| Polychlorinated biphenyls(PCBs)are a class of highly toxic organic pollutants that are widely distributed in the environment.PCBs have a typical environmental estrogen effect and can damage the animal's reproductive system.Since PCBs are difficult to degrade,they can persist in the environment for a long time.There are 209 homologs of PCBs,which can be divided into two categories according to their structure and toxicity,one is non-dioxin-like PCBs,which are similar in structure and physical and chemical properties to dioxins with higher toxicity.The chemical 2,3',4,4',5-pentachlorobiphenyl(PCB118)is an important dioxin-like PCB with strong toxicity.PCB118 can be accumulated in mammalian adipose tissue,serum and also breast milk.Higher concentrations of PCB 118 have also been detected in the follicular fluid of infertile women.At present,there is no relevant report on the effect and mechanism on offspring's germ cells of PCB 118 exposure during pregnancy of residual concentration in human milk.Therefore,this paper mainly conducted in-depth research on the effects of PCB 118 exposure during pregnancy at residual concentration in human milk on the development of germ cells and DNA methylation of imprinted genes in offspring mice.Methods and results:The mice in the control group and the treatment groups were given 0,20 and 100 ?g/kg/day of PCB118 at 7.5 to 12.5 days of pregnancy by gavage.When F1 generation female and male mice are 7-8 weeks old,their reproductive organs and germ cells were taken,and the effects of PCB 118 exposure during pregnancy on offspring mice were investigated by molecular biological analysis such as trace cell nucleic acid extraction and enzyme digestion analysis,sodium bisulfite sequencing,real-time fluorescent quantitative PCR,immunofluorescence localization,and transcriptome sequencing analysis.Molecular biological methods were used to explore the effects of PCB 118 on the female and male mice in the following aspects:1.Effects of PCB118 during pregnancy on the development and reproductive system of F1 male mice.(1)The results showed that PCB118 exposure during pregnancy did not significantly affect the body weight and the liver organ coefficient of the offspring male mice from birth to 7 weeks of age compared with the control group(P>0.05),but significantly increased the testicular and brain organ coefficients of F1 male mice(P<0.05).(2)The study also found that compared with the control group,the methylation pattern of the differentially methylated regions of the two imprinted genes,Snrpn and Igf2r,in the treatment groups in F1 male mice were not significantly altered(P>0.05),but the H19 and Gtl2 methylation levels in differentially methylated regions were significantly reduced(P<0.05).(3)Further analysis of the relative expression levels related to DNA methylation regulation genes,indicated that compared with the control group,the expression levels of DNA methyltransferases Dnmtl,Dnmt3a,Dnmt3b,Dnmt3l and epigenetic regulatory factors Uhrf1 in the testis tissue showed a significant down regulated trend(P<0.05).2.The effects of PCB118 exposure during pregnancy on the fetus growth and the epigenetic imprinting of germ cells in F1 female mice.(1)The study found that compared to the control group,PCB118 exposure during pregnancy did not significantly affect the body weight of F1 female mice from birth to 7 weeks of age(P>0.05),but significantly reduced the organ coefficients of female mice's liver,brain,and kidney(P<0.05).Subsequently,the methylation levels of four imprinted genes H19,Snrpn,Peg3 and Igf2r in the fully grown germinal vesicle(GV)stage oocytes of F1 female mice were detected.The methylation levels of the three imprinted genes Snrpn,Peg3,and Igf2r in the GV oocytes of F1 female mice were significantly reduced(P<0.05).(2)By analyzing the relative expression levels of the four imprinted genes,it was found that the relative expression levels of the H19 in the GV stage oocytes of treatment groups of mice did not change significantly compared with the control group(P>0.05),but significantly increased the relative expression levels of Snrpn,Peg3 and Igf2r(P<0.05).Further,we analyzed the relative expression levels of genes related to DNA methylation regulation.Compared with the control group,the relative expression of DNA methyltransferases in Dnrnt1,Dnmt3a and Dnmt3l decreased significantly,and the expression level of methylation regulatory factor Uhrf1 was also significantly reduced(P<0.05),while the expression level of demethylase Tet3 was significantly increased(P<0.05).(3)Immunofluorescence quantitative analysis was used to detect the GV oocytes global methylation levels in F1 mice.It was found that exposure to PCB118 during pregnancy significantly reduced the level of 5-methylcytosine in GV oocytes of F1 mice(P<0.05),and significantly increased the level of 5-hydroxy methylcytosine(P<0.05).3.Effect of PCB118 during pregnancy on GV stage oocyte maturation in F1 female mice.(1)The study found that exposure to PCB118 during pregnancy significantly reduced the in vitro maturation rate and first polar body extrusion rate of F1 mouse oocytes(P<0.05).At the same time,the in vitro fertilization rate of F1 mouse oocytes also showed a significantly downward trend in a dose-dependent manner(P<0.05).(2)The immunofluorescence localization experiment found that compared with the control group,the mouse oocyte spindle morphology and chromosome arrangement tended to be scattered.After statistical analysis,the number of oocytes with abnormal spindle morphology significantly increased in a dose-dependent manner(P<0.05).Furthermore,we analyzed the arrangement of chromosomes in mouse oocytes.According to statistics,whether in the 20?g/kg/day or 100 ?g/kg/day treatment group,the oocytes with abnormal chromosome arrangement significantly increased compared with the control group(P<0.05),and there was an obvious dose-dependent trend.(3)Sequencing of transcriptome analysis of bioinformatics revealed that compared with the control group,the gene expression in treatment groups on epigenetic regulation,apoptosis,and cytoskeleton protein assembling in the oocyte were significantly altered(P<0.05).Subsequently,bioinformatics was used to further analyze the genes related to actin aggregation and disaggregation in the oocytes of the offspring mice.It was found that compared with the control group,most of these genes showed an upward trend in expression.For brief summary,exposure of PCB118 at a concentration equivalent to that of human breast milk residues during pregnancy in maternal mice would reduce the level of DNA methylation of some imprinted genes in the germ cells of offspring mice,and also reduced the relative expression levels of Dnmts and Uhrf1 in testicular tissue and oocytes.After further analysis of the experimental results,we found that after mouse was exposed to PCB118 during pregnancy,the in vitro maturation rate and in vitro fertilization rate of the offspring mouse oocytes decreased,and the spindle morphology and chromosome arrangement was altered in the offspring mouse oocytes.Through transcriptome sequencing analysis of the offspring mouse oocyte,it was found that the gene expression levels related to actin aggregation and disaggregation in the treatment groups were disturbed.Therefore,exposure of PCB118 to female mice during pregnancy will not only affect the normal development process of the offspring mouse reproductive system,but also reduce the quality of the offspring mouse germ cells.This study provides a reference basis for clarifying the effect and mechanism of exposure to environmental estrogen during pregnancy on the epigenetic modification of progeny germ cells,and provides a theoretical basis for clinical treatment of infertility caused by environmental estrogen. |
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