Immunohistochemical demonstration of amelogenin penetration toward the dental pulp in the early stages of ameloblast development in rat molar tooth germs

Abstract

In order to examine the synthesis and secretion of enamel protein by ameloblasts in their early stages of development, immunohistochemical localization was carried out at light and electron microscopic levels using a monoclonal antibody produced in a preliminary experiment. Materials used were tooth germs of mandibular first molars of rats at 0-5 days after birth. Immunoblot analysis after two-dimensional electrophoresis revealed that antigen molecules recognized by the monoclonal antibody were amelogenins of 26-28 kDa (pI, 6.6-7.0). An immunohistochemical examination using this monoclonal antibody demonstrated that the presecretory ameloblasts in their early stages of differentiation both synthesized amelogenin and secreted through a classical merocrine secretory pathway. In some presecretory ameloblasts as well as ameloblasts we observed the distended cisternae of rough endoplasmic reticulum (rER) which demonstrated heterogenous immunolabelling. The immunolabellings were also detected in the predentin as well as the intercellular spaces of odontoblasts and dental pulp cells which indicated penetration of amelogenin from the presecretory ameloblast layer to the dental pulp. The presence of coated pits at the plasma membrane of odontoblasts in close proximity to enamel protein along with the immunolabelling of lysosomes of the odontoblasts suggests the phagocytosis of the enamel protein into the odontoblasts. These observations suggest the possibility that the penetration of enamel protein toward the dental pulp and odontoblasts plays a role in the interaction between ameloblasts and odontoblasts.

Regulation of amelogenin gene expression during tooth development

“…Snead et al (1984) isolated RNA from first molars of mice of various ages and found greatest hybridization to a radiolabeled amelogenin probe at birth and in 1 day postnatal mice. Results from immunohistochemistry experiments using anti-amelogenin antibodies have suggested that expression begins in pre-ameloblasts in rat molars (Inai et al, 1991;Bronckers et al, 1993). We, however, have not found evidence for expression in any part of the tooth that does not have some mineralized matrix present.…”

LAMB3 Mutations Causing Autosomal-dominant Amelogenesis Imperfecta

“…Laminin epitopes are lost as pre-ameloblasts initiate amelogenin secretion (Inai et al, 1991) and do not appear again until ameloblasts transition to the maturation stage (Nanci et al, 1993). In place of the basement membrane under secretory-stage ameloblasts is a mineralization front apparatus that initiates and elongates thin ribbons of amorphous calcium phosphate (Beniash et al, 2009;JP Simmer et al, 2012).…”

Reuptake of Extracellular Amelogenin by Dental Epithelial Cells Results in Increased Levels of Amelogenin mRNA through Enhanced mRNA Stabilization

“…Several laboratories showed that the amelogenin was detected in the cytoplasm lysosome and stippled material-like substance adjacent to the coated pits of plasma membrane in presecretory ameloblasts and odontoblasts, suggesting that it is likely that amelogenin might be taken up by an endocytic pathway (32)(33)(34). Recently, some integral membrane proteins binding amelogenin have been identified by using the yeast twohybrid assay (35).…”