Progesterone receptor activation mediates LH-induced type-I pituitary adenylate cyclase activating polypeptide receptor (PAC(1)) gene expression in rat granulosa cells

We have previously reported that the pituitary adenylate cyclase activating polypeptide (PACAP) gene is regulated in ovarian granulosa cells by the au tocrine and/or paracrine interaction between progesterone and its nuclear r eceptor progesterone receptor (PR). To initiate studies on the functional s ignificance of the progesterone-induced PACAP production in luteinizing gra nulosa cells, we sought to determine the expression and hormonal regulation of PACAP receptors in the rat ovary. The relative mRNA levels of three kno wn PACAP receptor subtypes (PAC(1), VPAC(1), and VPAC(2)) were determined i n ovaries of immature rats treated with gonadotropins, by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) assays. Results sh ow that all PAC(1), VPAC(1), and VPAC(2) transcripts are expressed at a det ectable level in immature rat ovaries. Importantly, the ovarian level of PA C(1), but not VPAC(1) or VPAC(2), mRNA notably changes during gonadotropin challenges. Ovarian PAC(1) mRNA expression decreases during the pregnant ma re's serum gonadotropin (PMSG)-induced follicular phase but substantially i ncreases during the human chorionic gonadotropin (hCG)-induced periovulator y period. Because the hCG-induced increase in ovarian PAC(1) mRNA expressio n is attributable to the hormone-induced PAC(1) mRNA expression in granulos a cells of the preovulatory follicles, we next examined whether hCG regulat es PAC(1) mRNA expression by directly acting on granulosa cells. When granu losa cells isolated from PMSG (40 h)-primed immature rats were challenged w ith hCG (or forskolin), PAC(1), but not VPAC(1) or VPAC(2), mRNA expression significantly increased within 6 h. Because the LH-induced PAC(1) mRNA exp ression (6 h) proceeds PR activation (3 h) in granulosa cells as the LH-ind uced PACAP mRNA expression (6 h) does, we further determined the cause-effe ct relationship among LH, PR activation and PAC(1) receptor gene expression , by examining the effect of PR antagonist, ZK98299, on the ability of LH t o increase PAC(1) mRNA levels in luteinizing granulosa cells. Results show that ZK98299 inhibited the stimulatory effect of hCG (or forskolin) on PAC( 1) mRNA expression, at the level of all known splice variants of PAC(1) mRN A in granulosa cells. In summary, our results demonstrating that PR activat ion is critical for the LH-induced PAC(1) gene expression in luteinizing gr anulosa cells suggest that PR activation regulates the finely tuned express ion of the PACAP/PACAP receptor genes in luteinizing granulosa cells and th us dictates the timing of the autocrine and/or paracrine function of PACAP in preovulatory follicles. (C) 2000 Academic Press.