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Optimized Protocols to Analyze Sphingosine-1-Phosphate Signal Transduction Pathways During Acrosomal Exocytosis in Human Sperm

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Book cover Sphingosine-1-Phosphate

Part of the book series: Methods in Molecular Biology ((MIMB,volume 874))

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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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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Authors and Affiliations

  1. Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología (IHEM-CONICET), Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Mendoza, Argentina

    Silvia A. Belmonte & Laila Suhaiman

Authors
  1. Silvia A. Belmonte
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  2. Laila Suhaiman
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Correspondence to Silvia A. Belmonte .

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Editors and Affiliations

  1. University of Melbourne, Parkville, Victoria, Australia

    Alice Pébay

  2. Sprott Centre for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, Smyth Road 501, Ottawa, K1Y 8L6, Canada

    Kursad Turksen

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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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  • DOI: https://doi.org/10.1007/978-1-61779-800-9_9

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-799-6

  • Online ISBN: 978-1-61779-800-9

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