Ultrastructure of quick-frozen secretory ameloblasts of the rat molar tooth

Ameloblasts in molar tooth germs from 4 to 12-day-old neonatal rats were quick-frozen and then freeze-substituted in acetone-tannic acid and acetone-osmium tetroxide. Following quick freezing and freeze-substitution, the preservation of ameloblasts was principally the same as seen in conventionally-fixed ones. Several differences, however, became apparent between two methods. Concerning enamel formation, the quick-freezing procedure greatly improved the preservation including that of the extrusion of secretory granules into the extracellular space. The exocytosis is a type of merocrine secretion. The precursor stippled material was demonstrated at the front of Tomes' processes and in the intercellular space of the distal terminal junction. In the more advanced stage, the stippled material tended to decrease gradually in amount. Prior to the formation of the typical Tomes' process, large secretory granules fused directly with the distal plasma membrane, which plays a role in the formation of prismless enamel. After the typical Tomes' process had formed, secretory granules containing electron-dense material instead of the large secretory granules accumulated in the distal portion of the Tomes' process. These latter secretory granules had two different destinations; i.e. the granules discharged their contents into the infoldings of the distal part of Tomes' process and into the infoldings of the lateral cell membrane. These types of secretion seem to have a functional role in the rod or inter-rod enamel formation. The ultrastructure of the stippled material consisted of 3-dimensionally interconnected strands after quick freezing, which is different from the appearance of granules embedded in amorphous material after conventional fixation. The stippled material was seen in the intercrystal space among the newly formed crystals. Also, an extensive tubulo-vesicular structure was preserved in both the cell body and Tomes' process. The present data suggest that the membrane of this structure showed a smooth contour opening to the extracellular space, and also an intimate relationship with the secretory granules.