| Background:Due to man-made influences,the eutrophication of freshwater body is getting more and more serious,leading to water blooms.The direct representation of water bloom is the overgrowth of cyanobacteria and the production of metabolites-Microcystins(MCs).As the most widely studied and most toxic microcystin,Microcystin-LR can induce impairment to the male reproductive and nervous system and will be passed on to offspring.However,very little is known about the inheritance of these effects to offspring and the mechanisms involved.Objective:Western blot and RT-PCR are used to explore the effects of parental MCLR(MicrocystinLR)exposure on the expressions of brain nerve development-related proteins,DNA methylases,hydroxymethylases and related gene in zebrafish and offspring.Through the analysis of global DNA methylation and hydroxymethylation,to explore the influence of parental MCLR exposure on the level of global methylation and hydroxymethylation in the parental brain,gonads and sperm.The effects of parental MCLR exposure on the levels of related gene methylation in the parental brain,sperm and offspring brain can be discussed through pyrosequencing.Behavioral experiments are used to investigate the influence of parental MCLR exposure on offspring’s behavior.To provide a theoretical basis for clarifying how the parental MCLR exposure causes parental and offspring nerve damage.Methods:1.About 10 800 embryos at 10 hpf were divided into the control groups,5 μg/L MCLR groups and 25 μg/L MCLR groups at random.These embryos belonging to different groups were simultaneously exposed to oxygenated water containing the MCLR at different concentrations.Three months later,the zebrafish reached sexual maturity and were separated by sex,then mate them and achieve the F1 embryos.Another three months later,the F1 zebrafish reached sexual maturity.The male and the female were separated,and mate them to achieve the F2 embryos.2.Calculate the hatching rate of F1 and F2 embryos,the survival rate of the juvenile zebrafish,the heart rate of embryos at 2dpf and juvenile zebrafish at 7dpf,and the body length and weight of juvenile zebrafish.The video tracking system was used to perform the behavioral studies of F1 juveniles at 30 dpf.3.Observe the changes in subcellular structure in the brains of F0 and F1 male zebrafish by electron microscope;PCNA antibody staining protocol for immunohistochemistry was used to observe the distribution of PCNA in F0 and F1 male zebrafish brains.4.The Global DNA Methylation Assay Kit and Global DNA hydroxymethylation Assay Kit were used to quantify the levels of global DNA methylation and hydroxymethylation in F0 male adult zebrafish brain,gonads,and sperm.Pyrophosphate methylation detection was used to quantify the levels of bdnf,drd1 a,gad1b,dio3 b and gabra1 genes in F0 and F1 male zebrafish brains and the levels of bdnf,drd1 a and dio3 b genes promoter methylation in F0 male zebrafish sperm.5.RT-PCR was used to detect the expressions of the genes related to neurodevelopment in F0 and F1 male zebrafish brain and F2 juveniles at 7 dpf,and the expressions of epigenetic modifying enzyme genes in F0 male zebrafish brain and sperm.6.Western blot was used to detect the expressions of neurodevelopment-related protein in F0 and F1 male zebrafish brain and DNMT1 protein in F0 male zebrafish brain.ResultsAfter MCLR exposure,the mitochondria in F0 male zebrafish brain became vacuolated and swelled,the myelin sheath collapsed and deformed,and the expression of PCNA decreased;the level of global methylation in brain,gonads and sperm increased(P<0.01),and the level of global hydroxymethylation in brain decreased(P<0.05);the levels of methylation of drd1 a and gabra1 in brain and drd1 a,bdnf and dio3 b in sperm and increased(P<0.05);the gene expressions of bdnf,syn2 a,gfap,mbp and elval3 were down-regulated(P<0.01),the gene expressions of dlg4 b and gap-43 were increased(P<0.01),and the gene expressions of dnmt5,dnmt8,tet1 and tet2 were increased too(P<0.01),while the gene expressions of dnmt1,dnmt7 and tet3 were down-regulated(P<0.01);the expression of proteins related to the brain BDNF/TRKB/PI3K/AKT signaling pathway was inhibited,the protein expressions of MBP and GFAP were decreased,and the protein expressions of GAP-43 and PSD95 were increased;the gene expressions of all dnmt3 s in sperm were significantly upregulated(P<0.01).After parental MCLR exposure,F1 embryos and juveniles had abnormal indicators related to growth and development;the juveniles at 30 dpf swim more slowly in the light and dark;the myelin sheath in F1 male zebrafish brain became collapsed,deformed and thinned,and the expression of PCNA was decreased;the gene expressions of bdnf,syn2 a,gfap,mbp,dlg4 b and elval3 in brain were inhibited;the expressions of proteins in BDNF/TRKB/PI3K/AKT signaling pathway and neurodevelopment-related proteins MBP,GAP-43,PSD95,GFAP in brain were inhibited;the levels of drd1 a,gabra1,gad1 b,dio3b and bdnf methylation in brain were increased.After MCLR exposure to F0 male zebrafish,F2 embryos and juveniles had abnormal growth and development-related indicators;the expressions of neurodevelopment-related genes bdnf,syn2 a,creb and elavl3 were down-regulated,and dlg4 b were increased.ConclusionAfter exposure to MCLR,the expressions of genes and proteins related to neurodevelopment were changed and the signal pathways related to neurodevelopment were inhibited in brain.The reproductive ability of F0 and F1 male zebrafish was damaged.These effects could be passed on to the offspring by changing the level of bdnf methylation in sperm,causing the abnormal gene and protein expressions related to neurodevelopment,leading to brain damage and abnormal behavior in the offspring. |
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