RBPJ in mouse Sertoli cells is required for proper regulation of the testis stem cell niche

Abstract

Stem cells are influenced by their surrounding microenvironment, or niche. In the testis, Sertoli cells are the key niche cells directing the population size and differentiation fate of spermatogonial stem cells (SSCs). Failure to properly regulate SSCs leads to infertility or germ cell hyperplasia. Several Sertoli cell-expressed genes, such as Gdnf and Cyp26b1, have been identified as being indispensable for the proper maintenance of SSCs in their niche, but the pathways that modulate their expression have not been identified. Although we have recently found that constitutively activating NOTCH signaling in Sertoli cells leads to premature differentiation of all prospermatogonia and sterility, suggesting that there is a crucial role for this pathway in the testis stem cell niche, a true physiological function of NOTCH signaling in Sertoli cells has not been demonstrated. To this end, we conditionally ablated recombination signal binding protein for immunoglobulin kappa J region (Rbpj), a crucial mediator of NOTCH signaling, in Sertoli cells using Amh-cre. Rbpj knockout mice had: significantly increased testis sizes; increased expression of niche factors, such as Gdnf and Cyp26b1; significant increases in the number of pre- and post-meiotic germ cells, including SSCs; and, in a significant proportion of mice, testicular failure and atrophy with tubule lithiasis, possibly due to these unsustainable increases in the number of germ cells. We also identified germ cells as the NOTCH ligand-expressing cells. We conclude that NOTCH signaling in Sertoli cells is required for proper regulation of the testis stem cell niche and is a potential feedback mechanism, based on germ cell input, that governs the expression of factors that control SSC proliferation and differentiation.

Keywords: Fertility; Mouse; NOTCH signaling; RBPJ; Spermatogenesis; Spermatogonial stem cell; Testicular microlithiasis.

Fig. 1.

Testis weights are significantly increased in non-atrophic knockout mice. (A) Body weights are not significantly altered in control (Rbpj^fl/fl) and Rbpj^SCKO (Amh-cre;Rbpj^fl/fl) mice throughout adulthood. (B) Non-atrophic testis weights are significantly increased in knockout mice beginning at 2 months of age and persist throughout adulthood. (C) Representative images of control and non-atrophic and atrophic Rbpj^SCKO testes at P150. Results are given as mean±s.e.m. *P<0.05, **P<0.01, ***P<0.005. Scale bar: 2 mm.

Fig. 2.

Tubule diameter and epithelial thickness are significantly increased in non-atrophic Rbpj^SCKO mice. Representative periodic acid-Schiff staining of bilateral control (Rbpj^fl/fl) (A), non-atrophic (B) and atrophic (C) Rbpj^SCKO (Amh-cre;Rbpj^fl/fl) mouse testes at P120. (D) von Kossa stain of atrophic Rbpj^SCKO mouse testes showing calcified tubules. (A′-D′) Higher magnification insets of the indicated region in A-D. Quantification of tubule diameter (E), epithelial thickness (F), lumen diameter (G) and percentage of interstitial space (H) in control and non-atrophic Rbpj^SCKO mouse testes at the indicated time points. Results are given as mean±s.e.m. *P<0.05. Scale bars: 100 µm.

Fig. 3.

Increase in testis weight is associated with an increase in number of germ cells, whereas the total number of Sertoli cells is unchanged in Rbpj^SCKO mouse testes. (A) In a representative example of anti-SOX9 antibody whole-mount staining of control (Rbpj^fl/fl) and Rbpj^SCKO (Amh-cre;Rbpj^fl/fl) tubules at P60, yellow dots highlight individual Sertoli (SOX9+) cells within a 25,000-µm² (100 µm×250 µm) tubule outer area. SOX9+ cells were quantified per 25,000 µm² (B), per mg testis (C) and per whole testis (D). (E) Representative periodic acid-Schiff (PAS/H)-stained tubule at P60. Red and blue dots highlight round spermatids and pachytene spermatocytes, respectively. Quantification of round spermatids and pachytene spermatocytes per tubule cross-section (F,H) and per unit seminiferous epithelium area (µm²) (G,I), respectively. Results are given as mean±s.e.m. ***P<0.005; ns, not significant. Scale bars: 100 µm.

Fig. 4.

The expression of Sertoli cell-expressed genes that are required for germ cell maintenance and differentiation is regulated by NOTCH signaling. Quantitative RT-PCR analysis of whole fetal (E14.5) overexpressor (Amh-cre;Rosa^NICD/+) and control (Rosa^NICD/+) gonads (A) and pure YFP+ FACS-sorted Sertoli cells from knockout (Amh-cre;Rbpj^fl/fl;Rosa^YFP/YFP) and control (Amh-cre;Rbpj^+/+;Rosa^YFP/YFP) testes (B). Results are given as mean±s.e.m. **P<0.01, ***P<0.005.

Fig. 5.

Spermatogonial stem cell numbers are increased in Rbpj^SCKO mouse testes. (A,B) Representative tubule cross-sections from control (Rbpj^fl/fl) and knockout (Amh-cre;Rbpj^fl/fl) testes at P27 stained for PLZF or GFRA1 (SSC markers), SOX9 (Sertoli cell marker) and TRA98 (germ cell marker). Arrows show representative PLZF+ or GFRA1+ cells. (C,D) Quantification of the number of PLZF+ or GFRA1+ SSCs per 100 µm tubule perimeter and (E,F) quantification of the number of PLZF+ or GFRA1+ SSCs per SOX9+ cell. (G) Representative anti-GFRA1 antibody whole-mount staining of control and knockout tubules. (H-J) Quantification of A_single, A_paired and A_aligned GFRA1+ cells per 25,000 µm² tubule surface. Results are given as mean±s.e.m. *P<0.05, **P<0.01, ***P<0.005. Scale bars: 100 µm.

Fig. 6.

Germ cells are the ligand expressing cells. (A,B) Pure GFP+ germ cells (Vasa-cre;Rosa^mTmG/+) and Sertoli cells (Vasa-cre;Rosa^mTmG/+) were isolated by FACS and subjected to qRT-PCR gene expression analysis. (A) Markers of germ cells and Sertoli cells were differentially expressed in these cell isolates. (B) All NOTCH ligands were more highly expressed, by 10- to 100-fold, in germ cells than in Sertoli cells. (C) Representative tubule cross-sections from wild-type testes at P60 stained for JAG1 (NOTCH ligand), SOX9 (Sertoli cell marker) and TRA98 (germ cell marker) showing JAG1 staining in spermatogonia but not in Sertoli cells. A magnified view of the indicated area is shown in the inset. The arrows show a representative JAG1+ germ cell; the arrowheads show a representative Sertoli cell that is JAG1−. (D,E) Sertoli cells from control (Rbpj^fl/fl, D) or knockout (Amh-cre;Rbpj^fl/fl, E) testes were cultured in vitro in the presence or absence of the NOTCH ligand JAG1; the lack of a significant difference in response showed that the Notch signaling response was ablated in the knockout testes. (F) Sertoli cells isolated from TNR-GFP mice were cultured in vitro in the presence or absence of germ cells, demonstrating NOTCH activation in the presence of germ cells. A magnified view of the indicated area is shown in the inset. (G) Quantification of GFP fluorescence in the cultures represented in F. Results are given as mean±s.e.m. *P<0.05, **P<0.01, ***P<0.005; ns, not significant. Scale bars: 100 µm.