Assessment of the vitality and acrosomal status of human spermatozoa using fluorescent probes.
The acrosomal status and vitality of human spermatozoa are generally assessed simultaneously using the lectins Pisums sativum agglutinin (PSA), Peanut agglutinin (PNA) and Concanavalin A (Con A) in conjunction with either Hoechst 33258 (H258; a fluorescent DNA-binding supravital stain with limited membrane permeability) or a hypo-osmotic swelling (HOS) test. In the present study, sperm vitality was assessed using H258 under different staining conditions and the HOS test. The sensitivity of PNA- and Con A-labelling methods were also compared by evaluating the acrosomal status of motile human spermatozoa after capacitation (1 and 4 h) in vitro followed by exposure to dimethylsulphoxide (DMSO) and a calcium ionophore. The E-N method employing eosin-staining for 15 sec, rather than for 30 sec, provided a more reliable estimate of sperm vitality. H258, as used in the H258/Con A-labelling method (with and without ethanol fixation) rather than under the staining conditions equivalent of H258/PNA-labelling, was as good for vitality assessment as the E-N method employing eosin-staining for 15 sec. However, the HOS test overestimated the proportion of dead spermatozoa compared to those obtained using different H258 and E-N methods. Further, the Con A-labelling method consistently scored a significantly lower percentage of spermatozoa undergoing the acrosome reaction compared to those estimated by the PNA-labelling method. It is concluded that the different sensitivity of these methods can be attributed to the different binding specificity of the lectins.(ABSTRACT TRUNCATED AT 250 WORDS)
Kinger
S
S
Department of Reproductive Biology, All India Institute of Medical Sciences, New Delhi.
Rajalakshmi
M
M
D000177:Acrosome / Q000502:physiology* / Q000648:ultrastructure*; D010367:Arachis; D006690:Bisbenzimidazole; D003208:Concanavalin A; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006801:Humans; D037102:Lectins; D008297:Male; D008856:Microscopy, Fluorescence; D019887:Peanut Agglutinin; D037121:Plant Lectins; D012016:Reference Values; D013081:Sperm Motility; D013094:Spermatozoa / Q000166:cytology* / Q000502:physiology*
D005456:Fluorescent Dyes; D037102:Lectins; D019887:Peanut Agglutinin; D037121:Plant Lectins; C036292:pea lectin; D003208:Concanavalin A; D016650:Fluorescein-5-isothiocyanate; D006690:Bisbenzimidazole