Epigenetic Modifier Supplementation Improves Mitochondrial Respiration and Growth Rates and Alters DNA Methylation of Bovine Embryonic Fibroblast Cells Cultured in Divergent Energy Supply

Abstract

Epigenetic modifiers (EM; methionine, choline, folate, and vitamin B12) are important for early embryonic development due to their roles as methyl donors or cofactors in methylation reactions. Additionally, they are essential for the synthesis of nucleotides, polyamines, redox equivalents, and energy metabolites. Despite their importance, investigation into the supplementation of EM in ruminants has been limited to one or two epigenetic modifiers. Like all biochemical pathways, one-carbon metabolism needs to be stoichiometrically balanced. Thus, we investigated the effects of supplementing four EM encompassing the methionine–folate cycle on bovine embryonic fibroblast growth, mitochondrial function, and DNA methylation. We hypothesized that EM supplemented to embryonic fibroblasts cultured in divergent glucose media would increase mitochondrial respiration and cell growth rate and alter DNA methylation as reflected by changes in the gene expression of enzymes involved in methylation reactions, thereby improving the growth parameters beyond Control treated cells. Bovine embryonic fibroblast cells were cultured in Eagle’s minimum essential medium with 1 g/L glucose (Low) or 4.5 g/L glucose (High). The control medium contained no additional OCM, whereas the treated media contained supplemented EM at 2.5, 5, and 10 times (×2.5, ×5, and ×10, respectively) the control media, except for methionine (limited to ×2). Therefore, the experimental design was a 2 (levels of glucose) × 4 (levels of EM) factorial arrangement of treatments. Cells were passaged three times in their respective treatment media before analysis for growth rate, cell proliferation, mitochondrial respiration, transcript abundance of methionine–folate cycle enzymes, and DNA methylation by reduced-representation bisulfite sequencing. Total cell growth was greatest in High ×10 and mitochondrial maximal respiration, and reserve capacity was greatest (p < 0.01) for High ×2.5 and ×10 compared with all other treatments. In Low cells, the total growth rate, mitochondrial maximal respiration, and reserve capacity increased quadratically to 2.5 and ×5 and decreased to control levels at ×10. The biological processes identified due to differential methylation included the positive regulation of GTPase activity, molecular function, protein modification processes, phosphorylation, and metabolic processes. These data are interpreted to imply that EM increased the growth rate and mitochondrial function beyond Control treated cells in both Low and High cells, which may be due to changes in the methylation of genes involved with growth and energy metabolism.

Effects of DNA Methylation on Gene Expression and Phenotypic Traits in Cattle: A Review

“…Meanwhile, the same sequence could possess different DNA methylation patterns in different tissues, regulating the specific expression of genes [117]. In addition, epigenetic modifiers such as methionine, choline, folic acid and vitamin B12 have been proven to participate in methylation responses during early embryonic development [118].…”

Maternal Mineral Nutrition Regulates Fetal Genomic Programming in Cattle: A Review

“…Although most of the fetal size increase takes place in the last trimester, the first third of gestation is a critical period for organogenesis and tissue hyperplasia, as well as placentation [21,34,45,46], as shown in Figure 1. The prenatal growth trajectory of the conceptus is controlled by maternal nutrition either directly, by the provision of the essential nutrients; indirectly, via placental function, which regulates the transport of these nutrients [47]; or by altering the epigenetic mechanisms [48][49][50][51]. Pregnancy recognition occurs 15 to 16 days after estrous in cattle [58] and initiates maternal uterine vasculature changes to allow for the proper respiratory gas exchange and nutrient transfer to the developing embryo.…”

DNA methylation dataset of bovine embryonic fibroblast cells treated with epigenetic modifiers and divergent energy supply

“…We performed a genome-wide DNA methylation analysis of bovine embryonic fibroblast cells to investigate the effects of glucose and epigenetic modifiers (EM) supplementation on cell epigenetic programming [1] . Herein, we describe the methylome datasets generated using Reduced Representation Bisulfite Sequencing (RRBS).…”

“…Data was generated from Bovine Embryonic Tracheal fibroblast cells treated with two glucose levels and 3 different concentrations of EM (3 samples per treatment). Table 1 shows a summary of the metadata, sample description, mapping statistics per sample, the experimental design, and the number of replicates per treatment as described elsewhere [1] . The (1) raw paired-end RRBS reads from 18 samples; (2) the metadata; and (3) the normalized DNA methylation levels are publicly available on the GEO database (GEO accession ID GSE180362); (4) Supplementary File S1 is the code used for DNA methylation data analysis as described in the methods section.…”