Histological and histomorphometric analysis
The left testis, epididymis and ovary were fixed in formalin-buffer 10% for one month. Tissue processing was done by tissue processor machine (model DS 2080/H, DID SABZ Co, Orumieh, Iran). After tissue processing, samples were embedded in paraffin. Testis and epididymis samples were cut in 5 µm using microtome and set on microscope slides. After rehydration in decreasing dilution of ethanol, they were stained with hematoxylin and eosin.
A camera (Dino-Lite®, New Taipei City, Taiwan) was set up on a light microscope. In Dino capture 2.0 (version 1.4.2.D) program the input magnification set on 725× and the objective lens was on 10× and unit set on 1 µm. For investigation of changes in spermatogenesis, some indices in seminiferous tubules were evaluated.
For the first index, lumen diameter and total diameter of five tubules per each slide were measured. Then according to the following formulas, cellular diameter, lumen area, cellular area and cross-section area were calculated.
Cellular diameter = (total diameter – lumen diameter)/2
Lumen area = 3.142 × (lumen diameter)²/4
Cellular area = 3.142 × (cellular diameter)²
Cross section area = 3.142 × (total diameter)²/4
Evaluation of the spermatogenesis index was performed on the basis of a modified scale of 0 to 7. For this purpose, the appearance of the spermatogenic cells throughout the seminiferous tubules was evaluated. Number of cell layers, types of cells, and the presence of late spermatids in the seminiferous tubules were considered in this index. The indices were as follows: 0, presence of no spermatogenic cells; 1, presence of only spermatogonia; 2, presence of spermatogonia and spermatocytes present; 3, spermatogonia, spermatocytes and round (early) spermatids with < 25 late spermatids per tubule; 4, presence of spermatogonia, spermatocytes, and round spermatids with up to 25–50 late spermatids per tubule; 5, presence of spermatogonia, spermatocytes, and round spermatids with up to 50–75 late spermatids per tubule; 6, presence of spermatogonia, spermatocytes, and round spermatids as well as up to 75–100 late spermatids per tubule; and 7, presence of all cell types present with > 100 late spermatids per tubule.
The last index was the number of tubules per 5×5 mm² of transverse sections of seminiferous tubules. Based on this number, the numerical density of tubules was calculated as fallowing formula.
Numerical density = number of profiles per unit area/(total diameter + average thickness of the section).
Left ovary was processed like testis with the difference that there are 10 slices (each 20 µm thickness) between 2 histology samples. Primary follicle was presented as one-layer cuboidal granulosa cells, secondary follicle was presented with two layer of cuboidal granulosa (with or without antrum), and tertiary follicle was presented as large antrum and several layers of granulosa cells. Number of primary and secondary follicles was measured by 40x objective microscopy and every area was observed with crinkle pattern. All data were obtained by a reproductive expert with blind observation. Tertiary and corpus luteum were measured by 10x microscopy. Area of follicle was measured by Dino capture 2.0 (version 1.4.2.D) tools. For calculating volume, we calculated radius of these area and gain circle diameter. If we suppose that 1 slice with its distance to another slice forms a cone frustum, we can use con volume equation for calculating total volume of a follicle.
In this equation, d is circle diameter and h is distance between 2 slices (according to our slicing pattern is 200 µm).
Luteinizing hormone (LH), follicle-stimulating hormone (FSH) measured by radioimmunoassay (RIA) kit (Padyab Teb Company, Tehran, Iran). and testosterone were analysis by ELISA kit (PGI Company, Tehran, Iran). For testosterone analysis, all reagents, samples, control and standards were first prepared as instructed. Standard and control samples were added to wells. Labaled HRP-conjugate was added to each well and incubated at 37°C. Then, they were washed and TMB substrate was added to each well and incubated at room temperature. Finally, stop solution was added to wells and read immediately. For FSH and LH hormonal analysis, briefly, buffer bottle was warmed to melt and diluted with 100 mL distilled water. Other components except separating reagent were added to assay buffer and replaced stopper. Working standards were prepared as company instruction. briefly, 200 µL assay buffer was pipetted to non-specific binding and 100 µL was pipetted to zero standard tubes. 100 µL antiserum was pipetted into all tubes except non-specific binding (NSB) and total count (TC) tubes and all tubes were vortexed and stored for 4 h at room temperature. Next, 100 µL tracer was pipetted into all tubes. TC tubes were stopped and put aside for counting. For second time, all tubes were vortexed and stored overnight at room temperature. Next, 400 µL second antibody was added to each tube except TC tube, vortexed and incubated at room temperature for 10 min. Then, all tubes were centrifuged for 10 min at 1500 ×g and the supernatant was discarded. The tubes were inverted on absorbent tissues to allow them drain for 5 min. The radioactivity present in each tube was determined by counting at last 60 sec in Gamma scintillation counter (Dream GAMMA counter-5, LabLogic Company, USA) and compered with a standard curve for determining concentration of LH and FSH in samples. A standard curve for LH was drawn with Y-axis, present of Radio activity and X-axis, concentration of determinate LH. Sensitivity of LH and FSH kit was determined as 0.22 mIU/mL and 0.09 mIU/mL, respectively.