Figure 3

AID null fertilised oocytes show increased paternal DNA methylation but equal levels of hydroxymethylation. (A) Diagrammatic illustration and representative 2D projections of Z-stack images of AID null oocytes fertilised by control sperm (AID KOxB6) pronuclear stage embryos (PN1 to PN5) simultaneously stained for DNA methylation (5mC) and hydroxymethylation (5hmC) clearly showing paternal loss of methylation during Phase I, corresponding to a pre-replicative state, but no apparent further demethylation during Phase II, when DNA replication is taking place, while showing a striking increase in DNA hydroxymethylation in the paternal pronucleus, similar to that observed in control (B6xB6) embryos. Scale bar 25 μm. f, female pronucleus; m, male pronucleus; pb, polar body. (B) Comparison of the changes of DNA methylation and hydroxymethylation between control (B6xB6) and AID null (AID KOxB6) mid-late zygotes. Box-and-whisker plots of the total immunofluorescence signal (3D imaging semi-quantification) ratio between the paternal and maternal pronuclei (male/female ratio) for 5mC and 5hmC, respectively, showing, on the left, a very significant increase in the levels of paternal DNA methylation but no significant difference in the levels of hydroxymethylation in AID null zygotes compared to controls, on the right. ****(P <0.0001, two-tailed Mann–Whitney test), ns (P = 0.6330, two-tailed unpaired t test with Welch’s correction).