Fig. 4

The homozygous mutation in ZCWPW1 generated high DNA fragmentation and loss of the DSB repair capability. A The Sperm Chromatin Dispersion (SCD) analysis of spermatozoa obtained from a control individual and the proband by the optical microscopy (Scale bars, 20 μm). B The histogram showed the difference in DNA fragmentation index (DFI) between normal spermatozoa and proband’s spermatozoa. C The western blot showed the lower level of γ-H2AX in WT-ZCWPW1 transfected cells than MUT-ZCWPW1 transfected cells and empty vector transfected cells after treated by hydroxyurea for 12 h. 40 μg protein of extracts was loaded in each lane. D The grayscale analysis of γ-H2AX in each groups was shown. Data represent the mean ± SD from three independent experiments. Student’s t-test, ***P < 0.001, ****P < 0.0001. E The neutral comet assay found out the more DNA tail in MUT-ZCWPW1 transfected cells and empty vector transfected cells than WT-ZCWPW1 transfected cells after treated by hydroxyurea for 12 h (Scale bars, 10 μm). F Bar plot showing the ratio of tail DNA in the neutral comet assays. Data represent the mean ± SD from three independent experiments. Student’s t-test, *P < 0.05. G The western blot revealed the higher level of H3K9ac in WT-ZCWPW1 transfected cells than MUT-ZCWPW1 transfected cells and empty vector transfected cells after treated by hydroxyurea for 12 h. 40 μg protein of extracts was loaded in each lane. H Bar plot showing the grayscale analysis of H3K9ac. Data represent the mean ± SD from three independent experiments. Student’s t test, *P < 0.05, **P < 0.01