Fig 2: NBS1 is required for chromosome synapsis and meiotic progression.a Immunofluorescence staining of SYCP3 and ?H2AX was used for analyses of stages of meiotic prophase in spermatocytes from control and Nbs1 vKO mice. Scale bar, 5 µm. b Percentage of spermatocytes at each stage of meiotic prophase from control and Nbs1 vKO mice. Error bars represent standard deviation, n = 3. L leptotene, Z zygotene, P pachytene, D diplotene. c Typical abnormal chromosome structures in Nbs1 vKO zygotene spermatocytes. White boxes indicate the sites of abnormal chromosomes, and are magnified on the right. Scale bar, 5 µm. d Number of abnormal chromosome structures per zygotene spermatocyte from control and Nbs1 vKO mice. Error bars represent SEM, n = 30. e Immunofluorescence staining of SYCP1 and SYCP3 were used for analyses of chromosome synapsis at late leptotene and zygotene stages of meiotic prophase from control and Nbs1 vKO mice. Scale bar, 5 µm. Defective chromosome synapsis was observed in all Nbs1 vKO spermatocytes (100%) but none of the control spermatocytes (0%).

Fig 3: Impaired DNA damage repair, abnormal synapses, and multigene expression changes in Chd2+/- mouse testes(A) Expression analysis of ?H2AX in frozen sections of mouse testes by immunofluorescence assay. Triangles represent the abnormal expression of ?H2AX. Data are shown as the mean ± SD. Student’s t-test, *p < 0.05. Scale bar, 50 µm.(B) Expression analysis of ?H2AX in meiosis by immunofluorescence assay. Scale bar, 5 µm.(C) Co-immunofluorescence staining of SYCP3 and SYCP1 in spermatocytes at pachytene from Chd2+/+ and Chd2+/- male mice. Scale bar, 5 µm.(D) The aberrant synapsis rate of Chd2+/+ and Chd2+/- male mice. Data are shown as the mean ± SD. Student’s t-test, *p < 0.05.(E) Global gene expression alterations with fold changes of =2 or =0.5 (p = 0.05) in Chd2+/- versus Chd2+/+ testes at 12 w.(F) Gene ontology analysis of differentially expressed genes identified by RNA-seq in Chd2+/- testes.

Fig 4: Knockdown of CSTF3 inhibits spermatogonial self-renewal(A) Cstf3 mRNA expression in mouse tissues by RT?qPCR. Data are shown as the mean ± SD.(B) CSTF3 expression analysis by western blot after CSTF3 knockdown in SSCs and C18-4 cells.(C–E) Cell proliferation analysis of SSCs and C18-4 cells with CSTF3 knockdown determined by CCK8, EdU, and colony-formation assays. Data are shown as the mean ± SD. Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar, 400 µm.(F) The impacts of CSTF3 knockdown on the sphere-formation ability of SSCs and C18-4 cells. Scale bar, 300 µm.(G) Immunofluorescence staining of SYCP3 after CSTF3 knockdown in SSCs. Scale bar, 20 µm.(H and I) RT?qPCR and western blot assays of the expression changes in the Oct4, Plzf, Ccnb1, and Ccnd2 genes in C18-4 cells. Data are shown as the mean ± SD. Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 5: Knockdown of CHD2 inhibits spermatogonial self-renewal(A) Detection of RNA interference efficiency for siCHD2 by western blot in SSCs and C18-4 cells.(B–D) Cellular proliferation in SSCs and C18-4 cells with CHD2 knockdown analyzed by CCK8, EdU, and colony-formation assays. Data are shown as the mean ± SD. Student’s t-test, *p < 0.05, ***p < 0.001. Scale bar, 400 µm.(E) The impacts of CHD2 knockdown on the sphere-formation ability of SSCs and C18-4 cells. Scale bar, 300 µm.(F) Immunofluorescence staining of SYCP3 after CHD2 knockdown in SSCs. Scale bar, 20 µm.(G and H) Expression changes of stem-associated genes, differentiation genes, and cell cycle genes in C18-4 cells with CHD2 knockdown determined by RT?qPCR and western blot assays. Data are shown as the mean ± SD. Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001; ns, no significance.