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Human Testis Phosphoproteome Reveals Kinases as Potential Targets in Spermatogenesis and Testicular Cancer*

  • Author Footnotes
    ‖ Current affiliation: Molecular Biology of Reproduction and Development Group, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Faculty of Medicine, University of Barcelona, Casanova 143, 08036, Barcelona, Spain.
    Judit Castillo
    Correspondence
    To whom correspondence may be addressed:Molecular Biology of Reproduction and Development Group, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Faculty of Medicine, University of Barcelona, Casanova 143, 08036, Barcelona, Spain. Tel.:+34 934021877;
    Footnotes
    ‖ Current affiliation: Molecular Biology of Reproduction and Development Group, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Faculty of Medicine, University of Barcelona, Casanova 143, 08036, Barcelona, Spain.
    Affiliations
    Lead Pharma BV, Pivot Park, Kloosterstraat 9, 5349 AB Oss, The Netherlands;
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  • Jaco C. Knol
    Affiliations
    OncoProteomics Laboratory, Department of Medical Oncology, VU University Medical Center, De Boelelaan 1117, 1081HV Amsterdam, The Netherlands;
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  • Cindy M. Korver
    Affiliations
    Center for Reproductive Medicine, Research Institute Amsterdam Reproduction and Development, Amsterdam UMC, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands
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  • Sander R. Piersma
    Affiliations
    OncoProteomics Laboratory, Department of Medical Oncology, VU University Medical Center, De Boelelaan 1117, 1081HV Amsterdam, The Netherlands;
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  • Thang V. Pham
    Affiliations
    OncoProteomics Laboratory, Department of Medical Oncology, VU University Medical Center, De Boelelaan 1117, 1081HV Amsterdam, The Netherlands;
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  • Richard R. de Goeij-de Haas
    Affiliations
    OncoProteomics Laboratory, Department of Medical Oncology, VU University Medical Center, De Boelelaan 1117, 1081HV Amsterdam, The Netherlands;
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  • Ans M.M. van Pelt
    Affiliations
    Center for Reproductive Medicine, Research Institute Amsterdam Reproduction and Development, Amsterdam UMC, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands
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  • Connie R. Jimenez
    Affiliations
    OncoProteomics Laboratory, Department of Medical Oncology, VU University Medical Center, De Boelelaan 1117, 1081HV Amsterdam, The Netherlands;
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  • Bastiaan J.H. Jansen
    Correspondence
    To whom correspondence may be addressed:Lead Pharma BV, Pivot Park, Kloosterstraat 9, 5349 AB, Oss, The Netherlands. Tel.:+31 (0)412782991;
    Affiliations
    Lead Pharma BV, Pivot Park, Kloosterstraat 9, 5349 AB Oss, The Netherlands;
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  • Author Footnotes
    * This work was supported by grants from EU-FP7-PEOPLE-2013-ITN 603568: “GROWSPERM” to A.M.M. van Pelt and B.J.H. Jansen.
    This article contains supplemental Figures and Tables. The authors declare no competing financial interest.
    ‖ Current affiliation: Molecular Biology of Reproduction and Development Group, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Faculty of Medicine, University of Barcelona, Casanova 143, 08036, Barcelona, Spain.
Open AccessPublished:January 25, 2019DOI:https://doi.org/10.1074/mcp.RA118.001278
      Spermatogenesis is a complex cell differentiation process that includes marked genetic, cellular, functional and structural changes. It requires tight regulation, because disturbances in any of the spermatogenic processes would lead to fertility deficiencies as well as disorders in offspring. To increase our knowledge of signal transduction during sperm development, we carried out a large-scale identification of the phosphorylation events that occur in the human male gonad. Metal oxide affinity chromatography using TiO2 combined with LC-MS/MS was conducted to profile the phosphoproteome of adult human testes with full spermatogenesis. A total of 8187 phosphopeptides derived from 2661 proteins were identified, resulting in the most complete report of human testicular phosphoproteins to date. Phosphorylation events were enriched in proteins functionally related to spermatogenesis, as well as to highly active processes in the male gonad, such as transcriptional and translational regulation, cytoskeleton organization, DNA packaging, cell cycle and apoptosis. Moreover, 174 phosphorylated kinases were identified. The most active human protein kinases in the testis were predicted both by the number of phosphopeptide spectra identified and the phosphorylation status of the kinase activation loop. The potential function of cyclin-dependent kinase 12 (CDK12) and p21-activated kinase 4 (PAK4) has been explored by in silico, protein-protein interaction analysis, immunodetection in testicular tissue, and a functional assay in a human embryonal carcinoma cell line. The colocalization of CDK12 with Golgi markers suggests a potential crucial role of this protein kinase during sperm formation. PAK4 has been found expressed in human spermatogonia, and a role in embryonal carcinoma cell response to apoptosis has been observed. Together, our protein discovery analysis confirms that phosphoregulation by protein kinases is highly active in sperm differentiation and opens a window to detailed characterization and validation of potential targets for the development of drugs modulating male fertility and tumor behavior.

      Graphical Abstract

      Spermatogenesis is a complex cell differentiation process that takes place in the testis, within the seminiferous tubules (
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      • Simorangkir D.
      • Wreford N.
      ). It includes tightly coordinated genetic, cellular, functional and structural changes to give rise to the highly specialized male gamete, the sperm cell (
      • de Kretser D.M.
      • Loveland K.L.
      • Meinhardt A.
      • Simorangkir D.
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      ,
      • Sutovsky P.
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      ,
      • Oliva R.
      Protamines and male infertility.
      ,
      • Kimmins S.
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      Chromatin remodelling and epigenetic features of germ cells.
      ,
      • Oliva R.
      • Dixon G.H.
      Vertebrate protamine genes and the histone-to-protamine replacement reaction.
      ,
      • Davies D.V.
      • Mann T.
      Functional development of accessory glands and spermatogenesis.
      ,
      • Fawcett D.W.
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      Changes in distribution of nuclear pores during differentiation of the male germ cells.
      ,
      • Oliva R.
      • Castillo J.
      Proteomics and the genetics of sperm chromatin condensation.
      ). Spermatogenesis is divided in three main processes - mitosis, meiosis, and spermiogenesis - and includes different germ cell types that are mechanically and nutritionally supported by the somatic Sertoli cells (
      • de Kretser D.M.
      • Loveland K.L.
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      • Wreford N.
      ,
      • Weber J.E.
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      • Wong V.
      • Peterson R.N.
      Three-dimensional reconstruction of a rat stage V Sertoli cell: II. Morphometry of Sertoli–Sertoli and Sertoli–germ-cell relationships.
      ). Spermatogonia are in the basal part of the germinal epithelium, and experience successive mitotic divisions to undergo either self-renewal or differentiation to spermatocytes. Thereafter, spermatocytes go through two consecutive meiotic divisions, which results in the generation of haploid round spermatids. During the last step of spermatogenesis, spermatids elongate, most of the cytoplasm is lost, chromatin is extensively remodeled, and specialized structures for fertilization are formed, such as the flagellum and the acrosome (
      • de Kretser D.M.
      • Loveland K.L.
      • Meinhardt A.
      • Simorangkir D.
      • Wreford N.
      ,
      • Oliva R.
      Protamines and male infertility.
      ,
      • Kimmins S.
      • Sassone-Corsi P.
      Chromatin remodelling and epigenetic features of germ cells.
      ,
      • Oliva R.
      • Dixon G.H.
      Vertebrate protamine genes and the histone-to-protamine replacement reaction.
      ,
      • Oliva R.
      • Castillo J.
      ,
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      • Castillo J.
      • de la Iglesia A.
      • Jodar M.
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      Mammalian sperm protamine extraction and analysis: a step-by-step detailed protocol and brief review of protamine alterations.
      ). At the end of the process, the spermatozoa are released to the lumen of the tubule, to continue the maturation in the epididymis (
      • de Kretser D.M.
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      • Meinhardt A.
      • Simorangkir D.
      • Wreford N.
      ,
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      Epididymosomes and prostasomes: their roles in posttesticular maturation of the sperm cells.
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      New insights into epididymal function in relation to sperm maturation.
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      • Sullivan R.
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      The human epididymis: Its function in sperm maturation.
      ,
      • Castillo J.
      • Jodar M.
      • Oliva R.
      The contribution of human sperm proteins to the development and epigenome of the preimplantation embryo.
      ).
      The process of spermatogenesis is very dynamic and disturbances in any of the steps would lead to fertility deficiencies. Therefore, it requires tight regulation at different levels. Although hormonal regulation of spermatogenesis by the hypothalamic-pituitary-testicular axis is well understood (
      • Cheng C.Y.
      • Mruk D.D.
      A local autocrine axis in the testes that regulates spermatogenesis.
      ,
      • Tilbrook A.J.
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      Negative feedback regulation of the secretion and actions of gonadotropin-releasing hormone in males.
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      Mechanisms of hormonal regulation of sertoli cell development and proliferation: a key process for spermatogenesis.
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      Peripheral and central mechanisms involved in the hormonal control of male and female reproduction.
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      • Alves M.G.
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      Hormonal control of Sertoli cell metabolism regulates spermatogenesis.
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      Regulation of spermatogenesis: An evolutionary biologist's perspective.
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      • Monaco L.
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      • LeMeur M.
      • Sassone-Corsi P.
      Impairing follicle-stimulating hormone (FSH) signaling in vivo: Targeted disruption of the FSH receptor leads to aberrant gametogenesis and hormonal imbalance.
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      The effect of a null mutation in the follicle-stimulating hormone receptor gene on mouse reproduction 1.
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      A Sertoli cell-selective knockout of the androgen receptor causes spermatogenic arrest in meiosis.
      ,
      • Holdcraft R.W.
      Androgen receptor function is required in Sertoli cells for the terminal differentiation of haploid spermatids.
      ,
      • Chang C.
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      • Yeh S.-D.
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      Infertility with defective spermatogenesis and hypotestosteronemia in male mice lacking the androgen receptor in Sertoli cells.
      ,
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      Androgen receptor roles in spermatogenesis and fertility: Lessons from testicular cell-specific androgen receptor knockout mice.
      ), other layers of regulation, such as signal transduction through phosphorylation, remain less well explored. Protein phosphorylation is well known to be involved in the regulation of cell cycle, cell growth, cell differentiation and cell death in many biological systems (
      • Fisher D.
      • Krasinska L.
      • Coudreuse D.
      • Novak B.
      Phosphorylation network dynamics in the control of cell cycle transitions.
      ,
      • Venerando A.
      • Cesaro L.
      • Pinna L.A.
      From phosphoproteins to phosphoproteomes: a historical account.
      ). Also, a role for protein phosphorylation in the regulation of testis-specific events, such as the maintenance of the Sertoli cell blood testis barrier and basal ectoplasmic specializations, has been observed (
      • Wan H.T.
      • Mruk D.D.
      • Tang E.I.
      • Xiao X.
      • Cheng Y.H.
      • Wong E.W.P.
      • Wong C.K.C.
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      Role of non-receptor protein tyrosine kinases in spermatid transport during spermatogenesis.
      ,
      • Wong C.H.
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      Mitogen-activated protein kinases, adherens junction dynamics, and spermatogenesis: A review of recent data.
      ,
      • Zhang H.
      • Yin Y.
      • Wang G.
      • Liu Z.
      • Liu L.
      • Sun F.
      Interleukin-6 disrupts blood-testis barrier through inhibiting protein degradation or activating phosphorylated ERK in Sertoli cells.
      ).
      By adding phosphate groups to substrate proteins, protein kinases are key regulators of cell functions, and therefore, good targets for the modulation of spermatogenesis and the identification of potential causes of male infertility. During the past decades, many studies have been focused on specific kinase families, such as the mitogen-activated protein kinases (MAPKs), which are critical for sperm development (
      • Wong C.H.
      • Cheng C.Y.
      Mitogen-activated protein kinases, adherens junction dynamics, and spermatogenesis: A review of recent data.
      ,
      • Walker W.H.
      Non-classical actions of testosterone and spermatogenesis.
      ,
      • Xia W.
      • Cheng C.Y.
      TGF-β3 regulates anchoring junction dynamics in the seminiferous epithelium of the rat testis via the Ras/ERK signaling pathway: An in vivo study.
      ). Other known kinases with roles in spermatogenesis are the cell cycle regulators POLO-like kinases (PLKs) (
      • Jordan P.W.
      • Karppinen J.
      • Handel M.A.
      Polo-like kinase is required for synaptonemal complex disassembly and phosphorylation in mouse spermatocytes.
      ,
      • Qi L.
      • Liu Z.
      • Wang J.
      • Cui Y.
      • Guo Y.
      • Zhou T.
      • Zhou Z.
      • Guo X.
      • Xue Y.
      • Sha J.
      Systematic analysis of the phosphoproteome and kinase-substrate networks in the mouse testis.
      ), the androgen receptor p21-activated kinase 6 (PAK6) (
      • Lee S.R.
      • Ramos S.M.
      • Ko A.
      • Masiello D.
      • Swanson K.D.
      • Lu M.L.
      • Balk S.P.
      AR and ER interaction with a p21-activated kinase (PAK6).
      ,
      • Liu X.
      • Busby J.
      • John C.
      • Wei J.
      • Yuan X.
      • Lu M.L.
      Direct Interaction between AR and PAK6 in Androgen-Stimulated PAK6 Activation.
      ,
      • Schrantz N.
      • Da Silva Correia J.
      • Fowler B.
      • Ge Q.
      • Sun Z.
      • Bokoch G.M.
      Mechanism of p21-activated kinase 6-mediated inhibition of androgen receptor signaling.
      ), and the members of the testis-specific serine/threonine-protein kinase (TSSK) family, which have a role during the last stage of spermatogenesis called spermiogenesis (
      • Kueng P.
      • Nikolova Z.
      • Djonov V.
      • Hemphill A.
      • Rohrbach V.
      • Boehlen D.
      • Zuercher G.
      • Andres A.C.
      • Ziemiecki A.
      A novel family of serine/threonine kinases participating in spermiogenesis.
      ). However, a large-scale identification of the phosphorylation events that occur in the human testis has not been conducted so far. The use of high-throughput techniques would provide an in-depth picture of the molecular regulation of spermatogenesis and identify additional kinases that might also be essential in the process. Phosphopeptide enrichment combined with MS has been used recently for the systematic analysis of the mouse testis phosphoproteome profile (
      • Qi L.
      • Liu Z.
      • Wang J.
      • Cui Y.
      • Guo Y.
      • Zhou T.
      • Zhou Z.
      • Guo X.
      • Xue Y.
      • Sha J.
      Systematic analysis of the phosphoproteome and kinase-substrate networks in the mouse testis.
      ). However, because of biological and genetic differences that exist between rodent and primate spermatogenesis, the development of such a study in human testis is warranted.
      In the present study we performed global phosphoproteomics on human testicular tissue with full spermatogenesis, to identify the most relevant signaling pathways taking place during the development of the male gamete. To this end, metal oxide affinity chromatography, using a highly efficient titanium dioxide (TiO2) method coupled to MS (
      • Kyono Y.
      • Sugiyama N.
      • Imami K.
      • Tomita M.
      • Ishihama Y.
      Successive and selective release of phosphorylated peptides captured by hydroxy acid-modified metal oxide chromatography.
      ,
      • Piersma S.R.
      • Knol J.C.
      • de Reus I.
      • Labots M.
      • Sampadi B.K.
      • Pham T.V.
      • Ishihama Y.
      • Verheul H.M.W.
      • Jimenez C.R.
      Feasibility of label-free phosphoproteomics and application to base-line signaling of colorectal cancer cell lines.
      ,
      • Sugiyama N.
      • Masuda T.
      • Shinoda K.
      • Nakamura A.
      • Tomita M.
      • Ishihama Y.
      Phosphopeptide enrichment by aliphatic hydroxy acid-modified metal oxide chromatography for nano-LC-MS/MS in proteomics applications.
      ), was carried out to profile the phosphoproteome of human testes with histologically complete spermatogenesis. To further investigate phosphoregulation, MS data was used to predict the most active kinases in the human testis. Our study contributes to the understanding of the regulation of human spermatogenesis that is orchestrated by protein phosphorylation. In addition, the identification of the most active kinases in the human testis opens the window to a detailed characterization and validation of potential good targets for the development of drugs modulating male germ cell development and testicular cancer progression.

      DISCUSSION

      In the present study we show for the first time a comprehensive analysis of the phosphoproteome of the human testis with full spermatogenesis. The advances in MS-based approaches make phosphoproteomics the most powerful technique nowadays for the global analysis of signaling networks in defined biological systems (
      • Macek B.
      • Mann M.
      • Olsen J.V.
      Global and site-specific quantitative phosphoproteomics: principles and applications.
      ). In the field of male reproduction, phosphoproteomics has been used to unravel the molecular mechanisms of sperm motility (
      • Amaral A.
      • Castillo J.
      • Ramalho-Santos J.
      • Oliva R.
      The combined human sperm proteome: Cellular pathways and implications for basic and clinical science.
      ,
      • Parte P.P.
      • Rao P.
      • Redij S.
      • Lobo V.
      • D'Souza S.J.
      • Gajbhiye R.
      • Kulkarni V.
      Sperm phosphoproteome profiling by ultra performance liquid chromatography followed by data independent analysis (LC-MS(E)) reveals altered proteomic signatures in asthenozoospermia.
      ). However, an in-depth study of the phosphoregulation in the human testis was not conducted so far. Through phosphopeptide enrichment using the TiO2 method combined with LC-MS/MS, we have identified 8187 phosphopeptides from 2661 proteins, resulting in the most complete report of the human testicular phosphoproteins to date. It is important to highlight that the global phosphopeptide enrichment approach conducted in this study has been proven to be highly reproducible and robust (
      • Piersma S.R.
      • Knol J.C.
      • de Reus I.
      • Labots M.
      • Sampadi B.K.
      • Pham T.V.
      • Ishihama Y.
      • Verheul H.M.W.
      • Jimenez C.R.
      Feasibility of label-free phosphoproteomics and application to base-line signaling of colorectal cancer cell lines.
      ). Therefore, the limited overlap between the three donors described here for the identification of both the phosphopeptides and the individual proteins, exposes the intrinsic biological heterogeneity between different donors.
      According to the results reported herein, phosphoproteins represent the 32.9% of the human testis proteome and are closely related to highly active processes in the male gonad, such as transcriptional and translational regulation, cytoskeleton organization, DNA packaging, cell cycle, and apoptosis. Of note, the term of spermatogenesis was also found overrepresented in the GO enrichment analysis conducted in the present study. This, together with the fact that the 32% of the human kinome was included in the human testicular phosphoproteome, confirms that phosphoregulation by protein kinases is highly active in cellular differentiation from spermatogonia to spermatozoa.
      Interestingly, the functional involvement of phosphoproteins in human sperm development is like rodent spermatogenesis, because comparable functional annotations were observed previously in the mouse testis phosphoproteome (
      • Qi L.
      • Liu Z.
      • Wang J.
      • Cui Y.
      • Guo Y.
      • Zhou T.
      • Zhou Z.
      • Guo X.
      • Xue Y.
      • Sha J.
      Systematic analysis of the phosphoproteome and kinase-substrate networks in the mouse testis.
      ). However, the main protein kinases orchestrating this regulation seem to be different between mouse and man. Although POLO-like kinases (PLKs) were found as highly active in the mouse testis (
      • Qi L.
      • Liu Z.
      • Wang J.
      • Cui Y.
      • Guo Y.
      • Zhou T.
      • Zhou Z.
      • Guo X.
      • Xue Y.
      • Sha J.
      Systematic analysis of the phosphoproteome and kinase-substrate networks in the mouse testis.
      ), no human PLKs were found in the top 20 after kinase ranking in this study. Instead, we found MAPK1 and MAPK3 (also known as ERK2 and ERK1, respectively) as two of the most active kinases in the human testis, indicated by the abundance of their phosphopeptides and the presence of phosphosites in their kinase activation loop. These data are consistent with several publications reporting that MAPK1 and -3 play critical roles in spermatogenesis. Specifically, they are known to be involved in the regulation of mitosis, meiosis, and the Sertoli-Sertoli and Sertoli-germ cell interface (
      • Wong C.H.
      • Cheng C.Y.
      Mitogen-activated protein kinases, adherens junction dynamics, and spermatogenesis: A review of recent data.
      ,
      • Walker W.H.
      Non-classical actions of testosterone and spermatogenesis.
      ,
      • Xia W.
      • Cheng C.Y.
      TGF-β3 regulates anchoring junction dynamics in the seminiferous epithelium of the rat testis via the Ras/ERK signaling pathway: An in vivo study.
      ). In addition, MAPK1 and -3 regulate cell proliferation after activation by Serine/threonine-protein kinase D1 (PRKD1) (
      • Jadali A.
      • Ghazizadeh S.
      Protein kinase D is implicated in the reversible commitment to differentiation in primary cultures of mouse keratinocytes.
      ), which was also identified in the human testis kinase ranking.
      Other highly prominent kinases in the human testis are known to be involved in the regulation of mRNA splicing in different cell types, such as the pre-mRNA processing factor 4B (PRPF4B) and cyclin-dependent kinases CDK12 and CDK13 (
      • Chen H.-H.
      • Wang Y.-C.
      • Fann M.-J.
      Identification and characterization of the CDK12/Cyclin L1 complex involved in alternative splicing regulation.
      ,
      • Chen H.H.
      • Wong Y.H.
      • Geneviere A.M.
      • Fann M.J.
      CDK13/CDC2L5 interacts with L-type cyclins and regulates alternative splicing.
      ,
      • Corkery D.P.
      • Holly A.C.
      • Lahsaee S.
      • Dellaire G.
      Connecting the speckles: Splicing kinases and their role in tumorigenesis and treatment response.
      ,
      • Schneider M.
      • Hsiao H.H.
      • Will C.L.
      • Giet R.
      • Urlaub H.
      • Lührmann R.
      Human PRP4 kinase is required for stable tri-snRNP association during spliceosomal B complex formation.
      ). Alternative splicing allows a single gene to encode different or multiple proteins (
      • Andreadis A.
      • Gallego M.E.
      • Nadal-Ginard B.
      Generation of protein isoform diversity by alternative splicing: mechanistic and biological implications.
      ) and this is particularly important for complex cell differentiation processes that require tight regulation, as is the case in spermatogenesis (
      • Huang X.
      • Li J.
      • Lu L.
      • Xu M.
      • Xiao J.
      • Yin L.
      • Zhu H.
      • Zhou Z.
      • Sha J.
      Novel development-related alternative splices in human testis identified by cDNA microarrays.
      ,
      • Venables J.P.
      Alternative splicing in the testes.
      ). In fact, testis is a tissue with one of the highest levels of alternative splicing in the body, together with brain and liver (
      • Yeo G.
      • Holste D.
      • Kreiman G.
      • Burge C.B.
      Variation in alternative splicing across human tissues.
      ), which is consistent with the overrepresentation of this pathway in the human testis phosphoproteomic profile. Further in silico, analysis of CDK12 protein-protein interactions have also suggested such a role for CDK12 in the male gonad, as well as potential involvements in regulation of transcription and the cell cycle. CDK12 is a cyclin-dependent kinase which requires the interaction with a cyclin-regulatory partner to become active, such as the cyclin K (
      • Chen H.-H.
      • Wang Y.-C.
      • Fann M.-J.
      Identification and characterization of the CDK12/Cyclin L1 complex involved in alternative splicing regulation.
      ,
      • Chen H.H.
      • Wong Y.H.
      • Geneviere A.M.
      • Fann M.J.
      CDK13/CDC2L5 interacts with L-type cyclins and regulates alternative splicing.
      ,
      • Blazek D.
      • Kohoutek J.
      • Bartholomeeusen K.
      • Johansen E.
      • Hulinkova P.
      • Luo Z.
      • Cimermancic P.
      • Ule J.
      • Peterlin B.M.
      The cyclin K/Cdk12 complex maintains genomic stability via regulation of expression of DNA damage response genes.
      ,
      • Loyer P.
      • Trembley J.H.
      • Katona R.
      • Kidd V.J.
      • Lahti J.M.
      Role of CDK/cyclin complexes in transcription and RNA splicing.
      ). In fact, the CDK12/cyclin K complex was found by others to be required for the regulation of DNA damage response genes through phosphorylation of the C-terminal domain of RNA polymerase II in mammalian cells (
      • Chen H.-H.
      • Wang Y.-C.
      • Fann M.-J.
      Identification and characterization of the CDK12/Cyclin L1 complex involved in alternative splicing regulation.
      ,
      • Blazek D.
      • Kohoutek J.
      • Bartholomeeusen K.
      • Johansen E.
      • Hulinkova P.
      • Luo Z.
      • Cimermancic P.
      • Ule J.
      • Peterlin B.M.
      The cyclin K/Cdk12 complex maintains genomic stability via regulation of expression of DNA damage response genes.
      ). Interestingly, potential roles of cyclin K in spermatogenesis were also suggested by others. In particular, cyclin K is known to be expressed in rodent testicular tissue in a developmentally regulated manner and ends with a specific localization in primary spermatocytes of adult testes (
      • Xiang X.
      • Deng L.
      • Zhang J.
      • Zhang X.
      • Lei T.
      • Luan G.
      • Yang C.
      • Xiao Z.X.
      • Li Q.
      • Li Q.
      A distinct expression pattern of cyclin K in mammalian testes suggests a functional role in spermatogenesis.
      ). Here we found CDK12 associated with specific structures from human mid-to-late pachytene spermatocytes at epithelial stages VII-XII, according to the stage classification proposed by Muciaccia and colleagues (
      • Muciaccia B.
      • Boitani C.
      • Berloco B.P.
      • Nudo F.
      • Spadetta G.
      • Stefanini M.
      • de Rooij D.G.
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      Novel stage classification of human spermatogenesis based on acrosome development1.
      ). However, transcriptional activity becomes dramatically reduced at post-meiotic steps, which could suggest additional roles for this kinase in the human testis. Therefore, because of its confined expression in the human testis, as well as its function in other cell types and tissues, two localizations could be envisioned for CDK12 in the male gonad: either (1) an association with the nuage, a germ-cell specific organelle that stores RNA and RNA binding proteins and plays crucial roles in spermatogenesis, such as the regulation of transposon elements (
      • Yokota S.
      Nuage proteins: their localization in subcellular structures of spermatogenic cells as revealed by immunoelectron microscopy.
      ,
      • Onohara Y.
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      Meiosis - Molecular Mechanisms and Cytogenetic Diversity.
      ,
      • Soper S.F.C.
      • van der Heijden G.W.
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      • Goodheart M.
      • Martin S.L.
      • de Boer P.
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      Mouse maelstrom, a component of nuage, is essential for spermatogenesis and transposon repression in meiosis.
      ), or (2) the association with pro-acrosomal vesicle-related structures, such as the Golgi apparatus. To determine the localization of CDK12 we conducted immunofluorescence analyses, which revealed the specific localization of CDK12 surrounding the Golgi apparatus marker GM130, whereas no co-localization was observed with the nuage component DDX4. In mammals, Golgi structures develop into the acrosome during the last stages of spermatogenesis, which is a unique organelle containing various hydrolytic enzymes which, when secreted, allow sperm penetration of the oocyte zona pellucida (
      • Berruti G.
      • Paiardi C.
      Acrosome biogenesis.
      ,
      • Leuchtenberger C.
      • Schrader F.
      The chemical nature of the acrosome in the male germ cells.
      ). Further, according to results reported by others (
      • Wang H.
      • Wan H.
      • Li X.
      • Liu W.
      • Chen Q.
      • Wang Y.
      • Yang L.
      • Tang H.
      • Zhang X.
      • Duan E.
      • Zhao X.
      • Gao F.
      • Li W.
      Atg7 is required for acrosome biogenesis during spermatogenesis in mice.
      ), CDK12 expression in the human testis seems to be limited to structures that might be identified as pro-acrosomal granules. Therefore, and although the potential involvement of CDK12 in the initiation of acrosome biogenesis demand deeper investigation, the results observed in this study, together with the high expression of CDK12 in testis compared with other tissues as indicated in The Human Protein Atlas, would suggest a determinant role for this kinase in sperm development and function. CDK12 might thus be a potential pharmacological candidate for the development of drugs that modulate male fertility.
      Protein kinases are heavily pursued targets for drug development because of their capacity to modulate signaling pathways in many diseases. Especially attractive candidates are those kinases that regulate cell survival and cell death, many of which have been identified in this study as highly abundant in the human testis. One of these is the serine/threonine-protein kinase PAK4, a member of the group II of the PAK family (
      • Wells C.M.
      • Jones G.E.
      The emerging importance of group II PAKs.
      ). Although PAK4 role has never been studied in the testicular environment, the results of the present study suggest a potential role in the regulation of cytoskeletal dynamics and cell survival in sperm development. Also, PAK4 was found to be expressed uniquely in human spermatogonia, which suggests an involvement of this kinase in the proliferative stage of spermatogenesis. Therefore, PAK4 might be involved in the initiation of sperm development and that its ablation would induce alterations in this process. In addition, PAK4 is known to protect cells from apoptosis, preventing the activation of caspase through two different mechanisms: (1) through phosphorylation of the pro-apoptotic protein Bad, which results in the deactivation of cytochrome-3 release and the blocking of the caspase cascade (
      • Gnesutta N.
      • Qu J.
      • Minden A.
      The serine/threonine kinase PAK4 prevents caspase activation and protects cells from apoptosis.
      ); and (2) by inhibiting caspase-8 in a kinase-independent manner (
      • Gnesutta N.
      • Minden A.
      Death receptor-induced activation of initiator caspase 8 is antagonized by serine/threonine kinase PAK4.
      ). In a similar fashion, PAK4 can also protect cancer cells from apoptosis, and thus, represents a potential therapeutic target for the treatment of malignancies. In fact, PAK4 is the only PAK family member that is considered oncogenic, and it has been found overexpressed in several tumor cell lines, such as those from breast and prostate (
      • Wells C.M.
      • Jones G.E.
      The emerging importance of group II PAKs.
      ,
      • Callow M.G.
      • Clairvoyant F.
      • Zhu S.
      • Schryver B.
      • Whyte D.B.
      • Bischoff J.R.
      • Jallal B.
      • Smeal T.
      Requirement for PAK4 in the anchorage-independent growth of human cancer cell lines.
      ). Similarly, here we show that PAK4-deficient embryonal carcinoma cells are less capable to resist to apoptotic stimuli, leading to a decrease number of metabolic active cells. Collectively, we suggest that PAK4 inhibitors would provide an interesting pharmacological target for the treatment of testicular cancer.
      Together, the comprehensive analysis of the human testicular phosphoproteome contributes to our molecular knowledge of human sperm development and allowed us to identify the main protein kinases involved in the phosphoregulation of spermatogenesis. Because human testis tissue from healthy fertile men for research purposes is scarce and difficult to obtain, the phosphoproteomic description of the human testis was conducted in testicular samples that, although showing morphological normal spermatogenesis, do not represent the population and age of healthy fertile men. Further studies are now required to identify age-derived changes as well as to relate the results described here with the fertilization capacity of sperm, which is also influenced by the genetic and epigenetic profile of the cells and post-testicular processes. Also, these results open a window to validate CDK12, PAK4, and additional predicted active kinases that have been identified in the human testis as potential candidates for pharmacological interventions in male fertility or testicular cancer.

      DATA AVAILABILITY

      The mass spectrometry data have been submitted to the ProteomeXchange Consortium (47) via the PRIDE partner repository, with the dataset identifier PXD010246 (https://www.ebi.ac.uk/pride/archive/projects/PXD010246).

      Acknowledgments

      We thank Dr Leendert H. J. Looijenga (Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands) for kindly providing the NCCIT embryonal carcinoma cell line.

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