Characterisation of the methylation pattern in the intragenic CpG island of the IGF2 gene in Bos taurus indicus cumulus cells during in vitro maturation
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The aim of this study was to characterise the methylation pattern in a CpG island of the IGF2 gene in cumulus cells from 1–3 mm and ≥ 8.0 mm follicles and to evaluate the effects of in vitro maturation on this pattern.
Genomic DNA was treatment with sodium bisulphite. Nested PCR using bisulphite-treated DNA was performed, and DNA methylation patterns have been characterised.
There were no differences in the methylation pattern among groups (P > 0.05). Cells of pre-IVM and post-IVM from small follicles showed methylation levels of 78.17 ± 14.11 % and 82.93±5.86 %, respectively, and those from large follicles showed methylation levels of 81.81 ± 10.40 % and 79.64 ± 13.04 %, respectively. Evaluating only the effect of in vitro maturation, cells of pre-IVM and post-IVM COCs showed methylation levels of 80.17 ± 12.01 % and 81.19 ± 10.15 %.
In conclusion, the methylation levels of the cumulus cells of all groups were higher than that expected from the imprinted pattern of somatic cells. As the cumulus cells from the pre-IVM follicles were not subjected to any in vitro manipulation, the hypermethylated pattern that was observed may be the actual physiological methylation pattern for this particular locus in these cells. Due the importance of DNA methylation in oogenesis, and to be a non-invasive method for determining oocyte quality, the identification of new epigenetic markers in cumulus cells has great potential to be used to support reproductive biotechniques in humans and other mammals.
KeywordsBovine Cumulus cells IGF2 Epigenetics DNA methylation In vitro maturation
Conflicts of interest
None of the authors have any conflict of interest to declare.
- 3.Altarescu G, Beeri R, Eiges R, Epsztejn-Litman S, Eldar-Geva T, Elstein D, et al. Prevention of lysosomal storage diseases and derivation of mutant stem cell lines by preimplantation genetic diagnosis. Mol Biol Int. 2012;797342.Google Scholar
- 14.de Pagter-Holthuizen P, Jansen M, van der Kammen RA, Vanschaik FM, Sussenbach JS. Differential expression of the human insulin-like growth factor II gene. Characterization of the IGF-II mRNAs and an mRNA encoding a putative IGF-II-associated protein. Biochim Biophys Acta. 1988;950:282–95.PubMedCrossRefGoogle Scholar
- 19.Fagundes NS, Michalczechen-Lacerda VA, Caixeta ES, Machado GM, Rodrigues FC, Melo EO, et al. Methylation status in the intragenic differentially methylated region of the IGF2 locus in Bos taurus indicus oocytes with different developmental competencies. Mol Hum Reprod. 2011;17:85–91.PubMedCrossRefGoogle Scholar
- 25.Krisher RL. The effect of oocyte quality on development. J Anim Sci. 2004;84:14–23.Google Scholar
- 35.Shim SW, Han DW, Yang JH, Lee BY, Kim SB, Shim H, et al. Derivation of embryonic germ cells from post migratory primordial germ cells, and methylation analysis of their imprinted genes by bisulfite genomic sequencing. Mol Cells. 2008;259:358–67.Google Scholar
- 37.Stojkovic M, Machado SA, Stojkovic P, Zakhartchenko V, Hutzler P, Gonçalves PB, et al. Mitochondrial distribution and adenosine triphosphate content of bovine oocytes before and after in vitro maturation: correlation with morphological criteria and developmental capacity after in vitro fertilization and culture. Biol Reprod. 2001;64:904–9.PubMedCrossRefGoogle Scholar