The Animal Care and Ethics Committee of Chonbuk National University (cuh-IACUC-2017-10-2) approved all experiments. All efforts were made to minimize animal suffering. Fifty sexually mature male and female SD rats (weight: 210–240 g; age: 8 weeks) were obtained from KOATECH, Jeonwi-ro, Jinwei-myeon, Pyeongtaek-si, Gyeonggi-do, Korea. They received a standard rat chow diet with free access to water ad libitum. They were maintained in the animal facility at a constant room temperature of 20 ± 2 °C with relative humidity of 50 ± 10% and a 12-h light/dark cycle. Rats were acclimated to that environment for the first week. All rats were placed in plastic cages (47 × 18 × 40 cm) with four rats per cage.
Chemicals and reagents
ADR was purchased from Tocris Bioscience (Tocris House, IO Center Moorend Farm Ave., Bristol, BS11 0QL, UK). All other chemicals were of analytical grade and purchased from standard commercial suppliers.
Preparation of DA-9401
DA- 9401 was prepared as previously described .
After 1-week of acclimatization, 8-week-old male SD rats weighing 220–240 g were randomly divided into five groups (10 rats per group): (1) control (CTR) group, (2) DA-9401 100 mg/kg/day p.o. group (DA 100), (3) ADR 2 mg/kg per week i.p. group (ADR), (4) ADR 2 mg/kg per week i.p. + DA-9401 100 mg/kg/day p.o. (ADR + DA 100), and (5) ADR 2 mg/kg per week i.p. + DA-9401 200 mg/kg p.o. group (ADR + DA 200). DA-9401 was dissolved in two different containers with sterile normal saline and administrated orally by gavage with a Zonde needle (JD-S-124, Jeungdo, Seoul, Korea) at a single dose of 100 or 200 mg/kg/day. The CTR group received normal saline (vehicle) for 56 days. ADR was dissolved in distilled water and received 2 mg/kg intraperitoneally once a week for 56 days. This dose is well documented to be able to induce testicular toxicity in rats . Fifty female rats were used to determine fertility parameters after natural mating. Each mating pair was kept in single case after 6 weeks of medication. After 2 weeks, female rats were separated from male rats and kept in a separate singled case. Male rats were proven fertility by producing offspring. All male rats were anesthetized 48 h after the last treatment. Rats were anaesthetized with mixture of ketamine (100 mg/ml) and 2% rumpin (20 mg/ml) at a dose of 170–230 μl/100 gm body weight . Blood samples were collected from rats’ vena cava. Testis tissues were collected and used for the following analysis.
Reactive oxygen species (ROS)/reactive nitrogen species (RNS) and malondialdehyde (MDA) level
ROS/RNS assay was determined using a fluorescence kit (STA-347, OxiSelectTM in vitro ROS/RNS assay kit, Cell Biolabs, Inc., San Diego, CA, USA) at excitation and emission wavelengths of 480 and 530 nm, respectively, with a SpectraMax Gemini XS Fluorimeter. To assess lipid peroxidation in SD rat testes, MDA levels in testis tissue homogenates were measured using a commercially available kit (NWLSSTM Malondialdehyde Assay kit; Northwest Life Science Specialties LLC., Vancouver, WA, USA) following the manufacturer’s instructions. MDA forms a pink complex in aerobic conditions after incubation with thiobarbituric acid (TBA) at 60 °C. Absorbance of the colored complex was measured by kinetic spectrophotometric analysis at 532 nm using a Spectra Max 180 (Molecular Devices, Sunnyvale, CA, USA). MDA concentration in the sample was analyzed by comparing the measured absorbance value to an MDA standard curve . MDA concentrations were normalized to total protein content .
Sperm motility and sperm count in the vas deferens and epididymis
The distal cauda of the epididymis and the entire length of the vas deferens were removed and placed in separate microcentrifuge tubes, minced, and suspended in pre-warm normal saline at 37 °C for 5 min. Sperm motility was evaluated by observing a sperm suspension within 3–5 min after being placed on a pre-warmed counting chamber (SEFI-Medical Instruments, Haifa, Israel). The number of motile spermatozoa within 10 squares of the grid were counted under a light microscope and mean sperm count was recorded. The percentage of motile spermatozoa was determined with the following formula: (mean number of motile spermatozoa/total number of spermatozoa) × 100%.
Spermatogenic cell density and Johnsen’s score
Testes tissues were immediately fixed in Bouin’s solution for 48 h and dehydrated through a graded ethanol series. These tissue samples were embedded in paraffin, sectioned (5 μm in thickness), deparaffinized, rehydrated, and stained with hematoxylin and eosin (H&E). Testis tissue was evaluated using standard light microscopy. Ten seminiferous tubules (ST) were randomly examined per section. Their diameters and germinal cell layer thicknesses (from the basal membrane towards the lumen of the tubule) were measured using an image analysis program (i-Solution; IMT i-solution Inc., Vancouver, BC, Canada). Spermatogenic cell density was determined by measuring the thickness of the germinal cell layer and the diameter of the seminiferous tubules. The seminiferous tubules of H&E-stained sections at X400 were graded by Johnsen’s score as previously described . Damaged tubules at edges of the section were excluded. A minimum of 20 seminiferous tubules from these slides were assessed according to the presence of spermatogenic cells and assigned a score from 1 to 10.
Terminal deoxynucleotidyl transferase-mediated (dUTP) nick-end labeling (TUNEL) staining
Small pieces of testis tissue from each group were fixed in Bouin’s solution in phosphate-buffered saline (PBS) and then processed via dehydration in a graded ethanol series, embedded, and sectioned at 5 µm in thickness on the serial coronal plane. Apoptotic activity within the seminiferous tubules was determined using TUNEL assays (Dead End™ Colorimetric TUNEL System for qualitative study; Promega, Madison, WI, USA). All procedures were carried out according to the manufacturer’s instruction. Two slides from each animal were used for quantitative study. In cross section, 100 seminiferous tubules from each group were counted for the number of apoptotic cells under a fluorescence microscope (20× objective). Positive nuclei stained dark-brown were visualized under a light microscope.
Determination of hormonal assay
Levels of sex hormones including serum testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were measured by enzyme-linked immunosorbent assay (ELISA) using commercial kits (55-TESMS-E01, Mouse/rat testosterone kit, ALOCO, 26-G Keewaydin Drive, Salem, NH, USA; E-EL-R0026, rat LH Elisa kit; E-EL-R0391, rat FSH Elisa kit; Elabscience, Houston, Texas, USA) following manufacturers’ instructions.
Determining concentrations of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase
Testis tissues (100 mg) were rinsed with 1X PBS (pH 7.4) to remove any red blood cells and clots. Concentrations of SOD, GPx, and catalase in whole tissue supernatant were measured using commercial kits (item no. 706002, superoxide dismutase kit; item no. 703102, glutathione peroxidase kit; item no. 707002, catalase assay kit, Cayman Chemical, Ann Arbor, MI, USA) following the manufacturer’s instructions. Values are expressed as per milligram protein.
Testis tissues (100 mg) were rinsed with 1X PBS (pH 7.4) to remove any red blood cells and clots. Tissues in 1 ml of 1X PBS were homogenized with a homogenizer on ice and stored at − 20 °C overnight. Two freeze–thaw cycles were then performed and the homogenate was centrifuged at 10,000×g for 15 min at 4 °C. The supernatant was used for assays. Concentrations of interleukin-6 (IL-6) and TNF-α were measured by enzymatic method using commercial kits (BMS625 IL-6 rat Elisa kit, BMS 622 rat TNF-α kit, Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. Values are expressed as per milligram protein.
Testis tissues were washed twice with cold PBS and homogenized using a cordless motor pellet pestles in extraction buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and 0.1% SDS) supplemented with protease inhibitor for 30 min on ice. Tissue lysates were centrifuged at 13,000×g for 30 min at 4 °C. The supernatant was collected and stored at − 80 °C. Protein concentration was determined by Bradford protein assay. Levels of ER stress marker [glucose-regulated protein-78 (GRP-78), phosphorylated inositol-requiring transmembrane kinase/endoribonuclease 1α (p-IRE1α), phosphorylated c-Jun-N-terminal kinase (p-JNK)], apoptosis markers [pro-caspase-3, cleaved caspase 3, BCL 2 associated X protein (Bax), B cell lymphoma 2 (Bcl-2)], steroidogenic acute regulatory protein (StAR), cation channels of sperm (CatSper), glycogen synthase kinase-3 (GSK-3), occludin, claudin 11, and zonula occludens-1 (ZO-1) were measured in testis tissue. A total of 30–60 μg of extracted protein from each sample was loaded per lane, subjected to 8–12% SDS–polyacrylamide gel electrophoresis, and electro blotted onto PVDF membranes with a trans-blot® SD semi-dry electrophoretic transfer cell (Bio-Rad, Hercules, CA, USA). For higher molecular weight proteins, overnight wet transfer was performed at 20 V. After protein transfer, the membrane was blocked with 5% bovine serum albumin (BSA) for an hour at room temperature and incubated with the following primary antibodies overnight at 4 °C: phosphorylated antibodies p-IRE1α (Abcam Cambridge, MA USA) and p-JNK (Santa Cruz Biotechnology, Dallas, TX, USA), non-phosphorylated antibodies GRP-78, pro-caspase-3, cleaved caspase 3, Bax, Bcl-2, StAR (Cell Signaling Technology, Beverly, MA, USA), occludin (Abcam Cambridge, MA USA), claudin 11, ZO-1 (Santa Cruz Biotechnology, Dallas, TX, USA), and GSK-3 (Thermo Fisher Scientific, Waltham, MA, USA) in the presence of 5% non-fat milk. The membrane was washed with Tris-buffered saline containing 0.05% Tween 20 (TBST, pH 7.2) three times prior to incubation with 1:5000 diluted secondary antibody [anti-mouse, anti-rabbit (Cell Signaling Technology, Beverly, MA, USA), or anti-rat IgG (Santa Cruz Biotechnology, Dallas, TX, USA)] at room temperature for 1 h. The membrane was washed three times with TBST. Antigen–antibody complexes were then visualized with an ECL system (Vilber Lourmat, France).
All data are expressed as mean ± standard error of the mean (SEM). A P-value < 0.05 was considered statistically significant by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. GraphPad PRISM (Version 5, GraphPad Software, San Diego, CA, USA) was used for the graph analysis. Statistical calculations were performed using SPSS version 22 (IBM, Armonk, NY, USA).