Study On The Immune Effect Of FSHR Peptides And The Association Of Polymorphisms In FSHR Gene With Male Infertility

As the rapid increase in population around the world and the development of social civilization, a majority of men would be willing to use a male contraceptive. In contrast, advances in male-directed methods have been confined to refinements in the type of condom and technique of vasectomy. Thus, it has become more and more urgent to develop a safe, effective, reversible and acceptable contraceptive for males. Researchers chose suitable target antigens based on the regulation of spermatogenesis as the vaccines. FSHR is expressed exclusively in testicular Sertoli cells and mediates the action of FSH during the spermatogenesis, so it is shown to be a suitable immunocontraception target. The present study aimed to produce the FSHR peptides vaccine and research its immune effect, which could provide the technical and theoretical bases for pilot scale productin and primate trial. Furthermore, we investigated whether polymorphisms in FSHR were associated with the risk of developing idiopathic male infertility and sperm apoptosis, which would help us analysize the etiology of male infertility and provide the theoretical bases for contraception vaccine.Part I The production of FSHR peptides and its immune effectThe present study aimed to produce the FSHR peptides vaccine and research its immuno-antifertility effects. In this research, an FSHR (1-57aa) cDNA was generated by PCR using the human testis cDNA as template, and then the cDNA was cloned into vector pET-28a(+) downstream of His6-Tag. To cut the His6-Tag, we added the enterokinase (EK) recognition site between His6-Tag and our target protein. Subsequently, the prokaryotic proteins were expressed under induction of 1 mM IPTG. After Ni-NTA purification, renaturation and EK digestion, we obtained FSHR peptides, which were existed in form of inclusion bodies. The purity of FSHR peptides is above 95%.To study the immune effects, we used the recombinant FSHR peptides, which were mixed with the aluminum adjuvant, to immunize male Balb/c mice. The specific anti-FSHR IgG could be detected in serum with the highest titre around 1:10000. Meanwhile, reduced sperm count, smaller testis and germ cells apoptosis were observed.In conclusion, the high purified FSHR peptides prepared by genetic engineering have powerfully immunogenicity after mixed with the aluminum adjuvant in mice, which could favor further research in pilot scale production and non-human primate trial. Part II Study on the association of polymorphisms in FSHR geneThis study aimed to investigate the effects of the FSHR polymorphisms on idiopathic male infertility in Han-Chinese. Three FSHR polymorphisms, -29(G>A), Thr307Ala(A>G) and Asn680Ser(A>G) were analyzed using Taqman allelic discrimination assay and PCR-RFLP in 285 fertile controls and 534 infertile patients. Association between the polymorphisms and risk of male infertility were estimated by univariate and multivariate logistic regression analysis. Furthermore, to analyze the association between FSHR polymorphisms and sperm apoptosis, we measured sperm apoptosis by TUNEL assay using flow cytometry in 337 infertile patients. The results showed that the ThrAla307 (TA307) and AsnSer680 (NS680) genotypes were associated with a significantly decreased risk of idiopathic asthenospermia (OR= 0.56 and 0.55, 95% CI = 0.37-0.85 and 0.36-0.83, P = 0.024 and P = 0.018 respectively) compared with TT307 and NN680 respectively. Haplotypic analysis showed a significantly decreased risk of asthenospermia associated with the A307-S680 haplotype compared with the T307-N680 haplotype (OR= 0.70, 95% CI = 0.51-0.96). However, no significant association was found between the -29(G>A) polymorphism and idiopathic male infertility. Analysis showed that patients with -29(G>A) AG genotype were associated with lower sperm apoptosis rate when compared to those with GG genotype (P=0.021). Our results suggest that the FSHR polymorphisms may contribute to the pathogenesis of idiopathic male infertility in the Han-Chinese population, but future mechanistic and epidemiologic studies are needed to confirm these findings...