Benzothiazolylphenol-Substituted Ketoester is a Useful Fluorescent Probe for Detection of the Mitochondrion in Sea Urchin Sperm

One of the ketoesters derived from benzothiazolylphenol-substituted dioxetane, benzothiazolylphenol-substituted ketoester (TPKE), demonstrates fluorescence in a 0.1 M NaOH 1) . In this study, the fluorescent staining of a living cell with TPKE was demonstrated by fluorescence microscopy. When sperm from two species of sea urchins—Pseudocentrotus depressus and Anthocidaris crassispina—were used as biological materials, TPKE showed a fluorescent signal in the midpiece that was composed of a single mitochondrion. The ratio of fluorescent signal intensity to background noise (S/N) was high in the sperm stained with 1.0-5.0 μg/ml TPKE in normal artificial seawater (pH 8.0). The S/N ratio decreased in acidic seawater (pH 6.0); acidic conditions repress respiratory activity in sea urchin sperm. Moreover, in the presence of the respiratory chain inhibitor antimycin A and the uncoupler carbonyl cyanide p--trifluoromethoxyphenyl-hydrazone, the sperm showed faint or no fluorescence in normal artificial seawater (pH 8.0). Sea urchin sperm stained with TPKE after fixation showed faint or no fluorescence. These results suggest that TPKE is a potential fluorescent probe of living sea urchin sperm mitochondria with high respiratory