Endocrine Cells in the Oesophagus of the Ascidian Styela clava,a cytochemical and immunofluorescence study

Summary Immunocytochemical studies have demonstrated the occurrence of an insulin-immunoreactive cell type in the oesophageal epithelium of the Ascidian Styela clava. Staining with aldehyde fuchsin has demonstrated a number of similar small, triangular, cells located on the basement membrane, which may have an endocrine function. Argyrophilic cells have also been found, suggesting the presence of a second endocrine cell type. The absence of argentaffin cells has led us to believe that the cells so far observed do not produce biogenic amines such as 5-HT (5-Hydroxytryptamine). The nature of these cells is discussed with reference to endocrine-like cells found in the digestive tracts of other protochordates.Animals were collected by courtesy of the Admiralty Marine Trials Station, Portsmouth. This research was carried out during the tenure of an S.R.C. grant no. B/RG 82919 to one of us (M.C.T.). —The localisation of polypeptide hormones in the pharynx and gut of protochordates     

Cell types of the endocrine pancreas in the shark Scyliorhinus stellaris as revealed by correlative light and electron microscopy

Summary In the pancreas of Scyliorhinus stellaris large islets are usually found around small ducts, the inner surface of which is covered by elongated epithelial cells; thus the endocrine cells are never exposed directly to the lumen of the duct. Sometimes, single islet cells or small groups of endocrine elements are also incorporated into acini. Using correlative light and electron microscopy, eight islet cell types were identified:Only B-cells (type I) display a positive reaction with pseudoisocyanin and aldehyde-fuchsin staining. This cell type contains numerous small secretory granules (Ø280 nm). Type II- and III-cells possess large granules stainable with orange G and azocarmine and show strong luminescence with dark-field microscopy. Type II-cells have spherical (Ø700 nm), type III-cells spherical to elongated granules (Ø450 × 750 nm). Type II-cells are possibly analogous to A-cells, while type III-cells resemble mammalian enterochromaffin cells. Type IV- cells contain granules (Ø540 nm) of high electron density showing a positive reaction to the Hellman-Hellerström silver impregnation and a negative reaction to Grimelius' silver impregnation; they are most probably analogous to D-cells of other species. Type VI-cells exhibit smaller granules (Ø250 × 500 nm), oval to elongated in shape. Type VI-cells contain small spherical granules (Ø310 nm). Type VII-cells possess two kinds of large granules interspersed in the cytoplasm; one type is spherical and electron dense (Ø650 nm), the other spherical and less electron dense (Ø900 nm). Type VIII-cells have small granules curved in shape and show moderate electron density (Ø100 nm). Grimelius-positive secretory granules were not only found in cell types II and III, but also in types V, VI, and VII. B-cells (type I) and the cell types II to IV were the most frequent cells; types V to VII occurred occasionally, whereas type VIII-cells were very rare.This work was supported by a fellowship from the Ministry of Education of Japan and the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg (La 229/8)     

An immunocytochemical study of the endocrine pancreas in the Australian fat-tailed dunnart (Sminthopsis crassicaudata)

Summary The endocrine pancreas of the Australian fattailed dunnart, Sminthopsis crassicaudata, was investigated by means of electron-microscopic immunocytochemistry using the protein A-gold technique on London resin (LR) white-embedded tissue. The primary antibodies used were raised against insulin, glucagon, somatostatin and pancreatic polypeptide. The morphology of the secretory granules differed in the four cell types. The insulin cells are pleomorphic, and the secretory granules composed of an electron-dense core surrounded by an electron-lucen halo. The glucago cells possess granules with an electron-dense core usually surrounded by a halo of less dense granular material. Somatostatin cells have large, less dense secretory granules. The pancreatic polypeptide cells show small, dense secretory granules. In order for an ultrastructural study to be considered reliable for the definite identification of endocrine cell types, it is essential that it be corroborated by immunocytochemical data at the light-or preferably electron-microscopic level. Recent developments in immuno-electron-microscopic techniques have contributed to a better knowledge of cells responsible for the secretion of a wide variety of hormones, as in this study.     

An ultrastructural study on gastric endocrine cells in the stomach gland patch of the koala Phascolarctos cinereus

The endocrine cells in the stomach gland patch of the koala (Phascolarctos cinereus) were studied ultrastructurally. They were classified into 3 types based on the ultrastructural profiles of their endocrine granules and tentatively categorized as type I, II, and III endocrine cells. Type I cells contained round granules that were for the most part larger than those observed in the other 2 cell types. The granules ranged from moderate to relatively high in electron density. Type II cells were angular in shape and characterized by the presence of granules that were polymorphous in profile. Contents of the endocrine granules in type II cells also showed a range of high to moderate electron density. Type III cells were oval or pyramidal in shape. They contained highly polymorphous granules that were round, oval, dumbbell-like or comma in shape and characterized by the presence of a clear space or halo separating the high to low electron-dense core from the limiting membrane of granules. Type III cells were observed most often whereas type I and II cells were a less frequent observation.     

The occurrence of argyrophilic and argentaffin cells in the gut of Ciona intestinalis L.

Summary Argyrophilic and argentaffin cells occur in the stomach and intestinal epithelium of the sea-squirt, Ciona intestinalis L.. These cells are characterized by their basal swelling which contains the nucleus surrounded by small secretory granules and by a filamentous cell-apex which reaches the gut lumen. The cells are scattered unevenly within the epithelium. Their number decreases rapidly towards the lower part of the intestine. The localization, size of granules and their shape are features which differentiate these cells from other secretory cells in the gut epithelium such as mucous cells. These cells are thought to possess an endocrine function.The excellent technical assistance of Mrs. R. Sprang is gratefully acknowledged     

Immunocytochemical study of the endocrine pancreas in the grey kangaroo (Macropus fuliginosus)

Summary The endocrine pancreas of the grey kangaroo,Macropus fuliginosus, was investigated by means of immunocytochemistry using the PAP method on the same section at the light- and electron-microscopic levels. Semithin plastic sections were stained individually with primary antibodies for insulin, glucagon, somatostatin and pancreatic polypeptide (PP), and then photographed. Sections were osmicated, re-embedded in BEEM capsules, and ultrathin sections made and examined. The same labelled cells as in the semithin sections were localised in the thin sections, photographs taken and the morphology of secretory granules studied. The insulin cells were pleomorphic; their secretory granules displayed an electron-dense core surrounded by an empty halo. The glucagon cells possessed granules with an electron-dense core usually surrounded by a halo of less dense granular material. Somatostatin cells had larger, less dense secretory granules. The PP cells showed small, dense secretory granules. In order for an ultrastructural study to be considered reliable for the definite identification of endocrine cell types, it is essential that it be corroborted by correlated immunocytochemical data at the light-and electron-microscopic levels.     

Ultrastructural characterization of 6 immunoreactive enteroendocrine cells in Barbus conchonius (Teleostei,cyprinidae)

Summary Using the semi-thin/ultra-thin technique six different immunoreactive endocrine cell types are ultrastructurally identified in 0.5% glutaraldehyde fixed gut of B. conchonius. In addition two of them (gastrin-and PP-immunoreactive cells) are also characterized with the immunogold method, showing that the immunoreactivity is only restricted to the secretory granules. Size distribution histograms and the average diameters of 30% (d30) of the largest granules are given, showing a gradual increase in granule size from unspecific immunoreactive cells, (d30=110 nm) via gastrin-(119 nm), VIP-like-(127 nm), met-enkephalin-(143 nm) and PP-(174 nm) to glucagon-immunoreactive cells (178 nm). The presence of PP-and glucagon-immunoreactivity in the same cells and the consequence for their granule size is discussed. In the distal part of the gut endocrine cells are found showing no immunoreactivity with the antisera used; their granules (d30=144 nm) were, although not significantly, larger then those of VIP-like-immunoreactive cells, also found in that part of the gut. It is supposed that they represent substance P-immunoreactive cells. Unfortanately, secretory granules of several cell types showed about 20% more shrinkage in 0.5% glutaraldehyde fixed tissue, than in osmicated tissue.     

Ontogeny of the endocrine pancreas in sea bass (Dicentrarchus labrax): an ultrastructural study. II. The big and secondary islets

The big and secondary islets of sea bass larvae were characterized ultrastructurally from, 25 to 60 days after hatching. From the 25th day, big islets consisted of inner type II and III, external type I and peripheral type IV cells. From the 55th day, type V cells appeared in limited peripheral areas. Secondary islets, first found in 32-day-old larvae, were made up of inner type II and III, external type I, and peripheral either type IV and V cells (type I islets), or only type V cells (type II islets). Type I cells contained secretory granules with a fine granular, low-medium electron-dense material, whereas the secretory granules of type II cells were smaller and had a high electron-dense core with diffused limits; needle and rod-like crystalloid contents were occasionally found. Type III secretory granules posessed a homogeneous, high or medium electron-dense material with or without a clear halo. Type IV cells had secretory granules with a polygonal dense core embedded in a granular matrix and granules containing a high or medium electron-dense material. Type V cells had secretory granules with a fine granular, high or medium electron-dense content. These cell-types correlated with cells previously identified immuno-cytochemically, as regards to their distribution in the islets, and related to those characterized ultrastructurally in adult specimens. Thus, types I, II, III, IV and V correspond to D₁, B, D₂, A and PP cells, respectively. From the 32nd day onwards, endocrine cells of all the different types were found grouped, type V cells also being observed in isolation close to pancreatic ducts and/or blood vessels. Small groups consisting of type I and II cells were found in 40-day-old larvae. A mitotic centroacinar ductular cell containing some secretory granules similar to those of type I cells, was seen adjacent to a type I cell. As the larvae grew older, the endoplasmic reticulum developed, the number of free ribosomes decreased, and the number and size of the secretory granules increased. Dark type I, II, III, IV and V cells were found in the islets and cell clusters from the 55th day onwards.     

Electron-microscopic immunocytochemical study of the endocrine pancreas of sea bass (Dicentrarchus labrax)

Insulin (B)-, somatostatin 25 (SST-25) (D1)-, somatostatin 14 (SST-14) (D2)-, glucagon (A)-, and glucagon PP/PYY/NPY (PP-like)-immunoreactive cells in islets of sea bass (Dicentrarchus labrax) were characterized according to their ultrastructure and immunogold labeling. Cells labeled with antisera to bonito and salmon insulin had numerous secretory granules with a small halo and round core, and a few with wide halo and round or crystalloid core. Gold particles were found throughout the granule in tissue labeled with the former but only in the core in tissue labeled with the latter. D1 cells had large granules with a medium electron-dense content and some with a darker core. D2 had smaller medium or high electron-dense secretory granules than D1 cells, located mainly in cell periphery. Glucagon-immunoreactive cells contained some granules with a polygonal core that was heavily labeled and other granules with a round core with no or hardly any labeling. Glucagon and PP-like immunoreactivity were co-localized in secretory granules, in which the gold particles showed no different distribution with the various antisera used. PYY-immunoreactive granules were also found in nerve endings. All the pancreatic endocrine cell types showing involutive characteristics are found.     

Ontogeny of the endocrine pancreas in sea bass (Dicentrarchus labrax): an ultrastructural study. I. The primordial cord and the primitive,single and primordial islets

The primordial cord and the primitive, single and primordial islets present in the 3 earliest stages of the developing endocrine pancreas of sea bass were studied ultrastructurally. The primordial cord consisted of type I and II cells and was included in the gut. Besides these cell types, X cells were seen in the primitive islet. The single islet was made up of type I, II, III and IV cells. A correlation between these endocrine cell-types and cells previously identified immunocytochemically, was established. Type I, II, III and IV cells, correlated respectively with SST-25-, insulin-, SST-14- and glucagon-immunoreactive cells, and could be related to the D₁, B, D₂ and A cells, respectively, of older larvae and adult sea bass. Each cell type shows characteristic secretory granules from its first appearance. A progressive development of the organelles and an increase in the number and size of the secretory granules, whose ultrastructure also varied, was observed in the endocrine cells of the primordial cord and the succeeding islets. In 25-day-old larvae at the beginning of the fourth developmental stage, the primordial islet, the first ventral islet found, was close to a pancreatic duct and blood vessel, and consisted of type I and II cells whose ultrastructure was similar to that of the type I and II cells in the primordial cord. These data suggest a ductular origin for the pancreatic endocrine cells in the ventral pancreas. It is suggested that although endocrine cells undergo mitosis, their increase in number during the earliest development stages is principally due to the differentiation of surrounding cells.     

Ultrastructural characterization of 6 immunoreactive enteroendocrine cells in Barbus conchonius (Teleostei, Cyprinidae)

Using the semi-thin/ultra-thin technique six different immunoreactive endocrine cell types are ultrastructurally identified in 0.5% glutaraldehyde fixed gut of B. conchonius. In addition two of them (gastrin- and PP-immunoreactive cells) are also characterized with the immunogold method, showing that the immunoreactivity is only restricted to the secretory granules. Size distribution histograms and the average diameters of 30% (d30) of the largest granules are given, showing a gradual increase in granule size from unspecific immunoreactive cells, (d30 = 110 nm) via gastrin- (119 nm), VIP-like- (127 nm), met-enkephalin- (143 nm) and PP- (174 nm) to glucagon-immunoreactive cells (178 nm). The presence of PP- and glucagon-immunoreactivity in the same cells and the consequence for their granule size is discussed. In the distal part of the gut endocrine cells are found showing no immunoreactivity with the antisera used; their granules (d30 = 144 nm) were, although not significantly, larger then those of VIP-like-immunoreactive cells, also found in that part of the gut. It is supposed that they represent substance P-immunoreactive cells. Unfortunately, secretory granules of several cell types showed about 20% more shrinkage in 0.5% glutaraldehyde fixed tissue, than in osmicated tissue.     

Ultrastructure and immunocytochemistry of endocrine cells in the midgut of the desert locust,Schistocerca gregaria (Forskal)

Summary The endocrine cells of the midgut epithelium of the desert locust are found dispersed among the digestive cells and are similar to those of the vertebrate gut. According to their reactivity to silver impregnation techniques and the ultrastructural features of the secretory granules (shape, electron-density, size, and structure) 10 types of endocrine cell have been identified, of which seven are located in the main segment of the midgut or in the enteric caeca, and the other three seem to be present only in the ampullae through which the Malpighian tubules drain into the gut. The endocrine cells have a slender cytoplasmic process that reaches the gut lumen, a feature that supports the receptosecretory nature postulated for this cellular type in insects as well as vertebrates. Antisera directed against mammalian gastrin, CCK, insulin, pancreatic polypeptide and bombesin reacted with some of the endocrine cells. This is the first time that insulin- and bombesin-like immunoreactive cells have been described in the midgut of an insect.     

An immunocytochemical and electron-microscopical study of endocrine cells in the gut and pancreas of a stomachless teleost fish,Barbus conchonius (Cyprinidae)

Summary Four immunoreactive endocrine cell types can be distinguished in the pancreatic islets of B. conchonius: insulin-producing B cells, somatostatin-producing A₁ (= D) cells, glucagon-producing A₂ cells and pancreatic poly-peptide-producing PP cells. The principal islet of this species contains only a few PP cells, while many PP cells are present in the smaller islets. Except for the B cell all pancreatic endocrine cell types are also present in the pancreatic duct.At least six enteroendocrine cell types are present in the gut of B. conchonius: 1. a cell type (I) with small secretory granules, present throughout the intestine, and possibly involved in the regulation of gut motility; 2. a C-terminal gastrin immunoreactive cell, probably producing a caerulein-like peptide; these cells are located at the upper parts of the folds, especially in the proximal part of the intestinal bulb; 3. a met-enkephalin-immunoreactive cell, present throughout the first segment; 4. a glucagon-immunoreactive cell, which is rare in the first segment; 5. a PP-immunoreactive cell, mainly present in the first half of the first segment; 6. an immunoreactive cell, which cannot at present be specified, located in the intestinal bulb. The latter four cell types are mostly located in the basal parts of the folds, although some PP-immunoreactive cells can also be found in the upper parts.Most if not all enteroendocrine cells are of the open type. The possible functions of all enteroendocrine cell types are discussed.Abbreviations BPP bovine pancreatic polypeptide - CCK cholecystokinin - GEP gastro-entero-pancreatic - GIP gastric inhibitory peptide or glucose-dependent insulin releasing peptide - PPP pig pancreatic polypeptide - VIP vasoactive intestinal polypeptide     

On the ultrastructure of polypeptide hormone-producing cells in the gut of the ascidian,Ciona intestinalis L. and the Bivalve,Mytilus edulis L.

Summary Ultrastructural evidence has been found for the presence of polypeptide hormone-producing cells in the gut of Ciona intestinalis L. and Mytilus edulis L. which do not appear to have been described before. Due to their localization and ultrastructural characteristics, it is suggested that the cells in Mytilus edulis probably produce an insulin-like substance and that some of these cells in Ciona intestinalis may produce 5-HT (5-Hydroxytryptamine). In each species only one granulated cell type can be observed. The granules, which are electron dense and membrane bound, also show a halo. The average diameter of the granules is 100–200 nm for Ciona and 200–400 nm for Mytilus.I thank Mr. G. Bargsten, M.A., Dept. of Marine Zoology, University of Kiel, for the supply of the animals     

Ultrastructural localization of chromogranin: a potential marker for the electron microscopical recognition of endocrine cell secretory granules

Summary Using a monoclonal antibody (LK2H10) directed against human chromogranin, we have been able to localize this soluble glycoprotein to the matrix of secretory granules from a wide variety of endocrine cells. In the gut, enterochromaffin, enteroglucagon, glucose-dependent insulinotropic peptide, gastrin, and neurotensin-containing cells exhibit chromogranin immunoreactivity. In our system, chromogranin-immunoreactive material was restricted to the halo of human pancreatic glucagon-containing secretory granules within A-cells. Chromogranin immunoreactivity was also localized to secretory granules in phaeochromocytomas, gastrinomas, medullary carcinomas of the thyroid and a carotid body tumour (chemodectoma). Chromogranin is proposed as a potential marker for the ultrastructural recognition of endocrine cell secretory granules.     

Ultrastructure of endocrine cells in the stomach of two teleost fish Perca fluviatilis L. and Ameiurus nebulosus L.

Summary In the gastric mucosa of two teleost species, the perch (Perca fluviatilis) and the catfish (Ameiurus nebulosus) three endocrine cell types were found, located predominantly between the mucoid cells of the gastric mucosa. A fourth cell type is present in the gastric glands of catfish. Each cell type was defined by its characteristic secretory granules. Type-I cells were predominant in both fish. These cells contained round or oval granules with a pleomorphic core. The average diameter of granules was 400 nm for the perch and 270 nm for the catfish. Type-II cells of both species displayed small, highly osmiophilic granules about 100 nm in diameter. The secretory granules of type-III cells (260 nm in the perch and 190 nm in the catfish) were round or slightly oval in shape and were filled with a finely particulate electron-dense material. Type-IV cells of the catfish were found in the gastric glands only. Their cytoplasm was filled with homogeneous, moderately electron-dense granules averaging 340 nm in diameter. The physiological significance of these different morphological types of gastric endocrine cells requires further investigation.     

Calcitonin-like cells in the pharynx of the ascidian Styela clava

Summary A small group of granulated endocrine cells have been described in the endostylar region of the pharynx in Styela. These cells are argyrophilic and exhibit calcitonin-like immunofluorescence. Tests with antisera to other peptides all proved negative. Cells from animals exposed to elevated calcium levels showed a degree of degranulation. The possibility that these cells may represent ancestral ultimobranchial C cells is discussed.The authors are very grateful to Julia Polak and Susan Van Noorden, Royal Postgraduate Medical School, London, for the kind gift of all antisera used. Animals were collected by courtesy of Mr. David Houghton and his staff of the Admiralty Marine Trials Station, Portsmouth. This research was carried out during the tenure of SRC grant no. B/RG 82919 to one of us (M.C.T.) — The localization of polypeptide hormones in the pharynx and gut of protochordates     

Ultrastructure of endocrine pancreatic granules during pancreatic differentiation in the grass snake,Natrix natrix L. (Lepidosauria,Serpentes)

We used transmission electron microscopy to study the pancreatic main endocrine cell types in the embryos of the grass snake Natrix natrix L. with focus on the morphology of their secretory granules. The embryonic endocrine part of the pancreas in the grass snake contains four main types of cells (A, B, D, and PP), which is similar to other vertebrates. The B granules contained a moderately electron‐dense crystalline‐like core that was polygonal in shape and an electron‐dense outer zone. The A granules had a spherical electron‐dense eccentrically located core and a moderately electron‐dense outer zone. The D granules were filled with a moderately electron‐dense non‐homogeneous content. The PP granules had a spherical electron‐dense core with an electron translucent outer zone. Within the main types of granules (A, B, D, PP), different morphological subtypes were recognized that indicated their maturity, which may be related to the different content of these granules during the process of maturation. The sequence of pancreatic endocrine cell differentiation in grass snake embryos differs from that in many vertebrates. In the grass snake embryos, the B and D cells differentiated earlier than A and PP cells. The different sequence of endocrine cell differentiation in snakes and other vertebrates has been related to phylogenetic position and nutrition during early developmental stages.     

Ultrastructure of the neurohypophysis of the teleost Poecilia latipinna in relation to neural control of the adenohypophysial cells

Summary The structure of the neurohypophysis of Poecilia latipinna (green molly, sailfin molly) was studied with the electron microscope. Profile diameters of neurosecretory granules in the non-myelinated neurohypophysial nerve fibres were measured and mathematically corrected for error due to section thickness. Six different types of nerve fibres could be distinguished by statistical classification of their granules and by other ultrastructural features. One fibre-type (type B) contained granules with a mean diameter of 85 nm, and the other five types (types Ala, Alb, A2, A3 and A4) all contained granules with mean diameters greater than 100 nm. Synaptic contacts were observed between type B fibres and all the adenohypophysial cell-types, although in the case of the ACTH cells the synapses were separated from the cell membrane by a continuous double basement membrane. Type A fibres were observed to contact the cells of the proximal pars distalis and pars intermedia, but did not form synapses. However, synapses occurred between type A fibres and pituicytes, and between type A fibres and the pericapillary basement membrane in the interior of the neurohypophysis. The possible roles of the different types of nerve fibres in controlling the adenohypophysial cells are discussed in the context of evidence from other teleosts.We thank Mr. W.A. Thomson and Mr. D.I. Hollingworth for technical assistance, and Dr. D.I.C. Pearson (Department of Physics, University of Nancy, Nancy, France) for advice on mathematical analysis and computer programs. The work was carried out during the tenure of an S.R.C. Research Studentship by T.F.C.B.