HORMONE INDUCTION OF LUTEINIZATION AND PROSTAGLANDIN ENDOPEROXIDE SYNTHASE-2 INVOLVES MULTIPLE CELLULAR SIGNALING PATHWAYS

To determine the cellular signaling pathways involved in granulosa cell luteinization, known activators of protein kinase-A (LH and FSH) and protein kinase-C [GnRH and phorbol 12-myristate 13-acetate (PMA)] as well as inhibitors of tyrosine kinases (AG18 and genistein) were tested in an in vitro system using specific markers of luteinization (cell hypertrophy, side-chain cleavage cytochrome P450, and progesterone) and ovulation [prostaglandin endoperoxide synthase-2 (PGS-2)]. When preovulatory follicles were incubated in the presence of an ovulatory (500 ng/ml) dose of LH or high GnRH (1 mum), the granulosa cells harvested from these follicles assumed and maintained a stable luteal cell phenotype in vitro. Granulosa cells harvested from follicles incubated in subovulatory doses of LH (5 and 50 ng/ml), lower doses of GnRH (5, 50, and 500 nm), or PMA alone were unable to form a stable luteal cell phenotype. When PMA was combined with subovulatory doses of LH, granulosa cells luteinized, and PGS-2 protein was induced. AG18 (or genistein) blocked agonist induction of luteinization and of PGS-2 mRNA and protein when present during the first 2 h (0-2 h) of follicle incubation, but failed to block these events if added for the last 2 h (5-7 h of incubation). Combined, these results provide evidence to support a primary role for cAMP and protein kinase-A, a supportive but essential role for protein kinase-C, and an obligatory role for tyrosine kinases acting at an early stage in the cascade of events required for luteinization and ovulation.