Stage-dependent regulation of ovarian pituitary adenylate cyclase-activating polypeptide mRNA levels by GnRH in cultured rat granulosa cells

The present study was designed to test whether GnRH regulates pituitary ade nylate cyclase-activating polypeptide mRNA levels in a stage-dependent mann er during follicle development in the rat ovary. The granulosa cells of pre ovulatory and immature follicles obtained from PMSG- and estrogen-treated r ats, respectively, were cultured in serum-free conditions in the presence o f various hormones. GnRH receptor mRNA expression was detected in both preo vulatory and immature granulosa cells and was down-regulated by gonadotropi ns. Treatment of preovulatory granulosa cells with GnRH agonist stimulated pituitary adenylate cyclase-activating polypeptide mRNA levels in a dose-de pendent manner. In situ hybridization analysis of cultured preovulatory fol licles revealed that GnRH-induced pituitary adenylate cyclase-activating po lypeptide signals were detected in granulosa cells, but not thecal cells. I n immature granulosa cells, cotreatment with GnRH agonist suppressed FSH-st imulated pituitary adenylate cyclase-activating polypeptide mRNA levels in a dose-dependent manner, whereas treatment with GnRH alone had no effect. F urthermore, treatment with GnRH antagonist inhibited LH-induced pituitary a denylate cyclase-activating polypeptide gene expression in preovulatory gra nulosa cells, whereas it stimulated FSH-induced pituitary adenylate cyclase -activating polypeptide gene expression in immature granulosa cells. Intere stingly, GnRH-stimulated pituitary adenylate cyclase-activating polypeptide mRNA levels in preovulatory granulosa cells was inhibited by arachidonyltr i fluoromethyl ketone, an inhibitor of phospholipase A(2), but not by an in hibitor of protein kinase A or C. Lastly, treatment of preovulatory follicl es with pituitary adenylate cyclase-activating polypeptide antagonist suppr essed GnRH-stimulated progesterone production during 6-9 h of culture. Take n together, these results demonstrate the stage-dependent regulation of pit uitary adenylate cyclase-activating polypeptide mRNA levels by GnRH, the st imulatory and inhibitory effect in granulosa cells of preovulatory and imma ture follicles, respectively.