The biosynthesis and secretion of precursor enamel protein by ameloblasts as visualized by autoradiography after tryptophan administration

Abstract

The incorporation of 3H-tryptophan into the inner enamel epithelium of newborn mouse incisor tooth organs has been studied in situ by light and electron microscopic autoradiography to determine the sites and kinetics of biosynthesis, migration, and secretion of precursor enamel protein during newborn mouse incisor tooth formation maxillary and mandibular incisor tooth amelogenesis was studied 5, 30, 60, 120, 240 minutes and 24 hours following the intraperitoneal injection of 3H-tryptophan. By 5 minutes, 40% of the total silver grains associated with the secretory ameloblasts were localized over the rough endoplasmic reticulum and 50% of the silver grains were localized over the Golgi apparatus. By 30 minutes, silver grains were observed predominately over condensing vacuoles and secretory granules within the forming Tomes' processes, and were also localized over the extracellular "granular" pre-enamel matrix. The enamel proteins were synthesized on membrane-bound polysomes, transferred within the cisternae of the rough endoplasmic reticulum and then accumulated in the inner saccules of the Golgi apparatus. The enamel proteins were than packaged in condensing vacuoles which subsequently became secretory granules which migrated to the lateral and apical secretory regions of the forming Tomes' processes. It was concluded from these in vivo studies that enamel protein were synthesized and subsequently secreted within 30 minutes. The initially secreted precursor enamel protein was localized over a material which demonstrated a granular or stippled ultrastructure. The labeled protein then was localized over the amorphous enamel matrix per se which contained the forming calcium hydroxyapatite crystals. We assumed, therefore, that there are two different ultrastructural forms of 3H-tryptophan containing extracellular enamel proteins and suggest that the granular or "stippled" form represents newly secreted precursor enamel protein.

Relationship between the quality of fixation and the presence of stippled material in newly formed enamel of the rat incisor

“…Some authors fixed by immersion in glutaraldehyde or glutaraldehyde-formaldehyde mixture (Garant and Nalbandian, 1968;Matthiessen and Bulow, 1969;Lester, 1970;Katchburian and Holt, 1972;Decker, 1973;Slavkin et al, 1976). Only a few studies used teeth fixed by whole body perfusion (Jessen, 1968;Kallenbach, 1971Kallenbach, , 1973Kallenbach, , 1976Kallenbach, , 1977.…”

Possible functions of mesenchyme cell-derived fibronectin during formation of basal lamina

“…Intact tooth organs were cultured in the presence of [3H]glucosamine (35 ,uCi/,umol; 1 Ci = 3.7 X 1010 becquerels; New England Nuclear) for 24 hr and then chased with 1 mM D-glucosamine for 1 hr. Labeled samples were prepared for autoradiography as described (30).…”

“…Sections were incubated at room temperature for 30 min in a humidified chamber with appropriate dilutions of affinity-purified primary;antibody to obtain a final protein concentration of 116 ,ug/ml. After three 10-mi washes with phosphate-buffered saline (pH-7.4), incubation was with a 1:20 dilution of fluorescein isothiocyanate-labeled goat anti-rabbit 7S globulin (Hyland, Costa Mesa, CA) for 30 (4,5,12,14,24,33). In contrast to a recent report by Osman and Ruch (14) (16 ,g); the RF of the band is 0.051.…”

Amelogenin Supra-Molecular Assembly in vitro Compared with the Architecture of the Forming Enamel Matrix

“…It has been proposed that the stippled material is a breakdown product resulting from poor fi xation [Nanci and Warshawsky, 1984b]. However, according to Slavkin et al [1976], following the intraperitoneal injection of 3H-tryptophan, the stippled material began to be labeled after 30 min, and labeling was gradually reinforced with time up to 24 h. They concluded that the stippled material represents newly secreted precursor enamel proteins. In addition to these diverging views, it was added by Chen and Eisenmann [1984] that in some experimental conditions, there was no relationship between the formation of stippled material and the fi xative procedure.…”