Deletion of the tyrosine phosphatase Shp2 in Sertoli cells causes infertility in mice

Abstract

The male's ability to reproduce is completely dependent on Sertoli cells. However, the mechanisms governing the functional integrity of Sertoli cells have remained largely unexplored. Here, we demonstrate that deletion of Shp2 in Sertoli cells results in infertility in mice. In Shp2 knockout mice (SCSKO), a normal population of Sertoli cells was observed, but the blood-testis barrier (BTB) was not formed. Shp2 ablation initiated the untimely and excessive differentiation of spermatogonial stem cells (SSCs) by disturbing the expression of paracrine factors. As a consequence, the process of spermatogenesis was disrupted, and the germ cells were depleted. Furthermore, Shp2 deletion impaired the cell junctions of the primary Sertoli cells and failed to support the clonal formation of SSCs co-cultured with SCSKO Sertoli cells. As expected, Shp2 restoration largely restores the cell junctions of the primary Sertoli cells and the clonal formation of SSCs. To identify the underlying mechanism, we further demonstrated that the absence of Shp2 suppressed Erk phosphorylation, and thus, the expression of follicle-stimulating hormone (FSH)- and testosterone-induced target genes. These results collectively suggest that Shp2 is a critical signaling protein that is required to maintain Sertoli cell function and could serve as a novel target for male infertility therapies.

Figure 1. Shp2 depletion in Sertoli cells impeded testes development in mice.

(A) Shp2 expression was detected with immunofluorescence staining in the seminiferous tubules at embryonic day 18 (E18). Arrowheads indicated Sertoli cells. The cell nucleus was stained with DAPI. (B) Shp2 protein levels were analyzed by Western blotting in primary Sertoli cells and germ cells isolated from 14-day-old mice. Wt1 is a marker of SCs and SCP3 is a marker protein of GCs. Tubulin was used as a loading control. The full-length blots are presented in Supplemental Figure S6. (C) The stereomicroscopy images of the testes from Shp2f/f and SCSKO male mice at 4 weeks. (D) The average weight of the testes from 2-, 3- and 4-week-old Shp2^f/f and SCSKO mice. The values are expressed as the mean ± standard error of the mean (SEM) from 8 mice. Statistical analysis was performed using Student’s t-test. Asterisks denote the statistical significance, **P < 0.01; ***P < 0.001.

Figure 2. Shp2 ablation caused abnormal seminiferous epithelium and the gradual loss of the germ cells.

(A) The histological structure of the seminiferous epithelium stained with hematoxylin and eosin (H/E) in Shp2^f/f and SCSKO mice at 1, 3, 4 and 16 weeks. Arrowheads indicated the abnormal phenotypes of the seminiferous epithelium. (B) Apoptotic cells in the seminiferous epithelium were detected by TUNEL staining. (C) The quantification of the number of apoptotic cells per tubule in the intersecting surface of the testes based on TUNEL analysis. (D) Sertoli cells labeled by Wt1 immunofluorescence staining in the seminiferous epithelium. (E) The quantification of the number of Sertoli cells per tubule in the intersecting surface of the testes. The cell nucleus was stained with DAPI. The values are expressed as the mean ± SEM from at least 5 mice from different litters. Statistical analysis was performed using Student’s t-test. Asterisks denote the statistical significance; **P < 0.01.

Figure 3. BTB integrity was disrupted by Shp2 deletion.

(A) BTB permeability was assayed with the biotin tracer (red) using confocal microscopy in two-week-old mice. Asterisk indicates the biotin tracer in the adluminal compartment of the seminiferous tubules. (B) Immunohistochemical staining of the BTB junction proteins Cx43, Claudin11 and JAMA in seminiferous tubules in two-week-old mice. (C) Altered BTB-related genes in the microarray gene database, and the protein levels of various genes were analyzed by Western blotting in testes from two-week-old mice. The full-length blots are presented in Supplemental Figure S7. (D) The quantity of the cell junctions between primary Sertoli cells isolated from two-week-old mice was measured by transepithelial resistance (TER) assays. The experiments were repeated at least three times, and one representative result was shown. (E) Shp2 expression and Erk and Akt phosphorylation in primary Sertoli cells used in the TER assays. Tubulin was used as a loading control. The full-length blots are presented in Supplemental Figure S8. Ad, adenovirus virus; f/f, Shp2^f/f cells; ko, SCSKO cells; Q79P, constitutively active Shp2 mutant. The values are expressed as the mean ± SEM. Statistical analysis was performed using Student’s t-test. Asterisks denote the statistical significance, **P < 0.01; ***P < 0.001.

Figure 4. Shp2 deficiency decreased the number of SSCs and initiated the untimely and excessive differentiation of SSCs in testes.

(A) Undifferentiated SSCs in the seminiferous tubules were identified by PLZF-positive immunofluorescence staining (green). (B) Proliferating SSCs in the seminiferous tubules were identified with PLZF and PH3-double positive immunofluorescence staining (yellow). Asterisks indicated positive SSCs (yellow). (C) Quantification of the proliferating SSCs (PLZF and Ph3 double stained cells) per tubule in the intersecting surface. The values are expressed as the mean ± SEM from at least 5 mice from different litters. Statistical analysis was performed using Student’s t-test. Asterisks denote statistical significance. **P < 0.01. (D) The differentiated SSCs in the seminiferous tubules were identified by c-Kit-positive immunofluorescence staining (green). The cell nucleus was stained with DAPI. The experiments were repeated at least three times, and one representative result is presented.

Figure 5. Shp2 deletion enhanced spermatogonia differentiation and altered the expression of proteins essential for SSC maintenance.

(A) The differentiated spermatogonia in the seminiferous tubules are denoted by stra8-positive immunofluorescence staining (green). (B) The spermatocytes were identified as Scp3 positive (red). Asterisks denote statistical significance. The cell nucleus was stained with DAPI. Tubulin was used as a loading control. The experiments were replicated at least three times, and one representative result is presented. *P < 0.05: **P < 0.01; ***P < 0.001. (C) Altered genes related to SSC maintenance in the microarray gene database, and the protein level of some of these genes was analyzed by Western blotting in primary Sertoli cells at postnatal day 10. The full-length blots are presented in Supplemental Figure S9.

Figure 6. The clone formation ability of SSCs seeded on primary Sertoli cells was impaired by Shp2 deletion in Sertoli cells.

(A) The clone formation of SSCs co-cultured with primary Sertoli cells isolated from 2-week-old mice. The arrowhead indicates the SSC clones. (B) Quantification of the number of stem cell clones identified by Thy (CD90) staining and flow cytometry. Ad, adenovirus; Q79P, constitutively active Shp2 mutant. The experiments were replicated at least three times, and one representative result is presented.

Figure 7. Shp2 deletion disturbed the activation of cytoplasmic signaling pathways and the expression of FSH- and testosterone-induced target genes.

(A,B) The expression of a few target genes and Erk and Akt phosphorylation in primary Sertoli cells treated with FSH (A) or testosterone (B). The full-length blots are presented in Supplemental Figure S10. (C) The expression of FSH- or testosterone-target genes in the testes from two-week-old mice. Tubulin was used as a loading control. The full-length blots are presented in Supplemental Figure S11. The experiments were replicated at least three times, and one representative result is presented.