Hyperglycaemia: A new player in oocyte epigenetics

摘要

DNA methylation differences in embryos are indicative for epigenetic changes imposed by culture conditions. Immunostaining is the preferred technique to assess methylation/hydroxymethylation status of zygotes because differences between paternal and maternal pronucleus can be assessed in single embryos. Antibodies anti-methylcytosine/hydroxymethylcytosine bind to ssDNA, therefore DNA denaturation is required. Most DNA-stains however, such as Hoechst, bind to dsDNA, and not to ssDNA. Ethidium homodimer 2 is a DNA dye able to bind ssDNA. The aim of this study was to optimize the DNA counterstaining for pronuclear stages in bovine. Bovine oocytes were matured in TCM 199 with 20 ng/ml EGF. At 12, 15, 18 and 22 h post fertilization, presumptive zygotes were fixed. After fixation, zygotes were permeabilized and denatured with 2 N (30, 45 min or 1 h) or 4 N (5, 10, 20, 30 min or 1 h) HCl. Zygotes were blocked and incubated with primary antibodies (mouse anti-5-MeC, rabbit anti-5-HmC), secondary antibodies (goat anti-mouse FITC, goat anti-rabbit Texas Red), and finally counterstained with Hoechst or EthD-2. When EthD-2 was used, RNase A treatment was performed. 5-MeC/5-HmC fluorescence after denaturation with 2 N HCl was only obtained after 1 h of denaturation, and this signal was weak. With 4 N HCl at least 30 min of denaturation were needed to obtain good resolution. For Hoechst, after 1 h denaturation with 2 N HCl the signal was faint or disappeared completely. After 4 N HCL treatment for 5 and 10 min the signal was present, whereas it was completely gone after 20 and 30 min. For EthD-2, after denaturation with 4 N HCl for 1 h the signal of the dye was still present, although weak, but it allowed pronuclear delineation. In conclusion, for bovine zygotes, denaturation of at least 30 min with 4 N HCl is needed. In these conditions Hoechst cannot be used as DNA counterstain, but EthD-2 can.