Fig. 1: Nonequilibrium pronuclear H3K9me3 is specified by LSM1.

a Immunostaining for H3K9me3 in nuclei from GV-, MI-, and MII-stage oocytes and 1-cell and 2-cell embryos. Scale bars, 20 µm. Representative images from 3 biological replicates, with each replicate containing ~10 oocytes/embryos. b Scheme for the Lsm1 oocyte-stage KD experiment. Lsm1 siRNA was injected at the GV stage after which in vitro maturation (IVM) and in vitro fertilization (IVF) were performed. Some of the fertilized oocytes were fixed at the zygote stage for immunostaining, and the others were cultured in vitro for embryo development assays. c Immunostaining for H3K9me3 in representative pronucleus of control (Ctrl) and Lsm1 knockdown (Lsm1_KD) zygotes. Scale bars, 20 µm. Representative images from 3 biological replicates, with each replicate containing ~10 zygotes. d Bar graph showing percentage of H3K9me3 positive male PN zygote in different groups. The statistical data are expressed as mean ± SEM, ***p < 0.001 (p = 0.0004) by two-sided Student’s t test for each comparison. Each treatment contains 3 biological replicates, with each replicate containing ~30 zygotes. e Scatterplot showing H3K9me3 fluorescence intensity mean values in pronucleus of Ctrl and Lsm1_KD groups. The statistical data are expressed as mean ± SEM, *p < 0.05 (p = 0.0289) and ns (p = 0.7834) means not significant by two-sided Student’s t test for each comparison. Each treatment contains 3 biological replicates, with each replicate containing ~10 zygotes. f Representative images of injected GV-stage oocytes, mature oocytes and blastocysts in the Ctrl and Lsm1_KD groups. Scale bars, 100 µm. Representative images from 3 biological replicates, with each replicate containing ~35 injected GV-oocytes. g Quantification of embryo development competence in response to Lsm1 KD with or without different versions of Lsm1 RNA. The statistical data are expressed as mean ± SEM, *p < 0.05 and ns means not significant by one-way ANOVA test. Each treatment contains 3 biological replicates, with each replicate containing ~35 embryos.