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

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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.


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The expression of BMP6 and its receptors, including ACVR1, BMPR1A, BMPR1B, ACVR2A, ACVR2B and BMPR2 in human Sertoli cells and testis.(A) RT-PCR displayed the expression of BMP6 and its receptors in human Sertoli cells. Water without DNA served as a negative control and ACTB was employed as a loading control for total RNA. (B) Western blots showed the expression of BMP6, ACVR1, BMPR1A, BMPR1B and BMPR2 in the isolated human Sertoli cells of three independent samples. Notes: 1, 2, 3 indicated three different samples. (C–H) Immunocytochemistry illustrated the expression of BMP6 (C), ACVR1 (D), BMPR1A (E), BMPR2 (F), and BMPR1B (G) in the isolated human Sertoli cells. Replacement of primary antibodies with PBS served as a negative control (H). (I) Double immunostaining showed the expression of BMP6 and VASA in human Sertoli cells. (J) Immunofluorescence showed the presence of VASA in male germ cells of OA patients. (K–P) Immunofluorescence revealed cellular localization of BMP6 (K), BMP6 and SOX9 (L), ACVR1 (M), BMPR1A (N) and BMPR2 (O) in human testis. Replacement of primary antibodies with PBS served as a negative control (P). Scale bars in C-P = 10 μm.
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f2: The expression of BMP6 and its receptors, including ACVR1, BMPR1A, BMPR1B, ACVR2A, ACVR2B and BMPR2 in human Sertoli cells and testis.(A) RT-PCR displayed the expression of BMP6 and its receptors in human Sertoli cells. Water without DNA served as a negative control and ACTB was employed as a loading control for total RNA. (B) Western blots showed the expression of BMP6, ACVR1, BMPR1A, BMPR1B and BMPR2 in the isolated human Sertoli cells of three independent samples. Notes: 1, 2, 3 indicated three different samples. (C–H) Immunocytochemistry illustrated the expression of BMP6 (C), ACVR1 (D), BMPR1A (E), BMPR2 (F), and BMPR1B (G) in the isolated human Sertoli cells. Replacement of primary antibodies with PBS served as a negative control (H). (I) Double immunostaining showed the expression of BMP6 and VASA in human Sertoli cells. (J) Immunofluorescence showed the presence of VASA in male germ cells of OA patients. (K–P) Immunofluorescence revealed cellular localization of BMP6 (K), BMP6 and SOX9 (L), ACVR1 (M), BMPR1A (N) and BMPR2 (O) in human testis. Replacement of primary antibodies with PBS served as a negative control (P). Scale bars in C-P = 10 μm.

Mentions: After isolation and identification of adult human Sertoli cells, total RNA was extracted from these cells of OA patients. BMP6 ligand and its multiple receptors, including ACVR1, BMPR1A, BMPR1B, ACVR2A, ACVR2B, and BMPR221, were determined in human Sertoli cells. RT-PCR showed that transcripts of BMP6 and its receptors ACVR1, BMPR1A, BMPR1B, ACVR2A, ACVR2B and BMPR2 were expressed in the freshly isolated human Sertoli cells (Fig. 2A). PCR with water but without cDNA served as a negative control, and PCR with ACTB was used as loading controls for total RNA (Fig. 2A). Western blots further showed that BMP6 and its receptors, including ACVR1, BMPR1A, BMPR1B and BMPR2, were present in the isolated human Sertoli cells (Fig. 2B). Furthermore, immunocytochemical staining displayed that BMP6 (Fig. 2C) and its multiple receptors, including ACVR1 (Fig. 2D), BMPR1A (Fig. 2E), BMPR2 (Fig. 2F), and BMPR1B (Fig. 2G), were present in the isolated human Sertoli cells. Replacement of primary antibodies with PBS served as a negative control, and no immunostaining was observed in these cells (Fig. 2H). Double immunostaining showed that BMP6 was expressed in the isolated cells whereas VASA was undetected in these cells (Fig. 2I). As a positive control, VASA immunostaining was observed in some human male germ cells from the testicular tissues after two-step enzymatic digestion (Fig. 2J). Immunofluorescence revealed that BMP6 was present in human Sertoli cells rather than male germ cells in human testis (Fig. 2K), and BMP6 was coexpressed with SOX9 (a marker for Sertoli cells22) in human Sertoli cells (Fig. 2L). ACVR1 (Fig. 2M), BMPR1A (Fig. 2N) and BMPR2 (Fig. 2O) were expressed in human Sertoli cells as well as human spermatogonia and spermatocytes. Replacement of primary antibodies with PBS served as a negative control, and no immunostaining was observed in human testis (Fig. 2P).


BMP6 Regulates Proliferation and Apoptosis of Human Sertoli Cells Via Smad2/3 and Cyclin D1 Pathway and DACH1 and TFAP2A Activation
The expression of BMP6 and its receptors, including ACVR1, BMPR1A, BMPR1B, ACVR2A, ACVR2B and BMPR2 in human Sertoli cells and testis.(A) RT-PCR displayed the expression of BMP6 and its receptors in human Sertoli cells. Water without DNA served as a negative control and ACTB was employed as a loading control for total RNA. (B) Western blots showed the expression of BMP6, ACVR1, BMPR1A, BMPR1B and BMPR2 in the isolated human Sertoli cells of three independent samples. Notes: 1, 2, 3 indicated three different samples. (C–H) Immunocytochemistry illustrated the expression of BMP6 (C), ACVR1 (D), BMPR1A (E), BMPR2 (F), and BMPR1B (G) in the isolated human Sertoli cells. Replacement of primary antibodies with PBS served as a negative control (H). (I) Double immunostaining showed the expression of BMP6 and VASA in human Sertoli cells. (J) Immunofluorescence showed the presence of VASA in male germ cells of OA patients. (K–P) Immunofluorescence revealed cellular localization of BMP6 (K), BMP6 and SOX9 (L), ACVR1 (M), BMPR1A (N) and BMPR2 (O) in human testis. Replacement of primary antibodies with PBS served as a negative control (P). Scale bars in C-P = 10 μm.
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Related In: Results  -  Collection

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f2: The expression of BMP6 and its receptors, including ACVR1, BMPR1A, BMPR1B, ACVR2A, ACVR2B and BMPR2 in human Sertoli cells and testis.(A) RT-PCR displayed the expression of BMP6 and its receptors in human Sertoli cells. Water without DNA served as a negative control and ACTB was employed as a loading control for total RNA. (B) Western blots showed the expression of BMP6, ACVR1, BMPR1A, BMPR1B and BMPR2 in the isolated human Sertoli cells of three independent samples. Notes: 1, 2, 3 indicated three different samples. (C–H) Immunocytochemistry illustrated the expression of BMP6 (C), ACVR1 (D), BMPR1A (E), BMPR2 (F), and BMPR1B (G) in the isolated human Sertoli cells. Replacement of primary antibodies with PBS served as a negative control (H). (I) Double immunostaining showed the expression of BMP6 and VASA in human Sertoli cells. (J) Immunofluorescence showed the presence of VASA in male germ cells of OA patients. (K–P) Immunofluorescence revealed cellular localization of BMP6 (K), BMP6 and SOX9 (L), ACVR1 (M), BMPR1A (N) and BMPR2 (O) in human testis. Replacement of primary antibodies with PBS served as a negative control (P). Scale bars in C-P = 10 μm.
Mentions: After isolation and identification of adult human Sertoli cells, total RNA was extracted from these cells of OA patients. BMP6 ligand and its multiple receptors, including ACVR1, BMPR1A, BMPR1B, ACVR2A, ACVR2B, and BMPR221, were determined in human Sertoli cells. RT-PCR showed that transcripts of BMP6 and its receptors ACVR1, BMPR1A, BMPR1B, ACVR2A, ACVR2B and BMPR2 were expressed in the freshly isolated human Sertoli cells (Fig. 2A). PCR with water but without cDNA served as a negative control, and PCR with ACTB was used as loading controls for total RNA (Fig. 2A). Western blots further showed that BMP6 and its receptors, including ACVR1, BMPR1A, BMPR1B and BMPR2, were present in the isolated human Sertoli cells (Fig. 2B). Furthermore, immunocytochemical staining displayed that BMP6 (Fig. 2C) and its multiple receptors, including ACVR1 (Fig. 2D), BMPR1A (Fig. 2E), BMPR2 (Fig. 2F), and BMPR1B (Fig. 2G), were present in the isolated human Sertoli cells. Replacement of primary antibodies with PBS served as a negative control, and no immunostaining was observed in these cells (Fig. 2H). Double immunostaining showed that BMP6 was expressed in the isolated cells whereas VASA was undetected in these cells (Fig. 2I). As a positive control, VASA immunostaining was observed in some human male germ cells from the testicular tissues after two-step enzymatic digestion (Fig. 2J). Immunofluorescence revealed that BMP6 was present in human Sertoli cells rather than male germ cells in human testis (Fig. 2K), and BMP6 was coexpressed with SOX9 (a marker for Sertoli cells22) in human Sertoli cells (Fig. 2L). ACVR1 (Fig. 2M), BMPR1A (Fig. 2N) and BMPR2 (Fig. 2O) were expressed in human Sertoli cells as well as human spermatogonia and spermatocytes. Replacement of primary antibodies with PBS served as a negative control, and no immunostaining was observed in human testis (Fig. 2P).

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