Animals
Forty eight male Sprague-Dawley rats (age, 6–8 weeks old; weight, 180-210 g) were purchased from the Animal Laboratory Center of Shiraz University of Medical Sciences. 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. All animal experiments carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). Also, the animal procedures were performed under the standard rules established by the Animal Care and Ethics Committee of Shiraz University of Medical Sciences (IR.SUMS.REC.1398.392).
Experimental design
The rats were randomly divided into eight groups (n=6); CONTROL group received distilled water orally per day for 56 days (spermatogenesis length), OLIVE OIL group received 0.5 ml/day Olive oil orally for 56 days, Carboxy methylcellulose (CMC) group received 1 ml of 10 g/l CMC orally [25] per day for 56 days, RES group received 100 mg/kg/day RES that was diluted in CMC and administered orally at a dosing volume of 1 ml [26, 27] for 56 days, BPA-LOW group received low dose of BPA (25 mg/kg/day) orally for 56 days, BPA-HIGH group received high dose of BPA (50 mg/kg/day) [28] for 56 days, BPA was diluted in olive oil and administered daily orally at a dosing volume of 0.5 ml. BPA-LOW + RES group received orally with low dose of BPA plus RES (100mg/kg/day) for 56 days, and BPA-HIGH + RES group received high dose of BPA plus RES (100mg/kg/day) orally for 56 days (Fig. 1).
It should be noted that the dosages of BPA (CAS 80-05-7, Sigma–Aldrich Co., St. Louis, USA) used in current study were based on the previously reported as maximum permissible dose that have no observable side effect on reproductive and developmental toxicity (50 mg/kg BW/day) in rats [13, 29].
Hormone measurements
At the end of the 8th week (on day 56), fasted rats were killed by cervical dislocation and blood samples were collected from the heart through a cardiac puncture 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 follicle-stimulating hormone (FSH; Category No. CK-30597), luteinizing hormone (LH; Category No. CK-E90904, and testosterone concentrations (Category No. E90243) were determined by rat ELISA kits (From East. Bio Pharm Company) using a microplate reader (Biotek, USA). Briefly, 100 μL of standard or sample was pipetted to each well and incubated for 2 hours at 37 °C. After removing any unbound substances, 100 μL of anti-biotin antibodies was added to the wells. After washing, 100 μL of avidin conjugated Horseradish Peroxidase (HRP) was added to each well and incubated for 1 hour at 37 °C. Then, 90 μL of 3,3'5,5'-Tetramethylbenzidine (TMB) substrate was added to each well and incubated for 20 minutes at 37 °C. Finally, the color development was stopped and the absorbance was determined at 450 nm using a microplate reader.
Spermatozoa counts, morphology and motility
Immediately after blood collection, the proximal part of the vas deferens just distal to the cauda epididymis (10 mm) was removed, and moved to a petri dish containing 3 mL normal saline solution. The suspension was gently shaken at 37°C for 5-10 min to diffuse the spermatozoa. The samples were counted in a hemocytometer. Ten fields were then randomly selected and evaluated for motility grading to distinguish the immotile sperms from those with progressive or non-progressive motility. Also, the sperm smears were stained with 1% eosin Y for assessing the morphology [30].
There is two types of progressive motility: 1- rapid progressive motility, 2- slow progressive motility. The efficient passage of spermatozoa through cervical mucus is dependent on rapid progressive motility.
We should add that it is necessary to distinguish between these two types of progressive motility. So that neglecting the distinction between two progressive sperm groups leads to ignoring the information in the semen sample, and the removal of such useful information would impoverish the semen analysis [31].
Stereological study
The left testis was removed and weighed. Then, according to the immersion method, it was immersed in isotonic saline-filled jar for measuring the primary volume “V (testicle)” [32]. Afterwards, the samples were fixed in 4% buffered formaldehyde solution for stereological studies. The orientator method was applied to obtain Isotropic Uniform Random (IUR) sections [32]. About 8-12 slabs in each testis were collected through this procedure. To estimate the shrinkage, a circle was punched out from a random testis slab by a trocar (diameter 5 mm), and the trocar radius was considered as the “area (before)” (πr2). After tissue processing, the area was calculated as the “area (after)”. After tissue processing and paraffin embedding, 5 and 25 μm sections were cut by the microtome and were stained using Hematoxylin-Eosin (H&E). The areas of the circles were measured before processing (unshrunk) and after processing (shrunk) and finally, the degree of shrinkage “d (shr)” was calculated by the following formula:
$$\mathrm d(\mathrm{shr})=1-{\lbrack\mathrm{Area}(\mathrm{after})/\mathrm{Area}(\mathrm{before})\rbrack}^{1.5}$$
Then, the total volume of the testis was evaluated with regard to tissue shrinkage [V(shrunk)] using the following formula:
$$\mathrm V(\mathrm{shrunken})=\mathrm V(\mathrm{unshrunk})\times\lbrack1-\mathrm d(\mathrm{shr})\rbrack$$
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 H&E-stained 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 [33, 34]. Finally, the total volume of each component was obtained by the following formula:
$$\mathrm V(\mathrm{structure})=\mathrm{Vv}(\mathrm{structure}/\mathrm{testis})\times\mathrm V(\mathrm{shrunk})$$
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 (H&E staining) [35], and calculated by the following formula:
$$\mathrm{Lv}=2\Sigma\mathrm{Q}/\lbrack\Sigma\mathrm{P}\times(\mathrm a/\mathrm f)\rbrack$$
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) [36].
$$\mathrm L(\mathrm{tubules})=\mathrm{Lv}\times\mathrm V(\mathrm{structure})$$
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 [35]. An average of 100 tubules were counted per testis.
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, round spermatids (steps 1–8 spermiogenesis), long spermatids (steps 9–16 spermiogenesis), Sertoli and Leydig cells.
The total number of the testicular cell types was calculated using the optical disector method applied on the H&E-stained sections (25μm thickness) [37]. 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 [38]. The numerical density (Nv) was estimated using the following formula:
$$\mathrm{Nv}=\Sigma\mathrm{Q}/(\Sigma\mathrm{A}\times\mathrm h)\times(\mathrm t/\mathrm{BA})$$
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):
$$\mathrm N(\mathrm{cells})=\mathrm{Nv}\times\mathrm V(\mathrm{structure})$$
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.
Statistical analysis
The data were expressed as mean ± standard error (SEM). 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.
