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S. C. MILLS and T. W. SCOTT

Previous studies revealed a significant decrease in the content of phospholipids and acyl-esters in spermatozoa during their passage through the epididymis (Scott, Voglmayr & Setchell, 1967). These results prompted us to investigate whether testicular and ejaculated spermatozoa could utilize fatty acids, which presumably become available to the spermatozoa as a result of phospholipid hydrolysis. The experiments described below show that both cell types were able to metabolize added 14C-labelled palmitic and oleic acids.

Testicular and ejaculated semen were obtained using procedures described by Voglmayr, Scott, Setchell & Waites (1967). The cells were incubated under aerobic conditions for 3 hr at 38° C in Krebs-Ringer bicarbonate buffer solution (pH 7·4) containing 14C-labelled fatty acid complexed to bovine serum albumin (using Complex B as described by Kessler,

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S. DasGupta, C. L. Mills and L. R. Fraser

Mammalian spermatozoa require extracellular Ca2+, some of which must be internalized, to undergo capacitation and acrosomal exocytosis. The mechanisms controlling the intracellular Ca2+ concentration are unclear, but current evidence suggests that a Ca2+-ATPase may be involved. Using treatments that potentially modulate enzyme activity, we investigated this possibility in human spermatozoa; the capacitation state and acrosomal integrity were monitored by chlortetracycline fluorescence. Incubation of cells in the presence of quercetin, a Ca2+-ATPase inhibitor, significantly accelerated the transition from the uncapacitated F pattern of chlortetracycline fluorescence to the capacitated, acrosome-intact B pattern within 1 h. This was followed by an increase in the number of cells displaying the capacitated, acrosome-reacted AR pattern. Since most Ca2+-ATPases in somatic cells are sensitive to calmodulin, we also investigated the effect of the calmodulin antagonist W-7 on chlortetracycline patterns. At 1–125 μmol l−1, W-7 significantly stimulated capacitation and acrosomal exocytosis. Furthermore, W-7 at 1 μmol l−1 proved to be more effective than W-5, a less potent antagonist, suggesting that the observed responses in human spermatozoa did reflect a calmodulin-sensitive mechanism. When the glucose concentration in the culture medium was varied (from 0 to 5.56 mmol l−1) to alter the availability of ATP for enzyme activity, it was found that a reduced concentration of glucose promoted capacitation more rapidly than did the standard concentration of 5.56 mmol glucose l−1. However, maximal changes, particularly in promoting the shift from the B to the AR pattern of chlortetracycline fluorescence, required millimolar concentrations of glucose during the last few minutes before assessment. Finally, the addition of partially purified mouse sperm decapacitation factor (proposed to activate a Ca2+-ATPase and thus maintain a low intracellular Ca2+ concentration) to capacitated human sperm suspensions caused a significant reversal in the capacitation state of cells (from the B to the F pattern). The F pattern of chlortetracycline fluorescence predominates in conditions favouring low concentrations of intracellular Ca2+. From these results, we suggest that a Ca2+-ATPase may play an important role during human sperm capacitation. A time-dependent decrease in endogenous enzyme activity would allow the intracellular concentration of Ca2+ to rise to a critical value necessary for initiation acrosomal exocytosis and subsequent successful fertilization.

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S. DasGupta, C. L. Mills and L. R. Fraser

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.