Autoradiographic Studies of Synthesis and Intracellular Migration of Glycoproteins in the Rat Anterior Pituitary Gland

Abstract

The incorporation of [aH]fucose in the somatotrophic and gonadotrophic cells of the rat adenohypophysis has been studied by electron microscope autoradiography to determine the site of synthesis of glycoproteins and to follow the migration of newly synthesized glycoproteins. The pituitaries were fixed 5 rain, 20 rain, 1 h, and 4 h after the in vivo injection of pH]fucose and autoradiographs analyzed quantitatively. At 5 rain after [~H]fucose administration, 80-90% of the silver grains were localized over the Golgi apparatus in both somatotrophs and gonadotrophs. By 20 min, the Golgi apparatus was still labeled and some radioactivity appeared over granules. At 1 h and 4 h, silver grains were found predominantly over secretory granules. The kinetic analysis showed that in both protein-secreting ceils (somatotrophs) and glycoprotein-secreting cells (gonadotrophs), the glycoproteins have their synthesis completed in the Golgi apparatus and migrate subsequently to the secretory granules. It is concluded from these in vivo studies that glycoproteins which are not hormones are utilized for the formation of the matrix and/or of the membrane of the secretory granules. The incorporation of [3H]fucose in gonadectomy cells (hyperstimulated gonadotrophs) was also studied in vitro after pulse labeling of pituitary fragments in medium containing [aH]fucose. The incorporation of [3H]fucose was localized in both the rough endoplasmic reticulum (ER) and the Golgi apparatus. Later, the radioactivity over granules increased while that over the Golgi apparatus decreased. The concentration of silver grains over the dilated cisternae of the rough ER was not found to be modified at the longest time intervals studied.Recent work on the synthesis of glycoproteins has demonstrated that the different sugars are added step by step to the preformed polypeptide chains, according to their position in the lateral side chains. Studies on the synthesis of thyroglobulin (Spiro and Spiro, 1966;Herscovics, 1969Herscovics, , 1970 and immunoglobulin (Swenson and Kern, 1968;Melchers, 1970; Schenken and Uhr, 1970) have shown that whereas glucosamine and mannose are incorporated during synthesis of the polypeptide chains, the terminal sugars, e.g., fucose, are added after the completion of the polypeptide backbone. Autoradiographic data have also shown that in thyroid cells (Haddad et al

Cytochemical and Three-Dimensional Studies on Golgi Apparatus and GERL of Rat Anterior Pituitary Cells by Transmission Electron Microscopy

“…In electron microscope autoradiography of sugars in anterior pituitary cells under both thyroidectomy and gonadectomy, Pelletier and his co-workers (25,27) found that radioactive fucose was incorporated in the rough endoplasmic reticulum as well as in the Golgi apparatus, though the latter was the only place for fucose incorporation in normal pituitary cells.…”

Effects of Estrogens on the Ultrastructural Characteristics of Female Rat Prolactin Cells as Evaluated by in situ Hybridization in Combination with Immunogold Staining Technique

“…Also, small fragments from the lateral wings were dissected out and sequentially postfixed in 2.5% glutaraldehyde (15 min) and 2% osmium tetroxide (30 min) before being dehydrated and flat-embedded in Araldite. Semithin (1 urn) and ultrathin (0.1 pm) sections were obtained from the outer 3 pm of the blocks and processed for light-and electron-microscopic autoradiography, using Kodak NTB-2 and Ilford L-4 emulsion, respec tively, as previously described [ 10). The ultrathin sections were immuno labeled for PRL using the protein A-gold technique [15] before being processed for autoradiography.…”

Cytological events involved in glycoprotein synthesis in cellular and syncytial trophoblast of human placenta. An electron microscope autoradiographic study of [3H]galactose incorporation.

“…These studies have demonstrated that some glycoproteins that are synthesized remain within the cell (5,6,15,37,38,43) while others are added either to the surface coat (3,4,6,23,28,29,41) or to the basal lamina (12, 13). In other cases, the glycoprotein synthesized is destined for secre-…”