Sphingosine 1-phosphate and sphingosine kinase 1 are involved in a novel signaling pathway leading to human sperm acrosomal exocytosis

SUHAIMAN, LAILA; DE BLAS, GERARDO A; OBEID, LINA M; DARSZON, ALBERTO; MAYORGA, LUIS S; BELMONTE, SILVIA A

Charleston, Carolina del Sur. Estados Unidos

Conferencia; 5th International Charleston Ceramide Conference; 2009

Charleston Ceramide Conference

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that regulates crucial physiological processes. S1P acts as an intracellular second messenger and as an extracellular signal through binding to five G protein-coupled receptors. Regulated secretion is a calcium-dependent membrane fusion process and a central issue for the specific function of many cells; for instance, mammalian sperm acrosomal exocytosis is essential for egg fertilization. Here we report a novel function for S1P, that of triggering acrosomal exocytosis in capacitated human sperm in a dose-dependent manner. By using a pharmacological approach in functional assays, we demonstrated that the fusion process triggered by S1P requires calcium influx from the extracellular medium through voltage-gated and store operated calcium channels as well as calcium efflux from the secretory vesicle (the acrosome). Single cell calcium measurements confirmed that S1P addition increases intracellular calcium levels in live sperm. Pertussis toxin blocked S1P-induced exocytosis suggesting that its effect is exerted through a Gi coupled receptor. S1P-induced acrosome reaction required PLC, PKA, and PKC activation. We provide the first piece of evidence implying Rab3A, a GTPase involved in exocytosis, downstream the signal transduction cascade initiated by S1P. Under physiological conditions, S1P is generated from phosphorylation of sphingosine by sphingosine kinase (SK). Thus, we hypothesized that S1P could come from two different sources, 1) S1P can be synthesized in the female genital tract or 2) sperm cells are able to produce S1P by themselves when challenged by a stimulus. Western blot assays indicated that SK1 is present in human sperm cells and translocates from cytosol to membranes when calcium or a phorbol ester (PMA) acrosome reaction-stimuli impact the cell. Indirect immunofluorescence showed that PMA causes a redistribution of SK1 to the acrosomal region. Furthermore, DMS and SKI, specific inhibitors of SK, inhibited the PMA-induced exocytosis. The last finding confirms that SK1 is active in sperm cells rendering S1P after a PMA stimulus, and suggests that the S1P exocytotic effect is exerted through an autocrine /paracrine action. Our results indicate that a SK1/S1P signaling pathway is implied in acrosome reaction and provide some clues about new molecules involved in exocytosis leading to a better understanding of how human spermatozoa fertilize eggs.