Sperm analysis by flow cytometry using the fluorescent thiol labeling agent monobromobimane

Abstract

The fluorescent labeling agent monobromobimane (mBBr) was used to label thiols and disulfides (after reduction of sperm disulfides by dithiothreitol) in intact spermatozoa. Bimane-labeled sperm of several mammalian species were analyzed by flow cytometry (FCM) and examined by fluorescent microscopy. FCM analysis showed sperm thiol oxidation to disulfides during epididymal maturation. FCM of labeled mature spermatozoa showed differences among species in the sperm thiol content. Heterogeneity in thiol content of sperm within individual samples was also observed. In addition, FCM patterns showed heterogeneity among and within samples in the content of disulfides and their resistance to reduction. FCM analysis reflected the microscopic appearance of the labeled spermatozoa. FCM analysis of bimane-labeled spermatozoa offers a convenient method for the study of sperm thiol-disulfide status and permits detection of sperm subpopulations within an individual sample. FCM analysis of mBBr-labeled spermatozoa may serve as a test to evaluate sperm quality.