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 demonstrated26.3-30.8%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.
Soluble adenylyl cyclase (sAC) plays a critical role in male fertility in mammals by regulating sperm hyperactivation. We aimed to study the mechanism of sAC in this phenomenon and to explore potential target sites for male contraception. In this study, in vivo electroporation and rete testis microinjection-mediated short hairpin (sh)RNA plasmids were adopted to silence sAC gene expression in male rats. The results showed that high transfection efficiency (shRNA717, 49.0% and shRNA4205, 65.0%) was achieved by shRNA plasmids injected directly into the rete testis. When the sAC was downregulated, the cyclic adenosine mono-phosphate (cAMP) content and protein phosphorylation level of spermatozoa both declined with a significantly lower hyperactivation rate compared with negative controls. The highest transfection efficiency occurred at 15 d and was obviously time dependent. Bioinformatic and experimental results showed that sAC and tmAC both belong to the AC family and might have analogous functions. ShRNA717 and shRNA4205 were the best targets for the sAC gene, suggesting that they could be candidates for male contraception. Thus, it appears feasible to achieve male contraception by silencing the expression of sAC, affecting sperm hyper-activation via a cAMP-mediated signaling pathway.
