Mouse gamete interactions during fertilization in vitro. Chlortetracycline as a fluorescent probe for the mouse sperm acrosome reaction.

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Abstract
An assay was developed for detecting the acrosome reaction in mouse sperm using chlortetracycline (CTC) as a fluorescent probe. Intact sperm with nonreacted acrosomes show CTC fluorescence in the presence of Ca2+ over the anterior portion of the sperm head on the plasma membrane covering the acrosome. Sperm which have undergone the acrosome reaction do not show fluorescence on the sperm head. Mouse sperm bind to zonae pellucidae of cumulus-free eggs in vitro in a Ca2+-dependent reaction; these sperm are intact by the CTC assay. Intact sperm bind to mechanically isolated zonae under the same conditions; the egg is apparently unnecessary for this initial reaction. Sperm suspensions, in which > 50% of the motile population completed the acrosome reaction, were prepared by incubation in hypersomolal medium followed by treatment with the divalent cation ionophore, A23187. Cumulus-free eggs challenged with such sperm suspensions preferentially bind intact sperm; acrosome-reacted sperm do not bind. The plasma membrane of the mouse sperm is responsible for recognition of the egg''s zona pellucida. The obligatory sequence of reactions leading to mouse gamete fusion is binding of the intact sperm to the zona pellucida, followed by the acrosome reaction at the zona surface, followed in turn by sperm penetration of the zona.