All procedures with animals were performed in accordance to protocols approved by the Institutional Animal Care and Use Committee of Chung-Ang University, Seoul, Republic of Korea.
Media and chemicals
All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise stated. Modified Tyrode’s medium (osmolality 300 ± 20 mOsm/kg, pH 7.2 ± 0.2) was prepared freshly and used as basic medium (BM) [6, 29]. The BM was pre-incubated 1 day prior to the experiment and supplemented with bovine serum albumin (BSA; 4 mg/mL). BPA was dissolved in dimethylsulfoxide (DMSO) and added to a final concentration to treat the spermatozoa. The control spermatozoa were treated with DMSO only. An estrogenic positive control was not considered in the present study because BPA has been reported to act via other signaling pathways together with ERs; thus, it is not obvious whether BPA and natural estrogens have similar effects [40, 41].
BPA dose selection
The exposure scheme consisted of four different doses of BPA: 0.0001, 0.01, 1, and 100 μM. The doses up to 1 μM were comparable to the acceptable human daily exposure levels . It has been demonstrated that low dose of BPA (~0.01 μM) advances early embryonic development, whereas a comparatively higher dose (~100 μM) decreases the development rate of the embryo [42, 43]. More recently, genotoxic and mitogenic effects of BPA has been demonstrated at the dose of 0.01–0.1 μM, in mammary cells . Therefore, range of concentrations (0.001–100 μM) was considered in current study to clarify in vitro adverse effect levels of BPA in spermatozoa.
Collection and preparation of spermatozoa and their exposure to BPA
Spermatozoa were collected from sexually mature ICR male mice (Nara Biotech, Seoul, Korea) following published procedures [6, 29]. Briefly, both cauda epididymides from each mouse were collected and the associated fat was removed. The epididymides were then placed in cell culture dishes with BM containing 0.4 % BSA, and punctured using a sterile needle to release spermatozoa. The released spermatozoa were incubated for approximately 10 min with 5 % CO2 in air at 37 °C to facilitate dispersal. An initial experiment was designed to determine the potential of BPA to alter a) sperm motility and b) viability. For this, the spermatozoa were incubated (5 % CO2 in air at 37 °C) with 100 μM BPA for different periods of time (ranging from 0.5 to 8 h at 30-min intervals) to investigate the potential shift of both parameters. Finally, 6 h was identified as the minimum effective period of BPA exposure. Therefore, the sperm suspension was incubated for 6 h under the same conditions in BM supplemented with various concentrations of BPA.
Assessment of sperm motility
Sperm motility was evaluated using computer-assisted sperm analysis (CASA) (SAIS plus version 10.1; Medical Supply, Seoul, Korea) according to described methods [6, 29]. The 10× phase contrast objective was used by the SAIS software to relay and analyze the spermatozoa. Five fields of each sample were randomly selected to evaluate the movement of at least 250 sperm.
Hypo-osmotic swelling test (HOST)
To evaluate sperm viability and functional integrity (membrane), we used the hypo-osmotic swelling test (HOST) as described previously . The sperm swelling patterns were classified broadly as viable and/or nonviable according to the WHO 2010 manual and by using the Microphot-FXA microscope (Nikon).
Detection of LDH
To determine cytotoxicity, the CytoTox 96® assay kit (Promega, Fitchburg, WI, USA), which is based on the colorimetric detection of LDH, was used according to the method described previously [6, 29]. The LDH activity was measured as the absorbance at 490 nm by using a luminometer (GEMINI EM, Molecular Devices Corporation) and was calculated using the SoftMax Pro 5 software. Activity is reported as the ratio of the fluorescence of BPA-treated samples to that of the control.
Detection of mitochondrial activity
Mitochondrial membrane potential, defined as mitochondrial activity, was measured by rhodamine 123 (R123) staining according to the manufacturer’s directions and the described method . The samples were analyzed by flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA) with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Ten thousand cells in each sample were considered to obtain R123 signal, and the signal was analyzed using the CellQuest software (Becton Dickinson). Mitochondrial activity was reported as the fluorescence of the BPA-treated samples compared with that of the control.
Detection of [ATP]i levels
[ATP]i was detected using an ATP Bioluminescence Assay Kit (CLS II; Roche Molecular Biochemicals, Mannheim, Germany) according to the described method . The luminescence signal was detected using a Microplate Multimode Reader (GloMax®-Multi; Promega, Madison, WI, USA). The [ATP]i is reported as the ATP (signal) of the BPA-treated samples compared with that of the control.
Preparation of spermatozoa for proteomic experiments
The BPA-treated and control spermatozoa after 6 h of incubation were washed twice by centrifugation (100 × g for 2.5 min) at room temperature (RT), re-suspended in BM, and allowed to swim-up at 37 °C for an additional 15 min. Swim-up was performed to separate the motile sperm fraction from immature spermatozoa and somatic cells [46–48]. The motile sperm fraction was then carefully collected, and the samples were checked for the nonappearance of immature spermatozoa and somatic cells using light microscopy and Hoechst staining (Additional file 2: Figure S3).
2-DE and gel-image analysis
To extract proteins from the spermatozoa, 50 × 106 cells were incubated in rehydration buffer for 1 h at 4 °C [17, 18, 48]. Then, 250 μg aliquots of sperm protein were placed in 450 μL of rehydration buffer in a rehydration tray with 24 cm-long NL Immobiline DryStrips (pH 3–11; Amersham, Piscataway, NJ, USA) for 12 h at 4 °C. The first-dimension electrophoresis (1-DE) was performed using an IPGphor IEF apparatus, and the strips were focused according to previously described settings [17, 18, 48]. After iso-electrofocusing, the strips were equilibrated for 15 min at RT by using equilibration buffer A, whereas the second equilibration was performed using equilibration buffer B [17, 18, 48]. Afterward, 2-DE was carried out on 12.5 % (w/v) SDS-PAGE gels with the strips at 100 V for 1 h and then at 500 V until the bromophenol blue front began to migrate off the gels. The gels were silver-stained for image analysis according to the manufacturer’s instructions (Amersham, Piscataway, NJ, USA). The silver-stain was used to conduct the present study because this method is highly sensitive, capable of detecting minimal variants of protein in the gel, and is compatible with downstream processing, such as mass spectrometry . The gels were scanned using a high-resolution GS-800 calibrated scanner (Bio-Rad, Hercules, CA, USA). Detected spots were matched and analyzed by comparing the gels from spermatozoa treated with BPA and the control using PDQuest 8.0 software (Bio-Rad, Hercules, CA, USA). Finally, the spot density was calculated and normalized as the ratio of the spot on the BPA-treated (spermatozoa) gel to that on the control gel.
Proteins were subjected to in-gel trypsin digestion according to the protocol established previously [17, 18]. Excised gel spots were destained with 100 μL of destain solution (30 mM potassium ferricyanide, 100 mM sodium thiosulfate) by shaking for 5 min. Then, the gel spots were incubated with 200 mM ammonium bicarbonate for 20 min. The gel pieces were dehydrated with 100 μL of acetonitrile and dried in a vacuum centrifuge. The above procedure was repeated thrice. Then, the dried gel pieces were rehydrated with 20 μL of 50 mM ammonium bicarbonate containing 0.2 μg of modified trypsin for 45 min on ice. Seventy microliters of 50 mM ammonium bicarbonate was added after removal of solution. Digestion was performed overnight at 37 °C. The peptide solution was desalted using a C18 nano column (homemade, Waters Corp, Milford, MA, USA).
Desalting and concentration
The custom-made chromatographic columns were used for desalting and concentration of the peptide mixture. A column consisting of 100–300 nL of Poros reverse phase R2 material (PerSeptive Biosystems, Framingham, MA, USA) was packed in a constricted GELoader tip (Eppendorf, Hamburg, Germany). The liquid was forced gently into the column using a 10-mL syringe. Thirty microliters of the peptide mixture from the digestion supernatant was diluted with 30 μL of 5 % formic acid, loaded onto the column, and washed with 30 μL of 5 % formic acid. Peptides were eluted with 1.5 μL of 50 % methanol/49 % H2O/1 % formic acid directly into a precoated borosilicate nanoelectrospray needle (New Objective, Woburn, MA, USA) for analysis by tandem mass spectrometry (MS/MS).
MS/MS of peptides generated by in-gel digestion was performed by nano-ESI on a MicroQ-TOF III mass spectrometer (Bruker Daltonics, Germany) at RT. A potential of 1 kV was applied to the precoated borosilicate nanoelectrospray needles (EconoTipTM, New Objective) in the ion source and combined with a nitrogen back-pressure of 0–5 psi to produce a stable flow rate (10–30 nL/min). The cone voltage was 800 V. The quadrupole analyzer was used to select precursor ions for fragmentation in the hexapole collision cell. Product ions were analyzed using an orthogonal TOF analyzer, fitted with a reflector, a micro-channel plate detector, and a time-to-digital converter. The data were processed using a peptide sequence system.
An MS/MS ion search was allocated as the ion search preference in the MASCOT software (Matrix 20 Science, Boston, MA, USA). Peptide fragment files were obtained from the peptide peaks in ESI-MS by ESI-MS/MS. Trypsin was selected as the enzyme with one potentially missed cleavage site. ESI-QTOF was selected as the instrument type. The peptide fragments were searched based on the database using the MASCOT (v2.4, Matrix Science) and FASTA search engine, and the search was limited to Mus musculus taxonomy in NCBInr, UniprotKB/TrEMBL and UniprotKB/Swissprot database. The mass tolerance was set at ± 1 and ± 0.6 Da for the peptides and fragments, respectively. High-scores were defined as those above the default significance threshold in MASCOT (p < 0.05, peptide score, >30).
Western blot analysis was performed as previously described [6, 29]. The antibodies were diluted in 3 % blocking agent: anti-phospho-p38 MAPK Antibody (1:1000, Cell Signaling, Danvers, MA), Anti-PI3K p85 (phosphor Y607) antibody (1:1000, Abcam, Cambridge, UK), anti-phospho-PKA substrate antibody (1:10,000; Cell Signaling Technology, MA, USA), anti-phosphotyrosine antibody (PY20, 1:2,500, Abcam), anti- GAPDH antibody (1:1000, Abcam), anti-UQCRFS1 antibody (1:15,000, LSBio, Inc.), anti- PRDX5 antibody (1:2000, Abcam), anti-GPX4 antibody (1:15,000, Abcam), anti-ACTB antibody (1:500, Abcam), and anti-GSTM5 antibody (1:5,000, Abcam). Anti-α-tubulin mouse antibody (1:1000, Abcam) was used as the loading control for all western blots. The proteins on the membranes were detected with by an enhanced chemiluminescence (ECL) technique using ECL detection reagents.
Pathway Studio (v 9.0, Aridane Genomics, Rockville, MD, USA) program was used to predict the signaling pathways and biological functions of BPA-mediated differentially expressed (>2-fold; p < 0.05) proteins according to previously described procedure [29, 48]. Briefly, differentially expressed proteins were entered into the Pathway Studio in order to determine the significantly matching pathways for each protein (p < 0.05). To fulfill the additional objectives, groups of proteins that significantly related to particular pathways were subject to a MedScan Reader (v5.0) search, and their functional affiliation with disease processes were predicted using Pathway Studio. The signaling pathways and biological functions were confirmed by the PubMed Medline hyperlink that was embedded in each node.
The data were analyzed by one-way ANOVA using SPSS program (v. 18.0, Chicago, IL, USA), and Tukey’s test was used to locate differences. P values of <0.05 were considered statistically significant. All data are expressed as mean ± SEM. The probabilities of the signaling pathways were determined using the Fisher’s exact test (p < 0.05).