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、功能和死亡是月经周期、不孕和避孕干预的关键方面。申请人的实验室最近的工作已经证明在非人灵长类动物中存在卵巢内盐皮质激素系统。在接受控制性卵巢刺激的恒河猴中，排卵刺激以时间依赖性方式增加21-羟化酶（CYP 21 A2）mRNA的表达。CYP 21 A2将孕酮转化为有效的盐皮质激素受体（MR）激动剂11-脱氧皮质酮（DOC），因此与排卵性hCG推注增加卵泡内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在黄体化颗粒细胞和黄体的细胞质中表达，直到黄体中期到中期-晚期，此时MR易位到细胞核。重要的是，黄体化过程中MR的拮抗作用减弱了hCG诱导的孕酮，表明细胞质MR在体外颗粒细胞的黄体化中起关键作用。该R21申请提出使用非人灵长类动物模型测试MR在黄体寿命期间至关重要的总体假设，具有两个特定目标。具体目标1将确定MR是否在使用MR拮抗剂的黄体发育中具有关键作用。预期体内MR的拮抗作用将阻止孕酮的临界升高，导致无排卵和不完全黄体形成。具体目标2将检验MR通过孕酮合成促进黄体功能，以及黄体内DOC水平与孕酮平行增加的假设。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在任何物种的卵巢生理学中的关键作用，并有望进一步加深我们对生物医学相关灵长类动物模型中黄体发育、维持和死亡的理解。公共卫生相关性：与卵巢相关的病理具有高发病率和死亡率，主要是由于难以检测，但也由于对基本卵巢过程的不完全理解而缺乏治疗。这些包括，例如，PCOS、卵巢癌和卵巢过度刺激综合征（OHSS）。此外，在美国，不孕症困扰着越来越多的夫妇（> 600万;疾病控制中心）;这种不孕症的一部分可能与黄体期P产生不足有关，尽管其根本原因完全未知。新的避孕方法仍然是一个问题，大部分北美妇女继续依赖复方口服避孕药的衍生物，看不到新的方法。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妇女的绝经过渡期。这些问题都支持卵巢生理学是一个具有深远健康影响的相关主题的观点。总的来说，这些探索性研究应该揭示灵长类动物卵巢中一种新的类固醇信号通路，这可能对避孕开发、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.