Premium
Flow cytometric analysis of mouse sperm using monoclonal anti‐sperm antibody A‐1
Author(s) -
Kawai Yuichi,
Ohnishi Shigeru,
Miyake Masaharu,
Hama Takao,
Mayumi Tadanori
Publication year - 1994
Publication title -
molecular reproduction and development
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.745
H-Index - 105
eISSN - 1098-2795
pISSN - 1040-452X
DOI - 10.1002/mrd.1080370308
Subject(s) - sperm , biology , epididymis , zona pellucida , andrology , acrosome , acrosome reaction , monoclonal antibody , antigen , antibody , flow cytometry , microbiology and biotechnology , immunology , embryo , oocyte , botany , medicine
We have previously prepared an anti‐mouse sperm monoclonal antibody (A‐1) which inhibited sperm penetration into the egg zona pellucida. By indirect immunofluorescence (IIF), the A‐1 antibody was shown to recognize an antigen localized in the acrosomal area of sperm. This antibody bound negligibly to fresh sperm, while binding to methanol‐fixed sperm was almost complete. After methanol fixation, no sperm that penetrated into the zona were immunoreactive for this antibody. In the present study we examined the localization and fate of A‐1 antigen during the acrosome reaction by IIF and flow cytometry (FCM). Cauda epididymal sperm were treated with either calcium ionophore A23187 or zona solution, immunostained indirectly, and subjected to FCM. Treatment with A23187 reduced the percentage of immunoreactive sperm to 59% from the 80% obtained in the untreated sperm. The treatment also reduced the average fluorescence intensity per fluorescence‐positive spermatocyte to 65 channels, while this intensity was 89 channels in the untreated sperm. A similar result was obtained from treatment with zona solution. The proportion of sperm that was immunoreactive with A‐1 antibody was reduced to 55% by incubation in zona‐containing media from the 80% obtained in zona‐free media. On the other hand, neither A23187 nor the zona solution affected the immunoreactivity or the fluorescence intensity of caput epididymal sperm, while the A‐1 antigen was present in both the immature sperm from the caput epididymis of adult mice and in the mature sperm from the cauda epididymis of the same mice. These findings suggest that the intramembrane antigen recognized by the A‐1 monoclonal antibody is released from sperm as a result of the acrosome reaction. © 1994 Wiley‐Liss, Inc.