Bovine sperm hyperactivation is promoted by alkaline-stimulated Ca2+ influx

Sperm hyperactivated motility is characterized by high flagellar bend amplitude and asymmetrical beating, which are detected by computer-assisted sperm motility analysis as increased curvilinear velocity and lateral head movement. It is required for sperm penetration of the oocyte zona pellucida during fertilization and is induced by an increase in flagellar Ca2+. Our objective was to determine whether pH plays a role in promoting Ca signaling of hyperactivated motility. The cell-permeant weak base NH4Cl increased curvilinear velocity and amplitude of lateral head movement of bovine sperm, indicative of hyperactivation. Fluorometric recordings of sperm loaded with BCECF-AM or fluo3-AM, revealed that NH4Cl evoked elevations of intracellular pH and Ca2+, respectively, with the rise in pH occurring more rapidly than that of Ca2+. Single-cell image analysis showed increased Ca2+ levels in the flagellum in response to NH4Cl. When extracellular Ca2+ was lowered with BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) prior to treatment with NH4CI, intracellular pH was increased, but elevation of Ca2+ and hyperactivation were diminished. This suggests that the rise in intracellular pH precedes an influx of Ca2+. The Ca2+ channel blocker Ni2+ also diminished NH4Cl stimulation of hyperactivation, demonstrating that Ca2+ entry is required for maximal expression of hyperactivation. Call ionophore produced an increase in Call that was 3-fold greater than that produced by NH4Cl; however, it produced a weaker hyperactivation response. These results indicate that a rise in pH increases intracellular Ca2+ and promotes hyperactivation primarily by stimulating Ca2+ influx, but also by other mechanisms.