Fig 2: Immunofluorescence analysis of follicular defects in TGFBR1-CAG9Cre mice. a-f Double immunofluorescence of DDX4 (red) and ACTA2 (green) using PD12 control (a-c) and TGFBR1-CAG9Cre (d-f) mice. Three independent samples per group were analyzed using immunohistochemistry and/or immunofluorescence. Note the presence of large follicles or abnormal follicle-like structures (dotted yellow lines) in the ovaries of TGFBR1-CAG9Cre mice in comparison with age-matched controls. Scale bar is representatively depicted in (a), and equals 100 µm (a-f)

Fig 3: Ablation of Cul4b in both germ cells and Sertoli cells leads to BTB defects. (A,B) IF staining and (C,D) confocal microscopy of tight junction marker CLDN11 in CTRL and Cul4bAmh;Vasa testis. The insets in A and B are magnified views of boxed areas. Basement membrane outlined by dashed lines in insets. (E,F) IF staining of pS6 (S235/236) showing its accumulation in the mutant tubules. (G,H) Confocal IF of pS6 (S235/236) (red) and SCP3 (green) showing localization of pS6 (S235/236) in CTRL spermatogonia (G, arrows), and ectopic activation in the mutant Sertoli cells (H, arrowheads). (I,J) IF staining of pS6 (S240/244) showing its accumulation in the mutant tubules. (K,L) Confocal IF of pS6(S240/244) (red) and SCP3 (green) showing its normal expression in spermatogonia (K, arrows) and ectopic activation in the mutant germ cells (L, arrowheads). (M,N) IF of a-catenin (CTNNA1) showing its accumulation in the mutant testis. S, spermatogonia; P, pachytene spermatocytes; Z, zygotene spermatocytes; Spg,. White dashed lines outline the seminiferous tubules. Scale bars: 200 µm in (A,B), (E,F), (I,J); 50 µm in the rest.

Fig 4: SPCs-somatic cells co-culture system was able to be maintained in vitro for short term. SPCs-somatic cells co-culture system stably expressed MVH ((a) bright filed, (b) MVH, (c) dapi, (d) merge), PLZF ((e) bright filed, (f) PLZF, (g) dapi, (h) merge), CD9 (i) bright filed, (j) WT1, (k) dapi, (l) merge), AR ((m) bright filed, (n) AR, (o) dapi, (p) merge) and WT1 ((q) bright filed, (r) WT1, (s) dapi, (t) merge). RT-PCR (n = 2) results exhibit the co-culture system expresses somatic markers ((u) 1.Gapdh, 2.Sox9, 3. Sma, 4.Coup-tf II, 5. Wt1, 6.Mvh), Ar (V. 1) and germline markers (v) 2.Scp3, 3.ckit, 4.Integrin-aV, 5.Cd9, 6.Integrin-ß1, 7. Integrin-a6, 8. Plzf, 9. Mvh) after 3 passages. Western blot result confirmed this co-culture system was sensitive to DHT or bicalutamide stimulation, 1, DMSO; 2, 10nM DHT; 3, 10nM bicalutamide, n = 3 (w)

Fig 5: Cul4 genes are essential for spermatogenic cell survival and germ cell homing. (A–H) Morphology of testes from E16.5, P1, P5 and P28 CTRL and Cul4a/bVasa dKO mice by H and E staining. Relative normal morphology was observed in neonatal dKO mutants, however, by P28 the mutant testes are filled with empty tubules. (I–P) IF staining of germ cell marker, VASA, in testicular sections of E16.5, P1, P5 and P28 CTRL and Cul4a/bVasa dKO mice. In neonate mutants, VASA-positive germ cells are present in the dKO seminiferous tubules, but show delay in homing. Insets in (K–N) show magnified view of boxed areas; dashed lines outline individual tubules. Note that clusters of germ cells are positioned in the mutant seminiferous tubule lumens (arrows, insets in L,N), whereas in the CTRLs they have migrated to the basement membranes (arrowheads, insets in K,M). By P28, VASA-positive germ cells are no longer detectable in dKO testes. Scale bars: 100 µm in (A–D), (O,P); 50 µm in (E–N).