Epigenetic remodeling of sheep oocytes and embryos induced by maternal methionine supplementation

Environmental factors can influence gene expression and developmental outcomes through epigenetic modifications. Although maternal diet influences offspring DNA methylation and phenotypes, its effects on the oocyte and the resulting embryonic epigenome remain poorly understood. Here, we investigated the effect of maternal methionine supplementation on DNA methylation patterns in oocytes and embryos in Polypay sheep. Whole-genome bisulfite sequencing (WGBS) was performed on oocytes collected from 16 twin ewe pairs (8 methionine-treated and 8 controls). These ewes were later bred to control rams, and embryos were flushed for WGBS as well. In oocytes, 2,056 differentially methylated cytosines (DMCs) were identified. Additionally, 17 mitochondrial DMCs were identified, with 12 hypermethylated and 5 hypomethylated. In embryos, 113 DMCs were identified. Mitochondrial DNA analysis revealed 22 hypermethylated DMCs. To assess the inheritance of methyl marks, we compared DMCs between oocytes and embryos. While no direct overlaps were found in nuclear DNA, 3 CpGs exhibited opposite methylation trends - hypomethylated in oocytes but hypermethylated in embryos. In contrast, 5 mitochondrial DMCs overlapped between oocytes and embryos. To functionally assess the role of differentially methylated genes, we performed siRNA-mediated knockdown of 2 embryo DMC-associated genes: <i>SCRIB</i> and <i>CERS3</i>. Knockdown of <i>SCRIB</i> led to a 16.4% average decrease in blastocyst formation rate (<i>p</i> = 0.001), while <i>CERS3</i> knockdown resulted in a 9.5% decrease (<i>p</i> = 0.005). These results demonstrate that maternal methionine supplementation alters nuclear and mitochondrial DNA methylation in oocytes and embryos, and that affected genes may play critical roles in early embryonic development, contributing to fetal programming.