Cadmium suppresses the proliferation of piglet Sertoli cells and causes their DNA damage, cell apoptosis and aberrant ultrastructure

Abstract

Objective: Very little information is known about the toxic effects of cadmium on somatic cells in mammalian testis. The objective of this study is to explore the toxicity of cadmium on piglet Sertoli cells.

Methods: Sertoli cells were isolated from piglet testes using a two-step enzyme digestion and followed by differential plating. Piglet Sertoli cells were identified by oil red O staining and Fas ligand (FasL) expression as assayed by immunocytochemistry and expression of transferrin and androgen binding protein by RT-PCR. Sertoli cells were cultured in DMEM/F12 supplemented with 10% fetal calf serum in the absence or presence of various concentrations of cadmium chloride, or treatment with p38 MAPK inhibitor SB202190 and with cadmium chloride exposure. Apoptotic cells in seminiferous tubules of piglets were also performed using TUNEL assay in vivo.

Results: Cadmium chloride inhibited the proliferation of Piglet Sertoli cells as shown by MTT assay, and it increased malondialdehyde (MDA) but reduced superoxide dismutase (SOD) and Glutathione peroxidase (GSH-Px) activity. Inhibitor SB202190 alleviated the proliferation inhibition of cadmium on piglet Sertoli cells. Comet assay revealed that cadmium chloride caused DNA damage of Piglet Sertoli cells and resulted in cell apoptosis as assayed by flow cytometry. The in vivo study confirmed that cadmium induced cell apoptosis in seminiferous tubules of piglets. Transmission electronic microscopy showed abnormal and apoptotic ultrastructure in Piglet Sertoli cells treated with cadmium chloride compared to the control.

Conclusion: cadmium has obvious adverse effects on the proliferation of piglet Sertoli cells and causes their DNA damage, cell apoptosis, and aberrant morphology. This study thus offers novel insights into the toxicology of cadmium on male reproduction.

Figure 1

Identification of the isolated piglet Sertoli cells. A: Oil red O staining showed that red lipid droplets (arrows) were presented near the nucleus or at the two poles of cytoplasm of the isolated piglet cells, which confirmed the identity of piglet Sertoli cells. Cell nuclei were counterstained with hematoxylin. B: Immunocytochemistry revealed that the isolated cells were positive for FasL (arrows), further verifying the identity of piglet Sertoli cells. Scale bars in A and B = 10 μm.

Figure 2

Effect of cadmium chloride and p38 MAPK inhibitor SB202190 on proliferation of piglet Sertoli cells. A: MTT assay showed cadmium chloride caused the proliferation inhibition of piglet Sertoli cells. B: MTT assay revealed that p38 MAPK inhibitor SB202190 alleviated the proliferation inhibition of piglet Sertoli cells caused by cadmium chloride. Compared to control group A, "*" indicated significant difference (p < 0.05), "**" indicated extremely significant difference (p < 0.01).

Figure 3

Effect of cadmium chloride on MDA content, GSH-Px, and SOD of piglet Sertoli cells and hepatocytes. A-B: Exposure of cadmium chloride to piglet Sertoli cells resulted in an increase of MDA content as well as a decrease of GSH-Px and SOD. C: Exposure of cadmium chloride to piglet hepatocytes led to an increase of MDA content and a decrease of GSH-Px. Compared to control group A, "*" indicated significant difference (p < 0.05), "**" indicated extremely significant difference (p < 0.01).

Figure 4

The single-cell gel electrophoresis (comet assay) showed the DNA damage of cadmium chloride on piglet Sertoli cells. A: DNA image of control group A. B: Comet assay revealed that the damaged DNA of cadmium chloride-treated group B contained strand breaks and migrated farther in the gel than intact DNA, creating an image resembling a celestial comet. Scale bars in A and B = 10 μm. C: Quantitation assay showed that DNA damage grades of piglet Sertoli cells by various concentration of cadmium chloride. Compared to control group A, "*" indicated significant difference (p < 0.05), "**" indicated extremely significant difference (p < 0.01).

Figure 5

Flow cytometry showed the apoptosis of piglet Sertoli cells without or with various concentration of cadmium chloride exposure. A: Control group A; B: Group B; C: Group C; D: Group D; and E: Group E.

Figure 6

TUNEL assay showed apoptotic cells in the seminiferous tubules of piglets with different doses of cadmium chloride treatment. A-D: TUNEL-positive cells in the seminiferous tubules of piglets without cadmium chloride treatment (A), or treated with cadmium chloride at 300 mg/kg body weight (B), 600 mg/kg body weight (C), or with 1200 mg/kg body weight (D). E: Quantitation assay showed that TUNEL-positive cells in the seminiferous tubules of piglets treated with various concentration of cadmium chloride. Compared to control group A, "*" indicated significant difference (p < 0.05), "**" indicated extremely significant difference (p < 0.01).

Figure 7

Effect of cadmium chloride on mRNA expression of transferrin and androgen binding protein of piglet Sertoli cells. A: RT-PCR showed the mRNA expression of transferrin and androgen binding protein on piglet Sertoli cells treated with various concentration of cadmium chloride. B: Quantitation assay showed mRNA expression of transferrin and androgen binding protein of piglet Sertoli cells by various concentration of cadmium chloride. Compared to control group A, "*" indicated significant difference (p < 0.05), "**" indicated extremely significant difference (p < 0.01).

Figure 8

Transmission election microscopy revealed ultrastructure of piglet Sertoli cells without or with various concentration of cadmium chloride exposure. Note: A: Control group A; B: Group B; C: Group D. Arrow indicated nucleus that were dissolved into several nuclear dense bodies with condensed chromatin. D: Group E. Arrow indicated apoptotic bodies. Scale bars in A and C = 5 μm; bars in B and D = 2 μm.