Stereological study on the effect of vitamin C in preventing the adverse effects of bisphenol A on rat ovary

Abstract

Background: Bisphenol A (BPA), an environmental pollutant, can generate free radicals which damages the reproductive system. Vitamin C is an antioxidant which may prevent the adverse effects of free radicals.

Objective: The aim was to investigate the effect of vitamin C on the ovary tissue in rats treated with BPA.

Materials and methods: In this experimental study, 24 female Wistar rats (200±20 gr) were randomly divided into 4 groups (n=6): control, BPA (60 µg/Kg/day), vitamin C (150 mg/Kg/day) and BPA + vitamin C and orally treated for 20 days. The left ovaries were taken out, fixed for tissue processing and studied using stereological methods. Data were analyzed with SPSS using one-way ANOVA, and the means were considered significantly different at (p<0.05).

Results: The total volume of ovary and cortex (p<0.01), medulla (p<0.05), the volume of corpus luteum (p<0.001) and the mean number of antral follicles (p<0.001) significantly reduced in BPA group compared with control, while the number of atretic follicles increased (p<0.05). The volume of oocyte (p<0.01) and its nucleus (p<0.001) in the antral follicles and the thickness of zona pellucida (ZP) in the secondary (p<0.05) and antral (p<0.001) follicles significantly decreased in BPA group compared with controls. The above parameters in the BPA + vitamin C group were compensated to control level.

Conclusion: Vitamin C can be used as a potential antioxidant in the case of BPA toxication.

Keywords: Bisphenol A; Ovary; Rat; Stereology; Vitamin C.

Figure 1

Orientator method: (A) The cylindrical paraffin block is placed on a φ clock. A random number between 0 and 9, for example 2 is selected and an appropriate cut was made along the selected number. (B).The block was then placed on the θ-clock along its cut surface on the 0-0 axis and a random number (for example 5) was selected and another cut was made along the selected number

Figure 2

Estimating the ovary volume using the Cavalieri principle. Resulting points from the randomly superimposed probe on the images were counted (magnification ×400

Figure 3

Estimating the number of follicles using the optical disector. A. 5 µm from the top and bottom of the sections was ignored as the guard area against the cutting artifacts. B, C. An unbiased counting frame superimposed on the selected field was used to sample the nucleoli profiles of the oocytes (magnification ×1000

Figure 4

Estimating the mean volumes of oocytes and their nuclei by using the unbiased stereological technique of the nucleator. For each sampled oocyte, an isotropic direction is generated from a random point within the nucleolus, and the distances in each direction out from the point to the boundary of the oocyte membrane (A) (to estimate the oocyte volume), and the nucleus (B) (to estimate the nuclear volume) are considered (magnification ×1000

Figure 5

Estimating the mean thickness of zona pellucida using orthogonal intercept method. (magnification ×1000

Figure 6

Micrographs of the ovary tissue in rats treated with BPA and vitamin C (H&E staining and 40 x magnification) representing the histopathologic variations of the ovarian tissue: (A) Control group representing the normal structure of the ovary tissue (B) BPA group showing an increase in the number of ovarian cysts indicated by ↗ (C) BPA+ vitamin C group showing the normal structure of the ovary