Hyperactivation is one of the most critical parts for fertilization. cAMP generated by soluble adenylyl cyclase (sAC) is necessary to activate sperm and is a prerequisite for sperm hyperactivation. The aim of this study is to investigate the function of sAC in hyperactivation in male rats. Four siRNAs of sAC gene were designed and separately transformed into rat sperm using electrotransformation method. Cultured for 12 and 24 h, physiological and biochemical indexes of these sperm were analyzed, and the expressions of some hyperactivation-related genes were detected using real-time PCR. We demonstrated 26.3-30.8% and 49.1-50.5% reduction in sAC at the protein by Western blot and mRNA levels by real-time PCR, respectively. The results showed that two siRNAs, Actb-717 and Actb-4205, were the best RNAi sites for silencing sAC. The VCL (curvilinear velocity) and ALH (amplitude of lateral head displacement) of RNA interference (RNAi)-transfected sperm were reduced. cAMP and protein phosphorylation in RNAi transfected sperm were also decreased. The hyperactivation-related genes, such as CatSper2, LDHC and PKA, were downregulated in the sperm, which sAC was knockdown. These findings demonstrated that sAC might play a critical role in cAMP signaling in the rat sperm hyperactivation, and downregulated sAC gene might prevent the expression of these hyperactivation-ralated genes resulting in sperm dysfunction. These findings suggest that these hyperactivation-ralated genes and sAC are functionally related in sperm hyperactivation and sAC falls into an expanding group of sperm proteins that appear to be promising targets for the development of male contraceptives.

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 μmol 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.