A biphasic pattern of 45Ca2+ uptake by mouse spermatozoa in vitro correlates with changing functional potential

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, spermatozoa incubated in 45Ca2+ exhibited a biphasic pattern of Ca2+ uptake, 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 supplied glucose was reduced from 5.56 mmol l−1 to 5.56 μmol l−1, the latter supporting capacitation but not fertilization, only the first peak of 45Ca2+ uptake was observed. Increasing the glucose to a millimolar concentration produced a second peak of uptake. We therefore propose that the first phase of 45Ca2+ uptake is associated with capacitation and the second phase with acrosomal exocytosis, which are both necessary prerequisites for fertilization. In micromolar glucose the rate of 45Ca2+ uptake during the first 30 min was 47% higher than in millimolar glucose, suggesting that the former conditions might promote a precocious rise in the intracellular Ca2+ concentration ([Ca2+]i) and hence accelerate capacitation. This hypothesis was confirmed by demonstrating both significantly accelerated transition from the uncapacitated F pattern of chlortetracycline (CTC) fluorescence to the capacitated B and AR patterns and significantly higher fertility in vitro in suspensions preincubated for 30 min in micromolar glucose, compared with those maintained continuously in millimolar glucose. These results suggest that an ATP-dependent mechanism, for example a Ca2+-ATPase, may be involved in maintaining a low [Ca2+]i. In micromolar glucose, available 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 suspensions in the presence of trifluoperazine (TFP), a calmodulin antagonist. TFP significantly accelerated both the initial uptake of 45Ca2+ and the transition from uncapacitated to capacitated CTC patterns, suggesting a role for a calmodulin-sensitive Ca2+-ATPase during capacitation. Finally, inclusion of the mitochondrial inhibitor azide had little effect on 45Ca2+ uptake, indicating that most of the observed uptake in this study was occurring in the sperm head.