Immunocytochemical and ultrastructural characterization of endocrine cells in chicken proventriculus

Summary The endocrine cells of the chicken proventriculus were investigated immunocytochemically, using the peroxidase-antiperoxidase technique on paraffin and semithin sections for light microscopy, and immunogold staining in osmium-fixed material for electron microscopy. The fixation procedure also allowed a detailed ultrastructural investigation. Twenty-three antisera were tested and 7 immunoreactive cell-types were identified: D-cells containing somatostatin-like peptide; EG-cells immunoreactive to anti-glucagon, anti-GLP1 and antineurotensin; NT-cells labelled only with anti-neurotensin; BN-cells containing bombesin-like material; ENK-cells showing met-enkephalin immunoreactivity; EC-cells reactive to anti-serotonin; and APP-cells positive to anti-avian pancreatic polypeptide. In addition, enterochromaffin-like (ECL) cells, were also detected by electron microscopy. The presence of ENK-cells and the ultrastructure of these and NT-cells are described for the first time in chicken proventriculus, and glucagon, GLP1 and neurotensin are shown to be colocalized in the EG-cells.     

Localization by immunoelectron microscopy of somatostatin-containing cells in the gastric mucosa of a teleost fish, Perca fluviatilis

Somatostatin-immunoreactive cells were localized on semithin and ultrathin sections of Epon-embedded samples of perch gastric mucosa, classically fixed with aldehydes and osmium tetroxide. On semithin sections, somatostatin cells were identified by using the immunoperoxidase method. The ultrastructural localization of somatostatin immunoreactivity was achieved using the colloidal gold method. Cells showing somatostatin immunoreactivity are found to be scattered among the surface mucous cells and the mucous neck cells. Somatostatin appears to be localized in cytoplasmic granules. Somatostatin-containing cells are identified as the type I cells which were described in a previous ultrastructural study. The present report also points out that tissue samples which have been classically processed for ultrastructural study could be in some cases suitable for immunocytochemical investigations.     

Electron microscopical identification of the immunofluorescent gastrin cells in the cat pyloric mucosa

Summary To solve the unsettled problem of the identification of the gastrin cells, a study has been undertaken on the electron microscopical characteristics of the gastrin-containing cells of the cat pyloric mucosa. Cells which, on semithin sections, were shown by an immunofluorescence method to contain gastrin, have been identified on serial ultrathin sections. The ultrastructural features of these cells are those which have been described as characteristic of the G cells of the antropyloric mucosa. Other non-entero-chromaffin endocrine cells, which have been recognized as the D cells of the gastro-intestinal mucosa, did not result to contain gastrin.     

Gastrin-cholecystokinin-like immunoreactivity in the central nervous system of the milkweed bug,Spilostethus pandurus. Ultrastructural aspects of the reactive A cells of the brain

Summary— All the ganglia belonging to the central nervous system of adults of the milkweed bug Spilostethus pandurus (Hemiptera) were screened immunohistochemically for vertebrate gastrin-cholecystokinin (CCK-8(s))-like peptides. Several large reactive perikarya are present in the median part of the protocerebrum, their processes extending to the dorsal ‘aorta’. These cell bodies are also paraldehyde fuchsin-positive, ie they are A-type cells. In the lateral part of the protocerebrum, in the deutocerebrum and tritocerebrum, and in the suboesophageal, prothoracic and abdominal ganglia, a few immunoreactive cell bodies send axonal processes into their respective neuropiles. The A-type cells reactive to CCK antiserum were identified, at the ultrastructural level, by combining paraldehyde fuschin staining of semithin sections with a post-embedding immunogold technique carried out on adjacent ultrathin sections. The neurosecretory cells contain numerous vesicles of elevated electron density. These data suggest that members of the CCK peptide family are present in the central nervous system of Spilostethus pandurus.     

Use of immunocytochemical staining of somatostatin for correlative light and electron microscopic investigation of D cells in the pancreatic islet of Xiphophorus helleri H. (Teleostei)

Summary Somatostatin-containing cells have been demonstrated by immunocytochemistry in semithin sections of the pancreatic islet of the teleost fish, Xiphophorus helleri. These cells were shown by correlative light and electron microscopy to be identical with D cells previously defined in this species by the silver impregnation method of Hellman and Hellerström.Supported in part by grants from the British Council and from the Medical Research Council of Great Britain     

Licht- und fluoreszenzmikroskopische Darstellung spezifischer Zellen und Strukturen des Binde- und Nervengewebes von Gastropoden im Semidünnschnitt

Zusammenfassung Im Binde- und Nervengewebe von Gastropoden, welche in Kunstharze eingebettet wurden, lassen sich nach Entfernung des jeweiligen Einbettungsmediums mit licht- und fluoreszenzmikroskopischen Färbeverfahren (Paraldehydfuchsin, Aldehydthionin, Alcianblau, Pseudoisocyanin, RF 500) mit hoher histochemischer Spezifität Zellen und Strukturen darstellen. Neben verschiedenen Zelltypen mit unterschiedlichen Funktionsstadien im Bindegewebe können vor allem die neurosekretorischen Zellen und ihre Fortsätze in den Cerebralganglien der untersuchten Schneckenarten bis in ihre Neurohämalbereiche differenziert angefärbt werden. Mit AT und PAF konnten dabei neurosekrethaltige Axone in der Cerebralkommissur von Planorbarius corneus erstmals sicher lichtmikroskopisch nachgewiesen werden. Die von uns verwendeten Verfahren erlauben eine gezielte elektronenmikroskopische Analyse der im Semidünnschnitt licht- und fluoreszenzoptisch selektiv histochemisch identifizierten Zellen und Strukturen.

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Light- and fluorescence microscopic investigations on specific cells and structures in semithin sections of the connective- and nervous tissues of gastropods

Summary In the present study specific cells and structures in the connective-and nervous tissues of some gastropods were demonstrated by light- and fluorescence techniques, performed on semithin sections of plastic embedded material. After dissolving the embedding medium (Epon or Styrene-methacrylate), the semithin sections are stainable by Gomori's Paraldehyde-Fuchsin, Aldehyde-Thionin, Alcian Blue and the fluorochromes Pseudoisocyanin and RF 500. Best results are obtained optically by staining with Pseudoisocyanin, because all fluorescing substances (i. a. the neurosecretory cells and fibres in the ganglia) appear on a practically dark background. For the first time, with Aldehyde-Thionin and Paraldehyde Fuchsin—but not with Pseudoisocyanin—neurosecretory fibres has been demonstrated in the cerebral commissure of Planorbarius corneus.The histochemical identifying of specific cells and structures in semithin sections by light- and especially fluorescence microscopic highly selective reactions gives the possibilities to analyse these target cells by electron microscopic investigations.

     

Demonstration of a somatostatin-like activity in retinal cells of the rat

Summary A somatostatin-like substance is demonstrated by light microscopic immunohistochemistry (PAP-method) in perikarya and cell processes of the retina of adult and infant rats. These perikarya are identified according to their size, arrangement and distribution. Each of the first two neuronal orders (receptors, bipolar cells, ganglionic cells) of the visual pathway can be associated with retinal cells reacting positively with anti-somatostatin. In the adult rat, perikarya and processes of (i) horizontal cells, (ii) amacrine cells and (iii) large neurons in the ganglionic layer are specifically labeled. The staining of middle-sized and small ganglion cells is probably caused by the close attachment of labeled fibers to non-reacting cells. Postnatally, the immunoreactive elements develop in parallel to the differentiation of the corresponding retinal layers. It is discussed whether the three types of retinal cells containing a somatostatin-like substance provide an inhibitory system to each of the two orders of retinal neurons.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr 569/2) and Stiftung Volkswagenwerk     

Development of the endocrine pancreas during larval phases of Rana temporaria

Summary The pancreatic endocrine component was studied at different stages of development in the tadpoles of Rana temporaria. The material was embedded in Epon, and serial semithin and thin sections were made in order to correlate ultrastructural features and tinctorial traits of the endocrine cells. Serial semithin sections were also stained with the peroxidase-antiperoxidase immunocytochemical method and with silver impregnations for argyrophilia and argentaffinity. In early larvae (legless tadpoles), A and B cells are present. Both can be found within ducts and exocrine tissue or, more frequently, in cellular clusters among the ducts and acini. These primitive islets are solid structures, surrounded but not penetrated by capillaries. Mitoses were observed in A and B cells. In the following phase (tadpoles with hindlegs), D and pancreatic polypeptide-immunoreactive cells are also present, as well as numerous endocrine cells scattered among exocrine tissue. There is also a change in the vascular-insular pattern: capillaries not only surround but also penetrate the endocrine group. The structure of the endocrine pancreas in older tadpoles is similar. Tinctorial traits and ultrastructural features of endocrine cells are described, and the origin of primitive islets is discussed.     

Distribution of 5HT-immunoreactivity in the pedal ganglion of Mytilus galloprovincialis

Summary Serotonergic cell bodies and fibers were identified in the pedal ganglia of Mytilus gattoprovincialis with a serum raised against serotonin and the unlabelled peroxidase-antiperoxidase pre- and post-embedding methods. Examination of Vibratome and serial semithin sections showed that most reactive perikarya are located in the ganglionic cortex, being mainly concentrated at the medial aspect of the postero-dorsal portion of the ganglia. Immunoreactive fibers form a dense network in the neuropil, extend throughout the commissure and run parallel in the nerves and connective tracts. The morphology of serotonin-positive cells compared with that of Golgi-impregnated neurons allows the identification of a main population of unipolar, probably projecting neurons and of smaller multipolar cells likely representing local circuit elements. The ultrastructure of labelled neurons is comparable to that of serotonergic cells described in both vertebrate and invertebrate nervous systems.Supported by Ministero Pubblica Istruzione (40%)     

Synaptophysin immunoreactivity in the mammalian endocrine pancreas

Summary Synaptophysin, a major membrane glycoprotein of small presynaptic vesicles in neurons, has also been found in microvesicles of endocrine cells, e.g., of the endocrine pancreas. In the present study, the endocrine pancreas in 9 mammalian species (man, dog, mink, bovine, rabbit, guinea pig, rat, mouse, gerbil) has been investigated immunohistochemically for synaptophysin immunoreactivity. Synaptophysin-positive cells have been identified and localized on semithin plastic sections. Our study demonstrates that, in all species examined, all pancreatic endocrine cell types are consistently synaptophysin-positive independent of their location within the tissue, or the conditions of tissue processing. In addition, a few cells that cannot be hormonally identified show synaptophysin immunoreactivity. Hence, synaptophysin appears to be a regular constituent of all pancreatic endocrine cells in mammals. In several species, a subpopulation of endocrine cells, consisting of glucagon-containing and/or pancreatic-polypeptide-containing cells, exhibits a significantly higher degree of synaptophysin immunoreactivity. In the gerbil, this heterogeneity can readily be detected from the day of birth onwards. Our findings indicate that closely related endocrine cell types may differ with respect to the content of synaptophysin.     

Ultrastructure of the Contractile Vacuole and Its Periphery in Amoeba proteus: Evolution of Vesicles During the Cycle1

ABSTRACT. The population of organelles in the periphery of the contractile vacuole of Amoeba proteus has been studied throughout the cycle using proper fixation and dehydration procedures on serial semithin and thin sections. Three distinct types of perivacuolar vesicles which arise successively from one another in the course of the cycle have been described. Size relationships have been determined by stereology.     

Immunohistochemical studies on the ontogenesis of some endocrine cells in the chicken antrum and duodenum

The time of appearance, morphology and topographic distribution of gastrin/CCK-, somatostatin-, 5HT-, and bombesin-like immunoreactive cells during embryonic and postnatal development were studied in chicken antrum and duodenum with immunohistochemical methods. Gastrin/CCK-like cells appeared on or about the 11th day in the antrum and duodenum, somatostatin-like cells around the 12th day in the antrum and the 11th day in the duodenum, bombesin-like cells appeared only in the antrum and only at hatching. In the early stages of development all the immunoreactive cells were localized in the surface epithelium, descending deeper into the glands as these form, although some cells could always be seen in the surface epithelium. Around the 17th day the number of gastrin/CCK-like cells and somatostatin-like cells in the antrum increases, while 5HT-like already become more numerous in the duodenum from the 13th day onwards. Two territories were recognized in the antrum of the adult: the first was near the duodenum where gastrin/CCK-like and somatostatin-like cells, often in close contact, were very numerous; the other territory was near the gizzard where bombesin-like cells were more numerous. Both regions contained 5HT-like cells in smaller number. In adult duodenum, 5HT-like cells were the most numerous, while somatostatin-like cells and gastrin/CCK-like cells, found in more superficial locations, were more scanty.     

Gonadotropic cells in the Atlantic salmon,Salmo salar

Summary The gonadotropin-producing cells (GTH-cells) in the Atlantic salmon were studied light and electron microscopically before, during and after spawning, and after injections of luteinizing hormone-releasing hormone (LH-RH). The double immunofluorescent technique was applied using rabbit anti-carp GTH as the first antibody. Numerous immunofluorescent cells were observed throughout the pars distalis, but very few in the pars intermedia. These cells are basophilic and PAS-positive, and ultrastructurally classified as globular gonadotropes. Only one gonadotropic cell type could be identified; its size, morphology and fine structure vary considerably. In the same specimen the GTH-cells can be predominantly globular or vesicular in appearance, depending on the reproductive phase of the fish. At spawning and after LH-RH injection, many GTH-cells reach a vacuolar stage; the content of the vacuoles is not immunofluorescent. Another cell type, which resembles GTH-cells in semithin sections, did not show gonadotropic properties; its nature and functional significance are unknown. In addition, the present study revealed an increase in the synthetic and exocytotic activity of prolactin cells after LH-RH injections. It is suggested that LH-RH mediates this effect via LH and eventually via estradiol.The authors are indebted to Dr. N. Johansson and superintendent N. Steffner for generously providing facilities at their institutes in Älvkarleby. They also wish to thank Mrs. Y. Lilliemarck and Mrs. V. Lundin for their skillful technical assistance. The grants made available by the Swedish Natural Science Research Council (No. 2124-037) are also gratefully acknowledged. Thanks are also due to Mrs. S.M. McNab for linguistic assistance     

Immunocytochemical and autoradiographic studies of the endocrine cells interacting with GABA in the rat stomach

Summary There are now increasing evidences suggesting that GABA is able of direct interaction with certain endocrine cells. In the present study, highly specific anti-GABA-glutaraldehyde antibodies and ³H-GABA uptake were used at the light and electron microscope levels to investigate the occurrence of cells containing endogenous GABA or taking up exogenous GABA in the mucosal antrum and corpus of the rat stomach. Only certain endocrine cell types of both regions were immunostained or grain-labelled. However, the morphology of their secretory granules did not allow to identify the nature of their hormone with certainty but suggested that somatostatin-like cells could interact with GABA. The combination of gastrin and somatostatin immunodetection with ³H-GABA uptake autoradiography at the light microscope level, revealed that a subpopulation of somatostatin-like cells and other still unidentified endocrine cells are able to take up GABA, while the gastrin-like cells are not. These results reinforce the hypothesis that certain endocrine cell types of the diffuse endocrine system of the digestive tract are able to directly interact with GABA.     

Ultrastructure and histochemistry of the foregut in<Emphasis Type="Italic"> Wirenia argentea</Emphasis> and<Emphasis Type="Italic"> Genitoconia rosea</Emphasis> (Mollusca,Solenogastres)

Wirenia argentea and Genitoconia rosea feed on Cnidaria like most representatives of the molluscan taxon Solenogastres (Aplacophora, Neomeniomorpha sensu Scheltema). The structure and histochemistry of the foregut are described based on histologic, semithin, and ultrathin section series. The ultrastructure was analyzed by means of transmission electron microscopy. There are two sets of unicellular glands: a narrow row of preoral gland cells opening to the preoral area, and pharyngeal gland cells in high numbers. Preoral gland cells produce serous secretions in W. argentea, but mucosubstances in G. rosea, whereas pharyngeal gland cells are similar in structure and histochemistry in both species. Based on the size and electron density of gland vesicles, five distinct types of pharyngeal gland cells can be defined. In contrast to earlier assumptions, all types of pharyngeal gland cells produce serous secretions, most probably representing digestive ferments, but no mucosubstances.     

FMRFamide-like immunoreactivity in the metathoracic ganglion of the locust (Schistocerca gregaria)

Summary The distribution of FMRFamide-like immunoreactivity in the metathoracic ganglion of the locust, Schistocerca gregaria, has been investigated in serial semithin transverse sections with the use of the peroxidase-antiperoxidase (PAP) technique. The topographical distribution of approximately 120 immunopositive neurons was established. Antiserum against bovine pancreatic polypeptide (BPP) stains the same ganglionic cells as FMRFamide-antiserum, yet this staining is largely blocked after preabsorption to FMRFamide. A comparison of these results with those from other studies suggests that there may be more than one type of endogenous RFamide-like peptide.     

Characterization of the somatostatin-like immunoreactivity extracted from an adrenal medullary tumour

Significant amounts of somatostatin-like immunoreactivity (SLI) were detected in the extract of a human catecholamine-secreting adrenal medullary tumour. After salt fractionation and reconstitution the major portion of SLI was purified by gel filtration and two HPLC steps; in all three systems it eluted in the position of somatostatin-14. The purified somatostatin-like peptide inhibited, in a dose-related manner, growth hormone release from stimulated perfused rat anterior pituitary cells in vitro. Amino acid analysis showed the purified peptide to have an identical composition to somatostatin found in other species.     

Immunofluorescent localisation of somatostatin-like cells in the esophagus of a protochordate

By means of the indirect immunofluorescence method, somatostatin-like immunoreactive (SLI) cells are detected in the esophagus of the ascidian Styela plicata. They are of the "open" type; they act probably by a paracrine mechanism on the esophageal secretin-like cells. The ascidian SLI cells are negative to all the specific cytochemical methods characteristic of vertebrate somatostatin (D) cells, which were applied in this work. In consequence, special SLI cells are probably present in ascidians.     

Immimocytological localization of a somatostatin-like substance in the brain of the giant slug,Limax maximus L.

Summary Immunocytological tests reveal the presence of a somatostatin-like substance in perikarya and axons in the brain of the giant slug Limax maximus L. Controls carried out on adjacent sections with absorbed antiserum or different antibodies raised against several biologically active peptides of vertebrates (ACTH-17-39, - and endorphin, - and MSH, methionin-enkephalin, TRH) demonstrate the specificity of the staining. However, some cells are both somatostatin- and FMRF-amide-positive. In the cerebral ganglia, the right Z-area cells, responsible for the synthesis of the maturation hormone (MH) are strongly somatostatin-positive. These results suggest a similarity between the MH and the somatostatin-like material contained in the Z-area cells. The simultaneous presence of two peptides in one and the same cell, the nature (elementary granules or soluble product) of the material, and its site of release are discussed.     

Ultrastructural characterization of fresh and cryopreserved in vivo produced ovine embryos

Controlled slow freezing and vitrification have been successfully used for ovine embryo cryopreservation. Selection of embryos for transfer is based on stereomicroscopical embryo scoring after thawing, but the subjectivity inherent to this selection step has been demonstrated by ultrastructural studies of controlled slow frozen, in vivo produced ovine morulae and blastocysts. These studies have shown that certain abnormalities remain undetected by stereomicroscopy only. In the present study, using ovine in vivo produced morulae and blastocysts, we have studied the ultrastructural alterations induced by open pulled straw vitrification (OPS) and controlled slow freezing, compared stereomicroscopical embryo scoring with light microscopy evaluation of embryo's semithin sections, and related the ultrastructural cellular damage with the embryo classification by stereomicroscopical embryo scoring of embryos’ and semithin section evaluation by light microscopy. The ultrastructural lesions found for OPS-vitrified and controlled slow frozen embryos were similar, independently of embryo stage. A significant higher number of grade 3 embryos was found at stereomicroscopical scoring after controlled slow freezing (P = 0.02), and a significant higher number of grade 3 blastocysts was found at semithin sectioning after OPS vitrification (P = 0.037). The extension of ultrastructural damage, especially of mitochondria and cytoskeleton, was related to the semithin classification but not to stereomicroscopical scoring at thawing. This suggests that semithin scoring is a useful tool for predicting ultrastructural lesions and new improvements in cryopreservation and thawing methods of ovine embryos are still warranted, including in the case of blastocysts cryopreserved by OPS vitrification.