双酚A斑马鱼雄性生殖毒性的表观遗传学基础

摘要

目的 从表观遗传学角度探讨双酚A(BPA)影响斑马鱼精子成熟与胚胎发育的机理.方法 将20 dpf斑马鱼幼鱼,暴露在乙醇(溶剂)对照、BPA0.5 mg/L、BPA1.5 mg/L至100 dph.选取各处理组雄鱼,荧光定量PCR检测性腺DNA甲基转移酶(DNMTs)表达量,免疫组化检测性腺全基因组甲基化水平,Western-blot、免疫组化检测性腺组蛋白3赖氨酸三甲基化(H3K9me3、H3K4me3、H3K27me3、H3K36me3)表达水平.各处理组雄鱼与正常雌鱼交配,观察胚胎期相关指标.结果 BPA处理组Dnmt1、Dnmt6、Dnmt7、Dnmt8 mRNA表达量明显升高,与对照组比较差别均具有统计学意义(P<0.05).BPA1.5 mg/L组中成熟精子DNA甲基化水平与H3K9me3水平均明显升高.与正常雌鱼交配,BPA 1.5 mg/组产卵量仅为对照组的63%±2.49%,12 h胚胎死亡率与对照组相比增加202.53%±6.21%,72 h胚胎畸形率与对照组相比增加399.08%±12.16%,差别均具有统计学意义(P<0.05).结论 双酚A诱导斑马鱼成熟精子DNA甲基化程度和组蛋白H3K9me3 表达水平增加,这些变化可能是BPA处理组后代胚胎发育异常的重要基础.%Objective To investigate the epigenetic modifications of zebrafish sperm maturation and embryo development by bisphenol A (BPA). Methods 20 dpf juvenile zebrafish were exposed to ethanol solution (control groups), BPA solution of 0.5mg/Land 1.5 mg/L (treatment groups) until 100 dph. As for the male selected from each group, the gonadal Dnmts mRNA expression levels were tested by quantitative PCR, the gonadal genome methylation levels were measured by immunohistochemistry. In addition, the levels of histone methylation including H3K9me3, H3K4me3, H3K27me3 and H3K36me3 were detected using Westem-blot and immunohistochemistry. Males of each group were mated with untreated female fish to observe and record the indicators relating to embryonic stage. Results The expression levels of Dnmtl、dnmt6> dnmt7、dnmt8 mRNA in BPA-treated groups increased significantly compared to the control group (P<0.05). Levels of genome DNA methylation and H3K9me3 in mature sperm of BPA 1.5 mg/L-exposed group increased significantly, while the number of eggs of female fish which mated with this group is reduced to 63%±2.49% of these fish with control group, the 12h embryonic mortality increased to 202.53%±6.21% and the 72 embryonic malformations increased to 399.08%±12.16%, these differences are of statistical significance (P<0.05). Conclusion BPA exposure selectively raised levels of DNA methylation and histone H3K9me3 in mature sperm of zebrafish, these changes may be an important foundation of the increased developmental abnormalities of Fl offspring.