Cd9基因表观遗传修饰影响超低温冷冻牛卵母细胞受精潜力的研究

Tetraspanin CD9 has been proved to be a key proten in the regulation of sperm-oocyte interaction during fertilization in our previous and other studies. Cd9 expression is regulated by epigenetic modification. After oocyte cryopreservation, the lower fertilization rate and decreased developmental capacity were observed, which can’t satisfy the needs in the in vitro embryo production and animal cloning in bovine. Therefore, epigenetic regulation of cd9 gene potentially plays an important role in the decreceased developmental potential of cryopreserved oocytes. However, it is not clear how the epigenetic regulation resulted in the alteration of CD9 expression in bovine MII oocytes and their lower developmental potential after in vitro fertilization. In this study, after in vitro maturation, some of the bovine oocytes were cryopreserved by Open-Pulled Straw (OPS) method and those with normal morphology after warming were selected for the following use (treated group), the others were used as a control. Firstly, sodium bisufite genomic sequencing were employed to detect the difference btween the treated group and the control in the distribution of DNA methylation in the gene cd9 promoter region. Secondly, the histone acetylation in the promoter region of cd9 were detected by Chromatin Immunoprecipitation-chip (ChIP-chip) and real-time fluorescence quantitative RT-PCR (Q-PCR). Thirdly, the Q-PCR was used to detect the expression of miRNAs which may regulate the gene cd9 in bovine oocytes after cryopreservation. And the key miRNAs were selected to transfect into the oocytes, followed by both western blot and in vitro fertilization. Taken together, the changes of DNA methylation, histone acetylation and miRNA regulation in the bovine oocytes after cryopreservation, and their relationship with both CD9 expression and the fertiliztion capacity of the oocytes would be obtained. The results would provide insight into the mechanism how to improve the in vitro fertilization of bovine oocytes after cryopreservation, and benefit from the wide application of animal biotechnology.

跨膜蛋白CD9是精卵质膜相互作用的关键蛋白，表达水平受表观遗传修饰的调控。牛卵母细胞超低温冷冻后，受精潜力下降，其原因之一是cd9基因表达水平下调。然而， cd9基因表观遗传修饰影响冷冻牛卵母细胞受精潜力下降的机制，尚不清楚。因此，本项目通过亚硫酸盐测序和CHIP-chip等技术分别检测牛卵母细胞冷冻前后cd9基因启动子区域甲基化与组蛋白乙酰化水平的变化，结合基因mRNA和蛋白表达及受精后的卵裂率和囊胚率的差异，确定DNA甲基化和组蛋白修饰与卵母细胞受精潜力的关系。通过定量PCR检测冷冻前后卵母细胞中调控cd9基因的miRNA表达水平，筛选出关键的miRNA，并转染至成熟卵母细胞中，分析miRNA表达在卵母细胞体外受精与早期胚胎发育中的作用。研究结果旨在揭示牛冷冻卵母细胞cd9基因的表观遗传调控机制及其与受精潜力下降的关系，为提高冷冻卵母细胞的受精潜力提供科学依据。

跨膜蛋白CD9是精卵质膜相互作用的关键蛋白，表达水平受表观遗传修饰的调控。牛卵母细胞超低温冷冻后，受精潜力下降，其原因之一是CD9基因表达水平下调。然而， CD9基因表观遗传修饰影响冷冻牛卵母细胞受精潜力下降的机制，尚不清楚。为此，课题组采用卵母细胞超低温冷冻保存与解冻、体外成熟培养、免疫荧光染色、孤雌激活、实时荧光定量PCR及转录组分析等实验方法，结果显示：1）牛生发泡（germinal vesicle，GV）期卵母细胞冷冻解冻后发育潜力下降，与DNA甲基转移酶基因（DNMT1，DNMT3B）发生变化，关联影响CD9与CD81基因表达，由于CD9与CD81基因在精卵结合中发挥重要作用，进而影响卵母细胞受精；2）牛GV期卵母细胞超低温冷冻后转录组发生变化，当GV期卵母细胞冷冻解冻后培养2小时，12个基因表达上调和19个基因表达下调（P<0.05），体外成熟培养24小时后，1519个基因表达上调，2162个基因表达下调（P<0.05）；冷冻GV期卵母细胞体外成熟后，相比于新鲜成熟卵母细胞，有47个基因表达上调，6个基因表达下调（P<0.05）；新鲜GV期卵母细胞及其体外成熟卵母细胞和冷冻GV期卵母细胞及其体外成熟卵母细胞共有的基因数为2405个，新鲜GV期卵母细胞及成熟卵母细胞中特有的1378个，冷冻GV期卵母细胞及成熟卵母细胞中特有的1276个，基因组学变化进而导致冷冻卵母细胞发育率下降；3）GV期卵母细胞玻璃化导致体外成熟过程中线粒体膜电位和ATP含量降低，ROS水平上升，GSH水平降低，纺锤体形态异常以及纺锤体组装检测点相关基因（BubR1，Mad1，Mad2，Mps1）mRNA表达紊乱，从而降低了卵母细胞的体外成熟；4）卵母细胞超低温冷冻后孤雌发育率下降与合子细胞周期进程发生改变有关；5）卵母细胞超低温冷冻过程中添加褪黑素可促进冻融卵母细胞的体外发育。研究结果有助于丰富和完善受精生物学理论，有助于提高卵母细胞冷冻保存效率和体外胚胎发育质量，最终应用于扩大优良奶牛种群，具有重要的实践意义。