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A time course study of capacitation and the acrosome reaction in human spermatozoa using a revised chlortetracycline
R L Perry1, M Naeeni, C L Barratt
1Department of Obstetrics and Gynaecology, University of Sheffield, United Kingdom.
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.