions might further contact inhibition by allowing the passage from cell to cell of small ions which can alter cytoplasmic motility. The whole area of overlap between the fibroblast leading lamellae described by Heaysman and Pegrum is probably a region of cohesion, with the specialized areas forming points of close adhesion, but the type of junction formed has not been identified. They may take the form of tight or gap junctions. Azarnia and Loewenstein ( 1977) have described functional junctions developing between normal liver cells in a time span similar to that described for contactinhibiting fibroblasts. They found electrical coupling in a substantial fraction of cell contacts after 5 minutes. SA-2 1 hepatoma cells, however, made junctions more slowly; the first electrical coupling was not found for 75-125 minutes. The . contrast was even more striking with regard to fluorescein transfer. Even after 48 hours, only 7% of the SA-21 cells transferred fluorescein, whereas all normals transferred it within 60 minutes. Other workers have described cancer cells which show a reduced ability to communicate directly with other cells, some that are good communicators; but then, as described above, tumor cells normally show some degree of contact inhibition toward each other and frequently toward normal cells. Recent work by Fentiman and Taylor-Papadimetrious (1977) suggests that malignant change may be associated not only with a loss of ability to communicate but also with a loss of specificity in communication. They suggest that both changes could be selec-

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tively advantageous for the metastasis of cancer cells enabling them, as noncommunicators to ignore inhibitory signals or, as nonselective communicators to receive stimulatory messages from abnormal sources such as the surrounding fibroblasts. The idea that the exchange of a signal through specialized contacts causes inhibition of the locomotory mechanism of the cell is an attractive one but, as Trinkaus et al. (1971) point out, this inhibition is local, and they question whether a signal, of whatever nature, would be so restricted in its action. Loewenstein (1975), however, showed that calcium entering a cell was very rapidly sequestered close to the point of entry, and other ions or even larger signal molecules may be treated similarly. An even more serious doubt is engendered by the fact that, in most cases of contact inhibition, the inhibition is reciprocal, both cells being inhibited. It is difficult to explain how a substance that must be inert while in one cell becomes active upon being transferred to a colliding cell. It is highly unlikely that each individual cell produces a different inhibitor and, even if this were so, one still has to explain the phenomenon of self-contact inhibition. Ebendal and Heath (1978) have described collisions between two lamellar processes extending from a single locomoting cell and showed that the response is similar to that produced when two cells collide, not only as far as the adhesion, paralysis, and retraction of lamellae seen in the light microscope are concerned, but also in regard to the associated changes in ultrastructure. It appears to be much more likely that contact inhibition results from a mechanical interaction. The surface structures involved in such an interaction might well exist in varying proportions in different types of cells, thus explaining the variation in the degree of contact inhibition shown. If we accept that, when contact is made by a cell with the substrate or another cell, polymerization of actin filaments may be triggered in the subjacent cytoplasm, and this has been suggested to happen not only in fibroblasts in culture (Abercrombie et al., 1971; Heaysman and Pegrum, 1973a) but also in platelets (Behnke et al., 1971), we may expect this reaction to pull the cell forward against the substrate or to pull the cells together against each other. Chick heart fibroblasts display this initial pulling together but, after approximately 10 minutes, they pull apart, often violently. It is difficult to determine the actual underlying cause of this separation. Cell-substrate plaques are thought of as being temporary structures, breaking down as the cell moves forward, and this separation could result from such a breakdown of c e l k e l l adhesions, its violent nature being due to the tension built up in the aligned microfibrils (Heaysman and Pegrum, 1973a). Alternatively, the cells could be pulled apart as a result of locomotion occurring in another area of the cell. Such locomotion almost always occurs when room is available around the colliding cells.

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This separation does not occur when two epithelial cells collide (Middleton, 1972). These cells display the other characteristics of contact inhibitionadhesion and paralysis-but once an isolated epithelial cell has made contact with another cell or a group of cells of the same type, it normally does not separate itself. Indeed, it exhibits the phenomenon of contact-induced spreading, described by Middleton (1976) as “an increase in spread area associated with contact.” The failure of epithelial cells to separate after a collision may be due either to the relatively limited locomotory ability of the epithelial cells allowing the adhesions to stabilize, or to the formation of stronger and more complex areas of adhesion than are found between fibroblasts. Such contacts have been described between epithelial cells which have been in culture for approximately 24 hours (Middleton and Pegrum, 1976), and a detailed study of the time course of the development of these adhesions comparable to that carried out for fibroblasts is in progress. Similar studies with other types of tissue cells may help us to understand more about the underlying ultrastructural components of the contact inhibition reaction.

VII. Conclusion In conclusion four main points need emphasizing. A reduction in overlap and the formation of a monolayer can be brought about in one of two ways. The first is by the classic contact inhibition response described as a process involving adhesion, paralysis, and contraction, and the second is the consequence of a stronger adhesion to the substrate than to the contacting surface. Which particular situation is operating can be decided only by monitoring individual collisions. It has been suggested in this article that the original term “contact inhibition of locomotion” should be retained for the first type of response and that the second type should be described as substrate-dependent inhibition of locomotion, as in this instance the reaction is probably not due to the formation of an actual contact but rather to a failure to form an adhesion to the colliding surface, whether it be the surface of another cell or of a nonliving substrate. Tumor cells are not properly described as being devoid of contact inhibition. Most, if not all, tumor cells exhibit homologous contact inhibition. From the point of view of invasion the heterologous inhibition of tumor cells by normal cells and the reciprocal inhibition of normal cells by tumor cells is probably of more importance than their individual homologous inhibitions. However, even here it may not be the absolute value of the heterologous inhibition that is important but the relative values of homologous and heterologous inhibition. Contact inhibition is not necessarily an all-or-none reaction; cells show different overlap indexes in different culture situations and with different cells,

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while overlap indexes vary among cells of different origin. Nonreciprocal inhibition may also occur. Many instances of apparent failure of contact inhibition may be due to a change in cell substrate adhesion such that less actual close contact occurs. In all cases a definite decision as to whether contact inhibition actually occurs, and its frequency, can be obtained only by the monitoring of individual collisions. The very variability of this response throws doubt on the theory that it is initiated by the exchange of signal substances through electrical coupling. The rapid formation of specialized adhesion areas at points of contact in some contact-inhibiting cells is of obvious interest, but the precise function of these areas is unknown. Finally, we do not know to what extent the cellular interactions we have described here play a part in the control of cellular locomotion in the in vivo situation. That they are, together with other phenomena, such as densitydependent inhibition of mitosis and contact guidance, of importance in both morphogenesis and carcinogenesis is highly probable. But we must be very careful when we attempt to extrapolate from the two-dimensional situation we generally study in tissue culture to the far more complex situation found in the organism.

ACKNOWLEDGMENTS My thanks are due to Michael Abercrombie for his kindness in reading and criticizing this text and for our many years of collaboration. I also wish to thank my colleagues Adam Middleton and Susan Pegrum for discussing with me and allowing me the use of as yet unpublished material.

REFERENCES Abercrombie, M. (1970). I n Virro 6, 128-142. Abercrombie, M. (1975). I n “Cellular Membranes and Tumor Cell Behaviour,” pp. 23-37. Williams and Wilkins, Baltimore, Maryland. Abercrombie, M., and Dunn, G. A. (1975). Exp. Cell Res. 92, 57-62. Abercrombie, M., and Heaysman, J . E. M. (1954). Exp. Cell Res. 6, 293-306. Abercrombie, M., and Heaysman, J. E M. (1976). J . Natl. Cancer Inst. 56, 561-570. Abercrombie, M., Heaysman, J. E. M., and Karthauser, H. M. (1957). Exp. Cell Res. 13, 276-291. Abercrombie, M., Lamont, D. M., and Stephenson, E. M. (1968). Proc. R. Soc. London, Ser. B 70, 349-360. Abercrombie, M., Heaysman, J. E. M., and Pegrum, S. M. (1970a). Exp. Cell Res. 60, 437444. Abercrombie, M., Heaysman, J. E. M., and Pegrum, S. M. (1970b). Exp. Cell Res. 62, 389-398. Abercrombie, M., Heaysman, J. E. M., and Pegrum, S. M. (1971). E q . Cell Res. 67, 359-367. Abercrombie, M., Heaysman, J. E. M., and Pegrum, S. M. (1972). I n “Cell Differentiation” (R. Hams, P. Allin, and D. Viza, eds.), pp. 4 6 4 8 . Munksgaard, Copenhagen. Albrecht-Buehler, G. (1973). Exp. Cell Res. 78, 67-71

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Albrecht-Buehler, G., and Chen, L. B. (1977). Nature (London) 266, 454-456. Armstrong, P., and Lackie, J. (1975). J . Cell B i d . 65, 439-463. Azamia, R., and Loewenstein, W. R. (1976). J . Membr. Biol. 30, 175-186. Barnett, R. E., Furcht, L. T., and Scott, R. E. (1975). Proc. Natl. Acad. Sci. U.S.A. 72, 1217. Barski, G., and Belehradek, J . (1965). Exp. Cell Res. 37, 464-480. Behnki, O., Kristensen, B. I., and Milsin, L. E. (1971). J . Ultrasrrucr. Res. 37, 351-369. Bell, P. B. (1972). J. Cell B i d . 55, 31a. Bellairs, R. (1978). In preparation. Boyde, A,, Grainger, F., and James, D. W. (1969). Z. Zellforsch. Mikrosk. Anar. 94, 46-55. Bray, D. (1970). Proc. Natl. Acad. Sci. U.S.A. 65, 905-910. Carter, S. B. (1967). Nature (London) 213, 256-260. Castor, L. N. (1971). Exp. CellRes. 68, 17-25. Curtis, A. S. G. (1964). J . Cell Biol. 20, 199-216. Di Pasquale, A. (1975). Exp. Cell Res. 94, 191-215. Di Pasquale, A., and Bell, P. B. (1974). J . Cell Biof. 62, 198-215. Dunn, G. A. (1971). J . Comp. Neurol. 143, 491-507. Ebendal, T., and Heath, J. P. (1978). Exp. Cell Res. 110, 469-473. Erickson, C. A. (1976). J . Cell Biol. 70, 250a. Fentiman, I. S . , and Taylor-Papadimetrious, J. (1977). Nature (London) 269, 156-157. Fine, R. E., and Taylor, L. (1976). Exp. Cell Res. 102, 162-169. Flaxrnan, B. A., Revel, J. P., and Hay, E. D. (1969). Exp. CeNRes. 58, 438-443. Guelstein, V. I., Ivanova, 0. Y.,Margolis, L. B., Vasiliev, J. M., and Gelfaud, I. M. (1973). Proc. Narl. Acad. Sci. U.S.A. 70, 201 1-2014. Hams, A. K. (1973a). Exp. Cell Res. 77, 285-297. Harris, A. K. (1973b). Dev. Biol. 35, 97-115. Hams, A. K. (1973~).I n “Locomotion of Tissue Cells” (R. Porter and D. W. Fitzsimons, eds.), Ciba Foundation Symposium (Discussion, p. 347). Elsevier, North Holland. Hams, A. K. (1974).In “Cell Communication” (R. P. Cox, ed.), pp. 147-185. Wiley, New York. Hams, A. K., and Dunn, G. A. (1972). Exp. Cell Res. 73, 519. Heaysman, J. E. M. (1970). Experientia 26, 1344. Heaysman, J. E. M., and Pegrum, S. M. (1973a). Exp. Cell Res. 78, 71-78. Heaysman, J. E. M., and Pegrum, S. M. (1973b). Exp. Cell Res. 78, 479-481. Heaysman, J . E. M., and Pegrum, S. M. (1973~).Diflerentiation 1, 191-198. Heaysman, J. E. M., and Turin, L. (1976). Exp. Cell Res. 101, 419-422. Ivanov, C. Y., and Margolis, L. B. (1973). Narure (London) 242, 200-201. Lochner, L. R., and Izzard, C. S. (1973). J . Cell Biol. 59, 199a. Loewenstein, W. R. (1975). I n “Cellular Membranes and Tumor Cell Behaviour,” p. 239. Williams and Wilkins, Baltimore, Maryland. Maroudas, N. G. (1975). Nature (London) 254, 695-696. Martz, E. (1973). J . Cell. Physiol. 81, 39-48. Martz, E., and Steinberg, M. S. (1972). J . Cell. Physiol. 79, 189-210. Martz, E.,and Steinberg, M. S. (1973). J . CeN Physiol. 81, 25-38. Martz, E., Phillips, H. M., and Steinberg, M. S. (1974). J. Cell Sci. 16, 401-409. Middleton, C. A. (1972). Exp. Cell Res. 70, 91-96. Middleton, C. A. (1976). Nature (London) 259, 311-313. Middleton, C. A., and Pegrum, S . M. (1976). J . Cell Sci. 22, 371-383. Oldfield, F. E. (1963). Exp. Cell Res. 30, 125-138. Poste, G. (1973). Exp. Cell Res. 77, 264-271. Projan, A., and Tannberger, S. (1973). Eur. J . Cancer 9, 703-709. Steinberg, M. S. (1970). J . Exp. 2001.173, 395434.

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INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 55

Morphological Correlates of Electrical and Other Interactions through Low-Resistance Pathways between Neurons of the Vertebrate Central Nervous System C. SOTELO AND H. KORN' Laboratoire de Neurornorphologie, Centre Medico-Chirurgical Foch, Suresnes, France

I. General Considerations . . . . . . . . . . . . . . . . A. Historical . . . . . . . . . . . . . . . . . . . B. Correlation between Low-Resistance Pathways and Gap Junctions . . . . . . . . . . . . . . . . . . . . 11. Ultrastructure of Electrical Synapses . . . . . . . . . . . A. Introduction . . . . . . . . . . . . . . . . . . B. Gap Junctions as Electrical Synapses . . . . . . . . . C. Gap Junction Conformational Changes and Functional State . D. Rectifying Electrical Synapses . . . . . . . . . . . . 111. Electrical Synapses in the CNS of Nonmammalian Vertebrates . A. Gap Junctions in Long-Projecting Pathways . . . . . . . B. Gap Junctions in Locally Operating Circuits . . . . . . . IV. Electrical Synapses in the Mammalian CNS . . . . . . . . A. Between Neuronal Perikarya . . . . . . . . . . . . B. Between Dendrites . . . . . . . . . . . . . . . C. Between an Axon Terminal and a Neuronal Perikaryon . . D. Between Dendritic Appendages in a Glomerular Formation . . V. Chemical Synaptic Control of Electrotonic Coupling . . VI. Mixed Synapses . . . . . . . . . . . . . . . . . VII. Gap Junctions and Molecular Transport . . . . . . . . . . A. Metabolic Coupling . . . . . . . . . . . . . . . B. Low-Resistance Pathways and Neuronal Differentiation . . . References . . . . . . . . . . . . . . . . , . . .

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I. General Considerations After the great advances brought about by the introduction of intracellular recording methods and electron microscopy, it became generally accepted that the long controversy involving electrical and chemical mechanisms of synaptic transmission had been resolved in favor of the latter (for a historical review, see 'Maitre de Recherches (INSERM, U 3).

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Eccles, 1964). A reflection of this current idea can be found in the articles in this series dealing with the morphology of synapses. For instance, in their respective reviews, De Robertis (1959) mentioned only chemical synapses, and Gray and Guillery (1966) devoted less than one-tenth of their paper to electrotonic transmission. However, more recent investigations have clearly emphasized the importance of electrotonic transmission in brain function, that is, in synaptic transmission, molecular transport, and perhaps neuronal differentiation. This chapter, which summarizes these new data, discusses electrical junctions in the vertebrate central nervous system (CNS). Since a large amount of information on electrotonic synapses has been obtained from invertebrate preparations, some of this work is also considered when necessary. This review makes it obvious that, on morphological and physiological grounds, a broadening of opinion is necessary, since electrical transmission appears to be a major mechanism of neuronal interaction in the CNS of vertebrates, including in mammals. A. HISTORICAL

The discovery in the middle of the nineteenth century that peripheral nerves were electrically excitable generated the idea that electricity was directly responsible for muscle contraction (Du Bois-Reymond, 1848) and that communication between nerve cells was electrically mediated. Later, however, Du BoisReymond (1874) favored the opposite hypothesis that a peripheral nerve releases an excitatory substance which is the cause of muscle contraction, but this somewhat obscure work cannot be considered a pioneering study on the chemical nature of synaptic transmission. Ironically this work was commonly cited by early supporters of the electrical transmission theory, and today it is considered by some to have been a “prediction” of the existence of both types of synaptic transmission. The question of the nature of synaptic transmission became crucial at the beginning of the twentieth century, after establishment of the neuron theory. A series of important discoveries made over 50 years, culminating in the early itudies using intracellular recording, resulted in a demonstration of the chemical nature of synaptic transmission at the neuromuscular synapse (Fatt and Katz, 1951) and in spinal cord motoneurons (see in Eccles, 1953). Therefore, early in the 1950s, the electrical transmission theory was almost completely abandoned. It was considered a rare kind of synaptic mediation almost exclusively observed in phylogenetically primitive forms (Eccles, 1964). However, more recent electrophysiological data have little by little demonstrated the existence of electrotonic transmission not only in invertebrates (Furshpan and Potter, 1959; Watanabe and Grundfest, 1961; Hagiwara and Morita, 1962; Levitan et al., 1970; Spira and Bennett, 1972) but also in vertebrates (Washizu, 1960; Bennett

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et al., 1963; Martin and Pilar, 1963; Furshpan, 1964; see other references in Bennett, 1972, and in Korn et al., 1977), including mammals (Baker and LlinBs, 1971; Korn et al., 1973; Llinas et al., 1974; LlinBs, 1975). It is now well established that synaptic transmission can be electrically or chemically mediated, and cases have been described in which both modes of transmission can operate between the same two neurons (see Section VI). It should be remembered that the physiological evidence supporting this conclusion often preceded the morphological data.

B. CORRELATION BETWEEN LOW-RESISTANCE PATHWAYS AND GAPJUNCTIONS The model of electrical mediation postulates that current flow spreads passively, or electrotonically, from the neuron in which it is generated to a neighboring one. In this way, transmission occurs without or with little of the synaptic delay typical of chemical transmission synapses (however, for a discussion of this principle, see Bennett, 1966, 1972). In addition, in the large majority of electrical synapses studied, the electrotonic spread of depolarizing current was bidirectional. Therefore the two properties which characterize electrotonic transmission are (1) high speed and (2) reciprocity, both of which contribute to a shorter latency response and to a more synchronous discharge of a group of neurons coupled together; for this reason Bennett (1972) proposed that they be called “synchronizing synapses.” Again with reference to the morphological requirements of electrical transmission, it should be kept in mind that neurons are independent units isolated from each other by glial envelopes. At sites where two or more different neuronal processes are in direct apposition, they are still separated from each other by their own plasmalemmas and by an extracellular space 150-300 A in width. The electrical resistance of neuronal membranes and of the extracellular space is thus great enough to prevent significant electrotonic coupling between cells (for a discussion, see Katz, 1966). Therefore electrical interactions between neurons must necessarily occur at specific sites on the membranes which could constitute low-resistance pathways. Such an ideal morphological structure could be represented by minute cytoplasmic bridges between neurons, which would allow maximum current flow between the cells but little or no leakage out into the extracellular space. Indeed, morphological studies have demonstrated that in most instances electrical transmission takes place at identifiable and morphologically distinct sites where the membranes of the coupled cells come together to form now classic gap junctions (Revel and Karnovsky, 1967). These junctions are intercellular communicating channels (see in Staehelin, 1974), and they constitute the ideal anatomical structure for the electrotonic spread of current between either excitable or nonexcitable cells. The correlation between the

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incidence of gap junctions and electrotonic coupling is now well established, and numerous examples of this correlation have been demonstrated in the vertebrate nervous system. (See Section 111.)

11. Ultrastructure of Electrical Synapses

A. INTRODUCTION In early ultrastructural studies of electrical synapses, the common feature observed in coupled neurons was the presence of specialized membrane regions where plasma membranes were in close apposition, constituting a junctional complex 150-170 A in width, suggesting that in this area the intercellular space was absent or very small. The appearance of these junctions varied substantially as methods of investigation and fixation became more sophisticated and precise. First, Farquhar and Palade (1963) described a new kind of junctional complex in mucosal epithelial cells, exhibiting a pentalaminar configuration, in which the external leaflets of the junctional membranes were fused, thus occluding the extracellular space; they called them “tight junctions” or “zonula occludens.” Subsequently, in most studies of electrical synapses, the close membrane appositions representing low-resistance pathways were referred to as tight junctions. In 1967, with new staining techniques (such as those using aqueous uranyl acetate solution prior to dehydration and embedding, or those employing alkaline lanthanum nitrate as a tracer for extracellular space), Revel and Karnovsky showed that there were two completely different types of so-called tight junctions. One of them corresponded to the original description of Farquhar and Palade (1963) of tight junctions and formed continuous beltlike regions in which there were no intercellular clefts, since they were not penetrated by the lanthanum. In contrast, the second type consisted of macular zones of variable diameter where the apposing plasmalemmas were not completely fused, since there was a narrow gap 15-20 8, in width between the membranes which was penetrated by the lanthanum and which therefore allowed communication between the extracellular spaces on either side. Thus this new type of intercellular junction was called a gap junction. Finally it was further established that tight junctions per se were transepithelial permeability barriers, whereas gap junctions represented intercellular communicating pathways (see references in Staehelin, 1974).

B . GAPJUNCTIONSAS ELECTRICAL SYNAPSES Since a thorough analysis of the morphology of gap junctions has already been presented in this series (Staehelin, 1974), we restrict this article to gap junctions of the vertebrate CNS.

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Excitatory electrical transmission in the CNS of vertebrates was first described in fish. Bennett et al. (1963) reported electrically mediated transmission between giant electromotor neurons of mormyrid fish and clearly established that this phenomenon was correlated with the presence of specific membrane junctions of the close apposition variety between the investigated cells. In the same year, Robertson (1963), working on the ultrastructure of the club endings synapsing on the lateral dendrite of the Mauthner cell of the goldfish, demonstrated most of the features which characterize the membranes involved at electrically transmitting synapses; a little later, Furshpan (1964) reported the existence of electrical transmission to the Mauthner cell and postulated that it was mediated through these specialized junctions. Comparing the appearance of apical ependymal gap junctions after various preparative methods, Brightman and Reese (1969) showed that these areas exhibited different morphological features depending on the method used: 1 . When the tissue was fixed in osmium tetroxide, dehydrated in alcohol, and embedded in plastic, and the thin sections stained with uranyl and lead, the gap junctions appeared to have five layers; the median lamina, instead of being continuous, appeared as a row of regularly spaced globules. The overall width of these junctions was 135-150 A. 2. If after osmic fixation, but prior to dehydration and embedding, the tissue was stained en bloc with an aqueous solution of uranyl acetate, the gap junctions exhibited, as first described by Revel and Karnovsky (1967), a seven-layered appearance and had an average width of 135-150 A. The median lamina was bisected by a narrow gap 15 -20 A in width. 3 . Finally, when the tissue was fixed in potassium permanganate, the junctions presented a five-layered appearance, and the electron-lucent layers of the heptalaminar pattern appeared electron-dense. The overall width of these fivelayered junctions was somewhat less than that of the seven-layered junctions. This conformational diversity explains the heterogeneity and the somewhat confusing descriptions reported in the early work on electrotonic junctions.

Today it is generally accepted that under favorable staining conditions, provided that the plane of section is perpendicular to the cell surfaces, neuronal gap junctions exhibit a heptalaminar configuration (Fig. 1). A unique feature almost invariably present is a cytoplasmic semidense material which undercoats the whole length of the inner surfaces of the junctional plasma membranes (Fig. 1). Occasionally, the distribution of this cytoplasmic undercoating is asymmetric, for example, in the electrical synapses between spinal motoneurons of the toadfish sonic muscle system (Pappas and Bennett, 1966) and in the gymnotid fish cerebellum (Sotelo and Llinhs, 1972). The undercoating and its occasionally asymmetric distribution have not yet been correlated with specific functions.

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FIGS.1 and 2. Ultrastructural features of neuroneuronal gap junctions.

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In favorable sections, a close examination of the low-resistance junctions discloses that the central narrow gap is not uniform, since there are spot contacts bridging the gap between the outer leaflets of the plasma membranes. The bridges have a periodicity of about 90 (Fig. 1). At some of the glial gap junctions, a complementary image of the central gap is observed after they are filled with lanthanum (Sotelo, 1977a). In these instances the stained extracellular space exhibits a beaded appearance, and neighboring opaque beads are separated by narrow light spots, the latter corresponding to the intermembranous bridges. Enface views of gap junctions in which the extracellular space is filled with lanthanum, resemble the description reported by Robertson (1963) for potassium permanganate-fixed material. The gap junction exhibits a honeycomb pattern composed of a system of lines disposed in a hexagonal network, the center of each hexagon being occupied by an electron-opaque spot. The best analyses of the membrane configuration at electrical synapses have been obtained by freeze-fracturing. With the use of this technique different examples of vertebrate gap junctions have been described (see Table I); in all cases they appear as polygonal lattices of particles protruding from the inner protoplasmic surface (P face) of the junctional membranes and complementary lattices of pits on the outer or extracellular surface (E face). The intramembranous particles measure about 80 A in diameter and are arranged in an almost hexagonal array with a center-to-center spacing of about 90 A (Fig. 2). Therefore these synapses exhibit the same intramembrane particle organization as gap junctions between nonexcitable cells (see references in Staehelin, 1974). The disposition of both junctional membranes is symmetric, such that the particles on one P face are in register with those on the other P face (Fig. 2). The particles represent proteins that completely traverse the lipid bilayer. Each of the opposing particles protruding into the extracellular space spans the 20-A gap which separates the two junctional membranes and forms with the opposite particle a continuous dimer, a “connexon” (according to the nomenclature introduced by Goodenough, 1976) which bridges the cytoplasm of the two connected cells. Since the centers of the proteins composing the gap junction particles are considFIG. 1. Axosomatic gap junction in the magnocellular mesencephalic nucleus of the weakly electric fish G. curapo. A layer of cytoplasmic material of moderate electron density underlies either side of the junction (arrows). The narrow gap between the two junctional membranes exhibits spot contacts which bridge the extracellular space, giving the gap a beaded appearance. The axon terminal (AT) contains vesicular profiles which are distant from the junctional membrane. X 210,000. (From Sotelo er ul., 1975.) FIG. 2. Freeze-fractured membrane of a mossy fiber (chicken cerebellum). Two aggregates of large P-face particles are present. The particles are arranged in the usual way which characterizes gap junctions (GJ).The fracture plane at the lower right steps from face E to face P. The E face (arrow) is formed by pits which are arranged in a hexagonal array and are complementary to the large P-face particles. x 82,500. (From Sotelo 1977a.)

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TABLE I ELECTRICAL SYNAPSES (GAPJUNCTIONS) STUDIED WITH THE FREEZE-FRACTURE METHODIN ADULTVERTEBRATE NERVOUS SYSTEM

THE

Nervous system of fish Isolated synaptic membranes of the goldfish medulla oblongata (Zampighi and Robertson, 1973) Lamprey spinal cord (Pfenninger and Rovainen, 1974) Gymnotid spinal cord (Bennett et al., 1977) Skate retina, between horizontal cells (Fain et al., 1976) Nervous system of amphibians Toad retina, between red rods (Fain et al., 1976) Nervous system of reptiles Turtle retina, between photoreceptors (Raviola and Gilula, 1973) Nervous system of birds Chick cerebellum, at the glomeruli (Cantino and Sotelo, present review) Chick tangential nucleus, at the spoon synaptic endings (D. Cantino, unpublished) Chick ciliary ganglion, at the calyceal synapse (Cantino and Mugnaini, 1975) Nervous system of mammals Rabbit olfactory bulb (Landis er al.. 1974) Outer plexiform layer of the primate retina (Raviola and Gilula, 1973, 1975)

ered hydrophilic pores, the structural disposition of the connexons confers on them the role of permeability channels which allow direct cytoplasmic communication between the two junctional cells. The nature of the interactions holding the two particles together in a connexon is still unknown. AND FUNCTIONAL STATE C. GAPJUNCTION CONFORMATIONAL CHANGES

Our understanding of the structural organization of gap junctions between nonexcitable cells has been extended in recent years to the molecular level (Dunia et al., 1974; Goodenough, 1974, 1976; Duguid and Revel, 1976). Although some discrepancies still exist, it seems that gap junctions have a major protein constituent with a molecular weight of about 34 X lo3 and, although studies on neuronal gap junctions are still lacking, it is likely that all gap junctions are similar since they share the same permeability properties and the same morphological features. Morphological data and x-ray diffraction experiments (Goodenough, 1976) support the idea that the gap junction subunits, or connexons, form the permeability channels through which intercellular coupling occurs. Although the observation that the hydrophilic pore present at the center of the connexons crosses the full thickness of the junction has not been confirmed, it is essential to determine if these channels act as passive holes through which all molecules can diffuse (if

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they are small enough) or if channel permeability is controlled by an active regulatory mechanism. Work done on the salivary glands of insects by Loewenstein and collaborators (references in Rose et al., 1977) suggests that the permeability of gap junctions is directly dependent on the concentration of unbound calcium in the cytoplasmic compartment. An increase in the concentration of free Ca2+ paralleled an increase in the coupling resistance. Experiments conducted on the electrical synapses between crayfish septate lateral giant axons (Pappas et al., 1971), in which functional uncoupling obtained by different experimental manipulations was accompanied by a morphological disruption of the gap junctions, seemed to indicate that uncoupling was associated with the disappearance of the low-resistance junctions. However, under other experimental circumstances an increase in coupling resistance took place without important changes in the structure of these gap junctions (Payton et al., 1969; Politoff and Pappas, 1972). Furthermore, Peracchia (1973a,b), in a morphological analysis of the same electrical synapse, described two different patterns of gap junction arrangements depending on the preparative procedure used. When they were studied in thin sections or in replicas obtained by freeze-fracture, the lattice of connexons exhibited a center-to-center spacing of about 200 (swollen form), but in negatively stained isolated membranes the spacing of the connexons decreased to 150-155 A (shrunken form). Peracchia proposed that the conformational changes involved in passing from the former to the latter configuration were the morphological expression of changes in the permeability of the junctional membranes, passing from low to high coupling resistance. In order to test this hypothesis (and that of Loewenstein on the role of unbound calcium in the regulation of gap permeability), Peracchia and Dulhunty (1976) carried out a series of electrophysiological and morphological experiments; the ventral nerve cord of the crayfish was treated in v i m with different solutions containing either a chelator of Ca2+ and Mg2+ or a metabolic inhibitor. The cytoplasmic concentration of unbound calcium increased under both conditions (treatment with EDTA followed by normal Van Harreveld’s solution for over 1-2 hours, or treatment with dinitrophenol), and electrical measurements confirmed that the coupling resistance was also greatly increased. A parallel morphological study showed that the electrical uncoupling corresponded to the shrunken conformational pattern of the gap junctions. The uncoupling was accompanied by a decrease in the connexon diameter. When the ventral nerve cord was put back in normal Van Harreveld’s solution, for over 4 hours, the change was found to be reversible; the coupling resistance went back to normal values, and the junctions resumed the swollen conformational pattern. These results strongly suggest not only that the permeability of gap junctions is under ionic control, but that changes in this permeability are paralleled by conformational changes in the connexon proteins.

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D. RECTIFYING ELECTRICAL SYNAPSES There are several examples in which electrically mediated transmission is unidirectional. The first one described, by Furshpan and Potter (1959), was the synapse between the giant fibers in the abdominal nerve cord of the crayfish and the motor giant fibers which innervate the fast-acting flexor muscles of the tail. Depolarizing current spreads passively and directly from the pre- to the postsynaptic fiber, but not in the opposite direction, whereas hyperpolarizing current crosses only from the post- to the presynaptic fiber. Thus these junctions allow synaptic transmission to occur in only one direction, as do chemical synapses. Other examples of rectifying electrotonic junctions include those between retinular and eccentric cells in the omnatidial eye of the horseshoe crab Limulus (Smith et al., 1965) and between the giant fibers and the motoneurons of the adductor muscles of the pectoral fins of the hatchet fish Gasteropefecus (Auerbach and Bennett, 1969). Only two of these junctions have been morphologically analyzed. Lasansky (1967) described close membrane appositions mainly between adjoining microvilli of retinular cells and also between eccentric cells and the tips of microvilli of retinular cells; the two apposed junctional membranes appeared to be fused, constituting five-layered junctions, and they were considered to be the morphological correlate of the electrotonic coupling between these cells. The giant motor synapses of the crayfish have been the subject of numerous morphological investigations. In early, conflicting ultrastructural studies they were described, according to the technical procedure used, as (1) close appositions in which the membranes of the two apposing axons were separated by a synaptic cleft about 100 A wide (Hama, 1961) or (2) fused together (Robertson, 1955). Both reports described the synapses as symmetric because, in spite of the absence of cytoplasmic differentiation on either side of the junction, there were vesicles about 400-600 A in diameter and tubular profiles of the smooth endoplasmic reticulum in both axons adjacent to the synaptic membranes. In more recent studies (Stirling, 1972; Keeter et al., 1974) this rectifying synapse was described (3) as asymmetric, because a few pleomorphic vesicles were found presynaptically (i.e., in the medial giant fiber) and a dense cytoplasmic differentiation was associated with the postsynaptic membrane (the giant motor fiber). Recently, Hanna et al. (1976), using the freeze-fracture technique, analyzed the membrane structure of the close appositions constituting these synapses. They concluded that the junctional membranes exhibited the typical gap junction appearance observed in nonrectifying lateral septa1 synapses. The only morphological difference reported between rectifying and nonrectifying electrical synapses in the crayfish is the presence, in the latter, of a few intracellular pleomorphic vesicles close to the presynaptic membrane, whereas in the nonrec-

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tifying one vesicles lie adjacent to both junctional sides. An attractive explanation for the rectification property of the synapse is that the vesicles have a role as C$+-sequestering organelles (D. D. Potter, mentioned by Peracchia and Dulhunty, 1976).. C$+ enters axons during the action potential and, if it is not sequestered from the hyaloplasm, an increase in its concentration may block the permeability of the gap junction. The presence of vesicles only in the presynaptic element prevents a high concentration of unbound calcium at this site. Since the postsynaptic side lacks vesicles, rectification may be dependent on an increase in the free C$+ concentration on this side of the synapse.

111. Electrical Synapses in the CNS of Nonmammalian Vertebrates

Instances in which electrical synapses have been demonstrated morphologically in the CNS of nonmammalian vertebrates are listed in Table 11. Their number, which has increased in recent years, that is, as soon as special interest was devoted to them by neurocytologists, proves that electrical transmission indeed represents a major mechanism of synaptic interaction. As in the case of chemically transmitting junctions, gap junctions can be present at various locations on the neuronal membrane; axosomatic, axodendritic, axoaxonic, somatosomatic, somatodendritic, and dendrodendritic gap junctions have been encountered in different regions of the CNS (see Table 11). From a functional viewpoint two different anatomical situations can be arbitrarily distinguished, as considered in the following discussion. A. GAPJUNCTIONS

IN

LONG-PROJECTING PATHWAYS

In some afferent pathways, communication between neurons is electrically mediated. Such is the case in electric fishes at several levels of the electromotor neuronal chain (Bennett, 1968) and of electrosensory systems (Szabo, 1974). In these pathways, neuroneuronal gap junctions are numerous (Bennett et al., 1967b,c,d; Meszler et al., 1974; Sotelo et al., 1975). Furthermore, electrical transmission is not restricted to such specialized organs, and in other pathways electrotonic mediation also occurs, at least at one of the synaptic steps. Two of these pathways are considered here, those of the vestibular system and of the cerebellum. 1. Vestibular Nuclei Electrophysiological as well as morphological evidence has accumulated in recent years in favor of electrical transmission between some primary vestibular fibers and neurons in the vestibular complex of all the nonmammalian vertebrates studied (see references in Korn et al., 1977). Gap junctions are established on

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C. SOTELO AND H. KORN TABLE I1 ELECTRICAL SYNAPSES (GAPJUNCTIONS) IN THE ADULTVERTEBRATE NERVOUS SYSTEM

Peripheral nervous system Ciliary ganglion of birds, axosomatic (Takahasbi and Hama, 1965; De Lorenzo, 1966; Koenig, 1967; Cantino and Mugnaini, 1975) Cardiac autonomic ganglion of the mud puppy, somatosomatic (McMahan and Purves, 1976) Labyrinth of the rat, axosomatic at the calyceal synapse of type I vestibular hair cells (Hamilton, 1968) CNS of fish Electromotor and electrosensory pathways Mormyrid spinal electromotor nucleus, dendrodendritic (Bennett er al. , 1967b) Mormyrid medullary relay nucleus, dendrodendritic (Bennett et al., 1967b) Catfish giant electromotor neurons, axosomatic and axodendritic (Bennett et al., 1967c) Gymnotid spinal electromotor nucleus, axosomatic and axodendritic (Pappas and Bennett, 1966; Pappas et al., 1975) Gymnotid medullary relay nucleus, axosomatic (Bennett et al., 1967d) Gymnotid pacemaker neurons, axosomatic and dendrodendritic (Bennett er al., 1967d) Electromotor neurons of the electric eel, axosomatic (Pappas and Bennett, 1966; Meszler ef al., 1972, 1974) Gymnotid magnocellular mesencephalic nucleus, axosomatic (Sotelo er al., 1975) Spinal cord Lamprey spinal cord, axosomatic and axodendritic (Rovainen, 1974a,b) Toadfish sonic muscle system, axosomatic (Pappas and Bennett, 1966) Goldfish and tench ventral horn, axosomatic and axodendritic (Gray, 1970; Schnitzlein and Brown, 1975) Atlantic puffer supramedullary neurons, axoaxonic (Bennett et al., 1967a) Brain stem Goldfish Mauthner cell, axodendritic, axosomatic, and axoaxonic (Robertson, 1963; Robertson ef al., 1963; Nakajima, 1974) Lamprey vestibular nuclei, axosomatic (Stefanelli and Caravita, 1970) Goldfish tangential nucleus, axosomatic (Hinojosa, 1973) Toadfish LVN, axosomatic and axodendritic (Korn et al., 1977) Teleost oculomotor nuclei, axosomatic (Kriebel et al., 1969; Waxman and Pappas, 1971) Selachian mesencephalic trigeminal nucleus, somatosomatic (Witkovsky and Roberts, 1976) Cerebellum Mormyrid cerebellum, axodendritic at the glomeruli (Kaiserman-Abramof and Palay, 1969) Gymnotid cerebellum, axodendritic at the glomeruli (Sotelo and Llinas, 1972) TONSsemicircularis Teleost torus semicircularis, axosomatic (Ito, 1974) Retina Skate retina, between horizontal cells (Fain ef al., 1976) Dogfish retina, between bipolar cell axons and between horizontal cells (Witkovsky and Stell, 1973) CNS of amphibians Spinal cord Frog, spinal cord, axosomatic and dendrodendritic (Charlton and Gray, 1966; Sotelo and Taxi, 1970; Sotelo and Grofova, 1976) (continued)

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TABLE I1 (Continued) Brain stem Frog oculomotor nuclei, axosomatic (Waxman and Pappas, 1971) Frog ventral nucleus of the VIII nerve, axosomatic (Sotelo, 1977b) Cerebellum Frog cerebellum, axodendritic at the glomeruli (Sotelo 1969, 1976) Retina Tiger salamander, between horizontal cell processes (Lasansky, 1976) Toad, between red rods (Fain et al., 1976) Axolotl, between photoreceptors (Custer, 1973) CNS of reptiles Cerebellum Viper cerebellum, axodendritic at the glomeruli (Sotelo, 1977a) Retina Turtle retina, between photoreceptors (Lasansky, 1972) CNS of birds Vestibular nuclei Chick tangential nucleus, axosomatic (Hinojosa and Robertson, 1967) Cerebellum Pigeon and chick cerebellum, axodendritic at the glomeruli (E. Mugnaini, personnal communication; Paula-Barbosa, 1976; Sotelo, this review) Retina Chick retina, between principal and accessory cones (Fujisawa et al., 1976b) CNS of mammals Brain stem Mesencephalic trigeminal nucleus, somatosomatic; mouse (Hinrichsen and Larramendi, 1968; Brightman and Reese, 1969); rat (Imamoto and Shimizu, 1970; Sotelo, 1975) Rat LVN, axosomatic (Sotelo and Palay, 1967, 1970; Kom et al., 1973) Rat inferior vestibular nucleus, axosomatic (Sotelo, 1975) Rat ventral cochlear nucleus, somatosomatic and dendrosomatic (Sotelo et al., 1976) Inferior olivary complex, dendrodendritic at the glomeruli; cat (Sotelo et al., 1974); opossum (King et al., 1975); monkey (Sotelo, 1975; Rutherford and Gwyn, 1977); rat (Sotelo, this review) Cerebellum Cat cerebellum, dendrodendritic (Sotelo and Llinas, 1972) Cerebellum of mutant mice (weaver and reeler), dendrodendritic (C. Sotelo, unpublished) Olfactory bulb Rat olfactory bulb, dendrodendritic at the glomeruli (Pinching and Powell, 1971) Neocortex Primate neocortex (somatic sensory cortex, area 3; motorcortex, area4), dendrodendritic (Sloper, 1972) Retina Inner plexiform layer of the primate retina (Dowling and Boycott, 1966) Outer plexiform layer of the primate retina (Dowling and Boycott, 1966; Raviola and Gilula, 1973, 1975) Outer plexiform layer of the cat retina (Kolb and Famiglietti, 1976)

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the soma of neurons of the tangential nucleus of the goldfish (Hinojosa, 1973), on the soma and dendrites of lateral vestibular neurons of the toadfish (Fig. 3) (Korn et al., 1977), on the spoon endings of the vestibular nuclei of the lamprey (Stefanelli and Caravita, 1970), on the soma and dendrites of the neurons of the ventral nucleus of the VIII nerve of the frog (Fig. 17) (Sotelo, 1977b), and on the soma of the neurons from the tangential nucleus of the chick (Hinojosa and Robertson, 1967). In addition, if the vestibular complex is considered to be composed of cells which receive primary vestibular afferents (Mehler, 1972), the Mauthner cell of fish and urodeles might be part of this system (see discussion in Korn et al., 1977), and indeed gap junctions have been observed between the lateral dendrite of this neuron and vestibular afferents in the goldfish (Robertson et al., 1963; Nakajima, 1974). Finally, no ultrastructural study has been made of the vestibular nuclei in reptiles, but Richter et al. (1975) obtained electrophysiological evidence in favor of electrical transmission between primary vestibular fibers and vestibular neurons in the lizard, which suggests that gap junctions are present here also. Electrical transmission also occurs in the vestibular system of small rodents. In fact, gap junctions are established between axon terminals and giant cells of the lateral vestibular nucleus (LVN) and in the inferior (or descending) vestibular nucleus of both the rat (Sotelo and Palay, 1967, 1970; Sotelo, 1975; see Fig. 4) and mouse (C. Sotelo, unpublished observation). In confirmation, electrophysiological evidence of electrotonic coupling (Korn et al., 1972, 1973) and electrical transmission (Wylie, 1973) was obtained in the LVN of the rat. Thus, from a comparative viewpoint, this mode of transmission from primary vestibular fibers to postsynaptic vestibular neurons is rather homogeneous, at least up to the rat. There are some morphological similaritiesin the mode of termination of afferent fibers on the vestibular neurons in all the above species. Primary vestibular fibers terminate in very conspicuous axonal formations of considerable size, all of which originate from thick myelinated fibers. Based on their shape, four different main'types of endings have been described:

FIG.3. LVN of the toadfish Opsanus tau. A large axon terminal (AT) containing rounded synaptic vesicles is in synaptic contact with the perikaryon of a giant vestibular neuron (N). At the synaptic interface three classes of junctional complexes are present: a gap junction (GJ),an attachment plaque (AP), and an active zone (AZ). The first- and last-mentioned thus constitute a mixed synapse. Note that a large portion of the axolemma is involved in junctional formation. X57,800. (From Korn et al., 1977.) FIG.4. Descending vestibular nucleus of the rat. A large axon terminal (AT) establishes a mixed synapse with the perikaryon of a vestibular neuron (N); an active zone (AZ), attachment plaques (AP), and a gap junction (GJ) are present at the same synaptic interface. Note that the amount of membrane free of junctions is much larger in this figure than in Fig. 3. ~63,000.

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FIGS.3. and 4. Examples of mixed synapses in vestibular nuclei.

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1 . Giant club endings establish synaptic contacts mainly with the lateral dendrite of the Mauthner cell (Robertson et al., 1963; Nakajima, 1974), but occasional club endings have been observed synapsing on lateral vestibular neurons in the toadfish (Korn et al., 1977) and on neurons of the tangential nucleus in the goldfish (Hinojosa, 1973). They are characterized by a myelin sheath that ends only a few microns from the synaptic interface, and by the fact that the distal unmyelinated region of the axon does not enlarge but keeps almost the same diameter as the myelinated region. 2. Spoon endings have been encountered i s the vestibular nuclei of the lamprey (Stefanelli and Caravita, 1970) and in the tangential nucleus of the chick (Hinojosa and Robertson, 1967). As described earlier by Ramdn y Cajal(1908), the majority of these endings are boutons en passant which originate as short, thick side branches from vestibular fibers and follow a path toward the LVN. When these expansions are observed in silver preparations, they have the shape of a microscopic spoon. 3. Calyciform endings are present in the tangential nucleus of the goldfish (Hinojosa, 1973). They were described by Bodian (1937), with his protargol method, as a variety of the spoon endings, because they do not have a spoon shape but rather take the form of a trefoiled calyx. 4. Large, spherical synaptic boutons with an average diameter of about 6 p m (almost similar to those of giant club endings) establish synaptic contacts with the lateral vestibular neurons of the toadfish (Korn et al., 1977). The ultrastructural features of all four classes of axon terminals are almost identical. They contain a large number of neurofilaments, intermingled with microtubules, small mitochondria, and cisterns of smooth endoplasmic reticulum. Near the axolemmal membrane facing the vestibular postsynaptic neuron are clusters of synaptic vesicles. In all four classes of terminals the latter have a constant rounded shape (Fig. 3). At the synaptic interface three types of junctional zones are present: attachment plates, gap junctions, and active zones (see Section VI). The location of these three classes of junctions is almost identical in the four kinds of terminals. The edges of the synaptic interfaces consist of active zones. The large majority of the remaining membrane is occupied by gap junctions interspersed within attachment plates, so that only a fraction of the membrane is free of junctional contacts (Fig. 3). The amount of membrane participating in gap junctions is larger than that establishing active zones; however, the relative amount of surface occupied by active zones in the LVN increases from fish to rats. Electrophysiological studies have demonstrated electrical mediation from eight nerve afferents in the LVNs of the fish (Korn et al., 1977), frog (Precht et al., 1974), lizard (Richter et al., 1975), pigeon (Wilson and Wylie, 1970), and rat (Wylie, 1973), and also that some vestibular neurons are electrotonically

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coupled. Thus LVNs are coupled in the fish (Korn et al., 1977), frog (Precht et al., 1974), and rat (Korn et al., 1972, 1973), but not in the cat (Ito et al., 1964). It should be pointed out that as indicated by electrophysiological results coupling is close between neurons in the fish and frog, since in these species electrotonic spread of current from excited to unexcited cells is sufficient to bring the membrane potential of vestibular neurons to their firing level and to generate action potentials in a mode analogous to that of chemical synaptic transmission (Precht et al., 1974; Kom et al., 1977). In contrast, coupling is weaker in the rat, since the spread of synaptic potentials and spikes from neighboring cells is not sufficient by itself to excite vestibular neurons. These data obviously parallel morphological ones and reinforce the correlation between gap junctions and electrotonic coupling; gap junctions are abundant in the LVN of the fish and frog, where neurons are closely coupled; they are much less abundant in the LVN of the rat, and they are absent in that of the cat (Mugnaini et al., 1967), where electrical transmission does not occur. It would be interesting to determine if other mammalian species more closely related to rodents than the cat also use electrical transmission in the first steps of the synaptic vestibular chain. This mode of synaptic transmission would result in a reduction in the latency of escape and balancing responses, and therefore, as discussed elsewhere (Korn et al., 1977), the saving would be more significant (and necessary) in cold-blooded forms because of the greater sensitivity of the release process of chemical transmission to temperature. 2. Sensory Afferents to the CerebelIum Some of the mossy fibers in the cerebellar glomeruli of fish (KaisermanAbramof and Palay, 1969; Sotelo and Llinas, 1972), frogs (Sotelo, 1969, 1976), reptiles (Fig. 5 ) , and birds (E. Mugnaini, personal communication; PaulaBarbosa, 1976; Sotelo, 1977a) establish gap junctions with their postsynaptic granule cell dendrites. Gap junctions at this location do not exist in the mammalian cerebellum; the functional significance of their constant presence in some of the glomeruli of nonmammalian species and their absence in mammals still remains unexplained. In contrast to the situation in the vestibular system, for which the physiology of the electrical transmission is well documented, nothing is known about the physiology of these cerebellar gap junctions, mainly because of the small size of the granule cells, which makes their electrophysiological study very difficult. This example illustrates once more the importance of morphological investigations in the study of electrical synapses. In the cerebellum of mormyrid and gymnotid fish, as well as in that of the frog, the glomeruli are considerably simpler than those in mammals. Two kinds of synaptic arrangements have been described. In one kind a mossy terminal is located at the center of the glomerulus and is completely surrounded by several granule cell dendrites (with a large range of diaineters). In the other, the mossy

C. SOTELO AND H. KORN

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FIGS.5-7.

Examples of gap junctions in cerebellar glomeruli.

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fiber terminal contacts only a few granule cell dendrites, and the remainder of the axonal surface is covered by glial processes. At the synaptic interface between the mossy fiber and !he dendrites two types of junctional zones can be observed. The first type, comprising the large majority of these junctions, is identical to the active zone. The second type consists of small plaques of gap junctions (see Section VI). In the viper cerebellum, and even more so in the pigeon and chick cerebellum, the glomeruli are more complex (Fig. 6), resembling those in mammals. The synaptic interface has three types of specialized junctions: active zones, gap junctions (Figs. 5 and 7), and attachment plates. Frequently, the attachment plates and the gap junctions are arranged in an alternating fashion (Fig. 6); in bird cerebellum, they commonly face different dendrites (Fig. 6), and only occasionally are they found side by side. Cantino and Sotelo (cited in Sotelo, 1977a,b, and described here) studied the cerebellar glomeruli of the chick with the freezefracture technique. Figure 8 illustrates one of these glomeruli; the center of the micrograph is occupied by the mossy terminal with only a small area of its axolemma (P face) exposed. In the axoplasm numerous synaptic vesicles can be clearly identified. The mossy fiber is surrounded by two granule cell dendrites whose E membrane faces are extensively exposed. Both of them have an area occupied by a small gap junction plaque marked by arrays of complementary pits. The junction at the top of the micrograph is illustrated at higher magnification in Fig. 9. Clusters of intramembrane particles encircle the gap junction plaque. Since the common type of junction observed in thin sections at lowresistance junctions are attachment plates, it is possible that these clusters of particles on the E face of the granule cell dendritic membrane correspond to the latter. Another possibility is that they represent the homogeneous particles associated with the E faces of excitatory postsynaptic membranes in chemically transmitting junctions described in the mammalian cerebellum (Landis and Reese, 1974). However, this possibility must be rejected because in thin sections active zones have never been found in such close contact with gap junctions, and FIG.5. Axodendritic gap junction in the cerebellum of the viper. A mossy fiber (MF) establishes a large gap junction with a granule cell dendrite (GD). Note the presence of attachment plaques (arrows) at the boundaries of this electrical synapse. X98,000.

FIG.6 . L ow magnification of a glomerulus in the cerebellum of the chicken; its center is occupied by a mossy rosette (MF) surrounded by granule cell dendrites (GD). Both neural elements are contacted by attachment plaques (arrows)and by a gap junction (GJ). X39,OOO. FIG.7. High magnification of the axodendritic gap junction illustrated in Fig. 6. Note its h e p talaminar appearance and the cytoplasmic dense material underlying the junctional membranes. X 115,600.

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FIGS.8-10.

Freeze-fracture of cerebellar glomeruli from the chicken,

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in replicas exposing the mossy fiber axolemma no signs of vesicle attachment sites (VAS) have been observed near the gap junction (Fig. 10). B . GAPJUNCTIONS

IN

LOCALLY OPERATING

CIRCUITS

Examples of gap junctions at somatosomatic, somatodendritic, and/or dendrodendritic locations are listed in Table 11. They are especially frequent in electromotor pathways of electric fish and in the retina of fish, amphibians, reptiles, and birds. Electrotonic coupling can also be indirectly mediated by way of the presynaptic axons. In fact, electrotonic spread of current has been demonstrated to occur between neurons that are not directly connected but where gap junctions are exclusively present between axon terminals and somata or dendrites (Bennett et al., 1964, 1967c; Bennett and Pappas, 1965; Bennett, 1966; Pappas and Bennett, 1966; Kriebel et al., 1969; Korn and Bennett, 1972, 1975; Korn et al., 1977). Figure 12 illustrates diagrammatically one of the possible forms of a prejunctional pathway for coupling. Two conditions are necessary: (1) there must be a short distance between the two coupled neurons; and (2) the same synaptic bouton or two nearly boutons belonging to the same prejunctional fiber must establish a gap junction with both coupled neurons. Anatomical demonstration of these two requisites has been reported in various regions of the CNS of vertebrates. Thus Bennet et al. (1967~)found large axon terminals in the giant electromotor nucleus of Malapterurus electricus, forming gap junctions with two different dendrites in single thin sections. A morphologically similar situation has been observed in the LVN of the toadfish (Korn et al., 1977). In both instances there is no definite proof that the two dendrites contacted by the axon terminal belong to two different neurons. However, in the electromotor nucleus of the spinal cord of Electrophorus electricus (Meszler et al., 1972) and of Sfernarchus albifrons (Pappas e f al., 1975), a single axon terminal establishes gap junctions with the cell bodies of two adjacent neurons and, as illustrated in Fig. 11, the same situation exists in the mesencephalic magnocellular nucleus of

FIG. 8. Low-magnification electron micrograph. A central mossy fiber (asterisk) has been cut through its cytoplasm; a small portion of its membrane (P face) can be identified on the right. The E faces of two granule cell dendritic membranes with arrays of pits characteristic of gap junctions (open arrows) are also exposed. ~ 5 0 , 0 0 0 . FIG. 9. High magnification of the E face illustrated at the top of Fig. 8. Note that the gap junction pits (GJ) are surrounded by clusters of large particles which correspond to the attachment plaques binding the mossy rosette and the granule cell dendrites (arrowheads). X68,OOO. FIG. 10. Freeze-fracture of another mossy fiber membrane. The fracture plane steps from the E face (E) to the P face (P) at the gap junction; note the absence of VAS close to the latter. X89,250. (From Sotelo, 1977b.)

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FIGS.11 and 12. Anatomical evidence for electrotonic coupling through prejunctional fibers.

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Gymnorus carapo (Soteloet al., 1975). There is no doubt that in this case the two processes contacted by the same axon terminal belong to two different cells, since the nucleus of each neuron is present in this micrograph. This article deals mainly with the morphological aspects of electrotonic junctions but, because of their now well-documented ubiquity in the vertebrate CNS, we must at least mention their functional importance. In brief, as shown by both direct and indirect methods of intracellular recording and stimulation, gap junctions constitute low-resistance pathways between nerve cells (references in Bennett, 1972, 1974; see also Korn and Faber, 1978) and behave generally as simple resistors connecting the interiors of the coupled cells. As a consequence, they are better fitted for the transmission of excitation. Since inhibition generally results in sign inversion (i.e., depolarization of the presynaptic neuron leads to hyperpolarization of the postsynaptic element), it cannot be commonly mediated by such junctions, except under special conditions involving the preferential transmission of a spike after hyperpolarization, or at rectifying junctions (references in Korn and Faber, 1975a); their mode of operation is now well documented on the basis of both experimental and theoretical considerations (see, for instance, Watanabe and Grundfest, 1961; Bennett, 1966). The role of gap junctions in controlling highly synchronized activity of neurons has been established in many specialized systems such as the sonic muscles of the toadfish and electric organs of electric fishes (Bennett, 1966; see also, 1972) and in more ordinary effectors such as the neurons innervating fish external eye muscles (Kom and Bennett, 1972, 1975). The speed of electrotonic transmission allows neurons (1) to interact very rapidly and (2) to achieve a degree of synchrony that could not be obtained with chemically transmitting synapses with their longer delay times. When cells are closely coupled, the spread of postsynaptic activity in adjacent neurons can be sufficient to initiate spikes in nearby cells, as observed for instance in the giant electromotor neurons of the electric catfish (Bennett et al., 1967c) and in the lateral vestibular neurons of the toadfish (Korn et al., 1977); in this case, the synchrony of firing is well achieved, because no further depolarization is required to fire some of the coupled cells than that brought about by the electrotonic coupling itself. Where

FIG. 1 I . Magnocellular mesencephalic nucleus of the weakly electric fish G . curupo. Two small neurons (N) are in contact with a single axon terminal (AT) which establishes gap junctions (asterisks) with both neuronal perikarya. Note that this terminal contains flattened synaptic vesicles (arrow). x53.600. (From Sotelo et al., 1975.) FIG. 12. Schematic model of electrotonic coupling between two lateral vestibular neurons by way of a presynaptic fiber. A prefiber branches and establishes gap junctions (dark areas) with both postsynaptic cells. The action currents set up by a spike in one neuron (A) flow through the presynaptic bridge to the second cell (Ell where a depolarizing potential (GAD) is thereby generated. (From Korn er al., 1973.)

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coupling is weak, the spread of activity from adjacent neurons can summate with subthreshold postsynaptic potentials (PSPs), thus still providing a significant synchronizing effect; that is, if a group of coupled cells is depolarized by synaptic inputs and some of these cells fire, their activity can spread to excite other neurons that otherwise would not reach their firing level, as demonstrated, for example, in fish oculomotor neurons (Kom and Bennett, 1975). As pointed out elsewhere (see Bennett, 1974), the action of electrotonic synapses that couple a group of cells can be viewed as both excitatory and inhibitory; a more depolarized cell depolarizes its less depolarized neighbors and simultaneously is made less depolarized by them. This mechanism results in positive feedback between neurons. Described as such, the behavior of gap junctions may seem rather rigid, but the above description of their effect does not take into consideration situations involving the spatial localization of chemical and electrical synapses allowing a given neuron to operate synchronously or asynchronously under appropriate physiological conditions (Korn and Bennett, 1975), or the more complex case of mixed synapses, which are far more abundant in the vertebrate CNS than purely electrotonic synapses (see Section 11). Furthermore, it must be pointed out that, although synchronization is also likely to be mediated by gap junctions when they are present in mammals (see Section IV), their role in such species has not yet been fully described. Finally, it should be mentioned that gap junctions are not the only means of electrical interaction between neurons, since they can also be mediated by field effects (references in Kom and Faber, 1975b), but since the latter do not require the presence of morphologically specialized junctions they are not considered here.

IV. Electrical Synapses in the Mammalian CNS Because electrical synaptic transmission has for years been thought to be almost exclusively present in primitive forms (see Section I), special emphasis must be given to gap junctions in the mammalian brain. The number of such junctions in the mammalian brain is considerable, including those described in primates (see Table 11), which indicates the importance of electrotonic mediation even in the CNS of these species. Furthermore, although electrophysiological confirmation of coupling is difficult to obtain for technical reasons (see Korn et al., 1973), the correlation of physiological and anatomical data has conclusively demonstrated the presence of electrotonic transmission in the mesencephalic trigeminal nucleus of the rat (Baker and Llinhs, 1971), in the LVN of the rat (Kom et af., 1972), and in the inferior olive of the cat (Llinas et al., 1974). The following discussion considers the types of electrotonic interactions to be expected based on the selective location of gap junctions.

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A. BETWEEN NEURONAL PERIKARYA Gap junctions have been described between somata of the large, monopolar sensory neurons of the mouse and rat mesencephalic trigeminal nucleus (Hinrichsen and Larramendi, 1968; Brightman and Reese, 1969; Imamoto and Shimizu, 1970; Sotelo, 1975). These cells are aggregated in clusters in which the perikaryal membranes of two adjacent cells exhibit large areas of direct apposition. Numerous attachment plates are present, but gap junctions are rare. The latter consist of small junctional plaques which occupy an extremely small area of the neuronal plasma membrane. The rarity of gap junctions correlates well with the electrophysiological data obtained by Baker and Llinas (1971), and the number of such coupled neurons is low, since in their study coupling was found only in about 10% of the penetrated cells. Somatosomatic gap junctions have been also encountered in the molecular layer of the cerebellum of the cat, between basket cells (Sotelo and Llinas, 1972), and between neurons of the anterior ventral cochlear nucleus of the rat (Sotelo et al., 1976). Cerebellar inhibitory interneurons, as well as cochlear neurons, are only uncommonly in direct apposition, being generally separated from one another by a zone of neuropil. This fact further explains why in both regions of the brain gap junctions, although present, are rather rarely observed.

B. BETWEEN DENDRITES This type of gap junctions has been described in the olfactory bulb of the rat (Pinching and Powell, 1971), in the rat and cat cerebellar cortex (Sotelo and LlinBs, 1972), and in the primate neocortex (Sloper, 1972). In the second case the dendrites have been identified as belonging to inhibitory interneurons Since these interneurons bear a few gap junctions at somatic and dendritic locations, it follows that they are electrotonically coupled and therefore that several of them can be activited as a closely knit group. It is probable that the synchronous activation of inhibitory interneurons thereby provided by gap junctions is of considerable importance in the organization of neuronal nets in the cerebellum. Abundant minute gap junctions, which are difficult to identify in thin sections, have been observed with the freeze-fracture method in the outer plexiform layer of the retina of the monkey (Raviola and Gilula, 1975). These junctions are present between the dendrites of bipolar cells (where they make synaptic contacts with the cone pedicle) and between processes of horizontal cells. Although there is no physiological evidence for electrotonic coupling between horizontal cells in the monkey retina, coupling was demonstrated in nonmammalian retinas (Kaneko, 1971; Simon, 1973; Lasansky, 1976) where gap junctions were also present (Fain et al., 1976; Lasansky, 1976). Therefore it can be postulated that

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horizontal cells in primates are also coupled. Finally, larger zones of gap junctions have also been reported between horizontal cell processes in the cat retina (Kolb and Famiglietti, 1976).

c.

BETWEEN AN AXONTERMINAL AND A NEURONAL PERIKARYON

Axosomatic gap junctions have been observed at the bipolar-ganglion cell junction of the primate retina (Dowling and Boycott, 1966), in the descending vestibular nucleus of the rat (Fig. 4) (Sotelo, 19751, and in the LVN of the rat (Sotelo and Palay, 1970). As noted in Section 111, physiological evidence suggests that the large axon terminals bearing gap junctions and synapsing on vestibular neurons are primary vestibular afferents (Wylie, 1973), and also that the giant cells of Deiters in the LVN of the rat are electrotonically coupled (Korn et al., 1973). Since these neurons are not directly connected by gap junctions, they constitute another example in which coupling must occur by way of the prejunctional axons. D. BETWEEN DENDRITIC APPENDAGES IN A GLOMERULAR FORMATION This precise location of neuroneuronal gap junctions is characteristic of the inferior olivary glomeruli of mammals. They were first reported in the cat (Sotelo et al., 1974) and later observed in the opossum (King et al., 1975), rhesus monkey (Sotelo, 1975), and squirrel monkey (Rutherford and Gwyn, 1977); they are described here in the rat (Figs. 14 and 15). With small variations, mainly due to the complexity of this synaptic arrangement in the different species (compare Figs. 13 and 14), the inferior olivary glomerylus is composed of a central core of dendritic elements surrounded by several axon terminals and wrapped in an astrocytic sheath. In the cat, the central dendritic core contains 4 to 10 dendritic profiles in the plane of the section (Fig. 13), whereas for instance in the rat only 2 to 4 of them are present (Fig. 14). These dendritic processes have an average FIG. 13. Glomerular arrangement in the neuropil of the inferior olive of the cat. The central core of this glomerulus is formed by numerous dendritic processes (D, to D4) probably belonging to different olivary neurons; its peripheral region is composed of axon terminals (AT) which establish chemical synapses (AZ) with the central dendrites. Two of the central dendrites, which are postsynaptic to the terminal, are directly connected by a gap junction (arrow). X49.300. FIG. 14. Glomerulus in the inferior olive of the rat. Note that the general arrangement is similar to that in Fig. 13, although the central core contains fewer dendritic processes (D1 to D2).X42.500. FIG. 15. High magnification of a gap junction established between two dendrites in the central core of an inferior olivary glomerulus of the rat; its heptalaminar configuration is evident. Note the large, unusual extent of the semidense cytoplasmic material undercoating the junctional membranes. x93,soo.

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FIGS.13-15.

Gap junctions in the mammalian inferior olivary nuclei.

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diameter of 0.5 pm; they contain a single mitochondrium and cisterns of the smooth endoplasmic reticulum. The origin of the dendritic protrusions forming the central core of these glomeruli has been determined in the cat (Sotelo et al., 1974). They generally originate from secondary dendritic branches as slender appendages; within the glomerulus, they twist and change their course and diameter, giving rise to small spheroid spines. A correlation between the ultrastructural features of the dendritic protrusions and their Golgi appearance indicates that the central dendritic core of a glomerulus is formed by the articulation of at least two of the racemose spinous protrusions observed in Ciolgiimpregnated material, each racemose formation probably deriving from a different inferior olivary neuron. The axon terminals forming the peripheral synaptic coverage range from medium-sized (4-2 p m in diameter) to smaller boutons (2-0.5 p m in diameter). The medium-sized boutons generally establish active zones with two or more different dendritic protrusions within the same section (Figs. 13 and 14). The dendrites composing the central glomerular core are associated through two types of junctional zones. The first type, which comprises the largest proportion of these junctions, is identical to the small attachment plates between dendrites described in other regions of the mammalian CNS (Peters et al., 1976). The second type is formed by gap junctions (Figs. 13-15) which, although small, are consistently present. The morphology again correlates well with the physiological results (Llinhs et al., 1974): the electrotonic coupling is rather extensive and is present and operative in nearly all cells which were investigated intracellularly. As in other systems, electrotonic coupling most likely generates synchronous activity of inferior olivary neurons. This is to be expected if the functional organization of the inferior olive requires that the olivocerebellar projection generate almost simultaneous activation of a set of cerebellar nuclear cells and their corresponding Purkinje cells; as discussed by Llinas et al. (1974), this type of synchrony may explain the high correlation between spontaneously occurring climbing fiber responses in neighboring pairs of Purkinje cells observed by Bell and Grimm (1969). Furthermore, Llinhs et al. (1974) have postulated that synchronous activation of inferior olivary neurons is induced by electrotonic coupling during the tremor produced by harmaline, since, under these conditions the inferior olive behaves as a lO/sec oscillator (Llinas and Volkind, 1973).

V. Chemical Synaptic Control of Electrotonic Coupling Synchronous as well as asynchronous modes of firing may occur in some motor systems between neurons which, however, are coupled together (references in Bennett, 1974). A specific mechanism of natural uncoupling neurons has been discovered in an invertebrate by Spira and Bennett (1972). In the buccal

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ganglia of Navanax, large identified neurons control the circumferential muscles of the pharynx. Under ordinary circumstances these motoneurons are electrotonically coupled; although coupling is relatively weak, it is sufficient to generate a synchronous firing. When the ipsilateral pharyngeal nerves are stimulated, the synaptic activity in the ganglia increases and the spread of current between neighboring adjacent motoneurons is almost completely abolished; during decoupling, even strong depolarization of one neuron fails to excite the other. Decoupling is associated with a decrease in cell resistance without a change in the coupling resistance. A tentative explanation of these results is that inhibitory synapses are strategically placed along the coupling pathway which, when firing, short-circuit the membranes of the coupled neurons (Spira and Bennett, 1972; Bennett, 1974). Such synaptic control of coupling provides the system with great flexibility, since it allows the same set of neurons to generate different behaviors. Similar observations have been reported by Carew and Kandel(1976). In Aplysia californica three motor neurons which mediate inking behavior are electrotonically coupled; increased conductance excitatory postsynaptic potentials (EPSPs) reduce the coupling which, by contrast, is increased during decreased conductance EPSP. Another central structure of the mammalian brain in which electrotonic interactions might be modulated by the shunting effects of adjacent chemical synapses is the inferior olive of mammals (Llinhs et al., 1974). In this region the gap junctions are strategically located between thin processes originating from dendrites of several neighboring neurons, which form the central core of the glomeruli (see Section IV,D). These dendritic processes are postsynaptic to the numerous axon terminals of the peripheral zone of the glomeruli, and frequently the same synaptic bouton forms active zones with two of the dendritic processes which are themselves linked by a gap junction (Figs. 13 and 14). This arrangement provides the best anatomical situation for synaptic control of electrotonic coupling. Thus a hypothetical uncoupling of inferior olivary cells might be due to increased conductance of the membrane of these two dendritic appendages during transmitter release from the single axon terminal synapsing on them. The functional significance of this uncoupling mechanism would be to increase the number of possible operational states of the inferior olivary neurons (Llinas et al., 1974).

VI. Mixed Synapses In early descriptions of axosomatic and/or axodendritic electrical synapses in the vertebrate CNS (Bennett et al., 1963; Robertson et al., 1963) it was clearly stated that in these axonal profiles vesicles of similar appearance that synaptic vesicles were always present; they were not associated with the electrotonic

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junctions themselves but with nearby differentiations exhibiting all the ultrastructural features of chemically mediating synapses, the active zones. Several years later, and on purely morphological grounds, Sotelo and Palay (1970) described a specific type of axon terminal synapsing on the giant cells of Deiters of the LVN of the rat. These were large endings originating directly from myelinated axons and bearing on their synaptic interfaces numerous dark patches which marked specialized junctional zones. According to their morphology, these investigators distinguished three main types of junctional complexes. The first, comprising only a small proportion of these junctions, was identical to the active zone and was considered to be the morphological counterpart of chemical synapses. The second type, which was the most numerous, was formed by small intermediate junctions or attachment plates consisting of zones where the cell membranes, lying straight and parallel, were separated by an extracellular space 250-305 8, wide bisected by a layer of semidense amorphous material. A band of dense material was closely applied to both cytoplasmic components of the unit membranes, giving these junctions a symmetric configuration. (The main criterion for differentiating these attachment zones from active zones was the presence of clustered synaptic vesicles associated with the presynatic membrane at the latter.) The third type of junction, the least abundant, consisted of close appositions of the gap junction variety, 0.05-0.5 p m long, and these were considered a low-resistance pathway. Since all three of these specializations occurred on the same synaptic interface, the synapses between the large axon terminals and the giant cells of Deiters were classified as mixed synapses. Electrophysiological studies on the same preparation indicated that the large axon terminals must belong to primary vestibular fibers, since some of them mediated the electrical EPSPs recorded in the vestibular neurons (Wylie, 1973). In the comparative study of the vestibular nuclei described in Section I11 we mentioned that in fact the primary vestibular fibers establish such mixed synapses with their target vestibular neurons in fish and other species up to rodents (Figs. 3, 4, and 17). As listed in Table 111, numerous mixed synapses have been described in the CNS of vertebrates. It is apparent that most of the axodendritic and axosomatic electrical synapses mentioned in Table I1 are in fact to be mixed synapses, and this raises an important question: Do the boutons with such junctions participate in both the chemical and electrical mode of transmission? Electrophysiological evidence of a dual mode of synaptic transmission was first obtained in the ciliary ganglion of the chick by Martin and Pilar (1963), who considered that the electrotonic coupling in this instance might be related to the large area of synaptic contact between the calyciform ending and the cell body. Morphological studies by Cantino and Mugnaini (1975), using the freeze-fracture method, later demonstrated the presence of sufficient membrane specialized areas of gap junctions to explain electrical transmission at the calyciform synapse. However, Bennett and co-workers (Pappas and Bennett, 1966; Bennett et al., 1967d), in a parallel

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TABLE I11 MORPHOLOGICALLY MIXEDSYNAPSES IN THE ADULTVERTEBRATE CNS Fish Electromotor and electrosensory pathways Mormyrid medullary relay nucleus (Bennett et at., 1967b) Catfish giant electromotor neurons (Bennett er at., 1 9 6 7 ~ ) Gymnotid medullary electromotor nuclei (Bennett et al., 1967d) Sternachus spinal motoneurons (Bennett et al., 1977) Gymnotid magnocellular mesencephalic nucleus (Sotelo et al., 1975) Spinal cord Sea lamprey giant axons with interneurons (Rovainen, 1974a,b; Christensen, 1976) Toadfish sonic muscle system (Pappas and Bennett, 1966) Goldfish and tench ventral horn (Gray, 1970) Brain stem Goldfish Mauthner cell (Robertson et a l . , 1963; Nakajima, 1974) Lamprey vestibular nuclei (Stefanelli and Caravita, 1970) Toadfish LVN (Korn et al., 1977) Spiny boxfish oculomotor nuclei (Waxman and Pappas, 1971) Goldfish tangential nucleus (Hinojosa, 1973) Cerebellum Mormyrid cerebellum (Kaiserman-Abramof and Palay, 1969) Gymnotid cerebellum (Sotelo and Llinas, 1972) Torus semicircularis Cyprinus carpio torus semicircularis (Ito, 1974) Amphibia Frog spinal cord (Sotelo and Taxi, 1970; Sotelo and Grofova, 1976) Frog oculomotor nuclei (Waxman and Pappas, 1971) Frog cerebellum (Sotelo, 1969, 1976) Frog ventral nucleus of the VIII nerve (Sotelo. 1977b) Reptiles Viper cerebellum (Sotelo, 1977a) Birds Pigeon and chick cerebellum (E. Mugnaini, personal communication; Paula-Barbosa, 1976; Sotelo, this review) Mammals Rat LVN (Sotelo and Palay, 1967, 1970; Korn et al., 1973) Rat inferior vestibular nucleus (Sotelo, 1975)

morphological and physiological study on gymnotid fish electromotor nuclei, and on the toadfish sonic muscle system, observed morphologically mixed synapses, but these investigators did not obtain any physiological evidence for a dual mechanism of transmission, since only electrically mediated PSPs were recorded from these cells. In other systems recently reviewed by Korn and Faber (1978), for instance, the lateral dendrite of the goldfish Mauthner cell and the toadfish LVN, mixed synapses were also observed, and stimulation of the afferents which establish such junctions evoked two types of PSPs which exhibited,

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respectively, all the expected characteristics of electrically and chemically mediated EPSPs (Diamond, 1971; Korn et al., 1977), but it was not demonstrated that this dual transmission occurred at the same presynatpic terminals. The best example of a dual mode of transmission is found in a mixed synapse between the giant axons of the Muller cells and identified spinal cord interneurons in the sea lamprey. Intracellular stimulation of the presynaptic Muller axon evokes composite potentials in the postsynaptic cells; the first one is obviously due to electrotonic coupling and the second one to chemical mediation, given, for instance, its synaptic delay or its disappearance after calcium is replaced by magnesium in the extracellular medium (see Rovainen, 1974a,b). A conclusive demonstration that the two potentials are brought about by the same terminals (and that therefore the second one is not mediated by an intercalated interneuron) was presented by Martin and Ringham (1975). These investigators sh3wed that the plot of EPSP amplitude versus presynaptic depolarization was the same for the decreasing action potential as for artificial depolarization of the axon with a current when tetrodotoxin was added to the extracellular fluid; thus the release of transmitter can be accounted for by depolarization of the same presynaptic axon as that coupled to the postsynaptic cell. In confirmation, in a freeze-fracture study of the sea lamprey spinal cord (Pfenninger and Rovainen, 1974) the presynaptic membrane of some large axons revealed areas exhibiting the conspicuous internal organization of active zones in chemically transmitting synapses and zones of gap junctions. In the active zones the axolemma contained a varying number of small protuberances 200 8,in diameter on its E face. In the complementary micrograph these protuberances appeared on the P face as small depressions, which were in direct contact with the synaptic vesicles and were referred to as VAS. In addition to VAS, the P face was characterized by the presence of numerous large intramembrane particles 80- 120 8, in diameter. Therefore the internal organization of these presynaptic membranes further favors the coexistence of a chemical release mechanism together with an electrotonic spread of current. Recently, one of the identified synapses of the spinal cord of the sea lamprey, between a giant Muller axon and the dendrites of an interneuron, was tridimensionally reconstructed by Christensen (1976). After electrophysiological identification of both synaptic partners, the postsynaptic neuron was injected with Procion brown, which is electron-dense and thus allows visualization under the electron microscope. The giant axon was traced in 5 pm-thick serial sections to the dendrites of the injected interneuron; this axon contacted three different dendrites of the interneuron in a spinal segment 25 p m in length. Of the large amount of axonal membrane in direct apposition to tile postsynaptic dendritic membrane, only a small fraction was involved in the formation of presumably functional contacts. The total number of active zones was 22, whereas only 4 gap junctions were encountered. From these results it is clear that the amount of membrane participating in active zones is over 5 times

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larger than the amount establishing gap junctions (the latter has been estimated to be about 0.8 pm'). These quantitative results, similar to those obtained by Cantino and Mugnaini ( 1 975) in the chick ciliary ganglion (where the membrane area of ganglionic neurons apposed to synaptic terminals was calculated to be about 16,000 pm2, of which approximately 9% was occupied by active zones and only 0.17%by gap junctions) favor a much greater efficiency of electrically transmitting junctions over chemical ones. Although these well-documented studies suggest that most, if not all, morphologically mixed synapses are probably capable of a dual mode of synaptic mediation, they still do not explain the mechanism of such a complex mode of transmission, whose functional significance thus remains hypothetical (see Korn and Faber, 1978). Obviously more electrophysiological work will be necessary in order to solve these important problems.

VII. Gap Junctions and Molecular Transport So far, gap junctions have been presented in this article only as low-resistance pathways between neurons. Although this type of cell interaction is of great functional importance, it is not the only role these junctions play in the CNS (Furshpan and Potter, 1968). The widespread occurrence of gap junctions between nonexcitable cells (see references in Staehelin, 1974) proves that electrical transmission is only one of their physiological properties. Another basic function ascribed to these junctions involves the fact that they provide intercellular communicating channels for the exchange of molecules in an intimate interactive process (Furshpan and Potter, 1968). Physiological evidence for this kind of cell-to-cell communication has been obtained by intracellular injection of fluorescent and colorant substances, which allows visualization of their passage from the injected to the coupled cell. Only a few cellular systems have been tested in adult animals with this method, mainly the salivary gland of Drosophila and Chironomus (Loewenstein and co-workers, see references in Simpson et al., 1977) and the electrical synapses between crayfish septate lateral giant axons (Bennett and co-workers, see references in Bennett, 1973), but the results obtained are of great value, because they indicate the size of the largest molecule to which the junctions are permeable, thus providing an indirect estimation of the size of the gap junction channels. The most widely used tracers are fluorescein, a molecule of 330 molecular weight, and Procion yellow (MW SO), but many other molecules have been also used to analyze the permeability of gap junctions. Recently, Simpson et al. (1977) have employed in the salivary gland of Chironomus different fluorescence-labeled amino acids and synthetic or natural peptides, ranging in molecular weight from 25 1 to 4 158. Molecules of about 380 molecular weight cross the junction easily

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and rapidly; within 1-10 minutes the fluorescence is equally distributed on the two sides of the junction. Larger molecules between 593 and 1158 molecular weight also succeed in crossing the junction but at a slower rate; at least 30 minutes are needed to equalize their distribution. Molecules of 1926 molecular weight and over do not cross the junction. Therefore a molecular weight of 1200 seems to represent the largest molecule that can permeate a junctional channel, which limits the channel diameter to 1-1.4 nm.

A. METABOLIC COUPLING The above-mentioned results suggest the possibility of an important metabolic coupling between cells through gap junctions. An indirect appraisal of such a chemical exchange can be obtained by degeneration experiments. The principle is as follows. If in long-projecting pathways the perikarya of origin of axon terminals bearing gap junctions are destroyed, the process of anterograde degeneration must affect the axon terminals, unless the material necessary for their survival is supplied by the postsynaptic cells to the terminals through gap junction channels. Preliminary results from experiments on the vestibular region of the frog (Rana esculentu) seem to confirm the existence of such a metabolic exchange (Sotelo, 1977b). Transection of the vestibular nerve, which results in the degeneration of axon terminals belonging to these fibers, was performed. In frogs with a maximal survival time of 7 days, some axon terminals were found to be in an advanced stage of dark degeneration, many of these remnants having already been engulfed by glia; but some of them still maintained synaptic relationships with postsynaptic partners. In these instances remnants of chemical synapses could be recognized at the synaptic interfaces (Fig. 16). In addition, large axon terminals, exhibiting all the characteristics of vestibular terminals described in Section I11 and bearing gap junctions, maintained a normal appearance (Fig. 17), indicating that the degenerative processes had been delayed, probably as the result of a metabolic coupling between ventral vestibular neurons and primary afferent terminals.

FIG. 16. Ventral vestibular nucleus 7 days after surgery. A dark, degenerating axon terminal partially engulfed by glia (G) still maintains a close relationshipwith its postsynaptic neuron (N).The arrows point to postsynaptic differentiations, remnants of active zones. X25,500. (From Sotelo, 1977b.) FIG. 17. Same material as in Fig. 16; another large vestibular axon terminal (AT) synapsing on a vestibular neuron (N) and bearing gap junctions (arrows), attachment plaques (asterisks), and an active zone (AZ), keeps its normal appearance although it has been separated from its cell body for 7 days. (See text for interpretation of this figure.) X59,500. (From Sotelo, 1977b.)

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FIGS.16 and 17. Persistence of some presynaptic vestibular axons following a vestibular nerve transection in the frog.

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B. LOW-RESISTANCE PATHWAYS AND NEURONAL DIFFERENTIATION It is generally accepted that the specificity of synapses cannot be accounted for on the basis of one-to-one biochemical matching, since there is not enough genetic material to program the great complexity of the nervous system organization (Changeux and Danchin, 1976). Among the different hypotheses suggested to explain how genetic information is spared during formation of the nervous system, one of them postulates that, prior to synapse formation, transient gap junctions between growing axons and neuroblasts occur. In these instances, the gap junctions may represent communicating pathways allowing informational exchange between neurons, providing some of the required specificity. In the visual system of a small crustacean, Daphnia magna, Levinthal and his group (Macagno et al., 1973; LoPresti et al., 1973, 1974) demonstrated that the first-order synaptic unit is a type of optic cartridge formed by eight optic fibers from the retinular cells in each ommatidium and five optic lamina neurons. Synaptic specificity is achieved by well-defined sequences of cell migration; only one of the eight optic axons develops an active growth cone, and it is the first to reach the five undifferentiated neuroblasts in the optic lamina; this axon is called the lead fiber. The lamina neuroblasts react to this fiber by wrapping around it in a glia-like fashion, and transient gap junctions develop between this lead fiber and the neuroblasts. Later, the seven follower axons, devoid of growth cones, reach the optic lamina and synapse on these neurons in a specific manner. A possible explanation is that arrival of the lead axon introduces order to the system by sequential specification. The informational exchange necessary for such specification may take place through the transient gap junctions. Investigating the development of synaptic connections in the mammalian brain, Van Buren et al. (1977) recently observed gap junctions between growing nerve processes and target neurons in the subfomical organ of the kitten. The junctions are present in 3-day-old animals, and they coexist with early, immature forms of active zones. They consist of small plaques 0.2-0.3 p m in diameter, which are difficult to visualize in thin sections but are obvious in replicas of freeze-etched material. These gap junctions no longer exist in the mature subfornical organ, and their presence raises the question as to whether they are precursors of chemical synapses. However, there is no evidence to sustain this hypothesis and, on the contrary, most of the morphological results from in vivo as well as in vitro synaptogenesis experiments indicate rather that chemical synapses develop independently of gap junctions. Although the role of gap junctions during synaptogenesis is still uncertain, it seems that they are of great importance in the acquisition of “positional information” in developing neuroblasts. In a series of experiments, using as a model the development of the retinotectal system of Xcnopus, Jacobson (see references in Jacobson, 1976) demonstrated that retinal ganglion cells must acquire their

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“locus specificity” at embryonic stages 29 to 31, which predisposes the future optic fibers to establish synaptic connections at determined loci within the optic tectum. In fact, inversion of the embryonic eye before stage 28 results in a realignment of the retinal axons, giving rise to a normal retinotectal projection. However, if inversion of the eye takes place after stage 3 1, realignment of the retinal axes no longer occurs, resulting in inverted retinotectal projections. Therefore retinal ganglion cells achieve their specification during the 7-hour period of embryonic stages 29 to 3 1. Jacobson also showed that this change in state was not due to extraocular conditions and that it started at the center of the retina when the first retinal ganglion cells withdrew from the mitotic cycle. The hypothesis proposed by Jacobson (1976) is that the transfer of positional information between developing cells must occur by means of intercellular communication channels, and that the gap junctions are possibly involved in this transfer. Ultrastructural studies on the embryonic retina of Xenopus (Dixon and CronlyDillon, 1972, 1974; Hayes, 1976) have demonstrated the existence of gap junctions not only between cells of the neuronal retina, but also between the retina and the pigment epithelium. The former are frequently seen up to embryonic stage 28 but disappear from the central region of the retina at stage 32. After stage 35 gap junctions are confined to the peripheral region, where new cells are formed throughout larval life. This temporal correlation between the acquisition of locus specificity and the change in gap junction distribution suggests a possible role for these communicating junctions in the process of retinal specification. Transient gap junctions in embryonic retinas have been reported in other species. Fujisawa er al. (1976a) observed them in chick embryos. At 3 days of incubation, gap junctions between neural retinal cells were infrequent. Their number increased to reach a maximum in 6-day-old embryos, and they disappeared by 9 days of incubation. By this time most of the retinal cells had withdrawn from the mitotic cycle. Therefore the same temporal correlation between the completion of cell proliferation and the disappearance of gap junctions as that described for the Xenopus retina occurs in the chick retina. Somewhat different results have been reported by Fisher and Linberg (1975) for the early human embryo. In 28- and 36-mm embryos all retinal cell types were linked by extensive gap junctions which eventually disappeared at some unknown time during development. However, the temporal correlation between ganglionic cell specification and the disappearance of gap junctions seems to fail in this material, because, if it is assumed that specification ‘occurs just before differentiation, ganglionic cells start differentiation as early as the 17-mm stage of development, and extensive gap junctions are still found in 36-mm embryos. The number of instances in which transient gap junctions have been observed has increased in the last year. They have been recently reported in the developing spinal ganglia of the chicken (Pannese et al., 1977). In 4-day-old embryos spinal ganglia neuroblasts are closely arranged and contacts are formed by such junc-

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tions, and at 10 days of incubation the satellite cells already ensheath the neuroblasts but the gap junctions have disappeared. Therefore, although much more work is needed to correlate their presence between neuroblasts with precise mechanisms of cell differentiation and specification, it is likely that these junctions are essential for the final development of the nervous system.

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Landis, D. M. D., and Reese, T. S. (1974). J. Comp. Neurol. 15.5, 93. Landis, D. M. D., Reese, T. S., and Raviola, E. (1974). J . Comp. Neurol. 155, 67. Lasansky, A. (1967). J. Cell Biol. 33, 365. Lasansky, A . (1972). Invest. Ophthalmol. 11, 265. Lasansky, A . (1976). Invest. Ophrhalmol. 15, 909. Levitan, H., Tauc, L., and Segundo, J. P. (1970). J. Gen. Physiol. 55, 484. Llinis, R. (1975). In “Golgi Centennial Symposium Proceedings’’ (M. Santini, ed.), p. 379. Raven Press, New York. Llinis, R., and Volkind, R. A. (1973). Exp. Brain Res. 18, 69. Llinis, R . , Baker, R., and Sotelo, C. (1974). J . Neurophysiol. 37, 560. LoPresti, V., Macagno, E. R., and Levinthal, C. (1973). Proc. Nutl. Acud. Sci. U.S.A. 70, 433. LoPresti, V., Macagno, E. R . , and Levinthal, C. (1974). Proc. Narl. Acad. Sci. U.S.A. 71, 1098. Macagno, E. R., LoPresti, V., and Levinthal, C. (1973). Proc. Natl. Acad. Sci. U.S.A. 70, 57. McMahan, U . J., and Purves, D. (1976). J. Physiol. (London) 254, 405. Martin, A . R., and Pilar, 0 . (1963). J. Physiol. (London) 168, 443. Martin, A. R., and Ringham, G. L. (1975). J. Physiol. (London) 251, 409. Mehler, W. R . ( I 972). Prog. Brain Res. 37, 5 5 . Meszler, R. M., Pappas, G. D., and Bennett, M. V. L. (1972). Bruin Res. 36, 412. Meszler, R. M., Pappas, G. D., and Bennett, M. V. L. (1974). J. Neurocytol. 3, 251. Mugnaini, E., Walberg, F., and Hauglie-Hanssen, E. (1967). Exp. Bruin Res. 4, 146. Nakajima, Y. (1974). J. Comp. Neurol. 156, 375. Pannese, E., Luciano, L., Iurato, S., and Reale, E. (1977). J. Ultrustrucr. Res. 60, 169. Pappas, G. D . , and Bennett, M. V. L. (1966). Ann. N. Y . Acad. Sci. 137, 495. Pappas, G . D., Asada, Y., and Bennett, M. V. L. (1971). J. Cell Biol. 49, 173. Pappas, G. D., Waxnian, S . G., and Bennett, M. V. L. (1975). J. Nruroc‘yfol. 4, 469. Paula-Barbosa, M. (1976). Experienria 32, 633. Payton, B. W., Bennett, M. V. L., and Pappas, G. D. (1969). Science 166, 1641. Peracchia, C. (1973a). J . Cell Biol. 57, 54. Peracchia, C. (1973b). J. Cell Biol. 57, 66. Peracchia, C., and Dulhunty, A . F. (1976). J . Cell Biol. 70, 419. Peters, A , , Palay, S. L . , and Webster, H. de F. (1976). “The Fine Structure of The Nervous System: The Neurons and Supporting Cells. ” Saunders, Philadelphia, Pennsylvania. Pfenninger, K. H., and Rovainen, C. M. (1974). Brain Res. 72, I . Pinching, A. J., and Powell, T. P. S. (1971). J. Cell Sci. 9, 347. Politoff, A , , and Pappas, G. D. (1972). Anat. Rec. 172, 384. Precht, W., Richter. A , , Ozawa, S., and Schimazu, S. (1974). Exp. Brain Res. 19, 377, Ram6n y Cajal, S. (1908). Truh. Lab. Invest. Biol. Univ. Madrid 6 , 1. Raviola, E., and Gilula, N. B. (1973). Proc. Nut/. Acad. Sci. U . S . A . 70, 1677. Raviola, E., and Gilula, N. B. (1975). J. Cell Biol. 65, 192. Revel, J . P., and Kamovsky, M. J. (1967). J. Cell Biof. 33, C7. Richter, A , , Precht, W., and Ozawa, S. (1975). Pfluegers Arch. 355, 85. Robertson, J. D. (1955). Exp. Cell Res. 8, 226. Robertson, J. D. (1963). J . Cell Biol. 19, 201. Robertson, J . D., Bodenheimer, T. S., and Stage, D. E. (1963). J . Cell Biol. 19, 159. Rose, B., Simpson, I . , and Loewenstein, W. R. (1977). Nature (London) 267, 625. Rovainen, C. M. (1974a). J. Comp. Neurol. 154, 189. Rovainen, C. M. (1974b). J. Comp. Neurol. 154, 207. Rutherford, J. G., and Gwyn, D. G. (1977). Brain Res. 128, 374. Schnitzlein, H. N., and Brown, H. K. (1975). Brain, Behav. Evol. 12, 207. Simon, E. J. (1973). J. Physiol. (London) 230, 199.

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INTERNATIONAL R E V E W OF CYTOLOGY. VOL. 55

Biological and Biochemical Effects of Phenylalanine Analogs D . N . WHEATLEY Department of Pathology. University Medical Buildings. Foresterhill. Aberdeen. Scotland

A subtle slippery knave; a finder out Of occasions; that has an eye can Stamp and counterfeit advantages. though True advantage never present itself . Iago. in Shakespeare’s Orhello. Act 2. Scene 1

I . Introduction . . . . . . . . . . . . . . . . . . . . A . Analogs in Molecular Biology . . . . . . . . . . . B . Phenylalanine Analogs . . . . . . . . . . . . . . C . Nature of an Analog . . . . . . . . . . . . . . D . Scope of This Article . . . . . . . . . . . . . . I1 . Analog Uptake . . . . . . . . . . . . . . . . . . A . Uptake and Intracellular Pool Formation . . . . . . . B . Exchangeability of Pools with External Medium . . . . 111. Incorporation into Protein . . . . . . . . . . . . . . A . Activation and Loading of tRNA . . . . . . . . . . B . Relative Efficiency of Incorporation . . . . . . . . . C . General Effects of Macromolecular Synthesis . . . . . D . Enzyme Induction. Repression, and Derepression . . . . IV . Analog Proteins: Molecular Effects . . . . . . . . . . A . Enzymes . . . . . . . . . . . . . . . . . . . B . Structural and General . . . . . . . . . . . . . . C . Thermolability . . . . . . . . . . . . . . . . . V . Analog Proteins: Cellular Effects . . . . . . . . . . . A . Cell Growth and the Cell Cycle . . . . . . . . . . B . Mitosis . . . . . . . . . . . . . . . . . . . C . In Vivo Studies with Tumors . . . . . . . . . . . D . Flagella . . . . . . . . . . . . . . . . . . . VI . Use of Analogs in Other Areas of Biological Research . . . A . Mutagenesis . . . . . . . . . . . . . . . . . . B . Aging . . . . . . . . . . . . . . . . . . . . C . Virus Development . . . . . . . . . . . . . . . D . Miscellaneous . . . . . . . . . . . . . . . . . VII . Degradation of Analog Proteins . . . . . . . . . . . . VIII . Concluding Remarks . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .

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I. Introduction A. ANALOGS IN MOLECULAR BIOLOGY Molecular biology surged forward in the late 1950s with the deciphering of the genetic code and the formulation of the “central dogma” by Crick (1970). About the same time several research groups began to explore the use of analog bases of nucleic acids (e.g., Heidelberger et al., 1957). The use of such molecules as 5-fluorodeoxyuridine, which could substitute for the homologous bases in DNA, offered a means of checking the fidelity of replication, the requirements for transcription, the stability of the “parent” macromolecules, and other properties of nucleic acids. Although DNA is the information bank of the cell, the availability of the information and its expression are controlled by proteins. With our increasing knowledge of the molecular biology of proteins, and the slower realization of its enormous potential and impact in the biological and medical sciences, which has been recently discussed by Perutz (1976), we are beginning to obtain a much clearer picture of the structure-function relationships of proteins. To some extent amino acid analogs have contributed to this understanding; they promise to be even more useful when the normal structures of more proteins have been elucidated by x-ray crystallography, and their functions and specificity need to be analyzed. The diversity and complexity of proteins as compared with nucleic acids makes this an awesome task. Much of the previous work with amino acid analogs was very much a hit-or-miss process, but it has provided a background against which more specific studies can be made on individual proteins with known substitutions of analog for homolog. With this in mind, it is of value to reassess earlier work. For an analog to be of use in such biochemical or biological studies, it must be cleverly disguised so as to be incorporated in place of a homolog, but it must also be able to report back to the investigator information about the configuration of the macromolecule it has entered and the functional ability of this macromolecule to interact with other molecules. As Iago says, it may not be able to tell the whole truth because it only “counterfeits advantage,” and there is a distinct possibility with proteins that even the smallest change can completely destroy the configuration and function of the molecule.

B. PHENYLALANINE ANALOGS A wide range of different amino acid analogs has been employed in biological and biochemical studies (e.g., Jacquez and Mottram, 1953; Richmond, 1962; Shive and Skinner, 1963; Fowden et al., 1967); rather than attempting to include all types of analogs as in earlier reviews, the intention here is to concentrate on

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analogs of one particular amino acid and to see how they have contributed to our knowledge. It quickly becomes evident from the literature that analogs of two amino acids have been used far more extensively than any others, namely, analogs of phenylalanine (Phe) and methionine (Met). The analog of the latter, ethionine, has received much attention because it not only substitutes for Met in proteins but also causes ethylation of other macromolecules, leading to mutation, liver injury, carcinogenesis, and so on. This has fostered much work on its metabolic effects and its carcinogenic behavior (e.g., see Farber, 1963). For this reason we do not concern ourselves with ethionine except where it can help in some comparative aspect of analog behavior. Phe contributes on average about 3 4 % of the residues in proteins, and its clustering and interaction along with other aromatic groups produces hydrophobic areas in the polypeptide chains, leading to the formation of a-helical regions, p-pleated sheets, and associations between subunits of proteins. Phe is therefore of considerable importance in determining the secondary, tertiary, and quaternary configuration of proteins, although it seldom is present at the active sites of enzymes. By substituting an analog of Phe, it should be possible to alter a protein with sufficient subtlety to make it adopt a slightly different configuration without necessarily destroying its function. However, this is a rationalization, since most of the effects of Phe analogs have been found empirically. They proved of use in elucidating metabolic pathways; they produce interesting cytological effects, particularly on mitotic cells; they interfere with virus maturation; and they slow cell growth, among many other properties. With these credentials, the analogs of Phe are worth examining more closely, in particular the extent to which their involvement in specific proteins produces some of these perturbations.

c. NATUREOF A N ANALOG Apart from creating more problems than those caused by substituting for homologs in proteins, for example, ethionine and ethylation, analogs can produce a deficiency status in organisms which have no endogenous method of synthesizing the homologous amino acids. A considerable amount of work has been done on the effects of withdrawal of an essential amino acid from cells and has covered such aspects as protein synthesis (e.g., Vaughan et al., 1971; Brunner, 1973; Christman, 1973), RNA synthesis (e.g., Hall and Gallant, 1973), DNA synthesis (e.g., Freed and Schatz, 19691, and tumor cell growth (Laissue et al., 1971). In many cases of analog treatment, the biological and biochemical effects compare closely with the patterns seen in these studies of amino acid deprivation. Even where endogenous synthesis might be expected to circumvent the effect of an analog, the feedback mechanism may be sensitive to the analog and thereby reduce synthesis of the homolog. In this article we are concerned

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more with the positive effects analogs have on cells than with these negative aspects. What, then, is an analog amino acid? Proteins are made up of about 20 familiar amino acids, of which about half are considered essential in higher organisms lacking endogenous synthetic mechanisms. Ari analog is any substance closely related chemically to these familiar amino acids. In a recent article (Wheatley and Inglis, 1977), three categories of analog amino acids were listed: 1. Structural analogs, which are substances with a chemical resemblance to amino acids but which to all intents and purposes behave in an entirely unrelated manner. 2. Interference analogs whose chemical similarity is sufficient to produce competition with homologs at some levels, for example, uptake, activation, feedback inhibition, and so on, but which do not themselves become incorporated into macromolecules. 3. Physiological analogs, which go further than type-2 analogs in substituting for homologs.

This categorization is quitc artificial but helps to distinguish three major types of analogs in the continuum from totally inactive to totally involved. The majority of biologically interesting analogs are of type 3, but analogs of type 2 are quite important in pharmacology. In dealing with Phe (I) derivatives, it is seen thatp-fluorophenylalanine@FPhe, 11) is one of the most outstanding substances, as it is obviously a type-3 analog. It is not always so easy to determine the relationships of some amino acid analogs. All Phe analogs might also be considered analogs of tyrosine (Tyr, 111). From chemical appearances alone, for example, thienylalanine (V) looks like an analog of Met (VI), but du Vigneaud et al. (1945) showed that it was exclusively a Phe analog. Phenylserine is chemically related to serine (Ser) and Phe but behaves like a Phe analog. More complex molecules containing a phenylalanyl group could also be included as analogs. One example is puromycin (IV) which is chemically similar to an activated Phe on tRNA. But for the most part, we must confine ourselves to the simpler amino acid derivatives and not to more complex molecules which might otherwise include phenylalanyl-modified oligopeptides related to hormones such as oxytocin or vasopressin. There are cases in which analogs of Phe not only act as false amino acids but also mimic other molecules; for example, 3,4,5-trimethoxyphenylalanine(VII) is obviously related to Phe, but in physiological terms it behaves like an analog of mescaline (VIII; Sethi et al., 1973). It is perhaps worth mentioning parenthetically that mescaline has been shown to act on dividing cells in a manner similar to that reported for Colcemid (Harrison et al., 1976). We do not suggest that mescaline and perhaps other Phe analogs act on mitotic cells in this way, as

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PHENYLALANINE ANALOGS I1

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seen in Section V,B, but note that such comparisons can serve as a means of increasing the frequency of making otherwise improbable associations. D. SCOPE

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Much of the earlier work with analogs was concerned with their effects on aromatic amino acid synthesis and helped to elucidate the various pathways and their interrelationships (e.g., Mitchell and Niemann, 1947; Bergmann et al., 1953). Since most of these have now been worked out in considerable detail, this aspect does not concern us here to any significant extent. Fowden et al. (1967) provide an extensive review of the details of these biosynthetic pathways of amino acids and the use of analogs in their analysis. The manner in which analogs become involved in protein synthesis, however, is much more topical

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and is considered in the next two sections. This discussion is followed by a section dealing with the properties of proteins which have incorporated analogs and their tendency to show enhanced thermolability. In this respect analogs can produce effects which relate closely to those found in temperature-sensitive mutants. In the next section the consequences of analog protein synthesis at the cellular level are discussed. One field in which amino acid analogs have featured prominently is cell growth. As far as bacterial growth is concerned, this subject has been well reviewed by Richmond (1962) and therefore requires little more than updating. The incorporation of analog amino acids into protein can lead to considerable changes, some of which may be permanent, and Section VI explores the use of Phe derivatives in the fields of genetics, aging, and virus development. Error proteins can be preferentially degraded by cells, according to Pine (1972) and Goldberg and St. John (1976). The production of analog proteins by cells provides an experimental approach with which one can test this hypothesis, and a brief orientation in this area can be found in Section VII. Amino acid analogs have also been extensively used by pharmacologists; in particular, p -chlorophenylalanine @ClPhe) has been employed as an irreversible inhibitor of phenylalanine hydroxylase (e.g., Ayling and Helfand, 1974). This has repercussions in that the resultant phenylalaninemia leads to an experimental model of phenylketonuria, which is a hereditary condition in humans due to a deficiency of this enzyme. The Phe analog L-dopa has been extensively used in Parkinson’s disease (see Barbeau and McDowell, 1970). In these pathological situations analogs antagonize certain key enzymes. Their action is not related to incorporation into protein, and therefore we do not go deeper into these areas of neuropharmacology which have already been reviewed (e.g., Ghl, 1972). No attempt is made to convert dosages of analogs or other compounds into common units, and therefore we switch from molar units to milligrams per milliliter. Many studies have been carried out by simply adding analogs at high concentrations to complete media or diets, and therefore dose levels cannot be used as a guide to efficacy or compared from one report to another. Similarly, chemical nomenclature has not been standardized; for example, both 3-fluorophenylalanine and mFPhe may be used in the following discussion.

11. Analog Uptake

A. UPTAKEAND INTRACELLULAR POOLFORMATION One site at which analogs might interfere with homologs is at the cell membrane during uptake from the external medium. The receptor sites of specific membrane “permeases” of individual (or groups of) homologs can be blocked

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by varying external concentrations of analogs and other naturally occurring amino acids, depending on the specificity of the sites (Conway et al., 1963). The mechanism of uptake and the subsequent pathways into proteins of an amino acid (homolog or analog) in different organisms are subjects on which there are many views but little agreement. In bacteria, for example, Britten and McClure (1962) suggest that an amino acid must pass through the intracellular pool before being incorporated into protein. In contrast, van Venrooij et al. (1974) have come to the conclusion that HeLa cells probably incorporate amino acids more directly from the external medium and do not preferentially incorporate them from an existing intracellular pool, that is, when the amino acids are readily available. This circumvention of the intracellular pool had been noted by Kipnis et al. (1961), who found that immediate, linear uptake of a labeled amino acid into protein took place on its addition to the medium, whereas there was a relatively slow equilibration with the existing intracellular pool to constant specific activity, These problems are raised here not only to draw attention to their importance and controversial nature, but because analog amino acids have been of considerable value in helping to resolve them and, as seen later, still have much to offer. In previous reviews, Fowden et al. (1967) dealt only with the uptake of proline (Pro) and its analogs in Escherichia coli, and Richmond (1962) and Cohen and Gros (1960) quoted Cowie and McClure (1959) extensively. Bacteria can concentrate amino acids from the external medium; this is especially true when the medium contains a very low level of amino acids, for example,
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Cells of higher organisms also concentrate amino acids from their bathing medium. Christensen et al. (1952) demonstrated a fourfold higher concentration of glycine (Gly) in duck erythrocytes compared with that in duck plasma. Subsequent work on ascites cells (Christensen er al., 1955) led to the hypothesis that the concentrating mechanism was a complexing of amino acids to form metal chelates and a reaction with pyridoxal to form Schiff bases. However, the process is seen as an active carrier mechanism coupled with this chemical process in which amino acids are retained as complexes once they have crossed the membrane. Pool formation is probably far more complex than the accumulation of amino acids, as a result of an active process of membrane transport. There is little doubt that the pool is heterogeneous (Conway et al., 1963; Berg, 1968) and that amino acids could have reached it by different pathways. Confusion has often arisen because the word “pool” is jargon and is difficult to define, and because the methods of estimating pools have varied considerably. It is impossible to deal with these complexities here but, posing some of the main questions might be helpful in unraveling some of the important steps: (1) To what extent do cells use membrane-associated enzymes to concentrate amino acids? (2) To what extent do they enter cells by way of an electrochemical or osmotic gradient? (3) Is an active process in the membrane essential for the formation of a trichloroacetic acid (TCA)-extractable intracellular pool? (4) Do analogs follow the same pathways as their homologs? The work on bacteria suggests that enzymes are involved in transport and that amino acids can often move up an electrochemical or osmotic gradient. The answer to the third question has not been ascertained, and for the fourth the work of Cowie (1959) suggests that the mechanism is the same but favors the homolog, that is, Phe rather thanpFPhe. The work on other species, notably sea urchin embryos (Berg, 1968) and mammalian cells (Kipnis et al., 1961; van Venrooij et al., 1974), is much more perplexing. There is clear evidence that mammalian cells given both an analog and its homolog (see Section II1,B) preferentially incorporate the latter into protein, but one can ask whether such cells behave like bacteria or yeast with regard to pool formation under these circumstances, as described by Cowie (1959). Despite the considerable number of studies on Phe analogs in eukaryotic systems, there has been almost no mention of pool development. For six years we have been actively engaged in this type of work with HeLa, Landschutz ascites tumor, and other cell types. Some results with HeLa cells using Phe, pFPhe, andpClPhe serve as an example of the potential of analogs in pool studies. These three substances were selected because Phe is a natural precursor used in protein synthesis, pFPhe is a good substitute in the absence of Phe but much poorer in the presence of Phe, and pClPhe to all intents and purposes does not enter protein (see Fig. 2a). Using a nonwashing technique, we

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estimated the total pools of the three ~ ~ - a m i n o -acids ~ H when individually added to cultures of HeLa cells shortly after resuspension in a Phe-deficient medium at 37°C (Wheatley et al., 1978a). The nonwashing technique, like the methods of earlier bacterial studies (e.g., Cohen and Rickenberg, 1956), prevented the loss of counts from inside cells, whereas other investigators washed cells usually to free them of extraneous counts from medium contaminants (Berg, 1968; van Venrooij et al., 1974). The cells were spun out as pellets, using inulin-14C as an estimate of trapped medium, with a pro rata correction being made for the contaminating extracellular a m i n ~ - ~acid. H After extraction with 10% TCA, the total soluble counts or uptake indicated that the cells came to equilibrium after concentrating the amino acids to between 1.5 and 2.0 times the medium level at 0°C (Fig. 1). When this experiment was carried out at 37°C or continued after 1 hour at 0°C for a further hour at 37°C (with the time taken for the temperature to rise being no more than 2-3 minutes), the internal concentration of amino acids (excluding those incorporated into proteins) reached 3 to 5 times the external concentration. The level of the Phe pool was usually lower

a

0

b

120

I

Min

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120

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FIG. 1 . (a) Uptake from a Phe-deficient medium containing dialyzed serum of pC1Phe3H (squares), pFPhe3H (open circles), and Phe3H (solid circles) of equivalent specific activity ( I pCi/ml, 0.2 mM DL form) by HeLa S-3 cells in suspension culture. The curves indicate the total intracellular pool formed (i.e., both freely and slowly diffusible) at 0°C for I hour on the left of the vertical line in the middle of each graph, and after further incubation at 37°C for I hour on the right. The dashed horizontal line is the level to which the cells would retain the labeled amino acids if a simple equilibrium with the external medium were reached at 0°C. (b) The same as in (a) but with 2 pg/ml cycloheximide present to prevent the flow of counts from the accumulating pools into protein.

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and, furthermore, fell after the first 30 minutes of incubation (Fig. la). The same experiments carried out at 37”C, but with cycloheximide added to prevent protein synthesis, resulted in the maintenance of high levels of all three amino acids (Fig. Ib). Uptake ~ f p F P h e - ~was H not impeded by the presence of 0.2 mM “cold” Phe added at the same time. Further analyses in the 37°C experiments were carried out with two parallel methods, one without the postincubation washing as described and one with three washes in a Phe-deficient medium. It was possible to distinguish in this way between the counts taken up as the freely diffusible pool and those which could no longer freely diffuse out, the slowly diffusible pool (Fig. 2b). The latter probably corresponds to the intracellular TCAextractable pool described by other workers (Piez and Eagle, 1958; van Venrooij et al., 1974) and shows the obvious features of being actively accumulated by its kinetics and temperature dependency.

a

b

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FIG.2. (a) The same as in Fig. la, but in this graph the corresponding counts which have become incorporated into protein have been plotted (same symbols as in Fig. 1). Note there has been no incorporation at 0°C. At 37°C the incorporation is seen in terms of the specific activities of the proteins labeled. It is of interest that even with a shift from the lower to the higher temperature there i s no appreciable lag in uptake into protein despite the rising pool size over this same time period (see Fig. la). (b) Demonstration of the extent to which three washes in a “cold” Phe-deficient medium can remove the pools accumulated by 60 minutes at 0°C (left) and by 60 minutes at 37OC (right) as a direct continuation of the analysis of Fig. la. This washing effectively removes the freely diffusible pool. Note that the 0°C pools fall to about 10%of their initial value but no lower. On the right it can be seen that the two analog pools have a larger slowly diffusible (TCA-extractable) component.

PHENYLALANINE ANALOGS

119

In brief, HeLa cells can concentrate Phe, pFPhe, andpClPhe to approximately equivalent levels at O”C, and also at 37°C. The size of the TCA-extractable pool bears no direct relationship to the protein-synthesizing activity of the cell or the ability of the amino acid to act as a precursor used in protein synthesis. Indeed, there appears to be a lowering at 37 “C of the pool size of Phe which is the amino acid most readily utilized in protein synthesis. In carrying out these experiments without washing, we made them comparable to those of Cowie (1959) and Cohen and Rickenberg (1956). There seems to be justification for several different pools of amino acids (analog or homolog) within the cells, as Cowie (1959) and Cowie and McClure (1959) suggested, that is, a “concentrating pool” which ( 1 ) expands with increasing external concentration of amino acids, (2) readily exchanges with the external medium, and (3) is sensitive to osmotic shock, and at least one other (a “conversion pool”) which (1) is less variable with external concentration, (2) exchanges much less readily with the medium, and (3) is insensitive to osmotic shock, all suggesting that the amino acids are retained in some altered state (see also Cohen and Gros, 1960). The difference appears to be that, in bacteria, a pool like the latter contains the precursor molecules used in protein synthesis (Britten and McClure, 1962), whereas in mammalian cells it can be bypassed. With this rapid equilibration of the freely diffusible pool, the question is raised as to whether the amino acids of the slowly diffusible pool are activated inside the cell, at the cell membrane, or both. B. EXCHANGEABILITY OF POOLS WITH EXTERNAL MEDIUM Although analogs and homologs can be taken up by mammalian cefls in considerable quantities at O”C, they discharge the greater part of them (- 90%) as rapidly when placed in a medium deficient in the amino acid or a balanced salt solution (Figs. 2b and 9). Thus the concentrating pool probably represents amino acids taken up by a free diffusion process, as it can be quickly discharged at 0°C. The equilibrium situation could favor a higher concentration inside the cells if loose complex formation occurred preferentially in this compartment. In many studies, the concentrating or freely diffusible pool is never seen, since it is washed away at the end of the incubation before the TCA extraction begins. The emphasis placed on washing extraneous labeled amino acids from cells by van Venrooij et al. (1974) shows that two to three treatments are necessary. This is “throwing out the baby with the bath water,” since most of the amino acids they claim to be washing off the outside come from inside the cells. Furthermore, apparently contradicting Piez and Eagle (1958), who showed that four washings with salt solution also reduced the TCA-extractable pool by as much as 30%,van Venrooij quotes several previous studies which indicate that ice-cold washes in salt solution do not extract TCA-soluble material. This discrepancy can be resolved, since it was found (Robertson et al., 1978) that the initial one-wash

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method of Piez and Eagle (1958) before TCA extraction did not remove all the freely diffusible pool. Two washes were required to reduce contaminating and freely diffusible pool counts to insignificant levels, and three washes to reach negligible levels. After this, the slowly diffusible pool sometimes fell slightly with continued washings at O”C, by about 10% over five or six washes. At a higher temperature (37°C) the TCA pool quickly disappeared, but little entered protein (Robertson et al., 1978). Exchanges of labeled analog and homolog pools with the external medium containing the “cold” amino acid or other amino acids have shown for the most part that mammalian cells can retain large amounts of many different amino acids without much interference between them. The rapid loss of labeled analog or homolog from the freely diffusible pool when cells are washed in a balanced salt solution can be largely prevented by raising the external concentration of the amino acid. For example, we compared the exchangeability of Phe-3H, pFPhe3H, a n d ~ C l P h e - ~from H HeLa cells washed in 4°C unlabeled medium containing 2 mM,0.2 mM(norma1 medium concentration),or 0 mM Phe (see Fig. 9b). Loss of the freely diffusible pool was virtually complete after two washes in deficient medium; slightly less was removed when 0.2 mM Phe (or pFPhe) was in the wash, and considerably less was removed with the 2 mM wash. Perhaps of more interest here was the difference in the amount of labeled analog and homolog removed when the loaded cells were washed in Phe-containing medium (medium was used in most of these studies because the cells were also to be chased in an investigation of intracellular protein degradation; see Section VII). Usually there was a greater retention of analog than homolog; that is, it was not as easily exchanged with the external medium of the homolog. Unlike the situation in mammalian cells, amino acid displacement and exchangeability clearly occur in bacteria. In this connection the most significant study on pool exchangeability with Phe analogs in bacteria is probably that of Conway er al. (1963), and this study is also particularly informative with regard to the controversy about the source of amino acids for protein synthesis discussed earlier. These investigators, estimating the total pools in E . coli 9723 by the method of Britten et al. (1955), showed that natural amino acids [Tyr and tryptophan (Trp)], as well as amino acid analogs which were growth-inhibitory @FPhe, /3-2-thienylalanine, 3-cyclohexane- 1 -alanine) and some that were not growth-inhibitory (0-and p-tolylalanine, 3-thianaphthenealanine), could affect Phe-I4C accumulation. The reduction in Phe-’*C pool size by as much as 85% had little or no effect on the rate of incorporation of Phe-14C into proteins, and it was concluded that only part of the total intracellular pool of Phe was involved in protein synthesis. Their work with phenylalanyl-tRNA synthetase further suggests that only a small fraction of the pool sustaining protein synthesis is actually associated with tRNA. The relationship of uptake of amino acids to pools is complex, and further discussion would take us beyond the scope of this article. The subject has been

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PHENYLALANINE ANALOGS

tRNA iynthetases

"Concentrating Pool"

Cell Membrane

Extracellular Fluid

I

Perms~lActivator(71

FIG. 3 . Hypothetical scheme of the relationships between extracellular amino acids and intracellular pools.

dealt with in detail for prokaryotes by Britten and McClure (1962), and a model for eukaryotes has been proposed by van Vernrooij et al. (1974). A scheme similar to the latter is proposed to explain some of the apparent discrepancies in the three-cornered relationships between uptake, pools, and protein synthesis in the light of analog studies (Fig. 3).

111. Incorporation into Protein A. ACTIVATION AND LOADING OF tRNA

Obligatory steps in the incorporation of amino acids into polypeptides are their activation and loading onto specific tRNAs. The steps involved are, first, activation to the S-adenosyl derivative, followed by charging of the tRNA by specific amino acyl-tRNA synthetases. There is some dispute about whether the two major reactions

+ ATP + E -+ E-AA-AMP + PP, E-AAA-AMP + AMP + tRNA + AA-tRNA + AMP + E AA

(1)

(2)

occur separately or in a concerted manner with only a cryptic intermediary formation of the E-AA-AMP complex (Loftfield and Eigner, 1969). Nor is it

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certain where the activation enzymes and synthetases are located (see the review of Neidhardt et al., 1975), except that they can be found in many cytoplasmic fractions. With regard to analogs, the questions which concern us are whether the same mechanisms of activation operate, the extent to which this occurs compared with the homologs, and the relationship of tRNA loading with analogs to the observed incorporation into protein. Since there is an absolute requirement for an amino acid to be loaded onto tRNA, any anlog not accepted by the appropriate tRNA synthetase(s) cannot become incorporated into protein. In this way Conway et al. (1963) showed that highly purified phenylalanyl-tRNA synthetase from E . coli could effectively load Phe and pFPhe, accept /3-2-thienylalanine about 50% as effectively, but handle other analogs only poorly. This corresponds well with the ability of these Phe analogs to serve as precursors for protein synthesis and confirms a similar set of observations made with Trp analogs by Sharon and Lipmann ( 1957). Amstein and Richmond (1964) studied the PhehFPhe activation ratio in vitro with enzyme preparations derived from rabbit reticulocytes. It was about 1.3, suggesting little selectivity against activation of the analog. Furthermore, this ratio was equivalent to the ratio between Phe and pFPhe incorporation into protein. There was a large difference, however, when these studies were carried out with Phe andpFPhe competing for the same enzymes. pFPhe was scarcely activated when as little as 0 . 2 M Phe was present. Conversely Phe activation was reduced to 50% only whenpFPhe was added in a 10-fold excess. Again, these changes in the incorporation ratio with competition are closely linked to the observed levels of incorporation of analog into protein under these conditions. It is recalled from Cowie (1959) that in bacteria a PhelpFPhe incorporation ratio of 100 was observed when the homolog and analog competed at equimolar concentrations. Once again this suggests greater discrimination against the analog in the bacterial system, but in prokaryotes there is the added coniplication of endogenous Phe production. From the studies of Richmond (1963) and Amstein and Richmond (1964) and others discussed later, it can be concluded that, once tRNA has been loaded with an analog, no further discrimination occurs in its incorporation into proteins, as would be deduced from tRNA anticodon becoming the only determining factor before entry into the polypeptide elongating on the mRNA in the ribosome. The only ways in which an analog could apparently affect the issue just before analog incorporation into protein are if (1) it is not an acceptable substrate for all the homolog tRNA synthetases, and (2) it is sufficiently intermediary in structure between two homolog amino acids to allow some loading onto other tRNAs. The first possibility has been elegantly explored for Phe and pFPhe by Dunn and Leach (1967). Having established that poly (U)and poly (U)-poly (C) stimulated the incorporation of pFPhe, as well as Phe, in a cell-free system from E . coli (Dunn and Leach, 1964), they demonstrated that the two amino acids were equally activated but loaded only about 60% of the tRNA with

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PHENYLALANINE ANALOGS

the analog as compared with the homolog. Through a series of elimination and competition experiments they concluded that the analog was utilized by only one of the two phenylalanyl-tRNA synthetases, whereas Phe was a substrate for both: E

+ Phe + ATP .1

E-Phe-AMP

+ ATP

.1

+ PP,

E-pFPhe-AMP

+ PPi

r. i

GAA-tRNA (anticodon)

AAA-tRNA (anticodon)

Phe-GAA-tRNA

Phe-AAA-tRNA

1 uuu

.1

uuc (rnRNA codon)

L

E +pFPhe

(rnRNA codon)

J Protein

pFPhe-AAA-tRNA

.1

uuu (rnRNA codon)

.1 Protein

With regard to the other possibility, some apparent discrepancies could arise because of the metabolic conversion of analogs to other amino acids. For example, bothpClPhe andpFPhe might be considered analogs of Tyr, since the former can be metabolized to m-chlorotyrosine (mClTyr, Guroff et al., 1966) and the latter to Tyr (Dolan and Godin, 1966). Thus these analogs may be found slowly loading tyrosinyl-tRNA. However, the amounts involved are probably very small, since there is little evidence to show that Phe analogs significantly replace Tyr in protein. As originally pointed out by Cowie (1959), Tyr is not a competitive inhibitor of the behavior of Phe analogs. The work of Dunn and Leach (1967) suggests that activation steps are separable in vitro and that the difference between analogs and homologs may not lie in the kinetics of amino acyl-tRNA synthetases but in the specificity of the different tRNA synthetases linked to the degeneracy of the triplet code (UUU and UUC) for Phe (see also Knorre et al., 1970). Work with mutants (see also Section VI,A) in which tRNA synthetases had altered affinities produced somewhat similar conclusions. Fangman and Neidhardt (1964a) showed that, although the phenylalanyl-tRNA synthetase for wild-type E . coli had approximately the same affinity for pFPhe and Phe, the analog-resistant mutant PFP-10 produced a phenylalanyl-tRNA synthetase with a normal affinity for Phe but a markedly reduced affinity for pFPhe. Equally important were two other findings: (1) The affinity of the mutant enzyme for other closely related analogs (oFPhe, mFPhe, and P-2-thienylalanine) was unaltered, and (2) it showed enhanced heat lability (Fangman et al., 1965). Other studies have shown changed affinities of specific amino acyl-tRNA synthetases inpFPhe-"adapted" cells (Wacker et al., 1964), and mutants; for example, both Santi and Danenberg (1971) and Hennecke and Bock (1975) confirmed the results of Fangman and Neidhardt in that mutants unable to loadpFPhe could still utilize o - and mFPhe with the affinities changing from the wild-type order ofp > m > o tom > o > p . Hennecke and Bock (1975)

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D. N . WHEATLEY

suggested an alteration in the a-subunit site of the synthetase which normally binds Phe. The specificity of normal synthetases may vary considerably. Old and Jones ( 1976) found a difference of one order of magnitude in the inhibitory action ofpFPhe on the loading of rat liver tRNAphecompared with E . coli tRNAPhe. The amount of an amino acid in a cell may be very high, but for protein synthesis to occur there must be a constant supply of amino-acylated tRNA. If the discussion above has validity, analog amino acids which become fully activated (onto tRNA) will become incorporated, as shown, for example, by Shearn and Horowitz (1969) and Horowitz et ul. (1970) in Neurosporu. Should any analog fail to conform, the validity of this principle would be questionable. Such an instance is cited by Wolfe and Hahn (1960) who found a Phe-deficient mutant of E . coli C-2 that could activate p-2-thienylalanine with about 60-70% of the efficiency of Phe under conditions from which it could be inferred that the same phenylalanyl-tRNA synthetase enzyme was involved. The analog apparently was not incorporated into protein, and these workers drew particular attention to this discrepancy. The experiments were inconclusive on this point since, first, they failed to show that the analog was not incorporated and, second, the “lack of protein synthesis” reported could simply have been the absence of a significant net increase in protein in the division-arrested E . coli. Our experience with analogs of Phe raises a further question which is pertinent to the model (Fig. 3). If amino acids move in and out of cells freely but are converted through energy-dependent steps from a freely diffusible pool to a TCA-extractable or slowly diffusible pool, by what process are they then converted to their amino acyl-tRNA form for incorporation into protein should only one concerted mechanism be involved? With pClPhe, for example, a large, slowly diffusible pool is produced at 37°C but is not utilized as a substrate for protein synthesis. Briefly, as shown in the model (Fig. 3), an activation process for amino acids occurs which converts freely diffusible amino acid into a slowly diffusible form (E-AA-AMP?) but does not automatically prepare it for complexing with tRNA. The fact that only a very small percentage of the total amino acid in cells is present as loaded tRNA also suggests the existence of several distinct pools of activated or complexed amino acids. Their relative levels obviously depend on the supply and demand at each step. In this way bacteria and yeast may differ from higher organisms. The tRNA synthetases would therefore be rate-limiting. Nisman and Hirsch (1958) showed that activation as measured by pyrophosphate exchange may still be of the order of 1 1: I in favor of Phe, which also suggests that step 1 of the equation for activation is separable from step 2. B. RELATIVE EFFICIENCY OF INCORPORATION In the previous section we saw that the incorporation of an analog can in general be related to the extent to which it becomes loaded onto the appropriate

125

PHENYLALANINE ANALOGS

tRNA. But many of these results relied on in vitro activating systems for comparison. The results of Conway er al. (1963) and others suggest that the presence of E. coli phenylalanyl-tRNA synthetase should lead to the incorporation of pFPhe with 95% of the efficiency of Phe, and of p-2-thienylalanine with 60%; this latter value agrees closely with the results of Wolfe and Hahn (1960). But these analogs are not incorporated with this degree of efficiency in vivo, or at least not for more than a few minutes after addition. The most obvious reason for this is that competition occurs in vivo for activation by the synthetases and favors the homolog, despite apparently equivalent rates of activation in vitro by these enzymes when analog and homolog are handled separately. In prokaryotes, competition for tRNA occurs between exogenously added analog and endogenously synthesized homolog, although the latter might be somewhat reduced by feedback inhibition by the analog, as originally shown by Munier and Cohen (1956). In cells of higher organisms, competition can arise because of retention of the homolog by the cells, the continual presence of the homolog in medium andor its serum supplement, and the ability of these cells to reutilize the natural amino acids from protein catabolism. Incorporation under controlled levels of competition has already been discussed, but two examples serve to show the relative levels of pFPhe-14C incorporation into HeLa cells (1) with differing Phe/pFPhe ratios (Fig. 4) and (2) from medium containing undialyzed or dialyzed serum (Fig. 5).

ip - FPhe

'1 p-

FPhe

60 min 120 rnin FIG. 4. Comparative uptakes of Phe-I4C (taken as 100%) andpFPhe-"C by HeLa cells, and also of the latter at different levels of competition with Phe from less than a thousandth of the pFPhe concentration (<10-9 to equimolarity (100).

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D. N . WHEATLEY

4 6 8 Hr FIG. 5 . Uptake of pFPhe-14C by HeLa cells over 7 hours from a medium containing dialyzed serum (open circles) as opposed to one containing undialyzed serum (solid circles) under conditions in which the final concentration of the analog had been adjusted to 0.2 mM of the DL form and the radioactivity was 0.4 pCi/ml.

2

It is the general consensus that the many biological effects of Phe analogs are completely abrogated by the presence of the homolog. Cowie (1959) found that with equimolar concentrations only about 1% ofpFPhe incorporation occurred in bacteria. From Fig. 4 it can be seen that, in mammalian cells, equimolar concentrations reduce pFPhe incorporation to 10-20% of the equivalent level of incorporation without competition. Because the pFPhe is not continually diluted out by further homolog synthesis in the latter, low levels of analog can still be incorporated. This may be insufficient to produce overt symptoms but could result in long-term changes which are discussed in Section V1,B. In addition, analogs can disturb the relative efficiency of their incorporation by progressively impairing biosynthetic pathways in the cell. c . GENERAL EFFECTSON MACROMOLECULAR SYNTHESIS Except for certain mutants which can survive and grow in the presence of pFPhe, cells which incorporate analogs tend to show a slowing of growth to more

PHENYLALANINE ANALOGS

127

linear kinetics (Munier and Cohen, 1956). Growth then slows progressively until it stops, and the cells die (Richmond, 1962; Previc and Binkley, 1964; Hardy and Binkley, 1967). As seen in Section IV, some analogs enter many proteins, and inevitably these include essential enzymes such as polymerases and synthetases for the synthesis of nucleic acids and proteins. The inference therefore is that the abnormal products of analog incorporation proliferate the mistakes in further biosynthesis and gradually slow down normal cell growth and functioning. While this appears to be a very plausible explanation and has been a basis for considerable work since the studies of Cohen and Munier (1959), there is as yet no definitive evidence for the accentuation of errors by aberrant biosynthetic enzymes (see also Caviness, 1966a,b). Apart from the possible feedback effects of analogs on the availability of precursors used in protein synthesis, which have been dealt with by Fowden et al. (1967), there may be several other ways in which analogs slow protein synthesis. A Phe analog behaving like puromycin could be inserted into an elongating polypeptide chain and cause premature release because of the inability of the ribosome to form further peptide linkages. This would produce many small polypeptide fragments which might be candidates for enhanced degradation (see Section VII). Although puromycin and the arginine (Arg) analog canavanine probably produce fragments, there is no evidence of a significant number of fragments being produced in HeLa cells by Phe analogs (D. N. Wheatley, J. H. Robertson, and M. R. Giddings, unpublished). Protein synthesis could be reduced by the suppression of mRNA synthesis by Phe analogs. However, the reverse is more likely the case; that is, the reduction of protein synthesis probably slows RNA synthesis. In relation to all types of RNA synthesis, analogs have been shown to reduce the number of active ribosomes and to suppress rRNA synthesis (Jeantet et al., 1968; Leick, 1969), but ribosome assembly processes are highly dependent upon protein, and analog proteins probably work less efficiently. Leick in fact demonstrated the involvement of pFPhe-14C-labeled proteins in ribosomes of analog-treated cells. In studies on polyribosomes 'in HeLa cells treated withpFPhe, there was a tendency for them to be less easily disassembled than controls (J. Y. Henderson, unpublished). The results of Caviness (1966a) with p-2-thienylalanine on rabbit macrophage RNA synthesis are inconclusive and suggest no effect of the analog on RNA synthesis and turnover, but changes in purine precursor turnover rates. This work was carried out at analog concentrations which did not affect protein synthesis. When an analog failed to be utilized and incorporated into protein, as in the auxotrophic mutant resistant to pFPhe (Fangman and Neidhardt, 1946b), RNA synthesis could not proceed, and Ezekiel (1965) substantiated this finding by showing that the effect was not due to false feedback with the analog shutting off Tyr and Try synthesis. Hardy and Binkley (1967) showed a definite dependency in synchronized cultures of the thymine auxotroph E . coli l5t- for both DNA-

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D. N. WHEATLEY

dependent RNA polymerase and DNA replicase activity, which was very sensitive to the addition of pFPhe. Finally, analogs may disturb the regulatory mechanism of protein synthesis in cells. Christopher et al. (1971) found that chick embryo cells recovering from cycloheximide inhibition had an overshoot above their normal rate of protein synthesis which subsided after 10-12 hours. When the cells were treated in the recovery phase with pFPhe, the overshoot effect was sustained. However, this same effect was not obtained with two other cell types.

D. ENZYMEINDUCTION, REPRESSION, AND DEREPRESSION Analogs have been used in studies on enzyme induction since Halvorson and Spiegelman (1952) recognized their merits: “The use of analogs of amino acids as agents to prevent the incorporation of free amino acids into proteins recommends itself immediately as one potentially fruitful method of analyzing the problem. ” pFPhe greatly retarded the growth of Saccharomyces cerevisiae and produced a long lag phase and a much slower induction of enzyme in response to maltose than in the controls. pClPhe and p-bromophenylalanine (pBrPhe) were ineffective, but mClPhe, mFPhe, and pFPhe were effective, particularly the last-mentioned. They were all ineffective in the presence of Phe. Although Halvorson and Spiegelman initially attributed the effects of the analogs to reducing the availability of the homolog, they noted that pFPhe could not “be functioning by preventing phenylalanine synthesis either primarily or by interconversion.” The real complexity of the problem soon became apparent and, of many subsequent studies, the work of JanaEek er al. (1959, 1960; JanaEek and Rickenberg, 1964; JanaEek, 1967; Spizek and JanaEek, 1969) on P-galactosidase has probably been the most intensive. Using an E. coli auxotrophic mutant strain 1431 Ser- Gly-, JanaCek et al. (1959, 1960) followed the effect on protein synthesis and particularly on lactose-induced P-galactosidase in the presence of p-threo-phenylserine. Enzyme formation was almost completely inhibited in this strain when in complete medium, but in minimal medium (i.e., medium lacking the required amino acids) the analog produced a stimulation of enzyme synthesis. These workers compared this with the responses of strain ML-48 Phe-; in this strain enzyme synthesis was not affected in complete medium, but in minimal medium (lacking Phe) there was no stimulation of enzyme induction although total protein was stimulated. These observations were explicable on the basis that phenylserine was, first, an analog of Phe and not Ser and, second, that the ML-48 Phe- mutant, incubated in the absence of Phe, used the analog to form an inactive product. Later JanaCek and Rickenberg (1964) incorporated P-thienylalanine into strain ML-48 Phe- and found, however, that even a 95% replacement of Phe did not significantly reduce the activity of E. coli

PHENYLALANINE ANALOGS

129

P-galactosidase, that their effects were not explicable on the basis of altered inducer uptake (although this could have implications; see Bowman et al., 1964), and that each analog produced its own characteristic effect on enzyme induction. Evidence was presented to show changes in the subunits of the enzyme in the presence of analogs and a failure of their ability to assemble into the comple enzyme. Analog enzyme was more sensitive to heat (Kepes, 1967) and other denaturing agents. Enzyme induction can be disturbed by the effects of analogs on aromatic acid biosynthesis, uptake of the inducer, overall protein synthesis and growth rates, and by the enzyme products themselves. With repressor proteins, further complications can be added. The lac repressor in E . coli has been studied in two mutants by Kang et al. (1968). Interestingly they came to much the same conclusion about the operation of lac repressor protein subunits when they contained pFPhe as JanaEek (1967) had reached with the enzyme itself; that is, these subunits did not assemble correctly. So in strain MC132 lac 11,2, which has a temperature-sensitive lac repressor, p-galactosidase was derepressed by pFPhe incorporation at 35°C but not at 25"C, whereas in the controls derepression did not occur at either temperature afterpFPhe incorporation. From temperature shift experiments, it was concluded that the lac repressor in the temperature-sensitive mutant (1) became inactive at 35°C but was still active at 25°C after it had incorporated pFPhe at these temperatures, (2) when formed at 25"C, remained stable and became active when the temperature was raised to 35"C, and, furthermore, (3) when formed at 35"C, changed from an inactive to an active form when the temperature was lowered to 25°C. This has many important implications in connection with the temperature sensitivity of both analog proteins and temperature-sensitive mutants and is referred to in Section IV,C. Earlier studies of Kang and Markovitz (1967a,b) described similar regulator gene products being affected by the Phe analog pFPhe in enzymes controlling polysaccharide synthesis and alkaline phosphatase in E . coli. Derepression of tyrosinase in rapidly growing vegetative cultures of Neurospora was triggered by several amino acid analogs, including pFPhe (Horowitz et al., 1970). In this work, however, the mechanism of derepression was not adequately determined, but the analogs were thought to reduce the effective level of a highly labile repressor protein.

IV. Analog Proteins: Molecular Effects A. ENZYMES There has been further evidence since Richmond (1962) showing that analogs, particularlypFPhe, can be incorporated into protein both in vivo and in vitro. In a

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D. N . WHEATLEY

recent study, an attempt was made to correlate the amount of a Phe analog incorporated into protein with a biological end point, namely, the inhibitory effect on the G2 to M progression in HeLa cells (Wheatley and Inglis, 1977). This correlation was based on the rather broad assumption that all analogs incorporated into protein are likely to affect this cell cycle parameter in a similar manner. It may not be too sweeping an assumption, however, because the work of Langridge (1968) showed that it is not so much a matter of the nature of the substituting residue as where in the protein the substitution is made. This is also true of proteins such as hemoglobin. Dacie et al. (1967) found that the fortysecond residue of thep chain, which is normally a Phe residue, was replaced by a Ser residue in hemoglobin Hammersmith. This resulted in far greater instability and insolubility of the hemoglobin and the development of severe hemolytic, Heinz body anemia. In this case, the switched residue is of critical importance, because the forty-second residue is normally in direct contact with the heme moiety. In less important cases, such as the surface of an extending polypeptide arm,a protein can hardly “feel” a wrong residue (Perutz, 1976). Phe is not often found at active sites of molecules but, if analogs replace residues in critical areas of folding such as hydrophobic regions, there will be a considerable effect on the conformation and assembly of protein subunits. This affects some enzymes more than others, as seen later. Table I lists some of the proteins and cell systems incorporating Phe analogs since or in addition to those reported by Richmond (1962). In some cases, enzymes have been synthesized with pFPhe and have retained their normal function, whereas in others function has been lost. Thus Horowitz et al. (1970) purified tyrosinase induced in the presence of pFPhe and showed that it retained its electrophoretic characteristics and specific activity but was much more sensitive to heat denaturation than the homologous enzyme. Enzyme function may vary depending on the degree of replacement of homolog by analog, as shown by Richmond (1960a,b). It also depends on the temperature at which the enzyme is assayed, as a few degrees can make a large difference (e.g., Kang et al., 1968; Wheatley, 1977). It is obviously worthwhile to work with proteins which are easily identified and in which sites of replacement are known. The absence of activity of a particular enzyme in cells treated with analogs cannot be accepted as evidence that the enzyme has not been synthesized. It is necessary to use alternative methods of detection such as purification, chromatography, electrophoresis, immunochemical demonstration, and so on, in these circumstances. Such techniques have been employed by Richmond (1960a,b), Vaughan and Steinberg (1960), Westhead and Boyer (1961), and Johnson and Kenney (1973). Nor can it be concluded that an analog has no effect on an enzyme if full function appears to be retained, since the possibility exists that the analog contribution is too small in relation to the total amount of enzyme to affect the assay significantly. This could in part be the reason for the apparent stability of the nuclear alkaline phosphatase

TABLE 1 PHE ANALOGS IN PROTEINS‘ Species or cell

Analog pFPhe pFPhe pFPhe and others pFPhe pFPhe pFPhe 2,5-Dihydrophenylalanine pFPhe pFPhe pFPhe pFPhe pFPhe Phenylserine, pFPhe, and 3-furanylalanine pFPhe pFPhe

Rabbit reticulocytes E . coli

HeLa cells HeLa cells HeLa cells Terrahymena E . coli. sarcoma-180 cells E . coli Rabbit reticulocytes Mouse Taper hepatoma cells Mouse hepatoma and adenocarcinoma cells Neurospora E . coli

E . coli E . coli

“Viral proteins have been excluded; see Section IV, C.

Proteins analyzed

Reference

Hemoglobin Alkaline phosphatase General Nuclear alkaline phosphatase General General General Polyphenylalanine General General General Tyrosinase

Kruh and Rosa Richmond (1963) Wheatley and Inglis Brown er al. (1975) Wheatley (1977) Zeuthen and Rasmussen Pine (1975) Dunn and Leach Arnstein and Richmond MacLean and Huber Ryan and Elliott Horowitz er al.

General, P-galactosidase Three cell fractions R2 gene product

Janatek (1967) Fangman and Neidhardt Kang ef al. (1968)

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D. N. WHEATLEY

of HeLa cells treated withpFPhe (Brown et a l . , 1975), although this enzyme was chosen because it turned over quickly. This objection cannot be leveled in studies with analogs of inducible enzymes, as seen in Section IV,D. Nomura et al. (1956) reported an interesting observation concerning the effect of analogs (pFPhe and ethionine) on the secretion of an amylase by Bacillus subtilis. With the intention of further reducing protein synthesis in washed bacteria resuspended in a nitrogen-free medium, they followed the amylase activity and found that it was unaffected. This was taken as evidence to support the idea that the enzyme was secreted from preformed zymogen inside the cells. Thangamani (1969) repeated this same type of experiment on Penicillium mold and observed a quite different result: the analogs largely depressed bacterial secretion of peptidase A without having significant effect on total cellular protein synthesis. These contradictory results are worth further study, because analogs offer a means of dissociating the time of synthesis of a protein from the time of its utilization. B. STRUCTURAL AND GENERAL Most of the proteins synthesized by growing cells are in fact structural. The obvious question is whether a Phe analog fed to an auxotrophic mutant or Phedeprived cells affects the general spectrum of proteins synthesized. Analog and homolog proteins have been compared by various techniques. Cowie et al. (1959), using DEAE-cellulose chromatography with a continuous gradient elution procedure, showed that norleucine and pFPhe incorporation gave a very similar pattern to the protein peaks in their separation. They concluded that there was no radical change in the protein profile in analog and homolog proteins of E . coli. A stricter comparison of labeled pFPhe-I4C and Phe-I4C incorporation was made with HeLa cells using Phe-3H proteins as a master control. The preparations were coelectrophoresed on three different polyacrylamide gel systems, all of which showed a high degree of correspondence between analog and homolog protein-labeling profiles (D. N. Wheatley , unpublished; see Fig. 6). Further analyses of other cell fractions gave a similar agreement. Much less work has been done at the molecular level with specific structural proteins containing Phe analogs than with enzymes, and only one storage protein has received attention, namely, ovalbumin produced by chicken oviduct (Vaughan and Steinberg, 1960). When the effects of analogs on mitotic spindles and flagella tubules are considered (Section V,B and D), it is evident that tektins can be affected. It has not been determined whether these anomalies produced are due to the formation of abnormal tektins or their associated enzymic proteins such as dynein. The tubulin peak of cytosol separated on disk gel electrophoresis becomes heavily labeled withpFPhe-14C in the absence of Phe, which is highly suggestive evidence that incorporation into this tektin occurs, but so far no

-

PHEN Y LALANINE ANALOGS C14 - pFPhe+ 3H - Plw

133

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(left) and Phe-14C-labeled (right) cytosol proteins from FIG.6 . Profiles of j~FPhe-'~C-labeled HeLa cells coelectrophoresed with a master control of Phe-3H-labeled cytosol proteins on three different gel systems. Top, 5% polyacrylamide with pH 8.3 buffer; middle, 8.5% polyacrylamide with a pH 7.2 buffer; bottom, 8.5% polyacrylamide containing 0.1% sodium dodecyl sulfate and 6 M urea with a pH 7 . 2 buffer.

analog tubulin has been purified. This is an inviting possibility, and a study of its behavior in in v i m assembly mixtures would be very interesting. C. THERMOLABILITY Analog proteins, like error proteins in which natural amino acids have been switched, consistently show increased lability. This is manifested as increased susceptibility to heat, urea, and trypsin (JanaCek, 1967). Heat has been the most widely used means of confirming that an alteration or error has occurred in a protein, to the point that in aging studies it has almost become a diagnostic test (Section V1,B).

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But do all analog proteins show increased thermolability? There is evidence that certain analog enzymes do not show greater thermolability, for instance, aldolase and glyceraldehyde-3-phosphatedehydrogenase (Westhead and Boyer, 1961) and alkaline phosphatase (Brown et al., 1975); but perhaps the appropriate range was not chosen to test the thermolability of these enzymes. There are many enzymes which can operate very effectively above the normal temperature range tolerated by an organism, and analogs of these enzymes may not show greater thermolability. However, if cells with analog proteins become extremely sensitive compared with their controls, substitution in key enzymes near or at active sites may have occurred. Proteins which undergo important conformational changes can be made particularly labile by Phe analogs. Thus, in HeLa cells, the G2 to M progression is affected by pFPhe incorporated into protein at 37°C (Wheatley and Henderson, 1974b), whereas Chinese hamster ovary (CH0)- 10 cells are almost unaffected. Yet raising the temperature by 2°C produces a marked inhibitory effect on both. Smith and Pardee (1970) found no effect on the growth of bacteria which had incorporated pFPhe at 37°C until they raised the temperature to 45°C. These findings, along with the demonstration of permissive and nonpermissive temperatures in bacteria (Kang et al., 1968) and mammalian cells (Wheatley, 1977), indicate that analogs are as useful in some types of biological investigations as temperature-sensitive mutants. Unfortunately, the permissive-nonpermissivecomparison cannot be extended to all organisms, as similar studies with Tetrahymena (Rasmussen et al., 1978) do not show the same phenomenon. We cannot conclude that analog substitution invariably makes proteins more thermolabile. Results with mutants (Langridge, 1968) suggest that the majority of alterations lead to enhanced thermolability. There is also the occasional possibility that a substitution will lead to increased hydrophobicity which might increase the stability of a protein, as suggested by the work of Yutani et al. (1977) on E. coli mutants with altered tryptophan synthetases. JanaEek (1967) examined parameters other than thermolability, and this may be a more valid approach, particularly in enzymological analyses. The extension to Phe analogs of such treatments as acid denaturation-renaturation, which showed clear differences between native alkaline phosphatase of E . coli and its mFTyr-substituted analog (Sykes et al., 1974), would be worthwhile.

V. Analog Protein: Cellular Effects A. CELL GROWTH AND CELL CYCLE

Cell growth is sooner or later hampered by any agent which reduces the availability of an endogenous substrate for protein synthesis, We have already referred to the ability of some analogs to inhibit endogenous substrate synthesis

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by a false feedback mechanism. In this and in other ways previously mentioned, analogs suppress growth in living organisms. Richmond (1962) reviewed analogs in relation to bacterial growth and noted that the majority of investigators had described a change in bacteria from exponential growth to more linear growth after an analog was added. Many bacterial studies are complicated by the experiments being carried out in the presence of the homolog, and in some cases by the use of wild types which can synthesize the homolog from simple carbon and nitrogen sources. Thus interpretation of the effects of an analog on bacterial cell growth can become difficult unless specific auxotrophic mutants are used. Eukaryotic cells, especially in mammalian systems, are entirely dependent upon exogenously supplied amino acids. This means that the effects of analogs on cell growth can be more easily interpreted. Surprisingly there has been no serious study of the growth effects of analogs on eukaryotes to compare with the detailed studies in bacteria by, for example, Atkinson et al. (1951), Cohen el al. (1958), and Binkley’s group (Previc and Binkley, 1964a,b;Hardy and Binkley, 1967). In the case of mammalian cell growth, the use of an analog in the absence of its homolog may produce no greater disturbance than an amino acid deficiency if it does not substitute for the homolog in macromolecular synthesis. The effects of simple amino acid deprivation on mammalian cell growth and metabolism have already been referred to (e.g., see Vaughan et al. 1967; also Section I,B for references). Briefly, some of the problems with analog growth studies which make collation of the findings difficult from one group to another include (1) the amount of competition occurring intentionally or unintentionally between the analog and the homolog in the organism being tested, (2) the rate at which endogenous synthesis of the homolog can circumvent utilization of the analog, (3) the homolog reserves and the resourcefulness of the cell in making the homolog available (e.g., reutilization), (4) the rate at which the analog can be converted into a useful substrate, ( 5 ) the rate of incorporation of the analog into protein, and (6) the extent to which analog proteins interfere with the normal functioning of the cell, among other factors. In the above-mentioned studies of Vaughan’s group on amino acid deprivation in mammalian cells, dialyzed serum was used, and therefore one would expect a quick response to deprivation of an essential amino acid. However, the cells did not stop quickly but continued to cycle for up to one generation time and grow a little, presumably by reutilizing their amino acids from less essential intracellular proteins or by the digestion of serum proteins. Eagle (1955) found that it was not until the fourth day after deprivation of Phe that HeLa monolayers began to die. 1. Prokaryotes

The argument that growth becomes linear in the presence of an amino acid analog in bacteria (and also in yeasts) has been debated since the work of Cohen and Munier (1959; Munier and Cohen, 1956, 1959). Richmond (1962) claimed

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that there was a slowing, but not to a linear growth rate, and suggested that changes in growth patterns of bacteria exposed to analogs could be used to predict the manner in which the analog interfered with cell metabolism: “If growth is immediately inhibited completely, yet the inhibition is reversible, it is likely that the antimetabolite inhibits some essential step in the biosynthetic pathway of the natural amino acid. In this case it is most unlikely to be incorporated into cell protein. On the other hand, onset of ‘linear’ growth. . . ,followed 4 to 5 hours later by complete inhibition, is characteristic of all the analogs known to replace natural amino acids in proteins.” This makes far too great a distinction between the main growth effects of analogs and begs the question of whether the growth kinetics of treated cultures can in fact reveal something about an analog. There are probably four features to observe: (1) how quickly a change in the growth rate occurs, (2) the subsequent shape of the growth curve, (3) the relative contributions of cell mass increase and cell number increase to the overall growth rate, and (4) the possibility that changes such as adaptation, mutation, and degeneracy may progressively and less predictably modify the growth curves with time. There is evidence from some of the work of Pardee and Prestidge (1958) with azatryptophan that bacterial growth rate alters immediately on addition of the analog. Collins and Richmond (1962) make a similar claim forB. cereus treated withpFPhe. Yet Richmond (1960a), using the same organism, found a 20-minute delay before a change in the growth rate. Previc and Binkley (1964b) found a 45-minute delay in the response of E. coli B after the addition ofpFPhe concentrations of up to 4 mM. But how soon is “immediate” as measured in these reports? For some prokaryotes, 20 or 45 minutes can amount to almost a whole cell cycle. This is not an immediate shutoff by an antimetabolite, even assuming that the bacteria possessed some homolog resources or were able to reutilize a considerable amount from salvable proteins. For several reasons it seems highly improbable that the cells will achieve a truly linear growth rate. If mass increased linearly, true linearity would be sustained only in the absence of cell division, or with division plus regulated cell death (see Richmond, 1962). The usual methods of measuring bacterial cell growth are based on the assumption that the average cell size remains constant, and therefore a simple technique such as turbidity or protein content can be used. To answer some of the questions raised in analog studies, additional techniques of analysis are necessary, including protein, RNA, DNA, cell size measurement, and cell counting. A method of estimating cell “dropout” would also be valuable. A more comprehensive study was carried out with pFPhe in E. coli by Previc and Binkley (1964a,b), Hardy and Binkley (1967), and Carpenter and Binkley (1968). Using phased E. coli they found that the speed with which cells responded with a change in growth rate depended upon their position in relation to an impending division when treated; that is, if cells had begun chromosome replication, growth was not disturbed until the next round of chromosome rep-

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lication began. This led to a synchronization of bacteria released from pFPhe inhibition (Brostrom and Binkley, 1969b). The analog-treated bacteria showed a progressive slowing up during the “linear” growth period which was accompanied by filamentation. From this quasi-linear phase, a slow logarithmic rate of growth was observed as either adaptation occurred or some resistant mutants began to overgrow the culture. This demonstrates that there was obviously a multifactorial effect of the analog on cellular processes, which makes interpretation of kinetics difficult. More recent work of Pine (1975) described the effects of another analog, 4,5-dihydrophenylalanine, on the growth of a Phe-requiring mutant of E . coli 9723F in similar terms, with cells eventually reaching a steady state or a slow logarithmic rate of increase when Phe was also available. Growth occurred for 5 minutes in cultures with only this analog present and thereafter tailed off and ceased within a matter of hours. With regard to isomers, Dittmer (1 950) found that P-3-thienylalanine was more effective than p-2-thienylalanine in inhibiting growth in yeast and bacteria. This contrasts with their effects in eukaryotic cells which indicated that these two isomers were of equal potency (Wheatley and Inglis, 1977). Interestingly Lewis and Dunn (1964) showed that P-3-furylalanine was more effective than p2-furylalanine in E . coli, but the reverse was true in Lactobacillus arabinosus. Analog studies on bacteria have been taken much further by Dunn (1958) and Smith and Dunn (1970), using more exotic derivatives of Phe, and tetra- and pentapeptides containing Phe or its analogs. They found that growth inhibition could often be produced in E . coli with a greater degree of potency by oligopeptides containing thienylalanine than by the analog amino acid itself. The permutations and combinations possible in the use of oligopeptides in growth inhibition and its alleviation can best be summed up by a quote from Dunn (1958), to which the reader should refer directly: A special function of peptides is further indicated by the finding that glycyl-p-tolylalanine can nullify growth inhibition in a medium containing glycyl-p-2-thienylalanine,phenylalanine and p1 -naphthylalanine or p-tolylalanine. This is an unusual situation in which the growth inhibition produced by an amino acid @-tolylalanine) appears to be nullified by its peptide (glycyl-ptolylalanine); however, the explanation appears to be that the growth inhibiting effect of p-tolylalanine and p-1-naphthylalanine is due to their interference with the utilization of phenylalanine (thus permitting glycyl-p-2-thienylalanineto inhibit growth) whereas the growth stimulating effect of glycyl-p-tolylalanine is due to its interference with the utilization of glycyl-p-2thienylalanine.

Although these modes of action and interaction have still to be verified, it is not a vain hope that this type of study will clarify rather than confuse the issue. 2. Eukaryotes Growth studies on eukaryotes may not have followed the same pattern as those on prokaryotes for two reasons. First, growth in eukaryotes systems has often

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been measured in the simplest of ways, not as growth curves but as end points, for example, the time taken for an analog-treated culture to attain confluency (Misefari et al., 1974). Second, their relatively long cell cycle compared with that of prokaryotes makes it possible to ask more specific questions about analog effects on mitosis, S-phase progression, or Gzevents. In this section we deal with the effects on cell growth and preparation for mitosis in terms of interphase events in the cell cycle, leaving the specific effects on mitosis and cytokinesis to Section V,B. After the early work on mammalian tumor growth in vivo and in vitro by Jacquez and others (Jacquez et al., 1953; Biesele and Jacquez, 1954), ciliates were some of the first eukaryotes to be more closely studied. Tetrahymena was in many respects an ideal organism, as Rasmussen and Zeuthen (1962) point out: “Tetrahyrnena cells which developed synchronous divisions in an inorganic medium lend themselves exceedingly well to the study of cell division by use of metabolic analogues of various sorts.” Many analogs of amino acids, nucleic acid bases, and steroids were tested by Holz et al. (1963). Two Phe analogs were included, pFPhe and p-2-thienylalanine. Both had significant growth inhibitory effects, the former at 0.15 mM and the latter at 0.5 mM. Growth was measured, however, by a single end point-the ability of the cells to reach a half-maximal growth concentration in 60 hours. Thus the concentrations of all substances in the growth medium including the homolog were restricted to producing this final cell concentration, but the actual concentrations were not given. When added 5 minutes after the last heat shock used to produce synchronized cultures, both analogs delayed division, pFPhe being about three times more effective than p-2-thienylalanine. Although the end point was division in these cases, the analogs disturbed the interphase processes which prepare cells for division, and the effects were probably on G2processes which are responsible for sending cells into division and not on division processes themselves (although this distinction cannot always be made, as will be discussed later. Rasmussen and Zeuthen (1962) addedpFPhe to synchronized Tetrahymena at varying times after the end of the heat shock synchronizing procedure with the cells in inorganic medium. Maximal cell division occurred at 85 minutes. Division delay occurred in treated cells, its length being related to the dose of analog. At the highest concentration used, cells were considerably delayed when the analog was added up to 37 minutes after heat shock, but the analog was ineffective when added later. They also determined dose-response curves for cells treated for short periods (mostly for 5 -35 minutes), following the effects of the analog on the growth of one of each pair of sister cells, the other acting as a control. This allowed greater precision in timing preparatory events for division, and the results clearly established the delaying effects of the analog up to a transition point. This careful study and others led to the formulation of a scheme to explain the preparation of cells for division and the actions of a wide range of

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different metabolic inhibitors ubon it. Precursors used in protein synthesis are incorporated into certain proteins which can be used to prepare cells for division. As time passes, more of these proteins become involved in an increasingly stabilized ‘‘division structure” which can permit division. If the flow of proteins into this structure is disturbed by analog or inhibitor treatment, the division processes are blocked except when the structure has already reached its stabilized form. “Setback” is caused by perturbing agents which disassemble the structure partly or completely, and extra time is needed to reassemble it after the perturbing influence has been removed. This model is set out more graphically in Williams and Zeuthen (1969). Following the work of Rasmussen and Zeuthen (1962), Frankel (1962) compared the effects of temperature andpFPhe on synchronized Tetrahymena proceeding toward division and provided evidence to support Zeuthen’s hypothesis. Loss of sensitivity to the different agents was closely correlated with completion of the new oral primordium. Some of the smaller differences in the agents were attributed to the time taken for each to develop its effect. He also followed the kinetics of redevelopment of the oral primordium on the removal of pFPhe from the medium. This study gives some credence to the idea that certain structural developments must occur prior to division and that setback may be related to the manner in which they collapse or are resorbed in the presence of inhibitors. Some of these studies with pFPhe were extended by Rasmussen and Zeuthen (1966). Tetrahymena was found (1) to adapt to pFPhe during continuous exposure to low concentrations (about 0.01 -0.04 mM) and eventually to go through division in the presence of the inhibitor, and (2) to adapt to higher concentrations of pFPhe when methionine treatment accompanied the analog. Although several possible explanations are given for these observations, including interference with pFPhe uptake by Met, the results do not offer support for the division protein hypothesis. In two subsequent studies (Rasmussen, 1967a,b)pFPhe was given to Paramecium aurelia, as in the Tetrahymena experiments, and it was concluded that it interfered with the development of preparations for cell division in this species until approximately 1 hour before a normal scheduled division. At about the same time these experiments were in progress, Mueller and Kajiwara (1966) were exploring the effects ofpFPhe on mammalian cells (HeLa) synchronized in S phase. The addition of 0.5 mM pFPhe (in the presence of normal culture medium levels of Phe, i.e., -0.1 mM L-Phe) at the time of reversal of amethopterin-blocked cells by the use of thymidine, reduced the final level of DNA synthesis by about 10-15% and inhibited cell division. This effect was reversible, but whenpFPhe treatment extended beyond the normal period of DNA synthesis, there was a delay of several hours before division occurred, suggesting that pFPhe added about 4-6 hours into S phase (normally 7-8 hours) did not prevent cell division. They interpreted this as evidence that the essential division proteins had already been synthesized by this time. These results are not

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easy to reconcile with pFPhe effects in HeLa cells, bacteria, and other organisms, possibly because of the difficulty in distinguishing between requirements for the completion of DNA replication and those for division. Later studies (K. Kajiwara, personal communication) indicated that pFPhe inhibition of cell division in synchronized HeLa could be brought about by preventing the last 2-3% of DNA replication. The effect on DNA synthesis itself is of interest because of Previc and Binkley's report (1964a) that this analog could inhibit the initiation of chromosome replication in bacteria but had no effect on its completion once started. Clearly, in Mueller and Kajiwara's experiment, the addition of pFPhe at reversal gave insufficient time for it to affect initiation proteins or enzymes concerned with DNA replication. Intrigued by the possibility that division proteins can be inhibited or mistranslated by analogs, and the possibility demonstrated by Smith and Pardee (1970) that analogs can be used to distinguish these proteins from other division-dependent processes including chromosome replication, investigators undertook further studies on the preparation of mammalian cells for mitosis. Experiments were devised to ensure that chromosome replication was over or assessable before examining the effect ofpFPhe in the absence of free Phe on the progression of HeLa cells into mitosis. The results reported in Wheatley and Henderson (1974a) showed that pFPhe had to be present in at least 0. I mM (L form) to produce inhibition, and at 0.2 mM to block division of G2 cell progression within 30 minutes of addition to HeLa cells. At least a fivefold greater concentration of analog had to be used to produce partial inhibition when 0.1 mM L-Phe was present. The inhibitory effect on the entry of cells into division was almost completely offset by blocking the incorporation of pFPhe into proteins. Thus it could be concluded that pFPhe inhibited cell progression by forming anomalous proteins, not by blocking protein synthesis. Using both HeLa and CHO-10 cells, Wheatley and Henderson (1974b) showed that the behavior of cells in response to pFPhe was temperature-dependent. For example, CHO-10 was not noticeably affected by pFPhe incorporation at 37°C but was inhibited at 39.5"C. Similarly HeLa cells incorporating pFPhe at 37°C were inhibited from entering mitosis, whereas cells treated in exactly the same way at 32°C were not. These differences did not correlate with changes in the incorporation of pFThe into proteins at the different temperatures. In fact, the reverse was true; there was better incorporation of analog into protein relative to controls at the permissive temperatures compared with that at the nonpermissive temperatures. The close correspondence of thepFPhe temperature effects in HeLa cells to the behavior of temperature-sensitive mutants is emphasized by further findings (Wheatley, 1977). Attempts were made (Wheatley and Henderson, 1975) to follow the effects of temperature shifts, for example, by incorporatingpFPhe at permissive temperatures and then raising the temperature to nonpermissive levels, and vice versa. The conclusions reached for HeLa cells were that (I) inhibitory pFPhe

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proteins made at 37°C (not permissive) did not become functional again at 32"C, and (2) noninhibitory products of pFPhe incorporation at 32°C did not become inhibitory at 37°C. In this connection, the findings of Kang et al. (1968) are particularly relevant, since their results suggest that there was asymmetry in the responses of analog proteins, possibly in the differential behavior of subunits during temperature shifts. Briefly they showed that, if lac repressor proteins were made with analogs at a lower temperature (25"C), they were stable and retained their repressor property at 35"C, while the converse was not true; that is, nonrepressing proteins made at the higher temperature become active repressors at the lower temperature. This latter instance correlates with the use ofpFPhe by Smith and Pardee (1970), who incorporated it into proteins at an early stage in the cycle and then later made them malfunction by raising the temperature. The inhibition of cell cycle progression by pFPhe suggests that, if a particular process essential for continued cycling were blocked, synchronization would occur to some degree in cells that had recovered in Phe-supplemented medium. In many studies on the recovery of pFPhe-treated HeLa cells, there was no unambiguous evidence of a parasynchronous wave (Wheatley and Henderson, 1975). In this respect, eukaryotes and prokaryotes may differ, but the former have not been examined for GI to S synchronization which would be more akin to the synchronization of chromosome replication of E. coli released from pFPhe inhibition, as shown by Brostrom and Binkley (1969b). The combined use ofpFPhe and temperature doubles the effectiveness of this analog technique in cell cycle studies. The studies of Rasmussen and Zeuthen (1962) and Frankel (1962) on pFPhe in Tetruhymena compared but did not combine heat treatment and analog treatment. Since those earlier experiments, attempts have been made by Rasmussen, Wheatley and Zeuthen (in preparation) to use a short-pulse treatment with temperature shifts in Tetruhymena. In Fig. 7 the effects ofpFPhe combined with temperature shifts are shown. They indicate a definite tendency for cells to respond to the inhibitory action of the analog when the temperature is raised. From these studies it became clear that the analog interfered with the correct assembly of proteins for division and that cells could be delayed until very late in the G2 phase. This could be due to the stabilization referred to earlier being relative and not absolute. Massive doses of analog are probably needed to overpower normal progression in these final stages before division. These studies and further analysis in HeLa cells suggest that there are two phases of analog sensitivity in cells progressing toward mitosis though G2. The first is almost certainly concerned with the production of an initially labile proteinaceous structure involved in division which is interfered with when analog proteins enter the pool of building units. The second follows as the more stable situation when this structure is complete or near complete. Here the analog can still exert an effect seemingly on the probability with which a cell will enter mitosis; that is, G2 may contain a deterministic phase followed by a stochastic

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FIG.7. Tetruhyrnenu pyriformis GL-C were synchronized by seven heat shocks at 34OC for 20 minutes at 40-minute intervals. On their final shift to the optimal growth temperature of 28°C (0 minutes) they were allowed to progress into division with a treatment from 20 to 35 minutes with 0.41 rnh4 pFPhe; the controls (squares) received Phe. The effects on the subsequent division peak were examined in cultures maintained at 28°C (lower graph) and in others raised to 3 1°C just 5-10 minutes before the cells were due to divide. It can be seen from the bottom graph that some delay occurred in cells reaching division after they had receivedpFPhe earlier and that division itself was also protracted (triangles). In the upper graph, the delaying effects of a pFPhe pulse has been considerably augmented by the small rise in temperature just before division (solid circles).

period during which cells may take up the option of proceeding into division. In the stochastic phase, the probability of the trigger event occurring within a given time is inversely related to the concentration of analog protein present.

B. MITOSIS The effects of amino acid analog incorporation throughout the cell cycle can cause disturbances in the mitotic process in higher cells. We have seen that cells treated with adequate levels of analog, particularly when it is given in the absence of homolog, fail to reach mitosis. However, in many studies analogs have had to compete with their homologs and therefore produced only partial inhibition of the progression of cells into division. Some processes more sensitive to analog incorporation actually occur within mitosis, and these have been described in detail.

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Jacquez and Mottram (1953) specifically attempted to disturb tumor cell growth, in relation to that of normal cells, with amino acid analogs. This work illustrated the predominance of Phe analogs as growth inhibitors, and in a subsequent study they dealt with the cytological aberrations seen in mitotic cells as a result of P-2-thienylalanine treatment (Jacquez ef al., 1953). Tumor cells experienced difficulty with anaphase, produced abnormal spindles, and frequently underwent abnormal cytokinesis to form multinucleate or polyploid cells. These anomalies quickly resolved themselves when the mice were taken off analog treatment, although exactly how this was achieved is not known. Their interpretation of the action of 0-thienylalanine was that it antagonized Phe, since there was evidence that the tumor tissue was unable to transaminate p -2-thienylalanine to produce L-Phe (Jacquez e f al., 1952)-a property which they believed was absent from tumor tissues but present in normal tissues. Biesele and Jacquez (1954) looked more closely at FPhe isomers and found the order of effectiveness as inhibitors of mitotic processes to b e p > o > m. The pFPhe analog was effective against sarcoma T241 both in vivo and in v i m . Mouse heart cells were sensitive, but skin cell cultures were not, nor were sarcoma-180 cells. They found that sarcoma-I80 cells became sensitive when cocultivated with liver cells, an effect which was further potentiated by adding ATP. The majority of cells delayed in division were slowed in metaphase and, in addition, many mitotic aberrations appeared including chromosome bridges, tripolar spindles, toroid metaphases, and fragmentation of chromosomes. No conclusions were reached as to the mechanism of analog damage. An important control was missing for this work in that the effect of the absence of an essential amino acid on cells was not observed. There is evidence that this in itself can cause chromosome breaks and endoreduplication in mammalian cells (Freed and Schatz, 1969). The subsequent work of Sisken’s group has done much to demonstrate the involvement of analogs in mammalian cell division. Just before cells enter mitosis, some protein synthesis must occur through the Gz period. Sisken and Wilkes (1967) showed that cultured human amnion cells allowed to incorporate pFPhe for several hours near this crucial time were unduly slow in completing metaphase. Using puromycin for comparative purposes, they concluded that the analog effect was due to its incorporation into “mitotic proteins” rather than the suppression of protein synthesis. Presumably the analog proteins disturbed the normal functioning of the mitotic apparatus or some other important part of the division process. By following these cells by time-lapse cinematography, they demonstrated that their eventual progeny were also slow in transversing mitosis; that is, they had presumably conserved sufficient analog proteins in the subsequent interphase so that the same problem arose again in a subsequent division. These workers point out that these results were obtained with cells treated with pFPhe at levels of up to 10 mM in the presence of a normal medium concentra-

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tion of Phe (0.1 mM). If analog proteins were of no use, the cells might still slowly produce the correct proteins. For the same reason, Sisken and Wilkes mention that it takes at least 2 hours of prior exposure to a concentration of 0.5 mM pFPhe before a mitotic delay is observed. Sisken and Iwasaki (1969) extended the above observation by exposing cells to 1.0 mM pFPhe continuously. The majority of the cells capable of reaching mitosis within the first 20 hours of treatment disintegrated after being arrested in metaphase for up to 8 hours. Lower concentrations had less drastic effects, but most cells still died after prolonged metaphase arrest, rarely dying in interphase. Thienylalanine (5 mM) prolonged metaphase in the first division cycle and produced a greater delay in a second mitosis. It also produced multipolar mitosis, as observed by Biesele and Jacquez (1954). Results with ethionine were complicated by interphase effects, and azatryptophan (5 mM) also produced general toxicity; but both compounds led to some accumulation of cells in metaphase, which became progressively more delayed. These findings suggest that the mitotic anomalies produced by analogs must be viewed cautiously if general cytotoxicity is enough to trap cells in metaphase. Finally, pFPhe (5 mM) produced a delay in metaphase in cells exposed for more than 10 hours, which also tended to increase stepwise with the subsequent duration of treatment. In the last-mentioned case, cells did not undergo abnormal mitosis and did not become pycnotic at this stage of the cycle. While the sensitivity of the cells to the effect of pFPhe suggests important repercussions of this analog on division proteins, the effect of pClPhe in particular, and of the o.ther analogs in general, on metaphase should not necessarily be ascribed to such direct involvement with the division protein. With increasing exposure of amnion cells topFPhe before the collection of metaphases by mechanical shake-off from roller bottles, Sisken et al. (1972) found that cells resuspended in an analog-free medium could not complete division so well. A brief exposure of 3 hours caused little delay in reversibility, whereas cells held in pFPhe for 6 hours before reversal had only a small percentage moving on through anaphase after 3 hours in fresh medium. Spindles of mitotic cells frompFPhe-treated cultures were shorter than normal by about 25% (Fig. 8), and appeared to have more than one center at each pole, even to the point of resembling a double spindle; other instabilities were also described. Sisken ( 1 973) subsequently made a careful study of metaphase-anaphasetelophase transitions in amnion cells under the influence of 1 mM pFPhe. The rate of movement of chromosomes poleward in the treated cells was directly proportional to the length of exposure to the analog. In all the cells given up to a 12-hour exposure, the eventual distance covered by the chromosomes was the same (approximately 13 pm), which does not seem to support an earlier contention that the spindles of treated cells were on the average 25% shorter. There may

FIG.8. Phase-contrast micrographs of unfixed human amnion cells in mitosis prepared in isolation medium after exposure to Phe X 1050. From Sisken et al. (1972) by kind permission of Dr.Sisken.

25,51, and 62 minutes (top, left to right) or to pFPhe for 18, 66, and 73 minutes (bottom, left to right).

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be no discrepancy, however, if anaphase does not begin until the spindle has reached its full length, or elongation of the spindle in anaphase occurs to a greater extent in the pFPhe-treated spindles then in the controls, thereby compensating for its shorter initial length. UsingpFPhe, Sisken (1973) also presented evidence that cytokinesis occurred at the same distance between the leading edges of the sister chromosome sets, irrespective of the time taken to reach this point. This is corroborated by a smaller comparative study relating speed of chromosome movement, distance apart, and onset of cytokinesis using temperature as the variable along the lines of previous work (Sisken et al., 1965). One obvious extension of these experiments would be to combine analog and heat treatments in future studies on mammalian cell mitosis. In conclusion it appears that many analogs given in sufficient concentration and for long enough disturb mitosis in cells which enter this phase. pFPhe and P-2-thienylalanine have been the most effective, probably through their incorporation into division proteins. However, no direct analysis of analog incorporation into mitotic proteins has actually been demonstrated, and this is obviously required. There is also a need for electron microscopic observation of aberrations in mitotic cells due to analogs.

c. In ViVO STUDIES WITH TUMORS Analogs were used on experimental tumors following the earlier reports of Jaquez et al. (1952). The initial promise of tumor tissue being selectively damaged by thienylalanine because of a greater difficulty in transaminating it was not borne out by subsequent in vivo observations (Jaquez et al., 1953). Attempts were made to reduce the toxicity of the analog in host cells by additional treatment with phenylpymvate, but without success. Several studies have since been carried out. The effectiveness of an analog in reducing tumor growth, in relation to its ability to enter protein, is definitely accentuated if animals are placed on a Phe-deficient diet before treatment. Lorincz and others have reported on the advantages of deprivation alone in obtaining a favorable response in tumors (Lorincz and Kuttner, 1965; Lorincz et al., 1969). In keeping with this idea, Ryan and Elliott (1968) fedpFPhe in a Phe-deficient diet to mice bearing a hepatoma or an adenocarcinoma. They obtained growth inhibition only when mice were treated with bothpFPhe and a low-Phe diet, but not when the diet or the analog alone was used. A preferential accumulation of free pFPhe in the hepatoma cells was found, whereas normal liver cells did not accumulate it. In a study on the Taper liver tumor in mice, MacLean and Huber (1971) were able to “retard its growth and prolong the life expectancy of the hosts.” This study was carried out with Phe in the diet. Their biochemical analysis showed, however, that the analog inhibited the uptake of

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Phe by the tumor cells and reduced tRNA Phe production. Apart from these two studies, most of the other tumor work with analogs of Phe has been carried out in vitro; there has been, however, a considerable amount of work with analogs of other amino acids in vivo. In investigating the Landschutz ascites tumor in mice, Inglis and Wheatley (in preparation) obtained clear evidence of the quick inhibition of tumor cells passing into mitosis by treatment withpFPhe. On the grounds that a well-established ascitic tumor containing about 108 cells/ml has a high requirement for Phe to maintain exponential growth, it was reasoned that it should be possible to incorporate a considerable amount of pFPhe into the cells. With the knowledge that such cells would probably show enhanced thermosensitivity , the animals were given periods of mild hyperthermia at an appropriate time after analog treatment in an attempt to selectively damage the tumor cells. This technique is still under development, but once again it emphasizes the fact that more might be accomplished in the chemotherapy of tumors by the combined use of analogs and temperature (mild hyperthermia). The uptake of analogs by tumor cells and other rapidly growing cells, and by tissues which synthesize large amounts of protein, has led to their use in screening procedures. Several radioisotopically labeled Phe derivatives have been studied includingpFPhe and iodophenylalanine (IPhe). Garzo et al. (1962) found pFPhe-3-I4C better than ethionine in revealing (on whole-animal autoradiographs) sites of rapid protein synthesis, with the pancreas, the salivary glands, and embryos prominent. Samal et al. (1963) showed the concentration of j33-thien~lalanine-~H in the kidney, liver, jejunal mucosa, and pancreas of rats bearing the Murphy-Sturm lymphosarcoma. IPhe-lz5Iwas intensively localized in the pancreas (Blomquist et al., 1969), but it was difficult to determine whether they had revealed a typical Phe-like incorporation with this analog. Their intention was to see if tumors could be “visualized” and, although some were, many others were not revealed against the normal pancreatic uptake. With the more appropriately labeled analog pFPhe-’*F Cottrall et al. (1973) came to the conclusion that, although there was some pancreatic localization of label, the method was inferior to the existing selenomethi~nine-’~Setechnique. These results offer little encouragement in the use of labeled analogs in future radiodiagnostic work on tumors.

D. FLAGELLA The molecular disturbance caused by analog incorporation into structural proteins has been studied to a significant degree only in flagella. In its simplest form, a flagellum consists of a long, cellular projection whose structure is maintained by tubulin polymerized into a 9+2 arrangement of microtubules and

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which is made to move by dynein (containing ATPase). Analogs can affect either of these two main types of protein. The first indication of an analog effect on flagella was observed by Kerridge (1959a) who showed that certain amino acid analogs interfered with the synthesis and development of bacterial flagella. Using mutants of Salmonella typhimurium, he supplied requirements according to whether the cells needed a particular amino acid for growth and for bacterial development. That is, if an amino acid was essential for growth but was not present in flagellin, the cells could develop flagella to some extent despite their overall inability to grow and divide (Kerridge, 1939b). Extending these observations, Kenidge (1960) found that the three analogs tested (pFPhe, P-2-thienylalanine, and ethionine) all failed to block flagella development completely in auxotrophic mutants, but the first distinguished itself by preventing the formation of functional flagella. They were shorter and had a wavelength about half that of normal flagella. No motility was associated with them, and their abnormality was not reversed when the bacteria were restored to Phecontaining medium. In a similar analysis on Salmonella, Iino (1969) used the abnormality produced by pFPhe in regenerating flagella structure to demonstrate that they grew with a definite polarity, new material being polymerized at the distal end. More recently, Suzuki and Iino (1977) reappraised the development of flagella in another bacterium, Pseudomonas aeruginosa, and showed that pFPhe induced straight end pieces after 2 0 4 0 minutes of regeneration in the presence of the analog, which confirmed their apical growth pattern. This straight-flagellum pattern suggests, but does not prove, that some abnormality is introduced in the flagellin itself by the presence of the analog rather than by an alteration in dynein. In view of the large amount of in vitro work on structural proteins from motile systems, it should be a simple and informative matter to examine the ability of analog-incorporated tektins to assemble into microtubules. An indirectly related application ofpFPhe to cell movement has been its use in analyzing chemotaxis in E. coli. Comparing it with chloramphenicol, Fleming (1967) came to the conclusion that the analog did not operate by inhibiting chemotactic responsiveness, as chloramphenicol does, but by reducing the proportion of motile bacteria and their rate of motility.

VI. Use of Analogs in Other Areas of Biological Research A. MUTAGENESIS pFPhe has the ability to alter the genetic complement within cells. Division and haploidization in Aspergillus was reported by Lhoas (1961), and in Saccharomyces by Emeis (1966); and many other forms of disorganization of

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chromosomal material in Saccharomyces and Schizosaccharomyces were seen by Stromnaes (1968) and Gutz (1966), respectively, and in fungi by Day and Jones (1971). McBride and Gowans (1969), using another Phe analog, 2amino-3-phenylbutanoic acid, found chromosomal aberrations in Chlamydomonas. They also showed that clones of resistant cells arose, the resistance being characterized as a mutation of a nuclear gene showing classic Mendelian segregation. The analog also induced streptomycin and neamine resistance. McBride and Gowans raise the following possibilities concerning how analogs might affect the genetic material of cells: (1) An analog becomes incorporated into chromosomal proteins; (2) metabolic interactions of the analog might release a moiety, for example, a methyl or ethyl group, which can alkylate nucleic acids; (3) an analog may become incorporated into DNA polymerase; (4) an analog could disturb informational suppressors or induce gene-controlling substances. For 2-amino-3-phenylbutanoicacid, the mechanism was left unresolved. With other analogs such as ethionine, ethylation of nucleic acid has been implicated in mutation in Coprinus, because mutagenesis was related to the degree of transethylation occurring, although Talamud and Lewis (1973, 1974b) could not conclude that the same mechanism was responsible in bacteria. These investigators clearly showed in the latter study that pFPhe-induced mutagenesis was closely related to incorporation of the analog into protein. This would lead to a lack of fidelity in DNA replication and repair. This argument is further supported by the observation that the two analogs have a synergistic effect when applied together. Further evidence indicates that both analogs produce mutations of the single base change type in DNA. In a previous study on Ustilago, Lewis and Tarrant (197 1) had noted the distinctly stronger mutagenicity of pFPhe as compared to that of ethionine in forward mutations and presented circumstantial evidence that altered protein configurations could be responsible (based on the thermolability of NADP-linked glutamine dehydrogenase after analog treatment). Other analogs were also used, including norleucine and canavanine. The complexity of the mutagenic action of analogs is increased by the variety of responses occurring in different organisms. Talamud and Lewis (1974a) examined the effect ofpFPhe on reversion frequency in E. coli mutants (Leu- and Trp-) and found no effect, likewise in a Leu- mutant of Pseudomonas and two phage mutants. This led to an incisive study on two strains of Succharomyces cerevisiae in which there were mitochondrial and nuclear markers (erythromycin sensitivity and an adenine or Met requirement, respectively). Treatment with pFPhe increased the back-mutation frequency of the nuclear genes without affecting the mitochondrial genes. These workers argued that there is a fundamental difference in the active site of DNA polymerase or repair enzymes in bacteria (mitochondrial) and yeast cells, although this is not the only possible explanation. One possibility is that the analogs affect information suppressors which would not result in a genetic mechanism. Evidence from Pardee and Prestidge

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(1958), Jenkins et al. (1974), and others favors the effect occurring only at the suppressor level. The problem of analog mutation is obviously in its infancy, but is worth analyzing more carefully because it offers an infinitely more subtle approach than the conventional mutagens such as nitrosoguanidine, ethylmethanesulfonate, and so on. A combination of these two approaches has already been tested (Johnston, 1975). The production of mutants with amino acids analogs has a reverse side, that is, the development of resistant forms either in response to treatment with the analogs themselves or with conventional mutagens. pFPhe-resistant mutants have been studied by Wacker et al. (1964), Waltho and Holloway (1966), Sinha (1970), Hannan (1972), Brooks et al. (1972), Barker and Lewis (1974), Rhodes and Wilkie (1979, and Hennecke and Bock (1 975) in several different organisms including bacteria, yeast, and fungi. This analog has also found use in distinguishing linkage groups in Aspergillus (McCully and Forbes, 1965). The changes which might confer resistance on an organism growing onpFPhe could be several, but in general the commonest explanation is based on a subtle alteration in the specificity of phenylalanyl-tRNA synthetase, as suggested by Fangman and Neidhardt (1964a), Fangman et al. (1965), Bock and Neidhardt (1967), and Hennecke and Bock (1975). The work of Printz and Gross (1967) is of interest in this connection, since they showed that the Leu-5 mutant of Neurospora is temperature-sensitive, the leucyl-tRNA synthetase being more flexible and less specific at higher incubation temperatures than at lower temperatures. A similar type of investigation with analog mutants would be informative. Mechanisms other than alterations in tRNA synthetase specificity are almost certainly involved. Barker and Lewis (1974), using pFPhe on Coprinus, suggested that mutants have an altered feedback regulation. The biochemical aspects of mutants of many lower organisms are complicated by the fact that they can often synthesize their requirements irrespective of the exogenously supplied analog. Finally, Rhodes and Wilkie (1975) did not come to a definite conclusion in their study on at least four nuclear gene alterations in Saccharomyces, but noted that the carbon source on which the cells were grown influenced the incorporation rate of analog into cytoplasmic but not into mitochondria1 proteins. The resistant mutants showed a lower incorporation of analog than the sensitive parental cells. B. AGING Of the many theories of aging one which has provided renewed impetus to research is that of Orgel (1963) or its amended form (Orgel, 1973). Briefly, it is argued that the machinery for protein synthesis in cells continually makes errors, and with the passage of time errors tend to increase if some of the important DNA, RNA, and protein synthesis enzymes themselves become aberrant. In this

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situation, the cells reach a point where too many abnormal proteins have arisen, leading to senescence and cell death in what is called an “error catastrophe.” Errors in protein translation presumably occur at different rates in different organism all the time. With the knowledge that analogs can be incorporated into proteins, this incorporation was an obvious choice for experimentally creating a far greater incidence of mistranslation of proteins. Early attempts with analogs, however, produced apparently contradictory results. Harrison and Holliday (1967) fed up to five different analogs to third-instar larvae of Drosophilu before returning them to unsupplemented medium to pupate. No abnormalities were found in larval development or in the anatomy of the imago except perhaps a slowing up of the former with 2 mM analog. A reduction in longevity was found. The treatment of larvae close to pupation, however, was associated with a separation of the resultant flies into two groups, one dying very soon (2-3 days) after emergence and the other having life-span characteristics similar to those described above. The experiments of Dingley and Smith (1969) showed thatpFPhe fed to Drosophilu for 6 days after emergence had no effect-apart from a small percentage of initial deaths-on longevity. These two studies are probably reconcilable on the grounds that adult flies are not so dependent upon de novo protein synthesis as larvae and no selection of cells can take place. Nevertheless, if Orgel’s theory has any substance, one might still have expected flies to die earlier in Dingley and Smith’s experiment. In neither study was there an attempt to determine the extent to which the analogs had become involved in protein or disturbed the synthesis and turnover of proteins in the larvae and adults during and after treatment; and there was no reason to expect a good correlation, because one analog acted on a developing system and the other on completely preformed cellular organization with cell division occurring only in the germ cells. In connection with the latter, it would be of interest to investigate the effects of analogs on gametogenesis and fertility, especially since sperm flagella may also be disturbed by analog incorporation (as seen in Section V,D). With evidence from other organisms favoring the idea that defective protein synthesis and senescence are related, as in the mutants of Neurosporu (Holliday, 1969), and that defective enzymes are highly thermolabile (Lewis and Holliday, 1970), two studies on mammalian cells were carried out, one utilizing the base analog 5-fluorouracil (5-FU) to induce errors (Holliday and Tarrant, 1972) and the other using analogs of amino acids as well as 5-FU (Lewis and Tarrant, 1972). These experiments showed that old cells produced more unstable enzymes, as predicted by Orgel. The latter report demonstrated that the amount of enzyme itself was not reduced, but that there was a fivefold decrease in the ability of old cells to discriminate between Met and ethionine compared with that of young cells. This suggests a change in the amino acyl-tRNA synthetase specificity, which has been discussed in Sections II1,A and VI,A. It would be of

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interest to follow the changes in these enzymes in cells with analog, temperature, and age. Results from Cristofalo’s laboratory (Ryan et al., 1974) seem to contradict the above in that pFPhe and ethionine treatment on WI-38 cells had no effect on expected longevity when dose levels were not maintained at toxic levels. Although they demonstrated the incorporation of both analogs into cellular macromolecules, they did not compare this with homolog incorporation or with relative incorporation of an independent amino acid in treated and control cultures. Their cells were treated in medium which presumably contained normal concentrations of the homologs, that is, with an extracellular ratio of about loo0 homolog molecules to 1 analog molecule for the nontoxic level, lo-‘ M. On this account alone, it would perhaps be a little surprising if the analog had had an effect. Similarly a short exposure to analogs at higher concentrations ( IF5and IF4M ) for 1 week did not affect longevity; that is, cells which recovered from the 7-day treatment lived out a normal life span. But their results suggest that cells exposed for 30 days or continuously to I .7 X 10-SMpFPheplus 0.92 x M ethionine, or to doses one order of magnitude higher, undergo fewer doublings. Their argument for toxicity is based on the analogs producing an immediate growth-inhibitory effect on the cells, but nevertheless these cells went through 60-70% of the doublings of controls. This point emphasizes the main weakness in such studies, that is, how to define toxic effects and to decide whether toxic sequelae should be considered part of the aging process or a transient disorder.

C . VIRUSDEVELOPMENT Ackermann and his colleagues found that polio virus formation by infected HeLa cells was inhibited bypFPhe, but the analog did not prevent development of the associated cytopathology of the infection (Ackermann et al., 1954). Using influenza virus on chick allantoic membrane, Ackermann and Maassab (1955) confirmed that pFPhe did not reduce the infectivity of the inoculated virus but only its intracellular proliferation and maturation stages. There are many ways in which Phe analogs can disturb virus production, as can be seen from the large number of studies on the subject (Table 11). The most obvious point to be drawn from this table is that, of the Phe analogs, pFPhe has been almost exclusively used, although there have been investigations with analogs of other amino acids, such as azatryptophan (Pardee and Prestidge, 1958) and methyltryptophan (Cohen and Anderson, 1946). With a general agreement that analogs do not appear to disturb initial infectivity either by incubating viruses with analogs or cells with analogs before mixing (e.g., Tanami and Pollard, 1962), analogs could prevent viral development by (1) preventing “uncoating” of the virus once inside the cell (Joklik, 1962), (2) inhibiting the synthesis of soluble antigen, (3) suppressing or interfering with

VIRAL SYSTEMS I N WHICH Virus

THE

TABLE I1 EFFECTOF PHE ANALOGS HAVEBEENINVESTIGATED

Host cell

Analog

Adeno Bacteriophase (MS-2)

HeLa E. coli

pFPhe pFPhe

Encephalomyelitis, murine Encephalomyelitis, western equine Fibroma Foot-and-mouth Fowl plaque

Mouse Chick fibroblasts Rabbit kidney Kidney Chick embryo

pFPhe pFPhe pFPhe pFPhe pFPhe

Influenza Mengo

Chick allantoic membrane L strain

pFPhe, methoxinine pFPhe

Polio

HeLa

pFPhe

Polyoma Psittacosis (now Chlamydia) Rabbit fox SV40 Vaccinia

Mouse embryo Human synovial HeLa Green monkey kidney HeLa

pFPhe pFPhe pFPhe pFPhe pFPhe

Reference Wilcox and Ginsberg (1963) Wray et al. (1970); Abdel-Hady and Leach ( 1972) Pearson et al. (1952) Wecker and Schonne (1961) Maloney and Minocha (1970) Brown et al. (1961) Zimmermann and SchSer( 1960); Scholtissek and Roti (1961) Ackermann and Maassab (1955) Baltimore and Franklin (1963); Venwoerd and Hausen (1963) Ackermann et al. (1954); Levintow and Darnell (1961); Levintow et al. (1962); Watanabe et al. (1962); Scharff et al. (1963, 1965); Hummeler and Wecker (1964) Munyon et al. (1964) Tanami and Pollard (1962) Appleyard and Zwartouw (1965) Rapp et al. (1965); Mayor et al. (1966) Salzman et al. (1963); Kit et al. (1963); Nielsen (1963); Loh and Payne (1965)

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nucleic acid-synthesizing enzymes, (4)inhibiting or interfering with viral coat proteins [or the associated maturation proteins factors of Watanabe et al. (1962)], (5) blocking virus-releasing mechanisms. Considerable differences have been found in analog inhibition of viral development; in some cases complete suppression of virus multiplication was reported (e.g., Wilcox and Ginsberg, 1963; Appleyard and Zwartouw, 1965). In other cases little suppression of viral nucleic acid was found, although no mature infectious particles were released (e.g., Levintow and Darnell, 1961, Hummeler and Wecker, 1964); and occasionally virus formation has gone to completion, but with a considerable delay in the release of infectious particles (Munyon et al., 1964). Inevitably many of these differences can be ascribed to the diversity of the virus-cell systems involved, but there may also be discrepancies arising from the way in which the analogs themselves have been used and the precautions taken to prevent the homolog from circumventing the analog effect. Some investigators have taken the trouble to remove Phe from the medium before adding the analog, which makes it possible to obtain significant effects at relatively low concentrations (-0.05 mM) and sufficiently rapidly to influence the late stages of viral development, as shown by the sensitive experiments of Levintow et al. (1962) and Salzman et al. (1963). These contrast strongly with less precise experiments involving pFPhe concentrations such as those of Munyon et al. (1964, over 1.1 mM), Loh and Payne (1965, over 1.65 mM), and Appleyard and Zwartouw (1965, over 2.2 mM). Treatment of cells withpFPhe at the time of or within about 1 hour of infection may suppress not only the synthesis of enzymes or proteins of the host cell (Venwoerd and Hausen, 1963) but also those concerned with the replication of viral nucleic acid (Brown et al., 1961; Kit et al., 1963; Wilcox and Ginsberg, 1963; Appleyard and Zwartouw, 1965). In some studies (e.g., Zimmermann and Schafer, 1960), host cells have been treated with the analog before viral infection with similar results. It is evident from other work, however, that analogs do not necessarily block nucleic acid synthesis from the start (Levintow and Darnell, 1961; Levintow et al., 1962; Wray et al., 1970; Abdel-Hady and Leach, 1972). The two explanations offered are: (1) The analog might act by suppressing protein synthesis necessary for the production of enzymes concerned with viral nucleic acid replication; and (2) the analog might be incorporated into the polymerase enzymes themselves and disturb the viral nucleic acid products. These mechanisms are not necessarily mutually exclusive as long as some protein synthesis continues in the presence of the analog. On this point, Kit et al. (1963) stressed thatpFPhe reduced overall protein synthesis by less than about 30%, yet it disturbed nucleic acid synthesis of vaccinia virus much more dramatically. This indicates the formation of appropriate but possibly unstable polymerase enzymes, a conclusion also borne out by the careful step-by-step study of polio virus development in HeLa cells by Scharff et al. (1965). Perhaps the clearest

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distinction between the suppression of viral nucleic acid synthesis and some other effect on replication comes from the observations of Abdel-Hady and Leach (1972) who found that RNA produced in the presence ofpFPhe had far lower infectivity (about 5 % ) than the control viral nucleic acid. In contrast, Levintow and Darnell (1961) and Levintow et al. (1962) found that extracted viral RNA produced by polio virus-infected HeLa cells was infectious, but that it did not form mature particles and was not released by the analog-treated cells themselves. If cells are left for a short time after infection beforepFPhe is added, viral nucleic acid synthesis becomes more difficult to suppress, although this may not be generally applicable. Timed exposures to analogs have proved a useful analytic method for sequencing the events of viral infection, as is well illustrated by the study of Zimmermann and Schafer (1960). When fowl plaque virus-infected chick embryo cells were left 2 hours before analog treatment, soluble antigen appeared, whereas earlier treatment with analog had prevented its appearance. However, infectious virus still did not appear. Increasingly larger doses ofpFPhe had to be given by 3 hours after infection to suppress its formation and release. Similar results are reported by Wilcox and Ginsberg (1963), Loh and Payne (1965), and others. In contrast, Scharff et al. (1963) blocked the late stages of viral RNA synthesis with pFPhe treatment of HeLa cells several hours after infection; although it took about 45 minutes before shutdown, inhibition of infectious virus production was observed. With certain DNA viruses, for example, SV40, the length and relative sensitivities of the various phases of development may be much more extended (Mayor et al., 1966), and pFPhe can still inhibit virus production when added a day after infection. In the final stages of viral maturation, capsomeres are assembled with the nucleic acid. There is evidence that the proteins (or some of the proteins) of the capsids can be abnormal if synthesized in the presence of pFPhe (Salzman et al., 1963; Hummeler and Wecker, 1964; Wray et al., 1970). Analog proteins can either prevent assembly of the capsomeres or disturb their proper orientation, as suggested by observations on negatively stained polio virus preparations with electron microscopy (Hummeler and Wecker, 1964). In RNA viruses, capsid proteins may be the cleavage products of larger precursor molecules. An interesting finding of Jacobson and Baltimore (1968) is that, whenpFPhe was incorporated into a precursor molecule of polio virus, it could no longer be cleaved to form the normal capsomere proteins. This work was extended in later studies with pFPhe and other amino acids (see Jacobson et al., 1970). The majority of studies withpFPhe on blocked viral development have shown that mature virus can be produced if Phe is restored to the cells. Two interesting observations have been made on this recovery. First, Salzman et af. (1963) found that the restoration of Phe resulted in the production of mature, infectious virus which contained none of the analog proteins made in the presence of pFPhe.

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Second, Levintow et al. (1962) showed a similar effect, but with the RNA made by polio virus in the presence ofpFPhe, where the recovery in homolog led to a new cycle of RNA synthesis, and only this RNA eventually appeared in the mature virus produced by the cells. In several cases, the presence of granules often close to the nuclear membrane of the host cell has been noted inpFPhe-treated virus-infected cells (Rapp et a l . , 1965; Mayor et al., 1966). These granules disappear soon after reversal of the analog effects. Finally, viral infection is associated with the production of interferon by the host cells, and even herepFPhe has been instrumental in revealing the timing of biochemical events. Friedman and Sonnabend (1964) showed that interferon synthesis was inhibited in cells treated withpFPhe. Once again the question is raised of whether synthesis of interferon was inhibited, or whether it was formed but was no longer recognizable as interferon, a point which remains to be resolved. Friedman (1966) pretreated chick cells withpFPhe during an overnight incubation at 4"C, or for 4 hours at 37"C, before infecting them with Sernliki Forest virus in the absence of pFPhe. There was stimulated protein synthesis under these recovery conditions when virus infection occurred, and more virus but less interferon was made. Buchan and Burke (1966), givingpFPhe along with infecting influenza virus to chick cells, markedly suppressed interferon production; puromycin produced similar results. However, when these two substances were given 26 hours after infection, pFPhe failed to suppress interferon production, whereas puromycin prevented its formation. This result suggests that the presence of pFPhe within cells near the time of infection interferes with protein-synthesizing mechanisms directly or indirectly concerned with the interferon response, and not necessarily with the actual synthesis or incorporation of analog into interferon itself.

D. MISCELLANEOUS 1 . Developmental Studies

Morphogenetic effects of analogs on Tetrahymena, and on flagellar regeneration, indicate that they also produce changes in whole embryos. In the plant world, there are well-known effects of analogs on seed generation and seedling growth (see Fowden, 1963). There have been, however, only a small number of studies concerned with the development of animal emlxyos after treatment with analog amino acids. Waddington and Perry (1958) retarded chick embryo growth with pFPhe at 1 mM and completely blocked development at 10 mM. This inhibition was about 10 times more effective on a molar basis than that observed with ethionine. They also showed that growth proceeded normally when Phe was

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present at the same concentration as pFPhe. Fiasconaro (1965) described antagonistic effects of Phe analogs on the development of ascidians. Kollar used thienylalanine to inhibit the development of whiskers from explants of mouse snout skin (Kollar, 1968) and tooth germs in jaw explants (Kollar and Baird, 1968). Phe analogs were among several inhibitors of protein synthesis which affected the development of preimplantation embryos of mice (Thomson and Biggers, 1966), but it is not certain from this study whether the mechanism was simply inhibition of protein synthesis. Puromycin and pFPhe produced their inhibitory effects in dissimilar ways, the latter probably by being incorporated into proteins, particularly cleavage proteins. This work is of interest, because there is in fact no net synthesis of protein in early embryos, and yet a definite requirement for de novo proteins for cleavage is involved (see Section V,B). In the neonatal animal it is possible to interfere with the formation of phenylalanine hydroxylase in the rat by feeding analogs. This in turn leads to excess Phe, with degradation resulting in simulated phenylketonuria. In human beings this condition can be caused by a hereditary deficiency of phenylalanine hydroxylase, and therefore animal models are of value. Greengard et al. (1976) have recently reported that a -methylphenylalanine induces a better simulation of phenylketonuria when fed to neonatal rats between days 3 and 15.

2. Immunology It is surprising to find little use of analogs in the study of immune responses in animals. The most concentrated effort in this field has been that of La Via’s group (La Via et al., 1960, 1972; Hothan-Iglewski and La Via, 1969) who followed the depression of sheep red blood cell response in rats on a Phedeficient diet containing P-3-thienylalanine. To reduce the response, the analog had to be given within the first 3 days of challenge and was correlated with a depression of the anticipated RNA synthetic response in the spleens of challenged rats, probably involving mRNA. RNA synthesis in other body tissue was not disturbed. The work of Caviness (1966a,b) has already been referred to, but it is of interest that the macrophage-which has now been assigned an important role in immune responses- was studied with P-2-thienylalanine. It would be of interest to see to what extent the interactions of macrophages and lymphocytes could be affected by analog treatment separately and in combination. 3 . Radiation Studies An intriguing observation was made by Dickie et al. (1968) on the effects of pFPhe on the radiosensitivity of E . coli wild type and on E . coli 6y,a radioresistant mutant. Treating these cells with pFPhe progressively reduced the radiosen-

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sitivity of the wild-type cells to irradiation; the 6y mutant, however, showed increased sensitivity to y-irradiation. The investigators explained the decreased radiosensitivity of the former as being due to their inability to reinitiate chromosome replication withpFPhe treatment but found it difficult to explain the latter phenomenon. Amino acid deprivation of 6y mutants made them considerably more, not less, radioresistant in the presence of pFPhe, and therefore these workers excluded the possibility that the analog produced its effect by inducing a deficiency condition. The possibility remains that analog incorporation led to faulty synthesis of DNA repair enzymes in the cells, which enhanced the damaging effect of irradiation. This has obvious implications with respect to potentiating the radiation-induced damage to tumor cells in the radiotherapeutic treatment of malignant disease. 4. Toxicology

Analogs of amino acids can be tolerated for long periods of time when cells or organisms have the homologs available or can synthesize them. In the absence of homologs, analogs produce cytotoxic effects because their incorporation into protein disrupts normal cellular functioning. We have already seen that many analog proteins can behave normally, but even if one essential protein becomes infiltrated by analog and malfunctions, the cell will eventually come to a halt. We have also seen that many analogs produce a false feedback inhibition of synthesis of amino acids or their metabolic conversion to other products. This could produce a much more rapid response in organisms if the availability of an essential substance were suddenly reduced. The toxic effect of mFPhe is of interest (Litzka, 1936; Koe and Weissman, 1967), operating by a slightly different mechanism. This analog can be a substrate for phenylalanine hydroxylase which converts it into pFTyr. In the rat, the latter is a powerful convulsant and is lethal. When the fluorine is in the para position, as inpFPhe, the convulsant Tyr derivative can no longer be formed, and it is noteworthy that this analog (and pC1Phe) can block phenylalanine hydroxylase activity, thereby preventing animals given mFPhe from producing the convulsant Tyr product. For the toxicity of Phe and Tyr analogs in rats, the work of Boyer et al. (1941) showed k2.5 mg/kg for mFTyr (minimum lethal dose) and 20 mg/kg forpFPhe. They also noted the convulsions produced by the former, but their study was designed to compare toxicity at half the minimum lethal dose with the effects of these and other Tyr analogs on basal metabolic rate, working on the assumption that the analogs interfere with thyroxine synthesis. Later, Armstrong and Lewis (1951) offset pFPhe toxicity with Phe in rats. In general, however, proper analyses of the toxicology of amino acid analogs of Phe and the mechanism of their toxicity have not been carried out, but some reports, for example, on tumor growth inhibition by analogs, include information on toxicity (Ryan and Elliott, 1968).

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5. Specijjcity of Hormone Action The interaction of a peptide hormone with its receptor must be highly specific in order to elicit the desired response. Although the specificity of interaction of large hormones (such as insulin) would be difficult to investigate, smaller oligopeptide hormones would be more amenable to analysis by chemical modification of its structure. Since Tracey and Gregory (1964) found that the four N-terminal amino acid fragment of hog gastrin retained the same biological activity as the whole heptadecapeptide, Morley (1968) was able to carry out the much simplified analysis of this ligand by substituting each of its amino acids in turn with analogs, and by altering the N- and C-terminal moieties. His comprehensive analysis showed how flexible each residue and terminal moiety could be, without losing biological activity, and from this it was possible to deduce the approximate configuration of the receptor surface. The ligand, Trp-Met-AspPhe-NH2, was synthesized with a variety of Phe analogs including p-nitro-, p -fluoro-, p-methoxy- and hexahydrophenylalanine, all of which allowed complete retention of biological activity. This contrasts withp-hydroxyl substitution, that is, replacing Tyr for Phe, which completely destroys activity. It is of interest that here, as in many other situations already discussed, this small switch of the para hydrogen to a hydroxyl group creates another amino acid whose behavior is so completely distinct from that of Phe that it rarely overlaps. The substitution of many different derivatives of each of the amino acids in the hog gastrin ligand led Old and Jones (1976) to examine the inhibitory effects of several of the analogs on the appropriate aminoacyl-tRNA synthetases of two distinctly different systems, rat liver and E . coli. They found that Phe and Met analogs produced similar effects in both systems whereas Trp analogs behaved differently (see also Section 111,A). There was little or no correlation between the action of an analog on aminoacyl-tRNA synthetase activity and its effect on biological activity when substituted in the ligand.

VII. Degradation of Analog Proteins We have seen instances in which analog proteins cease to have an interfering effect when homolog proteins can once again be made, for example, in viral proteins during maturation (Salzman et al., 1963). The rapid recovery of cells from the analog in such cases suggests that the interfering analog proteins can be quickly diluted out or destroyed. Some analog proteins are obviously not discarded quickly but are conserved and may exert effects on subsequent cellular functions (e.g., Sisken and Wilkes, 1967; Smith and Pardee, 1970). The question which concerns us here involves the rate of turnover of proteins, in particular whether analog protein is turned over or degraded faster than its corresponding

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homolog protein. There is no need to elaborate on the complexity of this problem which has received its fair share of attention recently (see reviews of Pine, 1972; Siekevitz, 1972; Goldberg and Dice, 1974; Goldberg and St. John, 1976). Although the answer to the question concerning analogs appears to be that they do turn over faster, the hypothesis essentially stems from the belief that the continual turning over of proteins has developed as a means of eliminating errors which would otherwise amplify themselves and lead to cell degeneration and death. In a sense, this is a counter-aging device which must break down or become overloaded if Orgel’s hypothesis (1963) of aging has validity. As Goldberg and St. John (1976) note, “It is clear that the fundamental role of intracellular protein degradation is to protect the cell against the accumulation of abnormal or potentially harmful polypeptides. In both animal and bacterial cells, proteins that are denatured or that deviate from normal configurations are rapidly hydrolyzed. Although the evidence for this role of protein degradation as a ‘cellular sanitation system’ is most extensive in bacteria, mammalian cells can also selectively degrade proteins that result from missense mutations, that contain amino acid analogs or that are incomplete. [Reproduced, with permission, from the Annual Review ofBiochernistry, Volume 45. @ 1976 by Annual Reviews Inc.] With some analogs, and in particular the Arg analog canavanine, the evidence is overwhelming that cells degrade the analog products faster than arginyl proteins (Pine, 1967; Goldberg, 1972; Kemshead and Hipkiss, 1974; Bradley et at., 1975; Knowles et a!., 1975; Hendil, 1976). In all cases, much less labeling occurred with the canvanine treatment than in the Arg controls, and there is some doubt as to the true nature of the analog products. Bacteria might appear to resist the effects of canavanine very well, but mammalian cells are very sensitive to this analog and cytotoxicity quickly develops. Canavanyl products also appear to be much more insoluble and form granular deposits in treated bacteria (Prouty et at., 1975), which are particularly rapidly degraded by proteolytic enzymes. pFPhe can also induce metachromatic granule formation in bacteria (Brostrom and Binkley, 1969a). The more rapid degradation of canavanyl products may be due to a mixture of these various factors in vivo. But there is no question that they are degraded faster, since this can clearly be demonstrated by in vitro incubation with trypsin or pronase (Goldberg, 1972; Bradley et al., 1975; Giddings and Wheatley, 1978). This fact nullifies any suggestion that there must be some kind of selective “sanitation system” operating in vivo to eliminate error proteins, since the error canavanyl proteins are simply better substrates for a common proteolytic enzyme system. In the case of Phe analogs, the majority of reports also support the conclusion that analog proteins are degraded faster. Pine (1967) clearly demonstrated that thienylalanine and pFPhe incorporated into E. coli showed increased rates of degradation. Goldberg (1972), citing unpublished work, made a similar claim. The evidence from eukaryotes also favors increased degradation (Bradley et al., ”

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1976; Hendil, 1976; Shakespeare and Buchanan, 1976). Extensive studies in our laboratory, however, have failed to produce similar evidence for HeLa cells labeled withpFPhe, both in vivo and in vitro and under conditions which show a faster degradation of canavanyl products (Wheatley et af.,1977a, 1977b). This is H cells labeled shown in Fig. 9a for cells labeled for 1 hour ~ i t h p F P h e - ~versus with Phe-3H. These results agree well with the findings of Johnson and Kenney (1973), who studied FTtp incorporation and degradation, and the lack of differential degradation in thienylalanine-treated cells reported by Caviness (1966b). There may be several reasons for the discrepancy between these findings, possibly due to certain differences in technique. These include the use of high concentrations of analogs, such as 5 mM (Bradley et al., 1976), and the washing of cells in amino acid-supplemented solutions (e.g., Hendil, 1976; Shakespeare and Buchanan, 1976), which can prevent the dissipation of amino acid pools from within cells rather than aid in the exchange with the medium (see

b

Hr of chase Min FIG. 9. (a) Fall in specific activity of proteins of HeLa cells labeled for 1 hour with Phe-3H (open squares) ~ r p F P h e - ~(solid H squares). The results are plotted as the percentage counts retained by the TCA-insoluble proteinaceous component of the cells. Careful analysis of these curves suggests that it is erroneous to plot them on semilogarithmic coordinates, as they are composite in nature with a tendency for the fastest fall to occur in the first hour or so of chase. Therefore linear coordinates.have been used here. The bars give one standard deviation from the mean of not less than four points at each sampling. (b) Demonstration ~singpFPhe-~H-labeled HeLa cells that washing at 4°C in media containing 2 mM (open squares), 0.2 mM (solid squares), or no Phe (open circles) can markedly affect the retention of counts by the cells. As soon as washing becomes a chase by resuspending the cells in medium at 37°C after the sixth wash, the residual pools are quickly dissipated. This occurs both in the presence and absence of cycloheximide.

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Fig. 9b). It is recalled from Fig. 1 that there is a marked tendency for analogtreated cells to accumulate larger total amino acid pools, especially the TCAextractable component, than control cells. Thus inappropriate washings may allow analog-treated cells to release greater amounts of soluble counts retained after washing than controls, and this could be construed as faster initial degradation. In conclusion, there are some analog proteins which can obviously become more susceptible substrates for intracellular proteolysis, but there are also others which do not necessarily show this alteration. The evidence from Yutani e f al. (1977) suggests that we should even be on the alert for altered proteins in which degradation occurs more slowly than normal. In this connection, the failure of some large macromolecular species to cleave into smaller functional proteins, as suggested by the work on viral proteins by Jacobson and Baltimore (1968), might well be relevant.

VIII. Concluding Remarks Analogs have made a valuable contribution to a diversity of biological and biochemical studies. Their earlier application in blocking amino acid biosynthesis, and therefore indirectly suppressing synthesis, led to wider horizons when it became apparent that some analogs were sufficiently similar to their homologs to be incorporated in proteins. The polypeptide products in some cases, such as those obtained after ethionine, norleucine, or canavanine incorporation, may not have been subtle enough to act as further probes in examining the functions of these macromolecules, but other analogs-notable fluorine-substituted aromatic amino acids-have obliged in this way. If they have been easily mistaken in biosynthesis, it is not unexpected that some of the resulting macromolecules may also be sufficiently normal to behave like their homologous proteins. Their ability to “counterfeit advantage” and approximate the mechanism of action of the homologs has in general taught us much about the role of proteins in virues, cell division, and aging. pFPhe has been the most outstanding analog, and it would obviously be worthwhile to develop and explore similar analogs in future; for example, dihydrophenylalanine looks promising (Pal et al., 1956; Snow et al., 1968; Genghof, 1970; Yamashitaet al., 1970; Pine, 1973, and there may be fluorine derivatives of other amino acids which are as effective aspFPhe. The particular advantage in using Phe analogs is that it allows one to separate in time the synthesis of protein from its utilization, as shown in the studies of Kang et al. (1968) and Smith and Pardee (1970). These experiments involved temperature shifts from a permissive state to a nonpermissive state, thus producing in analog studies a parallel to the use of temperature-sensitive mutants, which had made possible highly specific explorations into in vivo cellular function.

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However, there are some areas of work where far greater advantage could have been taken of the increased thennolability of analog proteins, for example, in virus maturation. More extensive studies with eukaryotic cells are required to analyze the way in which cell growth is affected by analogs, and not simply in terms of population kinetics. Also, the conservation of analog proteins suggests that they can become deeply entrenched in cell structure and cause persistent trouble (Sisken and Wilkes, 1967). The effect of pFPhe in tubulin could be studied at the cellular level and also in vitro where assembly into microtubules can be induced in the subunits under the appropriate conditions. In much the same way, synthetases, ligases, polymerases, and other enzymes important in the synthesis and repair of macromolecules could be analyzed more extensively in vitro for loss of fidelity after incorporating analogs. This would test the hypothesis that the deleterious effect of analogs is due to increased errors in these important enzyme systems and establish more firmly whether or not this is fundamental to the biological basis of aging and mutation. To produce an aberration through the incorporation of an analog, which can masquerade as the normal molecule under some conditions but which can be made to reveal itself under others, has provided us with a high-precision tool in biological science. With increasing familiarity coupled with our rapidly expanding knowledge of protein structure, there is a considerable profit to be gained in their future use. Up to the present, the majority of analog studies have dwelt more on the interference effects they produce. There is another side to this story which can also teach us much about cell functions, that is, the study of their recovery after the removal of interfering analog effects. Finally the question can be raised concerning the practical application of analogs. Their use in psychopharmacological research and treatment is well known, but there is little prospect of their early use in the field of cell growth and its control until we know much more about their behavior at the molecular, cellular, and organismal levels.

ACKNOWLEDGMENTS

I thank in particular Dr. Jesse E. Sisken for his encouragement and help. Much of the impetus for this work derived from close collaboration with Dr. Leif Rasrnussen and Professor Erik Zeuthen of the Carlsberg Biological Institute, Copenhagen, where we are continuing to explore the effects of analogs and temperature on synchronized Tetrahymena. My colleagues in Aberdeen, including J . Y. Henderson, M. R. Giddings, M. Inglis, M. Moffat, J . H. Robertson, J. D. A. Robertson, and E. Walker, have given much valuable advice. I thank my wife for translating some of the more abstruse passages of the manuscript into plain English, and Miss Ann Mackay for typing it. The work was partly supported by the Cancer Research Campaign.

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INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 55

Recent Advances in the Morphology, Histochemistry , Biochemistry, and Physiology of Interstitial Gland Cells of Mammalian Ovary SARDULS. GURAYA Deparrment of Zoology, Coliege of Basic Sciences and Humanities, Punjab Agricultural University, Ludhiana, Punjab, Indiu

I. Introduction . . . . . . . . . . . . . . . . . . . 11. Interstitial Gland Cells of the Developing and Maturing Ovary A . Fetal Ovary . . . . . . . . . . . . . . . . . . B. Postnatal Ovary . . . . . . . . . . . . . . . . 111. Interstitial Gland Cells of the Prepubertal and Adult Ovary . . A. Origin and Distribution . . . . . . . . . . . . . B . Cytology and Histochemistry . . . . . . . . . . . C. Biochemistry . . . . . . . . . . . . . . . . . D. Cytological, Histochemical, and Biochemical Changes after Gonadotrophic Stimulation . . . . . . . . . . E. Correlation of Cytological, Histochemical, and Biochemical Changes with Steroid Secretions . . . . . . . . . . F. Formation and Storage of Lipids and Other Organelles . . G . Dynamic and Degenerative Aspects . . . . . . . . . IV. General Discussion and Conclusions . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .

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I. Introduction It is now well established that the Leydig cells (also called the testicular interstitial gland cells) of the mammalian and nonmammalian vertebrate testis are the site of the biosynthesis and secretion of androgens that regulate the developpent and function of the male reproductive tract and external sex characteristics (Christensen, 1975; Neaves, 1975; Guraya, 1976b); the androgens also seem to promote spermatogenesis. Although the comparable interstitial gland cells of the mammalian ovary have been the subject of many investigations during the last several years, they are least understood in terms of steroidogenesis. Divergent opinions about their identity, origin, and function continue to be expressed by anatomists, reproductive physiologists, and pathologists (see references in Brambell, 1956; Guraya, 1973a, 1974b; Mossman and Duke, 1973a,b). The main reason for this controversy has been a general acceptance of traditionally 17 1

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established misconceptions and a failure to identify and differentiate properly the interstitial gland cells from the relatively embryonic fibroblast-like stromal cells of the ovary. It is somewhat difficult to distinguish the different tissue and gland cell types with routine histological techniques, and thus the presence of interstitial gland cells was denied in the ovaries of some mammals (Brambell, 1956). Because of the great variability in the number of ovarian interstitial gland cells in different mammalian species during the reproductive cycle (Mossman and Duke, 1973a,b) not much attention has been paid to them in textbooks and elsewhere. The other two compartments of the ovary-the maturing follicle and the corpus luteum-continue to dominate the research performed by reproductive biologists because of their relatively more conspicuous cyclic changes, and thus ovarian interstitial gland cells have received little attention with regard to their structure and function. Mossman and Duke (1973b), in summarizing the results of histological studies, have concluded that interstitial gland tissue occurs in all mammalian ovaries, in massive amounts in some and in very small amounts in others. According to these investigators, interstitial gland tissue of the mammalian ovary can tentatively be defined as any endocrine type of gland tissue found in the ovary or the mesovarium which is not thecal gland or luteal gland. During the last few years, the interstitial gland cells of the prenatal, postnatal, prepubertal, pubertal, and mature mammalian ovary have been submitted to modern electron microscope, histochemical, biochemical, and physiological techniques, and interesting information has been obtained from these studies; significant advances in ovarian interstitial gland cell morphology have been achieved at the subcellular level. In this article, it is intended to summarize and integrate the results of these techniques and then to discuss the functional significance of these correlations in relation to steroid hormone synthesis in ovarian interstitial gland cells. Comparisions of the histological details of interstitial gland cells are avoided here, as they have been presented in more detail by Mossman and Duke (1973b). Correlations of steroid hormone synthesis with the cellular morphology of ovarian interstitial gland cells are emphasized and discussed in more detail, as such correlations, which are an extremely important, are lacking in the previous literature. However, the morphological (including ultrastructural), histochemical, and biochemical aspects of testicular interstitial Leydig cells in mammalian and nonmammalian vertebrates have been reviewed and discussed in detail in relation to steroid hormone synthesis (Christensen, 1975; Neaves, 1975; Guraya, 1976b). The details of the morphological, histochemical, and biochemical actions of gonadotrophins (both endogenous and exogenous) on ovarian interstitial gland cells are described for the prenatal, postnatal, and mature ovary. The morphological, histochemical, and biochemical characteristics of ovarian interstitial gland cells are also compared and contrasted with those of well-established gonadal steroid gland cells such as the Leydig cells and luteal cells (Christensen and Gillim, 1969; Guraya, 1971,

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1976b; Christensen, 1975; Neaves, 1975). It is hoped that the discussion of integrated results of various morphological, histochemical, biochemical, and physiological studies on the interstitial gland cells of the mammalian ovary will also increase our knowledge of the biology of steroid gland cells in general and ovarian interstitial gland cells in particular.

11. Interstitial Gland Cells of the Developing and Maturing Ovary A. FETALOVARY

Previous light microscope studies have revealed that interstitial gland cells are usually distributed in the medulla and to some extent in the cortex of the late fetal ovary (Mossman and Duke, 1973b). They may be sparsely scattered, or they may be highly developed and hypertrophied and occupy almost the entire medulla and much of the cortex, as in the horse and zebra (see references in Mossman and Duke, 1973b). Recent correlative histochemical and electron microscope studies have clearly revealed the origin, development, and distribution of primary interstitial gland cells in the fetal ovary of the guinea pig (Fig. 1) (Gurayaet al., 1974; Guraya, 1977a). No such cells were described previously during the fetal life of the hamster (Weakley, 1967), mouse (Odor and Blandau, 1969a,b), rat (Guraya and Uppal, 1978), or ferret (Deanesly, 1970). However, Gondos and Hobel (1973), using the electron microscope, showed the presence of interstitial gland cells in ovarian tissue from human fetuses aged 12 to 20 weeks. These cells, having ultrastructural features typical of steroidogenic tissue, are distributed in the medullary region just beneath the cortical cords. Motta (1 972) also reported the presence of interstitial gland cells in the human fetal ovary, which possess ultrastructural features specific to steroidogenic tissues (see also Motta and Takeva, 1970). In both the guinea pig and human, fetal ovarian interstitial gland cells usually differentiate adjacent to blood vessels and capillaries. They contain smooth endoplasmic reticulum, large spherical mitochondria with prominent lamelliform or tubular cristae, well-developed Golgi complexes, and lipid bodies. These ultrastructural features are well known to be associated with steroidogenic activity. Besides the organelles typical of steroidogenic tissue, interstitial cells of human ovaries either immediately before or soon after delivery show various enzyme activities related to steroidogenesis (Brandau et al., 1968; Motta and Takeva, 1970). Therefore the source of steroidogenic activity in the human fetal ovary (Bloch, 1964; Jungmann and Schweppe, 1968; Resko, 1977) must be the interstitial gland cells demonstrated with electron microscope and histochemical techniques (Brandau et al., 1968; Motta and Takeva, 1969, 1970; Motta, 1972; Gondos and Hobel, 1973). Fetal granulosa cells lack the histochemical and ultrastructural features associated with steroidogenic activity (Gondos et

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FIG. 1. Histochemical preparation of a fetal 62-day guinea pig ovary, showing follicles (F)in the cortex and primary interstitial gland cells (IGC) filled with highly sudanophilic lipids in the medullary strorna (S). ~ 7 7 .

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al., 1971; Guraya et al., 1974; Guraya, 1977a), although they exhibit histochemical evidence of A5-3P-hydroxysteroid dehydrogenase (3P-HSDH) activity in the human fetal ovary (Goldman et a l . , 1966). Fetal interstitial gland tissue in the guinea pig oiary develops from a wave of differentiation which initially appears in the ovarian medulla and subsequently progresses toward the follicles of the corticomedullary regions (Fig. 1) (Guraya, 1977a). These interstitial gland cells clearly originate from undifferentiated stromal cells from fetal day 50 onward (Gurayaet al., 1974; Guraya, 1977a; see also Stegner, 1973). Cells which are cytologically intermediate between fibroblast-like cells and differentiated interstitial gland cells are present on day 50. These intermediate cells are similar in appearance to fibroblast-like cells, except for the presence of lipid droplets consisting mainly of phospholipids and large amounts of granular endoplasmic reticulum. The maximum development of this primary interstitial gland cell system during the fetal life of the guinea pig occurs between fetal days 55 and 65 (Fig. 1). Its size is further increased on postpartum days 1 and 2 (Fig. 2). During these days of ovarian development, almost the entire mass of stromal tissue, which is located between the follicles in the inner zone of the ovarian cortex and medulla, is composed of large interstitial gland cells. The latter lie in close contact with each other and form compact cell

FIG. 2 . Histochemical preparation of a postnatal 2-day-old guinea pig ovary, showing diffusely distributed sudanophilic lipids and highly sudanophilic lipid droplets (L) in the primary interstitial gland cells. x816.

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masses of variable size (Fig. 2). The identification of interstitial gland cells as a special group of hypertrophied fibroblast-like cells within the developing ovary of fetal guinea pigs and humans is based on their histochemical and ultrastructural similarities to the steroid-secreting cells of mature gonads, as discussed in detail in Section 111. The relatively immature interstitial gland cells of the fetal ovary do not show an appreciable development of smooth endoplasmic reticulum (or diffuse lipoproteins). Fibroblast-like cells, which give rise to interstitial gland cells, have granular endoplasmic reticulum, many free ribosomes, and mitochondria with simple cristae (Fig. 3) (Guraya et al., 1974; Guraya, 1977a). The presence of rough endoplasmic reticulum and ribosomes suggests that active protein synthesis is taking place during these intermediate stages of interstitial cell development. This protein synthesis may include steroidogenic enzymes. Between days 55 and 60, when the transformation process involves a great number of fibroblast-like cells, many individual cells are still seen in the intermediate stage of development in the guinea pig ovary (Guraya et al., 1974; Guraya, 1977a). It is shortly after birth that significant alterations occur in the cytoplasmic structures of interstitial gland cells. As a result of degranulation and cisternal dilation, the whorled structures of rough membranes are continuously transformed into short, interconnected tubules of smooth reticulum. But even in the most advanced stages of transformation they still bear some scattered clusters of ribosomes on their surfaces. The transformation of rough membranes into tubular and smoothsurfaced reticulum has also been observed in differentiating interstitial cells of the juvenile ovary of the mouse (Stegner, 1970; Quattropani, 1973) and rabbit (Davies and Broadus, 1968) and the fetal guinea pig testis (Black and Christensen, 1969). A similar sequence of events has also been found in Leydig cells of 2-day-old chicks following the exogenous stimulation of luteinizing hormone (LH) (Connel, 1972). Dallneret al. (1966), studying the development of smooth endoplasmic reticulum in hepatocytes, first observed a system of rough membranes but, as the cells matured, the attached ribosomes became more widely spaced and the profiles became tubular and smooth-surfaced. From this discussion, it can be concluded that the abundant granular endoplasmic reticulum of differentiating fibroblast-like cells (Fig. 3) may be involved in the genesis of smooth membranes of differentiated interstitial cells of fetal guinea pig and human ovaries. The appearance of smooth membranes, which indicates the structural maturation of interstitial cells, is also paralleled by other ultrastructural alterations such as the enlargement and swelling of mitochondria and the presence of myelin whorls (Stegner, 1973; Guraya et al., 1974). Corresponding to the ultrastructural alterations discussed above, during the differentiation of fibroblast-like cells into interstitial cells certain histochemical changes also occur, which consist of the development of lipid droplets and diffuse lipoproteins (Figs. 1 and 2) (Guraya et al., 1974; Guraya, 1977a); the

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FIG.3. Electron micrograph of a fetal 60-day guinea pig ovary, showing undifferentiated fibroblast-like cells and transitional stages of primary interstitial cell development. Abundant rough membranes (R) are concentrically arranged around lipid droplets (L). Mitochondria (M) are also seen. Note the presence of a Golgi complex (G) in the differentiating interstitial cell. x 107,100. (S. S. Guraya, H. E. Stegner, and C. Pape, unpublished.)

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diffuse lipoproteins have been presumed to constitute the ultrastructural membranes of increasing endoplasmic reticulum. The development of identifiable interstitial cells in the fetal ovary is clearly dependent on the hypertrophy and hyperplasia of certain organelles and lipid droplets (Figs. 1-3). The increasing lipid droplets develop abundant triglycerides (neutral fats) in addition to phospholipids which constitute the sparsely distributed lipid bodies of the fibroblastlike cells, granulosa cells, and oocytes of the developing ovary (Guraya, 1977a). These histochemical differences between the lipid droplets of fibroblast-like cells and interstitial cells are also supported by electron microscope studies (Stegner, 1973; Guraya et al., 1974). The lipid droplets of the interstitial cells of the fetal ovary show an electron-lucent core and an opaque periphery, whereas those of fibroblasts and follicle cells are uniformly electron-dense (see also Quattropani, 1973). Their electron-lucency in interstitial cells may be due to the accumulation of more triglycerides in the medullar than in the periphery. Quattropani (1 973) has suggested that the alteration in lipid granule structure is a reflection of qualitative changes in the lipid and a result of metabolic changes in the differentiating interstitial cells of the ovary. Also, Christensen ( 1 975) has noted that the density of staining exhibited by lipid droplets may reflect to some extent their unsaturated fatty acid content, since osmium tetroxide, a common fixative in electron microscopy, reacts with double bonds and is electron-opaque. However, the role of other factors, such as the extent of extraction of lipids, can be overlooked. The close spatial association seen between the smooth endoplasmic reticulum, mitochondria, and lipid droplets of fetal interstitial cells (Gondos and Hobel, 1973; Stegner, 1973; Guraya el al., 1974) implies a functional correlation. Lipids apparently pass freely across the interface between the lipid and the cytoplasm at the surface of the lipid droplet. The lipid droplet has been suggested to have a membrane at its surface (Flint and Armstrong, 1972; Armstrong and Flint, 1973), although this is not always clear in routine electron micrographs. Christensen (1 975) has stated that it is possible that the bounding layer of the lipid droplet resembles a half-membrane consisting of a phospholipid monolayer with protein associated with its hydrophilic (cytoplasmic) surface (see also Armstrong and Flint, 1973).

B. POSTNATAL OVARY The interstitial cells in the developing and maturing ovary of rodents have been classified as primary and secondary, depending upon their source and the sequence of their appearance (Dawson and McCabe, 1951; Rennels, 1951). The primary, being the first to appear, originate from fibroblast-like cells having no relationship with the follicular apparatus (Stegner, 1970, 1973; Stegner and Onken, 1971 ; Guraya et aZ., 1974; Guraya, 1977a; Guraya and Uppal, 1978), although cords of cells projecting from the follicle have also been proposed as

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their site of origin (Rennels, 1951). But recent electron microscope and histochemical investigations have revealed that the cells of these cords show ultrastructural and histochemical features similar to those of follicle cells and that the cords are delineated by a basal lamina (Brandau, 1970; Merchant and Zamboni, 1973; Quattropani, 1973; Guraya, 1977a). The histochemical findings of Baillie et ul. (1966) on the developing mouse ovary have revealed the differentiation of stromal mesenchymal cells into primary interstitial tissue (see also Ben-Or, 1963). In the ovary of the neonatal field rat (Millurdiu meltada), treated with histochemical techniques for lipids, primary interstitial cells are formed by the hypertrophy of fibroblast-like cells lying in the interfollicular spaces (Guraya and Uppal, 1978). Stegner and Onken (1971) observed that stromal cell complexes adjacent to the capillaries are the preferred sites of interstitial cell development. Precursor stromal cells show granular endoplasmic reticulum, elongated mitochondria with lamellar cristae, and lipid bodies, as already discussed for the ovary of the fetal guinea pig and human. During their transformation into interstitial cells, round mitochondria with tubular cristae, smooth endoplasmic reticulum, lipid bodies, and a well-developed Golgi complex develop (Stegner, 1970, 1973; Quattropani, 1973; Guraya et ul., 1974; Balboni, 1976; Guraya, 1977a; Guraya and Uppal, 1978). In the laboratory rat, field rat, and mouse primary interstitial cells appear during the neonatal or postnatal period instead of during fetal life, as already discussed for the ovary of the guinea pig, human, horse, and zebra. These differences in the time of appearance of primary interstitial cells may be due to differences in the gestation period, which is longer for the guinea pig, human, horse, and zebra. Even the time of appearance of primary interstitial cells varies among different species and strains of rats and mice. In the mouse strain C57 BL/6J, interstitial cells are first recognized on postnatal day 12 (Quattropani, 1973), although no distinction has been made between primary and secondary interstitial cells. In a study with C57 BL/6 Ra mice, the first appearance of interstitial cells was observed on postnatal day 10 (see Quattropani, 1973). Stegner (1970), using Swiss Albino mice, found similar cells on postnatal day 7. The primary interstitial cells in the ovary of the field rat can be distinguished on postnatal day 10 (Guraya and Uppal, 1978). Their maximum development occurs between postnatal days 10 and 16, when the interstitial cells form compact masses of various sizes lying in the interfollicular spaces, which are easily distinguished in frozen sections stained with Sudan black B. Secondary interstitial gland tissue originates from the thecal cells of atretic follicles (Rennels, 1951; Quattropani, 1973; Guraya, 1977a; Guraya and Uppal, 1978). Therefore it is designated theca-type interstitial gland tissue by Mossman and Duke (1973a,b). This type of tissue apparently makes up the majority of the prepubertal, pubertal, and adult interstitial gland tissue discussed in Section 111. Secondary interstitial cells of thecal origin begin to appear on postnatal days 17

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and 18 in the field rat (Guraya and Uppal, 1978) and mouse (Quattropani, 1973). The primary interstitial cells involute as the secondary interstitial cells accumulate (Guraya, 1977a; Guraya and Uppal, 1978). The origin of secondary interstitial cells is clearly related to the atresia of medium-sized follicles in the ovary of the field rat (Guraya and Uppal, 1978) and guinea pig (Guraya, 1977a). However, according to Quattropani (1973), the follicles around which these cells were distributed did not appear to be atretic. Apparently he did not distinguish the hypertrophied theca interna from the secondary interstitial cells of thecal origin, as the former always develop in the wall of normally growing follicles and the latter in association with atretic follicles. Secondary interstitial cells arising from the theca interna of the atretic follicles develop the cytological and histochemical characteristics of well-established steroid-secreting cells (e.g., luteal cells and Leydig cells), as already discussed in detail for the primary interstitial cells of the fetal ovary of the guinea pig and human.

111. Interstitial Gland Cells of the Prepubertal and Adult Ovary Maturation of the mammalian ovary is accompanied by the development and accumulation of interstitial cells derived from the theca interna of atretic follicles (Figs. 4 and 5) (Guraya, 1974a, 1977a; Guraya and Uppal, 1978). Follicular growth and atresia are greatly enhanced with the maturation of the ovary (Fig. 4). At the same time the theca-type interstitial cells accumulate (Fig. 5 ) , which are discussed in detail here from various viewpoints, since they have been studied more extensively in different mammalian species with various techniques. A.

ORIGIN AND

DISTRIBUTION

Besides the theca interna or thecal gland cells of Graafian follicles, and the luteal gland cells of the corpus luteum, the mammalian ovary contains several types of interstitial cells which have been tentatively classified as seven different types (Mossman and Duke, 1973a,b). The one type of interstitial gland that occurs universally in juvenile and adult mammals is that originating from the theca interna of atretic follicles (Figs. 4-7) (Guraya and Greenwald, 1964a,b, 1965; Guraya, 1%6a,b, 1967a,b,c, 1968a,b,c, 1971, 1972a,b, 1973a,b,c, 1974a,b,c, 1976c, 1977d; Mossman and Duke, 1973a,b; Balboni, 1976). These theca- or stroma-type interstitial gland cells are of necessity correlated in time of appearance with cycles of follicular atresia (Figs. 4-7). They also show a similar origin in ovaries of nonmammalian vertebrates (see Guraya, 1976a,d; Guraya and Chalana, 1976; Chalana and Guraya, 1977). Thus they first appear when the atresia of follicles large enough to have a theca interna first occurs.

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FIGS.4 and 5. Histochemical preparation of ovaries from immature and mature marmosets, respectively, showing the progressive development and accumulation of interstitial gland tissue (IGT) masses in the medullary regions during maturation of the ovary. Normal follicles (NF) and atretic follicles (RG) at various stages of growth and degeneration are seen in the cortex and corticomedullary regions. X20. (From Guraya, 1968a.)

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FIG.6. Histochemical preparation of a guinea pig ovary obtained on day 40 of pregnancy, showing the distribution of sudanophilic interstitial gland tissue masses (IGT) of variable sizes. Both normal and atretic follicles (F) are also seen. x96.

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FIG. 7. Histochemical preparation of a guinea pig ovary obtained on day 2 of lactation, showing sudanophilic lipids in the interstitial gland tissue masses (IGT) and corpus luteurn (CL) of lactation. Theca interna cells of degenerating follicles (DF) are seen to give rise to interstitial gland tissue. x96.

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Theca-type interstitial gland tissue therefore forms the most characteristic and important glandlike tissue whose presence has also been shown convincingly even in ovaries of nonpregnant humans (Fig. 8), rhesus monkeys, and buffalo (Figs. 9 and 10) (Mossmanet a l . , 1964; Guraya, 1966a, 1967c, 1968c, 1974a), in which its presence was denied in previous studies carried out with routine histological techniques. The presence of theca-type interstitial gland cells has also been demonstrated in ovaries of nonmammalian vertebrates (Guraya, 1976a,d; Guraya and Chalana, 1976; Chalana and Guraya, 1977). In its development, morphology, and distribution, it varies greatly among different mammalian species (see the numerous different figures in this article) (Guraya,

FIG.8. Histochemical preparation of an ovary from a nonpregnant woman, showing stroma and relatively undifferentiated interstitial gland tissue (IGT) surrounding the follicular cavity of an atretic follicle. Near the clear scar left after degeneration of the follicle, some residual interstitial cells (arrows) are still seen in the process of losing their lipids and cytoplasm. X40.

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FIG. 9. Histochemical preparation of part of an atretic follicle from the ovary of a nonpregnant buffalo, showing the differentiation of interstitial gland tissue (IGT) from the theca interna. The remnants of degenerating granulosa cells (DG) are also seen. Sudanophilic lipids are not seen in the stroma (S). X 100.

FIG. 10. High-power view of theca-type interstitial gland tissue (IGT) from the ovary of a nonpregant buffalo, showing deeply sudanophilic lipids which are not seen in the strorna (S). X400.

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FIG.11. Histochemical preparation of a human ovary at term, showing highly developed thecatype interstitial gland tissue (IGT) which can be easily distinguished from the undifferentiated stroma (S) because of the presence of abundant diffusely distributed sudanophilic lipids. X 240.

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FIG. 12. High-power view of greatly hypertrophied theca-type interstitial gland cells from a human ovary at term, showing abundant diffusely distributed sudanophilic lipids in their cytoplasm, which are not seen in the nuclei or in the stromal elements. X380.

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1973a,b,c, 1974a, 1978; Mossman and Duke, 1973a,b). The development and distribution of theca-type interstitial gland tissue also varies greatly with the ovarian cycle in the same species; the size of the individual gland cells also varies considerably from species to species and with the reproductive cycles of mammals and nonmammalian vertebrates. These differences in the development of interstitial gland cells probably represent responses to different hormonal stimuli during different reproductive stages. Alternatively, they may indicate a difference in ovarian response to external hormonal stimuli. Generally, in mammals this tissue is the most abundant and the most differentiated during proestrus, estrus, and throughout the implantation period of pregnancy, yet in humans it reaches its greatest abundance during the last month of gestation (Figs. 11 and 12) (Mossman et a l . , 1964; Guraya, 1966b). The ovaries of relatively immature marmosets do not show any appreciable development of hypertrophied interstitial gland cells (Guraya, 1974a). With the initiation of sexual maturation, there

FIG.13. Histochemical preparation of part of an ovary from an estrous rabbit, showing the highly sudanophilic theca- or stroma-type interstitial gland tissue which occupies the whole ovary, except the cortical region containing follicles of various sizes. x 148.

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FIG. 14. Histochemical preparation of part of an ovary from a pregnant hare, showing acid hematein-positive interstitial gland tissue (IGT) which occupies the whole ovary except the corpora lutea (CL) and the cortical region. X50.

occurs marked hypertrophy and the development of interstitial gland cells derived from the theca interna of degenerated follicles, which ultimately constitute large masses of variable size in the deeper medullary part of the ovary (Figs. 4 and 5). Guraya (1974a) has also observed complete atrophy of these masses of theca-type interstitial gland cells in ovaries of marmosets with underdeveloped corpora lutea. Interstitial cells also change with the season in annually breeding mammals (see Mossman and Duke, 1973b; Guraya and Gupta, 1978) and nonmammalian vertebrates (Guraya, 1976a; Guraya and Chalana, 1976; Chalana and Guraya, 1977, 1978). In the reduced ovary of the field rat during winter months when it does not breed, theca-type interstitial gland tissue disappears but is rebuilt before the initiation of reproductive activity (Guraya and Gupta, 1978). With the formation of interstitial cells from the theca interna of atretic follicles, the ovaries increase in size. Vascularization, along with gonadotrophins, seems to be the key to this cytoplasmic change which involves the hypertrophy of fibroblast-like cells (Guraya, 1968b). In comparison to other mammalian species, the rabbit has been the most extensively used in studying the cytology, histochemistry, biochemistry, and

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FIG. 15. Histochemical preparation of part of an ovary from Q nonpregnant hat, showing highly sudanophilic interstitial gland cells which occupy the whole ovary, except the cortex and follicles (F). X265.

physiology of ovarian interstitial gland cells, since in this animal they are highly developed, well-organized, and glandlike, forming lobules of various sizes (Fig. 13). Capillary plexuses occur between the gland cells. Interstitial gland cells occupy all of the rabbit ovary except for the extreme cortical portions which constitute the tunica albuginea and which contain small follicles (Brambell, 1956; Guraya and Greenwald, 1964a; Guraya, 1967a; Davies and Broadus, 1968; Mossman and Duke, 1973a,b). The undifferentiated stromal elements are poorly developed and divide the glandular tissue into lobules of various sizes (Fig. 13). Theca- or stroma-type interstitial gland cells also show extensive development in the ovaries of other mammalian species such as the hare Lepus nigricollis (Fig. 14), the bat Myotis grisescens (Fig. 1 3 , and the civet cat Puradoxurus hermuphroditus (Fig. 16). In contrast, the ovary of the nonpregnant human (Fig. 8), Indian mongoose Herpestes auropunctutus (Fig. 17), and buffalo (Fig. 18) shows sparse distribution of sudanophilic interstitial cells of thecal origin. Guraya and co-workers (Guraya and Greenwald, 1964a,b, 1965;

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FIG. 16. Histochemical preparation of part of an ovary from a nonpregnant civet cat during the follicular phase, showing highly developed sudanophilic interstitial gland tissue. X 32.

Guraya, 1974b) and Mossman and Duke (1973b) have detailed its histological variations in different mammalian species.

B. CYTOLOGY AND HISTOCHEMISTRY In ovaries fixed in formaldehyde-calcium, postchromed in dichromate calcium, and examined in frozen gelatin sections treated with Sudan black B, hypertrophied interstitial gland cells appear as spherical or polyhedral cells (Figs. 12 and 19-21). The size, amount, and distribution of individual gland cells vary considerably from species to species and with the reproductive cycle (Figs. 4-20) (Guraya and Greenwald, 1964a,b, 1965; Guraya, 1966a,b, 1967a,b,c,

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FIG. 17. Histochemical preparation of an ovary from a nonpregnant Indian mongoose during the follicular phase, showing the sparse distribution of sudanophilic interstitial gland cells (IGC) in the highly developed stroma (S). Part of a large Graafian follicle (F) with its sudanophilic theca interna cells is seen. X 100.

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FIG. 18. Histochemical preparation of part of an ovary from a nonpregnant buffalo, showing relatively undifferentiated interstitial gland celk (IGC) in the wall of two degenerated follicles (DF). The stroma (S) is highly developed. x50.

1968a,b,c, 1971, 1972a,b, 1973a,b,c, 1974a,b, 1976c, 1977d; Mossman and Duke, 1973a,b). There are many minor variations, usually involving the appearance and the extent of various cytoplasmic structures such as lipid droplets (Figs. 12 and 19-21). The number of lipid droplets seen in the cytoplasm varies greatly with the species and the physiological state (Figs. 4-21). These inclusions are particularly abundant in the rabbit, hare, bat, rat, hamster, cat, and guinea pig. Their number is relatively smaller in the cow, human, rhesus monkey, marmoset, dog, civet cat, and opossum (Guraya, 1974b). The greatly hypertrophied interstitial gland cells have abundant cytoplasm with a vesicular.or oval nucleus having one or more large nucleoli (Figs. 12 and 19-21). The heterochonnatin is usually found at the periphery of nucleus. It appears to represent regions of the chromosomes that remain tightly coiled.during interphase, or special portions of the chromosome that possess a high amount of satellite DNA (Lima-de-Faria, 1969). The bulk of the nucleus consists of euchromatin, in which chromosomes are dispersed and may be active in the transcription of mRNA required for the synthesis of enzymes and other proteins involved in steroidogenesis, as well as other functions of the interstitial gland cell. When interstitial gland cells are less active in steroid hormone synthesis, their cytoplasm is packed with spherical

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FIG. 19. High-power view of interstitial gland cells from the ovary of an estrous rabbit. The cytoplasm of the gland cells is packed with spherical lipid droplets (1). Vacuoles (v) involved in the release of lipid droplets are seen in the peripheral portions of the gland cells. Vesicular nucleus (n). Very few lipid droplets are seen in the intercellular spaces. ~ 9 9 0 (From . Guraya, 1967a.)

lipid droplets of variable size (Figs. 6, 7, 10, 13, 15, 19, and 21) consisting of phospholipids, phospholipids and triglycerides, or cholesterol and/or its esters, along with triglycerides and some phospholipids, depending on their physiological state (Guraya and Greenwald, 1964a,b, 1965; Guraya, 1966a, 1967a,b,c, 1968a,b,c, 1972b, 1974b, 1977d). The amount, size, and histochemical composition of lipid droplets are inversely related to the intensity of their secretory activity, since interstitial gland cells producing steroid hormones rapidly show few or no lipid granules (Figs. 1 1 , 12, 16, 17, and 20). Using histochemical techniques, other workers have also described lipids, and cholesterol and its esters, in the ovarian interstitial gland tissue of the rat, guinea pig, and rabbit (Claesson and Hillarp, 1947a,b; Claesson, 1954a,b, Falck, 1953; Deane, 1952, Jacoby, 1962). Their studies were mainly concerned with finding sites of steroid formation based on the appearance of cholesterol and/or its esters. The large lipid droplets, usually found in the outer cytoplasm of hypertrophied interstitial cells, are relatively uniform in size (Figs. 19 and 22). However, there are small lipid granules in the juxtanuclear Golgi area, which consist mainly of

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FIG.20. High-power view of fully differentiated interstitial gland cells from the ovary of a nonpregnant marmoset, showing the nuclei (N), Golgi zone (GZ), sparsely scattered, deeply and a diffusely distributed sudanophilic substance in the cytoplasm. sudanophilic lipid bodies (L), X880. (From Guraya, 1968a.)

phospholipids and some triglycerides. Histochemical studies by Guraya (1967a) clearly revealed a heterogeneity in the composition of different lipid inclusions in the same gland cell. The large lipid droplets in the gland cells are usually so close together that they obscure organelles such as the mitochondria and the Golgi elements. These organelles can be studied in material extracted with acetone and treated with Sudan black €3, as well as in material fixed in Regaud’s fluid and stained with iron hematoxylin; they can also be studied in material prepared according to Aoyama’s silver nitrate technique. In such sections some phospholipids continue to persist in the form of granules and duplex vesicles, indicating their location at the surface of growing lipid droplets (Guraya, 1967a). More mature lipid droplets contain abundant cholesterol andor its esters, triglycerides, and few phospholipids, since they dissolve completely in acetone. Mitochondria appear in the form of granules and rods distributed uniformly throughout the cytoplasm; they have the usual phospholipid-protein composition (Guraya, 1967a). In material treated with Aoyama’s silver nitrate technique, the multiple Golgi elements lie in the juxtanuclear cytoplasm which, because of their dense, organized nature stands out in sharp contrast to the outer cytoplasm. The entire

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FIG. 21. High-power view of interstitial gland cells from a guinea pig ovary obtained on day 40 of pregnancy, showing highly sudanophilic lipid accumulations in their cytoplasm. X960. (From Guraya, 1974b.)

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@ FIG. 22. Normal interstitial gland cells (ngc) and degenerating gland cells (dgc) from the ovary of an estrous rabbit. Normal gland cells are filled with spherical lipid droplets (1). Vacuoles (v) involved in the release of lipid droplets (1) are present; some are partly inside and partly outside the gland cells. Small lipid granules are distributed in the Golgi zone. Degenerating gland cells (dgc) are filled with relatively more coarse lipid droplets. Vesicular nucleus (n). (From Guraya, 1967a.)

cytoplasm, including the Golgi area consisting of hypertrophied interstitial gland cells, shows abundant, diffuse lipoproteins (Figs. 11, 12, and 20) (Guraya, 1966b, 1967b,c, 1968a,b,c, 1972a,b, 1974b, 1976c, 1977d). The amount of diffuse lipoprotein seems to vary greatly from species to species, as well as at different stages of differentiation of individual gland cells (Figs. 11, 12, and 20). The cytoplasm of interstitial gland cells also contains RNA (Guraya and Greenwald, 1964a). Proof that ovarian interstitial gland cells are the site of steroid hormone synthesis has come comparatively recently as the result of an important development. A histochemical technique for localizing the steroidogenic enzyme 3P-HSDH appeared in 1958 (Wartenberg, 1958; Levy et a l . , 1959). Since then enzyme activities indicative of steroid hormone synthesis have also been demonstrated in the cytoplasm of ovarian interstitial gland cells of different mammalian species (Taylor, 1961; Rubin et al., 1963a,b; Rennie and Davenport, 1965; Deane and Rubin, 1965; Davies et a l . , 1966; Davenport and Mallete, 1966; Pupkin et al., 1966; Baillie et al., 1966; Seth and Prasad, 1967; Boucek et al., 1967; Wiest and Kidwell, 1969; Motta et a l . , 1970; Motta and Bourneva, 1970a,b; Motta and

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Takeva, 1970, 1971 ; Bjersing, 1977). The interstitial gland cells or theca interna cells of atretic follicles in the human ovary during the menstrual cycle, pregnancy, and the postmenopausal period have also been shown to contain enzyme activities indicative of steroid hormone synthesis (Ikonen et al., 1961; McKay et al., 1961; Deane et al., 1962; Scully and Cohen, 1964; Novak et al., 1965; Brandau and Luh, 1965; Konig, 1965a,b, 1966; Koudstaal et al., 1966; Pesonen et al., 1968; Fienberg, 1969; Brandau, 1971; Guraya, 1971; Koudstaal and Jobsis, 1974; Oei et a l . , 1974; Brandau et al., 1974; Bjersing, 1977). Among the enzymes demonstrated, 3b-HSDH and 17P-HSDH are of importance in the biosynthesis of ovarian hormones (Appelgren, 1967; BjErsing, 1967, 1977; Deane et u l . , 1962, 1966; Wiest and Kidwell, 1969; Guraya, 1971; Koudstaal and Jobsis, 1974; Oei et al., 1974; Brandau et a l . , 1974). The histochemical variations in interstitial gland cells of different mammalian species, including humans, under different physiological conditions are mainly due to the varying amounts of diffuse lipids (lipoproteins), lipid droplets, RNA, and enzyme systems present in their cytoplasm, which can be easily studied with the light microscope. The lipid droplets, diffuse lipids (lipoproteins), enzyme activities indicative of steroidogenesis, and multiple Golgi elements described for interstitial gland cells are not present in the compressed stromal elements or fibroblast-like cells (Figs. 6, 7, 9, 11, 12, 16, and 17) which, however, contain a small Golgi area, RNA, and a few granular mitochondria. The observations on the presence of lipid droplets and various other organelles in the theca-type interstitial gland cells of the mammalian ovary as revealed with light microscopy and histochemical techniques have been confirmed and further extended by use of the electron microscope (Muta, 1958; Motta, 1966, 1972; Davies and Broadus, 1968; Motta et a l . , 1968, 1971, 1975; Nesci and Motta, 1969; Merker and Diaz-Encinas, 1969; Motta and Takeva, 1970; Carithers and Green, 1972a,b; Brandau et al., 1974; Balboni, 1976; Carithers, 1976). The nucleoli are usually large in the nucleus of hypertrophied interstitial gland cells, in spite of the fact that ribosomes are not so abundant in the cytoplasm. Their structure is typical, consisting of a network of nucleolonemata composed of fibrous areas, believed to be the site of rRNA synthesis, and granular areas in which nascent ribosomal precursors aggregate before moving to the cytoplasm (De Kobertis et al., 1970). There is also a pars amorpha of unknown physiological significance. The nuclear contents are enclosed by the nuclear envelope which consists of two membranes; the outer nuclear membrane is sometimes connected to nearby elements of the endoplasmic reticulum, indicating a strong possibility that the latter may be derived from the former by the process of evagination. The nuclear envelope shows nuclear pores which may be a site for the transport of materials from the nucleus into the cytoplasm, and vice versa. Ovarian interstitial gland cells possess the ultrastructural cytoplasmic features

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(Fig. 23) typical of most steroid-secreting cells (Fawcett et a l . , 1969; Christensen and Gillim, 1969; Christensen, 1975; Neaves, 1975; Guraya, 1976a). These are: (1) abundant agranular endoplasmic reticulum consisting of interconnected tubules and usually distributed throughout the cytoplasm; (2) pleomorphic mitochondria with a more complex system of internal cristae which may be lamellar, tubular, or villiform; (3) under certain physiological situations stored lipid droplets of variable size and ultrastructure; (4)free ribosomes; (5) scattered stacks of lamellae of rough endoplasmic reticulum which interconnect with the

FIG.23. Electron micrograph of interstitial gland cells from a rabbit ovary, showing abundant agranular endoplasmic reticulum (AER), mitochondria (M), sparsely scattered ribosomes in the peripheral portions, lipid droplets (L) of variable size, and a nucleus (N). X 107,000. (Courtesy of Dr. H. E. Stegner.)

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elements of smooth reticulum; (6) well-developed multiple Golgi elements consisting of stacks of closely packed cistemae and associated vesicles; (7) other cytoplasmic components (microfilaments, microtubules, dense bodies or lysosomes, lipofuscin pigment granules, and so on); and (8) a complicated plasma membrane in the region of the perivascular space. These ultrastructural features may show some variations in development, depending upon the physiological state of the interstitial cell. Typical whorls of smooth endoplasmic reticulum and their intimate morphological relationships with mitochondria and lipid droplets have been described (see Motta et al., 1971). The ultrastructural similarity suggests a similarity of function, and the available biochemical and histochemical literature indicates that in most mammals (Guraya, 1973a,b,c, 1974a), and indeed in most vertebrates (Guraya, 1976a,d; Guraya and Chalana, 1976; Chalana and Guraya, 1977), theca-type interstitial gland cells may be the source of steroid hormones, as discussed in detail in Section IIIC, D, E. Macrophages are common in interstitial gland cells. The differences in the ultrastructure of different lipid droplets as reported by Davies and Broadus (1968) may be attributed to their varying histochemical nature, incomplete fixation, or both, as already discussed. In agreement with the histochemical observations of Guraya (1967a), Davies and Broadus (1968) also found that lipid droplets appear to arise de nuvu within the cytoplasm, independently of cell organelles. The abundant diffuse lipoproteins demonstrated with histochemical techniques in the interstitial gland cells of different mammalian species (Figs. 11, 12, and 20) apparently derive from the extensively developed agranular endoplasmic reticulum seen in ultrastructural studies (Fig. 23). The tubules or cistemae of the agranular endoplasmic reticulum often lie very close to lipid droplets or to mitochondria (Fig. 23). These morphological associations, which also occur in other steroid gland cells (Christensen and Gillim, 1969; Christensen, 1975; Neaves, 1975), appear to be of great significance in view of the substrate transfer that takes place between these structures, as discussed in detail by Christensen (1975). Motta et al. (1975) have described an unusual arrangement of endoplasmic reticulum in the interstitial cells of the cat ovary. Depending upon the species as well as upon the physiological state, the ultrastructure of ovarian interstitial gland cells vanes quantitatively but not qualitatively, as the organelles related to steroid hormone synthesis show some variations in the degree of development and structure (see Davies and Broadus, 1968; Motta et al., 1971). Interstitial gland cells in early stages of differentiation are notable for their relative simplicity of cytoplasmic structure. They contain many free ribosomes (or RNA as shown by histochemical studies) and occasional elements of granular endoplasmic reticulum, but few elements of agranular endoplasmic reticulum (Davies and Broadus, 1968; Motta et d.,1971; Motta, 1972; Balboni, 1976; Guraya, 1977~).This stage is presumably associated more with protein synthesis and growth, as already discussed for the fetal ovary (Guraya, 1977~).Golgi areas are discrete but inconspicuous. Plasma membranes

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20 1

are uncomplicated, are in simple apposition with each other, and have occasional areas of contact. Mitochondria are few and have simple lamellar cristae. The potent stem cells (or undifferentiated stromal cells) for interstitial gland cells in the maturing mouse ovary and adult human ovary also have a few organelles such as small mitochondria, a few rough membranes, highly dispersed ribosomes, and endoplasmic vesicles scattered throughout the cytoplasm (Stegner, 1970; Stegner and Onken, 1971; Balboni, 1976; Guraya, 1977~).Now it can be concluded that the transformation of fibroblast-like cells into hypertrophied interstitial gland cells is accompanied by the synthesis and development of abundant agranular endoplasmic reticulum (or diffuse lipoproteins) and lipid droplets, and by structural alterations and an increase in the number of mitochondria (see also Balboni, 1976). Corresponding to these subcellular changes, during the transformation of stromal elements into gland cells, enzyme activities indicative of steroid hormone synthesis also develop, as already described. The majority of mammals are seasonal breeders. It will be interesting and rewarding to follow the ultrastructural changes during the differentiation of interstitial gland cells before the breeding season, since little or no work using the electron microscope has been done on the seasonal cycle of ovarian interstitial gland cells. The results of such ultrastructural studies should be correlated with the level of steroid hormones secreted by these cells during differentiation and involution. C. BIOCHEMISTRY The interstitial gland cells of the rabbit ovary have been used for most biochemical studies because of their relatively greater abundance. Claesson et al. (1 948) recovered cholesterol, phospholipids, acetalphospholipids, and residual fatty acids from the rabbit ovary. According to these workers, the source of these different lipids is exclusively the interstitial gland cells, since the larger Graafian follicles were removed before carrying out the biochemical analysis. The isolation of lipid droplets from the rabbit ovary has also shown that they are extremely stable over a wide range of time and physical conditions (Claesson, 1954b). About 74% of their lipid is esterified cholesterol, the remaining being fatty acids (23%), free cholesterol (lo%), and phospholipid (20%). Following acute gonadotrophic stimulation, the droplets become reduced in size and showed a decrease in esterified cholesterol to about 25%, a rise in fatty acids, and a rise in phospholipid to 5%. Armstrong and Flint (1973) isolated cholesterol ester storage granules from the luteinized rat ovary and rabbit ovarian interstitial tissue by centrifugal flotation and studied their structure and function. Cholesterol ester, protein, phospholipid, and unesterified cholesterol accounted for the dry weight of granules from the luteinized rat ovary. The protein and the phospholipid, which were resistant to removal by washing, are not due to contamination with endoplasmic reticulum. LH administered in vivo increases the phospholipid and unesterified cholesterol content of isolated granules relative to their cholesterol

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ester content and also tends to raise their protein content. This treatment decreases the ability of isolated granules to act as a substrate for cholesterol esterase in v i m and increases the activity of cholesterol esterase. Cycloheximide in vivo also raises the unesterified cholesterol/cholesterol ester ratio of isolated granules, and when given with LH acts synergistically to bring about a further increase. These results have suggested that cholesterol ester storage granules are surrounded by a membrane, that they arise by pinching off from the endoplasmic reticulum, and that they shrink on gonadotrophic stimulation of the tissue. The results of biochemical investigations are in good agreement with those from histochemical studies, which have clearly shown the histochemical composition of lipid droplets by demonstrahng phospholipids, triglycerides, and cholesterol and/or its esters (Guraya and Greenwald, 1964a; Guraya, 1967a). The fatty acids described by Claesson er al. (1948) and Claesson (1954b) may be derived from the triglycerides present in abundance in the lipid droplets of interstitial gland cells of estrous rabbits. Davenport and Mallete (1966) made a biochemical study of HSDH activity in the ovary (minus the corpus lutem) of the rabbit, which consists mainly of hypertrophied interstitial gland cells (Guraya and Greenwald, 1964a; Guraya, 1967a). They demonstrated activity with 3p-, 17p-, and 20a-hydroxysteroids. The preference of one enzyme system for NAD or NADP was observed, and some enzyme activities were seen in the microsomal fraction and others in the supernatant. These results obtained with biochemical techniques are in agreement with those obtained using histochemical techniques, as the interstitial gland cells of the rabbit ovary have been shown to possess enzyme activities indicative of steroid hormone synthesis (Rubin et al., 1963b; Davies et al., 1966). Flint and Armstrong (1973) investigated the activities of enzymes responsible for steroid biosynthesis and cholesterol ester metabolism in rabbit ovarian interstitial tissue, which include cholesterol esterase, cholesterol ester synthetase, cholesterol side-chain cleavage enzyme, and 3p-HSDH. Their activities were measured under a variety of conditions both in vivo and in vitru. It has been concluded that these enzymes are capable of catalyzing the conversions usually attributed to them. Flint er al. (1973) have provided biochemical evidence for the control of cholesterol ester biosynthesis in slices of rabbit ovarian interstitial tissue. The interaction of various biochemical factors such as steroid hormones, gonadotrophins (LH), CAMP,enzymes, and so on, is important in the control of cholesterol ester biosynthesis. AND BIOCHEMICAL CHANGES AFTER D. CYTOLOGICAL, HISTOCHEMICAL, GONADOTROPHIC STIMULATION

Various gonadotrophins such as human chorionic gonadotrophin (HCG), pregnant mare serum gonadotrophin (PMSG), LH, and interstitial cell-stimulating

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hormone (ICSH), which contain luteinizing factor, are well known to influence the morphology and metabolism of ovarian interstitial gland cells. After gonadotrophic stimulation, the first cytological change that occurs is the mobilization of cholesterol-positive lipid droplets from interstitial gland cells of rat, hamster, and rabbit ovaries (Guraya and Greenwald, 1964a,b, 1965; Guraya, 1967a, 197213, 1975c, 1977b). The interstitial gland cells of the rabbit ovary are favorable material for following the details of cytological changes after gonadotrophic stimulation. There is little visible release of cholesterol-containing lipid droplets from interstitial gland cells of estrous rabbit ovaries (Figs. 13, 19, and 22). It is accelerated greatly in preovulatory ovaries stimulated with gonadotrophins (Fig. 24) (Guraya and Greenwald, I964a; Guraya, 1967a). Similarly, ovarian interstitial gland cells of rat and hamster ovaries become depleted of lipid droplets after treatment with exogenous gonadotrophins (Figs. 25 and 26) which contain LH activity (Guraya and Greenwald, 1964b, 1965; Guraya, 1972b, 1975~).The interstitial gland cells of preovulatory ovaries of the rat, hamster, and guinea pig

FIG. 24. Histochemical preparation of interstitial gland cells from a rabbit ovary 4 hours and 25 minutes after the injection of HCG. The release of lipid droplets (1) from vacuoles (v) is most extensive in these ovaries. Many complex bodies consisting of lipid ( I ) , vacuoles (v), and cytoplasm, possibly with some of its organelles, have already been pinched off. The vesicular nuclei (n) of the gland cells are also seen. X990. (From Guraya, 1967a.)

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FIG.25. Histochemical preparation of part of hamster ovary removed on day 2 of estrous cycle, showing patches of interstitial gland tissue (IGT) filled with highly sudanophilic lipids not seen in the stroma (ST). X 4 4 0 . (From Guraya, 1972b.)

also become depleted of cholesterol-positive lipid droplets (Guraya and Greenwald, 1964b, 1965; Guraya, 1968c, 1972b, 1975c; Jacoby, 1962). After ovulation, the interstitial gland cells are again filled with lipid droplets of the same chemical nature as those observed before the preovulatory period. Their mobilization is apparently brought about by the high levels of endogenous LH which on secretion causes ovulation. The cholesterol-positive lipid droplets (or sterol granules) of the interstitial gland in the ovary of the guinea pig vary to a lesser extent than in the ovaries of the rat, hamster, and rabbit, because there is no extensive accumulation during the phases of the estrous cycle (Guraya, 1968c; Claesson and Hillarp, 1947b). Similarly, Claesson and Hillarp (1947b) and Deane (1952) have reported a reduction in the Schultz reaction in interstitial gland cells of preovulatory rat ovaries, showing mobilization of their cholesterol. Cholesterol-positive lipid droplets are maintained at a high level throughout pregnancy in the rat (Claesson and Hillarp, 1947b; Guraya, 1975c; Guraya and Gupta, 1978). They are present in lower amounts during lactation (S. S. Guraya and S. Chaudhry, unpublished observations). In the second half of pregnancy in the guinea pig, a marked accumulation of sudanophilic lipid droplets is seen (Figs. 6, 21, and 27), which consist of cholesterol and/or its esters, triglycerides,

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FIG. 26. Histochemical preparation of hamster ovary removed on day 2 of the estrous cycle, treated with 60 IU PMSG on day I , showing the depletion of sudanophilic, cholesterol-positive lipid droplets in the interstitial gland cells (IGT) seen of the untreated ovary (Fig. 25); the cytoplasm of individual cells shows diffuse sudanophilic lipids not seen in the nuclei (N). Lipid accumulations (LA) formed by the degeneration of granulosa cells of atretic follicles are not affected by gonadotrophin (PMSG). X445. (From Guraya, 1972b.)

and phospholipids (Claesson and Hillarp, 1947b; Guraya, 1977d); the number of cholesterol-positive lipid droplets decreases during lactation (Fig. 28). Estrogen and prolactin have been found to be ineffective in causing appreciable changes in cholesterol-positive lipid droplets of rat ovarian interstitial gland cells (Guraya, 197%). Guraya (1976e) studied the effects of LH and prolactin on cholesterol-positive lipid droplets in interstitial gland tissue of hypophysectomized or hypophysectomized PMSG-HCG-treated rats. Hypophysectomy in general causes a great decrease in cholesterol-positive lipid droplets which in intact rats show massive accumulation (Guraya and Greenwald, 1964b; Guraya, 1972b, 197%). LH alone or LH plus prolactin does not cause any appreciable change in cholesterol-positive lipid droplets in interstitial gland tissue of hypophysectomized PMSG-HCG-treated rats. The low levels of cholesterol-positive lipid droplets seen in interstitial gland tissue of hypophysectomized rats are completely depleted in hypophysectomized PMSG-HCG-treated rats. Choles-

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FIG.27. Histochemical preparation of part of a guinea pig ovary obtained on day 62 of pregnancy, showing the extensive development of theca-type interstitial gland tissue (IGT) which is highly sudanophilic. The stroma (S) does not contain many sudanophilic lipids. x96.

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terol-positive lipid droplets are also completely depleted from the interstitial gland tissue of the hamster ovary immediately after the administration of PMSG (Figs. 25 and 26) (Guraya and Greenwald, 1965; Guraya, 1972b, 1977b). With the disappearance of PMSG from the blood circulation, the interstitial gland cells start to store cholesterol-positive lipid droplets (Guraya, 1977b). The release of lipids, both in treated ovaries and ovaries of estrous rabbits, is accompanied by the development of vacuoles in the outer cytoplasm of gland cells, which gradually increase in size (Fig. 24); meanwhile, lipid droplets appear at the periphery. The lipid droplets, which coalesce with each other before their release, are also seen in the pockets of plasma membrane protruding between gland cells (Fig. 29). In this location the vacuole occupies the central portion of each cytoplasmic projection which finally is pinched off. These light microscope observations of Guraya (1967a) are further supported by the ultrastructural data of Motta and co-workers (1971; Motta, 1972) who demonstrated numerous cytoplasmic evaginations and microvilli in the interstitial cells. Many vacuoles associated with lipid droplets have already been pinched off and are seen between the gland cells. Motta et al. ( 1 97 1) also observed cell fragments containing organelles and lipid droplets distributed in the intercellular spaces. The release of lipids and possibly organelles by this mechanism is most pro-

FIG. 28. Histochemical preparation of part of a guinea pig ovary obtained on day 2 of lactation, showing a decrease in the number of sudanophilic lipid droplets in the interstitial gland tissue (IGT) in comparison to that in the second half of pregnancy (Fig. 21). x205.

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FIG.29. Interstitial gland cells from a rabbit ovary 4 hours and 25 minutes after the injection of HCG. The release of lipid droplets (I) from vacuoles (v) is very pronounced. Many cell fragments consisting of lipids (I), vacuoles (v), cytoplasm, and possibly organelles have already been pinched off (see also Fig. 24). Vesicular nuclei (n). (From Guraya, 1967a.)

nounced in preovulatory ovaries studied 1-5 hours after gonadotrophic stimulation (Guraya and Greenwald, 1964a; Guraya, 1967a); their vascularization also becomes far richer than that of estrous rabbit ovaries. Vascularization also increased in interstitial gland tissue of hamster and rat ovaries after treatment with gonadotrophins having LH activity. The release of lipids is accompanied by a loss of cytoplasm and organelles, as also reported by Motta et al. (1971); consequently the gland cells of the rabbit ovary are considerably reduced in size (Fig. 30). Complete depletion of lipid granules was not achieved because new lipid granules consisting mainly of phospholipid formed at the time of mobilization. Both HCG and PMSG treatments cause considerable luteinization and hypertrophy of the ovarian interstitial gland cells in the hamster and rat, which are accompanied by the development of more diffusely distributed sudanophilic lipids (lipoproteins; see Fig. 26); the diffuse lipoproteins are presumed to be derived from the abundant membranes of agranular reticulum. In rabbit ovaries, the released secretory lipid products are found in the form of discrete bodies between interstitial gland cells in regions adjacent to blood vessels, and in the lumen of the latter (Fig. 31). Mottaet al. (1971), using the electron microscope, also demonstrated the presence of cell fragments containing organelles or dispersed free mitochondria and lipid droplets in the intercellular and perivascular spaces and occasionally within the capillary lumen. According to these workers, these released cell fragments appear to be products of cytolysis. But Guraya (1967a) observed their presence after gonadotrophic stimulation. The mechanism by which the liberated lipids are transported into the circulation could not be determined (Guraya, 1967a). However, electron microscope observations have shown that the capillary wall is composed of flat cells connected by thin, digitiform microvilli (Motta et al., 1971). Numerous inward- or outward-directed pinocytotic vesicles and other slightly irregular evaginations are evident on the

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FIG. 30. Histochemical preparation of ‘part of a rabbit ovary 10 hours and 45 minutes after the injection of HCG. The interstitial gland cells have lost most of their cytoplasm and inclusions and consequently are reduced in size (compare with the cells shown in Fig. 19). Their vesicular nucleus (n) is surrounded by a thin layer of cytoplasm with a few inclusions. Some cells, having a Golgi zone (Gz),show relatively more lipid droplets. Some lipids lie in the intercellular spaces. X990. (From Guraya, 1967a.)

plasma membrane of the endothelial cells. A thin basal lamina is always present, but only a few collagen fibrils. The mobilization or depletion of “sterol granulae,” which are identical to the cholesterol-positive lipid droplets described by Guraya and co-workers (Guraya and Greenwald, 1964a; Guraya, 1967a), were also studied by Claesson and Hillarp (1947a) in the rabbit. Using biochemical techniques, Claesson and coworkers (Claesson and Hillarp, 1947a,b; Claesson et al., 1948, 1953; Claesson, 1954a,b,c), reported that the applied gonadotrophic stimulation probably mobilizes all the cholesterol stored as precursor. Claesson (1954a) quantitated the degree of cholesterol mobilization from interstitial gland tissue with the doses of gonadotrophin administered, noting that more than 80% of the total esterified fraction had disappeared by 12 hours after a dose of PMSG and that sterol levels

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remained maximally depressed for at least 24 hours. But the histochemical observations of Guraya and co-workers have shown that not only the cholesterol andlor its esters are mobilized, but also the triglycerides and phospholipids of lipid droplets (Guraya, 1967a, 1975c; Guraya and Greenwald, 1964a,b, 1965). The experiments of Solod et al. (1966) demonstrated the depletion of cholesterol in vivo and in vitro in rabbit ovarian interstitial gland tissue following LH treatment. Armstrong et al. (1969a) have described three distinct actions of LH on cholesterol metabolism in rabbit ovarian interstitial gland cells: (1) increased conversion of stored cholesterol to progestins (progesterone and 20ahydroxypregn-4-en-3-one),(2) decreased esterification of cholesterol with longchain fatty acids, and (3) decreased synthesis of monophosphate (CAMP). Dorrington and Kilpatrick (1 969) also made observations consistent with the hypothesis that CAMP is a mediator of LH action; both substances stimulated interstitial gland tissue to produce increased amounts of progestational hormones. According to Armstrong (1968), LH may stimulate steroidogenesis by facilitating the transport of cholesterol from cholesterol-containing lipid droplets to the mitochondria, the site of its utilization. But the histochemical studies of Guraya and Greenwald (1964a) and Guraya (1967a) revealed the release of cholesterol-containing Iipid droplets as such from the gland cells of the rabbit ovary after gonadotrophic stimulation (Figs. 22, 24, 29, and 31). Guraya ( 1 974a) has attributed the extensive development of hypertrophied interstitial gland cells to the secretion of endogenous LH or ICSH in the ovaries of maturing marmosets, which possess features typical of actively secreting steroid gland cells (abundant diffuse lipoproteins and a few lipid granules consisting of phospholipids; see Fig. 20); cholesterol andor its esters are absent. Guraya (1968b) has reported the histophysiological changes in interstitial gland tissue in the ovaries of mature nonpregnant marmosets, which occupies the second and third zones (or corticomedullary regions). The interstitial gland cells of the second zone (or cortical region) are relatively undifferentiated, as they are newly formed cells from the theca interna of atretic follicles. Their cytoplasm is filled with coarse lipid droplets consisting of cholesterol and/or its esters, triglycerides, and phospholipids. The third zone (the medullary part of the ovary) has large masses of greatly hypertrophied interstitial gland cells (Figs. 4 and 5) which are gradually formed from the small, quiescent cells of the second zone, apparently as a result of their shifting toward the medullary regions. Meanwhile, the interstitial gland cells are greatly stimulated, as evidenced by their greater hypertrophy, by the mobilization of their cholesterol-positive lipid droplets, and by the development of abundant diffuse lipoproteins (Fig. 20). These studies have indicated that, besides gonadotrophins, some intraovarian factors also play an important role in the histophysiology of interstitial gland cells. Cholesterol-positive lipid droplets are depleted in the extensively developed interstitial gland cells of pregnant bats, apparently as a result of high levels of endogenous gonadotrophins

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FIG. 31. Histochemical preparation of part of a rabbit ovary obtained 8 hours after the injection of HCG, showing aggregations of lipid droplets released from the interstitial gland cells, along the walls of blood vessels (bv), and several very fine lipid particles in the lumen of the latter (arrows). The gland cells show a spherical Golgi zone (Gz), lipid granules and spheres ( I ) , vacuoles (v), and a (From Guraya, 1967a.) nucleus (n). ~440.

having the properties of LH, formed by the pituitary, the placenta, or both, during pregnancy (Guraya, 1967b); but the gland cells of nonpregnant bats show a heavy accumulation of cholesterol-positive lipid droplets which obscure the other cell organelles in the cytoplasm (Fig. 15). During pregnancy the highly developed interstitial gland cells of the human ovary do not show an accumulation of lipid droplets (Figs. 11 and 12) (Guraya, 1966b, 1971, 1972a). The absence of cholesterol-positive lipid droplets and the presence of abundant diffuse lipoproteins in interstitial gland cells of pregnant women has been attributed to the prolonged effect of HCG secreted by the placenta. The normal theca-type interstitial gland cells in the ovaries of postmenopausal women also do not contain cholesterol-positive lipid droplets (Fig. 32), apparently as the result of some strong gonadotrophic stimulation (Guraya, 1976~).However, the diffusely distributed sudanophilic lipids (lipoproteins) presumably derived from the abundant membranes of smooth reticulum (Brandau et af., 1974) are developed in abundance in the cytoplasm of theca-type interstitial gland cells of both preg-

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FIG.32. Histochemical preparation of part of an ovary from a postmenopausal woman, showing greatly hypertrophied theca-type interstitial gland cells (IGT) in the wall of an atretic follicle, which show abundant sudanophilic lipids. Remnants of degenerting granulosa cells (DG)are also seen. X300.

nancy and the postmenopausal period (Figs. 1 1, 12, and 32). The ovaries of the nonpregnant civet cat, which contain large Graafian follicles, show a greater development of hypertrophied theca-type interstitial gland cells (Figs, 16 and 33). Their cytoplasm does not have an accumulation of cholesterol-positive lipid droplets, apparently because of high levels of ICSH, but diffusely distributed sudanophilic lipids are present (Fig. 33). The interstitial gland tissue is highly vascularized, as evidenced by the presence of many blood capillaries. Interstitial gland cells of hypophysectomized rabbits treated with LH are finestructurally indistinguishable from luteal cells with respect to the rich development of the smooth endoplasmic reticulum and the complex folding of the plasma membrane (see Davies and Broadus, 1968). This suggests that LH is responsible for the development and maintenance of the structure and function of the interstitial gland cells in the rabbit ovary. These observations are further supported by the fact that the ultrastructural alterations occurring in rat ovarian interstitial gland cells after hypophysectomy include a reduction in nuclear and cytoplasmic volume, a reduction in the extent of agranular endoplasmic reticulum and the Golgi complex, reorganization of mitochondria1 cristae from tubular to lamellar

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FIG.33. High-power view of interstitial gland cells from the civet cat ovary shown in Fig. 16, showing diffusely distributed sudanophilic lipids in the cytoplasm. X400.

forms, and a loss of lipid droplets (Carithers and Green, 1972a,b; Flerko et a l . , 1967; Guraya, 1976e). Treatment with PMSG effects a reversal of these ultrastructural changes (Carithers and Green, 1972b; Flerko et al., 1967). Within 6 hours after treatment with PMSG, marked dilation of the intercellular spaces, an accumulation of lipid, and nucleolar enlargement occur. The nuclear response is complete within 36 hours after injection, but reorganizational changes in all cytoplasmic organelles become more prominent at later times as the endoplasmic reticulum and mitochondria continue to proliferate for at least 72 hours (Carithers and Green, 1972b). A linear response of mitochondria after PMSG treatment has also been demonstrated morphometrically (Carithers and Green, 1968). In a subsequent study, Carithers (1976) determined the sequence in which ultrastructural responses of rat ovarian interstitial gland cells to purified LH occur. At the ultrastructural level, these responses include nuclear and nucleolar hypertrophy, an increase in cytoplasmic volume and number of ribosomes, and a proliferation of the membranous systems including the Golgi complex of the cytoplasm. The restoration of abundant agranular endoplasmic reticulum and tubular mitochondrial cristae typical of steroidogenic cells, which were lost after hypophysectomy, is initiated within 6 hours by LH treatment. Only mitochondria1 changes are continuous throughout the 72-hour observation period. Morphological responses

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to LH are prominent in these cytoplasmic components before any alteration is noticeable in the nuclei, but nuclear hypertrophy after LH treatment, although delayed, is very rapid and is completed before cytoplasmic membranogenesis ceases. These experiments have produced strong evidence that the structural integrity of ovarian interstitial gland cells is dependent upon the gonadotrophin having LH activity. Proliferation of agranular endoplasmic reticulum or diffuse lipoproteins has also been shown to be due to a significant subcellular response of interstitial gland cells and other steroid gland cells of the gonads to LH (see Davies and Broadus, 1968; Beltermann and Stegner, 1968; Christensen and Gillim, 1969; Guraya, 1971, 1976a,b; Neaves, 1975; Christensen, 1975). In a further experiment, Carithers (1975) observed that actinomycin D, an inhibitor of RNA synthesis, blocks the ultrastructural response to LH in the nucleus of rat ovarian interstitial gland cells without affecting membranogenesis in the smooth endoplasmic reticulum or Golgi complex for at least 24 hours. Gorski and Padnos (1966) have also suggested that the primary effects of LH occur at a cytoplasmic rather than at a nuclear level. These comments are based on studies showing that actinomycin D, in concentrations which decreased RNA synthesis to one-tenth of control values, had no inhibitory effect on LH-induced steroidogenesis in the rabbit ovary. Polysome formation, which occurs as early as 6 hours after LH treatment (Carithers, 1976), can be interpreted as evidence that renewed nuclear RNA synthesis or dispersion of polysomes during the regression of interstitial gland cells after hypophysectomy was due to a loss of mRNA. However, actinomycin D, while inhibiting chromatin dispersion and ribosome formation in interstitial gland cells in response to LH, does not affect polysome formation in existing ribosomes (Carithers, 1975), indicating that adequate mRNA remains for polysome formation without renewed synthesis. Mitochondria of ovarian interstitial gland cells seem to be unique in that, after treatment of hypophysectomized rats with LH and either actinomycin D or puromycin, they are prevented from reorganizing by the inhibition of either RNA or protein synthesis (Carithers, 1975). The recent findings of Carithers ( 1 976) have indicated that mitochondria1 reorganization is well under way before nuclei show visible changes, suggesting that new RNA may be synthesized in the mitochondria themselves. Endroczi et al. (1964) initiated enzyme activity indicative of steroid hormone synthesis in interstitial gland cells of 3-month-old rabbits after treatment with follicle-stimulating hormone (FSH) and LH; FSH may also contain Iuteinizing factor. However, Hilliard et al. (1969) have stated that LH stimulates cholesterol mobilization and steroidogenesis by the interstitial gland tissue of the rabbit ovary and that prolactin maintains the steroid-producing capacity and morphology of this tissue. According to Boucek et al. ( 1 967), the gonadotrophin intensified 3P-HSDH reactions in the interstitiurn of the rabbit ovary, while reducing the 17P-HSDH reaction. This may reflect increased synthetic activity of the interstitial gland cells for the class of steroids and decreased synthetic

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activity for the C19 class of steroids. A histochemical reaction for 20P-HSDH (but not 2h-HSDH) was intensified after treatment with gonadotrophin, suggesting that limited amounts of the 20P-hydroxy epimer are formed, and that the balance of the reactions favor increased formation of progesterone and 2 h hydroxy-pregn-4-en-3-one.

E. CORRELATION OF CYTOLOGICAL, HISTOCHEMICAL, AND BIOCHEMICAL CHANGES WITH STEROID SECRETIONS Ovarian interstitial gland cells of mammals possess the morphological (including ultrastructural), histochemical, and biochemical features of steroid-secreting cells and are therefore steroid secretors. The question then arises: What are the steroid hormones of ovarian interstitial gland tissue? Indirect observations have indicated that androgens, progestogens, and estrogens are the steroid secretions of interstitial gland tissue (see Harrison, 1962; Eckstein, 1962; Jacoby, 1962; Nalbandov, 1964), however, there is only a little direct evidence. The depletion of cholesterol-containing lipid droplets (steroid hormone precursors) during the preovulatory period in rodents including the hamster, rat, and rabbit indicates the formation of steroids. Claesson and co-workers (1948, 1953; Claesson, 1954a,b,c; Claesson and Hillarp, 1947a,b) have repeatedly stated that sterol granules (or cholesterol-containing lipid droplets) act as precursors in the formation of estrogenic hormones. However, Claesson presented no evidence for the formation of estrogenic hormones from the “sterol granulae” in any of his reports. Falck (1959) obtained evidence that estrogen is a product of the interstitial gland tissue of the rat ovary; however, Falck et al. (1962) reported an androgenic effect. The mobilization of cholesterol-containing lipid droplets from the interstitial gland cells of the rabbit ovary after gonadotrophic stimulation (Figs. 22, 24, 29, and 31), as described by Guraya and co-workers (Guraya and Greenwald, 1964a; Guraya, 1967a), is clearly correlated with the production of 2k-hydroxypregn-4-en-3-one and progesterone observed by several workers in the ovarian vein or peripheral blood of the rabbit after both exogenous and endogenous gonadotrophic stimulation (Forbes, 1953; Simmer et a l . , 1963; Hilliard et a l . , 1961, 1963, 1969; Hilliard and Sawyer, 1964; Armstrong et al., 1969a; Dorrington and Kilpatrick, 1969). These findings are consistent with those of Forbes (1953) who observed elevated levels of progestational steroid in the peripheral blood of female rabbits after mating but before ovulation. Similarly Guraya (19760 attributed the preovulatory release of progesterone to the interstitial gland cells of the rat and rabbit ovary, as their cholesterol-positive lipid droplets are simultaneously depleted (Guraya and Greenwald, 1964a,b). Domngton and Kilpatrick (1969) believe that a high level of steroid output after the injection of LH and FSH in the rabbit is due to an increase in de nova synthesis of progestational

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steroids and not simply to a release of stored steroids. They have also reported that CAMP may be an intracellular messenger involved in mediating the LHinduced increase in progestin synthesis by interstitial gland tissue slices of rabbit ovary. The experiments of Solod et al. (1966), in which the ovarian cholesterol pool was labeled by prior intravenous injection of cholester01-7a-~H,clearly demonstrated that the cholesterol which disappeared from the ovary following LH treatment was the principal source of the progestins secreted at increased rates. In these experiments, increased in vitro synthesis of progestins from cholesterol was also observed in interstitial gland tissue slices as a result of LH administration, whether gonadotrophins were added directly to the incubation medium or injected intravenously prior to removal of the ovaries for incubation. Some indirect evidence for progesterone production by interstitial gland cells of rat ovaries has also been presented by Taylor (1961) and Levy et al. (1959). These studies are further supported by the observations of Boucek et al. (1967) who have stated that the balance of the reactions in the cells of the interstitium of the rabbit ovary favors increased formation of progesterone and 20cu-hydroxypregn-4-en-3-one. They have also suggested the possibility of synthesis of androgens in interstitial gland cells. The experiments of Hammond and Marshall (1925) have indicated that the interstitial gland tissue of the rabbit ovary by itself is unable to release sufficient progesterone to maintain pregnancy. Human ovarian stroma has also been used to study the biosynthesis of steroid hormones in in vitro biochemical experiments (Savard et a l . , 1965; Rice and Savard, 1966; Leymarie and Savard, 1968; Guraya, 1971; Marsh et al., 1976). Stroma has been shown to have the capacity to form a number of steroid hormones from radioactive acetate. The steroids of the ovarian stroma differ in character from those formed by the follicle and corpus luteum of human ovary under similar conditions (see also Baird, 1974, 1976); androgenic steroids are the principal products of human ovarian stroma, suggesting that the latter forms a distinct gland of internal secretion. HCG increased the incorporation of I4Cacetate into steroid hormones, indicating that this compartment of the ovary is controlled by the gonadotrophic hormone, as already discussed. Channing (1969a) obtained human stromal tissue from normal ovaries, incubated it in tissue culture for 3 days, and found that 17-hydroxyprogesterone, progesterone, and androstenedione were the major secretory products. Guraya (1971) has attributed the biosynthesis of steroid hormones (androgens) by the human ovarian stroma in in vitro biochemial experiments to the presence of interstitial gland cells of thecal and stromal origin (Figs. 8, 11, and 12). These have been shown to possess the cytological and histochemical features specific to steroidogenic tissue. Ovarian stroma also forms the important site for the synthesis and secretion of androgens, under pathological conditions (Forti et al., 1976; Giusti ef al., 1976). According to Brandau et al. (l974), the postmenopausal ovaries, which contain theca- or stroma-type interstitial gland cells possessing the molpho-

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logical and histochemical characteristics of steroid-secreting cells (Fig. 32), also produce mainly androgens as precursors of estrogens. Plotz et al. (1967) also believe that postmenopausal ovaries produce mainly estrogen precursors that are then aromatized in the periphery. The secretion of significant amounts of hormones by the postmenopausal ovary having the stroma- or theca-type interstitial gland cells (Brandau et al., 1974; Guraya, 1976c) is further supported by the fact that in ovariectomized women there is a significant decrease in testosterone androstenedione and dehydroepiandrosterone (Judd et a l . , 1974a,b; VermeuIen, 1974). At present, it is still uncertain exactly what the interstitial gland cells (or the stromal compartment) of the human ovary contribute to the steroidal output of the ovary in vivo under different physiological conditions; however they are certainly stimulated by LH, HCG, or PMSG. AND STORAGE OF LIPIDSAND OTHERORGANELLES F. FORMATION

The replenishment of cytoplasm, lipid droplets, mitochondria, and Golgi rnaterial starts immediately in interstitial gland cells of rabbit ovary depleted of lipid droplets and organelles after gonadotrophic stimulation (Guraya, 1967a). The replenishment of cholesterol-positive lipid droplets has also been demonstrated in interstitial gland tissue of hamster and rat ovaries after gonadotrophic stimulation has decreased (Guraya and Greenwald, 1964b, 1965; Guraya, 1972b, 1977b). RNA, diffuse lipoproteins, and mitochondria consisting of lipoproteins are formed throughout the cytoplasm; a Golgi zone is usually located adjacent to the nucleus, though such organized areas are sometimes observed elsewhere in the cytoplasm. With replenishment of the cytoplasm and organelles, the cytoplasm increases in amount and correspondingly lipid inclusions also begin to increase throughout the cytoplasm (Fig. 3 I ) , without intimate association with mitochondria and the Golgi zone. They gradually grow into large droplets which also develop triglycerides, in addition to phospholipids. Lipid bodies in the same cell show considerable variation in size and cytochemistry; they range from large droplets to small granules (Fig. 3 1). The increase in the amount of phospholipid found biochemically by Claesson (1954a,b) can be attributed to the appearance and development of diffuse lipoproteins, several mitochondria, and small phospholipid granules in the interstitial gland cells of the rabbit ovary after gonadotrophic stimulation, as these organelles are well known to be rich in phospholipid. The problems of replenishment of cytoplasm and the origin of organelles and lipid inclusions can be better studied in rabbit ovaries removed after 6-8 hours of gonadotrophic stimulation, as well as in postovulatory ovaries (Guraya, 1967a). It is interesting to mention here that the interstitial gland cells of these ovaries also release newly formed lipid droplets. Thus both the storage and release of lipids begin to occur more or less simultaneously. But their release is not so intensive and extensive as that observed in the estrous rabbit ovary after 1-6

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hours of gonadotrophic stimulation (Guraya and Greenwald, 1964a; Guraya, 1967a). These newly formed lipid inclusions do not store cholesterol and/or its esters. Three days after ovulation in the rabbit, most of the lipid droplets are fully grown and have developed abundant cholesterol and/or its esters. In morphology and histochemistry , the interstitial gland cells begin to resemble those of estrous ovaries. With the disappearance of PMSG from the blood circulation, the interstitial gland cells of the hamster ovary also start to replenish their cholesterolpositive lipid droplets (Guraya, 1977b). Further studies using the electron microscope, combined with autoradiography and histochemistry , are needed to reveal the details of formation, replenishment, and the interrelationships of cytoplasm and its organelles and inclusions (cholesterol-positive lipid droplets). The results of such studies will enhance our knowledge of the biology of the steroid gland cell.

G. DYNAMIC AND DEGENERATIVE ASPECTS Theca-type interstitial gland cells show several variations in regard to pennanency, which seem to be related to the nature and amount of gonadotrophins, especially LH or ICSH. These gonadotrophins are well known to cause differentiation of interstitial gland cells, as well as to maintain their functional morphology and endocrinological function, as already discussed. However, some influence may also be exerted by nervous stimulation, as will be discussed. In the ovaries of some nonpregnant mammals (human, rhesus monkey, cow, buffalo, Indian mongoose, and so on), the theca-type interstitial gland cells are of very transient nature and, after existing in the wall of degenerating follicles (Figs. 8-10), they begin to lose their cytoplasm, organelles, and lipids and ultimately revert to the relatively embryonic fibroblast-like cells (Figs. 34 and 35) from which they originally differentiated during follicular atresia (Guraya, 1966a, 1967c, 1968a,c, 1973a, 1978; Mossman and Duke, 1973a); this cytoplasmic alternation is also accompanied by the loss of vascularization, indicating an important role in the maintenance of functional morphology of interstitial gland cells. These alterations also indicate that the stimulus causing the differentiation and accumulation of theca-type interstitial gland cells gradually disappeared along with the blood vascularity. The latter plays an important role in the regulation of steroid gland cell function (Niswender et al., 1976). Besides the gonadotrophins transported through blood vascularity, some influence may also be exerted by nervous stimulation, and Unsiker (1970a,b) suggested that adrenergic axons might affect the transformation of fibrocytes into interstitial gland cells in the mouse ovary. Neilson et al. (1970) have also proposed that neural influences regulate the differentiation of stromal cells into secretory types. The innervation of interstitial gland tissue in rat and guinea pig ovaries is exclusively adrenergic (Svensson et al., 1975; Lawrence and Burden,

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FIG. 34. Histochemical preparation of part of an ovary from a nonpregnant cow, showing interstitial gland tissue (IGT) in the wall of atretic follicles at various stages of differentiation and reversion to stromal elements. Remnants of degenerating granulosa cells (DG) are seen in early atretic follicles. (From Guraya, 1968a).

1976). Lawrence and Burden (1976) have suggested that increased ovarian norepinephrine during pregnancy in the rat may be related to functional or morphological changes in interstitial gland tissue during later stages of pregnancy. Fluorescence microscopy of freeze-dried, paraformaldehyde-treated rat ovaries has revealed adrenergic nerve fibers which run along with vessels and form a network among interstitial gland cells (Unsicker, 1974). Dahl (1970) demonstrated that in the fowl ovary axon terminals are in membranous contact with the thecal gland cells. Unsicker (1973) observed that testicular interstitial tissue in reptiles contains adrenergic nerve fibers, the amount and distribution of which varies considerably both in various species and in various stages of the reproductive cycle. The innervation of testicular and ovarian interstitial gland

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FIG.35. Histochemical preparation of part of an ovary from a nonpregnant buffalo, showing the reversion of theca-type interstitial gland cells (shown in Figs. 9 and 10) to stromal elements (S) by losing cytoplasm and its lipid inclusions and organelles. X50.

cells suggests that their final secretory state may depend on the balance of neural, gonadotrophic, and local metabolic stimuli (see also Balboni, 1976). Ultrastructural gap junctions observed between the ovarian interstitial gland cells may coordinate differentiation, dedifferentiation, and redifferentiation of this important ovarian gland (Lawrence and Burden, 1976). Interstitial gland cells do not show much permanency or accumulation in ovaries (Figs. 8, 18, 34, and 35) where they are rapid reverting to the original stromal elements. There is some sort of balance between the formation of new interstitial gland cells and their reversion to the original stromal tissue, thus leading to little accumulation in the ovary. This short duration of existence of theca-type interstitial gland cells led most earlier workers to deny their presence in the ovaries of the nonpregnant human, cow, buffalo, and rhesus monkey. In the ovaries of other mammals showing considerable development of theca-type interstitial gland cells (Figs. 4-7, 13-16, and 25-27), they begin to accumulate rather than reverting back so early to the original fibroblast-like cells. Thus they form the major portion of the ovarian stroma, as also demonstrated in many species of mammals by Mossman and Duke (1973b). During pregnancy the theca-type interstitial gland cells of the human ovary also accumulate, rather than reverting back to the original fibroblast-like cells (Figs. 11 and 12), as seen in the ovaries of nonpregnant women (Fig. 8). Mature interstitial gland cells are usually not observed in cell division, and yet their number increases after ICSH stimulation, indicating differentiation from another cell type. Many divergent views have been expressed about the identity

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of this stem cell (Motta, 1974). Most of the cell types of the ovary have been implicated at one time or another (see Mossman and Duke, 1973a,b). But it is well-established that both the primary and secondary interstitial gland cells of the mammalian ovary are derived from fibroblast-like cells, as already discussed (see also Balboni, 1976). However, in the rabbit ovary, there are indications that interstitial gland cells also arise from the surface epithelium of the ovary (Motta, 1974). It is possible that ovarian interstitial gland cells have different modes of origin, depending on the species, as discussed in detail by Mossman and Duke (1973a,b). Some interstitial gland cells are observed to undergo degenerative changes (Guraya, 1967a, 1968c, 1978; Davies and Broadus, 1968; Motta et af. 1971). Their degeneration usually involves single, isolated cells (Figs. 36 and 37) but is occasionally lobular in extent. In the ovaries of the guinea pig and civet cat degenerating cells are gradually shifted to the medullary part of the ovary (Figs. 38 and 39). Their cytoplasm is filled with coarse, dense lipid droplets which vary

FIG. 36. Histochemical preparation of part of a rabbit ovary 8 hours after the injection of HCG, showing liberated lipids in the intercellular spaces. Lipid droplets have been mobilized, except from the degenerating gland cells (dgc); the cytoplasm shows diffuse sudanophilic lipids. The nuclei (n) are sudanophobic. X275. (From Guraya, 1967a.)

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FIG.37. Histochemical preparation of part of a guinea pig ovary obtained on day 3 of lactation, showing normal interstitial gland cells (IGT) and degenerating gland cells (DGC) filled with coarselipid droplets in the deeper medullary regions. The stroma ( S ) does not contain many lipids. x 125.

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FIG.38. Histochemical preparation of part of a guinea pig ovary obtained on day 62 of pregnancy, showing normal interstitial gland tissue (IGT)masses and regressing gland cells (DGC)filled with coarse lipid droplets in the deeper medullary regions. X38.

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FIG. 39. Histochemical preparation of part of a civet cat ovary, showing highly sudanophilic degenerating gland cells (DGC) in the deeper medullary regions. The stroma (S) does not contain lipids. X400.

greatly in size and nature depending upon the stage of degeneration (Figs. 22 and 36-39). The problems of degeneration of theca-type interstitial gland cells, as well as their reversion to stromal elements, can be better studied in successive stages of degenerated follicles in postmenopausal ovaries (Figs. 32 and 40-42). The cytoplasm of regressing interstitial cells is gradually filled with sudanophilic lipid droplets of a coarse nature. Owing to degenerative changes, the lipid droplets develop pigments, in addition to cholesterol andor its esters and triglycerides (Guraya, 1967a, 1968c, 1976c, 1978). Electron microscope studies have revealed that their regression is accompanied by drastic ultrastructural alterations in the cytoplasmic components such as the mitochondria, endoplasmic reticulum, and lipid droplets (Davies and Broadus, 1968; Motta et al., 1971). The cyto-

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FIGS.40-42. Histochemical preparation of parts of an ovary from a postmenopausal woman, illustrating successive stages of lipid changes in the degenerating theca-type interstitial gland cells (DGC). Some cells degenerate, and other revert to stromal elements by losing their lipids. The stroma (S) does not contain many sudanophilic lipids. Fig. 40: X380. Figs. 41 and 42 (on following page): X80.

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FIG.41. See legend on preceding page.

FIG.42. See legend on preceding page.

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plasm of degenerating gland cells is filled with many autophagic structures (lysosomes, residual bodies, and so on), fragmented membranes of the endoplasmic reticulum, and a few swollen mitochondria (see also Balboni, 1976). The formation of concentric whorls of membranes having the unit membrane structure increases throughout the cytoplasm of degenerating gland cells, and they may ultimately become the predominant feature; these are the so-called myelin figures, apparently formed at the expense of smooth endoplasmic reticulum, which seem to correspond to the coarse, dense lipid bodies described in histochemical studies (Figs. 22 and 36-42) (Guraya, 1967a, 1968c, 1976c, 1977d). The nucleus of involuting gland cells develops an irregular condensation of chromatin or may be sharply pycnotic (Davies and Broadus, 1968; Motta et al., 1971). Degenerating gland cells can also be easily distinguished from normal gland cells after treatment with HCG, as the former become refractory to stimulation by gonadotrophic substances (Fig. 36). Cholesterol-positive lipid droplets also are not mobilized from degenerated granulosa cells after strong gonadotrophic stimulation (Fig. 26), but are mobilized from the normal interstitial gland cells of the same ovaries after such treatment (Figs. 25 and 26). The lipid droplets of degenerated cells are not released and converted into steroid hormones after gonadotrophic stimulation. In the early stages of degeneration, some vacuoles appear in the peripheral cytoplasm of rabbit interstitial gland cells after gonadotrophic stimulation, but there is no release of lipid droplets (Guraya, 1967a). The refractoriness of degenerating gland cells toward gonadotrophic stimulation may be due to changes in the transport mechanisms, as their degeneration is accompanied by drastic ultrastructural changes in the entire cytoplasm, including its organelles and plasma membrane (Balboni, 1976). The degenerating gland cells and their pigmented lipid inclusions gradually regress and disappear (Fig. 22, 36, and 4042).

IV. General Discussion and Conclusions The correlation of recent electron microscope, histochemical, and biochemical data has revealed the presence of interstitial gland cells in developing, maturing, and adult ovaries of mammals. Their presence has also been shown in the postmenopausal ovaries of women. However, the amount and distribution of interstitial cells vary greatly from species to species, as well as with the ovarian cycle. These variations may be due to endocrine, nervous, or genetic factors. The ovarian interstitial gland cells commonly occurring in the mammalian ovary have been placed in two broad categories, namely, primary and secondary interstitial gland cells, depending upon their origin and the sequence of their development. Primary interstitial gland cells, being the first to appear, originate from fibroblast-like cells and have no relationship with the follicular apparatus during

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fetal or postnatal life, depending upon the species. The most commonly occurring secondary interstitial gland cells originate from the theca interna and surrounding stroma of atretic follicles and have been called theca- or stroma-type interstitial gland cells (Mossman and Duke, 1973a,b). It is this class which apparently makes up the majority of the prepubertal, pubertal, and adult interstitial gland tissue, since the primary interstitial gland cells involute after performing their specific functions during fetal or early postnatal life (Guraya, 1977a,c); the extent of regression seems to vary with the species. Secondary interstitial gland cells formed from the theca interna cells of atretic follicles are present in the ovary from infancy to old age and thus appear to be the most important gland cells in the ovary. However, Mossman and Duke (1973a,b) identified additional interstitial cell types in the ovaries of some mammals, which had a different origin and occurred much less frequently. Their ultrastructural, histochemical, and biochemical characteristics are still to be determined. Both the primary and secondary interstitial gland cells of the mammalian ovary, as determined by the correlation of light and electron microscope, histochemical, biochemical, and physiological data, are typical steroid-producing cells. Their most striking features are: (1) abundant diffuse lipids (lipoproteins) in the cytoplasm, which apparently derive from the abundant ultrastructural agranular endoplasmic reticulum; (2) well-developed cell organelles, especially the multiple Golgi complex and pleomorphic mitochondria with a complex system of internal cristae; (3) the development of diffuse lipoproteins or abundant agranular endoplasmic reticulum accompanied by the appearance of enzyme activities indicative of the biosynthesis of steroid hormones; (4) under certain physiological conditions, stored lipid droplets in the cytoplasm, which consist of either phospholipids and triglycerides, or cholesterol and its esters, triglycerides, and phospholipids; and (4) the capacity to form steroid hormones in biochemical and physiological experiments in vivo and in vitro . Depending upon the species and its physiological state, these characteristics may show quantitative variations. The functional significance of structural variations in the organelles and inclusions involved in steroidogenesis is poorly unknown. But it is wellestablished that LH, ICSH, and other gonadotrophins (PMSG and HCG), which contain luteinizing factor, are essential to maintain these characteristics or a differentiated state of ovarian interstitial gland cells. The latter have been shown to possess receptors or binding sites for these gonadotrophins (see Han et al., 1974; Petrusz, 1974; Midgley el al., 1974). Centrifugal fractionation studies (Rajanierni et al., 1974) and electron microscope autoradiography (Han et al., 1974) of rat ovarian tissue have suggested that LH receptors are primarily located in the plasma membrane of steroid gland cells. That the LH receptor is a lipoprotein has also been indicated by evidence that binding is reduced by treatment of the receptor with proteolytic enzymes, phospholipase C, and phospholipase D (Lee and Ryan, 1972). The quantitative variations of interstitial gland cells in

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different species, as well as in different physiological states, may reflect a variable amount of LH under different physiological conditions, as also discussed for testicular interstitial cells (Christensen, 1975; Neaves, 1975). The important role played by LH in the development, differentiation, and functioning of ovarian and testicular interstitial gland cells indicates that its impact on steroid cell metabolism may be more extensive than has been determined. The physiological significance of diffusely distributed lipoproteins in the interstitial gland cells of the mammalian ovary, as well as in other steroid-secreting gland cell species, was generally overlooked by earlier workers, even by many of those who were especially concerned with luteinization. Guraya (1971, 1974c) has suggested that the appearance of diffuse lipoproteins (or abundant agranular endoplasmic reticulum) demonstrated during the transformation of granulosa cells into luteal cells in several mammals, including humans, may serve as a useful histochemical indicator of this process of hypertrophy or luteinization. The development of diffuse lipoproteins or membranes of smooth reticulum during the transformation of fibroblast-like cells into ovarian hypertrophied interstitial gland cells further supports this suggestion. The replenishment of diffuse lipoproteins and other organelles occurs in interstitial gland cells of the mammalian ovary that were depleted of them after gonadotrophic stimulation (Guraya, 1967a). Vascularization seems to be the key to this cytoplasmic change, since it supplies more oxygen, nutrient materials, and gonadotrophic hormones to the cells which, as a result, become hypertrophied (Guraya, 1971, 1974~).Channing (1969b), while studying the process of luteinization experimentally, arrived at a similar conclusion. Both exogenous and endogenous gonadotrophic hormones (especially hormones containing luteinizing factor) have been shown to cause an increase in the amount of diffuse lipoproteins or agranular endoplasmic reticulum in steroid gland cells, including ovarian and testicular interstitial gland cells (Guraya, 1971, 1974b; Christensen and Gillim, 1969; Christensen, 1975; Neaves, 1975). The hypertrophy or luteinization of steroid-secreting cells in gonads of nonmammalian vertebrates is also accompanied by the development of abundant diffuse lipoproteins or agranular endoplasmic reticulum (Guraya, 1976a,b). These comparative studies clearly indicate that the development of cytoplasmic machinery related to steroid hormone synthesis is apparently brought about by the same basic factors, including gonadotrophins and innervation. Since it is well established that the physiology of steroid gland cells is influenced by gonadotrophins in vivo and in vitro (see also Armstrong, 1968; Armstronget al., 1969a; Zarrow and Clark, 1969; Flint et af., 1973; Guraya 1975a,b,c), the development of primary interstitial gland cells having cytological and histochemical features typical of steroid gland cells also suggests a strong possibility of the presence of some gonadotrophic substance having LH-like activity during fetal or early postnatal life, depending upon the species. Experimental data support the possibility that the ovary of newborn

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animals is sensitive to gonadotrophic stimulation (see Guraya, 1977~).It is also well-established from biochemical and morphological studies that the LHinduced formation of ovarian proteins is closely related to steroid hormone biosynthesis (Guraya, 1977~).If differentiation of primary interstitial gland cells in fetal or early postnatal life depends on gonadotrophic stimulation, sex differences in the timing of their differentiation remain to be explained (Guraya, 1977~).The development of primary ovarian interstitial gland cells takes place later in the ovary than in the testis during fetal or early postnatal life. Are the abundant diffuse lipoproteins or membranes of smooth reticulum structural criteria of active steroid synthesis or are they only part of the hypertrophy or luteinization process and not necessarily associated with functional activity in steroid gland cells? An increase in progesterone synthesis and secretion by granulosa lutein cells, which closely corresponds to the development of diffuse lipoproteins or agranular endoplasmic reticulum, has been considered a good indicator of function (Guraya, 197I , 1974~).The synthesis and secretion of steroid hormones by the interstitial gland cells of the mammalian ovary also correlate well with the presence of diffuse lipoproteins or abundant agranular endoplasmic reticulum. This is further supported by the fact that interstitial gland cells of rabbit ovaries removed after 6-1 1 hours of gonadotrophic stimulation are reduced in size by the loss of cytoplasm and its organelles, including diffuse lipoproteins (Figs. 24, 29, and 30) (Guraya, 1967a), and consequently the synthesis and secretion of progestational steroids are decreased considerably during this period (see Fig. 2 in Hilliard et al., 1969). Correlated morphological and biochemical studies have shown recently that the membranes of smooth reticulum (or diffuse lipoproteins) play a broad role as sites for enzymes involved in cholesterol and steroid hormone synthesis (Christensen and Gillim, 1969; Guraya, 1971, 1976a,b; Christensen, 1975; Neaves, 1975). The abundant membranes of smooth endoplasmic reticulum or diffuse lipoproteins in steroid gland cells, including ovarian interstitial gland cells, provide a large surface area for these enzymes and for biochemical interactions. Biochemical analysis of cell fractions has revealed the detailed localization of enzymes of steroid biosynthesis within steroid gland cells. Christensen (1975) has discussed in detail the roles of various organelles in cholesterol and steroid hormone synthesis, as well as their travel routes within the cell, with special reference to Leydig cells, and thus details in this regard are not presented here. Since it has been shown that ovarian interstitial gland cells also possess ultrastructural and histochemical features specific to Leydig cells and other steroid gland cells, the basic mechanisms of their steroid hormone synthesis and secretion must also be similar to those of Leydig cells. The conclusions and suggestions of Christensen (1975) in regard to the sequential involvement of various organelles are further supported by the evidence summarized and discussed in this article, which indicates that the intense activity of enzymes involved in the biosynthesis of steroid hormones

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parallels the presence and distribution of abundant diffuse lipoproteins or agranular endoplasmic reticulum, mitochondria with complex cristae, and lipid droplets in the interstitial gland cells of the mammalian ovary. The presence of abundant sudanophilic lipids constituting the various organelles, and lipid droplets, in ovarian interstitial gland cells suggests that they play important fundamental roles in steroid biosynthesis.. This suggestion is further supported by the observation that fatty acids are the main initial precursor in steroid hormone synthesis in the gonads (Christensen, 1975), although glucose may also be a substrate. In addition to acting as a site for enzymes involved in the biosynthesis of steroid hormones, the diffuse lipoproteins or the membranes of smooth reticulum may also synthesize and accumulate cholesterol as a constituent of their lipid component, which is now well known to act as a precursor in the biosynthesis of steroids (Fawcett et al., 1969; Christensen and Gillim, 1969; Guraya, 1971, 1976a,b; Christensen, 1975; Neaves, 1975). In this way, precursor cholesterol would be stored close to enzymes associated with diffuse lipoproteins (or smooth reticulum) (Fawcett et al., 1969); these enzymes catalyze the conversion of cholesterol to biologically active steroid hormones. There is some indication in the previous literature (Christensen and Gillim, 1969) that the abundance of smooth membranes in a steroid gland cell reflects the extent to which the cell makes its own cholesterol rather than taking it up from the plasma. The amount of abundant diffuse lipoprotein in the interstitial gland cells of the ovary therefore may be in part an expression of the synthesis and accumulation of cholesterol, as also suggested for Leydig cells, luteal cells, and other steroid gland cells (Guraya, 1971, 1974b,c, 1976a,b; Christensen, 1975; Neaves, 1975). This masked cholesterol could not be demonstrated with histochemical tests. However, biochemical studies have shown that the membranes of smooth reticulum are rich in phospholipids and cholesterol (Christensen, 1975; Neaves, 1975). Since phospholipid micelles provide a suitable vehicle for solubilizing cholesterol (see Guraya, I97 I), the abundant phospholipids of the diffuse lipoproteins or smooth reticulum in the interstitial gland cells of the mammalian ovary may be one device for enhancing intracellular transport of cholesterol and possibly other steroids as well (see Guraya, 197 1, 1976a,b). Christensen (1975) has discussed in detail the problems of transport and travel of fatty acids, cholesterol, and steroid hormones within steroid gland cells. It is still not known precisely how substrates and products are transported through the plasma membrane and membranes of organelles in steroid gland cells. The movements of the agranular endoplasmic reticulum are believed to play important roles in transporting substances within the cytoplasm. Coated pits and vesicles are occasionally seen in association with the plasma membrane, which are usually believed to be involved in the uptake or discharge of substances. The plasma membrane of the steroid gland cell is believed to be the main site of action for LH which induces adenyl cyclase activity, the resulting CAMP moving into the cytoplasm as the actual

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mediator of LH action (see details in Eik-Nes, 1975). The various intracellular transport processes suggested by Christensen (1975) are apparently greatly affected by the degeneration of steroid gland cells, including ovarian interstitial gland cells, which is accompanied by alterations in their endoplasmic reticulum (or diffuse lipoproteins), mitochondria, lipid droplets, and so on, as already discussed. That changes take place in the transport mechanisms is strongly supported by the observation that the regressing or degenerating interstitial gland cells of the mammalian ovary become refractory to gonadotrophic stimulation in that their cholesterol-containing lipid droplets are not released (Fig. 36), whereas in the normal gland cells they are depleted. From this discussion, it can be concluded that the diffuse lipoproteins or membranes of smooth reticulum in steroid gland cells, including the ovarian interstitial gland cells, play important fundamental roles in steroidogenesis, as also suggested by Christensen (1 975). The increase in the size of mitochondria, as well as complex alterations in their inner profiles, concomitant with the development of diffuse lipoproteins (or smooth reticulum) reflects their participation in steroid metabolism, for example, as an energy source for steroid synthesis and cholesterol side-chain splitting (Fawcett et d.,1969; Christensen and Gillim, 1969; Guraya, 1971, 1976a,b; Christensen, 1975; Neaves, 1975). The cholesterol that enters the biosynthetic pathway leading to steroid hormone synthesis may be derived directly from de nuvo synthesis, from the splitting of cholesterol esters stored in lipid droplets, or from the plasma (see also Flint and Armstrong, 1972, 1973). It is uncertain at this time which source is the most important precursor. Whatever its source, cholesterol destined for steroid hormone synthesis first enters the mitochondria for cleavage of its side chain. The inner membrane and cristae of mitochondria have been shown to be the site of the localization of enzymes involved in cholesterol side-chain cleavage in the adrenal cortex (Christensen, 1975; Neaves, 1975). The cholesterol side-chain cleavage system of ovarian interstitial gland cells can also be presumed to be associated with the cristae of mitochondria which develop the configuration of mitochondria in well-established steroid gland cells. The pregnenolone formed is believed to leave the mitochondrion and to be acted upon by enzymes associated with the membranes of smooth endoplasmic reticulum (Christensen and Gillim, 1969; Fawcett et a l . , 1969; Neaves, 1975; Christensen, 1975). Flint and Armstrong (1973) determined the activities of various enzymes (cholesterol esterase, cholesterol ester synthetase, the cholesterol side-chain cleavage enzyme, and 3P-HSDH) which are responsible for steroid biosynthesis and cholesterol ester metabolism in rabbit ovarian interstitial tissue. The characteristic ultrastructure of mitochondria in ovarian interstitial gland cells is lost after hypophysectomy and restored by a LH-like hormone (Davies and Broadus, 1968; Carithers and Green, 1972b; Carither, 1976). Efforts to demonstrate a direct effect of LH or CAMP on various indexes of steroidogenic activity in isolated mitochondria have not been successful (Hamburger, 1968;

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Jackanicz and Armstrong, 1968). However, LH-sensitive adenylate cyclase activity has been found in outer mitochondria1 membranes (Sulimovici and Lunenfeld, 1974) and side-chain cleavage of cholesterol, which occurs in the mitochondria (Christensen, 1975), is widely believed to be a rate-limiting step in steroidogenesis (see also Savard, 1973). Only mitochondria have a steady rate of response throughout this course of treatment. Side-chain cleavage of cholesterol is believed to be controlled by LH and thus is a rate-limiting step, perhaps with CAMP as an intermediate. LH is also believed to influence other factors in 20a!-hydroxylation, such as the production of NADH, the activation of oxygen, and the removal of pregnenolone inhibition. There is increasing evidence that LH affects its target cells by triggering a variety of more-or-less distinct chains of events involving steroid-secreting cells (reviewed in detail by Christensen, 1975; Neaves, 1975; see also Flint et al., 1973), possibly through inactions with more than one type of receptor (Braendle ef a l . , 1973; Lee and Ryan, 1973; Moyle and Ramachandran, 1973). Jackanicz and Armstrong (1968) have even suggested that progesterone biosynthesis in rabbit ovarian interstitial gland cells occurs in the mitochondria. But the factors and mechanisms controlling the transport of cholesterol and steroid hormones, especially pregnenolone or progesterone, across mitochondrial membranes, are still very poorly understood (Christensen, 1975). Sometimes mitochondria may show an accumulation of lipids in their matrix, the significance of which is unknown. Extensive development of the Golgi complex in hypertrophied interstitial gland cells of ovaries suggests that it plays a role in steroid hormone secretion. This suggestion is further supported by evidence of a rapid proliferation of Golgi vesicles after LH treatment and the increased extent of its lamellae (Carithers, 1976). But the exact nature of its participation in steroid hormone secretion is still not known, and divergent views exist in this regard (reviewed by Christensen, 1975). However, ultrastructural studies of bovine luteal cells have revealed granules which appear to be formed in the Golgi region and transported to the cell membrane where their contents are expelled by exocytosis. Variations in granule formation and secretion are believed to reflect the pattern of progesterone secretion by the corpus luteum during the estrous cycle of the sheep (Gemell et a l . , 1974). Kramers et al. (1975) isolated a particulate fraction from bovine luteal tissue which was quite rich in progesterone. Morphological analysis of the gradient has revealed that progesterone-enriched fractions contain a large number of irregularly shaped organelles (granules) bounded by a unit membrane. They have a paracrystalline internal structure. These correlative morphological and biochemical data have suggested that progesterone may also be packed in the Golgi complex and secreted by the classic process described for protein hormones (Fawcett et al., 1969; Poste and Allison, 1974). Correlative histophysiological and biochemical studies indicate that cholesterol-positive lipid droplets of steroid gland cells are precursors in the synthesis of steroid hormones (Christensen and Gillim, 1969; Fawcett et al.,

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1969; Guraya, 1971, 1973a,b,c, 1974a,b, 1976a,b; Christensen, 1975; Neaves, 1975). They become depleted in response to strong gonadotrophic stimulation. Similarly, LH, HCG, and PMSG cause the mobilization of cholesterol-positive lipid droplets from ovarian interstitial gland cells (Figs. 24, 26, 29, 30 and 31). Histochemical and biochemical data have also demonstrated that these gonadotrophins stimulate steroidogenesis in interstitial gland tissue, but the exact mechanism of their action is still not known precisely. The treatment of rats with prolactin neither increases nor decreases the high levels of cholesterol-positive lipid droplets (Armstrong et a1 ., 1969b; Guraya, 1975~).In contrast, Zarrow and Clark (1969) reported that prolactin caused cholesterol accumulation not only in the luteal compartment but also in interstitial tissue. The interstitial gland tissue of the rabbit ovary has been extensively studied with morphological, histochemical, biochemical, and physiological techniques. There is little release of lipid droplets consisting of cholesterol and its esters, triglycerides, and some phospholipids from interstitial gland cells of estrous rabbit ovaries (Figs. 13, 19, and 22) (Guraya, 1967a), which is apparently responsible for the formation of basal amounts of 2Oa-hydroxypregn-4-en-3-one and progesterone in vivo (Hilliard et a l . , 1963, 1969; Hilliard and Sawyer, 1964; Solod et al., 1966) and in vitro (Dorrington and Kilpatrick, 1966; Flint and Armstrong, 1973; Ftint et a l . , 1973). The massive release of preformed lipid droplets rich in cholesterol and its esters from 1 to 6 hours after gonadotrophic stimulation (Figs. 24 and 29-3 1) is clearly correlated with the increased output of these progestational steroids described by several workers (Forbes, 1953; Simmer et al., 1963; Hilliard et al., 1961, 1963, 1969; Hilljard and Sawyer, 1964; Armstrong et a l . , 1969a; Dorrington and Kilpatrick, 1969). These correlative observations have shown that the steroids synthesized during the first 6 hours after stimulation of interstitial gland cells in the rabbit are derived from stored cholesterol (Fig. 24 and 29). From the various histochemical and biochemical studies as correlated above, it can be concluded that the lipid droplets of ovarian interstitial gland cells contain stored hormone precursor (cholesterol and its esters) (Armstrong and Flint, 1973), as also discussed for Leydig cells (Christensen, 1975; Neaves, 1975); the cholesterol esters of lipid droplets are utilized for the synthesis of progestational steroids collected from the ovarian vein in the rabbit. Cholesterol esters stored in the lipid droplets are possibly hydrolyzed by soluble esterase in the cytoplasm surrounding the lipid droplet, setting free more cholesterol for carrying out steroid hormone synthesis (Flint and Armstrong, 1973; Flint et al., 1973), as also suggested for Leydig cells (Christensen, 1975). The variable number and size of lipid droplets in ovarian interstitial gland cells of different mammalian species, or of the same species under different physiological conditions, suggests that cells that are more active in steroid biosynthesis apparently show fewer or smaller lipid droplets (as the substrates are being actively used in steroid hormone synthesis), whereas relatively less active cells

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show more lipid droplets. The variation observed in lipid droplets from cell to cell in the rabbit after HCG stimulation (Guraya, 1967a) indicates that the cells are in different phases of secretory activity (see also Motta et al., 1971). By utilizing the hormone precursor, further biosynthesis of steroid hormones occurs with the help of enzymes associated with the mitochondria and the membranes of smooth reticulum (or diffuse lipoproteins), as already discussed (see also details in Christensen, 1975). The questions then arises: What is the exact physiological significance of a massive release of lipid droplets from the interstitial gland cells of the rabbit ovary (Figs. 24 and 29-31), along with the cytoplasm and its organelles, after gonadotrophic stimulation? Two possibilities can be suggested in this regard. First the lipid droplets may also contain or be associated with a steroid hormone in addition to its stored precursor. These stored hormones are gradually released from the lipid droplets lying in the intercellular spaces as well as adjacent to the blood vessels (Fig. 31). This possibility can be confirmed or refuted by carrying out further biochemical studies on isolated lipid droplets from interstitial gland cells of estrous rabbits (Armstrong and Flint, 1973). The second possibility is that the conversion of hormone precursor (cholesterol and its esters) into progestational steroids continues to occur in the intercellular spaces as well as adjacent to the blood vessels where the released lipid droplets and the organelles form consqicuous aggregations (Fig. 31), as also observed by Motta et al. (197 1); the enzymes needed to carry out this biosynthesis outside the cell may be provided by the mitochondria and diffuse lipoproteins (or elements of smooth reticulum) which are simultaneously released along with lipid droplets from the interstitial gland cells as indicated by a great decrease in their size (Figs. 19, 22, and 30). The biosynthesis of steroid hormones outside the steroid gland cell is possible, as similar biochemical transformations utilizing cell-free systems have been carried out in test tubes (see Flint and Armstrong, 1973; Christensen, 1975; Neaves, 1975). After the release of stored hormone or hormone precursor the lipid droplets are apparently broken down, and their components including fatty acids pass into the lumen of blood vessels by pinocytosis, phagocytosis, or a combination of various mechanisms. It will be very interesting to determine the details of their passage into blood vessels by electron microscope techniques after gonadotrophic stimulation. Motta et al. (1971), using electron microscopy, demonstrated the presence of organelles and lipid droplets in the lumen of capillaries which, according to them, originate from the degeneration and cytolysis of interstitial cells. They have considered these cellular changes to be a sign of the intermittency of internal secretion of the interstitial cells. Cells showing partial or total cytolysis have been interpreted by Motta et al. (1 97 1) to be a morphological manifestation of the end phase of normal cyclic differentiation and secretion. Biochemical investigation should also be carried out on the composition of lipid contents in the ovarian vein before and after gonadotrophic stimulation. The differences in lipid contents will reveal the nature and amount of lipids derived

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from the released lipid droplets of ovarian interstitial gland cells after gonadotrophic stimulation. It is interesting to mention here that Rhodin (1971) has published electron micrographs showing the release of lipid droplets at the surface of the cell in the rat adrenal cortex; such profiles are more common after ACTH stimulation. His studies therefore support the observations of Guaya (1967a) in regard to the mechanism of release of lipid droplets from the interstitial gland cells of the rabbit ovary after HCG stimulation, as discussed above. These observations on different steroid gland cell species suggest that, under certain physiological conditions, lipid droplets may be involved in steroid hormone secretion. But ovarian interstitial gland cells and other steroid gland cell species do not have lipid droplets when they are actively involved in steroid hormone synthesis, suggesting the presence of some other mechanism for the secretion or release of steroid hormones from the cell surface. Motta et al. (1971) believe that numerous pinocytotic vesicles and cytoplasmic evaginations in the ovarian interstitial cell may be an indication of intense cellular exchange. Secretion of the final products appears to be carried out by a mechanism opposite pinocytosis, probably involving particles of molecular size. The morphology, biochemistry, and physiology of steroid secretion has not received much attention, as most of the previous studies dealt with the sites and pathways of steroid hormone synthesis (see Christensen, 1975; Eik-Nes, 1975; Neaves, 1975). The mechanisms by which steroid hormones, either free or conjugated, are secreted should be investigated with correlative morphological, biochemical, and physiological techniques. Further studies using the electron microscope are also needed to determine the exact mechanism of release of lipid droplets from ovarian interstitial gland cells after gonadotrophic stimulation, which apparently causes drastic physicochemical changes in relation to transport mechanisms in the peripheral cytoplasm including the cellular membranes of various organelles. Lytic changes in the plasma membrane and peripheral cytoplasm seem to occur, as suggested by the formation of vacuoles (Figs. 22, 24, and 29) (Motta et al., 1971). Such physicochemical changes do not occur in degenerating gland cells which become refractory to gonadotrophic stimulation, as their lipid droplets are not released (Figs. 22 and 36). In the early stages of degeneration vacuoles are formed in the outer cytoplasm, but the lipid droplets are not released (Guraya, 1967a). The nature of the forces causing the movement of liberated lipid droplets toward the blood vessels and capillaries could not be determined (see also Motta et al., 1971). Possibly a concentration gradient exists which establishes and facilitates the movement of lipid bodies through the intercellular spaces, as well as through the walls of blood capillaries and vessels. Corresponding to the low levels of progestational steroids observed in the ovarian vein after 7-11 hours of gonadotrophic stimulation in the rabbit (see Hilliard et a l , , 1963, 1969), after depletion of their stored lipid droplets and the

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loss of cytoplasm and its organelles, the reduced interstitial gland cells are seen to be actively engaged in the synthesis and replenishment of their cytoplasm, lipid granules, and other organelles. No cholesterol or cholesterol esters are seen in the newly formed lipid granules of such gland cells, or in those of 24-hour postovulatory ovaries. Three days after ovulation, the interstitial gland cells of the rabbit ovary morphologically and histochemically begin to resemble those of estrous ovaries, as abundant cholesterol-positive lipid droplets are stored in the cytoplasm (Guraya and Greenwald, 1964a; Guraya, 1967a). The control of cholesterol synthesis within the cell is poorly understood, not only in the ovarian interstitial gland cells but also in Leydig cells (Flint and Armstrong, 1972; Christensen, 1975; Neaves, 1975). Flint et al. (1973) made an attempt to study the nature of the biochemical factors involved in the synthesis, storage, and depletion of cholesterol esters in rabbit interstitial gland cells (see also Flint and Armstrong, 1973). It has been suggested that the steroid hormone produced regulates cholesterol synthesis through a feedback control system. The ultrastructural correlates of cholesterol storage and utilization in steroid hormone synthesis need to be investigated. The lipid droplets stored in ovarian interstitial gland cells are composed mainly of triglycerides, cholesterol esters, and a few phospholipids, as also described for other steroid gland cells (see Guraya, 1971, 1973a,b,c, 1974b, 1975a,b,c, 1976a,b). It is generally believed that, after the cholesterol has been synthesized within the steroid gland cell or taken in from the plasma, it is immediately esterified and accumulated in lipid droplets (Flint and Armstrong, 1972; Flint et al., 1973; Christensen, 1975; Neaves, 1975); cholesterol is generally esterified at position 3 with triglyceride fatty acids which simultaneously increase in amount. The fatty acids in turn may be derived either from the plasma or from de n o w synthesis by cytoplasmic enzymes (Johnson, 1970). In human ovarian interstitial gland cells, for example, the greatly hypertrophied gland cells of late pregnancy (Figs. 11 and 12) or the postmenopausal period (Fig. 32) (Guraya, 1966b, 1971, 1972a, 1976c), in which no storage of cholesterol-positive lipid droplets occurs, the synthesis or absorption of cholesterol from the plasma and its conversion into steroid hormone are balanced in such a way that no superfluous cholesterol is stored in the form of lipid droplets (see also Figs. 20 and 33). The absence of cholesterol-positive lipid droplets has been 'attributed to strong gonadotropic stimulation. Aldman et al. (1949a) found that the storage of cholesterol in ovarian interstitial gland cells is not an autonomous cellular process and that small amounts of HCG and PMSG in combination produce considerable storage. Aldman et al. (1949b) further showed that estradiol and progesterone have the same effect, in all probability because of a depression of pituitary gonadotrophin secretion to a low level. On the basis of these observations Aldman et al. (1949b) have assumed that the cholesterol storage process in these cells is directly regulated by gonadotrophins. Claesson et al. (1954a), while studying the effect of

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N-methylethylphenylbarbituricacid (Prominal) on the cholesterol content of the ovarian interstitial-gland cells of the rat, arrived at a similar conclusion. Cholesterol storage also does not occur in ovarian interstitial gland cells of hypophysectomized rats (Guraya, I976e). These observations have further confirmed that the cholesterol storage process in these cells is directly under the influence of pituitary gonadotrophins. The presence of RNA (or rough endoplasmic reticulum and free ribosomes) in steroid gland cells suggests that active protein synthesis takes place during the replenishment of cytoplasm and its organelles. A similar suggestion has also been made by Gillim et al. (1969) and Christensen (1975) in regard to human luteal and Leydig cells, respectively. These proteins may include steroidogenic enzymes and the enzymes presumably contained in the lysosome-like bodies described in electron microscope studies (Davies and Broadus, 1968; Christensen, 1975; Neaves, 1975). The rough endoplasmic reticulum may also be involved in the synthesis of membranes of smooth reticulum, mitochondria, and so on. Further work using the electron microscope and radioautography should be carried out to investigate the synthesis and replenishment of cytoplasm and its organelles in the interstitial gland cells of the rabbit ovary after gonadotrophic stimulation. This will increase our knowledge of the biology of the steroid gland cell. The correlation of various electron microscope, histochemical, biochemical, and physiological data has shown that both the primary and secondary interstitial gland cells of the mammalian ovary are steroid secretors. Similarly, the interstitial cells in the ovaries of nonmammalian vertebrates have been reported to be steroid-synthesizing sites (Guraya, 1976a,d; Guraya and Chalana, 1976; Chalana and Guraya, 1977). They also undergo cyclic changes, both in quantity and character, associated with their reproductive cycles. The building of interstitial cells is maximal during the prebreeding and breeding periods of nonmammalian vertebrates. Their formation in the prebreeding period has been considered a preparatory phase augmented before the onset of breeding activities in birds (Guraya and Chalana, 1976; Chalana and Guraya, 1977). Maximal activity of interstitial cells for the production of steroid hormones has been observed during the breeding period. The question now arises: What is the nature of steroid hormones formed by ovarian interstitial cells in vertebrates including mammals? Our knowledge of the pathways of steroidogenesis, including the nature of steroid hormones secreted by ovarian interstitial gland cells in mammals and nonmammalian vertebrates, is still limited; these pathways are, however, better known for the corresponding cells of the testis (see Christensen, 1975; Neaves, 1975; Guraya, 1976b). The understanding of pathways of steroidogenesis will demand detailed knowledge of the identity of all interstitial gland cell products of the ovaries. Clarification of the physiological role of steroid hormones in female

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reproduction, which are secreted by ovarian interstitial gland cells, is also urgently needed. The rabbit is the only species in which the synthesis and secretion of progestins has been attributed to interstitial gland cells. On the basis of their data, Hilliard et al. (1967) have, however, proposed that coitus causes a rise in the level of LH sufficient to produce an elevated 2k-hydroxy-pregn-4-en-3-one output, which in turn has a positive feeaback action on the pituitary, thus maintaining the elevated level of gonadotrophin necessary for ovulation. The rise in progesterone content demonstrated during the preovulatory period of some mammals has also been related to the release of preformed stores of cholesterol and/or its esters from ovarian interstitial gland tissue. In the previous literature, suggestions were made that ovarian interstitial gland cells secrete estrogens, progestins, or androgens, depending upon the physiological state and the species (see Hilliard et al., 1963; Nalbandov, 1964; Keyes and Nalbandov, 1968; Guraya, 1971, 1973a,b,c, 1974a,b, 1976a). Falck (1959) interpreted the results of his experiments to show that the theca interna and interstitial cells of the adult ovary are probably the cells that actually secrete estrogens, but that they can do so only under the influence of a substance originating from adjacent granulosa or corpus luteum cells. Studies with immature rats have indicated the presence of an extractable, biologically active estrogen within the ovary (Cieciorowska and Russfield, 1968). Incubations of ovaries from immature rats indicate an ability to convert progesterone to estrone and estradiol (Quattropani and Weisz, 1973). The amount of progesterone converted to estradiol at the various ages examined is directly correlated with the increase in recognizable interstitial gland celle which develop the characteristics typical of steroidogenic tissue. Similarly mouse ovaries have the ability to produce estrogens on postnatal day 16, when the ovaries contain interstitial gland cells (see Quattropani, 1973). The correlation of morphological and biochemical information at the subcellular level will help us to identify more precisely the nature of steroid hormones and the detailed localization of steroid biosynthetic steps within the organelles of interstitial gland cells of prenatal, postnatal, prepubertal, pubertal, and adult ovaries. The use of cytochemistry at the ultrastructural level will be especially helpful in solving these problems. The different roles of LH in steroid biosynthetic steps also form a promising area for future research, as they are poorly understood. It is also not known precisely whether or not LH synergizes with other pituitary hormones to regulate the function of ovarian interstitial gland cells which are present and fully differentiated, at least periodically, from birth to old age. Mossman and Duke (1973b) have suggested that the secretions of ovarian interstitial cells may be responsible for the development and maintenance of secondary female sex characters, just as testicular interstitial cell secretions are related to secondary male sex characters. This would involve mainly an estrogen or an estrogen analog.

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Motta, P., and Bourneva, V. (1970b).Acta Hisrochem. 38, 340. Motta, P.,and Takeva, 2. (1970).C. R. Acad. Bulg. Sci. 23, 883. Motta, P.,and Takeva, Z. (1971).Fertil. Steril. 22, 378. Motta, P., Nesci, E., and Fumagalli, L. (1968).Biol. Lat. 21, 225. Motta, P., Takeva, Z., and Bourneva, V. (1970).Experientia 26, 1128. Motta, P., Nesci, E., and Fumagalli, L. (1971).Arch. Anar. Histol. Embryo/. 54, 43. Mottd, P., Columbis, P., and Didio, L. J. A. (1975).Fertil. Steril. 26, 346. Moyle, W. R., and Ramachandran, J . (1973).J . Endocrinol. 93, 127. Muta, T. (1958).Kurume Med. J . 5 , 167. Nalbandov, A. V. (1964).“Reproductive Physiology.” Freeman, San Francisco, California. Neaves, W. B. (1975).Contraception 2, 571. Neilson, D., Jones, G. C., Woodruff, J. D., and Goldberg, B. (1970).Obster. Gynecol. 25, 889. Nesci, E.,and Motta, P. (1969). Biof. La?. 22, 1. Niswender, G. D., Akbar, A. M., and Nett, T. M. (1976).In “The Endocrine Function of the Human Ovary” (V. H. T. James, M. Serio, and G. Giusti, eds.), pp. 71-80. Academic Press, New York. Novak, E. R., Goldberg, B., Jones, G.S., and O’Toole, R. V. (1965).Am. J . Obstet. Gynecol. 93, 669. Odor, D. L.,and Blandau, R. J. (1969a).Am. J . Anat. 124, 163. Odor, D. L., and Blandau, R. J. (1969b).Am. J. Anat. 125, 177. Oei, H. F., Jobsis, A. C.. and Honnebier, W. J. (1974).Eur. J. Obstet., Gynecol. Reprod. Biol. 4, Suppl. s59. Pesoncn, S . , Ikonen, M., ProcopC, B. J . , and Saure, A. (1968).Acta Endocrinol. (Copenhagen)58, 364. Petrusz, P. (1974).Eur. J . Obsrer., Gynecol. Reprod. Biol. 4, Suppl., 53. Plotz, E. J., Wiener, M., Stein, A. A., and Hahn, R. D. (1967).Am. J . Obsrer. Gynecol. 99, 182. Poste, G . , and Allison, A. C. (1974).Biochim. Biophys. Acra 300, 421. Pupkin, M., Bratt, H., Weisz, J., Lloyd, C. W., and Balogh, K., Jr. (1966).Endocrinology79,316. Quattropani, S. L. (1973).Anat. Rec. 177, 569. Quartropani, S. L., and Weisz, J . (1973).Endocrinology 93, 1269. Rajaniemi, H. J., Hirshfield, A. N., and Midgley, A. R., Jr. (1974).Endocrinology 95, 579. Rennels, E. G . (1951).Am. J . Anat. 38, 63. Rennie, P. I. C., and Davenport, G. R. (1965).Proc. 47th Annu. Meet., Am. Endocrine Soc. Abstract. Resko, J . A. (1977).I n “Regulatory Mechanisms Affecting Gonadal Hormone Action. Advances in Sex Hormone Research” (J. A. Thomas and R. L. Singhal, eds.), Vol. 3, pp. 139-168.Univ. Park Press, Baltimore, Maryland. Rhodin, J. A. G. (1971).1. Ultrastruct. Res. 34, 23. Rice, B. F., and Savard, K. (1966).J . Clin. Endocrinol. Metab. 26, 593. Rubin, B. L., Deane, H. W., Hamilton, J. A., and Driks, E. C. (1963a).Endocrinology 72, 924. Rubin, B. L., Deane, H. W., and Hamilton, J. A. (1963b).Endocrinology 73,748. Savard, K. (1973).Biol. Reprod. 8, 183. Savard, K.,Marsh, 3. M., and Rice, B. F. (1965).Recent. Prog. Horm. Res. 21, 285. Scully, R. E., and Cohen, R. B. (1964).Obster. Gynecol. 24, 667. Seth, P.,and Prasad, M. R. N. (1967).Gen. Comp. Endocrinol. 9,383. Simmer, H.H., Hilliard, J., and Archiblad, D. (1963).Endocrinofugy 72, 67. Solod, E. A., Armstrong, D. T., and Greep, R. 0. (1966).Steroids 7, 607. Stegner, H. E. (1970).In “Gonadotrophins and Ovarian Development” ( W . R. Butt, A. C. Crooke, and M. Ryle, eds.), pp. 232-238.Livingstone, Edinburgh. Stegner, H. E. (1973).In “The Development and Maturation of the Ovary and its Functions” (H. Peters, ed.), pp. 84-94.Excerpta Med. Found., Amsterdam.

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Stegner, H. E., and Onken, M. (1971). Cytobiologie 3, 240. Sulimovici, S . , and Lunenfeld, B. (1974). FEBS Let?. 41, 345. Svensson, K. G . , Owman, C. Sjoberg, N . O., Sporrong, B., and Walls, B. (1975). Neuroendocrinology 11, 40. Taylor, F. B. (1961). Acta Endocrinol. (Copenhagen) 36, 361. Unsicker, K. (1970a). Z. Zellforsch. Mikrosk. Anat. 109, 46. Unsicker, K (1970b). Z . Zellforsch. Mikrosk. Anat. 109, 495. Unsicker, K. (1973). Z. Zellforsch. Mikrosk. Anat. 146, 123. Unsicker, K. (1974). Cell Tissue Res. 152, 513. Vermeulen, A. (1974). In “The Endocrine Function of the Human Ovary” (V. H. T. James, M. Serio, and L. Martini, eds.), Vol. 2, pp. 237-244. Academic Press, New York. Wartenberg, L. W. (1958). J. Histochem. Cytochem. 6, 225. Weakley, B. W. (1967). J. Anat. 101, 435. Wiest, W. G., and Kidwell, W. R. (1969). I n “The Gonads” (K. W. McKerns, ed.), pp. 295-325. Appleton, New York. Zarrow, M. X., and Clark, J. H. (1969). Endocrinology 84, 340.

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INTERNATIONAL REVIEW OF CYTOLOGY.VOL. 55

Correlation of Morphometry and Stereology with Biochemical Analysis of Cell Fractions R. P. BOLENDER Department of Biological Structure, School of Medicine, University of Washington, Seattle, Washington I. Introduction . . . . . . . . . . . . . . . . . . . . 11. The Technique of Stereological Analysis . . . . . . . . . A. Theoretical Considerations . . . . . . . . . . . . . B. Stereological Reference Systems . . . . . . . . . . . C. Practical Procedures . . . . . . . . . . . . . . . . 111. The Technique of Differential Centrifugation and Marker Enzyme Analysis . . . . . . . . . . . . . . . . . . . . . A. General Considerations . . . . . . . . . . . . . . B. Practical Procedures . . . . . . . . . . . . . . . . IV. The Integration of Stereological and Biochemical Data . . . . A. Stereology of Intact Tissue Correlated with Biochemistry of Fractions . . . . . . . . . . . . . . . . . . . . B. Stereology of Tissue Fractions Correlated with Biochemistry of Fractions . . . . . . . . . . . . . . . . . . . . C. Stereology of Intact Tissue and Fractions Correlated with Biochemistry of Fractions . . . . . . . . . . . . . V. Summary and Conclusions . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

247 248 248 256 263 27 1 27 1 272 275 275 279 283 287 287

I. Introduction One of the generalizations frequently applied to cells is that their behavior depends upon relationships of structure to function. While such relationships exist at several levels, those of particular interest to cell biologists occur when molecules are combined to form specific structures or organelles that can be identified biochemically and electron microscopically at the same time. Consider, for example, a membrane of the endoplasmic reticulum (ER). It may be thought of as an aggregation of biochemical constituents (molecules) arranged in “sheets” that can be characterized morphologically as a surface area. However, a peroxisomal granule containing molecules within a membrane package is described appropriately as a volume. 247

Copyright @ 1978 by Academic Ress, Inc. All rights of reproduction in any form reserved.

ISBN 0-12-364355-4

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But what relationships exist between structured organelles and the chemical activities they generate? The best operational answer to this question can be found in the postulates of de Duve (1971), wherein it is proposed that specific organelles contain uniform distributions (concentrations) of molecules that mark uniquely their presence in biochemical fractions. By following this type of reasoning considerable success has been achieved in assigning specific biochemical functions to cellular organelles, but we still seem to know relatively little about the quantitative relationships of organelle structure to function. It therefore seems appropriate to ask: Do quantitative relationships actually exist between the molecular constituents and the morphological components they produce? If so, what are they, how can they be determined, and what is the practical use of such information? The purpose of this article is to discuss how various workers have attempted to answer these questions by using stereology in combination with biochemistry. Within this discussion, the technique of stereological analysis is evaluated with respect to both theoretical and practical procedures, and special attention is given to the interpretation and integration of stereological data. This article considers primarily studies related to the liver but does not attempt a complete survey of the literature, and references have been chosen for purposes of illustration.

11. The Technique of Stereological Analysis A. THEORETICAL CONSIDERATIONS

1 . Stereological Data The mathematical proofs from which stereological equations are derived demonstrate that measurements taken from representative two-dimensional samples can be used to estimate quantitatively the internal structure of a three-dimensional object. The solutions to many of these equations are given in terms of morphological densities or concentrations, which relate the aggregate volume, surface area, and length, or the number of individual structural units of a specific internal structure to a standard unit of the object’s volume. This unit volume is called the reference or containing volume. For example, a surface density of 4 m Ycrn indicates that a unit volume equal to 1 cm3 of the object contains 4 m of membrane surface area. Or, similarly, a length density of 500 cm/cm3 might characterize the total length of microtubules in a similar 1 cm3 reference volume. Stereological data are relatively easy to collect, and descriptions of practical procedures abound (Chalkley, 1943; DeHoff and Rhines, 1968; Elias et a l . , 1971; Loud, 1962, 1968; Sitte, 1967; Underwood, 1970; Weibel, 1963, 1969; Weibel and Bolender, 1973). To a very large extent, however, data are collected

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from electron micrographs of anisotropic biological material using sections that are compressed and have a finite thickness-sample characteristics that violate the assumptions of many stereological proofs. Although the presence of these errors is widely recognized, the extent to which they influence stereological estimates continues to be one of the least well-defined areas of the technique. A favorite way to “argue away” errors associated with section artifacts, for example, has been to assume that all the data contain the same bias and then to make interpretations in terms of relative changes. This has been a good way to detect trends, but it is far less satisfactory when more precise morphological information is required, as is the case when looking for direct relationships between cell morphology and biochemistry. Quite simply, as one pursues more accurate stereological estimates, corrections are needed to satisfy the requirements of the theory. Unfortunately, problems associated with the application of stereological theory are not necessarily solved by accounting for the sampling errors referred to above. As soon as one departs from baseline studies and attempts to measure changes in cells, new difficulties in interpretation arise. This results from the fact that stereological densities can become ambiguous when changes related to 1 cm3 of reference volume are used to infer changes in cells whose volumes, and consequently reference systems, may not remain constant. These and other problems related to the interpretation of stereological data are explored as part of this article .

2 . Formulas a. Volume Density. In his attempts to quantitate the internal composition of rocks, Delesse (1847) discovered that for a random cross section the relative area occupied by each mineral was proportional to its volume in the entire rock (object). In other words, its area density (aggregate or compartmental area of the mineral within a unit reference area) is equal to its volume density (aggregate volume of the mineral within a unit reference volume). Mathematical proofs for the Delesse principle have been given by Chayes (1965), DeHoff and Rhines (1968), Miles (1976), Underwood (1970), and Weibel(1963), to mention only a few. How is this relative area of sectioned structures determined from a twodimensional sample? Several approaches are available (DeHoff and Rhines, 1968; Weibel and Elias, 1967; Weibel and Bolender, 1973, Underwood, 1970), but point-counting methods (Chalkley, 1943; Hilliard and Cahn, 1961; Weibel, 1969) seem to be most suitable when measurements are collected manually. Systematic point counting actually represents an abbreviated form of planimetry performed by counting squares. Instead of identifying the location of the entire square, only a point in the upper left-hand comer is used. The structural component directly beneath this point receives an area allocation equivalent to one

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square. Since this type of planimetry can be done quickly and easily, it becomes practical to analyze a large number of samples. A larger sample size, it has been shown (Hilliard and Cahn, 1961), is more important than the precision of individual measurements for obtaining stereological estimates (see Section II,C,2). In practice, the volume density VV of compartments (aggregates of similar components, i.e., organelles or spaces) can be determined by superimposing a systematic set of points on light or electron micrographs and then counting the number of points lying over the different components. The volume density is calculated by dividing the aggregate number of points Pt associated with a specific structure i (i in the general case) by the total number of points PT falling on the reference area (see Fig. 1). According to Glagolev (1934),

For example, if 1000 points were distributed over a representative sample of cells and if 150 of these points were on the nuclei, they would account for 15% (150/1000 = 0.15) of the total cell volume. If each point in the test system used to collect the data had an area allocation of I F 3 cm, the area density A A of nuclei would be 0.15 cm2/cm2,and the volume density Vv 0.15 cm3/cm3,according to the Delesse principle, where A A = V V . Since both numerical components VOLUME DENSITY AA *' Vvn'

Pp

(DELESSE, GLAGOLEV)

I '

' I SURFACE DENSITY

FIG. 1 . The relationship between collecting counts and calculating volume and surface densities is illustrated. The test system contains regularly arranged points P,each of which is assigned an area (diagonal lines) and two linear probes (left and top edge of each small square). The length of the linear probed (0.003 cm) is found by dividing the length of test systernD (1.5 cm) by the magnification M (X500) (see Bolender, 1974b). Two points (circled) out of 9 fall on the mitochondrial profile, and the 18 linear probes make 6 intersections (circled) with the outer mitochondrial membrane trace. These counts are used to calculate mitochondrial volume density Vvmi and outer membrane surface density SVomi,. Note: The small number of counts accommodates the figure, but not on actual analysis. See text for further details.

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25 1

of the volume density equation have identical units, whose quotient equals 1 (cm3/cm3 = l), a volume density in fact represents nothing more than a dimensionless ratio to which units may be applied arbitrarily. If, for example, a compartment represents 15% of an object’s unit volume, it will continue to account for 15% when the unit reference volume is 1 cm3, 1 mm3, or 1 pm3. When area measurements are derived from sections having a finite thickness, the sample is three-dimensional. This represents a departure from the twodimensional sampling requirement of stereological theory. Two-dimensional projected images of three dimensional sections usually contain too much information but, when some structures are hidden in the projection shadows of others or have too little contrast to be recognized, information may be lost. Generally speaking, over- or underestimates for volume density are related to (1) the staining characteristics of the components in the compartment being measured, (2) the distribution of components within the section, and (3) the average size of the components relative to that of the section thickness. A variety of corrections has been suggested for the volume density error produced by the finite section thickness (Hennig, 1957, 1969; Cahn and Nutting, 1959; Underwood, 1970; Weibel, 1976), the best known being that of Holmes (1927). Almost all these methods, however, assume a spherical model and, when applied to nonspherical organelles, are able to provide only an approximate correction. Figure 2 illustrates the “Holmes effect” for opaque spheres in a

GRANULE

L

1 I

a5 ID 1.5 2.0 2.5 SECTION THICKNESS ( T I /AVERAGE DIAMETER ( 0 ) OF SPHERE

FIG.2. The volume density correlation factor for opaque spheres Kvv, according to Holmes (1927) is plotted as a function of the relative section thickness (TID). Experimental examples are included to illustrate the effect of measuring the same organelles using section thicknesses frequently associated with light (1000 nm) and electron (40 nm) microscopy. A final estimate for volume density is obtained by multiplying the primary value by the correction factor.

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transparent matrix by plotting a volume density correction Kvv, as a function of the relative section thickness TID. Note, for example, that when the section thicknesdsphere diameter ratio = 1, the volume density of the compartment is overestimated by 60%. Increase the diameter of the same spheres by a factor of A t h e section thickness remaining unchanged-and the overestimate is reduced to 27%. In terms of a practical interpretation, this illustrates the fact that, by simply repackaging the same amount of material into larger granules, one obtaines a smaller estimate for volume density. A Holmes’ correction is usually worth considering whenever spherical organelles are undergoing size changes, or when their diameters approach that of the section thickness. However, Fig. 2 indicates that a TID ratio C0.1 produces an overestimate that amounts to only a few percent, and a correction may be unnecessary. The Holmes’ correction can be improved by also considering errors associated with the truncation effect, that is, errors produced by “lost” polar or cap sections of components (Weibel, 1976). b. Surface Density. With the use of a counting procedure somewhat similar to the one described for the volume density estimates, the surface density of a set of internal structures can be determined from two-dimensional samples taken at random from the three-dimensional object. Once again the variable, in this case surface area, is related to a unit of reference volume. Here “surface” is defined as an interface between an internal structure and its surrounding matrix. Such a definition for surface has been applied to biological membranes even though these structures display two surfaces which define an intervening volume. Whenever interfaces or two-dimensional membranes are sectioned, they can be identified in projected images as linear traces. The surface density Svi of a component i can be determined by applying a test system containing lines to micrographs of sections derived from an object and then counting the intersections between the test system lines and the underlying traces; planimetry by point counting determines the reference area. The surface density is calculated by dividing twice the number of intersections Zi with a component by the total length of the test line LT,according to Tomkeieff (1 945). The surface denisty equation is based on the fact that the length of the traces within a given area (i.e., an area density of traces, or boundary length within a reference area, B A ) is proportional to the three-dimensional surface area of the interface within the reference volume. The equation of Saltikov (1 958) defines this relationship: BA

The area density of traces B A is also proportional to the number of intersections the traces make with a test line of known length L T , according to Smith and Guttman (1953) and Saltikov (1958):

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By combining Eq. (2) and (3) and solving for the surface density SVa relationship is established between the traces identified in the two-dimensional samples and their intersections with the test lines (linear probes):

sv

21 =LT

(4)

Additional derivations for this formula are given by Duffin et al. (1953), Hennig (1956), Saltikov (1958), and Smith and Guttman (1953). Figure 1 illustrates a surface density calculation. Errors associated with surface density estimates are discussed in Section II,C,4. c. Length Density. The length density Lv is defined as the total length of randomly oriented lines per unit of reference volume. It can be calculated with the equation Ly = 2PA

where Lv is equal to twice the number of points representing intersections of the lines with the test plane-per unit of reference area P A . Derivations for this equation have been given by Saltikov (1958), Smith and Guttman (1953), and Underwood (1970). The equation is particularly useful in that it can also be used to estimate the length of tubular structures such as capillaries and microtubules. If the lines are randomly oriented, and if their cut ends are readily seen, section compression rather than section thickness (Hennig, 1963) represents the largest potential source of error when estimating length densities. d. Numerical Density. Calculations of numerical densities for biological structures rely on procedures far less satisfactory than those previously described for volumes, surfaces, and lengths. This stereological estimate requires information about shapes and size frequency distributions and assumes that the biological components being quantitated can be likened to geometrically defined models. Uncertainties associated with fitting generally nonconforming biological shapes to ideal spheres, ellipsoids, or cylinders can account for major sources of error in making numerical density estimates (Fig. 3). Several methods are available for determining numbers of biological components (reviewed by Underwood, 1970), but two approaches are generally followed when using sectioned material. The first, numerical density N v , estimates the number of components per unit volume directly, whereas the second first estimates the diameter or volume of an average geometric structure. In the latter case, the numerical density is found secondarily by dividing a compartmental volume density by the volume of the average structure. The second approach can provide the size frequency distribution of the diameters (for spheres or spherically reduced ellipsoids usually), and this

R. P.BOLENDER

254

2: I

1.4 I

I: I

b d

100

0.4

0.5 0:6

O:?

Ole

0:9

Ib

% FIG.3. Numerical density errors are produced by assuming ellipsoidal particles are spheres. When the ratio of the major (a) to the minor (b) elliptical axis is 2, for example, the use of a sphere correction factor @ = 1.382) instead of one characterizing the ellipsoids, produces a 15% overestimate for the numerical density. Changes in particle size frequency distributions, section thickness, and lost “cap” sections can contribute errors as well.

additional information can be useful for describing changes in organelle packaging. Corrections for section thickness errors can be introduced as described by Baudhuin (1968), Baudhuin and Berthet (1967), Floderus (1944), and Wibo et al. (1971). More often than not, changes in the numbers of biological components per unit volume are accompanied by changes in both their shape and size frequency distributions, while at the same time the numerical density reference volume itself can be affected by cellular changes (see Section II,B,l). Since an estimate for numerical density may require a consideration of several probable sources of error, many of which require assumptions difficult to confirm, the use of this method may involve a certain degree of uncertainty. Let us look at a few examples. When estimating the number of mitochondria by likening them to cylinders, an assumption is made that these organelles do not have reentrant shapes; that is, a given mitochondrion is represented by a single profile in a section. If, however, a mitochondrion does not conform to the assumption and instead bends or branches (see Fig. 4),a single organelle can be represented by more than one profile per section. This results in an overestimate for numerical density. Another problem arises when similar assumptions are applied to different sets of conditions. Average nuclear and cell volumes calculated from numerical densities that assume sphericity for nonspherical nuclei contain one error, but as soon as this assumption is applied uniformly to different experimental groups-each having

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FIG. 4. Hepatocytic mitochondria displaying bending. Arrows indicate the potential number completely separate profiles that can be obtained from a single mitochondrion sectioned perpendicular to the plane of the micrograph. Such “reentrant” mitochondria produce numerical density overestimates. X6000. (From Bolender et a l . , 1978.) (two) of

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unique degrees of nonsphericity-additional errors are incurred. The effect of these unknown errors on estimates for average cell values can be substantial.

B. STEREOLOGICAL REFERENCESYSTEMS 1 . Standard Unit of Reference Volume

Stereological densities relate a variable parameter (volume, surface, length, or number) to a standard unit of volume, usually 1 pm3 or 1 cm3. Although this type of reference volume is entirely suitable for baseline studies, it may become considerably less interpretable when used to describe changes. But what is meant by “change”? Characteristic of the biological standard unit reference system is the fact that, although it can detect change, it cannot identify its source. At least two factors, or some combination thereof, can produce a change in the cubic unit of reference volume: (1) a change in cellular organelles, or (2) a change in the number of cells per standard unit. Unfortunately, such interpretation difficulties are not necessarily limited to morphology. The standard unit volume of stereological data is quite similar to the standard unit weight of biochemical data (e.g., for rat liver 1 cm3 = 1.067 gm), and they both share the above ambiguities when used as references for detecting changes. It is particularly important to emphasize that, when one is interested in describing changes relative to cells, the use of a standard unit of weight or volume may or may not be an appropriate reference. Volume densities are uniquely difficult to interpret because they characterize a unit of reference volume as a percent composition. This means that a change in the relative volume of any one compartment produces an opposite change in the relative volumes of the remaining compartments. Figure 5 illustrates how very

Fic;. 5. Volume densities can be ambiguous when used to describe changes in cells, In the top row the nuclei (spheres) within the cubic units of reference volume actually increase in volume, whereas those in the bottom row maintain a constant volume and the number of cells packed into the same cubic unit increases from one to two to four. Although the cellular events producing the changes are quite different, the volume densities for both rows are identical. Similar ambiguities exist for surface, length, and number densities. Figures 6 and 7 continue the example.

CORRELATION OF STEREOLOGY AND BIOCHEMISTRY a2

0

NUCLEUS

257

I

I;

CM3 a5 00

FIG. 6 . Volume density ambiguities. No relative change can be the result of an absolute no change or change. Here the cube represents a cell and the sphere its nucleus. An analysis of point-counting data characterizing the two different situations in panels 2 and 3 would provide volume density estimates for the nuclear compartment that indicate no change in relative volume (panel 1). The ambiguity of these data results from the fact that at least two cellular events can be responsible for this finding. Relative (VVJ and absolute (cm3) changes in volume are used in the models to illustrate this point. In panels 1 and 2 both the relative and absolute changes are the same, whereas in panels 1 and 3 they are not. In panel 3 the relative volume of the nucleus remains constant only because its volume and that of the cell decrease proportionately to maintain the relative 10% nuclear volume.

different changes can produce identical volume densities, and Figs. 6-8 show the difficulties one can encounter when attempting to explain a change using volume densities.

2 . Average Cell Reference Volume A change may be somewhat easier to interpret when it is related to a cell rather than to an unknown number of cells which, as just shown, characterizes stereological densities. Furthermore, the cell is a convenient reference because it represents a biological unit of both structure and function. Since stereological data describe a cell population statistically, it is possible to relate the densities to a cell having an average volume. This average cell volume reference may be obtained from numerical densities of nuclei (Loud, 1968; Weibel et a l . , 1969) or from calculations of average nuclear volume (Bolender

R. P. BOLENDER

25 8

VV" 0.1

0.0

M3[:

ao

FIG.7. Volume density ambiguities. A relative change can be the result of an absolute change or no change. The observed volume densities (panel I ) indicate that the relative nuclear volume has increased from 5 to 20% of the cell's volume. Panels 1 and 2 illustrate how both relative V V , and absolute (cm3) changes can be the same, and panels I and 3 show how they can be different; both absolute events (panels 2 and 3), however, produce the identical relative estimates seen in panel 1. In panel 3, no absolute change in nuclear volume is detected as a relative change.

and Weibel, 1973; Bolender, 1974b). The Giger and Reidwyl(l970) or Wicksell (1925) procedures can be used to estimate this average nuclear volume, which in turn is used to calculate the average cell values. An average cell volume can be calculated simply from the average nuclear volume and the volume density of nuclei in the cells Vvnc:

vc

v,,

where

The volume of a compartment i per average cell volume ViiC

=

vevvi,c

v, becomes (7)

259

CORRELATION OF STEREOLOGY AND BIOCHEMISTRY

0.9 CYTCeLASMC MATRIX

0.7

\

8 04 2

I 0.4

I

1 1

u w

0.25

1

O.' 00

0.00

" B E R & CELLS PER C@

FIG. 8. Interpretation of relative volume density changes: the effect of changing the number of cells packed into a standard unit of reference volume ( 1 cm3). Different numbers of cells, identical with respect to their absolute nuclear and mitochondrial volumes but having variable cytoplasmic matrix volumes, are combined to give 1 cm3 of volume. Here, again, relative results can be misleading. In this example, the increased volume densities are not the result of an increase in nuclear or mitochondrial volume per cell (these remain exactly the same throughout) but reflect instead relative increases in compartmental volumes of these components as they relate to the l-cm3 reference. The primary event responsible for these relative changes can be traced to a decrease in the absolute volume of the cytoplasmic matrix. Notice, for example, that the apparent nuclear volume changes are greater than those of the mitochondrial compartment. Being larger to begin with, it assumes a proportionately larger percentage of the cell as the cytoplasmic matrix volume decreases. This illustration also attempts to emphasize the fact that a relative change in one volume compartment (e.g., the cytoplasmic matrix) is reflected in changes in all the remaining compartments.

and the surface of a compartment i per cell:

Although this appears to be an ideal reference system for studying cellular changes, in practice it is often difficult to obtain. The stereological methods used to calculate nuclear numerical densities and average nuclear volumes assume that the shape of the nuclei can be likened to a geometrically defined model, usually a sphere. This requirement places an unfortunate restriction on the use of this reference system, because most nuclei are pleomorphic and many cells contain more than one nucleus. The problem is further compounded in that the distribution and shapes of nuclei frequently change during an experiment. Shape and frequency distribution aside, the fact remains that the task of measuring nuclear profiles-ither directly or by fitting circles to their irregular outlines-invites a

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R. P. BOLENDER

certain degree of error. If one considers that an average nuclear diameter underestimated by as little as 10% leads to about a 30% underestimate for the average cell volume, the extent to which this reference system depends upon the original diameter estimate becomes more apparent (see Section II,C,4). One might suspect that a solution to this problem is to obtain an average cell reference without having to rely upon nuclear shapes and size distributions. One approach currently being considered is to use reference areas instead of volumes as the average cell reference (Bolender, 1977; Schofield et a f . , 1977). This method as it applies to membrane changes is described in the following section.

3 . Average Cell Reference Area A cell contains a total membrane compartment subdivided into individual compartments identified as specific organelles. The surface area ratio method assumes that, during the course of an experiment, the surface area of some membrane compartments changes while that of others remains constant. If the ones remaining constant can be identified, they can be used as a reference for the changing compartments. The method has two desirable features: First, it can be used to detect changes in cell membranes without having to know the average cell volume or the number of cells per cubic centimeter, and, second, it can be applied as a test to determine how well changes in surface densities reflect changes in average cells (Schofield et a l . , 1977). The method consists of calculating control and experimental ratios, which are subsequently compared: Control surface ratio:

The ratio SR of the surface of a membrane i to the surface of reference membrane of the control (C) is calculated simply from intersection counts Zi and Ireftaken from the same electron micrographs. The resulting ratio indicates the amount of surface area of membrane i relative to that of the reference membrane. Experimental surface ratio:

This ratio indicates the surface area of a membrane i relative to the area of the reference surface under experimental conditions. The percent change in membrane i associated with the experimental situation is obtained by comparing the two ratios. Equations (9) and (10) can be combined to give a single expression:

CORRELATION OF STEREOLOGY AND BIOCHEMISTRY SURFACE DENSITY

CDNT6‘OL

EXPTL

26 1

SURFACE RATIO

CONTROL

EXPTL

FIG. 9. Relative changes in parietal cell membranes determined with the surface density and surface ratio methods. Control membrane surface areas are compared to the membrane distributions seen in animals secreting H+ at a maximal rate. Plasma membranes are described as “free surface” when at the cell apex and “lateral-basal” when at the sides and base of the cell; the intracellular membranes are identified as the Golgi-tubulovesicular compartment. The dashed line assumes no change in mitochondria membrane surface area; see text for details. (Adapted from Schofield et al., 1977,)

A result equal to I indicates no change in the surface area of membrane i , greater than 1 indicates an increase, and less than 1 a decrease. Figure 9 illustrates the relative changes in membrane surface areas calculated with both the surface density and surface ratio methods. The ratio method in this example assumes that the actual surface area of the outer mitochondrial membrane compartment in an average cell is the same for both control and experimental animals. The results indicate that the two different methods detect very similar membrane changes. Such a result would be expected if (1) the outer mitochondrial membranes were in fact a stable reference and (2) if the number of cells per cubic centimeter remained essentially unchanged. Finally, if one were to adjust the estimates for the experimental surface densities so that the mitochondria1 membranes indicated no change (this would account for the apparent presence of a slight cellular swelling), the two sets of curves would become superimposed. Such agreement suggests that both methods detect changes relative to average cells. However, does this experimental finding contradict the earlier arguments that have attempted to disqualify surface density comparisons as a means for detecting membrane changes? Not at all. In this particular experiment, the ratio method supplied supporting evidence that the observed surface density changes were not being influenced to any great extent by changes in the number of cells

262

R. P. BOLENDER

per cubic centimeter. Quite the reverse was found in another study (Bolender, 1977). Changes in exocrine cell membranes observed with the ratio method were compared first to those based on the volume of an average cell (see Section 11,B,2); the difference between the two estimates averaged less than 6%. However, when compared to results from the ratio method, surface densities related to 1 cm3 of exocrine cell cytoplasm or to 1 cm3 of pancreatic tissue showed average differences of 9 and 15%; individual diflerences were as high as 47%. The greater variations in membrane changes detected when comparing surface deiisities could be explained in this experiment by changes in the number of cells filling the l-cm3 reference volume (see Section II,B, 1). 4. Analytical Models

Studies involving morphological kinetics can be interpreted within the framework of analytical models (Bolender, 1974a,b; Weibel et a / . , 1969) (see Fig. 10). In one type of data analysis, for example, the total surface area of the membranes found within an average cell is used as the reference instead of the average cell volume. A balance sheet is constructed and used to detect changes in the total and individual membrane compartments (organelles), as well as to describe movements of membrane from one compartment to another. Figure 11 illustrates how the surface area of the total membrane compartment ST associated

I&

NOW-EXOCRINE

IEXOCRINE CKLLS IExCI

IIITCCHOUDRIA

INTERSTITIAL OUCT L u m n B L W D VESSEL LUMINA

(nil

ZmOGEN GRANULES [ZGI

wopmsnxc

IUTRIX wynd GROUND SUBSTANCE LIPID DROPLETS LY80SCuES

FIG. 10. Stereological model for the guinea pig pancreas. The pancreas is divided into nonexocrine [cells (EExC) and spaces (EExS)] and exocrine (ExC) components. The cytoplasm of the exocrine cells (ExCCy) is then further subdivided into spaces and membranes. (From Bolender, 1974b.)

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263

FIG. 11. An average exocrine cell characterized by organelle volumes and surfaces. The total cell volume VT'is subdivided into organelle volumes including those of the cytoplasmic matrix (CyM), zymogen granules (ZG), condensing vacuoles (CoV), intracisternal granules (IG), rough-surfaced endoplasmic reticulum (RER), mitochondria (Mi), and nucleus (N). The total surface area of the cell membranes ST is subdivided into surfaces associated with individual organelle compartments: plasma membrane (PM), zymogen granules (ZG), Golgi apparatus (GA), condensing vacuoles (CoV), rough-surfaced ER (RER), mitochondria (Mi), and plasma membrane vesicles (PMV). (Adapted from Bolender, 1974b.)

with an average exocrine cell-as well as its total cell volumeV,+an be divided into individual organelle subcompartments. These control values may be thought of as representing the first frame in a series of quantitated pictures that can be generated stereologically to visualize morphological changes in membranes as a function of time.

C . PRACTICAL PROCEDURES 1 . Electron Microscopy A fixation and embedding procedure is chosen to ensure that the morphological components can be readily detected and identified. For studying membranes in intact tissue, osmium tetroxide fixation in a phosphate buffer (Bolender and Weibel, 1973; Bolender, 1974b) can fulfill these requirements satisfactorily, whereas brief fixation with glutaraldehyde in a cacodylate buffer followed by cacodylate-buffered osmium tetroxide has been described for cellular fractions (Deter, 1973; Baudhuin, 1974; Fleischer and Kervina, 1974; Bolen-

264

R. P. BOLENDER

der et a l . , 1977). In both cases, en bloc staining with uranyl acetate improves not only the membrane contrast, but also stains the membranes throughout the section thickness, a requirement for applying the section thickness corrections of Wibo (1971) and Weibel and Paumgartner (1978). An additional advantage of the uranyl acetate procedure is that it extracts material from the cytoplasmic "background," thereby further increasing the contrast of the membranes. In some instances, however, extraction may produce undesirable results. Cytoplasmic granules in cell fractions may lose part or all of their contents during fixation and become unidentifiable unless they are pretreated cytochemically to retain their contents (Baggiolini, 1972). Cytochemical methods have also proved to be most useful for identifying morphological components in fractions (Baudhuin et al., 1967; Leighton et al., 1968; Bolender, 1974a; Bolender et al., 1977). Microsomal membranes can be identified stereologically using freeze-fracture replicas (Losa et a l . , 1975). Electron micrographs used for a stereological analysis can be printed on 35- or 70-mm film and then projected onto frosted-glass screens containing test systems (Weibel, 1972) or simply recorded on photographic paper. Both approaches work well, but errors may be introduced by stretching the photographic paper during the drying procedure. Such an error can be avoided by printing the test system directly on the micrograph (Loud, 1968). 2 . Stereological Test Systems Whenever a stereological study is undertaken, test systems are chosen or designed to accommodate the sampling requirements of the problem. Stereological test systems can measure (1) areas from point counts which are subsequently used for volume density calculations, (2) boundary densities from intersections for calculating surface densities, (3) numbers of profiles for calculating length and numerical densities, and (4) the size of the reference areas to which all these measurements are related (see Fig. 1). Although methods for fitting test systems to micrographs of biological material are still largely empirical, the arrangement of the test probes depends upon several characteristics of the component profiles: (1) their magnification, (2) their relative sizes, and (3) their frequency and distribution within the reference areas. a. Magnification of Component Profiles. When three-dimensional structures are sectioned, they present many different profiles that must be detected and identified before they can be measured. A magnification is chosen to accommodate this primary requirement. Tangential sections of particles (granules) and sheets (membranes) introduce detection difficulties because the profiles frequently have a density similar to that of the surrounding cytoplasm and are effectively lost. Moreover, when these marginally visible profiles are detected, they may contain too little information to be identified. These problems can be minimized by keeping the sections as thin as possible, using a fixation procedure

CORRELATION OF STEREOLOGY AND BIOCHEMISTRY

265

that extracts some of the soluble proteins of the cytoplasm (this increases profile contrast), and by learning to recognize “difficult” profiles. Since a micrograph magnification is inversely proportional sampling area, it should be kept as low as possible to minimize the number of micrographs needed for an analysis. b. Size of Individual Component Profiles. The point and linear probes of a test system are used to measure profiles of sectioned structures. As the number of probes per unit of test area is increased (the sample magnification remaining constant), the precision in measuring the individual profiles in a micrograph also increases. At some point, however, an optimal relationship is obtained between the test probes and the profiles, and further precision becomes unnecessary. Hilliard and Cahn (1961) showed that the maximum efficiency for determining volume densities is obtained when the majority of the profiles contain a single test point. In practice, the minimum distance between adjacent test points is chosen to be about equal to the mean profile diameter when the component occurs frequently (Vv 3 lo%), and about one-half the mean profile diameter when the component is rare (Vv G 10%). Weibel (1969) has described a method for adjusting volume density test systems based on profile and test probe areas. Fitting test systems to profile traces for estimating surface densities is less well defined. Hilliard (1965) has indicated that the total number of intersection counts needed to obtain a given accuracy can be calculated from a preliminary estimate of surface density and variance cr:

IT -- 0.4(-) S”

2

c+SV

This equation presupposes that a correct relationship has already been established between the density of the linear probes and the underlying profile traces; note that a separate calculation for IT is required for each class of membrane traces. However, the lack of a definition for this relationship between probe and profile densities has created some problems. For example, if one collects surface density data from the same section at two well separated magnifications (the test system remaining constant), the estimate is sometimes larger at the higher magnification. It is not clear, for example, whether this larger estimate results from the fact that one is simply able to identify more of the structures at a higher magnification because of the increased resolution, or if it has something to do with the design of the test system as well. Keller et a l . (1976) estimated volume and surface densities of the same tissue at different magnifications using light and electron microscopy. As the magnification was increased, estimates of volume density decreased, whereas those of surface density increased. These workers concluded that stereological estimates were resolution-dependent. It might be noted, however, that these comparisons were based on raw data uncorrected for variable errors related to section thickness and compression.

266

R. P. BOLENDER

Since the relationships between probe and trace densities remain undefined, an empirical method has been used in an attempt to minimize the resolution errors while at the same time maintaining counting efficiency (Bolender, 1974b). A magnification is chosen at which membrane traces in an electron micrograph can be easily identified, and intersections are counted with a test system containing an excessive number of linear probes. The density of these probes is reduced stepwise as long as the reduction does not affect the surface density estimates. c. Frequency and Distribution of Component Profiles. If a component represents more than 10% of the reference volume and is distributed more or less uniformly, test systems can be designed as already outlined. However, when a component is rare or clustered, Weibel (1969) has suggested that the area of the test probe should be equal to or smaller than the average profile area to ensure that the majority of these profiles will be measured. Since biological material is characterized by components having a broad range of sizes and distributions, a single test system is usually not sufficient for measuring all the profiles encountered at a given magnification. A very satisfactory solution to this problem has been given by Weibel (1969). By using a double lattice, one of which is illustrated in Fig. 12, he accommodated two different ranges of profile sizes at the same time. Essentially, the test probe consists of large squares subdivided into smaller ones. The test systems are coherent, because a large square consists of an integral number of smaller ones. This characteristic of test system coherency provides considerable sampling flexibility. For example, when dealing with small, rare components, which usually occur in fewer than 10% of the small test square;;., a temporary increase in measuring precision may be required. In practice, however, it is inconvenient to

FIG. 12. A coherent test system illustrating the use of a movable test module. See text for details.

CORRELATION OF STEREOLOGY AND BIOCHEMISTRY

267

exchange one test system for another and, when the entire test system contains a dense array of lines, it is very unpleasant to use. A solution to this problem is to redesign a single large square having the appropriate number of smaller squares that can be moved freely about the stationary test system. The movable test module is simply aligned with each large square containing the rare components, and measurements are collected. The module data are related to the larger test system using the principle of coherency. An example of the procedure is given in Fig. 12. In this way, by intermittently increasing the measurement precision, one can reduce quite considerably the number of micrographs required to reach an acceptable sample size for a rare component.

3. Sampling for Stereology Stereological sampling is evaluated statistically, and standard methods can be used for making specific arguments when interpreting data. As far as sampling is concerned, one is usually interested first in how well a stereological estimate characterizes a parameter in a given animal and then how well the animal data characterize the animal population. The number of micrographs to be included within a unit statistical sample depends on the size and distribution of the components in the compartment being

al $1 b

W

i

OJ-

ii

- 36 r

n

NUMBER OF MICROGRAPHS

FIG. 13. The cumulative standard errors for the surface areasS of membranes per gram identified in the rat liver are illustrated. (Adapted from Bolender et al. 1978.)

268

CORRELATION OF STEREOLOGY AND BIOCHEMISTRY

quantitated. By changing the number of micrographs per unit sample, that is, by grouping data from two, three, four, five, or more micrographs, and then calculating a cumulative standard error using the grouped data, a reasonably good estimate can be obtained for a given animal variation. A plateauing standard error (S10% of the mean) has been used to indicate sampling adequacy, but such sampling precision may be impractical for certain rare organelles, and quite unnecessary for detecting large changes. In applying this type of sampling SURFACE DENSITY OF RER 4 ANIMALS

3 ANIMALS

u

0

WL

2 ANIMALS

no,

+++

FIG. 14. Control data from five animals describing the surface density of the RER of pancreatic exocrine cells. The complete data set (60micrographs for each of five animals) is used to calculate a mean value for the RER (dark line at 100%) and its standard error (dashed line at ?6%). Cornbinations of animals taken four, three, and two at a time from the group of five animals and 20,40, and 10 micrographs per animal are used to calculate mean and standard errors expressed as a percent of the complete data mean. The point of this analysis is to observe flucuations in the surface density estimates associated with a decrease in the number of animals and the number of micrographs per animal. When four animals are used, all the means for the different animal combinations remain relatively close to that of the complete data set. However, these control data show considerable variation when fewer than four animals are used, and significant differences between estimates may occur when only two animals are used to estimate the surface density ($ = p < .01; 4 = p < ,025; 3 = p <.05). (Adapted from Bolender, 1974b.)

CORRELATION OF STEREOLOGY AND BIOCHEMISTRY

269

analysis to several membrane systems (Blouin ef a l . , 1977; Bolender et al., 1978; Bolender, IY/7), the 10% level was often required to obtain sought-after recoveries and references. Figure 13 illustrates a cumulative standard error calculation. How many animals should be sampled? Figure 14 suggests that, for the surface density of the ER in pancreatic exocrine cells, the smallest number of animals needed to characterize an animal population is probably no fewer than four and preferably five or more. It should be emphasized that the number of animals required is influenced by the population variation, the required precision, and the method of sampling and analysis. Sampling analyses based on cumulative means have been described by Chalkley (1 943) and Burri et al. (1968). 4. Corrections for Sample Artifacts

Whenever stereological measurements are collected from sections having a finite thickness, instead of the usually required two-dimensional ones, the resulting density estimates are systematically altered. Furthermore, the fixation and embedding methods may also compromise stereological estimates by changing the volume of the original tissue or by introducing morphological artifacts. The growing interest in using stereology to integrate morphological and biochemical information seems to indicate that more attention should be given to these errors of measurement. a. Section Compression. When blocks containing biological material are cut for transmission electron microscopy, the surface area of the resulting section is often smaller than that of the original block face; the section is compressed. When a portion of such a section is projected onto a microscope film, the image may contain more information per unit area than found in a noncompressed section. Loud et al. (1965) reported that the amount of compression in a section can be estimated by comparing the average diameters of randomly oriented profiles measured parallel (d,) and transverse (dt) to the direction of cutting. A section area correction factor A , for compression is simply the ratio

This correction factor might be expected to improve length and numerical density estimates, but in the latter case the change in profile shape produced by the compression remains. One is still faced with the problem of how to measure compressed profiles for numerical density and average diameter estimates. If it can be safely assumed that the profiles actually came from spheres as, for example, in the case for rat liver nuclei, the maximum profile diameter may be the most suitable measurement. If, instead, an elliptical profile (compressed circle) is spherically reduced (corrected to a circle of equal area), an underestimate for an average diameter will be expected. Loud et at. (1965) indicate that

R. P. BOLENDER

270

the effect of compression on the lines (membrane traces) in a micrograph is to shorten their length when oriented parallel to the direction of cutting. This effect is likened to the reduction in the perimeter of a circle foreshortened by compression into an ellipse. They evaluated this change by elliptic integrals and prepared a correction factor L,. b. Section Thickness. Surface density estimates taken from thick sections, such as those used for transmission electron microscopy, contain a broad range of overestimates depending on the size and configuration of the membrane organelles (Underwood, 1970; Miles, 1976; Weibel, 1976; Blouin et a/., 1977; Bolender et al., 1978). Membranes estimated from intact tissue have one set of correction factors, but the same membranes after tissue homogenization can assume new shapes and sizes with a new set of corrections. Wibo et al. (1971) have described section thickness correction factors for microsomal membranes, and Weibel (1976) and Wiebel and Paumgartner (1978) have reported correction factors for intact tissue and the fractions described in Fig. 15; methods for estimating section thickness have been given by Williams and Meek (1966), Small (1968), Silverman er al. (1969), and Gillis and Wibo (1971). c. Fixation and Embedding. As discussed in Section II,B,l, stereological densities can be influenced by changes in their reference volumes. Specimen shrinkage usually results in overestimated densities, whereas swelling has the opposite effect. Both fixation (Weibel, 1963; Loud et a l . , 1965) and embedding (Luft, 1973) can change the volume of the original fresh specimen. Consequently, one cannot

LIVER

&

ExTR HOMOG .

NUCLEAR

HEAVY MITOCH.

LIGHT MITOCH.

SUPT.

MICROSOMAL

FIG. 15. Differential fractionation of rat liver (after de Duve et al., 1955). See text for details.

CORRELATION OF STEREOLOGY AND BIOCHEMISTRY

27 1

safely assume that a cubic centimeter of tissue characterized stereologically is equivalent to a cubic centimeter used for a biochemical assay. The problem here is a difficult one to generalize, in that morphological volume artifacts can be produced by virtually all the solutions used for specimen preparation. Although maintaining standard conditions for control and experimental samples is helpful in making relative morphological comparisons, integrating stereological and biochemical information requires that the reference volumes (e.g., cubic centimeters) be more correctly matched.

111. The Technique of Differential Centrifugation and Marker Enzyme

Analysis A. GENERAL CONSIDERATIONS

In his early attempts to study the function of cells, Claude (1946a,b) recognized the importance of isolating subcellular components as a first step in determining both their biochemical composition and biological activities. Furthermore, he realized that the interpretation of biochemical data had to be performed within an analytical framework, a reference system that would allow the isolated data of fractions to be related to the original material. In developing methods for analytical centrifugation, which consisted of breaking mammalian liver cells by homogenization and then stepwise isolation of subcellular components into fractions by applying different centrifugal forces, he emphasized the importance of expressing the enzymic activities found in each fraction in terms of the total activity exhibited by the unfractionated homogenate. By following this procedure, he calculated biochemical recoveries that were expected to approach 100% when the combined enzyme activities of the fractions were equivalent to the homogenate, the original material. When recoveries did not approximate loo%, he looked for technical errors or artifactual changes in enzyme activity. From the early studies of Claude (1946a,b), de Duve et al. (1955), and others, it became apparent that certain enzymes or groups of enzymes were concentrated predominantly within a single fraction, presumably because of their association with a single subcellular component. This observation led de Duve to propose that enzymes could be used as markers to indicate the presence of specific morphological components. This concept of marker enzymes is based on two postulates: (1) a marker enzyme has a single morphological location, and (2) a marker enzyme is uniformly distributed at a given location, In recent years, however, numerous examples of marker enzymes not obeying these postulates have been reported (Baudhuin et a l . , 1964; Dallner and Ernster, 1968; Farquhar et a l . , 1974; Glaumann and Dallner, 1970; Holtzman et a l . , 1970; Widnell,

272

R. P. BOLENDER

1972). Unfortunately, as soon as an enzyme distribution is found to be heterogeneous, interpretations based on assumptions of homogeneity may become somewhat problematic. If, for example, a membrane marker enzyme is heterogeneously distributed, it becomes difficult to extrapolate the data from a single fraction to that of the intact starting material, or to use enzymic data to correct a fraction for contaminating membranes. If specific membranes are in fact structurally and functionally heterogeneous and not merely apparently so because of contaminations or artifacts, an understanding of membrane phenomena may depend ever increasingly on an ability to identify and quantitate such heterogeneities. B. PRACTICAL PROCEDURES Several reviews have already dealt with the theoretical basis (de Duve, 1964; Deter, 1973) and practical applications of differential centrifugation (de Duve, 1971; Fleischer and Kervina, 1974), and these efforts are not duplicated here. Instead, an example of the fractionation method of de Duve et al. (1955) and Amar-Costesec et al. (1974) is used to illustrate how biochemical data are collected from liver fractions and subsequently interpreted. A brief introduction to these biochemical methods and a consideration of the data they produce serves to define the analytical framework of these methods. As discussed in Section IV,C, it is with this framework that morphological data seem to require a certain degree of compatibility when one is in pursuit of integratable relationships. 1 . Fractionation

A liver fractionation scheme is illustrated in Fig. 15. An adult rat is fasted for about 16 hours to deplete its glycogen which might otherwise interfere with the separation of organelles. The liver is then removed and immersed in a tared beaker containing an ice-cold 0.25 M sucrose solution to determine its weight. It is then homogenized, and six different fractions are collected by differential centrifugation (de Duve et a l . , 1955). These include the extract (E), nucleus (N), heavy mitochondria1 (M),light mitochondria1 (L), microsomal ( P ) , and supernatant (S). Together the extract and nuclear fractions equal the homogenate @ =E N) as well as the final five fractions (H = N + M L P S).

+

+ + +

2. Biochemical Recoveries In the analytical fractionation scheme illustrated in Fig. 15, the biochemical activity of a marker enzyme or chemical constituent (e.g., protein or phospholipid) found in the original material (homogenate) is compared with the sum of the activities found in the five fractions produced by the differential centrifugation. By dividing the total enzymic activity in the five fractions (N + M L P + S ) by that of the homogenate (E + N), one obtains a biochemical recovery,

+ +

CORRELATION OF STEREOLOGY AND BIOCHEMISTRY

273

which is expected to be 100%. Deviations either above or below this expected value may indicate changes either in enzyme activity resulting from the fractionation or assay procedure, or simply technical errors. The importance of calculating recoveries is that they can provide the information needed to define the limits within which data from a single or several fractions can be interpreted. Thoughtful evaluations of these analytical procedures have been given in reviews by de Duve and Berthet (1954), de Duve (1971), Baudhuin (1974), Deter (1973), and Fleischer and Kervina ( 1974).

3 . Biochemical Reference Systems Marker enzyme activities are frequently related to 1 gm of liver, to an associated chemical constituent (e.g., organelle protein or phospholipid), or to the total protein or phospholipid content of a fraction. a. One Gram of the Original Material. A biochemical constituent in a fraction can be related back to 1 gm of liver if the volume of the aliquot used for an assay and the various dilutions of the original tissue are known. But what does this gram of liver represent, and how can it be used as a reference for detecting changes? According to Blouin et a l . (1977), a rat liver consists of about 80% hepatocytes, 10% other cell types, and 10% vascular spaces. Consequently, when relating the activity of a marker enzyme associated with hepatocytes to I gm of liver, the reference system represents hepatocytes plus other cell types and vascular spaces. These additional compartments constitute the hepatocytic contaminations. If one recalls the discussion of the difficulties associated with interpreting morphological changes related to 1 cm3 of tissue (Section II,B,l), one can readily appreciate the fact that the interpretation of biochemical data using 1 gm of liver as a reference weight may be compromised by similar ambiguities. Since these biochemical data are a function of both a specific constituent and its reference system, they can be influenced by changes in either the constituent or in the number of cells per gram, or of course changes in both. b. Milligrams Protein or Milligrams of Phospholipid. Protein and phospholipid are constituents of organelles and membranes and are used frequently as reference systems for marker enzyme activities (Beaufay et a l . , 1974; de Duve, 1971; Glaumann and Dallner, 1968). The use of this reference quite often requires several purification steps, and the resulting information is subject to arguments associated with the suitability of the purification procedures. Contaminations, it is thought, consist of extraneous material that was not separated out during the purification and of foreign constituents adsorbed to the purified material. The extent of these contaminations is routinely monitored morphologically with the electron microscope and biochemically with marker enzymes, radioactive labels, and so on, but the interpretations may be problematic. In any case, the resultant biochemical data continue to be a function of both an enzyme activity and the total protein or phospholipid content of a fraction.

274

R. P. BOLENDER

c. Relative Specific Activity. A relative specific activity (RSA) plot relates the percent total marker enzyme activity to the percent total reference (Fig. 16); additional examples of this type of data presentation are given in reviews by de Duve (1971), Baudhuin (1974), and Deter (1973). Each fraction in the histogram is characterized by a rectangle; its width depends on the amount of reference material in the fraction, whereas its height (RSA) is a function of both enzyme activity and reference material. RSA indicates the extent to which a biochemical constituent has been concentrated above what is observed in the homgenate. At the risk of appearing repetitious, it is pointed out once again that the resultant data are a function of both the constituent activity and the “content” of the reference system. An increase in RSA, for example, can be produced by either a decrease in the protein content of a fraction or by an increase in enzyme activity. The point of the foregoing discussion of biochemical reference systems for enzyme activities has been to suggest that difficulties not unlike those of the morphological references can arise when attempting to interpret biochemical changes. If in fact changes-be they morphological or biochemical-are potentially a function of two variables, component or constituent and reference, difficulties in interpretation and integration are to be expected. If to study membranes undergoing changes ultimately involves dealing with multivariate organelle heterogeneities themselves influenced by contaminations

‘= I 5 14 0-

k; $

1.

c*

P

S

CORRELATION OF STEREOLOGY AND BIOCHEMISTRY

275

and unstable reference parameters, the question how one can deal with all this on-going complexity becomes an important one. Analysis systems based on integrated morphological and biochemical data may eventually provide a means for obtaining at least partial solutions to some of these problems, but this requires approaches capable of linking the two types of data together. Attempts to effect such a linkage are now discussed.

IV. The Integration of Stereological and Biochemical Data A. STEREOLOGY OF INTACT TISSUECORRELATED WITH BIOCHEMISTRY OF FRACTIONS Loud (1966, 1968) developed the first morphometric model for the rat liver and chose as his reference system the volume of cytoplasm associated with an average mononucleate hepatocyte. This was a particularly important step in that stereological data usually related to a geometric unit (per unit volume) could now be interpreted in terms of a biological unit, namely, the volume of a cell. In principle, the average cell reference is an excellent idea but, when applied to the liver, where cells can be both polyploid and multinucleate, the reference becomes somewhat less interpretable because a change can be produced by either a cytoplasmic event or simply a change in the distribution patterns of the hepatocytic nuclei. [See Carribre (1969) for a discussion of these nuclear variations in young and adult livers.] The “average cell” model was used in combination with biochemistry to consider the effects of cortisone treatment on rat liver mitochondria with respect to oxygen consumption, oxidative phosphorylation, and fine structure by Wiener et al. (1968). These investigators reported that cortisone was responsible for a 14-20% decrease in oxygen consumption per milligram of mitochondrial protein, that oxidative phosphorylation was uncoupled, and that the average mitochondrial volume increased while the number per average cell decreased. However, the mitochondrial volume and surface areas of the inner and outer membranes in the cytoplasm of both controls and experimentals remained essentially the same. They concluded that cortisone-treated mitochondria were larger but fewer in number, and biochemically defective. In this type of study, where stereological and biochemical data are related to different reference systems (cubic micrometers of hepatocytic cytoplasm versus milligrams of protein), comparisons are based on relative changes, and the experimental contact between the data types is somewhat indirect. Since the demonstration by Remmer and Merker (1965) that hepatocytes reacted to phenobarbital treatment by increasing both drugmetabolizing enzymes and the ER membranes to which they were attached, the liver has become a favorite tissue for studying membrane biosynthesis. Not until the study of

R. P. BOLENDER

276

Staubli et al. (1969), however, was an attempt made to correlate quantitatively enzymic changes with those of a membrane's surface area. During a 5-day period of phenobarbital treatment, these workers reported a linear relationship between the increases in the specific surface area of the smooth ER (SER)and the activities of cytochrome P-450,NADPH-cytochrome c reductase, and demethylase. In order to obtain this relationship, however, it was necessary to adjust both the surface area and enzymic data to account for membrane turnover. Figure I7 is a plot of their data comparing changes in ER surface area to those of the demethylase activity, using three different reference systems: specific dimensions, 1 cm3, and an average cell. The plot compares the amounts of membrane surface area to enzymic activity, in this case uncorrected for synthesis or tumover kinetics. The point this graph attempts to illustrate is that all three reference systems can be used to generate linear relationships but that the best linear correlation (r" = 0.97) is obtained with the average cell reference. Note also how the amount of change is influenced by the choice of reference system.

PHENOBARBITAL INDUCTION STI(UBLI ETAL. '69

U/IOOgEW vs W O O g EW

r'i.81

u/crn3

~/crn3

1'=.43

S/VceII

r' =.97

U/kll

VL

VS.

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200

ER MEMBRANE SURFACE AREA-% OF CONTROL

FIG. 17. Biochemical and morphological data are related to the same reference systems: specific dimensions [lo0 gm body weight (BW)], 1 cm' of liver volume, and the volume of an average cell S, surface area, V, volume, U = units of enzyme activity, f = coefficient of determination. The f values were calculated using the original data, but the results are presented as a percent of the control for purposes of illustration. The ? value is highest when the data are related to the volume of an average cell (f = .97), which indicates that this reference system provides the best linear correlation. Noteworthy is the fact that the 1 cm3 of liver (roughly equivalent to 1 gm) provides the poorest fit (? = .43);this is one of the commonly used reference systems for biochemical data. Experimentally, the curves indicate a parallel increase in the surface area of the ER and in the activity of a constituent drugmetabolizing enzyme. These changes were produced by an inductive effect of the drug phenobarbital on hepdtocytic membranes. Data adapted from Staubli et al. (1969).

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The removal of phenobarbital-induced membranes and enzymes from hepatocytes after cessation of treatment has likewise been studied stereologically (Bolender and Weibel, 1973) and biochemically (Orrenius and Ericsson, 1966). These data have indicated that the surface area of the ER membranes and their constituent demethylase activity return to control levels within 5 days after the last injection of phenobarbital. But how well do these changes correlate with one another? Figure 18 illustrates these data as regression lines calculated using using mixed reference systems; the biochemical data were related to I mg of protein, whereas the stereological data used the average cell, specific dimensions, and 1 cm3 as references. Although all three curves suggest linearity, each reference produces a very different slope for the same data. Noteworthy is the fact that the rate of change-as indicated by these curves-appears to be a function of both the data and the reference system to which the data are related. The mixed r

g4 8L 5 0

?

2 L 0 0 .

6 a I

4 300.

I >

t, 8200. W

2

5 looX 0

/mg PROTEIN vs, S/vell

rz = .71

U/mg PROTEN v s YloOg BW r2 =.66 U/WPROTEIN YS ~ / c m 3 r*=.82

278

R. P. BOLENDER

reference systems produced r2 values for the best fit somewhat lower than those observed in Fig. 17 where both morphological and biochemical data are related to the same reference. During this period of membrane withdrawal after phenobarbital treatment, the stereological data indicated large increases in the volume (800%)and number (96%) of autophagic vacuoles, organelles apparently capable of removing ER membranes preferentially (Bolender and Weibel, 1973). Since both the ER membranes and their attached enzymes presumably entered the autophagic vacuoles together, a parallel change in surface area and activity would be expected if this were an important turnover route. The data in Fig. 18 can be offered in support of a parallel removal, but this suggestion raises questions about the importance of the autophagic vacuole system as it applies to turnover mechanisms in general. Perhaps the biochemical turnover of membranes reflects the activity of several mechanisms, but the vacuole system plays a more prominent role when the hepatocytes undergo remodeling, as appeared to be the case after drug withdrawal. But what quantitative evidence actually links the changes in the membrane and vacuole compartments? An answer to such a question may depend on knowing something about the morphological half-lives of these organelles. This may not be an unrealistic approach. For example, when the surface areas of the excess induced ER membranes of this study are plotted as a function of time, the membrane removal (change in concentration) follows first-order kinetics and a half-life can be calculated (R. P. Bolender, unpublished data). If it were possible to demonstrate the extent to which the autophagic vacuoles consume these excess membranes, a quantitative link might be established. Pfeifer (1973) and Pfeifer and Scheller (1975) have already demonstrated that “stereological kinetics” can be used to study cellular autophagy. Several attempts have been made to correlate changes in the biochemistry and morphology of developing hepatocytes. The interpretation of stereological data derived from development models represents a challenging undertaking, because cells change with respect to size, number, relative distribution, and contents. Development as a quantitative entity can likewise be shown to be largely a function of the morphological and biochemical reference systems one chooses. Greengard et a / . (1972) compared the biochemical differentiation of the rat liver to changes in the volumes and numbers of hepatic cells. Parenchymal (hepatocytic), hematopoietic, and Kupffer cells were characterized during the gestational and postnatal periods by relating units of enzyme activity (per gram of liver) to 1 cm3 of parenchyma or to an average cell. These investigators indicated a preference for relating the enzyme activity to an average cell, but when the enzyme units are divided by the number of cells their reference becomes a unitless average cell, rather than the volume of an average cell as originally described by Loud (1968). Changes during liver development in hepatocytic glycogen synthetase did not correlate particularly well when compared with changes in enzyme units per unitless average cell (see their Fig. 4,Greengard et

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279

al., 1972). If, however, one substitutes the average cell of Loud for the unitless one, a remarkably good correlation results. Turning their attention to subcellular structures in developing hepatocytes, Herzfeld et af. (1973) attempted to correlate changes in enzyme activities with changes in organelle morphology. They compared marker enzyme activities (units per gram of liver) to milligrams of protein measured in specific organellecontaining fractions and to the volume density of organelles measured in the hepatocytic cytoplasm of intact tissue. They reported that only during the early postnatal development was there a parallel increase in some mitochondrial enzyme levels and in the mitochondrial protein. Formation of the ER marker enzyme glucose-6-phosphatase, however, did not coincide with changes in ER surface area when related to 1 cm3 of hepatocyte cytoplasm. Recalling the studies of Leskes et al. (1971), which indicated that during this development period glucose-6-phosphatase appeared simultaneously in all the ER of a given cell but not at the same time for all hepatocytes, one might suspect that any parallel changes in activity and surface area could have been obscured by the underlying heterogeneity of the hepatocytes.

CORRELATED WITH BIOCHEMISTRY OF B. STEREOLOGY OF TISSUEFRACTIONS FRACTIONS A second type of approach used to study relationships of organelle structure to function in the liver relies almost exclusively on morphological and biochemical properties of subcellular fractions. The interpretation of results usually includes the extrapolation of data from a single fraction to the whole liver and assumes (1) that a given marker enzyme has a single morphological location, and (2) that specific organelles in fractions are enzymically homogeneous. The enzymic homogeneity of organelles is defined with respect to either the protein content of fractions or to a morphological parameter such as volume, surface, or number (see recent review by Baudhuin, 1974). Fundamental to this approach has been development of the pellicle method by Baudhuin et al. (1967) for collecting representative morphological samples from tissue homogenates and fractions; the method has been recently reviewed and updated (Deter, 1973; Baudhuin, 1974). Baudhuin and Berthet (1967) used the pellicle system to characterize the mitochondrial content of a large granule fraction (M + L ) which contained about 78% of the cytochrome oxidase activity, a marker enzyme attached to the inner mitochondrial membrane. Extrapolating their data to the whole liver, they reported 5 X l@' mitochondria per gram, an aggregate mitochondrial volume of 0.14 ml/gm, and areas of 2.5 and 1 .O m2/gm for the inner and outer mitochondrial membranes, respectively. However, when mitochondrial morphology measured in these fractions is compared to similar parameters measured by other workers in the intact tissue, agreement has been

280

R. P. BOLENDER

notably lacking. Why? Rat liver mitochondria in intact tissue can occur as long-sometimes bent or branched-cylindrical structures (Fig. 4). During homogenization and fractionation, these threadlike mitochondria apparently become fragmented into smaller, spherical structures, as seen in electron micrographs of fractions (Fig. 19). When variations in animals and quantitating techniques are put aside, homogenization appears to bring about changes in mitochondrial morphology related to their shape (cylindrical to spheroidal), number (fragmentation), and identify [homogenization produces nonrecognizable mitochondrial membrane fragments (Bolender et al., 1976, 1978)]. All these factors could contribute to the difficulty of relating mitochondrial fraction data to those from the intact tissue. Leighton et al. (1968) have described the biochemical and morphological properties of purified rat liver fractions using animals pretreated with Triton WR- 1339. This treatment selectively decreases the equilibrium density of lysosomes in a sucrose gradient, thereby improving their separation from peroxisomes and mitochondria. Using this and additional methods for obtaining purified organelle preparations, they attempted to determine the contribution of mitochondria, peroxisomes, and lysosomes to the total protein content of an isolated fraction, as well as to the total liver protein. These calculations are based on the assumption that the marker enzymes associated with these organelles have the same specific activity in all the liver fractions, including subfractions thereof. This assumption accepts the idea that a direct quantitative relationship exists between organelle protein and marker enzyme activity. In other words, a doubling of organelle protein is expected to be accompanied by a doubling of enzyme activity. In the next step of their experiment, they determined the volume of an organelle (granule) compartment in a fraction stereologically and compared this volume to the organelle protein in the same fraction (Fig. 19; Leighton et al., 1968). If the organelles of a given type in a fraction are homogeneous with respect to their intra- and interprotein distributions (i.e., concentration), separate estimates for relative protein and volume are expected to be identical. In fact, this is essentially what was found (Fig. 19; Leighton et al., 1968). When these data are plotted as regression lines, the results are more easily compared to the postulated biochemical homogeneity (Fig. 20). Given the framework of the experimental procedures and analysis, which Leighton et al. cautiously point out are favorable to organelle homogeneity, most of the data adhere to the postulate. The organelles that agree, namely, peroxisomes and mitochondria, are almost exclusively hepatocytic, whereas the less adherent lysosomes are not. Blouin et al. (1977) have reported that only 57% of rat liver lysosomes are derived from hepatocytes, the remainder coming from endothelial ( 1 6.9%), Kupffer (25.8%), and fat-storing (0.27%) cells. Since liver lysosomes are quite heterogeneous with respect to their cellular origin, they would be expected to be one of the least homogeneous hepatic organelles. This is exactly what Fig. 20 indicates.

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28 1

FIG. 19. Heavy mitochondria1 fraction collected by differential centrifugation from a rat liver. ~48,000.(From Bolender et al., 1978.)

282

R. P. BOLENDER

DENSITY ORADIENT FRdEWNS LEIGHTON ZT AL. '68

100-

ED-

w

J

-

MITOCHONDRIA

f2

*.94

PEROXISDMES

f*

z.99

LYSOSOMES

r2 x.89

& ' ' ' ' I d 0

X ORGANELLE VOLUME

FIG. 20. The amount of protein and volume associated with a particular organelle in density gradient fractions are related directly to one another. If the organelles are all homogeneous with respect to their protein content, a given amount of protein would be expected to give an equivalent amount of organelle volume [theoretical homogeneity (45")]. When this criterion is used, the mitochondria appear to be the most homogeneous organelles, whereas the peroxisomes, being farther removed from the theoretical curve but with an r 2 = .99, are somewhat heterogeneous with respect to size. (The data were collected from four density gradient fractions which sort peroxisomes according to size.) The lysosomes show the greatest heterogeneity, which is not surprising in view of the fact that they have a heterogeneous cellular distribution in the liver: hepatocytes 57%, Kupffer cells 25.8%, endothelial cells 16.9%,and fat-storing cells 0.27% (Blouinet al., 1977). Data adapted from Leighton et al. (1968).

Glucagon-induced autophagy in the rat liver has been studied by Deter and de Duve (1967) in an attempt to locate the principal source of the acid hydrolysis (acid phosphatase) found within autophagic vacuoles. From their correlation of morphological and biochemical data, they concluded that preexisting pericanicular dense bodies (secondary lysosomes) provided most of the hydrolytic enzymes for autophages. Ericsson (1969) arrived at a similar conclusion using morphological markers, but Arstila and Trump (1968) have suggested that Golgi vesicles rather than dense bodies are the principal source of these autophagic vacuole enzymes. A more recent article by Deter (197 1) compares changes in enzyme activities of dense bodies and autophagic vacuoles to changes in the numbers and volumes of these organelles. The loss of dense bodies following glucagon treatment, as L fractions, correlates well with an increase in the osmotic measured in M fragility of a specific type of acid phosphatase-containing particles-presumably autophagic vacuoles. This experimental observation is consistent with the hypothesis that acid phosphatase is being transferred from particles less sensitive to osmotic shock (dense bodies) to those more sensitive (autophagic vacuoles).

+

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283

I

3.0 5 LL

a" N

E i

2.0 I

lrl

I

W

1.0

c

J

u)

x)

40 Minutes

50

FIG. 21. Data adapted from Deter (1971); see text for details.

The morphological data that fit this hypothesis most closely are those comparing changes in the numbers of particles to accessible (free) acid phosphatase activity (Fig. I ; Deter, 1971). Here the number of dense bodies decreases, while the number of autophagic vacuoles and accessible (free) acid phosphatase activity increases. Organelle volumes and surface areas, however, may offer additional interpretations (Fig. 21). Wibo et al. (197 1) characterized rat liver microsomes and microsomal subfractions using a combined stereological-biochemical approach. Their main purpose was to determine the distribution of ribosomes on microsomal vesicles, which they found to occur as a continuous spectrum ranging from ribosome-free vesicles to vesicles heavily coated with ribosomes. Their article is particularly important in that it was the first to deal rigorously with the effect of section thickness on membrane surface area estimates. Using the relative distributions of glucose-6-phosphatase and other marker enzymes, these workers estimated that the total area of the ER membranes in the homogenate was on the order of 7.5 m2/gm of liver.

c. STEREOLOGY OF INTACT TISSUEAND FRACTIONS CORRELATED WITH BIOCHEMISTRY OF FRACTIONS The third approach that attempts to integrate stereological and biochemical data represents a combination of the two just described. It includes an attempt to develop a maximum information system by collecting morphological and/or biochemical data from three different forms of the same liver: intact tissue,

R. P. BOLENDER

284

homogenate, and fractions. The integrative model for the rat liver illustrated in Fig. 22 uses the framework of analytical fractionation, in which recoveries are calculated for morphological and biochemical data as a means of evaluating data correlations. For example, by obtaining three independent estimates characterizing the same liver at various stages of separation, it is possible to observe the extent to which enzyme activity, organelle morphology, and membrane surface area are lost or conserved as a result of these preparation procedures. The results of such a comparison are given in Fig. 23. With the use of data corrected for both section thickness and compression, the recoveries indicate that almost all the total liver membrane surface area is conserved, but that a large portion of specific membrane organelles is no longer recognizable as a result of homogenization (Bolender et al., 1978). The experiments in which both the marker enzyme activity and total membrane surface area of the homogenate-fraction recoveries were found to be well into the 94-98% range provided some assurance that the fractions were a representative form of the liver suitable for attempting data integrations. However, before the activity of an enzyme in a fraction could be related to its associated membrane surface area, a morphological method was needed to identify and quantitate this membrane. A cytochemical procedure was used for this purpose (Bolender, 1974a), which is described in Fig. 24. Here, too, recoveries were used to determine the efficacy of the approach. In this example, where glucose6-phosphatase cytochemistry was used to estimate the surface area of the ER in

t

e

COWPARED WTH

HOMOCENATE

INTACT C F I I R

NTACT C E L L 8

N+M*L*P+S

FIG. 22. Biochemical and stereological data recoveries. A biochemical constituent rncasured in the homogenate (E + N) is considered the original material to which the aggregate data of the resultant fraction (N + M t-I . P + S) are compared, Stereological recoveries treat both intact cells and homogenates as the original material and calculate recoveries accordingly; here, however, only the organelle-containing fractions (N + M + L P) are used for the calculations. Enzyme densities are used to relate biochemical constituents to morphological references (Bolender, 1974a). See text for details and interpretations.

+

+

CORRELATION OF STEREOLOGY AND BIOCHEMISTRY Animal

285

I A

2.

1

3.

FRACTION HOHOGENAE

H~MLWNATE/TISWE

FRAcTrrlnSsUE

FIG. 23. Membrane surface area recoveries are used to compare components of the total hepatic membrane compartment-rough membranes, smooth membranes, and mitochondria1 membranesas they occur in intact, homogenized, and fractionated rat livers (see Fig. 22). From Bolender et al. (1978).

the fractions, the homogenate-fraction recovery was again in the 90-96% range. Still an additional check remains. Since the surface area of the ER can be readily identified and quantitated in the original tissue, these data effectively predict the amount of ER one would expect to find in both the homogenate and the fractions. A comparison of intact tissue ER to that cytochemically determined in the homogenate and the fractions produced recoveries approaching 90%. The fact that approximately the same surface area for the ER was found in intact tissue, homogenate, and fractions provided some assurance that the quantitative cytochemical test for the ER detected this organelle effectively in the fractions. Provided with this substantiating information, the surface area of the ER was used as a reference system for the glucose-6-phosphatase activity by calculating enzyme densities for each membrane-containing fraction (Bolender, 1974a; Bolender et d.,1976). The results of these calculations (Fig. 25) indicate that the distribution of enzyme activity per unit of ER surface area is not the same for all the fractions. The microsomal ER, for example, displays an enzyme density only about 50% of that of the ER in the nuclear or mitochondria1 fractions. This finding, supported by the recovery data, suggests that glucose-6-phosphatase activity is heterogeneously distributed across the surface area of the ER. This ER heterogeneity seems to express itself also as a variable morphology, which allows some separation even at the level of differential centrifugation. Perhaps the distribution of ribosomes on the rough ER (RER) as described by Wibo et al. (1971), or the relative amounts of RER and SER components in a given fraction are contributing factors. The fact that such a large heterogeneity has been observed after only differential centrifugation allows one to speculate that density gradient studies will reveal even greater differences.

286

R. P. BOLENDER

e ' LIVER

CELL8

IDENTIFIED

SCRFACE A R E A

---I---

-I

FRACTIONS

SCRFACE AREA

FUSITNE MEMBR.

SURFACE AREA

1 -1

FIG. 24. Cytochemical method for determining the surface area of a specific membrane compartment in the homogenate and fractions derived therefrom (Bolender, 1974a). For example, the surface area of the ER per gram of liver is determined using the intact tissue, and then again for the same liver two more times, after it has been homogenized and fractionated. These data from the disrupted tissue (homogenate and fractions) are then compared with that for the intact tissue, which serves as a control for the quantitative cytochemical method. The procedure consists of determining the total surface area of all the membranes in a fraction pellicle and then multiplying this area by the relative amount of membrane displaying a glucose-6-phosphataselead reaction product to obtain the surface area of the ER in the fraction. Recoveries can be calculated as illustrated in Fig. 22.

GLUCOSE 6 PHOSPHATASE U

FIG.25. The RSA of glucose-6-phosphatase is calculated using the surface area of the ER instead of the percent protein (see Fig. 16). The figure illustrates that glucose-6-phosphatase activity is heterogeneously distributed across the membranes of the ER. Data from Bolender (1974a) and Bolender er a / . (1976).

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287

V. Summary and Conclusions This article has focused on problems related to integrating and interpreting stereological and biochemical data. Procedures for choosing reference systems, collecting data, dealing with artefacts, and matching different types of data have been discussed. The fact that seems to emerge is that the fractionation system can supply stereological and biochemical data particularly well suited for pursuing quantitative correlations; they both can be expressed as concentrations, they both can be related to the same references, and they both can be interpreted analytically. But what are some of the incentives for pursuing data correlations in the first place? At least a partial answer seems to come from the cells themselves. Inherently complex to begin with, presumed homogeneous populations of cells or organelles may actually contain considerable inter- and/or intracellular heterogeneities. If this turns out to be true, data from fractions could conceivably reflect different combinations of several different variables. But what are the variables and how are they combined? With reference to the opening generalization that the behavior of cells follows relationships of structure to function, the power of the combined quantitative approach seems to exist in its potential for isolating and identifying such variables and combinations.

ACKNOWLEDGMENTS The preparation of this article was supported by USPHS Grant GM-22759 from the National Institutes of Health.

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INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 55

Cytophysiology of the Adrenal Zona Fasciculata GASTONE G. NUSSDORFER, GIUSEPPINA MAZZOCCHI, AND MENEGHELLI

VIRGIL10

Department of Anatomy, Laboratory of Electron Microscopy, University of Padua, Padua, Italy

I. Introduction . . . . . . . . . . . . . . . . . . . . 11. Fine Structure of Normally Functioning Adrenocortical Cells . , A. The Mammalian Zona Fasciculata . . . . . . . . . . B. The Interrenal Cells of Lower Vertebrates . . . . . . . . C. General Remarks . . . . . . . . . . . . . . . . . 111. Fine Structure of Hyperfunctioning and Hypofunctioning Adrenocortical Cells . . . . . . . . . . . . . . . . . A. Short-Term Stimulation . . . . . . . . . . . . . . B . Short-Term Inhibition . . . . . . . . . . . . . . . C. Long-Term Stimulation . . . . . . . . . . . . . . D. Long-Term Inhibition . . . . . . . . . . . . . . . IV. Morphological-Functional Correlations in Adrenocortical Cells . . A. Subcellular Localization of the Enzymes of Steroid Synthesis B. Mechanisms of Action of ACTH . . . . . . . . . . . C. The Mechanisms and the Functional Significance of the Ultrastructural Changes in Hyperfunctioning and Hypofunctioning Adrenocortical Cells . . . . . . . . . V. The Mechanism of Hormone Release by Adrenocortical Cells . . A. Holocrine Secretion . . . . . . . . . . . . . . . . B. Apocrine Secretion . . . . . . . . . . . . . . . . C. Endoplasmocrine Secretion . . . . . . . . . . . . . D. Secretion by Simple Diffusion . . . . . . . . . . . . E. Exocytotic Secretion . . . . . . . . . . . . . . . VI. Concluding Remarks . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

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I. Introduction Several excellent reviews have appeared concerning the morphology of the adrenal cortex and the techniques for its optimal preservation (Luse, 1967; Symington, 1969; Idelman, 1970; Neville and McKay, 1972; Malamed, 1975). Moreover, historical surveys (Gaunt, 1975) and discussions of hypotheses on the cytogenesis of this gland (Long, 1975) are available. 29 1

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However, in recent years many studies have been undertaken attempting to correlate morphological and biochemical findings in the adrenal cortex. Indeed, the development of suitable techniques for long-term in vitro culture of adult animal adrenocortical cells (Armato and Nussdorfer, 1972; Armato et al., 1974a; Armato, 1976) and the application of stereological procedures (Weibel, 1969) have made such correlations possible in many instances. Since biochemical and morphological studies have so far dealt mainly with the mechanism@) of ACTH action in regulating adrenal zona fasciculata activity, we discuss here the more recent findings on the cytophysiology of this zone. Although our concern is the mammalian zona fasciculata, the cytophysiology of the ACTH-dependent interrenal cells of some lower vertebrates is also briefly described.

11. Fine Structure of Normally Functioning Adrenocortical Cells It is well known that steroid-producing cells (granulosa lutein cells, adrenocortical cells, and Leydig cells) possess distinctive structural features: mitochondria containing tubular or vesicular cristae, well-developed smooth endoplasmic reticulum (SER), and an abundance of lipid droplets (Christensen and Gillim, 1969; Fawcett et al., 1969; Idelman, 1970; Malamed, 1975). Naturally differences exist in the fine structure of adrenocortical cells among various vertebrate species. However, certain ultrastructural characteristics described as physiologically significant in recent articles are now well known to be typical preservation artifacts (e.g., open mitochondria1 forms, SER in vesicular form, and light and dark cells). Therefore we consider the fine structure of the normally functioning adrenocortical cells of the various species as a basis for understanding their cytophysiology. A. THEMAMMALIAN ZONAFASCICULATA As nearly half of the studies on the ultrastructure of mammalian adrenocortical cells have concerned the rat, we first give a full description of the zona fasciculata in this animal and then a brief comparative description of adrenocortical cells in other species. 1. Rat

Idelman (1970), in presenting a comprehensive review of the pioneering work on the ultrastructure of the rat adrenal cortex (Brenner et al., 1953; Braunsteiner et a l . , 1955; Lever, 1955a,b; Belt and Pease, 1956; Sabatini and DeRobertis, 1961; Yamori et a l . , 1961), included a critical survey of fixation and embedding procedures. Accounts of sex dimorphism and age-dependent structural dif-

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ferences can be found in Sato (1967), Szaboet al. (1970), Nussdorfer (1970a,b), Dhom et al. (1971), and Maude (1971). Also available are ultrastructural stereological studies furnishing baseline data for the zona fasciculata cells of various rat strains (Nussdorfer et al., 1971a; Nussdorfer and Mazzocchi, 1972a, 1973; Rohr et al., 1975, 1976; Shilov et al., 1977). The zona fasciculata consists of 30 to 40 layers of cells whose volume averages, according to the strain, 1300-2000 pm3 (Nussdorfer et al., 1971a; Rohr et al., 1975; Shilov et al., 1977). The nucleus is round or slightly oval and always contains an obvious nucleolus (Rhodin, 1971). Mitochondria (Fig. I) are numerous, occupying about 26-36% of the cell volume (Nussdorfer et al., 1971a; Friihling et al., 1973; Rohr et al., 1975; Shilov et al., 1977). They are round or oval and quite variable in size, between 0.5 and 1.5 p m in diameter (Canick and Purvis, 1972; Nussdorfer et al., 1974a). Their cristae are invariably in the form of vesicles about 600-700 A in diameter (Kahri, 1966). Frequently, in the mitochondrial matrix, lipid inclusions can be observed (Friend and Brassil, 1970) (Fig. 2), as well as paracrystalline inclusions (Kjaerheim, 1967; Friihling et al., 1968; M. M. MagalhHes and MagalhCes, 1968; Saito and Fleischer, 1971); the latter result from the close association of fine tubules having an inner diameter of about 60 A and an outer diameter of about 120-130 A, and in cross section appear in a hexagonal array (Fig. 3). Some mitochondria have myelin-like protrusions (blebs) extending from the outer membrane and often penetrating an adjacent lipid droplet (Rhodin, 1971; Merry, 1975). The SER is abundant and occupies about 4 0 4 5 % of the cell volume (Nussdorfer et a1 ., 1971a; Friihlinget al., 1973). It is in the form of a network of anastomosing branching tubules which are in strict topographic relation to both mitochondria and lipid droplets (Idelman, 1970; Malamed, 1975) (Fig. 1). There has not yet been confirmation of the observation by Volk (1971b) of a direct continuation of SER tubules with the outer mitochondrial membrane, or of that by Yoshimura et al. (1968) of SER tubules opening into the intercellular space. True rough endoplasmic reticulum (RER) cisternae are absent, but free ribosomes and polysomes are numerous (Fig. 1). There are many lipid droplets occupying about 10-15% of the cell volume (Nussdorfer et al., 1971a; Friihling et al., 1973; Shilov et al., 1977). They have a 40-A-thick membrane, which has so far not been resolved as a trilaminar membrane (Rhodin, 1971). It is well established that lipid droplets contain cholesterol and cholesterol esters (Friihling et al., 1969; Moses et al., 1969; Sand et al., 1972). Sometimes near the lipid droplets there are crystalline inclusions of a rectangular shape, 0.7-1.5 p m in length and 0.08-0.15 p m in width (Szabo, 1968; Rhodin, 1971). The digitonin method of Okros (1968) shows that these inclusions, which are frequently completely extracted by the fixation procedures, contain cholesterol or cholesterol derivatives (Szabo, 1968).

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FIG. 1. Cell from the middle portion of a rat zona fasciculata. Round mitochondria contain vesicular cristae; SER and free ribosomes are well represented. Id, Lipid droplet; L, lysosome. X23.000.

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FIG. 2. Lipid inclusion in a mitochondrion from the rat zona fasciculata. x26,200. FIG. 3. Intramitochondrial paracrystalline inclusion in cross section. Note its hexagonal array. X 133,000. (From Friihling et a l . , 1968.) FIG.4. Cell from the rat zona fasciculata showing numerous Gomori-positive bodies (lysosomes). The arrow indicates a paracrystalline inclusion. ~ 3 1 , 5 0 0 .

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The well developed Golgi apparatus consists of many stacks of cisternae, usually in a juxtanuclear location, and of numerous vesicles, some of which, having a “coated” appearance, seem to arise from the dilated endings of the Golgi saccules (Propst and Miiller, 1966; Friend and Brassil, 1970; Nussdorferet a l . , 1971a; Rhodin, 1971). A few p-glycogen particles are scattered in the cytoplasm (Nussdorfer and Mazzocchi, 1970; Ueberberg, 1971, 1972). Numerous and uniformly distributed in the cytoplasm are dense bodies, displaying acid phosphatase (Penney and Barnett, 1964; Szab6 et al. 1967; Nussdorfer, 1969)

FIG. 5 . Portion of an adrenal cell from the rat zona fasciculata containing numerous DABpositive bodies (peroxisomes). Unstained Section. X47,600. (Courtesy of M. C. Magalhies.)

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(Fig. 4) and arylsulfatase-P activity (Rappay et a l . , 1973), which have been interpreted as primary lysosomes. Recently it became possible with Novikoff and Goldfischer’s technique (Novikoff and Goldfischer, 1969) to demonstrate the presence, especially in close relation to SER tubules, of small peroxidasepositive dense bodies which were assumed to be microbodies (microperoxisomes) (M. M. Magalhles and Magalhles, 1971; Beard, 1972) (Fig. 5). Another

FIG. 6. Septate zonulae adherentes in a rat zona fasciculata. (a) Potassium pyroantimonate (5% in the aldehyde fixative) encrusts the extracellular particles. X65.000. (b) Lanthanum permeates the cell attachment; note that the particles in the intercellular space form a continuous chain. The arrows indicate projections from the particles touching the cell plasma membrane. X260,OOO. (From Friend and Gilula, 1972.)

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ultrastructural feature, apart from the typical microtubules (Nussdorfer and Mazzocchi, 1972a), is the presence of a peripheral network of 40- to 80-A-thick microfilaments just beneath the cell membrane but occasionally penetrating more deeply into the cytoplasm to contact the lipid droplets and the mitochondria, as reported by Gabbiani et al. (1975). These investigators, using an immunofluorescence technique, demonstrated that the cortical portion of adrenocortical cells contained actin and suggested that the peripheral microfilamentous network was composed of actin filaments. Microvilli and coated pits (caveolae) are present at the plasma membrane (Nussdorfer et al., 1971a). Friend and Gilula (1972) made an accurate study of the cell-to-cell attachments between adjacent cortical cells and observed, in addition to exceptional focal tight junctions (zonulae occludentes), small desmosomes (maculae adherentes), intermediate junctions (zonulae adherentes), gap junctions (nexus), a new type of junction which they called “septate-like” zonulae adherentes and regarded as typical of steroid-producing cells. The adjacent plasma membranes are separated by a 210-w extracellular space penetrated by 100- to 150-8, particles; lanthanum and horseradish peroxidase permeate the extracellular space, and the particles are “encrusted” by pyroantimonate (Fig. 6a and b). Friend and Gilula (1972) hypothesize that this type of junction, besides functioning in adherence, may well maintain the width of the intercellular space, thus forming microchannels for the possible flow of secretory products into the blood stream. Moreover, the particles may be the site of a high cation concentration, presumably Ca‘+, which is involved in corticosteroid secretion (Rubin, 1974), and/or they may serve in the binding of ACTH or its carrier proteins. 2. Human Despite many investigations on the fine structure of the human adrenal cortex (Cam, 1958, 1959, 1962; Ross et a l . , 1958; Bachmann et al., 1962; Symington, 1962, 1969; Luse, 1967), little insight has been obtained, probably because of inadequate fixation and intraoperative ischemic damage to adrenocortical tissue, which is very susceptible to anoxemia (Horvath and Kovacs, 1973). This account is based on the articles of Long and Jones (1967b) and Kawaoi (1969), as well as on personal observations. Anoxic damage was avoided by fixing a small piece of the gland just before complete adrenalectomy.

FIG. 7. Cell from the middle portion of a human zona fasciculata. Mitochondria show vesicular cristae; a RER stack is located near the nucleus. X21,600. FIG. 8. Portion of a cell from an inner human zona fasciculata. Mitochondria contain short, tubular cristae. The SER is very abundant. The arrow indicates a few rough profiles. If, Lipofuschinic pigment. X20.300.

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The cells have a round nucleus and spherical or elongated mitochondria containing short, tubular (Long and Jones, 1967b) or vesicular cristae (Armato et a l . , 1974b) (Figs. 7 and 8). The SER is abundant, and juxtanuclear stacks of RER cisternae can be observed. Lipid droplets are quite numerous, as well as free ribosomes. The Golgi apparatus is well developed, and lysomes and small, lipofuschinic granules are present. Microvilli project into the subendothelial space. Magalhiies (1972) described a new type of cell occurring in the periendothelial spaces only in the male adrenal cortex and displaying the ultrastructural characteristics of typical mesenchymal elements. It contained, besides patches of filamentous structures in the Golgi area, crystalline bodies similar to the socalled Reinke’s crystals found in Leydig cells. MagalhHes (1972) suggested that these cells might be involved in testosterone secretion.

3. Monkey Two species were examined: rhesus monkey (Brenner, 1966) and squirrel monkey (Penney and Brown, 1971). The cells contained a round nucleus and mitochondria with tubulovesicular cristae. The SER was abundant, while RER was scarce or virtually absent. Free ribosomes and occasional polysomes were present in the cytoplasm. Lipid droplets were more abundant in the cells of the outer portion of the zona fasciculata. The Golgi apparatus was poorly developed. Lysosome-like dense bodies were numerous, and the microvillar apparatus was prominent. 4. Dog The ultrastructure of the dog adrenal cortex was described by Kaminsky et al. (i962) and Luse (1967), and in greater detail by Bloodworth and Powers (1968) who unfortunately employed only osmic fixation. Recently, two other contributions have appeared (Hart e? a l . , 1973; Powers et al., 1974), but as they were concerned with the effects of DDD, they merely provided a brief account of the fine structure of normally functioning adrenocortical cells. The cells have oval, round, or elongated mitochondria containing cristae of the tubulovesicular type. The SER is abundant, while only occasional profiles of RER and a moderate amount of free ribosomes are present. Lipid droplets are numerous, and the Golgi apparatus is prominent. Several small electron-dense bodies, which Bloodworth and Powers (1968) interpreted as being microbodies, are scattered in the cytoplasm. Microvilli project from the plasma membrane. 5 . Cat

Little attention has been paid to the ultrastructure of the cat adrenal gland. However, recent work from a laboratory where this gland is being studied at the biochemical level has provided some information (Gemmell et af., 1977a).

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FIG. 9. Cell from the zona fasciculata of a perfused cat adrenal gland treated with ACTH; pleomorphic mitochondria display tubulovesicular cristae. Note the numerous electron-dense granules which appear to originate in the Golgi zone ( G ) . X30,OOO. (Courtesy of R. P. Rubin.)

Mitochondria are spherical with tubulovesicular cristae. The SER is well developed, and lipid droplets are abundant. The Golgi apparatus is well represented, and electron-dense granules are associated with its cisternae. As seen in Section V,E, Gemmell et al. claim that these dense bodies are secretory granules (Fig. 9).

6. Ox

In addition to earlier contributions (Luft and Hechter, 1957; Weber et al., 1962, 1964), more recent articles with detailed descriptions have appeared (Friihling et al., 1971, 1973).

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The cells contain a round nucleus showing frequent indentations and intranuclear bodies (Weber et al., 1962, 1964). Mitochondria are spherical or pleomorphic and have tubular or vesicular cristae (Fig. 10). In the mitochondria1 matrix lipid inclusions are frequently observed (Friihling et al., 1971). The SER is well developed, occupying about 55% of the cell volume. Scattered RER cisternae and free ribosomes are intermingled with SER tubules. Lipid droplets are virtually absent (0.35% of the cell volume). The Golgi apparatus is prominent. Lysosomes and peroxisomes are found among the SER tubules (Goldenberg et al., 1975). 7. Rabbit Although this species is commonly employed in laboratory experiments, the fine structure of its adrenal glands has been little investigated. Early studies (Cotte, 1959; Cotte and Picard, 1959; Cotte and Cotte, 1961; Cotte et al., 1963a,b) described mitochondria with laminar cristae, a well-developed SER, and numerous lipid droplets. Since a recent examination of the fetal rabbit adrenal gland revealed that cell differentiation was coupled with the development of SER and a rearrangement of the mitochondrial lamellar cristae into a typical tubulovesicular pattern (Albano et al., 1976), it seemed worthwhile to reexamine the fine structure of the adult rabbit zona fasciculata. Our electron micrographs showed typical mitochondria with tubular or tubulovesicular cristae (Fig. 11). 8. Mouse There are several earlier papers (Zelander, 1957, 1959, 1968; Molbert and Amesen, 1960), but here we consider the more recent descriptions by Shelton and Jones (1971) and Nickerson (1975). For the ultrastructure of the mouse X zone and its involvement in androgenic hormone secretion, see Ross (1967), Sat0 (1968), Garweg et al. (1 97 1), Holmes and Dickinson (1971), and Hirokawa and Ishikawa (1974, 1975). The cells show large, spherical mitochondria which occupy about 20% of the cell volume. The organelles in the outer zona fasciculata have long, tubular cristae which branch and anastomose in a complex labyrinthine pattern, whereas in the inner portions of this zone they display vesicular cristae (Fig. 12). The

FIG. 10. Low-power electron micrograph of the middle portion of an ox zona fasciculata. In addition to a few lipid droplets (Id), almost completely extracted, the cytoplasm contains many mitochondria and SER profiles, which frequently are arranged in conspicuous patches (stars). Some mitochondria (m) are very elongated and have bizarre shapes. The arrows indicate points of thickening of the plasma membrane. G, Golgi apparatus; N, nerve fiber; S, Schwann cell. Fixation: osmium tetroxide (5%) in double-distilled water; staining: uranyl acetate and lead citrate. X 5900. (From Friihling et al., 1973).

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FIG. 11. Cell from a rabbit zona fasciculata. Ovoid mitochondria contain tubulovesicular cristae; the SER is well developed, as are free ribosomes. Id, Lipid droplets; arrows, electron-dense bodies probably of a lysosomal nature. X 17,500.

SER is well developed, and sometimes large lamellar collections of flattened smooth cistemae can be observed; according to Nickerson (1975) the SER occupies about 27% of the cell volume. Lipid droplets are plentiful, especially in the outer zona fasciculata cells (40% of the cell volume). The Golgi apparatus is extensive. Coated pits and microvilli are seen in both the subendothelial and intercellular spaces. 9 . Guinea Pig

The ultrastructure of the adrenals of this species has been investigated by Cotte et al. (1963b) and Sheridan and Belt (1964b). Recently a fuller account was presented by Friihling’s group (Friihling et al., 1973; Sand et al., 1973), who used stereological techniques also. The cells contain ellipsoidal mitochondria with laminar cristae (Fig. 13). In the outer portion of the zona fasciculata the SER is rather well developed (26.5% of the cell volume) and lipid droplets are abundant (28.3% of the cell volume), while in the inner portions of this zone the SER is much more abundant (40.7%

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FIG. 12. Cell from a mouse zona fasciculata, showing round mitochondria with vesicular cristae and many lipid droplets (Id). X28,OOO. (Courtesy of P. A. Nickerson.)

of the cell volume) and lipid droplets less numerous (19.6% of the cell volume). Among the SER tubules are scattered many rough profiles. At variance with Sheridan and Belt (1964b), Friihling and associates described a poorly developed Golgi apparatus. Black and Bogart ( 1973) demonstrated cytochemically numerous microperoxisomes and lysosomes intermingled with SER profiles in fetal and adult guinea pig adrenocortical cells. 10. Hamster DeRobertis and Sabatini (1958), Belt (1960), Cotte et al. (1963b), Yonetsu (1966), and Yates’ group (Yates, 1966; Arai et al., 1967) have examined this species. The most recent studies were limited to the innervation (Unsicker, 1969) and morphology of the sinusoidal wall (Dubois et a l . , 1971). According to Yates (1966) the cells contained elongated mitochondria with convoluted tubular cristae. The SER was much more developed than in other species; free ribosomes were present, but RER cisternae were rare. Lipid droplets

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FIG. 13. Cell from guinea pig zona fasciculata. Irregular-shaped mitochondria typically contain laminar cristae, which frequently are aggregated in patches of various sizes. SER and free ribosomes are abundant. L, Lysosome. X24,500. (Courtesy of J . Friihling.)

were almost completely absent. The Golgi apparatus was not well developed, dense bodies were present, and microvilli were abundant. 1 1. Mongolian Gerbil The ultrastructure of the adrenal cortex of this species was first described by Nickerson and co-workers (Nickerson and Curtis, 1969; Nickerson, 197l), with whom Kadioglu and Harrison (1975) agree in their more recent account. The cells contain numerous oval or pleomorphic mitochondria with a dense matrix and a convoluted tubular cristae occasionally arranged like a honeycomb (Fig. 14). The SER is abundant, and a few RER profiles and numerous free ribosomes are also observed. Lipid droplets are well represented. Microvilli and extensive cell contacts resembling the septate zonulae adherentes described by Friend and Gilula (1972) are observed in rat adrenocortical cells. An unique feature in this species is the presence of a distinct cell layer between the zona fasciculata and the zona reticularis, called by Nickerson (1971) the zona fasciculata-reticularis border. The cells of this zone have mitochondria similar to those of the zona fasciculata. Lipid droplets are present

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FIG. 14. Cell from a mna fasciculata of the Mongolian gerbil. Mitochondria show tubuloconvolute cristae; SER and lipid droplets (Id) are well represented. x26,400. (Courtesy of P. A. Nickerson.)

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FIG.IS. Zona fasciculata-reticularis border cell from the Mongolian gerbil adrenal cortex, which displays two concentric whorls of rough endoplasmic reticulum. M, Mitochondria; Id, lipid droplet; S, sinusoid. X26,400. (Courtesy of P. A. Nickerson.)

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in small amounts in some cells and apparently absent in others. There are no SER tubules, and the Golgi apparatus seems well developed. Typically RER is arranged in concentric whorls, composed of 6 to 12 paired arrays of rough membranes, usually located near the nucleus (Fig. 15). Nickerson (1970) postulates that the zona fasciculata-reticularis border cells represent a reservoir of elements which can transform into zona fasciculata cells upon appropriate stimulus (e.g., by ACTH). 12. Opossum According to Long and Jones (1967a, 1970), the zona fasciculata contains cells having pleomorphic mitochondria with predominantly long, tubular cristae. An elaborate SER occupies much of the cytoplasm, while only rarely are RER profiles encountered. Many free ribosomes are observed. Except in the outer zona fasciculata cells, lipid droplets are not very numerous. The Golgi area is prominent; lysosomes, microvilli, coated pits, and cell-to-cell attachments are also seen. 13. Other Mammalian Species Sporadic observations are available for other mammalian species. Hostetler et al. (1976) found in the insectivore Siamese shrew adrenocortical cells globular mitochondria with vesicular or tubulovesicular cristae, abundant SER sometimes organized into patches of highly ordered tubules (0.2-1 .Opm), and several lipid droplets. Seliger and Smith (1968) described, in the 13-lined ground squirrel, mitochondria with vesicular cristae. Unsicker (1971), in a study on innervation of the pig adrenal, presented electron micrographs showing zona fasciculata mitochondria with tubulobulbous or tubular, contorted cristae and lipid droplets. According to Romagnoli (1972) hedgehog zona fasciculata cells display spherical mitochondria with long, tubular parallel cristae, much SER, and many lipid droplets.

B . THEINTERRENAL CELLSOF LOWERVERTEBRATES 1. Agnatha The fine structure of the interrenal cells of two cyclostome species (suborder Petromyzontia), the marine lamprey (Petromyzon marinus) (Youson, 1972, 1973a,b) and the brook lamprey (Lampetra fluviatilis) (Hardisty and Baines, 1971; Hardisty, 1972a,b), has been described. The cells contain a round nucleus, mitochondria of various shapes either with tubulovesicular cristae and lipid osmiophilic intramatrix inclusions (Youson, 1972) or with mainly tubular cristae (Hardisty and Baines, 1971). There are abundant SER tubules and a few scattered RER profiles. Free ribosomes are

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numerous as well as lipid droplets which, according to Seiler et a l . (1970), contain cholesterol derived from the bloodstream (Youson, 1975). The Golgi apparatus is well developed. Dense bodies, which are possibly primary or secondary lysosomes, and numerous microvilli can also be observed (Youson, 1972).

2 . Chondrichthyes Two shark species (order Selachii) have been examined: Gynglymostoma cirratum (Taylor et a l . , 1975) and Scyliorhinus caniculus (Berchtold and Collenot, 1976). Mitochondria are elongated, displaying mainly tubulovesicular cristae; SER tubules and free ribosomes are abundant, while RER cisternae are absent. Lipid droplets are well represented, as well as the Golgi apparatus. P-Glycogen particles and peroxisomes can be observed (Berchtold and Collenot, 1976). The cytoplasm is strikingly rich in microfilamentous bundles, and the plasma membrane shows numerous microvilli, gap juctions, and a few coated pits. 3 . Osteichthyes Examples from all three superorders of the subclass Actinopterygii have been studied. a. Chondrostei. Youson and Butler (1976a) described the interrenal cells of the freshwater sturgeon (Acipenserfulvescens).The cells contained a spherical to oval nucleus with prominent nucleoli. Mitochondria were elongated with tubular or vesicular cristae and often showed in their matrix both lipid osmiophilic and paracrystalline inclusions. Extensive SER, occasional RER profiles, and numerous free ribosomes were also seen. Lipid droplets were abundant, and the Golgi apparatus was well developed. Some dense bodies were located near the plasma membrane. The cytoplasm contained several clumps of microfilaments (70-80 8, in diameter) and microtubules, especially in the Golgi area. Abundant microvilli, coated pits, desmosomes, and septate zonulae adherentes were also reported. b. Holostei. In the bowfin (Arnia cnlva), Youson and Butler (1976b) found many spherical mitochondria with tubular cristae, well-developed SER, small fragments of RER, and free ribosomes. The Golgi apparatus was prominent, and numerous dense granules were located near it. /3-Glycogen particles and microtubules were present. The plasma membrane showed microvilli, coated pits, and desmosomes. c. Teleostei. Ogawa (1967) examined the goldfish (Carassius auratus), and Wakisaka (1963) investigated the trout (Salmo irrideus). Mitochondria were elongated and contained tubulovesicular cristae and intramatrix osmiophilic inclusions. Both the SER and the Golgi apparatus were well developed; electrondense bodies were located near the latter. Lipid droplets were scarce. A characteristic feature of goldfish interrenal cells was the presence of many microfilaments and microtubules.

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4. Amphibia

Both the orders Urodela and Anura have been extensively investigated. a. Urodela. No significant differences were found between the interrenal cells of Salamandra and Triturus . According to Berchtold (1966, 1968, 1969a,b,c, 1970a,b, 1973) and Picheral (1968), in Salamandra salamandra the cells contained mitochondria displaying a pleomorphic cristal pattern: tubulovesicular cristae were more frequent, although the laminar type was also present. Some organelles had tubular cristae either closely packed in a hexagonal array (Picheral, 1968) or pursuing a tortuous course. Analogous findings were reported by Sheridan and Belt (1964a) in Necturus maculosus, Bunt (1969) in Taricha torosa, Hanke and Pehlemann (1969) and Fritsch et al. (1977) in Xenopus laevis , and Picheral (1970) in Pleurodeles walthii . SER tubules are abundant; numerous free ribosomes and many lipid droplets of various size are seen. The Golgi apparatus is well developed, except in the interrenal cells of N . maculosus (Sheridan and Belt, 1964a). In the Golgi area there are electron-dense bodies, which, as they show a positive reaction to acid phosphatase and thiamine pyrophosphatase, are assumed to be lysosomes (Berchtold, 1972). Cytochemically, Berchtold (1975a) demonstrated the presence of numerous microperoxisomes (Fig. 16), apparently arising from the SER tubules. At the cell surface microvilli, coated pits and some micropinocytotic vesicles were observed. b. Anura. The fine structure of the interrenal cells in the frogs and toads examined is essentially the same. In Rana catesbiana mitochondria are round or oval and contain small, tubular cristae (Volk, 1972), while in Rana pipiens (Burgos, 1959), Rana esculenta (Geyer, 1959), Rana temporaria (Pehlemann, 1968; Pehlemann and Hanke, 1968), and Bufo arenarum (Piezzi and Burgos, 1968) some mitochondria show cristae of the vesicular or tubulovesicular type. SER is found typically in small amounts, RER is absent, and free ribosomes are numerous. Lipid droplets are well represented. The Golgi apparatus is prominent. In the bullfrog, Volk (1972) described an abundance of microtubules and microfilaments. Microvilli have not been reported.

5 . Reptilia Only the order Squamata has been investigated. In the suborder Lacertilia, three species were examined: Lacerta sicula sicula (Varano et a l . , 1969), Lacerta vivipara (Dufaure, 19701, and the tropical lizard Cnemidophorus lemniscatus lemniscatus (Del Conte, 1976). Mitochondria were ovoid or elongated and contained tubular cristae sometimes following a tortuous course. SER tubules were abundant, and some RER cisternae and many free ribosomes were present. Lipid droplets were numerous, and the Golgi apparatus was well developed. In the cytoplasm there were abundant microfilaments, either sparsely distributed or in patches of varying size.

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FIG.16. Interrenal cell of T . crisrarus containing many DAB-positive bodies (peroxisomes). Note a typical mitochondrion exhibiting closely packed tubular cristae (star). Id, Lipid droplet; ES, extracellular space. X41 ,OOO. (Courtesy of J . P. Berchtold.)

In the suborder Ophidia only one species has been studied: the garter snake (Tharnnophissirtulis) (Sheridan, 1963). The ultrastmctural features are the same as in the Lacertilia, except that RER profiles are absent and numerous dense bodies are present in the Golgi area.

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6. Aves In the domestic fowl (Gallus domesticus) (Fujita, 1961; Fujita et a l . , 1963; Kondics and Kjaerheim, 1966; Kjaerheim, 1967, 1968a, 1969; Unsicker, 1973), the cells contain ovoid or elongated mitochondria with tubular cristae. Some organelles show intramatrix paracrystalline inclusions (Kjaerheim, 1967), as well as lipid osmiophilic bodies (Kjaerheim, 1968a). SER is well developed, RER is very scarce, and free ribosomes are well represented. Lipid droplets are abundant. In the Golgi area many electron-dense bodies can be found. P-Glycogen particles and microfilaments are also observed. The Pekin duck (Anus platyrhynchos) was examined by Unsicker (1973), Cronshaw et al. (1974), Bhattacharyya (1975a), and Pearce et al. (1977). The cells had ultrastructural features comparable to those of the fowl. The main differences were : (1) the absence of osmiophilic inclusions in the mitochondrial matrix; (2) the presence of microtubules and microfilamentous bundles; (3) the presence of several vesicles near the plasma membrane; (4) the presence of numerous cell-to-cell attachments, some resembling the septate zonulae adherentes. In the Japanese quail (Coturnix coturnix juponica) (Bhattacharyya, 1975b) and the domestic pigeon (Columbu liviu) (Unsicker, 1973; Bhattacharyya, 1975b), the interrenal cells show pleomorphic mitochondria with tubulovesicular cristae and occasional paracrystalline or osmiophilic inclusions in the matrix. The SER is well developed, and some RER cisternae and free ribosomes are present. Lipid droplets are abundant, and the Golgi apparatus is prominent. Dense bodies, microfilaments, and coated pits are said to be present at the plasma membrane. The ultrastructure of the brown pelican (Pelecanus occidentulis) interrenals is similar to that of other birds, according to Sheridan et al. (1963) and Belt et a l . (1965). However, the latter investigators noted a great number of electron-dense bodies near the plasma membrane and occasionally even in the subendothelial space. Unsicker (1973) reported on several other species (the domestic goose, gull, guillemot, rook, blackbird, starling, house sparrow, and chaffinch). The ultrastructure of their interrenal cells was much as described previously. C. GENERAL REMARKS

From the above survey it is clear that there are no conspicuous differences in adrenocortical ultrastructure among various species of vertebrates, and this conclusion is reasonable because the same fundamental steroid hormone pattern is laid down in the various animals (Sandor et a l . , 1976). The significance of the peculiar morphology of the mitochondrial cristae and the presence of a well-

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developed SER can be understood if we remember that the enzymes involved in steroid synthesis are located in both these organelles (see Section IV,A). Again the presence of lipid droplets, which contain cholesterol or cholesterol esters (Friihling et af., 1969; Moses et af., 1969; Sand et al., 1972), is easily explained, because cholesterol is the main precursor of steroid hormones. Other typical ultrastructural features of adrenocortical cells are a well-developed Golgi apparatus, lysosome-like dense bodies and peroxisomes, microtubules and microfilaments (which, however, seem to be more abundant in lower vertebrates, especially in fishes), and microvilli and coated pits at the plasma membrane. Although a septate zonula adherens is described in at least four species (rat, Mongolian gerbil, freshwater sturgeon, and Pekin duck), the hypothesis that this type of junction is found only in adrenocortical cells (Friend and Gilula, 1972) requires further investigation. The possibility that some dense bodies described in the Golgi area and also near the plasma membrane are true secretory granules is discussed in Section V,E. Finally, we stress that, in all other species except humans, adrenocortical cells contain only scattered RER cisternae which probably are temporary associations between smooth tubules and free ribosomes. Mitochondria1 morphology and a quantitative correlation between SER and lipid droplets merit further discussion. It has been previously noted that adrenocortical mitochondria in many species contain cristae of the vesicular type. Earlier, Sabatini and DeRobertis (1961) assumed that vesicular cristae originated from the inner mitochondrial membrane and floated free in the matrix of the organelles. However, Allmann et al. (1970), using calf adrenocortical mitochondria both in situ and isolated, reported that the so-called vesicular cristae were actually sections of tortuous tubular invaginations of the inner mitochondrial membrane, which were alternatively “ballooned-out” and “squeezed-down” (Fig. 17). This description, confirmed recently in the rat by Malamed (1975), was derived from an accurate analysis of high-magnification electron micrographs and is very convincing (Fig. 18). However, because the thickness of the tissue fragment sections (about 700-800 A) is much the same as the cristal vesicle diameter (600-700 A), we presume that the number of images of ballooned and squeezed tubules per mitochondrion is much higher than that demonstrated by Allmann and associates and by Malamed. Other features of the adrenocortical mitochondria common to many vertebrate species are intramatrix paracrystalline inclusions (in the rat, freshwater sturgeon, domestic fowl, Pekin duck, and Japanese quail) and lipid osmiophilic inclusions (in the rat, ox, marine lamprey, freshwater sturgeon, goldfish, trout, domestic fowl, Japanese quail, and domestic pigeon). With regard to the former inclusions, M. M. MagalhHes and Magalhiies (1968) hypothesized that they might be associated with the presence of the 1lp-hydroxylating system at the mitochondial cristae (Satre et al., 1969; Yago and Ichii, 1969; Dodge et al., 1970),

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FIG. 17. Stereogram of the inner membrane of an adrenocortical mitochondrion. (a) Tubular cristae; (b)-vesicular cristae according to the model of Sabatini and DeRobertis (1961); (c) vesicular cristae according to the model proposed by Allmann and associates (1970).

though other workers have interpreted them as postmorten artifacts, since their number appears to increase proportionally with the interval between sacrifice of the animal and fixation of the adrenal fragments (Dietert, 1969). The significance of the intramitochondrial osmiophilic inclusions is still not understood; however, the demonstration that hyperosmication did not result in a black deposit over these inclusions allowed Friend and Brassil(l970) to assume that they consisted of free cholesterol, but this was recently questioned by Kai et al. (1977). It has been seen that SER tubules and lipid droplets are typical features of adrenocortical cells of all species. However, on the basis of stereological evidence from three mammalian species (rat, ox, and guinea pig), Friihling et al. (1973) demonstrated that an inverse correlation existed between the volume of the lipid compartment and the quantity of SER. Such an inverse correlation can also be deduced from the stereological data presented by Nickerson (1 975) for mouse adrenocortical cells; even in hamster adrenocortical cells, in which the SER is exceedingly well developed, lipid droplets are almost completely lacking (Yates, 1966). In addition, Friihling and co-workers stress that the number of lipid droplets is in inverse proportion to the cell capacity during cholesterol

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FIG. 18. Mitochondrion with vesicular cristae from a rat zona fasciculata. The arrows indicate images supporting the hypothesis of Allmann er al. (1970). Star, lipid intramatrix inclusion. x36,OOO.

synthesis, which is known to occur in the SER (Christensen, 1965; Dorfman and Ungar, 1965; Olson, 1965; Chesterton, 1968); in fact, lipid droplets are abundant in the rat, scarce in the guinea pig, and virtually absent in the ox, in which endogenous cholesterol synthesis was found to be responsible, respectively, for about 10-15% (Ichii et al., 1967; Chevalier et al., 1968), 40% (Werbin and Chaikoff, 1961), and 90% (Goodman, 1965) of the total intracellular cholesterol. Hence it may be assumed that only cholesterol exogenously derived from the bloodstream is stored in the lipid compartment.

111. Fine Structure of Hyperfunctioning and Hypofunctioning Adrenocortical Cells It is well known that ACTH or stress, which increases ACTH release (review in Allen et al., 1973), exerts tropic (short-term) and trophic (long-term) effects

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on the mammalian zona fasciculata and on the interrenal cells of lower vertebrates (Barker JBrgensen, 1976). Tropic effects involve an immediate increase in corticosteroid hormone secretion, which begins after about 10 minutes and reaches a maximum after 15-30 minutes (Jaanus et al., 1970). In addition to normal maintenance, the trophic effects of ACTH involve an increase in the adrenal weight and in the steroidogenic capacity of adrenocortical cells; in other words, chronic ACTH stimulation induces several structural changes enabling adrenocortical cells to maintain a high rate of corticosteroid hormone output for longer periods (Kuo and Tchen, 1973; Tchen et al., 1977). Stimulation of adrenocortical cells was obtained with the administration of ACTH or its intracellular mediator CAMP (review in Halkerston, 1975), and indirectly with activation of the hypothalamohypophyseal axis by stresses of various types or unilateral adrenalectomy . Inhibition of adrenocortical cells was obtained either by hypophysectomy or by suppressing the hypothalamohypophyseal axis by the administration of corticosteroid hormones. A. SHORT-TERM STIMULATION We now consider the ultrastructural changes occurring in adrenocortical cells after a maximum of 6-8 hours after ACTH administration. Investigations of this subject would throw light on the mechanism(s) underlying adrenocortical hormone release, but unfortunately there are few such studies. The earliest reports (Yamori et al., 1961; Sabatini et al., 1962; Nishikawa et al., 1963) described vacuolization of SER tubules and the appearance of “open mitochondria” in rat zona fasciculata cells 30-1 20 minutes after ACTH administration. At present these changes can be considered due to poor fixation, although in recent articles SER vacuolization has also been reported to be a sign of cell hyperfunction (Kuo and Tchen, 1973). After a transient decrease at 20 minutes, rat adrenocortical nuclei seemed to become enlarged after 2-8 hours (Bannawarth et al., 1974; Frahm et al., 1974). In rat adrenocortical cells mitochondria did not display any qualitative change (Yoshimura et al., 1968; Mausle, 1971); however, after 1 hour of severe stress (chair restraint) disoriented tubular cristae were observed in the monkey’s mitochondria (Penney and Brown, 1971). Using stereological techniques, other workers reported mitochondrial hypertrophy in rat adrenals (Yago et al., 1971) and mitochondrial proliferation (Zoller and Malamed, 1975). Sekiyama and Yago (1972) reported both and also described an increase in the diameter and volume of the vesicular cristae. SER tubules seem to increase in number in rat adrenocortical cells and to become much more close to both mitochondria and lipid droplets (Mausle, 1971 ; Sekiyama and Yago, 1972). An opposite finding was obtained by Rhodin (1971) who described a decrease in the amount of SER as early as 10 minutes after ACTH intravenous administration in rats. This investigator interpreted the SER

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decrease as indicating that this organelle was rapidly utilized in endoplasmocrine secretion (see Section V,C). A similar finding was reported in the stressed monkey by Penney and Brown (1971) but was interpreted as a sign of cell exhaustion. Stereological investigations, however, did not show a significant variation in the SER surface area in rat adrenocortical cells 1 hour after ACTH administration (G. G. Nussdorfer, unpublished results). According to Penney and Brown (197 I), free ribosomes increased significantly in number. A severe decrease in lipid droplets was found to occur from 30 to 120 minutes after ACTH administration (Nishikawa et af., 1963; Yoshimura et al., 1968; Mausle, 1971; Penney and Brown, 1971; Sekiyama and Yago, 1972; Frahm et a l . , 1974; Zoller and Malamed, 1975). Frahm ef al. (1974) did not observe lipid depletion after 20 minutes, but Rhodin (1971) reported a noticeable decrease in lipid droplets as early as 10 minutes after ACTH administration. Our stereological findings confirmed Rhodin’s data (G. G. Nussdorfer, unpublished results). Most of the investigators did not comment on the Golgi apparatus; only Mausle (1971) showed hypertrophy of this organelle in the rat adrenocortical cells 30 minutes after ACTH stimulation. P-Glycogen particles seemed to be virtually absent (Rhodin, 197 l), and microvilli increased in number (Mausle, 1971). Numerous granules 200 8, in diameter were found in the intercellular spaces of the zona fasciculata of rats stressed by the injection of snake toxin (Harumiya, 1966), but Yoshimura et al. (1968) were not able to confirm this finding.

B , SHORT-TERM INHIBITION Rhodin (1971) described rat adrenocortical cells 16 to 25 hours after dexamethasone administration. Mitochondria did not display evident alteration. This investigator did not comment on the SER and reported a striking increase in the number of lipid droplets. The crystal-like inclusions described by Szabo (1968) increased in number and are frequently contained in the lysosomes. P-Glycogen particles showed a significant increase. STIMULATION C. LONG-TERM

The cell volume increases significantly as a function of the duration of stimulating treatment in the rat (Nussdorferet al., 1971a, 1974a; Nussdorfer and Mazzocchi, 1972a, 1973) and mouse adrenal zona fasciculata (Nickerson, 1975). Analogous results were described by Seelig and Rennels (1969) in unilaterally adrenalectomized rats and in both fetal rat (Kahri et al., 1970; Salmenpera, 1976) and human adult adrenocortical cells cultured in vitro (Kahri et al., 1976a; Nussdorfer et al., 1977a). However, according to Rohr et al. (1973, starvation stress (up to the ninth day) did not increase rat adrenocortical cell volume, which on the contrary decreased when the stress was prolonged. Stereology showed that

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prolonged ACTH treatment also caused an increase in the number of zona fasciculata cells in the rat adrenal gland (Nussdorfer et a l . , 1974a, 1977b). There is good agreement that chronic adrenal activation induces a significant increase in nuclear and nucleolar volumes in both mammalian (Weber et al., 1964; Starket a l . , 1965; Merkle, 1968; Palkovitz and Fischer, 1968; Seelig and Rennels, 1969; Mitro and Palkovitz, 1970; Heininger et a l . , 1971; Nussdorfer et a l . , 1971a, 1974a; Malendowicz, 1972; Nussdorfer and Mazzocchi, 1972a, 1973; Lutsenko et al., 1974; Nickerson, 1975; Sharawy and Penney, 1977) and submammalian (Kjaerheim, 1968c; Berchtold, 1970a,b; Hardisty, 1972b; Youson, 1973b; Kovacs, 1974) vertebrates. An increase in nucleolar size was also reported in many in vitro models employing rat (Kahri, 1970; Armato and Nussdorfer, 1972) and human (Kahri et a l . , 1976a) adrenocortical cells. In addition to the volumetric changes, an increase in the number of nuclear bodies was also described after ACTH treatment in both calf (Weber et al., 1964) and fowl (Kjaerheim, 1968c) adrenocortical cells. Mitochondria1 changes are also very conspicuous (Figs. I9 and 20); the bulk of the evidence indicates that chronic stimulation induces vesiculation of the cristae in hypophysectomized animals (Sabatini et a l . , 1962) and in human and rat adrenocortical cells cultured in vitro in the absence of ACTH (Kahri, 1966; Kahri et al., 1970; Milner and Villee, 1970; Armato and Nussdorfer, 1972; Milner, 1972b, 1973; O’Hare and Neville, 1973a; Armato et a l . , 1974a,b, 1975; Nussdorfer et al., 1977a; Suyama et a l . , 1977). However, this reaction was not elicited by ACTH in fetal human adrenocortical cells cultured in vitro (Stark et al., 1975; Kahri et al., 1976a). An apparent increase in the surface and volume of the vesicular cristae has also been described (Berchtold, 1970a,b; Sekiyama and Yago, 1972; Youson, 1973b). A significant increase in the surfaceof mitochondria1 cristae was stereologically demonstrated to be induced by prolonged ACTH or CAMP treatment in the rat (Nussdorfer et d . , 1971a, 1974a; Nussdorfer and Mazzocchi, 1972a, 1973; Rohr et al., 1975), in mouse adrenocortical cells (Nickerson, 1975), and in fetal rat (Salmenpera, 1976) and adult human (Nussdorfer et a l . , 1977a) adrenocortical cells cultured in vitro. An increased number of lipid intramatrix osmiophilic inclusions was also found (Kjaerheim, 1 9 68~;Youson, 1973b; Fritsch et al., 1977). On the basis of subjective examinations of electron micrographs many investigators reported that chronic adrenocortical cell activation caused an increase in the number of mitochondria per cell both in vivo (Youson, 1973b; Buuck et a / . , 1976) and in v i m (Milner, 1972b, 1973; Kahri et al., 1976a; Salmenpera and Kahri, 1977), as well as in their volume (Buuck et a l . , 1976). Kimmel et al. (1974) described an analogous finding in rats bearing a corticosterone-secreting Snell 494 adrenocortical carcinoma. Stereology has partly confirmed these results, showing that the volume of the mitochondrial compartment significantly increases as a function of the duration of adrenal stimulation (Nussdorfer et al.,

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1971a, 1974a; Nussdorfer and Mazzocchi, 1972a, 1973; Nickerson, 1975; Rohr et af., 1975), and that this increment is due to the increase both in the average volume of the organelles (Nussdorfer et a1 ., 1971a, 1974a; Sekiyama and Yago, 1972; Yago et af., 1972) and in the number of mitochondria per cell (Nussdorfer e t a l . , 1971a, 1974a; Canick and Purvis, 1972; Sekiyama and Yago, 1972; Yago et al., 1972). However, Canick and Purvis (1972) showed a decrease in the mitochondria1 average volume after ACTH administration to hypophysectomized rats, and Salmenpera (1976) confirmed this in fetal rat adrenocortical cells cultured in vitro. In contrast, Rohr et aZ. (1975) were not able to demonstrate a change in this parameter in stressed animals. There is much evidence that chronic activation of adrenocortical cells provokes a noticeable increase in the SER membranes, which frequently occupy large cytoplasmic portions lacking any other organelles (Kjaerheim, 1968c; Nickerson et al., 1970; Berchtold, 1970a,b, 1975b; Canick and Purvis, 1972; Sekiyama and Yago, 1972; Youson, 1973b; Kimmel et af., 1974; Kovacs, 1974; Rohr et al., 1975; Buuck et af., 1976); sometimes SER tubules are arranged around mitochondria to form structures resembling cisternae fenestratae (Nussdorfer and Mazzocchi, 1972a). Analogous findings have been reported in unilaterally adrenalectomized rats (Seelig and Rennels, 1969) and in hyperplastic adrenals from patients with Cushing’s syndrome (Mitschke et a l . , 1971). Using stereological techniques Nussdorfer and associates (197 1a, 1974a; Nussdorfer and Mazzocchi, 1972a, 1973) and Nickerson (1975) confirmed these data in both rat and mouse adrenocortical cells; Nussdorfer’s group also showed that the SER surface increased as a function of the duration of the stimulating treatment. SER proliferation has also been reported to occur during the ACTH-elicited differentiation of both rat and human adrenocortical cells cultured in vitro (Kahri, 1966, 1970; Armato and Nussdorfer, 1972; MiIner, 1972b, 1973; O’Hare and Neville, 1973a; Armato et al., 1974a,b, 1975) (Figs. 19 and 20). Moreover, in the human adrenocortical cells cultured in vitro , which contained numerous stacks of granular cisternae, SER proliferation was coupled with a dramatic RER decrease (Armato et af.,1974b; Kahri et af., 1976a). Similar findings have been reported in the fasciculata-reticularis border cells of the Mongolian gerbil adrenal cortex following ACTH administration (Nickerson, 1970) and in fetal human adrenocortical cells during their morphological-functional differentiation

FIG.19. Adult rat adrenocortical cell cultured in virro in the absence of ACTH. Elongated mitochondria contain laminar cristae; RER profiles (arrows) and free ribosomes are numerous. X 20,300. FIG.20. Adult rat adrenocortical cell cultured in vitro after a 7-day exposure to ACTH; ovoid mitochondria display vesicular cristae, and the SER is well developed. Id, Lipid droplet; arrows, microtubules. ~ 2 1 , 6 0 0 .

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(McNutt and Jones, 1970). Other studies have described an increased number of free ribosomes and polysomes in chronically stimulated adrenal cells both in vivo (Kjaerheim, 1968c; Youson, 1973b) and in vitro (Kahri et al., 1976a). Considerable disagreement exists regarding the changes in the lipid compartment following chronic adrenal activation; the greatest number of studies has reported a conspicuous decrease in the number and volume of lipid droplets (Kjaerheim, 1968c; Nickerson et a l . , 1970; Mitschke et al., 1971; Canick and Purvis, 1972; Sekiyama and Yago, 1972; Armato et a l . , 1974b; Berchtold, 1975b; Fritsch et a l . , 1977). However, some qualitative (Kahri et ul., 1970, 1976a; Szabo et a l . , 1970; O’Hare and Neville, 1973a; Kimmel et a l . , 1974; Buuck et a l . , 1976) and stereological quantitative (Nussdorfer et a l . , 197 1a, 1974a; Nussdorfer and Mazzocchi, 1972a, 1973) investigations demonstrated an evident increase in the volume of the lipid compartment of rat adrenocortical cells, at least after prolonged stimulation. By contrast, Nickerson (1975) was not able to demonstrate by stereological methods a significant change in this parameter in the adrenal zona fasciculata cells of mice bearing an ACTH-producing pituitary tumor. The Golgi apparatus is commonly found to hypertrophy in chronically activated adrenocortical cells (Kjaerheim, 1 9 6 8 ~ ;Berchtold, 1970a,b, 1972; Nussdorfer et a l . , 1971a; Nickerson, 1972, 1975; Nussdorfer and Mazzocchi, 1972a, 1973; Youson, 1973b; Fritsch et a l . , 1977); a high number of coated vesicles is frequently described in the Golgi area (Fig. 21). Similar findings have also been reported in in vitro cultured adrenocortical cells (Kahri el a l . , 1970; Armato and Nussdorfer, 1972; Armato et al., 1974b). Although P-glycogen particles are very scarce in normally functioning adrenocortical cells, some reports hold that glycogen is completely depleted by chronic ACTH treatment (Armato and Nussdorfer, 1972; Youson, 1973b; Armato et al., 1974b; Kahri et a l . , 1976a). Some lines of evidence indicate that lysosomes (dense bodies) increase in number after chronic ACTH administration (Nickerson, 1970, 1972; Szabo et a l . , 1970; Nussdorfer et a l . , 1971a). Accordingly, Szabo et a l , (1 967) showed an increase in the number of acid phosphatase-positive dense bodies in hypophysectomized rat adrenals after ACTH administration. Recently the effects of chronic activation on the number of peroxisomes were investigated in Triturus cristutus interrenal cells by Berchtold (1 975b), who demonstrated a noticeable increase. Microperoxisome proliferation has also been reported to parallel SER increase ~

FIG. 21. Cell from the zona fasciculata of a rat treated for eight consecutive days with ACTH. The juxtanuclear Golgi apparatus shows noticeable hypertrophy. X 19,800. FIG.22. Adrenocortical cell from the zona fasciculata of an ACTH-treated rat, showing an increased number of coated pits and pinocytotic vesicles at the plasma membrane. G, Golgi apparatus. X34,ooO.

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during morphological-functional differentiation of fetal guinea pig adrenocortical cells (Black and Bogart, 1973). Other ultrastructural changes in chronically stimulated adrenocortical cells involve plasma membrane specializations: (1) Microvilli seem to increase in number, forming an elaborate meshwork both in vivo (Kjaerheim, 1968c; Berchtold, 1970a,b; Nussdorfer et al., 1971a; Youson, 1973b; Kovacs, 1974) and in vitro (Kahri, 1966; Kahri et al., 1970, 1976a; Milner and Villee, 1970; Armato and Nussdorfer, 1972; O'Hare and Neville, 1973a; Stark et a t . , 1975). However, Milner (1972b) did not observe similar microvillar hypertrophy in fetal rat adrenocortical cells cultivated in vitro after CAMP exposure. (2) Micropinocytotic vesicles and coated pits are frequently seen to increase in number (Kjaerheim, 1968c; Nussdorfer ef al., 1971a; Youson, 1973b) (Fig. 22).

D. LONG-TERM INHIBITION The ultrastructural changes induced in adrenocortical cells by chronic suppression of the hypothalamohypophyseal axis are almost completely opposite those described in Section III,C. Adrenocortical cells show a significant decrease in average volume and frequently shrink, assuming irregular shapes, both in the rat (Sabatini ef al., 1962; Kimmel et al., 1974) and in human patients with a hydrocorticosterone-secreting adrenal tumor (Szabb et al., 1975). Similar findings have been reported for their nuclei, some of which sometimes show signs of pycknosis (Kjaerheim, 1968b; Nussdorfer, 1970c; Fujita, 1972; Berchtold, 1973, 1975b; Kimmel et al., 1974; Tchen et al., 1977). Regressive changes lead to the degeneration and death of some cells which, according to Tchen et al. (1977), was more rapid from the third to the seventh day after ACTH suppression. The stereological investigations of our group confirmed these data (Nussdorfer, 1970c; Nussdorfer and Mazzocchi, 1970, 1972a, 1973; Nussdorfer et al., 1975b). A noticeable decrease in nucleolar size was also found both in vivo (Kjaerheim, 1968b) and in vitro (Kahri, 1966; Kahri et a l . , 1970; Armato and Nussdorfer, 1972). Chronic ACTH suppression is found to cause extensive changes in the mitochondria; the organelles frequently assume bizarre shapes (Sharawy and Penney, 1973; Nussdorferet al., 1975b) and contain mainly tubular cristae, being apparently quite similar to the mammalian zona glomerulosa mitochondria (Sabatini et al., 1962; Picheral, 1970; Fujita, 1972; Berchtold, 1973; Nickerson, 1973; Kimmel et al., 1974). The transformation of vesicular cristae into tubular or laminar cristae is also usually described in rat (Armato and Nussdorfer, 1972; O'Hare and Neville, 1973a) and human adult (Armato et al., 1974a,b) adrenocortical cells cultured in vitro in the absence of ACTH. A decrease in the number and volume (Sekiyama and Yago, 1972), as well as in the surface area of vesicular cristae, was stereologically demonstrated in chronically inhibited ad-

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renocortical cells (Nussdorfer , 1970c; Nussdorfer and Mazzocchi, 1970; Nickerson, 1973; Nussdorfer er al., 1975b; Rohr er al., 1975). Several reports indicate that the average mitochondrial volume increases after chronic suppression of the hypothalamohypophyseal axis (Sekiyama et a l . , 1971; Canick and Purvis, 1972; Sekiyama and Yago, 1972), and giant mitochondria have been frequently described (Volk and Scarpelli, 1966; Kjaerheim, 1968b; Picheral, 1970; Fujita, 1972; Nickerson, 1972; Sharawy and Penney, 1973; Nussdorfer et a l . , 1975b). However, Rohr et al. (1975) stereologically demonstrated a significant decrease in the average mitochondrial volume in rats treated for 3 days with cortisol. Recently Nussdorfer and co-workers (1975b) showed that this parameter remained at a plateau up to the third day of dexamethasone treatment of adult rats and then decreased as a function of the number of days of ACTH suppression. Good agreement exists regarding the number of organelles per cell, which seems to decrease significantly in relation to the duration of the adrenocortical cell’s inhibition (Picheral, 1970; Canick and Purvis, 1972; Sekiyama and Yago, 1972; Mitschke and Saeger, 1973; Kimmel et al.,1974; Nussdorfer et al., 1975b). The SER decreases considerably in suppressed adrenocortical cells (Kjaerheim, 1968b; Picheral, 1970; Berchtold, 1973; Canick and Purvis, 1972; Fujita, 1972; Nickerson, 1972; Sekiyama and Yago, 1972; Mitschke and Saeger, 1973; Kimmel er al., 1974; Bhattacharyya er al., 1975a,b), and this finding has been confirmed by stereology (Nussdorfer, 1970c; Nussdorfer and Mazzocchi, 1970; Nickerson, 1973; Nussdorfer et al., 1975b; Rohr et d.,1975). Kjaerheim (1968b) reported that in the interrenal cells of dexamethasone-injected domestic fowls there was a decrease in the RER stacks and in free ribosomes; however, human adult (Armato et al., 1974a,b) and fetal (Kahri and Halinen, 1974) adrenocortical cells cultured in virro in the absence of ACTH showed a noticeable number of RER profiles, and hypophysectomy did not seem to affect RER concentric whorls in the zona fasciculata-reticularis border cells of Meriones unguiculatus adrenals (Nickerson, 1972). Contrasting findings have been reported with regard to the lipid compartment. An increase in the number and volume of the lipid droplets has been the more frequent report (Kjaerheim, 1968b; Berchtold, 1972, 1975b; Canick and Purvis, 1972; Fujita, 1972; Sekiyama and Yago, 1972; Nickerson, 1972; Mitschke and Saeger, 1973; Bhattacharyya et al., 1975a,b; Szabb er al., 1975). Other studies showed no changes in the lipid compartment (Yoshimura et al., 1968; Nickerson, 1973); our stereological findings indicated that the volume of the lipid compartment did not change during the first days of adrenal suppression and then displayed a slow decrease (Nussdorfer er al., 1975b) Much evidence indicates that chronic adrenal inhibition induces evident atrophy of the Golgi apparatus (Kjaerheim, 1968b; Nussdorfer, 1970c; Berchtold, 1973; Bhattacharyya et al., 1975a,b; Nussdorfer et al., 1975b). However, Nic-

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kerson (1973) reported no changes in this organelle in the atrophic adrenocortical cells of rats beating a corticosterone-secreting Snell 494 adrenocortical carcinoma. In chronically inhibited adrenocortical cells P-glycogen particles accumulate in small clumps (Nussdorfer and Mazzocchi. 1970; Ueberberg, 1971, 1972; Fujita, 1972; Nickerson, 1973) (Fig. 23), and a similar process has been described in adult human adrenocortical cells cultured in vitro in the absence of ACTH (Armato et al., 1974b). Lysosomes usually increase in number (Kjaerheim, 1968b; Nickerson, 1972; Sharawy and Penney, 1973), although Szabo et al. (1967) reported opposite findings. Berchtold (1975b) showed a decrease in the number of peroxisomes in the interrenal cells of hypophysectomized T . cristatus . Microvilli and coated pits appear to be occasional features of chronically inhibited adrenocortical cells (Nussdorfer and Mazzocchi, I970), and microvilli are observed only infrequently in adrenocortical cells from adult rats (Armato and Nussdorfer, 1972; O’Hare and Neville, 1973a) and adult humans (Armato et ul., 1974b3 cultured in vitro in the absence of ACTH.

FIG. 23. Cell from the zona fasciculata of a dexamethasone-treated rat. Note the intracytoplasmic accumulation of P-glycogen particles. L, Lysosome X22,OOO.

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IV. Morphological-Functional Correlations in Adrenocortical Cells In this section we correlate morphological featufes of normal and treated adrenocortical cells with their functional activity in light of the well-known biochemical pathways of steroid hormone synthesis and of the numerous investigations of the mechanism(s) of ACTH action. Here we consider articles dealing with the subcellular topology of enzymes of steroid synthesis, as well as with the mechanism(s) of the ACTH-provoked changes in the ultrastructure of adrenocortical cells, and discuss the possible involvement of the various subcellular organelles in steroid hormone synthesis. Hypotheses concerning the mechanism(s) of hormone release from adrenocortical cells are considered in Section V. A. SUBCELLULAR LOCALIZATION OF THE ENZYMES OF STEROID SYNTHESIS Several reviews are available on this subject, and the reader should consult them for comprehensive references (Tamaoki, 1973; Samuels and Nelson, 1975; Sandor et al., 1976). The bulk of the investigations carried out with differential centrifugation techniques, although frequently without accurate morphological monitoring of the subcellular fraction examined, indicates that the enzymes of steroid synthesis are located in the SER and mitochondria. The various steps in the biosynthesis of the principal steroid hormones secreted by adrenocortical cells under ACTH control ( i s , corticosterone and cortisol), as well as the more widely accepted subcellular localization of the enzymes involved, are depicted in Fig. 24. Briefly, free cholesterol, built up from acetate in the SER or taken up from the bloodstream, enters the mitochondria, where it is transformed into pregnenolone by hydroxylating enzymes which cleave its side chain. Pregnenolone is then transformed into progesterone by 3P -hydroxysteroid-A5-dehydrogenaselocated in the SER, where l7a-hydroxylase and 21-hydroxylase also convert progesterone into 11-deoxycorticosterone or 17a -hydroxydeoxycorticosterone. These intermediate products again penetrate into the mitochondria, where 1lphydroxylase transforms them into corticosterone and cortisol, respectively. This classic topology of steroid synthesis enzymes has been partly questioned in cytochemical investigations which showed that 3P-hydroxysteroid-A5-dehydrogenase in both calf (Snoeijing and Hardonk, 1973) and rat (Berchtold, 1977) adrenocortical cells was located not only in SER membranes but also in the mitochondrial cristae (Fig. 25). These findings lend support to the contention that 30 -hydroxysteroid-A5-dehydrogenase activity of the mitochondria1 fraction is not due only to microsomal contamination (Basch and Finegold, 1971; Kream and Sauer, 1976), as suggested for other steroid-producing cells by Sulimovici et al. (1973).

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acetate

SMOOTH ENDOPLASMIC RETICULUM

1

cholesterol c -synthesizing enzymes

a

MITOCHONDRIA

cholesterol cholesterol side-chain

c -cleaving

enzymes

pregnenolone

1-

a-

isomerase A5-3P- hydmxysteroid dehydrogenase &I5

1 i4eoxycorticostemne

-a

11-deoxycor t is01 I

~ phydmrylase -

CORTICOSTERONE

CORTISOL in- hydroxylase

ts-dehydrogenese

ALDOSTERONE FIG.24. Simplified scheme illustrating the pathways of the synthesis of the principal corticosteroid hormones and the subcellular topology of the enzymes involved.

From this brief account, it is clear that intermediate product molecules frequently switch between the SER and mitochondria before being transformed into definitive hormonal products. The morphological counterpart of this va et vient may be the frequent close interrelationships existing among mitochondria, lipid droplets, and SER membranes. In this context, the myelin-like blebs of the outer mitochondrial membrane, which sometimes penetrate lipid droplets (see Section II,A, l ) , the occasional wrapping of mitochondria around lipid droplets (Kadioglu and Harrison, 1972), and the arrangement of SER membranes to form structures around mitochondria resembling cisternae fenestratae (see Section III,C) must be borne in mind. As for the mitochondrial blebs, Merry (1975) claims that they may be significant in the transport of cholesterol from lipid droplets to the inner mitochondrial membrane, thus facilitating side-chain cleav-

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FIG.25. Ultrastructural localization of ~-hydroxysteroid-A5-dehydrogenase activity in an adrenocortical cell from a rat zona fasciculata. The reaction is conspicuous in the SER and very weak in the mitochondria; the arrows indicate a few grains located in the mitochondria1 cristae. X 4 I ,ooO. (Courtesy of J. P. Berchtold.)

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age of cholesterol to pregnenolone. However, we stress that the most probable hypothesis is that mitochondria1 blebs are fixation artifacts. The unusual content of adrenocortical mitochondria in steroid-hydroxylating enzymes associated with their cytochrome P-450 electron transport chain (for review, see Tamaoki, 1973; Samuels and Nelson, 1975) is related to the typical morphology of the cristae. Allmann et al. (1970) advanced the hypothesis that the repeating units in the ballooned-out segment of the cristae contain the steroidogenic electron transfer chain, whereas the repeating units in the collapsed sections contain the classic chain coupling electron transfer to ATP synthesis. Wang et al. (1974) and Pfeiffer et al. (1976), on the basis of studies of the phospholipid composition of bovine adrenocortical mitochondria and of a partially purified cytochrome P-450 fraction derived from mitochondria, suggested that vesicular cristal structure was a reflection of altered phospholipid composition, which in turn reflected the presence of the cytochrome P-450 electron transport chain. This suggestion would be reasonable if the zona glomerulosa mitochondria of almost all animal species (see Idelman, 1970, for review), as well as mitochondria from the zona fasciculata of many eutherian species and from the interrenal cells of numerous lower vertebrates (see Section II,A and B), did not contain only tubular cristae, in spite of their active involvement in steroid synthesis. Moreover, it is recalled that zona fasciculata mitochondria from guinea pig adrenals, although engaged in cortisol synthesis, have cristae of the laminar type (see Section II,A,9).

B. MECHANISMS OF ACTION OF ACTH As pointed out in Section 11, ACTH exerts tropic and trophic actions on the adrenal cortex, and both effects are mediated, at the intracellular level, by CAMP. The binding of ACTH to a cell membrane receptor activates adenylyl cyclase, which in turn catalyzes the formation of CAMPand pyrophosphate from ATP (for review, see Robison et al., 1971). CAMP, by displacing an inhibitory protein, activates a protein kinase (for review, see Halkerston, 1975) which exerts the following effects on adrenocortical cells. 1. Activation of glycogen phosphorylase, which promotes glycogenolysis and increases the intracellular concentration of glucose 1-phosphate and glucose 6-phosphate, thus increasing the substrate available for glucose-6-phosphate dehydrogenase. Subsequent activation of the pentose shunt results in increased availability of NADPH, a rate-limiting coenzyme in steroidogenesis (review in Haynes, 1968). 2. Activation of cholesterol esterase which, by hydrolyzing cholesterol esters stored in the lipid droplets (Friihlinget al., 1969; Moses et al., 1969; Sand et a l , , 1972), increases the intracellular free cholesterol available for steroid synthesis

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33 1

(Garren et al., 1965, 1971; Davis, 1969; Simpson et al., 1972; Naghshined et at., 1974; Trzeciak and Boyd, 1974; Sandor et a t . , 1976; Wallat and Kunau,

1976; Beckett and Boyd, 1977; Pittman and Steinberg, 1977). 3. Activation (phosphorylation) of ribosomes, resulting in increased translation of a rapid-turnover protein (half-life 10-12 minutes) (Garren et a l . , 1971) which affects steroidogenesis mainly at the mitochondrial level. The possibility is advanced that such a protein, which seems to activate cholesterol side chaincleaving enzymes (Farese, 1967, 1969), is involved in cholesterol or pregnenolone transport across the mitochondrial membrane. In this connection it is worth noting that cholesterol (Kanet al., 1972; Brownie and Paul, 1974, Lefevre and Saez, 1977) or pregnenolone (Kream and Sauer, 1977; Strott, 1977) carrier proteins have been isolated from bovine, guinea pig, and rat adrenal homogenates. 4. Some lines of evidence suggest that ACTH can enhance the uptake of plasma cholesterol into adrenocortical cells (Dexter et al., 1967a,b, 1970). Analogous results were found in adult rat adrenocortical cells cultured in vitro (Armato and Nussdorfer, 1972). It was demonstrated that this process did not depend upon protein synthesis, since it was not blocked by cycloheximide (Dexter et al., 1967a,b, 1970). It has not yet been determined whether or not this effect of ACTH is mediated by CAMP. These four effects of ACTH are presumably involved in its tropic action on adrenocortical cells. It is possible that the trophic action of the hormone, mediated by CAMP (Ney, 1969; Nussdorfer and Mazzocchi, 1972a, 1973), requires stimulation of the synthesis of both structural and enzymic proteins at the transcriptional and translational levels (Farese, 1968; Masui and Garren, 1970; Fuhrman and Gill, 1974, 1976, 1977). Recently the mechanism of ACTH in the maintenance of normal adrenal morphology and function was reviewed by Tchen et al. (1977). These investigators suggest that, in the normal adrenal cortex there is a normal rate of DNA damage, the repairing of which is dependent on an ACTH-CAMP activated DNA repairing enzyme. This process allows adrenal DNA to carry out its proper template activity.

c. THEMECHANISMS AND THE FUNCTIONAL SIGNIFICANCE OF THE ULTRASTRUCTURAL CHANGES IN HYPERFUNCTIONING AND HYPOFUNCTIONING ADRENOCORTICAL CELLS The data detailed in Section 111 show that endoplasmic reticulum, mitochondria, and lipid droplets are the subcellular organelles which display the most conspicuous variation in response to functional changes in adrenocortical cells. In addition we stress that stereology has demonstrated that the quantitative changes in the SER and mitochondria account almost completely for the cell

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volume increase or decrease in chronically stimulated or inhibited adrenal glands (Nussdorfer and Mazzocchi, 1972a; Nussdorfer et a l . , 1975b). Before discussing the functional significance of the changes in these organelles, we briefly consider the numerical changes in the zona fasciculata cell population observed after prolonged stimulatory or suppressive treatments (see Section II1,C and D). Chronic ACTH administration induces the proliferation of adrenocortical cells, and in fact some studies show that ACTH stimulates DNA replication in the adrenal gland in vivo (Imrie el al., 1965; Farese, 1968; Ney, 1969; Masui and Garren, 1970; Nussdorfer et al., 1974b; Schulster, 1974; Saez et a!., 1977). However, it has not yet been completely determined whether or not ACTH elicits the proliferation of zona fasciculata cells or stimulates mitotic activity in the zona glomerulosa and consequently the entry of newly formed cells into the zona fasciculata. This uncertainty reflects that concerning cytogenesis in the adrenal cortex; a discussion of this topic is beyond the scope of this article, and readers should consult the excellent review article by Long (1975), which deals with cell migration and functional zonation theories. Prolonged suppressive treatment causes the death of numerous zona fasciculata cells. According to Tchen et al. (1977), the adrenocortical cell death in the absence of ACTH may be the result of “template damage” (see Section IV,B), which impairs protein synthesis. Nuclear pycknosis found in chronically inhibited adrenocortical cells also suggests depressed RNA synthesis, since it is well known that the more condensed heterochromatin is not available for transcription. According to Tchen’s group, blockage of or reduction in protein synthesis provokes membrane damage, resulting in cell lysis without the involvement of lysosomes. 1. Endoplasmic Reticulum Section I11 illustrated that the SER, although it does not show changes in acutely stimulated or depressed adrenocortical cells, noticeably increases or decreases in chronically activated or inhibited adrenals. The ACTH-induced increase in SER surface implies a conspicuous enhancement of the synthesis of both structural and enzymic protein moieties which are to be assembled in the newly formed membranes. Numerous reports indicating that chronic ACTH treatment elicits nuclear DNA-dependent RNA and protein synthesis (see Section IV,B) correlate well with this view, which is also supported by studies showing that treatment with actinomycin D, puromycin (Kahri, 1968), or cycloheximide (Kahri, 1971) inhibits the ACTH-provoked SER proliferation in cultured fetal rat adrenocortical cells. It is recalled that changes in the nuclear and nucleolar size may be coupled with enhanced or depressed transcriptional processes (Stark et al., 1965; Palkovitz and Fischer, 1968). This is confirmed by the observation that the

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actinomycin-induced blockage of nuclear DNA-dependent RNA polymerase inhibits the ACTH-elicited hypertrophy of the nucleus and nucleolus in fetal rat adrenocortical cells cultured in vitro , while this does not occur with inhibitors of cytoplasmic translational processes (e.g., cycloheximide and puromycin) (Kahri, 1968, 1971). The bulk of the investigations on the mechanism(s) of SER proliferation were concerned with the hepatocytes of fetal, newborn, and phenobarbital-treated animals. Recently Cardell (1977) reviewed this subject and affirmed that the results of these studies could be extended to other cell types. Dallner et al. (1966a,b), studying developing rat hepatocytes by morphological and biochemical techniques clearly stated that SER membranes derived from the rough component of the endoplasmic reticulum. These and other similar studies have rendered reasonable the view that endoplasmic reticulum membranes are built up in the RER and then transferred to (or transformed into) the SER (Jones and Mills, 1973) although, as stressed by Cardell (1977), caution must be used since the enzymes involved in the synthesis of phospholipid moieties seem to be located exclusively in the SER (Higgins, 1974). This hypothesis was extended to the adrenocortical cells of the human fetus (McNutt and Jones, 1970; Fujita and Ihara, 1973) and fetal guinea pig (Black, 1972). However, it cannot be transferred sic et simpliciter to the ACTH-induced SER increase in adrenocortical cells of almost all adult animal species, since true RER has been found only in the adult human adrenal cortex (see Section 11,A,2). In the adrenocortical cells of species lacking RER cisternae, it is conceivable that temporary attachments of ribosome-polysomes to the smooth tubules may account for both the maintenance of and increase in SER. The increase in the number of ribosomes and polysome aggregates induced by short- or long-term ACTH stimulation (see Section II1,B and C), which seems to precede SER proliferation (Kahri et a l . , 1970), is in keeping with this view. The mechanism of SER proliferation was investigated in human adult adrenocortical cells cultured in vitro (Armato et d.,1974b), as well as in M . unguiculutus zona fasciculata-reticularisborder cells (Nickerson, 1970, 1972). In both an inverse proportion between SER and RER was found, since SER proliferation was coupled with a dramatic decrease in RER stacks. Recently Kahri et al. (1976a) confirmed this in human fetal adrenocortical cells cultured in vitro. Dallner and associates (1966a) proposed three processes to explain SER proliferation: (1) Attached ribosomes detach from the endoplasmic reticulum tubules after the synthesis of new membranes has been accomplished: (2) newly synthesized proteins are assembled into a definitive membrane at the ends of RER cisternae, and the newly built-up membrane “flows” from the RER to the SER at their points of continuity; (3) newly synthesized proteins are assembled into a definitive membrane between groups of attached ribosomes actively engaged in their synthesis.

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The first process seems to be no longer likely, at least in human adrenocortical cells cultured in vitro and treated with ACTH, since stereology showed that, in spite of the great increase in SER, the quantity of RER per cell remained unchanged (Armato et al., 1978), which suggests that an RER decrease in ACTHtreated cultured cells is only apparent, because of the dilution of RER in the hypertrophic cytoplasm. Armato et al. (1974b) showed that, up to the second day of treatment of cultures with ACTH, the space between adjacent polysomes attached to the endoplasmic reticulum membranes did not vary, which seems to exclude that the third process was operative at this experimental time. In addition, in cultured human adrenocortical cells undergoing differentiation, conspicuous RER stacks and SER tubules, although displaying many points of continuity, were segregated in distinct cytoplasmic areas. These observations suggest that the second mechanism proposed by Dallner and co-workers was involved in the onset of adrenocortical cell morphological differentiation. The third mechanism, however, might underlie the tumultuous proliferation of SER occurring after prolonged treatment of the cultures with ACTH; in fact, after 8 days of exposure to ACTH, the RER was completely disaggregated and consisted of a few tubules embedded in a well-developed mass of SER. Moreover, the average distance between two adjacent polysomes bound to the endoplasmic reticulum tubules was significantly increased (364 f. 67 versus 1054 +- 148 A, p < 0.01). These data fit well with the suggestion of McNutt and Jones (1970) that short, rough tubules are engaged in the rapid synthesis of SER membranes, whereas the more ordered stacks of rough cisternae are involved in SER maintenance, It should be pointed out, however, that in the adrenocortical cells of nonhuman species short, rough tubules are involved in both processes. In normally functioning adrenocortical cells the right quantity of SER is maintained by an adequate level of circulating ACTH and is an expression of the balance between the rates of assembly and degradation of SER membranes. Since the half-life of adrenocortical microsomal proteins ranges between 5 and 6 days (Canick and Villee, 1974; Ichii et a l . , 1974) and that of the smooth membranes, calculated by Purvis et al. (1973) from the data of Nussdorfer et al. (1971a), was found to average about 3 days, it is conceivable that in animals in which the hypothalamohypophyseal axis was chronically suppressed the considerable and rapid decrease in the SER might be interpreted as an expression of the normal degradation rate of SER membranes in the presence of a block in the synthesis of new membranes (Nussdorfer et al., 1975b). Based on what has been demonstrated in the hepatocytes of phenobarbitaltreated animals (Ernster and Orrenius, 1973; Jones and Mills, 1973), the accumulation of SER in chronically activated adrenocortical cells may depend not only on increased synthesis of smooth membranes, but also on a slowing down of their degradation rate. In support of this view are investigations indicating that chronic

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ACTH treatment significantly increases the half-life of microsomal proteins in rat adrenocortical cells (Purvis et a l . , 1973; Canick and Villee, 1974; Ichii et a l . , 1974). 2. Mitochondria The morphological changes in mitochondria elicited by short-term stimulation or inhibition of adrenocortical cells are slight; only an increase or decrease in the diameter of the cristae was reported (see Section 111,A). Although it is difficult to correlate this finding with the changed functional activity of the organelles, it is recalled that recent evidence showed that the transformation of 11deoxycorticosterone to corticosterone (see Section IV ,A) occurred inside the vesicular cristae (Rydstrom et a / ., 1976). The demonstration of osmium deposits at the level of the mitochondrial cristae in hyperosmicated adrenocortical cells, according to Friend and Brassil (1970), confirms the presence of steroid hormones inside them. Elaboration of this point requires further correlated stereological and biochemical investigations. Section I11 showed that there is good agreement among various research groups concerning chronically induced mitochondrial changes; the volume of the mitochondrial compartment and the surface of the cristae significantly increase or decrease, respectively, according to the long-term stimulation or inhibition of adrenocortical cells. The ACTH-provoked increase in the surface of mitochondria] inner membranes can be correlated in adrenocortical cells cultured in vitro with the assumption by the mitochondrial laminar cristae of a vesicular configuration, which is the morphological arrangement allowing the greatest concentration of membrane surface in the smallest volume of mitochondrial matrix (Kahri, 1966; Armato and Nussdorfer, 1972; Armato et a l . , 1974b; Salmenpera, 1976; Nussdorfer et a l . , 1977a). The ACTH-induced proliferation of mitochondrial inner membranes requires a parallel increase in the synthesis of structural and enzymic proteins as well as of the phospholipid moieties to be assembled in the newly formed membrane. It is well accepted that the synthesis of mitochondrial proteins is directed by nuclear and mitochondrial DNA (review in Ashwell and Work, 1970); some lines of evidence show that ACTH, in addition to eliciting nuclear DNA-dependent protein synthesis (see Section IV,B), also stimulates mitochondrial DNA-dependent RNA and protein synthesis (Ichii et a l . , 1970; Nussdorfer and Mazzocchi, 1971a, 1972b, 1973). Moreover, Ichii (1970) reported that ACTH caused a marked stimulation of the rate of incorporation of ~ h o l i n e - ~into H mitochondrial phospholipids of rat adrenals. That proliferation of the mitochondrial inner membranes is dependent upon adrenocortical RNA synthesis may also be inferred from the fact that the ACTH-induced rearrangement of tubular cristae into vesicular cristae is blocked by exposure of the cultures to actinomycin D (Kahri, 1968) or 5bromodeoxyuridine (Kahri et a l . , 1976b).

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Mitochondria1 DNA can code only a small number of proteins of low molecular weight (for review, see Ashwell and Work, 1970; Tzagoloff et al., 1973), and therefore the bulk of mitochondrial proteins is translated onto cytoribosomes and then transferred to the mitochondria (Kadenbach, 1967). The same also seems to apply to the phospholipid components of the mitochondrial membranes, since the enzymes involved in their synthesis are exclusively located at the SER level (see Cardell, 1977, for references). However, the assembly of newly synthesized proteins (and perhaps phospholipids) is possible only in the presence of protein molecules coded for by mitochondrial DNA (Henson et al, 1968; Mahleret al., 1968). Recent data from Purviset al. (1973) have confirmed this view as far as the adrenal cortex is concerned. The importance of the integrity of both nuclear and mitochondrial DNA-dependent protein-synthesizing machinery in the ACTH-induced increase in the surface of adrenocortical mitochondrial cristae was also evident in numerous in vitro investigations made by Kahri’s group and Milner’s group. It was found in fact that the effects of ACTH on cultured cell mitochondria were inhibited by both specific inhibitors of cytoribosomal protein synthesis (e.g., puromycin and cycloheximide) (Kahri, 1968, 1971) and inhibitors of mitochondrial transcriptional and translational processes (e.g., chloramphenicol and ethidium bromide) (Kahri, 1970; Milner, 1971, 1972a; Salmenpera and Kahri, 1977); the latter, however, did not interfere with the ACTH-induced SER proliferation. Oberholzer and Rohr (1973, by administering large doses of chloramphenicol to rats for 5 days, demonstrated a decrease in the surface density of mitochondrial inner membranes. Preliminary studies in our laboratory have extended these investigations using doses of chloramphenicol causing about 45-50% inhibition of l e ~ c i n e - ~incorporation H into adrenocortical mitochondrial proteins. It was stereologically demonstrated that this treatment caused a significant decrease in the surface of the mitochondrial cristae without affecting the SER tubule surface (Mazzocchi ef al., 1977b). Nussdorfer and associates (1975b) proposed that, in normally functioning adrenocortical cells, the integrity of the mitochondrial cristae was maintained by the ACTH-controlled balance between the rates of synthesis and degradation of the mitochondrial inner membranes and that blockage of new membrane synthesis in the absence of ACTH could easily explain the decrease in the mitochondrial cristal surface in hypothalamohypophyseal chronically suppressed animals. Nussdorfer et al. (1975b) showed that, in adrenocortical cells of chronically dexamethasone-treated rats, the rate of the decrease in the surface of mitochondrial cristae was lower than that of SER membranes, a finding which can be explained by the fact that the half-life of adrenocortical mitochondrial proteins is higher than that of microsomal proteins (7-8 versus 5-6 days) (Canick and Villee, 1974; Ichii et al., 1974). Recent research showed that chronic ACTH treatment significantly enhanced the half-life of mitochondrial proteins (Canick and Villee, 1974; Ichii et al.,

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1974; Neri et af., 1978) and phospholipids (Ichii et af., 1971). Therefore it is reasonable to assume that the increase in the surface of mitochondrial inner membranes in chronically stimulated adrenocortical cells may well be the expression not only of increased assembly of new cristal membranes but also of a decreased breakdown of these membranes. Stereological research has shown that chronic ACTH treatment causes a noticeable increase in the number of mitochondria per adrenocortical cell, but there is considerable disagreement concerning the changes in the average volume of the organelles (see Section 111,C). The results of the various investigators concerning mitochondrial average volume can be considered only apparently conflicting, based on the following data. 1. The increase in the number of mitochondria per cell is not due to the assembly of new organelles, but almost exclusively to the division of preexisting mitochondria; in fact, in chronically ACTH-stimulated adrenocortical cells numerous images which suggested mitochondria division were observed both in vivo and in vitro; these included complete mitochondria partition by a cristal septum associated with a mono- or bilateral continuation of the outer envelope (Idelman, 1964; Soeder and Themann, 1968; Nussdorfer et af., 1974a, 1977a) (Fig. 26) and, especially in cultured cells, organelles displaying evident strangling in their medial portion (Armato and Nussdorfer, 1972).

FIG. 26. An image suggesting mitochondria1 division (arrows) in a zona fasciculata cell of a rat chronically treated with ACTH. X31,OOO. (From Nussdorfer et al., 1974a).

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2. Canick and Purvis (1972) affirm that adrenocortical mitochondria undergo a growth cycle, increasing in size until they reach 1.5 p m in diameter before dividing into two organelles which begin the growth cycle again. This implies that the proliferation of mitochondria results in a decrease in the average volume of the organelles. As mitochondria possess a complete genetic apparatus (see Ashwell and Work, 1970, for review), it follows that mitochondrial growth is associated with duplication of their DNA. 3. Nussdorfer et al. (1974a) showed that rat zona fasciculata mitochondria average volume increased up to the ninth day of ACTH treatment, without a significant increase in the number of organelles per cell. On the twelfth day a great increase in the number of mitochondria per cell was found, coupled with a significant decrease in their average volume. From the twelfth to the thirty-sixth day the number of mitochondria per cell remained at a plateau, while their average volume again increased. These data, indicating that in ACTH-treated adrenocortical cells mitochondrial proliferation is a discontinuous process, were confirmed for the zona reticularis organelles, which displayed two phases of hypertrophy alternating with two proliferative bursts (on the sixth and twentyfourth days of treatment with ACTH), resulting in a significant decrease in the average volume of the organelles (Mazzocchi et al. 1976a). Analogous findings were described by Fritsch et al. (1977) in X . laevis interrenal cells after hypothalamohypophyseal axis chronic activation, but the proliferative bursts were found to occur on the third and thirty-fifth days. High-resolution autoradiographic studies showed that the ACTH-elicited mitochondrial proliferative burst was preceded by a significant increase in th~rnidine-~H incorporation into the mitochondrial compartment both in vivo , as reported by Nussdorfer et al. (1974b), and in vitro , as reported by Salmenpera and Kahri (1977). The latter, in addition, reported that this effect of ACTH was blocked when the cultures were exposed to ethidium bromide, a finding which demonstrated the validity of autoradiographic determination of mitochondrial DNA duplication. Based on the three lines of evidence considered here, it is likely that the effect of ACTH on the average volume of adrenocortical mitochondria depends upon the duration of the treatment employed. The data reported deserve, however, further consideration; it seems quite well established that in normally functioning adrenocortical cells the mitochondrial population synthesizes DNA at a constant rate, since each individual mitochondrion undergoes a genetic duplication cycle which is not in phase with that of other mitochondria (Nussdorfer and Mazzocchi, 1971a; Nussdorfer et al., 1974a). It is therefore likely that prolonged ACTH treatment, by accelerating the growth and DNA synthesis of adrenocortical mitochondria, induces noticeable synchronization of the growth cycle of the organelles.

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Nussdorfer et al. (1975b) proposed that in normally functioning adrenocortical cells the adequate number of mitochondria per cell is maintained by ACTH, which controls the balance between the rates of mitochondrial proliferation and destruction, and that in the absence of ACTH the number of mitochondria per cell decreases, for the degradative processes prevail over the proliferative ones. Nussdorfer and his associates, working on the assumption that DNA is an “extremely stable molecule, which seems to have no appreciable turnover” (Cleaver, 1967), investigated the half-life of adrenocortical mitochondria as intact units by following the radioactivity decay of the mitochondrial compartment of th~midine-~H-injected rats both by radioautographic and biochemical methods (Mazzocchi et al., 1976b; Nussdorfer et al., 1977~).They found that the half-life of zona fasciculata mitochondria averaged 11 days. Nussdorfer et al. (1975b) showed that in dexamethasone-treated rats the number of mitochondria per cell was reduced one-half in about 3.5-4.0 days. Therefore it follows that the decrease in the number of mitochondria per cell in chronically inhibited adrenocortical cells was too rapid to be accounted for by only the normal degradation rate of the organelles in the absence of the formation of new mitochondria. Furthermore, these workers described an unusual number of degenerate mitochondria and of autophagic vacuoles containing mitochondrial debris in suppressed adrenocortical cells. Based on these data Nussdorfer et al. (1975b) suggested that the decrease in the number of mitochondria in the dexamethasone-treated adrenocortical cells indicated the accelerated destruction of wornout organelles whose DNA-dependent protein synthesis was blocked in the absence of ACTH. Recently, Mazzocchi et af. (1977a) showed that the half-life of adrenocortical mitochondria in rats administered dexamethasone averaged 5 days. Evidence is available that prolonged suppressive treatment induces a decrease in the average volume of single mitochondria (Nussdorfer et af ., 1975b; Rohr et al., 1975), which can be explained as a result of the imbalance between the rates of synthesis and degradation of mitochondrial membranes. However, some articles report that ACTH suppression, at least initially, causes an increase in this parameter, and giant mitochondrial forms have been observed (see Section 111,D). In addition, images suggesting mitochondrial fusion were described in chronically suppressed adrenocortical cells (Canick and Purvis, 1972; Nussdorfer et al., 1975b), and giant mitochondria were also observed in cycloheximidetreated cultured adrenocortical cells (Kahri, 1971) and after chloramphenicol was administered to adult rats (Mazzocchi et a l . , 1977b). Canick and Purvis (1972), following Tandler et al. (1968), proposed that the fusion of mitochondria, which results in a relative decrease in the surface area of the outer mitochondrial envelope, was a mechanism for repairing the deficiency in the synthesis of mitochondrial membranes. This mechanism seems to be quite aspecific, since

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megamitochondria were also found in hepatocytes subjected to various injuries: protein starvation (Svoboda et a l . , 1966), cuprizone treatment (Wakabayashi and Green, 1974; Wakabayashi et al., 1975), ethidium bromide administration (Albring et al., 1973), or chronic dimethylbenz(a)anthracene treatment (Belloni et al., 1978). These observations allowed Nussdorfer et al. (1975b) to propose that, during the first days of dexamethasone treatment, the decrease in the number of zona fasciculata cell mitochondria is due not only to accelerated destruction of the organelles but also to their fusion. This may also account for the fact that the half-life of mitochondria of inhibited zona fasciculata cells, evaluated by isotopic methods, was higher than that calculated from stereological data (5 versus 3.54.0 days). Nussdorfer et al. (1977~)demonstrated that continuous ACTH administration caused a significant increase in the half-life of rat zona fasciculata mitochondria (11 versus 15 days) and hypothesized that the increase in the number of mitochondria per cell in chronically stimulated adrenocortical cells was due not only to the increased proliferation rate of the organelles but also to a slowing down of their rate of degradation. Since chloramphenicol was found to block this effect of ACTH (G. G. Nussdorfer and G. Neri, unpublished results), it is conceivable that the mechanism underlying it involves stimulation of the synthesis of new mitochondrial membranes. 3 . Functional Significance of the Changes in SER and Mitochondria The enzymes of steroid synthesis are located in both the SER and mitochondria (see Section IV,A), and the activity of several of these enzymes was found to be increased or depressed in stimulated or inhibited adrenocortical cells, for example, side chain-cleaving enzymes (Kimura, 1969), 21-hydroxylase (DeNicola, 1975), and 1lp-hydroxylase (Griffiths and Glick, 1966; Kowal, 1967, 1969; DeNicola, 1973; DeNicola and Freire, 1973). In addition, the quantity of cytochrome P-450 in rat adrenocortical cells was reported to be decreased after hypophysectomy and was restored by ACTH 4 months after the operation (Pfeiffer et al., 1972). Kahri’s group showed that the ACTH-induced differentiation of mitochondria in fetal human and rat adrenocortical cells cultured in vitro was associated with elevated activities of 1l p - and 18-hydroxylase contained in them (Kahri et al., 1970, 1976a; Salmenpera, 1976; Salmenpera and Kahri, 1976; Salmenpera et a l . , 1976). Similarly Milner and Villee (1970) reported that ACTH caused a rise in 17a-, 21-, and llp-hydroxylase in human fetal adrenocortical cells cultured in vitro. Furthermore, the ACTH-caused activation of llp- and 18-hydroxylation was found to be impaired by chloramphenicol (Milner, 197l), 5-bromodeoxyuridine (Kahri et a l . , 1976b), or ethidium bromide (Salmenpera and Kahri, 1977), all of which inhibit mitochondrial differentiation in cultured cortical cells. It was also shown that these compounds did not affect

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21-hydroxylation or inhibit the ACTH-induced SER hypertrophy. Based on these data it can be held that activation of mitochondrial hydroxylases is dependent on mitochondrial DNA replication and transcription, as well as on mitoribosomal translation. Other workers reported that in organotypic cultures of adrenal glands from 19-day-oldrat embryos the ACTH-induced transformation of mitochondrial cristae from a tubular to a vesicular configuration was coupled with a 10-fold rise in both aldosterone and corticosterone production (Manuelidis and Mulrow, 1973). Even in adult rat (O’Hare and Neville, 1973a,b) and human adrenocortical cells in primary culture (Nussdorfer et al., 1975a) morphological differentiation induced by ACTH and CAMP was found to be associated with an increased output of corticosterone and cortisol, respectively, in the growth medium. A correlation between cytochrome P-450 content and mitochondrial growth was demonstrated in regenerating rat adrenal cortexes, although stereology showed that membrane differentiation was not closely related to membrane growth (Yago et al., 1972). It is reasonable that, like the classic respiratory chain, the cytochrome P-450 electron transfer chain also requires an adequate steric arrangement for complete activity of the hydroxylating enzymes involved in steroid synthesis. Therefore, on the grounds of the evidence discussed here, we advance the hypothesis that the ACTH-induced increase in the surface of SER membranes and mitochondrial cristae provides an increased framework of basic membrane to which de novo synthesized enzymes of steroid synthesis can be added. An alternative, but not contrasting, hypothesis concerning the significance of SER proliferation has been proposed by Black (1972). She claims that, since SER is involved in cholesterol synthesis, it is conceivable that cholesterol may actually be incorporated into smooth membranes, which therefore serve as storage sites of steroid precursors awaiting further synthesis and release.

4. Lipid Droplets It seems well established that adrenocortical lipid droplets decrease in shortterm stimulation experiments (see Section 111,A); in this case, lipid depletion seems to be the morphological counterpart of the ACTH-enhanced activity of cholesterol esterase and of the increased utilization of free cholesterol in steroid synthesis (see Section IV,B). In contrast, there is considerable disagreement regarding the behavior of the lipid compartment during chronic stimulation or inhibition of adrenocortical cells (see Section III,C and D); the most studies have described a decrease or increase in the volume of the lipid compartment in chronically stimulated or depressed zona fasciculata cells, but Nussdorfer et al. (1977d) stressed that these changes might only be apparent, because of the marked cytoplasmic hypertrophy and atrophy caused, respectively, by the two treatments. In fact, stereological evaluations showed that chronic stimulation of adrenocortical cells significantly increased the volume of the lipid compartment,

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whereas chronic suppression did not cause evident change or, over more prolonged time periods, caused a slight decrease in this parameter. As pointed out by Armato et al. (1974b), the volume of the lipid compartment in adrenocortical cells is an expression of the balance of the rates of formation and utilization of lipid droplets and therefore is a result of the following processes: (1) endogenous synthesis of cholesterol from acetate and glucose, which occurs in the SER; (2) uptake of exogenous cholesterol from the bloodstream; (3) esterification of cholesterol and its storage in the lipid droplets as reserve material; (4) transformation of esterified cholesterol stored in the lipid droplets into free cholesterol by cholesterol esterase; (5) utilization of free cholesterol in steroid hormone synthesis. In the Section I,C, it was stated that the relative importance of processes 1 and 2 varies according to the species and that only exogenous cholesterol is stored in lipid droplets, since adrenocortical cells of the species in which process 2 is virtually absent lack lipid droplets (e.g., ox). Since process 1 seems to be controlled by ACTH (Sharmaet al., 1972), it can be speculated that these species can immediately adapt the rate of endogenous synthesis of cholesterol to the level required for steroid synthesis. The morphological counterpart of a greater capacity for endogenous synthesis of cholesterol is a well-developed SER and, in this connection, the hypothesis that SER functions as a site of free cholesterol storage (Black, 1972) should be borne in mind. ACTH stimulates processes 2, 4, and 5 (see Section IV,B) and possibly process I . In rat adrenocortical cells, in which endogenous synthesis produces no more than 10-15% of the total intracellular cholesterol (see Section I,C), the acute response to ACTH (Le., synthesis and output of corticosterone in 10-15 minutes) involves the utilization of intracellular stores of cholesterol (i.e., lipid droplets). We assume that in the rat the increase in the SER surface area induced by chronic ACTH treatment is coupled with a parallel rise in the endogenous synthesis of free cholesterol, which alone can adequately satisfy the precursor requirement for increased hormone synthesis. Under these conditions the processes leading to cholesterol intracellular accumulation prevail over those leading to its utilization, which entails an increase in the volume of the lipid compartment. Numerous investigations dealing with the effects of drugs inhibiting steroid synthesis support the idea that prolonged ACTH treatment causes an accumulation of lipid droplets in adrenocortical cells. Chronic treatment with Metopirone, an inhibitor of 1lp-hydroxylase (Dominguez and Samuels, 1963), and aminoglutethimide, an inhibitor of side chain-cleaving enzymes (Cash et al., 1967; Dexter et a l . , 1967a,b), by decreasing the levels of circulating definitive steroid hormones, causes a noticeable activation of the hypothalamohypophyseal axis, resulting in an increased level of circulating ACTH. In these experimental models an increase in the volume of the lipid compartment was found (Grignon et

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al., 1967; MagalhHes and MagalhHes, 1969, 1972; Racela et al., 1969; Marek et al., 1970; Itoh, 1971, 1977; Marek and Motlik, 1975; Kadioglu and Harrison, 1975), coupled with evident SER hypertrophy (Grignon et al., 1967; Kjaerheim, 1968d; M. C. MagalhHes and Magalhies, 1969, 1972; Idelman, 1970; Marek et al., 1971). A similar lipid accumulation was obtained by treating rat adrenocortical cells in vivo with inhibitors of protein synthesis (e.g., chloramphenicol and cycloheximide) (Szabo et al., 1971; Kovacs et al., 1972; Mazzocchi et al., 1977b), since the ACTH-elicited uptake of cholesterol from the bloodstream does not seem to be a process dependent on protein synthesis (see Section-IV,B). The absence of ACTH results in blockage of both the accumulation and utilization of intracellular cholesterol, and therefore in the invariability of the volume of the lipid compartment. Armato et al. (1 977) showed that dedifferentiated human adrenocortical cells, maintained in a monolayer culture in the absence of ACTH, were able to secrete discrete amounts of progesterone and cortisol into the culture medium. If this is also true for in vivo adrenocortical cells, the slight decrease in the volume of the lipid compartment in chronically suppressed cells can be easily explained. Armato et al. (1974b) reported almost complete lipid depletion in cultured adrenocortical cells after prolonged ACTH exposure. This finding fits well with the model we have proposed, since process 2, owing to the lack of a considerable amount of cholesterol in the culture medium, is absent or very low. In this connection, however, it is of interest to remember that Kahri’s group reported that morphological-functional differentiation was coupled with the appearance of lipid droplets in the cytoplasm of fetal rat and human cultured adrenocortical cells (Kahri, 1966; Kahri et al., 1970, 1976a). We cannot explain this discrepancy at present, except to point out that the Kahri culture technique was different from that employed by Armato and associates; it is conceivable that cholesterol or cholesterol precursors may derive from cells undergoing necrosis located in the central portion of the microexplants. We emphasize that investigations on the effects of ACTH on the volume of the lipid compartment in adrenocortical cells of animal species, in which the endogenous synthesis of cholesterol is higher than in the rat, may provide insight into this problem.

5. Golgi Apparatus The Golgi apparatus is present in the adrenocortical cells of all species and appears to be hypertrophic in stimulated cells both in vivo and in vitro (see Sections I1 and 111), as well as during morphological-functional differentiation of human fetal adrenocortical cells (Johannisson, 1968; McNutt and Jones, 1970). The role of the Golgi apparatus in steroid-producing cells is still under debate, although some authorities claim that it is “integral to steroidogenesis’ ’ (Christensen and Gillim, 1969; Fawcett et al., 1969).

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Long and Jones (1967a) proposed that this organelle might be involved in the modulation of steroid sulfation-desulfation processes in preparation for steroid synthesis andor secretion. This hypothesis is supported by evidence indicating that sulfated steroids are present in steroid-producing cells and that some intermediary products of steroid synthesis are transported in this form (Breuer and Rao, 1971; Notation and Ungar, 1972). In this connection, we mention an article by Rappay et al, (1973) describing arylsulfatase-P activity in the Golgi cisternae and lysosomes of rat zona fasciculata cells, and only occasionally, according to Schwartz and Spors (1970), in SER tubules or mitochondria1 cristae. Whether this finding can be correlated with the aforementioned role of the Golgi apparatus or simply with its involvement in lysosome production (see Cook, 1973, for review) is not known at present. Involvement of the Golgi apparatus in the intracellular transport of intermediate products or in the release of definitive steroid hormones, not only by a sulfation-desulfation mechanism, may be inferred from the high-resolution autoradiographic study of Magalhies et al. (1971, 1975), which followed the intracellular distribution of l e ~ c i n e - ~and H tyr~sine-~H in the zona fasciculata of intact and ACTH-treated rats. They reported that 10 minutes after the tracer injection the sites of protein synthesis were located mainly in the SER tubules and mitochondria; the tracer incorporation values for both subcellular compartments remained unchanged after 60 and 240 minutes, indicating that these were sites of synthesis of low-turnover proteins (e.g., structural and enzymic proteins). Incorporation values for the Golgi apparatus were low at 10 minutes, high at 60 minutes, and low again after 240 minutes after the tracer injection, suggesting the migration of a small fraction of the newly formed proteins through the Golgi apparatus and their subsequent discharge. These results, coupled with the demonstration in adrenocortical homogenates of camer proteins for cholesterol and pregnenolone (see Section IV,B), allow us to advance the hypothesis that the Golgi apparatus may play a role in the conjugation of steroid intermediates with their binding proteins. This hypothesis implies that steroid hormones pass into the Golgi cisternae, but a complete demonstration of this has not yet been achieved. By highresolution autoradiography Kahri et al. (1968) showed the accumulation of andor its metabolites) in the steroid-incorporatedradioactivity (pr~gesterone-'~C Golgi area, but Magalhies et al. (1974) were able to localize radioactive products only in the mitochondrial compartment of zona fasciculata cells of 11deoxycorticosterone-3H-injectedrats. Hyperosmication, which seems to retain and stain steroid hormones which, during routine fixation and dehydration procedures, are easily extracted, induces the formation of black osmium deposits in the Golgi cisternae of rat zona fasciculata cells (Friend and Brassil, 1970). Friihling and Pecheux (1976) attempted to determine the distribution of cholesterol, pregnenolone, progesterone, and corticosterone into the subcellular frac-

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tions of rat adrenal homogenates. These investigators showed that esterified cholesterol was present in the lipid droplets, and pregnenolone and progesterone mainly in the mitochondrial fraction, whereas corticosterone was present in the postmicrosomal supernatant. Friihling and associates suggest that the binding of corticosterone to any organelle is extremely weak and therefore that its release into the homogenation medium is instantaneous after the beginning of the preparative procedure. Possible involvement of the Golgi apparatus in the segregation of definitive hormones into secretory granules is discussed in Section V. Before concluding this discussion, however, we emphasize that the Golgi apparatus may be involved only in lysosome production, and that the notion that its hypertrophy or atrophy is simply the expression of the general stimulation or depression of adrenocortical cell metabolism cannot be disregarded at present. 6. Lysosomes Contrasting findings were obtained concerning the changes in the quantity of lysosomes in hyper- and hypofunctioning adrenocortical cells (see Section III), and this disagreement results in great uncertainty as to the function of these organelles in steroid-producing cells. Lysosomes may intervene in the normai turnover of the subcellular organelles and participate in the remodeling of cell structure, for instance, during ACTH suppression (e.g., autophagic processes involved in the decrease of SER and mitochondrial membranes) (Nussdorfer, 1969; Nickerson, 1972; Nussdorfer et a/., 1975b). This view explains the increase in the lysosome number in chronically suppressed adrenocortical cells. On the grounds of an evident proliferation of lysosomes and of their close spatial association with lipid droplets in the interrenal cells of dexamethasone- or medroxyprogesterone-treateddomestic fowls, Kjaerheim (1 968b) suggested that lysosomes might degrade lipid droplets which are not necessary for steroid synthesis. In this connection, it is of interest to recall that, according to Rhodin (1971), a conspicuous number of cholesterol-containing crystals seems to be present in lysosomes in adrenocortical cells of dexamethasone-treated rats, and that recently Szabb et al. (1977) suggested these crystals were due to the decreased or impaired utilization of cholesterol in steroidogenesis (e.g., aminogluthetimide-inducedinhibition of the cholesterol side chain-cleaving enzyme system). Several investigations, dealing with the effects of some inhibitors of cholesterol synthesis, seem to lend support to the contention that lysosomes are engaged in lipid metabolism in adrenocortical cells. Yates’ group (Yates, 1966; Arai et al., 1967) showed that chronic administration of triparanol (MER29), an inhibitor of A24-reductase, resulted in the appearance of numerous electron-dense bodies, frequently containing crystalloid inclusions, and SER proliferation (interpreted as a compensatory hypertrophy due to the blockage of

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cholesterol endogenous synthesis) in adrenocortical cells of the Syrian hamster. By differential centrifugation Arai et al. (1967) obtained a fraction containing these electron-dense bodies and showed that it contained mainly desmosterol (an intermediate product of cholesterol synthesis and a substrate for A24-reductase). Volk and Scarpelli (1964) reported only SER hypertrophy in the adrenocortical cells of triparanol-administered rats, but it must be taken into consideration that the effect of this chemical would be presumably scarce in the rat, in which endogenous cholesterol synthesis is poor. Dietert and Scallen (1969) showed cytoplasmic accumulation of dense bodies and SER proliferation in adrenocortical cells of mice chronically treated with triparanol, 20,25-diazacholesterol (SC- 12937), another inhibitor of AZ4-reductase, and AY-9944, an inhibitor of A'-reductase. These investigators demonstrated cytochemically that the dense bodies are lysosomes and proposed that these organelles are involved in the sequestration of cholesterol precursors. They stress that, since lysosomes arise from the Golgi-Endoplasmic Reticulum-Lysosomes (GERL) complex (see, for review, de Duve and Wattiaux, 1966; Cook, 1973), in which most of the enzymes of cholesterol synthesis reside, the sequestration of cholesterol preeursors into lysosomes would permit a remarkable economy of two different cellular activities. However, the hypothesis that lysosomes are involved in the degradation of cholesterol or its precursors presents some difficulties, because lysosomes seem to lack enzymes metabolizing the cyclopentane-perhydrophenanthrenering (see, for review, Barrett, 1969; Tappel, 1969). Lullmann etal. (1973), on the basis of evidence showing an increase in the number of dense bodies in rat adrenocortical cells after treatment with chlorphentermine, which, like triparanol, has amphiphilic characteristics, proposed a new hypothesis concerning the significance of these dense bodies. Since amphiphilic compounds bind to other amphiphilic molecules (e.g., certain phospholipids), they suggested that an interaction between triparanol and cellular phospholipids impairs the degradation of the lastmentioned molecules by phospholipases, resulting in an increased intralysosomal accumulation of phospholipids which may aggregate to form crystalloid arrays. A contrasting hypothesis was presented by Szabb et al. (1967), which was based on the demonstration of an increase in the number of lysosomes in adrenocortical cells after ACTH was administered to hypophysectomized rats. These workers suggested that lysosomes might intervene in the control of mobilization of steroid precursors stored in the lipid droplets (i.e., cholesterol esters). This view seems to be supported by the fact that lysosomes contain enzymes hydrolyzing ester bonds (e.g., carboxyl esterases) (for review, see Barrett, 1969; Tappel, 1969). However, recent cytochemical studies demonstrated the presence of various nonspecific carboxyl esterases only in SER tubules surrounding lipid droplets and never in lysosomes in both rat (Sugihara and Deimling, 1973, 1976) and human (Bocking el al., 1974) adrenocortical cells.

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7. Microbodies The widespread occurrence of microbodies in steroid-secreting cells, along with their numerical changes during differentiation (Black and Bogart, 1973) and stimulation or inhibition of adrenocortical cells (see Section 111, C and D), suggests that these organelles are involved, albeit in a still undefined fashion, in the specific functions of this type of cell. Black and Bogart (1973) proposed several possible functions for microperoxisomes, all related to the functions of SER, since microperoxisomes seem to arise by budding from smooth tubules (M. M. Magalhles and Magalhles, 1971; Black and Bogart, 1973; Berchtold, 1975a,b): 1 . Peroxisomes may intervene in NADPH generation, thus activating steroid hydroxylases, since they contain glucose-6-phosphate dehydrogenase (de Duve and Baudhuin, 1966), a key enzyme in the pentose shunt (see Section IV,B); alternatively, peroxisomes may be involved in the reoxidation of NADH to NAD, which is required for the activity of 3/3-hydroxysteroid-A5-dehydrogenase and I7a-hydroxysteroid dehydrogenase (de Duve and Baudhuin, 1966; de Duve, 1969). 2. Catalase is a powerful lipid peroxidation catalyst and, according to Boyd (1962), formation of fatty acid by peroxidation may lead to a rearrangement of cholesterol esters, thus facilitating the action of cholesterol esterase. This action of peroxisomes may affect both lipid droplets and SER membranes which, according to Black (1972), may function as stores of intracellular cholesterol. 3. Peroxisomes may function as intracellular translocators of intermediate products of steroid synthesis, “thus providing a possible link between organelles involved in steroid synthesis.”

0 . Other Organelles and Cell Inclusions The functional significance of the coated pits as well as of the microfilamentous and microtubular apparatus are discussed in Section V. As described in Section 111, an increase in P-glycogen particles was observed to follow both acute and chronic inhibition of adrenocortical cells. This ultrastructural change is easily explained by considering that one of the mechanisms of the action of ACTH is held to involve activation of glycogen phosphorylase, which promotes glycogenolysis (see Section IV, B). Blockage of glycogenolysis in the suppressed cells would clearly result in the intracytoplasmic accumulation of several clumps of @-glycogen particles.

V. The Mechanism of Hormone Release by Adrenocortical Cells To date, ultrastructural investigations have provided little insight into the mechanism(s) of steroid hormone release; electron microscopists have so far

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failed to demonstrate unequivocally a cellular organelle which may act as a vehicle for the export of steroid hormones into the extracellular fluid. Probably the difficulties reside in the fact that steroid-producing cells do not have the capacity for intracellular storage of definitive hormones; in fact, experiments demonstrating a latent effect (10 minutes) of ACTH on hormone secretion by adrenocortical cells led to the conclusion that the action of ACTH was exerted throygh enhancement of synthesis as well as through release of steroid hormones (Holzbauer and Newport, 1969; Jaanus et al., 1970). In this section we consider the various hypotheses proposed on this subject and criticisms of them. A. HOLOCRINE SECRETION This hypothesis was first advanced by Cotte’s group for rabbit adrenocortical cells (Cotte, 1959; Cotte and Cotte, 1961). It is based on the finding of degenerate cells and cell debris in the sinusoid lumina of the zona reticularis. Similar observations were made recently by Wassermann and Wassermann (1973) in the zona fasciculata of rats administered DDT, but it was questioned whether or not these features were poor fixation artifacts. Even more recently, the holocrine secretion hypothesis was readvanced by Chavin’s group (Taylor et ai., 1975). These studies distinguished in the interrenal gland of the shark three concentric layers called the external germinative zone, intermediate mature cell zone, and internal degenerating cell zone. The last-mentioned layer contains, in addition to several elements displaying the features typical of steroid-producingcells, a large number of cells appearing to be in the process of degeneration, and abundant cell debris in the sinusoids. Although the involvement of this zone in holocrine secretion cannot be excluded, we recall that, according to the cell migration theory (see Long, 1975, for review), this inner layer can be easily interpreted as a site of cell senescence and death, analogous to the zona reticularis of eutherian mammal adrenals. Recently, Wyllie et al. (1973a,b) described a process of cell deletion (apoptosis) i9 the rat zona reticularis, which ensures a normal turnover of adrenocortical cells. B. APOCRINESECRETION

This hypothesis seems to be advanced whenever a research group observes cell fragments in the adrenal sinusoid lumina: Brenner (1966) and Penney and Brown (1971) in the monkey zona glomerulosa; and Zelander (1959, 1963) and Luse (1967) in the mouse adrenals. More recently, Del Conte (1976) published micrographs suggesting that a mechanism of apocrine secretion operates in the inner mature zone of a lizard’s interrenal gland. This investigator found that the en-

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dothelial lining showed membrane-bridged fenestrations and free discontinuities, through which cytoplasmic pieces of interrenal cells projected into the bloodstream and were broken off. In this connection, we recall that Penney and associates described cytoplasmic projections (pseudopodia) of adrenocortical cells traversing the endothelial pori and projecting into the sinusoids in both normal and ACTH- or prostaglandin%-treated rat zona reticularis (Penney et al., 1972, 1973). Mitochondria, ribosomes, SER profiles, and lipid droplets are contained in these projections, and their ultrastructural features exclude the possibility that they are products of cellular degradation or autolysis. Since pseudopodia are always clearly in continuity with the parenchymal cell body, Penney and co-workers discredited that idea they are the expression of apocrine secretion and advanced the hypothesis that these parenchymal projections into the bloodstream may be an excellent mechanism by which circulating ACTH obtains access to plasma membrane receptors. Why pseudopodia are present only in the zona reticularis, however, remains an open question.

C. ENDOPLASMOCRINE SECRETION Recently, Rhodin (1971) proposed a new mechanism underlying hormone release by the zona fasciculata and zona reticularis cells of rat adrenals; endoplasmocrine secretion, which in his opinion is similar to the “microapocrine secretion” found in sweat glands by Kurosumi (1961). Rhodin’s mechanism was confirmed by Unsicker (1973) in bird interrenal cells. This revolutionary theory is based upon structural evidence which, however, seems not to be unequivocal and can summarized as follows. Lipid droplets originate from small SER profiles or from Golgi cisternae and gradually distend until they reach a state in which they can be recognized as small lipid vacuoles. SER tubules form a complete casing around the newly formed lipid droplets, and this process may be the morphological counterpart of the SER enzyme-mediated transformation of cholesterol stored in lipid droplets into intermediate products of steroid synthesis. The SER-lipid complex reaches the cell surface, the peripheral lamina of the membranous casing fuses with the plasma membrane, and the contents of the lipid droplet are released either by diffusion or by rupture of the droplet envelope. Subsequently, the boundary membrane and the inner lamina of the SER casing are extruded into the subendothelial space, where their remnants (ghosts) can frequently be observed. We emphasize that this hypothesis implies that all the steps of steroid synthesis occur inside the lipid droplets. Rhodin claims that only perfusion fixation techniques, allowing good preservation of the intercellular spaces, permit the visualization of steroid release, and his hypothesis is supported by the following data: (1) In addition to cortico-

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sterone, pregnenolone and progesterone are also secreted by rat adrenocortical cells (Klopper et al., 1957). This was confirmed by Nussdorfer et al. (1975a) in human adrenocortical cells cultured in vitro. Furthermore, some lines of evidence suggest that intermediary products of steroidogenesis accumulate in the lipid droplets: Holzbauer et al. (1973) detected pregnenolone in the lipid layer of supernatant homogenates of dog adrenals, and Magalhles et al. (1974) showed that 11-deoxycorticosterone accumulated in the lipid droplets when its 1lphydroxylation was prevented by the treatment of rats with Metopirone. By contrast, Friihling and Pecheux (1976) localized only cholesterol and cholesterol esters in the lipid fraction from normal rat adrenal homogenates. (2) After shortterm ACTH stimulation SER tubules seemed to have decreased, indicating their rapid utilization in endoplasmocrine secretion; our stereological findings have not confirmed this (see Section 111,A). The hypothesis of endoplasmocrine secretion has been subjected to further criticism: 1. If the lipid droplets originate from the SER or Golgi cisternae, they should be surrounded by a true trilaminar membrane. Rhodin’s explanation that either the contents of the droplet obscure the trilamination of the limiting membrane, or that the trilamination disappears through reorganization of the droplet boundary membrane as the lipid droplet increases in size, is not very convincing. 2. According to this hypothesis the lipid droplets contain only cholesterol derived from endogenous synthesis, while it has been demonstrated that an inverse correlation exists between the number of lipid droplets and the cell capacity during cholesterol synthesis (see Section 11,C). Moreover, how can the endoplasmocrine secretion mechanism be applied to ox adrenocortical cells which are virtually free of lipid droplets? 3. The decrease in the lipid droplets during the first 10 minutes after ACTH administration must be correlated with steroid synthesis rather than with steroid release (Jaanus et al., 1970). 4. The conversion of 11-deoxycorticosterone to corticosterone is catalyzed by 1lp-hydroxylating enzymes located on the mitochondria1 cristae (Satre et a l . , 1969; Yago and Ichii, 1969; Dodge et al. 1970) and/or on the outer mitochondrial membrane (Billiaretal., 1971). In any case, SERcasing would make contact between the enzyme and 11-deoxycorticosterone difficult. Moreover, evidence indicates that steroid substrates must penetrate the inner mitochondrial membrane to interact with the 1 lp-hydroxylating system (Rydstrom et a1., 1976). 5. According to Kuo and Tchen (1973), it is difficult to understand how intercellular canalicula collapse, which Rhodin assumes did not allow visualization of lipid droplet extrusion in immersion-fixed specimens, could “put the lipid droplets back inside the cells.”’

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6. The endoplasmocrine secretion hypothesis does not seem to apply to the rat zona glomerulosa which, however, shows ultrastructural features quite similar to those of the inner adrenal zones. These considerations cast doubt on the validity of Rhodin’s theory and, following Kuo and Tchen (1973), we suggest that the images referred to by Rhodin to support his hypothesis are artifacts of the perfusion-fixation technique. D. SECRETION BY SIMPLEDIFFUSION The most widely accepted theory for the release of adrenocortical hormones holds that they are free to diffuse throughout the aqueous cytoplasm and the lipid phase of the plasma membrane (Lever, 1955b; Porter and Bonneville, 1967; Christensen and Gillim, 1969). Although Pearce et al. (1977) note that “the physical properties of low polar steroid such as corticosterone do not preclude release by simple diffusion,” it has been suggested that the steroid hormone molecule must be modified prior to its release. The same applies to the frequent passing of intermediate products through the intracellular membranes of mitochondria and the SER. The mechanisms proposed are sulfation of steroid hormones andor their binding to a carrier protein (see Section IV,C,5). E. EXOCYTOTIC SECRETION The view that an exocytotic mechanism underlies hormone release in steroidproducing cells is now gaining more and more support, although the morphological demonstration of true secretory granules has not been possible to date. This hypothesis is based on the work by Rubin’s group (Laychock and Rubin, 1974; Rubin et al., 1974) showing that ACTH-induced steroid output in the perfused feline adrenal cortex was paralleled by increased secretion in the perfusate of “granular” proteins, which suggests that the mechanism of steroid hormone release shares certain basic features with the secretory mechanism of other endocrine cells such as adrenal medullary and neurophypophyseal cells (Rubin, 1974; Winkler and Smith, 1975). The presence of electron-dense granules, frequently located near the plasma membrane, was previously reported in the human corpus luteum during the secretory phase (Adams and Hertig, 1969a; Gillim e t a l . , 1969) or during the first trimester of pregnancy (Adams and Hertig, 1969b), in sheep lutein cells during the estrous cycle (Gemmell et a l . , 1974), in the inner layers (fetal zone) of the human adrenal cortex (Hatakeyama, 1966; Johannisson, 1968; Maude, 1972), in the outer zone cells of fetal human adrenals after cytodifferentiation (Fujita and Ihara, 1973), in the interrenal cells of the brown pelican (Belt eral., 1965), and in zona fasciculata cells from rats stressed by snake toxin (Harumiya, 1966).

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Rennels (1966) showed granules in the lutein cells of rats treated with pregnant mare serum and human chorionic gonadotrophin, but suggested that these granules were not related to steroid secretion, since they displayed the morphological features of proteinaceous secretory products. Volk (1 97 1a) reported the presence of similar granules in adrenocortical cells of rats whose corticosterone secretion was increased by chronic progesterone subcutaneous administration and proposed that they were true secretory organelles containing steroids bound to a carrier protein. More recently, Gemmell et a!. (1977a) demonstrated electron-dense granules (0.2-0.4 p m in diameter) in perfused cat zona fasciculata cells; the granules seemed to arise in the Golgi area and were prevalent near the plasma membrane. These workers excluded the possibility that these granules were lysosomes, since (1) their numerical increase was not associated with enhanced activity of certain typical lysosomal enzymes in an adrenal homogenate (e.g., /3 -glucuronidase and N-acetyl-B-glucosaminidase), and (2) the ACTH-elicited increase in the adrenal steroid content was of the same order of magnitude as that of the granular density per unit cytoplasmic area, as evaluated by stereological techniques. Gemmell and associates claim that most of the granules, previously interpreted as lysosomes or peroxisomes, are true secretory granules and suggest that the lack of images, suggesting exocytosis, is due to the paucity of secretory granules, since demonstration of exocytosis is not a simple task even in adrenal chromaffin cells, where large stores of catecholamine are invariably present. Evidence for an exocytotic mechanism of hormone release was also provided for the steroid-producing cells of the sheep, the goat (Gemmell et al., 1977b), and the duck (Pearce et al., 1977). As reviewed in Section 11, adrenocortical cells possess all the organelles known to function in the exocytotic release of secretory granules: microtubules and microfilaments (see Normann, 1976, for review). Close spatial associations between microtubules and “secretory granules” migrating from the Golgi area toward the plasma membrane (Gemmell et al., 1977a), and between microfilaments or microtubules and pinocytotic vesicles, were also described (Pearce et al., 1977). In this connection, it is recalled that CAMP-dependent protein kinase-induced activation of microtubules is involved in exocytotic secretion mechanisms (Soifer et al., 1975; Rappaport et al., 1975). Furthermore, exocytosis is usually coupled with pinocytosis in order to avoid the increase in cell surface caused by the surplus membrane derived from secretory granules during their extrusion (see Werb and Dingle, 1976, for review); the presence of many coated pits and vesicles associated with the plasma membrane of adrenocortical cells, as well as their increase after ACTH (see Section II1,C) or progesterone treatment (Volk, 1971a), can therefore be considered indirect evidence for an ACTH-dependent exocytotic mechanism. It is obvious that the difficulties encountered in visualizing secretory granules in adrenocortical cells can be ascribed to the fact that these elements lack any

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appreciable storage of secretion products, so that it is conceivable that secretory granules are released as soon as they are formed. Since microtubules were found to be involved in exocytosis (Normann, 1976), we thought that treatment with vinblastine, an antimicrotubular agent (Malawista et a t . , 1968), would lead to an intracellular accumulation of secretory granules by blocking their exocytotic release. This view was confirmed by recent evidence showing that colchicine can block progesterone secretion by sheep lutein cells (Gemmell and Stacy, 1977). Our preliminary results showed that intraperitoneal administration of 25 mg/kg of vinblastine to adult rats induced a significant decrease in the peripheral plasma corticosterone concentration in 1 hour, along with a significant rise in the hormone concentration in the adrenal homogenate. The administration of 10 IU/kg of ACTH to normal rats increases the concentration of circulating corticosterone, without a conspicuous increase in the intracellular level of the hormone. When ACTH is administered to vinblastine-treated animals, a 10-fold rise in the intracellular corticosterone concentration is obtained, without a significant increase in the plasma hormonal level. The data clearly indicate that the Vi'inca alkaloid

FIG.27. Cells from the inner portion of the zona fasciculata of a vinblastine-treated rat, containing two clumps of electron-dense granules. Id, Lipid droplet; s, sinusoid, ses, subendothelial space; P, pericyte. x7500.

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inhibits corticosterone release by adrenocortical cells, without impairing its synthesis. Electron microscopy showed that the rise in the intracellular concentration of corticosterone was associated with the appearance of numerous clumps of electron-dense granules usually located at the juxtasinusoidal pole of the cells (Fig. 27). The granules seemed to originate in the Golgi zone, and their number increased after ACTH was administered to vinblastine-treated animals. Although the granules displayed a weak positive reaction to acid phosphatate, we advance the hypothesis that they are secretory organelles; in fact, it is well known that immature secretory granules from several tissues contain low acid phosphatase activity (see Smith and Winkler, 1969, for review).

VI. Concluding Remarks The preceding sections have shown that in the last few years an imposing number of investigations has contributed to our knowledge of adrenal cortex cytophysiology. We now emphasize some points whose investigation should be the task of future research. First, the morphology of adrenocortical mitochondria1 cristae requires further studies to confirm Allmann’s hypothesis (see Section 11,C). It is possible that the study of stereoscopic images obtained by high-voltage electron microscopy will resolve this problem. It seems reasonable to consider that the increase in the surface of the SER and mitochondria1 inner membrane in hyperfunctioning adrenocortical cells is the morphological counterpart of the ACTH-enhanced activity of the enzymes of steroid synthesis contained in them (see Section IV,C,3); nevertheless, we stress that the biochemical investigations available did not discriminate clearly between the increase in the enzymic activity resulting from the activation of preexisting enzymes and that due to de n o w synthesis of enzymic moieties. In fact, in experiments dealing with the chronic stimulation of adrenocortical cells, it is not adequate to express the enzymic activity per milligram of protein, since ACTH causes an increase in the adrenal weight mainly by stimulating protein synthesis’ the enzymic activity assayed could appear unchanged, although the absolute amount of enzyme per cell is noticeably increased. By employing coupled biochemical and stereological techniques in a preliminary work we overcame this difficulty by expressing the enzymic activity per single cell (Nussdorfer et al., 1977e). Efforts in this direction are worthwhile in order to gain insight into this problem. The specific role, if any, played by the Golgi apparatus and lysosomes in steroid-secreting cells has not yet been determined; we suggest that isolation of the two organelles by differential centrifugation techniques and the biochemical characterization of their enzymic content are needed to ascertain their possible involvement in steroid synthesis and secretion.

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One other problem requiring further effort concerns the mechanism of steroid hormone release. The exocytotic theory is the best working hypothesis on which to base future investigations, and our opinion is derived not only from the evidence reviewed in Section V , but also from the following considerations. Research has shown that the administration of corticosterone to hypophysectomized rats inhibits the ACTH-enhanced growth of adrenocortical cells (Nussdorfer and Mazzocchi, 1970; Nussdorfer et at., 1971b) by impairing nuclear and mitochondria1DNA-dependent RNA and protein synthesis (Nussdorfer and Mazzocchi, 1971b). Moreover, corticosterone was found to prevent the ACTH-induced differentiation of fetal rat adrenocortical cells cultured in v i m (Kahri, 1973; Salmenpera, 1976). These data suggest that a negative feedback control mechanism operates at the adrenal level. In light of these results, the immediate release of newly formed steroid hormones, and their segregation into secretory granules, possibly bound to a carrier protein in a storage complex, probably involves a mechanism preventing the direct inhibitory action of steroid hormones on adrenocortical cells. Investigations employing in vivo and in vitro models, in which the possible exocytotic release of steroid hormones is blocked by antimicrotubular agents, would be fruitful. Efforts to isolate a reasonably pure granular fraction from vinblastine-treated adrenocortical cells are under way in our laboratory. In addition, we speculate that studies on the fate of tritiated precursors of steroid hormones by high-resolution autoradiography, using specimens processed for cryoultramicrotomy to prevent steroid molecule extraction (Christensen and Gillim, 1969; Weiller et al., 1977; Magalhiies, 1977), are needed to gain insight into this problem.

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I

Subject Index

A Adenosine triphosphatase, mitotic apparatus and, 36-38 Adrenocortical cells fine structure of hyper- and hypofunctioning, 316-317 long-term inhibition, 324-326 long-term stimulation, 31 8 -324 short-term inhibition, 318 short-term stimulation, 317-318 fine structure of normally functioning general remarks, 313-316 interrenal cells of lower vertebrates, 309313 mammalian zona fasciculata, 292-309 mechanism of hormone release, 347 -348 apocrine secretion, 348 -349 endoplasmocrine secretion, 349-35 1 exocytotic secretion, 35 1 -354 holocrine secretion, 348 secretion by simple diffusion, 35 1 morphological-functional correlations mechanism of action of ACTH, 330-33 1 significance of changes, 33 1 -347 subcellular localization of enzymes, 327330 Analog incorporation into protein activation and loading of tRNA, 121-124 enzyme induction, repression and derepression, 128-129 general effects on macromolecular synthesis, 126-128 relative efficiency of incorporation, 124126 molecular biology and, 110 nature of, 111-113 uptake exchangeability of pools, 119-121 intracellular pool formation, 114-1 19 use in other areas of biological research aging, 150-152 miscellaneous, 156-159 mutagenesis, 148-1 50 virus development, 152-156

Analog proteins cellular effects cell growth and cell cycle, 134-142 flagella, 147-148 in vivo studies with tumors, 146-147 mitosis, 142-146 degradation of, 159-162 molecular effects enzymes, 129-132 structural and general, 132-133 thermostability , 133-1 34

:

c

Chromatin active contemporary research on structure, 18-19 euchromatin and, 2-3 histone content, 9-10 fractionation, logic and logistics, 5-8 Contact inhibition failure between normal cells, 54-55 mechanism of action, 58-63 tumor cells and, 56-58 type 1, 51-53 type 2, 53-54

D Differential centrifugation and marker enzyme analysis technique of general considerations, 271-272 practical procedures, 272-275

E Egg, tubulin assembly into MTs, 26-29 pool of, 24-26 regulation of assembly in vivo, 29-3 I Electrical synapses in CNS of nonmammalian vertebrates gap junctions in local circuits, 87-90 gap junctions in long-projecting pathways, 77-87

367

SUBJECT INDEX

368

in mammalian CNS, 90 between an axonal terminal and a neuronal perikaryon, 92 between dendrites, 9 1-92 between dendritic appendages in a glomerular formulation, 92-94 between neuronal perikarya, 91 ultrastructure gap junction conformational changes, 74-75 gap junctions as electrical synapses, 70-74 rectifying electrical synpases, 76-77 Electrotonic coupling, chemical synaptic control of, 94-95 Euchromatin nuclear RNA and, 3-5 problem versus active chromatin, 2-3

G Gap junctions molecular transport and, 99-100 low-resistance pathways and neuronal differentiation, 102-104 metabolic coupling, 100

H Histone, active chromatin and, 9-10

correlation with gap junctions, 69-70 historical, 68-69

M Microtubules mitotic apparatus and, 33-36 role in mitosis actin and myosin, 4 3 4 4 anaphase chromosome movement, 39-40 induction of chromosome motion in isolated MA, 40-43 Mitosis, role of microtubules actin and myosin and, 4 3 4 anaphase chromosome movement, 39-40 induction of chromosome motion in isolated MA, 4 0 4 3 Mitotic apparatus, isolated adenosine triphosphatases, 36-38 isolation, 31-33 microtubules and tubulin, 33-36 response to heterologous tubulin, 38

N Nucleosome alteration of structure, transcription and, 15-18 paradigm of, 10-1 1 transcription and, 11-14

I

P Interstitial gland cells developing and maturing ovary fetal, 173-178 postnatal, 178-1 80 general discussion and conclusions, 227-239 prepubertal and adult ovary biochemistry, 201 -202 correlation of changes with steroid secretions, 215-217 changes after gonadotrophic stimulation, 202-2 15 cytology and histochemistry, 191 -201 dynamic and degenerative effects, 218-227 formation and storage of lipid, 217-218 origin and distribution, 180-191

L Low-resistance pathways between neurons

Phenylalanine, analogs of, 110-1 1 I

R Ribonucleic acid, nuclear, euchromatin and, 3-5

S Stereological analysis, techniques of practical procedures, 263-27 1 reference systems, 256-263 theoretical considerations, 248-256 Stereological data, integration and biochemical data intact tissue, 275-279 intact tissue and fractions, 283-2546> tissue fractions, 279-283 Synapses, mixed, 95-99

SUBJECT INDEX

T Transcription active units, morphology of, 14-15 alteration of nucleosorne structure. and, 15-18 nucleosorne and, 11-14 Tubulin

egg assembly into MTs, 26-29 pool of, 24-26 regulation of assembly in vivo, 29-31 heterologous, mitotic apparatus and, 38 mitotic apparatus and, 33-36 Tumor cells, contact inhibition and, 56-58

369

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Contents of Previous Volumes 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. AND H. FIRKET CH~VREMONT Gustatory and Olfactory Epithelia-A. F. AND G.H. BOURNE BARADI Growth and Differentiation of Explanted Tissues-P. J. GAILLARD Electron Microscopy of Tissue Sections-A. J. BRANDT DALTON Bacterial Cytology-K. A. BISSET A Redox Pump for the Biological Performance Protoplast Surface Enzymes and Absorption of of Osmotic Work, and Its Relation to the Suger-R. BROWN Kinetics of Free Ion Diffusion across Reproduction of Bacteriophage-A. D. HERMembranes-E. J. CONWAY SHEY A Critical Survey of Current Approaches in The Folding and Unfolding of Protein Molecules Quantitative Histo- and Cytochemistryas a Basis of Osmotic Work-R. J. GOLDDAVIDGLICK ACRE Nucleo-cytoplastmic Relationships in the DeNucleo-Cytoplasmic Relations in Amphibian velopment of Acetabularia-J. HAMMERLING Development-G. FRANK-HAUSER Report of Conference of Tissue Culture Workers Structural Agents in Mitosis-M. M. SWANN J. Held at Cooperstown, New York-D. Factors Which Control the Staining of Tissue HETHERINGTON Section with Acid and Basic Dyes-MARCUS AUTHOR INDEX-SUBJECT INDEX SINGER The Behavior of Spermatozoa in the Neighborhood of Eggs-Lorn ROTHSCHILD Volume 3 The Cytology of Mammalian Epidermis and Sebaceous Glands-WILLIAM MONTAGNA The Electron-MicroscopicInvestigation of Tis- The Nutrition of Animal Celk-CPHARITY WAYMOUTH sue Sections-L. H. BRETSCHNEIDER The Histochemistry of Esterases-4. GOMORI Caryometric Studies of Tissue CUltUreS-oTTO BUCHER AUTHOR INDEX-SUBJECT INDEX The Properties of Urethan Considered in Relation to Its Action on Mitosis-IvoR CORNMAN Volume 2 Giant Composition and Structure of Chromosomes-MAX ALFERT Quantitative Aspects of Nuclear NucleoproHow Many Chromosomes in Mammalian Soteins-HEwsoN SWIIT 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 ganiSm&TUART MUDDAND EDWARD D. Study of Tissue Enzymes-cHR. DE DUVE DELAMATER AND J. BERTHET Ion Secretion in Plants-J. F. SUTCLIFFE of Embryonic Enzymatic Aspects Multienzyme Sequences in Soluble ExtractsDifferentiation-TRYGGVE GUSTAFWN HENRY R. MAHLER

Volume 1

Some Historical Features in Cell BiologyARTHUR HUGHES Nuclear Reproduction-C. LEONARD HUSKINS Enzymic Capacities and Their Relation to Cell w. KIDDER Nutrition in Animd&EORGE The Application of Freezing and Drying Techniques in Cytology-L. G. E. BELL Enzymatic Processes in Cell Membrane AND W. WILPenetration-TH. ROSENBERG

37 1

312

CONTENTS OF PREVIOUS VOLUMES

Azo Dye Methods in Enzyme HistochemistryA. G. EVERSON PEARSE Microscopic Studies in Living Mammals with Transparent Chamber Methods-Roy G. WILLIAMS The Mast Cel1-G. ASBOE-HANSEN Elastic Tissue-EDWARDS w . DEMPSEYAND ALBERTI. LANSING The Composition of the Nerve Cell Studied with AND New Methods-!hEN-OLOE BRATTGARD HOLCERHYDEN AUTHOR INDEX-SUBJECT

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

INDEX

Volume 4 Cytochemical Micrurgy-M. J. KOPAC Amoebocytes-L. E. WAGGE Problems of Fixation in Cytology, Histology, and Histochemistry-M. WOLMAN Bacterial Cytology-ALFRED MARSHAK Histochemistry of Bacteria-R. VENDRELY Recent Studies on Plant Mitochondria-DAVID P. HACKETT MUHLEThe Structure of Chloroplasts-K. THALER

Histochemistry of Nucleic Acids-N.

The Results of Cytophotometry in the Study of the Deoxyribonucleic Acid (DNA) Content of the Nucleus-R. VENDRELYAND C. VEN-

B. KUR-

NICK

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<. 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

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 G . c. HIRSCH The Acrosome Reaction-JEAN C. DAN Cytology of Spermatogenesis-VisHwA NATH The Ultrastructure of Cells, as Revealed by the Electron Microscope-FIuTIoF S. SJ~STRAND AUTHOR 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 S t e d i n e Cell-sAJIRO MAKINO The Structure of the Golgi Apparatus-ARTHUR AND PRISCHIA F. 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 MZUIUII~~S-ELIO

BoRGHEsE Carbohydrate Metabolism and Embryonic Determination-R. J . O’CONNOR Enzymatic and Metabolic Studies on Isolated Nuclei-G. SIEBERTAND R. M. S . SMELLIE

373

CONTENTS OF PREVIOUS VOLUMES

The Cell Surface of P a r a m e c i u m P . F. EHRET AND E. L. POWERS The Mammalian RetiCUkXyt+LEAH MIRIAM LOWENSTEIN The Physiology of Chromatophores-MILTON FINGERMAN The Fibrous Components of Connective Tissue AUTHOR INDEX-SUBJECT INDEX with Special Reference to the Elastic Fiber-DAVID A. HALL CUMULATIVE SUBJECT INDEX (VOLUMES 1-5) Experimental Heterotopic Ossification-J. B. BRIDGES A Survey of Metabolic Studies on Isolated Volume 7 Mammalian Nuclei-D. B. ROODYN Some Biological Aspects of Experimental Trace Elements in Cellular Function-BERT L. L. HOCH VALLEEAND FREDERIC G. Radiology: A Historical Review-F. Osmotic Properties of Living Cells-D. A. T. SWAR DICK The Effect of Carcinogens, Hormones, and Vitamins on Organ Cultures-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 of the Mammalian The Structure Volume 9 Spermatozoon-DON W. FAWCETT The Lymphocyte-0. A. TROWELL The Influence of Cultural Conditions on BacteThe Structure and Innervation of Lamellibranch rial Cytology-J. F. WILKINWNAND J. P. Muscle-L. BOWDEN DUGUID Hypothalamo-neurohypophysial NeurosecreOrganizational Patterns within Chromosomestion-J. C. SLOPER HELEN GAY,A N D BERWIND P. KAUFMANN, Cell Contact-PAUL WEIS MARGARET R. MCDONALD The Ergastoplasm: Its History, Ultrastructure, Enzymic Processes in Ceh-JAY BOYDBEST and Biochemistry-FRANGolsE HAGUENAU The Adhesion of Ceh-LEONARD WEISS Anatomy of Kidney Tubules-JoHANNEs RHOPhysiological and Pathological Changes in DIN Mitochondria1Morphology-CH. ROUILLER Structure and Innervation of the Inner Ear SenThe Study of Drug Effects at the Cytological sory Epithelia-HANS E N G S T R ~AND M JAN Level-G. B. WILSON WERSKLL Histochemistry of Lipids in OogenesisThe Isolation of Living Cells from Animal VISHWANATH Tissues-L. M. RINALDINI Cyto-Embryology of Echinoderms and AmAUTHOR INDEX-SUBJECT INDEX phibia-KUTSUMA DAN The Cytochemistry of Nonenzyme ProteinsRONALD R. COWDEN Volume 8 Recent Approaches of the Cytochemical Study of Mammalian TiSSUeS-GEORGE H. HOGEBOOM, EDWARD L. KUFF, AND WALTER C. SCHNEIDER The Kinetics of the Penetration of Nonelectrolytes into the Mammalian ErythrocyteFREDABOWYER

The Structure O f CYtOplaSmxHARLES OBER-

AUTHOR INDEX-SUBJECT

INDEX

LING

Wall Organization in Plant Cells-R. D. PRE-

Volume 10

STON

Submicroscopic Morphology of apse-EDuARDo DE ROBERTS

the

Syn-

The Chemistry of Shiff's Reagent-FREDERICK H. KASTEN

374

CONTENTS OF PREVIOUS VOLUMES

Spontaneous and Chemically Induced Chromosome Breaks-ARuN KUMARSHARMAAND ARCHANA SHARMA The Ultrastructure of the Nucleus and Nucleocytoplasmic ReIationeAuL WISCHNITZER

In Vivo Implantation as a Technique in Skeletal Biology-WILLIAM J. L. FELTS The Nature and Stability of Nerve Myelin-J.

B.

FINEAN Fertilization of Mammalian Eggs in V i t r o 4 . R. AUSTIN Physiology of Fertilization in Fish EggsTOKI-oYAMAMOTO

The Mechanics and Mechanism of CleavageLEWISWOLPERT The Growth of the Liver with Special Reference AUTHOR INDEX-SUBJECT INDEX to Mammals-F. DOLJANSKI Cytology Studies on the Affinity of the CarVolume 13 cinogenic Azo Dyes for Cytoplasmic Components-YosHIMA NAGATANI The Coding HypOtheSiS-MARTYNAS YEAS Epidermal Cells in Culture-A. GEDEON Chromosome Reproduction-J. HERBERT MATOLTSY TAYLOR AUTHOR INDEX-SUBJECT INDEX Sequential Gene Action, Protein Synthesis, and CUMULATIVE SUBJECT INDEX A. FLICCellular Differentiation-REED (VOLUMES 1-9) KINGER The Composition of the Mitochondria1 Membrane in Relation to Its Structure and Volume 11 Function-Eiuc G. BALLAND CLIFFE D. Electron Microscopic Analysis of the Secretion JOEL Mechanism-K. KUROSUMI Pathways of Metabolism in Nucleate and AnuThe Fine Structure of Insect Sense Organscleate Erythrocytes-H. A. SCHWEIGER ELEANOR H. SLIFER Some Recent Developments in the Field of AlCytology of the Developing E Y ~ A L F R EJ.D kali Cation Transport-W. WILBRANDT COULOMBRE Chromosome Aberrations Induced by Ionizing The Photoreceptor Structures-J. J. WOLKEN Radiations-H. J. EVANS Use of Inhibiting Agents in Studies on Fertiliza- Cytochemistry of Protozoa, with Particular Reftion MechanismeHARLEs B. METZ erence to the Golgi Apparatus and the The Growth-Duplication Cycle of the Cell-D. Mitochondria-VISHWA NATH A N D G. P. M. PRESCOTT DUTTA L. Cell Renewal-FELIX BERTALANPPY Histochemistry of Ossification-RoMULO AND CHOCABRINI SEN LAU Cinematography, Indispensable Tool for AUTHOR INDEX-SUBJECT INDEX Cytology<. M. Pomerat AUTHOR INDEX-SUBJECT

INDEX

Volume 14

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

Volume 12

37 5

CONTENTS OF PREVIOUS VOLUMES Development of Drug Resistance by Staphylococci in Vitro 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 Waif-DOUGLAS E. SMITH

In Vivo Studies of Myelinated Nerve FibersCARLCASKEY 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 Trypanosomes: A AUTHOR INDEX-SUBJECT INDEX Critical Review Dealing Mainly with the Afric a Species in the Mammalian Host-P. J. WALKER Volume 15 The Blood Platelet: Electron Microscopic The Nature of Lampbrush Chromosomes-H. Studies-J. F. DAVID-FERREIRA The Histochemistry of MucopolysaccharidesG. CALLAN ROBERT c. 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 PlantsE ~ K A Interpretations-FELIX D. BERTALANFFY LEONARD MACHLIS AND RAWITSCHER-KUNKEL The Cells of the Adenohypophysis and Thejr Functional SignificanceMARc HERLANT The Cellular Basis of Morphogenesis and Sea AND L. AUTHOR INDEX-SUBJECT INDEX 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 thk 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. GOULD The Classification and Partial Tabulation of EnThe Behavior of Mast Cells in Anaphylaxiszyme Studies on Subcellular Fractions IsoIVAN MOTA 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 HyaluronidaseR. DAOUST B. Ribosomal Functions Related to Protein Cytoplasmic Deoxyribonucleic Acid-P. GAHANAND J. CHAYEN Synthesis-To% HULTIN Physiology and Cytology of Chloroplast Forma- Malignant Transformation of Cells in VirroKATHERINE 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 E. WOHLFARTH- FLAUMENHAFT, Ameboid Movement-K. J. J. KATZ BoTTERMA N Microbeam and Partial Cell 1madiation-C. L. The Use of Heavy Metal Salts as Electron RICHARDZOBELAND MICHAEL Stain-. SMITH BEER Nuclear-Cytoplasmic Interaction with Ionizing AUTHOR I N D E X 4 U B J E C T INDEX Radiation-M. A. LESSLER

376

CONTENTS OF 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- ExperimentalCytology of the Shoot Apical Cells SON during Vegetative Growth and Flowering-A. Synaptic Morphology on the Normal and DeNOUGAR~DE generating Nervous System-E. G. GRAY Nature and Origin of Perisynaptic Cells of the AND R. W. GUILLERY Motor End Plate-T. R. SHANTHAVEERAPPA 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 H A U ~ S. Phosphorus Metabolism in Plants-K. Current Techniques in Biomedical Electron ROWAN Microscopy-SAuL WISCHNITZER 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. FAUTREZConversion in Photosynthesis-RoDERIc B. FIRLEFYN AND J. FAUTREZ PARK Lymphopoiesis in the Thymus and Other TisControl of Chloroplast Structure by Lightsues: Functional Implications-N. B. LESTERPACKER AND PAUL-AND&S I E G E N ~ EVERETTAND RUTHW. TYLER(CAFFREY) THALER Structure and Organization of the Myoneural The Role of Potassium and Sodium Ions as Junction<. 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. SAHAISRIVASTAVA Volume 19

Roos

Cytology and Cytophysiology of NonMelanophore Pigment Cells-JOSEPH T. BAGNARA The Fine Structure and Histochemistry of Prostatic Glands in Relation to Sex HormonesDAVIDBRANDES Cerebellar Enzymology-LUCIE ARVY

AUTHOR I N D E X 4 U B J E C T INDEX

CUMULATIVE SUBJECT INDEX

(VOLUMES 1-21) Volume 23

Transformationlike Phenomena in Somatic Cells-J. M. OLENOV AUTHOR INDEX-SUBJECT INDEX Recent Developments in the Theory of Control and Regulation of Cellular ProcessesROBERTROSEN 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 SAKAI Microscopic Morphology of Ciliary Movement and Coordination in Electron Ciliates-BELA PARDUCA Oogenesis-ARNE NPRREVANG

CONTENTS OF PREVIOUS VOLUMES Dynamic Aspects of Phospholipids during Protein Secretion--LOWELL E. HOKIN The Golgi Apparatus: Structure and FunctionH. BEAMSAND R. G . KESSEL The Chromosomal Basis of Sex DeterminationKENNETHR. LEWISA N D BERNARD JOHN

w.

AUTHOR INDEX-SUBJECT

INDEX

377

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

INDEX

Volume 24

Volume 26

Synchronous Cell DifferentiatiOn-GEORGE M. PADILLA AND IVAN L. CAMERON Mast Cells in the Nervous System-YNwE

A New Model for the Living Cell: A Summary of the Theory and Recent Experimental Evidence in Its SUPPO~~-GILBERT N. LING The Cell Periphery-LEONARD WEISS Mitochondria1 DNA. Physicochemical Properties, Replication, and Genetic Function-P. BORSTAND A. M. KROON Metabolism and Enucleated CellS-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

OLSSON

Development Phases in Intermitosis and the Preparation for Mitosis of Mammalian Cells in vitro-BLAGOJE A. NESKOVIC Antimitotic Substances-GuY DEYSSON The form and Function of the Sieve Tube: A Problem in Reconciliation-P. E. WEATHER^ LEY AND 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 Celk-PETER G. TONER Liquid Junction Potentials and Their Effects on Potential Measurements in Biology Systems-P. C. CALDWELL 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 MitochondriaSYLvAN 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--SHELWN WOLFF

AUTHOR INDEX-SUBJECT

INDEX

Volume 27 Wound-Healing in Higher PlantS-JACQUES LIPETZ Chloroplasts as Symbiotic Organelles-D~~N~s L. TAYLOR The Annulate Lamellae-SAuL WISCHNITZER Gametogenesis and Egg Fertilization in Planarians-G. BENAZZILENTATI Ultrastructure of the Mammalian Adrenal C O ~ ~ ~ X - S IIDELMAN MON The Fine Structure of the Mammalian Lymphoreticular SYSkm-IAN CAM Immunoenzyme Technique: Enzymes as Markers for the Localization of Antigens and Antibodies-STRATIs AVRAMEAS AUTHOR INDEX-SUBJECT

INDEX

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

378

CONTENTS OF PREVIOUS VOLUMES

The Environment and Function of Invertebrate E. TREHERNEAND R. B. Nerve Cells-J. MORETON Virus Uptake, Cell Wall Regeneration, and Virus Multiplication in Isolated Plant Protoplasts-E. C. COCKING The Meiotic Behavior of the Drosffphila Oocyte-ROBERT c . KING The Nucleus: Action of Chemical and Physical Agents-REN6 SIMARD The Origin of Bone Cells-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. C. MOTT AUTHOR INDEX-SUBJECT

INDEX

Volume 29 Gram Staining and Its Molecular MechanismsB. B. BISWAS,P. S. BASU,A N D M .K. PAL The Surface Coats of Animal Cells-A. MART~NEZ-PALOMO Carbohydrates in Cell Surfaces-RICHARD J. WINZLER Differential Gene Activation in Isolated Chromosomes-MARKUS LEZZI Intraribosomal Environment in the Nascent Peptide Chain-HIDEKo KAJI Location and Measurement of Enzymes in Single Cells by Isotopic Methods Part I-E. A. RARNARD 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. JOHNSTON AND BETTYI. ROOTS Functional Electron Microscopy of the Hypothalamic Median Eminence-HIDESHI TOKUZO MATSUI,AND SUSUMI KOBAYASHI, ISHII Early Development in Callus Cultures-MICHAEL M. YEOMAN AUTHOR I N D E X 4 U B J E C T INDEX

Volume 30 High-Pressure Studies in Cell Biology-ARTHUR M. ZIMMERMAN Micrurgical Studies with Large Free-Living Amebas-K. W. JEONAND 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 TissuedHINJIRO 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-4. C. BUDD Morphological and Histochemical Aspects of Glycoproteins at the Surface of Animal Cells-A. RAMBOURG DNA Biosynthesis-H. S. JANSZ,D. V A N DER MEI, AND G. M. ZANDVLIET Cytokinesis in Animal Cells-R. RAPPAWRT The Control of Cell Division in Ocular Lens-C. V. HARDING,J. R. REDDAN, N. J. UNAKAR, AND M. BAGCHI The Cytokinins-HANS KENDE Cytophysiology of the Teleost PituitaryMARTINSAGEA N D 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 AND R. N. JONES Polarized Intracellular Particle Transport: Saltatory Movements and Cytoplasmic Streaming-LIONEL I. REBHUN The Kinetoplast of the Hemoflagellates-LARRY SIMPSQN

CONTENTS OF PREVIOUS VOLUMES

379

Transport across the Intestinal Mucosal Cell: Mechanisms of Ion Transport through Plan Cell AND Hierarchies of Function-D. S. PARSON Membranes-EMANUEL ERSTEIN C. A. R. BOYD Cell Motility: Mechanisms in Protoplasmic Wound Healing and Regeneration in the Crab Streaming and Ameboid Movement-H. Paratelphusa hydrodromous-RITA G. KOMNICK, W. STOCKEM, AND K. E. ADIYODI WOHLEPARTH-BOTTERMANN The Use of Femtin-Conjugated Antibodies in The Gliointerstitial System of MolluscsElectron Microscclpy~oUNCILMAN MORGAN GHISLAIN NICAISE Metabolic DNA in Ciliated Protozoa, Salivary Colchicine-Sensitive Microtubles--LYNN MAR^ GULIS Gland Chromosomes, and Mammalian Ce1ls-S. R. PELC AUTHOR INDEX-SUBJECT INDEX AUTHOR I N D E X 4 U B J E C T INDEX

Volume 35 Volume 33

The Structure of Mammalian ChromosomesELTONSTUBBLEFIELD Visualization of RNA Synthesis on ChromoA. Synthetic Activity of Polytene Chromosomessomes-0. L. MILLER,JR. AND BARBARA HANSD. BERENDES HAMKALO Cell Disjunction (“Mitosis”) in Somatic Cell Mechanisms of Chromosome Synapsis at MeiReproduction-ELAINE G. DIACUMAKOS, otic ROphas+hTER B . MOENS Structural Aspects of Ribosomes-N. NAN= AND PAULINE PECORA SCOTTHOLLAND, NINGA Neuronal Microtubles, Neurofilaments, and Microfilaments-RAYMOND B. WUERKER Comparative Ultrastructure of the Cerebrospinal AND JOELB. KIRKPATRICK Fluid-Contacting Neurons-B. VIGH AND I. VIGH-TEICHMANN Lymphocyte Interactions in Antibody Maturation-Inducing Substance in StdishesResponses-J. F. A. P. MILLER HARUOKANATANI Laser Microbeams for Partial Cell Irradiation-MZCHAEL w.BERNSAND CHRlSQ The Limonium Salt Gland: A Biophysical and TIAN SALET Structural Study-A. E. HILLAND B. S HILL Mechanisms of Virus-Induced Cell FusionToxic Oxygen Effects-HAROLD M. SWARTZ GEORGEPOSTE AUTHOR INDEX-SUBJECT INDEX Freeze-Etching O f BaCtefla4HARLES c . RE= MSEN AND STANLEY W. WATSON The Cytophysiology of Mammalian Adipose Volume 36 Cek-BERNARD G. SLAVIN Molecular Hybridization of DNA and RNA in AUTHOR INDEX-SUBJECT INDEX SitU-WOLFGANG HENNIG The Relationship between the Plasmalemma and Plant Cell Wdk-JEAN-CLAUDE ROLAND Volume 34 Recent Advances in the Cytochemistry and U1trastructure of CytoplasmicInclusions in MasThe Submicroscopic Morphology of the Interphtigophora and Opalinata (Protozoa+G. P. ase Nucleus-SAuL WISCHNITZER DUTTA The Energy State and Structure of Isolated Chloroplasts: The Oxidative Reactions Involv- Chloroplasts and Algae as Symbionts in of ing the Water-Splitting Step MUSCATINE AND MOIIUSCS-LEONARD Photosynthesis-ROBERT L. HEATH RICHARD W. GREENE Transport Neurospora-GENE A. SCARCE The Macrophage-hIMoN GORDON AND ZANa: BOROUGH VIL A. COHN

380

CONTENTS OF PREVIOUS VOLUMES

Degeneration and Regeneration of Neurosecretory Systems-HoRsT-DIETER DELLMANN AUTHOR INDEX-SUBJECT

INDEX

Cell Separation by Gradiant Centrifugation-R. HARWOOD SUBJECT 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 Spermiogenesis in Various Animal SpeCieS~ONPACHIRO YASUZUMI Morphology, Histochemistry, and Biochemistry of Human Oogenesis and Ovulation-SAmuL S . GURAYA Functional Morphology of the Distal LungKAYEH. KILBURN Comparative Studies of the Juxtaglomerular Apparatus-HIRoFUMI SOKABE AND MITE ZUHO OGAWA The Ultrastructure of the Local Cellular Reaction to Neoplasia-IAN CARR AND J. C. E. UNDERWOOD Scanning Electron Microscopy in the Ultrastructural 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-TuNG YUE WANGAND L.EROY M. NYBERC Nucleocytoplasmic Interactions in Development of Amphibian Hybrids-STEPHENSUBTELNY The Interactions of Lectins with Animal Cell SULfaCeSqARTH L. NICOLSON Structure and Function of Intercellular Junctions-L. ANDREW STAEHELIN Recent Advances in Cytochemistry and Ultrastructure of Cytoplasmic Inclusions in Ciliophora (ProtozoajG. P. DUTTA Structure and Development of the Renal Glomerulus as Revealed by Scanning Electron MiCrOSCOpy-FRANCO SPINELLI Recent Progress with Laser MicrobeamsMICHAEL W. BERNS The Problem of Germ Cell Determinants-H. W. BEAMSA N D R. G . KESSEL

AUTHOR INDEX-SUBJECT

INDEX

SUBJECT INDEX

Volume 40

B-Chromosome Systems in Flowering Plants and Animal Species-R. N. JONES Genetic Engineering and Life Synthesis: An In- The Intracellular Neutral SH-Dependent Protease Associated with Inflammatory troduction to the Review by R. Widdus and C. AUIt-JAMES F. DANIELLI Reactions-HIDE0 HAYASHI Progress in Research Related to Genetic En- The Specificity of Pituitary Cells and Regulation gineering and Life Synthesis-Roy WIDDUS of Their ACtiVitieS-VLADIMIR R. PANTIC A N D CHARLES R. AULT Fine Structure of the Thyroid Gland-HrsAo The Genetics of C-Type RNA Tumor VirusesFUJITA J. A. WYKE Postnatal Gliogenesis in the Mammalian Three-Dimensional Reconstruction from ProjBrain-A. PRIVAT ections: A Review of Algorithms-RICHARD Three-Dimensional Reconstruction from Serial GORDAN AND GABOR T. HERMAN SeCtiOnS-RANDLE w.WARE AND VINCENT LOPRESTI The Cytophysiology of Thyroid CellsVLADIMAR R. PANTIC SUBJECT INDEX The Mechanisms of Neural Tube FomationPERRYKARFUNKEL Volume 41 The Behavior of the XY Pair in MammalsALBERTO J. SOLARI The Attachment of the Bacterial Chromosome to Fine-Structural Aspects of Morphogenesis in AND the Cell Membrane-PAUL J. LEIBOWITZ Acetabu1aria-G. WERZ MOSELIO SCHAECHTER

Volume 38

CONTENTS OF PREVIOUS VOLUMES Regulation of the Lactose Operon in Escherichiu coli by c A M P 4 , CARPENTER AND B. H. SELLS Regulation of Microtubules in TetrahymenuNORMANE. WILLIAMS Cellular Receptors and Mechanisms of Action of Steroid Hormones~HuTsUNGLIAO A Cell Culture Approach to the Study of Anterior Pituitary Cells-A. TIXIER-VIDAL, D. GOURDJI,AND C. TOUGARD Immunohistochemical Demonstration of Neurophysin in the Hypothalamoneurohypophysial System-W. B. WATKINS The Visual System of the Horseshoe Crab Limulus polyphemus-WOLF H. FAHRENE BACH SUBJECT INDEX

Volume 42 Regulators of Cell Division: Endogenous Mitotic Inhibitors of Mammalian Cells-BISMARCK B. LOZZIO,ELENAG. B. LOZZIO,CARMEN BAMBERGER, AND STEPHEN V. LAIR Ultrastructure of Mammalian Chromosome AND WALTER Aberrations-B. R. BRINKLEY N. HITTELMAN Computer Processing of Electron Micrographs: A Nonmathematical Account-P. W. HAWKES Cyclic Changes in the Fine Structure of the Epithelial Cells of Human EndometriumGORDON MILDRED The Ultrastructure of the Organ of CortiS . KIMURA ROBERT Endocrine Cells of the Gastric Mucosa-ENRIco VASE SOLCIA, CARL0 CAPELLA, GABRIELE SALLO, AND ROBERTO BUFFA Membrane Transport of Purine and Pyrimidine Bases and Nucleosides in Animal CellsRICHARD D. BERLINAND JANETM. OLIVER

38 1

Biochemical Studies of Mitochondria1Transcription and Translation-C. SACCONE AND E. QUAGLIARIELLO The Evolution of the Mitotic Spindle-DONNA F. KUBAI Germ Plasma and the Differentiation of the Germ Cell Line-E. M. EDDY Gene Expression in Cultured Mammalian Cells-RoDY P. COX AND JAMESc. KING Morphology and Cytology of the Accessory Sex AND Glands in Invertebrates-K. G. ADIYODI R. G. ADIYODI SUBJECT INDEX

Volume 44 The Nucleolar StruCtUre-SIBDAS GHOSH The Function of the Nucleolus in the Expression of Genetic Information: Studies with Hybrid AND I. I. Animal Cells-". SIDEBOTTOM DEAK Phylogenetic Diversity of the Proteins Regulating Muscular COntraCtiOn-wILLIAM

LEHMAN Cell Size and Nuclear DNA Content in Vertebrates-HENRYK SZARSKI Ultrastructural Localization of DNA in Ultrathin Tissue SeCtiOns-ALAIN GAUTIER Cytological Basis for Permanent Vaginal Changes in Mice Treated Neonatally with Steroid Hormones-NoBORU TAKASUGI On the Morphogenesis of the Cell Wall of StaphylOCOCCi-hTER GIESBRECHT,J ~ R G WECKE, AND BERNHARD REINICKE Cyclic AMP and Cell Behavior in Cultured Cells-MARK C. WILLINGHAM Recent Advances in the Morphology, Histochemistry, and Biochemistry of SteroidSynthesizing Cellular Sites in the Nonmammalian Vertebrate OVXY-SARDUL S . GURAYA SUBJECT INDEX

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 AND RUDOLF A. RAFF

Volume 43

382

CONTENTS OF 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 Lymphocyteand Transitional Cell Populations of the Bone Marrow; Their Role in the Mediation of Immune and Hemopoietic Progenitor Cell FUnCtiOnS-cORNELIUSROSSE The Structure and Properties of the Cell Surface Coat-J. H. LUFT Uptake and Transport Activity of the Median M. Eminence of the Hypothalamus-K. M. F. KNIGGE,S. A. JOSEPH,J. R. SLADEK, NOTTER, M. MORRIS,D. K. SUNDBERG, M. AND L. A. HOLZWARTH, G. E. HOFFMAN, 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

SUBJECT INDEX

Volume 47

Volume 49

SUBJECT INDEX

Volume 48

Mechanisms of Chromatin Activation and AND VAUCE Repression-NoRblAN MACLEAN GHAN A. HILDER Origin and Ultrastructure of Cells in Vitro-L. SUBJECT INDEX A N D PATRICIA D. WILSON M. FRANKS Electrophysiology of the Neurosecretory Cel~-KINJI YAGI AND SHIZUKO IWASAKI Volume 46 Reparative Processes in Mammalian Wound Contractile Neurosecretion by Exocytosis-TOM CHRISTIAN Healing: The Role of Phenornena-GIuLIo GABBIANI NORMA" AND DENYS MONTANWN Genetic and Morphogenetic Factors in Hemoglobin Synthesis during Higher Vertebrate Smooth Endoplasmic Reticulum in Rat HepatoDevelopment: An Approach to Cell Difcytes during Glycogen Deposition and NIGON ferentiation Mechanisms-VICTOR Depletion-ROBERT R. CARDELL, JR. AND JACQUELINE GODET Potential and Limitations of Enzyme Cytophysiology of Corpuscles of Stannius-V. Cytochemistry: Studies of the Intracellular G. KRISHNAMURTHY Digestive Apparatus of Cells in Tissue Culture-M. HONDGEN Ultrastructure of Human Bone Marrow Cell AND F. Maturation-J. BRETON-GORIUS Uptake of Foreign Genetic Material by Plant REYES Protoplasts-E. C. COCKING Evolution and Function of Calcium-Binding The Bursa of Fabricius and Immunoglobulin Proteins-R. H. KRETSINGER Synthesis-BRUCE GLICK

Responses of Mammary Cells to Hormones-M. Cyclic Nucleotides, Calcium, and Cell Division-LIONEL I. REBHUN R. BANEWEE Recent Advances in the Morphology, His- Spontaneous and Induced Sister Chromatid Exchanges as Revealed by the BUdR-Labeling tochemistry, and Biochemistry of SteroidMethod-HATAo KATO Synthesizing Cellular Sites in the Testes of Nonmammalian Vertebrates4 ARDuL S. Structural, Electrophysiological, Biochemical, and Pharmacological Properties of GURAYA Neuroblastoma-Glioma Cell Hybrids in Cell Epithelial-Stromal Interactions in Development of the Urogenital TractaERALD R. CUNHA Culture-B . HAMPRECHT

383

CONTENTS OF PREVIOUS VOLUMES Cellular Dynamics in Invertebrate Neurosecretory Systems-ALLAN BERLIND Cytophysiology of the Avian Adrenal Medulla-AsoK GHOSH Chloride Cells and Chloride Epithelia of Aquatic Insects-H. KOMNICK Cytosomes (Yellow Pigment Granules) of Molluses of Cell Organelles of Anoxic Energy Production-IMRE ZS.-NAGY

Mechanism-HANS-GEoRG MANFRED SCHWEIGER

SCHWEIGER

AND

SUBJECT INDEX

Volume 52

Cytophysiology of Thyroid Parafollicular D. Cells-ELADIo A. NUNEZAND MICHAEL GERSHON Cytophysiology of the Amphibian Thyroid SUBJECT INDEX Gland through Larval Development and Metamorphosis-ELIANE REGARD Volume 50 The Macrophage as a Secretory Cell-Roy C. PAGE,PHILIPDAVIES,AND A. C. ALLISON Cell Surface Enzymes: Effects on Mitotic Activity and Cell Adhesion-H. BRUCEBOSMANN Biogenesis of the Photochemical Apparatus-TIMOTHY TREFFRY New Aspects of the Ultrastructure of Frog Rod Extrusive Organelles in Protists-KLAUS HAUSE Outer Segments-JORGEN ROSENKRANZ MANN Mechanisms of Morphogenesis in Cell c. BROWNAND RICHARDc. Cultures-J. M. VASILIEV AND I. M. GELE Lectins-JAY HUNT FAND SUBJECT INDEX Cell Polyploidy: Its Religion to Tissue Growth AND I. V. and Functions-W. YA. BRODSKY URYVAEVA Volume 53 Action of Testosterone on the Differentiation and Secretory Activity of a Target Organ: The Regular Arrays of Macromolecules on Bacterial Cell Walls: Structure, Chemistry, Assembly, Submaxillary Gland of the Mouse-MONIQUE and Function-UwE B. SLEYTR CHRBTIEN SUBJECT INDEX Cellular Adhesiveness and Extracellular Substrata-FREDERICK GRINNELL Chemosensory Responses of Swimming Algae Volume 51 AND D. c. and Protozoa-M. LEVANDOWSKY Circulating Nucleic Acids in Higher R. HAUSER Organisms-MAURICE STROUN, PHlLIPPE Morphology, Biochemistry, and Genetics of ANKER,PIERRE MAURICE,AND PETER B. Plastid Development in Euglena gracilis-V. GAHAN NIGONAND P. HEIZMANN Recent Advances in the Morphology, HisPlant Embryological Investigations and Fluorestochemistry, and Biochemistry of the Decence Microscopy: An Assessment of . L Integration-R. N. KAPILAND S . C. TIWARI veloping Mammalian O V ~ ~ A R DS U GURAYA The Cytochemical Approach to Hormone Morphological Modulations in Helical Muscles Assay-]. CHAYEN (Aschelminthes and Annelida)--GIuLIo SUBJECT INDEX LANZAVECCHIA Interrelations of the Proliferation and DifVolume 54 ferentiation Processes during Cardiac Myogenesis and Regeneration-PAvEL P. RUMYANTSEV The Kurloff Cell-PETER A. REVELL Circadian Rhythms in Unicellular Organisms: An Endeavor to Explain the Molecular

Microtubule Assembly and Nucleation-MARC W. KIRSCHNER The Mammalian Sperm Surface: Studies with K. Specific Labeling Techniques-JAMES KOEHLER

384

CONTENTS OF PREVIOUS VOLUMES

The Glutathione Status of CCh-NECHAMA S . Binding and Patterns of Axonal KOKIWERAND EDWARD M. KOKIWER Projections-DONALD W. PFAFFAND LILYC. Cells and SeneSCenCe-ROBERT RoseN A. CONRAD Immunocytology of Pituitary Cells from Teleost Ancient Locomotion: Prokaryotic Motility J. DOERR-SCHOTT, SySkm+LELENG P. TO AND LYNN MARFishes-E. FOLLBNIUS, AND M. P. DUBOIS GULlS Follicular Atresia in the Ovaries of Nonmamma- An Enzyme Profile of the Nuclear Envelope-I. lian Vertebrates-SRmIvAs K. SAIDAPUR B. ZBARSKY Hypothalamic Neuroanatomy: Steroid Hormone SUBJECT INDEX

A

B

c a D E F 6 H 1 J

9 O 1 2 3 4 5