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.