Cellular and Molecular Mechanisms of Mammalian Ovulation

描述（由申请人提供）：我们研究的长期目标是阐明LH诱导的信号在排卵前卵泡中的分子级联，从而导致排卵。LH峰刺激排卵前卵泡颗粒细胞中孕酮及其细胞内受体孕酮受体（PR）的合成。孕酮和PR之间的相互作用以自分泌/旁分泌的方式对排卵是必不可少的。然而，配体依赖性PR激活控制排卵从而控制正常生殖周期和生育力的确切机制尚不清楚。 为了深入了解PR介导的卵巢功能的分子机制，我们开始在黄体化颗粒细胞中克隆PR下游基因。我们已经表征为PR下游的两个基因是垂体腺苷酸环化酶激活多肽（PACAP）的配体-受体系统：PACAP及其受体1型（PAC 1）。配体PACAP的表达和分泌的时间和空间模式沿着受体PAC 1在卵巢中的细胞定位，主张该配体-受体系统对排卵过程的潜在意义。事实上，PAC 1的配体依赖性激活的药理学阻断似乎干扰LH和孕酮在引起排卵过程中的功效。因此，我们的工作假设是，PACAP诱导的PAC 1激活介导，至少部分，PR功能的卵泡破裂与减数分裂成熟的卵母细胞的释放至关重要。 本申请的直接目标是使用体内和体外方法确定排卵期期间排卵前卵泡内PACAP的功能重要性。在目的1中，我们将测试PR诱导的PACAP是否对卵泡破裂和排卵相关基因（包括蛋白水解酶）的表达至关重要。此外，我们将识别可能在卵泡破裂中发挥重要作用的PAC 1下游基因。在目标2中，我们将确定PR诱导的PACAP在黄体化颗粒细胞中调节的初始死亡/存活途径。在目标3中，我们将测试PR诱导的PACAP是否调节减数分裂成熟卵母细胞的聚腺苷酸化/翻译能力。拟定研究旨在提供排卵期PR诱导的PACAP与排卵前卵泡中PAC 1相互作用的功能终点。从我们的研究结果中获得的信息将使我们能够更好地管理生育，不育和内分泌疾病。

DESCRIPTION (provided by applicant): The long-term goal of our research is to elucidate the molecular cascades of LH-induced signals within preovulatory follicles, leading to ovulation. The LH surge stimulates the synthesis of progesterone and its intracellular receptors, progesterone receptors (PRs), in the granulosa cells of preovulatory follicles. Interaction between progesterone and PRs in an autocrine/paracrine fashion is essential for ovulation. However, the exact mechanisms by which ligand-dependent activation of PRs controls ovulation and thus normal reproductive cyclicity and fecundity are unknown. To gain insight into the molecular mechanisms underlying PR-mediated ovarian functions, we initiated cloning of PR downstream genes in luteinizing granulosa cells. The two genes we have characterized as PR-downstream are the ligand-receptor system for pituitary adenylate cyclase activating polypeptide (PACAP): PACAP and its receptor type 1 (PAC1). The temporal and spatial pattern of expression and secretion of the ligand PACAP along with the cellular localization of the receptor PAC1 in the ovary advocates the potential significance of this ligand-receptor system for ovulatory processes. Indeed, pharmacological blockade of ligand-dependent activation of PAC1 appears to interfere with the efficacy of LH and progesterone in bringing about ovulatory processes. Thus, our working hypothesis is that PACAP-induced activation of PAC1 mediates, at least in part, PR function critical for follicular rupture with release of a meiotically mature oocyte. The immediate goal of this application is to determine the functional importance of PACAP within preovulatory follicles during the periovulatory period, using in vivo and in vitro approaches. In Aim 1, we will test whether PR-induced PACAP is critical for follicular rupture and for expression of ovulation-related genes, including proteolytic enzymes. In addition, we will identify PAC1-downstream genes that may play an important role in follicular rupture. In Aim 2, we will determine the initial death/survival pathway(s) that is modulated by PR-induced PACAP in luteinizing granulosa cells. In Aim 3, we will test whether PR-induced PACAP regulates the polyadenylation/translation capacity of meiotically maturing oocytes. The proposed studies are designed to provide functional endpoint(s) of interaction between PR-induced PACAP and PAC1 in preovulatory follicles during the periovulatory period. Information derived from our results will allow us to better manage fertility, infertility, and endocrine-based disorders.