Analytical-grade chemicals were utilized. BSA (albumin bovine fraction V, pH 7.0), Immobilon-P membrane, Tween 20, Triton X-100 and Coomassie Brilliant Blue (CBB) R-250 were obtained from Serva (Germany). Gelatin, dithiothreitol (DTT), iodoacetamide, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulphonate (CHAPS) were from Sigma (Prague, Czech Republic); thiourea, urea and IPG buffer (pH 3–10) were purchased from Amersham Biosciences (Uppsala, Sweden). Protein standards were from Bio-Rad (Hercules, CA, USA), chemiluminescent substrate (SuperSignal, Pierce) was obtained from Rockford (USA) and VectaShield mounting medium for fluorescence with DAPI H-1200 from Vector Laboratories, (Burlingame, CA, USA).
Human ejaculated spermatozoa were obtained from the Iscare IVF Ltd., and Pronatal Ltd., Prague. All sperm donors gave their written informed consent with donating the sperm ejaculates for the purposes of the research project. The study was also approved by the institutional review board at the Institute of Biotechnology. The evaluation of semen density, motility and morphology was carried out in compliance with World Health Organization standards . Boar ejaculates were obtained from the Insemination Station Klimetice (Czech Republic). Mouse spermatozoa were obtained from the proximal fifth region of the left and right cauda epididymis of BALB/c mice (AnLab Ltd., Prague, Czech Republic). All procedures were approved by the Committee for Animal Welfare and Protection.
Monoclonal antibodies designated Hs-8 and ACR.2 were prepared in our laboratory by immunization of BALB/c mice with human ejaculated sperms. Hyperimmune spleen cells were fused with Sp2/0 myeloma cells. Positive clones were selected by ELISA with human sperm extracts and by indirect immunofluorescence with human spermatozoa. The immunization procedure and hybridoma technology were described in detail by Peknicova et al. . Monoclonal antibody Hs-8 reacted with intra-acrosomal human and boar sperms . Commercial mouse monoclonal antibody (MoAb) IgG1 to recombinant GAPDHS (ab57062, Abcam, UK) was used in verification tests. Monoclonal anti- progesterone (P4) was raised in our laboratory by immunization of mice with P4-BSA conjugate and selected with P4-OVA conjugate. Goat anti-mouse IgG (γ-chain specific) and IgM (μ-chain specific) antibodies conjugated with fluorescein isothiocyanate (FITC) (Sigma, Prague, Czech Republic), goat anti-mouse IgG-PE antibody (Santa Cruz Biotechnology, USA), horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (GAM/Px) from Bio-Rad (Prague, Czech Republic) were used as secondary antibodies.
Indirect immunofluorescence and co-localization
Human and boar ejaculated sperms and mouse epididymal spermatozoa were suspended into phosphate-buffered saline (PBS, pH 7.4) for 5 min at 37°C and centrifuged for 15 min at 200× g. Next, sperm cells were washed twice and diluted in PBS to a final concentration of 50 × 106 cells ml−1. Small drops of the cell suspension were smeared (10 μl) onto glass slides and the remaining spermatozoa were used for protein extraction.
Dried smears were fixed with acetone (10 min, room temperature), rinsed with PBS, and after blocking with bovine serum albumin (1 h, 2% bovine serum albumin in PBS) incubated for 1 h with Hs-8 MoAb (undiluted hybridoma supernatant, immunoglobulin (Ig) concentration < 20 μg ml−1) and with mouse monoclonal antibody to recombinant GAPDHS for 60 min at 37°C. For appropriate controls, smears were incubated with nonspecific monoclonal antibody, with the supernatant of myeloma cells, and with the FITC-conjugate only. After washes with PBS, the smears were incubated with secondary antibodies. In case of MoAb Hs-8, the secondary FITC-conjugated goat anti-mouse IgM (diluted 1:128 in PBS) was used and for anti-GAPDHS, FITC-conjugated goat anti-mouse IgG (diluted 1:64 in PBS) was used and incubated for 60 min at 37°C, washed in PBS, rinsed with distilled water and mounted in Vectashield medium. For double immunolabelling experiments, glass slides with the sperm smears were fixed and blocked as described above and incubated with MoAb Hs-8 and FITC-conjugated anti-IgM secondary antibody. The same slides were incubated subsequently with the commercial anti-GAPDHS antibody and goat anti-mouse IgG-PE secondary antibody.
Samples (200 sperm cells per slide) were evaluated and viewed with a Nikon Eclipse E400 fluorescent microscope equipped with 40x Nikon Plan 40/0.65 lenses and photographed with a CCD camera VDS1300 (Vosskühler, Osnabrück, Germany) with the aid of the NIS elements AR imaging software (Laboratory Imaging, Prague, Czech Republic).
Human fresh ejaculated sperm was separated by SupraSperm System (ORIGIO, Denmark), washed three times in PBS and fixed on ice for 30 min at 0°C in 3% paraformaldehyde and 0.1% glutaraldehyde in Sörensen buffer (SB; 0.1 M sodium/potassium phosphate buffer, pH 7.3), washed twice with SB (10 min each). Cells were then dehydrated in a series of ethanol solutions with increasing concentration of ethanol. Ethanol was then replaced in two steps by LR White resin (Polysciences Inc., Warrington, USA), and the resin was polymerized for two days at +4°C under UV light. After cutting 80 nm sections, nonspecific labelling was blocked by preincubation with 10% normal goat serum (British BioCell International Ltd., Cardiff, UK), 1% BSA and 0.1% Tween 20 in PBS for 30 min at room temperature (RT). For double immunogold labelling experiments, the sections were simultaneously incubated with Hs-8 and commercial anti-GAPDHS primary antibodies, washed three times in PBT (0.005% Tween 20 in PBS), and then incubated with 6 nm gold-conjugated Goat Anti-Mouse IgG (Fcγ fragment specific) and 12 nm gold-conjugated Goat Anti-Mouse IgM (μ chain specific) secondary antibodies (Jackson Immuno Research Laboratories, inc., USA), washed again twice in PBT, then twice in bi-distilled water, and air dried. Finally, sections were contrasted with a saturated solution of uranyl acetate in water (4 min) and observed in electron microscope Morgagni 268 (FEI, Czech Republic) operated at 80 kV. Control incubations without primary antibodies proved that the signal was highly specific and that there was no cross-reactivity in case of multiple labelling.
Isoelectric focusing, SDS-PAGE and Western blotting
Washed spermatozoa were diluted in PBS, centrifuged at 10000x g for 5 min, and extraction buffer was added to the sperm pellet (100 μl of the extraction buffer per 10 × 107 cells): SDS (2% m/v SDS (sodium dodecyl sulphate), 1% v/v glycerol, 50 mM Tris buffer titrated with HCl to pH 6.8) or RHB (rehydration buffer: 7 M urea, 2 M thiourea, 4% CHAPS,1% Triton X-100, 20 mM Tris). SDS extracts were vortexed, boiled in water bath for 3 min, cooled to 4°C and centrifuged (23,000x g, 5 min, 4°C). RHB extracts were incubated for 1 h at RT and centrifuged (23,000x g, 5 min, RT). The solubilized samples were divided into aliquots and stored at −80°C for electrophoresis and subsequent analysis. The pure GAPDHS recombinant protein (PO1) from Abnova (Oxford, UK) was directly used for the analysis.
The sperm samples were mixed with RHB, 2% (v/v) IPG buffer (3–10), 1% DTT and 0.005% bromophenol blue (added to the final concentration) and incubated for 1 h at RT. The solubilized proteins (200 μg of proteins in total volume 180 μl) were placed onto 7-cm, pI range 3–10, linear strips and rehydrated overnight (according to manufacturer’s instructions). Strips were focused at RT. For 2D electrophoresis, 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was used.
SDS-PAGE was carried out in 12% slab gels. The protein samples were mixed with reducing SDS sample buffer (50 mM Tris buffer titrated with HCl to pH 6.8, 1% v/v glycerol, 2% m/v SDS, 5% v/v 2-mercaptoethanol, 0.002% m/v bromophenol blue) and boiled for 3 min. Samples of proteins of the sperm extract (total quantity 25 μg) and recombinant protein GAPDHS (5 μg) were applied to the wells. Electrophoretic separation was carried out at constant current 16 mA for each gel in Tris-glycine electrophoretic buffer, pH 8.3 (25 mM Tris, 192 mM glycine), with 0.1% m/v SDS at 4°C. The relative molecular masses of the separated proteins were estimated using prestained Precision Plus Protein Standards run in parallel.
Further, the proteins were transferred onto Immobilon-P membrane for immunodetection. Electroblotting was carried out for 1.5 h at 500 mA and 4°C in TRIS-glycine buffer (pH 9.6) with 20% (v/v) methanol. The membrane was blocked with 5% (w/v) gelatin in PBS-T (0.05% Tween 20 in PBS) at 4°C overnight. After washing with PBS-T, the membrane was incubated with supernatant MoAb Hs-8 (1:15 dilution in 1% gelatin-PBS-T) or anti-GAPDHS MoAb (0.1 μg/μl) at RT for 1 h. Following a washing step, incubation with (GAM/Px) (diluted 1:3000 in 1% gelatin-PBS-T) was peformed for 1 h at RT. After washing, the membrane was developed in the dark with chemiluminescent substrate (SuperSignal) to visualize the corresponding bands.
CBB staining and sequence analysis
Gels intended for mass spectrometric analysis were stained with Coomassie Brilliant Blue (CBB) for visualization of all separated proteins. After SDS-PAGE, the gels were incubated at RT in a solution containing CBB (0.25% CBB R-250, 7% CH3COOH, 50% ethanol) for 1 h. After incubation with CBB, the gels were destained in 35% ethanol with 10% CH3COOH until the background disappeared and the separated proteins were clearly visible.
The mass of individual peptides obtained after tryptic digestion of Hs-8-detected protein was determined by the MALDI method. Mass spectra of peptides were measured using a MALDI-Time-of-Flight (MALDI-TOF) mass spectrometer, a peptide map was established and mass spectra were searched against the database using Profound software. Mass spectrometer BIFLEX II (Bruker-Franzen, Bremen, Germany) was equipped with a nitrogen laser (337 nm) and a gridless delayed extraction ion source. Ion acceleration voltage was 19 kV and the reflectron voltage was set to 20 kV. The spectrum was calibrated internally using the monoisotopic [M+ H]+ ions of trypsin autoproteolytic products. A saturated solution of α-cyano-4-hydroxy-cinnamic acid in 50% ACN/0.2% trifluoroacetic acid was used as a MALDI matrix. One microliter of matrix solution was mixed with 1 ml of the sample on the target and the droplet was allowed to dry at ambient temperature.
Isolation and culture of porcine oocytes
Porcine oocytes were recovered from fresh ovaries about 3 h after slaughter by puncturing and aspirating of 3 to 5 mm follicles. Oocytes were collected in BSA-PBS medium, placed in MPM (modified Parker medium) under paraffin oil, and incubated for 48 h at 37°C under 5% CO2 to complete maturation. After culturing, oocytes were transferred in a number of 40 to 50 pieces in 0.5 ml of 3 M DMSO in culture medium for 10 min at 4°C, and the tubes with oocytes were placed into liquid nitrogen vapours until use. On the day of binding assay, the tubes with frozen oocytes were thawed at 37°C, 5% CO2. Then the oocytes were washed three times in BSA-PBS, transferred to the drops of medium and overlaid with paraffin oil.
In vitro sperm-zona pellucida-binding assay
We examined the effect of monoclonal antibodies (Hs-8 and anti GAPDHS) during sperm/oocyte co-incubation. The oocytes (10–20) were transferred to 120 μl droplets containing monoclonal antibodies Hs-8 and GAPDHS, respectively, or monoclonal antibody against progesterone, ACR.2, or MPM medium only (control groups). Capacitated spermatozoa (50 μl) were added to the control and experimental groups of oocytes. After 30 min, co-incubation was stopped by adding 50 μl of 10% NaN3. Oocytes with bound spermatozoa were washed twice and fixed in 2.5% paraformaldehyde. After washing, the fixed oocytes were stained with Hoechst 33342 (Sigma, Prague) solution (0.3 mg Hoechst /10 ml BSA-PBS), rinsed twice and mounted in a very small droplet of 50% glycerol in PBS (pH 9.0), and Hoechst-labelled spermatozoa attached per oocyte were counted under a fluorescent microscope.
Experimental data were analysed and plotted using STATISTICA 6.0. and GraphPad Prism 5.04. Twenty oocytes were analysed per each control and experimental group (N = 20) in each experiment; the total number of analysed oocytes was 100. Sperm samples were obtained from two boars. The differences in the number of bound sperm cells among control and experimental groups were analysed by Kruskal-Wallis test, the post-hoc analysis was performed by Dunn’s multiple comparison test. The p value equal to or lower than 0.05 was considered to be significant, *p value ≤0.05 (**p ≤0.01 and ***p ≤0.001).