Lipid-induced acrosomal exocytosis analyzed in vivo by TIRFM.

PELLETAN, LEONARDO E; DE BLAS, GERARDO A; SUHAIMAN, LAILA; VITALE, NICOLAS; MAYORGA, LUIS S; BELMONTE, SILVIA A

Potrero de los Funes, San Luis

Congreso; XLVII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular. SAIB; 2011

SAIB

During fertilization, the spermatozoon penetrates the zona pellucida to reach the oolema. Only sperm that have completed the acrosome reaction (AR) can accomplish this task. The AR is a calcium regulated exocytosis where the membrane of the acrosome fuses to the plasma membrane. Sphingosine 1-phosphate, diacylglicerol and phorbol esters trigger the AR. The aim of this research was to analyze in vivo the dynamics of exocytosis in individual live spermusing TIRF, confocal microscopy and calcium variations in real time during exocytosis. Lysotracker allowed to localize the field in TIRFM and PSA-FITC stains the acrosome. We tested the response to different lipids in permeabilized and non-permeabilized cells (intact). Kinetics of acrosomal content losing between both groups varied greatly. In intact sperm AR started at the cell apex followed by a slow dispersal of acrosomal content. Permeabilized sperminitiated the AR at the ecuatorial segment and it finished in seconds. High resolution of TIRFM allowed the observation of different intensity spots representing fusion pores. Measurements ofintracellular calcium using Fluo-3 AM yielded an immediate calcium increase after stimuli in intact cells and the emptying of the acrosomal store in permeabilized sperm. Our data provide a real time view of lipid-activated exocytosis in a cell in which this fusion is a single and irreversible step.