Effects of exposure to bisphenol A during pregnancy and lactation on the testicular morphology and caspase-3 protein expression of ICR pups

Abstract

Bisphenol A (BPA), a xenoestrogen and endocrine-disrupting chemical, is a cause for concern due to its being a potential human carcinogen. The aim of this study was to investigate the effects of continued maternal exposure to BPA on the testicular structures and expression of caspase-3 protein in male ICR offspring during pregnancy and lactation and explore its possible mechanism. Pregnant ICR mice were divided into two control groups, which were either given or not given the solvent dimethyl sulfoxide (DMSO) and three treatment groups, which were gavaged with water-soluble BPA dissolved in DMSO at three different concentrations from gestational day 0 to weaning on postnatal day (PND) 21. The number of mice pups and ratios of males to females were recorded. On PND 21, male offspring were sacrificed to measure their wet weights and testicular coefficients. Electron microscopy was used to observe testicular morphological changes, Hoechst 33258 staining to detect cell apoptosis and immunohistochemistry to measure caspase-3 expression. Although there was no significant difference between offspring of the control group and the treatment group in litter size and male-female ratio (P>0.05), the testicular viscera coefficient in the latter decreased (P<0.01). Specifically, compared with offspring of the control group, in addition to increased cell apoptosis, those of the treatment groups were found to have changes in mitochondrial and endoplasmic reticulum in their spermatogenous, Sertoli, Leydig and peritubular myoid cells, which were concomitant with an elevated expression of caspase-3 in the cytoplasm (P<0.01). In conclusion, exposure of pregnant mice to BPA during pregnancy and lactation has some toxic effects on the testes of male ICR offspring and these may originate from increased apoptosis.

Keywords: bisphenol A; caspase-3; offspring mice.

Figure 1

Analysis of general parameters of reproduction and development. (A and B) There was no significant difference in the number and male:female ratio of litter mice exposed to bisphenol A (BPA) during pregnancy compared with the blank control group (P>0.05). (C) The testicular viscera coefficient decreased in BPA-exposed male offspring mice (P<0.001). Bc, blank control group; Sc, solvent control group; 10, 10 nmol/l BPA group; 100, 100 nmol/l BPA group; 1,000, 1,000 nmol/l BPA group.

Figure 2

Transmission electron microscopy. (A) Spermatogenous, (B) Sertoli, (C) Leydig and (D) peritubular myoid cells in the blank control group had no evident morphological abnormalities. (A1–D1) In the bisphenol A (100 nmol/l) groups, the (A1) Spermatogenous, (B1) Sertoli, (C1) Leydig and (D1) peritubular myoid cells exhibited alterations in the vacuoles. (B1) Smooth endoplasmic reticulum showing different degrees of expansion. (C1) There were additional and larger lysosomes compared with the blank control group. M, mitochondria; N, nucleus; ↗ indicates smooth endoplasmic reticulum; ⇦ indicates lysosomes; (A and A1) magnification, ×6,000; (B–D, B1–D1) magnification, ×12,000.

Figure 3

Hoechst staining results. (A) Blank control group. The nuclei of spermatogenous, Sertoli, Leydig and peritubular myoid cells show normal blue dye (magnification, ×400). (B) bisphenol A (100 nmol/l) group. Apoptotic nuclei of spermatogenous, Sertoli, Leydig and peritubular myoid cells, as indicated by the white arrow (magnification, ×400).

Figure 4

Immunohistochemical staining of caspase-3. The expression of caspase-3 is not evident in the (A) blank control and (B) solvent control groups. Caspase-3 expression became progressively evident in the (C) bisphenol A (BPA) (10 nmol/l), (D) BPA (100 nmol/l) and (E) BPA (1,000 nmol/l) groups (magnification, ×400).

Figure 5

Number of caspase-3-positive cells in testicular tissue. This number was less in the blank control (Bc) and solvent control (Sc) groups. However, the expression of caspase-3 was progressively evident in the bisphenol A (BPA) (10 nmol/l) group (10), BPA (100 nmol/l) group (100) and BPA (1,000 nmol/l) group (1,000). ^***P<0.001 vs. blank and solvent control group.