Abstract
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.
Highlights
We reported that transcripts for specific enzymes, most notably betaine-homocysteine methyltransferase (BHMT; EC 2.1.1.5), were not expressed in any of the somatic cell types found within the ovarian follicle, oocyte, and preimplantation embryo in the cow [14]
As a first step in establishing the extent to which 1C metabolic pathways operate within the ovarian follicle, the oocyte and preimplantation embryo, transcript expression for fourteen 1C related genes, representing the linked methionine, folate, and transsulfuration pathways [2], was determined in theca, granulosa, and cumulus cells, oocytes, and expanded blastocysts from the cow, sheep, and pig by quantitative real-time PCR
Quantification was based on normalization to a single reference gene (ACTB), which we previously had deemed stable in the different cell types studied [29,30], it is important to note that establishing transcript presence, rather than transcript abundance, was the primary goal of this phase of the study
Summary
One-carbon (1C) metabolism refers to a series of interlinking and closely associated metabolic pathways that serve to provide methyl groups (1C units) for the de novo synthesis and/or methylation of purines and pyrimidines, biogenic amines, proteins, and phospholipids; all of which are critical for cellular function [1,2]. These cellular processes are important during the periconceptional period, which witnesses sweeping epigenetic alterations to DNA and associated proteins during the latter stages of gametogenesis and early embryogenesis [3]. A number of studies across a diverse range of species, including humans, have demonstrated that disturbances to 1C metabolism brought about by parental malnutrition [4,5,6] and/or exposure to environmental chemicals [7,8] during the periconceptional period, or arising as a consequence of assisted reproduction (ART) [9,10,11], can lead to epigenetic alterations to chromatin with long-term consequences for offspring health and wellbeing
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