A NEW METHOD TO DETECT ACROSOME-REACTED SPERMATOZOA USING BIOTINYLATED SOYBEAN TRYPSIN-INHIBITOR

Objective: To develop a method to detect acrosome-reacted spermatozoa on human zonae pellucidae using only commercially available reagents and without need for sperm fixation.

Design: Sperm head labeling with biotinylated soybean trypsin inhibitor (SBTI-biotin) was compared with results of a known method using fluorescein isothiocyanate (FITC)-conjugated Pisum sativum agglutinin. The SBTI-biotin method was applied to sperm bound to human zonae pellucidae.

Setting and Subjects: Healthy sperm donors with normal semen characteristics were recruited by the Laboratory for Reproductive Medicine in a university medical center.

Main Outcome Measures: Soybean trypsin inhibitor-biotin binding patterns on nonfixed spermatozoa were visualized with avidin-Texas Red. The development in time of various patterns upon induction of acrosome reaction (AR) in suspension with Ca2+-ionophore A23187 was noted and compared with P. sativum agglutinin FITC labeling patterns. Soybean trypsin inhibitor-biotin labeling patterns of spermatozoa bound to human zonae pellucidae were determined.

Results: Soybean trypsin inhibitor-biotin bound specifically, via the SBTI-moiety, to an acrosomal factor as soon as AR started. Sperm-head labeling patterns could be assigned to defined stages of the AR process. The results were highly correlated to those obtained with P. sativum agglutinin FITC. The end point of the AR in suspension and on zonae pellucidae was SBTI-biotin binding confined to the equatorial segment.

Conclusion: The SBTI-biotin method can be used to detect nonfixed acrosome-reacted spermatozoa both in suspension and on zonae pellucidae.