"The effect of Dnmt1 on methylation modification and gene expression changes during in vitro cryopreservation of mouse oocytes using WGBS and RNA-seq analysis"

Understanding the molecular effects of in vitro cryopreservation of oocytes and their warming process is essential for the development and optimization of fertility preservation technologies, especially in the field of reproductive medicine. In this study, mature oocytes at the metaphase II (MII) stage were collected from mice and, after in vitro cryopreservation, were subjected to in-depth molecular studies using single-cell whole genome bisulfite sequencing (scWGBS) and single-cell RNA sequencing (scRNA-seq). The results showed that frozen oocytes had a significant decrease in global DNA methylation levels and gene expression compared to fresh oocytes. Of the genes identified, 1426 genes were upregulated and 3321 genes were downregulated. The up-regulated genes were mainly enriched in histone ubiquitination pathways, while the down-regulated genes were related to processes such as mitochondrial organization and ATP metabolism. Differentially methylated regions (DMRs) were mainly identified in the promoter, intron, and exon regions of genes, and the length of these regions was significantly shorter in frozen oocytes than in fresh oocytes. Examination of enzymes involved in methylation showed that the expression levels of DNA demethylases Tet1, Tet2, and Tet3 and methyltransferases Dnmt3a and Dnmt3b did not change significantly, but Dnmt1 and kcnq1ot1, which play a critical role in maintaining DNA methylation, were significantly reduced. Analysis of gene regulatory networks (GRNs) revealed that these two genes play key roles in regulating methylation and expression of downstream genes. Furthermore, the reduced expression of genes associated with oocyte quality in in vitro frozen oocytes raises concerns about the reduced growth and fertility potential of these oocytes. This study reveals for the first time the broad effects of in vitro freezing on gene expression and epigenetic patterns in mouse oocytes and provides valuable insights for improving cryopreservation and oocyte quality preservation methods. These results may help develop new methods to increase the efficiency of fertility preservation and reduce the negative effects of cryopreservation.