Effects of di-n-butyl phthalate on male rat reproduction following pubertal exposure

Abstract

Di-n-butyl phthalate (DBP) is an endocrine-disrupting chemical that has the potential to affect male reproduction. However, the reproductive effects of low-dose DBP are still not well known, especially at the molecular level. In the present study, pubertal male Sprague-Dawley rats were orally administered DBP at a wide range of doses (0.1, 1.0, 10, 100 and 500 mg kg⁻¹ day⁻¹) for 30 days. The selected end points included reproductive organ weights, testicular histopathology and serum hormonal levels. Additionally, proteomic analysis was performed to identify proteins that are differentially expressed as a result of exposure to DBP at low doses (0.1, 1.0 and 10 mg kg⁻¹ day⁻¹). Toxic effects were observed in the high-dose groups, including anomalous development of testes and epididymides, severe atrophy of seminiferous tubules, loss of spermatogenesis and abnormal levels of serum hormones. Treatment with low doses of DBP seemed to exert a 'stimulative effect' on the serum hormones. Proteomics analysis of rat testes showed 20 differentially expressed proteins. Among these proteins, alterations in the expression of HnRNPA2/B1, vimentin and superoxide dismutase 1 (SOD1) were further confirmed by Western blot and immunohistochemistry. Taken together, we conclude that high doses of DBP led to testicular toxicity, and low doses of DBP led to changes in the expression of proteins involved in spermatogenesis as well as changes in the number and function of Sertoli and Leydig cells, although no obvious morphological changes appeared. The identification of these differentially expressed proteins provides important information about the mechanisms underlying the effects of DBP on male rat reproduction.

Figure 1

Dose-dependent changes in testicular histopathology following di-n-butyl phthalate (DBP) exposure. Testis cross-sections from rats are shown following exposure to corn oil (control) or DBP at 0.1, 1.0, 10, 100 or 500 mg kg⁻¹ day⁻¹. (a–d) Control, 0.1, 1.0 and 10 mg kg⁻¹ day⁻¹, respectively. No obvious abnormalities in the seminiferous tubular structure and no loss of spermatogenesis; (e) 100 mg kg⁻¹ day⁻¹ resulted in slight damage to the seminiferous tubules (arrow); (f) 500 mg kg⁻¹ day⁻¹ resulted in severe damage to the seminiferous tubules, atrophy of the seminiferous tubules, vacuoles in Sertoli cells and loss of spermatogenesis (arrows). Scale bar=50 μm.

Figure 2

Germ cell numbers in postnatal rats 9 weeks after exposure to either corn oil (control) or five different doses of di-n-butyl phthalate (DBP). Values are expressed as mean±s.e. **P<0.01, in comparison to the respective control value (DBP, 0 mg kg⁻¹ day⁻¹).

Figure 3

Sertoli cell numbers in postnatal rats 9 weeks after exposure to either corn oil (control) or five different doses of di-n-butyl phthalate (DBP). Values are expressed as mean±s.e. **P<0.01 in comparison to the respective control value (DBP, 0 mg kg⁻¹ day⁻¹).

Figure 4

Representative two-dimensional electrophoresis (2-DE) images of testes from control and di-n-butyl phthalate (DBP)-treated rats. Proteins were extracted from rat testes, separated by 2D-PAGE, and visualized by silver staining. (a) Control; (b) 0.1 mg kg⁻¹ day⁻¹; (c) 1.0 mg kg⁻¹ day⁻¹; (d) 10 mg kg⁻¹ day⁻¹.

Figure 5

Cellular processes regulated by differentially expressed proteins. The involved cellular processes included spermatogenesis, spermatid development and the cell cycle. Proteins are shown as ovals; regulated processes are indicated by squares. ⊕represents positive-regulated, and ⊣ represents negative-regulated.

Figure 6

The effects of di-n-butyl phthalate (DBP) exposure on the expression level of proteins on two-dimensional electrophoresis (2-DE) gels. Data are presented as the ratio of the spot intensity in the treated group to that in the control group, and the results are expressed as mean±s.e. The bars with an asterisk are significantly different (*P<0.05), with an average change in spot intensity greater than twofold compared to the control group.

Figure 7

Western blot analysis of HnRNPA2/B1, vimentin and superoxide dismutase 1 (SOD1) protein expression in control and di-n-butyl phthalate (DBP)-treated rat testes. The left panel shows the results of western blot analysis, and the expression of GAPDH in corresponding tissues is displayed at the bottom as a loading control. The right panel shows the corresponding spots with the same molecular weight distributed in the two-dimensional electrophoresis (2-DE) gels. The expression tendencies were almost identical.

Figure 8

Immunolocalisation of HnRNPA2/B1, vimentin and superoxide dismutase 1 (SOD1) in normal rat testes. HnRNPA2/B1 was expressed in the nuclei of cells from spermatogonia to spermatocytes, vimentin was expressed in the cytoplasm of Sertoli cells and SOD1 was expressed in Leydig cells. Immunopositive sites are visualized as brown staining, and the positive signals are shown with arrows.