Fig. 1: C-terminal mutations of CHK1 cause pronuclear fusion failure and zygote arrest that can be rescued by CHK1 inhibitor.

a Pedigrees of four families with CHK1 mutations. The squares denote male family members; circles represent female family members; solid symbols represent affected subjects; open symbols represent unaffected ones; slashes indicate death; question marks unknown fertility status; and arrows indicate probands. W, wild-type. b Fertilized eggs of mice were injected with either WT or mutant hCHK1 cRNAs and then cultured in vitro for 18 h prior to fixation for immunofluorescence. Green, EGFP-tagged WT or mutant CHK1; blue, DAPI. Scale bar, 10 μm. c Schematic diagram of CHK1 protein showing its kinase ___domain, the C-terminal ___domain with SQ, CM1 and CM2 motifs, and the positions of altered amino acids. NES, nuclear export signal; NLS, nuclear localization signal. d A diagram showing the downstream pathway of CHK1 after activation. e HEK-293T cells were transfected with either WT or mutant hCHK1 constructs for 48 h in order to detect downstream CHK1 proteins. The WT group treated with camptothecin to induce DNA damage served as a positive control. f, g S216 in CDC25C and T14/Y15 in CDK1 were mutated to alanines. The CDC25C and CDK1 mutants were then respectively overexpressed in fertilized mouse eggs together with the CHK1 mutant F441fs*16. f Representative images revealing zygote cleavage in each group. WT, wild-type; MT, mutant. Scale bar, 100 μm. g Cleavage rate significantly increased in zygotes carrying mutated forms of CDC25C and CDK1 (P < 0.05; χ2 test). The total cleavage rates of three replicates (about 80 eggs) are shown above the column. h, i Mouse zygotes overexpressing either WT or mutant CHK1 (F441fs*16 or R379Q) were cultured with or without PF477736 (10 nM). Representative images showing embryo development in each group (h). Scale bar, 100 μm. PF477736 markedly increased blastocyst rates in mutated groups (i, based on an unpaired t-test). Data are presented as means ± SEM; ns, no significant difference. **P < 0.01; ***P < 0.001 j Representative image of the offspring (yellow dotted circle) in a mutant group (R379Q). k Time-lapse imaging showing development progress of the embryo in control group and PF477736 treatment groups (PF-1 and PF-3). Em, embryo. l Chromatograms of Sanger sequencing of embryo PF-1 and PF-3. W, wild-type, M, mutant. m Expression of human ESC markers in the two embryo stem cell lines derived from PF-1 and PF-3, including OCT4, SOX2, SSEA4, TRA-1-60 and TRA-1-81. Scale bars, 100 μm.