ABSTRACT
Subsequent to binding to the zona pellucida, mammalian sperm undergo a regulated sequence of events that ultimately lead to acrosomal exocytosis. Like most regulated exocytotic processes, a rise in intracellular calcium is sufficient to trigger this event although the precise mechanism of how this is achieved is still unclear. Numerous studies on mouse sperm have indicated that a voltage-operated Ca2+ channel plays some immediate role following sperm binding to the zona pellucida glycoprotein ZP3. However, there is also evidence that the mammalian sperm acrosome contains a high density of IP3 receptors, suggesting that the exocytotic event involves the release of Ca2+ from the acrosome. The release of Ca2+ from the acrosome may directly trigger exocytosis or may activate store-operated Ca2+ channels in the plasma membrane. To test the hypothesis that the acrosome is an intracellular store we loaded mammalian sperm with the membrane permeant forms of three Ca2+-sensitive fluorescent indicator dyes: fura-2, indo-1, and Calcium Green-5N. Fluorescence microscopy revealed that the sperm were labeled in all intracellular compartments. When fura-2 labeled sperm were treated with 150 microM MnCl2 to quench all fluorescence in the cytosol, or when the sperm were labeled with the low affinity dye Calcium Green-5N, there was a large Ca2+ signal in the acrosome. Consistent with the acrosome serving as an intracellular Ca2+ reservoir, the addition of 20 microM thapsigargin, a potent inhibitor of the smooth endoplasmic reticular Ca2+-ATPase (SERCA), to populations of capacitated sperm resulted in nearly 100% acrosomal exocytosis within 60 min (tau1/2 approximately 10 min), in the absence of extracellular Ca2+. Additionally, treatment of sperm with 100 microM thimerosal, an IP3 receptor agonist, also resulted in acrosomal exocytosis. Taken together, these data suggest that the mouse sperm acrosome is a Ca2+ store that regulates its own exocytosis through an IP3 Ca2+ mobilization pathway.
