LH Action in Ovarian Cell Differentiation

描述（由申请人提供）：排卵是一个独特的生物学过程，成熟的卵母细胞和周围的积云细胞，包括积云细胞-卵母细胞复合物（COC），从卵巢表面释放出来。这种由lh诱导的过程类似于炎症反应，需要特定基因的表达：egf样因子（Areg、Ereg、Btc）、基质因子（Has2、Ptgs2、Tnfaip6、Ptx3）和细胞因子（Il6）。AREG可激活培养COC中ERK1/2、COC扩增、卵母细胞成熟和il - 6的产生。然而，AREG/ERK1/2在体内特异性调控的基因以及破坏这些信号分子的后果尚未确定。因此，我们利用Erk1 （Erk1-/-）缺失小鼠、Erk2fl/fl小鼠和芳香化酶（Cyp19）启动子驱动表达Cre的小鼠，产生了Erk1/2双KO （Erk1-/-;Erk2fl/fl/;Cyp19-Cre）小鼠。在Erk1/2双KO小鼠中，卵泡发育正常，但排卵期前卵泡不能排卵，COC扩张受损。除了炎症相关基因外，LH/AREG还调节构成先天免疫细胞监视反应系统的特定基因的表达，包括toll样受体TLR2和TLR4以及该系统的组分（C1q、Cd14和Pdcd1）在积云细胞中唯一表达。TLR2和TLR4在积云细胞中发挥功能，并对已知的配体（包括基质因子）做出反应，导致IL6的产生，从而在缺乏AREG或PGE的培养中诱导COC扩增。初步数据表明，Tlr4-/-小鼠是弱育的。通过分析Erk1/2双KO小鼠和无COCs中表达的Areg靶基因（Tlr/42， Ptx3和Pdcd1）的信号级联和特定基因的表达，我们应该能够解剖和描绘一个控制积云细胞功能和排卵的新遗传程序。由于细胞因子（包括il - 6）的产生似乎对排卵至关重要，我们提出ERK1/2通路在排卵过程中控制着内分泌-免疫-细胞因子调节网络。因此，我们提出以下具体目的：具体目的一：确定ERK2 （MAPK1）和ERK1 （MAPK3）在体内调节颗粒细胞和积云细胞功能和排卵的分子机制。验证ERK1/2介导LH诱导排卵和积云细胞功能特异性效应的假说。特定目的II：确定在排卵COCs中高水平表达的AREG （ERK1/2）靶基因的功能，并影响排卵。验证在积云细胞中表达的特异性基质因子、il - 6和先天免疫细胞相关基因指导这些细胞独特命运的假设。公共卫生相关性：本申请中提出的研究旨在揭示卵巢卵泡在促性腺激素、促卵泡激素（FSH）和黄体生成素（LH）的作用下如何成熟和排卵的新信息。这一信息有助于我们了解月经周期异常、卵巢功能减退和无排卵的妇女不孕的基础。

DESCRIPTION (provided by applicant): Ovulation is the unique biological process by which a mature oocyte and surrounding cumulus cells, comprising the cumulus cell-oocyte complex (COC), are released from the surface of the ovary. This LH-induced process is similar to an inflammatory response and requires the expression of specific genes: the EGF-like factors (Areg, Ereg, Btc), matrix factors (Has2, Ptgs2, Tnfaip6, Ptx3) and cytokines (Il6). AREG potently activates ERK1/2, COC expansion, oocyte maturation and IL6 production in cultured COCs. However, the genes regulated specifically by AREG/ERK1/2 and the consequences of disrupting these signaling molecules in vivo have not been determined. Therefore, using Erk1 (Erk1-/-) null mice, Erk2fl/fl mice and mice expressing Cre driven by the aromatase (Cyp19) promoter, we have generated Erk1/2 double KO (Erk1-/- ;Erk2fl/fl/;Cyp19-Cre) mice. Follicular development is normal but preovulatory follicles fail to ovulate and COC expansion is impaired in the Erk1/2 double KO mice. In addition to inflammation-related genes, LH/AREG also regulate the expression of specific genes that comprise the innate-immune cell surveillance response system, including the Toll-like receptors TLR2 and TLR4 and components of this system (C1q, Cd14, and Pdcd1) uniquely expressed in cumulus cells. TLR2 and TLR4 are functional in cumulus cells and respond to known ligands (including matrix factors) leading to the production of IL6 that can induce COC expansion in culture in the absence of AREG or PGE. Preliminary data indicate that Tlr4-/- mice are subfertile. By analyzing the signaling cascades and the expression of specific genes in Erk1/2 double KO mice and in mice null for the Areg target genes (Tlr/42, Ptx3 and Pdcd1) that are expressed in COCs, we should be able to dissect and delineate a novel genetic program that controls cumulus cell function and ovulation. Because the production of cytokines, including IL6, appears to be critical for ovulation, we propose that the ERK1/2 pathway controls an endocrine-immune-cytokine regulatory network during ovulation. Therefore we propose these specific aims: Specific Aim I: Determine the molecular mechanisms by of ERK2 (MAPK1) and ERK1 (MAPK3) regulate granulosa cell and cumulus cell functions and ovulation in vivo. Test the hypothesis that ERK1/2 mediate specific effects of LH induced ovulation and cumulus cell function. Specific Aim II: Determine the function of AREG (ERK1/2) target genes that are uniquely expressed at high levels in ovulated COCs and that impact ovulation. Test the hypotheses that specific matrix factors, IL6 and innate immune cell-related genes expressed in cumulus cells direct the unique fate of these cells. PUBLIC HEALTH RELEVANCE: The studies being proposed in this application are designed to uncover new information about how ovarian follicles mature and ovulate in response to the gonadotropins, follicle stimulating hormone (FSH) and luteinizing hormone (LH). This information should help us understand the basis for infertility in women who have abnormal menstrual cycles, reduced ovarian function and are anovulatory.