Figure 2

TET3 null fertilised oocytes show increased paternal DNA methylation and reduced hydroxymethylation. (A) Diagrammatic illustration and representative 2D projections of Z-stack images of TET3 maternally deleted oocytes fertilised by control sperm (TET3 MAT 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, and during which there is complete failure in TET3 null oocytes to generate DNA hydroxymethylation in the paternal pronucleus. 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; Figure 1A) and TET3 maternally deleted (TET3 MAT 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 and, on the right, an equally significant decrease in the levels of hydroxymethylation in TET3 maternally deleted zygotes compared to controls. ****(P <0.0001, two-tailed Mann–Whitney test).