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Dibutyltin Dichloride Retards Leydig Cell Developmental Regeneration in Adult Rat Testis

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Dibutyltin Dichloride Retards Leydig Cell Developmental Regeneration in Adult Rat Testis

Xiande Huang et al. Front Pharmacol.

Abstract

Dibutyltin dichloride (DBTCl), widely used as plastic stabilizer, can cause comprehensive toxicity. The present study aims to investigate the effects of DBTCl on rat Leydig cell developmental regeneration and characterize the related mechanism. Adult male Sprague Dawley rats were randomly divided into four groups and gavaged with saline (control) or 5, 10, or 20 mg/kg/day of DBTCl consecutively for 10 days. At the end of the DBTCl treatment, all rats received a single intraperitoneal injection (i.p.,) of 75 mg/kg ethane dimethane sulfonate (EDS) to eliminate all the adult Leydig cells and to induce Leydig cell developmental regeneration. Leydig cell developmental regeneration was evaluated by measuring the levels of serum testosterone, luteinizing hormone, and follicle-stimulating hormone on days 7, 35, and 56 post-EDS. Leydig cell gene and protein expression levels, as well as cell morphology and cell counts were also carried out on day 56 post-EDS. The present study found that DBTCl significantly reduced serum testosterone levels on days 35 and 56 post-EDS, but increased serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels on day 56 at ≥ 5 mg/kg/day. The mRNA and protein levels of Leydig (Lhcgr, Scarb1, Star, Cyp11a1, Hsd17b3, and Hsd11b1) and Sertoli cells (Fshr, Amh, and Sox9) were significantly downregulated in the DBTCl-treated testes compared to the control. Immunohistochemical staining showed that DBTCl-treatment caused fewer regenerated Leydig cells and impaired Sertoli cell development and function in the testis on day 56 post-EDS. In conclusion, the present study demonstrates that DBTCl retards rat Leydig cell developmental regeneration by downregulating steroidogenesis-related enzymes at the gene and protein levels, inhibiting Leydig cell proliferation and impairing Sertoli cell function and development.

Keywords: developmental regeneration; dibutyltin dichloride; ethane dimethane sulfonate; leydig cell; testosterone.

Figures

FIGURE 1
FIGURE 1
Serum levels of testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) after dibutyltin dichloride (DBTCl) treatment. (A) Serum T levels on post-EDS (ethane dimethane sulfonate) days 7, 35, and 56. (B) Serum LH levels on post-EDS day 56. (C) Serum FSH levels on post-EDS day 56. Mean ± SEM, n = 6. , ∗∗, ∗∗∗ indicate significant difference when compared to the control at p < 0.05, 0.01, and 0.001, respectively.
FIGURE 2
FIGURE 2
Expression levels of Leydig and Sertoli cell genes in the testes of dibutyltin dichloride (DBTCl)-treated rats on the post-EDS day 56. Leydig cell genes: (A) Lhcgr, (B) Scarb1, (C) Star, (D) Cyp11a1, (E) Hsd3b1, (F) Cyp17a1, (G) Hsd17b3, and (H) Hsd11b1. Sertoli cell genes: (I) Fshr, (J) Amh, (K) Dhh, and (L) Sox9. Mean ± SEM, n = 6. , ∗∗, ∗∗∗ indicate significant difference when compared to the control (0 mg/kg DBTCl) at p < 0.05, 0.01, and 0.001, respectively.
FIGURE 3
FIGURE 3
Expression levels of Leydig and Sertoli cell proteins in the testes of dibutyltin dichloride (DBTCl)-treated rats on the post-EDS day 56. (A) Gel images. (B) Quantitative results. Leydig cell proteins: LHCGR, SCARB1, STAR, CYP11A1, 3β-HSD31, CYP17A1, 17β-HSD3, and 11β-HSD1. Sertoli cell proteins: FSHR, AMH, DHH, and SOX9. Mean ± SEM, n = 3. , ∗∗, ∗∗∗ indicate significant difference when compared to the control (0 mg/kg DBTCl) at p < 0.05, 0.01, and 0.001, respectively.
FIGURE 4
FIGURE 4
The effects of dibutyltin dichloride (DBTCl) on Leydig cell (LC) number and maturation in rat testes on post-EDS day 56. Leydig cells were identified by immunohistochemical staining of 11β-HSD1 and LC number was enumerated by the stereological method. 11β-HSD1 densities were determined by semi-quantitative assay. (A,B) for control group. (C,D) for 20.0 mg/kg DBTCl group. Bar = 50 μm. Black arrow points to Leydig cells. (E) Quantification of LC number (#). (F) Semi-quantitative assay of 11β-HSD1 densities. Mean ± SEM, n = 6. , ∗∗, ∗∗∗ indicate significant difference when compared to the control group at p < 0.05, 0.01, and 0.001, respectively.
FIGURE 5
FIGURE 5
The effects of dibutyltin dichloride (DBTCl) on Sertoli cell (SC) number and maturation in rat testes on post-EDS day 56. Sertoli cells were identified by immunohistochemical staining of SOX9 and SC number was enumerated by the stereological method. SOX9 densities were determined by semi-quantitative assay. (A,B) for control group. (C,D) for 20.0 mg/kg DBTCl group. Bar = 50 μm. White arrow points to Sertoli cells. (E) Quantification of LC number (#). (F) Semi-quantitative assay of SOX9 densities. Mean ± SEM, n = 6. , ∗∗, ∗∗∗ indicate significant difference when compared to the control group at p < 0.05, 0.01, and 0.001, respectively.
FIGURE 6
FIGURE 6
The Leydig cell size, nuclear size, and cytoplasmic size in rat testis sections from post-EDS day 56. (A) Leydig cell size. (B) Leydig cell nuclear size. (C) Leydig cell cytoplasmic size. Mean ± SEM, n = 6. , ∗∗, ∗∗∗ indicate significant difference when compared to the control at p < 0.05, 0.01, and 0.001, respectively.

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