Testis

Real-Time Observations of Individual Macaque Sperm Undergoing Tight Binding and the Acrosome Reaction on the Zona Pellucida

Abstract

For the first time in any mammalian species, changes in binding affinity, acrosomal status, and the corresponding changes in motility of living sperm on the zona pellucida were observed directly and analyzed with videomicroscopy. A single zona was air-dried and rehydrated on a microscope slide, and a cover slip supported by glass beads was added. Capacitated sperm were added together with SBTI-Alexa, a probe for acrosin which can detect the acrosome reaction. The heads of loosely attached sperm oscillated on the zona and the flagella beat symmetrically with a sigmoid-shaped waveform. After 16 sec, tight binding was observed as the sperm head became fixed in place on the zona. The shape of the flagellar beat simultaneously shifted to a more rigid, "c"-shaped waveform. Within 11 sec of tight binding, the first signs of the acrosome reaction were detected. Rapid flushing removed approximately 65% of sperm that were loosely attached but only 2% of tightly bound sperm. In the 2 min following the onset of tight binding, the lateral displacement of the flagellum increased by approximately 30% and the beat frequency decreased by 25%. Lignosulfonic acid (LSA) inhibited loose sperm attachment and the development of tight binding. LSA had no effect on the time of the acrosome reaction following tight binding or on changes in motility that followed tight binding. These data suggest that LSA affects initial attachment or "docking" of sperm to the zona, a step that may align or recruit specific zona receptor(s) responsible for mediating the acrosome reaction.