This study was conducted to investi- gate the pattern of DNA methylation in pronuclearstage mouse embryos derived from vitrified-warmed oocytes. Mouse oocytes at metaphase II （MII） stage of meiosis were allocated ra...This study was conducted to investi- gate the pattern of DNA methylation in pronuclearstage mouse embryos derived from vitrified-warmed oocytes. Mouse oocytes at metaphase II （MII） stage of meiosis were allocated randomly into three groups： 1） untreated （control）; 2 ） exposed to vitrification solution without being plunged into liquid nitrogen （toxicity）; and 3 ） vitrified by open-pulled straw （OPS） method （vitrification）. Oocytes were fertilized in vitro （IVF）. The level of DNA methylation was examined at 8 hpf （hours post-fertilization） by immunofluorescence using an anti-5-methylcytosine （5-MeC） monoclonal antibody and fluorescein isothiocyanate （FITC）-conjugated goat anti-mouse IgG. After IVF, rates of 2-cell embryos （ 51.39% ） and blastocysts （35.82%） for vitrified-warmed oocytes were lower （P〈0.01） than that for control （70.83%, 47.82% ） or vitrification solution treated （64.80%, 46.29% ） oocytes. At 8 hpf, there were more （P 〈 0.05） pronuclear-stage oocytes in which syngamy of pronuclei had not occurred in the vitrifica- tion group. In addition, 5-MeC fluorescent intensities for the female pronucleus and zygote were lower in the vitrification group （P 〈0.01 ） compared to pronuclear-stage embryos in the control and toxicity groups. In conclusion, oocyte vitrification causes a reduction in global genomic methylation in the female pronucleus and zygote, resulting in delayed fusion of pronuclei and compromising the developmental potential of mouse zygotes and embryos.展开更多
文摘This study was conducted to investi- gate the pattern of DNA methylation in pronuclearstage mouse embryos derived from vitrified-warmed oocytes. Mouse oocytes at metaphase II （MII） stage of meiosis were allocated randomly into three groups： 1） untreated （control）; 2 ） exposed to vitrification solution without being plunged into liquid nitrogen （toxicity）; and 3 ） vitrified by open-pulled straw （OPS） method （vitrification）. Oocytes were fertilized in vitro （IVF）. The level of DNA methylation was examined at 8 hpf （hours post-fertilization） by immunofluorescence using an anti-5-methylcytosine （5-MeC） monoclonal antibody and fluorescein isothiocyanate （FITC）-conjugated goat anti-mouse IgG. After IVF, rates of 2-cell embryos （ 51.39% ） and blastocysts （35.82%） for vitrified-warmed oocytes were lower （P〈0.01） than that for control （70.83%, 47.82% ） or vitrification solution treated （64.80%, 46.29% ） oocytes. At 8 hpf, there were more （P 〈 0.05） pronuclear-stage oocytes in which syngamy of pronuclei had not occurred in the vitrifica- tion group. In addition, 5-MeC fluorescent intensities for the female pronucleus and zygote were lower in the vitrification group （P 〈0.01 ） compared to pronuclear-stage embryos in the control and toxicity groups. In conclusion, oocyte vitrification causes a reduction in global genomic methylation in the female pronucleus and zygote, resulting in delayed fusion of pronuclei and compromising the developmental potential of mouse zygotes and embryos.
