Assessment of the acrosomal status and viability of human spermatozoa simultaneously using flow cytometry

Acrosomal status and viability were evaluated simultaneously on human spermatozoa using flow cytometry. Samples were divided into three aliquots and randomly assigned to one of three treatments: (i) cryopreservation; (ii) 10 microM calcium ionophore [A23187 in dimethylsulphoxide (DMSO)] or (iii) DMSO alone (control). Acrosomal status was evaluated using monoclonal antibodies recognizing MH61 and CD46, respectively. Fluorescein-conjugated goat anti-mouse immunoglobulin (IgG) was used as a second antibody. Sperm viability was assessed using Hoechst 33258 (H258) exclusion. The following factors were analysed: (i) the specificity of the monoclonal antibodies for the human acrosome; (ii) the relative effectiveness of flow cytometry and direct fluorescent microscopy scoring and (iii) the acrosomal status and viability of the control, ionophore-treated, and cryopreserved spermatozoa. Across all treatments, the MH61 and CD46 monoclonal antibodies resulted in acrosomal status values (acrosome-reacted/viable spermatozoa) which were not significantly 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 among treatments differed significantly (P < 0.01). Flow cytometric and direct fluorescent microscopy assessments were significantly correlated (r2 = 0.96, P < 0.01). These results indicate that flow cytometry, using an acrosome-specific monoclonal antibody and a supravital dye, provides an objective and efficient method to evaluate human sperm acrosomal and viability status simultaneously.