Shida M, Ito J, Inoue Y, Hara S, Shirasuna K, Iwata H
Deportment of Animal Science, Tokyo University of Agriculture, Funako 1737, Atsugi City, Japan.
J Assist Reprod Genet. 2025 Apr;42(4):1275-1286. doi: 10.1007/s10815-025-03415-7. Epub 2025 Feb 5.
Dimethyl sulfoxide (DMSO) alters DNA methylation in vitrified-warmed embryos and potentially affects subsequent development. This study aimed to examine possible countermeasures against DMSO-induced demethylation.
In vitro-produced bovine embryos (8-cell stage) were vitrified using a combination of DMSO and ethylene glycol (EG) or propylene glycol (PG) + EG. After warming, the lipid content and expression levels of 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), DNMTs, and TETs were examined. In addition, RNA-sequencing was performed on blastocysts derived from the vitrified embryos. Furthermore, the effect of supplementation with a vitrification medium containing DMSO and N-acetyl-L-cysteine (NAC, 5 mM) on the levels of 5mC in embryos was examined.
Vitrification decreased the levels of 5mC and increased the levels of 5hmC in 8-cell stage embryos. Low levels of 5mC persisted until the blastocyst stage in the DMSO group but increased in the PG group. The expression level of TET3A was higher in the DMSO group than in the fresh group, but not in the PG group. Both cryoprotectants reduced the lipid levels in post-warmed 8-cell stage embryos. The addition of NAC ameliorated DMSO-induced demethylation at both the 8-cell and blastocyst stages. RNA-seq analysis revealed that PG-specific pathways included ribosomes and mitochondria and that both DMSO and PG affected cGMP-PGK, MAPK, Wnt, and insulin secretion-related signaling. The K-medoids method predicted that DMSO affected cell adhesion molecules and that MAPK signaling was affected the most.
PG and NAC may antagonize DMSO-induced demethylation; however, PG exerts adverse effects on embryos.
二甲基亚砜（DMSO）会改变玻璃化冷冻复苏胚胎中的DNA甲基化，并可能影响后续发育。本研究旨在探究针对DMSO诱导的去甲基化的可能应对措施。
体外生产的牛胚胎（8细胞期）使用DMSO与乙二醇（EG）或丙二醇（PG）+EG的组合进行玻璃化冷冻。复苏后，检测脂质含量以及5-甲基胞嘧啶（5mC）、5-羟甲基胞嘧啶（5hmC）、DNA甲基转移酶（DNMTs）和TET蛋白的表达水平。此外，对来自玻璃化冷冻胚胎的囊胚进行RNA测序。此外，还检测了向含有DMSO和N-乙酰-L-半胱氨酸（NAC，5 mM）的玻璃化冷冻液中添加物质对胚胎中5mC水平的影响。
玻璃化冷冻降低了8细胞期胚胎中5mC的水平，并增加了5hmC的水平。在DMSO组中，低水平的5mC一直持续到囊胚期，但在PG组中有所增加。DMSO组中TET3A的表达水平高于新鲜组，但PG组中并非如此。两种冷冻保护剂均降低了复苏后8细胞期胚胎中的脂质水平。添加NAC改善了DMSO在8细胞期和囊胚期诱导的去甲基化。RNA测序分析表明，PG特异性途径包括核糖体和线粒体，并且DMSO和PG均影响cGMP-PGK、MAPK、Wnt和胰岛素分泌相关信号通路。K-medoids方法预测DMSO影响细胞粘附分子，且MAPK信号通路受影响最大。
PG和NAC可能拮抗DMSO诱导的去甲基化；然而，PG对胚胎有不良影响。
