title: Evaluation of acrosome reaction and viability of human sperm with two fluorescent dyes.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Obstetrics and Gynecology, Shimane Medical University, Izumo, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Ozaki
authors.forename: K
authors.initials: K
authors.lastname: Takahashi
authors.forename: H
authors.initials: H
authors.lastname: Kanasaki
authors.forename: K
authors.initials: K
authors.lastname: Miyazaki
mesh_terms: D020101:Acrosome Reaction / Q000187:drug effects; D006690:Bisbenzimidazole; D000001:Calcimycin / Q000494:pharmacology; D005069:Evaluation Studies as Topic; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D008297:Male; D037121:Plant Lectins; D013094:Spermatozoa / Q000187:drug effects / Q000502:physiology*
chemical_list: D005456:Fluorescent Dyes; D007476:Ionophores; D037121:Plant Lectins; C036292:pea lectin; D000001:Calcimycin; D016650:Fluorescein-5-isothiocyanate; D006690:Bisbenzimidazole