Serine protease (EMSP1) has been known to be synthesized by ameloblasts in late secretory- through early maturation stages of amelogenesis, and facilitate degradation of enamel matrix proteins. Recent observations further indicated EMSP1 expressions in ameloblasts at later stages of maturation, and also in odontoblasts at various developments. However, exact location of EMSP1 and its active sites in amelogenesis have not been fully understood. Objectives: and Methods: The aim of this study is therefore to establish precise mapping of EMSP1 and the sites of proteolysis in developing enamel. For this purpose, we used immunohistochemistry for EMSP1 and in situ zymography on undecalcified fresh-frozen sections of rat incisors. In zymography, sites of proteolysis could be identified by green fluorescence under the fluorescent microscope. Results: and Discussion: Immunoreactions for EMSP1 were located in the whole areas of secretory enamel. Iimmunoreactivity in the deepest and surface enamel layers was stronger than that in the intermediate layers. Distinct immunoreactions disappeared from enamel at early maturation almost concomitant with the timing of disappearance of amelogenin immunoreactions. However, moderate reactions for EMSP1 remained along the enamel surface up to mid stage of maturation. In situ zymography revealed intense fluorescence in the whole areas of immature enamel indicating active proteolysis throughout the growing enamel. Most intense reaction was depicted in early maturation. Unlike EMSP1 immunoreactions, fluorescence of DQ gelatin did not diminish immediately thereafter, and moderate ones persisted until mid maturation. EDTA pretreatment of sections caused significant reduction of fluorescence in secretory and mid maturation enamel. However, fluorescence in early maturation and the later ones in surface layers were unaffected. Conclusion: EMSP1 is present in the whole layers of immature enamel and persists in the surface layers up to mid maturation. Diminished fluorescence by EDTA pretreatment appears to represent the sites of proteolysis by metalloproteases.

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