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.     

Development of endocrine pancreatic islets in embryos of the grass snake Natrix natrix (Lepidosauria,Serpentes)

Differentiation of the pancreatic islets in grass snake Natrix natrix embryos, was analyzed using light, transmission electron microscopy, and immuno-gold labeling. The study focuses on the origin of islets, mode of islet formation, and cell arrangement within islets. Two waves of pancreatic islet formation in grass snake embryos were described. The first wave begins just after egg laying when precursors of endocrine cells located within large cell agglomerates in the dorsal pancreatic bud differentiate. The large cell agglomerates were divided by mesenchymal cells thus forming the first islets. This mode of islet formation is described as fission. During the second wave of pancreatic islet formation which is related to the formation of the duct mantle, we observed four phases of islet formation: (a) differentiation of individual endocrine cells from the progenitor layer of duct walls (budding) and their incomplete delamination; (b) formation of two types of small groups of endocrine cells (A/D and B) in the wall of pancreatic ducts; (c) joining groups of cells emerging from neighboring ducts (fusion) and rearrangement of cells within islets; (d) differentiated pancreatic islets with characteristic arrangement of endocrine cells. Mature pancreatic islets of the grass snake contained mainly A endocrine cells. Single B and D or PP–cells were present at the periphery of the islets. This arrangement of endocrine cells within pancreatic islets of the grass snake differs from that reported from most others vertebrate species. Endocrine cells in the pancreas of grass snake embryos were also present in the walls of intralobular and intercalated ducts. At hatching, some endocrine cells were in contact with the lumen of the pancreatic ducts.     

Development of the duct system during exocrine pancreas differentiation in the grass snake Natrix natrix (Lepidosauria,Serpentes)

We analyzed the development of the pancreatic ducts in grass snake Natrix natrix L. embryos with special focus on the three‐dimensional (3D)‐structure of the duct network, ultrastructural differentiation of ducts with attention to cell types and lumen formation. Our results indicated that the system of ducts in the embryonic pancreas of the grass snake can be divided into extralobular, intralobular, and intercalated ducts, similarly as in other vertebrate species. However, the pattern of branching was different from that in other vertebrates, which was related to the specific topography of the snake's internal organs. The process of duct remodeling in Natrix embryos began when the duct walls started to change from multilayered to single‐layered and ended together with tube formation. It began in the dorsal pancreatic bud and proceeded toward the caudal direction. The lumen of pancreatic ducts differentiated by cavitation because a population of centrally located cells was cleared through cell death resembling anoikis. During embryonic development in the pancreatic duct walls of the grass snake four types of cells were present, that is, principal, endocrine, goblet, and basal cells, which is different from other vertebrate species. The principal cells were electron‐dense, contained indented nuclei with abundant heterochromatin, microvilli and cilia, and were connected by interdigitations of lateral membranes and junctional complexes. The endocrine cells were electron‐translucent and some of them included endocrine granules. The goblet cells were filled with large granules with nonhomogeneous, moderately electron‐dense material. The basal cells were small, electron‐dense, and did not reach the duct lumen.     

Distribution of Na+,K+-ATPase in rat exocrine pancreas as monitored by K+-NPPase cytochemistry and [3H]-ouabain binding: a plasma membrane protein found primarily to be ductal cell associated

We investigated the distribution of Na+,K+-ATPase in rat exocrine pancreas. By use of enzymatic dissociation techniques, pancreatic acini (containing acinar cells and centroacinar ductal cells in a ratio of about 10:1) and all major classes of pancreatic ducts were isolated and analyzed for the presence of Na+,K+-ATPase using K+-NPPase cytochemistry and [3H]-ouabain binding assays. Ultrastructural analysis demonstrated a basolateral localization of ouabain-sensitive enzyme activity in all classes of pancreatic ducts, although the degree of activity varied among the various classes. Qualitative analysis (scale of 0 to + + +) indicated the following enzyme distribution: centroacinar ductal cells (+); intralobular ducts (+ +); interlobular ducts (+ + +); main duct (+ +). In contrast, no reaction product was associated with pancreatic acinar cells even when observed adjacent to enzyme-positive centroacinar ductal cells. Parallel experiments monitoring [3H]-ouabain binding supported the cytochemical studies. When expressed as femtomoles [3H]-ouabain/microgram DNA, the following values were obtained: whole pancreas, 100.3; ducts (pooled intralobular and interlobular), 337.0; acini, 48.2. The acinar value is complicated by the fact that acini contain both acinar and centroacinar cells, but in light of the cytochemical observations we suggest that most of the [3H]-ouabain binding is due to the few ductal cells present in acini. The results suggest that Na+,K+-ATPase is primarily associated with the ductal epithelium of the exocrine pancreas and is differentially distributed among the different classes of ducts.     

Immunocytochemical localization of elastase 1 in human pancreas

By light and electron microscopic immunocytochemistry the distribution is described of human pancreatic elastase 1 (E1) during ontogenesis, in adults, in cases of acute and chronic panceatitis, acute pancreatic ischaemia as well as pancreatic tumours. E1-positive cells were first detected in ductal sprouts in the 14th gestational week. Complete acini expressing E1 could be found from the 17th to the 20th week of gestation onwards. Scattered distinct E1-positive epithelia could be found in the ducts of fetal and adult pancreas. By immunoelectron microscopy, E1 was localized in rough endoplasmic reticulum, condensing vacuoles, zymogen granules of acinar epithelia and in acinar lumina. E1 appeared to be distributed homogeneously in zymogen granules. As specific markers of acinar cells, both monoclonal antibodies under study identified heterotopic pancreatic acini in peribiliar glands of the liver and also helped to visualize different damage patterns in pancreatitis. The acinar epithelia surrounding acute lipolytic necroses initially reacted more intensely with the E1-antibodies than undamaged pancreatic tissue. In acute ischaemia, acinar cells which are dissociated from intercalated ducts lost their immunocytochemical reactivity for E1. Pancreatic parenchyma involved in advanced acute pancreatitis as well as in chronic inflammation was detected only weakly by both E1-antibodies. However, atrophic lobules in post-inflammatory scars were stained more intensely by the E1-antibodies than normal parenchyma. Pancreatic tumours (adenomas, adenocarcinomas, solid-cystic tumours and islet cell tumours) were not labelled by these antibodies.     

Concanavalin a binding and cytodifferentiation in pancreatic acinar carcinoma of rat

The binding of concanavalin A to the plasmalemma of acinar carcinoma cells was characterized by electron microscopy utilizing horseradish peroxidase. Heavy labeling due to specific concanavalin A binding was detected on the plasmalemma of undifferentiated carcinoma cells lacking zymogen maturation, neoplastic cells of intermediate differentiation with only occasional zymogen granules, and highly differentiated acinar carcinoma cells containing numerous cytoplasmic zymogen granules. The plasmalemma of acinar carcinoma cells was also compared to the normal pancreatic acinar cell plasmalemma by measurement of specific ¹²⁵I-labeled concanavalin A binding. Although only about one-third of pancreatic acinar carcinoma cells demonstrate mature zymogen differentiation, the acinar carcinoma had a full complement of normal plasmalemma receptors for ¹²⁵I-labeled concanavalin A. It is concluded that, unlike normal pancreas, the presence of concanavalin A receptors on the plasmalemma of acinar carcinoma cells is not a specific membrane marker for differentiated cells containing zymogen granules.     

Effect of 5-bromodeoxyuridine on appearance of cell-surface saccharides in organ cultures of embryonic pancreas

Rat pancreatic rudiments from Day 15 embryos cultured for 11 days with 20 μM 5-bromodeoxyuridine (BrdU) show selective suppression of acinar cell cytodifferentiation with production of fluid-filled vacuoles lined by undifferentiated cells. Using a battery of lectin-ferritin conjugates (Con A, RCA I, WGA, SBA, and Ulex lectin) we have shown that the majority of these cells express cell-surface glycoconjugate patterns reminiscent of both protodifferentiated cells from Day 15 rudiments and of adult centroacinar (i.e., duct-like) cells. A smaller population of the undifferentiated cells which contain abundant elements of the rough-surfaced endoplasmic reticulum expresses surface saccharide patterns equivalent to those of acinar cells in Day 19 rudiments. These cells, however, lack zymogen granules characteristic of acinar cells in Day 19 rudiments and are similar morphologically to presumptive acinar cells in Day 17 rudiments. Culture of Day 14 pancreatic rudiments with BrdU leads to growth only of undifferentiated cells with duct-like cell-surface saccharide patterns. We interpret these results to indicate that (1) the differentiation program for the acinar cell plasmalemma is established earlier than that for its intracellular organelles; (2) these two developmental programs are independently regulated; and (3) the progenitor of the acinar cell in the protodifferentiated rudiment may be related to the centroacinar or duct-like cell.     

Postnatal development of the pancreas in the opossum. Light microscopy.

The pancreas of the newborn opossum consists of a central region of forming islets surrounded by primitive tubules that end in proacinar cells. Paratubular buds, which are outgrowths from the tubular epithelium, characterize the newborn pancreas and eventually give rise to both exocrine and endocrine units. 4 days after birth, definite intralobular ducts, acini and centroacinar cells are observed. In addition to the central expanding islets (primary islets), endocrine cells are observed singly or in small groups in the ductal epithelium. The endocrine cells are believed to originate from the terminal cells of the ductal epithelium and, throughout the entire postnatal period, retain a close association with the exocrine epithelium. With the simultaneous proliferation of both endocrine and exocrine components from the ductal system, the majority of the islets observed at 24 days (5.0 cm) appear to be surrounded by a single layer of acinar cells. As acini develop and the ducts expand toward the periphery, this layer of acinar cells separates from the developing islets, the majority of which have become localized within the centers of lobules to form the secondary islets by the 10.0-cm stage (59 days). A marked development of lobules is observed by the 13.0-cm stage and the majority of acinar cells now are filled with zymogen granules. Acinar cells continue to proliferate late into the postnatal period and the majority of acini exhibit a tubular form in the juvenile and adult opossum.     

Ultrastructural changes in acinar cells of hamster pancreas in chemically induced carcinogenesis

Ultrastructural changes arising in the pancreas of the Syrian golden hamster after treatment with N-nitrosobis (2-oxopropyl) amine (BOP) were studied at short intervals. Alterations were found in acinar cells i.e. loss of zymogen granules, dilatation of granular endoplasmic reticulum, depolarization, irregular nucleus and separation of lateral surfaces (intermembranary spaces). As a result, the compact morphology of normal acini switched towards a new structure resembling a pseudo-ductule. Such alterations occurred from the 3rd month and preceded tumor formation. It is noteworthy that ducts and islets of Langerhans appeared unaltered in all instances. These results are consistent with the hypothesis that BOP induced pancreatic adenocarcinoma in hamsters originates in the acinar cell.     

FINE STRUCTURAL CHANGES IN RESPONSE TO HORMONAL STIMULATION OF THE PERFUSED CANINE PANCREAS

The dog pancreas isolated in situ was perfused with oxygenated dog blood and stimulated with pancreozymin, secretin, or both. There were no significant changes in the fine structure of the acinar, centroacinar, or duct cells attributable to the perfusion. Combined glutaraldehyde and osmium fixation gave good preservation of the secretory products of the acinar cell. Before stimulation, the lumen of the acini is filled with material similar in texture to the content of the zymogen granules, but of somewhat lower density. Release of secretion commonly takes place by coalescence of the limiting membrane of zymogen granules with the plasmalemma, but one granule opening at the surface may frequently be joined by others coalescing with its membrane and forming an interconnected series all with contents having the same texture as the released zymogen. Such a mechanism seems to permit a more rapid release of secretory product than discharge of individual granules. Pancreozymin stimulation caused marked depletion of zymogen granules, but no obvious changes in the Golgi apparatus. It is clear, therefore, that this hormone exerts its effect upon release of granules rather than upon their formation. Secretin stimulation of water and bicarbonate secretion caused a marked washing out of the luminal contents, but had little detectable effect on cellular structure.     

Role of vesicular nucleotide transporter VNUT (SLC17A9) in release of ATP from AR42J cells and mouse pancreatic acinar cells

ATP is released from cells in response to various stimuli. Our previous studies on pancreas indicated that pancreatic acini could be major stores of secreted ATP. In the present study, our aim was to establish the role of the vesicular nucleotide transporter (VNUT), SLC17A9, in storage and release of ATP. Freshly prepared acini from mice and AR42J rat acinar cells were used in this study. We illustrate that in AR42J cells, quinacrine (an ATP store marker) and Bodipy ATP (a fluorescent ATP analog) co-localized with VNUT-mCherry to vesicles/granules. Furthermore, in acini and AR42J cells, a marker of the zymogen granule membranes, Rab3D, and VNUT co-localized. Dexamethasone treatment of AR42J cells promoted formation of acinar structures, paralleled by increased amylase and VNUT expression, and increased ATP release in response to cholinergic stimulation. Mechanical stimulus (pressure) and cell swelling also induced ATP release, but this was not influenced by dexamethasone, most likely indicating different non-zymogen-related release mechanism. In conclusion, we propose that VNUT-dependent ATP release pathway is associated with agonist-induced secretion process and downstream purinergic signalling in pancreatic ducts.     

Hollowing or cavitation during follicular lumen formation in the differentiating thyroid of grass snake Natrix natrix L. (Lepidosauria,Serpentes) embryos? An ultrastructural study

The mechanism of follicular lumen differentiation during thyroid gland morphogenesis in vertebrate classes is still unclear and the current knowledge regarding the origin and the mechanism of follicular lumen formation during thyroid differentiation in reptiles is especially poor. The present study reports on an ultrastructural investigation of thyroid follicle formation and follicular lumen differentiation in grass snake (Natrix natrix L.) embryos. The results of this study show that the earliest morphogenesis of the presumptive thyroid follicles in grass snake embryos appears to be similar to that described in embryos of other vertebrate classes; however, differences appeared during the later stages of its differentiation when the follicular lumen was formed. The follicular lumen in grass snake embryos was differentiated by cavitation; during thyroid follicle formation, a population of centrally located cells was cleared through apoptosis to form the lumen. This manner of follicular lumen differentiation indicates that it has an extracellular origin. It cannot be excluded that other types of programmed cell death also occur during follicular lumen formation in this snake species.     

Pancreatic atrophy in copper-deficient rats: Histochemical and ultrastructural evidence of a selective effect on acinar cells

Summary Copper deficiency had a differential effect between tissues in the rat pancreas. There was marked loss and atrophy of acinar cells, in which both hypertrophied and degenerating mitochondria were present. Cytochrome oxidase activity in acini was greatly depleted while monoamine oxidase activity was enhanced. Atrophy of acinar cells was accompanied by extensive degeneration of the rough endoplasmic reticulum, and by a failure of zymogen granule synthesis. These changes contrasted strongly with the appearance of non-acinar tissues, in which hypertrophy and degeneration of mitochondria were rarely observed. Islet tissue, pancreatic ducts and blood vessels showed no atrophic changes. Cytochrome oxidase activity in islet tissue, and in the epithelium of the main pancreatic ducts, appeared unaffected.     

Rab27A Is Present in Mouse Pancreatic Acinar Cells and Is Required for Digestive Enzyme Secretion

The small G-protein Rab27A has been shown to regulate the intracellular trafficking of secretory granules in various cell types. However, the presence, subcellular localization and functional impact of Rab27A on digestive enzyme secretion by mouse pancreatic acinar cells are poorly understood. Ashen mice, which lack the expression of Rab27A due to a spontaneous mutation, were used to investigate the function of Rab27A in pancreatic acinar cells. Isolated pancreatic acini were prepared from wild-type or ashen mouse pancreas by collagenase digestion, and CCK- or carbachol-induced amylase secretion was measured. Secretion occurring through the major-regulated secretory pathway, which is characterized by zymogen granules secretion, was visualized by Dextran-Texas Red labeling of exocytotic granules. The minor-regulated secretory pathway, which operates through the endosomal/lysosomal pathway, was characterized by luminal cell surface labeling of lysosomal associated membrane protein 1 (LAMP1). Compared to wild-type, expression of Rab27B was slightly increased in ashen mouse acini, while Rab3D and digestive enzymes (amylase, lipase, chymotrypsin and elastase) were not affected. Localization of Rab27B, Rab3D and amylase by immunofluorescence was similar in both wild-type and ashen acinar cells. The GTP-bound states of Rab27B and Rab3D in wild-type and ashen mouse acini also remained similar in amount. In contrast, acini from ashen mice showed decreased amylase release induced by CCK- or carbachol. Rab27A deficiency reduced the apical cell surface labeling of LAMP1, but did not affect that of Dextran-Texas Red incorporation into the fusion pockets at luminal surface. These results show that Rab27A is present in mouse pancreatic acinar cells and mainly regulates secretion through the minor-regulated pathway.     

Selective fluorescence of zymogen granules of pancreatic acinar cells stained with hematoxylin and eosin

Following staining with hematoxylin and eosin Y, paraffin sections of mouse pancreas were examined by transmitted light, epifluorescence and confocal laser scanning microscopy. Light microscopy revealed that the nuclei of pancreatic acinar cells were located basally, while the apices of the cells appeared eosinophilic, although the secretory granules were difficult to visualize. Under violet-blue light excitation, the zymogen granules at the apices of the acinar cells showed strong yellowish fluorescence; the other part of the cytoplasm was only faintly fluorescent and the nuclei and the supporting tissues were nonfluorescent. Confocal laser scanning microscopy resulted in clear pictures of the zymogen granules and their distribution within the cell. The fluorescent emission of zymogen granules was certainly the result of eosin Y staining, because hematoxylin is not a fluorochrome and the zymogen granules are not autofluorescent. Staining with eosin Y alone, however, did not result in clear fluorescent images of zymogen granules or any other cellular structures. Our observation shows that the fluorescence emission of eosin Y allows easy and precise recognition of zymogen granules of pancreatic cells.     

Selective fluorescence of zymogen granules of pancreatic acinar cells stained with hematoxylin and eosin

Following staining with hematoxylin and eosin Y, paraffin sections of mouse pancreas were examined by transmitted light, epifluorescence and confocal laser scanning microscopy. Light microscopy revealed that the nuclei of pancreatic acinar cells were located basally, while the apices of the cells appeared eosinophilic, although the secretory granules were difficult to visualize. Under violet-blue light excitation, the zymogen granules at the apices of the acinar cells showed strong yellowish fluorescence; the other part of the cytoplasm was only faintly fluorescent and the nuclei and the supporting tissues were nonfluorescent. Confocal laser scanning microscopy resulted in clear pictures of the zymogen granules and their distribution within the cell. The fluorescent emission of zymogen granules was certainly the result of eosin Y staining, because hematoxylin is not a fluorochrome and the zymogen granules are not autofluorescent. Staining with eosin Y alone, however, did not result in clear fluorescent images of zymogen granules or any other cellular structures. Our observation shows that the fluorescence emission of eosin Y allows easy and precise recognition of zymogen granules of pancreatic cells.     

Selective fluorescence of zymogen granules of pancreatic acinar cells stained with hematoxylin and eosin.

Following staining with hematoxylin and eosin Y, paraffin sections of mouse pancreas were examined by transmitted light, epifluorescence and confocal laser scanning microscopy. Light microscopy revealed that the nuclei of pancreatic acinar cells were located basally, while the apices of the cells appeared eosinophilic, although the secretory granules were difficult to visualize. Under violet-blue light excitation, the zymogen granules at the apices of the acinar cells showed strong yellowish fluorescence; the other part of the cytoplasm was only faintly fluorescent and the nuclei and the supporting tissues were nonfluorescent. Confocal laser scanning microscopy resulted in clear pictures of the zymogen granules and their distribution within the cell. The fluorescent emission of zymogen granules was certainly the result of eosin Y staining, because hematoxylin is not a fluorochrome and the zymogen granules are not autofluorescent. Staining with eosin Y alone, however, did not result in clear fluorescent images of zymogen granules or any other cellular structures. Our observation shows that the fluorescence emission of eosin Y allows easy and precise recognition of zymogen granules of pancreatic cells.     

Absence of the major zymogen granule membrane protein, GP2, does not affect pancreatic morphology or secretion

The majority of digestive enzymes in humans are produced in the pancreas where they are stored in zymogen granules before secretion into the intestine. GP2 is the major membrane protein present in zymogen granules of the exocrine pancreas. Numerous studies have shown that GP2 binds digestive enzymes such as amylase, thereby supporting a role in protein sorting to the zymogen granule. Other studies have suggested that GP2 is important in the formation of zymogen granules. A knock-out mouse was generated for GP2 to study the impact of the protein on pancreatic function. GP2-deficient mice displayed no gross signs of nutrient malab-sorption such as weight loss, growth retardation, or diarrhea. Zymogen granules in the GP2 knock-out mice appeared normal on electron microscopy and contained the normal complement of proteins excluding GP2. Primary cultures of pancreatic acini appropriately responded to secretagogue stimulation with the secretion of digestive enzymes. The course of experimentally induced pancreatitis was also examined in the knock-out mice because proteins known to associate with GP2 have been found to possess a protective role. When GP2 knock-out mice were subjected to two different models of pancreatitis, no major differences were detected. In conclusion, GP2 is not essential for pancreatic exocrine secretion or zymogen granule formation. It is unlikely that GP2 serves a major intracellular role within the pancreatic acinar cell and may be functionally active after it is secreted from the pancreas.     

Characterization of pancreatic-type tissue in the liver of rat induced by polychlorinated biphenyls

Pancreatic-type tissue induced in the livers of rats treated with polychlorinated biphenyls was characterized by transmission electron microscopy and high-resolution immunocytochemistry. The cells of pancreatic-type tissue were arranged as acini and in small groups. By electron microscopy the pancreatic-type tissue showed features very similar to normal pancreatic acinar tissue, such as well developed rough endoplasmic reticulum (RER), large numbers of mature zymogen granules, and a basally located nucleus. Protein A-gold imunocytochemical technique showed localization of amylase and trypsinogen over the zymogen granules and RER. These findings confirm that this tissue in the liver is morphologically and functionally identical to pancreatic acinar tissue.