Effect Of RNA Methylation M6A Modification On The Environmental Endocrine Disruptors Induced Immature Testicular Development Injury

Objective:To study the role of N~6-methyladenosine(m6A)modification in immature testicular damage induced by Di-(2-ethylhexyl)phthalate(DEHP)exposure from the perspective of RNA methylation epigenetics,and investigate the mechanism of Fat mass and obesity-associated protein(FTO)-mediated m6A modification in Leydig cell injury induced by Mono-2-ethylhexyl phthalate(MEHP),which is the main metabolite of DEHP.Methods:The first part:Male Sprague-Dawley(SD)rats at the prepuberty stage from postnatal day 22(PND 22)to PND 35,were administrated by oral gavage with corn oil in the control gourp,and 250,500mg DEHP per kg body weight per day in D250 group and D500 group,respectively.(1)Testicular histomorphology,testosterone concentrations and protein expressions of spermatogenesis markers were tested with hematoxylin eosin staining,enzyme-linked immunosorbent assay(ELISA)and western blot(WB),and sperm deformity rates were detected using epididymides in adult SD rats at PND 90,to evaluate DEHP-induced immature testicular injury.(2)Total antioxidant capacity,Nrf2-mediated antioxidant signaling pathway and proteins expressions related to apoptosis were detected by colorimetry,WB and in situ apoptosis detection to determine the oxidative stress state and apoptosis in the testes.(3)The changes of m6A modification in total RNA and the expressions of important regulators genes were detected by colorimetry and quantitative real-time PCR(qPCR)respectively,and the enrichment of m6A modification in the mRNA of Nrf2 was analyzed using methylated RNA immunoprecipitation(MeRIP)qPCR,to detect the role of m6A modification in DEHP-induced testicular development damage.The second part:TM3 mouse Leydig cells were treated with gradient concentrations of MEHP and FB23-2(the inhibitor of FTO)for 24 hours,respectively,and CCK8 assay was used to detect the cell viability of TM3cells and determine the appropriate concentration for this study.(1)Effects of 200μM MEHP on the expressions of m6A modification regulators in TM3 cells were detected with qPCR and WB.(2)0.1%DMSO,200μM MEHP and 20μM FB23-2 were used to treat TM3 cells,and they were divided into control group,MEHP group and FB23-2 group,respectively.Testosterone concentrations in the supernatant,cell apoptosis and level of m6A modification of TM3 cells were detected with ELISA,flow cytometry and colorimetry respectively,to evaluate the damage effect of MEHP and FB23-2 on Leydig cells.(3)Transcriptome sequencing combined with bioinformatics analysis were carried out to evaluate the molecular mechanism involved in the damage effect of MEHP and FB23-2 on Leydig cells.(4)MeRIP sequencing combined with bioinformatics analysis were applied to explore the mechanism of m6A-upregulated modification mediated by FTO inhibition in the process of Leydig cell injury.Results:The first part:(1)The testes from male rats from both D250and D500 group at PND 36 showed obvious damage in testicular histology with significantly decreased diameter of seminiferous tubules(P<0.001,P<0.001)and significantly decreased seminiferous cell layers(P<0.01,P<0.001).Decreased testosterone concentrations(P<0.001,P<0.001)and downregulated expression of spermatogenesis makers,including Plzf(P<0.001,P<0.001)and Stra8(P<0.001,P<0.001),were found in both D250group and D500 group at PND 36.And significantly increased sperm deformity rates were found at PND 90(P<0.05,P<0.001).(2)In the immature testes of D250 and D500 groups at PND 36,the total antioxidant capacity decreased significantly(P<0.05,P<0.001),the expression of Nrf2protein decreased significantly(P<0.05,P<0.01),the antioxidant signaling pathway mediated by Nrf2 was inhibited,and the number of apoptotic cells in testes increased significantly(P<0.001,P<0.001),the expression of important proteins in apoptosis pathway,such as Cleaved Caspase-3,Bcl-2and Bax,were found to be changed.(3)The global m6A modification in immature testis of SD rats in D250 and D500 groups increased significantly at PND 36(P<0.01,P<0.001).In m6A regulator genes,the expression of FTO decreased significantly(P<0.001,P<0.001)and YTHDC2 expression increased significantly(P<0.001,P<0.001)in both D250 and D500 groups.The enrichments of m6A in three sites of Nrf2 mRNA increased in D250group(P<0.001,P<0.001,P<0.001)and D500 group(P<0.001,P<0.001,P<0.001),and the enrichments of m6A in each site increased with the increases of DEHP exposure concentrations(P<0.001,P<0.001,P<0.001).The second part:The cell viability of TM3 cells treated with 200μM MEHP and 20μM FB23-2 was 90.588±5.667%and 100.991±4.343%,respectively.(1)The expressions of FTO in TM3 cells treated with 200μM MEHP were significantly decreased in both mRNA(P<0.01)and protein(P<0.05).(2)After TM3 cells were treated with 200μM MEHP and 20μM FB23-2 respectively,the concentrations of testosterone in the supernatant of TM3 cells decreased significantly(P<0.05,P<0.001),the level of apoptosis increased significantly(P<0.05,P<0.001),and the relative level of m6A modification increased significantly(P<0.001,P<0.001).(3)From transcriptome sequencing,212 overlapping differentially expressed genes were identified in TM3 cells treated with MEHP and FB23-2 respectively.The overlapping genes were significantly enriched in DNA replication(GO:0006260),cell cycle(mmu04110),regulation of kinase activity(GO:0043549),positive regulation of cell death(GO:0010942),regulated by Trp53(TRR01544),and apoptotic signaling pathway(GO:0097190).(4)From MeRIP sequencing,the classical RRACH motif were found in both TM3 cells treated with MEHP and FB23-2(P<0.001,P<0.001),and a total of 87 overlapped m6A-upregulated genes were identified.And the overlapping genes were mainly enriched in histone acetylation(GO:0016573),reactive oxygen species biosynthetic process(GO:1903409),MAPK signaling pathway(mmu04010),positive regulation of hormone secretion(GO:0046887),positive regulation of autophagy(GO:0010508),and male gonad development(GO:0008584).Conclusion:Increased m6A modification of RNA methylation contributes to the immature testicular injury induced by DEHP exposure at the prepuberty stage.The inhibition of FTO-mediated up-regulation of m6A may be involved in MEHP-induced Leydig cell injury.Based on our findings,there may be several associated physiological disorders involved with Leydig cell injury that are directly related to m6A-upregulation.