Role of progesterone receptor activation in pituitary adenylate cyclase activating polypeptide gene expression in rat ovary

It is well known that the pituitary gonadotropin surge induces progesterone receptor (PR) gene expression in luteinizing granulosa cells and that PR a ctivation is critical for successful ovulation. To further understand the m olecular mechanism(s) by which PR plays a role critical for granulosa cell functions, we wanted to identify progesterone-induced genes in granulosa ce lls. We employed a PCR-based subtraction cloning strategy to screen for gen es expressed differentially in granulosa cells that were challenged with fo rskolin in the presence of progesterone or ZK98299. One such differentially expressed clone was identified as the pituitary adenylate cyclase activati ng polypeptide (PACAP). To begin to understand the relationship between PR activation and PACAP gene expression in luteinizing granulosa cells, we exa mined whether PR and PACAP messenger RNA (mRNA) expression is temporally co rrelated. In cultured granulosa cells, both human CG and forskolin induced PR and PACAP mRNA levels in a dose-dependent manner, as determined by semiq uantitative RT-PCR assays. However, the peak expression for PR and PACAP mR NAs was observed at 3 h and 6 h after hormone treatment, respectively. This time difference in cAMP-responsive expression of the PR and PACAP genes is due, at least in part, to the requirement of ongoing protein synthesis for PACAP expression, as demonstrated by the inhibitory effect of cycloheximid e on cAMP-induced PACAP, but not PR, mRNA levels. To determine whether PR s ynthesis is prerequisite for PACAP expression, we examined the effect of ZK 98299, a specific PR antagonist, on cAMP-induced PACAP mRNA expression. Thi s compound blocked cAMP-induced PACAP mRNA expression in a dose-dependent m anner, indicating that PR activation is required for PACAP gene expression in granulosa cells. We then compared cellular localization and hormonal reg ulation of. ovarian PR and PACAP gene expression in immature rats treated w ith gonadotropins as well as in adult rats during the preovulatory period b y using in situ hybridization and semiquantitative RT-PCR assays. Results s how that both PR and PACAP mRNAs are induced in granulosa cells of preovula tory follicles by human CG, but that the PR gene is expressed before the PA CAP gene. Taken together, these results demonstrate that PRs mediate the LH -induced PACAP gene expression in rat granulosa cells.