DAG-induced acrosome reaction requires phosphatidic acid and phosphatidylinositol 4,5-biphosphate

LÓPEZ, CECILIA I; SUHAIMAN, LAILA; PELLETÁN, LEONARDO E; BELMONTE, SILVIA A; MAYORGA, LUIS S

Mar del Plata, Buenos Aires, Argentina

Congreso; XLIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2007

SAIB

The acrosome reaction (AR) is a particular Ca2+-regulated exocytosis. We have developed a SLO-permeabilization model where Ca2+ activates spermatozoa AR resembling the opening of SOC channels. By using this approach, we determined that PKC activators like diacylglicerol (DAG) and phorbol esters (PMA), triggered the AR in permeabilized spermatozoa. This effect was not inhibited by chelating extracellular Ca2+ suggesting that DAG plays a role after the opening of SOC channels. DAG-induced AR was abrogated by PKC inhibitors (cheleritrine, Ro317549) and PLD inhibitors (1-butanol, anti-PLD). These inhibitions were reversed by phosphatidic acid (PA) suggesting that PLD activity is required during the AR downstream PKC activation. In other cell types, PA activates a specific kinase leading to phosphatidylinositol 4,5-biphosphate (PIP2) production. We demonstrated by western blot that PMA stimulation leads to PLD membrane recruitment confirming the previous result. We determined that PIP2 depletion leads to the inhibition of PMA- and Ca2+-induced AR by using PLCdelta4-PH domains and anti-PIP2 antibodies. To confirm this hypothesis, we performed indirect immunofluorescence by using anti-PIP2 antibodies. We observed that PIP2 increases upon PMA stimulation. Altogether these results indicate that DAG, PA and PIP2 are part of the signaling cascade activated downstream the opening of SOC channels.