Progesterone mediates its anti-mitogenic and anti-apoptotic actions in rat granulosa cells through a progesterone-binding protein with gamma aminobutyric acidA receptor-like features

Progesterone (P-4) inhibits small granulosa cell (GC) mitosis and large GC apoptosis. These actions are steroid specific and dose dependent and are inhibited by the progesterone receptor (PR) antagonist, RU-486. However, these cells do not express the nuclear PR but rather an ill-defined P-4-binding protein (P4BP). This binding protein could function as a receptor and mediate P-4's actions in GCs. Therefore, a series of studies was designed to characterize this P4BP. First, an antibody directed against the ligand-binding site of the nuclear PR was used in a Western blot analysis. This analysis revealed the presence of a 60-kDa P4BP within ovarian and GC lysates as well as within an ovarian membrane preparation. This protein was not observed in lysates of cells derived from the ovarian surface epithelium. In addition, this P4BP was immunoprecipitated by an antibody to the alpha, chain of the gamma aminobutyric acid(A) (GABA(A)) receptor, suggesting that the P4BP could be the ovarian GABA(A) receptor. Since activation of the rat ovarian GABA(A) receptor increases intracellular CAMP levels, GCs were cultured with control medium supplemented with either 8-bromo-cAMP (8-br-CAMP), P-4, or muscimol (a GABA agonist). Increases in cAMP were detected by monitoring the CAMP-dependent phosphorylation of cAMP response element-binding protein (CREB). Phosphorylated CREB was not observed in control or P-4-treated cultures, but it was detected in the majority of both small and large GCs exposed to either 8-br-cAMP or muscimol. Since activation of the GABA(A) receptor with muscimol increases phosphorylated CREB but P-4 does not, this study indicates that P-4 does not activate the ovarian GABA(A) receptor. However, both bicuculline, a GABA(A) receptor antagonist, and the antibody to PR inhibited P-4's ability to prevent both insulin-dependent mitosis and apoptosis. Collectively, these studies suggest that P-4 mediates its antimitotic and anti-apoptotic effects through this 60-kDa P4BP, which has GABA(A) receptor-like properties and is localized within the surface membrane of GCs.