Mechanisms of PGRMC2 action in female reproduction

描述（由申请人提供）：孕酮（P4）是一种必需激素，在生殖轴的每个水平上发挥作用。在子宫中，P4减弱雌二醇诱导的上皮细胞增殖，促进细胞分化，为建立和维持妊娠做准备。在卵巢中，P4直接作用于颗粒细胞以抑制有丝分裂和凋亡，尽管这些细胞缺乏经典孕酮受体（PGR;即，PRA和PRB）。P4还促进黄体细胞的活力和类固醇生成潜力，并刺激其自身的分泌和胆固醇合成。P4在卵巢和子宫内的许多作用都是由PGR介导的。然而，并非P4的所有作用都可以通过PGR的激活来解释，因为许多不表达这种受体的细胞系以及Pgr缺失小鼠能够对P4做出反应。 我们最近对卵巢和子宫细胞的体外研究表明，P4的一些作用，如颗粒/黄体细胞活力，P4合成和子宫基质细胞分化，部分是通过P4结合蛋白，孕酮受体膜成分1（PGRMC 1）介导的。通过使用条件突变，我们已经确定，PGRMC 1是必不可少的正常生育能力的雌性小鼠缺乏PGRMC 1显示生育力低下的表型，并经历卵巢早衰（POF）。这种表型也在一些女性中观察到，其中较低的Pgrmc 1表达和单倍不足与POF和多囊卵巢综合征相关。Pgrmc 1条件性KO（cKO）雌性也有子宫增生，并出现子宫和卵巢囊肿。越来越多的证据表明，PGRMC 2在雌性生殖中也起着重要作用，因为在灵长类动物模型中，PGRMC 2降低与晚期子宫内膜异位症相关，并且可以作为分娩开始的标志物。这些累积研究沿着大量体外研究清楚地表明，PGRMC 1和PGRMC 2在调节子宫和卵巢功能中发挥重要的临床相关作用，并且其表达的改变导致女性生殖系统疾病状态的表现。在这项研究中，我们将通过使用Pgrmc 2 cKO小鼠来验证我们的假设，即Pgrmc 2在子宫和卵巢生理学中起着重要作用。我们还建议评估Pgrmc 1和Pgrmc 2缺陷的雌性小鼠（双cKO小鼠）的生育能力。最初将完成育种试验，以确定cKO小鼠的生育力状态。目标1中评价的其他参数包括子宫蜕膜化、排卵、卵巢类固醇生成、卵母细胞质量和胚胎着床。在目的2中，我们将评估Pgrmc 2在介导卵巢中P4的抗增殖和抗凋亡作用以及在雌激素刺激的子宫中的重要性。最后，从对PGR突变小鼠的研究中可以清楚地看出，PGR不是P4诱导的基因转录的唯一介质。在目标2中，我们计划使用RNA-seq评估P4诱导的Pgrmc 1/2双cKO小鼠子宫和卵巢中的转录应答。拟议研究的成功完成将为支持PGRMC 2在女性生殖生理学中的作用提供令人信服的证据。将PGRMC 2确定为特异性子宫和卵巢功能的介体将允许开发靶向PGRMC家族的新的和选择性类别的P4拮抗剂/激动剂。

DESCRIPTION (provided by applicant): Progesterone (P4) is an essential hormone that elicits its actions at each level of the reproductive axis. In the uterus, P4 attenuates estradiol-induced epithelial cell proliferation and facilitates cellular differentiation in preparation for the establishment and maintenance of pregnancy. In the ovary, P4 acts directly on granulosa cells to inhibit mitosis and apoptosis despite these cells lacking expression of the classical progesterone receptor (PGR; i.e., PRA and PRB). P4 also promotes the viability and steroidogenic potential of luteal cells and stimulates both its own secretion and cholesterol synthesis. Many of the actions of P4 within the ovary and uterus are mediated by the PGR. However, not all of the actions of P4 can be explained by activation of PGR, since a number of cell lines that do not express this receptors, as well as Pgr null mice, are able to respond to P4. Our recent in vitro studies of ovarian and uterine cells have revealed that some actions of P4 such as granulosa/luteal cell viability, P4 synthesis and uterine stromal cell differentiation are mediated in part through the P4 binding protein, Progesterone Receptor Membrane Component-1 (PGRMC1). Through the use of conditional mutagenesis, we have established that Pgrmc1 is essential for normal fertility in that female mice lacking PGRMC1 display a subfertility phenotype and undergo premature ovarian failure (POF). This phenotype is also observed in some women where lower Pgrmc1 expression and haploinsufficiency associate with POF and polycystic ovarian syndrome. Pgrmc1 conditional KO (cKO) females also have uterine hyperplasia and develop both uterine and ovarian cysts. There is mounting evidence that PGRMC2 also plays an important role in female reproduction in that decreased PGRMC2 associates with advanced endometriosis in a primate model and may serve as a marker for the onset of parturition. These cumulative studies along with numerous in vitro studies clearly demonstrate that PGRMC1 and PGRMC2 play important and clinically relevant roles in regulating uterine and ovarian functions and that alteration in their expression results in the manifestation of disease states in the female reproductive system. In this grant we will now test our hypothesis that Pgrmc2 plays a fundamental role in uterine and ovarian physiology through the use of Pgrmc2 cKO mice. We also propose to assess fertility in female mice that are deficient in both Pgrmc1 and Pgrmc2 (double cKO mice). Breeding trials will initially be completed to determine fertility status of cKO mice. Other parameters to be evaluated in Aim 1 include uterine decidualization, ovulation, ovarian steroidogenesis, oocyte quality, and embryo implantation. In Aim 2, we will evaluate the importance of Pgrmc2 in mediating the anti-proliferative and anti-apoptotic actions of P4 in the ovary, as well as in the estrogen stimulated uterus. Finally, it is clear from studies of PGR mutant mice that PGR is not the sole mediator of P4-induced gene transcription. In Aim 2, we plan to also evaluate P4-induced transcriptional responses in the uterus and ovary of Pgrmc1/2 double cKO mice using RNA-seq. The successful completion of the proposed studies will provide compelling evident to support a role for PGRMC2 in female reproductive physiology. Establishing PGRMC2 as a mediator of specific uterine and ovarian functions will allow for the development of a new and selective class of P4 antagonists/agonists that target the PGRMC family.