One-carbon metabolism and epigenetic programming of mammalian development

Clare, Constance E (2021) One-carbon metabolism and epigenetic programming of mammalian development. PhD thesis, University of Nottingham.

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One-carbon (1C) metabolism comprises a series of integrated metabolic pathways, including the linked methionine-folate cycles, that provide methyl groups for the synthesis of biomolecules and the epigenetic regulation of gene expression via chromatin methylation. Most of the research investigating the function of 1C metabolism pertains to studies undertaken in the rodent liver. Comparatively little is known about the function of 1C metabolism in reproductive and embryonic cells, particularly in domestic ruminant species. Periconceptional dietary deficiencies in 1C substrates and cofactors are known to lead to epigenetic alterations in DNA methylation in genes that regulate key developmental processes in the embryo. Such modifications can have negative implications on the subsequent development, metabolism and health of offspring. This thesis sought to improve current understanding of the regulation of 1C metabolism in the ruminant liver, ovary and preimplantation embryo through in vivo and in vitro nutritional supplementation experiments coupled with metabolomic, transcriptomic and epigenetic analyses.

The first part of this thesis (Chapter 2) assessed the metabolic consequences of dietary methyl deficiency using novel mass spectrometry–based methods that were developed for the quantification of B vitamins, folates and 1C-related amines in sheep liver. This study provided the first comparison of the relative abundance of bioactive 1C metabolites in liver harvested from methyl deficient sheep relative to a control study population of abattoir derived sheep. Relevant reductions in dietary methyl availability led to significant alterations in hepatic 1C metabolite concentrations. Large natural variations in the hepatic concentrations of individual metabolites in both sheep study populations reflected the dietary and genetic variation in our chosen outbred model species. These metabolomics platforms will be useful for investigating 1C metabolism and linked biochemical pathways in order to facilitate future dietary and genetic studies of metabolic health and epigenetic regulation of gene expression.

Based on the absence of methionine cycle enzyme transcripts (e.g. MAT1A and BHMT) in the bovine ovary and preimplantation embryo, the second part of this thesis (Chapter 3 and Chapter 4) addressed the hypothesis that ruminant reproductive and embryonic cells are highly sensitive to methyl group availability and, therefore, epigenetic programming during the periconceptional period. Transcript analyses confirmed MAT2A expression in the bovine liver, ovary and at each stage of preimplantation embryo development assessed to Day 8. Transcripts for BHMT isoforms (BHMT and BHMT2) were detected in the bovine ovary but were weak or absent in embryos, highlighting a key difference in methionine metabolism between hepatic and reproductive cells.

Bovine embryos were produced in vitro using custom-made media containing 0 (nonphysiological), 10 (low physiological), 50 (high physiological), and 500 µmol/L (supraphysiological) added methionine (Chapter 3). Gross morphological assessments of embryo stage, grade, cell lineage allocation and primary sex ratio revealed that culture in non- and supraphysiological methionine concentrations was detrimental for embryo development, whilst culture in the high physiological concentration appeared to be best. Reduced representation bisulphite sequencing (RRBS) of inner cell mass (ICM) and trophectoderm (TE) cells immunodissected from Day 8 blastocysts demonstrated that culturing embryos in low physiological methionine led to global hypomethylation within both cell lineages. Bioinformatic analyses of differentially methylated genes included gene set enrichment analyses (GSEA). Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that were enriched within the ICM were associated with protein catabolism and autophagy, and significant terms and pathways enriched within the TE were associated with cellular transport. Of particular biological interest was the loss of methylation within regulatory region (DMR2) of the paternally imprinted gene, IGF2R, in the TE following culture in low physiological methionine. Transcript analysis found no significant effect of methionine concentration on the expression of IGF2R or the antisense transcript, AIRN, in the primary cell lineages of the Day 8 bovine preimplantation embryo. Hypomethylation of IGF2R DMR2 has been associated with aberrant IGF2R expression and large offspring syndrome (LOS) in cattle and sheep that were subjected to embryo manipulation during assisted reproductive technology (ART) procedures, such as somatic cell nuclear transfer (SCNT) or non-physiological in vitro embryo culture environments.

Chapter 5 sought to evaluate the effect of somatic donor cell type on epigenetic reprogramming via DNA methylation in hepatocytes isolated from cloned sheep. RRBS facilitated the comparison of methylation reprogramming between Finn Dorset (D) clone hepatocytes and their mammary epithelial (OP5) donor cell line; and, Lleyn (L) clone hepatocytes and their Lleyn fetal fibroblast (LFF4) donor cell line. Methylation was most closely correlated between D and L clone hepatocytes than between clones and their respective donor cell lines. In general, hepatocytes were hypomethylated relative to their somatic donor cell nuclei. GSEA identified genes that encoded transcription factor proteins enriched within the ‘Sequence-specific DNA binding’ term (GO:0043565) as differentially methylated between clone hepatocytes and their donor cell lines. In addition, imprinted genes, including IGF2R, were differentially methylated in clone hepatocytes relative to somatic cell nuclei.

In summary, this thesis promotes and supports the importance of an optimal methyl balance to support periconceptional development in mammals. The experiments detailed herein provide an insight into the metabolic consequences of dietary methyl deficiency (and excess) in outbred populations of domestic ruminants, with a specific focus on the liver, ovary and preimplantation embryo. The results demonstrate that tissue- and species-specific features of 1C metabolism render ruminant embryonic cells sensitive to methionine inputs within a physiological range. The observation that in vitro embryo culture and manipulation techniques, such as somatic cell nuclear transfer, can cause epigenetic alterations to DNA methylation during preimplantation development provides a basis for further study into the safety and efficacy of emerging assisted reproductive technologies.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Sinclair, Kevin D
Alberio, R
Barrett, DA
Keywords: One-carbon metabolism, Methionine, Folate, Vitamin B12, Epigenetics, DNA methylation, Developmental origins of health and disease, Assisted reproduction
Subjects: Q Science > QP Physiology > QP1 Physiology (General) including influence of the environment
Faculties/Schools: UK Campuses > Faculty of Science > School of Biosciences
Related URLs:
Item ID: 64521
Depositing User: CLARE, Constance
Date Deposited: 31 Jul 2021 04:40
Last Modified: 31 Jul 2021 04:40

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