Evidence for a Negative Intrafollicular Role for Inhibin in Regulation of Estradiol Production by Granulosa Cells

Intrafollicular concentrations of inhibin A and estradiol vary inversely during development of dominant follicles in cattle. Thus, we hypothesized that inhibin has a negative autocrine or paracrine effect on estradiol production by granulosa cells. To examine this hypothesis, a homologous model system was used to test the effects of bovine antibovine inhibin antibodies, bovine inhibin, and a peptide fragment of bovine inhibin (bINH) on capacity of granulosa cells isolated from individual estrogen-active or -inactive dominant or subordinate follicles to produce estradiol during short-term (18 h) serum-free culture. Immunoblot analysis of media demonstrated that granulosa cells basally produce different molecular weight forms of inhibin, similar to those in bovine follicular fluid. Immunoneutralization of endogenous inhibin in culture with different doses (12.5-1000 microg) of highly purified bovine antibovine inhibin antibodies increased estradiol production 2- to 15-fold, compared with controls. Preadsorption of the anti-inhibin antibodies with bINH precursors or bovine pro-alpha(C) suppressed the capacity of anti-inhibin antibodies to enhance estradiol production by granulosa cells, compared with controls. Treatment of granulosa cells with an immunoaffinity-purified preparation of bINH suppressed basal estradiol production 60%, compared with controls. In contrast, treatment of granulosa cells with the bINH peptide increased estradiol production 14-fold, compared with controls. Based on these results, we concluded that both anti-inhibin antibodies and bINH blocked the suppressive local effects of basally produced inhibin on estradiol production during culture of granulosa cells and that inhibin has a negative autocrine or paracrine effect on the in vitro capacity of granulosa cells isolated from dominant or subordinate follicles to produce estradiol.