SPHINGOSINE 1-PHOSPHATE AND SPHINGOSINE KINASE ARE INVOLVED IN A SIGNALING PATHWAY LEADING TO ACROSOMAL EXOCYTOSIS

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

Buenos Aires, Argentina

Congreso; 4th International Meeting, Latin-American Society for Developmental Biology.; 2008

Sociedad Latinoamericana de Biología del Desarrollo

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid metabolite that regulates crucial physiological processes. S1P acts as a second messenger inside the cell, and as an extracellular mediator through binding to five G protein-coupled receptors (S1PR). A human spermatozoon is a specialized cell necessary to fertilize an oocyte and pass on the genetic information contained in its nucleus. In the proximity of the egg, zona pellucida glycoproteins stimulate mammalian sperm to release the content of the acrosomal granule, a key event in fertilization. The acrosome is a large membrane-limited granule that overlies the nucleus of the mature spermatozoon. Regulated secretion is a fundamental process underlying the function of many cell types. In particular, acrosomal exocytosis in mammalian sperm is essential for egg fertilization. Acrosomal exocytosis is an all-or-nothing event that involves the opening of multiple fusion pores between the outer acrosomal membrane and the plasma membrane. Here, we present evidences reporting a novel function for the sphingolipid: S1P triggers acrosomal exocytosis in capacitated human sperm in a dose-dependent manner (ED50, 10.94 nM). Our results suggest that S1P triggers exocytosis by a receptor-mediated mechanism rather than by an intracellular second messenger pathway. By using a pharmacological approach in a functional assay, we demonstrated that the fusion process triggered by S1P depends on extracellular calcium and requires extracellular calcium influx through Voltage Operated Calcium Channels and Store Operated Calcium Channels. We also demonstrate by using specific IP3-sensitive calcium channels inhibitors (2-APB and xestospongin C) that S1P-elicited exocytosis needs calcium efflux from intracellular stores. Single cell calcium measurements confirmed that S1P addition increases intracellular calcium in live sperm. Pretreatment of human sperm with pertussis toxin abrogated S1P-triggered exocytosis, suggesting that the sphingolipid utilizes a Gi protein-linked signaling pathway. Functional assays demonstrate that S1P-induced acrosome reaction requires PLC, PKA and, PKC activation. We also show the first evidence implying Rab3A, a GTPase involved in exocytosis in diverse cellular types and in sperm cells, downstream the signal transduction cascade induced by S1P. Under physiological conditions, S1P is generated from phosphorylation of sphingosine by the action of sphingosine kinase (SK). Because our results were obtained by addition of exogenous S1P we must consider two possibilities, 1) exogenous S1P can be synthesized in the female genital tract or 2) sperm cells can be able to produce S1P by themselves when challenged by a stimulus. Western blot assays showed that SK1 is present in human sperm cells and translocates from cytosol to membranes when calcium or phorbol ester (PMA) stimuli impact the cell. By indirect immunofluorescence assays, we demonstrated that under control conditions, most of the cells showed a postacrosomal or a diffuse immunolabeling pattern. If SK1 was involved in the acrosomal granule exocytosis we expected an acrosomal staining. Thus, we analyzed the staining pattern after a PMA stimulus. The fluorescent stain changed to the acrosomal region of most cells. These results indicate that PMA causes a redistribution of SK1 to the acrosomal region. To elucidate if S1P is produced by the spermatozoa and thus SK is involved in the acrosome reaction, we used DMS, a competitive inhibitor of SK, in functional assays. DMS abrogated PMA induced exocytosis suggesting the requirement of SK activity in PMA-induced exocytosis. Its effect was reversed by S1P confirming the involvement of SK and S1P in the diacylglycerol (DAG) exocytotic pathway. The last finding confirms that SK1 is active in sperm cells rendering S1P after a DAG stimulus, and suggests that the S1P exocytotic effect is exerted through its autocrine /paracrine action. Our results indicate that a SK1/S1P signaling pathway is implied in acrosome reaction. This discovery may have important implications to provide some clues about new molecules involved in exocytosis and to a better understanding of how human spermatozoa undergo acrosome reaction and consequently fertilize eggs.