Engineering Research Center of North-East Cold Region Beef Cattle Science & Technology Innovation, Ministry of Education, Yanbian University, Yanji, 133002, China; College of Agriculture, Yanbian University, Yanji, 133002, China; Jilin Engineering Research Center of Yanbian Yellow Cattle Resources Reservation, Yanji, 133002, China.
Tianjin Limu Biotechnology Co., LTD., Tianjin, 300456, China.
Theriogenology. 2024 Apr 1;218:8-15. doi: 10.1016/j.theriogenology.2024.01.025. Epub 2024 Jan 24.
To investigate the effects of limonin (Lim) on oxidative stress and early apoptosis in bovine oocytes during in vitro maturation (IVM), different concentrations of Lim (0, 10, 20, 50 μmol/L) were added to bovine IVM medium. Oocyte maturation rates and development 24 h after in vitro fertilization (IVF) were examined to determine the optimal Lim concentration. The optimal Lim concentration was added to the IVM medium, and 0 μmol/L Lim was used as the control. Immunofluorescence staining was used to detect the abnormal rate of spindle assembly, reactive oxygen species (ROS), glutathione (GSH), mitochondrial membrane potential (MMP) levels, mitochondrial distribution, and the fluorescence intensity of cathepsin B (CB)-active LC3 protein. RT‒qPCR was used to detect the mRNA expression levels of antioxidant-, apoptosis- and autophagy-related genes in oocytes. The total number of blastocysts and the proportion of apoptotic cells among blastocysts were detected. The results showed that the PBI ejection rate, cleavage rate and blastocyst rate of bovine oocytes in the 20 μmol/L Lim group were significantly higher than those in the control group (P < 0.05). Compared with those in the control group, ROS levels, abnormal mitochondrial distribution, the proportion of abnormal spindle assembly, CB activity and LC3 protein fluorescence intensity of oocytes in the 20 μmol/L Lim group were significantly decreased (P < 0.05), and GSH and MMP levels were significantly increased (P < 0.05). The expression of antioxidant genes (Prdx3, Prdx6, Sirt1) and antiapoptotic genes (Bcl-xl, Survivin) were significantly upregulated (P < 0.05), and the expression levels of proapoptotic genes (Caspase-4, BAX) and autophagy-related genes (LC3) were significantly downregulated (P < 0.05). The total number of cells among in vitro fertilized embryos was significantly increased (P < 0.05), and the apoptosis rate of blastocysts was significantly decreased (P < 0.05). Here, we show that Lim exerts positive effects on bovine oocyte IVM by regulating REDOX homeostasis, reducing spindle damage and enhancing mitochondrial function during IVM, thereby inhibiting oocyte apoptosis and autophagy.
为了研究柠檬苦素（Lim）对牛卵母细胞体外成熟（IVM）过程中氧化应激和早期凋亡的影响，将不同浓度的柠檬苦素（0、10、20、50μmol/L）添加到牛 IVM 培养基中。检查卵母细胞成熟率和体外受精（IVF）后 24 小时的发育情况，以确定最佳柠檬苦素浓度。将最佳柠檬苦素浓度添加到 IVM 培养基中，以 0μmol/L 的柠檬苦素作为对照。通过免疫荧光染色检测纺锤体组装异常率、活性氧（ROS）、谷胱甘肽（GSH）、线粒体膜电位（MMP）水平、线粒体分布和组织蛋白酶 B（CB）活性 LC3 蛋白的荧光强度。使用 RT-qPCR 检测卵母细胞中抗氧化、凋亡和自噬相关基因的 mRNA 表达水平。检测囊胚的总数量和囊胚中凋亡细胞的比例。结果表明，20μmol/L 柠檬苦素组牛卵母细胞的 PBI 排出率、分裂率和囊胚率显著高于对照组（P<0.05）。与对照组相比，20μmol/L 柠檬苦素组卵母细胞的 ROS 水平、异常线粒体分布、异常纺锤体组装比例、CB 活性和 LC3 蛋白荧光强度显著降低（P<0.05），GSH 和 MMP 水平显著升高（P<0.05）。抗氧化基因（Prdx3、Prdx6、Sirt1）和抗凋亡基因（Bcl-xl、Survivin）的表达显著上调（P<0.05），促凋亡基因（Caspase-4、BAX）和自噬相关基因（LC3）的表达水平显著下调（P<0.05）。体外受精胚胎的细胞总数显著增加（P<0.05），囊胚的凋亡率显著降低（P<0.05）。由此可见，柠檬苦素通过调节 REDOX 平衡、减少纺锤体损伤和增强线粒体功能，对牛卵母细胞 IVM 产生积极影响，从而抑制卵母细胞凋亡和自噬。
