The role of chemokines in cumulus oocyte expansion (C-OE) and oocyte maturation

描述（由申请人提供）：排卵是一种复杂的炎症样过程，其中完全发育的卵泡响应于周期中期促性腺激素激增的作用而破裂，释放卵丘-卵母细胞复合物（COC）进入生殖道并可能受精。在排卵前不久，促黄体生成激素（LH）激增诱导对生育力至关重要的过程，包括卵丘卵母细胞扩增（C-OE）和减数分裂的恢复。虽然已经确定了一些对这些事件很重要的旁分泌作用因子，但负责启动这种复杂过程的分子机制尚未完全了解。有限的已发表研究和PI的初步发现支持趋化因子调节C-OE和卵母细胞成熟所必需的事件的新作用。啮齿动物的研究表明，趋化因子信号调节卵丘细胞外基质的组装，从而受精。PI对家猫的初步结果表明，趋化因子MCP-1及其受体（CCR 2）在小窦卵泡COCs的卵母细胞和卵丘细胞中表达。在来自小窦卵泡和排卵前卵泡的猴COC中观察到类似的结果。刺激排卵后12小时，排卵前卵泡中MCP-1和CCR 2 mRNA水平升高，MCP-1在24和36小时恢复到hCG前水平，卵泡液中MCP-1蛋白含量高于mRNA水平，并在hCG后12小时达到峰值，特别是在恒河猴排卵前卵泡中C-OE所必需的事件开始和卵母细胞减数分裂恢复之前的时间段。基于这些发现，我们假设与趋化因子受体CCR 2（例如MCP-1）相互作用的趋化因子对促进C-OE和/或卵母细胞成熟具有直接作用。因此，为了验证这一假设，设计实验以：1）评价排卵前卵泡中趋化因子及其受体的LH依赖性调节，2）评估CCR 2信号传导对C-OE和/或卵母细胞成熟所必需的分子事件的调节的作用，3）确定COC中CCR 2信号传导的抑制是否阻止C-OE和/或卵母细胞成熟。该项目将在猫科动物模型中开发，该模型的优点是为理解生育所必需的人类COC事件提供了一个很好的替代品;因为猫卵母细胞与人类卵母细胞有几个共同的特征。猫还提供了一个独特的和有价值的模型来研究排卵前卵泡和COC内的分子过程，因为每只动物提供3至7个排卵前卵泡，自然选择处于“排卵准备”状态，等待发情期间的LH刺激，提供了大约7天的窗口，可以收集样品。因此，这些新的和创新的研究将有助于增加我们对受精所需事件的理解，从而导致识别避孕的新的非激素靶点，以及识别不孕症的可能原因和/或卵母细胞质量的良好分子标记。

DESCRIPTION (provided by applicant): Ovulation is a complex, inflammation-like process whereby a fully-developed follicle ruptures in response to the actions of the mid-cycle gonadotropin surge, releasing the cumulus-oocyte complex (COC) for passage into the reproductive tract and possible fertilization. Shortly before ovulation, the luteinizing hormone (LH) surge induces processes critical for fertility, including cumulus-oocyte expansion (C-OE) and resumption of meiosis. While some of the paracrine-acting factors important for these events have been identified, the molecular mechanisms responsible for initiating such complex processes are not fully understood. Limited published studies and the PI's preliminary findings support a novel role for chemokine regulating events necessary for C-OE and oocyte maturation. Rodent studies suggest that chemokine signaling regulates the assembly of the cumulus extracellular matrix and thus fertilization. The PI's preliminary results from domestic cats demonstrated that the chemokine MCP-1 and its receptor (CCR2) are expressed in the oocyte and cumulus cells of COCs from small antral follicles. Similar results were observed in the monkey COC from small antral and preovulatory follicles. Moreover, MCP-1 and CCR2 mRNA increased in the preovulatory follicle 12 hr after an ovulatory stimulus with MCP-1 returning to pre-hCG levels at 24 and 36 hr. MCP-1 protein content in the follicular fluid paralleled the mRNA levels and peaked at 12 hr post hCG, notably the period of time that is just prior to the initiation of events necessary for C-OE and the resumption of oocyte meiosis in the rhesus preovulatory follicle. Based on these findings, we hypothesize that chemokines that interact with the chemokine receptor CCR2 (e.g. MCP-1) have a direct effect on promoting C-OE and/or oocyte maturation. Therefore, to test this hypothesis experiments are designed to: 1) Evaluate LH-dependent regulation of chemokines and their receptors in preovulatory follicles, 2) Assess the effects of CCR2 signaling on the regulation of molecular events necessary for C-OE and/or oocyte maturation, 3) Determine whether the inhibition of CCR2 signaling in the COC prevents C-OE and/or oocyte maturation. The project will be developed in a feline model, which has the advantage of providing an excellent surrogate for understanding events the human COC that are necessary for fertility; as cat oocytes share several characteristics with human oocytes. The cat also provides a unique and valuable model to study molecular processes within the preovulatory follicle and COC, due to the fact that each animal provides between 3 to 7 preovulatory follicles, naturally selected in an "ovulation-ready" state waiting for the LH stimulus during estrus, providing a window of approximately 7 days that sample can be collected. Therefore, these novel and innovative studies will aid in increasing our understanding of events required for fertilization, thereby leading to the identification of novel non-hormonal targets for contraception as well as identification of possible cause of infertility and/or good molecular markers for oocyte quality.