LH Action in Ovarian Cell Differentiation

描述（由申请人提供）：排卵是一种独特的生物学过程，通过该过程，成熟卵母细胞和周围的卵丘细胞（包括卵丘细胞-卵母细胞复合体（COC））从卵巢表面释放。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（Has 2，Ptgs 2，Tnfaip 6，Ptx 3）和细胞因子（I16）。AREG有效激活培养的COCs中的ERK 1/2、COC扩增、卵母细胞成熟和IL 6产生。然而，由AREG/ERK 1/2特异性调节的基因以及在体内破坏这些信号分子的后果尚未确定。因此，使用Erk 1（Erk 1-/-）缺失小鼠、Erk 2fl/fl小鼠和表达由芳香酶（Cyp 19）启动子驱动的Cre的小鼠，我们已经产生了Erk 1/2双KO（Erk 1-/- ; Erk 2fl/fl/; Cyp 19-Cre）小鼠。在Erk 1/2双基因敲除小鼠中，卵泡发育正常，但排卵前卵泡不能排卵，COC扩增受损。除了炎症相关基因外，LH/AREG还调节组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TLR 2和TLR 4以及在卵丘细胞中独特表达的该系统的组分（C1 q，Cd 14和Pdcd 1）。TLR 2和TLR 4在卵丘细胞中是功能性的，并且响应于已知的配体（包括基质因子），导致产生IL 6，其可以在不存在AREG或PGE的培养物中诱导COC扩增。初步数据表明Tlr 4-/-小鼠生育力低下。通过分析Erk 1/2双基因敲除小鼠和COCs中表达的Areg靶基因（Tlr/42，Ptx 3和Pdcd 1）缺失小鼠中的信号级联和特定基因的表达，我们应该能够剖析和描述一种控制卵丘细胞功能和排卵的新遗传程序。由于细胞因子的产生，包括IL 6，似乎是排卵的关键，我们建议，ERK 1/2途径控制排卵期间的内分泌免疫细胞因子调节网络。因此，我们提出了这些具体的目标：具体目标一：确定ERK 2（MAPK 1）和ERK 1（MAPK 3）调节颗粒细胞和卵丘细胞功能和排卵的分子机制。验证ERK 1/2介导LH诱导排卵和卵丘细胞功能的特异性作用的假设。具体目标二：确定AREG（ERK 1/2）靶基因的功能，这些靶基因在排卵的COCs中以高水平表达并影响排卵。测试特定的基质因子，IL 6和卵丘细胞中表达的先天免疫细胞相关基因指导这些细胞的独特命运的假设。公共卫生相关性：本申请中提出的研究旨在揭示关于卵泡如何响应促性腺激素、促卵泡激素（FSH）和促黄体激素（LH）而成熟和排卵的新信息。这些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.