ASSESSMENT OF THE ACROSOMAL STATUS AND VIABILITY OF HUMAN SPERMATOZOASIMULTANEOUSLY USING FLOW-CYTOMETRY

Acrosomal status and viability were evaluated simultaneously on human spermatozoa using flow cytometry. Samples were divided into three aliq uots and randomly assigned to one of three treatments: (i) cryopreserv ation; (ii) 10 muM calcium ionophore [A23187 in dimethylsulphoxide (DM SO)] or (iii) DMSO alone (control). Acrosomal status was evaluated usi ng monoclonal antibodies recognizing MH61 and CD46, respectively. Fluo rescein-conjugated goat anti-mouse immunoglobulin (IgG) was used as a second antibody. Sperm viability was assessed using Hoechst 33258 (H25 8) exclusion. The following factors were analysed: (i) the specificity of the monoclonal antibodies for the human acrosome; (ii) the relativ e effectiveness of flow cytometry and direct fluorescent microscopy sc oring and (iii) the acrosomal status and viability of the control, ion ophore-treated, and cryopreserved spermatozoa. Across all treatments, the MH61 and CD46 monoclonal antibodies resulted in acrosomal status v alues (acrosome-reacted/viable spermatozoa) which were not significant ly different (P > 0.05): control, 1.0 +/- 0.3% and 1.5 +/- 0.6% (mean +/- SEM); A23187, 42.8 +/- 3.5% and 38.1 +/- 3.5%; cryopreserved, 8.2 +/- 2.0% and 9.9 +/- 1.3%; respectively. However, acrosomal status amo ng treatments differed significantly (P < 0.01). Flow cytometric and d irect fluorescent microscopy assessments were significantly correlated (r2 = 0.96, P < 0.01). These results indicate that flow cytometry, us ing an acrosome-specific monoclonal antibody and a supravital dye, pro vides an objective and efficient method to evaluate human sperm acroso mal and viability status simultaneously.