Delivery of CatSper2 siRNA into Rat Sperms by Electroporation Repressed Ca2+ Influx During Sperm Hyperactivation

Abstract

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 Ca²⁺ 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⁻¹ 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 mol L⁻¹ thimerosal or 5 mmol L⁻¹ procaine, it sharply increased the intracellular Ca²⁺ via two different channels. CASA and FCM detection indicated that intracellular Ca²⁺ 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 Ca²⁺ 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.