A total of 25 adult NMRI mice (15 females and 10 male) aged 8-10 weeks and weighting 25-30 g were used for breeding. These animals were obtained from animal house of Afzalipour faculty of Medicine, Kerman. They were housed in polypropylene cages and were kept under standard condition of animal house with a temperature of 23+2 ̊C and constant 12-hours light/dark cycle with free access to drinking water and standard pellet food.
Three female and one male mice (proportion of three females per male) were housed in one cage overnight and the vaginal plug was checked in the following morning. Presence of a vaginal plug was considered as gestational day (GD) 1. The pregnant mice were randomly allocated into 5 groups (n= 8) and treated daily as follows:
Vehicle group (Veh): the pregnant animals daily received 0.3 ml normal saline by intraperitoneal (i.p.) injection.
Nicotine group (Nic): the pregnant mice daily received nicotine tartrate (1 mg/kg, i.p., free base) 8213.
Ethanol group (Ethn): the pregnant mice daily received ethanol (3 gr/kg, i.p.) 53.
Nicotine + Ethanol group (Nic+Ethn): the pregnant mice daily received the same doses of nicotine and ethanol, simultaneously 68.
Treatments started from GD1, continued throughout the pregnancy and lactation periods until offspring weaning (postnatal day; PND 21). The male offspring were maintained in separate cages under the same controlled conditions until PND 90. In order to prevent from any gene homogeneity, only 2 male offspring of each dam were randomly assigned for each experimental group (n=8).
At PND 90, 8 pups of each group were anesthetized using ketamine ((5-10 mg/kg) /xylazine (5 mg/kg), and their blood was collected from left ventricle. Blood samples were centrifuged at 2500-3000 rpm for 10 minutes and the serum removed and held at -20 ˚C for further evaluations.
Testis and sperm collection
The anesthetized animals were authonized by cervical dislocation, their abdomen wall was cut and the left cauda epididymis of each mice were carefully removed, and were transferred into a petri dish containing 1 mL pre-warmed HTF media supplemented by 15 mg/mL bovine serum albumin. It was cut by a pair of scissors, to release sperm cells. The petri dishes were incubated at 37 ̊C for 15 minutes. Meantime, the left testes were removed and weighted by a digital scale. Testes dimensions including length, width and thickness, were measured using a standard digital caliper and then fixed by dipping in Bouin’s solution for 48 hours.
Sperm parameters analysis
After incubation of sperm cells for 15 minutes, the suspension was collected, quietly pipetted, 10 μL of sperm suspension was deposed on the pre-warmed slide, covered by coverslip and then the motility of at least 200 sperm cells was evaluated under a light microscope at × 200 magnification in 10 random fields. The motility rate was expressed as the number of motile sperm cells to total counted sperm cells ( × 100 83.
Five μL of sperm suspension was mixed with 5 μL of sperm fixative solution (formalin/sodium bicarbonate). The mixture was pipetted and the solution was placed on an improved Neubauer hemocytometer and the sperm heads were counted under a light microscope (Olympus BX 51, Tokyo, Japan) at × 200 magnification. The sperm number was expressed as million per one mL of suspension 84.
Ten μL of sperm suspension was mixed with an equal volume of 1% eosin Y/ 5% nigrosin staining solution in a microtube. After 2 minutes, a smear was prepared, air dried and at least 200 sperm cells were evaluated at × 1000 magnification under immersion oil. (Figure 2-D). The sperm viability rate was calculated as the number of unstained sperm cells (alive) to total counted sperm cells (white + red)× 100 85.
The sperm parameters were evaluated by an expert operator blinded to experimental groups.
Sperm DNA fragmentation assessment
Sperm DNA fragmentation (SDF) was assessed by sperm chromatin dispersion (SCD) test based on our previous study with some changes 86,87. Briefly, 30 μL sperm suspension was mixed with 70 μL 1% low gelling agarose, placed on a pre-coated slide with standard agarose (0.65%), covered by coverslip and transferred to refrigerator (4 ̊C) to solidify. After 5 minutes, the coverslip was carefully removed and the slide was suspended in 0.08 N HCl solution for 17 minutes at room temperature in darkness. Next, the slide was incubated in two consecutive lysis buffer solutions; lysis buffer solution 1 containing 0.4 M Tris, 0.8 M 2-mercaptoethanol, 1% SDS and 50 mM EDTA (pH 7.5) for 20 minutes followed by lysis buffer solution 2 containing 0.4 M Tris, 2 M NaCl and 1% SDS, (pH 7.5) for 15 minutes. The slide was finally incubated in Trisborate–EDTA buffer (0.09 M Trisborate and 0.002 M EDTA (pH 7.5) for 12 minutes. The slide was washed and dehydrated in increasing concentration of alcohol in water. The slide was stained by Diff-Quick solution for 3 minutes and air dried. Under light microscopy at × 400 magnification, 300 sperm cells were scored based on their halo size; big and medium-sized halo indicate sperm cells without DNA fragmentation, while small-sized halo and also sperm with no halo indicate sperm cells with DNA fragmentation. SDF rate was calculated as the number of sperm cells with small and no halo to total number of counted sperm × 100 88.
Testicular morphometric evaluation
The fixed testes were embedded in paraffin and sectioned by a rotary microtome (Leitz, Germany). Eight 5- μm thick sections of middle part of each testis was prepared at 10-μm intervals. The sections were stained by hematoxylin/eosin, the diameter and the area of 10 round seminiferous tubules in each section were evaluated. In order to evaluate the quality of spermatogenesis and maturity of germinal epithelium, Johnsen’s score was used as a semi-quantitative method in 80 seminiferous tubules per animal 89. The scores were summed up and their means were reported as Johnsen’s score (JS). All morphometric assessment of testicular tissue was performed in a blind fashion by an expert operator.
Malondialdehyde (MDA) determination
MDA level as a final product of lipid peroxidation in the serum of exposed pups (seven mice/ group) was assessed by thiobarbituric acid method using Nalondi™-Lipid Peroxidation Assay Kit-MDA (Navandsalamat Company, Urimia, Iran) based on the manufacture instructions. In short, 200 μL of serum was mixed with 1000 μL of working solution containing 2-thiobarbituric acid and trichloroacetic acid and then incubated at 95 °C for 45 minutes. The mixture was transferred on ice, incubated for 10 minutes and then, centrifuged at 1500 rpm for 15 minutes. Absorbance of the supernatant was read at 532 nm by a spectrophotometer (Alpha-1860, Thomas Scientific, USA). MDA concentration was expressed as nM/mL.
Total RNA extraction and complementary DNA synthesis
Total RNA was extracted from sperm cells by RNX plus solution (Cinnagen, Iran) based on the manufacturer's instructions. In short, 1 mL of cold RNX plus solution was added to 1 mL of sperm suspension, and incubated at room temperature for 5 minutes. Then, 200 μL of cold chloroform solution was added, shacked vigorously and centrifuged at 12000 rpm, at 4 °C, for 15 minutes. The supernatant was transferred into a clean microtube, equal volume of cold isopropanol solution was added and the microtube was transferred to a -20 °C freezer, overnight. Next day, the mixture was centrifuged at 12000 rpm, at 4 °C for 15 minutes. The upper phase was then removed, the pellet was washed by 75% ethanol, and again centrifuged at 12000 rpm, at 4 °C, for 15 minutes. In the next stage, the supernatant was removed and the pellet was placed under a laminar- air flow hood to dryness. The dried pellet was resuspended in 20 μL diethyl pyrocarbonate (DEPC)- treated water. The integrity and quantity of total RNA were verified by 1% agarose gel electrophoresis and the absorbance ratio at 260/280 nm with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, United States), respectively. One μg of total RNA was converted into complementary DNA (cDNA) with 1 μL of 200 U/μL SuperScript III Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Fermentas, Burlington, Canada), Four μL of 5X first-strand buffer, 0.5 μL of 40 U/μL RNase inhibitor, 1 μL oligo (dT) primer and 1 μL 10 mM dNTP were mixed in a 20 μL reaction. The samples were incubated at 42 °C for 60 minutes, then the enzyme was deactivated at 70 °C for 10 minutes using a thermocycler (Biometra, Germany) 90. The specific primers were designed by the primer3 program. The primer sequences are presented in Supplementary table S1. Conventional reverse transcriptase (RT)-PCR was done for optimizing annealing temperature for each primer set. The RT-PCR products were evaluated by agarose gel electrophoresis (2%).
Quantitative real time PCR (qRT-PCR)
Relative mRNA levels were quantified by real-time PCR with SYBR Green Master Mix
(Genaxxon bioscience, Ulm, Germany) using a Light Cycler Real-Time PCR System (MIC (Queensland, Australia) for the following genes: DNMT1, DNMT3A and DNMT3B, HDAC1 and HDAC2. Beta actin was used as the housekeeping gene to normalize the qRT-PCR reaction. For the amplification reaction,1 μL synthetized cDNA solution was mixed with a 1 μL specific primer and 10 μL SYBR Green I Master in a 20 μL reaction. The thermocycler cycling conditions was as follows: 95 °C for 15 minutes (1 rep), followed by 40 cycles of 95 °C for 15 seconds, 60-62 °C for 60 seconds and 72 °C for 30 seconds. Melting curves were checked for the validation of amplification of a single PCR product. The 2−ΔΔ cycle threshold method was used to assess gene expression levels.
Data analysis was conducted by using SPSS software (version 22). At first, data was analyzed for normality by using Kolmogorov-Smirnov test. Differences in parametric data were determined by One-way analysis of variance (ANOVA) test followed by Tukey post hoc test, while differences in non-parametric data were determined by using Kruskall-Wallis test. The data were presented as Mean ± SEM. The Graphpad Prism software was used for graphing.
Animal research was approved by the Animal Experiment Committee of the Kerman University of Medical Sciences of Iran (approval no.: EC/97-10/KNRC) and confirming that all animal study methods were conducted in strict accordance with the relevant guidelines and regulations. All experimental procedures were carried out in accordance with the ARRIVE guidelines.
All data generated or analyzed during this study are included in this published article (and its Supplementary Information file).