PGRMC1 function in female reproductive physiology

描述(申请人提供)：孕酮(P4)是一种调节整个生殖过程的基本激素。P4在整个生殖轴上有许多靶点，但两个主要的作用部位包括卵巢和子宫。卵巢在整个月经/发情周期中合成和分泌P4，其水平在怀孕期间升高。P4直接作用于卵巢颗粒细胞，抑制有丝分裂和细胞凋亡。此外，P4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在卵巢和子宫中的许多作用是由经典的核P4受体(PGRs)介导的。然而，并不是P4的所有作用都可以通过激活PGRs来解释，因为许多不表达这些受体的细胞系以及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。基于这些体外实验数据，我们推测PGRMC1在体内对卵巢和子宫功能的调节起重要作用。在这项资助中，我们将通过产生一只条件基因敲除小鼠来直接检验我们的假设，在条件基因敲除小鼠中，PGRMC1在卵巢的颗粒/黄体细胞和子宫的间质(间质)组织中特异性地被耗尽(特定目标1)。然后，我们将使用有条件的PGRMC1基因敲除小鼠来监测耗尽PGRMC1对1)女性生育力、2)卵巢功能(即卵泡生长、类固醇合成和排卵)和3)子宫功能(即子宫内膜蜕膜化和胚胎植入)的影响(特定目标2)。在特定目的3中，我们将使用卵巢和子宫组织的裂解物通过免疫沉淀分离PGRMC1结合蛋白，然后使用双向凝胶电泳法和随后的蛋白质组学来鉴定PGRMC1结合伙伴。这些研究的成功完成将为PGRMC1在雌性生殖生理中的作用提供有力的证据支持。建立PGRMC1作为特定卵巢和子宫功能的介体的重要之处在于，这将允许开发一类新的P4拮抗剂和激动剂。这些假定的PGRMC1调节剂将与目前的PGR拮抗剂完全不同，只会干扰通过PGRMC1而不是PGR介导的P4的作用。这些假定的PGRMC1调节剂可以应用于人类生育和潜在的卵巢癌的治疗，因为PGRMC1促进了这些癌症的生存。 与公共健康相关：黄体酮(P4)是一种调节整个女性生殖过程的基本激素。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。在这项资助中，我们将最终确定PGRMC1的作用，方法是产生一只条件基因敲除小鼠，然后监测耗尽PGRMC1对1)雌性生育、2)卵巢功能和3)子宫功能的影响。如果成功，拟议中的研究可能导致开发一类新的P4拮抗剂和激动剂，可用于治疗人类不孕不育以及潜在的卵巢癌和子宫癌。

DESCRIPTION (provided by applicant): Progesterone (P4) is an essential hormone that regulates the entire reproductive process. P4 has many target sites throughout the reproductive axis but two major sites of action include the ovary and the uterus. The ovary synthesizes and secretes P4 throughout the menstrual/estrous cycle with its levels increasing during pregnancy. P4 acts directly of the granulosa cells of the ovary to inhibit mitosis and apoptosis. In addition, the P4 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 classical nuclear P4 receptors (PGRs). However, not all of the actions of P4 can be explained by activation of PGRs, since a number of cell lines that do not express these 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). Based on these in vitro data, we hypothesize that PGRMC1 plays essential roles in regulating ovarian and uterine function in vivo. In this grant we will directly test our hypothesis by generating a conditional knockout mouse in which PGRMC1 is specifically depleted within the granulosa/luteal cells of the ovary and the mesenchymal (stromal) tissue of the uterus (Specific Aim 1). We will then use the conditional PGRMC1 knockout mouse to monitor the effects of depleting PGRMC1 on 1) female fertility, 2) ovarian function (i.e. follicular growth, steroid synthesis and ovulation) and 3) uterine function (i.e. endometrial decidualization and embryo implantation) (Specific Aim 2). In Specific Aim 3 we will isolate PGRMC1 binding proteins by immunoprecipitation using lysates of ovarian and uterine tissues and then using two- dimensional gel electrophoresis and subsequent proteomics to identify PGRMC1 binding partners. The successful completion of the proposed studies will provide compelling evident to support PGRMC1's role in female reproductive physiology. What is important about establishing PGRMC1 as a mediator of specific ovarian and uterine functions is that this will allow for the development of a new class of P4 antagonists and agonists. These putative PGRMC1 modulators would be completely different from the present-day PGR antagonists and would only interfere with the actions of P4 that are mediated via PGRMC1 and not the PGR. These putative PGRMC1 modulators could find application in the treatment of human fertility and potentially ovarian cancers, since PGRMC1 promotes the viability of these cancers. PUBLIC HEALTH RELEVANCE: Progesterone (P4) is an essential hormone that regulates the entire female reproductive process. 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). In this grant we will conclusively determine the role of PGRMC1 by generating a conditional knockout mouse and then monitoring the effects of depleting PGRMC1 on 1) female fertility, 2) ovarian function and 3) uterine function. If successful, the proposed studies could lead to the development of a new class of P4 antagonists and agonists that could be used to treat human infertility and potentially ovarian and uterine cancers.