Physiological Functions Of Sperm-specific LRRC52

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.