Study On The Function Of MicroRNA In Male Infertility

Infertility is becoming one of the most serious social problems troubling thousands of child-bearing age couples around the world. The World Health Organization (WHO) currently defines infertility as the inability of a sexually active couples (at least three times per month), not using contraception, to achieve pregnancy within one year. It is reported that10%-15%couples can not have babies via natural sexual activities, half of the causes are contributed by male factors. Although, as we have known, some factors which will lead to male infertility, such as inflammation of reproductive tracts, cryptorchidism, varicocele, sexual/ejaculatory inadequacy, obstruction of vas deferens, etc, most of etiologies are still unknown, which are defined as non-obstructive azooaspermia. What is more, patients with NOA have lower rates of spermatozoa retrieval, pregnancy and higher rate of DNA damage in spermatozoa compared with normal males. Therefore, to investigate pathogenesis and potential molecular mechanism of NOA will shed light and insights on clinical treatment of NOA patients.Spermatogenesis is a well-tuned and dynamic process occurring in seminiferous tubule, involving the transformation of spermatogonial stem cells to mature spermatozoa, including the stages of mitotic spermatogonia and meiotic spermatocytes. Later stage of spermatogenesis, transcription activity increases, however, translation activity is repressed. mRNAs transcribed at early stages of spermatogenesis are mediated and regulated by post-transcriptional factors, like microRNA (miRNA). MiRNAs are small non-coding RNAs with19-23nucleotides, regulate genes expression in two ways:degrade target mRNAs or repress translation of target genes by binding3’un-coding region (UTR) with themselves seed sequence at5’end of microRNAs.Male germ cells with aberrant DNA damage are the weighted factor contributing to male infertility. Mounting evidence shows that DNA damage in male germ cells impairs spermatogenesis and lowers fecundity. Our previous micro-array data shows that the expression of miR-383is notably down-regulated in testicular tissues of patients with MA, location of miR-383is expressed in early stages of spermatogenesis, like spermatogonia and spermatocytes, verified by ISH and real time RT-PCR. Due to mitosis and meiosis at early stages of germ cells, chromosomes are undergo remodeling with the high risk of DNA damage. However, is miR-383implicated in regulation of DNA damage during spermatogenesis? If yes, what is the exact mechanism of the regulation? The main purpose of this dissertation is to elucidate the molecular mechanism of DNA damage mediated by miR-383during spermatogenesis. First of all, we found the rates of DNA damage is almost tripled in testicular tissues of patients with NOA compared with that of normal control by testes spread. We found that miR-383inhibited the focal formation and abundance of yH2AX, which is the major marker of sites of DNA damage, with or without ultraviolet irradiation and cisplatin in testicular embryonal carcinoma(NT-2) cells. In addition, NT-2cells were remarkably sensitized to DNA damage reagent (cisplatin) by forcing expression of miR-383and silencing expression of protein phosphatase1, regulatory subunit10(PNUTS). Byconstructing Renilla luciferase reporters and co-transfecting miR-383and reporters in NT-2cells, we identified that PNUTS was a valid target ofmiR-383. Further results demonstrated that the repression of the phosphorylated form of H2AX by miR-383was due to independent depletion of PNUTS and cell cycle arrest. In conclusion, we found a novel function of miR-383in the DNA damage pathway. miR-383impairs the phosphorylation of H2AX by targeting PNUTS and inducing cell cycle arrest independently, as well as sensitizing NT-2cells to cisplatin.On the other hand, cryptorchidism is the weighted factor contributing to male infertility. The prevelance of cryptorchidism is about2%-4%of the population, and it occurs higher possibility in less developed countries. One of the main treatments to cryptorchidism is ochidopexy which is required to be conducted at the age of6months to2years. Some reports indicates that no matter how successful the surgery is, more than half of the patients with cryptorchidism will develop to male infertility. The detail and exact etiology of cryptorchidism mechanism is still unknown. Recent years, it is reported that the correlation between hormonal genesis axis, INL3/LGR8mutation between cryptorchidism, but the function of miRNAs in cryptorchidism is little known. Therefore, in order to investigate the function of miR-210in cryptorchidism, we build the mouse model with cryptorchidism.We found that miR-210is notably increased in human undescended testicles, as well as in mouse cryptorchidism model. There are significant DNA damage in undescended testicular tissues by immunofluorensce. And germ cells are separated from seminiferous tubule. In vitro experiment, we found that TNFa can induce the decrease of miR-210, which is inhibited by p65. However, the physiological consequence and molecular mechanism of increase of miR-210are about to do further investigation and research.