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INTERNATIONAL REVIEW OF CYTOLKIGY, VOL. 75

Structure and Function of Postovulatory Follicles (Corpora Lutea) in the Ovaries of Nonmammalian

Vertebrates

SRINIVAS K. SAIDAPUR’

Department of Zoology, Karnatak University, Dhnvar , India

I. Introduction . . . . . . . . . . . . . . . . . . . . 243 11. Comparative Review . . . . . . . . . . . . . . . . . 244

A. Cyclostomes . . . . . . . . . . . . . . . . . . 244 B. Cartilaginous Fishes . . . . . . . . . . . . . . . . 244 C. Bony Fishes. . . . . . . . . . . . . . . . . . . 250 D. Amphibians . . . . . . . . . . . . . . . . . . . 255 E. Reptiles . . . . . . . . . . . . . . . . . . . . 258 F. Aves . . . . . . . . . . . . . . . . . . . . . 262

111. General Discussion . . . . . . . . . . . . . . . . . 266 A. Structure and Life-span . . . . . . . . . . . . . . 266 B. Endocrine Capacity . . . . . . . . . . . . . . . . 269 C. Terminology . . . . . . . . . . . . . . . . . . 273 D. Functional Significance . . . . . . . . . . . . . . 273

IV. Concluding Remarks . . . . . . . . . . . . . . . . . 279 References . . . . . . . . . . . . . . . . . . . . 280

I. Introduction

The formation of postovulatory follicles andor corpora lutea following ovulation is a widespread phenomenon occurring in the vertebrate ovaries. There is an exhaustive body of literature dealing with the corpus luteum of mammals (references in Amoroso and Perry, 1977; Harrison and Weir, 1977; Rowlands and Weir, 1977). However, very little attention has been given to the study of postovulatory follicles and corpora lutea of nonmammalian vertebrates.

In the past there have been a few reviews dealing with some aspect or another of postovulatory Weal bodies in one or more classes of vertebrate ovaries (Boyd, 1940; Harrison, 1948; Hoar, 1955, 1969; Brambell, 1956; Miller, 1959; Dodd, 1960, 1972a,b, 1975, 1977; Amoroso and Finn, 1962; Franchi, 1962; Perry and Rowlands, 1962; Barr, 1968; Chieffi and Botte, 1970; Lofts and Bern, 1972; Callard et al., 1972a,b,c; Redshaw, 1972; Yaron, 1972; Browning, 1973; Lofts and Murton, 1973; Lofts, 1974; Guraya, 1976; Amoroso and Perry, 1977;

‘This article is dedicated to my “Guru” Dr. V. B. Nadkarni, Professor of Zoology, Karnatak University, Dharwar, India, who generated interest in me for the study of atretic and postovulatory structures in the ovaries of nonmammalian vertebrates.

243 Copyright @ 1982 by Academic F‘ress, Inc.

AU rights of repmduction in any form reserved. ISBN 0 - 1 2 - 3 ~ 7 5 - 5

244 SRINNAS K. SAIDAPUR

Callard and Lance, 1977; Lance and Callard, 1978a). It is apparent from these studies that there is much confusion and controversy with regard to (1) the mode of reorganization of the ovulated follicle, (2) the endocrine nature of postovulatory follicle and corpus luteum, and (3) the functional significances, if any.

This article deals with postovulatory follicle and corpus luteum in the ovaries of nonmammalian vertebrates based on the histological, histochemical, ultrastructural, and biochemical findings of recent years. The mode of reorganization, the endocrine capacity, the life-span, and the functional significance of luteal bodies, and their involvement in the reproduction of nonmammalian vertebrates are discussed. An additional objective of this report is to emphasize the need for more work and point out the lacunae that exist in our understanding of postovulatory follicles and corpora lutea of nonmammalian vertebrates. The problem of follicular atresia in the ovaries of nonmammalian vertebrates has been recently reviewed (Saidapur, 1978).

II. Comparative Review

A. CYCLOSTOMES

The class cyclostomata contains two orders, the Petromyzontia or lampreys and the Myxinoidea or hagfishes. The lampreys mature and spawn only once, whereas the hagfishes breed over several seasons. At ovulation, in lampreys the follicular membranes remain behind in the ovary and their subsequent fate is somewhat variable. The follicular tissues closeup and fluid accumulates in the cavity, but no further development of the resultant structure takes place before the animals die (Lewis and McMillan, 1965). Postovulatory follicles in the Myxine ovary are of two kinds. Some are solid masses of tissues derived from the follicle cells while others are described as fluid-filled cysts (Lyngnes, 1936). In the postovulatory follicles, which are short-lived, the presence of small quantities of cholesterol is demonstrable by the Schultz test (Chieffi and Botte, 1970). Ultrastructural studies on the postovulatory follicles of cyclostomes have not been made.

B. CARTILAGINOUS FISHES

The occurrence of postovulatory follicles in the ovaries of oviparous, ovoviviparous, and viviparous elasmobranchs is well known (Table I). Hisaw and Hisaw (1959), Chieffi (1962), Lance and Callard (1969), and Te Winkel (1972) have described the histological changes during the development and regression of the corpus luteum in four stages, the essential features of which are as follows:

POSTOVULATORY FOLLICLES 245

Stage 1 . This stage is characterized by an extremely thick theca and a folded granulosa which does not completely fill the center of the corpus luteum.

Stage 2. In this stage the granulosa elements completely fill the area enclosed by the thecal elements.

Stage 3. There is an onset of involution as indicated by vacuolization of the granulosa cells and connective tissue infiltration. The theca is much reduced in thickness.

Stage 4. In this stage involution is advanced. The corpus luteum is greatly reduced in size with extreme connective tissue infiltration and degeneration of the granulosa cells.

According to Hisaw and Hisaw (1959) there is very little difference between atresia of a large preovulatory follicle and the sequence of changes that occur in the formation of a corpus luteum subsequent to ovulation. The reaction of granulosa under the two situations is said to be identical. In both these processes there is elaborate folding and formation of phagocytic foam cells that actively ingest yolk and tissue detritus remaining in the ruptured follicle. The phagocytic action of the granulosa lutein cells ceases with the elimination of debris from the lumen, and the corpus luteum may become organized into a compact structure of irregular shape or is seen as a flattened baglike object on the surface of the ovary. When this stage of development is attained (stage 4) it is apparently impossible to distinguish between those derived from atresia of large follicles and those formed following ovulation (Hisaw and Hisaw, 1959; Lance and Callard, 1969). In the ovoviviparous T . murmorutu the postovulatory follicle does not luteinize but rapidly undergoes sclerosis and degenerates (Chieffi, 1962). Also, in this species postovulatory follicles clearly appear to be derived only from the small cells of the granulosa, whereas the theca interna supplies the stroma. By contrast the postovulatory follicle of Scyliorhinus (oviparous) develops from the granulosa and from cuboidal cells of the theca interna which remain separated from the granulosa cells by connective tissue (Dodd, 1960; Chieffi and Botte, 1961; Chieffi, 1962). In Rhinobutus the thecal tissue also contributes to the luteal cell mass (Samuel, 1943a) as also described for S . stelluris and several species of the genus Raja (Chieffi and Botte, 1970). The postovulatory follicles of C. maximum (Matthews, 1950) when fully developed are 4-5 mm in diameter, and they have a central cavity which contains lymphocytes and miscellaneous cellular debris.

Histochemical studies are very limited and the available data indicate species variation with regard to the response of postovulatory follicles to various histochemical tests. According to Chieffi (1962) the granulosa cells of postovulatory follicle in Torpedo show only a weak sudanophilia. The granulosa cells of postovulatory follicles of S. stelluris and S . cuniculu gave possitive results in the Schultz test for cholesterol and/or its esters and in the Ashbel Seligman test for

246 SRINIVAS K. SAIDAPUR

TABLE I NONMAMMALIAN VERTEBRATES IN WHICH POSTOVULATORY FOLLICLES (CORPORA LUTEA)

ARE REPORTED

Species References

Cyclostomes Petromyzon marinus Myxine sp.

Rhinobutus granulatus Squalus ucanthiaf'

Cartilaginous fishes

Cetorkinus m i m u 9 Squalus suckleyP Raja binoculutu Raja erinacea Hydrolugus colloeri Mustelus caniP

Torpedo mannorat& Torpedo ocelluta" Scyliorhinus stellaris

Sryliorhinus cunicula Several Raja species

Gustrosteus uculeutus Bony fishes

Fundulus heteroclitus Scomber scomber Mystus seenghala Pleuronectus platessa Heteropneustes fossilis Tor for Gobius giuris Xenentodon cuncila Eucaliu inconstans Sabustodes paucispini9 Clurias batrachus Acunthobruma terrae-sentae Cichlusomiu nigrofusciatum Glossogobius giuris Chunna gachua Gillichthys mirabilis

Lewis and McMillan (1965) Lyngnes (1936)

Samuel (1943a) Hisaw and Albert (1947);

Hisaw and Hisaw (1959); Lance and Callard ( 1 969)

Mathews (1950) Hisaw and Hisaw (1959) Hisaw and Hisaw (1959) Hisaw and Hisaw (1959) Hisaw and Hisaw (1959) Hisaw and Hisaw (1959);

Te Winkel (1972) a e f f i (1961, 1962) Chieffi (1961, 1962) Chieffi and Botte (196 I );

Chieffi ( 1962) Chieffi ( 1962) Botte (1963)

Craig-Bennet (193 1); Lam et al. (1978a)

Mathews (1938) Bara (1965a,b) Sathyansesan (1 962) Barr (1963) Nair (1963) Rai (1 966) Rajalakshmi ( 1966) Rastogi (1966) Braekevelt and McMillan (1967) Moser (1967) Lehri ( 1968) Yaron (1971) Nicholls and Maple ( 1972) Saksena and Bhargava (1972) Sanwal and Khanna ( 1 972) Vlaming (1972)

(continued)


TABLE I (Continued)

Species References

Brachidanio rerio

Oryzias latipes Hypseleotris galii Trachurus meditarraneus Mystus teengara Carussius auratus

Puntius sophore Oncorynchus kisutch Oncorynchus gorbuscha Salmo gairdneri

Cyprinus carpi0

Amphibians Triturus valgaris Xenopus laevis

Tarichu torosa Neciophrynoides occidentalif

Gastroiheca marsupiata Rana esculenta Triiurus cristatus

Salamandra atr& Salanandra tachetke‘ Salarnandra salamandrff

Rana catesbeiana Necturus maculosus R a m cyanophlyctis

Lambert et al. (1972); Lambert and van Oordt (1974); Van Ree (1976); Lambert (1978);

Iwasaki (1973) Mackay (1973) Bara (1 974) Guraya et al. (1975) Khoo (1975);

Nagahama et al. (1976); Lam et al. (1978b)

Agarwala and Dixit (1 977) Nagahama et al. (1978) Nagahama et al. (1 978) Hurk and Peute (1978);

Lambert et al. (1978); Szollosi et al. (1978)

Guraya and Kaur (1979)

Hett (1923) Cunningham and Smart (1934);

Redshaw and Nicholls (1971) Miller and Robins (1954) Lamotte and Rey (1954);

Lamotte et al. (1956); Gallien (1959); Vilter and Vilter (1960); Xavier (1 970a); Xavier et al. (1970); Xavier and Ozon (1971)

Amoroso et al. (1957) Chieffi and Botte (1963) Botte and Cottino (1964);

Saxena et al. (1977) Vilter and Vilter (1964) Joly (1 964) Joly (1965);

Guraya ( 1968) Kessel and Panje (1968) Saidapur and Nadkarni (1972a,b,

Joly and Picheral (1972)

1974b)

(continued)

248 SRINIVAS K . SAIDAPUR

TABLE I (Continued)

Species References

R a w tigrina Rona verrucosu Cacopus systoma Bufo melanostictus

h c e r f a agitis Lacerta viridis Amphibolxus muricatus Anguis fragiliJ" Zootoca vivipara" Egerina whiter" Lygmoma quoyi" Lygosoma quadridigitatunf Lygosoma weekisae" Lygosoma entrucastraexi" Natrix sipedoff

Reptiles

Crotalus terri9cu P Bothrops jarararff Bothrops alternard' Hoplodact);lus maculatuf' Hemidact+s flaviviridis

Terrapane caroiina Uta stanshunana stejnegeri Leolopismu rhomboid& Uma scoparra Takydromu F tachydromoides Dipsoraurus dorsalis Chelydra rerpentina

Saidapur and Nadkarni (1974a,b) Saidapur and Nadkarni (1974a,b) Saidapur and Nadkarni (1974a,b) Kanamadi and Saidapur (1980)

Hett ( 1924) Cunningham and Smart (1934) Weekes ( 1934) Cunningham and Smart (1934) Cunningham and Smart (1934) Weekes ( 1934) Weekes ( 1934) Weekes (1934) Weekes ( 1934) Weekes ( I 934) Rahn (1939);

Fraenkal et al . (1940) Fraenkal et al. (1940) Fraenkal ef al . ( 1940) Boyd (1 940) Dutta (1946);

Callard (1966)

Guraya and Varma (1976, 1978); Gouder and Nadkarni (1 976)

Altland (1951) Tinkle (1961) Wilhoft (1963) Mayhew ( 1966) Telford ( 1969) Mayhew (1971) Klicka and Mahmoud (1972, 1973);

Cyrus et al. (1978)

(continued)

carbonyl groups (Chieffi, 1962). Also, in the postovulatory follicle of S. stellaris a positive 3P-HSDH activity has been reported (Lupo et ul., 1965). However, enzyme activity could not be localized in the postovulatory follicles of T. mar- morutu (Lupo et al., 1965). Botte (1963) also reported positive histochemical demonstration of cholesterol in the postovulatory follicles of several species of Raja (all oviparous) but did not carry out any enzyme histochemistry. In S. acanrhias intense 3P-HSDH in the granulosa lutein cells of stage 1 postovulatory follicle has been reported (Lance and Callard, 1969). The enzyme activity de-


TABLE I (Continued)

Species References

Calotes versicolor

Chameleon calcuratus Lepidodactylus lugubris Chrysemys pictu Mabuya curinatu Psammophilus dorsalis Enhydrina schistosd Hydrophis cyunocinctud' Thumnophis radif Thamnophis sirtalif Xantusia vigil# Natrix rhombiferrP Chalicides ocellatud' Lacerta vivipara Sceloporus jarrovP Sceloporus cyunogenyf' Mabuya capensif Storeria dekayiP

Varma (1970); Varma and Guraya (1975); Gouder and Nadkarni (1976)

Gouder and Nadkami (1976) Jones et al. (1 978) Lance and Callard (1978b) Shekarappa and Sarkar (1978) Shivakumar et al. (1979) Samuel (1944) Samuel (1944) Cieslak (1945) Bragdon (1951, 1952) Miller (1948) Betz (1963) Badir (1 968) Morat (1969) Goldberg (1970) Callard et al. (1972a,b,c) Yaron ( 1 972) Colombo and Yaron (1976)

Aves Gallus domesticus Pearl and Boring (1918);

Stieve (1918); Hett (1923); Yocom (1924); Fell (1925); Davis (1942); Chalana and Guraya (1978)

Columba livia Agelaius tricolor Payne (1966) Agelaius phoenicus Payne (1966) Pica pica hudsona Erpino (1 969) Coturnix coturnix japonica Sayler et al. (1970) Parrus domesticus Guraya and Chalana (1976)

Dominic (1960); Bhujle et al. (1979a,b)

Live bearing (ovoviviparoushiviparous) .

creased in stages 2 and 3 and as the gestation proceeded. A similar pattern of distribution was also reported for the G-6-PDH enzyme (Lance and Callard, 1969). These authors also found an intense lipid staining with Fat red 7B in the granulosa lutein cells of the postovulatory follicles. Further, the cytoplasm of these cells contained discrete round lipid droplets. Cholesterol was also found in the granulosa cells of stage 1 to 4 postovulatory follicles. According to Hisaw and Hisaw (1959) the granulosa cells seem to continue to secrete yolk for some- time after the ovulation.


The postovulatory follicles of T . marmorata, when separated from the main ovarian mass and tested for biosynthetic capacity, did not produce progesterone but could synthesize estrogens whereas those of S . srellaris yielded progesterone and no estrogens under identical conditions (Lupo, 1968).

C . BONY FISHES

The histological and/or histochemical changes occurring during the formation and resorption of postovulatory follicles has been reported in several species of bony fishes (Table I ) . The organization of the postovulatory follicle and its fate subsequent to ovulation seems to vary considerably in different species. In a majority of the oviparous teleosts the postovulatory follicle does not become reorganized into a “corpus luteum-like” structure, but rather collapses and is rapidly absorbed. In some species the postovulatory follicle does develop into a glandular appearing structure (Fig. 1 ) . According to Barr (1963) in P . plafessa the ruptured follicle left after ovulation does not form a corpus luteum. It shrinks very considerably and appears as a pocket in epithelial lining of the ovarian cavity. The follicle is bounded by thecal cells which have an irregular arrangement due to contraction of the follicle. Mitotic divisions are absent in granulosa

FIG. 1. T.S. of C. batrachus ovary showing newly formed (8-10 hours after ovulation) postovulatory follicles (PF). Note the hypertrophied granulosa cells ( G ) which are columnar having basal spherical nuclei and prominent nucleoli. Hematoxylin and eosin. X 150.

POSTOVULATORY FOLLICLES 25 1

but are occasionally seen in the theca, but there is no indication that this tissue hypertrophies. About 2 months after spawning the follicles are present as small accumulations of thecal cells which eventually disappear completely. In G. giuris (Rajalakshmi, 1966) also the postovulatory follicles undergo resorption without forming a corpus luteum. Sathyanesan (1962) in M. seenghala, Nair (1963) in H. fossilis, and Mackay (1973) in H. galii have also denied the formation of functional corpora lutea. Barr and Nair even denied the hypertrophy of the follicular membranes after ovulation. In T. tor (Rai, 1966) there is a hypertrophy of the granulosa cells, and accompanied by the thecal tissues they fill in the evacuated lumen of the discharged follices. In C. batrachus (Lehri, 1968; D. K. Tikare and V. B. Nadkarni, unpublished observations) the granulosa and theca proliferate, hypertrophy (Fig. l ) , and invade the lumen of the postovulatory follicles and occupy most of the space. Similarly, in C. gachua (San- wal and Khanna, 1972) the follicular cells proliferate and vacuoles appear in them. These are ultimately absorbed by the ovarian tissues and disappear. In the goldfish C. auratus following ovulation, both thecal and granulosa cells hypertrophy, with more marked response in the granulosa (Khoo, 1975). The granulosa cells contain yellow luted pigments. According to Khoo (1975) the granulosa lutein cells eventually dzferentiate into oogonial cysts. Guraya and Kaur (1979) have described the histological changes occumng in the postovulatory follicles of C. carpio, the salient features of which are as follows:

Stage 1. After ovulation, the postovulatory follicle shows a large cavity surrounded by the hypertrophied granulosa and thecal layers. The granulosa cells are columnar in shape with basal spherical nuclei. The theca becomes thicker and consists of fibrous elements and connective tissues. Some of the thecal cells appear glandular. The theca is highly vascular.

Stage 2. In this stage the size of postovulatory follicles is reduced. The granulosa layer forms villus-like projections which are irregularly placed in the lumen. These cells are further hypertrophied and their cytoplasm is eosinophilic. There is an increase in the thecal vascularity.

Stage 3. In the third stage, the lumen of the postovulatory follicle is further reduced due to continued shrinkage. The granulosa cells appear the same as in the previous stage. However, there is no vascularization of the granulosa cells but they contain a few blood cells. The thecal gland cells become vacuolated.

Stage 4. The hypertrophied granulosa lutein cells form a mass and occupy most of the lumen which is drastically reduced. The postovulatory follicle becomes a multilayered structure and contains several blood cells. There is an increase in the pycnosis of granulosa cell nuclei. The histological features of the thecal layer remain the same as those described in the previous stages.

Stage 5. In this stage the postovulatory follicle is greatly reduced. The


granulosa cells are randomly arranged and seperated from each other suggesting dissolution of intercellular cohesion between them. All the nuclei are pycnotic. The thecal vascularity is decreased and it becomes more fibrous.

Stage 6. The postovulatory follicle in this stage is reduced to a very small structure. The number of granulosa lutein cells is decreased due to degeneration. The thecal layer invades the residual luted cells. The thecal vascularity is greatly reduced.

It is evident from the literature cited above that although there are numerous reports on the postovulatory follicles of fishes the descriptions of the histological changes are very brief. In a lone study by Szollosi et al. (1978), there is a good description of the thecal tissue of the pre- and postovulatory follicles in the trout S. gairdneri. Thecal cells in this species are morphologically smooth muscle as at the time of ovulation. The physiological behavior of this tissue confirms its smooth muscle nature. After ovulation the thecal cells start phagocytozing the adjacent collagen. The collagen bundles at first are partially surrounded by the cellular processes of the thecal cells and later they are interiorized. The number of collagen fibeis per phagocytic vesicle varies from one to many. The bundles can be morphologically recognized until 72 hours after ovulation, after which time they become indistinct indicating a possible hydrolysis. These authors (Szollosi et al . , 1978) have also shown the presence of a number of lytic enzymes in postovulatory follicles employing “Api-zim” tests in support of their morphological observations.

In recent years, the ultrastructural details of postovulatory follicles in C . nigrofasciatum (Nicholls and Maple, 1972), the goldfish C. auratus (Nagahama et al., 1976; Lambert et al . , 1978), 0. kisutch and 0. gorbuscha (Nagahama et al., 1978), B . rerio (Lambert, 1978), and S. gairdneri (Hurk and Peute, 1979) have become available.

1. Ultrastructure of Postovulatory Follicles of C. auratus 6 to 10 Hours after Ovulation

The postovulatory follicles at this stage are characterized by hypertrophied granulosa cells surrounding a cavity. These are cuboidal or columnar in shape and their cytoplasmic processes project into the follicular lumen. The nucleus with one prominent nucleolus is basally located. The granulosa cells are con- nected in apical regions by specialized attachments; wide lateral intercellular spaces are seen between them. These granulosa cells unlike those of the preovulatory follicles now contain numerous lipid droplets occurring mostly in the basal cytoplasm. The mitochondria are oval or elongated in shape and their cristae are usually lamelliform. They are mainly distributed in the basal cytoplasm together with the lipid droplets. A rough endoplasmic reticulum occurs throughout the cytoplasm but is more frequently seen in the basal cytoplasm around the nucleus. Smooth endoplasmic reticulum is rarely seen, in the apical


cytoplasm. The extensive Golgi apparatus containing lysosome-like bodies and microtubules occur in the apical cytoplasm.

In this stage, the thecal layer contains enlarged blood vessels with homogeneous spaces between the cells. The theca interna cells with deeply invaginated nuclei are irregular in shape and often have long cytoplasmic processes.

These cells appear somewhat oval in shape, seem to decrease in size, and lose some of the tubular cristae. The smooth endoplasmic reticulum appears reduced in volume when compared to the special thecal cells of preovulatory follicles. A fragmentary rough endoplasmic reticulum and Golgi apparatus are still present; lipid droplets are rarely seen.

The granulosa cells are collapsed into the follicular lumen forming an irregular cellular mass and showing various degenerative features. The mitochondrial cristae still show a typical tubular arrangement; the lipid droplets are irregular and vary in size and density. In some granulosa cells cellular organelles and inclusions fuse to form large masses of electron-dense materials. Many lysosome-like bodies appear in the Golgi area. The thecal layer is often vacuolated with many enlarged blood capillaries. The special theca cells exhibit more degenerative features than those of the previous stage.

a. Thecal Cells.

b. Special Theca Cells.

c. About 30 Hours after Ovulation.

2 . Postovulatory Follicles of 0 . kisutch and 0 . gorbuscha

The postovulatory follicles of salmonids are readily distinguishable from the remainder of the ovarian tissues by the naked eye; the follicles are flattened, opaquely white oval bodies with a maximum diameter of 2 to 3 mm. Ultrastruc- tures of postovulatory follicles are essentially identical in both the coho and pink salmon. The basic organization is similar to the preovulatory follicles; the granulosa layer remains separated from the thecal layer by a thick heavily convo- luted basement membrane. The most striking feature of the postovulatory follicles is the thickening of the thecal layer which ranges from 50 to 70 Fm and contains enlarged blood vessels and numerous fibroblastic cells with deeply invaginated nuclei. Granulosa cells are cuboidal with short cytoplasmic projections extending into the follicular lumen. The apical cytoplasm contains a few Gold bodies and numerous small spherical vesicles. Granulosa cells show cytoplasmic vacuoles of various sizes and many lysosome-like bodies which are indi- cations of degeneration. Cellular organelles and inclusions often coalesce to form large masses of electron-dense material. Various degenerative changes may be seen in the nucleus and mitochondria.

Special Thecal Cells. These are usually located close to the surface of the postovulatory follicles and often form small clusters consisting of several round or oval cells. Their nuclei are centrally located. Although most cellular organelles seem unchanged from those of preovulatory follicles, some differences are

2 54 SRINIVAS K. SAIDAPUR

seen; the endoplasmic reticular system becomes more prominent, and some of the peripherally situated smooth endoplasmic reticulum forms concentric whorls. Lipid droplets appear in some cells.

3 . Postowlatory Follicles of S . gairdneri and C . nigrofasciatum

The postovulatory follicles of S. gairdneri have a folded granulosa which is continuous with the germinal epithelium. A large number of special thecal cells are present in the theca of the postovulatory follicles. The granulosa cells of postovulatory follicles contain mitochondria with tubular cristae and some smooth endoplasmic reticulum. Similarly, special thecal cells of postovulatory follicles also contain mitochondria with tubular cristae and smooth endoplasmic reticulum. In C . nigrofusciaturn the mitochondria of granulosa cells have lamellar cristae but contain an abundance of smooth endoplasmic reticulum (Nicholls and Maple, 1972).

4. Histochemisuy

Histochemical studies to determine the steroidogenic ability of the postovulatory follicles of fishes are limited, the postovulatory follicles of H. gulii reacted negatively to the Schultz test suggesting the absence of steroid precursors in them

FIG. 2. Cryostat section of C . buwuchus ovary showing A5-3P-HSDH activity in the grmulosa lutein cells (G) of a postovulatory follicle (PF) (substrate used, Dehydroepiandrosterone). x 150. (Photomicrographs courtesy of Drs. D. K. Tikare and V. B. Nadkami, Kamatak University, Dhanvar, India.)


(Mackay, 1973). In 0. latipes and B . rerio only the granulosa cells of postovulatory follicles contain 3P-HSDH activity (Iwasaki, 1973; Lambert and van Oordt, 1974). In S. gairdneri in addition to the granulosa cells the special thecal cells of postovulatory follicles contain 3P-HSDH and G-6-PDH enzyme activities (Hurk and Peute, 1979). In others such as S . scomber (Bara, 1965a,b), T . mediterraneus (Bara, 1974), and C. auratus (Khoo, 1975; Nagahama et al . , 1976) both granulosa and theca cells possess 3P-HSDH activity. In T. mediter- runeus 1 1P-HSDH, 20cw-HSDH, and G-6-PDH have also been recorded in the postovulatory follicles (Bara, 1974). In B. rerio G-6-PDH, 3P-HSDH, and acid phosphatase activities were strong in the granulosa cells (stronger than that found in preovulatory granulosa) immediately after oviposition (2 hours after ovulation) and diminished in 2 or 3 days (Lambert and Oordt, 1974; Van Ree, 1976). Later, postovulatory follicles could not be recognized any more. In S. gairdneri 3p- HSDH was weak in granulosa but strong in the special theca cells of the postovulatory follicles whereas G-6-PDH was strong in both these cell types (Hurk and Peute, 1979). In C. butrachus the G-6-PDH and 3P-HSDH (Fig. 2) activities are maximum on the day of spawning and gradually decrease thereafter as reported for the zebrafish (D. K. Tikare and v . B. Nadkarni, unpublished observations).

D. AMPHIBIANS

The postovulatory follicles have been recorded in the ovaries of several amphibians (Table I). The histological changes that take place in the postovulatory follicles of oviparous Anura (Fig. 3) have been described in 3 or 4 stages (Cunningham and Smart, 1934; Guraya, 1968; Saidapur and Nadkarni, 1972a). The salient features of these stages are as follows:

Stage 1. The first stage is represented by the newly ruptured follicles, the follicular epithelium of which is almost similar to that of the preovulatory follicles; however close examination reveals that the granulosa cells have begun to hypertrophy. The cytoplasm may appear vacuolated and outlines are indistinct. The thecal wall consists of connective tissue containing scattered nuclei of normal appearance. The wall contains capillaries with blood, the tissue is vacuolated, and the inner layer is formed of a dense continuous membrane.

Stage 2. In this stage both granulosa and thecal cells are relatively more hypertrophied (Fig. 3). The follicle becomes collapsed and folded due to contraction. Nuclei of the granulosa lutein cells may show a vacuolated appearance or even be broken up into small particles. The thecal wall consists of fibrous tissue and blood capillaries. Its inner layer forms a distinct rather thick membrane.

Stage 3. In the third stage the granulosa becomes multilayered due to the further shrinkage or contraction of the follicle and the cavity of the postovulatory

1


follicles is almost filled up by the hypertrophied granulosa cells. The hypertrophy of the theca is maximum at this stage.

Stage 4. In this stage there is a further contraction of the follicle and the granulosa cells are separated from each other and are irregularly distributed. There is a further reduction in the size. The thecal wall is like a double membrane with a narrow space between, the inner wall being the theca interna. This wall is continued into a thicker mass of nucleated connective tissue forming a part of the ovarian wall where the aperture of rupture, now closed, was situated. The subsequent stages are marked by the degenerative changes.

The postovulatory follicles of ovoviviparous and viviparous urodeles also form in the same way as in oviparous anuran forms. However, the postovulatory follicles in these are larger in size and show a better organization (in terms of the hypertrophy of the theca and luteinization of the granulosa cells) and persist for longer duration.

It should be noted that the enlarged thecal cells remain at the periphery and do not invade the hypertrophied.granulosa cells at any stage of the development and regression of the postovulatory follicles in Amphibia.

The ultrastructural details of the postovulatory follicles have now become available for two species. Kessel and Panje (1968) have provided a detailed

FIG. 3. T.S. of R . verrucosa ovary showing the stage 3 postovulatory follicle attached to the ovarian epithelium. Note the hypertrophy of both granulosa (G) and theca (T) cells. Hematoxylin and eosin. ~ 1 5 0 .

F'OSTOVULATORY FOLLICLES 257

description of the postovulatory follicles of N. maculosus. The postovulatory follicle in this species consists of at least three types of cells each of which is active over a considerable period of time. Of these, two types of cells contain a predominantly smooth endoplasmic reticulum and mitochondria with tubular cristae, well-developed Golgi complexes, and lipid droplets. Similarly, electron microscopic studies of Joly and Picheral (1972) in S. salamandra show that the granulosa cells of the postovulatory follicle contain many liposomes, smooth endoplasmic reticulum, and mitochondria with tubular cristae, features typical of steroidogenic cells. The thecal cells consist of collagen fibers and fibroblasts. There is a thickening of theca and hypertrophy of the granulosa cells.

There are some good histochemical reports that deal with the steroidogenic ability of the postovulatory follicles in several species. According to Guraya (1968) in R. catesbeiana after ovulation the granulosa cells undergo transient hypertrophy or luteinization which is closely accompanied by the development of the diffuse lipoproteins throughout the cytoplasm. The cytoplasm of these cells contain lipid droplets consisting first of phospholipids, then phospholipids and triglycerides, and finally triglycerides, phospholipids, pigments, and some cholesterol or its esters. There was an increase in the lipid content from stage 1 to stage 4. Similarly, sudanophilic lipid droplets are found in the granulosa lutein cells of S. tachetee (Joly, 1964), N. maculosus (Kessel and Panje, 1968), N.

FIG. 4. Cryostat section of R . rigrina ovary showing A5-3P-HSDH activity in the granulosa (G) lutein cells of a newly formed (10-12 hours old) postovulatory follicle. The enzyme activity is absent in theca (T) (substrate used, pregnenolone). x50.


occidentalis (Xavier et al., 1970), S . salamandra (Joly and Picheral, 1972), and in the toad B. melanostictus (R. D. Kanamadi and S. K. Saidapur, unpublished). The postovulatory follicles of the above species also contain cholesterol or its esters as evidenced by the Schultz test. More recently 3P-HSDH andor other enzymes (such as 17P-HSDH, G-6-PDH etc.) involved in steroidogenesis have also been demonstrated in the granulosa lutein cells (Fig. 4) of R. esculentu and T . cristatus (Chieffi and Botte, 1963; Botte and Cottino, 1964; Saxena et al., 1977), N . maculosus (Kessel and Panje, 1968), S . salamandra (Joly, 1965; Joly and Picheral, 1972), X. laevis (Redshaw and Nicholls, 1971), N. occidentalis (Xavier et al . , 1970). R . cyanophlyctis, R. tigrina, R . verrucosa, and C . systoma (Saidapur and Nadkarni, 1972b, 1974b), and B. melanostictus (Kanamadi and Saidapur, 1980). Joly (1965) reported a weak 3P-HSDH activity in thecal cells of postovulatory follicles in the salamander.

Studies on the in vitro steroidogenic ability of isolated postovulatory follicles of amphibian ovary are lacking. However, 3P-HSDH and G-6-PDH enzyme activities of the postovulatory follicles have been biochemically determined in N. maculosus (Kessel and Panje, 1968).

E. REFWLES

There are several studies dealing with the postovulatory follicles or corpora lutea in the ovaries of oviparous, ovoviviparous, and viviparous reptiles (Table I). Descriptions of histological changes occurring during the formation and degeneration of corpus luteum are available for several reptilian species (Cunnin- gham and Smart, 1934; Weekes, 1934; Boyd, 1940; Miller, 1948; Altland, 1951; Betz, 1963; Goldberg, 1970; Guraya and Varma, 1976), the salient features of which are as follows:

Stage 1. The first stage is represented by a newly ovulated follicle consisting of a large cavity and having a wide opening on one side through which the egg has escaped. After ovulation the postovulatory follicle shrinks in size due to the contraction of its various layers resulting in the thickening and folding of its wall. The granulosa which was single layered in the preovulatory follicle becomes multilayered in the postovulatory follicle apparently owing to contraction since mitosis is not seen in its cells. The granulosa cells, which are oval or flat prior to ovulation, become spherical and increase in size due to hypertrophy. A great majority of them are of large size but interspersed between them are the small cells. The granulosa luteal cells have centrally located, rounded or ovoid nucleus which contains 1-3 nucleoli. Their abundant cytoplasm is deeply eosinophilic.

The thecal layers also undergo rapid morphological changes and form well- developed layers the thickness of which may vary in different places. The theca externa is thinner than the intema or equal at some places. The two thecal layers

F'OSTOVULATORY FOLLICLES 259

FIG. 5 . T.S. of C. versicolor ovary showing a developing corpus luteum. Note the presence of a slit (arrow) and its inner margins that curl in and project into the luted tissues (G). X50. (Courtesy of Dr. B. Y. M. Gouder, Karnatak University, Dharwar, India.)

are separated at some places by large spaces containing blood cells and macrophages. Some fibroblasts with deeply stained nuclei constitute the boundaries of these spaces. The theca interna is relatively more cellular than the fibrous externa and consists mainly of hypertrophied round cells and fibroblasts. Some collagen fibers and macrophages are also present. The blood cells and macrophages are abundant between theca interna and granulosa cells. Septa of collagen fibers arise from theca interna and invade the area of luteal cells.

Stage 2. The granulosa cell mass becomes multilayered and further hypertrophied.

Stage 3. The opening or slit at this stage narrows considerably and its inner margin curls in and projects into the luteal tissue (Fig. 5 ) . The postovulatory follicle is further reduced in size. There is also further hypertrophy of the granulosa luteal cells which spread toward the lumen and obliterate it. Septa of thecal origin along with blood capillaries invade the hypertrophied luteal cell mass.

Stage 4. The aperture is completely closed and the lumen is very much reduced. Large blood vessels filled with blood cells are found in the luted tissue in some species. The inner border of theca interna due to the presence of deeply stained nuclei is quite distinct as in the previous stages. The hypertrophied granulosa luted cells possess a large nucleus and abundant eosinophilic cytoplasm. The corpus luteum is now a compact solid structure having 3 distinct zones. The outermost zone is the theca externa which is of loose consistency. It


contains collagen, fibers, some hypertrophied cells with clear cytoplasm, and fibroblasts. The theca intema, the second zone, is twice as thick as the theca externa. The theca interna is more cellular, contains a large number of fibroblasts and some collagen fibers. Their nuclei stain deeply. The third or the innermost zone is composed of a solid mass of granulosa luteal cells which are very compactly arranged. The fully developed corpus luteum is richly vascularized (in some species).

Involution. The subsequent stages are characterized by the degnerative changes in the luteal cell mass. The most important sign of degeneration is the appearance of vacuoles in the cytoplasm of these cells. The nuclei become pycnotic. Macrophages become numerous in the degenerating luteal tissue. The theca interna cells also become vacuolated. The theca externa becomes thinner and fibrous. During advanced stages of degeneration the corpus luteum is greatly reduced in size and its central region is filled with a mass of vacuolated luteal cells with shrunken nuclei. The thecal layers become densely fibrous and can no longer be distinguished into two separate layers. The regression of corpus luteum is accompanied by the accumulation of pigments. Finally few cells remain en- tombed within the thin theca in the stroma of the ovary. Such structures are comparable to corpus albicans of the mammalian ovary.

There is some species variation with regard to the participation of theca interna and vascularization in the development of reptilian corpus luteum. The luteal elements are stated to be formed exclusively from the granulosa cells in most species although participation of the theca interna has been described in L . agilis (Hett, 1924) and 7'. carolina (Altland, 1951). The theca (interna) provides mostly the supporting tissue. The thecal layers remain distinct and do not undergo hypertrophy or luteinization to form paraluteal cells (or theca luteal cells). The septae of the theca interna alongwith blood vessels may invade the luteal cell mass which is formed by the luteinization of granulosa cells. However, the septa1 and vascular supply invasion of luteal tissue is believed to be absent in some forms (Weekes, 1934; Rahn, 1938; Boyd, 1940) which needs further confirmation according to Guraya (1976).

1 . Ultrastructure of the Corpus Luteum of the Snapping Turtle ( C . serpentina)

Cyrus el a f . (1978) made a detailed study of granulosa cells of pre- and postovulatory follicles of C . serpenrina. These authors have mainly described the presence or absence of the ultrastructural features known to be associated with steroidogenesis. Granulosa cells of the preovulatory follicle are elongated, contain large nuclei, and their endoplasmic reticulum is mainly granular in nature. Smooth endoplasmic reticulum is scarce. The mitochondria are elongated with lamellar cristae. A few lipid droplets are encountered. After ovulation the follicular wall becomes thicker and is thrown into folds. These folds disappear as the follicle is transformed into the disc-shaped corpus luteum. Luteinization of


granulosa cells begins soon after ovulation and is characterized by an increase in lipid droplets. The granulosa lutein cells lack blood vessels. Shortly after the formation of the corpus luteum, the theca interna produces septae composed of collagenous fibers, fibroblasts, and small blood vessels; the septae gradually penetrate into the region of granulosa lutein cells. Granulosa lutein cells ( 18 pm in diameter) are polygonal in shape with an eccentric nucleus. The nucleus is round or oval and contains one or two nucleoli. The endoplasmic reticulum is primarily agranular in nature. The mitochondria are spherical, oval, or rod shaped and possess tubulovesicular cristae. Often the mitochondria are swollen and are found associated with the lipid droplets. Golgi complexes are common and lie mostly near the nucleus. The lipid droplets are abundant.

The corpus luteum in C. serpentina lives for approximately 5 to 6 weeks. While the eggs are still in oviducts, degeneration of the corpus luteum begins and continues for about 2 weeks after oviposition. The first sign of degeneration is an increase in the amount of connective tissue reaching the granulosa lutein cells from the septae as well as the amount of connective tissue bordering the central cavity. As a result, most of the granulosa lutein cells are isolated into clusters by the surrounding connective tissue. With the onset of regression, there is a general disruption of the endoplasmic reticulum and mitochondria. The tubular endoplasmic reticulum becomes extremely vesiculated. The Golgi bodies disappear. There is no significant change in the number of lipid droplets in the degenerating granulosa lutein cells. During the final stages of degeneration the cells disinte- grate and are replaced by connective tissue. With the completion of regression the corpus luteum eventually disappears and the remaining connective tissue becomes part of the ovarian stroma.

2. Histochemistry

Histochemical studies have demonstrated the presence of various enzymes associated with steroid synthesis in the granulosa lutein cells of reptilian corpus luteum. Although, 3P-HSDH is the enzyme mainly recorded, other enzymes such as 17/3-HSDH, llP-HSDH, G-6-PDH (Fig. 6), LDH, and NADH dia- phorase have been recorded in some instances, and the species studied include L. sicula (Botte and Delrio, 1965; Callard et al., 1976), N . sipedon (Callard, 1966), L. vivipara and I/. aspis (Morat, 1969), S . cyanogenys (Callard et al., 1972c), M. capensis (Yaron, 1972), C. versicolor, H. jlaviviridis, and C . cal- caratus (Gouder and Nadkami, 1976), M. carinata (Sekarappa and Sarkar, 1978), and Psammophilus dorsalis (Shivakumar et al . , 1979).

The luteinization of granulosa cells following ovulation is accompanied by the synthesis of abundant diffuse lipoproteins containing free phosphilipids and discrete lipid bodies of varying sizes. Subsequently the luteal cells contain phospholipids, neutral lipids, cholesterol, and/or its esters and some pigments (Guraya, 1973; Varma and Guraya, 1975; Guraya and Varma, 1976). Thecal cells do not develop these features in H . jlaviviridis and C. versicolor (Guraya,


FIG. 6 . Cryostat section of C . versicolor ovary showing a newly formed corpus luteum and an intense C-6-PDH activity in the granulosa (C) lutein cells. The enzyme activity is weak in theca intema (arrow) cells and absent in theca extema (TE) cells. X50. (From Gouder and Nadkami, 1976, with permission from CSIR, New Delhi.)

1976). However, Callard et al. (1972c), observed intense 3P-HSDH activity in the inner thecal elements at least during the early part of gestation in iguanid lizards. Gouder and Nadkarni (1976) also noted a weak 3P-HSDH activity in the theca intema cells in the three species they studied.

Biochemical studies have shown that the corpora lutea in the snapping turtle, C. serpentinu, are capable of converting labeled cholesterol into pregnenolone and progesterone (Klicka and Mahmoud, 1972, 1973). Further, the postovulatory follicles and corpora lutea of the turtle produce significant amounts of progesterone in vitro in response to homologous as well as heterologous (ovine) gonadotropins (Licht and Crews, 1976). Similarly, Callard et ul. (1976) demonstrated that luteal tissue of C. picru converts pregnenolone into progesterone in ~ i t r o in response to the luteinizing hormone. Further, enzyme-dispersed luteal cells of C. picta secrete progesterone in response to dibutyryl cyclic AMP (Lance and Callard, 1978b). Interestingly, the corpus luteum of the snake S . dekayi can secrete 1 I-deoxycorticosterone (Colombo and Yaron, 1976).

E. AVES

Reports on the avian postovulatory follicles are limited to a few species only (Table I). The histological changes occurring during the life-span of the post-


ovulatory follicle may be classified into following stages which is mainly based on that given for the pigeon (Dominic, 1960; Bhujle et al., 1979a,b).

Stage 1. Following ovulation the newly formed postovulatory follicle (Fig. 7) shrinks in size rapidly due to the contraction of the theca. The granulosa cells begin to hypertrophy, and some of them get detached and lie freely in the lumen which is large. The membrana propria is thin and wavy lying between the theca interna and granulosa layers. The theca interna has an inner cellular and outer fibrous layers. Blood capillaries and nonvascular spaces are common in both the thecal layers but those in the inner layer are more gorged with blood. Erythro- cytes resulting from the hemorrhage of thecal capillaries may also be seen among the granulosa cells.

Stage 2. The hypertrophied granulosa cells become multilayered and have a syncytial appearance. Vacuoles appear in these cells. Yolk globules are some- time present in the lumen and in the granulosa cells bordering the lumen. An important feature of this stage is the thickening of the membrana propria which now appears thick and wavy between the theca interna and granulosa. This layer is broken up at many places and is seen to pucker into the granulosa cell mass. Theca interna cells also show a vacuolated appearance. Hemorrhage of thecal capillaries lets the erythrocytes into the follicular lumen as well as into the

FIG. 7. Portion of the newly formed postovulatory follicle of hen (T.S.) showing a large lumen (L), hypertrophied granulosa (G), and thecal cells. TI, theca interna: TE, theca externa. Hematoxylin and eosin. X 150.

264 SRINIVAS K. SAIDAF’UR

granulosa layers. The theca externa becomes more fibrous and its capillaries have a shrunken appearance.

Stage 3. There is a further shrinkage of the postovulatory follicle narrowing the lumen which is now filled with the granulosa lutein cells. The rupture point is closed. The granulosa cells are highly vacuolated and may contain pigments. The membrana propria which is thick at first begins to fragment and gradually disappears. The fibroblasts invade the theca interna and the granulosa lutein mass.

Stage 4. The postovulatory follicle is greatly reduced in size. The granulosa cells have pycnotic nuclei and reticulate cytoplasm, which is further vacuolated. Both thecal layers are very fibrous and are no longer distinguishable as separate layers. The thecal capillaries become greatly shrunken and are also not distinguishable.

Subsequent stages are more degenerative and often it is difficult to distinguish them from the last stages of atretic follicles. When the regression is complete the remains of the postovulatory follicles become part of the ovarian stroma.

According to Pearl and Boring (1918) “luteal” cells of postovulatory follicle in the hen arise from theca interna cells. Similarly, Guraya and Chalana (1975) in the sparrow and Chalana and Guraya (1 978) in fowl reported that both thecal and granulosa cells contribute to the formation of the corpus luteum. Also, granulosa luteal and thecal cells in these species show complete intermixing with each other in later stages.

Ultrastructural studies of avian postovulatory follicles are limited to G. dornes- ticus (Wyburn et al., 1966; Gilbert, 1968). Accordingly, in the newly formed postovulatory follicle the basement membrane becomes folded and granulosa cells clump together. The granulosa cells 24 hours later are filled with smooth endoplasmic reticulum and lipid droplets of irregular outline which also vary in size and density. Two days after ovulation the granulosa lutein cells are very randomly arranged, vacuolated, and possess large lipid droplets. However, these cells can be identified up to 72 hours. During the 72-hour period the cytoplasm becomes progressively replaced by lipid. These changes in intracellular structure may indicate a shift in function toward production of progesterone followed by fatty degeneration preceding their final disintegration (Wyburn et al., 1966).

The postovulatory follicle undergoes a series of morphological and histochemical changes leading first to its formation and subsequently to its involution. Sudanophilic lipids and cholesterol or its esters have been recorded mainly in the granulosa lutein cells and to some extent in the theca (interna) lutein cells of hen, sparrow, and blackbird ovaries (Botte, 1961, 1963; Floquest and Grignon, 1964; Chieffi and Botte, 1965; Payne, 1966; Woods and Domm, 1966; Guraya and Chalana, 1975: Chalana and Guraya, 1978). The newly formed postovulatory follicles (stages 1 and 2) in these species show the accumulation of diffuse


FIG. 8. Cryostat section of postovulatory follicle of hen showing intense A5-3P-HSDH activity in the granulosa lutein cells (G) and to some extent in the theca interna cells (TI). TE, theca externa (substrate used, dehydroepiandrosterone). X 150.

lipoproteins, and discrete lipid droplets consisting of triglycerides, cholesterol or its esters, and some phospholipids. In later stages which are characterized by degenerative changes, the luteal cells accumulate coarse lipid droplets, consisting of cholesterol, phospholipids, and pigments. Theca luteal cells of sparrow persist for some time after the degeneration of granulosa cells. The postovulatory follicles of hen, quail, and pigeon have also been shown to contain 3P-HSDH (Fig. 8), 17P-HSDH, 1 lP-HSDH, and G-6-PDH, enzyme activities which occur prin- cipally in granulosa cells but are weakly represented in the theca interna cells (Arvy, 1962; Chieffi and Botte, 1965; Woods and Domm, 1966; Sayler et al., 1970; Armstrong et al., 1977; Chalana and Guraya, 1978; Bhujle et al., 1979a,b). It should be emphasized that granulosa cells of the preovulatory follicle in the pigeon show weak reaction for the above enzymes but show intense reaction subsequent to ovulation and formation of the postovulatory follicle (Bhujle et al., 1979a,b).

The postovulatory follicles in the hen are known to contain progesterone and estrogens (Furr, 1969; Senior and Furr, 1975). Armstrong et al. (1977) demonstrated biochemically the presence of 3P-HSDH and G-6-PDH in both thecal and granulosa cells of the postovulatory follicles in the hen, the concentrations of which decline subsequently corroborating the histochemical observations. Botte et al. (1966) showed that postovulatory follicles are capable of steroid synthesis in vitro.


111. General Discussion

A. STRUCTURE AND LIFE-SPAN

In all vertebrates, after ovulation the follicular membranes remain behind in the ovary which give rise to postovulatory follicles and/or the corpora lutea. In oviparous species the postovulatory follicles are poorly organized and tend to regress rapidly especially in cyclostomes, teleosts, anurans, and avians. In live- bearing elasmobranchs and urodeles and in all reptiles there is an attempt to form a compact and glandular structure comparable in appearance to the mammalian corpora lutea. In a majority of the species studied so far, it is found that the granulosa cells contribute to the bulk of the corpus luteum while the theca provides the supporting elements. In almost all the species of nonmammalian vertebrates studied, so far, there is a hypertrophy and luteinization of granulosa cells both in oviparous and live-bearing animals during their early postovulatory phase. This is characterized histochemically by the development of diffuse lipoproteins, phospholipids, triglycerides, cholesterol or its esters, and certain enzyme activities such as 3/3-HSDH, G-6-PDH etc., which are involved in the synthesis of steroid hormones (see Section 11). Although electron microscopic studies are limited to five teleosts (Nicholls and Maple, 1972; Lambert, 1978; Nagahama et al., 1978; Hurk and Peute, 1979), two amphibians (Kessel and Panje, 1968; Joly and Picheral, 1972), one reptile (Cyrus et al . , 1978), and one bird (Wyburn et al., 1966) they provide ultrastructural evidence of luteinization of the granulosa cells following ovulation. This involves the formation of smooth endoplasmic reticulum, spherical mitochondria with tubular cristae, appearance of fine lipidic droplets, and well-developed Golgi zones, features indicative of steroid-secreting cells. These ultrastructural observations agree well with those of the histochemical findings. The theca becomes extremely thick which may be due partly to the contraction of the follicle and partly due to the hypertrophy of its elements, but remains at the periphery in most of the species investigated.

The organization of corpora lutea in all elasmobranch species studied is basically similar. The differences that are encountered in some cases are of a minor nature. This involves whether or not the thecal cells participate in providing theca lutein cells. The theca interna has been implicated in providing theca lutein or paralutein cells in Scyliorhinus (Samuel, 1943b), Rhinobarus (Chieffi and Botte, 1961, 1970) and several species of the genus Raja (Chieffi and Botte, 1970).

There is a difference of opinion with regard to the fate of the ovulated follicle in the teleost ovary. Several authors reported that ruptured follicles do not become reorganized into corpora lutea but instead collapse, become pycnotic, and are rapidly resorbed (Sathyanesan, 1962; Barr, 1963; Nair, 1963; Rajalaksmi, 1966; Mackay, 1973). However, it must be emphasized that these observations were made on the ovaries of fishes that had spawned in nature. Consequently,


one does not know how many houddays after spawning the fishes were collected. Fishes collected a day or two after spawning would contain degenerating postovulatory follicles. In fact, Nagahama et al. (1976), reported the onset of degeneration in 30-hour-old postovulatory follicle of goldfish. Sathyanesan (1 962), Barr (1963), Nair (1963), Rajalaksmi (1966), and Mackay (1973) reported an absence of hypertrophy of the follicular membranes after ovulation that might be due to the sampling errors or due to the fact that they were dealing with older/ degenerating corpora lutea. Others (Rai, 1966; Lehri, 1968; Yaron, 1971; Nich- 011s and Maple, 1972; Sanwal and Khanna, 1972; Lambert and van Oordt, 1974; Khoo, 1975; Nagahama et al., 1976; Van Ree, 1976; Lam et al., 1978a; Guraya and Kaur, 1979), however, reported the formation of corpora lutea characterized by the transient hypertrophy of both granulosa cells and thecal tissues. Therefore, studies on the postovulatory follicles of fishes induced to breed in laboratory conditions may be expected to shed more light on their structure and life-span. Corpora lutea are not formed in the viviparous fishes showing follicular gestation (Hoar, 1969).

Amphibian postovulatory follicles are somewhat better organized than those of the teleosts. Immediately after ovulation there is reorganization of the ruptured follicle to give rise to corpora lutea which are short-lived in oviparous species but persist longer in live-bearing forms. The mode of reorganization of the postovulatory follicles is similar in all amphibians. The theca also undergoes considerable hypertrophy but never invades the granulosa luteal cell mass. The thecal layer is composed of fibrous tissues and capillaries. The thecal layer both before and after ovulation is continuous with the external ovarian wall, though they may appear to be free and detached as a result of the section not passing through the region of connection.

Perhaps for the first time in vertebrate evolution true corpora lutea which closely resemble those of mammals appear in reptiles. They are remarkably large in size, compact, and very glandular in appearance. Following ovulation granulosa and thecal cells undergo a series of morphological and histochemical changes. There is a considerable hypertrophy of thecal layers as well as the granulosa cells. A fully formed corpus luteum is filled with the granulosa lutein cells surrounded by the hypertrophied theca interna and the theca externa layers which are clearly distinguishable. Further, in some species theca interna are considered to provide theca lutein or paralutein cells (Hett, 1924; Altland, 1951). Septae arising from the theca interna invade the luteal region to provide support.

It is evident from the literature that little attention has been given to the study of avian corpora lutea. There is confusion as to their formation among the few birds studied. In the pigeon (Dominic, 1960; Bhujle et al., 1979a,b), there is no mixing up of thecal and granulosa cells. In the hen and sparrow, at least at a later stage of corpus luteum development, there is a mixing up of theca lutein and granulosa lutein cells although they remain distinguishable due to their different


staining properties (Pearl and Boring, 1918; Guraya and Chalana, 1975; Chalana and Guraya, 1978).

Mitotic figures are rarely encountered either in granulosa or thecal cells of certain fishes (Barr, 1963), amphibians (R. D. Kanamadi and S. K. Saidapur, unpublished observations), and lizards (Weekes, 1934) but not in the pigeon (Dominic, 1960). Lehri (1968) in C. batrachus and Sanwal and Khanna (1972) in C. gachua reported the proliferation of granulosa cells during the postovulatory phase or the formation of a corpus luteum. However, it is doubtful whether the granulosa cell number really increases (irrespective of mitotically or amitoti- cally) during the formation of the corpora lutea. The feeling that the granulosa cell number increases during the formation of corpus luteum may be due to the fact that there is a contraction of the follicle resulting in the reduction of the follicular size concomitant with the hypertrophy of the granulosa cells which fill the lumen. However, more careful observations are needed to substantiate or dispute the proliferation of granulosa cells. Likewise, it is not clear whether the luteal cell mass becomes well vascularized in the various species studied. The thecal layers are no doubt vascular. The thecal invasion of the blood supply to the luteal cell mass is reported only for a few species (Weekes, 1934; Guraya and Chalana, 1975; Guraya, 1976; Guraya and Varma, 1976; Armstrong et al., 1977) which is denied in some other species (Weekes, 1934; Rahn, 1938; Boyd, 1940; Altland, 1951; Betz, 1963; Goldberg, 1970; Guraya, 1973; Varma and Guraya, 1973; Cyrus et al., 1978).

The life-span of the corpora lutea in the ovaries of nonmammalian vertebrates varies with the species and bears some relationship with their mode of reproduction. They may survive until oviposition in egg-laying and usually until parturition in live-bearing species. The teleosts, in which the eggs are generally shed within 10-12 hours after ovulation, the postovulatory follicles are very short lived. The time sequence studies of Lambert and van Oordt (1974), Nagahama et al. (1976), Lambert (1978), and D. K. Tikare and V. B. Nadkami (unpublished, personal communication) on some teleosts indicate that postovulatory follicles begin to degenerate within 24 hours of spawning but remain recognizable for 8-10 days.

The postovulatory luteal bodies of oviparous anuran species degenerate mark- edly within 2 days and are completely resorbed between 6 and 10 days (Cunnin- gham and Smart, 1934; Saidapur and Nadkami, 1972a, 1974b; R. D. Kanamadi and S. K. Saidapur, unpublished). However, in the ovoviviparous urodelans (Salamandra species), viviparous aniirans ( N . occidentalis), and oviparous anurans showing cutaneous gestation pouches (Pipa pipa, Gastrotheca marsupiata), the postovulatory follicles are more persistent (references in Lofts and Bern, 1972). In T. torosu the resorption of postovulatory follicles is complete 6-8 weeks after ovulation (Miller and Robins, 1954). In S. salamandra the postovulatory follicles become smaller in size a month after ovulation and continue to decrease in size subsequently but are active for at least 3 months (Joly and


Picheral, 1972). In N. occidentalis the postovulatory follicles are maintained throughout most of the 9-month gestation period and show signs of active secretion during the first 6 to 7 months (Lamotte and Rey, 1954; Vilter and Lugand, 1959; Xavier et al., 1970). In the ovoviviparous S. atra where the gestation period is of 4 years duration many corpora lutea persist for the first 2 years, but gradually become reduced toward full term (Vilter and Vilter, 1964). Informa- tion on the postovulatory follicles of apodan species is not available.

Reptilian corpora lutea persist for several days to several months depending on the species and the mode of reproduction. In oviparous forms there is a correlation between the longevity of the corpora lutea and the egg-laying or the egg- retaining habit of the species. Corpora lutea usually remain active until eggs are laid, but regression sets in immediately afterward (Dutta, 1946; Altland, 1951). In some species they begin to regress earlier than oviposition (Weekes, 1934; Altland, 1951) and the regression is completed at the time of oviposition or several days after (Cyrus et al., 1978). Among the viviparous reptiles, gestation ranges from 9 weeks in some species of Thamnophis (Cieslak, 1945; Bragdon, 1952) to 5 months in several genera of sea snakes (Bergman, 1943) and in the majority of them the corpora lutea persist throughout the pregnancy. The corpora lutea of S. cyanogenys are maintained for most of the 4 months gestation (Crisp, 1964), and S. jarrovi in which gestation is interrupted by a prolonged period of embryonic diapause, the corpora lutea persist until parturition (about 7 months after mating) and then degenerate. In C. ocellatus the corpora lutea are cytologi- cally active for 6 to 8 weeks, a period equivalent to about half of the gestation period (Badir, 1968). In L. quovi (Weekes, 1934) and X. vigilis (Miller, 1959) the regression of corpora lutea occurs before the end of pregnancy in both of which the gestation lasts 3 to 3.5 months. The postpartum regression of corpora lutea in live-bearing species is completed within 4 weeks. The degenerating luteal bodies in their advanced stages resemble the corpora albicantia described in the mammalian ovary.

In birds, which are exclusively oviparous the corpora lutea remain active for 3-4 days during which period the oviposition is completed. However, they remain recognizable for 10- 15 days.

The degeneration of postovulatory follicles and the corpora lutea is basically similar in all the species studied so far. This involves the vacuolization of granulosa lutein cells, pycnosis of nuclei, increase in coarse lipidic droplets, disintegration of the various cell organelles, and finally disappearance of the same. The connective tissue and erythrocyte infiltration may also occur in some species. The thecal elements regress and merge with the ovarian stroma and are perhaps recycled.

B. ENDOCRINE CAPACITY

The fact that the postovulatory follicle and/or the corpora lutea of oviparous species are generally very transient (excepting lizards and snakes) and in the

270 SRTNIVAS K. SAIDAPUR

absence of a definite theory to account for their possible role(s) they have been grossly neglected in the past. However, corpora lutea in viviparous species were believed to have some endocrine role (references in reviews by Franchi, 1962; Perry and Rowlands, 1962; Ban, 1968; Callard et at., 1972a,b,c; Lofts and Bern, 1972; Guraya, 1976; Lance and Callard, 1978a). Recent histochemical, ultrastructural, biochemical, and physiological studies indicate their steroidogenic capability (see Section II) and possible involvement in the reproduction of nonmammalian vertebrates. It is well known that luteinization of granulosa cells following ovulation in mammals is closely accompanied histochemically by the development of diffuse lipoproteins, phospholipids, triglycerides, sudanophilic lipids, cholesterol or its esters (steroid hormone precursors), enzymes involved in steroid hormone synthesis such as A5-3P-HSDH which oxidizes P-3P-hydroxysteroids to A4-3-ketosteroids, a step that occurs in the early biosynthetic pathway of all the biologically active steroid hormones (Samuel et al. , 1951; Baillie ef al . , 1966) and G-6-PDH which generates NADPH needed for steroid hydroxylations (McKerns, 1969). Likewise, ultrastructural transformations that occur during luteinization of granulosa cells in mammals include the development of abundant smooth endoplasmic reticulum, spherical mitochondria having tubular cristae, and prominent Golgi bodies. The tubular reticulum may form concentric whorls. Often, the mitochondira are found near the lipid droplets (Christensen and Gillim, 1969). In the past 15 years we have witnessed a great number of studies aimed at elucidating the steroidogenic potentiality of postovulatory luteal bodies in the nonmammalian vertebrates. Histochemical observations on the postovulatory follicles of representative species of cyclostomes, teleosts, amphibians, reptiles, and birds (examples and references in Section 11) suggest the development of diffuse lipoproteins, fine sudanophilic lipid droplets, cholesterol or its esters, various steroid dehydrogenases such as 3P-HSDH, 1 1P-HSDH, 17P-HSDH, and other enzymes (such as G-6-PDH, ICDH, LDH, and SDH) similar to that reported for the mammalian corpus luteum (references in Bjersing , 1977). The electron microscopic studies though limited clearly reveal the presence of varying amounts of smooth endoplasmic reticulum, mitochondria with tubular cristae, lipid droplets, and well-developed Golgi zones in the granulosa lutein cells of nonmammalian vertebrate species (Wyburn et al . , 1966; Kessel and Panje, 1968; Joly and Picheral, 1972; Nicholls and Maple, 1972; Nagahama et al . , 1976, 1978; Cyrus et al., 1978; Hurk and Peute, 1979). The ultrastructural studies thus agree well with the histochemical findings and provide additional support for the steroidogenic ability of postovulatory follicles in all classes of nonmammalian vertebrates.

The biochemical studies on the isolated corpora lutea of nonmammalian vertebrates are very limited. Lupo (1968) showed that the corpus luteum of S. stelluris produced progesterone in vitro but no estrogens. On the other hand, in the same


study it was found that the corpus luteum of T. marmorata produced estrogens and no progesterone in vitro. Lupo (1968) believes that the corpora lutea of T. marmorata do not undergo luteinization and hence do not synthesize progesterone. Studies on the isolated postovulatory follicles of bony fishes are lacking at present. The presence of 3P-HSDH in the corpora lutea of N. maculosus has also been biochemically confirmed (Kessel and Panje, 1968). Steroid production in vitro by the isolated postovulatory follices and corpora lutea has been well documented for some reptiles. Klicka and Mahmoud (1972,1973) have convinc- ingly demonstrated the conversion of labeled cholesterol or pregnenolone into progesterone by the homogenates of the corpus luteum of the snapping turtle C. serpentina. Subsequently, Licht and Crews (1976) in the same species showed that the luteal tissue can also produce progesterone in response to homologous as well as heterologous (ovine) gonadotropins. Interestingly, in these studies it was found that FSH was more potent than the LH in stimulating in vitro progesterone production by the turtle corpus luteum. Callard et al. (1976) working with a related species (C. picta) demonstrated that the enzyme-dispersed luteal cells mainly synthesize progesterone and very small quantities of estrone, whereas preovulatory follicular tisuse predominantly synthesized estradiol- 17P. Further, both mammalian and avian LH were stimulatory when incubated with cholesterol or pregnenolone and major steroid products were examined. Prolactin on the other hand, had no effect on luteal progesterone synthesis when used alone, but reduced the stimulatory effect of mammalian LH on progesterone synthesis. More recently, Lance and Callard (1978b) showed that luteal cells of C. picta secreted progesterone in response to dibutyryl cyclic AMP. The corpus luteum of the snake S . dekayi when incubated with labeled pregnenolone synthesized 17a-hydroxypregnenolone, progesterone, 1 l-deoxycorticosterone, andros- tenedione, and testosterone (Colombo and Yaron, 1976). Also, in the snake, steroid 21-hydroxylase is exclusively confined to the luteal tissue; the significance of this remains unknown (Colombo and Yaron, 1976). These above findings clearly indicate that reptilian corpora lutea are capable of progesterone synthesis. Biochemical studies on the avian postovulatory follicles are limited to G. domesticus. The enzymes 3P-HSDH and G-6-PDH in the postovulatory follicles of different ages have been biochemically assayed (Armstrong et al., 1977). Also, in vitro steroid synthesis by the postovulatory follicles of the hen has been demonstrated (Botte et al., 1966). These studies indicate that the postovulatory follicles in birds may be functional mainly during the first 24 hours of their formation.

There are several lines of indirect evidences that indicate steroid hormone (progesterone) secretion by the corpora lutea of nonmammalian vertebrates. For instance, Callard and Leathern (1965) demonstrated that the ovarian tissue of S. acanthias synthesized twice as much progesterone in vitro when the fishes are pregnant than when it was taken from nonpregnant animals. In N. occidentalis


the presence of corpora lutea during gestation or pseudogestation correlates with the synthesis of progesterone (Xavier and Ozon, 1971). Progestins have been demonstrated in extracts of ovaries containing corpora lutea (Porto, 1942; Valle and Valle, 1943) and in the plasma of pregnant viviparous snakes (Bragdon et al., 1954). Further, in the snake N . sipedon pictiventris there is an increased progesterone synthesis by the ovaries of pregnant animals as compared with nonpregnant females per unit tissue weight (Callard and Leathem, 1965). Likewise, ovaries of ovoviviparous forms ( N . sipedon, N . taxispilotu) were able to synthesize more progesterone than the oviparous species (Coluber C . con- structus). In N . sipedon pictiventris and s. dekayi steroid production by the ovaries of snakes during mid-pregnancy was greater than the ovaries from animals in early pregnancy (Callard and Leathem 1965; Colombo and Yaron, 1976). In V . uspis the sudanophilia and birefringency of newly formed corpora lutea diminish as the gestation period proceeds, suggesting a possible utilization of precursor material which would agree with the above findings (references in Lofts and Bern, 1972). Elevated progesterone levels recorded in pregnant snakes indicate that corpora lutea may be the source (Highfill and Mead, 1975a; Chan et al., 1973; Callard and Lance, 1977; Lance and Callard, 1978a). Progesterone has been identified in the plasma of a number of squamates and has been shown to correlate with the corpus luteum activity (Callard et ul., 1972a,b; Chan et al., 1973; Highfill and Mead, 1975a; Veith, 1974; Callard and Lance, 1977). In C.

pictu prior to and around the point of ovulation there is a surge in progesterone secretion which rapidly declines as the corpus luteum function declines. This progesterone surge lasts less than a week, as does the occurrence of histologically identifiable corpus luteum (Lance and Callard, 1978a). However, progesterone levels in squamates, as opposed to turtles, continue to rise after ovulation and remain high as long as the corpus luteum continues to function (Lance and Callard, 1978a). In the hen, removal of postovulatory follicles influences the retention time of eggs in the oviduct (Rothchild and Fraps, 1944b). Conner and Fraps (1954) show a quantitative relationship between the amount of postovulatory follicles and oviposition, so that the greater the proportion removed, the greater the retardation in oviposition. Also, the nesting behavior of laying hens induced by the injection of progesterone into estrogen-primed pullets is abolished when postovulatory follicles are ligated or removed (Wood-Gush and Gilbert, 1975). Tanaka and Nakada ( I 974) demonstrated that fowl postovulatory follicles secrete an unknown nonlipoidai component which is involved in genital tract motility and oviposition. Soliman and Walker (1975) considered this to be an oxytocin-like substance. However, Tanaka and Goto (1976) have denied the secretion of an oxytocic substance.

The studies outlined above strongly suggest that postovulatory luteal structures in nonmammalian vertebrates possess endocrine capacity and ability to synthesize steroid hormones which is reflected in their histochemical and ultrastructural features. Biochemical and in vitro studies though limited to a few species pro-


vide evidence for the steroid secretory activity during their functional phase. The duration of the functional phase in oviparous teleosts, anurans, and avians is limited to the first 24 hours after ovulation. In other oviparous, ovoviviparous, and viviparous species of elasmobranchs, urodeles, and reptiles the duration of the functional phase of the corpora lutea is much longer. It is also evident from the above studies that luteal structures may secrete mainly progesterone as in mammals. Corpora lutea of nonmammalian vertebrates also secrete progesterone in vitro in response to gonadotropins as shown in reptilian studies. Further, a recent study of Lance and Callard (1978b) shows that dibutyryl cyclic AMP as in mammals stimulates progesterone secretion by the turtle corpus luteum. How- ever, more studies on more representative species are needed to elucidate the nature of steroid hormone production and their regulation in the corpora lutea of nonmammalian vertebrates.

C. TERMINOLOGY

The structures arising out of the ovulated follicles in the ovaries of nonmammalian vertebrates have been variously termed “ruptured follicle, ” “discharged follicle, ” “postovulatory follicle, ” “corpus luteum, ” and “postovulatory corpora lutea” (as opposed to the “preovulatory corpora lutea”) by various authors (references in reviews by Chieffi and Botte, 1970; Browning, 1973; Dodd, 1977; Amoroso and Perry, 1977). This has been due mainly to the fact that their role(s) in reproduction is not well known. Second, it is doubtful whether they play roles similar to that of the mammalian corpus luteum. It is evident from the literature that previous authors have preferred the term “postovulatory follicle” in the case of oviparous species particularly, cyclostomes, teleosts, anurans, and birds and “corpus luteum” in the case of elasmobranchs, urodeles, and reptiles. It is a matter of opinion as to whether or not the term “corpus luteum” can be applied to these structures in nonmammalian vertebrates (Amoroso and Perry, 1977).

In recent years, the histochemical, ultrastructural, and biochemical studies on several species have provided evidence for the steroidogenic ability of postovulatory luteal structures (see Section 111,B). Besides, these structures arise in the same way as in mammals though poorly reorganized in many instances. Therefore, in light of these observations, to avoid confusion and to maintain uniformity of terminology, it is suggested that all structures arising out of the follicular membranes left behind after ovulation be designated as corpora lutea in nonmammalian vertebrates.

D. FUNCTIONAL SIGNIFICANCE

A survey of the early literature reveals that while corpora lutea of live-bearing elasmobranchs, urodeles, and reptiles were credited with a role in reproduction, the postovulatory follicles of egg-laying cyclostomes, teleosts, anurans, and aves


which are transient were quickly dismissed as having no function (references in reviews by Lofts and Bern, 1972; Browning, 1973). They were regarded as a mere consequence of ovulation which served to “tidy-up” the remains of yolk and debris in the postovulatory follicles following ovulation (Hisaw and Hisaw, 1959; Dodd, 1960, 1972a,b, 1975, 1977; Hoar, 1969; Chieffi and Botte, 1970; Lofts and Bern, 1972). Based on this assumption it was suggested that in lampreys which spawn once and die there is no need for “tidy-up” work and hence there is no reorganization of the discharged follicle (Lewis and McMillan, 1965; Dodd, 1972a,b, 1977). However, the hagfishes breed over several seasons and there is a need to study the role of postovulatory follicles in reproduction, if any. The role of postovulatory follicles and corpora lutea in other vertebrate classes is by no means well established.

There is a difference of opinion with regard to the role of corpora lutea in different species of elasmobranchs. Experimental studies are virtually lacking. Based on morphological and indirect evidence Matthews (1955) suggested that luteal hormones may stimulate the hypertrophy of the uterine mucosa to produce “trophonemata” (uterine folds) seen in live-bearing elasmobranchs during pregnancy. However, Chieffi (1961, 1962) found a correlation between the lengthen- ing of the trophonemata and the increasing number of corpora atretica in T. marmorata rather than with corpora lutea. Subsequently, Lupo (1968) demonstrated in v i m steroid production by the corpora atretica of this species supporting but not confirming Chieff’s view. Whether corpora lutea play any role in gestation is not known due to the paucity of studies. Hypophysectomy performed soon after the eggs had entered the uterus in M. canis had no effect on embryo development. The embryos continued to grow normally up to at least 3.5 months (Hisaw and Abramowitz, 1939). These authors work implies that pituitary stimulation may not be involved in the formation of a corpus luteum and also they suggest that corpora lutea are not essential for the maintenance of gestation in these fishes. However, the yolk sac of M. canis does not fuse with the uterine wall until the fourth month of gestation and therefore the above conclusion is open to doubt (Chieffi and Botte, 1970). Besides, to rule out the involvement of corpora lutea in gestation the authors should have performed ovariectomy or deluteinization which they did not. Obviously more studies are needed to under- stand the functional significance of corpora lutea in the cartilaginous fishes.

Only in recent years have the postovulatory follicles of teleosts been credited with some functions. It is the work of Lam et al. (1978a,b, 1979) which has shed new light on the possible role of the postovulatory follicles. In some teleosts there is a nesting and courtship behavior and such species are believed to have relatively long-lived postovulatory follicles (Lam et al. , 1978a,b, 1979). These authors postulate that steroids secreted by the postovulatory follicles function to maintain ovulated eggs within the ovarian cavity. The hormone(s) may do so directly and/or by stimulating the ovarian epithelium to secrete the cavity fluid


which normally bathes the iovulated eggs. Their studies indicate a relationship between the life of postovulatory follicles and the nature of reproductive behavior in teleosts. Both zebrafish and goldfish whose reproductive behavior is relatively simple and does not involve mate selection or nest building, possess short-lived postovulatory follicles. On the other hand, in the Kongo cichlid (C. nigrofus- ciutum) which shows elaborate reproductive behavior involving both mate selection and nest building, the life of postovulatory follicles is relatively long. It is conceivable that a prolonged preparatory phase prior to actual courtdhip in the behavioral repertoire of the male would increase the probability of a delay between ovulation and oviposition in the female. In this light, an extended life of postovulatory follicle would make phyisological sense if it functions in the maintenance of ovulated eggs (Lam et ul., 1978a). It is essential to extend such studies to more number of fishes particularly those exhibiting nesting and courtship behavior. Khoo (1975) has suggested that some of the older corpus luteal cells differentiate into oogonia. This is an interesting proposition but needs confirmation.

Anuran postovulatory follicles like those of teleosteans being very transitory are generally thought to have no functional significance (Cunningham and Smart, 1934; Thornton and Evennett, 1969; Lofts and Bern, 1972; Redshaw, 1972; Lofts, 1974). However, Galli-Mainini ( 1950), Guraya ( 1968), and Saidapur and Nadkarni (1974b) suggested that they may secrete steroid hormone@) which in turn may stimulate oviducal glands to secrete jelly to be placed around the eggs. Amphibian oviducts are known to secrete jelly in response to progesterone (Lodge and Smith, 1960). Studies by Thornton and Evennett (1969) on the toad B. bufo indicate that while progestins may cause both meiotic divisions of oo- cytes (maturation) and stimulation of oviducal glands to secrete the jelly, the source of the hormone is preovulatory follicle and not the postovulatory follicle since the jelly release begins several hours prior to ovulation. Later, Thornton (1972) showed the occurrence of the preovulatory progesterone surge in the toad. However, the control of jelly release by preovulatory secretion of progestin may not be a general phenomenon in Amphibia. In the anurans studied by Saidapur and Nadkarni (1974b) and R. D. Kanamadi and S. K. Saidapur (unpublished observations) on B. melanostictus, all the mature eggs do not undergo ovulation at the same time nor are the ovulated eggs shed at the same time. Consequently there is a time-gap between the eggs undergoing ovulation. Also, the eggs are shed over an extended period of several hours. Therefore, the ovualted eggs spend some time in the body cavity and later in the oviducts prior to their actual shedding. Unfortunately, the serum progesterone levels in these species during ovulation and spawning is not known. Histochemical observations cited earlier are indicative of the steroidogenic ability of the postovulatory follicles in these species (Saidapur and Nadkarni, 1974; R. D. Kanamadi and S. K. Saidapur, unpublished). There- fore, if the postovulatory follicle in such species secretes progestins it may help


in maintaining the ovulated eggs in the body cavity prior to their entering the oviducts and also help in sustaining the oviducal jelly secretion for prolonged periods. In addition the possibility that the progestins of postovulatory follicles may locally induce oocyte maturation and ovulation of the neighboring eggs is a tempting speculation in the absence of experimental evidence. In some species it seems that the mechanical stimulus provided by the eggs during their journey in the oviducts may cause the secretion of jelly (Waring et al., 1941).

In B. melanostictus it has been observed that some of the thecal cells of 3- to 4-day-old postovulatory follicles undergo mitosis and differentiate into oogonia (R. D. Kanamadi and S. K. Saidapur, unpublished observations). It is however necessary to extend such studies to more anuran species to determine whether postovulatory follicles play a role in contributing to new generation of oogonia during the postbreeding phase.

In ovoviviparous and viviparous urodeles, the endocrine capacity and the involvement of corpora lutea in reproduction seems accepted (review by Lofts, 1974). Lamotte and Ray (1954) and Vilter and Vilter (1960) credit the corpora lutea with the inhibition of ovulation and the control of oviducal secretions in N . occidentulis and the black salamander (S. a m ) , respectively. Vilter and Vilter ( I 964) consider that corpora lutea serve to maintain the activity of the pregnant uterus (S. utru) but their contention is not substantiated by any endocrinological data. The corpora lutea inhibit oogenesis and follicular growth for a period of 2 years or more so that the ovarian cycle would probably be a triennial event. Joly and Picheral (1972) are doubtful of the homology between the corpus luteum of salamander and those of mammals, and have shown that castration of S. salumundra at the beginning of pregnancy does not interfere with the development of larvae. In the newt N. occidentalis, ovariectomy terminated gestation only when it was carried out at the beginning of pregnancy in primigravid females (Xavier, 1970b; Xavier and Ozon, 1971). Xavier (1970b) has shown that though parturition is normal it is precocious in ovariectomized pregnant newts, and that there is an accelerated growth of the embryos. Further, it was found that progesterone implantation during gestation slows down development of embryos. Thus the role of progesterone arising from corpora lutea is implicated in checking the growth of embryos during the seasonal fasting of mothers (Xavier, 1970b). Based on these studies in the newt Xavier concluded that the role of corpora lutea is not to maintain pregnancy but to secrete a hormone which helps for a short period to make the oviduct favorable for the development of embryos.

Although a well-developed corpus luteum occurs in all the reptiles studied hitherto and that it is capable of steroid hormone synthesis (see Section III,B), its functional significance in both egg-laying and live-bearing species remains un- certain. It is now known that reptilian oviduct development and maturation requires both estrogen and progesterone (review by Yaron, 1972; Callard and Klotz, 1973). However, studies on the effect of steroids on the oviductal contrac-


tility are very limited. La Pointe (1969) failed to demonstrate the effect of estrogen or progesterone on contractility of the oviduct in Klauberina riversiana which is probably due to the use of animals during the nonbreeding season (Callard and Hirsch, 1976). Recently, however, Callard and Hirsch (1976) in the turtle C. picta showed that progesterone reduces oviductal contractility as in mammals. If this is true for other oviparous reptiles then progresterone of luted origin may facilitate egg retention by suppressing oviductal contraction. Regres- sion of corpus luteum around the time of normal oviposition, and the resulting drop in circulating steroid levels, may permit or induce oviducal contractions leading to oviposition. Indeed, deluteinization in S. undulutus resulted in early oviposition (Roth et al., 1973). In such an event the functional demise of corpus luteum is the key factor in determining the time of oviposition. Studies on some reptiles indicate that expulsion of eggs and young from the oviducts are con- trolled by the neurohypophyseal hormones (Clausen, 1940; Panigel, 1956; Mun- sick et al., 1960; LaPointe, 1964, 1969). However, the possible relationship between progesterone and neurohypophyseal hormones in reptiles is not well known.

Several investigators have examined the effect of progesterone and the roIe of corpora lutea on parturition in snakes (Clausen, 1940; Fraenkel et al., 1940; Bragdon, 1951) and lizards (Panigel, 1956; Callard et al., 1972a,b,c) and it is generally agreed that removal of corpora lutea or ovariectomy prevents parturition, presumably due to the absence of the ovarian steroids. It appears that early pregnancy in some snakes may depend on ovaries of corpora lutea, because ovariectomy or deluteinization (Calusen, 1935, 1940; Fraenkel et ul., 1940) in late pregnancy is not invariably followed by abortion. Significantly, 6 mg progesterone did not prevent abortion in the ovariectomized gravid water snake (Clausen, 1940). Ovariectomy or deluteinization delayed parturition in Zootoca (Panigel, 1956) and impaired parturition in S. cyanogenys (Lien and Callard, 1968). Parturition was normal in deluteinized C. ocellatus (Badir, 1968). Treat- ment with 3-5 mg progesterone in Z. vivipara and with progesterone + estradiol in S. cyanogenys did not modify the effects of ovariectomy or deluteinization, namely, apparent normal development of embryos but impaired parturition (Panigel, 1953, 1956; Lien and Callard, 1968). The studies of Rahn (1939) and Bragdon (1951) on the viviparous snakes Tharnnophis and Nutrix and those of Panigel (1956) on the ovoviviparous lizard Zootoca indicate that ovariectomy or hypophysectomy during gestation, even when performed as early as the first week, will not usually lead to abortion, but will interfere with the process of parturition, manifested by retention of the young in utero past full term. This was more effective after hypophysectomy rather than ovariectomy . Panigel ( 1953) reported that progesterone pellet implantation into pregnant lizards resulted in longer retention of embryos and a consequent marked delay in parturition, similar to that reported for N . occidentalis (Xavier, 1970a,b). Xavier reported that


implantation of progesterone diminished the growth rate of embryos and prolonged gestation by 2-3 months. Removal of ovaries accelerated embryonic growth and reduced gestation periods in these species. Therefore, another possible function of corpora lutea produced hormones may be in controlling the length of gestation.

The above studies indicate that progesterone secretion by the reptilian corpus luteum may not be necessary for maintaining pregnancy in the few viviparous species studied, although continued secretion of progesterone is gonadotropin dependent (reviews by Callard et al., 1972a,b,c; Yaron, 1972; Highfill and Mead, 1975b). Further, Yaron (1972) using labeled leucine demonstrated in Xanrusia that neither the corpora lutea nor other ovarian components influence the transport of an amino acid from the maternal circulation into the embryo. Therefore, several authors have concluded that in lizards, embryonic development up to the normal time of parturition is not dependent on any secretory product of the corpus luteum or indeed of the entire ovary (Panigel, 1956; Badir, 1968; Lien and Callard, 1968; Yaron, 1972).

Yet another function attributed to the reptilian corpora lutea concerns the inhibition of follicular development and ovulation during pregnancy (Cunnin- gham and Smart, 1934; Callard et al., 1972a,b,c) similar to that suggested for some urodeles (references in Lofts, 1974). However, in S. undularus the corpora lutea do not seem to inhibit follicular development (Roth et al., 1973).

Physiological studies to determine the role of avian postovulatory follicles in reproduction are limited to the hen. There are two lines of indirect evidence that indicate the involvement of postovulatory follicles in oviposition and nesting behavior. Removal of postovulatory follicles influence the retention time of eggs in the oviduct (Rothchild and Fraps, 1944b). Conner and Fraps (1 954) showed the existence of a quantitative relationship between the amount of postovulatory tissue and oviposition, so that the greater the proportion removed, the greater was the retardation in oviposition. Second, the nesting behavior of laying hens is abolished when postovulatory follicles are ligated or removed (Wood-Gush and Gilbert, 1975). Also, the injection of progesterone into estrogen-primed pullets induces nesting behavior (Wood-Gush and Gilbert, 1975). The postovulatory follicle in birds therefore seems to be associated with the control of oviduct motility, egg transport, oviposition, and nesting behavior (Rothchild and Fraps, 1944b; Layne et a/., 1957; Gilbert, 1971; Wood-Gush and Gilbert, 1975; Armstrong er al., 1977; Chalana and Guraya, 1978).

In summary, a variety of roles ranging from a simple tidying-up work, nesting behavior in fishes and birds, maintenance of ovulated eggs in the body cavity of certain fishes, stimulation and control of oviduct activity, and control of oviposition, gestation, and parturition in live-bearing forms have been ascribed to the luted structures of nonmammalian vertebrates. In addition, they are believed to inhibit follicular growth and ovulation during the gestation period in some


species. Although the above cited roles imply their association with reproduction as in mammals, the same are not proved with certainty. Nevertheless, they should be taken to provide insight for future investigations.

IV. Concluding Remarks

The past few years have witnessed a renewed interest in the study of postovulatory luteal structures occurring in the ovaries of nonmammalian vertebrates. Consequently, new light is being shed on the possible role of these structures. For instance, in fishes, particularly those showing nesting and courtship behavior, the corpora lutea seem to help in maintaining the ovulated eggs from overriping (Lam et al., 1978a,b, 1979). The validity of this hypothesis should be established by extending studies to more species. Likewise, the possibility that some of the luteal cells in their later stages may differentiate into oogonia suggested by Khoo (1975) in goIdfish and the probable neotransformation of some thecal cells into germ cells in the walls of the postovulatory follicles in toads (R. D. Kanamadi and S. K. Saidapur, unpublished observations) deserves attention. These propositions should be verified not only in other fishes and anurans but also in other vertebrates including mammals. One of the important functions of the corpora lutea especially in live-bearing forms and all reptiles seems to be associated with the inhibition of follicular growth and ovulation. However, more careful studies are needed to elucidate this and the mechanisms involved.

The histochemical and ultrastructural studies carried out in recent years indicate the possibility of steroid synthesis by the postovulatory follicles and corpora lutea in all representative species of submammalian vertebrates. Biochemical studies though limited to half a dozen species have provided evidence in support of the histochemical and ultrastructural observations, that granulosa cells undergo luteinization in a majority of the species studied, following ovulation and acquire steroidogenic capacity. Nevertheless, the nature of steroid hormone(s) produced, if any, and their regulation is not clear. In general, the regulation of structure and function of corpora lutea of nonmammalian vertebrates is far from clear due mainly to the paucity of studies. The studies on some snakes and turtles indicate that luteal steroidogenesis is gonadotropin dependent as in mammals (Lance and Callard, 1978a). More exhaustive studies involving more representative species are needed before a generalization is possible regard- ing the regulation of luteal function in submammalian vertebrates.

With regard to the functional significance of the luteal structures, it appears that they may not have the same function as in mammals, i.e., maintenance of gestation. Besides, the problem of viviparous nonmammalian vertebrates is not the same as in mammals. The presence of abundant yolk in the egg does not


necessitate the requirement of a placental transfer of nutrients and hence luted function is different. However, we have begun to realize that the postovulatory follicles and corpora lutea are involved in the reproduction of nonmammalian vertebrates.

It is hoped that future years will yield more meaningful results in terms of the role of the luteal structures whose function is certainly not limited to “tidy-up’’ work. Also, it is necessary to make careful observations as to whether the granulosa cells really proliferate and whether the postovulatory follicles and corpora lutea become truly vascular. With the availability of radioimmunoassay methods it is now possible to measure steroid levels with utmost accuracy and such studies if used widely would throw light on the luted functions in submammalian vertebrates in the years to come. Likewise, more biochemical and ultrastructural studies are needed.

REFERENCES

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