Effects Of Bisphenol A Exposure During Pregnancy On Testicular Damage And DNA Methylation In Male Offsprings

ObjectivesBisphenol A(BPA)is an environmental endocrine disruptor,which is widely used in daily necessities.Numerous studies have shown that BPA exposure can cause serious damage to male reproductive system.At present,the research on reproductive dysfunction caused by BPA exposure is mostly focused on adolescence or adulthood,but there are fewer reports on the damage to the offspring male reproductive development after BPA exposure during pregnancy.Epigenetic changes caused by BPA play an important role in the inheritance of male reproductive dysfunction.Therefore,this study investigated the effects of BPA exposure during pregnancy on testicular DNA methylation at different developmental stages of the offsprings,and the role of changes in DNA methylation,mRNAand protein expression levels of glial cell line-derived neurotrophic factor(GDNF)in testes of the offsprings in BPA-induced testicular injury,in order to provide a basis for the detection and treatment of early reproductive damage.Methods1.A total of 60 female healthy SPF-grade SD rats and 30 male SPF-grade SD rats,10 weeks old,were housed in cages in a ratio of 2:1.Vaginal smear was performed on the next morning,and the first day of gestation(GD0)was estimated by the presence of spermatozoids in vaginal smears.The successfully conceived female rats were randomly divided into 5 groups,and then were intragastrically administered with BPA at doses of 0,0.05,0.5,5,50 mg/kg/d at GD 5-20 once a day at a dosing volume of 5ml/kg.The testis index of the offspring mice was detected at postnatal day(PND)21 and PND56.2.The general toxicity of BPA exposure during pregnancy to male offsprings at PND21 and PND56 was observed.The damage of BPA to testicular tissue and ultrastructure was observed by H&E staining and transmission electron microscopy.3.The mRNA and protein expression levels of DNA methyltransferases(DNMTs)and GDNF in testes of the offsprings at PND21 and PND56 were detected by real-time PCR and Western blot respectively.4.Methylation-specific PCR(MSP)was used to detect the DNA methylation status of GDNF.Results1.Effects of BPA exposure during pregnancy on testicular damage and DNA methylation in male offsprings at PND21(1)The body weight,wet organ weight,and organ coefficient of male offsprings were measured.Compared with the control group,the body weights of the offsprings increased in each dose group,but the difference was statistically significant only in the 0.5 mg/kg dose group(P<0.05).There was no significant difference in anogenital distance,wet weights of liver,kidney and testis(P>0.05).Compared with the control group,there was no significant difference in testicular and renal organ coefficients,only liver organ coefficient significantly decreased in the 0.5 mg/kg dose group(P<0.05).(2)The results of H&E staining of testicular tissue showed that the structure of seminiferous tubules of the control group and the 0.05 mg/kg BPA group was,the spermatogenic cells were arranged in an orderly manner,there were more mature sperm cells in the lumen,and there were no shed spermatogenic cells.In the 0.5 and 5mg/kg BPA group,the local lumen was enlarged,the number of spermatogenic cell layers and spermatozoa were decreased.In the 50 mg/kg BPA group,the spermatogenic cells appeared irregular and sperm cells were degenerated and necrotic.Observation of testicular tissue under electron microscope revealed that no obvious abnormal changes were found in mitochondria in the 0.05 and 0.5 mg/kg BPA group;while in the 5 mg/kg BPA group,the mitochondrial ridge was blurred and broken,and in the 50 mg/kg BPA group,the disappearance of the mitochondrial ridge was disappeared.(3)The mRNA and protein expression levels of DNA methyltransferase in testes of the offsprings were detected.Compared with the control group,the mRNA level of DNMT1 was increased in the 5 mg/kg dose group and was significantly decreased in the 50 mg/kg dose group(P<0.05).The expression of DNMT1 protein was significantly reduced in the 50 mg/kg dose group(P<0.05).There was no significant difference in the mRNA and protein expression levels of DNMT3 a between the different dose groups and the control group(P>0.05).Compared with the control group,the expression levels of DNMT3 b mRNA and protein were significantly increased in the 0.5 mg/kg dose group(P<0.05).(4)Detection of DNA methylation levels of GDNF gene in testes of the offsprings showed that GDNF gene was partially methylated in the control group and0.05,0.5 mg/kg dose groups,while GDNF gene was fully methylated in the 5,50mg/kg dose groups.The results showed that BPA exposure could lead to increased methylation level of GDNF gene.Detection of GDNF mRNA and protein levels showed that compared with the control group,the mRNA and protein expression levels of GDNF decreased significantly in the 0.5 and 50 mg/kg dose groups(P<0.05).2.Effects of BPA exposure during pregnancy on testicular damage and DNA methylation in male offsprings at PND56(1)Compared with the control group,the weights of the offsprings in the groups of 5 and 50 mg/kg BPA increased significantly(P<0.05).The wet weights of liver and genitalia increased significantly in the groups of 5 and 50 mg/kg BPA(P<0.05).Kidney wet weight was only increased in the 5 mg/kg BPA group(P<0.05).After adjusting for body weight,only the liver coefficient increased in the groups of 0.5 and50 mg/kg BPA(P<0.05).(2)The H&E staining results of testicular tissue showed that the structure ofspermatogenic tubules in the control group were clear,with orderly arrangement of spermatogenic cells,more mature sperm cells in the lumen,and no exfoliated spermatogenic cells.Necrosis of sperm cells was observed in the 0.05 mg/kg BPA group.In the 0.5 mg/kg BPA group,the number of spermatogenic cell layers and spermatocytes were decreased significantly,and the lumen was enlarged.In the 5mg/kg BPA group,the wall of spermatogenic tubules became thinner,the number of spermatogenic cell layers was decreased,and the lumen was enlarged.Sperm cell necrosis was observed,and the nuclei of sperm cells were coagulated and fragmented.The multinucleated macrophages were also observed.In the 50 mg/kg BPA group,the spermatogenic tubules atrophied,the spermatogenic cells appeared irregular,sperm cells were necrotic,the lumen completely disappeared,and even a large amount of spermatocyte were necrotic.(3)The gene and protein expression levels of DNMT1,DNMT3 a and DNMT3 b in testes of the offsprings showed that,compared with the control group,the mRNA expression level of DNMT1 was significantly increased in the 0.05 mg/kg dose group,while was significantly decreased in the dose groups of 0.5 and 5 mg/kg(P<0.05).Compared with the control group,the expression level of DNMT1 protein was significantly decreased in the 5 and 50 mg/kg groups(P<0.05).The mRNA expression levels of DNMT3 a in 0.5,5 and 50 mg/kg groups were all lower than that in the control group(P<0.05).The protein expression levels of DNMT3 a in the 5 and50 mg/kg groups were significantly lower than that in the control group(P<0.05).Compared with the control group,the expression level of DNMT3 b mRNA was significantly increased in 0.05 and 0.5mg/kg dose groups,while was significantly decreased in 5 and 50 mg/kg dose groups(P<0.05).The expression level of DNMT3 b protein was significantly increased in the 0.5 mg/kg group(P<0.05).(4)DNA methylation level of GDNF in testes of the offsprings was detected,and methylation of GDNF occurred in each dose group.The mRNA and protein expression levels of GDNF showed a decreasing trend with the increase of the exposure dose,but the difference was not statistically significant(P>0.05).Conclusion1.BPA exposure during pregnancy can cause damage to the reproductive system in male offsprings,and the effects continue to different developmental stages.2.BPA exposure during pregnancy can cause abnormal changes in DNA methylation in testes of the offsprings at different periods.3.BPA exposure during pregnancy may reduce the expression of GDNF in testicular Sertoli cells of the offsprings at PND21 through abnormal changes in DNA methylation,which may cause testicular damage and spermatogenesis disorder,and even cause male reproductive disorders,but the effect at PND56 is not obvious.The specific mechanism needs further research.