Calcium-dependent binding of mouse epididymal spermatozoa to the zona pellucida

The minimal requirements and characteristics of epididymal sperm binding to the zona pellucida of the mouse egg were investigated using a new stop-fix centrifugation technique. This assay provided a precise physical definition of the association between the spermatozoon and the zona and permitted quantitation of the binding reaction at short time intervals. The results demonstrated that Ca 2+ is an essential physiological component required for binding to occur. Sperm preincubated for 60 min in a simplified medium lacking Ca 2+ did not acquire the ability to bind to eggs. In contrast, if sperm preincubation occurred in this medium supplemented with 1.7 m M Ca 2+, binding was identical to that observed following sperm preincubation in the complete culture medium which supports both capacitation and fertilization in vitro. The Ca 2+-dependent binding reaction was rapid, reversed by EGTA, specific for Ca 2+, and did not require the transport of Ca 2+ into the cell. Sperm bound to the zona surface following preincubation with Ca 2+ were capable of fertilization in vitro when the eggs were subsequently transferred to the culture medium. It is proposed that this binding reaction represents a part of capacitation and not the acrosome reaction.