Study On Testis Spermatogenesis-related Gene

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 and Oligosperm. More and more evidences suggest that genetic factors such as mutations of relevant genes are involved in spermatogenic failure. Spermatogenesis and its regulation is a complex process mediated by multiple genes. Identification the regulators and mechanism of spermatogenesis might help exploring new therapy approaches for male infertility and male contraception. Spermatogenesis is an important characteristic in multicellular animals, yet the lack of in vivo and in vitro models limit our understanding of its molecular mechanism. Identifying new genes involved in spermatogenesis and studying their functions may help understanding of male infertility and the development of new approaches on both male infertility and male contraceptive.We conducted preliminary study on the functions of several spermatogenesis-related genes and report here as three chapters.Chapter 1 mutation screening on new spermatogenesis-related genes BCLT, GSSTP1,KLHL10, MET1 and hGPAT4BCLT, GSSTP1,KLHL10, MET1 and hGPAT4 are newly cloned spermatogenesis-related genes.But it is not understood yet whether its genetic variations are associated with idiopathic male infertility. Using PCR, DHPLC and sequencing techniques, we collected blood samples from patients with idiopathic azoospermia or severe oligozoospermia, and conducted mutation screening on the above 5 genes on 325 of them with comparison to 100 normal controls.In the DHPLC assays on 12000 PCR products, we found 8 new SNPs but no mutation or deletion. It is therefore concluded that mutations in these genes are not likely the major contributor to those patients with azoospermia and oligospermia in Chinese population.Chapter 2 The clone and functional exploration of spermatogenesis-related gene GPAT4 in mice spermatogenesis cellsWe cloned spermatogenesis-related gene GPAT4 in mice spermatogenesis cells (and human homologue hGPAT4) from EST BE644543 in our mice testis apoptosis model.Bioinformatics analysis reveals that mice GPAT4 locates at 8A1.3.,ranges 3838bp including 1371bp complete ORF.The gene codes 456 amino acids. The non-secretary protein locates in cytoplasm, has phosphate acyltransferase domain and belongs to phosphate acyltransferase family, capable of synthesizing lipid. The human homologue hGPAT4 locates at 8 p11.21., comprises a 2592 bp cDNA and codes 456 amino acids.Similarly it has phosphate acyltransferase domain thus belongs to the phosphate acyltransferase family. RT-PCR and real-time quantitation PCR on various tissues from mice revealed that GPAT4 are highly expressed in testis tissues.In situ hybridization demonstrated that GPAT4 mRNA is mainly expressed in spermatocytes and round spermatids.We collected total testis RNA from mice along development course and analyzed using real-time quantitation PCR and found that GPAT4 was first expressed in mice at 2 weeks postnatally. Expression was abundant from 3 weeks, plateaued at 5-6 weeks and was then maintained at a high level in the adult. In pEGFP-C3,we observed that GPAT4 expresses in GC-lspg, consistent with the prediction by bioinformatics.After transfection with constructed pET-28a(+)/GPAT4 prokaryotic expression plasmid, there was specific and efficient expression of target protein in E. coli following IPTG-induced. ISH revealed that GPAT4 gene expressed abundantly in spermatocytes and round spermatids during meiosis division,but not in elongated spermatids in spermiogenesis phase. Also, GC-lspg cells showed a marked increase in proliferation after over-expression of GPAT4 gene and the percentage of cells in G0/G1 phase decreased and S phase increased in our cell cycle analysis.GPAT4 gene may play an important role in the spermatogenesis process through its catalyzed products lysophosphatidic acid. However, additional experiments are necessary to unravel the detailed molecular mechanism.Chapter 3 Functional study of spermatogenesis-related gene mTSARG3 in mice spermatogenesis cellsThe functions of some spermatogenesis-related genes in mice spermatogenesis cells that we newly identified have not yet fully been understood. In this study we focus on mTSARG3 in our in vitro and in vivo models.It has been shown that the ORF of TSARG6 and mTSARG3 are sharing 85% similarity, including 8 exons and 7 introns among which the amid 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 38th base of exon-2 in TSARG6, resulting a serin to proline mutation at the 36th amino acid.We propose that a study on the molecular and cellular changes after site-directed mutagenesis at exon-2 of mTSARG3 gene might shed light to the understanding of mTSARG3 normal functions and facilitate illustration of its role in spermatogenesis.We constructed mTSARG3 mutant gene by site-directed mutation at 38 codon of exon-2,and subsequently cloned it into eukaryotic expression plasmid pcDNA3.1/Hygro(-).The plasmid was transfected with GC-1 spg via Cationic Liposome.GC-1 spg cell lines carried wild type or mutant mTSARG3 gene were established after hygromycin B screening. We subsequently studied the characteristics of this cell line and the anti-apoptosis role of mTS ARG3 gene in spermatogenesis using heat shock test. Western blot revealed that both wild type and mutant cell lines expressed mTSARG3 protein.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, suggesting there be no anti-apoptosis affect from mTSARG3 gene.When FCM assay were performed for G1,S and G2 cells, statistics signs showed no significant differences among mTSARG3 wild type cells, mTSARG3 mutant type cells, GC-1 spg cells and empty vector cells.It suggested that mTSARG3 gene has no the capability of promoting cell proliferation by regulating the cell cycle.In summary, we screened BCLT,GSSTP1,KLHL10,MET1,hGPAT4 genes by using PCR, DHPLC and sequencing and found 9 new novel SNPs but no mutation or deletion. We cloned two new genes GPAT4/hGPAT4 and found that they are new members of the phosphate acyltransferase family, might be involved in maintenance of normal spermatogenesis in testis.GPAT4 are highly expressed in testis tissues.In situ hybridization demonstrated that GPAT4 mRNA is mainly expressed in spermatocytes and round spermatids.GPAT4 gene may play an important role in the spermatogenesis process through its catalyzed products lysophosphatidic acid.mTSARG3 mutant gene was constructed by site-directed mutation at 38 codon of exon-2,and subsequently cloned it into eukaryotic expression plasmid pcDNA3.1/Hygro(-).Heat shock test results suggested that there be no anti-apoptosis affect from mTSARG3 gene and the mTSARG3 gene might be involved in developmental regulation rather than heat shock response.