氧化应激状态下受精卵细胞周期停滞的Chk1/Cdc25分子机制

The development of zygotes can be affected by culture environment, while the mechanism remains unknown. In our previous studies, we found that zygotes fertilized with oxidative stress-inducing DNA-damaged sperm showed mitotic delay with Chk1 and Cdc25 activation. Antioxidant epigallocatechin-3-gallate (EGCG) promoted the development of embryos fertilized with DNA-damage sperm. The repair of damaged sperm DNA relies on the proteins and mRNA stored in zygotes. Chk1 is the critical cell cycle checkpoint kinase and Cdc25 is closely associated with cell cycle arrest. Therefore, is it possible that the arrest of zygotes induced by oxygen radical is due to Chk1 and Cdc25 activation, and how does Cdc25 mediate G2/M arrest? We proposed that the mitotic delay of zygotes in adverse environment might be attributed to the cell cycle arrest mediated by Chk1/Cdc25 in zygotes under oxidative stress and this may have biological significance to error correction in individual zygote. Based on the mouse zygote cultivation system which our group has set up, we use a variety of interventions and testing means to investigate the cell cycle G2/M arrest in zygotes which is modulated by Chk1/Cdc25 under oxidative stress, to explore the protective mechanism of antioxidants for zygotes, to perfect the theory of zygote damage repair, and finally, to provide a theoretical basis to optimize human ART.

培养环境影响受精卵发育。前期研究发现：氧化应激性DNA损伤精子受精后受精卵分裂延迟，细胞周期检验点激酶Chk1、底物Cdc25激活；抗氧化剂EGCG促进DNA损伤精子受精后胚胎发育。由于精子DNA损伤依赖于受精后受精卵内修复，Chk1是检验点关键信号分子，Cdc25与细胞周期停滞关系密切。那么：受精卵受氧自由基损害的分裂延迟，是否与Chk1和Cdc25的激活有关? Cdc25如何介导细胞周期G2/M期停滞？我们设想：不良培养环境中受精卵分裂延迟可能涉及氧化应激状态下受精卵细胞周期停滞，且Chk1/Cdc25参与了G2/M期调控，对受精卵细胞个体纠错具有生物学意义。本课题拟在已建立的小鼠受精卵培养体系中，采用多种干预措施及检测手段，研究Chk1/Cdc25调控的氧化应激状态下受精卵细胞周期停滞的分子机制；探讨抗氧化剂对受精卵保护机制。完善受精卵损伤修复理论，为优化人类ART技术提供理论依据。

由于培养环境中的温度、氧、PH值和能量物质等与体内不同容易生成过多活性氧类物质ROS，受精卵细胞直接受ROS损害时出现的分裂延迟现象的机制不清。本研究模拟人类胚胎体外发育特点，采用0.03mM H2O2 成功建立了氧化应激性DNA损伤受精卵模型,针对氧化应激状态下受精卵细胞发育延迟现象的机制展开研究。研究发现：受精卵细胞G2/M期（即S期结束至第一次有丝分裂M期结束）比对照组约延迟3.05h，且ROS高、MMP下降，胞核有γH2AX表达；受精卵核内或核周出现Chk1(pSer-345)、pSer323-Cdc25B、pSer216-Cdc25C蛋白表达，胞质中可见Cdc2p34(Try15)荧光信号；Chk1干预有改变、抗氧化剂有保护作用，等，证实：氧化应激性受精卵存在细胞周期检验点激活、G2/M期延长；并启动了DNA损伤反应，Chk1、Cdc25参与其中。但与我们前期研究：氧化应激性DNA损伤精子受精后受精卵中获得的结论有差异：Cdc25亚型胞核胞浆分布不一致现象；囊胚形成率下降、囊胚细胞凋亡增加；G2/M期延长时间较氧化应激性DNA损伤精子组1.8小时长；受精卵1-，2-细胞期均有γH2AX表达。提示：H2O2处理受精卵同时可能也损伤了母系效应基因的DNA损伤修复功能。对此，开展了进一步研究，获得初步结果：有丝分裂期发挥监督作用的纺锤体组装检查点SAC及相关分子MAD2、Aurora B、H3S10P、TTK，等也同样因此受到影响；同时在如何改善卵母细胞质量方面开展了相关研究。下一步研究重点探讨改善卵母细胞质量措施等情况下，氧化应激状态下受精卵细胞的DNA损伤反应，与SAC交联的分子机制研究（已获后续基金资助）。本研究有助于理解氧化应激状态下受精卵损伤修复理论，为优化人类辅助生殖技术提供理论依据，具有重要的理论价值和广阔的应用前景。具体数据、图表，等详见发表SCI论文、研究生毕业论文，等。

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