Washington National Primate Research Center, University of Washington, Seattle, WA 98121, USA.
Hum Reprod. 2010 Oct;25(10):2465-74. doi: 10.1093/humrep/deq225. Epub 2010 Aug 20.
The inadequacies of oocyte in vitro maturation (IVM) systems for both non-human primates and humans are evidenced by reduced fertilization and poor embryonic development, and may be partly explained by significantly lower glutathione (GSH) contents compared with in vivo matured (IVO) oocytes. As this influence has not been fully explored, this study investigated the effect of the GSH donor, glutathione ethyl ester (GSH-OEt), on the IVM and development of macaque oocytes as a model of human oocyte IVM.
Macaque oocytes derived from unstimulated ovaries were cultured in mCMRL-1066 alone or supplemented with 3 or 5 mM GSH-OEt. In vitro matured oocytes were subjected to the GSH assay, fixed for the assessment of spindle morphology or prepared ICSI. Embryo development of zygotes cultured in mHECM-9 was assessed up to Day 9 post-ICSI. RESULTS Supplementation of the maturation medium with GSH-OEt significantly increased oocyte maturation and normal fertilization rates compared with control oocytes, but only 5 mM GSH-OEt significantly increased the oocyte and cumulus cell GSH content. Confocal microscopy revealed significant differences in the spindle morphology between IVO and control in vitro matured metaphase II oocytes. Oocytes matured with 5 mM GSH-OEt exhibited spindle area and spindle pole width similar to that seen in the IVO oocyte. While no significant differences were observed in blastocyst rates, addition of 3 mM GSH-OEt during IVM significantly increased the proportion of embryos developing to the 5-8 cell stage while 5 mM GSH-OEt significantly increased the proportion of morula-stage embryos compared with controls.
Supplementation of the IVM medium with GSH-OEt promotes better maturation and normal fertilization of macaque oocytes compared with non-supplemented medium. However, further improvement of the primate oocyte IVM culture system is required to support better blastocyst development of oocytes derived from unstimulated ovaries.
非人类灵长类动物和人类的卵母细胞体外成熟（IVM）系统的不足表现在受精率降低和胚胎发育不良，这可能部分归因于与体内成熟（IVO）卵母细胞相比，其谷胱甘肽（GSH）含量明显较低。由于这种影响尚未得到充分探讨，本研究以猕猴卵母细胞为模型，研究了 GSH 供体谷胱甘肽乙酯（GSH-OEt）对 IVM 和发育的影响。
从未刺激的卵巢中获得的猕猴卵母细胞在 mCMRL-1066 中单独培养或添加 3 或 5mM GSH-OEt 培养。体外成熟的卵母细胞进行 GSH 测定，固定评估纺锤体形态或准备 ICSI。在 mHECM-9 中培养的受精卵胚胎发育至 ICSI 后第 9 天进行评估。
与对照组卵母细胞相比，成熟培养基中添加 GSH-OEt 可显著提高卵母细胞成熟和正常受精率，但仅 5mM GSH-OEt 可显著提高卵母细胞和卵丘细胞 GSH 含量。共聚焦显微镜显示，IVO 和对照组体外成熟中期 II 期卵母细胞的纺锤体形态存在显著差异。用 5mM GSH-OEt 成熟的卵母细胞表现出与 IVO 卵母细胞相似的纺锤体面积和纺锤体极宽度。虽然囊胚率没有显著差异，但在 IVM 中添加 3mM GSH-OEt 可显著增加发育至 5-8 细胞阶段的胚胎比例，而与对照组相比，添加 5mM GSH-OEt 可显著增加桑椹胚阶段胚胎的比例。
与未添加 GSH-OEt 的培养基相比，IVM 培养基中添加 GSH-OEt 可促进猕猴卵母细胞更好地成熟和正常受精。然而，需要进一步改进灵长类动物卵母细胞 IVM 培养系统，以支持来自未刺激卵巢的卵母细胞更好的囊胚发育。
