Reprogramming of Sertoli cells to fetal-like Leydig cells by Wt1 ablation

Abstract

Sertoli and Leydig cells, the two major somatic cell types in the testis, have different morphologies and functions. Both are essential for gonad development and spermatogenesis. However, whether these cells are derived from the same progenitor cells and the mechanism regulating the differentiation between these two cell types during gonad development remains unclear. A previous study showed that overactivation of Ctnnb1 (cadherin-associated protein, beta 1) in Sertoli cells resulted in Sertoli cell tumors. Surprisingly, in the present study, we found that simultaneous deletion of Wilms' Tumor Gene 1 (Wt1) and overactivation of Ctnnb1 in Sertoli cells led to Leydig cell-like tumor development. Lineage tracing experiments revealed that the Leydig-like tumor cells were derived from Sertoli cells. Further studies confirmed that Wt1 is required for the maintenance of the Sertoli cell lineage and that deletion of Wt1 resulted in the reprogramming of Sertoli cells to Leydig cells. Consistent with this interpretation, overexpression of Wt1 in Leydig cells led to the up-regulation of Sertoli cell-specific gene expression and the down-regulation of steroidogenic gene expression. These results demonstrate that the distinction between Sertoli cells and Leydig cells is regulated by Wt1, implying that these two cell types most likely originate from the same progenitor cells. This study thus provides a novel concept for somatic cell fate determination in testis development that may also represent an etiology of male infertility in human patients.

Keywords: Leydig cells; Sertoli cells; Wt1; transdifferentiation; β-catenin.

Fig. 1.

Leydig-like cell tumor development in Wt1 and Ctnnb1^ex3 double KO mice. (A) The morphology of testes from Wt1^–/floxAMH-Cre, Ctnnb1^+/flox(ex3)AMH-Cre, and double KO mice (Wt1^–/floxCtnnb1^+/flox(ex3)AMH-Cre). (B–E) Histological analysis of testicular tumors from Ctnnb1^+/flox(ex3)AMH-Cre and double KO mice by H&E staining. Immunohistochemical analysis of testis tumor sections using antibodies to WT1 (F and H), 3β-HSD (I and J), and P450SCC (K and L), showing that WT1 protein was present in tumor cells from Ctnnb1^+/flox(ex3)AMH-Cre mice (H) but not in tumor cells from double KO mice (F). 3β-HSD (I and J) and P450SCC (K and L) were expressed in tumor cells from double KO mice (I and K) but not Ctnnb1^+/flox(ex3)AMH-Cre mice (J and L).

Fig. 2.

Leydig-like tumor cells in double KO mice derived from Sertoli cells. (A–C) In control testes, Leydig cells were labeled with 3β-HSD (A, arrows) and ORO (C, arrows), and Sertoli cells within seminiferous tubules were labeled with X-Gal (B, arrows). (D–F) In double KO testes, adjacent serial sections were stained with 3β-HSD (D), X-Gal (E), and ORO (F). (G) The tumor cells in double KO testes were double-labeled with X-Gal and 3β-HSD. (H) Adjacent serial section with X-Gal staining but not with 3β-HSD as a negative control.

Fig. 3.

3β-HSD was expressed in the remnant seminiferous tubules of Wt1^–/floxAMH-Cre mice. (A–C and G–I) In control testes, 3β-HSD was detected only in the Leydig cells from E13.5 to P5 (arrows). (D–F and J–L) In Wt1^–/floxAMH-Cre testes, in addition to Leydig cells (arrows), 3β-HSD was also detected in the remnant seminiferous tubules (F and J–L, arrowheads).

Fig. 4.

Sertoli cell-derived 3β-HSD–positive cells were detected in the testes of adult Wt1^–/floxRosa26R^+/floxAMH-Cre mice. In control testes, Sertoli cells were labeled with X-Gal (A, arrows), whereas Leydig cells were labeled with ORO (C, arrows) and 3β-HSD (E, arrows). In Wt1^–/floxRosa26R^+/floxAMH-Cre testes, serial sections were prepared and stained with X-Gal (B, arrows) and ORO (D, arrows). (F) X-Gal and 3β-HSD double-positive cells (red asterisks) were observed in Wt1^–/floxRosa26R^+/floxAMH-Cre testes.

Fig. 5.

The expression of Leydig cell and Sertoli cell-specific genes in Wt1-deficient Sertoli cells. (A) Leydig cell-specific genes were abundantly expressed in Wt1-deficient Sertoli cells at levels that were comparable to those of control Leydig cells. (B) The expression of Sertoli cell-specific genes was dramatically reduced in Wt1-deficient Sertoli cells. Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01.

Fig. 6.

Differentially expressed genes in Wt1-overexpressing Leydig cells. (A) The efficiency of adenovirus virus transfection of Leydig cells. (B) Relative mRNA level of Wt1 after adenovirus transfection. (C) The mRNA levels of steroidogenic enzymes were significantly reduced in Leydig cells after Wt1 transfection. (D) The expression of Sertoli cell-specific genes was significantly increased in Wt1-overexpressing Leydig cells. Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01.