Ca2+‐Regulating mechanisms that modulate bull sperm capacitation and acrosomal exocytosis as determined by chlortetracycline analysis

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Abstract
We have used chlortetracycline (CTC) analysis to investigate mechanisms that may play important roles during bull sperm capacitation in a culture medium (containing glucose, heparin, and caffeine) known to promote capacitation and fertilization in vitro. In initial experiments employing the Ca2+ ionophore A23187, we identified three discrete CTC patterns so similar to those described for mouse and human sperm that we have employed the same nomenclature: “F,” characteristic of uncapacitated, acrosome‐intact cells; “B,” characteristic of capacitated, acrosome‐intact, cells; “AR,” characteristic of capacitated, acrosome‐reacted cells. Over a 60‐min period, A23187 stimulated significant increases in B and AR pattern cells, with concomitant decreases in F pattern cells, suggesting a very rapid transition from the uncapacitated to the capacitated state and then on to exocytosis. Without ionophore, significant changes in the proportions of F and B pattern cells were also observed, but the maximum responses required 4 hr; the proportion of AR cells was consistently ∼ 15% throughout, indicating a low incidence of spontaneous acrosome loss. Analysis of cells in media with altered composition indicated that the inclusion of either heparin or caffeine significantly promoted capacitation to about the same extent, but together, heparin plus caffeine had an even more stimulatory effect. Despite this, none of these treatments triggered acrosome loss above the levels seen in media lacking these constituents. In the presence of caffeine, with or without heparin, the inclusion of glucose had little effect on responses, but in the presence of heparin there were fewer B cells. In the presence of either quercetin, a Ca‐ATPase inhibitor used at 50–200 μM, or W‐7, a calmodulin antagonist used at 5–125 μM, capacitation per se was accelerated, as evidenced by significant decreases in F and significant increases in B pattern cells; only the highest concentration of each caused significant increases in AR cells. In addition, 25 and 125 μM W‐7 markedly stimulated motility, both quantitatively and qualitatively. Finally the Na+ ionophore monensin at 500 μM significantly accelerated both capacitation and acrosomal exocytosis. The addition of the dihydropyridine calcium channel blocker nifedipine at 10 nM, just prior to monensin, did not inhibit capacitation (F to B transition) but blocked acrosomal exocytosis (B to AR transition). We suggest that Ca2+ is required for functional changes in bull sperm, with a Ca2+‐ATPase modulating intracellular Ca2+ during capacitation and calcium channels controlling the Ca2+ influx required for acrosomal exocytosis.