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

Yang Y, Han C - BMC Cell Biol. (2010)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China.

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.

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Related in: MedlinePlus

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.
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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.

Mentions: Highly purified ISC cultures from 4-5-day-old mice were acquired through several passages of testicular cells, which were maintained in serum-free DMEM/F12 medium. The Sertoli cell-specific protein vimentin [22-25] was detected by immunostaining to evaluate the purity of the culture. After immunocytochemical staining, the numbers of vimentin-positive cells and DAPI stained nuclei were counted. As shown in Figure 1A, more than 95% (data not shown) of the cells were vimentin-positive Sertoli cells.


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

Yang Y, Han C - BMC Cell Biol. (2010)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2967512&req=5

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.
Mentions: Highly purified ISC cultures from 4-5-day-old mice were acquired through several passages of testicular cells, which were maintained in serum-free DMEM/F12 medium. The Sertoli cell-specific protein vimentin [22-25] was detected by immunostaining to evaluate the purity of the culture. After immunocytochemical staining, the numbers of vimentin-positive cells and DAPI stained nuclei were counted. As shown in Figure 1A, more than 95% (data not shown) of the cells were vimentin-positive Sertoli cells.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China.

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.

Show MeSH
Related in: MedlinePlus