Delivery of CatSper2 siRNA into Rat Sperms by Electroporation Repressed Ca^(2+) Influx During Sperm Hyperactivation

作     者：ZHANG Zhen ZHOU Xuan LI Hui-xia CUI Qun-wei YU Jing WANG Gen-lin 

作者机构：College of Animal Science and Technology Nanjing Agricultural University Nanjing 210095 PR.China 

出 版 物：《Agricultural Sciences in China》 (中国农业科学（英文版）)

年 卷 期：2011年第10卷第12期

页      面：1958-1967页

核心收录：

学科分类：0710[理学-生物学] 071010[理学-生物化学与分子生物学] 081704[工学-应用化学] 07[理学] 08[工学] 0817[工学-化学工程与技术] 0905[农学-畜牧学] 09[农学] 090501[农学-动物遗传育种与繁殖] 

基　　金：supported by the National Key Technology R&D Program of China (2006BAD04A12) 

主　　题：sperm hyperactivation motility CatSper2 Ca2+ channel RNAi electroporation (EP) 

摘      要：CatSper is a unique Ca2＋ channel-like protein family exclusively expressed in the testis and sperm, and plays important roles in sperm motility, capacitation, acrosome reaction and sperm-egg interactions. Here we studied the mechanism of regulation of CatSper2-dependent Ca〉 influx, extracellular and intracellular Ca2＋on sperm hyperactivated motility. The siRNA duplexes were transfected into the sperm cells by electroporation （EP） to silence the expression of CatSper2. The results for targeted disruption of CatSper2 showed the highest silence efficiency 77.7% （P〈0.05）, the hyperactivated sperm rate calculated by computer-assisted semen analysis （CASA） analysis of interferenced sperm was significantly lower 11.1% than the control 99.2%. Flow cytometry （FCM） detection of the intracellular Ca2＋ concentration of interferenced sperm was 50 nmol L-t higher than the normal. It was remarkably lower than hyperactivated sperm with 200-1 000 nmol L-1 （P〈0.05）. It was not sufficient to evoke hyperactivation. To trigger hyperactivation, the sperm were incubated in 50 pmol L-t thimerosal or 5 mmol L-1 procaine, it sharply increased the intracellular Ca2＋ via two different channels. CASA and FCM detection indicated that intracellular Ca〉 is required for initiating hyperactivation while extracellular Ca2＋ is essential to maintain the process. We concluded that to mediate sperm hyperactivation, it is essential to inhibit Ca〉peak present with targeted disruption of CatSper2 expression. This provides important prospective for further exploration of signal channel of sperm hyperactivated motility, potential factors for male infertility and provide further reference to the exploration of male contraceptive drug targets of male reproduction.