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The present work on two freshwater teleosts, Cyprinion watsoni and Barilius vagra (Cyprinidae, Teleostei) was conducted to study (1) monthly steroid profile in relation to seasonal changes in ovarian development, (2) relative importance of various ovarian steroids in inducing maturation of oocytes in vitro as well as the role of human chorionic gonadotropin (hCG) in this process, and (3) metabolic potential of the ovary of Cyprinion watsoni in vitro in the presence of selected steroid substrates, given singly or jointly with hCG. The study shows that both C. watsoni and B. vagra 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 GSI. 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 vitellogenic progress that began in January/February and reached its peak in April, the vitellogenic and maturing oocytes coexisted during the active spawning months. The determination of ovarian steroids in vivo and in vitro was done by solid phase extraction of steroids from ovarian samples and the incubation medium, using Sep-Pak C 18 and resolution by reverse phase gradient mobile HPLC at 254 and 280 nm. The steroids identified in vivo during the year were estriol (E: estra-l,3,5 (10)-triene-3-16α,17β-triol), estrone (El: estra-l,3,5 (10)-triene-3-01-17-one), estradiol 17 β (E2: estra-l,3,5 (10)- triene-3, 17 β-diol), testosterone (T: 17 β- hydroxy-4- androsten- 3- one), androstenedione (AD: 4-androstene-3, 17-dione), 11β-hydroxyandrostenedione (11B-OHA: 11 β-hydroxy-4-androstene-3, 17-dione), 19-hydroxyandrostenedione (19-OHA: 19 β-hydroxy-4-androstene-3, 17-dione), progesterone (P4: pregn-4-ene-3, 20-dione), 17-α-hydroxyprogesterone (17-OHP: 17 α-hydroxypregn-4-ene-3, 20-dione), 17 α-20 β-dihydroxypregn-4-ene-3-dione (17,20 β P: 17, 20 β -dihydroxypregn-4-ene-3-one), 11-deoxycorticosterone (DOC: 21-hydroxypregn-4-ene-3,20-dione), corticosterone (B:11β, 21-dihydroxypregn-4-ene-3, 20-dione), cortisol (F: 11 β, 17α. 21-trihydroxypregn-4-ene-3, 20-dione) and aldosterone (ALDO: 11 β, 21-dihydroxypregn-4-ene-3, 20-dion-18-al). The levels of the three estrogens increased in parallel with the initial vitellogenic progress during January to April, with peak levels in March/April followed by only gradual decline in the spawning season (May to August). El appeared as a prominent estrogen in both species and matched E2 both in level and persistence at appreciably high levels during the spawning season. The seasonal profile of T and AD was also similar to that of estrogens, suggesting a role in vitellogenesis as well as maturation of the oocytes. 11β -OHA and 19-OHA waxed and waned during the year and were the dominant androgens in both species. The concentration of P4 was generally low during the year in both species. 17 -OHP and 17, 20 βP 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 oviposition). The presence of DOC, B and F throughout the year in the two species demonstrated that their ovaries are capable of synthesizing these steroids. In both species, DOC and B appeared as dominant corticoids. Peak: levels of DOC and B in C. watsoni matched the early to late recrudescent phase; while in B. vagra high levels of these steroids occurred in the periovulatory and spawning season. The levels of F were generally and relatively low throughout the year in both species. Species differences were evident in that its concentration was slightly higher in B. vagra, whereas in C. watsoni 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 in vivo profile collectively showed a pattern that matched the asynchronous ovarian development and affirms 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 determine 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. The results of the in vitro work on oocyte maturation reveals that 17, 20 βP is the most potent MIS in the two species. While the estrogens tested in vitro were entirely ineffective in causing significant GVBD, both the androgens and progestins caused significant GVBD, the response being both time and dose-dependent. However, only 17, 20 βP caused the earliest GVBD and with near physiological dosage (0.01 ug/ml, 24 hr incubation, C. watsoni). In B. vagra, even DOC had a significant GVBD effect but again 17, 20 βP turned out to be the most potent MIS. Human chorionic gonadotropin (hCG) also caused slight but significant GVBD in both species and strongly promoted the GVBD response to all exogenous steroids, especially the progestins. Treatment of the ovarian follicles of C. watsoni with hCG alone or jointly with the various steroid substrates and recovery of metabolites in the incubation medium demonstrated existence of a repertoire of enzyme pathways in the fully grown ovarian follicles. Whereas the in vitro and in vivo analyses support existence of at least cytochrome P450aromatase, 17-hydroxysteroid dehydrogenase (17-HSD), 20 β-hydroxysteroid dehydrogenase (20β-HSD), 11-hydroxylase, 16-hydoxylase, aldosterone synthetase (P450aldo) and sulphuronyl and glucuronyl transferases, the presence of a number of unknown peaks in the chromatograms suggests existence of other enzymes as well. Further implication of this latter observation is that while 17, 20 βP appears to be the most potent MIS in vivo and in vitro in the two species, definitive conclusion in this regard must be deferred until further work has been done to identify these as yet unknown steroids, some of which may be triols or tetrols. The presence of conjugated estrogens, androgens and progestins both in vivo and in vitro 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 steroids have other biologically important functions, pheromonal or other, in fish reproduction. The present study was undertaken against the background of total neglect in this country of a very fundamental area of reproductive physiology of fishes. It thus represents a “ground-breaking� effort to overcome technical handicaps and paves the way for further in-depth work on these two species and other local commercially important species

Item Type: Thesis (Doctoral)
Uncontrolled Keywords: female reproductive cycle, ovarian steroid, cyprinids, barilius vagra, cyprinion watsoni, teleostei, human chorionic gonadotropin, ovarian follicles
Subjects: Q Science > QH Natural history > QH301 Biology
Depositing User: Muhammad Khan Khan
Date Deposited: 08 Sep 2016 05:13
Last Modified: 08 Sep 2016 05:13
URI: http://eprints.hec.gov.pk/id/eprint/1100

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