Effect of vitrification at the germinal vesicle stage on the global methylation status in mouse oocytes subsequently matured in vitro
Effect of vitrification at the germinal vesicle stage on the global methylation status in mouse oocytes subsequently matured in vitro
复制标题
生泡阶段玻璃化冷冻对随后体外成熟的小鼠卵母细胞整体甲基化状态的影响
DOI：
10.3760/cma.j.issn.0366-6999.20141383
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发表时间：
2014-12
期刊：
影响因子：
--
通讯作者：
Qiao J
中科院分区：
文献类型：
--
作者：
Yan J;Zhang L;Wang T;Li R;Liu P;Yan L;Qiao J
背景目前还不清楚玻璃化冷冻过程本身是否与卵母细胞玻璃化冷冻过程中异常DNA甲基化的发生有关。本研究的目的是通过分析小鼠卵母细胞在体外成熟（IVM）后的成熟期和未成熟期玻璃化冷冻后的DNA甲基化水平，评价其表观遗传学特征。方法取成年雌性小鼠中期II（M II）期和生殖泡（GV）期卵母细胞，分别进行玻璃化冷冻。评估M II卵母细胞的冷冻存活和总体DNA甲基化。评估GV卵母细胞的冷冻存活，仅体外培养存活的GV卵母细胞，用于随后评估成熟卵母细胞中的总体DNA甲基化。未进行玻璃化冷冻的体内成熟的新鲜M II卵母细胞用作对照。然后在激光扫描共聚焦显微镜下，使用抗5-甲基胞嘧啶（抗5-MeC）单克隆抗体和异硫氰酸荧光素（FITC）标记的山羊抗小鼠IgG，通过免疫荧光检查M II卵母细胞中的整体DNA甲基化水平。结果玻璃化冷冻对成熟卵母细胞DNA甲基化水平的影响：M Ⅱ ‐v组90例（90/90）卵母细胞均存在高甲基化，其中63.3%（57/90）的卵母细胞DNA甲基化水平极高，25.6%（23/90）的卵母细胞DNA甲基化水平较高，GV‐v组129个成熟卵母细胞中，67.4%（87/129）的卵母细胞DNA甲基化水平极高，23.3%（30/129）的卵母细胞DNA甲基化水平较高，9.3%（12/129）为中度。两组间差异无统计学意义（P>0.05），与对照组相似：68.6%（83/121）的新鲜M Ⅱ期卵母细胞DNA甲基化水平为极高，21.5%（26/121）为高，9.9%（12/121）为中等水平（P >0.05）。IVM对成熟卵母细胞整体DNA甲基化状态的影响方面，在体内成熟卵母细胞组中，所有检测的卵母细胞（94/94）均存在高甲基化，其中80.9%（76/94）的卵母细胞表现为极高水平的DNA甲基化，19.1%（18/94）的卵母细胞表现为高水平的DNA甲基化，而在体外成熟卵母细胞组中，69个卵母细胞中有56个（85.2%）的卵母细胞DNA高度甲基化，11个（11.9%）的卵母细胞DNA高度甲基化，2个（2/69）的卵母细胞DNA中度甲基化。这一结果与体内成熟的新鲜M Ⅱ期卵母细胞相似（P >0.05）。结论GV期玻璃化冷冻不会引起小鼠卵母细胞DNA甲基化状态的广泛改变。
Background It is still unclear whether the vitrification procedure itself is associated with the incidence of abnormal DNA methylation during oocytes vitrification. The purpose of this study was to evaluate the epigenetic profile of mouse oocytes, which went through vitrification either at a mature stage or at an immature stage following in vitro maturation (IVM) by analyzing the global DNA methylation. Methods Metaphase II (M II) stage and germinal vesicle (GV) stage oocytes were collected from adult female mice and were vitrified respectively. The M II oocytes were assessed for cryo‐survival and global DNA methylation. The GV oocytes were assessed for cryo‐survival and only the surviving GV oocytes were cultured in vitro for subsequent assessment of global DNA methylation in mature oocytes. In vivo matured fresh M II oocytes without undergoing vitrification were used as control. The level of global DNA methylation in the M II oocytes was then examined by immunofluorescence using an anti‐5‐methylcytosine (anti‐5‐MeC) monoclonal antibody and fluorescein isothiocyanate (FITC)‐conjugated goat anti‐mouse IgG under a laser scanning confocal microscope. Results In terms of the effect of vitrification on global DNA methylation status in matured oocytes, in the M II‐v group, all the examined oocytes (90/90) were found with hypermethylation, including 63.3% (57/90) of them displaying DNA methylation of a very high level, 25.6% (23/90) with a high level, and 11.1% (10/90) with an intermediate level, whereas in the GV‐v group, all the matured oocytes (129/129) were also examined with hypermethylation, including 67.4% (87/129) of them displaying DNA methylation of a very high level, 23.3% (30/129) with a high level, and 9.3% (12/129) with an intermediate level. Statistically, it was similar between both groups, which were similar to the control: 68.6% (83/121) of fresh M II oocytes displayed DNA methylation of a very high level, 21.5% (26/121) with a high level, and 9.9%(12/121) with an intermediate level (P >0.05). In terms of the effect of IVM on global DNA methylation status in matured oocytes, in the in vivo matured oocytes group, all oocytes examined (94/94) were found with hypermethylation, including 80.9% (76/94) displaying DNA methylation of a very high level and 19.1% (18/94) with a high level, whereas in the in vitro matured oocytes group, all oocytes examined (69/69) were also found with hypermethylation: 85.2% (56/69) of them displayed with DNA methylation of very high level, 11.9% (11/69) with high level, and 2% (2/69) with intermediate level. This result was similar to that in in vivo matured fresh M II oocytes (P >0.05). Conclusion The vitrification procedure at GV stage does not induce widespread alteration of global DNA methylation status of mouse oocytes subsequently matured in vitro.
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影响因子：
6.7
作者：
Huang, Jack Y. J.;Chen, Hai Ying;Chian, Ri-Cheng
通讯作者：
Chian, Ri-Cheng
影响因子：
6.7
作者：
Xueming Zhao;Jing-Jing Ren-Jing;W. Du;H. Hao;Dong Wang;T. Qin;Yan Liu;Huabin Zhu
通讯作者：
Xueming Zhao;Jing-Jing Ren-Jing;W. Du;H. Hao;Dong Wang;T. Qin;Yan Liu;Huabin Zhu
影响因子：
3.8
作者：
Kageyama, Shun-ichiro;Liu, Honglin;Aoki, Fugaku
通讯作者：
Aoki, Fugaku
影响因子：
6.7
作者：
Goud, A;Goud, P;Dhont, M
通讯作者：
Dhont, M
DOI：
10.1016/s8756-5005(08)79109-1
发表时间：
2008
期刊：
--
影响因子：
--
作者：
P. Patrizio
通讯作者：
P. Patrizio