Therefore, the intracellular calcium concentration regulated by the above mentioned proteins might be crucial for the SNARE complex-complexin-synaptotagmin interactions and thus directly or indirectly be involved in sperm AE. In the present study, we investigated whether or not the trimeric trans-SNARE complexes, which we have previously found aggregated in membrane rafts in capacitated sperm, are stabilized by XAV939 complexin and/or other interacting proteins. Moreover, the SNARE complex and interacting proteins found in mixed vesicles that are formed after calciumionophore induced AE were identified. The role of sperm membrane rafts in the stabilization of SNARE complexes and the relevant SNARE associating proteins that were found by proteomic detection are discussed with respect to their putative functions in sperm-zona binding and the membrane fusion events involved in AE. Results Acrosomal exocytosis and the formation of mixed vesicles is calcium dependent and facilitated by bicarbonate AE was induced in incubated sperm cells treated with 5 mM Ca2+ ionophore A23187 in both control non-activated and bicarbonate capacitated sperm, albeit that the efficiency of AE in capacitated sperm was much higher. The resulting formation of unilamellar vesicles contained both PM and OAM material and hence are called mixed vesicles. These MVs could be isolated from other sperm structures via differential centrifugation, and their ultrastructural appearance was visualized with transmission electron microscopy. MVs obtained under both conditions had a unilamellar membrane structure. However, in the absence of bicarbonate, MVs were less homogeneous in size and in shape Nilotinib compared with the MVs formed in the presence of bicarbonate. Besides the differences in their ultrastructure, it appeared that in ionophore treated cells in the presence of bicarbonate, acrosomal material was present at only one side of the MVs. This was in contrast to the ionophore treated sperm in the absence of bicarbonate that resulted in a more homogeneous distribution of this material around the entire MVs surface. To elucidate whether membrane rafts play a role in the SNARE proteins- complexin interaction, the widely applied detergentbased isolation protocol to isolate membrane rafts was used. We first characterized membrane raft fractions from these Triton-treated sperm samples with the raft marker protein flotillin 1. The 40 kDa complexin positive band reflects the germ-line specific stable dimeric form of complexin. We speculate that the 79 kDa complexin positive complex represents the recently reported pre-fusion SNAREpin-complexin complex that consists of two QSNARE proteins and complexin. To investigate this possibility, we performedWestern-blotting of the sucrose gradient fractions from sperm samples with antibodies against potential SNAREpin forming candidates.