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

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One of the ketoesters derived from benzothiazolylphenol-substituted dioxetane,benzothiazolylphenol-substituted ketoester (TPKE), demonstrates fluorescence in a 0.1 MNaOH 1). In this study, the fluorescent staining of a living cell with TPKE was demonstratedby fluorescence microscopy. When sperm from two species of sea urchins—Pseudocentrotusdepressus and Anthocidaris crassispina—were used as biological materials, TPKE showed afluorescent signal in the midpiece that was composed of a single mitochondrion. The ratioof fluorescent signal intensity to background noise (S/N) was high in the sperm stained with1.0–5.0 μg/ml TPKE in normal artificial seawater (pH 8.0). The S/N ratio decreased inacidic 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 uncouplercarbonyl cyanide p--trifluoromethoxyphenyl-hydrazone, the sperm showed faint or nofluorescence in normal artificial seawater (pH 8.0). Sea urchin sperm stained with TPKEafter fixation showed faint or no fluorescence. These results suggest that TPKE is apotential fluorescent probe of living sea urchin sperm mitochondria with high respiratoryactivities.
神奈川大学の論文