活性氧对猪卵母细胞SUMO-1表达及其质量的影响

本研究旨在调查活性氧过氧化氢（hydrogen peroxide，H₂O₂）对猪卵母细胞体外成熟（in vitro maturation，IVM）过程中蛋白质类泛素SUMO-1表达及精-卵结合能力的影响。试验分为0（control）、10、50、75和100 μg/mL H₂O₂处理组。利用Western blotting、流式细胞术、实时荧光定量PCR、Hoechst染色等方法检测猪卵母细胞体外成熟、蛋白质类泛素SUMO-1含量、细胞活力、凋亡基因mRNA表达、透明带（zona pellucid，ZP）溶解度和精子-卵母细胞结合的表达。结果表明，75 μg/mL H₂O₂组与对照组、10和50 μg/mL H₂O₂组比较卵母细胞体外成熟率及细胞活力显著降低；75 μg/mL H₂O₂组与其他H₂O₂组相比ZP溶解时间显著延长，并减少了精子黏附在成熟卵母细胞透明带上的数量（P<0.05）。在77和18 ku处出现SUMO-1蛋白标记，75 μg/mL H₂O₂组与对照组、10和50 μg/mL H₂O₂组比较SUMO-1蛋白含量显著降低（P<0.05）。75 μg/mL H₂O₂与对照组、10和50 μg/mL H₂O₂组比较显著下调了Bcl-2基因，而Caspase-3基因表达与对照组和10 μg/mL H₂O₂组比较显著升高（P<0.05），50 μg/mL H₂O₂与对照组和10 μg/mL H₂O₂组相比显著上调了Bax基因水平（P<0.05）。综上所述，H₂O₂能调控猪卵母细胞体外成熟过程中类泛素化水平以及精-卵结合能力。

Effects of ROS on Expression of SUMO-1 and Quality of Porcine Oocytes

The purpose of this study was to investigate the effects of reactive hydrogen peroxide (H₂O₂) on the expression of the protein ubiquitin SUMO-1 and the ability of sperm-oocyte binding of porcine oocyte during in vitro maturation (IVM).The test was divided into 5 groups:0 (control),10,50,75 and 100 μg/mL of H₂O₂ treatment group.Western blotting,flow cytometry,Real-time quantiative PCR,Hoechst staining and other methods were used to detect porcine oocyte in vitro maturation,content of protein SUMOylation SUMO-1,cell viability,expression of apoptotic gene mRNA,solubility of zona pellucida (ZP) and sperm-oocyte binding.The results showed that 75 μg/mL H₂O₂ group compared with the control,10 and 50 μg/mL H₂O₂ groups,the in vitro maturation rate and cell viability of oocytes decreased significantly,while 75 μg/mL H₂O₂ group compared with other H₂O₂ groups,the ZP lysis time prolonged significantly,and the number of sperm adhering to the ZP of mature oocytes was reduced (P<0.05).SUMO-1 protein markers appeared at 77 and 18 ku,and the content of SUMO-1 protein was significantly reduced in 75 μg/mL H₂O₂ group when compare with the control,10 and 50 μg/mL H₂O₂ groups (P<0.05).75 μg/mL H₂O₂ group compared with the control,10 and 50 μg/mL H₂O₂ groups,Bcl-2 gene was significantly down-regulated (P<0.05),while 50 μg/mL H₂O₂ significantly up-regulated Bax gene level when compared with the control and 10 μg/mL H₂O₂ groups (P<0.05).In summary,H₂O₂ could regulate the SUMOylation level and sperm-oocyte binding ability of porcine oocytes during in vitro maturation.