Kinetics of onset of mouse sperm acrosome reaction induced by solubilized zona pellucida: Fluorimetric determination of loss of pH gradient between acrosomal lumen and medium monitored by Dapoxyl (2-aminoethyl) sulfonamide and of intracellular Ca2+ changes monitored by fluo-3

The onset of the zona pellucida-induced acrosome reaction in mouse sperm is marked by loss of the pH gradient existing in acrosome-intact sperm betwee n the acidic acrosomal lumen and the suspending medium, due to pore formati on between outer acrosomal and plasma membranes. In earlier work, it was sh own that this pH gradient loss occurred in single sperm bound to structural ly intact zonae pellucidae with a half-time of 2.1 min; the extended kineti cs of this loss determined in a sperm population bound to intact zonae was due to a 180-min range of variable lag times. We hypothesized that this lag time range was due to steric constraints imposed by the three-dimensional structure of the structurally intact zona pellucida, and that this constrai nt should be removed in solubilized zonae. The fluorescent probe, Dapoxyl(T M) (2-aminoethyl)sulfonamide (DAES) allowed a test of this hypothesis in a population of sperm cells. It is a weak base that is non-fluorescent in aqu eous solution, but which accumulates in the acidic acrosomal compartment du e to the pH gradient with highly enhanced fluorescence; loss of the pH grad ient leads to a decrease in fluorescence The half-time for DAES fluorescenc e loss in a population of capacitated, acrosome-intact sperm in response to solubilized zona pellucida protein was 2.13 +/- 0.10 min (SEM, n = 9). The agreement between single cell and cell population kinetics validates the h ypothesis of steric constraint in the structurally intact zona pellucida. T he change in intracellular Ca2+ concentration in response to solubilized zo na pellucida, as monitored with fluo-3, was a rapid increase, followed by a decrease, with a half-time of 0.85 +/- 0.09 min (SEM, n = 6) to a steady s tate level higher than the initial level, indicating this Ca2+ transient as the precursor reaction to onset of the zona-induced acrosome reaction. Mol . Reprod. Dev. 55:335-349, 2000. (C) 2000 Wiley-Liss, Inc.