Abstract
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.
Highlights
Phosphoproteomic and Functional Analyses Reveal Sperm-specific Protein Changes Downstream of Kappa Opioid Receptor in Human Spermatozoa*□S
Amaral et al [20] have presented the most complete catalogue of human sperm proteins with 6198 reported protein IDs, in the present study we have identified over the 68% of the total sperm proteome because the human spermatozoa is estimated to be composed by 7500 different proteins [20]
Regarding proteins linked to G-protein coupled receptors (GPCRs), we found proteins associated to different signaling pathways
Summary
Phosphoproteomic and Functional Analyses Reveal Sperm-specific Protein Changes Downstream of Kappa Opioid Receptor in Human Spermatozoa*□S. 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]. 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; ¶Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, University of the Basque Country (UPV/EHU), Vitoria-Gasteiz, Araba, Spain, 01006; ʈDepartment of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark, 5320; **Instituto de Investigaciones Químicas, CSIC, Sevilla, Spain, 41092
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