Evaluation of acrosome reaction and viability of human sperm with two fluorescent dyes.

Abstract

To evaluate the usefulness of the Hoechst 33258/FITC-Pisum sativum agglutinin (FITC-PSA) staining for simultaneous assessment of viability and acrosome reaction rate (%AR) of human sperm. Fresh sperm was collected 13 fertile donors provided fresh semen. We used motile sperm selected by the "swim-up" procedure using modified HTF. Hoechst 33258 was added and co-incubated with sperm for 10 min. Samples were washed free of unbound stain and the sperm were mounted as smears on glass slides. After drying, sperms were incubated with FITC-PSA for 30 min. Sperm were examined by fluorescence microscopy. Also, FITC-Concanavalin A (FITC-ConA) staining and vital staining with yellowish eosin were performed simultaneously. The correlation of viability and %AR were analyzed. Four different staining patterns were observed and clearly distinguished as follows: a) Viable acrosome-reacted sperm, b) Viable acrosome-intact sperm, c) dead acrosome-reacted sperm, d) dead acrosome-intact sperm. There was significant correlation between the results obtained by Hoechst 33258 and vital staining methods in viability of human sperm (r = 0.927, P < 0.001). There was significant correlation between the two methods (FITC-PSA and FITC-ConA) in %AR of human sperm (r = 0.92, P < 0.001). Viability and acrosome status of a human sperm can be easily assessed simultaneously by Hoechst 33258/FITC-PSA staining method. This combination method is considered useful to evaluate sperm function.