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 [1-14C]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 microM) reported to inhibit human phospholipases A2 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 Bx (IC50 < or = 10 mg/kg) or by pretreatment of the enzyme with 4-bromophenacyl bromide. Human sperm phospholipase A2 (10 micrograms) 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 microM prostaglandin Bx. Sperm penetration of zona pellucida-free hamster oocytes was inhibited in a dose-dependent fashion when sperm were incubated with prostaglandin Bx (IC50 approximately 15 microM) during capacitation; sperm motility was not affected by this treatment. Capacitation in the presence of prostaglandin Bx 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.
Fry
M R
MR
Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University, Richmond 23298.
Ghosh
S S
SS
East
J M
JM
Franson
R C
RC
D000177:Acrosome / Q000187:drug effects / Q000502:physiology; D000704:Analysis of Variance; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004487:Edema / Q000139:chemically induced; D005227:Fatty Acids / Q000096:biosynthesis; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000502:physiology*; D054467:Phospholipases A2; D011108:Polymers / Q000494:pharmacology*; D011456:Prostaglandins B / Q000494:pharmacology*; D013084:Sperm-Ovum Interactions / Q000187:drug effects / Q000502:physiology*
D005227:Fatty Acids; D011108:Polymers; D011456:Prostaglandins B; C034467:prostaglandin B1; C024303:prostaglandin Bx; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium