Fig. 4

Oxidative stress in sperm impairs active DNA demethylation on the paternal pronucleus. a Representative immunofluorescence images showing the levels of 5mC (green) and 5hmC (red) in PN3/4 zygotes using 5mC- and 5hmC-specific antibodies. Zygotes were analysed 20 h after IVF. Quantifications of 5mC (b) and 5hmC (c) signals are shown as ratio of the signal of the mean intensity of the paternal pronucleus (pPN) over the mean intensity signal of the maternal pronucleus (mPN) after background subtraction. Each data point represents an independent zygote (5mC control n = 27 zygotes and 5mC +H₂O₂ = 21 zygotes, three independent experiments; 5hmC control n  = 52 and 5hmC +H₂O₂ n = 48, three independent experiments). Statistical analysis was performed using Student’s t-test (two-tailed). Error bars indicate s.d., ns: non-significant **P < 0.01. Maternal pronucleus: mPN/♀; paternal pronucleus: pPN/♂. Scale bars, 50 μm. d Quantification of 5mC/dG by LC–MS of two-cell embryos obtained upon IVF with control sperm and sperm treated with H₂O₂. Data are from two independent experiments. Each data point represents the mean of two technical replicates of a pool of 50 embryos. e Quantification of 5mC and 5hmC by LC–MS of bovine sperm untreated or treated with H₂O₂ and MII oocytes. Data are from three independent experiments. Each data point represents the mean of two technical replicates