Study On The Role Of MiRNA And Genetical Factors In Male Infertility

Infertility affects10-15%of couples who are trying to conceive, and half of the cases are due to male factors. Despite several decades of efforts to elucidate the etiologic mechanism of male infertility, most cases are idiopathic. For example, a significant proportion of male infertility is diagnosed as unexplained non-obstructive azoospermia (NOA). Furthermore, patients with NOA usually had a significantly lo-wer sperm retrieval rate and a clinical pregnancy rate. Therefore, elucidating the underlying molecular pathogenesis of NOA may help to improve treatment outcomes and therpies in these patients.Spermatogenesis is a tightly regulated process during which spermatogonial stem cells develop into highly differentiated spermatozoa. The meiotic and haploid phases of spermatogenesis are characterized by high transcriptional activity but repressed translational activity. Translational repression of mRNAs in these phases must be achieved through a posttranscriptional regulatory mechanism, one of which is mediated by microRNAs (miRNAs). Our group had reported that miRNA-383expression is down-regulated in the testes of infertile men with maturation arrest (MA). So the first aim of this study was to understand the underlying mechanisms of miR-383involved in the pathogenesis of MA. miR-383targeted a tumor suppressor, interferon regulatory factor-1(IRF1), which in turn resulted in suppression of proliferation, cell-cycle arrest in G1phase and induction of apoptosis, a reduction of Cyclin D1, CDK2and p21levels, and an inhibition of pRb phosphorylation. These results suggest that miR-383functions as a negative regulator of proliferation by targeting IRF-1, in part, through inactivation of the pRb pathway. Abnormal testicular miR-383expression may potentiate the connections between male infertility and testicular germ cell tumor.Although miR-383was expression is downregulated in the testes of infertile men with MA, how miR-383’s functions and targeting were regulated in spermatogenesis still remained unknow. So in the second part of this study (Chapter Three), we were trying to elucilate the regulating mechanisms of miR-383during spermatogenesis. We found that FMRP was associated with88miRNAs in mouse testis including miR-383. FMRP enhanced miR-383-induced suppression of cell proliferation by decreasing the interaction between FMRP and miR-383and then affecting miR-383binding to the3’UTR of its target genes IRF1and Cyclin Dl. Furthermore, FMRP levels were also down-regulated by miR-383by targeting Cyclin D1directly. Reduced miR-383 expression, dysregulated CDK4expression and increased DNA damage were also observed in the testes of Fmrl knockout mice and of MA patients with a down-regulation of FMRP. A potential feedback loop between FMRP and miR-383during spermatogenesis is proposed, and FMRP acts as a negative regulator of miR-383functions. Our data also indicate that dysregulation of the FMRP-miR-383pathway may partially contribute to human spermatogenic failure with MA.On the other hand, genetic factor also can affect male infertility. Kallmann syndrome (KS) is an inherited developmental disorder defined as the association of hypogonadotropic hypogonadism (causing male infertility) and anosmia or hyposmia. Due to olfactory bulb agenesis during embryonic development, the neuroendocrine GnRH cells fail to migrate from the nose to the forebrain along the olfactory nerve fibers.Various genes are involved the axonal pathfinding of the neurons, and KS has been shown to be a genetically heterogeneous disease with different modes of inheritance. However, mutations in any of the causative genes identified so far are only found in approximately one third of KS patients, thus indicating that other genes or pathways remain to be discovered. In the third part of this study, we report a large Han Chinese family with maternally inherited KS. The synergistic effect of mutations in KAL1(146G>T (p.49Cys>Phe))and mitochondrial tRNAcys(mt.5800A>G) can account for the occurrence of the disorder in this family. Furthermore, the disturbance of the mitochondrial cysteinyl-tRN A pathway can significantly influence the migration of GnRH cells in vitro. Also, Cars2is essential for the migration of GnRH3neurons, survival of larvae and sense of smell in zebrafish. Therefore, we propose that the mitochondria play important roles in the migration of GnRH cells and provide valuable clues to the genetic etiology of KS.In summary, the results in this study revealed the role of miR-383/FMRP feedback pathway in control of spermatogonial proliferation and apoptosis, and mitochondrial cysteinyl-tRNA pathway in Kallmann syndrome. These results could help to further understanding of the underlying mechanisms of spermatogenesis failure and defects in GnRH nueron development in NO A and KS patients.