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Phosphoproteomic and Functional Analyses Reveal Sperm-specific Protein Changes Downstream of Kappa Opioid Receptor in Human Spermatozoa*

  • Author Footnotes
    §§ These authors contributed equally to the work.
    Itziar Urizar-Arenaza
    Footnotes
    §§ These authors contributed equally to the work.
    Affiliations
    From the ‡Department of Physiology, Faculty of Medicine and Nursing, University of the Basque Country (UPV/EHU), Leioa, Bizkaia, Spain, 49840;

    Biocruces Bizkaia Health Research Institute, Barakaldo, Bizkaia, Spain, 48903;
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  • Author Footnotes
    §§ These authors contributed equally to the work.
    Nerea Osinalde
    Footnotes
    §§ These authors contributed equally to the work.
    Affiliations
    Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, University of the Basque Country (UPV/EHU), Vitoria-Gasteiz, Araba, Spain, 01006;
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  • Vyacheslav Akimov
    Affiliations
    Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark, 5320;
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  • Michele Puglia
    Affiliations
    Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark, 5320;
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  • Luz Candenas
    Affiliations
    Instituto de Investigaciones Químicas, CSIC, Sevilla, Spain, 41092
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  • Francisco Maria Pinto
    Affiliations
    Instituto de Investigaciones Químicas, CSIC, Sevilla, Spain, 41092
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  • Iraia Muñoa-Hoyos
    Affiliations
    From the ‡Department of Physiology, Faculty of Medicine and Nursing, University of the Basque Country (UPV/EHU), Leioa, Bizkaia, Spain, 49840;

    Biocruces Bizkaia Health Research Institute, Barakaldo, Bizkaia, Spain, 48903;
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  • Marta Gianzo
    Affiliations
    From the ‡Department of Physiology, Faculty of Medicine and Nursing, University of the Basque Country (UPV/EHU), Leioa, Bizkaia, Spain, 49840;

    Biocruces Bizkaia Health Research Institute, Barakaldo, Bizkaia, Spain, 48903;
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  • Roberto Matorras
    Affiliations
    Biocruces Bizkaia Health Research Institute, Barakaldo, Bizkaia, Spain, 48903;
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  • Jon Irazusta
    Affiliations
    From the ‡Department of Physiology, Faculty of Medicine and Nursing, University of the Basque Country (UPV/EHU), Leioa, Bizkaia, Spain, 49840;
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  • Blagoy Blagoev
    Affiliations
    Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark, 5320;
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  • Author Footnotes
    §§ These authors contributed equally to the work.
    Nerea Subiran
    Correspondence
    To whom correspondence may be addressed:Department of Physiology, Faculty of Medicine and Nursery, University of the Basque Country, 48940 Leioa, Bizkaia, Spain. Tel.:+34 946015673;
    Footnotes
    §§ These authors contributed equally to the work.
    Affiliations
    From the ‡Department of Physiology, Faculty of Medicine and Nursing, University of the Basque Country (UPV/EHU), Leioa, Bizkaia, Spain, 49840;

    Biocruces Bizkaia Health Research Institute, Barakaldo, Bizkaia, Spain, 48903;
    Search for articles by this author
  • Author Footnotes
    §§ These authors contributed equally to the work.
    Irina Kratchmarova
    Correspondence
    To whom correspondence may be addressed:Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5320 Odense, Denmark. Tel.:+45 65502494;
    Footnotes
    §§ These authors contributed equally to the work.
    Affiliations
    Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark, 5320;
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  • Author Footnotes
    * IU-A is supported by a fellowship from the University of the Basque Country (UPV/EHU). IM-H is supported by a fellowship from the Basque Government. I.K. is supported by a grant from the Danish Medical Research Council.
    This article contains supplemental material.
    §§ These authors contributed equally to the work.
Open AccessPublished:January 08, 2019DOI:https://doi.org/10.1074/mcp.RA118.001133
      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.

      Graphical Abstract

      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 (
      • 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.
      ). It is well documented that ionotropic modulation through rapid responses are the main regulators of sperm physiology (
      • Hille B.
      ). However, the presence of a high number of G-protein coupled receptors (GPCRs)
      The 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 (
      • Spehr M.
      • Schwane K.
      • Riffell J.A.
      • Zimmer R.K.
      • Hatt H.
      Odorant receptors and olfactory-like signaling mechanisms in mammalian sperm.
      ).
      The GPCRs are seven transmembrane receptors that represent approximately the 1% of the human genome and are one of the best therapeutic targets (
      • Nambi P.
      • Aiyar N.
      G protein-coupled receptors.
      ). 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 (
      • 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.
      ,
      • Schaefer M.
      • Hofmann T.
      • Schultz G.
      • Gudermann T.
      A new prostaglandin E receptor mediates calcium influx and acrosome reaction in human spermatozoa.
      ,
      • 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.
      ,
      • 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.
      ,
      • 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.
      ). 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 (
      • 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.
      ). 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 (
      • 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.
      ). Moreover, because spermatozoa are transcriptionally and translationally silent cells, it has been suggested that they may possess unique, sperm-specific signaling pathways (
      • 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.
      ,
      • 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.
      ). 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 (
      • 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.
      ), 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.

      DATA AVAILABILITY

      The mass spectrometry proteomics data have been deposited to ProteomeXchange Cinsortium via the PRIDE partner repository (http://proteomecentral.proteomechange.org) with data identifier PXD011290. Annotated MS/MS spectra can be accessed through MS-Viewer (
      • Baker P.R.
      • Chalkley R.J.
      MS-viewer: a web-based spectral viewer for proteomics results.
      ) http://msviewer.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msviewer with the following search keys: yfsnn6olid. Project Title: Phosphoproteomic and functional approaches reveal changes in sperm-specific proteins downstream KOR in human spermatozoa. Project ID: PXD011290.

      Acknowledgments

      We thank Dr. S. Sidoli for the help with the analysis of the data. Basque Government and University of the Basque Country (UPV/EHU).

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