| The mammal male germ cell development is a complex biological process. This process is regulated by multi-level precise regulation in vivo and in vitro, and the correct expression of related genes of germ cell development in the whole process plays an important role. Studying the genes in regulation and related functions during development of male germ cells will help us to further elucidate the molecular regulation mechanism of spermatogenesis, and provide new ideas for targeting to solve the problem of livestock breeding and male sterility.Heterogeneous nuclear ribonucleoprotein K(Hnrnpk) participates widely in chromosome repackaging, transcription, shear and the translation process, has very important cell functions, is closely associated with spermatogenesis. DNA methylation reconstruction was accompanied by spermatogenesis process, and abnormal DNA methylation regulated by DNA methylation enzyme(Dnmts) influences germ cell development and spermatogenesis. So we chose Hnrnpk and Dnmts as the breakthrough point of the research, the molecular structure characteristics, expression distribution and functions of Hnrnpk have been carried on the preliminary discussion, to reveal the roles playing in the process of proliferation and differentiation of spermatagonial cells. At the same time, to set up rats Dnmts dynamic expression profiles in different developmental periods, and explore the regulation factors behind. The main research results are as follows:(1) We isolated the pig Hnrnpk gene, its full length c DNA of 2712 bp, contains 1392 bp open reading frame, encoding 463 amino acids, initiation codon is located at 230-232 bp, termination codon is located at 1622-1624 bp, the 5’UTR contains 229 bp, the 3’ UTR contains 1091 bp. According to the sequence alignment and analysis of genetic structure, inferred that the pig Hnrnpk gene length of about 12.3 kb, contains 16 exons and 17 introns, connected to the gene exons and introns bases in accordance with laws of GT/AG. Through RACE and further PCR amplification, found there are at least 8 different transcriptions in the pig, is mainly due to the alternative splicing of exon 1, 8 and 17. And eight different transcripts are corresponding to four kinds of proteins.(2) Hnrnpk had relatively high expression in testis, skeletal muscle and fat tissue; lung, kidney, ovary, and brain tissue took the second place; and the expression is relatively low in heart and liver tissue. And Hnrnpk expressed higher in early development periods of swine testis tissue, and the results have been further validated in rats from different development periods. Orientation distribution results showed that Hnrnpk mainly locate in spermatogonias, spermatocyte nucleus and spermatid cytoplasm, Sertoli cells and myoid cells in three different development periods in swine testicular tissue seminiferous tubule. All the results may suggest that Hnrnpk will play an important role in the process of germ cell development.(3) 3 si RNAs were designed and synthesized, transfection to C18-4 and GC-1spg cells, which get si RNA-Hnrnpk interference efficiency is above 80%. Cell phenotype results indicated that cells numbers with si RNA-Hnrnpk transfection group was declined, discrete distribution in the field of vision, and more floating cells. Compared with si RNA-NC and Mock cell transfection group. Transfection group cells was significantly lower than the control group after transfection for 48 h through cell counting method(P < 0.05), and Cell Titer-Glo cell proliferation test results showed that Hnrnpk downregulation correlated well with the decrease in cell viability, and apoptosis detection showed that TUNEL positive cells was significantly higher than that of control group(P < 0.05). These data suggest that Hnrnpk knockdown can inhibit the proliferation and promote the apoptosis of GC-1spg cells.(4) Differentially expressed genes from the knocked down GC-1spg transcriptome sequencing found 24 genes were related to cell proliferation, 15 genes were involved in the regulation of cell apoptosis. The positive control proliferation genes Icmt, Oaz1, Nanog, Fntb expression levels were decreased,and the negative control proliferation genes Nos2, Ptpn6, Serpine1, Spry1 were raised. In addition, the antiapoptotic genes Xiap, Rap1GAP1, Sirt1 were decreased,and the apoptosis genes Caspase2, Caspase3 were raised. These results suggested that Hnrnpk may regulate the proliferation and apoptosis related genes expression to exert its biological functions.(5) The dynamic expression profile of Dnmts from the different development period showed that expression levels of Dnmt3 a and Dnmt3 l were abundant before birth and were present at the highest levels at 18.5 days postcoitus(18.5 dpc) testes, which was much higher than at any other time point(P < 0.01), and gradually decreased from 0 day postpartum(0 dpp) to 90 dpp, and Dnmt1 and Dnmt3 b expression reached a peak after birth(P < 0.01), and then gradually reduced until adulthood. Results suggested that Dnmt3 a and Dnmt3 l may coordinate with each other to play a role in DNA methylation reconstruction process, and Dnmt3 b and Dnmt1 may affect DNA methylation maintaining.(6) mi R29 expression trend is opposite to that of Dnmt3 a and Dnmt3 b through LHCD detection. GC-1spg cell line was transfected by mi R29 inhibitor validated that the expression of Dnmt3 a and Dnmt3 b was upregulated by mi R29 inhibitor.The results suggested that Dnmts may be mediated by mi R29 in playing a role in the development of rat testis.In a word, Hnrnpk expressed in the process of male germ cell development, which could decide the cells fate by regulating the expression of genes related proliferation and apoptosis. Dnmts presented dynamic expression pattern in rat testicular tissue development, Dnmt3 a and Dnmt3 l interaction may coordinate to establish DNA methylation patterns, and Dnmt3 b and Dnmt1 may participate in DNA methylation. At the same time, mi R29 may play the roles in the process through regulating negatively the expression levels of Dnmts. |
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