Measurement of intracellular pH in human spermatozoa by flow cytometry with the benzo[c]xanthene dye SNAFL-1: a novel, single excitation, dual emission, molecular probe

In numerous animal species the acrosome reaction of spermatozoa has been linked to elevations in intracellular pH (pHi). However, whether or not this is merely a passive consequence of calcium ion influx is not known. Studies into the fluctuations in pHi in sperm cells have been hampered by the lack of a pH-sensitive probe that could be used in conjunction with flow cytometry. In this study, flow cytometric analysis of pHi in human spermatozoa was accomplished by using one of the new benzo[c]xanthene dyes (SNAFL-1). SNAFL-1 was then observed in situ with conventional fluorescent microscopy and was found to be located in the post-acrosomal cytoplasm of the head. It was then used to measure the differences in pHi between acrosome-intact populations of spermatozoa, and populations that had been induced to acrosome-react with human follicular fluid or the calcium ionophore A23187 to mimic the calcium influx. It was concluded that the human sperm acrosome reaction is also accompanied by a rise in pHi and the natural agonist-induced rise could not be accounted for by calcium ion influx alone.