DAG STIMULATES ACROSOMAL EXOCITOSIS AND INCREASES PIP2 IN A PKC, PLD AND ARF6 DEPENDENT PATHWAY

LOPEZ, CECILIA I; PELLETAN, LEONARDO E; SUHAIMAN, LAILA; 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

Fertilization is the process in which the egg and the spermatozoa fuse and a zygote is formed. When sperm cell reaches the egg must undergo acrosomal exocytosis (AE) to get through the zona pellucida and fertilize the egg. During AE outer acrosomal membrane and plasma membrane get fused in multiple points, acrosomal enzymes are released and inner acrosomal membrane is exposed. Multiple signaling pathways are involved finally leading to SNARE protein complexes assembly and membrane fusion. Because spermatozoa are transcriptional and translationally inactive, powerful tools like overexpresion or interference RNA cannot be used. To overcome these difficulties we developed a SLO permeabilized spermatozoa model, which permits us to study the role of different proteins in AE. Permeabilized spermatozoa can be stimulated with calcium resembling the opening of SOC channels. Considering that diacylglycerol (DAG) is a well-known AE inducer and that its signaling pathway is still unveiled, we decided to explore the signal transduction cascade stimulated by DAG downstream the opening of SOCCs. First, we determined that DAG and its analog PMA stimulate AE in permeabilized spermatozoa in a calcium-independent manner. Because DAG interacts with PKC through its C1 domain, we tried the PKC inhibitors cheleritrin and Ro-31-7549 in a functional assay. Both inhibitors abrogated calcium and DAG-elicited AE suggesting that PKC is involved in exocytosis induction. PLD1 interacts with PKC. In some cellular systems, is a PKC alpha effector but in others is upstream the kinase. Here, we demonstrated by indirect immunofluorescence that PLD1 is present in the acrosomal region of human spermatozoa. 1-butanol, a specific anti-PLD1 antibody and a phosphatydic acid binding domain (PABD) inhibited calcium and PMA-triggered AE. Phosphatydic acid (PA) rescued PKC activity inhibition thus we conclude that PLD1 is downstream of PKC in DAG induced AE. Western Blot demonstrated that PMA and calcium ionophore A23187 led to PLD1 membrane recruitment, confirming that both inducers activate the phospholipase. Because Arf6 has been described as a PLD1 activating protein, we decided to analyze its role in AE. By using Western blot and indirect immunofluorescence we demonstrated that Arf6 is present in human spermatozoa and is localized at the acrosomal region. Myristoilated and GTPgammaS loaded Arf6 triggered AE meanwhile myristoilated and GDPbetaS loaded Arf6 inhibits calcium-triggered AE. A monoclonal anti-Arf6 antibody inhibits calcium and DAG-induced AE indicating that Arf6 is involved in calcium and DAG-induced AE. PLD1 inhibitors abrogated Arf6-triggered AE which was reversed by PA indicating that PLD is downstream Arf6 in the signal transduction cascade and that Arf6 probably stimulates PLD1 activation. In other cell types, a kinase involved in PIP2 synthesis is activated by PA and Arf6, so we hypothesized that PMA-triggered exocytosis could be dependent of PIP2. PLC delta 4-PH domain and a PIP2 specific antibody inhibited calcium, PMA and Arf6-triggered AE which were reversed by PIP2. Moreover, PIP2 rescued PKC, PLD1 and, Arf6 inhibition consisting with the activation of the kinase involved in PIP2 synthesis. To elucidate if phospholipids were produced during the AE we performed a TLC for 32P labeled spermatozoa phospholipids and we determined that calcium, PMA, PA and Arf6 leads to a rise of PIP2 and PIP3. Indirect immunofluorescence against PIP2 showed that PMA stimulation leads to a rise in fluorescence intensity. In our laboratory we have collected strong evidence suggesting that two principal pathways are activated during stimulation of AE, one leads to SNARE complex assembly while the other produces an IP3 dependent acrosomal calcium release necessary for membrane fusion. IP3 dependent calcium channel blocker, xestospongine C, avoided Arf6-dependent AE indicating that this pathway is activated before IP3 dependent acrosomal calcium release. The IP3 analog, adenophostin, rescued PLD1 and Arf6 inhibition. These results lead us to conclude that PIP2 increase is necessary to produce IP3 and acrosomal calcium release. Taking in account all the results presented here, we propose a model where DAG triggers a pathway leading to keep a pool of PIP2 in spermatozoa necessary for IP3 dependent acrosomal calcium release.