title: Assessment of the vitality and acrosomal status of human spermatozoa using fluorescent probes.
abstract: 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)
authors.affiliation: Department of Reproductive Biology, All India Institute of Medical Sciences, New Delhi.
authors.forename: S
authors.initials: S
authors.lastname: Kinger
authors.forename: M
authors.initials: M
authors.lastname: Rajalakshmi
mesh_terms: 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*
chemical_list: D005456:Fluorescent Dyes; D037102:Lectins; D019887:Peanut Agglutinin; D037121:Plant Lectins; C036292:pea lectin; D003208:Concanavalin A; D016650:Fluorescein-5-isothiocyanate; D006690:Bisbenzimidazole