A Study of Annual Female Reproductive Cycle: the Ovarian Steroid Profile in the Cyprinids, Barilius vagra and Cyprinion watsoni (Teleostei)

Abstract

The present work on two freshwater teleosts, CyprilliOIl watson; and Ba,.tllII.~ l'ugra (Cypnnldae, releostei) was conducted to study (I) monthly steroid profile in relation to seasonal changes in ovarian development, (2) relative imponancc of various ovarian steroids in inducing maturation of oocyles III vitro as well as the role of human chorionic gonadotropin (heO) in this process, and (3) metabolic potential orthe ovary of Cypriru'oll watsoll; ill vitro in the presence of selected steroid substrates, given singly ai' jointly with heG. The study shows that both C. walso"i and B. lIagra are asynchronous spawners with a breeding season that lasts from May to August. The histological picture of the ovaries of these fish matched the seasonal OS!. The mean GSI in the two species was low in the postspawning months, reached a peak in April and gradually declined during the spawning season (May to August) to its lowest values in the postspawning months of September to December. Following initial vitellogellic progress that began in JanuaryIFebruary and reached its peak in April , the vitellogcnic and maturing oocytes coex isted during the active spawning months. The detenninnlion of ovarian steroids ill vivo and in vitro was done by solid phase extraction of steroids from ovarian samples and the incubation medium, using Sep~Pak CI8 and resolution by reverse phase gradient mobile HPLC at 254 and 280 run. The steroids idenl ified ill vivo during the year were estriol (E: estra~ I .3,5 (I0)-trienc-3. 16a, 17P-triol), estrone (E,: estra- I ,3,5 (I 0)-tri ene-3-01-17-one), estradiol 17P (E, : estra-I ,3,5 (10)- trienc-3, 17P- diol), testosterone (T: 17p- hydroxy- 4- androsten- 3- one), androstenedione (AD: 4-androstene-3, 17-dione), IIP-hydroxyandrostenedione (11BOl- IA: 11 p-hydroxy-4-androstene-3, 17·dione), 19-hydroxyandrostenedione (L9-0HA: 19f3·hydroxy-4-androstene-3, 17·dione), progesterone (P 4: pregn ~ 4-ene·3, 20-dione), 17- a-hydroxyprogesterone (17-0HP: 17a-hydroxypregn-4-ene-3, 20-dione), 17a-20pdihydroxypregn- 4-ene-3-dione (17,20 ~P : J 7, 20P-dihydroxypregn-4-ene-J-one), 11- deoxycorticosterone (DOC: 21.hydroxypregn-4-ene-3.20-dione), corticosterone (B: 110, 2 1.dihydroxypregn-4-cnc-3, 20-dione), cortisol (F: 11 p, 17(1, 21·trihydroxypregn-4-cne. 3, 20-dione) and aldosterone (ALDO: II p, 21 -dihvdroxypregn-4-ene-3. 20-dion-18-al). The levels orthe three estrogens increased in para llel with the initial vitellogcll ie progress during JfUluary to April, with peak levels in March/Apri l followed by only gradual declme III !he spawmng season (May to August). EI appeared as a prominent estrogen in both species and matched El both in level and persistence at appreciably high levels during the spawning season. The seasonal profile ofT and AD was also similar to that of estrogens, suggest ing a role in vitellogenesis as well as maturation of the oocytes. 11 pOHA and 19-0HA waxed and waned during the year and were the dominant androgens in both species. The concentration of P" was generally low during the year in both species. 17-0HP and 17, 20PP showed peak concentration coincident with vitellogenic peaks and onset of the spawning season and prevailed at appreciably high levels during major part of the spawning season (period of oocyte maturation/ovulation and ovipositiOll). The presence of DOC, Band F throughout the year in the two species demonstrated that their ovaries are capable of synthesizing these stero id s. In both specie~, DOC and B appeared as dominant corticoids. Peak levels of DOC and B in C. watsoll; matched the early to late recrudescent phase; while in B. vagra high levels of these sterOids occurred 111 the penovulatory and spawning season. The levels of F were generally and re latively low throughout the year in both species. Species differences were evident in that its concentration was slightly higher in B. vagra, whereas in C. watson; it persisted for a longer duration. The detection of ALDO was not surprising since it has also been identified in a few other species. The ill vivo profile collectively showed a patlem that matched the asynchronous ovarian development and affinns the view that the estrogens principally support vitellogenic growth and the progestins oocyte maturation. The androgens appear to be associated with both vitellogenesis and maturational progress. There is need to experimentally detemline the precise physiological significance of 11-oxoandrogens and the corticoids identified presently in the two species. It is further noted that a more precise definition of the association between the ovarian steroids and the ovarian stages may be achieved by analysis at shorter sampling intervals than has been possible in this study. potent MIS 111 the two species. While the estrogen:1 tested itl vitro were entirely ineffective ill causing significant GVBD, both the and rogens !1nd progestins caused .,ignilicant GVBD. the response being both time and dose-dependent. However, only 17, 20f}P causeu the earl iest GVBO and with near physiological dosage (0.01 ug/ml. 24 hr incubation, C. watsolli), In B, vagI'''. evell DOC had a significant GVBD effect but again 17, 20PP turned out to be the most polent MIS, Human chorionic gonadotropin (heG) also caused slight but significant GVSO in both species and strongly promoted the GYSO response to all exogenous steroids, especially the progestins. Treatment of the ovarian follicles of C. watsoni with heG alone or jointly with the various steroid substrates and recovery of metabolites in the incubat ion medium uemonstrated ex istence of a repertoire of enzyme pathways in the fully grown ovarian folli cles. Whereas the ill vilro and ill vivo analyses support existence of at least cytochrome P450aromatase, 17-hydroxysteroid dehydrogenase (17-HSD), 20phydroxy, teroid dehyd rogenase (20P-HSD), II-hydroxylase, 16-hydoxylase, aldosterone synthetase (P450aldo) and sulphuron.yJ and glucuronyJ transferases, the presence of a lIumber of unknown peaks in the chromatograms suggests existence of olher enzymes as well. Further implication of this latter observation is that while 17. 20PP appears to be the Illast potent MIS ill vivo and ill vitro in the two species, definitive conclusion in this regard must be deferred until further work has been done to ident ify these as yet unknown steroids, some of which may be trials or tetral s. The presence of conjugated estrogens, androgens and progestins both ill vivo and III vifro not only reflects the ability of the ovaries to intramurally deactivate and excrete these steroids but also invites attention for future investigations to determine whether the conjugated s teroids have other biologically important functions, pheromonal or other, in fish reproduction.