Abstract
Regulated secretion is a central issue for the specific function of many cells; for instance, mammalian sperm acrosomal exocytosis is essential for egg fertilization. Sphingosine 1-phosphate is a bioactive sphingolipid that regulates crucial physiological processes. We have recently reported that sphingosine 1-phosphate and sphingosine kinase are involved in a novel signaling pathway leading to acrosomal exocytosis (Suhaiman L et al., J Biol Chem 285:1630–16314, 2010). Acrosomal exocytosis in mammalian sperm is a regulated secretion with unusual characteristics. We therefore employed biochemical functional assays to assess the sphingolipid signaling in both permeabilized and nonpermeabilized sperm. The exocytosis of the acrosomal content is regulated by Ca2+. During exocytosis, changes in [Ca2+]i occur induced by either Ca2+-influx or Ca2+-mobilization from intracellular stores. By using single cell [Ca2+] measurements, we detected intracellular Ca2+ changes after sphingosine 1-phosphate treatment. Additionally, measuring sphingosine kinase activity, we determined that sphingosine 1-phosphate levels increase after an exocytotic stimulus.
This chapter is designed to provide the user with sufficient background to analyze sphingosine 1-phosphate signal transduction pathways during acrosomal exocytosis in human sperm.
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References
Suhaiman L, De Blas GA, Obeid LM, Darszon A, Mayorga LS, Belmonte SA (2010) Sphingosine 1-phosphate and sphingosine kinase are involved in a novel signaling pathway leading to acrosomal exocytosis. J Biol Chem 285:16302–16314
Darszon A, Acevedo JJ, Galindo BE, Hernandez-Gonzalez EO, Nishigaki T, Trevino CL, Wood C, Beltran C (2006) Sperm channel diversity and functional multiplicity. Reproduction 131:977–988
Yanagimachi R (1994) Mammalian fertilization. In: Knobil E and Neill JD (ed) The physiology of reproduction. Raven, New York, pp 189–317
Mayorga LS, Tomes CN, Belmonte SA (2007) Acrosomal exocytosis, a special type of regulated secretion. IUBMB Life 59:286–292
Kirkman-Brown JC, Barratt CL, Publicover SJ (2003) Nifedipine reveals the existence of two discrete components of the progesterone-induced [Ca2+]i transient in human spermatozoa. Dev Biol 259:71–82
Jungnickel MK, Marrero H, Birnbaumer L, Lemos JR, Florman HM (2001) Trp2 regulates entry of Ca2+ into mouse sperm triggered by egg ZP3. Nat Cell Biol 3:499–502
Breitbart H (2003) Signaling pathways in sperm capacitation and acrosome reaction. Cell Mol Biol (Noisy-le-Grand) 49:321–327
Darszon A, Beltran C, Felix R, Nishigaki T, Trevino CL (2001) Ion transport in sperm signaling. Dev Biol 240:1–14
Zanetti N, Mayorga LS (2009) Acrosomal swelling and membrane docking are required for hybrid vesicle formation during the human sperm acrosome reaction. Biol Reprod 81: 396–405
Spiegel S, English D, Milstien S (2002) Sphingosine 1-phosphate signaling: providing cells with a sense of direction. Trends Cell Biol 12:236–242
Kohama T, Olivera A, Edsall L, Nagiec MM, Dickson R, Spiegel S (1998) Molecular cloning and functional characterization of murine sphingosine kinase. J Biol Chem 273: 23722–23728
Taha TA, Hannun YA, Obeid LM (2006) Sphingosine kinase: biochemical and cellular regulation and role in disease. J Biochem Mol Biol 39:113–131
Hla T, Lee MJ, Ancellin N, Paik JH, Kluk MJ (2001) Lysophospholipids-receptor revelations. Science 294:1875–1878
Spiegel S, Milstien S (2003) Sphingosine-1-phosphate: an enigmatic signalling lipid. Nat Rev Mol Cell Biol 4:397–407
Hannun YA, Obeid LM (2008) Principles of bioactive lipid signalling: lessons from sphingolipids. Nat Rev Mol Cell Biol 9:139–150
Taha TA, Argraves KM, Obeid LM (2004) Sphingosine-1-phosphate receptors: receptor specificity versus functional redundancy. Biochim Biophys Acta 1682:48–55
Yatomi Y (2006) Sphingosine 1-phosphate in vascular biology: possible therapeutic strategies to control vascular diseases. Curr Pharm Des 12:575–587
Rosen H, Goetzl EJ (2005) Sphingosine 1-phosphate and its receptors: an autocrine and paracrine network. Nat Rev Immunol 5:560–570
Pébay A, Toutant M, Premont J, Calvo CF, Venance L, Cordier J, Glowinski J, Tence M (2001) Sphingosine-1-phosphate induces proliferation of astrocytes: regulation by intracellular signalling cascades. Eur J Neurosci 13:2067–2076
Sanchez T, Hla T (2004) Structural and functional characteristics of S1P receptors. J Cell Biochem 92:913–922
Spiegel S, Milstien S (2003) Exogenous and intracellularly generated sphingosine 1-phosphate can regulate cellular processes by divergent pathways. Biochem Soc Trans 31:1216–1219
Anelli V, Bassi R, Tettamanti G, Viani P, Riboni L (2005) Extracellular release of newly synthesized sphingosine-1-phosphate by cerebellar granule cells and astrocytes. J Neurochem 92:1204–1215
Kajimoto T, Okada T, Yu H, Goparaju SK, Jahangeer S, Nakamura S (2007) Involvement of sphingosine-1-phosphate in glutamate secretion in hippocampal neurons. Mol Cell Biol 27:3429–3440
Lopez CI, Belmonte SA, De Blas GA, Mayorga LS (2007) Membrane-permeant Rab3A triggers acrosomal exocytosis in living human sperm. FASEB J 21:4121–4130
Arnoult C, Villaz M, Florman HM (1998) Pharmacological properties of the T-type Ca2+ current of mouse spermatogenesis cells. Mol Pharmacol 53:1104–1111
Fox AP, Nowycky MC, Tsien RW (1987) Single-channel recordings of three types of calcium channels in chick sensory neurones. J Physiol 394:173–200
Gonzalez-Martinez MT, Galindo BE, de De La TL, Zapata O, Rodriguez E, Florman HM, Darszon A (2001) A sustained increase in intracellular Ca(2+) is required for the acrosome reaction in sea urchin sperm. Dev Biol 236:220–229
Yunes R, Michaut M, Tomes C, Mayorga LS (2000) Rab3A triggers the acrosome reaction in permeabilized human spermatozoa. Biol Reprod 62:1084–1089
Yunes R, Tomes C, Michaut M, De BG, Rodriguez F, Regazzi R, Mayorga LS (2002) Rab3A and calmodulin regulate acrosomal exocytosis by mechanisms that do not require a direct interaction. FEBS Lett 525:126–130
Belmonte SA, Lopez CI, Roggero CM, De Blas GA, Tomes CN, Mayorga LS (2005) Cholesterol content regulates acrosomal exocytosis by enhancing Rab3A plasma membrane association. Dev Biol 285:393–408
De Blas GA, Roggero CM, Tomes CN, Mayorga LS (2005) Dynamics of SNARE assembly and disassembly during sperm acrosomal exocytosis. PLoS Biol 3:e323
Roggero CM, Tomes CN, De Blas GA, Castillo J, Michaut MA, Fukuda M, Mayorga LS (2005) Protein kinase C-mediated phosphorylation of the two polybasic regions of synaptotagmin VI regulates their function in acrosomal exocytosis. Dev Biol 285:422–435
Branham MT, Bustos MA, De Blas GA, Rehmann H, Zarelli VE, Trevino CL, Darszon A, Mayorga LS, Tomes CN (2009) Epac activates the small G proteins Rap1 and Rab3A to achieve exocytosis. J Biol Chem 284:24825–24839
De Blas G, Michaut M, Trevino CL, Tomes CN, Yunes R, Darszon A, Mayorga LS (2002) The intraacrosomal calcium pool plays a direct role in acrosomal exocytosis. J Biol Chem 277:49326–49331
Roggero CM, De Blas GA, Dai H, Tomes CN, Rizo J, Mayorga LS (2007) Complexin/synaptotagmin interplay controls acrosomal exocytosis. J Biol Chem 282:26335–26343
Castillo BJ, Roggero CM, Mancifesta FE, Mayorga LS (2010) Calcineurin-mediated dephosphorylation of synaptotagmin VI is necessary for acrosomal exocytosis. J Biol Chem 285:26269–26278
World Health Organization (2010) WHO laboratory manual for the examination and processing of human semen. World Health Organization, Geneva, Switzerland
Mendoza C, Carreras A, Moos J, Tesarik J (1992) Distinction between true acrosome reaction and degenerative acrosome loss by a one-step staining method using Pisum sativum agglutinin. J Reprod Fertil 95:755–763
Pickett JA, Campos-Toimil M, Thomas P, Edwardson JM (2007) Identification of SNAREs that mediate zymogen granule exocytosis. Biochem Biophys Res Commun 359:599–603
Deeney JT, Branstrom R, Corkey BE, Larsson O, Berggren PO (2007) 3H-serotonin as a marker of oscillatory insulin secretion in clonal beta-cells (INS-1). FEBS Lett 581:4080–4084
Waselle L, Gerona RR, Vitale N, Martin TF, Bader MF, Regazzi R (2005) Role of phosphoinositide signaling in the control of insulin exocytosis. Mol Endocrinol 19:3097–3106
Vitale N, Mukai H, Rouot B, Thierse D, Aunis D, Bader MF (1993) Exocytosis in chromaffin cells. Possible involvement of the heterotrimeric GTP-binding protein G(o). J Biol Chem 268:14715–14723
Alouf JE, Geoffroy C (1988) Production, purification, and assay of streptolysin O. Methods Enzymol 165:52–59
Bhakdi S, Tranum-Jensen J, Sziegoleit A (1985) Mechanism of membrane damage by streptolysin-O. Infect Immun 47:52–60
Sekiya K, Satoh R, Danbara H, Futaesaku Y (1993) A ring-shaped structure with a crown formed by streptolysin O on the erythrocyte membrane. J Bacteriol 175:5953–5961
Sekiya K (1995) Electron-microscopic observation of pore formation in the erythrocyte membrane by streptolysin O. Nippon Saikingaku Zasshi 50:509–517
Sekiya K, Akagi T, Tatsuta K, Sakakura E, Hashikawa T, Abe A, Nagamune H (2007) Ultrastructural analysis of the membrane insertion of domain 3 of streptolysin O. Microbes Infect 9:1341–1350
De Blas GA, Darszon A, Ocampo AY, Serrano CJ, Castellano LE, Hernandez-Gonzalez EO, Chirinos M, Larrea F, Beltran C, Trevino CL (2009) TRPM8, a versatile channel in human sperm. PLoS One 4:e6095
Chavez JC, De Blas GA, De la Vega-Beltran JL, Nishigaki T, Chirinos M, Gonzalez-Gonzalez ME, Larrea F, Solis A, Darszon A, Trevino CL (2011) The opening of maitotoxin-sensitive calcium channels induces the acrosome reaction in human spermatozoa: differences from the zona pellucida. Asian J Androl 13:159–165
Anelli V, Gault CR, Cheng AB, Obeid LM (2008) Sphingosine kinase 1 is up-regulated during hypoxia in U87MG glioma cells. Role of hypoxia-inducible factors 1 and 2. J Biol Chem 283:3365–3375
Makler A (1980) The improved ten-micrometer chamber for rapid sperm count and motility evaluation. Fertil Steril 33:337–338
Cardona-Maya W, Berdugo J, Cadavid A (2008) Comparing the sperm concentration determined by the Makler and the Neubauer chambers. Actas Urol Esp 32:443–445
Imade GE, Towobola OA, Sagay AS, Otubu JA (1993) Discrepancies in sperm count using improved Neubauer, Makler, and Horwells counting chambers. Arch Androl 31:17–22
Harrison RA, Gadella BM (2005) Bicarbonate-induced membrane processing in sperm capacitation. Theriogenology 63:342–351
Visconti PE (2009) Understanding the molecular basis of sperm capacitation through kinase design. Proc Natl Acad Sci USA 106:667–668
Acknowledgments
We wish to thank Dr. L. Mayorga for valuable discussions and critical reading, graduate student L. Pelletán for art work, Drs. C. Tomes, G. De Blas, and J. Castillo Bennett for critical reading, graduate students M. Bustos and L. Pelletán for extensive contribution in protocol details and critical reading. This work was supported by grants from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina and Secretaría de Ciencia, Técnica y Postgrado, National University of Cuyo, Argentina (to SAB).
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Belmonte, S.A., Suhaiman, L. (2012). Optimized Protocols to Analyze Sphingosine-1-Phosphate Signal Transduction Pathways During Acrosomal Exocytosis in Human Sperm. In: Pébay, A., Turksen, K. (eds) Sphingosine-1-Phosphate. Methods in Molecular Biology, vol 874. Humana Press. https://doi.org/10.1007/978-1-61779-800-9_9
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