title: Exposure of bovine oocytes and embryos to elevated non-esterified fatty acid concentrations: integration of epigenetic and transcriptomic signatures in resultant blastocysts.
abstract: Metabolic stress associated with negative energy balance in high producing dairy cattle and obesity in women is a risk factor for decreased fertility. Non-esterified fatty acids (NEFA) are involved in this pathogenesis as they jeopardize oocyte and embryo development. Growing evidence indicates that maternal metabolic disorders can disturb epigenetic programming, such as DNA methylation, in the offspring. Oocyte maturation and early embryo development coincide with methylation changes and both are sensitive to adverse environments. Therefore, we investigated whether elevated NEFA concentrations affect establishment and maintenance of DNA methylation in oocytes and embryos, subsequently altering transcriptomic profiles and developmental competence of resultant blastocysts.
  
  
  
  Bovine oocytes and embryos were exposed to different NEFA concentrations in separate experiments. In the first experiment, oocytes were matured in vitro for 24 h in medium containing: 1) physiological ("BASAL") concentrations of oleic (OA), palmitic (PA) and stearic (SA) acid or 2) pathophysiological ("HIGH COMBI") concentrations of OA, PA and SA. In the second experiment, zygotes were cultivated in vitro for 6.5 days under BASAL or HIGH COMBI conditions. Developmental competence was evaluated by assessing cleavage and blastocyst rate. Overall gene expression and DNA methylation of resultant blastocysts were analyzed using microarray. DNA methylation data were re-evaluated by pyrosequencing. HIGH COMBI-exposed oocytes and embryos displayed a lower competence to develop into blastocysts compared to BASAL-exposed counterparts (19.3% compared to 23.2% and 18.2% compared to 25.3%, respectively) (P < 0.05). HIGH COMBI-exposed oocytes and embryos resulted in blastocysts with altered DNA methylation and transcriptomic fingerprints, compared to BASAL-exposed counterparts. Differences in gene expression and methylation were more pronounced after exposure during culture compared to maturation suggesting that zygotes are more susceptible to adverse environments. Main gene networks affected were related to lipid and carbohydrate metabolism, cell death, immune response and metabolic disorders.
  
  
  
  Overall, high variation in methylation between blastocysts made it difficult to draw conclusions concerning methylation of individual genes, although a clear overview of affected pathways was obtained. This may offer clues regarding the high rate of embryonic loss and metabolic diseases during later life observed in offspring from mothers displaying lipolytic disorders.
authors.affiliation: Laboratory of Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Wilrijk, Belgium. karolien.desmet@uantwerpen.be.
authors.forename: K L J
authors.initials: KL
authors.lastname: Desmet
authors.affiliation: Laboratory of Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Wilrijk, Belgium.
authors.forename: V
authors.initials: V
authors.lastname: Van Hoeck
authors.affiliation: Centre de Recherche en Biologie de la Reproduction (CRBR), Département des Sciences Animales, Université Laval, Québec, Canada.
authors.forename: D
authors.initials: D
authors.lastname: Gagné
authors.affiliation: Centre de Recherche en Biologie de la Reproduction (CRBR), Département des Sciences Animales, Université Laval, Québec, Canada.
authors.forename: E
authors.initials: E
authors.lastname: Fournier
authors.affiliation: British Columbia Cancer Agency, University of British Columbia, Vancouver, Canada.
authors.forename: A
authors.initials: A
authors.lastname: Thakur
authors.affiliation: School of Agriculture and Food Science, University College Dublin, Dublin, Ireland.
authors.forename: A M
authors.initials: AM
authors.lastname: O'Doherty
authors.affiliation: Centre for Molecular Biosciences, School of Biomedical Sciences, University of Ulster, Coleraine, UK.
authors.forename: C P
authors.initials: CP
authors.lastname: Walsh
authors.affiliation: Centre de Recherche en Biologie de la Reproduction (CRBR), Département des Sciences Animales, Université Laval, Québec, Canada.
authors.forename: M A
authors.initials: MA
authors.lastname: Sirard
authors.affiliation: Laboratory of Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Wilrijk, Belgium.
authors.forename: P E J
authors.initials: PE
authors.lastname: Bols
authors.affiliation: Laboratory of Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Wilrijk, Belgium.
authors.forename: J L M R
authors.initials: JL
authors.lastname: Leroy
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000378:metabolism*; D002417:Cattle; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects; D004622:Embryo, Mammalian / Q000187:drug effects / Q000378:metabolism*; D044127:Epigenesis, Genetic / Q000187:drug effects*; D005230:Fatty Acids, Nonesterified / Q000633:toxicity*; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D006657:Histones / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D059467:Transcriptome / Q000187:drug effects*; D055517:snRNP Core Proteins / Q000235:genetics
chemical_list: D005230:Fatty Acids, Nonesterified; D006657:Histones; D055517:snRNP Core Proteins; D004247:DNA
keywords: DNA methylation; Embryo; Epigenetics; Fertility; Free fatty acids; Maternal metabolism; Oocyte