弱精症相关hPAQR7受体在人成熟精子孕酮信号转导中的作用机制

Progesterone, one of the most important steroid hormone for reproductive events, can increase the concentration of intracellular Ca2+ ([Ca2+]i) of human spermatozoa, enhance sperm motility, initiate sperm hyperactivation, activate acrosome reaction and induce chemotaxis towards the egg. This non-genomic progesterone action has been regarded to correlate with sperm specific CatSper calcium channel. However, whether progesterone directly acts on CatSper still remains obscure. Recent studies on human spermatozoa have identified a novel G protein-coupled receptor (GPCR)-like membrane protein hPAQR7 (human progestin-adiponectin Q receptor 7) which can specifically bind the progesterone. Our present studies demonstrated that the hPAQR7 antibody significantly inhibits the progesterone-induced [Ca2+]i increasing in human sperm. What’s more, the contents of hPAQR7 mRNA and protein as well as the progesterone-induced [Ca2+]i increasing were significantly decreased in the spermatozoa from several asthenozoospermia donors compared with the normal male donors. On the basis of these results, we hypothesize that the hPAQR7 plays a important role in the non-genomic progesterone action of human sperm. In this study, we combine the molecular, cellular and physiological biology techniques such as sperm-hPAQR7 antibody co-incubation assay, sperm function analysis, Western blot, co-immunoprecipitation, sperm single cell calcium imaging and patch-clamp: 1) to systematically characterize the relationship between hPAQR7 and asthenozoospermia under the large scale of clinical samples; 2) comprehensively reveal the functional role of hPAQR7 in the non-genomic progesterone action of human sperm; 3) to illustrated hPAQR7 related intracellular signaling, especially focusing on the interaction between hPAQR7 and CatSper and its mechanism. The results of the subject will not only make the important modifications and additions for the present mechanism of progesterone-CatSper, but also provide a novel molecular target for the diagnosis and treatment of asthenozoospermia.

重要生理激素孕酮对精子功能的调节效应与精子特异性CatSper钙通道密切相关，而孕酮是否直接作用于CatSper是当前争议焦点。hPAQR7（人孕酮-脂联素受体7）是能特异性结合孕酮的G蛋白偶联受体，但其在人成熟精子中的功能仍不清楚。我们最新结果显示，hPAQR7抗体显著抑制孕酮导致的人精子内钙增加效应；更重要的是，hPAQR7在部分弱精症人精子中含量显著减少，且此类精子对孕酮的响应也相应降低。据此我们推测：hPAQR7在人精子孕酮效应中扮演重要角色。本项目拟利用单精子钙成像和精子功能分析等技术：1）系统揭示hPAQR7在人成熟精子孕酮效应中的作用及其与弱精症的内在联系；2）多角度阐明hPAQR7相关胞内信号通路；3）重点探讨hPAQR7与CatSper之间可能的相互作用及机制。课题的完成将补充和完善倍受关注的孕酮-CatSper作用理论，并且有望为弱精症的诊疗提供新的分子靶标。

重要生理激素孕酮对精子生理功能的调节作用已被揭示多年，且近来来发现与精子特异性CatSper钙通道密切相关，但孕酮是否直接作用于CatSper是当前争议焦点。本课题以能特异性结合孕酮的G蛋白偶联受体hPAQR7为切入点，在前期验证了其参与人精子孕酮效应的前提下，利用大样本筛查、精子膜片钳、单细胞钙成像等方法，进一步研究hPAQR7介导人精子孕酮效应的胞内信号机制及其与CatSper之间的关系。结果显示：1）western blot大样本（300例弱精子症和200例正常人）筛查证实弱精症精子中hPAQR7含量显著低于正常人且hPAQR7含量与精子前向运动率成正相关；2）hPAQR7含量降低的弱精症病例的精子对孕酮响应能力下降，且抗hPAQR7胞外氨基酸片段的特异抗体LID2显著抑制精子对孕酮的响应能力，这些结果均表明hPAQR7在介导孕酮诱导的CatSper电流和内钙增加以及精子功能的激活中起到必不可少的作用；3）通过LID2阻断人精子中hPAQR7功能研究hPAQR7可能下游通路的结果揭示了增强型G蛋白-cAMP-PKA通路在hPAQR7介导人精子孕酮效应中起重要作用；4）免疫共沉淀实验和免疫荧光共定位实验数据支持人成熟精子中hPAQR7与CatSper互作，且膜片钳结果显示LID2抑制孕酮增加的CatSper电流。综上所述，hPAQR7在介导人精子孕酮-CatSper-内钙通路中起到至关重要的作用。以上部分结果已发表在Human reproduction、cellular physiology and biochemistry、BBRC等国内外知名期刊，部分内容处于论文撰写、专利申请准备中，且延续性工作已得到2017年国家自然科学基金面上项目的资助。这些工作对补充和完善倍受关注的孕酮-CatSper作用理论，开发以hPAQR7为靶点的男性不育诊断试剂盒具有重要意义。