Centre of Reproductive Medicine, Inner Mongolia People's Hospital, Hohhot, Inner Mongolia, 010021, China.
The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, College of Life Sciences, Inner Mongolia University, Hohhot, Inner Mongolia, 010020, China.
Theriogenology. 2022 Jan 1;177:11-21. doi: 10.1016/j.theriogenology.2021.09.032. Epub 2021 Oct 1.
Understanding the molecular level changes of oocyte cryopreservation and the subsequent warming process is essential for improving the oocyte cryopreservation technologies. Here, we collected the mature metaphase II (MII) oocytes from mice and vitrified. After thawing, single-cell whole-genome bisulphite sequencing (scWGBS) and single-cell RNA sequencing (scRNA-seq) were used to investigate the molecular attributes of this process. Compared to the fresh oocytes, the vitrified oocytes had lower global methylation and gene expression levels, and 1426 genes up-regulated and 3321 genes down-regulated. The 1426 up-regulated differentially expressed genes (DEGs) in the vitrified oocytes were mainly associated with the histone ubiquitination, while the 3321 down-regulated genes were mainly enriched in the mitochondrion organisation and ATP metabolism processes. The differentially methylated regions (DMRs) were mainly located in promoter, intron and exon region of genes, and the length of DMRs in the vitrified oocytes were also significantly lower than that of the fresh oocytes. Notably, there were no significant difference in the expression levels of DNA demethylases (Tet1, Tet2 and Tet3) and methyltransferases (Dnmt3a and Dnmt3b) between two treatments of oocytes. However, Dnmt1 and kcnq1ot1, which are responsible for maintaining DNA methylation, were significantly down regulated in the vitrified oocytes. Gene regulatory network (GRN) analysis showed the Dnmt1 and kcnq1ot1 play a core role in regulating methylation and expression levels of downstream genes. Moreover, some genes associated with oocyte quality were significantly down-regulated in the vitrified oocytes. The present data provides a new perspective for understanding the impact of vitrification on oocytes.
了解卵母细胞冷冻保存的分子水平变化及其随后的解冻过程对于提高卵母细胞冷冻保存技术至关重要。在这里，我们从小鼠中收集成熟的中期 II (MII) 卵母细胞并进行玻璃化冷冻。解冻后，采用单细胞全基因组亚硫酸氢盐测序 (scWGBS) 和单细胞 RNA 测序 (scRNA-seq) 来研究这一过程的分子特征。与新鲜卵母细胞相比，玻璃化冷冻的卵母细胞的整体甲基化和基因表达水平较低，有 1426 个基因上调和 3321 个基因下调。玻璃化冷冻卵母细胞中上调的 1426 个差异表达基因 (DEG) 主要与组蛋白泛素化有关，而下调的 3321 个基因主要富集在线粒体组织和 ATP 代谢过程中。差异甲基化区域 (DMR) 主要位于基因的启动子、内含子和外显子区域，且玻璃化冷冻卵母细胞中的 DMR 长度也明显低于新鲜卵母细胞。值得注意的是，两种处理方式的卵母细胞中 DNA 去甲基酶 (Tet1、Tet2 和 Tet3) 和甲基转移酶 (Dnmt3a 和 Dnmt3b) 的表达水平没有显著差异。然而，负责维持 DNA 甲基化的 Dnmt1 和 kcnq1ot1 在玻璃化冷冻卵母细胞中显著下调。基因调控网络 (GRN) 分析表明，Dnmt1 和 kcnq1ot1 在调节下游基因的甲基化和表达水平方面发挥核心作用。此外，一些与卵母细胞质量相关的基因在玻璃化冷冻卵母细胞中显著下调。本研究数据为理解玻璃化对卵母细胞的影响提供了新的视角。
