WCRB2014 POSTER PRESENTATIONS (1) (335 abstracts)
Postovulatory ageing influences methylation marks and development in bovine oocytes and early embryos
Julia Heinzmann 1 , Felix Mattern 2 , Patrick Aldag 1 , Andrea Lucas-Hahn 1 , Christine Wrenzycki 3 , Thomas Haaf 2 & Heiner Niemann 1
1Institute of Farm Animal Genetics (FLI), Neustadt, Germany; 2Julius-Maximilians-University, Wuerzburg, Germany; 3Justus-Liebig-University, Giessen, Germany.
Introduction: Oocytes that are not fertilized in a specific time-frame after ovulation undergo a time-dependent process of degradation and loss of developmental potential that can be referred to as postovulatory ageing. This process occurs in vivo and under specific in vitro settings. Here, we studied epigenetic changes related to postovulatory ageing.
Materials and methods: Bovine oocytes were collected from slaughterhouse ovaries and matured in vitro either for 24 h (standard protocol) or 48 h (postovulatory ageing) in TCM199 medium supplemented with BSA and Suigonan®. Maturation, fertilization, cleavage and blastocyst rates were determined. Correct spindle formation was determined by immunhistochemistry. Matured oocytes and 4-8 cell stage embryos were used for gene specific methylation analysis using the limiting dilution assay and direct sequencing. RT-qPCR was performed with developmentally important genes in single oocytes and early embryos.
Results and discussion: Maturation rates did not differ between the two groups; spindle formation was not impaired, but aged oocytes showed distinct detachment of cumulus cells. Fertilization rates did not differ significantly between the two groups; presumptive zygotes derived from aged oocytes showed significantly reduced cleavage rates and very low development to the blastocyst stage (1.3%). Analysis of relative transcript abundance of a panel of genes did not reveal significant differences. Gene specific methylation analysis of DNMT3Lo revealed a significant increase of aberrantly methylated alleles in postovulatory aged oocytes (P=0.03), other imprinted and non-imprinted genes were not affected. Results indicate that postovulatory ageing severely influences development and may affect offspring due to aberrant epigenetic marks.
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ISSN 2052-1472 (online)
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