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 i ncubation in vitro using the chlortetracycline (CTC) assay with a revi sed fluorescent pattern classification. Design: Fertile donor spermato zoa 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 th e acrosome reaction. Setting: The University Clinic, Jessop Hospital f or Women, Sheffield, United Kingdom. Patients: Donors participating in the Donor Insemination Program. Main Outcome Measures: Eight fluoresc ent patterns identified by the CTC assay and acrosome-reacted spermato zoa detected by indirect immunofluorescence using 18.6 monoclonal anti body. Results: Using a statistical model defined by analysis of devian ce allowed rationalization of the CTC pattern classification by groupi ng together patterns that showed a similar and significant change over time. In addition, spontaneous and A23187 induced acrosome-reacted sp ermatozoa identified by the CTC assay were shown to be correlated sign ificantly to those identified by indirect immunofluorescence. Conclusi on: 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.