Progesterone Receptor Activation Mediates LH-Induced Type-I Pituitary Adenylate Cyclase Activating Polypeptide Receptor (PAC1) Gene Expression in Rat Granulosa Cells

Abstract

We have previously reported that the pituitary adenylate cyclase activating polypeptide (PACAP) gene is regulated in ovarian granulosa cells by the autocrine and/or paracrine interaction between progesterone and its nuclear receptor progesterone receptor (PR). To initiate studies on the functional significance of the progesterone-induced PACAP production in luteinizing granulosa cells, we sought to determine the expression and hormonal regulation of PACAP receptors in the rat ovary. The relative mRNA levels of three known PACAP receptor subtypes (PAC1, VPAC1, and VPAC2) were determined in ovaries of immature rats treated with gonadotropins, by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) assays. Results show that all PAC1, VPAC1, and VPAC2 transcripts are expressed at a detectable level in immature rat ovaries. Importantly, the ovarian level of PAC1, but not VPAC1 or VPAC2, mRNA notably changes during gonadotropin challenges. Ovarian PAC1 mRNA expression decreases during the pregnant mare's serum gonadotropin (PMSG)-induced follicular phase but substantially increases during the human chorionic gonadotropin (hCG)-induced periovulatory period. Because the hCG-induced increase in ovarian PAC1 mRNA expression is attributable to the hormone-induced PAC1 mRNA expression in granulosa cells of the preovulatory follicles, we next examined whether hCG regulates PAC1 mRNA expression by directly acting on granulosa cells. When granulosa cells isolated from PMSG (40 h)-primed immature rats were challenged with hCG (or forskolin), PAC1, but not VPAC1 or VPAC2, mRNA expression significantly increased within 6 h. Because the LH-induced PAC1 mRNA expression (6 h) proceeds PR activation (3 h) in granulosa cells as the LH-induced PACAP mRNA expression (6 h) does, we further determined the cause–effect relationship among LH, PR activation and PAC1 receptor gene expression, by examining the effect of PR antagonist, ZK98299, on the ability of LH to increase PAC1 mRNA levels in luteinizing granulosa cells. Results show that ZK98299 inhibited the stimulatory effect of hCG (or forskolin) on PAC1 mRNA expression, at the level of all known splice variants of PAC1 mRNA in granulosa cells. In summary, our results demonstrating that PR activation is critical for the LH-induced PAC1 gene expression in luteinizing granulosa cells suggest that PR activation regulates the finely tuned expression of the PACAP/PACAP receptor genes in luteinizing granulosa cells and thus dictates the timing of the autocrine and/or paracrine function of PACAP in preovulatory follicles.