| In 1973, James and Smith found ethanol exposure during pregnancy led to offsprings’prenatal and postnatal growth deficiencies, termed fetal alcohol syndrome (FAS). Since then, more and more scientists focused on the effects and mechanisms of pregnancy ethanol exposure on offspring. However, a very few studies has been done on the paternal alcohol consumption influence on offspring development. Animal models have evaluated the effects of paternal ethanol exposure on offspring and some of the results have been inconsistent. These may because that the duration and concentration of ethanol the mice were exposure to were different. Previous study advised ethanol as teratogens had multiple effects on birth defect by involving in the epigenetic modification. But so far, the epigenetic mechanism in paternal ethanol exposure leading to FAS is rarely reported.In our study, a possible direct link between paternal ethanol exposure and epigenetic changes affected offspring development would be established. Firstly, mice model of FAS related to chronic paternal ethanol exposure could be built. Then to provide a possible epigenetic mechanism in FAS, we used Methyl-Sensitive Cut Counting technology and Expression Assays to check the methylated status of F0, Fl and the mRNA expression. Our research wanted to answer whether epigenetic variation has been passed on to offspring through male sperm, lead to the birth defect.Study contents as follows:Section 1:Male KM, C57 and Aldh2-/- mice were all randomly assigned to three groups:3.3g/kg EtOH-exposed group, 1.1g/kg EtOH-exposed group, and distilled water group:Og/kg. Mice were then intubated intragastrically with ethanol and water every two days for 4 weeks. To explore the poisonous effects of ethanol on the reproductive system in male mice, the following data were collected:body weight, the weight of testis and epididymidis, sperm activity and concentration.Section 2:One month after dosing were completed, male mice were let to breed with untreated females within 2-3 days. When F1 were born, the following data were collected:number of live litters, sex ratio, weight and percent of malformation. In addition,8 weeks after birth, behavioral tests was conducted to F1 to determine the cognitive and mood changes.Section 3:DNA methylation at the DMR of selected imprinted genes were quantified using Sequenom MassARRAY EpiTYPER analysis. In the paternal sperm, the DMRs of 1 paternally (H19) and 3 maternally methylated (Peg3, Ndn and Snrpn) imprinted genes were analyzed. We also analyzed the methylation and the mRNA levels of these four imprinted genes in cerebral cortex of offspring.Section 4:Methyl-Sensitive Cut Counting (MSCC) was used to analyze the whole genome DNA methylation of C57 mice, which included the sperm DNA of the alcohol treatment mice and the hippocampus DNA of their offspring. Then, the gene expression profile was used to analyze the mRNA expression level of the offspring’s hippocampus. Finally, we used bioinformatics methods to find out some genes, which showed the same changed methylation in DNA promoter from FO to Fl were consisted with their mRNA expression. MassARRAY and QRT-PCR methods were used to verify the results in C57 mice and Aldh2-/- mice’s FO and F1 generation.Study results as follows:Section 1:In male KM mice, compared with the control group,3.3g/kg EtOH-exposed dams gained less weight (p<0.05), and the decreased trend in sperm concentration and motility were also observed. In C57 and Aldh2-/- mice, decreased body weight and the decreased weight of testis and epididymidis were also observed in these mice (p<0.05). And we also found the increased ratio of sperm apoptosis in C57 and Aldh2-/- mice. Compared to the C57, the Aldh2-/- mice had an higher increased motality with an increased ethanol concentration.3.3g/kg EtOH exposed Aldh2-/- mice had a decreased T, VAP and VCL.Section 2:The Fl of three kinds of mice had a lower survival rate (p<0.05), and ethanol did impair their cognitive and mood. In KM mice, a specific circling behavior and deafness were observed in ethanol exposed offspring. The F1 of KM mice showed a dose-related learning and memory impairment, and also had an anxiety and depressive behavior (p<0.05). Compared to C57 mice, Aldh2-/- mice showed a higher sensitive to ethanol. It showed that the offspring of Aldh2-/- mice had a significant decrease in learning and memory (p<0.05).Section 3:One paternally (H19) and three maternally (Peg3, Ndn and Snrpn) imprinted genes were tested in paternal sperm and offspring’s cerebral cortex in KM mice. In the H19 DMR of FO sperm, the DNA methylation was significantly decreased in 3.3g/kg EtOH-exposed mice than that in control group (p<0.05). Specifically, there was significantly increased methylation at the Peg3 DMR in sperm DNA of paternal 3.3g/kg ethanol exposure. However, only two increased methylation of CpG sites at Peg3 DMR (CpG7 and CpG11) were also found in the cerebral cortex DNA of 3.3g/kg ethanol exposed offspring (p<0.05). In addition, no difference was observed at Ndn and Snrpn DMR in paternal sperm between treatment group and control group. But the significant decreased methylation was found at the Snrpn DMR in F1 and F2 cerebral cortex of 3.3g/kg ethanol treatment offspring (p<0.05).Section 4:Methyl-Sensitive Cut Counting (MSCC) was used to analyze the whole genome DNA methylation of C57 mice, which included the sperm DNA of the ethanol treatment mice and the hippocampus DNA of their offspring. Then, the gene expression profile was used to analyze the mRNA expression level of the offspring’s hippocampus. Finally,12 genes which showed the same changed methylation in DNA promoter from FO to F1 and consisted with their mRNA expression were screened. Then we used MassARRY and QRT-PCR and enlarged samples in C57 and Aldh2-/-mice FO and F1 generation to verify these 12 genes. Finally, Syt2 gene which had a significant expression changed was screened. And the further mechanism and verification researches were underway to confirm our results.In summary, we confirmed that chronic paternal ethanol exposure in mice impaired its reproductive system. Significant changed methylation were observed in paternal sperm. And these abnormal change could be passed on to their offspring altered some genes expression, which may be the reason for the birth defect in F1... |
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