Changes in the steroidogenic pathway in medaka (Oryzias latipes) ovarian follicles during vitellogenesis and oocyte maturation were investigated in vitro by incubation of follicles with several radiolabeled steroid precursors, followed by thin layer chromatography (TLC) fractionation and recrystallization. When vitellogenic follicles collected at 18 hr before the expected time of spawning were incubated with ³H-labeled pregnenolone, the major metabolites were 17α-hydroxypregnenolone, 17α-hydroxyprogesterone, and androstenedione. Incubations of vitellogenic follicles with androstenedione produced testosterone and estradiol-17β By contrast, when maturing follicles (postvitellogenic follicles undergoing maturation) collected at 10 hr before spawning were incubated with ³H-labeled pregnenolone, the major metabolites were 17α-hydroxypregnenolone, 17α-hydroxyprogesterone, and 17α, 20β-dihydroxy-4-pregnen-3-one (17α, 20β-DP, maturation-inducing hormone of medaka); androstenedione was not detected. Neither vitellogenic and maturing follicles produced progesterone when they were incubated with ³H-labeled pregnenolone, suggesting that in medaka ovarian follicles both estradiol-17β and 17α, 20β-DP are synthesized by the ⁵Δ-steroid pathway. Thus, there is a distinct shift in the steroidogenic pathway from estradiol-17β to 17α, 20β-DP production in medaka ovarian follicles, and it is suggested that the decrease in C17, 20-lyase activity is responsible for this shift.

The phosphodiesterase inhibitor IBMX enhanced androstenedione production in incubations of vitellogenic follicles with ¹⁴C-labeled progesterone. Calcium ionophore A23187 and the phorbol ester TPA (a protein kinase C activator) blocked the stimulatory actions of IBMX on androstenedione production. These findings suggest that multiple signalling pathways may participate in the regulation of ovarian steroidogenesis, and further emphasize the importance of calcium as a regulator of P-450c17 activity.