冷冻温度和CPAs浓度影响牛GV期卵母细胞发育能力的机理研究

The cryopreservation of GV stage oocytes is of great significance to the conservation of animal genetic resources and the preservation of fertility for women with cancer radiotherapy, but a satisfactory method of freezing has not been established. Our previous studies have shown that the developmental ability of vitrified oocytes is different with different freezing protocols, but the mechanism is not clear. Although preliminary studies have found that there are differences in cell structure and maternal gene expression in different vitrified oocytes, however, more regulatory pathways have to be explored to understand the mechanisms by which different protocols affect oocyte development. In this project, bovine GV stage oocytes will be vitrified in different temperatures and concentrations of cryoprotective agents (CPAs). The transcriptional sequence of oocytes will be sequenced by RNA-Seq. The differentially expressed genes (DEGs) will be screened and the regulatory pathways of key DEGs will be identified by bioinformatics. The proteins expression of key DEGs is going to be verified by western blot after qRT-PCR be used to verify the expression level of DEGs. At the same time, the effects of freezing temperature and CPAs concentration on oocyte development, cytoskeleton and organelle will be observed by in vitro fertilization, immunofluorescence and electron microscopy. The mechanism of the effect of freezing temperature and CPAs concentration on the oocyte development ability will be analyzed synthetically to provide the theoretical basis for improving the oocyte freezing technology.

GV期卵母细胞冷冻对动物遗传资源保护和癌症放疗妇女生育能力保存具有重要意义，但尚未建立令人满意的方法。我们的前期研究证明，不同冷冻方案冷冻卵母细胞的发育能力不同，但机制不清楚。尽管初步研究发现，不同方案冷冻卵母细胞的细胞结构和母源基因表达有差异，但必须挖掘更多的调控通路才能弄清不同方案影响卵母细胞发育能力的机制。本项目拟将牛GV期卵母细胞在不同温度、不同浓度保护剂（CPAs）条件下玻璃化冷冻，用RNA-Seq进行转录组测序，用生物信息学筛选差异表达基因（DEGs）并确定关键基因调控通路，用qRT-PCR验证关键基因，用Western blot验证蛋白表达；同时，分别用体外受精、免疫荧光和电镜观察冷冻温度和CPAs浓度对卵母细胞发育能力、细胞骨架和细胞器的影响；综合分析冷冻温度和CPAs浓度影响卵母细胞发育能力的机理，为改进卵母细胞冷冻技术提供理论依据。

按照项目任务书完成了如下研究：.（1）不同VTs和CPAs浓度对牛GV期卵母细胞冷冻后发育能力及胚胎移植效果的影响。发现液氦玻璃化冷冻可以明显改进牛GV期卵母细胞冷冻效果；随着VTs的提高，CPAs的浓度可以适当降低，从而为解决玻璃化冷冻中高浓度CPAs损伤难题提供了一条新途径；胚胎移植试验结果表明：牛GV期卵母细胞无论是否经过玻璃化冷冻，或者无论VTs和CPAs是否不同，只要经过冷冻后获得的桑椹胚和早期囊胚达到移植标准，就可以获得正常犊牛。.（2）VTs和CPAs浓度对牛GV期卵母细胞玻璃化冷冻后转录组、部分蛋白表达、细胞骨架和细胞器的影响。用RNA-Seq、qRT-PCR和生物信息学筛选出了与VTs和CPAs浓度相关的基因；用Western blot验证了与VTs和CPAs浓度相关的关键蛋白表达水平；用免疫荧光技术研究了VTs和CPAs浓度对卵母细胞细胞骨架和发育能力的影响，发现更低的VTs和CPAs浓度明显提高骨架蛋白表达水平，降低微丝、微管异常分布和缺失；电镜观察了VTs和CPAs浓度对卵母细胞细胞器和发育能力的影响，发现更低的VTs和CPAs剂浓度提高了卵母细胞的抗凋亡能力，改善卵母细胞超微结构的损伤，从而有效地提高了牛GV期卵母细胞玻璃化冷冻后的发育能力。.（3）不同VTs和CPAs浓度下冷冻牛GV期卵母细胞的发育能力与凋亡和线粒体相关基因mRNA表达的关系。发现降低VTs和CPAs浓度通过改变凋亡基因和线粒体基因mRNA表达水平而增加囊胚率。.（4）LncRNA介导的VTs和CPAs对玻璃化冷冻牛GV期卵母细胞发育能力影响的分子机制。发现不同的VTs和CPAs显著影响牛GV期卵母细胞发育能力和lncRNA表达；受不同VTs和CPAs浓度影响的lncRNA可能通过参与细胞骨架、细胞周期、蛋白质代谢、免疫、脂质代谢和跨膜转运等生物学过程来调控玻璃化冷冻后卵母细胞的发育。.（5）羟基磷灰石（HA）纳米颗粒改进牛GV期卵母细胞玻璃化冷冻技术。发现在VSⅡ中加入0.05%HA纳米颗粒能够提高牛GV期卵母细胞玻璃化冷冻后的发育能力，这种发育能力的改进可能与加入HA纳米颗粒后卵母细胞活性氧水平的降低和线粒体膜电位水平的提高密切相关。