Epigenetic Remodeling of Sheep Oocytes and Embryos Induced by Maternal Methionine Supplementation

Environmental factors, including diet, can influence gene expression and developmental outcomes through epigenetic modifications. While maternal diet is known to affect offspring DNA methylation and phenotypes, its impact on the oocyte and subsequent embryo epigenome remains less understood. Here, we investigated how maternal methionine supplementation affects DNA methylation patterns in oocytes and embryos of Polypay ewes. Whole genome bisulfite sequencing (WGBS) was performed on oocytes collected from 16 twin ewe pairs (8 methionine-treated and 8 control). 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, with 2,034 hypomethylated and 22 hypermethylated. Additionally, 17 mitochondrial DMCs were identified, with 12 hypermethylated and 5 hypomethylated. In embryos, 113 DMCs were identified, with 112 hypermethylated and only one hypomethylated. 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, three CpGs exhibited opposite methylation trends—hypomethylated in oocytes but hypermethylated in embryos. In contrast, five mitochondrial DMCs overlapped between oocytes and embryos. To functionally assess the role of differentially methylated genes, we performed siRNA-mediated knockdown of two embryo DMC-associated genes: SCRIB and CERS3. Knockdown of SCRIB led to a 16.4% average decrease in blastocyst formation rate (p = 0.001), while CERS3 knockdown resulted in a 9.5% decrease (p = 0.005). These results demonstrate that maternal methionine supplementation alters both nuclear and mitochondrial DNA methylation in oocytes and embryos and that affected genes may play critical roles in early embryonic Overall design: Whole genome bisulfite sequencing (WGBS) was performed on oocytes collected from 16 twin Polypay ewe pairs (8 methionine-treated and 8 control). These ewes were later bred to control rams, and embryos were flushed for WGBS as well.