Epigenetische Analyse der Eizell- und Embryonalentwicklung am Modellorganismus Rind
Gynäkologie und Geburtshilfe
Zusammenfassung der Projektergebnisse
Assisted reproductive technologies (ARTs), including in vitro maturation of oocytes, in vitro fertilization and in vitro culture of embryos are associated with an increased incidence of epigenetic aberrations. Due to the high degree of similarities between bovine and human oocyte and embryo development, bovine reproduction is increasingly considered as a valuable model for human reproduction. During the first funding period, we studied epigenetic effects of different maturation conditions, by using two different IVM systems. Using a limiting dilution bisulfite sequencing assay, no significant differences in the methylation patterns of representative imprinted genes were found between in vitro and in vivo matured oocytes, indicating that current IVM protocols have no or only marginal effects on these critical epigenetic marks. However, different mRNA expression profiles were found between in vivo matured oocytes and their in vitro matured counterparts for a panel of developmentally important genes. During the second funding period methylation and expression analyses were extended to different sources of oocytes, i.e. prepubertal, adult and aged oocytes. In the second funding period, DNA methylation and mRNA expression analyses were extended to different sources of oocytes, including prepubertal, adult and aged bovine oocytes. It was found that cAMP regulates in vitro maturation in prepubertal and adult bovine oocytes in a similar manner with impact on epigenetic marks and acquisition of developmental comptetence. Results also revealed differences in the methylation profile of DNA satellite sequences, bovine testis satellite 1 (BTS 1) and bovine Taurus ɑ satellite (BTɑS) between oocytes derived from prepubertal versus adult donors. The methylation levels of satellite sequences were affected by origin and prior treatment of the oocyte. In an effort to mimic postovulatory ageing, the in vitro maturation phase was extended to 48 hours and effects were compared to the conventional 24 hours. Effects on developmental potential, expression of a panel of developmentally important genes, putative pertubations in gene specific epigenetic marks were investigated. Cleavage and blastocyst rates were significantly reduced using aged oocytes. No significant differences were observed in the methylation of entire alleles in oocytes for the genes bH19, bSNRPN, bZAR1, bOct4 and bDNMT3A. However, differentially methylated CpG sites were found in the DNMT3LS locus in aged oocytes and embryos derived thereof compared to controls. Embryos derived from the aged oocyte group were significantly less methylated in CpG5 and 7 compared to controls. Methylation of CpGs 5 to 8 of bDNMT3LS was significantly lower in oocytes of the 24 hours control group compared to the aged oocytes. Results indicate that extended IVM leads to ageing like alterations and demonstrates for the first time that epigenetic mechanisms are critically involved in ageing of bovine oocytes. This warrants further study into epigenetic mechanisms involved in ageing of female germ cells including humans. Overall, research funding within the Research Group 1041 (Germ Cell Potential) has yielded significant new knowledge into bovine oocyte development which could be published in a series of peer reviewed publications in leading journals of the field.
Projektbezogene Publikationen (Auswahl)
- (2011) Characterization of differentially methylated regions in 3 bovine imprinted genes: a model for studying human germ-cell and embryo development. Cytogenet Genome Res 132:239-247
Hansmann T, Heinzmann J, Wrenzycki C, Zechner U, Niemann H, Haaf T
(Siehe online unter https://doi.org/10.1159/000322627) - (2011) Epigenetic profile of developmentally important genes in bovine oocytes. Mol Reprod Dev 78:188-201
Heinzmann J, Hansmann T, Herrmann D, Wrenzycki C, Zechner U, Haaf T, Niemann H
(Siehe online unter https://doi.org/10.1002/mrd.21281) - (2011) Limiting dilution bisulfite (pyro)sequencing reveals parent-specific methylation patterns in single early mouse embryos and bovine oocytes. Epigenetics 6:1176 – 1188
El Hajj N, Trapphoff T, Linke M, May A, Hansmann T, Kuhtz J, Reifenberg K, Heinzmann J, Niemann H, Daser A, Eichenlaub-Ritter U, Zechner U, Haaf T
(Siehe online unter https://doi.org/10.4161/epi.6.10.17202) - (2012) DNA methylation and mRNA expression profiles in bovine oocytes derived from prepubertal and adult donors. Reproduction 144:319-330
Diederich M, Hansmann T, Heinzmann J, Barg-Kues B, Herrmann D, Aldag P, Baulain U, Reinhard R, Kues W, Weissgerber C, Haaf T, Niemann H
(Siehe online unter https://doi.org/10.1530/REP-12-0134) - (2013) Epigenetic disturbances in in vitro cultured gametes and embryos: implications for human assisted reproduction. Fertil Steril 99:632-641
El Hajj N, Haaf T
(Siehe online unter https://doi.org/10.1016/j.fertnstert.2012.12.044) - (2014) Epigenetic disorders and altered gene expression after use of Assisted Reproductive Technologies in domestic cattle. Epigenetics 9:803-815
Urrego R, Rodriguez-Osorio N, Niemann H
(Siehe online unter https://doi.org/10.4161/epi.28711) - (2015) Effects of different oocyte retrieval and in vitro maturation systems on bovine embryo development and quality. Zygote 23:367-77
Bernal SM, Heinzmann J, Herrmann D, Timmermann B, Baulain U, Großfeld R, Diederich M, Lucas- Hahn A, Niemann H
(Siehe online unter https://doi.org/10.1017/S0967199413000658) - (2015) Extended in vitro maturation affects gene expression and DNA methylation in bovine oocytes. Mol Hum Reprod 21:770-82
Heinzmann J, Mattern F, Aldag P, Bernal-Ulloa SM. Schneider T, Haaf T, Niemann H
(Siehe online unter https://doi.org/10.1093/molehr/gav040) - (2016) Cyclic AMP affects oocyte maturation and embryo development in prepubertal and adult cattle. PLOS One 11:e0150264
Bernal Ulloa SM, Heinzmann J, Herrmann D, Hadeler KG, Aldag P, Winkler S, Pache D, Baulain U, Lucas-Hahn A, Niemann H
(Siehe online unter https://doi.org/10.1371/journal.pone.0150264)
