Electron microscopic and cytochemical studies on the so-called zymogen granules in the rat sublingual glands

Abstract

The present study of the rat sublingual gland was undertaken to clarify the relationship between the fine structure of the secretory granules and the digestive enzymes, especially amylase activity by the cell fractionation procedures.For electron microscopic study, small pieces which were removed from sublingual gland of male Wistar strain rats differing in age from 1 day to 2 years were employed. The pieces were immersed in Caulfieled's solution at pH 7.4 (0°C) for 1.5 hours. The individual fractions which were obtained by the fractionation methods (SCHRAMM et al.) were examined for the identification of their contents after fixed in 5% ice cold glutaraldehyde solution (pH 7.4) for 1 hour and again in Caulfield's for 17 hours. The ultrathin sections from the materials embedded in epoxy resin (Luft) were stained with uranyl acetate and examined with the electron microscope.As to the histological findings, glandular epithelial elements of new born rats were more loosely arranged within lobules and they were smaller in size than in adults. In the course of postnatal development, the lobules were recognized to increase conspicuously in diameter at about 3 to 4 weeks. The secretory granules in mucous cells and serous demilune cells showed significant differences in shape, size and electron density throughout the postnatal period. The granules in the demilune cells resembled the zymogen granules found in the parotid gland or pancreas; they were round, homogeneous and osmiophilic granules with a single limitting membrane. The product of the mucous cells was amorphous and electron lucent substance containing fine granules, and was similar in structure to that of intestinal goblet cells.In the biochemical study of amylase in the glands, the individual fractions and supernatant obtained from cell fractionation were examined in the distribution of enzymatic activity. Postnatal changes for amylase activity were studied simultaneously, during the period from 1 day to 2 years. Amylase activity was determined by Fuwa's modified method. As for the distribution of amylase activity of the individual fractions, F-2(“zymogen”granule fraction), F-4 (microsomal fraction) and final supernatant showed a considerably higher activity than others, but there were no significant differences in the intensity of the activity during the postnatal development. In the electron microscopic findings, F-2 was mainly composed of round, homogeneous and osmiophilic granules with about 1μ diameter; a little amount of mitochondria were contained in this fraction. A high amylase activity in F-4 suggested the possibility that it might be biosynthesised in microsomes of demilune cells. In the F-2, after sonication (0°C for 10min., 10KC) and centrifugation, the amylase activity had almost been transferred to the supernatant and the“zymogen”granules, when observed under the electron microscope, had been destroyed to pieces. Thus it was concluded that the granules were the very sites where amylase was contained.The round osmiophilic granules in the demilune cells are believed to be real zymogen granules as it is the case in the pancreas and parotid gland from morphological as well as biochemical viewpoints. From the localization of the granules in the demilune cells, it seems probable that these cells synthesize and store amylase in the zymogen granules.