Physiological induction of the acrosome reaction in human sperm: Validation of a microassay using minimal volumes of solubilized, homologous zona pellucida
Purpose: To develop a method that could accommodate microvolumes of solubilized human zona pellucida (ZP) and sperm for assessing the induction of the acrosome reaction. Methods: A microassay using 1 μl of 2.5, 1.25, 0.6, 0.3, and 0.125 ZP/μl incubated with 1 μl of a highly motile sperm suspension for 60 min. As a control and parallel to the microassay a standard acrosome reaction technique was performed. Results: No significant differences were observed between the percentage acrosome reacted sperm reported by the two assays under basal conditions (spontaneous) or after induction with a Ca2+ ionophore or solubilized ZP. At a ZP concentration of 0.6 ZP/μl, the percentages of acrosome-reacted spermatozoa in both techniques were significantly higher compared to the spontaneous acrosome reaction results, namely, 18% and 17%, compared to 10% and 10%, respectively. An approximately 30% level of acrosomal exocytosis was induced with 2.5 ZP/μl in both methods. Conclusions: This newly devised microtechnique is easy and rapid to perform, is repeatable and facilitates the use of minimal volumes of solubilized human ZP (even a single ZP) for assessment of the inducibility of the acrosome reaction of a homologous sperm population.