Evaluation of mouse sperm acrosomal status and viability by flow cytometry

Authors

  • Jun Tao,

    1. Reproductive Biology Laboratory, Methodist Hospital of Indiana, Inc., Indiana University Medical Center, Indianapolis, Indiana
    Search for more papers by this author
  • Elizabeth S. Critser,

    1. Reproductive Biology Laboratory, Methodist Hospital of Indiana, Inc., Indiana University Medical Center, Indianapolis, Indiana
    2. Department of Biophysics/Physiology and Department of Obstetrics and Gynecology, Indiana University Medical Center, Indianapolis, Indiana
    Search for more papers by this author
  • John K. Critser PhD

    Corresponding author
    1. Reproductive Biology Laboratory, Methodist Hospital of Indiana, Inc., Indiana University Medical Center, Indianapolis, Indiana
    2. Department of Biophysics/Physiology and Department of Obstetrics and Gynecology, Indiana University Medical Center, Indianapolis, Indiana
    • Reproductive Biology Laboratory, Methodist Hospital of Indiana, Inc., 1701 N. Senate Blvd., P.O. Box 1367, Indianapolis, IN 46206–1367
    Search for more papers by this author

Abstract

A procedure was developed to evaluate mouse sperm acrosomal status and viability simultaneously utilizing flow cytometry. Four fluorescein isothiocyanate (FITC)-conjugated lectins, peanut agglutinin (PNA), concanavalin agglutinin (ConA), Pisum sativum agglutinin (PSA), and soybean agglutinin (SBA), were investigated, with PNA providing the greatest sensitivity and specificity in distinguishing acrosome-present and acrosome-absent mouse spermatozoa. To expose lectin binding sites, digitonin (20 μM at room temperature for 10 min) was used to permeabilize sperm plasma membranes. Sperm cell viability was determined by Hoechst 33258 (H258) exclusion. To prevent permeabilized cells from staining with H258, salmon sperm DNA (SS-DNA) was applied to bind excess dye in the solutions after supravital staining. Calcium ionophore (A23187; 5 or 20 μM) was used to induce acrosome reactions. The results of flow cytometric analyses were compared with epifluorescence microscopic observation and were highly correlated (r = 0.999; P < 0.001). The method developed provides an objective and efficient procedure to estimate simultaneously both acrosomal status and viability of mouse spermatozoa. © 1993 Wiley-Liss, Inc.

Ancillary