EVIDENCE FOR CA2-DEPENDENT ATPASE ACTIVITY, STIMULATED BY DECAPACITATION FACTOR AND CALMODULIN, IN MOUSE SPERM()

Membrane preparations from mouse sperm heads and tails were used in a gamma(32)P-ATP hydrolysis assay to investigate Ca2+-dependent ATPase a ctivity. In membranes from sperm heads, but not tails, a Ca2+-dependen t ATPase that was further stimulated by calmodulin was detected. The a ddition of partially purified mouse sperm decapacitation factor (DF) t o head membrane preparations significantly stimulated Ca2+-ATPase acti vity, this effect being further increased in the presence of DF plus c almodulin; in contrast, no response was observed when the same treatme nt was applied to tail membranes. Sperm preincubated in the presence o f trifluoperazine (TFP), a calmodulin antagonist, were significantly m ore fertile than cells from the same males incubated in the absence of TFP, indicating that inhibition of calmodulin accelerates capacitatio n. When sperm cells were preincubated briefly, then gently centrifuged to remove DF and resuspended in medium containing Ca-45(2+) +/- DF, t heir ability to accumulate Ca-45(2+) was significantly lower in the ea rly stages after resuspension in the presence of DF than in its absenc e. These data correlated with chlortetracycline analysis of the sperm functional state. When cells were centrifuged and resuspended in mediu m only, there was a noticeable shift from the F pattern (characteristi c of uncapacitated cells) to the B pattern (characteristic of capacita ted cells), but the reintroduction of DF caused a significant reversio n to the F pattern. Finally, using a monoclonal antibody to somatic ce ll Ca2+-ATPase, we have obtained evidence that the enzyme is particula rly localized to the postacrosomal region of the mouse sperm head; spe cific binding was observed only in permeabilized cells, indicating tha t the epitope involved in the binding has an intracellular location. B ased on these various pieces of evidence, we propose that when present on mouse sperm, DF stimulates calmodulin-sensitive Ca2+-ATPase activi ty and thus ensures maintenance of a low intracellular Ca2+ concentrat ion. As capacitation proceeds, DF is lost and Ca2+-ATPase activity dec lines, allowing intracellular Ca2+ to rise and promoting capacitation- related changes. The fact that inhibitors of Ca2+-ATPase and calmoduli n appear to accelerate capacitation in several mammalian species, as d etermined by chlortetracycline analysis, suggests that Ca2+-ATPase act ivity may play an important role in modulating capacitation in many or even all mammals. (C) 1996 Wiley-Liss, Inc.