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Separation of calcium effects on motility and zona binding ability in mouse spermatozoa.

The ionic specificity of Ca+2 requirements for binding to the zona pellucida and maintenance of motility were studied in mouse gametes by comparing the effects of four cations: Ca+2, Sr+2, La+3, and spermine cation (Sp+4). Cumulus-free mouse eggs were inseminated in vitro with sperm which had been preincubated for 60 min in Tris-NaCl buffer containing one or more of the above cations. Motility was assessed microscopically and zona binding, by the stop-fix technique, utilizing centrifugation through a discontinuous density gradient. The sperm were motile upon release from the epididymis, and all sperm maintained their motility in complete culture medium. Sperm incubated in Tris-NaCl buffer differed in motility among samples as follows: The majority of sperm samples remained motile in Tris-NaCl containing 1.7 mM Ca+2, but lost motility if Ca+2 was absent. The remaining samples could be divided into two populations: those which maintained motility in the absence of Ca+2. Support of Zona binding required Ca+2 in all samples examined. Equimolar Sr+2 could substitute for Ca+2 in maintaining motility but not in supporting binding. Sp+4and La+3 did not support motility. If Sr+2 were present to maintain motility, both Sp+4 and La+3 supported binding as well as Ca+2. Sr+2 did not complete with any of the other cations for support of zona binding. Ca+2 and Sp+4 together in the medium produced a synergism in the number of sperm bound. La+3 would substitute for Ca+2 in this synergism. These results demonstrate for the first time that mouse sperm have Ca+2 requirement for the motility maintenance and that binding to the zona and maintenance of motility are two different Ca+2-dependent processes whose active sites can be distinguished by cation substitution.