MODULATION OF GRANULOSA-CELL DEOXYRIBONUCLEIC-ACID SYNTHESIS AND DIFFERENTIATION BY ACTIVIN

FSH stimulates follicular growth through inducing granulosa cell proli feration. Our working hypothesis is that mitogenesis is facilitated by a locally produced growth/differentiation factor(s) that modulates FS H action in developing granulosa cells. The present study was undertak en to examine the effect of the related gonadal peptides activin and i nhibin on granulosa cell proliferation. Monolayer cultures of granulos a cells isolated from preantral/early antral follicles in immature rat ovaries were established by incubation overnight in serum-free medium 199. Treatment was initiated with serum-free medium containing recomb inant human (rh) activin and/or rh-inhibin in the presence or absence of FSH. After incubation for 18 h, medium was collected for progestero ne determination (as a marker of cell differentiation), and the cell m onolayers were incubated for 2 h in the presence of [H-3]thymidine to measure DNA synthesis. Activin dose dependently (1-100 ng/ml) stimulat ed DNA synthesis (minimal effective dose, 1 ng/ml), whereas inhibin or FSH alone was without effect. When activin (1 ng/ml), but not inhibin , was present with FSH, the gonadotropin caused dose-dependent increas es in [H-3]DNA synthesis over a wide range of FSH concentrations (1-10 0 ng/ml). This property of activin was unaltered by the additional pre sence of inhibin (1-100 ng/ml). To analyze the role of cAMP in mediati ng the mitogenic action of FSH in the presence of activin, experiments were repeated substituting a membrane-permeable cAMP agonist, 8-bromo -cAMP (8br-cAMP; 0.1-3 mM). Similar to FSH, 8br-cAMP had no effect on granulosa cell DNA synthesis in the absence of activin. However, in th e presence of activin (1 ng/ml) 8br-cAMP was stimulatory. The dose res ponse to 8br-cAMP revealed a biphasic effect on DNA synthesis and diff erentiation: DNA synthesis rose to a maximum in the presence of 0.5 mM 8br-cAMP and declined thereafter. Progesterone synthesis only started to increase in the presence of 0.1 mM 8br-cAMP, rising to a maximum a t 3 mM 2 8br-cAMP, at which concentration DNA synthesis was fully supp ressed. We conclude that activin induces DNA replication in rat granul osa cells. In the presence of activin, FSH and 8br-cAMP are mitogens. These actions of FSH and 8br-cAMP occur at doses too low to stimulate steroidogenesis, emphasizing the role of intracellular cAMP tone in gr anulosa cell proliferation and differentiation.