Phosphoproteomic and Functional Analyses Reveal Sperm-specific Protein Changes Downstream of Kappa Opioid Receptor in Human Spermatozoa
2019; Elsevier BV; Volume: 18; Linguagem: Inglês
10.1074/mcp.ra118.001133
ISSN1535-9484
AutoresItziar Urizar‐Arenaza, Nerea Osinalde, Vyacheslav Akimov, Michele Puglia, Luz Candenas, Francisco M. Pinto, Iraia Muñoa-Hoyos, Marta Gianzo Citores, Roberto Matorras, Jon Irazusta, Blagoy Blagoev, Nerea Subirán, Irina Kratchmarova,
Tópico(s)Hypothalamic control of reproductive hormones
ResumoG-protein coupled receptors (GPCRs) belong to the seven transmembrane receptor superfamily that transduce signals via G proteins in response to external stimuli to initiate different intracellular signaling pathways which culminate in specific cellular responses. The expression of diverse GPCRs at the plasma membrane of human spermatozoa suggests their involvement in the regulation of sperm fertility. However, the signaling events downstream of many GPCRs in spermatozoa remain uncharacterized. Here, we selected the kappa-opioid receptor (KOR) as a study model and applied phosphoproteomic approach based on TMT labeling and LC-MS/MS analyses. Quantitative coverage of more than 5000 proteins with over 3500 phosphorylation sites revealed changes in the phosphorylation levels of sperm-specific proteins involved in the regulation of the sperm fertility in response to a specific agonist of KOR, U50488H. Further functional studies indicate that KOR could be involved in the regulation of sperm fertile capacity by modulation of calcium channels. Our findings suggest that human spermatozoa possess unique features in the molecular mechanisms downstream of GPCRs which could be key regulators of sperm fertility and improved knowledge of these specific processes may contribute to the development of useful biochemical tools for diagnosis and treatment of male infertility. G-protein coupled receptors (GPCRs) belong to the seven transmembrane receptor superfamily that transduce signals via G proteins in response to external stimuli to initiate different intracellular signaling pathways which culminate in specific cellular responses. The expression of diverse GPCRs at the plasma membrane of human spermatozoa suggests their involvement in the regulation of sperm fertility. However, the signaling events downstream of many GPCRs in spermatozoa remain uncharacterized. Here, we selected the kappa-opioid receptor (KOR) as a study model and applied phosphoproteomic approach based on TMT labeling and LC-MS/MS analyses. Quantitative coverage of more than 5000 proteins with over 3500 phosphorylation sites revealed changes in the phosphorylation levels of sperm-specific proteins involved in the regulation of the sperm fertility in response to a specific agonist of KOR, U50488H. Further functional studies indicate that KOR could be involved in the regulation of sperm fertile capacity by modulation of calcium channels. Our findings suggest that human spermatozoa possess unique features in the molecular mechanisms downstream of GPCRs which could be key regulators of sperm fertility and improved knowledge of these specific processes may contribute to the development of useful biochemical tools for diagnosis and treatment of male infertility. Ejaculated mammalian sperm cells are immature and infertile and must undergo many physiological and biochemical modifications to become fertilization competent. These processes as the acquisition of sperm motility, capacitation, hyperactivation and acrosome reaction occur sequentially inside the female reproductive tract and are considered key functions in the control of the reproduction as well as essential for spermatozoa to become fertile (1.Visconti P.E. Westbrook V.A. Chertihin O. Demarco I. Sleight S. Diekman A.B. Novel signaling pathways involved in sperm acquisition of fertilizing capacity.J. Reprod. Immunol. 2002; 53: 133-150Crossref PubMed Scopus (289) Google Scholar). It is well documented that ionotropic modulation through rapid responses are the main regulators of sperm physiology (2.Hille B. Ionic channels of excitable membranes. 2nd edition. Sinauer Associates Inc, Sunderland, MA1992Google Scholar). However, the presence of a high number of G-protein coupled receptors (GPCRs) 1The abbreviations used are: GPCR, G-protein coupled receptor; KOR, Kappa opioid receptor; SACY, soluble adenylate cyclase; tmAC, transmembrane adenylate cyclase; cAMP, cyclic AMP; PKA, protein kinase A; PKC, protein kinase C; MAPK, mitogen activated protein kinase; TMT, Tandem mass-tag; LC-MS/MS, Liquid chromatography tandem mass spectrometry; FDR, False discovery rate.1The abbreviations used are: GPCR, G-protein coupled receptor; KOR, Kappa opioid receptor; SACY, soluble adenylate cyclase; tmAC, transmembrane adenylate cyclase; cAMP, cyclic AMP; PKA, protein kinase A; PKC, protein kinase C; MAPK, mitogen activated protein kinase; TMT, Tandem mass-tag; LC-MS/MS, Liquid chromatography tandem mass spectrometry; FDR, False discovery rate. described over the last decade in human spermatozoa, suggests that metabotropic mechanisms could also be important in the acquisition of the sperm fertilizing capacity (3.Spehr M. Schwane K. Riffell J.A. Zimmer R.K. Hatt H. Odorant receptors and olfactory-like signaling mechanisms in mammalian sperm.Mol. Cell. Endocrinol. 2006; 250: 128-136Crossref PubMed Scopus (80) Google Scholar). The GPCRs are seven transmembrane receptors that represent approximately the 1% of the human genome and are one of the best therapeutic targets (4.Nambi P. Aiyar N. G protein-coupled receptors.Drug Discovery. 2003; 1: 305-310Google Scholar). In somatic cells, the canonical (G-protein dependent pathways) and noncanonical (G-protein independent pathways) are the major signaling pathways initiated downstream GPCRs and have an important role in the regulation of basic cellular activities as well as in the coordination of cell actions. Over the last 20 years, several GPCRs have been described in human spermatozoa providing clear evidences of their involvement in the regulation of sperm fertility (5.Köhn F.M. Dammshäuser I. Neukamm C. Renneberg H. Siems W.E. Schill W.B. Aumüller G. Ultrastructural localization of angiotensin-converting enzyme in ejaculated human spermatozoa.Hum. Reprod. 1998; 13: 604-610Crossref PubMed Scopus (52) Google Scholar, 6.Schaefer M. Hofmann T. Schultz G. Gudermann T. A new prostaglandin E receptor mediates calcium influx and acrosome reaction in human spermatozoa.Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 3008-3013Crossref PubMed Scopus (81) Google Scholar, 7.Jiménez-Trejo F. Tapia-Rodríguez M. Cerbón M. Kuhn D.M. Manjarrez-Gutiérrez G. Mendoza-Rodríguez A. Picazo O. Evidence of 5-HT components in human sperm: implications for protein tyrosine phosphorylation and the physiology of motility.Reproduction. 2012; 144: 677-685Crossref PubMed Scopus (38) Google Scholar, 8.Rossato M. Popa F.I. Ferigo M. Clari G. Foresta C. Human sperm express cannabinoid receptor Cb1, the activation of which inhibits motility, acrosome reaction, and mitochondrial function.J. Clin. Endocrinol. Metab. 2005; 90: 984-991Crossref PubMed Scopus (193) Google Scholar, 9.Pinto F.M. Cejudo-Román A. Ravina C.G. Fernández-Sánchez M. Martín-Lozano D. Illanes M. Tena-Sempere M. Candenas M.L. Characterization of the kisspeptin system in human spermatozoa.Int. J. Androl. 2012; 35: 63-73Crossref PubMed Scopus (67) Google Scholar). Further, different components from the G-protein dependent transduction pathways such as the cAMP-dependent and the Ca2+/PKC signaling cascades have been described to influence aspects of sperm function such as sperm motility, capacitation and acrosome reaction (10.Hess K.C. Jones B.H. Marquez B. Chen Y. Ord T.S. Kamenetsky M. Miyamoto C. Zippin J.H. Kopf G.S. Suarez S.S. Levin L.R. Williams C.J. Buck J. Moss S.B. The "soluble" adenylyl cyclase in sperm mediates multiple signaling events required for fertilization.Dev. Cell. 2005; 9: 249-259Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). In addition, in respect to the noncanonical signaling pathway, β-arrestin that promotes desensitization and internalization of the GPCRs via a G-protein independent signaling pathway, has been demonstrated to act as a signal transducer, capable of modulating the sperm motility and acrosome reaction (11.Almog T. Lazar S. Reiss N. Etkovitz N. Milch E. Rahamim N. Dobkin-Bekman M. Rotem R. Kalina M. Ramon J. Raziel A. Brietbart H. Seger R. Naor Z. Identification of extracellular signal-regulated kinase 1/2 and p38 MAPK as regulators of human sperm motility and acrosome reaction and as predictors of poor spermatozoan quality.J. Biol. Chem. 2008; 283: 14479-14489Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Moreover, because spermatozoa are transcriptionally and translationally silent cells, it has been suggested that they may possess unique, sperm-specific signaling pathways (10.Hess K.C. Jones B.H. Marquez B. Chen Y. Ord T.S. Kamenetsky M. Miyamoto C. Zippin J.H. Kopf G.S. Suarez S.S. Levin L.R. Williams C.J. Buck J. Moss S.B. The "soluble" adenylyl cyclase in sperm mediates multiple signaling events required for fertilization.Dev. Cell. 2005; 9: 249-259Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 11.Almog T. Lazar S. Reiss N. Etkovitz N. Milch E. Rahamim N. Dobkin-Bekman M. Rotem R. Kalina M. Ramon J. Raziel A. Brietbart H. Seger R. Naor Z. Identification of extracellular signal-regulated kinase 1/2 and p38 MAPK as regulators of human sperm motility and acrosome reaction and as predictors of poor spermatozoan quality.J. Biol. Chem. 2008; 283: 14479-14489Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). In 2006, although we described for the first time the presence of functional Mu-, delta- and kappa- opioid receptors (MOR, DOR and KOR, respectively) in human spermatozoa (12.Agirregoitia E. Valdivia A. Carracedo A. Casis L. Gil J. Subiran N. Ochoa C. Irazusta J. Expression and localization of δ-, κ-, and μ-opioid receptors in human spermatozoa and implications for sperm motility.J. Clin. Endocrinol. Metab. 2006; 91: 4969-4975Crossref PubMed Scopus (73) Google Scholar), the signaling events downstream of these receptors remain uncharacterized. Taking all this into account, the aim of this study was to elucidate the existence of sperm-specific molecular mechanisms of GPCRs signaling in human spermatozoa. For this purpose we combined for the first time phosphoproteomic approaches together with functional analyses in human spermatozoa, by using the kappa-opioid receptor as a study model. Ethical approval for this study was obtained from the Ethics Committee of the University of the Basque Country (CEISH-UPV/EHU (M10/2016/254)). Freshly ejaculated semen was collected from patients undergoing routine semen analysis at the Cruces University Hospital (Bilbao, Spain). The donors had normal sperm parameters according to World Health Organization standards (13.World Health Organization WHO laboratory manual for the examination and processing of human semen. Fifth Edition. WHO Press, World Health Organization, Geneva, Switzerland2010Google Scholar). Semen samples were obtained by masturbation after 3–4 days of sexual abstinence and immediately processed on liquefaction (at 37 °C for 30 min). Spermatozoa were capacitated by the swim-up procedure and resuspended in G-IVF (Vitrolife, Goteborg, Sweden) supplemented with 1% bovine serum albumin for 3 h at 37 °C under 5% CO2. Isolated spermatozoa were treated at 37 °C and 5% CO2 in G-IVF culture media. The spermatozoa were treated with 1 μm U50488H (the specific agonist of the receptor) (Sigma-Aldrich, Madrid, Spain) for 1 and 60 min independently for proteomic and functional analyses. For functional analyses the samples were co- incubated with U50488H (Sigma-Aldrich) and the different activators and inhibitors of different signaling pathways. To study the calcium signaling pathway we used U73122 (3 μm) (Sigma-Aldrich), a phospholipase C inhibitor; Mibefradil (30 μm) (Tocris Biosciences, Bristol, UK), a calcium channel activator and NNC55–0395 (10 μm)(Sigma-Aldrich), a CatSper specific calcium channel inhibitor. To study the cAMP/PKA signaling pathway we used the SQ2336 (200 μm) (Sigma-Aldrich), the transmembrane adenylate cyclase (tmAC) inhibitor; Forskolin (50 μm) (Sigma-Aldrich), the tmAC activator and the HCO3− (50 mm)(Sigma-Aldrich), the SACY activator. To study the MAPK signaling pathway we used the BARK1 (126 μm)(Calbiochem, Darmstadt, Germany), the GRK inhibitor and IBMX (0.5 mm)(Sigma-Aldrich), the phosphodiesterases inhibitor. Human spermatozoa that had normal parameters were treated with 1 μm U50488H (the specific agonist of the receptor) for 1 and 60 min independently for proteomic and functional analyses. For proteomic analyses, we adopted a Tandem Mass-Tag (TMT) 6-plex isotopic labeling strategy followed by phosphopeptide enrichment by consecutive incubations with titanium dioxide beads (TiO2) and generated samples for LC-MS/MS. The experiment was performed in three biological replicas. Peptide and protein searches were performed using the Andromeda search engine (integrated in MaxQuant, version 1.5.3.30) at a FDR threshold of 1%. Perseus software (v.1.6.0.7) was employed for the calculation of the statistical significance (two-sample student′s t, test) and fold changes between U50488H-treated and Control samples. We considered as U50488H-dependent phosphosites the ones that were consistently regulated presenting a 1.5-fold change (U50488H/Ctr > 1.5 or U50488H/Ctr < 0.67 and p, value < 0.05) in the three replicas for each time point and protein fraction. The followed proteomic strategy is summarized in Fig. 1A,. Three biological replicas of each untreated (Ctrl) and treated spermatozoa (U50488H) were used for 1 and 60 min. For soluble and insoluble protein extraction, treated sperm cells were lysed using ice-cold RIPA buffer (50 mm Tris-HCl pH 7.5, 150 mm NaCl, 1% NP-40, 1 mm EDTA, 0.25% sodium deoxycholate, 1 mm sodium pervanadate, 5 mm beta-glycerophosphate, 5 mm NaF, complete protease inhibitor mixture. (Roche Basel, Switzerland). After protein homogenization and sonication (20% amplitude, 10 pulses, three times), proteins were separated in soluble and insoluble fractions after 15 min centrifugation at 13,000 × g, 4 °C. To solubilize the insoluble fraction, we resuspended proteins into 8 m Guanidinium chloride and sonicated them (20% amplitude, 10 pulses). Both soluble and insoluble protein fractions were reduced and alkylated using 2 mm dithiothreitol and 11 mm chloroacetamide respectively. Then the soluble proteins were precipitated using acetone to further resuspend them in urea buffer (8 m urea, 10 mm TrisHCl pH 7,5) and estimate the protein abundance by bicinchoninic acid (BCA) method (Pierce, Thermo Fisher). After adjusting all the samples to 1:1:1:1:1:1 in each time point and protein fraction (soluble or insoluble), the samples were subjected to in-solution digestion using LysC (4 h) and Trypsin (overnight). Proteolytic digestion products were desalted on a Sep-Pak C18 cartridge (Waters, Milford, MA) for further sample processing. Digested peptides were labeled by Tandem Mass-Tag (TMT) 6-plex isotopic label reagent set (Thermo Scientific, Rockford, IL) to study the total proteome. After the peptide drying, each condition was resuspended in 100 mm Triethylammonium Bicarbonate Buffer (TEAB) (Sigma-Aldrich). Peptides were incubated with each label for 1 h at room temperature (RT) and reactions were quenched using 8 μl of 11% Lysine, following the manufacturer′s recommendations. After further 15 min incubation, the peptide solutions were acidified 1:10 v/v with 10% trifluoroacetic acid (TFA, Sigma-Aldrich). Then we mixed the independent samples in 1:1:1:1:1:1 proportion and fractionated the samples into 17 different fractions following the stepwise high- pH reversed phase fractionation protocol. Each fraction was loaded onto a C18 Stage Tip (made in house using Empore disc -C18 Agilent Life Science, Santa Clara, CA) for further proteomic analyses. A part of the digested peptides (1/10) were TMT labeled and then fractionated into 17 different fractions following the Stepwise high-pH reversed phase fractionation protocol. The samples were adjusted to pH 10 with 10 mm ammonium hydroxide and transferred to a home-made tip containing ReproSil-Pur, 1,9 μm, 120A° beads (Dr. Maisch, Ammerbuch-Entringen, Germany). Sequential elutions were made using 1.75, 3.5, 5.25, 7, 8.75, 10.5, 12.5, 14, 15.5, 17.5, 21, 24.5, 28, 31.5, 35, 50, and 70% ACN. Each fraction was loaded onto C18 StageTips. Peptides were eluted with 60% ACN to further continue with mass spectrometry analysis. The 9/10s of the peptides were enriched in phosphopeptides by consecutive incubations with titanium dioxide beads (TiO2). After all the samples were brought to 60% acetonitrile (ACN) and 1% TFA, the beads were added and incubated for 15 min rotating. Beads were then transferred to home-made C8 Stage Tips (Empore disc -C8 Agilent Life Science) and washed with 60% ACN/1% TFA. Phosphopeptides were eluted with 5% ammonia and 25% ACN/10% ammonia and loaded onto C18 StageTips. Peptides were eluted with 60% ACN and subjected to TMT labeling as previously described. Finally, the samples were analyzed by mass-spectrometry. Acidified peptide mixtures were separated by online C18- reverse-phase nanoscale liquid chromatography and analyzed by tandem mass spectrometry (LC-MS/MS). MS analysis was performed on an Q-Exactive HF mass spectrometer (Thermo Scientific, Bremen, Germany) connected to an EASY-nanoLC 1000 System (Thermo) using a nanoelectrospray ion source (Proxeon Biosystems, Odense, DK). Survey full-scan MS spectra (m,/z, range, 300–1700; resolution 60,000 at m,/z, 400) were acquired in the Orbitrap followed by the fragmentation of the twelve most intense multiply charged ions. Ions selected for MS/MS were placed on a dynamic exclusion list for 45 s. To improve mass accuracy, internal real time lock mass calibration was enabled. Additional mass spectrometric parameters included a spray voltage of 2.3 kV, no sheath and auxiliary gas flow, and the temperature of the heated capillary was 275 °C. All raw files were searched against combined human database 2015.08 UniProt (with 42,122 sequence entries) and TrEMBL (with 49496 sequence entries) using MaxQuant platform version 1.5.3.30 with an Andromeda search engine. Precursor and fragment tolerances were 4.5 and 20 ppm, respectively. A peak list was generated using the Quant element of MaxQuant using the following parameters. A maximum of 2 missed cleavages was allowed and enzyme specificity was set to trypsin. In addition, carbamidomethyl (C) was chosen as fixed modification and variable modifications included oxidation (M), deamidation (NQ) and Phospho_STY (STY). The peptide and protein FDR 0.01; site FDR 0.01; max. peptide PEP, 1; min. peptide length 7; min unique peptides and peptides, 1. For protein quantitation, only unmodified peptides and peptides modified by acetyl (protein N terminus), oxidation (Met) and deamidation (NQ) were used. According to the protein group assignment done by MaxQuant, the identified proteins were determined after removing the contaminants, reverses and those proteins only identified by site. Moreover, we considered those proteins with ≥ 2 identified peptides and ≥1 unique peptides. On the other hand, the phosphopeptide data was filtered by FDR < 1% and only the phosphosites displaying a localization probability above 0.75 were considered as confident phosphorylated sites (Class I sites). The Perseus software (v.1.6.0.7) was employed for the calculation of the statistical significance and Fold changes between U50488H-treated and Control samples. To identify the U50488H-dependent phosphosites only the data of the quantified phosphosites and proteins were considered and used further. First, the data was normalized to correct the labeling variability and then it was normalized depending on phosphosite reporter ion intensities/protein reporter ion intensities. We considered as U50488H-dependent phosphosites the ones that were consistently regulated presenting a 1.5-fold change (U50488H/Ctr > 1.5 or U50488H/Ctr < 0.67 and p, value < 0.05) in the three replicas for each time point and protein fraction. The PANTHER (v13.1) functional annotation tool (http://geneontology.org/) was used to detect the overrepresented gene ontology (GO) term "biological process" within the total identified proteins and Class I identified phosphoproteins in human spermatozoa. The Homo Sapiens dataset was used as reference. To construct the signaling pathways regulated by the kappa-opioid receptor, GENEMANIA (http://genemania.org/) the interactive functional association network tool was used. Human spermatozoa were capacitated and adjusted to a concentration of 50 × 106 cells/ml. Motility analysis was conducted by computer-assisted sperm analysis (CASA) (Sperm Class Analyzer, S.C.A., Microptic, Barcelona, Spain) following the WHO guidelines. For that purpose we examined the percent of motile sperm being by the manufacturer as appropriate for human species: progressive motility (rapidly progressive with velocity ≥35 μms/s at 37 °C (grade "a") + slow sluggish progressive with velocity ≥10 μms/s but <35 μms/s (grade "b"), nonprogressive motility with velocity <10 μms/s and immotile motility. Moreover, the following kinematics parameters were also measured: curvilinear velocity (VCL, μm/s); straight-line velocity (VSL, μm/s), average-path velocity (VAP, μm/s); amplitude of lateral head displacement (ALH, μm); linearity of progression (LIN = VSL/VCL × 100); straightness (STR = VSL/VAP × 100); motility and hyperactivated motility. The percentage of hyperactive cells was defined following parameters above (11.Almog T. Lazar S. Reiss N. Etkovitz N. Milch E. Rahamim N. Dobkin-Bekman M. Rotem R. Kalina M. Ramon J. Raziel A. Brietbart H. Seger R. Naor Z. Identification of extracellular signal-regulated kinase 1/2 and p38 MAPK as regulators of human sperm motility and acrosome reaction and as predictors of poor spermatozoan quality.J. Biol. Chem. 2008; 283: 14479-14489Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar): VCL 100 m/s, LIN 60% and ALH 5 μm. To investigate the effects of drugs, sperm samples were divided in several aliquots and treated with a single concentration of U50488H (1 μm) (Sigma). Sperm motility was measured 5 min before U50488H addition (initial value) and after a contact time of 1 and 60 min. Additional experiments were performed in similar conditions to evaluate the effects of Mibefradil (30 μm), U73122 (3 μm) and NNC55–0395 (10 μm) and their co-incubations with the U50488H agonist. Acrosome reaction was evaluated by using the anti-CD46 antibody by Flow cytometry. The FACScalibur flow cytometer (Becton, Dickinson, San Jose, CA) was used to study the role of the kappa-opioid receptor in human sperm acrosome reaction. Spermatozoa were treated with U50488H for 1 and 60 min, and the different activators and inhibitors of different signaling pathways. The progesterone (10 μm) was used as an internal control for the acrosome reaction. For the staining of the cells the Fluorescein IsoTioCyanate (FITC) antihuman CD46 antibody (BioLegend, CA) (5 μl) was used for 60 min at room temperature as acrosome reaction molecular marker. As indicator of cell viability, we used 0.1 μg/ml Hoechst 33258 for 2 min. Finally, samples were washed twice by centrifugation in PBS at 800 g for 5 min, suspended in PBS and kept in the dark until analysis. Fluorescence data from at least 10.000 alive sperm cells were analyzed and the green fluorescence belonging to spermatozoa was measured. Histograms were analyzed using the Summit v4.3 software (Beckman Coulter, CA). For Western blotting analyses, whole cell extracts (500,000 cells) were diluted in 1× Laemmly sample buffer containing Dithiothreitol (DTT) (%10v/v) and boiled for 5 min. Samples were loaded onto 12% resolving gels and separated by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to polyvinylidene fluoride membranes (PVDF) (Amersham Biosciences Hybond, Sigma) using the Mini Trans-Blot electrophoretic transfer system (Bio-Rad Laboratories, Hercules, CA). Then, membranes were blocked with Blotto (20 mm Tris-HCl, pH 7.5, 0.15 m NaCl, 1%Triton X-100) containing 5% Bovine Serum Albumin (BSA) for 1 h and then incubated with a 1:1000 dilution of the monoclonal mouse 4G10 anti-phosphotyrosine antibody (05–231, Millipore, Darmstadt, Germany) as a molecular marker of the human sperm capacitation (69.Leclerc P. de Lamirande E. Gagnon C. Regulation of protein-tyrosine phosphorylation and human sperm capacitation by reactive oxygen derivatives.Free Radic Biol Med. 1997; 22: 643-656Crossref PubMed Scopus (254) Google Scholar), the polyclonal rabbit anti-Kappa opioid receptor (ABN456, Millipore) (1:500), the polyclonal rabbit anti-phosphorylated protein kinase C substrates (#2261, Cell Signaling, Danvers, MA) (1:750), the polyclonal rabbit anti-phosphorylated protein kinase A substrates (#9624,Cell Signaling) (1:750), the monoclonal mouse anti-phosphorylated MAP kinase substrates (#2325, Cell Signaling) (1:750) and the monoclonal mouse anti-alpha tubulin (T5168, Sigma, 1:4000). After washings (3 × 5 min) in Blotto buffer, the membranes were incubated for 1 h at RT with peroxidase-conjugated goat anti-rabbit (Blotto + %5 BSA 1:1000) (Goat anti-rabbit IgG HRP, sc-2004; Santa Cruz Biotechnology) and donkey anti-mouse IgG antibodies (Blotto+ %5 BSA 1:2000) (Donkey anti-mouse IgG HRP, sc-2314; Santa Cruz Biotechnology). After washing (3 × 5 min) blots were revealed for peroxidase activity by enhanced chemoluminiscence (ChemiDoc XRS detector, Bio-Rad). Results were analyzed by semiquantitative Western blots densitometry analysis using Image J (Image Processing and analysis in Java) software. Isolated human sperm cells were fixed in 4% paraformaldehyde for 10 min, permeabilized in 0.5% Triton X-100 for 10 min and blocked for 30 min with 10% (v/v) fetal bovine serum (FBS) in PBS. After, samples were incubated overnight at 4°C with the polyclonal rabbit anti-Kappa opioid receptor (ABN456, Millipore) (1:500). Secondary antibody incubations included Alexa Fluor 488 donkey anti-rabbit IgG (1:200) (Molecular Probes, OR). At the same time, controls for the specificity of the secondary antisera were performed by omitting the primary antiserum before addition of the second antisera. Nuclei were stained with Hoechst 33258 at 10 μg/ml and slides were assembled with Fluoromont G (Molecular Probes). Finally, the samples were examined using confocal microscopy (Zeiss, Apotome 2, Jena, Germany) at the High Resolution Facility (SGIKER UPV/EHU). The image analysis was conducted using the ImageJ software. Changes in [Ca2+]i were monitored using the Fura-2 as previously described by Cejudo-Roman et al., (15.Cejudo-Roman A. Pinto F.M. Subirán N. Ravina C.G. Fernández-Sánchez M. Pérez-Hernández N. Pérez R. Pacheco A. Irazusta J. Candenas L. The voltage-gated sodium channel Nav1.8 is expressed in human sperm.PLoS ONE. 2013; 8: 1-13Crossref Scopus (21) Google Scholar). Briefly, spermatozoa were adjusted to a concentration of 10 × 106 cell/ml in human serum albumin (HSA) medium. They were then incubated with the acetoxymethyl ester form of Fura-2 (Fura-2/AM 8 × 10−6 m; Molecular Probes) for 60 min at room temperature in the presence of the noncytotoxic detergent pluronic acid (0,1%; Molecular Probes). After loading, the cells were washed and resuspended in G-IVF solution and used within the next 2–7 h. Sperm aliquots (1 ml) were placed in the quartz cuvette of a spectrofluorometer (SLM AMinco-Bowman, Series 2; Microbeam, Barcelona, Spain) and magnetically stirred at 37 °C. The emitted fluorescence was measured at 510 nm. To measure [Ca2+]i, samples were alternatively illuminated with two excitation wavelenghts (340 nm and 380 nm) and the fluorescent light from the two excitation wavelengts was measured by a photomultiplier through a 510 nm filter. After subtracting the autofluorescence signal, obtaining by adding 5 nm MnCl2 at the end of the experiment, the F340/F380 ratio was used an indicator of [Ca2+]i. The effect of U50488H was studied on sperm aliquots incubated with this peptide at different doses (1 μm). Mibefradil (30 μm) was added to the same sperm aliquots to analyze the effect of U50488H of the mibefradil-induced intracellular calcium level. Progesterone (1 μm) was added to the same sperm aliquots as a control. Calibration of [Ca2+]i was achieved adding Triton X-100 (5%) to obtain the maximal response, followed by the addition of ethylene glycol tetra-acetic acid (EGTA) (40 nm) to obtain the minimal response. Sperm motility data were normalized as [(Treatment -Control)/(Control)] ×100, and acrosome-reacted data were normalized as [(Treatment- Control)/(Progesterone-DMSO)] x100 and evaluated using Students t, test and one-way ANOVA with post-hoc Bonferroni test. These procedures were undertaken using the IBM SPSS Statistics program (version 22). Differences were considered significant at *p, < 0.05 and highly significant at **p, < 0.01. Data are expressed as mean ± S.E. using the GraphPad PRISM (version 6.0) (GraphPad Company, CA) program. To gain insights into the human sperm proteome, we followed mass-spectrometry (MS) based proteomic and phosphoproteomic approaches in parallel. Isolated spermatozoa samples, untreated or stimulated with U50488H, were divided into soluble and insoluble protein fractions and subjected to the workflow described in Fig. 1A,. Once we excluded reverse hits and common contaminants and accounting for the reporter ion intensities and number of peptides (≥ 2 identified peptides and ≥ 1 unique peptides), a total of 5109 proteins were identified of which 5070 were confidently quantified (supplemental Table S1). To obtain an overview of the biological functions of the identified proteins, Gene Ontology (GO) analyses were performed using the Panther classification system (Fig. 1B,). The most enriched functions were related to the protein and tRNA transport, metabolic processes, nuclear organization or processes related to sperm function. Specifically, we found a wid
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