Effects of activators of different intracellular signaling pathways on steroid production by goldfish vitellogenic ovarian follicles

Abstract

For the present study, calcium ionophore A23187 and phorbol 12-myristate 13-acetate (PMA) were used to investigate the possible roles that changes in intracellular calcium content and protein kinase C activation play in steroid production by goldfish vitellogenic ovarian follicles (0.2-0.7 mm in diameter) incubated in vitro. While largely ineffective when tested alone, PMA and A23187 exhibit synergism leading to increased testosterone and 17 beta-estradiol production. Production of both steroids was also increased by the addition of either human chorionic gonadotropin (hCG) or forskolin, a direct activator of adenylate cyclase. However, hCG- and forskolin-stimulated testosterone and 17 beta-estradiol production was attenuated by PMA and A23187 either alone or in combination. Both drugs blocked the stimulatory actions of hCG on adenosine 3',5'-cyclic monophosphate (cAMP) formation. PMA also influenced steroid production at sites distal to cAMP formation as it blocked the actions of dibutyryl cAMP a permeant analog of cAMP. This action was prior to cholesterol side-chain cleavage as PMA had no effect on the metabolism of 25-hydroxycholesterol to testosterone or 17 beta-estradiol. By comparison, A23187 had no effects on either dbcAMP- or 25-hydroxycholesterol-stimulated steroid production. Separation of vitellogenic and prematurational, full-grown (0.9-1.1 mm in diameter) ovarian follicles from the same fish lead to the demonstration of opposing actions of A23187 in the two follicle types. Whereas A23187 inhibited hCG-stimulated steroid production in vitellogenic follicles, it had an additive effect on the stimulatory action of hCG on testosterone production by prematurational, full-grown ovarian follicles. In conclusion, these studies on vitellogenic ovarian follicles demonstrate specific regulatory actions of calcium and protein kinase C at multiple steps in the steroidogenic cascade and that ovarian responsiveness to calcium changes during the course of follicle development in the goldfish.