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. Fertil Steril 1995;64:150-9 Key Words: Capacitation, acrosome reaction, chlortetracycline (CTC) assay, indirect immunofluorescence, human spermatozoa, A23187. InThe chlortetracycline (CTC) assay has been reported to be a suitable method to study calciumdependent events in fertilization, such as capacitation and the acrosome reaction of spermatozoa (1). Chlortetracycline binds specifically to membrane-as- Received July 27, 1994; revised and accepted February 13, 1995. * Supported by the Infertility Research Trust, University of Sheffield, Sheffield, United Kingdom. sociated calcium, resulting in a series of fluorescent patterns on the head of sperm, which show a change over time. In nonhuman studies, CTC fluorescent patterns have been classified according to the species studied, in mouse (2), and horse (3). The CTC assay provides a method by which calcium-associated membrane changes on the head of sperm, which occur during the capacitation process, can be observed directly. Hence, the CTC assay provides an area of great interest in reproductive biology. Lee and co-workers (4) first described a classification for CTC patterns in human sperm and were able to quantify the changes in patterns during time course studies. The classification consists off our major patterns: early fresh (EF) for un capacitated sperm, shown by a bright band of fluorescence in

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The present study was designed to investigate the effect of human cervical mucus on capacitation and the acrosome reaction of human spermatozoa and compare its effect to that of a cervical mucus substitute, sodium hyaluronate (Healonid). Spermatozoa from donors of proven fertility were isolated from semen using cervical mucus, Healonid or a direct swim-up (acting as the control). Sperm capacitation and the acrosome reaction were monitored by the chlortetracycline assay. In the mucus-treated group, there was a significantly higher percentage of capacitated spermatozoa, but a low incidence of spontaneous and A23187-induced acrosome reactions compared to the control. The use of Healonid during sperm isolation mimicked the effect of mucus relatively successfully. Since mucus and Healonid show very little chemical similarity, this finding would imply that cervical mucus exerts a physical effect during its interaction with spermatozoa, although a chemical effect cannot be completely dismissed. In conclusion, this study confirms early reports describing the ability of cervical mucus to capacitate spermatozoa but at the same time conserve sperm function. The finding that Healonid exerts an almost identical effect on spermatozoa would lend support to its use as a cervical mucus substitute during in-vitro fertility assessments and research studies.

The aim of this study was to investigate the effect of an internal calcium ATPase inhibitor, TBQ, on human sperm capacitation and the acrosome reaction during incubation in a calcium-depleted media. Sperm were isolated into and incubated for up to 6 hr in media depleted of Ca2+ and two Ca(2+)-containing media controls. At set time intervals, sperm in each media group were treated with 100 microM TBQ for 5 min. Afterwards, sperm were induced to acrosome react using the divalent cation ionophore, A23187, as a measure of sperm fertilizing potential. It was established, using the Chlortetracycline assay, that incubation of sperm in a Ca(2+)-depleted media inhibited or delayed sperm capacitation resulting in fewer spontaneous or A23187-induced acrosome reacted sperm. However, incubation of sperm in a Ca(2+)-depleted media did not appear to inhibit sperm motility. The treatment of sperm with TBQ during their incubation in Ca(2+)-depleted media was found to have very little effect resulting in low numbers of capacitated and acrosome reacted sperm. The results from this study suggest that human sperm have an obligatory requirement for extracellular calcium during capacitation and the acrosome reaction, but may require either very little extracellular Ca2+ to maintain motility or possess internal Ca2+ stores sufficient for their requirements. In addition, TBQ did not increase the number of capacitated and acrosome reacted sperm during incubation in a Ca(2+)-depleted media suggesting that the TBQ-effect of accelerating sperm capacitation is dependent on presence of extracellular Ca2+.

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