Figure 5: Ablation of Tet proteins diminishes the loss of 5mC signal in the paternal genome of G2-phase zygotes.
From: 5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming
(a) Experimental setup according to Okada et al.19. Briefly, siRNA injected metaphase II oocytes were cultured for 8 h and fertilized by intracytoplasmic sperm injection (ICSI). G2-phase zygotes were fixed and analysed for 5mC and 5hmC by immunofluorescence. (b) Representative images of 5mC (green mouse monoclonal, gift from Dirk Schübeler) and 5hmC (red rabbit polyclonal from Active Motif) immunostainings on G2-stage zygotes derived from Tet1-3 siRNA injected oocytes fertilized by ICSI. As control IVF-derived zygotes and zygotes derived from scrambled siRNA injected and ICSI fertilized oocytes are shown. Red arrows indicate the diminished loss of 5mC signal in paternal pronuclei of Tet KD-derived zygotes. Note the decreased 5hmC signal in Tet1–3 and Tet3 KDs. (c) Quantification of 5hmC signal of G2-phase zygotes derived from scrambled and Tet siRNA injected oocytes. (d) Paternal to maternal pronuclear 5hmC signal ratios. (e) Quantification of 5mC signal of G2-phase zygotes derived from scrambled and Tet siRNA injected oocytes. (f) Paternal to maternal pronuclear 5mC signal ratios. Blue bars represent signals from paternal pronuclei and red bars represent signals from maternal pronuclei. A total of 8–9 precisely staged ICSI-derived zygotes per group were analysed. Asterisks show significant changes of paternal or maternal 5hmC/5mC signals calculated using Student's t-tests (*=P<0.05, **=P<0.01, ***=P<0.001). Error bars represent standard deviations. ♂, male pronucleus; ♀, female pronucleus; Pb, polar body; scale bar, 20 μm.
