title: Extended in vitro maturation affects gene expression and DNA methylation in bovine oocytes.
abstract: To mimic post-ovulatory ageing, we have extended the in vitro maturation (IVM) phase to 48 h and examined effects on (i) developmental potential, (ii) expression of a panel of developmentally important genes and (iii) gene-specific epigenetic marks. Results were compared with the 24 h IVM protocol (control) usually employed for bovine oocytes. Cleavage rates and blastocyst yields were significantly reduced in oocytes after extended IVM. No significant differences were observed in the methylation of entire alleles in oocytes for the genes bH19, bSNRPN, bZAR1, bOct4 and bDNMT3A. However, we found differentially methylated CpG sites in the bDNMT3Ls locus in oocytes after extended IVM and in embryos derived from them compared with controls. Moreover, embryos derived from the 48 h matured oocyte group were significantly less methylated at CpG5 and CpG7 compared with the 24 h group. CpG7 was significantly hypermethylated in embryos produced from the control oocytes, but not in oocytes matured for 48 h. Furthermore, methylation for CpG5-CpG8 of bDNMT3Ls was significantly lower in oocytes of the 24 h group compared with embryos derived therefrom, whereas no such difference was found for oocytes and embryos of the in vitro aged group. Expression of most of the selected genes was not affected by duration of IVM. However, transcript abundance for the imprinted gene bIGF2R was significantly reduced in oocytes analyzed after extended IVM compared with control oocytes. Transcript levels for bPRDX1, bDNMT3A and bBCLXL were significantly reduced in 4- to 8-cell embryos derived from in vitro aged oocytes. These results indicate that extended IVM leads to ageing-like alterations and demonstrate that epigenetic mechanisms are critically involved in ageing of bovine oocytes, which warrants further studies into epigenetic mechanisms involved in ageing of female germ cells, including humans.
authors.affiliation: Institute of Farm Animal Genetics (FLI), Mariensee, 31535 Neustadt, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Heinzmann
authors.affiliation: Institute of Human Genetics, Julius Maximilians University, 97070 Würzburg, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Mattern
authors.affiliation: Institute of Farm Animal Genetics (FLI), Mariensee, 31535 Neustadt, Germany.
authors.forename: Patrick
authors.initials: P
authors.lastname: Aldag
authors.affiliation: Institute of Farm Animal Genetics (FLI), Mariensee, 31535 Neustadt, Germany.
authors.forename: Sandra Milena
authors.initials: SM
authors.lastname: Bernal-Ulloa
authors.affiliation: Institute of Human Genetics, Julius Maximilians University, 97070 Würzburg, Germany.
authors.forename: Tamara
authors.initials: T
authors.lastname: Schneider
authors.affiliation: Institute of Human Genetics, Julius Maximilians University, 97070 Würzburg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Haaf
authors.affiliation: Institute of Farm Animal Genetics (FLI), Mariensee, 31535 Neustadt, Germany heiner.niemann@fli.bund.de.
authors.forename: Heiner
authors.initials: H
authors.lastname: Niemann
mesh_terms: D000483:Alleles; D000818:Animals; D002417:Cattle / Q000235:genetics*; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000235:genetics; D018899:CpG Islands / Q000235:genetics; D004247:DNA / Q000235:genetics; D019175:DNA Methylation; D015257:DNA-Cytosine Methylases / Q000235:genetics / Q000378:metabolism; D004624:Embryo Transfer; D005260:Female; D005307:Fertilization in Vitro; D018507:Gene Expression Regulation, Developmental; D059471:In Vitro Oocyte Maturation Techniques / Q000662:veterinary*; D009865:Oocytes / Q000166:cytology / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D013997:Time Factors; D014158:Transcription, Genetic
chemical_list: D004247:DNA; D015257:DNA-Cytosine Methylases
keywords: DNA methylation; epigenetics; gene expression; imprinting; oocyte maturation