Limits...
BMP6 Regulates Proliferation and Apoptosis of Human Sertoli Cells Via Smad2/3 and Cyclin D1 Pathway and DACH1 and TFAP2A Activation

View Article: PubMed Central - PubMed

ABSTRACT

Sertoli cells are essential for regulating normal spermatogenesis. However, the mechanisms underlying human Sertoli cell development remain largely elusive. Here we examined the function and signaling pathways of BMP6 in regulating human Sertoli cells. RT-PCR, immunocytochemistry and Western blots revealed that BMP6 and its multiple receptors were expressed in human Sertoli cells. CCK-8 and EDU assays showed that BMP6 promoted the proliferation of Sertoli cells. Conversely, BMP6 siRNAs inhibited the division of these cells. Annexin V/PI assay indicated that BMP6 reduced the apoptosis in human Sertoli cells, whereas BMP6 knockdown assumed reverse effects. BMP6 enhanced the expression levels of ZO1, SCF, GDNF and AR in human Sertoli cells, and ELISA assay showed an increase of SCF by BMP6 and a reduction by BMP6 siRNAs. Notably, Smad2/3 phosphorylation and cyclin D1 were enhanced by BMP6 and decreased by BMP6 siRNAs in human Sertoli cells. The levels of DACH1 and TFAP2A were increased by BMP6 and reduced by BMP6 siRNAs, and the growth of human Sertoli cells was inhibited by these siRNAs. Collectively, these results suggest that BMP6 regulates the proliferation and apoptosis of human Sertoli cells via activating the Smad2/3/cyclin D1 and DACH1 and TFAP2A pathway.

No MeSH data available.


Related in: MedlinePlus

The effect of BMP6 on the proliferation, DNA synthesis and apoptosis of adult human Sertoli cells.(A,B) CCK-8 assay showed the standard curve (A) and growth curve of human Sertoli cells treated with various doses of human recombinant BMP6 factor (B) for 5 days. (C,D) Immunocytochemical staining showed the EDU incorporation in human Sertoli cells without treatment (control) or treated with 100 ng/ml BMP6 for 48 hours. Cell nuclei were counterstained with Hoechst 33342. The percentages of EDU-positive cells were counted out of 500 total cells from three independent experiments. (E–G) Annexin V-APC/PI and flow cytometry analysis showed the apoptosis in human Sertoli cells by 100 ng/ml BMP6 or 0.1% BSA (control) at 72 hours. *Indicated statistically significant differences (p < 0.05) between the control and BMP6-treated group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5384448&req=5

f3: The effect of BMP6 on the proliferation, DNA synthesis and apoptosis of adult human Sertoli cells.(A,B) CCK-8 assay showed the standard curve (A) and growth curve of human Sertoli cells treated with various doses of human recombinant BMP6 factor (B) for 5 days. (C,D) Immunocytochemical staining showed the EDU incorporation in human Sertoli cells without treatment (control) or treated with 100 ng/ml BMP6 for 48 hours. Cell nuclei were counterstained with Hoechst 33342. The percentages of EDU-positive cells were counted out of 500 total cells from three independent experiments. (E–G) Annexin V-APC/PI and flow cytometry analysis showed the apoptosis in human Sertoli cells by 100 ng/ml BMP6 or 0.1% BSA (control) at 72 hours. *Indicated statistically significant differences (p < 0.05) between the control and BMP6-treated group.

Mentions: We probed the effect of BMP6 on the proliferation and apoptosis of human Sertoli cells. CCK-8 assay was performed in human Sertoli cells using recombinant human BMP6 factor and RNA interference8. Standard curve indicated a good correlation between the absorbance and the concentrations of BMP6 in human Sertoli cells (Fig. 3A). CCK-8 assay showed that recombinant human BMP6 promoted the proliferation of human Sertoli cells in dose- and time- dependent ways (Fig. 3B). BMP6 induced a significant increase of cell number of these cells at 100 ng/ml for 5 days. We further examined the influence of BMP6 on DNA synthesis in human Sertoli cells utilizing the EDU incorporation assay. Compared to the control (56.2% ± 4.8% of EDU-positive cells), BMP6 increased the EDU-positive cells up to 64.8% ± 3.5% (Fig. 3C,D), reflecting that BMP6 promotes DNA synthesis of human Sertoli cells. We also probed the influence of BMP6 on the apoptosis of human Sertoli cells, and we found that the average percentage of apoptosis of Sertoli cells was decreased by 7.7% ± 1.9% after treatment with BMP6 at 72 hours (Fig. 3F,G), compared with control group (Fig. 3E,G).


BMP6 Regulates Proliferation and Apoptosis of Human Sertoli Cells Via Smad2/3 and Cyclin D1 Pathway and DACH1 and TFAP2A Activation
The effect of BMP6 on the proliferation, DNA synthesis and apoptosis of adult human Sertoli cells.(A,B) CCK-8 assay showed the standard curve (A) and growth curve of human Sertoli cells treated with various doses of human recombinant BMP6 factor (B) for 5 days. (C,D) Immunocytochemical staining showed the EDU incorporation in human Sertoli cells without treatment (control) or treated with 100 ng/ml BMP6 for 48 hours. Cell nuclei were counterstained with Hoechst 33342. The percentages of EDU-positive cells were counted out of 500 total cells from three independent experiments. (E–G) Annexin V-APC/PI and flow cytometry analysis showed the apoptosis in human Sertoli cells by 100 ng/ml BMP6 or 0.1% BSA (control) at 72 hours. *Indicated statistically significant differences (p < 0.05) between the control and BMP6-treated group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384448&req=5

f3: The effect of BMP6 on the proliferation, DNA synthesis and apoptosis of adult human Sertoli cells.(A,B) CCK-8 assay showed the standard curve (A) and growth curve of human Sertoli cells treated with various doses of human recombinant BMP6 factor (B) for 5 days. (C,D) Immunocytochemical staining showed the EDU incorporation in human Sertoli cells without treatment (control) or treated with 100 ng/ml BMP6 for 48 hours. Cell nuclei were counterstained with Hoechst 33342. The percentages of EDU-positive cells were counted out of 500 total cells from three independent experiments. (E–G) Annexin V-APC/PI and flow cytometry analysis showed the apoptosis in human Sertoli cells by 100 ng/ml BMP6 or 0.1% BSA (control) at 72 hours. *Indicated statistically significant differences (p < 0.05) between the control and BMP6-treated group.
Mentions: We probed the effect of BMP6 on the proliferation and apoptosis of human Sertoli cells. CCK-8 assay was performed in human Sertoli cells using recombinant human BMP6 factor and RNA interference8. Standard curve indicated a good correlation between the absorbance and the concentrations of BMP6 in human Sertoli cells (Fig. 3A). CCK-8 assay showed that recombinant human BMP6 promoted the proliferation of human Sertoli cells in dose- and time- dependent ways (Fig. 3B). BMP6 induced a significant increase of cell number of these cells at 100 ng/ml for 5 days. We further examined the influence of BMP6 on DNA synthesis in human Sertoli cells utilizing the EDU incorporation assay. Compared to the control (56.2% ± 4.8% of EDU-positive cells), BMP6 increased the EDU-positive cells up to 64.8% ± 3.5% (Fig. 3C,D), reflecting that BMP6 promotes DNA synthesis of human Sertoli cells. We also probed the influence of BMP6 on the apoptosis of human Sertoli cells, and we found that the average percentage of apoptosis of Sertoli cells was decreased by 7.7% ± 1.9% after treatment with BMP6 at 72 hours (Fig. 3F,G), compared with control group (Fig. 3E,G).

View Article: PubMed Central - PubMed

ABSTRACT

Sertoli cells are essential for regulating normal spermatogenesis. However, the mechanisms underlying human Sertoli cell development remain largely elusive. Here we examined the function and signaling pathways of BMP6 in regulating human Sertoli cells. RT-PCR, immunocytochemistry and Western blots revealed that BMP6 and its multiple receptors were expressed in human Sertoli cells. CCK-8 and EDU assays showed that BMP6 promoted the proliferation of Sertoli cells. Conversely, BMP6 siRNAs inhibited the division of these cells. Annexin V/PI assay indicated that BMP6 reduced the apoptosis in human Sertoli cells, whereas BMP6 knockdown assumed reverse effects. BMP6 enhanced the expression levels of ZO1, SCF, GDNF and AR in human Sertoli cells, and ELISA assay showed an increase of SCF by BMP6 and a reduction by BMP6 siRNAs. Notably, Smad2/3 phosphorylation and cyclin D1 were enhanced by BMP6 and decreased by BMP6 siRNAs in human Sertoli cells. The levels of DACH1 and TFAP2A were increased by BMP6 and reduced by BMP6 siRNAs, and the growth of human Sertoli cells was inhibited by these siRNAs. Collectively, these results suggest that BMP6 regulates the proliferation and apoptosis of human Sertoli cells via activating the Smad2/3/cyclin D1 and DACH1 and TFAP2A pathway.

No MeSH data available.


Related in: MedlinePlus