All chemicals, including BPS (CAS: 80-09-1, cat. No. 103039) were purchased from Sigma-Aldrich (USA), unless stated otherwise.
2.1 Animals
All animal procedures were done in accordance with the Protection of Animals against Cruelty (Act No. 246/1992) under the supervision of the Animal Welfare Advisory Committee at the Ministry of Education, Youth, and Sports of the Czech Republic. Adult 7-week-old ICR male mice were purchased from Velaz Ltd. (Prague, Czech Republic), housed in standard cages in groups of 3 and maintained in a 12/12-h light/dark cycle at 21 ± 1°C with a relative humidity of 60%. Bisphenol contamination was reduced using intact polysulfonate cages and glass drinking bottles. Mice were maintained on a phytoestrogen-free diet (1814P Altromin, Altromin Specialfutter GmbH & Co., Germany) with ultrapure water available ad libitum.
2.2 BPS dosage and sample collection
Mice were randomized into four experimental groups and allowed to adapt for 1 week. Vehicle control (0.1% ethanol; VC) and BPS for three treatment groups were administered through drinking water at final concentrations of 0, 0.0038, 3.8, and 380 µg/L, respectively, for 8 weeks (8–16 weeks of age). The following dosages were presumed [0, 0.001, 1, and 100 µg/kg body weight (bw)/day] with actual exposure estimated based on the knowledge of recorded body weight and water intake as previously reported (17). A wide range of doses and the route of exposure have been chosen appropriate to the real human exposure; doses of experimental animals through the drinking water have been used with respect to the welfare of animals. Hereafter, experimental groups will be stated as BPS1, BPS2 and BPS3.
Nine mice per group were included in three individual independent experiments (n = 36). Animal weights were recorded at the end of the experiments mice euthanised by cervical dislocation. Blood samples were collected by cardiac puncture, and serum was stored at -80°C until hormonal assay performance. Left and right testes were collected, weighed, and processed for histology and proteomics, respectively.
2.3 Sperm isolation and assessment
From the mice described above, the cauda epididymidis was dissected in 0.5 mL Whitten’s medium (Suppl. table S1), and sperm were allowed to swim out for 30 min. Thereafter, sperm concentration and motility were evaluated using Makler chamber and light microscope (Olympus CKX 41; Germany) equipped with a 10× objective (CAchN NA 0.25). 10 µl of sperm suspension was pipette to the Makler chamber, thereafter spermatozoa were counted in 3 lines, each of 10 squares and divide by 3 to obtain average sperm concentration in million per milliliter. Simultaneously, each spermatozoon across the counted area was identified either as motile or immotile. Accordingly, the sperm motility was expressed as the ratio of motile to immotile spermatozoa. The analysis was performed blindly to avoid bias
2.4 Hormonal profiling
Blood serum samples in three independent experiments (n=5 mice per group) were assayed with Immunobeads Milliplex MAP kit (HPTP1MAG-66K, MSHMAG-21K; Merck Millipore, USA) for the following hormone levels: adrenocorticotropic hormone, follicle-stimulating hormone, growth hormone, luteinising hormone, thyroid-stimulating hormone, cortisol, progesterone, testosterone, triiodothyronine, and thyroxine.
2.5 Quantitative and qualitative analyses of testes
One testis from each animal (n = 9 per group) was fixed in Bouin solution, embedded in paraffin wax with random orientation, and sectioned completely into 10-µm-thick slides. The total testis volume, total germ epithelium volume, and interstitium volume were estimated according to the Cavalieri principle (18). The fractions of spermatogenesis (pre-spermiation stages I–VI; middle spermiation stages VII–VIII; post-spermiation stages IX–XII) were found using the point grid approach (19,20). To determine the precision and accuracy of the stereological analysis, the coefficient of error was estimated (Suppl. Tab. S3) (18). Qualitative analysis of seminiferous tubes was performed according to the methods described by the Society of Toxicologic Pathology (21,22) to assess the following abnormalities: missing germ cell layers and germ cell depletion, retained spermatids (spermiation failure), multinucleate and apoptotic germ cells, and exfoliation of spermatogenic cells into the lumen. At least 100 seminiferous tubules were evaluated blind to treatment group for each testicular cross section. The quantitative assessment was performed on a Nikon Eclipse Ti-U microscope (Nikon, Japan) equipped with a motorised stage (Prior, UK) using a 10× objective (Plan Fluor, NA 0.3) and Stereologer 11 software (SRC, Biosciences Tampa, FL, USA) for histopathological evaluation was performed using a 40× objective (UPlanFl, NA 0.75).
2.6 Western blot
Testicular tissue and sperm were dissolved in lysis buffer (40 mM Tris base, 7 M urea, 2 M thiourea, 4% CHAPS, 120 mM dithiothreitol), enriched with Complete Mini Protease Inhibitor Cocktail (Roche, Switzerland), for 30 min on ice. Sperm samples of three individuals belonging to the same experimental group were pooled. Thereafter, samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis on 4–15% separating Mini-PROTEAN precast gels and blotted using a Trans-Blot Turbo Transfer System onto polyvinylidene difluoride membranes (Bio-Rad Laboratories, France). The membranes were blocked in 1% bovine serum albumin in TBS with 0.5% Tween-20 for 60 min at room temperature and incubated overnight at 4°C with primary antibodies diluted in blocking buffer. The following primary antibodies were used: anti-acetyl lysine antibodies (cat. no. ab80178; Abcam, UK), anti-phospho-tyrosine antibodies (cat. no. ab10321; Abcam), anti-acetylated α-tubulin antibodies, and anti-γH2AX antibodies. Mouse monoclonal anti-α-tubulin antibodies (cat. no. T6199; Sigma, St. Louis, MO, USA) and rabbit monoclonal anti-histone H3 antibodies (cat. no. D1H2; Cell Signaling Technology, Danvers, MA, USA) were used as the loading control for γH2AX and acetylated α-tubulin, respectively. Horseradish peroxidase-conjugated secondary antibodies (goat anti-mouse or anti-rabbit IgG; dilution: 1:15,000; Invitrogen, Carlsbad, CA, USA) were applied for 60 min at 22 °C. Target proteins were visualised using ECL Select Western Blotting Detection Reagent (GE Healthcare Life Sciences, UK) and a ChemiDoc MP System (Bio-Rad). Alternatively, proteins were visualised using a colorimetric Opti-4CN substrate kit (Bio-Rad), followed by matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry (MS) for peptide detection in the dissected bands.
2.7 Proteome profiling
Testis lysates from animals in the experimental groups were collected for complete proteomic analysis. Nano-liquid chromatography-MS (nano-LC-MS) was used for protein identification and quantification, as described previously (7). The acetylome and phosphorylome were analysed separately.
2.8 Statistics
The data were processed with GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA). Based on Shapiro-Wilk normality distribution tests, analysis of variance (ANOVA) and Kruskal-Wallis tests were used for normally and non-normally distributed data. In cases of significant overall findings, differences between individual group pairs were assessed by Tukey’s and Dunn’s post-hoc tests, respectively. Results with P less than 0.05 were considered statistically significant. Normally and non-normally distributed data were expressed as means and medians, respectively.