The pathway and time course of fucose-containing glycoprotein synthesis and intracellular translocation in osteoclasts of the mice maxillary alveolar bone were investigated by electron microscopic radioautography.
Male Balb-C mice weighing 17gm were anesthetized with Nembutal and injected via the external jugular vein with 2.5 mCi of L-[6-3H]-fucose (specific activity 16.8 mCi/mmol) in 0.1 ml of sterile saline solution. At 5, 10, 20, 35 minutes and 8 hours after administration of the 3H -fucose, animals were killed by intracardiac perfusion of 3Oml of 2% glutaraldehyde in a modified Tyroid solution, pH 7.4. The maxillae were then removed and further fixed in Karnovsky fixative for an additional 3-4 hours. After rinsing in O.1M cacodylate buffer for 10 minutes, the maxillae were demineralized for 2 weeks at 4°C in ethylene diamine tetraacetate containing 2% glutaraldehyde. The first interdental areas were mesiodistally sectioned into slices of 1mm thickness and postfixed in osmium tetroxide. Tissues were then dehydrated and embedded in Poly Bed. To prepare electron microscopic radioautography, the dipping method of Kopriwa (1973) was employed. Thin sections were coated with a crystalline monolayer of ILford L4 photographic emulsion.
After exposure for 4 months at 4C, the sections were developed Kodak Microdol-X and Phenidon (for compact grains), fixed in 30% sodium thiosulfate, stained with uranyl acetate and lead citrate and examined in the electron microscope (JEOL 1200 EX).
At 5, 10 and 20 minutes after injection, 3H-fucose was concentrated in Golgi cisternae of the
osteoblasts. By 35 minutes the labels were observed over small vesicles in the suprannclear area of osteoclasts. At 8 hours, numerous silver grains were located on the ruffled border and cell membrane of osteoclasts.
These results indicate that fucose molecules are added in the Golgi apparatus and small vesicles appear to be responsible for translocation of the glycoproteins to the marginal portion of osteoblasts. The glycoproteins are distributed on the osteoclast cell surface and especially over the ruffled border.
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