Glycoprotein secretion in the hypothalamo-neurohypophyseal system of the rat

Summary Although the secretory products of the hypothalamoneurohypophyseal system are not glycoproteins, synthesis and migration of these macromolecules occur within its secretory neurons. After being labeled with ³H-fucose in the Golgi apparatus, newly synthesized glycoproteins migrate to secretion granules, lysosomes and the plasma membrane of the secretory neurons, as demonstrated by quantitative electron-microscopic radioautography. Secretion granules bearing newly synthesized glycoproteins migrate to the pars nervosa, the labeling pattern of which was studied in rats killed from 4 h to 14 days after the isotope injection. Most of the silver grains were observed to overly the secretory axons. Labeling of pituicytes was negligible and the number of silver grains over the perivascular spaces was about 10% of the total at certain postinjection intervals. In the secretory axons, most of the silver grains were seen to overly the secretion granules. The proportion of silver grains over the different portions of the secretory axons changed with time. At the longer intervals, the percentage of silver grains increased over the nerve swellings (including Herring bodies) and decreased concomitantly in the undilated portions of the axons and in the nerve endings. This labeling pattern conforms with observations on the secretion products. Water deprivation increased the release of neurosecretion as well as glycoproteins from the pars nervosa. However, glycoproteins inside the Herring bodies were not easily releasible. There was a parallel decrease in the amount of secretion granules and ³H-fucose-labeled glycoproteins indicating that the glycoproteins are predominantly a constituent of the granule content. Some newly synthesized glycoproteins were probably also used in the renewal of the axonal membrane. The labeling of smooth vesicles in nerve endings was discussed. In conclusion, most of the glycoproteins synthesized in the perikarion of the hypothalamic secretory neurons migrate inside secretion granules along the axon to the pars nervosa where they are secreted.     

AUTORADIOGRAPHIC STUDIES OF SYNTHESIS AND INTRACELLULAR MIGRATION OF GLYCOPROTEINS IN THE RAT ANTERIOR PITUITARY GLAND

The incorporation of [³H]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 min, 20 min, 1 h, and 4 h after the in vivo injection of [³H]fucose and autoradiographs analyzed quantitatively. At 5 min after [³H]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 cells (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 [³H]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.     

The site of incorporation of sialic acid residues into glycoproteins and the subsequent fates of these molecules in various rat and mouse cell types as shown by radioautography after injection of [3H]N- acetylmannosamine. I. Observations in hepatocytes

To study the site of incorporation of sialic acid residues into glycoproteins in hepatocytes, we gave 40-g rats and 15-g Swiss albino mice a single intravenous injection of [3H]N-acetylmannosamine (8 mCi) and then sacrificed them after 2 and 10 min. To trace the subsequent migration of the labeled glycoproteins, we injected 40-g rats with 4 mCi of [3H]N-acetylmannosamine and sacrificed them after 20 and 30 min, 1, 4, and 24 h, and 3 and 9 d. Concurrent biochemical experiments were carried out to test the specificity of injected [3H]N-acetylmannosamine as a precursor for sialic acid residues of glycoproteins. In radioautographs from rats and mice sacrificed 10 min after injection, grain counts showed that over 69% of the silver grains occurred over the Golgi region. The majority of these grains were localized over the trans face of the Golgi stack, as well as over associated secretory vesicles and possibly GERL. In rats, the proportion of grains over the Golgi region decreased with time to 37% at 1 h, 11% at 4 h, and 6% at 24 h. Meanwhile, the proportion of grains over the plasma membrane increased from 4% at 10 min to 29% at 1 h and over 55% at 4 and 24 h; two-thirds of these grains lay over the sinusoidal membrane, and the remainder were equally divided over the lateral and bile canalicular membranes. Many silver grains also appeared over lysosomes at the 4- and 24-h time intervals, accounting for 15-17% of the total. At 3 and 9 d after injection, light microscope radioautographs revealed a grain distribution similar to that seen at 24 h, with a progressive decrease in the intensity of labeling such that by 9 d only a very light reaction remained. Because our biochemical findings indicated that [3H]N-acetylmannosamine is a fairly specific precursor for the sialic acid residues of glycoproteins (and perhaps glycolipids), the interpretation of these results is that sialic acid is incorporated into these molecules in the Golgi apparatus and that the latter then migrate to secretion products, to the plasma membrane, and to lysosomes in a process of continuous renewal. It is possible that some of the label seen in lysosomes at later time intervals may have been derived from the plasma membrane or from material arising outside the cells.     

The site of incorporation of sialic acid residues into glycoproteins and the subsequent fates of these molecules in various rat and mouse cell types as shown by radioautography after injection of [3H]N- acetylmannosamine. II. Observations in tissues other than liver

Biochemical evidence from the preceding paper indicated that [3H]N- acetylmannosamine may be used as a fairly specific precursor for the sialic acid residues of glycoproteins (and perhaps glycolipids) in radioautographs of rat liver and duodenum. In order to study the site of incorporation of this label in cell types of various tissues, we gave 40-g rats and 15-g Swiss albino mice a single intravenous injection of 8 mCi of [3H]N-acetylmannosamine and sacrificed them after 2 and 10 min. To trace the subsequent migration of the labeled glycoproteins, we injected 40-g rats with 4 mCi of [3H]N- acetylmannosamine and sacrificed them after 20 and 30 min, 1, 4, and 24 h, and 3 and 9 d. Light microscope radioautographic analysis revealed that in a great variety of cell types the label was initially localized to the Golgi region. Electron microscope radioautographic analysis of duodenal villous columnar and goblet cells, pancreatic acinar cells and Paneth cells, from rats and mice sacrificed 10 min after injection, showed that the silver grains were localized over Golgi saccules (and adjacent secretion granules). In kidney proximal and distal tubule cells reaction was initially localized to the Golgi apparatus in some areas of the kidney cortex whereas in other areas it was more diffuse. In all cells, the proportion of silver grains over the Golgi apparatus decreased with time after injection while an increasing number of grains appeared over secretion products in secretory cells or over the plasma membrane in other cell types. Lysosomes also became increasingly labeled at later time intervals. The above results suggest that in most cell types sialic acid residues are incorporated into glycoproteins (and perhaps glycolipids), primarily in the Golgi apparatus. With time, these newly synthesized molecules migrate to secretion products, to the plasma membrane, or to the lysosomes.     

Radioautographic study of glycoprotein biosynthesis and renewal in the ovarian follicles of mice and the origin of the zona pellucida

Summary L-fucose-³H was injected intravenously into mice which were killed at several time intervals after injection and semi-thin sections of their ovaries were processed for radioautography and analysed quantitatively. At the same time the specific activity of serum glycoproteins was determined. Glycoprotein biosynthesis was demonstrated in the oocytes, granulosa and stromal cells. The silver grain density of the follicular fluid in large follicles reached a peak at 4 h, remained high at 8 h after injection and decreased steadily at the subsequent intervals. It was demonstrated that the labeling pattern of the follicular fluid depends on the secretory activity of the granulosa cells and also on the specific activity of serum glycoproteins. The collapsed zonae pellucidae which represent the highest degree of follicular atresia are able to take up glycoprotein macromolecules. Based on this finding and also on the labeling pattern of the large follicles it was shown that there is very little synthesis of specific glycoproteins for the zona pellucida in large follicles. A more specific labeling of the zona pellucida occurred in the medium follicles. Following the growth of these follicles having a previously labeled zona pellucida, it was demonstrated that this extracellular structure is secreted by the oocyte.     

Incorporation of L-(3H)-fucose into glycoproteins of the adrenal gland of mice. Light microscope radioautographic study on semi-thin sections

Summary To study the biosynthesis and intracellular migration of glycoproteins in the adrenal gland, adult mice were injected intravenously with L-(³H) fucose and killed from 10 min to 14 days after injection. Semi-thin sections of the adrenal glands were then processed for radioautography. Incorporation of labeled fucose occurred in the steroid-secreting cells of the three zones of the cortex as well as in the adrenalin (A) and noradrenalin (NA) cells of the medulla. At short intervals after injection, the main site of incorporation was the paranuclear region of the cells, suggesting uptake by the Golgi apparatus. Subsequently, labeled glycoproteins migrated from the paranuclear region to other cell sites. The labeling pattern observed in the adrenocortical parenchyme strongly suggests that the glycoproteins are transferred to lysosomes, lipofuscin granules and the cell coat (glycocalyx). Counts of silver grains clearly indicate that these glycoproteins undergo renewal. The qualitative and quantitative analysis of the radioautographs also suggest that glycoproteins, acting as intracellular carriers of steroids, may be released to the extracellular environment together with the hormones. Most of the glycoproteins synthesized by the A and NA cells of the adrenal medulla seem to be transferred to secretion granules in which they may play some role in the cytophysiology of these structures. It is likely that glycoproteins are released from the cells during exocytosis of secretory granules.     

Cellular localization of 3H-oestradiol in the hypophysis

Summary The pituitaries of male and female rats given 0.3 g of 6.7-³H-oestradiol-17 per 100 g body weight were examined by autoradiography in order to 1) identify the cells responsible for the uptake of the hormone, 2) determine the intracellular distribution of the hormone and quantify the proportions localized within the cytoplasm and nucleus by silver grain counting, and 3) see if sex differences existed in the cellular and intracellular distribution of the hormone. The animals were killed at intervals varying from 1 minute to 8 hours following intravenous or intramuscular injection.A large proportion of pituitary cells having the morphologic characteristics of acidophils, basophils and chromophobes contained radioactive material. Castration cells and acidophils of gonadectomized and lactating rats showed marked labelling. In male and female rats killed 10 minutes after intravenous injection, 84.4 and 83.6 per cent of the cells were labelled. One hour after intramuscular injection, 86.6 and 76.1 per cent of the cells were labelled in males and females, respectively. Thus, a small proportion of the cells remained unlabelled.Labelled cells showed silver grains both in the cytoplasm and over the cell nuclei, but the major proportion of the radioactive material was invariably associated with the cell nuclei in all cell types and at all time intervals. About 65 per cent of the radioactive material was associated with the cell nuclei in animals killed five minutes or one hour after intravenous or intramuscular injection of the hormone. The silver grains appeared to be randomly distributed in both the cytoplasm and over the cell nuclei.In the intermediate lobe and the neurohypophysis, only sparse labelling with random distribution was observed. At the border between the intermediate lobe and the neurohypophysis, labelling of single cells or clusters of cells similar to those in the adenohypophysis was found.The results, which were essentially the same in male and female rats, appear to indicate a direct effect of oestradiol at the pituitary level.This work was supported by grants from the Norwegian Cancer Society and by Nordisk Insulinfond. The skilful assistance of Miss Helga Friedl and Mrs. Jane Larsen is gratefully acknowledged.     

Migration of glycoprotein from the Golgi apparatus to the surface of various cell types as shown by radioautography after labelled fucose injection into rats

A single intravenous injection of L-[³H]fucose, a specific glycoprotein precursor, was given to young 35–45 g rats which were sacrificed at times varying between 2 min and 30 h later. Radioautography of over 50 cell types, including renewing and nonrenewing cells, was carried out for light and electron microscope study. At early time intervals (2–10 min after injection), light microscope radioautography showed a reaction over nearly all cells investigated in the form of a discrete clump of silver grains over the Golgi region. This reaction varied in intensity and duration from cell type to cell type. Electron microscope radioautographs of duodenal villus columnar cells and kidney proximal and distal tubule cells at early time intervals revealed that the silver grains were restricted to Golgi saccules. These observations are interpreted to mean that glycoproteins undergoing synthesis incorporate fucose in the saccules of the Golgi apparatus. Since fucose occurs as a terminal residue in the carbohydrate side chains of glycoproteins, the Golgi saccules would be the site of completion of synthesis of these side chains. At later time intervals, light and electron microscope radioautography demonstrated a decrease in the reaction intensity of the Golgi region, while reactions appeared over other parts of the cells: lysosomes, secretory material, and plasma membrane. The intensity of the reactions observed over the plasma membrane varied considerably in various cell types; furthermore the reactions were restricted to the apical surface in some types, but extended to the whole surface in others. Since the plasma membrane is covered by a "cell coat" composed of the carbohydrate-rich portions of membrane glycoproteins, it is concluded that newly formed glycoproteins, after acquiring fucose in the Golgi apparatus, migrate to the cell surface to contribute to the cell coat. This contribution implies turnover of cell coat glycoproteins, at least in nonrenewing cell types, such as those of kidney tubules. In the young cells of renewing populations, e.g. those of gastro-intestinal epithelia, the new glycoproteins seem to contribute to the growth as well as the turnover of the cell coat. The differences in reactivity among different cell types and cell surfaces imply considerable differences in the turnover rates of the cell coats.     

Radioautographic tracing of 3H-proline in the endodermal cells of the parietal yolk sac as an indicator of the biogenesis of basement membrane components

The biogenesis of basement-membrane components was investigated in the endodermal cells of the rat parietal yolk sac in 12.5-day pregnant rats; 3H-proline was injected into conceptuses. After various time intervals, the parietal yolk sac, including endodermal cells and the associated Reichert's membrane, was removed and processed for electron-microscopic radioautography. Silver grains were counted over endodermal cell organelles and Reichert's membrane. At 2 and 5 min after 3H-proline injection, endodermal cells showed heavy labeling in rough endoplasmic reticulum (rER). Silver grain density over the rER decreased from 2 to 20 min and then remained at a plateau. Grain density was moderate over the Golgi apparatus initially but rose to a peak at 2 hr and decreased by 4 hr and later. Grain density was negligible over secretory granules at 2 and 5 min and increased moderately with time to reach a maximum at 8 hr. Thus, radioautographic peaks occurred sequentially in rER, Golgi apparatus, and secretory granules. By 4 hr and later, silver grains accumulated over Reichert's membrane. These results indicated that endodermal cells incorporated labeled proline into substances which were processed from the rER through the Golgi apparatus, transported from there to the cell surface by secretory granules, and released for export to Reichert's membrane. To clarify the nature of the exported substances, the amount of label present in proline and hydroxyproline residues after 3H-proline injection was measured in Reichert's membrane with or without the associated endodermal cells. Within the cells, 61.8% of the labeled proteins were classified as "sedentary" and 38.2% as "exportable." Of the label exported to Reichert's membrane, 66.3% consisted of type IV collagen and the rest of other basement-membrane components. The results obtained with this model suggest that basement-membrane proteins, including type IV collagen, are elaborated by the associated cells through the classical pathway: rER-Golgi apparatus-secretory granules.     

Light- and electron-microscopic radioautographic study of glycoprotein secretion in the granular duct of the submandibular gland of the male mouse

Summary L-³H-fucose was injected intravenously into adult male mice, after which, at different time intervals, the submandibular glands were removed and processed for light-and electron-microscopic radioautography. This radio active hexose was taken up by newly synthesized glycoproteins in the cells lining the granular ducts which were maximally labeled at 4 h after injection. Between 4 and 72 h the amount of labeled glycoproteins decreased moderately indicating that these macromolecules undergo a slow renewal. The main subcellular site of incorporation of 3 H-fucose into glycoproteins was the Golgi apparatus. From this organelle labeled glycoproteins were transferred to small secretory granules (diameter up to 1.0 m) located not only near the Golgi region but also throughout the apical cytoplasm. At 1 h after injection the concentration of label reached a maximum in the small secretory granules and labeling of medium (diameter between 1.1 and 2.0 m) and large (diameter over 2.0 m) granules was very low. At this postinjection interval the secretion product inside the lumen of the duct was already labeled. Between 1 and 72 h after injection the concentration of radioactivity in the small secretory granules decreased intensely while increasing in the medium and in the large ones. The concentration of fucose label reached a maximum in the medium secretory granules at 24 h and in the large ones at 72 h after injection. Additional experiments using mice previously injected with 4 intraperitoneal doses of ³H-fucose given 3 h apart demonstrated that the large granules undergo a very slow renewal. Some were found to be labeled as long as 28 days after administration of ³H-fucose. Recorded in this latter series of experiments was the labeling pattern of dense bodies that were regularly visualized in the cells lining the granular ducts. Their significance in the secretory process is discussed. In conclusion, newly synthesized glycoproteins are transferred from the Golgi apparatus to small secretory granules which carry a readily releasible pool of these macromolecules to the lumen of the duct. The small secretory granules also transfer newly synthesized glycoproteins to medium and large secretion granules which store a pool that is released very slowly. This characterizes the large secretory granules as the intracellular sites of storage of secretion products. The results of this investigation were correlated with the knowledge about the chemical composition of the different macromolecules that are known to be synthesized by the secretory cells of the granular ducts of the submandibular gland of the mouse.     

Synthesis and migration of 3H-fucose-labeled glycoproteins in the ciliary epithelium of the eye: effects of microtubule-disrupting drugs

3H-fucose was injected intravenously or intravitreously into albino rats. After time intervals of 10, 40, and 50 min, 1, 1.5, and 4 hr, 1, 3, and 7 days, and 1, 2, and 4 weeks after injection, the animals were sacrificed by intracardiac perfusion with glutaraldehyde. Samples of the ciliary body were prepared for light and electron microscope radioautography. Light microscope autoradiographs showed that the cells of both the inner and outer layers of ciliary epithelium actively incorporated 3H-fucose label in a reaction that peaked in intensity at 4 hr after injection, and then progressively declined. Electron microscope radioautographs revealed that, at early time intervals, most of the label was localized to the Golgi apparatus. With time, the plasma membrane of both cell types became increasingly labeled, and accounted for 60-70% of the total silver grains at 4 hr after injection. Adjacent to the basal cell surface of the inner layer cells, the fibers of the zonula became increasingly labeled from 1.5 hr onwards, providing strong evidence that these cells secrete glycoproteins to the zonula. When vinblastine was administered 30 min before 3H-fucose injection, followed by sacrifice 1.5 hr later, a much larger proportion of label remained localized to the Golgi apparatus than in controls, and the plasma membrane and zonula were much less labeled. These results suggest that, as documented in other cell types, microtubules may play a role in the intracellular transport of membrane and secretory glycoproteins in these cells.     

Ultrastructural radioautography of synthesis and migration of proteins and catecholamines in the rat adrenal medulla

Summary The synthetic pathways of proteins and catecholamines in the rat adrenal medullary cells were compared systematically at the ultrastructural level, within a 24 h period, with 2 tracers, L-tyrosine 3,5-³H and L-3,4-dihydroxy [ring 2,5,6-³H] phenylalanine (L-dopa³H). Young rats were injected with either of these tracers and sacrificed in pairs at close time intervals. With L-tyrosine ³H, the label was about equal over rough endoplasmic reticulum (RER) and secretory granules at 2 min after injection and remained almost constant in intensity over the secretory granules throughout the period of observation. A peak of radioactivity was also observed in the Golgi complex between 5 and 20 min after injection. This indicates that L-tyrosine ³H participates in the synthesis of both granule proteins and catecholamines as confirmed by the results obtained after injection of L-dopa ³H. With this tracer, radioactivity over RER, Golgi complex, cytosol and cell surface remained very low at all times and was undetectable at several time intervals. In contrast, radioactivity over secretory granules was very high at all time intervals. The present results thus confirm that in both adrenaline- and noradrenaline-storing cells, the protein moiety of chromaffin granules is synthetized in the RER, packaged in the Golgi complex and rapidly found in newly formed secretory granules. Following either L-tyrosine ³H or L-dopa ³H injection, catecholamine synthesis occurs only in or in close vicinity to chromaffin granules in both cell types at all time intervals. Acknowledgements. This work was supported by a grant from the Medical Research Council of Canada to the Multidisciplinary Research Group of Hypertension of the Clinical Research Institute of Montreal and by the Canadian Heart Foundation     

Synthesis and migration of proteins and glycoproteins in juxtaglomerular cells of sodium-deficient rats

Summary Sections of juxtaglomerular cells from sodium-deficient rats were subjected to radioautography after a single intravenous injection of L-tyrosine3,5 ³H or of L-fucose ³H to identify the sites of synthesis and to follow the migration of newly-formed proteins and glycoproteins. As early as 2 min after injection of L-tyrosine ³H, the label was highest in the rough endoplasmic reticulum (RER), suggesting that cisternal ribosomes are sites of protein synthesis. By 60 min, much of the label had migrated from the RER to the Golgi complex. Some radioactivity was already present over specific granules by 2 min but a peak was reached at 4h. The label over myofilaments was evident at all time intervals, indicating a certain incorporation of tyrosine into their contractile and/or structural proteins. The label over the cell surface peaked at 4h. After injection of L-fucose ³H, there was an early and important relative specific radioactivity in the Golgi complex at 5 min with a peak at 20 min and a decrease thereafter. The label increased slightly but steadily in secretory granules and cell surface to reach maxima at 4 h. A low level of radioactivity was recorded in mitochondria at all time intervals. After injection of both fucose ³H and tyrosine ³H, the label was detected at relatively low levels in the cytosol. These results suggest that renin, as the major secretory glycoprotein of juxtaglomerular cells, is synthetized in the RER, packaged in the Golgi complex and found relatively rapidly in newly-formed secretory granules. Part of the fucose and tyrosine labels is also associated with the thick cell coat of these cells.Recipient of a summer fellowship from the Kidney Foundation of Canada     

Electron microscopic autoradiography on the localization of serotonin in the frog median eminence

Summary For the identification of the nerve fiber containing serotonin (5-hydroxytryptamine) in the frog median eminence, an electron microscopic autoradiography was performed with 5-hydroxytryptophane-³H which is the precursor of serotonin. At 1 and 24 hours after the intraperitoneal injection, most silver grains were located over the nerve fibers and endings, and a few were also found over the glia cell and the perivascular space. A large number of silver grains were located over the type 3 nerve endings (Nakai, 1971) containing small dense granules about 600–1000 Å in diameter 1 and 24 hours after the injection. Some silver grains were localized over the nerve endings containing intermediate-size dense granules 1100–1700 Å in diameter. Silver grains were also frequently observed over the nerve fibers in the inner layer of frog median eminence. There is no significant difference in the pattern of distribution of silver grains between tissues of 1 hour and 24 hours after the injection.The authors wish to thank Prof. H. Fujita for his advice and criticism.     

Presence of glycoproteins in the cell nucleus as shown by radioautographic studies after administration of [3H]fucose and [3H]galactose

Dorsal root ganglia were removed from adult bullfrogs and incubated with [3H]fucose for intervals from 15 min to 1 h, followed by fixation. Some ganglia were post-incubated in the absence of [3H]fucose for up to 17 h. In additional in vivo experiments, young frogs were injected with [3H]fucose, and killed 30 min or 1 h later, and then ganglia were removed and fixed. Electron microscope radioautographs of the ganglia revealed an intense radioautographic reaction over the nuclei of Schwann and satellite cells as early as 5 min after initial exposure to [3H]fucose. At time intervals up to 2 h after initial exposure to [3H]fucose, the silver grains were evenly distributed over both the periphery and internal regions of the nucleus, while at 18 h they were localized to the cell periphery. In occasional cells, the perinuclear space was expanded in some areas and was the site of reaction. In young rats, injected with [3H]galactose and killed 15 min to 5 h later, electron microscope radioautographs revealed heavy reaction over the nuclei of duodenal villous and crypt columnar cells, in which the grains were evenly distributed over both the peripheral and internal regions. In mitotic cells, grains appeared to be associated with the condensed chromatin of forming chromosomes. These results provide strong evidence that glycoproteins exist in the nuclei of the above cell types and that they are actively renewed. The rapid appearance of nuclear reaction after initial exposure to [3H]fucose or [3H]galactose indicates that either these sugars are added to glycoproteins within the nucleus itself or that they migrate rapidly to this site after having been glycosylated elsewhere.     

RADIOAUTOGRAPHIC STUDY OF IN VIVO AND IN VITRO INCORPORATION OF FUCOSE-3H INTO THYROGLOBULIN BY RAT THYROID FOLLICULAR CELLS

The incorporation of fucose-³H in rat thyroid follicles was studied by radioautography in the light and electron microscopes to determine the site of fucose incorporation into the carbohydrate side chains of thyroglobulin, and to follow the migration of thyroglobulin once it had been labeled with fucose-³H. Radioautographs were examined quantitatively in vivo at several times after injection of fucose-³H into rats, and in vitro following pulse-labeling of thyroid lobes in medium containing fucose-³H. At 3–5 min following fucose-³H administration in vivo, 85% of the silver grains were localized over the Golgi apparatus of thyroid follicular cells. By 20 min, silver grains appeared over apical vesicles, and by 1 hr over the colloid. At 4 hr, nearly all of the silver grains had migrated out of the cells into the colloid. Analysis of the changes in concentration of label with time showed that radioactivity over the Golgi apparatus increased for about 20 min and then decreased, while that over apical vesicles increased to reach a maximum at 35 min. Later, the concentration of label over the apical vesicles decreased, while that over the colloid increased. Similar results were obtained in vitro. It is concluded that fucose, which is located at the end of some of the carbohydrate side chains, is incorporated into thyroglobulin within the Golgi apparatus of thyroid follicular cells, thereby indicating that some of these side chains are completed there. Furthermore, the kinetic analysis demonstrates that apical vesicles are the secretion granules which transport thyroglobulin from the Golgi apparatus to the apex of the cell and release it into the colloid.     

Autoradiographic observations on the incorporation of amino acids into oesophageal,gastric and pancreatic glands in the leopard frog,Rana pipiens

Summary The uptake and incorporation of L-³H-leucine and L-³H-serine in the oesophageal, gastric and pancreatic exocrine glands of Rana pipiens was examined using light microscopic autoradiographic techniques. At 1/2 h, 1 1/2 h and 3 h following injections of amino acids, silver grains are observed over the basal region of the cells comprising the glands. At 6 h after injection, silver grains occur over the apical region of the cells and by 24 h the grains can be observed over the cytoplasm of the cells adjacent to the lumen of the glands, collecting ducts, oesophagus and stomach. These observations indicate that the amino acids are being incorporated into proteins that are then discharged into the lumen of the pancreatic ducts, oesophagus and stomach. The secretory picture observed in the oesophageal glands closely resembles that of the gastric glands. This autoradiographic study supports the suggestion that the two glands are homologous and that they both secrete pepsinogen granules.     

Intracellular pathway and kinetics of protein secretion in the coagulating gland of the mouse

The coagulating gland of male rodents is part of the prostatic complex. Various mechanisms of secretion have been postulated, in part because organelles commonly involved in the secretory process possess unusual features, such as extreme distension of the rough endoplasmic reticulum. In the present study, the pathway, kinetics, and mode of secretion in the coagulating gland of the mouse were studied by electron microscope autoradiography at intervals between 5 min and 8 h after administration of 3H-threonine. The percentage of grains associated with the rough endoplasmic reticulum was initially high and generally decreased throughout the experiment, while a pronounced rise in the proportion of grains associated with the Golgi apparatus and secretory granules was observed 6 h after injection of precursor. In addition, there was a smaller elevation in the percentage of grains over the Golgi apparatus and secretory granules between 1 and 4 h, and radioactive material first reached the lumen of the gland 4 h after injection of the precursor. Although the general pathway of intracellular transport of secretory protein resembles that in other cells, the results indicate that there are several unusual aspects to the secretory process in the coagulating gland. First, the rate of transport was markedly slower than in most other exocrine gland cells, since the bulk of the labeled protein did not reach the Golgi apparatus and secretory granules until 6 h after administration of precursor. This reflected prolonged retention of secretory products in the endoplasmic reticulum. Second, in addition to the major bolus of labeled material that traversed the cells at about 6 h, a smaller wave of radioactivity appeared to pass through the Golgi apparatus and secretory granules and reach the lumen earlier, within the first few hours after the injection. Finally, the primary mode of secretion in the coagulating gland appears to be merocrine because the secretory granules contained much labeled protein.     

Biosynthesis and transport of glycoproteins in the small intestinal epithelium of rats: I. Developmental change and effect of hypophysectomy

The biosynthesis and intracellular transport of glycoproteins in duodenal absorptive cells of intact rats at 6 and 24 days and hypophysectomized rats at 24 days of age were studied after 20 min intralumenal pulse-labeling of d-[³H]galactose, l-[³H]fucose, or d-[³H]mannose. Autoradiographic studies showed that the incorporation of sugars increased significantly in intact rats between 6 and 24 days. When rats were hypophysectomized at 6 days of age, the intestinal epithelium at 24 days incorporated d-[³H]galactose at a level significantly lower than that of intact rats at 24 days. Hypophysectomy also interfered with the developmental increase in d-[³H]mannose, but not in l-[³H]fucose, incorporation. Biochemical study indicated that the radioactivity in the lipid-free acid-precipitable glycoproteins in the intestine of 24-day-old intact rats at 20 min after d-[³H]galactose injection was 129% and 97% higher than that in 6-day-old rats and in 24-day-old hypophysectomized rats, respectively. The patterns of intracellular transport of newly synthesized galactosylated or fucosylated glycoproteins in all animal groups were similar; the labeled glycoproteins were initially present in the Golgi and were transported through the smooth endoplasmic reticulum to either the lateral membrane or the brush-border membrane within 60 min after the injection of labeled sugars. The proportion of labeled glycoproteins that migrated to the brush-border membrane, however, increased about twofold in the intact rats between 6 and 24 days of age at 60–240 min after d-[³H]galactose injection. Hypophysectomy interfered with developmental increase in the transport of glycoproteins from the apical cytoplasm to the brush-border membrane. It was concluded that the incorporation of monosaccharide precursors into glycoproteins and the porportion of newly synthesized galactosylated or fucosylated glycoproteins transported to the brush-border membrane increase during postnatal development. The developmental changes are regulated, at least partially, by the pituitary gland.     

Binding and internalization of native gonadoliberin (GnRH) by anterior pituitary gonadotrophs of the rat

Summary To identify anterior pituitary cell types containing GnRH binding sites and to study the internalization process of this peptide by target cells under physiological conditions, autoradiography was performed on rat anterior pituitaries removed at specific time intervals (2–60 min) after intravenous injection of mono-radioiodinated ¹²⁵I-GnRH into intact males. At electron-microscopic level, gonadotrophs and lactotrophs appeared to contain silver grains. Concomitant administration of an excess of unlabeled GnRH with the radioiodinated hormone prevented this localization indicating the specificity of the reaction. The time-course study in gonadotrophs showed that 2 min after injection silver grains could be found over the plasma membrane, secretory granules and nuclear membrane. Similar results were observed 5 and 15 min after injection. Extensive label was observed over the nucleus and nuclear membrane 15 to 60 min after injection. The injection of a radioiodinated GnRH agonist [D-Trp⁶, Pro⁹ (Net), DesGly¹⁰]-GnRH produced comparable results. In contrast, the injection of ¹²⁵I-[D-pGlu¹, D-Phe², Trp^3,6]-GnRH, an antagonist of GnRH, produced positive labeling only at the plasma membrane without internalization. These results indicate that, after binding with receptors on the plasma membrane, GnRH is rapidly internalized, accumulating in secretory granules, and localizing over the nuclear membrane and later, in the nucleus. Association of radioactivity with secretory granules could be related to a specific action of GnRH at this level or to receptor recycling, and presence of label in the nucleus may be related to stimulation of neosynthesis of LH and GnRH receptors.