Interspecific Variation in One-Carbon Metabolism within the Ovarian Follicle, Oocyte, and Preimplantation Embryo: Consequences for Epigenetic Programming of DNA Methylation.

One-carbon (1C) metabolism provides methyl groups for the synthesis and/or methylation of purines and pyrimidines, biogenic amines, proteins, and phospholipids. Our understanding of how 1C pathways operate, however, pertains mostly to the (rat) liver. Here we report that transcripts for all bar two genes (i.e., BHMT, MAT1A) encoding enzymes in the linked methionine-folate cycles are expressed in all cell types within the ovarian follicle, oocyte, and blastocyst in the cow, sheep, and pig; as well as in rat granulosa cells (GCs) and human KGN cells (a granulosa-like tumor cell line). Betaine-homocysteine methyltransferase (BHMT) protein was absent in bovine theca and GCs, as was activity of this enzyme in GCs. Mathematical modeling predicted that absence of this enzyme would lead to more volatile S-adenosylmethionine-mediated transmethylation in response to 1C substrate (e.g., methionine) or cofactor provision. We tested the sensitivity of bovine GCs to reduced methionine (from 50 to 10 µM) and observed a diminished flux of 1C units through the methionine cycle. We then used reduced-representation bisulfite sequencing to demonstrate that this reduction in methionine during bovine embryo culture leads to genome-wide alterations to DNA methylation in >1600 genes, including a cohort of imprinted genes linked to an abnormal fetal-overgrowth phenotype. Bovine ovarian and embryonic cells are acutely sensitive to methionine, but further experimentation is required to determine the significance of interspecific variation in BHMT expression.

Comprehensive and quantitative profiling of B vitamins and related compounds in the mammalian liver.

A method for the simultaneous quantification of B vitamins and related amines in one-carbon (1C) metabolism would benefit the study of diet and genetic/epigenetic regulation of mammalian development and health. We present a validated method for the simultaneous quantitative analysis of 13 B vitamers and four related 1C-pathway amine intermediates in liver using hydrophilic interaction chromatography (HILIC) coupled to electrospray ionization tandem mass spectrometry. Frozen sheep liver samples (50 mg) were homogenized in cold 50% acetonitrile containing 1% acetic acid with the addition of two isotope labelled internal standards. Hot acid hydrolysis was applied to release the protein-bound forms. The separation of 17 analytes was achieved using a pHILIC column with a total run time of 13 min. Detection was achieved in electrospray positive ionisation mode. Limits of detection for the majority of analytes were within the range of 0.4-3.2 pmol/g. The method was applied to 266 sheep liver samples and revealed that adenosylcobalamin, methylcobalamin, pyridoxic acid, flavin adenine dinucleotide and thiamine were the major forms of the B vitamers present with pyridoxal 5'-phosphate and thiamine pyrophosphate being detected at lower concentrations. Trimethylglycine and methylglycine were the predominant 1C-related amines measured. As anticipated, the B vitamin status of individuals varied considerably, reflecting dietary and genetic variation in our chosen outbred model species. This method offers a simple sample extraction procedure and provides comprehensive coverage of B vitamins coupled with good sensitivity and reliability.

One-Carbon Metabolism: Linking Nutritional Biochemistry to Epigenetic Programming of Long-Term Development.

One-carbon (1C) metabolism comprises a series of interlinking metabolic pathways that include the methionine and folate cycles that are central to cellular function, providing 1C units (methyl groups) for the synthesis of DNA, polyamines, amino acids, creatine, and phospholipids. S-adenosylmethionine is a potent aminopropyl and methyl donor within these cycles and serves as the principal substrate for methylation of DNA, associated proteins, and RNA. We propose that 1C metabolism functions as a key biochemical conduit between parental environment and epigenetic regulation of early development and that interindividual and ethnic variability in epigenetic-gene regulation arises because of genetic variants within 1C genes, associated epigenetic regulators, and differentially methylated target DNA sequences. We present evidence to support these propositions, drawing upon studies undertaken in humans and animals. We conclude that future studies should assess the epigenetic effects of cumulative (multigenerational) dietary imbalances contemporaneously in both parents, as this better represents the human experience.