Curnow E C, Ryan J, Saunders D, Hayes E S
University of Washington National Primate Research Center, University of Washington, Box 357331, Seattle, WA 98195, USA.
Reprod Fertil Dev. 2008;20(5):579-88. doi: 10.1071/rd08041.
Glutathione (GSH) is the main non-enzymatic defence against oxidative stress and is a critical intracellular component required for oocyte maturation. In the present study, several modulators of intracellular GSH were assessed for their effect on the in vitro maturation (IVM) and intracellular GSH content of bovine metaphase (MII) oocytes. Of the five GSH modulators tested, only the cell-permeable GSH donor glutathione ethyl ester (GSH-OEt) significantly increased the GSH content of IVM MII oocytes in a concentration-dependent manner without adversely affecting oocyte maturation rate. The GSH level in IVM MII oocytes was greatly influenced by the presence or absence of cumulus cells and severely restricted when oocytes were cultured in the presence of buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. The addition of GSH-OEt to cumulus-denuded or BSO-treated oocytes increased the GSH content of bovine MII oocytes. Supplementation of the maturation medium with bovine serum albumin (BSA) or fetal calf serum (FCS) affected the GSH content of IVM MII oocytes, with greater levels attained under BSA culture conditions. The addition of GSH-OEt to the maturation medium increased the GSH content of IVM MII oocytes, irrespective of protein source. Spindle morphology, as assessed by immunocytochemistry and confocal microscopy, displayed distinct alterations in response to changes in oocyte GSH levels. GSH depletion caused by BSO treatment tended to widen spindle poles and significantly increased spindle area. Supplementation of the IVM medium with GSH-OEt increased spindle length, but did not significantly alter spindle area or spindle morphology. GSH-OEt represents a novel oocyte-permeable and cumulus cell-independent approach for effective elevation of mammalian oocyte GSH levels.
谷胱甘肽（GSH）是对抗氧化应激的主要非酶防御物质，是卵母细胞成熟所需的关键细胞内成分。在本研究中，评估了几种细胞内GSH调节剂对牛中期（MII）卵母细胞体外成熟（IVM）和细胞内GSH含量的影响。在所测试的五种GSH调节剂中，只有细胞可渗透的GSH供体谷胱甘肽乙酯（GSH-OEt）以浓度依赖的方式显著增加了IVM MII卵母细胞的GSH含量，且未对卵母细胞成熟率产生不利影响。IVM MII卵母细胞中的GSH水平受卵丘细胞存在与否的极大影响，当卵母细胞在谷胱甘肽合成抑制剂丁硫氨酸亚砜胺（BSO）存在下培养时，GSH水平会受到严重限制。向去除卵丘或经BSO处理的卵母细胞中添加GSH-OEt可增加牛MII卵母细胞的GSH含量。用牛血清白蛋白（BSA）或胎牛血清（FCS）补充成熟培养基会影响IVM MII卵母细胞的GSH含量，在BSA培养条件下可达到更高水平。无论蛋白质来源如何，向成熟培养基中添加GSH-OEt均可增加IVM MII卵母细胞的GSH含量。通过免疫细胞化学和共聚焦显微镜评估的纺锤体形态显示，其对卵母细胞GSH水平的变化有明显改变。BSO处理导致的GSH耗竭倾向于使纺锤体极变宽并显著增加纺锤体面积。用GSH-OEt补充IVM培养基可增加纺锤体长度，但未显著改变纺锤体面积或纺锤体形态。GSH-OEt代表了一种新型的可渗透卵母细胞且不依赖卵丘细胞的方法，可有效提高哺乳动物卵母细胞的GSH水平。
