DNA demethylation pattern of in-vitro fertilized and cloned porcine pronuclear stage embryos.
DNA demethylation pattern of in-vitro fertilized and cloned porcine pronuclear stage embryos.
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DOI：
10.1016/j.cca.2017.07.025
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发表时间：
2017-10
期刊：
影响因子：
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通讯作者：
X. Nie;Qiang Liu;Ronggen Wang;Wenjie Sheng;Xiaokang Li;Manling Zhang;Yong Jin;Lihua Zhao;Daorong Hou;Ning Yang;Zhaoqiang Wu;Yifan Dai;Rongfeng Li
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文献类型：
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作者：
X. Nie;Qiang Liu;Ronggen Wang;Wenjie Sheng;Xiaokang Li;Manling Zhang;Yong Jin;Lihua Zhao;Daorong Hou;Ning Yang;Zhaoqiang Wu;Yifan Dai;Rongfeng Li
最近对小鼠的研究表明，10 - 11易位酶（TET）家族参与了活跃的DNA去甲基化。同种型TET-3在小鼠胚胎原核阶段负责5mc（5-甲基胞嘧啶）向5hmc（5-羟甲基胞嘧啶）的转化。本研究旨在探讨TET家族在猪体外受精（IVF）和体细胞核移植（SCNT）胚胎脱甲基过程中的甲基化变化模式和作用。采用亚硫酸氢盐测序PCR （BSP）和定量PCR前的DNA糖基化和酶切（qglams -PCR）来评估猪原核阶段甲基化的确切变化。结果表明，体外受精胚从原核2期（PN2）到原核5期（PN5）， 5hmc的检测量呈上升趋势，5mc的检测量呈下降趋势。此外，免疫荧光染色显示，在卵母细胞中也检测到5hmc信号，从受精到PN2，两个原核中5hmc信号明显增加。从PN2到PN5，雄性原核中5hmc的数量持续上升，而雌性原核中5hmc的数量下降到很低的水平。上述结果表明，雌性原核可能仅在原核早期发生活跃的去甲基化。另一方面，雄性原核可能在整个原核阶段都经历活跃的去甲基化。检测TET-1、TET-2、TET-3三种TET同型的表达，发现TET-3是表达量最高的同型。免疫荧光染色主要在雄性原核中观察到高浓度的TET-3，提示TET-3可能是催化5mc转化为5hmc的主要酶。相比之下，雌性原核在整个原核阶段均未检测到TET-3信号。SCNT胚胎的去甲基化模式与体外受精胚胎的雄性原核相似，表明猪克隆胚胎可能发生活跃的去甲基化。
Recent studies in mice showed that the Ten-eleven translocation Enzymes (TET) family is involved in the active DNA demethylation. The isotype TET-3 is responsible for the conversion of 5mc (5-methylcytosine) to 5hmc (5-hydroxymethylcytosine) at the pronuclear stages of mouse embryo. This study was performed to investigate the pattern of methylation change and the role of TET family in the demethylation process of porcine in-vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) derived embryo. Bisulfite-sequencing PCR (BSP) and DNA glucosylation and digestion before quantitative PCR (qGluMS-PCR) were done to evaluate the exact change of methylation during porcine pronuclear stages. The results showed that the amount of 5hmc detected increased whereas 5mc decreased in IVF embryo from pronuclear stage 2 (PN2) to pronuclear stage 5 (PN5). In addition, Immunofluorescent staining showed that the 5hmc signal, also detected in oocytes, significantly increased in both pronucleus from fertilization to PN2. The amount of 5hmc continued to rise in male pronucleus but decreased to a very low level in female pronucleus from PN2 to PN5. The above results indicate that female pronucleus might undergo active demethylation only at early pronuclear stages. On the other hand, male pronucleus might undergo active demethylation throughout all pronuclear stages. The expression of three TET isotypes (TET-1, TET-2, TET-3) were tested and TET-3 was found to be the highest expressed isotype. High TET-3 concentrations observed mainly in male pronucleus using immunofluorescent staining, implying that TET-3 might be the main enzyme which catalyzes the conversion of 5mc to 5hmc. In contrast, no TET-3 signal was detected in female pronucleus through the pronuclear stages. The demethylation pattern of SCNT embryos resembled that of the male pronucleus of IVF embryos, suggesting that active demethylation might happen in porcine cloned embryo.