A BIPHASIC PATTERN OF CA-45(2-VITRO CORRELATES WITH CHANGING FUNCTIONAL POTENTIAL() UPTAKE BY MOUSE SPERMATOZOA IN)

Mouse sperm capacitation in vitro, leading to hyperactivated motility, acrosomal exocytosis and rapid fertilization, takes approximately 120 min in a medium containing sufficient Ca2+. During that period, sperm atozoa incubated in Ca-45(2+) exhibited a biphasic pattern of Ca2+ upt ake, with the first and lower peak occurring from 10 to 50 min and the second and higher peak from 60 to 90 min. When the exogenously suppli ed glucose was reduced from 5.56 mmol l-1 to 5.56 mumol l-1, the latte r supporting capacitation but not fertilization, only the first peak o f Ca-45(2+) uptake was observed. Increasing the glucose to a millimola r concentration produced a second peak of uptake. We therefore propose that the first phase of Ca-45(2+) uptake is associated with capacitat ion and the second phase with acrosomal exocytosis, which are both nec essary prerequisites for fertilization. In micromolar glucose the rate of Ca-45(2+) uptake during the first 30 min was 47% higher than in mi llimolar glucose, suggesting that the former conditions might promote a precocious rise in the intracellular Ca2+ concentration ([Ca2+]i) an d hence accelerate capacitation. This hypothesis was confirmed by demo nstrating both significantly accelerated transition from the uncapacit ated F pattern of chlortetracycline (CTC) fluorescence to the capacita ted B and AR patterns and significantly higher fertility in vitro in s uspensions preincubated for 30 min in micromolar glucose, compared wit h those maintained continuously in millimolar glucose. These results s uggest that an ATP-dependent mechanism, for example a Ca2+-ATPase, may be involved in maintaining a low [Ca2+]i. In micromolar glucose, avai lable ATP would be limited and hence the ATPase activity would decline , allowing [Ca2+]i to rise. The possibility that such an ATPase might be calmodulin-sensitive was investigated by incubating sperm suspensio ns in the presence of trifluoperazine (TFP), a calmodulin antagonist. TFP significantly accelerated both the initial uptake of Ca-45(2+) and the transition from uncapacitated to capacitated CTC patterns, sugges ting a role for a calmodulin-sensitive Ca2+-ATPase during capacitation . Finally, inclusion of the mitochondrial inhibitor azide had little e ffect on Ca-45(2+) uptake, indicating that most of the observed uptake in this study was occurring in the sperm head.