Artificial induction of exocytosis in bull sperm

We have investigated an exocytotic event, the acrosome reaction (AR), induced by treatment of bovine sperm with vesicles composed of dilauroyl phosphatidylcholine (PC12). Cell membrane permeability barriers (dye exclusion), acrosomal status (pisum sativum (PSA) lectin binding), and intracellular Ca2+ (Fluo3 fluorescence) were evaluated utilizing flow cytometry and fluorescence microscopy. By these methods the AR is resolved into four kinetically distinct steps: (a) PC12 transfer to the sperm plasma membrane (PM); (b) increased permeability of the PM to extracellular Ca2+; (c) localized leakage of acrosomal contents at the anterior tip of the sperm; and (d) vesiculation of sperm membranes and complete exposure of acrosomal contents. Evidence for PC12 transfer to sperm includes transfer of a fluorescent PC12 analogue from vesicles to cells and the absence of detectable vesicle--cell fusion. The fusion inducing properties of PC12 appear to reside in the lipid head group as neither dilauroylphosphatidylethanolamine nor dilauroylphosphatidylglycerol stimulated the AR. The effect of PC chain length on AR induction closely parallels the aqueous phase solubility of the lipid tested. The rate and extent of the AR depend on the extracellular calcium concentration. Cells treated in the absence of calcium do not undergo the AR, but do so rapidly (less than 1 min) upon subsequent addition of calcium. This role of Ca2+ is partially filled by Sr2+, but not by Ba2+ or Mg2+. The rate of the AR decreases with decreasing temperature and the AR occurs very slowly below 27 degrees C. Simultaneous evaluation of intracellular calcium and acrosomal status reveals the kinetic relationship between Ca2+ influx and the exposure of acrosomal contents. N-Ethylmaleimide preincubation arrests PC12-treated sperm at an intermediate stage in the AR, characterized by punctate PSA binding over the tip of the sperm head. The AR, a developmentally regulated, receptor-mediated fusion event, synchronously induced here in vitro, provides a useful model for mechanistic studies of exocytosis.