Argyrophile and argentaffin reactions in individual granules of enterochromaffin cells of reserpine treated guinea pigs

Summary The individual granules of enterochromaffin cells of normal and reserpine treated guinea pigs have been studied by staining slides of the duodenum first by an argentaffin method and subsequently by an argyrophile method. Some argentaffin cells can be shown to contain not only argentaffin granules, but also granules that are purely argyrophile. The relative number of such argentaffin cells is greatly increased following administration of reserpine, as depletion of their 5-hydroxytryptamine content converts argentaffin granules into purely argyrophile ones. On the basis of this finding it is confirmed that the argyrophile granule is merely an argentaffin granule depleted of its 5-HT content, and that the argyrophile (nonargentaffin) and the argentaffin cells represent different phases of a secretory cycle.This investigation was supported by a grant from the Indian Council of Medical Research. I am grateful to Ms. Ciba of India Ltd. for making available reserpine (Serpasil) and solvent for reserpine. It is a pleasure to thank Mr. Anand Parkash for technical assistance and Mr. M. L. Sharma for the photographs.     

On the alleged presence of non-argyrophile argentaffin cells in the human gastro-intestinal tract

Summary The relationship between the argentaffin and argyrophile cells of the human gastrointestinal tract has been studied, in foetal and adult material, by a technique involving the staining of sections first by an argentaffin method (Gomori-hexamine silver, Schmorl, Diazonium) and subsequently by an argyrophile method (Bodian). A comparison of the cells staining by the two methods shows that all argentaffin cells of the human gastrointestinal tract are also argyrophile and that there is no evidence to support the claim of Hellweg (1952) and of HamPerl (1952) regarding the presence of non-argyrophile argentaffin cells.W. H. O. fellow from the Department of Anatomy, Medical College, Rohtak, India. — I am very grateful to Professor J. D. Boyd and to the World Health Organisation for having made it possible for me to carry out this research at the Anatomy School, Cambridge. I am indebted to Dr. G. A. Gresham and his staff for their very willing cooperation in providing material from surgical resections. My thanks are also due to Mr. J. F. Crane for the photographs and to Mr. J. W. Cash and Mr. R. Smith for helpful discussions on staining techniques.     

Further observations on the alleged presence of non-argyrophile argentaffin cells in the human gastro-intestinal tract

Summary The relationship between the argentaffin and argyrophile cells of the human gastro-intestinal tract has been studied by staining paraffin sections first by the Masson-Hamperl method and then by the Bodian method. The investigations confirms the author's earlier observation (Singh 1963) that all argentaffin cells of the human gastro-intestinal tract are also argyrophile and that there is no evidence to support the claim of Hellweg (1952) and of Hamperl (1952) regarding the presence of non-argyrophile argentaffin cells.     

The relative proportion of pre-enterochromaffin (argyrophile) and enterochromaffin (argentaffin) cells in the gastro-intestinal tract of human foetuses

Summary The proportion of argyrophile cells that are argentaffin varies considerably over different parts of the gastro-intestinal tract of the human foetus. In the small intestine plots of the number of argentaffin cells against the number of argyrophile cells, at various cranio-caudal levels, reveal no correlation. The percentage of argyrophile cells that are argentaffin decreases in a caudal direction in the proximal part of the small intestine, but markedly increases in the distal part. In the large intestine there is a positive correlation between the numbers of argyrophile and argentaffin cells, but the percentage of argyrophile cells that are argentaffin also varies with cranio-caudal level. The pattern of this variation is not consistent in the various foetuses studied.     

Electron microscopic classification of amine-producing endocrine cells by selective staining of ultra-thin sections

Summary The argyrophil, argentaffin and chromaffin reactions were performed directly on ultra-thin sections for examination in the electron microscope. Glutaraldehyde fixation was appropriate for the argentaffin and chromaffin reactions; additional fixation with osmium tetroxide, however, caused impairment of these reactions. Fixation with formaldehyde, but not with glutaraldehyde, was adequate for the argyrophil reaction; post-fixation with osmium tetroxide did not affect this staining. At the light microscopic level the staining reactions were correlated with fluorescence histochemistry according to the method of Falck and Hillarp. The techniques described were used to study certain amine-producing endocrine cell systems: adrenal medullary cells and thyroid parafollicular cells of the mouse, gastric endocrine cells from the oxyntic gland area of the mouse, rat and rabbit. All these cells stained argyrophil. The adrenal medullary cells and one cell type in the oxyntic gland area of the rabbit were strongly argentaffin and chromaffin. The remainder of the cells were non-argentaffin and non-chromaffin but could be induced to give an argentaffin (and chromaffin) reaction after injection of the animals with l-3,4-dihydroxyphenylalanine or l-5-hydroxytryptophan, a treatment which is known to result in the accumulation of the highly reducing dopamine and 5-hydroxytryptamine, respectively, in these endocrine cells. Without exception the precipitates formed in all the staining reactions accumulated selectively over the secretory granules of the cells.The techniques described permit differential staining of consecutive ultra-thin sections for electron microscopic characterization of one and the same cell. They will provide information necessary for correlative studies of the stainable cells at the light and electron microscopic levels.     

The depletion of the granules of argyrophile cells of dog duodenum by reserpine

Summary The influence of reserpine on the argentaffin and non-argentaffin argyrophile cells of the duodenum has been studied in 8 dogs. In two dogs that were given 5 mg/kg of reserpine there was a complete disappearance of both types of cells. In another dog given 5 mg/kg and in dogs given 2.5 mg/kg and 1 mg/kg reserpine, the number of argentaffin cells was reduced to almost nil while the number of non-argentaffin argyrophile cells showed a statistically significant reduction. The degree of depletion of non-argentaffin argyrophile cells was proportional to the dose of reserpine given. The findings show that the non-argentaffin argyrophile cells are closely related to 5-hydroxytryptamine and their distinction from argentaffin cells is only a quantitative one.     

Peptide and amine producing endocrine-like cells in the chicken thymus

Summary The thymus of the chicken contains at least two types of endocrine-like cells predominating in the juxtacortical medulla. One type stores 5-hydroxytryptamine and is stained by the argentaffin, chromaffin and Schmorl methods. Treatment with reserpine markedly reduces its 5-hydroxytryptamine content. The other cell type is devoid of 5-hydroxytryptamine; if supplied withl-dopa it can produce and store dopamine. Both cell types stain with the argyrophil method of Grimelius and with HCl-basic dye methods believed to reflect the presence of peptides with masked carboxyl groups. In the electron microscope both cell types were found to contain numerous cytoplasmic 2000–3000 ? granules similar to those seen in polypeptide hormone-producing cells elsewhere. The cytoplasmic granules in one of the two endocrine-like cell types are argentaffin and chromaffin, indicating that they are the storage site for 5-hydroxytryptamine. It is suggested that the main secretory products of the two endocrine-like cell types are peptides, possibly regulating lymphatic tissue function.     

Ultrastructure of the neurohaemal region of the toad median eminence

Summary According to the internal structure and size of the granules, six types of nerve endings can be distinguished in the toad median eminence: 1. Endings containing mostly dense granules of 600 Å in diameter; 2. Endings containing dense granules of about 800 Å in diameter; 3. Endings which contain dense granules 1,000–2,000 Å in diameter, with the peak at 1,200–1,400 Å; 4. Endings containing granules with a characteristic structure, which differentiate them from the other three types; 5. Scarce endings containing granules 2,000 to 3,800 Å in diameter; and 6. Endings containing only vesicles 400–500 Å in diameter. Types 3 and 4 endings are mainly found in the outer pericapillary zone, and are probably responsible for the strong Gomori-positive reaction observed in this zone. The other four types of endings occur mainly in the inner pericapillary zone, and appear to be Gomori-negative.The probable origins of the different types of endings, and their possible relations with the different releasing factors is discussed.The subendothelial basement membrane has numerous long processes which form a complicated network in contact with all the nerve endings, some nerve fibres and glial cells.Two types of glial cells are described. Pinocytotic vesicles are frequently seen at the points where these cells contact the basement membrane. All the ultrastructural features suggest that these cells are carrying out transport functions.Fellow of the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina.The author is very grateful to Professor H. Heller for his continued encouragement and criticism and to Mr. J. Lane and Mr. P. Heap for their valuable help.     

Ultrastructural studies of endocrine cell populations showing an argentaffin reaction and/or serotonin immunoreactivity in the rat antral mucosa

Summary On the basis of staining results in closely related semi-thin sections from rat antral mucosa immunostained with polyclonal serotonin antibodies and silver-stained for the argentaffin reaction, respectively, three different cell populations could be distinguished. One of these cell populations showed both serotonin immunoreactivity and an argentaffin reaction, a second one serotonin immunoreactivity alone, and a third one only an argentaffin reaction.These cell populations were studied electron microscopically in ultra-thin sections located between the stained semi-thin sections. The cell population displaying an agentaffin reaction and serotonin immunoreactivity showed secretory granules of the enterochromaffin cell type. A similar granular appearance was observed in cells which only exhibited an argentaffin reaction. Serotonin immunoreactivity in the absence of an argentaffin reaction was evident in some G (gastrin) cells. and in some D₁ and possibly also some D (somatostatin) cells; but not all the endocrine cells of the non-enterochromaffin type displayed serotonin immunoreactivity. The significance of the different reactions in the three cell populations is discussed.     

The effect of castration upon the ultrastructure of the rat hypothalamus

Summary The neurosecretory hypothalamic nuclei and the inner zone of the median eminence of castrated rats were studied under the electron microscope. After one month of castration all the neurosecretory neurons of both nuclei show signs of hyperactivity characterized by dilated cisternae of the endoplasmic reticulum containing a macromolecular filamentous material and an increase in the number of ribosomes. After six months of castration, some neurosecretory neurons show an increased number of neurotubules and larger lysosomes than in the controls. Other neurons show a very significant hypertrophy of the endoplasmic reticulum, with large amounts of intracisternal filamentous material. These cells have few neurosecretory granules and in the adjacent synapses the number of granulated vesicles is increased. In the supraoptic nucleus there are two kinds of neurosecretory axons: the clear ones, which are similar to those that appear in control animals and the dark ones, which have smaller elementary granules. In the inner zone of the median eminence the axons show an increase in the number of neurosecretory granules with respect to the controls. After supplementary administration of sexual hormones, all the modifications produced by castration disappear. The ultrastructural changes observed in the neurosecretory nuclei after castration are discussed in relation to those previously described in the neurohypophysis under the same experimental conditions. A feedback regulatory action of sex hormones on hypothalamic neurosecretory neurons is postulated.Supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas and by the Air Force Office of Scientific Research (AF-AFOSR 963-67).We are deeply indebted to Mrs. Defilippi-Novoa and Mr. Alberto Saenz for their skillful assistence.     

The distribution of enterochromaffin cells in the human small intestine

Summary The distribution of argyrophile and argentaffin cells has been studied in the small intestine of five human adults. In proceeding cranio-caudally the characteristic feature of their distribution is the presence of eight to ten waves of rising and falling density. A progressive decrease in density of cells from duodenum to terminal ileum (described by previous workers) is not present.Re-examination of findings reported earlier in the small intestines of human foetuses shows that a predominant U shaped pattern of distribution is present in younger foetuses. This changes to the adult pattern by full term. The appearance of the adult pattern occurs earlier for argyrophile cells than for argentaffin cells.     

Quantification of endocrine cells and ultrastructural study of insulin granules in the large intestine of opossum Didelphis aurita (Wied-Neuwied, 1826)

This study aimed to investigate the distribution of argyrophil, argentaffin, and insulin-immunoreactive endocrine cells in the large intestine of opossums (Didelphis aurita) and to describe the ultrastructure of the secretory granules of insulin-immunoreactive endocrine cells. Fragments of the large intestine of 10 male specimens of D. aurita were collected, processed, and subjected to staining, immunohistochemistry, and transmission electron microscopy. The argyrophil, the argentaffin, and the insulin-immunoreactive endocrine cells were sparsely distributed in the intestinal glands of the mucous layer, among other cell types of the epithelium in all regions studied. Proportionally, the argyrophil, the argentaffin, and the insulin-immunoreactive endocrine cells represented 62.75%, 36.26%, and 0.99% of the total determined endocrine cells of the large intestine, respectively. Quantitatively, there was no difference between the argyrophil and the argentaffin endocrine cells, whereas insulin-immunoreactive endocrine cells were less numerous. The insulin-immunoreactive endocrine cells were elongated or pyramidal, with rounded nuclei of irregularly contoured, and large amounts of secretory granules distributed throughout the cytoplasm. The granules have different sizes and electron densities and are classified as immature and mature, with the mature granules in predominant form in the overall granular population. In general, the granule is shown with an external electron-lucent halo and electron-dense core. The ultrastructure pattern in the granules of the insulin-immunoreactive endocrine cells was similar to that of the B cells of pancreatic islets in rats.     

Fine structure of argyrophil and argentaffin cells in the gastro-intestinal tract of the fowl

Summary The fine structure of granular cells in the proventriculus gizzard and intestine of the fowl has been described. The proventriculus and gizzard have only one type of granular cell, the pure argyrophil cell. In the intestine two cell types are shown. One corresponds to the argyrophil cell seen in the proventriculus and gizzard. The other, more common, is considered to be the argentaffin cell of the intestine. Structural differences between these two cell types confirm the known differences in histochemical reaction. Acknowledgement. I wish to thank Mr. R. N. C. Aitken, Department of Veterinary Histology and Embryology for supplying the birds used in this study.     

Observations on the fine structure of giant mast cells produced by microwave radiation of the peritoneal fluid

Summary An electron microscopic examination of the fine structure of giant mast cells in the peritoneal fluid of the rat was made. The giant mast cells were produced by irradiating rats with a microwave apparatus generating energy of a frequency of 2,425±25 megacycles.The most obvious features of the giant mast cell appeared to be abundant swelling of the cytoplasm, which increased the distance between the granules, and the partial disappearance of the granules. Disruption of the mast cells, with escape of the granules, was not observed. Whether the formation of the giant mast cells is a reversible process or not cannot be answered on the basis of the present study.It is a pleasure to acknowledge the skilful assistance of Miss Tellervo Huima and Mr. Mauri Nyholm, M. Sc.This study is supported by the PAULO Foundation.     

Particles resembling microbodies in normal and neoplastic perianal glands of dogs

Summary By electron microscopy, the parenchymal cells of the perianal glands of dogs contain granules which have the morphological features of microbodies (peroxisomes) including marginal plates and, occasionally, dense nucleoids. Like microbodies, they are occasionally attached to the endoplasmic reticulum. Histochemical evidence is presented suggesting that they contain at least one of the peroxisomal enzymes, L--hydroxy acid oxidase. The granules of a perianal gland adenoma showed abnormal morphologic variations.Mrs. Murtie Still, Mrs. Bertha McClure and Mr. Bob White gave valuable technical assistance.     

Enterochromaffin cells in the chicken duodenum during prenatal development

Summary The development of enterochromaffin cells (EC) was studied in the duodenum of the chicken during prenatal development.Basally granular cells, i.e. EC were first found at the 14th incubation day and thereafter their number increased greatly during further development, as did also the number of specific osmiophilic granules in the cells.First empty or pale granules were found in the cytoplasmic area showing various kinds of saccules and vacuoles, possibly representing the poorly developed Golgi area. The primitive granules seemed to turn rapidly in to granules with high affinity for osmium tetroxide.The differentiation of EC occurred in the epithelial lining of the intestinum and no morphologically similar cells were found in other intestinal layers.This work was supported by a grant from the Yrjö Jahnsson Foundation, Helsinki. The electron microscopic observations were made in the Electron Microscope Laboratory, University of Helsinki; the skillful assistance of Mr. M. Nyholm is acknowledged.     

Endocrine cells of the human gastric mucosa

Summary By light and electron microscopy investigation of the human gastric mucosa five types of ultrastructurally different endocrine cells have been detected: 5-hydroxytryptamine storing enterochromaffin (EC) cells, gastrin storing G cells, and functionally undefined ECL, D and D₁ cells. By direct application of Masson's argentaffin reaction as well as of Sevier-Munger's and Grimelius' argyrophil method to electron microscopy specimens, selective deposition of silver grains upon the endocrine granules of such cells was obtained. In particular, only EC cell granules reacted to the argentaffin method, granules of both EC and ECL cells heavily reacted to Sevier-Munger's technique, granules of EC, ECL, G and D₁ cells reacted to Grimelius' technique, while D cell granules failed to react either to argentaffin or argyrophil methods. By the application of the same silver methods to paraffin sections as well as by other selective staining methods for endocrine granules (5-hydroxytryptamine techniques, lead-haematoxylin, HCl-basic dye method), at least four of the above cell types were also identified under light microscope. This opens the way for extensive studies of such cells in conventional histologie specimens.This investigation was supported in part by grant N.70.01022.04 from the Italian Consiglio Nazionale delle Ricerche.     

Cytochemistry of 5-hydroxytryptamine at the electron microscope level

Summary The nerves of rat pineal gland are known to contain norepinephrine and 5-hydroxytryptamine. With the glutaraldehyde-dichromate reaction for the cytochemical localization of unsubstituted catechol- and indoleamines, dense reactive granules could be demonstrated in such endings. A similar reaction was observed in the adrenergic nerves supplying the vas deferens and storing exclusively norepinephrine. Formaldehyde fixation, prior to the glutaraldehyde-dichromate treatment, interferes with the reaction given by catecholamines not affecting the indolic reactive sites. After this combined procedure pineal nerves still exhibited the dense reactive granules, while these were not found in the nerves of the vas deferens. Following bilateral cervical sympathectomy reactive granules disappeared from the perivascular processes of the pineal gland. No reaction could be observed in the cytoplasm of parenchymal cells neither in their perivascular processes.These cytochemical results suggest that both catecholamines and 5-hydroxytryptamine are contained within the granulated vesicles of pineal nerves.This work has been supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina and U.S.Air Force (AF-AFOSR 963-66).Fellow of the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina. We want to express our gratitude to Prof. E. De Robertis for his constant help and encouragement; and to Miss Nélida Fernández Oranges, Mr. Raúl Castelli and Mr. Alberto Sáenz for their skillful technical assistance.     

The fine structure of the light organ of the glowworm Lampyris noctiluca

Summary The four main parts of the glowworm light organ are the cuticle, the hypodermis, the photocyte layer and the reflector cell layer. The hypodermis is one cell thick and it contains hypodermic glands. These glandular cells have a lumen that opens to the outside of the cuticle. Projecting into the lumen are numerous microvilli. Between the hypodermis and photocytes are typical insect tunicated nerve fibres. They pass down between the photocyte and reflector layer cells. They do not appear to innervate the photocytes and they are thought to innervate adjacent muscle fibres or to be sensory. Tracheoles are commonly present between the photocytes but no tracheolar end organs are found. The photocytes contain amorphous granules, mitochondria, photocyte granules and a vesiculated reticulum. All, except the mitochondria, are absent from the reflector layer and so probably have some connection with light production. The reflector layer contains glycogen granules, clear spaces thought to be the sites of urate crystals, and membranous granules. The latter granules are sometimes found in photocytes adjacent to the reflector layer whilst amorphous granules are sometimes absent from these adjacent cells. So a cell layer with some features of the photocyte and reflector layer cells is present. These morphological findings are discussed with regard to the unknown function of the reflector layer and the control of light emission. Acknowledgments. We would like to thank Professor J. Z. Young and Dr. E. G. Gray for their advice and encouragement, Mrs. Jane, Astafiev for drawing fig. 1, Mr. S. Waterman for photographic assistance, Miss Cheryl Martin for secretarial assistance, and many colleagues for help in collecting specimens of glowworms.     

Fluorescence and electron microscopy of amine-storing enterochromaffin-like cells in tracheal epithelium of mouse

Summary The tracheo-bronchial mucosa of the mouse has been found to contain an extensive system of argyrophilic epithelial cells. In the trachea the cells morphologically resemble enterochromaffin cells. Normally, these enterochromaffin-like cells contain no fluorogenic amine, as revealed by the Falck-Hillarp formaldehyde technique. On the other hand the cells have the capacity to take up and decarboxylate 3,4-dihydroxyphenylalanine (DOPA) or 5-hydroxytryptophan (5-HTP); the amine formed is stored in the cytoplasm in a reserpine-sensitive store. This capacity to produce and store amines under experimental conditions may reflect the presence in the tracheal enterochromaffin-like cells of an amine which can not be demonstrated with available fluorescence histochemical techniques. In the electron microscope the tracheal enterochromaffin-like cells were identified by a positive argyrophil reaction and by their capacity to accumulate radioactivity after administration of ³H-DOPA or ³H-5-HTP as revealed by autoradiography. The radioactive labelling was associated with cytoplasmic electron-dense granules (800–1000 Å), suggesting that the amine formed was stored in these granules. Accordingly, the granules stained argentaffin after DOPA-pre-treatment of the animal. It is suggested that, like similar cells in the gastric mucosa, these argyrophilic enterochromaffin-like cells constitute an endocrine system in which amines are of cytophysiological importance.