Autoradiographic Investigations on Cell Proliferation in the Digestive Mucosa of Fishes

The labelling index (TLI) of the digestive mucosa of some fish species was determined following a pulse labelling with tritiated thymidine ([³H]TdR) and light microscopic autoradiography. In the oesophageal epithelium, proliferation was observed to occur in non mucus-secreting cells. In the intestine, both undifferentiated and absorptive cells incorporated [³H)TdR within 1 h after injection. Statistically significant differences in [³H]TdR incorporation were observed between the upper intestine region and both the middle and lower parts on the one hand, and between the middle and lower parts on the other hand. Mucus-secreting cells seemed unable to proliferate. In the stomach, significantly fewer labelled nuclei were counted; they were located in the isthmus epithelium. No significant difference was observed between the TLI of these regions in the different species.     

Intestinal epithelium in Anarhichas lupus L., with emphasis on cell renewal

Histomorphological studies on the dynamics of intestinal epithelium in larvae, juveniles and adult stages of common wolffish Anarhichas lupus showed that the major structural development took place during the first 8 weeks after hatching ( c . 320 day degrees). This development preceeded a period of increase in relative growth. The mucosa of the proximal and distal intestine gradually developed from simple primary mucosal folds into a complex structure of primary and secondary folds with regularly distributed crypts. Cell proliferation, based on the observations of the presence of immature cells, mitotic figures and proliferating cell nuclear antigen reacting cells, was limited to the crypts after 8 weeks of age. The observation of bands of epithelial cells showing apoptotic and necrotic changes, suggested the existence of extrusion zones, where effete cells were eliminated by both shedding and phagocytosis. In the proximal intestine of adult common wolffish, increasing amounts of lipids were observed from the upper part of the crypts to the surface epithelium, probably reflecting an increased maturation of epithelial cells.     

Epithelial cell migration on small intestinal villi in the neonatal rat. Comparison between [3H] thymidine and cytoplasmic labelling after Pu-citrate ingestion

This study compares, in 2-d-old rats, the migration rates of epithelial cells on villi of the small intestine, using two labelling methods: a single [3H] thymidine injection; and cytoplasmic labelling by a single ingestion of Pu-citrate. Histoautoradiography showed negligible diffusion of Pu after the initial retention, which was mostly confined to the epithelial cells of the villi. However, after sloughing of labelled cells in the intestinal lumen, Pu was reabsorbed by the distal epithelial cells. In segments in which Pu reabsorption was negligible, the migration rates of Pu- and 3H-labelled cells were very close. These rates, expressed in micrometers, were almost constant along the length of the villus, and the Pu and 3H labelling edges reached the top of the villi in about 5 and 7 d, respectively. Once Pu retention had reached its maximum in 9 equal segments cut along the small intestine, tissue counting showed an exponential Pu release of 30-40%/d from each segment until the end of the experiment at d16. This constant release might reflect a constant cell migration rate during the period from Pu ingestion until d16.     

Intestinal epithelial cell proliferation and migration in Atlantic salmon, Salmo salar L.: effects of temperature and inflammation

A 28-day feeding trial was carried out to characterise intestinal epithelial cell (IEC) turnover in Atlantic salmon (Salmo salar L.) post-smolts in seawater. Four groups of fish raised at two temperatures of 8°C or 12°C and fed two different diets were investigated. The diets included a reference maize gluten and fishmeal-based diet (FM) and an experimental enteropathy-causing diet containing 20% extracted soybean meal (SBM). IEC proliferation and migration were investigated by labelling cells with the in vivo proliferation marker 5-bromo-2′-deoxyuridine (BrdU). Proliferating cell nuclear antigen (PCNA) labelling was used as a control for identifying proliferating cells. Samples of the proximal (PI), mid (MI) and distal (DI) intestinal regions were collected at five time points (3 h–28 days) over the experimental period. Histologically, FM-fed fish had normal mucosa, whereas the SBM-fed fish developed DI enteropathy. Major zones of cell proliferation were observed in the mucosal fold bases for all intestinal regions. Over time, BrdU-labelled cells migrated up mucosal folds to the tips before being lost. Migration rates were dependent on intestinal region, temperature and diet. Highest migration rates were observed in the PI followed by the MI and DI for FM-fed fish. Diet and temperature barely affected migration in the PI and MI. Migration in the DI was most sensitive to diet and temperature, with both SBM and the higher water temperature increasing proliferation and migration rates. The slow IEC turnover in the DI might help to explain the sensitivity of this region to dietary SBM-induced enteropathy.     

Cell proliferation kinetics of the bile duct epithelium of the rat

Abstract. Cell proliferation kinetics of the extrahepatic bile duct were studied by flash and cumulative labelling methods and immunohistochemical techniques. We compared the cell kinetics of the epithelium of the intra- and extra-pancreatic bile ducts and of the bile duct of the ampulla in rats administered intraperitoneally with bromodeoxyuridine (BrdUrd). After a single injection of BrdUrd (flash labelling), labelled cells appeared in the lower portion of the downgrowths of the epithelium in the intra-and extra-pancreatic bile ducts. A gradual accumulation of the labelled cells at the surface epithelium was observed during the cumulative labelling. After cumulative labelling the labelled cells gradually decreased in number and were finally confined to the degenerative cell zone of the surface epithelium 30 days later. Similarly, after a single injection of BrdUrd, the labelled cells in the bile duct of the ampulla appeared at the lower half of the crypt from where they migrated to the upper portion during cumulative labelling. These findings indicate that epithelial cells of the bile duct are renewed at the lower portion of the downgrowths of the epithelium, or crypt, and shed from the surface epithelium or upper portion of the fold. The labelling indices reached 23.83 ± 7.47% in the intra-pancreatic bile duct, 14.74 ± 7.99% in the extra-pancreatic bile duct and 43.42 ± 4.40% in the bile duct of the ampulla at the end of 70 h cumulative labelling. The fluctuating values of the labelling index were higher in the bile duct of the ampulla than in the intra- or extra-pancreatic bile ducts. These results indicate that the bile-duct epithelium undergoes a slower renewal rate than the other parts of the gastrointestinal tract, and that the renewal time of the epithelial cells is shorter at the bile duct of the ampulla than at the intra- or extra-pancreatic bile ducts.     

Prenatal and postnatal development of the small intestine in the insectivore Suncus murinus

The development of the small intestine in the insectivore Suncus murinus was noted during the period from 21 days' gestation to 20 days after birth. At 21 days of gestation, the proximal small intestine exhibited the beginning of villus formation, whereas the distal small intestine preserved the stratified epithelium. Stratified epithelium in the distal small intestine changed into a single layer by 24 days' gestation. At 26 days' gestation, each epithelial cell was immature; but by 28 days mature-looking epithelial cells were found. The shape of the villi changed from cuboid to columnar during the same period. The connective-tissue cores of the villi began to develop at 7 days after birth in the proximal small intestine and at 15 days after birth in the distal small intestine. Crypts appeared at 15 days after birth. Endocytosis of epithelial cells took place at 28 days of gestation. In the proximal small intestine, supranuclear vesicle clusters were observed first at birth; they began to decrease both in number and size at 10 days' gestation and then disappeared completely by 20 days after birth. In the distal small intestine, large supranuclear vacuoles were observed first at 28 days of gestation. Although these vacuoles invariably were found up to 15 days after birth, they also disappeared completely by 20 days. Epithelial cells showed a structure similar to those of the adult after weaning.     

Apoptosis, cell proliferation and serotonin immunoreactivity in gut of Liza aurata from natural heavy metal polluted environments: preliminary observations

In the present paper, the effect of natural environment non-lethal heavy metal concentration on cell renewal of Liza aurata intestinal epithelium, was studied by the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling) method and anti-PCNA (proliferating cell nuclear antigen) immunohistochemistry, in order to detect, respectively, apoptosis and cell proliferation. In addition, the presence and distribution of the cell renewal regulator, serotonin, was immunohistochemically investigated. In order to reduce variability, only immature specimens were considered. The results indicated that in the control specimens from non-polluted areas, the PCNA immunoreactive nuclei of the proximal intestinal epithelium were only located at the bottom of the intestinal folds, together with a few TUNEL-positive nuclei, and goblet mucous differentiated cells. In the specimens from polluted areas, the number of PCNA immunoreactive cells was greatly enhanced, and they extended along the mid portion of the intestinal folds; the number of TUNEL-positive nuclei was enhanced as well, but they were almost exclusively detected in the third apical portion of the intestinal folds. Serotonin immunoreactive nerve elements were more frequently detected in the intestinal wall of L. aurata specimens from polluted areas, and besides that, some serotonin immunoreactive endocrine cells were also present. Variations in distribution and frequency of TUNEL-positive nuclei, PCNA immunoreactive nuclei, and serotonin immunoreactivity put in evidence an alteration of cell renewal with an enhancement of cell proliferation, probably leading to morphological intestinal fold changes.     

The influence of diet on the regional distribution of glutathione S-transferase activity in channel catfish intestine

There is evidence that glutathione conjugates are the major metabolites formed following systemic uptake of carcinogenic contaminants from the intestine. The effect of commercial diet versus a semi-purified diet on the distribution of glutathione S-transferase (GST) activity was examined in proximal, medial, and distal sections of catfish intestine. The bulk of GST activity with 1-chloro-2,4-dinitrobenzene, ethacrynic acid, and 3H-benzo[a]pyrene-4,5-oxide, and the percent cytosolic protein cross-reacting with anti-catfish GST-pi were in the more proximal segments and dropped off distally in the two diet groups. However, the total GST-pi cross-reacting protein in the proximal section was significantly higher in fish fed a chow diet. Western blot analysis revealed pi-class GST to be expressed principally in the proximal intestine. Cytosol samples cross-reacted with antibodies to human GST-alpha, -mu, and -pi, but not -theta, classes. Alpha-like GST isoforms of MW 26,200 and 24,600, absent in sections from fish fed a purified diet, were differentially expressed only in the distal section of chow-fed fish. These results indicate that diet significantly elicits regional differences in GST protein levels, that components of the commercial chow affect GST protein expression in the distal intestine, and that maintenance diet should be taken into consideration during dietary exposure studies.     

Autoradiographic and histochemical study of the intestinal epithelium of the Baltic lamprey]

The intestinal epithelium of the Baltic lamprey and its larva was studied. Glandular cells and absorbing cells simultaneously capable to synthesize polysaccharides were found histochemically in the composition of the epithelial layer. The cranial zone where glandular cells analogous to the cells of the pancreas were concentrated was revealed in the medial intestine of the larva. The rest of the intestine was devoid of specialized glandular cells. Using H3-thymidine as a precursor it was shown that in the cranial zone of the intestine the cambial cells were scattered diffusely throughout the whole epithelial layer and in other portions of the medial intestine the cambial zone was distinctly seen in the lateral portions of the intestine (the area of the fornix) where labelled nuclei were formed and mitoses took place. After injection of H3-thymidine poor incorporation of the isotope was found in the nuclei of solitary cells of some adult lampreys going to spawning. Uneven incorporation of S35-methyonine, used as a precursor, in the epithelium of the intestine and the liver of the adult lamprey was shown.     

The fine structure of the elasmobranch renal tubule: intermediate, distal, and collecting duct segments of the little skate.

Sharks, skates, and rays (Elasmobranchii) have evolved unique osmoregulatory strategies to survive in marine habitats. These adaptations include a complex renal countercurrent system for urea retention. The fine structure of the complete renal tubular epithelium has yet to be elucidated in any species of cartilagenous fish. The present study, which is a companion to our recent paper describing the ultrastructure of the neck and proximal segments of the elasmobranch nephron, uses thin sections and freeze-fracture replicas to elucidate the fine structural organization of the intermediate, distal, and collecting duct segments of the little skate, Raja erinacea, renal tubule. The epithelium of the intermediate, distal, and collecting duct segments consists of two major cell types: nonflagellar cells, the major epithelial cell type; and flagellar cells, described elsewhere. The intermediate segment consists of six subdivisions lined by cuboidal-columnar cells with variously elaborated microvilli and interdigitations of lateral and basal cell plasma membranes, as well as some subdivisions with distinctive vesicles and granules. The distal segment consists of two subdivisions, both of which are lined by a simple epithelium, and are distinguished from each other by their distinctive contents; dense bodies and granules. The collecting duct segment also has two subdividions, the first lined by a simple columnar epithelium and the second by a stratified columnar epithelium. Both subdivisions have apical secretory granules. The present findings show a more highly specialized and diverse epithelium lining the renal tubule of these cartilagenous fish than is found in either of the "adjacent" phylogenetic taxa, Agnatha or Ostheichthyes, suggesting significant differences among these groups in transepithelial transport mechanisms and renal function.     

Stereological studies on the small intestinal epithelium of the rat

Summary Quantitative macroscopic, light-microscopic and electron-microscopic studies were performed on the small intestine of fasted and non-fasted adult, male Sprague-Dawley rats. In non-fasted rats the small intestine was longer than in fasted rats. Due to the presence of villi the surface area in the duodenum and the jejunum was enlarged about six times. The microvilli on the villous crests caused a surface enlargement by 13 times in the duodenum (value corrected for overestimation due to section thickness), and 19 times in the jejunum of the fasted rats. At the base of the villi these values were about 50% lower. It was calculated that, in the fasted rats, the total enlargement of the luminal surface area — due to villi and microvilli — was 63 times in the duodenum and 81 times in the jejunum (corrected for section thickness).Differences between the villous crest epithelium and the villous base epithelium were also found with regard to the mean cell height, and the volume densities of the absorptive cell nuclei, the mitochondria, and the paracellular channels.Supported by grants from the Swedish Medical Research Council (Project No. 12X-2298), from the Swedish Group-Insurance Co. Förenade Liv, from Tore Nilson's Fund for Medical Research and from the Medical Faculty, University of Umeå     

Microautoradiographic study of cell proliferation in compensatory renal growth.

DNA synthesis in the renal parenchyma was studied by electron microscopic autoradiography 48 and 72 hours after unilateral nephrectomy in mice and weanling rats. The proportion of labelled nuclei belonging to the epithelium, endothelium, interstitial areas, or circulating cells was evaluated. Most of cells showing DNA synthesis were epithelial but many belonged to stroma. All the nephron segments were found to participate in compensatory hyperplasia, with a greater contribution, however, of the proximal convoluted tubules. DNA synthesis by the capillary endothelial cells occurred later than the peak epithelial mitotic activity. The site of DNA synthesis in nuclei was the euchromatin, the silver grains being uniformly distributed throughout the nuclear areas with condensed chromatin, and more seldom in the nuclear envelope areas or that of the perinucleolar satellites.     

An autoradiographic study of macromolecular syntheses in the epithelium of the ductus epididymidis in the mouse

Summary Glycoprotein dynamism in the mouse epididymis was studied by means of histoautoradiography after injection of l-fucose-1-³H. The label was detected, at thirty minutes p.i., in the area occupied by Golgi apparatus in the epithelial cells. At 4 h p.i. the label was already present in the lumen of ductus epididymidis. At this time interval, the luminal labelling was highest in the initial segment of the epididymis and decreased against the more distal segments considerably.At ten days p.i. very high labelling was detected in the luminal contents in the terminal segment of the ductus epididymidis and in ductus deferens, the labelling in the proximal segments of the epididymis being much lower. These observations suggested a wave of labelled glycoprotein in epididymal plasma passing through the epididymis after a fucose pulse.Higher labelling was detected in so-called clear cells than in the neighboring principal cells.Association of the labelled material was seen in epididymal and uterine spermatozoa, mostly in sperm tail region.     

Localization of actin during differentiation of the ameloblast, its related epithelial cells and odontoblasts in the rat incisor using NBD-phallacidin

Using NBD-phallacidin, which specifically binds to F-actin, we investigated changes in the localization of actin during the differentiation of ameloblasts, related epithelial cells and odontoblasts in rat incisors. In cryosections treated with NBD-phallacidin, intense fluorescence was observed in undifferentiated epithelial cells in the apical loop and at the proximal extremity of undifferentiated inner enamel epithelial cells. During differentiation, the distal extremity began to exhibit strong fluorescence. In cross-sections of secretory ameloblasts, the fluorescence took the form of polygons of uniform intensity at the proximal end, and of rectangles of non-uniform intensity at the distal end. At the distal end, the fluorescence was more intense at right angles to the long axis of the incisor. At the distal end, this pattern was established just before the appearance of the enamel layer. These patterns were maintained during the secretory stage of ameloblasts. The location, pattern and time of appearance of these sites were identical to those of the terminal webs in ameloblasts. NBD-phallacidin weakly labelled the peripheral cytoplasm of the cell body of ameloblasts, and also labelled Tomes' process. The cells forming the stratum intermedium were mainly labelled at their periphery (i.e. forming larger polygons), while the overlying epithelial cells exhibited labelling throughout their cytoplasm. Except for the terminal webs, the cell bodies of odontoblasts were weakly labelled throughout the period of differentiation. Young odontoblasts secreting predentin were first labelled on the terminal web, with the fluorescence becoming gradually more intense as the thickness of the dentin increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphological characteristics of the Pterodoras granulosus digestive tube (Valenciennes, 1821) (Osteichthyes,Doradidae)

Little is known about the digestive tube (DT) morphology of the fish Pterodoras granulosus. Therefore, macro‐, meso‐ and microscopic aspects of 15 P. granulosus DTs were analysed. The muscular layer was composed of striated skeletal muscle in the oesophagus and smooth muscle in the other segments. The epithelium progressed from a stratified pavement in the oesophagus to a simple column in the other segments, with a flat striated border in the intestine. A large number of mucus‐secreting periodic acid‐Schiff (PAS)‐positive cells were observed in the oesophagus. In the stomach, the number of glands in the region decreased towards the cardiac–fundic region, and none were found in the pylorus. The intestine showed an epithelium with absorption cells and an increasing number of PAS‐positive caliciform cells towards the distal region. Tests showed that the oesophagus is adapted for passing and preparing food for the chemical digestion that occurs in the stomach, which also has storage functions without grinding action. The proximal intestinal region was consistent with fat absorption, and the medium region, with the absorption of other nutrients. The distal region was short and consistent with a role in absorption for osmoregulation as well as in the formation, storage and disposal of faeces.     

Electron microscopic studies on the small intestine of rats after mechanical intestinal obstruction.

The morphologic changes of the small intestine after the mechanical obstruction were studied by light and electron microscopy. After the ligation of the upper small intestine, segments of jejunum, both proximal and distal to the site of obstruction, were removed at intervals varying from 45 min to 24 h. The essential changes were found in the epithelial cells of the tips of villi, and few morphologic differences were recognized between samples proximal and distal to the site of obstruction. The most remarkable changes in the mucosa were the pseudopodlike extension of the cytoplasm, vacuolar alteration of the villus epithelial cells, desquamation of these degenerated cells, and the dilatation of the epithelial intercellular spaces. A few epithelial cells showed hypertrophy of the smooth endoplasmic reticulum. In the submucosa, vascular stasis and edema were observed throughout the course. The mechanism of fluid loss into the intestinal lumen was discussed briefly.     

T-cell factor 4 (Tcf7l2) maintains proliferative compartments in zebrafish intestine

Previous studies have shown that Wnt signals, relayed through beta-catenin and T-cell factor 4 (Tcf4), are essential for the induction and maintenance of crypts in mice. We have now generated a tcf4 (tcf7l2) mutant zebrafish by reverse genetics. We first observe a phenotypic defect at 4 weeks post-fertilization (wpf), leading to death at about 6 wpf. The phenotype comprises a loss of proliferation at the base of the intestinal folds of the middle and distal parts of the intestine. The proximal intestine represents an independent compartment, as it expresses sox2 in the epithelium and barx1 in the surrounding mesenchyme, which are early stomach markers in higher vertebrates. Zebrafish are functionally stomach-less, but the proximal intestine might share its ontogeny with the mammalian stomach. Rare adult homozygous tcf4(-/-) 'escapers' show proliferation defects in the gut epithelium, but have no other obvious abnormalities. This study underscores the involvement of Tcf4 in maintaining proliferative self-renewal in the intestine throughout life.     

Morphological and histochemical features of the digestive tract of Leiarius marmoratus (Gill, 1870)

Leiarius marmoratus, a freshwater catfish from Pimelodidae family, shows great biological and commercial relevance because of its geographic distribution and adaptation to fish-farm. The knowledge of the morphological characteristics of the digestive tract is fundamental to the understanding of fish physiology and nutrition, which helps in the planning of diets to provide better management and success in fish farming. Thus, this work described the morphology and histochemistry of the digestive tract of L. marmoratus adults. After euthanasia, the animals were dissected for analysis of the digestive tract. The oesophagus is a short and distensive organ with longitudinal folds that allow the passage of large food, e.g., other fishes. Oesophageal mucosa layer shows a stratified epithelium with goblet cells and club cells. The secretion of goblet cells is composed of neutral and acidic mucins that are anchored in the epithelium luminal face by epithelial cells fingerprint-like microridges, lubricating the surface to facilitate the food sliding. Club cells have protein secretion that can be involved in alarm signals when epithelium is damaged and in immunological defence. The saccular stomach is highly distensible to store large food. Gastric mucosa layer is composed of epithelial cells with intense secretion of neutral mucin to protect against self-digestion of gastric juice. Cardiac and fundic regions of stomach show well-developed gastric glands composed of oxynticopeptic cells. These cells have numerous mitochondria, highlighting their intense activity in the synthesis of acid and enzymes. The intestine is divided into three regions: anterior, middle and posterior. Although it is a short tube, intestine shows longitudinal folds and microvilli of enterocytes to increase the contact surface. These folds are higher in the anterior region of the intestine, highlighting their function in digestion and absorption. Intestinal goblet cells have acidic and neutral mucins that lubricate the epithelium and aid in digestive processes. These cells increase in number towards aboral, and they are related to the protection and lubrication to expulsion of faecal bolus.     

Immunolocalization of EGF receptor (EGFr) in intestinal epithelium: recognition of apoptotic cells

The EGF-like family of growth factors are known to be involved in the control of the intestinal epithelium. The intracellular events are mediated by the EGF receptor (EGFr), a transmembrane glycoprotein which is overexpressed in many malignancies and also in many radiosensitive cell types. The precise mode of action of the receptor in controlling proliferation and whether the factor is also involved in controlling apoptosis in this tissue is not clear. Using polyclonal antibodies raised against a cytoplasmic region of the receptor distant to the phosphorylation site and one raised against the peptide sequence DVVDADEYLIPQ, which is present in the cytoplasmic tail phosphorylation site of the EGFr, we have examined the immunostaining in normal and irradiated murine intestine. The former antibody labelled the basolateral membranes of the epithelial cells in the proliferative zones of both the small intestine and colon, in both control and irradiated tissue. The latter antibody however, strongly labelled the Goblet cells and the microvilli of the enterocyte apical membrane in control tissue. Following irradiation\ the apical labelling redistributed and was localized in the apical cytoplasm and in a paranuclear region. Furthermore, strong labelling was now seen in many of the apoptotic cells of the small intestinal epithelium. The greatly differing results with the two antibodies indicates that interpretation of such immunostaining must be viewed with caution and may relate to the availability of each particular epitope. These results also suggest that antibodies to DVVDADEYLIPQ may be a useful marker of apoptotic calls and could imply a correlation between high levels of epitope availability, the radiosensitive (frequently p53 expressing) cells of the crypt epithelium and the induction of apoptosis.This work was supported by the Cancer Research Campaign.