氧化应激在哺乳动物卵母细胞非整倍体形成中的作用机制

Infertility has become one of the major health problems in human beings since the past few decades and increased numbers of couples have to reley on assisted reproductive technology (ART) to achieve the pregnancy. However, even with the advanced technologies in ART, the implantation rates of embryos and livebirth rates of babies after ART are still low. There are many factors are responsible for those low rates. One of the major factors is the embryonic aneuploidy that causes miscarriage and congenital birth defects in humans. It has been found that the incidence of aneuploidy in the embryos derived from ART can be up to 50-70% of the embryos and the majority is resulted from abnormal chromosome segragation of oocytes during meiosis, especially meiosis I. Maternal age is the major factor causing chromosomal abnormalities in the oocytes. However, the mechanism(s) by which the formation of aneuploidy in the oocytes are still not well understood. It is believed that errors may occur during oocyte development and/or meiosis. When mammalian oocytes were examined for chromosome segragation, it has been noticed that meiotic spindle formation failure, defect spindle assembly checkpoint and/or DNA damages are all associated with chromosome abnormalities. It is an important research subject to study the mechanism(s) of aneuploidy formation in mammals. One of the hypotheses is that the oxidative stress, i.e excessive production of reactive oxygen species (ROS), induces cell dysfunctions including abnormal chromosomal segregation. ROS is mainly produced in mitochondria and is supposed to be removed by antioxidants, such as glutathione, within the cells. If cell produces too much ROS and/or cells does not have sufficient antioxidants, excessive ROS would harm organelles, proteins and DNA in the cells. Heavy metal accumulation during life is a major factor to induce excessive ROS production and extra amount of antioxidants are required to remove the excessive ROS, thus the levels of antioxidants are reduced in the cells exposed to ROS. Our hypothesis is that if ROS production can be reduced in the oocytes by adding metal chelators, and glutathione level can be increased by food supply, it would be able to protect the oocytes from oxidative stress, as a result, the rate of aneuploidy could be reduced. In order to prove this hypothesis, in the present research project, we will study the mechanism(s) of maternal aging-associated oocyte aneuploidy formation in mammals (mouse and human). Experiments will be designed to examine ROS production, mitochondria activity, ATP level, meiotic spindle formation, spindle assembly checkpoint, DNA methylation and chromosome structure under various experimental conditions. Our goal is to provide further information for better understanding the mechanisom(s) by which oocyte aneuploidy formation during mammalian aging.

辅助生殖技术已经成为治疗人类不孕不育的一个重要手段和途径。然而，由于染色体异常胚胎（非整倍体胚胎）的比例相当高（约50-70%），通过辅助生殖技术移植后的胚胎着床率仍然较低。非整倍体是导致胚胎发育停滞，早期流产和胎儿畸形的主要原因。非整倍体胚胎主要来源于卵子，而且其比率随着女性年龄的增加而增加。造成卵子染色体异常的原因和卵子内的信号通路及分子机理仍然不太清楚。由于重金属可导致细胞氧化应激，而氧化应激不但可以降低细胞内抗氧化剂的含量，又是造成染色体异常分裂的一个主要原因。因此，卵子非整倍体的形成很有可能与卵子内活性氧的水平和抗氧化剂的含量有关。本项目将在动物个体水平和细胞水平两个方面通过用耦合摄入的重金属和增加摄入的抗过氧化物来探讨哺乳动物个体衰老与卵子非整倍体形成之间的关系。并从氧化应激、能量代谢、线粒体活性、纺锤体形态、纺锤体检测机能以及DNA结构上做进一步深入的机理性研究。

辅助生殖技术已经成为治疗人类不孕不育的一个重要手段和途径。然而，通过辅助生殖技术移植后的胚胎着床率和活产率仍然较低，卵子的老化明显影响着IVF成功率和移植后胚胎的发育能力。而普遍认为氧化应激是卵子老化的一个重要原因，导致受精率和胚胎发育率低。卵子在体内或体外成熟过程中可以被活性氧（ROS）损伤，卵子体内成熟后质量下降主要与母体年龄相关；而体外成熟的卵子由于暴露于高氧环境，使得卵子数量和质量都明显下降，甚至导致凋亡。卵子内活性氧水平和抗氧化剂的平衡对于细胞器的功能很重要，如染色体组装、线粒体功能、纺锤体整合、ATP水平和DNA甲基化等。当线粒体产生过多的ROS，而没有足够的抗氧化剂去移除它们，ROS和抗氧化剂的平衡就会被打破，凋亡程序就会加速。这些都可能就是老化卵子质量低下的重要原因。同时重金属也可导致细胞氧化应激，氧化应激不但.可以降低细胞内抗氧化剂的含量, 又是造成染色体异常分裂和细胞器功能异常的一个主要原因,从而影响卵子的质量。因此，本项目旨在通过动物个体水平和细胞水平两个方面通过用耦合摄入的重金属和增加摄入的抗过氧化物来探讨哺乳动物个体衰老与卵子细胞器功能、非整倍体形成等之间的关系。并从氧化应激、能量代谢、线粒体活性、纺锤体形态、纺锤体检测机能以及DNA损伤上做进一步深入的机理性研究。. 我们的研究发现，在小鼠个体水平上，饮用水中适当添加抗氧化剂硫辛酸和乙酰肉碱对生殖细胞确有延缓老化过程的作用,而重金属耦合剂EDTA 和柠檬酸钠未见有明显的相似作用。在细胞水平上，我们通过体外老化的未成熟小鼠卵子，并在培养液中添加EDTA、柠檬酸钠、硫辛酸和乙酰肉碱，发现这些药物对体外老化的未成熟小鼠卵子都有明显的抗氧化作用。无论是体外或体内实验，我们都发现重金属耦合剂和抗氧化剂有提高卵子数量，维持小鼠卵子正常的纺锤体形态，提高正常线粒体分布，降低了DNA损伤的发生的重要作用，解除由氧化应激导致的不良效应。再者，EDTA，柠檬酸、钠硫辛酸和乙酰肉碱混合物在体外培养过程中有更加强大的抗氧化效应。。. 总的来说，我们的结果显示，通过饮用水中/培养液中添加重金属耦合剂和抗氧化剂，有助于尝试提高老化卵子质量，为老化卵子的成熟和受精延长时间，同时，对防止机体卵子的老化，对高龄妇女采取的辅助生殖策略等方面有重要意义。