Masala, Laura (2016) Study of DNA methylation dynamics in a model of differential developmental competence in the ovine species. Doctoral Thesis.
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DNA methylation is an important epigenetic mark that plays a role in gene regulation by the addition of a methyl group to CpGs in the DNA. Despite being relatively stable in somatic cells, DNA methylation is subject to reprogramming during embryo development and gametogenesis.
Aim of this work was to evaluate different aspects of DNA methylation in relation to oocyte quality in the ovine species. A model of differential developmental competence consisting in ovine oocytes and in vitro produced (IVP) embryos derived from prepubertal (P) and adult (A) donors was used. The methylation dynamics were analysed in terms of gene expression of a panel of genes involved in DNA methylation reprogramming (DNA methyltransferases (DNMTs), ten-eleven translocation dioxygenases (TET) and methylbinding proteins (DMB) and immunolocalization of TET3 protein, 5-methylcytosine (5-mc) and 5-hydroxymethylcitosine (5-hmc). Gene relative quantification was performed by RNA reverse transcription followed by Real-Time PCR. Pools of growing oocytes at different diameter (70/90 µm, 90/110 µm, 110/130 µm), germinal vesicles (GV), IVM metaphase II (MII) oocytes, IVP embryos at two (2C) and four cell (4C) stage and blastocysts derived from P and A donors were analysed. The analysis in growing oocytes revealed that DNMT1, DNMT3B, TET1 and TET3 transcript abundance increases in relation to oocyte diameter, suggesting that synthesis and storage occur during the final stage of oocyte growth. P-GV oocytes present lower expression level of TET1, TET2 and TET3 (ANOVA; P< 0.05). DNMT1 expression is lower in 4C embryos from prepubertal oocytes and MBD1 and MBD3 expression decreases during the transition from 2C to 4C stage in embryos derived from P-animals. No significant differences were observed in embryos at the blastocyst stage (ANOVA; P>0.05). These observations confirm different gene expression patterns in our model.
Immunolocalization was performed by direct immunofluorescence. TET3 immunostaining revealed the presence of the protein only in the cytoplasm of early embryos (2C and 4C) from both P and A animals. The analysis of global DNA methylation showed high levels in all analyzed samples. Interestingly, pronuclear (PN) stage zygotes displayed a decrease in 5-mC signal in male PN compared to maternal PN from both young and adult animals; conversely, hydroxymethylation signal was scattered evident throughout the chromatin of all analyzed samples; the analysis of zygotes revealed 5-hmc signal only in male PN. Observations demonstrate that male PN undergoes a decrease in methylation and an increase in DNA hydroxylation suggesting that also in sheep an active DNA demethylation occurs soon after fertilization.
Our results are in accordance with the recent hypothesis that the acquisition of epigenetic modifications may be involved in the gaining of oocyte competence during its growth. They indicate that epigenetic mechanisms may affect oocyte quality and pave the way to better understanding methylation dynamics during sheep pre-implantation development.
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