Regulation Of TET1-SP1 During Spermatogonia Self-renewal And Proliferation

Background Currently,the proportion of male infertility caused by primary causes is increasing year by year,mainly due to abnormal spermatogenic waves caused by abnormal spermatogonia.As the source of spermatogenic waves,abnormal spermatogenic cells can cause abnormal spermatogenic waves and eventually azoospermia,oligospermia and other spermatogenic disorders.Among them,the self-renewal of spermatogonia serves as the basis for maintaining the spermatogenesis process.A closely regulated balance between self-renewal and differentiation of spermatogonia can maintain continuous sperm production,which in turn can be influenced by many factors,among which epigenetic regulation plays an important role in the maintenance of spermatogonia self-renewal.Epistatic modifications are involved in the process of spermatogenesis and affect the fate of spermatogonia through the regulation of reproduction-specific genes.Tet methylcytosine dioxygenase 1(TET1)is an important epistatic modifying enzyme that hydroxylates the methylation sites of specific genes,enabling the process of DNA demethylation and regulating gene expression,or by forming protein complexes with certain proteins,thereby regulating the transcriptional activation or silencing of genes.However,the key differential genes affected by the specific action of TET1 in spermatogonia and the mechanism of interaction between key differential genes are not clear.Objective To investigate the effects of TET1 on the self-renewal and proliferation of spermatogonia,to identify the key genes and their regulatory pathways that influence the specific effects of TET1 on spermatogonia,and to provide new ideas for studying spermatogenesis,revealing the causes of spermatogenic disorders and elucidating their mechanisms.Methods1,The biological properties of TET1 overexpression group(MYC-TET1)and control(MYC)spermatogonia were detected using quantitative real-time fluorescence PCR(QRT-PCR)analysis,western blot(WB)analysis,immunofluorescence analysis and flow cytometry.2,Analysis of key differential genes affected by TET1-specific effects using transcriptome sequencing.BSP methylation assay,Co-IP,protein sequencing and protein interaction analysis were used to verify the mode of TET1-SP1 interaction and the mechanism of interaction between key differential genes.3,Constructed SP1 overexpression stable cell line by lentivirus infection,followed by QRT-PCR analysis and WB analysis to detect the biological characteristics of SP1overexpression group(p CDH-SP1)and control group(p CDH)spermatogonia.4,SP1 expression was interfered by transfection of si RNA,and the biological properties of SP1-interfered group(SP1~-)and control group(NC)spermatogonia were examined by using QRT-PCR analysis,WB analysis,immunofluorescence analysis,flow cytometry,TMT proteomics analysis and protein interaction analysis,as well as the analysis of the interaction mechanism.5,The spermatogenic disorder model rats were prepared by using white elimination solution,and the testes were transplanted with SP1 overexpressing cells after 4 weeks,and testicular tissues were collected after 8 weeks for morphological examination of spermatogenic tubules,which were verified by hematoxylin-eosin staining(HE staining)and immunohistochemistry for spermatogenic cell animal experiments.Results1,TET1 is involved in the regulation of expression of spermatogonia self-renewal and affects the expression of key differential gene SP1.2,SP1 overexpression promotes cell proliferation,accelerates the process of spermatogenesis and promotes sperm production.3,SP1 expression down-regulation can reduce the transcriptional activity of spermatogonia,decrease proliferative activity,induce cell cycle arrest,and make cells tend to apoptosis.4,TET1-SP1 maintains the self-renewal of spermatogonia and promotes cell proliferation.Conclusion The results show that TET1 maintains self-renewal of mouse spermatogonia,promotes cell transcription and enhances cell activity,and identifies the key differential gene SP1 affected by the specific action of TET1,and the regulatory role of SP1 on spermatogonia proliferation,providing a scientific basis for studying spermatogenesis,revealing the causes of spermatogenic disorders and elucidating their mechanisms.