精子特异性LRRC52作为调控雄性生育力分子靶标的探索性研究

Clarifying the molecular mechanisms by which sperm functions are regulated is crucial for the treatment of male infertility and the development of novel contraceptives. Ion channels are critical regulators for sperm function. Previous reports from our laboratory showed that the sperm-specific Slo3 potassium channel plays a vital role in male mouse fertility, and heterologous expression of LRRC52 with Slo3 exhibits a current with features approximate to the native KSper（PNAS,2011）. However, whether LRRC52 is the auxiliary subunit of SlO3 in sperm is yet to be confirmed. Our preliminary studies have found that LRRC52 was located at principal piece of sperm tail, and LRRC52 antibody could reduce sperm activity in mice. Thus, we hypothesize that LRRC52 may be a native auxiliary subunit with functional effect on Slo3. Taking advantage of our extended research experience in reproductive physiology（Reprod Biol Endocrinol, 2010.etc) and sperm patch-clamp technique, we will analyze the correlation between the expression levels of LRRC52 and the changes of sperm motility, investigate the effects of LRRC52 gene deficit or LRRC52 antibody application on sperm function,and explore the underlying mechanisms of LRRC52 in regulating sperm activities. Taken together, we expect to provide a new target for the regulation of sperm function. Furthermore, the results obtained from human sperm in this study may offer scientific basis for future clinical applications.

阐明精子功能调控机制是诊疗男性不育、研发新型避孕药的前提。离子通道是决定精子功能的关键因子。实验室前期研究表明，Slo3是生理条件下调控精子功能最重要的K+通道，离体Slo3结合LRRC52后具有接近于精子钾电流的特征（PNAS,2011），然而LRRC52是否为精子Slo3的在体调节亚基亟待证实。申请者初步研究发现，LRRC52 亦定位于精子尾部主段，抗体可降低小鼠精子活性，提示LRRC52可能是Slo3的在体辅助亚基。依据良好的生殖生理学基础（Reprod Biol Endocrinol, 2010等）和精子膜片钳技术，申请者拟以小鼠和人精子为实验对象，分析LRRC52表达变化与精子活力变化的相关性；观察LRRC52缺失或抗体封闭对精子生理功能的影响；研究LRRC52调控精子钾电流及其活性的具体机制，以期为精子功能调控提供新靶标，而从人精子获得的结果则可为临床应用提供直接理论依据。

阐明精子功能调控机制是诊疗男性不育、研发新型避孕药的前提。Slo3通道在精子获得运动、受精能力，参与顶体反应和精卵融合等过程中，发挥着必不可少的作用，是决定精子功能的关键因子，然而其具体生理调控机制并不清楚。本课题以小鼠和人精子为实验对象，主要包含以下内容:①制备抗LRRC52 胞外段多克隆抗体（LID1），并利用该抗体,观察LRRC52、SLO3在正常小鼠和人精子中的定位与表达，分析LRRC52、SLO3之间相关关系。②利用精子膜片钳技术，观察小鼠和人精子LRRC52亚基经特异性抗体封闭后，其精子膜电流（IKSper和ICatSper）变化。③通过特异性抗体封闭的方式，观察LRRC52对小鼠和人成熟精子生理功能的影响作用。此外，④初步探讨环境重金属镉离子对于小鼠成熟精子功能损伤的相关机制。研究结果发现：①LRRC52和Slo3在小鼠精子中存在着共表达，且二者密切相关构成复合物。②小鼠精子LRRC52 亚基经特异性抗体封闭后，其精子膜电流IKSper出现明显抑制，但ICatSper无明显变化。③通过特异性抗体封闭的方式，LRRC52 对小鼠精子活率、平均曲线速度（VCL）无明显影响，但平均路径速度（VAP）、平均直线速度（VSL）等各项运动指标均显著下降；LRRC52抗体不影响小鼠精子自发顶体反应发生率，但对A23187诱发顶体反应发生率却有显著抑制作用；LRRC52抗体显著抑制精子的体外受精能力。④人精子中同样存在LRRC52 和Slo3共表达，LRRC52和Slo3均分布于人精子尾部，尤以中段明显；人精子LRRC52 亚基经特异性抗体封闭后，其精子膜电流IKSper出现明显抑制，但ICatSper无明显变化。⑤环境重金属镉离子通过CatSper而非Slo3机制致小鼠成熟精子功能损伤。这些结果提示：①LRRC52亚基是小鼠精子Slo3通道的在体调节亚基、是构成小鼠精子KSper的关键组分。②LRRC52也是介导人精子KSper的分子组分，并可能承担着与小鼠精子中类似的生理功能。③LRRC52亚基可作为设计筛选精子功能调控化合物的分子靶点。