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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 chlortetracy-cline (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. |
