Steroidogenesis of porcine granulosa cells from small and medium-sized follicles: effects of follicle-stimulating hormone, forskolin, and adenosine 3,'5'-cyclic monophosphate versus phorbol ester.

ABSTRACT

We studied the effects of porcine FSH, forskolin, and (Bu)2cAMP [agents that stimulate steroidogenesis via the adenylate cyclase-cAMP pathway (cAMP system)] either alone or with concomitant addition of phorbol 12-myristate 13-acetate (TPA; a phorbol ester that activates protein kinase-C) on steroidogenesis in porcine granulosa cells cultured from small (less than 3 mm) and medium-sized (3-6 mm) ovarian follicles. We attempted to determine if granulosa cells from different maturational states had different responses to these agonists and antagonists. Cells were cultured in serum-free medium 199 supplemented with insulin (10 micrograms/ml), transferrin ( 5 micrograms/ml), and androstenedione (2.5 X 10(-7) M) for 48 h. Levels of progesterone (P) and estradiol (E2) were determined in spent medium by RIA. We found that FSH, forskolin, and cAMP all stimulated secretion of E2 and P in a dose-dependent manner in both developmental groups. When TPA was added alone to cultures, P levels were stimulated at low doses of TPA but inhibited at higher doses in granulosa from both sized follicles, whereas cells from both small- and medium-sized follicles demonstrated reductions in E2. TPA was also found to inhibit FSH-, forskolin-, and cAMP-induced steroidogenesis in a dose-dependent manner in cells from the two groups of follicles. The stimulatory effects of any of the secretagogues on E2 secretion were inhibited by TPA to a significantly greater extent in granulosa cells from small follicles. Although inhibition of FSH- and forskolin-induced P secretion by TPA was also greater in granulosa cells from small follicles, cAMP-treated cells did not show this differential inhibition. Thus, it appears that modulators of the protein kinase-C system regulate steroidogenesis differently in granulosa cells from small and medium follicles. These differences may involve alterations in the interplay between the protein kinase-C and cAMP pathways.