Stage-Dependent Regulation of Ovarian Pituitary Adenylate Cyclase-Activating Polypeptide mRNA Levels by GnRH in Cultured Rat Granulosa Cells

Abstract

The present study was designed to test whether GnRH regulates pituitary adenylate cyclase-activating polypeptide mRNA levels in a stage-dependent manner during follicle development in the rat ovary. The granulosa cells of preovulatory and immature follicles obtained from PMSG- and estrogen-treated rats, respectively, were cultured in serum-free conditions in the presence of various hormones. GnRH receptor mRNA expression was detected in both preovulatory and immature granulosa cells and was down-regulated by gonadotropins. Treatment of preovulatory granulosa cells with GnRH agonist stimulated pituitary adenylate cyclase-activating polypeptide mRNA levels in a dose-dependent manner. In situ hybridization analysis of cultured preovulatory follicles revealed that GnRH-induced pituitary adenylate cyclase- activating polypeptide signals were detected in granulosa cells, but not thecal cells. In immature granulosa cells, cotreatment with GnRH agonist suppressed FSH-stimulated pituitary adenylate cyclase-activating polypeptide mRNA levels in a dose-dependent manner, whereas treatment with GnRH alone had no effect. Furthermore, treatment with GnRH antagonist inhibited LH-induced pituitary adenylate cyclase-activating polypeptide gene expression in preovulatory granulosa cells, whereas it stimulated FSH-induced pituitary adenylate cyclase-activating polypeptide gene expression in immature granulosa cells. Interestingly, GnRH-stimulated pituitary adenylate cyclase-activating polypeptide mRNA levels in preovulatory granulosa cells was inhibited by arachidonyltri fluoromethyl ketone, an inhibitor of phospholipase A(2), but not by an inhibitor of protein kinase A or C. Lastly, treatment of preovulatory follicles with pituitary adenylate cyclase-activating polypeptide antagonist suppressed GnRH-stimulated progesterone production during 6--9 h of culture. Taken together, these results demonstrate the stage-dependent regulation of pituitary adenylate cyclase-activating polypeptide mRNA levels by GnRH, the stimulatory and inhibitory effect in granulosa cells of preovulatory and immature follicles, respectively.

Gonadotropin-dependent regulation of bovine pituitary adenylate cyclase-activating polypeptide in ovarian follicles prior to ovulation

“…Previous studies and the present work identified high/ovulatory levels of gonadotropins as a physiological regulator of PACAP in preovulatory follicles (Gras et al 1996, Lee et al 1999. Results from granulosa cell cultures also indicated that the expression of PACAP transcripts is increased by the treatment with LH, FSH, GnRH, FSK and cAMP analogs (Lee et al 1999, Park et al 2001, Wang et al 2003. The present study confirms that the expression of PACAP was significantly stimulated by FSK treatment or cPKA overexpression, but inhibited by PKA inhibitor H89 treatment or PKI overexpression, suggesting that activation of adenylyl cyclase/cAMP/ PKA induces PACAP expression in bovine granulosa cells.…”

Investigation of pituitary adenylate cyclase activating polypeptide in human gynecological and other biological fluids by using MALDI TOF mass spectrometry

“…PACAP has been shown to have several functions in follicular development. PACAP is expressed stage‐specifically in granulosa cells of large mature follicles before ovulation, but weaker expression has also been shown in the wall of immature antral and pre‐antral follicles 19, 32, 33. PACAP receptors have also been demonstrated in developing follicles 34–36.…”