RNA Interference-mediated Downregulation Of SAC Gene Affects Sperm Hyperactivation In Male Rat

The spermatozoa undergo several essential activation processes,including the initiation of motility on the release from the epididymis,induction of hyperactivated motility,capacitation,and acrosome reaction,to fertilize eggs.Hyperactivation is one of the most critical parts for fertilization,which may help spermatozoa swim faster and generate enough force,and is required for penetration of the zona pellucid to fertilize eggs.Hyperactivation is characterized by high amplitude,asymmetrical flagellar bending.Recently,the study of sperm hyperactivation is becoming a research hotspot.But the molecular mechanism triggering hyperactivation is not fully understood.Moreover,the study of sperm hyperactivation will promote the development of male contraceptives.The aim of this study is to clear the molecular mechanisms of s.AC in the sperm hyperactivation using RNAi technology.First,predicted functions of sAC were analyzed by bioinformatics methods.Then four siRNAs of the sAC gene were designed,and were subsequently transformed into rat sperm using electro-transformation method.Cultured for 12 and 24 hours,physiological and biochemical indices,and the expressions of other hyperactivation-related genes were investigated using real-time PCR,Western blot and ELISA methods.Furthermore,the sperm mobile parameters were analyzed using CASA method.Based on the results in vivo,we constructed two shRNA plasmids.In vivo electroporation(EP)and rete testis microinjection-mediated shRNA plasmid were adopted to silence sAC expression.This study demonstrated that it was feasible to achieve male contraception by silencing the expression of sAC,affecting sperm hyperactivation by cAMP-signaling pathway.The results were as follows:1.Bioinformatic analysis of rat sACAlternative splicing 46 KDa sAC was more stable than the full-length sAC.The 46 KDa sAC is hydrophobic and contains more a-helix structures for the formation of stable secondary structure.The predicted results of sAC tertiary structure indicated that two Guanylate_cyc domains were close together,forming a small cavity,which was important for accommodating a target molecule in 46 KDa sAC.Therefore,the 46 KDa sAC was considered to be the main present form and functional molecule.Phylogenetic analysis showed that ACs and GCs were divided into four subgroups,including transmembrance guanylate cyclases(tmGC),soluble guanylate cyclases(sGC),tmAC and sAC.sACs and tmACs contain two Adenylate and Guanylate cyclase catalytic domain(Guanylate_cyc),but sGCs and tmGCs only contain one Guanylate_cyc domain.From the NJ tree,we could imply that sACs were the ancient of tmACs and GCs.In the eukaryotes,the sAC and sGC cyclase domains are most similar to ACs.Based on the evolutionary tree of sACs,we investigated that from Reptiliomorpha,the sAC was duplicated once,becoming two copy genes,sAC and sAC-like.Thus,one sAC was lost in some species including human,rat and so on.2.siRNAs of sAC affect sperm hyperactivation in vitroThe aim of this study is to investigate the function of sAC in hyperactivation in male rats.Four siRNAs of sAC gene were designed and were separately transformed into rat sperm using electrotransformation method.The transfection efficiency was about 90%using the electronic transfermation parameters of 400 V,200 ?s and 3 times.Cultured for 12 and 24 hours,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 and ALH of 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 spenn,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.3.Knockdown of sAC affects sperm hyperactivation by cAMP-signaling pathway in male ratWe 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 monophosphate(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 days 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 hyperactivation via a cAMP-mediated signaling pathway.In conclusion,knockdown of rat sAC by electroporation-mediated shRNAs and siRNAs could effectively interfere with fertility in male rats and thus could provide prospective candidates for male contraception.However,this method could not completely inhibit sAC protein expression.Furthermore,two siRNAs,Actb-717 and Actb-4205,were identified to be the best RNAi sites for silencing sAC.We propose that sAC is a central factor in controlling rat sperm hyperactivation via a cAMP-mediated signaling pathway and predict that the downregulation of sAC will reduce the expression of other hyperactivation-related genes.