褪黑素对小鼠卵母细胞氧化应激及孤雌发育的影响

doi:10.11843/j.issn.0366-6964.2013.07.007

畜牧兽医学报 ›› 2013, Vol. 44 ›› Issue (7): 1037-1042.doi: 10.11843/j.issn.0366-6964.2013.07.007

褪黑素对小鼠卵母细胞氧化应激及孤雌发育的影响

姜园园，张宝修，孙婧陶，李钟淑，方南洙^*

（延边大学农学院动物遗传育种与繁殖实验室，延边 133002）

收稿日期:2012-12-24 出版日期:2013-07-23 发布日期:2013-07-23
通讯作者: 方南洙，教授，E-mail: nzfang@ybu.edu.cn
作者简介:姜园园（1989-），女，内蒙古赤峰人，硕士生，主要从事动物遗传育种与繁殖研究，E-mail: jiang2006322@126.com
基金资助:
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）

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

JIANG Yuan-yuan，ZHANG Bao-xiu，SUN Jing-tao，LI Zhong-shu，FANG Nan-zhu^*

(Laboratory of Animal Genetic Breeding and Reproduction，Agricultural College of Yanbian University，Yanji 133002，China)

Received:2012-12-24 Online:2013-07-23 Published:2013-07-23

摘要/Abstract

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（>250 μmol·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^-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。

Abstract:

Mouse oocyte were cultured in maturation medium to investigate melatonin(MT)could affect mouse oocyte maturation during exogenous H₂O₂.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 (MⅡstage oocytes with a first polar body) was significantly decreased by the addition of exogenous 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 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.

中图分类号:

Q132.4

姜园园，张宝修，孙婧陶，李钟淑，方南洙. 褪黑素对小鼠卵母细胞氧化应激及孤雌发育的影响[J]. 畜牧兽医学报, 2013, 44(7): 1037-1042.

JIANG Yuan-yuan，ZHANG Bao-xiu，SUN Jing-tao，LI Zhong-shu，FANG Nan-zhu. The Effect of Melatonin on the Oxidative Stress of Oocyte and Parthenogenetic Development in Mouse[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(7): 1037-1042.

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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

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