Light microscope radioautographic study of glycoprotein secretion in the hypothalamic-neurohypophysial system of the rat,after L-fucose-3H injection

Summary Fucose-³H was injected into the cerebral ventricle of rats that were killed at several time intervals after injection. Semi-thin sections of the supraoptic nucleus and neurohypophysis were processed for radioautography and analysed quantitatively. Silver grains indicating the site of fucose-labeled glycoproteins were first located at the perinuclear region of the secretory neurons. The highest silver-grain density in these cells was observed at 2 h after injection, declining afterwards. Silver grains over the neurohypophysis were observed from 2 h on, reached a peak at 1 day after injection and decreased in the subsequent time intervals. The distributions of the silver grains over the neurohypophysis fitted Poissonian distributions and these were shown to be heterogeneous at the several time intervals. Pituicytes were not labeled. The percentage of silver grains over the Herring bodies increased with time. In rats deprived of water after fucose-³H injection there was a great increase in the release of labeled glycoproteins from the neurohypophysis. These results indicate that the glycoproteins synthesized by the secretory neurons of the hypothalamic nuclei are secreted in the neurohypophysis.     

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.     

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.     

Fine structure of the median eminence and the pars nervosa of the bullfrog,Rana catesbeiana

Summary The hypothalamic neurosecretory system of the bullfrog, Rana catesbeiana, was studied with light- and electron microscopy. The median eminence is roughly divided into two portions. The upper portion mostly consists of ependymal cells, glial cells and preoptico-hypophysial nerve tract, whereas in the lower portion, neurosecretory axons, glial cells, processes of glial and ependymal cells, and fine blood vessels of the hypothalamic portal vein are located. A part of the neurosecretory axons of the preoptico-hypophysial tract proceeds to the lower portion of the median eminence. These axons are arranged perpendicularly to the capillaries of the hypothalamic portal vein. The glial cells are densely located in the area of the median eminence where neurosecretory material is abundant. The neurosecretory material in the neurosecretory cells, their axons, the median eminence and the pars nervosa of the bullfrog shows a positive reaction to PAS treatment.The neurohemal area of the median eminence is occupied by many neurosecretory and non-neurosecretory axons, containing neurosecretory granules and/or synaptic vesicles. The axonal portions with the synaptic vesicles which are considered to be the nerve endings abut on the capillaries of the portal system. The size of synaptic vesicles in the axon terminals containing few neurosecretory granules is larger than those in the endings with many neurosecretory granules. Infrequently glial and ependymal processes are interposed between the nerve endings and the capillary wall.In the hilar region of the infundibulum, synapses are frequently observed between the thin fibers with or without neurosecretory granules and dendrites of non-neurosecretory neurons. The probable functions of these synapses are briefly discussed on the basis of our findings. Both in the hilar region of the infundibulum and in the pars nervosa, electron-dense neurosecretory granules of two different sizes were observed. The median eminence contains only one type of granules.The fine structure of the pars nervosa shows similar structures to those of the median eminence. Both in the median eminence and the pars nervosa, the fenestrated endothelium of the capillaries was frequently observed. The thick perivascular connective tissue space containing fibroblasts and collagen fibrils was observed both in the median eminence and the pars nervosa. Vesicles in the cytoplasm of the endothelial cells which appear to take a part in the transendothelial transport were observed.This investigation was supported in part by United States Public Health Service Research Grant, No. A-3678, to Hideshi Kobayashi from the National Institute of Arthritis and Metabolic Diseases and partly by a grant for Fundamental Scientific Research from the Ministry of Education of Japan. The authors wish to express their thanks to Prof. K. Takewaki for his kind encouragement.     

Glycoprotein secretion in the mouse submandibular gland as revealed by radioautography after L-3H-fucose injection

Summary Glycoprotein secretion in the mouse submandibular gland was investigated by light microscope radioautography of semi-thin sections after the administration of L-³H-fucose. The incorporation of the precursor in the acini was negligible. ³H-fucose was taken up in the paranuclear region of the cells lining the intercalated, secretory, striated and excretory ducts. This labeling pattern was interpreted as addition of the precursor to glycoproteins within the Golgi apparatus. Incorporation in the intercalated duct was restricted to the cells with fine cytoplasmic granules. The glycoproteins synthesized by the intercalated and secretory ducts were transported to the saliva by the secretion granules. It is assumed that the glycoproteins synthesized in the striated and excretory ducts are plasma membrane glycoproteins which seem to renew continuously. Quantitation of the radioautographs supplied data concerning the incorporation of ³H-fucose into newly synthesized glycoproteins as well as the renewal of the labeled macromolecules in each duct.     

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.     

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.     

Glycoconjugate localization with lectin and PA-TCH-SP cytochemistry in rat hypophysis

Glycoconjugates were localized by light microscopy with lectin-peroxidase conjugates and by electron microscopy with the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) sequence in immunocytochemically or morphologically identified cell types in rat pituitary. Lectin histochemistry demonstrated sialic acid and glycoconjugates with N-glycosidically linked oligosaccharides in gonadotrophs, thyrotrophs, and corticotrophs. Galactose penultimate to sialic acid was observed mostly in gonadotrophs. The terminal galactose-N-acetylgalactosamine disaccharide was detected in a few gonadotrophs and in a moderate number of mammotrophs. Fucose was localized in only corticotrophs with two fucose-binding lectins and in thyrotrophs with another. Several different monosaccharides were seen in glycoconjugates in melanotrophs and in Herring bodies. Melanotrophs displayed heterogeneous staining with fucose-binding lectins. A small number of nonsecretory cells were also visualized in the pars distalis by virtue of their glycogen content. PA-TCH-SP staining revealed complex carbohydrates in secretory granules and some Golgi cisternae in all types of hormone-producing cells in the pars distalis except for the somatotrophs. Melanotrophs of pars intermedia exhibited stained secretory granules and irregular dense bodies containing a stained meshwork. Corticotrophs of the pars distalis lacked the latter bodies, although they form the same glycoprotein precursor hormone as melanotrophs. Lectin conjugates and the PA-TCH-SP sequence stained some groups of secretion granules in Herring bodies, possibly representing vasopressin-containing granules as well as other cell types in the pars nervosa.     

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.     

Electron microscopic and pharmacological studies on the rat median eminence

Summary The rat median eminence contains at least three kinds of granules or vesicles: 1. large electron-dense granules (perhaps carriers of neurohypophysial hormones), 2. small electron-dense granules with or without haloes (perhaps carriers of catecholamines) and 3. synaptic vesicle-like structures (perhaps carriers of acetylcholine). The former two electrondense granules exist in separate axons but they coexist with the latter vesicles in the same axons.The pars nervosa shows basically a similar structure to the median eminence. However, the axons containing the small electron-dense granules are very few. In the pars tuberalis, there are at least two types of cells: the cells of one type contain much cytoplasm with large round nuclei and those of the other type contain a small amount of cytoplasm with polymorphic nuclei. The cells of the former include multivesicular bodies and secretory granules, but those of the latter do not. Some of capillaries of the primary plexus are surrounded by the cells of the pars tuberalis on one side and by neurosecretory axon endings on the other side.The median eminence contains high concentration of acetylcholine or an acetylcholine-like substance and shows neurohypophysial hormone activity.Aided by Grant A-3678 from the United States National Institute of Arthritis and Metabolic Diseases. The authors are indebted to Dr. Welsh, Harvard University, for the kind gift of Mytolon.     

Histology and ultrastructure of the neural lobe of the lizard,Klauberina riversiana

Summary The pars nervosa of Klauberina riversiana belongs to a primitive tetrapod type which is characterized by the deep penetration of the infundibular recess, a thin-walled structure, and the virtual absence of pituicytes. The differential response of this gland to aldehyde fuchsin and periodic acid Schiff suggests the presence of two types of neurosecretory nerve endings. Ultrastructurally four kinds of nerve endings are distinguishable. Type I, probably a cholinergic nerve ending, contains only small clear vesicles ca. 400 Å in diameter. The relative abundance of cholinergic nerve endings in this pars nervosa may be related to the necessity of transporting hormone through the ependymal cell. Type II, containing granulated vesicles about 1,000 Å in diameter and probably aminergic, is very rare. The two remaining types apparently secrete neurohypophysial hormones. They are Type III, containing dense granules ca. 1,500 Å in diameter and Type IV containing pale granules ca. 1,500 Å in diameter. Evidence is reviewed which suggests that Type III nerve endings may secrete arginine vasotocin while Type IV endings may secrete (an)other hormone(s).All these axons end only on the ependymal cells, the vascular processes of which form a continuous cuff over the basement membranes of the blood vessels. Hence the ependymal cells link the cerebrospinal fluid, the nerve endings and the blood vessels. Particles resolvable with the electron microscope are traced through a possible transport pathway from the granules, through the ependymal cells to the basement membrane. It is suggested that pituicytes replace ependymal cells and assume their transport functions in animals with massive neural lobes containing large numbers of nerve endings and blood vessels.Fellow of the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina.This investigation was supported in part by a Public Health Service fellowship 1 FZ HD 32,949-01 REP from the national Institute of Child Health and Human Development.The authors wish to thank Professor H. Heller for his constant interest and constructive criticism.     

Seasonal immunocytochemical and ultrastructural changes in the neurohypophysis of the migratory Canada goose

Immunocytochemical localization of arginine vasotocin (AVT) in the neurohypophysis of the migratory Canada goose was studied during six periods (Spring premigratory, Spring postmigratory, breeding, moulting, Fall or Autumn premigratory, and Fall postmigratory) of its annual life cycle. Ultrastructural changes in the pars nervosa, with emphasis on the axonal endings, were also examined during the above periods. In the Spring premigratory period, the staining intensity for AVT was greater in the anterior median eminence than in the pars nervosa where the density of axonal secretory granules was lower than that in the preceding period, in both sexes. During moulting females seemed to be under greater osmotic stress than males as indicated by low AVT staining intensity and secretory granule concentration in the pars nervosa. The occurrence of considerable amounts of lipofuscin bodies in the pars nervosa of both sexes during moulting was indicative of high lysosomal activity in the pituicytes. AVT release is recognized as a significant event in initiating Spring migration.     

Unstimulated amylase secretion is proteoglycan-dependent in rat parotid acinar cells

It is well-known that amylase is secreted in response to extracellular stimulation from the acinar cells. However, amylase is also secreted without stimulation. We distinguished vesicular amylase as a newly synthesized amylase from the accumulated amylase in secretory granules by short time pulse and chased with ³⁵S-amino acid. The newly synthesized amylase was secreted without stimulation from secretory vesicles in rat parotid acinar cells. The secretion process did not include microtubules, but was related to microfilaments. p-Nitrophenyl β-xyloside, an inhibitor of proteoglycan synthesis, inhibited the newly synthesized amylase secretion. This indicated that the newly synthesized amylase was secreted from secretory vesicles, not via the constitutive-like secretory route, which includes the immature secretory granules, and that proteoglycan synthesis was required for secretory vesicle formation.     

Ultrastructural studies on the hypothalamic neurosecretory neurons of the rat

Summary The general ultrastructural features of the hypothalamo-neurohypophysial system in rats with hereditary hypothalamic diabetes insipidus (DI-rats, Brattleboro strain) are described. There is no decisively distinguishing difference between the neurons of the supraoptic and paraventricular nuclei. The neurons of both nuclei show signs of active protein synthesis. The perikarya of the neurons are markedly hypertrophic, the nuclei are large and the nucleoli prominent. In the cytoplasm there are numerous ribosomes, abundant rough-surfaced endoplasmic reticulum and extensive Golgi complexes. However, very few neurosecretory granules are to be seen. The axons of the hypothalamo-neurohypophysial tract are likewise enlarged and the paucity of neurosecretory granules is a striking feature also in the area of the tract. The majority of nerve endings in the posterior pituitary of DI-rats are devoid of neurosecretory granules. Microvesicles are abundant in the nerve endings and there are findings which suggest that microvesicles are involved either in endoor exocytosis. The signs of active protein synthesis and the concomitant paucity of neurosecretory granules are interpreted to imply transportation of the secretory proteins in an extragranular phase. The possible mode of release of the secretory proteins from the nerve endings and the role of microvesicles therein are discussed.This study has been supported by grants from the Finnish Cultural Foundation and the Sigrid Jusélius Foundation. The collaboration of Professors Antti Arstila and Tapani Vanha-Perttula is gratefully acknowledged.The Brattleboro-rats were kindly provided by Dr. Heinz Valtin, to whom we express our thanks.     

DISTRIBUTION OF LEUCINE-3H DURING AXOPLASMIC TRANSPORT WITHIN REGENERATING NEURONS AS DETERMINED BY ELECTRON-MICROSCOPE RADIOAUTOGRAPHY

The distribution of leucine-³H in neurons was determined by electron-microscope radioautography after infusion of label into the spinal cord or sensory ganglia of regenerating newts. In the nerve cell bodies 3 days after infusion, the highest concentration of label per unit area occurred over the rough-surfaced endoplasmic reticulum. In the large brachial nerves, the silver grains were not distributed uniformly in the axoplasm, indicating that the labeled materials are restricted in their movement to certain regions of the axon. Almost all of the radioautographic grains observed in myelinated nerves could be accounted for by the presence of a uniformly labeled band occupying the area 1500–9000 A inside the axolemma. This region of the axon was rich in microtubules and organelles while the unlabeled central core of the axon contained mainly neurofilaments. This observation supports the hypothesis that microtubules are related to axonal transport. In small, vesicle-filled nerve terminals in the blastema, labeled material was restricted to a thin zone a short distance beneath the plasma membrane while the central region of the terminal was largely unlabeled. The peripheral pattern of labeling in the nerve endings is consistent with successive addition of newly synthesized proteins at the periphery of the growth cone and release of substances such as trophic factors at the nerve terminal.     

Ultrastructural Analysis of the Neuroendocrine Apparatus of Oncopeltus fasciatus (Heteroptera)

• 1 In Oncopeltus fasciatus, the A-cells of the pars intercerebralis and their tracts are stainable in situ with the performic acid-victoria blue (PAVB) method. The axons from these cells, after traversing the corpus cardiacum, terminate in the anterior part of the aorta which thus serves as the neurohemal organ.
• 2 Ultrastructurally, four types of secretory neurons are distinguishable in the pars intercerebralis region: pic-I with granules measuring 1000–3000 Å in diameter; pic-II with granules of irregular size and shape, the elongate ones showing mean dimensions of 2400 × 1400 Å; pic-III with less electron-dense granules measuring 1000–2700 Å in diameter; pic-IV, present not only in the pars intercerebralis but also in adjacent regions of the brain, with variable proportions of granules measuring 700–1800 A and dense-cored vesicles measuring 1000–2400 Å.
• 3 The nervi corporis cardiaci contain at least three types of neurosecretory axons, based on granule content, presumably representing pic-I, pic-II and pic-III neurons.
• 4 The wall of the aorta contains endings of at least three distinct types, again representing pic-I, pic-II and pic-III neurons, and thus provides the neurohemal site for these three types of protocerebral neurosecretory cells. Axons of pic-IV neurons appear to enter the cerebral neuropil.
• 5 The corpus cardiacum is composed of two types of parenchymal secretory cells, with electron-dense granules measuring 1300–3000 Å and 1000–2300 Å in diameter, respectively. The corpus cardiacum also contains interstitial cells and some axons of extrinsic origin, with and without granules.
• 6 The corpus allatum may be paired or median, and receives a small number of at least two types of axons. The corpora allata of some reproducing females show a large number of PAVB-stainable inclusions which appear to be modified cytoplasmic organelles, but are definitely not neurosecretory material.
• 7 The hypocerebral ganglion is composed of two types of secretory-appearing neurons and glial cells. The two neuronal types contain secretory granules, 1000–3000 Å and 900–2100 Å in diameter, respectively. Axons of the recurrent nerve also may contain occasional granules.
• 8 In this heteropteran insect, the two principal functions of the corpus cardiacum appear to be spatially separated: the neurohemal function is subserved by the aortic wall, which permits release of material into both the aortic lumen and the hemocoel, and the intrinsic endocrine function is possessed by the parenchymal cells.

     

Orexin-B-like immunoreactivity localizes in both luteinizing-hormone-containing cells and melanin-concentrating hormone-containing fibers in the red-bellied piranha (Pygocentrus nattereri) pituitary

We examined orexin-like immunoreactivity in the pituitary of the red-bellied piranha (Pygocentrus nattereri). Orexin-B-immunoreactive (IR) cells corresponded to luteinizing hormone (LH)-containing cells in the pars distalis, and orexin-B-IR fibers corresponded to melanin-concentrating hormone (MCH)-containing fibers in the pars nervosa. In the pars distalis, orexin-B-IR puncta that were also immunoreactive for MCH were observed around the orexin-B-IR cells. In the ventral hypothalamus, orexin-B-IR and MCH-IR neurons were found in the nucleus lateralis tuberis. Immunoelectron-microscopic analysis revealed that the orexin-B-like substance co-localized with LH in secretory granules and with MCH in MCH-containing neurons. Some of the MCH secreted in the pituitary might participate in the modulation of LH secretion from the gonadotrophs, together with orexin-B, leading to food intake by the stimulation of growth hormone secretion from the somatotrophs.     

Glycoprotein synthesis and migration in beta cells of the islets of Langerhans as shown by quantitative light- and electron-microscopic radioautography

Summary L-3H-fucose was injected intravenously into rats that were killed from 10 min to 7 days after isotope administration. Semi-thin and thin sections of the islets of Langerhans were processed for light- and electron-microscopic radioautography, respectively, and analyzed quantitatively. L-3H-fucose was incorporated into newly synthesized glycoproteins in the Golgi apparatus of the beta cells and subsequently labeled glycoproteins migrated to secretory granules and plasma membrane. Therefore, some of the glycoproteins synthesized by the beta cells of the islets of Langerhans are destined for the renewal of plasma membrane. Although the labeling of the secretory granules was clearly demonstrated, it was not possible to decide if the newly formed glycoproteins are incorporated into the content or into the membrane of the granule. Thus, the fate as well as the function of secretory-granule glycoproteins could not be determined precisely. Several hypotheses concerning the presence of glycoproteins in the secretory granules in relation with insulin metabolism are considered.     

Morphometric classification of the neurosecretory granules in the rat pars nervosa

Summary The distribution of the diameters of the neurosecretory granules in the rat pars nervosa (measured from electron micrographs taken at 40 000 × ) was compared among axons by nonparametric statistical methods and the axons were classified into five groups with median granule diameters of 143, 155, 167, 180 and 193 nm. We suggested that these five axon types carried different secretory substances contained in the pars nervosa. This investigation is supported by a grant from the Population Council, New York and grant from the Ministry of Education. Authors are grateful to Japan Electron Optics Laboratory Company for their technical assistance with the electron microscopy and to Miss Kazue Yamamoto for her help in preparing the figures.     

Ultrastructural relationship between monoamine- and TRH-containing axons in the rat median eminence as revealed by combined autoradiography and immunocytochemistry in the same tissue section

Summary The correlation of dopamine (DA)-, noradrenaline (NA)- or serotonin (5HT)-containing neurons and thyrotropin releasing hormone (TRH)-containing neurons in the median eminence of the rat, as well as the coexistence of monoamines (MA) and TRH in the neurons, were examined by subjecting ultrathin sections to a technique that combines MA autoradiography and TRH immunocytochemistry. The distribution and localization of silver grains after ³H-MA injection were examined by application of circle analysis on the autoradiographs.TRH-like immunoreactive nerve terminals containing the immunoreactive dense granular vesicles were found to have an intimate contact with monoaminergic terminals labeled after ³H-DA, ³H-NA or ³H-5HT infusion in the vicinity of the primary portal capillaries in the median eminence. Synapses between TRH-like immunoreactive axons and MA axons labeled with silver grains, however, have not been observed to date. Findings suggesting the coexistence of TRH and MA in the same nerve terminals or the uptake of ³H-MA into TRH-like immunoreactive nerve terminals, where silver grains after ³H-MA injection were concurrently localized in TRH-like immunoreactive nerve terminals, were rarely observed in the median eminence. Percentages of the nerve terminals containing both immunoreactive granular vesicles and silver grains after ³H-MA injection to total nerve terminals labeled after ³H-MA infusion silver grains were equally very low in ³H-DA, ³H-NA or ³H-5HT, amounting to less than 6.1%.This work was supported in part by grant-in-aid for scientific research from the Japan Ministry of Education (No. 557018).