Molecular mechanism of otolith microstructure formation : immunohistochemical localization of two matrix proteins and expressions of tneir mRNAs

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1. Identification of otolith-matrix-producing cells and localization of matrix proteins in the otolith : Specific antibodies against the major proteins of otolith matrix, OMP-1 and otolin-1, were raised and immunohistochemistry was conducted. The squamous epithelial cells and the transitional epithelial cells of the inner ear sacculus were identified as OMP-1-producing cells, whereas the cells located at the most periferal region of the sensory epithelium were identified as otolin-1-producing cells. The OMP-1 was contained in the otolith only, whereas the otolin-1 was included in both the otolith and the otolith membrane (gelatinous layer). The electron microscopy revealed that the otolith was composed of two types of the organic matrices, a dense sheet-like matrix and a fine meshwork matrix. Both OMP-1 and otolin-1 were detected in the meshwork. Differences in the density of the meshwork matrix makes daily rings, thus it is possible that the daily changes in the rate of production and … More /or deposition of OMP-1 and otolin-1 closely relate to the formation of the daily rings.2. Development of real-time quantitative PCR system for OMP-1 and otolin-1 mRNA expression : We have developed the real-time PCR system to quantify OMP-1 and otolin-1 mRNA expression levels from the left and right sacculi of the individual rainbow trout. The daily changes of the expression levels of OMP-1 and otolin-1 mRNA were now under the investigation.3. Determination of the supersaturation ratio of the inner ear endolymph : Otolith is composed of calcium carbonate poly crystals crystallized from the endolymph. Therefore, the growth rate of the otolith calcium carbonate must be highly dependent on the supersaturation ratio (S_a) of the endolymph. We have measured individually the ionic composition of the endolymph and calculated the S_a. The endolymph S_a was 2.027-4.303, clearly indicating that the endolymph is supersaturated with respect to the aragonite. The S_a was closely related to the pH of the endolymph, thus, pH-regulation of the endolymph is crucial for the otolith growth. Less