Semen collection and processing
This experimental study was carried out on discharged semen samples of IVF (in vitro fertilization) clinic of Imam Khomeini Hospital of Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran, during a period from August 2016 to March 2017. This research was approved by the Ethics Committee of the Research Deputy of Ahvaz University of Medical Sciences (IR.AJUMS.REC.1395.235). Seventeen human semen samples were randomly collected and transferred to the Cellular and Molecular Research Center. After liquidation, samples were loaded on slides for analyzing sperm parameters (count, motility, morphology, and leukocytes percentage) and selecting normal and asthenozoospermic samples according to WHO’s 2010 guidelines [10]. It should be noted that samples of men who had underlying disorders such as varicocele and infection, as well as cigarette smokers and those who were taking medications were excluded from this study. Each sample was washed twice with 1 ml fresh sperm wash solution and centrifuged at 300×g for 5 min. Then, 2 ml fresh sperm wash was added to the cell plate which was then kept at a 45° angle in an incubator at 37 °C for 45 min in order to have the motile sperms swim up [11].
Vitamin D3 treatment
The supernatant of each sample was divided into two groups: One was considered the control group and the other received 20 μmol of Vit.D3 (vitamin D3 (cholecalciferol) 300,000 IU/ml—Caspian company) as experimental group, and they were incubated for 1 h [11]. After incubation, 5 μl of a well-mixed sample was placed on a glass slide and checked under a microscope at × 400 magnification for sperm motility assessment. According to the WHO’s definition [10], motility of spermatozoa is classified into the following groups: progressive motility (PR), spermatozoa moving actively, either linearly or in a large circle, regardless of speed; non-progressive motility (NP), all other patterns of motility with the absence of progression, e.g. swimming in small circles, the flagellar force hardly displacing the head, or when only a flagellar beat can be observed; and immotility (IM), no movement of spermatozoa whatsoever.
Western blotting
Approximately 1.0 × 107 spermatozoa were mixed with lysis buffer (170 mg urea, 56 mg thiourea, 14 mg CHAPS, and 76 mg Tris in 18 ml PBS final volume) and 1X protease inhibitor solution on ice for 1 h and then centrifuged at 16000×g for 20 min. The supernatant was frozen at 80 °C.
SDS-PAGE was performed on a 12% Bis-Tris gel [12]. Electrophoresis and electrotransfer to PVDF membrane were done for each step at 100 V for 70–100 min. After transfer of proteins, PVDF membranes were blocked with 5% milk powder for 1 h at room temperature while being shaken. In the next step, the membranes were incubated with primary monoclonal anti-HSP70 (sc-373867) (1/500) at 4 °C overnight and then with goat anti-mouse IgG-HRP secondary antibody (sc-2005) (1/5000) for 1 h at room temperature with shaking. Finally, the bands were detected by a molecular imaging apparatus. Anti-β actin (sc-47778) was used as internal control [13].
Immunocytochemistry
For detection of HSP70 by immunocytochemistry, several smears were created from the samples and fixed with 4% paraformaldehyde for 10 min. All the steps were carried out at room temperature in a humidified chamber. The slides were incubated for 5–10 min in 0.1–1% hydrogen peroxide and were blocked with 1.5% blocking serum. Then, primary and secondary antibodies (1:200) were applied to each slide for 1 h, respectively. Negative control slides were treated with PBS alone during the primary antibody step; after that, slides were incubated in 1–3 drops of peroxidase substrate. Finally, the slides were checked under a microscope at × 400 magnifications. Then, 100 cells were counted according to the staining intensity measurement method in three classes, including high staining, low staining, and colorless.
Measurement of lipid peroxidation
Lipid peroxidation in sperm cells was evaluated by thiobarbituric acid reactive substances (TBARS) method, which was used to measure malondialdehyde (MDA) level. Initially, sperm samples were lysed by the method of doing rapid freeze to − 80 °C and thawing to 35 °C for at least three times. Then, the samples were centrifuged at 4000×g for 10 min and the supernatant was used for the MDA assays [14].
Two hundred and fifty microliters of supernatant were added to a solution made from 1 ml of 20% trichloroacetic acid and 1 ml of 0.5% thiobarbituric acid. The mixture was then incubated in a boiling water bath at 95 °C for 1 h. After cooling, the samples were centrifuged at 4000×g for 15 min and the MDA content was measured by a spectrophotometer at 532 nm. The MDA intensity of spermatozoa was determined by using various concentrations (5–100 μmol/dl) of tetraethoxypropane as standards and the results were expressed as μmol/dl [15].
NBT test
Nitroblue tetrazolium (NBT) test was employed for assessing the level of intracellular ROS. Briefly, 200 μl of a sperm sample was washed twice with 1 ml PBS at 300×g for 10 min. The washed spermatozoa was resuspended in 100 μL PBS and mixed with an equal volume of 0.1% NBT, and the mixture was shaken (10 mg of nitroblue tetrazolium chloride powder in 10 ml PBS) at 37 °C for 45 min. After washing, the intracellular formazan crystals were solubilized in 60 μl of 2 mol/l of KOH and 2 mol/l of DMSO. After 5 min, the resulting color of the supernatant was measured by an ELISA reader at 655 nm [16].
Statistical analysis
Data analysis was performed using SPSS 23.0. All the values were expressed as mean ± standard error. Due to the fact that data distribution was not normal, bootstrap test was used for comparison of mean values according to 95% confidence interval (CI) in 95% level. In order to analyze the results of all the western blot experiments, the protein bands’ density was quantified by Image J software.
