Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 8, 704
eCollection

A Short-Term Exposure to Tributyltin Blocks Leydig Cell Regeneration in the Adult Rat Testis

Affiliations

A Short-Term Exposure to Tributyltin Blocks Leydig Cell Regeneration in the Adult Rat Testis

Xiaolong Wu et al. Front Pharmacol.

Abstract

Background: Tributyltin (TBT) is widely used as an antifouling agent that may cause reproductive toxicity. The mechanism of TBT on Leydig cell development is still unknown. The objective of the present study was to investigate whether a brief exposure to low doses of TBT permanently affects Leydig cell development and to clarify the underlying mechanism. Methods: Adult male Sprague Dawley rats were randomly assigned into four groups and gavaged normal saline (control), 0.1, 1.0, or 10.0 mg/kg/day TBT for a consecutive 10 days, respectively. At the end of TBT treatment, all rats received a single intraperitoneal injection of 75 mg/kg ethane dimethane sulfonate (EDS) to eliminate all of adult Leydig cells. Leydig cells began a developmental regeneration process on post-EDS day 35. The Leydig cell regeneration was evaluated by measuring serum testosterone, luteinizing hormone, and follicle-stimulating hormone levels on post-EDS day 7, 35, and 56, the expression levels of Leydig cell genes, Leydig cell morphology and number and proliferation on post-EDS day 56. Results: TBT significantly reduced serum testosterone levels on post-EDS day 35 and 56 and increased serum luteinizing hormone and follicle-stimulating hormone levels on post-EDS day 56 at ≥1 mg/kg/day. Immunohistochemical staining showed that there were fewer regenerated Leydig cells in the TBT-treated testis on post-EDS day 56. Further study demonstrated that the mRNA or protein levels of Leydig (Lhcgr, Cyp11a1, Hsd3b1, Cyp17a1, and Hsd17b3) and Sertoli cells (Fshr, Dhh, and Sox9) were significantly down-regulated in the TBT-treated testes when compared to the control. Immunofluorescent staining showed that TBT inhibited Leydig cell proliferation as judged by the reduced number of proliferating cyclin nuclear antigen-positive Leydig cells on post-EDS day 35. Conclusion: The present study demonstrated that a short-term TBT exposure blocked Leydig cell developmental regeneration process via down-regulating steroidogenesis-related proteins and inhibiting the proliferation of Leydig cells.

Keywords: Leydig cell; ethane dimethane sulfonate; regeneration; tributyltin.

Figures

FIGURE 1
FIGURE 1
Serum levels of testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) after tributyltin (TBT) exposure. (A) Experimental plan; (B) Serum T levels on post-EDS (ethane dimethane sulfonate) day 7, 35, and 56; (C) Serum LH levels on post-EDS day 56; (D) Serum FSH levels on post-EDS day 56. Mean ± SEM, n = 6. , ∗∗, ∗∗∗ indicate significant difference when compared to the control (CON) at p < 0.05, 0.01, and 0.001, respectively.
FIGURE 2
FIGURE 2
The effects of tributyltin (TBT) on Leydig cell (LC) and Sertoli cell (SC) number. Leydig cells were identified by immunohistochemical staining of 3β-hydroxysteroid dehydrogenase 1 (HSD3B1) and Sertoli cells were identified by staining of SOX9 in rat testis sections from post-EDS day 56 and enumerated by the stereological method. Images of HSD3B1 staining (with inserts): (A) control; (B) TBT 10.0 mg/kg dose. Bar = 50 μm. Black arrow points to Leydig cells. (C): quantification of Leydig cell (LC) number (#). Images of SOX9 staining (with inserts): (D) control; (E) TBT 10.0 mg/kg dose. Bar = 50 μm. White arrow points to Sertoli cells. (F): quantification of Sertoli cell (SC) number (#). Mean ± SEM, n = 6. , ∗∗, ∗∗∗ indicate significant difference when compared to the control (TBT 0 mg/kg) at p < 0.05, 0.01, and 0.001, respectively.
FIGURE 3
FIGURE 3
Expression levels of Leydig and Sertoli cell genes in the tributyltin (TBT)-treated testis on the post-EDS day 56. Leydig cell genes: Lhcgr, Cyp11a1, Hsd3b1, Cyp17a1, and Hsd17b3. Sertoli cell genes: Fshr, Dhh, and Sox9. Mean ± SEM, n = 6. , ∗∗, ∗∗∗ indicate significant difference when compared to the control (TBT 0 mg/kg) at p < 0.05, 0.01, and 0.001, respectively.
FIGURE 4
FIGURE 4
Expression levels of Leydig and Sertoli cell gene products in the tributyltin (TBT)-treated testis on the post-EDS day 56. (A):Gel images; (B): quantitative result. Leydig cell proteins: LHCGR, SCARB1, CYP11A1, HSD3B1, CYP17A1, and HSD11B1. Sertoli cell proteins: FSHR, AMH, DHH, and SOX9. Mean ± SEM, n = 3. ∗, ∗∗, ∗∗∗ indicate significant difference when compared to the control (TBT 0 mg/kg) at p < 0.05, 0.01, and 0.001, respectively.
FIGURE 5
FIGURE 5
The semi-quantitative assay of 3β-hydroxysteroid dehydrogenase 1 (HSD3B1) and SOX9 in rat testis sections from post-EDS day 56. Images of HSD3B1 staining: (A) control; (B) TBT 10.0 mg/kg dose. Images of SOX staining: (D) control; (E) TBT 10.0 mg/kg dose. Black arrow points to HSD3B1 staining (cytoplasm); and white arrow points to SOX9 staining (nucleus). (C,F): quantification of density of HSD3B1 and SOX9, respectively. Mean ± SEM, n = 6. , ∗∗, ∗∗∗ indicate significant difference when compared to the control (TBT 0 mg/kg) 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 (TBT 0 mg/kg) at p < 0.05, 0.01, and 0.001, respectively.
FIGURE 7
FIGURE 7
The percentage of PCNA (proliferating)-positive and 11β-hydroxysteroid dehydrogenase 1 (HSD11B1, Leydig cell)-positive cells in rat testis sections from post-EDS day 56. Images: (A) control; (B) TBT 0.1 mg/kg dose; (C) TBT 1 mg/kg dose; (D) TBT 10 mg/kg dose. PCNA = red color in the nucleus; HSD11B1 = green color in the cytoplasm. (E) Quantification of data for the percentage of PCNA+-HSD11B1+ cells in all HSD11B1+ cells. Mean ± SEM, n = 6. , ∗∗, ∗∗∗ indicate significant difference when compared to the control (TBT 0 mg/kg) at p < 0.05, 0.01, and 0.001, respectively.

Similar articles

See all similar articles

Cited by 1 article

References

    1. Aluoch A. O., Odman-Ghazi S. O., Whalen M. M. (2007). Pattern of MAP kinases p44/42 and JNK activation by non-lethal doses of tributyltin in human natural killer cells. Arch. Toxicol. 81 271–277. 10.1007/s00204-006-0155-4 - DOI - PubMed
    1. Barrionuevo F., Bagheri-Fam S., Klattig J., Kist R., Taketo M. M., Englert C., et al. (2006). Homozygous inactivation of Sox9 causes complete XY sex reversal in mice. Biol. Reprod. 74 195–201. 10.1095/biolreprod.105.045930 - DOI - PubMed
    1. Cooke G. M., Forsyth D. S., Bondy G. S., Tachon R., Tague B., Coady L. (2008). Organotin speciation and tissue distribution in rat dams, fetuses, and neonates following oral administration of tributyltin chloride. J. Toxicol. Environ. Health A 71 384–395. 10.1080/15287390701801653 - DOI - PubMed
    1. Davidoff M. S., Middendorff R., Enikolopov G., Riethmacher D., Holstein A. F., Muller D. (2004). Progenitor cells of the testosterone-producing Leydig cells revealed. J. Cell Biol. 167 935–944. 10.1083/jcb.200409107 - DOI - PMC - PubMed
    1. Fent K. (1996). Ecotoxicology of organotin compounds. Crit. Rev. Toxicol. 26 1–117. 10.3109/10408449609089891 - DOI - PubMed
Feedback