精子功能调控的膜蛋白机制研究

Both Slo3 potassium channel and CatSper calcium channel are sperm-specific,alkalization-activated ion channels important for mammalian sperm function. The applicant's previous reports (PNAS,2011a,2011b) have demonstrated that Slo3 is the dominant potassium channel in mouse sperm to regulate male fertility, and the deletion of Slo3 gene results in infertility in male mice. However, barely is it known regarding the in vivo expression, biophysical properties and physiological functions of human Slo3. By combining biophysical studies on heterologously expressed hSlo3 and electrophysiological studies in human sperm with traditional in vitro fertilization methods, this project aims to reveal the properties and possible functions of Slo3 in human sperm. Furthermore,taking advantage of the applicant's unique techniques of completing electrophysiological research directly in sperm, by comparing the expression level and functional differences between normal sperm and sperm from infertile men, this project is also anticipated to illustrate the intrinsic relationship between ion channel disorder and male infertility. On the other hand, we will examine the coupling mechanisms between the above critical ion channels and pH regulatory transporters in sperm. Overall, the success of this project will systematically reveal the physiological and pathological roles of ion channels in human sperm, forming basis for developing creative diagnosis and treatment methods for male infertility or screening novel male contraceptives.The applicant and co-applicant have extended research experience in the related field with publications in a series of prestigious journals.

Slo3钾通道和CatSper钙通道是精子特异性、pH敏感的两种重要离子通道。申请人前期研究表明（PNAS，2011a，2011b），Slo3 是控制小鼠精子生育力的关键钾离子通道。然而，有关Slo3在人精子中的特性和功能的了解几乎是空白。申请人拟结合hSlo3的异体生物物理研究、在体电生理研究、以及传统生殖学方法等，揭示hSlo3的特性和可能的生理功能。更进一步，通过比较正常和某些不育症患者精子细胞上hSlo3和CatSper表达和特性的差异，以期揭示精子特异性离子通道表达和功能异常与男性不育的内在联系。同时，申请人将探讨上述关键离子通道和精子上pH调控转运体之间的耦联机制。本课题的完成对深入认识精子生理、诊疗男性不育和开发新型避孕药具有重要意义。申请人和合作者的相关工作已发表在Nature, NSB, PNAS, JN, JGP，JP，PLoS One等杂志上。

世界范围内约12-15%的育龄夫妇存在生育障碍，其中50%与男性相关，而精子功能缺陷为主要原因。因此，从分子水平深入认识成熟精子功能调控的机制是诊断、治疗男性不育症的基础。pH敏感的KSper钾通道和CatSper钙通道是目前仅知的两种精子特异性重要离子通道，基因敲除小鼠均表现为雄性不育。揭示KSper在人精子中的分子组成与功能、这两种通道如何相应外界的刺激信号、其异常与男性不育的相关性等是本课题的主要目标。本课题的主要发现包括：1）证明了LRRC52亚基不仅是构成小鼠KSper的必要组分、也可能是构成人KSper的重要组分，KSper是调节人精子功能的关键因子；2）通过与膜上不同GPCR偶联从而激活CatSper是人精子响应不同外界刺激信号的普遍机制，得到验证的配体/受体对有hDEFB1/CCR6、孕酮/hPAQR7、bourgeonal/OR1D2、ATP/P2Y2；3）虽然G蛋白与cAMP是否参与了 GPCR对CatSper的增强效应存在明显争议， bourgeonal/OR1D2增强CatSper电流的信号过程中需要激活G蛋白、升高cAMP水平；4）生理可测浓度的双酚A能通过影响CatSper的表达与功能而损害精子功能，精子特异性离子通道还参与了铅、氯胺酮、苦参碱、大黄素等对精子功能的损害作用；5）CatSper钙通道异常导致特发性男性不育、其结合膜蛋白hPAQR7表达低下则与弱精症密切相关。需强调的是，有关精子膜上GPCR的种类、特性和功能的了解非常有限，我们的结果表明生理配体与相应GPCR结合后激活CatSper可能是精子响应外界刺激的普遍途径。总之，这些结果对于认识精子功能调控的分子与胞内信号途径、揭示男性不育的病理机制、发展诊断与治疗男性不育的新方法具有重要意义。