Combined use of fluorescent peanut agglutinin lectin and Hoechst 33258 to monitor the acrosomal status and vitality of human spermatozoa.
Fluorescein-conjugated peanut agglutinin (PNA) lectin-labelling is an established procedure for assessing the status of the human sperm acrosome. However, unlike the triple-stain technique, PNA-labelling does not provide a simultaneous assessment of cellular vitality. We have therefore evaluated the use of the fluorescent dye Hoechst 33258 (H33258) as a vital stain for use in combination with PNA-labelling. Human sperm populations were stained for 1 min with 1 microgram/ml H33258 in culture medium and then washed through 2.0 (w/v) polyvinylpyrollidone columns and air-dried onto microscope slides. H33258 was found to provide vitality assessments comparable to those obtained using the standard eosin-exclusion method. However, best results were obtained with an ethanol fixation step between air-drying and PNA-labelling. This vitality assessment was found to be more reliable than that provided by Trypan blue staining under conditions equivalent to the triple-stain technique. There was no alteration of PNA-labelling due to the H33258 although ethanol fixation actually provided more uniform PNA-labelling than previously obtained without ethanol fixation. Consequently, we have stopped using the triple-stain technique for assessing human sperm acrosome reactions and now use the H33258/PNA procedure routinely.
Gamete Biology Research Unit, University of Calgary Health Sciences Centre, Alberta, Canada.
D
D
Mortimer
E F
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Curtis
A R
AR
Camenzind
D000177:Acrosome / Q000502:physiology*; D001562:Benzimidazoles; D006690:Bisbenzimidazole; D004338:Drug Combinations; D005456:Fluorescent Dyes; D006801:Humans; D066298:In Vitro Techniques; D037102:Lectins; D008297:Male; D019887:Peanut Agglutinin; D013081:Sperm Motility; D013094:Spermatozoa / Q000502:physiology*; D013194:Staining and Labeling / Q000379:methods
D001562:Benzimidazoles; D004338:Drug Combinations; D005456:Fluorescent Dyes; D037102:Lectins; D019887:Peanut Agglutinin; D006690:Bisbenzimidazole