Fig 2: Distinct forms of ubiquitin regulation by RNF8 and SCML2: Immunostaining with FK2 antibody, which recognizes both mono- and polyubiquitinated conjugates.(A) Schematic of spermatogenesis. (B) Model of distinct forms of regulation of ubiquitination by RNF8 and SCML2. (C-F) Immunostaining of SYCP3 and FK2 on meiotic chromosome spreads. Dotted circles: sex chromosomes. Scale bar: 10 µm. Representative images are shown for at least 30 spermatocytes from each substage, from at least 3 independent mice per mouse model. The intensity of immunostaining is quantified by densitometry across the indicated path (‘ to”) and plotted in a relative intensity range of 0–1, which is normalized among the samples at the same stage. (G) Schematic of ubiquitin targets recognized by the FK2 antibody in each mouse model. A pink circle denotes RNF8-dependent ubiquitination and an orange circle denotes events mediated by a different E3 ubiquitin ligase.

Fig 3: Ppp6c cKO mice arrest spermatogenesis at step 7-8 spermatids.A Determination of stages (I–XII) and identification of cell types using a combination of IHC and PNA-lectin histochemistry. Three-µm-thick sections were double-immunostained for PLZF (red) and SOX9 (white), then stained with PNA-lectin histochemistry (green) and DAPI (blue). Scale bar: 10 µm. B Determination of stages (I–XII) and identification of cell types using a combination of IHC and PNA-lectin histochemistry. Three-µm-thick sections were double-immunostained for SYCP3 (red) and SOX9 (white), then stained with PNA-lectin histochemistry (green) and DAPI (blue). Scale bar: 10 µm. C Quantification of spermatogenic cell types in Ppp6cWT and Ppp6ccKO mice based on the images shown in A-B. For each cell type, at least 150 tubules from three mice were counted. The average numbers of cells per tubule were converted to ratios and compared between Ppp6cWT and Ppp6ccKO mice. SG-A, type A spermatogonia; SG-In, type In spermatogonia; SG-B, type B spermatogonia; lepSC/zygSC leptotene spermatocytes/zygotene spermatocytes, pacSC/dipSC pachytene spermatocytes/diplotene spermatocytes, rST round spermatids, eST elongating spermatids, Se Sertoli cells.

Fig 4: RNF8-dependent establishment of H3K27ac and its inhibition by H2AK119ub.(A-D) Immunostaining of SYCP3 and H3K27ac on meiotic chromosome spreads. Dotted circles: sex chromosomes. Scale bar: 10 µm. Representative images are shown for at least 30 spermatocytes from each substage, from at least 3 independent mice per mouse model. The intensity of immunostaining is quantified by densitometry across the indicated path (‘ to”) and plotted in a relative intensity range of 0–1, which is normalized among all pictures in A-D. (E) Model of the regulation of H3K27ac. (F) Immunostaining of SYCP3 and H3K27ac on wild-type and Rnf8-KO round spermatids using slides that preserve the relative nuclear organization of spermatogenic cells. Dotted circles: post-meiotic sex chromatin. Scale bar: 10 µm. Representative images are shown for at least 30 round spermatids from at least 3 independent mice.

Fig 5: Crossover number and length of SC are reduced in Ewsr1 CKO. (A) Presence of bivalents without crossovers in Ewsr1 CKO. MLH1 (green) and SYCP3 (magenta) staining in Ewsr1 control and CKO chromosome spread. White arrow, autosome with no crossover; yellow arrow, sex chromosomes with no crossover. Scale bar, 10 µm. (B) Distribution of the number of MLH1 foci in pachytene spermatocytes. (C) Distribution of the synaptonemal complex lengths. (D) Cumulative plots of crossovers on chromosome 1 (left) and chromosome 11 (right). Bars in C and D are mean ± SD. ***p < 0.001 by Student’s t test.