Protease regulation of ovarian recrudescence

描述(申请人提供)：卵巢功能，包括卵泡发育、排卵和黄体形成/退化，依赖于组织重塑事件，其中许多与锌依赖内肽酶家族有关，即基质金属蛋白酶(MMPs)。在光周期物种中可以观察到卵巢组织的极端重塑，其中日长的季节性变化可以抑制或刺激下丘脑/垂体GnRH/促性腺激素的分泌，导致卵巢功能的萎缩或恢复。因此，对光敏感的个体是基本卵巢功能的极佳模型，因为日长的改变自然会导致卵巢萎缩，然后逆转。在光周期的西伯利亚仓鼠中，由日长变化引起的卵巢重塑与MMPs的差异表达有关。事实上，在体内应用广谱基质金属蛋白酶抑制剂GM6001后，光刺激恢复卵巢功能(复发)可以受到阻碍。虽然这暗示了这些蛋白酶的重要作用，但MMPs在复发过程中的作用以及静止的卵巢在萎缩数周后如何恢复周期的过程尚不清楚。这项建议假设：1)GM6001抑制复发的发生是因为正常情况下介导卵巢功能恢复的MMP底物的切割受到阻碍，2)GM6001治疗的卵巢未能恢复功能，因为关键的卵巢过程，如血管生成、颗粒细胞增殖和类固醇生成依赖于MMP的活性，以及3)由于明胶酶(MMPs-2/9)是卵巢周期中的关键角色，并且明胶酶活性在GM6001给药后显著下调，大部分重塑可以归因于明胶酶。最初的实验将使用假说驱动的蛋白质组学方法，通过比较GM6001处理的卵巢和对照卵巢的蛋白质组，确定MMPs在复发期间切割的底物。对血管生成的关键标记物(例如CD34、VEGF-R1)、颗粒细胞增殖(PCNA)和类固醇生成(例如Cyp19、32HSD)的mRNA和蛋白的检测将揭示MMPs在复发过程中的作用机制，并确定MMPs在这些过程中的作用，将GM6001处理的组织与对照组进行比较。最后，一种明胶酶特异性抑制剂(SB-3CT)将在光刺激复发期间在体内应用，以提供明胶酶在恢复的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基质金属蛋白酶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，有助于阐明如何用非激素机制关闭(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。 与公共卫生相关：卵巢复发是一个自然过程，由光周期(每天光照的小时数)的变化刺激，萎缩和无功能的卵巢在2-8周内恢复到完全功能的器官。虽然荷尔蒙对复发的启动已经被彻底研究，但发生在卵巢水平的细胞变化仍然未知。随着临床寻求新的非激素避孕方法和辅助生殖技术，理解哺乳动物卵巢复发的细胞/分子途径，如基质金属蛋白酶(MMPs)的作用，是新的和及时的。我们的研究将为“开启”和“关闭”卵巢功能的临床研究提供潜在的非激素靶点。

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.