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The extracellular Ca2+ requirements for support of capacitation and the spontaneous acrosome reaction (AR) in human spermatozoa have been evaluated. Motile suspensions were prepared using a swim-up method, incubated for up to 24 h in media of various Ca2+ concentrations, fixed and assessed for occurrence of the AR using transmission electron microscopy. Results indicated that the AR response was significantly lower after incubation in Ca2+-deficient medium (generally less than 10% reacting cells) than in 1.80 mm-Ca2+-containing medium (approximately 15%). In the latter the majority of cells were fully reacted, while in Ca2+-deficient conditions the majority were at intermediate stages of the AR. Subsequent experiments indicated that a maximum AR response required the continuous presence of millimolar Ca2+; preincubation in the presence of micromolar Ca2+ did not prepare the spermatozoa to undergo rapid AR upon increase of Ca2+ to millimolar concentrations, suggesting that capacitation requires relatively high concentrations of extracellular Ca2+. Incubation in elevated Ca2+ (3.60 mM) promoted an even greater response (mean of 24-35% reacting cells compared with 12% for 1.80 mM-Ca2+). The ability of the divalent cations Ba2+, Mg2+ and Sr2+ (each at 1.80 mM) to substitute for 1.80 mM-Ca2+ in promoting the AR was also assessed. Of these, only Sr2+ provided a response greater than that observed in unsupplemented Ca2+-deficient medium. In Sr2+ the proportion of responding cells after 24 h (approximately 13%) was similar to that obtained in Ca2+ (approximately 15%), although a majority of those in Sr2+ were at intermediate stages. In 3.60 mM-Sr2+ the response was significantly higher than that observed in both 1.80 mM-Ca2+ and 1.80 mM-Sr2+, but significantly lower than that in 3.60mM-Ca2+. Under all conditions motility was maintained at greater than 90% for 24 h. The introduction of the Ca2+ ionophore ionomycin, in the presence of 1.80 mM-Ca2+, induced the AR in a concentration-dependent but preincubation time-independent manner, with the maximum response of approximately 60% being obtained with 30 microM-ionomycin. Finally, incubation in the presence of 1.80 mM-Ca2+ and verapamil, generally considered to be a calcium channel antagonist, resulted in a concentration- and incubation time-dependent increase in the AR, the maximum response in all groups being observed only after 24 h incubation. Recent evidence from other species suggests that this may represent an agonistic interaction with calcium channels.(ABSTRACT TRUNCATED AT 400 WORDS)

Divalent cations, capacitation and the acrosome reaction in human spermatozoa