褪黑素对小鼠卵母细胞氧化应激及孤雌发育的影响  

The Effect of Melatonin on the Oxidative Stress of Oocyte and Parthenogenetic Development in Mouse

作　　者：姜园园[1] 张宝修[1] 孙婧陶[1] 李钟淑[1] 方南洙[1] 

机构地区：[1]延边大学农学院动物遗传育种与繁殖实验室,延边133002

出　　处：《畜牧兽医学报》2013年第7期1037-1042,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：吉林省科技厅重点项目"人血清白蛋白转基因延边奶山羊新品系培育"(20100228)

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、10.0ng·mL-1)后均能阻止H2O2的抑制作用,其中10.0ng·mL-1的MT作用效果显著(P<0.05);(3)取GV期卵母细胞经H2O2(500μmol·L-1)和MT(0.000、0.001、0.010、0.100ng·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0.100ng·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10-7～10-3 mol·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 02. the parthenogenetic embryos were incubated with different concentrations of MT（0,10.9 , 10-7 , 10 s, 10 3 mol ·L 1 ）, every stage of development would be analyzed. The results showed. （1） The percentage of the mature oocytes （M II stage oocytes with a first polar body） was significantly decreased by the addition of exoge- nous H2O2 in a dose-dependent manner （〉250 μmol · 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 μmol · 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 （500 μmol · L-1） and MT （0. 000,0. 001,0. 010,0. 100 ng ·mL-1 ） ,the increased fluorescence intensity of oocytes incubated with H202 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 during10-3tool. L-1 of MT. Oxidative stress was harmful for ooeytes 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 f

关 键 词：褪黑素 卵母细胞 H2O2 抗氧化剂 孤雌发育 

分 类 号：Q132.4[生物学—普通生物学]