Forskolin-Induced Differentiation of Cultured Rat Granulosa Cells: New Evidence for an Intermediary Role of Adenosine 3′,5′-Monophosphate in the Mechanism of Action of Follicle-Stimulating Hormone*

Abstract

The intermediary role of cAMP in the mechanism of action of FSH was reinvestigated in vitro using forskolin, a highly specific adenylate cyclase probe. Granulosa cells from immature hypophysectomized diethylstilbestrol-treated rats were cultured for 3 days in the absence or presence of forskolin. Treatment with increasing concentrations (10-7-10-4 M) of forskolin led to dose-dependent increments in the accumulation of extracellular cAMP, with an apparent median effective dose of 1.6 .+-. 0.5 .times. 10-5 M. Concomitant blockade of cAMP phosphodiesterase activity further enhanced the forskolin effect. Treatment with forskolin also brought about dose- and time-dependent increments in progesterone and estrogen accumulation. Granulosa cells not pretreated with forskolin displayed negligible LH/hCG [luteinizing hormone/human chorionic gonadotropin] binding and remained unresponsive to luteotropic (LH/hCG), .beta.2-adrenergic (terbutaline), or lactogenic (prolactin) stimulation. In contrast, forskolin (10-5 M)-pretreated granulosa cells displayed significant increased over controls in LH/hCG binding (46-fold) as well as in progesterone accumulation stimulated by hCG (3.3-fold), terbutaline (1.9-fold), and prolactin (1.8-fold). Furthermore, concomitant treatment with a functionally inert low dose (10-7 M) of forskolin, substantially potentiated the FSH-stimulated accumulation of extracellular cAMP, progesterone, and estrogen as well as the FSH-mediated increase in LH/hCG binding. Forskolin, like FSH, is capable of inducing the differentiation of cultured rat granulosa cells by itself, and a functionally inert low dose of forskolin can potentiate FSH hormonal action. Inasmuch as forskolin-simulated and forskolin-potentiated hormonal action are acceptable as novel criteria of cAMP dependence, cAMP may be an intracellular 2nd messenger of FSH.