【作者】 郑莉萍；

【导师】 曾旭辉； 李葆明；

【作者基本信息】 南昌大学 ， 动物学， 2013， 博士

【摘要】 阐明精子功能调控机制是诊断和治疗男性不育、研发新型男性避孕药的前提。离子通道是调控精子功能的关键因子。精子特异性SL03通道是生理条件下调控精子功能的主要K＋通道。离体实验表明,睾丸特异性表达的LRRC52(Leucine-Rich-Repeat-Containing protein52)具有调节SL03通道的功能。然而,LRRC52是否为SL03的在体调节亚基尚不清楚。本研究设计、制备抗LRRC52胞外段的多克隆抗体,对小鼠和正常人精子LRRC52生理功能及其机制进行研究。应用分子生物学、免疫荧光、电生理及精子功能分析等手段,我们发现LRRC52是SL03通道的在体调节亚基,是介导精子钾电流(IKSper)的关键组分。此外,我们的结果还提示,LRRC52可作为设计精子功能调控化合物的分子靶点。第一部分LRRC52在小鼠成熟精子的表达、定位及与SLO3的关系为获得能从细胞外特异性作用于LRRC52的化合物,我们设计制备了抗LRRC52胞外段的多克隆抗体(LID1)。通过Western blot、精子凝集实验、免疫荧光和免疫共沉淀等技术,我们验证了所获抗体的特异性,研究了在体条件下LRRC52与SLO3的相关性。结果表明：1)小鼠精子蛋白中可检测到LRRC52, LID1孵育的精子发生明显的凝集反应；2) LRRC52分布于小鼠精子尾部,尤以中段明显,并与SLO3存在明显的共定位；3)小鼠精子LRRC52与SLO3构成蛋白复合体。这些结果提示,LRRC52是SLO3的潜在调节亚基。第二部分LRRC52对小鼠成熟精子膜电流的影响为阐明LRRC52的生理功能,我们应用全细胞膜片钳技术研究了LID1对精子膜电流的影响。结果表明：1)LID1显著抑制小鼠IKSper但不影响精子钙电流(ICatSper);2) LID1对IKSper电导.电压曲线(G-V曲线)的影响与异体表达系统中LRRC52亚基对SL03的作用相符。这些结果提示LRRC52是构成精子钾通道、介导精子钾电流的重要组分。第三部分LRRC52对小鼠成熟精子生理功能的影响应用计算机辅助精子分析(CASA)、金霉素(CTC)染色、体外受精(IVF)、调节性体积下降(RVD)检测等实验手段,我们观察了LID1对精子活力、顶体反应(AR)、体外受精能力及RVD等相关生理功能指标的影响。结果表明：1)LID1显著抑制小鼠精子总运动力、直线速度(VSL)、曲线速度(VCL)和平均路径速度(VAP)；2)LID1不影响自发性AR的发生率,但显著抑制A23187诱发的AR发生率；3)LID1孵育对精子RVD能力的影响不显著；4)LID1显著抑制小鼠精子的体外受精率。这些结果提示,LRRC52在精子生理功能中扮演重要角色,是开发调控精子功能化合物的潜在分子靶点。第四部分LRRC52在人成熟精子的表达及其生理功能介导人精子IKSper的离子通道分子组成尚不清楚。由于用于制备LID1的LRRC52抗原片段在小鼠与人完全相同,我们用LID1初步观察LRRC52在人精子的表达及其可能的生理功能。结果表明：1) LRRC52和SLO3均在人精子蛋白中有表达,二者主要分布于精子尾部中段；2)LID1降低人精子顶体反应的发生率；3)LD1抑制人精子总运动力和曲线速度(VCL)。这些结果提示,LRRC52可能扮演与其在小鼠精子上类似的功能角色。更多还原

【Abstract】 Elucidating the molecular mechanisms underlying the regulation of sperm functions is crucial for treatment of male infertility and development of novel contraceptives. The ion channels in the cytosolic membrane are critical regulators of sperm functions. Previous studies have shown that the sperm-specific SLO3potassium channel plays a vital role in fertility of male mouse. In addition, studies in heterologous expression system have shown that the testis-specific LRRC52(Leucine-Rich-Repeat-Containing protein52) holds the ability to regulate SLO3channels. However, it remains to be determined whether LRRC52is a native auxiliary subunit for SLO3in sperm. In this study, we designed a polyclonal antibody recognizing an extracellular domain of LRRC52and investigated the physiological functions of LRRC52and the related mechanisms in both mouse and human sperm, using a diversity of methods including biochemistry, immunofluorescence, electrophysiology and sperm function analysis. Our results suggest that LRRC52is a physiologically important interacting partner of SLO3to mediate potassium current in sperm (IkSper), and LRRC52may serve as a potential molecular target for developing regulatory compounds of sperm functions.PART1. Expression, localization of LRRC52and its interaction with SLO3in mouse spermIn order to obtain an agent that can get access to LRRC52from the extracellular side, we designed a polyclonal antibody (LID1) recognizing an extracellular domain of LRRC52. Using western blot, sperm agglutination test, immunofluorescent staining and co-immunoprecipitation (Co-IP), we observed the specificity of LID1, and then investigated the co-localization and interaction between LRRC52and SLO3in mouse sperm. The results showed that:1) LRRC52was detected from mouse sperm protein extracts, and sperm incubated with LID1exhibited significant agglutination;2) LRRC52was specifically localized at the sperm flagellum, especially at the mid-piece region where SLO3was located;3) there was co-assembly between LRRC52and SL03in mouse sperm. These results indicate that LRRC52is a potential auxiliary subunit for native SLO3channel.PART2. Effects of LRRC52on membrane currents in mouse spermTo clarify the physiological importance of LRRC52, we investigated its effects on sperm-membrane currents by whole-cell patch-clamp technique. The results showed that:1) LID1inhibited IKSper in a dose-dependent manner, with no effect on calcium current (Icatsper);2) LID1displayed a similar effect on the conductance-voltage curve (G-V curve) of IKSper with that in heterologous expression system. These results indicate that LRRC52constitutes a functional component of the channel mediating IKSper.PART3. Effects of LRRC52on physiological functions of mouse spermTo further verify the physiological function of LRRC52, we examined the effects of LID1on sperm viability, AR (acrosome reaction), fertilization capacity and RVD (regulatory volume decrease) capacity, using computer-assisted sperm analysis (CASA), chlortetracycline (CTC) staining, in vitro fertilization (IVF) and sperm morphology analysis, respectively. The results indicated that:1) LID1incubation significantly reduced sperm viability, straight progressive velocity (VSL), curvilinear velocity (VCL) and average path velocity (VAP);2) LID1significantly inhibited the percentage of AR induced by A23187, leaving the spontaneous AR unaffected;3) LID1did not influence the RVD ability of sperm;4) LID1reduced sperm fertility in vitro. Taken together, these results strongly support that LRRC52is a physiologically regulatory subunit of SLO3, and may serve as a potential molecular target for developing regulatory compounds of sperm functions. PART4. Existence and physiological functions of LRRC52in human spermThe molecular composition of ion channels mediating potassium current in human sperm has been of a great scientific interest. Since the sequence of amino acids for the antigenic determinant of LRRC52is completely conserved in mouse and human, we attempted to study the expression profile and possible physiological functions of LRRC52in human sperm using LID1. The results were:1) both LRRC52and SLO3were expressed in human sperm and located in the mid-piece of sperm flagellum;2) LID1significantly reduced the occurrence of both spontaneous and induced AR in human sperm;3) LID1inhibited the motility and VCL of human sperm. These results suggest that LRRC52may play similar physiological roles in human sperm, as in mouse sperm.更多还原