Mouse Sperm Acrosome Reaction Research Articles

Detection and kinetics of the normal acrosome reaction of mouse sperm

AbstractA procedure is described for quantifying the percentage of normal acrosome reactions in fixed mouse sperm. This technique, which is a modification of the triple stain technique (TST) devised for human sperm [Talbot and Chacon, 1981a, b], utilizes trypan blue exclusion to identify the percentage of dead sperm in culture and subsequent staining of fixed sperm to differentiate the acrosome. Four classes of mouse sperm can be distinguished with the TST: (1) “live,” unreacted sperm, (2) “live,” acrosome‐reacted sperm (normal reactions), (3) dead, unreacted sperm, and (4) dead, acrosome‐reacted sperm (degenerative acrosome reactions). We have used this technique to study the time course of the normal acrosome reaction of mouse sperm in vitro. Our results with TST show that a low percentage of mouse sperm undergo normal acrosome reactions during the first 30 min of incubation; a maximum level of reactions is reached by 2 hr. Calcium was required in the medium for occurrence of normal reactions.

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

We have developed an assay for detecting the acrosome reaction in mouse sperm using chlortetracycline (CTC) as a fluorescent probe. Sperm known to be intact 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 inital reaction. Sperm suspensions, in which greater than 50% of the motile population had completed the acrosome reaction, were prepared by incubation in hyperosmolal 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. We conclude that the plasma membrane of the mouse sperm is responsible for recognition of the egg's zona pellucida and that the obligatory sequence of reactions leading to fusion of mouse gametes 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.