The Primary Investigation Of Several Spermatogenesis-related Genes

Infertility happens on about 15% of married couples, among which male infertility contributes to around 50% of the etiology. Although there are a variety of mechanisms inducing male infertility, spermatogenic failure is considered to be the dominant one, predominantly manifesting as azoospermia, oligospermia, teratozoospermia. Spermatogenesis is a complex process of cell differentiation involving spermatogenic cell growth, division, differentiation and signal transduction and so on, resulting in a significant number of molecular and morphological changes, and ultimately the formation of mature sperm. This process requires a series of precise regulation of gene expression. The genetic research of male infertility found that many genes in both autosomal chromosomes and sex chromosomes play an important role in spermatogenesis, the genes in Y chromosome has been studied and separated the candidate azoospermia gene family: RNA binding motif (RBM) gene and Gene deleted in azoospermia (DAZ), a number of autosomal genes associated with spermatogenesis are reported , including DAZLA (DAZ homologous autosomal gene), CREM, HSPA2 (human homologous gene of HSP70-2); Proteomics study also found a number of spermatogenesis-related genes by comparing normal and abnormal sperm, In addition, due to the specific disease of male infertility, there are not many patients, many spermatogenesis-related genes reported are mostly derived from animal experiments, gene knockout mouse model is an important research tool for spermatogenesis, many spermatogenesis-related genes clone from the knockout mice. Although the discovery of so many genes associated with spermatogenesis, the process of the molecular mechanism of spermatogenesis remains to understand.At present, on the one hand, further study on the function of spermatogenesis-related gene cloned, on the other hand make full use of existed method of cloning to find the relevant gene and to explore the role of new genes in the process of spermatogenesis, which not only helps to understand the molecular mechanism of male infertility, but also lays a theoretical basis on the treatment of male infertility and the development of new contraceptive drugs. In this study, several genes related to spermatogenesis are preliminary studied. Content is divided into two parts, the main experimental methods and experimental results are as follows:Chapter 1,2 Search for mutations of gene GOPC,Hrb,Csnk2a2, SPATA16 involved in globozoospermiaGlobozoospermia is a kind of male infertility due to spermatogenic failure, its incidence rate is less than 0.1%, the main feature of the disease is acrosomal deformities, according to the missing degree of acrosome globozoospermia is divided into typeI and typeII, globozoospermia typeI show the complete absence of acrosome, globozoospermia typeII performance as a residual element of the existence of the acrosome. Due to the lack of normal acrosome, globozoospermia patients are sterile. Previous studies showed that globozoospermia is one kind of hereditary diseases, especially a number of reported cases of brothers with globozoospermia further support the view, although there are different gene knockout mouse models resemble in human globozoospermia, the molecular mechanism of globozoospermia remains to elucidate. Previous research has reported three kinds of genes such as GOPC, Hrb, Csnk2a2 knockout mice have symptoms similar to human globozoospermia, in addition,homozygous mutation in SPATA16 is found in a Dutch family with human globozoospermia patients.To study the relationship of the candidate genes and globozoospermia by gene scanning,the three patients as the test group and sperm donors as the control group in this study,the candidate genes(GOPC,Hrb,Csnk2a2,SPATA16) were determined by PCR and DNA sequencing,and the effect of the candidate genes on globozoospermia were analyzed. The scanning results of candidate genes in the test group are mostly polymorphism,and have much in common with the identified polymorphism in the past.The concordance between the scanning polymorphism in the test group and the reported polymorphisms shows the identified polymorphisms are not resulted from the difference of district and race.Further investigation has yet to be performed into the relationship of these polymorphisms and globozoospermia when many patients with globozoospermia are found.Chapter 3 Functional study of spermatogenesis-related gene mTSARG3 in mice spermatogenesis cellsThe functions of some testis and spermatogenesis cell apoptosis related genes in mice spermatogenesis cells that we newly identified have not yet fully been understood. The study focuses on the function of mTSARG3. It has been shown that the ORF of TSARG6 and mTSARG3 are sharing 85% similarity, including 8 exons and 7 introns among which the middle 6 exons are identical in length. The two genes both code 36KD protein, sharing 89% similarity. In our screening on patients diagnosed as idiopathic male infertility, a patient with cryptorchidism was found carrying a T-C miss-sense mutation at the 38^th base of exon-2 in TSARG6, resulting a serin to proline mutation at the 36^th amino acid.Previous studies of this study complete the construction of eukaryotic vectors containing both normal mTSARG3 gene and mutant mTSARG3. Through transfection of mouse spermatogenic cell line GC-1 spg establish the expression of wild-type gene and the expression of mutant gene mTSARG3 GC-1 spg cell lines.However heat shock test results revealed there were no significant differences of the apoptosis cells between mTSARG3 mutant type cell line and that in wild type, When FCM assay were performed for G1, S and G2 cells, statistics signs showed no significant differences among mTSARG3 wild type cells and mTSARG3 mutant type cells, We infer that a single point mutation seems not enough to affect the function of the gene, so we are ready to build the gene without specific domain to further study the function of gene...