The morphological features of tooth enamel and enameloid in actinopterygian fish are reviewed to provide basic data concerning the biomineralization of teeth in lower vertebrates. Enameloid, which covers the tooth surface, is a unique well-mineralized tissue and usually has the same functions as mammalian tooth enamel. However, the development of enameloid is different from that of the enamel produced by dental epithelial cells. Enameloid is made by a combination of odontoblasts and dental epithelial cells. An organic matrix that contains collagen is provided by odontoblasts, and then dental epithelial cells dissolve the degenerate matrix and supply inorganic ions during advanced crystal growth in enameloid. It is likely that enameloid is a good model for studying the growth of well-mineralized hard tissues in vertebrates. Some actinopterygian fish possess a collar enamel layer that is situated at the surface of the tooth shaft, indicating that the origin of tooth enamel is found in fish. Collar enamel is thought to be a precursor of mammalian enamel, although it is thin and not well mineralized in comparison with enameloid. In Lepisosteus and Polypterus, both of which are living actinopterygians, both enameloid and enamel are found in the same tooth. Therefore, they are suitable materials for examining the developmental processes of enameloid and enamel and the relationship among them.

This is the first detailed report about the collar enamel of the teeth of Polypterus senegalus. We have examined the fine structure of the collar enamel and enamel organ of Polypterus during amelogenesis by light and transmission electron microscopy. An immunohistochemical analysis with an antibody against bovine amelogenin, an antiserum against porcine amelogenin and region-specific antibodies or antiserum against the C-terminus, middle region and N-terminus of porcine amelogenin has also been performed to examine the collar enamel matrix present in these teeth. Their ameloblasts contain fully developed Golgi apparatus, rough endoplasmic reticulum and secretory granules. During collar enamel formation, an amorphous fine enamel matrix containing no collagen fibrils is found between the dentin and ameloblast layers. In non-demineralized sections, the collar enamel (500 nm to 1 μm thick) is distinguishable from dentin, because of its higher density and differences in the arrangement of its crystals. The fine structural features of collar enamel in Polypterus are similar to those of tooth enamel in Lepisosteus (gars), coelacanths, lungfish and amphibians. The enamel matrix shows intense immunoreactivity to the antibody and antiserum against mammalian amelogenins and to the middleregion- and C-terminal-specific anti-amelogenin antibodies. These findings suggest that the proteins in the enamel of Polypterus contain domains that closely resemble those of bovine and porcine amelogenins. The enamel matrix, which exhibits positive immunoreactivity to mammalian amelogenins, extends to the cap enameloid surface, implying that amelogenin-like proteins are secreted by ameloblasts as a thin matrix layer that covers the cap enameloid after enameloid maturation.

The fine structure of collar enamel and the cells constituting the enamel organ during amelogenesis in Lepisosteus oculatus was observed by light, scanning electron and transmission electron microscopy. In the enamel, slender crystals were arranged perpendicular to the surface and the stripes that were parallel to the surface were observed, suggesting that the enamel in Lepisosteus shares common morphological features with that in sarcopterygian fish and amphibians. Ameloblasts containing developed Golgi apparatus, rough endoplasmic reticulum (rER) and secretory granules were found in the secretory stage. In the maturation stage, a ruffled border was not seen at the distal end of the ameloblasts, while many mitochondria and lysosome-like granules were obvious in the distal cytoplasm. The enamel organ consisted of the outer dental epithelial cells, stratum reticulum cells and ameloblasts, but there was no stratum intermedium. It is likely that the ameloblasts have less absorptive function in comparison with the inner dental epithelial cells facing cap enameloid.

Enameloid is a well-mineralized tissue covering the tooth surface in fish and it corresponds to the outer-most layer of dentin. It was reported that both dental epithelial cells and odontoblasts are involved in the formation of enameloid. Nevertheless, the localization and timing of secretion of ectodermal enamel matrix proteins (EMPs) in enameloid are unclear. In the present study, the enameloid matrix during the stages of enameloid formation in spotted gar, Lepisosteus oculatus, an actinopterygian, was examined mainly by transmission electron microscopy-based immunohistochemistry using an anti-mammalian amelogenin (AMEL) antibody and antiserum. Positive immunoreactivity with the antibody and antiserum was found in enameloid from the surface to the dentin-enameloid junction just before the formation of crystallites. This immunoreactivity disappeared rapidly before the full appearance of crystallites in the enameloid during the stage of mineralization. Immunolabelling was usually found along the collagen fibrils but was not seen on the electron-dense fibrous structures, which were probably derived from matrix vesicles in the previous stage. In inner dental epithelial (IDE) cells, the granules in the distal cytoplasm often showed positive immunoreactivity, suggesting that the EMP-like proteins originated from IDE cells. EMP-like proteins in the enameloid matrix might be common to the EMP-like proteins previously reported in the collar enamel of teeth and ganoine of ganoid scales, because they exhibited marked immunoreactivity with the same anti-mammalian AMEL antibodies. It is likely that EMP-like proteins are involved in the formation of crystallites along collagen fibrils in enameloid.

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