The spermatozoon is specialized for a single vital role in fertilization. Past studies show that Ca2+ signals produced by the opening of plasma membrane entry channels initiate several events required for the sperm to reach and enter the egg but reveal little about how resting Ca2+i is maintained or restored after elevation. We examined these homeostatic mechanisms by monitoring the kinetics of recovery from depolarizing stimuli under conditions intended to inhibit candidate mechanisms for sequestration or extrusion of Ca2+ from the cytosol. We found that the Ca2+-ATPase pump of the plasma membrane performs the major task of Ca2+ clearance. It is essential in the final stages of recovery to achieve a low resting Ca2+i. With immunomethods we found a ∼130-kD plasma membrane Ca2+-ATPase protein on Western blots of whole sperm extracts and showed immunolocalization to the proximal principal piece of the flagellum. The plasma membrane Na+-Ca2+ exchanger also exports Ca2+ when Ca2+i is elevated. Simultaneous inhibition of both mechanisms of extrusion revealed an additional contribution to clearance from a CCCP-sensitive component, presumably sequestration by the mitochondria. Involvement of SERCA pumps was not clearly detected. Many aspects of the kinetics of Ca2+ clearance observed in the presence and absence of inhibitors were reproduced in a mathematical model based on known and assumed kinetic parameters. The model predicts that when cytosolic Ca2+ is at 1 μM, the rates of removal by the Ca2+-ATPase, Na+-Ca2+-exchanger, mitochondrial uniporter, and SERCA pump are ∼1.0, 0.35, 0.33, and 0 μmole l−1 s−1, rates substantially slower than those reported for other cells studied by similar methods. According to the model, the Na+-Ca2+ exchanger is poised so that it may run in reverse at resting Ca2+i levels. We conclude that the essential functions of sperm do not require the ability to recover rapidly from globally elevated cytosolic Ca2+.
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