Mikio Ishiyama

The structure and development of the collar enameloid in two teleost fishes, Halichoeres poecilopterus and Pagrus major.

SummaryHistologically the outer layer of the collar enameloid obviously differs from the inner layer, and it has a degree of mineralization nearly as high as the cap enameloid which has the highest. In the stage of matrix formation, the organic matrix of the collar enameloid contains a number of collagen fibers, and odontoblasts display features suggesting that these cells actively synthesized and secreted collagen. A number of cell processes, matrix vesicles and some cell debris which were probably derived from the odontoblasts were observed in the organic matrix of the collar enameloid. We consider that the majority of the organic matrix in collar enameloid originates from the odontoblasts. In the stage of maturation, collagen fibers were not observed in the outer layer of the collar enameloid in demineralized specimens. In the IDE cells during this stage, the complex infoldings of cell membranes developed in the distal portion, and several lysosomal granules and irregular-shaped granules containing many tubular structures, were observed in the distal cytoplasm. In the ODE cells, abundant labyrinthine canals appeared in the cytoplasm, and capillary vessels were found close to the outer surface of the ODE cells. We assume that the higher mineralized outer layer of the collar enameloid is made possible by the absorptive and transport functions of the epithelial cells during the stage of maturation. It is considered that the collar enameloid in this study was initially produced by the odontoblasts and then reconstructed by the epithelial cells, so that the collar enameloid differs from true enamel.

Fine structural and cytochemical observations on the dental epithelial cells during cap enameloid formation stages in Polypterus senegalus, a bony fish (Actinopterygii).

Tooth germs during cap enameloid formation stages in Polypterus senegalus were investigated by transmission electron microscopy and enzyme histo- and cytochemistry. Enameloid formation was divided into three stages: matrix formation, mineralization, and maturation. The enamel organ consisted of the inner dental epithelial cells, stellate reticulum, and outer dental epithelial cells during cap enameloid formation stages, but no stratum intermedium was found. During the matrix formation stage, the tall inner dental epithelial cells contained well-developed Golgi apparatus, abundant cisternae of rough endoplasmic reticulum and mitochondria. Spindle-shaped vesicles containing a filamentous structure were seen in the distal cytoplasm. During mineralization and maturation stages, many ACPase positive lysosomes were present in the cytoplasm, whereas the organelles were decreased in number. The infoldings of the distal plasma membrane of the inner dental epithelial cells were visible in the mineralization stage but were not marked in the maturation stage. The activity of nonspecific ALPase, Ca-ATPase, and K-NPPase was detected at the plasma membrane of the inner dental epithelial cells during the stages of mineralization and maturation. The results of fine structure and enzyme cytochemistry suggested that the dental epithelial cells were mainly involved in the degeneration and removal of enameloid matrix and in material transportation during the enameloid mineralization and maturation stages, rather than in the enameloid matrix formation. The results also showed that the structure of the dental epithelial cells in Polypterus was different from that in teleosts and gars, but that the function of the dental epithelial cells was similar to that in teleosts possessing well-mineralized cap enameloid.

Fine Structure and Ca-ATPase Activity of the Stratum Intermedium Cells During Odontogenesis in Gars, Lepisosteus, Actinopterygii

This is the first report on the stratum intermedium in vertebrates other than mammals. The aim of this study is to elucidate the fine structure and cytochemical features of the stratum intermedium during the stages of enameloid formation in Lepisosteus . Inner dental epithelium, stratum intermedium, stellate reticulum, and outer dental epithelium are consistently present in the tooth germs of Lepisosteus . The stratum intermedium cells are oval in shape, contain elliptical nuclei, and extend many small processes. It is implied that the structure of the enamel organ is different among actinopterygians, and that constitution of the enamel organ in Lepisosteus resembles that in higher vertebrates. Marked Ca-ATPase activity is observed at the cell membrane of the stratum intermedium cells, suggesting that the cells are involved in calcium transport during the stages of enameloid formation.

Immunohistochemical and Western blot analyses of collar enamel in the jaw teeth of gars, Lepisosteus oculatus, an actinopterygian fish.

Abstract Although most fish have no enamel layer in their teeth, those belonging to Lepisosteus (gars), an extant actinopterygian fish genus, do and so can be used to study amelogenesis. In order to examine the collar enamel matrix in gar teeth, we subjected gar teeth to light and electron microscopic immunohistochemical examinations using an antibody against bovine amelogenin (27 kDa) and antiserum against porcine amelogenin (25 kDa), as well as region-specific antibodies and antiserum against the C-terminus and middle region, and N-terminus of porcine amelogenin, respectively. The enamel matrix exhibited intense immunoreactivity to the anti-bovine amelogenin antibody and the anti-porcine amelogenin antiserum in addition to the C-terminal and middle region-specific antibodies, but not to the N-terminal-specific antiserum. These results suggest that the collar enamel matrix of gar teeth contains amelogenin-like proteins and that these proteins possess domains that closely resemble the C-terminal and middle regions of porcine amelogenin. Western blot analyses of the tooth germs of Lepisosteus were also performed. As a result, protein bands with molecular weights of 78 kDa and 65 kDa were clearly stained by the anti-bovine amelogenin antibody as well as the antiserum against porcine amelogenin and the middle-region-specific antibody. It is likely that the amelogenin-like proteins present in Lepisosteus do not correspond to the amelogenins found in mammals, although they do possess domains that are shared with mammalian amelogenins.

Microstructural features of dipnoan tooth enamel

Tooth plates of extant lungfishes were examined by contact microradiography, light microscopy and TEM, to elucidate the nature and origin of the hypermineralized surface layer. TEM indicated that the matrix of this layer consisted of organic filamentous material, and no collagen fibres were seen in the matrix. The matrix appeared to be secreted by oral epithelial cells of the inner fold surrounding the surface of the tooth plate, and deposited in the area between the cell membrane and basal lamina-like structure of these epithelial cells. Therefore, it is suggested that the hypermineralized surface layer of lungfish plates is enamel of ectodermal origin.

Ultrastructure of Pleromin, a Highly Mineralized Tissue Comprizing Crystalline Calcium Phosphate Known as Whitlockite, in Holocephalian Tooth Plates

The gross structure and microstructure of holocephalian tooth plates were initially investigated by Bargmann (1,2) and Brettnacher (3). Orvig (4,5,6) subsequently reported in the detail microstructural characteristics of both extant and fossil holocephalian tooth plates. These studies indicate the holocephalian tooth plate to consist of bony tissue and hypermineralized pleromin (4), with the hardness of pleromin comparable to that of enamel and enameloid. Ishiyama et al. (7) found the crystalline material of pleromin to be not hydroxyapatite but whitlockite by a powder X-ray diffractometry, and pleromin to be the sole sound dental tissue consisting of whitlockite in vertebrates.

Immunohistochemical and Western Blotting Analyses of Ganoine in the Ganoid Scales of Lepisosteus oculatus: an Actinopterygian Fish.

In order to compare its characteristics with those of jaw tooth collar enamel, normally developing and experimentally regenerating ganoine from ganoid scales of Lepisosteus oculatus (spotted gar), an actinopterygian fish species, was examined by Western blotting and immunohistochemistry. Amelogenin, a major enamel matrix protein (EMP), is widely found from sarcopterygian fish to mammals. Therefore, we used antimammalian amelogenin antibodies and antisera: an antibody against bovine amelogenin; antiserum against porcine amelogenin; and region-specific antibodies or antiserum against the C-terminus, middle region, or N-terminus of porcine amelogenin in this study. Positive immunoreactivity with the antibody against bovine amelogenin, antiserum against porcine amelogenin, and the middle and C-terminal region-specific antibodies was detected in both normally developing and regenerating ganoine matrix, as well as in granules found within inner ganoine epithelial cells. These immunohistochemical analyses indicated that the Lepisosteus ganoine matrix contains EMP-like proteins with epitopes similar to mammalian amelogenins. In Western blotting analyses of regenerating ganoid scales with the antibovine amelogenin antibody, two protein bands with molecular weights of approximately 78 and 65 kDa were detected, which were similar to those found in Lepisosteus tooth enamel. Our study suggests that in Lepisosteus, EMP-like proteins in the ganoine matrix corresponded to those in tooth enamel. However, it was revealed that the 78 and 65 kDa EMP-like proteins were different from 27 kDa bovine amelogenin.

Immunolocalization of Enamel Matrix Protein-Like Proteins in the Tooth Enameloid of Actinopterygian Bony Fish

Tooth enameloid in bony fish is a well-mineralized tissue resembling enamel in mammals. It was assumed that the dental epithelial cells are deeply involved in the formation of enameloid. However, unlike enamel matrix which fully consists of several ectodermal enamel matrix proteins (EMPs), whether enameloid matrix contains ectodermal EMPs has been debated for a long time. In the present study, transmission electron microscopy-based immunohistochemical examinations, using the protein A-gold method with antibodies and antiserum against mammalian amelogenin, were performed in order to search for EMP-like proteins in the cap enameloid of basic actinopterygians, Polypterus and gar. Positive immunoreactivity was detected in the cap enameloid matrix just before the appearance of many crystallites along collagen fibrils, indicating that the cap enameloid contains EMP-like proteins. Immunolabelling was usually found along the collagen fibrils but was not seen on the electron-dense fibrous structures. Therefore, it is conceivable that the ectodermal EMP-like proteins in cap enameloid are involved in crystallite formation along collagen fibrils.

Immunolocalization of enamel matrix protein-like proteins in the tooth enameloid of spotted gar, Lepisosteus oculatus, an actinopterygian bony fish

ABSTRACT 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 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 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 cells, the granules in the distal cytoplasm often showed positive immunoreactivity, suggesting that the enamel matrix protein-like proteins originated from inner dental epithelial cells. Enamel matrix protein-like proteins in the enameloid matrix might be common to the enamel matrix protein-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 amelogenin antibodies. It is likely that enamel matrix protein-like proteins are involved in the formation of crystallites along collagen fibrils in enameloid.

Histochemical and immunohistochemical examination of odontoblasts (petroblasts) in petrodentine formation of lungfish

OBJECTIVE Petrodentine, the core of the lungfish tooth plate, is a well-mineralized tissue similar to mammalian enamel and analogous to enameloid in fish teeth. Petrodentine is formed solely by petroblasts, which are specialized odontoblasts, whereas enameloid is a composite tissue produced by both odontoblasts and dental epithelial cells. To clarify the details of petrodentine formation, petroblasts were investigated using histochemical and immunohistochemical techniques. METHODS Extant lungfish (Lepidosiren paradoxa) were used in this study. Tooth plates during the stage of petrodentine formation were observed by means of histochemistry and immunohistochemistry. Commercial kits were used to detect enzyme activity. Correlative sections were immunostained using antibodies against selected peptides. Routine staining such as periodic acid-Schiff (PAS) reaction to identify glycogen and Elastica van Gieson staining for the detection of elastic fibers in histological sections were performed. In addition, conventional transmission electron microscopy was used for observing the fine structure. RESULTS Petroblasts showed marked acid and alkaline phosphatase activities, and positive immunoreactivities against anti-nestin, anti-V-ATPase, and anti-Ca2+-ATPase, during the maturation stage, but in the matrix formation stage, reactions were much weaker than that of the maturation stage. During the maturation stage, petroblasts showed intense PAS reactivity, and glycogen particles were observed in petroblasts by transmission electron microscopy. Glucose transporter 1-immunoreactivity was observed in petroblasts in the matrix formation stage and the initial to mid part of the maturation stage. CONCLUSIONS The results in this study suggested that petroblasts have two functional stages, matrix formation and maturation, and glycogen plays an important role in the modulation of petroblasts.