International Review of Cytology: A Survey of Cell Biology, Volume 105

INTERNATIONAL

REVIEW OF CYTOLOGY A SURVEY OF CELLBIOLOGY VOLUME105

ADVISORY EDITORS H. W. BEAMS HOWARD A. BERN GARY G. BORISY PIET BORST BHARAT B. CHATTOO STANLEY COHEN RENE COUTEAUX MARIE A. DIBERARDINO CHARLES J. FLICKINGER OLUF GAMBORG M. NELLY GOLARZ DE BOURNE YUKIO HIRAMOTO YUKINORI HIROTA K. KUROSUMI GIUSEPPE MILLONIG ARNOLD MITTELMAN AUDREY MUGGLETON-HARRIS DONALD G. MURPHY

ROBERT G. E. MURRAY RICHARD NOVICK ANDREAS OKSCHE MURIEL J. ORD VLADIMIR R. PANTIC W. J. PEACOCK DARRYL C. REANNEY LIONEL I. REBHUN JEAN-PAUL REVEL L. EVANS ROTH JOAN SMITH-SONNEBORN WILFRED STEIN HEWSON SWIFT K. TANAKA DENNIS L. TAYLOR TADASHI UTAKOJI ROY WIDDUS ALEXANDER YUDIN

INTERNATIONAL

Review of Cytology A SURVEYOF CELLBIOLOGY

Editor-in-Chief

G. H. BOURNE St. George’s University School of Medicine St. George’s, Grenada West lndies

Associate Editors

K. W. JEON

M. FRIEDLANDER

Department of Zoology University of Tennessee Knoxville, Tennessee

The Jules Stein Eye Institute UCLA School of Medicine Los Angeles, California

VOLUME105

1986

ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers

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(alk. paper)

PRI\TtUlhTHI I ~ I I F D \ 1 4 T F S O F 4 h l ~ R l ( ~

Xh 87 88 89

9 8 7 6

5 4 3

2 I

Contents

Remodeling of Nucleoproteins during Gametogenesis, Fertilization, and Early Development DOMINICPOCCIA I. 11. 111. IV.

... .. Introduction . . . . . . . . . . Chromatin Structure and n ............... ............... Embryonic Histone Variants and Posttranslational Modifications . . . . . . . . . . . . . Sperm Nuclear Proteins and Transitions during Spermatogenesis . . . . . . . . . . . . .

VI.

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

......

1 2 3 15 39

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

53 54

Toward a Comprehensive Three-Dimensional Model of the Contractile System of Vertebrate Smooth Muscle Cells ROLANDBAGBY

I. 11. 111. IV. V.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Two-Dimensional Information Three-Dimensional Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Three-Dimensional Analysis and Reconstruction . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

67 70 97 111

121 124

Neuroendocrine Controi of Secretion in Pancreatic and Parotid Gland Acini and the Role of Na+,K+-ATPase Activity SETHR. HOOTMAN

I. 11.

111. IV. V.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stimulus-Response Coupling in Pancreatic Acini . . . . . . , . . . . . . . . . . . . . . . . . . Stimulus-Response Coupling in Parotid Acini . . . . . . . . . . . . . . . . . . . . . . . . . . . . Morphology of Membrane Domains in the Exocrine Pancreas and Parotid Gland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Intercellular Junctions in Pancreatic and Parotid Acini . . . . . . . . . , . . . . . . . . . . . V

129 130 135 138

141

vi

CONTENTS

VI . VII . VIII .

IX . X. XI

Molecular Characteristics of Na+ .K +.ATPase and Presence in the Pancreas ................................ and Parotid Gland . . . . . . . . . . . . Cytochemical Localization of Na TPase . . . . . . . . . . . . . . . . . . . . . . . . . . Autoradiographic Localization of Na+ K + -ATPase . . Determination of Na+ .K +.ATPase Activity in Viable Cells . . . . . . . . . . . . . . . . Effects of Secretagogues on Na+ .K+.Pump Activity in Pancreatic and Parotid Gland Acinar Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

144 147 154 155 157 172 176

Automated Individual Cell Analysis in Aquatic Research CLARICE M . YENTSCHAND SHIRLEYA . POMFWNI I.

I1 . I11 . IV . V.

VI . VII . VIII .

............................................. ntation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell Separation and Enrichment ............................. Introduction . . . . .

Cellular Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Standards, Controls, Data Interpretation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phylogenetic Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Addressing Aquatic Research Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SummarylConcluding Remarks ............................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

183 190 201 210 217 218 232 239 239

Establishment of the Mechanism of Cytokinesis in Animal Cells R . RAPPAPORT I

I1

111. IV

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Early Studies and Speculation ......................................... Results of Experimental Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

245 246 250 274 278

The Circumventricular Organs of the Mammalian Brain with Special Reference to Monoaminergic Innervation CLAUDEBOUCHAUD AND OLIVERBOSLER

I I1 111 IV

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anatomofunctional and Cytofunctional Aspects of the Mammalian Circumventricular Organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Monoaminergic Innervation of the Mammalian Circumventricular Organs ..... Concluding Remarks: On the Role of Monoamines in the Integrative Functions of the Circumventricular Organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

283 285 295 318 321 329

INTERNATIONAL REVIEW OF CYTOLOGY. VOL. 105

Remodeling of Nucleoproteins during Gametogenesis, Fertilization, and Early Development DOMINICPOCCIA Departmenf of Biology, Amherst College, Amherst, Massachusetfs 01002

I. Introduction The chromosomes of virtually all eukaryotic cells consist minimally of DNA and histone proteins. However, it has long been known that the DNA-associated proteins of many mature sperm cells may be radically different from the histones characteristic of somatic cells (Miescher, 1897). In fact, the only known exceptions to the ubiquity of histones occur in male germ cell lineages, where histones are sometimes dispensed with altogether in postreplicative, posttranscriptive spermatids. In other cases, sperm-specific histone variants replace or supplement somatic-type histones. The near ubiquity of the histones and their unusually high degree of amino acid sequence conservation during evolution serve as evidence for a critical role in normal cellular physiology. Therefore, the unusual nucleoprotein composition of the mature sperm cell might be expected to be reversed soon after fertilization if the male chromatin is to behave properly in subsequent cell cycles. Substitution of one or more histone variant subtypes by other variants or basic DNA-binding proteins represents a fundamental remodeling of the chromatin. Histone remodeling is likely to have major effects on the structure of the chromatin, since histone “function is structure, the proper dynamic packaging of DNA in the nucleus” (Simpson and Bergman, 1981). In addition to the more extreme types of nucleoproteins or sperm-specific histone variants found in sperm cells, nonallelic variants of histones have been demonstrated in different somatic tissues and at different stages of embryonic development (Cohen et al., 1975; Zweidler, 1984). Models have been devised for how chromatin composition might change in cell lineages in which different histone variants are synthesized at different times (Newrock et al., 1978b; Weintraub et al., 1978). Such replication-dependentremodeling differs from the major switching of histone or basic protein variants occurring in single-cell types without replication, for example, during spermatogenesis or pronuclear development (Dixon, 1972; Poccia et al., 1984). In this article, I will review what is known about the transformation of nu1

Copyright Q 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

3

DOMINIC POCCIA

cleoprotein types during gametogenesis, fertilization, and early development, outline chromatin structural changes which accompany these nucleoprotein transitions, and speculate on some possible functions of the unique nucleoproteins and histone variants associated with gamete, zygote, and early embryonic nuclei.

11. Chromatin Structure and Histone Variation

The basic structure of the 10-nm-chromatin fiber is now well established and has been reviewed extensively (Elgin and Weintraub, 1975; Komberg, 1977; Lilley and Pardon. 1979; Lewin, 1980; McGhee and Felsenfeld, 1980; IgoKemenes et a / . , 1982; Weisbrod, 1982; Reeves, 1984). Two each of the four core histones (H2A, H2B, H3, and H4; molecular weights 10,000-16,000) form an octamer which protects 146 base pairs of DNA from micrococcal nuclease digestion. This DNA wraps about the octamer 1.75 tums to form the core nucleosome (Richmond et al., 1984; Burlingame et a l . , 1985). The central portion of the fifth histone, H1, is believed to bind to the DNA as it enters and exits the nucleosome to complete two full tums, conferring protection from micrococcal nuclease digestion on an additional 15-20 base pairs (bp) (Simpson, 1978; Allan et a/., 1980). The approximately 160-bp unit containing the core histone octamer and H 1 has been termed the chromatosome (Simpson, 1978). H 1 is also believed to associate with the variable amount of linker DNA which connects one nucleosome to the next along the chromatin fiber (Noll and Kornberg, 1977). The amount of linker DNA determines the average nucleosomal spacing or repeat length revealed by limited nuclease digestion. Linker DNA may also associate with N-terminal regions of core histones (Allan et a / . , 1982). The 10-nm fiber organizes as a 30-nm fiber for which there are several models (Felsenfeld and McGhee, 1986). Above the level of the 30-nm fiber, little is known about the way in which the chromatin is packed. The most densely packed interphase chromatin is found in some sperm nuclei (Pogany et a ] . , 1981; Green and Poccia, 1985) and equals or exceeds in compaction metaphase chromosomes. Histones are not as conserved as is often assumed. The most highly conserved histone, H4, which suffers just two conservative amino acid substitutions out of 102 residues between calf and pea (de Lange et af., 1969), shows much greater variation (9-22%) in yeast, Neurospora, and Tetrahymena (Woudt et al., 1983; Hayashi et a!., 1984; Glover and Gorovsky, 1979). In general, H3 is the next most conserved, H2B and H2A show greater variability, and H1 is the least conserved. This order also holds generally for variation within different tissues of a given organism. Since most organisms have multiple genes for histones, nonallelic variants are possible. For example, yeast has two genes coding for H2A subtypes different at 2 out of 131 amino acids (Choe et a l . , 1982). Non-

REMODELING OF NUCLEOPROTEINS

3

allelic histone variants can be of the same size (homomorphic), differing in sequence, or of different sizes (heteromorphic) related by various insertions or deletions (West and Bonner, 1980). Certain regions of histones are less conserved than others. In general, most differences are seen in the N-terminal portions of core histones, thus conserving the C-terminal regions which are involved in histone-histone interactions (Isenberg, 1979). In H1, variation is greatest on both sides of a more or less centrally located conserved segment (Allan et al., 1980). Nonallelic homomorphic variants often differ in their hydrophobic regions and are usually best resolved using polyacrylamide gel electrophoresis in the presence of the nonionic detergent Triton X-100 (Zweidler, 1978). Protein sequencing and the sequencing of DNA clones continue to give more information on the range of histone variants found in nature. The list is far from complete for most organisms. Although the synthesis of histones is often tightly linked to DNA synthesis, this is not always the case (Coffin0 et al., 1984; Wu et al., 1984). Zweidler (1984) has classified mammalian histone variants as replication dependent, partially replication dependent, replication independent, minor, and tissue specific. For example, the HI variant H5 is erythrocyte specific; the Hlo variant is associated with tissues that have ceased cell division. Such behavior suggests that different histone variants may serve different functions and that some might be restricted to subregions of the genome. In addition to primary sequence variation, histones suffer various postranslational modifications such as phosphorylation, acetylation, methylation, ubiquitination, and ADP-ribosylation, which can alter their charges, conformation, and strengths of binding to DNA. Most of these modifications take place in the N-terminal regions of core histones and most likely modulate the affinity of the histones for DNA (Isenberg, 1979). Secondary modifications may play a role in gene activation (Allfrey, 1977; Weisbrod, 1982).

111. Embryonic Histone Variants and Posttranslational Modifications

A. HISTONEVARIANTS AND CHROMATIN REMODELING IN EARLYDEVELOPMENT Changes in HI subtypes during early development have been reported for several organisms, but changes in core histones are apparently less common. However, the demonstration of core histone variants generally requires sensitive electrophoretic techniques which have not always been employed (Fig. 1). The best documented cases of histone remodeling in the early embryo are from the sea urchin. A list of references to known sequences of histones expressed in oocytes or early embryos is given in Table I.

4

DOMINIC POCCIA

cs1-

1

0

CS2AP,r*82A Sp2A+ 0 2 A

m

y T f cs2e M

U 4' 4

FIG. I . Diagram of a two-dimensional gel electrophoretic separation of histones found in sea urchin development. Sp variants are found in mature sperm cells, CS proteins in oocytes and cleavage stages, and histones designated with Greek letters in embryos. Protein M is the H2A variant H2A.Z, N is phosphorylated Sp H I , and 0 and P are phosphorylated Sp H2Bs. From Poccia et ul. (1981).

TABLE I SEQUENCES FROM HISTONE GENESEXPRESSEDI N OOCYTES OR EARLYEMBRYOS Histone

PIDO

Reference

Chicken (GaNus domesticus) H1 H2A H2A.F H2AlH2B H2B H3 H4

Sugarman et al. (1 983) D'Andrea er a/. (1981) Harvey ef a/. (1 983) Harvey et a / . (1982) Grandy et al. (1982) Engel et a / . (1982) Sugarman er al. (1983)

Newt (Norophrhalmus viridescens) Hi H2A H2B H3 H4

Stephenson e t a / . (1981) Stephenson et a / . (1981) Stephenson et a / . (1981) Stephenson er al. (1981) Stephenson et al. (1981)

5

REMODELING OF NUCLEOPROTEINS TABLE I (Continued) Histone Frog (Xenopus laevis) H2A H2B H3 H4 H4

Sea urchin HI

P/Da

D D D D D

Reference

Moorman et al. (1982) Moorman er al. (1982) Moorman et al. (1980); Ruberti et al. (1982); Moorman et al. (1981) Turner and Woodland (1982) Turner and Woodland (1982); Moorman et al. (1981); Zernik et al. (1980)

D

Levy et al. (1982)

H2A H2A H2A H2A

Eb (Strongylocentrotus purpuratus) E (S.purpuratus) E (Parechinus miliaris) E ( P . miliaris) E ( P . miliaris)

D D D D

H2A H2B 1

Lc (P. miliaris) E ( P . miliaris)

D D

H2B2 H2B H2B H3 H3 H3 H4 H4 H4 H4

E E L E E L E L E E

D D D D D D D D P D

Sures et al. (1976); Sures et al. (1978) Busslinger et al. (1980) Schaffner et al. (1978) Grosschedl et al. (1981); Birchmeier et al. (1982) Busslinger and Barberis (1985) Busslinger et al. (1980); Schaffner et al. (1978) Busslinger et al. (1980) Sures et al. (1978); Sures et al. (1976) Busslinger and Barberis (1985) Sures et al. (1978) Busslinger et al. (1980) Childs et al. (1982) Grunstein et al. (1981) Childs et al. (1982) Wouters-Tyrou et al. (1976) Bussingler et al. (1980)

P

Rodrigues et al. (1985)

( P . miliaris) (S.purpuratus) ( P . miliaris) ( S . purpuratus) ( P . miliaris) (L.pictus) ( S . purpuratus) (Lytechinus pictus) ( P . miliaris) (P. miliaris)

Wheat (Triticum aestivum) H2A(1)

aP, from protein sequence and D, from DNA sequence. bEarly. cLate.

1. HI Histones

a. Sea Urchin. The first report of modulations of HI species during sea urchin (Strongylocentrotus purpurutus) development was by Hill et al. (197 1). The predominant species at blastula was augmented by a species with faster mobility on acid-urea gels. It was not clear whether the H l s appearing in early and late embryogenesis were different variants or forms differing in phosphoryla-

6

DOMINIC POCCIA

tion state as found in Arbacia lixula (Ruiz-Carillo and Palau, 1973). It was later shown in S. purpuratus that the two H1 species were synthesized at different times (Seale and Aronson, 1973). Similar switches in HI subtype were demonstrated for Lytechinus picrus, A . punctulafa (Ruderman and Gross, 1974), and Parenchinus angufosus (Brandt et a / ., 1979). Labeled early blastula H 1 is retained almost quantitatively in larval chromatin (Ruderman and Gross, 1974). After its synthesis ceases, the fraction of the total H1 complement contributed by early H1 decreases with the same kinetics as the fraction of total cell number (nuclear DNA) contributed by blastula to any given stage (Poccia and Hinegardner, 1975) which is consistent with a lack of turnover k i n g development. Early H1 accumulates in embryonic cells which cease division early and therefore presumably make no late Hls to “dilute” the preexistent species (Pehrson and Cohen, 1985). The switch in H1 subtypes between early and late stages was shown to be transcriptionally regulated by Arceci et a f .(1976). In an in vitro cell-free translation system, RNA from unfertilized eggs codes for only early HI, but postgastrula RNA codes predominantly for late HI. These data also suggested that the HI subtypes were not merely forms differing in secondary modifications but transcripts of different genes. This suggestion was confirmed by Newrock et al. (1978a) who showed that mRNAs extracted from polyribosomes from different embryonic stages in S. purpuratus code for three different HI variants in an in v i m translation system in which secondary modifications were absent. The early HI was called olHl and the two later forms were named P and y. The switch from early to late HI is not affected by preventing cleavage (Brookbank, 1978), disrupting the cell cycle with hydroxyurea or polyspermy (Harrison and Wilt, 1982), nor by separating the 16-cell embryo into micro-, macro-, and mesomeres (Arceci and Gross, 1980a). Others, however, have claimed that histone synthesis is shut off in cells dissociated at the swimming blastula stage (di Liegro el al.. 1978). The H1 switch is apparently sensitive to the drug cordycepin (Brookbank, 1980). Late H1 (postblastula) consists of at least two species (Rudennan and Gross, 1974; Poccia and Hinegardner, 1975; Gineitis er al., 1976). Pehrson and Cohen (1984) report that the two late forms of HI (6 and y) are retained in adult tissues, in addition to another HI (A) which has a low molecular weight and is not expressed before feeding larva. Sequence data are available for the embryonic and adult HIS of P . angulosus (Brandt et a [ . , 1979; de Groot et a / . , 1983). The H1 switches in S. purpuratus are not completely coordinate. Of the two electrophoretically resolved species of early Hl in S. purpuratus, the H l a , ceases synthesis at about 400 cells (hatching blastula) and the H l a , stops at about 700 cells (early gastrula) (Harrison and Wilt, 1982). Synthesis and incorporation into chromatin of the late Hls begin at about the 200-250 cell blastula (for Hly) and the 250-300 cell stage (for HIP).

REMODELING OF NUCLEOPROTEINS

7

Senger et al. (1978) have reported that the early H1 of A . punctulata is made up of two variants whose synthetic patterns show a transient change at the 8-cell stage. An unusual H1-like molecule is synthesized even before the early Hls (Newrock et al., 1978b). This species, called cleavage-stage (CS) H1, has solubility properties, staining characteristics, low Triton X- 100 affinity, and amino acid composition which place it in the H1 class. It has a rather high molecular weight for an H1, originally estimated at 24,000-28,000, but probably closer to 34,000 (Newrock et al., 1978b; Poccia, 1986). Cleavage-stage H1 reacts with HI-specific antibodies (Pehrson and Cohen, 1984). It is discussed further in Section V. b. Other Organisms. Switches in H1 subtypes in the early embryos of other organisms are fairly common. In the echiuroid worm, Urechis caupo, two H1 variants have been detected (Das et al., 1982; Franks and Davis, 1983). Germinal vesicles and cleavage-stage nuclei are enriched in the maternal Hlm, whereas the embryonic form Hle becomes predominant in the later embryo. This shift is reflected in a shift of synthesis of H1 from oocytes to early embryos. The surf clam, Spisula solidissima, has RNA coding for two H1 subtypes whose synthesis switches at the 32-64 cell stage (Gabrielli and Baglioni, 1975, 1977). In the snail Zlyanassa obsoleta, several H1 subtypes were identified which show differential synthetic patterns during development (Mackay and Newrock, 1982). Most workers have reported no synthesis or change in the set of H1 histone variants of the frog Xenopus laevis in early development (Destrke et al., 1973; Byrd and Kasinsky, 1973a,b; Adamson and Woodland, 1974; Cassidy and Blackler, 1978; Flynn and Woodland, 1980). Others have claimed that a shift in H1 subtypes detectable on Triton gels is seen in a comparison of histones iabeled from the 8-cell stage to blastula and late blastula to neurula (Koster et al., 1979). However, these shifts may result from differences in secondary modifications (van Dongen et al., 1983). Several H1 variants were found in later embryos and adult tissues and two were apparently adult specific, possibly related to H5 or Hlo (Risley and Eckhardt, 1981; Moorman and de Beer, 1985). Genes for several different Xenopus H 1 variants, which exist in different arrangements, have been isolated (Destrie et al., 1984). One gene cluster is expressed in oocytes, gastrula stage, and erythroblasts. An Hlo/HS-like variant has been detected cytochemically in many adult tissues of Xenopus, but not in oocyte or mature sperm nuclei (Moorman and de Boer, 1985). It was, however, present in spermatogenic cells. 2. Core Histones a. Sea Urchin. An elaborate developmentally regulated program of core histone variant incorporation into chromatin takes place in early sea urchin embryos. The urchin has been the most intensively studied and may be the

8

DOMINIC POCCIA

organism which possesses the most extreme diversity of histones. For example, at least 24 histone variants are known in P. angulosus, not counting CS subtypes (Schwager et al., 1983; Brandt et al., 1979). It is worth reviewing the progress made in identifying histone subtypes in the sea urchin since few other organisms have received the kind of scrutiny that it has. Early experiments were hampered by the inadequacy of electrophoretic systems used and contamination problems, particularly in early stages. For example, Orengo and Hnilica (1970) reported typical histones in hatching blastula and gastrula stages, but unusual arginine-rich proteins in the 4- to 8-cell stage. Johnson and Hnilica (1970) could not find typical histones in the chromatin before the @-cell stage, although they reported histone synthesis and therefore suggested a lag in the incorporation of the histones into nuclei. Benttinen and Comb (1971) found nonstoichiometric ratios of core histones. Crane and Villee (197 1) and Thaler et a / . (1970) compared gastrula, sperm, and unfertilized egg histones, but inadequate resolution and contamination problems make interpretation of these patterns difficult. Changes in core histone patterns between blastula and pluteus could not be distinguished by Marushige and Ozaki (1967). Vorob’yev et a/. (1969) suggested that there were quantitative differences in the arginine-rich histones between blastula and gastrula. Clear histone patterns on high-resolution gels were obtained by Hill et al. (1971). These gels revealed multiple-core species with one H2A increasing and one H3 decreasing from blastula to pluteus. Whether these were differences in modified forms or in primary structure could not be demonstrated. Seale and Aronson (1973) found no differences in core histones between the 16-cell stage and pluteus and found only H2A and H2B before this stage. Ruiz-Carillo and Palau (1973) found quantitative differences in core histone fractions between blastula and gastrula and heterogeneity in the H3 and H4 fractions due to acetylation. Poccia and Hinegardner ( 1 975) found differences in the H2A and H2B fractions, with the apparent loss of two H2B species by late larval stages. Similar results were reported by Gineitis et al. (1976) who also showed that the patterns remained the same in animalized, vegetalized, or normal embryos. Much of the confusion regarding the designation of histone bands on gels due to primary structural variation or secondary modification was eliminated only with the introduction of gel systems of greater resolution (Zweidler and Cohen, 1972; Cohen et al., 1973; SaviC and Poccia, 1978), of in v i m translation systems (Arceci et al., 1976; Newrock er a / ., 1978a), and of protein sequencing (von Holt et al., 1984). By pulse-labeling histones of S. purpururus with [3H]leucine and the analysis of acid-extracted chromatin at various stages on gels containing the nonionic detergent Triton X-100,Cohen et al. (1975) demonstrated convincingly that a set of stage-specific switches in core histone variant synthesis occurred in early sea urchin development. Histones incorporated into early chro-

REMODELING OF NUCLEOPROTEINS

9

matin did not turn over extensively and late forms were not derived from early forms. They suggested that these forms differed in primary structure. This work was extended by Newrock et al. (1978b) who identified additional components. Similar switches of core variants have been demonstrated in the chromatin of other sea urchin species (Treigyte and Gineitis, 1979; Brandt et al., 1979; von Holt et al., 1984) The known core variants are in the H2A and H2B classes (Figs. 1 and 2); all sequenced sea urchin genes or proteins of H3 or H4 are identical within each class (Childs et al., 1982). The first synthesized histone variants are CS histones (Newrock et al., 1978b). These are synthesized between fertilization and morula, after which their synthesis ceases. A second set of variants (a)is synthesized by at least the third S phase after fertilization until the blastula stage. Another set of variants (p, y, E) begins to be made during blastula with the late forms of H2A and H2B synthesized slightly later than the late forms of H1 (Harrison and Wilt, 1982). As a consequence, the composition of the chromatin changes throughout the cell cycles of early development as new histone variants are incorporated, while the preceding variants are, for the most part, retained (Rudeman and Gross, 1974; Poccia and Hinegardner, 1975; Cohen et al., 1975; Newrock et al., 1978b; Arceci and Gross, 1980b). That the histone variants in early sea urchin development actually differ in primary structure was proved by in vitro translation of mRNA from different stages of development (Newrock et al., 1978a; Weinberg et al., 1977; Hieter et al., 1979; Childs et al., 1979). Newrock et al. (1978a) showed that in vitro translation of morula RNA produces only a variants, blastula polysomal RNA gives almost entirely a variants, but gastrula stage RNA codes predominantly for later types. Typical modified forms due to acetylation are not made in the in vitro system. No late forms were seen in the translation of mRNA from the total RNA of early embryos. Childs et al. (1979) showed that the transitions of mRNAs in the early to late histone switches are not abrupt, since small amounts of late mRNAs could be detected in early stages and small amounts of early mRNAs were synthesized during gastrula. An additional early (a)and a late (E) H2A variant were found, and CS H2A mRNA assayed by cell-free translation was found in late stages as well as in unfertilized whole egg RNA (Fig. 2). Low levels of late histone mRNAs can be detected in oocytes (Knowles and Childs, 1984; Busslinger and Barberis, 1985). Spinelli et al. (1979) confirmed the shift from early to late mRNAs in Parechinus lividus but did not detect any CS variant mRNAs. However, they selected newly synthesized histone mRNAs with a recombinant probe containing (Y histone genes, so CS mRNAs might have escaped detection. They suggested that either CS transcripts were present in very low concentrations, were subtypes of a mRNAs, or were synthesized in early but not mature oocytes. Shifts in mRNA populations from early to late stages are also seen for

10

DOMINIC POCCIA

REMODELING OF NUCLEOPROTEINS

11

histone genes that code for identical proteins such as H4s (Grunstein, 1978). In situ hybridization experiments have shown that the ci mRNAs are uniformly distributed in cleaving embryos, whereas, at blastula, cells in certain regions become depleted (Cox et al. 1984). The shift from early to late histone mRNAs is not regulated differently in various cell blastomeres and subsequent differences in the ratio of early to late mRNAs may be a simple reflection of variation in cellcycle progression for different lineages (Angerer et al., 1985). To date, early and late sea urchin histone variant genes have been isolated but CS genes have not (Kedes and Birnstiel, 1971; Overton and Weinberg, 1978; Maxson et al., 1983b; Childs et al., 1982; Busslinger and Barberis, 1985). Most of our knowledge of embryonic histone amino acid sequences is derived from these isolated genes. The expression of sea urchin histone genes has been extensively reviewed elsewhere (Kedes, 1976, 1979; Hentschel and Birnstiel, 1981; Weinberg et al., 1983; Maxson et al., 1983a,b). The ci and later variants are similar in size and sequence (see Table I for references; also von Holt et al., 1979, and Schwager et al., 1983, for partial sequences including H2B from the adult intestine). The most variable regions occur at the ends of the molecules. Within the H3 or H4 classes, early and late genes code for identical proteins and the sequences determined for different species are also identical (Childs et al., 1982). The CS proteins differ most radically on gels from the later embryo histones of their classes. CS H2A has the high Triton affinity characteristic of H2As, is larger than somatic or embryonic H2As (heteromorphic), and reacts with an H2A-specific antibody that recognizes ci and all later H2As except an H2A variant called Y6, Z, or M (Newrock et al., 1982; Newrock et al., 1978b; Wu et al., 1982; Poccia et al., 1981). CS H2B is essentially the same size as somatic or embryonic H2Bs (homomorphic) but has a higher Triton affinity. CS H2A and CS H2B behave as expected for core nucleosomal proteins upon digestion of chromatin with micrococcal nuclease (Shaw et al., 1981) and during replication (Poccia et al., 1981, 1984). The designation CS is somewhat of a misnomer, since CS proteins are synthesized in oocytes (Herlands et al., 1982). CS histones accumulate in a storage pool in sea urchin eggs (see Section V,B). The pool does not contain 01 or later subtypes (Poccia et al., 1981; Salik et al., 1981). In addition to the rather extensive switching of histone variants in sea urchins, most of the histones are actively modified posttranslationally during develop-

FIG. 2. Developmental expression of sea urchin histone genes. The pattern of expression of S. purpurutus histone genes in early development is indicated by thick lines for abundant components

and thin lines for relatively minor components. Broken lines indicate uncertainty about the synthesis of a species. Data are based on in vivo protein synthesis, in vitro cell-free translation, or the pulselabeled in vivo mRNA experiments of Newrock et al. (1977) and Childs et d . (1979). From Childs et ul. (1979).

12

DOMINIC POCCIA

ment. Although mature sperm (Sp) variants show no microheterogeneity due to secondary modifications (Easton and Chalkley, 1972), Sp H1 and Sp H2B become phosphorylated after fertilization (Green and Poccia, 1985). Acetylation accounts for most of the heterogeneity of embryonic H3 and H4 fractions (Burdick and Taylor, 1976; Treigyte and Gineitis, 1979). In an in vitro system, Horiuchi et al. (1984) found that the rates of histone acetylation and deacetylation in isolated sea urchin nuclei remained at a high and constant level between morula and gastrula stages. Chambers and Shaw (1984), however, found that the amount of diacetylated H4 declined with development and suggested a correlation with the decreasing rate of cell division seen in the early embryo. H2A and H2B, but not H1 variants, also seem to be acetylated (Horiuchi et al., 1984; Chambers and Shaw, 1984) but these were not directly investigated. b. Other Organisms. Switches in core histone subtypes analogous to the sea urchin switches have been found in a limited number of organisms. During development of the mud snail, I. obsoleta. there appears to be an H2A and an H2B component which are synthesized in oogenesis but not during early embryogenesis, an H2A and H2B synthesized only during early embryogenesis, and an additional set of H2Bs which corresponds to sea urchin late histones appearing in the veliger larva stage (Mackay and Newrock, 1982). In the chicken, new H2B and H3 variants appear during somite formation (Urban and Zweidler, 1983). All variants present at this stage continue to be expressed in the adult. Most chicken histone genes isolated so far are expressed in the embryo but not in the adult chicken (Engel, 1984). These might be specific to embryonic cell types or to cells undergoing rapid proliferation. The chicken variant H2A.F appears to be activated relative to the major H2A variant (H2A. 1) during early development and expressed in some but not all adult tissue. It is 40% divergent from H2A.1 (Harvey et al., 1983). Engel (1984) suggests that because of sequence divergence, not all chicken genes may have been accounted for in searches using embryonic or heterologous probes. Wheat embryos contain at least three histone H2A variants, one of which has been completely sequenced (Rodrigues el ul., 1985). This one is unusual in that it has a 19 amino acid C-terminal extension which has some sequence homology with Hls. In some organisms, no major changes are detected in the core histone complement in early development. Imoh (1978) could find no differences on Triton gels in histones of the newt Trifuruspvrrhogaster from blastula to tail bud. No one has found core histone variants inX. luevis (Woodland, 1980, 1982). Although it is not certain whether some special variants are synthesized in the first several cell cycles in this large egg, it is likely that Xenopus possesses limited heterogeneity within its histone classes. Hybrid selected mRNAs from ovary, blastula, or neurula, when translated in a cell-free system, all code for the same set of

REMODELING OF NUCLEOPROTEINS

13

histone variants as judged by electrophoresis in a variety of systems (van Dongen et a f . , 1983). Other histone genes active before blastula or in minor amounts could not be excluded. In Drosophifa, no stage-specific histone mRNA variants could be detected by size or by fidelity of hybridization to cloned DNA (Anderson and Lengyel, 1984). These methods should have distinguished mRNAs as different as early and late sea urchin histone variant mRNAs, but it is not clear that they would have been successful at detecting the more divergent CS-type mRNAs present in relatively low amounts. No changes were seen in core histones of U . caupo between mid-cleavage stage and gastrula (Franks and Davis, 1983). However, it is difficult to rule out the presence of histone variants in the isolated germinal vesicles which contain many unidentified spots in the core histone region of two-dimensional gels. In the mouse, all somatic variants appear to be expressed from neurula until birth. The proportions of the variants differ in different tissues and can be correlated with rates of cell division (Zweidler, 1980, 1984). However, major differences in the proportions of histone variant mRNAs occur between oocytes and CS embryos (Graves et al., 1985). The histone composition of early embryo mouse nuclei is not known.

3. Summary Switches in H1 subtypes seem to be of general occurrence in early embryos. Available data do not indicate that the switching of core histone subtypes, as seen most prominently in the sea urchin, is as widespread. Since some late embryo histone variants may be present in adult tissues (Lennox and Cohen, 1984; Busslinger and Barberis, 1985), one must entertain the possibility that the only histone variants that represent truly embryonic subtypes in the sea urchin are the a and CS proteins. Since these appear predominantly during very early stages when cell numbers are low, it is possible that other organisms also have embryo-specific histones which, because of technical limitations, have escaped detection. Even in the sea urchin, especially favorable for studies of early embryonic nuclei, the realization that variant switches occurred, or even that a full complement of histone classes was present in early embryos, was a long time in coming. Comparative data are not yet sufficient to determine how general the occurrence of early embryo-specific histone variants might be. B. EFFECTSAND POSSIBLE FUNCTIONS OF HISTONE VARIANT MODULATIONS IN EARLYDEVELOPMENT Variations of histone subtype between blastula and pluteus stages in the sea urchin confer altered physical properties upon core nucleosomes. Differences

13

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were found in thermal denaturation, ionic strength-dependent unfolding, DNase I cutting site specificity, and the rate of digestion of nucleosomes (Simpson and Bergman, 1980. 1981; Simpson, 1981). In general, the embryonic chromatin core particles are more easily unspooled or totally disrupted than adult nucleosomes (Simpson and Bergman, 1981). It has been suggested that such alteration is necessary during replication or transcription and that the in vivo instability may be affected both by the type of histone variants present and by other factors, such as secondary modifications (Simpson and Bergman, 1981). Positive correlation has been made between the types of histone variants and rates of replication in sea urchin embryos (Cohen et al., 1975; Lennox and Cohen, 1984). In this view, a (or CS) histones might facilitate rapid replication, and later subtypes restrict or be incompatible with it. In contradiction to this hypothesis, more recent data on the distribution of H1 in cell lineages during early development demonstrate that aHI is most concentrated in cells which have ceased or slowed division before the early to late histone switch, whereas late H l s predominate in cells that are still dividing (Pehrson and Cohen, 1985). Roles for histone variants in modulating the average nucleosomal repeat length have been suggested for both H I (Keichline and Wasserman, 1977, 1979; but see Savii et a / . , 1981) and core histones (Savik ef a/.. 1981; Shaw et al., 1981; Poccia et al., 1984). In general, repeat lengths in various sea urchin species increase from the two-cell stage to larval stages as histone subtypes are modulated (Keichline and Wasserman, 1977, 1979; Arceci and Gross, 1980b,c; Savii ef al., 1981; Shaw et al., 1981; Spinelli ef al., 1982). A role in generating special genomic subregions which might have informational content and be involved in the generation of defined cell lineages has been proposed (Weintraub et al., 1978). Assuming switches in histone variant synthesis, declining numbers of variable replication origins used during early development, and reasonable models of histone deposition during replication, Weintraub et af. (1978) have shown how variegated chromosome structures (containing regions of different variant composition) can be generated, resulting in divergent chromosome compositions in different cell lineages. Assuming fixed origins of replication and a restriction of histone subtype synthesis, the model also shows how a dividing stem cell might generate two sets of daughter chromosomes, one of which is identical to and one of which is different from the maternal chromosome set in histone composition. Richards and Shaw (1984) offer evidence that nucleosomes become increasingly heterogeneous as development proceeds in the sea urchin. Whether this is a reflection of divergence of cell type or includes changes within a given cell type could not be determined. The switch from early to late mRNA subtypes does not appear to be coordinate in all cells (Angerer et al., 1984a). Whether this difference is cell-type specific or cell-cycle dependent is not known.

REMODELING OF NUCLEOPROTEINS

15

IV. Sperm Nuclear Proteins and Transitions during Spermatogenesis A. MATURESPERM Mature sperm cells exhibit a wide range of sizes and shapes (Fig. 3; Yasuzumi, 1974; Fawcett et a l . , 1971). Their nucleoprotein contents are almost as diverse. The variety of nucleoproteins in mature sperm cells was reviewed by Bloch (1969) and a revised catalog was subsequently prepared (Bloch, 1976). Bloch organized sperm nucleoproteins into five classes: (1) monoprotamine or “salmon-type’ ’ (containing arginine but no lysine), (2) protamine-like or ‘‘mouse or grasshopper-type” (arginine-rich, containing oxidized cysteine), (3) intermediate or ‘‘Mytilus-type” (both histone and protamine properties), (4) somatic histone or “Rana-type” (typical histone), and ( 5 ) nonbasic or “crab type” (no histones or protamines). Much of this categorization was based upon cytochemical data and is therefore of limited specificity. Thus, Bloch’s valuable compendia need to be supplemented with the many biochemical studies subsequently reported. Recent biochemical work demonstrates relationships between some of the categories and subtle differences within them. In the biochemical sense, the catalog is still incomplete. In particular, some of the biochemical studies have suffered from difficulties in isolating pure populations of mature sperm cells and must be taken as tentative. I shall consider sperm nucleoproteins in three relatively clear categories and combine Bloch’s classes (3) and (4) into a rather heterogeneous fourth grouping. A list of references of the amino acid sequences of histones and other nucleoproteins from mature sperm or testis is given in Tables II-V. 1, Nonbasic Proteins The most unusual group of sperm nuclear proteins draws representation entirely from crustaceans whose sperm is generally aff agellate and nonmotile, and whose sperm nuclei are not highly condensed (Fig. 3c; Yasuzumi, 1974). Using cytochemical criteria, the sperm of several crabs and two crayfish seem to lack basic nuclear proteins altogether (Bloch, 1976). Vaughn and Hinsch (1972) isolated crab chromatin from Libinia emarginata and could find neither histones nor protamines by gel electrophoresis. The chromatin thermal denaturation profiles showed no stabilization over that of naked DNA and the protein copurifying with the DNA was acidic. However, another crustacean with aflagellate sperm and relatively diffuse chromatin, the shrimp Palaemon serratus, is reported to contain histones resembling the somatic type and no protamines (Sellos and LeGal, 1981). Chauviere et al. (1982) reported a full set of histones from testis of the crab Cancer pagurus, but the cell types present were not reported.

16

DOMINIC POCCIA ,-

1

A

a

C

b

d

Fic. 3 . Various spermatozoa. (a) From animals: (A) beetle (Copris),(B) insect (Calathus), (C) bird (Phyllopneusre), (D)bird (Muscicupu), (E) bullfinch, (F) gull (Larus). ( G I ) Tadorna. (J,K) snail (Paludina),and (L) snake (Coluber);(b) from mammals: (A,B) badger, (C-E) bat (Vesperugo. Rhinoloph~.~), (F) pig, (GIopossum, (H)opossum, double spermatozoon, and (I) rat; (c) unusual spermatozoa: (A-C) crustacean (Pol~phemus).(D,E) crab (Dromia), (F) Ethusia, ( G ) Maja, (H) Inachus, (I) lobster (Hon~arus), and (J) crab (Porctlluna);and (d) from plants: (A) alga (Fucus), (B) liverwort (Ptllia),(C) moss (Sphagnum), (D) Marsilia. (E) fern (Angioprtris). and (F)fern (Phegopteris). From Wilson (1898).

17

REMODELING OF NUCLEOPROTEINS

SPERM OR

TABLE I1 TEsns HISTONESEQUENCES

Histone

P/Da

Reference

P P P P P P

Macleod et al. (1977) Bailey and Dixon (1973) Kootstra and Bailey (1976; 1978) Candido and Dixon (1972d) Candido and Dixon (1972d) Candido and Dixon (1971)

Sea urchin (Parechinus angulosus) H1 H1 (Echinolampas crassa) (Strongylocentrotus gram1losus) H1 H2A (Parechinus miliaris) H2A (P. angulosus) H2A (P. miliaris) H2B 1 (P. angulosus) H2B2 (P. angulosus) H2B3 ( P . angulosus) H2B 1 ( P . miliaris) H2B2 (P. miliaris)

P P P P P D P P P D D

Strickland et al. (1980b,c) Strickland et al. (1980a) Strickland et al. (1982b) Wouters et al. (1978) Strickland et al. (1980a) Busslinger and Barberis (1985) Strickland et al. (1977a) Strickland et al. (1977b) Strickland et al. (1978b) Busslinger and Barberis (1985) Busslinger and Barberis (1985)

Starfish H2A (Asterias rubens) H2B (Marthasterias glacialis) H2B (A. rubens)

P P P

Martinage et al. (1983) Strickland et al. (1980d) Martinage et al. (1985a)

Cuttlefish H2A (S.officinalis)

P

Wouters-Tyrou et al. (1981)

Limpet H2B

P

van Helden

P P

Kmikcik et al. (1985) Kmitcik et al. (1985)

Trout HI H2A H2B H3 H4

(Salmo gairderii)

(S. gairderii) (S. (S. (S. (S.

strutfa) gairderii) gairderii) gairderii)

(Patella granatina)

Polychaete Hla (Plafynereis dumerlii) Hlb ( P . dumerlii)

et

al. (1979)

"P,from protein sequence and D, from DNA sequence.

2. Protamines The classic protamines (from fish) are small proteins rich in arginine, generally lacking lysine, and always lacking cysteine. They are about 30 amino acids long (molecular weights about 3000) with clusters of polyarginine (4-6 residues) scattered throughout. They are found in many teleosts and Bloch's survey (1976)

18

DOMINIC POCCIA TABLE III PROTAMINE SEQUtNCES

Organism

Number of residues

Reference

Tuna Thynnin Y2 Thynnin Z1 Thynnin 2 2

34 34 34

Bretzel (1972) Bretzel (1973a) Bretzel (1973b)

Caspian sturgeon Sturine B Stellin A

27 27

Yulikova et a/. (1976) Yulikova et a/. (1979)

Northern pike Protamine I

32

Speckert et a/. (1983)

Chum salmon Salmine A l Salmine A2

32 33

Ando and Watanabe (1969) Ando and Watanabe (1969)

Rainbow trout lridine IB pRPT59;pTPI I:CII Iridine I1 pRTP43;CIII pRTP94;pRTPI 78 pTP4 pRTP242;pTP8

33 33 32 30 30 30 21

Ando and Watanabe (1969) Gedamu et a/. (I98 I); Jenkins ( 1 979) Ando and Watanabe (1969) Gedamu e t a / . (1981) Gedamu et al. ( 198I ) Jenkins (1979) Gedamu et a/. (1981); Jenkins (1979)

Pacific herring Clupeine YII Clupeine Z Clupeine YI

30 31 31

Suzuki and Ando (1972) Iwai et a/. (1971) Suzuki and Ando (1972)

reports them in earthworm, centipede, pillbug, wolf spider, whelk, land snail, slipper, congo eel, lizard, snake, opossum, marsupial rat, liverwort, and club moss. The latter, however, were all cytochemically determined. Protamine sperm is nonnucleosomal (Honda et al., 1974). Protamines have been extensively reviewed elsewhere (Ando er d . ,1973; Hnilica, 1972). The references for protamine sequences are given in Table 111. 3. Mammalian Protamines Mammalian protamines are related to typical protamines. They are slightly larger, also arginine rich, and contain little or no lysine but always contain

REMODELING OF NUCLEOPROTEINS

19

TABLE IV MAMMALIAN PROTAMINE SEQUENCES

Organism Mouse Ram Bull Boar Human“ Rat* Horse“

Number of residues

50 50 47 50

Reference Kleene et al. (1985) Sautitre et al. (1984a) Coelingh et al. (1972) Tobita et al. (1983) Gaastra er al. (1978) Kistler et al. (1976) Monfoort et al. (1973)

aPartial sequence.

cysteine. The four known sequences of mammalian protamines, from bull, boar, ram, and mouse, and three partial sequences, from horse, rat, and human (see Table IV) show a definite relationship to the classic fish protamines. The eutherian mammalian protamines are 47-50 amino acids long and contain three domains. The central protamine-like stretch (approximately 25 residues) is highly arginine rich, containing 3-4 clusters of polyargine (each 4-7 units long) separated by neutral amino acids. In size, charge, and arginine content, this central portion resembles fish protamine and is immunologically cross-reactive (Rodman et al., 1984). The amino-terminal regions of mammalian protamines (approximately 15 residues) are highly homologous to each other. Each begins with four identical amino acids and each is the same in at least 10 of the first 15 sites. The C-terminal end is less conserved. The cysteine residues are more or less evenly distributed at relatively conserved positions. The relationship of the mammalian and fish protamines is discussed by Coelingh and Rozijn (1975). Some workers have claimed that mammalian sperm chromatin is nucleosomal (Wagner et al., 1978; Gusse and Chevailler, 1980a; Wagner and Yun, 1981). Others, however, report that such structures disappear in late spermatids so that mature mammalian sperm chromatin lacks nucleosomes (Kierszenbaum and Tres, 1975; Cech et al., 1977; Young and Sweeny, 1979; Loir et al., 1985). The mature mouse sperm nucleus is characterized by a very high packing density of chromatin fibers which, by calculation, is inconsistent with nucleosomal structure (Pogany et al., 1981). “Mammalian” protamines have been reported in several insects, a mollusc, and some elasmobranches as well as mammals (Bloch, 1976). In these nonmammalian cases, the evidence was entirely cytochemical so the relationship of these protamines to mammalian protamines is not clear. For example, insect protamines may not contain sulfur (Bloch, 1969). Since they are not yet well

20

DOMINIC POCCIA

characterized, nonmammalian protamine sperm will be discussed in the next section. 4. Somatic Histones and Intermediate Types Bloch’s two categories of somatic histones and intermediate types probably represent a continuum from the histones to the protamine-like molecules. At one extreme, R a m and goldfish sperm have been reported to contain only somatic type histones, although the former may have a sperm-specific basic protein and neither set of proteins was fractionated on Triton gels to search for spermspecific histone variants. Some intermediate type sperm, such as those of the sea urchin and starfish, lack protamine but contain sperm-specific histone variants. Others, such as from many molluscs and frogs, apparently have typical somatic histones with additional protarnines or other basic proteins. a. Molluscs. Most mollusc sperm fit cytochemically into Bloch’s categories ( 1 ) or (3), i.e., protamine or intermediate. Subirana et al. (1973) studied a large group of molluscs, including squid, octopus, abalone, mussel, chiton, surf clam, and limpet, by biochemical criteria. They found that some had protamines (snail, abalone, squid, octopus), although these molecules appeared larger than fish protamines (about 4000-8000 Da). Some had both histones and intermediate proteins (mussel and chiton). Others had intermediate-type proteins which were larger than histones (surf clam, limpet, Ostrea). Efedone, a cephalopod, had cysteine-containing proteins. Unfortunately, the amount of contamination from immature sperm and somatic cells is not often easily monitored. This is particularly troublesome when choosing between protamine-like and intermediate-type sperm since contaminating cells are likely to be histone containing. Balhorn et al. (1979) claim that in carefully prepared populations of mature abalone sperm only protamines are present. However, Colom and Subirana (1981) claim that the abalone, Haliotus tuberculata, and the marine snail, Gibbula divaricata, each contains protamine and, in addition, a single histone which has a similar amino acid composition to H2B, though it is different in molecular weight. The limpets, Patella granatina and Patella vulgata, had no histones. In the surf clam, Spisula solidissimu, Ausio and Subirana (1982a) report a high-molecular-weight basic protein with properties intermediate between protamines and HI histone as the major sperm protein. It is rich in lysine and arginine and, together with alanine and serine, the four amino acids account for 84% of the residues in 33,500-Da protein. The chromatin is nonnucleosomal and the histones may originate in contaminating immature spermatids (Herlands and Ausio, 1979). Anodonta piscinalis, a fresh water bivalve, has both somatic HI of 187 residues and an extremely basic sperm-specific histone of 224 residues with 59 lysines and 50 arginines (Rozov et al., 1985). Two marine bivalves, Swijtopectin swifti and Glycymeris yessoensis, have sperm-specific arginine-rich H 1s and

REMODELING OF NUCLEOPROTEINS

21

low-molecular-weightbasic proteins (S proteins) in addition to somatic histones in their sperm (Zalenskaya et al., 1985). Their chromatin is organized into nucleosomes of repeat lengths 226 and 223 bp respectively, approximately 30 bp longer than somatic tissues. In a study of sperm from the razor shell, Ensis minor, in which contamination was carefully monitored, core-type histones were found to coexist with an H1 histone variant and a protamine-like molecule (Giancotti et al., 1983). Its chromatin is organized into nucleosomes with a 200-bp repeat length. The HI, which contains a typical trypsin resistant central domain, is 50% basic with a high alanine and serine content, but no proline. It resembles the protein reported for surf clam by Ausio and Subirana (1982a). The protamine component resembles that from D . trunculus (Colom and Subirana, 1979), Mytilus edulis (Subirana et al., 1973), and Crenomytilus grayanus and M . dzflcilus (Odintsova et al., 1982). It is larger than typical fish protamine and contains lysine. Ausio and Subirana (1982b) claim that the mussel, M . edulis, contains a sperm-specific H2B in addition to a protamine and sperm-specificH1. However, based on its immunological cross-reactivity, Uschewa et al. (1985) suggest that the putative H2B is really an H1. Mussel sperm of three species have a fraction of their chromatin, corresponding to the histone-containingportion, organized in nucleosomes (Zalensky and Avramova, 1984), although the purity of cell type in these experiments was not documented. Oyster sperm from Crassostrea gigas contain no protamine and have two H 1 variants rich in arginine and serine in addition to a typical complement of core histones (Sellos, 1985). No comparison was made to somatic tissue. In summary, molluscs display typical somatic histones, sperm-specific histones, and protamine-like molecules different from fish or mammalian protamines. In some species, all three types of nucleoproteins are present. Sperm with histones and intermediate proteins can possess highly condensed chromatin, as in, for example, Chiton (Russell-Pinto et al., 1983). b. Arthropods. In the horseshoe crab, Limulus polyphemus, the sperm histones appear to be very similar to calf thymus, but 2-3% is comprised of a protein (Hlc) with properties intermediate between HI and H2B and resembling Hlo (Munoz-Guerra, 1982b). It is not known whether this variant is sperm specific. Limulus sperm chromatin is not completely condensed (Fahrenbach, 1973). Electrophoretic analysis of mature sperm from the cricket Acheta domesticus shows at least four low-molecular-weight species and no histones (McMasterKaye and Kaye, 1976). The chromatin is devoid of nucleosomes (Kierszenbaum and Tres, 1978; McMaster-Kaye and Kaye, 1980). c. Annelids. The polychaete worm, Platynereis dumerlii, has somatic histones, two protamine-like species, and two sperm-specific H1 variants in its sperm chromatin (Sellos and KmiCcik, 1984). The H1 variants have been sequenced (KmiCcik et al., 1985). They are unusually short (121 and 119 residues)

22

DOMINIC POCCIA

for Hls but have a typical central globular domain. Platynereis sperm chromatin has a nucleosomal repeat length of only 165 bp (KmiCcik et a l . , 1985). Sperm of the marine worm, Chaetopterus variopedatus, is reported to have an arginine- and lysine-rich protamine of about 5600 Da and an arginine-rich H1 of 22,000 Da (de Petrocellis et a l . , 1983). No core histones were detected. d. Echinoderms. Cytochemically, echinoderms exhibit sperm histones of the intermediate type. Biochemically, there are differences between the echinoderm classes. The sea cucumber, Holothuria tubulosu, contains five somatic type histones, a sperm-specific HI, and an H1-like molecule called $, (Subirana, 1970; Azorin et al., 1983). The $<,molecule is 78 amino acids long, highly basic, and similar to the C-terminal regions of somatic Hls. The sperm-specific H1 has a high arginine content (Phelan et al., 1972). Sea cucumber sperm chromatin is organized into nucleosomes with a repeat length of 227 bp and, upon micrococcal nuclease digestion, the insoluble fraction is associated with $<,(Azorin rt a l . , 1983, 1985). On the basis of three-dimensional reconstruction from electron micrographs, Subirana et al. (1985) have suggested that sea cucumber sperm chromatin is organized as 30-nm fibers formed from a zigzag arrangement of nucleosomes and that these are organized into layers. Sea urchin sperm contain only histones, but the sperm-specific Sp H2B is considerably larger than a typical somatic or embryonic H2B. Sp H2B consists of two or more variants (Subirana and Palau, 1968; Easton and Chalkley, 1972; de Petrocellis el a l . , 1980; Strickland et a l . , 1977a,b, 1978b). The number of sperm H2B variants is different for different species and sometimes for different individuals (Geraci et a l . , 1979; de Petrocellis et a l . , 1980; Strickland et a l . , 1978b, 1981; Zalenskaya and Zalensky, 1980). The sperm-specific Sp H1 is also larger than embryonic or somatic Hls (Hnilica, 1967; Palau et d., 1969; Paoletti and Huang, 1969; Ozaki, 1971; Strickland et a l . , 1980b, 1982a,b). Both sea urchin sperm histone variants are richer in arginine than their somatic counterparts (Subirana and Palau, 1968; Shirey and Huang, 1969; Palau et al., 1969). Sea urchin sperm also contain an H2A electrophoretically different from blastula aH2A (Easton and Chalkley, 1972). It is, however, identical in sequence to a variant expressed in the late embryo (Busslinger and Barberis, 1985). Most of the extra size of the Sp H1 and Sp H2B proteins is due to N-terminal extensions. These extensions are largely comprised of tandemly repeated tetrapeptides (for H1) or pentapeptides (for H2B) (Strickland et a l . , 1977a,b, 1978a,b, 1980b,c, 1982a,b; von Holt et a l . , 1979). Sea urchin sperm chromatin is organized into nucleosomes which are separated by the longest linker length of any chromatin yet measured. Reported repeat lengths for sea urchin sperm chromatin are 241 bp for A . lixula (Spadafora et a l . , 1976), 237 bp for Strongylocentrotus intermedius, 239 or 248 bp for L . pictus (Arceci and Gross, 1980~:Savid et a l . , 1981), 260 bp for A . punctulata

REMODELING OF NUCLEOPROTEINS

23

(Keichline and Wasserman, 1977), and 243 or 250 bp for S . purpuratus (Keichline and Wasserman, 1979; SaviC et al., 1981). Starfish sperm (Aphelusterias japonica) have five histones. H2A is slightly smaller and H1 is much larger than typical somatic histones (Zalenskaya et al., 1981a). The other sperm histones resemble calf thymus histones. Starfish sperm H2B (Asterias rubens) has been sequenced and has an unusual blocked (dimethylated) proline at its N-terminus (Martinage et al., 1985a). Unlike the sea urchin, starfish Sp H2B lacks N-terminal repeating pentapeptides and consists apparently of only a single variant. The repeat length of A . japonica sperm chromatin is 224 bp (Zalenskaya et a l . , 1981a,b). e. Fish. Sperm cells of bony fish, especially of the Clupeiformes, have been taken as possessing the archetypal protamines (Bloch, 1976). However, even these may contain small amounts of residual histones. Trout sperm was recently reexamined for the presence of histones (Avramova et al., 1983; Tsanev and Avramova, 1983). In preparations of sperm heads in which no somatic or spherical spermatid nuclei could be seen, small amounts of histones were detected after extraction of protamines with high salt, urea, and mercaptoethanol. These were tightly bound to the DNA, extractable in SDS (sodium dodecyl sulfate), and had electrophoretic mobilities identical to liver core histones. They were also recognized by antibodies to liver histones. The sperm of some fish retain histones and lack protamines altogether. For example, the goldfish, Carassius auratus, apparently contains no protamines and its sperm histones are of the somatic type (Munoz-Guerra et al., 1982a). Goldfish chromatin is beaded and shows nucleosomal digestion patterns with a repeat length of about 205 bp. Grass carp (Ctenopharyngodon idella) sperm chromatin has somatic-type histones (Kadura et al., 1983a) with a modest increase of an HI subfraction occurring during maturation (Kadura et a l . , 1985). Its repeat length is 200 bp (Kadura et al., 1983b). Although the sperm of both fish contain somatic-type histones, neither was analyzed on Triton X-100 gels for variants. In the winter flounder, Pseudopleuronectes americanus, mature sperm contain typical core and H1 histones and, in addition, a family of at least 16 highmolecular-weight basic proteins. These are 80,000-200,000 Da and are rich in arginine, serine, lysine, and proline (75% of total) with no cysteine (Kennedy and Davies, 1980, 1981, 1982). They are unphosphorylated in the mature sperm (Kennedy and Davies, 1982). The amino acid composition of these proteins is reminiscent of the N-terminal extensions of sea urchin sperm histone H1 and H2B discussed above, although the exact sequence may be slightly different (Kennedy and Davies, 1985). The chromatin is organized into nucleosomes with a repeat length of 222 bp (Kennedy and Davies, 1982). The dogfish Scylliorhines caniculus, an elasmobranch, has several low-molecular-weight sperm nuclear proteins called scylliorhinines (Gusse et al., 1983) (Table V). One of these (23) resembles fish protamine (Sautihre et ul., 1981). A

24

DOMINIC POCCIA TABLE V MISCELLANEOUS TESTIS-SPECIFIC NUCLEOPROTEIN SEQUENCES

Organism

Number of residues

Reference

Chicken (galline) Dogfish (scylliorhinine 2 3 ) Dogfish (scylliorhinine S4) Dogfish (scylliorhinine 22) Rat (TP) Boar ( H i t )

65 31 32 46 54 21 1

Nakano et a / . (1 976) Sautitre er a / . (1981) Sautitre el al. (1984b) Martinage er a/. (1985b) Kistler et a / . (1975) Cole er al. (1984)

second (22) is cysteine and arginine rich and 46-residues long (Martinage et al., 1985b). Another (S4) is an arginine- and lysine-rich protein of 32 residues with 4 cysteines, thus reminiscent of the mammalian protamines, although its sequence is unique (Sautikre et al., 1984b). Dogfish chromatin has been reported to have a beaded appearance in electron microscope spreads at low ionic strength after reduction and alkylation and treatment with an anionic detergent (Gusse and Chevaillier, 1980a, 1980b). f. Other Vertebrates. Frog sperm display a range of nucleoprotein types. Bufo sperm has a high-molecular-weight protein in addition to histones (de Leon and Vaughn, 1970). Bols and Kasinsky (1973) compared anuran sperm histones by gel electrophoresis. X. luevis sperm contained three rapidly migrating species and Bufo americanus sperm contained one more rapidly migrating species which comigrated with authentic protamine, but was later shown to contain lysine and histidine (Kasinsky et a f . , 1985). These proteins were not found in liver. The electrophoretic patterns of the rapidly migrating proteins in Xenopus varied with species (Mann et al., 1982). Rana pipiens showed only histones. While Rana sperm has long been classified as a “somatic type,” testis histones apparently contain a sperm-specific H1 (Alder and Gorovsky, 1975) which is lysine rich (Kasinsky et al., 1985). Reptiles (snakes and lizards) contain two testis-specific rapidly migrating bands similar to protamines (Kasinsky et al., 1978). Snake and lizard sperm chromatins do not seem to be stabilized by disulfide bonds (Bedford and Calvin, 1974). The one known avian sperm nucleoprotein sequence, from the chicken, seems to be a form that is intermediate between fish and mammalian protamine types (Nakano et al., 1976). It is 65 residues long, has a 14-residue N-terminal region with the same initial tetrapeptide as the mammalian sequences, and possesses a C-terminal arginine-rich region with six arginine clusters similar to fish protamines. The molecule, however, does not contain cysteine. g. Plants. Protamine-like molecules have been reported in algal, bryophyte, and fern sperm (Reynolds and Wolfe, 1984). These motile sperm have low-

REMODELING OF NUCLEOPROTEINS

25

molecular-weight, arginine-rich proteins, but contain lysine as well. In the case of the liverwort, Marchantiu polymorpha, where pure mature sperm could be obtained, no histones were found. 5 . Summary It is extremely rare to find sperm chromatin which is identical to somatic Chromatin in histone composition. With the exception of crustacean sperm, most sperm contain proteins more arginine rich than somatic histones. These may be sperm-specific histones, low-molecular-weighttypical protamines, cysteine containing protamine-like molecules, high-molecular-weight basic proteins, and many as yet ill-defined molecules. Most sperm chromatins are highly condensed. Nucleosome structure is usually retained except in cases where substitution is extreme, such as protamine and mammalian-protamine sperm. It seems likely that the more information accumulated on sperm nucleoproteins, the less applicable will be the initial five categories offered by Bloch (1969). For example, the somatic and intermediate categories could perhaps be subdivided into several classes. Already alternative classification schemes have been devised (Subirana, 1983; Kasinsky et al., 1985). Clear evolutionary trends are not obvious (see Kasinsky et ul., 1985, for a discussion of the vertebrates). B. SPERMATOGENESIS Since most mature sperm contain sperm-specific nuclear proteins, somatic histones must be replaced or augmented at some point in the developmental pathways leading to the spermatozoon. The most detailed work on nuclear protein transitions during spermatogenesis has been done on fish and on rodents, and, by extrapolation, it seems most likely that the sperm-specific nuclear proteins of most species are incorporated into the chromatin during late stages of spermatogenesis (spermiogenesis). In addition, testis-specific histone variants are sometimes present in earlier (meiotic) stages. For stages of rat spermatogenesis, see Fig. 4.Some different mammalian testicular cell types are shown in Fig. 5. 1. Teleosts Almost a century ago, Miescher showed that immature salmon sperm contain predominantly histones, whereas mature sperm contain largely protamines (Miescher, 1897). Alfert (1956) showed, cytochemically, that the transition from histone to protamine occurred in very late stages of salmon spermatogenesis. This work was most elegantly extended through biochemical analysis of the testicular cells of the trout, size (stage) separated by Dixon and colleagues (Ingles and Dixon, 1967; Marushige and Dixon, 1969, 1971; Marushige et al., 1969; Ling et al., 1969, 1971; Ling and Dixon, 1970; Candido and Dixon, 1971,

26

DOMINIC POCCIA

!

m-/s / J - / s

9-12

FIG. 4. Spermatogenic cells of the rat. Mitotic and meiotic cells are indicated by m and M, respectively. Proliferation of spermatogonia is followed by meiotic stages (spermatocytes). Spermiogenesis includes the postreplicative differentiation of spermatids to spermatozoa. From Meistrich er nl. (1981).

1972a,b,c; Sung and Dixon, 1070; Louie and Dixon, 1972a,b; Louie et al. 1973a,b) and is reviewed in detail by Dixon (1972) and Louie et a / . (1973a). Spermatogonia in the trout are large cells active in DNA and RNA synthesis. Spermatocytes engaged in meiosis are postreplicative but active in RNA synthesis. Spermatids are inactive in RNA synthesis (Marushige and Dixon, 1969; Gillam et al., 1979). The decrease in RNA synthesis is paralleled by a decrease in RNA polymerase I and I1 activities and, at least for RNA polymerase 11, this is due to a loss of the enzyme itself as assayed by a-amanitin binding (Gillam et a / . , 1979). Spermatogonia, spermatocytes, and early spermatids contain histones which are extensively acetylated and phosphorylated. In mid-stage spermatids, the histones are replaced by phosphorylated protamines which become dephosphorylated as the nuclei condense to their mature state. Histones are actively synthesized only in spermatogonia and spermatocytes. Protamines are synthesized at intermediate stages when histone synthesis is turned off and no nuclear protein synthesis occurs in the final stages of spermiogenesis. During the transitional period, histones appear to be removed starting with H4 and ending with H I . The sites of phosphorylation were determined for histones and protamines and all were serines. Most phosphorylation of histones occurs in early cells, during DNA synthesis. Little histone phosphorylation is seen in spermatids. Acetylation is active in spermatids, mostly on preformed histones. Hyperacetylation (tri- and tetraacetylation)of histones, especially H4, occurs in late spermatids, whereas in

REMODELING OF NUCLEOPROTEINS

27

FIG.5 . Separation of hamster testis cell types. (a) Unseparated suspension, (b) spermatozoa, (c) round spermatids, and (d) pachytene spermatocytes. Cells were separated from dissociated testis by elutriation. From Meistrich (1977).

preprotamine stages, H4 exists only in mono- and diacetylated forms (Christensen and Dixon, 1982). Trout testis apparently contains a single H1 variant, although other trout tissues show heterogeneity in this fraction (Seyedin and Cole, 1981). Histone methylation during trout spermatogenesis has also been demonstrated (Honda et al., 1975b,c). Histone-containing nuclei of maturing trout testis show a repeat length of approximately 200 bp (Honda et al., 1975a). By the end of spermatogenesis,

28

DOMINIC POCCIA

there is no evidence for nucleosomal structure by nuclease digestion (Honda et af., 1974), although globular structures have been reported as electron microscope observations (Gusse and Chevailler, 1980a). In the winter flounder, histones are not replaced by protamines, but additional basic proteins of high molecular weight accumulate during spermatogenesis (Kennedy and Davies, 1980, 1981, 1982, 1985). During early spermatogenesis, histones apparently identical to liver histones are present in the nuclei. H3 and H4 are highly acetylated and H4 and H2A are highly phosphorylated. In midspermatid nuclei, the high-molecular-weight basic proteins accumulate as phosphoproteins until they reach about 25% of the weight of the basic nuclear protein complement. The high-molecular-weight basic nuclear proteins, which are virtually completely phosphorylated (80% of the serines), become dephosphorylated, as do histones H2A and H4. At this time, the average repeat length of the chromatin increases from 195 to 222 bp. The sites of phosphorylation of the high-molecular-weight basic proteins lie in a consensus sequence X-Ser(P)-XSer(P)-Pro where X is lysine or arginine (Kennedy and Davies, 1985). The limited complexity of their peptide maps suggests that these proteins are closely related and contain many repeats of the simple sequences. In contrast to the situation in trout, flounder histone mRNAs (H3 and H4) are expressed transiently in mid-spermatids at the time of chromatin reorganization (Kennedy et ul., 1985).

2. Rodents Several aspects of rodent spermatogenesis have been reviewed previously (Bellvt, 1979; Meistrich et a / . , 1981). During rodent spermatogenesis, at least three classes of nucleoproteins appear in addition to somatic-type histones. These are testis-specific or testis-enriched histone variants (THs) of spermatocytes, transitional proteins (TPs) of the spermatids, and mammalian protamines of the mature sperm. a. Mouse. In the mouse, spermiogenesis can be divided into early spermatid (stages I-@, mid-spermatid (stages 9- 12), and late spermatid (stages 13-16) periods. The immature spermatids contain beaded, presumably nucleosomal, chromatin which is being transcribed as judged by electron microscopy and autoradiography (Monesi, 1964; Moore, 1971; Kierszenbaum and Tres, 1975). The transcripts probably include protamine mRNA which accumulates postmeiotically (Erickson et al., 1980). The chromatin undergoes a transition to a smooth form parallel to a decrease in transcription (stages 11-14) and the synthesis of an arginine-rich protein fraction (Lam and Bruce, 1971). The cessation of RNA synthesis probably precedes histone replacement. Nonround spermatids may contain both nucleosomal (beaded) and nonnucleosomal chromatin fibers (Fig. 6). A similar transition has been demonstrated for ram sperm by electron microscopy, nuclease digestion, and X-ray diffraction analysis (Loir et ul., 1985).

REMODELING OF NUCLEOPROTEINS

29

FIG. 6 . Chromatin fibers from cricket spermatids. (a) Beaded fibers from early spermatids, (b) mixture of smooth and beaded fibers from early spermatids, and (c) smooth fibers from later spermatids. From Kierszenbaum and Tres (1 978).

30

DOMINIC POCCIA

Histones are replaced by arginine-rich proteins in late spermatids as judged cytochemically (Monesi, 1965). This transition takes place in at least two steps, with a set of nonhistone transitional proteins appearing after the histones and before the protamines. The chromatin then condenses into the state characteristic of the mature head which contains proteins rich in arginine and cysteine (BellvC and Carraway, 1978). There are two species of mouse protamines (BellvC and Carraway, 1978). The two differ in amino acid composition, especially in cysteine, histidine, and tyrosine. The ratio of the protamine forms differs in late spermatids and epididymal sperm in the mouse (Balhorn et al., 1978). The same is true of humans (Tanphaichitr et al., 1978), who have three protamine forms. The synthesis of the two mouse forms is asynchronous during spermiogenesis. Their synthesis and deposition parallel the acquisition of sperm head sonication resistance at stages 12-15 (Balhorn el al., 1984). Preceding protamine accumulation in mouse spermatids is a set of TPs (Kistler et al ., 1973). The synthesis of TPs is concomitant with the synthesis of the protamines and it was suggested that TPs might have a role in protamine deposition or might even be protamine precursors (Balhorn et al., 1984). In support of the latter idea, isolated mRNA from mouse testis codes in virro only for TP, not protamine (Elsevier, 1982). Prior to spermiogenesis, testis-specific histone variants are found in the mouse. These are HI.S, H2A.S, H2B.S, and H3.S, which correspond to TH histones in the rat as discussed below. H2A.S and H3.S, along with a spermatocyte-enriched minor somatic variant H2A.4 (H2A.X), increase during early meiotic prophase, whereas H1.S and H2B.S appear in late prophase (Zweidler, 1984; but see Meistrich et al., 1985). All of these transitions occur after replication. H2B.S has deletions in its N-terminal portion and a minimum of seven conservative substitutions of hydrophobic amino acids. H3 .S differs in several positions in the C-terminal portion compared to somatic H3 (Zweidler, 1980). Testis-specific histones participate in nucleosome formation (Bhatnagar and Faulkner, 1983). b. Rar. In the rat, early round spermatids are stages 1-8, early elongating spermatids are stages 9- 12, mid-elongated spermatids are stages 13- 15, and elongated late spermatids are stages 16-19 (Fig. 7). During spermiogenesis, spermatids become increasingly resistant to chemical and physical disruption by sonication, trypsin-DNase, and SDS in stage 12, stage 15, and epididymal sperm, respectively (Meistrich el a [ . , 1976). As in the late spermiogenesis of other mammals, disulfide cross-links are formed in the epididymal sperm which cross-link the chromatin and make it exceptionally stable to thermal denaturation, trypsin digestion, or SDS extraction (Calvin and Bedford, 1971; Krzanowska, 1982; Meistrich et a l . , 1976). The full extent of disulfide stabilization is not achieved until the last stages of spermiogenesis (Loir and Lanneau, 1984).

REMODELING OF NUCLEOPROTEINS

31

J U J P

8:

>. 0

a

;$

oa n

0

I

0

10 20 30 40 50 60 DAYS

FIG. 7. Correlation of nucleoprotein and cell type in rat spermatogenesis. Time, cell type, stage of spermatogenesis, and appearance of testis-enriched or testis-specific histones, transitional proteins, and protamine are shown. High mobility group (HMG) proteins are also shown. Used with permission from Dr. Marvin Meistrich (unpublished).

Nucleoprotein changes in rat spermatogenesis have been reviewed by Meistrich et al. (1981). As in the mouse, one set of transitions involving histones takes place in early spermatogenesis and a second set involving TPs and protamines occurs in the spermatids (see Fig. 7). The TPs of the rat are well characterized. There are five basic, low-molecularweight proteins (600-20,000) which appear in elongated spermatids (stage 1319) devoid of histones (Kistler et al., 1975; Grimes et al., 1977; Platz et al., 1975; Meistrich et al., 1978, 1980). These are TP, TP2, TP3, TP4, and S1. All but TP contain appreciable quantities of cysteine. One has been sequenced (TP) and is a 54 amino acid long basic protein (6200 Da) containing 10 arginines, 10 lysines, and 8 serines, with a carboxyl end that is relatively nonbasic (Kistler et al., 1975). TP, TP2, and TP4 are found at stages 13-15; S1 and traces of TP3 are found in stages 16-19. Only S1 is found in the epididymal spermatozoa and it is the rat protamine of the mature sperm (Kistler et al., 1973). RNA synthesis is turned off before the last synthesis of arginine-rich nucleoproteins (Soderstrom and Parvinen, Soderstrom, 1985). Elongation of the head initiates before TP substitution, but condensation of the chromatin is closely correlated with and has been suggested as a role for the TP proteins (Meistrich et al., 1981). The chromatin becomes trypsin-DNase resistant when TP3 and S1 replace the other TP proteins (Meistrich et al., 1976). The mature rat sperm chromatin is highly condensed with a lamellar appearance (Koehler et a/. 1983). Transitional proteins are preceded by the testis-specific histones H1t (see Bucci et al., 1982; Seyedin and Kistler, 1983), TH2A (Trostle et al., 1979; Trostle-Weige et al., 1982), TH2B (Branson et al., 1975; Shires et al., 1976;

32

DOMINIC POCCIA

Tanphaichitr et al., 1978), and TH3 (Trostle-Weige et al., 1984), and by the testis-enriched histones Hla (Bucci et al., 1982; Seyedin and Kistler, 1983) and H2A.X (Trostle et al., 1979; Trostle-Weige et al., 1982). [In earlier work, Hlt and Hla were taken together as TH1, and TH2A was considered to be both X2 (now H2A.X) and X9 (now TH2A). H2A.X (West and Bonner, 1980) is also called M2 (Urban et al., 1979).] Spermatogonia synthesize TH3 in addition to Hla, H2A.X, and a variety of somatic histones (Meistrich et al., 1985). In spermatocytes, several other histone variants appear (Brock et al., 1980). TH2A and TH2B begin to be made in preleptotene spermatocytes and H 1t is synthesized initially at pachytene stage (Meistrich et al., 1985). Chromatin containing TH variants is nucleosomal (Trostle-Weige et al., 1982). TH variants are not present in elongated spermatids (Brock et al., 1980). Partial biochemical characterizations are available for rat H 1t (Seyedin and Kistler, 1980),TH2B (Shires et al., 1976), TH2A (Trostle-Weige et al., 1982), and human TH2B (Wattanaseree and Svasti, 1983; Yongvanich and Svasti, 1984). Hlt has been found in humans (Seyedin and Kistler, 1983), rabbits, boars, bulls, and mice, in addition to rats (Seyedin et al., 1981). In the boar, the complete sequence of Hit is known (Cole et al., 1984). It is a 22,058-Da protein with the usual H1 domains. It is more similar to standard somatic Hls than to Hlo in its conserved central domain. It is also more arginine rich and has a somewhat shorter N-terminal domain than a typical somatic HI. Histones H2B and TH2B, H2A, H3, and H4 are acetylated in all stages of rat spermiogenesis from spermatocyte to late spermatid (Grimes and Henderson, 1983, 1984; Grimes et al., 1975a.b). H4 hyperacetylation occurs more specifically in mid-spermatids undergoing replacement by TP proteins and is accompanied by a decreased thermal stability of the chromatin (Grimes and Smart, 1985). Roles in recombination, meiotic mechanics, and other meiotic transitions have been suggested for TH histones (Meistrich et al., 1981, 1985). Bucci et u1. (1982) have suggested a role for Hla in replication. Lennox and Cohen (1984) argue that H la and H Ic allow extended chromatin for recombination events and HI t permits chromatin condensation. 3. Other Organisms Much less is known about nucleoprotein transitions during spermatogenesis in other organisms. Cytochemical studies in the leopard frog, Rana, show no change in the basic protein during spermatogenesis (Zirkin, 1970), although mature sperm contain a sperm-specific HI (Alder and Gorovsky, 1975; Kasinsky et ul., 1985). Cytochemical studies show that arginine-rich proteins accumulate in the spermatids of snail (Bloch and Hew, 1960a,b), Urechis (Das et al., 1975), squid (Bloch, 1962), Drosophifa (Das et al., 1964a,b), and grasshopper (Bloch

REMODELING OF NUCLEOPROTEINS

33

and Brack, 1964). In the case of Urechis, RNA synthesis ceases in the spermatocyte stage (Das et al., 1967). In the sea cucumber, histones of the sperm are similar to somatic histones (Subirana et al., 1970), but a new H1 variant accumulates during spermatogenesis and mature sperm acquire an additional small basic protein (Comudella and Rocha, 1979). The repeat length remains constant at 227 bp during spermatogenesis. The nuclei do not completely condense (Pladellorensand Subirana, 1970, 1975). In the sea urchin, the incorporation of newly synthesized proteins into the nuclei decreases with later spermiogenesis (Nicotra et al., 1984). In the dogfish, S. caniculus, typical histones are present in round spermatids, two new basic proteins (S1 and S2) appear in elongating spermatids which replace the histones, then four high mobility proteins (Zl, 22, 23, and S4) replace these in the mature sperm, and are subsequently linked by disulfide bonds (Gusse and Chevaillier, 1981). In mature sperm, Z1 and 2 2 appear to be monophosphorylated while 23 appears to be unphosphorylated. In unripe sperm, Zl is monophosphorylated, 2 2 has one or two phosphates, and 23 has 1-3 phosphates (Martinage et al., 1985b). In the house cricket, Acheta dornestica, spermatocyte and early spermatid nuclei contain only somatic histones. By the late spermatid stage, they have been replaced by two testis-specific histones, which are then replaced by two other testis-specific histones (Kaye et al., 1978). Very late spermatids contain a complex mixture of protamine-size molecules, and the sperm from the female spermatheca contain four such molecules (McMaster-Kaye and Kaye, 1976). Chromatin fibers lose their beaded appearance in the late spermatid stages while they still contain histones and the protamine-containingfibers condense through sideto-side alignment and regular foldings into packaging units (McMaster-Kaye and Kaye, 1980; Kierszenbaum and Tres, 1978). Some striking patterns of insect sperm chromatin condensation are seen in Fig. 8. As in the trout and the rat, high levels of histone acetylation have been reported in rooster (Oliva and Mesquita, 1982), locust (Bouvier and Chevailler, 1976), and cuttlefish (Wouters-Tyrou et al., 1981) spermatids.

4. Summary From the few well-studied systems, it is perhaps premature to speculate on general mechanisms operating in the remodeling of chromatin proteins during spermatogenesis. Somatic histones may be replaced directly by sperm-specific histones or indirectly through intermediary sperm-specific histones or TPs. Most chromosomal protein remodeling takes place after meiotic DNA synthesis, thus offering an example of the uncoupling of replication and basic nucleoprotein synthesis. RNA synthesis is turned off before the final nucleoproteins are in place. Postranslationalmodifications of histones are probably extensive and may aid in the removal and replacement of DNA-bound proteins. The final deposition

34

DOMINIC POCCIA

Fic. 8. Condensing chromatin in insect sperrnatids. (a) Grasshopper (Melanoplus), (b) treehopper (Ceresa), (c) leafhopper (Tvlozygres). and (d) robber fly (Leptogasrer). From Fawcett et al. ( 197 1 ).

REMODELING OF NUCLEOPROTEINS

35

of proteins of the mature sperm occurs before, or simultaneously with, chromatin condensation.

C. EFFECTSAND POSSIBLEFUNCTIONS OF SPERM-SPECIFIC PROTEINS Several potential roles for the sperm-specific nucleoproteins were discussed almost two decades ago by Bloch (1969). Most were dismissed. A few deserve reevaluation. 1. Sperm Chromatin Structure There are many levels at which histone variants and other basic nuclear proteins might affect chromatin structure. Variants might affect (1) the stability of nucleosomes, (2) the spacing of nucleosomes (the repeat length), ( 3 ) the structure of the 30-nm-chromatin fiber, and (4) the packing of these fibers into the mature sperm head. There is circumstantial evidence for all of these effects. Nucleosomal structure may be lost or retained in sperm. In sperm chromatin containing histone variants, stability of the core DNA with respect to thermal denaturation or ionic unwinding and its susceptibility to DNases are altered (Simpson and Bergman, 1980, 1981). In protamine-containing sperm, nucleosomes may be lost altogether. Protection of the DNA with respect to DNases and heat is radically altered in protamine sperm (Honda et al., 1974; Willmitzer et al., 1977a,b). In the unusual crustacean sperm, the loss of histones results in the destabilization of the DNA to heat (Vaughn and Hinsch, 1972). Protamine-containing sperm lack nucleosomes and therefore repeat lengths (Honda et al., 1974). In some cases, repeat length changes occur during spermatogenesis as somatic histones are replaced or supplemented. In winter flounder, the nucleosomal repeat length increases from 195 to 222 bp during spermiogenesis, while the high-molecular-weight sperm basic nuclear proteins accumulate and the H1 and core histone composition remains constant (Kennedy and Davies (1982). Zalenskaya et al. (1981a), in comparing starfish and sea urchin sperm which differ in the size of their H2B complement and average repeat length (224 vs 237, respectively, for A . japonica and Strongylocentrotus intermedius) but are very similar in their other histones, suggest that Sp H2Bs have a role in determining repeat length. An unusual 500 bp cutting periodicity in the sea urchin L. pictus was reported by Arceci and Gross (1980~)which was ascribed to the unusual sperm H1. In the sea cucumber, no change in repeat length takes place in spermatogenesis, although additional sperm-specific nucleoproteins appear (Cornudella and Rocha, 1979). Sea cucumber sperm chromatin does not completely condense as does sea urchin sperm chromatin (Pladellorens and Subirana, 1970, 1975). The most striking change associated with nucleoprotein transitions in the maturing spermatid is the condensation of the chromatin. The final degree of

36

DOMINIC POCCIA

chromatin DNA condensation in sperm is variable: from little or none, nonnucleosomal (crustaceans), to partially condensed, nucleosomal (sea cucumber), to fully condensed, nucleosomal (sea urchin), to extremely condensed, nonnucleosomal (mouse). In addition, there are several different patterns by which condensation may proceed (Walker, 1971; Yasuzumi, 1974; Fig. 8). A role for disulfides in the cross-linking of mammalian sperm chromatin resulting in increased mechanical and chemical stability associated with the condensed state is likely (Calvin and Bedford, 1971). The inclusion of cysteine in mammalian protamines permits an increased stability not possible for true protarnine-containing sperm. Kennedy and Davies (1982) suggest a cross-linking function for high-molecular-weight nucleoproteins of flounder sperm. If H 1 is extracted with salt and sperm chromatin extensively degraded with micrococcal nuclease, very few nucleosomes are released until the nucleoproteins are broken down with trypsin. Under the same conditions, prespermatid nuclei release most of their chromatin (70%). Similarly, in sea urchin sperm, nuclease does not release nucleosomes into the supernatant until Sp H1 is removed (Simpson and Bergman, 1980). Under other conditions, supranucleosomal particles released from sea urchin sperm upon brief digestion with micrococcal nuclease were larger than those from chicken erythrocytes or liver, and Zentgraf and colleagues have suggested a possible link with sperm variants (Zentgraf et al., 1980; Zentgraf and Franke, 1984). The sea urchin Sp H1 and H2B molecules interact with DNA in vitro in ways that differ from their calf thymus counterparts. Mature sperm H1 is better than calf thymus HI or red cell H5 at inducing large-scale fiber-type DNA aggregates (Russo et al., 1983). Mature sperm H 1 preferentially interacts with superhelical SV40 DNA and is more effective at inducing its aggregation than calf thymus H 1 (Osipova er a [ . , 1985). Reconstituting SV40 DNA with core histones results in large intermolecular aggregates when Sp H2B, but not calf thymus H2B, is used (Osipova et al., 1982). Therefore, Osipova et al. suggest the two sperm-specific histones participate in higher order chromatin structure. Using amino acid sequence data, von Holt and colleagues have postulated a cross-linking role for Sp H2B in stabilizing sea urchin sperm chromatin (von Holt et al., 1984). Based upon similar in vivo behavior, a role has been suggested for sperm Hi and H2B variants in cross-linking chromatin fibers in the mature sperm to stabilize and condense the chromatin (Poccia et al., 1984; Green and Poccia, 1985; Poccia, 1987). It was proposed that the function of the long repeat length in sea urchin sperm is to provide extra DNA for the binding of these histones, through their long N-terminal regions (and the C-terminus of HI), to different strands of chromatin. This model is consistent with the observation that s M i s h sperm chromatin, lacking extended H2B molecules, has a shorter repeat length than sea urchin (Zalenskaya et al., 1981a) and that Platynereis sperm,

REMODELING OF NUCLEOPROTEINS

37

lacking extended H2Bs and possessing HI molecules lacking both N- and Cterminal portions, has a minimal repeat length of 165 bp (Kmiecik er al., 1985). In the latter case, condensation is presumably mediated by protamines (Sellos and Kmiecik, 1984). Other roles have been postulated for the N-terminal regions of Sp H2B by Simpson and Bergman (1981). They attribute differences in isolated core particle stability and DNase I cutting site specificity to the Sp H2B and Sp H2A. A role for sperm-specific proteins not only in condensation but also in nuclear shaping has been proposed by Fawcett et al., (1971). They have ruled out a general role for microtubules in nuclear shaping and instead propose that selfassembly of nucleoprotein subunits is responsible for the particular shape of the sperm head. However, a correspondence between nucleoprotein type and head shape is not obvious (Bloch, 1969). 2. Suppression of Genetic Activity A role in the suppression of RNA synthesis during spermatogenesis has been suggested for the sperm-specificnuclear proteins (see Bloch, 1969). However, in the best documented cases, transcription apparently ceases well before the final deposition of these proteins, thus ruling out a primary role in transcriptional inactivation. Whether the sperm-specific nuclear proteins are compatible with transcriptional activity in vivo must be answered by following pronuclear reactivation. A related suggestion is that sperm nucleoproteins “erase” the developmental history of the chromosomes (see Bloch, 1969). From a current perspective, this might be understood in terms of altering the special features of chromatin structure associated with active, potentially active, or formerly active genes, such as those features revealed by DNase I sensitivity or hypersensitivity (Groudine and Weintraub, 1982). A study of chicken chromatin suggests that such hypersensitive sites are lost in sperm for all genes, but that those genes which are constitutively expressed are marked during spermatogenesisby hypomethylation at sites of future hypersensitivity (Groudine and Conkin, 1985). It is of considerable interest to extend such studies to other organisms, especially to those which have nucleosomal sperm or may not use hypomethylation mechanisms for gene control (Bird, 1984). 3. Displacement of Somatic Nucleoproteins Some sperm-specific proteins, such as TPs, may facilitate the replacement of somatic histones by the nucleoproteins characteristic of the mature sperm. A similar role could be played by postranslational modifications. Sung and Dixon (1970) and Grimes and Smart (1985) proposed that histone modifications facilitate the substitution of protamines or TPs for histones in the trout and rat, respectively.

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Dixon and colleagues (Dixon, 1972; Louie et al., 1973a) postulated two roles for the enzymatic modification of histones and protamines during spermatogenesis: (1) in the correct binding of histones to DNA following their synthesis and (2) in the replacement of histones by protamines. In the former case, charge neutralization would lower the affinity of histones for DNA until correct binding was achieved; in the latter case, reduced affinity of histones for DNA would allow its displacement from the chromatin, whereas dephosphorylation of protamine would result in the tightly bound, stable nucleoprotamine complex characteristic of the mature sperm head. Such events might be reversed in male pronuclei following fertilization (see Section V,C,3). Protamine binds electrostatically to DNA much more strongly than histone (Bode et al., 1977). Phosphorylation of protamine results in an in vitro decrease in binding affinity and a greater packing density of protamine on DNA. Willmitzer and colleagues (Bode et al., 1977; Willmitzer et al., 1977a,b) suggest that phosphorylation of protamine serines allows binding of the phosphate to arginines, thus condensing the protamine and allowing more molecules to bind to DNA. Dephosphorylation would then allow the protamine to expand and displace the histone. They suggest that phosphoserine-arginine bonds could form a cross-linked network of nucleoprotamine.

4. Other Roles A protective role against a hostile environment has been proposed for sperm nucleoproteins. No straightforward correlation of nucleoprotein type with factors such as internaUexterna1 fertilization, seasonaUcontinuous reproduction, or sperm longevity could be made (Bloch, 1969). One view is that, freed from constraints imposed by normal cellular mechanics, spermatid nucleoproteins are merely products of evolutionary drift. It has been proposed that, in.organisms in which sex is genetically determined, sperm nucleoprotein genes may be localized in the male chromosomes and active only during spermatogenesis (Bloch, 1969). Thus, these genes escape from the constraints imposed on somatic histone genes. They are free to evolve into the variety observed since they are not important in an inert nucleus. The only requirement being basicity, a drift toward arginine occurs because of the larger number of codons for arginine than for lysine or histidine. (See Kasinsky et al., 1978, for a further discussion of this theory.) However, selection could operate on several aspects of spermatogenesis, mature sperm function, or postfertilization phenomena. Many events occurring during spermatogenesis and pronuclear development are not well understood. They could provide a variety of constraints, within which sperm nucleoproteins would have evolved. For example, the need to rapidly replace sperm nucleoproteins prior to embryonic development will likely depend on the particular timing of postfertilization events. The timing will vary for eggs which are fertilized at

39

REMODELING OF NUCLEOPROTEINS

different meiotic stages or cleave at different rates. It might be necessary to balance a stable or hydrodynamically efficient nucleus in the mature sperm with a structure that can be rapidly remodeled in a particular egg. The resulting nucleoprotein complement might not maximize either demand. What is clear is that the variety of nucleoproteins tolerated by sperm cells is much greater than that tolerated by any other cell type, and this is probably permitted by the absence of constraints imposed by normal chromatin functions, such as replication, transcription or mitosis in maturing sperm and early pronuclei.

V. Nuclear Protein Transitions following Fertilization A. MATERNAL PRONUCLEAR HISTONES Limitations on the amounts and purity of nuclei available have made the study of female pronuclear histones difficult. Most of the data available are for the sea urchin. Hnilica and Johnson (1970) could not detect typical lysine-rich histones in the unfertilized egg nucleus of S. purpurutus. Thaler et ul. (1970) reported histones in A . punctulutu egg nuclei. Evans and Ozaki (1973) reported the presence of a typical H4 and no sperm-type or blastula aHls. An H3 of equivalent mobility to that in the sperm or blastula was absent. A band with slower mobility than Sp H1 was postulated to be a maternal pronuclear-specific H1. Limited resolution of the gel system did not permit definition of the other histone classes. Carroll and Ozaki (1979) found H2A and H2B histones corresponding to zygote variants and no H1, Sp, or a variants. Green and Poccia (1985) observed CS H2A, CS H2B, and H3 and H4 proteins similar to those acquired by the male pronuclei, but CS H1 was absent. Convincing controls to eliminate contamination by the maternal histone storage pool were not included in any of these studies. Rodman et ul. (1981), on the basis of immunocytochemical criteria, claim that mouse oocyte chromosomes have associated sperm nucleoprotamines, which are lost from the pronuclei soon after fertilization. More data are needed to establish if oocyte nuclei generally undergo transitions involving nonsomatic type nucleoproteins during oogenesis.

B . SOURCES OF CHROMOSOMAL PROTEINS IN

THE

EGG

There are several sources of histones after fertilization. Histones may be translated from new embryonic transcripts or from maternal stored mRNAs following fertilization. In addition, they may be synthesized during oogenesis and stored in the egg. Histone synthesis following fertilization from maternal and embryonic transcripts has been extensively reviewed elsewhere (Weinberg,

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1977; Woodland, 1980, 1982) and discussed above (Section III,B,2). The following is an account of histone storage in oocytes. Pools of maternal histones, in excess of the expected complement of maternal chromosomes, are known to exist in the oocytes of frog, mouse, Urechis, Drosophila, snail, and sea urchin eggs and so may be a general phenomenon. The size of the pools vanes greatly and may reflect the particular physiology of the system. In Xenopus oocytes, about 20,000 diploid equivalents of histone are stored (Adamson and Woodland, 1974, 1977). These may be stored in both the germinal vesicle and the cytoplasm (Woodland, 1979, 1980, 1982). Histones of the maternal storage pool are used to make up for an insufficient production of histones from maternal mRNAs and embryonic transcripts during the rapid cleavages of early development in the large frog egg. Xenopus histones can assemble purified DNA into chromatin with a 200 bp spacing when used in a lysate containing an acidic cofactor, nucleoplasmin (Laskey et a f . , 1977; 1978). A similar extract from Drosophila eggs can assemble exogenous DNA into nucleosomes (Nelson et al., 1979). However, the assembly of DNA and core histones into correctly spaced nucleosomes has been achieved in vitro using just synthetic acidic polypeptides as cofactors (Stein and Bina, 1984). Oocyte nuclei contain non-chromatin-bound histones H3, H4, and H2B (Kleinschmidt and Franke, 1982). Little H2A was detected electrophoretically and two isoelectric variants of H3 were found. H3 and H4 are associated with 1 LO kDa acidic proteins, but not nucleoplasmin (Kleinschmidt and Franke, 1982). Recently, two storage forms were separated in Xenopus (Kleinschmidt et al., 1985), the complexes reported originally with H3 or H4 bound to one of two acidic polypeptides and larger complexes containing any core histone bound to nucleoplasmin. Earnshaw er a]. (1982) detected a Xenopus storage form in unfertilized eggs which reacts with antibodies that recognize octameric but not single histones and therefore suggest that the histones are bound to an anionic factor. The antibody interferes with chromatin assembly. These complexes contained H3, H4, H2B, and perhaps H2A in a “hidden” form. Earnshaw et al. (1982) offer data to suggest that the anionic component is not nucleoplasmin. Using histochemical procedures. Cowden ( 1966) detected arginine-rich basic proteins in sea urchin oocytes. Benttinnen and Comb (1971) reported an electrophoretic gel band corresponding to H4 in unfertilized eggs and postulated a histone pool. By comparison of lysine-labeled proteins extracted from oocytes with morula and gastrula stage histones, Cognetti et al. (1974) concluded that histones were synthesized during sea urchin oogenesis (vitellogenesis). In subsequent work, Cognetti et al. (1977) showed that one band had identical mobility and amino acid composition with an H2B fraction from gastrula. A store of CS histones was implied by the results of Newrock et a / . (1978b), based upon the specific activities of labeled CS and a histones in the early embryo.

REMODELING OF NUCLEOPROTEINS

41

A pool of assembly competent histones was demonstrated using polyspermically fertilized sea urchin eggs blocked in protein synthesis (Poccia et al., 1981). At least 25 haploid equivalents of DNA could be assembled into chromatin (Salik et al., 1981). Quantification of spots, whose identities were established by two-dimensional electrophoretic gel comigration with standards and antibody binding, indicates an upper limit of several hundred haploid equivalents for CS H1, CS H2A, CS H2B, an H3 variant, and H4. No Sp, a,P,y, or 6 histone variants were seen in extracts of whole eggs (Poccia et al., 1981; Salik et al., 1981). The presence of the proteins in enucleated egg halves suggested a cytoplasmic store of at least a fraction of the pool. Recruitment of H1 and core histones from the pool into chromatin in vivo is not coordinate (Poccia et al., 1981; Poccia et al., 1984; Green and Poccia, 1985). A store of nonhistone chromosomal proteins in the urchin egg is suggested by the work of Kuhn and Wilt (1980). As in the frog (Woodland 1980, 1982), the pool of stored histones in the unfertilized sea urchin egg is synthesized in the oocyte. Since sea urchin oocytes contain mRNA for both CS and a histones (Arceci et al., 1976; Hieter et al., 1979; Childs et al., 1979) but only CS forms accumulate in the storage pool, a histones are either not synthesized in the oocyte or rapidly degraded. Unfertilized eggs were shown to synthesize CS but not ci histones, while both forms were synthesized following fertilization (Herlands et al., 1982). This implies that synthesis of a histone variants in the sea urchin is under translational control. A possible explanation for the lack of mRNA translation in the unfertilized eggs is the sequestering of these RNA sequences in the maternal pronucleus. Several studies show that mRNA for histones is, essentially, completely localized in the maternal pronucleus and released to the cytoplasm at about the time of pronuclear envelope breakdown (Venezsky et al., 1981; Showman et d., 1982; de Leon et al., 1983; Raff, 1983; Cox et al., 1984; Angerer et al., 1984a,b). As mentioned, Drosophila eggs contain a store of histones capable of assembling DNA into chromatin (Nelson et al., 1979). Wasserman and Mrozak (1981) showed that mouse oocytes synthesize 2-3 diploid equivalents of H4 during oogenesis and that this excess histone is stored in the germinal vesicle. A pool of histones in Ilyanassa eggs was inferred from labeling patterns of early embryo histones (Mackay and Newrock, 1982). Urechis eggs have a pool of at least 3264 diploid equivalents in the oocyte germinal vesicle (Franks and Davis, 1983), whereas they apparently do not store maternal histone mRNAs (Franks and Davis, 1985). As in spermiogenesis, histone synthesis in oocytes and early embryos is uncoupled from DNA synthesis (see Woodland, 1980; Shih et al., 1980; Nishioka and Mazia, 1977; Arceci et al., 1976; Cognetti et al., 1974, 1977). Such uncoupling occurs, though to a lesser extent, in adult somatic cells (Coffin0 et al., 1984; Wu et al., 1984).

42

DOMINIC F'OCCIA

In summary, histone storage in oocytes may be universal, but the amounts stored vary considerably between large, externally developing eggs and small, internally developing eggs. For eggs with large histone pools, extensive assembly of pronuclear and early histone chromatin can take place without embryonic histone synthesis.

C. FATEOF SPERM-SPECIFIC NUCLEARPROTEINS As a result of nucleoprotein transitions during spermatogenesis, most sperm nuclei enter egg cytoplasm with proteins which may, to varying degrees, be incompatible with subsequent chromosomal activities such as replication, transcription, recombination, and mitotic condensation. Elements which are incompatible or not optimal are likely to be replaced soon after fertilization. 1, Cytoplasmic Control of Pronuclear Development

Several aspects of this subject have been reviewed previously (Longo, 1981; Longo and Kunkle, 1978). The eggs of different organisms may be fertilized at various stages of meiosis. The ability of the sperm nucleus to transform to a male pronucleus (Fig. 9) and for its chromatin to decondense is usually dependent on the state of the cytoplasm it invades. Often, the ability of egg cytoplasm to support pronuclear development is acquired after germinal vesicle breakdown. In the brittle star, Amphiphofis kochii (ophiuroid), fertilization occurs at the first meiotic metaphase. Male pronuclear decondensation proceeds toward the acrosome. Decondensation occurs while the female chromosomes continue through meiotic condensations and then decondense (Yamashita, 1983). Male decondensation occurs in two steps, both before and after pronuclear envelope formation. The second step can be inhibited (Yamashita, 1985). In the starfish Asteria pectinifera, immature oocytes can be polyspermically fertilized, but the male chromatin fails to decondense. If, in the same eggs, maturation is induced with I-methyladenine and the germinal vesicle breaks down, normal pronuclear development ensues (Hirai et al., 1981). In the sea urchin, A . punctularu, normally fertilized after both meiotic divisions are complete (Longo and Anderson, 1968), immature previtellogenic and vitellogenic eggs do not support male chromatin decondensation upon fertilization (Longo, 1978). If the eggs are fertilized during meiotic division, a limited decondensation takes place. Sea urchin eggs retain the ability to decondense sperm heads at various stages of the first cell cycle (Krystal and Poccia, 1979; Longo, 1980, 1983b). Microinjection of whole sperm into sea urchin eggs results in a lack of pronuclear development, suggesting that plasma membrane removal may be an important step in pronuclear development (Hiramoto, 1961; see Longo, 1981). Oocytes of the teleost, Oryzias latipes, from which the germinal vesicles have

REMODELING OF NUCLEOPROTEINS

43

FIG. 9. Stages of pronuclear development in the sea urchin, Arbacia. (a) Condensed sperm nucleus (SM) just entering the cytoplasm (R, ribosomes; F, filament; CF, centriolar fossa; SN, nucleus; C, centriole; AC, activation calyx; and FC, fertilization cone); (b) partially condensed male pronuclear chromatin (FDC, finely dispersed chromatin; CC, condensed chromatin; and CDC, coarsely dispersed chromatin); and (c) zygote nucleus after fusion of male pronucleus containing with female pronucleus. From Longo and Anderson (1968). paternal chromatin (FC)

been removed, can be matured in v i m and fertilized. The male chromatin is able to decondense, although it is not able to transform into a normal nucleus since chromosome condensation fails and, eventually, the chromatin turns into a pycnotic mass which disappears (Iwamatsu and Ohta, 1980). Germinal vesicle breakdown is needed to promote frog (Rana) sperm decondensation after injection into toad (Bujii) oocytes (Skoblina, 1976). In mouse oocytes, pronuclear formation is supported by nucleated and enucleated egg halves if germinal vesicle breakdown has occurred, but only in nucleated halves if it has not (Balakier and Tarkowski, 1980). Artificially activated eggs develop conditions which promote sperm nucleus decondensation for about 1.5 hours after activation, but then the conditions are lost (Komar, 1982). In the golden hamster, the ability of egg cytoplasm to decondense sperm chromatin

44

DOMINIC POCCIA

begins at about the time of germinal vesicle breakdown and continues throughout maturation, decreasing after fertilization (Usui and Yanagimachi, 1976). The ability of mammalian somatic cells to decondense sperm nuclei is severely limited (Bendich et al., 1976; van Meel and Pearson, 1979b; Elsevier and Ruddle, 1976; Phillips et al., 1976). Attempts at reconstituting in v i m conditions for sperm nuclear decondensation using Xenopus extracts are promising (Lohka and Masui, 1983a,b; 1984a,b; Gordon et al., 1985). Chromosomal protein transitions in these nuclei, if any, have not been characterized and comparison to in vivo nucleoprotein transitions is not yet possible. In summary, pronuclear development is not merely a consequence of the invasion of foreign cytoplasm by sperm. The ability of oocytes to support pronuclear development is stage dependent. Usually, but not always, the state of condensation of the male and female chromosomes in common egg cytoplasm is coordinated. In many cases, the germinal vesicle (GV) must break down to allow male pronuclear development, suggesting that the GV may contain required factors. Whether special histone variants or modifying enzymes are sequestered in GVs is not known.

2 . Cytochemistry of Male Pronuclear Proteins For technical reasons, information about transitions in sperm-specific nuclear proteins following fertilization has come almost exclusively from cytochemical procedures. Most studies suggest that a period occurs between loss of spermspecific nuclear proteins and the acquisition of “typical” adult histones during which the male chromatin has proteins different from either. Until methods are developed for investigating male pronuclear proteins in a variety of organisms biochemically, these studies, with all their limitations, constitute the best case for transitional proteins analogous to CS histones in organisms other than the sea urchin. In the snail, Helix aspersa, fertilized during meiotic metaphase I, comparison of alkaline fast green (AFG), eosin, bromophenol blue (BPB), and ammoniacal silver staining reactions indicates that the sperm nucleus loses its protamines soon after fertilization (Bloch and Hew, 1960b). Protamines seem to be replaced by weakly basic histones which can be found in both male and female pronuclei, early polar body chromosomes, and cleavage and morula nuclei. By the gastrula stage, these are, in turn, succeeded by adult-type histones. The sperm nuclei of the marine echiuroid worm U . caupo contain protaminelike molecules which stain with BPB. Within 15 minutes of postfertilization of the eggs in meiotic prophase I, the BPB reaction disappears and the male nuclei stain with AFG (Das etal., 1975; Das and Barker, 1976). During this period, the male nuclei decondense. They then recondense as the female nucleus progresses through meiosis.

REMODELING OF NUCLEOPROTEINS

45

In the fruit fly, Drosophifa mefanogaster, fertilized during meiotic metaphase I, the sperm nuclei contain arginine-rich molecules which stain with BPB and AFG. Pronuclei and the syncitial CS nuclei seem to contain atypical “histones” that stain with BPB but not AFG (Das et al., 1964a,b). Typical histone-staining patterns occur just before blastula formation. Using the criteria that protamines will stain with BPB both before and after acetylation and that histones will stain with AFG before but not after acetylation, Vaughn (1968) showed that crab sperm contain neither histone nor protamine. Following fertilizatjon in the sand crab, Emerita anuloga, the male pronuclei stain with neither AFG nor BPB after acetylation but bind BPB before acetylation, implying the acquisition of weakly basic protein. Egg chromosomes stain for histones throughout maturation. At the time of pronuclear fusion, both male and female pronuclei stain for histones in the same way as cleavage nuclei. In the sea urchin, A . punctufata, sperm nuclei show an increase in ammoniacal silver staining immediately following fertilization, even before decondensation, then a decrease in staining during chromatin dispersion (Longo, 1983a). The black color associated with sperm and early male pronuclei is taken as an indication of arginine-rich protein. The female pronucleus stains yellow, suggesting a more lysine-rich protein composition. The increase in male pronuclear staining upon fertilization was interpreted as either an increase in arginine-rich protein content or an unmasking of sites in the sperm proteins. It may be related to the phosphorylation events of H 1 exchange analyzed biochemically (Green and Poccia, 1985; see Section V,C,3). In the mouse, whose eggs are fertilized at meiotic metaphase 11, sperm nuclei contain protamine-like molecules cross-linked by disulfide bridges (Calvin and Bedford, 1971). Cytochemical work indicated that the arginine-rich protein stained with AFG is lost or modified after fertilization (Alfert, 1956). The sperm nucleoproteins labeled with [3H]arginine during spermatogenesisdisappear from male pronuclei soon after fertilization, while the egg is completing meiotic division and before pronuclear fusion (Ecklund and Levine, 1975; KopeEnY and Pavlok, 1975). Through the use of immunochemical-staining techniques, sperm basic nuclear proteins were also demonstrated in oocyte chromosomes during maturation, but were lost from both female and male pronuclei soon after fertilization (Rodman et al., 1981). The decondensing male nuclei entered a period in which they were recognized by neither antibodies to protamines nor to histones H2B or H4, suggesting a transitional period of unusual chromatin composition. Miller and Masui (1982) have reported changes in stainability with Giemsa and in the binding of N-[3H]ethylmaleimide occurring before decondensation which suggests accessibility of the dye to DNA and that reduction of nucleoprotamine disulfide bonds may precede decondensation and the protamine-histone transition in mouse. Taken together, these results suggest that soon after fertilization, some or all

46

DOMINIC POCCIA

of the sperm-specific nucleoproteins are removed from the male chromatin. Special nucleoproteins, showing staining properties that distinguish them from somatic histones, may appear before the transition to typical nucleohistone. These may be histone variants or histones which have been postranslationally modified. The exchange of basic proteins may be a general occurrence in the reactivation of dormant nuclei, as in the case of the replacement of protamine by H1 histone following fusion of human sperm cells with mouse fibroblasts (van Meel and Pearson, 1979a), of H5 with HI following fusion of chicken erythrocytes with HeLa cells (Appels et a l . , 1974; Ringertz et al., 1985), or the replacement of sperm HI with CS H 1 following fertilization in the sea urchin egg (Poccia et al., 1978, 1981).

3 . Biochemistry of Male Pronucleur Proteins a. Variant Protein Transitions. Attempts to study nuclear proteins in the first cell cycle directly by biochemical means have been limited almost exclusively to the sea urchin. No comparable data exist for other organisms. Johnson and Hnilica (1970) could not find histones in the sea urchin nuclei prior to blastula stage. Carroll and Ozaki (1979) compared histones in zygote nuclei 60 minutes after fertilization (following one round of replication) with nuclei of unfertilized eggs and gastrula in S . purpuratus. The gel patterns lacked detectable Sp H1, Sp H2B, and Sp H2A, and did not have the corresponding forms characteristic of gastrula (p, ?,or 6 variants). The authors concluded that the egg and zygote nuclei have unique H2A and H2B histones and might lack H I . Imschenetzky et al. (1980) compared unfertilized egg nuclei with zygote nuclei entering the first round of replication in Tetrapygus niger and noted a lack of Sp HI but also noted some additional bands in the HI region. In detehnining the fate of sperm-specific histones, studies of zygote nuclei are hampered by the dilution of these components by an equal mass of maternal chromosomal histones before replication and the doubling of histones during replication, so that at the end of the first cycle, the sperm histones, even if fully retained, can contribute only 25% of the embryonic nuclear histone complement. In addition, only small quantities of early embryo nuclei can be isolated and contamination by the histone storage pool tends to give an underestimate of the male pronuclear histone contribution. It is still not clear whether, in normal embryos, sperm-specific core histones are retained but diluted in the chromosomal histone complement during cell division or whether they turn over (Poccia et al., 1984). These problems are largely circumvented by the use of moderately polyspermic eggs, which increases the yield of male pronuclei and minimizes the contribution of the maternal chromosomal and storage histones to the nuclear isolate. The feasibility of such a system was demonstrated by Poccia et a / . (1978) who

REMODELING OF NUCLEOPROTEINS

47

showed that unmodified Sp H1 was lost from the male pronuclear chromatin within 10 minutes following fertilization. This work was extended to allow a detailed description of male pronuclear histone transitions taking place in the first cell cycle of the sea urchin (Poccia et al., 1981; Poccia, 1982). The transitions are complex and apparently do not require normal progression of the cell cycle. The transitions take place in two phases separated by a relatively quiescent period as summarized in Fig. 10. Within 10 minutes after fertilization, Sp H1 is phosphorylated, then greatly diminished in the chromatin. The CS variant, CS H1, is gained from the maternal pool. In addition, the Sp H2B complement is phosphorylated during this period (Green and Poccia, 1985). Otherwise, the histones appear to be similar to sperm histones. A relatively quiescent period ensues for about 20 minutes. Beginning at replication (30 minutes), new histone variants make their appearance in the male chromatin. These are CS H2A, CS H2B, and a species of H3 which might be a CS variant (Fig. 11). These forms accumulate and become the predominant forms in their classes by the end of the first cycle. None of the histone transitions seems to be sensitive to the inhibition of protein synthesis, thus providing evidence for a functional CS histone storage pool in sea urchins (Poccia et al., 1981). Because of the inherent synchrony of the polyspermic sea urchin system and the homogeneity of the male pronuclear population, it is possible to study these transitions within a single-cell type with a degree of detail that is not available in the later embryo with its divergence of cell types and cell cycle times. The apparent linking of replication and accumulation of CS core histone variants, for example, is eliminated in polyspermic eggs treated with the DNA polymerase inhibitor aphidicolin. With replication blocked by 295%, the accumulation of CS forms still proceeds (Poccia et al., 1984), although at a somewhat reduced rate. In addition, since the CS forms predominate under conditions in which no newly synthesized DNA is available for complexing, it appears that Sp core histone variants can be displaced from the chromatin in a process which is in essence a reversal of the transition presumably taking place without replication during spermiogenesis. Male pronuclei are able to support transcription within the first cell cycle (Poccia et a f . , 1985). Among the major transcripts are those coding for a histones. Pronuclear chromatin containing substantial amounts of Sp H2B can therefore be transcribed, suggesting that, at least in its phosphorylated form, Sp H2B is not incompatible with RNA synthesis. b. Histone Variant Modifications in the First Cycle. During the first cell cycle, histone phosphorylation events are numerous. Ord and Stocken (1978) showed that an H1-containing fraction isolated from zygote nuclei of Echinus was labeled from 10 to 30 minutes by radioactive phosphate. Krystal and Poccia (1981) showed that CS H1 is phosphorylated throughout the first cell cycle in S. purpuratus. Pronuclear CS H1 is phosphorylated to a greater extent than in

TIME POSTFERTILIZATION ( MIN 1

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49

FIG. 11. Two-dimensional gel electrophoresis of sperm and male pronuclear histones. (a) Sea urchin male pronuclear histones at 45 minutes postfertilizationand (b) sperm histones. First dimension is acid-urea-Triton; second dimension, SDS. Protein N is phosphorylated Sp HI; 0 and P are phosphorylated Sp H2Bs. Green and Poccia (unpublished).

blastula chromatin and exhibits increasing phosphorylation as the cell cycle progresses, reaching a maximum at metaphase. In this way, the rather unusual CS H1 behaves as a typical H1 (Poccia, 1987). By introducing sperm nuclei into the egg cytoplasm for equivalent intervals but at different phases of the artificially activated maternal cell cycle, it was shown that the degree of phosphorylation was dependent on the stage of the cycle rather than the time of egg cytoplasm. Prematurely condensed male chromosomes (from sperm fertilizing the egg at prometaphase) show levels of phosphorylation equivalent to metaphase chromosomes. If chromosomes are prevented from condensing, phosphorylation proceeds to the same extent, thus uncoupling the two events. The sites of phosphorylation were not determined. Green and Poccia (1985) studied phosphorylation events taking place during pronuclear decondensation. Within 5 minutes after fertilization, Sp H 1 and Sp H2B are phosphorylated and converted to proteins previously identified (Poccia et al., 1981) as N (Sp H1) and 0 and P (Sp H2Bs). No other male pronuclear histones are phosphorylated during this period, although later in the cycle most of the other histones become labeled (Green, Herlands, and Poccia, unpublished). Peptide mapping established the relationships of N, 0, and P to the Sp variants and localized the sites of phosphorylation to the N-terminal portion of Sp H2B but to both sides of Sp H1. Cleavage-stage H1 enters the chromatin as a phosphoprotein from the maternal pool as phosphorylated Sp HI leaves. Only serine is phosphorylated in the Sp variants, although phosphothreonine is also detected in CS H1. These phosphorylation events have recently been reviewed (Poccia, 1987).

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D. EFFECTSAND POSSIBLE FUNCTIONS OF HISTONE VARIANTSAND THEIR MODIFICATIONS FOLLOWING FERTILIZATION 1 . Histone Vuriunts and the Repeat Length Transition

Many sperm have longer nucleosomal repeat lengths than do somatic cells (Sections IV,A and 6). Sea urchin sperm has the longest average nucleosomal repeat length known. Its value declines by 50-60 bp within the first or second cell cycle following fertilization (Savid et al., 1981; Chambers et al., 1984). During the period in which Sp H1 is replaced by CS H1 and Sp H2B is phosphorylated, little change occurs, eliminating these as sufficient conditions for the repeat length transition. Most of the decline occurs during replication but does not require DNA synthesis (Fig. 12; Savii et al., 1981; Poccia et al., 1984). A reasonable correlation exists between the amount of CS core histones incorporated into the chromatin and the repeat length (Poccia et a / . , 1984). Increases in repeat length occur later in development as other histone variants are incorporated (Keichline and Wasserman, 1979; Arceci and Gross, 1980a,b), but, by then, heterogeneous populations of cell types exist in the embryo, making correlations difficult. 2 . Histone Variants, Protamines, and Decondensation Mechanisms The decondensation of mammalian sperm has received much attention deriving from the observation that mammalian sperm heads are extremely difficult to swell in vitro and that their protamines are not extractable unless disulfide bonds are first reduced (Calvin and Bedford, 1971; Krzanowska, 1982). Disulfide reduction (Calvin and Bedford, 1971; Lung, 1972), proteolysis (Gall and Ohsumi, 1976; Marushige and Marushige, 1975), and phosphorylation (Willmitzer, et al. 1977a,b; Wiesel and Schultz, 1981) have all been suggested as playing a role in the removal of mammalian protamine following fertilization (see Zirkin et al., 1985). The effects of reduction have been studied both in viva and in vitro. Perreault et a / . (1984) showed that the injection of the sulfhydryl-blocking agents, iodoacetamide or giutathione oxidant diamide, into mature hamster oocytes inhibited decondensation of injected sperm nuclei in a dose-dependent fashion while having no effect if the sperm nuclei were first reduced with dithiothreitol (DTT).If D?T-treated sperm nuclei or disulfide-poor early (testicular) spermatid nuclei are injected into germinal vesicle or pronuclear stage eggs, they decondense, but untreated mature sperm nuclei do not. The authors suggest that decondensation requires disulfide reduction of the mature sperm nucleus and that immature oocytes and pronuclear stage eggs lack sufficient reducing power to support decondensation. (Zirkin et a / ., 1985). On the other hand, Rodman et u / . (1982) have argued, on the basis of in vitro studies, that reduction is not sufficient for decondensation but that protamine removal must occur.

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FIG. 12. Change in nucleosomal repeat length between sperm and male pronuclear chromatin in the absence of DNA synthesis as seen in micrococcal nuclease digests of sea urchin sperm and pronuclear chromatin. The pronuclear sample was taken from polyspermic embryos blocked in DNA synthesis with aphidicolin. (A) Sperm DNA, (B) molecular weight markers, (C) sperm DNA, and (D) pronuclear DNA. Multimer numbers are given. Poccia and Greenough (unpublished).

Rabbit sperm nuclei incubated with DTT and Triton X-100 exhibit autodigestion of protamine which precedes evident decondensation in v i m (Zirkin and Chang, 1977; Chang and Zirkin, 1978). However, this activity is eliminated by removal of the acrosome (Young, 1979). Bull sperm incubated with 2-mercaptoethanoi show degradation by acrosin, an acrosomal enzyme, in v i m (Marushige and Marushige, 1978). The relatively nonbasic ends of the protamines are degraded with no effect on decondensation. As the arginine-rich central portions

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of the molecules degrade, the chromatin decondenses. Similar observations were made for rabbit, guinea pig, and hamster but not rat sperm, and it was suggested that proteolysis may have an in vivo role (Marushige and Marushige, 1975; 1978). It is not obvious that such degradation is physiologically significant and Young (1979) has presented several arguments against the notion. Phosphorylation of protamines, as a reversal of changes occumng during fish spermiogenesis, has also been offered as a mechanism for modulating condensation/decondensation (Willmitzer, 1977a). A rabbit egg kinase active on protamine with a pH optimum of 8.0-8.5 has been described by Wiesel and Schultz f 198l ) , who also assayed glutathione reductase activities in these eggs. Dean (1983) reduced and alkylated isolated mouse sperm nuclei, which makes them somewhat accessible to micrococcal nuclease. When polyglutamic acid is added, as much as 85-90% of the DNA can be digested. Adding core histones results in regaining some of the nuclease resistance and the formation of 150-bp ladders, suggesting closely packed nucleosome assembly. Using similar conditions, nucleosomes may be assembled in vitro with correct spacing (Stein and Bina, 1984). Dean (1983) suggests that acidic molecules might mediate assembly of chromatin (and disassembly of nucleoprotamine). Such acidic molecules and histone/acidic protein complexes are known to exist in amphibian eggs (Earnshaw ef al., 1982; Kleinschmidt et af., 1985). In the sea urchin, phosphorylation and pH have been proposed as being involved in male pronuclear decondensation. The decondensation of sea urchin sperm depends on the state of maturity of the oocyte as described above (Longo, 1978). Carron and Longo (1980) reported that shifting fertilized sea urchin eggs to Na+ -free sea water at 30 second postfertilization prevented extensive male pronuclear decondensation and attributed this inhibition to the prevention of the alkalinization that normally follows fertilization. The effect is reversible by raising the internal pH by adding ammonia to the sea water. The critical period for inhibition of decondensation is 6 minutes following fertilization. Phosphorylation of Sp H1 and Sp H2B variants in the sea urchin following fertilization was proposed as a prerequisite for decondensation by Green and Poccia (1985). The N-terminal extensions of Sp HI and Sp H2B have in common tetrapeptide sequences (Ser-Pro next to two basic amino acids) which are known phosphorylation sites in H1 molecules, but are rarely found in other histones or in any proteins (Poccia, 1987). In addition to providing phosphorylation sites, these regions, due to their amino acid compositions, probably exist in an extended conformation (Strickland et a l . , 1978b; Poccia, 1987). It has been postulated that they bind to and cross-link the long linker DNA of sperm chromatin. Upon phosphorylation, their affinity for linker should decrease, thus permitting the chromatin fibers to move apart. Thus, histone phosphorylation would have an effect similar to protamine reduction in mammalian systems, weakening the stabilizing cross-links.

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3 . Histone Variants as Postfertilization Transitional Proteins Just as a complex set of nucleoprotein and histone variant transitions occurs during spermatogenesis in rodents, the transitions following fertilization in sea urchin male pronuclei involve both variant substitution and histone modifications. Could CS histones be considered transitional proteins between spermspecific histones and more typical embryonic or somatic variants? Sequences are not yet available for CS proteins, but their electrophoretic behavior suggests they are very different from the histones that succeed them in embryogenesis. Cytochemical data are consistent with the hypothesis that postfertilization transitional proteins are of widespread occurrence.

VI. Conclusions Clearly, more comparative data on nucleoprotein remodeling are needed. Most of the information on early development comes from the frog and sea urchin, most of our detailed knowledge of spermatogenesisis based on the rodent and fish, and most of the data on male pronuclear transformations are on the sea urchin. The sea urchin shows the widest repertoire of histone variants during its development. Since, however, late histone variants may be adult forms, only the (Y forms may be embryo specific. Late histones may be minor variants of the sort seen in the adult tissues of most organisms. The CS forms, while not incompatible with rapid replication, may serve as transitional proteins between spermspecific and embryo-specific forms. The biochemical evidence for transitional proteins is scanty for other organisms, but cytochemical studies suggest they may be widespread. Transitional nucleoproteins in spermatogenesis, between somatic and sperm-specific proteins, exist in some organisms; few have been studied in detail. Transitional proteins might be utilized before or after fertilization for more effective histone replacement or might serve a more specific structural role during meiosis or pronuclear decondensation. Although fundamental conservation of histone sequence and structure remains the rule in chromosome construction, variation on histone forms, including their extreme replacements by other types of nucleoproteins, continue to offer interest and possible insight into nucleoprotein function.

ACKNOWLEDGMENTS I am indebted to Dr. G . R. Green and Prof. P. Williamson for helpful discussions, Dee Cinner for graphics, Ruth Davidon for editing, and to the Protein Identification Resource of the National Biomedical Research Foundation and BIONET (1U41 RR-01685-02)for sequence databases. Special

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thanks to Dr. Daniel Mazia for introducing me to polyspermy and Bloch’s review in 1972. This work was supported by a grant from the National Institutes of Health (HD 12982).

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INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 105

Toward a Comprehensive Three-Dimensional Model of the Contractile System of Vertebrate Smooth Muscle Cells ROLANDBAGBY Department of Zoology, University of Tennessee, Knoxville, Tennessee 37996

I. Introduction A. PROBLEMS WITH ULTRATHIN SECTIONS

The study of vertebrate smooth muscles has benefited greatly from the advances in knowledge gained from the study of much more highly ordered striated muscles and comparisons between the two have always been a fruitful avenue of research. However, it is time to realize that some of the methods used in studying the structure of striated muscles have been useful because of their inherent order and that quite different and, in some cases, much more sophisticated methods need to be applied to vertebrate smooth muscles to determine whether any recognizable order exists. For example, the use of ultrathin sections, which is essentially a high-magnification, two-dimensional (2-D) approach, has been widely used to study both striated muscle and smooth muscle fiber organizations. The 2-D aspect is valid if there is some plane, roughly longitudinal, in which a functional contractile unit is wholly represented so that one can see all the contractile components, their spatial relationships to other components, and the mechanical attachments which produce force and/or movement. The high-magnification aspect is valid if there is a high degree of order and redundancy so that one needs to see the disposition of contractile filaments in only one small part of the cell in order to understand the organization in the whole cell. This approach has worked well in skeletal muscles where myofibrils run roughly longitudinally and are composed of regularly repeated units (sarcomeres), but it has not worked nearly as well with smooth muscles. Because a recognizable organization was not usually seen in ultrathin sections of smooth muscles, many investigators (myself included) had come to the conclusion that there was no regular organization of contractile filaments. An alternate conclusion, which I now think is more valid, is that the 2-D, high-magnification approach is inadequate for determining the organization of the contractile system in vertebrate smooth muscle cells and that the three-dimensional (3-D) observations of larger portions of cells, or whole 61 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

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cells, are more likely to reveal how the contractile machinery is organized within to produce force and movement appropriate to their functions in their respective tissues. Justification of the latter conclusion will, I think, be apparent from the material presented in the review. B. THEROLEOF THE ISOLATED CELLPREPARATION Much of the recent progress in the study of the 3-D organization of smooth muscle cells has been due directly or indirectly to the development of isolated cell preparations. Until the development of a viable isolated cell preparation (Bagby et al., 1971) that could be used to study structural changes during contraction (Fay and Delise, 1973; Small, 1974; Fisher, 1974; Fisher and Bagby, 1977), there was no means of determining the whole-cell organization of the contractile system in living cells. Shortly afterward, “contractile units” or fibrils were described in isolated cells by Small (1974) and it was agreed by several investigators that contractile filaments change their orientation from roughly parallel to oblique (with respect to the cell axis) during contraction (Fay and Delise, 1973; Small, 1974; Fisher, 1974; Fisher and Bagby, 1977). In addition, some of these investigators noted that the cell seemed to have a helical organization of the contractile system which became more obvious when the cell contracted Fisher, 1974; Small, 1974, 1977a; Fisher and Bagby, 1977). More recently, freshly isolated cells have been shown to display striated fibrils when stained with antimyosin (Bagby and Pepe, 1978) or anti-a-actinin (Bagby, 1980; Fay et al., 1983). These new features called for a reappraisal of our former concepts of organization, and new speculative models appeared which incorporated the new features (Fay and Delise, 1973; Fisher, 1974; Small, 1977a; Bagby and Abercrombie, 1979; Bagby, 1983; Bagby and Corey-Kreyling, 1984; see especially Bagby, 1983, for a critical review of cell models). With few exceptions, these features had not been noted in previous ultrastructural studies using whole tissues, and there was, and to some extent still is, an understandable reluctance to accept the validity of these findings from isolated cells. However, it has caused those using tissues to look at their own findings in a new light so that indirecrly, as well as directly, the isolated cell preparation has resulted in progress in determining the organization of the contractile system. In later years, additional isolated smooth muscle cell preparations have been developed to study the pharmacological, hormonal, and electrophysiological responses of smooth muscle cells, and most of these preparations are also suitable for contraction/structure studies. In these, the cells are isolated in a more or less relaxed state and will respond to physiological stimuli. Since one purpose of the review is to stimulate future research, Table I lists single cell preparations suitable for 3-D organization studies.

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TABLE I ISOLATEDSMOOTHMUSCLECELLPREPARATIONS SUITABLE FOR STRUCTURAL STUDIES

Tissue

Bufo marinus circular mus-

Cell length (Pm) 260

Physiologically responsive Yes

References for preparative techniques

Guinea pig taenia coli

450

Yes No, but responds to ATP Yes

Guinea pig fundic stomach muscularis Rabbit jejunum longitudinal muscularis Hog carotid media

125

Yes

Bagby et al. (1971); Fay et al. (1982) Caffrey and Anderson (1979) Kominz and Groschel-Stewart (1973); Small (1974); Bagby and Pepe (1978) Small (1974); Bagby el al. (1976); Momose and Gomi (1977) Bitar and Makhlouf (1982)

200-400

Yes

Benham and Bolton (1983)

110 (diameter 6 wn) 240

Yes

Driska and Porter (1985)

Yes

Warshaw (1985)

cularis Amphiuma stomach Chicken gizzard

Bovine carotid media

1000-2000 ?

C. THE SCOPEOF

THE

REVIEW

The purpose of this review is to summarize the recent progress made in determining the 3-D organization of the contractile system of vertebrate smooth muscle cells and to point to fruitful avenues for future research. As the title of this review indicates, there is as yet no model which is universally accepted nor is any model “comprehensive.” A comprehensive model, as I have chosen to define it, is one in which the whole-cell distribution of contractile proteins and the structural entities which contain them are known. Since the development of force is dependent upon the attachment of contractile proteins to the cell framework, the distribution of attachment proteins and structures also needs to be determined to complete the model. Such a model would enable one to determine whether the cellular distribution of the contractile system components is random or ordered and whether the distribution is compatible with the prevalent theory of muscle contraction, i.e., the sliding filament theory (Huxley and Hanson, 1954; Huxley, 1972), which is based almost entirely on striated muscle observations. “Three-dimensional model” will be used rather loosely to mean any representation of the whole cell which shows the relative positions, in three-dimensions, of the contractile components and attendant cytoskeletal components. ‘‘Contractile

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system” includes all the proteins known to participate in muscle contraction and the structures which contain them, as well as other structures to which contractile units attach and on which they depend for transmission of force. The review is limited to vertebrate smooth muscles, although the term “smooth muscle” has sometimes been used to describe any nonstriated muscle in the animal kingdom. The structure of these other smooth muscles is often quite different from that of vertebrate smooth muscles and thus inappropriate to the review. The tacit assumption has been made by some investigators that all vertebrates smooth muscles have the same cellular organization, while research by others, notably Gabella (1976a, 1983a, 1984), has shown some specific differences between vertebrate smooth muscles. At some point in time, specific models for each smooth muscle cell type will have to be constructed, but for now we will consider vertebrate smooth muscle cells as being homogeneous enough so that a single model of cell organization will be sufficient to show aspects common to all. This review can only be a progress report since the work is far from finished; in terms of 3-D analysis and reconstruction, the work -has only just begun. Although 3-D information is necessary to the completion of the model, there has been a tremendous amount of work dealing with the 2-D distribution of muscle proteins and with 2-D studies of ultrastructure, and it would be foolish to ignore it altogether, especially when comparable 3-D information.on some aspects is not yet available. Therefore, the early part of the review will cover 2-D information and its contribution to our understanding of cell organization. Many aspects of smooth muscle cell organization have been covered in earlier reviews (Small and Sobieszek, 1980; Somlyo, 1980; Groschel-Stewart and Drenckham, 1982; Bagby, 1983; Cooke, 1983; Gabella, 1983a, 1984). The reader is urged to read these earlier reviews for additional details and background.

11. Two-Dimensional Information

In looking at 2-D information, information from transmission electron microscopy (TEM) will be discussed first, partly because TEM of tissues was used long before the isolated cell techniques but also because it allows a progression from the individual molecular components of the contractile system to the higher level of organization in the whole cell. Since the subject of this review is 3-D organization, the distribution of the contractile components and their relationship to other contractile and to cytoskeletal components will be emphasized rather than the structure of individual components. The reader is urged to read other recent reviews which deal in more detail with the molecular structure of contractile proteins (Groschel-Stewart and Drenckham, 1982; Small and Sobieszek, 1983).

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A. TRANSMISSION ELECTRON MICROSCOPY OF THINSECTIONS 1. General Comments It is appropriate to begin the section on 2-D information with a review of TEM of ultrathin sections of smooth muscle material because in the ultrathin section we have the closest approach to truly 2-D objects. In fact, besides making the object thin enough for conventional TEM beam penetration, the aim of making sections ultrathin is to eliminate information from other planes. In the studies of Huxley and Hanson on skeletal muscles, it was very important to cut sections thin enough so that one was sure that only one layer of thick filaments was included within a section (Hanson, 1956; Huxley, 1957) so that the interactions of individual thick and thin filaments could be seen. If one wished to establish the nature of the interaction of individual thick and thin filaments in smooth muscles, it would seem that ultrathin longitudinal sections would be necessary in these tissues as well. However, it was soon found that it was extremely difficult to obtain thin sections where thick filaments were wholly within the plane of one section. Although some success was obtained in seeing thick-thin filament interaction with ultrathin longitudinal sections (Somlyo et al., 1973), it was soon realized that thicker longitudinal sections were more useful in a tissue where filament orientation could not be determined prior to sectioning (Ashton er al., 1975). As a result of the difficulty in obtaining ultrathin, strictly longitudinal sections, the transverse plane became the most useful plane of section for most studies of smooth muscle because the different filament types and their distributions are easily seen in both thin and thick sections.

2. ‘ ‘Normal” Distribution of Contractile System Components a. General Morphology. Figure 1 shows transverse sections of the smooth muscle cells which have been used the most in recent years in studies of the 3-D organization of contractile entities and the localization of muscle proteins in single cells and tissues, namely, Bufo marinus circular stomach muscle, guinea pig taenia coli, and chicken gizzard. Guinea pig taenia coli is the only one of the three which had been studied extensively with TEM prior to the use of its tissue for the study of isolated smooth muscle cells (Rice et al., 1970; Cooke and Fay, 1972; Small et al., 1972; Fay and Cooke, 1973; Cooke, 1976; Gabella, 1976b; Shoenberg and Needham, 1976). The other two have become important due to their extensive use in isolated cell studies. Currently, the laboratories most active in using isolated smooth muscle cells to study structure are Fay’s laboratory, which uses B . marinus stomach almost exclusively, Small’s laboratory, which concentrates on guinea pig coli, and our own laboratory, which uses chicken gizzard, so it is likely that new cell models will come from one of these tissues. Some TEM information is available on these tissues (Bennet and Cobb, 1969; Fay and Delise, 1973; Bagby and Fisher, 1979; Bagby, 1983; Cooke, 1983;

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73

Bailey et al., 1984) but more needs to be done. All these tissues have thin, thick, and intermediate filaments, dense plaques (dense bands), and cytoplasmic dense bodies, as do all other smooth muscles whose structure has been studied (individual references are too numerous to mention, but see reviews by Shoenberg and Needham, 1976; Somlyo et a l . , 1977; Somlyo, 1980; Small and Sobieszek, 1980; Bagby, 1983; Cooke, 1983; Gabella, 1984). It can readily be seen, however, that the distribution and the structure of these entities are not the same in all these cells, a fact to which Gabella, especially, has referred (1976a, 1983a, 1984). However, despite differences, there are some patterns of distribution which have been recognized and seem to apply to all vertebrate smooth muscles. Many early studies and even some more recent ones have tended to fix smooth muscles without regard to muscle length or contractile state. I have lumped these studies into the category of “normal” since it is likely that there was some uniformity of fixation conditions, namely, the muscles were in a relaxed state with a mild degree of stretch, conditions considered optimal for most studies. b. Thick Filaments. Thick filaments have irregular profiles in transverse section and their diameter is usually greater than 15 nm. They occur as either “rods” or “ribbons,” depending upon conditions before and during fixation (Somlyo et al., 1971, 1973; Somlyo, 1980; Bagby and Fisher, 1979; Small and Sobieszek, 1980). Rods were originally thought to be bipolar filaments with a middle “bare” zone analogous to those in striated muscles (Somlyo et a l . , 1977), while ribbons have been shown to be “face”-polar with no middle bare zone and bridges have opposite polarity on either face (Small and Squire, 1972; Craig and Megerman, 1977; Hinssen et a l . , 1978). Although it is generally agreed that the ribbon profile is obtained with extreme stretch or high osmolality in the prefixation solution (Somlyo et al., 1971, 1973; Somlyo, 1980; Small and Sobieszek, 1980), both of which seem to favor lateral aggregation of myosin, it is not at all clear how the transition is made. Is the transition made by an intermediate step involving disaggregation into monomers or dimers, or is the ribbon simply a lateral aggregation of existing rod filaments? If the transition is by simple lateral aggregation, then it implies that the rods must also be facepolar, since there does not seem to be any way that face-polar ribbons could be formed by an aggregation of bipolar filaments (Bagby, 1983). It has generally been assumed that thick filaments in smooth muscles are composed mainly of myosin, but TEM confirmation of direct labeling in situ with antimyosin has ~~

~

FIG. 1. Transverse sections of (a) B . morinus circular stomach, muscularis, (b) guinea pig taenia coli, and (c) young chicken gizzard lateral muscle, the tissues most widely used for preparing isolated smooth muscle cells. All tissues have the same components in the contractile system, thick filaments (large arrows), thin filaments (small arrows), intermediate filaments (double-headed arrows), membrane-associated dense bodies (m), and cytoplasmic dense bodies (c), but their distribution and organization are different. X 90,000.

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been done only recently (Small et al., 1985). Another proof of the presence of myosin is the in vitro growth of synthetic thick filaments from myosin suspensions (Wachsberger and Pepe, 1974; Nonomura and Ebashi, 1980; Shoenberg and Stewart, 1980), which share most of the ultrastructural characteristics of the in situ filaments. Other proteins, such as C protein, may exist in thick filaments but their presence has not been shown directly. Thick filaments are unattached to other filaments except for presumed lateral interaction with thin filaments by means of cross-bridges. Their length has been determined by 3-D means (see Section 111) to be 2.2 p m and lengths are reasonably uniform in the muscles used for measurement (Ashton et al., 1975). Although there is a tendency for groups of thick filaments to be in register, there do not appear to be any lateral connections between thick filaments analogous to the M-line connections between thick filaments in striated muscles. Thick filaments seem to be fairly evenly distributed throughout the profile of the transversely sectioned cell. There is little tendency for thick filaments to form bundles excluding other filaments, in contrast to the frequently seen lattice bundles of thin filaments (Rice et al., 1970 Heumann, 1973; Oki et a!., 1980; Gabella, 1984; Bagby and Corey-Kreyling, 1984). Cooke has referred to the distribution of thick filaments as “parenchymal” to the thin filament bundles (Cooke, 1983). However, studies by Somlyo et al. show a more even distribution of thick and thin filaments (Somlyo et al., 1973, 1977; Somlyo, 1980). The distribution of thick filaments among the thin filaments is known to be affected by the contractile state and/or length (Heumann, 1973; Bagby and Corey-Kreyling, 1984), and differences in the literature may be dependent on the conditions in the muscle when it was fixed. The effects of length and contractile state are discussed in a later section. c. Thin Filaments. Thin filaments have rounded profiles in transverse section and diameters of 4-8 nm. It has been determined by heavy meromyosin (HMM) labeling that in situ thin filaments contain actin (Bond and Somlyo, 1982; Tsukita er al., 1983). Thin filaments are assumed to contain tropomyosin as well (Small and Sobieszek, 1983; Marston and Smith, 1985) but not troponins, although a calcium-regulated protein has been suggested (Ebashi, 1980) and a recent report shows staining of smooth muscles by antiskeletal muscle troponin-T (Lim et al., 1984). Caldesmon, a 155-kDa protein, binds to actin and seems to exercise Ca2 -dependent regulation over actin-myosin interactions (Sobue et al., 1982). Marston and Smith (1984, 1985) describe a 120-kDa protein which is a component of thin filaments isolated from smooth muscles. This component confers a Ca2 sensitivity to the thin filaments similar to that of caldesmon and, despite the difference in reported M,,it may be the same protein. These findings raise the possibility that there may be a type of thin-filamentlinked regulation of contraction. Thin filaments have been shown to attach at one +

+

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end to either membrane-associated attachment sites (MADBs), also known as dense bands (Ashton et al., 1975; Somlyo et al., 1977; Gabella, 1979), or to cytoplasmic dense bodies (CDBs) (Somlyo et al., 1977; Bond and Somlyo, 1982; Tsukita et al., 1983). HMM labeling produces “arrowhead” structures along the thin filament which point away from attachment points (Bond and Somlyo, 1982; Tsukita et al., 1983). Thus, dense bands seem to be equivalent to terminal Z-lines of myofibrils and the CDBs seem to be analogous to the intramyofibrillar Z-lines of striated muscles. However, it is by no means certain that every thin filament attaches to a dense band or a CDB, and models have been proposed which suggest that not all thin filaments are attached in this way (Small, 1977a,b, 1980; Cooke, 1983). Filamin also binds to actin, and Small’s group has suggested that there are two distinct populations of thin filaments, one of which contains caldesmon and interacts with myosin filaments, while the other contains filamin and is in the cytoskeletal domain (Small et al., 1986; Furst et al., 1986; see Section II,B,2 for a complete discussion of these findings). Thin-to-thick filament ratios have been measured from the transverse sections of several smooth muscles, with values of 12:1 for the rabbit portal-anterior vein (Devine and Somlyo, 1971; Somlyo et al., 1981) and the rat small intestine (Bois, 1973); 15-18:l for the rabbit main pulmonary vein (Somlyo et al., 1973) and the rabbit portal-anterior mesenteric vein (Berner et al., 1981b), and 2530:l in freeze-substituted guinea pig taenia coli (Tsukita et al., 1982). Unpublished data from our laboratory give ratios of 12-14:l for guinea pig taenia coli and 20-22:l for B . marinus stomach circular muscularis. Compared to the ratios found in vertebrate skeletal muscles of 2:l (Huxley, 1972), the ratios in vertebrate smooth muscles are quite high. Since the values are obtained from cross-sectioned profiles in transverse sections, it is possible that one may not be counting the ratio between numbers of filaments. Murphy (1979) has discussed the mechanical consequences of models in which thin filaments are either much shorter, the same length, or much longer than thick filaments. He has speculated that thin filaments five times longer than thick filaments would allow one to have the same actual filament ratio (2:l) as in striated muscles while having an apparent filament ratio (from transverse section filament profiles) of 10:1 . This arrangement leads to an increase in force generated per myosin filament by putting more myosin filaments in parallel rather than in series (Murphy, 1979), and it would end perplexing questions about how 12-30 thin filaments interact with a single thick filament. A longer thin filament length would also lead to a longer sarcomere length. If we assume thick filaments in all muscles are 2.2 pm long, according to measurements by Ashton et ul. (1975), then thin filaments could be as long as 1 1 pm and sarcomeres would be as long as 24 pm. Unfortunately, accurate thin filament length measurements are much more difficult to obtain than those of thick filaments, and at present we have no reliable measure-

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ments of thin filament length. Clearly, the measurement of thin filament length is critical for determining the functional organization of the contractile system in smooth muscles. Thin filaments are found rather evenly distributed throughout the transversely sectioned cell profile except where the nucleus and other organelles occur in the central region. They rarely occur singly, but are found in rows or rosettes around thick filaments (Somlyo et al., 1973, 1977; Somlyo, 1980) or in lattice-like bundles virtually free of thick filaments (Rice et al., 1970; Heumann, 1973; Oki et al., 1980; Bagby and Corey-Kreyling, 1984; Gabella, 1984). Are the latticelike bundles equivalent to the I bands of striated muscles? This question is better addressed in the section which deals with structural changes associated with perturbations of length and contractile state. d. Intermediate Filaments. lntermediate filaments (IF) have very crisp, rounded profiles in transverse section and a remarkably uniform diameter in a particular tissue, measuring 10 nm in smooth muscles (Uehara et al., 1971; Fay and Cooke, 1973; Ashton et a f . , 1975; Small and Sobieszek, 1977, 1983; Small and Celis, 1978; Campbell et al., 1979). Intermediate filaments were the third type discovered in smooth muscles (Uehara et a / . , 1971), and their diameter being intermediate to that of thin and thick filaments gave rise to their name. Intermediate filaments are composed of desmin, vimentin, or possibly copolymers of both ( G a d and Lazarides, 1980; Berner et al., 1981a; Geisler and Weber, 1981; Schmid et a / . , 1982). Although it was first thought that vimentin IF were restricted to vascular smooth muscles (Gabbiani et al., 1981), there are enough cases in which this is not true (Berner er al., 1981a; Schmid et al., 1982) that it is not feasible to classify smooth muscles on the basis of IF proteins. Since their discovery in embryonic smooth muscle by Uehara et al. ( 197l), IF function has been assumed to be cytoskeletal, but their mechanical and functional relationship to the contractile proteins has been controversial. Their usual distribution is in a ring just peripheral to a cytoplasmic dense body (Somlyo et al., 1973, 1977; Somlyo, 1980; Ashton et al., 1975; Small and Sobieszek, 1980; Cooke, 1983) or, less noticeably, at the cytoplasmic border of dense bands (Cooke, 1976; Small, 1977b; Small and Sobieszek, 1980; Gabella, 1983a, 1984). Aggregations of IF are routinely found in the axial region of some smooth muscles (Heumann and Weigold. 1978; Asmussen, 1980) where they may form loops and whorls. In other tissues, they are found in aggregations in embryonic tissue (Uehara et al., 1971; Bailey et al., 1984), in hypertrophying smooth muscle (Berner et al., 1981b; Gabella, 1979), or in response to microtubuledisrupting agents such as colchemide (Fellini et al., 1978). Small has developed a technique (Small ef al., 1985) that seems far superior to the use of cryoultramicrotomy for immunocytochemistry. Tissues are aldehyde fixed, embedded in polyvinyl alcohol, sectioned, and then immuno-gold stained after removal of the embedding media. All filaments are easily recognizable and

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FIG.2. Transverse sections of chicken gizzard smooth muscle stained with 10 nm gold-labeled antifilamin. The gold particles are seen to be localized in the intermediate filament domain. Myosin filaments (my), actin filaments (act), intermediate filaments (if), dense body (db). X 9 7 , 5 0 0 . From Small et al. (1986), by copyright permission of the Rockefeller University Press.

the gold label is quite easily discerned because of its electron density. Figure 2 shows a cross section of guinea pig taenia coli labeled with antifilamin. The filamin seems to be colocalized with IF and is excluded from contractile domains where thick and thin filaments intermingle. Filamin label and 1F are especially noticeable at the periphery of CDBs and in the subsarcolemma adjacent to MADBs. Because of their association with dense bodies, some investigators have proposed a cytoskeleton of IF and CDBs with the cytoskeleton attached by IF to the membrane-associated dense bodies (Cooke and Fay, 1972; Cooke, 1976; Bagby, 1983). In these models, the attachment of this IF-CDB cytoskeleton is assumed to be primarily to the MADBs in the terminal regions of the cell (Cooke, 1976;

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Cooke and Fay, 1972) and there is disagreement as to whether contrackile proteins interact mechanically with this cytoskeleton (Bagby and Corey, 1981; Bagby, 1983) or not (Cooke and Fay. 1972; Cooke, 1976; Small, 1977b). The evidence for each of these points of view can be found in Section II,A,3. e. Membrane-AssociatedDense Bodies and CytoplasmicDense Bodies. Membrane-associated dense bodies are amorphous, subsarcolemmal dense regions which lie between the areas combining caveolae (Ashton et al., 1975; Gabella, 1976, 1979, 1983a, 1983b, 1984; Somlyo et al., 1977; Somlyo, 1980; Gabella and Blundell, 1978). Although some MADBs clearly have a structure that can be described as dense bands, I prefer the generic term, MADBs, which does not imply a particular shape unless the shape is known. In freeze-fractured cells, the openings of caveolae form longitudinally oriented rows in areas 2-3 rows wide, and dense bands are recognized as relatively smooth, generally longitudinal areas between the caveolar domains (Gabella and Blundell, 1978). In grazing tangential sections, caveolar and dense band areas form alternate longitudinal rows (Ashton etal., 1975;Gabella, 1979, 1984;Somlyo, 1980). The width of the dense bands is 0.1-0.4 pm and their length is indeterminant (Gabella and Blundell, 1978). In some smooth muscles, MADBs may protrude for a considerable distance into the cytoplasm (Oki et al., 1980; Gabella, 1983b, 1984), while in most the MADBs form a thin layer paralleling the sarcolemma. MADBs occupy 30-50% of the cell profile in the middle portions of the cell, with higher percentages, sometimes as much as loo%, in the terminal portions (Gabella, 1984). Cytoplasmic dense bodies are amorphous dense regions in the cytoplasm. Their size and shape are variable, but they are thought to be ovoid with their main axis roughly longitudinal. Are MADBs and CDBs equivalent except for localization? Both have been shown to be attachment sites for thin filaments and in both cases HMM decoration of attached thin filaments revealed that they were attached at the “barbed” end of the filaments (Bond and Somiyo, 1982; Tsukita et al., 1983). However, immunocytochemical localization studies have shown that while anti-a-actinin stains both types (Schollmeyer et al., 1976; Geiger et al., 1980; Small, 1985) antivincuiin stains only MADBs (Geiger ef al., 1980; Small, 1985), indicating at least one biochemical difference and implying functional differences. Are all CDBs equivalent? It was proposed by some groups that CDBs were attachment sites for thin filaments (Somlyo et al., 1973, 1977, 1980; Ashton et al., 1975; Bond and Somlyo, 1982), while other groups maintained that their main function was attachment of IF (Cooke, 1976; Small, 1977b). A good part of the problem was the difficulty in seeing both filaments interacting with the same dense body in one section due to IF interacting laterally with CDBs while thin filaments interacted axially. The study by Bond et al. (1982), where the cell was swollen to allow one to see interactions more clearly, shows both thin filaments and IF interacting with the CDB (Fig. 3). IF take looping paths to contact

FIG. 3. Longitudinal section of rabbit portal-anterior mesenteric vein smooth muscle cell. The tissue was saponin treated in relaxing solution and then fixed in the presence of tannic acid. The saponin treatment causes the cell to swell, revealing relationships between dense bodies (db) and filaments. Thin filaments (arrows) insert into both sides of the dense bodies. Intermediate filaments (arrowheads) are associated with the lateral aspects of dense bodies and sometimes interconnect a series of dense bodies to form a chain. X70,OOO. From Bond and Somlyo (1982), by copyright permission of the Rockefeller University Press.

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adjacent CDBs at their lateral aspects, while thin filaments take straight, axial paths into the matrix of CDBs. There is still the question of whether all CDBs are intramyofibrillar, acting as Z-lines in a sarcomere, or whether some are extramyofibrillar and act purely as cytoskeletal elements. Their attachment to thin filaments with opposite polarity at opposite ends of the CDB (Bond and Somlyo, 1982; Tsukita et al., 1983) argues for a myofibrillar role, but recent evidence from immunocytochemical studies (Small ef al., 1985) argues for a cytoskeletal role (see Section II,B,2). Are all MADBs equivalent? Attachment of IF to MADBs is less well documented than attachment to CDBs, but the argument has been made (Cooke and Fay, 1972; Cooke 1976) that IF attach preferentially to terminal MADBs. Since these regions of the cell are less studied than other regions, it might account for IF attachment to MADBs being reported less frequently than thin filament attachment to MADBs, which is almost universally reported (Somlyo et al., 1973, 1977; Somlyo, 1980; Ashton et al., 1975; Gabella, 1976a,b, 1979, 1984; Gabella and Blundell, 1978; Small and Sobieszek, 1980). 3 . Changes in Organization with Changes in LengthIContractile State a. Fixation of All Components. Although some early studies found that thick filament preservation was achieved only in muscles fixed while actively contracting (see Shoenberg and Needham, 1976, for a review), later studies have shown that thick filament preservation is not affected by contractile state if the fixation methods are adequate and that thick filaments are well preserved even in relaxed smooth muscles (Somlyo et al., 1973, 1977; Somlyo, 1980; Ashton et al., 1975). Our laboratory measured thin-to-thick filament ratios in muscles whose length andlor contractile state varied, and we found no significant differences between treatments (Bagby and Kreyling, 1983). Therefore, it is feasible to look at changes in the filament organization of smooth muscles fixed in different contractile states without worrying about effects on filament preservation. An early study by Heumann (1973) showed that the thin filaments tended to be found in lattice-like areas which excluded thick filaments in the relaxed state, while in the shortened contracted state there was a more homogeneous distribution of thick and thin filament profiles. Cooke and Fay (1972) made a careful study of changes in distribution of contractile and cytoskeletal elements in guinea pig taenia coli with attention to the effects of both length and contractile state, whereas previous experiments had often equated shortening and contraction, leading to some confusion in causeand-effect relationships. They concluded that contractile state had no effect; that is, all effects they saw were due to length perturbations. These effects were striking, but largely unappreciated at the time. The most consistent feature was the redistribution of dense bodies with stretch, CDBs changing from a more or less random distribution in transverse sections of shortened tissue to a markedly

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axial concentration in highly stretched tissue. Another effect was a “layering” of filaments such that thin filaments formed a peripheral ring, thick filaments were found in the next layer, and IF and CDBs occupied the axial area. The authors paid scant attention to the thick and thin filament redistribution, possibly because some experiments were done in the presence of EDTA which led to the disappearance of thick and thin filaments (see also Fay and Cooke, 1973). Instead, the redistribution of CDBs and IF with stretch was used to construct a model of the cytoskeleton where CDBs and IF formed a network which was connected to MADBs in the terminal region of the cell by IF (Cooke and Fay, 1972; Cooke, 1976). Since contraction had, in their experiments, not changed the distribution of CDBs, they also concluded that the cytoskeleton was independent of the contractile system. The redistribution of thick and thin filaments to the peripheral areas during stretch was attributed to exclusion of these filaments from the axial area due to the concentration of IF and CDBs (Cooke and Fay, 1972). This independence of cytoskeletal and contractile elements was echoed in models by Small (1977a,b). Our laboratory repeated the experiments of Cooke and Fay (1972) because we were interested in the changes in thick and thin filament distribution with stretch. We studied B . marinus stomach muscularis in addition to guinea pig taenia coli and we used acetylcholineto produce contraction instead of KC1 because KCI led to the disappearance of thick filaments (Jones et al., 1973), but aside from these modifications we used the same techniques. However, we did not obtain the same results. Although we saw the same axial concentration of CDBs in relaxed stretched muscles, CDBs had a nearly random distribution in contracted stretched muscles (Bagby and Corey-Kreyling, 1984). From this data we constructed a model of the cytoskeleton similar to that of Cooke and Fay (1972), except that in our model (Fig. 4) the cytoskeletonprovided attachment for one end of a myofibril with the other end being attached to an MADB (Bagby and Corey, 1981; Bagby, 1983). The layering of filaments in passively stretched muscles was also consistent with attachment both axially and peripherally, since thin filaments were found primarily near the periphery and again near the CDBs in the axial region, with the area in between occupied by a mixture of thick and thin filaments. This distribution was also consistent with the obliquely organized myofibril described in that study (Bagby and Corey-Kreyling, 1984), and in retrospect the data also favor a myofibril only one sarcomere long. In addition to changes in distribution throughout the cell due to contraction, the contractile filaments also appear to change their orientation with respect to the cell axis during shortening (Fay and Delise, 1973; Small, 1974; Fisher, 1974; Fisher and Bagby, 1977). The change in orientation is quite apparent in single cells studied with the electron microscope (Fay and Delise, 1973) and with the light microscope (see Section 11,B). However, reorientations that occur in whole tissues have been disputed (Shoenberg and Needham, 1976; Gabella, 1976b,

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FIG. 4. A model of the smooth muscle cell, showing the distribution of cytoskeletal and contractile components in the stretched, relaxed state. The model’s construction shows the innermost components in (a) and progresses to the outennost with (b) showing an axial cytoskeleton of CDBs and IF according to Cooke and Fay (1972). In (c) a few myofibrils are shown. These are attached by one end to MADBs and follow roughly helical paths until they attach at the other end to CDBs in the axial cytoskeleton. (d) Transverse sections of the model at two levels. When the myofibrils contract, CDBs are moved toward the periphery of the cell. From Stephans (1983).

1979, 1984) because there is always the pohsibility that the coiling or bending of an inactive cell may be caused by an actively contracting neighboring cell. This has been shown to be the case in sipunculid nonstriated muscles, where a regular folding of one cell type occurs due to the contraction of another cell type (Abercrombie and Bagby, 1984; Abercrombie et al., 1984), so it is only in single-cell preparations that a bona fide active reorientation of filaments can be proven, from nearly parallel to the cell axis in the relaxed state to a large angle

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with respect to the cell axis in highly shortened cells (Fay and Delise, 1973; Fisher, 1974; Small, 1974; Fisher and Bagby, 1977). Although it is doubtful that the same extent of shortening of cells and reorientation of filaments could occur in intact tissues, the fact that such reorientation of filaments does occur in isolated cells argues against an arrangement of filaments where contractile units are attached only at either end of the cell. Although thin-to-thick filament ratios from random transverse sections should not change as a result of length changes (Bagby and Kreyling, 1983), the amount of overlap of thick and thin filaments should change, so that sections through the A band of a muscle should show marked changes in thin-to-thick filament ratio as overlap changes. In vertebrate striated muscles, thin-to-thick filament ratios in the A band should be 0 : 1 at 1.7L0 (Lois rest length of muscle) where there is no overlap, 2 : 1 at 1.OLo where there is optimal overlap and values approaching 4 : 1 at 0.5L0 where opposing thin filaments overlap each other. We studied the thinto-thick filament ratios in B . marinus stomach muscularis and guinea pig taenia coli at different muscle lengths and we found very little change in whole-cell ratios (Bagby and Kreyling, 1983). A refinement of the study (unpublished data) designated areas where thick filaments were concentrated as A band areas. Surprisingly, when taenia coli shortened from 0.91L0 to 0.56L0, A band thin-tothick filament ratios went from 7.0 rt 1.2 to 8.7 ? 1.9 with no significant difference. B . marinus stomach muscularis showed no change, A band ratios being 13.6 ? 2.4 at 1.17L0 and 13.6 +- 3.2 at 0.58L0, with no significant difference. These data argue against the conventional sarcomere arrangement seen in vertebrate skeletal muscles. Bagby and Kreyling (1983) originally favored obliquely organized myofibrils as the best explanation and they proposed a model of the oblique sarcomere (Bagby and Corey-Kreyling, 1984), but the data are also consistent with other possibilities including very long thin filaments (Murphy, 1979; Small and Sobieszek, 1980; Groschel-Stewart and Drenckham, 1982) or noninteracting thin filaments (Cooke, 1983). Cooke (1983) has an interesting model which is worth considering here. As Fig. 5 shows, the model features networks of thin filament bundles rather than fibrils, and the thick myosin filaments are located in the interstices of thin filament bundles. Presumably, only the outer thin filaments of the bundles could interact with the myosin in the thick filaments. Small et al. have evidence for two distinct actin domains, only one of which contains thin filaments which interact with myosin filaments (Small et al., 1986; Furst et al., 1986; see Section 11,B,2), supporting the idea of noninteracting thin filaments. Unfortunately, the different explanations for the filament ratios are not mutually exclusive and, without additional data, none of the possibilities may be ruled out. b. Selective Removal of Contractile Components. Under certain prefixation and fixation conditions, selected components of the contractile system may be removed. Although it has been argued that certain components may disappear in the living state (Fay and Cooke, 1973; Ohashi and Nonomura, 1979, 1984), it is

FIG. 5 . Illustration of the 3-D relationships between the three classes of filaments based upon serial reconstructions of taenia coli smooth muscle fibers. The thin filaments are grouped into bundles that are integrated into a spatially complex network through branches and anastomoses between adjacent bundles. The thick filaments are parenchymal to the bundles of thin filaments and interdigitale with some bundles of the network that assume the form of rosettes. The intermediate filaments are complexed with the dense bodies to form a cytoskeletal network extending throughout the muscle fiber. From Cooke (1983).

more probable that the prefixation and/or fixation conditions lead to the components being soluble during fixation and dehydration and that this is the reason for their absence in electron micrographs. The removal of actin and myosin by prolonged EDTA treatment prior to fixation (Fay and Cooke, 1973) makes the distribution of remaining components easier to determine. This treatment was used to show the axial distribution of the IF and CDBs in relaxed, highly stretched muscles (Cooke and Fay, 1972; Cooke, 1976). Removal of thick and thin filaments also made it easier to trace connections between the cytoskeleton and the plasma membrane MADBs (Cooke, 1976). However, the presence of EDTA precludes similar observations during the contracted state, so the technique has its limitations. Prolonged soaking in KC1 prior to fixation has been shown to lead to loss of thick filaments in smooth muscle tissues processed for EM (Jones et al., 1973). The loss of thick filaments is apparently related to the swelling and/or dilution of internal constituents, since the addition of sucrose or MgC1, to the bathing medium prevents the disappearance of thick filaments (Jones et al., 1973; Ohashi and Nonomura, 1984). High K is also used to induce contractions, and Ohashi and Nonomura (1984) used these two effects of KCI in a very interesting study. They found that fixation during the phasic period of contraction or early tonic contraction gave preservation equal to that in the resting state. However, fixation after at least 30 minutes in K + , while tonic contraction was still maintained, led to the loss of thick filaments. Other muscles were kept in the high K + , but diltiazem was used to reverse contraction. Both groups lost thick filaments, +

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FIG.6 . Transverse section of guinea pig taenia coli 60 minutes after the onset of K+-induced contraction. Thick filamentshave disappeared.Thin filaments and CDBs occur only in the peripheral part of the cell in the large cell profiles, but in the small profiles cut nearer the ends of the cell thin filaments and CDBs are found throughout the cytoplasm. X15.120. Inset, X 7 5 , 6 0 0 . From Ohashi and Nonomara (1984).

showing that the effect on them is not dependent on contraction, but thin filament distribution was contraction dependent; contracted muscles had thin filaments only in peripheral and terminal regions of the cell, leaving a central region devoid of both thick and thin filaments (Fig. 6). The thin filament density in the peripheral and terminal regions was unchanged from that of the control or re-

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laxed cells, implying that thin filaments did not move from a central to a peripheral location; they merely disappeared from the central region. Although it is not mentioned by the authors, it appears that many CDBs are also lost from the central regions in contracted cells. Even without the loss of filaments, contraction caused a redistribution since in KCl solutions made hypertonic by sucrose or MgCI,, thick filaments were condensed in the central region of the cell in contracted muscles. I feel that the striking results of Ohashi and Nonomura (1984) are quite relevant to our understanding of the organization of the contractile system, although the investigators were not primarily interested in organization, but rather in mechanism. I would like. to propose the following explanation of their results. It seems obvious that there is an underlying peripheral-to-central organization that would have to be present to see the central redistribution of thick filaments and the preferential removal (or movement) of central thin filaments with KCI contraction. It is also probable that the KCI treatment does more than just solubilize thick filaments. The preferential removal (or movement) of thin filaments and CDBs from the central area only during contraction could also be due to dissolution or weakening of the axial cytoskeleton, allowing the entire contractile apparatus attached to the MADBs to contract toward the periphery. The thin filament density is the same in thin filament-containing areas of both KCI-contracted tissues and resting tissues. However, the thin filament density appears to be quite reduced in the KC1-diltiazem-relaxed tissues compared to the KCI-contracted tissues, suggesting a loss of thin filaments. It is possible that movement of central thin filaments to the periphery may have occurred during contraction, which would account for the higher density of thin filaments at the periphery. Later, the fixation and dehydration procedure would remove thick filaments. This scenario is consistent with a model which proposes that myofibrils attach at one end to MADBs and at the other to CDBs in an axial cytoskeleton (Bagby and Corey, 1981; Bagby, 1983).

1. Some Insights from Developmental Studies One of the difficulties in dealing with the 3-D structure of smooth muscles is the number of components in the cells. We reasoned that it might be easier to determine some facets of organization in a simple system. The developing smooth muscle cell in chick embryonic gizzard was thought to be such a system. Besides, to use an analogy, it is easier to find out how a car is put together by going to the car factory than to take apart an already assembled car. Hirai and Hirabayashi (1983) have shown the sequence of appearance of contractile proteins, and Bennet and Cobb (1969) had previously shown a sequence of structural component triariufacture. We found, indeed, that in the early stages "protofibrils" were seen which contained only CDBs and thin filaments (Fig. 7) (Bailey et a l . , 1984). Bennet and Cobb (1969) also saw thin filaments and CDBs as the earliest components of the contractile system. There are no MADBs at this stage, day seven, although desmosomes do occur (Bennet and Cobb, 1969).

FIG. 7. A “protofibril” from a 7-day-old tembryonic chick gizzard cell. Thin filaments are attached to regularly spaced CDBs. No thick filaments are present. X45,OOO. Micrograph provided by C. Bailey. FIG. 8. In 14-day embryonic chicken gizzard, the contractile material is concentrated in the axial region as shown in this transverse section. Thin filaments, thick filaments, intermediate filaments, and CDBs are present by this stage, but MADBs are infrequently seen. X27,OOO. Micrograph by C. Bailey.

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Somewhat later stages show an axial aggregation of contractile components, including thick filaments, with no attachment to MADBs (Figs. 8 and 9). This axial concentration of contractile components may also be seen in micrographs of embryonic chicken gizzard tissue by Cooke (1982), but this feature is not noted in the publication. The latest embryonic stages we examined (17 days of incubation) show peripheral additions to the axial contractile system and the beginnings of contractile system attachment to MADBs. Very few ultrastructural studies of embryonic smooth muscle cell organization have been made. However, Konishi et al. (1984), who studied development of fetal human uterus, and Bennett and Cobb (1969), who studied embryonic gizzard, also found that CDBs were found early in development, while MADBs did not appear until much later. Other studies simply do not mention any sequence in appearance of CDBs and MADBs (Yamauchi and Burnstock, 1969; Uehara et al., 1971; Campbell and Chamley, 1975; Cooke, 1983). Most studies argue that thin filaments precede thick filaments. When the 14-day embryonic stage is compared to the 3-day posthatching organization (Fig. Ic), it can be seen that a tremendous amount of change must occur in the interim period, since the 3-day posthatching structure is typical of adult tissue. We had hoped to find a stage in which a few isolated myofibrils with both thick and thin filaments would be seen and terminal attachments determined. No such stage was found. However, the developing smooth muscle did give some important insights into 3-D organization. For example, the study was consistent, with at least some myofibrils having one attachment centrally and the other peripherally (on MADBs). There was also evidence for the periodic arrangement of some contractile components in the protofibrils, and the CDBs seemed to be equivalent to Z-lines, as was shown earlier by others (Bond and Somlyo, 1982; Tsukita el al.. 1983).

B. LIGHTMICROSCOPY I . Living Cells The emphasis on the 3-D organization of smooth muscle cells really began once techniques were developed for isolating living cells from smooth muscle tissues. For the first time, one could look at the whole 3-D object instead of just sections of small portions. Although our most valuable information has come from immunofluorescence studies of fixed cells, it is doubtful that these results would be trusted if corroborating features had not been seen in living cells where the artifacts of fixation are avoided. The living smooth muscle cell seen with phase-contrast microscopy confirms some of the general features which had already been surmised from sectioned matenal. 'The isolated cell has the shape of a double spindle with the nucleus

FIG. 9. Longitudinal section of 14-day embryonic chicken gizzard. Thick filaments (large arrows), thin filaments (small arrows), and cytoplasmic dense bodies (c) are seen axially. Microtubules (mt) are seen peripherally. X30,OOO. Micrograph by C. Bailey.

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FIG. 10. Contraction of an isolated B . murinus stomach muscle cell, as shown in a movie taken with phase-contrast optics. Rior to stimulation (a) the cell membrane is smooth. At 0.63 second after stimulus (b), there is a slight shortening and a detectable undulation. At 0.75, 0.87, and 1 .OOsecond after stimulus (c, d, and e, respectively), the undulation becomes more pronounced until the end of the cell appears like a corkscrew. Calibration bar represents 5 pm. X2200. From Fisher and Bagby ( 1977). FIG. 11, A broken cell from freshly isolated cells of chicken gizzard stained with fluorescent antimyosin. Faint striations may be seen in the myofibrils protruding from the broken end of the cell (left side of the micrograph). Calibration bar represents 5 pm. X2600. From Bagby and Pepe (1978).

occupying a medial, central location and organelles, including most of the mitochondria, forming cones on either end of the nucleus, resulting in a central area devoid of contractile material for some distance. Contractile material appears as dense, mostly longitudinally oriented material which is sometimes seen as separate fibrils (Small, 1974, 1977a,b). When movies are made of shortening cells viewed with phase contrast (Fisher and Bagby, 1977), the ends of the cell are the first places to show evidence of contraction. Within 0.75 second, the formerly straight cell end begins to show a helical or corkscrew appearance which be-

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comes tighter as the cell contracts (Fig. 10). In some, but not all, cells this helical appearance is accompanied by a rotation of the end of the cell. Polarizing optics revealed the contractile material with much greater clarity and definite fibrils were first described by Small (1974). Their 3-D arrangement in living cells was later shown by Bagby and Abercrombie (1979) and is shown in Fig. 23, Section 111. Small recognized that the fibrils were oriented slightly obliquely to the cell axis even in relaxed cells and showed that the fibrils reoriented during shortening to a greater angle (Small, 1974). Although Fisher did not see individual fibrils in isolated B . marinus stomach cells, he made movies using polarizing optics and a ‘‘sensitive tint” filter which translated angle changes into color changes (Fisher and Bagby, 1974) to show that the decrease in birefringence during shortening was largely due to a change in the angle of birefringent entities (Fisher, 1974; Fisher and Bagby, 1977). Both he (Fisher, 1974) and Small (1977a) devised cell models based on the helical appearance of the cells as the contractile filaments changed their orientation during shortening. At the time, a reasonable explanation for the cell’s change in appearance was a model in which contractile units were attached to opposite sides of the cell with attachment sites forming a helix on the plasmalemma. Such an arrangement would produce the change in angle of contractile material seen during shortening and would also give the helical appearance seen with both phase-contrast and polarizing optics. During the same time period, Fay and Delise (1973) proposed a more conservative model where the fibrils attached at both ends to membrane attachment sites, but in some cases both sites were on the same side of the cell; in other cases the fibril crossed to the opposite side. (For a more detailed critique of these models, see Bagby, 1983.)

2 . Immunocytochemistry a. Thick Filament Proteins. While studies with isolated living cells were used to show that fibrillar structures were present, it was not known whether the birefringent fibrils were composed of contractile or cytoskeletal proteins. Studies with cultured smooth muscle cells have shown that fluorescent anti-myosin staining led to “interrupted” staining patterns along fibrils (Groschel-Stewartet al., 1975a, 1975b; Chamley et al., 1977a; Chamley-Campbell et al., 1979). However, the staining offreshly isolated cells led to stained fibrils throughout the entire cell interior, except for the nucleus and other organelles, and interrupted patterns were sometimes recognized in areas where tom cells exposed individual myofibrils (see Fig. 11) near the periphery of cells and in bundles of myofibrils released from cells (Bagby and Pepe, 1978). This finding confirmed the fibrils seen in living cells as entities which contained myosin and justified their being termed “myofibrils.” Anti-M-protein staining also led to interrupted fibrils in cultured smooth muscle cells (Schollmeyer et al., 1976), but the identity of these structures in relation to those seen in the in situ cells is by no means certain. In

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view of the fact that interrupted or “striated” myofibrils are not seen under all conditions, it is still controversial whether the myofibrils have sarcomeric units. b. Thin Filament Proteins. There have been no reports of freshly isolated smooth muscle cells stained with antiactin. Cultured smooth muscle cells have been stained with antiactin by several investigators (Chamley et al., 1977a,b: Chamley-Campbell et al., 1979; Franke et al., 1980; Groschel-Stewart, 1980). Actin-stained fibrils occur throughout the cell, but none has shown the interrupted pattern seen with antibodies to other contractile proteins (Chamley et al., 1977a,b; Chamley-Campbell et al., 1979; Groschel-Stewart, 1980). This lack of an interrupted pattern cannot be ascribed to superimposition, etc., since cultured cells provide the clearest visibility of fibrils due to their thin, flattened shape. The actin-staining pattern is not consistent with the presence of a sarcomere organization, unless thin filaments are extremely long and/or constantly overlap each other. Freshly isolated cells have not been stained with antitropomyosin, but cultured cells show staining of fibrils with a weakly interrupted pattern (Chamley-Campbell er al., 1977). In view of the noninterrupted staining with antiactin, it is surprising to see any indications of striations with antitropomyosin. Small has proposed that thin filaments may not be equivalent throughout the cell (Small e f al., 1986). He divides the cytoplasm into two domains, the conventional actomyosin domain and the IF-actin-filamin domains in which thin filaments may contain filamin. Filamin is an actin binding protein with MW of 500,000 (Wang, 1977). Antifilamin has been shown to be colocalized with IF domains (see Fig. 2) by electron microscopic immunocytochemistry. Immunofluorescently stained longitudinal sections (Fig. 12) reveal filamin to be localized in fibrillar material in the cytoplasm and as longitudinal “ribs” just beneath the sarcolemma (Small et ul., 1985). Small has proposed that the filamin may be involved in linking contractile and cytoskeletal domains or may even play a role in maintaining stress with low expenditure of energy, i.e., an alternative to the “latch-bridge’’ hypothesis (Dillon et al., 1981). The presence of large numbers of thin filaments which do not interact with myosin would help explain the lack of expected change in thin-to-thick filament ratios with length changes (see Section II,A,3) and would support the organizational model proposed by Cooke (1983). c. Intermediate Filament Proteins. Campbell et al. (1979) are the only ones to have stained freshly isolated cells with antibodies to intermediate filament proteins although Small et al. (1986) have stained it in fixed-tissue sections. The antiskeletin (desmin) staining revealed a dense network of mostly longitudinal fibers throughout most of the cell (see Fig. 12) with a concentration near the nucleus. Studies of vimentin localization have been done only with cultured cells, but the same pattern seems to be present (Franke et al., 1980). d. Dense Body Proteins. Two proteins, a-actinin and vinculin, have defi-

FIG. 12. Ultrathin sections of polyvinyl alcohol-embedded smooth muscle stained with single antibodies or combinations as indicated. The longitudinal sections (a-f) are from chicken gizzard and the transverse sections 6-1) are from guinea pig taenia coli. A homogeneous label is obtained with actin, but the other antibodies used singly show fibrillar structure. The filamin + desmin combination (e, k) looks little different from filamin alone (d, j) or desmin alone (c, i), but the filamin + myosin combination (f, 1) gives a continuously stained cell unlike either myosin alone (b, h) or filamin alone (d, j). Thus, filamin and desmin appear to colocalize while filamin and myosin appear to occupy different domains. X990. From Small et al. (1986), by copyright permission of the Rockefeller University Press.

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FIG. 13. An isolated chicken gizzard smooth muscle cell double stained with rhodarnine-labeled antimyosin (a) and fluorescein-labeled anti-a-actinin (b). Note the undulating niyofibrils revealed by antimyosin. Some of the anti-a-actinin stained areas exclude myosin, leading to voids in the antimyosin pattern (arrowheads). Calibration bar represents 5 pm. X 1600. From Bagby (1980).

nitely been localized in CDBs and/or MADBs (Schollmeyer el af., 1976; Geiger et al., 1980). When structures stained by anti-a-actinin or antivinculin are seen with immunofluorescence, it is therefore assumed that these structures are dense bodies of some sort. The first study utilizing anti-a-actinin staining of freshly isolated cells was done by Bagby (1 980) in combination with antimyosin staining. Most anti-a-actinin staining in chicken gizzard cells was in the form of long strips located near the cell surface, whereas myosin stained fibrillar components throughout the cell (Fig. 13). Fibrillar material was sometimes faintly stained by anti-a-actinin in a vague interrupted pattern, but it could not be determined whether the a-actinin and myosin were localized within the same fibrils. Fay et ai. (1983) used anti-a-actinin to stain freshly isolated cells from the B . murinus stomach. A pattern of ovoid fluorescent bodies throughout the cytoplasm was seen, with occasional areas where the fluorescent bodies throughout the cytoplasm was seen, with occasional areas where the fluorescent bodies seemed to form a “string” (Fig. 14). Some of these strings could be traced to the plasmalemma where larger, brighter patches of anti-a-actinin staining indicated attachment areas for the strings. Although the 2.2 Fm spacing of bodies in strings is suggestive of Z-line spacing in sarcomeres and this spacing decreased to 1.4 Fm in contracted cells, it is not known whether these strings also contained myosin, since counterstaining with antimyosin was not utilized. One of the biggest surprises has been the antivinculin staining seen in isolated guinea pig vas deferens cells by Small (1985). The vinculin forms parallel longitudinal “ribs” along the plasmalemma, with many ribs appearing to go the length of the cell (Fig. 15). a-Actinin was also found in parallel ribs near the cell surface. The ribs remain parallel to the cell axis even in shortened cells. This

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FIG. 14. Photomicrographs of cells isolated from B. marinus circular muscularis and stained with fluorescent anti-a-actinin. Fusiform-stained bodies occupy the cytoplasm and larger, more irregularly stained elements are found on the cell margin. Several of the fusiform elements appear to be part of a stringlike array (triangles) characterized by a relatively regular spacing between elements. Some of these strings appear to terminate on the larger plaques (large arrows) along the cell margins. Calibration bar represents 10 pm. x 1479. From Fay et al. (1983), by copyright permission of the Rockefeller University Press.

behavior makes models depending upon helically arranged attachment sites (see Bagby, 1983) invalid since vinculin and a-actinin are components of the attachment sites. Cross-sectioned, stained tissue shows that vinculin is confined to plasmalemmal sites (presumably MADBs), while a-actinin is found both in the plasmalemma and in the cytoplasma, confirming that MADBs and CDBs are not biochemically equivalent. e. Microtubules. Microtubules have been reported at times in smooth muscles (Sachs and Daems, 1966; Ashton et al., 1975; Cooke, 1976; Makita and Kiwaki, 1978; Moriya and Miyazaki, 1979; Oki et al., 1980; Somlyo, 1980; Cameron et al., 1982), but their particular function in these cells is not known.

FIG. 15. Isolated guinea pig taenia coli cells stained with fluorescent monoclonal antivinculin. Note the longitudinal course of vinculin-stained structures in an extended cell (a) and in shortened cells (b, c). X7.59. From Small (1985).

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FIG. 16. Cells isolated from B . murinus stomach muscularis stained with fluorescent antitubulin. Tubulin seems to be located in elements which are longitudinal in extended cells (a) but follow an undulating course in contracted cells (b). Nucleus (N). Calibration par represents 10 pm. X9.50. From Fay et ul. (1983), by copyright permission of the Rockefeller University Press.

Microtubules are very abundant in embryonic smooth muscles, where they generally parallel the course of myofilaments (Bailey et al., 1984). Fay el ul. (1983) stained isolated cells with fluorescent antibodies to tubulin, the protein of microtubules. They found a fibrillar pattern of staining with fibrils parallel to the longitudinal axis in relaxed cells and fibrils in coils and undulations in shortened cells (Fig. 16). f. Fibrils. Although ultrathin sections viewed with TEM give little or no indication of whether the contractile material was organized into fibrils, light microscopy of isolated cells, both living and immunofluorescently stained, gives ample evidence for fibrils containing myosin (Bagby and Pepe, 1978; Bagby and Abercrombie, 1979; Bagby, 1980), actin (Small, 1985), tropomyosin (ChamleyCampbell et al., 1977), and/or a-actinin (Bagby, 1980; Fay et ul., 1983). However, it is by no means certain that all these proteins are found within the same fibrils since appropriate evidence from double-stained preparations is lacking. “Myofibril” has been used to describe fibrils containing myosin (Bagby and Pepe, 1978; Bagby, 1980, 1983) and this term is entirely appropriate regardless of the organization within the myofibrils. Although it is assured that rnyofibrils also contain actin due to the ubiquitous presence of antiactin staining in the cytoplasm (Chamley et al., 1977a,b; Chamley-Campbell et al., 1979; Groschel-Stewart, 1980; Small et al., 1985), it is not certain that all of the actin is found in myofibrils. Indeed, the evidence from thin-to-thick filament ratios (see Section II,A,3) indicates a greater number of thin filaments than necessary for interaction with thick filaments, suggesting that some actin might be cytoskeletal. What is the function of the cytoplasmic a-actinin, myofibrillar or cytoskeletal? The specific attachment of thin filaments to dense bodies argues for a myofibrillar role for some cytoplasmic dense bodies (Bond and Somlyo, 1982;

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Tsukita et al., 1983) while the evidence of others (Cooke and Fay, 1972; Cooke, 1976; Bagby, 1983; Bagby and Corey-Kreyling, 1984) argues that some cytoplasmic dense bodies have a cytoskeletal role. Recent experiments by Small et al. (1986) may serve to resolve the issue. He found that although actin was found in both the myofibrillar and cytoskeletal domains, a-actinin was found only in the domain containing actin, IF, and filamin. This type of distribution, with all cytoplasmic a-actinin being found in cytoskeletal domains, would tend to preclude the usual sarcomere arrangement in the myofibrils, but it might well give rise to a periodicity reminiscent of Z-lines in the cytoskeletal fibrils. These findings by Small are bound to raise more questions than they answer since they are in conflict with most current thinking about the organization of contractile machinery, but it is also clear that they cannot be ignored. The localizations need to be confirmed by others; if confirmed, then new models are in order.

111. Three-Dimensional Information A. THENEED FOR THREE-DIMENSIONAL INFORMATION Three-dimensional techniques are required for many measurements. Contractile components are often not parallel to the cell axis, making it difficult to cut sections in a suitable plane. Errors result from judgments about whether a filament has terminated or merely passed out of the plane of the section, and some components are too large to be contained within one thin section. The use of thicker sections overcomes these problems, but superimposition of similar components can cause confusion. The solution is to use stereo-tilt to take micrographs of thick sections from more than one angle so that continuity can be distinguished from contiguity by viewing stereo pairs (Ashton et al., 1975; Bond and Somlyo, 1982). The stereo views of thick sections also allow one to correct for errors of measurement that arise because the object is not parallel to the plane of the section. Another useful solution for measuring objects too large or long to be contained in one section is reconstruction from serial sections. The preceding section provided a great deal of data on the structures of individual components, but the relationships among components and how they are organized in the whole cell are a matter of speculation. In fact, several speculative models have been proposed, based largely on 2-D information (see Bagby, 1983, for a critique of speculative models). Some of these models have been shown to be invalid by the new data presented in this review, and, undoubtedly, new speculative models will spring up to take their place. However, 3-D techniques provide an additional tool for the understanding of structural organization: the reconstructed model. The reconstructed model attempts to duplicate the exact organization found in a particular cell and therefore requires exact

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information on the location of a component in three planes. For most 3-D reconstructions, serial sections are required, either serial sections cut by a microtome or serial optical sections. Serial sections may be used to make concrete models or tracings may be made of serial sections and used in a variety of ways. Perhaps the most sophisticated reconstruction process is computer-aided reconstruction in which the models are reconstructed on video monitors for study from any perspective. A very recent innovation is the use of holography to reconstruct a 3-D image from serial sections. The main use of all these reconstructions is to study spatial relationships between components in a way unmatched by other techniques. The reconstructed model will not replace the speculative model, but it will serve as a means of making the authors of speculative models more accountable. Whole isolated cells are ideal for 3-D techniques with the light microscope. The limited depth of focus of the light microscope allows one to make serial optical sections from different focal planes. These are ideal for reconstructing whole cells. Also, the angle of viewing can be changed with a technique developed by Osborn et al. (1978) so that stereo viewing may be used to reproduce the cell in 3-D. The recent proliferation of intermediate high-voltage electron microscopes has made viewing whole critical-point dried smooth muscle cells feasible. The depth of focus of electron optics is too great to allow one to make serial electron-optical sections, but stereo-tilt micrographs allow one to view the whole cell as a 3-D object. Three-dimensional information thus greatly enhances our ability to make accurate measurements and allows us to see spatial relationships more clearly. The most sophisticated product of 3-D information is the 3-D reconstruction. The remainder of this section deals with how 3-D information is obtained. The analysis and reconstruction of these data are discussed in the last section.

B . TRANSMISSION ELECTRON MICROSCOPY OF SERIAL SECTIONS The earliest types of 3-D smooth muscle studies used serial sections (Ashton et af., 1975; Cooke, 1976). They were used by Somlyo’s group to follow thin filaments into cytoplasmic dense bodies (Ashton et af., 1975; Somlyo et al., 1977; Somlyo, 1980) and to confirm the length of thick filaments (Ashton et al., 1975). Cooke (1976) used serial sections to show that thin filaments and intermediate filaments did not go in the same directions and later to show the branching of thin filament bundles (Cooke, 1982, 1983). The technique is indispensible for tracing the pathways of structures at high resolution, but it is very exacting and there are situations in which it is inappropriate. Figure 17 shQws6 transverse serial-thin sections of a muscle cell in B . marinus stomach muscularis out of a series of 60 80-nm-thick sections. The sections were originally made for the purpose of measuring thin filament lengths. The large number of sections was

FIG. 17. Six adjacent serial transverse sections (a-f, about 80 nm thick) from a series of 60 sections of the terminal region of a B . marinus stomach muscle cell stretched to 160%Lo to align filaments. Thick filament lengths may be easily estimated from such a series. X71,610. Micrographs by M. Kreyling.

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needed in case their length was much longer than the 2.2 pm length ascribed to thick filaments in smooth muscles (Ashton et al., 1975), but, in fact, thin filament length could be as much as 11 pm according to one of the alternative models proposed by Murphy (1979), in which case the 6 pm series would be insufficient to follow individual filaments from one end to the other. However, it is exceptionally difficult to obtain enough 80-nm-thick sections to span 11 pm. Thicker sections mean fewer sections but then another problem arises; since the thin filaments are so closely packed, it would be easy to mismatch filaments in successive sections. The thicker the section, the greater the chance of mismatch. Another problem is the accuracy of the z-axis measurement since it is based on section thickness. Unless each section thickness is measured individually, estimates of the length of filaments based on the number of sections in which a filament occurs are subject to considerable error. This is why Ashton et al. (1975) used longitudinal sections for measuring thick filament lengths (2.2 0.14 pm) and merely used serial sections for confirmation. Therefore, the series represented by Fig. 17 is probably unsuitable for measuring thin filament length, but it may be used for tracing bundles of thin filaments or dense bodies or for estimating thick filament lengths. Thick filament length measurements from this series had a mean of 1.2 p m with a range of 0.6-2.1 pm. Serial action data may also be used to make 3-D reconstructions, as discussed in Section IV.

*

C. TEM STEREO-TILT VIEWSOF THICKSECTIONS AND WHOLECELLS Thick sections present a distinct advantage in that individual filaments may more easily be followed for their entire length, even if the sectioning plane is not perfectly longitudinal. However, thick sections present their own problems, penetration and superimposition. The penetration problem is overcome by using a higher accelerating voltage, by using a more intense source of electrons such as the LaB,, gun, or both. Most investigators prefer to use the higher accelerating voltage because the specimen interacts less with higher energy electrons and specimen heating is less of a problem, even though specimen contrast is considerably less with higher accelerating voltages. The superimposition problem is overcome by using a tilt stage to make micrographs at different angles to the beam. When two such tilted images are viewed with a stereoscope, the 3-D image allows one to distinguish whether contiguous structures are superimposed. Ashton et al. ( I 975) used this approach to follow individual thick filaments and determine their length. Unfortunately, thin filaments are so closely packed that even stereo viewing does not allow one to follow individual filaments and their length has not yet been determined. The stereo-tilt technique has been used more recently to show that thin filaments run into dense bodies rather than coursing above or below them (Bond and Somlyo, 1982). Figure 18 shows stereo pairs from a longitudinal section of smooth muscle which has been swollen by saponin

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FIG. 18. Stereo-tilt view of a longitudinally sectioned CDB and its attached thin filaments. Rabbit PAMV cells were swollen by saponin treatment and labeled with S1. S1-decorated thin filaments can be seen inserting into the CDB, and the arrowhead structure formed by SI (white arrowheads) is of opposite polarity on thin filaments attached to opposite sides of the CDB. This is entirely consistent with a Z-line function for CDBs. X80,OOO. From Bond and Somlyo (1982), by copyright permission of the Rockefeller University Press.

treatment and labeled with S1 to decorate the thin filamenu. It can be seen that thin filaments have opposite polarity at either end of the CDBs and that they insert into the CDBs. We have recently had success viewing whole, Triton-X-extracted, demembranated cells from the chicken gizzard with 200 kV. Cells with intact membranes did not reveal intracellular details, but demembranated cells clearly show the presence of filament bundles (fibrils) which are roughly parallel to the cell axis. These same cells, stained with antimyosin and viewed with a fluorescence microscope, revealed fibrils of the same dimensions as those subsequently seen with TEM (Frierson and Bagby, 1985). Therefore, at least some of the fibrils seen

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FIG. 20. Scanning electron micrograph of a portion.of an isolated guinea pig taenia coli smooth muscle cell. Although one can only see the surface of the cell, there are undulating ridges which probably represent the course of fibrillar structures beneath the plasmalemma. Calibration bar represents 5 Fm. X3060. From Obara (1984).

with TEM are myofibrils. The TEM also revealed that the sometimes helical appearance seen in isolated cells with the light microscope (Small, 1974, 1977a,b; Fisher and Bagby, 1977; Small and Sobieszek, 1980) was due to the curved paths of filaments. Some cells were oriented so that their axis coincided with the axis of the tilting stage and a series of micrographs were made as the cells were rotated on their axes. Figure 19 shows a portion of a cell rotated from -20 to +20". From such rotation series, the paths of individual fibrils may be traced. Their paths are rarely in one plane and some fibrils appear to terminate in the cytoplasm. CDBs are not seen with this technique, so it is not clear whether they are within the fibrils, nor can it be determined whether fibrils terminate on CDBs. We feel that this technique, in combination with immunocytochemical labeling, will be a powerful means of determining 3-D relationships among ultrastructural entities. D. SCANNING ELECTRON MICROSCOPY Because scanning electron microscopy (SEM) uses solid objects and the coating process leads to shadows that give depth cues, SEM gives 3-D information whether stereo pairs are used or not. Some studies have looked at the exterior of isolated cells using SEM (Fay and Delise, 1973; Fay et al., 1983; Obara, 1984). For the most part, since only the surface features are visible, this technique does not reveal much about the contractile system. However, in some studies, such as in Fig. 20 which is taken from work by Obara (1984), the shape of the underlying ~

~~

FIG. 19. Stereo-rotation of an isolated chicken gizzard smooth muscle cell demembranated by Triton X and critical point dried (micrograph by K. Frierson and J. Dunlap). The cell was rotated on its axis at lo" intervals with a-e representing -20, -10, 0, +lo, and +20" tilt, respectively. Adjacent micrographs are stereo pairs. Most filaments are found within fibrils, at least some of which contain myosin (Frierson and Bagby, 1985). Some fibrils are straight but many undulate and are nonplanar. The ovoid bodies are mitochondria. Calibration bar represents 1.0 pm. X 10,OOO.

FIG.21. Field-emission scanning electron micrographs of a guinea pig taenia coli smooth muscle cell tom after critical-point drying to reveal the cell interior (from Sawada, 1981, with permission). (a) Inner surface of the plasmalemma. Fibrils run between rows of caveolae. (b) Smooth muscle cell interior. The filaments form bundles in a network. Calibration bar represents I pm. X29.000. From Sawada (1981).

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contractile material may be seen by the shape of the plasmalemma. This cell is interesting since it shows that the contractile material follows curved paths, even in a relaxed cell. Sawada (1981a,b) has introduced some innovative SEM techniques for looking at muscles. He has used a field-emission source of electrons which gives much higher resolution than the tungsten filament source used in most SEM work. Also, he has used mechanical tearing of specimens to reveal the cell interiors. This combination of techniques allows one to see the organization of thin filaments within a smooth muscle cell. Figure 21a shows the interior of the cell just below the plasmalemma. Rows of caveolae and bundles of thin filaments between the caveolar areas may be seen. Figure 21b shows a deeper portion of the cell interior. Bundles of thin filaments anastomose and split to form a network. Individual bundles can be followed for only very short distances. This structure is very similar to the model depicted by Cooke (1983).

E. ROTARY-SHADOWED REPLICAS OF FREEZE-FRACTURED, DEEP-ETCHED TISSUES This technique has only recently been applied to the study of the smooth muscle contractile system (Sawada, 1981b; Somlyo and Franzini-Armstrong, 1985). Specimens are fixed, frozen, fractured, and then deep-etched under vacuum. While frozen, the tissue is rotary shadowed with platinum and replicated with carbon. When the replicas are viewed, there is a sense of depth in the specimen which is present even without stereo viewing with stereo pairs. The depth perceived rivals that seen with SEM, but the resolution is far better. For 3D imaging at high resolution, this technique is probably the best of all. Somlyo and Franzini-Armstrong (1985) have used the technique to show the attachment of S 1-labeled thin filaments to MADBs and CDBs and the looping attachment of IF to the exterior surface of CDBs. Perhaps, the most potentially useful application of the technique was an attempt to use S1-labeled rabbit was deferens in rigor to determine the polarity of bridges on thick filaments, the rationale being that S l-labeled thin filaments would reveal their polarity and thus the polarity of bridges linking them to thick filaments. Figure 22 shows this attempt. Unfortunately, the fracture does not always follow the same filaments for long distances. Although the polarity of bridges is clear, not enough of the filament was revealed to determine whether thick filaments are biopolar. F. SERIALOPTICALSECTIONS OF ISOLATED CELLS

No 3-D technique has been as useful as serial optical sections of isolated cells. The technique, in a rather qualitative form, was introduced by Bagby and Abercrombie (1979) with living B . marinus stomach cells. A movie camera was used

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FIG. 22. Stereo pair ('7" tilt) of a portion of rabbit vas deferens smooth muscle which was freeze-fractured and deep-etched in the rigor state with S1 labeling of thin filaments. Since the polarity of thin filaments is revealed by SI labeling, the polarity of cross bridges (arrows) on an interacting thick filament (arrowhead) may also be determined. A dense body (db) with accompanyin@ intermediate filament (white arrow) is also seen. X147,900. From Somlyo and FranziniArmstrong (1985).

to photograph a cell viewed with polarizing optics while the microscope was used to focus at different levels in the cell. Figure 23 shows that the orientation of fibrils changes at different focal levels within the cell and that fibrils appear to travel in helical paths around the cell axis. As interesting as this observation is, a more quantitative approach is needed for 3-D reconstruction. Also, one would like to use a more specific technique than birefringence to view the cell. Recent developments have allowed very specific, quantitative techniques to be applied to the smooth muscle cell. z-Axis resolution comparable to that possible in the x and y axes has been achieved by at least two means. Fay's group has used an eddy current sensor with

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a z-axis resolution of 0.025 pm (Fay et al., 1983, 1985). We have used a sensor which matches diffraction gratings with a z-axis accuracy of 0.1 pm. Since outof-focus information from the material above and below the focal plane limits the z-axis resolution, a z-axis resolution of objective-to-object of 0.25 pm is about the best that can actually be achieved. Immunofluorescencegives highly specific information about the localization of protein components, but rapid photobleaching used to make it almost impossible to make time exposures of several focal planes within the same cell. These problems have been largely overcome in our laboratory with the use of p-phenylenediamine to reduce bleaching (Platt and Michael, 1983) and by push-processing to reduce exposure times. Fay’s group has further reduced bleaching by using a video-intensification microscope and video recording (Fay et al., 1983, 1985). Image processing is used to improve zaxis resolution (Fay et al., 1985). Figure 24 shows serial optical sections of a B . marinus stomach cell stained

FIG. 23. Serial sections series of a portion of an isolated living smooth muscle cell from the B. marinus stomach viewed with polarizing optics. In (a) a lone fibril on the surface runs from 10 to 4 o’clock. In subsequent micrographs (b, c, d, e) other fibrils tend to run from 8 to 2 o’clock. This pattern is consistent with fibrils which run helically around the cell axis. Calibration bar represents 5 pm. X 1300. From Bagby and Abercrombie (1979).

I08

FIG. 25. Serial optical sections of the end of an isolated chicken gizzard smooth muscle cell stained with fluorescent anti-a-actinin. The subsequent micrographs show sections separated in the z axis by about 0.2 pm. Calibration bar represents 5 pm.

FIG. 24. Serial optical sections of an isolated smooth muscle cell from B. marinus stomach muscularis stained with fluorescent anti-a-actinin. The distance between focal planes is about 0.5 pm. The area marked off in (a) is shown at higher magnification in (b). In (c) are tracings of the respective micrographs seen in (b). Circled elements in (c) show elements which occurred in more than one plane. Nucleus, N. The calibration bar represents 10 pm. From Fay et al. (1983) by copyright permission of the Rockefeller University Press. 109

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FIG.26. Stereo pairs of two B . marinus stomach smooth muscle cells stained with fluorescent anti-a-actinin. Note the stringlike arrays of stained fusiform elements in the cytoplasm. In several regions, elements in adjacent strings are in lateral register. The calibration bar represents 10 pm. From Fay rr a!. (1983). by copyright permission of the Rockefeller University Press.

with anti-a-actinin and the digitized images of separate planes of focus. The digitized image planes are then used to reconstruct a 3-D cell model (see Section IV). Note the rather uniform size of stained structures and the uniform distribution throughout the cytoplasm, with a dark void showing the position of the unstained nucleus. Figure 25 shows serial optical sections of a chick gizzard cell stained with

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anti-a-actinin. The appearance is similar to that seen in the B . murinus stomach cell. The original images, without image analysis, are then used to make a holographic 3-D reconstruction (see Section IV).

G. STEREOPAIRSOF IMMUNOFLUORESCENTLY STAINED CELLS Osborn et al. (1978) developed a technique which results in two different views of a cell without tilting the cell or the microscope. The technique consists of using a slider at the back of the objective which covers half the objective field at a time. By covering first the right and then the left halves of the field, one obtains views of the object from a slightly different angle so that photographs of the two views seen with a stereoptical device gives one a 3-D image of the object. This technique has recently been used by Fay et al. (1983) to show the relationships among anti-a-actinin-stained components (Fig. 26). We have found that by using a slider to cover alternate halves of the field in the filter holder in the body of our Nikon microscope we can accomplish the same effect. Figure 27 shows stereo pairs of a chick gizzard cell stained with antimyosin to show the paths of myofibrils. Note the hollow appearance of the cell near the nuclear poles where myofibrillar material is absent and the more undulating paths of myofibrils near the cell ends.

IV. Three-Dimensional Analysis and Reconstruction Three-dimensional information is either in the form of different points of view of the same object or as serial sections of the object. Quite different analysis and reconstruction techniques are used for the two types of 3-D data. Some of the techniques for analysis and reconstruction which are presently being used are presented in this section, along with the new information gained thereby.

A. STEREOPAIRS Stereo pairs are formed when micrographs are taken from two viewing angles. When the two views are seen with separate eyes, the brain uses the two views to reconstruct a 3-D object. Thus, stereo pairs are unique in allowing reconstruction directly from the micrographs without the necessity for an analysis step. The disadvantage of this method is that it is purely qualitative. However, stereo pairs may also be analyzed to obtain quantitative information. There are devices which project a pinpoint of light that one can move by micrometers until the point matches the location of a point seen with stereo viewing. One then reads the coordinates of the point from the micrometers used to move the light. In this way, the x, y, and z coordinates of particular points and their spatial relationships to other points may be determined. The analysis may be taken a step further by a

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method of Olins et al. (1983), which has not yet been applied to smooth muscle, but is worthy of note. Sections of 0.2 pm were coated on both sides with a suspension of colloidal gold particles which acted as fiduciary marks. A series of micrographs taken at 6” intervals over a 120” rotation of the specimen were analyzed by a computer program, which first determined the common rotation axis and then calculated x, y , and z coordinates. These coordinates were then used to generate a 3-D model which could be “sectioned” in any plane ( O h et al., 1983). This same group has used another innovative technique to increase one’s appreciation of stereo pairs. Using the 120” tilt series, adjacent micrographs (stereo pairs) were rephotographed with red and green filters to make single frames on color movie film. When the resulting movie is projected and viewed with glasses having one green and one red lens, one sees the rotation of the object in 3-D (D. Olins, personal communication)! This technique would greatly enhance the stereo-tilt series of isolated cells viewed with TEM. The stereo micrographs from light microscopes using the Osborn et al. (1978) technique have some disadvantages to stereo pairs obtained from TEM or SEM. In micrographs obtained by tilting the specimen, one knows the exact angle of tilt and can use stereo analysis for quantitation, whereas with the light microscope one would have to use some independent means to determine the angle difference between the two micrographs before quantitative analysis could be used. Also, since only two viewing angles are possible, it is not possible to do the type of analysis used for a rotation-tilt series. Nevertheless, their technique is quite useful for qualitative purposes.

B. SERIALSECTIONS Reconstruction of some type is a sine qua non before serial sections reveal the 3-D character of the original object. Separate image planes and the 3-D information they contain are not easily appreciated by the brain until the separate planes are rejoined to form a solid object. A possible exception is the manufacture of a movie in which each section becomes a separate frame and the viewer gets a 3-D illusion as he “walks through” the object (Macagno et al., 1979). Reconstruction by serial sections provides the most accurate representation of the real object, and because all points may be assigned x , y , and z coordinates it is more easily used for quantitative analysis than stereo pairs. Reconstruction techniques are quite diverse. They are described in chronological order of use with smooth muscle tissue.

FIG. 27. Stereo pairs of two chicken gizzard smooth muscle cells stained with fluorescent antimyosin. (a) Near the nucleus the myofibrils are relatively straight but near the cell ends (b) myofibrils are often curved. Calibration bar represents 5 pm. X 1600.

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FIG. 28. Three-dimensional “concrete models” of the concourse of lattice-like bundles of thin filaments from a series of consecutive thin sections illustrating the axial disorientation, convolution, and branching with 2 pm-long segments. The models suggest that the bundles interconnect through convergent and divergent segments to form a spatially complex network that is comparable to the pattern of stress fibers in nonmuscle cells. From Cooke (1982).

1. Concrete Models

In this method, a “concrete” or real model is constructed by tracing around the object of interest (as seen in individual planes) onto uniform-thickness material, cutting around the tracings, and then “stacking” the separate tracings to form the object. The object may then be turned in space to appreciate its 3-D shape. By making tracings on transparent material and stacking the tracings, one can determine the relationships among several 3-D objects. This technique was the only method available in the not-too-distant past and it has been used recently by Cooke (1982) to show the branching that occurs in thin filament bundles (Fig. 28). 2 . Photographic Reconstruction

In this technique one begins by tracing the subject, as seen in different sections, onto transparent sheets. The transparent sheets are then stacked in exact registration and the composite is photographed to make one photograph of a stereo pair. A second photograph is made after shifting each plane 0.5 mm with respect to the preceding plane and rephotographing the composite. The two photographs, when viewed stereoscopically, give a 3-D image faithfully reproducing the 3-D relationships between different components. This technique was developed and used by Bond and Somlyo (1982) to show the shape of cytoplasmic dense bodies and their relationship to surface dense bodies (Fig. 29). They found that cytoplasmic dense bodies were elongated and oblique to the cell

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axis. In some regions they saw chains of dense bodies which terminated at the cell membrane. Reconstruction of relatively large objects does not require as precise an alignment of successive sections as does the reconstruction of very small objects. Registration becomes a major problem in reconstructing filament paths unless a reliable fiduciary mark can be found. Cooke (1976) used microtubules traveling the same direction as filaments as fiduciary marks for tracing the paths of thin and intermediate filaments. 3. Computer-Aided Reconstruction Several programs for computer-aided reconstruction have been used for serial electron micrographs of other tissues (Macagno et al., 1979; Marino et al., 1980; Moens and Moens, 1981; Prothero and Prothero, 1982; Gras and Killman, 1983; Nierzwicki-Bauer et al., 1983), but none of them has been used for reconstruc-

FIG. 29. Photographic 3-D reconstruction of dense bodies from 13 serial 80-nm longitudinal sections of rabbit portal-anterior mesenteric vein. A stereo pair was made from transparent tracings of sections as follows: the transparencies were exactly superimposed and photographed. A second photograph of the transparencies was made with each tracing shifted 0.5 mm with respect to the tracing below it. The two photographs make a stereo pair which show the 3-D relationships between dense bodies. Arrows point to dense bodies which can be followed through several serial sections. Chains of CDBs often converge on MADBs (arrowheads). From Bond and Somlyo (1982), by copyright permission of the Rockefeller University Press.

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tion of smooth muscle tissue. Thus far, Fay’s group is the only one which has used computer-aided reconstruction with smooth muscle tissue, and they have utilized serial optical sections of isolated cells (Fay et al., 1983). There are several reasons why serial optical sections of isolated cells have been singled out for this technique: 1. There is no alignment problem for registration of successive sections. All have the same optical axis. 2. There is no distortion of dimensions such as that seen with serial plastic sections. 3. Section thickness can be measured with greater ease than with serial plastic sections. 4. Presently, it is of greater interest to determine the gross organization of components in the whole cell than to determine the 3-D relationships between filaments in a small portion of the cell. There is one major problem, however. Out-of-focus material may contribute to several image planes, leading to a point-spread function as much as 2 pm above and below the object (Fay et al., 1983). Initially, this problem was dealt with by assigning the object to the median plane in a series of planes where the object seemed to occur. More recently, a computer-driven iterative restoration technique was used, which, after 30 “guesses,” reduced the point-spread function to more acceptable limits (Fay et al., 1985). Thus far, only fluorescent anti-a-actinin-stained cells have been used for computer-aided reconstruction. This is not because it is any harder to obtain serial optical sections of cells stained with other fluorescent antibodies, but because the analysis of the position of discrete objects is easier than positional analysis of more continuously stained objects. Anti-a-actinin staining also results in a relatively uniform size of the stained objects. Therefore, one needs to determine only the body center of each stained object and assign a structure to that position. On the other hand, following a continuously staining fibril (e.g., as seen with antimyosin staining) requires one to assign coordinates to several closely spaced regions along the fibril to define its path. Fay’s group has gone farther than just assigning coordinates to body centers; they have developed a program for assigning vectors to each ovoid a-actinin-stained entity (Fay el al., 1983, 1985; Coggins, 1983). This was done by passing the objects through conical computer-generated filters which determine the orientation of individual bodies. The 3-D vectors may then be used to determine whether neighboring aactinin bodies are oriented in a sufficiently similar manner to be considered part of a “string” of a-actinin-stained bodies. In this way, strings of a-actinin bodies from different section planes were recognized within cells (Fay et al., 1983, 1985). Once the positions and vectors of the fluorescent bodies are known, each plane is digitized either by hand or automatically. The computer program then

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reconstructs a 3-D model from the separate digitized planes (Fig. 30). The 3-D nature of the model is shown by the fact that one can view it from any point of view either inside or outside the model. An attractive feature of many programs is “real-time rotation,” which means that the model is rotated while you view it on the monitor. In this way many relationships between components may be recognized. Some programs also have “hidden-line removal,” which means that lines which should not be seen in a solid model are removed. The models shown in Fig. 30 are “open’ models; that is, one can see all the points regardless of depth and two views at different angles of rotation are used to make a stereo pair. Viewing stereo pairs gives additional information about the 3-D relationship of components. Another feature shown in Fig. 30 is the ability to intensify or “highlight” features of interest. In this case, ‘‘strings’’ of fluorescently stained bodies, presumably cytoplasmic dense bodies, are shown. In Fig. 30 the string appears to terminate on larger stained plaques on the cell surface. These types of relationships might never have been seen with other techniques. Computer-aided reconstruction can be of incalculable utility not only because of its precise geometrical relationships, which far surpass any other method of reconstruction, but also because it makes it possible to view the cell from angles which could not be achieved by other techniques. For example, not even the most complex concrete model would allow us to see the cell from the inside nor would it allow us to view sections of the model taken at whatever level and orientation we desire. On the other hand, one needs to be aware of some of the limitations involved. For example, in the program used by Fay (Fay et al., 1983, 1985), it was assumed that each stained body had a uniform length and shape for ease in digitization, resulting in a model which shows every stained cytoplasmic body as being identical, differing only in position and orientation. It would be more accurate if each stained body were represented as it actually is, but to do so would cause an enormous increase in the task of digitization. Thus, detail must suffer somewhat in order to show the larger scale relationships. As computer memory capacity increases and procedures for digitization become automated to a greater extent, it may be possible in the future to incorporate more of the detail from the original micrographs in the final model. Certainly, the technique has provided important structural insights and will be a major tool in future work. 4. Holographic Reconstruction

Holograms are recorded by splitting a coherent light beam, such as that issuing from a laser, into an object beam, which illuminates the object and is reflected onto a film plate, and a reference beam, which illuminates only the film. The object and reference beams produce interference patterns which are recorded as a hologram on film. When coherent light is used to “play back” the hologram, an image of the original object is projected which faithfully reproduces the 3-D

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characteristics of the original object in great detail (Kock, 1969; Caulfield, 1979). The faithfulness of the 3-D reproduction would make this technique ideal for reproducing cell organization except for one major drawback. The image is reproduced at exactly the same size as the original object, meaning that the reconstructed hologram needs to be played back and viewed with a microscope. Despite this drawback, a holographic microscope has been developed (Smith, 1982) and is being used for comparison of biological specimens to museum standards. However, another form of holographic reconstruction is more suited to data obtained from fluorescently stained cells. In this method, transparencies of serial sections are used to make multiple exposures on one holographic plate by a procedure known as “multiplexing” (Bailey et al., 1972; Blackie et al., 1986; Caulfield, 1979; Higgins, 1983). When transparencies of serial sections are positioned so that successive sections are moved a distance scaled to their separation on the z-axis, the resultant hologram, when played back, reproduces the 3-D object at a magnification which is dependent upon the magnification of the transparencies, thereby overcoming the major difficulty of holograms of microscopic objects. If “volume” holograms are made using the multiplexing technique, one can obtain a reflected 3-D image which is played back using a white light point source instead of coherent light (Kock, 1969; Caulfield, 1979). The holographic image shows the parallax present in the original scene and, unlike the stereoscopic image seen with stereo pairs, as one views the hologram from different angles different parts of the hologram change their spatial relationship to other parts just as when viewing the real object. Also, it displays both horizontal and vertical parallax. Figure 31 shows several views of a multiplexed hologram reconstructed by S. D. Dover and L. Wright from the serial optical sections shown in Fig. 25, using the apparatus described by Higgins (1983). The spacing between planes has been exaggerated to make the parallax properties more evident. Closer spacings of the transparency positions during exposure of the hologram would have resulted in a less obvious separation of the object into separate image planes and would have led to a more solid-appearing image. This reconstruction method has both advantages and disadvantages compared to computer-aided reconstruction. It is much faster, since it is done using micrographs directly without any need for analysis or digitization. It is also more ~

FIG.30. Two stereo pairs of computer-aided reconstructions of smooth muscle cells stained with anti-a-actinin. Computer displays were rotated 12”on the cell axis to give the two views comprising stereo pairs (a and a’, b and b’), CDBs are represented by single vectors, MADBs by crossed vectors. In a and a’, a stringlike array of CDBs has been intensified. In b and b’, CDBs in lateral registration have been highlighted. A relaxed cell is shown in a and a’, while b and b’ show a contracted cell. Note the change in vectors of a-actinin-containing bodies in the contracted cell. From Fay et al. (1983), by copyright permission of the Rockefeller University Press.

FIG. 31. The serial optical sections shown in Fig. 25 were used to make a multiplex hologram. With the hologram mounted and illuminated so that the axis of the reconstructed cell image was vertical, a camera mounted on a tripod was moved radially over a 25" arc (75, 80, 85, 90,95, and 100" to the plane of the holographic plate) and at two tripod heights (the camera perspective was 108 and 121" to the axis of the cell image), giving 12 perspectives to illustrate that the holographic image has the 3-D qualities of a real object. Specifically, the image has both horizontal and vertical parallax, and any two adjacent views, whether separated by a horizontal or vertical angle, may be used to form a stereoscopic pair. Since the spacing between serial sections was exaggerated during multiplexing to increase the apparent depth of the cell in the viewing axis and to demonstrate parallax more easily, the separation between the original sections is much more obvious than if the spacing was to the same scale as the magnification of the cell. The undulating course of anti-a-actinin-stained structures is more apparent in this figure than in Fig. 25. X 1760. (Multiplexed hologram made by S . D. Dover, Dept. of Biophysics. King's College, London, and L. Wright. Dept. of Medical Physics, Royal Sussex County Hospital. Brighton. England.)

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faithful to the data, since no assumptions about the shape or size of objects is required, and no information is lost by deblurring or other image-analysis techniques. However, there are also many disadvantages. The brightness of the hologram is dependent upon the number of exposures (number of planes) used. The brightness decreases with the number of exposures so that holograms of large numbers of serial sections are not practical. The image produced is monochromatic, making it difficult to represent different components, whereas the computer can code different components by color and display them in different colors on the monitor. It is difficult to enhance or delete specific areas in a holographic reconstruction, whereas both can easily be done with the computer. The computer-aided reconstruction is easily adapted to measurement since all points have x, y , and z coordinates. Coordinates can be obtained from holograms, but only by the use of clumsy, time-consuming stereographic methods (Schmandt, 1982). I believe that there is a place for all types of reconstructions and the choice of techniques depends upon the use intended for the reconstruction.

V. Conclusions A. SUMMARY It was stated at the beginning of this review that we did not as yet have sufficient information to construct a comprehensive 3-D model of the contractile system of vertebrate smooth muscle cells. However, some major features of previous models have been reinforced or invalidated by the data reviewed and it would be useful at this point to summarize the findings. 1. It was shown by several whole-cell techniques that contractile material is organized into bundles or fibrils (Small, 1974; Bagby and Pepe, 1978; Bagby and Abercrombie, 1979; Bagby, 1980; Small et al., 1986) that may run for some distance. Some of the fibrils could be called myofibrillar because they contain myosin (Bagby and Pepe, 1978; Bagby, 1980). On the other hand, the SEM of cracked cells (Sawada, 198la,b) revealed that, although superficial bundles are clearly defined, the interior bundles of the cell tend to form networks. 2. It is clear from immunofluorescently stained cells (Bagby and Pepe, 1978; Bagby and Abercrombie, 1979; Small, 1985; Small et al., 1986) and whole cells viewed with TEM (see Fig. 19) that the helical appearance seen earlier in isolated cells is due to curved paths of myofibrils. 3. The identification of thin filaments with opposite polarity at either end of CDBs (Bond and Somlyo, 1982; Tsukita et al., 1983) argues for CDBs being analogous to Z-lines.

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4. CDBs are found in strings (Bond and Somylo, 1982; Fay et al., 1983; Bailey et al., 1984) with a periodic spacing (Bagby, 1980; Fay et al., 1983). At least some of these strings are attached to MADBs (Bond and Somlyo, 1982; Fay et al. , 1983). Both points three and four are consistent with a sarcomere arrangement of contractile materials. However, recent evidence by Small et nl. (1985) argues that the a-actinin is not found in the myosin-containing fibrils.

5. Studies of thick and thin filament distribution with changes in length and/or contractile state (Section II,A,3) have revealed behavior unlike that expected if the filaments are organized into sarcomeres similar to those in skeletal muscles. The behavior could be due to extremely long thin filaments or some bundles of thin filaments which never interact with thick filaments. Cooke (1982) and Small et al. (1986) favor the latter explanation (see Cooke’s model, Fig. 5). 6. CDBs and MADBs are not biochemically equivalent since vinculin is found only in MADBs (Geiger et al., 1981; Small, 1985). 7. Attachment sites for thin filaments on the plasma membrane, as revealed by antivincufin and anti-a-actinin staining (Small, 1985). are longitudinal in all stages of contraction. Therefore, all cell models dependent upon helically arranged attachment sites (Fisher, 1974; Small, 1977a) are invalid. 8. There is strong evidence for a peripheral-to-central organization of contractile material (Cooke and Fay, 1972; Bagby, 1983; Bagby and Corey-Kreyling, 1984; Bailey et al., 1984; Ohashi and Nonomura, 1984). In most cases, contractile material is peripheral while cytoskeletal material is axial (Cooke and Fay, 1972; Cooke, 1976; Bagby, 1983; Bagby and Corey-Kreyling, 1984) (see model by Bagby, 1983, Fig. 4). 9. Several new techniques for 3-D data collection, analysis, and reconstruction have been introduced in recent years (Osborn et al., 1978; Fay et al., 1983, 1985; Blackie et a / ., 1986; Bagby, this review). They are too new to have made much of an impact on our understanding of 3-D organization as yet, but they have great promise. B. ADDITIONAL DATANEEDED The above findings have greatly improved our understanding of 3-D organization but it should be noted that there are several critical pieces of information which are badly needed to test present models: I . The length of thin filaments in relation to the length of thick filaments in the same cell. 2. The attachment points of both ends of the myofibril. At present it is certain that at least one end is attached to MADBs, but where does the other end attach?

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The model of Bagby (1983) speculates that one attachment point is a CDB while others (Fay and Delise, 1973; Fisher, 1974: Small, 1977a) have both attachments on MADBs. 3. Determining whether all fibrils are equivalent. If not, what proteins are found in different types? This will require many double-immunofluorescent staining experiments. Until these points are cleared up, no purpose is served by proposing yet another speculative model.

MODELSVERSUS RECONSTRUCTIONS C. SPECULATIVE A “speculative model” is any model in which the disposition and orientation of structural elements are not known precisely. Until techniques for 3-D reconstruction were devised, all models were speculative models. Speculative models need not reproduce the organization in a particular cell. In fact, speculative models tend to be generalizations of how structural elements are arranged and the data used to generate them come from very diverse techniques. The aim is to be consistent with as much of the data in the literature as possible. Their power lies in their general nature which allows them to be applied to many smooth muscle preparations. The speculative model is limited by the ability of the human brain to perceive and imagine spatial relationships between structural elements. It is also limited in its accuracy, and inaccuracies can be corrected only by a comparison to the data from specific cells. A “reconstruction,” on the other hand, is highly specific. It is an accurate representation of the disposition and orientation of structural elements in a specific cell. The aim of a reconstruction is to show how structural elements are related to each other in one cell. Its power lies in its precision. It is, however, limited because of the limited data one might collect from one cell. For example, current dye and filter combinations limit one to localizing only three different proteins in the same cell by immunofluorescence, and it is extremely tedious to obtain data from a whole cell and then obtain thin-section data from the same cell. Until recently, reconstructions were made by hand from serial sections, an extremely tedious process attempted by only a few smooth muscle investigators (Ashton et al., 1975; Cooke, 1976, 1982, 1983) and these reconstructions were limited to small portions of cells. The advent of the laboratory computer and the availability of the isolated smooth muscle cell preparation for immunofluorescent protein localization have led to the first computer-generated reconstructions of whole smooth muscle cells from serial optical sections (Fay et a l . , 1983, 1985). Holographic techniques (Baily et a l . , 1972; Caulfield, 1979; Higgins, 1983) have allowed reconstructions of whole cells from serial sections also. The future looks bright for the continued application of these techniques to reconstruction.

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Which type of model is best? It should be obvious that neither type of model is self-sufficient. Both types are necessary. The ultimate model will almost have to be a speculative model since it will have to incorporate data from so many different types of observations, but reconstructions will have to be used to give precise spatial information if future speculative models are to have any accuracy. It is interesting to see this interaction of speculation vs analysis because the human brain is supposed to use one cerebral hemisphere for “intuitive” tasks while the other hemisphere is used for “analytical” tasks (Springer and Deutsch, 1981). It is clear that the task of making comprehensive 3-D models requires both halves of the brain to operate or, at the very least, requires the cooperation of individuals who tend to excel in using the different halves of their brain.

ACKNOWLEDGMENTS

I would especially like to thank Margaret Kreyling who did an excellent job as my research associate for the past three years. Two graduate students, Kerek Frierson and Connie Bailey, have graciously allowed me to use their micrographs for illustrations prior to their appearance in publications. I thank them for this courtesy. I would like to thank S. D. Dover and L. Wright for their production of holograms for this review. I also want to thank all the other investigators who contributed illustrations and provided preprints of their recent work.The review was greatly enriched by their contributions. I am grateful to the Department of Zoology, University of Tennessee for giving me time off to write the review and for the use of their wordpmessor and secretaries, without which this manuscript could not have been done. My especial thanks go to F. S. and H. S. for providing inspiration and guidance. Support for our laboratory’s research which is reported herein was provided by NlH Research Grant HL-18077 and by a Grant-in-Aid from the American Heart Association, Tennessee Affiliate.

REFERENCES Abercrombie. R. K., and Bagby, R. M. 11984). Comp. Riochern. Phy.riol. 77A, 31-38. Abercrombie, R. K . . Bagby. R. M., and Matsumoto. Y. (1984). Comp. Biochem. Phvsiol. 77A, 2329. Ashton, F. T., Somiyo, A. V., and Somlyo, A. P. (1975). J. Mol. Biol. 98, 17-29. Asmussen, I. (1980). J . Submicrosc. Cjtol. 12, 673-680. Bagby, R. M. (1980). Hisrochemisrry 69, 113-130. Bagby, R. M . (1983). In “The Biochemistry of Smooth Muscle” (N. L. Stephens, ed.), Vol. 1, pp. 1-84. CRC Press, Boca Raton, Florida. Bagby, R. M.,and Abercrombie, R. K. (1979). In “Motility in Cell Function” (F. A. Pepe, ed.), pp. 427-431. Academic Press. New York. Bagby, R. M.. and Corey, M. D. (1981 1. Physiologist 24, 89. Bagby. R . M., and Corey-Kreyling, M. D. (1984). In ”Smooth Muscle Contraction” (N. L. Stephens. ed.). pp. 47-74. Marcel Dekker. New York. Bagby. R . M., and Fisher. B. A. (1979). Eur. J. Celt B i d . 19, 196-200.

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INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 105

Neuroendocrine Control of Secretion in Pancreatic and Parotid Gland Acini and the Role of Na+ ,K+ ATPase Activity SETHR. HOOTMAN Department of Physiology, University of California, San Francisco, San Francisco, California 94143

I. Introduction The mammalian salivary glands and exocrine pancreas both produce secretory fluids rich in electrolytes. The flow rate and composition of saliva and pancreatic juice are regulated by a number of neurohumoral factors which exert their effects both on the epithelia of secretory endpieces (acini) and on the anastomosing system of ducts which provides a conduit for delivery of secreted ions and macromolecules to the duodenum (pancreas) and oral cavity (salivary glands). Based on studies of saliva formation by the human parotid gland, Thaysen et al. (1954) formulated the now generally accepted two-stage secretory hypothesis to explain fluid secretion in salivary glands, a model that has since been modified to encompass also pancreatic secretory properties. The basic premise of the hypothesis is that acini secrete a primary fluid of plasma-like composition and that this fluid is modified by both secretion and reabsorption of ions during its passage along the ductal tree. Thus, the transport properties of duct epithelial cells are, to a large extent, responsible for the composition of the final secretory effluents characteristic of each organ, although the rate of production of primary fluid by the acinar epithelium also plays a determining role. At low flow rates, the primary effluent will remain in contact with the duct epithelium for longer intervals and will be more greatly altered from its original composition, whereas the reverse is true at high flow rates. A comparison of fluid samples obtained by micropuncture of the duct system immediately proximal to acini in pancreas and parotid and submandibular glands, with final saliva and pancreatic juice at different flow rates, has clearly demonstrated the validity of this principle (Schulz et af., 1969; Mangos et af., 1973; see also the review by Young and Van Lennep, 1979). Modulation of the rate of primary fluid formation thus regulates not only the volume of effluent delivered to the excretory duct orifice, but its composition as well. The volume of primary fluid secreted per unit time from each acinar unit is regulated by levels of circulating hormones and I29 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

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by release of neurotransmitters from intraglandular nerve terminals. These interact with specific receptor proteins located on the membrane surface of acinar cells, triggering cascades of intracellular events that result in alterations in the electrolyte transport properties of the acinar epithelium. This process of stimulus-response coupling, a term originally coined by Douglas (1968), results in the accelerated translocation of ions, principally Na and C1- , from the serosal to the luminal epithelial surface, with water following passively by paracellular and possibly transcellular routes. Efforts to elucidate how these changes are effected have progressed rapidly from the analysis of responses at the level of the intact organ to the level of single molecular components of the acinar transport machinery. The following discussion will concentrate on one such component, the enzyme Na ,K -activated adenosine triphosphatase (Na ,K -ATPase). Recent studies have demonstrated that Na+ ,K -ATPase activity in pancreatic and parotid acini is modulated by both hormones and neurotransmitters. It is the purpose of this article to explore how these changes are effected and to suggest how they may contribute to acinar secretion of fluid and electrolytes. +

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Cholecystokinin (CCK) and acetylcholine (ACh) are the primary secretagogues responsible for the stimulation of fluid and digestive enzyme release by the pancreatic acinus, although in some species secretin and vasoactive intestinal peptide (VIP) also elicit secretion. Specific receptors for both ACh (Larose et al., 1981; Dehaye et al., 1984; Hootman et al.. 1985b) and CCK (Jensen et al., 1980; Sankaran et al., 1980; Sakamoto et al., 1984) have been demonstrated and at least partially characterized through the use of labeled agonists and antagonists. Occupation of either receptor is followed within seconds by activation of phospholipase C, which catalyzes the hydrolysis of a minor phospholipid constituent of the plasma membrane, phosphatidylinositol-4,5-bisphosphate (PIP,) (Halenda and Rubin, 1982; Putney et al., 1983; Orchard er al., 1984). Two products are released at the inner surface of the plasma membrane by PIP, breakdown. One, diacyl glycerol (DAG), is an endogenous activator of protein kinase C (Nishuzuka, 1984). The other, inositol- 1,4,5-trisphosphate (lP3), elicits the release of Ca2+ from the rough endoplasmic reticulum (Streb et al., 1983, 1984; and see review by Berridge, 1984). Earlier studies with 45Ca2+ and isolated dispersed acinar cells or acini had established that CCK and cholinomimetics such as carbachol (CCh) cause a biphasic pattern of Ca2+ fluxes, a rapid efflux phase wherein approximately 20% of cell Ca2 is lost followed by a slower phase during which prestimulus Ca2+ levels are reestablished (see reviews by Schulz and Stolze, 1980; Williams, 1980). Chelation of extracellular Ca2 abolishes the second phase, but is +

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without effect on the first. The first phase is interpreted as resulting from extrusion by plasma membrane Ca2+-ATPases of Ca2+ released from the plasma membrane during PIP, breakdown as well as Ca2+ released from endoplasmic reticulum by IP,. The second phase is the result of Ca2+ influx by an as yet undefined mechanism from the external environment. More recent studies utilizing the fluorescent Ca2+ chelator quin-2 have shown that coincident with the first phase, cytoplasmic Ca2+ rises several-fold from a resting level of 100-200 nM (Ochs et al., 1983, 1985; Powers et al., 1985). The peak of Ca2+ concentration is reached within 3-5 seconds after initial agonist exposure and then slowly declines to a lower concentration roughly two-to-three times that of prestimulus levels. When acini loaded with quin-2 are incubated in a Ca2+-free medium, the initial response to CCK or CCh is not reduced, but declines more rapidly to resting levels. Current evidence indicates that this rise in cytoplasmic Ca2 levels provides the intracellular stimulus for both protein and electrolyte secretion. The exact mechanisms by which elevated Ca2 triggers exocytosis are not fully understood at present, although much attention is now focused on changes in phosphorylation of cellular proteins as crucial intermediaries in the response. Aside from protein kinase C, Ca2 -activated, calmodulin-dependent protein kinases and phosphatases have been demonstrated in pancreatic acini (Gorelick et al., 1983; Burnham and Williams, 1984; Burnham, 1985). Stimulation of acini by CCK or CCh causes both increases and decreases in phosphorylation of specific acinar proteins (Burnham and Williams, 1982; Freedman and Jamieson, 1982), as determined by polyacrylamide gel electrophoresisand autoradiography of lysates of acini incubated with 32Pin the presence and absence of these secretagogues. Phosphorylation of integral membrane proteins in preparations of purified zymogen granules can be increased by Ca2 -activated kinases (Peiffer et al., 1984; Wren, 1984; Bumham et al., 1985), although the significance of these changes to initiation of exocytosis remains to be determined. Additionally, the phosphorylation states of several proteins in pancreatic acini are altered by the Ca2 ionophore A23187, which also elicits digestive enzyme release (Bumham et al., 1986). These phosphoproteinsrepresent a subset of the larger group whose phosphorylation is altered by CCh and CCK. However, the identity of these proteins, except for ribosomal protein S6, has not been established, so while there is general consensus that phosphorylation changes must play a role in regulating exocytosis, it is not clear at what steps in the sequence of intracellular events that couple receptor activation to fusion of zymogen granules with the luminal-plasma membrane this regulatory input is expressed. The situation is only slightly less obscure with respect to secretagogue-elicited electrolyte transport events. Secretion of primary fluid by pancreatic acini in response to CCK or cholinomimetics is dependent on extracellular Ca2 (Case and Scratcherd, 1974; Petersen and Ueda, 1977; Scratcherd et al., 1981). The +

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extensive electrophysiological studies of Petersen and co-workers on rodent pancreatic acinar cells (reviewed by Petersen et al., 1981; Petersen, 1982) have demonstrated that ACh or its analogs and CCK cause rapid depolarization of the basolateral plasma membrane by 10-15 mV from a resting potential of about -40 mV accompanied by an abrupt decrease in input resistance. Although these studies have not defined the nature of the secretagogue-related conductance pathway, depolarization was ascribed primarily to simultaneous efflux of K and influxes of Na+ and C1- along their respective electrochemical diffusion gradients. In isolated rat pancreatic acinar cells, Putney et al. (1980a) showed that CCh and caerulein (a CCK analog) stimulated 22Na+ uptake. This stimulation was dependent on extracellular Ca2+. A similar Ca2 -dependent stimulation of 36c1- uptake was also observed (Putney and Van De Walle, 1980a). Electron probe X-ray microanalysis of resting and stimulated pancreas in vivo has confirmed these redistributions of ions. In rat pancreas, carbachol increased Na+ in the basal cytoplasm of acinar cells from 81 to 140 mmol/kg dry weight and C1- from 119 to 139 mmol/kg dry weight (Roomans and Wei, 1985). Potassium decreased slightly from 533 to 501 mmol/kg dry weight. In the canine pancreas, pilocarpine, a cholinergic agonist, caused increases in cytoplasmic Na+ and C1- from 4.8 to 15 and 14 to 25 mmol/kg wet weight, respectively (Nakagaki et al., 1984). Here, also, cytoplasmic K declined, from 132 to 119 mmol/kg wet weight. Despite this evidence for substantial secretagogue-evoked changes in pancreatic Na +,K +,and C1- content, other authors have reported either very small changes or no changes. Total Na , K , and C1- contents of isolated mouse pancreatic acini were not altered by CCh (Preissler and Williams, 1981), nor did ACh evoke any measurable change in K + activity in superfused mouse pancreatic fragments (Poulsen and Oakley, 1979). However, O’Doherty and Stark (1983) reported ACh-induced increases in Na+ and C1- activities in mouse pancreatic acinar cells of 3.9 and 4.1 mM, respectively. There remains, therefore, some uncertainty regarding the magnitude of changes in acinar cell electrolyte content induced by secretagogues. Understanding of the nature of the membrane elements that mediate changes in pancreatic electrolyte distribution has been advanced significantly by application of the technique of patch clamping to the acinar cell. High resolution current recording from isolated patches of the basolateral plasma membrane of rat and mouse pancreatic acini revealed a cation channel having a mean conductance of 30 pS (Maruyama and Petersen, 1982a). The frequency of channel opening was increased by Ca2+ applied at the cytoplasmic aspect of the membrane patch, but was not affected by induced changes in membrane potential. Both Na+ and K + penetrated the channel freely. Cholecystokinin applied to patch-clamped intact acinar cells also increased the opening of this nonselective, Ca2 -activated +

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cation channel (Maruyama and Petersen, 1982b). Further studies of mouse pancreatic acinar cells (Maruyama and Petersen, 1984) indicated that activation occurred at submicromolar Ca2 concentrations. These channels would thus represent a physiologically relevant conductance pathway. Further studies of this nature on porcine and human pancreatic acinar cells, however, have complicated the picture. In superfused pig pancreatic fragments, ACh evoked a sustained 10-15 mV hyperpolarization from a resting potential of -30 mV. Removal of Ca2+ from the bathing medium decreased this to only a transient response (Pearson et al., 1984). Patch-clamp studies on pig pancreatic acini (Maruyama et al., 1983b) demonstrated, instead of a nonselective channel of small unitary conductance, a population of highly selective K + channels (2560/cell) with a single channel conductance of 200-250 pS. The open-state probability of these large conductance channels was increased both by internal Ca2 and by membrane depolarization. CCK and ACh also evoked the Ca2 dependent opening of the K + conductance pathway. Efflux of K+ but not influx of Na+ through these channels was interpreted as the basis of the observed hyperpolarization. More recent observations on human pancreatic acinar cells (Petersen et al., 1985) have identified two separate Ca2 -activated, K + -selective channels in basolateral plasma membranes, with unitary conductances of 250 and 50 pS. The opening of both channels was voltage dependent. It seems likely from these studies that an initial step in the process of electrolyte and fluid secretion elicited by CCK, ACh, and related secretagogues is Ca2 modulation of cation-selective channel opening, which results in passive redistribution of Na+ and K + across the basolateral plasma membrane. The nature of the channel(s) activated is clearly species specific and on this basis it may be expected that the transport processes that modulate anion conductances at this cell surface could vary as well. Petersen and Philpott (1980) demonstrated a substantial ACh-elicited C1- current in superfused mouse pancreatic acinar cells, which they suggested resulted from the opening of relatively nonselective anion channels. The marked depolarization of the basolateral plasma membrane caused by openings of cation channels would support a compensatory passive influx of C1- if the electrochemical diffusion potential for the anion was then inwardly directed. However, others have suggested that C1- uptake may be mediated not by the opening of a channel, but by a facilitated diffusional carrier similar to the furosemide-sensitiveNa /C1- (or Na /K /2C1-) cotransporter described in other epithelial tissues (Frizzell et al., 1979). Using ion-selective microelectrodes, O’Doherty and Stark (1983) measured both resting and AChstimulated C1- activities in mouse pancreatic acinar cells. Resting C1- activity was 69 mM, which increased stepwise with increasing concentrations of agonist to 73 mM at 10 pA4 ACh. Sodium activity increased in parallel with C1- and with a 1:l stoichiometry. Since intracellular C1- activity was above the calcu+

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lated Nernst equilibrium potential in both resting and stimulated cells, a secondary active transport process linked to the favorable transmembrane potential for Na+ uptake was suggested. Pearson et al. (1984) also invoked Na + /K + /C lcotransport in their model of stimulus-response coupling in the pig pancreatic acinus, although no data supporting its presence were presented. Clearly, the nature of the mechanisms that mediate ACh and CCK stimulation of C1- (and to a lesser extent N a+ ) uptake at the basolateral surface of pancreatic acinar cells remains to be fully defined. Even less is known, however, regarding the mechanisms by which secretin and VIP elicit secretion from acinar cells. Both of these compounds increase acinar cell cyclic AMP levels (Robberecht et al., 1976, 1977), although only in a few species, such as the guinea pig, do they elicit significant increases in digestive enzyme release (Gardner et al., 1982). Receptors for both VIP (Bissonnette et al., 1984) and secretin (Jensen ef al., 1983) have been characterized by radiolabeled ligand binding on pancreatic acini. Acinar cells contain a cyclic AMP-dependent protein kinase (Jensen and Gardner, 1978; Burnham and Williams, 1984) and exposure of acini to secretin, VIP, or exogenous derivatives of cyclic AMP induces changes in protein phosphorylation (Freedman and Jamieson, 1982; Roberts and Butcher, 1983; Vandermeers et al., 1984). Intravenous administration of secretin to rats (Blomfield and Settree, 1980) or sheep (Blomfield et al., 1982) also caused alterations in the ultrastructural appearance of pancreatic acinar cells. In both species, injection of secretin induced enlarged acinar lumina and the appearance of electron-lucent vesicles in the apical cytoplasm of individual acinar cells. However, despite these indications of the stimulatory effects of secretin and VIP on pancreatic secretion, very little is known of the changes in membrane conductance that might be evoked by these secretagogues. Petersen and Ueda (1975) reported that neither secretin nor dibutyryl cyclic AMP had any effect on either membrane potential or input resistance in acinar cells of the rat pancreas. Although substantial progress has been made to date in defining the complement of receptors, ion channels, and other transport processes present in the acinar cell basolateral plasma membrane, very little progress has been made in elucidating transport components present at the apical cell surface. Morphometric studies (Bolender, 1974) indicate that the apical plasma membrane domain comprises about 5% of the total surface area of the acinar cell. Isolation of this cell surface by conventional fractionation schemes, therefore, has proven impractical. Cytochemical techniques, however, have yielded some information as to its composition. The apical plasma membranes of acinar cells avidly bind limulin, a lectin from horseshoe crab specific for sialic acid residues (Muresan et al., 1982). They also stain more heavily with dialyzed iron than do basolateral plasma membranes (Katsuyama and Spicer, 1977), an indication of the abun-

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dance of anionic sites. Another indication of the different composition of apical and basolateral membrane domains has been provided by labeling with the polyene antibiotic, filipin, a putatively specific probe for cholesterol (Orci et al., 1983). Freeze-fracture of pancreatic fragments fixed in the presence of filipin showed a dense distribution of filipin-cholesterol complexes on the basolateral acinar cell surface and on both the apical and basolateral surfaces of centroacinar cells. Luminal membranes of acinar cells were labeled only very sparsely. The luminal surface also exhibits a much lower density of intramembranous particles than lateral plasma membranes (Meldolesi et al., 1978). While these studies emphasize compositional differences between the two acinar cell surface domains, they tell little about the functional properties of either. Because the ultimate destination for secreted ions and proteins is the acinar lumen, it is likely that important transport events occur at the apical acinar cell plasma membrane. In other NaC1-secreting epithelia, including frog and rabbit cornea (Zadunaisky, 1966; Klyce and Wong, 1977) and dogfish rectal gland (Greger et al., 1985), agonists induce an increase in apical plasma membrane anion conductance. Whether such pathways exist in the luminal membranes of pancreatic acinar cells is unknown. However, in recent studies on ion permeabilities of rat pancreatic zymogen granules, DeLisle and Hopfer (personal communication) have observed both anion exchange and conductance pathways. Since zymogen granules are transiently integrated into the luminal plasma membrane during exocytosis, these transport elements may be inserted. This mechanism could potentially couple protein and electrolyte secretion. Although pancreatic zymogen granules have been purified and their integral membrane proteins visualized by SDS-polyacrylamide gel electrophoresis (Paquet et al., 1982; LeBel and Beattie, 1984), the functional identities of these proteins have not been defined. It remains, therefore, for future investigations to establish their roles in exocytosis and ion transport.

111. Stimulus-Response Coupling in Parotid Acini As with the exocrine pancreas, both compounds that mobilize intracellular Ca2+ and those that elevate cyclic AMP act as potent secretagogues in the parotid gland. However, an important distinction exists in that in most species, the former appear to be more effective in stimulating fluid secretion while the latter elicit a more vigorous release of secretory proteins. Specifically, agonists acting through P-adrenergic or VIP receptors induce a limited flow of saliva rich in amylase and other proteins, while a-adrenergic and cholinergic agonists and substance P evoke a copious secretion of low protein content. The alternative

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roles of cyclic AMP and Ca2+ as intracellular messengers in the parotid gland have been reviewed more fully by others (Schramm and Selinger, 1975; Young and Van Lennep, 1979; Butcher and Putney, 1980). Receptors for muscarinic cholinergic (Ludford and Talamo, 1980; Putney and Van De Walle, 1980b; Hootman et al., 1985b) and a-adrenergic (Strittmatter et al., 1977) agonists and substance P (Liang and Cascieri, 1980, 1981; Putney et al., 198Ob) have been characterized by radiolabeled ligand binding on parotid acinar cells. Agonist occupation of any of these receptors results in PIP, hydrolysis and the generation of IP, and DAG by a mechanism analogous to that outlined above for the pancreatic acinar cell (Weiss et al., 1982; Aub and Putney, 1984, 1985). Parotid acinar cells also show biphasic permeability changes to Ca2 following exposure to these secretagogues. In Ca2 -containing media, phenylephrine, an a-adrenergic agonist, and CCh cause transient decreases in total cellular Ca2 which are followed by replenishment of depleted Ca2+ pools from environmental Ca2* (Butcher, 1980; Putney et al., 1981; Poggioli and Putney, 1982). In Ca2+-free medium, these agonists evoke a sustained loss of cellular Ca2 . By analogy to the pancreas, it is presumed that cytosolic Ca2+ activity increases, although this parameter has not been measured. The sites of secretagogue-sensitive Ca2 sequestration and release in parotid acinar cells have not been identified, although cytochemical studies have implicated the plasma membrane (Sampson et al., 1983). While the mechanism responsible for the decrease in plasma membrane Ca2 following secretagogue challenge is not known with certainty, a likely possibility is that Ca2+ is bound to the acidic headgroups of PIP, molecules in the inner leaflet of the bilayer and that hydrolysis of the phospholipid releases this Ca2 . Support for an analogous mechanism has been gained from studies on human platelets (Broeckman, 1984). Both Ca2 - and cyclic AMP-mediated secretagogues elicit changes in the phosphorylation of several proteins in parotid acinar cells (Jahn et al., 1980; Baum et al., 1981; Jahn and Soling, 1981; Horio et al., 1984; Spearman et al., 1984). As in the pancreas, both decreases and increases in 32Plabeling have been reported. Subcellular fractionation has provided diverse localizations for these secretagogue-sensitive phosphoproteins. Isoproterenol, a P-adrenergic agonist, alters phosphorylation of proteins in cytoplasm, endoplasmic reticulum, and secretory granules (Spearman et al., 1984). Only one of these kinase and phosphatase substrates, ribosomal protein S6, has been identified (Jahn and Soling, 1983), although indirect evidence suggests that a second secretagogue-responsive phosphoprotein localized in the rough endoplasmic reticulum may be involved in Ca2+ sequestration (Plewe et al., 1984). Direct phosphoprotein involvement in either exocytosis or changes in membrane permeability has not been demonstrated. Electrophysiological studies of rodent parotid acinar responses to secre+

+

+

+

+

+

+

+

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tagogues have pointed out a key difference between these cells and the acinar cells of the rodent exocrine pancreas. While CCK and cholinergic receptor occupancy elicits membrane depolarization accompanied by a decrease in resistance in the latter, in parotid acinar cells ACh and a-adrenergic agonists cause a transient hyperpolarization (Pedersen and Petersen, 1973; Petersen and Pedersen, 1974; Nishiyama et al., 1980). f3-Adrenergicreceptor activation has been reported to both hyperpolarize (Iwatsuki and Nishiyama, 1982) and depolarize (Petersen and Pedersen, 1974) the parotid acinar cell. Iwatsuki and Petersen (1981) showed in mouse parotid gland that ACh-induced effects on membrane potential and resistance were not a consequence of exocytosis and most probably represented a preceding sequence of ionic events triggered by receptor occupancy. Muscarinic cholinergic or a-adrenergic agonists elicit concomitant increases in Na+ influx (Putney and Parod, 1978; Landis and Putney, 1979) and K + efflux (Batzri et al., 1973; Putney and Van De Walle, 1980b; Danielsson and Sehlin, 1983) in parotid acinar cells. The presence of extracellular Ca2+ is essential to both responses (Selinger et al., 1973; Landis and Putney, 1979). Patch-clamp studies have clarified the reason for this dependence and the nature of the membrane element responsible for mediating K + efflux. In mouse parotid acinar cells, Maruyama et al. (1983a) demonstrated the presence in the basolateral plasma membrane of a population of large conductance (250 pS) K channels whose open-state probability was both Ca2 and voltage dependent. They suggested that K release following occupancy of cholinergic or a-adrenergic receptors via these channels could serve to activate influx of Na+ , K + , and C1- by the cotransporter previously invoked to rationalize Na+ and C1- uptake in pig pancreatic acinar cells (see above). Evidence for the involvement of a cotransport mechanism in parotid secretion was obtained by Poulsen and Kristensen (1982). In isolated rat parotid acini, CChstimulated uptake of 22Na+ was partially reduced by C1- removal from or furosemide addition to the extracellular medium. Furosemide similarly reduced both ACh-stimulated fluid secretion from the isolated perfused rat submandibular gland (Martinez and Cassity, 1983) and uptake of 36Cl by dispersed submandibular acini (Martinez and Cassity, 1985). In contrast to the growing body of information relative to secretagogue-regulated transport processes present in the basolateral plasma membrane of the parotid acinar cell, understanding of secretory events occurring at the apical cell surface has not advanced appreciably. CCh elicits a substantial decrease in intracellular C1- content of rat parotid acini (Poulsen and Kristensen, 1982) and this response may reflect the opening of anion conductance pathways in the luminal plasma membrane. Apical plasma membranes have been partially purified from rat parotid gland (Arvan and Castle, 1982) and shown to be enriched in y-glutamyl transpeptidase. A second enzyme, dipeptidyl peptidase, has been localized to the luminal acinar surface by immunocytochemical techniques (Sa+

+

+

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SETH R . HOOTMAN

hara, 1985). However, aside from these two proteins, other constituents of the apical membrane domain have not been characterized.

IV. Morphology of Membrane Domains in the Exocrine Pancreas and Parotid Gland The exocrine pancreas and parotid gland are morphologically similar in that the majority of the secretory parenchyma of both organs consists of groups of up to 30 cells concentrically arranged about a common luminal space. Issuing from each such secretory unit or acinus is a tube-like duct which anastomoses with ducts leading from other acini to form an extensively arborized tree of successively enlarged segments, terminating in a single excretory duct. The main pancreatic excretory duct empties into the duodenum while the paired parotid ducts open into the vestibule of the oral cavity. Although pancreatic morphology is similar in most mammalian species, parotid gland morphology, especially as it pertains to secretory endpieces, vanes greatly (see reviews by Young and Van Lennep, 1978; Pinkstaff, 1980). Here, the discussion will focus primarily on the structural characteristics of guinea pig parotid and pancreatic acini as they relate to fluid and electrolyte secretion. In the guinea pig, as in most mammals, pancreatic acinar cells are shaped like truncated pyramids, with the flattened apex represented by the luminal cell surface and the base and sides representing basal and lateral plasma membranes, respectively (Fig. 1 ) . Acinar cells are identifiable by zymogen granules clustered in the apical cytoplasm and by arrays of basally localized rough endoplasmic reticulum. Mitochondria are moderately abundant in these cells and are often associated with the cisternae of endoplasmic reticulum. The apical cell surface is characterized by a few short microvilli. The basal plasma membrane, however, shows little evidence of elaboration. Adjacent acinar cells are closely apposed along their lateral surfaces and display only a few membrane folds. Sharing the acinar lumen and and forming junctional contacts with acinar cells are centroacinar cells, which represent the terminal extensions of intralobular ducts that penetrate into the center of the acinus. Centroacinar cells do not appear to contact the basal lamina of the acinar epithelium and can be distinguished in electron micrographs by the low density of their cytoplasm and by their numerous mitochondria. Microvilli are similar in form and density to those of acinar cells, although folding of centroacinar cell basolateral surfaces is more pronounced. Short folds or plicae decorate the plasma membrane at frequent intervals and, where two centroacinar cells abut, usually form a sequence of interdigitating processes. Intralobular duct cells share similar morphological hallmarks, forming, once outside the acinus proper, a simple cuboidal epithelium. Unlike the pancreatic acinus, centroacinar cells are not present in guinea pig

FIG. 1. Portion of a guinea pig pancreatic acinus. Acinarcells (AC) are identifiable by their large size and abundant zymogen granules (ZG) and rough endoplasmic reticulum (rER). A centroacinar cell (CC), characterized by numerous mitochondria and electron lucent cytoplasm, also borders the acinar lumen (L). Cell surfaces here are outlined by the inclusion of potassium ferrocyanide in the osmium solution employed for tissue fixation. Note the absence of significant membrane elaboration at the acinar cell basal and lateral surfaces. The basolateral plasma membrane of the centroacinar cell pictured displays a slight enhancement of elaboration relative to acinar cells. X4880. Scale bar = 5 CLm.

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FIG. 2. Slightly oblique section through a guinea pig parotid gland acinus. Secretory granules ISG) are numerous In acinar cells. Adjacent cells are separated at their lateral borders by prominent intercellular channels (arrows) which extend from the epithelial basal lamina to the level of the adluminal junctional complexes. Lateral plasma membranes are folded and these folds extend into the channel spaces. Intemellularcanaliculi (IC) extend from the central acinar lumen (L) along the lateral borders of acinar cells. Slender microvilli characterize these canalicular surfaces. x4880. Scale bar = 5 km.

parotid acini. Acinar cells are roughly pyramidal, but less regular in appearance than in the pancreas (Fig. 2). Mitochondria are approximately as abundant, but rough endoplasmic reticulum is less so. Two major differences are also observed

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when the cell surface is examined. In common with pancreatic acinar cells, basal surfaces of parotid acinar cells are usually flat. However, lateral plasma membranes display regular arrays of plicae that interdigitate with those of neighboring cells, creating prominent intercellular channels. These extend from the basal lamina of the epithelium to the junctional complexes that delimit the acinar lumen, where the second major difference is apparent. Lumina are characterized by numerous long, finger-like microvilli. In addition, canaliculi extend from the central luminal space out between acinar cells, some extending to within a few microns of the contraluminal epithelial surface. Thus, membrane elaboration at both the apical and basolateral surfaces of parotid acinar cells is significantly greater than at the corresponding surfaces of acinar cells in the pancreas. Intercalated ducts in the guinea pig parotid gland consist of cuboidal cells with no conspicious membrane elaboration at their basal surfaces, few lateral folds, and sparse microvilli. As their name indicates, these ducts intercalate between acini and striated ducts. These latter are numerous in guinea pig parotid lobules and their component cells are identifiable by abundant mitochondria and pronounced folding of the basolateral plasma membranes.

V. Intercellular Junctions in Pancreatic and Parotid Acini Much evidence now indicates that transepithelial conductances are governed, to a large extent, by the permeability properties of the paracellular shunt pathway. The main barrier to permeation of ions and other small solutes between cells in an epithelium is the zonula occludens or occluding junction which forms a band-like arrangement about each epithelial cell and attaches it to neighboring cells. Claude and Goodenough (1973) first noted a correlation between the number of strands in freeze-fracture images of occluding junctions and the electrical properties of the epithelium, with epithelial resistance and strand number increasing in parallel. Although other factors such as total junctional depth, linear extent of junctions, and arrangement of strands also appear to be important (Claude, 1978), the initial correlative premise has not been refuted. Typically, epithelia such as the gallbladder and proximal tubule of the kidney, which display low transmural resistances (<100 L? cm2), have occluding junctions consisting of networks of four or fewer strands, while in toad urinary bladder and other high resistance epithelia (>1000 L? cm2) up to 11 strands may comprise the junction. To date, it has not been possible to directly measure the transepithelial resistance of the pancreatic or parotid acinus due to the relative inaccessibility and small size of the luminal space. However, based on the observed correlation between strand number in the occluding junction and the resistance of other epithelia, junctional resistance in these two tissues can be predicted from morphological observations.

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FIG. 3. Freeze-fracture replica of the luminal surface of rat parotid acinar cells. Occluding junctions appear as shallow networks of two to four strands on the P face (arrows) or grooves on the E face (arrowheads) of the cleaved acinar cell plasma membrane. The P face of the apical cell surface (P)exhibits numerous large intramembranous particles and cross-fractured stumps of microvilli. A secretory granule (SG) beneath the patch of apical plasma membrane identifies this as an acinar cell. Lumen (L). X60,OOO. Scale bar = 0.25 pm. From Simson and Bank (1984) with permission.

Freeze-fracture images of pancreatic acinar occluding junctions were first presented by Friend and Gilula ( 1972) and since then by Metz et al. (1978) and Meldolesi et al. (1978). Although so far confined to rat and guinea pig pancreas, these studies revealed typical junctions consisting of loosely organized networks of two-to-four strands in both species. De Camilli et al. (1976), Mazariegos et al. (1984), and Simson and Bank (1984) have described junctions of similar structural organization in rat parotid glands (Fig. 3). Simson and Bank quantitated freeze-fracture data and arrived at a mean of 2.5 sealing strands for acinar occluding junctions, 2.5 for intercalated ducts, and 6.0 for striated ducts. Similar values had been determined earlier by Shimano et al. (1980) for rat sublingual

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gland acini and striated ducts, respectively. In this latter study, however, intercalated ducts displayed an intermediate value of 4.9 strands per occluding junction. These morphological observations suggest that junctional resistance in pancreatic and salivary gland acini may be quite low, a supposition borne out by studies wherein passage across these organs of solutes that do not readily penetrate cells has been monitored. Using the isolated rabbit pancreas, Jansen et al. (1979) demonstrated that urea, glycerol, and several sugars including lactose and sucrose readily enter the effluent collected from the cannulated duct following their addition to the medium bathing the organ. The relative permeability of these solutes was inversely related to molecular size. Moreover, addition of carbachol to the medium elicited an increase in the concentration of each solute in the effluent, although none of the compounds tested appeared at a concentration greater than that in the medium. In subsequent studies, Jansen et aE. (1980a,b) showed that the paracellular permeability of the rabbit pancreas to Ca2+ and Mg2+ was also increased by CCh or by CCK, and that the increase in permeability to both Ca2+ and sucrose elicited by CCh could be blocked by 2,4,6triaminopyrimidine, a compound that had previously been shown to block cation permeation across occluding junctions in other epithelia (Moreno, 1975). Since CCK and cholinergic agonists primarily affect acinar secretion, these results indicate that the paracellular shunt in the pancreatic acinus provides a low resistance pathway for passive entry of solutes of a wide range of molecular sizes into the primary secreted fluid. Further, the demonstrated increase in permeability evoked by CCh and CCK demonstrates that neurohumoral agents can modulate the resistance properties of the shunt pathway. The effects of secretagogues on junctional permeability to tracers have also been assessed in parotid and submandibular glands. Garrett et al. (1982) induced secretion from canine submandibular glands by parasympathetic nerve stimulation and demonstrated that horseradish peroxidase (MW 40,000) injected into the lingual artery appeared in the collected saliva. Mazariegos et al. (1984) and Mazariegos and Hand (1984) utilized a number of tracer molecules including horseradish peroxidase that ranged in size from microperoxidase (MW 1900) to lactoperoxidase (MW 82,OOO) in studies of occluding junction permeability in the rat parotid gland. Here, retrograde infusion of tracer solutions into the cannulated excretory duct was employed and secretion was stimulated by the injection of isoproterenol, methacholine, or methoxamine. In resting glands, only horseradish peroxidase and lactoperoxidase penetrated acinar junctions and entered interstitial spaces. However, when stimulated with isoproterenol, all tracer molecules up to a molecular weight of 34,500 as well as horseradish peroxidase and lactoperoxidase were found extraluminally. Methacholine, a cholinergic agonist, and methoxamine, an a-adrenergic agonist , did not increase junctional permeability to the tracers tested. Aside from the anomalous behavior of lactoperox-

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SETH R. HOOTMAN

idase and horseradish peroxidase, which the authors attributed to membrane damage caused by these enzymes, the results indicate that occluding junctions in the parotid gland may become transiently permeable to quite large solutes during stimulation by P-adrenergic agonists. The failure of agonists that activate either cholinergic or a-adrenergic receptors to increase junctional permeability suggests that the observed response is in some manner occasioned by enzyme secretion, which, as noted above, in the parotid gland is more strongly stimulated by P-adrenergic receptor activation. Whether the passive permeability of small organic solutes such as sucrose and urea as well as divalent cations might be potentiated by activation of either cholinergic or a-adrenergic receptors in the parotid acinus is unknown. A further demonstration of the permeability of parotid acinar cell occluding junctions has been provided by a recently developed lead-ion tracer technique (Simson and Dom, 1983; Simson and Bank, 1984). In this procedure, a leadcontaining acetate buffer solution is perfused into the vasculature and, following excision and fixation of selected tissues, deposits of the heavy metal ion are localized in thin sections by electron microscopy. In parotid acini, the tracer was localized at the luminal cell surface and between adjacent acinar cells at the sites of occluding junctions. Lead deposits were occasionally observed in the lumens of intercalated ducts, but were absent from striated duct lumena, although deposits were frequently seen in the spaces between adjacent striated duct cells. The relative permeability of occluding junctions in acini and ducts of the parotid gland, as assessed by this tracer technique, thus agreed with predictions from junctional morphology. The authors concluded that occluding junctions in the parotid acinus were very permeable to cations and that the paracellular pathway therefore could provide the transepithelial route for Na secretion. +

VI. Molecular Characteristics of Na ,K +-ATPase and Presence in the Pancreas and Parotid Gland +

Na+ ,K -ATPase (EC 3.6.1.3) was first recognized as a discrete enzymatic entity by Skou (1957) and has since been the subject of numerous studies. Consequently, much is now known of the enzyme’s molecular structure, interactions with ligands, and physiological significance. Reviews updating research in the area appear frequently (Skou, 1965, 1975; Dahl and Hokin, 1974; Jorgensen, 1975, 1980, 1982; Robinson and Hashner, 1979; Cantley, 1981; Kaplan, 1983; Anner, 1985). It is now well established that Na+ ,K+-ATPase activity represents the biochemical manifestation of the active transport mechanism present in animal cells that exchanges Na+ for K + across the plasma membrane. The energy of ATP hydrolysis is utilized to “pump” these two ions uphill against considerable electrochemical diffusion gradients. This activity has earned the +

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.Mg* ATP Mg ATP

3Nq

2Ki

Ouabain

k

Pi, Mg

I

*Dp{

(~K)E~-P*M~TE~-P*M~ 2KO

f 3Nao

(3Na)E1-p* Mg

FIG. 4. Schematic representation of the reaction cycle of Na+ ,K+-pumps in intact epithelial cells. El and E2 represent different conformational states of the pump enzyme. Binding and release of cytoplasmic Na+ and environmental K + are sequential, with the respective ions translocated across the plasma membrane in a ratio of 3:2 during each turnover of the Na+ ,K+-pump. Hydrolysis of ATP is accompanied by formation of a phosphorylated enzyme intermediate designated by E-P. Ouabain and other cardiotonic steroids inhibit Na+ ,K+ -pump turnover through binding to the E2-P conformation.

enzyme its other common sobriquet, the Na+ ,K+ -pump. The functional ATPase is a tetramer consisting of two alpha or catalytic subunits and two beta or glycoprotein subunits (cx2p2). The former have an apparent molecular weight ranging from 90,000 to 110,000 and contain the sites for Na+ and K + binding and sequestration, and for ATP hydrolysis. The significance of the P-subunits, which have a protein backbone of M, = 30,000-40,000 and display another M, = 10,000-20,000 of glycosyl residues, is not clear at present since they do not contain binding sites for any of the known physiologically relevant ligands. Both subunits are integral membrane proteins, with at least the a-subunit spanning the bilayer. Fourier transforms of data from electron micrographs of highly purified Na ,K -ATPase crystallized in the presence of vanadate and magnesium have been recently used to construct a three-dimensional model of the functional enzyme unit (Herbert et al., 1985). In this model, the unit contains two slightly flattened rod-like protomers of about 10 nm length symmetrically associated so that a deep cleft is formed along the central unit axis. Each protomer protrudes about 4 nm on the cytoplasmic side of the lipid bilayer and 2 nm on the extracellular side. Other studies have demonstrated that the site of ATP binding is located on an M, = 60,000 tryptic digest fragment of the a-subunit (Jorgensen et al., 1982), which probably represents this cytoplasmic extension. The cyclic reaction mechanism of Na+ ,K+ -ATPase entails several conformational transitions of the enzyme induced by the sequential binding and release of the different ligands. A simplified version of the reaction cycle as it appears to operate in intact cells is presented in Fig. 4. The stoichiometry of Na+ and K + +

+

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exchanged per ATP hydrolyzed is 3:2, although it is now clear that variable stoichiometries can be induced in v i m and may also occur in siru (Levitt, 1980; Cantley, 1981; Jorgensen, 1982). An important aspect of Na ,K -ATPase biochemistry is its inhibition by cardiac glycosides, an interaction first noted by Post et al. (1960). Ouabain, digitoxin, and related cardiotonic steroids bind with high affinity and specificity to a site on the external aspect of the catalytic subunit of the enzyme (Forbush, 1983; Anner, 1985). Binding is potentiated by N a + , Mg2+, and ATP and antagonized by K , which has led to the supposition, recently confirmed (Yoda and Yoda, 1982), that the E,P configuration is the cycle intermediate to which binding preferentially occurs. Ouabain binding to Na+ ,K+ -ATPase in both membrane fractions and intact cells is reversible, although the affinity of the enzyme for the ligand varies considerably. Na+ .K+ -ATPase in epithelial tissues from rats, mice, and some other rodents is notoriously insensitive to ouabain, millimolar concentrations being required to effect complete inhibition, while in other species, Na ,K -ATPase activity may be abolished by submicromolar concentrations of the drug. The affinity of Na ,K -ATPase in a given tissue for ouabain can be assessed both by inhibition of enzyme activity and by binding of radiolabeled ouabain. In tissues where the equilibrium dissociation constant (K,) for the ATPase-ouabain interaction is below 5-10 pM, binding assays with 13H]ouabain can be used to elucidate the effects of other ligands on binding, as well as kinetic aspects of the binding reaction, and to quantitate specific binding sites. As discussed below, these properties have been used to examine modulation of Na+ .K+-ATPase activity in the exocrine pancreas and parotid gland. In 1969, Ridderstap and Bonting (1969a) demonstrated an ATPase in canine pancreatic homogenates that was activated by Na+ and K + and inhibited by ouabain. Maximal activation was achieved at Na+:K+ ratios of 10-2O:l and 50% inhibition of ATPase activity (I(&) occurred at 100 nM ouabain. In this study, the authors also determined the effects of ouabain on pancreatic secretion. Secretin-stimulated secretion of fluid from the cannulated main pancreatic duct of anesthetized dogs was inhibited 70% by injection of ouabain into the celiac axis, a direct circulatory source for the pancreas. Similar studies also were carried out on rabbit pancreas (Ridderstap and Bonting, 1969b). In pancreatic homogenates, an ATPase activated by Na+ and K was demonstrable. Ouabain inhibited this activity with an IC,, of 4 pM. To assess the effects of ouabain on pancreatic secretion in this species, Ridderstap and Bonting utilized isolated pancreatic preparations (Rothman, 1964). The addition of ouabain to the perfusing bath inhibited fluid production almost completely with an IC,, of 4 pM, identical to that obtained for inhibition of homogenate Na+ ,K+ -ATPase activity. Electrolyte concentrations in the secreted fluid were not significantly altered by ouabain, although with the reduced volume of secretory effluent produced, net electrolyte secretion was greatly diminished. These results established the presence of Na ,K -ATPase in the pancreas +

+

+

+

+

+

+

+

+

+

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and emphasized the importance of its activity to pancreatic secretion of fluid. However, distribution of the enzyme between acinar and duct elements was not determined. Schulz (1980) addressed this question by separation of acinar and duct cells from the rat pancreas using centrifugal elutriation and measurement of activity of several enzymes including Na+ ,K -ATPase in the separated cell populations. Although both cell types exhibited appreciable ouabain-sensitive activity, purified duct cells were enriched by a factor of 2-3 times more than unseparated cell suspensions. Individual duct cells therefore appear to possess a greater complement of ouabain-binding sites than acinar cells, although the glycoside would be expected to interact with Na ,K -ATPase complexes on both cell types. Inhibition of both acinar secretion of primary fluid and augmentation of this fluid by duct secretory activity could therefore be expected. The distribution of Na ,K -ATPase activity between acini and ducts in the exocrine pancreas has not been determined biochemically in other species, although it is likely that the abundance of the enzyme will be strongly correlated with the relative contribution of acinar and duct secretion to the final secretory volume, which is strikingly species specific. As discussed by Case et al. (1980) and Schulz (1981), in some mammalian species (rodents, rabbits, sheep) acinar secretion appears to contribute over one-half of the final volume of pancreatic juice produced, while in others (human, cats, pigs) acinar secretion is minimal with fluid secretion being almost exclusively the province of the duct system. Na+ ,K+ -ATPase also has been characterized biochemically in the canine parotid gland (Schwartz et al., 1963) and the rat submandibular gland (Schwartz and Moore, 1968). Total ATPase activity of canine parotid microsomes measured in the presence of Na+, K + , and Mg2+ was inhibited 66% by 0.1 mM ouabain. In the more highly purified rat submandibular ATPase preparation, 0.1 mM ouabain reduced the activity by 55%. Studies by Schneyer and Schneyer (1965) demonstrated ouabain inhibition of fluid secretion from both rat parotid and submandibular glands in situ, although only moderate decreases, especially in parotid glands, were observed. Thus, as in the exocrine pancreas, the presence of Na+,K+-ATPase was confirmed in salivary glands and its importance in supporting fluid secretion suggested, although the distribution of enzyme activity between acinar and duct elements remained unknown as did the subcellular location of the transport ATPase. More recently, cytochemical procedures have been developed that provide localization of Na ,K -ATPase activity and these have been utilized to address these questions. +

+

+

+

+

+

+

VII. Cytochemical Localization of Na ,K -ATPase +

+

Early attempts to localize sites of Na+ ,K+-ATPase activity in epithelia utilized a variation of the heavy metal capture reaction pioneered by Gomori (1952) and further developed by Wachstein and Meisel (1957). Depositions of reaction

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product were, in some epithelia, localized to plasma membranes (Ashworth et ul., 1963; Farquhar and Palade, 1966). However, later biochemical investigations indicated that the phosphohydrolase activity localized by the WachsteinMeisel procedure was not attributable to Na+ ,K+ -ATPase (Emst and Philpott, 1970; Hokin et af., 1973). Additionally, precipitates obtained by this procedure were localized to the external surfaces of plasma membranes, the side of the bilayer opposite from that at which phosphate release occurs during ATP hydrolysis by Na+ ,K+-ATPase (Ernst and Philpott, 1970; Emst, 1972a,b, 1975). The physiological significance of this phosphatase localization is still unknown (see Firth, 1978, 1980). Failure of the Wachstein-Meisel technique to provide unequivocal localization of Na ,K -ATPase activity led Emst (1972a,b) to develop a different cytochemical procedure based on the potassium-dependent phosphatase activity of the enzyme, which is equivalent to the dephosphorylation step of the complete ATPase reaction sequence (see reviews by Dahi and Hokin, 1974; Robinson and Flashner, 1979; Cantley, 1981). In this technique, tissue sections were incubated in an alkaline cytochemical medium (pH 9.0) containing p-nitrophenyl phosphate @-NPP) as the substrate and strontium as the capture ion. During hydrolysis of p-NPP, both inorganic phosphate and the colored nitrophenolate ion are released, a circumstance exploited by Ernst to quantitate spectrophotometrically the effects of fixatives, heavy metal capture ions, activating ions (Na+ , K + , Mg2+), and ouabain on the enzymatic reaction. In studies utilizing slices of duck salt gland, a tissue rich in Na ,K -ATPase, Ernst demonstrated abundant electron-dense precipitates lining the basolateral plasma membranes of the principal cells of the secretory parenchyma. Apical plasma membranes, however, were devoid of reaction products. Precipitates on basolateral plasma membranes were confined to the cytoplasmic surfaces of the bilayer and were greatly reduced by ouabain, by deletion of K + or Mg2+ from the reaction medium, or by substitution of p-NPP by P-glycerophosphate, a substrate not hydrolyzed by Na+ , K + ATPase. Thus, it appeared that valid localization of the enzyme had been achieved. Indeed, the Emst technique has been widely accepted and used to localize Na+ ,K+-ATPase activity in a number of tissues (see reviews by Emst et al., 1980; Ernst and Hootman, 1981). The original Ernst procedure was subsequently altered by Mayahara et al. ( 1980) who substituted lead chelated with citrate for strontium, added Ltetramisole, an inhibitor of alkaline phosphatase, to the incubation medium, and included dimethyl sulfoxide (DMSO), which shifts the pH optima of K + NPPase from a neutral to an alkaline range (Albers and Koval, 1972). Due largely to its simplicity, the Mayahara technique has found favor with an increasing number of researchers and has to some extent superceded the Emst technique on which it is both conceptually and procedurally based. In tissues in which both procedures have been carried out, identical localization of K -dependent +

+

+

+

+

NEUROENDOCRINE CONTROL OF SECRETION

149

NPPase activity has been afforded and the newer variation appears to satisfy criteria for validation, such as sensitivity of reaction product deposition to ouabain or to K deletion. Bogart (1975), using the Ernst technique, fist demonstrated in rat submandibular gland ouabain-sensitive deposition of reaction product on acinar cell basolateral plasma membranes. The two figures shown in his paper illustrate lateral intercellular channels with abundant membrane plications as the primary foci of precipitate deposition. Observations on K + -NPPase localization in intercalated and striated ducts, if made, were not reported. Localization of K + NPPase activity has also been reported for mouse and human submandibular glands. In the former study, which was confined to light microscopic observations, Sims-Sampson et aZ. (1984), using the Mayahara technique, demonstrated intense staining of striated ducts, staining of intermediate intensity in basal regions of granular convoluted tubules, and light staining along the basolateral margins of both acinar and intercalated duct cells. Luminal membranes in all cell types were uniformly devoid of precipitates. Staining of basolateral membranes in granular convoluted tubules was also more intense in female than in male mice. By contrast, Cossu et al. (1984) were unable to demonstrate, again using the Mayahara procedure, any deposition of reaction product on membranes of cells in secretory endpieces of either the human parotid or submandibular gland, although ouabain-inhibitable deposition of precipitates was demonstrated on cytoplasmic surfaces of basolateral plasma membranes in striated ducts. One reason for the absence of reaction products in acini in this study may have been the relatively high glutaraldehyde concentration (0.5%) and the long time (60 minutes at room temperature) employed for fixation of tissues. In rat parotid, Speight and Chisholm (1984), using the Ernst procedure, demonstrated abundant electron-dense precipitates along the lateral plicated borders of acinar cells. Plasma membranes of intercellular canaliculi and the central acinar lumen gave no evidence of reactivity. The other conspicuous location of precipitates was the basolateral plasma membrane complex of striated duct cells, where reaction product deposition was heavy. Precipitation in both locations was reduced by 10 mM ouabain or by omission of K + or Mg2+ from the cytochemical medium. We have also employed a variation of the Mayahara procedure to localize sites of Na+ ,K+-ATPase activity in guinea pig pancreatic and parotid gland acini. Isolated acini were prepared from both organs and fixed for 5-10 minutes at room temperature with 2% formaldehyde-0.125% glutaraldehyde in 30 mM PIPES buffer (pH 7.2) containing 5% DMSO. Further cytochemical incubations and processing of acini were carried out according to Yamamoto et al. (1984). Figure 5 illustrates the appearance of parotid acini incubated for 30 minutes at 37°C in the cytochemical medium. Secretory granules are extracted and their limited membranes broken in many cases. Otherwise, cellular morphology is acceptable. The primary site of K+-NPPase reactivity is on lateral membrane +

SETH R. HOOTMAN

150

FIG. 5 . K -NPPase localization in guinea pig parotid acinus. Acini were prepared from parotid glands of fasted guinea pigs and fixed and processed according to the procedure of Mayahara et al. (1980) as modified by Yamamoto el 01. (1984). Cytochemical incubation was for 30 minutes at 37" C. Precipitates attributable to the activity of K + -NPPase are localized to the cytoplasmic surfaces of lateral plasma membrane extensions (arrows). Membranes of nuclei (N), luminal microvilli (Mv), and secretory granules (SG) are devoid of precipitates. Unstained section. X 15,400. Scale bar = 1 CLm. +

plications. The unelaborated basal cell surface displays few if any precipitates, nor are nuclear, mitochondrial, or endoplasmic reticulum membranes reactive. Membranes of luminal microvilli display only a light scattering of nonspecific background precipitation. Striated duct fragments are occasionally observed in preparations of parotid acini, one of which is illustrated in Fig. 6. Here, also, the deposition of K -NPPase reaction products is principally confined to basolateral plasma membranes while the luminal cell surface is devoid of precipitates. +

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NEUROENDOCRINE CONTROL OF SECRETION

In guinea pig pancreatic acini, localization of K -NPPase activity required longer incubation times due to to the low level of glandular Na+ ,K -ATPase. Figure 7 illustrates portions of a centroacinar cell and several acinar cells incubated for 60 minutes at 37°C in the Mayahara medium. Precipitates are distributed along the surfaces of both cell types and are somewhat more heavily +

+

FIG. 6. Localization of K+-NPPase activity in a fragment of striated duct from guinea pig parotid gland. Fixation and processing were as in Fig. 5. Enzyme activity is prominent on highly folded basolateral plasma membranes (arrows), but is absent from the luminal epithelial surface (arrowheads). Unstained section. X6210. Scale bar = 4 pm.

152

SETH R. HOOTMAN

FIG. 7. Part of a pancreatic acinus fixed and processed for localization of K+-NPPase activity. Cytochemical incubation was for 60 minutes at 37°C. Portions of five acinar cells (AC) and a single centroacinar cell (CC) are shown. Enzyme activity is demonstrable on the basolateral surface of each cell and is most apparent on extensions of the centroacinar cell surface. A light scattering of nonspecific precipitation is visible over mitochondria (M) and cisternae of rough endoplasmic reticulum (rER). Unstained section. X 16,300. Scale bar = 1 pm.

deposited in the former. Luminal cell surfaces within the acinus were unreactive and precipitation on lateral surfaces of acinar cells was substantially heavier than on basal surfaces (Fig. 8).

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FIG. 8. Luminal area of a guinea pig pancreatic acinus incubated for localization of K + -NPPase activity. Portions of several acinar cells are visible. Reaction products are. present on the cytoplasmic surfaces of lateral plasma membranes, especially where folding occurs (arrows), but is absent from membranes of zymogen granules (ZG) and the luminal plasma membrane (L).Sixty minute incubation at 37°C. Unstained section. X 14,600. Scale bar = 1 km.

As shown in Fig. 9, the inclusion of 5 mM ouabain in the cytochemical incubation medium virtually abolished K -NPPase-reaction product deposition on plasma membranes of parotid gland acinar cells. A similar reduction was observed on membranes of striated ducts and pancreatic acinar and centroacinar cells. +

154

SETH R. HOOTMAN

FIG. 9. Reduction in K -NPPase activity in a guinea pig parotid acinus incubated for 30 minutes at 37°C in cytocheniical medium containing 5 mM ouabain. Precipitation is virtually abolished on lateral plasma membrane extensions (arrows). Compare with Fig. 5 . Acinar lumen (L). Secretory granules (SG). Unstained section. X 10,300. Scale bar = 2 wm. +

VIII. Autoradiographic Localization of Na ,K -ATPase +

+

As previously noted, ouabain and related cardiotonic steroids bind to Na+ ,K+-ATPase with high affinity, a circumstance that has potentiated their use in radiolabeled form to localize sites of enzymatic activity in various tissues by autoradiographic analysis. However, ouabain and its congenitors are freely soluble in acetone and ethanol, which mediates against the use of standard dehydration techniques prior to embedding of labeled tissues. This problem was solved by Stirling (1972), who lyophilized tissue slices after incubation with 13H]ouabain,fixed the slices with osmium vapors, and embedded the fixed slices directly into epoxy resin. Autoradiographs of semithin sections were prepared by conventional techniques utilizing commercial liquid emulsion. This technique

NEUROENDOCRINE CONTROL OF SECRETION

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and its application to the IocaIlzation of compounds soluble in organic solvents have been reviewed (Stirling, 1976; Emst and Mills, 1980). Specificity of localizations can be demonstrated by incubation of tissues in the presence of high concentrations of unlabeled ouabain or K , both of which inhibit binding of the labeled glycoside. Quantitative studies of [3H]ouabain binding to tissues as diverse as rabbit kidney (Shaver and Stirling, 1978), duck salt gland (Emst and Mills, 1977), and frog skin (Mills et al., 1977), in conjunction with autoradiographic analysis, have clearly demonstrated that this technique faithfully localizes sites of Na+ ,K+ -ATPase activity. Criteria for unequivocal localizations and limitations of the [3H]ouabain binding procedure have been discussed more fully elsewhere (Ernst and Hootman, 1981). Although sites of [3H]ouabain binding have not been localized in the parotid gland, Bundgaard et al. (1977, 1981) have provided such localization in feline submandibular gland and pancreas. Silver grain densities in autoradiograms of [3H]ouabain-labeled submandibular glands were highest over the contraluminal halves of striated duct cells, coincident with localization of the arrays of extensively folded basolateral plasma membranes. Demilunar cells in secretory endpieces displayed a lighter scattering of grains located primarily over lateral and basal plasma membranes, while intercalated duct cells showed very few grains. Grain counts above luminal cell surfaces, including secretory canaliculi, were not above background. In the cat pancreas, [3H]ouabain again labeled the basolateral but not luminal surfaces of both acinar and duct cells. A quantitative measure of the relative abundance of Na+ ,K+-ATPase in these cell types was afforded by counting grains located above specific cell surfaces in autoradiograms. Although acini were moderately labeled, grain densities over basolateral surfaces of intercalated and interlobular ducts were 12 and 10 times as great, respectively. Centroacinar cells were as heavily labeled as duct cells outside the acinus proper. +

IX. Determination of Na+ ,K+-ATPase Activity in Viable Cells Measurement of Na ,K -ATPase activity in homogenates or subcellular membrane fractions gives an estimate of the tissue density of the enzyme, while cytochemical and autoradiographic techniques reveal sites of enzyme localization. However, since cell viability is compromised by each of these approaches, they cannot be utilized to determine how Na ,K -ATPase activity is modulated in situ in response to neurohumoral agents. Consequently, alternate techniques have been devised that allow measurement of ATPase activity in living cells. All are based on the specific affinity of ouabain for the enzyme. The most widely used indicators of Na ,K -ATPase (Na ,K -pump) activity are ouabain-sensitive oxygen consumption and 86Rb uptake. Ouabain-inhibitable hydrolysis of +

+

+

+

+

+

+

+

+

SETH R. HOOTMAN

156

p-NPP in tissue slices also has been claimed to represent Na+ ,K+ -pump activity (Shi et al., 1980). Recently, we developed a fourth procedure to assess pump activity based on the kinetics of binding of ['Hlouabain to isolated intact cells. Each of these techniques relies on a separate property of the transport enzyme and gives information of a different nature than the others. The use of ouabain-sensitive oxygen consumption as a measure of Na ,K pump activity is based on the utilization of ATP by the enzyme. The exchange of Na+ for K + across the plasma membrane entails pumping each ion uphill against a large electrochemical diffusion gradient, a process that is supported by the free energy of hydrolysis of ATP. The constant pump activity necessary to maintain physiologically compatable transmembrane Na and K gradients utilizes a substantial amount of the ATP produced in mitochondria by oxidative phosphorylation. Changes in Na ,K -pump activity cause local alterations in cytoplasmic ATP/ADP ratios, which in turn elicit compensatory increases or decreases in oxidative phosphorylation and thus in oxygen uptake. The contribution of Na ,K -ATPase activity to the cellular aerobic energy budget can therefore be determined in practice by incubating tissue slices or isolated cells in physiological media with or without ouabain and secretagogues in the closed chamber of a respirometer or oxygen electrode and measuring oxygen uptake. The difference in uptake in the presence and absence of ouabain represents oxygen that is utilized to fuel cell surface Na+,K+-pumps. This technique, while indirect, has the advantage of methodological simplicity and can provide insights into the metabolic control of active transport. Ouabain-sensitive 86Rb uptake has also been widely used as an indicator of K + influx mediated by Na+ ,K+-ATPase. Rubidium is only slightly less effective than K + in activating Na+ ,K+-ATPase and is readily transported by the enzyme (Dahl and Hokin, 1974; Robinson and Flashner, 1979; Cantley, 1981). Its isotopic form, 86Rb+ is a strong p particle emitter and can readily be quantitated by standard liquid scintillation techniques or be measuring Cerenkov radiation. Since it has a half-life of 18.7 days, it is more convenient to use than 42K+ (r1,2 = 12.4 hours). Measurement of 86Rb+ uptake in the absence or presence of ouabain in dispersed cells or tissue slices gives perhaps the most direct estimate of Na ,K -pump activity. A trace amount of 86Rb is usually added to K containing media and, if the concentration of both cations is known, total K + flux through cellular Na ,K -ATPases can be determined. A third technique for measuring Na ,K -ATPase activity in intact cells was introduced by Shi et al. (1980) and entailed monitoring the hydrolysis of p-NPP. In these studies, slices of rat submandibular gland were incubated with the substrate and release of the colored nitrophenolate ion was measured spectrophotometrically. Ouabain decreased the basal rate of p-NPP hydrolysis by 10% and the authors attributed this difference to substrate utilization by Na+ ,K + -ATPase. However, despite the simplicity of this procedure, it has been little used. +

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

NEUROENDOCRINE CONTROL OF SECRETION

157

While each of these three techniques provides estimates of Na+ ,K+-pump activity under various conditions, they cannot be used to quantitate cellular pump sites. In an effort to provide information on the numbers of Na+ ,K+ -pumps in guinea pig pancreatic and parotid gland acinar cells, we began studies using [3H]ouabainas a specific probe for the transport enzyme. During these investigations, we discovered that when the concentration of the radiolabeled ligand was adjusted to suitable low levels, changes in pump activity evoked by secretagogues were mirrored by changes in both the initial rate of binding and the equilibrium level of [3H]ouabainbinding attained (Hootman et al., 1983, 1985a; Hootman and Williams, 1985). Thus, estimation of the size of the cellular Na ,K -pump population and of the effects of secretagogues on pump turnover can be obtained by slight variations in binding conditions. Both the methodological details and rationale underlying these techniques have been reviewed (Hootman and Emst, 1986). When ouabain interacts with Na+ ,K+-pumps in the plasma membranes of intact cells, an equilibrium is established between free ligand and pump sites and bound ouabain-pump complexes. The relative percentage of pumps bound at equilibrium depends on the respective concentrations of both ouabain and pump sites and on the affinity of pumps for the glycoside. At ouabain concentrations substantially above that of the Kd for the interaction, most pump sites will be bound at any given time. Under these conditions, r3H]ouabain binding reflects the size of the pump population. Conversely, at very low [3H]ouabainconcentrations, only a small percentage of total Na+ ,K+ -pump sites is bound once equilibriumbinding conditions are achieved, and the amount of ligand bound reflects not only the number of pump sites present, but also their rate of turnover or cycling. This latter property results from three important aspects of the ouabain-enzyme interaction, (1) that the reaction mechanism of the Na+ ,K+-pump is cyclical, (2) that ouabain binds primarily to the phosphorylated E,P intermediate, and (3) that binding is reversible (Fig. 4). Consideration of these parameters indicates that the magnitude of binding at [3H]ouabain concentrations substantially below the Kd will depend on the frequency of presentation of the E,P configuration. We predicted, based on this rationale, that secretagogue-induced changes in Na+,K+-pump activity in pancreatic and parotid gland acinar cells could be monitored using [3H]ouabain.This prediction has since been confirmed. +

+

X. Effects of Secretagogues on Na ,K +-Pump Activity in Pancreatic and Parotid Gland Acinar Cells +

Most early in vitro studies of pancreatic and parotid gland secretory mechanisms were carried out using gland slices or fragments. However, the presence of multiple cell types and multiple intracellular and extracellular tissue compartments in slices complicated measurements of ligand binding and radionuclide

158

SETH R . HOOTMAN

Fic. 10. Suspension of monodispersed guinea pig pancreatic acinar cells. X3100. Scale bar = 5 eLm.

uptake and efflux and inevitably raised questions of cell viability. These problems spurred the development of procedures whereby suspensions of isolated, physiologically responsive acinar cells could be obtained. The first successful isolation of pancreatic acinar cells was reported by Amsterdam and Jamieson (1973) and of parotid acinar cells by Mangos et al. (1975). Both procedures used a combination of enzymatic digestion (collagenase, hyaluronidase, and trypsin or chymotrypsin), divalent cation chelation, and mechanical disruption to effect

NEUROENDOCRINE CONTROL OF SECRETION

159

dissociation of the secretory parenchyma, and both yielded suspensions of monodispersed acinar cells. Subsequently, it was discovered that intact acini could be prepared through the use of purified collagenase and by omitting chelation of Ca2 . Acini have been prepared in this manner from rat parotid gland (Schmidt et al., 1980) and from mouse (Williams et al., 1978) and guinea pig (Peiken et al., 1978; Schultz et al., 1980) pancreas. Acinar cells prepared by the latter procedure retain their in situ polarity and remain attached to adjacent cells by occluding junctions, which are disrupted by Ca2+ chelation during the preparation of monodispersed cell suspensions. Enzyme release in response to secretagogues is usually more vigorous in pancreatic and parotid acini than in isolated acinar cells, a circumstance that has been attributed to the retention of luminal cytoarchitectural features in the former (Williams, 1984). In our studies of [3H]ouabain binding, we have utilized suspensions of monodispersed cells in order to assure complete access of the probe to the entire cell surface. These are prepared by digestion of guinea pig pancreata in a HEPESbuffered Ringer solution containing collagenase, hyaluronidase, and a-chymotrypsin, followed by incubation in Ca2 ,Mg2 -free Ringers containing EDTA and mechanical dispersion (Hootman et al., 1983). As was reported originally by Amsterdam and Jamieson (1973), final suspensions consisted of 90-95% acinar cells (Fig. 10). Although the polarity of the isolated cells is lost, over 95% routinely exclude the vital dye trypan blue and display normal morphological features at the electron microscopic level. In such preparations, duct cells account for 2-3% of the total and erythrocytes, endothelial cells, endocrine cells, and connective tissue cell types comprise the remainding 2-3%. An isolated intralobular duct or centroacinar cell is illustrated in Fig. 11. Surprisingly, when the same protocol was used to dissociate guinea pig parotid glands (Hootman and Williams, 1985), acini rather than single cells were produced (Fig. 12). In these preparations, cell viability was usually greater than 95%. Small fragments of intercalated ducts and straited ducts were also occasionally present in these suspensions. Dispersed acinar cells prepared from both guinea pig pancreas and parotid gland specifically bind [3H]ouabain. As shown in Fig. 13, following ligand addition to suspensions of pancreatic acinar cells incubated in a HEPES-buffered Ringer solution (HR) at 37"C, a hyperbolic increase in cell-associated [3H]ouabain is observed. If a large excess of unlabeled ouabain is added after equilibrium binding conditions have been attained, the [3H]ouabain bound falls to negligible levels. Similar binding and dissociation curves have been observed for parotid acinar cells (Hootman and Williams, 1985). The ability of the unlabeled inhibitor to rapidly displace virtually all of the previously bound [3H]ouabain indicates that the latter is not internalized to an appreciable extent following binding and that the binding reaction is reversible. The Kd for [3H]ouabain binding to dispersed pancreatic acinar cells, calculated from the ratio of the +

+

+

160

SETH R . HOOTMAN

FIG. 1 1. Isolated centroacinar or intralobular duct cell from guinea pig pancreas. These comprise up to 3% of acinar cell suspensions and can be readily distinguished from acinar cells by their small size, abundant mitochondria and paucity of secretory granules or rough endoplasmic reticulum. X8140. Scale bar = 2 pn.

backward and forward unidirectional rate constants, k - and k , respectively, was 0.9 pM. For parotid acinar cells, the kinetically determined Kd for ouabain binding was 2.2 pM. The Kds for the binding reaction were also determined in both acinar cell preparations by saturation-binding analysis. Here, acinar cells were incubated in HR at 37°C with concentrations of [3HJouabain from 0.1 to 10 pJ4 for time periods long enough to allow attainment of equilibrium binding conditions. Scatchard analysis of the binding curves thus generated was used to determine both the Kd and maximal binding level for each cell type. Figure 14 illustrates Scatchard plots of these binding data. In both parotid and pancreatic acinar cell suspensions, a single class of high-affinity-binding sites was revealed. The Kds

FIG. 12. Suspension of acini prepared from guinea pig parotid glands. X540. Scale bar = 25 Pm.

0.1 mMouabain

#

180-

-0

‘IP

40

20

I 10

30

80

00

120

150

180

Time (minutes) FIG. 13. Time course of binding and release of [3H]ouabain from guinea pig pancreatic acinar cells. Cells were incubated at 37°C in HR containing 0.1 p M 13H]ouabain. Points represent means 2 SD of four separate experiments. Modified from Hootman er al. (1983).

SETH R. HOOTMAN

162

0.5

1.0

1.5

2.0

2.5

[3H]ouabain bound ( X 1OO/cell) FIG. 14. Scatchard analysis of saturation binding of ['Hlouabain to dispersed guinea pig panand parotid) . ( acinar cells. Cells were incubated at 37°C in HR with concentrations of creatic (0) ['Hlouabain from 0.1 to 10 Points represent mean values from four (parotid)or five (pancreas) experiments. Calculated Kds for pancreas and parotid acinar cells were 0.9 and 2.1 ~JM,respectively. B,,, values were 7.8 X 105 and 2.9 X lo6 [3H]ouabainbinding sites/cell.

a.

for pancreatic and parotid acinar cell ['Hlouabain binding were 0 . 9 and 2.1 pM. essentially the same as was determined kinetically. Although the affinity of cellular Na ,K +-pumps for ouabain was slightly greater in pancreatic than in parotid acinar cells, there were far fewer individual pump sites. The maximal binding level (B,,,,,) for parotid acinar cells was 2.9 x lo6 ouabain-binding sitesicell while pancreatic acinar cells averaged only 7.8 x lo5 siteslcell. These values correspond well with biochemical estimation of N a + , K + -ATPase activity in the two organs. In unpublished studies, we determined N a + ,K+-ATPase levels in homogenates of guinea pig pancreas and parotid gland as previously described (Hootman and Emst, 1980). The mean specific activity of Na+ ,K+-ATPase in four pancreas preparations was 2.75 t 0.28 pmol P,/mg proteidhour and in four parotid homogenates 7.52 It 0.29 pmol P,/mg proteidhour. As predicted from biochemical estimation of Na ,K+-ATPase activity in glandular homogenates, ease of cytochemical demonstration of K NPPase activity, and morphological correlates (i.e., the relative amount of basolateral plasma membrane elaboration), density of Na ,K -pumps in acinar cells of guinea pig parotid gland is several-fold greater than that in pancreatic acinar cells. +

+

+

+

+

163

NEUROENDOCRINE CONTROL OF SECRETION A

120

0

Control Epinephrine (10 JIM) Carbachol (10 AM)

h

+Awna

(~OOJIM)

Atropine (1OOyM)

+

Phrntdrmlnc (1OOyM)

0

30

60

so

120

Time (minutes) FIG. 15. Effects of CCh and epinephrine on [3H]ouabain binding to guinea pig parotid acinar cells incubated at 37°C in HR. Indicated concentrations of the two secretagogues were added to cell suspensions 5 minutes prior to addition of 0.1 pkf [3H]ouabain. From Hootman and Williams (1985).

Specific binding of [3H]ouabain to intact secretory epithelial cells, including pancreatic and parotid acinar cells, can be accelerated by physiological secretagogues. This observation was first made in studies with dispersed duck salt gland cells (Hootman and Emst, 1981), in which the cholinomimetic methacholine stimulated the initial rate of [3H]ouabain binding at 23°C by 50-60%. In these initial experiments, 2 pA.4 [3H]ouabain was used. Since this concentration is substantially greater than the Kd for ouabain binding to salt gland cell Na+ ,K -pumps (0.2 pA.4), the methacholine-induced initial increase in binding disappeared as larger percentages of the pump population were bound. As previously noted, we have since determined that the simple expedient of lowering the free ligand concentration allows secretagogue-evokedchanges in initial rates of [3H]ouabainbinding to be extended to equilibrium binding conditions. Figure 15 illustrates this principle. When 0.1 [3H]ouabainwas added to suspensions of guinea pig parotid acinar cells in HR containing either CCh or epinephrine at 37"C, both the initial rate of binding and equilibrium binding level attained were markedly increased. The ratio of stimulated-to-controlbinding was the same at all times at which binding was assessed. That the noted increases in binding arise +

164

SETH R. HOOTMAN

secretagogue (MI FIG. 16. Concentrationdependence of stimulation of [3H]ouabain binding to guinea pig parotid acinar cells by four agonists. Cells were incubated for 60 minutes at 3TC in HR containing 0.1 pM [3Hloubain and tbe indicated concentrationsof agonists. Points representthe means of four separate experiments. From Hootman and Williams (1985).

from receptor occupancy and activation was demonstrated by the addition of specific receptor antagonists to cell suspensions 60 minutes after [3H]ouabain addition. Atropine, a classic inhibitor of cellular responsiveness mediated via muscarinic cholinergic receptors, abolished the CCh-elicited increase in [3H]ouabain biding. Similarly, the a-adrenergic antagonist, phentolamine, reversed the increase in binding evoked by epinephrine. Atropine had no effct on the increase in cell-associated r3H]ouabain induced by epinephrine, nor did phentolamine effect binding in the presence of CCh. These experiments demonstrated rather convincingly that Na * ,K -ATPase activity in parotid acinar cells could be increased at least 2-fold by cholinergic and a-adrenergic agonists. The pump response was very rapid and was sustained as long as receptor activation was maintained. Desensitization of the response to either agonist was not observed for at least 60 minutes after their addition to cell suspensions. Measuring the increase in equilibrium binding of 13H]ouabain evoked at different concentrations also allows assessment of the relative potency and efficacy of different agonists as stimulators of acinar cell Na+ .K+ -pump activity. For parotid acinar cells, CCh. epinephrine, phenylephrine. and isoproterenol were all +

165

NEUROENDOCRINE CONTROL OF SECRETION

equally effective, each stimulating basal activity by 100- 150% (Fig. 16). Epinephrine was the most potent of the four agonists tested, followed by CCh, phenylephrine, and isoproterenol. The stimulation evoked by isoproterenol was seen at relatively high concentrations and was abolished by phentolamine, but not by the selective P-adrenergic antagonist, propranolol, indicating that isoproterenol was, in this instance, interacting with a-adrenergic receptors (Hootman and Williams, 1985). As noted previously, muscarinic cholinergic and a-adrenergic receptor activation in parotid glands elicits a substantially greater fluid flow than does activation of p-adrenergic receptors. The same relative potency was seen for Na ,K -pump activation in vitro. Similar relationships were also established for activation of Na ,K -pumps by secretagogues in guinea pig pancreatic acinar cells (Hootmanet ul.. 1983). Carbachol, CCK8, secretin, and VIP each elicited increases in t3H]ouabain binding, but with characteristically different potencies and efficacies (Fig. 17). +

+

+

’“c 180

+

T

s-ewmw

(M)

FIG. 17. Concentration dependence of stimulation of [3H]ouabain binding to guinea pig pancreatic acinar cells by CCKS (01, secretin VIP (=), and CCh (0). Cells were preincubated with indicated Concentrations of secretagogues in J3R for 5 minutes, then a further 15 minutes with the addition of 0.1 phf [3H]ouabain. Points represent means 2 SD of four experiments. Modified from Hootman er ul. (1983).

(a),

1 66

SETH R . HOOTMAN

Atropine (1OOyM)

23

0 n

.-Ca

clrbchol (lOpM)

n a

6

Standard HR (1.3 mM Ca + ) oCa 2+-free HR (0.2 m M EGTA) 0

10

30

90

60

Time (minutes) FIG. 18. Effect of extracellular Ca2+ removal on the stimulation of ["Iouabain binding by CCh. Guinea pig parotid acinar cells were incubated at 37°C in the indicated media, each containing 0.1 p M [3H]ouabain. From Hootman and Williams (1985).

CCK8 and CCh were equally effective, stimulating [3H]ouabainbinding by 6080%, although CCK was several orders of magnitude more potent. Secretin augmented binding by 40-50%, while the increase in binding caused by VIP rarely exceeded 20% over controls. Subsequent studies have demonstrated increases in [%H]ouabain binding to guinea pig pancreatic acinar cells elicited by cyclic AMP analogs, by the phosphodiesterase inhibitors theophylline and isobutylmethylxanthine, and by forskolin, a diterpene activator of adenylate cyclase (Hootman et al., 1985a). It appeared from these studies that parotid acinar cell Na ,K -pump activity is regulated primarily by secretagogues that utilize Ca2 as an intracellular messenger, while in the pancreatic acinus both Ca2 -mediated agonists and those whose effects are mediated via changes in cellular cyclic AMP levels are important pump modulators. In order to assess the dependence of Na + , K+ pump stimulation by specific agonists on C a 2 + , we incubated parotid acinar cells in either standard HR (1.3 mM Ca2+)or in Ca2+-freeHR (0.5 mM EGTA) and followed [3H]ouabainbinding in the absence and presence of CCh or epinephrine (Fig. 18). Deletion of CaZ+from the incubation medium had no apprecia+

+

+

+

167

NEUROENDOCRINE CONTROL OF SECRETION

ble effect on [3H]ouabain binding in the absence of either secretagogue. On addition of CCh or epinephrine to the cell suspension in standard HR, binding rapidly increased and a new equilibrium was established after 10-20 minutes at a binding level 2- to 3-fold higher. Atropine and phentolamine reversed the increases elicited by CCh and epinephrine, respectively. In Ca2 -free medium, CCh and epinephrine each elicited only a small increase in [3H]ouabain bound, and this quickly returned to prestimulus levels. However, readdition of millimolar Ca2+ to the medium after either agonist caused a response equal to that seen in standard HR. Results from [3H]ouabain binding studies were also corroborated through measurements of ouabain-sensitive oxygen uptake. Figure 19 illustrates typical recordings obtained when parotid acinar cells were incubated in the chamber of a polarographic oxygen electrode. Addition of CCh to acinar cells in HR resulted in an abrupt increase in the rate of oxygen utilization which, in the absence of further drug addition, was maintained for up to 20 minutes. If, however, atropine was added to the incubation chamber, oxygen uptake quickly returned to the previously established basal rate. Acinar cells incubated in Ca2+-free HR with +

C

8 2 0

0

5

10

15

20

Time (minutes) FIG. 19. Oxygen consumption by guinea pig parotid acinar cells incubated at 37°C in the chamber of a polarographic oxygen electrode. In A, cells were suspended in standard HR (1.3 mM Ca*+). In B, acinar cells were suspended in Ca*+-free HR containing 0.5 mM EGTA. Additions were as follows: (1) 10 p M CCh, (2) 100 pA4 ouabain, and (3) 10 pM atropine.

168

S E W R. HOOTMAN

FIG.20. Effects of secretagogues and inhibitors on oxygen uptake by guinea pig parotid acinar cells. Results are means SE of ttuee-tefive separate experiments. Columns, from left to right, represent oxygen uptake in the presence of secretagogue only, m t a g o g u e plus 10 pb4 atropine (CCh) or 10 fl pbentolamine (epinephrine), secretagogue plus 100 flouabain, and secretagogue in Caz+-free HR (0.5 mM EGTA). Basal oxygen uptake is represented as 10096.

*

EGTA showed only a small and transient response to CCh or epinephrine, although ouabain caused a decrease in oxygen uptake equal to that observed in standard HR. The mean results from several experiments are presented in Fig. 2O.Ouabain-sensitive oxygen uptake accounted for 53% of basal uptake, indicating that in the resting acinar cell at least half of the ATP produced during oxidative phosphorylation is utilized by cell surface Na ,K -ATPase. Similarly, epithelial cells isolated from rabbit kidney (Eveloff er al., 1981), duck salt gland (Hootman and Emst, 1980), and sheep liver (McBride and Milligan, 1985) utilize about half of ther aerobic energy budget to fuel plasma membrane Na+,K+-pumps. In parotid acinar cells, CCh and epinephrine increased this component of respiration by 160 and 139%, respectively. The magnitude of stimulation of [3H]ouabain binding and ouabain-sensitive oxygen uptake by these two agonists was therefore roughly equal. The average stimulation of ouabain-sensitive oxygen uptake evoked by CCh or epinephrine in Ca2 -free HR was 40 and 27%, and, as illustrated in Fig. 19, these small responses disappeared within 5 minutes. Together, results from [3H]ouabain binding and oxygen uptake studies indicate a strict dependence on extracellular Ca2+ for the prolonged increases in parotid Na ,K -pump activity induced by muscarinic cholinergic and a-adrenergic agonists. The transient increases in ouabain-sensitive oxygen uptake and 13H]ouabain binding that follow addition of CCh or epinephrine to suspensions +

+

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+

+

169

NEUROENWCRINE CONTROL OF SECRETION

of parotid acinat cells in Ca2+-free HR most probably reflect release of the limited pool of Ca2+ that is sequestered in the plasma membrane and rough endoplasmic reticulum. Since the pump response is manifested immediately, it would appear that recruitment of new pump units into the membrane is not an important part of the response mechanism. It seems more likely that the inmases noted result from an increased turnover of Na+ ,K+-pumps already present in the plasma membrane. However, activation of quiescent pumps cannot be excluded. The close correspondence between the magnitude of stimulation of [3H]ouabainbinding and ouabain-sensitive oxygen consumption evoked by epinephrine and CCh also suggests that Na ,K -pumps are the primary energyutilizing transport element activated by agonist Occupation of muscarinic cholinergic or a-adrenergic receptors in parotid acini. As shown in Fig. 17, guinea pig pancreatic acinar cell Na+ ,K+-pump activity can be stimulated by CCh, CCKS, secretin, and to a small extent by VIP. We recently determined the Ca2+ dependency of the pump response to the &t three secretagogues by [3H]ouabain binding and ouabain-sensitive =Rb uptake (Hootman er al.. 1985a). Results of these studies are shown in Figs. 21 and 22. Incubation of pancreatic acinar cells in Ca2 -free HR containing 0.5 mM EGTA +

+

+

+

180-

--

Eg

.

0

170160c

E D

L

T

*

150 -

c

140-

-c

130-

z a

I

6 n

-I3

m -

120110100-

secretin (100 nM)

FIG. 21. Effect of Ca2+ removal on secretagogue stimulation of [3H]ouabain binding to guinea pig pancreaticacinar cells. cells were incubated for 30 minutes at 37°C either in standard HR (0) or in Ca2+-free HR with 0.5 mM EGTA 0containing 0.05 (LM [3H]ouabaii and indicated secretagogues. Results represent means 2 SE of three experiments. Modified from data in Hootman et al. (1985a).

170

SETH R. HOOTMAN

+ 8

150-

COE

8

140-

s . c .f n o

3 -8

.-C L a

9

130120 -

T

m

5

110-

100

t (10pM)

CCK 8

Secretin

(300pM)

(100 nM)

.,

FIG. 22. Effect of Ca2+ removal on secretagogue stimulation of ouabain-sensitive s6Rb+ uptake by guinea pig pancreatic acinar cells. Cells were preincubated for 5 minutes at 23°C in the ouabain before the addition of 3-5 fl x6Rb+. presence of secretagogues with or without 100 Uptake was determined at 60 seconds after x6Rb+ addition. Results represent means f SE of four to six experiments. IZ. Standard HR (1.3 mM CaZt). Ca2+-free HR (0.5 mh4 EGTA). Data modified from Hootman cf a/. (1985a).

reduced the increase in ['Hlouabain binding elicited by either CCh or CCK8 8595%, but reduced secretin-stimulated binding only 13%, a nonsignificant decrease. Secretagogue-stimulated, ouabain-sensitive 86Rb uptake by acinar cells incubated in standard HR was of the same magnitude as [3H]ouabain binding. Removal of extracellular Ca' + decreased CCh- and CCK8-stimulated uptake 80-90%, but again resulted in only a small decrease (1 1%) in secretin-stimulated, ouabain-sensitive 86Rb uptake. These results indicate the presence of two separate mechanisms for the modulation of Na+ ,K+-pump activity in guinea pig pancreatic acinar cells, one dependent on external Ca2+ and one Iargely independent of Ca' but involving cyclic AMP. Although not shown here, dibutyryl cyclic AMP and forskolin likewise elicit Ca' -independent increases in ['Hlouabain binding. Results from both [3H]ouabain binding (Hootman et a / . , 1983) and 86Rb+ uptake studies (Hootman et al., 1985a) indicate that the Ca2 - and cyclic AMP-dependent pathways stimulate acinar cell Na ,K -pumps independently. The increase in pump activity induced by a maximally effective concentration of CCh, as measured by either technique, cannot be augmented by CCK, but is enhanced in an additive fashion by secretin. +

+

+

+

+

+

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NEUROENDOCRINE CONTROL OF SECRETION

Similarly, VIP does not enhance secretin’s effects on Na+ ,K+ -pump activity, but both CCK and CCh are effective in this regard. These studies demonstrate how monitoring changes in Na ,K -pump activity in vitro can reveal which intracellular messengers are involved in modulating the response to particular secretagogues. Since alterations in pump activity elicited by neurohumoral agents appear to represent a secondary response, measuring the changes induced under different environmental conditions can potentially give information about transport events that follow more proximally the initial activation of membrane receptors. An illustration of this principle is shown in Table I. In many epithelia, a transport component has been described that mediates the coupled uptake of Na , K , and C1- (Frizzell et al., 1979). The energetic basis for this electrically silent cotransport (Na+, K + , 2C1-) derives from the inwardly directed electrochemical diffusion gradient for Na maintained by cellular Na+ ,K+-pumps. If this transport component is involved in mediating the initial secretagogue-induced uptake of Na+ by acinar cells, one might predict that the compensatory stimulation of Na+ ,K -pump activity that follows would be reduced in Cl--free media. Conversely, if Na+ entry is facilitated through a pathway independent of C1- (e.g., a cation-selective channel), the secretagogue-induced Na ,K -pump response should not be diminished initially. Incubation of guinea pig pancreatic acinar cells in C1--free HR had no appreciable effect on the initial rate of [3H]ouabain binding to cells in either the absence or presence of CCh, CCK8, or secretin (Table I). By contrast, C1- removal from medium bathing isolated guinea pig parotid acinar cells +

+

+

+

+

+

+

+

TABLE I EFFECTOF CHLORIDE DEPLETION ON AGONIST STIMULATION OF [3H]OUABAIN BINDING TO GUINEA PIG PANCREATIC AND PAROTID GLANDACINARC E L L S ~ [3H]Ouabain bound (percentage of control) Cell type

Agonist

Parotid gland 10 Pancreas

None CCh

None 30 pJ4 CCh 300 pM CCK8 30 nM secretin

Standard HR

CI - -free HR

100 201 ? 13

73f 6 128 ? 15

100 162 t 7 159 ? 9 143 ? 10

1042 166 t 158 2 137 f

3 11 8 6

a[3H]Ouabainbound was measured either as initial rate of binding at 23°C (pancreas) or as equilibrium binding at 37°C (parotid). In standard HR, CI- concentration was 127 mM. In CI--free HR, CI- was replaced by SO4*- and osmolarity was adjusted with mannitol. Values represent means f SE of three experiments.

I72

SETH R. HOOTMAN

caused a small decrease in the binding of [3H]ouabain in the absence of secretagogues and a substantial reduction in the increase in binding induced by CCh. These results suggest the presence in guinea pig parotid gland but not in pancreas of a Na+ , K + , C1- cotransport mechanism.

XI. Summary and Conclusions The results of our investigations into the localization of Na ,K -pump activity in pancreatic and parotid acinar cells and the effects of hormones and neurotransmitters on pump turnover can be integrated with data on other aspects of stimulus-response coupling to construct models of the neurohumoral control of protein, fluid, and electrolyte secretion (Fig. 23). In both tissues, Ca2+ and cyclic AMP serve as intracellular messengers. In pancreatic acinar cells, the Ca2 dependent pathway activated by the occupation of CCK or cholinergic receptors provides the primary stimulus for digestive enzyme secretion. Cyclic AMP plays a comparatively minor role; VIP and secretin are much less effective stimulators of protein secretion. Conversely, cyclic AMP levels in parotid acinar cells, which are modulated primarily through occupation of p-adrenergic receptors, are a major determinant of enzyme secretion. Activation of the Ca2+dependent pathway by cholinergic or a-adrenergic agonists or substance P is less important. The presence of dual control processes in each gland suggests that the observed differences in effectiveness of cyclic AMP- versus Ca2 -dependent secretagogues may reflect not different mechanisms, but rather a shift in the relative emphasis placed on each pathway. This emphasis could conceivably result from subtle variations in the interaction between cellular protein kinases and phosphatases and their phosphoprotein substrates. Electrolyte secretion, on the other hand, appears to involve both discrete and common entities. In pancreatic acinar cells from rodent species, cholinergic or CCK receptor occupancy elicits a Ca2 -dependent increase in the open-state probability of nonselective cation channels in the basolateral plasma membrane. The resultant influx of Na and efflux of K is most probably the factor which activates Na+,K+-pumps. Based on electron probe studies of the effects of cholinergic agonists on acinar cell Na+ and K + contents discussed earlier, a transient reduction in the intracellular K+/Na+ ratio of up to 4-fold may occur. A shift of this magnitude in the cytoplasmic microenvironment of the Na+ ,K pump clearly would have a stimulatory influence (see discussion by Jorgensen, 1980). In addition, Ca2+ itself may have direct effects on Na+,K+-pump activity. Calcium at levels much above 1 fl progressively inhibits Na+ , K+ ATPase activity (Tobin et al., 1973; Yingst and Polasek, 1985). In unstimulated guinea pig pancreatic acinar cells, Ca2 , measured by quin-2 fluorescence was 161 13 nM (Hootman et al., 1985a) which increased to a maximal concentra+

+

+

+

+

+

+

+

*

+

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NEUROENDOCRINE CONTROL OF SECRETION

A

B

FIG. 23. Schematic diagrams illustrating neurohumoral control of protein and electrolyte secretion in acinar cells of the exocrine pancreas (A) and parotid gland (B). For details, see text.

tion of 803 ? 122 nM following CCh stimulation. Thus, intracellular free levels of Ca2+ are not of a sufficient magnitude in pancreatic acinar cells to substantially inhibit Na ,K -pump activity even after CCh or CCK exposure. Based on their studies with isolated rat hepatocytes, Berthon et al. (1983) suggested that Ca2 -mobilizing hormones stimulate Na+ ,K -pumps by releasing Ca2 sequestered in the plasma membrane, which in the resting cell exerts an inhibitory effect on pump turnover. Whether such an interaction is physiologically signifi+

+

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SETH R. HOOTMAN

174

cant in the pancreatic acinar cell is not known, although cytochemical studies (Haase et al, , 1984) recently demonstrated a substantial reduction in plasma membrane-associated Ca2+ following CCh or CCK treatment of rat acinar cells. As discussed above, the release of Ca2 from the plasma membrane may coincide with PIP, hydrolysis. Secretin, forskolin, and cyclic AMP analogs also stimulate Na+ ,K+ -pump activity in guinea pig pancreatic acinar cells, although the mechanism responsible for this increase is unclear. It is distinguishable from the increase elicited by CCK and CCh by its lack of dependence on extracellular Ca2+. We recently found that secretin stimulates ouabain-insensitive s6Rb uptake by pancreatic acinar cells as well (Hootman et al., 1985a), indicating that a pathway for cation transport across the plasma membrane is effected by cyclic AMP that is separate from the ouabain-inhibitable Na+ ,K+ -pump. Regulation of metabolic events by cyclic AMP-mediated secretagogues is effected principally by cyclic AMP-dependent protein kinase activation, so phosphorylation of channel proteins or other endogenous regulatory proteins may play roles in pump activation. Although the or-subunit of Na ,K -ATPase can be phosphorylated at a site separate from the site of catalytic ATP hydrolysis (Mardh, 1979, 1983; Ling and Cantley, 1984), the physiological signficance of this effect is not known. Secretin, forskolin, and VIP induce phosphorylation of several common proteins in guinea pig pancreatic acinar cells as assessed by two-dimensional polyacrylamide gel electrophoresis (D. B. Bumham and S. R. Hootman, unpublished results). However, none is within the molecular weight range of the a-subunit of the Na+ ,K -pump. The mechanism by which cyclic AMP activates Na ,K ATPase activity, therefore, is uncertain at present. Since acinar cell Na+,K+-pumps are localized to lateral and, to a lesser extent, basal cell surfaces, the net effect of a secretagogue-induced increase in pump turnover will be a more rapid extrusion of Na+ into paracellular spaces. As acinar-occluding junctions appear to be very permeable to small cations, Na+ should be able to diffuse freely into the luminal compartment. However, in order to effect net transepithelial movement of Na+ by a diffusive route, a separate driving force must be present. One possibility would be the presence of a favorable transepithelial potential difference established by anion transport into the acinar lumen. Electrical potential differences across either the resting or stimulated acinus have not been measured because of the difficulty of positioning electrodes in the small luminal space. Nor is much known about the C1- transport properties of either cell surface. In recent unpublished studies, we have ~ from preloaded pancreatic acinar observed secretin stimulation of W 1 efflux cells. This stimulation was not affected by bumetanide or DIDS, and it remains to be determined at what surface of the acinar cell this pathway is located and the molecular nature of the pathway. There is precedence in other CI--secretory epithelia for the presence of secretagogue-regulated anion conductance pathways +

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NEUROENDOCRINE CONTROL OF SECRETION

in the apical plasma membrane (Zadunaisky, 1966; Greger et al., 1985). If transport components are present similar to those indicated in Fig. 23 in the pancreatic acinar cell, transcellular C1- transport could provide the driving force for movement of Na+ along paracellular channels. In the model presented in Fig. 23, centroacinar cells have been omitted. Although they appear to have a higher Na ,K -pump content than acinar cells, it is not clear what contribution they make to acinar fluid secretion. As terminal extensions of the intracellular duct system, they are expected to possess similar characteristics, including activation of transport processes by secretin as opposed to CCK and CCh. It is likely that acinar-centroacinar cell interactions are important in regulating secretion, but these have not, to date, been discerned. In the parotid acinus, cyclic AMP appears to play a major role in regulating secretion of macromolecules while Ca2 mediates fluid and electrolyte transport. In rodents, uptake of Na+ and Cl- may be at least partially mediated through a cotransport process secondarily activated by the Ca2 -dependent opening of K -specific channels in the basolateral plasma membrane. As in the pancreas, Na ,K -pumps are localized primarily along lateral cell borders and would be activated by the concomitant efflux of K + and influx of Na+ induced by Ca2 -mediated secretagogues. Routing of Na to the acinar lumen would occur by a similar pathway. Here, also, occluding junctions appear to be both cation selective and highly permeable. The presence of anion transport pathways in the luminal plasma membrane is hypothetical. Together, the cytochemical and physiological studies described in this article indicate an important role for plasma membrane Na ,K -ATPase in mediating electrolyte transport across the epithelia of both pancreatic and parotid acini. Activity of the enzyme is regulated by secretagogues that utilize Ca2+ as an intracellular messenger and those whose effects are mediated by cyclic AMP. The sensitivity of this regulation allows the acinar cell Na+ ,K -pump population to maintain homeostatic control of cytoplasmic Na and K -t concentrations in the face of repeated agonist challenge and to channel Na+ to intercellular locations that potentiate its transport into the luminal compartment. These observations demonstrate the dual role played by Na ,K -pumps in the acinus, which forms the foundation on which primary fluid productions is based. +

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ACKNOWLEDGMENTS

I wish to thank Drs. Stephen A. Emst, Duane L. Ochs, and John A Williams for collaborations in the laboratory and many long and fruitful discussions; Dr. Williams and Drs. Craig D. Logsdon and Robert C. DeLisle for their comments during preparation of this manuscript; and Ms. Eileen Roach for excellent technical assistance. This research was funded by National Institutes of Health Grants AM32708 and AM32994 and by a grant from the Cystic Fibrosis Foundation.

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INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 105

Automated Individual Cell Analysis in Aquatic Research CLARICE M. YENTSCH"AND SHIRLEY A. POMPONI~~' *Bigelow Laboratory for Ocean Sciences, McKown Point, W . Boothbay Harbor, Maine 04575, and Department of ChemistrylBiochemistry, Bowdoin College, Brunswick, Maine 0401 1 and ?University of Maryland, Horn Point Laboratories, Cambridge, Maryland 21613

I. Introduction Most living organisms in aquatic systems exist as independent, single-cell entities in a fluid medium. In even the most nutrient-impoverished oligotrophic waters of the world, very small cells occur at nearly one million cells per liter. In more eutrophic waters, numbers per unit volume are several orders of magnitude higher. It is no wonder that the examination and characterization of individual cells were preoccupations of early naturalists interested in aquatic ecosystems. For the most part, cells, whether occurring as solitary units or as multicellular organisms, are in the 1-100 pm size range (Fig. 1). Generally in open waters, the smaller the cells, the more numerous they are (Fig. 2). Microscopes were essential tools for examining cells. Classical taxonomic and systematic studies flourished in the 1800s and early 1900s. Much of this work is definitive and remains unchallenged today. Major leaps in progress resulted from the development of improvements in microscopes. Each progression, from a single lens (lox), to a compound microscope (lOOOX), to a scanning electron microscope (5O,OOOX), to a transmission electron microscope (1OO,OOOX), precipitated a wealth of new information and understanding. Additional developments, such as phase contrast, oil immersion, Nomarski, and epifluorescent illumination, brought markedly expanded insights. In its infancy, the era of aquatic research was paralleled by an explosion of chemical assessment methods. Thus, there was a thrust to improve biological sampling through chemical measurement in dynamic aquatic systems. Many aquatic scientists were proponents of the application of rapid chemical assays which could be quantitative and intercalibrated in various parts of the world. Yet these represented the average, or mean, of the populations at hand, thus giving no information about the distribution and properties among cells. These chemical bulk assays became highly developed and popular. Multiple extracts were easily 'Present address: 2024 Maryellen Lane, State College, Pennsylvania 16803. 183 Copyright Q 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

INOdNOd .V AEllXIHS a N V H3S.LN3A 'M 331tIV13

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-15

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FIG.3 . Pump profile of temperature, chlorophyll, sigma-t, and nitrate data on September 6, 1984, at 43"20'N, 69"W in the Gulf of Maine aboard the R/V Cape Hatteras (pg atoms . liter- 1 or pmol).

run from the samples, and information became available while researchers were still at sea. On the research platform, cells, from hundreds to thousands of milliliters of seawater and freshwater, were collected on filters and made into extracts yielding highly informative data sets. In vivo methods were found to be even more rapid and permitted real-time assessment of the organisms in the water mass via continuous flow and analysis systems, which were also compatible with the physical scientist's conductivitytemperature-depth (CTD) measurements, the chemical scientist's autoanalyzer nutrient measurements, and the optical scientist's light transmission measurements. Combining these methodologies remains very useful for data acquisition from aquatic systems today (Fig. 3). Bulk analyses yielding additional information about the organisms, per se, in these water masses were at a premium. These included both biomass estimates (chlorophyll, ATP, and protein) and rate estimates (14C and 15N uptake; enzymatic activity; and cell duplication rates, DNA, and tritiated thymidine).

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While interpretation and quantitation of in vivo fluorometry are less straightforward than that from extracted material, the gains in speed and the continuous nature of the technology offset the limitations. Additionally, satellites were launched with optical sensing systems such as the Coastal Zone Color Scanner (CZCS) which yielded data from which the total plant pigment in surface waters could be estimated. Remote systems were also produced and operated from small aircraft. Remote sensing represents the high water mark to date in synoptic analysis systems. The four basic drawbacks of bulk analyses are that they do not give any appreciation for (1) the contribution to the optics of the water mass, (2) the contribution by rare events and forms, (3) the contributionfrom the smallest cells (1-3 pm and less), and (4)the distribution of properties among cells. Bulk analyses were never believed to be the cure-all. Several biases and drawbacks were always appreciated, but were deemed secondary when considering the gains made by these synoptic approaches. At the time, the task was to map global regions, and the timing of high and low productivity in lakes, estuaries, and Ocean water masses. Now, as the interests of aquatic scientists expand beyond the biogeographical distribution of high biomass and turnover rates, the limitations of the bulk analyses are becoming more obvious and thus should be appreciated in some detail. Optics is a simple aspect to appreciate. One can have a chlorophyll biomass level of 10 pg/liter packaged as one part of a green macroalga or as several million individual cells. The optics of the water mass are changed markedly merely by the quality of the material (refractive index) and the way the material is packaged (Fig. 4). Rare events. cells occurring at 1/ lo00 or 1/ 100,000of the population, contribute insignificantly to the mean of the population. Yet we are aware that these cells are distinct genetic lines or DNA parcels. Thus, prediction using the mean can, at times, be misleading. An example is the red-tide dinoflagellates which can be identified by individual cell analysis as sometimes being less than 1/ 1000 of the population, yet, if present at all, can lead to a “bloom” and a subsequent toxic event. Many chemical measurements have been reported without any observation or appreciation of even the dominant organisms in the population, irrespective of some rare forms. The smallest cells have only recently become popular objects of study. As late as the 1970s and 1980s, aquatic scientists began to realize that organisms of bacterial size, numbering lo5- lo6 per liter [the prokaryote cyanobacteria, plus the small (<3 pm) eukaryotes] were very numerous (Johnson and Sieburth, 1979; Waterbury et al., 1979; Yentsch, 1983; Murphy and Haugen, 1985). Many were passing through the filters used for most standard bulk analyses. Additionally, these same cells were destroyed or rendered unidentifiable using standard preservation techniques (Murphy and Haugen, 1985). This awakening

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LIGHT PENETRATION

FIG.4. Percentage of light transmitted at various depths for particles at concentrations of 10" ml- I . (+) Refractive index of 1.03 relative to seawater, 5 pm diameter, (0)refractive index 1.01, 5 pm, ( X ) refractive index 1.03, 2 pm, and (0)refractive index 1.01, 2 pm. These values are for scattering contribution only; absorbing particles, such as phytoplankton, would decrease transmission further. Courtesy of R. Spinrad from Yentsch and Yentsch (1984a).

was once again the result of improved microscopy-in this case, the advent and use of epifluorescence. Perhaps the most significant loss with bulk measurements is any index of variability. There is no appreciation of the variability or distribution of individuals within a population or of properties among cells. Figure 5 depicts schematically how the same mean can represent many population distributions. Variability is a reality which is only recently starting to be appreciated as having

CLARICE M. YENTSCH AND SHIRLEY A. POMPON1

188

mean for eoch population

0

12b

256 chonnel number

FIG. 5 . Distributions of four different particle populations representing fluorescence intensity as channel number on the x axis, and the number of events which fall into each channel on the y axis. The mean fluorescence intensity for each population is identical. This illustrates the power of histograms from individual particle analysis. (a) A bimodal population as one would find in DNA analyses, (b) typical for standard calibrations, (c) broad, but normal, distribution as for chlorophyll in a synchronized algal culture, (d) common for chlorophyll fluorescence in an asynchronous logarithmically growing algal culture.

ecological significance to the microorganism researcher. According to Conrad ( 1983): Variability of data is still viewed as a nuisance by most experimental and field biologists. According to adaptability theory, nuisance phenomenon is especially pronounced in biological materials because of its great importance for life. There is an interesting analogy to the situation in physics. Originally the variability of data was viewed as an extraneous nuisance. But now it is known that at least some uncertainty in measurement is due to quantum fluctuations and that such fluctuations, rather than being a nuisance, are responsible for the forces which hold our universe together. The variabilities of biological systems are essential to their integrity in different but equally significant ways. In both physics and biology there must therefore be a point at which the paradigm of a prototype reality masked by error becomes inappropriate.

Now in the mid-1980s we find ourselves at a crossroad. interest in individual cells is regaining favor. We ask the following questions. How much does each part of a population contribute to the productivity and the biomass? Does an increase by a factor of two in “growth rate” reflect some parts of the population increasing several-fold while other parts are unchanged, or are all cells increasing by a factor of two? Is the variability witnessed in aquatic systems ubiquitous? Are distribution patterns representative of genetic characteristics via evolution, or environmental characteristics via adaptation? Predictive ecology will depend upon an understanding of the various species components and their variability, not merely a mean value for a population or community.

AUTOMATED INDIVIDUAL CELL ANALYSIS

189

We are fortunate today that there are several instrumental techniques available which combine the rapid, quantitative, multiple-parameter, and run-at-sea advantages of bulk assays, yet retain individual cell identity. The best of both bulk analyses and individual cell measurements can be a reality. It is these relatively new approaches which we will describe in this article. Some are being actively used by aquatic researchers. Other approaches are available, yet await exploration and adaptation to aquatic problems. The aspects described are evolving so rapidly that many advances undoubtedly will be made between the time of this writing and eventual publication. Many of the methods described are still under development and, thus, any person with a sincere interest should do an appropriate search on the very latest revisions and applications. Two important concerns demand special consideration and merit presentation here. One is extended data analysis, and the other is the need for strict standards and controls. The only way to maximize the wealth of data and thereby have new information from added data sets is to make strides in both data analysis and the use of strict standards and controls. Reducing the data from 100, 1000, 10,000, or 100,000 individual cells to a strictly mean value does not merit the expense involved in acquisition and operation of the instruments which are capable of collecting these data sets for individual cells. True, one gets the mean statistically not procedurally. Thus, the coefficient of variation (CIV) and standard deviation (SD) are also obtainable, but the power of simultaneous data from large numbers of cells is lost unless extended data analysis as a mathematical science is also supported. We now have the opportunity for new flexibility and experimentation and the possibility to manipulate the data sets themselves, which can and should advance theoretical approaches. The temptation to simplify this highly complex data analysis using multiple, simultaneous parameters is great for the biological scientist. We are simply inexperienced and ill-prepared to cope with large, complex data sets. Thus, we contend that collaboration with experts in multivariate analysis is essential. The other critical aspect of individual cell approaches is the need for strict standards and controls. This need is urgent and only remedially and partially addressed at present-but it is sure to cause either the success or lack of such with the approaches discussed in this chapter. Failure to work with adequate standards and controls will render the data into little more than a series of hasty, preliminary observations. Again, this would be a waste of technological capability and the aquatic community’s limited financial resources. In these economic-conscientious times, these instruments can be money savers but only with strict adherence to proper controls. Precision, high sensitivity, intercalibration, and absolute quantification are all feasible at this time, but demand far more than the casual experiment. So, we remain cautious and note that the venture into

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CLARICE M. YENTSCH AND SHIRLEY A. POMPON1

exploratory technology involves the responsibility of using the instrumentation and data creatively in addressing theoretical questions. There are numerous aspects of aquatic biological studies which would be sizably advanced by implementing the emerging techniques described. These aspects include (1) allometry, the measurement of size and generalization on the basis of size; (2) ataxonomy, the enumeration by discrimination and characterization of subpopulations on the basis of pigmentlfunctional groups vs genera and species; (3) distinctive properties, for example, cell surface characteristics and sinking rates; (4)cell metabolism, the rates of photosynthesis, respiration, protein synthesis, and measurement of membrane potential; (5) cell growth, the measurement and description of change of cell size and chemical composition with the growth and aging processes; (6) cell duplication and division, the measurement of the rate of increase of DNA, cell cycling, and the environmental influence on cell cycling; (7) cell differentiation, the transformation of one general cell type to a specialized cell type; and (8) cell-to-cell interaction, the responsiveness of cells to various stimuli and communication between cells.

11. Methodology and Instrumentation

A. STATICSYSTEMS 1. Microscope Photometq Microscope photometry permits high-resolution, quantitative studies of spatial and spectroscopic characteristics of individual cells. There is a new system (model UMSP manufactured by Carl Zeiss) which offers an extension of the range of applications from the visible spectra into the infrared spectral region. This system is capable of image scanning and wavelength scanning in the UV and visible range and up to 2100 nm with only minor system changes. This quantitative system is capable of measuring transmittance or reflectivity. Data are acquired by either solid state camera or a photomultiplier tube (PMT). A 10 kHz scanning stage is standard. There are several ways to upgrade the system depending on user requirements. One model has an image-side visual/infrared grating monochromator to record transmittance, reflectance, and photoluminescence spectra in the range of 3802100 nm. Spectral resolution goes down to 0.5 nm and spatial resolution into the submicron range. A variety of software enhances data handling. An additional illumination-side monochromator is adapted to permit sequential spectral scanning in the 240-2100 nm range. Thus, employing both monochromators, excitation and emission spectra for photoluminescence analysis can be recorded for individual cells or even parts of cells.

MD

A

C

EPI FLUORESCENCE MICROSCOPE

/

Footrwifch

FIG. 6. Image-analyzed epifluorescence microscope system composed of an Olympus BHT-F epifluorescence microscope fitted with the Artek model 810 image analyzer. (A) A schematic diagram in which a fluorescent image is formed in the epifluorescence microscope and detected by an enhanced video camera equipped with a sensitive chalnicon tube. The image is sent as a video signal (VS) to the model 982 counter, digitized, and displayed on the image monitor, then sent to the model 940 Silhouette Memory Unit for storage, editing, and analysis. A footswitch causes the binary image (BI) to be stored, and the image can be displayed on the image monitor as enhancements (IE). Editing on the image monitor can be done with a light pen. An Apple 11+ microcomputer provides control (C) of the editing functions, processes the measurements, and counts data (MD). It also stores the data on a disk and can print it on the printer. (B) Photograph of the system in the laboratory in the same arrangement as that of A. From Sieracki et al. (1985).

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CLARICE M . YENTSCH AND SHIRLEY A . POMPON1

2. Image Analysis This offers a wide variety of advanced approaches to individual cell analysis. Some systems are manual, while others are fully automated. Semiautomated systems involve an “observer,” and thus a human discriminatory process which is not achieved by current flow methods. The system of Sieracki et al. (1985) is one example (Fig. 6). Systems include both dedicated hardware and software. Computer systems as small as personal computers have generated successful image analysis. The major advantage of employing large computers is the ability to analyze a far greater number of cells with rapid speed. Most systems are based on Fourier transformation of shapes resulting in pattern recognition. Only “positive” recognition is recorded, thus, when an “unknown” is encountered, it goes unrecorded. Image analysis has several advantages over totally manual microscopic analysis. These include the following: 1. It is easier to work with fluorescent particles since the fluorescence is recorded by an image which can be analyzed and studied over minutes, hours, or even days. No fading will occur in the image, yet the fluorescence in the cells, per se, decays in a very obvious manner under microscope epiillumination in a matter of seconds to minutes. 2. The system can be programmed to recognize shape, which, in the case of microalgae and microzooplankton, offers a far greater range of possible unique combinations than does normalization of cell volume as equivalent-spherical diameters. 3. Cells do not need to be physically separated into individuals to be analyzed as single cells. Therefore, cells in tissues can be effectively analyzed. 4. The cell suspension does not need to be free of contaminants since only the “positives” are recorded. This is useful for such things as resting cyst detection in sediments and elimination of detritus. 5. “Sorting” by selection is possible in an innovative manner. Basically, some investigators (M. Schindler, personal communication) use one low-power laser to “scan” and another, much higher power laser of the same wavelength to “burn-out” the cells selected against. The Meridian instrument (Fig. 7) is one example. 6. Cells can be cultured in microtiter plates or on microscope slides and located and relocated by “address” and an addressable stage. Therefore, one can study the development or change in identified individual cells over time.

B. DESCRIPTION OF FLOWSYSTEMS, OTHERMETHODOLOGIES 1. Flow Cytometry

Flow cytometry is a general term for the rapid measurement of particles in a moving fluid. Information is obtained through the use of various optical proper-

AUTOMATED INDIVIDUAL CELL ANALYSIS

193

FIG. 7. The ACAS 470 Workstation for analysis of single cell fluorescence and clonal cell selection in a stationary system. Manufactured by Meridian Instruments, Inc., Okemos, MI 48864.

ties of particles in the 1-150 pm size range. A variety of measurements were developed during the 1960s, 1970s, and 1980s (Horan and Wheeless, 1977; Melamed et al., 1979; Kruth, 1982; Muirhead et al., 1985). In these and later instruments, the particles in a stream flow are illuminated by an intense light source. The fluorescence and/or 90" light scatter is detected by PMTs. Forward angle light scatter (FALS), which is some function of cell size, is detected by a photodiode. The use of lasers permits the selection of specific coherent excitation wavelengths at high intensity which maximizes fluorescence emission. Epifluorescence illumination or laser-based flow cytometry, pigment autofluorescence, stain-induced fluorescence, and light scatter are used as probes to quantify subpopulations of phytoplankton cultures and natural populations. This technique, when combined with biochemical selective stains and immunofluorescence technologies, makes possible simultaneous measurement of multiple parameters (e.g., chlorophyll, protein, DNA, forward angles, plus 90" light scatter) on individual cells/particles. Measurements are made at rates in excess of loo0 cells/second. The ability to process large numbers of particles permits rigorous statistical analysis for even heterogeneous natural samples. Figure 8 is a portion of the configuration of a flow cytometer. The simul-

194

CLARICE M. YENTSCH AND SHIRLEY A . POMPON1

FIG.8. Signal split by dichroic mirror (D). Short wavelengths go into one PMT; long wavelengths into the other PMT. Simultaneous measurements result when this system is used in flow cytometry.

taneous measurement of multiple parameters on individual cells, providing information on correlation among, or cmcurrence of parameters at, the cellular level should not be underestimated. By judicious selection of excitation wavelength, dichroic mirrors and barrier filters, two natural pigment systems, or some combination of a stain and a natural pigment system, or two stains, can be excited by a single laser, and each emission measured by a separate photomultiplier tube. A second laser allows for additional measurements and flexibility. Applications of autofluorescence to pigment group discrimination are given in Yentsch el a/. (1983a, 1986). Flow cytometry requires careful handling and/or preparation of cells and/or particles, sophisticated instrumentation using a variety of current models ranging from epifluorescence microscope adaptations (Olson et a/., 1983) to three-laser systems (National Flow Cytometry Center, Los Alamos, NM), and extended data analysis. The procedure is summarized in Fig. 9. The EPICS V (Fig. 9A and B) is manufactured by Coulter Electronics (Hialeah, FL) and the epifluorescent FACS Analyzer (Fig. 10A and B) is manufactured by Becton Dickinson (Sunnyvale, CA). The advent of single cell analysis has permitted fast (data acquisition rates in excess of lo00 per second), accurate (quantitative but relative, therefore the data axes must be calibrated by traditional methods), and nearly continuous (thus FIG.9 (A) Flow cytometric two-color analysis, forward angle light scatter, and subsequent sorting. Fate of cell and resulting signals obtained via flow cytometric analysis. IGF, Integrated "green" fluorescence: IRF, integrated "red" fluorescence; and FALS, forward angle light scatter. (B) Photograph of EPICS V Coulter flow cytometer/sorter with 5W argon-ion laser. Courtesy of Yentsch er a / . . 1986.

-- + computer

:@I deflection plates

+

+ \

beads

cel Is

196

CLARICE M. YENTSCH AND SHIRLEY A. POMPON1

Emissions I

Volume Sensin Somple Flow

FIG. 10. (A) Schematics of the FACS analyzer. (B) Photograph of FACS analyzer flow cytometer manufactured by Becton Dickinson. Courtesy of Yentsch e r a / . , 1986.

AUTOMATED INDIVIDUAL CELL ANALYSIS

A

B

197

C

FIG. 11. Beyond instrumentation. Flow cytometry in aquatic research is more limited by cell collection, cell preparation, and extended data analysis than current instrument design and capabilities (in stippled area). Cells passing through a deflection plate can be separated into negatives (A), rejects (B), or positives (C).

permitting rate experiments) measurements. The degree to which these measurements are informative and can thus advance our understanding of the oceanic microenvironment is just beginning to be investigated and appreciated, and the quality of data will depend on cell collection and preparation as well as extended data analysis (Fig. 11). There has been a recent revolution in fluorescent probes. In addition to nature’s chromophores (chlorophyll a, b, and c, phycoerythrin, phycocyanin, fucoxanthin, peridinin, luciferin, flavins, NADP, and NADPH), a large variety of stains, many vital, have become highly useful. Immunofluorescent probes (Ward and Perry, 1980; Campbell et al., 1983), as well as more specific monoclonal antibodies and flow cytoenzymology (Dolbeare and Smith, 1979), are highly promising new ventures. By quantifying single cells, subpopulations and variance within subpopulations can be identified and studied in cultures and in natural populations. Previously, in aquatic sciences, individual particle measurements have been restricted to characterization by electronic counting. The addition of fluorescence as a simultaneous measurement has resulted in a new generation of microchemical tools. Presently, information derived from this new technology is being compared with the traditional bulk measurements which, to date, have typified description of plankton rate processes in the oceanic environment. Optimal fluorescent yield, minimum interference from autofluorescence, minimum energy transfer to pigments, and uniform uptake of stains are imperative

198

CLARICE M. YENTSCH AND SHIRLEY A . POMPON1

for the central dogma-that fluorescence is proportional to concentration-to be true. Of course, this limitation, too, has potential for exploitation. Removal of the pigments from fixed cells has been approached by photooxidation (Yentsch et al., 1983b) and solvent extraction (Olson et al., 1983). Development of extended data analysis has lagged behind instrument development. Rarely have biologists encountered these vast sums of data. Simply reducing the up to six-parameter fingerprints of each cell to one-parameter histograms, two-parameter scattergrams, or three-dimensional plots with frequency of events on the z axis is a waste of a valuable information base and defeats the goal of multiple parameterization. New models now need to be constructed to account for this multivariant approach-heralded as desirable by ocean ecologists (Legendre and Legendre, 1983).

2 . Fluorescence-Activated Cell Sorting In addition to fluorescence analysis, some of the cytofluorometric instruments are equipped with fluorescence-activated cell sorting capabilities, a technique originating with Bonner et al. (1972). The cell sorting capability of flow cytometry is critical both for the validation of signals from aquatic particles and for the isolation of subpopulations for conducting further study. Successful sorting experiments have been accomplished at sea using a Coulter EPICS V (Olson et al., 1985). Sorting is accomplished by setting up a sort logic (up to 24 questions to which “yes” must be the proper response in each case). Gates are easily set on fluorescence and/or light scatter criteria. The cells are in a saline sheath. Individual droplets are broken off by vibration of a piezoelectric crystal. A time delay is calculated between analysis and sort mode. Then the droplet passes through a charging collar. Droplets containing a cell and meeting preset criteria are charged, others are not charged. The droplets are then passed through a deflection plate and cells sorted right (+) or left (-) (Fig. 9A), depending upon the charge, or rejected into a center reject vial. The time required and the purity of the sort are controlled by flow rate, concentration of desired particles in the sample, and differentials in fluorescence signals; 99% purity sorts are not uncommon. The instrument operator must determine the level of purity of sort tolerable for a given experiment and must verify sort purity using epifluorescence microscopy with excitation and emission filter combinations most comparable to the flow cytometer/sorter. 3. Flow-Vision Analysis Flow-vision analysis is a new image analysis approach which combines the advantages of flow analysis with the ability to visualize the particles being analyzed. It is principally a sizing and counting system which places the signal

AUTOMATED INDIVIDUAL CELL ANALYSIS

199

FIG. 12. Set-up of Flow Vision Analyzer for cell sizing and enumeration, as well as fluorescence, showing a unique combination of image analysis and flow cytometry.

into various channels (Fig. 12), but fluorescence options are available. The Flow Vision Analyzer (Flow Vision, Inc., Clifton, NJ 07015) permits on-line, realtime display with counting and size distribution analysis of particles in a moving stream or surface. The basic components of the system include ( 1 ) a compact bundle of 50,000 optical fibers leading from a light source (of almost any sort) and providing collimated light to the lucite-flow chamber, across the optical path of common 1/ 16-in-inner diameter tygon tubing (compensations for imperfections is done by the computer moving past an analysis region and selecting out only images which change), (2) a sapphire lens in hastalloy C stainless steel, (3) an optical fiber bundle at 180", 90", or both, taking the image to a TV camera, (4) computer-to-process image (thus substracting nonmoving objects) prior to (5) video monitor image presentation, and (6) an analysis window which can be

200

CLARICE M. YENTSCH AND SHIRLEY A. POMPON1 VIDEO MONrrOR

(up fo ILXT) KilomefersJ

optiml fiber bundle (limit SmefersJ

*detecting

region

x* expond aperture to effectively “concentrote“ sample and reduce operture to‘bilute“

sopphire lens in hastalloy stainless steel

FIG. 13. Schematic of component parts of Flow Vision Analyzer

expanded to effectively “concentrate” the sample, or reduced to effectively “dilute” the sample analyzed (Fig. 13). This system can be adapted to an in situ device by pressure encasing the light source, flow cell, and TV camera, and transmitting the image back to the ship via telecommunication-type optical fibers (that is a wave transmitting mode). The flow cell is customarily built to withstand 500°C and 7000 psi. This development to an in situ device is underway by Kilham and Yentsch. Features include visual display of particles showing shape and speed of travel and particle count per minute or per hour tally as actual count or weighted averages. The optimum range for sizing is 25-600 pm, but detection from 2 to lo00 pm is possible. Flow rates at 1 ml min- are routine, with no clogging due to chains, filaments, etc. The analyzer can connect directly on-line to all micro-, mini-, or main-frame computers. While the computer, display, and principal electronics packages are located in any convenient location such as the lab or control room, the optics can be located up to thousands of meters away below the water surface, and no intake, accumulation, or removal of water is required, making it ideal for submarine use. An advantage for the aquatic sciences community is the ability to make a videotape of the images inflow. The instrument does work well at sea as demonstrated in 1985 in surface samples (Kilham and Yentsch, unpublished).

AUTOMATED INDIVIDUAL CELL ANALYSIS

20 1

111. Cell Separation and Enrichment A. CELLDISSOCIATION Analysis of individual cells for flow cytometry requires that the cells be monodisperse or in single-cell suspension. Most aquatic organisms studied to date using flow cytometry (FCM) are unicellular. Multicellular plants and animals must be dissociated into single cells prior to flow analysis. No single dissociation method is suitable for all tissue types. The appropriate technique for each tissue must be determined by experimentation to arrive at a method which yields the greatest number of viable cells. The most common dissociation techniques are physical, chemical, or a combination of the two, and are far from trivial aspects of research. Physical techniques include (1) mincing or chopping to separate tissues into small pieces or cell clumps, (2) forcing small pieces of tissue through mesh or screens, (3) pipetting or forcing tissue through needles or syringes, (4) stirring or shaking to disperse loosely bound cells, and (5) sonication. Physical techniques are useful when dissociating loosely bound cells, such as chains of diatoms or previously dissociated cells which have reaggregated. Disadvantages of physical dissociation methods include damage to a large percentage of cells, the probability of operating in a selective rather than random manner, and separation into clumps or aggregates rather than single cells. Chemical separation techniques act upon the physical and chemical bonds between cells. These include chelators, such as EDTA, EGTA, and citrate, and enzymes, such as collagenase, trypsin, and protease (Brattain, 1979). Aquatic organisms are bathed in a calcium- and magnesium-free medium to which may be added chelators (Hynes and Gross, 1970; Flick and Bode, 1983) or enzymes (Muller and Zahn, 1973; McClay, 1974). For example, a technique routinely used by one of us (Pomponi) involves a combination of physical and chemical techniques to dissociate sponge cells. Sponge samples are fist minced with scissors in calcium- and magnesium-free artificial seawater (CMF) (Spiegel and Rubinstein, 1972). Aggregates smaller than 2 mm3 are bathed in CMF with 0.25% ( w h ) trypsin or Pronase, a nonspecific protease from Sfrepfomyces griseus (Foley and Aftonomos, 1973; Gwatkin, 1973; McClay, 1974). In our experience with sponge cells, Pronase yields fewer cell clumps than trypsin. Digestions are performed for 20- to 30minute periods, at pH 7, and at either 4°C for temperate species or at the ambient temperature of the water from which the organism was collected. The sample is gently stirred on a magnetic plate during digestion. Aggregates may periodically be gently pipetted to disperse loosely bound clumps. After each digestion period, dissociated cells are filtered through an appropriate-sized nylon mesh (10-30 p,m for sponge cells) to remove cell clumps. The cells are then either centrifuged or

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CLARICE M. YENTSCH AND SHIRLEY A . POMPON1

allowed to sediment at unit gravity, and the dissociation medium is decanted. As an alternative to repeated centrifugation and rinsing, fetal calf serum may be added to inhibit the activity of trypsin. Pronase, however, is not inhibited by serum and must be removed by rinsing (Foley and Aftonomos, 1973). To maintain a monodisperse suspension, cells are resuspended in CMF. Other combinations of physical and chemical dissociation techniques have been used on sponges (Humphreys, 1963; Moscona, 1963; De Sutter and Van de Vyver, 1977; Kartha and Mookerjee, 1979; Burkart et a f . , 1979; Muller, 1982; Bretting and Jacobs, 1982; Dunham et a f . , 1983), whole coelenterates (Flick and Bode, 1983), various tissues from bivalve molluscs (Stanley et al., 1981; Allen, 1983), and echinoderm embryos (Hynes and Gross, 1970). B. SAMPLEENRICHMENT Samples of natural unicellular populations (such as phytoplankton and microheterotrophs) or cells dissociated from multicellular organisms consist of a heterogeneous assemblage and are often too dilute with respect to the cells targeted for analysis. Such samples must be enriched, concentrated, or separated into less heterogeneous subsamples. A number of enrichment techniques have been used with aquatic samples-filtration, centrifugation, density gradient sedimentation, unit gravity sedimentation, continuous flow centrifugation, centrifugal elutriation, and flow sorting. Two additional sorting techniques, affinity chromatography and cell electrophoresis, have not, to our knowledge, been used to date with aquatic samples but are promising and thus will be described briefly (Table I).

I . Filtration Techniques Filtration techniques include the use of screens or filters of different mesh size to concentrate dilute samples or to selectively retain or transmit cells according to size. This technique is used extensively to concentrate natural seawater samples containing phytoplankton (Sheldon and Sutcliffe, 1969; Malone et al., 1979; McCarthy et a f . , 1974; Derenbach and Williams, 1974). Limitations of filtration techniques are (1) delicate cells may be damaged, (2) cells retained on filters may become caught within meshes, and (3) cells with small size differences are difficult to separate. The advantages of filtration techniques are that they are simple, inexpensive, and relatively rapid. 2 . Concentration Centrifugation Concentration centrifugation of dilute samples is another simple and relatively rapid technique. The major disadvantage is that cells may be damaged as a result of pelleting or high g forces. Also centrifugation cannot separate heterogeneous cell samples into less heterogeneous fractions unless the cells have relatively large differences in sedimentation velocity.

TABLE I COMPARISON OF CELLSAMPLE ENRICHMENT TECHNIQUES ~

Basis of separation

Advantages

~~~~

Disadvantages

Method Filtration Centrifugation Density gradient centrifugation Unit gravity sedimentation Continuous flow centrifugation Centrifugal elutriation Flow sorting Cell affinity chromatography Cell electrophoresis

X

x x

X

X

X

x x x

x x x

x x

x x X

x x x X

x

x

x

x x x x x

x x x

x x

X X

x x x

x x x

x x x

X

X

X

X

X

X

X

X

x x x

x

x

x x x

X X X

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CLARICE M. YENTSCH AND SHIRLEY A. POMPON1

Isopycnic density gradient centrifugation results in cell separation. Unit gravity sedimentation, continuous flow (or rate-zonal gradient) centrifugation, and centrifugal elutriation separate cells on the basis of the sedimentation rate of the cells. According to Stokes’ law, the sedimentation rate (dxldt) of a particle of and density (dc),suspended in a medium of density shape (S), diameter (D), (d,,,), and viscosity (p,), with an acceleration ( a ) , is described by the equation

(Tolles, 1979; Sheeler and Doolittle, 1980). From this equation, it can be seen that the two primary physical properties that cause cells to be separated by sedimentation are their diameter (0)and density (dc).Isopycnic density gradient centrifugation is based on differences in the density of the cells, whereas velocity sedimentation is based on the differences in both diameter and density. Density gradients include sucrose, Metrizamide, Ficoll, and Percoll. Physical properties of each of these gradient solutes are described by Sheeler (1981). Sucrose was the primary density gradient used in early studies, however it caused severe osmotic stress to the cells and has generally been replaced with Ficoll and Percoll, which are less damaging or neutral to the cells. 3. Isopycnic Density Gradient Centrifigation In this technique, cells are layered on top of a fluid column consisting of discrete density bands or a continuous density gradient, increasing from top to bottom. In the presence of centrifugal acceleration, cells sediment to the layer of density equal to their own. The advantages of density gradient separation are that it is relatively simple and requires no specialized instrumentation other than a centrifuge. The disadvantages are (1) density gradient media are expensive (-$I60 per liter) if used in large volumes, (2) cell yields are relatively low, (3) gradient media must usually be rinsed from the harvested cells, resulting in a further decrease in cell yield, and (4) cells may be subjected to osmotic shock. Isopycnic density gradient centrifugation has been a popular cell separation technique in aquatic science. Percoll density gradient centrifugation has been used to separate mixtures of pure cultures of freshwater (Oliver et al., 1981) and marine (Price et al., 1978b) phytoplankton, as well as phytoplankton collected from the marine environment (Ortner et al., 1983). Dissociated metazoan cells have also been separated using this technique. Fractions of sevtml types of freshwater sponge cells have been obtained by centrifugation on discrete gradients of Ficoll (DeSutter and Van de Vyver, 1977; De Sutter and Buscema, 1977; De Sutter and Van de Vyver, 1979; Bretting, 1979; Bretting and Konigsmann, 1979) (Fig. 14). One of us (Pomponi) has had limited success obtaining homogeneous fractions

1

205

AUTOMATED INDIVIDUAL CELL ANALYSIS

A

-

6

sample

.:.. . .:. ....(.. ..

A

. . . .

. . . .

$

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I

8

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0

............ ... ... -archaeocytes ...I..

1 FIG. 14. Distribution of sponge cells through a discrete (A) and continuous (B) Ficoll density gradient after centrifugation using calcium- and magnesium-free artificial seawater (CMF). Redrawn with permission from DeSutter and Van de Vyver (1979).

of marine sponge cells with Ficoll gradient sedimentation, but has succeeded in obtaining nearly homogeneous fractions of marine sponge cells with discrete gradients of Percoll. Monospecific, single cell suspensions ( lo6- lo9 cells/ml) of several temperate and tropical species of the boring sponge Cliona were layered onto discrete gradients of 10,40, and 70% Percoll/CMF, and centrifuged at 2000 rpm for 30 minutes. The results are detailed in Table 11. Isopycnic density gradient centrifugation has also been used to separate cells dissociated from sea urchin embryos for subsequent developmental studies (Hynes and Gross, 1970; Spiegel and Rubinstein, 1972). Velocity sedimentation techniques (unit gravity sedimentation, continuous flow or rate-zonal centrifugation, and centrifugal elutriation) are based on both the density and diameter of the cells and, therefore, often result in a better separation than with isopycnic density gradient centrifugation, particularly if the cells overlap in either diameter or density. TABLE I1 RESULTS OF PERCOLL GRADIENT CENTRIFUGATION OF DISSOCIATED CELLS OF MARINE SPONGES (Cliona spp.) Percoll concentration

Cell types

~ _ _ _ _ _ _

O/ 10% band 10/40% band 40/70% band Pellet

aFrom Pomponi (1979).

Choanocytes Archaeocytes Cells with inclusions (spherulous cells, gray cells, etc.)a Cell clumps, spicules, debris

CLARICE M.YENTSCH AND SHIRLEY A. POMPON1

206 t

i

Cdshlon

Overlay

t

a

C

b

d

e

FIG. 15. Unit gravity sedimentation chamber. (a) Light gradient pumped into tilted chamber and underlayered by denser cushioning liquid. (b) Cell suspension loaded over gradient by reversing direction of flow, (c) chamber reoriented to horizontal, and cells allowed to sediment, (d) separation of cells after sedimentation, and (e) chamber tilted and separated fractions collected. Redrawn with permission from Wells (1982).

4. Unit Gravity Sedimentation In this technique, cells are layered onto a fluid column in a sedimentation chamber (Tulp and Bont, 1975; Tulp et al., 1980a,b; Wells, 1982) (Figs. 15 and 16). The fluid column consists of a light gradient (e.g., a lower concentration of Ficoll) formed to stabilize the column. The cells sediment for a certain period of time and become distributed according to their sedimentation rates as described by the equation

where g is the earth's gravitational acceleration constant (980 cm/sec2) and other variables are defined as in Eq. (1) (Sheeler and Doolittle, 1980).

207

AUTOMATED INDIVIDUAL CELL ANALYSIS

Fractions are collected by slow drainage of the gradient from the bottom of the sedimentation chamber. This method allows a finer resolution of cells with small differences in size or shape. For example, separation of cells of the freshwater sponge Ephydatia fluviatilis using Ficoll isopycnic density gradient centrifugation resulted in three bands of cells (De Sutter and Van de Vyver, 1979). Using unit gravity sedimentation, cells from this same species were separated into seven fractions (De Sutter and Tulp, 1981). Burkhart et al. (1979) combined unit gravity sedimentation with fluorescence-activated cell sorting on a flow cytometer to separate cells of the marine sponge Microciona prolgera. The major disadvantage of unit gravity sedimentation is that separation can take up to 4 hours, during which time cells may undergo physiological changes or begin to reaggregate (De Sutter and Tulp, 1981). A modification of the unit gravity chamber has been devised (Fig. 16) to allow sedimentation in as little as 10 minutes (Tulp et al., 1980b). One other disadvantage is that a density gradient is required, which may need to be rinsed from harvested cells prior to subsequent experimentation.

5 . Continuous Flow {or Rate-Zonal Gradient) Centrifugation In rate-zonal gradient centrifugation, cells are continuously injected into a separation chamber containing a shallow density gradient (e.g., 4-8% Ficoll) (Fig. 17). As the cell suspension passes through the rotor, cells sediment primarily on the basis of their diameter and density [Eq. (l)] and remain in the chamber. After the entire sample is loaded, the rotor is stopped and removed, and fractions are collected (Wells, 1980; Pretlow and Pretlow, 1982). Continuous flow centrifugation has been used in aquatic science primarily to concentrate phytoplankton from natural seawater samples. The advantage of this technique to such an application is that large sample volumes can be processed.

I

1.5cm

15cm

5 cm

FIG. 16. Sedimentation chambers for separation of cells at unit gravity (a) and 10 g (b). Hatched areas represent flow deflectors which allow sample layering without disrupting gradient. i, Inlet; 0, outlet; c, s, cylinders and screw for attachment of flow deflector; r, O-ring; A, reservoir for antiturbulence glass beads. Redrawn with permission from Tulp et al. (1980a.b).

208

CLARICE M . YENTSCH AND SHIRLEY A . POMPON1

b

i-

*

Grodient Loaded at Rest (LigMEnd R r s t ) I

I

Lwding a m p l e

- I

Reorientation During kcelemtion

-

-

Reartentatton During Acceleration

Separation of Particles

Unlooding

FIG. 17. Rate-zonal gradient centrifugation technique. Redrawn with permission from E. I. duPont de Nemours & Co., Inc.

The disadvantage, of course, is that a density gradient medium must be used and then rinsed from the cells. 6. Centrifugal Elutriation This technique is similar in principle to continuous flow centrifugation in that cells are separated according to their sedimentation velocity, the flow rate of the medium, and centrifugal acceleration. Thus, cells with overlapping diameters or densities can be separated into subpopulations. The major difference between continuous flow centrifugation and centrifugal elutriation is that a density gradient medium is not required for the latter method. Cells are separated in an isotonic medium, such as culture medium or filtered seawater. Medium is pumped into the elutriator rotor (Beckman Instruments) (Fig. 18a), through a rotating seal connection into the separation chamber and toward the center of the rotor. At the center, the medium flows through another rotating seal connection to the collection tubing. The sample of cells is injected into a sample reservoir, and cells flow in the medium to the separation chamber, where they are distributed according to their physical properties, the flow rate of the medium, and the centrifugal acceleration of the elutriator rotor (Fig. 18b). Stepwise increases in flow rate or decreases in rotor speed cause slower sedimenting cells to flow to the innermost end of the elutriation chamber, where the converging walls increase the fluid velocity and the cells flow out of the chamber (Sanderson and Bird, 1977; Keng et al., 1981). Centrifugal elutriation has been used in preliminary experiments with phytoplankton cultures and sponge cells (Pomponi, 1983). Continued method development (Pomponi and Cucci, 1986) has demonstrated the separation of

AUTOMATED INDIVIDUAL CELL ANALYSIS

209

asynchronous cultures of the chlorophyte Dunaliella tertiolecta into cell cycle stages, as well as the separation of mixtures of cultures of Dunaliella tertiolecta and Phaeodactylum tricornutum into species groups. Research currently in progress in our laboratory indicates that elutriation is an effective technique for concentrating phytoplankton in natural seawater samples (Pomponi and Cucci, 1985). By continuously loading the sample at a low flow rate or high rotor speed, cells can be concentrated within the elutriation chamber. The rotor and fluid flow are then stopped, the chamber is removed from the rotor, and the concentrated sample is removed from the chamber. This concentrated sample can then be reelutriated, if desired, to separate it into subpopulations. In addition to its separation capabilities, elutriation thus offers an alternative to concentration techniques routinely used with phytoplankton samples

a

Centrifuaal Force

+

b FIG.18. (a) Elutriator rotor. Cells in medium are pumped through tubing into the rotating elutriator rotor. Arrows indicate direction of flow. Redrawn with permission from Beckman Instruments, Inc. (b) Elutriation chamber. (1) Cells suspended in seawater medium are pumped into the chamber; (2) with centrifugal and fluid forces in equilibrium, cells are distributed according to sedimentation velocity; (3) as flow is increased, slower sedimenting cells are elutriated out of chamber, equilibrium conditions change, and remaining cells are redistributed.

210

CLARICE M. YENTSCH AND SHIRLEY A . POMPON1

(e.g., filtration and centrifugation). Quantification of the relative percentage of the population at outflow vs inflow continues to be a problem (Pomponi and Cucci, 1985). In addition to the advantages discussed above, elutriation causes little or no cell damage since cells are not subjected to high centrifugal force or pelleting, and relatively large numbers of cells ( lo7- lo9) can be separated and collected in a short period of time (less than 1 hour) (Mitchell and Tupper, 1977). The disadvantages of elutriation are that it requires moderately expensive instrumentation (the elutriator rotor), and that establishing elutriation conditions for different cell types can be time consuming. 7. Flow Cytometer and Sorter As described above, this method can also be used to enrich samples for subsequent analyses (Horan and Wheeless, 1977). Discrete populations are identified using FCM, and appropriate sorting windows are established to separate different subpopulations. The advantage of this method is that resolution of cells with extremely small physical, chemical, or physiological differences is excellent. The major disadvantage is that, compared with other separation methods, it is relatively slow. Separation of lo6 cells can often take one to several hours (Sweet, 1979). 8. Afiniry Chromatography This is a separation technique based on cell surface characteristics and the assumption that differences in these properties are related to differences in functional properties of the cells (Wilchek and Bayer, 1978; Ward and Kilpatrick, personal communication). Cells are passed through a column of glass or Sephadex beads which may be coated with a variety of substances, such as antigens, antibodies, adhesion inhibitors, or activators. Cell yields from this method are relatively low, but resolution of specific cell types can be high. 9. Cell Electrophoresis This is a separation technique based on the cell surface charge and the motility of charged cells in an electrical field. The technique is reviewed by Pretlow and Pretlow (1979) and Todd et a1 (1979).

IV. Cellular Probes Implicit in the various methods of individual cell analysis described in this review is the hypothesis that certain structural and functional characteristics of the cell can be measured and quantified. These characteristics include, but are not limited to size, shape, volume, index of refraction, texture or surface structure, the absolute or relative content of a compound (e.g., nucleic acids, pro-

AUTOMATED INDIVIDUAL CELL ANALYSIS

21 1

PROKARYOTES

I

"

sPECIA LI z ED" cyonobacterioI structures phycoerythrin )

EUKARYOTES

I

"SPECIALIZED" microolqol

icrobody (outof luorescence flovins ) mitochondrion (rhodomine 123 stoin )

FIG. 19. Basic features of prokaryotic and eukaryotic cells which can be exploited for fluorescence detection by image analysis and/or flow cytometry. Prokaryotes: phycobilisomes, autofluorescent; membrane potential; DNA stains. Eukaryotes: (1) chlorophylasts, autofluorescent; (2a,b) membranes for membrane potential; (3) DNA-numerous stains available; (4) mitochondrion, rhodamine 123 stain is useful; ( 5 ) ribosomes, site of protein synthesis, many stains available for various protein and subprotein molecules; (6) Golgi apparatus. Cell wall is present in plant eukaryotes. Reproduced by permission from Yentsch et al., 1986.

teins, antigens, pigments), membrane characteristics (e.g., surface charge, fluidity, permeability), viability, and cell function (e.g., enzyme activity, uptake and metabolism of compounds, cycling, differentiation, intracellular pH, endocytosis, phagocytosis). Natural, "specialized" features to exploit in prokaryotic and eukaryotic cells are given in Fig. 19. Cellular probes are used to produce an end product which can be detected and measured. Detection parameters include fluorescence, absorbance, scatter or polarization of light, radioactive decay, electrical charge, and acoustical sensing. Probes resulting in an optically detectable end product are generally cytochemi-

212

CLARICE M. YENTSCH AND SHIRLEY A. POMPON1

cal, based on the chemical and/or physical interactions between the probe and the cellular substrate. For the past century, cytochemical stains have been routinely used to study various tissues. Often, the stains employed were neither specific nor quantitative. The physical and chemical mechanisms underlying staining reactions were, in general, poorly understood. At the level of optical microscopy, however, these routine cytochemical probes were adequate and the results of staining reactions were predictable. As the level of resolution increased with better optics, fluorescence microscopy, and electron microscopy, we developed a need for more specific and quantitative probes. In the case of electron microscopy, chromogenic substances were of little use and specific electrondense probes were developed to react with lipids in membranes, enzymes in organelles, nucleic acids, proteins, glycogen, and mucopolysaccharides (Hayat, 1975). We have begun to reexamine the classical absorptive staining techniques of optical cytochemistry in an effort to understand the physical and chemical basis for staining reactions and thus be better able to modify or develop chromophores which are quantitative and specific for the cellular components we wish to study (Horobin, 1980). Absorbance of compounds often results in changes in the light scatter characteristics of cells and organelles, and thus allows the differentiation of one cell type from another. Such differentiation is not always from the detection of color, but rather from detection by optical sensors of absorbance or scatter. An example of an absorptive stain used in automated detection and analysis systems is the classic vital stain, trypan blue, which is excluded by live cells but stains dead cells, resulting in an increase in light scatter (Melamed et al., 1969). Haugland (1984a,b), Shapiro (1983, 1985), and Muirhead ef al. (1985) have recently reviewed the application of fluorescent probes for some biomolecules (Fig. 20). Haugland (1984b) divides extrinsic fluorescent probes into two types of noncovalent probes which form a reversible association with a combination of hydrophobic, dipole-dipole, and ionic interactions. The other probes-covalent probes which form much stronger chemical bonds with specific atoms on the biomolecule and are essentially irreversibly bound-are the major subject of his article. Of special note are the following three chemical moieties: (1) the reactive group of the biomolecule (protein, cell membrane, nucleic acid, polysaccharide, etc.), (2) a chemically reactive “handle” which is usually part of the fluorescent probe, and (3) a fluorescent chromophore with spectral characteristics appropriate to the problem under study. Each of these aspects is considered in detail, as are the solubilization, stoichiometry, removal of excess probe, and other considerations in appropriate labeling. Haugland’s treatment ( 1984b) of selective modification reactions, namely thiol, methionine, histidine, amine, tyrosine, and carboxylic acid modifications,

213

AUTOMATED INDIVIDUAL CELL ANALYSIS (nm) 300

400

DYE FLUORESCENCE SPECTRA:

SOURCE EMISSION (nm)300 I

HELIUM-CADMIUM LASER HELIUM-NEON LASER

500

Excitation

600

Emission

700

800

-

400

500

600

700

800

I

I

1

I

t

: k

FIG. 20. Spectral characteristicsof some fluorescent dyes and excitation sources usable for flow cytometry. Excitation.and emission lines are compressed; the top line of each defines a 15 nm region around the peak, the middle line connects the half-maximum points, and the bottom line connects points falling to one-tenth of maximum. Abbreviations: DAPI, 4’,6-diamidino-2-phenylindole; SITS, 4-acetamido-4‘-isothiocyanatostilbene-2,2’-disulfonic acid; DANS, dansyl chloride; FITC, fluorescein isothiocyanate;TRITC, tetramethylrhodamineisothiocyanate. Reproduced by permission from Shapiro (1983).

FIG. 21. tesy of J . I .

Actual light scattering pattern for a single Sfaurastrtcm (A) and Osciltuforia (B). Cour-

M . Forrest. Ispra, Italy.

AUTOMATED INDIVIDUAL CELL ANALYSIS

215

as well as modification of other residues in proteins, nucleic acids, polysaccharides, and glycoproteins, and modification by photoactivated fluorescent probes is careful. The reader is directed to Haugland's (1984b) and Shapiro's (1985) reviews for details of these important considerations. Although most of the effort in probes developed for static and flow analysis has been in the area of specific fluorescent compounds, we should not overlook the potential of using traditional absorptive stains to analyze cells on the basis of light scatter characteristics. Light scattering of cells is definitive (Fig. 21) and can be used for cell sizing in flow cytometry. Care must be taken in the choice of scattering angles and angular resolution, which differ markedly (Fig. 22) depending on the effectiveness of discrimination among subclasses of cells in a heterogeneous population (Salzman et al., 1979; Salzman, 1982; Trask et al., 1982; Spinrad, 1983, 1984; Spinrad and Yentsch, 1986). Polarization studies add a new dimension. As Salzman states, a biological cell is an optically active scattering object because it contains asymmetric molecules. Polarization phenomena can be exploited to explore changes in intracellular molecular conformations. There are four variables involved in polarization transformations: (1) the total intensity, (2) the degree of linear polarization of 0 or 90" with respect to the scattering plane, (3) the degree of linear polarization at k45" to the scattering plane, and (4) the degree of left or right circular polarization. These variables are modified by the beam's interaction with the cell and are subsequently detected. The parameters can be combined into a four-component vector and the interac-

FIG.22. Logarithmic scattering diagram showing angular variability. After data from Kullenberg (1968). Cell is at center. Light is from left.

216

CLARICE M . YENTSCH AND SHIRLEY A. POMPON1

tion with the cell described by a four-by-four Mueller matrix (Mueller, 1948). Investigators (Voss, personal communication) are examining Mueller scattering matrices for nonspherical particles. Circular intensity differential scattering (CIDS), pioneered at the National Flow Cytometry Center, Los Alamos, NM, exploits the fact that asymmetric structures and molecules preferentially absorb and scatter circularly polarized light (Atkins and Barron, 1969; Barron et af., 1973). Salzman and Maestre (1981) (in Salzman, 1982) have used a microscope system to show that the circular dichroism is sensitive to changes in DNA conformation as a function of cell cycle position. ANTIBODY RESEARCH

As early as 1933, visualization of an antigen-antibody reaction was achieved using fluorescent dyes (Visser and Van den Engh, 1982). Early problems with high background fluorescence, nonspecific staining, and considerable loss of serological specificity of the labeled antibody are now routinely overcome. In addition, the design of microscopes, filters, and flow cytorneters permits new approaches to both cell-surface and histochemical antibody detection. In the past decade there has been a rapid rise in the popularity of the antibodystaining technique in cell biology. The success rests in three aspects: (1) specific antibodies to virtually any macromolecule can be produced experimentally, (2) the technique is very sensitive, and (3) the fluorescent images allow one to map out the molecular anatomy of the cell to a resolution of about 0.5 pm (Fujiwara and Pollard, 1980). Monoclonal antibodies have received considerable attention and, indeed, offer many advantages. Basically, the antibodies are produced in cell culture lines (hybridomas), typically a hybrid cell from a mouse spleen which is producing an antibody and an easily grown cell culture line. Thus, the production of antibodies is independent and continuous after the animal initially injected is sacrificed. Monoclonal antibodies are also typically screened for complete specificity. The screening process is very labor intensive and the initial product amount is generally far less than traditional polyclonal antibodies. Choosing monoclonal antibodies is, therefore, not always an advantage. Published antibody research in biological oceanography, to date, has been limited to surface labeling of antibodies, basically reacting to, and thereby discriminating, nitrifying bacteria (Ward and Perry, 1980) and various cyanobacteria (Campbell et a/., 1983). in each case, different antibody reactive types have been identified and mapped both vertically and horizontally. Such approaches offer great promise. Research in progress involves raising antibodies to intracellular enzymes, a

217

AUTOMATED INDIVIDUAL CELL ANALYSIS

feat routinely accomplished in analytical cytology. The antibodies penetrate the plasma membrane and react within the cell. Ribulose-biphosphate carboxylaseAb (RUBPCase-Ab) is being produced (M. J. Perry, personal communication) with promising applications to primary productivity research. An antibody to algal nitrate reductase is under production (W. M. Balch, B. Reguera, and C. M. Yentsch, unpublished). Enzyme antibodies permit characterization and quantification of the enzymes present against time. This feature, over time, permits assessment of turnover rates.

V. Standards, Controls, Data Interpretation Appropriate interpretation of individual particle analysis in the aquatic sciences depends on strict adherence to protocols for standards and controls (Phinney et al., 1986). The more the observer is removed from direct observation, i.e., away from image analysis to flow cytometry, the more critical the appreciation and use of standards and controls in a rigorous fashion. Indeed, an understanding of an “event” is paramount. Knowledge of how an event is created and stored is important. There are good vs bad events and indirect methods for discrimination exist (Fig. 23). Instrument noise can result from electronic noise and/or sheath background particles. Instrument drift can be monitored using instrument standards. Additionally, controls are essential to ensure proper analysis. Controls used in biomedical research on mammalian cells [e.g., chick red blood cells (CRBC)] while useful in analyses of some aquatic metazoan cell types are inappropriate for phytoplankton. In the case of a DNA stain, various species of phytoplankton react in a far different manner from each other as well as from CRBC (Phinney and Yentsch, unpublished data). 12E

-A-

-B-

4 c

+

u)

t

t

aJ 0) w

u-

0

t

n

E,

Z

Relotive Chlorophyll Fluorescence

FIG. 23. FCM analysis with EPICS V. “Population” A is, in fact, noise and would be misinterpreted without sorting for verification and appropriate standards and controls. From September 6, 1984, at 43”20’N, 69”W, 11 m, in the Gulf of Maine aboard the RIV Cape Hatteras.

218

CLARICE M. YENTSCH AND SHIRLEY A. POMPON1

Additionally, the photosynthetic pigments must be extracted, or compensated for, in analyses. Some absolute measurements are now becoming feasible due to volume calibration beads and fluorescent equivalent beads for chlorophyll and phycoerythrin. These are under development (FCM Standards, Research Triangle Park, NC) and assessment (Phinney and Cucci, unpublished).

VI. Phylogenetic Overview A. PROKARYOTES

Aquatic Bacteria

There have been studies of aquatic bacteria which involved the staining of various cellular components, such as DNA, RNA, and protein. These studies approach the characterization of cellular types based on cellular constituents. Robertson and Button (personal communication) distinguish cellular types. Sheldon (personal communication) identifies the less than 0.5 pm equivalent spherical diameter fraction achieved via a Coulter counter as bacteria. This distinction is of particular importance in that he separates out size/trophic levels via filtration and then permits growth vs grazing to occur over a time frame. Time course samples provide data on the in situ growth/division rates. Studies by Ammerman et at. (1984) and Sieracki et al. (1985) have provided new insights. An interesting innovative approach for the characterization of bacteria is an immunofluorescent assay of the nitrifying bacteria (Ward and Perry, 1980). A fluorescently tagged antibody permits automated characterization, quantification, and distribution patterns of the particles of interest. The Ward and Perry research was the introduction of antibody techniques into biological oceanography and limnology. Its importance was to set the stage for the utility of the immunochemical approach. The antibody in this instance was polyclonal to surface protein antigen. B. CYANOBACTERIA

Campbell et al. (1983) pioneered the immunochemical approach to autotrophic cells, namely the cyanobacteria. Again, the antigen was surface proteins. In fact, over five antisera were produced permitting typing of both clonal cultures and natural coastal and oceanic populations. Oceanographic mapping of the distribution of these antibody-reactive types has yielded horizontal, vertical, and seasonal patterns of great interest. When fluorescent antibodies are used with autotrophic cells, the excitation and emission spectra of the fluorescent label must not overlap to any great extent with any photosynthetic pigment for two reasons. First, discrimination, especially automated discrimination, requires a unique spectral signature to avoid ambigu-

AUTOMATED INDIVIDUAL CELL ANALYSIS

219

ity. Second, energy transfer can occur between the pigment and the probe, or vice versa, resulting in either false high or false low intensity for the fluorescence of the fluorochrome-antibody complex or for the photosynthetic pigment. Therefore, great care must be taken in the selection of the fluorochrome and the filters for the sensors of automated systems. For the most part, antibody preparations are successfully preserved in glutaraldehyde or formalin. Researchers observe little change if the analysis is within a few months after sample collection. Pigment autofluorescence is not as successful, however, particularly with the water-soluble phycobilin pigments. The phycoerythrins, phycocyanins, allophycocyanins, and phycourobilins are the major pigments of interest. It is these same water-soluble phycobilin pigments present in the cyanobacteria which are now ever-increasinglyused as fluorescent tags for antibody molecules, particularly in biomedical applications. The phycobilin pigments each have a variety of molecular forms, each with distinct excitation and emission spectra. Therefore, flow cytometric characterization of various cyanobacterial strains/forms is automatically distinguishable with prudent use of sharp cut-off filters and laser excitation. One such example is the research of Wood in Yentsch et al. (1983a) with further sophistication by Wood et a/. (1985) (Fig. 24). Olson et al. (1985) have taken a Coulter EPICS V flow cytometer/sorter to sea with particular attention to the distributions of cyanobacteria. This was relatively easy to accomplish due to the fact that cyanobacterial concentrations, even in the open ocean, are nearly at the ideal range (lo5 to lo6cells/ml) for flow cytometric analysis without prior concentration of the cells. Absolute cell densities were determined by the addition of known quantities of standard beads which fall into channels outside the normal range of the cells of interest. This study reports pigment content, as estimated by fluorescence of phycoerythrin, and is measured as opposed to calculated on a per cell basis. They found that cells near the bottom of the euphotic zone had more fluorescence per cell than cells near the surface. Whether this represents the photoadaptation of one species or the replacement of one species by another species of cyanobacteria was not determined, but it is one of the most important questions posed in aquatic biology today. Image analysis is the automated-techniqueapproach used by Sieracki and coworkers at the University of Rhode Island and at the Virginia Institute of Marine Science. Since this is a static as opposed to a flow handling system, fewer cells can be analyzed, yet additional parameters, such as particle size, can be accurately determined. One other major advantage of the image analysis approach is the differentiation of cyanobacteria cells vs detritus. The visual image is semiautomatically screened and involves an operator who observes the images. This is a very important feature, particularly in the cell size range of less than 3 p,m diameter. The discrimination of importance to aquatic ecosystem interpretation here is under human control.

220

CLARICE M. YENTSCH AND SHIRLEY A. POMPON1

Log Green Fluorescence

Log Red Fluorescence

I

a, 2000~:

a

II I000 a,

0

Channel Number

FIG. 24. Distribution of cells in exponentially growing populations of phycoerythrin (PE)-containing Synechococcus spp. with respect to green fluorescence (A,C,E) and red fluorescence (B,D,F). A linear increase in channel number corresponds to a logarithmic increase in fluorescence intensity. Each line represents a different clone; those containing the Type I PE are shown with a solid line and those containing the Type I1 PE are shown with a dotted line. In A and B, the 514 nm laser line was used for excitation, green fluorescence was measured between 540 and 590 nm, and red fluorescence was measured from 590 to 700 nm. In C and D, green and red fluorescence were measured as above, but the 488 nm line was used for excitation. In E and F, the 488 nm laser line was used for excitation, green fluorescence was measured between 540 and 560 nm, and red fluorescence was measured from 560 to 700 nm. In all cases, laser power was lo00 nm, forward angle light scatter (FALS) amplifier gain was 5; green PMT amplifier gain was 20; and red PMT amplifier gain was 10. High voltage for the red PMT was set at 700 in all cases. For the green PMT, it was 700 when samples were excited at 5 14 nm and 850 when samples were excited at 488 nm. Fifty thousand cells were analyzed for each panel (Wood er al.. 1985).

C. EUKARYOTES

Microalgae

Microalgal species are most commonly distinguishable by cellular volume (Sheldon and Parsons, 1967; Sheldon et al., 1972; Sheldon, 1978), cellular configuration (Salzman, 1982; Uhlmann et al., 1978; Salzman et al., 1979; Spinrad and Yentsch, 1986; Tsuji and Nishikawa, 1984), accessory pigment

AUTOMATED INDIVIDUAL CELL ANALYSIS

22 1

content (Yentsch and Yentsch, 1979), or a combination of the above (Trask et al., 1982; Paau et al., 1978, 1979). Staining for internal cellular components has been undertaken (Falkowski and Owen, 1982; Trask et al., 1982; Yentsch et al., 1983a,b; Olson et al., 1983). In some cases, unique nonfluorescent molecules became fluorescent under altered conditions, as in the case of cellular saxitoxin in toxic dinoflagellatesupon the addition of H202 (hydrogen peroxide) (Yentsch, 1981). To date, antibodies to cell surface proteins of eukaryotes have not been exploited, however, the approach is indeed promising and some antibody generation is in the process at the time of this writing (Yentsch and Horan, unpublished). It is likely that some groups, such as coccolithophores with their calcium carbonate cell wall, will never have antibodies produced to the cell surface. Cellulose, glucopolysaccharide, and sporopollenin are good candidates for antibody production, however. A generalized table of cell surface materials is presented in Table 111. Successful polyclonal antibody production for intracellular enzymes has been accomplished and is in a final phase of development and application. The first such antibody is to the enzyme RUBPCase (Perry, personal communication), an enzyme essential in photosynthetic carbon fixation. Cell-by-cell analysis of this enzyme plus chlorophyll fluorescence per cell and cell volume will have major implications in expanding our understanding of cells in the aquatic environment. Yentsch and colleagues (unpublished) are attempting to prepare antibodies to the enzymes involved in nitrogen transport [nitrate reductase (NR)] and assimilation [namely glutamine synthetase (GS) and glutamine synthesis dehydrogenase (GDH)]. Preparation of antibodies to the enzymes involved in phosphorus transport and assimilation is an obvious extension in cellular studies from freshwater. These new approaches offer promise for biochemical measurement on individual cells. Special attention should be paid to variability and distribution of cells within the population. TABLE I11 COMFWSITION OF CELLWALLSOF VARIOUS MICROALGAE: A REPRESENTATIVE LISTING Alga

Common name

Chlorophyta Chrysophyta Cyanophyta Euglenophyta Phaeophyta MOPhYta Rhodophyta

Greens Diatoms Blue-greens Euglenoids Browns Dinoflagellates Reds

Cell wall composition Cellulose, pectin, occasionally with CaC03 Cellulose, pectin, silicon Cellulose, pectin None Cellulose, pectin, algin, fucoidin Cellulose, pectin Cellulose, pectin, mucilages (like agar), mannan, some with CaC03 or MgC03

222

CLARICE M. YENTSCH AND SHIRLEY A . POMPON1

Internal cellular pH stains (Dixon and Webb, 1964) and membrane potential stains (Waggoner, 1979) offer great promise for the microalgal researcher. Again, care must be exercised in the selection of stains so that the spectra do not overlap with pigment spectra. Researchers have suggested photobleaching of pigments (Yentsch et a [ . , 1983b) or methanol extraction (Olson et al., 1983) to bypass the interference problems, but obviously this limits the researcher to nonliving, preserved cells. For measurement of most turnover rates, healthy viable cells are essential, hence the preference for vita! stains. A few researchers have had some success with special attention and preservatives which permit cell-by-cell detection of pigment fluorescence on the nonliving samples (Haas, 1982). Based on our experience, summarized in Table lV, from Yentsch et a/. (1986), we are not enthusiastic about preservatives. Currently, there is major emphasis on species-specific growth rate, cell division rates, photosynthetic rates, nutrient uptake rates, etc. R. Knoechel has achieved increased understanding of individual species of the phytoplankton natural assemblage by track autoradiography. He and co-workers have refined track autoradiography on microscopy slides to permit bright field species identification of the cell plus emulsion development of the numbers of tracks emanating from each cell. In this way he derives a species-specific 14C uptake. This approach is perfect for extension to image analysis. The technique is most successful on cells greater than 3-5 p m . Knoechel has demonstrated the practicality of this approach and concludes that a major proportion of the carbon fixed is by a relatively small number of cells in the population. Knoechel has worked in both temperate eutrophic and tropical oligotropic waters with far greater success in the former (personal communication). B . Sweeney, as early as 1960, was conducting research on individual cells of Gonyaulaxpo/yedra (Sweeney, 1960) (Fig. 25). Hastings et al. (1962) followed

TABLE IV ATTLMITS

A I PRtSERVATlON Ol- VARIOUS PHYTOPLANKTON PIGMENT GROUPS AND

S~.BSEQUENT FLOWCYTUMETRIC ANALYSIS Greens. diatoms, dinoflagellates

Lugol's with subsequent bleaching Glutaraldehyde + cacodylic acid Glutaraldehyde" Liquid N?( BNG, not good; OK, acceptable. hFrom Haas cf a / . (1983). 'From K . Davis (personal communication)

NGi OK" OK OK

Cryptonionads

Cyanobacteria

NG NG

NG NG NG OK

?

NG

AUTOMATED INDIVIDUAL CELL ANALYSIS

0

1

2 3 4 5 CELLS/ VIAL

223

6

FIG. 25. Pyrocystis noctiluca. Carbon uptake as a function of number of isolated P . nocriluca per scintillation vial during a 1.2-hour in situ incubation at the depth of the 42% isolume at Station 3 (28 Aug. 1980). Each value is mean -f- SEM (n = 12-18). From Rivkin et al. (1982).

along these lines of investigation and continued with bioluminescence measurements of single cells. Rivkin (1985) and co-workers (1979, 1982, 1985) have progressed the furthest in nonautomated individual cell analysis. Their ambitious research has given insight into details of diatoms (Rivkin et al., 1986) and dinoflagellates by using single cells, or very low number single species isolates, from the natural environment. After measurement of carbon assimilation, a regression is performed (Fig. 26). The picture that is emerging shows that species differences can account for up to three orders of magnitude variability in cellular rates, cellular composition, and cellular volume. If aquatic researchers fail to acknowledge or consider this, an incomplete understanding of both marine and freshwater systems is certain to result. Single-celled measurement of the flash from bioluminescence has been studied more frequently. This originated with the pioneering, but definitive, work of Sweeney and Hastings (1958) (Fig. 25). It has been expanded upon by Krasnow et al. (1981) (Fig. 27) and Lapata and Losee (1984). The advent of video has revolutionized much of the microscopic and macroscopic analysis. It is most essential when following a single cell in work such

224

CLARICE M. YENTSCH AND SHIRLEY A. POMPON1

HOURS AFTER DAWN -I

0

1000

2000

3000

LIGHT INTENSITY IN FOOT CANDLES FIG. 26. Early example of information obtained by single cell measurements. Photosynthetic rhythm in single cell of Gonyaular polydra. From Sweeney (1960).

as behavior studies and even differentiation studies. The features of time control, slow motion, fast forward, and frame-by-frame playback permit measurement rates of activities such as feeding (F. J. R. Taylor, personal communication; G. Gaines, personal communication), swimming (P. Ortner, personal communication), pigment fluorescence bleaching (C. M. Yentsch, unpublished), and stain reactions. Unspecialized recording-playback systems including video camera and video cassette recorder (VCR) can be purchased for less than $1000 (US). For microscopy adaptation, this is no more than doubled. If fluorescence vs bright field resolution is necessary, then one needs a low light detector and the cost is substantially increased, as is the case for highly controlled, accurately timed frame-by-frame VCRs. One author (C. M. Yentsch) has tested several models for color fluorescence resolutions. The best system viewed to date is the Medicam unit by Zeiss. Twocolor fluorescence was apparent on cells of the large (30 Fm) dinoflagellate Protogonyaulux rumurensis var. excavuta (clone GT-429, CCMP) with the chloroplasts appearing red and the accumulation body bright yellow. At present, this system would not be advisable for much smaller cells. In our laboratory, clonal cultures have been established after sorting, yet data indicate that anywhere from a 10 to 50% decrease in cell physiology of phytoplankton I4C uptake (Cullen and Yentsch, unpublished; Rivkin et al., 1986) can be expected from postsorting, depending on laser power.

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FIG. 27. (A) Four “events” from a single isolated cell at different times during the 12 hour constant dark cycle (after the L phase of the 12:12 L:D cycle). Measurements were made by computer-aided photon counting, as described in text, with single cells micropipetted and placed in a 3 ml vial suspended before the light-collection optics of a Packard scintillation counter. The electronics of the scintillation counter were bypassed, and the phototube signal was sampled by an ORTEC photon counting apparatus, which was, in turn, controlled and read by a NOVA-1200 computer. These events are notable for their similarity to “classic” flashes (but contain smaller numbers of photons), as well as for the temporal structure they exhibit (the variations are not due to counting statistics). (B) An example of a long-lived event from a single cell. (C) Record of spontaneous luminescence from single isolated cell, showing flashes and glow. The same protocol was used as in A. Flashes are not to scale (in the high gain mode used to measure glow, flashes are counted modulo 9999). The glow curve is remarkably similar to that of population. Not all cells had a glow peak, nor did all cells flash. Examples were found where either, neither, or both glow peaks and flashing occurred. Courtesy of Krasnow et al., 1981.

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The basic flow cytometerlsorter instrumental design, a result of the objective of sort purity, has resulted in computer-controlled, anticoincidence feedback loops which result in highly pure sorts to the right and to the left. The reject container (Fig. 11, part 3B), however, is in no way a “pure” sample. Contents will include (1) particles failing to meet sort criteria, (2) particles which meet sort criteria but are rejected due to coincidence and/or improper spacing between droplets, (3) debris, and (4) excess sheath fluid. To clear the reject container of particles which meet sort criteria but are rejected due to coincidence, the reject container must be resorted, a process which, in some cases, must be done repeatedly. Investigations of the evolutionary and ecological consequences of genetic variation in plankton (Murphy and Guillard, 1976; Gallagher, 1982; Brand et al., 1981) has been facilitated by the combination of single cell analysis, clonal culture techniques, and quantitative genetics. Single cell analysis of individuals within a clonal culture provides an estimate of the phenotypic mean and variance associated with a genotype. When analyzed for multiple clones using standard quantitative genetic techniques, these data can be used to estimate variance components, the heritability of different characters, and the degree to which a given character can be influenced by natural selection. This approach has been used to estimate environmental and genetic components of variance in diatom frustule morphology (A. M. Wood, unpublished; R. S. Lande and G. A. Fryxell, unpublished; Lynch, 1984a,b) and in studies of zooplankton evolution.

D. HETEROTROPHIC NANOPLANKTON Stoecker et al. (1986) demonstrated that flow cytometry is useful in studying the grazing preferences of three species of dinoflagellates by ciliates. Techniques for the enumeration of heterotrophic and phototrophic nanoplankton using epifluorescence microscopy have been published by Shen and Sherr (1982) and Caron (1983).

E. METAZOANS As discussed above, individual cell analysis of aquatic metazoans generally requires that the organisms or tissues be dissociated into single cells. Exceptions include the analysis of blood and reproductive cells, which, in most instances, are already monodisperse. Research on reproductive cells, larvae, and blood or coelomic cells is not within the scope of this review. Such studies are included only if cells have been analyzed using the techniques described above. The groups of metazoans to which the analytical methods described in this review have been applied include sponges, cnidarians, crustaceans, molluscs, and fishes. The general objectives of individual cell analysis in these groups have been to examine cell recognition, reaggregation, differentiation, and development; to

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determine the functions of specific cell types; to study selective feeding activity; to examine the pathology of commercially important fish and shellfish; to measure ploidy levels; and to determine karyotype.

F. SFQNCES By virtue of their primitive level of organization, sponges are perhaps the metazoans best suited for individual cell analysis. Dissociated cells remain viable and will reaggregate into a functional sponge. Since the early work of Wilson (1907), sponges have been the classic model of multicellular systems for the study of invertebrate cell recognition and immune responses, reaggregation, differentiation, and development. Most of these studies, however, do not involve automated cell analysis and are thus not within the scope of this review. They are thoroughly reviewed by Van de Vyver (1975, 1979), Turner (1978), Muller (1982), and Simpson (1984). Burkart and colleagues (1979) used fluorescence-activated cell sorting to sort fractions of autofluorescent gray cells of Microciona prolifera for subsequent studies of surface proteins and lipid content (Fig. 28). Research is in progress in our laboratories to develop techniques for differentiating cell types using flow cytometry. Preliminary analyses of crude cell preparations of Anthosigmella varians resulted in discrimination of sponge cells from algal symbionts on the basis of chlorophyll autofluorescence, and choanocytes from larger cells (archaeocytes, gray cells, and spherulous cells) on the basis of forward angle light scatter and fluorescence of phagosomes stained with acridine orange (Yentsch et al., 1983a; Pomponi, 1983). We have also successfully distinguished subpopulations of spherulous cells of Cliona truitti on the basis of fluorescence of lipid

FIG. 28. Cytogram of Microciona proliferu cells based on scatter signal (S) and fluorescence (F). From Burkart et al. (1979).

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inclusions stained with phosphine 3R. The ultimate goal of these studies is to understand the role of different cell types in the excavation of calcium carbonate substrates by boring sponges (Pomponi, 1979, 1980).

G. CNIDARIA The model system for the study of cnidarian cells is the hydroid Hydra spp. Methods used to dissociate, culture, and analyze Hydra cells are summarized in Lenhoff ( 1983) and include research on quantitative cytology, aggregation, differentiation, and development, structure and function of nematocysts, and the physiology of symbiotic relationships. Most of the Hydra research, however, employs only the dissociation and separation techniques reviewed above. Automated analytical methods have been used in only a few studies (Arndt-Jovin and Jovin, 1974; Amdt-Jovin er al., 1976). Recognizing that most differentiating cell systems are heterogeneous mixtures in which not even cells of the same type may be synchronously cycling, ArndtJovin and colleagues ( 1976) developed staining techniques to selectively measure different cell types and to distinguish the position of cells during reassortment. They differentiated interstitial from ectodermal cells on the basis of cytoplasmic mass using rhodanile blue, labeled vacuoles in endodermal cells with dansyl amide and dansyl azridine which are specific for sulfhydryl groups on proteins, and selectively stained nematocytes and outer cell walls of ectoderma1 cells with fluorescamine. Cells were analyzed with a cell separator on the basis of polarized fluorescence emission and forward angle light scatter (ArndtJov& and Jovin, 1974). Their work is significant not only for the technical developments in the study of invertebrate cell systems, but also for the potential to use such methods to address questions in invertebrate cell recognition, differentiation, and development.

H. CRUSTACEA Feeding activity of marine zooplankton has been studied using both Coulter counters and flow cytometry. Copepods are fed cultures of phytoplankton of different concentrations and size ranges. Feeding rates are determined by Coulter counts of the media in which the zooplankton are feeding (Frost, 1972, 1975, 1977; Cowles, 1979; Cowles and Strickler, 1983). Selectivity varies directly with the concentration of food and the availability of food particles (phytoplankton species) of different sizes. Algal species preferences of the copepods are determined by measurement of algal pigment autofluorescence in media preand postfeeding. Jeffries and co-workers (1981, 1984) have used image analysis to size, count, and identify zooplankton.

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I. MOLLUSCS

1. Selective Feeding Selective feeding by filter-feeding bivalve molluscs has been analyzed by flow cytometry (Cucci et al., 1985a,b; Shumway et a f . , 1985). Mixed cell suspensions of algal cultures were fed to Ostrea edulis, Ensis directus, Placopecten magellaniius, Arctica islandica (Shumway et al., 1985), and Mytilus edulis (Cucci et al., 1985b). By FCM analysis of autofluorescent algal photosynthetic pigments in media pre- and postfeeding, gut contents, pseudofeces, and feces, selective mechanisms were discovered to occur during ingestion, digestion, and egestion (Fig. 29). In addition, in the presence of the toxic dinoflagellate Gonyaulax tamarensis, bivalve feeding rates decreased (Cucci et al., 1985a). 2. Pathology Reinisch and colleagues (1983) were the first to generate monoclonal antibodies to marine invertebrate neoplastic cells. Antibodies were raised to hematopoietic neoplasms in the soft shell clam Mya arenaria. The antibodies were screened by flow cytometry, and subsequent samples of clams were analyzed for neoplasms by microscopy and flow cytometry of the immunofluorescent neoplastic hemocytes (Fig. 30). They suggest that an important application of this technique is the identification of structural cell surface molecules and their relationship to cell differentiation and cell recognition. Cheng and colleagues (1980) identified surface receptors on oyster hemocytes by separating the cells on sucrose density gradients. Surface receptors of several subpopulations of hemocytes were analyzed by agglutination with plant and animal lectins. Subpopulations initially separated on the basis of diameter and density could be further divided on the basis of differences in specific surface binding sites. Further research on a hemocyte membrane-associated lectin suggests that this protein may function as a receptor in recognition of self (Vasta et al., 1982). 3. Pofyploidy The market quality of commercially important shellfish deteriorates during the breeding season. Since polyploid individuals are unable to reproduce, growth and market quality can be improved by induction of polyploidy (Stanley et al., 1984; Tabarini, 1984). Additionally, polyploid individuals often grow more rapidly than diploid individuals (Hidu, personal communication). To determine if polyploidy was established in individual oysters (Stanley et al., 1984) and scallops (Tabarini, 1984) treated with cytochalasin B to block meiosis, hemolymph cells were fluorescently stained for DNA content and analyzed using flow cytometry. Previous methods involved microscopic selection of embryos in

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FIG 29 Summary of the percentage of the total cell count within an algal mixture of the clones Phaeo, diatom Phaeodacijlitm rricornutum (stnped bars), 3C, cryptomonad Chroomonas salinas (stippled bars), and Exuv, dinoflagellate Prorocenrrrrm mmimum (solid bars) at times 0 , 30, and 60 minutes within the pseudofeces (PF) and feces (F) dunng the grazing experiments using six marine hlvalve species

which chromosomes could be seen well enough to distinguish diploidy from polyploidy (Stanley ef al., 1981). Flow cytometric analyses have increased both the efficiency and reliability of polyploid counts.

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FI uorescence Intensity

FIG. 30. HN tumor cells (10 X lo6) were incubated with NMS (- - -) or monoclonal antibodies 3H5C (-), 5A4A (...), 5A6A (-), or ID7A (---). The cells were then incubated with FITCrabbit anti-mouse Ig. From Reinisch (1983).

J. FISHES 1 . Polyploidy Automated individual cell analysis in ichthyology has been concentrated in two areas, screening for polyploidy and cytogenetics. Polyploidy measurements are similar to those of shellfish described previously. Blood cells from salmon (Utter et al., 1983; Johnson et al., 1984), rainbow trout (Solar et al., 1984; Thorgaard et al., 1982), and carp (Allen and Stanley, 1983) were stained for DNA and measured using flow cytometry (Fig. 31). One study (Johnson et al., 1984) actually compared the reliability of a Coulter counter with a flow cytometer in measuring ploidy. Their conclusions were that while both techniques could be used to screen populations for polyploidy, flow cytometry unequivocally identified polyploidy, while there was 11% error with Coulter counts. They suggested that because of availability, Coulter counters be used as preliminary screening devices.

2. Cytogenetics A powerful application of flow cytometry in mammalian research is the analysis of chromosome preparations to the study of karyotypes and genetic disorders. This suggests the application of FCM methodologies to the study of systematics, evolution, and genetics in other groups of organisms. Gold and Price (1985) have used scanning microspectrophotometry of nuclear DNA content to examine genome size variation among five closely related species of North American cyprinid fishes. Their results suggest that there is no direct relationship between

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FIG.31. Flow cytometric histograms of DNA fluorescence of diploid (F2n) and triploid (F3n) fish erythrocyte nuclei. Chick red blood cell nuclei (CRBC) are used as an internal standard. All nuclei types are in one suspension. Modal channel number (inside histograms) depends on ploidy of fish assayed. Redrawn with permission from Solar er a/. (1984).

genome size variation and speciation within this group. Their study is the beginning of a long-term investigation of genome size variation. Research in progress employs flow cytometric techniques for karyotyping (Gold, unpublished).

VII. Addressing Aquatic Research Questions Biological oceanographers have been convinced of the importance of partitioning trophic levels for decades. Sakshaug (1980) summarizes this nicely in his review chapter entitled “Problems in the Methodology of Studying Phytoplankton. He states that pelagic communities constitute numerous groups of organisms: bacteria, phytoplankton, zooplankton, etc., with a wide range of sizes within each group. This feature raises persistent problems with regard to the determination of chemical properties for the various groups of organisms. The problem of separation between groups of organisms and detritus is far from adequately resolved. Sakshaug continues: ”

In phytoplankton ecology, in particular when dealing with chemical methods, it is crucial to separate groups of organisms from each other and from detritus (dead organic matter) so that chemical data can be assigned correctly to the various groups of organisms and a correction made for detritus. This is one of the most difficult methodological problems in phytoplankton ecology. . . . Problems increase with maturity of a community. Thus, the simplest case to

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handle is a unialgal bloom and the most difficult one the complex oligotrophic warm water community close to climax.

Partitioning of natural populations, although heretofore restricted to fractionation based on size/filter retention, has advanced our understanding of the marine ecosystem (Derenbach and Williams, 1974; Durbin et al., 1975). Now, fluorescence-activated cell sorting is possible. To date, there have been successful sorting experiments to partition autotrophs and heterotrophs, based on exploiting chlorophyll plus phycoerythrin autofluorescence, prokaryotes and eukaryotes, based on phycoerythrin content of prokaryotic cyanobacteria, two distinct forms/species of cyanobacteria from each other, based on differences in phycobilin chromophores present in the cells (Wood et al., 1985), and viable vs nonviable cells, based on the exclusion of nucleic acid stain propidium iodide by live cells. So, in approaching aquatic oceanographic questions, we must be aware of the following basic constraints: 1. We can have two relatively pure sorts per pass of sample through the instrument, yet some of the desired particles may appear in the reject container. 2. Sample volumes are not conserved due to sheath dilution, thus sheath fluid should be of identical osmotic and chemical characterization to the sample. 3. While the instrument is exceedingly rapid, time is required for sorting. Just what sort time is permissible (seconds, minutes, hours, days) for appropriate experimentation will certainly vary according to experimental objective. In combination with the capability to partition natural populations by fluorescence activation, there is another significant advantage to flow sorting. We know precisely how many cells or particles are sorted into either the left or right vessel. This is important for all subsequent measurements on the sorted cells. Thus, the 10-20% error common in cell counting via microscopic estimates of small aliquots is eliminated. To reiterate, we know precisely the number of sorted particles and the fluorescence intensity upper and lower limits of each; however, the sample volume (not to be confused with cell volume) is neither measured nor conserved. Sorting has been used for experimentation even when using a clonal culture so that cell numbers can be known precisely. Such sorting is essential to establish the minimum number of cells on which reliable bulk measurements can be attempted. This is especially important for intercalibration among various flow cytometers, image analyzers, and with more traditional methods for C, N, and chlorophyll analysis per cell. Questions regarding the variance of marine microbes have not surfaced in a major way to date, primarily because oceanographers have lacked the tools to analyze individual cells/particles (Yentsch and Yentsch, 1984). The sole excep-

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tions are the Coulter particle volume detector which categorizes particles strictly by a change in resistivity (Coulter volume), and microspectrophotometers and microspectrofluorometerswhich permit quantitative measurements on individual cells based on absorption or fluorescence capabilities. Coulter volume sizing has resulted in major observational and theoretical advances as has microspectral research. Yet, the data, for the most part, have not been subjected to rigorous statistical analysis of variance. Major limitations of the use of Coulter volume singly are (1) contamination by detritus and (2) inability to resolve trophic level (i.e., autotrophs vs heterotrophs), or even biogenic vs abiogenic origin. Major limitations in microspectral work are the small population size measured in which n is frequently less than 100 individuals. Recent work suggests that variance and variability are key dimensions in biologically important chemicals, cells, organisms, populations, and ecosystems (Conrad, 1983). While the supporting theory of adaptability has been tested in some ecosystems, it has received little attention in the marine ecosystem to date. This is not benign neglect, but instead, due to the inconvenience of the tools with which to explore such phenomena. To be convinced of the utility of flow cytometry, one must be informed of the time scales of fluorescence from photosynthetic pigments per se, transient changes of fluorescence, and the assumptions made based on the knowledge of time of fluorescent reactions and time in the flow cytometer “interrogation region.” In Fig. 32, the time scale of fluorescence changes noted for photosynthetic pigments in phytoplankton are given on a log scale based on 1 second. The rate of fluorescence generation in the singlet state of chlorophyll requires lop9seconds. The rate of supply of one quanta to one molecule of chlorophyll is lo-* seconds. Transient effects occur on a time scale of seconds and fractions thereof. Reversible shielding from high light effects, that is, near full sun and above (the so-called sun glass effect of Sakshaug et al., unpublished; Vincent, 1979) occurs over minutes. Changes in the chlorophyll and/or accessory pigments per cell induced by suboptimal light occur after one cell division, which, on the time scale of a generation, is days or fractions thereof. It is important to point out that the length of time a cell is in the light field of a flow cytometer (the time between fluorescence excitation and fluorescence emission) is approximately seconds. Therefore, it is true that using flow cytometry, an accurate assessment of the chlorophyll fluorescence state at the time the cell enters the light field is achieved. This is not true using image analysis, fluorescence spectrophotometry, or in vivo fluorometry on a bulk sample; in all of these techniques the cells are exposed to the light field for seconds or multiples thereof. It is important to realize that reversible fluorescence effects, transient effects, DCMU, or other photosystem inhibitor effects are measurable using flow cytometry but only when the variable treatment is applied prior to the entry of the cell into the flow cytometer instrument itself.

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Chonges In chlorophy// and/or occesswy pjgmenfsper eel/

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FIG.32. Time scale (as fraction and multiples of a second) of fluorescence changes noted for photosynthetic pigment in microalgae.

Traditionally, process-oriented sea-going research often involves a suite of measurements made over hours to days in a region assumed to be homogeneous with respect to species assemblages. Observed variations in metabolic rates and biomass within vertical profiles or throughout the course of a day are commdnly attributed to environmental factors such as nutrients and light. Metabolic rates and biomass measured on the whole population may change with time in complex patterns which are difficult to interpret. Yet, inferences made from bulk measurements of the whole assemblage of organisms are attributed to the individual cells comprising the populations. If the relative abundance of species is changing during the course of these

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measurements (Fig. 33), then the observed variation may not apply to any individual. Indeed, the individual cells may exhibit little or no variability in cell properties (Fig. 34), metabolic rates, or biomass, or their variability may be predictable. Now, individual cell measurements can be made at sea. Studies concentrating on the simultaneous measurement of properties in individual cells, with special reference to the effects of nutrients and light, are now underway and represent the first comprehensive, systematic approach to further aquatic researchers' attempts to acknowledge and understand heterogeneity in phytoplankton populations with the goal of eventually predicting chlorophyll a fluorescence from oceanic variables. Because patterns at the individual lcvel may be simpler than composite population patterns, both theorcfical and empirical research may be unnecessarily complicated by failure to recog nize the effects of heterogeneity. (Vaupel and Yashin, 1985)

Spatial and temporal variability in oceanic phytoplankton may be attributed to two related but distinct causes-one is the response of individual cells to environmental changes in light and nutrients, and the other is the variability in species assemblages. On time scales of weeks to months and spatial scales of hundreds of kilometers or more, differences in the complexity and composition of the marine food webs account for most of the variability. On smaller time and space scales it becomes more difficult to separate these two causes of variability (J. W. Campbell, personal communication). Instrumentation has changed dramatically in the past few years and the biomedical research tools. the image analyzer and the flow cytometer/sorter, are

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rapidly being employed and accepted in biological oceanographic studies (Yentsch et al., 1983a,b; Olson et al., 1983; Yentsch and Yentsch, 1984a), both in the laboratory and in field studies. They are rapid, sensitive, and capable of measuring multiple parameters simultaneously. Automated individual particle analysis methods should become tools of major importance for autoecological studies on pure cultures of bacteria, phytoplankton, and protozoa, and for natural phytoplankton assemblages. The instrument can analyze cellular concentrations and changes in these relative concentrations as a function of time. Combined with the use of fluorescent antibody reagents

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FIG. 34. A laboratory experiment involving a culture of the diatom clone 3H (Thalussiosiru pseudonuna). The cells were grown in low light and then placed into high light. Summing the FACS analyzer individual particle data (A) for this population showed a downward trend in chlorophyll a fluorescence per cell. The same trend would be expected from bulk analysis. By extended data analysis, it became evident that a subpopulation of low fluorescent cells was increasing and a subpopulation of high fluorescent cells was decreasing over the days of the experiment. The two subpopulationsshowed no change in fluorescence intensity per cell (C). This is a clear demonstration of the power of individual particle analysis coupled with the appropriate extended data analysis. Courtesy of E. Sakshaug, S. DemerS, and C. M. Yentsch (unpublished data).

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PARTICLES

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FIG.35. Scheme of separating oceanic particles. From Yentsch et a/. (1983).

and stains specifically for enzymes, substrates, or products, the analysis of specific cell constituents and rates of synthesis or turnover is possible on a cellby-cell basis. Flow cytometry offers potential for phytoplankton, bacterial, and grazing studies on natural populations. A general scenario for resolving component particles in a natural sample is given in Fig. 35 which utilizes specific fluorescent stains in addition to autofluorescence. Immediate scientific gains in the aquatic sciences are clearly visible; the longterm gains may be more impressive. Present contradictions between community models that consider only one type of variable (size, chemical composition, etc.) could be resolved by developing multivariate models in which size, chemical composition, and rate measurements are incorporated simultaneously.

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.

VIII Summary/ Concluding Remarks As the interests of aquatic scientists expand beyond the biogeography of regions of high biomass and turnover rates, the incomplete understanding resulting from bulk analysis is becoming painfully obvious (Yentsch and Phinney , 1985). Predictive ecology will depend on an understanding of the various species components and their variability, not merely a mean value for the population. The promise of a broad range of techniques designed to measure properties and rates of processes of individual cells has been reviewed in a cursory manner. Appropriate application to many pressing questions should result in new interpretations and understanding of allometry, ataxonomy, distinctive properties, cell metabolism, cell growth, cell duplication and division, cell recognition, differentiation and development, and cell-to-cell interaction.

ACKNOWLEDGMENTS The authors acknowledge the patience and assistance in collecting many marvelous assemblages of mammalian cells, namely by Terry L. Cucci and David A. Phinney, coexplorers in the learning process; Janet W. Campbell, Paul K. Horan, Katharine Muirhead, Richard W. Spinrad, and Charles S. Yentsch, for expanding our knowledge in various aspects; and Wendy Korjeff-Bellows, Pat Boisvert, Mary Lou Gillmor, Jim Rollins, and Pam Shephard for manuscript details. Funding from ONR NOOO14-81C-0043 and N00014-79-C-0916, NSF OCE-84-16217, Maryland Sea Grant R/F-42, and NASA NAGW-410 bas supported our exploration. This is Bigelow Laboratory for Ocean Sciences contribution number 85026.

REFERENCES Allen, S. K., Jr. (1983). Aquaculture 33, 317-328. Allen, S. K., Jr., and Stanley, J. C. (1983). Transact. Am. Fish. SOC. 112, 431-435. Ammerman, J. W., Fuhrman, J., Hagstrom, A., and Azam, F. (1984). Mar. Ecol. Prog. Ser. 18, 31-39. Amdt-Jovin, D. J., and Jovin, T. M. (1974). FEBS Lett. 44, 247-252. Arndt-Jovin, D. J., Ostertag, W., Eisen, H., Klimek, F., and Jovin, T. M. (1976). J . Hisrochem. Cytochem. 24, 332-347. Atkins, P. W., and Barron, L. D. (1969). Mol. Phys. 16, 453. Barron, L. D., Bogaard, M. P., and Buckingham, A. D. (1973). J . Am. Chem. SOC. 95, 603. Bonner, W. A., Hulett, K. R., Sweet, R. G., and Herzenberg, L. A. (1972). Rev. Sci. Instrum. 43, 404-410. Brand, L. E., Murphy, L. S., Guillard, R. R. L., and Lee, H.-t. (1981). Mar. Biol. 62, 103-110. Brattain, M. G. (1979). In “Flow Cytometry and Sorting” (M. R. Melamed, P. F. Mullaney, and M. L. Mendelsohn, eds.), pp. 193-215. Wiley, New York. Bretting, H. (1979). “Biologie des Spongiares.” Colloq. Inr. C.N.R.S. 291, 247-255. Bretting, H., and Jacobs, G. (1982). In “Lectins-Biology, Biochemistry, Clinical Biochemistry,” Vol. 11, pp. 91-103. de Gruyter, Berlin. Bretting, H., and Konigsmann, K. (1979). Cell Tissue Res. 201, 487-497.

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INTERNATIONAL REVIEW OF CYTOLOGY. VOL. 105

Establishment of the Mechanism of Cytokinesis in Animal Cells R. RAPPAPORT Department of Biological Sciences, Union College, Schenectady, New York 12308 mid The Mt. Desert Island Biological Laboratory, Salsbury Cove, Maine 04672

I. Introduction Early recognition of the importance of cell division led to many descriptive studies of mitosis and cytokinesis in different animal cells and much speculation concerning the physical processes underlying the visible events. The initial expectation was that the mitotic apparatus would be involved in the physical events of cytokinesis to the same extent that it was involved in the physical events of mitosis, although there was no unanimity of opinion concerning the nature of mitotic apparatus involvement in either process. Many alternative physical mechanisms that were proposed to explain the physical process of cytokinesis were eliminated when it was shown that division proceeded despite removal (Yatsu, 1912; Hiramoto, 1956) or chemical disruption of the mitotic apparatus (Beams and Evans, 1940) and that, shortly before the onset of cleavage, nothing in the subsurface region or its organization was necessary for furrowing activity (Hiramoto, 1965; Rappaport, 1966). These results implied that the physical mechanism is located in the cell surface. The longestablished correlation between the position and orientation of the mitotic apparatus and the furrow was accounted for by postulating that the mitotic apparatus determines where the furrow is to form before division begins. The determination was presumed to occur when the mitotic apparatus altered a region of the surface or subsurface environment by some form of stimulatory activity. The idea of a stimulus is both old (Butschli, 1876) and useful, but there is still little information concerning its nature and some controversy about the region where it acts. In order to determine where and when the furrow develops, the mitotic apparatus must evoke regional changes in the surface behavior and physical properties. The majority of evidence concerning this function of the mitotic apparatus has been obtained by experiments in which the normal relation between it and the surface was altered while the evocation was in progress. The results have provided information concerning (1) the length of the period when interaction between the mitotic apparatus and the surface is possible; (2) the length of the 245 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

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interaction period necessary for surface alteration; (3) the time (relative to mitosis and real time) when the alteration becomes irreversible; (4) the relation of the distance between the mitotic apparatus and the surface to furrowing activity; ( 5 ) the parts of the mitotic apparatus that are necessary to establish furrows; and (6) the identificationof the surface region most affected by the mitotic apparatus. 11. Early Studies and Speculation

The reviewer of information concerning a central event in the life of the cell that has interested cytologists for more than 100 years carries a historical burden. Although some understanding of the older work in this area is desirable for an understanding of the factors which direct present day investigations, a general history of the study of animal cell division is not the purpose of this article. This article is primarily concerned with the results of attempts to analyze by experimentation the nature of the relation between the mitotic apparatus and the surface and its immediate consequences. The literature concerning cell division from before 1950 is notable both for its volume and for the infrequency of its citation in present-day publications. In the early period (beginning in the 1870s), the approach was primarily encyclopedic. Dividing cells, both living and fixed, from many different animals were studied and numerous hypothetical mechanisms were invented to explain the visible events. While some investigators based their hypotheses on the observations of cells, others placed greater emphasis on the study of models that appeared to simulate division activity. Those who studied cells were often unable to distinguish between active and passive events. Consequently, each of the following major visible events could be, and was in time, considered the principal mechanical cause of division: “mitotic elongation” (Gray, 1924; Chambers, 1924; Dan and Dan, 1947), surface increase (Schectman, 1937; Swann and Mitchison, 1958), equatorial diameter decrease (Butschli, 1876; Ziegler, 1898b; Lewis, 1942), and displacement of cytoplasm from the equator to the region of the poles or cell center (Teichmann, 1903). Those who emphasized the study of models, on the other hand, had no assurance that the forces and mechanical factors which made their models work existed in the cell. They also showed a strong tendency to explain cell events by precisely the same steps that made the model work. For instance, those who studied liquid-drop models which divided when the surface tension at the poles of the drops was rapidly decreased usually transferred the idea of polar surface tension decrease directly to the cell. They rarely discussed the idea that the important concept might be the difference in tension at the surface between the poles and the equator and that it was equally possible that the difference could originate because of increase at the equator. Animal cell division does not appear to be a complicated event and this simplicity has been one of the sources of its fascination. Despite the fact that numerous models based upon different physical principles can simulate the general process, there has been a

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tendency for each inventor to assume that, because his model works, the possibility that the cell operates according to the same principles as the model is enhanced. This reasoning might be justified if only one model worked but when several different models can simulate the same event the criterion of workability loses validity. Models of cell division certainly have a fascination of their own, whether they be mechanical or computer simulations. Perhaps Heilbrunn (1928) put it too strongly when he stated that “There is no field of biological thought that has suffered more from the use of analogies than has the theoretical study of cell division.” However, it seems apparent that until the nature of events inside the cell is better known, models will have the potential for being deceptive as well as instructive. The ultimate test of a model is the cell itself. When models are sufficiently versatile to simulate the alterations in shape and proportion that can be imposed on the living, dividing cell, then the ability of the model to predict the outcome of the experiment can be used to test the validity of the assumptions upon which the model is based. Investigations involving both models and manipulated cells may provide further insights concerning cell division mechanisms. The hypotheses that were formulated during that speculative era were consistent with the visible events of cell division, rational, and usually physically possible. Theorists borrowed bits of each other’s theories and, without any recognition of the law of parsimony, some hypotheses became quite elaborate and multifaceted. No one commented that, given the limited kind of data then available, nearly all were equally possible. Many authors seemed compelled to comment on the mechanism of cell division whether or not the paper containing the comments concerned cell division and whether or not the author worked in the field. Succinctly stated theories appeared in footnotes and were buried in discussion sections. As a rule, authors supported one theory at a time, although they might shift their support from one theory to another. In time, all of the mechanisms that could possibly divide a cell were proposed, some of them several times, and the diligent student of the older literature realizes that no one now living can realistically consider himself the first proposer of a major cell division theory. This circumstance should enhance the objectivity of current discourse and diminish intense personal attachment to ideas. At present, the first proposers of major cell division ideas are infrequently cited and the lesson in this may be that theorization that does not lead quickly to a testable hypothesis is destined for a short life. Despite the nineteenth century successes of Bernard and Pasteur in solving complex problems with carefully designed and controlled experiments, the desirability of experimentation on dividing cells was rarely discussed. Ziegler (1898a) and McClendon (1908), in their studies of cleavage in a cell without chromosomes, and Yatsu (1912), in his demonstration that furrowing continued despite surgical separation of the mitotic apparatus from the remainder of the cell, clearly demonstrated the effectiveness of studying the division of altered cells. However, their work was rarely cited by their contempo-

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raries, their methods of analysis were not adopted by others until many years had passed, and hypothetical mechanisms that were inconsistent with their findings were still being proposed four decades later. These circumstances resulted in a generally inconclusive, sometimes redundant, body of writings contributed by a number of respected biologists. They may explain why Wilson barely mentioned the literature on animal cytokinesis mechanisms in the second edition of “The Cell” (Wilson, 1900). In the third edition (Wilson, 1928), he described many of the theories, but in his discussion of observations that different authors cited in support of their ideas, he appeared to be convinced by interpretations that were inconsistent with each other. It is easy to gain the impression that in the 50 years before the third edition of “The Cell little progress was made despite intense effort and frequent publication. Each theorist appeared to be motivated by the desire to be the first to propose what would eventually prove to be the actual mechanism. Very little consideration seems to have been given to divising means for discriminating among the numerous alternatives. Heilbrunn (1928) stated: “Usually it is easier to make a new theory of cell division than to test an old one.” Perhaps that perception may have been an important factor in the shaping of the approach to the problem. There may also have been greater satisfaction (and recognition?) in exposition than in analysis. Whatever the cause, the time lag between the development of techniques and equipment suited to experimental analysis of cell division mechanism and the systematic application of those techniques to the problem is surprisingly long. Early speculations concerning the mechanisms of animal cell division revolved around the identification of the physical roles of the visible cell components. Theories postulating an active physical role for the mitotic apparatus usually fell into one of two major categories. According to one group of theories, the mitotic apparatus accomplished division directly as it distorted or deformed the cell or its surface by mechanical means, such as traction or pushing the poles apart (Platner, 1886; Rhumbler, 1903; Heilbrunn, 1928; Dan, 1943, 1948). These hypothetical activities were correlated with changes in the dimensions of the mitotic apparatus that normally take place during the cycle. In the alternative group of theories, the mitotic apparatus was thought to play its role by redistributing and altering the cytoplasm into two masses around the cell centers (Erlanger, 1897; Teichmann, 1903; Danchakoff, 1916; Chambers, 1919; Gray, 1924). According to this mechanism, furrow formation became a nearly passive event which took place when the equatorial cytoplasm moved elsewhere. This proposal was consistent with observable cyclical changes in the size of the centrosomes and asters and with accepted contemporary data concerning local intracellular “viscosity” changes. Elements of the latter mechanism are more frequently encountered in theoretical discussions than are elements of the former. Some theories combined both mechanisms. Hypotheses which proposed an active physical role for the cell surface fell into three genera1 categories. According to one group of theories, the existing surface ”

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accomplished division by active expansion (Schechtman, 1937; Marsland, 1939; Swann and Mitchison, 1958), contraction (Butschli, 1876; Ziegler, 1898b; Yatsu, 1912; Lewis, 1939; Marsland and Landau, 1954), or relaxation (Lillie, 1903; Robertson, 1909; McClendon, 1912; Conklin, 1912; Just, 1922; Wolpert, 1960; Schroeder, 1981a; White and Borisy, 1983). These proposals were consistent with information concerning regional changes in surface area, but the observations on which they were based did not permit differentiation between active and passive events. In the third category of hypotheses, formation of the furrow was attributed to a reorganization of the subsurface cytoplasm in the equatorial plane that eventually resulted in de novo formation of furrow surface. The most commonly proposed form of subsurface division-related structure is the diasteme, a sheetlike layer of modified cytoplasm that appears to extend outward from the metaphase plate toward the surface in fixed, sectioned cells (Andrews, 1897; Motomura, 1950; Selman and Waddington, 1955; Zotin, 1964). A similarly positioned sheet of vesicles was also described in several early ultrastructural studies of dividing animal cells (Buck and Tisdale, 1962; Humphreys, 1964; Thomas, 1968). The diasteme was thought to form a new surface in the furrow by splitting. The vesicles were postulated to achieve the same effect by fusion. The idea was satisfying, because it suggested that the fundamental process of cytokinesis was accomplished by very similar mechanisms in higher plant and animal cells. More recent ultrastructural studies have failed to confirm the presence of either the diasteme (Selman and Perry, 1970) or sheets of vesicles (Schroeder, 1970), and it is possible that the earlier studies involved some misinterpretation and artifact, although Selman (1982) believes that the diasteme cannot be entirely dismissed as an artifact. After it was shown that furrowing could take place following the physical removal of the mitotic apparatus, it was apparent that the roles of the mitotic apparatus in mitosis and cytokinesis were very different. The cell’s ability to divide despite replacement or disruption of the subsurface cytoplasm focused attention on the preexisting surface as the formation site of the division mechanism. It is reasonable to assume that a multistep processes is involved. In the first step the mitotic apparatus alters the surface and then becomes dispensable. In the second step the altered surface organizes the division mechanism and in the last step the division mechanism actively constricts the cell. Many of the experiments which will be described in the following sections were undertaken in the belief that a better understanding of the circumstances of the interaction of the mitotic apparatus and the surface would help elucidate the nature of the division mechanism and the way in which it is established. Although increased use of experimentation concentrated attention on testable hypotheses and helped distinguish between cause and effect, it also resulted in a great reduction in the variety of cells that were studied. The durability, predictability, transparency, and regularity of form of cleaving marine invertebrate eggs, especially those of the echinoderms, are temptingly convenient to the investigator who must also cope with problems of experimental design and instrumenta-

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tion. When subjected to similar experimentation, vertebrate tissue cells and cleaving invertebrate eggs respond in much the same way (Rappaport and Rappaport, 1968, 1974) but, in their shape immediately before division and in the relative size of the mitotic apparatus and its major components, they are different. It is desirable to compare the results of experiments on these different kinds of cells whenever possible.

111. Results of Experimental Analysis A. TIMERELATIONS

1. When Is the Division Mechanism Fixed in the Surjke?

The position of the mitotic apparatus relative to the surface is stabilized long before division. The time when the furrow is fixed in the surface has been determined by inactivating or removing the mitotic apparatus at different times before furrowing is anticipated; the subsequent development of a functioning furrow shows that the minimum essential interaction has occurred. Two time criteria have been used, mitotic time and real time. Determination of mitotic time involves ascertainment of the relation between the mitotic state at the time of experimental intervention and the cell’s subsequent ability to furrow. Echinoderm eggs, in which the mitotic apparatus has been dissociated with colchicine, reveal that at some point during anaphase the presence of a normal mitotic apparatus is no longer required (Beams and Evans, 1940, late anaphase; Swann and Mitchison, 1953, mid anaphase; Hamaguchi, 1975, early anaphase). Hiramoto (1956) found that echinoderm eggs from which he had physically removed the mitotic apparatus at anaphase or later subsequently divided. The apparent correlation with anaphase suggests some causal relationship between the position of the chromosomes and furrow establishment, an idea that was incorporated into cell division theory (Swann and Mitchison, 1958). However, Ziegler (1898a) and McClendon (1 908) observed cleavage in eggs lacking chromosomes, and it will be subsequently shown that the time of furrow establishment can be dissociated from mitotic events. It may be important to point out that when the chromosomes are in anaphase the achromatic part of the mitotic apparatus is generally considered to have attained maximum size (Wilson, 1928). Similar experiments on cleaving eggs of amphibia and sturgeon, in which the mitotic apparatus was shifted before the anticipated cleavage time, indicate that in these cells also the furrow position is determined at anaphaseearly telophase (summarized in Selman, 1982). However, when these results are expressed in Detlaff units (D, the time interval between the onset of first and second cleavage) the interval between determination and cleavage is about 0.1 D for echinoderm eggs and 0.4 D for the eggs of amphibia and sturgeon. It is also possible to determine the real-time interval between the appearance of the cleavage furrow and the time when the mitotic apparatus becomes unneces-

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sary. These experiments were carried out on echinoderm eggs at second cleavage when the divisions of the two blastomeres are normally precisely synchronous. Hamaguchi (1975) quickly destroyed the mitotic apparatus of one of the blastomeres by colchicine injection. He found that furrowing occurred in operated cells when the mitotic apparatus was destroyed 10 minutes or less before the midpoint of cleavage of the control cell. The midpoint of cleavage was reached 4-7 minutes after the beginning of furrowing. In a similar experiment, cells were confined in short capillaries which reshaped them into cylinders that controlled and standardized their dimensions (Rappaport, 1981). The mitotic apparatus was aspirated from one blastomere at different times before control cleavage. Cells from which the mitotic apparatus was removed 4 minutes or less before the beginning of control furrowing were still able to cleave. These experiments show the time (or point in mitosis) when the mitotic apparatus becomes unnecessary-that is, when the effect of the mitotic apparatus on the surface was sufficient to promote local, active shape change. They signify that surface alteration was taking place before that time, very possibly before the mitotic apparatus had expanded to maximum size. 2. How Long Must the Mitotic Apparatus Act upon the Surface to Establish the Division Mechanism? The experimental designs described above can show when the minimum effective period of interaction between the mitotic apparatus and the surface ends. They do not show when it begins. In order to determine how long the mitotic apparatus must act upon the surface, it is necessary to control the time when the period begins either by moving the mitotic apparatus to a different position in the cell or by moving a part of the surface from a location where it would not normally form a furrow into a relationship where furrows are normally produced. Following centrifugal displacement of the contents of an egg in early cleavage, a furrow subsequently appears in normal relationship to the new position of the mitotic apparatus (Harvey, 1935; Rappaport and Ebstein, 1965). This technique, however, does not provide precise information concerning timing. The mitotic apparatus can also be shifted by compressing the spherical egg between the side of a needle and the bottom of the operation chamber (Rappaport and Ebstein, 1965). The mitotic apparatus can be shifted several times in the same mitotic cycle and, following each shift, a new furrow appears in about 2 minutes. The same results were obtained with eggs that had been preshaped into cylinders by fertilizing them when they were in extruded form (Rappaport, 1975; Isaeva and Presnov, 1983). The possibility that the outcome of these experiments might be affected by local surface stress was avoided in subsequent experiments by confining the eggs in capillaries so that they were reshaped into cylinders. Then the mitotic apparatus was shifted by alternately pushing the poles inward. In these experiments also, the mitotic apparatus elicited multiple furrows in a single mitotic cycle and the new furrow appeared 1-2 minutes after the mitotic apparatus was shifted (Rappaport, 1985). The ability of the mitotic apparatus to affect

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the surface is distance limited (see more extensive discussion below), so that, when it is naturally or experimentally located eccentrically in the cell, a furrow forms only in the part of the margin that is closest to its equatorial region (Rappaport and Conrad, 1963). When the distant margin is pushed inward so that it is the same distance from the mitotic apparatus as the closer margin, a furrow also forms in the distant margin. By moving the distant margin in and out of the effective range of the mitotic apparatus and noting whether a furrow subsequently forms, the minimum time necessary for the mitotic apparatus to fix the division mechanism in the surface can be estimated. Experiments of this kind revealed that the minimum interaction time which resulted in furrowing was about I minute. Between the end of the interaction period and the beginning of furrowing there was an interval of about 2.5 minutes (Rappaport and Ebstein, 1965). In these experiments, furrows formed only when the distant equatorial margin was pushed into the zone between the asters. Attempts to elicit furrows in other parts of the surface by pushing them into different regions of the mitotic apparatus were unsuccessful. The 2.5 minute interval before the cell shape changes may be required for organization of the division mechanism. Since the total period between exposure of surface to the mitotic apparatus and the beginning of furrowing is usually not more than 3 or 4 minutes under experimental conditions, it is implied that in normal cleavage the interaction period may begin about 5 minutes before the beginning of cleavage. If that is true, it is not correlated with any distinctive morphological event.

3 . HOWLong Are the Mitotic Apparatus and the Surface Cupuble oj ItiteraciingP In most cells, the relation between the mitotic apparatus and the surface is stable over a long period before division. The information discussed above suggests, however, that furrow establishment may take place only shortly before division begins. Two questions then arise. What determines when the interaction begins and how long are the mitotic apparatus and the surface able to interact? It is possible to advance the time of furrowing. Experiments involving the formation of unilateral furrows which implied that the effect of the mitotic apparatus is distance related (Rappaport and Conrad, 1963) suggested that furrows might appear sooner if the distance between the mitotic apparatus and the surface was reduced. Sand dollar eggs were flattened before the second cleavage and the mitotic apparatus was isolated in a narrow cylinder of cytoplasm made by two cuts parallel to the mitotic axis. In these experiments, furrows developed in the operated cells 7 minutes before they developed in the control cells (Rappaport, 1975). It is also possible to prolong the period during which the mitotic apparatus and the surface can interact. Normally, division increases the distance between the asters and isolates them in the daughter cells. Both of these events can render the mitotic apparatus incapable of furrow establishment (see the following sections). The problem can be avoided by moving the mitotic apparatus each time division

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begins. Experiments in which the mitotic apparatus was shifted by squeezing a cylindrical cell each time the early furrow appeared revealed that the mitotic apparatus could establish a new furrow 17 minutes after the appearance of the first furrow (Rappaport, 1975). In later experiments, in which cells were confined in cylindrical capillaries, the mitotic apparatus was moved by alternately pushing the poles inward. In this circumstance, the same mitotic apparatus could establish as many as 13 furrows over a 24.5-minute period following the appearance of the first furrow (Rappaport, 1985). These results indicate that the surface and the components of the mitotic apparatus can establish a furrow for some time after their normal period of interaction. It is unclear what terminated the activity in these experiments, although the asters become more widely separated with successive shifts. These results indicate that, under modified geometrical conditions, the mitotic apparatus and the surface can play their roles in furrow formation for a considerably longer period than is used in normal circumstances. They suggest the possibility that, in the normal cell, the interaction period could begin when the mitotic apparatus has grown to attain some required distance from the surface and it may end because the active components of the mitotic apparatus are separated and sequestered in separate cells. 4. How Fast Does the Cleavage Stimulus Move toward the Su@ace? The information thus far discussed is consistent with the idea that a factor that alters the surface moves from the central, axial parts of the mitotic apparatus toward its periphery and thus toward the cell surface. If this is true, then within the effective range of the mitotic apparatus, the time when the furrow appears should be related to the distance from the mitotic apparatus to the part of the surface in which the furrow appears. Analysis of this relationship requires measurement of the distance from the mitotic apparatus to the surface but, because the perimeter of the mitotic apparatus is indistinct, it is necessary to make the measurements from a line joining the astral centers. Also, because time differences were expected to be small and eggs resulting from the same fertilization are not precisely synchronous, the appearance of two furrows located at different distances from the same mitotic apparatus in the same cell were compared by timing the appearance of furrows in opposite equatorial margins of flattened eggs in which the mitotic apparatus was located eccentrically. Under these conditions, the time difference between the appearance of the furrow in the closer and distant margins was proportional to the difference in distances from the mitotic axis to the two marginal surfaces. In two separate investigations the rates of movement were calculated to be 6.3 t I .8 Kmlminute (Rappaport, 1973) and 7.4 3 pm/minute (Rappaport, 1982). The findings are consistent with the concept of a surface-altering factor that moves or spreads peripherally from the vicinity of the mitotic axis. They do not provide any information concerning the nature of the factor. They could be explained by the radial movement of a substance or component along elements of

*

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the mitotic apparatus, or by the extension of certain components of the mitotic apparatus, or they may simply reflect the normal expansion of the mitotic apparatus as a whole.

B. ESSENTIALSTRUCTURAL COMWNENTS OF THE MITOTICAPPARATUS At the same time that proponents of an active physical role for the mitotic apparatus were theorizing different ways in which it could provide the forces necessary for cytokinesis, proponents of an active physical role for the surface were speculating about ways in which the mitotic apparatus could stimulate the surface activity that they believed accomplished the same process. In time, a central indispensable role was attributed to each of the visible components of the mitotic apparatus. Wilson (1928) accorded important roles to the central bodies, the astral rays, the spindle, and the chromosomes. Despite Ziegler’s (1898a) and McClendon’s ( 1908) demonstrations that cells without chromosomes could cleave and Chambers’s (1921a) description of furrows forming between isolated cytasters in eggs submitted to excessive parthenogenetic treatment, an essential role for the chromosomes was considered possible some years later (Swann and Mitchison, 1958). More recently, an understanding of the way in which the mitotic apparatus acts has been obtained by attempts to determine its minimal essential components. A part of the mitotic apparatus is essential only if its removal or inactivation during the time the furrow is established blocks subsequent furrow formation or, conversely, its nonessentiality is demonstrated when division takes place subsequent to its removal or inactivation during the interaction period. Ziegler’s (1898a) description of the cleavage of a blastomere lacking chromosomes would appear to rule out an essential role, but the fact that the cleavages lagged behind those of blastomeres with chromosomes allowed the possibility that the chromosomes supplied something required for the completely normal process. The second division of torus-shaped sand dollar eggs, in which the furrow forms between the polar surfaces of the asters of two different mitotic apparatus, showed that cleavage without chromosomes can be synchronous with normal cleavages if the positions of the asters relative to the surface in which the furrow forms are approximately normal (Rappaport, 1961). Hiramoto (1971) also showed that in spherical sea urchin eggs, aspiration of the spindle and chromosomes did not block cleavage. These results clearly indicate that the spindle and its contents are not necessary. They concern not only the chromosomes, but also the spindle and kinetochore microtubules and any other substances or structures which are normally found in the spindle. It should be kept in mind, however, that the experiments on which these conclusions are based were performed on spherical, cleaving embryonic cells in which the asters are large and the spindle is far from the equatorial surface. In tissue cells, the positions and proportions of the division-

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related parts may be different. The asters of adult tissue cells are also relatively smaller and the spindle is relatively larger and closer to the equatorial surface. Furrows form in surface brought close to or in contact with the spindle under conditions where contact with the asters is unlikely in echinoderm eggs, vertebrate tissue cells (Rappaport and Rappaport, 1974), and grasshopper neuroblasts (Kawamura, 1977). These findings imply that in echinoderm eggs, the asters are the effective components in furrow establishment, even though the spindle shares the capacity. However, in differently proportioned tissue cells, the spindle is probably the effective component. The asters of the intact mitotic apparatus are fixed relative to each other by the spindle and they may be considered to have equatorial and polar regions. No part of the aster appears more effective than another in furrow establishment. The polar parts of the asters have been shown to be effective in torus-shaped cells (Rappaport, 1961) and the “sides” of the asters in cells of other shapes (Rappaport and Ebstein, 1965). The property that elicits the furrow is not peculiar to the spindle region of the mitotic apparatus where the furrow normally appears, but it seems to pervade the entire achromatic apparatus. It does not therefore appear fruitful to search within the equatorial region of the mitotic apparatus for a special localized structure or division-related activity. In cleavage cells, where the aster is usually large and the linear components of the mitotic apparatus appear to extend close to the periphery, it appears probable that the entire cell surface is affected by the same mitotic apparatus component that fixes the furrow at the equator, and that the furrow region is determined by quantitative rather than qualitative factors. Most of the aster-like structures which develop in response to injection of microtubules, centrioles, and other components can cause localized furrowing (Maller, et al., 1976; Iwamatsu et al., 1976; Forer et al., 1977; Heidemann and Kirschner, 1975, 1978; Hirano and Ishikawa, 1979). The regression of furrows that may occur in this circumstance can be due to the size and geometrical arrangement of the asters as well as their mode of origin. At this time, the identification of the ultrastructural mitotic apparatus components that are actively involved in cytokinesis is conjectural. The microtubules have been favored for several reasons. Colchicine and agents which disperse microtubules can block furrow establishment without blocking cyokinesis (Beams and Evans, 1940; Swann and Mitchison, 1953). The ability of the mitotic apparatus to establish furrows appears to travel in straight lines as do microtubules (Dan, 1943; Rappaport, 1968). The effect of agents that reduce the size of the mitotic apparatus and, at the same time, reduce its ability to establish furrows can be reversed by microtubule-enhancing agents (Rappaport, 197la). The rate of movement of the stimulus appears to approximate that of the rate of in vivo microtubule elongation (Aronson and Inout, 1970; Rappaport, 1973). The invention of methods for demonstrating microtubules has resulted in numerous

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investigations which, taken as a group, convey the impression that they are the most important constituents of the mitotic apparatus. On the other hand, other information provides less support for the idea of a central role for microtubules. Modified methods of preparation have demonstrated prominent vesicular components of the mitotic apparatus (Harris, 1975; Paweletz and Finze, 1981; Paweletz and Fehst, 1984; revieved, Paweletz, 1981; Hepler and Wozniak, 1984) which may play a role in local ion regulation and have the same possibility for involvement in cytokinesis as microtubules. An ultrastructural study of microtubule arrangement reveals no convincing evidence of the quantitative difference in regional relation to the surface that presumably is required to cause regional functional surface differentiation (Asnes and Schroeder, 1979). Micromanipulation measures which repeatedly disrupt microtubules during the interaction period do not block furrow establishment, although the possibility of very rapid reextension between disruptions has not been eliminated (Rappaport, 1978). Fully effective interaction can occur in the presence of agents that inhibit microtubule assembly (Hinkley et al., 1982; Hinkley and Chambers, 1982; Rappaport, 1971a; Rappaport and Rappaport, 1984).

STIMULUS C. THENATUREOF THE CLEAVAGE The types of experiments emphasized in this article are not suitable for obtaining an understanding of the molecular events of cleavage furrow establishment, although they can serve to focus efforts on organelles and regions where division-related molecular events occur. There is considerable opportunity for complexity in any hypothetical explanation of events basic to the establishment of the contractile region that constitutes the division mechanism. In the interpretation of results, it may become difficult to separate contraction-related activity from establishment-related activity. Attempts to learn more of the nature of the stimulus by manipulating the subsurface environment while the interaction is in progress have yielded a small amount of information in sea urchin eggs. Neither constant cell shape alteration by repeated extrusion (Rappaport, 1961), constant stirring with a needle between the mitotic apparatus and the equatorial surface, nor vibrating the equatorial surface blocks furrowing (Rappaport, 1978). These results were interpreted as suggesting that the normal distribution of any furrow-promoting stimulus from the mitotic apparatus is not the consequence of simple free diffusion. In sea urchin or other invertebrate eggs, there has been no report of successful transfer of furrow-promoting substance from one region to another or from cell to cell. In the larger and in some ways more convenient amphibian egg, however, an extensive series of experiments based upon a transferrable furrow-inducing substance has been reported. Sawai et d.(1969) found that cytoplasm beneath the advancing furrow tip induces contractile activity when transplanted to other

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parts of the same egg. The substance is effective across species lines (Sawai, 1972, 1983), provided that the cortex is in a reactive state (Sawai, 1976). Already functional furrow cortex transplanted to an egg that is between division cycles will continue its division activity, but the furrow does not progress beyond the limits of the graft. The chemical nature of the furrow-inducing substance is unknown. Because the contractile mechanism which accomplishescytokinesis appears to resemble the better known contractile mechanism of muscle cells in which Ca2 plays an important role, it would be surprising if cytokinesis were not affected by measures that manipulate Ca2+ generally or locally. Timourian et al. (1972) found that the effect of local application of EDTA-filled capillary probes during metaphase varied according to the region in which the probe was applied. Application of the probe to the polar surface had no effect. Application to the equatorial surface resulted in a change of the cleavage plane, or division directly into four cells, or failure of cleavage. Since the same treatment of the equatorial surface after the beginning of division did not affect furrowing, they reasoned that the treatment altered the mechanism that determines the site of the furrow rather than the division mechanism. The authors concluded that the only effect of the EDTA was to sequester Ca2+ that would otherwise have accumulated under the periphery. The results make it clear that, at the time of the experiment, the equator was more susceptible to perturbation than the poles. It would be interesting to redesign these ingenious experiments to incorporate changes consistent with current information concerning the way in which Ca2+ chelators function. Schantz (1985) recently observed transient fluctuations of free calcium ion concentration correlated with cleavage in a fish embryo. Various other aspects of possible involvement of Ca2 in different facets of cytokinesis have recently been reviewed by Harris (1981), Nagle and Egrie (1981), and Sisken (1981). The cleavage stimulus is often considered to be a discrete chemical entity which moves radially in association with the linear elements of the mitotic apparatus until it reaches the surface where it affects local contractile elements. So little information concerning this aspect of the process exists, however, that possible alternative mechanisms must eventually be tested. The important effect of the mitotic apparatus appears to be the creation of regionally different subsurface environments. The differentiation could be accomplished by removal of components from the affected region as readily as by addition. The ability of microtubules to promote bidirectional movement is now well documented (Allen et al., 1985). The substance moved by the mitotic apparatus may be actively contractile rather than an activating substance. If the force-producing elements are mobile and if they move up a tension gradient (Schroeder, 1981a; White and Borisy, 1983) (see Section III,E,3), then anything which increases the contractility in one part of the web of subsurface contractile elements will initiate a localized accumulation of elements that would be self-enhancing. Transport of a +

+

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relatively small amount of contractile material to the equatorial subsurface could thus trigger accumulation of sufficient contractile material to establish an equatorial contractile ring that could create its typical ultrastructure by the play of forces within itself (Rappaport, 1975; Fujiwara er al., 1978). Conversely, local accumulation of relaxing substance at the poles could cause similar events at the equator (Schroeder, 1981a; White and Borisy, 1983). The fact that the contractile mechanism must be attached to the cell surface in order to cause the typical division shape changes has led to the idea that the active component in the stimulation process must be transported directly to the surface. Asnes and Schroeder ( I 979), for instance, assumed that microtubules which end more than 5 p n from the surface are not effective. At present, it is not necessary to postulate that such an intimate, precise arrangement is necessary because furrow establishment can occur during manipulations which would be expected to disrupt the normal cytoplasmic organization (Rappaport, 1978). The mitotic apparatus could be effective if it altered a relatively large, subsurface region that lacks sharp limits. The precision of subsequent furrow formation could result from the disposition of its precursor elements and the results of their interaction.

D. IDENTIFICATION OF THE SURFACE REGION AFFECTEDBY THE MITOTICAPPARATUS Cytokinesis is accomplished by an equatorial band of superficial cytoplasm that contracts with greater force than the rest of the surface and develops special ultrastructural features (reviewed, Arnold, 1976; Schroeder, I981b). Before interaction with the mitotic apparatus, all parts of the surface have the same physical properties. All parts of the surface can interact with the mitotic apparatus to form a furrow. The asters do not function in complementary pairs. The capacity to establish furrows appears to be present in the entire achromatic apparatus. The dimensions of most cells are such that all parts of the surface are close enough to the mitotic apparatus to be affected to some extent at the time of the interaction. Furrow establishment can occur when the distance from the spindle to the equatorial surface is 70% greater than normal (Rappaport, 1982). It is apparent that in order to establish a regionally restricted structure such as cleavage furrow, the mitotic apparatus must affect some parts of the surface differently from the way it affects other parts. In the absence of regional predisposition in the surface or polarity in the asters, the regional differences are most likely attributable to differences in intensity of interaction with the asters which, in turn, are probably a consequence of the geometrical relation between the surface and the mitotic apparatus. Many of the speculations concerning the geometrical relations between the mitotic apparatus and the surface appear to have been secondary to the physical

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division mechanism. That is, after a hypothetical physical division mechanism was devised, the geometrical relation necessary to establish it in the surface was proposed. Information concerning the physical properties of the surface and the cell division mechanism have been used as the basis for inferences concerning the geometrical relations and vice versa. Some problems may arise, however, when reasoning from changes in physical properties and behavior if the identity of the active and passive processes is uncertain. It is also possible to analyze the process by experimentally altering the geometrical relations between the mitotic apparatus and the surface while the interaction is in progress. Because fewer assumptions need be made, interpretation of the results from this kind of experiment is often simpler. Experimental analysis of the geometrical relations of the interacting cell parts has been facilitated by several factors. Cell shape can be drastically altered without harmful effect. The mitotic apparatus can be moved and it can be broken into major components apparently without reducing the ability of the components to interact with the surface. The behavior of the equatorial and polar surfaces can become different if one of them is changed. The major question is whether the key alteration takes place at the poles or at the equator. Presently, two alternative mechanisms are under consideration. According to the polar stimulation hypothesis, the polar or subpolar surfaces are changed. According to the equatorial stimulation hypothesis, it is the equatorial surface that is changed. The two hypotheses are clear-cut, simple alternatives. No other alternatives are now seriously considered although their possible existence cannot be ruled out. They can be tested by altering the geometrical relations between the mitotic apparatus and the surface. Although direct proof of a hypothesis is more satisfying, progress by disproof of alternatives is more usual (Platt, 1964). When an experimental design has the potential to disprove either hypothesis, then the one that is not disproven is made more likely and convincing by the failure of the other. The polar stimulation hypothesis has been proposed many times (Lillie, 1903; McClendon, 1912; Just, 1922; Marsland and Landau, 1954; Swann and Mitchison, 1958; Wolpert, 1960; Schroeder, 1981a;White and Borisy, 1983). White and Borisy (1983) have presented the most recent and complete exposition. They made the following assumptions: (1) the stimulatory activity of the asters is spherically symmetric; (2) the stimulatory activity of the asters can combine additively both at the poles and the equator (presumably both polar surfaces are affected by both asters); (3) the magnitude of the stimulation varies as an inverse power law of the distance from the astral center to the cortex; and (4) the stimulus mechanism ceases when cleavage begins. Applying these assumptions, they found that the combined stimulatory activity at the equatorial surface was less than at the poles. The equatorial stimulation hypothesis has also been proposed many times, beginning over a century ago (Biitschli, 1876). It includes two simple ideas. The

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first is that when the asters are sufficiently close to each other their effect is additive. The second is that the surface closest to the region where the asters are confluent is subjected to the highest level of stimulation and the division mechanism forms by direct response in the stimulated region. In a spherical cell, there are differences between the relation of the mitotic apparatus to the polar and equatorial surfaces. The equatorial surface is closer to the region of the spindle. The angle of the linear elements to the surface is different in the equatorial and polar regions. The polar surfaces are closer to the astral centers. The equatorial surface is positioned so that both asters could act with equal intensity upon it if their action is distance related but the polar surfaces are not so positioned. The equatorial surface is a spherical segment with two bases that approximates a cylinder. The polar surfaces are spherical segments of one base that approximate a hemisphere. Depending upon the way in which the mitotic apparatus establishes the furrow, some of these relations and circumstances must be important to the process of furrow establishment and others must be unimportant. 1. Does Stimulatory Activity Cease at the Beginning of Cleavage?

The importance of this question relates to the assumption in the polar stimulation hypothesis that the effect of the asters depends upon their distance from the surface. As the equatorial constriction of normal cleavage progresses, the cell must pass through a phase in which the distance from the astral centers to the equatorial and polar surfaces is equal. This would mean that the polar and equatorial surfaces would be equally affected by the asters so that they would behave in the same way and the result would be cessation of cleavage. If, for instance, asters caused the surface to relax (Wolpert, 1960; Schroeder, 1981a; White and Borisy, 1983), then equatorial constriction would cease and furrowing would stop when the furrow entered the area that promoted relaxation. It has been known for some time that when the mitotic apparatus is shifted after the beginning of furrowing, another furrow forms in association with the new position of the mitotic apparatus (Harvey, 1935). Experiments described in Section II,A,3 demonstrated that the mitotic apparatus can continue to establish furrows long after the time when division would normally have been completed. These results demonstrate that the stimulatory activity of the asters does not cease when division begins. This finding is inconsistent with a fundamental assumption of the polar stimulation hypothesis.

2 . How Do Blocks between the Surface and Different Regions of the Mitotic Apparatus Affect Cleavage? According to the role postulated for the mitotic apparatus, interposition of blocks between the surface and different parts of the mitotic apparatus during the interaction period could affect furrowing. The nature of the effect would depend

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26 1

FIG. 1. Cleavage of a flattened Tripneusfes gratilla egg perforated parallel to the mitotic axis across the equatorial plane. Left, shortly after the operation; right, 28 minutes later. Reprinted by permission from the Journal of Experimental Zoology 227,213-227, copyright 1985, Alan R. Liss, Inc.

upon whether the polar or the equatorial stimulus mechanism was operating. Blocks consisting of punctures (Dan, 1943; Rappaport, 1968), oil drops, and glass needles (Rappaport and Rappaport, 1983) have been used with consistent results. Blocks of any size placed under any part of the surface outside of the equatorial region have no effect on division. The effect of blocks introduced below the equatorial surface is size related. Larger blocks result in greater inhibitory effect (Rappaport and Rappaport, 1983). When the cell is perforated in the equatorial plane, a block is interposed between the spindle and the equatorial surface. In these cells, a furrow forms in the proximal (with respect to the mitotic apparatus) surface of the perforation, but not in its distal surface nor in the original cell margin distal to it (Fig. 1) (Dan, 1943; Rappaport, 1968). It could be argued that the persistence of furrowing despite the blocks under the polar surface was due to the failure to create sufficient blockage in the right area. However, the effectiveness of blocks of the same size in the equatorial area is not predicted by the polar stimulation hypothesis which postulates that the equatorial surface initiates contraction because it was not directly affected by the asters. Failure of furrowing on the distal margin of perforations in the equatorial region is also not predicted by the polar stimulation hypothesis for the same reason. Formation of a furrow on the proximal margin of the same perforation shows that furrowing is possible in that region. The effectiveness of blocks between the mitotic apparatus and the equatorial surface and the ineffectiveness of similar blocks beneath the polar surfaces are predicted by the equatorial stimulation hypothesis but not the polar stimulation hypothesis.

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3 . How Does Manipulation of the Cell Outside of the Equatorial Region Affect Furrow Formation?

In brief, alteration of the polar and subpolar areas has little or no effect on furrow establishment. Sequestration or immobilization of the polar surface does not interfere with division (Rappaport, 1981), but immobilization close to the functioning furrow does (Rappaport and Ratner, 1967), probably because it reduces the amount of surface available to yield when the furrow deepens. Aspiration of cytoplasm between the mitotic apparatus and the polar surface is without effect, as is sequestration of the polar and subpolar parts of the asters in pipets (Rappaport. 1981). Neither changing the shape of the polar surface nor pushing it closer to or moving it farther away than normal from the astral center altered furrowing behavior (Rappaport, 1981). If the critical, distance-related effect of the mitotic apparatus were imposed on the surface outside of the equator (as the polar stimulation mechanism postulates), then some of these manipulations would be expected to affect the division process. 4 . Is the Abifiv of the Mitotic Apparatus to Establish Furrows Affected by the Distance between the Astral Centers?

In a spherical cell, when the interastral distance is increased, the distance from the astral centers to the polar surface decreases. According to the polar stimulation hypothesis, this change should not affect that cell’s ability to divide. However, many experiments have shown that when the interastral distance is increased division is inhibited. Sluder and Begg (1983) reported that when the spindle was cut and the half-spindles were separated no cleavage occurred. However, when the asters of the half-spindles were pushed together again after they were separated, division followed. The same results were obtained when isolated asters were pushed together in cylindrical cells (Rappaport and Rappaport, 1985b). Asters that are too far apart to establish a furrow in an equatorial surface located the normal distance from the mitotic apparatus can establish furrows when the equatorial surface is closer than normal (Rappaport, 1969). The loss of ability to establish furrows by reason of increased interastral distance is predictable according to the equatorial stimulation hypothesis because the zone in which the effect of the asters is additive would be reduced. It follows from this reasoning that pushing the equatorial surface closer to abnormally distant asters restores their ability to establish a furrow because the surface is moved into the reduced effective zone. The loss of ability to establish furrows when the asters are moved closer to the poles is not predicted by the polar stimulation hypothesis and neither is the finding that the deficiency caused by excessive distance between the asters can be remedied by decreasing the distance from the equatorial surface to the mitotic axis.

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b 192.9

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FIG.2. Dimensions of spherical (a) and artificially constricted (b) sand dollar eggs 5 minutes before the beginning of furrowing, at the time the position of the furrow is established. Dimensions are in microns; dots indicate positions of astral centers. Reprinted by permission from the Journal of Experimenrul Zoology 231, 81-92, copyright 1984, Alan R. Liss, Inc.

5 . Is the Normal Difference in the Distance between the Astral Centers and the Polar and Equatorial Surfaces Essential for Division? According to the polar stimulation hypothesis, the reason that the polar surface behaves differently from the equatorial surface is that the polar surface is closer to the astral center and the lesser distance results in more intense interaction. Therefore, it should be possible to make the surface behavior pattern abnormal by changing the distance between the mitotic apparatus and the surface in different regions. The normal distances in a sand dollar (Echinarachnius parma) egg are shown in Fig. 2a, which demonstrates that the astral centers are normally farthest from the equator and closest to the poles. When these eggs were artificially constricted by an 80-pm-diameter glass loop in the equatorial plane, the poles bulged outward while the equator was pushed inward. Since the length of the mitotic apparatus was not changed significantly, the dimensions changed to those shown in Fig. 2b. When compared to the distance relations in the normal spherical cell (Fig. 2a), there was a reversal; the astral centers in constricted cells were farthest from the poles and closest to the equator. According to the White and Borisy (1983) hypothesis, this change should reverse the normal pattern of surface behavior and cause contraction at the poles and relaxation at the equator,

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which would effectively block cleavage. Cleavage in this circumstance is, however, normal (Rappaport and Rappaport, 1984). The furrow forms at the midpoint between the asters, regardless of the relation of the constriction to the mitotic apparatus, and there is no contraction in the constriction plane unless it lies between the astral centers (Rappaport, 1964). The polar stimulation hypothesis, as stated, predicts that the dimensional changes described should block cleavage. The equatorial stimulation hypothesis predicts that cleavage would be unaffected. White (1985) has proposed that these results can be explained by revising his hypothesis to include a provision that the effect of the asters is not spherically symmetrical, but the data do not support his contention (Rappaport, 1969), and he does not discuss the effects of the revision on the original model. 6. What Dimensional Changes Compensate for Reduction in Size of the Mitotic Apparatus? Wilson (1901) found that ether blocked cleavage and reduced the size of the mitotic apparatus. Other anesthetics, volatile and nonvolatile, were subsequently shown to have the same effect (Harvey, 1956) (Fig. 3a). The size reduction is apparent not only in the living cell but it is also reflected in the dimensions of the mitotic apparatus isolated from treated cells (Hinkley et al., 1982). Because blockage of division caused by urethane treatment can be reversed by pushing the entire mitotic apparatus closer to the surface (Rappaport, 1971a), failure of furrow formation has been attributed to the greater-than-normal distance between the periphery of the reduced mitotic apparatus and the surface. Urethane-blocked cells provide an opportunity to determine which dimensions are critical to furrow establishment because they make it possible to selectively reduce the distance from different parts of the mitotic apparatus to different parts of the surface. Spherical sand dollar eggs fail to divide in 0.06 M urethane. However, when they are reshaped into cylinders by inserting them into 80-pmi.d. capillaries, the same urethane concentration fails to block cleavage. The reshaping decreases the distance from the astral centers to the equatorial surface and increases the distance from the centers to the polar surfaces (Fig. 3b). Bilaterally constricting treated cells in the polar and subpolar regions to 80 pm (Fig. 3c) and pushing the polar surfaces toward the astral centers had no effect on urethane blockage. However, urethane-treated cells that were artificially constricted in localized areas by insertion into 80-pm glass loops divided, provided the plane of constriction bore a certain relation to the mitotic apparatus (Rappaport and Rappaport, 1984). Whenever the constriction plane was positioned between the astral centers, cleavage occurred (Fig. 3d). When the mitotic apparatus was shifted so that the constriction plane failed to achieve that relationship, the cells failed to cleave even though the constriction partially bisected the cell (Fig. 3e and f). Although an artificial constriction imposed on the equatorial side of the aster results in a furrow, the same degree of constriction imposed the same

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e

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(2

FIG.3. Dimensions and proportions of urethane-treated sand dollar eggs. Dots indicate positions of astral centers; dimensions are in microns. (a) Spherical egg 5 minutes before anticipated cleavage time. These eggs did not cleave. (b) Proportions of egg confined in 80-pm-i.d. capillary. These eggs cleaved. (c) Proportions of egg artificially constricted to 80 pm diameter perpendicularto the mitotic axis in the planes of the astral centers. These eggs did not cleave. (d) Dimensions of eggs artifically constricted to 80 pm diameter in the equatorial plane. These eggs cleaved. (e) Proportions of eggs artificially constricted to 80 pm diameter so that the volume of cytoplasm on either side of the constriction was about equal and the reduced mitotic apparatus was positioned outside the constriction plane and perpendicular to it. These eggs did not divide. (f) Proportions of eggs artificially constricted as in (e), in which the mitotic axis was parallel to the constriction plane. These eggs did not divide. Reprinted by permission from the Journal of Experimental Zoology 231, 81-92, copyright 1984, Alan R. Liss, Inc.

distance from the astral center on the polar side of the aster is ineffective. The degree of constriction and the distance from the astral center in the two types of experiments are the same. The difference cannot be explained by simply supposing that the constriction predisposes the surface to furrow formation (Greenspan, 1977; White, 1985). The results indicate that there is a difference between the subsurfaces in the equatorial and polar regions. If the critical distance relationship were between the mitotic apparatus and surface regions outside of the

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equator, then the block should have been reversed by constrictions in the subpolar and polar areas and equatorial constriction should have had no effect. The equatorial stimulation hypothesis predicts that only equatorial constriction will be effective.

E. CONSEQUENCES OF INTERACTION BETWEEN APPARATUS AND

THE

THE

MITOTIC

SURFACE

Although the mechanism used to carry out the visible events of cytokinesis in animal cells is not as simple as it might be, it certainly could be more complex. Each of the major visible events that take place during division has been proposed as the active, driving mechanism of the process, including spindle elongation (Platner, 1886; Dan, 1948), increases in astral size (Gray, 1924), surface increases (Swann and Mitchison, 1958), equatorial constriction (Biitschli, 1876), and displacement of cell contents toward the poles (Robertson, 1909). Meves ( 1897) proposed that several of these mechanisms participated, some simultaneously, others in sequence. Fortunately, according to existing information, a single event-active constriction at the equatorial surface-can account for nearly all of these activities. At present, there is some controversy concerning the mechanism by which the contracting region is established. Two alternative hypothetical mechanisms have been proposed. According to one, the direct effect of the mitotic apparatus is to create, in the surface, a localized contractile region that ultimately carries out cytokinesis. According to the alternative hypothesis, the direct effect of the mitotic apparatus is to create localized relaxed regions which may permit the movement of mobile force-producing units to other, unrelaxed, regions where the contractile mechanism is assembled. Existing ultrastructural information is not useful for resolution of the controversy. A brief statement that immediately before cleavage the concentration of microfilaments under the polar surface is less than that under the equatorial surface (Usui and Yoneda, 1982) provides no information concerning the cause of the difference. Mobile force-producing units could be shifted either by contraction at the equator or by relaxation at the poles. 1. Early lndicutions of Local Contraction

In echinoderm eggs with large pigment granules embedded in the cortex, one of the first visible events of cleavage is the formation of a pigmented stripe in the equatorial region. The stripe is caused by regional isotropic shrinkage of the distance between the granules (Scott, 1960). In contrast with Scott (1960), Fischel ( 1906), and Rappaport and Rappaport ( 1976), Schroeder ( 1981b) believes that stripe formation takes place after furrowing begins, but the point is not important for the purposes of this discussion. The importance of the event is that it reveals a localized band of surface that behaves differently from that of the rest

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of the cell. The difference must be a consequence of interaction between the surface and the mitotic apparatus. In Arbacia lixula, the band is initially 22 pm wide and it comprises about 32% of the egg surface. It subsequently shrinks 34% to form a band 15 pm wide (Rappaport and Rappaport, 1976). It was postulated that the shrinkage could cause both an increase in microfilament density and their circumferential rearrangement (Rappaport, 1975; Fujiwara et al., 1978). 2. Effects of Experimental Surface Alteration The surface as well as the mitotic apparatus (see Section III,B) can participate in furrow establishment despite extensive alteration or disruption. The surface of nucleated exovates expressed from A. lixula eggs is formed by stretching the original egg surface over 100-fold. They furrow synchronously with control cells. When the mitotic apparatus was forced from a cell into an exovate after the cell initated cleavage, a new furrow appeared in the exovate in about 4 minutes (Rappaport, 1976). The “precipitation membrane” which forms rapidly over the surface of decorticated endoplasmic fragments (Chambers, 1917a, 192lb; Costello, 1932) also forms furrows in the presence of an active mitotic apparatus (Rappaport, 1983). These results strongly suggest that the normal organization of neither the surface nor the mitotic apparatus is required for their interaction and the subsequent formation of the division mechanism.

3. Origin of the Contractile Mechanism Contractile activity precedes development of an ultrastructurallydemonstrable contractile ring characterized by circumferentially oriented microfilaments (Schroeder, 1972; Usui and Yoneda, 1982). Actin has been demonstrated in cleavage furrows (Perry et al., 1971; Schroeder, 1973), as have myosin (Fujiwara and Pollard, 1976, Fujiwara eta!., 1978), and a-actinin (Fujiwara et al., 1979). The contractile ring may be recruited from elements from within as well as from outside the zone of initial contraction. If some of the units of the contractile ring moved into the equatorial region from elsewhere and actin is an important functional component, a higher concentration of actin in the furrow would be expected. However, there is some question whether the concentration of actin is greater in the furrow than elsewhere in the cortex (Herman and Pollard, 1978; Aubin et al., 1979). The idea that localized contraction could affect the distribution of force-producing components has been conjectured for some years and was recently restated (Schroeder, 1981a; White and Borisy, 1983), but experimental results that may possibly demonstrate such activity have only recently been obtained. When the mitotic apparatus of a cylindrical cell is shifted after furrowing begins, the original furrow is obliterated. The mode of obliteration depends upon how far the mitotic apparatus is moved. When the distance is 45 pm or less, the constriction appears to slide, intact, to the midpoint between the asters in their new position. When the mitotic apparatus is moved 90

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pm, the original furrow fades after the new one develops at the midpoint of the relocated mitotic apparatus and there is no appearance of sliding (Rappaport, 1985). Since furrow formation takes place and cleavage is completed when the mitotic apparatus is entirely removed 4 minutes before cleavage begins in these geometrical circumstances (Rappaport, 1981), the reaction cannot be due to simple dislocation of the mitotic apparatus from the vicinity of the furrow. And, since the furrow can slide intact, its force-producing elements have not been dispersed so as to bring about a relaxation. Cylindrical cells can support the formation of multiple furrows that divide them simultaneously into multiple cells so that the phenomenon cannot be attributed to a mechanism that limits the cell to one furrow at a time. It was suggested that the new furrow may contract with greater force than the old furrow and that, in this circumstance, force-producing units may be shifted from the old to the new furrow (Rappaport, 1985). Further analysis of this phenomenon is necessary. The idea that force-producing units could be recruited from outside the equator was revived by Schroeder (1981a) and White and Borisy (1983) as part of a hypothetical mechanism in which an equatorial contractile mechanism is constructed at the equator by a relaxation phenomenon which takes place at the poles. Lewis (1942) pointed out that force-producing substances could accumulate at the equator as a consequence of mitotic apparatus interaction with either the equatorial or the polar surfaces. If the hypothetical recruitment is real, it can be accounted for by a difference in tension in the equatorial and polar regions (White and Borisy, 1983). There is a major question whether the difference arises by a decrease in tension at the poles or an increase in tension at the equator. The basis of the difference cannot be determined by observing its consequences in intact cells.

4. The Immediate Consequences of Mitotic Apparatus-Surjiace Interaction The different hypothetical stimulus mechanisms described in Section II1,D require that the immediate consequences of the interaction between the mitotic apparatus and the surface be different. If the direct effect of the mitotic apparatus on the equatorial surface is responsible for cleavage, then it must cause contraction. Equatorial relaxation cannot cause the visible events of furrowing. However, if division results from the action of asters upon the poles, its immediate consequence cannot be contraction if the normal events of furrowing are to be produced. In this case, it was proposed that the immediate effect is relaxation which, combined with mobile contractile elements, could result in the formation of an equatorial contractile ring (Schroeder, 1981a; White and Borisy, 1983). Thus the two hypotheses differ not only in which part of the surface is affected but also in the postulated nature of the immediate effect upon the physical behavior of the surface. The two postulated mechanisms operating in the normal dimensional relations of a spherical cell produce identical outcomes because both

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D

FIG. 4. Cleavage of a torus-shaped echinoderm egg. (A) and (B) Immediately before and after the first cleavage; (C) and (D) immediately before and after second cleavage. Each pair of asters is joined by a spindle, resulting in uninucleate cells. The central circle represents a glass sphere. Reprinted by permission from the International Review of Cytology 31, 169-213, copyright 1971, Academic Press.

hypotheses were designed to explain the same event under the same circumstances. However, when experiments in which the interaction takes place under unusual geometrical circumstances are designed, the results predicted by the alternative mechanisms can be different. If the immediate consequence of astral influence were to reduce the tension of force-producing units in the proximity of the asters so that the units move up a tension gradient and accumulate in regions more distant from the asters (White and Borisy, 1983), then it would be possible to arrange conditions so that one mitotic apparatus would be predicted to establish two furrows. A spherical sand dollar egg may be reshaped into a torus by holding it against a flat surface and forcing a small sphere through its center (Rappaport, 1961). In this circumstance, the mitotic apparatus is confined to a restricted region of the torus but the mitotic cycle of the reshaped cell remains synchronous with that of the controls. If the furrow were established by localized relaxation near the asters and movement of force-producing units toward more distant surface regions, then there should be furrowing activity between the polar surfaces of the asters as well as between their equatorial surfaces. At the first cleavage, however, only a single furrow develops between the equatorial surfaces of the asters (Fig. 4B). At the

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second cleavage of torus-shaped cells, when the polar surfaces of the asters of the two mitotic apparatus are closer together, a furrow forms in regions where neither spindle nor chromosomes are present (Fig. 4D). In fact, the single binucleated torus cell at that time forms three furrows simultaneously which suggests that failure to form more than one furrow at first cleavage was not due to lack of contractile mechanism precursor material. In this experiment, the only significant difference between the first and second cleavage was the proximity of the asters to each other. The actual outcome of the first cleavage of torus-shaped cells is predicted by the equatorial stimulation hypothesis but not by the polar stimulation hypothesis. In spherical cells, single asters (Hiramoto, 1971) and pairs of asters that are situated at a great distance from each other (Sluder and Begg, 1983) do not elicit furrows but may promote surface irregularities (Hiramoto, 1971). In cylindrical cells, in which the asters are closer to the surface than in spherical cells of equal volume, asters can establish furrows when they are farther apart than normal (Rappaport and Rappaport, 1985b). In cylindrical cells, also, the relation of furrowing and constriction activity to the asters would be different depending upon whether the aster directly causes relaxation and relocation of force-producing units or, alternatively, regional surface contraction. The polar relaxation mechanism predicts that when the asters are close to the poles of an elongate cell the astral centers will be isolated by two furrows in spherical cytoplasmic masses which are separated by a single, anucleate mass (White and Borisy, 1983). The equatorial constriction hypothesis predicts that if the distance from the astral center to the pole equals the distance from the astral center to the cylindrical surface, then the cell should form a hemispherical contracting zone at the pole, which reduces the radius of curvature and shrinkage away from the nearby cylindrical surface, but it need not necessarily form a furrow. The result is as predicted by the equatorial stimulation hypothesis (Rappaport and Rappaport, 1985b) because, despite the visible consequences of the predicted shrinkage, no furrow forms. When one aster has been removed from a cylindrical cell and the remaining aster is located some distance from the pole, furrow-like constrictions which sometimes become permanent form. In most cases, the furrow deepens in the plane of the astral center and may bisect the aster (Rappaport and Rappaport, 1985b) (Fig. 5 ) . This result would be expected if the direct astral effect were to cause nearby surface constriction. If the aster caused local relaxation, then any furrow which might form would not cut into the aster because the local surface would promptly recede owing to the aster’s relaxing effect. There appears to be no way in which single asters could bring about the experimental results just described by causing local relaxation and transfer of force-producing units. If, on the other hand, asters caused local constriction in the nearby surface, the actual outcome would be predicted. There is no justification for proposing that the effect of a single aster on the surface is qualitatively different from the effect of

CYTOKINESIS IN ANIMAL CELLS

27 1

FIG. 5 . Local surface constriction in the plane of the center of an isolated aster in a sand dollar egg confined in an 82-pm-i.d. capillary. Linear elements of the companion aster are visible in the region of the arrowheads. Reprinted by permission from the Journal ofExperimenru1 Zoology 235, 217-226, copyright 1985, Alan R. Liss, Inc.

each of the asters of the intact mitotic apparatus. If one aster can cause local constriction, it is logical to propose that a pair of asters acting jointly upon an intervening surface region should have the same effect. When cylindrical cells in which the asters have been separated by breaking the mitotic apparatus are treated with ethyl urethane, the size of the asters is reduced and their ability to cause astral constrictions is affected. Low concentrations of the substance can abolish astral constriction formation in cells with abnormally distant asters yet permit cleavage in cells containing an intact mitotic apparatus. The two groups of cylindrical cells being compared were subjected to the same treatment and the shortest distance from the astral centers to the cylindrical surface was the same. They differed only in that the asters of the intact mitotic apparatus are closer together and farther from the polar and subpolar surfaces. Astral constriction and normal furrowing appear to be attributable to the same physical mechanism. The finding that reduced asters can cause surface constric-

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tion only when they are close together strongly suggests that, in that circumstance, their effects are addititive and that, even though the effect of their joint action on the surface is diminished, it is sufficient to cause visible activity (Rappaport and Rappaport, 1985b).

5 . Effect of Distance from the Mitotic Apparatus on Furrow Establishment and Function When the mitotic apparatus is shifted in the equatorial plane, the linear elements in the equatorial region are longer than normal and, when the sea urchin egg is distorted, they may be longer than they could possibly be if the egg were spherical (Harvey, 1935). A similar capacity of linear elements to extend farther than normal can be seen between the asters and the poles of cylindrical eggs in which the distance from the astral center to the polar surface is increased by polar distension. Observations of this kind imply that the length of the linear elements in the normal, spherical cleaving egg is not determined by factors within the elements but rather by the boundary imposed by the surface and cortex. Asnes and Schroeder (1979) found that relatively few microtubules extended to within 5 pm of the surface. In the sand dollar egg, furrows can form when the distance between the axis of the mitotic apparatus and the equatorial surface is increased by 70% (Rappaport, 1982). At greater distances, furrowing becomes unpredictable. Temporary indentations usually form in the region where the furrow would be expected but not progressive, permanent furrows. There is no evidence of a well-defined threshold of response. More distant equatorial margins form apparently feebler furrows, which are unable to exert the tension necessary to completely and permanently divide the cell. Within the effective range of the mitotic apparatus, the time when the furrow appears in the equatorial margin is correlated with the distance between the margin and the mitotic axis (Section III,A,4). There is also a correlation between the initial distance from the mitotic apparatus to the equatorial surface and the rate of furrow progress. Furrows formed on closer surfaces move faster. An inverse proportionality exists between the ratio of the rates of the slower and the faster furrows and the ratio of the distances from the mitotic apparatus to the closer and more distant margins (Rappaport, 1982). When the rates of progress of two furrows within the same cell are compared, the shape and resistance to deformation of the cell regions encountered by the furrows are the same. The difference in rates appears to be due to the difference in the amount of force that the furrows can exert. The surface located farther from the source of the cleavage stimulus forms weaker furrows. It is implied that the effectiveness of the stimulus decreases with distance, but whether it does so as a power function, as White and Borisy (1983) assume, is unknown. The position of the mitotic apparatus can be so eccentric that the furrow forms only on the closer equatorial margin. This is the normal condition in a number of cleaving eggs. In some Cnideria, Ctenophora and Arnphibia which cleave ini-

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tially into two cells, the mitotic apparatus is relatively small as well as eccentric. After cleavage begins, the furrow continues to progress through areas that were not directly affected by the mitotic apparatus (Yatsu, 1912; Rappaport and Conrad, 1963). A mitotic apparatus that normally establishes a unilateral furrow will establish a symmetrical furrow when it is centered in the protoplasmic mass and a mitotic apparatus that normally produces symmetrical furrows will cause unilateral furrows when pushed into an eccentric location (Rappaport and Conrad, 1963). In some Cnidaria and Insecta, the fxst cleavage results in more than two cells because it is preceded by a period in which several mitotic cycles occur without intervening cytokinesis. After the population of nuclei reaches a certain size, multiple furrows simultaneously partition the nuclei into separate cells. Fullilove and Jacobsen (197 1) correlated the development of furrows with the proximity of the asters to each other and to the surface. Wilson (1903), on the other hand, found that simple amputation experiments performed on the eggs of Renillu, the sea pansy, which should have brought the mitotic apparatus closer to the surface, did not advance the time of cytokinesis.

6. Polar Body Formation Because it contrasts in some respects with the characteristics of ordinary cell division, the formation of the polar bodies which occurs during meiotic divisions of the oocyte has fascinated students of cytokinesis for many years. In polar body formation, there is great disparity in the size of the daughter cells, and the mitotic apparatus is relatively small and oriented so that its axis is perpendicular to the nearest surface. It is usually firmly attached to the surface by the polar region of one aster after the beginning of anaphase. However, despite some of the special circumstances associated with the event, the achromatic apparatus that is usually associated with meiosis can establish a furrow of normal appearance and function if it is shifted away from the surface (Conklin, 1917; Morgan, 1937). Observers have divided the process into two phases. In the first phase, the surface in the immediate vicinity of the mitotic apparatus bulges outward. Yatsu (1909) and many others attributed the bulge to local decrease in tension at the surface caused by the nearby aster or centriole. Nuclear material is carried outward in the bulge. In the second phase, the contents of the polar body are permanently separated from the oocyte by events which closely resemble ordinary cytokinesis. At the time that electron microscopists saw sheets of vesicles in cleavage furrows, they also saw them in the plane of separation of the polar body (Humphreys, 1964). More recently, however, bands of microfilaments resembling the contractile ring have been described (Burgess, 1977). Attempts have been made to analyze the nature of the relationship between the mitotic apparatus and the surface by experimentation. Chambers (1917b) found that when the daughter nucleus remaining in the oocyte cytoplasm after the first meiotic division was pushed into a new location, it subsequently moved toward the nearby surface and at the next division cycle became involved in polar body

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formation at some distance from the location of the first polar body. When a portion of the attached mitotic apparatus was tom away with a needle and pushed under a more distant surface, a protuberance appeared in association with the new location in about 1.74 minutes (Rappaport and Rappaport, 1985a). There can be little doubt that the bulge is formed in response to the material of the mitotic apparatus but, in view of the fragmented nature of the material that was moved, it seems unlikely that the bulge was formed by the pushing of the growing internal aster against the yolk granules as Burgess (1977) proposed. In order to throw more light on the question of whether the protuberance forms because of local surface weakening, pressure inside the oocyte was increased and the effects on polar body size were observed (Rappaport and Rappaport, 1985a). Neither flattening the cell nor increasing its volume osmotically increased the size of the polar body or enhanced the cytoplasmic flow associated with its formation. Incipient polar bodies appeared to snap back into the body of the occyte when the pressure was mechanically increased, but the same treatment caused cytoplasmic outflow into surface blebs caused by cytochalasin B treatment. The osmotic rupture that resulted from immersion in dilute sea water showed no tendency to appear in the area of the incipient polar body. Although Longo (1972) found that the protrusion phase of polar body formation in a molluscan egg could not be blocked with cytochalasin B as could the division phase, both phases of the process in starfish were susceptible to cytochalasin B (Rappaport and Rappaport, 1985a). Burgess (1977) found that a filamentous layer formed adjacent to the plasma membrane near the peripheral aster after it was close enough to the surface to displace the intervening material. The bulge formed in the surface associated with the filamentous layer. Dan and Ito (1984) found that the surface to which one aster of the mitotic apparatus was attached was more resistant to dispersion by detergent treatment. These observations would not be expected in an area that has been physically weakened by the presence of an aster. They are, however, consistent with the results of experiments which demonstrated no local weakening or decreased resistance to deformation (Rappaport and Rappaport, 1985a). It is possible that the protuberance is due to local differences in surface-associated cytoskeleton which develop close to the asters. There are other examples of protuberances that form in association with asters. The formation of bulges or caps is associated with the syncytial nuclei of Drosophilu embryos (Warn et ul., 1984).

IV. Summary The division mechanism is fixed in the surface during anaphase or about 4 minutes before furrowing begins in cylindrical cells. Under experimental conditions, the minimum time that the mitotic apparatus must act upon the surface is

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about 1 minute. The stimulus period is followed by a latent period of 2-3 minutes. The time of furrow formation can be advanced or delayed by manipulating the surface and the mitotic apparatus. Since furrows can be elicted long after normal division would have been completed, it is suggested that the brevity of the normal interaction period is not a consequence of the constitution of the interactants. The component of the mitotic apparatus that establishes the furrow moves from the region of the mitotic axis to the surface at 6-8 Fmlminute. The components of the mitotic apparatus that are essential for furrow establishment are confined to the achromatic regions. In spherical cells with large asters, the spindles are not required, although the spindle’s ability to establish furrows in spherical cells can be demonstrated by changing the cell’s geometry. In nonspherical cells with small asters, the spindle is probably the normal active agent. Although the ability of the mitotic apparatus to establish furrows can be diminished or abolished by measures that reduce its overall size, there are no decisive data concerning which of its ultrastructural components play essential roles in cytokinesis. The effect of changing the geometrical relation between the mitotic apparatus and the surface differs according to the region affected. Division can be blocked or impeded only by changing the relation between the equatorial surface and the mitotic apparatus. The ability of the mitotic apparatus to establish furrows is diminished by increasing the distance between the astral centers and also by increasing the distance between the mitotic axis and the equatorial surface. The cleavage block that results from reduction in size of the mitotic apparatus can be reversed only by decreasing the distance from the mitotic axis to the equatorial surface. Artificial constrictions imposed in other regions are ineffective. The normal distance relation between the astral centers and the equatorial and polar surfaces in spherical eggs is not required for division. Cleavage can occur when the dimensional relations are reversed. Both the surface and the mitotic apparatus can interact to establish furrows after exposure to measures that disrupt their normal organization. Single, isolated asters can cause furrow-like constrictions. Their immediate effect is to cause local contraction in nearby surface. The rate of progress of the furrow is in inverse proportion to the initial distance between the mitotic apparatus and the equatorial surface. This finding suggests that, beyond the normal distance from the mitotic axis to the equator, the intensity of stimulus activity decreases with distance. The final separation of the polar body from the oocyte closely resembles normal cytokinesis. The preceding protrusive phase cannot be shown to be associated with local surface weakening, and a possible role for cytoskeletal components is suggested.

TABLE I SUMMARY Ob EXPERIMENTS I N V O L V I N G ALTERATION OF NORMAL GEOMETRICAL RELATIONSBETWOEN T H t MITOTIC APPARATUS A N D THE SURFACE Predicted outcome Description of experiment 1. Interposition of blocks between the as-

ters and the polar and subpolar surfaces (Dan, 1943; Rappaport, 1968; Rappaport and Rappaport, 1983) 2. Interposition of blocks between the mitotic apparatus and the equatorial surface (Dan, 1943; Rappaport, 1968; Rappaport and Rappaport, 1983) 3. Manipulation of polar and subpolar parts of asters, cytoplasm between asters and surface, and surface (Rappaport and Ratner, 1967; Rappaport, 1981) 4. Relocation of asters farther from equator, closer to poles (Sluder and Begg, 1983; Rappaport, 1968, 1981; Rappaport and Rappaport, 1985b) 5. Relocation of asters in distant positions in 4 closer to equator (Sluder and Begg, 1983, Rappaport and Rappaport, 1985b) 6. Relocation of polar and subpolar surfaces away from astral centers (Rappaport, 1981; Rappaport and Rappaport, 1984) 7. Relocation of equatorial surface closer to mitotic axis in cells with asters too far apart to establish furrows (Rappaport, 1969) 8. Artificial constriction at equator and distension of poles to equalize or reverse distance relations to astral centers (Rappaport and Rappaport, 1984) 9. Artificial constriction at equator and distension of poles in urethane-blocked cells (Rappaport and Rappaport, 1984) 10. Relocation of polar and subpolar surfaces closer to astral center in urethaneblocked cells (Rappaport and Rappaport, 1984) 11. Relocation of the mitotic apparatus after the furrow develops (Rappaport and Ebstein. 1965; Rappaport, 1975; Rappaport, 1985)

Polar stimulation

Equatorial stimulation

Actual result

No furrow

Furrow

Furrow

Furrow

Interference with furrow formation

Interference with furrow formation

Interference with furrow formation

No interference, furrow

Furrow

Furrow

No furrow

No furrow

No furrow

Furrow

Furrow

Interference with furrow formation

Furrows

Furrows

No furrow

Furrow

Furrow

No furrow

Furrow

Furrow

No furrow

Furrow

Furrow

Furrow

No furrow

No furrow

No furrow after the first

Possible multiple furrows

Multiple furrows

277

CYTOKINESIS IN ANIMAL CELLS TABLE I (Continued) Predicted outcome Description of experiment !. First cleavage of a torus-shaped cell

(Rappaport, 1961)

Location of isolated aster close to pole of elongate cell (Rappaport and Rappaport, 1985b)

1.

1. Location of isolated aster distant from poles in cylindrical cell (Rappaport and Rappaport, 1985b)

Location of equatorial surface next to spindle in circumstances where close asterpolar relation difficult to visualize (Dan, 1943; Rappaport, 1968; Rappaport and Rappaport, 1974; Kawamura, 1977)

i.

Equatorial stimulation

Actual result

Furrows at equator and between polar regions of asters Furrow on side of aster away from pole (White and Borisy, 1983)

Furrow at equator only

Furrow at equator only

Constriction of polar and cylindrical surface, possibly no furrow

Possible constriction not in plane of astral center No furrow

Constriction in plane of astral center

No furrow, constriction at polar and cylindrical surface (Rappaport and Rappaport, 1985b) Constriction in plane of astral center

Furrow

Furrow

Polar stimulation

Experimental results have usually been used to characterize and localize processes, events, and circumstances that are essential to division. They can also be used to evaluate hypothetical explanations of different aspects of the process. From a hypothesis, testable predictions can be deduced. If the predicted results do not occur, the validity or usefulness of the hypothesis is diminished, if it is not altogether refuted. The visible events of cytokinesis are the consequence of a chain of events that can be broken in many places. A complete hypothetical division mechanism concerns factors that organize the division mechanism and set it in motion as well as the nature of the physical process that divides the cell. If one of the links of the logic chain fails because it does not predict the results of an experiment, then the entire hypothesis may be refuted. For instance, the astral cleavage theory in its various forms (Chambers, 1919; Gray, 1924) is logical and it provides a clear, rational basis for correlating the changes in form and behavior of the mitotic apparatus, surface, and subsurface during cleavage. However, if cells are divided by an astral cleavage mechanism, the continued presence of the asters would be required during division. The fact that removal of the asters shortly before the beginning of cytokinesis does not prevent division refuted the

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theory, despite the fact that it otherwise satisfactorily explained the visible events of the process. In Table I is listed a series of experiments that were described previously. The outcome of the experiments would have been different depending upon whether an equatorial stimulation-contraction mechanism or a polar stimulation-relaxation mechanism operated. Most of the experiments involved manipulation of the geometrical relation between the mitotic apparatus and the surface. The polar stimulation-relaxation hypothesis predicts none of the results accurately, while the equatorial stimulation-contraction hypothesis predicts all of the results accurately. The polar stimulation-relaxation hypothesis appears to be deficient in ways that cannot be remedied by tinkering. Serious proponents of the hypothesis now must obtain new supporting data. The idea of mobile force-producing units is still tenable as their redistribution could be caused by increased tension at the equator as well as by decreased tension at the poles.

ACKNOWLEDGMENTS The author’s work was supported by grants from the National Science Foundation and this project was supported by NSF Grant PCM 8404174. The expert and dedicated assistance of Barbara N . Rappaport is gratefully acknowledged.

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INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 105

The Circumventricular Organs of the Mammalian Brain with Special Reference to Monoaminergic Innervation CLAUDEBOUCHAUD AND OLIVIER BOSLER Universite‘ Pierre et Marie Curie, Faculte‘ des Sciences, Institut des Neurosciences, UA CNRS 1199, De‘partement de Cytologie, 75230 Paris Cedex 05, France, and CNRS, Laboratoire de Neurobiologie, Equipe de Neuromorphologie Fonctionnelle, 13402 Marseille Cedex 09, France

I. Introduction Vertebrate brains contain specialized regions which establish special relationships with both the cerebrospinal fluid (CSF)’ and the blood supply. These regions, most of which lie outside the blood-brain barrier (BBB), belong to the group called “circumventricular organs” (CVO) (Hofer, 1958, 1965), which have been shown to be present in all vertebrate species, tending toward simplification in the superior vertebrates (see Vigh, 1971; Kelly, 1982). In mammals, moving in the rostrocaudal direction, they classically comprise the organum vasculosum laminae terminalis (OVLT), the median eminence (ME) and neural lobe (NL) of the neurohypophysis, the subfornical organ (SFO), the subcommissural organ (SCO), the pineal gland, and the area postrema (AP). According to some authors, the choroid plexuses, the paraventricular organ, and the collicular recess organ, three morphologically specialized ependymal structures, should also be included in the CVO group (Palkovits, 1965; Vigh, 1971; Stumpf et al., 1977), but they will not be considered in this article. The CVO are located in strategic positions for integrating humoral, hormonal, and neural messages and their morphological organization is optimal for such functions, which has led Knigge (1975) to collectively call them the “windows of the brain. These organs are indeed readily accessible to CSF-borne and blood-borne signals and a release of bioactive substances into the blood is known ”

‘Abbreviations: Ad, adrenaline; AP, area postrema; BBB, blood-brain bamer; CRF (corticotropin-releasing factor), corticolibenn; CSF, cerebrospinal fluid; CVO, circumventricular organ; DA, dopamine; DBH, dopamine P-hydroxylase; 5-HT, 5-hydroxytryptamine (serotonin); LH-RH (luteinizing hormone-releasing hormone), luliberin; ME, median eminence; NA, noradrenaline; NL, neural lobe; 6-OHDA, 6-hydroxydopamine; OVLT, organum vasculosum laminae terminalis; PNMT, phenylethanolamine N-methyltransferase; SCO, subcommissural organ; SFO, subfomical organ; SRIF (somatotropin release inhibiting factor), somatostatin; TH, tyrosine hydroxylase; TRH (thyrotropin releasing hormone), thyroliberin. 283 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

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or presumed to occur at their level (Weindl, 1973). All CVO have a neuroglial parenchyma. Their internal surface is covered with a modified ependyma, which differs morphologically and biochemically from typical ependyma (see Knowles, 1969; Didier et af., 1985a). Among the glial elements which can be recognized in the mammalian CVO, four types are particularly noteworthy: (1) the tanycytes, which establish an anatomical link between the ventricular cavity and the vascular compartments in the ME, the OVLT, and the SFO, (2) the pituicytes, which constitute the specialized glia of the NL, (3) the pinealocytes, which predominate among the pineal parenchymal cells and have been found to elaborate indoleamines, especially melatonin, and peptidergic compounds, and (4) the SCO ependymocytes, which synthesize and release into the CSF a complex mucopolysaccharide-protein secretion constituting Reissner’s endoventricular fiber. From a physiological point of view, all CVO actually participate in regulating homeostatic functions. The ME and the NL of the neurohypophysis are two neuroendocrine interfaces receiving projections from the parvo- and magnocellular neurosecretory systems elaborating the various hypophysiotropic and neurohypophysial hormones. Among the CVO, the neurohypophysis, along with the pineal gland, is the most thoroughly investigated structure to date and its physiological roles, mainly in neuroendocrine regulation, have been well documented. The OVLT has also been found to be a site of neurohormonal release. It is thought to carry out important roles, especially in the regulation of gonadotropic function (Kawakami and Sakuma, 1976; Wenger, 1976a,b; Wenger and KerdelhuC, 1979; Samson and McCann, 1979; Piva et al., 1982), and also to be involved in osmotically and angiotensin 11-induced drinking and vasopressin secretion (Phillips, 1978; Knowles and Phillips, 1980; Thrasher er al., 1982; also see Ramsay et a/., 1983). The pineal gland, which is an important photoreceptor center in lower vertebrates (see Collin, 1969), seems in mammals to be exclusively involved in endocrine and/or neuroendocrine mechanisms (Ptvet, 1983) although, in some mammalian species such as the mole and the bat, rudimentary photoreceptor cells are known to persist. The SFO and the AP might be more specifically concerned with the reception of peripheral, blood-borne signals, with the former (SFO) being a key region for the regulation of thirst, saltwater balance (Palkovits, 1966; Summy-Long et al., 1976; Miselis, 1981), and possibly blood pressure, while the latter (AP) is chiefly believed to be a chemoreceptor trigger zone involved in vomiting, osmoreception, and cardiovascular regulation (Borison and Wang, 1949; Wise and Ganong, 1960; Borison et af., 1974; Barnes et af., 1984). Although various observations have also shown the SCO to be involved in osmoregulation (see reference in Vullings and Diederen, 1985), this organ is probably the most obscure of all the CVO with regard to its physiological functions. The SCO, which displays an ependymal secretory activity known to be subject to periodic variations (Didier et al.,

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1985a), differs from the other CVO in that it does not lack the BBB and is thus the site of liquido-tissular rather than hemato-tissular exchanges. To better understand the nervous mechanisms subserving the regulation of CVO functions, a number of works have dealt with the neurochemical identification of the neural elements constituting the CVO. Since it has been found that these organs are densely innervated with monoaminergic fibers, they have been taken as models for investigating the morphological substrate of both neural and neurohormonal monoamine activity (see Calas et al., 1978). This review article, which will be devoted to some cytofunctional topics relating to the CVO, will in fact focus on the monoaminergic innervation of these organs as well as on the possible involvement of monoamines in CVO mechanisms, especially in terms of their interactions with the other transmitter-specific systems, mainly peptidergic systems, which have been identified in the CVO (Weindl and Sofroniew, 1978, 1981).

11. Anatomofunctional and Cytofunctional Aspects of the Mammalian Circumventricular Organs The CVO were first investigated through their ability to be selectively stained by colloidal acid dyes administered via the general route (see for instance Wislocki and Putnam, 1920; Putnam, 1922). The locations of the CVO in the brain and their anatomical properties allow them to integrate information conveyed by the nervous system and both the vascular and ventricular compartments. This section will be concerned particularly with the description of the morphological substrate underlying such interactions. A. ANATOMICAL CONSIDERATIONS All CVO are located around the midline ventricular system (Fig. 1). 1. The Neurophypophysis The neurohypophysis constitutes the central interface for the co-operation of the nervous and endocrine systems. The ME is a differentiation of the floor of the third ventricle where multiple hypophysiotropic factors are released from the nerve endings of the so-called “tubero-infundibular system” in a primary vascular plexus that drains into the portal vessels of the pituitary gland. The tuberoinfundibular system connects several hypothalamic nuclei to the external neurovascular zone of the ME. Precise information concerning the exact location of the projecting neuronal perikarya within these nuclei has recently been gained in an elegant study using horseradish peroxidase microiontophoresis (Lechan et al., 1980).The brainstem as well as the nucleus raphis dorsalis and, to a lesser extent,

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FIG. 1. The circumventricular organs of the mammalian brain from a midsagittal section, depicted schematically. AP, Area postrema; ME, median eminence; NL, neural lobe; OVLT, organum vasculosum laminae terminalis;P, pineal gland; SCO, subcommissural organ; and SFO. subfornical organ.

the substantia nigra also contribute to the innervation of the ME (see below). The NL constitutes a distinct neurosecretory organ, having lost direct contact with the cerebral ventricular cavity. It receives the terminals of the supraoptico- and paraventriculo-hypophysialtracts, which discharge their neurosecretory products (oxytocin and vasopressin) into the general circulation. A tubero-posthypophysial tract has also been identified (see below).

2 . The UVLT The OLVT, originally named the “supraoptic crest,” lies immediately above the optic chiasma and closes the cavity of the third ventricle rostrally, at the level of the preoptic recess. Like the ME, it is essentially a site of distal integration where various types of neurohormones (luliberin, LH-RH; somatostatin, SRIF; thyroliberin, TRH; and corticoliberin, CRF) might be released into the blood to reach still unknown targets. Anatomical studies using the axonal transport of horseradish peroxidase have shown that the OVLT receives afferent fibers from several hypothalamic nuclei (mainly from the medial preoptic nucleus, the ventromedial nucleus, and the lateral hypothalamus) as well as from extrahypothalamic regions, the midbrain central gray, the locus coeruleus, and another CVO, the SFO (Palkovits e t a / ., 1977; Camacho and Phillips, 1981). Kato et al. (1984) have also reported, on the basis of retrograde axonal Iabeling with wheat germ agglutinin that, within the hypothalamus, the periventricular, paraventricular, and dorsomedial nuclei also send off important projections to the OVLT. Using anterograde labeling with tritiated amino acids, projections from the cholinergic limbic system, mainly the septum (Shute and Lewis, 1967; Lewis and Shute, 1967), and from the nuclei raphe dorsalis and centralis superior (Moore, 1977) have eventually been evidenced. Nerve cell bodies projecting to

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the limbic system (Camacho and Phillips, 1981) and to the supraoptic nucleus (Tribollet et al., 1985) have also been shown to exist. 3. The SFO and the AP The SFO and the AP display some comparable features. Both establish a close relationship with the anterior and posterior margin of the choroid plexus and are composed of a neuroglial tissue containing a significant proportion of nerve cell bodies, unlike that of the NH and the OVLT. The SFO is a small median nodule which bulges into the third ventricle between the interventricularforamina while the AP, a paired bilateral structure which is fused into a single midline organ in rodents, is found at the obex overlying the junctions between the fourth ventricle and the central spinal canal. The SFO, initially described by Putnam (1922) as the intercolumnar tubercle, has been reported to receive projections, such as the OVLT from the cholinergic limbic system (Lewis and Shute, 1967), a major afferent input from the medial preoptic nucleus (Hemesniemi et al., 1972), a pivotal component of which has been termed the “anteroventral third ventricle” (AV3V) area (Brody and Johnson, 1980). Recently, evidence has been obtained for the existence of an angiotensin-containing neural input to the SFO arising from the lateral hypothalamic area and adjacent structures (e.g., the rostra1 zona incerta) (Lind et al., 1984, 1985). In the SFO, Summy-Long et al. (1984) have also identified arginine vasopressin fibers given off by the neurons of the hypothalamo-neurohypophysial system. Efferent pathways from the SFO and the sites of terminal fields have also been evidenced (Miselis, 1981). The SFO innervates the preoptic region including the AV3V area, at the core of which lies the OVLT (Camacho and Phillips, 1981, see above). Findings from recent double-labeling studies employing immunostaining in combination with retrograde transport of fluorescent tracers have suggested that at least some of the SFO afferents to the medial preoptic nucleus belong to angiotensin-producing cells (Lind et al., 1985). A second pathway from the SFO goes to the neurosecretory hypothalamus, especially the supraoptic and paraventricular nuclei (Miselis, 1981; Lind et al., 1982; Renaud et al., 1983; Tanaka et al., 1985). Some of the cells projecting to the paraventricular nucleus have been found to be angiotensin immunoreactive (Lind et al., 1984) or sensitive (Tanaka et al., 1985). Electrophysiological studies have established that SFO efferents to hypothalamic magnocellular nuclei influence predominantly the activity of the neurohypophysial projecting oxytocin and vasopressin neurons but also affect the excitability of a number of neurons that project to the ME and the dorsomedial medulla (Ferguson, 1984; Ferguson er al., 1984; Sgro et al., 1984; Renaud et al., 1985). The neural connections of the AP have recently been reviewed by Leslie and Gwyn (1984) and by Shapiro and Miselis (1985). The AP receives both peripheral and central input. Peripheral afferent fibers come from the carotid sinus, aortic and vagal nerves, and from

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thoracic and abdominal viscera conveying baroreceptive and visceral information respectively, while the central input originates in the nucleus of the solitary tract, the A-2 catecholamine-cell group, and the paraventricular and dorsomedial nuclei of the hypothalamus (Hosoya and Matsushita, 1981; Shapiro and Miselis, 1985). Two efferent projections from the AP have been described: a major projection to the parabrachial nuclei and a minor one to the medial solitary nucleus and the internal solitary zone (Van der Kooy and Koda, 1983).

4 . The Pineal Gland The pineal gland, being part of a family of diencephalic roof appendages classically referred to as the “pineal complex” in the lower vertebrates, is a single organ in mammals (epiphysis cerebri) which, like the NL of the hypophysis, has no relation to the ventricular CSF. It varies greatly in size, being generally well developed in mammals living at high altitudes (PCvet, 1983). The pineal gland also varies morphologically among the species (see, for instance, Vollrath, 1979). Its main cell component is the pinealocyte which in mammals has lost the morphological structures for direct light perception, showing a secretory rather than a sensory activity. This secretory function is responsible for the conversion of light input into hormonal output. In lower vertebrates, the existence of neural connections between the brain and the pineal gland has been well established, but in mammals most authors have long considered the pineal body to be exclusively innervated via its stalk by numerous sympathetic and rare parasympathetic fibers (Ariens Kappers, 1960, 1965; see review in PCvet, 1983). As early as 25 years ago, however, Barry (1961) suggested that the pineal Gomori-stained fibers come from the hypothalamus. Since then, experimental evidence has been accumulated, all of which indicates a direct innervation of the pineal gland from the brain via the pineal stalk, at least in some species (see Korf and Moller, 1984). Orthosympathetic fibers coming from the superior cervical ganglia nevertheless constitute the major input to the pineal. Some of these afferents end in the superficial part of the pineal body, mainly in the perivascular spaces, and others reach the deep pineal and, via the stalk, travel to the posterior and habenular commissures (Nielsen and Moller, 1975; GuCrillot et al., 1979; Moller and Korf, 1983). The nonorthosympathetic afferent fibers include (1) parasympathetic fibers, present mainly in the rostra1 part of the gland and thought to arrive via the petrosal nerve (Romijn, 1973, 1975a,b), and (2) myelinated fibers coming from the brain as demonstrated by David and Herbert (1973) in the ferret and by Gutrillot et a f . (1982) in the rat. By means of retrograde horseradish peroxidase tracing, fibers from various other central regions (colliculi, amygdala, dorsal lateral geniculate nucleus, hypothalamic paraventricular and suprachiasmatic nuclei, preoptic area, and olfactive centers) have also been identified in the cat and the gerbil (GuCrillot et al., 1982; Moller and Korf, 1983). Guerillot et af. (1979) have reported that the pineal stalk in the rat is in

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fact composed of at least two types of fibers, afferent fibers originating in the superior cervical ganglia, directed toward the pineal and the habenula, and efferent fibers arising from the habenula and posterior commissures. In a recent paper, Ariens Kappers (1985) pointed out the physiological significance of this minor central innervation. PCvet (1983) has found recent immunohistochemical results using antineuropeptide antibodies to be in keeping with the presence of extrahypothalamic fibers in the pineal. 5 . The SCO The SCO, which can easily be recognized by its horseshoe shape in transverse sections, is situated under the posterior commissure at the junction of the roof of the third ventricle and the cerebral aqueduct and constitutes an ependymal structure with a secretory activity even in adult humans, where it is quite small (Wislocki and Roth, 1958; Palkovits el al., 1962). The SCO columnar ependymocytes form a pseudostratified epithelium separated from the posterior commissure by a hypendyma, the thickness of which varies from one species to another (Lenys, 1965). The SCO is formed early in fetal life (Rakic and Sidman, 1968). Its secretory potentialities can be demonstrated in the mammal fetus (Marcinkiewicz and Bouchaud, 1983). There are many interrelations at the level of the posterior roof of the brain, particularly through the lamina intercalaris, which connects the epithalamus area to the basis of the pineal stalk. The SCO might be related in function to the pineal complex (Oksche, 1962) but this anatomical relationship does not appear to be generalizable to the lower vertebrates. Embryologically, the anlages of the SCO and the pineal gland are contiguous. In mammals, numerous relationships are maintained between these two structures. In some species, the SCO receives an innervation from the raphe nuclei (see below). According to Wiklund (1974), the rat SCO innervation could arrive via the lamina intercalaris.

COMPOSITION OF THE CIRCUMVENTRICULAR B. NEURONAL ORGANS (GENERAL CYTOLOGICAL FEATURES) Since the pioneering investigations of Brettschneider (1956) on the rat, a great deal of information has been gained from various species regarding the fine structure of the ME under normal physiological conditions as well as after experimental manipulations inducing changes in adenohypophysial activity (for review, see Kobayashi et al., 1970). In mammals, the ME is now classically defined as a neurohemal area from which neuronal perikarya and dendrites are excluded. Its neural cytological elements are essentially composed of nerve fibers which, in the inner zone (fiber layer), may be gathered into bundles that form the hypothalamo-hypophysial tract and are of unmyelinated axonal varicosities. These include some Herring bodies, but consist mostly of nerve endings

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containing neurosecretory granules (up to 150 nm in diameter) and/or large granular vesicles (100 nm) associated with small clear vesicles (30-50 nm). On the basis of the size and profile of the granules and vesicles they contain, Kobayashi and Matsui (1969) divided the nerve endings of the ME into four types, two of which consisted of typical neurosecretory terminals. It is well known, however, that neurosecretory granules and vesicles can change their size and/or shape depending on the activity of the axons, making any neurochemical classification impossible. A detailed description of the ultrastructure of the NL and the OVLT, the two CVO which have been found to be the most similar to the ME from a cytological point of view (Weindl and Joynt, 1973), can be found in Boudier er af. (1970), Rohlich and Wenger (1969), and Le Beux (1971, 1972). In the OVLT, dendritic profiles as well as some nerve cell bodies receiving synaptic inputs can be found (at least in certain species) within the anatomical limits of the OVLT, which is therefore now held to be not only a distal but also a proximal site of integration. In both the NL and the OVLT, a great number of neurosecretory terminals can be found containing, in addition to electron lucent microvesicles (40 nm), a high proportion of granules which are presumably carriers of neurohormones (80- 160 nm). As in the ME, the relative proportion of electron-lucent and granulated vesicles as well as the size of the latter in both the NL and the OVLT can vary according to the physiological condition of the animal (see, for example, Boudier etaf., 1970; Krisch, 1974, 1975; Wenger, 1976a,b). In addition to neurosecretory terminals, nerve endings exhibiting a majority of clear synaptic vesicles (4060 nm) with sometimes an eccentric dense dot, and generally associated with pleomorphic dense cored vesicles, can be seen in the NL and the OVLT. These nerve endings and the neurosecretory terminals can be considered as belonging to the two main categories of axonal varicosities present in these organs, although more precise classifications have been proposed (Rodriguez, 1971; Le Beux, 1972; Ishii et al., 1973). Neurosecretory terminals in the NH and the OVLT are mostly encountered in the neurohemal contacting zone (in the so-called reticular and palisade layers within the ME). At this level, they are frequently associated with the processes of tanycytes, pituicytes, and/or glial cells, forming what has been called “synaptoid contacts” (see Giildner and Wolft, 1973). These contacts are characterized by a thickening of the plasma membrane facing the glial element associated with an aggregation of synaptic vesicles. In the ME, synaptoid differentiations have also been reported to occur occasionally between two contiguous nerve terminals (Fuxe and Hokfelt, 1969). A common and striking feature of the nerve endings in the neurovascular zone of the NH and the OVLT is their frequent association with the parenchymal basement membrane limiting the perivascular space, which is considered to be the morphological structure subserving the neurohemal release known to take place at these levels. Also worthy of note is the fact that

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neurosecretory nerve endings have been observed protruding into the lumen of the infundibular and preoptic recesses at the levels of the ME and the OVLT (Weindl and Schinko, 1975; Rodriguez, 1976; Scott et al., 1977; Ugrumov and Mitskevich, 1981). This indicates that these two organs could be sites not only of vascular but also of ventricular neurosecretion. The fine structural organization of the SFO has recently been reviewed by Dellmann (1985). Since the presence of nerve cell bodies intimately associated with glial cells was established in the SFO, these have been classified into several types (Dellmann and Simpson, 1979). In view of their morphological and electrophysiological characteristics (Buranarugsa and Hubbard, 1979), some of the SFO neurons appear to be neurosecretory in nature. It is noteworthy that neuronal perikarya may also be present supraependymally at the level of the SFO. Afferent fibers make frequent axosomatic and axodendritic synapses; most presynaptic terminals contain numerous clear vesicles (50 nm, on average) and occasional large granular vesicles (80- 100 nm). Some neurosecretory axons have also been described (Dellmann and Simpson, 1979). Interestingly, typical “crest synapses” have been described in the cat SFO by Akert et al. (1967). Axonal dilatations are found in the parenchyma as well as within the ventricular lumen and the perivascular connective tissue. A striking feature of axon terminals at various stages in the regression-restitution (Dellmann and Simpson, 1975) or degeneration-involution cycle (Bouchaud, 1974b) is when they show an increase in size (measuring up to 15 pm in diameter) and a change in content (dense lamellar bodies, numerous clear vesicles). As in the SFO, the neuronal composition of the AP, the structure of which has recently been reviewed by Brizzee and Klara (1984), consists of neuronal perikarya and nerve fibers. These elements are separated from each other by glial processes and the neural processes are always unmyelinated, at least in the rat (Dempsey, 1973). The nerve fibers establish axodendritic and, more rarely, axosomatic synapses. The varicosities contain both clear and dense cored vesicles. It has been consistently observed that neurons and neuronal processes are frequently located in the perivascular sheaths of the AP blood vessels (Dempsey, 1973) but, at least in the squirrel monkey, always with an intervening glial process (Klara and Brizzee, 1975). The reader will find a didactic scheme of AP cytological organization in Pickel and Armstrong ( 1984). In the pineal of some species, intraparenchymal neurons have been described in very variable numbers (see PCvet, 1983). Numerous unmyelinated varicose fibers run between the parenchymal cells. Their vesicular content shows a circadian evolution (Matsushita and Ito, 1972). Axonal varicosities have frequently been found adjacent to perivascular spaces (Pellegrino de Iraldi et al., 1965; Taxi and Droz, 1966). This innervation is described below. No nerve cell bodies have been described in the mammalian SCO, whereas nerve fibers have been demonstrated in the rat (Stanka, 1964), the mongolian

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gerbil (Wiklund et a/., 1977), and, more recently, in the cat (Sakumoto et a f . , 1984). In these species, the SCO ependymocytes and hypendymocytes receive an innervation from an hypendymal plexus. In the rat, the unmyelinated axons display numerous varicosities (0.1-0.6 pm, mean diameter) containing clear vesicles and less than 1% of large granular vesicles; they establish numerous asymmetrical synapses with the somata of the SCO hypendymal cells or with the latero-basal parts of the SCO ependymal cells (Bouchaud and Arluison, 1977; Mdlgird and Wiklund, 1979).

c. CIRCUMVENTRICULAR ORGANS AND CEREBRAL BARRIERS 1. Inter.$ace between the Blood and the Brain: The Endothelium

The lack of a BBB constitutes a distinctive feature of the CVO, with the exception of the SCO and the rostra1 part of the SFO (Weindl and Joynt, 1973; Bouchaud, 1974a, 1975). This can be evidenced by histological and histochemical methods, for instance, after vascular administration of trypan blue (complexed in vivo with plasmatic proteins), horseradish peroxidase, tritiated monoamines or after oral administration of silver nitrate complexed in vivo with plasmatic proteins (Figs. 2 and 3, see review in Bouchaud, 1983).The rich vascular supply to the CVO consists of essentially sinusoidal capillaries (Wislocki and King, 1936; Wislocki and Leduc, 1952; Dempsey and Wislocki, 1955; Van Breeman and Clemente, 1955; Duvernoy and KoritkC, 1964; Weindl, 1965; Bouchaud, 1975), which generally penetrate deeply into nervous tissue and are surrounded by unusually wide perivascular connective tissue spaces due to the labyrinthine expansions of the outer basal lamina into the parenchyma (Spoeni, 1963; RiveraPomar, 1966; Kobayashi er af., 1970; Schwendemann, 1973; Bouchaud, 1975). These features promote an increase in surface area for neurohemal release and/or for contact with circulating elements in systemic blood. The endothelium of most capillaries in the CVO, with the exception of the SCO, shows the typical fenestrations found in endocrine glands, which are thought to be the microanatomical substrate for the exchanges of substances (proteins, peptides, monoamines, etc.) through the vascular wall. Unfenestrated capillaries are also present

FIG. 2. Micrograph from a horizontal section of the habenular region in a mouse, which had received 0 . 1 5 9 silver nitrate in drinking water ad libifurnto localize brain areas deprived of a bloodbrain barrier. The SFO and adjacent choroid plexus (arrow) can be seen to selectively accumulate silver in this experimental argyria apposed to the SCO (arrowhead). Scale bar = 580 p m . FIG. 3. Subfomical organ of a rat treated as in Fig. 2. Only the caudal part of the organ, together with the choroid plexus, shows silver accumulations. V . Ventricle. Scale bar = 200 pm. FIG.4. Freeze-fracture replica illustrating the loosely interconnected tight junction network between SFO ependymal cells. No gap junctions are visible. The arrow indicates the shadowing. V , Ventricle; PF, protoplasmic face. Scale bar = I pm.

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and these are surrounded by a narrow, simple perivascular space. Besides, especially in the OVLT (see Fig. 16) and the SFO (Rohr, 1966), arterioles and unfenestrated capillaries possess numerous vesicles evoking a possible transendothelial transport normally absent from brain capillaries. 2 . Interface between the CSF and the Brain: The Ependyma

The characteristics of the ependyma of the CVO differ fundamentally from those of the adjacent ependymal lining both in their morphology and in the nature of their junctional relationships. CVO ependymocytes constitute an atypical ependyma which is almost deprived of supraependymal ‘‘innervation” (Calas er al., 1978). Their noteworthy features are their size and shape, the presence of apical microvillosities, and the quasi lack of cilia. These cells (tanycytes) have long basal processes which terminate in foot-like endings on the parenchymal basement membrane limiting the perivascular space, as evidenced at least in the ME, the OVLT, and the SFO. The glial apparatus supporting the perivascular basement membrane is also formed by processes of intraparenchymatous cells. Within the AP in particular, “glialoid” cells resembling astroblasts and giving rise to multiple vascular podia have been described (Brizzee and Klara, 1984). Such arrangements are also found in the SFO as well as on the lateral edges of the OVLT. The junctions between typical ependymocytes are invariably a “gap” and desmosome type, whereas “tight” junctions are present at the ventricular surface of the CVO, as found in the choroid plexus (Brightman, 1967; Brightman and Reese, 1969). This has been shown to be the case in the ME and the AP (Reese and Brightman, 1968). More recently, the freeze-fracture method has thrown light on the spatial configuration of these fibrils constituting the tight junctions in various CVO (ME: Brightman et al., 1975) (SCO: Kimble et al., 1973; Madsden and MdlgArd, 1979). As illustrated in Fig. 4,which shows the aspect of such junctions in the rat SFO ependyma, the junctional fibrils constitute a loose network and they are frequently disrupted. The CVO ependyma constitute a barrier preventing the leakage of the plasmatic proteins into the CSF [the proteins of the CSF amount to only 0.46% of their plasmatic level (Rapoport, 1976)] and the tight junctions are responsible for this barrier. Monoamines or proteins administered into the CSF through the ventricular route will circumvent the BBB (Brithman, 1965) and diffuse into the intercellular spaces. In the CVO, the tight junctions are relatively permeable, allowing for instance the penetration of intraventricularly injected horseradish peroxidase and monoamines into the parenchyma (Brightman et al., 1975; Richards, 1978; Calas et al., 1978). This relative permeability, which has been useful in many in vivo autoradiographicexperiments (Bosler and Calas, 1982), could be accounted for by the frequent disruptions of the junctional fibrils. It should, however, be noted that the administration into the ventricular CSF of high concentrations of

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molecules which are normally absent (or present at very low concentrations)may alter the normal physiology of the liquido-tissular barrier.

111. Monoaminergic Innervation of the Mammalian Circumventricular Organs In the search for morphological correlates for the functions of monoamines in the brain, some studies have been published which focused on the topographical and cytological characteristics of the catecholaminergic and/or serotoninergic innervation of the CVO. The first data were obtained using fluorescence histochemistry, but most of our current knowledge on the ultrastructural features of the monoaminergic innervation of the CVO derives from autoradiography after the uptake of [3H]monoamines administered through the ventricular system, and from immunocytochemistry, using antibodies directed against catecholaminesynthesizing enzymes or against serotonin (5-HT). The available data on each CVO will be reviewed successively. A. NEUROHYFQPHYSIS Much information is now available on the general organization, morphological features, and cellular interrelationships of the monoaminergic supply of the neurohypophysis, especially the ME of which all the rostrocaudal subdivisions are innervated by dense catecholaminergic and serotoninergic plexuses. Most of our knowledge about the origin and topographical distribution of the catecholaminergic innervation of the neurohypophysis is from the Swedish school (Bjorklund et al., 1970, 1973, 1974; Jonsson et al., 1972; Ajika and Hokfelt, 1973, 1975; Lofstrom et al., 1976). The dopaminergic tubero-infundibular and tubero-hypophysial systems originating in the arcuate-periventricular complex of the hypothalamus [A-12 cell group, according to Dahlstrom and Fuxe (1964)l and projecting to the ME and the NL, respectively, were described by means of fluorescence histochemistry in combination with microspectrofluorimetricanalysis and stereotaxic lesions. Whether or not dopaminergic afferents to these organs might originate from the same neurons cannot yet be determined. According to Bjorklund et al. ( 1974), tubero-infundibular neurons terminate in all layers of the ME and the pituitary stalk and can be divided into two groups on the basis of the topographical organization of their projections. One of these cell groups also includes those dopaminergic neurons projecting to the NL. According to Kizer et al. (1976), a nigro-hypothalamic projection may also contribute to the dopaminergic innervation of the ME. In the NL, dopaminergic fibers are homogeneously distributed throughout the parenchyma but in the ME they easily predominate in the external zone, es-

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pecially in the lateral portions of the organ at its central and caudal levels. In this neurohemal contact zone, dopaminergic terminals, the topographical distribution pattern of which overlaps that of various peptidergic as well as y-aminobutyric acid (GABA)ergic endings (see review in Hokfelt et af., 1978), establish close relationships with the portal vascular plexus. According to the results of Bjorklund et al. (1974), there is also a noticeable dopaminergic innervation of the inner zone of the ME where catecholamine-histofluorescentvaricosities are closely related to the deep capillary loops of the portal vessels. This was contradictory to the findings reported by Jonsson et af. (1972) and Liifstrom et al. (1976), who assigned a noradrenergic identity to the catecholamine varicose axons detected at this level. In autoradiographic experiments after the intraventricular administration of [3H]dopamine(t3H]DA>,Bosler (1983) observed a noticeable axonal labeling in both the inner and outer zone of the ME (Fig. 5 ) , even in rats previously subjected to selective 6-hydroxydopamine (6-OHDA) lesions on ascending noradrenergic and adrenergic afferent fibers, which agreed with the conclusions of Bjorklund et af. The noradrenergic supply to the ME comes mainly from a reticulo-infundibular system originating within the pontine medullary reticular formation, essentially the region referred to as A-1 in the nomenclature by Dahlstrom and Fuxe (1964), but it also includes sympathetic fibers from the superior cervical ganglia (Gallardo et af., 1984). The precise topographical distribution of the noradrenergic terminals of the reticulo-infundibular system is still a controversial subject (cf. Bjorklund et af., 1974). According to some authors, these afferents must be localized exclusively within the inner layer (Jonsson e? al., 1972; Swanson and Hartman, 1975) and, according to others, they also seem to be present in the outer layer, particularly the medial part (Lofstrom et af., 1976; Hokfelt et al., 1978). As for the NL, it also seems to have a dual noradrenergic innervation, of central and peripheral origin (Bjorklund et al., 1973; Alper et al., 1980; Saavedra, 1985). The reticulo-infundibular system also contributes to an adrenergic innervation

Fics. 5-8. Light-microscopic autoradiographs of semithin frontal sections after intraventricular administration of ['HIDA (Figs. 5 and 7) or 5-[3H]HT (Figs. 6 and 8). Scale bars = 80 pm. FIGS.5 AND 6. Arcuate median eminence region. A strong axonal [3H]DA labeling is found associated with portal capillaries in the outer zone of the median eminence. The inner part of the organ as well as the adjacent arcuate nucleus where dopaminergic cell bodies have selectively accumulated the tracer (arrows) also show typical axonal reactions (dense clumps of silver grains). By contrast, as shown in Fig. 6, 5-[3H]HT labeling of purely axonal origin is homogeneously distributed throughout the arcuate-median eminence complex. FIGS.7 A N D 8. Organum vasculosum laminae terminalis. A few reactive varicosities are visible after 13H]DA uptake (Fig. 7). They are mainly distributed around the vascular plexus of the organ. Most of the 5-[3H]HT-accumulating terminals shown in Fig. 8 (ventral part of the OVLT) are located at the upper vascular pole.

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of the ME (Hokfelt et al., 1974), originating essentially from nerve cell bodies of the region designated as C-1 (Palkovits et al., 1980). Using antibodies directed against phenylethanolamine N-methyltransferase (PNMT), the specific enzyme converting NA to adrenaline (Ad), a moderately dense immunoreactive network could be seen in the inner layer of the ME with only a few fibers extending into the outer layer (Hokfelt er al., 1984). The pituitary stalk also contained few PNMT-positive fibers. In contrast, no Ad-synthesizing fibers have been identified to date in the NL. The whole NH also receives serotoninergic fibers originating in the nucleus raphis dorsalis. In the ME, where they were first identified by Calas et al. (1974) using 5-[3H]HT autoradiography, they terminate in a more “diffuse” manner than their catecholaminergic counterparts, being ubiquitously distributed within the whole thickness of the organ (Steinbusch and Nieuwenhuys, 1981; Bosler et al., 1982) (Fig. 6). In the guinea pig, however, serotoninergic fibers supplying the ME have been reported to be present only rarely in the external layer (Tramu et al., 1983). They continue through the pituitary stalk to reach the NL. As a rule, serotoninergic fibers are plentiful in the pituitary stalk but become less numerous as they enter the NL itself where they consist of fine varicose fibers either investing the whole organ or concentrated in the zone adjacent to the intermediate lobe, depending on the species (Steinbusch and Nieuwenhuys, 1981; Sano et af., 1982; Kawata etal., 1984; Payette et af., 1985; Calas, 1985). The first electron microscopic data on monoaminergic fibers in the neurohypophysis were obtained by cytochemical studies using false neurotransmitters (Hokfelt, 1967; Mazzuca and Poulain, 1971; Baumgarten et al., 1972), but most of our current knowledge of the ultrastructural features is derived from autoradiographic investigations after selective labeling using [3H]catecholamines or 5-[3H]HT. Most catecholaminergic varicose profiles are similar to their serotoninergic counterparts (see Fig. 14). In addition to mitochondria and fragments of smooth endoplasmic reticulum, both types (0.8 nm in transverse diameter) contain “synaptic” vesicles (40-60 nm), sometimes with a small dense core (or dense dot) generally associated with a few large granular vesicles (80-1 10 nm) (Ajika and Hokfelt, 1973; Cuello and Iversen, 1973; Calas et al., 1974; Chetverukhin et a f . , 1979; Ajika, 1980) (Figs. 9-13). Recent autoradiographic observations (0. Bosler and L. Descarries, unpublished data), conducted after [3H]DA in vivo labeling and fixation of the brain using the double-successive vascular perfusion technique with both glutaraldehyde and osmic acid, a procedure which allowed adequate retention of the exogenous amine in its sites of uptake and storage (Descarries et al., 1980), have indicated that, in the ME, some catecholaminergic terminals belong to other morphologically different types. These authors observed (1) a few axonal varicosities displaying an unusually high number of large dense core vesicles, sometimes typical of neurosecretory granules which were present in the outer vascular

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zone of the organ, and (2) some large axonal dilatations (up to 2 pm in mean diameter) which, in addition to clear synaptic vesicles, contained a high number of granulations and/or dense core vesicles as well as, in some cases, multilamellar bodies and/or myelinic figures. Such monoaminergic varicosities, which were detected exclusively in the inner zone, have also been found to selectively accumulate [3H]adrenaline ([3H]Ad) (Bosler and Descarries, 1983). Finally, [3H]DA as well as [3H]Ad-accumulatingelements in the inner layer of the ME also include rare myelinated fibers (Chetverukhin ef al., 1979; Bosler and Descanies, 1983; Bosler, 1983). Recently, using PNMT immunocytochemistry, Bosler and Beaudet (in preparation) carried out a specific identification of adrenergic afferents in the arcuateME complex. Immunoreactive varicosities in the ME, which were strictly confined to the inner zone, belonged to either of the two categories of catecholaminergic axon terminals identified at this level (Fig. 11). Myelinated axons, however, never showed any PNMT immunostaining. This reinforces the hypothesis that large axonal swellings found to be capable of accumulating either [3H]DA or [3H]Ad in the inner zone of the ME might be truly adrenergic elements. It is noteworthy that monoaminergic nerve fibers, like any other type of axon terminals, have never been observed making morphologically defined synaptic junctions in either the ME or the NL. It is thought, however, that different types of neuroactive molecules mutually interact through axoaxonic contacts at these distal sites of integration. In the external zone of the ME, especially within its reticular layer, such contacts are particularly frequent and, in spite of the lack of true synaptic complexes between the different partners, are considered to be potentially active sites at which one factor can influence the synthesis and/or FIGS. 9- 14 (pp. 300-301). Electron microscopic preparations from the median eminence. Scale bars = 1 pm. FIGS. 9 AND 10. Two catecholaminergic axonal varicosities having selectively accumulated either [3H]DA (Fig. 9) or [3H]Ad (Fig. 10)given intraventricularly are illustrated in the inner zone of the organ. Both of them contain numerous clear synaptic vesicles and a few large granular vesicles. Note that the [3H]DA-labeled terminal is completely engulfed in the apical cytoplasmic portion of a tanycyte. V, Ventricular lumen; N, nucleus of a tanycyte. FIG. 11. An axonal varicosity is significantly immunostained for PNMT. This adrenergic terminal is closely associated with the cell body of a tanycyte within the ependymal layer. FIGS. 12 AND 13. Presumed dopaminergic terminals are shown in the outer vascular zone of the ME, where they abut with unidentified terminals (*) and tanycytic processes on the parenchymal basement membrane. They were identified by means of TH immunocytochemistry as shown in Fig. 12 and [3H]DA autoradiography as shown in Fig. 13. Arrows point to typical penetrations of the capillary on the endothelium. t, Tanycyte. FIG. 14. Combined detection of serotoninergic and catecholaminergic terminals in the outer zone of the ME using 5-[3H]HT autoradiography and TH immunocytochemistry. Radiolabeled and immunoreactive terminals belong to separate axonal properties. Note that they display comparable morphological features.

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release of another. Although in the reticular layer of the ME, monoaminergic nerve endings are frequently engaged in such axoaxonic relationships, in the palisade layer, they are more often isolated by tanycytic processes among which dopaminergic terminals frequently insinuate themselves, together with many peptidergic as well as GABAergic axon terminals, toward the external perivascular basement membrane (Figs. 12 and 13). This feature provides the morphological correlate to the known neurohormonal release of DA in the portal vessels as a prolactin-inhibiting factor (see McLeod, 1976). Whether or not noradrenergic and/or serotoninergic terminals can also abut on the peripheral basement membrane, as found in birds in the latter case (Calas, 1975), still has to be fully demonstrated. Some of the varicosities directly apposed to monoaminergic terminals in the neurohypophysis could be identified through electron microscopic double-labeling experiments. In the external zone of the ME, axoaxonic contacts between catecholaminergic, presumably dopaminergic, and LH-RH terminals were illustrated by Ajika (1980), who performed combined tyrosine hydroxylase (TH) and LH-RH immunocytochemical staining on the same ultrathin sections. More recently, by means of combined autoradiography and immunocytochemistry after in vivo injections of either [3H]DA or 5-[3H]HT (Fig. 14), a cytological basis for direct intercellular interactions between catecholamines, 5-HT, SRIF, and TRH, at the same level has also been reported (Bosler et al., 1982; Nakai et al., 1983; Bosler and Beaudet, 1985). Using a similar approach in the NL, Pelletier (1983) has observed the close proximity between dopaminergic terminals (selectively labeled after [3H]DA uptake) and immunoreactive vasopressin neurosecretory terminals. These data provide the morphological substrate for some of the intercellular interactions known to occur in the neurohypophysis between monoamines and neuropeptides. In particular, DA has been shown to stimulate in vitro SRIF release from hypothalamic synaptosomes and ME fragments (Wakabayashi et al., 1977; Negro-Vilar et al., 1978) as well as LH-RH secretion from slices of the rostra1 ME (Rotsztejn et al., 1977). In the NL, it has been reported to have a direct inhibitory effect on the evoked release of vasopressin in vitro (Lightman et al., 1982). It is also conceivable, however, that interactions between monoamines and neuropeptides, in addition to involving axoaxonic relationships, might be triggered by “remote control” after the release of the rnonoamines into the intercellular space. Concordant with this view is the fact that [3H]DA-labeled catecholaminergic terminals were found to be separated more frequently from SRIF-immunoreactiveneurosecretory axons than directly apposed to them in the external zone of the ME (Bosler et af., 1982). Lastly, at the same level, [3H]DA and 5-[3H]HT have been shown to be accumulated by some SRIF (Bosler et al., 1982) and substance P (Calas, 1985) axon terminals, which can be said to reflect the intraaxonal coexistence of the mediators concerned or, rather, to indicate some inaacellular action of monoamines on the release of neuropeptides.

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One striking feature of monoaminergic fibers within the neurohypophysis is their frequent associations with nonnervous elements, i.e., tanycytes in the ME and pituicytes in the NL, both of which might be involved in the control of neurohormone release (Rodriguez, 1976; Tweedle and Hatton, 1980). Tanycytes have also been thought to form a functional link between the CSF and portal vessels (see Knigge et al., 1972) and, in addition, might be a cellular site of 5HT synthesis (Sladek and Sladek, 1978). Both catecholaminergic and serotoninergic terminals have frequently been observed to make “synaptoid-like’ ’ contacts with tanycyte and pituicyte processes, which could represent the morphological substrate of a monoamine-mediated regulation of their activity. Some arguments have been presented by Sladek et al. (1978) favoring the view that the catecholaminergic ‘‘tanycytic innervation” of the ME could be dopaminergic in nature. According to these authors, DA might exert an inhibitory influence on 5HT synthesis in ME tanycytes (and/or on its release). Such DA/5-HT interactions would thus occur in a manner reminiscent of the catecholamine-indoleamine interactions taking place in the pineal gland (see below). VASCULOSUM LAMINAETERMINALIS B . ORGANUM In keeping with biochemical data on the endogenous content of monoamines and monoamine-biosynthetic enzyme activities in the CVO (Saavedra et al., 1976), the monoaminergic innervation of the mammalian OVLT is predominantly serotoninergic. As demonstrated in the rat by fluorescence histochemistry using the glyoxylic acid method (Moore, 1977), autoradiography after intraventricular 5-[3H]HT administration (Bosler, 1978; Parent et al., 1981), and 5HT immunohistochemistry (Jennes et al., 1982; Takeuchi and Sano, 1983), serotoninergic afferents form an extensive plexus of varicose fibers throughout the organ (Fig. 8). In the guinea pig, however, only a moderate 5-HT innervation of the OVLT has been reported (Tramu et al., 1983). In view of the autoradiographic observations by Moore (1977), these probably originate in the nuclei raphe dorsalis and centralis superior of the midbrain. The area between the upper vascular pole and the preoptic recess of the third ventricle, which, by analogy to the ME can be referred to as the inner zone, is supplied with a particularly dense population of serotoninergic fibers, as evidenced particularly in autoradiographic (Bosler et al., 1982) and immunohistochemical (Takeuchi and Sano, 1983) preparations from sagittal sections. Also, the presence of many serotoninergic fibers around the vascular plexus which penetrate deeply into the organ, i e . , into the outer neurohemal contact zone, has also been emphasized as the conspicuous feature giving some morphological support to the distal involvement of 5-HT in the control of secretion of peptidergic factors. It has been observed, however, that such perivascular arrangements are more evident at the dorsal than at the ventral level of the organ

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(Bosler er al., 1982) and that, caudally, serotoninergic fibers remote from the ependyma are preferentially detected within the lateral edges (Takeuchi and Sano, 1983), as previously observed in the duck (Bosler, 1977). The whole rostra1 penventricular preoptic region is supplied with a dense plexus of catecholaminergic afferents, but most of these do not terminate within the OVLT proper. This has been clearly illustrated using fluorescence histochemistry (Moore, 1977) as well as TH, DBH, and PNMT immunohistochemistry (Jennes et al., 1982, 1983; Hokfelt et af., 1984). Accordingly, only scarce autoradiographically labeled axonal varicosities could be seen within the OVLT after intraventricular administration of [3H]DA (Fig. 7) or L3H]Ad followed by glutaraldehyde and osmic acid fixation using the successive vascular perfusion procedure (Bosler et al., 1982; Bosler and Descarries, 1983). Catecholaminergic terminals supplying the OVLT seem to predominate in the neurohemal contact zone, around the vascular plexus of the organ, suggesting a possible involvement of catecholamines in the axoaxonic modulation of neuropeptide release at this level, as indicated by the results obtained by Gerendai et al. (1980) who concluded that stimulatory role effects are exerted by NA on LH-RH release in the OVLT. However, as recently stressed by Jennes er al. (1982), given the scarcity of the catecholaminergic terminals present at this level, any catecholamine/ peptide interactions in the OVLT would certainly be limited. According to present experimental evidence, these terminals are likely to be composed of both dopaminergic and noradrenergic fibers. On the one hand, the presence of a dopamine P-hydroxylase (DBH) immunoreactivity (Jennes et al., 1982) acFIGS. 15- 18. Electron microscopic autoradiographs from the organum vasculosum laminae terminalis after intraventricular administration of [3H]DA (Figs. 15 and 16) or 5-[3H]HT (Figs. 17 and 18). Scale bars = 1 pm. FIG. 15. An axonal varicosity having significantly accumulated [3H]DA is located in the immediate vicinity of the perivascular space (pvs) in which fenestrated capillaries (arrows) can be observed. This catecholaminergic, presumably dopaminergic, terminal is isolated from the parenchymal basement limiting membrane by a glial (tanycytic) process which completely surrounds it. FIG. 16. A sympathetic perivascular nerve fiber can be seen to have been intensely labeled within the perivascular space. Note that the clustering of silver grains overlies two varicose parts of this fiber, which is filled with small clear and dense cored vesicles. The internal features of these varicosities are more similar to those of sympathetic pineal nerve fibers (compare with Figs. 24 and 25) than to those illustrated for monoaminergic terminals of a central origin in any other CVO. Note that the endothelium and adventitia of the neighboring arteriole show numerous endocytotic vesicles. FIG. 17. Two serotoninergic radiolabeled axon terminals are illustrated. One of them (right comer) is contiguous to a nonreactive, presumably peptidergic, varicosity containing a high proportion of granular vesicles. The other one displays a synaptoid contact (arrow) with a tanycytic process, which also receives another unlabeled afferent, presumed to be of a peptidergic nature. A synaptoid differentiation is also visible at the site of contact between this neurosecretory axon and the tanycytic element (arrowhead). FIG. 18. The labeled terminal is apposed to four dendritic profiles (dl-d4). With one of these (d4) it establishes a typical synaptic contact.

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counts for a noradrenergic innervation of the OVLT in view of the virtual absence of any PNMT-positive, i.e., Ad-synthesizing fibers, at this level (Bosler, unpublished data). On the other hand, the fact that DA is present in the OVLT at a concentration twice as high as NA probably rules out the possibility that it could merely be involved as a precursor of the latter amine (Saavedra et al., 1976). Accordingly, Bosler and Descarries (unpublished data) did not observe any striking difference between the pattern of autoradiographic axonal labeling observed in normal rats given [3H]DA intraventricularly and that obtained with rats having received the tracer under the same experimental conditions 2 weeks after selective lesion of ascending noradrenergic bundles by bilateral microinjection of 6-OHDA on either side of the decussation of the superior cerebellar peduncles (Thierry et al., 1973; Reader et al., 1981). This seems, in fact, to indicate that the catecholaminergic innervation of the OVLT is predominantly dopaminergic. It must also be mentioned that, as in the case of the ME, a sympathetic noradrenergic input also contributes to the catecholaminergic innervation of this organ (see Fig. 7). The monoaminergic innervation of the OVLT has been investigated under electron microscopy using high-resolution autoradiography after in vivo selective labeling with either 5-[3H]HT or [3H]DA (Bosler, 1978; Bosler, 1983; Bosler and Descarries, in preparation). Serotoninergic terminals in the OVLT have been described as axonal varicosities 0.2-1 p,m in transverse diameter containing, in addition to mitochondria, mainly clear synaptic vesicles with an occasional dense dot and a few large granular vesicles (Figs. 17 and 18). Most catecholaminergic, presumably dopaminergic, varicosities displayed similar ultrastructural features, but, occasionally, large dilatations (up to 3 p.m in transverse diameter) were also encountered in the inner, subependymal zone of the OVLT and these contained, scattered throughout a relatively clear and fibrillous matrix, clear vesicles and profiles of smooth endoplasmic reticulum sometimes associated with dense core vesicles. Some of the serotoninergic terminals were found to be engaged in synaptic junctional complexes of a symmetrical or asymmetrical variety, with dendrites or dendritic spines (Fig. 18). These might belong to peptidergic neurons, including LH-RH neurons which are known to be present at the vicinity of the OVLT (King er al.. 1982) and even within the organ proper in the monkey (Mazzuca, 1977). In contrast, catecholaminergic profiles have never shown any morphologically defined synaptic differentiation, which was also true of their serotoninergic counterparts detected within the neurohemal contact zone. At this level, monoaminergic profiles were seen juxtaposed to other unidentified varicosities, including neurosecretory terminals, and/or to tanycytic processes with which they sometimes established, as in the ME, synaptoid-like contacts (Fig. 17). They were often situated at very short distances from the perivascular space, usually being separated from the basement limiting membrane by the endings and cleft-

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like invaginations of the tanycyte processes (Fig. 15). Occasionally, however, serotoninergic and dopaminergic terminals could be found contacting the perivascular basement membrane, indicating that a neurohormonal release of 5-HT and DA might take place in the OVLT, as demonstrated with DA in the ME (Bosler and Descarries, in preparation). Finally, within the perivascular space, the sympathetic noradrenergic fibers could be readily identified using [3H]catecholamine autoradiography (Bosler and Descarries, 1983; Fig. 16) and these were found to innervate small arterioles. C. SUBFORNICAL ORGANAND AREAPOSTREMA 1. Subfornical Organ

Neurochemical techniques have established that the SFO contains catecholamines, 5-HT, histamine, and the related enzymes (Saavedra et al., 1976). As early as 1967, Lichtensteiger demonstrated fluorescent indoleaminergic, presumably serotoninergic, fibers in the mouse SFO, particularly in the caudal part of the organ where fenestrated capillaries are present. Since then, a few serotoninergic fibers have been discovered by immunohistochemical studies in the rat and guinea pig SFO (Takeuchi and Sano, 1983; Tramu et al., 1983). SFO serotoninergicterminals have been characterized by electron microscopic autoradiography after intraventricular administration of 5-[3H]HT (Calas et al., 1978). These fibers, which amount to less than 1% of the SFO terminals in the rat, are localized under the ependyma of the caudal part of the organ (Fig. 19) as well as in the immediate vicinity of capillaries near the endothelial basal lamina, where they are generally larger (1-2 vs 0.7 pm in mean diameter, Fig. 20). In the perivascular spaces, serotoninergic fibers sometimes display dystrophic aspects which make them similar to Herring bodies (see Fig. 4 in Calas et al., 1978). These terminals are characterized by an abundant population of clear vesicles (40-50 nm). Occasionally, intraparenchymal serotoninergic terminals show synaptic differentiations (Fig. 20). Much less is known about the catecholaminergic innervation of the SFO. Surprisingly, to our knowledge, no histofluorescent, autoradiographic, or immunocytochemical studies aimed at specifically identifying catecholaminergic fibers have referred to the SFO. Dellmann and Simpson (1975, 1979) simply state that axon terminals displaying the regression-restitution cycles (see above) could have a catecholaminergic identity. 2. Area Postrema It has been known for a long time that the AP contains considerable quantities of NA and 5-HT (Vogt, 1954; Amin et al., 1954). Using Falck’s method, Fuxe and Owman (1965) described, in the AP of several mammals, small nerve cells

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as well as scarce terminals exhibiting the green fluorescence characteristic of primary catecholamines. They also noticed that the surrounding cerebral tissue was very rich in monoamine-containing terminals. Using autoradiography after in vivo t3H]NA uptake, Sotelo (1971) further observed a labeling of some rare nerve cell bodies and many axon terminals in the AP. More recently, catecholaminergic neurons have been identified in the AP by means of TH and DBH immunohistochemistry (Torack and LaValle, 1973; Armstrong et al., 1981). In fact, the AP is part of the noradrenergic region referred to as A-2 in the nomenclature by Dahlstrom and Fuxe (1964). In a very recent work, after incubation of serial sections in TH, DBH, and PNMT antisera, Kalia et al. (1985) have emphasized the fact that the AP contains not only noradrenergic but also dopaminergic neurons. By contrast, no Ad-synthesizing cells could be demonstrated within the AP, PNMT-positive cells being present only at the limit of the organ in the dorsal subnucleus of the nucleus of the tractus solitatius. An interesting feature of catecholaminergic, TH-positive neurons of the AP is their anatomical relationship with the perivascular and subpial spaces (Pickel and Armstrong, 1984). In their earlier histofluorescence study, Fuxe and Owman (1965) also described some indoleaminergic, presumably serotoninergic, neurons in the rat AP, the location of which was more ventral than that of the catecholamine-containing cells. Since then, the presence of 5-HTcells has been confirmed by immunohistochemistry (Steinbusch, 1981; Tramu et al., 1983; see Fig. 21). These are thought to constitute a population of some 300-400 neurons in the rat. Using autoradiography after intraventricular administration of 5-r3H]HT, we, ourselves, did not detect any radiolabeled perikarya in the AP but only axonal varicosities located either in the parenchyma, where they frequently showed axodendritic synaptic differentiations (Fig. 22), or within the pericapillary spaces. Pickel and Armstrong (1984) have also found 5-[3H]HT-labeled processes in the form of free endings within the ventricular spaces. After combined autoradiographic and immunocytochemical experiments, Armstrong et al. (1984) have reported that enkephalin-like immunoreactivity was occasionally present in some 5-[3H]HT-accumulating terminals, indicating a possible colocalization of the peptide and 5-HT. By combining 5-r3H]HT uptake autoradiography with TH immunohistochemistry, Pickel et al. (1984) have demonstrated the existence of serotoninergic ~~

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FIGS. 19 AND 20. Electron microscopic autoradiographs of the rat subfornical organ after intraventricular administration of 5-[3H]HT showing labeled serotoninergic terminals. Scale bars = 1 pM. Especially noticeable in Fig. 20 are the clear synaptic vesicles tightly packed within these varicosities. Also note that the reactive terminal illustrated in this micrograph lies in the vicinity of a capillary and establishes at least one synaptic contact (arrows) with a dendritic profile, which receives another unlabeled synaptic afferent (*).

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axodendritic synaptic terminals on catecholaminergic as well as noncatecholaminergicneurons. After the combination of TH immunocytochemistry and autoradiographic detection of anterogradely transported labeled proteins from the nodose ganglion, Pickel and Armstrong (1984) have shown that vagal afferents also established synaptic contacts with dendritic profiles, some of which were identified as catecholaminergic. In view of the fact that the nodose ganglion comprises a serotoninergic neuronal population (Gaudin-Chazal et al. (1981), it remains to be determined whether or not the vagal afferents anterogradely labeled by Pickel and Armstrong (1984) included serotoninergicterminals. In this connection, it should be mentioned that Gaudin-Chazal et al. (1982) have demonstrated by selective retrograde transport of 5-[3H]HT a direct serotoninergic projection from the nodose ganglion to the nucleus of the solitary tract contiguous to the AP in the cat.

D. PINEALGLAND The mammalian pineal gland is densely supplied with noradrenergic fibers of the sympathetic type (Ariens Kappers, 1965). These fibers, which originate in the superior cervical ganglia, were found to be located both in the pineal parenchyma, where they regulate pinealocyte secretion, and in the perivascular spaces, where they might influence the capillaries of the gland. Sympathetic nerve fibers are known to nonsynaptically contact the pinealocytes, whereas synaptic junctions between nonsympathetic terminals of an unidentified neurochemical identity and pinealocyte processes have recently been described in the gerbil (Moller, 1985). Pineal sympathetic processes were autoradiographically labeled by Wolfe et al. (1962) and by Taxi and Droz (1966) after intravenous administration of [3H]NA (Figs. 23 and 24). These authors were then able to report on the ultrastructure of noradrenergic postganglionic fibers, having shown that the labeled terminals contained both clear vesicles and dense cored vesicles. Interestingly, some authors have described variations in the content of the varicosities following pharmacological treatments (Pellegrino de Iraldi et al., 1965) or depending on the circadian rhythm (Matsushita and Ito, 1972).

FIG. 21. Immunohistochemical preparation from the region of the rat area postrema after staining for 5-HT. 5-HT immunoreactive neurons are conspicuously demonstrated within the AP proper. Also note the reactivity of intraventricular fibers at the surface of the ependymal lining of the fourth ventricle (V). Scale bar = 250 pm. FIG. 22. Electron microscopic autoradiography of rat AP after intraventricular administration of 5-[3H]HT. The labeled varicosity contains pleomorphic clear and dense core vesicles. It is synaptically linked to a dendritic spine with an asymmetrical junctional complex (arrows). Scale bar = 1

m.

FIGS.23 AND 24. Electron microscopic autoradiographs of the rat pineal gland after intravascular [3H]NA administration. Scale bars = I pm. Courtesy of J. Taxi. Figure 23 illustrates a bundle of labeled unmyelinated autonomic fibers. These contain the typical small clear and dense cored vesicles found in monoaminergicperipheral fibers. Figure 24 shows a higher magnification of a labeled ending adjacent to a pinealocyte.

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Phylogenetically and ontogenetically, it is well known that the cells of the receptor line (CRL) are represented by pinealocytes sensu strictu (Collin 1969, 1979; Oksche, 1971; Juillard and Collin, 1980; Collin and Oksche, 1981). As recalled by Collin (1979) and Collin and Juillard (1980), the cells of the receptor line may be classified as members of either the diffuse neuroendocrine system (Pearse and Takor, 1979) or the class of paraneurons (Ueck and Wake, 1977; Fujita and Kobayashi, 1979). In mammals, the pinealocytes elaborate indoleamines, especially melatonin (Balemans, 1981), and peptidic compounds (PCvet, 1983). High intracellular endogenous levels of 5-hydroxytryptophan (5HTP) and 5-HT are found in some species. In fact, two pools of 5-HT have been identified within the cells of the receptor line, one in the cytosol and the other in dense cored vesicles. Depending on the species, numerous variations exist, one pool being more or less predominant (for details see Collin, 1979; Collin and Oksche, 1981). One part of the 5-HTis used as a precursor for the active pineal methoxyindoles (melatonin, 5-methoxytryptophenol, 5-methoxytryptamine). The conversion of 5-HT into melatonin appears during the dark period of the light-dark cycle. In the 20-day-old rat and in the adult rat, the pineal rhythm is regulated by the nervous system since superior cervical ganglionectomy abolishes both 5-HT and N-acetyltransferase rhythms (Klein et al., 1971). Sometimes, from birth to the sixth day in the rat, the rhythm exists although the innervation level is very faint. An intrinsic regulation of the 5-HT circadian rhythms in immature rats (1 1- to 15-day-old rats) persists when pineals are placed in organ culture (Brammer, 1979), providing evidence for a spontaneous circadian rhythmicity. When the gland is innervated, the circadian 5-HT fluctuation comes under the control of the noradrenergic sympathetic input. Finally, although intrapineal nerve fibers have been shown to display the characteristic yellow fluorescence of 5-HT with Falk and Hillarp’s technique, it is now thought that there is no serotoninergic innervation in the pineal gland, since the sympathetic fibers are able to accumulate 5-HT released from the pinealocyte (Owman, 1964). Interesting, in this context, Taxi and Droz (1966) have observed that the noradrenergic terminals are able to take up 5-[3H]HTP. In the pineal of numerous species, 5-HT might also diffuse and be stored in the interstitial cells. E. SUBCOMMISSURAL ORGAN

Since a zinc-iode positive plexus was first observed in the rat SCO by Stanka (1964), numerous contributions have specified the cytochemical characteristics of SCO innervation. They show no indication that the organ receives any substantial catecholaminergicinput (see Bosler and Descanies, 1983; Hokflet et al., 1984) (Fig. 25), but a massive serotoninergic innervation of SCO ependymo-

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cytes has been evidenced in the rat by means of fluorescence histochemistry (Fuxe et al., 1968; Bjorklund et al., 1972; Wiklund, 1974; Bouchaud and Arluison, 1977; Md1gzk-d et al., 1978). This has been confirmed by autoradiography (Fig. 26) and immunocytochemistry (Fig. 27) (Bouchaud and Arluison, 1977; Steinbusch, 1981; Takeuchi and Sano, 1983). Takeuchi and Sano (1983) have proposed that a continuity might exist between the supraependymal and SCO subependymal serotoninergicplexuses, which does not, however, agree with our own observations. Recent experimental data by LCger et al. (1983) have established that the rat SCO receives its serotoninergic innervation from the nucleus raphis dorsalis, the nucleus raphis centralis superior, and possibly the nucleus raphis pontis. These fibers come via the periventricular serotoninergic system, as shown by Parent et al. (1981). Using 5-HT immunohistochemistry,it can be seen that they reach the rat SCO by the rostral part of the organ at the level of the lamina intercalaris (Fig. 27). Recent observations on the rat from the age of 3 days to 1 month have suggested a progressive growth of serotoninergic afferents in a rostrocaudal direction (Marcinkiewicz and Bouchaud, 1983). Using high-resolution 5-[3H]HT autoradiography or neurotoxic drugs known to induce degeneration of serotoninergic axons, it has been demonstrated that serotoninergic terminals establish true axoglandular synaptic contacts with both ependymocytes and hypendymocytes (Bouchaud and Arluison, 1977; MolIgPrd et al., 1978; Mgllgird and Wiklund, 1979). The formation of characteristic synaptic axoglandular synapses has been observed on the third day of life even though synapses were very rare at this age. On the twenty-first day of life, the axoglandular innervation was already found to be morphologically analogous to that found in the adult (Marcinkiewicz and Bouchaud, 1985). Axoglandular synaptic contacts have always been found to be of the asymmetrical type and have invariably been observed near the cell nuclei of the ependymocytes (0.1 p,m away). In the SCO, 5-[3H]HT-accumulating terminals were found to represent not less than 80% of all boutons (Bouchaud, 1979). They include occasional varicosities with a dystrophic appearance (Fig. 28). As their labeled counterparts, unlabeled terminals were frequently observed in synaptic contact with more than one of the glandular ependymocytes, indicating that these cells receive a polyneuronal innervation (Fig. 28). Based on the autoradiographic FIGS.25-27. Rat subcommissural organ. A very dense plexus of serotoninergic nerve fibers innervates the basal part of the SCO, as demonstrated by autoradiography after intraventricular administration of 5-[3H]HT (Fig. 26, frontal section) and by immunohistochemistry using an anti-5HT antiserum (Fig. 27, sagittal section, arrows). In Fig. 27 note the positive reaction of pinealocytelike cells in the lamina intercalaris (li). Figure 25 is an autoradiograph obtained after injecting [3H]Ad in the presence of nonradioactive 5-HT.It does not demonstrate any catecholaminergicinnervation in the SCO. h, Habenula; pc, posterior commissure; and V, ventricle lumen. Scale bars = 160 km (Figs. 25 and 26) and 110 pm (Fig. 27).

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results obtained by Gamrani et al. (1981) after [3H]GABA administration, GABAergic afferents might contribute to the innervation of SCO ependymocytes. Interestingly, in this study, part of the [3H]GABA-accumulating varicosities were reported to be of a serotoninergic rather than a GABAergic nature (also see Belin et al., 1980). The presynaptic elements of axoglandular innervation are small varicosities containing rare large granular vesicles and clear vesicles (Fig. 28-30). In the serotoninergic varicosities, clear vesicles are often pleomorphic (40-55 nm in diameter) and are generally devoid of the eccentric dense dot described in other serotoninergic terminals after treatment with inhibitors of monoamineoxidase (IMAO) and 5-HT. In nonserotoninergic, unidentified terminals, the clear vesicles sometimes display a “paracrystalline” arrangement as shown in Fig. 30. After lesion of serotoninergic systems using specific neurotoxins, MAlgArd et al. (1978), then MgllgArd and Wiklund (1979), have reported on a nonmonoaminergic reinnervation of the SCO which could be accounted for by a local sprouting of persistent fibers. These authors have also shown that 5-HT denervation induced changes in the SCO secretory activity, which indicated that afferent serotoninergic fibers inhibit the synthetic and secretory functions of the organ. Along the same lines, Marcinkiewicz and Bouchaud (1983) have reported that the noninnervated fetal rat SCO produces conspicuous amounts of secretov material, unlike the innervated adult rat SCO. According to the observations of Uger et al. (1983) the nucleus raphis dorsalis seems in fact to inhibit synthesis of the SCO secretory products whereas the nucleus raphis centralis superior may inhibit its release. The embryonic differentiation of the glandular ependymocytes of the mammalian SCO is independent of serotoninergic innervation, indicating that 5-HT is not in fact a differentiating signal. The onset of serotoninergic innervation inhibits the secretory activity of the organ, particularly through the endoplasmic FIGS.28-30. Electron microscopic autoradiographs of the rat subcommissural organ. Note, in all micrographs, the short distance between the synaptic complexes and the nuclei of the ependymocytes. FIG.28. In addition to a small nerve terminal filled with clear synaptic vesicles (arrow), an atypical axonal dilatation showing a dystrophic appearance has strongly accumulated 5-[3H]HT. Both labeled varicosities contact the same ependymocyte onto which they are suspected of making synapses (arrowheads). Note that the reactive serotoninergic terminal on the left is also apposed to an unlabeled one that displays similar morphological features. FIG. 29. A serotoninergic terminal, identified here after interspecific uptake of [3H]Ad (no cold 5-HTadded in the radioactive solution-see Bosler and Descarries, 1983), is shown inserted between two ependymocytes. It is unequivocally engaged in a synaptic junctional complex (arrows) with one of these. FIG.30. An axon terminal which does not exhibit any reactivity after 5-[3H]HT labeling is presynaptic to an ependymocyte (arrows). The round, clear synaptic vesicles it contains show a characteristic crystalline arrangement.

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reticulum (Marcinkiewicz and Bouchaud, 1983). Certain cytological modifications take place at the very same time as synaptic contacts appear and develop. These modifications could alternatively be interpreted as indicating that the SCO ependymocyte must reach a certain level of development before it can signal the serotoninergic axon to make contact (Marcinkiewicz and Bouchaud, 1986).

IV. Concluding Remarks: On the Role of Monoamines in the Integrative Functions of the Circumventricular Organs All the CVO eventually contribute toward integrating nervous and humoral messages. Their monoaminergic innervation might thus have a common role having to do with the regulation of a wide range of homeostatic functions. Most remarkable in this context is the fact that DA released from the terminals of the dopaminergic tubero-infundibular neurons in the neurovascular zone of the ME can directly convey true hormonal messages to the pituitary gland via the portal vessels. As mentioned above, a similar neurohemal release is also being envisaged for other monoamines in the ME, especially 5-HT but also Ad (see Johnston er al., 1983; Gibbs, 1985), and for DA and 5-HT in the OVLT. It must also be noted that in the AP, serotoninergic neurons might provide supraependymal innervation indicates a possible neurohormonal release of 5-HT into the CSF, which may influence still unknown target cells (see Calas ef al., 1978). In some CVO, monoamines are also assumed to participate in neuroendocrine regulations by behaving like true neurotransmitters acting on proximal and/or distal levels of neuronal targets. Distal interactions occur in the neurohypophysis where monoaminergic neurons are known to modulate hypophysiotropic and posthypophysial hormone release through axoaxonic contacts with neurosecretory terminals. Let us note, in this context, that it is conceivable this modulatory action of monoamines need not necessarily be bound to such structural appositions. The OVLT is another site of vascular neurosecretion at which monoaminergic fibers probably assume similar distal neuroendocrine functions but it also constitutes, together with the SFO and the AP, a potential site of proximal neuroendocrine and/or nonneuroendocrine regulation in which synaptic serotoninergic endings are likely to play an important part. For example, the OVLT, SFO, and AP might be important target organs for the action of 5-HT in the control of cyclic gonatotropin secretion (Limonta ef a l . , 1981; Piva et al., 1982), water-electrolyte balance regulation (Knowles and Phillips, 1980; Thrasher er al., 1982), and cardiovascular functions (Barnes et al., 1984). In discussing the functional role and mode of action of monoamines in CVO, special attention should also be paid to the interrelationships that monoaminergic fibers establish with nonneuronal cells, namely the tanycytes and/or pituicytes in

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the NH and OVLT, the pinealocytes in the pineal gland, and the secretory ependymocytes of the SCO, which are assumed to carry out important functions in the physiological activities of the organs concerned. The fact that monoaminergic nerve endings frequently establish synaptoid and/or true typical synaptic contacts with these elements provides strong evidence that catecholamine and 5-HT influence their functioning. This has now been fully demonstrated in the case of the pinealocytes and SCO ependymocytes whose secretory activities, as outlined above, undergo noradrenergic and serotoninergic control, respectively. Experimental data concerning tanycytes have been presented indicating the existence of a direct link between their coverage of portal capillaries and the functional state of tubero-infundibular dopaminergic neurons, reinforcing the assumption that they “may undergo continuous functional adjustments in response to changes in the activity of neurons projecting to the ME” (Lichtensteiger et al., 1978). In the NL, the “dopaminergic innervation” of pituicytes could similarly play an important functional role in the dynamic interactions known to occur between these nonneuronal elements and neurosecretory axons (Tweedle and Hatton, 1980). According to this hypothesis, monoamines (especially DA) could then control the release of hypophysiotropic and posthypophysial hormones not only through direct axoaxonic contacts with neurosecretory axons but also via a regulation of tanycyte and pituicyte functions. Conceivably, the monoaminergic fibers innervating the NH and even the OVLT could also have important regulatory effects on the other speculative functions that have been ascribed to tanycytes, in particular, synthesis of substances (Knowles, 1972; Sladek and Sladek, 1978), vascular (Knowles and Anand Kumar, 1969) and ventricular (LCvCque et al., 1966) secretion, receptor function (Wittkowski, 1967), and transcellular transport of substances (especially neurohormones) to capillaries of the portal plexus after selective absorption from the CSF (Knigge and Scott, 1970; Kobayashi et al., 1970). The latter possible functional role of tanycytes, however, is beginning to be considered highly questionable (see Pilgrim, 1978). The CVO are in a position to not only emit but also receive fluid-borne information that cannot cross the BBB and are thus key regions for neurohumoral integration. In some CVO, monoaminergic processes are ideally situated to play a role in such integrative functions. This is the case with the ME where dopaminergic terminals can constitute central targets for circulating hormones, namely prolactin, in view of their frequent association with the parenchymal basement membrane limiting the perivascular space. Similar receptive functions can be attributed to the monoaminergic processes present within pericapillary and subpial spaces at the level of the AP (Pickel and Armstrong, 1984). Chemoreception is in fact indispensable in those physiological processes in which the AP, the OVLT, and the SFO are synergically involved, namely thirst and cardiovascular regulations. In view of the extensive projections of these CVO to various central

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structures, humoral signals entering the brain at their level can be conveyed to other brain regions provided with a BBB. This is the case with the blood-borne messages mediated by angiotensin I1 (Simpson and Routtenberg, 1978), a peptide primarily involved in the regulation of thirst and cardiovascular functions. Since they are able to translate hormonal stimuli into new neural activity, the AP, OVLT, and SFO can be viewed as neuroendocrine transducers, as recently pointed out in the case of the latter by Gross (1985). There is an anatomical substrate for monoamines taking part in the synaptic regulation of transduction mechanisms at the level of each of these three CVO. Interestingly, in this context, in vitro extracellular recordings have indicated that SFO neurons can be excited not only by angiotensin I1 and carbachol, a cholinergic drug, but also by 5-HT (Buranarugsa and Hubbard, 1979). Similar data were obtained on OVLT explants from which extracellular recordings have shown excitatory responses to 5-HT as well as to DA (Sayer ef al., 1984). In the AP, however, 5-HTand DA were reported to be without effect on neuronal unit discharge frequency (Brooks et al., 1983). It has also been argued that angiotensin 11-induceddrinking would require the adequate functioning of catecholaminergic neurons (Fitzsimons and Setler, 1975). The destruction of these neurons has been reported to reduce water intake induced by angiotensin I1 in satiated rats (Gordon et al., 1979), while direct injections of NA into the rat SFO caused an increase in water intake (Menani et al., 1984). In the AP, monoamines could even participate in transduction itself. Circulating cholecystokinin, a gastrointestinal peptide involved in food intake regulation, might indeed influence AP serotoninergic neurons, some of which project to the ventromedial hypothalamus known to be a satiety center (for review see Morley, 1980). These neurons would then relay cholecystokinin-mediatedmessages from the periphery for transduction into a neural activity which, according to Willis et al. (1984), might induce a local release of the peptide into the ventromedial hypothalamus. Finally, while precise information has been gained in the last decade on the structural organization and morphofunctional properties of monoaminergic systems in CVO, the specific functions of monoamines in regulatory processes involving these organs are often still obscure. In the brain, CVO can in fact be said to be microcenters of integration where multiple transmitter interactions do occur. A precise characterization of these interactions would certainly be a useful step toward achieving a better understanding of monoamine function in CVO.

ACKNOWLEDGMENTS

Part of the personal work presented in this paper was supported by grants from the Direction des Recherches et Etudes Techniques (DRET) (C.B.) and from the Institut National de la Santi et de la Recherche Mbdicale (INSERM, CRE No. 856003)(O.B.).

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The authors are greatly indebted to Drs. A. Calas, J. P. Collin, H. D. Dellmann, and C. Gudrillot for their helpful discussions. They also thank Mrs. B. Derderian for typing the manuscript and Mr. A. Yvinec for photographic work.

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Index

A Acetylation. of histones and protamines during spermatogenesis. 26-28. 32 Acetylcholine. secretion by pancreas and, 130. 133 by parotid and. 137 Actin cleavage and. 267 thin filaments of smooth muscle and. 74. 92 fibrils and, 96-97 a-Actinin. 110, I l l computer-aided reconstruction of smooth muscle and. 116 smooth muscle dense bodies and. 92. 94-95 cytoplasmic. 96-97 fibrils and. 96-97 Adrenergic innervation. of neurohypophysis. 296.299 Affinity chromatography. sample enrichment and. 203. 210 y-Aminobutyric acid. subcommissural organ and. 317 Anaphase, establishment of division furrow and. 250 Anatomical considerations. circumventricular organs and neurohypophysis. 285-286 organum vasculosum laminae terminalis, 286-287 pineal gland. 288-289 subcommissural organ. 289 subfornical organ and area postrema. 287288 Anesthetics. cleavage and. 264. 271-272 Annelids, sperm. somatic and intermediate type histones of, 21-22 Antibody research, aquatic samples and. 216. 219. 229 Aquatic environments. study of. 183-186 drawbacks of bulk analysis. 186-189 new techniques. 189-190 329

Aquatic research addressing questions in. 232-238 cell separation and enrichment cell dissociation. 201-202 sample enrichment. 202-210 cellular probes and. 210-216 antibody research and. 216-217 methodology and instrumentation description of flow systems and other methodology. 192-200 static systems. 190-192 phylogenetic overview cnidaria. 228 crustacea. 228-229 cyanobacteria. 219-22 I eukaryotes. 221-226 fishes. 231-232 heterotrophic nanoplankton. 226 metazoans. 221 molluscs. 229-231 prokaryotes. 218 sponges, 227-228 standards. controls, data interpretation. 217218 Area postrema anatomical considerations, 287-288 monoaminergic innervation of. 307. 309. 311

neuronal composition of. 291 Arthropods. sperm. somatic and intermediate type histones of, 21 Aster(s) furrow formation and. 252. 255. 270 single. furrow formation and, 270-271 Abtral centers distance between. ability of mitotic apparatus to establish furrows and. 262 normal distance between polar and equatorial surfaces. division and. 263264 4tropint, ouabain binding and, 164 Autofluorescence. tlow cytometry and. 194

330

INDEX

B Bacteria. aquatic. studies on, 218 Blood-brain barrier. circumventricular organs and, 292 C

Calcium ions cleavage furrow and. 257 secretion by pancreas and. 130-131. 132133. 169-170 secretion by parotid and. 135-136. 166-170 Cardiac glycosides, Na + ,K -ATPase and. 146-147 autoradiographic localization of. 154- 155 Catecholaminergicfibers in area postrema. 309. 311 in OVLT. 304. 306-307 Cell cycle. separation of stages of, 209 Cell dissociation. for flow cytometry. 201-202 Cell electrophoresis. sample enrichment and. 203. 210 Cells of receptor line, in pineal gland. 313 Cell surface consequences of interaction with mitotic apparatus early indication of local contraction. 266267 effect of distance from mitotic apparatus on furrow establishment and function. 272-273 effect of experimental surface alterations. 267 immediate consequences. 268-272 origin of contractile mechanism, 267-268 polar body formation, 273-274 cytokinesis and. 249 identification of region affected by mitotic apparatus. 258-260 blocks between surface and different regions of mitotic apparatus and cleavage. 260-261 difference in distance between astral centers and polar and equatorial surfaces. 263-264 divisional changes compensating for reduction in size of mitotic apparatus. 264-266 duration of stimulus for cleavage. 260 +

effect of distance between astral centers on ability of mitotic apparatus to establish furrows, 262 manipulation of cell outside equatorial region and effect on furrow formation, 262 rate of movement of cleavage stimulus toward, 253-254 time of fixation of division mechanism in, 250-251 time mitotic apparatus is capable of interacting with, 252-253 time required for action of mitotic apparatus to establish division mechanism. 251252 Centrifugal elutriation. sample enrichment and. 203. 208-210 Cerebral barriers. circumventricular ogans and. interface between blood and brain endothelium. 292-294 ependyma. 294-295 Chicken, sequences from histone genes expressed in oacytes or early embryos. 4. 12 Cholecystokinin, secretion by pancreas and. 130-134 by parotid and, 337 Chromatin structure histone variation and, 2-3 sperm-specific proteins and. 35-37 Chromatosome, histone of, 2 Chromosomes. establishment of division furrow and. 254 Circular intensity differential scattering. aquatic research and, 215-216 Circumventricularorgans functions, 283-284 integrative role of monoamines in, 318320 mammalian anatomical considerations. 285-289 cerebral barriers. 292-295 components of. 283 neuronal composition, 289-292 monoaminergic innervation of neurohypophysis, 295-303 organum vasculosum laminae terminalis. 303-307 pineal gland, 311-313

INDEX

33 1

subcommissural organ, 313-318 results of experimental analysis subfornical organ and area postrema, consequences of interaction between mito307-311 tic apparatus and surface, 266-274 Cleavage, see also Cytokinesis; Division essential structural components of mitotic blocks between surface and different regions apparatus, 254-256 of mitotic apparatus and, 260-261 identification of surface region affected by cessation of stimulatory activity and, 260 mitotic apparatus, 258-266 Cleavage stimulus nature of cleavage stimulus. 256-258 nature of, 256-258 time relations, 250-254 rate of movement toward surface, 253-254 Cnidaria, aquatic, studies, on. 228 Computer-aided reconstruction. of serial secD tions of smooth muscle, 115-117, 121 Concentration centrifugation, sample enrichDecondensation mechanisms, histone variants ment and, 202-204 and protamines and, 50-52 Concrete models, of smooth muscle, 114 Dense bodies, membrane-associated and Continuous flow centrifugation, sample enrichcytoplasmic of smooth muscle, 78-80 developmental studies and, 86-88 ment and, 203,207-208 Contractile mechanism, for cleavage, origin immunochemistry of, 92-95 of. 267-268 Deoxyribonucleic acid Contractile state, changes in organization with replication changes in length of smooth muscle histone synthesis and, 3 fixation of all components, 80-83 histone types and, 14 selective removal of contratile components, Desmin, smooth muscle intermediate filaments 83-86 and, 92 Developmental studies, insights into smooth Contractile system, normal distribution of muscle stmcture and. 86-88 components of smooth muscle Diacyl glycerol, secretion general morphology, 71-73 intermediate filaments, 76-78 by pancreas and, 130 membrane-associated and cytoplasmic dense by parotid and, 136 bodies, 78-80 Diasteme, cytokinesis and, 249 thick filaments, 73-74 Dimensional changes, reduction in size of thin filaments, 74-76 mitotic apparatus and, 264-266 Distance, from mitotic apparatus, effect on Core histones, variants in early development other organisms, 12-13 furrow establishment and function, 272273 sea urchins, 7-12 Corticoliberin, 286 Division. see also Cleavage; Cytokinesis normal distance between astral centers and Crustacea aquatic, studies on, 228-229 polar and equatorial surfaces and, 263sperm nuclear proteins of, 15 264 Cuttlefish, sperm histone of, 17 Division mechanism, time of fixation to cell Cyanobacteria, aquatic, studies on, 219-221 surface, 250-251 Dopaminergic fibers, in neurohypophysis, 295, Cyclic adenosine monophosphate secretion by pancreas and, 134 298-299, 302-303 secretion by parotid and, 135-136 Cysteine residues, in protamines, chromatin S t N C t U R and. 36, 50 E Cytokinesis, see also Cleavage; Division early studies and speculation, 246-250 Echinoderms, sperm, somatic and intermediate historical background, 245 type histones of, 22-23

332

INDEX

Egg cytoplasm, sperm pronuclear development. 42-44 source of chromosomal proteins in. 39-42 Electrolytes. transport in pancreas, 131-134 in parotid. 137 Embryonic development. histone variants and chromatin remodeling in, 3-5 core histones. 7-13 HI histones. 5-7 summary, 13 Endothelium, interface between blood and brain in CVO and, 292-294 Ependyma, interface between blood and brain in CVO and. 294-295 Eukaryotes, aquatic. studies on microalgae, 221-226 Excitation sources. suitable for flow cytometry, 213 Exocrine pancreas. morphology of membrane domains in, 138

F Fibrils, of smooth muscle, 101. 103 immunochemistry of, 96-97 Filamin, smooth muscle thin filaments and, 92 fibrils, 97 Filtration techniques. sample enrichment and. 202-203 Fish protamines of, 17-18 sperm, somatic and intermediate type histoms Of, 23-24 studies on cytogenetics. 23 1-232 polyploidy, 23 1 Flow cytometer and sorter, sample enrichment and, 203. 210 Flow cytometry, aquatic research and, 192-198 Flow systems, for aquatic research, 233 flow cytometly, 192-198, 234 flow-vision analysis, 198-200 fluorescence-activated cell sorting. 198 Flow-vision analysis, aquatic research and. 198-200 Fluorescence-activated cell sorting, aquatic research and, 198

Fluorescent dyes. spectral characteristics of, 213 Fluorescent probes. aquatic research and. 197198 Frog. sequences from histone genes expressed in oocytes or early embryos, 5 , 7, 12 Furrow formation distance between astral centers and. 262 manipulation of cell outside equatorial region and, 262 Furrow-inducing substance, in dividing eggs, 256-251

G Genes core histones and, II HI histones and, 6, 7 Genetic activity. suppression. sperm-specific proteins and, 37

H HI histone cell division and, 14 variants in early development other organisms, 7 sea urchin, 5-7 H2A histones, early development and, 9. 10, 11, 12 H2B histones. early development and. 9-12 Histones conservation of structure of, 2-3 in eggs, 39-42 embryonic effects and functions of modulations, 13-14 variants and chromosome remodeling. 313 of maternal pronucleus. 39 somatic and intermediate type in sperm, 2025 annelids, 21-22 arthropods, 21 echinoderms, 22-23 fish. 23-24 molluscs, 20-21 other vertebrates. 24 plants. 24-25

333

INDEX

variants as postfertilization transitional proteins. 53 variants during spermatogenesis other organisms. 32-33 rodents. 28-32 summary. 33, 35 teleosts. 25-28 variant modification in first postfertilization cell cycle. 47-49 variation, chromatin structure and. 2-3 Heterotrophic nanoplankton, aquatic, studies on, 226 Holographic reconstruction, of serial sections of smooth muscle, 117-121 5-Hydroxytryptamine, in pineal gland. 313

I Image analysis. aquatic research and, 192 Immunochemistry, of smooth muscle proteins dense body, 92-95 fibrils. 96-97 intermediate filaments, 92 microtubules, 95-96 thick filament, 91-92 thin filament, 92 Innervation. of pineal gland, 288-289 Inositol-l,4,5-trisphosphate. secretion by pancreas and, 130, 131 by parotid and, 136 Intercellular junctions, in pancreatic and parotid acini, 141-144 Intermediate filaments of smooth muscle, TEM of thin sections and. 76-78 immunochemistry of, 92 Isolated cell preparations, model of smooth muscle and, 68-69 Isopycnic density gradient centrifugation, sample enrichment and, 204-206

L Light microscopy, of smooth muscle immunochemistry, 91-97 living cells, 88-91 Limpet, sperm histone of, 17 Lulibarin, 286

M Mammals circumventricularorgans, components of. 283 protamines of, 18-20 Median eminence anatomical considerations, 285-286 monoaminergic innervation of, 295-296. 298-299.302 neuronal composition of, 289-290, 291 Membrane domains of exocrine pancreas, morphology of, 138 of parotid gland, morphology of. 138, 140141 Metazoans, aquatic, studies on, 227 Microalgae, aquatic, studies on, 221-226 Microfilaments, cleavage and, 266-267, 273 Microscope photometry, aquatic research and, 190-191 Microtubules furrow establishment and. 255-256 of smooth muscle, immunochemistry of, 95-96 Mitotic apparatus alteration of normal geometrical relations with surface, summary of results, 276277 consequences of interaction with surface early indications of local contraction, 266-267 effect of distance from mitotic apparatus on furrow establishment and function, 272-273 effect of experimental surface alterations, 267 immediate consequences, 268-272 origin of contractile mechanism, 267-268 polar body formation, 273-274 cytokinesis and, 248-249 essential structural components, 254-256 identification of region of cell surface affected by, 258-260 blocks between surface and different regions of mitotic apparatus and cleavage, 260-261 difference in distance between astral centers and polar and equatorial surfaces, 263-264

334

INDEX

divisional changes compensating for reduction in size of mitotic apparatus. 264-266 duration of stimulus for cleavage. 260 effect of distance between astral centers on abiilty of mitotic apparatus to establish furrows. 262 manipulation of cell outside equatorial region and effect on furrow formation. 262 time required for action on surface to establish division mechanism, 251-252 time surface is capable of interacting with. 252-253 Models of cytokinesis. 246-247 of Na ,K -ATPase, 145 Modification. of histones. early development and. I2 Molluscs aquatic. studies on pathology. 229 polyploidy. 229-231 selective feeding. 229 sperm. somatic and intermediate type histones of. 20-21 Monoaminergic innervation. of circumventricular organs neurohypophysis. 295-303 organum vasculosum laminae terminalis. 303-307 pineal gland, 311-313 subcommissural organ, 313-318 subfomical organ and area postrema. 30731 I Monoamines, role in integrative functions of circumventricularorgans, 318-320 Mouse. spermatogenesis. nucleoproteins and. 28-30 Myosin in thick filament of smooth muscle. 73-74. 91-92 in fibrils. 96. 101

Organum vasculosum laminae terminalis anatomical considerations. 286-287 monoaminergic innervation of, 303-307 neuronal composition of, 290 Ouabain. binding by pancreatic and parotid acinar cells. 159-162 secretagogues and. 163-172 Oxygen consumption. ouabain-sensitive, measure of Na .K -ATPase activity in viable cells and, 156

N

P

+

+

Na .K -Adenosine triphosphatase autoradiographic localization of. 154-155 +

+

cytochemical localization of. 147-154 determination of activity in viable cells. 155-157 molecular characteristics and presence in pancreas and parotid gland, 144-147 Neurohypophysis anatomical considerations. 285-286 monoaminergic innervation of, 295-303 neuronal composition of, 290 Neuronal composition. of circumventricular organs, 289-292 Newt, sequences from histone genes expressed in oocytes or early embryos, 4 Noradrenergic fibers in neurohypophysis. 296 in pineal gland, 311 Nuclear proteins. of mature sperm. I5 mammalian protamines. 18-20 nonbasic proteins. 15-17 protamines, 17-18 somatic histones and intermediate types, 2025 summary. 25 Nucleoprotein(s). somatic, displacement by sperm-specific proteins. 37-38 Nucleosome repeat length. histones and. 14 sperm-specific proteins and. 35-36 StNCtUre of. 2 Nucleus raphis dorsalis. 285. 315

0

+

+

Pancreas, Na+ ,K+-ATPase in, 146-147 localization of 151-153. 155

335

INDEX Pancreatic acini intercellular junctions in, 141-143 ouabain binding by, 159-162 secretagogues and, 163-172 preparation of monodispersed suspensions of, 159 stimulus-response coupling in, 130-135 Parotid acini intercellularjunctions in, 143-144 ouabain binding by, 159-162 secretagogues and, 163-172 stimulus-response coupling in, 135-138 Parotid gland morphology of membrane domains of, 138, 140-141 Na ,K -ATPase in, 147 localization of, 149-150 preparation of dispersed acini of, 159 Phentolamine, ouabain binding and, 164 PhenylethanolamineN-methyltransferase. in median eminence, 299 Phosphatidylinositol-4.5-bisphosphate, secretion by pancreas and, 130, 131 by parotid and, I36 Phospholipase C, secretion by pancreas and. I30 Phosphorylation, of histones and protamines after fertilization, 47,49,52 during spermatogenesis, 26-28, 38 Photographic reconstruction, of serial sections of smooth muscle, 114-115 Pineal gland anatomical considerations, 288-289 monoaminergic innervation of, 311-313 neuronal composition of, 291 Plants, sperm, somatic and intermediate type histones of, 24-25 Plasma membrane, of pancreatic acinar cells, 134-135 Polar body, formation of, 273-274 Polarization phenomena, aquatic research and, 215 Polychaete, sperm histone of, 17 Polyploidy in fishes, 231 in molluscs, 229-232 Prokaryotes, aquatic studies on, 218 Pronuclear development, cytoplasmic control of, 42-44 +

+

Pronuclear proteins, male biochemitry of, 46-49 cytochemistry of, 44-46 Protamines of mature sperm, 17-18 mammalian, 18-20 synthesis during spermatogenesis. 26 Protein(s) nonbasic, of mature sperm, 15 nuclear, transitions following fertilization effects and possible function of histone variants, 50-53 fate of sperm-specific nucleoproteins. 4249 maternal pronuclear histones. 39 sources of chromosomal protein in egg. 42-49 sperm-specific, effects and possible functions of displacement of somatic nucleoproteins. 37-38 other roles, 38-39 sperm chromatin structure, 35-37 suppression of genetic activity, 37 Protein kinase C. secretion by pancreas and, 130. 131

R Rat, spermatogenesis, nucleoproteins and, 3032 Receptors, on parotid acini. 136 Repeat length transition, histone variants and, 50 Resolution, z-axis, of isolated smooth muscle cells, 106-107 Ribonucleic acid, messenger, histones and, 9I1 Rotary-shadowed replicas, of freeze-fractured, deep-etched smooth muscle, 105 Rubidium, radioactive, assay of Na ,K+-ATPase activity in viable cells and, 156 +

S

Sample enrichment, for aquatic research affinity chromatography, 210 cell electrophoresis, 210 centrifugal elutriation, 208-210 concentration centrifugation, 202-204

336

INDEX

continuous flow Centrifugation. 207-208 filtration techniques. 202 flow cytometer and sorter. 210 isopycnic density gradient centrifugation. 204-206 unit gravity sedimentation, 206-207 Scanning electron microscopy, of smooth muscle, 103-105 Sea urchin sequences from histone genes expressed in oocytes or early embryos. 5-7 core histones, 7-12 HI histone, 5-7 sperm histone of, 17 Secretagogues effects on Na+ .K+-pump activity in pancreatic and parotid acinar cells, 157I72 ouabain binding and, 157 effects on Na .K -pump activity in pancreatic and parotid acinar cells. 157 Secretin, 130 pancreas and, 134 Serial optical sections, of isolated smooth muscle cells, 105-111 Serial sections, three-dimensional analysis and reconstruction of smooth muscle computer-aided, 115-117 concrete models, I14 holographic, 117-121 photographic, 114-115 Serotoninergic fibers in area postrema, 309,311 in neurohypophysis, 298 in OVLT, 303-304.306 in subcommmissural organ, 313-315, 317318 in subfomical organ, 307 Smooth muscle three-dimnensional analysis and reconstruction serial sections, 113-121 stereo pairs, 111-113 three-dimensional information need for, 97-98 rotary-shadowed replicas of freeze-fractured, deepetched tissues, 105 scanning electron microscopy and. 103+

105

+

serial optical sections of isolated cells. 105-111

stereo pain of immunofluorescently stained cells, 1II transmission electron microscopy of serial sections, 98-100 transmission electron microscopy of stereo-tilt views of thick sections and whole cells, 100-103 three-dimensional model, 69-70 additional data needed, 122-123 problems with ultrathin sections, 67-68 role of isolated cell preparations, 68-69 speculatiye models versus reconstructions. 123-124 summary, 121-122 two-dimensional information, 70 light microscopy, 88-97 transmission electron microscopy of thin sections, 71-88 Somatostatin, 286 sperm mature mammalian protamines. 18-20 nonbasic proteins, 15-17 prolamins, 17-18 somatic histones and intermediate types. 20-25 summary, 25 nuclear proteins effects and possible functions of, 35-39 mature sperm. 15-25 spermatogenesis, 25-35 Spermatogenesis, histone variants and other organisms, 32-33 rodents. 28-32 summary. 33, 35 teleosts, 25-28 Spindle, establishment of division furrow and, 254-255 Sponge cells dissociation of, 201-202 isopycnic density gradient centrifugation of. 204-205 studies on, 227-228 unit gravity sedimentation of, 207 Staining techniques. cellular probes and. 212215 Starfish, sperm histone of, 17

337

INDEX Static systems. for aquatic research image analysis, 192 microscope photometry. 190-191 Stereo pairs. of immunofluorescently stained smoot muscle cells, 111 three-dimensional analysis and reconstruction, 111-113 Stimulus-response coupling in pancreatic acini, 130-135 in salivary acini, 135-138 Subcommissural organ anatomical considerations, 289 monoaminergic innervation of, 313-318 neuronal composition of, 291-292 Subfomical organ anatomical considerations, 287-288 monoaminergic innervation of, 307 neuronal composition of, 291 Submandibular gland, localization of Na+,K+-ATPase in, 149, 155 Substantia nigra, 286

T Tanycytes, in neurohypophysis. 303 Teleosts, histone variants during spermatogenesis in, 25-28 Thick filaments of smooth muscle immunochemistry of, 91-92 loss or retention during fixation, 84-86 TEM of thin sections of, 73-74, 80-81. 83 Thin filaments of smooth muscle. TEM of thin sections of. 74-76.80-81.83 immunochemistryof, 92 length of filaments and, 75-76 TEM of serial sections of, 98-100 Thyroliberin, 286 Time relations, of cytokinesis how fast does cleavage stimulus move to surface?, 253-254

how long are mitotic apparatus and surface capable of interacting?, 252-253 how long must mitotic apparatus act?, 251252 when is division mechanism fixed in surface?, 250-251 Transition, of male pronuclear proteins. 46-47 Transmission electron microscopy of serial sections of smooth muscle. 98-100 stereo-tilt views of thick sections and whole cells of smooth muscle, 100-103 of thin sections of smooth muscle changes in organization with changes in contractile state, 80-86 general comments, 71 insights from developmental studies. 8688 normal distribution of contractile system components, 71-80 Tropomyosin, smooth muscle thin filaments and, 92 fibrils and, 96 Trout, sperm histone of, 17 Tubulin, in smooth muscle, 96

U Ultrathin sections, probelms with, in smooth muscle, 67-68 Unit gravity sedimentation, sample enrichment and. 203, 206-207

V Vasoactive intestinal peptide, 130 pancreas and, 134 Vertebrates, sperm, somatic and intermediate type histones of, 24 Vimentin, smooth muscle intermediate filaments and, 92 Vinculin, smooth muscle dense bodies and. 92. 94-95

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