METABOLISM OF PROGESTOGENS DURING INVITRO MEIOTIC MATURATION OF FOLLICLE-ENCLOSED OOCYTES OF THE GOLDFISH (CARASSIUS-AURATUS)

Progestogens are known to elicit meiotic maturation of fish oocytes in vitro. Exogenous 17-alpha-hydroxyprogesterone (HP), as assessed by radioimmunoassay (RIA), decreased exponentially to less than 10% of the initial level in media during a 48 hr incubation with large (i.e., 1 mm dia.) ovarian follicles of goldfish undergoing meiotic maturation in vitro. During follicle incubation, media, into which [H-3]-HP was introduced, showed exponential reduction of [H-3]-HP with a concomitant increase in the number of [H-3]-HP metabolites. Analysis of follicle extracts revealed that small amounts of [H-3]-HP were taken up by the follicles early in the incubation (i.e., 3 hr) and converted to several metabolites; the major one of these, which accumulated in the follicle, comigrated with 17-alpha, 20-beta dihydroxy 4-pregnen 3-one (DHP). Autoradiography of extracts run on TLC revealed an increasing number of metabolites with time, i.e., 9 major spots for media, 6 major spots for follicles incubated 24 hr with [H-3]-HP. Three of the more prominent metabolites, DHP, 3-alpha,17-alpha-dihydroxy5-beta-pregnan 20-one, and 5-beta-pregnan3-alpha,17-alpha,20-beta-triol, were identified tentatively by comigration with standards on TLC and crystallization to constant specific activity. HPLC of extracts revealed a selective accumulation of DHP in follicles over time i.e., this steroid was the major metabolite peak associated with follicles by 24 hr incubation. Nevertheless, the process of oocyte maturation had already been initiated before the peak level of DHP had been reached. By contrast, HP diminished with time in follicles despite the relatively high levels occurring in the media. This research indicates that a complex steroid milieu at the level of individual, fully grown, ovarian follicles is produced in vitro from exogenous HP; significant HP metabolism occurs by 6 hr incubation, well before the process of oocyte maturation (i.e., germinal vesicle migration and dissolution) has begun.