Cellular sites of steroid synthesis in the oviparous teleost fish (Cyprinus carpio L.) : A histochemical study

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1 Proc, Indian Acad. Sci. (Anirn. ScL), Vol. 91, Number 6, November 1982, pp Printed in India. Cellular sites of steroid synthesis in the oviparous teleost fish (Cyprinus carpio L.) : A histochemical study SARDUL S GURAYA and SURINDERPAL KAUR Department of Zoology, College of Basic Sciences and Humanities, Punjab Agricultural University, Ludhiana , Punjab, India MS received 30 December 1981 ; revised 26 July 1982 Abstract. Histochemical techniques for lipids were employed to study the steroid synthesizing ceilular sites in the ovary of teleost fish (Cyprinus carpio L.). The ceilular sites of steroid biosynthesis appear to be the ovulating corpora lutea, interstitial cells, and special thecal cells of developing follicle. They possess the cytological and histochemical features of well-established steroid gland cells. The functional significance of histochemical changes in the granulosa cells of postovulatory follicles in the teleost ovary has been discussed in the light of recent researches on corresponding cells in the ovaries of other vertebrates. The corpora atretica are merely the large yolky eggs in the process of their degeneration and resorption. Keywords. Cellular sites; steroid synthesis; histochemistry ; lipids; ovary; teleost. 1. Introduction Histochemical techniques mainly for.6.5-3,b-hydroxysteroid dehydrogenase (3ft-HSDH) and electron microscopy have been employed for the study of cellular sites of steroid synthesis in the ovaries of some teleosts (see Bara 1965; Guraya 1976, 1978a, 1979 ; Nagahama et al1978 ; Kagawa et ai1981). However, the precise roles of atretic yolky eggs (corpora atretica or corpora lutea of atresia), postovulatory follicles (corpora lutea of ovulation) and the scarcely developed stroma, together with its interstitial (or thecal gland) cells, in relation to steroid biosynthesis, are still not known. In most of the previous studies carried out with the routine histological techniques the corpora atretica have been claimed to be the main site of steroid biosynthesis in the teleost ovary (Ball 1960 ; Chan et al 1967). This opinion was not shared by others (Dodd 1960 ; Polder 1964 ; Guraya et al 1975, 1977). No any attempt has been made so far to study the nature of lipid changes during the involution of postovulatory follicles or corpora lutea in teleosts (see Guraya 1979 ; Kagawa et ai1981). This study using histochemical techniques for 587

2 588 Sardul S Guraya and Surinderpal Kaur lipids describes the lipid changes of follicles, postovulatory follicles, corpora lutea of ovulation, corpora atretica or preovulatory corpora lutea and interstitial (thecal gland) cells in the ovary of the scale carp, Cyprinus carpio L. 2. Materials and methods The ovaries of oviparous teleost fish (scale carp Cyprinus carpio Linn.) were used. The fish were obtained from the fishery pond of the Punjab Agricultural University, Ludhiana. This fish usually breeds in the months of February and March in the Punjab waters. During the breeding season, two mature males and a female were put in the hapa. The spawning usually occurred in the early hours of the morning. Weeds were also added in the hapa for the attachment of eggs. The recovery of eggs from hapa was the cri\erion for ovulation. After spawning, the ovary was removed and transferred into physiological saline solution. The ovarian material was collected at 12 hr intervals from different females. It was also collected after an interval of one day. Thereafter, the material was collected on alternate days. These time intervals were taken into account from the time of spawning. After washing off the blood in physiological saline the ovary was immediately fixed in freshly prepared fixing fluids. The details of histochemical techniques used were the same as those reported previously (Guraya 1968). 3. Observations and discussion The vascularized thecal layer of follicle during the preovulatory period consists of fibroblasts and does not show any appreciable development of sudanophilic lipids-containing cells which could easily be demonstrated with the light microscope. The distribution and histochemical nature of lipids in the follicular epithelium are the same as those reported for the teleost Channa (Guraya 1976, 1978a). The cytoplasm of follicle (or granulosa) cells shows some sparsely scattered, deeply sudanophilic lipid droplets which give positive reactions for phospholipids, no Schultz-positive substances (cholesterol and/or its esters) are observed. The follicular epithelium of the developing follicle in the ovaries ofsome teleosts may show a positive 3fl-HSDH activity which is also accompanied by the presence of enzymes of the citric acid and the penrose-phosphate cycles (Guraya 1976, 1978a, 1979). The two enzymes 3f3- and 17f3-HSDH are not only present in the granulosa cells but also in the cortical cytoplasm during the last phases of follicle growth in the fish ovary. It is still not known whether the presence of these enzyme systems indicates synthesis and secretion of steroid hormones by the granulosa cells of developing follicle in vivo, or simply indicates their potentialities for steroidogenesis Postovulatory follicles The granulosa cells after ovulation develop sudanophilic lipid droplets of variable size during different stages of evolution and involution of postovulatory follicles

3 Histochemistry of teleost ovary and steroidogenesis 589 Figures Portion of postovulatory follicle in stage I showing sudanophilic lipids in the follicular epithelium (FE) and some thecal gland cells (TC) of thecal layer. Lumen (L) is seen. x Portion of postovulatory follicle in stage 2 showing sudanophilic lipids in granulosa luteal cells and thecal gland cells (TOC). Thecal layer (TL) shows some vacuolated cells (vc). Lumen (L) is reduced. x Portion of postovulatory follicle in stage. 5, showing heavy accumulation of sudanophilic lipid droplets in the granulosa luteal cell mass (OLM). The cells have separated from each other. x Postovulatory follicles in stage 6, showing heavy accumulation of sudanophilic lipids in degenerated granulosa luteal cells (OOLC). The amount of sudanophilic lipids has also increased in the thecal layer (TL). x Higher magnification of portion of postovulatory follicle shown in figure 4 showing accumulation of sudanophilic lipids in degenerated granulosa luteal cells (OOLC). The thecal layer also shows cells (TOC) filled with sudanophiliclipids. x A portion of ovary showing sudanophilic lipids in residual granulosa luteal cells (ROC) and interstitial cells (IC) distributed in the stroma (s). The latter does not show such lipids. x 100.

4 590 Sardu/ S Guraya and Surinderpa/ Kaur Figure 7. A portion of ovary showing sudanophilic lipids in residual granulosa luteal cells (aoc) and interstitial cells (IC). Stroma proper (s) does not show such Ii pids. X 400.

5 Histochemistry of teleost ovary and steroidogenesis 591 (figures 1-7), which consist offirst phospholipids, then phospholipids and triglycerides, and finally triglycerides, cholesterol and/or its esters and some phospholipids (table 1). Besides the lipid droplets, they also develop diffusely distributed sudanophilic lipids (lipoproteins) in their cytoplasm (figures 1, 2). Similar lipid droplets and diffuse lipoproteins are also developed during the transformation of granulosa cells into the luteal cells in other vertebrates (see Guraya 1976). Diffusely distributed lipoproteins apparently.derive from the membranes of smooth reticulum described for the granulosa cells of postovulatory follicles in teleosts and other vertebrates (Hoar and Nagahama 1978; Nagahama et a11976, 1978; Guraya 1976, 1979; Kagawa et al 1981). The thecal layer of postovulatory follicle in the present fish shows some hypertrophied cells with sudanophilic lipid droplets and diffuse lipoproteins (figures 1, 2) which correspond to the special thecal cells in the spent follicles of other teleosts (Nagahama et al1978 ;. Kagawa et ai1981). These special thecal cells give a positive reaction for 3,B-HSDH and show ultrastructural features of steroid gland cells such as greatly developed agranular endoplasmic reticulum and numerous large mitochondria with tubular cristae (see Nagahama et al 1978; Kagawa et al 1981). Generally when lipid droplets are abundantly present in the steroid gland cells, storage is taking place and when the amount is less, the steroid hormone is being released (Guraya 1976, 1978a, b, 1979). According to this concept, the granulosa cells in stages 1,2, 3, which contain a few lipid droplets of the postovulatory follicles (table 1), may be functioning in the secretion of some steroid hormone (see also Guraya 1976, 1979). This suggestion is also supported by the fact that the granulosa cells during these stages show organelles and enzyme activities related to steroid biosynthesis (see Guraya 1976, 1979; Hoar and Nagahama 1978 ; Nagahama et al 1976, 1978 ; Kagawa et al 1981). The degenerating granulosa cells in stages 4, 5 and 6 of postovulatory follicle (table 1) apparently function in the storage of hormone precursor, as supported by the accumulation of highly sudanophilic, cholesterol-positive lipid droplets (figures 3-7) which constitute the precursor material stored within the steroid gland cells (Guraya 1976, 1978b, 1979). The regression of steroidogenesis during the later life of postovulatory follicle in teleost ovary is also supported by the gradual disappearance of its enzyme activities and alterations in organelles related to steroidogenesis (Lambert and van Oordt 1974 ; Guraya 1976, 1979 ; Nagahama et al 1976, 1978). The minimum enzyme 3,B-HSDH activity is reached between the third and fourth day after spawning in the zebra fish (Lambert and van Oordt 1974), indicating a short functional life of postovulatory follicle in the oviparous teleosts (Guraya 1976, 1979) as also supported by the results of the present study Corpora atretica (or preovulatory corpora lutea) The first change, which occurs during the atresia of ripe yolky eggs, is the development of large sudanophobic vacuoles in the region of cortical vacuoles (figures 8,9). As the atresia advances, highly sudanophilic fatty yolk elements coalesce to form

6 Table 1. Summary of lipid changes in the granulosa and thecal cells during successive stages of postovulatory follicle in the fish Cyprinus carpio. Technique Fixation Reference Cell types Lipid droplets Diffuse lipids Stage 1 Stage 2 Stage 3 Stage 4 Stages 5, 6 Remarks Sudan black B(SBB) in 70% ethanol suu" PB Nile blue sulphate FCA + PC After Baker GC 1946, 1956 (as cited in TC Pearse 1968) WB+ pb l'ca + PC Sudan III and FCA + PC Sudan IV Acid FCA + PC haernatin (AB) AH. PB WB + PB Schultz FCA + PC test Cited in GC Pearse 1968 TC After Cain GC 1947 (cited in TC Pearse 1968) As cited in GC Pearse 1968 GC Baker 1946 cc TC (cited in GC Pearse 1968) TC Pearse 1968 GC TC + blue + blue P + to p+ +to + + to + + p+ p+ + + P P + + p p + to + P p + + to + Showing the presence of diffuse lipid and discrete lipid droplets in granulosa cells (oc) and hypertrophied thecal cells at different stages of postovulatory follicle Revealing the increasing development of neutral fats in the lipid droplets of granulosa and thecal cells at different stages. Revealing the increasing devement of triglycerides in lipid droplets during the successive stages Revealing the presence of phospholipid in the lipid droplets of granulosa and thecal cells Revealing the accumulation of cholesterol and/or esters in granulosa luteal cells in stages 4. 5,6 Key to abbreviations :..., after treatment with; FCA + PC, formaldehyde-calcium and postchromed in dichrcrnate-calcium ; P, pink, WB + PB, Weak Bouin followed by hot pyridine extraction; +, weak reaction j, moderate reaction j +, strong reaction;. very strong reaction; -. negative.

7 Histochemistry of teleost ovary and steroidogenesis 593 Figures Yolky eggs in its early stage of atresia, showing sudanophobic vacuoles at the periphery (cv). Follicular epithelium and zona pellucida are still intact. Germinal vesicle (ov) is also seen. x Corpus atreticum in stage 3 showing accumulation of sudanophilic lipids in granulosa cells. The yolk has been ingested by the granulosa cells. x Showing degenerated corpora (AF) in stage 4, filled with sudanophilic lipids. X 400.

8 Histochemistry of teleost ovary and steroidogenests 595 highly sudanophilic masses (figures 8,9, 10). The yolky contents of atretic follicles are gradually digested and removed by the granulosa cells which, at the same time, store lipids consisting of triglycerides and some phospholipids. Similar lipids also accumulate in the granulosa cells of atretic follicles in other vertebrates (Guraya 1976). In their later stages of resorption, atretic yolky eggs in the present teleost also store cholesterol and/or its esters. The corpora atretica or ' pre-ovulatory, corpora lutea of lower vertebrates are usually believed to secrete steroid hormones (see references in Browning 1973). But Guraya (1976) believes that they are merely the large eggs in the process of their resorption as also observed in this study. The various enzyme cytochemical investigations have also shown that the corpora atretica of teleost ovary are merely the large yolky eggs in the process of degeneration and resorption (Guraya 1976, 1979). The dense lipid and cholesterol accumulations demonstrated in the atretic yolky eggs of the present carp may be due to degenerative changes Interstitial cells The interstitial cells in the ovaries of present teleost occur singly or in groups in the stroma (figures 6, 7). Some of them appear to derive from the persisting hypertrophied thecal gland cells of postovulatory follicles as well as from their residual granulosa cells (figures 6, 7). They are filled with sudanophilic lipid droplets consisting of triglycerides, cholesterol and/or its esters and some phospholipids. The thecal gland cells, described by Nicholls and Maple (1972) in the wall of the cichlid fish follicles, may be the interstitial (or thecal) gland cells having the cytological features of steroid-secreting cells. These special thecal cells are believed to be the only cells responsible for steroid production in the thecal layer of salmonid fishes (Nagahama et al 1978 ; van den Hark and Peute 1979). Kagawa et al (1981) have also identified special thecal cells in the wall of preovulatory follicle of S. leucomaenis, which show mitochondria with tubular cristae and both agranular and granular forms of endoplasmic reticulum, the latter being more prominent. The results of these various studies have indicated that interstitial cells form the important component of the teleost ovary. With the growth of vitellogenic follicles, they get sparsely distributed in their walls and then are called as special thecal cells or thecal gland cells. The presence of interstitial cells has also been demonstrated in the ovaries of different vertebrates (see Guraya 1976). The ovarian interstitial cells of these vertebrates possess the cytological and histochemical features of well-established steroid gland cells (Guraya 1976). It can also be presumed that the interstitial (or thecal gland) cells ofpresent teleost ovary are steroid secretors as they contain cholesterol-positive lipids in their cytoplasm under certain physiological situations, and are associated with blood vessels. This suggestion is further supported by the presence of 3p-HSDH activity indicative of steroidogenesis in the ovarian interstitial (or thecal gland) cells of teleosts (Lambert and van Oordt 1974 ; Guraya 1976, 1978a, 1979) as well as by the various electron microscope studies (see Nagahama et al 1978 ; Kagawa et al 1981). In the ovaries of teleosts investigated by Lambert and van Oordt

9 596 Sardul S Guraya and Surinderpal Kaur (1974), the 3,B-HSDH positive interstitial cells arc mainly distributed in the stroma, as well as against the follicle wall. They show conspicuous fluctuations in their distribution and enzyme contents with the ovarian cycle. The physiological significance of the interstitial cells remains doubtful as in the Swordtail where they show clear cytochemical indications ofsteroid metabolism, but in the zebra fish they lack any glucose-6-phosphate dehydrogenase activity (Lambert and van Oordt 1974). Kagawa et al (1981) have attributed the secretion of high progesterone levels to the special thecal cells of postovulatory follicle in the white-spotted char rather than to its granulosa luteal cells. References Ball J N 1960 Reproduction in female bony fishes; Symp, Zool. Soc. London Bara G 1965 Histochemical localization of t>5-3p-hydroxysteroid dehydrogenase in the ovaries of a teleost fish Scomber scomber L.; Gen. Compo Endocrinol Browning H C 1913 The evolutionary history of the corpus lateum ; Bioi. Reprod Chan S T H, Wright A and Phillips J G 1961 The atretic structures in the gonads of the rice-field eel (Monopteru9 a/bus) during natural sex reversal; J. Zool. (Lond.) Dodd J M 1960 Gonadal and gonadotrophic hormones. In Marshall's physiology of Reproduction (ed.) A S Parkes (London: Longmans, Green and Cc.) Vol. I Part II pp Guraya S S 1968 Histochemical study of granulosa (follicular cells) in the preovulatory and postovulatory follicles of amphibian ovary; Gen. Compo Endocrinol, Guraya S S 1976 Recent advances in the morphology, histochemistry and biochemistry of steroid-synthesizing cellular sites in the non-mammalian vertebrate ovary; Inter. Rev. Cytol, Guraya S S 1978a Maturation of the follicular wall of non-mammalian vertebrates. In The vertebrate ovary (ed.) R E Jones (New York: Plenum Press) pp Guraya S S 1918b Recent advances in the morphology, histochemistry. biochemistry and physiology of interstitial gland cells of maminalian ovary; Int. Rev. Cytol, Guraya S S 1919 Recent advances in the morphology and histochemistry of steroid-synthesizing cellular sites in the gonads of fish; Proc, Indian nom. Sci. Acad Guraya S S, Kaur R and Sr.xena P K 1915 Morphology of ovarian changes during the reproductive cycle of fish, Mystus tengara (Ham.) ; Acta Anat Guraya S S, Toor H S and Kumar S 1917 Morphology of ovarian changes during the reproductive cycle of the fish Cyprinus carpio communis (Linn.) ; Zool. Baitrage Hark R van den and Peute J 1979 Cyclic changes in the ovary of the rainbow trout, Salmo gairdneri, with special reference to sites of steroidogenesis; Cell Tissue Res Hoar W S and Nagaham~ Y 1918 The cellular sources of sex steroids in teleost gonads; Ann. Bioi. Anim. Biochim, Biophys Kagawa H, Takano K and Nagahama Y 1981 Correlation of plasma estradiol-17ft and progesterone levels with ultrastructure and histochemistry of ovarian follicles in the whitespotted char, Salvelinus leucomaenis ; Cell Tiss. Res Lambert J G D and van Oordt P G W 1914 Ovarian hormones in teleosts; Fortschr, Zool, Nagahama Y, Clarke W C and Hoar W S 1916 Ultrastructure of putative steroid producing cells in the gonads of coho (Oncorhynchus kisutch) and pink salmon (Oncorhynchus gorbuscha i Can, J, ~ool ~8-113?

10 Histochemistry of teleost ovary and steroidogenesis 597 Nagahama Y, Chan K and Hoar W S 1978 Ultrastructure of putative steroid producing cells in the gonads of coho (Oncorhynchus kisutch) and pink salmon (Oncorhynchus gorbuscha) ; Can. J. Zool Nicholls T J and Maple G 1972 Ultrastructural observations on possible sites of steroid biosynthesis in the ovarian follicular epithelium of two species of cichlid fish, Cichlasoma nigrofasciatum and Haplochromis multicolour; Z. Zellforscb Pearse AGE 1968 Histochemistry (London: Churchill Ltd.) Polder J J W 1964 On the occurrence and significance of atretic follicles (preovulatory corpora luteal in ovaries of Bitterling Rhodeus amarus (Bloch); Prod. Kon. Ned. Akad. Net