Subjects
In this experimental study semen samples were collected from 30 healthy men, aged 18–35 years old, and referred to cytogenetic clinic in Shiraz, Iran. All the samples were considered normal based on World Health Organization (WHO) guidelines [17]. All the participants were asked to abstain from ejaculation 2-3 days before sample collection. The study design was approved by the Local Medical Ethics Committee of Shiraz University of Medical Sciences (IR.SUMS.REC.1396.S808). All the participants were informed about nature and purpose of the study and an informed consent form was obtained from all of them.
Swim-Up Method for Sperm Preparation
30 semen samples were allowed to liquefy at 37°C for 30 minutes. 1mL of each liquefied semen samples was transferred to a sterile conical centrifuge tube, and mixed with 2mL of Ham’s F10 (Sigma) supplemented with 20% Human Serum Albumin (HSA) (Sigma). Then, sperm suspensions were centrifuged at 300g for 10 minutes. After centrifugation, the supernatant was decanted; then, 1 mL of the Ham’s F10 was added to each pellet and the tubes were placed gently at 45° angle for 1hour at 37°C and 5% CO2 to allow swimming -up of motile spermatozoa. Motile spermatozoa swam into the supernatant. This fluid was carefully aspirated and its volume was adjusted to 1 mL using Ham’s F10. The samples were prepared individually and all experiments were performed for each individual sample separately.
Experimental Design
Each prepared sample was divided into three equal parts: Control group, 0.25 mL of the sample and 0.25 mL of Ham’s F10 containing HSA; KP-treated group, 0.25 mL of the sample and 0.25 mL of 20 μM Kisspeptin-13 (Human KP, at final concentration of 10 μM, Sigma) [9,11]; and Reduced Glutathione (GSH)- treated group, 0.25 mL of the sample and 0.25mL of 2 mM GSH (at final concentration of 1mM,Sigma) [18]. All aliquots were incubated at 37°C for 30 minutes.
Each group was evaluated for motility according to the WHO guideline. Based on WHO criterion, the sperm motility can be classified into three categories: progressive in which the spermatozoa move forward linearly or in a large circle; non-progressive in which the spermatozoa move in site or slowly without any forward progress such as moving in small circles or beating flagellum; and immotile which no movement is seen among the spermatozoa [17]. The sperm motility was evaluated using light microscope through employing 10 randomly selected microscopic fields. In each field, number of progressive, non-progressive, and immotile sperms was counted and percentages of progressive, non-progressive, and immotile spermatozoa were calculated.
Sperm Cryopreservation and Thawing
A half mL of treated and control aliquots were diluted (1:1) with cryopreserved medium (Life Global, USA) dropwise in cryotubes at room temperature. The cryotubes containing the samples and freezing medium were exposed to liquid nitrogen vapor by locating them horizontally at 5 cm above the liquid nitrogen surface for 20 minutes; then, they were quickly plunged into liquid nitrogen for longer storage.
After 48 hours, the cryotubes were thawed at 37°C for 10 min; then, 1mL of fresh Ham’s F10 was added to each sample. Finally, the tubes were centrifuged at 300g for 10 min. Spermatozoa were re-suspended in Ham’s F10 containing 20% Human Serum Albumin (HSA, Kedrion, Italy). After thawing, the sperm motility was re-examined.
Assessment of Sperm Plasma Membrane Integrity
Lectins Histochemistry: In the present study, Wheat Germ Agglutinin (WGA), Peanut Agglutinin (PNA) and Concanavalin A (ConA, All purchased from Sigma, USA) were used to detect non-capacitated, the acrosome intact, and acrosome-reacted spermatozoa.
The smears were prepared from all thawed samples, fixed with 2% paraformaldehyde for 20 minutes and subsequently washed with Phosphate Buffer Saline (PBS) for 30 minutes. The samples were incubated with Fluorescein isothiocyanate-conjugated WGA, ConA, and PNA at a dilution of 10 μg/mL for 2 hours at darkness. After washing with PBS, the samples were double stained with Hoechst (Sigma) for 5 minutes. The slides were evaluated using a fluorescent microscope (Nikon, Eclipse, E600).
Lectins Flowcytometry: Thawed samples were washed with 800 mL of PBS, were centrifuged at 170 g for 10 minutes and were fixed with 2% paraformaldehyde for 30 minutes at 4°C. Thereafter, the aliquots were centrifuged and the pellets were re-suspended in PBS. The aliquots were exposed to FITC-conjugated WGA, PNA, and ConA at a dilution of 10 μg/mL for 2 hours at 37°C in humidified environment and darkness. Frequency of the lectin-reacted spermatozoa was assessed, using FL1 channel of BD FACSCaliberTM flow cytometer (BD Biosciences, USA). The data were analyzed using FlowJo software.
Assessment of Sperm DNA Quality
The smears were prepared from each study group to record DNA and chromatin status, using Acridine Orange (AO), Aniline Blue (AB), Chromomycin A3 (CMA3), and TUNEL staining. A total of 200 sperms were evaluated per smear in randomly selected microscopic fields in all tests.
Acridine Orange (AO) Test: Thawed samples were spread on glass slides and allowed to air-dry. All the smears were fixed in methanol/acetic acid (3:1). Then, the slides were stained with 2–3 mL of 19% AO solution in citrate-phosphate buffer for 10 minutes. Stained spermatozoa were evaluated using fluorescence microscope (Nikon, Eclipse, E600). Three types of staining patterns were identified: green (double-stranded DNA), yellow (partially denatured DNA), and red sperms (completely single-stranded DNA) [8].
Sperm DNA Structure Assay: The sperm aliquots stained with AO (Sigma) were analyzed by flowcytometry as well. Briefly, 0.2 mL of thawed samples were diluted with TNE buffer (0.01 M Tris–HCl, 0.15 M NaCl, and 1 mM EDTA with a pH of 7.4) containing 10% glycerol at a final sperm concentration of 1-2×106 cell/mL. Then, the samples were immediately admixed with 0.4 mL of the detergent solution containing 0.1% Triton X-100, 0.15 M NaCl, and 0.08 N HCl with a pH of 1.2, at 4°C. After a short incubation of 30s, the cells were incubated in 1.2 mL of AO at the concentration of 6μg/mL in a solution containing 0.037 M citric acid, 0.126 M Na2HPO4, 0.001 M disodium EDTA, 0.15 M NaCl with a pH of 6.0 at 4°C. Then, the cells were analyzed by a FACSCaliberTM flow cytometer (BD Biosciences, USA). Strong green fluorescence (FL-1) and negative red fluorescence (FL-2) depicted regular sperm integrity. A sample of acid-treated sperms was used as positive control. DNA Fragmentation Index (DFI) was calculated using the following formula and obtained value was expressed as percentage:
DFI= Mean value of red fluorescence/mean value of red fluorescence + green fluorescence[18].
AO as a cationic metachromatic dye shows green fluorescence in monomeric state (when it bonds to DNA) and shows red fluorescence in polymeric state (when it bonds to RNA or denaturated single -stranded DNA). Cells with High DNA Stain ability (HDS) present in upper quarters of the dot blot chart represent immature cells indicating the cells with higher histone and lower protamine content [18]. The sperms with higher histone and lower protamine content have larger size.
Aniline Blue (AB) Staining
Air-dried smears were fixed in 3% buffered glutaraldehyde for 30 minutes. Then, the slides were stained with 5% aqueous Aniline Blue (AB) in 4% acetic acid (pH 3.5) for 7 minutes. Staining intensity of the sperm head was divided into three categories: unstained (gray/white), partially stained, and entire sperm head stained dark blue.
Chromomycin A3 (CMA3) Staining
For microscopic evaluation, the smears were fixed in methanol/glacial acetic acid (3:1) at 40°C for 5 minutes. Each slide was treated for 20 minutes with 100 µL of 0.25 mg/mL CMA3 in McIlvaine buffer containing 10 mM MgCl2 (pH 7.0). The slides were rinsed in PBS and mounted using buffered glycerol. The samples were observed by fluorescent microscope (Nikon, Eclipse, E600). Two types of staining patterns were identified, namely bright green fluorescence of the sperm head (abnormal chromatin packaging), and dull green staining (normal chromatin packaging).
TUNEL Staining
To detect the DNA breaks, Dead End Fluorometric TUNEL System kit (Promega, United States) was used according to the manufacturer’s protocol. Briefly, the samples were fixed in 4% buffered formaldehyde for 25 minutes at 4°C, were washed twice in PBS and then were treated with 0.2% Triton X-100 in PBS for 5 minutes. Then, the samples were then incubated in equilibration buffer for 10 minutes. They were incubated in the reaction mix which was prepared according to the manufacturer’s instructions. Staining was performed at 37°C for 60 minutes in humidified chamber protected from light. The reaction was stopped by adding 2× Saline-Sodium Citrate (SSC) buffer after 15-min incubation and then was counterstained using Hoechst (0.1 mg/mL) in PBS for 10 minutes. The slides were evaluated using fluorescent microscope (Nikon, Eclipse, E600).
Statistical Analysis
Data normality was checked using Shapiro–Wilk test. Comparisons were performed using one-way Analysis of Variance (ANOVA) and least significant difference (LSD) or Tukey tests if the data were normally distributed, and Kruskal–Wallis and one-way ANOVA test were used if the data were not normally distributed. All data were expressed as mean values ± Standard Deviations (SD). The level of statistical significance was set at ≤0.05. SPSS software version 24 for windows was used to analyze the data. The graphs were illustrated by GraphPad Prism software (version 6.0).