Fig. 2

Asf1a, but not Asf1b, was required for histone H3.3 assembly in paternal genome. A, B Nuclear localization of Asf1a and Asf1b were examined by immunofluorescence staining in zygotes at 2, 4, 6, 8 and 10 hpi, respectively. 11–36 embryos were analyzed in each group. C, D Representative images of zygotes stained with antibodies against Asf1a and Asf1b. Asf1a-MO, Asf1b-MO or Control-MO was microinjected in zygotes at 2 hpi and the knockdown efficiency was examined in zygote at 8 hpi by immunofluorescence staining. E, F Quantification of the fluorescence intensities of Asf1a and Asf1b in zygotes. G, J Representative images of zygotes stained with antibodies against histone H3.3. Asf1a-MO, Asf1b-MO or Control-MO was microinjected into zygote at 2 hpi and immunostained with H3.3 antibody at 6 and 8 hpi, respectively. H, K Quantification of H3.3 fluorescence intensity in the Asf1a KD and Asf1b KD zygotes at 6 hpi and 8 hpi, respectively. I, L Quantification of the size of the paternal pronuclei in zygotes. Each dot represents a paternal pronucleus examined. Control-MO, Asf1a-MO or Asf1b-MO was microinjected in zygote at 2 hpi and the diameter of the paternal pronuclei was measured at 6 hpi and 8 hpi, respectively. Three horizontal lines from top to bottom represent the upper quartile, median and lower quartile, respectively. M Representative images of zygotes stained with antibody against EGFP. MII oocytes were collected from  H3.3B-EGFP knock-in mice and fertilized in vitro. Asf1a-MO or Control-MO was microinjected into zygote at 2 hpi and immunostained with EGFP antibody at 8 hpi. N Quantification of the EGFP fluorescence intensity in the male and female pronuclei. Data were presented as mean ± SEM, and analyzed by Student’s t-test. N represents the number of embryos or blastomeres examined