Fig. 5

H3K4 trimethylation is a prerequisite for H3T3 phosphorylation and meiotic cell cycle progression. a Western blot of H3T3ph in WT and Cxxc1^oo−/− oocyte at 6 h after in vitro culture. b Immunofluorescence of H3T3ph on WT and Cxxc1^oo−/− oocyte chromosome spreads prepared at MI stage. Centromeres and DNA were labeled by CREST immunofluorescence (green) and DAPI staining (blue), respectively. Scale bar, 5 μm. c Western blot of the indicated proteins in WT oocytes ectopically express Flag-tagged histone H3.3^WT and H3.3K4^K4R. d Quantification of H3T3ph signals in (e). Error bars, S.E.M. ***P < 0.001 by two-tailed Student’s t tests. n.s., non-significant. e Immunofluorescence of Flag-tagged histone H3.3 (H3.3^WT and H3.3K4^K4R) and H3T3ph on chromosome spreads prepared at MI stage. Fully grown WT oocytes at the GV stage were microinjected with mRNAs encoding histone H3.3^WT or H3.3^K4R and were released to resume meiotic maturation at 12 h after microinjection. Scale bar, 5 μm. f Co-immunofluorescence of H3K4me3 and H3T3ph on MI chromosome spreads prepared from WT oocytes. Scale bar, 5 μm. g Rates of GVBD, PB1 emission, and normal spindle assembly in oocytes cultured in vitro. Fully grown WT oocytes at the GV stage were microinjected with mRNAs encoding histone H3.3^WT or H3.3^T3E and were released to resume meiotic maturation at 12 h after microinjection. Error bars, S.E.M. ***P < 0.001, **P < 0.01, and *P < 0.05 by two-tailed Student’s t tests. n.s., non-significant. h Immunofluorescent staining of α-tubulin showing spindle assembly in WT oocytes microinjected with mRNAs encoding Flag-tagged histone H3.3^WT or H3.3^T3E. Scale bar, 20 μm