INTERNATIONAL
REVIEW OF CYTOLOGY VOLUME 57
ADVISORY EDITORS H. W. BEAMS HOWARD A. BERN GARY G. BORISY ROBERT W. BRIGGS STANLEY COHEN RENE COUTEAUX
ROBERT G. E. MURRAY
MARIE A. DIBERARDINO
JEAN-PAUL REVEL
CHARLES J. FLICKINGER
WILFRED STEIN
M. NELLY GOLARZ DE BOURNE K. KUROSUMI MARIAN0 LA VIA GIUSEPPE MILLOMG
ELTON STUBBLEFIELD
ARNOLD MIlTLEMAN
ROY WIDDUS
DONALD G . MURPHY
ALEXANDER L. W D I N
ANDREAS OKSCHE
VLADIMIR R. PANTIC W. J. PEACOCK DARRYL C. REANNEY LIONEL I. REBHUN
HEWSON SWIFT DENMS L. TAYLOR TADASHI UTAKOJI
INTERNATIONAL
Review of Cytology EDITED BY
G . H. BOURNE
J. F. DANELLI
St. George’s University School of Medicine St. George’s, Grenada West Indies
Worcester Polytechnic Institute Worcester, Massachusetts
ASSISTANT EDITOR K. W. JEON Department of Zoology University of Tennessee Knoxville, Tennessee
VOLUME 57
ACADEMIC PRESS New York San Francisco London A Subsidiary of Harcourt Brace Jovanovich, Publishers
1979
COPYRIGHT @ 1979, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART O F THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.
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United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London NW1 IDX
LIBRARY OF CONGRESS CATALOG CARD NUMBER:52-5203 ISBN 0-12-364357-0 PRINTED IN THE UNITED STATES OF AMERICA
79808182
9 8 7 6 5 4 3 2 1
Contents LISTOF CONTRIBUTORS .......................................................
vii
The Corpora Allata of Insects PIERRECASSIER I. Introduction ............................. ................. II. The Embryonic Origin of Corpora Allata .................................... IU. Morphological Types ..................... ............... IV. Innervation and Tracheal Supply of Corpora Allata ..................... V. Histological Characteristics and Types ........... ............ VI. Cytological and Infrastructural Characteristics of Co ................. VII. Conclusions ....................................... ............ References ....... ...................................
1 4 6 9 11 21 65 66
Kinetic Analysis of Cellular Populations by Means of the Quantitative Radioautography J.-C. BISCONTE
I. Introduction.. ..........................................................
II. Quantitative Radioautography .
..................... III. Kinetics of Cell Proliferation.. ............................................ IV. Migration and Chronoarchitectony V. Concluding Remarks . . . . . . . .
.............
.............
References .................
75 77 92 112 118 118
Cellular Mechanisms of Insect Photoreception F. G. GRIBAKIN
........................
.................. ............................ UI. Electrical Basis for Insect Photoreception. IV. Conclusion .............................. ....................... References ......................................... .............. Note Added in Proof ...................... ........... and photoreceptor Optics . . .
127 128
159 177 178 184
Oocyte Maturation YOSHIOMASWAND HUGH1. CLARKE I. Introduction ............................................................ II. Hormonal Control of Maturation.. ......................................... V
186 191
vi
CONTENTS
III. IV. V. V1. W.
Progression of Maturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Initiation ofOocyte Maturation ............................................ Cytoplasmic Control of Oocyte Maturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nucleocytoplasmic Interaction during Oocyte Maturation . . . . . . . . . . . . . . . . . . . . . . . Control of Meiosis and MitosisConcluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . References .............................................................
205 222 240 256 267 271
The Chromaffin and Chromaffh-like Cells in the Autonomic Nervous System JACQUES TAXI
I. Terminology ........................................................... II. Techniques ............................................................
III. CCL Cellsin Mammals .................................................. IV. CCL Cells in Nonmammalian Vertebrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Concluding Remarks . . . . . . . . . . . . . . . . . . . . ...... References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
283 285 287 327 335 336
The Synapses of the Nervous System A. A. MANINA
I. Introduction.. . . . . . . . . . . . . . . . . . II. The Mechanisms of Synaptic Transmission . .
.........................
345 347 35 1 351 352 357 359 361
................ ....... IV. Classification of .......................... .. ..... V. Structural-Functi e Synaptic Contact.. . . . . . . . . . . . . . . . . . . . . . . VI. The Role of Neurospecific Roteins.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . W. The Role of Glycoproteins . . . . . . . . . . . . . . . . . . . . ..................... Wr.The Enzymic Activity of ATPase and Adenyl Cyclase Reactions in Synapses . . . . . . IX. Autoradiographic Investigations of the Synthesis of Biopolymers in the Synapses . . . X. The Structurd-Functional Features of the Axospinal Apparatus. . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . ......................
368 379
SUBJECT INDEX .............................................................. CONTENTSOFPREVIOUSVOLUMES ..............................................
385 387
,
364
List of Contributors Numbers in parentheses indicate the pages on which the authors’ contributions begin.
J.-C. BISCONTE (75), Luboratoire de Neurobiologie Quantitative, Centre Hospitalier et Universitaire de Bobigny, Universiti de Paris XIII, France
PIERRE CASSIER (l), Universite Pierre et Marie Curie, ERA 620, Cytophysiologie des Arthropodes, 105 Boulevard Raspail, 75006 Paris, France
HUGHJ . CLARKE ( 185), Department of Zoology, University of Toronto, Toronto M5S l A l , Ontario, Canada F. G . GRIBAKIN (127), The Luboratory of Evolutionary Morphology, Sechenov institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, 194223 Leningrad, USSR
A. A. MANINA(343, Laboratory of Cytology, institute of Experimental Medicine, USSR Academy of Medical Science, Leningrad, USSR YOSHIOMASIJI(185), Department of Zoology, Universiv of Toronto, Toronto M5S 1A1, Ontario, Canada
JACQUES TAXI(283), Luboratoire de Neurocytologie, Universitt Pierre et Marie Curie, 12 Rue Cuvier, 75005 Paris, France
Vii
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INTERNATIONAL,
REVIEW OF CYTOLOGY VOLUME 57
This Page Intentionally Left Blank
INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 57
The Corpora Allata of Insects PIERRE CASSIER Universite' Pierre ef Marie Curie, Cytophysiologie &s Arthropodes, Paris, France
I. Introduction . . . . . . . . . . . . . . . . . . . 11. The Embryonic Origin of Corpora Allata . . . . . . . . 111. Morphological Types . . . . . . . . . . . . . . . A. The Lateralized Type . . . . . . . . . . . . . . B. The Distal Lateralized Type . . . . . . . . . . . C. The Semicentralized Type . . . . . . . . . . . D. The Centralized Type . . . . . . . . . . . . . . E. The Annular Type . . . . . . . . . . . . . . . IV. Innervation and Tracheal Supply of Corpora Allata . . . . . A. Innervation . . . . . . . . . . . . . . . . . . B. The Tracheal Supply of Corpora Allata . . . . . . . . V. Histological Characteristics and Types . . . . . . . . . A. Histological Types . . . . . . . . . . . . . . . B. Volume of the Gland, Mitosis, and Pycnosis . . . . . . C. Sexual Dimorphism . . . . . . . . . . . . . . . VI. Cytological and Infrastructural Characteristics of Corpora Allata A. BasalLamina . . . . . . . . . . . . . . . . . B. Glandcells . . . . . . . . . . . . . . . . . . C. Neurosecretory Fibers . . . . . . . . . . . . . . VII. Conclusions . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .
.
1 4
6 6
7
.
8
.
. . .
. . . . . .
. .
9 9 9 9 10
I1 11 14 19 21
23 25 63 65
66
I. Introduction
Insect corpora allata (Heymons, 1897a,b, 1899) or corps allates (Cazal, 1948) are endocrine glands which were previously called paired posterior visceral ganglia (Hofer, 1887), ganglia allata (Heymons, 1895), or corpora incertae (Meinert, 1861), owing to their being confused with the sympathetic cerebral structures constituting the stomatogastric system. These glands were first identified in the ant (Meinert, 1861; Forel, 1874); however, the endocrine function of these structures was first reported by Nabert (1913), It0 (1918), and Muller (1829), and was experimentally demonstrated by Wigglesworth (1935), Bouhniol (1936a,b, 1937a,b,c, 1938a,b,c,d,e,f), m u g 1
Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-364357-0
2
PIERRE CASSIER
felder (1937a,b), Piepho (1938a,b,c,d), Bodenstein (1938a,b,c,), and WeedHeiffer (1936a,b). Since these early experiments corpora allata glands have been found in many insect species. In his comprehensive topographic, histological study, which is still often referred to, P. Cazal presented both the data of previous workers and his own personal observations on more than 130 species (Cazal and Guerrier, 1946; P. Cazal, 1948). The definitions of structural types are used even today, although Cazal admitted that he lacked certain knowledge about Grylloblattidae, Zoraptera, Strepsiptera, Raphidioptera, and Plecoptera. His observations on Apterygota, which often were lacking in details, were later completed by other workers (Chaudonneret, 1949; Bitsch, 1962; Watson, 1964; Rohdendorf, 1965; Cassagnau and Juberthie, 1967; Rohdendorf and Watson, 1969; Palevody, 1976; Palevody and Grimal, 1975). The secretion of corpora allata is designated by the generic name “juvenile hormone” (JH) Vable I). Actually, three different JHs have been identified (Roller et al., 1967; Dahm et al., 1967; Meyer et al., 1968; Judy et al., 1973a,b). With one exception (Lanzrein er al., 1975; Luscher and Lanzrein, 1976) it is not yet clear, however, whether any one of these hormones has a particular morphogenetic or gonadotropic effect. A possible explanation of the physiological effects that have been established (Roller and Dahm, 1968) may be that, for the criteria measured, there are different activity thresholds or different induction capacities for these three hormones (Table 11). Thus, in the final larval stage of Galleria, the injection of 1p1 of JH-I caused a larval molt in more than 50% of the cases, 10 pg of JH-I1was less effective, and following an injection of 100 p g of JH-I11 most of the larvae had a normal pupal molt. Such an estimation TABLE I
THEJUVENILE HORMONES R
cis
0
R’
trans
’k,
OCH3
trans
H
R
Source
~~
Hyalophora cecropia Hyalophora cecropia Manduca sexta
R = R’ = CH,CHI R = CH3CH,; R‘ = CH, R = R‘ = CH,
JH-I (CilJH) JH-II (C 17 JH) JH-111 (C 16 JH) ____
methyl-2(E),6(E)-lO(R),1 l(S)-lO,Il-epoxy-7-ethyl-3,l l-dimethyl-2,6-tridecadienoate (trans-trans-cis).JH-11: 7-methyl analog of JH-I. JH-111 = methy1-2(E),6(E)-lO(R), 1 l(S)-10,lla JH-I:
epoxy-3,7,11 -tnmethyL2,6,dodecadienoate.
3
THE CORPORA ALLATA OF INSECTS
Smcinc ACTIVITIES OF RACEMIC JHs
TABLE II Gulleriu WAXTESTAND IN
IN THE
THE
Tenebrio TEST"
Juvenile hormone
Galleria units per microgramb
Tenebrio units per microgramc
JH-I JH-11 JH-III
200 x 103 200 x 1 0 3 2 x 103
8.00 30 0.05
"From Dahm et al., 1976. bGalleriu unit: Amount required to elicit a positive response in 50% of the animals scored. =Tenebrio unit: Amount required to elicit a positive response in 40%of the treated pupae.
is a delicate one and varies according to the tests being used. With a single exception (Lanzrein et al., 1975) JH-I and JH-I1 have been identified only in Lepidoptera (Roller et al., 1967; Meyer et af., 1968; Dahm et al., 1976; Dahm and Roller, 1970; Roller and Dahm, 1970; Schooley et al., 1973; Judy et af., 1973a; Jennings et af., 1975a,b). JH-111 alone was found in orthopteran, coleopteran, and hymenopteran species (Judy et d., 1973b; Trautmann et d., 1974b, 1976) and also in cultures of orthopteran and coleopteran corpora allata (Judy et al., 1973b, 1975; Peter and Dahm, 1975; Miiller e l af., 1974; Dahm et af., 1976). These findings suggest that the original JH of insects is JH-III and that JH-I and JH-I1 are special evolutionary achievements in Lepidoptera and possibly in other insect orders. In adult insects JH-I11 is the most common form. This may indicate which is the first form in the biosynthetic system of terpenoids; the synthesis of JH-I and JH-11, which are noteworthy since they contain ethyl radicals, requires the presence of enzyme systems and particular precursors. These JHs are transported in the hemolymph, presumably by lipoprotein carriers (Gilbert, 1974). The endocrine activity of the corpora allata and the mode of action of the different forms of JH are well known and retain a central position in entomological research (Scheme 1). They are concerned with morphogenesis (inhibition of metamorphosis), activation of follicular cells and sexual accessory glands, control of polymorphism, and so on, general physiology (synthesis of proteins, respiration, effect on fat body, oenocytes, water balance, imaginal diapause, coloration, pheromone secretion), behavior, and so on. The different aspects of classic endocrinology have been extremely well documented in several reviews which the reader should consult (Wigglesworth, 1964, 1965; Engelmann, 1968, 1970; Novak, 1966; Cassier, 1967; Gilbert, 1963, 1964, 1974, 1976; Menn and Beroza, 1972; Wyatt, 1972; Gilbert and King, 1973; Doane, 1973; de Wilde, 1964; de Wilde and de Loof, 1973; S l h a et al., 1974; Willis, 1974; Steel, 1975). The interest in this field of research is further demonstrated by the amount of research
4
PIERRE CASSIER
Controlling mechanisms
I
Secretion , into 6
Summation of covert effects
Over effects
Decav of covert effects SCHEME I. Mechanisms of control of the activity of corpora allata (Williams, 1976; Ohtaki et al.. 1968). The activation may be neuroendocrine and the inhibition of a neural nature (Sehnal and Granger, 1975). An inhibitory factor from the blood (see Williams, 1976) and an antigonadotropin (Liu and Davey, 1974) may be also involved.
carried out on juvenoids, growth regulator and juvenile hormone mimetics with specific short-range action affecting insects which are economically important (cf. Henrick et al., 1976; Zurflueh, 1976). However, histological and, even more important, cytological observations, whether they are based on optical or electron microscopy separately or combined, have not been subjected to comparative analysis. They are therefore presented here in the hope that this article will provide a better understanding of corpora allata activities and of their regulatory modalities, since in spite of all the efforts made, the formation and excretion of JH have not yet been clarified with regard to binding modalities of precursors, the site of synthesis, sites of release, and functional cooperation between the organelles, namely, between the chondriome and endoplasmic reticulum. The number of species investigated by electron microscopy is limited; thus attempts at interpretation and, even more so, at generalization might be subject to error and must be treated with caution. 11. The Embryonic Origin of Corpora Allata
That the corpora allata are ectodermal in origin is definite; this was established by Heymons in 1895 and since then has been frequently verified (cf. Haget,
THE CORPORA ALLATA OF INSECTS
5
1977, in Traite de Zoologie). The site of appearance of the buds (Fig. l), at first paired and symmetrical, differs slightly from species to species. The corpora allata may be formed by invaginations at the anterior part of the mandibular somite (Corynodes; Paterson, 1936), at its posterior part (Pieris; Eastham, 1930), between the mandibular and the maxillary segments (Silpha; Smereczyrisky, 1932; Locusta, Roonwald, 1936, 1937; Maldte, 1962; Carausius, Wiesmann, 1926; F'flugfelder, 1937a) or even in the maxillar somite (Forficula and Gryllus, Heymons, 1895). In Apis they appear to be derived from lateral outgrowths of the epithelium of the transverse bar of the tentorium (Pflugfelder, 1937a; Nelson, 1915). The conspicuous differences are due, first, to the amplitude of morphogenetic movements which affect the ventral part of the gnathal region and, second, to the timing of bud formation or appearance. Examination of the available data allowed Haget (1977) to confirm that the corpora allata are intersegmental organs originating in a region anterior to the maxillar segment.
0 FIG.1. The embryonic origin of corpora allata in C. morosus. (A) Sagittal section showing corpora allata (C.A.) bud. (B) Corpora allata bud becoming vesiculous. Mx., maxillae; Md., mandible; A., antenna; Dt., deutocerebrum; Pr., protocerebrum; Coe., antennal celom (after Pflugfelder, 1937).
6
PIERRE CASSIER
Subsequently, the buds of the corpora allata form two coherent cellular masses. During the time they are in contact with the ectoderm from which they originate they become pediculous; then they subsequently become globular and migrate in dorsal and mesa1 directions. They follow the anterior branch of the tentorium and finally attach themselves at the ventrolateral angles of the celomic sacs of the antenna1 segment. At this level the corpora allata fuse with the hypocerebral ganglia and the corpora cardiaca. Throughout the class Insecta and throughout its various orders, the more highly evolved the species, the more marked the migration of the corpora allata. Thus, in Thysanura, the corpora allata or corps jugaux (Chaudonneret, 1946, 1949; Bitsch, 1962) still occupy the ventral position. In Odonata they are anterior and ventral (Hanstrom, 1940a,b; Cazal, 1948; Schaller, 1968), in contact with the circumesophageal connectives and even between the connectives and the esophagus. In the majority of Heterometabola they flank the esophagus laterally and remain distinct. However, in numerous species of Dermaptera, Hemiptera, and Holometabolathe corpora allata fuse on the ventral surface of the aorta. Last, in superior Diptera they form a single mass above the aorta. Exceptionally, the corpora allata are situated extremely posteriorly. Thus in Sialis, Japyx, and the larvae of Lampyris they are located in the prothorax. In the embryo of Carausius (Wiesmann, 1926) the buds of corpora allata very rapidly become vesiculated (Figs. 4 and 5 ) .
111. Morphological Types
At the moment of hatching the corpora allata occupy different positions according to the group or species. Their mutual relations, as well as those with neighboring organs, also vary considerably. Following his numerous observations P. Cazal(l948) described five important morphological types whose essential characteristics are presented in Fig. 2. A. THELATERALIZED TYPE The lateralized type is the most common and is found in the primitive forms of most groups. It is characterized by the presence of two corpora allata, symmetrically arranged on each side of the digestive tube, connected by completely individualized allatocardiac nerves (N.C.A. 1). Beginning with this type, the evolution always follows the same pattern. The corpora allata reach the dorsal surface of the esophagus, join side to side and fuse either beneath or above the aorta. Thus two corpora allata beneath the aorta
THE CORPORA ALLATA OF INSECTS
I
C.A
A
D
F FIG.2. Retrocerebral glands in various groups of insects. (A) The lateralized type ( e . g . , Japyx); (B) the ventral type (e.g., Ephemera vulgara); (C) the semicentralized type (e.g., Blatta orientalis); (D) the centralized type (e.g., Pyrrhocoris apterus); (E) the distal lateralized type, primitive stage (e.g., Sphynx ligustri); (F) the distal lateralized type, evolved stage ( e . g . , Hydrous piceus); (G)the annular type (e.g., C. erythrocephala). Solid areas, corpora allata (C.A.); densely dotted areas, corpora cardiaca (C.C.); lightly dotted areas, peritracheal glands, molting or prothoracic glands (G.M.); Ao., aorta; N.C.C., nervi corporis cardiaci; Gg.SS.Oe., subesophageal ganglia; Cerv., cerebral ganglia.
may still be observed in Isoptera, Phasmidae, Heteroptera, Cryptocera, and Cicadidae.
B. THE DISTALLATERALIZED TYPE The distal lateralized type seen in several Diptera and Coleoptera differ from the lateralized type by the attachment of homolateral corpora cardiaca and corpora allata.
8
PIERRE CASSIER
Tr. -
FIG.3. Diagram of an ideal transverse section through the ring gland of a third-instar larva of Drosophila. Ao., aorta; Ax., axons from corpus cardiacum to corpus allaturn; C.A., corpus allatum; Tr.t., transverse trachea; Tr., trachea; R.G., prothoracic gland; C.C., corpus cardiacum (after King et al., 1966a).
C. THESEMICENTRALIZED TYPE
This type, found in Paleoptera and primitive Neoptera (Dictyoptera, Orthoptera, and so on), is characterized by a sagittal fusion of the corpora cardiaca (or corpus paracardiacum) which are attached directly to the aorta and the hypocerebral ganglia; the corpora allata remain distinct, while the N.C.A. 1 may remain separate (Orthoptera)or not (Dictyoptera), depending on the closeness of the relationship between the corpora cardiaca and corpora allata. This semicentralized type, which is very common, seems to be the most primitive.
THE CORPORA ALLATA OF INSECTS
9
D. THECENTRALIZED TYPE In this type (Embioptera, Plecoptera, Dermaptera, Psocoptera, various gymnocerate Heteroptera, Cicadidae, Aphididae) there is a development of the previous type, since the corpora allata also fuse and form a separate mass located beneath the aorta, in direct contact with the corpora cardiaca so that it seems as if they were actually inside the corpora cardiaca. In Oncopeltus the incomplete fusion of corpora allata may give rise to a bilobed structure (Novak, 1951; Unnitham et al., 1971).
E. THEANNULAR TYPE The annular type (ring-shaped type; Fig. 3) characteristic of higher Diptera (Brachycera, Cyclorhapha) results from a fusion above the aorta of the corpora allata, which together with the ventral corpora cardiaca and the lateral peritracheal glands form a ring around the aorta known as Weissmann’s ring. Formation of this ring takes place in the larval stage, and the N.C.A. 1 are not visible; they become apparent during metamorphosis, from the onset of the imaginal molt as the ecdysial glands (peritracheal glands) degenerate. Furthermore, the corpora allata in contact with the brain of the larvae migrate posteriorly to reach the prothorax of the imago.
IV. Innervation and Tracheal Supply of Corpora Allata
A. INNERVATION The most complex innervation of the corpora allata is double. In fact, these glands are connected both to the brain, by the superior allatocardiac nerve (N.C.A.l), and to the subesophageal ganglion (N.C.A.2), either directly or by means of the tritocerebral paracardiac nerve (N.C.C.1V). The superior allatocardiac nerve (N.C.A. 1) is formed from ordinary fibers and neurosecretory fibers which arise in the protocerebrum and traverse the corpora cardiaca. In Schistocerca cancellata (=paranensis) (Strong, 1965a,b) the neurosecretory fibers arise exclusively from lateral protocerebral neurosecretory cells via the external paracardiacal nerves (N.C.C.11). In Locusta (Cassier and Fain-Maurel, 1970) they arise also from median protocerebral neurosecretory cells (pars intercerebralis) along the slope of the internal paracardial nerves (N.C.C.1).
10
PIERRE CASSIER
The relationships between the pars intercerebralis (a neurosecretory center), the corpora cardiaca (a neurohemal organ), and the corpora allata (an endocrine gland) are frequently compared with those in the hypothalamic-hypophyseal complex, particularly at the level of the adenohypophysis. In insects with the lateralized type and some semicentralized type (Paleoptera, Orthoptera) the N.C.A. 1 are completely individualized. They enter the corpora allata at the level of the hilum, they lose their sheath, and then the fibers ramify between the glandular cells. However, in Dictyoptera and in species of the centralized and annular types, the N.C.A. 1 are morphologically indiscernible because of the coalescence of the corpora allata and corpora cardiaca. It is only by histological studies that the constitutive fibers may be recognized. They frequently form a periglandular plexus and then enter the gland by several hila and ramify between the glandular cells (Schultz, 1960; Fukuda et al., 1966; Tombes and Smith, 1970; Dorn, 1973; Baehr el al., 1973; Melnikova and Panov, 1975; Paledovy and Grimal, 1975; Morohoshi et al., 1976a). In Hyalophora (Waku and Gilbert, 1964) only one part of corpora allata is covered by the neurosecretory fibers. Since the origin of corpora allata is ventral, the relationship with the superior centers must be acquired secondarily. However, the subesophageal innervation (N.C.A.2) is primitive; it exists only in Ephemeroptera (Hanstrom, 1940; Cazal, 1948, Bouhniol et aE., 1953), Thysanura (Cazal, 1948), Collembola (Cassaugnau and Juberthie, 1967), and in young stages of Odonata where the cerebral innervation appears only in aged larvae (Cazal, 1948). In Locusta the N.C.A.2 consist of neurosecretory fibers which emerge from the subesophageal cells (Chalaye, 1965, 1966), and the pattern is the same in Blattidae and Culicidae (Fuller, 1960; Harker, 1960). In Thysanura (Chaudonneret, 1949) the corps jugaun are entirely covered with maxillary nerves but are directly innervated by the subesophageal ganglion. In Machilidae (Bitsch, 1962) the corpora allata are innervated by both maxillary and mandibular neuromers. The afferent and efferent nerves are surrounded by a thick (3-4 pm), stratified neural sheet and by a perilemma consisthg of a layer of thin (1-3 p m ) glial cells (the lemmoblasts of Edwards et al., 1958; the Schwann cells of Hess, 1958; the neuroglial cells of Trujillo-Cenoz, 1962). Extensions of these cells enclose the isolated fibers or bundles of fibers. They are united by septate desmosomes. Their ovoid nucleus (4-6 p m in diameter) contains in general only one nucleolus; the fragmented chromatin clumps are attached to the nuclear membrane. B. THETRACHEAL SUPPLY OF CORPORA ALLATA
Corpora allata are abundantly supplied with tracheae; their distribution seems to follow the same pattern as that of the nerve fibers. Thus, where N.C.A.l are
THE CORPORA ALLATA OF INSECTS
11
entirely individualized (e.g., Locustu), they run side by side with the nerve and enter the gland at the level of the hilum and ramify between the gland cells. However, when these nerves lose their individuality there is a network of tracheae surrounding the gland, which then ramify and run into the glandular parenchyma (Cazal, 1948; Busselet, 1968; Baehr et al., 1973). In the latter example the tracheae are often enclosed between the basal laminae and the allata cells.
V. Histological Characteristics and Types A. HISTOLOGICAL TYPES Structural study of corpora allata shows that there are at least six distinguishable types of cells which correspond mainly to glandular cells (undifferentiated, normal, polyploid; Ozbas, 1957; Joly, 1976; Mendes, 1948) and to associated elements (axons, neurosecretory fibers, glial cells, tracheoblasts, peripheral conjunctive cells). Furthermore, these glands are often surrounded by fat-body tissue and pericardial cells. The distribution of these different cellular types, their abundance, and their morphometric and histological characteristics provide so many criteria that P. Cazal (1948) was able to identify four principal types of corpora allata. 1. The Pseudolymphoid (or Lymphoid) Type
The pseudolymphoid type is found in Paleoptera (Ephemera, Odonata) and is characterized by the presence of many cells packed closely one against another. They have a reduced amount of cytoplasm, which is slightly basophilic, and do not have any apparent secretory activity. The small nuclei (7-9 pm in diameter) are round or oval. There are a few mitochondria; the Golgi apparatus is not well developed, and it is difficult to distinguish any secretory products or secretionrelated phenomena. 2 . The Small-Cell Type The small-cell type is fairly common, appearing in Blattidae, Mantidae, Orthoptera, Dermaptera, Paraneoptera, and in numerous Oligoneoptera. It is characterized by the presence of many gland cells which are small, irregularly shaped, and particularly rich in mitochondria, Golgi apparatus, and secretory grains. In Acrididae and Coccinellidae the peripheral cells are more or less palisade, whereas the central cells are irregularly shaped; many intercellular cavities whose size varies according to the physiological state of the insect and size of the gland may also be seen.
12
PIERRE CASSIER
Fusion of these intercellular cavities sometimes gives rise to a large central cavity. This is the case in senescent males of Locusta where this phenomenon is even more marked because of degeneration of many of the central cells (Cassier, 1965b; Fain-Maurel and Cassier, 1969, 1970). The nuclei are generally average-sized (10-12 p m in diameter), ovoid or elongated, and contain many chromatin blocks. The cytoplasm is clear and basophilic. The well-developed chondrion consists of granular or filifonn mitochondria. Each cell contains a 5 to 12 thick ring- or scale-like Golgi areas.
3 . The Macrocell Type The macrocell type observed in Panorpu is characteristic of Trichoptera, Lepidoptera, Hymenoptera, and some cycloraphous Diptera. It differs greatly from the above-mentioned types and is apparently the most highly evolved. The allate cells are few in number and are large: Hyphantria, 20 to 25 cells (Melnikova and Panov, 1975); Aphis craccivora, 10 to 12 cells (Elliot, 1976); Speophyes and Diaprysius, 16 to 18 cells (Deleurance and Charpin, 1971); Troglodromus bucheti gaveti, 50 cells (Jkleurance and Charpin, 1972); Hyperu postica, 85 cells (Tombes and Smith, 1970); DrosophiZa melanogaster, 20 to 24 cells (Poulson, 1945; King et al., 1966a). In Bombyx mori (Fukuda et al., 1966) the peripheral cells are the largest (50 X 90 pm) and are certainly more active than those in the center (30 x 50 pm) of the organ. Occasionally the limited
FIGS.4 and 5 . Corpora allata of an adult female C. morosus (P. Cassier, unpublished). FIG.4. In the central cavity, the product of secretion forms an amorphous matrix; a nodule of dense material is frequently associated with structured, concentrically disposed bodies.
THE CORPORA ALLATA OF INSECTS
13
number of cells may be correlated with the small size of the insect: Folsomia cundidu (Collembola), 3 cells (Paledovy and Grimal, 1975; Paledovy, 1976). The nuclei are hypertrophied and branched in appearance. The cytoplasm is abundant and contains many inclusions. 4 . The Vesicular Type The vesicular type is noteworthy for an epithelial arrangement of the glandular cells in a single layer or several layers surrounding a central cavity. Many
FIG. 5 . Part of a glandular cell showing an active Golgi apparatus with numerous dense granules of secretion and rough endoplasmic reticulum.
14
PIERRE CASSIER
investigators consider this arrangement a reappearance of primitive characteristics. This type is found in Phasmidae (Figs. 4 and 5 ) where simple, high epithelium lines a subspherical cavity containing a PAS-positive mass, the central body apparently being formed by five to six chitinous lamellae concentrically disposed. Each lamella may correspond to an exuvium removed during each of the molts which occur during the postembryonic development of Phasmidae. These exuvial structures, which are inevitably confined, persist during the imaginal stage (cf. Joly, 1976). Such an interpretation deserves careful study. The corpora allata of Psyllidae, Mallophagidae, Anoplura (Cazal, 1948), Thysanura (Chaudonneret, 1949), Machilidae (Bitsch, 1962), and Blattidae (Diplapteru punctaru) (Engelmann, 1970) are also of the vesicular type. In Psocoptera (Badonnel, 1934) each corpus allatum contains three cavities and three central bodies; however, these are not present in Mallophaga or in Anoplura. In Japyx the nuclei of allate cells are grouped on the periphery of the organ in a type of envelope. In Embioptera the cells have a radial arrangement. In Isoptera the glandular cells are spindle-shaped. The outer extremity is connected to the basal laminae; the inner one, which is more elongated and chromophilic, is wound in a whorl at the center of the corpus allatum (Cazal, 1948). All these structural characteristics are reported in Table 111 following a hypothetical evolutionary process. B.
VOLUME OF THE
GLAND,MITOSIS,and PYCNOSIS
During the postembryonic development and imaginal life of insects the volume of corpora allata is subject to two types of changes. TABLE Ill EVOLUTIONARY STAGES OF CORPORA ALLATA BASEDON THEIRANATOMICAL, HISTOLOGICAL, AND CYTOLOGICAL CHARACTERISTICS"
Intermediate type. ~~~~
~
~
~
Subesophageal innervation Ventrolateml position Pseudolymphoid structure Vesicular type (remnants of embryonic characteristics) "From Cazal, 1948.
~
Developed type
~~
Subesophageal and cerebral innervation Dorsolateral or dorsal position Microcellular type
Subesophageal and cerebral innervation Fusion below or above aorta Macrocellular type
-
-
THE CORPORA ALLATA OF INSECTS
15
1. Allomerric Growth .This regular increase, independent of any physiological event, may or may not be accompanied by modifications in form and is related to insect growth. An allometric type of growth follows the mitotic events (hyperplasia) and that of cell enlargement (hypertrophy) whose relative importance varies according to species. In the majority of species (e .g .,Blattidae) these two phenomena coexist during their larval and imaginal stages; in Acrididae, particularly in Locustu, this coexistence occurs only during the larval stage, whereas during the imaginal stage it results only from an increase in the cytoplasmic mass, from dilatation of intercellular spaces, and from endopolyploidy in a relatively limited number of cells (Ozbas, 1957; Cassier, 1965b; Joly, 1968). In small insects the corpora allata contain a limited number of cells (e.g., F. candidu, three cells) and growth depends exclusively on cytoplasmic and nuclear hypertrophy which may or may not be associated with endopolyploidy (Paledovy and Grimal, 1975; Paledovy, 1976). However, in Thermobiu domestica growth is associated with an increase in the number of cells (Rohdendorf and Watson, 1969). In Oncopeltusfusciutus an increase occurs 3-4 days after the imaginal molt and continues during the egg-laying period (from day 8 to day 38), thus the rate of increase is 30. The increase in size is only partially due to divisions (Novak, 1951; Johansson, 1958b). In Calliphora erythrocephalu (Lea and Thomsen, 1969; Thomsen and Thomsen, 1970) the volume of corpora allata is increased three- or fourfold between the first (200 X lo3 pm3) and the fourth day of imaginal life, which results exclusively from growth of the various cells. In flies fed a protein-deficientdiet, the volume of corpora allata was reduced (100 x lo3 pm3). In general, resumption of the activity of the corpora allata after the imaginal molt, during sexual maturation, or after diapause, is associated with an increase in their volume. This has been observed in Anacridium (Girardie and Granier, 1973), Pterostichus nigritu (Hoffmann, 1970), Nebriu brevicollis (Ganagarajah, 1965), Macrodytes marginalis (Joly, 1945), Carabus nemoralis (Klug, 1959), Galeruca tanaceti (Siew, 1965), Leptinotarsa (de Wilde and de Boer, 1969), and Aulacophora foveicollis (Saini, 1966). 2 . Periodic Growth In most insects the corpora allata undergo cyclical changes of variable importance during larval development and as adults. These changes, which result in fluctuations of corpora allata activity, affect in particular the nucleocytoplasmic relations. a. in Larvae. These changes are synchronous with molt cycles. This has been established in Holometabola and in Heterometabola (Novak, 1954). Thus, in Oncopeltus (Novak, 1951, 1954; Johansson, 1958b) at the onset of each larval stage the volume of corpora allata is reduced, but it progressively increases and
16
PIERRE CASSIER
reaches maximal values in the middle of the intermolt cycle which is the phase of intense endocrine activity; later, the volume decreases in parallel to the slowing down of this endocrine activity. Although these glands are considered nonactive during the last stage, their volume still increases, but to a smaller degree than during the earlier stages; this certainly corresponds only to allometric growth. In B. mori (Legay, 1950)the increase in volume occurs at each larval molt and to a lesser extent during the intermolts. The synchronous changes in the molt cycles depend partly on a hyperplasia (mitotic crisis), which has been established in Curuusius (Pflugfelder, 1937b)
FIGS.6 and 7. Corpora allata (iugal bodies) of a thysanuran insect, Petrobius rnaririmus Leach. Inactive corpora allata during the postimaginal molting process. (P. Cassier, unpublished.) FIG. 6. Inactive allata cells contain a pycnotic nucleus and fingerprint-like bodies.
THE CORPORA ALLATA OF INSECTS
17
and in Rhodnius (Wigglesworth, 1936, 1948) and more particularly on a cytoplasmic hypertrophy which is shown by a high reduction in the nucleocytoplasmic ratio ( R = N / C ) . b. In the Imaginal Stage. The cyclic fluctuations are synchronous with ovarian cycles and are manifested by variations of the gonadotrophic activity of the corpora allata (Wigglesworth, 1948, 1964; Engelmann, 1957, 1959, 1970; Strangeways-Dixon, 1961, 1962; Highnam, 1962, 1964; Cassier, 1967; Scharrer and von Harnack, 1958; Strong, 1965a; Johansson, 1958; Nayar, 1958; Siew, 1965; Ganagarajah, 1965; Thomsen, 1947; Wilkens, 1968; Pratt and Davey, 1972; Weaver et al., 1974). In Musca dornesticu (Adams et al., 1968; Adams, 1970) a small corpus allatum releases the JH, whereas a large one stores it; this may depend on the action of an oostatic hormone, which is contrary to Lea’s ideas (1975). These phenomena have been particularly well studied in Leucophaeu muderae, a viviparous cockroach (Scharrer and von Harnack, 1958; von Harnack, 1958; Engelmann, 1957, 1958). Glandular activity (vitellogenesis, a period of rapid growth) is associated with an increase in the number of cells (mitosis); during nonactive periods and particularly during periods of gestation, the number of cells diminishes following pycnosis. These two processes (mitosis, pycnosis) do in fact, coexist, but their relative importance varies,
FIG. 7. High magnification of a fingerprint-like body. Note the presence of numerous membranes, dense material, and a lipid droplet.
18
PIERRE CASSIER
mitosis being predominant during vitellogenesis and pycnosis being characteristic of gestation periods. Similar phenomena were observed in the corps jugaux of adult Petrobius maritimus (Figs. 6 and 7) (P. Cassier, unpublished) and in Acridium (Girardie and Granier, 1973; Warkievi-Granier and Leonide, 1971). The factors contributing to sexual maturation, to egg laying, and to an increase in fecundity (mating, enforced activity, feeding, excitatory pheromones, wounds, amputations) also stimulate the postimaginal growth of the corpora allata. Conversely, factors which delay sexual maturation and cause a decline in fecundity (gestation, parasitism, inhibitory pheromones, imaginal diapause, overcrowding, the presence of mature eggs in the ovarioles) are marked by reduced activity and size of the corpora allata (cf. Cassier, 1967). The corpora allata of ovariectomized female Acheta domesticus (Huignard, 1964), Melanoplus differentialis (Pfeiffer, 1945), L . maderae (Scharrer and von Harnack, 1961), Rhodnius prolixus (Wigglesworth, 1936), Calliphora vicina (Thomsen, 1940), and D . melanogaster (Vogt, 1942; Dome, 1961), and of sterile females (the Drosophila fes mutant; Vogt, 1942; King et al., 1961, 1966b), are abnormally large. 3. Volumetric Changes and Polymorphism In Acrididae (phase polymorphism) the corpora allata of solitary locusts are larger than those of gregarious individuals (Nickerson, 1954; P. Joly, 1949, 1967; L. Joly, 1960; Carlisle and Ellis, 1959; Staal, 1961). Their hypertrophy is associated with increased activity and contributes to the development of phase polymorphism (chromotropic, gonadotropic, and morphogenetic effects; cf. Cassier 1967, 1974). In Schistocerca the level of circulating JH causes the differentiation and functioning of integumentary glands (Cassier and DelormeJoulie, 1973, 1974, 1975, 1976a,b,c; Cassier, 1977a,b) which are responsible for the secretion of a stimulatory specific sexual pheromone (Norris, 1952, 1954; Loher, 1958, 1960, 1961). In the honeybee (Apis mellifera) (social polymorphism) queens possess large corpora allata, while those of the workers are average-sized and those of the drones are small. Even in the larval stages the differences between queens and workers are discernible; they become even more noticeable during the pupal and adult stages (Lukoschus, 1955a). In adult workers (sterile females) there are periods of considerable growth of the corpora allata, which are in particular related to the activity of the hypopharyngeal glands (6-12 days), achievement of the first orientation flight (9-10 days), secretion of the wax glands (14-15 days), and acquisition of behavior in field bees. In this last case the sudden increase in the level of JH causes degeneration of the hypopharyngeal glands and a fall in the level of vitellogenic and hemolymphatic proteins (Rutz et al., 1976). Sub-
THE CORPORA ALLATA OF INSECTS
19
sequently, the corpora allata activity decreases (Pflugfelder, 1937a,b, 1948, 1958; Hanstrom, 1942). 4. Further Remarks Several workers, having considered the above-mentioned observations, have concluded that the degree of activity of the corpora allata may be assessed by measuring their size and nucleo-cytoplasmic ratio (see Highnam, 1964; Wigglesworth, 1964; P. Joly and L. Joly, 1970). Such a correlation has been confirmed in many cases, but it is not a general rule, and in the literature there are various examples of cases in which no relationship exists between the size of the corpora allata and the development of oocytes (Johansson, 1958a;Mordue, 1967; Engelmann, 1968; Lea and Thomsen, 1969; Tobe and Pratt, 1975; Gillot and Dogra, 1972; Brousse-Gaury, 1971c, 1972). Thus bilateral sectioning of antennae between the scape and the pedicel in Blaberafusca (Brousse-Gaury et al., 1973; Brousse-Gaury and Cassier, 1975) causes a retardation of ovulation and reduced ovarian activity but results in an increase in the size of the corpora allata. C. SEXUAL DIMORPHISM Male and female corpora allata are generally comparable during the juvenile or larval stage. However, following the imaginal molt the process of allometric growth results in a difference in their size, independent of any morphometric difference. This sexual dimorphism results in either hyptertrophic male or hypertrophic female corpora allata. 1. Hypertrophic Corpora Allata of Females This is the most common type of sexual dimorphism. It has been described in Orthoptera (Fain-Maurel and Cassier, 1969b), in Coccidae (Cazal, 1948; Mugfelder, 1936), in Isoptera (Holmgren, 1909; Pflugfelder, 1937; Stringberg, 1913), and in Aphenogaster (Cazal, 1948) and has been particularly well studied in B. mori (Fukuda et al., 1963, 1966) (Fig. 8). In the last-mentioned case the corpora allata cells of the female may be distinguished Erom those of the male by the presence of many osmiophilic vacuoles of varying size (0.3-14 p m in diameter) which are particularly large (12 pm) and abundant in the circumferential cells (Figs. 4 and 5). These inclusions are not formed when the corpora allata are separated from the brain (Fukuda and Takeuchi, 1965). The inclusions are associated with lamellar structures or rod-shaped mitochondria with welldeveloped cristae of the tubular type. In males the osmiophilic bodies are infrequent and small, and the chondriosomes (cup- or ring-shaped) have many lamellar cristae, particularly well-
20
PIERRE CASSIER S.E.R.
N.
B.L.
A
N.
I
L.
I
L.
I
B.L.
I
D.-
S.E.R.
.L.
L.S.-
B FIG.8. Schematic drawing of peripheral cells of corpora allata of the male (A) and female (B), 50 hours after emergence, in B . rnori. N.F., neurosecretory fiber; D., desmosome; L.S., laminated structure; S.E.R., smooth endoplasmic reticulum; B.L., basal laminae; N . , nucleus; I.S., intercellular space; L, lipid droplet; H . , hemocyte. (After Fukuda et nl., 1966.)
THE CORPORA ALLATA OF INSECTS
21
developed endoplasmic reticulum may be seen. Degenerating cells may be observed at the center of the corpora allata. The sexual differences are basically evident immediately after the imaginal molt but are well-established 50 hours later. In Anacridium (Girardie and Granier, 1973) sexual dimorphism during the active period concerns the size and the mitotic index (female corpora allata are larger and show more mitoses than those of males).
2 . Hypertropic Corpora Allata of Males This type of sexual dimorphism is less common than that in females; it has, however, been reported in different Lepidoptera: Ephestia Kuhniella (Schrader, 1938), Hyalophora cecropia (Gilbert and Schneiderman, 1961b), and Galleria mellonella (Rehm, 1951). IV. Cytological and Infrastructural Characteristics of Corpora Allata Only a few species have been studied by electron microscopy (see Table IV); in addition, these observations do not form a cohesive picture, since in some species juvenile forms and larvae have been studied, while in others adult insects have been examined. This variation is largely dependent on whether the researcher’s principal concern has been problems related to postembryonic development or those of reproductive activity. There are very few systematic, complete studies, and even in these the results are sometimes not easy to compare, since different techniques have been used (osmium fixation, double fixation with gluteraldehyde-osmium or paraformaldehyde-osmium), the differences in inclusion media markedly affect the preservation of organelles, especially the endoplasmic reticulum. Nevertheless, the available results have as a common denominator the fact that the research was carried out to find fine cytological criteria which act as markers for the synthetic activity of corpora allata and the organelles involved in the production of JH. The studies are carried out indirectly. Corpora allata removed in the middle of the intermolt phase, in mature males or in females during vitellogenesis are considered active according to the classic criteria (Scharrer, 1961, 1964; Fukuda et al., 1966; Odhiambo, 1966b; Fain-Maurel and Cassier, 1969b; Melnikova and Panov, 1975). However, corpora allata removed at the beginning or at the end of an intermolt, during the last larval stage, immediately after the imaginal molt, during egg laying, in viviparous females during gestation, in sterile castes (workers, soldiers), and in insects undergoing an imaginal diapause, are considered inactive (Waku and Gilbert, 1964; Odhiambo, 1966c; Panov and Bassurmanova, 1970; Tombes and Smith, 1970; Guelin and Darjo, 1974). These categories may be completed by including insects in which corpora
22
PIERRE CASSIER TABLE IV MAINSPECIES STUDIED BY ELECTRON MICROSCOPE PROCEDURES
Larvae
Adults
+
+
P. Cassier (unpublished)
-
+
Palevody and Grimal (1975) Palevody ( 1976)
+
+ +
Scharrer (1961, 1962a. 1964a, 1970, 1971) Brousse-Gaury and Cassier (1973), ( 1975)
?
?
Meyer and Pflugfelder (1958) Smith (1968)
Locusta migratoria L.
+ +
+ +
Locusta migratoria migratorioides (var. Kazalinsk) Anacridium aegyptium L.
Odhiambo (1966b,c) Papillon et al. (1976) L. Joly e t a l . (1967) L. Joly et al. (1968, 1969) P. Joly and L. Joly (1970) Fain-Maurel and Cassier (1969a,b, 1970) Cassier and Fain-Maw1 ( 1970) Guelin and Darjo (1974)
+
+
Girardie and Granier (1974a)
Species Thysanura Petrobius maritimus Leach Collembola Folsomia candiah Willem Dictyoptera Leucophaea maderue Fabr. Blabera fusca Br.
Cheleutoptera Carausius morosus Br. Orthopteroida Schistocerca gregaria Forsk
Dennaptera Labidura riparia Hemiptera Eurygaster integriceps Put. Rhodnius prolixus StHl Oncopeitus fasciatus Dallas
-
C. Caussanel and P. Cassier (1978) -
-
+ + -
Aphis craccivora Koch Myzus persicae Pass.
Coleoptera Diaprysius seruliazi Peyer Diaprysius fagei Jean Hypera postica Gyllenhal
Reference
+ + +
-
+ + -
+
+ + -
+
Panov and Bassunnanova Baehr et al. (1973) Busselet (1969) Dorn (1973) Unnitham et al. (1971) Elliot (1976) Bowers and Johnson (1966) Deleurance and Charpin (1971) Deleurance and Charpin (1971) Tombes and Smith (1970, 1972) Tombes (1970)
23
THE CORPORA ALLATA OF INSECTS TABLE IV (continued)
Species
Larvae
Adults
Speophyes lucidulus Delar Choleva cisteloides Friil
+
+
Diptera Chironomus melanotus Drosophila melanogaster Meig.
-
+
+ +
DeleuranceandCharpin(1971,1973) Charpin (1973) Kiimmel (1969) King et a / . (1966a,b)
+
Calliphora erythrocephala
Reference
Agganval and King (1 969) Thomsen and Thomsen (1969,1970)
Meig. Calliphora stygia F.
Hymenoptera Apis mellifera mellifera L. Lepidoptera Antheraea pernyi Guer. Hyphantria cunea Drury Celerio lineata L. Hyalophora cecropia L. Bombyx mori L. Miscellaneous (Ostrinia nubilalis, Diatraea grandiosella, Diatraea saccharalis, Chilo plejadellus, Calleria mellonella, Plodia urtapunctella
Johnson (1966)
+
Wirtz (1973) Busselet (1969) Melnikova and Panov (1975) Schultz (1960) Waku and Gilbert (1964) Fukuda er al. (1966) Morohoshi et al. (1976a,b) Nishiitsusuji-Uwo (1961) Yin and Chippendale (1977)
allata are inactive because of a mutation (e.g., the fes mutant in Drosophila; King et al., 1966a) or because of experimental intervention such as parsectomy (Joly et al., 1967, 1969; Baehr et al., 1973), fasting (Thomsen and Thomsen, 1969, 1970; Brousse-Gaury et al., 1973), modifications of the sensory afferents (Brousse-Gaury et al., 1973; Brousse-Gaury and Cassier, 1975), or castration (P. Cassier, unpublished). Only elements of corpora allata (see Section V) having functional significance are discussed (glandular cells, axons, neurosecretory fibers); the associated elements (glial cells, tracheoblasts, conjunctive cells) are not considered here.
A. BASALLAMINA The noncellular layer enclosing the endocrine glands of insects has been given different names: the mesenchymatic envelope (Cazal, 1948), the conjunctive tissue (Willey and Chapman, 1960; Scharrer, 1963), the stroma (Scharrer, 1964a; Nonnann, 1965; Thomsen and Thomsen, 1970), the basal membrane
24
PIERRE CASSIER
(Odhiambo, 1966b), the tunica (King et al., 1966a), the cytoplasmic membrane (Fukuda et al., 1966), and the tunica propria (Beaulaton, 1964; Busselet, 1969). The designation “basal lamina” seems to be the most appropriate one and does not lead to presumptions regarding its origin or function. The presence of the basal lamina is a constant structure of insect corpora allata; it is involved in glandular cohesion. It is continuous with that of the allatocardiac nerves (N.C.A.l) when they are individualized, and with that of the corpora cardiaca when the N.C.A.l are joined. In the latter case, the distribution of nerve, neurosecretory fibers, and tracheae (see Section IV) is such that the basal lamina envelop these structures also (Baehr et al., 1973). According to Cazal (1948) the basal lamina is rarely continuous, and thus some of the glandular cells are in direct contact with the hemolymph. No discontinuity has been established at the infrastructural level, although hemocytes are capable of traversing the membrane (Busselet, 1969; Fukuda et al., 1966). The basal lamina frequently has, on its exterior, a discontinuous layer of flattened mesenchymal cells. These cells have a dense nucleus and a few organelles. The cells or lemnoblastscontain short rough endoplasmicreticular cisternae, free ribosomes, mitochondria, and dictyosomes, and in general have no smooth endoplasmic reticulum. The thickness of the basal lamina varies greatly from one species to another, being 0.6-1 or 2 p m in Antheraea (Busselet, 1969), 0.02-2 p m in larvae, and 0.1-0.2 p m in adults of Rhodnius (Busselet, 1969; Baehr et al., 1973), 0.1 p m in Calliphora (Thomsen and Thomsen, 1970), 0.25 pm in Aphis (Elliot, 1976), and 0.1-1 p m in Hyalophora (Waku and Gilbert, 1964). The thickness of the basal lamina also varies with the physiological state of the insect; as a general rule it is relatively thin, slightly tortuous, and directly apposed to the cytoplasmic membrane in active corpora allata. However, it is thicker, has scallop shapes which may be more or less marked, and may be separated from the glandular cells by a 3- or 4-pm-wide space in nonactive corpora allata (Busselet, 1969; Baehr et al., 1973; Thomsen and Thomsen, 1970). The basal lamina of the corpora allata of Leucophaea, Blaberus craniifer (Harper et al., 1967), B. fusca (Brousse-Gaury et al., 1973), and Schistocerca (Odhiambo, 1966b,c) contain collagen fibers with characteristic periodic transverse striations. These fibers are rich in hydroxyproline and hydroxylysine. The texture of the basal lamina is also variable. It may be homogeneous and granulofilamentous, as in Aphis (Elliot, 1976), Folsomia (Paledovy and Grimal, 1975; Paledovy, 1976), and HyaZophora (Waku and Gilbert, 1964), or composed of layers of lamellae each 0.02-0.2 p m thick, as in Antheraea and Rhodnius (Busselet, 1969; Baehr et al., 1973). Finger-like projections of the basal lamina penetrate to a greater or lesser depth into the cavities between the cells of the glandular parenchyma (Scharrer, 1961, 1963, 1964; Thomsen and Thomsen, 1970; Willey, 1961; Waku and Gilbert,
THE CORPORA ALLATA OF INSECTS
25
1964; Odhiambo, 1966c; Melnikova and Panov, 1975; Brousse-Gaury et al., 1973; Brousse-Gaury and Cassier, 1975). B. Scharrer (1961, 1962a,b, 1963, 1964) stressed in particular the importance of this structure which, in the absence
of a circulatory apparatus, seems to provide an effective means of supplying nutrients and removing the secretory product, JH, from the corpora allata. The basal lamina, which may be stained with aniline blue (Cazal, 1948) or PAS is composed of neutral and acid mucopolysaccharides associated with a protein component. No lipid has been detected. It may contain inclusions of varying sizes which are homogeneous in Calliphora (Thomsen and Thomsen, 1970) and in larvae of Rhodnius (Busselet, 1969), in which they measure from 1200-1500 A to 6000-6500 A in diameter, and more frequently from 2000 to 2500 A In neither case has the origin of these inclusions or their significance been elucidated. In Leucophaea (Scharrer, 1971), dense, “cribriform, ” pleomorphic inclusions measuring up to 1.6 p m have been seen. They are formed in the Golgi apparatus (C-body material) and released by exocytosis into intercellular spaces, although part of the C-body material may be captured by the multivesicular bodies. Cribriform bodies are particularly numerous in the extracellular stroma of inactive corpora allata, which may simply suggest a relationship with the endocrine functions of these glands.
B. GLAND CELLS 1. Light and Dark Cells
As is true for many tissues, the examination of corpora allata under the conditions of electron microscopy fixation frequently allows two types of glandular cells to be distinguished by their different electron density, namely, light and dark cells (Fig. 9). According to Busselet (1969), Thomsen and Thomsen (1969), Johnson (1966), and Deleurance and Charpin (1971, 1972), these distinctions have no functional or structural significance; they are an artifact due to the variable penetration of fixatives. However, Dorn (1973) considered that the differences of electron density observed in Oncopeltus had a functional significance. He proposed that the dark cells, which predominate during the last larval stage and in young adults, are inactive. The light cells, which are characteristic of males and mature females, are active; they are larger than the dark cells, and their nuclei have more indentations. 2. Plasma Membranes and Membrane Differentiation a. Plasma Membranes. One of the essential contributions of electron mic-
roscopy studies has been the demonstration of plasma membranes. Previous observations using the light microscope have led to a hypothesis suggesting the
FIG.9. Corpus allatum of a mature adult male desert locust, S. gregaria Forsk. Two types of glandular cells may be apparently distinguished by their different electron density, namely, light and dark cells; but the presence of a clear area inside the mitochondria and of vesicles in the vicinity of the plasma membrane indicate that the distinction has no functional or structural significance. (P. Cassier, unpublished.)
THE CORPORA ALLATA OF INSECTS
27
syncytial nature of corpora allata or of the existence of multinuclear cells (see P. Cazal, 1948, for references). Plasma membranes are universally distributed, and this rule has no known exception in the case of corpora allata. The difficulties encountered in their study are essentially due to their meandering path since the allata cells have extremely complex forms (branched or star-shaped), particularly those located at the center of large, compact glands. In contact with the peripheral basal lamina and its finger-like projections (see Section V1,A) the plasma membrane projects numerous fine or stocky digitations which are supported by microtubules oriented parallel to their longitudinal axis or reinforced at their extremities by hemidesmosomes. In Schistocerca, independently of these digitations (Odhiambo, 1966a,b), there also exist crypts provided with microvilli formed by invaginations of the plasma membrane of certain cells or are developed in the zone of junction of several glandular cells (Papillon et al., 1976). These crypts are similar to the glandular epidermal cells (Noirot and Quennedey, 1974); the elements of the basal lamina may penetrate them. Generally, the course of the plasma membrane varies according to the physiological condition of the corpora allata. Thus, in Aphis craccivora (Elliot, 1976), Hypera postica (Tombes and Smith, 1970, 1972), H . cecropia (Waku and Gilbert, 1964), Schistocercu gregaria (Odhiambo, 1966a), R . prolixus (Busselet, 1969; Baehr et al., 1973), Antheraea pernyi (Busselet, 1969), L. muderae (Scharrer, 1964a), and Celerio lineata (Schultz, 1960), the plasma membranes of active corpora allata are much less sinuous than those of inactive corpora allata. The increase in size of the cytoplasmic mass associated with cellular activity causes distension of the membranes. Thus, in Schistocerca (Papillon et al., 1976) the above-mentioned crypts are clearly visible in the inactive gland immediately after the imaginal molt but are subsequently found less frequently and are less well-developed (Figs. 10-12). b. Intercellular Junctions. Glandular cohesion of the corpora allata is essentially ensured by the peripheral basal lamina (see Section VI,A) and the interdigitations of the projections of adjacent cells. The differentiations of junctional membranes are not well-developed. In the region of contact of the basal lamina and the finger-like process of the glandular cells the extremities of these cellular digitations are generally reinforced by hemidesmosomes associated with microtubules (Girardie and Granier, 1974a; Tombes and Smith, 1970; Melnikova and Panov, 1975). In B. fusca the hemidesmosomes may be up to 2.5 p m in length. In H . cecropia the accumulation of dense material corresponding to the hemidesmosomes is visible only in active glands; this may be related to the secretion of JH (Waku and Gilbert, 1964). In Hyphantriu (Melnikova and Panov, 1975) no specialized system of junctions between the glandular cells has been observed. In B . mori (Fukuda et al., 1966), larval stages of Rhodnius (Busselet, 1969), Locusta migrutoria L. (Fain-Maurel and Cassier, 1969b), and
FIGS. 10 and 1 1 . Corpora d a t a of the adult desert locust, S. gregaria Forsk. (From Papillon et al., 1976.) FIG. 10. In inactive corpora allata (male adult reared at 28°C). intercellular crypts are well developed.
FIG. 11. Adult male, 23 days old, reared at 28°C. The glandular cell are in a moderate state of activity; rough and smooth endoplasmic reticulum are poorly developed. The mitochondria form a heterogeneous population. 28
FIG. 12. Corpora allata of the adult desert locust, S . gregaria Forsk. Active corpus allatum of an adult male desert locust reared at 33°C. The smooth endoplasmic reticulum frequently associated with the Golgi apparatus is well developed. ( F h m Papillon et al., 1976.)
29
30
PIERRE CASSIER
Oncopeltus (Dom, 1973) the junctional system consists of occasional desmosomes of the zonula or macula adherens type; it consists of numerous macula adherens in A. pernyi (Busselet, 1969). In Anacridium (Girardie and Granier, 1974a) and Drosophila (King el al., 1966j gap junctions (tight junctions) and desmosomes are associated with microtubules. In R. prolixus, during imaginal life (Baehr et al., 1973), and in B. fusca (Brousse-Gaury et al., 1973), this junctional system is particularly complex, because it consists of gap junctions, maculae adherens, and septate desmosomes. c. Pinocytotic Vacuoles. Pinocytotic vacuoles notable for the radial ornamentation of their cytoplasmic surface have frequently been observed both during their formation when they are associated with or are close to the plasma membrane and when they are scattered throu out the cytoplasm (coated vesicles). They are particularly large (1000-2000 in diameter) in Rhodnius (Baehr et al., 1973) and in Blubera (Brousse-Gaury et al., 1973).
x
3. Intercellular Spaces and Microtubules a. Intercellular Spaces. The intercellular spaces deserve particular attention, since in corpora allata they constitute an important pathway not only for the supply of structural or energetic material and hormonal precursors but also for the transport of JH into the hemocoel. In resting glands or glands where the level of activity is low, the cells are contiguous, the plasma membranes are apposed, and the intercellular spaces are narrow and moniliform. However, during the peak of hormonal activity the intercellular spaces become much wider (Schultz, 1960; Waku and Gilbert, 1964; Scharrer, 1964a; Pasteels and Deligne, 1965; King et al., 1966a; Odhiambo, 1966b; Joly, 1968; Brousse-Gaury et al., 1973; Brousse-Gaury and Cassier, 1975; Baehr et al., 1973). b. The Intercellular Microtubules. In the fourth-instar larvae of B. mori (Morohoshi et al., 1976a) the intercellular spaces contain numerous microtubules which form connections between the glandular cells and axons and even penetrate the basal lamina. These unusual structures may participate in the intercellular transport of material or even of the brain hormone which, in this species, enters the hemolymph in the region of the corpora allata (Morohoshi et al., 1975; Morohoshi and Shimada, 1975). In Locusta migrutoria migrutorioides (R. and F. ) (P. Cassier, unpublished) (Figs. 13-16) the intercellular spaces frequently contain an electron-dense substance which is usually amorphous and does not have the appearance of the entangled fibrils (40A) characteristic of the basal lamina, is able to reorganize into macrofilaments, as is indicated by the coexistence of these two forms. In its amorphous form the substance is homogeneous and electron-dense; it is distributed in an irregular fashion, and this gives the spaces separating the glandular cells a varicose appearance. It even penetrates the globular, filiform, or cap-shaped part of the intercellular spaces.
THE CORPORA ALLATA OF INSECTS
31
FIGS.13-16. Corpora allata of the female African migratory locust, L . rnigruforiu migrntorioides (R. and F.). (P. Cassier and M. A. Fain-Maurel, unpublished.) FIG. 13. Intercellular spaces with dense material and bundles of tubules. FIG. 14. Intercellular spaces with thick, tortuous filaments.
32
PIERRE CASSIER
FIG. 15. Expanded intercellular spaces with thin filaments. FIG.16. Crossed intercellular spaces with mixed tubules and dense material.
In its structured form the substance is composed of macrofilaments which can be described by their diameter and distribution as follows: (1) Long, flexible filaments, 100-120 A in diameter, are irregularly distributed in large spaces (1-2 pm) which are not electron-dense. (2) Thicker filaments (160-200 A in diameter), very electron-dense and equidistant from one another (250 A from center to center), are grouped in compact bundles in the distended zones of the intercellular spaces, which in other regions are narrow and regular. These bundles have the shape of the winding outline of the adjacent cells and intersect each other in regions where several cellular dilatations converge. The appearance of filaments (100-120 A in diameter) in the amorphous intercellular material, which may lose locally, its electron density may be an indication of differentiation of this substance into solid elements. The origin and significance of the intercellular material and tubules is still conjectural; they may play a role in glandular cohesion, secretion, or intercellular transport. The first hypothesis is a likely one, since the material is similar to the intercellular oriented fibrillar material which appears during the reaggregation of dissociated embryonic cells (Overton, 1969). 4. The Nucleus
Although allata cells are uninuclear, their nucleus has not been the subject of much research. In corpora allata of the lymphoid (see Section V,A,1) or microcellular type (Section V,A,2) the very numerous nuclei are regular in shape, being spherical or ovoid, and of small dimensions: 7-9 p m and 10-12 p m in diameter, respectively. However, in the macrocellular type the nuclei are less frequent but are large, lobed, and branched or star-shaped. The size and appearance of the nuclei, regardless of the type, vary according to the level of glandular activity. Cellular activity of corpora allata is accompanied by nuclear swelling, dilatation of the perinuclear space, and dispersion of
THE CORPORA ALLATA OF INSECTS
33
chromatic clumps (Panov and Bassurmanova, 1970; Elliot, 1976; Thomsen and Thomsen, 1970; Waku and Gilbert, 1964; Baehr et al., 1973). For example, in Lewophaea (Scharrer, 1964a) the nuclear dimensions increase from 11.7 X 8.8 pm in inactive glands to 15 x 11 p m in the active state. The dilatations of the perinuclear space, which measures 150-200 A at rest, are frequently accompanied by a proliferation of the external rough endoplasmic reticular layer of the nuclear sheath, thus increasing the amount of endoplasmic reticulum. This phenomenon, comparable to that resulting in the production of the annulated lamella, has been reported in Hyphantria (Melnikova and Panov, 1975), Drosophila (Agganval and King, 1969), Calliphora erythrocephala (Thomsen and Thomsen, 1970), and Bathyscinae (Coleoptera) (Deleurance and Charpin, 1971, 1972); it may be the source of reticular, pseudocrystalline structures or “twisted areas” (Deleurance and Charpin, 1971, 1972). The number of nucleoli is a specific characteristic; it varies according to species from one (Rhodnius, Triatoma, Eurygaster), to two (Leucophaea) or three, to five (Anrheraea, Bombyx, Hyalophora). In inactive glands they are large and compact. In active glands they have a tendency to fragment (e.g., Rhodnius; Baehr et al., 1973) or to take on a very characteristic annular configuration (e.g., Eurygaster; Panov and Bassurmanova, 1970; B. mori: Fukuda et al., 1966). The presence of nuclear inclusion has been reported. In A. pernyi the single inclusion is large (1.75 X 1.75 pm), is Feulgen-positive, and contains a large quantity of RNA and DNA (Busselet, 1969); in Leucophaea it is a spherical body (7000 A in diameter) of an unknown nature.
5 . Mitochondria Mitochondria are an important element in the cell, and their study can provide information about the structure and the activity of the corpora allata. In inactive glands mitochondria are in general scarce and are represented principally by globular (0.15-0.3 p m in diameter) or rod-shaped (0.5-1 p m x 0.3-0.5 pm) elements which are uniformly distributed in the cytoplasm (on average, 10.4 per 25 pmZ in Hyalophora). These organelles contain lamellar cristae with no preferential orientation in a dense matrix and zero, one, or two opaque bodies 1000-1500 A in diameter (Wakuand Gilbert, 1964; Scharrer, 1964a; Odhiambo, 1966b; Panov and Bassurmanova, 1970;Thomsen and Thomsen, 1970; Baehr et al., 1973; Papillon et al., 1975). Several cup-shaped elements have also been observed in Hyphantria (Melnikova and Panov, 1975). In B. fuscu the mitochondria are frequently found close to the nucleus (BrousseGaury et al., 1973). Important modifications correlated with the cycle of activity have frequently been observed; these generally consist of a considerable increase in the mitochondrial population (39.7 per 25 pm2 in Hyalophora) (Waku and Gilbert,
34
PIERRE CASSIER
FIGS. 17-18. Corpus allatum of an adult male African migratory locust, L . migrutoria migratorioides (R. and F.). The mitochondria are all of the same type and are characterized by their light matrix, numerous lamellar cristae, and ovoid (Fig. 17), globular, or bilobulated form (Fig. 18). (From M. A. Fain-Maurel and P. Cassier, 1969b.)
1964) and of an increase in the size of the mitochondria which also frequently undergo a change in shape, becoming dumbbell-, cup- or ring-shaped or give rise to macromitochondria as a result of the matrix becoming opaque and the number of cristae increasing (Scharrer, 1964a; Fukuda et al., 1966; Odhiambo, 1966b; Joly et al., 1968; Baehr et al., 1973; Browse-Gaury et al., 1973; BrousseGaury and Cassier, 1975; Dom, 1973; Melnikova and Panov, 1975; Joly, 1976). Mitochondria frequently appear as globular elements (0.1-0.2 p m in diameter) arranged in small chains, which is a sign of mitochondrial division (Busselet, 1969; Baehr et al., 1973; Brousse-Gaury et al., 1973; Brousse-Gaury and Cassier, 1975). Cup- or ring-shaped mitochondria frequently form concentric structures in which they regularly alternate with cisternae of smooth endoplasmic reticulum (e.g., Bombyx: Fukuda et al., 1966; Locusta: Fain-Maurel and Cassier, 1969b; Schistocerca: Odhiambo, 1966b).
THE CORPORA ALLATA OF INSECTS
35
FIG. 18. See legend on p. 34.
During the transition from an active phase to a resting phase a considerable number of mitochondria disintegrates or is incorporated into lytic bodies (see Scharrer, 1964a). Locusta migratoria migratorioides deserves particular mention; in this species sexual dimorphism is associated with mitochondrial pleomorphism (Fain-Maurel and Cassier, 1969b). After the imaginal molt each sexual type (male or female) and even each physiological state (females during maturation and mature females) may be defined and individualized by the type of mitochondria. In males, the activity of the corpora allata is continuous, and the mitochondrial population is homogeneous (Figs. 17 and 18). The mitochondria are characterized by their swollen appearance and their large size, by the increasing transparency of the matrix material, and by the large number and regular arrangement of the cristae. In females the activity of the corpora allata is cyclic, and the mitochondrial population is heterogeneous. It consists of (Figs. 19-23) (1) a strain of small mitochondria with an electron-dense matrix substance, which are permanent source elements in the glandular cells, and (2) a strain characteristic of each physiological stage. During sexual maturation a line of chondriosomes differentiates from the permanent line. These mitochondria contain a clear matrix
36
PIERRE CASSIER
FIGS. 19-21. C o p r a allata of a mature female African migratory locust, L. migruroriu migrarorioides (R.and F.). (From M. A. Fain-Maw1 and P. Cassier, 1969b.) FIG.19. Giant mitochondria of a female during sexual maturation.
FIG. 20. Association of extended mitochondria and endoplasmic reticulum at the beginning of an ovarian cycle.
THE CORPORA ALLATA OF INSECTS
37
FIG.21. An annular mitochondria endoplasmic reticulum complex may be observed at the end of each ovarian cycle.
substance with a small number of fine lamellar rectilinear cristae; some of these mitochondria evolve into giant mitochondria (1.5-2 p m x 2-3 pm) which are characterized by marked proliferation of their lamellar cristae which form loops and rings (see also Anacridium: Girardie and Granier, 1974a). However, during ovarian cycles individualization of the mitochondria occurs at the expense of the permanent line; very elongate mitochondria which contain an electron-opaque matrix substance, but become dumbbell- or later cup-shaped, take part by their association in the construction of a large mitochondrial complex-the chondriosphere of De Robertis and Sabatini (1958). They are frequently found in organs involved in the synthesis of steroids (Christensen and Chapman, 1959; Idelman, 1964, 1970), as well as in the prothoracic glands (Beaulaton, 1968a,b, 1969; Cassier and Fain-Maurel, 1970, 1971a,b). 6 . The Endoplasmic Reticulum Of all the organelles in allata cells the endoplasmic reticulum has most attracted the attention of cytologists, especially in early ultrastructural investigations when the chemical nature of JH was still unknown. Indeed, taking into account the frequent existence of a relationship between structure and function, the endoplasmic reticulum could, because of its abundance, provide an exact index of the level of glandular activity, which in turn could provide investigators
38
PIERRE CASSIER
FIGS.22-23. Corpus allatum of the African migratory locust, L . migratoria migratorioides (R. and F.). FIG.22. Corpus allatum of a female at the end of an ovarian cycle, containing numerous large associations of annular mitochondria and smooth endoplasmic reticulum concentrically disposed.
with a means of evaluating the consequences of their experiments and thus the mechanisms which control the activity of corpora allata. This criterion could replace the measurements of the volume or the nucleocytoplasmic ratio of these
glands. By its appearance the endoplasmic reticulum also suggests that the hormone formed by the corpora allata is a protein (rough endoplasmic reticulum), a lipid, or even a sterol (smooth endoplasmic reticulum). The first of these two working hypotheses has in general been verified; one should, however, take into account the existence of possible asynchrony in the activity of the allata cells in corpora allata, and in the organism of a separation of the phases of optimal synthetic activity and the peaks of hormonal activity
THE CORPORA ALLATA OF INSECTS
39
FIG. 23. Naked terminal part of a neurosecretory axon showing mitochondria, synaptic vesicles, synaptoid area, and vesicles of pinocytosis.
measured in the hemolymph. This last reservation poses crucial problems in the synthesis, release, and storage of JH, which will probably be resolved by the use of a sensitive, precise radioimmunological assay which has been developed in our laboratory (Baehr et al., 1976, 1977a,b). The second hypothesis has been only partially confirmed. Although JHs are lipid-soluble sesquiterpenoids (Roller ef al., 1967; Meyer et al., 1968; Judy et al., 1973a,b), their synthesis is not necessarily linked to the formation of an abundant smooth endoplasmic reticulum, as may be seen on examination of the different groups shown in Table V. Thus, in insects belonging to the first group, during peaks of hormonal activity, glandular cells have shown evident enrichment in rough endoplasmic structures (Figs. 24-28). However, in insects belonging to groups I1 to V, under the same conditions, the study of corpora allata reveals an increase in smooth endoplasmic reticulum, which, in the extreme case (group V) may invade the cell and form structures which may be pseudocrystalline, lenticular, whorled, or concentric. Moreover, on examining Table V it can be seen that there is no direct relationship between the type of endoplasmic reticulum, the systematic position (Apterygota, Pterygota, Hemimetabola, Holometabola), or the physiological
40
PIERRE CASSIER TABLE V NATUREOF THE END~PLASMIC RETICULUM IN ACTIVE CORPORAALLATACELLS" Species
Order
Stage
Reference
Group 1: Smooth endoplasmic reticulum absent, hypertrophy of the rough endoplasmic reticulum Carausius morosus Phasmodea Smith (1968); P. Cassier (unpublished) Eurygaster sp. Put. Heteroptera Adult Panov and Bassunanova ( 1970) Rhodnius prolixus StiU Heteroptera Adult Baehr et al. (1973) Heteroptera Larva Busselet (1969) Diaprysius serullazi Peyer Coleoptera Larva Deleurance and Charpin (1971) Diaprysius fagei lean Coleoptera Larva Deleurance and Charpin (1971) Antheraea pernyi Guer. Lepidoptera Larva Busselet (1969) Group 2 Smooth endoplasmic reticulum slightly developed, predominant rough endoplasmic reticulum Oncopeltus fasciatus Dallas Heteroptera Adult, Dorn (1973) larva Homoptera Adult, Elliot (1976) Aphis craccivora Koch larva Hypera postica Gyllenhal Coleoptera Adult Tombes and Smith (1970, 1972) Group 3: Smooth endoplasmic reticulum and rough endoplasmic reticulum present Petrobius maritimus Br. Thysanura Adult P. Cassier (unpublished) Collembola Adult Palevody and Grimal (1975); Fofsomia candida Willem Palevody ( 1976) Leucophaea maderae Fabr. Blattodea Larva, Scharrer (1964a, 1971) adult Blabera fusca Br. Blattodea Adult Browse-Gaury et al. (1973); Browse-Gaury and Cassier (1975) Apis mellifera mellifera L. Hymenoptera Larva Wirtz (1973) Group 4: Smooth endoplasmic reticulum, preponderant but unorganized Chironomus melanotus Diptera Larva Kiimmel (1969) Group 5 : Rough endoplasmic reticulum negligable, smooth endoplasmic preponderant and organized Anacridium aegyptium Orthoptera Larva, Girardie and Granier (1974a) adult Locusta migratoria L. Orthoptera Larva, Joly et al. (1967, 1968); adult Fain-Maurel and Cassier (1969b); Cassier and FainMaurel (1970); Guelin and Darjo (1974) Schistocerca gregaria F. Orthoptera Adult Odhiambo (1966b); Papillon e t a l . (1976) (continued)
41
THE CORPORA ALLATA OF INSECTS TABLE V (continued)
Species
Order
Oncopeltus fasciatus
Heteroptera
Lobidura riparia
Dermaptera
Speophyes lucidulus
Coleoptera
Choleva cisieloi‘des Troglodromus brucheti gaveti Isereus colasi Bathysciola schiodtei Drosophila melanogaster
Coleoptera Coleoptera Coleoptera Coleoptera Diptera
Calliphora erythrocephala Hyalophora cecropia
Diptera Lepidoptera
Celerio lineata Bombyx mori
Lepidoptera Lepidoptera
Hyphantria cunea Miscellaneous
Lepidoptera Lepidoptera
Stage Larva, adult Adult Larva, adult Adult Adult Adult Adult Adult, larva Adult Pupa, adult Adult Adult, larva Larva
Reference Unnitham
et
al. (1971)
C. Caussannel and P. Cassier (unpublished) Deleurance and Charpin (1971, 1973) Charpin (1973) Deleurance and Charpin (1972) Deleurance and Charpin (1972) Deleurance and Charpin (1972) King et al. (1966a,b) AggarwaI and King (1%9) Thomsen and Thomsen (1 970) Waku and Gilbert (1 964) Schultz (1960) Fukuda et al. (1966); Morohoshi et al. (1976a,b) Melnikova and Panov ( 1975) Nishiitsusuji-Uwo (1960- 1961)
“After Melnikov and Panov, 1975. bAccordmg to published papers the endoplasmic structures of H . cecropia should be considered of the smooth endoplasmic reticulum type.
state of an insect (larva, pupa, adult). It is worth noting that insects belonging to groups 1V and V and, to a certain extent, group 111 are the most common, are large, or have a holometabolic type of postembryonic development. Those belonging to the first group are heterometabolous or larvae of holometabolous insects, and it is unfortunate that insufficient studies have been made and that these are incomplete. Interference in an evolutionary process within the class Insecta and within its various orders is possible. This was very well understood by Deleurance and Charpin (1971, 1972) in their comparative study of different coleopteran Bathyscinae. The structural differences of the corpora allata of primitive species which have small eggs (extended development) and those of the most evolved species which have large eggs (direct, contracted development) are essentially dependent on the amount of endoplasmic reticulum present. In Bathysciola and Speophyes species which have small eggs the granular reticulum is abundant, while the smooth endoplasmic reticulum i s less abundant and is represented by tubules and scarce or grouped vesicles which nevertheless show a tendency to organize into
FIGS.24 and 25. Corpus allatum of the adult female of the blood-sucking bug R . prolixus Stiil. (From Baehr et al., 1973, by permission of Arch. Zool. Exp. Gen., Paris.)
THE CORPORA ALLATA OF INSECTS
43
FIGS.26-28. Corpus allatum of the adult female of the blood-sucking bug R. prolixus StB1. (From Baehr et al., 1973, by permission of Arch. Zool. Exp. Gen., Paris.) FIG.26. Corpus allatum of a female provided with a blood meal. Increased cytoplasmic areas contain numerous extended mitochondria; rough endoplasmic reticulum forms frequently annular membranous structures (arrow),and nucleoli often are fragmented. These symptoms of glandular activity are particularly discernible after ovariectomy or late parsectomy.
more-or-less coalescent lamellar structures. In Troglodromus and Isereus species which have large eggs, the rough endoplasmic reticulum is not well developed, but the smooth reticulum is abundant and formed of lamellae which organize to form well-individualized lenslike structures (6 x 3 p m in diameter) in Isereus which is the most developed species. The males of these species undergo the same evolution. ~~
FIG. 24. Topographic distribution of the retrocerebral organs. Ao, aorta; CA, corpus allatum; CC, corpus cardiacum; LB, basal laminae; Tr, tracheolae. FIG.25. Part of the glandular parenchyma of the inactive corpus allatum of a fasting female. Nuclei with regular outline shows a large nucleolus. The less-developed cytoplasm contains randomly distributed mitochondria of reduced size and scarce rough endoplasmic reticulum structures.
44
PIERRE CASSIER
FIG.27. The glandular cells of an active corpus allatum contain numerous dense granules originating from the Golgi apparatus. FIG.28. Part of a cytoplasmic area in an active corpus allaturn, showing moniliform association of globular mitochondria (arrows) well-developed rough endoplasmic reticulum, and dense granules.
In addition the absence of smooth endoplasmic reticulum or the presence of simple smooth vesicles is not an established fact, because this organelle is particularly fragile and is frequently destroyed or denatured during cytological experiments; so the use of an osmium fixative alone should be avoided (cf. Fawcett, 1961). Finally one cannot neglect the effect of subjectivity on observations. Thus, in Oncopeltus, according to Unnithan et al. (1971) the smooth endoplasmic reticulum was abundant, while Dorn (1973) found that it was not well developed. The presence of ribosomes either scattered or associated in polysomes has been reported in the corpora allata in all species studied. Their number increases in parallel with the level of glandular activity and with development of the cytoplasmic mass (Scharrer, 1964a; Waku and Gilbert, 1964; Fukuda et al., 1966; Odhiambo, 1966b; King et al., 1966a; Busselet, 1969; Fain-Maurel and Cassier, 1969b; Thomsen and Thomsen, 1970; Melnikova and Panov, 1975). However, in Eurygaster (Panov and Bassumanova, 1970) the number of ribosomes decreases during the active phase but, in parallel, the ergastoplasmic membrane system begins a considerable development. Independent of their participation in formation of the ergastoplasmic membrane systems probably involved in protein synthesis, the ribosomes take part in
THE CORPORA ALLATA OF INSECTS
45
FIGS.29-30. Corpora allata of an adult female African migratory locust, L . migratoria migratorioides (R. and F.). Evolution of the endoplasmic reticulum during an ovarian cycle. (From Fain-Maurel and Cassier, 1969a. f FIG.29. Onset of an ovarian cycle, length of the terminal oocytes is 0.5-0.7 mm. Glandularcells contain numerous ribosomes, polysornes, and flattened cisternae of rough endoplasmic reticulum. Dense bodies may represent residual bodies of the endoplasmic reticulum developed during the last ovarian cycle.
46
PIERRE CASSIER
FIG.30. Part of a glandular cell during the first day of an ovarian cycle (length of the terminal oocyte is about 1 mm). Numerous smooth vesicles buds are present at the level of the cisternae of the rough endoplasmic reticulum. Note the presence of numerous microtubules.
periodic cellular growth by synthesizing constitutive proteins which are not released. The chronological succession of different forms of endoplasmic reticulum which represent stages of the cycle of activity associated with the development of larval stages or with ovarian cycles has been confirmed. Thus in solitary female adults of Locusta (Fain-Maurel and Cassier, 1969a) at the time when the terminal oocytes measure 0.5-0.7 mrn in length (low-growth phase) (Figs. 29 and 30) the reticulum consists of ribosomes, polysomes, and flattened cisternae of rough endoplasmic reticulum from which small, smooth vesicles become detached. At the onset of vitellogenesis (oocytes ranging from 0.7 to 1.2 mrn in length) (Figs. 31 and 32) allata cells show correlatively an increase in the amount of rough or granular endoplasmic reticulum. During the high-growth phase (oocytes ranging from 1.2 to 6.5 mm in length) (Figs. 33-35) the smooth endoplasmic reticulum (vesicles, tubules, cisternae) takes up a large proportion of the cell volume, forming entangled aggregates which at first are not oriented (oocytes 3 mm in length) but later become concentrically organized. During oviposition and the following 2 days, the concentric formations are transformed into lamellar bodies which frequently engulf numerous mitochondria and finally take on myelinic form. Similar patterns have been observed in the males of Schistocerca (Odhiambo, 1966b) and Locustu (Fain-Maurel and Cassier, 196913) during sexual maturation, in females of Locusta in the gregarious phase (Joly et d.,1967, 1968), in B. fusca (Browse-Gaury et al., 1973; Brousse-Gaury and Cassier, 1975), in Leucophaea (Scharrer, 1964a), and during the larval stages of Hyphantria (Melnikova and Panov, 1975). Such a differentiation is never seen to occur synchronously in all the allata cells, instead there is an average condition. An
FIGS.31 and 32. Corpora allata of an adult female African migratory locust, L. migratoria migratorioides (R. and F.). Evolution of the endoplasmic reticulum during an ovarian cycle. At the beginning of the ovarian cycle (oocytes about 1-1.2 mm long) the smooth endoplasmic reticulum proliferates (Fig. 31) and progressively invades large cytoplasmic areas (Fig. 32). (From M. A.
Fain-Mauml and P. Cassier, 1969a.)
48
PIERRE CASSIER
FIGS.33-35. Corpora allata of an adult female African migratory locust, L. migratoria migratorioides @. and F.). Evolution of the endoplasmic reticulum during an ovarian cycle. (M. A. Fain-Maurel and P. Cassier, 1969a.) FIG.33. At the end of an ovarian cycle (oocytes 2-6.5 mm long), the smooth endoplasmic reticulum progressively forms large areas of stmctures frequently concentrically disposed around mitochondria and vesicles.
active corpus allatum is distinguished from an inactive one by the larger number of active cells rich in smooth endoplasmic reticulum. In Anucridium (Girardie and Granier, 1974a) the ultrastructural appearance of the corpora allata does not change during oocyte maturation. The sequence of the different ultrastructural appearances of the endoplasmic reticulum is not temporal, as in Locustu, but spatial; it occurs simultaneously in each corpus allatum, regardless of the stage of vitellogenesis. Oocyte maturation in Anucridium does not result from a single cycle of activity of the corpora allata but from a succession of secretory cycles which continue asynchronously in all the cells of the organ. In L. rnigrutoria migrutorioides (Kazalinsk strain) Guelin and Darjo (1974) observed a distinct distribution of various forms of reticulum in each allata cell,
THE CORPORA ALLATA OF INSECTS
49
which enabled them to determine the cellular polarity exactly and to define three zones: a perinuclear A zone rich in mitochondria, tubules, ribosomes, polysomes, and short rough endoplasmic reticulum tubules, containing a crown of dictyosomes; an internal, lobed B zone specialized with secretory processes and having abundant smooth reticulum associated with the dictyosomes. An external C zone composed of a group of digitations which serve to anchor the cell and in which there is no smooth reticulum and only a few dictyosomes. Such polarity may be observed during a period of activity in the allata cells of B. fusca (Brousse-Gaury et al., 1973; Brousse-Gaury and Cassier, 1975) (Fig. 36). Indeed, during the imaginal stage the progressive increase in gonadotropic activity of the corpora allata is manifested by an increase in the glandular cells of ergastoplasmic formations which, in the perinuclear zone, are involved in the formation of large rolls of membranes and also of agranular tubular reticulum scattered inside the peripheral cytoplasmic digitations (Figs. 37-40). Similar observations have also been made in Leucophaea (Scharrer, 1964a).
RGS.34 and 35. At the moment of egg laying and during the first day of the next ovariancycle,an area of concentrically disposed smooth endoplasmic reticulum generates fingerprint-like structures and then myelinic structures.
FIG. 36. Inactive corpus allatum of a young adult dictyopteran insect, B . fusca, 1 day after the imaginal molt.
THE CORPORA ALLATA OF INSECTS
51
FIGS.37-40. Active corpora allata of an adult female B . fusca. (From Browse-Gaury et a[., 1973.) FIG. 37. Mitochondria are frequently elongated and rough endoplasmic reticulum cisternae are well developed.
FIG. 38. Nearby the basal laminae (LB), elongated mitochondria, and smooth endoplasmic reticulum tubules are mixed.
There may be a regional distinction. Thus the peripheral allata cells of Calliphora (Thornsen and Thomsen, 1970), Bombyx (Fukuda et al., 1966), Schistocerca (Odhiambo, 1966b,c), Hyphantria (Melnikova and Panov, 1975), and Locusta (Cassier and Fain-Maurel, 1970; Fain-Maurel and Cassier, 1969b, 1970) are richer in reticulum and inclusions and more active than the central cells. The opposite is seen in Anacridium (Girardie and Granier, 1974a); this may be linked to the fact that the axonal endings of the allatocardiac nerve are present in the medullar zone.
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FIG. 39. Rough endoplasmic reticulum cistemae are concentrically disposed. FIG.40. Peripheral cytoplasmic infoldings contain numerous smooth endoplasmic reticulum vesicles.
Experimental evidence suggests that both smooth and rough endoplasmic reticulum may be involved in the biosynthesis of JH. In Drosophila (King e f al., 1966a) the allatum bodies, the characteristic piles of lamellar smooth reticulum do not develop in sterile females (the fes mutation). In Leucophaea (Scharrer, 1970) the hypertrophied corpora allata of ovariectomized females are particularly rich in smooth endoplasmic reticulum. There is no smooth reticulum nor are there lenticular associations in the corpora allata of young coleopteran females of Bathyscinae (Deleurance and Charpin, 1971, 1972). In B.fusca (Brousse-Gaury et al., 1973) (Figs. 41 and 42) cauterization of the right ocellus in isolated females results in a large and significant increase in gonadotropic activity associated with hypertrophy of the right corpus allatum; the allata cells contain long, flexible, curved, or moniliform ergastoplasmic cistemae and a large number of large mitochondria. In Locustu the disconnection of male active corpora allata rich in endoplasmic reticulum or their implantation in larvae or females, results in less than 8 days in aplasia of the gland, its inactivation, and disappearance of the reticulum. This agrees with experimental data (Cassier, 1963, 1964a,b,c, 1965a,b, 1967; Albrecht and Cassier 1964), although the observations made by Joly e f al. (1969) are in this respect more equivocal.
THE CORPORA ALLATA OF INSECTS
53
A reduction in the diurnal temperature from 33" to 28°C in the breeding colonies of Schistocerca (Papillon et al., 1972; Cantacuzene et d . , 1972) results in male sterility and a decline in female fecundity, a reduction in volume, persistence of intercellular crypts, a delay in the appearance of the criteria by which glandular activity is measured, and a noticeable reduction in the amount of different forms of endoplasmic reticulum (Papillon et al., 1976). Finally, in Locusta ovariectomy causes hypertrophy of corpora allata associated with proliferation of the endoplasmic reticulum. In Eurygaster (Panov et ul., 1972), the corpora allata of females parasitized by Clytomyia have the appearance of inactive glands and degenerate. This last finding was also confirmed by Girardie and Granier (1974b) in females of Anacridium parasitized by Metucemyia calloti during nondiapausing periods; nevertheless certain cells maintained the criteria of activity. The transition from an active phase to a period of inactivity is associated with elimination of the endoplasmic reticulum in the form of lamellar bodies or pseudomyelinic structures (Fain-Maurel and Cassier, 1969a,b; Deleurance and Charpin, 1972; Palevody and Grimal, 1975). According to Girardie and Granier (1974a), pseudomyelinization is a characteristic of corpora allata activity and may denote the rapid renewal of structures concerned with synthesis. It does not appear in the physiologically inactive corpora allata of Anacridium and Locusta. In insects belonging to groups IV and V (see Table V) the proliferation of smooth endoplasmic reticulum may give rise to structures of variable size and appearance: lenslike bodies in coleopteran Bathyscinae (Deleurance and Charpin, 1971, 1972), allatum bodies in Drosophila (King et d., 1966a,b), paracrys-
FIGS.41 and 42. Right hyperactive corpora allata of a female B . fusca after right ocellus cauterization. (From Brousse-Gaury et al., 1973.) FIG. 41. Hypertrophied cytoplasmic areas contain numerous ergastoplasmic structures (arrow) and granules with a granular matrix (star).
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FIG. 42. The cells are characterized by much homogeneous hyaloplasm, associated ergastoplasmic cistemae, and large mitochondria.
talline bodies in Hyphantria (Melnikova and Panov, 1975), concentrically disposed areas in Locusta and Schisiocerca, twisted areas in Leucophaea (Scharrer, 1964a) and B. fusca (Brousse-Gaury ei al., 1973), and aggregates or networks of tubules in Calliphora (Thornsen and Thomsen, 1970) and Hyalophora (Waku and Gilbert, 1964). Such structures may be seen also in Labidura (C. Caussanel and P. Cassier, unpublished) (Fig. 43).
THE CORPORA ALLATA OF INSECTS
55
These structures are certainly associated with the synthesis of JH, since they characterize active corpora allata. Furthermore: 1. They are frequently associated with organelles (mitochondria, rough endoplasmic reticulum, Golgi apparatus) or inclusions (lipid droplets, osmiophilic bodies) and even closely disposed near the nucleus whose granular outer membrane seems to be involved in their formation. 2. An area of sinuous tubules, vacuoles, or vesicles with a clear content frequently surrounds the structures and seems to migrate toward the plasma
FIG.43. Active corpus allatum of the dermapteran insect Labidura riparia, showing a very characteristic StNCtUre composed of a strict association of vesicles and tubules of smooth endoplasmic reticulum near the nucleus. (By courtesy of C. Caussanel, unpublished.)
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membrane (Thomsen and Thomsen, 1970; Melnikova and Panov, 1975). 3. They are not specific for corpora allata; similar structures have been observed in the steroidogenic organs of vertebrates (Berchtold, 1969, 1970; Picheral, 1968; Christensen, 1965), the seminal vesicles of Schistocerca (Cantacuzene et al., 1972), the oenocytes of Locusta (Fain-Maurel and Cassier, 1969a, 1972), and the digestive tube of Petrobius (Fain-Maurel and Cassier, 1972). Formation of these structures seems to be correlated to the synthesis of lipid or small molecules. In conclusion, although morphological analysis of corpora allata is fragmentary, it demonstrates that, in spite of the chemical identity and similarity of JH synthetized, there is a great disparity in the evolution of the organelles involved, particularly of the endoplasmic reticulum. The qualitative (rough or smooth) and quantitative differences in the endoplasmic reticulum are related to the level of activity of the corpora allata, which depends upon the relative size of insects and that of the corpora allata, physiological processes (morphogenesis, vitellogenesis), and the intensity of these processes (production of large or small eggs). Moreover, such differences suggest that there is a dissociation of the two stages of glandular activity: on the one hand formation of synthesizing structures and secretory product, and on the other the release of this product and regression of the structures participating in its synthesis. Thus, according to the hypothesis of Panov and Bassurmanova (1970), which was further developed by Brousse-Gaury et al. (1973), the appearance of the gland in a given physiological state is a result of the balance, which varies according to species, between the rate of JH synthesis and its release, which in the case of Locusta confirms the suggestion of Joly et al. (1968): Our photographs suggest that the production of juvenile hormone is associated with the formation of smooth endoplasmic reticulum vesicles. In the active glands these vesicles are expelled progressively as they are produced and the juvenile hormone diffuses into the hemolymph. However, in the inhibited glands the rate of production of the smooth endoplasmic reticulum vesicles would be greater than the rate of their expulsion, thus causing the accumulation observed. Subsequently the membranes of these vesicles are incorporated in oriented fields, then in whorls which become progressively smaller and are finally resorbed into the cytoplasm. The increase in cellular smooth reticulum may indicate the absence of juvenile hormone release which coincides with the physiological inactivity of corpora allata.
To adopt this point of view would be to admit implicitly the possibility that JH is stored in situ, which is plausible (Brousse-Gaury, 1971a,b,c, 1972; BrousseGaury et al., 1973; Brousse-Gaury and Cassier, 1975) but has not been observed. The hypertrophy of corpora allata caused by ovariectomy may indicate an accumulation of JH (King et al., 1966a,b).
THE CORPORA ALLATA OF INSECTS
57
The use of fine-analytical methods for the assay of cellular fractions, or immunocytological or radiographic techniques, should enable workers in the field to clarify these problems and to localize the JH and the function of the endoplasmic reticulum. The incorporation of tritiated farnesic acid in the form of methyl farnesoate and of C16JH (JH-111) into the corpora allata of Schistocerca adult females was studied by optical and electron microscopy using autoradiographic methods (Tobe et al., 1976). The granules of silver were almost entirely localized upon the surfaces of the smooth endoplasmic reticulum and in the intercellular spaces, which correlates with the rapid conversion of the precursor into JH.Participation of the Golgi apparatus and the mitochondria cannot be excluded; they were occasionally radioactively labeled. However, the use of methionine-methyl-l4C, in vitro demonstrated that there was no satisfactory correlation between the size of the corpora allata of Schistocerca and the level of their activity. It was also shown that the rate of secretion varied proportionally with the synthetic capacity, which excludes the possibility of an accumulation of JH under these conditions. The highest allata activity appears to coincide with the onset of the previtellogenic growth phase (slow growth) and not with the period of vitellogenesis vobe and Pratt, 1974,1975a,b; Pratt and Tobe, 1974). It seems therefore that the activity of corpora allata results from a modulation of synthesis rather than of secretion.
7. The Golgi Apparatus The secretory nature of the corpora allata has led researchers to focus their attention on the dictyosomes to determine if modification of these organelles is associated with the activity of the glandular cells. In general, this has not been the case. The dictyosomes are unobtrusive structures, simple masses of vesicles or aggregates of rare, short, flattened saccules and vesicles (600-1200 8, in diameter) with a smooth or coated wall (transition vesicles, multivesicular bodies); their appearance does not vary greatly (Scharrer, 1964a,b; Fain-Maurel and Cassier, 1969a,b; Thomsen and Thomsen, 1970; Panov and Bassurmanova, 1970; Dom, 1973; Brousse-Gaury et al., 1973; Browse-Gaury and Cassier, 1975; Elliot, 1976). In male adult Schistocerca sexual maturation is accompanied by growth of the dictyosomes, which is noticeable from the fifth day of imaginal life (Odhiambo, 1966b). In Celerio the Golgi apparatus is very active (Schultz, 1960). In Leucophaea (Scharrer, 1971) it participates in the elaboration of cribriform structures which have already been described; these structures may correspond to those reported in Folsomia (Paledovy and Grimal, 1975). In Rhodnius (Baehr et al., 1973) opaque granules (0.1 p m in diameter) were Seen associated with the Golgi apparatus, scattered in the cytoplasm, or even attached to the plasma
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membrane; although their number increased with the activity of the corpus allatum (nutrition, severing nerves, extirpation of the pars intercerebralis, ovariectomy), their function remains unknown. They may be hypothetically grouped with the primary lysosomes which participate in the degradation of structures implicated in the synthesis of JH. In Carausius morosus (P. Cassier, unpublished) the secretory activity of the allata cells is very noticeable but is irregularly distributed inside these vesicular organs. In certain cells it is manifested by hypertrophy of ergastoplasm and dictyosomes and the elaboration of numerous large granules composed of glycoproteins, which are released into the central cavity. 8. The Secretion The chemical nature of low-molecular-weight JHs and their solubility in organic solvents makes it impossible for them to be preserved and visualized at the ultrastructural level using conventional techniques. No morphological parameter can be used to localize the hormone in the allata cells or to determine exactly the mechanism of its release into the hemolymph (vesicles or clear vacuoles) (Ito, 1918; Mendes, 1948; Nayar, 1956a,b; Highnam, 1958; Elliot, 1976). It is reasonable to imagine that, during the phase of normal activity, JH is released by diffusion in proportion to its production; this has been experimentally demonstrated in vitro in Schistocerca (Tobe and Pratt, 1974). At the level of the light microscope a secretion product may be observed. According to Cazal (1948) the use of hematoxylin after osmium fixation clearly shows that the secretory product begins at the center of the gland and moved progressively outward toward the periphery. Hematoxylinophilic globules appear near the granular mitochondria and at the extremities or in the concavities of filamentous mitochondria. The secretion progressively fills the allata cells, becomes osmiophilic, and passes into the intercellular spaces or diffuses into the hemocoel. No ultrastructural observation has demonstrated the presence of secretion either free or in granules. The secretory process observed in Leucophaea (Scharrer, 1971) and Rhodnius (Busselet, 1969; Baehr et al., 1973) are negligible. However, one may assume that the description summarized by Cazal (1948) indicates an enrichment of cells in lipids, lipoproteins, lipofuchsins, and chromoproteins, which correspond to the lipids, reticular structures, lysosomes, and residual bodies observed with electron microscopy (cf. Pasteels and Deligne, 1965). The situation is quite different for vesiculated corpora allata (see Section V,A,4) possessing one or more central bodies. The cavity of the corpsjugaux of Thysanura (Chaudonneret, 1949) contains a hyaline secretion which weakly retains the light-green dye. In C. morosus the secretion is PAS-positive, lamellar, and composed of glycoproteins formed inside the Golgi apparatus and
THE CORPORA ALLATA OF INSECTS
59
ejected by exocytosis at the apex of the cells which are notable for their large number of microvilli. The presence of the lamellar structures described in light microscopy studies cannot be confirmed, but the central body contains remarkably structured bodies which are comparable to the cribriform inclusions described by Scharrer (1971). 9. Reserve Substances: Glycogen and Lipids a. Glycogen. It seems that the high glycogen content of corpora allata depends essentially upon the nutritional condition of the insect; it does not correlate well with the cycle of activity (Thomsen and Thomsen, 1970; Dorn, 1973; Scharrer, 1971). Melnikova and Panov (1975) found high glycogen levels in the allata cells of Hyphantria during nonactive periods, while during active periods these cells contained no glycogen. In Rhodnius (Busselet, 1969) no glycogen was found in fasting larvae, but it appeared after a meal. b. Lipids. One of the essential characteristics of the Calliphora corpus allatum (l’homsen and Thomsen, 1970) (Figs. 44-47) is the presence during the active periods of numerous spherical or round, dense, coalescent lipid droplets
FIGS.44-45. Corpus allatum of the female blowfly, C. eryrhrocephala Meigen. (From Thomsen and Thomsen, 1970.) FIG.44. Active corpus allatum of the sugar fly (glutaraldehyde,osmic acid-ethyl gallate). Numerous blackened lipid droplets.
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FIG.45. In corpus allatum profile of axon (A) containing neurosecretoly granules and mitochondria (M). EX, exocytosis of a granule. Another granule seems to adhere to the axolemma behind the first one.
which have no limiting membrane. These droplets, which are particularly abundant at the periphery of the gland, and are capable of invading certain cells. They may measure as much as 0.5 x 1.5 pm in diameter. During nonactive periods they are small and either rare or absent. Similar observations have been made in Hyalophora (Waku and Gilbert, 1964) where numerous lipid droplets were seen during the active period, but not in diapausing animals. In Bombyx (Fukuda et al., 1966) numerous lipid globules are characteristic of the active period and of females (see Section V,C). They have also been reported in Antheraea (Busselet, 1969) and Diatraea grandiosella (Yin and Chippendale, 1977). Except in these Holometabola the lipids are not much developed. In Acrididae the central part of concentric fields of endoplasmic reticulum are frequently occupied by a lipid droplet.
FIG. 46. Corpus allatum of the female blowfly, C . erythrocephala Meigen. Active corpus allatum of l-day-old sugar fly, showing numerous lipid droplets (L) scattered in the cytoplasm and one accumulation of large droplets (Ll). Note the irregular shape of the cells. A, axon; AO, aorta wall; H, hemocoel; N. nucleus. (From Thomsen and Thomsen, 1970.)
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FIG.47. Corpus allatum of the female blowfly, C. erythrocephala Meigen. Active corpus allatum. Aggregate of tubular agranular reticulum (AR) partly surrounded by membrane-bounded, electron-lucent vacuoles (V) and an axon (A) with neurosecretory granules, mitochondria, and neurotubules. (From Thomsen and Thomsen, 1970.)
THE CORPORA ALLATA OF INSECTS
63
Taking into account the lipid solubility of the JH one may consider, as did Thomsen and Thomsen (1970), that lipid globules represent sites of accumulation of JH or may be a means of transport and release for these substances. In the case of Hyphandria (Melnikova and Panov, 1975) this interpretation is only partially valid, because the lipid droplets appear in large numbers only at the end of the last larval stage which is the nonactive period. 10. Microtubules Microtubules (250-300 A in diameter) are abundant in allata cells; in general, they are randomly distributed, without any preferential orientation (Busselet, 1969; Thomsen and Thomsen, 1970; Tombes and Smith, 1970; Scharrer, 1971; Deleurance and Charpin, 1972; Dorn, 1973;,Baehr et al., 1973; Brousse-Gaury et al., 1973; Girardie and Granier, 1974a;Morohoshi et al., 1976a,b), except at the level of the cytoplasmic peripheral infolding (Odhiambo, 1966b,c; Guelin and Darjo, 1974). In the fusiform allata cells of Folsornia (Palevody and Grimal, 1975; Palevody, 1976), the microtubules are arranged parallel to the major axis of the cells. These two last-mentioned findings corroborate the cytoskeletal function of microtubules. 11. The Lytic Structures Independently of the lamellar formations which participate, by pseudomyelinization at the end of each active cycle, in the elimination of reticulum involved in JH synthesis, the allata cells, like any active cells, contain lytic structures (primary lysosomes, cytosegresomes or autophagic vacuoles, multivesicular bodies, residual bodies). Lysosomes (dense bodies) are frequently found in Schisrocerca (Odhiambo, 1966 ), Calliphora (Thomsen and Thomsen, 1970), Leucophaea (Scharrer, 1964 ), Drosophila (King et al., 1966a,b), Aphis (Elliot, 1976), and Hypera (Tombes and Smith, 1970). In Oncopeltus Dorn (1973) found that they were particularly frequent after the imaginal molt and in senescent adults. Similar results were found during the active period in Hyalophora (Waku and Gilbert, 1964).
The multivesicular bodies which have classic structures have been studied in particular in Rhodnius (Busselet, 1969; Baehr er al., 1973) and in Spheophyes lucidulus (Deleurance and Charpin, 1971).
C. NEUROSECRETORY FIBERS
The presence of neurosecretion and neurosecretory fibers in the corpora allata has been established many times using light microscopy (see Cassier, 1967 for references). Electron microscope studies have confirmed these results which
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have been found in all cases examined. This observation is important on the functional level, since an allatotropic neurosecretory activity having been demonstrated (Girardie, 1967a; Baehr et al., 1973). The neurosecretory fibers penetrate the corpora allata through one or several hila and then ramify at the center of the glandular parenchyma and rapidly lose their glial sheaths. The naked, dilated endings are characterized by the presence of neurosecretory granules free or attached to the plasma membrane, synaptic vesicles (350-600 A) isolated or grouped in the synaptic areas, pinocytic vesicles, and numerous mitochondria. The process of pinocytosis has been observed by several investigators (Cassier and Fain-Maurel, 1970; Busselet, 1969; Brousse-Gaury et al., 1973; Girardie and Granier, 1974a,b; Thomsen and Thomsen, 1970; Fukuda et al., 1966). It seems that only one type of granule is present in Anacridium (1000-2000 A in diameter) (Guelin and Darjo, 1974), Oncopeltus fasciatus (Unnithan et al., 1971), and B. mori (750-2000 (Fukuda et al., 1966; Morohoshi et al., 1976a,b)(wherethe fibers may be distinguished by the presence or absence of the grains ?), Troglodromus and Cytodromus dapsoides (900 (Deleurance and Charpin, 1972; Deleurance, 1967), Hyalophora (500-1500 (Waku and Gilbert, 1964), and Calliphora (1100-2000 (Thomsen and Thomsen, 1970) p i g . 47). It appears that two, three, or more types of granules of variable diameter and density exist in Hyphantria (Melnikova and Panov, 1975), A . craccivora (Elliot, 1976), Celerio (lOOO-3OOO A) (Schultz, 1960), Schistocerca (800-1850 A)(Odhiambo, 1966b), Rhodnius (Busselet, 1969), Oncopeltus @om, 1973), and Blabera (Browse-Gaury et al., 1973; Brousse-Gaury and Cassier, 1975). In Locusta (Fain-Maurel and Cassier, 1970; Cassier and FainMaurel, 1970) all the parameters were taken into consideration, and four fiber types have been identified, three of which are also present in the internal (NCC.1) and external (NCC.11) paracardiac nerves, the corpora cardiaca, and the allatocardiac nerves (NCA.1). Type-l fibers contain dense granules which have a maximal diameter of 2500 hi; type-2 fibers have dense granules of diameter 3000 bi and gray granules with a diameter of 3000-3500 A; type-3 fibers contain dense granules of diameter 5000-8000 bi. The fourth category (type 4), found only in the corpora allata, contains a few dense granules (2500-3000 in diameter) and bright, spherical or ovoid vesicles comparable to the “empty neurosecretory granules” described by Odhiambo (1966b) in Schistocerca. The last-mentioned type may derive from type 1. This description is in good agreement with that of M. Cazal (1971). From the examination of these data it seems obvious that the microgranular neurosecretory granules (1000-2500 bi in diameter) or the fibers containing this type of granule are well-represented in insect corpora allata. It is possible that they are aminergic in nature and are involved in the inhibitory or stimulatory mechanisms which regulate the activity of the corpora allata. Indeed, in Locustu,
A)
A)
A) A)
THE CORPORA ALLATA OF INSECTS
65
in the corpora cardiaca which contain the same types of fibers as the corpora allata, Lafon-Cazal (1976; Lafon-Cazal and Arluison, 1976), using a technique of induced fluorescence (according to Falck and Owman, 1965) and highresolution autoradiography, demonstrated the presence of indolamines (serotonin) and catecholamines in type-1 fibers, and dopamine in types 1 and 2. A variation in the number of granules and synaptoid areas in relation to the physiological conditions of the insect has been reported, but the findings are not very convincing and, at the ultrastructural level, quantitative variations should be considered with caution. Common nerve fibers which have no neurosecretory-type dense granules are associated with neurosecretory fibers. With certain exceptions the integrity of the innervation of the corpora allata is indispensable for the maintenance and regulation of their activity, as well as for their postimaginal growth. Thus in Schistocerca, Highnam et al. (1963), Pener (1965, 1967), and Strong and Amerasinghe (1977) found that implanted corpora allata were incapable of inducing sexual receptivity and ovarian development in allectomized females. In L . migratoria migratorioides restoration of these faculties required either a massive implantation (Albrecht and Cassier, 1964) or repeated implantations (Cassier, 1964a,b, 1965a,b, 1967; Cassier and Papillon, 1968). A similar situation exists in Locusta migratoria cinerascens (Girardie, 1967b). All these operations carried out in normal females give rise to temporary aplasia of the corpora allata. Conversely, the ablation of a corpus allatum induced compensating hypertrophy of the symmetrical gland (Cassier, 1965a,b, 1966a,b,c). Similar results have been reported by Quo Fu (1965), Muller (1965a), and Johansson (1958). In vitro, in the presence of a radioactive precursor (methi~nine=rnethyl-'~C) of JH-I11 (C-16) (Pratt and Tobe, 1974) the activity of the corpora allata of adult females is transient, being 5 hours in Diploptera punctata (Tobe and Stay, 1977) and only 3 hours in Periplaneta americana (Pratt et al., 1975).
VII. Conclusions
In spite of extensive studies on the structure and function of the corpora allata of numerous insects, no clear relationship between morphological characters and the production of JH has been revealed. Therefore studies using cellular fractions would now be useful. Thus the biosynthesis of JH-III from exogenic precursors was carried out in the presence of a corpora allata homogenate of Manduca sexta (l0,OOO x g supernatant) (Reibstein et al., 1976). Hammock et al. (1975) has also succeeded in JH-111 synthesis in the presence of a corpora allata microsomal fraction of Blaberus giganteus, at least at the final stage of epoxidation.
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INTERNATIONALREVIEW OF CYTOLOGY,VOL.57
Kinetic Analysis of Cellular Populations by Means of the Quantitative Radioautography J.-C. BISCONTE Laboratoire de Neurobiologie Quantitative, Centre Hospitalier et Universitaire de Bobigny, Universite' de Paris X l l l , France
I. Introduction . . . . . . . . . . . . . . . . . . . . 11. Quantitative Radioautography . . . . . . . . . . . . . A. Histological and Radioautographic Processes . . . . . . . B. Observation and Measurement . . . . . . . . . . . . 111. Kinetics of Cell Proliferation . . . . . . . . . . . . . . A. Precursors . . . . . . . . . . . . . . . . . . . B. TheCellCycle . . . . . . . . . . . . . . . . . C. Applications . . . . . . . . . . . . . . . . . . IV. Migration and Chronoarchitectony . . . . . . . . . . . . V. Concluding Remarks . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .
75 77 77 83 92 92 96 105 112 1 18 1 18
I. Introduction Segmentation in frog ova was first described by Rusconi in 1826, but it was not until 1875, with Mayzel's observations, that the descriptive bases of cell proliferation were firmly established. Not long after, numerous studies resulted in the systematization of mitosis and its division into four main stages: prophase, metaphase, anaphase, and telophase. The possibility of a tissue increasing, differentiating, or decreasing depends to a large extent on the ability of its cells to proliferate. Generally, three types of tissues are discerned according to proliferative ability: (1) tissues that proliferate continuously, (2) tissues whose cells do not proliferate, and (3) tissues whose cells are usually nonproliferating but can begin mitosis under the action of a specific factor. The kinetic characteristics of a tissue can be determined according to these three types by calculating the ratio of the number of cells in which mitoses are seen to the total number of cells (the mitotic index). In 1951, a new method was employed by Howard and Pelc to describe more precisely the steps separating 75 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form resewed ISBN 0-12-364357-0
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two successive mitoses. Using 32P as a marker, they showed that DNA was synthesized only during a relatively short interphase period preceding mitosis. This work corroborated Swift’s observations (1950) based upon cytophotometrical measurements obtained by the Feulgen method. At this time, DNA was proposed as the support for genetic material (Hershey and Chase, 1952; Watson and Crick, 1953). In 1953, Howard and Pelc separated into four stages the interval between the division of the mother cell and that of the daughter cells: (1) the pre-DNA synthetic stage (GI), (2) the period of DNA replication (S), (3) the post-DNA synthetic and premitotic phase (G2),and (4) mitosis (M). The best method for determining the number of cells in these different phases, and even measuring these phases, is based on the use of DNA radioactive precursors. Although different molecules can be employed (thymidine, thymine, deoxyuridine) combined with various isotopes CH, 14C, IZ5I), the most commonly employed combination is thymidine labeled with tritium (HTdR). The total radioactivity can be detected by counting in liquid scintillation or, at the cellular level, with light or electron microscopy, using a sensitive emulsion; this is the radioautographic technique, first applied to biological specimens by Lacassagne and Lattks in 1924, and improved by Belanger and Leblond in 1946 by the use of liquid emulsions. The ability to detect cells that have incorporated a radioactive marker into their DNA provides applications that largely exceed the problem of the cell cycle. Thus cells that lose their ability to divide, such as neurons, can be labeled in an indelible way if the radioactive precursor is provided during the S phase that precedes their last division. It is then possible to follow these cells during their migration and to locate them at their final site. Therefore it is possible to correlate very closely the architectonicsof a tissue such as that comprising the central nervous system (CNS) with the time of the last division of its cells; this is called chronoarchitectony (Bisconte, 1973). In practice, the radioautographic technique adds a dimension hardly accessible with classic histology: the time dimension (Leblond, 1965). M a n y other methods compete with radioautography in applications to cell kinetics. This is true in the case of cytofluorometric measurements on a slide or in a flux; these measurements allow one to determine immediately the amount of DNA in a cell or in a cell population. Recently, fluorescent markers that can be bound to DNA have been envisaged for use instead of radioactive thymidine; these components should be more rapidly detectable because of lack of usefulness of the radioautographic exposure. It is possible that such approaches will make the radioautographic technique useless, but perhaps the multiplication of techniques will allow specialization and combination among them. It is therefore necessary to have a comprehensive knowledge of the radioautographic method as applied to kinetics, especially when considered from a quantitative viewpoint.
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11. Quantitative Radioautography A. HISTOLOGICAL AND RADIOAUTOGRAPHIC PROCESSES Radioautography or autoradiography is a method which enables one to study the distribution of radioactive products, making use of their ability to affect photographic emulsions. In biology, the data are qualitative, as they show the localization of the incorporation, but they can be quantitative if they permit a correlation between the number of grains revealed and the quantity of radioactive precursor absorbed. Another quantitative approach allows the study of the kinetics of the biological events by using radioautographs which differ in (1) time of precursor injection, (2) duration of this precursor disponibility, (3) in vivo, the date of sacrifice, or (4) any combination of these three parameters. This shows, according to Leblond (1963, that “radioautography gives histology a fourth dimension, time. The usual isotopes, I4C and particularly 3H, are p- particle emitters whose spectra are not monoenergetic. For 3H, Em,, is 0.018 MeV with a mean value of 0.006 MeV. In the case of I4C, Em,, is 0.155 MeV, with a mean value of 0.05 MeV. The radioactivity half-life of these isotopes is so long compared to the duration of a cell kinetics experiment that a correction for this phenomenon is not necessary. Electrons crossing a silver bromide crystal weaken in the internal bonding and can even break it; this is the way latent images form. This subject is discussed in specialized publications (Mott and Gurney, 1950; Mees and James, 1970). After exposure to ionizing radiations, the sensitive layer is developed in order to increase the size of the latent images or to transform them into metallic silver. The latent images change with time, mainly by gradually disappearing (Rogers, 1973); however, if the exposure is less than 2 months, this fading phenomenon is negligible. The photographic emulsion is a detector which converts radioactive events into grains. Like any detector, it is not only sensitive to radioactivity but also to light, shocks, and chemical substances (see Section II,B,6), and it has its own noise-causing undesirable grains. All these phenomena lead to a background which, in radioautography with 3H p particles, must not exceed 3 grains/100 pm2. It is in fact the value of the signal/background ratio which must be considered, and the experimenter should try to decrease the background value and to increase the detection yield so that this ratio is as high as possible. The main purpose in quantitative radioautography, especially when applied to cell kinetics, is to obtain radioautographs that are comparable from one experiment to another. This implies (1) maximum standardization of the radioautog”
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raphic process, (2) knowledge of the factors which may modify the yield, and (3) determination of the cause of artifacts. All these parameters involved have been discussed in the literature, especially in Kogers’ work (1973). Their appelations follow, and the most important ones for the radioautographic technique are discussed. 1. Fixatives
DNA is easily rendered insoluble by a range of fixatives. However, the choice of fixative depends on its nonextractive properties and also on its ability to preserve of tissue. For many tissues, an acidified or neutral alcohol which allows contrasted nuclear images after staining is convenient. In nerve tissue many aldehyde fixatives are commonly used, such as acrolein, Formalin, and glutaraldehyde. Vanha-Perttula and Grimley (1970) showed that radioisotope losses were influenced not only by fixatives but also by buffer washes. Formadelhyde induces greater extraction than glutaraldehyde.
2. Section Thickness The choice of an embedding material is not critical for radioautography, whereas the section thickness is very critical in quantitative studies. When this parameter varies, the grain count may change. With HTdR the count increases when the section thickness (glycol methacrylate) vanes from 0.5 to 2.5 pm (Sidman , 1970). When the section is more than 2.5 pm thick, the strength of the 3H p- particles does not allow recording of the disintegration for all molecules situated more than 2.5 p m below the section surface; for example, in quantitative radioautography the correction for the grain count is minimal for a section thickness of 2.5 p m or more (Caviness and Barkley, 1971). 3. Staining Staining can be performed prior to application of the emulsion (cytochemistry, Feulgen, reduced-silver methods) or after development of the emulsion (cresyl violet, toluidine blue, Sudan dyes, hematoxylin-eosin). Staining before the radioautographic process sometimes requires interposing a layer between the tissue and emulsion to avoid artifacts (Benastad, 1977). In other cases these treatments may eliminate part of the radioactivity, especially when Feulgen staining is used (Bryant, 1969). 4. Emulsions
In radioautography there are three main types of emulsions: (1) stripping film emulsions (e.g., Kodak AR lo), which are reinforced with gelatin and can then be removed from their glass support and placed on the section; they are delicate
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to use but offer the advantage of a standardized sensitive layer; (2) emulsions on a plastic (Kodirex)or glass support, which can be applied to the section and, after exposure, separated for development; these are the easiest to use, but their sensitivity is low and, most of all, the recombination between the radioautographic image and the tissue is not precise; (3) liquid emulsions (e.g.. Ilford K2 and Kodak NTB2), which are easy to use and allow one to treat a great number of samples; to a certain extent, the dilution and the drying conditions permit one to adjust the thickness of the layer; depending on their type, these emulsions have different grain sizes and are more or less adapted to a certain range of particle energy.
5 . Physical Surroundings and Coating The emulsion is laid on the biological specimen in a darkroom or in inactinic light. The temperature and humidity conditions are important when the emulsion is laid on the slide either by gravity or by centrifugation (Bisconte, 1974). In our laboratory the physical environment is 28°C and 80% relative humidity, but it is very important to obtain reproducible conditions from one experiment to another. When developing, the room should be at a temperature close to the one necessary for the baths (e.g., 18°C). In the literature, many different methods have been proposed for covering the sample with the emulsion: the use of a roller or a paint brush, a dropper, or platinum loop (Car0 and Van Tubergen, 1962), or by dipping (Belanger and Leblond, 1946; Joftes and Warren, 1955; Kopriwa and Leblond, 1962). Dipping is the usual procedure, especially in light microscopy. One of the problems is thickness regularity which is obtained only under standardized conditions (see above). Many devices have been developed for dipping the slides in a controllable manner (Kopriwa, 1967), or for treating them at the same time (Coleman, 1965; Traurig, 1967; Eide, 1968;McGuffee et al., 1977). With these procedures, the emulsion flows down because of gravity, or a centrifugal system is used. In the last-mentioned method, the histological slides are placed in groups of 4 in plastic clamps (Fig. 1). After immersion in a radioautographic liquid emulsion in a darkroom, the slides are centrifuged, 16 at a time, in the 4 compartmentsof a centrifuge. The slides, still in groups of 4, in the clamps are placed in individual containers, dried by nitrogen gas, and exposed at 6°C. The developing, fixing, and staining are carried out in holders, which allows simultaneous processing of 24 slides. Photometric analysis of the density of the emulsion applied to the specimen slides shows that the spread obtained is uniform. In this respect the method complies with the requirements of quantitative radioautography. Its main advantages over traditional methods are speed of treatment (200 slides completely processed per hour) and improved homogeneity during the developing process.
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Centrifugation
ma-]
Drying by Nitrogen
=I3*
FIG. 1. Sequence of radioautographic operations. The histological slides are placed in plastic clamps, immersed in a liquid emulsion in a darkroom, and then centrifuged. The final drying is done in containers filled with nitrogen gas. Exposure takes place in airtight containers in the presence of dry Drierite. Developing, fixing, and rinsing are carried out in holders, thus allowing grouped processing of the slides.
6 . Development Whether the radioautographic analysis is automatic or not, it has become more and more necessary to make quantitative evaluations. Such procedures require that, under identical experimental conditions, scattering of the radioautographic measurements as a result of technical factors be minimal. This means that unavoidable variations in these factors should have little influence on (1) the number of silver grains developed, (2) their size, (3) their reflectivity, and (4) their distribution. The most important factor is certainly the developer. Its chemical action is to reveal a signal (grains exposed to radioactivity) as well as a noise (nonexposed grains). The best ones must (1) develop all grains which have been irradiated, (2) give the best signalhoke ratio, and (3) be the least sensitive to temperature differences and time of action, and conform to the four conditions noted above. For an emulsion such as Ilford LA, the best developer is Dektol, because it gives
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an identical response between 2 and 4 minutes without any background. It also provides the best development yield. Kodak D19 offers an equivalent yield, but the safety margin is lower than 1 minute (Neely and Combs, 1976). Other developers, such as Microdol-X, do not show a good efficiency, 50% less than the others, but give a constant response between 5 and 15 minutes after application. In this case errors due to development are reduced, but such developers need radioautographs containing a lot of information. Developers do not supply exactly the same yield with emulsions such as Ilford LA and Kodak NTB2, as shown by Neely and Combs (1976). Their experiments also prove that the mean size of the grains, as well as the distribution about this mean value, changes with the developer. From this point of view, when using Ilford LA emulsion, Acufme and Rodinal are the best developers. Microdol-X is not recommended because the grains become too small. Electron microscope examination of nondeveloped emulsions confirms that the problem of scattering of grain sizes is not due to an important range of size of nondeveloped grains but is linked to the fact that grains are generally formed by twisted filaments which are variable in size and in shape (James and Mees, 1966; Kopriwa, 1967; Salpeter and Bachmann, 1964; Rogers, 1972). The measurable size of these grains depends upon the type of measurement, reflection or transmission (Neely and Combs, 1976). In electron microscopy, the large size and the irregular shape of the silver grains are important disadvantages. In 1966, Lettri and Paweletz proposed development with Phenidone which gives very regular and compact silver grains but unfortunately with a low efficiency, about 4%, which is half that obtained with Kodak D19 (Bouteille et al., 1976). Elon ascorbic acid development is also used, and in this case gold latensification (James, 1948) gives an acceptable yield (Wisse and Tates, 1968). In the study of Neely and Combs the slides were exposed to visible light. However, even if it has been proved that the sensitivity of emulsions to X rays and to visible photons is identical (James and Mees, 1966), it is certain that the action of electrons emitted by 3Hor 14C is very different (Rechenmann and Wittendorp, 1976). The spectrum of the energy released by these electrons is very wide and, for this reason, it can lead to a scattering of the results by creating latent images of different values. Many signal and background curves have been published by many investigators for different emulsions and developers; this makes them hardly comparable, and spurs every experimenter to verify them under his or her own experimental conditions (Rogers, 1972; Rechenmann and Wittendorp, 1976). It is very frequently observed that, in numerous cases, the response of the emulsion to a given developer does not show the plateau indicating a constant response within a safety interval for the development; under these conditions, it is difficult to find a quantitative study. Nevertheless, the background can be increased in the same proportions, and then the important factor is the signdnoise ratio. If this ratio remains constant, the quantitative conditions
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will be fulfilled if the background measurement is subtracted from each signal measurement. Some investigators thought that the developer might have a double effect, for example, transformation of the latent image by the reduction of silver bromide grains but also the destruction by oxidization of certain not very active centers of development (Reinders, 1934; Mees and James, 1970). For this reason and, from a more general point of view, to increase the yield of the development, Rechenm2nn et al. (1969) suggested a procedure called “activation, applicable to nuclear emulsions, which is different from methods based on temperature (Fig. 2). Since 1931, numerous studies have attempted to increase the sensitivity of the photodetector (Crabtree and Muehler, 1931; Sheppard et al., 1945), and gold salts, mercury vapors, and other substances have been used. With nuclear emulsions triethanolamine is often employed (Demers, 1958). Rechenmann and Wittendorp have recently proposed a procedure which is applicable to most emulsions (Rechenmann, 1970; Rechenmann and Wittendorp, 1972; Wittendorp, 1973). Its action induces neither background nor change in the size of grains. On the contrary, the great increase in the yield allows one to reduce the time of exposure and the doses of radioactivity. ”
a)
-activation b)
I
C
FIG.2. Schematic representation of a transverse section through the emulsion layer over a histological specimen. (a) Trajectory of a p particle. Latent image in the microcrystals before (b) and after (c) activation. (d and e ) Result of development in each case. (Redrawn from Rechenmann and Wittendorp, 1976.)
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B. OBSERVATION AND MEASUREMENT I . Observation with Light Microscopy Light microscopy allows three main types of observation: observations in transmitted light, which are simple but hardly applicable to quantification, those in incident light, and those in mixed light. Very often, the grain density of the radioautographs, is measured with a microphotometer. This is a very sensitive machine whose detection ability is not as limited as that of the human eye which makes unacceptable errors as soon as the density exceeds 35 grains/100 p m 2 (Rogers, 1973). For photometric measurements in transmitted light, it is absolutely necessary that the density read be due only to grains; thus this type of measurement requires the subjacent tissue to be transparent, eliminating radioautographs whose sections are stained. The incident light uses the reflective and relatively selective characteristics of the silver grains (Gullberg, 1957; Rogers, 1961). In this case, the grains appear bright on a dark background. The properties of observation against a dark background enable one to use thinner grains (resulting in improved resolution, better detection of cytological details, and delayed saturation of the emulsion). This process does not totally eliminate the influence of interfering sources such as light diffused by the tissue or reflected by grains outside the measurement field (Dormer et al., 1966). Moreover, the major disadvantage attendant upon this method is the need, for each measurement, to alternate between an incident light (dark field) observation necessary for photometric measurement and a mixed light observation which alone allows one to determine the correlation between the grains and the labeled material. This problem has been solved by spectral lighting based upon a combination of filters permitting continuous observation of the preparation in transmitted light and, simultaneously, selective measurement of the silver grains (Bisconte, 1973; Bisconte and Marty, 1974). The preparation is illuminated by yellow transmitted light, and a blue filter is placed in the incident beam.The sensitivity range of the photomultiplier when a blue spectral filter is interposed in front of it is then limited to the light reflected by the grains, whatever the intensity of the transmitted light. Consequently the observer perceives both silver grains, appearing in blue, and cells whose cytological details are well brought out by the yellow light; at the same time the microphotometer, and the galvanometer associated with it, continuously measure the light reflected by the labeled cells which are at the center of the field. The filters must be chosen taking the following three factors into account: the ratio of the light reflected by the grains to the light diffused by the tissue must be as high as possible; the photometer must selectively measure the light reflected by the grains and must not be sensitive to transmitted light; the observer should discern both silver grains and subjacent cytological details (Bisconte and Marty, 1974). On Lei& microscopes, different sets of objectives are used for metallography (Opak-Ultropak),
84
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and an illuminator and objectives (Opak-Autoradiography) specific for radioautography are now available.
2. Grain Measurement All the atoms of a radioactive molecule have the same probability of undergoing decay within a given time. It is well known that disintegrations occur conforming to the statistical Poisson distribution in which the variance is approximately equal to the mean. In radioautography the grain count over a series of identical radioactive sources must show a Poisson distribution. In many cases, in cell kinetics, after HdTR labeling the nuclei may be considered similar sources. This hypothesis has been tested by Car0 (1961), Campbell et al. (1967), England and Rogers (1970), and Bisconte and Marty (1975a). Their results agree with this statistical distribution, but in some cases it was necessary to make corrections because of cell diameter variations. However, some results have shown “unpredictable” distributions, which are probably due to technical considerations (such as radioautography or measurement) and/or to biological variations in the sources (Chemick and Evans, 1968; England and Rogers, 1970). The silver grain count of radioautographs can be made either indirectly from a photograph or directly under the microscope. Generally, it is possible to count labeled cells by eye with the help of an integrative ocular or of a tracing device, but in cell kinetics it is practically impossible to determine in this way the quantity of grains over each cell. Thus different electronic systems are used to solve this problem. Two types of detectors are now mainly employed: a photometer (see above) and a television camera. The photometer is connected to an analog or a digital recorder (Fig. 3A). It is possible to measure the cells by surrounding the labeled nucleus with a measurement diaphragm. With this rather long procedure, it is necessary to take into account (1) the diffused light and (2) the error introduced by the nuclear size differences which require either the adjustment of the diaphragm to each nucleus or the choice of a sufficiently wide diaphragm aperture so that a nucleus of any size can enter it. In both cases, corrections are necessary (Dormer et al., 1966; Bisconte and Marty, 1975a, 1975b). Another way of using the photomultiplier is to close the diaphragm up to a 1-pm diameter and to analyze the preparation step by step, either displacing the section by means of a motor-driven x-y stage, or moving the diaphragm. The theory of scanning photometry is due to Mertz and Gray (1934) and has been applied to DNA cytometry (Caspersson, 1936, 1950; Sandritter and Kiefer, 1965) and to radioautographs as well (Vendrely, 1971; Gray, 1974; Rogers, 1974; Entingh, 1974; Brugal, 1976). The Nipkow disk which is sometimes used (Brugal, 1976) scans the preparation in front of the photomultiplier window with a 4 - w frequency. ~ This measurement frequency is much higher than that obtained by a motor-driven step-by-step scanning stage (100-200 Hz). Recently,
KINETIC ANALYSIS OF CELLULAR POPULATIONS
85
FIG. 3. (a) Microphotometric system. R, xy recorder; L, argon laser; M, microphotometric module MPV 2. (b) Texture analysis system. C, Control module; T, television camera and microscope; I, individual logic; E, electronics.
this system has been improved, and the frequency can exceed 5 kHz for a one-axis scanning. However, this scanning of a field requires alternate displacements causing a slowdown, and the efficient frequency is much lower. In both cases, a moving stage or a moving diaphragm, the important frequency of measurements requires the use of a computer to pilot the scanning and to process the data. Generally, these data are expressed in the form of histograms (Fig. 4) and/or graphics, for instance, maps of grain density per cell. The considerable advantage of the photomultiplier is its rapidity and, above all, its sensitivity which enables a real discrimination between at least 100 gray levels in the image. The major disadvantage is that it detects only one field, and so the analysis requires a relative displacement of the image and of the diaphragm, which is rather slow with the electromechanics now used. The combination of grain counting with DNA cytofluorometry provides additional data on the quantitative cytochemistry of DNA. Recently, improvement
86
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0
50
100 Photometric Values
FIG.4. Microphotometric analysis of a labeled cell. A, No label; B, very slight density corresponding to surfaces either lightly labeled or illuminated by light diffused by neighboring grains; C, majority corresponding to the labeled cell nucleus.
of the microphotometric technique has allowed simultaneous measurement of DNA and grain counting (Fujita et al., 1974). Applications are numerous, for example, in vitro, when it is necessary to determine cell cycle parameters without disturbing the culture. Microphotometric measurement of the DNA content in an individual cell supplemented by radioautographic counting and cell size determination fulfills this purpose (Kimball et al., 1971). More generally, it is easy to use a microphotometer to combine grain counting and fluorometry of different substances such as fluorescent antibodies (Karb and Goldstein, 1971). Image detection by a television camera is much quicker, but unfortunately the number of gray levels separated is only 20 to 30. In radioautography this disadvantage is not very great, because the contrast between the grains and the tissue is important with incident light (Fig. 3b). In television microscopy the video system can use any of several different camera tubes, generally a vidicon or a plumbi-
KINETIC ANALYSIS OF CELLULAR POPULATIONS
87
con. With high numerical apertures and powerful objectives, the resolution is about 0.5 p m (Dyer, 1973); but under these conditions, light and contrast may be insufficient, and the experimenter has to obtain good stains and appropriate illumination to provide the best yield @chardson, 1971). This type of measurement has been achieved using image analysis systems such as Quantimet or Leitz T.A.S which record the image through a camera (Polig, 1975; Prensky, 1971). According to whether the magnification is high or low, grains can be counted separately @article counting; see Fig. 5 ) or brightness levels evaluated (microdensitometric study; see Fig. 6). At high magnification, in automatic analysis there is a problem of keeping the focus when the slide is displaced. There are autofocusing devices based on the detection of maximum contrast, but they cannot be easily used for radioautographic preparations whose grains are located at different levels above the uppermost plane of the histological section. There are also devices included in none of these categories, which are based on photodiode sensors (Krekule ef al., 1978) or photodiode matrices (Le G6 and C. Favier, unpublished, 1978). In this case, analysis of the data requires a computer or a microprocessor. Generally, the study of cell kinetics by quantitative radioautography employs light microscopy, but sometimes electron microscopy is necessary-scanning
FIG. 5 . Labeled neurons after image analysis. The silver grains are selected, and their number, size, and distribution can be computed.
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0
10
20
30
40
50
60
70
Gray values
FIG.6 . Microdensitometric analysis of labeled fields by the use of the T.A.S. The values of the reflected light (gray values) are different for highly labeled fields (C), slightly labeled areas (B), and unlabeled fields (A).
electron microscopy (SEM) or in transmission electron microscopy (TEM). In SEM (Fig. 7), the grains inside the gelatin matrix are identifiable (Hodges and Muir, 1974), and the use of simultaneous x-ray spectroscopy allows both the identification and measurement of the silver found in an area (Hodges and Muir, 1975). This methodological approach is possible with ' E M and can provide an automatic quantitative analysis (Lebiedzik et al., 1973). Usually, in TEiM the analysis of the radioautographs is made on micrographs, and the counting is manual [see the review on quantitative evaluations by Bachmann and Salpeter (1965)l with or without the help of stereological grids (Weibel, 1969; Schmid and Rohr, 1976). However, new devices are a great help in this work. They generally involve digitizer tablets on which the micrographs are laid; with an electronic pencil the outlines of organelles and silver grains can be recorded. Then a microcomputer or microprocessor processes these data according either to the algorithms of the experimenter or to stereological programs loaded in memory (Exner, 1978). In some cases, the topographic distribution of the labeled cells inside a tissue provides valuable data, such as for CNS tissue which is characterized by a nonrandom distribution of the cells according to their last division age. Light microscopy does not enable one to solve the problem of detecting and mapping labeled cells. We have proposed that the displacement transducers be connected to the microscope stage (Bisconte et al., 1968). Coordinates can be semiautomat-
KINETIC ANALYSIS OF CELLULAR POPULATIONS
89
ically replotted by an xy recorder. Tracing by the plotter can be driven by a signal from the microphotometer. Numerous applications can be made in light microscopy, especially when microscopic details must be located and mapped to reflect their positions inside the outlines of tissue sections (Fig. 8), for example, fluorescence, degeneration, and axonal flow radioautography (Bisconte and Marty, 1974). 3. Autoabsorption
Generally, the microcompartment which contains the radioactive material is heterogeneous and the trajectory length of the p- particles varies greatly in relation to the density of cell components. Maurer and Primbsch (1964) calculated that “infinite thickness” for tritium was 0.05 gm/cm2,for example, 0.5 p m through the nucleolus, 2 p m through the cytoplasm, and 3 p m through the nucleoplasm. The consequences of this heterogeneity were studied by Pelc and Welton (1967), who reported that about six times more p- particles emerged toward the emulsion from a 3Hnuclei smear as from 4-pm sections of the same nuclei. 4. Saturation, Coincidence, and Overlapping The sensitive emulsion is a detector which converts and integrates radioactive events. So its capacity for reacting is not infinite, and it can become saturated.
FIG.7 . Radioautography of brain cells in primary culture. The same field has been observed under incident light (a) and scanning electron microscopy (b). The silver grains (arrow) can be recognized in both cases. (By courtesy of C. Derbin.)
90
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FIG. 8. Distribution of labeled neurons in normal (a) and in reeler mutant (b) mouse brain. These two animals came from the same mother which was injected with HTdR on the fifteenth day of gestation. The reeler neocortical chronoarchitectony is inverted (arrows). (By courtesy of M. Courtois.)
The curve representing the number of grains which can be developed beyond a determined point versus the number of particles emitted at this point grbws linearly at the beginning and tends afterward to a plateau. This result depends mainly on the isotope and on the nature and thickness of the emulsion. For a 2-pm-thick Ilford K2 emulsion, a good response range on a cell nucleus is 3 to approximately 100 grains/100 p m 2 . Another phenomenon is the coincidence @ormer and Brinkmann, 1970) which can be explained by the successive action of particles on a latent image already created. Finally, with microphotometric and even visual counting, there is also a loss of information when the grains are superimposed and mask each other. These three phenomena act in the same direction (a loss of yield) and require corrections when the quantitation is made on cells whose radioautographic labeling is very different.
KINETIC ANALYSIS OF CELLULAR POPULATIONS
91
5 . Resolution This problem has been extensively studied, in theory and in practice, by Car0 (1966) and Rogers (1973). It should be remembered that, if the radioisotope molecules are not displaced, they can be localized in light microscopy to about 2 p m in the case of 14C(Robertson et al., 1959) and to about 0.3 pm with 3H (Hill, 1962). With 3H, in electron microscopy, a resolution to about 0.1 p m is attainable (Bachmann and Salpeter, 1965; Caro, 1966).
6. Artifacts In any experiment based on radioautography the question is whether or not the silver grains really correspond to the radioactivity to be detected. A certain number of factors may give rise to an unwanted photographic reaction. Cosmic radiations could create artifacts, but in practice their effects may be negligible with the usual emulsions and the classic exposures (Rogers, 1973). In electron microscopy, uranyl may cause traces which are easily identifiable (Droz, 1976). Artifacts are frequently observed when the emulsion coats an uneven surface. Thus in the depressions found in a deparaffined tissue section, for example, grains may accumulate. This reflects both the excessive thickness of the emulsion and the mechanical strains related to these thickness irregularities. More generally, edge artifacts can often be observed along the borders of sections, ventricles, vessels or over cell aggregates in culture. Sawicki and Pawinska (1965) demonstrated that this background increased after fast drying. It is caused by the rapid shrinkage of a wet emulsion, which results in an increase in the intraemulsion pressure. These artifacts can be avoided by careful and gradual radioautographic processing, particularly in the drying stage (Sawicki and Pawinska, 1965; Boyenval and Fischer, 1976). These mechanical artifacts, which include unexpected contacts with the emulsion, are easily identifiable, whereas the chemographic artifacts are more insidious. They are produced by the action, on the emulsion, of still chemically active substances which are contained in tissue sections. They result in an undesirable presence of grains or, on the contrary, in the disappearance of some latent images (Droz, 1976). Prevention involves interposing a thin protective layer between the tissue and the emulsion. These artifacts are easily identifiable in studies with HTdR, where attention is concentrated on nuclei, but they might constitute a problem with other radioactive molecules less precisely located. 7 . Calculation Measurements in radioautography are complicated by many factors such as artifacts, statistical variability of disintegrations, and background corrections. In practical terms, these parameters require repeated measurements over nuclei and over unlabeled cells to estimate the natural background. However, it is necessary
92
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(1) to adjust the count in each case (England and Miller, 1970), and (2) to rectify these factors in order to attain the best accuracy. The relation between the number n of atoms in a layer of nucleus of thickness t and volume V and the total number of atoms N in a nucleus of volume V can be expressed according to Appleton et al. (1969) by the relation
n
=
(Nv/V)(N3r211‘/4r311)= N3t/4r
Since the grain count is proportional to the radioactivity present, this relationship can be used to modify the grain count according to the change in nuclear size. The arithmetic mean grain count in radioautography is more frequently utilized than the median or the geometric mean as a measure of the average amount of labeling. Chernick and Evans (1968) and Fried (1970) demonstrated that the choice of the best test was dependent upon the type of grain distribution. In the processing of radioautographs, cells are schematically classified as labeled or unlabeled. This binary classification is reflected in the model proposed by Howard and Pelc (1953). In fact, labeled cells exhibit a range of labeling intensities in relation to technical parameters (see above), biological parameters such as variations in HTdR availability and variations in DNA synthesis rate. As a result, exposure duration and background threshold might have important effects on the measurement of cycle phases and labeling indexes (Stillstrom, 1965). For example, Clarkson et al. (1967) reported that, when the emulsion exposure time was doubled from 2 to 4 weeks, the labeling index rose from a 5-grain threshold to a 7-to-8-grain threshold. Benassi et al. (1973) suggested an iterative method to calculate the labeling time even when the background appears to be incompatible. This computer program displays grain count distributions before or after correction and calculates the labeling index and the average grain count per labeled cell. Recently, Shackney (1975) proposed the development of a functional relationship between nuclear tritium content and radioautographic labeling intensity. This “radioautographic transfer function” can be applied to nonstandardized conditions.
111. Kinetics of Cell Proliferation
A. PRECURSORS
1. Chemical Purity The purity of the radioactive compound is an important parameter. For example, Evans and Bayly (1965) showed that radioactive compounds could undergo chemical as well as radiochemical decomposition. The rate of self-radiolysis during storage depends on the length of storage and on the temperature. For
KINETIC ANALYSIS OF CELLULAR POPULATIONS
93
example, 25-30% of HTdR stored in aqueous 70%alcohol at -2°C decomposes within 2.5 months. The rate is 10%higher at -20°C than at -2°C but there is no significant radiolysis at - 190°C @vans, 1966). Wand et al. (1967) demonstrated that the degradative products of HTdR were incorporated into cytoplasm, and treatment with RNase and DNase did not remove this radioactivity.
2. Disponibility Thymidine, labeled with a radioactive isotope and injected in vivo, is transported by the blood and then distributed to the cells and taken up by those which are in S phase. A quick uptake is observed, since less than 15 minutes after the injection labeling is found both in the cytoplasm and in the nucleus (Asboe and Levi, 1962). In the rat, as Fulcrand et al. (1968) showed the maximum amount of HTdR appears in the blood less than 10 minutes after the injection, whatever the type of injection (intramuscular, intraperitoneal, or intravenous). At best, only 20% of the precursor is found in the vascular compartment; this indicates the rapidity of the cell absorption mechanisms combined with degradation and clearance. Three hours after injection, 95% of the radioactivity of the plasma is found as tritiated water, confirming the observations of Rubini et al. (1960, 1962). About 30%of the initial radioactivity is thus transformed and disappears within a 2-week period (Fulcrand et al., 1968). Within a short time, various metabolites can be identified (P-aminoisobutyric acid and thymine). One hour after the injection of HTdR, only 2.6% of the radioactivity related to thymidine and its metabolites remains. In practice, with usual doses (1 pCi/gm body weight in the rat), efficient cell labeling takes place during the first 20 minutes after the injection. Rubini et al. (1960) estimated it to be 10 minutes in humans, and Hughes et al. (1958) reported between 30 and 60 minutes in mice. The amount of HTdR incorporated can be more than 15-20% of the injected dose. Cleaver (1967) and Harris (1 976) noted an increase in the labeling index between 30 and 60 minutes after injection of the labeled precursor into mice. Finally, HTdR elimination, the size of the precursor pools, and the blood-tissue barrier phenomena influence the extent of incorporation of exogenous HTdR into DNA. For example, to obtain the same labeling over embryonic neuron nuclei as over cells of other tissues, the initial dose of precursor must be about 10 times higher than the ones commonly used in the study of these tissues (Sidman, 1970). 3 . Radiotoxicity
One of the problems encountered when using radioactive precursors is the action of radiation on the cell functional state. Another concerns the genetic perturbations caused by the radioactive compound. Numerous facts and studies show the toxicity of radiation toward all cells, and in particular toward those
94
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undergoing proliferation (Oakberg, 1968). HTdR, in vivo or in vitro, modifies the length of the G2 phase (Fraccaro, 1968), and cessation of cell divisions can even be observed when specific activities are important (Grouse and Schrier, 1977). Burki and Okada (1970) have reviewed the killing of cultured mammalian cells by exposure to HTdR radiation. The effects become important above 1 pCi/ml (when the concentration is lower than 1 pCi/ml, 100%cell survival is observed). This is probably due to thymidine dimer formation, chain breakage, and chromosome aberrations (Burki et al., 1975; Huang et al., 1975). These factors limit the ability to obtain high-specific-activity tritiated DNA (Grouse and Schrier, 1977). There are numerous opinions regarding experiments in vivo. Doses of 1 or 2 pCi/gm body weight cause radiation effects (Mendelsohn, 1960; Smith et al., 1962; Baserga et al., 1966b; Johnson and Cronkite, 1967). In contrast, Altman (1966) recorded no toxic consequences after injecting 10 pCi/gm in long-term experiments in rats. The most extensive study on the effects of long-span action of tritium during embryonic life was made by Fliedner et al. (1968). According to Harris (1976), an injection of HTdR at a concentration of 3 pCi/gm body weight has no toxic effect on mouse lymphoid tissue. Leblond and Cheng (1976) used high doses of HTdR to cause cell death in mice. They noted that this result could be partially obtained with very low doses of 1.5-2 ,uCi/gm body weight, that intestinal cell sensitivity was not the same from one cell to another inside the crypt, and that the crypt base cells (stem cells) were the most sensitive. Potten (1977) has recently confirmed that, in the crypts, there is a very radiosensitive subpopulation which cannot be identified by means of the usual morphological criteria. This hypersensitivity could be explained as being related either to a certain period of the cell cycle and, in fact, it seems that cells in the G, and M phases are particularly sensitive (Sinclair, 1974), or to a differentiation phase. It is significant that in Potten’s experiments (1977), 10%of the sensitive cells were destroyed by one radiation dose, with external irradiation. According to R. Masse (personal communication, 1978), a correlation between irradiation and HTdR can be established: One rad is equal to 100 ergs/cm3, and a cell nucleus is cm3. If we assume that each disintegration corresponds to 1 keV absorbed by the nucleus, the equivalent of 1 disintegration will be 0.15 rad 3H(a coefficient of 2). The results of Potten (1977) show that the LDS0is reached with 7-8 rads; therefore it can be estimated that with tritium such an effect is obtained for about 20 to 25 disintegrations (without considering a possible effect of the dose rate). In radioautography the efficiency is about 1-20% (grains per disintegration). A 30-grain-labeled nucleus after an exposure of 1 month therefore corresponds to 3000 disintegrations, that is, 4 per hour. Without taking into account a possible loss of radioactivity during the histological treatments, it is noted that in the living cell a critical dose is easily reached during the interphase or even the G2 phase. In embryonic cells, with a short G,
KINETIC ANALYSIS OF CELLULAR POPULATIONS
95
phase and a brief generative cycle (10-20 hours), the effects could paradoxically be reduced if the radiosensitivity were limited to a specific phase, and if the daughter cells do not linearly “remember” the perturbations the stem cell was subjected to. However, the injection of HTdR into pregnant mice must be important enough to obtain a sufficient labeling of the embryos. The doses are about 3-10 pCi/gm body weight. The gestating female may suffer some perturbations without any discernible consequences for the embryos which are perhaps submitted to less important labeling (placental barrier, degradations, accelerated uptake by a certain population, and so on). It is also possible that in such “embryogenesis ” experiments there exist cell deficits; but they concern only limited cell generations, and for this reason are difficult to estimate in the adult CNS. Finally, the fact that radiotoxicity seems not to affect cell lines uniformly may be an explanation of the great differences in the radiotoxicity limits found in the literature. 4. Specificity of DNA Labeling The study of cell kinetics using a radioactive precursor of DNA is based upon the postulate that the cell labeling obtained is specific and determines the premitotic state of the cell. However, investigators have demonstrated that it is possible to label nonproliferative cells too; but in this case, very long exposures (sometimes more than 6 months) are necessary. Pelc (1972) showed that this phenomenon occurred in neurons in adult mouse brain after he had proved it in cardiac muscle (Pelc, 1964) and in mouse liver (Pelc, 1969). He has concluded that there can exist a DNA synthesis unrelated to premitotic syntheses. More recently, Harris (1975, 1976) has confirmed the relation between these DNA syntheses and the immune response in the mouse spleen. His experiments have also illustrated the importance of the reutilization of DNA after cell death. Manuelidis and Manuelidis (1974) confirmed that some nondividing cells, especially Purkinje cells, could integrate a small amount of HTdR. Ultraviolet irradiation during the various phases of the cycle shows that incorporation of the precursor into DNA in response to radiation may occur in GI as well as in S phase (Henry et al., 1971), or in the other phases (Rasmussen and Painter, 1964). Sakharov et al. (1976) found that with ultraviolet microirradiation the labeling was located only over the irradiated zone. This is due to unscheduled DNA syntheses which can be explained by the excision and replacement of bases damaged by irradiation. Although such phenomena occur spontaneously, their importance must not be exaggerated in regard to errors of interpretation appearing in radioautographic investigations on cell kinetics. With the most frequently used exposures, they are not discernible from the usual background. However, the treatment of tissues for radioautography with fixatives, dehydrating solvents, clearing agents, and embedding, removes unincorporated HTdR and its metabolic products (Appleton, 1964), and the main radioactivity is located only
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1
cell death
FIG.9. Cell cycle of continuously dividing cells [G,, (GI)', (GI)"]. Nonproliferative cells (Go cells) can be stimulated to enter the S phase.
over nuclei. However, small amounts of labeling over cytoplasm may be attributed to mitochondria1 DNA or to small amounts of thymidine or its metabolites that are bound to other macromolecules.
B. THECELLCYCLE
I. Nomenclature in Cell Kinetics The most used nomenclature is derived from the first study of Quastler (1963) describing the compartments and parameters of cell renewal (Fig. 9). M S GI
G2
C (for cycle) G O
Tr F Size, N i
Transit time, Ti, T , , T ,
cells in mitosis cells during DNA synthesis, usually equal to cells taking up specific tracers and incorporating them into DNA postmitotic presynthetic period postsynthetic premitotic period M + G , S G, = the generative compartment fertile cells not actually proliferating cells in transition from proliferative to mature functional (mature) cells number (or mass, and so on) of cells in a compartment, usually as a fraction of the size of the system time elapsed between entering and leaving a compartment, for example, S time, mitosis time, and cycle time (time for the cells to complete an entire proliferative cycle)
+ +
KINETIC ANALYSIS OF CELLULAR POPULATIONS
the inverse of transit time; cells passing through a compartment per unit time per number of cells in the compartment, for example, cycle rate (preferred to generation rate) number of cells passing a particular boundary in a particular direction (from i to j ) per unit time total flux in or out across all boundaries of a given compartment
Transit rate, Rb R c
Flux, K U Influx, efflux, CKs, SKU I
97
1
influx or efflux per cells in compartment turnover rate for a proliferative system or part of a system
Tumover rate Proliferation rate (birate) Doubling time LI
time necessary for a cell population to double labeling index; proportion of labeled cells fraction of labeled mitoses state in which the cell number of a compartment is constant in relation to cell loss (intestinal epithelium) or lack of cell division (neurons in brain) mitotic index; proportion of cells in mitosis
FLM steady state
MI
When proliferation is homogeneous and asynchronic and when the population is in a steady state, Tccan be calculated by the following relation (Sisken, 1964):
Tc = (T,ILI)X 100 In the case of an exponentially growing population the following relationship exists between the different parameters of the cell cycle:
LI =
w,, + TM)/TCI/(ZRC)
1 a3 u)
0
.I 4.a
0
8 0.5 (0
-I r
0 C
.-0 4.a
0
z
Y
0 0
1.0 Period
2.0 (TC)
FIG. 10. Ideal curve of FLM after a pulse injection of HTdR as a function of the cycle periods. (Symbols are defined in the text.)
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T , can be calculated if LI, T,, and TG + T Mare known (Lennartz and Maurer, 1964; Cleaver, 1965). For mathematical considerations, see Rubinow (1975).
2 . Cell Cycle (T,) and FLM method The cell cycle time varies from one cell to another within a population, over a widely distributed statistical range. The different methods employed to measure T , allow one only to estimate the average cell cycle length. The duration of the cell cycle has been regarded as the average time for one cell division (Christensen and Giese, 1956), as the time necessary for doubling a population (Merchant et al., 1964), and as the time between two successive divisions seen by time-lapse cinematography (Sisken, 1963). This parameter is sometimes obtained on the basis of the metaphase accumulation rate after the blocking of proliferation by colchicine (Leblond, 1959; Puck and Steffen, 1963), or with 5-bromodeoxyuridine (Pera et al., 1977). But the most used methods are based on radioautography. The FLM method was proposed by Quastler and Sherman in 1959 (Figs. 10 and 11). Since then, it has played a crucial role in the analysis of human tumors.
FtG. 11. Radiaautography of cultured brain cells observed under transmitted light. Heavily labeled aggregate after a 1 pCi/ml HTdR incubation for 2 hours.
KINETIC ANALYSIS OF CELLULAR POPULATIONS
0
99
~ ~ ~ ~ " " " " ~ " " " ' ~ ' " ' '
3
6
12
18 Time
24 (hr)
FIG. 12. Curves of labeled mitoses (FLM) in embryonic CNS after labeling with HTdR. Solid line is for control animals. Dashed-dotted line represents animals whose temperature was progressively lowered under the secondary effects of anesthesia. If the temperature is artificially maintained near normal after anesthesia, the cycle remains subnormal (dashed line) (Bisconte, 1977).
The basis of the FLM method is pulse-labeling of cells with a radioactive precursor during the DNA synthesis phase (S phase) and the identification of labeled cells by radioautographic detection during the mitotic phase. In an ideal population, uniformly cycling, the FLM curve can have the shape shown in Fig. 10. In this case, it is very simple to deduce the parameters of the cell cycle. According to Quastler and Sherman (1959) the phase limits can be found by locating the intersections of the curve with the 0.5 level of FLM. Bresciani (1965) and Mendelsohn (1965) introduced the area method in which T , , T , + O S M , and Tc, + M Tol are measured by integration. However, it appears that the success of the FLM method is dependent on the determination of at least two successive waves. Although a rough estimate of the average T , time can be made with poorly defined waves, they must be determined more accurately if an estimate of the variance is to be obtained (Barrett, 1966; Takahashi, 1968). Fried (1972) proposed a method for determination of the median and variability of T , in the growth fraction and for detection of the number of resting cells. In cell kinetics some computer simulation models have been developed and reported in the literature (Bisconte, 1973; Donaghey, 1976). For mammalian cells cultivated at 37"C, the values for the S, G, and M phases are relatively stable (Watanabe and Okada, 1967), whereas the GI phase is quite variable. Some workers have attributed these variations to a genetic program (Defendi and Manson, 1963), but it is now well-known that epigenetic factors
+
100
J.-C. BISCONTE
can have considerable consequences. In many cases, it was observed that the GI phase was greatly affected by temperature (Sisken, 1965; Watanabe and Okada, 1967), but Rao and Engelberg (1965) found that HeLa cells appeared to be an exception, since the M phase was in this case the most thermosensitive stage (Fig. 12). Under appropriate conditions, the increase in TG,may change a proliferative cell state to a nonproliferative one. The cells are in GI arrest or Go.In vitro this interesting and important class of cells normally exists as a pool of nonproliferating cells in which the balance between the increase in GI, the arrest in Go, and the reinitiation of DNA synthesis is mediated by a multiplicity of interacting operations of an ill-defied nature. In vitro one can obtain a simpler model system in which large quantities of proliferating cells can be reversibly induced to enter a nonproliferative state. Numerous data show that in many cell types DNA replication and protein synthesis are coordinated, at least in mammalian cells (Young, 1966; Hodge et al., 1969). Thus each change in culture medium causing a decrease in the protein synthesis capacity should be expected to inhibit DNA synthesis. This happened with an F-10 medium from which isoleucine was omitted in a monolayer culture of Chinese hamster cells pobey and Ley, 1971; Tobey, 1973). After addition of a certain amount of isoleucine to the medium, initiation of DNA synthesis and division take place. The isoleucine deficiency-induced GI arrest method is not specific to a given cell type. For example, it was possible to accumulate mouse L cells, BHK, and CHK cells in GI (Tobey and Ley, 1971; Tobey, 1973). However, other arrest methods are available, based on physical processes (cold storage, Newton and Wildy, 1959) or various antimetabolites (5-fluoro-2’deoxyuridine, Schindler, 1960; hydroxyurea, Sinclair, 1967; excess thymidine, Xeros, 1962). Control of synchronization or arrest is accomplished by radioautography and more and more often by flow microfluorometry (for other details, see Section II1,C). 3 . S Phase and the Double-Labeling Method During S phase, replication of the genetic material takes place (DNA, histones, and chromosomal proteins). This excludes any other DNA synthesis occurring outside the nucleus, or inside it for unscheduled DNA synthesis (e.g., the repair following radioactive damage). The most useful precursor and also the one generally used is the deoxyribonucleoside HTdR. Before its integration, the thymidine is converted to its triphosphate derivative which is then incorporated into DNA, together with the triphosphate derivatives of the other deoxyribonucleosides (dATP, dGTP, and dCTP). Although, positive identification of HTdR incorporation into nucleus reflects DNA synthesis, it does not provide complete knowledge of all the important pathways that contribute to DNA synthesis. The double-labeling method was first used to determine the S-phase duration (Hilscher and Maurer, 1962; Wimber and Quastler, 1963). This determination is
KINETIC ANALYSIS OF CELLULAR POPULATIONS
101
based upon two injections, one of HTdR and one of thymidine-14C,separated by a delay. This method is essential for the measurement of cell fluxes through compartments, and for estimation of the beginning and end of the S phase. Knowing the cell fluxes enables one to determine the growth mechanism of cell populations (Burholt et al., 1976). The double-labeling mitoses curve shows the total generation time and the phase duration. Discrimination between the p- particles of 3H and those of 14C requires two emulsion layers. Different methods have been proposed combining stripping films and liquid emulsions in various ways (Krause and Plaut, 1960; Baserga, 1961; Pilgrim et al., 1966; Harriss and Haelzer, 1971a,b); Schultze et al. (1976) uses a first thin coat (2 pm) of Ilford K2 emulsion obtained by dipping. An inert layer of gelatin (5 pm) is interposed before the second dipping. The thickness will then be up to 15 pm. After development in Amidol and a 50-minute fixation, the preparation is mounted with Entellan (Merck 7960). In the first emulsion layer, because of the very different characteristics of the 3H and 14C radiations, there is no ambiguity in the high grain density provided by the tritium and the low grain density produced by the I4C emission. Instead of using I4C and 3H, double labeling can be performed with HTdR at two very different doses (for instance 1 and 10 p Ci/gm body weight). In most cases distinction between the lightly labeled cell population and the heavily labeled cell population is possible. Sometimes there may be ambiguity, for example, when polyploidy is found. However, in the strongly labeled population, there are always cells which have taken up only a small quantity of precursor because they were at the end of the S phase at the beginning of the pulse. Statistically, only a few cells are involved in this phenomenon, and the method is still useful (Lesch and a h le r t, 1970; Galand and CMtien, 1969; Heenen and Galand, 1971; Westermark, 1973). T , duration may be obtained from the relation (Lennartz and Maurer, 1964)
NHlN, = TJt where N H is the number of heavily labeled cells, N L is the number of lightly labeled cells, T , is the duration of the S phase, and t is the time between injections. The main disadvantage of the double-labeling method using only HTdR is that a relatively large first dose is required. Such a dose may possibly have adverse effects on the cell cycle and affect subsequent incorporation of the second dose (Lord, 1970). All the results obtained show that the S phase is the least variable Fable I). 4. G , Phase
According to Howard and Pelc (1953) the G2phase is the premitotic phase during which certain biochemical processes are essential for the successful initiation of mitosis. As for the other phases, the existence of these events has been
102
J.-C. BISCONTE TABLE I DURATION
S-PHASE
S duration
Type of cell Mouse Embryo primitive ependymal cells Esophageal epithelium Mammary gland, normal alveoli In intact females In estrogen-treated females Rat Cells of splenic germinal centers Human Bronchus Esophageal carcinoma Basal cell carcinoma of the skin Human diploid cells in culture
(hours)
5.5 8.5 21.7 9.2 4.5 11.3 22-25 19.0 7.5
Reference
Atlas and Bond (1965) Blenkinsopp (1969) Bresciani ( 1965) Bresciani (1965) Fliedner er al. (1964) Fabrikant (197Ob) Fabrikant (1970b) Malaise et al. (1967) Defendi and Manson (1963)
demonstrated with cell lines grown in vitro. Nevertheless, the results concerning biochemical events in G, (or more generally in all the phases of the cycle) are comparable for normal cells in vivo and those grown in vitro. The quiescent compartment is thought to be essentially composed of cells in GI and Go prolonged phases. However, some experiments have shown that there are G2blocked cells (Gelfant, 1962, 1963; Tobey et al., 1971). The mechanisms involved in the G2 versus G, arrest decision are unknown. The same problem concerns the specific stage during which a cell enters a prolonged GI or Go phase, or proceeds once more through the cell cycle. Perhaps the programming occurs in the S phase. The biochemical events that perform this program take place in the G2phase. G2 processes would then be expected to quite differ for cells preparing for GI and cells preparing for Go. For all these reasons, the G2 phase is quite variable but not as variable as GI (Table 11).
M Phase The presence of mitotic figures in histological sections indicates that cell proliferation occurs. In 1892 Bizzozero observed that mitosis in the crypts of the small intestine was very active and suggested that daughter cells must be pushed up onto the epithelium of the villi. Other areas of intense mitotic activity exist in adult mammals, for example, in reproductive systems and epithelia. The MI is the percentage of cells in a population that are in a certain stage of mitosis at a given time (all the different phases of mitosis must be taken into account and not only metaphases). The mitotic incidence observed in tissue sections gives the true proportion of cells in mitosis only if the diameters of mitotic and interphase 5.
103
KINETIC ANALYSIS OF CELLULAR POPULATIONS
nuclei are the same. To obtain the ratio of metaphases to total nuclei, the MI must be multiplied by a correction factor k:
k = L (interphase) + M L (metaphase) + A4 where L is the nucleus diameter (Abercrombie, 1946; Simnett, 1968). The use of colchicine, or other spindle poisons, increases considerably the number of metaphases and allows an accurate estimate of the turnover time in the cell population without using thymidine (Stevens-Hoopex, 1961). Other methods have been used to determine cell turnover and renewal, for example, x-irradiation (Oakberg, 1956). Polyploidy or polygenomy may result from incomplete mitotic cycles, in general by the omission of mitosis. Polyploidizing tissues are characterized by the retention of proliferative ability in maturing and mature cells. This problem has been extensively reviewed by Brodsky and Uryvaeva (1977). The length of mitosis in some mammalian cells is relatively stable, for example, about 1 hour (TM= 1.38, crypt epithelium of the mouse, Fry et al., 1961; T M = 0.43, same tissue in the rat, Wright et al., 1972). 6. G I Phase ( T c , ) The postmitotic G , phase has been well studied in v i m essentially in synchronized cells. Tests for synchrony are based upon the measurement of mitoses and of cell growth, often made by radioautography. As shown elsewhere (Table III), the GI phase is the most variable phase of the cell cycle. Several investigations demonstrate that the G1phase is heterogeneous and, for example, Baserga et al. discovered an actinomycin D-sensitive step (generally considered a step requiring RNA synthesis) in the GI period 3 hours before the beginning of DNA synthesis (Baserga et al., 1966a; Doida and Okada, 1972). TABLE II G,-PHASEDURATION Type of cell
Mouse embryo primitive ependymal cells Regenerating rat liver Human epithelial cells, colon Human metastatic carcinoma of the ovary Mouse L cells Human diploid fibroblasts Cultured human lymphocytes
G2 duration (hours)
1.0 2.5 1 .0
3-16 4.8 4.0 3.5
Reference
Atlas and Bond (1 965) Fabrikant (1968) Lipkin et al. (1962) Clarkson et nl. (1965) Cleaver ( 1965) Defendi and Manson (1963) Cave (1966)
J .-C . BISCONTE
104
TABLE UI DURATION OF G I IN REPRESENTING MAMMALIAN CELLS"
Type of cell Mouse embryo primitive ependymal cells Mouse L cells Sheep wool follicles Mouse mammary gland, alveoli Mouse ascites tumor Human diploid fibroblasts HeLa S3 Rat polychromatic erythroblasts
G duration (hours)
3.5 6.2 9 38 2.5-15 6 8 1.o
Reference
Atlas and Bond ( 1965) Cleaver (1965) Domes et al. (1966) Bresciani (1968) Frindel et al. (1968) Macieira-Coelho et al. (1966) Terasima and Tolmach (1963) Hanna et az. (1969)
"From Baserga (1976).
7. Cells in G o The concept of Gocells was introduced by Gilbert and Lajtha (1965) to qualify quiescent cells which do not synthesize DNA and consequently do not divide, but which can be stimulated to do so by the application of a particular stimulus (Tables IV and V). In adult animals cells such as hepatocytes which normally do not synthesize DNA can also be induced to divide by appropriate stimuli. But for several investigators the distinction between GI and Go reflects an artificial subdivision, and Go cells are simply cells with a very long GI phase. Recently, it has been clearly shown that Gois discernible from prolongated G, on a functional basis (Epifanova and Terskikh, 1969; Smets, 1973; Baserga et al., 1973; Baserga, 1976). TABLE IV OF CELLPROLIFERATION DURING G o PHASE(in V i m ) STIMULATION
Type of cell
Stimulus
Reference
Chick embryo fibroblasts
Neuraminidase Papain Isoleucine Serum Phytohemagglutinin Concanavalin A Sodium I 0, Cell fusion with transformed fibroblasts Hormones CelI fusion
Vaheri et al. (1973) Vaheri et al. (1973) Tobey and Ley (1971) Ponten et al. (1969) Nowell (1960); Cooper (1971) Powell and Leon (1970) Zatz et al. (1972)
Chinese hamster cells Human glia cells Lymphocytes
Macrophages Mouse mammary gland Quiescent nuclei
Croce and Koprowski (1974) Lockwood et al. (1967) Harris (1967)
KINETIC ANALYSIS OF CELLULAR POPULATIONS
105
TABLE V STIMULATION OF CELLPROLIFERATIONDURING G o PHASE(in Vivo) Tissue
Stimulus
Reference
Adrenal glands, guinea pig Growth cartilage, rat Kidney, rat Liver, rat and mouse Liver, fasted rat Mammary gland, mouse Mammary gland, rabbit Pancreas, mouse Prostate, castrated rat
ACTH Growth hormone Lead acetate Partial hepatectomy High-protein diet Estrogens Prolactin Glucagon plus T3 5-Dihydrotestosterone Croton oil EthyIphenylpropionate
Masui and Garren (1970) Kember ( 1971) Choie and Richter (1972) Grisham (1962); Bucher (1967a,b) Short et al. (1973) Bresciani ( 1971) Bourne et al. (1974) Malamud and Pemn (1974) Lesser and Bruchovsky (1973) Hennings and Boutwell (1970) Raick and Burdzy (1973)
~~~~~~~~~~
Skin, mouse
In conclusion, it is possible to distinguish cells which are directly engaged in the cell cycle, those. which are only linked to it (prolonged GI cells), those which are conditionally engaged (Gocells), and finally, those which are nonproliferative (resting cells).
C. APPLICATIONS
1. Cell Cycle and Cell Diflerentiation The problem of cell differentiation has been studied for a long time, and numerous data demonstrate the basic role of exogeneous molecules and cell-cell interactions in multipotential cells (Hunt, 1974). In contrast, other works suggest that the main mechanism is endogeneous. Holtzer and Holtzer (1976) have demonstrated the preeminent role of endogenous programming, particularly the strong correlation between cell differentiation and the cell cycle. Their studies are based on the following assumptions: (1) fundamental information unavailable in the mother cell becomes available in daughter cells (Holtzer et al., 1973); ( 2 ) genetic controls of constitutive molecules are independent of differentiation mechanisms (Dienstman and Holtzer, 1975); (3) each cell type is committed to a single, limited program of synthesis (Holtzer, 1968, 1970); and (4) for transition in a developmentalprogram, the number of options never exceeds two (Abbott et al., 1974). Dienstman and Holtzer (1975) have also stressed the importanceof cell lineages in differentiation . . .: (1) All embryonic cells at all times are members
of some functional lineage; (2) The cells in one “compartment” of a lineage have an ongoing program of synthesis distinct from programs of synthesis in cells in antecedent or subsequent compartments; (3) The sequence of compartments in a lineage is obligatory, that is, a terminal cell type
106
J.-C. BISCONTE
appears only as the result of the appearance of an invariable line of precursors; and (4) No single cell type has the option of producing more than 2 new cell types as immediurepropeny. According to this view, the induction of competent cells is a permissive not an instructive event for the transition from one compartment to the next one in a lineage (Holtzer, 1968). Only if there are 2 or more generations, acollection of “bipotential” cells can exhibit the properties of a “multipotential” system. . . . Ideally, an embryonic cell in one compartment of a lineage may be doubly characterized: (1) by its unique ongoing program of synthesis, and (2) by its options, or potentiality, of yielding one, or maximally, two particular types of divergent progeny. It has been proposed that when transitions occur in a lineage that leads to daughter cells with programs of synthesis different from that of the mother cells they are cell-cycle-dependent events (H. Holtzer, 1963; Holtzer et al., 1972; H. Holtzer, 1970; Abbott et (11.. 1974).
Myogenesis provides the most completely studied model. Many such experiments using a DNA synthesis inhibitor, 5-fluorodeoxyuridine or cytosine arabinoside, and a mitosis inhibitor, Colcemid, lead to two conclusions: (1) The transition from the presumptive myoblast compartment to the myoblast compartment requires DNA synthesis or at least one mitosis, and (2) the presumptive myoblasts blocked at the GI-S interface, or in M phase, cannot start to synthesize myosin, actin, or tropomyosin (Dienstman and Holtzer, 1975). The notion of critical divisions in going from one differentiation step to another has been called the “quantal cell cycle” by Holtzer (1970). Dienstman and Holtzer (1975) have concluded that transition from the presumptive myoblast compartment to the myoblast compartment “requires a quantal cell cycle such that the withdrawal from the cell cycle in the myoblast compartment cannot be imitated by any inhibition of DNA replication in the presumptive myoblast compartment. ” Cytochalasin B blocks cytokinesis; its application to presumptive myoblasts does not impede the beginning of division but does impede the separation of daughter cells, which gives rise to binucleated cells (Sanger and Holtzer, 1972). Cytokinesis, whether blocked by cytochalasin B or not, is not necessary for myotube formation or for cell transfer from the presumptive myoblast compartment to the myoblast compartment. However, the experiments of Bischoff and Holtzer (1968) demonstrate that myogenic cell fusion can only take place in GI phase. Finally, other experiments lead to the conclusion that, on the one hand, the presumptive myoblast and myoblast compartments exist and that, on the other hand, fusion and myofibril differentiation are two independent phenomena which can only occur during the myoblast state. All these facts show that myogenesis is one of the best examples of an apparent relation between the cell cycle and the expression of differentiation. However, data are still insufficient to make a general rule. 2. Growth Control Many theories have been proposed to explain the mechanism of growth control. Two kinds of theories may be distinguished; they are based on special
KINETIC ANALYSIS OF CELLULAR POPULATIONS
107
growth-controlling substances and on the functional adaptation of molecules normally involved in cell homeostasis. (1) In vivo, some investigations suggest that cell proliferation is controlled by mitotic inhibitors (Weiss and Kavanau, 1957); such factors, named chalones (Bullough, 1965), have been found in several cell renewal systems (Houck and Hennings, 1973; Brugal and Pelmont, 1975); many and varied compounds have been included in the chalone category, and their precise nature remains unknown. (2) The presence of hormones is not necessary for completion of the cell cycle, as well demonstrated in cell culture (Fig. 13). Nevertheless, there is considerable evidence that, in vivo, mammalian hormones have strong effects on mitosis and on the cell cycle (Epifanova, 1971; Sibley et al., 1974). For example, numerous studies have demonstrated the effects on the uterus of estrogens which increase the number of cells engaged in the cell cycle, with a great number of Gocells entering the S phase (Bresciani et al., 1974). 3. Growth and Nutrition It is well known that a protein-calorie deficiency significantly affects tissue growth, particularly in early infancy (Widdowson and McCance, 1963). In
FIG. 13. Primary brainculture showing the incorporation of HTdR into carpet cells (arrows). The surface neuronlike cells are unlabeled.
108
J.-C. BISCONTE TABLE VI CELLCYCLEIN EMBRYOS Tissue
Frog neurula Chick neuroepitheliae cells 18-32 horn Chick mesencephalon, 1 day Chick mesencephalon, 6 days Chick muscle, 9 days Mouse cleavage Mouse neural tube, 10 days Mouse neural tube, 11 days Rat tongue muscle, 17 days Rat intestinal epithelium, 20 days
TG.
Tr
TG- T M
Tr
5.5
6
2
-
13.5
Flickinger et al. (1967)
None
5
2.5
0.4
8
Langman et aE. (1966)
-
-
-
0.4
5
Fujita (1963)
6
6
2
1
16
Fujita (1963)
5.9 3-3.5
1.7
-
-
10.5 6-24
Marchok and Herrmann (1967) Samoshkina (1968)
2
4.6
0.6
1.3
8.5
Kauffman (1968)
2.7
5.4
1.2
1.2
10.5
Kauffman (1968)
7
6
3
2.5
19
Zhinkin and Andreeva (1963)
5.6
5.6
1.8
0.6
13.5
Wegener e t a l . (1964)
3.9
-
Reference
protein-deficient monkeys (Deo et al., 1965) and rats (Rose et al., 1971), there is prolongation of the DNA synthetic phase and an increase in the life cycle of the jejunal crypt cells after the injection of HTdR and radioautographic processing. In the hair follicle a protein-calorie deficiency induces an increase in the duration of the cell cycle, although all phases are not affected (Mathur and Deo, 1976; G, is not affected, S is prolonged by 57%, and G, by 100% (Rose er al., 1971). There is a metabolic clock in the organization of every animal with a sense of time (McCance, 1962; Burton, 1971). According to this concept a particular event, for example, the beginning of differentiation, occurs at a determined time, or rather after a specific number of divisions (the notion of quanta1 division, Holtzer, 1970; see above). In the external granular layer (EGL) of the rat cerebellum, the proliferation of grains normally completely stops on the twenty-first postnatal day (Altman, 1969). Barnes and Altman (1973) demonstrated a deficit of 26% of the microneurons during the same period for undernourished rats. Another experiment (Deo et al., 1975) showed that proliferation in the EGL still occurred in these animals. The malnutrition could also affect the number of stem cells @eo et al., 1975). However, in aged and in germ-free mice, there is delayed migration in the intestine (Lesher et al., 1964). 4. Kinetics According to Age As a tissue ages there is generally a simultaneous decrease in the growth fraction; a nonproliferative subpopulation, an increase in the length of the genera-
109
KINETIC ANALYSIS OF CELLULAR POPULATIONS
tive cycle phases, and sometimes a circadian rhythm in cell proliferation are observed. In the mouse liver, the MI increases to a maximum of 80% at about the eleventh day of gestation, falls to about 30% on the eighteenth day, and finally reaches only 1-5% on the first postnatal day (Zavarzin, 1967; Grisham, 1969a,b; Stticker et al., 1972). This decrease can be correlated with a change in the distribution of proliferative cells which, from a random distribution, tends to a peripheral one in the liver (Grisham, 1973). In this organ these considerable changes in the MI do not seem to be mainly due to an increase in the cell cycle phases but are essentially correlated with an important transfer of cells to the quiescent compartment. However, numerous studies have shown that the generative cycle increases with age. In the rat CNS during the fetal period, the cycle time changes from 8 to 15 hours between the eleventh and the sixteenth day (Dememes, 1969; Bisconte, 1973). Kauffman (1968) measured an increase from 8.5 to 10.5 hours between the tenth and the eleventh day in the mouse CNS, but most workers reported that the S phase was more constant and that it lasted 4-6 hours (Atlas and Bond, 1965; Langman et al., 1966; Bisconte and Marty, 1968; Martin, 1967; Kauffman, 1968; Dememes, 1969; Bisconte, 1973) (Tables VI and VII). After birth, this phenomenon continues. In the liver, a month after birth, the increase leads to a 20-to-26-hour GI period, a 6-hour S period, and a 3.5-to-4-hour G2 + M period (Grisham, 1969b; Laguchev et al., 1972). Experiments have proved that cell proliferation sometimes has a nychthemeral or a circadian rhythm. This has been demonstrated for the MI of the rat liver (HouTABLE VII CELLCYCLETIME,T , T , duration Type of cell Mouse, qdnyo Trophoblasts of an 8-day placenta Neural tube Tail Mouse, young Colon crypt Pharynx epithelium Jejunum crypt Mouse, adult Esophagus epithelium Jejunum crypt Adrenal cortex Other mammals Hamster jejunum crypt Bovine lymphocytes Human colon crypt
Olom) 9.4 8.4 9
12.7 84 10.1-13.8 87 11.2 1875
13.0 7 24
Reference
Cameron (1964) Kauffman (1966) Wimber ( 1963) Galand (1%7) Galand (1967) Galand (1 967) Maurer et al. (1965) Fry et af. (1961) Maurer er al. (1965) Betts et of. (1966) Vincent etal. (1969) Lipkin et af. (1962)
110
J.-C. BISCONTE
brechts and Barbason, 1972) and for the LI of the epidermis of hairless mice (Grunbe et al., 1970). 5 , Other Applications
The applications of cell kinetics are countless. They concern all vegetal and animal tissues from bacteria to humans. For example, Vermeulen and Venema (1974) showed the relations existing between the cell cycle and the competence or noncompetence of Bacillus subtilis cells. Lin et al. (1971), also using radioautography, determined the mechanisms of cell division in Escherichia coli. The proliferation of the most diverse tissues has been studied in different animals and in humans: rabbit gall bladder (Kaye et al., 1966), Langerhans cells (Schellander and Wolff, 1967), and mandibular condyle in mice (Frommer et a l . , 1969). Lufti (1970) investigated cell proliferation in the cartilage by a combined application of HTdR and colchicine, and the cell kinetics of osteogenesis has been reviewed by Owen (1969) and Kember (1971). Kim and Benirschke (1971) studied the kinetics of cells in the human placenta. Other works concern hematopietic tissue (Meuret et al., 1971), the stomach (Hattori, 1974; Lehy and Willems, 1976), the intestinal epithelium, and more generally all the epithelia, since they are quickly renewed tissues. Radioautography was also performed on glial cell proliferation in studies on the maturation of the CNS (Fulcrand et al., 1968; Skoff et al., 1976) and on cell degeneration (Fulcrand and Marty, 1973). Many studies were done with the aim of investigating the rejection phenomenon in vivo (Inoue, 1969; Porter and Calne, 1960) and in vitvo (Pickens, 1973); others determined the effects of denervation in muscle (Hassler, 1970) or the effects of stress in neuroglia and sympathetic neurons. Dropp and Sodetz (1971) demonstrated that there was stress-stimulated DNA synthesis; however, this phenomenon seems to be due to specific gene amplification rather than to cell proliferation. Quantitative radioautography in culture has been extensively applied to cytogenetics, including studies involving the accurate identification of specific chromosomes, the organization and segregation of the genetic material, and the control of gene duplication and transcription. A technique has been developed allowing repeated radioautography of isotope distribution in the chromosomes of a single cell (Stubblefield, 1965). Densities were measured with a recording microdensitometer in order to obtain graphs reflecting the thymidine distribution along each chromosome. For Takagi and Sandberg (1968) the cumulative integral graphs of autosomes approach a linear term throughout most of the S period (excluding the very early and very late S phase). Kuroiwa and Tanaka (1970) studied the behavior of chromosomes during the G , phase by electron microscope radioautography. Wright et al. (1970) determined the rate of DNA synthesis in X chromosomes of bovine females. They demonstrated that this rate differed in relation to the different steps of the S
KINETIC ANALYSIS OF CELLULAR POPULATIONS
111
phase. Henry et al. (1971) showed that pulverization, uncoiling, and nuclear changes were not only morphologically distinguishable but were induced at different stages of the cell cycle. In human cytogenetics, many investigations apply these techniques to quantitative radioautography and statistics. Obviously, investigations in humans are fewer and, above all, they involve human cells in culture. For example, radioautographic determination of the proliferative pattern of neurons in the human fetal brain at different stages of gestation in different regions is not practicable. However, some data have been gathered with the use of “supravital” labeling (Rakic and Sidman, 1969) and neural explant cultures (Choi and Lapham, 1974). One of the most important applications in cell kinetics concerns malignant cells and cancer therapy control. All tissue grows on the basis of variations in three parameters: (1) the length of the cell cycle in the proliferative compartment, (2) the growth fraction, that is, the fraction of the cell population engaged in the cell cycle, and (3) the rate of cell loss. In cancer growth, the most important disturbance is probably the recruiting of Gocells into the proliferative pool. This fact reflects the disturbance of control in the growth fraction. In this field, three kinds of labeling are employed. (1) The FLM method after pulse-labeling is limited to situations where multiple biopsies are feasible. The long radioautographic procedure renders the results hazardous in planning therapy for the patient. (2) The double-labeling technique for the determination of T , has been adapted for use in human tumors by incorporation of a second precursor, thymidine-14C, in vitro into disaggregated cells from tumors labeled with HTdR in situ (Vincent et al., 1969). Helpap and Maurer (1969) performed experiments with normal tissues and tumors in vivo and in vitro, applying the double-labeling method with HTdR and thymidineI4C. They conclude that the radioautography of excised tissue samples incubated with thymidine represents the conditions of in vivo labeling. (3) Continuous labeling may indicate the surviving fraction of tumor cells. Some workers used a combination of radioautography and external detection of the loss rate in mice with radioiodine125 deoxyuridine (Jenkinson et al., 1975). Fabrikant and Cherry (1970) employed hyperbaric oxygen to increase the uptake of HTdR and to accelerate the experimental procedure. Recently, Braunschweiger et al. (1976) improved the double-labeling radioautographic method by gold activation and reduced the interval between the sampling and the diagnosis. The results of irradiation in vitro demonstrate that the greatest radiosensitivity occurs during different phases depending on the type of cells cultured and the irradiation levels, but in general the effects are greater in the M phase and lower in the S phase. In vivo, the results are not as clear as those found in vitro. The difficulties in interpretation are mainly due to the nonsynchronization of tissues (Frindel and Tubiana, 1971). In these cases, HTdR has been widely used to demonstrate every event that interrupts the orderly progress of a proliferative cell
112
J.-C. BISCONTE
through the cycle. The best known effect of radiation on a proliferative cell is blocking in the G2phase. Other experiments demonstrate that the growth fraction increases after irradiation (Song and Tabachnick, 1969). In the stem cells of normal bone marrow, 80-90% of the cells are in GO(Becker et al., 1965), and after irradiation the proportion of stem cells in the DNA synthetic phase increases, showing that a fraction of Go cells enters the mitotic cycle (Frindel and Tubiana, 1971). The effects on solid tumors show that irradiation does not produce significant prolongation of the cell cycle. Unfortunately, radioautography does not allow one to make a distinction between viable cells and unviable ones still able to divide once or twice more. In cancer chemotherapy, the major aim is to learn how to choose the appropriate drugs and their doses. In the future, cancer therapy will perhaps combine chemotherapy, radiotherapy, immunotherapy, and hyperthemy, but it will probably always be necessary to measure the parameters of cell kinetics.
IV. Migration and Chronoarchitectony
1. General Considerations
Because of radioautography with HTdR, much information was obtained concerning migration and chronoarchitectony. This type of investigation requires more cells to be slowly dividing or clearly nondividing. Under these conditions, cells which were labeled previously do not lose their labeling by dilution. The nervous system, especially the CNS, is closest to the ideal model. The CNS is certainly the most complex tissue; billions of neurons combine size, form, function, and position so each of them is almost unique. The patterns relating the neurons to one another, inside laminations or nuclei, are constant for a given species. These patterns progressively develop during phylogenesis, from simple structures which become more and more complicated, and finally reach the great complexity of the mammalian brain. However, it is well known that neuron proliferation is essentially restricted to the prenatal period. Thus neuron prenatal HTdR labeling allows one to establish a close correlation between the date of the last division of a neuron and its final position in the adult brain. This approach is a very powerful investigative tool which has been extensively used in a reconstruction of CNS architectonic history (Bisconte, 1973). This method has not been employed much with other tissues, probably because they are proliferative compartments and, above all, each one is very often composed of identical cells whatever their localization inside the tissue. The basic method used in these studies with HTdR or thymidine-I4Cconsists either of single or multiple injections into pregnant animals on different days of
KINETIC ANALYSIS OF CELLULAR POPULATIONS
113
gestation, killing the fetuses, newborns, or adults at different times, and determining the pattern of migration of the labeled cells by radioautography. In the case of neurons, the labeling is permanent when the mother cells have been labeled during their last period of DNA synthesis. Specific information such as the chronoarchitectony within the germinal zone, the time of final DNA synthesis of neurons, the pattern of migration, the final destination, and the chronoarchitectony of the adult CNS can then be obtained. Investigations in this area have been extensively reviewed (Altman, 1969; Sidman and Rakic, 1973; Berry, 1973).
2 . Migration HTdR radioautography has been used to map the positions of migrating neural cells in the brain at successive stages of embryogenesis and postnatal development and to trace certain migrations. For example, neural crest cells migrate from their origin to many regions of the vertebrate embryo. To follow the early migratory behavior of avian cephalic neural crest cells, neural fold tissue was transplanted orthotopically from HTdR-labeled donors, into unlabeled hosts. The hosts were killed at subsequent stages, and the position of labeled cells was plotted (Johnston, 1966; Noden, 1975). In the neural tube, the peculiar behavior of ventricular cells has been observed by the HTdR radioautographic method (Sidman et al., 1959; Fujita, 1963) (Fig. 14). These cells move alternately between an external region in which they are in S phase and the ventricular region where they divide. These stem cells supply the nondividing neuroblasts and the proliferating glioblasts. The neurons reach their final position following specific pathways (Berry et al., 1964; Hinds, 1968; Rakic, 1977). The speed of their movement has been determined by Hicks and d’Amato (1968). Altman et al. (1968), combining x-irradiation and radioautography, demonstrated that the migrating cells and prospective migratory cells were extremely radiosensitive, while stationary differentiating cells and mature cells were not affected. Other tissues were examined according to the same procedure by Kobberling (1965), for example, in the thymus. However, as shown before, the decrease in labeling by mitosis renders this method rarely useful in most tissues. 3 . Chronoarchitectony When HTdR is injected every few hours, an increase in the LI can be observed. This method is thoroughly discussed by Wimber (1963). Fliedner et al. (1968) used this particular approach to investigate the cytokinetics of the rat brain. It has been shown that all the cells of a newborn animal can be labeled in their nuclear DNA if HTdR is available for all the DNA synthetic phases which occur during development of the fetus. This condition can be realized by continuous perfusion of the precursor during the critical period of gestation when the organs are being formed. In theory, after birth the labeling intensity of the cells is
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FIG. 14. Rat embryo neural tube after HTdR labeling in vivo; the animal was killed 1 hour later. Ventricular cells in the deep regions are labeled, whereas mitotic cells are not.
diluted as a function of their proliferative activity based partially on the time of their last division. Another method is based on the extreme radiosensitivity of undifferentiated cells; with this technique the identification of newly forming
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FIG. 15. Hypothalamic neurons labeled in adult rat brain. The pulse injection of HTdR was made on the twelfth day of embryonic life (mixed illumination).
cells is possible by exposing the brain to ionizing radiations (Hicks and d’Amato, 1966; Berry and Eayrs, 1966). In fact, the most commonly used method is based on pulse-labeling during embryonic life. Consequently, neurons whose last division occurred not long after the HTdR injection appear as the most heavily labeled cells in adult brain sections pigs. 15 and 16). As a consequence, it is theoretically possible to give each neuron a date of ultimate division called the time of origin or birth day. The first studies of Sidman et al. were made in 1959. The whole rat brain and principally the mouse brain were then studied zone by zone: cerebellum (Sidman and Miale, 1959), neocortex (Angevine and Sidman, 1961; Bisconte and Marty, 1975c), hippocampal formation (Angevine, 1963), brain stem Vaber, 1963), olfactory bulb (Hinds, 1966), diencephalon (Johnston and Angevine, 1966; Bis-
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FIG.16. Radioautographic patterns of rat brain when the injection date of I4CTdR vaned from embryonic day 10 to embryonic day 17.
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HTdR El5
:h,10
LI
=33
yo
MG = 2 0
0
L Grains
LI
=25
MG =13.5
FIG. 17. Patterns of labeled cells in three different areas of rat neocortex (HTdR injection on embryonic day 15). In this brain region the chronoarchitectonic gradient is inside-outside. Histograms vary considerably according to whether the cells are contemporaneous with the injection (area I) or with later generations (areas I1 and 111).
conte and Marty,1968), and ventral cochlear nuclei (Taber-Pierce, 1966). In this first step, analysis has been only more or less precise, for two principal reasons: (1) the great difficulty in mapping labeled neurons without an appropriate system, and (2) the existence, in the adult brain, of all the intermediaries between heavily labeled neurons and those that are not labeled at all, which requires arbitrary choices. In any case, the results revealed that neurons organized themselves according to main gradients. Particularly, the neocortex gradient, which is inside-out, has been demonstrated to be opposite the classic concepts of Tilney (1933). Recently, these studies have been resumed with the help of automated cartography and of neuron kinetics modeling (Bisconte, 1973; Bisconte and Marty, 197%) which has given rise to a more extensive but also a more precise
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analysis (Smart, 1973; Smart and Smart, 1977, Rakic, 1973; Lawson er af., 1974; Bisconte, 1974); for the data on mutant mouse histogenesis, see also Caviness and RakiC, 1978). The bases of the chronoarchitectony concept are neuron time origin data, but also the quantitative and spatial relationships between the successive cellular generations in a given area of adult tissue. In most cases, neurons belonging to the same generation can be linked by an isochrone curve. The histogram representing the number of labeled neurons versus the number of grains per nucleus is an exact representation of the chronoarchitectonic characteristics of the tissue (Fig. 17). These characteristics can be deduced by calculation (Bisconte, 1974).
V. Concluding Remarks Quantitative radioautography is suitable for several different purposes, such as understanding cell syntheses, obtaining knowledge of the CNS connections, and studying cell kinetics. Nevertheless, quantitative radioautography has been applied most in the last-mentioned case. In order to maintain the full significance of the quantitative dimension, precautions must be taken during the radioautographic process, and the causes of errors must be determined. The automatization of measurements, by microphotometry and computerized image analysis, is a very decisive factor in the development of a method which ordinarily is very long. However, recent devices allowing one to measure simultaneouslyDNA content and cell size on slides as well as in cellular flow will certainly eliminate some present radioautographic applications; but in most cases, all these methods will become complementary and will then provide a better understanding and use of the kinetic clock.
ACKNOWLEDGMENTS
I thank Sylvie Margules, Robert Gardette, Raymonde Joubert, and Michel Farez for technical assistance, and Yvonne Huguenin for typing the manuscript. This work was supported in part by CNRS, DGRST, and the Fondation pour la Recherche Medicale Francaise.
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INTERNATIONAL REVIEW OF CYTOLOGY,VOL. 57
Cellular Mechanisms of Insect Photoreception F. G. GRIBAKIN The Laboratory of Evolutionary Morphology, Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. USSR
I. Introduction . . . . . . . . . . . . . . . . . . . . 11. The Compound Eye and Photoreceptor Optics . . . . . . . . A. Light and the Eye . . . . . . . . . . . . . . . . B. The Compound Eye . . . . . . . . . . . . . . . . C. Visual Pigments and the Photoreceptor Membrane . . . . . D. Absolute Light Sensitivity of the Compound Eye . . . . . E. Color Vision . . . . . . . . . . . . . . . . . . F. Sensitivity to Polarized Light . . . . . . . . . . . . G. Final Remarks . . . . . . . . . . . . . . . . . . 111. Electrical Basis for Insect Photoreception . . . . . . . . . A. Introduction: The Compound Eye as a Volume Conductor . . B. Dc Parameters of the Compound Eye in the Dark and in the Light C. Photoresponses and Their Cellular Mechanisms . . . . . . D. The Role of Compartmentalization . . . . . . . . . . IV. Conclusion . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . Note Added in Proof . . . . . . . . . . . . . . . . .
127 128 128 129 129 138 153 157 158 159 159 159 169 177 177 178 184
When you go after honey with a balloon, the great thing is not to let the bees know you’re coming. Now, if you have a green balloon, they might think you were only part of the tree, and not notice you, and if you have a blue balloon, they might think you were only part of the sky, and not notice you, and the question is: Which is most likely? A. A. Milne, Winnie-the-Pooh
I. Introduction The photoreceptor of insects is a highly specialized sensory cell whose principal function is to transform information on the arrival of a light quantum into a receptor signal. At the same time, all the specific machinery of the insect photoreceptor is undoubtedly based on general cellular mechanisms underlying not 127
Copyright Q 1979 by Academic Press, Inc. All rights of reproduclion in any form reserved. ISBN 0-12-364357-0
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only sensory cell functioning but, to some extent, the functioning of a neuron. However, unlike the neuron, which has many neuronal (or synaptic) inputs, the photoreceptor cell has only one input which is physical. Such a peculiarity of the photoreceptor has determined the logic of this article which discusses the cellular mechanisms of photoreception in insects from the absorption of a light quantum to generation of the receptor signal. Both the start and the end of the insect photoreceptor process have been studied fairly well, however, many intermediate stages are still unknown, and, we will emphasize those aspects of the problem we believe to be of paramount importance, at least, at the end of the 1970s. We apologize to those whose original works are not referred to in this article, since the number of references had to be reduced to a reasonable minimum. This article is addressed not only to specialists in cell biology but also to investigators dealing with the nervous system, since the compound eye of arthropods can serve as an excellent model for various neurophysiological studies.
11. The Compound Eye and Photoreceptor Optics
A. LIGHTAND THE EYE Undoubtedly, all the morphological and physiological characteristics of the eye, as well as of its photoreceptors, have been totally predetermined by the nature of light. As an electromagnetic wave, light can be characterized by energy (or intensity), wavelength, polarization, and direction of propagation. A photoreceptor must display absolute, spectral, polarization, and directional sensitivities which at the whole-eye level are known as light sensitivity, color vision, polarized light discrimination, and image perception. Investigation of these characteristics, based on the fundamentals of cell biology (cytology) and physical optics, forms the subject of a relatively new branch of science called photoreceptor optics, the birth of which was recently announced at a special workshop by Menzel and Snyder (1975). Here we consider the absolute, spectral, and polarization sensitivities of the photoreceptors of the photopic compound eye in terms of photoreceptor optics. Directional sensitivity and image perception have been omitted from this account, since these subjects are closely related not to cell biology but rather to three-dimensional space perception, a field in which we have had no personal experience. The term “scotopic” and “photopic” are used here instead of “superposition” and “apposition,” since we accepted terminology of Post and Goldsmith (1965) who suggested to discriminate between compound eyes with short proximal rhabdoms and migratory pigment (scotopic) and those with long rhabdoms and no longitudinally migrating pigments (photopic).
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B. THECOMFQUND EYE The compound eye consists of structural units called ommatidia. The three main elements of an ommatidium are the dioptric apparatus, retinula, and pigment sheath. In turn, the dioptric apparatus includes a corneal lens (the biconvex transparent cuticule region) and a crystalline cone produced by four transparent cone cells. Nearly round in cross section, the retinula is formed by several (usually eight) visual cells or photoreceptors, each producing a membraneous photoreceptive structure termed a rhabdomere. In many insects rhabdomeres fuse axially in the retinula to form a rhabdom. A photoreceptor sends its central process, or axon, through the basal membrane (or basal lamina) toward higher nerve centers-optic ganglia (the lamina, medulla, and lobula). The pigmented sheath of an ommatidium is composed of primary (or comeagenous or iris), secondary (or accessory), and sometimes basal pigment cells. It should be emphasized that the compound eye is encapsulated within chitinous walls and proximally separated from underlying structures and optic centers by a basement lamina. Thus the extracellular space of the compound eye should be considered an extracellular compartment isolated from the rest of the insect (Heisenberg, 1971). We omit further description of the compound eye structure and classification, as well as ultrastructural details, since they can be easily found in the literature (see, for review, Mazokhin-Porshnyakov, 1969; Goldsmith and Bernard, 1974).
C. VISUAL PIGMENTS AND THE PHOTORECEPTOR MEMBRANE 1. Introduction Since the retina is intended to absorb light, it cannot be as transparent as common nerve tissue. The stronger its coloration, the greater the amount of light absorbed and the greater its absolute sensitivity. Dealing with photoreception, natural selection might realize four ways to get the photoreceptor cell colored; it might stain (1) cell cytoplasm, (2) intracellular inclusions and organelles, (3) the plasma membrane, or (4) all three. Thanks to the efforts of many workers, most of all G. Wald, we know that the third possibility has been realized in vision, and that the absorbing substance of a photoreceptor-the visual pigment-is an integral part of specialized areas of the outer, or plasma, membrane of the cell, which therefore has been called the photoreceptor membrane. Current knowledge of the photoreceptor membrane derives mainly from studies on the retina of vertebrates. However, all we know about the arthropod photoreceptor membrane indicates that both have much in common. Thus, for simplicity, we discuss the visual pigments and optical properties of these membranes as though they were the same "universal" photoreceptor membrane (Gribakin and Govardovskii, 1975), and particular emphasis is placed upon their differences when appropriate.
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2. Visual Pigments a. Retinal as a Chromophoric Group in Insect Rhodopsin. A visual pigment molecule is believed to consist of a lipoprotein moiety to which a chromophoric group is attached. The first visual pigment studied in detail was rhodopsin extracted from retinal rods of cattle and frogs and having a maximal absorption A,,, at 500 nm. At present all visual pigments are usually termed rhodopsins, no matter what class of animals they belong to or where their A,, is located. The basis for this generalizationis the chromophoric group 11 &-retinal (a derivative of pcarotene) which is the same in almost all pigments known; only porphyropsins found in some vertebrates, which have 3,3’-dehydroretinal as a chromophoric group, are exceptions to this rule. In dark-adapted vertebrate photoreceptors the chromophoric group is bound to the phospholipid phosphatidylethanolamine (Abrahamson and Wiesenfeld, 1972). After absorption of a light quantum and subsequent cis-trans isomerization, the chromophoric group is transferred from the lipid to a lysine group on a protein (opsin), and this coincides with the change from metarhodopsin I (A,, = 478 nm) to metarhodopsin I1 (A,, = 380 nm). Of all the intermediate transitions (which occur in the dark and at room temperature), this is the first to require water, and it is thought that the metarhodopsin transition gives rise to the generation of a late receptor potential, or the receptor signal (Abrahamson and Wiesenfeld, 1972). This is not true for cephalopod rhodopsins where the retinal binding site is located on a protein, and light-evoked transitions stop at a stage of metarhodopsin formation (Hubbard and St. George, 1958). It means that, unlike the final product of vertebrate rhodopsin transitions-N-retinylidene opsin, which readily hydrolyzes to yield free retinal and opsin (so-called bleaching of the pigment)-cephalopod metarhodopsin is resistant to hydrolysis (for more details, see Abrahamson and Wiesenfeld, 1972). Further, since in cephalopods water is involved in the lumirhodopsin-metarhodopsin transition, this stage may be considered an analog of the vertebrate metarhodopsin I-metarhodopsin I1 reaction responsible for initiation of the receptor signal. Visual pigments of arthropods resemble cephalopod rhodopsins rather than those of the vertebrate retina in having stable or long-lived metarhodopsins (see, for review, Goldsmith, 1972). According to the evolutionary classification of Eakin (1965, 1972), both cephalopod molluscs and arthropods have rhabdomeric photoreceptors, so the similarity in photochemistry may reflect a fundamental property of the rhabdomeric photoreceptor. The small dimensions of the insect compound eye make it difficult to investigate its visual pigments in vitro. Nevertheless, Goldsmith (1958) was the first to show that retinal occurred in honeybee heads but not in abdomens and thoraxes. The same proved to be true for six species of other orders: Hymenoptera, Orthoptera, Odonata, Coleoptera, and Lepidoptera (Briggs, 1961). Retinal has also been found in Diptera (Wolken et al., 1960) and Blattoptera (Wolken and Scheer, 1963). Free retinol has been
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131
reported in light-adapted bees, although retinal usually appears to prevail in a state of dark adaptation (Goldsmith and Warner, 1964). The absolute sensitivity of the eye of carotene-depleted flies (Musca dornestica) has been found to drop by about four orders of magnitude in both the visible and ultraviolet regions possibly because of a shortage of chromophoric substance-retinal (Goldsmith et al., 1964; Goldsmith and Fernandez, 1966). Paulsen and Schwemer (1972) demonstrateddirectly that retinal in the 1 1 -cis form was actually the chromophoric group of Ascalaphus macaronius ultraviolet-sensitive rhodopsin-the first visual pigment of insects isolated and photochemically characterized (for its history, see Gogala and Michieli, 1965; Gogala, 1967; Gogala et al., 1970; Hamdorf et al., 1971; Schwemer et al., 1971). Thus there is ample reason to extend the above findings to the whole class of insects and to suggest that all insect rhodopsins incorporate 11 -cis-retinal (never 3-3’dehydroretinal) as a chromophoric group. b. Absorption Spectra of Znsect Rhodopsins. In vertebrates visual pigments incorporating 11-cis-retinal range in A,, from 433 to 575 nm (Dartnall and Lythgoe, 1965; Liebman and Entine, 1968). Between these points the absorption maxima of about 200 known vertebrate rhodopsins are not randomly distributed, but cluster about certain points of the spectrum about 7 nm apart (Dartnall and Lythgoe, 1965). As judged from in vivo spectrophotometry and, mainly, electrophysiology, insect rhodopsins show a greater diversity of A,, values (Goldsmith and Bernard, 1974; Menzel, 1975b). The red limit for A,, in insects does not seem to exceed the 575 nm reported for vertebrates (e.g., 567 nm in the green receptors of Notonecta, Bruckmoser, 1968), and the presence of redsensitive pigments can not be inferred as yet from the data available from electrophysiology (Goldsmith, 1965; Swihart, 1972; Post and Goldsmith, 1969; Autrum and Kolb, 1968; also see review of Menzel, 1975b). The most distinctive feature of insect rhodopsins (as well as of arthropod rhodopsins in general) is the presence of retinal-based pigments with a A,,, in the near ultraviolet region (340-360 nm) and with practically no absorption in the visible region. Theory predicts both bathochromic (to the longwave side) and hypsochromic (to the shortwave side) shifts of the A,,, from 435 to 440 nm inherent in essential visual pigment of vertebrates ( N - 1 1-cis-retinylidene phosphatidylethanolamine). These may result from twisting about a double bond in the chromophore, which depends upon the molecular microenvironment of the chromophore, or from interaction of the chromophore with a closely located negative charge (Abrahamson and Wiesenfeld, 1972). It appears, however, that even twisting cannot explain the full range of A,, values reported in vertebrates (433-575 nm) let alone in the ultraviolet region, whereas a model calculation of the effect produced by a negative point charge placed 0.45 nm above the plane of that are both bathothe chromophore molecule demonstrates shifts in A,, chromic (to 530 nm when positioned at the fifth carbon of retinal) and hypsochromic (to 356 nm when displaced toward the nitrogen atom of the binding site)
132
F. G. GRIBAKIN
(see Table 6 in Abrahamson and Wiesenfeld, 1972). An alternative possibility is the linkage of retinal with the sulfhydryl group of cysteine, or with both the sulfhydryl and amino groups of opsin (Goldsmith, 1972). Thus the existence of retinal-based ultraviolet-sensitive rhodopsins may be postulated from theory (see also Morton and Pitt, 1969). All the data available on color receptors in insects are presented in Fig. 1. Although their A,, values may not coincide exactly with those for their rhodopsins for many reasons (Section 11,E; Menzel, 1975b), we can infer that longwave (green and blue) receptors of insects located within the same broad spectral band as vertebrate retinal-based photoreceptors do (412-567 nm in insects versus 433-575 nm in vertebrates). Only 2 of about 50 color receptors have sensitivity peaks between 380 and 420 nm, which may imply an “exclusion principle” for this region, and, in turn, this may have a real physical or physiological basis. As judged from Fig. 1, visual pigments of insects tend to cluster around four distinct points of the spectrum, 350, 430, 470, and 520 nm; the reason for this is still unknown. c. Energetics of the Photoexcitation and Regeneration of Insect Rhodopsin. Visual pigments of animals originate from p-carotene synthesized in plants. But, unlike the situation in plants, in which the energy of light absorbed by chlorophyll, carotenoids, or accessory pigments is used metabolically, in animals the energy of a light quantum absorbed by a rhodopsin molecule does not appear to serve as the energy supply for further stages of the visual process. The only function of a light quantum in vision is to release from the photoreceptor cell an appropriate “quantum” of metabolic energy which has been stored in it as electricity. Thus the molecular evolution of visual pigments has changed the light quantum function from that of an energy supplier in plants to that of an information carrier in animals (Vinnikov, 1974).
iII N=50
400
5
I
,
600
500 A , nm
FIG. 1 . Distribution of insect color receptors over the spectrum. (Maxima positions are mainly from Goldsmith and Bernard, 1974.)
CELLULAR MECHANISMS OF INSECT PHOTORECEPTION
133
A molecule of bovine rhodopsin-498, upon absorption of a light quantum, transforms through an intermediate-prelumirhodopsin-543 (still with an 11-cis chromophoretinto lumirhodopsin-497 (with an all-trans chromophore) (Morton, 1972). The energy levels of both rhodopsin-498 and lumirhodopsin497 are nearly the same (see, for instance, Abrahamson and Wiesenfeld, 1972), so the energy of a light quantum is necessary only for the molecule to overcome the potential barrier between the two states encoding either the absence of light (rhodopsin) or the presence of light (lumirhodopsin). All the excessive energy liberated during the “fall” of the molecule from the potential barrier to the lumirhodopsin state dissipates as heat (see discussion in Falk and Fatt, 1972). The other mechanisms which could help the molecule to dispose of the unnecessary energy do not seem to be of any significance (e.g., the quantum yield of the fluorescenceof rhodopsin is as small as 0.005; see Guzzo and Pool, 1968). Thus, after absorption of a light quantum (photoexcitation) followed by cis-trans isomerization (photoisomerization), the rhodopsin molecule, which has already triggered a mechanism for signal generation, leaves the field through several spontaneous (thermal) transitions ending with its breakdown into free retinal and opsin. In order to keep the absolute sensitivity of the photoreceptor high enough, a continuous renewal, or resynthesis, of rhodopsin molecules is needed, and two methods of regenerating rhodopsin are known-enzymic and photochemical. Enzymic regeneration is most characteristic of vertebrates. However, of all the vertebrates studied, only the rat seems to have an effective isomerizing system in the retina (Cone and Brown, 1969). In other vertebrates (frogs, cattle) isomerizing systems are thought to be located mainly in pigment epithelium, where 11-cis-retinol esters dominate other retinol isomeric derivatives (see, for review, Baumann, 1972). Photoregeneration of rhodopsin in vertebrates can be demonstrated in three ways. First, strong illumination or a brilliant flash is used so that the photoexcited molecule of rhodopsin can absorb a second quantum during its short stay in the lumi- or metarhodopsin state, that is, before its thermal breakdown begins. The second quantum thus absorbed can convert the intermediate molecule into its native state-rhodopsin-or into isorhodopsin with 9cis-retinal as a chromophore [see the experiments of Dowling and Hubbard (1963) with the rat retina]. Second, the temperature can be lowered so as to stablize the intermediate. In this case prolonged illumination of rhodopsin preparations produces an equilibrium mixture composed of 11-cis, 9-cis, and all-trans modifications of visual pigment (Yoshizawa, 1972). Third, shortwave light, blue or violet, with its wavelength lying within the absorption band of all-trans-retinal can be used. Under these conditions, light bleaches rhodopsin, but all-trunsretinal liberated from bleached pigment undergoes photoisomerization resulting in 11-cis isomer formation, and the latter can combine freely with opsin. Thus, partial regeneration of rhodopsin (to 15-20%) is again possible (Hubbard and Wald, 1952). From this consideration one can infer that photoregeneration of
134
F. G. GRIBAKIN
rhodopsin in vertebrates demands very specific light or temperature conditions and, for this reason, can hardly be of any importance in their visual cycle. On the contrary, in insects as well as in other arthropods, and also in cephalopod molluscs (all having rhabdomeric eyes), photoregeneration prevails in the restoration of visual pigments. As mentioned above, light-evoked changes in rhodopsins in these groups result in metarhodopsins (Hubbard and St. George, 1958, for the squid; Hamdorf et al., 1968, for the octopus; Wald, 1967, for the crayfish; Gogala et al., 1970; Hamdorf et al., 1971; Schwemer et al., 1971, for the insect Ascalaphus). The same appears to be true for Calliphora, Drosophila, and Deilephila, and in all these species photoregeneration of rhodopsin from long-lived or thermally stable metarhodopsin has been demonstrated (see, for review, Hamdorf and Schwemer, 1975). Since the absorption coefficients of both rhodopsin and metarhodopsin yR(A) and yM(A), respectively, depend on the wavelength of the illumination, absolute quantities of both pigments in the photoreceptor depend only on wavelength and quantum yields of direct and reverse photoreactions. Assuming quantum yields to be equal and illumination prolonged enough, one can obtain a rhodopsidmetarhodopsin ratio at a given Ao, as [R]/[M] yM(AO)/yR(Ao), which results in a maximal rhodopsin content when A. corresponds to the A,, of the metarhodopsin absorption spectrum, and in a maximal metarhodopsin content when A. is the A,, of the rhodopsin absorption spectrum. Thus prolonged illumination with monochromatic light (under experimental conditions) or wide-band light (in a natural environment) inevitably leads to a photoequilibrium between the rhodopsin and metarhodopsin in the photoreceptor. As experiments with Deilephila elpenor (Lepidoptera) have shown, the same metarhodopsin (M480) forms from different rhodopsins (R350, R450, and R545) in different color receptors of the same eye (Schwemer and Paulsen, 1973). There is evidence that the energy of a light quantum absorbed by metarhodopsin is needed only to convert all-trans-retinal to 1 1-cis-retinal but is not necessary to produce reverse conformational changes in the protein from the metarhodopsin to the rhodopsin state (Hamdorf and Schwemer, 1975). Also, metarhodopsin, no matter what its amount in the photoreceptor, has no influence on signal transduction which is totally defined by rhodopsin function and amount (Hamdorf and Schwemer, 1975), although it may affect the optical properties of the photoreceptor by optical screening of rhodopsin. Dark regeneration of rhodopsin (probably enzymic) from metarhodopsin has also been shown to occur in photoreceptors of invertebrates, although this process is slow. So, to convert half the initial amount of metarhodopsin into rhodopsin about 10 minutes at 20°C is needed in the octopus Eledone moschata (M520 to R470, Schwemer, 1969), about 25 minutes at room temperature in Calliphora (M580 to R495, Stavenga et al., 1973), 15-45 minutes at room temperature in the butterfly Aglais urticae (M480 to R535, Stavenga, 1975), and several minutes in the lobster Homarus (M490 to R515, Goldsmith and Bruno, 1973). At the
-
CELLULAR MECHANISMS OF INSECT PHOTORECEPTION
135
same time, Brown and White (1972) failed to find dark regeneration of rhodopsin R515 from M480 in larvae of the mosquito Aedes aegypti. Thus photoregeneration of visual pigments seems to play a leading part in the insect visual cycle (as well as in the visual cycles of other invertebrates), whereas dark (or metabolic or enzymic) regeneration is probably a continuously working standby mechanism which, although it is not so powerful as photoregeneration, could activate the visual system into a “ready-to-see’’ state under poor light conditions or even in the dark when photoregeneration is impossible (cf. Stavenga, 1975). 3 . The Photoreceptor Membrane In 1962 G. Wald formulated the concept of a photoreceptor membrane: “It is at least very reasonable to expect that porphyropsin . . . is located in the membranes” (Wald et al., 1962). Since photoreceptor membranes of different species have much in common, the variety of light-sensitive organelles known in the animal kingdom can be considered as having evolved from the originally flat universal photoreceptor membrane (Gribakin and Govardovskii, 1975; Laughlin et al., 1975; Menzel and Snyder, 1975). Rhodopsin molecules are inlaid in this membrane, and in vertebrates they are free to undergo Brownian rotation and translation (Brown, 1972; Cone, 1972; Po0 and Cone, 1974; Liebman and Entine, 1974). Thus the photoreceptor membrane conforms to the model of a fluid mosaic membrane with protein molecules floating in a lipid bilayer (cf. Singer and Nicholson, 1972; Edidin, 1974). The rod outer segment may be considered a “pure preparation” of photoreceptor membrane, and its lipid and protein content well reflects the composition of the photoreceptor membrane. The lipid content is 40% of the total dry weight of the rod outer segment, and proteins comprise about 60%. The protein moiety of rhodopsin-opsin-constitutes up to 80-90% of the total protein content in the frog rod outer segment and 15-20% in cattle (Abrahamson and Wiesenfeld, 1972). Rhodopsin molecules are believed to be located in the inner (facing the cytoplasm) lipid layer of the photoreceptor membrane, that is, on the outside in rod disks and on the inside in rhabdomeric microvilli (Blasie, 1972; Daeman, 1973; Mason et al., 1974; Jan and Revel, 1974, for outer segments; Hamdorf and Schwemer, 1975; Fernandez and Nickel, 1976; Eguchi and Waterman, 1976; Nickel and Menzel, 1976, for rhabdomeres). The surface concentration of rhodopsin in outer segments is about (2 X 1 0 4 ) / ~ m z (a “square lattice” with molecules 7 nm apart; see, for instance, Daeman, 1973); calculated for a crayfish rhabdomere this is about half less, that is, (1 x 104)/pm2 [a 9 x 9 nm square lattice as calculated by Gribakin and Govardovskii (1975) from the data of Hays and Goldsmith (1969)l. Freeze-etch study of the crayfish rhabdomeric membrane gives a similar surface density of protein particles on its inner side-about 8000/pm2 (Eguchi and Waterman, 1976). Nearly the same value, 7000/,um2, has been obtained for the ant (Nickel and Menzel, 1976) and
136
F. G. GlUBAKIN
the crayfish Procamburus (6600/pm2, Fernandez and Nickel, 1976). There is much evidence that these particles correspond to rhodopsin molecules in vertebrate photoreceptors (see Jan and Revel, 1974). This is also thought to be true for the rhabdomeric membrane (Fernandez and Nickel, 1976; Nickel and Menzel, 1976), and the change in surface density of particles observed in carotenedepleted flies strongly supports this view because of the numerical coincidence between the decrease in rhodopsin content (about eight times) and in the surface density of particles (nearly six times) (Boschek and Hamdorf, 1976). The diameter of a vertebrate rhodopsin molecule (with a molecular weight of 40,000 daltons) has been suggested to be 4-5 nm (Wald er al., 1962), and the globular substructures in the disk membrane appear to have the same size (Daeman, 1973). This implies that at above surface concentrations [(2 x lo4)/ pm2] about 60% of the membrane surface is free of rhodopsin (it would be only 21 or 14% for square or hexagonal packing). So at this concentration there is no need for rhodopsin molecules to be packed in any kind of crystalline lattice; instead, they are seemingly free to locate at random. The larger diameter of particles in the invertebrate eye membrane-mean 8 nm (Eakin and Brandenburger, 1975, for the snail; Fernandez and Nickel, 1976, for the crayfish; Nickel and Menzel, 1976, for the anttsuggests a molecular weight of the pigment as high as 200,000 daltons, and only if the rhodopsin molecule in the particle were surrounded by two layers of lipids could this value be lowered to 52,000 daltons, close to that of vertebrate rhodopsins (Nickel and Menzel, 1976). Another explanation of the large size of particles suggests that the rhodopsin molecule is a dimer, or even a multimer (Nickel and Menzel, 1976), however, this is not consistent with optics from which a surface density of absorbing centers (i.e., rhodopsin chromophores) of about (1 X 104)/pm2has been obtained (see above), while a hypothetical dimer or multimer would have shown absorption two or several times greater than that measured by Hays and Goldsmith (1969). Vertebrate rhodopsin molecules are free to rotate and translate, but strict limitations exist as to spatial orientation of their rotation axes so that tumbling “head over heels” is highly improbable. Owing to these restrictions, the rhodopsin molecule behaves as if it were suspended in its molecular environment from two points-a hydrophilic “top” jutting out into the aqueous phase (cytoplasm) and a hydrophobic “bottom” embedded in the lipid phase of the membrane (cf. Singer and Nicholson, 1972; Laughlin et al., 1975). This rotational suspension of the molecule in the photoreceptor membrane can be considered one of the principal attainments in the molecular evolution of rhodopsin which made it possible to orient all the molecules (about 3 x 10’ in the frog rod outer segment, for instance) in a standard position providing maximal absorption of the light normally incident upon a flat membrane. [Maximal and isotropic absorption results from keeping the absorbing dipoles of properly suspended rhodopsin molecules at a right angle to the suspension axes, i.e., in a plane of the photoreceptor membrane, a fact first discovered by Schmidt (1938), Wald er al.
CELLULAR MECHANISMS OF INSECT PHOTORECEPTION
137
(1962), and Liebman (1962).] Rolling up of the flat universal membrane, which is necessary to form a microvillus, puts additional restrictions on rhodopsin Brownian translation (Wehner and Goldsmith, 1975) and rotation (Goldsmith and Wehner, 1975; Laughlin et al., 1975). Using microspectrophotomet, Wehner and Goldsmith (1975) observed no translational diffusion of metarhodopsin produced by a light flash in crayfish photoreceptor membranes. In addition, the absorbing dipoles of rhodopsin are partially aligned along the axes of microvilli in the squid and crayfish (microspectrophotometry, Hagins and Liebman, 1963; Goldsmith, 1975), which follows from the dichroic ratio of a microvillus, which is about 2.5-2.7 (mean value) in the crayfish and 6 in the squid, instead of the expected 2.0 if the ideal dipoles were oriented at the plane of the membrane at random (the model of Moody and Parriss, 1961). Several physical reasons responsible for the above restrictions were put forward. First, a lower proportion of long-chain polyunsaturated fatty acids with more than 20 carbons (27.3% of the total fatty acids in squid rhabdoms versus 42% in vertebrate rods), as well as a higher amount of cholesterol (with a molar ratio to phospholipids in the squid of about 1:2 versus 1:20 in vertebrate rods), make the rhabdomeric membrane more rigid or stiff, and consequently more viscous (Goldsmith, 1975; Hamdorf and Schwemer, 1975). Second, since the surface of a microvillus is curved, a boundary between different phases of the membrane (hydrophilic and hydrophobic) would be disturbed periodically during rhodopsin molecule rotation, and this would be energetically unfavorable. Thus bending of the membrane might prevent molecules from free rotation in the membrane, though oscillations or wobbling may occur (Goldsmith and Wehner, 1975; Laughlin et al., 1975). Third, the molecule may be asymmetric in the plane of the membrane, hence if subjected to viscous drag (e.g., from hypothetical cytoplasmic flows within the microvillus; see Goldsmith, 1975), it might move or oscillate with its long axis directed along the microvillus (Laughlin et al., 1975). Fourth, curvature of the microvillar membrane seems to prevent circumferential translation energetically (Laughlin et al., 1975). Fifth, taking into account the location of rhodopsin molecules inside the microvillus, the calculated area per molecule can be lowered from about 80 nm2 (for an outer diameter of the microvillus of 50 nm) to 65 nm' (for an inner diameter of about 40 nm). Then, for 8-nm particles one can obtain a surface density closely corresponding to a square lattice with little room for free diffusion. Last, an interaction between closely positioned molecules may occur which could stabilize the positions of the molecules and eventually activate the microvillar photoreceptor membrane into a state similar to the crystalline state. Most of the above restrictions may apply to a greater extent in microvilli of smaller diameter (Laughlin et al., 1975). Thus the rhabdomeric photoreceptor membrane has evolved to incorporate rhodopsin molecules in more-or-less fixed positions, and this arrangement seems to be a consequence of the bending (or rolling up) of an originally flat photo-
138
F. G. GRIBAKIN
receptor membrane to form a tubule or microvillus. In this situation photoregeneration has proved to be the more universal way of restoring rhodopsin, because of the easy accessibility of any molecule to light, while it does not seem that natural selection could easily supply every rhodopsin molecule fixed in the membrane with a quickly operating enzymic regeneration mechanism of its own.
D. ABSOLUTE LIGHTSENSITIVITY OF THE COMPOUND EYE 1. Principal Problems In order to design an ideal photoreceptor evolution (or natural selection) had to solve at least two principal problems: first, how to transduce information on every photon absorbed to the proper discrete receptor signal (one-to-one transduction) and, second, how to maximize the ability of a receptor to absorb photons. The first problem is known to have been successfully solved both in vertebrate and invertebrate photoreceptors which can act as photon counters (Hecht et al., 1942; Sakitt, 1972, for humans; Fuortes and Yeandle, 1964; Scholes, 1965; Borsellino and Fuortes, 1968; Kaplan and Barlow, 1976, for arthropods). The efficiency of counting is limited by the quantum yield of rhodopsin which, being 0.67 in vertebrates (see Dartnall, 1972), means that one light quantum from the three absorbed fails to evoke bleaching and apparently the unit electrical response. In arthropods, unit electrical responses were first described by Yeandle (1958) in Lirnulus, and at present they are known as quantum “bumps,” discrete potentials, or miniature potentials in all the arthropod species studied. So, it appears that the first general problem of vision-one-to-one transduction from a quantum of light to a “quantum of electricity”-was solved early in evolution and that since then the second problem has appeared-how to increase the probability of absorption of a photon. Unlike a one-to-one transduction mechanism which must be considered in terms of membrane physiology (Section III,C), the evolution of light absorption by photoreceptors is totally within the sphere of photoreceptor optics.
2. The Beer-Lambert Law as Applied to Rhabdomeric Photoreceptors Quantitatively transmission of light (and consequently its absorption) is expressed by the well-known Beer-Lambert law for an absorbing layer: Zt = Zo exp
-
a (A, 40) cl
(1)
where It and Zo are the intensities of the transmitted and incident light, respectively, a (A, cp) is the extinction (absorbance)coefficient depending in general on the wavelength A and the angle cp between the E vector of t b incident wave and the axes of the absorbing dipoles, c is the concentration of the absorbing substance (pigment), and I is the path length, or the thickness of the absorbing layer. Equation (1) can be written in another form using Napierian logarithms:
CELLULAR MECHANISMS OF INSECT PHOTORECEPTION
139
or, using common logarithms, = log,o(~t/~o)
E
(A, cp)cl
(3)
where, for 1 in centimeters and c in moles per liter, E (A, cp) has dimensions of liters per centimeter-mole and is termed the molar extinction (or molar absorbance). The optical density (or absorbance) D is often used, since it is more convenient:
D
= -log&/&)
=
E
(A, cp)d
(4)
The greater D, the greater the absorption, so in order to increase the absolute sensitivity of a photoreceptor (i.e., to maximize the probability of photon absorption), all three factors, namely, E (A, cp), c, and 1 must be made as large as possible. a. Molecular Extinction Coeficient, Molar Extinction, and the Orientation of Rhodopsin Molecules. If the path length 1 is expressed in centimeters and the concentration c in number of molecules per cubic centimeter, a (A, cp) is the molecular extinction coefficient having the dimensions of area. a(A, cp) and the molar extinction E (A, cp) are connected by a simple relation:
(A, cp) = (2.303
X
103) [ E (A,
cp)/Nl
(5)
where N is Avogadro’s number. In terms of physics, a (A, cp) is related to the effective area of the absorbing site of the molecule multiplied by the probability that a photon striking the molecule within this area will be absorbed. The change in this probability with wavelength forms the natural basis for the absorption spectrum, while its dependence upon the angle cp reflects dichroic properties of the molecule. At the maximum of its absorption spectrum (Amax) randomly oriented vertebrate rhodopsin (e.g., in a solution) has an average (Y (A,,, p) of 1.56 x cm2 (Dartnall, 1972). For linearly polarized light with A = A,, and (c = 0 or cp = T (when all the absorbing dipoles are parallel to the E vector), the highest value of the molecular extinction coefficient is three times greater, that is, 4.68 x cm2, since only one-third of the dipoles were parallel to the E vector when measured in a solution. This situation resembles that reported for rhabdomeric membranes with predominant dipole alignment along the microvilli. However, for unpolarized light, half the uniformly oriented dipoles make no contribution to absorption and, in order to obtain maximal absorption in this case, the dipoles should be allowed to arrange at random (or to rotate) in the plane perpendicular to the light path; this has been found to be true for vertebrate rod disks. The above considerations are illustrated by Fig. 2 and Table I where four theoretically possible modes of orientation are given. Several important conclusions can be made based on these models. First, the value of a (A, cp) for
140
F. G . GRIBAKIN
Y/
FIG.2. Scheme illustrating the dependence of the average molar extinction of rhodopsin on the orientation of absorbing dipoles. A unit volume is filled with a rhodopsin solution with concentration c and partial concentrations c, c , , and c , ; I, is the intensity of the incident light; E, and E, are the components of the E vector. Self-screening has been neglected. Quantitative analysis is given in Table I.
rhodopsin measured in solution is three times less than the maximal value. The latter is the most appropriate parameter of the absorbing dipole when one deals with pigment orientation in the photoreceptor membrane (4.68 X cm2 or emax= 12.18 x lo4 literkm-mole; cf. 1.56 X cm2 and emax = 4.06 x lo4 literkm-mole when in solution; see Dartnall, 1972). Second, twodimensional orientation of absorbing dipoles (“disks”) produces a 1.5-fold increase in the average molar extinction (as compared to that measured in solution) for both polarized and unpolarized light. Such a system fails to discriminate between polarized and unpolarized light when light travels normal to the disk plane. Third, an all-parallel mode of orientation of absorbing dipoles gives maximally a 3-fold gain in the average extinction coefficient for polarized light and a 1S-fold gain for unpolarized light. Thus all-parallel orientation gives no gain in absolute sensitivity to unpolarized light as compared to disk. However, it allows the polarization plane position to be detected by the receptor. This is in line with the statement of Laughlin et al. (1975) that the orientation of dipoles along the microvilli is necessary mainly to obtain maximal absolute sensitivity to unpolarized light in rhabdomeric photoreceptors, and that their polarized light sensitivity appears to be no more than an extremely useful by-product of the evolution of absolute sensitivity. Fourth, when the disk membrane is rolled up to form a microvillus (see Moody and Parriss, 1961; Laughlin et al., 1975; Gribakin and Govardovskii, 1975), the latter is only 1.13 times more sensitive than solution, and only 0.75 as effective as disk for unpolarized light; even for linearly polarized light, with sensitivity of a microvillus being maximum, this system cannot be more sensitive than the disk (see Table I). According to Dartnall (1972), of many colored substances only astacene has a amax greater than that of
141
CELLULAR MECHANISMS OF INSECT PHOTORECEflION
cm2 versus 4.68 x cm", and this implies that the rhodopsin (9.9 X in both pigments approaches unity probability term of light absorption at A, which is the theoretical limit. Thus rhodopsin seems to be one of the most intensive stains known in organic chemistry. In conclusion, it can be said that the main factor contributing to the optical density of the photoreceptor (hence to its absolute sensitivity), namely, the molar TABLE I THEORETICAL DEPENDENCE OF AVERAGE MOLAREXTINCTION OF RHODOPSIN ON ORIENTATION OF ABSORBING DIPOLESFOR POLARIZEDAND UNPOLARIZED LIGHT" Average molar extinction at peak absorption, proportional to I,c,
+ luc, + Izcx
~~~~~~~~~~~
Microvillus model
Mode of orientation of dipoles Components of concentration (Fig. 1) Polarized light I , = I , (or zero); I, = I, = 0 (or I, = l o ) Extinction for polarized light (literskm-mole) Unpolarized light I, = I, = I J 2 ; I, = 0 Extinction for unpolarized light (literdcm-mole) Maximal gain in extinction as compared to solution Polarized light Unpolarized light
Solution model c z = c, = C L = c/3
'h
IOC
Disk model c, = 0
c,
= cz = c/2
'h I,c
40,600
60,900
$5 I &
'h I,c
40,600
60.900
Made of disk membrane c, = c/4 cy = c/2 c, = c/4 'h I o c or % I,c
60,900or 30,450
Ideal (all-parallel)
c, = 0 cy = c c, = 0
I o c or zero
121,800,or zero
%? I O C
45.675
60.900
1 .o
1.5
1.5
3.0
1.o
1.5
1.13
1.5
"The total c of pigment mobcules in the unit volume (see Fig. I) can be oriented either randomly in the volume (solution model) or randomly in the yOz plane (disk model) or all parallel to the y-axis (ideal microvillus). The intensity I , in the case of unpolarized light can be considered as consisting of two components I, = I, = 1,/2 with mutually perpendicular E vectors. In the case of linearly polarized light either the I, or I, component is equal to the full intensity I o, whereas the other two are zero. Numerical values for molar extinction are from the basic value determined for a solution as 40,600literslcm-mole by Wald and Brown (1953).
142
F. G. GRIBAKIN
extinction E (A, (o) of rhodopsin, has a very high value. Absolute sensitivity of the rhabdomeric eye strongly depends on the orientation of the rhodopsin molecules in the photoreceptor membrane and, in the case of ideal alignment of the molecules along the axes of the microvilli, the value of the average molar extinction (and the same is true for the molecular extinction coefficient) is equal to that of vertebrate rod and cone disks for unpolarized light. Also, sensitivity to polarized light arises which can be considered a by-product of evolutionary growth in absolute sensitivity of the compound eye photoreceptors (Laughlin et al. 1975). In the above discussion the molar extinction of insect rhodopsin was considered the same as that in vertebrates and cephalopods (see Goldsmith, 1972). b. Concentration of Rhodopsin. The concentration of rhodopsin in vertebrate photoreceptors is known to be 2-2.5 mM (see Liebman, 1972), whereas for an arthropod Hays and Goldsmith (1969), taking the molar extinction to be 40,000, obtained 1.4 mM. Specific absorbances (per unit length of rhabdomere) at,,,A appeared to be nearly the same in different arthropod species (with all the values determined for the E vector parallel to microvilli): 0.0073 in the crab Libinia (Hays and Goldsmith, 1969), about 0.0085 in the crayfish Orconectes (calculated from the data of Waterman et a/., 1969), and 0.0075 in the fly (Kirschfeld, 1969). From this similarity one can assume that the rhodopsin concentration in the photoreceptors of different arthropods must be nearly equal (about 1.5 mM). In favor of this assumption is a similar density of protein particles in the photoreceptor membranes of different arthropods (7000/pm2; see Section I1,C). The upper limit of rhodopsin concentration in the rhabdomeric membrane can easily be determined. Let the unit volume (1 dm3 or liter) be completely filled with spherical rhodopsin molecules 8 nm in diameter (like protein particles in a microvillar membrane) forming a hexagonal or cubic lattice. Then the molar concentration of these molecules is c = k (VO/VIn) (1/N)
(6)
where c is the molar concentration, V ois the unit volume (1 liter = lP4nm3),Vm is the volume of one molecule (2.7 x 10' nm3), N is Avogadro's number, and k is the relative volume occupied by spherical molecules within the unit volume. The value of k can easily be calculated and is rr *I6 = 0.74 for a hexagonal lattice and T I6 = 0.52 for a cubic lattice. The calculated rhodopsin concentrations given by Eq. (6) are 4.56 and 3.25 mM for hexagonal and cubic lattices, respectively. However, in a rhabdomere consisting of microvilli 50 nm in diameter only 0.48 of the volume is taken up by the photoreceptor membrane (Gribakin and Govardovskii, 1975). Then the upper limit for the rhodopsin concentrations in the rhabdomere must be reduced to 2.18 and 1.56 mM, respectively. The second value appears to be very close to the above microspectrophotometricdata. Thus the second factor contributing to the optical density D, namely, the rhodopsin concentration c, is about 1.5 mM in arthropods, and this value is only
CELLULAR MECHANISMS OF INSECT PHOTORECEPTION
143
30% less than that characteristic of the most dense hexagonal lattice and conforms rather to a square lattice. c. The Length of the Rhabdomere. The length of the rhabdomeres in arthropods may range from several micrometers to several hundred micrometers; for instance, it is 400 p m in the crab Callinectes (Eguchi and Waterman, 1966), and 400 pm in the drone bee (Perrelet, 1970). With a specific absorbance of 0.0078/pm (Hays and Goldsmith, 1969) and a length of 300 pm, the transmission of the rhabdomere is 4.5 x l o + , or less than 0.5%, which means a relative absorption of more than 0.995; thus the length of the rhabdomere in arthropods seems to be the most changeable factor of the three related to optical density, and consequently to the absolute sensitivity of the photoreceptor. In order to provide high absolute sensitivity of the photoreceptor, a rhabdomere can evolve to become long enough so that it can absorb every quantum entering its distal end with a probability approaching unity. 3. Waveguide Effects and Sensitivity Control In Section II,D,2 no special mention was made of a mechanism which could confine a light wave, once it enters a thin absorbing fiber (the rhabdomere), to within this fiber, thus causing it to travel the full fiber length. Dielectric waveguide theory, the fundamentals of which were elaborated in the 1960s as the basis of fiber optics technology and exploration, provides a clue to this mechanism. As applied to photoreceptors of insects and other arthropods, the theory was advanced by A. W. Snyder and his co-workers in about 30 papers published during the last 10 years. The general principles of waveguide optics as applied to absolute light sensitivity of the rhabdomeric photoreceptor are considered briefly (for full theory, see Snyder, 1975a,b) in the following discussion. a. General Ideas of Waveguide Optics. Any optical fiber made of a material with a refractive index n1 and immersed in or surrounded by a medium with a refractive index n2 < n , may serve as a light guide if illuminated end on. When transmitted along the fiber, light, being an electromagnetic wave, forms specific field patterns termed dielectric waveguide modes. Every mode is a consequence of resonant or interference effects derived from internal reflections of the light wave within the fiber and can be characterized by the field distribution in a cross section of the fiber (Snitzer, 1961; Enoch, 1963; Snyder, 1975b). Only a fraction of the light energy is transmitted by a given mode within the fiber; the rest is transmitted outside the fiber, though along it, and fails to contribute to absorption in the fiber. The most important parameter of the fiber determining the number of modes and the fraction of energy transmitted within it is the so-called characteristic waveguide parameter I/: where d is the diameter of the fiber, h is the wavelength, and n , and n2 are the refractive indexes of the fiber and medium, respectively. A limiting value of V
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F. G. GRIBAKIN
FIG.3. Relative power P channeled through the optic fiber versus the waveguide characteristic parameter V for the angle of incidence 0 = 0. Replotted from the data of Snyder (1975b).
exists for each mode (the so-called cut-off V or V c ) , and at V < V , the given mode fails to propagate. Snyder (1975b, see his Fig. 9.8) has calculated a fraction of the total light power P(V,8) transmitted by all the modes within a fiber versus V and the angle of incidence 8. From his data, taking 8 = 0 (an axial light beam), we can plot P(V,t?) as shown in Fig. 3 and then evaluate the fraction of light channeled through real rhabdomeres and accessible to interact with rhodopsin. b. Waveguide Properties of the Rhabdomere. The refractive indexes of photoreceptor structures lie between those of the bilipid layer (1.66) and of Ringer’s solution (1.336) (Table 11). In arthropods the highest refractive index for a rhabdomere has been reported to be 1.405, and the lowest 1.347 (Table 11). From these extreme values the characteristic waveguide parameter V is calculated as given in Table I11 for h = 500 and 350 nm. Since for V 2 4 more than 80% of the light energy is transmitted within the rhabdomere (Fig. 3), we can infer that, first, the waveguide properties of the rhabdomere do not reduce markedly the absolute senstivity of a photoreceptor at V 2 4 and, second, since they provide confinement of light to within the rhabdomere, the Beer-Lambert absorption law remains applicable for the rhabdomere, taking its length as the absorbing layer thickness (see Section II,D,2). Thus the optical properties of the rhabdomere allow it to function as a photon counter at low light intensities, and the problem arises of how to lower the absolute sensitivity of the photoreceptor at high intensities. 4. Optical Mechanisms of Absolute Sensitivity Control
The effects of light adaptation on the microenvironment of the rhabdomere are well known, and they are, first, dispersion of the principal endoplasmic cistern (PEC) into the peripheral cytoplasm (“palisade” movement) with mitochondria migrating to the rhabdom and, second, radial migration of screening pigment
CELLULAR MECHANISMS OF INSECT PHOTORECEPTION
145
granules to the rhabdom (for review, see Walcott, 1975). The former results in a refraction change in the medium surrounding the rhabdom (or rhabdomere), while the latter modifies both the refraction and, mainly, absorption properties of the rhabdom “cladding.” Both mechanisms are intended to reduce the fraction of light absorbed in the rhabdomere. a. Refraction Waveguide Mechanism of Sensitivity Control. As seen in Fig. 3, high absolute sensitivity of a photoreceptor can easily be reduced by a decrease in V, which means that a fraction of incident light power is allowed to leave the rhabdomere or even pass by it. Perhaps during palisade migration (porridge and Barnard, 1965), of all the terms contributing to V, only n2 changes; this has two consequences, namely, a change in V and in the acceptance angle. Since for efficient n,-dependent sensitivity control V must be less than 4 (see Fig. 3), we can express this condition, from Eq. (7), as
V which can be rewritten as
=
(.rrd/A)(n12- n22)1’25 4
(8)
(n2/n1)22 1 - (4A/r dn,)2
(9) This means that, the greater the diameter of the rhabdom (i.e., the greater the dlX ratio), the less the difference between n2 and n, must be to provide sensitivity TABLE II REFRACTIVE INDEXES OF PHOTORECEFTOR STRUCTURES ~~
Refractive index Bilipid layer Ringer’s solution Rod outer segment Rod outer segment photoreceptor membrane Rod outer segment cytoplasm Bee rhabdomere Fly rhabdomere
Bee cytoplasm Fly cytoplasm Surrounding media Bee PEC Fly axial cavity Effective surroundings in fly (cavity plus cytoplasm)
1.66 1.336 1.41
Reference‘‘ a b C
1.475 1.365 1.347 1.349 1.365 1.390-1.405 1.343 1.340 1.339 1.336
d e
1.339
f
“(a) Finean and Engstrom (1967); (b) Liebman er al. (1974); (c) Sidman (1957); (d) Varela and Wiitanen (1970); (e) Seitz (1968); (0 Stavenga (1974); (g) Kirschfeld and Snyder (1975).
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F. G. GRIBAKIN
TABLE III WAVEGUIDE CHARACTERISTIC PARAMETER V CALCULATED FOR SEVERAL ARTHROPOD SPECIES" Initial parameters Waveguide (rhabdomere) diameter (pm) n , = 1.405; n 2 = 1.339 For A = 500 nm For A = 350 nm n = 1.347; n 2 = 1.339 For A = 500 nm For A = 350 nrn
,
~~~~~~~~~~~~~~~~~~~~
Bee
fly
Cricket
Crayfish
1
2
2
4
15
2.54 3.64
5.08 7.28
5.08 7.28
10.16 14.56
38 55
0.91 1.30
1.81 2.59
1.81 2.59
3.62 5.18 ~~~~~~~
13.5 19.4 ~~
"The highest and lowest n , - n 2 differences are from Table 11. Diameters of rhabdomeres (rhabdoms) are from Trujillo-Cenoz and Melamed (1966) for the fly, Gribakin (1967) for the bee, Polyanovskii (1976) for the cricket Gryffus, and Eguchi and Waterman (1966) for the crayfish Orconectes.
control through a change in the refraction of the surroundings. For instance, for n , = 1.4 and d = 10 pm, V can be made less than 4 only if n2/nl = 0.9990 (a 0.1% difference in refraction), whereas for d = 2 pm, n 2 / n 1must be as large as 0.9900 (with a 1% difference between n2 and nl). From Table I1 one can see that the real difference between n , and n2 is about 1%, hence sensitivity control through a change in the refraction of the surroundings works efficiently only in rhabdoms no more than 3-4 p m in diameter. For instance, consider the bee rhabdom (see Table 11) with nl = 1.347 and a diameter of 2 p m (for wavelength 500 nm). Substitution of the dark-adapted refractive index n2 = 1.339 (bee endoplasmic cistern) for n2 = 1.343 (bee cytoplasm) in the light causes a decrease in V from 1.81 to 1.28 and consequently a change in the relative amount of light power absorbed from 0.44 to 0 . l b n e a r l y a three-fold decrease in sensitivity. (Note that nl = 1.347 is only 1% more than n = 1.336 for Ringer's solution). This mechanism probably provides sensitivity control over a wider range when organelles having a refractive index higher than that of cytoplasm (e.g., mitochondria) surround the rhabdom in the light, and these structural changes have been found in the light-adapted photoreceptors of locusts (Homdge and Barnard, 1965) and crickets (Petrosyan, 1977a). Simultaneously with a decrease in the fraction of light power confined to the rhabdom, the acceptance angle is changed. As found experimentally, the acceptance angle of the photopic eye is reduced approximately from 6"-7" in the dark to 2.5"-3.5" in the light (Tunstall and Honidge, 1967, locust; Butler and Hor-
CELLULAR MECHANISMS OF INSECT PHOTORECEPTION
147
ridge, 1973, cockroach). This should bring about attenuation of the relative light power within the rhabdom nearly proportional to the sine squared of the acceptance angle, or approximately 5 to 10 times to that in the dark. Theoretical analysis of angular sensitivity can be found in Snyder (1975b). Thus the refraction mechanism of absolute sensitivity control might lower the absolute sensitivity by at least one order of magnitude, or even more,, and this seems to be of importance in insects with no radial pigment migration (e.g., the locust). The refractive index of the bee rhabdom (1.347) may appear very low in comparison with that of the fly (formerly 1.340 which has been corrected to 1.365 and even to 1.405; see Table 11). Nevertheless, from these values the solid content of rhabdomeres can be determined: c, = [(nl - n , ) / a ] x 100
(10)
where c , is the solid content in percent, n, and n , are the refractive indexes of the rhabdomere and of water, and a is the refractive index increment (0.18 for biological tissues; see Finean and Engstrom, 1967). Thus the solid content of the bee rhabdom is as low as 6.1%, while those for fly rhabdomeres and rod outer segments are similar-38 and 41%, respectively. Further, for the bee rhabdom, assuming a rhodopsin concentration of c = 1.5 mM and a molecular weight as low as M = 50,000 daltons (Section LII,D), we obtain the solid content: c, = (Mc/IooO) x
loo = 7.5%
(11)
which means that the refractive index for the bee rhabdom is likely to require correction, since the dry weight of only rhodopsin (7.5%) exceeds the total solid content of the rhabdom found with refractometry (6.1%) performed by Varela and Wiitanen (1970). Nevertheless, the refractive mechanism of sensitivity control seems to work successfully in insects and other arthropods, however the refractive indexes of their rhabdomeres are defined. b. Absorption Waveguide Mechanism of Sensitivity Control. This mechanism works when screening pigment granules migrate to the rhabdom in the light, hence both the refraction and absorption of optical fiber caldding are changed. According to the analysis of Snyder and Homdge (1972) and Snyder (1975b) as applied to the cockroach rhabdom, only 25% of a 10-fold decrease in sensitivity in the light is due to the refraction mechanism, while the rest is produced by the absorption of light transmitted outside the rhabdom in pigment granules. The absorption waveguide mechanism of sensitivity control was first described in terms of photoreceptor optics by Kirschfeld and Franceschini (1969) in Musca. In this insect it takes only a few seconds for the sensitivity to decrease and pigment granules to congregate close to the rhabdomeres in cells Nos. 1-6 after the light is switched on. Recently, Stavenga and Kuiper (1977) and Stavenga et al. (1977) showed experimentally that the same mechanism operates in hymenopterans and in butterflies where it covers about 3 log units of sen-
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F. G. GIUBAKIN
sitivity with a time constant of only a few seconds as in the fly. The time constant in hymenopterans appears to be about 10 seconds. The absorption waveguide mechanism of sensitivity control has been termed as “insect pupil mechanism” by Stavenga and co-workers, since in the light screening pigment forms a kind of “longitudinal pupil” around the rhabdom. Screening pigment material of cockroach photoreceptors has been calculated to be 70 times more absorptive than the material of the rhabdomere (Snyder, 1975b) though, as one can judge from the data of Langer (1975), the specific zbsorbance of single primary pigment cell granules is lower-about 0.0275/pm in Gerris and 0.1000/pm in Apis, which is, respectively, 3.5 and 13 times greater than that of the rhabdomere (about 0.0075/pm; see Section 111,D). Unfortunately, data on the specific absorbance of individual pigment granules of photoreceptors are not available because of their small dimensions. Thus two waveguide mechanisms provide a means for absolute sensitivity control and, since these manifest themselves as ultrastructural changes in the perirhabdomeric region of the photoreceptors, it is reasonable to consider both mechanisms in terms of cell biology. 5. Cell Biology and Sensitivity Control a. Cellular Manifestations of Sensitivity Control. In general, cellular manifestations of absolute sensitivity control (or dark-light adaptation) is not confined to migration of the palisade, mitochondria, and pigment granules in photoreceptors themselves-the effects most characteristic of the photopic compound eye. Other known structural adaptations are longitudinal migration of screening pigment in photoreceptors and pigment cells (both primary and secondary), movement of photoreceptor cell bodies and their rhabdomeres, and movement of cone cells and their crystalline tracts (see, for review, Horridge, 1975; Walcott, 1975). The time necessary for the above processes to be accomplished after the light is switched on are quite different: from a few seconds (fly, Kirschfeld and Franceschini, 1969) to about a minute (wasp and bumblebee, Stavenga and Kuiper, 1977) for radial pigment migration in photoreceptors; 10-15 minutes for palisade movement (locust, Horridge and Barnard, 1965; Tunstall and Homdge, 1967); 10-30 minutes for longitudinal pigment migration (moth, Post and Goldsmith, 1965); and 40-60 minutes for cone and photoreceptor cell movement (Eckert, 1968; Walcott, 1971). Apart from more-or-less detailed descriptions, nothing is known at present about the nature of these photomechanical mechanisms of sensitivity control, and only the initial steps have been made in understanding probably the simplest mechanism-light-evoked migration of pigment granules and palisade movement (see Miller, 1975). b. Light-Dependent Pigment Migration. When illuminated, pigment granules in both photoreceptors and pigment cells change their positions; they tend to congregate close to the rhabdom in photoreceptors of the photopic eye and move proximally from a distal dark position to form a continuous pigment
CELLULAR MECHANISMS OF INSECT PHOTORECEPTION
149
sheath around the retinula in the secondary pigment cells of the scotopic eye. The general features of pigment migration in both the photopic and scotopic eye appear to be similar, and they are as follows (see, for review, Goldsmith and Bernard, 1974; also Bernhard and Ottoson, 1964; Post and Goldsmith, 1965; Hoglund, 1966; Kirschfeld and Franceschini, 1969; Butler, 1971; Menzel and Lange, 1971; Franceschini, 1972; Kolb and Autrum, 1972, 1974; Menzel and Knauth, 1973, etc.). The movement of pigment is restricted to the illuminated ommatidia, or even to individual illuminated cells within the same ommatidium (including cells selectively adapted with monochromatic light), and this opposes any humoral or neuronal control of pigment migration. Carbon dioxide and narcosis make pigment granules move to light-adapted positions in both photopic and scotopic eyes, and so does cold in scotopic eyes. The extent of pigment displacement to the light-adapted position depends on the intensity of the light. Using action spectra of pigment movement dependent on wavelength and polarization, Franceschini (1972) has stated that light-induced pigment migration in the photoreceptors of the photopic eye is triggered by visual pigment itself, but little is known about the nature of the mechanism of motion. The light-induced depolarization of the photoreceptor is believed to evoke pigment migration, and consequently two hypotheses have been put forward to explain the forces which could drive pigment granules in the cell (Kirschfeld and Franceschini, 1969). According to the first (the passive movement hypothesis), granules are forced to move to the rhabdom by an electric field dependent on light-induced depolarization of the cell (see also Stavenga, 1971; Walcott, 1975). An alternative mechanism (the active movement hypothesis) is thought to be connected with the activity of microtubules (see also Miller, 1975) in a manner similar to that observed, for instance, in fish melanophores (e.g., Bikle et al., 1966). While there is no evidence to support the first view as yet, Miller and Cowthon (1974) and Miller (1975) stimulated light-induced palisade movement and radial pigment migration in photoreceptors of Limulus using colchicine, and a simultaneous decrease in the number of radially orientated microtubules was observed. However, treatment with colchicine did not mimic other anatomical changes generally evoked by light in Lirnulus photoreceptors, namely, an increase in the number of small multivesicular bodies and a decrease in the thickness of the rhabdomeres. Colchicine, being an antimitotic agent, exerts disruptive effects on microtubules in vivo (e.g., Wikswo and Novales, 1972), and its lightlike action upon pigment movement might mean that the microtubular system of the photoreceptor, when disrupted by colchicine, cannot confine pigment granules to the periphery of the cell any longer as it can in the dark. A mechanism triggering pigment movement in the secondary pigment cells of the scotopic eye has not been described, and many arbitrary possibilities can be offered. For instance, movement could be due to absorption of light by the
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F. G. GRIBAKIN
pigment granules themselves, a change in the ionic composition of the extracellular space between the photoreceptor and the pigment cell which might modify the resting potential of the pigment cell, the release of a chemical transmitter or messenger from the photoreceptors, and so on. It is interesting, however, that in the dark all the pigment granules congregate to form a dense mass in the distal region of the cell that is in its perinuclear region. Similarly, in the dark, fish melanophores show “contraction,” and their pigment also is confined to the perinuclear region of the cell (Bikle ef al., 1966). In connection with this it is useful to recall briefly what is known about pigment migration in fish melanophores (Bikle et al., 1966; Egner, 1971). In isolated scales from the dorsal side of Fundulus heteroclitus 0.1 M potassium chloride and adrenaline ( 10-3-10-5 M) cause “contraction” of melanophores, whereas 0.1 M sodium chloride causes “expansion. ” Usually, granules move along more-or-less limited paths in the cytoplasm of the melanophore processes. It seems that granules on parallel paths move nearly independently, while those. on the same path can move as single units (chains) consisting of three to five granules. Occasionally, during a short period of time units on neighboring paths may move in opposite directions. Electron microscopy shows that, in the processes of melanophores, granules are lined up along microtubules, and this is probably due to the motive function of the latter. Microtubules diverge radially from a certain locus in the cell body where a centriole is usually located, though direct connection between microtubules and the centriole has not been found. The activity of melanophores is controlled by the nervous system, and the membrane potential does not seem to be a factor causing granules to move (Egner, 1971). With reference to secondary pigment cells, an assumption should be made that the dark-adapted position of the pigment imposes a greater requirement upon the metabolic energy of the cell (cf. carbon dioxide, narcosis, and light action; see also Goldsmith and Bernard, 1974). Whether microtubules control pigment movement in these cells is still unknown. c. Palisade Movement. The term “palisade,” introduced by Horridge and Barnard (1965), is not exact and from a cytological point of view this formation is the principal endoplasmic cistern (PEC) of the photoreceptor (Gribakin, 1975). However, workers in the field often call the PEC a palisade in the literature. From the refractive index of the PEC (1.339 in the bee; see Table 11) one can infer its solid content to be about 1.7%,while that of the cytoplasm is 4% (n = 1.343). Also, the potassium content of the PEC is lower than that of the cytoplasm (Gribakin et al., 1976; Burovina et al., 1978). Thus the PEC content is mainly water, and PEC contraction and dispersion into smaller cisterns in the light, termed palisade movement, is probably accompanied by water transport. It is interesting that other processes similar to pigment and palisade movement are believed to be connected with the transport of water in different tissues from Calliphora salivary glands to the toad bladder (Berridge and Prince, 1972; Taylor
CELLULAR MECHANISMS OF INSECT PHOTORECEPTION
151
et al., 1973). These processes are also related to the action of microtubules and microfilaments which on being inhibited by antimitotic agents (colchicine, vinblastine, podophyllotoxin) cease responding to the antidiuretic hormone vasopressin (Taylor et al., 1973). The fact that in vivo transport of water is also cyclic-AMP- and calcium-dependent suggests that the mechanism of light action on both pigment migration and palisade movement has a striking resemblance to the action of hormones. The existence of a light-dependent extracellular palisade, discovered by Meyer-Rochow (1974) in the compound eye of the beetle Creophilus erythrocephalus, makes this idea even more intriguing. d. Energetics of Sensitivity Control and Entropy of the Compound Eye Structure. The above discussion of the cellular manifestations of sensitivity control closely related to optical mechanisms shows that the compound eye, when darkadapted, displays more ordered fine structure (lower entropy) than when lightadapted. Indeed, in the course of adaptation to the dark, the photopic eye photoreceptor seems to pump liquid (water?) into its perirhabdomeric cistern, and the PEC thus formed (like a balloon) presses pigment granules andor mitochondria back from the rhabdom. This process should require metabolic energy supplied by the cell. In the light, the PEC liquid become free to disperse into the cytoplasm as small cisterns, and pigment andor mitochondria are allowed to approach the rhabdom. Similarly, during dark adaptation the pigment cell of a scotopic eye retracts all the pigment granules back into its distal region where a dense pigment cluster forms, and again this process should show an energy demand, since the resulting distribution is far from equlibrium. In the light, pigment granules become more fkee to drift toward the proximal end of the cell, the distribution approaching uniformity and the process resembling diffusion. Thus, considering the change in morphology alone, one could already come to the important conclusion that, in order to realize the high absolute sensitivity inherent in the light-absorbing structures of the photoreceptor, an insect must continuously supply the eye with metabolic energy, thus providing perfect structural, hence optical, conditions for absorption. In other words, energy consumption in the dark should be more than that in the light. Two experimental findings support this view. First, as Autrum and Tschamtke (1962) have found, oxygen consumption in the dark is higher than in the light in the locust and cockroach compound eye, that is, in insects showing well-expressed movement of the palisade, pigment, and mitochondria. This is also true for the bee eye, but only at temperatures not exceeding 25°C. The opposite has been found for the Calliphoru eye (see also Hamdorf and Schwemer, 1975), which has no palisade movement but only radial pigment migration, and for the bee eye at 34°C. Second, carbon dioxide, narcosis, and cold make the screening pigment pass into the light-adapted position, and this is likely due to a deficit in metabolic energy (cf. Goldsmith and Bernard, 1974).
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F. G. GRIBAKIN
Thus we can infer that, when dark-adapted, the compound eye exhibits the lowest structural entropy and an energy consumption higher than when lightadapted, and in connection with this a conclusion may be given: the adaptation of the insect eye to the dark is the process requiring the continuous metabolic energy influx, which is necessary for the formation and preservation of perfect optical structures for a prolonged period of time; the adaptation of the eye to the light is the light-protective response to the excessive stimulation of the photoreceptors, whose normal function is photon counting. The light-protective mechanisms are on automatically, and no additional energy seems to be required. This conclusion emphasizes the principal difference between two main photobiological processes-photosynthesis and vision. Using the former, the organism utilizes the light energy. By contrast, the latter requires the energy from the organism (cf. Section II,C,2,c and Vinnikov, 1974). e. Light-Evoked Structural Changes Connected with Rhabdomeres. There are many light-evoked structural changes in rhabdomeric photoreceptors which seem to have a lesser beating on active sensitivity control; they are swelling of rhabdomeric microvilli (Rohlich and Torok, 1962; Rohlich and Toro, 1965; Gribakin, 1969a,b, 1975), diminution of the rhabdomere (White, 1967, 1968; White and Lord, 1975; Miller and Cowthon, 1974), and an increase in number of cytoplasmic organelles such as multivesicular bodies, lamellar bodies, and mixed vesicular-lamellar bodies (Eiguchi and Waterman, 1967, 1968; White 1967, 1968; Eguchi et al., 1973; Miller and Cowthon, 1974; Miller, 1975; Nemanic, 1975; Behrens and Krebs, 1976). All these changes are thought to be connected with destruction of the photoreceptor membrane in the light and its renewal during dark adaptation (Burnel et al., 1970). Destruction of the photoreceptor membrane in arthropods is believed to occur by phagocytosis at the bases of the rhabdomeric microvilli. The mechanism of photoreceptor membrane renewal is still totally obscure (Behrens and Krebs, 1976). The above changes in structure are usually obtained using high light intensities andlor prolonged illumination. For this reason, they could hardly be related to mechanisms which control absolute sensitivity in the natural light environment of the given species. Nevertheless, ultrastructural changes in rhabdomeres produced by intense and/or prolonged illumination proved to be of much use, since they have made it possible to discover cellular bases for color vision and polarized light perception in arthropods (J3guchi and Waterman, 1968; Gribakin, 1969a,b, 1975; Eguchi et al., 1973). 6. Conclusion The compound eye intended to operate in the light environment usual for given species has evolved rhabdomeres which can normally work as photon counters. To attenuate a light flux reaching its rhabdomeres from a brighter environment, the eye triggers cellular mechanisms, which optical and electrical functions are
CELLULAR MECHANISMS OF INSECT PHOTORECEPTION
153
to decrease its absolute sensitivity so that the amount of light which interacts with visual pigment within the rhabdomeres can be reduced to a level close (but not identical) to the original one. As quantitative comparison has shown (Kirschfeld, 1974), an insect rhabdom receives nearly the same (or even a greater) number of quanta as a vertebrate photoreceptor when operating in the same light surroundings, however, the compound eye pays for high absolute sensitivity, combined with extremely small size, with a loss in angular resolution (Kirschfeld, 1974).
E. COLORVISION 1. General Approach
It seems evident that the great variety of insect visual pigments considered in Section II,C,2 is a prerequisite for color vision in these animals. Moreover, since all insect visual pigments include retinal as a chromophoric group, their absorption spectra have to follow Dartnall’s nomogram (Dartnall, 1953). However, photoreceptors incorporating Dartnall’s visual pigments usually show spectral properties different from those of their native pigment, and this difference is due totally to geometry (or, which is the same, morphology) of both the rhabdom and its rhabdomeres. Since the theoretical principles of these effects have been elaborated rather well (Snyder, 1975a,b; Bernard, 1975; Gribakin and Govardovskii, 1975), only a brief account of the cellular mechanisms of color vision in insects is given here.
2. Isolated Rhabdomeres and Self Screening of Visual Pigmen2 An isolated rhabdomere is usually referred to as a “fly rhabdomere” (Snyder, 1975a,b; Gribakin and Govardovskii, 1975), and this is considered an isolated absorbing optic fiber. It is obvious from Eq. (I) (Section II,D,2) that the shape of the absorption spectrum of an isolated rhabdomere strongly depends not only on the pigment extinction coefficient a ( X , cp) but also on the product c x I (concentration times rhabdomere length). In the limit, at c X I -+ 03, all the light in the pigment absorption band could be absorbed by the rhabdomere, and consequently the absorption spectrum of the rhabdomere would show no dependence on wavelength (so-called self-screening). In other words, the greater is c X I , the greater the absolute sensitivity, but the less the spectral selectivity of the photoreceptor (see Snyder, 1975a,b; Gribakin and Govardovskii, 1975). This statement is quantitatively illustrated in Fig. 4 and 5 where real values for the extinction coefficient and pigment concentration have been taken from the results in Section I1,D. Thus the above discussion indicates that the perfect spectral selectivity necessary to evolve color vision is in conflict with the high absolute
154
F. G. GRIBAKIN
400
500
A . nm
600
FIG.4. Relative absorption A of an isolated rhabdomere versus wavelength A calculated for different rhabdomere lengths-50, 100, 200, and 300 p n . The molar extinction of rhodopsin at A ,,,ax = 500 nm has been taken equal to 40,600 litedcm-mole (Table I), and the rhodopsin concentration as M (from Section III,D,2). Waveguide effects have been ignored. 1.5 x
sensitivity requirement. In many insects this situation has been mastered by the evolution of a fused rhabdom. 3 . Fused Rhabdoms, Lateral Filtration, and Color Vision
A fused rhabdom consisting of rhabdomeres with the same visual pigments has no advantage in spectral selectivity, and from this viewpoint is totally identical to an isolated rhabdomere. This is not true if the rhabdomeres comprising the fused rhabdom contain different rhodopsins. The first experiment demonstrating different spectral cell types in the ommatidium of the honeybee worker was that of Gribakin (1969a,b). The same appeared to be true for other insects (Mote and
300
400
600
500 A . nm
FIG. 5. The relative absorption spectra A in Fig. 4 normalized to their maxima at 500 nm to illustrate the deterioration of spectral selectivity with increase in rhabdomere length (respectively, 50, 100, 200, and 300 pm). Rh is the relative absorption of rhodopsin.
CELLULAR MECHANISMS OF INSECT PHOTORECERION
155
Goldsmith, 1971; Butler, 1971, for the cockroach; Menzel and Knauth, 1973, for the ant; Kolb and Autrum, 1974, for the bee). The first theory concerning the fused rhabdom was elaborated by Snyder el al. (1973) who showed that the presence of more than one spectral type of rhabdomere within a fused rhabdom narrowed the spectral sensitivity curve of a photoreceptor because neighboring rhabdomeres acted as colored light filters for each other. Since every filter extends the full length of the rhabdom, the effect has been termed lateral filtration (Snyder et al., 1973). Further details can easily be found in the original paper, and we strongly emphasize the fact that the fused rhabdom formed by rhabdomeres with different visual pigments proved to be one of the most efficient developments in the evolution of the insect visual system which allowed perfect color vision to evolve without any loss of absolute sensitivity (Snyder et al., 1973). 4. Tiered Rhabdoms
Tiered rhabdoms are known to exist in many insects, but their physiology is not clear yet, nor is their optics. With respect to color vision, longitudinal filtration may take place in tiered rhabdoms with effects somewhat similar to those of lateral filtration. If one tried to calculate the relative absorption curves for a tiered rhabdom consisting of rhabdomeres with the same visual pigment, one would obtain curves like those shown in Fig. 6, which have never been seen experimentally. When investigating spectral sensitivity curves for the cricket Gryllus domesricus, we failed to find different color receptors and, since the animal has a typically tiered rhabdom (Polyanovskii, 1976), one would expect to obtain curves like those in Fig. 6. However, we have never seen them either (F.
h , nm
FIG.6 . Calculated normalized relative absorption spectra of proximal rhabdomeres, showing the probable effect of longitudinal filtration in a tiered rhabdom. The proximal rhabdomere is taken to be 100 k m in length, while the distal one is 50 or 1 0 0 pm long. Both rhabdomeres contain the same rhodopsin.
156
F. G. GRIBAKIN
G. Gribakin and T. M. Vishnevskaya, unpublished). Perhaps the two-peaked receptors with both peaks in the visible region (Eguchi, 1971, tiered rhabdom of a dragonfly Aeschna) can be explained in this way (Gribakin and Govardovskii, 1975) but, as measured by Eguchi, the peaks proved to be much sharper than the theory predicted, and consequently further study along these lines is needed. An alternative explanation of double-peaked sensitivity curves having no relation to the tiered rhabdom is off-axis illumination (Snyder, 1975b).
5 . Comblike (‘ ‘Crustacean-Like”)Rhabdoms Comblike rhabdoms are not typical of insects, though they have been found in several species (Meyer-Rochow, 1974, staphylinid beetle Creophilus; Frantsevich et al., 1977, the lamellicornian beetle Lethrus). Nothing is known about spectral cell types of the Creophilus retinula, while Lethrus has been found to have color vision (Frantsevich e f al., 1976, 1977) which is mediated by longwave and ultraviolet receptors. Unfortunately, we failed to observe structural changes in the Lethrus retinula during selective color adaptation (A. D. Polyanovskii and F. G. Gribakin, unpublished), and this is why the interdigitation of microvillar layers in the Lethrus rhabdom cannot be explained at present in terms of color vision. In crustaceans, however, both longwave and shortwave receptors contribute to both the sets of mutually perpendicular rhabdomeric microvilli (Eguchi et al., 1973). Since in the crayfish retinula color receptors of the same modality occupy no more than two quadrants of the square rhabdom cross section, one can assume that lateral filtration takes place in comblike rhabdoms as well. Thus we seemingly have another example of a photoreceptor system displaying color vision combined with high absolute sensitivity.
6. Double-Peaked Sensitivity Problem Wasserman (1973) has paid special attention to broad-banded double-peaked photoreceptors with the peaks located usually in the visible and ultraviolet regions, or in the green and blue regions (beta cells, unlike “tuned” alpha cells). Beta cells do not seem to be due to artifactual recordings from two different cells simultaneously, and three of many explanations may be valid: (1) self-screening of visual pigment (Section II,E,2 and Wasserman, 19731, (2) electrical coupling (Snyder et al., 1973), and (3) sensitising pigments’ (Kirschfeld et al., 1977). Broad-banded beta cells might give an advantage in a poor light environment when every quantum absorbed, no matter what its energy (wavelength), provides information on the presence of a light source. Comparative analysis of light quanta by their energies, that is, color vision, has no meaning under these conditions because of the shortage of light quanta to be compared (cf. scotopic and photopic vision in humans). Indeed, we think that the much more serious problem in color vision is to obtain narrow-banded receptors, since every visual pigment, apart from its a-absorption peak in the visible region, must display a
CELLULAR MECHANISMS OF INSECT PHOTORECEPTION
157
P-absorption peak in ultraviolet light. Nothing is known about how this problem was solved early in evolution in both vertebrates and invertebrates. Gribakin and Govardovskii (1975) have suggested that rhodopsin molecules, known to have their a-and @absorbing dipoles nearly perpendicular, might be incorporated into the photoreceptor membrane so that their f3 dipoles can avoid interaction with light waves (e.g., lie in parallel with the light path). Perhaps still unknown filtering effects suppress p absorption, or insects have mastered the technology of making “tuned” visual pigments with no additional maxima (as they have in the case of ultraviolet-sensitive rhodopsin; see Section I1,C). 7. Conclusion As the above brief survey has shown, physiological characteristics of insect color vision systems are closely related to the morphological organization of the ommatidium and its photoreceptor membrane, and such complicated evolutionary developments as fused, comblike, and probably tiered rhabdoms serve to overcome contradictory requirements for color vision and absolute sensitivity.
F. SENSITIVITY TO POLARIZED LIGHT Polarization sensitivity (PS) in arthropods is thought to be a by-product of the high absolute sensitivity demand in the compound eye with rhabdomeric photoreceptors (Laughlin et al., 1975; see Section II,D) and is totally due to the orientation of rhodopsin molecules in the photoreceptor membrane or, in other words, to dichroic properties of the membrane. The theory of polarized light perception in arthropods has recently been presented in detail (Snyder, 1973, 1975a,b; Gribakin, 1973, 1975; Gribakin and Govardovskii, 1975; Waterman, 1975; Menzel, 1975a), and the reader is referred to the literature for the fundamentals of the theory. Here we give only a simplified outline of the problem. An individual rhabdomeric microvillus made of a universal photoreceptor membrane should show a dichroic ratio of 2 (Moody and Parriss, 1961). In the case of an isolated rhabdomere, the longer the rhabdomere, the lower its dichroic ratio, because of rhodopsin self-screening (Shaw, 1969b). Thus an isolated rhabdomere made of a disk membrane fails to display a PS of more than 2, and this is true for cells 1 through 6 of the dipteran retinula (Kirschfeld and Snyder, 1975). Waveguide effects may increase the PS of a thin, isolated rhabdomere, and this probably occurs in central dipteran rhabdomeres (R7 and R8) which are as small as 1 Fm in diameter (Snyder, 1973). A comblike (crustacean-like) rhabdom makes it possible to gain PS identical to the dichroic ratio of a single microvillus without any loss in absolute sensitivity (plus color vision) (Shaw, 1969b; Snyder, 1973). The fact that photoreceptors with comblike rhabdoms in insects mainly show a PS of about 2 (Meyer-
158
F. G. GRlBAKW
Rochow, 1974, Creophilus; Gribakin, 1979, Lethrus) is in favor of a universal membrane covering the microvilli. A fused rhabdom is difficult to analyze, since the PS of a given photoreceptor contributing to the rhabdom depends on morphological arrangement, crosssectional area, dichroic ratio of the microvilli, and the spectral sensitivities of all the rhabdomeres forming the rhabdom (Snyder, 1973), as well as on the twisting of the rhabdom (Grundler, 1974; Wehner et al., 1975; Wehner, 1976). Owing to optical coupling between the rhabdomeres of the fused rhabdom, the PS of a given photoreceptor may theoretically vary from a value much less than the dichroic ratio of a microvillus to a value greater than the dichroic ratio (Snyder, 1973). The most complete study of PS has been performed on the honeybee compound eye. Started in our laboratory as a morphological study more than 10 years ago (Gribakin, 1967a,b), it now shows that the low PS measured in honeybee photoreceptors (Menzel and Snyder, 1974) can be explained by twisting of the rhabdoms (see, for instance, Wehner, 1976), which rules out the hypothesis on electrical coupling suggested by Menzel and Snyder (1974). It appeared that only a small basal cell (the ninth cell), which seemed to be an ultraviolet light receptor (Gribakin, 1972), could display the high PS (up to 9) experimentally found by Menzel and Snyder (1974, also see Menzel, 1975a). Twisting of the rhabdom eliminates the filtering effect of distal ultraviolet receptors upon the ninth cell, and this makes the filter mechanism of polarized light perception in the bee suggested by Gribakin (1973) rather questionable, although it may operate in central rhabdomeres of dipterans (see also Snyder, 1973). If so, one must assume that in the ninth cell rhabdomere rhodopsin molecules are preferentially oriented to provide a dichroic ratio much higher than 2. Actually, a microvillus produced by rolling up the universal photoreceptor membrane must show a dichroic ratio of less than 2, that is, 1.64 (Gribakin and Govardovskii, 1975) or 1.67 (Laughlin et al., 1975). The theoretical limit for the dichroic ratio of a microvillus is 20 when ideal dipoles are all aligned along the microvillus, and about 6.5 in the case of nonideal dipoles (Laughlin et al., 1975). The most complete theory of the microvillar dichroism was advanced by Goldsmith and Wehner (1977). These authors also experimentally showed that the absorbing dipoles were aligned within k 50" of the microvillar axes in the crayfish Orconectes. In conclusion, we think that a systematic investigation of PS mechanisms in insects is needed not only to understand neural mechanisms of astroorientationin insects but mainly for a better understanding of cell membrane structure and function.
G. FINALREMARKS Light has made it possible for humans to unravel many mysteries of the structure of matter. Similarly, photoreceptor optics as applied to the cellular
159
CELLULAR MECHANISMS OF INSECT PHOTORECEPTION
mechanisms of photoreception in insects, provides a clue to the functional morphology of insect photoreceptors and, in particular, their photoreceptor membrane. However, both the functional morphology and structural lability of insect photoreceptors remains to be further investigated using methods of cell physiology. With this in mind we proceed to the electrical events resulting from the absorption of a photon by the photoreceptor membrane.
111. Electrical Basis for Insect Photoreception
A. INTRODUCTION: THECOMFOUND EYEAS
A
VOLUME CONDUCTOR
Unlike a common neuron which transmits a signal as a local change in membrane conductivity traveling along the full length of its axon and manifested locally as a nerve impulse or spike, the arthropod photoreceptor transmits a signal electrotonically, that is, by a change in direct current flowing through its axon, and this has been unambiguously proved by Shaw (1972). In the transmission of a signal by direct current, one could consider the compound eye a volume conductor with many similar embedded electrical sources (i.e., cells, in both the biological and electrical sense) as well as with many similar electric (ionic) currents produced by these cells. Then the light effect on the compound eye extracellularly manifested as an electroretinogram (ERG) may be thought of as a disturbance of the electrical equilibrium peculiar to the eye in a state of dark adaptation. Thus we must first consider the potentials, currents, and resistances inherent in the eye in the dark and their changes during illumination.
B. DC PARAMETERS OF THE COMPOUND EYEIN
THE
DARKAND IN
THE
LIGHT
1. Extracellular dc-Parameters of the Eye a. Dc Potentials. Our present knowledge of extracellular dc potentials (DCP) is much more limited than that of ERGS, and only several studies dealing with phenomenology rather than with the nature of DCPs have been performed (Burtt and Catton, 1964a,b; Cosens, 1967; Mote, 1970; Heisenberg, 1971). On reinvestigating DCPs in our laboratory, we have confirmed many previous findings and so are able to discuss the DCP problem with regard to our own experience. A typical DCP profile characteristic of the locust (Burtt and Catton, 1964a) is shown in Fig. 7. Two more curves are superimposed on the DCP profile. The first is the voltage difference A I/ measured between two electrode tips separated constantly in depth when penetrating an eye through which dc pulses have passed (Shaw, 1975); this curve reflects the axial resistance of the eye tissue. The
160
F. G. GRIBAKIN
V
4014aV
ERG mV
FIG. 7. Dc-characteristics of the locust compound eye. The solid line is the DCP profile V ( x ) , where x is the depth from the eye surface (Locusra migratoria, modified from Fig. 2a of Bum and Catton, 1964a). The dashed line is the voltage difference A V between two electrode tips separated by 115 p m in depth as 5-pA dc pulses pass through the eye from the cornea toward the basal membrane. The dashed-dotted line is the ERG peak amplitude. (Both curves modified from Shaw (1975) for Schistocerca gregaria). Top inset: Schematic sections of the Locusta (L) and Schistocerca (S) compound eyes according to Bum and Catton (1964a) and Shaw (1977). d, Dioptric apparatus (lens and cone); r, retinula; bm, basal membrane; lam, lamina; med, medulla; hc, hemolymph channel.
second curve is the ERG amplitude with depth (also, according to Shaw, 1975). In spite of the fact that the DCP profile has been measured in Locusta, and A V and the ERG in Schistocerca, there is not much difference in the size of the ommatidia in the two species, since, first, their basal membranes are located within a narrow range of depth (550-650 pm) and, second, the null point of the ERG is a clear indication of a distal border of the first negative voltage peak, and this is seen in Fig. 7. [For the null point of the ERG see, for instance, Fig. 2 in Cosens (1967); also, F. G. Gribakin and A. M. Petrosyan (personal observation) have found that the null point of the ERG is inevitably located at a level where the first peak reaches about half its amplitude, both in the locust and cricket]. According to Bum and Catton (1964a), the DCP profile of the eye may show three negative peaks coinciding with the first (lamina), second (medulla), and third (lobula) optic ganglia, and we have observed all three peaks in the locust, cricket, and cockroach (see Petrosyan, 1977a). The nature of all three peaks seems to be similar, and we deal only with the first. In brief, the properties of the first peak are as follows (Burtt and Catton, 1964a; Cosens, 1967; and personal
CELLULAR MECHANISMS OF INSECT PHOTORECEPTION
161
observations of Gribakin and Petrosyan). The first peak has no relation to the basal membrane, and its rising phase (i.e., the fall in potential in Fig. 7) coincides exactly with the distal border of the lamina. The highest peak value can be recorded only once-when the eye is first penetrated, though with a thick micropipet (a resistance of about 1 megaohm); during withdrawal, a second insertion, or when the electrode tip is left at the depth of the peak for several minutes, the peak displays strong damping, so that the potential profile can be totally smoothed out. However, if the eye is penetrated once again, but about 50 or 100 p m away, the negative peak is undisturbed. Thus the first peak is generated by local axial electrical sources situated at the outer border of the lamina; axial damage to the tissue from the electrode tip can abolish the peak only locally and the damaging effect does not spread radially to neighboring ommatidia. However, the peak can be greatly diminished over the whole eye by anoxia (to some extent, reversibly), while mechanical trauma to the lamina abolishes it irreversibly (Bum and Catton, 1964a). In general we recorded many normal three-peaked DCP profiles, though some of them showed no negativity near the cornea. Instead, a gradual increase in negativity along the full length of the ommatidium was observed. The effect of light on the DCP profile depends on where the reference electrode is placed. If it is in the insect body (in hemolymph), the DCP profile “seesaws” (Cosens, 1967) about the null point of the ERG profile, with more light-evoked negativity distal to the null point and with more positivity proximal to this point. In other words, the profile of the quasi-static ERG is the difference between the dark and light DCP profiles. With the reference electrode placed just beneath the cornea, the DCP profile can be shifted by light only positively, with no “seesaw” and with practically no change in potential until a depth of 400-500 p m is reached (in the locust). This means that using a corneal reference electrode no ERG can be recorded up to 400-500 pm. The effect of the reference electrode position on both the light DCP and ERG profiles could easily be explained by the presence of hemolymph channels just beneath the basal membrane, and these might serve as a ground point (the axis of the “seesaw”) when the reference electrode is located far away in hemolymph. The existence of these channels was postulated four years ago by A. D. Cherkasov of Moscow State University (personal communication) as an explanation of the ERG change in sign with depth, and experimentally the channels have recently been observed by Shaw (1977). The hemolymph channels appear to transfix the visual tract of the locust anterioposteriorly in between the basal membrane and the lamina where numerous trachea also have been found (Burtt and Catton, 1964a). Thus both the eye and the lamina have not only an excellent feeding system and oxygen supply, but also the ground point for ERG. There is no doubt that light-evoked changes in the DCP profile are produced by extracellular photocurrents of photoreceptors, as well as by the ‘‘response
162
F. G. GRIBAKIN
currents" of laminar neurons which respond to light with hyperpolarization (2ettler and Jhilehto, 1971), and the next question is what currents flow extracellularly within the compound eye in the dark and in the light. b. Dark Current and Photocurrent. In order to determine the direction of extracellular current flowing axially in the dark within the eye (i.e., parallel to its ommatidia), one must record the DCP profile V ( x ) ,where x is the depth from the eye surface. Then a profile of the axial electric field can be found as E ( x ) = -dV(x)/dx, where the minus sign means that the current is defined as a flux of positive charges forced to move by the electric field E ( x ) which flows in the direction in which V ( x ) decreases. The calculated profile E ( x ) (Fig. 8) clearly shows that the extracellular axial current directed by E (x) has to reverse its sign somewhere in the depth of the eye. (Note that E ( x ) does not depend on a shift of the whole V ( x ) curve along the V axis, hence does not depend on where the reference electrode was placed when V ( x )was recorded.) It appears that, even in the dark, three current loops exist within the eye zone considered (Fig. 8). If distal negativity is absent, the first loop disappears and the second one spreads, covering the whole retinula and possibly the cone cells. Since every current loop is to be closed, the photoreceptors and neurons of the lamina serve as the return
- 9 1
, & ' I'
"
.+. -i. -l -- -- .---,[4 - -., ..--*.,..U' E
5-
"
"
1 -*-'
I-,
V
mV
FIG.8 . Dc characteristics of the locust compound eye. The solid curve is the smoothed DCP profile V ( x ) , as in Fig. 7. The dashed curve is the electric field E ( x ) given as a derivative of V ( x ) with respect to depth from surface x taken with a reverse sign since E(x) = - dV(x)/&. The graph of E ( x ) characterizes the direction of the current flowing along the ommatidia but outside them. Top inset: Three possible current loops in the dark resulting from the sign of the graph E ( x ) obtained.
CELLULAR MECHANISMS OF INSECT PHOTORECEPTION I
'
'
'
'
'
f
'
'I
-- -- - --
1
I
'
' I '
I
'
'
163
I
- - - I
It *---\--+.:-,\--*.-I
c-
I _ _ _ _ -
- 1 5 ERG
"wires" for the extracellular currents and probably as their sources. A quantitative evaluation of the dark currents in the compound eye demands an accurate knowledge of tissue resistance, however, their approximate values may be given as follows. Near the basal membrane the electric field E is about 0.13 V/cm (taken from Fig. 7 or 8). Taking into account that pigment cells may serve as a conductive medium (which is thought to be true for the glial processes in the brain; see Ranck, 1963) with a specific resistance p of about 80-100 ohm-cm, we obtain the dark cumnt density as j = E / p = 1.3 x Alcm2. At the basal membrane the axonal bundle of a locust retinula has a diameter d of about 4 pm, and the mean distance D between the axes of adjacent retinulas is about 8 p m (F'etrosyan, 1977a). Then a conductive extraretinular cylinder has the crosssectional area S = d 4 (D2- &) = 3.8 X lo-' cm2, and the extraretinular dark current i d per retinula about the basal membrane is i d = j X S = 5 X A, that is, 0.5 nA per retinula (or, divided by eight cells, 0.06 nA per photoreceptor). A whole eye consisting of 8000 ommatidia (locust) would show a dark current of 4.0 pA at the basal membrane. In the light, the extracellular dark current changes, and this change can be termed the extracellular photocurrent. The additional voltage drop produced by photocurrent flowing through extracellular (more exactly, extraretinular) resistance can be measured as an ERG. From the ERG amplitude profile with depth (Fig. 7), one can determine that extracellular photocurrent underlying the ERG flows toward the cornea, that is, opposite the dark current. In other words, light diminishes the dark current (Fig. 9). If the specific resistance of the lamina were of the same order of magnitude as that between the cornea and basal membrane, one could expect maximal photocurrent density somewhere in the lamina where
164
F. G . GRIBAKIN
FIG10. A possible current path for pulses passed througb the eye to measure its resistance. The current electrode A is at the cornea, and the current electrode B is in the hemolymph. The resistance profile can be obtained in two ways: (a) two electrodes with tips separated longitudinally move from the cornea along the x-x line in order to measure the potential difference A V arising in tissue when current pulses pass from A to B (see Shaw, 1975); (b) the reference electrode is located near the electrode A, and the probe electrode is inserted by steps along the x-x line (Cribakin and Petrosyan, hitherto unpublished). Both potential profiles reflecting the eye resistance are shown qualitatively as curves a and b. R, Retinula; PC, pigment cells; L, lamina; H, hemolymph; bm, basal membrane.
the electric field of the ERG is maximum (Fig. 9). A maximum ERG amplitude, and consequently zero axial photocurrent, may indicate that these are the terminals of axons through which all the photocurrent produced by photoreceptors flows outward in the synaptic region. A quantitative evaluation of the ERG photocurrent cannot be made, since the number of stimulated ommatidia is not known as a rule from the studies available. However, from the fact that dark current and photocurrent produce similar voltage drops in the extracellular medium of the eye in routine experiments with ERG recordings, one can infer that the photocurrent at the basal membrane is within the same limits as the dark current, that is, about 0.1 nA per receptor (though it strongly depends on the light intensity). Comparisons between the dark current loops (Fig. 8) and the photocurrent loops (Fig. 9) show that it is the second dark current loop which shares a current path with the photocurrent at the retinular level, and this indicates that the receptor signal can be interpreted as a modulation of the dark current within the second loop. Thus the principal problem to be solved concerns what emf sources are responsible for this second loop current. Of course, not only photoreceptors but, equally, neurons of the lamina may appear to be involved. c. The Resistance Profile. The actual resistance of the compound eye is not known exactly, and only two attempts to determine it have been made. Shaw (1975) measured the voltage drop A V ( x ) between two electrodes with tips 115
CELLULAR MECHANISMS OF INSECT PHOTORECEPTION
165
p m apart in depth when 5-pA current pulses were passed axially through the eye of a locust (Fig. 7). The resistance profile found by Shaw can easily be interpreted if again the existence of hemolymph channels (Shaw, 1977) is taken into account. Assume that the paired electrodes are inserted into the eye along the x-x line as shown in the model in Fig. 10. If the basal membrane is a resistance barrier (Shaw, 1975), the current will flow from the pigment cells and extracellular space into the photoreceptors, and this will cause a decrease in AV with depth. However, as soon as one of the electrodes penetrates a hemolymph channel, a sharp rise in the potential AV will be measured. Then, when it is inserted deeper, A V must change its sign, since the current pulse in the lamina has the opposite direction between the outer border of the lamina and the terminals of retinular axons. When the electrode is inserted still deeper, A V becomes positive again (cf. Figs. 10 and 7). However, if the reference electrode is placed near the current electrode A (Fig. lo), and the probe electrode is inserted by steps into the eye, a different profile like that shown in Fig. 10 (curve b) can be recorded (F. G. Gribakin and A. M. Petrosyan have used this second method in our laboratory; see Fig. 11). Then, a sudden jump in potential at the basal membrane reflects the penetration of a hemolymph channel but, when the electrode reaches the lamina, the potential difference thus measured drops again. The value of the postbarrier potential divided by the pulse current might be taken as the approximate resistance of all the photoreceptors connected in parallel. Indeed, if the axons are the only conductors inside the eye connected to the inside of the lamina (for current
FIG.1 1 . The voltage drop V between a corneal reference electrode and a probe electrode inserted stepwise into the eye when current pulses have passed through the eye as shown in Fig. 10. Note that the voltage drop appeared to be nearly the same in three preparations beyond the resistance barrier. This voltage divided by a 5-c~.A cumnt pulse gives a cornea-lamina resistanceof about 4-5 kilohms. (F. G . Gribakin and A. M. Petrosyan, hitheno unpublished; Locusta preparations 17-L, 18-L and 19-L.)
166
F. G. GRIBAKIN
pulses which then flow out of the lamina through its envelope), then all the photoreceptors have plasma membranes (or input resistances) connected in parallel. Taking the number of photoreceptors in the locust eye as about 60,000 and the input resistance of a single photoreceptor as about 10 megaohms (Shaw, 1969a), we obtain the eye resistance for a uniform current pulse of 170 ohms. Then, taking the average axon length equal to 200 p m and its diameter 1 pm, we obtain a single axon resistance, 100 megaohms (with a specific axoplasm resistance of 100 ohm-cm). Further, if connected in parallel, all 60,000 axons would show a resistance of 2 kilohms. Subtracting this value from the total cornealamina resistance (Fig. l]), we obtain 2-3 kilohms which are probably due to synaptic membranes. As a rule, a potential difference of 10-15 mV exists in the dark between the extracellular interior of the eye and that of the lamina. This voltage difference is probably due to total dark current flowing through the axons, and again we obtain the total dark current of the eye equal to 2.5 p A , taking a parallel axonal resistance of 4 kilohms and a voltage difference of 10 mV (cf. 1.5-2 p A in previous paragraph). d. Conclusion. The compound eye must be considered a volume conductor showing a complicated pattern of extracellular DCPs and currents existing even in the dark. Nothing is known at present about the cellular mechanisms responsible for this dark electrical activity which does not seem to reflect exclusively the metabolism of the compound eye but undoubtedly underlies its principal physiological function. 2. Intracellular Dc Parameters of the Photoreceptors and Accessory Cells a. Resting Potentials. Resting potentials of photoreceptors have not been measured systematically, though a precise knowledge of the potential difference between the inside of a photoreceptor and its extracellular environment is necessary for an understanding of the ionic mechanisms of both dark emf’s and the photoexcitation process. To fill the gap, in part, we have made measurements of resting potentials of the photoreceptors and the cone cells in three insect species and have found that the resting potentials, first, are quite different in the photoreceptors and cone cells of the same species (though both cells are surrounded by the same extracellular medium) and, second, differ within the same cell type in different species (Gribakin and Petrosyan, 1979; see Table IV). Relatively low resting potentials of photoreceptors might be due to a slight permeability of their membranes to sodium in the dark, as Fulpius and Baumann (1969) have suggested in order to explain the deviation of the drone bee photoreceptor resting potential from the Nernst equation produced by a change in external potassium. Also, appreciable sodium permeability might explain why resting potentials of photoreceptors is lower in species with a higher sodium level in the hemolymph (Gribakin and Petrosyan, 1979). Here two possibilities are considered to explain
167
CELLULAR MECHANISMS OF INSECT PHOTORECEPTION TABLE IV RESTINGPOTENTIALS OF ~OTORECEFTORS AND CONECELLS~ ~
~
~~~~~~~~
Resting potential (mV) Photoreceptors Insect species Locusra rnigratoria Periplaneta americana Gryllus domesticus
Cone cells
Extreme values
Mean T SD
Extreme values
Mean fSD
15-75
42?13(n=40)
20-95
6 0 k 16(n = 51)
10-65
32?lO(n=72)
30-70
55 k 10(n = 56)
5-45
23 f 1 1 (n = 45)
15-85
49 4 16(n = 36)
the relatively low photoreceptor resting potential, and the photoreceptors of the house cricket G. domesticus are taken as an example. The first possibility is based on two statements: (1) the lower resting potential of the photoreceptor is due totally to the noticeable permeability of its plasma membrane to sodium ions, and (2) extracellular concentrations of sodium and potassium are the same as those in the hemolymph. An alternative explanation rests upon the assumption that sodium permeability in the dark is negligible and that statement 2 is not true since the extracellular ionic content in the nerves and ganglia of many insects greatly differs from that of their hemolymphs (see review of Treherne and Pichon, 1972). For the first possibility, the sodium/potassium permeability ratio may be calculated from the Goldman equation:
Em = 58 log,,
W+I,+ b"a+I,
[K+],
+ b[Na+Ii
where E m is the average resting potential measured (see Table IV); [K+],, [K+II, "a+],, and [Na+II are the concentrations (or, to be more exact, activities) of potassium and sodium inside and outside the photoreceptor; and b is the sodiudpotassium permeability ratio. The potassium concentration in the cytoplasm [K+Ii is taken equal to 120 m M as revealed by x-ray microprobe analysis (Gribakin et al., 1976; Burovina et al., 1978). The sodium content [Na+Ii is taken equal to 55 mM, since flame photometry data have given a concentration of 55 mM as the mean for the whole eye (Petrosyan et al., 1977), while electron cytochemistry has demohstrated sodium pyroantimonate precipitate, mainly in the photoreceptors (Petrosyan, 1977a,b). "a+], and [K+], are taken as equal to
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208 and 8 mM for cricket hemolymph (Petrosyan et al., 1977). For the mean E = 23 mV (Table IV), the value of b = 0.21 is obtained which appears to be too high in comparison with the smaller b value postulated from the experiments of Fulpius and Baumann (1969) in which the total removal of external sodium resulted in only slight hyperpolarization of the drone bee photoreceptor (about 5 mV). For the second explanation, “a+], and “a+]* can be totally ignored, since sodium permeability in the dark is assumed to be negligible. Then, a similar potassium content in the extracellular medium might be expected when calculated from the resting potentials of either cone cells or photoreceptors as both having the same extracellular surroundings. From our x-ray microprobe data, averaged [K+] is 170 mM in the cone cells and 120 mM in the cytoplasm of a photoreceptor (Burovina et al., 1978). Using &. (12), with b = 0, one can find that [K+], for the cricket cone cell (resting potential 49 mV; see Table IV) and the photoreceptor (resting potential 23 mV) are 24 and 48 mM, respectively. This 2-fold difference in calculated values of [K+], is probably due to many reasons and, particularly, to the fact, that we used concentrations, not activities, here which are still unknown. There is much evidence that neither photoreceptors of insects (drone bee, Fulpius and Baumann, 1969) nor their neurons (Treherne and Pichon, 1972) are potassium electrodes, since their E m versus [K+], plots show a lesser slope than that calculated using Nernst’s equation. However, H. M. Brown (1976), using potassium-sensitive intracellular microelectrodes, showed that, in Balanus photoreceptor, [K+] i increased with growth in external potassium concentration [K+],, and this accurately explained the difference between the experimental and theoretical Em versus [K+], plots. The principal conclusion of H. M. Brown was that the plasma membrane of the balanus photoreceptor does behave as a potassium-sensitive electrode. In conclusion, we think that the relatively low resting potentials of insect photoreceptorsmay serve as an indication of a higher extracellular potassium content in the eye rather than of their noticeable permeability to sodium in the dark. It should be noted that the higher resting potentials of the cone cells make them similar to glial cells which inevitably display resting potentials higher than those of adjacent neurons; this is thought to be due to their possible role as a mechanism controlling the extracellular potassium level in nerve tissue (see review of Somjen, 1975). b. Dark Current of a Single Photoreceptor. In Section III,B, 1 ,b extracellular axial dark current in the compound eye has been described and the photoreceptors were considered the return wires. Practically, the reverse is more convenient with a photoreceptor as the direct conductor through which two intracellular dark currents flow; the first flows proximally in its distal region, and the second distally in its proximal region, including the whole axon (Fig. 8). To avoid misunderstandings connected with the terms “proximal” and “distal,
”
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which are related to both region and direction, we term these currents the receptor dark current (with its receptor loop) and the axonal dark current (with its axonal loop). Nothing can be said at present about the cellular origin and ionic carriers of these currents, though the receptor current might be related to an ionic pump mechanism which seems to be inherent in the distal third of the photoreceptor where the bulk of its mitochondria is located (seee.g., review of Gribakin, 1969~).The axonal dark current can be considered to be generated by the electrical source responsible for the first DCP peak, and consequently located in the lamina. Thus the axonal dark current does not seem to be due to photoreceptor electrical activity but probably to that of the first neuron in the lamina. From this an interesting speculation arises that the receptor dark current reflects mainly metabolic functions of the photoreceptor necessary to provide its high absolute sensitivity (and, probably, the ionic gradient needed), while it is the first neuron which continuously sends its “inquiry current” through the receptor axon to determine the state of the receptor. If so, the receptor might modulate this “inquiry current” upon absorption of a photon, and this modulation might be what we know as the receptor signal. Only further studies will c o n f i either the above speculation or the more prosaic conclusion that the axonal dark current is no more than a simple background phenomenon having nothing in common with the receptor signal. At any rate, at present we can confirm the presence of two intracellular dark currents in a photoreceptor and calculate the axonal dark current to be 0.06 nA in the locust (Section III,B), which corresponds to about 4 x 108 unit charges per second. A voltage drop produced by the dark current through the axon can be as small as 6 mV, taking the dark current as 6 x lo-” A and the resistance of the axon as 10’ohms. A voltage drop characteristic for the synaptic membrane of the locust photoreceptor in the dark is obtained as the total dark current of the eye (4 pA) times the total synaptic membrane resistance (2-3 kilohms), which gives 8-12 mV, negative inside.
C. PHOTORESFONSES AND THEIR CELLULAR MECHANISMS 1. Photoresponses
Several types of photoresponses inherent in the compound eye can be recorded intra- and extracellularly. First, intracellular miniature potentials, discrete waves, or quantum “bumps,” according to different terminology. We use the latter term “bumps,” which is more felicitous (cf. Levinson, 1972) and concise, though “miniature potentials” apparently is more correct (e.g., Tsukahara and Homdge, 1977). Second, also intracellular, is the receptor potential, and this term is thought to be more suitable in comparison with the term “generator potential” used, for instance, by Fuortes and O’Bryan (1972a,b); in our opinion,
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the term “generator potential” reflects rather an active, regenerative process, hence it should be used to designate the receptor repetitive spikes, if any. In turn, four components of the receptor potential are, consecutively, the spike, transient, plateau, and prolonged depolarization afterpotential (PDA) (Goldsmith and Bernard, 1974; Wulff and Mueller, 1975; Hamdorf and Razmjoo, 1977). Third, the extracellularly recorded photoresponse is the ERG which occurs as a voltage drop produced by net photocurrents of the receptors in the extracellular medium of the eye. In this section we consider these photoresponses only from the viewpoint of their cellular origin.
2. Quantum Bumps Quantum bumps in arthropod photoreceptors are thought of as electrical manifestations of single photon events (see, for review, Fuortes and O’Bryan, 1972b), but whether or not these are one-to-one transformations is still not clear. Rare bumps are clearly seen even in complete darkness (Yeandle and Spiegler, 1973, for Limulus; Tsukahara and Horridge, 1977, for the locust), and with an increase in illumination they fuse to give rise to the receptor potential. If considered voltage noise during the receptor potential, bumps correspond to a standard deviation of 1.55 mV in the cockroach, 0.7 mV in the fly, and 0.6 mV in the bee (Smola, 1976). On the assumption that one light quantum triggers a mechanism generating one bump, Levinson (1972) has defined the energy gain as bump energy related to photon energy and has obtained the value of lo6 for Limulus photoreceptors. According to another definition, the gain is expressed as an increase in “particles,” or current carriers, and in this sense the gain in Limulus photoreceptors appears to be about 6 x lo8 unit charges per absorbed photon (Levinson, 1972). For the locust, the energy gain can be approximately evaluated, taking the bump amplitude as 3 mV, the duration of the bump as 0.1 second Psukahara and Horridge, 1977), and the cell input resistance of 10 megaohms (Shaw 1969a) as 2.5 x lo5 per photon with a wavelength of 500 nm; this value is of the same order of magnitude as that in Limulus. The amplification thus found favors the presence of a yet unknown process which has to intervene between photon absorption by the photoreceptor membrane and a change in potential across this or, perhaps, the plasma membrane of the cell. The transduction process is seemingly mediated by one or more intracellular transmitters. Pak et al. (1976) demonstrated that, in norpd Drosophila mutants with phototransduction blocked totally or in part, neither rhodopsinmetarhodopsin photoisomerizationnor bump-generating machinery had been disturbed. They concluded that the norp-A mutation affected a step which preceded bump production. This step may, for example, be involved in the release of a substance which in turn produces quantum bumps (pak et al., 1976). The fact that bumps remain rather a long time after a strong light is switched off (and consequently when there are no light quanta to be absorbed) may also be in favor
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bump generation through an intracellular transmitter produced by light in excess, which continues to act for some time in the dark (Minke et al., 1975; Tsukahara and Horridge, 1977). According to Martinez and Srebro (1976), a decrease in extracellular calcium leads to an increase in the latency of the bumps and their variability (this effect is probably due to a subsequent decrease in intracellular calcium). Lowering the temperature has given the same result. However, since the quantum efficiency of the process remains the same in both cases, Martinez and Srebro have concluded that calcium probably affects the rate constant of one of the intermediate reactions. From the fact that, the higher the extracellular calcium, the lower the amplitude of the bumps (Millecchia and Mauro, 1969; Martinez and Srebro, 1976), one can assume that another function of calcium is to reduce bump amplitude in the light, for bumps are known to be smaller in the light (Dodge et al., 1968; Fuortes and O’Bryan, 1972b; Srebro and Behbehani, 1974, for Limulus; Tsukahara and Homdge, 1977, for the locust). On the contrary, in trp mutants of Drosophila bumps show no decrease in amplitude in the light, and so Minke et al., (1975) suggested a lower efficiency of bump production in the light. Thus there is no clear distinction so far between a diminution of the electrical response per photon absorbed at higher intensities (i.e., a fall in bump amplitude) and a decrease in the relative number of photons (divided by incident) per rhodopsin molecule provided by cellular mechanisms of absolute sensitivity control (see Section II,D, and Tsukahara and Horridge, 1977). The last stage of bump generation is generally beIieved to be the activation of sodium channels by a hypothetical transmitter, but whether the number of the transmitter molecules in one transmitter quantum is a constant and, if so, the number of channels it can activate simultaneously are not known (Tsukahara and Horridge, 1977). It also remains to be clarified whether bumps really fuse to form the receptor potential or the two electrical responses are based on different cellular mechanisms, even with different cellular membranes involved, so that bumps are simply masked by the larger receptor potential at higher intensities. At any rate, the trp mutant of Drosophila has shown a “bumpy” receptor potential with no fusion of bumps (h4inke et al., 1975). 3 . Receptor Potential a. Spike. An initial spike has been found to contribute to the receptor potential in many arthropods (see review of Goldsmith and Bernard, 1974; Wulff and Mueller, 1975; Baumann, 1975), though it does not seem to be a requirement in insects. For example, we have observed the spike in the beetle k t h r u s but never in the locust (neither have Tunstall and Honidge, 1967) or the cricket. In the drone bee the spike is due totally to a regenerative inflow of sodium ions into the photoreceptor, since it is abolished in a sodium-free solution (Fulpius and Baumann, 1969) and can be blocked reversibly by tetrodotoxin (Baumann, 1968,
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1975). Upon withdrawal of the recording microelectrode from the cell, the spike does not change its polarity, unlike the other components of the receptor potential, and this means that the spike is generated by cellular elements other than the plasma membrane (Baumann, 1968, 1975). According to Naka and Eguchi (1962), the spike originates in the axon, though the photoreceptor membrane might be a better candidate for this response (Wulff and Mueller, 1975, for Limulus). If so, the fact that the spike is the earliest of the light-evoked ionic processes in the photoreceptor might result from a sodium permeability increase in the photoreceptor membrane itself, whereas both the transient and plateau develop as the result of an intracellular transmitter action upon the peripheral plasma membrane of the photoreceptor. b. Transient, Plateau, and Related Cellular Mechanisms. The plateau is the third component of the receptor potential on the time scale (after the spike and transient), however, it is the first resulting from bump fusion at intensities which still remain low in order to initiate spike and transient generation. Only at comparatively high intensities is the plateau preceded by a transient and, when a firing level of depolarization is reached, the transient in turn may be preceded by a spike. Wulff and Mueller (1975) hold that in the lateral eye of Limulus the plateau originates in the photoreceptor membrane of the rhabdomere, as the spike does, while the transient is generated in the plasma membrane of the receptor. In favor of this is the fact that the reversal potential for the spike and plateau is the same-about +16 mV, while that for the transient is only +4 mV. From the positive sign of the reversal potential, Wulff and Mueller (1975) have deduced that not only the spike but both the transient and the plateau are also due mainly to sodium influx in the light. Apart from the Limulus lateral eye, a sodium permeability mechanism seems to operate in Limulus ventral photoreceptors, the barnacle ocellus, the compound eye of the crayfish and hermit crab, and the compound eye of the drone bee (for review, see Wulff and Mueller, 1975). A controversy, however, has developed concerning the well-known fact that photoreceptors of arthropods are capable of maintaining the receptor potential, though it is markedly decreased, in sodium-free solutions (Fulpius and Baumann, 1969; Baumann, 1975; Wulff and Mueller, 1975); and in general this ability of arthropod photoreceptors is not unique among the sensory end organs of animals (see, for instance, Brown and Ottoson, 1976). In order to resolve this contradiction, an assumption has been put forward that even in a sodium-free solution a significant amount of sodium is preserved close to the plasma membrane of the receptor (Stieve, 1964, for arthropod photoreceptors; Ottoson, 1964, for muscle spindles). This assumption has proved to be very useful, and sodium pump activity has been suggested as a factor responsible for maintaining the sodium concentration gradient across the photoreceptor cell plasma membrane even in a sodium-free solution (Wulff et al., 1975, for Limulus). However, Wulff et al. (1975) note that the inherent weakness of their hypothesis is that “rigorous proof
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rests with the demonstration of sodium concentrations in the various compartments associated with the Limulus lateral eye retinular cells, the techniques for which are not at hand. ” Indeed, the principal demand of the above assumption is that sodium be stored in either accessory cells of the eye (pigment cells or cone cells) or in the photoreceptors themselves. According to our measurements, the sodium content of the insect compound eye is relatively large, about 55 meqkg wet weight in the house cricket and 14 meqkg wet weight in the locust (Petrosyan et al., 1977). When tested by electron cytochemistry using a potassium pyroantimonate technique to precipitate sodium, the bulk of the deposit was found in the photoreceptors and not in the other cells of the eye (Petrosyan, 1977a,b, for cricket)’. X-ray microprobe analysis has shown the sodium content in cricket photoreceptors to be 40-70 mM (Gribakin et al., 1977; Burovina et al., 1978). The intracellular sodium activity has not been measured in insect photoreceptors, but in those of the barnacle it appears to be about 10 mM in the dark and 18 mM after illumination with bright light (Brown and Comwall, 1975b; Brown and Ottoson, 1976) or, according to other data, about 25 mM in the dark with no more than a 5-mmole increase in the light (Brown, 1976). Thus we suggest that the photoreceptors themselves are capable of storing sodium, hence they can supply their own ionic pump with internal sodium for a prolonged time even in a sodium-free solution (Gribakin et al., 1977; Petrosyan, 1977a). The “sodium mechanism, consequently, can be accepted for insect photoreceptors, and this may underlie generation of both the transient and the plateau. It should be noted that the concepts of transients and plateaus are convenient but formal descriptions of the receptor potential components. Indeed, the drop from the transient to the plateau is mediated by intracellular calcium. After intracellular injection of EGTA the transient fails to drop to the plateau, and the receptor potential remains at the transient level as long as the light is on (Lisman and Brown, 1975; Bader et al., 1976). These experiments, as well as those with intracellular injection of sodium and calcium (Lisman and Brown, 1972; Bader et al., 1976), allow the transient-plateau transition to be interpreted as a result of the action of a calcium-mediated feedback intended to provide absolute sensitiv”
‘According to Petrosyan (1977a,b), all deposits should be related to sodium (not calcium), since (1) all sodium in the cricket photoreceptors is accesible to water, while calcium is practically not (see Burovina et al., 1978), and (2) sodium concentration as well as its activity in the cytoplasm, (10-50mM, see text for further details) is several orders of magnitude higher than that of calcium (no more than M ,see Borle, 1973; Bygrave, 1978). Thus calcium could hardly be responsible for the abundant deposit found in the cricket photoreceptors. Similarly, demonstration of calcium localization in the drone bee photoreceptors by Perrelet and Bader (1978) using pyroantimonate technique seems to be doubtful for these reasons (in addition, in the latter experiments, the treatment of the eye with the pyroantimonate solution had been preceded by prolonged fixation with glutaraldehyde solution; this procedure inevitably led to a redistribution of elements under study and possibly to their fractional or even total removal from the tissue; x-ray microprobe analysis in this case might indicate calcium still bound in vivo, not necessarily deposited by the reagent).
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ity control at the level of the receptor potential. In this interpretation, oscillations of the receptor potential often observed during the transient-plateau drop at high-intensity stimuli (e.g., see Fig. 12 in Fulpius and Baumann, 1969) are identical to those arising in electrical and mechanical oscillatory systems with nearly critical damping. Thus the plateau level of the receptor potential is a result of the interaction of two opposing ionic mechanisms: first, the light-induced sodium influx which depolarizes the cell membrane to the transient level and, second, a calciummediated mechanism (probably triggered by the former process) which tends to reduce this depolarization (antitransient repolarization). This interaction using a calcium-mediated feedback provides a wider dynamic range of receptor functioning at the plateau level in comparison with the transient level (see Fig. 2 in Fulpius and Baumann, 1969; Fig. 2 in Stieve et al., 1976). The light-induced calcium increase in the photoreceptor directly demonstrated by Brown and Blinks (1974) in Limulus ventral photoreceptors using aequorin may be due to the liberation of calcium ions from intracellular stocks activated by a small light-induced increase in intracellular sodium (Bader et al., 1976). In the arthropod photoreceptor calcium may be stored intracellularly, not only in mitochondria (see, e.g., Borle, 1973) but also within the pigment granules where it does not seem to be directly accessible to water (Burovina et al., 1978). Consequently, both mitochondria (Carofoli et al., 1974; Bader et al., 1976) and pigment granules might serve as intracellular calcium reservoirs (or depots) [Burovina et al., 1978; cf. the light-induced calcium release from the pigment granules of Aplysia neurons reported by Brown et al. (1975)]. In this respect, the migration of mitochondria and pigment granules to the rhabdom in the light considered previously to be the optical mechanism of absolute sensitivity control (Section II,D) may have a double function, the second purpose being to provide a photoreceptor zone of the cell with additional calcium to prevent a fatal increase in permeability to sodium under bright illumination. The PEC can hardly be considered an ionic depot, as suggested by Bader et al. (1976), because of its watery content (Section II,D,S,c). Moreover, we failed to find in the PEC an increased amount of any cation; on the contrary, potassium has a concentration in the PEC nearly half that in the photoreceptor cytoplasm (Gribakin et al., 1976; Burovina et al., 1978). However, participation of the PEC in the transduction process cannot be excluded, taking into account the potassium gradient on its membrane. In conclusion, the transient and plateau-principal components of the receptor potential-need to be interpreted as the algebraic sum of continuous depolarization (with the amplitude of the transient) produced by sodium influx and a subsequent opposing process-more slowly developing calcium-mediated repolarization. Both the transient and plateau probably originate in the plasma
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membrane of the photoreceptor, but not in its rhabdomere, and are triggered by a still unknown intracellular transmitter. c. Prolonged Depolarization Afterpotential. After the strong or prolonged light stimulation (especially with monochromatic light) ceases the plateau fails to fall sharply to a resting potential. Instead, the depolarizing afterpotential is seen to last from several seconds to many hours depending on the light stimulus (wavelength, intensity, time) and the species (Baumann and Hadjilazaro, 1972; Nolte and Brown, 1972; Hochstein et aE., 1973; Brown and Cornwall, 1975a,b; Hamdorf and Razmjoo, 1977; Tsukahara et al., 1977; Hillman et al., 1977). This effect has been termed prolonged depolarization afterpotential (PDA). A mechanism generating a PDA was first suggested by Baumann and Hadjilazaro (1972); “The retinula cell produced some unknown substance which, by acting on its membrane, leads to depolarization. Strong or prolonged illumination might lead to the accumulation of this substance and, owing to its relatively slow disappearance at the end of the flash, to a prolongation of the response into the dark.” Using the early receptor potential (ERP) as a measure of the rhodopsin concentration, Minke et al. (1973) and Hochstein et al. (1973) demonstrated the existence of two thermally stable states of rhodopsin in the barnacle eye, as well as a correlation between metarhodopsin formation and the presence of PDA, so that PDA is restrained by the amount of metarhodopsin produced by a light flash. Since PDA can easily be abolished by a flash with a wavelength corresponding to the metarhodopsin absorption maximum, to the early suggestion of Baumann and Hadjilazaro (1972) can be added the idea that removal of an unknown substance accumulated during rhodopsin conversion is prolonged when the pigment is in a metarhodopsin state Psukahara et al., 1977). An ionic mechanism underlying PDA is thought to be the same as that underlying the plateau, that is, a prolonged increase in sodium permeability, since both responses display the same dependence on ionic environment and temperature (Baumann and Hadjilazaro, 1972, drone bee; Brown and Cornwall, 1975b, barnacle). The role of calcium in PDA fading or abolition is still unknown (Brown and Cornwall, 1975b). Like the ERP, PDA does not seem to arise in natural light environments (see Hochstein et al., 1973) and, for this reason, has no real physiological function or significance; but it can be considered a tool for the study of visual pigments and their conversions triggering the ionic mechanisms that generate the receptor signal. 4. Photoresponses of Accessory Cells of the Eye a. Pigment Cells. Pigment cells are capable of responding to light with a depolarization which develops slower than the depolarizing response of the photoreceptors (Bertrand et al., 1972; Baumann, 1975). The resting potential of the pigment cells in the drone bee is lower than that of the photoreceptors (45 mV
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average to 50 mV average in the photoreceptors) and depends mainly on the potassium concentration in the extracellular medium. The response to light decreases when the potassium concentration in the extracellular medium is increased. The resistance of the pigment cell plasma membrane (or input resistance) does not change in the light, unlike that of a photoreceptor. Because of this, the pigment cell response is thought to be due to an increase in extracellular potassium leaving the photoreceptors in the light (Bertrand et al., 1972; Baumann, 1975). Pigment cells can be thought of as regulators of the extracellular ionic concentration (Baumann, 1975), and this function may appear similar to that suggested for neuroglia (see Somjen, 1975). b. Cone Cells. Like pigment cells, cone cells respond to illumination by slow depolarization lasting for 15-20 seconds after the light goes off (Vishnevskaya and Mazokhin-Porshnyakov, 1969; Cherkasov e f al., 1976; Vishnevskaya e f al., 1977), and this response is also assumed to be due to an increase in extracellular potassium in the light, as in the case of pigment cells (Vishnevskaya et al., 1977; Petrosyan, 1977a). An ability of cone cells to respond to light has also been demonstrated by electron cytochemistry. Petrosyan (1977a,b) found that numerous granules of sodium pyroantimonate inherent in cricket cone cells in a state of dark adaptation disappeared from these cells when they became light-adapted. However, both the mechanism and significance of this response are still obscure. Spectral efficiency characteristics of cone cells coincide with those of the whole eye, as measured by ERG, and can change during color adaptation in the grasshopper Teftigonia which has at least two spectral types of photoreceptors (Vishnevskaya and Mazokhin-Porshnyakov, 1972; Cherkasov et al., 1976). This finding favors the above assumption that the cone cell response is triggered by the activity of the photoreceptors. Similar to what has been assumed for the ion-controlling function of pigment cells, cone cells might also be involved in this regulation, and four cone processes going along the photoreceptors and terminating at the basal membrane as pigment-filled expansions (Gribakin, 1967, 1975; Wachmann e f d.,1973; Polyanovskii, 1976) may form a morphological basis for this function.
5 . Electroretinogram The ERG is defined as a light-evoked potential difference recorded between two electrodes one of which is placed just beneath the cornea while another is inserted elsewhere in the insect body. The ERG was the first electrical response of the compound eye to be recorded, and the history of its investigation as well as more details on ERG interpretation can easily be found in the literature (Mazokhin-Porshnyakov, 1969; Heisenberg, 1971; Goldsmith and Bernard, 1974). Being a mass response the ERG is produced by all the extracellular currents flowing in the illuminated eye and generated mainly by photoreceptors and laminar neurons. Consequently, the two principal components of the ERG
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are the receptor and lamina responses. The ease with which the ERG can be recorded is rather a warning to workers of the difficulties arising in ERG interpretation in terms of lightevoked changes in extracellular photoreceptor dark currents or, to be more exact, in terms of cellular mechanisms of insect photoreception. At any rate, many questions concerning ERGS clearly formulated by Heisenberg (1971) are still being investigated. D. THEROLEOF COMPARTMENTALIZATION The role of compartmentalizationwas first noticed by Heisenberg (1971), who suggested that the compound eye be considered an insect body compartment. This is in line with the ideas developed by Treheme and Pichon (1972) that the ionic composition of the extracellular medium of an insect nerve or ganglion should be stabilized to provide reliable functioning of the nerve system independently of the inconstant life conditions of the insect. Similarly, the extracellular medium of the eye can in no way be considered identical to the hemolymph and needs to be specially studied. The discovery of the resistance barrier (Shaw, 1975) and diffusion barrier (Shaw, 1977) in the compound eye, as well as direct studies of ionic composition of the eye and its elements (Gribakin et af., 1976, 1977; Petrosyan 1977a,b; Petrosyan et al., 1977; Burovina et al., 1978), have clearly shown that the compound eye is really a compartment of the insect nerve system which in turn contains “second-order” compartments (i.e., different cells) among which photoreceptors are strictly compartmentalized so that even their potassium is far from being uniformly distributed in the cell (Gribakin et al., 1976; Burovina et al., 1978). A correlation between ionic and morphological compartmentalizationmay favor various cellular sources of different receptor potential components and perhaps provide the clue to understanding the cellular mechanisms of receptor signal generation.
IV. Conclusion In this section several important principles and ideas following from this account are briefly summarized. Adaptation of the insect eye to the dark is an active process showing a continuous demand for energy to create and maintain a highly efficient optical structure ensuring perfect absorption properties. In a poor light environment the insect photoreceptor is able to operate as a photon counter, and so light-protectivemechanisms had to be evolved to provide normal functioning under brighter illumination. Since the insect photoreceptor responds to light by depolarization, a fatal decrease in the resting potential down to zero might be caused by a strong light
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(e.g., direct sunlight), and again a need for protection from excessive light has arisen. At least two kinds of light-protection mechanisms are known at present: first, those diminishing the relative absorption of the rhabdomere, that is, optical mechanisms of sensitivity control; and, second, those controlling the transduction process electrically and chemically somewhere between the rhabdomere and the cell plasma membrane. In this respect vertebrate photoreceptors responding by hyperpolarization appear to have an advantage, since in the brightest environment all the sodium channels are closed, the membrane potential increases to obtain a steady level (light level), and the cell keeps all its energetic potency although its response to light ceases. Rhodopsin is a membrane protein, and in order for absolute sensitivity to evolve, the photoreceptor membrane had to increase in area. This should cause an inevitable increase in ionic leakage and consequently an increase in noise level. Thus it was of vital importance for natural selection to prevent generation of the receptor signal from being triggered by dark- or light-evoked sodium permeability changes in the photoreceptor membrane itself. An intracellular transmitter appears to have resolved this problem, so that no receptor potential can be generated by the cell plasma membrane until excited by the direct action of the transmitter. The nature of the transmitter is still unknown. The compound eye can be considered an insect body compartment, and the ionic composition of its extracellular medium is seemingly regulated by the activity of the photoreceptors themselves. As in vertebrates, there exists noticable dark current in insect photoreceptors, and total dark current in the eye can reach several microamperes. The clear spatial regularity of the insect eye allows one to consider this organ a ‘‘biological crystal, ” and workers investigating its integral properties can hope to obtain information on the properties of its constituents, that is, the photoreceptors and accessory cells. The crystallinity of the insect eye gives an advantage to a worker in neuromorphology and neurophysiology, and it is hoped that several general problems can be better understood using this model, for instance, principles of compartmentalizationof inorganic ions, mechanisms controlling the extracellular ionic composition of nerve tissue, antidromic axonal transport connected with direct (in this case, dark) current, and so on.
REFERENCES Abrahamson, E. W., and Wiesenfeld, J. R. (1972). Handb. Sens. Physiol. 7 , Part 1, 69-121. AUtNm, H . , and Kolb, G. (1968). 2. Vergl. Physiol. 60, 450-477. AutNm, H . , and Tschamtke, H. (1962). Z. Vergl. Physiol. 45, 695-710. Bader. C. D . , Baumann, F., and Bertrand, D. (1976). J . Gen. Physiol. 67, 475-491.
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NOTEADDEDIN PROOF Recent findings of P. G. Lillywhite strongly support the view that quantum bumps are unit responses of the locust photoreceptors to single photons [Lillywhite, P. G. (1977). J . Comp. Physiol. 122, 189-200; (1978). J. Comp. Physiol. 125, 13-27].
INTERNATIONAL REVIEW OF CYTOLOGY. VOL . 57
Oocyte Maturation YOSHIOMASUI AND
HUGHJ . CLARKE Department of Zoology. University of Toronto. Toronto. Ontario. Canada
I . Introduction
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A . Concept of Maturation . . . . . . . . . . . . . . . B . Maturation and Ovulation . . . . . . . . . . . . . . C . Maturation and Fertilization . . . . . . . . . . . . . Hormonal Control of Maturation . . . . . . . . . . . . A . Gonadotropins . . . . . . . . . . . . . . . . . . B . Amphibian Oocyte Maturation . . . . . . . . . . . . C . Starfish Oocyte Maturation . . . . . . . . . . . . . D. Fish Oocyte Maturation . . . . . . . . . . . . . . E . Mammalian Oocyte Maturation . . . . . . . . . . . F . Oocyte Maturation in Other Animals . . . . . . . . . G . The Role of Follicles in Oocyte Maturation . . . . . . . Progression of Maturation . . . . . . . . . . . . . . . A . Chronology . . . . . . . . . . . . . . . . . . . B . Morphological Changes . . . . . . . . . . . . . . C . Biochemical Changes . . . . . . . . . . . . . . . Initiation of Oocyte Maturation . . . . . . . . . . . . . A . Maturation-Inducing Substance . . . . . . . . . . . . B . The Role of Ca Ions . . . . . . . . . . . . . . . C . Changes in Electrophysiological Properties . . . . . . . Cytoplasmic Control of Oocyte Maturation . . . . . . . . . A . Maturation-Promoting Factor . . . . . . . . . . . . B . Phosphorylation of Proteins . . . . . . . . . . . . . C . Arrest of Meiotic Division . . . . . . . . . . . . . Nucleocytoplasmic Interaction during Oocyte Maturation . . . A . Chromosome Condensation . . . . . . . . . . . . . B . Development of the Pronucleus . . . . . . . . . . . C . Development of Motile Systems . . . . . . . . . . . Control of Meiosis and Mitosis2oncluding Remarks . . . . A . RoleofCa . . . . . . . . . . . . . . . . . . . B . RoleofcAMP . . . . . . . . . . . . . . . . . . C . Phosphorylation of Cellular Proteins . . . . . . . . . . D. SH Cycle . . . . . . . . . . . . . . . . . . . E . Cytoplasmic Control Factors . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .
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185 Copyright 0 1979 by Academia press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-364357-0
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I. Introduction A. CONCEPT OF MATURATION Possibly the outstanding phenomenon associated with sexual reproduction in animals is that every cell of an individual derives from a single germ cell. Accordingly, it is thought that germ cells, in contrast to somatic cells, retain developmental totipotentiality throughout the life of the individual. Nonetheless, it is also true that germ cells become as highly differentiated as somatic cells during ontogenesis. In female germ cells, this specialization begins during the very early stages of life. The cells enter meiosis at the fetal or larval stage. Before they begin to grow, meiosis proceeds to the terminal stage of the first meiotic prophase, the diplotene stage. Growing primary oocytes have an enormously enlarged nucleus called the germinal vesicle (GV). Characteristically, it contains lampbrush chromosomes which have been actively engaged in RNA synthesis. Toward the end of the growth period the loops of the lampbrush chromosomes regress after which the oocytes enter a stationary state which persists until ovulation. The duration of the stationary state is consequently dependent on the time of sexual maturity of the animal and on the period of its reproductive cycle. Oocytes in this stationary state have lost their ability to proliferate and will eventually perish if allowed to remain in the ovary. If the oocytes are to continue to live, they must emerge from the stationary state and undergo various changes. These changes occur shortly before or shortly after ovulation. In the normal process of sexual reproduction they occur as a result of maturation and fertilization. In this article, the term “maturation” is used to describe the completion of meiosis as defined by Wilson (1925, pp. 397-398), who stated that maturation is accomplished in the animal oocyte by means of two successive meiotic divisions in the course of which the oocyte buds forth two polar bodies. It represents “the ripening or final stages of the formation of the germ cells. Though often applied to the nuclear changes (meiosis) it properly includes also the cytoplasmic” (p. 1136). Maturation is interrupted by suspension of meiotic division in many species, and its resumption is triggered by insemination. Oocytes which complete maturation independently of sperm penetration must be inseminated in order to begin mitosis. Thus the change oocytes undergo following insemination is a prerequisite for them either to complete maturation or to initiate mitosis. This change, which is caused by insemination, has been designated “activation. Therefore the conversion of quiescent oocytes into active zygotes involves two major pro”
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cesses, maturation and activation. There is, however, one exception to this rule. In parthenogenesis, quiescent oocytes are converted into mitotically active cells without the aid of insemination and often even without two successive meiotic divisions (Tyler, 1941; Graham, 1974). However, we can assume that these oocytes have undergone the same change in physiology that takes place during the maturation and activation of normal oocytes. Although we follow Wilson’s definition of “maturation” throughout this article, we should comment on the usage of this word to avoid confusion in the following discussion. The term “maturation” has been used in various ways by different researchers. Usually, invertebrate zoologists have considered that “maturation” encompasses the entire process of oogenesis (Highman and Hill, 1977). In order to limit the implications of the word, Schuetz (1969) proposed the term “meiotic maturation” to refer to the meiotic process following the release of the oocyte from prophase arrest. In many species, however, meiosis is again arrested at metaphase of the first or second meiotic division (metaphase I or I1 arrest), at which time the oocytes are fully fertilizable. Consequently, many investigators have referred to these oocytes as “mature oocytes, ” implying that they have completed maturation. This usage of the word may be misleading when the progression of oocyte maturation is compared in different species, since oocytes become fertilizable at different stages of maturation in different species. In fact, Wilson (1925) recognized this difficulty, stating that the maturity of the oocyte required for its fertile union with the sperm should not be confused with (the) consequences of maturation (p. 404). Recently the term “prematuration” has been introduced to refer to the process of maturation by which oocytes reach a certain intermediate stage of maturation and become fertilizable (see Schroeder and Hermans, 1975, p. 108). In past years, the problems of oocyte maturation have been reviewed by several investigators from numerous points of view. Early studies of oocyte maturation were reviewed by Schuetz (1969) and Smith and Ecker (1970a). Biochemical aspects of the maturation of the amphibian oocyte have been discussed by Smith (1975) and by Wasserman and Smith (1978a). Reviews by Redshaw (1972), Schuetz (1974), and Baulieu et al. (1978) of amphibian oocyte maturation and those of Channing and Tsafriri (1977) and Tsafriri (1978) concerning mammalian oocyte maturation are particularly relevant to the problems of endocrinological control. Oocyte maturation in fish was reviewed by Jalabert (1976) and Wallace and Selman (1978), and described in starfish by Kanatani (1973, 1975, 1978). Descriptionsof oocyte maturation in marine invertebratesare also found in the books edited by Giese and Pearse (1975). Recently an extensive review of various aspects of oocyte maturation in different species was published in the USSR (Dettlaff, 1977). This article emphasizes cellular and comparative aspects of oocyte maturation.
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B. MATURATION AND OVULATION Various temporal relationships between ovulation and maturation have been observed in different species. In some molluscans, such as clams (Spisula), echiuroids (Urechis), and annelids (Nereis), ovulation occurs before the initiation of maturation. Conversely, in sea urchins, oocytes are not ovulated until after they have completed maturation. However, in many other species the initiation of maturation and ovulation occurs almost simultaneously during normal reproductive periods. In such cases, one might well speculate that ovulation and maturation are closely linked by common systemic factors which may correlate the physiology of the ovary and the oocytes, or that maturation and ovulation are causally linked in some manner. However, it has been shown that maturation and ovulation can occur independently. In mammals, fully grown oocytes often show signs of initiating maturation in the ovary; this is followed, however, not by ovulation, but by atretic degeneration (Baker, 1972). Furthermore, it has been demonstrated (in the LT strain of mice) that oocytes can complete maturation within their follicles and spontaneously develop into embryos (Eppig et ul., 1977). These results indicate that maturation can occur without ovulation. As we discuss later, mammalian oocytes can be induced by gonadotropins to mature in follicles cultured in vitro, but in no case has it been reported that the oocytes have been ovulated. Conversely, Ryan and Grant (1940) reported that follicles of Rana pipiens cultured in Ringer’s solution containing a pituitary suspension ovulated oocytes which showed no signs of maturation. Similar results were reported by Subtelny et al. (1968). And recently, Mom11 and Bloch (1977) found that an antiovulatory drug, ethynylestradiol, blocked maturation but not ovulation when it was applied together with progesterone to isolated follicles of R. pipiens. In the fish Salmo, maturation without ovulation can be induced in cultured follicles by various steroid hormones, but when follicles are exposed to coelomic fluid collected from females which have previously been induced to ovulate by gonadotropins, these follicles ovulate oocytes showing no signs of maturation (Jalabert et al., 1972). Ovulation of immature oocytes can also be induced by exposing follicles to prostaglandin F,, (Jalabert, 1976). Dissociation of ovulation from oocyte maturation has also been reported in starfish. In Asterias, ovarian extracts prepared out of the spawning season were found to contain steroid glycosides (asterosaponin A and B) which suppressed ovulation (Ikegami, 1976). Ikegami also reported that asterosaponin A and B suppressed ovulation but not maturation of ovarian oocytes treated with 1-methyladenine(1-MA) when both chemicals were applied to an ovary obtained during the spawning season. However, ovaries exposed to Ca-free seawater for a short time ovulate immature oocytes upon their return to normal seawater. All these observations provide clear evidence that the processes of ovulation
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and maturation are not causally connected in a wide variety of species, although under normal circumstances these two events are regulated by common systemic factors such as gonadotropins.
C. MATURATION AND FERTILIZATION The timing of sperm entry or insemination in relation to the progression of meiotic maturation under normal conditions is characteristic for any given species. Animals may be classified into four groups (Rothschild, 1956) according to the stage of maturation at which insemination normally occurs. In class I, which is represented by Echiuroidea and Platyhelminthes, oocytes are inseminated before maturation begins. For these oocytes, activation by the sperm triggers maturation. Class I1 includes animals in which oocyte maturation proceeds to metaphase I before the sperm enters the oocyte. Insects and ascidians are members of this class. The animals belonging to class I11 are vertebrates whose oocytes are inseminated at metaphase 11. In the last-mentioned two groups (classes I1 and 111), activation causes the resumption of meiotic progression which had been arrested at metaphase. Animals belonging to class IV include sea urchins and coelenterates. Their oocytes complete maturation before sperm entry occurs. Here, activation triggers the initiation of mitosis by the zygotes. Finally, it has been noted that oocytes of some animals, such as starfish, are inseminated at any stage of maturation after GV breakdown (GVBD) (Rothschild, 1956; Stevens, 1970). This classification of animals appears to have no correlation with their phylogenetic order. For instance, Ascaris (nematode), Spisula (mollusc), and Myzostoma (annelid) all belong to class I, and ascidians and insects to class 11. At the same time, two closely related animals, the polychaetes Nereis and Chaetopterus are classified in classes I and 11, respectively. Among the mammals, canines are known to belong to class I, while the others are members of class 111. A most intriguing finding is that two species of the same genus, Arenicolu crisrata (Okada, 1941) and A. marina (Howie, 1963) belong to classes I and 11, respectively. Changes in the fertilizability of oocytes during the course of maturation have been studied using artificial insemination in mice (Iwamatsu and Chang, 1971), pigs (Leman and Dziuk, 1971; Motlik and Fulka, 1974), dogs (Mahi and Yanagimachi, 1976), rabbits (Overstreet and Bedford, 1974), hamsters (Usui and Yanagimachi, 1976), sea urchins (Franklin, 1965; Longo, 1978), frogs (R. pipiens) (Elinson, 1977), and toads (Bufo bufo) (Katagiri, 1974). All these investigations have indicated that sperm can penetrate oocytes at any stage of maturation. However, the incidence of polyspermy is higher in oocytes inseminated at earlier stages of maturation than in those inseminated at the stage at
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which fertilization normally takes place. But no significant correlation has been demonstrated between the number of sperm per oocyte and the stage of maturation of the oocyte. The consonant results obtained by these investigators suggest that the development of the mechanism for blocking polyspermy concomitant with oocyte maturation is a general phenomenon. Niwa and Chang (1975), working on rats, found that polyspermy did not occur at any stage of maturation but pointed out that this might be due to the relatively low concentration of sperm suspension used in their experiment. Mahi and Yanagimachi (1976) also found no evidence of polyspermy, using canine oocytes. Since in dogs the sperm normally enters the oocyte before the onset of maturation, a blockage of polyspermy would not be expected to require maturation. The mechanism underlying developmentof the polyspermy blockage in maturing oocytes is not fully understood at present. However, an increasing capacity of oocytes undergoing maturation to support formation of the fertilization membrane or the zona reaction upon insemination has been generally found to play an important role in polyspermy blockage. In mice, Iwamatsu and Chang (1971) observed only a weak zona reaction in oocytes which had been penetrated by sperm at early stages of maturation, suggesting that the lack of a zona reaction was responsible for polyspermy. Usui and Yanagimachi (1976) in hamsters, and Soupart and Strong (1974) in humans, demonstrated that zona-free oocytes underwent polyspermy regardless of their stage of maturation. The ability of starfish oocytes to form the fertilization membrane upon insemination develops as maturation progresses. Hirai et al. (1971) and Cayer et al. (1975) determined that in Asterias pectinifera this ability appeared at the time of GVBD. Although Schuetz (1975b) in Asteriasforbesii, and Lee et al. (1975) in Pisaster giganteus, found that elevation of the vitelline membrane could be induced in oocytes with an intact GV by insemination, Schuetz (1975b) also demonstrated that these oocytes could not develop normally after the induction of maturation by 1-MA. Possibly oocytes with an intact GV failed to block polyspermy. Rosenberg et al. (1977) have noted that oocytes of Pisaster, exposed to 1-MA, undergo structural changes in the surface of the vitelline membrane as maturation progresses, changes which they postulate to be responsible for development of the blockage of polyspermy. Thus it is likely that development of the polyspermy-blockingmechanism during oocyte maturation is associated with development of the ability of the oocyte to give rise to a genuine fertilization membrane upon insemination. The change in the vitelline membrane following insemination has been regarded as a result of oocyte activation by sperm entry, possibly caused by enzymes released from the cytoplasm (Carroll and Epel, 1975a). Ample evidence has accumulated to indicate that in nonmammalian species (Vacquier et al., 1972; Schuel et al., 1973; Carroll and Epel, 1975b) and in the rabbit (Flechon et al., 1975) cortical granule breakdown (CGBD)plays a major role in changing the properties of the vitelline membrane following insemination. Therefore it seems
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reasonable to ascribe the failure of precociously inseminated oocytes to develop effective polyspermy blockage to the absence of a cortical response to sperm entry. There is evidence that, at least in sea urchins, a close association of the cortical granules (CGs) with the plasma membrane is important for CGBD to occur (Millonig, 1969; Longo and Anderson, 1970). Longo (1978) found that the CGs in sea urchin oocytes did not move to the plasma membrane until maturation had reached a certain stage and suggested that this was at least partly responsible for the absence of CGBD in inseminated immature oocytes. Experiments with frogs by Btlanger and Schuetz (1975) and by Cloud and Schuetz (1977) showed that oocytes which had not matured to metaphase I1 did not activate after insemination or pricking. Yet the divalent cation ionophore A23187 can induce activation reactions such as CGBD and vitelline membrane elevation in these oocytes. It is known that this ionophore is a ubiquitous parthenogenetic agent (Steinhardt et al., 1974), and that it induces Ca release from the egg (Steinhardt et al., 1977), thereby causing CGBD (Vacquier, 1975). Therefore it seems likely that development by the oocyte of the ability to block polyspermy also depends on its increasing capacity to release Ca in response to sperm entry. The variability among species in the timing of sperm entry into the oocyte relative to the meiotic progression of the nucleus may indicate that these cytoplasmic changes, occurring during the course of maturation, do so rather independently of the nuclear changes. Iwamatsu (1966, 1971) showed that this was the case in the fish Oryzias lutipes (medaka). He demonstrated that oocytes could become fertilizable and develop into haploid embryos even when the GV was kept intact in the yolk mass by centrifugal displacement. Amphibian oocytes from which the GV has been removed also develop the ability to undergo parthenogenetic activation when they are treated with progesterone (Smith and Ecker, 1969; Skoblina, 1969). Skoblina (1974) and Katagiri and Moriya (1976) further demonstrated that sperm could enter and activate these enucleated oocytes. Experiments on starfish oocytes by Hirai et ul. (1971) also indicated that enucleated oocytes became activatable upon insemination 20-30 minutes after I-MA treatment. All the results cited above strongly suggest that cytoplasmic maturation can occur independently of the presence of the nucleus (the GV). 11. Hormonal Control of Maturation
A. GONALIOTROPINS
In most animals, other than those whose oocytes are induced to mature by insemination, the initiation of oocyte maturation is dependent on ovarian function. Generally, only oocytes which are fully grown mature in response to ovari-
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an stimulus. The systemic factors controlling oocyte maturation are gonadotropins. It is well known that the administration of gonadotropins to female animals having fully grown oocytes in their ovaries causes oocyte maturation, with concomitant ovulation. In vertebrates, gonadotropins are secreted by the pituitary gland (hypophysis). Two different polypeptides, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), can be distinguished in mammals (Papkoff et al., 1973), birds (Farmer et al., 1975), reptiles (Licht and Papkoff, 1974a), and amphibians (Licht and Papkoff, 1974b), but only one gonadotropic hormone (GTH) has been found in fish (Fontaine and Gerard, 1963; Donaldson et al., 1972). The gonadotropin(s) found in invertebrates has not been chemically characterized, except for that of starfish. In starfish, a peptide hormone analogous to vertebrate gonadotropin, known as gamete-shedding substance (GSS), is secreted by the supporting cells of the radial nerves (Kanatani et al., 1971). Gonadotropins act directly on the ovary. Heilbrunn et al. (1939), using R. pipiens, demonstrated for the first time that oocyte maturation and ovulation could be induced in ovarian fragments incubated in Ringer’s solution containing pituitary extracts. This method has been widely used since then to study the effects of gonadotropins on oocyte maturation and ovulation in numerous species. It will become apparent in the following sections that, because the role of gonadotropins in oocyte maturation has been studied in many different species by many different investigators, conflicting results have been obtained, making it difficult to define a generalized mode of gonadotropin action. Nevertheless, information gained from studies using frogs and starfish, which are probably the most thoroughly analyzed animals with regard to this problem, points to a rather simple common principle, namely, that gonadotropins primarily act on the follicle (granulosa) cells to induce maturation of the oocytes in these follicles. B. AMPHIBIAN OOCYTEMATURATION
The experiment by Heilbrunn and his associates (1939) showed that gonadotropins acted on the ovarian follicle to induce maturation in R. pipiens. However, if follicles are incubated with progesterone, ovulation (Wright, 1961) and oocyte maturation (Schuetz, 1967a) are induced. In R. pipiens, it was found that gonadotropin had no effect on the oocyte when the follicle cells were completely removed following treatment with Ca-free medium (Masui, 1967) or pronase (Smith et al., 1968). However, these follicle-free oocytes were shown to undergo maturation in response to progesterone (Masui, 1967; Schuetz, 1967b; Smith et al., 1968) or, when they were incubated with follicle cells, to gonadotropin (Masui, 1967). Hence it has been hypothesized by Masui (1967) and by
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Smith et al. (1968) that gonadotropins induce the follicle cells to secrete a hormone, possibly progesterone-like, which in turn acts on the oocyte to initiate! maturation. Although progesterone is the most potent steroid inducer of oocyte maturation, other hormones such as deoxycorticosterone (DOC) and testosterone, but not estradiol or its derivatives, are also effective in R. pipiens (Schuetz, 1967a; Smith et al., 1968) and in Xenopus laevis (Jacobelli el al., 1974; Schorderet-Slatkine,-1972). The above hypothesis has been substantiated by recent work. Fortune ef al. (1975) and Thibier-Fouchet et al. (1976) demonstrated in Xenopus that both Xenopus gonadotropin and human chorionic gonadotropin (HCG) induced follicles to convert pregnenolone into progesterone and induced oocytes to mature. However, isolated oocytes did not carry out this steroid conversion (ThibierFouchet et al., 1976). Furthermore, drugs which suppress this steriod conversion, such as cyanoketone and eliptin, inhibit gonadotrophicinduction of maturation in follicleenclosed oocytes of R. pipiens (Wright, 1971; Snyder and Schuetz, 1973) and X. Zaevis (Fortune et al., 1975). But these inhibitors do not affect progesterone-induced oocyte maturation. Information concerning hormonal control of oocyte maturation in urodeles is rather scarce. In these animals, complete control of ovulation and oocyte maturation in vitro has not yet been achieved. Methods which induce oocyte maturation in anurans are not successful when applied to urodeles. Two examples should suffice. Progesterone is an effective inducer of maturation in Notophthalmus viridescens only when isolated oocytes are primed with gonadotropin (Pilone and Humphries, 1975). And only oocytes obtained from Pleurodeles waltlii collected during the breeding season are responsive to progesterone treatment (Brachet, 1974; Ozon et aZ., 1975). Perhaps the inefficiency of progesterone in these urodeles can be explained by a consideration of their natural breeding habits, which are different from those of anurans. In many urodele species, only a few eggs, which have completed growth, are spawned each day; those remaining in the ovary quite possibly have not yet grown sufficiently to be able to initiate the maturation process. C . STARFISH OOCYTEMATURATION
Studies by Chaet (1966) and by Kanatani and Ohguri (1966) demonstrated that isolated fragments of the starfish ovary ovulated upon transfer to seawater containing radial nerve factor (RNF or GSS) and that the oocytes underwent maturation concomitant with ovulation. Further research (Schuetz and Biggers, 1967; Kanatani and Shirai, 1967) indicated that oocytes freed from follicle cells did not respond to GSS. At the same time, these investigators also showed that isolated oocytes matured when they were exposed to seawater in whichisolated follicles
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had been incubated with GSS. Thus it has become clear that GSS, or RNF, acts by inducing follicles to secrete a maturation-inducing substance (MIS). Kanatani et al. (1969) purified MIS and identified it as 1-MA. At a concentration of lo-' M, this substance can induce oocyte maturation in all species of starfish so far examined (see Kanatani, 1973). 1-MA is also produced in the ovary of echinoids other than starfish, but an effect on the oocytes of these species has not been confirmed (Kanatani, 1975). Investigation into the mechanism of 1-MA production in the starfish ovary has disclosed that this substance is a derivative of a purine compound which contains a methyl group at the N- 1 position. GSS stimulatesmethylationof this compound,utilizingS-adenosylmethionine (Shirai et al., 1972), to form 1-methyladenosinemonophosphate, which is then hydrolyzed to give rise to 1-MA through the formation of 1-MA riboside (Schuetz, 1970; Shirai and Kanatani, 1972).
D. FISHOOCYTE MATURATION Research on oocyte maturation in fish has produced complicated results with respect to the role of gonadotropins. This may be due primarily to the fact that, excepting studies on the sturgeon and the trout, few experiments have been performed using follicle-free oocytes. Sturgeon oocyte maturation has been investigated by Dettlaff and her colleagues (Dettlaff and Skoblina, 1969). Fully grown sturgeon oocytes are invested with a jelly coat secreted by the follicles and can easily be separated from them without damaging the oocytes. Progesterone treatment induces maturation both in follicle-enclosed and follicle-free oocytes, while gonadotropin is effective only in follicle-enclosed oocytes. Similarly, trout oocyte maturation is induced in isolated follicles by steroids and by gonadotropin (Jacobelli et al., 1974). And, as in the sturgeon, trout gonadotropin is effective only in follicle-enclosed oocytes. Not many steroid hormones affect follicle-free oocytes; 17a,20fl-dihydroxyprogesteroneand 20phydroxyprogesterone are the only potent steroid inducers of maturation of isolated oocytes (Fostier et al., 1973; Jalabert, 1976). Some steroids are effective to a certain extent when applied to follicle-enclosedoocytes of trout, goldfish, or pike. Consequently, it has been suggested that these steroids are metabolized to 17a,20P-di- or 20P-hydroxyprogesterone (Jalabert, 1976). In other fish species, the effects of hormones on oocytes have not been conclusively resolved. In the medaka, cortisol appears to be a more potent inducer of maturation than progesterone when intact follicles are cultured until maturation in a medium containing the steroid (Hirose, 1972). But progesterone is the most potent inducer when the follicles are only briefly exposed to steroids (Iwamatsu, 1974). Recently, Hirose (1976) reported that neither gonadotropin nor corticosteroids were effective in inducing maturation of medaka oocytes
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cultured in vitro after removal of the follicular tissue by ethylenediamine tetraacetic acid (EDTA) or trypsin treatment. Pointing out that cortisol can be synthesized by the ovary in some fish (Colombo et al., 1973;Hirose et al., 1975),he concluded that the maturation process in the medaka, stimulated by the pituitary-ovarian axis, may be mediated by a second substance produced in the ovary during corticosteroid metabolism. Effects of mammalian and fish gonadotropins and various steroid hormones on fish ovarian follicles were investigated by Goetz and Bergman (1978a,b) using yellow perch (Perca flavescens), walleye (Stizostedion vitreum), and brook trout (Salivelinus fontinalis). These investigators found that most of the hormones tested were effective in inducing maturation of follicle-enclosed oocytes, and also suggested that corticosteroids facilitated the efficacy of gonadotropins. An enhancing effect of corticosteroids on the efficacy of gonadotropin and I7~~,20/3-dihydroxyprogesteroneaction in the induction of maturation of follicle-enclosed trout oocytes has been noted by Jalabert (1976).Dettlaff and Davydova (1974)observed that triiodothyronine increased the gonadotropin sensitivity of follicles isolated from cold-stored sturgeon. These results strongly suggest that hormones other than gonadal steroids serve to sensitize follicles to gonadotropins. Recently, Wallace and Selman (1978) succeeded in inducing maturation of follicle-enclosed oocytes of the marine fish Fundulus hereroclitus in vitro using HCG, DOC, and progesterone. These workers consider that the physiological condition of the cultured follicle is an important factor determining its response to hormones. Although the studies discussed above appear to indicate that fish gonadotropin stimulates the ovarian follicle to produce progesterone-like steroids which in turn trigger oocyte maturation, another route of hormonal action has been suggested in catfish (Sundararaj and Anand, 1972). Isolated follicles of this fish respond neither to gonadotropins nor to gonadal steroids, although in vivo administration of LH effectively induces oocyte maturation and ovulation. The effect of the gonadotropin is, however, attenuated by the drug Metopirone which blocks corticosteroid synthesis in the interrenal gland; this inhibition can be overcome by hydrocortisone (HC) or DOC administration. These corticosteroids are also effective in inducing the maturation of follicle-enclosed oocytes in vitro (Goswami and Sundararaj, 1971), while gonadotropin is effective only when the follicles are also incubated with interrenal tissue (Sundararaj and Goswami, 1974). And minced interrenal tissue is reported to increase corticosteroid synthesis, primarily that of DOC, in the presence of gonadotropic hormone (Sundararaj and Goswami, 1969).Finally it was recently reported that the ovary of the catfish was unable to synthesize DOC (Unger et al., 1977). Taken together, these results have been interpreted as indicating that gonadotropic hormone stimulates the interrenal gland to secrete corticosteroids which induce oocyte maturation (Sundararaj and Goswami, 1977).
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However, this interpretation should not be considered definitive. The results obtained with the catfish may be interpreted in a different way. The failure of gonadotropin to induce oocyte maturation in isolated follicles may be attributable to a low sensitivity of the tissue per se toward the hormone; corticosteroids may act to increase the sensitivity of the follicular tissue. Second, it should be noted that the action of steroids in the induction of oocyte maturation generally is not as specific as that of most hormonal action. For example, although DOC is not produced by the amphibian ovarian follicle, it has been found to be as potent an inducer of oocyte maturation as progesterone (Smith et al., 1968; SchorderetSlatkine, 1972). All in all, we feel it would be premature to consider the female of this species an exception to the pituitary-gonadalaxis principle of reproductive control which has been so often demonstrated-in fact, even in the male catfish (Sundararaj and Nayar, 1967).
OOCYTEMATURATION E. MAMMALIAN Maturation of mammalian oocytes enclosed in follicles is induced under the influence of LH, both in vivo and in vitro. As shown by Ayalon et al. (1972) in rats, follicle-enclosed oocytes remain at the dictyate (diplotene) stage if the follicles are explanted from the female before the preovulatory LH surge and cultured in a hormone-free medium, whereas oocytes undergo maturation if the follicles are isolated after the LH surge. Later, Hillensjo et al. (1974) confirmed these results using prepubertal rats injected with pregnant mare serum (PMS). In vitro experiments by Baker and Neal (1972) with mice, Tsafriri et al. (1972) with rats, Hay and Moor (1973) with sheep, Thibault and Gerard (1973) with rabbits, Thibault et al. (1975a) with monkeys and calves, and Gwatkin and Andersen (1976) with hamsters have all indicated that addition of LH to the medium in which follicles are cultured causes the oocytes within these follicles to initiate maturation. However, the ability of LH to induce follicle-enclosedoocytes to mature does not appear to be due to its specific steroidogenic action on the follicles. Experiments using rats (Tsafriri et al., 1972) and rabbits (Thibault and Gerard, 1973) have demonstrated that FSH is also capable of inducing maturation of follicleenclosed oocytes cultured in vitro. The possibility that the effect of FSH might be caused by contaminating LH in the hormone preparation can be ruled out, since antibody made against the p chain of LH does not abolish the effect of FSH, but only that of LH (Tsafriri et al., 1972; Hillensjo et al., 1976). It is also known that the effects of FSH and LH on steroidogenesis in the granulosa cells are different (see Channing and Tsafriri, 1977). Therefore the effect of gonadotropin on the induction of maturation in follicleenclosed oocytes does not appear to be mediated by hormone-stimulated steroidogenesis.
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Studies using rats (hfriri e l al., 1972) and cows and pigs (Foote and Thibault, 1969) have shown that the addition of steroids to follicle culture medium has no effect on oocyte maturation. Furthermore, inhibition of LHinduced steroidogenesis in rat follicles by cyanoketone or aminoglutethiimide does not prevent LH induction of oocyte maturation in the follicles (Lieberman er al., 1976). These results suggest that the maturation of mammalian oocytes is not necessarily induced by steroid production following follicular stimulation by gonadotropins. The initiation of oocyte maturation appears rather to be dependent on the general physiological condition of the follicles. In the monkey, while LH normally induces only oocytes enclosed in fully grown follicles to mature, it also induces maturation of oocytes in small follicles if the follicles have undergone atresia (Thibault et al., 1975b). It is known that oocytes in atretic follicles even spontaneously initiate maturation (Foote and Thibault, 1969). Recently, Moor and Trounson (1977), using sheep ovaries, showed the LH induced oocyte maturation when it was applied to nonatretic large follicles or to atretic small follicles, but that it had no effect on atretic large follicles or on nonatretic small follicles. They also found that oocytes in atretic follicles, both large and small, did not undergo maturation spontaneously when the follicles were incubated under hyperbaric conditions. It was further shown that oocytes within atretic follicles which had been cultured in the presence of FSH and 17P-estradiol possessed the same capability as those in nonatretic follicles, in spite of follicular deterioration, to mature and develop normally following insemination in the oviduct of recipient animals. These results indicate that LH-induced oocyte maturation in atretic small follicles is not due to deterioration of the oocytes, but primarily to deterioration of the follicles. Thus it can be inferred that induction of oocyte maturation within the follicle is brought about by perturbation of the follicular physiology. One of the first indications of atresia is degeneration of the cumulus cells (Hay et al., 1976). Perhaps significantly, the effect of a gonadotropin on follicles first appears in the cumulus cells. It induces both the dispersion of (Thibault et al., 1975a) and hyaluronic acid secretion by (Hillensjo et al., 1976) the cumulus cells. Thus it may be that gonadotropins alter the physiology of the cumulus cells, producing a change in their relationship with the oocytes, which in turn stimulates the latter to initiate maturation. In mammals, it is now well known that oocytes removed from the follicular environment always tend to undergo maturation. This phenomenon was first demonstrated in 1935 by Pincus and Enzmann, using rabbit oocytes, and later confirmed by Chang (1955). Edwards (1965) pointed out that the maturation of oocytes following isolation from their follicular environment was almost universal among mammals-found in the mouse, sheep, cow, pig, monkey, and human. The following species have been added to this list since then: rat (Magnus-
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YOSHIO MASUI AND HUGH J. CLARKE
son et al., 1977), guinea pig (Jagiello, 1969; Yanagimachi, 1974), hamster (Haidri et al., 1971), and dog (Mahi and Yanagimachi, 1976). In most species, maturation of follicle-free oocytes can take place in a simple medium such as Krebs-Ringer solution containing pyruvate, kept in a 5 % COP95% air mixture (Biggers et al., 1967; Donahue, 1968). Oocytes of some species such as hamsters (Gwatkin and Haidri, 1973, 1974) and sheep (Hay and Moor, 1973) require a lower 0, pressure or addition of amino acids to the culture medium. However, neither hormones nor tissue factors are necessary. These findings might lead one to speculate that the intrafollicular milieu plays an important role in maintenance of the physiological stability of oocytes at the dictyate stage. In this respect, any gonadotropin-induced alteration in the physiological activity of the cumulus cells might cause an interruption of the stabilizing function of the follicles, resulting in maturation of the oocytes. Such physiological perturbation of the follicles might be brought about by gonadotropic activity stimulating follicular activities other than steroidogenesis. For instance, LH stimulates prostaglandin (PG) production in the follicle (Chasalov and Pharriss, 1972; Bauminger et al., 1975), and application of PGE, to cultured follicles induces oocyte maturation (Tsafriri et al., 1972). However, indomethacin, which suppresses PGE, production and also inhibits ovulation in rats (Armstrong and Zamecnik, 1975), fails to suppress LH-induced oocyte maturation (Tsafriri et al., 1972). Thus it is clear that the action of LH on the induction of maturation in follicle-enclosed oocytes is not mediated by PG production, but rather that both act simultaneously and independently to cause physiological changes in the follicles. Both LH and PG are known to stimulate adenyl cyclase to increase the level of cyclic adenosine monophosphate (CAMP) in the follicle (Lamprecht et al., 1973). Reasoning that this might provide a clue as to their function, several groups have tested the effects of cAMP on rat oocytes. Tsafriri et al. (1972) reported that, while both cAMP and its dibutyryl derivative (dbcAMP) exerted no inductive effect on oocyte maturation when applied extrafollicularly, dbcAMP had a positive effect when injected into the follicle. The possibility that the injection procedure itself may have induced the oocytes to mature was ruled out by control experiments showing that injected 5'-AMP had no effect on the oocytes. Moreover, cholera toxin, known to stimulate cAMP production in various types of cells, also effectively induced maturation of follicle-enclosed rat oocytes (Tsafriri et al., 1972). However, derivatives of cAMP have been found to have an inhibitory effect on oocyte maturation at concentrations greater than M. Hillensjo et al. (1978) in rats, and Nekola and Smith (1975) in mice, showed that addition of dbcAMP to the medium in which follicles were cultured inhibited the inducing effect of LH on oocyte maturation. Nonetheless, the changes in the cumulus cells which occurred in the presence of dbcAMP, with or without gonadotropin, were identi-
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199
cal to those observed during LH-induced maturation (Hillensjo, 1977). An inhibitory effect of dbcAMP on spontaneous maturation of follicle-free oocytes has also been observed in mice (Stem and Wassarman, 1974; Cho et al., 1974) and rats (Magnusson and Hillensjo, 1977). Therefore it is highly probable that the inhibition of maturation of follicle-enclosedoocytes caused by dbcAMP is due to direct action on the oocytes, an action counteracting signals from the cumulus cells to release the oocytes from meiotic arrest. If so, it is conceivable that CAMP or its derivatives could induce maturation of follicle-enclosed oocytes if their action is localized in the cumulus cells. A key to understanding the mechanism which initiates maturation of follicular oocytes is a determination of the nature of the follicular milieu which prevents oocytes from maturing. Analyses of the intrafollicular factors responsible for the stabilization of dictyate oocytes have been carried out by several groups. The possibility that follicular factors which prevent oocytes from initiating maturation affect the physiological activity of the oocytes in general has been speculated upon by Zeilmaker et al. (1972). He points out that spontaneous maturation of rat oocytes can be prevented by hypoxia, suggesting that the reduced oxygen supply to follicular oocytes may be the factor responsible for the suspension of meiosis. However, this simple hypothesis cannot explain the complicating results obtained in other species. For example, oxygen supplied at its atmospheric partial pressure impedes the maturation of follicle-free hamster oocytes (Gwatkin and Haidri, 1974) and causes spontaneous maturation and atretic changes in follicleenclosed sheep oocytes (Hay and Moor, 1973). The existence of a specific follicular factor which prevents oocytes from spontaneously maturing was suggested by Chang (1955) based on his experiments in which isolated rabbit oocytes were cultured in a medium containing follicular fluid. Foote and Thibault (1969) observed that, while follicle-free porcine oocytes spontaneously underwent maturation, oocytes cultured with follicle wall hemispheres failed to do so. Experiments by Tsafriri and Channing (1975a,b) demonstrated that a graded addition of cumulus cells to a culture of follicle-free oocytes inhibited their maturation in a dose-dependent manner but that the inhibition could not be removed by LH. However, Tsafriri et al. (1977), carrying out a similar experiment, found that LH was indeed capable of reversing the inhibitory effect of cumulus cells. Using porcine oocytes, Tsafriri and Channing (1975a,b) determined that isolated oocytes, cultured in a medium containing 50% porcine follicular fluid, underwent maturation with a frequency about half that of oocytes cultured without follicular fluid. The inhibitory effect of follicular fluid on the spontaneous maturation of follicle-free oocytes has been reported to be non-species-specific. Oocytes of the mouse (Channing and Tsafriri, 1977), rat Vsafriri et a/., 1977), and sheep and cow (Jagiello et al., 1977) cultured in media containing porcine follicular fluid were found to undergo maturation with a low frequency compared to those
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YOSHIO MASUI AND HUGH J . CLARKE
cultured without follicular fluid. In addition, the frequency of maturation of hamster follicle-free oocytes cultured in medium containing bovine follicular fluid was found to be decreased to the same extent as those cultured in medium containing hamster follicular fluid (Gwatkin and Andersen, 1976). The effects of bovine follicular fluid on hamster oocytes (Gwatkin and Andersen, 1976) and of porcine follicular fluid on porcine flsafriri et al., 1976b) and rat (Tsafriri et al., 1977) oocytes have recently been examined under a variety of physical and chemical conditions. These experiments revealed several facts concerning the nature of the maturation inhibitor. Its ability to block oocyte maturation is dose-dependent and is abolished by the addition of LH but not by the addition of dbcAMP. It is associated with a substance whose molecular weight lies between lo00 and 2000 daltons. Finally, it is trypsin-sensitive but resistant to heating at 60°C for 20 minutes. Yet, up to now, no tests have been carried out on the physiological activity of oocytes exposed to the follicular fluid inhibitor. With regard to the reversibility of the inhibitory effect of follicular fluid, some preliminary work by Stone et al. (1978) revealed that, among rat oocytes exposed to porcine follicular fluid for 24 hours, a certain proportion was capable of resuming maturation, but of those exposed to the inhibitory influence for 28 hours or more none resumed maturation. All in all, the evidence cited above indicates that oocyte maturation is caused by an abrupt cessation of the follicular function acting to stabilize the oocyte physiology. In view of the facts that mammalian oocytes spontaneously undergo maturation when they are freed from surrounding follicle cells, and that the tight junctions between oocytes and cumulus cells are lost when oocytes begin to mature following gonadotropin action (Szollosi, 1978; Gilula et al., 1978), it appears likely that maturation of follicle-enclosed oocytes also results from the loss of communication between oocytes and cumulus cells, which may follow an alteration in physiological activities of follicle cells by the gonadotropin action. From this point of view the recent finding by Tsafriri and Bar-Ami (1978) that maturation of follicleenclosed rat oocytes can be induced by Ca-deficient media without hormones may be interpreted in such a way that Ca deficiency brings about oocyte maturation by destabilizing the association between oocytes and cumulus cells. If so, the implication of this finding may be that no inhibitor exists in the follicular fluid to prevent oocytes from maturation or, if the inhibitor is present, its effect must be Ca-dependent. The latter possibility may be tested. However, in view of the following observations, it would be premature to deny totally the possibility of an action of gonadotropin and steroids on oocyte activity in relation to maturation. Recently, Jagiello and Ducayen (1977) demonstrated, in human, monkey, and sheep ovaries, a dense accumulation of HCG1251and LH on oocyte chromatin and in the cytoplasm, using autoradiographic and immunocytochemical techniques, and that these hormones also accumulated in follicle-free oocytes in vitro. Furthermore, it has been demonstrated in rats
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that there is a significant difference in the timing of GVBD in follicle-free oocytes cultured in the presence of LH and in those cultured in its absence (hpata et al., 1977). In the former, GVBD occurs less than 1 hour after isolation but not until after at least 2 hours in the latter case. The role of steroid hormones secreted by the follicle cells in promoting oocyte maturation has been studied by several workers. Baker and Neal (1972) noted the synergistic action of estrogen and gonadotropin. Hunter et al. (1976) found that pig oocytes ovulated following HCG administrationoften failed to undergo maturation if ovulation was induced when 17pestradiol levels in the follicle were low (day 17 of the estrous cycle), while those ovulated when hormone levels were high successfully completed maturation. Corroborative evidence comes from Moor and Trounson (1977) who found that the addition of 17P-estradiol to a culture of sheep follicles, in the presence of low levels of gonadotropin, increased the percentage of oocytes which could support normal embryonic development, although it did not affect the percentage which underwent maturation. McGaughey (1977), using pig oocytes, also reported that spontaneous maturation of follicle-free oocytes progressed beyond metaphase I more frequently in the presence of 170-estradiol and progesterone than in their absence. These observations seem to favor the view that follicular steroids also play a significant role in the maturation of mammalian oocytes, although the ability of gonadotropins to alter the physiological condition of the follicle is sufficient to allow the oocyte, meiotically arrested at the diplotene stage, to resume meiosis by initiating maturation.
F. OOCYTEMATURATION IN OTHER ANIMALS In many invertebrates, oocyte maturation is triggered by sperm penetration, but in some species the oocytes begin to mature following completion of their growth period, usually coincident with ovulation. The mechanism which induces oocyte maturation has not been elucidated except in the starfish (see Section KC). In many marine invertebrates, oocytes complete their growth in the coelom before beginning maturation. Oocyte maturation in polychaetes and sipunculoids begins and progresses to metaphase I while the oocytes are in the coelom. In Arenicola marina (a polychaete), it has been shown that removal of the brain (prostomiurn) 3 weeks before spawning prevents the onset of maturation, while the injection of brain homogenate into decerebrated animals causes the resumption of oocyte maturation as well as spawning (Howie, 1963, 1966). Recently Meijer and Durchon (1977) have reported that immature oocytes isolated from the coelom are induced to mature in vitro by exposure to seawater containing brain (prostomiurn) extracts in a dose-dependent manner.
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YOSHIO MASUI AND HUGH J. CLARKE
In Pectinaria gouldii (Cistemides), Tweedell (1962, 1966) found that oocyte maturation normally occurred only during the natural spawning season; it was characterized by oocytes undergoing GVBD after they were collected in the nephromixium. However, in oocytes collected during nonspawning periods, maturation can be induced by the injection of extracts obtained from various tissues into the coelom (K. S. Tweedell, personal communication). Since tissue extract injection induces the oocytes first to move into the nephromixium, where GVBD later occurs, it may be possible that the tissue factor changes the properties of the oocytes so that they become capable of being drawn into the nephromixium. Although these observations suggest that factors residing in the nephromixium play an important role in triggering oocyte maturation, the factors may not be specific ones since, when the animals are shaken mechanically in seawater, immature oocytes are ovulated and these oocytes spontaneously undergo GVBD in seawater 12-18 minutes after ovulation (Tweedell, 1962; Austin, 1963). Similar observations by Rice (1966, 1975) have been recorded in Sipuncula oocytes. In this species, oocytes begin to mature inside the coelom during the breeding season. Oocytes collected from the coelom of animals during nonbreeding periods will undergo maturation if treated with extracts from various tissues. Although it is premature to speculate on the mechanism initiating oocyte maturation in these species, it is possible that the coelomic fluid of these animals exerts an inhibitory effect on oocytes which prevents them from maturing during nonbreeding seasons. The inhibitory activity is removed as a result of tissue secretion during the breeding season, and consequently maturation is triggered. The lack of tissue specificity shown by the maturation-inducing factor suggests that it is a substance distributed to all tissues. Recent experiments by Peaucellier (1977) indicate that this is indeed the case in the polychaete Subellaria alveoluta. When oocytes of this species are carefully isolated, so that they have no contact with the cloaca, they fail to mature in seawater. But the oocytes thus isolated mature upon exposure to cloaca1 secretion. The factor responsible has been identified as a protease; apparently proteases from any source are effective.
G . THEROLEOF FOLLICLES IN
OOCYTE
MATURATION
Maturation does not normally take place during the growth period of oocytes, with the exception of a few groups of animals. In these species, maturation begins before the oocytes are fully grown. Oocytes of the spmge Hippospongia communis undergo maturation before the period of major growth begins (Tuzet and Pavans de Caccaty, 1958), and those of the insect Drosophila reach metaphase I before their growth ceases (Mahowald, 1977).
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Oocytes in the growth phase rarely respond to maturation signals from the outside. Reynhout et al. (1975) reported that follicle-enclosed oocytes of Xenopus must be at least 1.2 mm in diameter to be responsive to gonadotropic or HCG stimulation. However, smaller oocytes, having diameters as small as 0.9 mm, can be induced to mature in vitro by progesterone, provided they have been removed from their follicles. Small follicleenclosed oocytes become responsive to gonadotropins after repeated progesterone administration. Similar observations were recorded by Sakum (1961, 1972, 1975) in the trout. She found that repeated gonadotropin administration caused growing small oocytes to begin maturation, although the maturation process was generally abnormal and resulted in atypical condensation and arrangement of the oocyte chromosomes. In mammals, the ability to initiate maturation appears to be restricted to oocytes enclosed in follicles of some minimum size. No oocytes removed from preantral follicles of prepubertal mice (younger than 14 days) undergo maturation (Szybek, 1972; Erickson and Sorenson, 1974). A graded increase in the tendency of oocytes to undergo maturation was found to occur with the progression of follicular growth in pigs (Tsafriri and Channing, 1975b). As pointed out by Iwamatsu and Yanagimachi (1975) in the hamster and by Sorensen and Wassarman (1976) in the mouse, the tendency of a follicle-free oocyte to begin maturation is directly related to oocyte growth. According to their observations, the frequency with which spontaneous maturation occurs increases linearly in proportion to the diameter of the oocytes, up to a certain size, 80 p m in the hamster and 68 p m in the mouse. Furthermore, maturation of the smaller oocytes is often abortive, being arrested at metaphase I. Development of the tendency toward spontaneous maturation of mouse oocytes occurs in the follicles in the absence of gonadotropic influences. According to Eppig (1977), follicles of 8-day-old mice, cultured for 1 week in hormone-free media, contain growing oocytes capable of initiating maturation upon isolation from the follicles. He also pointed out the importance of a close relationship between the cumulus cells and the oocytes for the latter to grow and mature. The spontaneous maturation of follicle-free oocytes in mammalian species may be indicative of a certain physiological instability of oocytes which have reached a certain stage of growth. Perhaps the physiological state of these oocytes is so unstable that, upon removal from the stabilizing influence of the follicles, they tend to undergo changes which result in maturation. In nonmammalian species, fully grown oocytes appear to be relatively stable, so that external stimuli are needed to initiate maturation. However, their responsiveness to such stimuli may be conditioned by some degree of intrinsic physiological instability. Thus it is possible that the increasing tendency of growing oocytes to undergo maturation is a reflection of increasing physiological instability. From studies of the gonadotropin control of oocyte maturation in various
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forms of animals we have learned that changes in the physiological activities of the follicle cells are crucial for the initiation of oocyte maturation. However, the maturation process of amphibian, starfish, and sturgeon oocytes can take place without follicles following treatment with follicular hormones. In these animals, follicular hormones such as progesterone and 1-MA act on the oocyte in the same manner regardless of the presence or absence of the follicle; that is, both folliclefree and follicle-enclosed oocytes, once induced to mature, exhibit the same developmental capacity as naturally ovulated oocytes. In R. pipiens, both oocytes which have been ovulated in vitro from isolated follicles after gonadotropin treatment (Ryan and Grant, 1940) and those induced to mature by progesterone following defolliculation (Smith et al., 1968) develop normally if they are inseminated after passing through the oviduct of recipient females. Similar results were obtained with sturgeon oocytes (Dettlaff and Skoblina, 1969). Finally, in starfish, oocytes matured either with or without follicles cleave normally (Kishimoto and Kanatani, 1973) and develop at least to the bipinnaria stage (Guenier et al., 1978). However, oocyte maturation in mammalian species is highly dependent on the follicle. Follicle-free oocytes which have undergone spontaneous maturation are found to lack complete developmental potentiality as compared with follicleenclosed oocytes induced to mature by gonadotropin. Thibault (1972) found that follicle-free oocytes of the rabbit underwent maturation spontaneously in culture but that they did not exhibit the ability to transform sperm nuclei into pronuclei after insemination. Oocytes matured within follicles cultured in the presence of gonadotropin acquired the ability to form male pronuclei. It has also been reported, in the pig, that meiosis which takes place in folliclefree oocytes during spontaneous maturation usually results in considerable chromosomal aberration (McGaughey and Polge, 1971). Studies using rabbits (Chang, 1955) and mice (Cross and Brinster, 1970; Mukherjee, 1972) have shown that oocytes which have undergone maturation without follicles rarely develop beyond the early cleavage stages after insemination, and Van Blerkom and McGaughey (1978b) observed that only a small proportion (13%) of rabbit oocytes matured in vitro could reach the blastocyst stage. Recently Eppig (1978) using oocytes of LT/Sv hybrid mice has noted that follicle-free oocytes, which spontaneously matured in vitro and activated, can initiate cleavage parthenogenetically, but fail to develop further. To attain the potential to develop to blastocysts the oocytes must mature within follicles for 8-9 hours after gonadotropin administration. In sheep, Moor and Trounson (1977) demonstrated that oocytes cultured without follicles failed to develop beyond the blastocyst stage, whereas those matured within follicles, which were cultured in the presence of LH, FSH, and 17P-estradio1, developed normally. When the oocytes of this latter group were fertilized following transfer into recipient females, 73% developed to the blastocyst stage and 63% into newborn lambs. These observations
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clearly indicate that an indispensable role is played by the follicles in the process of normal oocyte maturation in mammals.
111. Progression of Maturation
A. CHRONOLOGY
Traditionally the process of oocyte maturation has been described referring to changes in chromosome morphology during meiotic progression. Before maturation starts, the oocyte contains a GV with a few large or many small nucleoli (Fig. 1). Chromosomes in the GV are extended and widely dispersed (Fig. 2). With the initiation of maturation, the GV breaks down and the chromosomes begin to condense. As maturation progresses, the chromosomes further contract and become arranged in the middle of a spindle in pairs-metaphase I (Fig. 3). Separation of the paired homologous chromosomes (Fig. 4) is followed by formation of the first polar body (Fig. 5 ) . Then the chromosomes remaning in the oocyte become aligned in the metaphase plate-metaphase I1 (Fig. 6). When the second meiotic division begins, the daughter chromosomes separate (Fig. 7) and become partitioned between the oocyte and the second polar body. The chromosomes remaining in the oocyte after separation decondense to form a nucleusthe pronucleus stage (Fig. 8). Any comparison of the time course of oocyte maturation in different species or under different conditions imposes several difficulties. First, determination of the time at which maturing oocytes move from one stage to another is entirely dependent on one's definition of the two stages, which necessarily involves a certain degree of arbitrariness. From a morphological standpoint, metaphase is the only stage which can be clearly defined. Even if each stage could be clearly defined, arbitrarily or otherwise, abnormalities in the maturation process, which often occur under experimental conditions, make it difficult to judge whether or not the oocytes have reached a certain stage. In addition, since morphological changes in an oocyte are continuous, it is not clear exactly when transitions between stages occur. Second, the progression of maturation in members of an oocyte population is not synchronous. Therefore a statement regarding its time course can be made only on a statistical basis. For example, the time at which oocytes reach a certain transient stage of maturation can only be defined as the time when the proportion of the population at that stage reaches a maximum. Or, the time at which oocytes reach a stage where meiosis is suspended indefinitely, such as metaphase I1 of the vertebrate oocyte, may be defined as the time when half the population has reached that stage. Similar but more complicated considerations may be neces-
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YOSHIO MASUI AND HUGH J. CLARKE
FIGS.1-8. Chromosome changes during progression of oocyte maturation. Follicle-free mouse oocytes were fixed at different times following culture in an alcohol-acetic acid mixture and stained with Giemsa. FIG. 1. An oocyte with an intact GV and a nucleolus. FIG. 2. Chromosomes in the GV. FIG. 3. Condensed chromosomes at metaphase I. FIG.4. Expulsion of polar body I and oocyte chromosomes and a spindle (arrow). FIG. 5 . Segregation of oocyte chromosomes (aggregated) and polar body I chromosomes (scattered).
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207
s q if a proportion of the population fails to complete maturation because of physiological deterioration. Third, inaccuracy in determining the time course of oocyte maturation could be introduced by ignorance of its exact time of initiation. In nonmammalian species this initiation time may be defined as the time when a maturationinducing agent, such as a hormone or sperm, is applied to the oocytes in vitro. Similarly, in mammals the time at which follicle-free oocytes initiate maturation in vitro may be defined as the time at which oocytes are isolated from their follicles or at which a maturation inhibitor, such as dbcAMP, is withdrawn. Determination of the time at which follicle-enclosed oocytes initiate maturation is a more complex problem. It is generally agreed that, in the normal estrous cycle of mammals, maturation begins at the time of the LH surge, since oocytes enclosed in follicles fail to initiate maturation if they are removed before the LH surge occurs (Ayalon et al., 1972; Hillensjo, 1976; see also Section 11,E). The timing of the LH surge and of the initiation of maturation may be determined at the same time if LH secretion is suppressed at various times using a drug such as Nembutal. The time at which Nembutal administration fails to inhibit oocyte maturation can be considered the point at which oocyte maturation begins. During natural ovulation, rat oocytes reach metaphase I1 10 hours after the LH surge (hfriri and Kraicer, 1972). When ovulation is induced by HCG treatment, oocytes also reach metaphase I1 about 10 hours after HCG injection ( a i l maker et al., 1974). These observations suggest that any delay in the initiation of oocyte maturation following HCG administration is negligible. In fact, it was found that, when HCG was injected into animals, the hormone could be detected within 5 minutes of its administration (Jagiello and Ducayen, 1977). Therefore it may be assumed that the administration of a gonadotropin, as well as the natural surge in vivo, immediately stimulates follicles to initiate oocyte maturation. On the basis of the considerations above, we discuss differences in the time course of oocyte maturation observed under different experimental conditions and among various species. In rodents, the interval between the initiation of maturation and metaphase I1 is similar in both hormone-induced maturation of follicle-enclosed oocytes and spontaneous maturation of follicle-free oocytes (Table I). Thus it may be assumed that the initial events of maturation occur virtually simultaneously in hormonally stimulated follicles and in isolated oocytes. However, a considerable delay (McGaughey and Polge, 1971) or acceleration (Motlik and Fulka, 1976) in the progression of maturation was reported for spontaneously maturing pig ooFIG.6. Metaphase II chromosomes. FIG. 7. Segregation of oocyte chromosomes and polar body I1 chromosomes following activation with ionophore A23187 (anaphase II). FIG. 8. Female pronucleus and polar body II chromosomes. (Brazill, 1977.)
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YOSHIO MASUI AND HUGH J. CLARKE
TIME
TABLE I COURSE OF OoCYTE MATURATION^
In vivo maturation of follicleenclosed oocytes (hours)
Animal: Stimuli: Stage GV PMI MI
Rabbit Coitus
Rat HCG
Rat
LH surge
Mouse HCG
Hamster HCG
Ram pipiens Pituitary
0 0 0 0 0 0 4.0-7.0 3.0-3.5 4.0-5.0 6.5 6.0 6.5 7.0-8.0 22 MII 9.0-9.5 10.5 10.00 13.00 11.0-12.0 38 MVMII 0.44-0.51 0.62 0.60 0.49 0.62-0.66 0.58 Reference Thibault Zeilmaker Tsafriri and Edwards Usui and Smith et al. (1972) et al. Kraicer and Gates Yanaghachi (1966) (1 976) (1974) (1972) (1959) In v i m maturation of oocytes (hours) Animal: Rabbit Rabbit Mouse Hamster Sturgeon Starfish Stimuli: Isolation Isolation Isolation Isolation Gonadotropin 1-MA Stage 0 0 0 0 0 GV 0 6.0-7.8 6.0-9.0 12.2-12.6 PMI MI 3.3-3.6 5.0-6.0 9.0 9.0 13.4-14.8 1 .o MII 8.1-8.5 9.0-11.0 13.0 c12.0 18.4 11.5 MI/MII 0.41-0.42 0.55 0.69 (0.78) 0.72 0.67 Reference Thibault Chang Donahue Iwamatsu and Vassetzky Ikeda et al. (1972) (1955) (1968) Yanagimachi (1970) ( 1976) ( 1975) Relative duration (7) of different phases in first meiosis and first mitosis**.‘ First mitosis
First meiosis Animal
Rat
Hamster
Mouse
Acipenser
Oyster
Acipenser
Stage PMI
MI
Al TI Total ~~~~
1.5 (20) 2.0 (27) 2.0 (27) 2.0 (27) 7.5 (100)
1.0 (21) 2.3 (48) 0.25 (5) 1.28 (27) 4.8 (100) ~~~~
~~~
1.5 (18) 4.4-4.5 (55)
1.5-2.0 (21) 0.5 (6) 7.5-9.5 (100) ~~~
2.2 (25) 3.7 (43) 1.7 (20) 1.0 (12) 8.6 (100) ~
~
13-14 (26-27) 12-19 (25) 13-14 (29) 7-9 (19)
0.1 (20) 0.2 (40) 0.14 (28) 0.05 (12) 0.5 (100)
-
~
“GV, Immature stage; PMI, prometaphaseI; MI, metaphase I; Al,anaphase I; TI, telophase I; MII, metaphase II, 7 , duration of the first cleavage cycle. bVassetzky ( 1977). CPercentageis given in parentheses.
cytes as compared with hormone-induced in vivo maturation. The time required for follicle-free oocytes to reach metaphase I1 in in vitro culture following their isolation ranges from 42.5 to 55 hours (McGaughey and Polge, 1971), whereas that for follicle-enclosedoocytes maturing in vivo following HCG administration lies between 36 and 37 hours (Hunter and Polge, 1966).
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209
In different species, oocyte maturation proceeds at different rates, even in members of the same phylogenetic class (Table I). However, the relative times required for oocytes to reach different stages of maturation appear to be fairly constant among species. For example, the ratio between the times required for oocytes to reach metaphases I and 11, recorded from the onset of maturation, is similar in different species. This trend seems to suggest that each step of oocyte maturation is coordinated under a principle common to oocytes of different species. This notion may be strengthened by the observation that the relative durations of prometaphase, metaphase, anaphase, and telophase are approximately the same among species and even in meiosis and mitosis (Vassetzky, 1970, 1973, 1977). In addition, evidence has been presented that coordination also exists between different maturational events occumng in the cytoplasm of the oocyte. In X. Zuevis, the time of GVBD, again taken from the onset of maturation, that is, progesterone application to the oocytes, and the time required for progesteronetreated oocytes to become resistant to protein synthesis inhibitors have been found to be variable among oocytes from different females, but the ratio between these two values is always constant, that of the former to the latter being 0.65:l.OO (Wasserman and Masui, 1975a). In R. pipiens oocytes, the times required for GVBD to occur and for an oocyte to become responsive to activation stimuli vary among oocytes obtained from different females. There is also a seasonal dependence. In the fall or early winter, GVBD takes place 20-24 hours following progesterone application at 18"C, while in the spring or early summer it occurs after only 10-14 hours. Likewise, the time required for oocytes to become responsive to activation stimuli varies between 30 and 50 hours. Nonetheless, the ratio of the time elapsed before the appearance of the ability to activate to that elapsed before GVBD was found to remain fairly constant at 2.7 ? 0.2 (Lohka, 1978). These observations may indicate that sequential events taking place during oocyte maturation, either cytoplasmic or nuclear, are closely coordinated within each oocyte. B. MORPHOLOGICAL CHANGES
The progression of oocyte maturation is accompanied by fundamental changes in cytoplasmic as well as in nuclear structures. The recent technical development of oocyte culture has enabled investigators to obtain oocytes at various stages during maturation, allowing detailed study of these changes with light and electron microscopy in mammalian, amphibian, and fish oocytes. Information from these studies has been carefully compared with that from oocytes at the diplotene stage in the ovary. The chromosome configuration of oocytes at the diplotene stage varies from species to species. In mammals, which have been the most extensively studied,
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the chromosomes, condensed during the early phase of meiosis, become more or less diffuse when the oocytes enter the stationary phase. In mouse oocytes at this stage, the nucleus (the GV) contains thin chromatin strands which are occasionally heterochromatinized, clusters of granules ranging between 400 and 900 8,in diameter, and a large spherical nucleolus (Szollosi et al., 1972; Calarco et al., 1972). However, the chromosomes of primate oocytes remain in a partially condensed state and are scattered in the GV (Zamboni et al., 1972). Baker and Franchi (1966, 1972) have observed many short loop projections on these chromosomes, similar to those of lampbrush chromosomes of amphibian oocytes. The chromosomes of fully grown oocytes of lower vertebrates remain in a more condensed state than those of mammalian oocytes and are grouped in a small area of the GV. Light microscopy of chick oocytes has revealed that these discrete chromosomes are confined to an area 20 p m in diameter within the discoidal GV which itself measures approximately 100 p m in depth and 300-400 p m in diameter (Olsen and Fraps, 1950). In amphibian oocytes the GV contains lampbrush chromosomes with many projecting loops. Chromosomes in anuran (R. pipiens) fully grown oocytes are more contracted and project shorter loops (Duryee, 1950) than those of urodele (Norophthalmus viridescens) oocytes (Pilone and Humphries, 1975). This difference in chromosomal morphology between the two amphibian species may reflect a difference in the state of growth of preovulatory oocytes, which might be due to their different breeding habits (see Section 11,B). Studies of fish oocyte chromosomes during oocyte maturation are rather limited. In sturgeons, Dettlaff and Skoblina (1969) showed that the chromosomes in fully grown oocytes were partially condensed and aggregated near the center of the GV. Observations of invertebrate oocyte chromosomes during oocyte maturation are also few, though many investigators have studied the chromosomes of growing oocytes. Das (1976) noted that, in Urechis caupo oocytes, meiosis progressed from the diplotene stage to diakinesis before fertilization triggered maturation. Transition from the diplotene stage to the first meiotic division is initiated by condensation of the chromosomes in the GV. In mammals, chromosome condensation begins near the inner membrane of the nuclear envelope where the chromatin first adheres (Calarco et al., 1972; Motlik et al., 1978). In lower vertebrates, the chromosomes gather in the middle of the GV, where they begin to condense. The process of condensation, which in urodeles involves rapid regression of the loops of the lampbrush chromosomes (Pilone and Humphries, 1975) and progressive contraction of the entire chromosome, has been observed in anurans by Dettlaff and Skoblina (1969) and Brachet et al. (1970) and in fish by Aisenstadt and Dettlaff (1976). These condensing chromosomes also migrate toward the animal pole concurrently with GVBD. Light microscope observations by Vassetzky (1973) of oyster oocyte maturation showed similar chromosome behavior.
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The behavior of nucleoli during the early phase of GVBD has been described both in mice (Zamboni, 1972) and in amphibians (Brachet et al., 1970). In mammals the nucleolus increases in size and accumulateselectron-dense material before disintegrating, while in amphibians the nucleoli condense into a giant nucleolus before disintegration occurs. It was pointed out by Brachet (1965) and Brachet et al. (1970) that DNA cores in the nucleoli remained after nucleolar disintegration, being discharged into the cytoplasm as Feulgen-positive bodies. Undulation of the nuclear envelope is observed shortly before GVBD in oocytes of Chaetopterus (Merriam, 1961), mice (Szollosi et al., 1972), amphibians (Brachet et al., 1970), and sturgeons (Dettlaff and Skoblima, 1969). This undulation of the nuclear envelope heralds GVBD. Cinematographic observations of rat oocyte maturation by Lopata et al. (1977) clearly indicate that only oocytes showing this nuclear envelope undulation undergo GVBD. According to Szollosi et al. (1972), the undulating nuclear envelope in mouse oocytes is eventually folded to form nuclear envelope doublets which are then fragmented. Most of the fragments thus formed later separate into single cistemae at or before metaphase I and eventually become indistinguishablefrom smooth endoplasmic reticulum. In amphibian oocytes, this undulating movement of the nuclear envelope, as well as its disintegration, begins in the vegetal half of the GV (Dettlaff and Skoblina, 1969; Brachet et al., 1970). A classic study of GVBD in chick oocytes by Olsen and Fraps (1950) reported further that the nuclear envelope, once disintegrated at the time of GVBD, was reconstituted during diakinesis as a smaller envelope surrounding an aggregation of the chromosomes discharged from the GV. In the process of GVBD, varied interactions occur between the GV and the cytoplasm. In mice (Zamboni, 1972), amphibians (Brachet et al., 1970), and loach (Iwamatsu and Ohta, 1977), it has consistently been observed that mitochondria increase in number, particularly in the area close to the actively undulating nuclear envelope-more specifically, in the indentations of the envelope. In amphibian oocytes, cytoplasmic granules rich in /3-glycogen accumulate near the disintegrating nuclear envelope and, as GVBD progresses, the glycogen granules, as well as the mitochondria move into the nucleus. Here they surround the meiotic spindle which has a high &glycogen content (Brachet et al., 1970). At the same time, in both mouse (Calarco et al., 1972) and amphibian oocytes, the development of prominent microtubules running perpendicularly to the envelope can be observed. These later penetrate the nuclear envelope. In addition, numerous cytasters form in oocytes of these species. In amphibians (Balinsky and Dans, 1963), sturgeon (Aisenstadt and Dettlaff, 1972), and mouse and human (Zamboni, 1972) oocytes the stacked lamellae disintegrate concomitantly with dissolution of the nuclear envelope. The vesicles formed from them migrate peripherally via Golgi apparatus to give rise to secretory granules. The cortical granules formed inside the cytoplasm before oocyte maturation then migrate toward the periphery of the oocyte as maturation
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proceeds. A similar observation has been reported in sea urchins (Long0 and Anderson, 1970). Changes in the peripheral structures of oocytes undergoing maturation have been studied in amphibians (Van Gansen and Schram, 1968; Kemp and Istock, 1967) and in mammals (Zamboni, 1972). In amphibian oocytes, the number and size of the surface microvilli are progressively reduced during GVBD. This change in surface fine structure may cause macroscopic changes as GVBD progresses, which include increased surface luster and weakened attachment to the vitelline membrane (Schuetz, 1974). However, in mammalian oocytes, the surface microvilli increase and are maintained during the period of maturation (Zamboni, 1972). C. BIOCHEMICAL CHANGES
1 . Energy Metabolism The respiration of oocytes was one of the classic subjects which attracted the attention of “chemical embryologists” a few decades ago (see Needham, 1942; Brachet, 1950). However, until recently, when extensive mammalian research was undertaken, the field remained undeveloped. Much of the original work on oocyte respiration was done using marine invertebrates. Ironically, however, this remains the most confusing area when one attempts to determine the relationship between maturation and respiration changes. Boell et al. (1940) found no change in the O2 consumption rate of starfish ( A . forbesii) oocytes during maturation. But Lindahl and Holter (1941) and Borei (1948) reported a decrease in the case of sea urchin (Psammechinus, Paracentrotus) oocytes. A similar decrease in O2consumption during maturation was reported for the medaka (Nakano, 1953) and recently for the loach (Ozernyuk et al., 1973). Conversely, an increase in the level of respiration during maturation was observed in starfish by Borei (1948), Houk (1974), and Schulz and Lambert (1973), and in sea urchins (Arbacia) by Boell et al. (1940). Among the vertebrates, Brachet et al. (1975a) found that the O2consumption of progesterone-stimulated oocytes of the toad X. laevis gradually increased following GVBD. Spontaneous maturation of follicle-free oocytes of the rat is also accompanied by a gradual increase in the O2 consumption of the oocytes following GVBD; this rise does not occur in oocytes which fail to undergo GVBD (Magnusson et al., 1977; Magnusson and Hillensjo, 1977). These conflicting results, especially those concerning marine invertebrates, may in part be explained by the fact that the oocytes used in some experiments were completely devoid of follicles, while in others they may have been invested with follicle cells. In the latter instance, the values obtained for 0, consumption clearly reflect changes occurring both in the oocyte and in the surrounding cells.
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In fact, as shown by Hillensjo et al. (1975) and Dekel et a f . (1976), the rate of O2 consumption of rat follicle-oocyte complexes decreases during oocyte maturation following LH stimulation. Since by far the greater proportion of the respiratory activity of the complex is due to respiration of the follicle cells surrounding the oocyte (Hillensjo et al., 1975; Magnusson et al., 1977), it is important to realize that, while the 0, consumption of the oocyte may increase during maturation, this would be hidden by a greater decrease in the O2consumption of the surrounding follicle cells. Experiments with mammalian follicles have demonstrated an increase in the rate of lactic acid release as well as of glucose uptake by the follicles after LH stimulation (Nilsson, 1974; Hillensjo, 1976; Tsafriri et al., 1976a). However, ailmaker and Verhamme (1977) recently measured the lactate present within the follicle itself and found that there was no significant change in its level as maturation proceeded to metaphase I. Thus it appears that the level of lactate present in the follicle before LH stimulation is sufficient to support the initiation of oocyte maturation. In other words, increased production of lactic acid by follicles stimulated by LH is not obligatory for the initiation of oocyte maturation. In fact, the lactic acid production of follicles following LH stimulation can be suppressed by treatment with iodoacetate without inhibiting the initiation of oocyte maturation (Tsafriri et al., 1976a). Thus it seems that the stimulatory effect of LH on oocyte maturation is independent of any enhancement of glycolytic activity of the follicle. A wide variety of glucose metabolites, such as pyruvate, lactate, oxaloacetate, succinate, and fumarate, as well as glucose itself, can be used by the follicle to support oocyte maturation, though with varying degrees of effectiveness. However, as first pointed out by Biggers et al. (1967) in mice, follicle-free oocytes can effectively utilize only pyruvate and oxaloacetate as substrates. Lactate and other glucose derivatives can be utilized effectively provided the oocytes are incubated with follicle cells, which are capable of metabolizing lactate. This is apparently a universal phenomenon found not only in the oocytes of other mammalian species such as the monkey (Brinster, 1971), cow (Rushmer and Brinster, 1973), and rat (ailmaker and Verhamme, 1974) but also in amphibians such as X. laevis (Eppig and Steckman, 1974, 1976). In fact, Zeilmaker and Verhamme (1974) suggested that, because of the limited availability of 0, in the follicle, lactate rather than pyruvate can be expected to be the major energy source available to follicle-enclosed oocytes. It has been suggested (Sorensen, 1972; Zeilmaker et al., 1972) that the inability of follicle-free oocytes to consume lactate may result from a lack of endogenous nicotinamide adenine dinucleotide phosphate (NAD) necessary to convert lactate to pyruvate via the activation of lactic acid dehydrogenase (LDH). This suggestion is based on observations that addition of NAD causes the maturation of follicle-free oocytes in a medium containing only lactate as an energy source. However, Eppig and Steckman (1976)
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raise the possibility that the ineffectiveness of substrates other than pymvate and oxaloacetate as energy sources in Xenopus oocytes in culture may be due to differences in the permeability of the oocyte to different substrates, in view of the fact that pyruvate is taken up 30 times faster than any of the other glucose metabolites. In their experiments, these investigators noticed that several days are required before the differential effects of various energy sources became manifest, indicating that the oocytes were using an endogenous energy source. Legname and Buhler (unpublished) reported that Bufo ovarian oocytes contained more citrate than fumarate during the winter months, but that the ratio was reversed in the spring or when winter oocytes were induced to mature by progesterone treatment in the absence of exogenous nutrients. Although there is not yet complete agreement with respect to the mechanisms underlying the energy metabolism of maturing oocytes, there is no doubt that oocyte maturation is an energy-consumingprocess. Most investigators agree that GVBD cannot take place under conditions inhibiting aerobic metabolism, such as lack of oxygen (Zeilmaker and Verhamme, 1974; Gwatkin and Haidri, 1974) or the presence of a respiratory inhibitor such as KCN (Brachet et al., 1975a). Schulz and Lambert (1973) noted that the maturation of starfish oocytes involved a decrease in AMP and ATP levels, but an increase in the level of ADP, and suggested that the increased 0, consumption observed could be interpreted as an indicator of enhanced oxidative phosphorylation acting to restore ATP to its prematuration level. This increase in 0, consumption of the oocyte has been found to occur after GVBD in starfish (Schulz and Lambert, 1973; Houk, 1974), Xenopus (Brachet et al., 1975a), and rats (Magnusson et al., 1977). In this connection, it is interesting that neither anaerobic conditions nor the presence of KCN interfere with progression of the second meiotic division in rat oocytes (Zeilmaker and Verhamme, 1974). Perhaps the increased level of O2 consumption following GVBD is a result of mechanisms operating to replenish ATP consumed during GVBD, thus enabling oocytes to proceed with meiotic division. One of the energy-requiring processes occumng during oocyte maturation is the synthesis of protein. This dependence seems logical intuitively; two observations are mentioned here. First, there is a substantial reduction in the level of protein synthesis during maturation of R. pipiens oocytes under anaerobic conditions (Smith and Ecker, 1970b). Second, the rise in O2consumption observed in maturing X . laevis oocytes coincides with the time of increased amino acid incorporation into oocyte protein (Brachet et al., 1975a). 2. Protein Synthesis An increase in the protein synthesis activity of oocytes during maturation is apparently a general phenomenon among animals. However, the fact that protein synthesis can be measured in a variety of ways under a variety of conditions makes it difficult to compare accurately the results obtained by different inves-
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tigators. Smith et al. (1966) injected R. pipiens oocytes with radioactive leucine and then extracted the oocytes with a saline solution following incubation for a given period of time. The extracts were then precipitated with hot trichloroacetic acid ("CA), and the leucine incorporated into this fraction measured. Obviously, this approach gives no information concerning proteins insoluble in the saline solution. Some investigators, however, have exposed oocytes to a labeled amino acid for a certain length of time, collected all the material precipitated by TCA directly from the oocytes, and measured the radioactivity in the precipitate. This fraction may represent the total protein content of the oocytes. However, our experience has led us to believe that with amphibian oocytes this method often introduces some error into the determination, since free leucine in TCA precipitates is difficult to remove, while the method using perchloric acid (PCA) instead of TCA gives more accurate results. Moreover, it should be noted that an accurate determination of the size of the amino acid pool in the oocyte, which may change during maturation, is difficult and yet crucial for comparison of the rates of protein synthesis at different stages of maturation. Nevertheless, some reports provide no data concerning the amino acid pool size in maturing oocytes at different stages. In spite of these shortcomings, we state as a general rule that changes in protein synthesis activity occur during the maturation of oocytes of all species studied to date. The rate of synthesis usually increases as a result of treatment with a maturation-inducing hormone. In R. pipiens, protein synthesis increases by a factor of about 10, about 18-24 hours after hormone treatment, following GVBD, and reaches its maximum level after metaphase I (Smith et ul., 1966; Smith and Ecker, 1970a). However, recent reinvestigations of protein synthesis in X. laevis (O'Connor and Smith, 1976) and R. pipiens (Shih et al., 1978) oocytes have indicated that the increase in the rate of total protein synthesis during maturation is at most two-fold when amino acid pool sizes and diffusion rates of labeled amino acids are taken into consideration. Protein synthesis activity in oocytes of A. forbesii (Wassarman, 1971) and U.caupo (Blankstein and Kiefer, 1977) increases two to four times, again after GVBD. However, it was reported that although treatment of X . laevis oocytes resulted in a stimulation of protein synthesis 1-4 hours after treatment, which was before GVBD, this stimulation was only temporary and protein synthesis was sharply reduced, eventually to 50% of the maximum level attained (Baltus et al., 1973; Brachet et al., 1974). Invertebrate studies by Houk and Epel (1974), using Patiria niniata, demonstrated that protein synthesis in starfish oocytes began to increase 12 minutes after 1-MA stimulation, before GVBD occurred. By prophase 11, it had reached a level five times that of untreated oocytes. This change in protein synthesis activity following 1-MA stimulation is unaffected by fertilization. Changes in the pattern of oocyte protein synthesis also occur as maturation progresses. Smith and Ecker (1971), using R. pipiens oocytes at different stages of maturation, analyzed the incorporation of radioactive leucine into various
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protein fractions following separation by one-dimensional gel electrophoresis and discovered characteristic changes occurring during the progression of maturation. Pennequin et al. (1975) and Schorderet-Slatkine and Beaulieu (1977), using the technique of double-labeling electrophoresis, observed differences in the synthetic activity of various protein fractions extracted from hormone-treated and untreated Xenopus oocytes labeled with 3H and '*C, respectively. Protein synthesis by mammalian oocytes has been studied by one- or two-dimensional gel electrophoresis followed by autoradiography in mice (Golbus and Stein, 1976; Schultz and Wassarman, 1977a,b; Schultz et al., 1978), pigs (McGaughey and van Blerkom, 1977), sheep (Warnes et al., 1977), and rabbits (van Blerkom and McGaughey, 1978a). While the studies done on amphibian (Smith and Ecke, 1971; Pennequin et al., 1975) and mouse (Golbus and Stein, 1976; Schultz and Wassarman, 1977a,b) occytes showed significantchanges in protein synthesis patterns only after GVBD, the study of sheep oocytes revealed changes occuring before GVBD (Warnes et al., 1977). These conflicting results may lead us to question whether the oocyte protein synthesized during maturation is required for the progression of maturation. Early studies showed that protein synthesis inhibitors, such as cycloheximide and puromycin, acted as strong inhibitors of oocyte maturation in amphibians (X. laevis, Brachet, 1967; R . pipiens, Smith and Ecker, 1969; R . temporaria and B . bufo, Dettlaff, 1966) and in fish (Acipenser, Dettlaff and Skoblina, 1969). These reports consistently indicated that GVBD, chromosome condensation, and nucleolar dispersion were all inhibited at concentrations of the inhibitor which suppressed virtually all protein synthesis. However, Smith and Ecker (1970b) and Ecker and Smith (1971a) showed that a substantial reduction in the level of protein synthesis under anaerobic conditions did not prevent oocytes from maturing, provided they had undergone GVBD. Baltus et al. (1973) also obtained evidence that maturation could proceed when protein synthesis was 50% inhibited by a 5-hour cycloheximide treatment. In mammals, treatment with protein synthesis inhibitors such as puromycin or cycloheximide arrests oocytes at metaphase I or at the circular bivalent chromosome stage, but GVBD and chromosome condensation are unaffected (Stem et al., 1972; Golbus and Stein, 1976; Wassarman and Letourneau, 1976b; Schultz and Wassarman, 1977b). However, it should be noted that protein synthesis was not completely inhibited in these experiments. It is possible that the small amount of synthesis still occurring in the presence of the inhibitors may provide the proteins necessary for the early events of maturation. The results obtained by researchers working with invertebrates are not quite so uniform. Houk and Epel (1974) found pactamycin to be a very effective inhibitor of protein synthesis in P. rniniata oocytes-a dose of 100 pg/ml eliminated all incorporation into TCA-precipitable protein within 15 minutes of its application. Despite the complete lack of protein synthesis, GVBD occurred and the oocytes
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developed a spindle capable of organizing chromosomes. Maturation was not arrested until metaphase I or anaphase I. Brachet and Steinert (1967), conducting experiments with the starfish Asterias rubens, found that puromycin (50 pg/ml) and cycloheximide (20 pg/ml) did not inhibit GVBD but did prevent chromosome migration and spindle formation. The work of Zampetti-Bosseler et al. (1973), using Asterias glacialis, demonstrated that fusidic acid or puromycin prevented formation of the mitotic apparatus required at metaphase I, but not GVBD. In this case, protein synthesis appears to have been reduced to about 20% of control levels. The same group also studied Chaetopterus oocytes, finding that fusidic acid greatly suppressed protein synthesis and stopped maturation before GVBD. Puromycin, however, while inhibiting protein synthesis by 50%, did not affect maturation, implying that only some of the protein synthesized during maturation was actually required for maturation (cf. amphibian results above). According to Blankstein and Kiefer (1977) incubation of U.caupo with cycloheximide (50 pg/ml) inhibits protein synthesis but does not prevent fertilized eggs from undergoing fertilization membrane elevation, GVBD, two meiotic divisions, and pronuclear formation. These eggs are unable to cleave, though. Apparently, in this species, all the proteins required for meiotic maturation are present before the onset of maturation, and the proteins synthesized during maturation are needed for later developmental events. Thus two questions arise. First, what portion of the protein synthesized by the maturing oocyte is responsible for the progression of maturation and, second, at what time are the proteins necessary for each step of maturation synthesized? Mom11 et al. (1975), using R. pipiens, reported that the proteins responsible for GVBD, which occurs 8-9 hours following gonadotropic stimulation of follicleenclosed oocytes, were synthesized within 5 or 6 hours after hormone treatment. Inhibition of protein synthesis by cycloheximide after this time failed to inhibit GVBD. Wasserman and Masui (1975a) showed that the inhibitory action of cycloheximide on GVBD in X. laevis oocytes was effective when the inhibitor was applied during the first two-thirds of the time period between progesterone stimulation and GVBD. Thus, in amphibians, as suggested by Smith and Ecker (1970b) and Ecker and Smith (1971a), it seems likely that the low level of protein synthesis occurring during the early period before GVBD reflects the production of proteins necessary for the events of maturation per se to occur, while the much higher level observed later represents proteins required for processes occurring later in development. However, it must be pointed out that the effect of protein syndesis inhibitors preventing oocytes from initiating maturation does not necessarily signify a requirement for new protein synthesis by the oocytes to initiate maturation, since it is possible that the inhibitors also interfere with ongoing synthesis of preexisting, but short-lived, proteins in the oocytes to deprive them of the ability to respond to maturation-inducing agents. Schultz and Wassarman (1977a), using mice, have suggested that inhibition of
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protein synthesis during the first 5 hours of maturation-up to the circular bivalent chromosome stage-does not affect maturation, but that inhibition during the second 5-hour phase-from the circular bivalent chromosome stage to metaphase I-prevents any further progression of maturation. These results suggest a situation different from that in amphibians. Nevertheless, the two can be reconciled to the extent that certain proteins are synthesized during specific phases of maturation and that they are essential to the maturation process at some subsequent point. However, in P. miniuta, Houk and Epel (1974) noted that the maturation of pactamycin-treated oocytes, compared to that of controls, was retarded at all stages before finally stopping. Based on this observation, they postulate that the blocking of maturation by protein synthesis inhibition may be a meiosis-specific, but not stage-specific, response. In other words, meiosis is arrested not because a specific protein required at a specific stage is unavailable, but rather because a general lack of protein synthesis causes maturation to slow down as the reserves of available protein are exhausted. Eventually, maturation stops when there is not enough protein available to support any further progression. Changes in oocyte protein synthesis activity have been studied under the influence of other compounds which inhibit maturation. Drugs such as theophylline (O’Connor and Smith, 1976) and papaverine (Bravo et al., 1978) have been shown to inhibit oocyte maturation in X. laevis, as does dbcAMP (Stem and Wassarman, 1974) in mice. According to Bravo et al. (1978), aclose correlation exists between the inhibitory effects of the compounds on Xenopus oocyte protein synthesis and their capacity to block maturation. However, mouse oocytes which fail to undergo GVBD, because of treatment with dbcAMP, also fail to exhibit only the changes in protein synthetic pattern which occur during normal maturation (Stem and Wassarman, 1974; Schultz and Wassarman, 1977b). Stimulation of protein synthesis following the initiation of maturation does not appear to require the presence of the GV in amphibians. Smith and Ecker (1969) and Ecker and Smith (1971a) showed, in R. pipiens, that normal oocytes and those from which the GV had been removed (enucleated)exhibited quantitatively and qualitatively almost identical patterns of leucine incorporation into proteins following progesterone treatment. In mice Schultz et ul. (1978), using labeled methionine and two-dimensional electrophoresis, found that there was no difference in the protein synthesis pattern between nucleated and enucleated oocytes when they were cultured in the presence of dbcAMP which prevents nucleated oocytes from GVBD. However, when oocytes were cultured without dbcAMP, the difference became manifest approximately at the time at which nucleated oocytes underwent GVBD. From these observations they proposed that certain protein synthesis-stimulating factors which have appeared in the oocyte cytoplasm in the initial period of maturation, are stored in the GV until they are released into the cytoplasm at GVBD. Here they direct the observed changes in
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protein synthesis. If, as in enucleated oocytes, these factors remain and accumulate in the cytoplasm, the pattern of protein synthesis in the cytoplasm may be changed when the level of the factors reaches a critical point. This point clearly may be reached earlier than it is reached in intact oocytes. Whatever the case, it would be expected that GV removal just prior to GVBD ought to delay these changes in the protein synthesis pattern until concentrations of the proteinstimulating factors reach the required cytoplasmic levels. At any rate, the fact that the changes in the protein synthesis pattern during cytoplasmic maturation of oocytes can occur independently of the GV indicates that these changes are not dependent on transcriptional activity of the nucleus. This explains why destruction of the chromosomes in the oocyte by X ray (Masui, 1973a,b), or their inactivation by RNA synthesis inhibitors (Smith and Ecker, 1970a; Ziegler and Masui, 1976a), does not affect the course of cytoplasmic maturation in amphibian oocytes. 3. RNA Synthesis Early work with amphibians consistently showed that gonadotropin-induced maturation of follicle-enclosed oocytes was inhibited by the RNA synthesis inhibitor, actinomycin D (Dettlaff, 1966; Brachet, 1967; Brachet and Steinert, 1967; Schuetz, 1967b). Another inhibitor, a-amanitin, is also effective, reducing RNA synthesis in follicles by 80% or more (Wasserman and Masui, 1974). Curiously, though, these RNA synthesis inhibitors occasionally fail to suppress maturation of follicle-enclosedoocytes (Merriam, 1972; Wasserman and Masui, 1974). The reason for this is unclear. It may be that these follicles were exposed to gonadotropins at subthreshold levels in vivo (Wasserman and Masui, 1974). In rats, LH-induced maturation of follicle-enclosed oocytes cannot be inhibited by actinomycin D vsafriri et al., 1972). It is interesting to note that ethidium bromide which is known to inhibit mitochondria1 RNA synthesis in eukaryotic cells is always the most potent inhibitor of gonadotropin-induced maturation of follicle-enclosed oocytes in amphibians (Wasserman and Masui, 1974; Schmerling and Skoblina, 1978). These observations suggest that the effect of gonadotropin on follicles in initiating oocyte maturation may involve the induction of RNA synthesis under certain physiological conditions, since all RNA synthesis inhibitors fail to inhibit maturation when follicle-freeoocytes are induced to mature, as discussed later. Progesterone-induced maturation of follicle-free amphibian oocytes is inhibited neither by actinomycin D (Schuetz, 1967b; Smith and Ecker, 1969; Baltus et al., 1973) nor by a-amanitin (Baltus ef al., 1973; Wasserman and Masui, 1974; Brachet et al., 1974). Although actinomycin D does not prevent oocytes from initiating maturation and has even been observed to exert a favorable effect (Baltus et al., 1973), the progression of maturation is not normal. For instance, the behavior and morphology of the chromosomes in actinomycin D-treated
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oocytes are quite irregular, either becoming pycnotic or failing to condense following GVBD (Baltus et al., 1973; Ziegler and Masui, 1976a). Maturation of follicle-free mouse oocytes proceeds to metaphase I or to the circular bivalent chromosome stage in the presence of low concentrations of actinomycin D, but at concentrations higher than 1 pg/d it is inhibited before GVBD (Bloom and Mukhejee, 1972; Golbus and Stein, 1976) or is accompanied by severe chromosomal aberrations (Alexandre and Gerin, 1977). Isolated oocytes of Asterias and Chaetopterus have been found by different investigators to initiate maturation in the presence of actinomycin D in all species examined. Maturation of A. forbesii oocytes occurs normally at actinomycin D concentrations as high as 10 pg/ml, as scored by the occurrence of normal fertilization (LaMarca et al., 1971). Asterias glacialis and Chaetopterus oocytes undergo normal maturation at actinomycin-D concentrations of 20 pg/ml (Zampetti-Bosseler et al., 1973). However, Brachet and Steinert (1967) found that a concentration of 20 pg/ml prevented polar body formation in A . rubens. It should be noted, though, that actinomycin D reduced RNA synthesis in the oocytes only by about 50% at the concentrations used in the experiments of LaMarca et al. (1971) and Zampetti-Bosseler et al. (1973). It is not known how completely RNA synthesis was inhibited in the experiment of Brachet and Steinert (1967). Although the results cited above appear to indicate that oocyte maturation is not highly dependent on RNA synthesis, this does not necessarily imply that there is no RNA synthesis during oocyte maturation. The pioneering work of Brown and Littna (1964) with X. laevis indicated that fully grown oocytes, stimulated to ovulate by HCG, synthesized not only rRNA but also a significant amount of heterogeneous RNA. The latter may include mRNA and mitochondrial RNA (mtRNA). Oocyte RNA synthesis is stimulated during a brief period between the onset of maturation following progesterone treatment and GVBD in Xenopus (LaMarca et al., 1975; Webb et al., 1975) and R . pipiens (Morrill et al., 1975). However, this progesterone-induced rise in RNA synthesis has not been observed in R. pipiens (Smith and Ecker, 1970a). At the time of GVBD, a sharp decrease in rRNA synthesis occurs, and only the synthesis of heterogeneous RNA, which corresponds to 1-2% of prematurational levels of RNA synthesis, continues (Webb et al., 1975). In mice, Rodman and Bachvarova (1975, 1976) showed that RNA synthesis continued at least up to the last 2 hours before GVBD. Experiments by Wassarman and Letourneau (1976a) also indicate that RNA synthesis occurs in fully grown oocytes containing an intact GV. These results contradict earlier work by Oakberg (1968) and Moore et al. (1974). Wassarman and Letourneau (1976a), who injected radioactive precursor directly into the follicle, suggest that the failure of the earlier workers to detect labeled RNA in fully grown oocytes may
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have been due to low intrafollicular levels of the precursor which was injected intraperitoneally. Alexandre and GBrin (1977) hypothesize that the RNA synthesized early in maturation, before GVBD, may include mRNA. During and after GVBD, there is a dramatic reduction in the rate of RNA synthesis directed by the nucleus (Rodman and Bachvarova, 1976). A similar reduction in nuclear RNA synthesis also occurs in starfish and Chaetopterus oocytes following the onset of maturation (Boylan et al., 1973; Zampetti-Bosseler et al., 1973). With respect to the nature of the RNA synthesized after GVBD, there is evidence suggesting that the RNA is not of ribosomal origin. In Asterias (LaMarca et al., 1971; Boylan et al., 1973), as well as in Xenopus (Webb et al., 1975), the major portion of the RNA synthesized comprises two RNA populations of different S values, 4-5 and 15-19S, while the minor portion is heterodispersed RNA.
4. DNA Synthesis Experiments with X . laevis oocytes by Hanocq et al. (1974) and by Brachet et al. (1974) have shown that there is no detectable DNA synthesis in the nucleus or chromosomes during progesterone-induced maturation, and that various agents which inhibit DNA synthesis, such as hydroxyurea, deoxyadenosine, cytosine arabinoside, ethidium bromide, X rays, and dimethyl B rifampicin, exert no inhibitory effect on maturation. Although treatment with these agents leads to pycnotic chromosomes in the oocytes, it is difficult to assess whether this is due to DNA synthesis inhibition or to other effects of the agents. Huez et al. (1972) and Zampetti-Bosseler et al. (1973), working with starfish oocytes, obtained results similar to those for Xenopus oocytes. In their experiments, maturation, scored by polar body formation, was unaffected by DNA synthesis inhibitors, though synthesis was not completely inhibited. Experiments with U . caupo oocytes by Blankstein and Kiefer (1977) demonstrated that normal chromosome condensation and pronuclear formation were not inhibited by DNA synthesis inhibitors. While it is thus apparent that DNA synthesis is not required during maturation, it is equally true that some amount of DNA synthesis occurs during the maturation process. In amphibian oocytes, Hanocq et al. (1974) noted that DNA synthesis occurred in the cytoplasm, since DNA synthesis could be detected in oocytes from which the GV had been removed. This DNA synthesis is possibly of mitochondria1 origin and a continuation of the low level of mitochondrial DNA synthesis taking place throughout oogenesis. During maturation, DNA synthesis of an undetermined nature has also been reported to take place in starfish (Wassannan, 1971; Huez el al., 1972; Zampetti-Bosseler et al., 1973) and U . caupo (Blankstein and Kiefer, 1977) oocytes. Wassarman (1971) found
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that the DNA synthesized during maturation of A. forbesii was of nuclear origin, whereas mitochondria1DNA synthesis did not occur until the 128-cell stage. We were unable to find any reports which examined DNA synthesis during the maturation of mammalian oocytes, except for two dealing with repair synthesis (Masui and Pedersen, 1975; Brazil1 and Masui, 1978). In conclusion, then, we state that any DNA synthesis which occurs during oocyte maturation is not obligatory for the progression of maturation.
IV. Initiation of Oocyte Maturation A. MATURATION-INDUCING SUBSTANCE 1. Specificity
As discussed in Section I1,A-F, there is a growing body of evidence indicating that hormones play an essential role in the induction of oocyte maturation in a wide variety of animals. As yet, though, there are only a few groups of animals in which a chemically defined substance is known to act directly on oocytes as a MIS. These are amphibians and starfish. In amphibians, progesterone appears to be the natural MIS causing oocyte maturation, although other steroids are known to be as effective as progesterone; these include DOC in R. pipiens (Schuetz, 1967a; Smith and Ecker, 1971) and aldosterone and testosterone in X. laevis (Jacobelli et al., 1974; SchorderetSlatkine, 1972), but not estradiol or its derivatives in either species. A comparison of the potency of various steroids as inducers of maturation in R. pipiens oocytes led Morrill and Bloch (1977) to suggest that a special arrangement of substituents on the upper (p) surface of the steroid molecule was of critical importance with respect to its potency as a MIS, 3,20-dione, 21-01 forms being the most active. However, introduction of a polar group oriented toward the lower (a)surface abolishes the activity of a MIS. A similar comparative study of the maturation-inducing potency of various steroid using medaka oocytes has led Iwamatsu (1978) to the conclusion that the steroids effective in inducing maturation have in common a C=O (or a-OH)group at 3C, and a P-OH group at 17C in the C19-steroids,and a C=20 (or P-OH) group at 3C and a C=O (or a-OH) group at 20C in the C2,-steroids, in addition to a A4 or A5unsaturated or 5-saturated configuration. Progesterone is known to be metabolized by amphibian oocytes. To determine its metabolic products, oocytes were incubated with radioactive progesterone, and the derivatives produced by the oocytes were chromatographicallyanalyzed. As shown in Table II, oocytes of R. pipiens (Reynhout and Smith, 1973), X. laevis (Fouchet et al., 1975), P . waltlii (Fouchet er al., 1975; Ozon et al.,
TABLE II METABOLISM OF PROGESTERONE IN AMPHIBIAN OOCYTES Species R a m pipiens,
R . temporaria Xenopus laevis
Triturus alpestris
Pleurodeles waltlii
Metabolite Sa-Pregnan-20a-ol-3-one Sa-Pregnane-3,20-dione Sa-Pregnane-3P2Oa-diol 17a,2Oa-Dihydroxypregn-4-en-3-one 17a-Hydroxy-4-pregnene-3,20-dione CAndrostene-3,17-dione Sa-Pregnene-3,20-dione SP-Regnene-3.20-dione 17a-Hydroxy-4-pregnene-3,2O-dione Sa-Pregnene-3,2O-dione SP-Pregnene-3,20-dione 2OP-Hydroxypregn-4-en-3-one Sa-Pregnane-3,20-dione 3a-Hydroxy-Sa-pregnan-20-one 3P-Hydroxy-Sa-pregnan-20-one
Enzyme Sa-Reductase 20a-Hydroxylase 17a-Hydroxylase 19,2I-desmolase Sa-Reductase 5P-Reductase 1701-Hydroxylase Sa-Reductase 5P-Reductase 20P-Hydroxysteroidoxidoreductase (soluble) Sa-Reductase (microsomal) 3a-Hydroxysteroidoxidoreductase(microsomal) 3p-Hydrox ysteroid oxidoreductase (microsomal)
Reference Reynhout and Smith, 1973; Antilla, 1977 Fouchet e t a l . , 1975 Antilla, 1977
Antilla, 1977 Ozon et a l . , 1975
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1975), and R. temporaria and Triturus alpestris (Antila, 1977) all possess 5a-or SP-reductase, which converts progesterone into 5a- or SP-reduced derivatives such as 5a-pregnandione. In addition, 17a-hydroxylase was found in Xenopus and Triturus oocytes. Pleurodeles oocytes contain 3a-, 3p-, and 2Qphydroxysteroid oxidoreductases. Thus different steroid derivatives of progesterone are the major products of its metabolism in the oocytes of different species; for instance, Sa-pregnandione in R. pipiens and androstenedione in X. luevis. However, all these derivatives are less potent than progesterone as MISS when applied to the oocytes of these species. Thus it seems quite unlikely that the metabolic conversion of progesterone plays a significant role in the initiation of maturation. In starfish, it has been shown beyond doubt that 1-MA is the natural MIS. Although this chemical possesses the highest maturation-inducing activity among compounds used with Asterius oocytes (Kanatani and Shirai, 1971), recently an artificially synthesized adenine derivative, 1-benzyladenine (1-BA), has been shown to be a more potent MIS in Marthasterias glacialis oocytes (Doree et al., 1976a,b). After comparing the activity of various adenine derivatives in this species of starfish, Doree et al. (1976a) concluded that the nature of the group in the N-1 position, and the absence of additional groups in the N-7 and N-9 positions, were of critical importance for MIS activity. An analysis of the metabolism of 1-MA in the oocytes of A . forbesii was carried out by Toole and Schuetz (1974). They found that radioactive 1-MA applied to oocytes was metabolized into a biologically inactive compound, the nature of which was unknown. Thus it may be concluded that 1-MA acts as a MIS prior to chemical modification.
2. Action Site The necessity for stereochemical specificity of the molecules which act as MIS suggests that these molecules must interact with a certain site on a specific reacting molecule or a molecular unit in the oocyte. In order to determine the location of such reacting molecules in the oocyte, the capacity to bind MIS has been compared in different subcellular components of the oocyte. In amphibian oocytes, Ozon and Belle (1973) found that the component containing the melanosome fraction showed the highest progesterone-binding capacity. Further experiments indicated that progesterone was the steroid hormone with the strongest affinity for melanosomes (Ozon et al., 1975; Belle et al., 1977a). When melanosomes incubated with progesterone are injected illto Xenopus oocytes, some of the changes occurring during the early phase of maturation, such as GV migration toward the animal pole and its partial disintegration, can be seen in the recipient oocytes, although they eventually undergo degeneration (Jacobelli et al., 1974). On the basis of these observations, the suggestion was made that progesterone-bound melanosomes may play a role in
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the initiation of oocyte maturation in amphibians. However, the discovery of a mutant albino strain of X . laevis completely devoid of melanosomes and their precursors, which produces fertile oocytes (Bluemink and Hoperskaya, 1975), has made it difficult to assign any significant role to melanosomes with respect to the initiation of oocyte maturation. The nature of the subcellular components in starfish oocytes which interact with 1-MA has been investigated in A. forbesii by Jeffery (1977). He compared the ability of various enzymes to deprive the oocytes of the capacity to respond to 1-MA, finding RNases and proteases to be the most effective among those tested. He also noted that enzyme-treated oocytes recovered from the loss of reacting capacity within 90minutes following transfer to enzyme-freemedium and that this recovery was not suppressed by an RNA synthesis inhibitor (actinomycin D) or a protein synthesis inhibitor (emetin). Although it would be premature at the present time to ascribe the observed effects of enzyme treatments to the specific action of the enzymes used, it may be suggested that interaction of the molecular unit with 1-MA involves RNA. However, in the starfish M . glacialis it was found that mild treatment of the oocytes with Triton X-100 (0.01-0.02%) abolishes their responsivenessto 1-MA @ode et al., 1976b). This was confirmed by Kanatani (1978), using A . pectinifera. Kanatani (1978) and Morisawa and Kanatani (1978) further reported that a Triton X-100 wash of follicle-free oocytes contained a heat-stable, nonprotein substance, and that oocytes incapacitated following Triton treatment had their responsiveness to 1-MA restored when incubated with this substance. These observations strongly suggest the existence of a substance in starfish oocytes that interacts with 1-MA, thus initiating maturation. Smith and Ecker (1969) and Masui and Markert (1971) using R. pipiens, and Kanatani and Hiramato (1970) using A. pectinaris, showed that a MIS, progesterone or 1-MA, always failed to induce oocyte maturation when it was microinjected into oocytes. These MISS are effective only when applied to the outer surface of oocytes. Accordingly, it has been hypothesized that a MIS primarily interacts with molecules located on or near the outer surface of the oocyte. There is one report indicating that some steroids, for example, hydrocortisone, effectively induce maturation if injected into X. laevis oocytes (Schorderet-Slatkine, 1972). However, this observation does not appear to contradict the above hypothesis, since the possibility that the injected steroid may have leaked from the oocyte and exerted a surface action was not ruled out. The surface action hypothesis has recently gained strong support from the experiments of Baulieu and his associates (Baulieu et al., 1978; Godeau et al., 1978). They succeeded in inducing Xenopus oocytes to mature by exposing them to a polymer-conjugated steroid 3-0x0-4-androstein-17P-amido polyethylene oxide (ACA-poly EO), which has a molecular weight of over 20,000 daltons. This compound is as effective as progesterone in inducing the maturation of
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Xenopus oocytes when it is applied to the oocyte surface, whereas it is totally ineffective when injected into oocytes. In additon, uptake of this chemical by treated oocytes is negligible compared to that of free steroids, and no degradation of the compound occurs during the incubation period with the oocytes. Conversely, neither the carrier polymer (poly EO) alone, nor its conjugate with an inactive steroid, such as estradiol, has a maturation-inducing effect. Therefore it is almost certain that the active polymer-conjugated steroid acts on the surface of the oocytes to induce maturation, although, strictly speaking, proof of its actual surface localization by electron microscope autoradiography is still required. Recent experiments by Shida and Shida (1976) suggest that the molecules reacting with 1-MA may also be localized on the oocyte surface. Their results demonstrate that a-(1+6)-heterogalactan, a polysaccharide, reversibly inhibits the maturation-inducing action of 1-MA. Because of its large size (1.55S), it probably interacts mainly with molecules on the surface of the oocytes to interfere with 1-MA action. The distribution of reaction sites for a MIS on the surface of the oocyte has been investigated in both amphibians and starfish. Recently, Cloud and Schuetz (1977) found that the sensitivity of R. pipiens oocytes to MISS was higher in the animal half than in the vegetal half. This demonstration involved the application of progesterone to a restricted area (ranging from 13 to 15% of the total surface area) of a single oocyte by tightly fitting it into a conical tube with the open end exposed to a medium containing progesterone. During the course of exposure, progesterone was found to accumulate on the exposed hemisphere. When the oocytes were exposed to progesterone at the animal pole, 55% underwent GVBD within 24 hours, whereas none of those exposed to progesterone at the vegetal pole underwent GVBD. This result supports the idea that the molecule or molecular unit responsive to progesterone is more concentrated in the animal hemisphere than in the vegetal hemisphere. Local application of 1-MA to starfish oocytes has been carried out by Shirai (1978). In her experiment, an oocyte was tightly fitted into a capillary tube, and one hemisphere was stained with Nile blue and the other with neutral red. When both hemispheres of the oocytes thus stained were exposed to 1-MA, the polar body was given off by each of the two hemispheres equally frequently. However, when only one of the hemispheres was exposed to 1-MA, the polar body was usually given off by the hemisphere to which 1-MA had been applied. From these observations, it may be concluded that 1-MA acts equally on all regions of the starfish oocyte, but that its differential application determines the site of polar body formation.
3. Mode of Action Investigations into the nature of the interaction between a MIS and reacting oocytes molecules appear to have been hampered by limited evidence concerning
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the existence of competitive chemical analogs of MISS. Although it has been reported that the steroid analogs ethynylestradiol and MK 665 inhibit the maturation of progesterone-treated R. pipiens oocytes to some extent (Morrill and Bloch, 1977), the presence of follicle cells in this experiment makes it difficult to assess the direct action of the steroids on the oocytes. In starfish, it was shown that I-MA uptake by oocytes was competitively inhibited to a high degree by its analog, 1,9-dimethyladenine, which itself has little MIS activity @ode et al., 1976b). Nevertheless, no competitive inhibition of 1-MA-induced maturation is caused by this chemical analog. Both in starfish and in amphibians, xanthine derivatives have an inhibitory effect on oocyte maturation. But, although xanthine derivatives are chemically analogous to 1-MA, their inhibitory effect in starfish is observed only when they are applied at concentrations lo4 times higher than 1-MA (Doree et al., 1976a). Similarly, in amphibian oocytes, xanthine derivatives, such as caffeine and theophylline, inhibit maturation at high concentrations (0'Connor and Smith, 1976). Obviously, the inhibition cannot be due to the derivatives acting as competitive analogs of the MIS (progesterone). Rather, it is likely that the inhibitory effect of xanthine derivatives on oocyte maturation, both in amphibians and in starfish, is brought about through their well-known effects on phosphodiesterase and Ca movements in the cell. The reaction of an oocyte to a MIS appears to involve two distinct steps. Marot et al. (1977) found that, when Xenopus oocytes were exposed to progesterone for 20 hours at subthreshold concentrations, the oocytes began GVBD at much earlier times following a second treatment with maturation-inducing levels of progesterone. However, the facilitating effect of subthreshold progesterone treatment does not last more than 24 hours after the oocytes are returned to hormone-free medium. The induction of maturation in the starfish ovary also appears to be a two-step process. Using M. glacialis, Guerrier and Dorke (1975) showed that oocytes exposed to lo-' M 1-MA for at least 4.5 minutes, or for two 2.5-minute periods separated by no more than 7.5 minutes, matured. The short treatments became ineffective when the interval between the two exceeded 7.5 minutes, indicating that the oocyte reaction to subthreshold doses of 1-MA was reversible. These observations seem to indicate that, in amphibians and starfish, the first response of oocytes to a MIS is a reversible reaction which is followed by an irreversible reaction that actually triggers maturation. The reversibility of the initial step of maturational change induced by a MIS in oocytes suggests that this change may not depend on the formation of a stable complex between the MIS and its receptor on the oocyte. If the formation of a receptor-inducer complex is a prerequisite for the subsequent process of maturation, it should be expected that some minimum amount of MIS must be taken up by the oocyte before it initiates maturation. However, at least in the induction of maturation in Xenopus oocytes, this is not the case. Bell6 et al. (1976) found
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that, as the concentration of progesterone to which the oocytes were exposed was decreased, the duration of exposure required to induce maturation increased, but the amount of steroid taken up by each oocyte before initiating maturation was greatly reduced. Apparently, the critical factor for the induction of maturation in this case was not the number of complexes formed between the inducer and its oocyte receptor, but the number of interactions between them. Perhaps the MISreceptor molecule interaction involves the induction of a permanent change in the receptor molecule located on the oocyte surface, without the formation of a stable MIS-receptor complex. Maturation would be induced by the cumulative effect of many molecules undergoing this change. The nature of the change in the oocyte surface molecules necessary to initiate maturation may be inferred from the effects of various chemicals which mimic the effect of a natural MIS. The molecules involved in this change may be proteins with sulfhydryl (SH)or disulfide (S-S) residues. Kishimoto and Kanatani (1973) observed that S-S reducing agents, such as dithiothreitol (D'IT) and 2,3-dimercapto-l-propanol(BAL), acted as MISs in starfish oocytes and that SH-blocking agents such as p-chloromercuribenzoate (PCMB) reversibly inhibited 1-MA-induced maturation. Furthermore, the SH content of the proteins isolated from the oocyte cortex rapidly increased following 1-MA treatment (Kishimoto et al., 1976). Opposing this notion, however, is the work on X. laevis by Brachet et al. (1975b), who found that SH-reducing agents were ineffective in inducing maturation but that some organomercurials, such as p-hydroxymercuriphenylsulfonate (PHMPS) and p-hydroxymercuribenzoate (PHMB), acted as potent MISs. Recently, Pays et ul. (1977) found that the SH-oxidizing agent cysteamine was also an effective inducer of maturation and that the effects of both it and the mercurial compounds were reversed by D'IT, while other SH-oxidizing agents, such as diamide and dithionitrobenzene, were ineffective. It should be noted that SH reagents active as MISs do not induce maturation when they are injected into oocytes, suggesting that their sites of action are on the oocyte surface (Brachet et al., 1975b;Pays et al., 1977). The findings discussed above are consistent with the idea that the reaction of the oocyte to a MIS involves a change in the conformation of the surface protein, a change including either the formation or dissociation of S-S bonds. The notion that the initial oocyte reaction to a MIS consists primarily of conformational changes in the surface protein may be of general significance. It has been shown that Chaetopterus oocytes, which have been prevented from maturing following isolation in Ca-free seawater, begin to mature upon treatment with trypsin (Goldstein, 1953; Ikegami et al., 1976) or with PHMPS (Brachet and Denis-Donini, 1977). Similarly, oocytes of U. caupo can be induced to mature by treatment with trypsin (Paul, 1975) or with mersalyl acid (sarylgan) (Johnston and Paul, 1977). The induction of oocyte maturation by proteases has also been successful in Sabellaria (Peaucellier, 1977a) and Spisulu (Ashton,
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1959). All these observations, taken together, suggest that an alteration in oocyte protein character, induced either by proteolytic enzymes or by SH reagents, may be the initial step of maturation. Thus it is possible that the primary action of a MIS is to cause alterations in the conformation of oocyte surface protein.
B. THE ROLEOF Ca IONS The induction of oocyte maturation is dependent on the presence of divalent cations in the external medium to various degrees in different animals. Chaetopterus (Goldstein, 1953; Ikegami et al., 1976; Brachet and Denis-Donini, 1977), Spisula (Schuetz, 1975a), and U . caupo (Johnston and Paul, 1977) oocytes fail to initiate maturation in Ca-free seawater. Oocytes of amphibians, such as X . luevis, fail to undergo maturation when cultured in Ca- and Mg-free Ringer’s solution after treatment with progesterone (Merriam, 1971a,b), while the addition of these or other divalent cations, such as Ba and Sr, has been found to support the maturation of oocytes (Wasserman, 1976; Marot et al., 1976). In R. pipiens, oocyte maturation can be induced by progesterone in Ca- and Mg-free Ringer’s solution if the oocytes are exposed to the hormone shortly after isolation from the follicles (Eicker and Smith, 1971b). However, they become unable to respond to the hormone if they are stored for 2 hours or more in Ca- and Mg-free medium at 4°C (Kostellow and Morrill, 1979). In all these cases, it has been observed that oocytes kept in medium lacking divalent cations become capable of initiating maturation upon their return to normal conditions. That is, oocytes are reversibly incapacitated by being deprived of external divalent cations. However, the oocytes of some marine invertebrates, such as starfish (Shirai and Kanatani, 1974; Guerrier et al., 1978) and Sabellaria (Peaucellier, 1977), can be induced to mature in seawater lacking divalent cations. The role of internal Ca ions in maturation has been tested by injecting ethylene glycol bis(Zaminoethy1ether)-N,N’-tetraacetic acid (EGTA) into oocytes. This type of experiment showed that Xenopus oocytes injected with EGTA were unable to initiate maturation following progesterone treatment even when divalent cations were present in the external medium (Wasserman, 1976; Masui et al., 1977). Similarly, starfish oocytes injected with EGTA failed to respond to 1-MA (Guemer et al., 1978; Moreau et al., 1978). Thus it is clear that removal of internal Ca ions from oocytes inhibits their maturation, whether or not maturation is dependent on the presence of divalent cations in the external medium. This observation implies that intracellularCa ions play an indispensable role in the initiation of oocyte maturation. Apparently, oocytes which do not require the presence of Ca in the external medium for maturation possess sufficient endogenous reserves of Ca ions to provide that necessary when the oocytes are stimulated by a MIS. It may be that these oocytes release Ca from an internal
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reservoir upon stimulation by a MIS. However, the initiation of maturation in oocytes which require the presence of Ca in the external medium appears to depend on an influx of ions from the medium following MIS stimulation. The release of Ca ions from the internal reservoir of the oocyte following stimulation by a MIS has recently been examined. O’Connor et al. (1977), using Xenopus oocytes stimulated by progesterone, and Johnston and Paul (1977) using inseminated or trypsin-treated oocytes of U.caupo, demonstrated that oocytes preloaded with 45Carapidly released a considerable amount of Ca into the external medium when they were stimulated to initiate maturation. However, the rate of Ca release soon decreased, returning to a level comparable to that occurring in unstimulated oocytes. Eventually, shortly before GVBD, the rate of efflux of Ca from the preloaded stimulated oocytes became less than that from the unstimulated oocytes indicating a greater degree of sequestration of Ca in the stimulated oocytes. The rate of Ca influx into oocytes was also studied in stimulated and unstimulated oocytes of both species. The data indicate that the amount of 45Cataken up from the external medium increases at a much faster rate in stimulated oocytes than in unstimulated controls, reaching the maximum rate shortly before GVBD. These observations indicate that oocytes induced to mature first release stored Ca ions but, as maturation progresses, sequester Ca ions more actively than those which have not been induced to mature. This may explain an early observation that R. pipiens oocytes which had been ovulated contained more Ca than those in the ovary (Morrill et al., 1971). Marot et al. (1976) showed that Xenopus oocytes undergoing maturation following stimulation by mercurial compounds also accumulated Ca from the external medium, although no significant change in the influx or efflux of Ca was observed in progesterone-stimulated oocytes. It appears likely that oocytes, when stimulated by a MIS, activate a mechanism which mobilizes Ca ions. Direct evidence of this Ca mobilization was recently provided by Moreau et al. (1978), who injected a Ca-sensitive luminescent protein, aequorin, into oocytes. They found that aequorin injected into starfish (M.glacialis) oocytes kept in Ca-free seawater luminesced less than 1 second after the oocytes were exposed to 1-MA. This suggests a rapid release of endogenous Ca, sequestered in the oocytes before their exposure to 1-MA, into the free space in the cell where the aequorin was introduced. When the oocytes were immersed in Ca-free seawater containing aequorin, no light flash was detected following the addition of 1-MA to the seawater, indicating that there was no release of endogenous Ca into the external medium upon stimulation by 1-MA. When EGTA was injected into oocytes before or during the appearance of the Ca peak, the emission of light by previously injected aequorin and GVBD were both suppressed, whereas when EGTA was given after the Ca peak had appeared, GVBD took place.
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A similar approach was used by Belle et al. (1977b) to study Ca mobilization in Xenopus oocytes following MIS stimulation. In this case, however, no significant light emission was observed. This negative result may have been due to inadequate optical properties of the surface of Xenopus oocytes with respect to the detection of light emitted from the inside. The observation of Ca mobilization using the aequorin method in starfish oocytes strongly suggests that an increase in the internal concentration of Ca is a prerequisite for the initiation of maturation. In this respect, the Ca efflux observed in Xenopus and Urechis oocytes following MIS stimulation can be regarded as representing a discharge of excess ions released during the transient Ca surge. An active role for Ca ions in the initiation of oocyte maturation was suggested by classic experiments on parthenogenesis in marine invertebrates such as Hydroida (Pasteels, 1935) and Cumingia (Hollingsworth, 1941). However, the exceedingly high Ca concentrations, usually over 100 mM, required to stimulate the oocytes in these experiments makes doubtful the interpretation that the Ca ions applied to the oocytes acted in a specific way to control cell physiology. In fact, Guerrier et al. (1978) found that starfish (M. glacialis) oocytes isolated from the ovary in Ca-free seawater could be induced to mature without 1-MA stimulation if they were exposed to high Ca concentrations (75-300 mM). Thus it appears that Ca ions applied at high concentrations act as a nonspecific triggering agent which secondarily causes the release of internal Ca. Evidence for a specific role of Ca ions in the initiation of oocyte maturation has, however, been obtained from studies of the effects of certain chemicals known to interfere with the physiological action of Ca in a variety of cells, including neural, muscle, and secretory cells. In Xenopus oocytes, amphiphilic cations, including phenothiazine neuroleptics, tricyclic antidepressants, anorexiants, local anesthetics, P-adrenergic blocking agents such as propanolol and the tertiary amines D200 and D600, have been found to be capable of inducing maturation (Schorderet-Slatkine and Schorderet, 1976; SchorderetSlatkine er al., 1977a). It is known that these agents concentrate in cellular membranes, disturbing their phospholipid turnover and resulting in a concomitant release of Ca from its binding site in phosphatidic acid (Feinstein, 1964; Seeman, 1972). In addition, La ions, known to displace external membrane Ca, thus blocking Ca flux and releasing sequestered internal Ca, also effectively induce oocyte maturation in X . laevis (Schorderet-Slatkineet al., 1976). La ions have also been found to act as a MIS in R. pipiens, but D600 has neither an inhibitory nor an inducing action on oocyte maturation, although both effectively block Ca uptake by oocytes (Kostellow and Momll, 1979). Recently, gammexane, which specifically suppresses Ca mobilization in excitatory cells, has been found to inhibit effectively maturation of progesterone-treated Xenopus
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oocytes (Schorderet-Slatkineet al., 1977b). D600, isoptin, procaine, Mn ions, and methylxanthine all inhibit 1-MA-induced maturation in starfish oocytes, and these agents have also been found to suppress the light emission usually seen following 1-MA stimulation of aequorin-injectedoocytes (Moreau et al., 1978). Finally, oocyte maturation in Chaetopterus can be induced by tetracaine (Ikegami el al., 1976). All these findings, taken together, indicate beyond doubt that the mobilization of Ca is deeply involved in the initiation of oocyte maturation in many species. The mode of Ca action, however, is not yet fully understood. It may differ among species, depending on the specific molecular organization of the oocyte membranes. For instance, the antibiotic, ionophore A23187, which facilitates the transportation of divalent cations across membranes (Reed and Lardy, 1972), acts as a MIS for the oocytes of several species, but under differing ionic conditions. Among marine invertebrates, the ionophore is known to induce oocytes of Nereis (Chambers, 1974), SpisuZa (Schuetz, 1975a), and Chaetopterus (Brachet and Denis-Donini, 1977) to mature, provided that Ca ions are present in the ambient medium. The ionophore can also induce oocyte maturation in X . laevis, if the Mg or Ca ion concentration in the external medium is greater than 10 mM, but it cannot induce R. pipiens oocytes to mature (Wasserman and Masui, ,1975b). In this connection, it is interesting to note the recent finding by Baltus et al. (1977) that fully grown Xenopus oocytes, when cultured in a medium supplemented with an excess of Ca (20 mM) or Mg (40 mM) for 9-10 hours, could initiate maturation without the addition of ionophore or hormones to the medium. Furthermore, they reported that medium-sized oocytes, normally unresponsive to progesterone, initiated maturation in response to the hormone when they were cultured in Ca- or Mg-fortified medium. Although the observations cited above appear to indicate that inward mobilization of Ca or Mg ions from the external medium into oocytes, which can be facilitated by ionophore A23 187, effectively induces maturation, it should be noted that the ionophore cannot induce Xenopus oocytes to mature at excessively high external Ca levels, though its activity is unaffected by high Mg levels (Masui et al., 1977). Peaucellier (1977b) found that Sabellaria oocyte maturation could be induced by the ionophore in the absence as well as in the presence of divalent cations, and that simple treatment with EDTA also induced maturation. Furthermore, Ca-mobilizing agents which have been found to be effective in inducing oocyte maturation in X . laevis had no effect on Sabellaria oocytes at nontoxic doses except for La ions (Peaucellier, 1978). Finally, it should be pointed out that the ionophore is completely ineffective as an inducer of oocyte maturation in starfish (Schuetz, 1975b), even though photometric determination of the intracellular Ca ion level using aequorin has revealed that the ionophore actually induces a sizable Ca surge, to a level 20 times higher than that induced by 1-MA (Moreau et al., 1978). These facts may imply that the transient increase in the intracellular
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level of Ca ions preceding the initiation of maturation must be regulated within an appropriate range in order for the actual process of maturation to take place. The site of action of Ca ions in triggering oocyte maturation may be near the surface of the oocyte. Moreau et al. (1976b) reported that a very small amount of Ca introduced by iontophoresis into areas less than 0.2 mm below the surface induced maturation of Xenopus oocytes, while introduction into areas deeper than that was ineffective. They also found that this effect of iontophoresis was ion-specific; that is, no ions other than Ca were able to induce maturation, and EGTA iontophoresis counteracted the effect of Ca. In this experiment, the effectiveness of Ca iontophoresis depended on the presence of relatively high Mg concentrations (10-20 mM), suggesting a synergism between Ca and Mg in the initiation of oocyte maturation. Furthermore, with respect to the surface action hypothesis, it is interesting to note that La, which may mobilize Ca in oocytes, can induce maturation only when applied to the external surface of Xenopus oocytes, failing to do so if injected into the oocytes (Schorderet-Slatkine et al., 1976). Consideration of these results, together with those from the experiments using Ca iontophoresis, certainly suggests that the site of the reaction of Ca ions with oocytes is near the surface membrane.
IN ELECTROPHYSIOLOGICAL PROPERTIES C. CHANGES
1. The Membrane Potential of Immature Oocytes In all animals studied to date, measurements of the electropotential difference between the outside and inside of an oocyte, called the membrane potential, have shown that the inside of a fully grown ovarian oocyte is negatively charged with respect to the outside; that is, the membrane is inwardly negative. Recent studies of the membrane potential in amphibian oocytes have revealed that the resting potential changes when oocytes are isolated from their follicles. In R. pipiens, Ziegler and Mom11 (1977) showed that the membrane potentials of follicle-enclosed and of follicle-free oocytes are -36 5 2 and -77 -+ 2 mV, respectively. Similar results were obtained in X. Zaevis by Wallace and Steinhardt (1977), who reported that the membrane potentials of follicleenclosed and follicle-free oocytes were -27 -t 2 and -65 f 2 mV,respectively. The hyperpolarization caused by removal of the follicles has been attributed to the activation of Na,K-dependent ATPases in the oocyte membrane, since ouabain significantly inhibits the hyperpolarization (Wallace and Steinhardt, 1977). However, in Xenopus it was found that there were oocytes in the ovary which did not undergo hyperpolarization following defolliculation. Thus two types of fully grown oocytes must exist in the ovary. Generally, oocytes which are hyperpolarized by defolliculation are of average size, less than 1.25 mm in diameter, while those whose membrane potential is the same before and after defollicula-
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tion tend to be larger, greater than 1.25 mm in diameter (Wallace and Steinhardt, 1977). Such variations in the physiological properties of oocytes have not been observed in R . pipiens, the oocytes of which grow synchronously in the ovary. Both in Rana (Tupper and Maloff, 1973; Ziegler and Morrill, 1977) and in Xenopus (Wallace and Steinhardt, 1977) it was observed that, when the concentration of K ions in the external medium was increased, the membrane was gradually depolarized, and no difference in the electropotential between the inside and outside was observed when the K concentration was increased to 200 mM. Thus the internal concentration of K ions in the amphibian oocyte may be estimated to be 200 mM. This concentration is significantly higher than that calculated from measurements of K and water content of the oocyte using atomic absorption methods (130-140 mM), as well as that determined by measurements of intracellular K activity with a K-selective electode (106 mM) (Ziegler and Morrill, 1977). Ziegler and Morrill (1977) hence suggested that one factor responsible for this discrepancy might be a nonuniform distribution of K ions in the oocyte, as suggested by Horowitz and his associates (Century and Horowitz, 1974; Horowitz and Paine, 1976). However, substitution of tris(hydroxymethy1 amino)methane (tris) for Na in the external medium causes hyperpolarization (Tupper and Maloff, 1973), indicating that a Na influx contributes to depolarization of the oocyte membrane. Studies of Na ion exchange using radioactive isotopes have shown that there is indeed a constant exchange of Na taking place in the oocytes of R . pipiens (Mom11 et al., 1977b) and X. laevis (O’Connor et al., 1977). When the oocytes are maintained in Ca-free solution, the Na influx is increased, but the K influx is decreased (Ecker and Smith, 1971b; Tupper and Maloff, 1973; Morrill et al., 1977b). Consistent with this observation, marked depolarization of the membrane has been observed in oocytes following the removal of Ca from the external medium in the case of both Rana (Tupper and Maloff, 1973) and Xenopus (Bell6 et al., 1977b). These results strongly suggest that the selective permeability of amphibian oocytes to K and Na ions is highly dependent on external Ca. Furthermore, Bell6 et a1. (1977b) showed that the depolarization induced by deprivation of external Ca exceeded that induced by ouabain, indicating that a major portion of Na-K transport in amphibian oocytes was regulated by Ca. In the starfish Nordora punctiformis (Hagiwara and Takahashi, 1974) and A. pectinifera (Miyazaki et al., 1975a) it was found that, as in X. Laevis, two types of oocytes, differing in membrane potential characteristics, existed in an oocyte population. The depolarized type had a low membrane potential, -14.6 -+ 5.5 mV, and the hyperpolarized type had a high membrane potential, -72.7 +- 3.6 mV. The membrane potential of oocytes of the depolarized type, however, can be induced to become more negative by replacing Na in the seawater by tris,
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suggesting that the reduced (i.e., less negative) membrane potential of oocytes of this type is due to a change in permeability to Na ions. Miyazaki et a2. (1975a) found that, in the hyperpolarized type, the resting membrane potential was virtually unaffected by changing the levels of Na, Ca, and Mg ions in the external medium but was strongly affected by changing the K levels, and in fact complete depolarization occurred when the K concentration was raised to 196 mM. This suggests that the internal K concentration of oocytes is about 200 mM. Thus it appears that the membrane potential of starfish oocytes is due almost exclusively to K diffusion across the membrane and, as opposed to the situation in amphibian oocytes, is not influenced by external divalent cations. Measurements of steady currents passing through the oocyte membrane at various voltage levels have indicated that there is an ingoing rectification (i.e., current flowing into the oocyte) at voltages near the resting potential (-70 mV), an outgoing rectification (i.e., current flowing out of the oocyte) above -20 mV, and a high negative resistance at voltages between these two rectification regions (Fig. 9). Shen and Steinhardt (1976), studying oocytes of P. miniata, noted that two types of oocytes distinctly different in resting membrane potential existed, as in Asterias and Xenopus. The depolarized type showed membrane potentials ranging from - 10 to -25 mV, and the hyperpolarized type showed potentials ranging from -65 to -90 mV. The membrane potentials of both types of oocytes were also influenced by the concentration of K ions in the external medium. However, these investigators found the oocytes of this species to exhibit peculiar electrophysiological characteristics as compared with those of Asterias. First, since the membrane could not be completely depolarized until the external K concentration approached 400 mM, the internal K concentration of the oocytes was estimated to be about 400 mM. This value is extraordinarily high compared to that of the oocytes of other species. Second, the membrane potential did not change when the Na and Mg concentrations in the external medium were changed but became more negative-that is, the membrane was hyperpolarized when the Ca concentration was increased or when the C1 concentration was decreased. In addition, ouabain had no effect on the value of the membrane potential. Measurement of the steady current passing through the oocyte membrane at different voltages indicated that there was a relatively high resistance near the resting potential level and above 0 mV, and a negative resistance between these two levels (Fig. 9). Data for mammalian oocytes are rather limited. Although some work has been carried out on the membrane potential of metaphase I1 oocytes of the mouse, which has indicated properties similar to those observed in amphibian oocytes (Powers and Tupper, 1974, 1975), no detailed studies on ovarian oocytes have been published.
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n
FIG.9. Current ( I ) and voltage ( V )relationship in immature oocytes and maturing oocytes. A,, Immature oocyte ( A . pectinifera. 0 minute after hormone treatment); A2, maturing oocyte (A. pectinifera. 30 minutes after treatment); P,,immature oocyte (P.mininta, 0 minute after treatment); P,, maturing oocyte ( P . miniata, 30-40 minutes after treatment); M,. maturing oocyte (Mus musculus. metaphase 11). A, and AZ (Miyazaki et al., 1975b); P, and P, (Shen and Steinhardt, 1976); M2 (Powers and Tupper, 1974). Note an increase in the slope of curves in the inward current region, indicating the disappearance of the inward rectification and an increase in the resistance to an inward directed current, occurring during the course of maturation.
From the brief survey of the literature summarized above, it is clear that, before initiating maturation, an oocyte is polarized negatively inward, and this polarization is mainly due to a selective permeability to K ions. However, it should be emphasized that the membrane potential appears to be controlled in a different manner in different species. In amphibians, Ca appears to control Na and K transport, both of which are involved in establishing the resting potential, whereas in starfish, except Patiria, the regulation of the membrane potential is due primarily to K ions.
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2. Changes in Electrical Properties during Maturation Measurements of the membrane potentials in amphibian oocytes have revealed that fully grown oocytes in ovarian follicles are hyperpolarized compared to ovulated oocytes in the uterus (Maeno, 1959; Mom11 and Watson, 1966). This result suggests that depolarization of the membrane occurs during maturation. The membrane potential of progesterone-treatedoocytes of Xenopus (Belle et al., 1976, 1977b; Moreau et al., 1976c; Wallace and Steinhardt, 1977) and Rana (Ziegler and Momll, 1977) becomes less negative, usually to less than -20 mV and sometimes reaching 0 mV, before GVBD. A similar depolarization of the membrane has also been observed when maturation is induced by ionophore A23187 or PHMB (Moreau et al., 1976~).However, very large oocytes of the depolarized type do not undergo further depolarization following progesterone treatment (Wallace and Steinhardt, 1977). When 1-MA is applied to oocytes of the starfish A . pectinifera, the membrane becomes depolarized within 15 minutes (prior to GVBD) and reaches a steady potential ranging from -20 to 0 mV (Miyazaki et al., 1975b). However, in this species, the membrane later hyperpolarizes once again. In P. miniata, the membrane potential becomes more negative with the initiation of maturation, causing hyperpolarization from -80 to -95 mV (Shen and Steinhardt, 1976). Recently, the membrane potential change in mouse oocytes during maturation was examined by Powers (Biggers et al., 1977; Powers and Biggers, 1976). It was found that the resting potential in dictyate stage oocytes was about -35 mV and was reduced to -25 mV at the time of GVBD. Further depolarization occurred as the oocytes proceeded to metaphase 11, at which time it reached - 14 mV (Powers and Tuppers, 1974, 1975). During maturation, membrane resistance is also changed. In Xenopus oocytes the resistance to ingoing current increases well before GVBD, shortly before depolarization takes place (Belle et al., 1977b). Membrane resistance, however, then decreases just before GVBD occurs (Moreau el al., 1976~).As seen in Fig. 9, during starfish ( A . pectinifera) oocyte maturation, the current-voltage slope (resistance) becomes steeper in the rectification regions, especially in the inward rectifying region near the resting potential level, and the negative resistance previously observed at voltages above it disappears before GVBD begins (Miyazaki et al., 1975b). A similar change in membrane resistance has been observed in Patiria oocytes. In this species, although no depolarization of the membrane occurs during maturation, membrane resistance at voltages near the resting potential and in the inward rectifying region increases before GVBD begins (Shen and Steinhardt, 1976). Mouse oocytes at metaphase I1 also exhibit a steep voltage-current slope over a wide range of voltages (Powers and Tupper, 1974).
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3 . Changes in K and Nu Ion Permeability The changes in the electrophysiologicalproperties of oocytes during the initial period of maturation described above may be interpreted to be a result of changes in the membrane permeability to K ions, and possibly also to Na ions, since the resting potential of the oocyte membrane depends mainly on its selective permeability to Na and K, as discussed at the beginning of this section. Studies by O’Connor et al. (1977) of ion transport in Xenopus oocytes, using radioactive K and Na, have shown that both the influx and the efflux of K ions are considerably increased shortly (2 hours) after progesterone treatment; however, this is followed by a continuous decrease beginning before GVBD. However, the rate of Na influx steadily increases, starting almost immediately after exposure to progesterone, but subsequently decreases following GVBD. Na efflux also increases, and it continues to do so for several hours after progesterone treatment. In Xenopus oocytes, the permeability ratio between Na and K ions (PNa/PK), calculated from ion flux rates, first decreases from 0.10 to 0.05 in the period following progesterone stimulation and then increases to 0.43 at GVBD (O’Connor et al., 1977). Similarly, in R. pipiens the rate of Na uptake increases after progesterone treatment but declines to the unstimulated level after GVBD, whereas the rate of K uptake steadily declines (Ziegler and Morrill, 1977), producing overall an increase in the total Na content and a decrease in the total K content per oocyte. However, since oocyte water content increases from 50% to 65% of the total weight of the oocyte when it reaches metaphase 11, the Na concentration does not rise proportionally to its uptake, while the decrease in K concentration is augmented, resulting in depolarization. The depolarization of mouse oocytes during maturation may also be ascribed to an increase in the permeability to outgoing K ions, as suggested by Powers and Biggers (1976). In starfish (A. pectinifera) 1-MA-treated oocytes reduce the conductance of ingoing K current (Miyazaki et al., 1975b). In batstar (Patiria) oocytes, however, the hyperpolarization induced by l-MA is augmented in Na-free media, suggesting an increase in inward-directed Na conductance during maturation, while the internal K concentration is rather increased and no marked change occurs in the ratio between Na and K ion permeability (PNJPK) (Shen and Steinhardt, 1976). This is in contrast to the trend found in oocytes of other animals, in which all evidence suggests a decrease in internal K concentration during maturation. Except for the case of Patiria, changes in ion transport during maturation in various animals may be summarized as follows. The depolarization of the membrane during the initial period of maturation is accompanied by a loss of selective K permeability and perhaps also by an increase in inward directed Na diffusion, which result in a decrease in the internal K concentration and an increase in Na concentration in the oocyte.
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4. Significance of Changes in Electrophysiological Properties
The question of whether changes in ion flux into oocytes are a necessary step for the initiation of maturation has been examined by several groups of investigators. Dorke et al. (1976b) showed that the maturation of starfish oocytes following 1-MA treatment was inhibited by treatment with tetraethylammonium PEA), which is known to block K channels in the cell. In the mouse (Powers and Biggers, 1976) it was found that treatment with valinomycin, which acts as an ionophore for monovalent cation transport, induced hyperpolarization of the oocytes and strongly retarded the progression of maturation. This effect was interpreted as being a result of the increase in membrane permeability to K ions induced by the ionophore, which may temporarily increase internal K levels. In accordance with this interpretation is the finding by Baltus et al. (1977) that Xenopus oocytes exposed to valinomycin in a K-free medium could initiate GVBD without stimulation by a MIS. In their experiment, the K ion content of gocytes was reduced by 40% as compared with that of controls kept in normal Ringer’s solution. Vitto and Wallace (1976) found that removal of K ions from the external medium or treatment with ouabain to inhibit the selective permeability to K of Xenopus oocytes facilitated their maturation. Conversely, maturation of R. pipiens treated with progesterone can be inhibited by increasing the K concentration in the external medium in the absence of divalent cations (Ecker and Smith, 1971b). All this evidence is consistent with the view that a reduction in internal K concentration following the loss of selective K permeability is a necessary step in the initiation of maturation by a MIS. The question of whether changes in electrical properties of oocytes are necessary for them to initiate maturation has been examined. Doree et aE. (1976b), who voltage-clamped starfish oocytes following 1-MA treatment, found that oocytes clamped at -40 mV were unable to undergo maturation. They also observed that depolarization of the membrane induced in the starfish oocyte following a short treatment with 1-MA (2.5 minutes) was reversible and that oocytes with the membrane potential reversed did not initiate maturation. These results appear to suggest a correlation between the depolarization induced by a MIS and the initiation of maturation. However, there is evidence that apparently contradicts this view. In starfish (M.glacialis), oocyte maturation can be induced with 1-ethyladenine, as well as with 1-isopropyladenine, without changing the membrane potential of oocytes (Doree et al., 1976b). Moreover, the fact that Patiria oocytes can be induced to mature by 1-MA treatment without undergoing any depolarization, but rather hyperpolarization, may present the most serious challenge to the theory that assumes the existence of a strict causal relationship between changes in the membrane potential and the initiation of maturation. The observation that a reduction in Na concentration of external media brings
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about a retardation of maturation in Xenopus (O’Connor et al., 1977) and mouse (Powers and Biggers, 1976) oocytes suggests that not only K but also Na ions play a role in the regulation of ionic conditions necessary for oocyte maturation. Therefore, it may be assumed that the intracellular ionic conditions of an oocyte required for the initiation of maturation can be fulfilled by changing the membrane conductance to these two ion species without causing observable depolarization of the membrane potential.
5. Roles of ATPase Changes in the rate of K and Na ion flux occurring early in maturation suggest that corresponding changes in membrane ATPase activity occur in oocytes, which regulate the transport of these ions. Mom11 et al. (1974) found that the ATPase activity of whole homogenates of R. pipiens oocytes increased sharply prior to GVBD. They noted that this rise in ATPase activity appeared to coincide in time with the characteristic membrane potential change. However, they also found that cycloheximide treatment of oocytes had no effect on the rise in ATPase activity, while it completely inhibited GVBD. It may be that oocyte protein which must be synthesized for GVBD to occur is not related to the measured ATPase activity. According to Mom11 et al. (1971, 1974), before the initiation of maturation R. pipiens oocytes possess ATPases which can be activated by Mg, Na, and K and strongly inhibited by ouabain and by strophantidin, whereas ATPases in oocytes at metaphase I1 are activated by Ca in the presence of Mg. However, in X . laevis, Pays et al. (1977) found that ATPase activities detectable both on the surface of intact ovarian oocytes and in homogenate preparations were virtually unaffected by ouabain and strongly activated by Ca and Mg but not by Na or K. This observation apparently contradicts those of Morrill et al. (1971). Pays et al. (1977) also found that the ATPase activities of homogenates of progesteronetreated oocytes and of oocytes treated with PHMPS sharply rose before GVBD took place, while the surface ATPase activity of intact oocytes did not show any significant change during the course of maturation. In view of these results, it may be premature to speculate on the correlation between changes in the ATPase activity of oocytes and in their ion transport activities during the process of maturation.
V. Cytoplasmic Control of Oocyte Maturation A. MATURATION-PROMOTING FACTOR 1. Origin and EfSects
In amphibian and starfish oocytes it has been repeatedly shown, as reviewed in Section IV,A, that a MIS is effective only when it is applied externally so as to
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act on the mcyte surface. In addition, discovery of the early change in the membrane potential of oocytes following MIS Stimulation strengthens the View that the initial maturational event takes place in the plasma membrane of the wcyte. Therefore it appears that the action of a MIS is primarily directed toward the surface membrane, as opposed to the cytoplasm or the nucleus (the GV). A logical consequence of this notion is that the signal given by a MIS to the oocyte surface must be transmitted to the nucleus by a cytoplasmic messenger. Whatever the mechanism of transmission, there is evidence that transmission of the signal through the cytoplasm of the oocyte occurs. Iwamatsu (1966), using fish (medaka) oocytes, demonstrated that GVBD could be inhibited when the GV was displaced from the hyaline cytoplasm near the surface of the animal pole into the yolk-filled vegetal hemisphere. Masui (1972) obtained similar results with R. pipiens oocytes. In his experiment oocytes were constricted in the equatorial zone to various degrees, using thread, after the GV had been displaced into the vegetal hemisphere by centrifugation. The oocytes were then exposed to progesterone. The occurrence of GVBD when the GV was in the vegetal hemisphere was considerably delayed compared to when it was in the animal hemisphere, and the frequency of GVBD was markedly decreased as the cytoplasmic connection between the animal and vegetal hemispheres was made narrower by increased constriction. Thus it seems likely that the cytoplasmic activity causing GVBD develops first in the animal hemisphere and then spreads to the vegetal hemisphere, especially in view of the fact that the animal hemisphere is more sensitive to progesterone than the vegetal hemisphere (Cloud and Schuetz, 1977). It was at one time thought that oocyte maturation could be initiated by the direct action of gonadotropin on the GV. Dettlaff and her associates (1964), using amphibians, demonstrated that substances in the GV of follicleenclosed oocytes treated with gonadotropin became capable of breaking down the GV itself when injected into untreated oocytes. They cautioned, however, that the active factor inducing GVBD might be a cytoplasmic product, since the material transferred into the recipient oocytes contained a small amount of cytoplasm surrounding the GV. Gurdon (1967) found that gonadotropin failed to induce GVBD when it was injected directly into oocytes, suggesting that its action was indirect. Evidence of indirect hormonal action on the GV has been produced by Masui and Markert (1971), who demonstrated that the cytoplasm of progesteronestimulated R. pipiens oocytes exhibited the ability to induce GVBD when injected into untreated oocytes before the donor oocytes underwent GVBD. They also found that this cytoplasmic activity appeared in oocytes treated with progesterone after their GV had been removed. It thus became clear that the cytoplasm of progesterone-treated oocytes developed the ability to induce GVBD independently of the presence of the GV. Further observations of the oocytes induced to
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undergo GVBD by the injection of cytoplasm from progesterone-treated oocytes have revealed that the recipient oocytes undergo further maturational changes, including development of surface contractility, condensation of chromosomes to the metaphase state, and polar body formation. Thus it has been postulated that a cytoplasmic factor responsible for promoting maturational events in general appears in the oocyte as a result of its surface stimulation by a MIS; this cytoplasmic factor has been designated maturation-promoting factor (MPF) (Masui and Markert, 1971). The GV-independent origin of MPF strongly suggests that it is a product of cytoplasmic activities which do not require genomic function. MPF has been found in oocytes of other amphibian species, such as X. laevis (Schorderet-Slatkine and Drury, 1973) and Ambystoma mexicanum (Reynhout and Smith, 1974), and also in the sturgeon (Dettlaff et al., 1977). In starfish oocytes, Kishimoto and Kanatani (1976) found that the cytoplasm of 1MA-treated oocytes, injected into untreated oocytes, was capable of inducing maturation of the recipient oocytes, and that the maturational events induced in the recipients followed the normal time course of 1-MA-treated oocytes. Reynhout and Smith (1974) showed that oocytes of a given species of amphibians could be induced to mature by microinjection of an appropriate amount of cytoplasm from maturing oocytes of other species. This finding demonstrates that the MPF of oocyte cytoplasm is effective in other species, suggesting that the active factor is not species-specific. Similarly, Kishimoto and Kanatani (1977) demonstrated that the interspecific transfer of cytoplasm from 1-MA-treated oocytes among different species of starfish resulted in the induction of maturation in the recipients. MPF can be found in the cytoplasm of oocytes induced to mature by MISS other than steroid hormones. It has been detected in Xenopus oocytes treated with mercurials (Brachet et al., 1975b), ionophore A23187 (Wasserman and Masui, 1975b), valinomycin (Baltus et al., 1977), and La ions (SchorderetSlatkine et al., 1976). These results suggest that MPF is not a product of the reaction between receptor molecules on the oocytes and steroid hormones. The effects of MPF on young oocytes at various stages of oogenesis which are unable to respond to MIS have been tested by Hanocq-Quertier et al. (1976) using X. laevis. It was found that MPF obtained from maturing oocytes caused GVBD and chromosome condensation in these young oocytes, though meiotic division could not take place because of failure to form a spindle. Similar effects of maturing oocyte cytoplasm on small, immature oocytes have been demonstrated in mice by Balakier (1978), who fused maturing oocyte cytoplasm with small oocytes, using inactivated Sendai virus, and observed GVBD as well as chromosome condensation to the metaphase state. These results clearly indicate that MPF is an ubiquitous cytoplasmic factor which promotes meiotic changes in the nucleus.
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The effect of MPF has been shown to be dose-dependent by Masui and Markert (1971) using amphibian oocytes, and by Kishimoto and Kanatani (1976) using starfish oocytes. Both these groups found that the frequency with which GVBD occurred in oocytes following the injection of maturing oocyte cytoplasm was almost linearly proportional to the volume of cytoplasm injected. This dose dependence of the effect of MPF has enabled us to assay its activity in a semiquantitative fashion.
2 . Time Course In amphibian oocytes, MPF appears shortly before GVBD and attains its maximum activity at GVBD. The activity remains at a high level until the oocytes are activated, at which time it begins to decrease rapidly (Masui and Markert, 1971). However, it should be emphasized that MPF activity can be detected even in the cytoplasm of cleaving blastomeres, although by this time it is quite weak (Masui and Markert, 1971). In starfish (A. pectinifera), Kishimoto and Kanatani (1976) found that MPF activity appeared 13 minutes after I-MA stimulation of oocytes. It reached its highest level between 20 and 40 minutes and then declined rapidly, disappearing by 80 minutes after hormone treatment. Since GVBD and formation of the second polar body take place 30 and 80 minutes after treatments, respectively, in the oocytes of this species, the change in MPF activity relative to meiotic progression shows a similar time course in Rana and Asterias; that is, MPF rises during karyokinesis and falls during pronuclear formation. The maturation of Xenopus oocytes induced by the injection of maturing oocyte cytoplasm containing MPF cannot be inhibited by protein synthesis inhibitors (Wassennan and Masui, 1975a; Drury and Schorderet-Slatkine, 1975). The time at which the maturation process becomes resistant to the inhibition of protein synthesis coincides with the time at which MPF activity can first be detected in the oocytes (Wasserman and Masui, 1975a). The time required for oocytes to develop MPF activity, measured from the time of progesterone stimulation, is approximately 65% of the time required to initiate GVBD. Since MIS-induced maturation of amphibian oocytes is always inhibited by protein synthesis inhibitors (Section III,C), it seems likely that the process preceding the appearance of MPF is the one which requires protein synthesis. Thus Wasserman and Masui (1975a) hypothesize that the process leading to the initial increase in MPF activity involves synthesis of a new protein, called the “initiator,” but that the action of MPF to induce GVBD does not. Furthermore, MPF action does not appear to be dependent on Ca ions. Masui et al. (1977) showed that the injection of EGTA into progesterone-stimulated oocytes did not inhibit their maturation when the injection took place after MPF had appeared in the oocytes. Guerrier et al. (1976) found that maturation of Xenopus oocytes
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induced by MPF injection was also resistant to proteolytic enzyme inhibitors such as antipain and leupeptin, both of which inhibit the maturation of progesterone-treated Xenopus oocytes and 1-MA-treated starfish oocytes (Clark and Kanatani, 1975). Thus it may be concluded that the action of MPF involves neither the synthesis nor the degradation of proteins.
3. Amplification MPF has been found to develop in oocytes induced to mature by the injection of cytoplasm from maturing oocytes. In order to examine the mechanisms which underly this secondary development of MPF activity in the recipient oocytes, a serial transfer of cytoplasm was carried out in R. pipiens oocytes (Masui and Markert, 1971). In this experiment, 60 nl of cytoplasm, which represents about 3% of the volume of an oocyte, was transferred from progesterone-treateddonor oocytes to untreated recipient oocytes, and then from these first recipients to the second recipients and from the second to the third, at 24-hour intervals. The cytoplasm thus transferred was able to induce maturation in the recipients after every transfer with similar frequencies, ranging from 75 to 90%, despite the fact that the cytoplasm of the original progesterone-treateddonors was clearly extensively diluted by the serial transfers. Therefore the cytoplasm transferred appears to stimulate development of MPF in the recipient oocytes, increasing its activity to the same level as that of the donor within the following 24 hours. Based on this observation, it has been hypothesized that MPF is produced by autocatalytic amplification (Masui and Markert, 1971). Similar experiments have been carried out with oocytes of X . laevis (Reynhout and Smith, 1974; Drury and Schorderet-Slatkine, 1975), the starfish A . pectinifera (Kishimoto and Kanatani, 1976), and the sturgeon Acipenser stellatus (Dettlaff et al., 1977). In all these experiments, cytoplasm of maturing oocytes could be serially transferred to recipient oocytes as many as 5 to 10 times without any resulting loss of MPF activity. In order to examine the requirement of the secondary development of MPF activity for protein synthesis in recipient oocytes injected with MPF, experiments involving the transfer of cytoplasm from progesterone-treated oocytes into a series of cycloheximide-treated recipients have been carried out. Drury and Schorderet-Slatkine(1975), using X . laevis oocytes, transferred the cytoplasm at 2-hour intervals. Their results showed that the frequencies with which recipient oocytes underwent GVBD following cytoplasmic injection declined during the transfer, and that GVBD could not be induced after three successive transfers. In contrast to this result, Wasserman and Masui (1975a), also using Xenopus oocytes, showed that serial transfers caused no appreciable decrease in the frequency of GVBD among cycloheximide-treated recipient oocytes when the transfers were carried out at 7-hour intervals. The latter result was recently confirmed by Dettlaff et al. (1977), using X . laevis oocytes. In their experiment, each cyto-
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plasmic transfer was carried out after the donoroocyte had undergone GVBD, and it was found that high MPF activity could still be detected in the cycloheximidetreated recipient oocyte cytoplasm even after cytoplasm was transferred five times. The experiments cited here were performed under similar conditions except for the difference in the duration of the interval between successive transfers. The amount of cytoplasm injected in each transfer ranged from 4 to 5% of the average volume of the oocyte, and the doses of cycloheximide applied to recipient oocytes were found to be sufficient to inhibit more than 90%of the protein synthesis occurring in untreated oocytes undergoing maturation. Therefore it may be that the apparent discrepancy between the results of the first experiment and those of the second and third can be reconciled if the rate of MPF amplification is taken into account. Since cycloheximide-treated recipient oocytes may take more time than untreated recipients to amplify MPF to the same level as that in the donors, it is reasonable to expect that a continuous decrease in MPF activity might result from successive cytoplasmic transfers taking place at short intervals insufficient for the cycloheximide-treated oocytes to produce a maximum concentration of MPF. In starfish and mammals, it may be supposed that the MPF amplification process, if it occurs, is independent of protein synthesis, since in these animals oocyte maturation is not inhibited by protein synthesis inhibitors up to metaphase 1 (see Section 111,C). However, it has been reported that MPF-induced maturation in sturgeon oocytes is sensitive to protein synthesis inhibitors (Dettlaff et al., 1977). In this fish, although MPF amplification as well as GVBD fails to occur following injection of maturing oocyte cytoplasm into recipients treated with cycloheximide, the oocytes acquire the ability to undergo cortical changes, such as vitelline membrane elevation and CGBD, following activation. 4. Nature In order to determine the cytoplasmic localization of MPF in R. pipiens oocytes, Masui (1972) assayed MPF activities in different fractions of oocytes stratified by a mild centrifugal force. In his experiments, oocytes which had been induced to mature by progesterone were placed in the interface between a 40% Ficoll solution and Ringer’s solution and centrifuged. The cytoplasmic contents of the oocytes separated into five layers-the lipid, fluid hyaline, gel hyaline, pigment, and yolk layers. MPF activity was found mainly in the fluid hyaline and gel hyaline layers. A similar experiment with starfish oocytes treated with 1-MA has recently been carried out by Kishimoto and Kanatani (1977), who found that MPF activity was primarily localized in the hyaline layer, which consists of multivesicular bodies, Golgi apparatus, and homogeneous cell sap. The extraction of MPF from amphibian oocytes was hampered by unexpected difficulties. MPF activity is so unstable that it is easily lost if the oocytes are homogenized. Baltus et al. (1973), however, found that extracts from homoge-
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nates of Xenopus oocytes exhibited the ability to induce atypical reactions, called pseudomaturation, in oocytes injected with the extracts. Electron microscope studies by Steinert et al. (1974) revealed that the pseudomaturation thus induced involved lobulation of the cortical cytoplasm, hyperdevelopment of internal membranes, and folding and disruption of the nuclear envelope following the migration of the GV toward the animal pole. These pseudomaturational events were not followed by meiotic progression, however. In order to avoid the adverse effect of homogenization on MPF activity a new extraction procedure was developed (Masui, 1974; Wasserman and Masui, 1976). This procedure consists of a rapid crushing of oocytes in a tube by direct application of a centrifugal force to the oocytes, followed by a quick separation of the extract from the particulate fractions. Wasserman and Masui (1976) found that MPF remained active in R. pipiens oocyte extracts thus prepared with a phosphate-buffered sucrose solution containing NaCl and MgSO,. Furthermore, they found that the activity became relatively stable when EGTA was added to the extraction medium, and that the presence of Mg was essential for its maintenance; the presence of Ca at a concentration as low as M rapidly inactivated MPF. These investigators suggest that MPF activity is associated with macromolecules of 4, 13, and 30S, which are heat-labile, protease-sensitive, and RNase-resistant. Further stabilization of MPF was recently achieved by Drury (1978), who extracted MPF from homogenates of Xenopus oocytes using a new extraction medium which consisted of a glycerophosphate-buffered sucrose solution containing NaF or ammonium molybdate. The presence of these chemicals is essential for stabilizing MPF activity. Furthermore, he also found that MPF was irreversibly destabilized by dilution in the absence of ATP. These results may indicate that MPF activity is maintained by phosphorylation of its molecules, and that NaF and ammonium molybdate, which are known to be inhibitors of phosphatases, may act as inhibitors of dephosphorylation of MPF, while ATP may enhance its phosphorylation. Gel filtration experiments by Druly (1978) have indicated that MPF is a macromolecule having a molecular weight between 0.6 and 1.0 x lo6 daltons. Furthermore, he found that the molecules could be sedimented from Xenopus oocyte extracts by a 30% saturated solution of (NH4)$04. Thus tentatively the nature of MPF may be assumed to be that of a phosphoprotein. B . PHOSPHORYLAT~ON OF PROTEINS 1. Phosphokinesis The characteristics of MPF revealed by recent experiments appear to point to a key role for protein phosphorylation in oocyte maturation. According to Monill
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and Murphy (1972), incorporation of 3'P into the protein of R. pipiens oocytes rapidly increases during maturation. Maller et al. (1977) found that protein phosphorylation of Xenopus oocytes rose sharply a few hours after progesterone treatment and reached its highest level at the time of GVBD. The protein phosphorylated during oocyte maturation is not yolk protein (Wallace, 1974). According to Maller et al. (1977), the predominant fraction is a protein molecule of 5.5 x lo4daltons. These workers note that the rise in protein phosphorylation in individual oocytes after progesterone treatment is always followed by GVBD, suggesting a close relationship between protein phosphorylation and GVBD. Further, they found that protein phosphorylation occurred in enucleated oocytes treated with progesterone and that not only progesterone, but also the injection of maturing oocyte cytoplasm, induced a burst of protein phosphorylation in normal oocytes which was followed by GVBD. Maller et al. (1977) also showed that protein phosphorylation induced by progesterone was inhibited by cycloheximide, whereas that induced by cytoplasm injected from maturing oocytes was not. Since protein synthesis of the oocytes had been suppressed by 95%, this result clearly indicates that phosphorylation occurred on preexisting protein, though its initiation by progesterone may require protein synthesis. These features of protein phosphorylation, which are quite similar to those of MPF activity, may be indicative of a close relationship between protein phosphorylation and the activation of MPF molecules. A similar pattern of protein phosphorylation has been discovered by Guerrier et al. (1975) in 1-MA-stimulated oocytes of M. glacialis. In this member of the starfish family, protein phosphorylation in the oocyte increases dramatically 5 minutes after hormone treatment. Their results further indicate that there is a differential distribution of phosphokinase activities in the oocyte, that is, histone and nonhistone phosphokinase activities are detected in the soluble and particulate fractions, respectively. Later experiments by Guerrier et al. (1977) revealed that protein phosphorylation following 1-MA treatment first increased in the cortex where it reached a maximum 8- 10 minutes after hormone treatment, but it increased rather slowly in the endoplasm, reaching a maximum 30 minutes after treatment, after which phosphorylation activity decreased until it reached control levels 1 hour after treatment. Interestingly, the time course of the phosphorylation activity appeared to follow that charted by Kishimoto and Kanatani (1976) for MPF. There is a close relationship between the level of protein phosphorylation in a given oocyte population and the frequency with which GVBD is induced in that population. Furthermore, various maturation inhibitors, such as SH oxidants, inhibit protein phosphorylation in oocytes treated with 1-MA (Guerrier et al., 1977). That oocyte maturation is induced by stimulation of protein phosphokinase is a possible interpretation of the results of Wiblet et al. (1975) in Ambystoma mexicanum and of Moreau et al. (1976a) in X . laevis, both of whom injected a
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phosphokinase preparation obtained from heterologous tissues into oocytes and succeeded in inducing GVBD in the recipients.
2. Role of CAMP The phosphorylation of certain proteins has been shown to be dependent on the level of cAMP in various types of cells. Thus it appears quite likely that changes in protein phosphorylation activity occurring in an oocyte during the course of maturation are correlated with changing levels of cAMP in the oocyte. Early experiments by Pays-de Schutter et al. (1975) to determine the levels of cyclic nucleoside monophosphates (CAMPand cGMP) in Xenopus oocytes revealed no marked differences between oocytes in the ovary and those at metaphase 11. However, the work of Speaker and Butcher (1977), using R. pipiens, showed that cAMP and cGMP levels, which are 0.6 and 0.8 pM respectively in fully grown ovarian oocytes, decrease to about half that 5 hours after hormone treatment, but then increase in the next 5 hours until shortly before GVBD occurs. The cAMP level decreases again after GVBD, reaching a minimum level when the first polar body is given off, and then returns to the same level as that of ovarian oocytes when they reach metaphase 11. The cGMP level, however, does not change as markedly as the cAMP level during the post-GVBD maturation period. Similar changes in cAMP levels in follicle enclosed oocytes of R. pipiens have been reported by Mom11 et al. (1977a). The decrease in cAMP level immediately following oocyte stimulation may be necessary for the initiation of maturation. As indicated in Section II,E, in mammals, high concentrations of dbcAMP inhibit maturation of both follicle-free oocytes and those enclosed in follicles. In amphibians, O’Connor and Smith (1976) showed that maturation of Xenopus oocytes failed to occur when cAMP degradation was inhibited by a xanthine derivative, theophylline, but this inhibitory effect was not observed when the chemical was injected into the oocytes. Morrill et al. (1977a), using follicle-enclosed oocytes of R. pipiens, reported that dbcAMP and theophylline inhibited maturation, without suppressing the protein synthesis stimulated by progesterone, when these chemicals were applied to oocytes incubated in a Ca-containing medium. These results, though difficult to interpret at present, seem to imply that a decrease in the cAMP level of oocytes is required for the initiation of maturation. Corroborating this notion is the recent finding by Godeau et al. (1977) that cholera toxin, known to activate cellular adenyl cyclase to increase cAMP levels in the cell, antagonizes the action of progesterone in Xenopus oocytes, thus inhibiting their maturation when applied externally. In starfish, it is known that xanthine derivatives have an inhibitory effect on the maturation of 1-MA-treated oocytes (Dorke et al., 1976a). It has been pointed out that cAMP derivatives added to mammalian oocyte culture do not interfere with general biochemical activities, such as protein synthesis (Stem and Wassarman, 1974; Schultz and Wassarman, 1977a,b) or
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respiration (Magnusson and Hillensjo, 1977; Magnusson et al., 1977; Hillensjo et al., 1977), of oocytes occurring prior to the initiation of maturation, and that theophylline applied to amphibian oocytes similarly does not interfere with protein synthetic activity prior to the onset of maturation (Morrill et d.,1977a). Rather, these compounds affect only the changes in biochemical activities which accompany the initiation of maturation. Therefore it appears likely that the maturation-inhibiting effect of these chemicals is brought about by raising cAMP levels in oocytes. If so, a high level of cAMP in ovarian oocytes probably prevents them from altering their biochemical activities in a manner which would initiate maturation. It has been shown beyond doubt that the role of CAMPin cellular activities lies mainly in regulation of the activities of protein phosphokinases. Maller and Krebs (1977) demonstrated that the catalytic (C) subunit of CAMP-dependent protein kinase, when injected into X. laevis oocytes treated with progesterone, inhibited the maturation of these oocytes, whereas the regulatory (R) subunit, as well as the protein inhibitor (I) of the enzyme, when injected into untreated oocytes, triggered the initiation of maturation. Since C subunits increase CAMP-dependent protein phosphorylation in the cell, while R subunits and I protein inhibit it by interacting with the C subunits, Maller and Krebs (1977) hypothesize that a high level of cAMP liberates C subunits from binding with R subunits and thus enhances the protein phosphorylation responsible for preventing oocytes from maturing. Conversely, inhibition of the C subunit, either by a decrease in the cAMP level or by the addition of R subunits or I protein, stops this protein phosphorylation and releases the oocytes from arrest at the diplotene stage. Ozon et al. (1978), using X . laevis and Discoglossus pirtus, demonstrated that the injection of I protein into oocytes initiated maturation. These experiments seem to indicate that an alteration in the pattern of protein phosphorylation in oocytes is the factor initiating their maturation. Thus the decrease in the CAMP level in oocytes in response to a MIS may lead to an alteration in their protein phosphorylation pattern by causing, on the one hand, suppression of cAMP-dependent protein kinase and, on the other, enhancement of phosphorylation of different kinds of protein. The initial decrease in the cAMP level of oocytes in response to a MIS may be caused by an increase in phosphodiesterase (PDE) activity. The observation that the PDE inhibitor, theophylline, has no inhibitory effect on oocyte maturation when it is injected into oocytes (O’Connor and Smith, 1976) suggests that the PDE responsible for the initiation of oocyte maturation is localized near the surface of oocytes. In this regard, Ca ions appear to play an important role in activating surface membrane-bound PDE. Maller and Krebs (1977) have postulated the existence of a Ca-dependent regulatory (CDR) protein that regulates PDE activity in amphibian oocytes. This CDR protein, upon reacting with Ca, changes its conformation and becomes capable of activating PDE. According to
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J. L. Maller (personal communication) the CDR protein isolated from beef heart can induce maturation in Xenopus oocytes when it is injected into oocytes after treatment with Ca. At this point it might be useful to summarize the foregoing discussion into some form of a working hypothesis in order to explain the initiation mechanism of oocyte maturation (Fig. 10). (1) The action of a MIS on the surface of oocytes causes a release of Ca ions which have been sequestered in the oocytes. (2) The released Ca ions act on the CDR protein which in turn causes activation of PDE. (3) The activated PDE degrades CAMP, resulting in a decreased CAMPlevel. (4) Thus CAMP-dependent protein phosphorylation is discontinued, and CAMPindependent protein phosphorylation increases. (6) This phosphorylated protein itself may represent active MPF, and its nonphosphorylated form the inactive precursor of MPF. If so, the protein is a phosphorylase kinase which catalyzes the phosphorylation of its own precursor and activates the precursor, thus autocatalytically amplifying its own activity. A protein of this kind may actually exist (Belle et al., 1976; Singh and Wang, 1977). (7) In amphibian oocytes, the
Gy 8
1
MIS
Protein 6-P
2
MWactiye)
1
;
ProteinA-p (inactive)
.L
GV FIG. 10. Scheme of the initiation of maturation in Xenopus oocytes. 1, MIS induces a conformational change in surface receptors; 2, Ca is released; 3, PDE is activated, thus lowering the level of CAMP; 4, CAMP-dependent protein kinase, which has been phosphorylating protein A, is inactivated; 5, phosphorylation of protein A, which has been constantly synthesized and degraded in the oocyte is terminated, and thus free protein A (the initiator) appears; 6, the initiator catalyzes phosphorylation of protein B (MPF precursor) to form active MPF; 7, active MPF phosphorylates autocatalytically precursor MPF (protein B); 8, MPF acts as a GVBD inducer. The scheme is a synthesis of previous hypotheses (Wasserman and Masui, 1975a; Maller and Krebs, 1977).
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CAMP-independent phosphorylation of MPF may require synthesis of a protein (initiator), as hypothesized by Wasserman and Masui (1975a). However, there are at present no theories concerning exactly how MPF causes GVBD.
C. ARRESTOF MEIOTICDIVISION 1. Metaphase Arrest
Except for species whose oocytes complete maturation before fertilization (coelenterates and echinoids) and those in which maturation is initiated by fertilization (echiuroids and some marine invertebrates), oocyte maturation is generally arrested either at metaphase I or 11. The arrested meiotic process is resumed following fertilization. These facts have been interpreted as suggesting that oocytes build up a self-inhibitory factor during the course of their maturation, and that their activation can be regarded as a mechanism for removing the inhibitory factor (see Monroy, 1965). Early investigators (Bataillon and Tchou-Su, 1930; Dalcq et al., 1936) considered the oocytes of amphibians to be “intoxicated” with COz. Heilbrunn and his associates (Heilbrunn et al., 1954) and Osanai (1967) suggested that an accumulation of acid polysaccharide was responsible for the arrest of meiotic progression prior to fertilization. However, the mechanisms underlying the meiotic block at metaphase I or I1 have not been the subject of intensive experimental analysis until recently. Humphries (1961) observed that coelomic oocytes of Triturus viridescens which had initiated maturation during ovulation sometimes advanced past metaphase I1 unless they were invested with a jelly coat while passing through the oviduct. Thus he suggested the potential significance of a contribution from the oviducal secretion to establishment of the meiotic block. However, since a majority of the oocytes without jelly investment still remained at metaphase 11, he acknowledged the possibility that a metaphase-blocking mechanism intrinsic to the oocytes existed.
2. Cytostatic Factor If an inhibitory factor blocking meiotic divisions at metaphase exists in unfertilized eggs, and its removal allows the eggs to proceed toward the completion of meiosis and the initiation of mitosis, it might be expected that the reintroduction of suck an inhibitory factor into fertilized eggs would arrest the progression of their cell cycles. And, in fact, Masui and Markert (1971), using R. pipiens, injected unfertilized egg cytoplasm (in volumes ranging from 30 to 120 nl) into one of the blastomeres of two-cell embryos and demonstrated that the cell cycle of the recipient emZlryos was indeed amsted a€ metaphase (Figs. 11 and 12). They found that the frequency with which metaphase arrest was induced and the
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YOSHIO MASUI AND HUGH J. CLARKE
FIG.11. An arrested blastomere of a two-cell embryo injected with the extract of an unfertilized egg (R. pipiens). (Meyerhof and Masui, 1977.) FIG.12. Metaphase arrest in a blastomere injected with the extract of an unfertilized egg (R. pipiens). (Meyerhof and Masui, 1977.) FIG. 13. Dark-field photomicrographs showing grain density over chromosomes induced to condense in enucleated oocytes injected with leucine-labeled GV contents. (Ziegler, 1979.) FIG.14. Bright-field photomicrographs showing the absence of grain accumulation over chromosomes induced to condense in enucleated oocytes injected with ovarian oocyte cytoplasm. (Ziegler, 1979.)
time at which the division of the recipient blastomeres was arrested were both correlated with the amount of cytoplasm injected and with the time of injection during the cleavage cycle; that is, the larger the cytoplasmic volume injected, and the earlier the stage of the recipient blastomeres, the earlier the blastomeres were arrested-hence the larger the arrested blastomeres. In a control series of experiments, cytoplasm obtained either from activated eggs which had been pricked with a glass needle, or from two-cell embryos, was injected into recipient blastomeres. Since cytoplasm from the activated donor eggs exhibited little ability to arrest the cell cycle in the recipient blastomeres, they concluded that unactivated eggs possessed a cytoplasmic factor which caused metaphase arrest of the cell cycle during the mitotic division of blasto-
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meres, and that its inhibitory activity could be removed by the activation process. This cytoplasmic factor was designated “cytostatic factor” (CSF) by Masui and Markert (1971). Furthermore, they postulated that it was CSF which was responsible for the metaphase block during meiosis, since its activity disappeared concomitantly with the resumption of meiosis triggered by egg activation. According to these investigators, CSF activity develops in the oocyte during maturation. It is undetectable in the oocyte before GVBD but appears shortly after GVBD and remains at a high level until activation. The development of CSF does not depend on the presence of the GV, since oocytes enucleated prior to the initiation of maturation also develop CSF activity following progesterone treatment. Meyerhof (1978) found that Xenopus oocytes induced to mature in vitro by progesterone developed CSF activity following GVBD; this activity was detectable when the cytoplasm was injected into blastomeres of R. pipiens two-cell embryos. No activity could be detected after the donor oocytes were activated by electric shock. However, very little CSF activity could be demonstrated in Xenopus eggs ovulated in vivo when their cytoplasm was injected into blastomeres of two-cell embryos of the same species. Also, failure to find evidence of CSF activity has been reported by Chulitzkaya (1970) and Chulitzkaya and Feulgengauer (1977), who injected cytoplasm from unfertilized eggs of R. temporaria and A . stellatus into eggs of the same species at various times following fertilization. Recently, however, Meyerhof (1978) found that cytoplasm of unfertilized Xenopus eggs obtained following HCG-induced ovulation consistently showed CSF activity when injected into blastomeres of two-cell embryos of the same species, provided the donor eggs were injected with a small quantity of EGTA (50 nmoledegg) prior to withdrawal of the cytoplasm to be tested. In control experiments cytoplasm from two-cell blastomeres which had been injected with the same dose of EGTA exhibited no CSF activity when injected into recipient blastomeres. Therefore the previous failure to demonstrate CSF activity in naturally ovulated Xenopus eggs can be attributed to a loss of CSF activity by the cytoplasm, probably caused by a release of Ca by the egg cytoplasm during the injection procedure. Similar reasoning may also explain the failure of Chulitzkaya and her associate to detect CSF activity in R. temporaria and Acipenser eggs. Interestingly, evidence supporting the existence of CSF activity in mammalian oocytes may be found in the results of recent experiments by Balakier and Czolowska (1977) who fused fragments of cytoplasm of mouse oocytes, which had been induced to mature in vitro, with blastomeres of two-cell embryos using Sendai virus. They found that the blastomere cells began to undergo mitosis following cytoplasmic fusion and reached metaphase, but the majority of these cells arrested at this point and failed to divide.
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Interspecific transfer of unfertilized egg cytoplasm into blastomeres of twocell embryos has been carried out between R . pipiens and X . laevis by Meyerhof (1978), whose technique included EGTA injection into donor eggs prior to the transfer. He found that cytoplasm of unfertilized Xenopus eggs strongly inhibited the cell cycle of R . pipiens blastomeres at the metaphase stage, whereas the cytoplasm of unfertilized Rana eggs generally failed to arrest Xenopus blastomeres. As expected, the cytoplasm of each species showed a CSF effect on cleaving blastomeres of its own species. It may be conjectured that Xenopus eggs have relatively higher CSF activity than Rana eggs or that Xenopus blastomeres have a stronger resistance to the activity of CSF than Rana blastomeres. In any case, this observation, taken together with previous results, suggests the possibility that CSF is a cytoplasmic factor involved in the arrest of both meiosis and mitosis at the metaphase stage in oocytes and embryonic cells of various species.
3. C a Effects on CSF CSF has been extracted from unfertilized eggs of R . pipiens by crushing the eggs using centrifugal force in a manner identical to that used for MPF extraction. Masui (1974) noted that CSF activity in the extracts was as unstable as MPF, disappearing within 24 hours on cold storage; but a more stable CSF activity, lasting at least a few weeks in cold storage, was obtained when Ca was added to the extraction medium. The effects of Ca ions on CSF activity have been further investigated by Meyerhof and Masui (1977). In contrast to the previous result, they found that the high CSF activity in the extracts made with freshly ovulated R . pipiens eggs could be maintained by increasing the Mg concentration in the extracts as well as by the addition of EGTA. However, the addition of EDTA was found to abolish CSF activity completely. Thus it became apparent that CSF activity of fresh oocyte extracts was dependent on Mg ions and became relatively stable after the removal of Ca ions. At the same time, Ca ions apparently destabilized the activity, since addition of Ca to the extracts at a concentration as low as M rapidly abolished CSF activity (Meyerhof and Masui, 1977). This finding, an apparent contradiction to the previous one, indicates that Ca-sensitive CSF activity is a entity different from that previously found to be stabilized by Ca. Study of the time course of the CSF activity in the extracts prepared from freshly ovulated R . pipiens eggs has revealed that the extract loses CSF activity, either in the presence or absence of Ca, and then regains this activity during cold storage. The second CSF activity is resistant to Ca, and its development is accelerated when Ca ions are present in the extract. Meyerhof and Masui (1977) named the CSF in fresh egg extracts, whose activity is Casensitive, “primary” CSF, distinguishing it from that developed secondarily in aged extracts, “secondary” CSF, whose activity is Ca-resistant. Although secondary CSF may be an artifact created during the aging process
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of the extract, the effects of primary and secondary CSF on progression of the blastomere cell cycle are indistinguishable, arresting mitosis at metaphase in the same fashion. Because of its highly stable nature, secondary CSF has been partially characterized and found to be associated with macromolecules which can be precipitated with @H4)2S04 at 30% saturation, can be passed through BioGel 15M, and are RNase- and heat-sensitive (Masui, 1974; Y. Masui, unpublished). 4. Inactivation of CSF and E g g Activation During fertilization, eggs arrested at metaphase I1 resume meiosis, forming the pronucleus and then proceeding to mitosis. The process of egg activation must involve inactivation of CSF. The ability of activated oocytes to inactivate CSF has been tested by injecting unfertilized egg cytoplasm or its fresh extracts into fertilized eggs at various times following insemination. Meyerhof and Masui (1977) found that cleavage and further development of the recipient eggs were unaffected when the injection took place within the first 45 minutes following insemination, indicating that the eggs were capable of inactivating CSF in the initial 30 minutes of activation, since all eggs had been penetrated by sperm by 15 minutes after insemination. The ability of activated oocytes to inactivate CSF appears to develop in the cytoplasm without participation of the nucleus, since the CSF developed in oocytes which had been induced to mature after removal of the GV disappeared following pricking with a glass needle or electric shock (Masui and Markert, 1971). This demonstration of cytoplasmic autonomy in the process of egg activation is consistent with the observations by Smith and Ecker (1969) and Skoblina (1969) that oocytes induced to mature after GV removal underwent the same surface changes as observed in normal eggs when they were activated. The high sensitivity of primary CSF to Ca may be an indication that its inactivation during egg activation is caused by Ca ions, whose concentration rapidly increases at the time of activation, as demonstrated by Steinhardt er al. (1977) in sea urchin eggs, and by Ridgway et al. (1977) in medaka eggs. The Ca release induced by sperm penetration appears to be a factor causing egg activation. In fact, the resumption of meiosis, arrested at metaphase 11, can be induced by ionophore A23 187 which releases Ca sequestered by the membrane system in the eggs of various species (Steinhardt et al., 1974). Also injection of Ca ions by iontophoresis activates mouse oocytes arrested at metaphase I1 to resume meiosis (Fulton and Whittingham, 1978). However, it has been noted that egg activation in the mouse is difficult to induce by ionophore treatment when Mg ions are present in the external medium (Masui et aE., 1977). In this mammal, activation of eggs by the ionophore is highly dependent on the relative strengths of the antagonistic effects of Ca and Mg present in the external medium, and only when the Ca effect exceeds the Mg effect can eggs be activated (Masui and Miller,
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YOSHIO MASUI AND HUGH J. CLARKE
unpublished). This antagonism between these two divalent cations may be attributed to the opposite effects exerted by these ions on the activity of CSF, Mg having a stabilizing effect and Ca a destabilizing one. Thus it may be concluded that the decrease in the free Ca level by cytoplasmic sequestration during oocyte maturation in vertebrates favors the development of MPF and CSF, both of which are inactivated by Ca and stabilized by Mg, whereas its increase during activation favors the disappearance of both factors upon fertilization.
5 . Metaphase Arrest in Other Species Meiotic arrest at metaphase I is a widely observed phenomenon in oocyte maturation in invertebrates. In these animals, whether or not the arrest is brought about by mechanisms similar to those postulated in vertebrate oocytes is open to speculation. It has been suggested by von Borstel (1957) that metaphase arrest during oocyte maturation in the parasite wasp Habrabracon juglandis is a genetically controlled process. His hypothesis is based on the following observations. In this species, maturation is initiated shortly after the oocytes are ovulated from the ovariole, but meiotic division is arrested at metaphase I during the period when the oocytes are in the uterine sac. At the time of oviposition, meiosis is resumed and the eggs develop parthenogenetically into haploid males (telytokous). However, in animals of a certain genetic strain, the oocytes continue meiosis, without metaphase arrest, and develop parthenogenetically in the uterine sac. The mechanism responsible for resumption of the meiotic division arrested at metaphase I has been analyzed recently by Pijnacker and Ferwerda (1976) in another telytokous insect, Carausius morosus (stick insect). In this animal meiosis is normally arrested at metaphase I for 5.5 days until the eggs are released from the uterine sac by oviposition. However, as long as the eggs are held in the uterine sac artificially by obstruction of the cloaca, they never resume meiosis. Only when they are exposed to the air can meiosis be resumed. In fact, meiosis in eggs laid in an atmosphere lacking 0, is arrested until the eggs are exposed to air. Thus it may be concluded that the factor overcoming meiotic arrest is the activation of aerobic respiration in oocytes.
VI. Nucleocytoplasmic Interaction during Oocyte Maturation
A. CHROMOSOME CONDENSATION 1. Chromosome Condensation Activity
Cytoplasmic transfer experiments involving immature and maturing oocytes have demonstrated that the behavior of the GV during maturation is under the
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control of oocyte cytoplasmic activities which occur independently of nuclear functions. The effects of oocyte cytoplasm can be observed not only in the GV but also in nuclei introduced from other cells. Classic observations by Brachet (1922) on sea urchin oocytes, and by Bataillon and Tchou-Su (1934) on amphibian oocytes, showing that sperm nuclei introduced into oocytes by precocious insemination conformed to the behavior of the female nucleus in the oocyte, suggested the significant influence of the cytoplasm on the nucleus. The effects of oocyte cytoplasm on somatic cell nuclei were first investigated by Gurdon (1967, 1968) using X. Zaevis. He found that nuclei isolated from blastulas or from adult brain and exposed to oocyte cytoplasm underwent changes in morphology similar to those occurring in the GV. This was confirmed by Ziegler and Masui (1973), using R. pipiens, who found that the changes induced in the foreign nuclei followed the same time course as those in the oocyte nucleus. These observations strongly suggest that cytoplasmic control over the nucleus is not tissue-specific. The ability of oocyte cytoplasm to condense chromosomes to the metaphase state has been studied by Ziegler and Masui (1973, 1976a,b), who introduced nuclei isolated from adult R. pipiens brain into oocytes which had been treated with progesterone but prevented from undergoing activation by the use of a phosphate buffer @H 6.2 or lower) in the injection procedure. They found that the cytoplasmic activity causing condensation of the chromosomes appeared in the oocytes shortly after GVBD and persisted until they were activated. Using sperm nuclei, Moriya and Katagiri (1976) investigated the time course of chromosome condensation activity (CCA) in B . bufo oocytes undergoing maturation. These workers treated sperm with a dilute solution of Triton X-100, making them permeable to cytoplasmic factors, before introduction into the oocytes. They found that the sperm chromosomes condensed to the metaphase state upon exposure to the cytoplasm of oocytes which had undergone GVBD, but that sperm injected into activated eggs failed to do so. This CCA of the oocyte cytoplasm has not been demonstrated in normally ovulated uterine eggs. In this connection, it was noted by Brun (1974) that X. laevis oocytes ovulated in vivo were unable to condense chromosomes of transplanted nuclei, in contrast to oocytes induced to mature in vitro after progesterone treatment. He suggested that the development of the ability of oocytes to respond to activation stimuli is due to an influence of oviducal secretion on the oocytes (Brun, 1975). The fact is that oocytes induced to mature by progesterone in vitro are able to condense the chromosomes of transplanted nuclei when oocyte activation is prevented by external application of EGTA buffer during the transplantation operation, whereas oocytes ovulated in vivo cannot condense chromosomes. However, recently Meyerhof (1978) has discovered that CCA can be demonstrated in oocytes matured in vivo as well as in CSF-arrested blastomeres if nuclei are injected together with EGTA and Mg,
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whereas CCA is not detectable in oocytes under any conditions after they are activated. Thus it appears that jelly-invested oocytes matured in vivo have a more unstable cytoplasm than jelly-free oocytes induced to mature in vitro by progesterone administration, so that CCA disappears in the former because of cytoplasmic perturbation caused by the injection procedure. Since EGTA stabilizes CCA in normally ovulated oocytes, it is highly probable that this cytoplasmic perturbation includes a discharge of Ca which is responsible for the rapid loss of CCA. The necessity of RNA and protein synthesis for the appearance of CCA has been investigated by Ziegler and Masui (1976a) using R . pipiens oocytes induced to mature in vitro by progesterone. They treated these oocytes with cycloheximide or a-amanitin at concentrations high enough to suppress protein or RNA synthesis, respectively, and at the same time injected the oocytes, which were at various stages of maturation, with isolated adult brain nuclei. The injected nuclei formed metaphase chromosomes in spite of the inhibition of protein and RNA synthesis when the injection took place at metaphase 11 (48 hours after hormone treatment), whereas those injected at metaphase I (24 hours after treatment) failed to undergo chromosome condensation. Since in both cases the recipient oocytes could be shown to have possessed CCA before the nuclei were transplanted, the failure of the nuclei injected into the recipients at metaphase I to condense must be attributed to a deficiency in protein and RNA synthesis which would normally occur following nuclear injection. However, no new protein or FWA synthesis was necessary for the chromosome condensation induced at metaphase 11, indicating that all the proteins and FWA required for chromosome condensation had been provided by the host oocytes by that time.
2 . Roles of the GV The possible importance of substances in the GV in chromosome condensation was suggested by Ziegler and Masui (1973), based on their early observation that no condensed brain nuclei chromosomes could be found 3-4 hours after the nuclei were injected into enucleated oocytes induced to mature by progesterone. It was noted, however, that the number of nuclei remaining in these oocytes was greatly reduced compared to the number in oocytes containing GV material. In later experiments therefore Ziegler and Masui (1976b) examined the number of nuclei remaining in the oocytes as well as the percentage of nuclei with condensed chromosomes at various times following injection. They found that chromosome condensation occurred both in nuclei injected into oocytes lacking GV material and in those injected into normal oocytes containing GV material when the nuclei were first examined, after 2 hours of exposure to oocyte cytoplasm. However, the number of clusters of condensed chromosomes was found to be markedly decreased after a 3-hour exposure, and no condensed chromosomes were found at 4 hours in oocytes lacking GV material, while the number of
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condensed chromosome clusters increased in nOITnal OOCYteS Over this time period. This observation strongly suggests that substances in the GV are not required for the induction of chromosome condensation in transplanted nuclei, but that the maintenance of condensed chromosomes following nuclear brane breakdown is highly dependent on the presence of GV substances. Whether or not material which accumulates in the GV during oogenesis associates with the chromosomes is a question several groups have undertaken to answer. Smith and Ecker (1970a) pointed out that proteins synthesized in ovarian oocytes of hibernating R. pipiens tended to accumulate in the GV, but these proteins were released into the cytoplasm following GVBD and redistributed into the zygote nuclei and into nuclei transplanted into activated eggs. According to Wassarman and Letourneau (1976b), 3H-labeled proteins which accumulate in the GV of mouse oocytes are found to associate with condensing chromosomes when GVBD takes place. They suggest that these proteins are predominantly histones, since protein labeled with try~tophan-~H neither accumulates in the GV nor associates with condensing chromosomes. This observation is in accordance with others indicating that the synthesis of histones is highly stimulated both in Xenopus (Adamson and Woodland, 1977) and mouse (Wassarman and Letourneau, 1976b) oocytes undergoing maturation. In mouse oocytes, Wassarman and Letourneau (1976b), using sodium dodecyl sulfate (SDS) gel electrophoresis, showed that F, histone was synthesized within 5 hours of isolation and was also phosphorylated. Recently Ziegler (1978) and Masui et al., (1979), using R. pipiens oocytes, analyzed the association of GV and cytoplasmic proteins with condensed chromosomes in oocytes. Progesterone-treatedoocytes, from which the GV had been removed, were injected with GV material obtained from immature oocytes previously labeled with Ie~cine-~H. The recipient oocytes were then injected with brain nuclei, while at the same time their protein synthesis was inhibited by cycloheximide. Autoradiographs of the nuclei injected in these oocytes revealed that the labeled material accumulated on the condensed chromosomes of the nuclei (Fig. 13). In the control experiment, labeled cytoplasm, instead of GV material, was injected together with brain nuclei into oocytes at metaphase I1 after protein synthesis had been inhibited. In contrast to the results for the experimental series, there was no accumulation of label on the condensed brain chromosomes (Fig. 14). In a second set of experiments, enucleated oocytes were treated with progesterone and labeled for 48 hours, at which time they reached a stage of maturation equivalent to metaphase 11. Cytoplasm from these oocytes was then injected, together with brain nuclei, into normal metaphase I1 oocytes which had been treated with cycloheximide. Under these conditions, the condensed chromosomes of the brain nuclei accumulated the label. Taken together, these results indicate that chromosomes undergoing condensation in maturing oocytes accumulate not only GV proteins but also cytoplasmic proteins which are
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synthesized by the oocytes during the course of maturation, whereas proteins found in the cytoplasm of immature oocytes with an intact GV do not accumulate on injected chromosomes. The association of GV proteins with condensed chromosomes may reflect a role of GV proteins in maintenance of the chromosomes in the oocyte cytoplasm; more specifically, they may act to protect chromosomal DNA from disintegration by cytoplasmic enzymes. Recently, Wyllie et al. (1977) showed that DNA injected into Xenopus oocytes remained inact when it was inside the GV, whereas DNA injected into the cytoplasm was degraded quickly. In this regard, it is interesting to note a recent report by Clark and Merriam (1977) that the GV contains a large amount of nonpolymerized actin which acts as a strong DNase inhibitor.
B. DEVELOPMENT OF THE PRONUCLEUS 1 . Male Pronucleus Growth Factor There are significant changes in cytoplasmic activities as a result of oocyte activation as discussed in Section V,A, and C. In amphibian oocytes, for instance, CCA disappears during activation, nuclei exposed to activated egg cytoplasm increase their volume, and their chromosomes become diffuse and initiate DNA synthesis (Graham et a l . , 1966; Gurdon, 1967, 1968). Changes in the cytoplasmic activity of mammalian oocytes during the course of maturation and activation have been studied by the insemination of oocytes at various stages of maturation and activation. Usui and Yanagimachi (1976), using hamster oocytes, found that sperm nuclei could be incorporated into zona-free oocytes at any stage of maturation as well as into zygote cells. An examination of the behovior of the incorporated sperm chromatin showed that chromatin incorporated into the oocytes before GVBD remained in a condensed state, whereas that incorporated at any time after GVBD decondensed even though the female chromosomes were undergoing condensation. Similar results have been reported by Iwamatsu and Chang (1972) using mice, and Niwa and Chang (1975) using rats, both of whom observed that decondensation of sperm nuclei incorporated into oocytes generally occurred only after GVBD. Only in the dog (Mahi and Yanagimachi, 1976) is there evidence that sperm nuclei incorporated into oocytes can undergo decondensation before GVBD. Generally, mammalian sperm nuclei incorporated into maturing oocytes at post-GVBD stages undergo decondensation independently of oocyte activation. These results apparently contradict the observations of Skoblina (1974, 1976) and Katagiri and Moriya (1976) that sperm nuclei injected into amphibian 00cytes remained in a condensed state until the oocytes were activated.
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However, in the sea urchin, while Long0 (1978) also observed swellkg of spem nuclei incoprated into meiotically dividing oocytes, he noted that these swelling nuclei lacked a pronuclear envelope and were composed of a central conoid mass of chromatin. This observation suggests that, although sperm nuclei incorporated into meiotically dividing mammalian or sea urchin oocytes may undergo a certain amount of swelling, this does not necessarily imply that a male pronucleus in being formed. On the contrary, recent studies of precocious insemination of R. pipiens OOcytes by Elinson (1975,1977) have revealed that sperm nuclei incorporated before oocytes become activatable develop into metaphase chromosomesembedded in a well-developed spindle following decondensation to a limited extent. He considers that a genuine pronucleus can be formed only after the oocytes have been activated by parthenogenetic stimuli. This has been confirmed by Ziegler (1979) and Lohka (1978), who observed a transient decondensation of sperm nuclei chromosomes injected into unactivated R. pipiens oocytes prior to condensation into the metaphase state. The ability of mammalian oocyte cytoplasm to induce development of the male pronucleus has been found to be dependent on the conditions under which the oocytes are induced to mature. Thibault and his associates (Thibault, 1972; Thibault et al., 1975a,b) showed in a variety of species, including rabbits, cows, and sheep, that foIIicIe-free oocytes which had undergone spontaneous maturation in vitro were unable to induce male pronuclear formation, while follicleenclosed oocytes which had been induced to mature by gonadotropins were able to support development of the pronucleus. They accordingly hypothesized that the failure of spontaneously maturing oocytes to induce pronuclear formation was due to lack of a cytoplasmic factor, named “male pronucleus growth factor” (MPGF). Mandelbaum et al. (1977) found that hamster oocytes developed MPGF only if they were kept in follicles for at least 6 hours following gonadotropin stimulation. Hunter et al. (1976) and Moor and Trounson (1977), working with pigs and sheep, respectively, have suggested that development of MPGF depends on the presence not only of gonadotropin but also of 17P-estradiol in the follicular environment, based on their finding that follicle-enclosed oocytes became capable of inducing male pronuclear formation following fertilization only if the oocytes matured in a medium containing sufficiently high levels of 17pestradiol. 2. Preparation for DNA Synthesis The importance of GV substances in the formation of, and DNA synthesis by, the pronucleus has been demonstrated by Skoblina (1974, 1976) and Katagiri and Moriya (1976) in R. temporaria and B . bufo oocytes, respectively. They found
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that sperm injected into OOCyteS underwent pronuclear transformation and initiated DNA synthesis once the recipients had reached metaphase a and had been activated. However, when the GV of a recipient oocyte was removed prior to injection, the sperm nuclei failed to swell and did not synthesize DNA. Katagiri and Moriya (1976) confirmed the active role of GV substances in promoting sperm nuclear transformation by introducing the karyoplasm of the GV into enucleated oocytes, which restored the ability of the oocytes to induce swelling as well as DNA synthesis in the injected sperm nuclei which would otherwise have remained inactive. They also found that the GV contents exerted the same effect whether they were taken from progesterone-treated or untreated oocytes, indicating that essentially the activity of the nucleoplasm did not change following progesterone stimulation. However, precocious rupture of the GV and subsequent mixing of the nucleoplasm and cytoplasm did not result in acquisition by the oocytes of the ability to promote DNA synthesis in transplanted nuclei (Gurdon, 1967). Clearly then, cytoplasmic maturation is also necessary for development of the oocyte activity promoting DNA synthesis in the nucleus. Gurdon (1967) transplanted adult brain nuclei into maturing oocytes of Xenopus and found that the cytoplasmic activity initiating DNA synthesis in the nuclei appeared after GVBD. This activity was also manifested when purified DNA was introduced into the oocytes (Gurdon and Speight, 1969). Furthermore, it was found that, not only did a marked increase in the overall activity of DNA polymerases in oocytes occur during maturation but, in addition, significant activity of a new DNA polymerase, distinguishable from those existing in immature oocytes, became detectable (Grippo and LoScavo, 1972; Grippo et al., 1975; Benbow et al. 1975). A recent study by Grippo et al. (1977) has indicated that DNA polymerase activity in Xenopus oocytes increases by 50% before GVBD (2-4 hours after hormone treatment), and that the presence of the GV is essential for this increase to occur, since only a 10%increase in activity occurs in oocytes deprived of their GV. Qualitative changes with respect to the factors and conditions controlling the initiation of DNA synthesis have been found to occur in Xenopus oocytes during maturation. Benbow and Ford (1975) found that DNA polymerases in extracts of ovarian oocytes were capable of initiating DNA synthesis only when supplied with denatured (single-stranded) DNA as a template, whereas extracts of uterine eggs could promote DNA synthesis using native (double-stranded) DNA templates. They attributed this capability of uterine oocyte extracts to the presence of a protein factor in the extracts which opened the template DNA and formed the initiation ‘‘eye. ’’ The ability of an oocyte to synthesize DNA for repairing damage to chromosomal DNA changes during maturation. In mouse oocytes, this unscheduled DNA synthesis can be induced at various stages of maturation by ultraviolet light irradiation (Masui and Pedersen, 1975) and by carcinogenic
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agents such as 4-nitroquinoline oxide (4NQO) (Brazil1 and Masui, 1978). Autoradiographic studies have indicated that the highest activity occurs in oocytes with an intact GV, regardless of the agent applied. When oocytes are treated with ultraviolet light or 4NQ0 after GVBD, the levels of DNA synthesis induced in the oocyte chromosomes are reduced by half, and little DNA synthesis is observed in the chromosomes in the polar body. Consequently, it was suggested that the levels of enzymic activities involved in repair DNA synthesis decreased during maturation of mouse oocytes, especially in the polar body. However, some alkylating agents such as methylmethane sulfonate (MMS) induce DNA synthesis at fairly constant levels in the oocyte and the polar body chromosomes throughout the maturation period. These observations may indicate that the activities of various enzymes involved in DNA synthesis in mouse oocytes are differentially altered during the progression of maturation. These changes may represent the initial step in a general decline in DNA repair activity in mouse cells, which continues throughout early life until by adulthood all somatic cells show very little activity in repairing damaged DNA.
c . DEVELOPMENT OF MOTILESYSTEMS 1. Surface Contractility In order for a mature oocyte to begin development, it must be able to undergo cleavage following fertilization. Various chemical and physical agents activate unfertilized eggs which subsequently begin to cleave. A growing body of evidence suggests that the primary agent responsible for triggering surface contraction of activated eggs is the C L ion (Gingell, 1970; Schroeder and Strickland, 1974; Brachet, 1977). Classic experiments by Delage (1901), Wilson (1903), Yatsu (1905), Chambers (1921), and Costello (1940) with oocytes of marine invertebrates suggested that the ability of the oocyte cortex to cleave depended on GV substancess distributed throughout the cytoplasm following GVBD. A similar conclusion was reached by Tchou-Su and Yu-Lan (1958), Dettlaff et al. (1964), and Smith and Ecker (1970a), all of whom examined the surface contractility of amphibian oocytes undergoing maturation. Their experiments, essentially the same as the classic ones performed on marine invertebrates, showed consistently that the ability of the oocytes to initiate cleavage upon introduction of a nucleus, either by fertilization or by nuclear transplantation, became manifest only after GVBD had taken place, and that oocytes deprived of GV substances by enucleation failed to develop this ability. However, Smith and Ecker (1970a) noted that an equatorial constriction could occur in enucleated R. pipiens oocytes following activation by pricking with a glass needle, indicating that a certain degree of surface contractility could be
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acquired by the OOCYteS without GV substances. Recently, Hollinger and Schuetz (1976) found that OoCYtes of R . pipiens undergoing maturation could induced to ckave if Ca was locally injected underneath the cortex, and further that the cleavage furrow always occurred in a definite orientation with respect to the location of the Ca injection. This ability of oocytes to react to Ca injection occurred in those which had undergone GVBD but not in those with an intact GV. However, oocytes deprived of GV substances by enucleation also became capable of reacting to Ca injection in the same manner as normal oocytes if they were matured for the same length of time. Also, interestingly, Iwamatsu (1971), using medaka oocytes, found that oocytes with an intact GV, which had been displaced into the yolk mass by centrifugation and consequently failed to break down during maturation, became capable of cleaving and developing into haploid embryos upon fertilization. He obtained similar results using oocytes from which the GV had been surgically removed. These results suggest that, contrary to the classic notion, GV substances are not necessary for the development of surface contractility by oocytes. It has been shown that cytochalasin B (CB) can induce cleavage of oocytes under certain conditions. In Sabellaria alveolata (polychaete), Peucellier et al. (1 974) found that cleavage occurred in unfertilized eggs (arrested at metaphase I) treated with CB, despite the fact that meiosis did not resume. Furthermore, Wassaman et al. (1976) have shown that CB can induce cleavage of fully grown mouse oocytes with an intact GV which have been prevented from undergoing maturation by dbcAMP. Electron microscope studies have revealed that underlying the CB-induced cleavage furrow are microfilaments of a contractile ring similar to those found in normal cleavage furrows in developing embryos (Wassarman et al., 1977). Thus it may be that the surface contraction of oocytes induced by CB requires neither the release of GV substances into the cytoplasm nor activation of the cortical cytoplasm. The observations described here seem to be consistent with the notion that oocytes are equipped with a surface contractile system before they undergo maturation. This contractile system must remain unresponsive to activation stimuli until the oocytes have reached a certain stage of maturation. Maturation of the oocyte cytoplasm somehow induces the surface contractile system to become responsive to the activation stimuli. The fact that oocytes deprived of GV substances are unable to cleave normally upon insemination, as observed in toad (Katagiri and Moriya, 1976), sturgeon (Dettlaff and Skoblina, 1969), and starfish (Hirai et al., 1971; Lee et al., 1975), strongly suggests an indispensability of the contributions from the GV to the development of endoplasmic mechanisms involved in the coordination of karyokinesis and cytokinesis. One key endoplasmic structure involved in this coordination is the aster, as pointed out by Rappaport (1971) and Kubota (1969).
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2 . Aster Formation
In the sea urchin it has been observed that oocytes with an intact GV, either at the previtellogenic or vitellogenic stage, fail to form an aster after insemination. The inability of these growing oocytes to develop an aster has been attributed to a lack of necessary cytoplasmic components (Franklin, 1965). Recent research by Longo (1978) has shown that aster formation can take place only after oocytes begin meiotic division, that is, after GVBD. Skoblina (1974), 1976) in R. temporaria, and Katagiri and Moriya (1976) in B. bufo, consistently observed aster formation in the cytoplasm of oocytes matured after GV removal, following insemination or injection of Triton X100-treated sperm nuclei. Clearly, such aster formation occurred independently of any GV contribution. Heidemann and Kirschner (1975) studied aster formation in Xenopus oocytes microinjected with various cellular components, such as nuclei, basal bodies, or centrioles from a variety of sources. They found that aster formation could be induced in uterine eggs but not in ovarian oocytes possessing an intact GV. According to these investigators aster formation requires the presence of GV substances (Heidemann and Kirschner, 1978). However, Elinson (1977) has suggested that the ability to form asters may be correlated with changes in cytoplasmic conditions induced by egg activation, rather than by maturation itself. He reported that, when maturing oocytes of R. pipiens were inseminated after GVBD, the sperm nuclei incorporated into the oocytes failed to develop asters but instead formed spindles around their chromosomes. However, when these oocytes were activated, large astral rays developed around the spindles. Recently, Hanocq-Quertier et al. (1978) showed that aster formation could also be induced by the presence of DzO in the cytoplasm of Xenopus oocytes undergoing maturation, and that this D,O-induced aster formation could be observed even in medium-sized oocytes, which are unresponsive to progesterone, after GVBD had been induced by MPF injection. Furthermore, these workers found that, while no cytaster or spindle formation could be induced by DzO in small oocytes, even if GVBD and chromosome condensation were induced by MPF injection, the formation of these fibrillar systems could be induced after the injection of protamine. They suggest that the ability of protamine to enhance fibrillation in young oocytes is due to its action in precipitating an excess of soluble RNA present in small oocytes which would otherwise inhibit formation of microtubules (Bryan et al., 1975). However, contrary to the observations of Skoblina (1974, 1976) and Katagiri and Moriya (1976) concerning aster formation by sperm nuclei, these investigators found that DzO-induced cytaster formation did not occur under any circumstances if the GV was removed from the oocytes before treatment, consistent with the observation by Heidemann and Kirschner (1978).
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3 . GV Material Knowledge of the various physiological functions as well as of the molecular characteristics of the GV may be of paramount importance in the study of oocyte maturation in view of the essential role played by the GV during the course of oogenesis and early development of animals. The nuclear envelope of the GV shows a wide variation in permeability to different types of molecules, ranging from ions to macromolecules. Especially important is the fact that proteins of low or medium molecular weight can freely pass through the envelope (Century and Horowitz, 1974; Bonner, 1975a; Feldherr, 1975). Certain classes of proteins have been found to accumulate predominantly in the GV and, when these proteins are labeled and injected into the cytoplasm of recipient oocytes, they again migrate into and remain in the GV (Bonner, 1975b). Recently Feldherr and Pomerantz (1978) have shown that the accumulation of specific nuclear proteins in the GV is not controlled by the nuclear envelope but rather by selective binding within the nucleoplasm, since a loss of the nuclear permeability barrier does not affect the distribution of nuclear proteins. Hill et al. (1974) using Triturus oocytes, and Merriam and Hill (1976) using Xenopus oocytes, resolved the proteins present in the GV into as many as 68 polypeptides by SDS gel electrophoresis. Most (85%) of these proteins can readily be dissolved in saline solution, but the remainder form a gel which can be dissolved only in alkaline salt solutions. One of the two major components of the gel appears to be actin (Clark and Merriam, 1977). This component shows the same electrophoretic mobility and peptide-mapping pattern as muscle actin, reacts with antiactin antibodies, and binds with DNase I to inhibit its activity. According to Clark and Merriam (1977) these actin molecules are present in a nonpolymerized form in intact oocytes but are readily polymerized upon exposure to Mg and K ions. The other major component of GV proteins is a myosinlike protein weighing about 250,000 daltons (Clark and Merriam, 1977). The gel component of the GV contracts slowly in the presence of ATP and a low concentration of Ca, forming microfilaments 70-80 A in diameter which can be decorated by heavy meromyosin. Actin has also been suggested to be present in the GV of mammalian oocytes as a result of studies by Amsterdam et al. (1977) using immunofluorescent-cytochemical techniques. These observations make it tempting to speculate that it is actin in the GV which is responsible for protecting DNA from nucleolytic degradation (Wyllie et al., 1977) and that the GV contributes actin which aids spindle formation and chromosome condensation and protection in oocytes following GVBD, thus making karyokinetic movement of the chromosomes possible, as suggested by Forer (1974). Finally, Burzio and Koide (1976), using Xenopus oocytes, found that the GV contains poly-(ADP-ribose) synthetase having an extremely high activity, and that its activity further rises sharply following progesterone treatment of the oocytes, but before GVBD (Burzio and Koide, 1977). These workers
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postulate that, since this nuclear enzyme adds ADP ribose from NAD to chromosome proteins (Honjo and Hayashi, 1973; Sugimura, 1973), probably causing alterations in chromosomal conformation, it is highly probable that the progesterone-stimulated increase in the activity of the enzyme in the GV is related to the appearance of CCA during oocyte maturation. L. 0. Burzio @ersonal communication) recently found that inhibition of poly-(ADP ribose) synthetase by its substrate (NAD) also inhibits maturation of progesterone-treated oocytes in Xenopus.
VII. Control of Meiosis and Mitosis-Concluding Remarks Oocytes arrested at the diplotene stage are in the G2 phase of the cell cycle. They proceed into the GI phase as zygotes, following maturation and activation. Hence oocyte maturation and activation as a whole appear to be analogous to the transition process of a cell, which includes mitosis, and take it from the G2to the G, phase. This concluding section considers several ways in which the control mechanisms of oocyte maturation and those of mitosis in somatic cells are analogous.
A. ROLE OF Ca Although the agents which induce the transition from G2 to GI in oocytes, that is, MISS, are generally ineffective in inducing mitosis in other cells, some of them, for example, proteolytic enzymes (Burger et al., 1972) and ionophore A23187 (Maino et al., 1974) are known to be active mitogenic agents in certain types of cells such as lymphocytes. The common effect induced by all these agents in the cell is a rise in the intracellular Ca level. That Ca plays an important role in regulation of the cell cycle is suggested by the periodic changes in free Ca level in the cell occurring during the cell cycle. Clothier and Timourian (1972), for instance, showed that a cyclic influx and efflux of Ca occurred during the cell cycle of cleaving zygotes of the sea urchin. In this cell system, three peaks of Ca mobilization are observed: the first during interphase, the second just before the beginning of mitosis, and the third at the initiation of cleavage. This observation agrees well with recent observations by Holmes and Stewart (1977) on the cell cycle of Physarumpolycephalum. They showed that a Ca efflux, which involved more than half of the total cellular Ca, occurred over a short period of time, ranging from 3 to 4 minutes, at the beginning of metaphase. This efflux was followed by an influx which continued until the commencement of anaphase. The Ca incorporated into the cell during metaphasic progression was then released.
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In oocytes, such as those of Xenopus and starfish, a release of cytoplasmic bound Ca occurs following MIS treatment (Section IV,B). This release of Ca is soon followed by a continuous uptake of Ca by the cell, which may be interpreted as active sequestration, thus lowering the free Ca level in the oocyte. This condition may be a prerequisite for the appearance of MPF and CSF in the cell, since both these cytoplasmic factors are sensitive to Ca ions. The relationship between the movement of Ca ions during the cell cycle and during mitosis is an interesting topic for speculation. First, Ca ion levels probably regulate the assembly of microtubules in the cell, since it is known that Ca ions inhibit their assembly in vitro (Weisenberg, 1972; Borisy and Olmsted, 1972). Second, Ca ions activate or inactivate numerous cellular enzymes. In oocytes, however, since Ca or Ca-mobilizing agents effectively induce maturation only when they are applied to the surface of oocytes (Section IV,B), it is difficult to ascribe the primary effect of Ca mobilization on oocytes to its action on the microtubule systems in the cell. Rather, it appears more likely that the action of the mobilized Ca is exerted on enzymes associated with the surface membrane of oocytes, causing a change in their activities. These enzymes probably include ATPases, PDE, and adenyl cyclase. At present, we know little about the action of these enzymes in animal oocytes, and the data currently available, for instance concerning ATPase activity of the oocytes, have given us no significant information with respect to the initiation of oocyte maturation. However, the importance of changes in the enzyme activities which regulate cAMP levels in the oocytes is suggested by observations that PDE inhibitors and cAMP derivatives have a marked inhibitory effect on oocyte maturation in numerous kinds of animals. Again, these inhibitors are effective only when applied to oocytes externally. These results strongly suggest that Ca plays an important role in controlling the activities of enzymes located on the surface membrane of oocytes, which are involved in the regulation of oocyte cAMP levels. It may be that, as put forth in Maller and Krebs’ (1977) hypothesis, the role of Ca present on the surface of the oocyte is to directly or indirectly activate PDE.
B. ROLEOF cAMP Studies on the level of cAMP during the cell cycle of various cell types have revealed that it always decreases sharply when cells enter mitosis from the G2 phase (Burger et al., 1972; Sheppard and Prescott, 1972; Yasumasu et al., 1973; Zeilig et al., 1974), while the changes in the cAMP levels of cells undergoing transitions between other phases of the cell cycle vary among different cell types
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(see Prescott, 1976). In contrast to the behavior of CAMP,cGMP levels appear to remain rather constant throughout the cell cycle, although some fluctuation has been observed (Seifert and Rudland, 1974). It has been also observed that dbcAMP (Freedman et al., 1975), as well as PDE inhibitor (Burger et al, 1972; Millis et al., 1974), prevents cells at interphase from entering mitosis. Analogies concerning cAMP function can be drawn between mitotically dividing somatic cells in culture and meiotically dividing oocytes, since in the latter it has also been observed that cAMP levels are sharply decreased upon initiation of maturation and that dbcAMP and PDE inhibitors prevent oocytes from undergoing maturation (see Section V,B).
C. PHOSPHORYLATION OF CELLULAR PROTEINS The finding that the inhibition of CAMP-dependentprotein kinase by its R subunit leads to the initiation of oocyte maturation, and that addition of the C subunit of the enzyme to the oocyte causes its suppression, strongly suggests that the processes by which primary oocytes eventually become zygotes are initiated by an interruption of the protein phosphorylation catalyzed by CAMP-dependent protein kinases. This interruption may cause an alteration in the pattern of protein phosphorylation in the cell. It has been established that, during mitosis, nuclear or chromosomal proteins are phosphorylated. For example, the phosphorylation of F, histone has been well-documented in various types of cells (Bradbury et al., 1974; Gurley et al., 1974). This phosphorylation has been suggested to be correlated with chromosome condensation. Alterations in the pattern of protein phosphorylation of cells undergoing the transition from a mitotically quiescent to an active state have been investigated in different types of cells. Generally, it has been found that type I and type I1 CAMP-dependentprotein kinases display different activities at different phases of the cell cycle (Costa et al., 1976). For example, in lymphocytes, a mitogen stimulates the cells to activate type I CAMP-dependent protein kinase, whereas dbcAMP activates both type I and type 11, thereby causing an inhibition of cell proliferation (Byus et al., 1977). It appears that type I1 protein kinase exerts a negative influence on the proliferation process of the cell. Corroborating this notion are the recent findings of Kletzien et al. (1977) using the baby hamster kidney cell line. They demonstrated the existence of a protein which was phosphorylated only in mitotically active cells and showed that cAMP suppressed phosphorylation of this protein, while at the same time enhancing phosphorylation of other proteins, the net result being an inhibition of cell proliferation.
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These observations suggest that meiotic divisions in oocytes and mitotic divisions in somatic cells are both controlled by common cellular mechanisms involving phosphorylation of a certain category of proteins.
D. SHCYCLE The finding that oocyte maturation can be induced by SH-blocking or SHreducing agents only when these agents are applied externally suggests that the initiation of maturation involves changes in the SH content of surface proteins (Section IV,A). This has been verified by measurements of the SH content of the cortex of starfish oocytes before and after the induction of maturation by 1-MA or SH-reducing agents (Kishimoto and Kanatani, 1973). Furthermore, Ikeda et al. (1976) showed that the amount of cortical SH reached a peak a few minutes before the expulsion of each polar body and then dropped sharply. This cyclic change in the SH content of the cortex is comparable to that observed during the cleavage cycle in sea urchin zygotes, in which the maximum level occurred shortly before each cleavage division (Sakai, 1968). This parallel change in cortical SH values during the course of oocyte maturation and of zygote cleavage seems to further strengthen the view that the cellular processes of meiosis and mitosis are governed by a common control system involving oxidoreduction of SH groups in the cortex proteins.
E. CYTOPLASMIC CONTROL FACTORS It has been established beyond question that maturing oocytes develop cytoplasmic factors which induce nuclear membrane breakdown, chromosome condensation, and metaphase arrest. These cytoplasmic factors have proven to be effective in inducing the same changes in somatic cell nuclei introduced into oocytes as in the native germ cell nucleus (Section VI, A-C). Furthermore, the existence of similar cytoplasmic factors in somatic cells has been well documented by cell fusion experiments in which nondividing (G or G 2 )cells are fused with cells in mitosis, resulting in premature chromosome condensation (Johnson and Rao, 1971; Matsui et al., 1971). Thus it appears that the nuclear events in oocytes undergoing meiotic divisions, and those in somatic cells undergoing mitotic divisions, are under the control of the same cytoplasmic factors. In this connection, we recall the observation of Masui and Markert (1971) that MPF, which is abundant in maturing oocytes of R. pipiens, can also be detected in blastomeres actively engaged in mitosis. Wasserman and Smith (1978b) also showed that MPF appeared in the cleaving blastomeres of amphibian oocytes
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during mitosis. Furthermore, W. J. Wasserman (personal communication) found that MPF capable of inducing maturation in Xenopus oocytes existed in the cytoplasm of cultured mammalian cells in mitosis. Recent experiments by Y. Masui (unpublished), involving the transfer of cytoplasm from sea urchin (Strongylocentrotuspurpuratus) oocytes into R . pipiens oocytes, have indicated that sea urchin zygotes produce a cytoplasmic factor during mitosis which causes oocyte maturation in frogs.
ACKNOWLEDGMENTS
We thank Mr. William A. Welch for his valuable contribution to the preparation of the manuscript for this article. A portion of the work cited here was supported by grants from the National Cancer Institute of Canada and the National Research Council of Canada, awarded to Y.M. for the period 1972-1977.
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Wassarman, P. M., Ukena, T. E., Josefowicz, W. J., Letourneau, G. E., and Karnovsky, M. J. (1977). J. Celt Sci. 26, 323-337. Wasserman, W. J. (1976). Ph.D. Thesis, University of Toronto. Wasserman, W. I., and Masui, Y. (1974). Biol. Reprod. 11, 133-144. Wasserman, W. J., and Masui, Y. (1975a). Exp. Cell Res. 91, 381-388. Wasserman, W. I., and Masui, Y. (1975b). J . Exp. Zool. 193, 369-375. Wasserman, W. J., and Masui, Y. (1976). Science 191, 1266-1268. Wasserman, W. J., and Smith, L. D. (1978a). In “The Vertebrate Ovary” (R. E. Jones, ed.), Plenum, New York (in press). Wasserman, W. J., and Smith, L. D. (1978b). J . Cell Biol. 78, R15-22. Webb, A. C., LaMarca, M. J., and Smith, L. D. (1975). Dev.Biol. 45, 44-55. Weisenberg, R. C. (1972). Science 177, 1104-1105. Wiblet, M., Baltus, E., and Brachet, J. (1975). C.R. Acad. Sci., Ser. D 281, 1891-1893. Wilson, E. B. (1903). Wilhelm Roux’ Arch. Enlwicklungsmech. Org. 16, 411-461. Wilson, E. B. (1925). “The Cell in Development and Heredity.” McMillan, New York. Wright, P. A. (1961). Gen. Comp. Endocrinol. 1, 20-23. Wright, P. A. (1971). Gen. Comp. Endocrinol. 16, 511-515. Wyllie, A. H., Gurdon, J . B., and Price, J. (1977). Nurure (London) 268, 500-502. Yanagimachi, R. (1974). J . Reprod. Ferril. 38, 485-488. Yasumasu, I., Fujiwara, A,, andIshida, K. (1973). Biochem. Biophys. Res. Commun. 54,628-632. Yatsu, N. (1905). J . Exp. Zool. 2 , 287-312. Zamboni, L. (1972). In “Oogenesis” (J. D. Biggers and A. W. Schuetz, eds.), p. 545. Univ. Park Press, Baltimore, Maryland. Zamboni, L., Thompson, R. S., and Moore-Smith, D. (1972). Biol. Reprod. 7 , 425-457. Zampetti-Bosseler, F., Huez, G., and Brachet, J. (1973). Exp. Cell Res. 78, 383-393. Zeilig, C. E., Johnson, R. A., Sutherland, E. W., and Friedman, D. L. (1974). Fed. Proc. Fed. Am. SOC. Exp. Biol. 33, 1391. Zeilmaker, G. H., and Verhamme, C. M. P. (1974). Biol. Reprod. 11, 145-152. Zeilmaker, G. H., and Verhamme, C. M. P. (1977). Acfu Endocrinol. (Copenhagen) 86, 380-383. Zeilmaker, G. H., Hulsmann, W. C., Wensinck, F., and Verhamme, C. (1972). J . Reprod. Fertil. 29, 151-152. Zeilmaker, G. H., Vermeiden, J. P. W., Verhamme, C. M. P. M., and van Vliet, A. C. W. (1974). Eur. J. Obsret. Gynecol. Reprod. Biol. 4, 15-24. Ziegler, D. H. (1979). Ph.D. Thesis, University of Toronto. Ziegler, D. H., and Masui, Y. (1973). Dev. Biol. 35, 282-292. Ziegler, D. H., and Masui, Y. (1976a). J . Cell Biol. 68, 620-628. Ziegler, D. H., and Masui, Y. (1976b). I n “Progress in Differentiation Research” (N. Muller-BCrat et ul.. eds.), pp. 181-188. North-Holland Publ., Amsterdam. Ziegler, D. H., and Momll, G. A. (1977). Dev. Biol. 60, 318-325.
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INTERNATIONAL REVIEW OF CYTOLOGY VOL. 57
The Chromaffin and Chromaffin-like Cells in the Autonomic Nervous System JACQUES
TAXI
.
Laboratoire de Neurocytologie Universiti Pierre et Marie Curie. Paris. France
I. Terminology
. . . . . . . . . . . . . . . . . . . .
II. Techniques . . . . . . . . . . . . . . . . . . . . A . Fluorescence Method . . . . . . . . . . . . . . . B . Electron Microscopy . . . . . . . . . . . . . . . 111. CCL Cells in Mammals . . . . . . . . . . . . . . . . A . Distribution of CCL Cells in the Autonomic Nervous System . B . Morphology of CCL Cells . . . . . . . . . . . . . C . Histochemistry of CCL Cells . . . . . . . . . . . . D . Action of Drugs on CCL Cells . . . . . . . . . . . . E . CCL Cells in Culture . . . . . . . . . . . . . . . F . CCL Cells and Development . . . . . . . . . . . . G . Relations of CCL Cells with Surrounding Structures . . . . H . Morphological Evidence for Endocrine Function . . . . . I . CCL Cell Types . . . . . . . . . . . . . . . . . J . Functional Role of CCL Cells . . . . . . . . . . . . IV . CCL Cells in Nonmammalian Vertebrates . . . . . . . . . A . Birds . . . . . . . . . . . . . . . . . . . . . B . Reptiles . . . . . . . . . . . . . . . . . . . . C . Amphibians . . . . . . . . . . . . . . . . . . . D . Fishes . . . . . . . . . . . . . . . . . . . . . E . Cyclostomes . . . . . . . . . . . . . . . . . . V . Concluding Remarks . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .
283 285 286 286 287 281 289 299 302 306 306 308 318 322 325 327 327 328 329 334 335 335 336
.
I Terminology It has been known for a long time that there were clusters of chromaffin cells scattered along and within the nerves and ganglia of the autonomic nervous system (see the classic reviews of Kohn. 1902. 1903. and more recently. Coupland. 1965b). when new interest in these cells was aroused about 1960 because they could represent a structural basis for explanation of special features of the synaptic transmission in the sympathetic ganglia. But. as they were studied further. these initially supposed chromaffii cells were given different names by different workers who investigated them with 283
Copynght 0 1979 by Academic Press. Inc . All rights of reproduction in any form reserved. ISBN 0-12-364357-0
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different techniques. Thus Hamberger et al. (1963), using the histochemical fluorescence technique for catecholamines and indolealkylamines recently worked out by Falck et al. (1962), noted in the cat “cells located in clusters adjacent to the capsule in the prevertebral ganglia exhibiting a very strong fluorescence in the yellow-green. ” They were considered chromaffin cells, as their shape and fluorescence was identical to those of the adrenomedullary cells. At the same time Eranko and Hakonen (1963) observed in the rat superior cervical ganglion (SCG) ‘‘occasional small cells exhibiting an extremely bright yellow fluorescence.” Such cells were seen again by Norberg and Hamberger (1964) in the same ganglion and later called small, intensely fluorescent (SIF) cells by Norberg et al. (1966). Soon after these observations, Siegrist et al. (1966) and Grillo (1966) described with electron microscopy, also in the rat SCG, small cells containing granular vesicles; these cells were considered chromaffin cells by Siegrist et al. (1966) and to have characteristics of both neurons and chromaffin cells by Grillo (1966). In fact, similar cells were probably seen for the first time by Viragh and Porte (1961) in the rat heart and ganglia and called cellules particulieres, microvesicles containing cells considered chemoreceptors. In 1967 Williams observed that such cells in the rat SCG were provided with both afferent and efferent synapses and proposed that they be considered the interneurons of the sympathetic ganglia. In order to avoid confusion, other neurons of the ganglia were called principal neurons by Matthews and Raisrnan (1969). The identity of the SIF cells seen with the fluorescence method with the chromaffin cells of Siegrist et al. (1966, 1968) or with the interneurons of Williams (1967), described in more detail by Williams and Palay (1969) and Matthews and Raisman )1969), was rather obvious. However, direct evidence was given only in 1974 by Grillo et a / . , who first localized the SZF cells by the fluorescence method and then observed the same section with electron microscopy. The term “chromaffin cells,” introduced by Kohn in 1899, has an indubitable chronological precedence for the denomination of these cells. But difficulty arises from the fact that certain SIF cells (in the rat SCG, for instance) are not stained by the standard chromaffin reaction (Lempinen, 1964; Eranko and Hiirkonen, 1965b; Norberg et al., 1966; Matthews and Raisman, 1969). Even an improved chromaffin reaction did not allow Santer et al. (1975) to stain more than a limited number of SIF cells in the ganglia of the rat, confirming the opinion of E r a 0 (1976) who stated that most SIF cells probably were nonchromaffin because the catecholamine they contained was less firmly bound than in adrenomedullary cells. Thus the overlapping of the two concepts, chromaffin and SIF cells, is not complete. If all chromafin cells exhibit a strong fluorescence with Falck’s method or its derivatives, the converse is not true. However, because of precedence and in order to not complicate the nomencla-
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tUE, many workers used the term “chromaffin” for chrornaffin as well as for nonchromaffin cells. For instance, Jacobowitz (1967, 1970) indicated that the “ChrOmaffm cells” of the cardiac ganglia he observed with the fluorescence method exhibited a chromaffin reaction only in the cat, and not in the rat, guinea pig, or mouse, but he justified the use of the term “chromaffin” by the fact that these cells all contained a large number of granular vesicles, as do adrenomedullary cells (Coupland, 1965a). But this point of view may lead to awkward situations, such as speaking of “nonchromaffm chromaffin cells, and it was not adopted by Williams and Palay (1969), who stated, in agreement with 0. Erankii and Eranko, (1971): “The term chromaffin cannot be used properly on the basis of electron microscopic appearance.” The designation “SIF cells” is better, as it corresponds to a property actually found in each cell. But it is arguable whether the fluorescence reaction by itself allows us to identify SIF cells with certainty. Confusion might occur with sympathetic neurons, which display great individual variations in size and intensity of fluorescence (Norberg and Hamberger, 1964). Thus El-Badawi and Schenk (1968) wondered whether the cells described as small adrenergic neurons located within the adrenergic terminal plexus of smooth muscles or other tissues could in fact be SIF cells. They considered the presence of varicose processes a definite criterion for identifying neurons, but it is now well established that SIF cells may have very long, fairly varicose processes, of which Furness and Costa (1976) gave remarkable examples, and the conclusion of El-Badawi and Schenk remains questionable. Also, Chamley er al. (1972b) Eranko et al. (1976) questioned whether highly fluorescent cells seen in cultures of sympathetic ganglia were SIF cells or neurons. Those who have studied these cells exclusively or predominantly with electron microscopy often used the term ‘‘small granule-containing” (SG) cells, introduced by Matthews and Raisman (1969), although actually the characteristic organelles are granular (dense-cored) vesicles rather than granules. Recently Kobayashi and Coupland (1977) introduced the term “small granule chromaffin” ( S G C ) cells for a special type of adrenal cell obviously similar to the cells we are dealing with in this article. Finally, it is clear that any designation based in fact on one character is open to criticism, as noted by Coupland (1976). In this article we attempt to reconcile precedence with other considerations, using the term “chromaffin and/or chrornaffin-like” (CCL) cells, which is of course also criticizable. Occasionally other terms mentioned above are also ernployed, especially “SIF cells. ”
”
11. Techniques
All cytological and cytochemical techniques can be applied to CCL cells. Only those which are of special interest are presented here.
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Chromaffm cells can be identified with the classic chromaffin reaction (see Coupland, 1965b), but recently Lever et al. (1974) and Santer et al. (1975) emphasized that using primary fixation with glutaraldehyde (Coupland et al., 1964) instead of formaldehyde, the reaction is more sensitive, producing reacting cells in certain ganglia (rat SCG, for instance) which are completely negative with the classic technique. This could be due to a better preservation of catecholamines other than noradrenaline @A).
A. FLUORESCENCE METHOD As certain CCL cells are not stained with the chromaffin reaction, the fluorescence method is more convenient for obtaining a complete picture of CCL (SIF) cells in a tissue. The classic method of Falck et al. (1962) is described in detail in Falck and Owman (1965). It involves freezing-drying of the tissue (air-drying is sufficient for thin materials), which is now generally accomplished with rather simple devices, in a primary vacuum in the presence of phosphorus pentoxide. Then specific fluorophoresare formed by the condensationof catecholamineswith formaldehyde vapors at 80°C. More recently, Bjorklund et al. (1972) and Axelsson et al. (1972) introduced the use of glyoxylic acid for primary stabilization of catecholamines in tissues before air-drying and subsequent vapor treatment. It appears from comparative counts made by Chiba and Williams (1975) with the classic method and the glyoxylic method (Lindvall and Bjorklund, 1974) that the latter shows fewer cells, but these workers believe that this is due to better specificity and that there is less diffusion. Recently Furness et al. (1977) propsed a “FAGLU” fixation (4% formaldehyde plus 0.5% glutaraldehyde) which should be more sensitive than the glyoxylic acid method.
B. ELECTRON MICROSCOPY
The study of CCL cells in tissues prepared directly for electron microscopy by routine techniques requires a time-consuming search for CCL cells in semithin sections stained by a basic dye. CCL cells are distinguished from principal neurons by their arrangement in clusters, their individual size, the high ratio of nucleus to cytoplasm volume, the chromatin repartition in the nucleus, and the lack of basophilia in the cytoplasm (Matthews and Raisman, 1969). This process operates rather efficiently in materials rich in CCL cells, such as the rat SCG, but becomes prohibitive in materials poor in SIF cells, such as the cat SCG. In order to select the tissue before embedding, Grillo et al. (1974) propose an improvement of the method of Eranko (1952, 1955), which consists of primary fixation of the tissue by a formaldehyde-glutaraldehyde mixture giving a water-insoluble
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fluorophore as well as adequate fixation for ultrastructural studies. Thus the fixed tissue can be cut without inclusion or freezing with a tissue chopper or a Vibratome. The sections are observed under a fluorescence microscope, and the CCL cells localized. The selected areas are then processed routinely for electron microscopy. All procedures giving water-insoluble fluorophores, such as the glyoxylic acid method or the FAGLU mixture, can be used for primary stabilization or fixation. 111. CCL Cells in Mammals
A. DISTRIBUTION OF CCL CELLS IN THE AUTONOMIC NERVOUSSYSTEM 1. Anatomical Data It is obvious that all information available from the numerous studies dealing with the localization of chromaffin cells is directly utilizable. There are exhaustive reviews on chromaffin cells and paraganglia by Kohn (1902, 1903), Iwanow (1932), and Watzka (1942), and the excellent book of Coupland (1965b) which gives a detailed description of abdominal, especially paraaortic, paraganglia in young and adult laboratory animals. As certain CCL cells are not chromaffin, the descriptions based on the fluorescence method must be expected to be more complete. There are no monographs on the distribution of CCL cells studied with the fluorescence method in a given species, but the same ganglia have been studied in different species, allowing interesting comparisons, as reviewed by Jacobowitz (1970). CCL cells were first described in the rat SCG (Eranko and Hiirkonen, 1963, 1965b), where there are no cells stained by the classic chromaffin reaction, as well as in other adult rat ganglia (Vincent, 1910). At the same time they were seen also in the sheath of the prevertebral ganglia of the cat (Hamberger et al., 1963). In the SCG CCL cells have been found in all species studied so far: rat (Eriinko and Harkonen, 1963, 1965; Taxi et al., 1969; 0. Eranko and Eranko, 1971); cat (Hamberger et al., 1964; Jacobowitz and Woodward, 1968); mouse (Olson, 1967); rabbit (Libet and Owman, 1974); guinea pig (Elfvin et aE., 1975); ox (Dermirjian, Aghajanian, and Kebabian, unpublished observations, 1976, in Kebabian, 1976); monkey (Chiba et al., 1977); human fetus (Hervonen and Kanerva, 1972); and human adult (Chiba, 1977, 1978). A systematic search in sympathetic tissues related to the reproductive organs was made by Sjostrand (1965) and Owman and Sjostrand (1965). In the guinea pig, rabbit, cat, dog, and monkey, SIF cells were found in the small ganglia scattered along the branches of the hypogastric nerve to the effectors, also referred
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to as pelvic ganglia (Falck et al., 1965; Doteuchi et al., 1969), in the hypogastric ganglia in the guinea pig (Watanabe, 1971) and in the nerve plexus accompanying the ovarian vessels in the rat (Payer, 1978). In the cat, dog, and monkey (Falck et al., 1965; Doteuchi et al., 1969), as well as in the bull (Owman and Sjostrand, 1966), there are also nests of SIF cells in the capsule of the prostate, in the external sheath of the vas deferens, in the wall of the seminal vesicles, and on the inside surface of the trigone of the bladder (Furness and Malmfors, 1971). A more detailed description of the CCL cells in the mesenteric and hypogastric regions in the guinea pig was made from stretched preparations by Costa and Furness (1973), who showed CCL cells isolated along the blood vessels or in groups apart from the nerve cells to which they send branched processes. Falck et af. (1965) pointed out CCL cells in the small ganglia annexed to the coagulating glands of the guinea pig. CCL cells with short processes were observed in the vaginal muscular wall of the rabbit (Owman and Sjoberg, 1966) and in the paracervical (Frankenhauser’s) ganglia of the female cat (Rosengren and Sjoberg, 1967). In the wall of the urinary bladder paraganglia or isolated CCL cells were frequently seen in cats, in the vicinity of small groups of nerve cells @ixon and Gosling, 1974), and in humans, in this case also in the connective tissue surrounding the urogenital organs (Hervonen et al., 1976). Alm et al. (1967) found CCL cells in an intrapancreatic ganglion composed of nonfluorescent nerve cells. In the heart, CCL cells were encountered within or in the vicinity of atrial ganglia or nerve trunks of the rat, cat, guinea pig, and mouse (Jacobowitz, 1967), cat (Ellison, 1974), rabbit (Friedman et al., 1968), calf (Vogel et al., 1969), human fetus @ail and Palmer, 1973), as well as of hibematorshedgehog, bat, and ground squirrel (Nielsen and Owman, 1968). CCL cells were noted in the mediastinum (Winckler, 1969a,b), in the epicardium, and surrounding adipose tissue (Nielsen and Owman, 1968). CCL cells were described along the thoracic and abdominal portions of the vagus nerves of the rat, guinea pig, and rabbit, along the mesorectum of the guinea pig, the peribronchial nerves of rat lung, and in two cases, a newborn rabbit, and a newborn cat, in association with Auerbach’s plexus of the intestine (Gabella and Costa, 1968). CCL cells were also observed in the parasympathetic ganglia of calf and dog lung (Jacobowitz et al., 1973). CCL cells were soon identified as SIF cells in the carotid body, in groups of species known to contain either chromaffin cells (dog, human, rabbit, cat) or nonchromaffin (chromaffin-like) cells (rat, mouse) (Coupland, 1965b; Kobayashi, 1971). As these cells are apparently related more to a receptor organ than to the autonomic nervous system, they are not discussed here except for comparative purposes.
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‘I’hese observations with the fluorescence method confirm that CCL cells are present in most (if not all) of the parasympathetic and sympathetic regions.
2. Quantitative Direrences among Ganglia in the Same Animal a& among Species It appears clearly from studies performed thus far that there are important differences in the number of CCL cells in the different ganglia of the same animal, and also in the number and characteristics of CCL cells in homologous regions of different species. For instance, it is obvious that CCL cells are more numerous in the rat SCG than in the cat SCG (Jacobowitz, 1970). Moreover they are localized in the cat mainly at the caudal end of the ganglion, whereas in the rat they are much more numerous in the anterior two-thirds of the ganglion, with one or several large clusters at the origin of the postganglionic nerve (Matthews and Raisman, 1969). In the stellate ganglion they are numerous in the rat (Norberg and Sjoqvist, 1966), but there are few in the same ganglion of the dog (Jacobowitz, 1970). In the cat, Norberg and Sjoqvist (1966) found that CCL cells were more numerous in the abdominal ganglia than in the SCG . Autillo and Seite (1976) and Autillo (1977, 1978) reported that the number of SIF cells was about five times greater in the coeliac ganglion than in the SCG; this is probably why Csillik et al. (1967) did not find CCL cells in the SCG but only in the coeliac, stellate, and inferior mesenteric ganglia. A comparative study was recently made by Chiba and Williams (1975) Williams et al. (1976b) in some species. Although the numbers of CCL cells were probably underestimated because of use of the glyoxylic acid method instead of the classic fluorescence technique, there is no doubt about the differences between the rat and the other species: reported for 1 mg of wet ganglion, the number of SIF cells was approximately 5 in the cow, 4 in the cat, 2 in the monkey, and 323 in the rat. For absolute values, 0. Erlinko and Eranko (1971) found between 429 and 986 cells in 5 adult rat SCGs with the classic fluorescence method, whereas Chiba et al. (1977) found, respectively, 37 and 98 cells in the monkey SCG. Noticeable differences in the number of CCL cells in the rat SCG appeared even among different strains, such as white Wistar (Santer et al., 1975) and Sprague-Dawley (0.Erlinko and Erhko, 1971). B. MORPHOLOGY OF CCL CELLS 1. Light Microscopy For studying the shape and size of CCL cells, the fluorescence method is the most suitable because it gives the most complete picture. As indicated by the often-used name “SIF cells,” they are small cells compared to principal
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neurons. They are spherical or oval when isolated (Plate I, Fig. 3), and polyhedral when clustered (Plate I, Fig. 1). There are variations in their size according to the species. Er*o and Hkkonen (1965b), Norberg e l al. (1966), Siegrist et al. (1968), Matthews and Raisman (1969), and others indicated a mean diameter of 6-18 p m in the rat SCG, whereas Watanabe (1971) found 10-20 pm in the hypogastric plexus of the guinea pig and 20-40 p m in the dog inferior mesenteric ganglion. The size of cell clusters is highly variable, from a few to 50 cells or even more. Certain clusters are surrounded by a connective capsule of fibrocytes, which can take on a greenish fluorescence by diffusion (Jacobowitz, 1970). These clusters are often located near a small artery or a capillary. Certain CCL cells have one, two, or even more processes, 1-3 p m in diameter (Owman and Sjostrand, 1965; Norberg et al., 1966). Processes are often 30-40 p m in length in sections 10 p m thick but have been reported to be up to 100 pm in favorable sections of the dog stellate ganglion (Norberg et al., 1966), rabbit SCG (Libet and Owman, 1974), human ganglia (Chiba, 1978), and even to be more than 500 pm in the whole amounts of the guinea pig hypograstric plexus (Furness and Costa, 1976). Processes have usually a more-or-less beaded appearance and have been seen to terminate in a large varicosity (plate I, Fig. 5 ) . When they originate from neighboring cells, they can remain associated in a loose bundle (Plate I, Fig. 2). Some processes, especially from isolated SIF cells, are in intimate contact with the perikaryon of principal neurons (Plate I, Fig. 4). The functional meaning of such contacts cannot be determined with light microscopy. Other SIF cells send their processes toward a blood vessel. Libet and Owman (1974) and Libet (1976) thought that the bulk of the beaded catecholamine-containingfibers winding around the principal cells in the rabbit SCG represented processes issuing from CCL cells rather than processes of principal neurons, as other workers believed (Norberg and Hamberger, 1964; Norberg et al., 1966; Jacobowitz and Woodward, 1968;Taxi etal., 1969).They argue that the network is maintained after several hours in vitro, when the fluorescence of principal cells and thin axons has quite disappeared. Moreover, the remaining fluorescence at this time should be due to dopamine @A), as for the CCL cells, and not NA, as for principal neurons. The first argument does not appear very strong, because it is well known that the fluorescence of perikarya is noticeably weaker than that of fibers, and it is not surprising that under unfavorPLATE I. Rat and Cat SCG. Fluorescence method. FIG.^. Rat SCG. CCL cells arranged in clusters and devoid of processes. X320. FIG. 2 . Rat SCG. A group of CCL cells provided with processes forming a kind of bundle. X520. FIG. 3. Cat SCG. Isolated CCL cell with a process. ~ 5 2 0FIG. . 4. Rat SCG. A CCL cell whose processes surround a principal neuron. X520. FIG.5. Rat SCG. A CCL cell with a short process ending with a swelling in contact with a principal neuron. X520.
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PLATE II. FIG.6 . Rat SCG. General view of the cytoplasm of aCCLcell. Dense-cored vesicles are localized mainly just beneath the membrane; there are only a few mitochondria1 sections which are elongated, because mitochondria are mainly spherical. At the arrow the cell surface is separated from the intercellular medium by only a basement membrane. X 16,000. (From Taxi, 1973.)
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able conditions it disappears first. Moreover, it is well known that the binding of catecholamines to structures is weaker in perikarya than in fibers (Axelrod, 1965). If the interpretation of Libet and Owman (1974) is correct, CCL cells processes should be found rather frequently in ultrathin sections of the rabbit SCG. From our observationson the ultrastructure of young rabbit SCGs, this seemed not to be the case; but recent results obtained by Dail and Evan (1978) on ganglia treated by 5-hydroxydopamine brought support to Libet and Owman’s ideas.
2 . Ultrastructure of CCL Cells The papers by Siegrist et al. (1966, 1968), Matthews and Raisman (1968, 1969), Williams and Palay (1969), Tamarind and Quilliam (1971), Taxi et al. (1969), and Taxi (1973) on the CCL cells of the rat SCG have been taken as a basis for this description. The nucleus of CCL cells is usually spherical or oval, with chromatin condensations along the nuclear envelope which are especially distinct after glutaraldehyde fixation. The nucleolus is infrequently conspicuous. This nuclear structure is very useful in distinguishing CCL cells from small neurons in semithin sections, especially in embryos or in tissue culture, when principal neurons may contain rather numerous granular vesicles @rMo ef al., 1976). The cell surface bears a limited number of microvilli (0.1 X 0.5 pm), localized in restricted areas; there are coated vesicles in various relationships with the plasma membrane (Becker, 1972). The mitochondria are usually round or oval, much less elongated than those of the neurons, with numerous parallel, narrow cristae which are regularly spaced (Plate 11, Fig. 6; Plate III, Fig. 7). These mitochondria appear rather sensitive to fixation and easily become swollen, even when the rest of the cell appears well preserved. The endoplasmic reticulum is poorly developed. Isolated granular cisternae are scattered in the cell, and free ribosomes are numerous. However, small cisternae regularly arranged as in Nissl bodies are occasionally found (Siegrist et al., 1968). Close relations between endoplasmic reticulum and mitochondria have also been observed, particularly in CCL cell processes (Williams and Palay, 1969) (Plate 111, Fig. 7). Multivesicular bodies are rather numerous. Dense bodies, morphologically similar to lysosomes, are few (Plate 11, Fig. 6). A well-developed Golgi apparatus lies in the juxtanuclear region, sometimes forming an arc. In certain cells, granular vesicles are numerous in the vicinity of the dictyosomes (Siegrist et al., 1968; Polonyi et al., 1976). Microtubules and microfilaments are rather frequent (van der Zypen, 1974), disposed apparently at random except in the processes where they are roughly parallel to the length, but are not so numerous as in nerve processes (Plate IV,
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Fig. 9). In the cat SCG, bundles of microfilaments are scattered in the cytoplasm (Autillo, 1977). Occasionally sections pass through a centriole, but it is not known whether this organelle is usually present. However, the frequency of a cilium in CCL cell sections suggests that centrioles are probably present in all cells (plate V, Fig. 10). Glycogen granules dispersed throughout the cytoplasm or grouped in small clusters were described by Benitez e l al. (1974) in their studies of cultured CCL cells. We have found CCL cells rich in glycogen only in experimental (autografted) rat SCGs (unpublished observations). The most conspicuous organelles of the CCL cells, first mentioned by Eranko and Hiirkonen (1965b) and illustrated by Grill0 (1966) and Siegrist et al. (1966), are the granular (dense-cored or granule-containing) vesicles. Notwithstanding their vesicular structure, they have sometimes been called granules, the internal portion having been confused with the whole structure. This confusion must be avoided, because it is important to be sure that the same structure is being dealt with when the size of organelles is measured, for instance. These vesicles are scattered within the cytoplasm with local aggregations but form a rather regular peripheral crown beneath the membrane (Plate 11, Fig. 6). The dense core is variable in size, position, and electron density. In many cases it occupies all the space inside the vesicle except a narrow halo beneath the membrane. Some micrographs clearly show that the vesicular contents are heterogeneous, with a matrix of low density and a more-or-less large area of high density, presumably due to osmium precipitation by the catecholamine (plate In, Fig. 8). Granular vesicles have often been described as being similar to those of the NA-containing adrenomedullary cells. This is true only in certain ganglia of certain animal species, in relation to the presence of several CCL cell t y p s (Section 1141). In the rat SCG the granular vesicles are smaller on the average than those of the adult adrenal medulla and are similar to those of embryonic cells @her, 1965). There are important differences in the values reported in the literature for vesicle size, as well as the density, shape, and position of contents. These differences can be related to differences in CCL cell origin (species, ganglion), different fixatives, and different physiological states. The values for size are ~
~
~~~
~
~
~~~~
~~
PLATE III. FIG. 7. Rat SCG. In a CCL process, two mitochondria are almost completely surrounded by a flat saccule of smooth endoplasmic reciculum. X41,000. FIG. 8. Rat SCG. Heterogeneity of vesicular contents after osmium tetroxide fixation. Except in a clear peripheral halo, there is a matrix of moderate electron density and a more-or-less eccentric granule of high density x 120.000.
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Usually rough aPProximatiOnS not supported by statistics. It is still likely that the smaller values represent only tangential sections of larger vesicles. some of the values given for vesicle diameter follow: in the rat SCG 65-120 nm (Matthews and Raisman, 1969), 70-100 nm (Taxi et al., 1969; Dail and Palmer, 1973), and 40-140 nm (Tamarind and Quilliam, 1971) in osmium tetroxide-fixed ganglia; 80-120 nm (Hokfelt, 1969) and greater than 100 nm (Eriinko, 1976) using potassium permanganate; mean values of 140 nm (Williams and Palay, 1969), 150-250 nm (VanOrden et al., 1970), 100-150 nm,and 200-400 nm (Siegrist et at., 1968) after aldehyde fixation followed by osmium tetroxide; in the inferior mesenteric ganglion of the rabbit 70-300 nm, and mostly between 150 and 200 nm (Elfvin, 1968); in the paracervical ganglion of the rat 80-140 nm and 200-300 nm only in certain cells (Hervonen et al., 1972b; Kanerva and Teraviiinen, 1972); in the hypogastric ganglion of the guinea pig 100-250 nm (Watanabe, 1971); in the coeliac ganglion of the cat 120-220 nm; in the abdominal paraganglia of the newborn rat, mouse, and hamster 50-200 nm (Mascorro and Yates, 1970); in the ganglia of the rabbit 150 nm as an average (Brundin and Nilsson, 1965; Brundin, 1966). The vesicles seem especially polymorphic in the human ganglia (100-300 nm in diameter according to Chiba, 1978). These values are mainly for tissues first fixed with glutaraldehyde. Except when they are in continuity with the cell soma, the identification of CCL processes in ultrathin sections is based on the presence of a certain number of granular vesicles which allows us to distinguish them from dendrites, as both contain in various proportions endoplasmic reticulum, ribosomes, mitochondria, clear vesicles, microfilaments, and microtubules (Plate IV, Fig. 9). According to Siegrist et al. (1968) the smaller vesicles, 100-150 nm in diameter, are found only in the processes. Siegrist et al. (1968) referred to axonic and dendritic processes of CCL cells, but they admitted that they could not be distinguished on a morphological basis. However, Watanabe (1971) identified dendritic processes, short and large, which had the same ultrastructural features as somata, and longer axons containing large granules, mitochondria, microtubules, and microfilaments. Unfortunately there are no physiological data available to support this point of view, and more information is necessary to accept it as more than a rather adventurous hypothesis. As for the satellite cells, they are often in continuity with those of the neurons and exhibit a morphology which has been described (Elfvin, 1963; Taxi, 1965).
PLATE IV. FIG. 9. Rat SCG. Longitudinal section of a CCL cell process with manydense-cored vesicles. It shares the same glial sheath with presumed neuronal processes to which it is apposed (arrows) and which contains many microtubules. x41,OOO. (From Taxi, 1973.)
PLATE V. FIG. 10. Rat SCG. A long contact between two CCL cells, with several attachment plaques (arrows). A cilium in a CCL cell is cut in a longitudinal section. X32,OOO.
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C. HISTOCHEMISTRY OF CCL CELLS 1. Fluorescence Histochemistry and Related Data Under cumnt conditions employing the histochemical fluorescence technique for catecholamines and indolealkylamines palck et al., 1962; Falck and OWman, 1965) CCL cells exhibit an intense yellow-green fluorescence. For this reason E r W o and Hiirkonen (1965b) wondered whether they contained serotonin, which was reputed to give yellow fluorescence, or a catecholamine, because the same yellow color is obtained with fluorescence of adrenomedullary cells. It was proposed by Caspersson et al. (1966) that the yellow fluorescence observed instead of the expected green was due to a secondary reaction related to a high concentration of a primary catecholamine. In fact, the fluorescence can vary in the same ganglion from an unequivocal green to bright yellow ( E r W o and H&konen, 1963), orange (Dail and Palmer, 1973), or even red (Santer et al., 1975), always more intense than that of principal neurons. The fluorescence is homogeneous in the CCL cell cytoplasm, the nucleus being often hardly distinguishable, whereas the fluorescence of principal neurons is heterogeneous, with many bright granules, a fact emphasized by Er2inko and Hiirkonen (1963). CCL cells seem devoid of autofluorescent granules (lipofuscin), which are numerous in many principal neurons. However, the analysis of excitation and emission spectra of CCL cells by microspectrophotometry have led Norberg et al. (1966) to conclude that the spectra are the same in principal neurons and CCL cells and that the substance responsible is a primary catecholamine, DA or NA excluding serotonin and adrenaline. On the basis of certain secondary properties of the fluorophore, Owman and Sjostrand (1965) suggested that the fluorescence could be due to adrenaline in CCL cells of the genital accessory organs (Section III,A,l). In 1968 Bjorklund et al. described a new method which permits discrimination between DA and NA fluorophores. Bjorklund et al. (1970) applied the method to CCL cells of the rat, cat, and pig SCG and concluded that these cells and those of the dog prostrate contained DA; they co n f i i e d biochemically in the pig and cat that the cervical-thoracic sympathetic chain contained a certain amount of DA, about one-seventh of the amount of NA. The same conclusion was reached for the CCL cells of the rabbit SCG by Libet and Owman (1974). However, using the same method, 0. Er"dnkij and E&o (1971) reported that the SIF cells of the rat SCG contained NA. In 1973 King and Angelakos described a fluorescence method using trihydroxyindole (THI) which gives fluorophores with different emission spectra for NA and adrenaline, and no fluorescent compound with DA. Using this new method in parallel with immunocytochemical techniques (Section III,C,3),
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Rybarczyck et al. (1976) concluded that CCL cells contained DA in the rat SCG, and NA in pelvic ganglia. In the heart, where DA was detected by dosage in relatively important amounts in the region of the sinoatrial node (Angelakos et al., 1963), it was proposed that it was localized in CCL cells which were rather numerous in this region (Ehinger et al., 1968). Also, indirect correlations may be speculated between the high amount of DA found in the cervix of the rat uterus (Swedin and Brundin, 1968) and the presence of the paracervical ganglion containing CCL cells (Kanerva, 1971). Further indirect additional evidence was put forward by Chiba et al. ( 1977), who demonstrated the existence of a DA receptor-adenyl cyclase complex in rhesus monkey sympathetic ganglia. But this does not prove by itself that CCL cells contain DA. Koslow et al. (1975) and Koslow (1976) combined a very sensitive mass fragmentography dosage of catecholamines (Koslow et al., 1972) with pharmacological treatments and concluded that CCL cells contained DA and perhaps adrenaline.
2 . Chromafin Reaction The term chosen here, “CCL cells, refers to the fact that some of these cells are chromaffh and contain NA or adrenaline, and that the others are chromaffinlike. Probably the most studied CCL cells were those of the rat SCG, which are not stained by the chromaffin reaction in its classic form involving fixation with formaldehyde ( E r M o and Hiirkonen, 1965b; Matthews and Raisman, 1969). Recently, Santer et al. (1975), using a modified chromaffin reaction with primary fixation with glutaraldehyde (Coupland et al., 1964), stained some of the CCL cells in the rat SCG (much less than the total). A comparative statistical study led Kemp et al. (1977) to conclude that chromaffin cells probably corresponded to the yellow CCL cells in fluorescence, whereas nonchromaffin cells corresponded to the green ones. This can be explained by the view of 0. Eriinko and Eranko (1971), according to which the difference between chromaffin and nonchromafh cells is due to different amounts of catecholamines and to the size of granular vesicles, the storage structures. Moreover, there are probably also differences in the nature of the catecholamines. According to Lever et al. (1976), cells containing a high concentration of DA do not give a positive chromaffin reaction. In as much as certain cells are stained in the rat SCG, it was concluded that they probably contained an amine other than DA. In favor of such a duality of CCL cells in the rat are the observations of Lempinen (1964), who showed that paraaortic nonchromaffin tissue in the newborn rat became chromaffin under the action of hydrocortisone, which caused an increase in phenylethanolamine N-methyltransferase (PNMT) and adrenaline. With electron microscopy simple mophological observations may provide informationon the nature of catecholamines in a cell, since clear differences have been established by Coupland and Hopwood (1966) between NA- and ”
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adrenaline-containing cells in the adrenal medulla after glutaraldehyde fixation followed by osmium tetroxide. Under these conditions, adrenaline-containing granules are of low electron density, whereas NA-containing granules are of high electron density. This distinction should also be applied to CCL cells. Unfortunately the caSe of DA was not examined. 3. Immunocytochemistry Interesting evidence on the nature of CCL cell catecholamines has been inferred from the localization of synthesis enzymes of catecholamines by immunocytochemistry, using specific antibodies prepared against these enzymes: tyrosine hydroxylase (TH), DOPA decarboxylase (DDC), two enzymes common to all catecholamine-containingcells; dopamine P-hydroxylase @-/3-H), which catalyzes the transformation of DA into NA and therefore is present in NA- and adrenaline-containing cells; and PNMT which catalyzes the transformation of NA into adrenaline and is restricted to adrenaline-containing cells. Using the immunofluorescence technique, Fuxe et al. (1971) did not find D-P-H in the CCL cells of the rat SCG, while the enzyme was demonstrated in the principal neurons. Thus these cells cannot synthesize NA, and it was concluded that they contained DA. This result was confirmed and extended to CCL cells of the coeliac ganglion by Hartman (1973). However, D-/3-H was observed in CCL cells of the male and female rat pelvic ganglia (Rybarczyck et al., 1976; Baker et al., 1977), using the peroxidase-antiperoxidase visualization technique (Sternberger, 1973). An extensive study was made on ganglia of the guinea pig by Elfvin et al. (1975). In the SCG, CCL cells exhibit a reaction with anti-TH and anti-D-P-H, but not with anti-PNMT (as some of the adrenal cells do), suggesting that they contain NA. In the inferior mesenteric ganglion, many CCL cells react with anti-D-P-H and are presumed to contain NA, but some react with anti-PNMT, and these do contain adrenaline. These interpretations are in agreement with the biochemical data of Crowcroft et al. (1971), who found NA and adrenaline in this ganglion in a ratio of 4:1, and no DA. Thus, according to the comparison between the fluorescence method micrographs and those obtained with immunohistochemical reactions, it seems that DA, NA, or adrenaline may be present in CCL cells, and perhaps sometimes there are two compounds stored in the same cell (Elfvin et al., 1975). 4. Uptake and Storage Properties In as much as CCL cells are interneurons (Williams, 1967), it was interesting to test their properties of uptake and storage of exogenous catecholamines and compare them to those of principal neurons. With the fluorescence method Libet and Owman (1974) observed loading by exogenous DA of CCL cells previously depleted by a-methyl-p-tyrosine. Using tritiated DOPA, DA, or NA, Taxi and Mikulajova (1976) did not obtain
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labeling of CCL cells after DA or NA, although labeling appeared over small granular vesicles within dendrites of principal neurons, showing clearly that there was no technical mistake. Only DOPA-3H gave moderate labeling of CCL cells. As it is generally accepted that granular vesicles are the storage site of catecholamines in the cell, it seems unlikely that DA is not stored in granular vesicles if it enters the CCL cell, since it is stored in the small, dense-cored vesicles in the neurons. Thus it is suggested that the lack of labeling is due to the fact that DA (or NA) is not taken up by the CCL cells membrane, or is taken up in very small amounts. However, an uptake of catecholamines by adrenal chromaffin cells was established biochemically (Iversen, 1975), and recently labeling of mouse adrenal cells by DA was obtained by Hirano et al. (1977). Probably these discrepanciescould be related to the catecholamineconcentration, which is unknown at the cellular level. The difference between uptake properties of CCL cells and principal neurons could be evidenced only at a low concentration, involving the uptake one of Iversen (1975).
5 . Varia Monoamineoxidase activity was estimated to be low in CCL cells by Costa and Furness (1973). Kanerva et al. (1972) reported that SIF cells in the rat paracervical ganglion did not exhibit acetylcholinesteraseactivity, contrary to principal neurons. They have only a low nonspecific cholinesterase activity.
D. ACTION OF DRUGS ON CCL CELLS 1 . Reserpine A pronounced fall in the catecholamine content of rabbit paraganglia after reserpine treatment was first observed by Tominaga (1967) using the chromaffin reaction. However, Norberg (1965) noted that CCL cells were resistant to depletion by reserpine or metaraminol as ascertained by the fluorescence method. Jacobowitz (1967) obtained a decrease in fluorescence in rat heart ganglia after 3 days of treatment. Furness and Costa (1976) found a delayed partial loss of fluorescence after repeated injections, but even after four daily injections CCL cells were not completely depleted. In fact, quantitative evaluations by the fluorescence method seem highly questionable (Jonsson, 1971; Kopin et al., 1974). Van Orden et al. (1970) also observed only slight variations in the fluorescence of CCL cells or the electron density of their vesicular contents after one injection of reserpine in a dose sufficient for almost total depletion of principal neurons. The improved chromaffin reaction of Coupland et al. (1964) combined with a fluorometric measurement allowed Lever et al. (1976) to detect, after a
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single injection of reserpine, variations in amine content which were not detectble with the fluorescence method. The same conclusion was reached by Koslow et al. (1975) after mass fragmentography determination of the m i n e content of newborn rat CCL cells. A depletion of catecholamine after reserpine was also inferred from a significant reduction in the proportion of chromaffin-positive cells in rat coeliac-mesenteric ganglia (Santer et al., 1976). At the ultrastructural level, Mascorro and Yates (1971) reported a progressive reduction in the intensity of the vesicular contents after two or three daily subcutaneous injections of reserpine. They noted important variations in density from one granule to another, but the number of granules was much lower after three injections, and they assumed that all granules probably would be depleted with a longer treatment. These results are quite similar to those obtained by Yates (1963) for the adrenal medulla. 2. Inhibitors of Catecholamine Synthesis Treatment with an inhibitor of TH, a-methyl-p-tyrosine, produced in the rat SCG and inferior mesenteric ganglion a progressive loss of electron density in large vesicles within 24 hours (Van Orden et al., 1970). However, modifications are difficult to ascertain in fluorescence preparations. This result indicated a very low turnover time for catecholamines, contrary to that in principal neurons, which has been estimated to be about 24 hours. This conclusion was also made by Furness and Costa (1976). P-Methyl-m-tyrosine gives the same results as reserpine. Compound 48/80, a condensation product of p-methoxyphenethylmethylamine and formaldehyde, which is a potent histamine inhibitor, was reported by Behrendt et al. (1976) to have an effect on the ultrastructure of CCL cells of the rat SCG. They noted hypertrophy of the Golgi apparatus, an increase in the number of dense-cored vesicles, and an accumulation of such vesicles in cell processes, especially at afferent synapses. P-Chlorophenylalanine, an inhibitor of serotonin synthesis, was tested on newborn (Heym, 1975) and adult rat SCGs (Heym et al., 1974; Heym, 1976) with similar results. The fluorescence of CCL cells shifted from yellow to green. A gentle swelling of mitochondria was observed. Specific granules were decreased in number and sometimes almost completely lacking, but their morphology was not modified. These effects were assumed to be related to interference with the first step of catecholamine synthesis rather than to an effect on serotonin synthesis. 3. Guanethidine Guanethidine causes a loss of catecholamines from sympathetic ganglia and sympathetically innervated tissues (Sanan and Vogt, 1962). It produces chemical sympathectomy in newborn and adult rats (Jensen-Holm and Juul, 1970) and hyperplasia of CCL cells in the ganglia of newborn rats (L. Eriinko and EIidnko,
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1971) but apparently does not affect these cells in adult rats (Burnstock et al., 1971). With the fluorescence method L. E r h k o and Eriinko (1971) showed that after a 1-month treatment of newborn rats the number of principal neurons was dramatically decreased, whereas the size of CCL cells clusters was increased, and some micrographs suggest a proliferation of CCL cell processes. CCL cells appear three to five times more numerous than normal, suggesting proliferation by CCL cell division. This phenomenon does not occur in adult rats, in which ganglia can be destroyed by repetitive injections of guanethidine. There is some similarity between the effect of guanethidine on CCL cells and that of nerve growth factor (NGF) on principal neurons. Higher concentrations yield variable effects, probably because of a depletion of catecholamines. Results reported by Heym and Grube (1975) were at variance with the preceding ones. Short-term treatment (15 days) affects only mitochondria. After 30 days significant decreases in the number and size of dense-cored vesicles were noted. It was not determined whether the decrease in catecholamine storage was due to synthesis inhibition or blockage of the storage process. Guanethidine and disulfiram (tetraethylthiuram disulfide), an inhibitor of D-P-H, have similar effects, according to Heym (1976). In certain CCL cells granular vesicles decreased in number, whereas they increased in others, with a swelling of vesicles and an eccentric location of their dense core, whatever the fixative. This difference in response of CCL cells might be related to the existence of different types of CCL cells, as proposed by Lu et al. (1976). 4. Glucocorticoids The effect of hydrocortisone on chromaffin tissue of newborn rats was first described by Lempinen (1964), who showed that this drug prevented the normally intervening degeneration of paraaortic ganglia (organ of Zuckerkandl) in young rats. Costa et al. (1973) observed with the fluorescence method that extraadrenal CCL cells increased in number and developed processes in various ganglia where they were normally present in limited number. A detailed study was made on sympathetic ganglia of young rats by L. Eriinko and E r s o (1971, 1972b) and L. Eriinko et al. (1972). After a 5-day treatment with hydrocortisone, numerous clusters of highly fluorescent cells, interpreted as CCL cells, appeared everywhere in the rat SCG, giving a total increase about 10 times the normal number; these newly formed cells had no processes. Similar results were obtained in pelvic ganglia by Hervonen et al. (1972a), using another glucocorticoid, prednisolone. With electron microscopy granular vesicles were not found at first, probably for reasons related to sampling, but then Eranko et al. (1973) observed typical granular vesicles in presumably newly formed CCL cells. The number as well as the size of vesicles was increased in every CCL cell, compared to control ani-
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mals. It had been previously established that hydrocortisone stimulated the formation of adrenaline in the extraadrenal chromaffin cells which normally contained NA (Eranko et al., 1966), but there is no clear morphological correlate of this fact (ErBnko et al., 1973). More recently Liuzzi et al. (1977) showed that dexamethasone increased PNMT activity and adrenaline in CCL cells, unlike NGF, which affects only slightly the number of CCL cells. Hydrocortisone had no appreciable effects in adult rat ganglia (L. E r h k o and Erhko, 1972b). According to Ciaranello et al. (1973) no effect was detectable when the treatment was delayed until 12 days after birth. However, an increase in the fluorescence of SIF cells of the adult rat glomus caroticum was reported by Korkala et al. (1973). There are similarities in the effects of hydrocortisone and guanethidine, but these compounds do not act in the same way (L. E r h k o and Eranko, 1972b). Hydrocortisone seems to have a true inductive action on undifferentiated stem cells, as new CCL cells appear in areas where they are normally absent (L. E r h k o and Eranko, 1971; Hervonen, 1971), whereas guanethidine stimulates proliferation of already differentiated CCL cells or satellite cells, because new CCL cells are often tightly apposed on the surface of neurons (Kanerva, 1972a). Although the formation of large clusters of CCL cells suggests that division of CCL cells occurs, no mitosis was seen on electron micrographs (Hervonen and Kanerva, quoted from Hervonen et al., 1972a). Testosterone, estrogen and progesterone had no detectable effect on paracervical ganglia (Hervonen et al., 1972a). Also, Kanerva et al. (1972) were unable to confirm with the fluorescence method the earlier observations of Blotevogel (1925, 1928) with the chromaffin reaction, according to which there is a significant increase in chromaffin cells in the paracervical ganglion during estrus and at the end of pregnancy. Kanerva and Hervonen (1976) assume that there is an increase in the catecholamine content of certain cells which renders the chromaffin reaction perceptible in cells previously not reactive. 5. 6-Hydroxydopamine This drug destroys sympathetic terminals flranzer and Thoenen, 1967b; Thoenen and Tranzer, 1968) and, in newborn rats, sympathetic neurons (Angeletti and Levi-Montalcini, 1970). According to L. Er5nko and E r W o (1972a), 6-hydroxydopamine (6-OH-DA) in newborn rats does not affect the number, form, or fluorescence intensity of CCL cells. However, Kanerva (1972a,b) and Papka (1973) reported that at least some CCL cells were sensitive to a daily injection of 6-OH-DA during 1 week in newborn rabbits but not in adult animals. Kanerva et al. (1974) noted striking ultrastructural changes in SIF cells located near the capillaries, such as the swelling of endoplasmic reticulum and formation of large vacuoles in the cytoplasm. Moreover, the density of intravesicular granules was reduced, suggesting a drop in catecholaminecontent.
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E. CCL CELLSIN CULTURE If studies on cultured sympathetic ganglia are rather numerous, only a few concern CCL cells. Organotypic cultures from various sympathetic ganglia of newborn rats and guinea pigs were obtained by Chamley et al. (1972a,b), L. Eriinko et al. (1972), and Eranko et al. (1972a, 1973), using the same culture medium. They noted that CCL cells exhibited moderate fluorescence in the cultures, whereas principal neurons were not fluorescent. Chamley et al. (1972b) observed processes up to 300 pm in length and noted that CCL cells had limited property or potentiality of migration in contrast with principal neurons. Masurovsky et al. (1972), Benitez et al. (1974), and Murray (1976), using their own media, distinguished CCL cells from principal neurons in living cultures. With electron microscopy the morphology of CCL cells was rather similar to that found in vivo. However, E r h k o et al. (1972b, 1973, 1976) reported that CCL cells in culture showed large variations in the shape and size of granular vesicles; there were elongated, dense-cored vesicles, 40-150 nm in cross section and 150-200 nm in length, but the number of vesicles per cell was substantially lower than in vivo (Heath et al., 1973). This difference could be related to different culture media or different concentrations of glutaraldehyde used for fixation. Growth of CCL cells in culture does not require NGF (L. E r h k o and Eranko, 1972a), which is necessary for principal neurons (Olson, 1967; Aloe et al., 1975). However, NGF does not seem prejudicial to cultured CCL cells. Effects of various drugs were tested in organotypic cultures. Guanethidine, even at a low concentration (1 mg/liter) induced an increase in CCL cell number (L. Eranko and Eranko, 1972a). It was expected from the toxic action of guanethidine and 6-OH-DA on principal neurons that a pure culture of CCL cells could be obtained, but the concentrations needed in vitro for destruction of the principal neurons were too high and had a general toxic effect. Hydrocortisone causes an increase in catecholamine synthesis, as ascertained by the fluorescence method and by the number of CCL cells, which may occur by cell division or by the differentiation of immature forms. However, the number of large clusters suggests a division process (L. Eriinko and Eranko, 1972b; L. Eranko et al., 1972). Ultrastructural studies showed that the cytoplasm was crowded with dense-cored vesicles of larger size than those in the control cultures, especially in the processes (Erhko et al., 1973, 1976). CCL cells also survive 1 month or more in autografts and homografts of ganglia beneath the capsule of the testicle (Coujard et al., 1975). F. CCL CELLSAND DEVELOPMENT In developing sympathetic ganglia Owman et al. (1971) observed that catecholamine-containing cells always appeared as ‘‘SIF cells with short pro-
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cesses,” some of which become adrenergic neurons while others remained SIF cells, their processes becoming shorter or disappearing. The recognition of CCL (SIF) cells in newborn animals may be difficult with fluorescence microscopy, because at this stage their size is not different from that of principal neurons, nor do their staining properties differ in semithin sections. Only their disposition in clusters near the vessels allows us to distinguish them, for instance, in the rat paracervical ganglion (Kanerva, 1972a,b). Principal neurons increase in size in the days following birth and discrimination becomes progressively easier from 2 weeks on, the more so because of a slight decrease in CCL cell size. The same state as in the adult is reached about 1 month after birth; with electron microscopy, the size of the granular vesicles is the same at birth as later (Kanerva, 1972a,b,c). However, Eranko (1972) indicated that CCL cells were easily distinguished in both fluorescence and electron microscopy in the newborn rat SCG, and that there were no intermediary forms. it has been established for a long time that chromaffii tissue is well developed at birth in many mammals and then decreases (Coupland, 1965b). In the rat (Lempinen, 1964; Costa et al., 1973) and in the rabbit (Bmndin, 1966) chromaffin cells were identified in several abdominal ganglia (superior mesenteric,suprarenal, and lumbar) at birth, which disappeared more or less completely several days later. This postnatal reduction was also noted by Gabella and Costa (1968). On the contrary, in the rabbit heart CCL cells are numerous at 29 days of gestation in the connective tissue and ganglia located near the origin of the main arterial trunks and seem not to regress later (Friedman er al., 1968). in the paracervical ganglia Kanerva (1970, 1971) observed that both degenerating and mitotic CCL cells were found in newborn rats but not later than 8 days after birth. According to de Champlain et al. (1970) and Owman et al. (1971), certain CCL cells could have been transformed into principal cells during this period. On the contrary 0. Eranko and E h k o (1971), E r h k o (1972), and Santer er al. (1975) noted than in newborn rat SCGs CCL cells were often solitary, in contrast to those of adult animals, which were frequently in clusters. In this material it seemed that CCL cells increased in number during the postnatal period. Division of a granule-containingcell was observed in a 6-day-old rat abdominal paraganglion by Mascorro and Yates (1970). Such divisions could explain the formation of clusters after birth. They could occur even later if CCL cells have properties similar to those of adrenomedullary cells, which were seen dividing 60-80 days after birth (Malvaldi et al., 1968). The ultrastructure of developing mouse abdominal paraganglia (Viragh and Kodnyi Both, 1967) is similar to that of the adrenal medulla of the rat embryo (Diner, 1965) and to that of adult CCL cells of the rat SCG, while in the adrenal medulla the dense-cored vesicles are larger. Becker (1972) noted that the endoplasmic reticulum was more developed in CCL cells of very young mice. Santer
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et al. (1975) observed that the chromaffinity was not the same in embryonic as in adult CCL cells. They were not able to obtain a positive reaction in embryonic CCL cells with their improved chromaffin method, which stains adult CCL cells. This failure was attributed to an insufficient concentration of catecholamine or an unstable mode of storage. The development of CCL cells in sympathetic ganglia of the rabbit was followed by Papka (1972). CCL cells were recognized in 14-day-old fetuses (the total duration of fetal development is 31 days in the rabbit) by their granular vesicles, 12-14 nm in diameter at this stage and then 15-18 nm. The endoplasmic reticulum is well developed at this time and is progressively reduced. Cell processes are already developed. These observations differ from those of Coupland and Weakley (1968) in which the size of vesicles was constant in CCL cells (6.5 nm) and increased in adrenal cells. Synaptic connections, afferent or efferent, were not encountered. In the rat heart Owman et al. (1971) pointed out that CCL cells were the first aminergic structures. At this stage they had processes, which disappeared later on. According to Dail and Palmer (1973) in the human fetal heart CCL cells are always associated with nerve trunks; they are first (8-13 weeks) more numerous in the adventitia of large vessels (aorta, pulmonary artery, . . . ) and then (17-18 weeks) more numerous in the interatrial septum. Recently, Tennyson and Mytilineou (1976) identified ultrastructurally precursors of SIF cells and other paraganglionic cells (including adrenomedullary cells which soon migrated out of the sympathetic chain) by their size and vesicular contents as early as 11-12 days of gestation, but they were not distinguishable from principal neurons by fluorescence microscopy at this stage.
G . RELATIONSOF CCL CELLS WITH
SURROUNDING STRUCTURES
1. Sheath Cells
Isolated or clustered CCL cells are usually covered by a sheath of cells similar to those of perineuronal glia. This sheath is not continuous, but lacking at various locations on the cell surface, on the cell soma or on a process. At these sites the CCL cell membrane is separated from the intercellular medium only by a PLATE VI. FIG. 1 1. Inferior mesenteric ganglion of a guinea pig; the animal was injected with 5-hydroxydopamine. A synapse between a noradrenergic axon (NA) and a chromaffin cell body (C) is shown. Note the presence of humerous granulated vesicles in the presynaptic axon, the presynaptic dense projections, and the postsynaptic dense bars (arrows). X75,000. (From Furness and Sobels, 1976.) FIG. 12. Inferior mesenteric ganglion of a guinea pig. Ultrastructural evidence for the extrusion of granules from chromaffm cells. Two granulated vesicles (arrows) appear to have fused with the membrane of the chromaffin cell and to be in the process of releasing their contents into the pericapillary space. X60,OOO. (From Fumess and Sobels, 1976.)
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basement membrane, which is in continuity with that which always covers the outer glial surface. Such a situation is very infrequent in the principal neurons of sympathetic ganglia (Elfvin, 1963; Taxi, 1965), but it occurs rather commonly in the parasympathetic neurons of Auerbach’s plexus (Taxi, 1959). The naked surface was estimated by Yokota (1973) to be about 1.2% of the total surface. Becker (1972) reported a layer of dense cytoplasm appearing as a membrane thickening at this level. Attachment plaques were observed by van der Zypen (1974) between sheath cells and CCL cells in the rat heart. 2 . Interrelationships between CCL Cells When CCL cells are in a cluster, they are frequently in direct apposition, separated by a cleft 20 nm in width, without interposed glia. Surfaces directly apposed to another CCL cell were estimated by Yokota (1973) to average 25% of the total cell surface. Some specializations appear along these contacts. At some sites the cleft is increased up to 0.8 pm, and interdigitations (microvilli) develop (Matthews and Raisman, 1969; van der Zypen, 1974). Frequently there are also attachment plaques (also called desmosome-like structures), characterized by symmetric dense layers against the internal face of each apposed membrane, and often a line of dense material in the middle of the cleft (Plate V, Fig. 10). In the SCG of the rat the attachment plaques may represent from 1.7 to 2.3% of the cell surface (Yokota, 1973). However, they were rarely found between SIF cells of cardiac ganglia, according to Papka (1976). Becker (1972) and others mentioned desmosomes between SIF cells or between SIF cells and their sheath cells but, since the cleft did not exceed 20 nm at this level, these structures must be called attachment plaques. Extension of the attachment plaques caused Furness and Sobels (1976) to wonder whether these structures could be responsible for electrical coupling between SIF cells. Such an hypothesis presently lacks physiological support. 3 . Afferent Synapses
There were contradictory conclusions from studies with silver impregnation, which are well summarized in Coupland’s book (1965b), several of them strongly suggesting innervation of paraganglia. With electron microscopy, synapses are identified by the presence of synaptic vesicles in the presynaptic endings, associated with membrane differentiations forming synaptic complexes (Palay, 1958) or active zones (Couteaux, 1961). According to these criteria, there are obvious afferent synapses on CCL cells (Williams, 1967; Siegrist et al., PLATE W. Rat SCG. Afferent synapses on CCL cells. FIG. 13. Attachment plaques are well developed along the contact. X41,OOO. (From Taxi and Mikulajova, 1976.)Frcs. 14 and 15. The postsynaptic densities along the membrane are less developed than the presynaptic dense patches. Fig. 14. X40,OOO. Fig. 15. X20,OOO.
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1968; Matthews and Raisman, 1969) exhibiting the main features of the preganglionic synapses of the sympathetic neurons as described by Elfvin (1963) and Taxi (1965), which are known to be cholinergic. However, they have some peculiar characteristics. The synapses on CCL cells are predominantly axosomatic. This is just the opposite of the synapses of principal neurons, which are almost exclusively axodendritic at least in the SCG (Elfvin, 1963, 1971; Taxi, 1957, 1965). This distribution probably has some physiological significance in the case of principal neurons; in CCL cells, the processes are much less developed, and synapses occur preferentially on somata perhaps only because the surfaces available for making contacts are larger. Attachment plaques between presynaptic endings and CCL cells are much more frequent than in ganglionic synapses (Matthews and Raisman, 1969; Yokota, 1973; Taxi and Mikulajova, 1976) (Plate VII, Fig. 13). This can be related to the length of the contacts, which are often longer than in ganglionic synapses, and also to a peculiar ability of CCL cells to form attachment plaques, as seen in contacts between CCL cells themselves. Yokota (1973) also noticed that there were frequently two or more synaptic complexes per contact, whereas there was generally only one per ganglionic synapse. The functional meaning of these features could be ascertained by physiologists, but it can perhaps be explained merely by the larger size of the synaptic endings. Matthews and Raisman (1969) and Taxi and Mikulajova (1976) reported that membrane thickenings of the synaptic complexes were often symmetric (Plate VII, Figs. 14 and 15), the postsynaptic dense layer being rather poorly developed. However, Yokota (1973) found no differences between afferent synapses of CCL cells and those of principal neurons. It is possible that a difference in fixation can explain this discrepancy, inasmuch as the first investigators used osmium tetroxide fixation, while Yokota used aldehyde fixation. Also, confusion might have occurred involving the attachment plaques so frequent at these synapses. It seems quite unlikely that there are two types of afferent synapses on CCL cells. Matthews and Raisman (1969) noted that afferent endings often occurred in pairs, located closely on the same CCL cell and thus determining an afferent pole of the cell. But this idea does not seem to be of general application, as Yokota (1973) found in a semiserial section study that afferent and efferent synapses may be located near each other and certain cells may receive afferent synapses at sites far from each other. Williams and Palay (1969)emphasized that afferent synapses were not numerous, whereas Siegrist et al. (1968) found many of them. In fact, from our own experience there are large variations in their number in different CCL groups of the rat SCG. However, in a study of four CCL cells in semiserial sections Yokota (1973) observed that they were all provided with afferent synapses in a limited
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number (one to three per cell). This suggests that the terminal arborization of the afferent fibers is probably densely packed in certain zones. Their precise form is not known, nor is it known whether afferent fibers are specialized, certain ones innervating only principal neurons and others innervating CCL cells. It is not certain that CCL cells are innervated at every location. Thus Mascorro and Yaks (1973) did not find synapses on CCL cells of abdominal paraganglia in the Syrian hamster, although they observed vesicles containing nerve fibers in contact with CCL cells. This rather surprising situation may be related to the fact that they studied young animals (up to 10 days old). It may be that synapses are not yet fully developed at this age, and the development of innervation and its extension might be related in some way to the dramatic decrease in paraganglia from the embryo to the adult, which is general in mammals although occurring to a different extent according to the species (Section 1,F). According to Autillo (1977), SIF cells in clusters in the cat coeliac ganglion are not innervated; only isolated SIF cells are innervated. The origin of afferent synapses is well established (Taxi ef al., 1969; Matthews, 1971; Matthews and Ostberg, 1974; Dail and Evan, 1978a), as they completely disappear within a few days after division of the preganglionic trunk and are not found in organotypic culture (Benitez ef al., 1974). These observations rule out the possibility that presynaptic endings have a local origin, and it can be assumed that they are cholinergic fibers originating from the spinal cord like other preganglionic fibers. But it has not been established whether these fibers are of small diameter (C fibers) and endowed with slow conduction, as observed by Dunant (1967) in the preganglionic trunk of the SCG. Recently, Furness and Sobels (1976) and Furness and Costa (1976) described two types of afferent synapses on CCL cells of the inferior mesenteric ganglion of the guinea pig after injection of 5-hydroxydopamine, which reinforces the vesicular contents of NAcontaining fibers (Tranzer and Thoenen, 1967a). One type is similar to that described above, with asymmetry of membrane thickenings and clear vesicles; the endings are large, like those described for NA cells of the adrenal medulla by Grynszpan-Winograd (1974). The second type is characterized by vesicles containing a dense granule. Other specializationstypical of synapses are also present, sometimes including a subsynaptic dense layer (Plate VI, Fig. 11). The sizes of the dense-cored vesicles are larger and more varied than those of the empty vesicles of the presumably cholinergic fibers. The section of lumbar splanchnic and intermesenteric nerve causes the degeneration of all presumably cholinergic endings, but presynaptic areas containing dense-cored vesicles and thought to be noradrenergic remain intact. This suggests a local origin from noradrenergic neurons, which could be also concluded from the degeneration experiments of Mustonen and Teravainen (1971) and Becker (1972) on uterine (Frankenhauser's) ganglion. Only Watanabe (1971) believes that some of the afferent
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synapses are axo-axonic; this interpretation remains questionable, as it is not supported by precise illustrations. Apparently the origin of this second type of ending from another CCL cell was not taken in consideration by investigators. Acetylcholinesterase activity was localized in a discontinuous network between the CCL cells by Jacobowitz (1970) and Hervonen (1971). Although the existence of afferent innervation of CCL cells seems general, Muscholl and Vogt (1964) were unable to demonstrate an effect of the stimulation of afferent nerve fibers on catecholamine release from chromaffin cells of the inferior mesenteric ganglion of the dog. Moreover, the fluorescence of SIF cells remained unaffected after the division of preganglionic fibers ( E r M o and Harkonen, 1965a; Dail and Evan, 1978).
4. EfSerent Synapses These synapses were first described by Williams (1967) in the rat SCG, and his description was confirmed by Siegrist et al. (1968), Matthews and Raisman (1969), Williams and Palay (1969), and others. Their identificationwas based on the presence of groups of dense-cored vesicles associated with dense projections attached to the CCL cell membrane, and a postsynaptic membrane thickening facing the presynaptic dense-cored vesicles Plate VIII, Figs. 16 and 18). Siegrist et al. (1 968) reported an increase in the intercellularcleft to 15-20 nm at this level; in fact, these values are quite normal. The presynaptic element is either the soma or a process of a CCL cell. The postsynapticprofiles have been interpreted as dendritic branchlets of principal neurons by the above-cited workers, who considered that CCL cells were the interneurons of the rat SCG. The most peculiar feature of the presynaptic zones, in comparison with the synapses described elsewhere in the nervous system, is the varied size of the synaptic vesicles, the majority of which contain a dense granule. Their size varies usually from 40 to 80 nm. The smallest vesicles of the CCL cells are especially numerous, if not exclusively located at these presynaptic areas; they are identical to those described by Richardson (1962) in sympathetic fibers. Their preferential location rules out the possibility that they are tangential sections of larger vesicles (Plate VIII, Figs. 17 and 18). In certain contacts some synaptic vesicles appear empty and this would be related to some release process (Taxi, 1971). It was well established by Richardson (1966) that clear vesicles found in sympathetic fibers were only vesicles whose dense granule had not been preserved by fixation, perhaps because of a low concentration of catecholamine. PLATE VIlI. FIG. 16. Rat SCG. Efferent synapse between a CCL process and a presumed dendritic branchlet of a principal neuron. X 3 1 ,ooO.FIGS.17 and 18. Efferent synapses between a CCL cell process and presumed dendritic branchlets, showing the variety of dense-cored vesicles sizes at these sites. In Fig. 17 the vesicles are. comparable to the so-called small, dense-cored vesicles of noradrenergic neurons. Fig. 17. X38,500. Fig. 18. X32,OOO.
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Such a situation may also explain the empty vesicles in CCL cells, especially since CCL cells are considered to contain DA, and it is well known that, at least in the case of the nucleus caudatus (Hokfelt, 1968), DA does not usually give rise to a dense core in vesicles after conventional fixation for electron microscopy. Ivanov (1974) distinguished in the coeliac ganglion of the rat several types of efferent synapses according to the size of their vesicles, one type having only large, granulated vesicles. But it is difficult to be sure that they do not correspond simply to different planes of section of synapses containing heterogeneous populations of vesicles. A subjunctional body was seen by Matthews and Raisman (1969), but it seems that this organelle is infrequent. The presynaptic area is often a region of the CCL cell soma; Matthews and Raisman (1969) reported that, among 60 synapses, only 11 were undoubtedly formed by CCL cell processes. They observed that the dense-cored vesicles were always in a single row beneath the cell membrane, except in presynaptic areas where they were arranged in several rows. In most instances the postsynaptic elements of these synapses are small profiles lacking specific cytoplasmic features or connections which would allow determination of their nature using objective criteria (Siegrist et al., 1968; Taxi et al., 1969; Chiba et al., 1977). Although aware of this difficulty, Williams (1967) hypothesized that these elements were dendritic expansions of the principal neurons and thus that the CCL cells were interneurons of the sympathetic ganglia. Taxi et al. (1969) emphasized the lack of evidence concerning the neuronal nature of the postsynaptic element and described "synaptoid zones. " At present they have abandoned this position because of evidence on the neuronal nature of the postsynaptic element. For instance, Matthews and Raisman (1969) reported a postsynaptic profile directly related to a broader process, which was in continuity with a neuronal perikaryon. Matthews and Nash (1970) described a soma-to-soma contact with synaptic specializations between a CCL cell and a principal neuron (Plate IX, Fig. 19). In fact, such relations seem very rare. Yokota (1973) studied in semiserial sections the connections of four CCL cells, two of which were proved to have efferent synapses. The postsynaptic areas contain tubules and vacuoles of irregular size, and, exceptionally, small, dense-cored vesicles. One of these profiles was seen in continuity with a perikaryon of a principal neuron and thus can be identified as dendritic. Unfortunately the enlargement of the figures illustrating this fact does not allow all the details to be seen. Yokoya considered that certain postsynaptic profiles may represent terminal branches of recurrent collateral axons, because of the presence of small, dense-cored vesicles. But such vesicles are rather frequent within the dendrites of principal neurons (Taxi, 1965, 1973; Grillo, 1966; Taxi et al., 1969) and recurrent synapses have not yet been demonstrated unquestionably in the rat SCG (Taxi et al., 1969; Matthews, 1971). Thus it seems more probable that these processes are dendritic. They also could be interpreted as CCL cell processes, but this would be in opposition to the generally
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PLATE M.FIG. 19. Rat SCG. An afferent synapse between the perikaryon of a CCL cell (SC) and that of a principal neuron (PN). Scale represents 1 p m in (a) and 0.5 p m in @), which is an enlargement of the region outlined in (a), taken in an adjacent serial section. sat, Satellite cell cytoplasm; d, desmosome-like linkage; s-s, region of synapse. (From Matthews and Nash, 1970.)
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accepted view that there are no synapses between CCL cells (Siegrist et al., 1968). The Occurrence of efferent synapses seems to be limited to certain ganglia in certain species. In the rat they have been found in the SCG (see above), the coeliac ganglion (Ivanov, 1974), the major pelvic ganglia @ail et al., 1975; Dail, 1976), the plexus surrounding ovarian vessels (Payer, 1978), but not in the paracervical (Frankenhauser's) ganglion (Kanerva and Teravainen, 1972; Kanerva and Hervonen, 1976) nor in the same ganglion in the mouse (Becker, 1972). In the guinea pig such synapses were not found: mesenteric ganglia (Ostberg, 1970; Elfvin et al., 1975), hypogastric ganglia (Watanabe, 1971), the SCG (Elfvin et al., 1975), cardiac ganglia (Ellison and Hibbs, 1974). In the cat coeliac ganglion the interpretation of certain images as efferent synapses (Autillo, 1977) is questionable. In the monkey SCG, they were recently described by Chiba et al. (1977). It is recalled that isolated sections of CCL cell processes containing a mixture of clear vesicles of the synaptic type and large, dense-cored vesicles of moderate size cannot be distinguished from sections of preganglionic endings.
5. Reciprocal Synapses Reciprocal synapses were described between afferent nerve endings and CCL cell somata by Yamauchi el al. in rat (1975b) and turtle (1975a) heart ganglia. The synaptic complexes polarized in opposite directions were separated by less than 0.5 p m in the turtle and 0.3 pm in the rat, and several were found on one ending (Plate X, Fig. 20). This structure is rather frequent; Yamauchi et al. found that 16 out of 25 CCL cells in the turtle and 6 out of 23 in the rat had reciprocal synapses. The same structure has also been found in rabbit cardiac ganglia (Papka, 1976) and thus seems to be peculiar to the heart. Reciprocal, albeit not adjacent, synapses between axons of unknown origin and CCL cells were described by Kondo (1977) in the rat SCG serially sectioned. H. MORPHOLOGICAL EVIDENCE FOR ENDOCRINE FUNCTION CCL cells are often grouped near precapillary arterioles, veinules, or capillaries, especially fenestrated capillaries Eriinko and Harkonen, 1965b; Matthews and Raisman, 1969), the fenestrations of which are sealed by a single-layered membrane, as in the adrenal medulla (Elfvin, 1965). The vascularization of a CCL cell cluster was studied by the injection of an india ink-gelatin solution by Winckler (1969a) and by a fluorescent dye by Lever et al. (1976), who showed that there was a kind of capillary glomerulus associated with the cell cluster. The vascularity of CCL cells is more developed than that of principal neurons. Cell somata or processes abutting on pencapillary spaces are often devoid of a glial sheath (Furness and Sobels, 1976). In the inferior mesenteric ganglion of
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PLATE X. FIG.20. Turtle heart ganglion. Both top sections are serial sections of reciprocal synapses between a cholinergic axon (C) and a CCL cell (G). The C-to-G synapse is better seen in the bottom figure, in the middle of the contact; the G-to-C synapse is developed on the right of the top figure. X 18,700. Each bottom image represents half of reciprocal synapse polarized from CCL cell to cholinergic axon. X26,500 and 29,000. (From Yamauchi et al., 1975a.)
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the guinea pig, it is the only structure related to the release of the catecholamine from the CCL cells (Elfvin et al., 1975; Furness and Sobels, 1976). However, in certain ganglia, for instance, the paracervical ganglion of the mouse, Becker (1972) noted that the cell clusters were very closely associated with blood capillaries, which, however, were fenestrated. The relationships with fenestrated capillaries have been diversely interpreted. They may suggest an endocrine function (Matthews and Raisman, 1969; Chen and Yates, 1970; Jvanov, 1974) or a role of modulation in the chemoreceptive function (Biscoe, 1971; MacDonald and Mitchell, 1975), especially in the glomus caroticum but eventually at other locations. The hypothesis of an endocrine function of the CCL cells would be strongly supported by figures showing or suggesting release of the vesicular contents at the level of the plasma membrane. The micrographs easiest to interpret are those showing exocytosis. Unfortunately they are very rare under normal conditions. In several papers it was mentioned that no exocytosis was visible (Matthews and Raisman, 1969; Becker, 1972). However, in guinea pig ganglia, Elfvin et al., (1975) and Furness and Sobels (1976) interpreted inpocketings of plasma membrane facing a capillary and containing an amorphous material as the effects of exocytosis of vesicular contents by the fusion of vesicular and cell membranes (Plate VI, Fig. 12). More unexpected were the observations of Polonyi et al. (1976), who described intact vesicles in the intercellular space surrounding the CCL cells or within the lumen of certain vessels. The way such vesicles are released has not been established; it is not clear from the micrographs whether the whole vesicle is expelled or only its dense core, in which case it could be merely exocytosis. Recently, Matthews (1977) found clear images of exocytosis in the rat SCG (Plate XI, Fig. 21), which is under control of preganglionic stimulation. Such images are very rare in normal ganglia, and thus their role in ganglion function remains questionable .But it may only be due to the duration of the phenomenon and its frequency, which might render highly improbable to fix it. If micrographs of exocytosis are rare, there are other cytological images which can be related to the release of intravesicular material (Taxi et al., 1969; Taxi, 1973; Taxi and Mikulajova, 1976; Matthews, 1976). These structures appear as dense patches attached to the cell membrane (Plate XII, Fig. 22). Similar patches were first described along the presynaptic membrane of synapses in the spinal cord by Gray (1963). They are thought to be related in some way to neurotransmitter release (Akert et al., 1969). In CCL cells they are localized at efferent synapses (Plate VU, Fig. 14), but also in places where it is unlikely to assume a synaptic connection, inasmuch as the facing element, another CCL cell or a glial layer, does not have a postsynaptic “membrane thickening. These patches might play a role in the attachment of vesicles during the release of their contents, as they were presumed to do in synapses; they might also be interpreted as transient aspects of vesicles emptying through the membrane (Taxi and ”
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PLAm XI. Fro. 21, Rat SCG removed 12 1/2 hours after preganglionic denervation and fmed in 1% osmium teaoxide. In top section, what appears to be an extruded granule core is shown lying extracellularly between a CCL cell and its satellite cell wrapping (S)in anindentationofthe surface membrane of the CCL cell. Arrow indicates collagen fibers in transverse section. x60,750. Bottom section is an adrenal chromaffi cell from a rat perfused with aldehydes. The core of a chromflin granule is seen lying extracellularly. X42,OOO. (From Matthews, 1977.)
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PLATE XU. FIG.22. Rat SCG. A contact between two CCL cells. The thick arrow indicates two dense patches which are interpreted as structures related to a vesicular release phenomenon. The thin arrow indicates a vesicle close to the membrane X40,OOO.
Mikulajova, 1976). In all cases such aspects are probably related in some way to the release of vesicular contents. There are physiological arguments in favor of an endocrine role for these cells. Muscholl and Vogt (1964) correlated the high catecholamine contents of the venous effluent of the dog hypogastric ganglion with the presence of chromaffin cells in it.
1. CCL CELLTYPES The occurrence of several types of CCL cells was first proposed based on purely morphological criteria by Siegrist et al. (1968) in the rat SCG. They distinguished two types of CCL cells according to the predominant size of the vesicles they contained: (1) small vesicles, 100-150 nm in diameter, with a
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central dense core, and (2) large vesicles, 200-400 nm in diameter, with a dense mass lying against the membrane in the shape of a full, half-, or quarter-moon. These two types were found only after glutaraldehyde primary fixation; no variations in shape and size of vesicles appeared after osmium tetroxide fixation alone. Although each cell contained both types of vesicles, one type was usually predominant, defining a CCL cell type. Cells having more of the large granules were fewer. Because of their equal density afrer glutaraldehyde-dichromate treatment, both large and small vesicles were thought to store the same catecholamine, which was presumed to be NA, because the large granules were rather similar to those of the NA-storing cells of the adrenal medulla. The functional meaning of this difference in size of vesicles was not explained. In the paracervical ganglion of the mouse Becker (1972) distinguished two types of vesicular contents: (1) spherical granules, 50-250 nm in diameter, with highly dense contents, which lack certain small, clear vesicles; (2) spherical, ovoid, hemispherical, or scaled-shaped granules, 50-300 nm in diameter, in a more-or-less regular envelope, up to 450 nm in diameter, within which the granule is eccentric. The predominance of one of these types determines each type of CCL cell. In the rat paracervical ganglion Kanerva (1972a) also distinguished two types of CCL cells, one with 80- to 140-nm vesicles and the other with 200- to 300-nm vesicles, both containing larger granular vesicles 200-600 nm in diameter. In the hypogastric ganglion of the guinea pig, Watanabe (1971) recognized four types of CCL cells: (1) type I-round vesicles, 200-250 nm in diameter, with a spherical, ovoid, angular, often eccentric granule; (2) type 11-round vesicles, 100-150 nm in diameter, with a central or eccentric granule of high electron density; (3) type 111-polymorphous, elongated vesicles; and (4) type IV-round vesicles, 200-250 nm in diameter, with a granular or filamentous core of low-electron density. With reference to the differences observed by Coupland and Hopwood (1966) between adrenomedullary cells, type IV was presumed to contain adrenaline, and types I through I11 to contain NA. Watanabe (1971) wondered whether types I through I11 were not merely functional stages of the same type. In a comparative study of the rat SCG and coeliac-mesenteric ganglia, Lu et al. (1976) and Lever et al. (1976) distinguished three types of CCL cells: (1) type I-round vesicles, 50-150 nm in diameter, with a core of low to moderate density; (2) type 11-polymorphic vesicles, 100-300 nm in diameter, with a highly electron-dense core; (3) type III-elongated vesicles, average dimensions 170 x 50 nm, with a core of moderate to low density. Only type I is provided with efferent synapses. It is quite predominant in the SCG (93%), while it makes up only 32% in the coeliac mesenteric ganglion, as against 40% for type I1 and 28% for type III. Moreover, the same investigators (Lever et al., 1974; Santer et al., 1975) confirmed the fact that certain CCL cells give a modified chromaffin
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reaction with the well-known differences in fluorescence color, yellow and green. By a statistical analysis Kemp et al. (1977) concluded that chromaffinpositive cells probably corresponded to yellow cells. Electron probe analysis (Lever et al., 1977) showed that, among the three types of CCL cells, only type I1 cells gave peaks for chromium. These peaks appeared in cytoplasmic areas rich in granular vesicles but not in other regions of the cell. Inasmuch as NA is known to be more reactive than DA (Hopwood, 1971), it was proposed that type I1 cells contained NA, whereas type I and 111 contained DA. Type I1 cells greatly resemble NA-containing adrenomedullary cells as described by Coupland and Hopwood (1966). Only two types of granule-containing (CCL) cells were recognized by Elfvin et al. (1975) in the inferior mesenteric ganglion of the guinea pig, and by Dail et al. (1975) in pelvic ganglia of the rat. These types, which differ in the electron density of the vesicular contents, correspond to the adrenaline-containing and NA-containing cells of the adrenal medulla (Coupland and Hopwood, 1966). However, Elfvin et al. (1975) noted that many cells contained both types of granules, the NA type being more numerous, and they concluded that these cells probably contained NA and adrenaline; such a conclusion agrees with the biochemical data establishing the presence of NA and noradrenaline, but not DA, in this ganglion (Crowcroft et al., 1971). Besides the tentative distinction among CCL types according to the size and shape of vesicles and their contents, Chiba and Williams (1975) and Williams et al. (1975, 1976b) distinguished two types on the basis of their arrangement and connections as seen with the glyoxylic acid fluorescence method. Type I corresponds to isolated cells with long processes; they are interneurons. Type I1 corresponds to cells in clusters without processes; they are secretory cells. The proportion of both types is highly variable according to the tissue; for instance, in the SCG of different species the proportion of type I is: cat, 1.9%; rabbit, 74.5%; monkey, 48.5%; and ox, 24%.But these types do not seem applicable to the rat and guinea pig (Autillo, 1977). If we take into consideration all the points of view from which different types of CCL cells can be distinguished, it seems that the most fundamental difference concerns the efferent connections, which allows us to distinguish interneurons, or type I, from purely endocrine cells, or type 11, a kind of paraneuron (Fujita, 1976, 1977), designated as endocrine “brothers of neurons” (Fujita, 1976). Morphological definition of types is complicated by the obvious large differences among species, even those taxonomically not remote (e.g., rat and guinea pig): (1) type I, which predominates in the rabbit or rat SCG, probably contains DA in vesicles of moderate size, and (2) type 11, which predominates in the cat or guinea pig, probably contains NA and eventually adrenaline in vesicles of larger size and high electron density when containing NA .
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Other types described by different workers, as seen above, presumably correspond to functional stages of one type or the other, but much more experimental and comparative work is needed to reach clear, definitive conclusions on CCL cell types.
J. FUNCTIONAL ROLEOF CCL CELLS Most of the interest in CCL cells in the last decade came from the need for a structural basis to explain special features of the synaptic transmission in sympathetic ganglia, which are briefly summarized here. More complete accounts can be found in the papers of Laporte and Lorente de No (1950), Eccles (1952), Eccles and Libet (1961), Libet (1970), Volle (1969), Volle and Hancock (1970), Libet and Owman (1974) and Tosaka and Kobayashi (1977). In the sympathetic ganglia of certain mammals (rabbit), as well as in other vertebrates (turtle), the normal synaptic potential is followed by slow potentials. The usual synaptic potential, called an N (negative) wave when recorded extracellularly, or an excitatory postsynaptic potential (EPSP) is followed, at least in certain neurons, by a slow P (positive when recorded extracellularly) wave or a slow inhibitory postsynaptic potential (S-IPSP), and finally by an additional LN (late negative) wave or slow EPSP (S-EPSP). This triphasic sequence is obtained by stimulation of preganglionic fibers under certain conditions, or by the acetylcholine, whose action is blocked by antinicotinic drugs, while the generation of slow postsynaptic potentials is blocked by antimuscarinic agents. The intervention of a catecholaminergic step was demonstrated in various experiments after Marazzi (1939) observed the depressant action of adrenaline on postganglionic discharges. Later it was shown that the P wave was reduced by an inhibitor of a-adrenergic receptors, dibenamine (Eccles and Libet, 1961), and that catecholamines, especially DA, applied on sympathetic neurons gave a hyperpolarizing response similar to an S-IPSP (Libet and Tosaka, 1970). Other data were provided by the study of ganglionic cyclic AMP (CAMP), which increases during synaptic transmission (McAfee et al., 1971) and mimics the action of DA. Exogenous DA increases the CAMPcontent of neurons in the bovine SCG similarly to stimulation (Kebabian and Greengard, 1971; Greengard and Kebabian, 1974), which might be mediated by DA-containing CCL cells (Dermirjian, Aghajanian, and Kebabian, unpublished observations, 1976, in Kebabian, 1976). However, cAMP levels are also raised by NA, but in higher concentrations than DA (Kebabian, 1976). Similar experiments and pharmacological data showed that cAMP increased in the same manner as S-IPSPs in the rabbit SCG (McAfee, 1976). In contrast, in the feline SCG Black ef al.
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(1974) were unable to find a significant increase in CAMP when exogenous DA or NA was added. Libet and Owman (1974) established in the rabbit SCG very good correlations between variations affecting the DA content, which is localized in CCL cells, and the values of S-IPSPs. For instance, the depletion of DA by prolonged stimulation, by inhibition of catecholamine synthesis, or by the action of bethanechol (a muscarinic drug) reduced markedly and selectively the S-IPSP, which is restored by the addition of DA to CCL cells, as ascertained by the fluorescence method. Thus Libet and Owman (1974) provided strong physiological support for the hypothesis of Williams (1967), already reinforced by the data of Matthews and Raisman (1969) and Yokota (1973), that CCL cells are interneurons having an inhibitory “feed-forward” action (Yamauchi, 1976) on principal neurons following preganglionic stimulation. The neuronal functions of CCL cells were recently rendered still more plausible by the demonstration that even chromaffin cells of the adrenal medulla were able to generate action potentials (Brandt et al., 1976). But even in ganglia where interneurons exist, all CCL cells are not interneurons (Yokota, 1973). Other CCL cells are restricted to an endocrine function, which is suggested by images of exocytosis or more subtle relations of vesicles with the plasma membrane (Section II1,I). The secretion product is a catecholamine, DA, NA, or adrenaline, and perhaps two compounds are produced by the same cell (Elfvin et d.,1975); moreover, other components of vesicles are probably released together with the mine, as it occurs in the adrenomedullary cells. The secretion is controlled by afferent innervation when it exists (Matthews, 1977). This innervation can bring about a certain synchronization in the discharge of cells of one cluster or of different clusters of the same ganglion. The secreted substances may have a local action through mere diffusion, which is probably severely restricted by the uptake properties of the principal neurons. Further action may involve the blood vessels, as many investigators have mentioned the proximity of CCL cells to capillaries. However, Lever et al. (1976) indicated that CCL cells were not frequently directly apposed to pericapillary space without the interposition of collagen or other connective tissue elements; this arrangement is similar to that of the glomus caroticum and may be interpreted as suggesting a receptor function, as recently assumed by Yamauchi (1977). However, it is emphasized that the role of catecholamine-containing cells (CCL cells) as receptors in the glomus caroticum is still questioned by certain specialists working with this organ. As a general conclusion, it seems highly probable that CCL cells as interneurons are restricted to certain ganglia in certain species (such as the rat or rabbit). In any case, CCL cells are small in number (a few hundred versus several thousands of principal neurons). Inasmuch as each CCL cell has a limited number of efferent synapses (Yokota, 1973), even taking into account processes
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which contribute only a little to efferent junctions (Matthews and Raisman, 1969), it seems that interneurons are involved only in a limited number of special circuits within the ganglia.
IV. CCL Cells in Nonmammalian Vertebrates This article discusses only data based on the fluorescence method and electron microscopy. Data obtained with the chromaffin reaction were thoroughly reviewed by Coupland (1965b).
A. BIRDS
Although chick sympathetic ganglia were often used for organ or dissociated cell culture of sympathetic tissues, studies of these ganglia and especially of their CCL cells are limited. In their study of the development of the chick Enemar et al. (1965) observed with the fluorescence method in 6-day embryos highly fluorescent cells in the sinusoatrial region as well as sometimes in the pericardium, which were not identified as CCL cells but were presumably of this type. In the SCG (Bennett, 1967) and in the heart of the domestic fowl (Bennett and Malmfors, 1970) presumed CCL cells were infrequently observed as “bright, yellow fluorescent cells,” about 5-8 p m in diameter, similar to adrenal cells seen with fluorescence microscopy. With the electron microscope Wechsler and Schmekel(l966, 1967) found in a 4%-day embryo granular vesicles in the perikaryon and processes of all the cells of the primary ganglia. At 6-7 days, the structure of all sympathicoblasts is similar to that of adult CCL cells. CCL cells of a typical size and with typical processes were identified by Costa (1969) in adult sympathetic ganglia, beneath the capsule of the adrenal glands or within the periadrenal ganglia of several birds species (Peking duck, chaffinch). The possibility of intermediary forms between typical chromaffin cells and neurons in the adrenal gland was raised by Unsicker (1976). He described groups of small polygonal cells having a high nucleus/ cytoplasm ratio, with concentrations of dense material along the nucleus membrane. Some contained granular vesicles 50-300 nm in diameter, greatly resembling A vesicles but of varying electron density. The question of the nature of such cells remains open, because they can contain granular vesicles or not. Moreover, Unsicker (1976) described several types of fibers, some of which might be CCL cell processes. Paravertebral ganglia of chick embryos were the first structures in which CCL cells were studied in organotypic culture (Lever and Presley, 1971). They con-
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tain dense-cored vesicles 160 nm in diameter and also a rich endoplasmic reticulum and numerous ribosomes. Organotypic cultures with different culture media were also studied by Chamley et al. (1972a,b), Benitez et al. (1973, 1974), and Hervonen and E r W o (1975). All these workers observed clusters of highly fluorescent cells which were somewhat larger than CCL cells of mammals, some of them having richly branched processes. In long-term cultures of dissociated sympathetic ganglia of 11-day chick embryos, Jacobowitz and Greene (1974) observed a constant proportion of about 5% CCL cells. Their processes grow slowly up to 500 p m in length. These workers thought they had obtained cultures of pure CCL cells using a medium without NGF, but it seems that sympathetic neurons of chick embryos can survive several days under these conditions and thus the identity of the cells considered CCL cells remains questionable (Burnham et al., 1972; Varon and Raiborn, 1972). According to Benitez et al. (1974) CCL cells can be distinguished from principal neurons among highly fluorescent cells because they resist depletion by reserpine or by a catecholamine synthesis inhibitor, a-methyl-p -tyrosine. Ultrastructural studies revealed that the cytoplasm of the cells considered CCL cells, which are in limited number in cultures, is crowded with dense-cored vesicles, but their nuclei are spherical and clear and they have a well-developed ergastoplasm (L. E r W o et al., 1972; Heath et al., 1973). Hydrocortisone in the culture medium causes a substantial (about 10-fold) increase in the number of highly fluorescent cells (Hervonen and E r W o , 1975; Santer et al., 1976). These cells have the same ambiguous ultrastructural features as mentioned above; in addition, their granular vesicles appear more polymorphic, especially by their varied size, than in control cultures (Eranko et al., 1976). This effect was observed in cultures of 12-day embryos and not in embryos younger than 9 days (Benitez et al., 1974; Hervonen, 1975). From all the observations made with fluorescence microscopy, as well as with electron microscopy, on normal and hydrocortisone-treated cultures of ganglia, it seems that in chick embryos there is a new type of cell, intermediary between principal neurons and typical CCL cells as they appear in mammals. Mitosis was observed in these cells in 9 to 12-day embryos (Cohen, 1974; Hervonen and Eranko, 1975). This could be related to a critical stage in the differentiation of sympathicoblastsinto CCL cells or neurons under the control of glucocorticoids which are first produced in 11-day embryos (Eranko et al., 1976).
B . REETILES There are only a few data on reptiles, restricted to the turtle heart. Numerous CCL cells were found in the sinus venosus wall in the heart ganglia of the turtle
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Pseudemys crypta elegans. They have the typical features of mammalian CCL cells (Chiba and. Yamauchi, 1973). Moreover, they form reciprocal synapses which were studied in serial sections (Plate X, Fig. 20) (Yamauchi et al., 1975a), and are identical to those of the rat heart (Yamauchi et al., 1975b) (Section III,G,5). In these ganglia vagotomy demonstrated that afferent synapses were of postganglionic origin, while preganglionic fibers terminating on principal neurons were of central origin. This disposition results in a feedback inhibitory action on principal neurons (Yamauchi et al., 1975b; Yamauchi, 1976).
C. AMPHIBIANS
1 . Urodeles In the Urodeles, it is well established that chromaffin cells are associated with sympathetic ganglia (see Coupland, 1965b). An ultrastructural study of the posterior subclavian ganglion was performed by Lentz (1967). In this ganglion, the central region is occupied by interrenal cells, and CCL cells are mixed with ganglionic neurons in the outer part. The CCL cells, enveloped or not by a layer of glial cells, are of two types. The first, more abundant type is characterized by vesicles containing highly electron-dense granules 150 nm in diameter. The other contains larger vesicles, 250 nm in diameter, containing a less dense granule. Rare CCL cells contain both types of granules. These granules seem to correspond, respectively, to noradrenaline- and adrenaline-containing cells of the mammalian adrenal medulla (Coupland, 1965a; Wood and Barrnett, 1964). These CCL cells are innervated by unmyelinated, preganglionic fibers. No intermediary types between CCL cells and neurons, nor CCL cells endowed with long processes were described. The heart of the mud puppy (Necturus maculosus) revealed to McMahan and Purves (1976) a fascinating type of CCL cells in the small parasympathetic ganglia located within the vagal branches or the wall of the sinus venosus. In these ganglia the proportion of CCL cells which were clearly identified as interneurons is especially high (one out of two or three cells). This proportion was evaluated directly in vivo by differential interference contrast optics or after a fluorescence reaction. As to the nature of the catecholamine, it was not determined whether it was DA, NA, or adrenaline. These CCL cells possess two to four processes ranging from 1 to 5 p m in diameter. They branch one to three times and reach a length of a few hundred micrometers. Usually the processes remain within the cluster of cells in which the perikaryon is located. There are varicosities along the processes, and often a terminal varicosity. It was demonstrated by electron microscopy that at least some of these boutons make specialized contacts with principal neurons. They correspond to less than 10% of the endings received by these neurons; they contain predominantly granular
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vesicles of various sizes and shapes, some of which are focused on dense projections attached to the plasma membrane, like the efferent synapses of CCL cells in mammals. Such endings may be located directly on the cell body of a principal neuron. Moreover, close membrane appositions interpreted as gap junctions occur at sites along the contacts between CCL cells and principal neurons. Afferent synapses on interneurons are infrequent and localized on processes. The contacts are smaller than those of principal neurons (0.5-1 pm) and have a typical appearance, but with rather symmetric membrane specializations (cf. Section 111,G, 3). 2 . Anurans Groups of small cells were identified as chromaffin cells in toad (Bufo vulgaris japonicus) ganglia on the basis of their ultrastructure (Fujimoto, 1967) and in the pararenal (paraaortic or coeliac ganglion and coeliac plexus) of Bufo arenarum (Piezzi and Rodriquez-Echandia, 1968). Honma (1970) in B . vulgaris japonicus and Jacobowitz (1970) in the bullfrog Rana catesbiana, using the fluorescence method, found CCL cells in the ganglia of the paravertebral chain and their connective sheath. Weight and Weitsen (1977), in an analysis of 10 ganglia in serial sections, found a maximum of 13 clusters of CCL cells, (a total of 64 cells) in the bullfrog. It was emphasized that CCL cells were especially numerous in the region near the initial portion of the coeliac artery (coeliac plexus), confirming classic observations with the chromaffin reaction (Coupland, 1965b). CCL cells are also numerous in the sympathetic ganglia (V, VI, VII) close to this region, according to the observations of Hill et al. (1975) made on the frog Limnodynastes dumerilii. CCL cells with processes were rarely seen, only in the sheathes of ganglia (Jacobowitz, 1970; Hill et al., 1975; Taxi, 1976) (Plate XIII, Fig. 24). However, they are probably not constant in all ganglia or in all individuals, since Norberg and McIsaac (1967) did not find them in the last dorsal ganglia of Rana temporaria. They seem absent from frog heart (Angelakos et al., 1965). CCL cells are 10-20 prn in diameter. They are usually in clusters of variable size (Plate XIII, Fig. 23), sometimes isolated, and located within a ganglion or frequently within its sheath. Hill et al. (1975) distinguished four types of CCL cells in the Australian frog L. dumerilii: 1. Type I (the most numerous) contained dense-cored vesicles, 60-700 nm in diameter, in the cell body and the processes; the vesicular contents were highly electron-dense, separated from the vesicular membrane by a narrow, clear halo; continuities were seen between smooth endoplasmic reticulum and vesicles partially filled with a less electron-dense material. 2. Type I1 contained vesicles of comparable size, but the contents were of variable electron density, usually lower.
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PLATE Xm. Sympathetic ganglia. Ram esculema. Fluorescence method. FIG.23. General view of an abdominal ganglion, showing weakly to moderately fluorescent principal neurons and some clusters of CCL cells. x 115. FIG. 24. Two CCL cells with processes within a frog SCG X260.
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3. Type I11 contained vesicles 100-300 nm in diameter, often elongated; the contents were of variable electron density, with a more-or-less wide halo. The smooth endoplasmic reticulum was well developed, and the vesicles less numerous than in the preceding types. It is suggested that this type corresponds to functional stages of type I or 11. 4. Type IV cells were isolated or in small groups. Each cell had its own glial sheath, not shared with other cells of the same type, as usually occurs in clusters of other CCL cell types. Granular vesicles were only 100-150 nm in diameter and spherical to elongated in shape. The cytoplasm contained parallel arrays of rough endoplasmic reticulum, that is, small Nissl bodies. In their special study of this type of cell, Watanabe and Burnstock (1976) distinguished two types of processes, axons and dendrites. The axons were smaller in diameter and contained densely packed microtubules, microfilaments, and granular vesicles, whereas the dendrite contents were similar to those of the perikaryon. In the vicinity of these cells, they distinguished two types of vesicle-containingfibers. One contained clear vesicles mixed with larger dense-cored vesicles, as in preganglionic endings (Taxi, 1965). The other contained only large, dense-cored vesicles and were considered processes of type IV cells. The first type of fibers made synaptic contacts with type IV cells very similar to those located on the principal neurons. The similarity of these cells to neurons is striking, being greater that of any type of CCL cell described elsewhere. They could be interpreted as neurons more differentiated in their ability to store biogenic amines or as an intermediary step between neurons and chromaffin cells.
In their recent study Weight and Weitsen (1977) distinguished only two types of CCL cells, which corresponded to two types of staining in semithin sections with toluidine blue in 1% sodium borate solution; one type was blue and the other green. It seems that these two types correspond to types I and I1 of Hill et ul. (1975), although the measured vesicle sizes were rather different in different species and correspond to the two types present in the adrenal medulla. The most intriguing fact is that no afferent synapses were found on CCL cells in R. catesbiuna, whereas they were found in other species (Fujimoto, 1967; Taxi, 1976) (Plate XIV, Fig. 25).
3 . Functional Role It is remarkable that thus far the situation has appeared rather different in urodeles and in anurans, but urodeles have been much less studied, and the ganglia studied were not homologous in both groups. In urodeles, the role of CCL cells as interneurons is well established morphologically (McMahan and Purves, 1976), although afferent endings appear less frequently than efferent synapses. In anuran sympathetic ganglia, no fluorescent pericellular apparatus nor efferent synapses from CCL cells onto sympathetic neurons were seen; thus
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PLATE XIV. FIG.25. Sympathetic ganglion in R . esculenru. View of apart of a CCLcell, showing the main cytoplasmic organelles and an afferent synapse at the upper left. x32,OOO.
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CCL cells cannot be considered interneurons. This does not necessarily imply that they are not involved in the formation of S-IPSPs recorded in certain neurons (Tosaka et al., 1968). Anuran ganglia are especially suitable for study of the late potentials in ganglionic transmission, because unlike the situation in mammals the S-IPSPs and the S-EPSPs are not produced in the same neurons but in two different types, called B and C (Nishi et al., 1965). Moreover, the S-IPSPs differ from those of mammalian sympathetic neurons in their longer latency, which might be related to diffusion of a catecholamine to receptor sites of the neurons. However, an antagonist of a-adrenoreceptors, dibenamine, which blocks S-IPSPs in mammalian ganglia, has little effect in the frog (Libet et al., 1968). A direct hyperpolarizing action of catecholamines was nevertheless observed on B cells as well as on C cells (Tosaka et al., 1968), whereas under normal conditions S-IPSPs are produced only in C cells and are antagonized by atropine (Libet et al., 1968). Considering these facts, the absence of efferent synapses (Weitsen and Weight, 1973) and their own observations on the properties of S-IPSPs, Weight and Pajden (1973) proposed that CCL cells were not involved in the formation of S-IPSPs in the anuran ganglia. They may have only a general effect on the excitability of all neurons, and other effects of a diffuse endocrine gland, although several investigators reported that their associations with blood vessels did not seem especially close, as they are in mammals. However Tosaka and Kobayashi (1977) considered that CCL cells play a comparable role in the ganglia of Amphibians and Mammals.
D. FISHES Very few data on CCL cells in fishes have been published since the review of Coupland (1965b). With the fluorescence method some CCL cells were observed along the course of large blood vessels within the wall of the large intestine of the trout by Read and Burnstock (1968). Recently, Campbell and Gannon (1976) described CCL cells isolated or in clusters within the splanchnic nerve and the splanchnic plexus extending between the coeliac artery and the gut wall. Many of these cells bear branching processes. In elasmobranchs it is interesting to note that granular vesicles containing cells, very similar in ultrastructure to the cells discussed in this paper, appear devoid of catecholamines as ascertained by the chromaffin reaction or fluorescence technique (Selmer Saetersdal et al., 1975). The question arises whether these cells, which receive afferent innervation, are precursors of the cardiac CCL cells observed in air-breathing vertebrates.
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E. CYCLOSTOMES The autonomic nervous system of cyclostomes is not differentiated anatomically. Data available to date concern exclusively the heart. Ganglion cells and nerve fibers spreading within the wall of the sinus venom of the lamprey were described by Augustinsson et al. (1956) using silver impregnation; a part of these cells, the smallest ones, were chromaffin and could be considered as CCL cells, which were recognized long ago in this material (see Coupland 1965b). The fluorescence method yielded images very similar to those of SIF cells of the rat SCG, with well-developed processes (Bloom et al., 1961; Dahl et al., 1971; Lignon and Le Douarin, 1978). In electron microscopy large dense-cored vesicles were observed in the cytoplasm of these cells by Ostlund et al. (1960), by Dahl et al. (1971) and Shibata and Yamamoto (1976) in the adult lamprey, by Ostlund et a2. (1960) and Leak (1969) in the hagfish, and by Lignon and Le Douarin (1978) in the ammocoete. In none of these works were the presence of nerve elements in a more or less close association with CCL cells and/or relationships between CCL cells and myocardic cells reported, and thus it seems likely that CCL cells in cyclostomes heart are purely endocrine.
V. Concluding Remarks There is a striking diversity in the shape and connections, and probably in the functions, of CCL cells located within the autonomic nervous system or in close association with it. The present state of research is still far from covering all the vertebrate groups equally, and the variety of the situations already described suggests that a more extensive analysis will again reveal new models. This rather complex situation probably reflects a very high sensitivity of sympathicoblaststo certain environmental factors, such as hormones, neurotransmitters, and other unknown factors. The sympathicoblast has several possible means of differentiation: it can become a typical neuron, a type of chromaffin cell (adrenaline-containingcell, NA or DA) or an intermediary cell more or less enriched in organelles specialized for the storage of catecholamines. Presumably the differentiationdepends on very subtle variations in the environmentalfactors intervening in due time. Precise conditions are probably then required for the maintenance of each newly differentiated cell, since a regression of chromaffin cells takes place in certain regions while CCL cells are maintained or multiply elsewhere. The sensitivity of the cells to factors influencing differentiation is emphasized by the fact that, in certain species, modified chromaffin cells having long processes containing vesicles of the synaptic type were recently described in
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the adrenal medullary gland, among typical adrenaline- and NA-containing cells (Unsicker, 1976; Unsicker et al., 1978; Kobayashi and Coupland, 1977; Kobayashi, 1977). Another interesting fact was reported by Olson (1970), who observed that adrenal cells grafted into the anterior chamber of the eye grew processes within denervated smooth muscles. The result is that various CCL types can comprise various systems of more-orless precise control of neuronal activity in sympathetic and parasympathetic ganglia. Only in certain ganglia of some species are CCL cells integrated in “feed-forward” circuits (Yamauchi, 1976), which control a limited number of principal neurons. These circuits coexist with a more diffuse hormonal action, originating from the same cells and/or other CCL cells. Thus there are at least two types of influences on ganglionic activity (not taking into account the special case of reciprocal synapses in certain heart ganglia); several hypotheses were proposed by Williams et al. (1976a) to explain the modalities of CCL cell intervention in ganglionic transmission. The variability among species is very surprising. How can it be predicted that there is more similarity between the circuitry of the cardiac ganglion of the mud puppy and that of the rat SCG than between rat and guinea pig SCGs? This may correspond to either extremely precise control of the excitability of certain ganglionic neurons, based on some unknown physiological requirements or, on the contrary, to an overlapping of two redundant processes without any clear gain or disadvantage in functioning. The CCL cells are included in the so-called amine precursor uptake and decarboxylase cells, a concept introduced by Pearse (1969, 1976). The concept of paraneurons, recently proposed by Fujita (1976, 1977), emphasizes that these cells are related to neurons by origin, metabolism, and connections. The existence of morphological intermediaries emphasizes the relationship of CCL cells to the sympathetic neurons, which themselves have morphological claims (Taxi et al., 1969; Taxi, 1973) for a release of noradrenaline at certain places of the surface of the dendrites and/or of the perikaryon, as CCL cells do for NA, DA, or even adrenaline.
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INTERNATIONAL. REVIEW OF CYTOLOGY, VOL. 57
The Synapses of the Nervous System A. A. MANINA Laboratory of Cytology, Institute of Experimental Medicine, USSR Academy of Medical Science, Leningrad, USSR
. . . . . . . . . . . . . . . . . . General Principles of the Synapse Ultrastructure . Classification of Synapses . . . . . . . . . A. Chemical Synapses . . . . . . . . . . B. Electrical Synapses . . . . . . . . . .
I. Introduction
11. The Mechanisms of Synaptic Transmission
III. IV.
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V. Structural-Functional Properties of the Synaptic Contact . . . . VI. The Role of Neurospecific Proteins . . . . . . . . . . . VII. The Role of Glycoproteins . . . . . . . . . . . . . . VIII. The Enzymic Activity of ATPase and Adenyl Cyclase Reactions in Synapses . . . . . . . . . . . . . . . . . . . . . A. The Activity of ATPase . . . . . . . . . . . . . . B. The Activity of Adenyl Cyclase . . . . . . . . . . . IX. Autoradiographic Investigations of the Synthesis of Biopolymers in the Synapses . . . . . . . . . . . . . . . . . . . . X. The Structural-Functional Features of the Axospinal Apparatus . References . . . . . . . . . . . . . . . . . . . .
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I. Introduction A unique property of nerve cells is the ability to form interneuronal contacts (synapses) while still retaining their structural individuality, and to transfer information. Development of the principal elements of all systems of the brain and the organization of its neuronal connections are genetically determined and programmed in embryogenesis, that form a precise junction of nerve endings (synapses) with the corresponding parts of different sections of the central nervous system (CNS) (Sperry, 1959, 1971). The nerve cells of the CNS are differentiated at very early stages of development (Attardi, 1963) and making connections with millions of other neurons or their processes. The formation of such connections is possible only during development of the CNS. Neurons form connections which are genetically determined. The same mechanism is likely to be responsible for the involvement of the synapses which participate in the transmission of information by micropotentials arriving at the presynaptic mem345
Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-364357-0
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brane. These neuronal connections are formed with the participation of neurospecific proteins involved in synaptic chemical transmission, that is, enzymes participating in the synthesis, metabolism, and inactivation of the transmitter“biosynthetic enzymes or satellite enzymes” (Blaschko, 1971). Catecholamines serve as hormones or transmitters-satellite proteins. Together with ATP they participate in the recording, storage, and reproduction of information. During the stimulation of adrenergic neurons satellite protein (chromogranin A) was found to accumulate in the neuron body, localized in the amine-carrying particles (Geffen, 1969; Hopwood, 1968). According to Blaschko (1971), chromogranin A is an oligomer. During CNS development many interneuronal contacts are formed, and oligomers are involved. During this period any hindrance can interfere with the development of brain connections and result in irreversible defects. The experiments of Gaze (1970) and Jacobson (1970) on the transection and subsequent regeneration of the retinal ganglion showed that ganglion cell neuroblasts obtained a chemical coding for their exact tectal disposition within 10 hours of differentiation. The critical period coincides with the time of mitotic differentiation of neuroblasts developing into retinal ganglion cells. In mammals the regenerative property of brain tissue at the cellular level is completely lost after this period. Throughout life, however, the human memory continues to accumulate information as a result of the appearance of new functional associations (e.g., during conditioning) due to activation of the available neuronal structures (synapses). The chemistry of the complex synaptic membranes is determined by the neuron genome (Crain, 1970). In vitro as well as in vivo, beginning at the neuroblast stage, the synaptic cleft substance and postsynaptic specializations gradually become differentiated in the process of ontogenesis which is intimately related to functional development. In the process of functioning each neuron, by means of a synapse, makes chemical contacts with other nerve cells, involving a great number of neuronal assemblies. This system maintains the dynamic state of the organized brain structure. Eccles (1973) and SzentAgothai (1972) call this state the trophic resonance of a mutually penetrating specific chemical communication process. Homeostasis of synapses (Katz and Miledi, 1967) is maintained through micropotentials independent of the impulses arriving at the axon. Information is transferred from one area of the brain to another, and from one neuron to another, along multiple connecting channels, the synapses, involving the simultaneousactivation of thousands of nerve cells. This article describes the principal subcellular characteristics of nervous system synapses. Some of them are considered in detail, while others are only briefly mentioned because recent reports are available, for example, on the genesis of the synaptic vesicles (Gray, 1976) and on the characteristics and role of transmitters (Gray, 1974; Iversen et al., 1973; Elfvin, 1977).
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11. The Mechanisms of Synaptic Transmission Standard nervous impulses are the only means for transferring all information about external stimuli obtained by the receptor system of an organism. For example, auditory cortex neurons receive acoustic information in several switching steps. Anokhin (1975) and Kostiuk (1975) have shown that, after receiving a signal the nerve cell becomes excited and transmits the signal further. There are about 200 such neurons. They form a network which receives and differentiates acoustic signals. The longest latent period of excitation determines the maximum time the network is active. It is, however, the first wave of excitation which causes inhibition of about 60% of the neurons; the percentage of inhibited cells increases as the wave spreads. It is as if the inhibition concentrates the sound signal in the group of excited neurons, preventing it from spreading. In the
FIG. 1 . Axodendritic synapses containing synaptic vesicles (SV) that differ in StmctllTe and function, as well as glycogen granules (G).
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cerebral cortex excitation and inhibition are coordinated and their unity organizes the structure of the neuronal network concerned with the analysis of stimuli arriving from outside. In this connection we believe that the active zones of synapses possess a universal genetic mechanism responsible for the coordination of excitation and inhibition in the activation and synthesis of the corresponding transmitter biopolymers and macromolecules. This is confirmed by the role of the adenyl cyclase system. It has been established that cyclic AMP (CAMP),present in the synaptic membranes of the CNS, exerts an excitatory influence. The inhibitory effect is affected by guanylate cyclase and cyclic GMP (cGMP), as well as by synapses localized in certain portions of membranes responsible for the depression or involvement of the genes related to the synthesis of inhibitors (Whittaker, 1965). The function of adenyl cyclase in synaptic transmission is dealt with in Section VIII, B. Such an approach prevents division of the CNS synapses into excitatory and inhibitory types based on the shape of the synaptic vesicles and the type of mediation and other features, since in the brain there are synapses containing simultaneously all kinds of vesicles (Fig. 1) and their intermediate structural and functional forms (Manina, 1971, 1976, 1978). Some investigators suggested that excitation and inhibition not be explained only in terms of the activity of the synaptic membranes or the functioning of vesicles containing a transmitter (O’Brien etal., 1972; Cartaud et al., 1973; Elfvin, 1977; Angaut and Sotelo, 1973). Some workers classify synapses as excitatory or inhibitory according to the type of mediation: purinergic, having ATP as a transmitter (Burnstock, 1972), monoaminergic, (Dahlstrom, 1973), and absorbing labeled glycine (natus and Dennison, 1972). However, this is in disagreement with the ideas of physiologists about the mechanisms of higher nervous activity and excitatory and inhibitory processes in the CNS. Multisynapses are a reflection of the integration of synaptic activity in the cerebral cortex at the subcellular level (Fig. 1). Electron micrographs show that the dendritic branches, while performing an integrating function in the cerebral cortex, form a very complicated system of connections involved in receiving and processing vast amounts of information arriving simultaneously. The active zones of synapses are excited, and other zones are inhibited, when the synaptic cleft substance is distributed homogeneously and hinders the directional spread of a nerve impulse. In the postsynaptic area the reception and transmission of information are accompanied by energetic and metabolic processes; the activation of protein synthesis (polysomes) and the conformational transformation of mitochondria are clearly seen (Fig. 2). A new approach to this key problem of synaptology is proposed by neuromorphologists and neurophysiologists on the basis of new developments. Anokhin (1975) has substantiated new interpretations of the mechanisms of synaptic transmission differing from those of classic neurophysiology,according to which excitation potentials are summed on the neuronal surface. This process is based
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FIG.2. Axodendritic synapses of the rat motor coltex in different structural-functionalstates. In the center the dendrite (D) has a large mitochondrion (m). ribosomes (r), and polysomes (p). On the dendrite there are axon endings (S) with synaptic vesicles (SV) and mitochondria (m).
on structural-functionalunity and integrated neuronal activity. Excitation, conduction, and the processing of information are interrelated in the ultrastructures of neurons and synapses. Of greatest importance in these reactions are subcellular changes in the active zones of the synapse, especially postsynaptic specialization. Schmitt ef al. (1976) propose that previous concepts, according to which neurons transmit information one way when integrative capabilities are focused at the axon hillock, should be revised. The main p i n t s of their hypothesis are as follows. 1. The dendrite is not only a positive receptor but can also transmit information to other neurons, acting as a presynaptic synapse for other dendrites. Such neurons form many connections involving a wide network in the information process. Information is transmitted to other brain regions by projection neurons (neurons with long axons).
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2. The transmission of information between neurons is associated with the propagation of spike action potentials. It has been established that small, graded changes in the potential of one neuron can synaptically influence the electrical activity in other neurons. The interaction distances in the dendritic network are measured in micrometers, millimeters or centimeters being used for spike propagation. Neurons may interact ephaptically without the help of a spike, with a membrane potential less than a millivolt changing the synaptic transmission (Bloom et al., 1970; Burt, 1971). 3. Rapid bivalent transport and biochemical signals between neurons of the brain provide metabolic support and carry out the transmission of information between neurons. In the interaction of neurons in the region of the nervous system devoted to spike-mediated and controlled long-distance transmission, information is processed electrotonically through graded changes in the membrane potential. The resulting electrotonic currents are transmitted through dendrodendritic synapses along short axons, as well as by aphaptic means (Schmitt et al., 1976). These new interpretations are in complete agreement with our electron micro-
FIG. 3. Quantum release of mediator in the axodendritic synapse takes place only in certain portions of the synapse zones (arrows). Histochemical reaction to cholinergic drugs. pre, Presynaptic pole; post, postsynaptic pole; SV, synaptic vesicles; SC, synaptic cleft; S, synapse. (Iawayama, 1968.)
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graphs showing, in portions of the cytolemma, possible contacts between the neuron and its dendrites that are both synaptic and ephaptic (Bogolepov, 1975; Manina, 1976). At the present time the chemical theory of synaptic transmission is generally recognized. It is based on quantum release of the mediator (Fig. 3) from synaptic vesicles and its interrelation with receptor macromolecules on the postsynaptic membrane @e Robertis, 1959; Gray and Whittaker, 1962; Eccles, 1966; Katz, 1968; Lenkov, 1972; Zakusov, 1973). Katz (1968) believes that release of the mediator from synaptic vesicles is controlled by highly specialized structures present on the internal surface of the presynaptic terminal. Bernhard and Schade (1967) have established that an impulse can induce excitation of one neuron and inhibition of another. Accordint to Tauc and Gerschenfeld (1962) postsynaptic membranes within the same ganglion may have a response opposite that of acetylcholine. Attempts to determine the nature of the morphological and neurophysiological mechanisms of synaptic transmission are currently in progress.
111. General Principles of the Synapse Ultrastructure All the chemical synapses of the CNS consist of three discrete regions: presynaptic and postsynaptic areas, and a synaptic cleft which is 25-40 nm wide. The presynaptic area contains synaptic vesicles and mitochondria. The synaptic cleft is limited by the presynaptic and postsynaptic membranes. The postsynaptic area contains dense fibrous or granular material (Gray, 1959). In combination with membranes at the site of contact of nerve endings it forms an osmiophilic zone called a synaptic complex (F‘alay, 1958). Couteaux (1961) referred to this region of the synapse as the “active zone.” It is here that the accumulation of syanptic vesicles occurs and the mediator is released. The term “active zone” is now generally accepted, and information is thought to be transmitted within this area. Synaptic densities of the synapse active zone may often be discontinuous.
IV. Classification of Synapses A. CHEMICAL SYNAPSES The subcellular classification of synapses that chemically transmit nerve impulses to the CNS (Gray, 1959) was based on morphological signs of their contact: length of the active zone, width of densities (filaments) in the area of pre- and postsynaptic membranes, width of the synaptic cleft, and shape of
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synaptic vesicles. Later this classification was supplemented. Much literature is devoted to the ultrastructure and function of synapses (Couteaux, 1961; Gray and Whittaker, 1962; Van der Laos, 1963; De Robertis, 1967; De Robertis et al., 1973; Eccles, 1966; Gray, 1959, 1974, 1976; Akert et al., 1971; Roitback, 1969; Peters and Walsh, 1972; Peters et al., 1972; Kostiuk, 1972; Lenkov, 1972; Anokhin, 1975; Bogolepov, 1975; Sakharov, 1975; Kositsyn, 1976), and it is useful to cite these findings again. It is noteworthy that different investigators classify synapses mainly according to the following features: (1) topographic interaction of the pre- and postsynaptic regions of the junction; (2) ultrastructural features referred to by Gray (1959) as type I and type 11: the shape of synaptic vesicles and the type of mediation in synaptic vesicles (e.g., cholinergic and adrenergic); (3) function-inhibitory or excitatory. Now such subdivisions are being criticized. Each of these groups can in turn have great morphological variety, although without principal distinctions in ultrastructure even in different organisms. The nervous system p&sesses all types of synapses. Schmitt believes that synapses formed by local short-axon chains in the cerebral cortex are related to higher neuronal activity.
B. ELECTRICAL SYNAPSES In lower vertebrates (fishes), as well as in invertebrates, there are functioning synapses that transmit nerve impulses electrically. Such synapses are characterized by a desmosomal type of junction; chemical mediators are not released here. They have close contacts without a synaptic cleft; their dense material and septa are regularly disposed and are 5-8 nm wide and 16-22 nm long (Gray, 1963; Bennett, 1972; Chalcroft and Bullivant, 1970). Based on their ultrastructure, such synapses are considered the inhibitory type. Such types of synapses are observed in the nervous systems of mammals: mice, rabbits, rats, monkeys (Hinrichsen and Larrarnendi, 1968; Sotelo and Palay, 1970; Pinching and Powell, 1971; Sotelo and Llinas, 1972; Sloper, 1972). Recent findings in this area have been reviewed by Elfvin (1977). However, it should be noted that the current division of synapses into excitatory and inhibitory types, as well as into chemical and electrical types, in the nervous system of vertebrates requires further clarification and should be correlated with their structural features.
V. Structural-Functional Properties of the Synaptic Contact According to modem views membranes of the active zones of brain synapses have a detailed submicroscopic organization of pre- and postsynaptic areas. They
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receive and transmit information and participate in the formation of memory engrams. Mediators, enzymes, and sialoglycoproteins, as well as neurospecific proteins, are involved in these processes. The active zones of synapses are the sites of sodium and potassium ion exchange. They have a complete set of glycolytic enzymes (Taranova, 1968; Barondes and Squire, 1972; Bosmann, 1972; Deutsch, 1971; Kosower, 1972; Hydkn, 1974; H y d h and Rdnnback, 1975; Donato, 1976; Tumanova, 1976). In synapses there are autonomic protein- and glycoprotein-synthesizingsystems. Three types of proteins are synthesized in the membranes of the synapse active zone (Bosmann, 1972; Barondes and Squire, 1972; Grasso et al., 1977a,b). These membranes are the site of incorporation of le~cine-~H, glu~osamine-~H, and methi~nine-~H within 30 minutes and within 1 hour after administration (Manina et al., 1975a, 1976a,b; Nadtochy, 1976). The synaptosomal complex, when functioning, shows dynamic ultrastructural changes. Proteolipid has been isolated from membranes of the synapse active zone. It is dispersed subsynaptically and is present only in the synapse active zone membranes, which distinguishes them from other membranes of the CNS. The conformation of this proteolipid is changed under the influence of substances acting on the synaptic transmission @e Robertis et al., 1973). Because of the position of the enzymes, fixed in the synaptic membrane, enzymic reactions have a vector character. Their regulation is associated with the specificity of the enzyme and mediator ultrastructure. The interaction between the mediator and the satellite receptor protein underlies the transmission of excitation through the synaptic membrane. A special molecular “superstructure” has been found which consists of molecules of receptor proteins fixed in the membrane of the synapse active zones and is capable of specifically intericting with the mediator (Vinnikov, 1971). The ultrastructural model of synaptic membranes (Singer and Nicolson, 1972) consists mostly of multimolecular complexes (O’Brien et al., 1972). Universal systems of neurofilaments perform integrative regulation in the synapse active zone. When contrasted with phosphotungstic acid, the membranes remain light, while all three areas of the synapse contain large basic protein and glycoprotein macromolecules and have structural-molecular specializations (Fig. 4).
On the inner surface of the synaptic membrane are neurofilaments forming dense projections (Pfenninger et al., 1969). Many investigators (Taxi, 1971; Taxi and Droz, 1970; Hebb, 1972; Akert and Pfenninger, 1970; Akert, 1973; F‘fenninger et al., 1969) showed that the presynaptic membrane of a nerve terminal was built of strictly regular hexagonal structures with holes in the center for neurotransmittersof any nature to reach the synaptic cleft. According to some investigators (Pfenninger, 1971;Akert, 1973), the synaptic vesicle, with a neurotransmitter, is formed in several steps. On the
FIG.4. Histochemical characteristics of active zones of a synapse (S). PTA. (a) General view of the synapse. @) In the presynaptic area neumfilaments (no form dense projections (pv). pre, Presynaptic pole; post, postsynaptic pole.
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presynaptic membrane there is a “thorn” which breaks the synaptic vesicle. The thorn may contain proteolytic enzymes. They specifically digest the protein-lipid membrane of the synaptic vesicles, releasing their contents into the synaptic cleft. The synaptic cleft is 15 nm wide and contains granular material consisting of macromolecules of basic proteins with gangliosides. They show dynamic plasticity during the functioning of the synapse (Bloom and Aghajanian, 1968; Pfenninger, 1971; Kositsyn, 1973, 1976; Manina, 1976; Manina et d.,1976a). The myosin-like proteins of the synaptic vesicle membranes may interact during this process (Streit et al., 1972; Glebov and Kryzhanovski, 1975). This is in agreement with data on the localization of contractile proteins in synaptic membranes and their cytochemistry (Puszkin et al., 1972; Berl et al., 1973; Walter and Matus, 1975; Hydkn, 1974). The outer membrane of the postsynaptic pole has a system of neurofilaments covered with a glycocalyx. The neurophilaments run from the membrane to the synaptic cleft, being as long as 5 nm, and control the structural-functional relationships (Fig. 5 ) . They can block calcium channels, inhibiting the influx of
FIG.5 . A synaptosome of the rat cerebral cortex. The neurofilaments running from the postsynaptic membrane are directed into the synaptic cleft (arrows). pre, Presynaptic pole; post, postsynaptic pole. FTA.
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mediator. This relationship is very strong. The synaptic contact cannot be broken even by centrifugation (Fig. 6). Another system of neurofilaments is formed by the inner membrane of the postsynaptic pole and consists of inwardly running components of a subsynaptic network, 50 nm in length. The neurofilaments are channels about 8 nm diameter. This is the site where the synaptic receptor apparatus is concentrated. The bismuth-iodine-uranyl lead method has demonstrated that the cleft has two layers of electron-dense material separated by a 2-nm-wide band (Akert et d.,1972; Pfenninger, 1973). Each of these layers has a fluffy lining adjacent to the synaptic membrane. Regular, dense bands or filaments crossing the cleft alternate with a light zone. They penetrate into the postsynaptic cleft from the postsynaptic membrane. Neuraminidase and histochemical methods with lanthanum hydroxide, ruthenium red, and ferrous hydroxide were used to reveal a discrete mosaic localization of carbohydrate groups in the synaptic complex (Fig. 7). These groups are composed of glycoproteins and gangliosides built into the membranes (Leninger, 1968). They form a glycocalyx cover for the outer surface of the synaptic membranes and their filaments. These carbohydrate-containingbiopolymers of the synaptic membranes serve as acceptors for certain mediators (Wiegandt, 1972). The increase in the polysialogangliosidefractions (GTand G,) of synapto-
FIG.6 . A synaptosome of the rat cerebral cortex with two zones of activity. At the presynaptic pole there are electron-denseprojections, and at the postsynaptic pole there are neurofilaments (nf); the synaptic cleft (SC) is limited by light membranes; the synaptic contact is not disturbed. M, membrane; pv, dense projections.
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FIG.7. The topography of the discrete mosaic distribution of glycoprotein groups (the glycocalyx) on the outer surface of the synapse membranes in the cerebral cortex of the rat. pre, &synaptic pole; post, postsynaptic pole; gx, glycocalyx.
somes during stimulation and learning in animals observed in our laboratory might be associated with the activation and increase of the receptor function of the post-
synaptic membrane, which is in agreement with electron micrographs of postsynaptic specializations (Nadtochy, 1976). Thus the synaptic contact in the active zones of the synapse is manifested in the chemical continuity of the oriented macromolecular groups of basic proteins and gangliosides which make up the subsynaptic contact and form a common integrated structural-molecularsystem of subsynaptic units (see Fig. 2). They are formed by dense projections of the synaptic pole, neurofilaments, and macromolecules of the synaptic cleft and the postsynaptic pole with its receptor zone. Changes in the conformation of the subsynaptic unit are dependent on the functioning of the synapse and its activity. During excitation of the synapse neurofilaments and charged macromolecules of the synaptic cleft substance provide the passing of a mediator to the postsynaptic pole. In an inactive state neurofilaments and macromolecules of the synaptic cleft form an amorphous mass which prevents passage of the mediator.
VI. The Role of Neurospecific Proteins The integration of information transmission with the active zone of the synapse is controlled by structural-molecular mechanisms with the participation of the
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neurospecific S-100 protein and glycoproteins. The neurospecific S-100 protein is found only in the brain. Hyden (1 974), using microsurgicaltechniques, has revealed neurofilaments on the inner surface of the neuron membrane. Their network is closely connected with the postsynaptic membrane and contains Ca2+-activated ATPase. The neurofilaments are capable of undergoing contraction and relaxation. The S-100 protein comes into contact with calcium ions, the neurofilaments relax, and the synaptic cleft diminishes in size, facilitating the conduction of a nerve impulse through it. If calcium ions are bound to a actomyosin-like protein of the neurofilarnents, they contract and enlarge the size of the synaptic cleft, making passage of the impulse more difficult. Thirty percent of the amino acid S-100 protein is acidic (glutamic and aspartic acids) (Hyden, 1974; Hyden and RBnnback, 1975). Approximately 90% of the S-100 protein is dissolved, and about 10%is bound with the membranes of the neuron and synapses, where it is mosaically distributed on their outer surface. S-100 protein possesses molecular heterogeneity (Viazovaya et al., 1975). It has been established that S-100 protein activates RNA polymerase of neuron nuclei but does not exert an analogous influence on other organs (Donato et al., 1975). It is possible that the main function of S-100 protein is to participate in controlling the gene activity of nucleic processes in the transcription of neurons including synapses and to code for specific information (Klementjev et al., 1975; Donato and Michetti, 1974). Hyden (1974) believes that S-100 protein combines with histones in DNA molecules and involves some portions of DNA in transcription by DNAdependent RNA polymerase. The acid S-100 protein may combine with basic histones. The histones leave DNA molecules to free sites on structural genes for the reception of DNA-dependent RNA polymerase. The S-100 protein in the nuclei is a depressor in the transcription of information related to memory. This problem, however, must be further investigated. The detection of S-100 protein in the animal brain is possible only during the development of functional connections in the CNS. Its absence during embryogenesis (Hyden, 1974; Donato et al., 1975; Stewart, 1975) indirectly confirms the relation of s-100 protein to information and learning. Koppiak et al. (1976) showed by an immunological method that the antiserum to S-100 protein and the synaptic membranes is incorporated at the sites of formation of memory engrams, changing the EEG pattern. It may be assumed that antibodies specifically block the sites on the surface of the synaptic membranes where S-100 protein is distributed mosaically, limiting the recording of engrams. Our investigations have established that the neurospecific S-100 protein is present in nerve endings of the cerebral cortex, making up 15-20% of all available low-molecular-weight protein. In complex synaptic membranes the neuro-
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specific S protein is also present, though in a smaller quantity than in synaptosomes. Of considerable interest is the fact that in the process of learning the quantity of S-100 protein is changed both in synaptosomes and in active zones of the synapse. During stimulation of the CNS with Phenamine, and during learning, the number of protein bands in fractions of heavy synaptosomes increases from 9 to 12. Additional protein bands move along with the acid proteins to the anode. Disc microelectrophoresis in capillaries has revealed two types of specific changes: (1) the appearance in the motor cortex of a band of protein with a mobility equal to 0.42 in experiments with conditioning, which was not found in controls. It may indicate a high regional specificity and a chemical character of protein synthesis in learning. It cannot be attributed to neurospecific acid proteins of the brain but seems to be a neurospecific 14-3-2 protein (Grasso et al., 1977a,b) involved in the fixation and storage of memory engrams; (2) the appearance of considerable molecular heterogeneity in the zone of acid neurospecific proteins in the region of migration of S-100 protein. This fact, being related to rearrangement of the protein apparatus of neuronal elements during the period of consolidation of memory traces, might be associated with an increased level of functional activity in the neurons and synapses (Manina et al., 1975a, 1976a; Turovsky et al., 1976a,b). Our findings on the anode mobility of S-100 protein are in agreement with those of other investigators (Shapiro and Vinuela, 1967) and show its specific role in addition to the synaptic modulation function. Thus, according to modem views, S-100 protein is an important component involved in the specific regulation of synapses and in coding information.
VII. The Role of Glycoproteins Glycoproteins as well as neurospecific proteins play a substantial part during conditioningof the brain and serve as connectors between individual neurons and their chains in controlling specific functions in the reception and synthesis of biogenic amines and are also acceptors of mediators. Glycoproteins are believed to perform an important function in forming memory engrams during conditioning (Bogoch, 1968; Barondes and Squires, 1972; Bosmann, 1972). Specific biopolymers (lop-glycoprotein, a-glycoprotein) have been isolated only in the brain during the formation new connections and are not found in other organs. Glycoproteins of the synaptic contact contain many carbohydrate chains which are rich in highly acidic sialic acid residues. They control specificity and molecular recognition during synaptic regulation in ontogenesis. Bogoch (1968) and Barondes (1968) believe that enzymic changes in terminal glucoside and fucoside groups are responsible for the plasticity of synaptic membranes in the consolidation of information.
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Synthesis of sialo-containing biopolymers is evidenced by the presence of enzymes (sialidase, sialyl transferase, and sialyl synthetase) in their membranes, as well as by the incorporation of radioactive precursors by a synaptosome fraction (Maccioni et al., 1971; Bosmann, 1972; Manina, 1976; Manina et al., 1976a; Nadtochy, 1976). Data are available on the relation of sialogangliosides (SGs) to noradrenaline (Robinson, 1963), which confirm their possible role in the transport and reception of mediators of synaptic transmission. The synaptosome fractions isolated from the rat cortex can bind a~etylcholine-'~C when incubated in vitru. Acetylcholine is set free under the influence of neuraminidase which splits off the sialic acid from gangliosides. The amount of SG in fractions of the synaptic endings correlates with that in acetylcholine (Lapetina and De Robertis, 1968), serotonin (Wolley and Gommi, 1965), noradrenaline (Robinson, 1963), histamine @e Robertis et al., 1973), and y-aminobutyric acid in synapses ( h w d e n and Wolf, 1964; Kuhn and Wiegandt, 1963). These data suggest that sialo-containing biopolymers of the synaptic membranes can act as receptors for biogenic amines. SGs occupy a strategic position in the receptor zones of postsynaptic membranes (Wolley and Gommi, 1965; Manukhin, 1968). Bondareff and Sjostrand (1969) found sialic acid among biopolymers of the synaptic cleft. This was confirmed by electrophoretic mobility studies (Bosmann, 1972). SGs are involved in the mechanisms of transport and release of mediator in the synapse (Tumanova, 1976). In the synapse active zones paramembrane structures in the synaptic subunit system fulfill a specific function in diffusion of the transmitter substance through the synaptic cleft (Jones and Brearley, 1972; Lenkov, 1972). Electrophoretic analysis of gangliosides has shown that in synapses they contain tri-, di-, and mono-SGs. Their fractional composition is greatly changed after stimulation with Phenamine. In the heavy fraction of synaptosomes an additional (sixth) fraction of mono-SGs is found. Light synaptosomesretain their fractional composition and increase the mono-SG content (in the third and fourth fractions), probably as a result of the transformation of tri- and digangliosides. We have observed the rearrangement and transformation of the external paramembrane layers (the glycocalyx) which are associated with shifts in the location of saccharide portions of gangliosides and glycoproteins fixed in the synaptic membranes. Stress leads to changes in the distribution of SGs on the outer surface of membranes of the synaptosomal complex and spine apparatus (Turovsky et al., 1976b; Nadtochy, 1976) (Fig. 7). At present it is established (Cotman and Taylor, 1974; Turovsky et al., 1976a) that the synaptic cleft substance, which is glycoprotein in nature, can serve as a molecular sieve for the ion stream and quanta of neurotransmitters as far as the receptor on the postsynaptic membrane, hence the polymers of the spine apparatus. In learning during the period of consolidation of trace processes a distinct increase in the number of glycoproteins can be seen in phoregrams of the
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a
FIG.8. Densitograms characterizing the electrophoretic mobility of glycoprotein zones in the rat visual cortex. normally (a) and in learning (b).
visual cortex (Fig. 8). The transformation of supramolecular paramembrane structures of the synapse glycocalyx was observed in the same animals (Gilerovich et al., 1977). Glycoproteins as well as neurospecific proteins are of great importance when the brain undergoes conditioning.
VIII. The Enzymic Activity of ATPase and Adenyl Cyclase Reactions in Synapses During intensive metabolic and information-acquiringprocesses the impulse activity of the CNS synapses increases depending on their involvement in functioning, followed by intensification of enzymic reactions of ATPase and adenyl cyclase. A. THEACTIVITYOF ATPASE It is known that in nerve tissue, especially at high energy expenditure, ATP hydrolysis increases. The energy of the phosphate bonds formed in the course of
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biochemical reactions is used for different intracellular transformations (Straub, 1965; Nakao, 1974). Nerve tissue contains magnesium-activated ATPase fulfilling special functions (Kharchenko et al., 1973; Daniel and Guth, 1975; Agafonov, 1976). [Detectionof ATPase activity is based on the formationof an electrondense sediment of lead phosphate resulting from an interaction between the residue of phosphoric acid removed from ATP (due to the enzyme reaction) and lead ions in the presence of K +,Na+, and Mg2+ (Sabatini et al., 1968). ATP is used as a substrate. Control experiments have shown that no reaction takes place when the incubation medium does not contain ATP.] ATPase activity is closely related to the formation of pre- and postsynaptic potentials of different functional significance and to the transsynaptic exchange of macromolecules. Morgan and Gombos (1976) found that the activity of transport ATPase at the synaptic contact was 10 times higher that in the neural plasmalemma. Investigations in our laboratory have shown that in synapses the level of ATPase activity is characterized by great diversity; the ATPase activity and the topographic distribution of the reaction product are different in different synapses. In one group of synapses the presynaptic pole does not contain the reaction product at all; in another group there are solitary granules of lead phosphate; in the third group-less numerous-the concentration of the product is more pronounced.
FIG. 9. The intensity of a reaction to ATPase in the synapses of the neuropile of the rat brain; accumulation of the reaction product in the postsynaptic pole (post). pre, Presynaptic pole.
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The synaptic cleft is almost devoid of lead phosphate granules. The extremely high activity of ATPase, comparable in concentration to that in the neural nucleolus only, is observed at the postsynaptic pole; the reaction product is concentrated on the inner membrane, in the postsynaptic filaments of the receptor zone, and in its specializations. A considerably increased concentration of lead phosphate granules, as compared with normal in all structures of the postsynaptic field, is observed under CNS stress conditions caused by stimulating neurotropic drugs (Etipirol, Phenamine), and in learning (Fig. 9). The process of learning is responsible for the two-fold increase in ATPase activity in subcellular fractions of the synaptic membranes of the cerebral cortex. The relationship between the concentration of the reaction product and the structural-functional state of the synapse can be determined. High synaptic activity is correlated with a more pronounced ATPase reaction, especially in axospinal synapses (Manina et al., 1976a; Turovsky et al., 1976b; Manina, 1977; Gilerovich et al., 1977). The increased ATPase in the postsynaptic area when the CNS is excited is probably associated with the increased flow of information and the intensified transmembrane function of synaptic apparatuses.
B. THE ACTIVITY OF ADENYL CYCLASE The role of oligopeptides in information transmission in the CNS is becoming increasingly obvious. Ungar (1973) showed that scotophobin formed in the rat brain is a connector. The regulatory function of adenyl cyclase is based on its interaction with the enzymes and proteins of the cell. It is performed by the universal adenyl cyclase system of the cell representing a protein oligomer which consists of two subunits. One of them contains a receptor center facing the extracellular space, and the other contains a catalytic one located on the inner surface of the membrane (Greengard et al., 1971; Greengard, 1976; Burkard, 1975; Brown and Stabrovskaya, 1974). Protein kinases are activated by binding of the nucleotide (CAMP) with its regulatory subunits. The synaptic function during the fixing of traces is controlled by the same mechanisms (Weiss and Crayton, 1970; Greengard et al., 1971; Greengard, 1976; Florendo et al., 1971; Kometiani, 1974; Brown and Stabrovskaya, 1974; Podoprigora, 1975). The neurotransmitterentering the postsynaptic membrane activates adenyl cyclase in the postsynaptic membrane, resulting in the synthesis of CAMP. It stimulates protein kinase which exerts stimulatingeffects on the phosphorylation of proteins of synaptic membranes. Under the influence of protein kinases of the nerve endings (Palladin et al., 1972) stimulated by CAMP the protein is split into separate oligopeptides, facilitating the processing of information and closing connections of neurons.
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FIG. 10. Activity of adenyl cyclase in the membranes of axodendritic synapses of the rat cerebral cortex; the reaction product granules are disposed on the inner surface of synaptic membranes (mows).
S-100 protein of the postsynaptic membrane may be a potential substrate, forming many specific oligopeptides capable of closing the connections of neurons concerned with the transmission of information in the brain (Greengard et al., 1971; Greengard, 1976; Tumanova, 1976). Our histochemical studies have shown the product of the enzyme reaction of adenyl cyclase to accumulate on the inner surface of the synaptic membrane, mostly in its active zone, which morphologically characterizesthe vector disposition of the receptor and active parts of the enzyme. The activity of adenyl cyclase when the CNS is excited in animals during stimulation and learning is sharply increased, especially at the postsynaptic pole (Fig. 10).
M. Autoradiographic Investigations of the Synthesis of Biopolymers in the Synapses Electron microscope autoradiography added new details to the structuralfunctional characterization of the synapses of the nervous system. In axonal terminals of the brain of different animals this method has revealed the presence of dopamine (Berry et al., 1975), glutamate (Evans, 1974), noradrenaline (Taxi and Droz, 1970; Prijmak, 1975), and serotonin (Ducros, 1975). The amount and spatial distribution of cholinergic receptors on the surface of the postsynaptic membrane of the neuromuscular synapse were established. The axonal current
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and its role in the renewal of protein macromolecules in nerve endings in the brain hemispheres were demonstrated (Schonbach and Chenod, 1973; Droz, 1973; Porter and Bernard, 1975). Gambetti etal., (1973) showed the transport of nucleotides, labeled with ~ r i d i n e - ~ H to ,synapses of the visual cortex. The injection of fructo~e-~H and gluco~amine-~H into a chicken has revealed the migration ofglycoprotein precursors to the synapses of the ciliar ganglion (Bennett, 1972). It was noted that increased synaptic activity was responsible for the renewal of a receptor protein, rhodopsin, in the retina (Krauhs et al., 1976). Our autoradiographic studies have revealed the local synthesis of protein and bioprotein biopolymers on the ultrastructure of the cortical synapses. The incorporation of label is observed within 20-30 minutes or 1 hour after the injection of methi~nine-~H and l e ~ c i n e- ~(Fig. H 11). The stimulation within the first 1-2 hours increases the intensity by 20%. The radioactive label of incorporated leucine shows that synthesis takes place in the pre- and postsynaptic areas of membranes of the active zone without the involvement of mitochondria (Fig. 12). The learning that occurs during conditioning causes some quantitative and qualitative changes in this synthesis (Nadtochy, 1976). The incorporation of l e ~ c i n e - ~and H glu~osamine-~H into different structures of dendrites and axons within 30 minutes or 1 hour after administration indicates
FIG. 1 1. Electron micrograph of a contrast autoradiographof a neuropile of the rat cerebral cortex (methi~nine-~ H); traces are disposed over the synapse ultrastructures ( m w s ) .
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FIG. 12. Electron micrograph of trace autoradiographs of brain synapses of the rat. (a) Incorpora(b) . Incorporation of glucosamine. tion of l e ~ c i n e - ~ H
Ftc. 13. Trace autoradiographs. Incorporation of label. (a) Gluco~amine-~H in the axoplasm. (b) L e ~ c i n e - ~in H the dendroplasm. A, Axon; D, dendrite.
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local synthesis of biopolymers (Fig. 13). The axocurrent transport of labeled biopolymers from the neuron perikaryon fails to reach the nerve endings, since the quantitative distribution of label in the cytoplasm, dendrites, axon, and synapses remains the same. This provides a possibility for instant acceptance and processing of the information on the interneuronal contact surface at any site on the axon and dendrite, which is especially important for a rapid change in excitation and inhibition. This does not exclude possible renewal of biopolymer macromolecules at the synapses by the transport of axonal flow. In the postsynaptic area the spine apparatus is labeled more intensively than the presynaptic pole-the labeling exceeding the level in the mitochondria. Experiments after incubation in vitro have demonstrated the predominance of l e ~ c i n e - ~in H the presynaptic area as compared with glu~osamine-~H. Thus our original data on the local synthesis of biopolymers of a protein and glycoprotein nature at the ultrastructural level of the synapse and the spine apparatus and their changes during motivation are of importance in local macromolecular homeostasis, flexibility, and the dynamic character of the synapse in information transmission and trace fixation.
X. The Structural-Functional Features of the Axospinal Apparatus Many investigators (Manina, 1971, 1975; Manina et al., 1976a,b; Kositsyn, 1973, 1976; Eccles, 1973; Anokhin, 1975) believe that the axodendritic synapses on the spine (axospinal synapses) are of prime importance in the performance of complex functions of integration and analysis. Ramon y Cajal (1890) was the first to discover spines and to show their important functional role. The spines represent spiculae of the dendrite, its fine terminal branches making contacts mainly with axons (Sukhanov, 1899; Poliakov, 1953; Sarkisov and Bogolepov, 1967). Gray was the first to describe spine ultrastructure in the visual cortex of rats. The axospinal synapse has all the components of the usual axodendritic contact (Manina, 1971, 1972; Kositsyn, 1973). The axospinal synapse with the spine apparatus, however, has specific features: an ultrastructural and chemical similarity of the subcellular organization of the spine apparatus to that of the subsynaptic field (Fig. 14); constant intimate relationships of the presynaptic neurofilaments with the spine apparatus; high morphological and biochemical specialization of the spine apparatus-its ability to synthesize proteins and glycoproteins locally both in vivo and in vitro (Fig. 15); plasticity of the spine apparatus-its ability to proliferate membrane complexes and specializations (Fig. 16a and b). Spine apparatuses develop from the membranes of the postsynaptic pole within the limits of such structures of the brain as spines Fig . 16c). The structural-functional plasticity of the spine apparatus during motivation is
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FIG. 14. An axospinal synapse with a spine apparatus in the motor cortex of the rat (in learning). One can see a dendritic terminal (D), a spine (Sp), a spine apparatus (SA), a presynaptic pole @re), a postsynaptic pole (post), and an active zone; structural and chemical homogeneity of the postsynaptic receptor zone, spine apparatus, and its development are seen. Different stmctural-functionalstates of the synapse are shown in (b).
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FIG. 15. Electronmicrograph of trace autoradiographs. (a) Incorporation of label in the spine apparatus in vivo, Ie~cine-~H. (b) Gl~cosamine-~H incorporation in vitro.
related to the increased energy potential and to the increased activity of ATPase (Fig. 17a) and adenyl cyclase (Fig. 17b) involved in the regulatory role of information. A special feature of the spines is their predominance in the cerebral cortex over other neuronal structures. Some investigators (Ramon y Cajal, 1935; Van der Loos, 1963; Kositsyn, 1976) have calculated that the number of spines along the dendrite is 8 to 10 per 10 pm, and that there are hundreds of millions of spines in the cerebral cortex (Manina, 1975).
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FIG. 16. (a) Proliferation of membranes of the axospinal synapse of the rat cerebral cortex. (b) Postsynaptic specialization (stimulation with Phenamine and learning). (c) Genesis of the spine apparalus (SA). pre, Presynaptic pole; post, postsynaptic pole; Sp, spine; D, dendrite; S, synapse; mt, microtubules.
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FIG. 16c. For legend see p. 371.
In axospinal and other contacts of the cerebral cortex one synapse forms several active zones (Fig. 16a) or one presynaptic pole makes simultaneous contacts with two or more synapses. Active zones of one synapse can transfer information simultaneously or in successive order and are responsible for excitatory or inhibitory states. The spine apparatus ultrastructure being different in density, osmiophilic nature, and development of membranes, conformational transformations are likely to depend on its functional state. There appear to be spine apparatuses in continuous operation and others that are used rarely, at least there are intact spines having the capacity to develop and specialize. The active state of the constantly working spine apparatus depends on the degree of specialization-the number of membrane complexes. Most information axospinal synapses are those with increased postsynaptic specialization-spine apparatuses. The structural-molecularhomogeneity of the spine apparatus with the receptor zone of the postsynaptic membrane (Fig. 18) can be detected by contrasting with phosphotungstic acid (PTA) and manifests itself in an affinity with protein and glycoprotein components. It may suggest the functional unity of receptor activity. The layer adjacent to the membrane of the spine apparatus and the active synaptic zone consists of protein macromolecules bound to gangliosides. The substance of the synaptic cleft and spine apparatus
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FIG. 17. (a) Enzymic activity of the spine apparatus. ATPase reaction. (b) Adenyl cyclase activity. pre, Presynaptic pole; post, postsynaptic pole; D, dendrite.
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FIG. 18. Distinct ultrastructural and cytochemical similarity of the spine apparatus to the neurofilaments of the postsynaptic receptor zone. pre, Presynaptic pole; post, postsynaptic pole; SA, spine apparatus.
can serve as a regulator of synaptic function in processing information and maintain the constancy of the synaptic contact at the expense of micropotentials independent of the impulses traveling along the axon (Katz and Miledi, 1967). The contact of the postsynaptic area with the spine apparatus is clearly detected by the chemical continuity of the common structural-molecular system. Several workers believe that the synaptic membranes are sites of storage of memory engrams (Deutsch, 1971; Kosower, 1972). So far, however, no facts confirming these data have been reported. Studies at our laboratory (Manina, 1971, 1972, 1975, 1976; Manina and Kucherenko, 1971; Manina et al., 1975a, 1976a; Turovsky et al., 1976a; Nadtochy, 1976; Gilerovich et al., 1977) have shown that, during stimulation of the CNS and during learning, in the process of conditioning as a form of manifestation of memory, the structural-functional activity of axospinal synapses increases (Fig. 20). Quantitative analysis with the computer Minsk-32 has shown that during learning in rats the number of spine apparatuses is increased by SO%, and during learning against the background of stimulation it rises to 200%. In monkeys the number of spine apparatuses is increased twofold, and the size of
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the active zones of the synapse is increased by 40% (Fig. 20b). A structural molecular transformation of the synaptic complex is observed. In the presynaptic area dense projections are pronounced. At the presynaptic pole of different animals a regular specific reaction of proliferation of complex membranes forming the spine apparatus can be observed, their being 30 and more. The spine apparatus is highly specialized, and information can arrive simultaneously through several channels. In this case the synapse may be involved in the fixing of traces (Fig. 21). In humans the spine apparatus has an analogous structure. Thus the spine apparatus membranes with their neurofilaments, possessing ultrastructural and chemical characteristics similar to those of the postsynaptic area and having immediate contacts with it, contain specific complex chemical components and have local protein- and glycoprotein-synthesizingsystems. They are responsible for both the reception and transmission of information and undoubtedly for its processing and, possibly, storage. Dense projections of the presynaptic pole, together with the neurofilaments of the receptor zone, form a single integrated structural molecular system of subsynaptic units (Fig. 22). Stimulation and learning cause changes in their conformation and structure. The spine apparatus membranes can be a potential site of storage of memory engrams. Spines contain a large pool of precursors. During increased functional
FIG. 19. Structural-functionalrelationship of the spine apparatus (SA) to a postsynaptic receptor zone of cortical synapse. pre, Presynaptic pole: post, postsynaptic pole.
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FIG.20. Axospinal synapses of the cerebral cortex in learning. (a) Rat. (b) Monkey. pre, Presynaptic pole; post, postsynaptic pole, Sp, spine; SA, spine apparatus.
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FIG.21. Scheme illustrating development of the spine apparatus (SA) in motivation. pre, Presynaptic pole; post, postsynaptic pole; SSE, subsynaptic unit, D, dendrite.
activity they form polymer chains from protein monomers. These chains act as a framework for the membranes of the spine apparatus with simultaneous synthesis of glycoproteins, being a part of this organelle. Undoubtedly, the memory trace must be fixed on the structures functioning as long as the neuron does. We believe that spine apparatus membranes are of this
FIG.22. Scheme illustrating the integrated system of subsynaptic units responsible for information transmission. pre, Presynaptic pole; post, postsynaptic pole; SA, spine apparatus;D, dendrite.
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nature. They perform certain functions of autoreproduction. All processes of synthesis on the template related to the function of nucleic acids during the reproduction and transmission of hereditary information are organized and fixed in the membranes. The integrative functions of the membranes are performed by supramolecular systems: (1) the system that generates energy, (2) the transport system, (3) the system governing partial autoreproduction (Fernhdez-Moran, 1954; Kreps, 1970; De Robertis et al., 1973). The structural-functionalcorrelation of the synaptic membranes was established by Harrison and Lunt (1975). Regular changes in the ultrastructure of synaptic apparatuses in the spines occur in connection with the functional activity of the brain-learning. The brain uses its systems economically, by activating numerous synapses and spines through the formation of spine apparatus. It reflects common biological regularity in the complexity of its ultrastructure and in the developmentof membranes within the limits of the available structural systems without the formation of new structures. It is quite reasonable that in the loss of physiological functions and morphological structures there occurs a formation of new connections. The data obtained enable us to put forward ideas concerning the mechanism of formation of memory engrams in the spine apparatus. The specialized activity of the spine apparatus of cerebral nerve cells might be based upon a unique mechanism involving the copying of functioning areas of the postsynaptic membrane and a subsequent storage of membranes-copies as steady and stable ultrastructural formations. Such molecular processes (asymmetric duplication of certain loci within the limits of the whole structure) are under detailed investigation in other organelles of the cell: symmetric reduplication of the genes controlling rRNA synthesis in the nucleolar organizer of cells of higher and lower animals, formation of “puffs” in chromosomes of amphibians and dipterans, selective synthesis of elements of the inner and outer membranes of mitochondria, and so on. It is possible that the mechanism of selective copying can be applied to the functioning of axospinal synapses. Our subcellular data show that the ultrastructure of a functioning synapse is characterized by the following features: ( 1 ) the local, regional synthesis of macromoleculeson the surface of the postsynaptic membrane and the formation of a superficial pattern of zones of active incorporation of amino acids and glucosamine labels; (2) a joint secondary new formation of a membrane complex inside the postsynaptic terminal; it is noteworthy that there is a morphologically distinct relationship-some structural continuity-between active centers of biosynthesis on the postsynaptic membranes and newly developing membrane elements inside the postsynaptic terminal; (3) differences in the local pattern of active biosynthesis on the postsynaptic membrane in different synapses. These facts suggest that the coding of information signals in the synapses is related to the spatial organization of the activation of biosynthesis on the postsynaptic membrane surface.
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At the next stage of recording, that is, during coding of the information passing through the synapse, there occurs a complementary spatial recoding of the primary pattern of chemical activity on the membrane surface onto the screen membrane structure of the spine apparatus. This structure has the same mosaic pattern of activity as an excited postsynaptic membrane. At the molecular level this ultrastmctural process is based on strict autoreduplication of the postsynaptic membrane elements. Experiments with labeled glucosamine show that during modification of synapses the glycoprotein complexes of the membranes are actively involved in structural transformation. On the surface of the membranes they have a discrete mosaic distribution. The process of autoreduplication of membrane glycoprotein complexes in the course of active formation of cerebral neurons might be tested by examining the kinetics of the incorporation of labeled carbohydrate precursors. It is possible, however, that the incorporation of labeled glucosamine reflects only a stable chemical modification of precursor membrane glycoproteins by means of addition or direct chemical changes in the polysaccharide radicals of synaptic membrane glycoproteins.
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Subject Index A Adenosine triphosphatase, synapse and, 361363 Adenyl cyclase, synapse and, 363-364 Amphibians chromaffin cells in, 329-334 oocyte maturation in, 192-193 Axospinal apparatus, stmctural-functional features of, 368-379
B Birds, chromaffin cells in, 327-328
c Calcium ions, oocyte maturation and, 229-233 Cell cycle quantitative radioautography and, 96-105 Chromafh cells in mammals action of drugs on, 302-305 cell types, 322-325 culture of, 306 development and, 306-308 distribution in autonomic nervous system, 287-289 functional role, 325-327 histochemistry. 299-302 morphologicalevidence for endocrine function, 318-322 morphology, 289-298 relations with surrounding structures, 308318 in nonmammalian vertebrates amphibians, 329-334 birds, 327-328 cyclostomes, 321 fishes, 334 reptiles, 328-329 techniques, 285-286 electron microscopy, 286-287 fluorescence method, 286 terminology, 283-285 385
Corpora d a t a cytological characteristics, 21-23 basal lamina, 23-25 gland cells, 25-63 neurosecretory fibers, 63-65 embryonic origin, 4-6 histological types, 11-14 historical background, 1-4 innervation, 9-10 morphological types annular, 9 centralized, 9 distal lateralizd, 7-8 lateralized, 6-7 semicentralized, 8 sexual dimorphism, 19-21 Y 10-11 a h 4 S U ~ P ~Of, volume, mitosis and pycnosis, 14-19 cyclostomes, chmmaffin cells in, 325
F Fish chromaffin cells in, 334 myte maturation in, 194-196 Follicles, oocyte mahlration and, 202-205
G Glycoproteins, role in synapse, 359-361 Gonadotropins, oocyte maturation and, 191-192
I Insect photoreception absolute light sensitivity, 138-153 color vision, 153-157 compound eye and, 129 electrical basis compound eye as volume conductor, 159 Dc parameters in light and dark, 159-169 photoresponses and cellular mechanisms, 169- 177 role of compartmentalization, 177 light and the eye, 128
386
SUBJECT INDEX
sensitivity to polarized light, 157458 visual pigments and photoreceptor membrane, 129-138
M Mammals, oocyte maturation in, 196-201
0 Oocyte maturation biochemical changes, 212-222 chronology of, 205-209 concept of, 186-187 control of meiosis and mitosis cytoplasmic control factors. 270-271 phosphorylation of cellular proteins, 269270 role of calcium, 267-268 role of CAMP, 268-269 SH cycle, 270 cytoplasmic control of arrest of meiotic division, 251-256 maturation-promoting factor, 240-246 phosphorylation of proteins, 246-25 1 fertilization and, 189-191 hormonal control in amphibians, 192-193 in fish, 194-196 gonadotropins and, 191-192 in mammals, 196-201 in other animals, 201-202 role of follicles, 202-205 in starfish, 193-194 initiation of changes in electrophysiological properties, 233-240 maturation-inducing substance, 222-229 role of calcium ions, 229-233 morphological changes, 209-212
nucleocytoplasmic interaction chromosome condensation, 256-260 development of motile systems, 263-267 development of pronucleus, 260-263 ovulation and, 188-189
P Protein(s), neurospecific, role in synapse, 357359
R Radioautography historical background, 75-76 kinetics of cell proliferation applications, 105-1 12 cell cycle, 96-105 precursors, 92-96 migration and chronoarchitectony, 112-1 18 quantitative histological and radioautographical processes, 77-82 observation and measurement, 83-92 Reptiles, chromaffin cells in, 328-329
S Starfish, oocyte maturation in, 193-194 Synapse. adenylcyclase and, 363-364 ATF'ase and, 361-363 biopolymer synthesis in, 364-368 classification chemical, 351-352 electrical, 352 ultrastructure, general principles, 351 Synaptic contact, structural-functional properties, 352-357 Synaptic transmission, mechanismsof, 347-35 1
Contents of Previous Volumes Volume 1
The Nature and Specificity of the Feulgen Nucleal Reaction-M. A. LESLER Some Historical Features in Cell BiologyQuantitative Histochemistly of PhosARTHUR HUGHES phataseS-wILLIAM L. DOYLE Nuclear Reproduction-€. LEONARD HUSKINS Enzymic Capacities and Their Relation to Cell Alkaline Phosphatase of the Nucleus-M. AND H. FIRKET CH~VREMONT Nutrition h AnimddEORGE W. KIDDER Gustatory and Olfactory Epithelia-A. F. The Application of Freezing and Drying TechAND G. H. BOURNE BARADI niques in Cytology-L. G. E. BELL Growth and Differentiation of Explanted Enzymatic Processes in Cell Membrane Tissues-P. J. GAILLARD AND W. WILPenetration-TH. ROSENBERG Electron Microscopy of Tissue Sections-A. J. BRANDT DALTON Bacterial Cytology-K. A. BISET A Redox Pump for the Biological Performance Protoplast Surface Enzymes and Absorption of of Osmotic Work, and Its Relation to the Suger-R. BROWN Kinetics of Free Ion Diffusion across Reproduction of Bacteriophage-A. D. HERMembranes-E. J. CONWAY SHEY A Critical Survey of Current Approaches in The Folding and Unfolding of Protein Molecules Quantitative Histo- and Cytochemistryas a Basis of Osmotic Work-R. J. GOLD DAVIDGLICK ACRE Nucleo-cytoplastmic Relationships in the DeNucleo-Cytoplasmic Relations in Amphibian velopment of Acetabularia-J. HAMMERLING Deve1opment-G. FRANK-HAUSER Report of Conference of Tissue Culture Workers Structural Agents in Mitosis-M. M. SWANN J. Held at Cooperstown, New York-D. Factors Which Control the Staining of Tissue HETHERINGTON Section with Acid and Basic Dyes-MARcus AUTHOR INDEX-SUBJECT INDEX SINGER The Behavior of Spermatozoa in the Neighborhood of Eggs-Lorn ROTHSCHILD Volume 3 The Cytology of Mammalian Epidermis and Sebaceous Glands-WILLIAM MONTAGNA The Electron-Microscopic Investigation of Tis- The Nutrition of Animal Celk-cHARlTY WAYMOUTH sue Sections-L. H. BRETXHNEIDER The Histochemistry of E s t e r a s e s 4 . GOMORI Caryometric Studies of Tissue Cultures4mo BUCHER AUTHOR INDEX-SUBJECT INDEX The Properties of Urethan Considered in Relation to Its Action on Mitosis-IVoR CORNMAN Volume 2 Composition and Structure of Giant Chromosomes-MAX ALFERT Quantitative Aspects of Nuclear NucleoproHow Many Chromosomes in Mammalian Soteins-HEWsoN Swwr matic Cells?-R. A. BEATTY Ascorbic Acid and Its Intracellular Localization, with Special Reference to Plants-J. CHAYEN The Significance of Enzyme Studies on Isolated Cell Nuclei-ALEXANDER L. DOUNCE Aspects of Bacteria as Cells and as OrThe Use of Differential Centrifugation in the ganiSm&TUART MUDDAND EDWARD D. Study of Tissue EnzymesxHR. DE DUVE DELAMATER AND J. BERTHET Ion Secretion in Plants-J. F. SUTCLIFFE Enzymatic Aspects of Embryonic Multienzyme Sequences in Soluble ExtractsDifferentiation-TRYGGVE GUSTAFSON HENRYR. MAHLER
387
388
CONTENTS OF PREVIOUS VOLUMES
Azo Dye Methods in Enzyme HistochemistryA. G. EVERSON PEARSE Microscopic Studies in Living Mammals with Transparent Chamber Methods-Roy G. WILLIAMS The Mast Cell-G. ASBOE-HANSEN Elastic Tissue-EDWARDS w. DEMPSEYAND ALBERTI. LANSING The Composition of the Nerve Cell Studied with New Method&VEN-oLOE BRATTGARDAND HOLGER HYDEN AUTHOR INDEX-SUBJECT
INDEX
Volume 4 Cytochemical Micrurgy-M. J. KOPAC Amoebocytes-L. E. WAGGE Problems of Fixation in Cytology, Histology, and Histochemistry-M. WOLMAN Bacterial cytology-ALFRED MARSHAK Histochemistry of Backria-R. VENDRELY Recent Studies on Plant Mitochondria-DAVID P. HACKER The Structure of Chloroplasts-K. MUHLETHALER
Histochemistry of Nucleic Acids-N. B. KURNICK
S. Structure and Chemistry of Nucleoli-W. VINCENT On Goblet Cells, Especially of the Intestine of some Mammalian Species-HARALD MOE Localization of Cholinesterases at Neuromuscular Junctions-R. COUTEAUX Evidence for a Redox Pump in the Active Transport of Cations-E. J. CONWAY AUTHOR INDEX-SUBJECT
INDEX
Volume 5 Histochemistry with Labeled AntibodyALBERTH. COONS The Chemical Composition of the Bacterial Cell Wall<. s. CUMMINS Theories of Enzyme Adaptation in Microorganisms-J. MANDELSTAM The Cytochondria of Cardiac and Skeletal Muscle-JOHN w . HARMON The Mitochondria of the NeUTOn-wARREN ANDREW
The Results of Cytophotometry in the Study of the Deoxyribonucleic Acid (DNA) Content of AND c . VENthe Nucleus-R. VENDRELY DRELY
Protoplasmic Contractility in Relation to Gel Structure: Temperature-PressureExperiments on Cytokinesis and Amoeboid MovementDOUGLAS MARSLAND Intracellular pH-PETER C. CALDWELL The Activity of Enzymes in Metabolism and Transport in the Red Cell-T. A. J. PRANKERD
Uptake and Transfer of Macromolecules by Cells with Special Reference to Growth and Development-A. M. SCHECHTMAN Cell Secretion: A Study of Pancreas and Salivary C. J. JUNQUEIRA AND G. C. Glands-'. HIRSCH The Acrosome Reaction--JEAN C. DAN Cytology of Spermatogenesis-VIsHwA NATH The Ultrastructure of Cells, as Revealed by the Electron Microscope-FRITIoF S . SJOSTRAND AUTHOR INDEX-SUBJECT
INDEX
Volume 6 The Antigen System of Paramecium aureliaG. H. BEALE The Chromosome Cytology of the Ascites Tumors of Rats, wtih Special Reference to the Concept of the Stemline Cell-SAmo MAKINO The structure of the Golgi Apparatus-ARTHUR AND PRISCHIAF. POLLISTER W. POLLISTER An Analysis of the Process of Fertilization and Activation of the Egg-A. MONROY The Role of the Electron Microscope in Virus Research-ROBLEY C. WILLIAMS The Histochemistry of PolysaccharidesJ. HALE ARTHUR The Dynamic Cytology of the Thyroid GlandJ. GROSS Recent Histochemical Results of Studies on Embryos of Some Birds and Mammals-ELI0 BORGHESE Carbohydrate Metabolism and Embryonic Determination-R. J. O'CONNOR Enzymatic and Metabolic Studies on Isolated Nuclei-G. SIEBERTAND R. M. S. SMELLIE
CONTENTS OF PREVIOUS VOLUMES Recent Approaches of the Cytochemical Study of Mammalian Tissues--GEoRGE H. HOGEBOOM, EDWARD L. KUEF, AND WALTER C. SCHNEIDER The Kinetics of the Penetration of Nonelectrolytes into the Mammalian ErythrocyteFREDABOWYER
389
The Cell Surface of Parameciurn-C. F. EHRET AND E. L. POWERS The Mammalian ReticUlocyte-LEAH MIRIAM LOWENSTEIN The Physiology of Chromatophores-MILTON
FINGERMAN The Fibrous Components of Connective Tissue AUTHOR INDEX-SUBJECT INDEX with Special Reference to the Elastic CUMULATIVE SUBJECT INDEX Fiber-DAVID A. HALL (VOLUMES 1-5) B. Experimental Heterotopic Ossification-J. BRIDGES A Survey of Metabolic Studies on Isolated Volume 7 Mammalian Nuclei-D. B. ROODYN Some Biological Aspects of Experimental Trace Elements in Cellular Function-BERT L. Radiology: A Historical Review-F. G. VALLEEAND FREDERICL. HOCH SPEAR Osmotic Properties of Living Cells-D. A. T. ‘ h e Effect of Carcinogens, Hormones, and DICK Vitamins on Organ Cultures-ILsE LASNITZKI Sodium and Potassium Movements in Nerve, Muscle, and Red Cells-I. M. GLYNN Recent Advances in the Study of the Kinetochore-A. LIMA-DE-FARIA Pinocytosis-H. HOLTER AUTHOR INDEX-SUBJECT INDEX Autoradiographic Studies with S35-Sulfat+D. D. DZIEWIATKOWSKI The Structure of the Mammalian Volume 9 Spermatozoon-DON w. FAWCETT The Lymphocytes. A. TROWELL The Influence of Cultural Conditions on BacteThe Structure and Innervation of Lamellibranch rial Cytology-J. F. WILKINSONAND J. P. Muscle-L. BOWDEN DUGUID Hypothalamo-neurohypophysial NeurosecreOrganizational Patterns within Chromosomestion-J. C. SLOPER BERWNDP. KAUFMANN, HELENGAY, A N D Cell Contact-PAUL WEISS MARGARET R. MCDONALD The Ergastoplasm: Its History, Ultrastructure, Enzymic Processes in Cells-JAY BOYDBEST and Biochemistry-FRANcOIsE HAGUENAU The Adhesion of Cells-LEONARD WEIss Anatomy of Kidney Tubules-JOHANNES RHOPhysiological and Pathological Changes in DIN Mitochondria1 Morphology-CH. ROUILLER Structure and Innervation of the Inner Ear SenThe Study of Drug Effects at the Cytological sory Epithelia-HANS ENGSTROMAND JAN L e v e l - G . B. WILSON WERSALL Histochemistry of Lipids in OogenesisThe Isolation of Living Cells from Animal VISHWANATH Tissues-L. M. RINALDINI Cyto-Embryology of Echinoderms and AmAUTHOR INDEX-SUBJECT INDEX phibia-KUTSUMA DAN The Cytochemistry of Nonenzyme ProteinsRONALDR. COWDEN Volume 8 The structure of CYtOplaSmxHARLES OBER-
AUTHOR I N D E X 4 U B J E C T INDEX
LING
Wall Organization in Plant Cells-R.
D. PRE-
Volume 10
STON
Submicroscopic Morphology of apSe-EDUARDO DE ROBERTS
the Syn-
The Chemistry of Shiff‘s Reagent-FREDERICK H. KASTEN
390
CONTENTS OF PREVIOUS VOLUMES
Spontaneous and Chemically Induced Chromosome Breaks-ARUN KUMARSHARMA AND ARCHANA SHARMA The Ultrastructure of the Nucleus and Nucleocytoplasmic RelationsSAuL WISCHNITZER
In Vivo Implantation as a Technique in Skeletal Biology-WILLIAM J. L. FELTS The Nature and Stability of Nerve Myelin-J. B. RNEAN Fertilization of Mammalian Eggs in Vitro-C. R. AUSTIN Physiology of Fertilization in Fish E g g s TOKI-o YAMAMOTO
The Mechanics and Mechanism of CleavageLEWISWOLPERT The Growth of the Liver with Special Reference AUTHOR INDEX-SUBJECT INDEX to Mammals-F. DOWANSKI Cytology Studies on the Affinity of the CarVolume 13 cinogenic A m Dyes for Cytoplasmic The Coding Hypothesis-MARTYNAS YEAS Components-YosHIMA NAGATANI HERBERT Epidermal Cells in Culture-A. GEDEON Chromosome Reproduction-J. MATOLTSY TAYLOR AUTHOR INDEX--SUBJECT INDEX Sequential Gene Action, Protein Synthesis, and CUMULATIVE SUBJECT INDEX Cellular Differentiation-REED A. FLIC(VOLUMES 1-9) KINGER The Composition of the Mitochondria1 Membrane in Relation to Its Structure and Volume 11 G. BALL AND CLIFFE D. Function-Eruc Electron Microscopic Analysis of the Secretion JOEL Mechanism-K. KUROSUMI Pathways of Metabolism in Nucleate and AnuThe Fine Structure of Insect Sense Organscleate Erythrocytes-H. A. SCHWEIGER ELEANOR H. SLIFER Some Recent Developments in the Field of Alcytology of the Developing Eye-ALFRED J. kali Cation Transport-W. WILBRANDT COULOMBRE Chromosome Aberrations Induced by Ionizing The Photoreceptor Structures-J. J. WOLKEN Radiations-H. J. EVANS Use of Inhibiting Agents in Studies on Fertiliza- Cytochemistry of Protozoa, with Particular Reftion MechanismdHARLEs B. METZ erence to the Golgi Apparatus and the The Growth-Duplication Cycle of the Cell-D. Mitochondria-VIsHwA NATH AND G. P. M. PRESCOTT DUTTA Histochemistry of Ossification-RoMuLo L. Cell Renewal-FELIX BERTALANFFY AND CHOCABRINI SEN LAU Cinematography, Indispensable Tool for AUTHOR INDEX-SUBJECT INDEX Cyto1ogyy-C. M. Pomerat AUTHOR INDEX-SUBJECT
INDEX
Volume 14
Inhibition of Cell Division: A Critical and ExVolume 12 perimental AnalysidEYMouR GELFANT Sex Chromatin and Human Chromosomes Electron Microscopy of Plant Protoplasm-R. -JOHN L. HAMERTON BUVAT Chromosomal Evolution in Cell PopulationsCytophysiology and Cytochemistry of the Organ T. C. Hsu of Corti: A Cytochemical Theory of Chromosome Structure with Special Reference AND L. K. TITOVA Hearing-J. A. VINNIKOV to the Role of Metal IOIIS-DALE M. STEF- Connective Tissue and Serum Proteins-R. E. FENSEN MANCINI Electron Microscopy of Human White Blood The Biology and Chemistry of the Cell Walls of Cells and Their Stem Cells-MARCEL BESIS Higher Plants, Algae, and Fungi-D. H. AND JEAN-PAUL THIERY NORTHCOTE
39 1
CONTENTS OF PREVIOUS VOLUMES Development of Drug Resistance by Staphylococci in Vitro and in V~VO-MARY BARBER Cytological and Cytochemical Effects of Agents Implicated in Various Pathological Conditions: The Effect of Viruses and of Cigarette Smoke on the Cell and Its Nucleic Acid ZECILIE LEUCHTENBERGER AND RUDOLF LEUCHTENBERGER The Tissue Mast W~~I-DOUGLAS E. SMITH
I n Vivo Studies of Myelinated Nerve F i b e r s
CARLCASKEY SPEIDEL Respiratory Tissue: Structurt, Histophysiology, Cytodynamics. Part I: Review and Basic Cytomorphology-FELIX D. BERTALANFFY AUTHOR INDEX-SUBJECT
INDEX
Volume 17 The Growth of Plant Cell Walls-K. WILSON Reproduction and Heredity in Trypanosomes: A AUTHOR INDEX-SUBJECT INDEX Critical Review Dealing Mainly with the African Species in the Mammalian Host-P. J. WALKER Volume 15 The Blood Platelet: Electron Microscopic The Nature of Lampbrush Chromosomes-H Studies-J. F. DAVID-FERREIRA The Histochemistry of MucopolysaccharidesG. CALLAN c. CURRAN ROBERT The Intracellular Transfer of Genetic Respiratory Tissue Structure, Histophysiology, Information-J. L. SIRLIN Cytodynamics. Part 11. New Approaches and Mechanisms of Gametic Approach in PlantsInterpretations-FELIX D. BERTALANFFY ERIKA LEONARD MACHLIS AND RAWITSCHER-KUNKEL The Cells of the Adenohypophysis and Their Functional Significance-MARC HERLANT The Cellular Basis of Morphogenesis and Sea AUTHOR INDEX--SUBJECT INDEX Urchin Development-T. GUSTAWNAND L. WOLPERT Plant Tissue Culture in Relation to Development Volume 18 cytOlOgy
392
CONTENTS OF PREVIOUS VOLUMES
Electromyography: Its Structural and Neural Basis-JOHN v. BASMAJIAN “Metabolic” DNA: A Cytochemical Study-H. Cytochemical Studies with Acridine Orange and ROELS the Influence of Dye Contaminants in the The Significance of the Sex ChromatinStaining of Nucleic Acids-FREDERICK H. MURRAYL. BARR KASTEN Some Functions of the Nucleus-J. M. MITCHI- Experimental Cytology of the Shoot Apical Cells SON during Vegetative Growth and Flowering-A. Synaptic Morphology on the Normal and DeNOUGARPDE generating Nervous System-E. G. GRAY Nature and Origin of Perisynaptic Cells of the AND R. W. GUILLERY Motor End Plate-T. R. SHANTHAVEERAPPA Neurosecretion-W. BARGMANN AND G. H. BOURNE Some Aspects of Muscle Regeneration-E. H. AUTHOR INDEX-SUBJECT INDEX BETZ, H. FIRKET,AND M. REZNIK The Gibberellins as Hormones-P. W. BNAN Volume 22 Phototaxis in PhtS-WOLFGANG H A U ~ S. Phosphorus Metabolism in Plants-K. Current Techniques in Biomedical Electron ROWAN Microscopy-s AUL WISCHNITZER
Volume 19
AUTHOR INDEX-SUBJECT
INDEX
AUTHOR INDEX-SUBJECT
INDEX
The Cellular Morphology of Tissue Repair-R. M. H. MCMINN Structural Organization and Embryonic Volume 20 Differentiation-GAJANAN V. SHERBETAND The Chemical Organization of the Plasma MemM. S. LAKSHMI brane of Animal Cells-A. H. MADDY The Dynamism of Cell Division during Early Subunits of Chloroplast Structure and Quantum Cleavage Stages of the Egg-N. FAUTREZConversion in Photosynthesis-RoDERIC B. FIRLEFYN AND J. FAUTREZ PARK Lymphopoiesis in the Thymus and Other TisControl of Chloroplast Structure by Lightsues: Functional Implications-N. 9. SlEGENLe ~ LESTERPACKER AND P A U L - A N D EVERETT A N D RUTHW. TYLER (CAFFREY) THALER Structure and Organization of the Myoneural The Role of Potassium and Sodium Ions as Junction-C COERS Studied in Mammalian Brain-H. HILLMAN The Ecdysial Glands of Arthropods-WILLIAM Triggering of Ovulation by Coitus inthe RatS. HERMAN CLAUDE ARON,GITTAASCH,AND JAQUELINE Cytokinins in Plants-B. I. SAHAISRIVASTAVA Roos AUTHOR INDEX-SUBJECT INDEX Cytology and Cytophysiology of Non- CUMULATIVE SUBJECT INDEX Melanophore Pigment Cells-JOSEPH T. (VOLUMES 1-21) BACNARA The Fine Structure and Histochemistry of ProstaVolume 23 tic Glands in Relation to Sex HormonesDAVIDBRANDES Transformationlike Phenomena in Somatic Cerebellar EnZymOlOgy-LUCIE ARVY Cells-J. M. OLENOV Recent Developments in the Theory of Control and Regulation of Cellular ProcessesROBERTROSEN Volume 21 Contractile Properties of Protein Threads from Sea Urchin Eggs in Relation to Cell DiHistochemistry of Lysosomes-P. 9.GAHAN vision-HIKoICHl SAKAI Physiological Clocks-R. L. BRAHMACHARY of Microscopic Morphology Ciliary Movement and Coordination in Electron Oogenesis-ARNE N ~ R R E V A N C Ciliates-BELA PARDUCA
CONTENTS OF PREVIOUS VOLUMES
393
Dynamic Aspects of Phospholipids during Protein Secretion-LowELL E. H o r n The Golgi Apparatus: Structure and F u n c t i o e H. W. BEAMS AND R. G. KESSEL The ChromosomalBasis of Sex DeterminationKENNETHR. LEWISAND BERNARD JOHN
Isozymes: Classification, Frequency, and Significance-CmmEs R. SHAW The Enzymes of the Embryonic NephronLUCIEARVY Protein Metabolism in Nerve Cells-B. DROZ Freeze-Etching-HANS MOOR
AUTHOR INDEX-SUBJECT
AUTHOR I N D E X 4 U B J E C T INDEX
INDEX
Volume 24
Volume 26
Synchronous Cell Differentiation-GEoRGE M. AND IVANL. CAMERON PADILLA Mast Cells in the Nervous System-YNGVE
A New Model for the Living Cell: A Summary of the Theory and Recent Experimental EviN.BLING ERT dence in Its S U P ~ O ~ ~ ~ I L The Cell Periphery-LEONARD WEIS Mitochondrial DNA: Physicochernical Properties, Replication, and Genetic Function-P. BORSTAND A. M. KROON Metabolism and Enucleated Ceh-KoNRAD
OLSON
Development Phases in Intermitosis and the Preparation for Mitosis of Mammalian Cells in ViWeBLAGOJE A. NESKOVIC Antimitotic Substances-Guu DEYSSON The form and Function of the Sieve Tube: A Problem in Reconciliation-P. E. WEATHERE LEY AND R. P. C. JOHNSON Analysis of Antibody Staining Patterns Obtained with Striated Myofibrils in Fluorescence Microscopy and Electron Microscopy-FRANK A. PEPE Cytology of Intestinal Epithelial Celk-F’!2TER G. TONER Liquid Junction Potentials and Their Effects on Potential Measurements in Biology Systems-P. C. CALDWELL AUTHOR INDEX-SUBJECT
Volume 25
INDEX
KECK Stereological Principles for Morphometry in Electron Microscopic Cytology-Ev&iLD R. WEIBEL Some Possible Roles for Isozymic Substitutions during Cold Hardening in Plants-D. W. A. ROBERTS AUTHOR INDEX--SUBJECT INDEX
Volume 27 Wound-Healing in Higher Plants-JACQUES LIPETZ Chloroplasts as symbiotic organelles-DENNIS L. TAYLOR The Annulate Lamellae4AuL WISCHNITZER Gametogenesis and Egg Fertilization in PIanarians-G. BENAZZILENTATI Ultrastructure of the Mammalian Adrenal COrteX--SIMONIDELMAN The Fine Structure of the Mammalian Lymphoreticular SySteIW-IAN CARR Immunoenzyme Technique: Enzymes as Markers for the Localization of Antigens and AntibodiesSTRATis AVRAMEAS
Cytoplasmic Control over the Nuclear Events of Cell Reproduction-NOEL DE TERRA Coordination of the Rhythm of Beat in Some Ciliary Systems-M. A. SLEIGH The Significance of the Structural and Functional Similarities of Bacteria and MitochondriaAYLVAN NASS The Effects of Steroid Hormones on Macrophage Activity-B. VERNON-ROBERTS AUTHOR INDEX-SUBJECT INDEX The Fine Structureof Malaria Parasites-MARIA A. RUDZINSKA The Growth of Liver Parenchymal Nuclei and Its Volume 28 Endocrine Regulation-RITA CARRERE The Cortical and Subcortical Cytoplasm of LymStrandedness of ChrOmOSOmeS~HELDON naea Egg--cHRISTlAAN P. RAVEN WOLFF
394
CONTENTS OF PREVIOUS VOLUMES
The Environment and Function of Invertebrate Nerve Cells-J. E. TREHERNEAND R. B. MORETON Virus Uptake, Cell Wall Regeneration, and Virus Multiplication in Isolated Plant Protoplasts-E. C. COCKING The Meiotic Behavior of the Drosophila OOCyte-ROBERT c . KING The Nucleus: Action of Chemical and Physical Agents-RENfi SIMARD The Origin Of Bone Cells-MAUREEN OWEN Regeneration and Differentiation of Sieve Tube Ekments-WILLIAM P. JACOBS Cells, Solutes, and Growth: Salt Accumulation in Plants Reexamined-F. C. STEWARD AND R. L. MOTT AUTHOR INDEX-SUBJECI
Volume 29 Gram Staining and Its Molecular MechanismsB. B. BISWAS,P. S. BASU,AND M. K. PAL The Surface Coats of Animal Cells-A. MARTfNEZ-PALOMO Carbohydrates in Cell Surfaces-RICHARD J. WINZLER DifferentialGene Activation in Isolated Chromosomes-MARKUS LEZZI Inuaribosomal Environment in the Nascent Peptide Chain-HIDEKO KAJI Location and Measurement of Enzymes in Single Cells by Isotopic Methods Part I-E. A. BARNARD Location and Measurement of Enzymes in Single Cells by Isotopic Methods Part 1 1 4 . C. BUDD Neuronal and Glial Perikarya Preparations: An Appraisal of Present Methods-PATRICIA v . AND BETTYI. ROOTS JOHNSTON Functional Electron Microscopy of the Hypothalamic Median Eminence-HIDEsH1 KOBAYASHI, TOKUZOMATSUI,AND Susuhn *
ISHII
AUTHOR INDEX-SUBJECT
INDEX
High-Pressure Studies in Cell Biology-ARTHUR M. ZIMMERMAN Micrurgical Studies with Large Free-Living Amebas-K. W. JEONAND J. F. DANIELLI The Practice and Application of Electron Microscope Autoradiography-J. JACOB Applications of Scanning Electron Microscop) in Biology-K. E. CAM Acid Mucopolysaccharides in Calcified TissuesSHINJIRo KOBAYASHI AUTHOR INDEX-SUBJECT
INDEX
CUMULATIVE SUBJECT INDEX
(VOLUMES 1-29)
Volume 31
INDEX
Early Development in Callus -MICHAEL M. YEOMAN
Volume 30
Cultures-
Studies on Freeze-Etching of Cell Membranes-KURT M~~HLETHALER Recent Developments in Light and Electron Microscope Radioautography4. C. BUDD Morphological and Histochemical Aspects of Glycoproteins at the Surface of Animal Cells-A. RAMBOURG DNA Biosynthesis-H. S. JANSZ,D. VAN DER MEI, AND G. M. ZANDVLIET Cytokinesis in Animal Cells-R. RAPPAFORT The Control of Cell Division in Ocular Lens-C. V. HARDING, J. R. REDDAN, N. J. UNAKAR, AND M. BAGCHI The Cytokinins-HANS KENDE Cytophysiology of the Teleost PituitaryA. BERN MARTIN SAGEAND HOWARD AUTHOR INDEX-SUBJECT
INDEX
Volume 32 Highly Repetitive Sequences of DNA in Chromosomes-W. G. FLAW The Origin of the Wide Species Variation in Nuclear DNA Content-H. REES AND R. N. JONES
Polarized Intracellular Particle Transport: Saltatory Movements and Cytoplasmic Streaming-LIONEL I. REBHUN The Kinetoplast of the Hemoflagellates-LARRY SIMP~ON
395
CONTENTS OF PREVIOUS VOLUMES Transport across the Intestinal Mucosal Cell: AND Hierarchies of Function-D. S. PARSON C. A. R. BOYD Wound Healing and Regeneration in the Crab Purutelphusu hydrodromous-RrrA G. ADIYODI The Use of Femtin-Conjugated Antibodies in Electron Microscopy-CouNcILmN MORGAN Metabolic DNA in Ciliated Protozoa, Salivary Gland Chromosomes, and Mammalian Cell&. R. PELC AUTHOR INDEX-SUBJECT
Mechanisms of Ion Transport through Plan Cell Membranes-EmNuEL ERSTEIN Cell Motility: Mechanisms in Protoplasmic Streaming and Ameboid Movement-H. KOMNICK, W. STOCKEM,AND K. E. WOHLEFARTH-BOTTERMANN The Gliointerstitial System of MollusesGHISLAIN NICAISE Colchicine-SensitiveMicrotubles-LYNN MARCX GULIS AUTHOR INDEX-SUBJECT
INDEX
INDEX
Volume 35 Volume 33
The Structure of Mammalian ChromosomesELTONSTUBBLEFIELD Visualization of RNA Synthesis on ChromoA. Synthetic Activity of Polytene Chromosomessomes-0. L. MILLER,JR. AND BARBARA HANSD. BERENDES HAMKALO Cell Disjunction (“Mitosis”) in Somatic Cell Mechanisms of Chromosome Synapsis at MeiB. MOENS Reproduction-ELAINE G . DIACUMAKOS, d C hphas-BTER NAN^ Structural Aspects of Ribosomes-N. AND PAULINE PECORA SCOTTHOLLAND, NlNGA Neuronal Microtubles, Neurofilaments, and Microfilaments-RAYMOND B. WUERKER Comparative Ultrastructure of the Cerebrospinal Fluid-Contacting Neurons-”. VIGHAND I. AND JOELB. KIRKPATRICK VIGH-TEICHM ANN Lymphocyte Interactions in Antibody Maturation-Inducing Substance in StarlishesResponses-J. F. A. P. MILLER HARUOKANATANI Laser Microbeams for Partial Cell Irradiation-MICHAEL w. BERNSAND CHRISO? The Limonium Salt Gland: A Biophysical and Structural Study-A. E. HILLAND B. S HILL TIAN SALET Toxic Oxygen Effecb-HAROLD M. SWARTZ Mechanisms of Virus-Induced Cell FusionAUTHOR INDEX-SUBJECT INDEX GEORGE POSTE Freeze-Etching of BaCtena-CHARLES C. RECX MSEN AND STANLEY W. WATSON The Cytophysiology of Mammalian Adipose Volume 36 Celk-BERNARD G. SLAVIN Molecular Hybridization of DNA and RNA in AUTHOR INDEX-SUBJECT INDEX SI’tU-WOLFGANG HENNIG The Relationshipbetween the Plasmalemma and Plant Cell WPl1-JEAN-CLAUDE ROLAND Volume 34 Recent Advances in the Cytochemistry and U1trastructure of Cytoplasmic Inclusions in MasThe SubmicroscopicMorphology of the Interphtigophora and Opalinata (Protozoaj-G. P. ase N u c l e u s S ~ u LWIsCHNITZER DUTTA The Energy State and Structure of Isolated Chloroplasts: The OxidativeReactions Involv- Chloroplasts and Algae as Symbionts in MOIIUSCS-LEONARD MUSCATINE AND of ing the Water-Splitting Step RICHARD W GREENE Photosynthesis-ROBERT L. HEATH AND ZANm Transport in Neurospora-GENE A. SCARm The Macrophage-hIMoN GORDON BOROUGH VIL A. C o r n
396
CONTENTS OF PREVIOUS VOLUMES
Degeneration and Regeneration of Neurosecretor)’ Systems-HoRsT-DIETER DELLMANN
Cell Separation by Gradiant Centrifugation-R. HARWOOD
AUTHOR INDEX-SUBJECT
SUBJECT INDEX
INDEX
Volume 37
Volume 39
Units of DNA Replication in Chromosomes of Eukaroytes-J. HERBERT TAYLOR Viruses and Evolution-D. C. REANNEY Electron Microscope ‘Studieson Spermiogenesis in Various Animal SpeCieS4ONPACHIRO YASUZUMI Morphology, Histochemistry, and Biochemistry of Human Oogenesis and OvulationSARDuL S. GURAYA Functional Morphology of the Distal LungKAYEH. KILBURN Comparative Studies of the Juxtaglomerular Apparatus-HIRoFuMl SOKABE AND MITCE ZUHO OGAWA The Ultrastructure of the Local Cellular Reaction to Neoplasia-IAN CARR AND J. c. E. UNDERWOOD Scanning Electron Microscopy in the Ultrastructural Analysis of the Mammalian Cerebral E. SCOTT,G. P. Ventricular System-D. AND M. N. SHERIDAN KOZLOWSKI,
Androgen Receptors in the Nonhistone Protein Fractions of Prostatic Chromatin-TuNG YUE WANGAND LEROYM. NYBERG Nucleocytoplasmic Interactions in Development of Amphibian HybnddTEPHEN SUBTELNY The Interactions of Lectins with Animal Cell SUrfaCeS-GARTHL. NICOLSON Structure and Function of Intercellular Junctions-L. ANDREWSTAEHELIN Recent Advances in Cytochemistry and Ultrastructure of Cytoplasmic Inclusions in Ciliophora (Protozoa)-G. P. DUTTA Structure and Development of the Renal Glomeruhs as Revealed by Scanning Electron Microscopy-FRANC0 SPINELLI Recent Progress with Laser MicrobeamsW. BERNS MICHAEL The Problem of Germ Cell Determinants-H. W. BEAMSAND R. G. KESSEL
AUTHOR INDEX-SUBJECT
Volume 40
INDEX
SUBJECT INDEX
B-Chromosome Systems in Flowering Plants Volume 38 and Animal Species-R. N. JONES Genetic Engineering and Life Synthesis: An In- The Intracellular Neutral SH-Dependent Protroduction to the Review by R. Widdus and C. tease Associated with Inflammatory Auk-JAMES F. DANIELLI Reactions-HIDE0 HAYASHI Progress in Research Related to Genetic En- The Specificity of Pituitary Cells and Regulation gineering and Life Synthesis-Roy WIDDUS of Their Activities-VLADIMIR R. PANTIC AND CHARLES R. AULT Fipe Structure of the Thyroid Gland-HisAo The Genetics of C-Type RNA Tumor VirusesFUJITA Postnatal Gliogenesis in the Mammalian J. A. WYKE Brain-A. PRIVAT Three-Dimensional Reconstruction from ProjThree-Dimensional Reconstruction from Serial ections: A Review of Algorithms-RICHARD AND GABOR T. HERMAN GORDAN Sections-DANDLE w . WARE AND VINCENT The Cytophysiology of Thyroid C e l l s LOPRESTI VLADIMAR R. PANTIC SUBJECT INDEX The Mechanisms of Neural Tube FormationPERRY KARFUNKEL Volume 41 The Behavior of the XY Pair in M a m m a l s J. SOLARI ALBERTO The Attachment of the Bacterial Chromosometo the Cell Membrane-PAUL J. LEIBOWITZ Fine-Structural Aspects of Morphogenesis in AND Acetabularia-G. WERZ MOSELIO SCHAECHTER
CONTENTS O F PREVIOUS VOLUMES
397
Regulation of the Lactose Operon in Escherichiu Biochemical Studies of Mitochondria1Transcription and Translation<. SACCONEAND E. cofi by c A M P 4 . CARPENTER AND B. H. QUAGLIANELLO SELLS The Evolution of the Mitotic Spindle-DONNA Regulation of Microtubules in TetrahymenuF. KUBAI NORMANE. WILLIAMS Cellular Receptors and Mechanisms of Action of Germ Plasma and the Differentiation of the Germ Cell Line-E. M. EDDY Steroid HOrmOneS~HLJTSUNGLIAO A Cell Culture Approach to the Study of An- Gene Expression in Cultured Mammalian Cells-RoDY P. COX AND JAMESc. KING tenor Pituitary Cells-A. TIXIER-VIDAL,D. Morphology and Cytology of the Accessory Sex GOURDJI,AND C. TOUGARD Glands in Invertebrates-K. G. ADIYODI AND of Immunohistochemical Demonstration R. G. ADIYODI Neurophy sin in the SUBJECT INDEX Hypothalamoneurohypophysial System-W. B. WATKMS The Visual System of the Horseshoe Crab Volume 44 Limulus polyphemus-WOLF H. FAHRENC~ The Nucleolar StrIICtUre~lBDASGHOSH BACH The Function of the Nucleolus in the Expression SUBJECT INDEX of Genetic Information: Studies with Hybrid Animal Cells-E. SIDEBOTTOMAND I. I. DEAK Volume 42 Phylogenetic Diversity of the Proteins RegulatRegulators of Cell Division: Endogenous Mitotic ing Muscular Contraction-WiLLIAM Inhibitors of Mammalian Cells-BISMARCK LEHMAN B. Lozzro, CARMENB. LOZZIO,ELENAG. Cell Size and Nuclear DNA Content in BAMBERGER, AND STEPHEN v . LAIR Vertebrates-HENRYK SZARSKI Ultrastructure of Mammalian Chromosome Ultrastructural Localization of DNA in Ultrathin AND WALTER Aberrations-B. R. BIUNKLEY Tissue Sections-ALAIN GAUTIER N. HITTELMAN Cytological Basis for Permanent Vaginal Computer Processing of Electron Micrographs: Changes in Mice Treated Neonatally with W. A Nonmathematical Account-P. Steroid Hormones-NoBoRIJ TAKASUGI HAWKES On the Morphogenesis of the Cell Wall of Cyclic Changes in the Fine Structure of the Staphylococci-PETER GIESBRECHT,JORG Epithelial Cells of Human EndometriumREINICKE WECKE,AND BERNHARD MILDREDGORDON Cyclic AMP and Cell Behavior in Cultured The Ultrastructure of the Organ of CortiCells-MARK C. WILLINCHAM ROBERTS. KIMURA Recent Advances in the Morphology, HisEndocrine Cells of the Gastric Mucosa-ENRIco tochemistry, and Biochemistry of SteroidSOLCIA,CARLOCAPELLA, GABRIELEVAXX Synthesizing Cellular Sites in the NonmamSALLO, AND ROBERTO BUFFA malian Vertebrate O V ~ ~ ~ ~ A RS.D U L Membrane Transport of Purine and Pyrimidine GURAYA Bases and Nucleosides in Animal CellsSUBJECT INDEX RICHARDD. BERLINAND JANETM. OLIVER SUBJECT INDEX
Volume 45 Approaches to the Analysis of Fidelity of DNA Repair in Mammalian Ceh-MICHAEL w. LIEBERMAN The Evolutionary Origin of the Mitochondrian: A Nonsymbiotic Model-HENRY R. MAHLER The Variable Condition of Euchromatin and Heterochromatin-FRIEDRICH BACK A N D RUDOLFA. RAW
Volume 43
398
CONTENTS OF PREVIOUS VOLUMES
Effects of 5-Bromodeoxyuridine on Tumorigenicity, Immunogenicity , Virus Production, Plasminogen Activator, and Melanogenesis of Mouse Melanoma CellsSELMA SILAGI Mitosis in Fungi-MELVIN S. FULLER Small Lymphocyte and Transitional Cell Populations of the Bone Marrow; Their Role in the Mediation of Immune and Hemopoietic Progenitor Cell Functions-cORNELIuS ROSSE The Structure and Properties of the Cell Surface Coat-J. H. LUFT Uptake and Transport Activity of the Median Eminence of the Hypothalamus-K. M. KNIGGE,S. A. JOSEPH,J. R. SLADEK,M. F. NOTTER,M. MORRIS,D. K. SUNDBERG, M. G. E. HOFFMAN,AND L. A. HOLZWARTH, O’BRIEN
Chemical Nature and Systematization of Substances Regulating Animal Tissue GrowthVICTORA. KONYSHEV Structure and Function of the Choroid Plexus and Other Sites of Cerebrospinal Fluid Formation-THOMAS H. MILHORAT The Control of Gene Expression in Somatic Cell Hybrids-H. P. BERNHARD Precursor Cells Of Mechanocytes-ALEXANDER J. FRIEDENSTEIN
SUBJECT INDEX
SUBJECT INDEX
Volume 47
Volume 49
SUBJECT INDEX
Volume 48
Mechanisms of Chromatin Activation and AND VAUCC Repression-NORMAN MACLEAN GHAN A. HILDER Origin and Ultrastructure of Cells in Vifro-L. SUBJECT INDEX M. FRANKSA N D PATRICIAD. WILSON Electrophysiology of the Neurosecretory Cell-KINJI YAGI A N D SHIZUKO IWASAKI Volume 46 Reparative Processes in Mammalian Wound Neurosecretion by Exocytosis-ToM CHRISTIAN Healing: The Role of Contractile NORMANN Phenomena-GrutIo GABBIANIA N D DENYS MONTAND~N Genetic and Morphogenetic Factors in Hemoglobin Synthesis during Higher Vertebrate Smooth Endoplasmic Reticulum in Rat HepatoDevelopment: An Approach to Cell Difcytes during Glycogen Deposition and NIGON Depletion-ROBERT R. CARDELL, JR. ferentiation Mechanisms-VICTOR AND JACQUELINE GODET Potential and Limitations of Enzyme Cytophysiology of Corpuscles of Stannius-V. Cytochemistry: Studies of the Intracellular G. KRISHNAMURTHY Digestive Apparatus of Cells in Tissue Ultrastructure of Human Bone Marrow Cell Culture-M. HUNDGEN Maturation-J. BRETON-GORIUS AND F. Uptake of Foreign Genetic Material by Plant REYES Protoplasts-E. C. COCKING Evolution and Function of Calcium-Binding The Bursa of Fabricius and Immunoglobulin Proteins-R. H. KRETSINGER Synthesis-BRUCE GLICK
Responses of Mammary Cells to Hormones-M. Cyclic Nucleotides, Calcium, and Cell R. BANERIEE Division-LIONEL I. REBHUN Recent Advances in the Morphology, His- Spontaneous and Induced Sister Chromatid Exchanges as Revealed by the BUdR-Labeling tochemistry, and Biochemistry of SteroidSynthesizing Cellular Sites in the Testes of Method-HATAo KATO Nonmammdian VertebratesSARDuL S. Structural, Electrophysiological, Biochemical, GURAYA of and Pharmacological Properties Epithelial-Stromal Interactions in Development Neuroblastoma-Glioma Cell Hybrids in Cell Culture-B. HAMPRECHT of the Urogenital TraCt-ciERALD R. CUNHA
CONTENTS OF PREVIOUS VOLUMES Cellular Dynamics in Invertebrate Neurosecretory SyStems-hLAN BERLIND Cytophysiology of the Avian Adrenal Medulla-AsoK GHOSH Chloride Cells and Chloride Epithelia of Aquatic Insects-H. KOMNICK Cytosomes (Yellow Pigment Granules) of Moliuses of Cell Organelles of Anoxic Energy Production-IMRE ZS.-NAGY
Mechanism-HANS-GEoRG SCHWEIGER MANFRED SCHWEIGER
399 AND
SUBJECT INDEX
Volume 52
Cytophysiology of Thyroid Parafollicular Cells-ELADIo A. NUNEZAND MICHAELD. GERSHON Cytophysiology of the Amphibian Thyroid SUBJECT INDEX Gland through Larval Development and Metamorphosis-ELIANE REGARD Volume 50 The Macrophage as a Secretory Cell-Roy C. PAGE,PHILIPDAVIES,AND A. C. ALLISON Cell Surface Enzymes: Effects on Mitotic Activity and Cell Adhesion-H. BRUCEBOSMANN Biogenesis of the Photochemical Apparatus-TIMOTHY TREFFRY New Aspects of the Ultrastructure of Frog Rod Extrusive Organellesin Protists-KLAUS H A U ~ Outer Segments-JORGEN ROSENKRANZ MANN Mechanisms of Morphogenesis in Cell C. BROWN AND RICHARDC. Cultures-J. M. VASILIEVAND I. M. GELIX kctins-JAY HUNT FAND INDEX Cell Polyploidy: Its Religion to Tissue Growth SUBJECT AND I. V. and Functions-W. YA. BRODSKY URYVAEVA Volume 53 Action of Testosterone on the Differentiation Regular Arrays of Macromolecules on Bacterial and Secretory Activity of a Target Organ: The Cell Walls: Structure, Chemistry, Assembly, Submaxillary Gland of the Mouse-MONIQUE and Function-Urn B. SLEYTR CHROTIEN SUBJECT INDEX Cellular Adhesiveness and Extracellular Substrata-FREDERICK GRINNELL Chemosensory Responses of Swimming Algae Volume 51 AND D. c . and Protozoa-M. LEVANDOWSKY Circulating Nucleic Acids in Higher R. HAUSER Organisms-MAURICE STROUN, PHILIPPE Molphology, Biochemistry, and Genetics of ANKER,PIERRE MAURICE,AND PETER B. Plastid Development in Euglena gracilis-V. GAHAN NIGONAND P. HEIZMANN Recent Advances in the Morphology, HisPlant Embryological Investigationsand Fluorestochemistry, and Biochemistry of the Decence Microscopy: An Assessment of veloping Mammalian O V ~ - S A R D U LS. Integration-R. N. KAPIL AND s. c. TIWARI GURAYA The Cytochemical Approach to Hormone Morphological Modulations in Helical Muscles Assay-J. CHAYEN (Aschelminthes and AnnelidajGIuLIo SUBJECT INDEX LANZAVECCHIA Interrelations of the Proliferation and Differentiation Processes during Cardiac Volume 54 Myogenesis and Regeneration-PAvEL P. Microtubule Assembly and Nucleation-MARC RUMYANTSEV W. KIRSCHNER The Kurloff Cell-hTER A. REVELL The Mammalian Sperm Surface: Studies with Circadian Rhythms in Unicellular Organisms: Specific Labeling Techniques-JAMES K. An Endeavor to Explain the Molecular KOEHLER
400
CONTENTS OF PREVIOUS VOLUMES
The Glutathione Status of Ceh-NECHAMA s. Biological and Biochemical Effects of Phenylalanine Analogs-D. N. WHEATLEY AND EDWARD M. KOSOWER KOSOWER Recent Advances in the Morphology, HistoCells and .%neScenCe-ROBERT ROSEN chemistry, Biochemistry, and Physiology of Immunocytology of Pituitary Cells from Teleost J. DOERR-SCHOTT, Interstitial Gland Cells of Mammalian OvaryFishes-E. FOLL~NIUS, SARDUL S. GURAYA AND M. P. DUBOIS Follicular Atresia in the Ovaries of Nonmamma- Correlation of Morphometry and Stereology with Biochemical Analysis of Cell Fractionslian Vertebrates-SRrNrvAs K. SAIDAPUR R. P. BOLENDER Hypothalamic Neuroanatomy: Steroid Hormone Cytophysiologyof the Adrenal Zona FasciculataBinding and Patterns of Axonal ProjectionsGASTONE G. NUSSDORFER,GIUSEPPINA DONALD w . PFAFF AND LILYc . A. CONRAD MAZZOCCHI, A N D VIRCILIO MENECHELLI Ancient Locomotion: Prokaryotic Motility SyStemS-LELENG P. TO A N D LYNNMAR- SUBJECT INDEX CVLIS
An Enzyme Profile of the Nuclear Envelope-I. B. ZBARSKY
Volume 56
SUBJECT INDEX
Volume 55 Chromatin Structure and Gene Transcription: Nucleosomes Permit a New SynthesisTHORUPEDERSON The Isolated Mitotic Apparatus and Chromosome Motion-H. SAKAI Contact Inhibition of Locomotion: A Reappraisal-JOAN E. M. HEAYSMAN Morphological Correlates of Electrical and Other Interactions through Low-Resistance Pathways between Neurons of the Vertebrate Central Nervous S y s t e m - C SOTELO A N D H. KORN
Synapses Of CephalopodS-cOLETTE DVCROS Scanning Electron Microscope Studies on the Development of the Nervous System in Vivo and in Vitro-K. MELLER Cytoplasmic Structure and Contractility in AND Amoeboid Cells-D. LANSING TAYLOR JOHNS. CONDEELIS Methods of Measuring Intracellular CalciumANTHONY H. CASWELL Electron Microscope Autoradiography of Calcified Tissues-ROBERT M. FRANK Some Aspects of Double-Stranded Hairpin Structures in Heterogeneous Nuclear RNAHIROTONAORA Microchemistry of Microdissected Hypothalamic Nuclear Areas-M. PALKOVITS SUBJECT INDEX
