Epigenetic Modification Of Tet1 In Dairy Goat Male Germline Stem Cells’ Self-renewal And Proliferation

The dairy goat is one kind of important livestock in China, while the unstable culture system and low reprogramming rate are obstacles to optimize new varieties of dairy goat. The study of dairy goat male germline stem cells(mGSCs) are increasing. The epigenetic modification plays key role in spermatogenesis, DNA methylation dynamic in specific gene regions is important in sustaining normal spermatogenesis. Ten-eleven translocation 1(Tet1) is a key demethylase. The dynamic of Tet1 is involved in epigenetic modification during spermatogenesis, the study of epigenetic modification in mGSCs is good for the optimization of adult stem cell culture system in vitro, and the improvement of sperm quality, heredity and breeding of selected livestock.In our study, we firstly analyzed the expression pattern of Tet1 and 5hmC in tissues and dairy goat testis at different stages, and the expression pattern of dimethyl histone H3 lysine 9(H3K9me2), tri-methyl H3 lysine 9(H3K9me3) and tri-methyl H3 lysine 27(H3K27me3) in the testis and cultured mGSCs. Secondly, we analyzed functional domains of Tet1 protein and mouse Tet1(mTet1) overexpression in goat mGSCs, then we analyzed the morphology, gene expression, DNA methylation and histone methylation dynamic in mGSCs and mGSC-mTet1 cells. Finally, we analyzed the function by in vivo transplantation and explored the functional mechanism of Tet1 in goat mGSCs. The results showed as follows:1 Compared with other tissues, Tet1 protein showed relatively low level in testis, in mGSCs, Tet1 protein showed relatively low level in adolescent-stage goats, whereas Tet1-positive mGSCs were localized within seminiferous tubules of adult stage and sertoli cell. 5hmC has almost the same distribution as Tet1. The H3K9me2 and H3K9me3 showed different expression pattern, H3K9me2 level increased in early pachytene and then began to decrease. It reached the peak in adolescent stage and began to reduce in adult stage. H3K9me3 amount increased more than H3K9me2 and was maintained until adult-age dairy goats. The mGSCs cultured in vitro showed almost the same tendency as in vivo. Tet1 and 5hmC were absent in differentiated mGSCs cultured in vitro, which showed that Tet1 was involved in mGSCs self-renewal. Moreover, H3K9me2 and H3K9me3 were detected in mGSCs cultured in vitro, which showed higher level in the differentiated cells.2 Bioinformatics analysis showed that the Tet1 has the same conservative domains in different species, we used mTet1 to screen immortalized dairy goat mGSC cell line bearing mTet1(mGSC-mTet1) cells with optimal Dox concentrations(0.50 μg/mL), which showed better proliferation ability than wild-type mGSCs, the mGSCs-specific markers such as Gfra1, PCNA, CCND1, Ki67, ETV5 and REC8 were dynamic and endogenic Tet1, Tet2 were upregulated. The histone methylation level of H3K9me2, H3K9me3 and H3K27me3 were decreased, H3K9me3 changed its location from dotted to uniform distribution, H3K27me3 changed its location from uniform in the nuclear to perinuclear distribution, which showed significant epigenetic modification.3 In order to search the function in vivo, we used mTet1 to screen lentiviral vector with mTet1 stably expression. The embryoid body(EBs) was made and made it towards spontaneous differentiation, it showed that the mGSC-pCDH-mTet1 cells got better EBs formation and lower differentiation rate. Cells were transplantated into the seminiferous tubule of mice with reproductive defects, 4 weeks later, the analysis of mGSCs-specific markers such as PGP9.5, VASA, PCNA and Ki67 were positive, which showed that mGSC-pCDH-mTet1 cells in seminiferous tubule sustained the self-renewal ability and proliferative capability.4 The assay of Tet1 functional mechanism in goat mGSCs showed that Tet1 could bind to promoters to demethylate the genes, Tet1 could also upregulate JMJD3 to decrease the amount of H3K27me3, which also showed suppress to MEK-ERK pathway. Besides, Tet1 could work with other proteins by forming protein complex with PCNA to increase the proliferation of mGSCs, it also changed the histone acetylation level by forming complexes with Hdac1.Conclusively, the results provided comprehensive analysis of Tet1 during dairy goat spermatogenesis and gene regulation of mGSCs mediated by Tet1, which showed not only better research ideas about Tet1 modification, but also stronger footing of epigenetic modification in livestock reproduction.