GDNF stimulates the proliferation of cultured mouse immature Sertoli cells via its receptor subunit NCAM and ERK1/2 signaling pathway

Abstract

Background: The proliferation and final density of Sertoli cells in the testis are regulated by hormones and local factors. Glial cell line-derived neurotrophic factor (GDNF), a distantly related member of the transforming growth factor-β superfamily, and its receptor subunits GDNF family receptor alpha 1 (GFRα1), RET tyrosine kinase, and neural cell adhesion molecule (NCAM) have been reported to be expressed in the testis and involved in the regulation of proliferation of immature Sertoli cells (ISCs). However, the expression patterns of these receptor subunits and the downstream signaling pathways have not been addressed in ISCs.

Results: In the present study, we have reported that the proliferation of cultured ISCs was significantly enhanced by GDNF. The receptor subunits GFRα1 and NCAM but not RET were expressed in ISCs, and the stimulatory effect of GDNF on the proliferation of ISCs was significantly reduced by anti-NCAM antibody blocking or siRNA that specifically targets NCAM mRNA. Additionally, the ERK1/2 inhibitor, PD98059, completely abolished the mitogenic effect of GDNF on ISCs.

Conclusions: GDNF stimulates the proliferation of ISCs via its receptor subunit NCAM and the consequent activation of the ERK1/2 signaling pathway.

Figure 1

GDNF enhances the proliferation of cultured ISCs. (A) The identity and purity of cultured ISCs was confirmed by immunostaining with an antibody against Sertoli cell-specific vimentin protein. (B-C) BrdU-positive ISCs in control (B) and GDNF treated (C) groups. (D) Quantitative analysis of ISC proliferation as indicated by the percentage of BrdU-positive cells in control and GDNF treated groups. (E) Quantitative analysis of TM4 cell proliferation as indicated by the percentages of BrdU-positive cells in GDNF treated and control groups. Statistically significant differences (p < 0.05) among groups are indicated by an asterisk. At least three separate experiments were carried out using the ISC and TM4 cells, with 150-200 cells counted in each experiment. Scale bars indicate 10 μm.

Figure 2

Expression of GDNF and its receptor subunits in cultured ISCs and TM4 cells. (A) RT-PCR results exhibited the expression of GDNF, GFRα1 and NCAM but not RET mRNAs in cultured ISCs and TM4 cells. G3PDH and the Sertoli cell-specific gene CLU were used as positive controls. (B) RET expression in SSCs but not cultured ISCs or TM4 cells was detected by RT-PCR. (C-E) Western blotting demonstrated the expression of NCAM (C) and GFRα1 (D) but not RET (E) proteins in cultured ISCs and TM4 cells. The SY5Y cell line was used as the positive control for the RET protein. The β-actin protein was used as a positive control for each sample.

Figure 3

The proliferation stimulating effect of GDNF on ISCs is mediated by NCAM. (A-C) BrdU-positive ISCs in response to GDNF stimulation with cells pre-treated with non-specific IgG (A) and NCAM polyclonal antibody (B) and the quantitative comparison (C). (D-G) NCAM mRNA (D, E) and protein (F, G) levels in ISCs and TM4 cells were significantly reduced by NCAM-specific siRNA but not by the negative control siRNA when compared with control groups (1, normal culture control; 2, negative control siRNA; 3, NCAM siRNA). G3PDH and β-actin served as the loading controls for RNA and protein respectively. Expression values of NCAM mRNA (E) or protein (G) were normalized against G3PDH or β-actin signals, respectively. (H-L) BrdU-positive ISCs with negative control siRNA (H) and NCAM siRNA groups (I) stimulated with GDNF and in normal culture controls (J) and the NCAM siRNA group (K) not treated with GDNF, as well as the quantitative comparison among all groups (L). The data is presented as means ± SD from three independent experiments. Statistically significant differences (p < 0.05) among groups are indicated by * or ** (p < 0.01). Scale bar indicates 10 μm.

Figure 4

Phosphorylation of ERK1/2 but not AKT in ISCs is upregulated by GDNF. (A) Time-course of ERK1/2 phosphorylation levels in cultured ISCs stimulated with GDNF and the first bar (0) is cells without GDNF treatment. Total ERK1/2 was the internal control in the semi-quantitative densitometry analysis (right panel). (B) Time course of AKT phosphorylation levels in ISCs stimulated with GDNF, with total AKT as an internal control in the semi-quantitative densitometry analysis (right panel). (C) Time course of ERK1/2 phosphorylation level in PD98059 pre-treated (10 μM) ISCs stimulated with GDNF and the bar (control) is cells without GDNF or PD98059 treatment, with total ERK1/2 as the internal control in the semi-quantitative densitometry analysis (right panel). (D) Time course of AKT phosphorylation levels in PD98059 pre-treated (10 μM) ISCs stimulated with GDNF, with total AKT as the internal control in the semi-quantitative densitometry analysis (right panel). The data were presented as means ± SD from three independent experiments. Statistically significant differences (p < 0.05) among groups are indicated by an asterisk.

Figure 5

Proliferation enhancement effects of GDNF on cultured ISCs is completely abolished by the ERK1/2 inhibitor PD98059. (A-D) BrdU-positive cells in control (A), PD98059 treated (B), GDNF stimulated (C) and pre-treated with PD98059 plus GDNF stimulation groups (D). (E) Quantitative analysis results. The data were presented as means ± SD from three independent experiments. Statistically significant differences (p < 0.05) among groups are indicated by an asterisk. Scale bar indicates 10 μm.