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

ABSTRACT: 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

ORGANISM(S): Ovis aries

PROVIDER: GSE304764 | GEO | 2025/11/18

REPOSITORIES: GEO

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Project description:An important facet of the oocyte to embryo transition in mammals is the rapid elimination of a subset of the maternal products that got accumulated during oogenesis. RNA studies support a view that the transition occurs quite rapidly. Whether this applies also for proteins remains to be determined beyond the very few cases known to date. We report that COPS3, the 3rd subunit of the COP9 signalosome complex, forms a large protein deposit in mouse oocytes (94th percentile of the riBAQ distribution). The one and only knock-out study had previously shown that Cops3 null (-/-) mouse embryos obtained from heterozygous intercrosses arrested after 5.5 dpc and were resorbed by 8.5 dpc mainly due to increased cell death in the epiblast (PMID: 12972600). However, more recent studies from our group ascribed Cops3 gene with a developmental role already at the 2-cell stage. Specifically, the sister blastomeres differ from each other in Cops3 mRNA levels and distribution, preceding the discordance of epiblast formation in derivative twin blastocysts, and implicating Cops3 in the molecular underpinnings of totipotency. This discrepancy between original knock-out result and recent observations can be resolved if we posit that the first requirement for Cops3 gene function was bridged by maternal protein that outlived the locus excision, consistent with the observation that Cops3 -/- blastocysts had the same immunostaining intensity as the +/- or +/+ counterparts in the original knock-out study. Thus, these contradicting observations support a hypothesis that 5.5 dpc may not have been the first time when the gene function was needed in development, but the second time. To test this hypothesis, pronuclear-stage mouse oocytes were subjected to an immunological method that directly targets the protein of interest by way of proteasomal degradation of the COPS3-antibody-TRIM21 complex. Briefly, pronuclear-stage mouse oocytes were microinjected with a mixture of the COPS3-specific purified monoclonal IgG antibody, mCherry-Trim21 mRNA and Oregon Green dextran beads (inert tracer). Injected oocytes were then allowed to develop and collected for lysis 24 hours later, when they reached the 2-cell stage but were not able to progress further. In addition to the COPS3, we targeted two additional proteins, OCT4 and TEAD4. The following seven experimental groups were examined by liquid chromatography-mass spectrometry (LC-MS/MS) using the pipeline described previously by Israel et al.: 1) non-manipulated 2-cell embryos; 2) 2-cell embryos from oocytes injected with Oregon Green dextran beads; 3) 2-cell embryos from oocytes injected with mCherry-Trim21 mRNA and Oregon Green; 4) 2-cell embryos from oocytes injected with mCherry-Trim21 mRNA, Oregon Green and anti-COPS3 (Abcam 79698); 5) 2-cell embryos from oocytes injected with anti-COPS3 (Abcam 79698) and Oregon Green; 6) 2-cell embryos from oocytes injected with mCherry-Trim21 mRNA, Oregon Green and anti-TEAD4 (Abcam 58310); 7) 2-cell embryos from oocytes injected with mCherry-Trim21 mRNA, Oregon Green and anti-OCT4 (Abcam 181557).

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

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