College of Life Science, Inner Mongolia University, 235 West Univ. Road, Hohhot, 010021, Inner Mongolia, China.
Department of Geriatric Medical Center, Inner Mongolia People's Hospital, 20 Zhaowuda Road, Hohhot, 010021, Inner Mongolia, China.
Theriogenology. 2021 Oct 1;173:144-155. doi: 10.1016/j.theriogenology.2021.07.025. Epub 2021 Aug 8.
The beneficial effect of glutathione (GSH) on the in vitro maturation (IVM) of bovine/porcine oocytes has been confirmed; however, the antioxidant effect of exogenous GSH supplementation on the IVM of ovine oocytes has not been determined. In this study, ovine cumulus oocyte complexes (COCs) were classified into three groups according to the layer number of cumulus cells (the Grade A group has more than five layers, the Grade B group has three to four layers and the Grade C group has less than three layers). After in vitro culture of COCs in the presence of exogenous GSH, the meiotic competence of ovine oocytes was assessed by analyzing nuclear maturation to metaphase II (MII) stage, cortical granules (CGs) dynamics, astacin like metalloendopeptidase (ASTL) distribution, histone methylation pattern, reactive oxygen species (ROS) production, mitochondrial activities and genes expression. After in vitro fertilization (IVF), assessments of embryonic development were conducted to confirm the effects of exogenous GSH supplementation. The results showed that exogenous GSH not only enhanced the maturation rates of the Grade B and Grade C groups but also promoted CGs dynamics and ASTL distribution of the Grade A, B and C groups (p < 0.05). Exogenous GSH increased the mitochondrial activities of the Grade A, B and C groups and decreased the ROS production levels of oocytes (p < 0.05), regardless of the layer number of cumulus cells. Moreover, exogenous GSH promoted the expression levels of genes related with oocyte maturation, antioxidant activity and antiapoptotic effects in the Grade B and Grade C groups (p < 0.05). The expression levels of H3K4me3 and H3K9me3 in the Grade B and Grade C groups were promoted after exogenous GSH supplementation (p < 0.05), consistent with the expression levels of genes related with histone methylation (p < 0.05). In addition, exogenous GSH strongly promoted the embryonic developmental competence of Grade B and Grade C groups (p < 0.05). Taken together, our findings provide foundational evidence for the free radical scavenging potential of exogenous GSH in the in vitro developmental competence of ovine oocytes, especially oocytes from COCs lacking cumulus cells. These findings, which demonstrated the potential for improving the quality of ovine oocytes during IVM, will contribute to researches on GSH applications and the efficiency of assisted reproductive technology for ovine breeding.
谷胱甘肽（GSH）对牛/猪卵母细胞体外成熟（IVM）的有益作用已得到证实；然而，外源性 GSH 补充对绵羊卵母细胞 IVM 的抗氧化作用尚未确定。在这项研究中，根据卵丘细胞层数将绵羊卵丘-卵母细胞复合物（COC）分为三组（Grade A 组有超过五层，Grade B 组有三到四层，Grade C 组少于三层）。在存在外源性 GSH 的情况下，将 COC 进行体外培养后，通过分析中期 II（MII）期核成熟、皮质颗粒（CG）动力学、天冬氨酸蛋白酶样金属内肽酶（ASTL）分布、组蛋白甲基化模式、活性氧（ROS）产生、线粒体活性和基因表达来评估绵羊卵母细胞的减数分裂能力。进行体外受精（IVF）后，进行胚胎发育评估以确认外源性 GSH 补充的效果。结果表明，外源性 GSH 不仅提高了 Grade B 和 Grade C 组的成熟率，还促进了 Grade A、B 和 C 组的 CG 动力学和 ASTL 分布（p<0.05）。外源性 GSH 增加了 Grade A、B 和 C 组的线粒体活性并降低了卵母细胞的 ROS 产生水平（p<0.05），而与卵丘细胞的层数无关。此外，外源性 GSH 促进了 Grade B 和 Grade C 组与卵母细胞成熟、抗氧化活性和抗凋亡作用相关的基因表达水平（p<0.05）。外源性 GSH 补充后，Grade B 和 Grade C 组的 H3K4me3 和 H3K9me3 表达水平升高（p<0.05），与与组蛋白甲基化相关的基因表达水平一致（p<0.05）。此外，外源性 GSH 强烈促进了 Grade B 和 Grade C 组的胚胎发育能力（p<0.05）。综上所述，本研究结果为外源性 GSH 在绵羊卵母细胞体外发育能力中清除自由基的潜力提供了基础证据，特别是在缺乏卵丘细胞的 COC 卵母细胞中。这些发现表明，改善 IVM 期间绵羊卵母细胞质量具有潜力，将有助于 GSH 应用和绵羊繁殖辅助生殖技术效率的研究。
