HistochernicalJournal, 11 (1979), 253-265
Steroid synthesizing cellular sites in the ovary of the domestic pigeon Columba livia (Gmelin)" a histochemical study
BHAGYASHRI V. BHUJLE*, VINAYAK B. N A D K A R N I and M. APPASWAMY RAO Department of Zoology, Karnatak University, Dharwad 580 003, Karnataka State India
Received 20 April 1978 and in revised form 4 December 1978
Synopsis. The ovary of the domestic pigeon, Columba livia, has been assayed histochemically for the localization of A s-3~-hydroxysteroid dehydrogenase (A s-3/3-HSDH), 17fi-hydroxysteroid dehydrogenase (17fi-HSDH), 1 l~-hydroxysteroid dehydrogenase (ll~-HSDH), glucose-6-phosphate dehydrogenase (G6P-DH)and NADH-diaphorase activities during different periods of the reproductive cycle. As-3~-HSDH, 17i3-HSDH, 11/3-HSDH, G6P-DH and NADH-diaphorase activity was found in the theca interna of growing, atretic and postovulatory follicles, the granulosa of ovulatory, atretic and postovulatory follicles, and interstitial gland cells during the pre-incubation and the laying periods. During the incubation and squab feeding periods only As-3~-HSDH, G6P-DH and NADH-diaphorase activities were observed in the above mentioned cells. The steroidogenic potential of atretic follicles depends upon the type of atresia a follicle undergoes.
Introduction
In recent years it has been shown that the avian ovary, like its mammalian counterpart, is a source of androgenic, oestrogenic and progestogenic secretion (Layne et al., 1958; Ozon, 1965; Hohn & Cheng, 1967; O'Grady, 1968; O'Malley etal., 1968; Guraya, 1976a). The steroidogenic cells of the avian ovary in a few species have been identified by the histochemical localization of lipids, cholesterol and some hydroxysteroid dehydrogenases (Marshall & Coombs, 1957; Chieffi & Botte, 1965; Chieffi, i967; Arvy & Hadjiisky, 1970; Boucek & Savard, 1970; Guraya, 1976a). Divergent views have been expressed about the cellular sites of steroid biosynthesis in the avian ovary. Further, it seems that the inconsistency in the results obtained by previous workers *Present address: Department of Biology, S. P. Chowgule College, Margao 403 601, Goa, India. 9 1979 Chapman and Hall Ltd. Prin ted in Great Britain.
254
Bhufle, Nadkarni and Rao
might be due to the fact that in all these studies the phase of the reproductive cycle during which the ovaries were examined is not taken into consideration. Boucek & Savard (t970) have suggested that some steroid,zonverting enzymes are found in the ovary of the fowl during a particular phase of the cycle, but these are not present in another phase. Therefore, the present study was undertaken to follow the histochemical distribution of hydroxysteroid dehydrogenases in the ovary of the domestic pigeon, Columba lMa, during different periods of the reproductive cycle, particularly the pre4ncubation, incubation and squab feeding periods. FoUicular atresia, a widespread phenomenon occurring in the ovaries of all vertebrates, is a very common feature of the avian ovary (Lofts & Murton, 1973). Atretic follicles are generally considered to be degenerating elements of doubtful endocrine function (Lofts & Bern, 1972; Dodd, 1977). However, it has been suggested that follicular atresia can be considered as an integral part of ovarian function (Guraya, 1976a; Weir & Rowlands, 1977). Further, it is suggested that the atretic follicles in their early stages may have a transient endocrine activity (Saidapur, 1978). Hence, the localization of hydroxysteroid dehydrogenases has been studied in different types of atretic follicles found in the ovary of the pigeon in an attempt to find out whether they have steroidogenic potential or not.
Materials and methods Adult female pigeons reared in the pigeon colony of the Zoology Department were selected for the study. A minimum of ten pigeons from each of the pre-incubation, laying, incubation and squab feeding periods were used. They were killed by decapitation and parts of the ovary were frozen over dry ice vapour. Cryostat sections, 12/~m thick, were incubated aerobically in appropriate media having different substrates (listed in Tables 1 and 2 for different enzyme activities) at a final concentration of 1 mg/ml dissolved in dimethylformamide, t3-NAD (1.5 mg/ml) and Nitro BT (1 mg/ml) dissolved in phosphate buffer at pH 7.3. The details of the histochemical procedure were as described by Baillie et al. (1966). Parallel control sections were incubated in a medium that individually lacked either the co-factor NAD, NADP or the corresponding substrate. Alternative sections from frozen tissues were also ftxed in neutral formalin and stained with Haematoxylin-Eosin to study the histological details of the different ovarian follicles, atretic follicles in particular. The remaining parts of the ovary from the pigeon during all the periods were fixed in Bouin's fluid and processed for histological studies. The tissues were embedded in paraffin and 6/~m sections were stained with Haematoxylin-Eosin. All substrates were obtained from Sigma Chemical Co, USA. Remits
(a) GROWING FOLLICLES s s-3~-HSDH activity As-3/3-HSDH activity was not found in the granulosa ceils of the previtellogenic follicles, but a weak activity was found in some cells of theca interna of early viteUo-
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Steroid synthesizing cellular sites in the ovary of the domestic pigeon Columba livia (Gmelin)" a histochemical study
BHAGYASHRI V. BHUJLE*, VINAYAK B. N A D K A R N I and M. APPASWAMY RAO Department of Zoology, Karnatak University, Dharwad 580 003, Karnataka State India
Received 20 April 1978 and in revised form 4 December 1978
Synopsis. The ovary of the domestic pigeon, Columba livia, has been assayed histochemically for the localization of A s-3~-hydroxysteroid dehydrogenase (A s-3/3-HSDH), 17fi-hydroxysteroid dehydrogenase (17fi-HSDH), 1 l~-hydroxysteroid dehydrogenase (ll~-HSDH), glucose-6-phosphate dehydrogenase (G6P-DH)and NADH-diaphorase activities during different periods of the reproductive cycle. As-3~-HSDH, 17i3-HSDH, 11/3-HSDH, G6P-DH and NADH-diaphorase activity was found in the theca interna of growing, atretic and postovulatory follicles, the granulosa of ovulatory, atretic and postovulatory follicles, and interstitial gland cells during the pre-incubation and the laying periods. During the incubation and squab feeding periods only As-3~-HSDH, G6P-DH and NADH-diaphorase activities were observed in the above mentioned cells. The steroidogenic potential of atretic follicles depends upon the type of atresia a follicle undergoes.
Introduction
In recent years it has been shown that the avian ovary, like its mammalian counterpart, is a source of androgenic, oestrogenic and progestogenic secretion (Layne et al., 1958; Ozon, 1965; Hohn & Cheng, 1967; O'Grady, 1968; O'Malley etal., 1968; Guraya, 1976a). The steroidogenic cells of the avian ovary in a few species have been identified by the histochemical localization of lipids, cholesterol and some hydroxysteroid dehydrogenases (Marshall & Coombs, 1957; Chieffi & Botte, 1965; Chieffi, i967; Arvy & Hadjiisky, 1970; Boucek & Savard, 1970; Guraya, 1976a). Divergent views have been expressed about the cellular sites of steroid biosynthesis in the avian ovary. Further, it seems that the inconsistency in the results obtained by previous workers *Present address: Department of Biology, S. P. Chowgule College, Margao 403 601, Goa, India. 9 1979 Chapman and Hall Ltd. Prin ted in Great Britain.
254
Bhufle, Nadkarni and Rao
might be due to the fact that in all these studies the phase of the reproductive cycle during which the ovaries were examined is not taken into consideration. Boucek & Savard (t970) have suggested that some steroid,zonverting enzymes are found in the ovary of the fowl during a particular phase of the cycle, but these are not present in another phase. Therefore, the present study was undertaken to follow the histochemical distribution of hydroxysteroid dehydrogenases in the ovary of the domestic pigeon, Columba lMa, during different periods of the reproductive cycle, particularly the pre4ncubation, incubation and squab feeding periods. FoUicular atresia, a widespread phenomenon occurring in the ovaries of all vertebrates, is a very common feature of the avian ovary (Lofts & Murton, 1973). Atretic follicles are generally considered to be degenerating elements of doubtful endocrine function (Lofts & Bern, 1972; Dodd, 1977). However, it has been suggested that follicular atresia can be considered as an integral part of ovarian function (Guraya, 1976a; Weir & Rowlands, 1977). Further, it is suggested that the atretic follicles in their early stages may have a transient endocrine activity (Saidapur, 1978). Hence, the localization of hydroxysteroid dehydrogenases has been studied in different types of atretic follicles found in the ovary of the pigeon in an attempt to find out whether they have steroidogenic potential or not.
Materials and methods Adult female pigeons reared in the pigeon colony of the Zoology Department were selected for the study. A minimum of ten pigeons from each of the pre-incubation, laying, incubation and squab feeding periods were used. They were killed by decapitation and parts of the ovary were frozen over dry ice vapour. Cryostat sections, 12/~m thick, were incubated aerobically in appropriate media having different substrates (listed in Tables 1 and 2 for different enzyme activities) at a final concentration of 1 mg/ml dissolved in dimethylformamide, t3-NAD (1.5 mg/ml) and Nitro BT (1 mg/ml) dissolved in phosphate buffer at pH 7.3. The details of the histochemical procedure were as described by Baillie et al. (1966). Parallel control sections were incubated in a medium that individually lacked either the co-factor NAD, NADP or the corresponding substrate. Alternative sections from frozen tissues were also ftxed in neutral formalin and stained with Haematoxylin-Eosin to study the histological details of the different ovarian follicles, atretic follicles in particular. The remaining parts of the ovary from the pigeon during all the periods were fixed in Bouin's fluid and processed for histological studies. The tissues were embedded in paraffin and 6/~m sections were stained with Haematoxylin-Eosin. All substrates were obtained from Sigma Chemical Co, USA. Remits
(a) GROWING FOLLICLES s s-3~-HSDH activity As-3/3-HSDH activity was not found in the granulosa ceils of the previtellogenic follicles, but a weak activity was found in some cells of theca interna of early viteUo-
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