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Human Reproduction, Vol. 5, No. 1, pp. 99-103, 1990
© 1990 European Society of Human Reproduction and Embryology

research-article

Combined use of fluorescent peanut agglutinin lectin and Hoechst 33258 to monitor the acrosomal status and vitality of human spermatozoa

D. Mortimer, E.F. Curtis and A.R. Camenzind

Gamete Biology Research Unit, Reproductive Medicine Research Group, University of Calgary Health Sciences Centre 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada

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 [GenBank] ) as a vital stain for use in combination with PNA-labelling. Human sperm populations were stained for 1 min with 1 µg/ml H33258 [GenBank] in culture medium and then washed through 2.0 (w/v) polyvinylpyrollidone columns and air-dried onto microscope slides. H33258 [GenBank] 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 [GenBank] 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 [GenBank] /PNA procedure routinely.

Key words: spermatozoa/human sperm/acrosome/vitality/lectin


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