Ca2+-related changes in the capacitation state of human spermatozoa assessed by a chlortetracycline fluorescence assay

摘要

Chlortetracycline (CTC) fluorescence patterns were used to assess Ca -related changes in the capacitation state of human spermatozoa incubated under conditions that would affect their intracellular Ca2+ levels. Initial experiments were designed to identify consistently occurring patterns and to correlate these with acrosomal status. Incubation for up to 1 h with the ionophore A23187 (10 μmol l?1), known to promote capacitation and acrosomal exocytosis, allowed the identification of three different CTC staining patterns which were very similar to those described for mouse spermatozoa. For this reason, they were given the same nomenclature: 'F' – characteristic of uncapacitated, acrosome-intact cells; 'B' – characteristic of capacitated, acrosome-intact cells; and 'AR' – characteristic of capacitated, acrosome-reacted cells. The distribution of the three patterns in the ionophore-treated suspensions was very different from that in control suspensions treated with dimethylsulfoxide only, with a significantly higher proportion of cells displaying the B and AR patterns and a significantly lower number of cells displaying the F pattern in the ionophore-treated group at all times. A strong concordance was found between the acrosomal status of cells determined using both CTC and fluorescein-conjugated Pisum sativum agglutinin (PSA) staining methods on the same cells. Verification of PSA staining patterns with acrosomal status was obtained by means of transmission electron microscopy. The proportion of cells with uniform fluorescence in the acrosomal region correlated with acrosome-intact cells; those with only equatorial segment staining correlated with fully-reacted cells, and those exhibiting equatorial fluorescence and patchy fluorescence over the rest of the acrosomal region correlated with cells in intermediate stages of exocytosis. Having established and verified the morphological basis for the CTC staining patterns, we then incubated cells in medium containing standard (1.80 mmol l?1) and high (3.60 mmol l?1) CaCl2. In both media the proportion of F cells decreased with time, whereas the B and AR patterns increased, but the high Ca2+ treatment significantly accelerated the change from F to B to AR at all time points. In contrast, when spermatozoa were incubated in a Ca -deficient medium for up to 22 h, the majority of cells displayed the uncapacitated F pattern. The introduction of millimolar Ca2+ during the final 15 min of incubation failed to alter the CTC patterns, thus confirming the fact that human spermatozoa require the continuous presence of extracellular Ca to undergo capacitation and the acrosome reaction. These results suggest that changes in CTC fluorescence patterns indicate Ca2 +-related changes in the functional state of human spermatozoa and therefore that CTC assessment may prove useful in clinical assessment of human sperm fertilizing potential.