CONTROL OF RAT GRANULOSA-CELL MITOSIS BY PHORBOL ESTER-17-BETA-DEPENDENT, CYCLIC AMP-17-BETA-DEPENDENT AND ESTRADIOL-17-BETA-DEPENDENT PATHWAYS

The present studies examined the effect of 8-bromo-cAMP (8-Br-cAMP), p horbol ester (TPA), and estradiol- 17beta (E2) on the capacity of rat granulosa cells (GC) to undergo mitosis. In the first series of experi ments, GC were either maintained within immature rat ovaries in perifu sion culture or isolated and placed in tissue culture. GC were culture d for 24 h with 1) control medium, 2) 8-Br-cAMP, 3) TPA, or 4) 8-Br-cA MP plus TPA in the presence of H-3-thymidine (H-3-T). In perifusion cu lture, 8-Br-cAMP stimulated both H-3-T incorporation (p < 0.05) and E, secretion (p < 0.05) while TPA increased H-3-T (p < 0.05) without alt ering E. secretion (p > 0.05). Simultaneous exposure to 8-Br-cAMP and TPA enhanced 3H-T incorporation and suppressed E2 as compared to 8-Br- cAMP treatment alone (p < 0.05). In tissue culture, 8-Br-cAMP did not increase H-3-T incorporation or cell number. TPA increased both, H-3-T incorporation (p < 0.05) and cell number (p < 0.05), while 8-Br-cAMP suppressed both of these TPA-induced responses. In the presence of tes tosterone, 1 ) TPA's mitogenic action was also observed, 2) basal E2 s ecretion ranged between 30 and 35 pg/ml, 3) neither 8-Br-cAMP nor TPA stimulated E2 secretion over basal levels, and 4) Rp-cAMP, a cAMP anta gonist, blocked TPA-induced cell proliferation. E2 at 250 pg/ml also b locked TPA's mitogenic action. In a second series of experiments, GC w ere collected from eCG-treated rats. Although E2 levels ranged from 25 0 to 350 pg/ml, TPA induced mitosis that was inhibited by 8-Br-cAMP. T hese data demonstrate that cAMP- and TPA-dependent pathways interact t o determine whether the GC will undergo mitosis. Specifically, low end ogenous levels of cAMP synergize with TPA-dependent pathways to promot e mitosis, while high levels of 8-Br-cAMP inhibit proliferation. An E2 -dependent mechanism also exists that attenuates TPA'S Mitogenic actio n in GC isolated from immature rats but not eCG-treated rats. Since 8- Br-cAMP blocks mitosis in the absence of androgen, it is unlikely that cAMP mediates its action by increasing E, secretion. It appears then that the anti-mitogenic actions of cAMP and E2 are mediated through re dundant, independent mechanisms that prevent GC mitosis. Further, the anti-mitogenic properties of E2 but not cAMP diminish as a result of g onadotropin-induced GC maturation.