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Testosterone regulates the autophagic clearance of androgen binding protein in rat Sertoli cells.

Ma Y, Yang HZ, Xu LM, Huang YR, Dai HL, Kang XN - Sci Rep (2015)

Bottom Line: Dysregulation of androgen-binding protein (ABP) is associated with a number of endocrine and andrology diseases.Further studies identified that the autophagic clearance of ABP might be selectively regulated by testosterone, independent of stress (hypoxia)-induced autophagic degradation.These data demonstrate that testosterone up-regulates ABP expression at least partially by suppressing the autophagic degradation.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biobank, Renji Hospital, School of Medicine, Shanghai JiaoTong University, Building 1, 1630 DongFang Road, Shanghai. 200127, China [2] Department of Urology, Renji Hospital, School of Medicine, Shanghai JiaoTong University, Building 7, 1630 DongFang Road, Shanghai. 200127, China.

ABSTRACT
Dysregulation of androgen-binding protein (ABP) is associated with a number of endocrine and andrology diseases. However, the ABP metabolism in Sertoli cells is largely unknown. We report that autophagy degrades ABP in rat Sertoli cells, and the autophagic clearance of ABP is regulated by testosterone, which prolongs the ABP biological half-life by inhibiting autophagy. Further studies identified that the autophagic clearance of ABP might be selectively regulated by testosterone, independent of stress (hypoxia)-induced autophagic degradation. These data demonstrate that testosterone up-regulates ABP expression at least partially by suppressing the autophagic degradation. We report a novel finding with respect to the mechanisms by which ABP is cleared, and by which the process is regulated in Sertoli cells.

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Stress-induced autophagy does not degrade ABP.(A–C), Primary Sertoli cells were cultured in hypoxia (1%) or serum deprivation medium for 24 h, LC3B and P62 were determined by Western blots (A). LC3B was also assessed by immunofluorescence (B). A high concentration of CQ (100 uM) was used before the immunofluorescence assay to promote the accumulation of LC3. The average number of puncta per cell was calculated from 10 random fields (C) (n = 3, **p < 0.01). (D–G), Cells were treated with CQ (50 uM) or rapamycin (10 nM) in the presence or absence of hypoxia for 24 h. ABP, LC3B and Beta actin were determined by Western blots (D). The bands in ABP immunoblots were analysed (E) (n = 3, *p < 0.05, **p < 0.01). ELISA was used to determine the ABP expression in the supernatants (F) (n = 3, *p < 0.05). ABP was also assessed by immunocytochemistry (G). (H–I), Cells were exposed to hypoxia and treated with testosterone (10 nM), CQ (50 uM) or rapamycin (10 nM) as indicated, ABP and LC3B were determined by immunoblots (H). The densitometric analysis of the bands in ABP immunoblots is shown in (I) (n = 3, *p < 0.05 when compared with any other group).
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f4: Stress-induced autophagy does not degrade ABP.(A–C), Primary Sertoli cells were cultured in hypoxia (1%) or serum deprivation medium for 24 h, LC3B and P62 were determined by Western blots (A). LC3B was also assessed by immunofluorescence (B). A high concentration of CQ (100 uM) was used before the immunofluorescence assay to promote the accumulation of LC3. The average number of puncta per cell was calculated from 10 random fields (C) (n = 3, **p < 0.01). (D–G), Cells were treated with CQ (50 uM) or rapamycin (10 nM) in the presence or absence of hypoxia for 24 h. ABP, LC3B and Beta actin were determined by Western blots (D). The bands in ABP immunoblots were analysed (E) (n = 3, *p < 0.05, **p < 0.01). ELISA was used to determine the ABP expression in the supernatants (F) (n = 3, *p < 0.05). ABP was also assessed by immunocytochemistry (G). (H–I), Cells were exposed to hypoxia and treated with testosterone (10 nM), CQ (50 uM) or rapamycin (10 nM) as indicated, ABP and LC3B were determined by immunoblots (H). The densitometric analysis of the bands in ABP immunoblots is shown in (I) (n = 3, *p < 0.05 when compared with any other group).

Mentions: The above results allow us to speculate that the autophagic clearance of ABP is selectively regulated by testosterone. Hypoxia and/or nutrition deprivation induce autophagy in most cell types2526. To investigate if stress induces autophagy in primary Sertoli cells, we treated cells with hypoxia or nutrition deprivation. The results showed that only hypoxia induced obvious autophagy in primary Sertoli cells (Fig. 4A), as indicated by the analysis of LC3B and P62 levels. Hypoxia can accelerate the turnover of LC3 protein; to promote the accumulation of LC3, a high concentration of CQ was used before the immunofluorescence assay in both the control and hypoxia groups, and we found that cells treated with hypoxia had more LC3 puncta (Fig. 4B, 4C).


Testosterone regulates the autophagic clearance of androgen binding protein in rat Sertoli cells.

Ma Y, Yang HZ, Xu LM, Huang YR, Dai HL, Kang XN - Sci Rep (2015)

Stress-induced autophagy does not degrade ABP.(A–C), Primary Sertoli cells were cultured in hypoxia (1%) or serum deprivation medium for 24 h, LC3B and P62 were determined by Western blots (A). LC3B was also assessed by immunofluorescence (B). A high concentration of CQ (100 uM) was used before the immunofluorescence assay to promote the accumulation of LC3. The average number of puncta per cell was calculated from 10 random fields (C) (n = 3, **p < 0.01). (D–G), Cells were treated with CQ (50 uM) or rapamycin (10 nM) in the presence or absence of hypoxia for 24 h. ABP, LC3B and Beta actin were determined by Western blots (D). The bands in ABP immunoblots were analysed (E) (n = 3, *p < 0.05, **p < 0.01). ELISA was used to determine the ABP expression in the supernatants (F) (n = 3, *p < 0.05). ABP was also assessed by immunocytochemistry (G). (H–I), Cells were exposed to hypoxia and treated with testosterone (10 nM), CQ (50 uM) or rapamycin (10 nM) as indicated, ABP and LC3B were determined by immunoblots (H). The densitometric analysis of the bands in ABP immunoblots is shown in (I) (n = 3, *p < 0.05 when compared with any other group).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4352847&req=5

f4: Stress-induced autophagy does not degrade ABP.(A–C), Primary Sertoli cells were cultured in hypoxia (1%) or serum deprivation medium for 24 h, LC3B and P62 were determined by Western blots (A). LC3B was also assessed by immunofluorescence (B). A high concentration of CQ (100 uM) was used before the immunofluorescence assay to promote the accumulation of LC3. The average number of puncta per cell was calculated from 10 random fields (C) (n = 3, **p < 0.01). (D–G), Cells were treated with CQ (50 uM) or rapamycin (10 nM) in the presence or absence of hypoxia for 24 h. ABP, LC3B and Beta actin were determined by Western blots (D). The bands in ABP immunoblots were analysed (E) (n = 3, *p < 0.05, **p < 0.01). ELISA was used to determine the ABP expression in the supernatants (F) (n = 3, *p < 0.05). ABP was also assessed by immunocytochemistry (G). (H–I), Cells were exposed to hypoxia and treated with testosterone (10 nM), CQ (50 uM) or rapamycin (10 nM) as indicated, ABP and LC3B were determined by immunoblots (H). The densitometric analysis of the bands in ABP immunoblots is shown in (I) (n = 3, *p < 0.05 when compared with any other group).
Mentions: The above results allow us to speculate that the autophagic clearance of ABP is selectively regulated by testosterone. Hypoxia and/or nutrition deprivation induce autophagy in most cell types2526. To investigate if stress induces autophagy in primary Sertoli cells, we treated cells with hypoxia or nutrition deprivation. The results showed that only hypoxia induced obvious autophagy in primary Sertoli cells (Fig. 4A), as indicated by the analysis of LC3B and P62 levels. Hypoxia can accelerate the turnover of LC3 protein; to promote the accumulation of LC3, a high concentration of CQ was used before the immunofluorescence assay in both the control and hypoxia groups, and we found that cells treated with hypoxia had more LC3 puncta (Fig. 4B, 4C).

Bottom Line: Dysregulation of androgen-binding protein (ABP) is associated with a number of endocrine and andrology diseases.Further studies identified that the autophagic clearance of ABP might be selectively regulated by testosterone, independent of stress (hypoxia)-induced autophagic degradation.These data demonstrate that testosterone up-regulates ABP expression at least partially by suppressing the autophagic degradation.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biobank, Renji Hospital, School of Medicine, Shanghai JiaoTong University, Building 1, 1630 DongFang Road, Shanghai. 200127, China [2] Department of Urology, Renji Hospital, School of Medicine, Shanghai JiaoTong University, Building 7, 1630 DongFang Road, Shanghai. 200127, China.

ABSTRACT
Dysregulation of androgen-binding protein (ABP) is associated with a number of endocrine and andrology diseases. However, the ABP metabolism in Sertoli cells is largely unknown. We report that autophagy degrades ABP in rat Sertoli cells, and the autophagic clearance of ABP is regulated by testosterone, which prolongs the ABP biological half-life by inhibiting autophagy. Further studies identified that the autophagic clearance of ABP might be selectively regulated by testosterone, independent of stress (hypoxia)-induced autophagic degradation. These data demonstrate that testosterone up-regulates ABP expression at least partially by suppressing the autophagic degradation. We report a novel finding with respect to the mechanisms by which ABP is cleared, and by which the process is regulated in Sertoli cells.

Show MeSH
Related in: MedlinePlus