Fig 2: Zcwpw1 is essential for male meiotic recombination.(A to U) Immunostaining for SYCP3 (red) and recombination protein (green) was performed on Zcwpw1+/+ and Zcwpw1-/- spermatocytes from PD25 mice. Representative images of spermatocytes at the leptotene, zygotene, and pachytene (pachytene-like) stages are shown. Each dot represents the number of DNA repair protein foci per cell, with black dots indicating Zcwpw1+/+ spermatocytes and red dots indicating Zcwpw1-/- spermatocytes. Solid lines show the mean and SD of foci number in each group of spermatocytes. (A to G) RPA2 foci. (H to N) RAD51 foci. (O to U) DMC1 foci. P values were calculated by Student’s t test. (V to X) Immunostaining for SYCP3 (red) and TRF1 (green) was performed on Zcwpw1+/+ and Zcwpw1-/- spermatocytes. Representative images of spermatocytes at the pachytene (pachytene-like) stages are shown. Zcwpw1-/- spermatocytes had comparable numbers of TRF1 foci on equator images as compared to Zcwpw1+/+ spermatocytes. n.s., no statistical significance.

Fig 3: DSB repair defects in Meilb2-/- male mice. a Spermatocytes from wild-type (WT) (+/+) males stained with the indicated antibodies and 4,6-diamidino-2-phenylindole (DAPI). b Spermatocytes from Meilb2 knockout (KO) (-/-) males stained with the indicated antibodies and DAPI. c–e Zygotene spermatocytes from WT (+/+) and Meilb2 KO (-/-) males stained with SYCP3 in red, DAPI in blue, and RPA2 (c), DMC1 (d), or RAD51 (e) in green. The images in the other stages are shown in Supplementary Figs. 4d and 5a. The graph shows the number of RPA2 (c), DMC1 (d), or RAD51 (e) foci associated with the chromosome axes. The mean value is shown as a red bar. n shows the analyzed spermatocyte number pooled from three mice for each genotype. f Immunoblots of mouse testis extracts (PD90) from WT (+/+) and Meilb2 KO (-/-) males using the indicated antibodies. g Spermatocytes from WT (+/+) and Meilb2 KO (-/-) males stained with the indicated antibodies and DAPI. Each meiotic prophase I substage is shown. The graph shows the number of SPATA22 foci associated with the chromosome axes. The mean value is shown as a red bar. n shows the analyzed spermatocyte number pooled from four mice for each genotype. h Zygotene spermatocytes from WT, Spo11-/-, and Dmc1-/- males stained with the indicated antibodies and DAPI. The graph shows the number of MEILB2 foci associated with the chromosome axes in zygotene spermatocytes. The mean value is shown as a red bar. n shows the analyzed spermatocyte number pooled from two mice for each genotype. Lep leptotene, Zyg zygotene, Pac pachytene, Dip diplotene. All analyses were with two-tailed t tests. N.S. not significant. ***p < 0.001, ****p < 0.0001. Scale bars, 5 µm. Source data are provided as a Source Data file

Fig 4: BRCA2 localization during meiotic prophase I. a, b Wild-type (WT) spermatocytes expressing GFP-BRCA2-C (a) or GFP-BRCA2-MBD (b) stained with the indicated antibodies and 4,6-diamidino-2-phenylindole (DAPI). c WT zygotene spermatocytes expressing GFP-BRCA2-MBD stained with the indicated antibodies and DAPI. The quantification of co-localization was performed using nine zygotene cells pooled from three electroporated mice. The axis-associated foci were counted. d, e Zygotene spermatocytes from WT, Meilb2-/-, and Dmc1-/- males expressing GFP-BRCA2-C (d) or GFP-BRCA2-MBD (e) stained with the indicated antibodies and DAPI. The graph shows the frequency of the green fluorescent protein (GFP)-positive zygotene spermatocytes with punctate GFP foci. The mean values of three independent experiments from three different electroporated mice are shown (GFP-BRCA2-C: 63 cells in WT, 57 cells in Meilb2-/-, and 85 cells in Dmc1-/-. GFP-BRCA2-MBD: 51 cells in WT, 53 cells in Meilb2-/-, and 76 cells in Dmc1-/-). Error bars show SD. f Schematic of the hierarchical loading of meiotic double-strand break (DSB)-associating proteins. After DSB formation by SPO11, the double-strand DNA is resected into single-strand DNA (ssDNA). RPA is loaded onto the ssDNA, which is epistatic to the localization of MEILB2. MEILB2 is then recruited to the DSBs, likely through binding to RPA or SPATA22-MEIOB, and this in turn recruits BRCA2 through the MEILB2–MEILB2-binding domain (MBD) interaction. BRCA2 then facilitates the loading of the RAD51 and DMC1 recombinases onto the ssDNA, resulting in the formation of a nucleoprotein filament that promotes DNA strand invasion. Lep leptotene, Zyg zygotene, Pac pachytene, Dip diplotene. All analyses used two-tailed t tests. N.S. not significant. ****p < 0.0001. Scale bars, 5 and 1 µm (magnified panel). Source data are provided as a Source Data file

Fig 5: The H3K4me3 reader function of ZCWPW1 is required for synapsis and meiotic recombination.(A) Hematoxylin staining of adult C57BL/6 wild type, Zcwpw1-/-, and Zcwpw1KI/KI testes (left panel) and epididymides (right panel). Adult Zcwpw1-/- and Zcwpw1KI/KI testis sections showed near complete arrest of spermatogenesis. Arrows, apoptotic spermatocytes; arrowheads, empty seminiferous tubules; asterisks, seminiferous tubules lacking post-meiotic spermatocytes. The spermatogenic arrest led to empty epididymides in adult Zcwpw1-/- and Zcwpw1KI/KI mice. (St) Seminiferous tubules, (Ep) Epididymides. Adult mice (6–8 weeks) with n = 5 for each genotype. (B) Chromosome spreads of spermatocytes from the testes of adult WT (upper panel), Zcwpw1-/- (middle panel), and Zcwpw1KI/KI (lower panel) males were immunostained for the SC marker proteins SYCP1 (green) and SYCP3 (red). The arrow indicates a pachytene spermatocyte in WT mice, with completely synapsed chromosomes, and the arrowheads indicate the pachytene-like spermatocytes in adult Zcwpw1-/- and Zcwpw1KI/KI mice with incompletely synapsed chromosomes. (C) Chromosome spreads of spermatocytes from the testes of adult WT and Zcwpw1KI/KI males were immunostained for DMC1 (green) and SYCP3 (red). Representative images of spermatocytes at zygotene and pachytene in WT and at zygotene and pachytene-like stages in Zcwpw1KI/KI are shown. (D) Each dot represents the number of DMC1 foci per cell, with black dots indicating WT spermatocytes and red dots indicating Zcwpw1KI/KI spermatocytes. Solid lines show the mean and SD of foci number in each group of spermatocytes. P values were calculated by Student’s t-test. N represents the number of cells counted, with black indicating WT spermatocytes and red indicating Zcwpw1KI/KI spermatocytes. (E) Chromosome spreads of spermatocytes from the testes of adult WT and Zcwpw1KI/KI males immunostained for RAD51 (green) and SYCP3 (red). (F) Each dot represents the number of RAD51 foci per cell, with black dots indicating WT spermatocytes and red dots indicating Zcwpw1KI/KI spermatocytes. Solid lines show the mean and SD of the foci number for each group of spermatocytes. P values were calculated by Student’s t test. Adult mice (6–8 weeks) with n = 3 for each genotype.Figure 1—source data 1.Number of DMC1 and RAD51 foci in Figure 1D and F.