Progesterone receptor activation mediates LH-induced type-I Pituitary Adenylate Cyclase Activating Polypeptide receptor (PAC₁) 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 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 resuits 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. (C) 2000 Academic Press.