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Metabolic Regulation During Early Embryo Development

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Metabolic Regulation During Early Embryo Development Metabolic regulation during early embryo development Paul Joseph McKeegan, BSc (Hons), PGCE A thesis submitted for the degree of PhD The University of Hull and the University of York Hull York Medical School January 2015 i Abstract The preimplantation embryo must satisfy dynamic changes in energy demand during development to the blastocyst stage. Energy is provided through regulated metabolic pathways including glycolysis, β-oxidation and oxidative phosphorylation. Oxygen consumption rate (OCR), representing overall oxidative metabolism, has been reported in several species but few studies have examined the bioenergetics of embryo development. Several methods were optimised to measure components of OCR by individual embryos. On average, 66% of blastocyst OCR was coupled to ATP synthesis, the majority being complex I-dependent. A further 13% was of non-mitochondrial origin, while maximal OCR was 189% of basal, providing a spare respiratory capacity of +89%. This profile allows re- interpretation of existing data to estimate ATP production by the bovine embryo. The endogenous triglyceride store of the oocyte is increasingly considered a vital energy source in preimplantation development. In the present study, β-oxidation was manipulated during embryo culture. Inhibition of β-oxidation led to i) increased OCR ii) increased lipid content, iii) increased pyruvate uptake and iv) decreased lactate release at the blastocyst stage. Enhancing β-oxidation caused i) OCR at blastocyst stage to fall, ii) decreased lipid content during early cleavage, iii) decreased pyruvate consumption and iv) increased lactate release. Neither treatment affected blastocyst development rate or differential cell count, while both led to mitochondrial depolarisation. These metabolic observations were hypothesised to have legacy effects on gene expression. Groups of 10 blastocysts with similar metabolic profiles were analysed using transcription and DNA methylation microarray platforms. Following manipulation of β- oxidation, gene transcripts involved in mitochondrial function, metabolism, key signalling cascades, recognition of pregnancy, stress response, protein modification and transcription were differentially expressed. Genes involved in transcription, protein modification, key signalling cascades and disease were differentially methylated, potentially linking dysregulated β-oxidation to deleterious conditions in later development. These data highlight the plasticity of metabolic regulation in the embryo, allowing successful preimplantation development despite an apparently deleterious phenotype, yet indicate that metabolic activity has subtle effects on development. ii Contents Abstract ............................................................................................................................................... ii Contents ............................................................................................................................................. iii List of tables ....................................................................................................................................... xi List of figures .................................................................................................................................... xiii List of abbreviations ........................................................................................................................ xvii Acknowledgements ......................................................................................................................... xxv Author’s declaration ...................................................................................................................... xxvi 1 Introduction ................................................................................................................................ 1 1.1 Bovine oocyte and embryo development ........................................................................... 2 1.1.1 In vivo oocyte development ........................................................................................ 2 1.1.2 The ovarian cycle......................................................................................................... 3 1.1.3 Follicle development ................................................................................................... 5 1.1.4 Oocyte maturation ...................................................................................................... 6 1.1.5 Fertilisation ................................................................................................................. 8 1.1.6 Pre-implantation embryo development ..................................................................... 9 1.1.7 Predicting oocyte and embryo viability .................................................................... 10 1.2 Eukaryotic energy metabolism ......................................................................................... 12 1.2.1 Glycolysis ................................................................................................................... 12 1.2.2 Fatty acid metabolism ............................................................................................... 14 1.2.3 Tricarboxylic acid cycle .............................................................................................. 14 1.2.4 Oxidative phosphorylation ........................................................................................ 17 1.2.5 Productive and non-productive oxygen consumption ............................................. 18 1.2.6 Mitochondrial function ............................................................................................. 18 iii 1.3 Bovine oocyte and embryo metabolism ........................................................................... 20 1.3.1 Glucose, pyruvate and lactate .................................................................................. 22 1.3.2 Fatty acids ................................................................................................................. 24 1.3.3 Amino acids ............................................................................................................... 25 1.3.4 Oxygen consumption ................................................................................................ 26 1.3.5 Oxidative damage ..................................................................................................... 29 1.4 Aims................................................................................................................................... 31 2 General materials and methods ............................................................................................... 32 2.1 Bovine embryo In Vitro Production (IVP) .......................................................................... 33 2.1.1 Source of reagents .................................................................................................... 33 2.1.2 Preparation of glassware and consumables for embryo culture .............................. 33 2.1.3 Preparation of IVP media .......................................................................................... 33 2.1.4 Holding medium stocks ............................................................................................. 35 2.1.5 FSH:LH stock .............................................................................................................. 35 2.1.6 EGF:FGF stock ............................................................................................................ 36 2.1.7 Maturation additives................................................................................................. 36 2.1.8 Preparation of holding and bovine maturation media ............................................. 37 2.1.9 Preparation of in-vitro fertilisation media ................................................................ 38 2.1.10 Preparation of Percoll® gradient ............................................................................... 40 2.1.11 Preparation of in vitro embryo culture media .......................................................... 42 2.1.12 Preparation of amino acid supplement .................................................................... 44 2.1.13 Collection and In Vitro Maturation (IVM) of bovine oocytes. .................................. 45 2.1.14 In Vitro Fertilisation (IVF) .......................................................................................... 46 2.1.15 In Vitro Culture (IVC) ................................................................................................. 46 2.2 Measurement of Oxygen Consumption Rate (OCR) ......................................................... 48 2.2.1 Measurement of OCR by the Becton-Dickinson Oxygen Biosensor System ............. 48 2.2.2 Measurement of OCR by nanorespirometry............................................................. 49 iv 2.3 Energy substrate assays .................................................................................................... 51 2.3.1 Pyruvate .................................................................................................................... 54 2.3.2 Glucose ...................................................................................................................... 55 2.3.3 Lactate ....................................................................................................................... 56 2.4
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