CA2-REGULATING MECHANISMS THAT MODULATE BULL SPERM CAPACITATION AND ACROSOMAL EXOCYTOSIS AS DETERMINED BY CHLORTETRACYCLINE ANALYSIS()

We have used chlortetracycline (CTC) analysis to investigate mechanism s that may play important roles during bull sperm capacitation in a cu lture medium (containing glucose, heparin, and caffeine) known to prom ote capacitation and fertilization in vitro. In initial experiments em ploying the Ca2+ ionophore A23187, we identified three discrete CTC pa tterns so similar to those described for mouse and human sperm that we have employed the same nomenclature: ''F,'' characteristic of uncapac itated, acrosome-intact cells; ''B,'' characteristic of capacitated, a crosome-intact cells; ''AR,'' characteristic of capacitated, acrosome- reacted cells. Over a 60-min period, A23187 stimulated significant inc reases in B and AR pattern cells, with concomitant decreases in F patt ern cells, suggesting a very rapid transition from the uncapacitated t o the capacitated state and then on to exocytosis. Without ionophore, significant changes in the proportions of F and B pattern cells were a lso observed, but the maximum responses required 4 hr; the proportion of AR cells was consistently similar to 15% throughout, indicating a l ow incidence of spontaneous acrosome loss. Analysis of cells in media with altered composition indicated that the inclusion of either hepari n or caffeine significantly promoted capacitation to about the same ex tent, but together, heparin plus caffeine had an even more stimulatory effect. Despite this, none of these treatments triggered acrosome los s above the levels seen in media lacking these constituents. In the pr esence of caffeine, with or without heparin, the inclusion of glucose had little effect on responses, but in the presence of heparin there w ere fewer B cells. In the presence of either quercetin, a Ca-ATPase in hibitor used at 50-200 mu M, or W-7, a calmodulin antagonist used at 5 -125 mu M, capacitation per se was accelerated, as evidenced by signif icant decreases in F and significant increases in B pattern cells; onl y the highest concentration of each caused significant increases in AR cells. In addition, 25 and 125 mu M W-7 markedly stimulated motility, both quantitatively and qualitatively. Finally the Na+ ionophore mone nsin at 500 nM significantly accelerated both capacitation and acrosom al exocytosis. The addition of the dihydropyridine calcium channel blo cker nifedipine at 10 nM, just prior to monensin, did not inhibit capa citation (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 require d for acrosomal exocytosis. (C) 1995 Wiley-Liss, Inc.