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

“…The two region‐specific antibodies against the C‐terminus and middle region of porcine amelogenin were produced using synthetic peptides. The same antibodies and antisera were previously used for studies of the tooth collar enamel of Lepisosteus (Sasagawa et al, ) and Polypterus species (Sasagawa et al., ). They were used in the present study to compare the results to those of our previous study on the collar enamel (Sasagawa et al., ).…”

“…The same antibodies and antisera were previously used for studies of the tooth collar enamel of Lepisosteus (Sasagawa et al, ) and Polypterus species (Sasagawa et al., ). They were used in the present study to compare the results to those of our previous study on the collar enamel (Sasagawa et al., ). Among several tetrapods amelogenin molecules, the N‐terminal region contains an abundance of amino acids in common and the C‐terminal region has several amino acids in common, but the middle region contains only a few shared parts (Fig.…”

“…Among several tetrapods amelogenin molecules, the N‐terminal region contains an abundance of amino acids in common and the C‐terminal region has several amino acids in common, but the middle region contains only a few shared parts (Fig. , Sasagawa et al., ). Detailed information about the antibodies and antisera are provided in previous papers (Sasagawa et al., , ).…”

“…In polypterids, these scales mainly consist of three layers, a bony plate, dentin, and ganoine, whereas living lepisosteids lack the dentin layer (Sire, , ). Lepisosteus and Polypterus also have typical teeth composed of dental hard tissues, that is, dentin constituting the body of the tooth, and two kinds of well‐mineralized covering, cap enameloid and collar enamel (Ishiyama et al., ; Sasagawa et al., , ). Therefore, these species might be a suitable model for examining the relationship between teeth and ganoid scales.…”

“…In the polypterids Polypterus senegalus and Calamoichthys calabaricus , both preganoine and collar enamel exhibited immunoreactivity with an antimammalian amelogenin antibody (Kogaya et al, ’97; Zylberberg et al, ; Sasagawa et al., ). In the lepisosteid L. oculatus , marked immunoreactivity with antimammalian amelogenin antibodies was also observed in the collar enamel matrix, indicating that the collar enamel of teeth contains EMP‐like proteins (Ishiyama et al., ; Sasagawa et al., ). However, for the preganoine of Lepisosteus scales, there is no information about the localization of EMP‐like proteins or their relationship to proteins in the enamel of jaw teeth.…”