Optimized protocols to analyze sphingosine 1-phosphate signal transduction pathways during acrosomal exocytosis in human sperm

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 recently reported that sphingosine 1-phosphate and sphingosine kinase are involved in a novel signaling pathway leading to acrosomal exocytosis. 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 non-permeabilized 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.