Construction And Phenotype Analysis Of Sp3111 Transgenic RNAi Mouse Model

sp3111 gene is a gamete-specific gene identified by our lab previously. SP3111 protein was originally identified to be specifically expressed in the acrosome of rat sperm, and therefore named sperm specific protein 3111 (SP3111). Its amino acid sequence indicated that SP3111 might be a membrane protein with four transmembrane motifs. It was subsequently found that, SP3111 protein is specifically localized not only to the acrosome of sperm, but also the cell membrane of egg and zygote, indicating it is a gamete-specific gene. And the results of in vitro fertilization (IVF) experiment showed that, the treatment of mouse sperm with anti-SP3111 antibody (Ab2438) in vitro could significantly inhibit the fertilization and the development of fertilized eggs. Thus, it was suspected that, SP3111 protein might play critical roles in sperm-egg recognition and binding, as well as in fertilization and early embryo development.RNA interference (RNAi) has been proved to be a powerful tool to investigate the biological functions of certain gene in vitro or/and in vivo, and extensively applied in life science. Delivering small hairpin RNA-expressing constructs into animals'genome by transgenic technology can generate the transgenic RNAi animal with the down-regulated expression of the target gene in vivo. Therefore, in order to further explore the biological function of SP3111 in reproduction, we herewith successfully established the sp3111-knockdown male mouse model by transgenic technology, and it has been stably passaged to F4 genetation. Then, these sp3111 transgenic RNAi mice were used to assess the sp3111 mRNA expression level by Real-time RT-PCR analysis. The results showed that, the suppression efficiency of 7 lines of transgenic RNAi mice on the testis expression of sp3111 mRNA in vivo was more than 30%.Subsequently, the phenotype analysis of sp3111 transgenic RNAi male mice was performed. Both transgenic and wild type male mice were successively mated with the same batch of fertile wide type females. It was observed that, the sizes of litters from wild-type females×transgenic males were significantly reduced compared to wild-type females×wild-type males, indicating the down-regulation of sp3111 gene expression could reduce the male reproductive activitiy. Sperms collected from sp3111 transgenic RNAi male mice and wild-type male mice were respectively fertilized with the same batch of wild-type female eggs in vitro to assess the quality of sperms. The results showed that, compared to the wild-type male mice, a decreased fertilization rate of sperms and an increased abnormal fragmentation rate of fertilized eggs were observed in sp3111 transgenic RNAi males, which were consistent with our preliminary data of in vitro fertilization experiments using Ab2438 (anti-SP3111)-treated wild-type sperms. These studies further verified that SP3111 protein might play important roles in fertilization and early embryo development.In conclusion, it was suggested that, SP3111, as a gamete-specific protein, might paly essential roles in the processes of sperm-egg recognition and interaction, as well as the early development of fertilized eggs. The suppression of sp3111 gene expression could reduce the male fertility in mice. A better insight into the molecular mechanisms underlying the roles SP3111 played during fertilization and early embryo development would undoubtedly be contributive to the improvement on the clinical treatment of male infertility and the development of novel contraception.