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Updated: Jul 27, 2025

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy
Published on: August 15, 2014
Low-Na
Tosikazu Amano1, Yoshihito Okita1, Motonori Hoshi1
1Department of Life Science, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuda, Midori-ku, Yokohama 227, Japan.
Abstract:
Egg jelly induces the degradation of histones as well as the acrosome reaction in the spermatozoa of Asterina pectinifera. Much similar degradation of histones without any apparent morphological changes such as the acrosome reaction was induced in the spermatozoa by merely dispersing them into Na+ -free seawater. It required external Ca2+ much less than the jelly-induced one in normal seawater, and was not susceptible to Ca2+ -channel antagonists, verapamil and diltiazem. Once spermatozoa were incubated with egg jelly in Ca2+ -free seawater, they did not undergo the histone degradation even after subsequent addition of Ca2+ , but Na+ -free seawater rescued such blockage. Spontaneous acrosome reaction occurred in seawater containing 10-30 mM Na+ in a Ca2+ -dependent manner. This reaction was accompanied by a rapid increase in intracellular pH (pHi) followed by a large pHi decrease. Diltiazem blocked a large decrease in pHi but scarcely inhibited the acrosome reaction induced by low-Na+ seawater. Increasing K+ inhibited both pHi changes and the acrosome reaction induced by low-Na+ seawater. Decreasing pH of seawater also inhibited the pHi changes but did not affect the acrosome reaction. Strontium was also effective to induce a rapid increase, followed by a gradual decrease, in pHi and the acrosome reaction.
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