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首页> 外文期刊>Epigenetics & Chromatin >Dynamics of 5-methylcytosine and 5-hydroxymethylcytosine during pronuclear development in equine zygotes produced by ICSI
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Dynamics of 5-methylcytosine and 5-hydroxymethylcytosine during pronuclear development in equine zygotes produced by ICSI

机译：ICSI产生的马合子原核发育过程中5-甲基胞嘧啶和5-羟甲基胞嘧啶的动力学

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Background Global epigenetic reprogramming is considered to be essential during embryo development to establish totipotency. In the classic model first described in the mouse, the genome-wide DNA demethylation is asymmetric between the paternal and the maternal genome. The paternal genome undergoes ten-eleven translocation (TET)-mediated active DNA demethylation, which is completed before the end of the first cell cycle. Since TET enzymes oxidize 5-methylcytosine to 5-hydroxymethylcytosine, the latter is postulated to be an intermediate stage toward DNA demethylation. The maternal genome, on the other hand, is protected from active demethylation and undergoes replication-dependent DNA demethylation. However, several species do not show the asymmetric DNA demethylation process described in this classic model, since 5-methylcytosine and 5-hydroxymethylcytosine are present during the first cell cycle in both parental genomes. In this study, global changes in the levels of 5-methylcytosine and 5-hydroxymethylcytosine throughout pronuclear development in equine zygotes produced in vitro were assessed using immunofluorescent staining. Results We were able to show that 5-methylcytosine and 5-hydroxymethylcytosine both were explicitly present throughout pronuclear development, with similar intensity levels in both parental genomes, in equine zygotes produced by ICSI. The localization patterns of 5-methylcytosine and 5-hydroxymethylcytosine, however, were different, with 5-hydroxymethylcytosine homogeneously distributed in the DNA, while 5-methylcytosine tended to be clustered in certain regions. Fluorescence quantification showed increased 5-methylcytosine levels in the maternal genome from PN1 to PN2, while no differences were found in PN3 and PN4. No differences were observed in the paternal genome. Normalized levels of 5-hydroxymethylcytosine were preserved throughout all pronuclear stages in both parental genomes. Conclusions In conclusion, the horse does not seem to follow the classic model of asymmetric demethylation as no evidence of global DNA demethylation of the paternal pronucleus during the first cell cycle was demonstrated. Instead, both parental genomes displayed sustained and similar levels of methylation and hydroxymethylation throughout pronuclear development.
机译：背景技术整体表观遗传重编程被认为在胚胎发育过程中建立全能性至关重要。在首次在小鼠中描述的经典模型中，全基因组DNA去甲基化在父本和母本基因组之间是不对称的。父本基因组经历了十一个11易位（TET）介导的活性DNA去甲基化，该过程在第一个细胞周期结束之前完成。由于TET酶将5-甲基胞嘧啶氧化为5-羟甲基胞嘧啶，因此推测后者是DNA脱甲基化的中间阶段。另一方面，保护母体基因组免于主动去甲基化，并进行依赖复制的DNA去甲基化。但是，由于在两个亲本基因组的第一个细胞周期中都存在5-甲基胞嘧啶和5-羟甲基胞嘧啶，因此几个物种并未显示出此经典模型中描述的不对称DNA脱甲基过程。在这项研究中，使用免疫荧光染色评估了在体外产生的马合子中整个前核发育过程中5-甲基胞嘧啶和5-羟甲基胞嘧啶水平的总体变化。结果我们能够证明5-甲基胞嘧啶和5-羟甲基胞嘧啶在整个前核发育过程中都明确存在，在两个亲本基因组中强度相似，在由ICSI产生的马合子中。然而，5-甲基胞嘧啶和5-羟甲基胞嘧啶的定位模式不同，其中5-羟甲基胞嘧啶均匀地分布在DNA中，而5-甲基胞嘧啶倾向于聚集在某些区域。荧光定量显示母体基因组中从PN1到PN2的5-甲基胞嘧啶水平增加，而在PN3和PN4中没有发现差异。在父本基因组中未观察到差异。在两个亲本基因组的所有前核阶段中，保留了5-羟甲基胞嘧啶的标准化水平。结论总之，这匹马似乎没有遵循经典的不对称去甲基化模型，因为没有证据表明在第一个细胞周期中父核的整体DNA脱甲基。相反，两个亲本基因组在整个前核发育过程中均显示出持续且相似的甲基化和羟甲基化水平。

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