Shanghai Ji Ai Genetics & IVF Institute, Obstetrics & Gynecology Hospital, Fudan University, Shanghai, 200011, People's Republic of China.
Reproductive Medical Center, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200025, People's Republic of China.
J Assist Reprod Genet. 2018 Jul;35(7):1169-1178. doi: 10.1007/s10815-018-1215-4. Epub 2018 Jun 6.
Oocyte vitrification is currently used for human fertility preservation. However, vitrification damage is a problem caused by decreasing ooplasmic levels of glutathione (GSH). The GSH donor glutathione ethyl ester (GSH-OEt) can significantly increase the GSH content in oocytes. However, it is difficult to obtain oocyte from woman. To overcome this, we used mouse oocytes to replace human oocytes as a model of study.
Oocytes from B6D2F1 mice were preincubated for 30 min with 2.5 mmol/L GSH-OEt (GSH-OEt group), without GSH-OEt preincubation before vitrification (control vitrification group) or in nonvitrified oocytes (fresh group). After thawing, oocytes were fertilized for evaluating the developmental competence of embryos in vitro and in vivo. Immunofluorescence, Polscope equipment and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to analyze damage, including mitochondrial distribution, reactive oxygen species (ROS) levels, spindle morphology, and gene expression levels (Bcl-2, BAX, and MnSOD).
The rates of fertilization, 3-4 cell, blastocyst formation and expanded blastocysts were significantly higher (p < 0.05) in the GSH-OEt group (90.4%; 91.1%; 88.9% and 63.0%) than in the control (80.0%; 81.4%; 77.7% and 50.5%). Provided embryos overcame the 2-cell block and developed to the blastocyst stage, birth rates of all groups were similar. Vitrification altered mitochondrial distribution, increased ROS levels, and caused abnormal spindle morphology; GSH-OEt preincubation could improve such damage. RT-qPCR showed that the expression of Bcl-2 was lower in the control group compared with the GSH-OEt group; BAX and MnSoD expression levels were higher in the control group than in the GSH-OEt group (p < 0.05).
The beneficial effect of GSH-OEt preincubation occurred before the 2-cell stage.
卵母细胞玻璃化目前用于人类生育力保存。然而，玻璃化损伤是由卵浆中谷胱甘肽（GSH）水平降低引起的问题。GSH 供体谷胱甘肽乙酯（GSH-OEt）可以显著增加卵母细胞中的 GSH 含量。然而，从女性中获得卵母细胞是困难的。为了克服这一问题，我们使用小鼠卵母细胞代替人卵母细胞作为研究模型。
B6D2F1 小鼠的卵母细胞在预孵育 30 分钟后用 2.5mmol/L GSH-OEt（GSH-OEt 组）处理，在玻璃化之前不进行 GSH-OEt 预孵育（对照玻璃化组）或在非玻璃化卵母细胞中（新鲜组）。解冻后，通过体外和体内受精评估胚胎的发育能力。免疫荧光、Polscope 设备和定量逆转录聚合酶链反应（RT-qPCR）用于分析损伤，包括线粒体分布、活性氧（ROS）水平、纺锤体形态和基因表达水平（Bcl-2、BAX 和 MnSOD）。
GSH-OEt 组的受精率、3-4 细胞、囊胚形成率和扩展囊胚形成率均显著高于对照组（p<0.05）（90.4%、91.1%、88.9%和 63.0%）。提供的胚胎克服了 2-细胞阻滞并发育至囊胚阶段，所有组的出生率相似。玻璃化改变了线粒体分布，增加了 ROS 水平，并导致异常的纺锤体形态；GSH-OEt 预孵育可以改善这种损伤。RT-qPCR 显示对照组中 Bcl-2 的表达低于 GSH-OEt 组；对照组中 BAX 和 MnSoD 的表达水平高于 GSH-OEt 组（p<0.05）。
GSH-OEt 预孵育的有益作用发生在 2-细胞阶段之前。
