M6A Is Involved In DEHP Exposure-induced Mouse Spermatocyte GC-2 DNA Damage Response

Bis(2-ethylhexyl)phthalate(DEHP)is an important part of phthalates,which is an environmental endocrine disruptor.According to previous reports,DEHP exposure can cause male reproductive toxicity and impair spermatogenesis.The previous studies on male reproductive toxicity caused by DEHP exposure mainly focused on the effects of DEHP exposure on the oxidative stress and apoptosis of testicular tissue and spermatogenic cells,and the research on sperm quality.The existing understanding of the mechanism of DEHP-induced spermatogenesis is still very shallow,and it cannot reveal the impact of DEHP exposure on spermatogenic cells.For example,meiosis is an important part of the spermatogenesis process,but the effect of DEHP exposure on the physiological processes of spermatocytes is not clear.N6-methyladenosine(m6A)as a post-transcriptional modification is essential for regulating gene expression,and studies have shown that m6 A regulates the normal progress of spermatogenesis and is indispensable for spermatogenesis.However,the regulatory role of m6 A in spermatocyte damage caused by DEHP exposure remains to be explored.This study intends to investigate the role of m6 A in DEHP-induced genotoxicity of mouse spermatocytes through in vitro cell experiments,in order to further study the toxic mechanism of DEHP-induced spermatogenesis.By establishing a DEHP exposure model of mouse spermatocytes GC-2,observing the effect of DEHP on the DNA replication activity of GC-2 cells,and combining the detection of cell cycle and intracellular reactive oxygen species(ROS)to understand the effects of DEHP on GC2-Genotoxicity of cells,and explore the corresponding toxicity mechanism.On this basis,the level of DNA double-strand breaks(DSBs)in GC-2 cells and the possible repair pathways of the cells to DSBs were detected,and the effect of DEHP on the genome of GC-2 cells was further explored.And by observing the changes in the level of m6A methylation modification of total RNA in the GC-2 cell model exposed to DEHP and the effect of DEHP exposure on the expression of m6A-related proteins in GC-2 cells,and analyzing the relationship between DNA damage-associated RNAs m6A modification level changes and DEHP-induced DDR(DEHP-induced DDR)we explored the mechanism of RNA epigenetics in DEHP-induced genotoxicity in GC-2 cells.The main contents of this research are as follows:1.DEHP exposure induces DNA double-strand breaks in GC-2 cellsIn this study,a mouse spermatocyte GC-2 model was used to give 50μM,100μM,200μM,300μM,and 400μM DEHP exposure for 24 hours.Through the detection of cell proliferation and viability,the general toxic effect of DEHP on GC-2 cells was explored;Combined the Ed U experiment,this research analyzed the effect of DEHP exposure on the DNA replication ability of GC-2 cells.Flow cytometry and laser confocal microscopy was used to detect DEHP-exposed ROS levels in GC-2 cells.q PCR experiment was used to detect m RNA expression of cell cycle and DNA damage response(DDR)related gene.Western Blot and cell immunofluorescence experiments are used to detect the expression and Intracellular localization of DNA damage response related repaire factors.The experimental results showed that DEHP exposure significantly inhibited the proliferation ability of GC-2 cells,and flow cytometry and laser confocal microscopy showed that DEHP induced a large amount of ROS in GC-2 cells,indicating that the level of intracellular oxidative stress increased.Ed U experimental results showed that DEHP exposure caused the DNA replication ability of GC-2 cells to be significantly inhibited,indicating that DEHP may have a detrimental effect on the genomic stability and integrity of GC-2 cells.In view of this,this study was further to tested the cell cycle,and it was found that DEHP exposure caused the GC-2 cell cycle to be blocked in G0/G1 phase,and the number of cells transitioning to S phase was significantly reduced,indicating that in order to prevent DEHP-induced genome damage from being passed down,through self-regulation cell cycle,GC-2 cells block cell cycle in G0/G1 phase to repair damaged DNA.After further testing the damage of DEHP to the genome,it was found that DEHP exposure induced DSBs in GC-2 cells.The expression of two repair factors,RAD51 and XRCC5,was up-regulated due to DEHP exposure.They appeared co-localization with the DSBs marker γH2AX indicating that the two repair pathways of homologous recombination(HR)and non-homologous end binding(NHEJ)are involved in the repair of DEHP-induced DSBs of GC-2 cells.2.m6 A is involved in DEHP-induced DNA damage response of GC-2 cellsStudies have shown that exposure to environmental endocrine disruptors can affect the level of m6 A methylation modification in tissue and cells.That m6 A was involved the male reproductive toxicity induced by DEHP and its metabolite mono(2-ethylhexyl)phthalate(MEHP),but the specific mechanism about that is still unclear.In this study,the Dot Blot experiment was used to detect the m6 A methylation modification level of the total RNA of GC-2 cells exposed to DEHP.q PCR and Western Blot were used to detect the m RNA and protein expression levels of m6A-related regulatory factors.The intracellular localization of m6 A modified RNA,METTL3,YTHDC1 with γH2AX was analyzed by the immunofluorescence experiment to demonstrating the role of m6 A in DEHP-induced DDR of GC-2 cells.This study found that 100μM and 200μM DEHP exposure significantly increased the level of m6 A methylation modification of total RNA,and the expression of m6 A methyltransferase METL3 and m6 A binding protein YTHDC1 were also significantly upregulated by DEHP exposure.The results of the study showed that METL3 was recruited to DEHP-induced DNA damage sites due to DEHP exposure,which indicated that METLL3 may be involved in the repair process of DEHP-induced DSBs by GC-2 cells.As a regulator of m6 A methylation modification,we speculate that m6 A methylation modification may be involved in the repair process of DEHP-induced DSBs.After detection,it was found that the m6 A methylation modification RNA in the nucleus and the DSBs marker γH2AX formed a co-localization.This indicates that m6 A methylation-modified RNA may play an important role in DEHP-induced DDR in GC-2 cells and participate in the repair of DSBs.YTHDC1,as the m6A binding protein,is responsible for the recognition of m6A methylation modification sites and mediates the physiological functions of m6A.The experimental results show that DEHP exposure significantly up-regulates the expression level of YTHDC1 and co-localizes YTHDC1 andγH2AX,which indicates that YTHDC1 It may be recruited to the DNA damage site due to DEHP exposure and mediates the repair of DEHP-induced DSBs by m6A methylated RNA of GC-2 cells.The results of this study indicate that DEHP exposure induces DSBs in GC-2 cells,the level of m6 A methylation modification of total RNA increases due to the DEHP,and the m6 A methylation modification of DNA damage-associated RNAs and m6 A regulatory factors may be involved in DEHP-induced DDR to participate in the repair of damaged DNA in GC-2 cells.