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Ca(2+) signaling in spermatozoa plays a crucial role during processes such as capacitation and release of the acrosome, but the underlying molecular mechanisms still remain unclear. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca(2+)-releasing second messenger in a variety of cellular processes. The presence of a NAADP synthesizing enzyme in sea urchin sperm has been previously reported, suggesting a possible role of NAADP in sperm Ca(2+) signaling. In this work we used in vitro enzyme assays to show the presence of a novel NAADP synthesizing enzyme in human sperm, and to characterize its sensitivity to Ca(2+) and pH. Ca(2+) fluorescence imaging studies demonstrated that the permeable form of NAADP (NAADP-AM) induces intracellular [Ca(2+)] increases in human sperm even in the absence of extracellular Ca(2+). Using LysoTracker, a fluorescent probe that selectively accumulates in acidic compartments, we identified two such stores in human sperm cells. Their acidic nature was further confirmed by the reduction in staining intensity observed upon inhibition of the endo-lysosomal proton pump with Bafilomycin, or after lysosomal bursting with glycyl-l-phenylalanine-2-naphthylamide. The selective fluorescent NAADP analog, Ned-19, stained the same subcellular regions as LysoTracker, suggesting that these stores are the targets of NAADP action.

Original publication

DOI

10.1016/j.bbrc.2013.12.011

Type

Journal article

Journal

Biochem Biophys Res Commun

Publication Date

10/01/2014

Volume

443

Pages

531 - 536

Keywords

ADP-ribosyl cyclase, Acidic stores, Calcium, NAADP, Sperm, cADPR, Calcium, Calcium Signaling, Cells, Cultured, Humans, Male, NADP, Spermatozoa