EVIDENCE AGAINST A FUNCTIONAL ATP-DEPENDENT CALCIUM EXTRUSION MECHANISM IN BOVINE EPIDIDYMAL SPERM

Bovine epididymal sperm resuspended in ionic buffers take up relativel y large amounts of calcium. This uptake, which is almost entirely mito chondrial, apparently bypasses the sperm cytosol. The direct mitochond rial loading is an unusual aspect of sperm calcium uptake, which sugge sts that the plasma membrane region surrounding the mitochondria shoul d be highly permeable to calcium, whereas the membrane domains surroun ding the head and tail regions of sperm should be impermeable. This st udy was undertaken to determine the role of a plasma membrane calcium ATPase in sperm calcium homeostasis. Kinetics of calcium (Ca-45(2+)) u ptake into intact and permeabilized caudal epididymal sperm confirmed that mitochondrial calcium uptake occurs with virtually no resistance from the surrounding plasma membrane. Cytoplasmic calcium accumulation by sperm depleted of intracellular ATP, measured in the presence of m itochondrial calcium uptake inhibitors, showed no increase upon energy depletion as would be expected if an ATP-dependent calcium extrusion mechanism were present. Furthermore, lowering the incubation temperatu re to further reduce the activity of the calcium ATPase in these energ y-depleted sperm was also without effect on calcium accumulation. The calcium ATPase inhibitor vanadate, even at high concentrations, failed to increase intracellular Ca-45(2+) accumulation. However, vanadate w as effective in inhibiting motility showing that the compound was accu mulated into sperm to inhibit flagellar dyenin ATPase. Therefore, the lack of effect of vanadate on Ca-45(2+) accumulation was not due to it s inability to enter sperm. Other calcium ATPase inhibitors such as qu ercetin, thapsigargin, and cyclopiazonic acid, which readily demonstra te ATP-dependent calcium extrusion in other somatic cells, were also w ithout effect on sperm calcium accumulation. These results with Ca-45( 2+) were also confirmed using single-cell calcium distribution measure ments with a fluorescent indicator. Our data strongly suggest that spe rm, contrary to previous assumptions, do not possess a functional plas ma membrane, ATP-dependent, calcium extrusion mechanism. (C) 1994 Wile y-Liss, Inc.