褪黑素对小鼠卵母细胞氧化应激及孤雌发育的影响

摘要

通过小鼠卵母细胞自发成熟体外培养模型研究了褪黑素(MT)对外源性H2O2刺激的小鼠卵母细胞体外成熟及其孤雌发育的影响.结果表明:(1)随着H2O2剂量依赖性的增加,卵母细胞PB1的排出率下降,当增加H2O2(＞250μmol·L-1)时,显著下降(19.4％ vs.92.0％,P＜0.05)；(2)培养液中添加MT(0.1、1.0、10.0 ng·mL-1)后均能阻止H2O2的抑制作用,其中10.0 ng·mL-1的MT作用效果显著(P＜0.05)；(3)取GV期卵母细胞经H2O2(500 μmol·L-1)和MT(0.000、0.001、0.010、0.100 ng·mL-1)联合培养后孵育在2',7'-二氯荧光素二乙酸酯(DCF-DA)中,荧光显色表明,MT清除了由于H2O2引起的细胞内大量的活性氧(Reactive oxygen species,ROS),其中0.100 ng·mL-1的MT效果最显著(P＜0.05),极大程度的降低了氧化应激损伤,提高了成熟率；(4)统计卵母细胞孤雌激活后的发育情况,MT浓度为10-9～10-3 mol·L-1组培养的孤雌胚2-细胞发育情况无显著差异(88.2％、91.4％、91.8％、88.8％ vs.88.1％,P＞0.05),MT浓度为10-7～10-3mol· L-1组的4～8-细胞率差异显著(58.8％、73.8％、43.6％ vs.23.4％,P＜0.05),MT浓度为10-9～10-5 mol· L-1均明显促进囊胚发育(24.6％、46.6％、49.2％vs.8.0％,P＜0.05).氧化应激对卵母细胞的成熟是不利的,MT保护了卵母细胞免受氧化应激的影响,MT浓度为10-5 mol·L-1为卵母细胞孤雌发育能力的最适浓度.由此可推出MT作为细胞内的自由基清除剂,可能是提高卵母细胞和胚胎质量的重要条件之一.%Mouse oocyte were cultured in maturation medium to investigate melatonin(MT)could affect mouse oocyte maturation during exogenous H2 O2.the parthenogenetic embryos were incubated with different concentrations of MT(0,10-9,10-7,10-5,10-3 mol · L-1),every stage of development would be analyzed.The results showed:(1) The percentage of the mature oocytes (M lⅡ stage oocytes with a first polar body) was significantly decreased by the addition of exogenous H2O2 in a dose-dependent manner (＞250μmol · L-1,19.4％ vs.92.0％,P＜0.05).(2) When oocytes were incubated with MT(0.1,1.0,10.0 ng · mL-1) in the presence of H2O2 (250 tμ mol · L-1),MT dose-dependently blocked the inhibitory effect of H2O2 on oocyte maturation,there was a significant effect at the concentration of 10.0 ng · mL-1 of MT.(3)GV stage oocytes were incubated with 2',7'-dichlorofluorescein (DCF-DA) after co-culture of H2O2 (500μmol · L-1) and MT (0.000,0.001,0.010,0.100 ng · mL-1),the increased fluorescence intensity of oocytes incubated with H2O2 was significantly decreased by MT treatment(0.100 ng · mL-1).(4) There was no significant difference in the groups of 10-9-10-3 mol · L-1 than the control group during two-cell phase (88.2％,91.4％,91.8％,88.8％ vs.88.1％,P＞0.05),the rate of four-eight-cell was improved when added 10-9M of MT (30.5％ vs.23.4％,P＞0.05).There was very significant difference among the rates of 10-7-10-3 mol · L-1 than control group(58.8％,73.8％,43.6％ vs.23.4％,P＜0.05),Blastocyst rates were increased in the higher concentrations 10-9-10-5 mol · L-1 (24.6％,46.6％,49.2％ vs.8.0％,P＜0.05),but it decreased during 10-3 mol · L-1 of MT.Oxidative stress was harmful for oocytes maturation which were protected by MT.The optimum concentration of MT for development of mouse oocytes after parthenogenetic activation was 10-5 mol · L-1.As a direct free radical scavenger,MT could become one of the most important conditions for improving oocyte and embryo quality.