title: Assessment of the acrosomal status and viability of human spermatozoa simultaneously using flow cytometry.
abstract: 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.
authors.affiliation: Reproductive Biology Laboratory, Methodist Hospital of Indiana, Inc., Indianapolis 46202.
authors.forename: J
authors.initials: J
authors.lastname: Tao
authors.forename: J
authors.initials: J
authors.lastname: Du
authors.forename: E S
authors.initials: ES
authors.lastname: Critser
authors.forename: J K
authors.initials: JK
authors.lastname: Critser
mesh_terms: D000177:Acrosome / Q000502:physiology*; D000911:Antibodies, Monoclonal; D000001:Calcimycin / Q000494:pharmacology; D002470:Cell Survival; D015925:Cryopreservation; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D008297:Male; D008856:Microscopy, Fluorescence; D013094:Spermatozoa / Q000502:physiology* / Q000648:ultrastructure
chemical_list: D000911:Antibodies, Monoclonal; D000001:Calcimycin; D004121:Dimethyl Sulfoxide