ROLE OF HUMAN SPERM PHOSPHOLIPASE-A2 IN FERTILIZATION - EFFECTS OF A NOVEL INHIBITOR OF PHOSPHOLIPASE-A2 ACTIVITY ON MEMBRANE PERTURBATIONS AND OOCYTE PENETRATION

Phospholipase A2 was isolated from human sperm and its potential role in the membrane fusion events of fertilization was examined. Highly purified enzyme hydrolyzed the phospholipids of [1-C-14]oleate-labeled Escherichia coli optimally at neutral to alkaline pH with 5 mM CaCl2 and 150 mM NaCl (specific activity = 20 mumol/min/mg). Activity was inhibited in a dose-dependent manner by an oligomer of prostaglandin B1 (IC50 = 1.5 muM) reported to inhibit human phospholipases A, in vitro and in situ. Sperm phospholipase A2 injected into mouse foot pad induced a dose-dependent edema that was inhibited by oral administration of prostaglandin B(x) (IC50 less-than-or-equal-to 10 mg/kg) or by pretreatment of the enzyme with 4-bromophenacyl bromide. Human sperm phospholipase A2 (10 mug) induced fusion of phosphatidylserine vesicles in the presence of 1 mM calcium chloride by approximately 80% (+/- 10%) as determined by monitoring turbidity (O.D.400) and efficiency of fluorescence resonance energy transfer. This enzyme-induced fusion was accompanied by phospholipid hydrolysis, and both fusion and phospholipid degradation were inhibited by more than 60% when enzyme was preincubated with 5 muM prostaglandin B(x). Sperm penetration of zona pellucida-free hamster oocytes was inhibited in a dose-dependent fashion when sperm were incubated with prostaglandin B(x) (IC50 almost-equal-to 15 muM) during capacitation; sperm motility was not affected by this treatment. Capacitation in the presence of prostaglandin B(x) had little to no effect on the in vitro acrosome reaction. These results suggest that sperm phospholipase A2 and its modulators may contribute to membrane fusion events in mammalian fertilization.