基于精子特异性嗅觉受体OR1D2研究人精子趋化机制

Study on the mechanisms underlying sperm chemotaxis is important for male reproduction physiology. The relatively sperm-specific olfactory receptor OR1D2, which can be activated by the synthetic odorant called bourgeonal, plays key role in mediating the chemotaxis of human spermatozoa. However, the physiological ligand of OR1D2 and the intracellular signaling pathways after OR1D2 activation are far from being illuminated. Based on our recent results that bourgeonal dramatically increased the current level of sperm-specific calcium channel CatSper and that effect was significantly inhibited by anti-OR1D2 antibody, this application hypothesizes that OR1D2 mediates human sperm chemotaxis by regulating CatSper. Combining patch clamping, calcium signal measurement and sperm chemotaxis analysis, this project intends to: 1) elucidating the intracellular signaling pathways underlying human sperm chemotaxis mediated by OR1D2; 2) screening the possible physiological ligand of OR1D2; 3) revealing the relationship between OR1D2 abnormality and male infertility. The completion of this project is expected to contribute to elucidate the mechanism of human sperm chemotaxis, clarify the activation mechanism of CatSper and discover new factors related to male infertility. The applicant has outstanding research records (Nature, 2012; PNAS, 2015, 2011) in the related field, which provides a solid base for the successful completion of this application.

精子趋化机制研究是雄性生殖生理的重要内容。人精子上特异表达的嗅觉受体OR1D2是介导人精子趋化效应的关键分子，其能被合成香味剂bourgeonal激活。然而，有关OR1D2介导的胞内信号途径、其生理配体等远未阐明。根据bourgeonal显著增加精子特异性钙电流CatSper、该增强效应被抗OR1D2抗体显著抑制等前期结果，本申请假设OR1D2激活后通过调节CatSper而介导趋化效应。结合精子膜片钳、钙信号测量、精子趋化分析等手段，本申请拟：1）系统阐明OR1D2所介导的人精子趋化效应的胞内信号途径；2）筛选OR1D2可能的生理配体；3）探讨OR1D2途径异常与男性不育的相关性。本课题有望在阐明人精子趋化机制、完善CatSper激活机制、发现新的男性不育因素等方面作出贡献。申请人良好的研究记录（Nature，2012；PNAS，2015,2011）为本申请提供了保障。

精子趋化机制研究是雄性生殖生理的重要内容。人精子上特异表达的嗅觉受体OR1D2是介导人精子趋化效应的关键分子。然而，有关OR1D2介导的胞内信号途径、其生理配体等远未阐明。本申请假设OR1D2激活后通过调节CatSper而介导趋化效应。结合精子膜片钳、钙信号测量、精子趋化分析等手段，围绕上述假设本申请主要从三个方面开展研究：系统阐明OR1D2所介导的人精子趋化效应的胞内信号途径；筛选OR1D2可能的生理配体；探讨OR1D2途径异常与男性不育的相关性。相应地，主要发现有：1）发现bourgeonal/OR1D2通过G蛋白途径激活mAC-cAMP-PKA、从而增加CatSper电流与胞内钙浓度而最终导致精子趋化，胞外cAMP分子则可直接激活CatSper；2）CatSper与pH调节分子钠氢交换器存在功能偶联； 3）趋化效应分子孕酮与NPPC对CatSper或其它通道的激活不依赖于OR1D2、因此这些分子应该不是OR1D2的生理配体；4）部分不育样本其OR1D2表达水平与bourgeonal所导致的钙增加显著降低、提示OR1D2途径异常可能是男性不育的致病因素。这些工作第一次揭示了一条较完整的人精子趋化信号通路。此外，也是第一次证明胞外刺激物能通过胞内信号过程激活CatSper。鉴于CatSper对于精子功能调控的重要性，且精子膜上还有众多G蛋白偶联受体，我们的结论对于认识精子功能调控机制以及男性不育病理机制具有普遍意义。在本项目资助下，共发表了一篇综述以及6篇SCI论文，包括JBC、Human Reproduction、IJMS、Theriogenology等。而且，主体工作正在尝试投往具有良好影响力的杂志。此外，我们已申请一份专利、另外两份专利申请即将递交。本项目执行中共有三名博士研究生、多名硕士研究生毕业，多名年轻科研人员获得训练。总体上，本项目达到了预期目标。