Hiroyuki Yokosuka

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.

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.

Occurrence of gustducin-immunoreactive cells in von Ebner's glands of guinea pigs

An immunohistochemical examination of guinea-pig taste buds in vallate papillae revealed gustducin-immunoreactive cells in the area of von Ebner’s glands, minor salivary glands. Since there have been no reports describing those cells in these locations for other species, we investigated these glands in order both to localize the cells and compare their immunoreactive characteristics with corresponding cells in the vallate taste buds. The gustducin-immunoreactive cells coincided with cells containing no secretory granules in the end portion of the glands, which was supported by the electron-microscopic immunocytochemistry. Double immunofluorescence microscopy confirmed these cells to be entirely immunopositive to type III inositol 1,4,5-triphosphate receptor (IP3R-3), phospholipase Cβ2 (PLCβ2), and villin and also partly immunopositive to neuron-specific enolase (NSE) and calbindin D-28K. The gustducin-immunoreactive cells in the vallate taste buds exhibited completely the same immunoreactivities for these five molecules. Accordingly, the present results give credence to a consideration that the gustducin-immunnoreactive cells in both locations are identical in function(s) e.g., chemo-reception.

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.