Therefore, this unusual stability may due to the additional capacitation-dependent protein-protein interactions or the raftspecific binding of complexin with the trans-SNARE complex. The weak detection of the raft specific complexin-containing complex might be explained by the alteration of the binding epitode or the reduced accessibility for the antibody during the DRM isolation where it may have become cryptic for the antibody under the artificial Triton-treated conditions. From the proteomic analysis of the MVs, we found a number of proteins from the OAM and luminal matrix are only recovered in the MVs from sperm that were bicarbonate treated and subsequent challenged with Ca2+ ionophore. Some of the identified proteins have affinity for glycosylated proteins and thus may serve to establish the firm secondary sperm-zona binding after AE. The fact that such proteins were not detected in MVs from control sperm provides an indication that the sperm surface rearrangement and the concomitant reordering of the interacting OAM are functionally relevant for secondary R428 spermzona binding. The proteomic data also identified a number of proteins that are known to be involved in the regulation of SNARE-mediated exocytosis. Indeed, apart from the proteomic detection of above mentioned SNARE regulators, we observed the similar capacitation-dependent redistribution of these Staurosporine PKC inhibitor regulating proteins to the same apical area of the sperm head where SNARE proteins and the raft marker protein flotillin 1 have been detected during sperm capacitation. Moreover, SNARE regulators were no longer detected at the apical ridge in the acrosome reacted sperm which strongly suggests their incorporation with the SNARE protein complex and their release from the sperm surface during the shedding of the MVs in which they are then captured after AE. In summary, we have detected a specific set of SNARE proteins that form a trimeric SNARE complex upon the induction of AE. This newly identified SNARE complex differs from the previously identified syntaxin 1B/SNAP 23/VAMP 3 complex that is responsible for the stable docking of acrosome to the PM. The binding and the dissociation of complexin 2 from this VAMP 2 containing SNARE complex demonstrates the dynamic interactions between different trimeric SNARE complexes and complexin 2 upon capacitation and AE. Moreover, apart from interacting with trimeric SNARE complexes, a separate complexin sub-population interacted with the prefusion SNAREpin and, by doing so, likely serves to prevent the preliminary fusion of the membranes at the non-apical sperm head area. When AE was induced by a Ca2+ ionophore, complexin 2 dissociates from the SNARE complex and allows the participation of complementary R-SNARE for the completion of AE. We postulate that the specific docking of the acrosome with the sperm surface is required to recruit certain secondary zona binding proteins at the surface as soon as AE is initiated by the ZP.