Protease regulation of ovarian recrudescence

描述（由申请人提供）：卵巢功能，包括卵泡发育、排卵和黄体形成/降解，依赖于组织重塑事件，其中许多与Zn+依赖性内肽酶家族，基质金属蛋白酶（MMPs）相关。在光周期物种中观察到卵巢组织的极端重塑，其中白天长度的季节性变化可以抑制或刺激下丘脑/垂体分泌GnRH/促性腺激素，导致卵巢功能萎缩或恢复。因此，光反应性个体是卵巢基本功能的优秀模型，因为白昼长度的改变会自然诱导并逆转卵巢萎缩。在光周期西伯利亚仓鼠中，白昼长度变化引起的卵巢重塑与MMPs的差异表达有关。事实上，在体内施用广谱MMP抑制剂GM6001后，光刺激卵巢功能的恢复（复发）可能会受到阻碍。虽然这表明这些蛋白酶发挥着重要作用，但MMPs在复发过程中的作用以及卵巢在萎缩数周后如何恢复休眠的过程尚不清楚。该研究假设：1)GM6001对复发的抑制是因为通常介导卵巢功能恢复的MMP底物的切割受到阻碍，2)GM6001处理的卵巢未能恢复功能，因为卵巢的关键过程，如血管生成、颗粒细胞增殖和类固醇生成依赖于MMP活性，3)明胶酶（MMP -2/-9）是卵巢周期的关键参与者。并且明胶酶活性在服用GM6001后显著下调，大部分重塑可归因于明胶酶。初始实验将使用假设驱动的蛋白质组学方法，通过比较gm6001处理与对照卵巢的蛋白质组，确定MMPs在复发期间切割的底物。检测血管生成（如CD34、VEGF-R1）、颗粒细胞（PCNA）增殖和类固醇生成（如Cyp19、32HSD）的关键标志物的mRNA和蛋白，将揭示MMP在复发过程中的作用机制，并将GM6001处理组织与对照组织进行比较，确定MMPs在这些过程中的作用。最后，明胶酶特异性抑制剂（SB-3CT）将在光刺激复发期间在体内给予，以提供明胶酶在恢复卵巢中的作用和功能的直接证据。总之，这些研究应该为卵巢功能复发的细胞和分子调控提供新的见解。此外，这些数据将为卵巢周期恢复时MMP的作用靶点提供第一个证据。通过使用光周期模型了解MMPs在哺乳动物卵巢功能中的作用，有助于阐明如何通过非激素机制关闭（避孕）和重新启动（辅助生殖，过早绝经）卵巢活动的关键临床问题。

DESCRIPTION (provided by applicant): Ovarian function, including follicle development, ovulation, and corpus luteum formation/degradation, is dependent upon tissue remodeling events, many of which are associated with a family of Zn+dependent endopeptidases, the matrix metalloproteinases (MMPs). Extreme remodeling of ovarian tissue is observed in photoperiodic species where seasonal changes in day length can either inhibit or stimulate hypothalamic/ pituitary secretion of GnRH/gonadotropins, leading to atrophy or resumption of ovarian function. Photo- responsive individuals are therefore excellent models for basic ovarian function, because modifications of day length naturally induce and then reverse ovarian atrophy. In photoperiodic Siberian hamsters, ovarian remodeling prompted by changes in day length is associated with differential expression of MMPs. Indeed, photostimulated return to ovarian function (recrudescence) can be impeded following in vivo administration of a broad-spectrum MMP inhibitor, GM6001. While this suggests an important role for these proteases, the action of MMPs during recrudescence and the process of how the quiescent ovary can resume cycling following weeks of atrophy is unknown. This proposal hypothesizes that 1) inhibition of recrudescence in response to GM6001 occurs because cleavage of MMP substrates that normally mediate return to ovarian function is impeded, 2) that the GM6001-treated ovary fails to return to function because key ovarian processes such as angiogenesis, granulosa cell proliferation, and steroidogenesis are dependent on MMP activity, and 3) that because gelatinases (MMPs-2/-9) are key players in ovarian cyclicity, and gelatinase activity is significantly down regulated following GM6001 administration, much of the remodeling can be attributed to gelatinases. Initial experiments will use a hypothesis-driven proteomics approach to identify substrates cleaved by MMPs during recrudescence by comparing the proteome of GM6001-treated vs. control ovaries. Examination of both mRNA and protein for key markers of angiogenesis (e.g., CD34, VEGF-R1), proliferation of granulosa cells (PCNA), and steroidogenesis (e.g., Cyp19, 32HSD) will reveal a mechanism of MMP action during recrudescence, and define a role for MMPs in these processes as GM6001 treated tissue is compared to controls. Finally, a gelatinase-specific inhibitor (SB-3CT) will be administered in vivo during photostimulated recrudescence to provide direct evidence of gelatinase action and function in the recovering ovary. Together, these studies should provide novel insight into the cellular and molecular regulation of recrudescence of ovarian function. In addition, these data will provide the first evidence of the targets of MMP action as ovarian cyclicity returns. Understanding the role that MMPs play in mammalian ovarian function by using a photoperiodic model helps to elucidate critical clinical questions of how to shut down (contraception) and restart (assisted reproduction, premature menopause) ovarian activity with non-hormonal mechanisms. PUBLIC HEALTH RELEVANCE: Ovarian recrudescence is a natural process stimulated by changes in photoperiod (the number of hours of light per day) whereby the atrophied and non-functional ovary returns to a fully functional organ in 2-8 weeks. While the hormonal initiation of recrudescence has been thoroughly examined, the cellular changes that occur at the level of the ovary remain unknown. Understanding the cellular/molecular pathways of mammalian ovarian recrudescence, such as matrix metalloproteinase (MMP) action, is novel and timely, as new non-hormonal methods of contraception and assisted reproduction techniques are clinically sought. Our studies will provide potential non-hormonal targets for clinically- geared studies focused on "turning on" and "turning off" ovarian function.