A time course study of capacitation and the acrosome reaction in human spermatozoa using a revised chlortetracycline pattern classification

OBJECTIVE: To study the time course of capacitation, spontaneous, and A23187-induced acrosome reaction of human spermatozoa during 8 hours incubation in vitro using the chlortetracycline (CTC) assay with a revised fluorescent pattern classification.

DESIGN: Fertile donor spermatozoa were isolated by direct swim-up and incubated in Earle's balanced salt solution for up to 8 hours. At hourly intervals, spermatozoa were stained with CTC before and after the addition of A23187 to induce the acrosome reaction.

SETTING: The University Clinic, Jessop Hospital for Women, Sheffield, United Kingdom.

PATIENTS: Donors participating in the Donor Insemination Program.

MAIN OUTCOME MEASURES: Eight fluorescent patterns identified by the CTC assay and acrosome-reacted spermatozoa detected by indirect immunofluorescence using 18.6 monoclonal antibody.

RESULTS: Using a statistical model defined by analysis of deviance allowed rationalization of the CTC pattern classification by grouping together patterns that showed a similar and significant change over time. In addition, spontaneous and A23187-induced acrosome-reacted spermatozoa identified by the CTC assay were shown to be correlated significantly to those identified by indirect immunofluorescence.

CONCLUSION: The CTC assay using a revised pattern classification offers a more precise description of human spermatozoa capacitation in vitro. Also, CTC-identified acrosome reaction (both spontaneous and A23187 induced) was confirmed independently by indirect immunofluorescence.