Low doses of Bisphenol S affect post-translational modifications of sperm proteins in male mice

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.

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.

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.

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. S2) [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).

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.

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.

At the end of 8-week exposure to actual doses of BPS, the body and testes weights were recorded and relative testes weights (mg/g bw) were determined. There were no differences between the experimental groups and the vehicle control (Table 1). Hormonal assays showed no significant differences in plasma hormone levels between the BPS-treated and vehicle control groups (Suppl. Table S3). Moreover, the spermatozoa count was not affected by BPS exposure (Fig. 1a), although treatment with 0.001 μg/kg bw BPS1 decreased the portion of motile spermatozoa (Fig. 1b).

Histological assessment was performed to evaluate the impact of actual BPS doses on testicular tissues. Stereological analysis showed no differences between groups in terms of testis volume, germinal epithelium volume (Fig. 2a, b), interstitium volume, germ layer volume fraction, and interstitium volume fraction. To investigate the effects of BPS treatments on spermatogenesis, individual stages of the seminiferous epithelium were identified and no differences between experimental groups were found (Fig. 2c, c’). Histopathological analysis of testicular tissues from BPS-exposed male mice showed an increased incidence of abnormalities in mice treated with the highest BPS dose (BPS3; Fig. 2d). In addition to vacuolisation of germ layer cells and enlarged multi-nuclear germ cells, the atypical residual bodies demonstrated the effects of BPS3 on testicular tissues (Fig. 2d–g). There were fewer mature spermatozoa in the germ layer in the BPS2 experimental group (Fig. 2h). Representative images of individual histopathologies are shown (Fig. 2d’–h’).

Based on the different modes of action of BPS at various doses, whole-proteome profiling of testicular tissues was performed. In total, 3044 proteins were detected. Unique protein expression in the control and BPS-treated groups is shown in the Venn diagram in Fig. 3a. However, after quantification of the levels of 1886 proteins, followed by subsequent principle component analysis (PCA), no distinct clusters of mice (n = 24) from individual groups were observed, thus indicating a lack of a consistent proteome pattern (Fig. 3b). In addition to total protein analysis, acetylated (n = 15) and phosphorylated (n = 26) peptides were quantified (Fig. 3c, d), and no significant differences were observed. Moreover, the deleterious effects of BPS3 were elucidated using antibodies against the phosphorylated form of H2AX (γH2AX) to label DNA double-stranded breaks; γH2AX is a representative PTM that can be used to identify DNA damage and cellular stress. Consistent with the increased incidence of abnormalities in seminiferous tubules in the BPS3 group, we observed an increase in the γH2AX signal as well (Fig. 3e).

In accordance with whole-proteome analysis of BPS-treated testes, analyses of the sperm acetylome and phosphorylome were performed using western blotting and MALDI-TOF MS. Because of the low protein amounts in sperm lysate extracts, sperm samples from three individuals belonging to the same experimental group were pooled, and three independent experiments were performed. After loading equal amounts of protein per well, we found that the acetylation of proteins with molecular weights of approximately 37, 40, and 50 kDa were affected by treatment with BPS1 (Fig. 4a, b). Moreover, BPS1 also modified the phosphorylation of sperm proteins (37, 40, 85, and 100 kDa; Fig. 4c, d, d’). Candidate acetylated and phosphorylated proteins are summarised in Fig. 4 (e, f), and the results indicated the involvement of housekeeping proteins (ATP synthase subunit, hexokinase-1) and enzymes (DNA repair protein, E3 ubiquitin-protein ligase). In accordance with previous findings, demonstrating that BPS1 suppressed sperm motility, cytoskeletal factors (i.e., tubulin chains, actin; Fig. 4e) seems to be underwent to acetylation. Therefore, an antibody against acetylated α-tubulin (acTubulin) was used for a verification of tubulin as a candidate BPS target.

Next, we evaluated the densitometry of bands representing acetylated tubulin after treatment with BPS1 (Fig. 4g). Decreased tubulin acetylation was observed; however, the difference was not statistically significant, suggesting that other targets (such as ATP synthase and actin) may be related to sperm motility.