Dynamics of genomic 5-hydroxymethylcytosine during mouse oocyte growth
Dynamics of genomic 5-hydroxymethylcytosine during mouse oocyte growth
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DOI：
10.1111/gtc.12164
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发表时间：
2014-08-01
期刊：
影响因子：
2.1
通讯作者：
Kono, Tomohiro
中科院分区：
文献类型：
--
作者：
Sakashita, Akihiko;Kobayashi, Hisato;Kono, Tomohiro
最近对小鼠受精卵中去甲基化过程的研究表明，5-甲基胞嘧啶（5 mC）首先被母体10 - 11易位3（TET 3）酶转化为5-羟甲基胞嘧啶（5 hmC）或进一步氧化的胞嘧啶。这个过程对于正常的胚胎发生是至关重要的，我们的目的是阐明Tet 3在雌性生殖系发育过程中对母体基因组的影响。免疫荧光分析表明，5 hmC是明确存在于完全生长的卵母细胞，但不是在nongrowing和早期生长阶段的卵母细胞。在DNA甲基转移酶3样酶（Dnmt 3L）无效的卵母细胞及其受精卵中，母亲基因组中的5 hmC是清楚地检测到的，虽然Dnmt 3L是卵母细胞中DNA甲基化所必需的。用酶消化法分析表明，5 hmC存在于卵母细胞生长后期的LTR反转录转座子中。定量RT-PCR分析表明，Tet 3的表达增强在卵母细胞的生长和nongrowing和完全生长的卵母细胞之间表现出约40倍的增加。我们的研究结果表明，5 hmC产生自卵母细胞生长阶段，伴随着Tet 3的上调; 5 hmC主要位于LTR反转录转座子，表明5 hmC产生于生长阶段的卵母细胞是负责受精后全基因组去甲基化。
Recent studies of the demethylation process in murine zygotes have shown that 5-methylcytosine (5mC) is first converted into 5-hydroxymethylcytosine (5hmC) or further-oxidized cytosines in the paternal genome by the maternal ten-eleven translocation 3 (TET3) enzyme. This process is crucial for normal embryogenesis, and our aim was to elucidate the effect of Tet3 on the maternal genome during female germ-line development. Immunofluorescence analysis showed that 5hmC was clearly present in fully grown oocytes but not in nongrowing and early growth-stage oocytes. The 5hmC in the maternal genome was clearly detectable in DNA methyltransferase 3-like enzyme (Dnmt3L)-null oocytes and their fertilized zygotes, although Dnmt3L is essential for DNA methylation in oocytes. An analysis using an enzyme digestion-based method showed that 5hmC was present in LTR retrotransposons from the late growth period of oocytes. Quantitative RT-PCR analysis showed that Tet3 expression was enhanced during oocyte growth and exhibited an approximately 40-fold increase between nongrowing and fully grown oocytes. Our results show that 5hmC is generated since the oocyte growth stage, accompanied by up-regulation of Tet3; 5hmC is located mainly in LTR retrotransposons, indicating that 5hmC generated in growth-stage oocytes is responsible for genomewide demethylation after fertilization.