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

总结 排卵是一个复杂的，炎症样的过程，其中一个充分发展的卵泡破裂，以回应 周期中期促性腺激素激增的作用，释放卵丘-卵母细胞复合体（COC）进行传代 进入生殖道并可能受精排卵前不久，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， 引发这种复杂过程的分子机制尚未完全了解。有限 已发表的研究和PI的初步发现支持趋化因子调节事件的新作用 C-OE和卵母细胞成熟所必需的。啮齿类动物的研究表明，趋化因子信号调节 卵丘细胞外基质的组装，从而受精。PI的国内初步结果 猫证明趋化因子MCP-1及其受体（CCR 2）在卵母细胞中表达， 小窦状卵泡COCs的卵丘细胞。在来自小细胞的猴COC中观察到类似的结果。 窦卵泡和排卵前卵泡。此外，MCP-1和CCR 2 mRNA在排卵前卵泡12中增加， 在用MCP-1刺激排卵后24小时和36小时，MCP-1蛋白含量恢复到hCG前水平。 卵泡液中的mRNA水平在hCG后12小时达到峰值， 正好在恒河猴中C-OE和卵母细胞减数分裂恢复所必需的事件开始之前 排卵前卵泡基于这些发现，我们假设趋化因子与细胞因子相互作用， 趋化因子受体CCR 2（例如MCP-1）对促进C-OE和/或卵母细胞 成熟因此，为了验证这一假设，实验设计为：1）评估LH依赖性 排卵前卵泡中趋化因子及其受体的调节，2）评估CCR 2信号传导的作用 对C-OE和/或卵母细胞成熟所必需的分子事件的调节，3）确定 COC中CCR 2信号传导的抑制阻止了C-OE和/或卵母细胞成熟。该项目将 在猫科动物模型中开发，其优点是提供了一个很好的替代理解 事件的人类COC是必要的生育能力;因为猫卵母细胞与人类共享几个特征， 卵母细胞猫还提供了一个独特的和有价值的模型，以研究分子过程中， 排卵前卵泡和COC，由于每只动物提供3至7个排卵前卵泡， 自然选择在“排卵准备”状态等待LH刺激在发情期，提供了一个窗口， 大约7天，可以收集样品。因此，这些新颖和创新的研究将有助于 增加了我们对受精所需事件的理解，从而导致识别新的 避孕的非激素靶点以及确定不孕症的可能原因和/或良好的 卵母细胞质量的分子标记。

SUMMARY 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.