Goblet Cells and Mucus Types in the Digestive Intestine and Respiratory Intestine in Bronze Corydoras (Callichthyidae: Teleostei).

The structure and histochemical properties of the intestine in bronze corydoras (Corydoras aeneus), a stomach-containing teleost, are described, with emphasis on goblet cells and mucin types. The proximal intestine displayed a normal structure for teleosts, whereas the distal intestine was wide, translucent, thin-walled, richly vascularized and constantly filled with air, suggesting an important respiratory role. Goblet cells were common throughout the entire intestine and displayed a variable, but mainly faint metachromatic colour after toluidine blue. They were moderately coloured by alcian blue at both pH 2.5 and 0.2 and displayed no colour after periodic acid followed by Schiff's solution (PAS), but a distinct purple-brown colour after high iron diamine followed by alcian blue (pH 2.5). Together, these results suggest that the mucin in the intestine goblet cells consists mainly of sulphated proteoglycans. Further, the results from the present lectin and neuraminidase tests suggest that these mucins contain much N-acetylglucoseamines and some N-acetylgalactosamines and sialic acid, but seem to lack glucose and mannose. They also contain some galactose-N-acetylgalactosamines sequences, normally hidden by sialic acid. The distinct brush border and mucus layer on the epithelial cells in the respiratory intestine may indicate some digestive roles, such as absorption of water, ions and simple carbohydrates. As sulphated proteoglycans are tough and attract much water, this mucus may play important roles in the protection against mechanical and chemical damages and in the defence against micro-organisms throughout the entire intestine, but in the respiratory intestine it may impede significantly the oxygen uptake. However, as this part of the intestine usually contains no digesta, but is completely filled with air, frequently renewed by dry air from the atmosphere, and the main function of the mucus may be to protect the respiratory epithelium against a destroying and dangerous desiccation.

Goblet cell types in intestine of tiger barb and black tetra (Cyprinidae, Characidae: Teleostei).

Histochemical properties of goblet cells in intestine of a stomach-less teleost, tiger barb (Puntius tetrazona), and a stomach-containing teleost, black tetra (Gymnocorymbus ternetzi), are described and compared. The intestine goblet cells were mostly wide in both species, but in tiger barb, some of them were markedly thinner. In black tetra, all the intestine goblet cells displayed magenta colour after PAS, whereas in the tiger barb, only the thinner goblet cells displayed such affinity. The latter cell type was coloured strongly magenta when the tissue was treated with alcian blue (pH 2.5) followed by PAS, whereas the wide goblet cells in tiger barb and all goblet cells in black tetra displayed mainly a blue colour after such treatment. Further, the goblet cells in both species were coloured cleanly blue after high iron diamine followed by alcian blue (pH 2.5). The intestine goblet cells in both species displayed a moderate affinity to WGA and concanavalin A lectins and no affinity to DBA. Most of the goblet cells displayed no affinity to PNA, but some of them in the tiger barb displayed a moderate or strong affinity to this lectin. The affinity to WGA was somewhat strengthened after pre-treatment with neuraminidase. These results suggest that tiger barb contains two types or variants of intestinal goblet cells: high numbers of wide cells filled by acidic, non-sulphated mucin and some thinner cells filled by neutral mucin. The intestine goblet cells in black tetra were filled by variable amounts of neutral and acidic mucin, but the total number of such cells is much less than in tiger barb. The present lectin and neuraminidase results suggest that the intestinal mucins in both species contain significant amounts of N-acetylglucosamine, sialic acid and glucose/mannose, but seem to lack N-acetylgalactosamine. However, some of these cells in tiger barb contain moderate to large amounts of galactose. Together, these results suggest significant species-specific features of the intestine goblet cells and mucin types in tiger barb and black tetra. In conclusion, the present results suggest that the diet and feeding habits in stomach-less teleosts compared with stomach-containing teleosts, greatly influence the number of intestine goblet cells and type of mucin in these cells.

Blood cleansing cells in head kidney and spleen in Buenos Aires tetra, Hyphessobrycon anisitsi (Eigenmann), (Characidae: Teleostei).

The general structure and cell types in kidney and spleen in Buenos Aires tetra, Hyphessobrycon anisitsi, family Characidae, are described. The capability and capacity of these organs to clean foreign ferritin from the blood stream are analysed and compared. Head kidney was mainly composed of neutrophils, macrophages, lymphocytes and other white blood cells, whereas unmatured and matured red blood cells were few in number. Spleen often contained much red pulp, that is mainly matured red blood cells between splenic cords, often with some macrophages and neutrophils in the latter. Occasionally, this pulp contained large volumes of unmatured red blood cells, particularly in the periphery of the spleen. The splenic white pulp consisted of ellipsoids composed of an inner endothelial layer covered by a thick sheet of white blood cells, which in the periphery consisted mainly of macrophages. Erythrocytes occupied nearly the entire splenic volume in some specimens, whereas up to half of this volume was filled by ellipsoid macrophages, neutrophils, lymphocytes and other white blood cells in other specimens. The macrophages and sinusoidal endothelial cells in kidney and spleen from ferritin-injected specimens were tightly packed by yellow-brown granules or Prussian blue precipitations, in tissue treated with Mallory stain or acid ferrocyanide, respectively, suggesting a large uptake of foreign ferritin. In the present tetra large amounts of white blood cells are developed in head kidney, where macrophages and sinusoidal endothelial cells play important roles in the cleansing of scavenger and foreign molecules and particles from the blood stream. The spleen seems primarily to be a site for iron recycling and production and storage of red blood cells. Sometimes, however, it was rich in macrophages, neutrophils, lymphocytes and other white blood cells, suggesting functions like blood cleansing and non-specific and specific defence in such specimens.

Carbohydrates in rod and cone light absorbing segments in the firemouth cichlid Thorichthys meeki retina.

Carbohydrates in photoreceptor segments in the retina of the firemouth cichlid Thorichthys meeki are described and compared. The present periodic acid-Schiff (PAS) and lectin results revealed the occurrence of neutral carbohydrates, composed mainly of glycosamine and mannose and glucose, in the light absorbing segments in rods and cones in this species. Unlike in mammals, the retina in this teleost seems to be poor in galactose and galactose-galactosamine units in the light absorbing segments.

Mucus cells in the digestive tract in prenatal platyfish larvae (Poeciliidae, Teleostei).

The appearance and histochemical properties of mucus cells in various parts of digestive tract in prenatal larvae of platyfish (Xiphophorus maculatus), a stomachless teleost, are described and compared. Such cells occurred regularly throughout the entire epithelial cell layer in mouth, pharynx, oesophagus and intestine in 6 mm long larvae i.e. approximately 1 week before delivery, but were very scarce in 4 mm long larvae. The mucin in these cells was strongly stained by alcian blue at pH 2.5 and weakly coloured by this dye at pH 1.0. It displayed a clean blue colour after high iron diamine followed by alcian blue (pH 2.5), except in some very few cells which displayed a purple-brown colour after this treatment. The mucus cells displayed various colours between purple-blue and magenta when the alcian blue (pH 2.5) was followed by periodic acid-Schiff (PAS) solution; the magenta-coloured cells were mainly confined to oesophagus. Together these findings suggest that the mucin in the digestive tract of prenatal platyfish consists mainly of non-sulphated, carboxylated glycoconjugates. The observations from the present lectin histochemistry suggest that the amount of galactosamine and glucosamine in this mucin varies much from cell to cell. Also, the various colours after PAS suggest a significant cellular heterogeneity in the mucus cell population. This may reflect different stages in development and maturation or a true and permanent cellular heterogeneity, suggesting that this mucus is able to play a number of important roles even a week before the larvae start to feed and digest.

Histochemical study on the intestine goblet cells in cichlid and poecilid species (Teleostei).

Histochemical properties of intestine goblet cells in firemouth cichlid, zebra mbuna, freshwater angelfish and platyfish are described. Goblet cells occurred regularly in the epithelial cell layer throughout the entire intestine, they were strongly coloured by alcian blue at pH 2.5. This colour got gradually weaker when the pH was reduced, but still after alcian blue at pH 0.2 these cells displayed a distinct blue colour. When the goblet cells were treated with periodic acid-Schiff (PAS), they displayed a strong purple-magenta colour. The findings that a number of goblet cells displayed various colours between blue and purple-magenta when acidic alcian blue was followed by PAS, and between blue and red-brown when acidic alcian blue was followed by neutral red, may reflect different ages or stages of development and differentiation for these cells. However, such results may also suggest a true cellular heterogeneity in the present population of goblet cells, reflecting that the intestine mucus layer has a number of roles in teleosts like lubrication, protection, immunological defence, digestion and absorption. In the ferritin injected specimens of firemouth cichlid and platyfish, a number of macrophage-like cells in intestine wall displayed Prussian blue precipitations in tissue treated with acid ferrocyanide, suggesting that these cells play a cleansing role in the intestinal wall. No ferritin uptake was seen in the intestine goblet cells and eosinophilic granule cells.

Structural and histochemical studies on the teleostean bulbus arteriosus.

The structure and histochemical properties of the bulbus arteriosus in two species from an evolutionary old teleost family, Characidae, and in three modern teleosts, family Cichlidae, are described. The bulbar wall was composed of an outer layer, a middle layer and a strongly folded inner layer covered by a thick, granule-rich endothelial cell layer towards the lumen. One of the cichlid species (Thorichthys meeki) was injected intraperitoneally with horse ferritin; the endothelial cell layer of the heart atrium and ventricle displayed high ability to endocytose ferritin particles from the blood stream, but the corresponding layer in the bulbus arteriosus displayed no such uptake. This finding suggests that the bulbar endothelial cell layer plays no scavenger or immunological blood cleansing roles in this species. The bulbar endothelial cell granules were strongly coloured by periodic acid-Schiff (PAS) in the present cichlids, but weakly coloured by PAS in the present characids. These cell layers were uncoloured by alkaline carmine in ethanol in both cichlids and characids. The negative carmine test combined with a positive PAS test for the bulbar endothelial cell layer in the present cichlids indicates that these cells contain only small amounts of polysaccharides. The weak PAS-colouring for the bulbar endothelial cell layer in characids indicates a very low content of sugars in these cells. These findings together with the fact that this cell layer in the present cichlids and characids was nearly uncoloured when treated with orcein, Heidenhain's Azan or Schmorl's solutions for elastic materials suggest that the bulbar endothelial granules do not play any role in the blood cleansing or in the rebuilding or maintenance of the ground substance or elastic material in the bulbar wall. Probably, the granules in the bulbar endothelial cell layer in the present species contain mainly proteins, connected to some PAS-positive polysaccharides to enhance their solubility.

On the structure of intestine and presence of endocytic cells in the lamina propria of platy and black tetra (Teleostei).

The structure of the intestine in platy (Xiphophorus maculatus) and black tetra (Gymnocorymbus ternetzi) and the capability of cells within the intestinal wall to endocytose intraperitoneally injected horse-spleen ferritin, are described. The intestinal epithelial layer has about the same thickness in both species, but the width of the lamina propria and tunica muscularis in black tetra was only about 1/5 of that in platy. Ferritin was taken up by numerous cells within the lamina propria throughout the entire length of the platy intestine. The uptake was demonstrated as large and strongly coloured intracellular Prussian blue granules in sections treated with acid ferrocyanide. There was no such uptake by the lamina propria in black tetras. We suggest that the high numbers of endocytic cells within the intestinal lamina propria of platies provide a local defence against foreign cells and particles. Such a functional role may to some extent compensate for the lack of an HCl-based defence in the digestive system of this stomach-less species.

Histological studies on the lymphoid organs in prenatal larvae of platy, Xiphophorus maculatus L (Poeciliidae: Teleostei).

The thymus was very large and tightly packed by lymphocytes in prenatal larvae of platy, Xiphophorus maculatus L., whereas the lymphoid tissues in the spleen and trunk kidney were poorly developed at these stages. We therefore suggest that the specific cellular defence is developed much earlier than the corresponding humoral defence mechanisms in this species.

Endocytosis of ferritin and hemoglobin by the trabecular endocardium in swordtail, Xiphophorus helleri L. and platy, Xiphophorus maculatus L. (Poecilidae: Teleostei).

The cardiac atrium and ventricle of swordtail, Xiphophorus helleri L. and platy, Xiphophorus maculatus L., are spongious, consisting of muscle trabeculae covered by endocardial cells. The cardiac trabecular endocardium is able to take up and store large amounts of horse-spleen ferritin and bovine hemoglobin from the blood stream. No such uptake was registered in endocardial cells lining the cardiac valves, atrio-ventricular junction and ventriculo-bulbar junction. The trabecular endocardium in these species seems to be unable to accumulate latex beads or bovine myoglobin, cytochrome C and holotransferrin from the blood stream. It is proposed that the trabecular endocardium in these species is able to clear the blood stream of some types of waste macromolecules; i. e. this tissue may have a scavenger function. The present results indicate that the uptake of foreign ferritin in bony fish endocardium can be clearly demonstrated at the light microscopic level in deparaffined sections by means of acid ferrocyanide or Mallory solutions. A similar uptake of hemoglobin is demonstrated by means of Mallory stain.

Dissociation of muscle cells from the bony fish heart.

A method for dissociating myocardial cells from the bony fish heart is presented. Beating hearts of Pleuronectes platessa were perfused by an aerated medium containing 0.1% collagenase and 0.1% hyaluronidase. After 60 min, the hearts were quite soft and easy to dissociate. The cardiac muscle cells were then collected on Nucleopore membranes, and their surface structure was revealed by scanning electron microscopy.

Endocytosis of horse-spleen ferritin by bony fish endocardium.

The endocytic uptake of horse-spleen ferritin by the endocardial cells in 2 bony-fish species, Xiphophorus helleri and Pollachius virens, is described. In specimens of X. helleri injected intraperitoneally by a ferritin solution 1/10 h before the sacrifice, the endocardial bristle-coated vesicles, cytoplasmic tubules, and endosomes (smooth vesicles of variable size) contain a number of ferritin particles. These particles are taken up by the bristle-coated vesicles, transferred through the cytoplasmic tubules, and emptied into the endosomes. The latter get more tightly packed by ferritin and increase in size with the time elapsed between the injection and sacrifice. After 9 h, most ferritin-packed endosomes (4 to 6 micron) contain fragments of those inclusion bodies (0.5 to 1.5 micron) which normally occur in the teleostean endocardium, and are therefore regarded as lysosomes. The ferritin-rich lysosomes increase greatly in size with time and display a width of 6 to 12 micron after 28 h. The number of cytoplasmic tubules declines rapidly with time, whereas there is a constant production of new endosomes, which probably are derived from the former. The ferritin particles are not accumulated in the endothelium of the bulbus arteriosus, whereas the hepatic endothelial cells take up some small amounts of ferritin. A similar uptake of ferritin as described above was also observed in the heart of P. virens when perfused by a ferritin solution for 3 h. The present results are discussed and compared with those previously reported for the uptake of ferritin in various tissues in fishes and mammals.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural and ultrahistochemical studies on the ventral aorta in larvae of a teleost, Poecilia reticulata.

The ultrastructure of the ventral aorta of Poecilia reticulata is described in larvae of variable age. In postnatal larvae this structure is located within the ventral pharyngeal wall. The tunica media consists of elongated smooth muscle cells, surrounded by a network of elastic fibrils (10 to 20 nm). The muscle cells are rich in free and membrane associated ribosomes, and contain a well developed Golgi apparatus surrounded by membrane-bound, electron dense bodies. During the first 21 d of postnatal life, the number of ribosomes declines gradually, whereas there is a corresponding increase in the amount of contractile material. In addition, the number of intercellular, elastic fibrils seems to increase with advancing age. No elastic lamina was seen in the present tissue, but in the 21 d old larvae the subendothelial elastic fibrils are sometimes aligned and may therefore make up an elastic lamina at a later stage of development. Probably, the elastic fibrils are composed of a central string (2-4 nm) of unknown chemical nature, covered by a layer of elastin. Thus, the elastic fibrillar network in young and adult bony-fish may be regarded as heavily reticulose elastin laminae, resembling those of the early fetal aorta in mammals. The present intercellular elastin is shown to be intensely stained with phosphotungstic acid at low pH and may therefore be rich in basic proteins. In 3 mm prenatal larvae the heart and the posterior part of the aorta are located at the outside of the body, whereas the anterior part occurs in the ventral pharyngeal wall. The aortic endothelium and tunica media consist of undifferentiated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Fine structure and endocytic properties of subendothelial macrophages in the bulbus arterious of two bony fish species.

The ultrastructure and endocytic properties of the subendothelial macrophages in the bulbus arteriosus of 2 teleosts, Cichlasoma severum and Xiphophorus helleri, are described. These cells show a diameter of 6 to 10 microns and contain a number of membrane-bound inclusion bodies, which vary greatly in size, shape, and electron density. Frequently, these bodies display myelin figures. Further, in the peripheral part of the cell there regularly occur bristle-coated vesicles. In specimens of X. helleri, injected intraperitoneally by a ferritin solution 20 to 400 h before the sacrifice, the subendothelial macrophages in the bulbus arteriosus contain large, ferritin-packed lysosomes. Similar inclusions were also seen in monocytes in the bulbar lumen. In the bulbus arteriosus of C. severum, there regularly occur mast cells beneath the endothelial lining, whereas the bulbar tissue in X. helleri lacks such cells. The present results are discussed and compared with those reported previously on the fine structure of free macrophages in mammals.

Ultrastructural and ultrahistochemical studies of post-mortem changes and effects of hypoxia in the bony fish heart.

The ultrastructural and ultrahistochemical properties of post-mortem changed and hypoxic heart tissue of Gadus virens, Gadus morhua, and Poecilia reticulata are described. When incubated for 0.5 h at 20 degrees C the cardiac tissue is rich in vacuoles and endocardial blebs. After 1 h of incubation, the myocardial mitochondria contain some amorphous, flocculent densities (50-100 nm), which seem to increase in number and size with advancing incubation time. Furthermore, the mitochondria in heart perfused by a calcium-containing solution for 7 h at hypoxic conditions contain large numbers of highly electron dense annular-granules (35-50 nm). Numerous myocardial mitochondria exclude tannic acid in tissue incubated or perfused for up to 1.5 h, whereas all mitochondria seem permeated by this substance when incubated for 5 h or perfused at hypoxic condition for 7 h. The amount of myocardial glycogen in P. reticulata is greatly reduced in the hypoxic heart compared with the normal heart. The mitochondria and contractile material, however, seem to tolerate oxygen depletion remarkably well. The present results are compared with those reported previously for the post-mortem changes and hypoxic hearts in fishes and mammals.

Ultrastructure of the parietal pericardium in teleosts.

The ultrastructure of the parietal pericardium in Melanogrammus aeglefinus, Paracheirodon innesi, and Poecilia reticulata is described in larvae of variable age. This structure is composed of a mesothelial layer facing the pericardial cavity and an outer layer of collagen tissue. The latter is continuous with the surrounding tissue, i.e. mainly skeletal muscles. Generally, the parietal pericardium is attached to loose connective tissue dorsally, whereas its ventral and lateral parts are connected to muscles and tendons. Posteriorly, it is fused with the peritoneum. In prenatal larvae of P. reticulata the parietal pericardium is in contact with yolk material. The parietal mesothelial cells are attenuated and contain numerous ribosomes, a well developed Golgi apparatus, and a number of membrane-bound inclusion bodies (0.1-0.4 micron) of moderate electron density. In addition, numerous pinocytotic vesicles (80-100 nm) occur in differentiated mesothelial cells, whereas they are scarce in undifferentiated cells. Furthermore, bristle-coated vesicles are very rare in differentiated mesothelial cells, whereas they are seen regularly in undifferentiated cells. The collagen fibre bundles of the parietal pericardium are often orientated perpendicularly to each other, and in postnatal larvae of P. reticulata a one cell thick layer of melanophores occurs within the fibrous tissue. These cells contain numerous membrane-bound, highly electron-dense inclusion bodies (0.3-0.6 micron). They are occasionally seen in Paracheirodon innesi also, whereas they are absent in 2 and 6 days old larvae of M. aeglefinus and prenatal larvae of P. reticulata. Macrophages, capillaries, and nerve bundles occur in the pericardial and subpericardial connective tissue. The present results are discussed and compared with those previously reported for mammalian parietal pericardium.

Ultrastructure of bony-fish heart tissue treated with beta-glycerophosphate/lead citrate or adenosine triphosphate/lead nitrate.

The ultrastructure of bony-fish heart tissue is described when subjected to beta-glycerophosphate/lead citrate (9.2 less than or equal to pH less than or equal to 9.4) or adenosine triphosphate/lead nitrate (pH = 7.3). When treated in the former way, electron dense precipitates occur in the endocardial and myocardial endoplasmic/sarcoplasmic reticulum, Golgi apparatus, and nuclear intermembranous space. Such precipitates are absent in the control tissue and reflect therefore probably alkaline phosphatase activity. In tissue treated with adenosine triphosphate/lead nitrate the precipitates are irregularly distributed, and may also occur in the control tissue. In the present material, they therefore seem not to reflect adenosine triphosphatase activity. The results are compared and discussed with those revealed in the present study for the bony-fish intestine and with those previously reported for various mammalian tissues.

Calcium in myocardial tissue and isolated mitochondria from a teleost.

A method is presented for isolating cardiac mitochondria from bony-fish. Calcium levels in ventricular whole tissue and isolated mitochondria of Gadus virens L. are determined by atomic absorption flame spectroscopy, and were found to be about 8 and 16 nmolCa/mg prot., respectively. In conclusion, the calcium concentration within the myocardial mitochondria in this species may be nearly three times higher than at the outside, and probably these structures serve as a "calcium sink". The results are compared with those previously reported for mammals.