Fig 2: Cul4 genes are critical to maintain male germ cell proliferation. (A–D) IF staining of phospho-histone H3 (pHH3, red) in P5 CTRL and Cul4a/bVasa dKO testes. Arrows point to pHH3-positive G2 phase cells. (E) Quantification of pHH3-positive cells in seminiferous tubules revealed significant reduction in number of pHH3+ cells in the dKO, particularly in cells at G2 phase. Inset shows typical pHH3 staining pattern of cells at prophase (P), metaphase (M) and G2 phase. M/T, metaphase/telophase. Total: CTRL 57.5 ± 5.3, dKO 24.0 ± 5.3, p = 2.5 × 10 -5; G2: CTRL 25.8 ± 5.1, dKO 4.8 ± 1.3, p = 3.9 × 10 -5; P: CTRL 20.2 ± 3.3, dKO 13.0 ± 2.7, p = 0.007; M/T: CTRL 11.5 ± 3.1, dKO 13.0 ± 2.7, p = 0.07; n = 4 for CTRL and n = 5 for dKO. (F–I) Double IF staining of pHH3 (red) and PLZF (green) of P5 CTRL and dKO testicular sections. Solid white lines circle out pHH3+; PLZF+ proliferating germ cells, dashed white lines circle out pHH3+; PLZF- cells. (J) Quantification of pHH3/PLZF double IF cells revealed significant decrease in number of double positive cells. pHH3+; PLZF+: CTRL 22.8 ± 7.7, dKO 7.5 ± 1.0, p = 8.8 × 10 -4; pHH3+; PLZF-: CTRL 28.8 ± 9.1, dKO 25.1 ± 5.1, p = 0.42; n = 5 for CTRL and n = 6 for dKO. Scale bars: 50 µm in (A–D), 20 µm (F–I).

Fig 3: Cul4 genes are required for male germ cell survival. (A–D) IF staining of CUL4A (red) and SCP3 (green) of P28 testis. While primary spermatocytes expressing both CUL4A (A) and SCP3 (B) are present in the CTRL testis, this population is completely missing in the dKO mutant testis (C,D). (E–H) IF staining of CUL4B (red) and SCP3 (green) of P28 testis. CUL4B is highly expressed in the Sertoli cells (E, arrowheads) and round spermatids (Spd) of CTRL testis (E,F); whereas in the Cul4a/bVasa dKO, CUL4B-positive Sertoli cells are the only cell type remaining in the seminiferous tubules (G,H arrowheads). (I–N) IF staining of AR (red) and CTNNB1 (green) of P28 testicular sections. Wild-type testis show AR-high myoid cells (I, arrows) and AR-low Sertoli cells (I, arrowheads), and well organized germ cell/Sertoli cell network outlined by CTNNB1 staining (J). The dKO mutant testis retains myoid and Sertoli cells (L, arrows and arrowheads, respectively), with disorganized residual CTNNB1 staining (M). DAPI counterstain is shown in (K) and (N). Spd, spermatids; Spct, spermatocytes; Spg, spermatogonia; Int, interstitial cells. (O,P) IF staining of CLDN11 (red) and SCP3 (green) of P28 testis. At this stage in the wildtype testis, tight junctions have formed apical to the spermatogonial stem cells to separate the basal and adluminal compartments (O, arrowheads). CLDN11 was only detected in the basal-lateral membranes of dKO Sertoli cells (P, arrows). Scale bars: 50 µm.

Fig 4: CUL4A and CUL4B co-localize in fetal gonocytes. (A–F) Immunofluorescence (IF) staining of CUL4A (red) and CUL4B (green) in E16.5 wild-type testis. Both CUL4A (A) and CUL4B (B) are highly expressed in the gonocytes (arrowheads), with CUL4B more prominent in the nuclei and CUL4A in both nuclei and cytoplasm. (C) shows merged CUL4A and CUL4B staining, and (D) shows DAPI counterstain. (E,F) are higher-magnitude views of the boxed areas in (A,B), respectively, with DAPI staining overlaid. Arrowheads point to gonocytes. Dashed lines outline seminiferous tubules. Ser, Sertoli cells; Gc, gonocytes. Scale bar 50 µm.