ASSESSMENT OF THE VITALITY AND ACROSOMAL STATUS OF HUMAN SPERMATOZOA USING FLUORESCENT-PROBES

The acrosomal status and vitality of human spermatozoa are generally a ssessed simultaneously using the lectins Pisum sativum agglutinin (PSA ), Peanut agglutinin (PNA) and Concanavalin A (Con A) in cor?junction 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 H25 8 under different staining conditions and compared with that estimated using eosin-nigrosin (E-N) under different staining conditions and th e HOS test. The sensitivity of PNA- and Con A-labelling methods were a lso compared by evaluating the acrosomal status of motile human sperma tozoa after capacitation (1 and 4 h) in vitro followed by exposure to dimethylsulphoxide (DMSO) and a calcium ionophore. The E-N method empl oying eosin-staining for 15 sec, rather than for 30 sec, provided a mo re reliable estimate of sperm vitality. H258, as used in the H258/Con A-labelling method (with and without ethanol fixation) rather than und er the staining conditions equivalent to H258/PNA-labelling, was as go od 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 m ethods. Further, the Con A-labelling method consistently scored a sign ificantly lower percentage of spermatozoa undergoing the acrosome reac tion compared to those estimated by the PNA-labelling method. It is co ncluded that the different sensitivity of these methods can be attribu ted to the different binding specificity of the lectins. Since PNA and Con A specifically label the outer and inner acrosomal membrane, resp ectively, spermatozoa in the early stages of the acrosome reaction, un dergoing modification of the outer acrosomal membrane but retaining th eir acrosomal contents, would be scored as acrosome-reacted by the PNA -labelling method but as acrosome-intact by the Con A-labelling method . The lower sensitivity of the Con A-labelling method could also be du e to the use of aldehyde-fixed spermatozoa for Con A-labelling. Also, the PNA-labelling requires less time to complete than does the Con A-l abelling.