Study On The Variants And Down-regulated Expression Of Eppin Gene In Male Reproductive Function

As a potential candidate for male immunocontraception vaccine, EPPIN (Epididymal protease inhibitor) has attracted the attention of many researchers in the recent decade. This protein is expressed specifically in the testis and epididymis. Many studies have demonstrated its importance in sperm function and male fertility. In the present study, we sought to investigate the function of EPPIN in male fertility from two aspects of molecular epidemiology and animal models, which could provide some new clues for the etiology of male infertility and clarify the molecule mechanism of EPPIN's biological function from new perspective. Part I The association study between variants in the EPPIN gene and idiopathic male infertilitySingle nucleotide polymorphisms (SNPs) represent the most abundant class of inheritable human gene mutations, and have profound influence on gene expression and function. For the EPPIN gene, although a number of SNPs were listed on the website http://www.ncbi.nlm.nih.gov/projects/SNP/, there is no related report to date on the roles of EPPIN genetic variants on semen quality and male infertility in the general male population. Therefore, in order to clarify the association between EPPIN variant and semen quality and male infertility, we first analyzed the association between four EPPIN tagSNPs (rs6124715, rs2231829, rs2227290 and rs11594) and semen quality in 473 males with definite idiopathic infertility. Then, to clarify the association between EPPIN genetic variants and idiopathic male infertility, we expanded the sample capacity by adding a control group consisting of 198 fertile men and analyzed the genotype distribution of each SNP, evaluated the influence of these genetic variants on serum testosterone level. Additionally, we explored that how the positive SNPs play the biological functions by bioinformatics methods. We have demonstrated significant associations between genetic variant rs2231829 and sperm number, and between variants rs6124715 and rs11594 and sperm motility characteristics. In the analysis of SNPs to male infertility risk, we found variants rs2231829 / rs11594 are associated with decreased / increased male infertility risks, while the patients with abnormal semen parameters might be at lower / higher risk. Almost similar serum testosterone levels among different EPPIN genotypes were observed for each group. The bioinformatics analysis found that variant rs2231829 could alter transcription factor binding site, variant rs11594 could change the mRNA structure, and in turn affect the sperm maturation process and male fertility. Whether this indeed is the case requires further investigation. Part II The down-regulated expression of EPPIN gene on sperm motilityWe know from existing research that EPPIN participates in the process of spermatogenesis and sperm maturation, but to date, there was no effective in vitro or animal model to confirm its function in these processes. In the present study, we investigated the effect of down-regulated expression of EPPIN gene to mouse sperm motility in a mouse model by the utilization of RNA interference in vivo with the combination of testis microinjection and patch clamp, and explored the possible mechanism. We found the sperm motility declined significantly when the EPPIN gene was down-regulated, which was mainly manifested as the decline of motility, VAP, VSL and VCL. Further studies demonstrated that, according with the decline of sperm motility, the T-type calcium current and the T-type calcium channel mRNA level of spermatogenic cells also declined significantly. Therefore, we concluded that the low expression of EPPIN gene induced the decline of T-type calcium current and the T-type calcium channel mRNA level, which in turn resulted in the decline of sperm motility. However as we know, the acquisition of sperm motility is an extremely complex process, which is associated with not only the sperm structure but also the external environment. Although the calcium ion plays a key role in sperm motility, we needs further study to elucidate how the alteration of T-type calcium channel affects the sperm motility.