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.     

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.     

Identification of enterochromaffin cells in adjacent Epon-embedded sections at light and electron microscopic levels

Summary Methods for light and electron microscopic comparison of individual argentaffin and argyrophil enterochromaffin cells (EC) in the sheep duodenal mucosa are described. These silver procedures were applied for light microscopy to Epon-embedded sections. The adjacent sections were examined with the electron microscope. The most specific characteristics of the argentaffin and argyrophil EC in electron microscopy are highly osmiophilic cytoplasmic granules. In one cell type these granules are smaller and more roundish than in the another type. These two cell types are stainable both by the argentaffin and argyrophil reactions. No essential difference can be observed in the localization of these elements. It is suggested that both cell types belong to the enterochromaffin system. Both silver methods are also suitable for the light microscopic identification of other intestinal structures in sections adjacent to that sectioned for electron microscopy.This work was supported by a grant from the Yrjö Jahnsson Foundation, Helsinki, Finland.The electron microscopic observations were carried out in the Electron Microscope Laboratory, University of Helsinki.     

Histochemical and ultrastructural studies on the enterochromaffin-like cell in the gastric mucosa of the opossum Didelphis albiventris (Marsupialia)

Summary An ultrastructural study of enterochromaffin-like (ECL) cells in the gastric mucosa of the white-belly opossum Didelphis albiventris (Marsupialia) was carried out. In parallel, histochemical methods were used at the light-microscopical level to demonstrate argentaffin cells, argyrophilic cells, and serotonin- and histamine-immunoreactive elements. Argentaffin and serotonin-immunoreactive cells were scattered, and argyrophilic cells were numerous, within the full thickness of the mucosa. Argyrophilic cell distribution was similar to that of histamine-immunoreactive elements. At the electron-microscopical level, the oxyntic mucosa of D. albiventris presented endocrine cells with secretory granules morphologically similar to those of the ECL cell of eutherian mammals. However, in this marsupial, the ECL cell exhibited a variable mixture of two distinct types of secretory granules: (1) granules with the morphological appearance of the eutherian ECL cell, and (2) granules morphologically similar to those of the eutherian enterochromaffin (EC) cells. Based on this morphological pattern of the ECL cell granules, it is proposed that in the oxyntic mucosa of the opossum D. albiventris, the EC and ECL cells represent distinct steps in the same line of cell differentiation; the ECL cell should also be a site of histamine storage.     

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.     

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.     

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.     

A novel endocrine cell type in the guinea-pig gastric mucosa: cellular source of pro-enkephalin-derived peptides. A histochemical, immunohistochemical, and electron microscopical characterization

A novel endocrine cell type has been identified in the guinea-pig gastric mucosa which preferentially occurs in the oxyntic area. Cells of this type exhibit immunoreactivities for bovine adrenal medulla dodecapeptide (BAM-12P) and in many cases for Met-enkephalin and are thus presumed to contain a pro-enkephalin-like precursor protein. Systematic immunohistochemical investigations show that these cells do not contain immunoreactivities for various enteric hormones, neuropeptides and biogenic amines (serotonin, histamine). However, they do contain immunoreactivity for chromogranin A, an acidic glycoprotein which is common to the majority of entero-endocrine cells. Using silver impregnation techniques BAM-12P immunoreactive cells prove to be argyrophil, but fail to react argentaffin. On the electron microscopical level, these cells contain a well-developed endoplasmic reticulum and Golgi apparatus and numerous polymorphous secretion granules which measure about 290 nm in diameter. The secretion granules are ovoid or pear-shaped but largely plump compared to those of enterochromaffin cells. Light and electron microscopical findings indicate that BAM-12P immunoreactive cells constitute an endocrine cell population of the gastric epithelium in addition to the "established" endocrine cells hitherto known in this location.     

Light and electron microscopic identification of several types of endocrine cells in the gastrointestinal mucosa of the cat

Summary The following histological methods, previously proved to be useful in selective light microscopic detection of endocrine cells, were applied to the cat gastrointestinal mucosa: for the identification of biogenic amines, diazonium, ammoniacal silver and xanthydrol methods; for granules identification, methyl green-red acid dyes, toluidine blue, HCl-basic dye, lead-haematoxylin, phosphotungstic haematein and argyrophil methods. Results were compared with those of an extensive electron microscopic investigation.Five types of endocrine cells were identified in the gastric mucosa. Three types were found in the pyloric mucosa: the previously described 5-hydroxytryptamine-producing enterochromaffin cell, the gastrin producing G cell and a cell with an unknown function, labelled in this paper the X cell. Four types were found in the fundic mucosa: enterochromaffin cells (rarely observed), enterochromaffin-like cells secreting a 5-hydroxyindole but showing some ultrastructural and staining differences from true enterochromaffin cells (numerously present), A-like cells (few), resembling A cells of the pancreatic islets, and X cells, resembling those in the pyloric mucosa.In the intestinal mucosa, at least three endocrine cell types were distinguished in its duodenal part: enterochromaffin cells and two types of polypeptide-producing cells — some with smaller granules (S cells) and others with larger granules (L cells). Only two types were found in the mucosa of terminal ileum: enterochromaffin cells and numerously-occurring cells with large granules resembling in part duodenal L cells. The possibility of a relationship between S and L cells and the production respectively of the intestinal hormones secretin and cholecystokinin-pancreozymin was discussed.This investigation was supported by a grant N. 115/1139/0/4715 of the Italian Consiglio Nazionale delle Ricerche.     

On the occurrence of argyrophil cells in salivary glands

Summary Salivary glands (parotid, submandibular and sublingual glands) of nine mammalian species were investigated with respect to presence and localization of argyrophil and argentaffin cells. With the exception of the parotid gland of the rat, no positive staining was observed within the examined glands. In the rat parotid distinctly argyrophil cells could be demonstrated in the intercalated ducts. Histochemical studies of the cells, ultrastructural analysis of their cytoplasmic granules as well as their reactions to certain drugs indicate that these cells are of exocrine rather than of endocrine nature. After a subcutaneous injection of pilocarpine, the intensity of the argyrophil staining was markedly reduced. No specific catecholamine fluorescence could be detected within the cells, not even after pretreatment of the animals with high doses of L-DOPA. The membrane-bounded cytoplasmic granules of the intercalated duct cells furthermore displayed a strong positive staining reaction after treatment of ultrathin Vestopal sections with the periodic acid-chromic acid-silver technique of Rambourg et al. (1969).Supported by grants from the Swedish Medical Research Council (Project No. 12X-718), and the Medical Faculty of the University of Umeå. The skilful technical assistance of Miss Siw Domeij is gratefully acknowledged     

SGC (small granule chromaffin) cells in the mouse adrenal medulla: light and electron microscopic identification using semi-thin and ultra-thin sections.

Adrenal glands of the mouse, fixed either in glutaraldehyde followed by osmium tetroxide or in a mixture of potassium dichromate and glutaraldehyde, and embedded in Epon 812, were investigated by light and electron microscopy. An argentaffin reaction was applied to semi-thin sections for light microscopy and to ultra-thin sections for electron microscopy. Since the mature secretory granules in the Small Granule Chromaffin (SGC) cell were argentaffin and were mainly located along the cell membrane, this cell was clearly distinguishable under the light microscope both from the A (adrenaline) cell whose secretory granules were non-argentaffin and from the NA (noradrenaline) cell whose cytoplasm was rich and was filled with large, strongly argentaffin granules. Chromaffinity of the SGC cell was demonstrated under the light microscope. The SGC cell was intensively stained with toluidine blue without revealing metachromasia. It was demonstrated at the EM level that not only the secretory granules but also the synaptic-like vesicles in the SGC cell contained argentaffin substances. Possible functional relationship between the secretory granules and the synaptic-like vesicles was discussed.     

Light and electron microscopic identification of the histamine-storing argyrophil (ECL) cell in murine stomach and of its equivalent in other mammals

Summary A peculiar type of cell, the ECL cell, accounts for a large portion of nonenterochromaffin endocrine cells in the gastric oxyntic glands of several mammals, including rat, mouse, guinea pig, rabbit, cat, dog, pig, and man. The ECL cell is characterized mainly by its secretory granules with irregular cores heavily reacting to Grimelius' silver, Sevier-Munger silver and phosphotungstic acid, while failing to react, in all species but the cat and rabbit, to Masson's argentaffin method. In the rat and mouse, the ECL cell seems to correspond exactly to the argyrophil non-argentaffin histamine-storing enterochromaffin-like cell of Håkanson and Owman (1967); in the remaining species, ECL cells seem to account for only a part of the gastric argyrophil enterochromaffin-like cells described by Håkanson and coworkers. Besides sporadic amines, ECL cell granules store non-amine components, whose possible nature is discussed.This investigation was supported in part by Farmitalia S.p.A., Milano.     

Ultrastructural localization of serotonin and polypeptide YY (PYY) in endocrine cells of the human rectum

This study was performed with the aim of ultrastructurally localizing serotonin and polypeptide YY (PYY) in the endocrine cells of the human rectum. Existing basic methods for immunolocalization of antigenic sites in ultrathin sections were tested and modified to allow reproducible results with distinct localization of marker (colloidal gold probes coupled either to IgG or protein A). Probes signifying presence of serotonin were distinctly localized over all heteromorphous granules in argentaffin cells and, in addition, over some of the more monomorphous, rounded granules in a second cell type whose granules all were covered by probes showing localization of the PYY antigen. The results suggest that serotonin in endocrine cells of the gut is not confined to the enterochromaffin type but may also be present in trace amounts in non-enterochromaffin endocrine cells storing peptide hormones. Since probes marking sites of PYY were deposited over some heteromorphous granules in enterochromaffin cells, the evidence obtained also suggests that PYY may occur in low concentration in these cells. The distribution of probes in the sections indicated that antigenic sites were confined to granules in the cells.     

A novel endocrine cell type in the guinea-pig gastric mucosa: cellular source of pro-enkephalin-derived peptides

Summary A novel endocrine cell type has been identified in the guinea-pig gastric mucosa which preferentially occurs in the oxyntic area. Cells of this type exhibit immunoreactivities for bovine adrenal medulla dodecapeptide (BAM-12P) and in many cases for Met-enkephalin and are thus presumed to contain a pro-enkephalin-like precursor protein. Systematic immunohistochemical investigations show that these cells do not contain immunoreactivities for various enteric hormones, neuropeptides and biogenic amines (serotonin, histamine). However, they do contain immunoreactivity for chromogranin A, an acidic glycoprotein which is common to the majority of entero-endocrine cells. Using silver impregnation techniques BAM-12P immunoreactive cells prove to be argyrophil, but fail to react argentaffin. On the electron microscopical level, these cells contain a well-developed endoplasmic reticulum and Golgi apparatus and numerous polymorphous secretion granules which measure about 290 nm in diameter. The secretion granules are ovoid or pear-shaped but largely plump compared to those of enterochromaffin cells. Light and electron microscopical findings indicate that BAM-12P immunoreactive cells constitute an endocrine cell population of the gastric epithelium in addition to the established endocrine cells hitherto known in this location.This study was supported by grants of the Deutsche Forschungsgemeinschaft (EN 65/15-2)For this work the author was awarded the Wolfgang-Bargmann-Price 1990 of the Anatomische Gesellschaft     

Serotonin-storing cells of the chicken duodenum: light,fluorescence and electron microscopy,and immunohistochemistry

Summary In an attempt to identify duodenal endocrine cells emitting formaldehyde-induced fluorescence (FIF), chicken duodena were studied by combined fluorescence, ultrastructural, silver impregnation and immunohistochemical methods in the same or consecutive sections. Our results show that: (1) Almost all the cells emitting yellow fluorescence by both the Falck-Hillarp and the Furness methods exhibit an immunohistochemical reaction with serotonin (5-HT) antiserum. (2) Almost all cells radiating yellow fluorescence by the Furness method stain with toluidine blue in Epon-embedded sections but, by high-voltage electron microscopy, can be subdivided into two types of cell containing either small round or polymorphous types of granules. (3) In the sections from which resin had been removed, all the cells emitting yellow FIF show argentaffinity by the Singh method, but not all cells display argyrophilia with the Grimelius method. (4) Cells exhibiting both argyrophil and argentaffin reactions in deresined serial sections are also separated into two types of cell, containing either small spherical or polymorphous types of granules by conventional electron microscopy in thin sections. Therefore, chicken enterochromaffin cells emit yellow FIF, store 5-HT, show both argentaffinity and argyrophilia, but are ultrastructurally classified into two types of granule-containing cells which may be related to polypeptides coexisting with 5-HT.     

On argyrophil reactions of endocrine cells in the human fetal pancreas

Summary The endocrine cells in the pancreas of five human fetuses with gestational ages of 18–20 weeks were examined by light and electron microscopy with special regard to argyrophil reactions. B-cells and typical A and D-cells were easily identified electron microscopically on the basis of their typical secretory granules. In the Grimelius argyrophil silver stain, a concentration of silver grains over the less electron dense peripheral mantle of the A-cell secretory granules was observed by electron microscopy. In the Hellerström and Hellman modification of the argyrophil Davenport alcoholic silver stain, silver grains were concentrated over the internal structures of the D-cell secretory granules. With this stain an accumulation of silver grains was also seen at the surface of the A-cell secretory granules. The argyrophil reaction of the A-granules was less pronounced than in the D-cells. In addition to B-cells and A- and D-cells, two other types of endocrine cell were observed by electron microscopy. These cells were argyrophil with the silver impregnation method of Grimelius. The electron microscopic findings at least partly explain the frequent overlapping between the two staining methods observed at the light microscope level.This study was supported by the Swedish Medical Research Council (Project No. 102)     

Age and changes in the number of endocrine cells in the stomach and their role in senile atrophy of the gastric glands

In the gastric mucous membrane of 40 rats representing 4 age groups (pubertal, mature, old and very old age) per 10 animals in each, thickness of the mucous membrane has been measured by the argyrophil method of Grimelius and the argentaffin method of Masson - Hamperl and the number of the endocrine cells has been counted. The thickness of the mucous membrane and the length of its glands, as well as the number of argyrophil cells increase from the pubertal towards the mature age. From the mature up to the old age the number of argyrophil cells rises in the antral part, and in the acid-producing zone it significantly decreases. The number of argentaffin cells in the stomach does not change from the pubertal towards the old age. In the very old age, against the background of atrophy of gastric glands, the number of both argyrophil and argentaffin cells decreases in both parts of the stomach studied. The data obtained are discussed taking into consideration functional role of various types of endocrine cells, producing biologically active peptides and monoamines.     

Ultrastructural identification of argyrophil and argentaffin cells of the gastric mucosa in the rat

Ultrastructure of endocrine cells impregnated in the rat gastric mucosa by Grimelius method (identification of argyrophilia) and by Masson--Hamperl method (identification of argentaffinity) and influence of various fixatives on the structure and properties of the secretory granules in these cells have been studied. Fixation of the material in paraformaldehyde or glutaraldehyde varies in its effect on the granule structure of EC-, D1- and ECL-cells, while its influence on the granule structure of G-, D- and AL-cells is identical. The granules of EC-, ECL- and G-cells are argyrophil, and only those of EC-cells are argentaffin. Weak argyrophilia, which is evidently not appearant at the light-optical level, is specific for granules of D1- and AL-cells. Fixation in paraformaldehyde and especially the subsequent treatment in osmium tetroxide results in increasing argyrophilia of the endocrine cells, as compared to fixation in glutaraldehyde. Varieties in the effect of the fixatives do not prevent ultrastructural and histochemical identification of the endocrine cell types.     

Neuron-specific enolase in mucosal endocrine cells and carcinoid tumours of the small intestine: A comparative study with neuron-specific enolase immunocytochemistry and silver stains

Summary Endocrine cells of human small intestinal mucosa, small intestinal carcinoids and carcinoid liver metastases were stained with an immunocytochemical technique using an antiserum against neuron-specific enolase (NSE), with the argyrophil technique of Grimelius and with the argentaffin technique of Masson. In the normal mucosa, scattered NSE-immunoreactive cells were seen mainly in the deeper parts of the crypts. These cells, as shown in the same sections, corresponded to the argentaffin and/or argyrophil cells indicating that they were of endocrine type.All intestinal carcinoids (16 cases) displayed NSE immunoreactivity. However, this reaction did not correlate on the cellular level with the silver techniques employed. Thus, many tumour cells were NSE immunoreactive but lacked an argentaffin or argyrophil reaction and vice versa. On the light microscopical level the silver techniques reveal the presence of neurohormonal granules in the tumour cells, while the NSE immunoreactivity appears to disclose neuroendocrine differentiation of the tumour cells irrespective of their hormone and granular content.Out of 13 carcinoid liver metastases, eight displayed strong NSE immunoreactivity, three were weakly stained and two were unreactive. Consecutive or the same tumour sections showed an argentaffin and argyrophil reaction in all carcinoid metastases. Since silver staining provides one type of information and NSE immunocytochemistry another, they provide in combination a good discriminator for neuroendocrine tumours.     

Endocrine cells of the duodenal glands in various representatives of Primates]

The duodenum of four species of monkeys, belonging to the genera Macaca and Papio, has been investigated. In order to reveal argyrophil and argentaffin Ec-cells, Grimelius and Masson-Hamperl methods have been used, respectively. In all the species studied in the terminal parts and in the ducts of the duodenal glands presence of endocrine cells is noted; their amount, however, is considerably less than in crypts. Open and close types of endocrine cells++ have been revealed in the glands. A part of the endocrine cells population is argentaffin Ec-cells (about 40% in Papio and 80% in Macaca). This demonstrates participation of serotonin they produce in regulation of the duodenal glands functional activity. Dependence between taxonomic position of the species and degree of endocrine cells++ development in the duodenal glands has been followed. In particular, in representatives of Macaca amount of argyrophil and argentaffin in the glands is greater than in Papio. Possible reasons of the differences are discussed. Comparing the results on investigation of the glands in the primates with those previously obtained for representatives of Carnivora, a suggestion is made concerning some general principles in organization of the proper endocrinic apparatus of the mammalian duodenal glands.