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

Abstract

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 between the acidic acrosomal lumen and the suspending medium, due to pore formation between outer acrosomal and plasma membranes. In earlier work, it was shown that this pH gradient loss occurred in single sperm bound to structurally intact zonae pellucidae with a half-time of 2.1 min; the extended kinetics 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 constraint should be removed in solubilized zonae. The fluorescent probe, Dapoxyl(TM) (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 aqueous solution, but which accumulates in the acidic acrosomal compartment due to the pH gradient with highly enhanced fluorescence; loss of the pH gradient leads to a decrease in fluorescence. The half-time for DAES fluorescence 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 hypothesis of steric constraint in the structurally intact zona pellucida. The change in intracellular Ca(2+) concentration in response to solubilized zona 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 state level higher than the initial level, indicating this Ca(2+) transient as the precursor reaction to onset of the zona-induced acrosome reaction.

Expression, localization and functions in acrosome reaction and sperm motility of CaV3.1 and CaV3.2 channels in sperm cells: An evaluation from CaV3.1 and CaV3.2 deficient mice

“…These values fall in the range of previously described ZP‐induced kinetics. Indeed, a wide range of ZP‐induced calcium increase kinetics have been reported in the past and peaks were reached from few seconds (Shirakawa and Miyazaki, 1999; Rockwell and Storey, 2000; Fukami et al, 2003) to minutes (Arnoult et al, 1996; Fukami et al, 2003). This range is likely due to experimental differences between groups, like ZP concentration, capacitation time or medium composition.…”

“…All experiments were carried out at 37°C in a continuous flow of medium. Solubilized zona pellucida, prepared as precisely described (Rockwell and Storey, 2000), was introduced in the recording medium by adding 10 µl of a solution containing 20 solubilized ZP by µl (final volume 100 µl). Thus, the final solubilized ZP concentration is 2 ZP/µl.…”

Zona pellucida from fertilised human oocytes induces a voltage-dependent calcium influx and the acrosome reaction in spermatozoa, but cannot be penetrated by sperm

“…Using spectrofluorimetry, we found that the calcium response to hZP may be biphasic or monophasic, depending on the sample. Previous studies using spectrofluorimetry and a similar probe showed that the Ca 2+ response to solubilised mouse ZP [33] or recombinant hZP3 [5,6] was biphasic, with a first transient peak and a second sustained phase. However, in nine of the 21 cases analysed in our study, the calcium response appeared to be monophasic, with only sustained increase in calcium concentration.…”