Ruan Z, Zhao X, Qin X, Luo C, Liu X, Deng Y, Zhu P, Li Z, Huang B, Shi D, Lu F
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning, Guangxi, China.
Guangxi High Education Laboratory for Animal Reproduction and Biotechnology, Guangxi University, Nanning, Guangxi, China.
Reprod Domest Anim. 2018 Feb;53(1):203-212. doi: 10.1111/rda.13093. Epub 2017 Oct 27.
The DNA methylation of imprinted genes is an important way to regulate epigenetic reprogramming of donor cells in somatic cell nuclear transfer (SCNT). However, the effects of sexual distinction on the DNA methylation of imprinted genes in cloned animals have seldom been reported. In this study, we analysed the DNA methylation status of three imprinted genes (Xist, IGF2 and H19) from liveborn cloned buffaloes (L group, three female and three male), stillborn cloned buffaloes (S group, three female and three male) and natural reproduction buffaloes (N group, three female and three male), using bisulphite sequencing polymerase chain reaction (BS-PCR). The expression levels of these imprinted genes were also investigated by quantitative real-time PCR (QRT-PCR). The DNA methylation levels of H19 were not significantly different among the groups. However, the Xist in female and IGF2 in male of the S group were found to be significantly hypomethylated in comparison with the same sexual buffaloes in L group and N group (p < .05). Furthermore, the expression levels of Xist, IGF2 and H19 in the stillborn female cloned buffaloes of S group were significantly higher than that of the female buffaloes in the L group and N group (p < .05). The expression levels of IGF2 and H19 in the stillborn male cloned buffaloes in the S group were significantly higher than that of the male buffaloes in the L group and N group (p < .05). These results indicate that Xist may be associated with the viability of female cloned buffaloes, and IGF2 may also be related to the viability of male cloned buffaloes.
印记基因的DNA甲基化是调节体细胞核移植（SCNT）中供体细胞表观遗传重编程的重要方式。然而，性别差异对克隆动物中印记基因DNA甲基化的影响鲜有报道。在本研究中，我们使用亚硫酸氢盐测序聚合酶链反应（BS-PCR）分析了存活的克隆水牛（L组，3只雌性和3只雄性）、死产的克隆水牛（S组，3只雌性和3只雄性）和自然繁殖水牛（N组，3只雌性和3只雄性）中三个印记基因（Xist、IGF2和H19）的DNA甲基化状态。还通过定量实时PCR（QRT-PCR）研究了这些印记基因的表达水平。H19的DNA甲基化水平在各组之间没有显著差异。然而，与L组和N组中的同性水牛相比，发现S组雌性中的Xist和雄性中的IGF2显著低甲基化（p <.05）。此外，S组死产雌性克隆水牛中Xist、IGF2和H19的表达水平显著高于L组和N组中的雌性水牛（p <.05）。S组死产雄性克隆水牛中IGF2和H19的表达水平显著高于L组和N组中的雄性水牛（p <.05）。这些结果表明，Xist可能与雌性克隆水牛的活力有关，而IGF2也可能与雄性克隆水牛的活力有关。
