Phospholipase A2 activity and exocytosis of the ram sperm acrosome: regulation by bivalent cations

Previous work has shown that the sequence leading to exocytosis of the sperm acrosome involves at least three Ca(2+)-requiring processes, the first one probably represented by breakdown of the polyphosphoinositides and the final one by membrane fusion. We have investigated whether phospholipase A2 (PLA2) represents the intermediate Ca(2+)-requiring event by stimulating ram spermatozoa with the ionophore A23187 and various bivalent cations. Spermatozoa prelabelled with [14C]arachidonic acid and treated with ionophore and millimolar Ca2+ showed a considerable release of arachidonic acid; parallel sperm samples similarly treated underwent acrosomal exocytosis. Mn2+ was capable of completely substituting for Ca2+, even if residual Ca2+ in the system was chelated with EGTA: both arachidonic acid release and acrosomal exocytosis took place after treatment with A23187, EGTA and Mn2+. Neither Mg2+ nor Ba2+ promoted arachidonic acid release or exocytosis. The effects of Sr2+ were more complex and allowed us to probe the sequence of events leading to membrane fusion. Both arachidonic acid release and exocytosis occurred after treatment with A23187 and Sr2+ but none of these responses were seen if EGTA was also included. These results suggest that residual micromolar Ca2+ is either needed for Sr2+ to fully promote PLA2 activity, or that micromolar Ca2+ is needed for one or more upstream events that may in turn serve to activate PLA2. Evidence for or against the first possibility was sought by examining PLA2 activity in sperm sonicates. Enzyme activity was maximal in the presence of any bivalent cation and it was not reduced (in the case of Sr2+) or only reduced slightly (Mg2+, Mn2+, Ba2+) if residual Ca2+ was chelated with EGTA; this indicates that Sr2+ can promote PLA2 activity in the total absence of Ca2+. The second possibility was explored by treating spermatozoa with A23187 for 5 min (to allow for complete phosphoinositide breakdown; Roldan and Harrison (1989) Biochem. J. 259, 397-406), and then adding EGTA and Sr2+. This resulted in neither arachidonic acid release nor exocytosis, thus indicating that another as yet unidentified Ca(2+)-dependent event may occur before PLA2 activation.