Role of mineralocorticoid receptor in primate ovarian function

描述（由申请人提供）：灵长类动物黄体的形成、功能和消亡是月经周期、不孕症和避孕干预的关键方面。申请人实验室最近的工作证明了非人灵长类动物卵巢内矿化皮质激素系统的存在。在接受控制卵巢刺激的恒河猴中，排卵刺激以时间依赖性的方式增加21-羟化酶（CYP21A2） mRNA的表达。CYP21A2将孕酮转化为强效的矿物皮质激素受体（MR）激动剂11-脱氧皮质酮（DOC），因此与排卵期hCG丸增加卵泡内DOC水平的观察结果相关。为了验证DOC的卵巢起源，在排卵刺激之前分离颗粒细胞并在hCG存在下培养，导致DOC的培养基浓度急剧增加。MR在黄体生成素颗粒细胞和黄体的细胞质中表达，直到黄体中期到中晚期，此时MR被转移到细胞核。重要的是，MR在黄体生成过程中的拮抗作用减弱了hcg诱导的黄体酮，这表明细胞质MR在体外颗粒细胞的黄体生成中起关键作用。这个R21应用程序提出了一个总体假设，即MR在黄体的生命周期中是至关重要的，使用非人类灵长类动物模型有两个特定的目的。特异性目的1将确定MR是否在使用MR拮抗剂的黄体发育中起关键作用。预计MR在体内的拮抗作用将阻止黄体酮的临界上升，从而导致无排卵和黄体不完全形成。具体目标2将验证MR通过黄体酮合成促进黄体功能的假设，以及黄体内DOC水平与黄体酮水平平行增加的假设。在这两个目标中，MR的特定基因靶点将被阐明。这些研究将首次证明MR在任何物种卵巢生理中的关键作用，并有望在生物医学相关的灵长类动物模型中进一步了解黄体的发育、维持和死亡。公共卫生相关性：与卵巢相关的病理具有很高的发病率和死亡率，主要是由于难以发现，但也由于对卵巢基本过程的了解相当不完整而缺乏治疗。例如，多囊卵巢综合征、卵巢癌和卵巢过度刺激综合征（OHSS）。此外，不孕不育困扰着越来越多的美国夫妇（600万；疾病控制中心）；这种不孕症的一部分可能与黄体期P分泌不足有关，尽管其根本原因尚不清楚。新的避孕方法仍然是一个问题，大部分北美妇女继续依赖于联合口服避孕药的衍生品，没有新的方法。在美国，衰老和生殖显然是一个日益增长的话题，更好地了解卵巢的基本过程将有助于理解女性的更年期过渡。这些问题都支持卵巢生理学是一个具有深远健康结果的相关主题的观点。总的来说，这些探索性研究应该揭示灵长类动物卵巢中新的类固醇信号通路，这可能对避孕药的发展、不孕症和潜在的卵巢癌具有临床意义。

DESCRIPTION (provided by applicant): The formation, function, and demise of the primate corpus luteum are critical aspects of menstrual cyclicity, infertility, and contraceptive intervention. Recent work by the applicant's laboratory has demonstrated the presence of an intra-ovarian mineralocorticoid system in the non-human primate. In rhesus monkeys undergoing controlled ovarian stimulation, an ovulatory stimulus increases the expression of 21-hydroxylase (CYP21A2) mRNA in a time-dependent manner. CYP21A2 converts progesterone to the potent mineralocorticoid receptor (MR) agonist 11-deoxycorticosterone (DOC), and thus correlates with the observation that the ovulatory hCG bolus increases intra-follicular levels of DOC. In order to verify an ovarian origin for DOC, granulosa cells were isolated prior to an ovulatory stimulus and cultured in the presence of hCG, resulting in a dramatic increase in media concentrations of DOC. MR is expressed in the cytoplasm of both luteinizing granulosa cells as well as the corpus luteum until the mid to mid-late luteal phase, at which time MR is translocated to the nucleus. Importantly, antagonism of MR during luteinization attenuates hCG-induced progesterone, indicating that cytoplasmic MR has a key role in luteinization of granulosa cells in vitro. This R21 application proposes to test the overall hypothesis that MR is critical during the lifespan of the corpus luteum with two specific aims using the non-human primate model. Specific aim 1 will determine if MR has a critical role in the development of the corpus luteum using an MR antagonist. It is expected that antagonism of MR in vivo will prevent the critical rise in progesterone, leading to anovulation and incomplete luteal formation. Specific aim 2 will test the hypothesis that MR promotes luteal function through progesterone synthesis, and that intra-luteal levels of DOC increase in parallel with progesterone. In both aims, specific gene targets of MR will be elucidated. These studies will be the first to demonstrate a key role for MR in the ovarian physiology of any species, and are expected to further our understanding of corpus luteum development, maintenance, and demise in a biomedically relevant primate model. PUBLIC HEALTH RELEVANCE: Pathologies associated with the ovary carry a high morbidity and mortality, largely through difficulty in detection, but also from a lack of treatment stemming from a rather incomplete understanding of basic ovarian processes. These include, for example, PCOS, ovarian cancer, and ovarian hyperstimulation syndrome (OHSS). In addition, infertility plagues a growing number of couples in the United States (>6 million; Center for Disease Control); a proportion of this infertility can be linked to insufficiency of P production during the luteal phase, although the underlying causes of this are completely unknown. Novel contraception remains a problem, with a large preponderance of North American women continuing to rely on derivations of combined oral contraception and no new methods in sight. Aging and reproduction is clearly a growing topic in the United States, and a better knowledge of basic ovarian processes will facilitate understanding the menopausal transition in women. These issues all support the idea that ovarian physiology is a relevant topic with profound health outcomes. Overall, these exploratory studies should reveal a novel signaling steroid pathway in the primate ovary that could have clinical significance for contraceptive development, infertility, and potentially ovarian cancer.