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 Pisum 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 compared with that estimated using eosin‐nigrosin (E‐N) 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 to 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. Since PNA and Con A specifically label the outer and inner acrosomal membrane, respectively, spermatozoa in the early stages of the acrosome reaction, undergoing modification of the outer acrosomal membrane but retaining their 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 due 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‐labelling. Copyright © 1995, Wiley Blackwell. All rights reserved