Subjects
Fresh semen samples were obtained from 245 men presenting for semen evaluation at our hospital’s Assisted Reproduction Unit (Lille University Medical Center, Lille, France). All samples were evaluated with regard to (i) conventional parameters (semen volume, sperm concentration, sperm count per ejaculate, motility, and vitality), according to the WHO guidelines [4], and (ii) sperm morphology, according to David’s modified classification [7].
Extraction of sperm proteins
Each fresh semen sample was prepared for protein analysis as described previously [6]. Seminal plasma, immature germ cells, and non-sperm cells were removed by centrifugation (350×g, for 20 min at room temperature) through 50% PureSperm (Nidacon, Mölndal, Sweden)/FertiCult medium (FertiPro N.V., Beernem, Belgium). The sperm pellet was washed once by resuspension in 1 mL of Tris-buffered saline (10 mM Tris-HCl pH 7.6, 100 mM NaCl) and further centrifugation (350×g, for 20 min at room temperature). The sperm pellet was then resuspended in 500 μL of lysis buffer containing 20 mM Tris, 2% SDS and 1% Nuclease Mix (GE Healthcare Life Sciences, Piscataway, NJ, USA). Lysates were sonicated on ice (40 Hz, 60 pulses) and centrifuged (14,000×g, for 20 min at 4 °C). The supernatants were recovered, and the pellets were discarded. The protein concentration was assayed according to Bradford’s method (BioRad, Hercules, CA, USA) by following the kit manufacturer’s instructions. Protein lysates were stored at − 80 °C prior to analysis.
One-dimensional PAGE
Fifteen μg of extracted proteins from each prepared sperm sample were diluted in lithium dodecyl sulfate buffer (LDS 2×, Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions. Protein homogenates were boiled for 10 min, quickly centrifuged at 500×g (using a bench centrifuge), and loaded onto a 4–12% polyacrylamide gel (NuPage® Bis-Tris PreCast 12 wells, Life Technologies). Molecular weight markers (Novex and Magic Marks, Life Technologies) were loaded into the first well. Electrophoresis was performed at a constant voltage of 200 V for 1 h. Polyacrylamide gels were stained with Coomassie Blue reagent (0.1% Blue G250 BioRad, Hercules, CA, USA, 50% ethanol (v/v), 10% acetic acid (v/v) and H20). The protein bands were visualized after destaining with 7% (v/v) acetic acid and 10% ethanol in H2O. The gels were digitized with a Perfection V750 Pro scanner (Epson France, Levallois-Perret, France) and Photoshop Element 9 software (Adobe, Paris, France). To evaluate a potential effect of the protein concentration, larger amounts of total protein (i.e. 20, 25 and 30 μg) from sperm samples were loaded onto 4–12% acrylamide gels and stained with Coomassie Blue reagent. To inhibit seminal proteases, sperm samples were treated with a protease inhibitor cocktail (cOmplete™, Roche Diagnostics GmbH, Mannheim, Germany), according to the manufacturer’s instructions. One-dimensional PAGE was performed twice on each independent sample. The densitometric intensity of the molecular weight bands between 110 and 80 kDa was quantified using ImageJ software (version 1.47c, NIH). Data were expressed in arbitrary units.
Protein identification
Coomassie-Blue-stained protein bands were trypsinized, and nanoseparation was performed with a U3000 nano-high-performance liquid chromatography (LC) system (Dionex-LC-Packings, Sunnyvale, CA, USA). After a pre-concentration step (using a C18 cartridge, 300 μm, 1 mm), peptide samples were separated on a Pepmap C18 column (75 μm, 15 cm) by applying an acetonitrile/0.1% TFA gradient: 0% acetonitrile for 3 min, 0 to 15% for 7 min, 15 to 65% for 42 min, 65 to 90% for 5 min and, lastly, 90% acetonitrile for 6 min. The flow rate was set to 300 nL/min, and the fractions were automatically collected every 30 s (giving 110 fractions in total) on an AnchorChip™ MALDI target by using a Proteineer™ FC fraction collector (Bruker Daltonics, Bremen, Germany). Two μl of α-cyano-4-hydroxycinnamic acid matrix (0.3 mg/mL in acetone:ethanol; 0.1% TFA-acidified water, 3:6:1 v/v/v) were added during the collection step. Mass spectrometry (MS) and MS/MS measurements were performed off-line using an Ultraflex™ II time-of-flight (TOF)/TOF mass spectrometer (Bruker Daltonics). The spectrometer’s parameters are given in the following section. Peptide fragmentation was driven by Warp LC™ software (Bruker Daltonics), using the following parameters: signal-to-noise ratio > 15, more than three MS/MS steps per fraction if an MS signal was available, a 0.15 Da tolerance for peak merge, and the elimination of peaks that appeared in more than 35% of the fractions.
Measurements on an Ultraflex™ II TOF/TOF instrument were performed in automatic mode (using FlexControl™ 3.0 software, Bruker Daltonics) for molecular mass determination, in reflectron mode for MALDI-TOF peptide mass fingerprinting (PMF), and in “LIFT” mode for MALDI-TOF/TOF peptide fragmentation fingerprinting (PFF). External calibration over a 1000–3200 mass range was performed using [M + H] + monoisotopic ions of bradykinin 1–7, angiotensin I, angiotensin II, substance P, bombesin and adrenocorticotropic hormone (clip 1–17 and clip 18–39), using a peptide calibration standard kit (Bruker Daltonics). Briefly, MS spectra were obtained with an accelerating voltage of 25 kV, a reflector voltage of 26.3 kV, and a pulsed ion extraction of 160 ns. Each individual spectrum was produced by accumulating data from 800 laser shots. A maximum of five precursor ions per sample was chosen for LIFT-TOF/TOF MS/MS analysis. Precursor ions were accelerated to 8 kV and selected in a timed ion gate. Metastable ions generated by laser-induced decomposition were further accelerated by applying 19 kV in the LIFT cell, and their masses were measured in reflectron mode. Peak lists from MS and MS/MS spectra were generated using Flexanalysis™ 3.3 software (Bruker Daltonics). Combined PMF and PFF searches in the UnitProt 2012_06_database (ProteinScape™ 2.1, Bruker Daltonics) were performed with Mascot 2.3.02 (Matrix Science Ltd., London, UK). Taxonomic analysis was restricted to human sequences. A mass tolerance of 75 ppm and 1 missing cleavage site (for PMF) and an MS/MS tolerance of 0.5 Da and 1 missing cleavage site (for MS/MS) were allowed. Carbamidomethylation of cysteine and oxidation of methionine residues were considered to be fixed and variable modifications, respectively. The relevance of protein identities was judged according to the probability-based molecular weight search score; the threshold for statistical significance was set to p < 0.05.
Statistical analysis
Statistical analysis was performed using SAS software (version 9.3, SAS Institute Inc., Cary, NC, USA). Quantitative data were presented as the median [interquartile range (IQR)]. The normality of the data distribution was checked with a Shapiro-Wilk test. Quantitative semen variables were compared by applying Student’s t test. Quantitative densitometry values for the gels were compared in a Kruskal-Wallis test. Potential associations between the densitometry values and the semen variables were analyzed using Spearman’s test. The threshold for statistical significance was set to p < 0.05.