5-甲基胞嘧啶到5-羟甲基胞嘧啶的转变过程参与小鼠雄原核的DNA主动去甲基化

Conversion of 5mC to 5hmC is involved in active DNA demethylation in mice paternal pronucleus

利用5-甲基胞嘧啶(5-methylcytosine,5mC)和5-羟甲基胞嘧啶(5-hydroxymethylcytosine,5-hmC)特异性抗体对小鼠原核时期胚胎进行免疫荧光染色。同时使用荧光定量PCR方法检测Tet(Ten eleven translocation)基因在小鼠早期胚胎中的表达。免疫荧光染色结果显示早期原核阶段(PN1到PN3),雄原核中5mC的含量逐渐减少,而5hmC的含量逐渐增加。但是雌原核中5mC和5hmC的含量基本不变。在原核后期阶段(PN4到PN5)5hmC主要存在于雄原核中。荧光定量结果显示在小鼠MⅡ卵母细胞和植入前胚胎中Tet1和Tet2基因表达量较低,但是Tet3在卵母细胞和原核时期表达量较高,随着胚胎的发育其表达量逐渐降低。结果表明,在小鼠原核时期阶段5mC到5hmC的转变过程主要是由TET3蛋白催化的,并且此过程参与小鼠雄原核的DNA主动去甲基化。

Mouse M II oocytes and zygotes were collected and used for immunofluoresence （IF） staining. IF results showed that during early pronuclear embryonic stages, the fluorescence intensity of 5-hydroxymethyleytosine （5hmC） in the paternal pronueleus increased from PN1 to PN3,while 5hmC staining of maternal pronucleus exhibited no obvious differences. In the later pronuclear stages, 5hmC was mainly present in the paternal pronucleus. The expression of Tet genes in M II oocytes and embryos was examined by qRT-PCR. The qRT-PCR results showed that there was moderate expression of Tetl and Tet2 in the eight-cell stage and morula stage embryos,but trace levels of Tetl and Tet2 expression were present in zygotes and two-cell stage embryos. However in fertilized embryos, Tet3 showed high expression levels in M II oocytes and zygotes,but its expression was reduced during the two-cell stage. Our results indicated that the conversion between 5mc to 5hmc is mainly catalyzed by TET3 in zygotes,and it is involved in the active demethylation of paternal genome.