Figure 1.
Inactivation of Wt1 results in seminiferous tubule atrophy and testis hypoplasia.
Reproductive tracts of control (A) and Wt1−/flox; Cre-ERTM (B) males 3 weeks after Tamoxifen-induced Wt1 ablation. Compared to the control mice, the size of Wt1−/flox; Cre- ERTM testes is dramatically reduced (C). (D–I) Cross-sections of control and Wt1−/flox; Cre- ERTM testes at 1 (D, G), 2 (E, H), and 3 (F, I) weeks after Tamoxifen treatment. Vacuolization was detected in a small number of tubules at 1 week after Tamoxifen induction (G) and severe epithelial vacuolization (arrows) was noted at 2 weeks after Tamoxifen induction in Wt1−/flox; Cre- ERTM testis (H). Massive cell loss with empty tubules (asterisks) was observed in Wt1−/flox; Cre- ERTM testis at 3 weeks after Tamoxifen treatment (I).
Figure 2.
Loss of Wt1 results in germ cell death: IHC and H & E staining of testes 3 weeks after Tamoxifen treatment.
In control testes, the seminiferous tubules are filled with GCNA1 positive germ cells (C), whereas, very few GCNA1 positive germ cells (D, black arrows) are noted in the Wt1−/flox; Cre- ERTM testes and some tubules are completely void of germ cells (D, asterisks). Wt1 positive Sertoli cells are present in both control (A) and Wt1−/flox; Cre- ERTM (B) testes. The cauda epididymes of control mice are filled with mature sperms (E, black arrows); in contrast, only immature spermatocyte and cell debris (arrow heads) are present in the cauda epididymes of Wt1−/flox; Cre-ERTM mice and no mature sperm are detectable (F).
Figure 3.
Electropherogram showing single-nucleotide mutation of WT1 in patients with non-obstructive azoospermia.
Figure 4.
The integrity of the BTB is damaged in Wt1−/flox; Cre-ERTM testis 1 week after Tamoxifen treatment.
In control testes, biotin tracer is restricted to the testicular interstitium and the basal compartment of the seminiferous tubules, with no tracer observed in the tubular lumen (A). Biotin tracer is detected along the Sertoli cell plasma membranes from the basement membrane to the lumen in some seminiferous tubules (asterisks) of Wt1−/flox; Cre- ERTM testes (B). By TEM a normal BTB structure (E) and apical ES ultrastructure with well-organized actin bundles(C, arrows) is observed in control testes, but Wt1 mutant testes display abnormal apical ES structures (D, arrows) and the BTB structure is disrupted with numerous blisters (F, asterisks and arrow heads).
Figure 5.
Deletion of Wt1 causes morphologic changes and loss of tight junction formation in cultured SCs.
Control SCs display a typical epithelial morphology (A and E), while Wt1-ablated SCs display a mesenchyme-like morphology (C and E). The tight junctions between SCs were assessed by ZO-1 staining. ZO-1 protein is present along the boundary between control SCs (B, white arrows). In contrast, ZO-1 protein is diffused in the cytosol and not detected along the cell boundary in Wt1-deficient SCs (D). (F) FITC-dextran flux assays indicated increased permeability in Wt1-deficient SCs compared to control SCs. * p<0.05.
Figure 6.
Real time PCR and Western blot verification in SC cultures of altered expression of cell polarity and Wnt signaling genes following Wt1 ablation.
(A) The expression of cell polarity related genes (E-cadherin, Par6b, Cdc42bp5, Sfn) and Wnt signaling genes (Wnt4 and Wnt11) is significantly decreased in Wt1-deficient SCs (white bars). (B) E-cadherin and Espin protein expression is significantly reduced in Wt1-deficient SCs, whereas the expression of occludin, N-cadherin, Zo-1, and β-catenin is not changed. (C) The expression of E-cadherin, Par6b, Wnt4, Wnt11, and Cdc42ep5 in Wt1-deficient SCs was rescued by transfection with Wt1 expressing adenovirus. (D) The expression of Par6b was induced by Wnt4 and Wnt11 expressing adenovirus in Wt1-deficient SCs. (E) The expression of E-cadherin was not rescued by Wnt4 and Wnt11 expressing adenovirus in Wt1-deficient SCs. (F) Wnt4 was knocked down by two different siRNAs (siWnt4-2 and siWnt4-3) in SCs and the expression of E-cadherin and Par6b was significantly decreased compared to control cells (NC) treated with a scrambled siRNA. * p<0.05.
Figure 7.
Functional analysis of WT1 mutations detected in NOA patients.
(A) The expression of Wnt4, Wnt11, and E-cadherin in Wt1-deficient SCs was rescued by wild type Wt1 expressing adenovirus, but not Wt1R362Q and Wt1K386R expressing adenovirus. Co-transfection of Wt1R362Q and Wt1K386R in Wt1-deficient SCs did not affect Wt1 induced Wnt4, Wnt11, and E-cadherin expression. (B) Schematic image of Wnt4 promoter, black oval indicated the predicted Wt1 binding site. (C) A 211 bp band in Wnt4 promoter was amplified after pulled down with Wt1 antibody in Wt1 expressing adenovirus transfected HepG2 cells, but not in Wt1R362Q and Wt1K386R expressing adenovirus transfected HepG2 cells. Histone3 antibody and input was used as positive control, and IgG and vector was used as negative control.