玻璃化冷冻后卵母细胞发育潜能与H1FOO表达水平关联机制的研究

Mature oocyte vitrification technology and the establishment of egg bank is the most promising way to reserve the female fertility. To improve the efficiency and safety of oocyte cryopreservation technology is one of the focuses of attention in the field of assisted reproductive technology. On the basis of the our research result that the expression of H1foo in oocytes is reducing after oocyte vitrification and the development of early stage embryo is depressed due to the epigenetic mechanism, in consideration of that H1foo is essential for meiotic maturation of oocytes, nuclear reconstruction, expression of early stage embryo genes and modification of chromatin, We proposed the relationship between the H1FOO expression in oocyte and the embryo genome activation for the first time. In this study, we change the expression H1FOO in mouse oocytes and observe the level of histone acetylation, RNA polymerase II phosphorylation status and imprinted genes IGF2/IGF2R in the early stage of the embryos after fertilizaiton in order to discuss the relationship between H1FOO expression in oocytes and the embryo genome activation after fertilization, and reveal the mechanism of the relationship of H1foo expression and early stage embryo development. The study may not only provide a new theoretical basis to improve oocyte cryopreservation, but also has important theoretical significance for understanding the molecular mechanism of early embryonic development.

成熟卵母细胞冻存技术和建立卵子库是保存女性生育储备力最具前景的途径，完善卵母细胞冻存技术、提高其效率和安全性是辅助生殖技术领域所关注的热点。我们在前期卵母细胞特异链接组蛋白H1FOO 表达降低，早期胚胎发育减慢停滞增多，玻璃化冷冻对于卵母细胞在表观遗传层面的影响与后续早期胚胎发育潜能降低相关的研究结果基础上，首次提出卵母细胞H1FOO 表达改变与卵母细胞受精后胚胎合子基因正常启动的关联，本课题以鼠卵母细胞为研究材料，通过干扰H1FOO 表达变化后，观察卵母细胞受精后受精卵和各胚胎发育时期直至囊胚期的组蛋白乙酰化水平、RNA 多聚酶II 磷酸化状态和印记基因IGF2/IGF2R 状态，探讨H1FOO表达变化和胚胎基因组启动的关系，揭示H1FOO 与早期胚胎发育潜能的关联机制，不仅对于合理改善卵母细胞冷冻保存技术提供新的理论根据，并且对于理解早期胚胎发育的分子机制具有重要的理论意义。

成熟卵母细胞冷冻保存技术和建立卵子库是保存女性生育储存力最具前景的途径，而目前该技术的效率仍较胚胎冷冻低下，完善卵母细胞冻存技术、提高其效率和安全性是辅助生殖技术领域所关注的热点。本课题以小鼠卵母细胞为研究材料，检测新鲜卵母细胞和和冷冻后卵母细胞及受精后各发育阶段的胚胎直至囊胚期的H1Foo表达改变及组蛋白乙酰化水平、印记基因IGF2状态，并对各期胚胎进行了单细胞RNA测序分析，分析玻璃化冷冻对于后续胚胎发育中基因表达的影响；并且研究流体静力是否提高玻璃化冷冻卵子发育潜能，研究抗氧化剂谷光甘肽在玻璃化冷冻前的预孵育在提高卵母细胞发育潜能的作用。结果如下：.1、H1foo-α的表达在玻璃化冷冻后的卵母细胞和后续发育的2胚胎中的表达较新鲜是卵母细胞是降低的，H1foo-β在玻璃化冷冻卵母细胞的合子期核2细胞期表达是降低的，而组蛋白H1h1f0在卵和合子期都降低，但是在2细胞期由于H1foo的表达降低而表达上升。在2细胞期以后的各期胚胎发育中未见差异。acH4在玻璃化冷冻后的卵母细胞及后续发育胚胎中未见明显差异。.2、单细胞RNA测序结果表明，在2细胞期冷冻组和对照组间有1575个基因表达的差异，但是到融合期胚胎，只有613个基因差异表达，最显著差异表达的基因是有关氧化还原过程，说明玻璃化冷冻对于卵母细胞后续发育的影响主要在于对于氧化还原的影响。.3、MII卵子玻璃化冷冻前经流体静力压处理，其在冷冻过程中的平衡时间要显著降低，相对于对照组卵子的ROS产生降低，透明带硬度降低，THP处理不影响玻璃化冷冻卵子的纺锤体结构及DNA拷贝数。.4、谷胱甘肽预处理组的卵母细胞玻璃化冷冻后受精率和后续胚胎发育较对照组显著改善。胚胎跨越2细胞阻滞发育成囊胚后，最终小鼠出生率并无明显区别。玻璃化冷冻导致卵母细胞线粒体分布的改变，ROS水平上升以及纺锤体异常形态的增加。GSH-Oet预孵育可以改善上述损伤。RT-qPCR显示，冷冻对照组Bcl-2水平低于GSH-OEt组，BAX和MnSoD表达均高于GSH-OEt组。.本课题的研究在基因表达、ROS、氧化还原等各方面揭示了玻璃化冷冻对于卵母细胞和后续胚胎发育的影响，并且对改善卵母细胞冷冻结局进行了探索。