Electron microscopic identification of the histamine-storing argyrophil (enterochromaffin-like) cells in the rat stomach

Summary In the oxyntic gland area of the rat stomach the histamine-containing epithelial cells (also referred to as enterochromaffin-like cells because of their morphologic similarity with the 5-hydroxytryptamine-storing enterochromaffin cells) constitute the system of argyrophil cells in this area as previously shown by the combined use of fluorescence and light microscopic techniques. By performing the argyrophil staining reaction directly on ultra-thin sections it could be demonstrated in the electron microscope that the argyrophil cells have features suggesting that they are endocrine. Based on the ultrastructure of their secretory granules at least two such endocrine cell systems—both argyrophil—could be recognized in the oxyntic glands. The silver deposits were accumulated over the secretory granules of both these cell systems.It is well known that after injection of 1-3,4-dihydroxyphenylalanine, the histamine-storing (enterochromaffin-like) cells of the oxyntic glands store also dopamine. Under these conditions the enterochromaffin-like cells stain argentaffin, which has been shown at the light microscopic level. Also this reaction could be performed directly on ultra-thin sections. By electron microscopy it was then established that the two endocrine cell systems of the oxyntic gland area stained argentaffin upon treatment with 1-3,4-dihydroxyphenylalanine, and that the staining was confined to the secretory granules.The results clearly show that the enterochromaffin-like cells of the rat oxyntic gland area (which is devoid of 5-hydroxytryptamine-containing enterochromaffin cells) are identical with cells characterized as endocrine by ultrastructural criteria, and that gastric non-mast-cell histamine occurs in at least two separate systems of enterochromaffin-like cells.     

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.     

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.     

Osmium dependent argentaffin staining of lysosomes

Summary Lysosomes stain with the argentaffin reaction after fixation with glutaraldehyde followed by osmium tetroxide. The reaction works well both at the level of the light and electron microscope. Control experiments show that this argentaffinity is caused by reduced osmium tetroxide. No staining could be observed in freeze-dried material, in tissues fixed only with glutaraldehyde, or after bleaching of the sections with hydrogen peroxide solutions. In the electron microscope, the population of lysosomes appears heterogeneous as related to the density of silver deposits over the organelles. No correlation is found between size and argentaffinity of lysosomes. X-ray microanalysis of sections from glutaraldehyde/osmium tetroxide fixed material reveals significantly higher amounts of osmium in lysosomes, as compared to other cell organelles (e.g. peroxisomes or mitochondria). A significant peak for silver is observed in lysosomes after treatment of the sections with ammoniacal silver solution, whereas the signal for osmium is reduced. Amounts of sulphur are too low to be detected in lysosomes. It is concluded that argentaffin staining of lysosomes is an osmium dependent reaction.Parts of these results have been presented as a poster during the 20th Congress of Electron Microscopy, joint session of the Austrian Society of Electron Microscopy and the German Society of Electron Microscopy, August 23–28, 1981, Innsbruck, Austria     

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.     

Osmium tetroxide/p-phenylenediamine staining of nucleoli and Balbiani Rings in Chironomus salivary glands.

This paper deals with the application of the osmium tetroxide fixation followed by p-phenylenediamine treatment to salivary gland cells from Chironomus larvae. After this procedure, cytoplasm, nucleoli and Balbiani rings show a high degree of staining both in light and electron microscopy, while chromatin remains unstained. Ethanol fixation followed by osmium tetroxide/p-phenylenediamine does not modify the above mentioned staining pattern. Under these conditions, extractive procedures for lipids do not affect the osmiophilia of nucleoli and Balbiani rings, while RNase or trichloroacetic acid treatment decreaes the staining degree of these structures. In osmium tetroxide/p-phenylenediamine treated salivary glands, the highest contrast within nuclei is seen to occur in the pars granulosa from normal or segregated nucleoli, as well as in Balbiani ring granules, which appear either as hollow granules or with a bipartite or horseshoe-like structure.     

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.     

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.     

Preservation of arachidonoyl phospholipids during tissue processing for electron microscopic autoradiography

To facilitate autoradiographic subcellular localization of arachidonoyl phospholipids, the retention of radioactivity during tissue processing of murine fibrosarcoma cells labeled in vitro with 3H-arachidonate was assessed. Approximately 94% of cell radioactivity was incorporated into phospholipids. During tissue processing, extraction of radioactivity was monitored by liquid scintillation spectrometry. Fixation of cells in glutaraldehyde-tannic acid, postfixation in osmium tetroxide, en bloc staining in uranyl magnesium acetate, dehydration in ethanol, and embedding in Epon resulted in preservation of 93.5% of total tissue radioactivity. Analysis of extracted radioactivity by thin layer chromatography revealed that no specific class of phospholipids was selectively extracted. Fixation with osmium tetroxide alone was nearly as effective as the complete fixation protocol and resulted in retention of 90.0% of radioactivity. However, fixation with glutaraldehyde-tannic acid alone without osmium tetroxide post-fixation led to extraction of 69.8% of total cell radioactivity. Thus, osmium tetroxide is crucial in the preservation of arachidonoyl phospholipids and presumably forms extensive cross-links between polyunsaturated acyl residues. This degree of preservation of arachidonoyl phospholipids is indicative of spatial fixation of the radiolabeled moieties and will permit quantitative studies of subcellular loci of eicosanoid metabolism by electron microscopic autoradiography.     

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     

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.     

Application of silver stains to cytologic specimens of neuroendocrine tumors metastatic to the liver

Cytologic specimens of neuroendocrine tumors metastatic to the liver were examined with regard to their silver staining properties after the application of argentaffin and argyrophil staining techniques (Masson, Grimelius and Sevier-Munger). In tumors with a content of serotonin (small intestine carcinoids), the presence of this substance was demonstrated cytologically as an argentaffin reaction in individual tumor cells; however, formalin fixation was a prerequisite for positive staining. Melanin in malignant melanoma cells displayed a positive argentaffin reaction, irrespective of the fixation used (air drying, formalin, Bouin's fluid or acetone-alcohol). Thus, serotonin and melanin can be distinguished in cytologic samples of neuroendocrine tumors by the use of the Masson argentaffin reaction with different fixatives. The nonargentaffin-positive neuroendocrine tumor cells were weakly stained or unreactive with the Grimelius argyrophil technique. The Sevier-Munger argyrophil technique was negative or gave a disturbing nonspecific background staining reaction that was difficult to interpret in the cytologic samples. Thus, the Grimelius method appears to be the most useful silver stain for identifying neuroendocrine tumor cells in cytologic material, irrespective of their hormone content, since both argentaffin-positive and argentaffin-negative cell samples were stained at least to some degree.     

Immunocytochemistry with osmium-fixed tissue. I. Light microscopic localization of growth hormone and prolactin with the unlabeled antibody-enzyme method.

Growth hormone and prolactin were localized on thin plastic sections of rat anterior pituitary gland and mammosomatotropic tumor MtTW15 that were fixed with osmium tetroxide (alone,mixed with aldehydes, or after aldehydes). Intense immunocytochemical staining for both antigens was obtained after plastic was removed from sections with an alcoholic solution of sodium hydroxide. The results indicated that antigenic determinants of rat prolactin and growth hormone were not completely destroyed or inactivated by fixation with osmium and embedment in epoxy resin, and that removal of the polymerized epoxy resin was necessary to obtain light microscopic postembedding immunocytochemical staining of these antigens. The results also demonstrated that tissues which have been conventionally processed for morphological evaluation by electron microscopy may be suitable for postembedding immunocytochemical staining of some antigens for light microscopy.     

Ultrastructural cytochemistry of the secretory granules of the hamster submandibular gland

Summary The ultrastructure and cytochemistry of the secretory granules of the male hamster submandibular salivary gland were studied. After fixation in glutaraldehyde followed by osmium tetroxide the granules exhibit a characteristic bipartite substructure, with an electron lucid crescenteric rim and a more dense central core. A differentiation into two regions of the granules could also be visualized in specimens primarily fixed in Millonig's osmium tetroxide or in potassium permanganate. The electron lucid peripheral portion of the membrane bounded secretory granules further displays a strong positive reaction after staining of ultrathin sections with the periodic acid-chromic acid-(PA-CrA)-silver technique. The strong periodate reactivity of the rim relative to the core, suggests a difference in mucin composition of the two granule regions. With the PA-CrA-silver staining technique a positive reaction was also observed within the Golgi apparatus of the acinar cells.     

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.     

Effect of different fixatives on Con A surface receptors of mouse peritoneal macrophages

The effects of glutaraldehyde, formaldehyde, or osmium tetroxide fixation on the number of labeled Con A surface receptors on mouse peritoneal macrophages were compared. Gold-labeled Con A receptors were found to be isolatedly arranged and evenly distributed on cell surfaces independent of the fixative used. Only cells preincubated with Con A and subsequently fixed by osmium tetroxide showed arrangement of labeled receptors in clusters. Significant differences were found in the number of Con A receptors per cell depending on the fixative used. The fluorescence intensity of FITC-Con A staining was detected spectrophotometrically, the characteristic X-rays of gold-labeled Con A receptors were determined by means of electron beam-induced X-ray microanalysis. The experimental results obtained both at light and electron microscopic level pointed to formaldehyde being the best fixative also for this purpose.     

Effect of different fixatives on Con A surface receptors of mouse peritoneal macrophages

Summary The effects of glutaraldehyde, formaldehyde, or osmium tetroxide fixation on the number of labeled Con A surface receptors on mouse peritoneal macrophages were compared. Gold-labeled Con A receptors were found to be isolatedly arranged and evenly distributed on cell surfaces independent of the fixative used. Only cells preincubated with Con A and subsequently fixed by osmium tetroxide showed arrangement of labeled receptors in clusters. Significant differences were found in the number of Con A receptors per cell depending on the fixative used. The fluorescence intensity of FITC-Con A staining was detected spectrophotometrically, the characteristic X-rays of gold-labeled Con A receptors were determined by means of electron beam-induced X-ray microanalysis. The experimental results obtained both at light and electron microscopic level pointed to formaldehyde being the best fixative also for this purpose.     

Osmium tetroxide/p-phenylenediamine staining of nucleoli and balbiani rings in Chironomus salivary glands

Summary This paper deals with the application of the osmium tetroxide fixation followed by p-phenylenediamine treatment to salivary gland cells from Chironomus larvae. After this procedure, cytoplasm, nucleoli and Balbiani rings show a high degree of staining both in light and electron microscopy, while chromatin remains unstained. Ethanol fixation followed by osmium tetroxide/p-phenylenediamine does not modify the above mentioned staining pattern. Under these conditions, extractive procedures for lipids do not affect the osmiophilia of nucleoli and Balbiani rings, while RNase or trichloroacetic acid treatment decreaes the staining degree of these structures. In osmium tetroxide/p-phenylenediamine treated salivary glands, the highest contrast within nuclei is seen to occur in the pars granulosa from normal or segregated nucleoli, as well as in Balbiani ring granules, which appear either as hollow granules or with a bipartite or horseshoe-like structure.     

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)     

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.