Mechanisms of PGRMC2 action in female reproduction

描述(申请人提供)：黄体酮(P4)是一种重要的激素，它能在生殖轴的每一级产生作用。在子宫中，P4抑制雌激素诱导的上皮细胞增殖，促进细胞分化，为妊娠的建立和维持做准备。在卵巢中，P4直接作用于颗粒细胞，抑制有丝分裂和凋亡，尽管这些细胞缺乏经典的孕激素受体(PGR；即PRA和PRB)的表达。P4还促进黄体细胞的活性和类固醇生成潜力，并刺激其自身分泌和胆固醇合成。P4在卵巢和子宫内的许多活动都是由PGR介导的。然而，并不是P4的所有作用都可以通过激活PGR来解释，因为许多不表达这种受体的细胞系以及PGR缺失的小鼠都能够对P4产生反应。 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/黄体细胞活性、P4合成和子宫基质细胞分化，部分是通过P4结合蛋白-孕酮受体膜组件-1(PGRMC1)介导的。通过条件突变，我们已经确定了PGRMC1对于正常生育是必不可少的，因为缺乏PGRMC1的雌性小鼠表现出亚生育表型并经历卵巢早衰(POF)。这种表型在一些女性中也可以观察到，在这些女性中，PGRMC1表达降低和单倍体功能不全与POF和多囊卵巢综合征相关。PGRMC1条件性KO(CKO)女性也有子宫增生，并同时发展为子宫和卵巢囊肿。越来越多的证据表明，PGRMC2在雌性生殖中也起着重要作用，因为在灵长类动物模型中，PGRMC2降低与晚期子宫内膜异位症有关，并可能作为分娩开始的标志。这些累积的研究以及大量的体外研究清楚地表明，PGRMC1和PGRMC2在调节子宫和卵巢功能方面发挥着重要的临床相关作用，并且它们的表达变化导致女性生殖系统的疾病状态的表现。在这项资助中，我们现在将通过使用Pgrmc2 CKO小鼠来检验我们的假设，即Pgrmc2在子宫和卵巢生理中发挥重要作用。我们还建议评估同时缺乏PGRMC1和Pgrmc2的雌性小鼠(双CKO小鼠)的生育力。育种试验将初步完成，以确定CKO小鼠的生育状况。在目标1中要评估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植入。在目标2中，我们将评估Pgrmc2在介导P4在卵巢和雌激素刺激的子宫中的抗增殖和抗凋亡作用中的重要性。最后，对PGR突变小鼠的研究表明，PGR不是P4诱导基因转录的唯一中介。在目标2中，我们还计划使用RNA-seq来评估P4在PGRMC1/2双CKO小鼠子宫和卵巢中诱导的转录反应。拟议研究的成功完成将提供令人信服的证据，支持PGRMC2在女性生殖生理中的作用。将PGRMC2确立为特定子宫和卵巢功能的介体，将允许开发一类新的、选择性的P4拮抗剂/激动剂，针对PGRMC家族。

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.