Study On The Effect Of TET1 And Mechanism On Male Reproductive Injury Induced By Low Dose Bisphenol A

BackgroundStudies have found that exposure to BPA in animals and humans has many adverse effects on the reproductive system.Our group has previously confirmed that BPA exposure can induce the increase of the total methylation level in mouse GC-2 cells.As a very important demethylase,TET1 is closely related to a variety of biological processes.Combined with the latest literature reports,we speculate that TET1 may be involved in the process of germ cell injury induced by BPA exposure through regulating the dynamic balance of DNA demethylation and methylation of downstream molecules.Its specific mechanism needs to be further studied.In this study,three kinds of cells related to mouse reproductive system combined with human samples were used for experimental design to explore the epigenetic regulation and its specific mechanism of TET1 mediated key molecules of downstream regulatory pathway in the process of male germ cells injury induced by BPA exposure.This study will provide a new idea for finding more sensitive molecular markers of male reproductive damage from the epigenetic point of view.Objective1.To screen BPA toxicity related genes,explore possible pathways based on CTD database.2.To explore the role and regulatory mechanism of TET1 in male reproductive damage induced by low dose BPA.Methods1.The genes related to BPA toxicity were screened by CTD database,and these genes were analyzed by enrichment analysis.2.Mouse GC-2 cells,TM3 cells and TM4 cells were treated in the final concentration of 0μM,20μM,40μM and 80μM BPA for 72h.The survival rate of cells was measured by CCK-8 method.3.The survival rate of cells was measured by CCK-8 method.The intracellular calcium levels of GC-2 cells treated with different doses of BPA were observed by laser confocal microscope.q RT-PCR and Western blot were used to detect the expression level of these molecules.4.The cell model of differential expression of TET1 gene was established in GC-2cells.The changes of apoptosis,cell cycle and intracellular calcium level were detected by flow cytometry.5.hMeDIP and MeDIP experiments were used to detect the（hydroxy）methylation level of genes after TET1 overexpression/interference.6.The correlation between gene expression and semen quality parameters was analyzed by SPSS25.0.Results1.The GO functional enrichment analysis showed that BPA toxicity related genes were involved in molecular biological processes such as reproductive system development and reproductive structure development.These genes were significantly enriched in HIF-1signal pathway and PI3K-AKT signal pathway（P<0.01）.2.In GC-2 cells exposed to BPA,the m RNA expression levels of PI3K and JAK were significantly down regulated,while P53 and BCL2 were significantly up-regulated.The protein levels of PI3K and BCL2 were significantly down regulated,while P53 was significantly up-regulated（P<0.05）.In BPA treated TM3 cells,the m RNA expression levels of PI3K and HIF1-αwere significantly down regulated,the m RNA expression levels of BCL2 and BAX were significantly up-regulated（P<0.05）.In TM4 cells exposed to BPA,the m RNA expression level of P53 was significantly down regulated,the m RNA expression levels of HIF1-α,P300/CBP and BAX were significantly up-regulated（P<0.05）.3.The expression levels of TET1 gene and key molecules of Catsper calcium signaling pathway were significantly down-regulated in mouse reproductive system cell injury induced by low-dose BPA.（1）With the increase of exposure dose,the survival rate of three kinds of cells decreased significantly（P<0.001）.（2）The m RNA expression of TET1 was significantly down-regulated in GC-2 and TM3 cells exposed to BPA after 72hours;After 72 hours of exposure to BPA in TM4 cells,the m RNA expression of TET1was significantly up-regulated（P<0.05）.（3）With the increase of the dose of BPA,the intracellular calcium level of GC-2 cells decreased significantly（P<0.01）.The expression levels of Catsper1-4 showed a downward trend.4.TET1 gene participated in the process of cell injury in mouse reproductive system induced by low dose BPA through promoting cell proliferation and increasing calcium concentration.（1）The proliferation rate increased significantly in GC-2 cells with overexpression of TET1 gene（P<0.001）.（2）Compared with GC-2 cells in control group,the apoptosis was inhibited in TET1 overexpression group（P<0.001）.（3）The intracellular Ca²⁺was significantly increased in the TET1 overexpression group（P<0.001）.The expression levels of Catsper1-4 also increased significantly（P<0.05）.While in the TET1interference group of GC-2 cells,the Ca²⁺level was significantly down-regulated（P<0.001）.The expression levels of Catsper1-4 are also significantly reduced（P<0.05）.5.TET1 involved in the process of cell injury of mouse reproductive system induced by low dose of BPA Through hydroxymethylation modification.（1）After overexpression of TET1 gene,the hydroxymethylation levels of Catsper1 and Catsper3 were changed.After the low expression of TET1 gene,the methylation levels of Catsper2 and Catsper4were changed（P<0.05）.（2）In the 80μM BPA group,the relative growth rate of cells in the TET1 gene overexpression group was higher than the control group（P<0.05）.The relatively growth rate was lower in the TET1 gene knockdown group than the control group（P<0.01）.6.Relationship analysis between TET1 gene expression and human semen quality parameters.（1）The expression level of Catsper1-4 was positively correlated with the expression level of TET1 gene in spermatozoa（P<0.01）.（2）The sperm concentration,the proportion of fast forward movement and the total motility of sperm in the group with qualified forward movement ratio were significantly increased（P<0.05）,while the resting sperm was significantly decreased（P<0.001）.Conclusion1.Different doses of BPA exposure induced different degrees of damage to GC-2,TM3 and TM4 cells,and PI3K-AKT signaling pathway may play an important role in the process.2.Low dose BPA exposure could induce apoptosis of GC-2 cells,decrease the level of intracellular calcium and down-regulate the expression of Catsper1-4.3.It is found for the first time that TET1 gene regulates the expression of Catsper calcium signal pathway related molecules through its hydroxymethylation modification and affects the calcium level,thus participating in the process of low-dose BPA induced male reproductive toxicity.