小鼠孤雌胚与体内正常胚H3K27三甲基化模式的差异

Different H3K27 Trimethylation Patterns in Parthenogenetic and In vivo Mouse Embryos

为了考察小鼠(Mus musculus)孤雌激活胚胎H3K27三甲基化模式与体内正常胚胎之间的差异,以及曲古抑菌素A(TSA)对孤雌胚H3K27三甲基化水平的影响,探究表观遗传修饰对孤雌胚胎发育的作用。首先,用H3K27me3特异性抗体对MⅡ期卵母细胞染色,利用激光共聚焦对其荧光强度进行检测,结果发现该时期的甲基化荧光强度相对较低。接着,采用同样的方法对小鼠孤雌胚胎和体内正常胚胎植入前各时期的H3K27me3模式进行比较,结果显示,从2-细胞到囊胚期孤雌组呈现逐渐升高的趋势,与体内组变化趋势完全相反,且总体平均荧光强度较体内组普遍偏低。孤雌胚胎经TSA处理后,处理组和未处理组在前三个时期虽然没有显著性差异(P>0.05),但是处理之后的H3K27三甲基化水平有所提高,囊胚期与未处理组相比有显著性差异(P<0.05)。以上结果表明,小鼠孤雌胚胎的H3K27三甲基化模式与体内胚胎之间存在着巨大的差异,这可能是造成孤雌胚胎发育能力差的重要原因之一。TSA处理对H3K27me3模式造成了一定的影响,使体外培养环境有所改善,这可能对提高孤雌胚胎发育能力具有一定的意义。

The purpose of this study is to investigate the differences of H3K27 trimethylation pattern between parthenogenetic and in vivo mouse （Mus musculus） embryos. The effect of trichostatin （TSA） on the level of H3K27me3 in parthenogenetic embryos, and the impact of TSA on parthenogenetic embryonic development were also studied. Firstly, we used the indirect immunofluorescence with specific antibody against H3K27me3 and showed that there was a mild intensity staining in the metaphase oocyte. We activated the metaphase oocytes by strontium chloride and then collected the embryos at different developmental stages. The in vivo embryos were collected by flushing the uterus at different times after injecting pregnant mare serum gonadotrophin and human chorionic gonadotropin. Different patterns of H3K27me3 staining were detected in normal fertilized embryos and parthenogenetic embryos. The relative fluorescence intensities of the different stage embryos were determined by laser scanning confocal microscopy. Moreover, the pronuclear embryos which were activated by strontium chloride were treated with TSA for 20 hours. The un-treated parthenogenetic embryos were cultured in a medium containing dimethyl sulfoxide （0.05%） as a control. The results showed that there was a trend of increasing fluorescence intensity in parthenogenetic embryos, reaching the highest in blastocysts, while it was opposite in the in vivo embryos from 2-cell to the blastocyst stage. However, the mean fluorescence intensity of the parthenogenetic embryos was generally lower than that of normal embryos. After the treatment with TSA, though there was no significant difference in early embryo development （P〉0.05） between the treated and the non-treated groups, the level of H3K27me3 in parthenogenetic embryos increased. There was significant difference （0.015±0.002 vs.0.004±0.000 5,P〈0.05） in blastocyst formation. These results indicated that the pattern of the H3K27me3 in parthenogenetic embryos differ significantly from that of the in vivo embryos, which may be one of the reasons causing the low development ability of the parthenogenetic embryos. The TSA treatment of parthenogenetic activation embryos causes a certain effect on the H3K27me3 pattern, improving the in vitro embryo development.