Two-color fluorescence staining of lectin and anti-CD46 antibody to assess acrosomal status

Objective: To examine potential methods for distinguishing between the acrosome reaction and acrosomal loss. Design: Prospective randomized study. Setting: Department of Obstetrics and Gynecology, Osaka University Hospital, Suita, Japan. Patient(s): Five healthy volunteers and 34 patients with normozoospermia who were participating in an IVF program. Intervention(s): Semen samples were collected from the volunteers before the hamster egg penetration assay and from the patients at the time of IVF. Main Outcome Measure(s): The numbers of oocytes penetrated and spermatozoa bound were determined with the hamster egg penetration assay. Acrosomal status was assessed with two-color fluorescence staining using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and MH61 (anti-CD46 monoclonal antibody) with Texas red-conjugated antimouse immunoglobulin G antiserum. Result(s): The MH61 monoclonal antibody inhibited the penetration of human spermatozoa into hamster oocytes but did not reduce the number of spermatozoa bound to the zona-free hamster oocytes. Two-color fluorescence staining revealed four staining patterns of the acrosomal region. The percentage of PSA-negative/CD46-positive spermatozoa increased to a greater extent than that of PSA-negative/CD46-negative spermatozoa with an increase in the incubation time. Conclusion(s): Two-color fluorescence staining with FITC-PSA and the anti-CD46 monoclonal antibody may be useful for distinguishing between the acrosome reaction and acrosomal loss. (Fertil Steril(R) 1999;71:497-501. (C)1999 by American Society for Reproductive Medicine.).