Figure 7: Altered genomic 5′-methylcytosine and 5′-hydroxymethylcytosine dynamics in phICSI-13.

(a) Immunofluorescence showing nuclear 5′-methylcytosine (5mC, green) and 5′-hydroxymethylcytosine (5hmC, red) 30 h after the start of SrCl₂ treatment or sperm injection (ICSI). Parthenogenetic haploid, ph; bar, 50 μm. Insets (bar, 2 μm) show merged nuclear images with line plots of relative intensities (rightmost). (b) Ratios (±s.e.m.) of relative paternal and maternal 5hmC fluorescence in individual (left) or pooled (histograms) 10 h after ICSI (upper; n=16), or 30 h after ICSI (2-cell embryos, I2; n=12) or the start of SrCl₂ treatment in either parthenogenesis (ph; n=14) or phICSI-13 (phICSI; n=15). Experiments were on 2 days and differences are indicated where P<0.05 (two-tailed, unpaired t-test). (c) Protocol for generating microbeads conjugated to 5mC-containing DNA (5mC-beads), showing (below) electrophoresis of 5mC-containing DNA following treatment with the CpG DNA methyltransferase, M.SssI (M.SssI+) or unmethylated DNA (M.SssI-), challenged with the methylation-sensitive restriction endonuclease, HpaII. (d) Fluorescence images showing 5mC or 5hmC (green) in beads conjugated to control, non-methylated DNA (DNA) or to 5mC-containing DNA (meDNA) 7 h after injection into in mII oocytes (scale bar, 20 μm), with insets showing close-ups (bar, 3 μm). DNA is stained with propidium iodide (PI). For DNA 5mC, n=7; meDNA 5mC, n=7; DNA 5hmC, n=7; meDNA 5hmC, n=10. (e) Fluorescence images of haploid parthenogenotes containing latex microbeads conjugated to non-methylated DNA (DNA-beads; n=7) or 5mC-containing DNA (meDNA-beads; n=7), stained with PI (DNA, red) or anti-5hmC antibodies (green). Beads were either injected into mII oocytes (mII) followed by Sr²⁺ activation and analysis 6 h (ph-6, 1-cell; n=10) or 20 h (ph-20, 2-cell; n=8) later, or ph-13 parthenogenotes were injected with beads followed by analysis at 20 h (+7 h) (n=7). Scale bar, 50 μm. Arrowheads indicate DNA-beads (magnified in insets; bar, 3 μm). (f) Fluorescence intensity quantification of 7≤n≤10 embryos of e showing P-values following one-way ANOVA followed by a Tukey–Kramer test. (g) Fluorescence staining with PI (DNA, red) or anti-5mC antibodies (5mC, green) showing relative paternal and maternal 5mC in ICSI and phICSI-13 embryos 6 h after sperm injection. Scale bar, 20 μm. (h) Pixel quantification (±s.e.m.) of samples (for ICSI, n=12; phICSI, n=11) of g and 1nP (n=10) and ICSI 2-cell embryos (n=14) 6 h post-cleavage, showing fluorescence levels normalized against PI (DNA). Experiments were on 2 days and differences are indicated where P<0.05 (two-tailed, unpaired t-test).