腐胺提高高龄小鼠卵母细胞质量及其机制的研究

Putrescine improves oocyte quality of aging mice and its mechanism

目的本研究探讨腐胺是否通过抗氧化应激的机制提高体外培养的高龄卵母细胞的质量。方法从9月龄C56/B6J雌鼠获取GV期卵丘-卵母复合体(COC),分为对照组、0.5mmol/L腐胺处理组、0.5mmol/Lα-二氟甲基鸟氨酸(DFMO,腐胺抑制剂)组。三组COC体外培养液16～18h,收集MⅡ期卵母细胞。计算各组高龄卵母细胞成熟率,检测卵母细胞mtDNA拷贝数,JC-1荧光分析染色线粒体膜电位,并分析卵母细胞内氧自由基和超氧化物歧化酶1(SOD_1)的表达。结果腐胺组卵母细胞成熟率显著高于对照组[(87.57±1.70)%vs.(66.02±4.05)%,P<0.01]。DFMO处理组线粒体DNA拷贝数显著高于对照组(P<0.05)。与对照组相比,腐胺组卵母细胞内氧化应激水平显著下降(P<0.01);DFMO组氧化应激水平显著升高(P<0.01)。与对照组比较,腐胺组卵母细胞线粒体膜电位显著升高(P<0.01),而DFMO组显著下降(P<0.05)。与对照组比较,腐胺组卵母细胞内SOD_1蛋白表达显著增高(P<0.05),DFMO组显著下降(P<0.05)。结论体外培养的高龄卵母细胞,腐胺通过抗氧化应激、改善线粒体功能提高卵母细胞质量和胚胎发育潜能,具有潜在的临床应用价值。

Objective:To investigate whether putrescine improves the quality of oocytes in older mice cultured in vitro through the mechanism of anti-oxidative stress.Methods:The GV oocytes obtained from 9-month old C56/B6 Jfemale mice were in vitro cultured in the IVM medium as control group;those were cultured in the IVM medium with 0.5 mmol/L putrescine as the putrescine group;and those were treated with 0.5 mmol/Lα-difluoromethylornithine(DFMO,an inhibitor of putrescine production)as the DFMO group.After cultured for 16-18 hours,MII oocytes were collected.The maturity rate was calculated,and the mitochondrial DNA(mtDNA)copy number was detected by RT-PCR.The mitochondrial membrane potential was tested by JC-1 fluorescence staining,while the levels of ROS and expression of superoxide dismutase(SOD1)protein were measured.Results:The maturity rate of oocytes in the putrescine group was significantly higher than that in the control group[(87.57±1.70)%vs.(66.02±4.05)%,P<0.01].The mtDNA copy number in DFMO group was significantly higher than that in control group(P<0.05).Compared with the control group,ROS level in the putrescine group was significantly decreased(P<0.01),while that in the DFMO group was significantly increased(P<0.01).When compared with the control group,the mitochondrial membrane potential in the putrescine group was significantly increased(P<0.01),while that in the DFMO group was significantly decreased(P<0.01).The expression of SOD1 was significantly increased in the putrescine group(P<0.05),but that in the DFMO group was significantly decreased(P<0.05).Conclusions:The aging mice oocytes were cultured in vitro,putrescine can improve the oocyte quality and developmental potential of embryo derived from aging oocytes through decreasing oxidative stress and promoting mitochondrial function.It has potential clinical application value.