Fine structure of transforming-type granules in mucous cells in the early postnatal rat parotid gland when processed by rapid freezing followed by freeze-substitution fixation

The present study was designed to clarify the more precise ultrastructural feature of granules, especially mucous granules in the early postnatal rat parotid gland by using rapid freezing followed by freeze-substitution fixation. The parotid gland of Wistar rats (aged 0-10 days) was removed under anesthesia and immediately underwent cryofixation followed by substitution with osmium tetroxide. After fixation, the samples were embedded in Epon-Araldite, cut into ultrathin section, and then examined by transmission electron microscopy. Electron microscopy showed that the mucous granules of samples treated by freeze-substitution fixation had low electron density and were almost spherical in shape with a clear limiting membrane. By Day 5, granules that were a little more electron dense than mucous granules, granules with a more electron dense portion at their periphery, and heterogeneous granules with an internal highly electron dense portion were found. Moreover, cells containing both homogeneous granules with a high electron density similar to that of mature serous granules and heterogeneous granules were observed. These findings demonstrated that the morphology of the transforming-type mucous granules by chemical fixation in the previous study was an artifact and, as a result, strongly suggested that on the sequential morphological changes of transitional mucous/serous granules by rapid freezing method in the present study, the mucous cells in the early postnatal rat parotid gland undergo transformation to serous cells.