Fish otoliths provide a record of age, growth, and environmental influences. In both trout and turbot, spatial chemical investigation of the endolymph surrounding the otolith (sagitta) showed a lack of uniformity. Proteins, [Formula: see text], and Mg2+ were significantly more concentrated in the proximal (facing the macula) than distal zone, whereas the opposite was observed for K+ and total CO2(totCO2). Na+ concentration ([Na+]) was 20% higher in the proximal zone in trout but not in turbot. Total Ca and Cl− contents were uniformly distributed in both species. We propose that the endolymphatic gradients of protein and totCO2 concentration within the endolymph are involved in the otolithic biocalcification process. Microchemical analyses of otolith sections by wavelength dispersive spectrometry showed a lack of spatial uniformity in the K/Ca and Na/Ca ratios, whereas the Sr/Ca ratio was uniform. There is a clear relationship between endolymph and otolith [K+], but the interpretation of the results for [Na+] needs further investigation. Thus the lack of uniformity in the otolith composition must be taken into account when investigating otolith microchemistry.

This paper compares the organic compositions of the otolith and endolymph of trout and turbot. Irrespective of the method of demineralization (0.5 M EDTA or acetic acid), trout otoliths were found to be largely composed of proteins (48%), collagens (23%), and proteoglycans (29%). Collagen was only detectable in the EDTA-insoluble (0.30 microg/mg) and in the acetic acid-soluble fractions (0.53 microg/mg). The same compounds were found in the endolymph but in different proportions (proteins 85%, collagens 12%, and proteoglycans 3%). It was shown that the distribution of these compounds was not uniform within the endolymph. Proteins, collagens, and amino acids were 4, 10, and 3 times, respectively, more concentrated in the proximal (facing the macula) than the distal side whereas proteoglycans were 10 times more concentrated at the distal side. SDS PAGE analyses of proximal and distal samples of endolymph showed similar patterns suggesting that the spatial gradient of protein is quantitative and not qualitative. SDS PAGE comparison of endolymph and otolith samples showed only two proteins with similar molecular weights. We propose that collagen and protein gradients are involved in the organic matrix formation and otolith calcification process. Endolymphs from both trout and turbot display inhibitions of in vitro calcification although these inhibitions were 50 and 80 times, respectively, less than that of the otoliths. The inhibitory factor probably plays a significant role in the regulation of otolith calcification.

SUMMARYIonic and organic parameters of the otolith calcification process in the trout Oncorhynchus mykiss were analysed in plasma and endolymph over the day:night cycle. Plasma pH remained constant and total CO2concentration was significantly lower (by 21%) during the day than at night. Calcifying parameters (total CO2, total calcium concentration) were measured in the proximal and distal endolymphs and were unchanged in the latter during the day:night cycle, but fluctuated in the former. Non-collagenous protein and collagen concentrations in endolymph were higher(1.5- and 10-fold, respectively) during the day than at night. As there was no change in total calcium concentration, we propose that Ca2+increases during the dark period and was maximal by the end of the night when the total CO2 concentration has also increased (by 14%). Measurements of endolymph pH in situ revealed significant differences between samples from proximal and distal endolymph (7.38 and 7.87,respectively), but no variation between values obtained during the day and at night. Thus, the saturation state of aragonite (Sa) in the proximal endolymph should fluctuate around unity during the day:night cycle, and CaCO3precipitation should occur when supersaturation is reached. The electrophoretic pattern of proximal endolymph showed variations in both major and minor components. Immunoblotting of endolymph, using a rabbit antiserum raised against the otolith soluble organic matrix revealed an increase in the expression of two proteins (65 kDa and 75 kDa) during the day period. We propose that organic matrix and calcium carbonate deposition on the otolith vary antiphasically: organic matrix deposition begins by the end of the day period, when the concentration of organic precursors is maximal in the endolymph, whereas CaCO3 precipitation starts once the solubility of CaCO3 is exceeded.

The soluble organic matrix (OM) of various biominerals (red coral skeleton, oyster shell, sea urchin test, turbot otolith, chicken eggshell) was extracted after demineralization with acetic acid. The protein content of the OM varies strongly from 0.02 to 1.6 microg/mg biomineral whereas proteoglycans present less variations (from 0.7 to 1.4 microg/mg biomineral). Electrophoresis of biominerals OM shows differences in their protein pattern although several bands are present in all matrices. OM of all biominerals shows carbonic anhydrase activity but no activity was detectable in the endolymph. OM of all biominerals also displays an anticalcifying activity. After separation of the OM extracts by chloroform-methanol, 80% of the anticalcifying activity was found in the methanol phase except in the urchin test. After OM precipitation with trichloracetic acid, 70% of the activities was found in the supernatants. Partial biochemical characterization suggests that the anticalcifying factor is a polyanionic and water-soluble molecule, which could be proteoglycans. The endolymph surrounding the otolith also displays an anticalcifying activity although its inhibitous activity was 50 times lower than that of the otolith OM. However, the anticalcifying activity of the endolymph is assumed by a proteic structure (80% activity precipitated with TCA treatment). Our results suggest that both carbonic anhydrase and anticalcifying activities are widespread and play a significant role in the regulation of biomineral formation. Results are discussed in relation to the calcification process that takes place at the fluid-mineral interface.