Study On The Role Of Male Infertility-related Protein MLH1 And MiR-383 In Spermatogenesis

Male infertility is a worldwide reproductive health problem. Moreover, recent research provides strong evidence that infertile men have an increased risk of subsequently developing testicular cancer. However, most cases of non-obstructive azoospermia (NOA) remain unexplained (idiopathic infertility) and medical treatment has limited. Some studies indicated that defects in meiotic homologous recombination and/or imbalance between proliferation and apoptosis of germ cells are the etiological factors underlying spermatogenesis arrest in NOA patients. However, the molecular mechanisms of these defects are currently poorly documented.Homologous recombination involves the generation of crossovers (COs, marked by MLH1 in pachytene) between homologous pairs of chromosomes, which ensure the proper segregation of chromosomes during meiosis. In humans, the distribution of COs is affected by positive interference, by definition, means that the occurrence of one CO discourages the formation of other COs in its vicinity, which leads to widely spaced COs along chromosomes. To study the extent of human male CO interference, immunofluorescence techniques combined with centromere-specific multicolor fluorescence in situ hybridization (cenM-FISH) were used to identify the frequency and location of COs in specific chromosomes of pachytene cells, and the interference parameterÏ…estimated by fitting the frequency distribution of inter-CO distances to the gamma model served as measure the strength of CO interference.CO interference was observed to act continue uninterrupted across the centromere. Significant inter-individual, inter-chromosomal and intra-chromosomal variations in the levels of CO interference were found, with smaller chromosomes and inter-arm COs exhibiting stronger interference than larger chromosomes and intra-arm COs, respectively. Furthermore, synaptonemal complexs (SCs) were found to play an important role in regulating CO interference levels. The evidence for this includes:1) the interchromosomal effect on interference levels occurred among chromosomes with similar SC length; 2) chromosome synapsis anomalies such as discontinuous regions (gaps) and unsynapsed regions (splits) in chromosome 9 had both cis and trans effects on CO interference levels. The present report is the first to demonstrate CO interference shows significant heterogeneity across the whole genome and that, at least in the human male, anomalies in chromosome synapsis can affect the levels of CO interference.The next aim is to identify the signals contributing to the defects in NOA patients with mixed patterns of maturation arrest (MA). As a unique class of small non-coding RNA molecules, microRNAs (miRNAs) are indicated to play essential roles in spermatogenesis. To identify the potential functional miRNAs, the miRNA expression profiles of testes of NOA patients and normal controls were performed by using microarray technologies. Widespread deregulation of miRNAs in NOA patients was found, with 154 differentially down-regulated and 19 up-regulated miRNAs. Among the differentially expressed miRNAs, miR-383 whose expression is restricted to the spermatogonia and spermatocytes, was significantly down-regulated in the testes of MA patients. Loss of miR-383 was associated with hyperactive proliferation of germ cells in MA patients. Silencing of miR-383 in NTera-2 (testicular embryonal carcinoma) cells enhanced proliferative activity, promotes G1/S transition and inhibits basal apoptosis, while overexpression of miR-383 reversed these effects.Furthermore, miR-383's functional effects were mediated through targeting a tumor suppressor, interferon regulatory factor-1 (IRF1), and miR-383 expression was negatively correlated with IRF1 protein expression in vivo. miR-383 inhibited IRF1 expression, which consequently down-regulated critical cell cycle-related genes, cyclin D1, CDK2 and p21 at the transcriptional level. Down-regulation of IRF1 or cyclin D1, but not CDK2, enhanced miR-383-mediated effects, while silencing of p21 partially inhibited miR-383'effects. On the other hand, miR-383 increases proteasome-dependent degradation of CDK4, and ultimately leads to down-regulation of pRb phosphorylation. Therefore, abnormal miR-383-IRF1-pRb pathway may contributes to the dysregulation of spermatogonial proliferation in MA patients and potentiate the connections between male infertility and testicular cancer.In conclusion, the results in this study revealed the normal distribution pattern of meiotic interference among MLH1 foci and the role of miR-383 in spermatogonial proliferation. These results could help to further understanding of the underlying causes of the defects in homologous recombination and/or imbalance between proliferation and apoptosis of germ cells in NOA patients.