Forty eight male Sprague-Dawley rats (180–210 g) were purchased from the Animal Laboratory Center of Shiraz University of Medical Sciences (SUMS). The animals were kept under standard conditions at room temperature (22 ± 2°C), with normal humidity and 12–12 h light-dark cycles. They also had free access to standard food and water. The animal procedures were performed under the standard rules established by the Animal Care and Ethics Committee of SUMS (IR.SUMS.REC.1398.392).
2.2. Experimental design
The rats were randomly divided into eight groups (n = 6); Group I: received distilled water as control (CON), Group II: Olive oil (0.5 ml/ day), Group III: Carboxy methylcellulose (CMC) (1 ml of 10 g/l), Group IV: RES (100mg/kg/day) suspended in CMC, Group V: low dose of BPA (25 mg/kg/day) dissolved in olive oil, Group VI: high dose of BPA (50 mg/kg/day) dissolved in olive oil, Group VII: low dose of BPA plus RES (100mg/kg/day), and Group VIII: high dose of BPA plus RES (100mg/kg/day). It should be noted that the animals in all groups were treated orally by gavages per day for 56 days.
2.3. Hormone measurements
At the end of the 8th week, Blood samples were collected and stored in heparin-free tubes. Then, the samples were centrifuged at 3500 rpm for 15 min. The serum was obtained and stored at -70°C for subsequent hormone evaluation. The serum levels of FSH, LH, and testosterone concentrations were determined by rat ELISA kits using a micro plate reader (Biotek, USA).
2.4. Spermatozoa counts, morphology and motility
The rats’ ductus deferens were evaluated for semen analysis as previously described (Aminsharifi et al. 2016).
2.5. Stereological study
On day 56, the left testis was removed and weighed. Its primary volume “V (testicle)” was measured by the immersion method (Mandarim-de-Lacerda 2003). Then, the samples were fixed in 4% buffered formaldehyde solution for stereological studies. The orientator method was applied to obtain Isotropic Uniform Random (IUR) sections (Mandarim-de-Lacerda 2003). About 8–12 slabs in each testis were collected through this procedure. A circle was punched out from a random testis slab by a trocar for estimation of the tissue shrinkage. After tissue processing and paraffin embedding, 5 and 25 µm sections were cut by the microtome and were stained using Hematoxylin-Eosin. The areas of the circles were measured before processing (unshrunken) and after processing (shrunken) and finally, the degree of shrinkage “d (shr)” was calculated by the following formula:
d (shr) = 1- [Area(after)/ Area(before)]1.5
Then, the total volume of the testis was evaluated with regard to tissue shrinkage [V(shrunken)] using the following formula:
V(shrunken) = V(unshrunken) × [1 − d (shr)]
2.5.1. Estimation of the testicular components volume
The volume density of the testis sections was analyzed by a video microscopy system. In doing so, the point grid was superimposed on the microscopic images of the sections (5µm thickness) on a monitor by the software designed at the Histomorphometry and Stereology Research Center. The volume density “Vv (structure/testis)” of the testicular components, including seminiferous tubules, interstitial tissue, and germinal epithelium, was estimated by the point counting method (Tschanz et al. 2014). Finally, the total volume of each component was obtained by the following formula:
V(structure) = Vv (structure/testis) × V(shrunken)
2.5.2. Estimation of the length and diameter of seminiferous tubules
The length density (Lv) of the seminiferous tubules was measured on the sampled tubules in an unbiased counting frame applied on the 5 µm thick sections (Dalgaard et al. 2002), and calculated by the following formula:
Lv = 2ΣQ / [ΣP × (a/f)]
Where “ΣQ” is the total number of the selected tubules, “ΣP” represents the total points superimposed on the testis, and “a/f” indicates the area of the counting frame. The total length of the seminiferous tubules “L(tubules)” was calculated by multiplying the lengths density (Lv) by V(structure) (Howard and Reed 2004).
L(tubules) = Lv × V(structure)
The diameter of the seminiferous tubules was also measured on the sampled tubules in the counting frame. The diameter was measured perpendicularly to the long axis of the tubules where the tubules were widest (Dalgaard et al. 2002). An average of 100 tubules were counted per testis.
2.5.3. Estimation of number of testicular cell types
A computer linked to a light microscope (Nikon E200, Japan) with 40× oil lens (NA = 1.4) was used to assess the total number of testicular cell types, including spermatogonia (A and B), spermatocytes, spermatids (round and long), sertoli and leydig cells. The total number of the testicular cell types was calculated using the optical disector method applied on the 25 µm thick sections (Wreford 1995). In so doing, the microscopic fields were scanned by moving the microscope stage at equal distances in X and Y directions based on systematic uniform random sampling. The movement in Z direction was also performed using a microcator (MT12, Heidenhain, Germany) fixed on the microscope stage. The Z-axis distribution from the sampled cells in different focal planes was plotted to determine the guard zones and disector’s height (von Bartheld 2012). The numerical density (Nv) was estimated using the following formula:
Nv = ΣQ/ (ΣA×h) × (t/BA)
Where “ΣQ” was the number of each cell type nuclei coming into focus, “ΣA” indicated the total area of the unbiased counting frame, “h” represented the disector’s height, “t” was the mean section thickness, and “BA” was the microtome block advance. Finally, the total number of the testicular cell types was calculated by multiplying the numerical density (Nv) by V(structure):
N(cells) = Nv × V(structure)
Where, V(structure) was the total volume of the germinal epithelium for the germinal layer cells and the total volume of the interstitial tissue for the leydig cells.
2.6. Statistical analysis
The results were analyzed by one-way analysis of variance (ANOVA) and Tukey’s post hoc test using Graph Pad Prism 6 software (San Diego, CA, USA). P < 0.05 was considered to be statistically significant.