DAG stimulates acrosomal exocitosis and increases PIP2 in a PKC, PLD and ARF6 dependent pathway

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

Buenos Aires, Argentina

Congreso; 4th International Meeting of the Latin American Society of Developmental Biology; 2008

Latin American Society of Developmental Biology

When sperm cell reachs 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 fuse in multiple points. Several signaling pathways lead to SNARE protein complexes assembly and membrane fusion. We developed a SLO permeabilized spermatozoa model where calcium can stimulate AE resembling the opening of SOC channels. By using this model we explored the signal transduction cascade stimulated by DAG downstream the opening of SOCCs. We determined that DAG and PMA stimulate AE in permeabilized spermatozoa in a calcium-independent manner. As PKC is a target for DAG we tried PKC inhibitors and they abrogated calcium and DAG-elicited AE. It has been described that PLD1 and PKC con interact. We demonstrated by indirect immunofluorescence that PLD1 is present in the acrosomal region of human spermatozoa. PLD1 inhibitors and quenching of phosphatydic acid (PA) inhibited calcium and PMA-triggered AE. PA rescued PKC activity inhibition suggesting that PLD1 is downstream of PKC. Western blot demonstrated that PMA and calcium ionophore A23187 led to PLD1 membrane recruitment. Because Arf6 has been described as a PLD1 activating protein, we analyzed 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 GTPγS loaded Arf6 triggered AE meanwhile Arf6 inhibition avoids calcium and DAG-induced AE. PLD1 inhibitors abrogated Arf6-triggered AE which was reversed by PA suggesting that Arf6 stimulates PLD1 activation. In other cell types, a kinase involved PIP2 in synthesis is activated by PA suggesting that Arf6, so we hypothesized that exocytosis could be dependent of PIP2. Quenching PIP2 of inhibited calcium, PMA and Arf6-triggered AE. 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 profermed a TLC for 32P labeled spermatozoa phopholipids 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 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 acrosomal calcium release. The IP3 analog, adenophostin, rescued PLD1 and Arf6 inhibition. These results leads 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.