Light and electron microscope observations on the gastric mucosa of the frog (Rana esculenta)

Summary The structure of the frog gastric and esophageal mucosa was studied in the course of a complete hibernation period and compared with that in summer frogs (see preceding article).It appeared that especially chief cells and parietal cells are liable to cytoplasmic remodelling. Thus, in chief cells the rough endoplasmic reticulum (RER) undergoes disorganization, the number of free ribosomes increases and the Golgi system becomes transformed into a compact vesicular structure. The number of pepsinogen granules in chief cells of late winter frogs is only 20% of that in frogs studied at the onset of hibernation. The loss of pepsinogen granules is at least partly due to autophagy. In addition, lysosomes are involved in focal degradation of the cytoplasm, which may ultimately result in complete degeneration of some chief cells. The presence of zymogen granules containing fibrocyte-like cells in the tunica propria proved heterophagocytosis by these cells.In parietal cells, the area occupied by smooth endoplasmic reticulum becomes reduced. The basal cytoplasm of both chief cells and parietal cells contains numerous lipid droplets, which, in contrast to those in summer frogs, are continuous with RER cisternae. The juxtaposition of lipid droplets and mitochondria seen in summer frogs is eventually lost in hibernating animals.Apart from the appearance of supra-nuclear lipid droplets, the mucous cells of the surface epithelium show no striking alterations. However, in the glandular pits both surface mucous cells and mucous neck cells contain less mucous granules than in summer frogs.The results are discussed in connection with parallel biochemical work and available literature, and in the light of our previous studies on the exocrine pancreas in hibernating frogs.     

Autoradiographic study of the renewal rate of neutrophils and thrombocytes in summer and winter frogs

In early August frogs were injected with 3H-thymidine and observed during 10 months under conditions close to natural. Individual changes of neutrophil and thrombocyte contents in the blood, those of the number of labeled cells among them, and the density of labeling were studied. The life span of neutrophils in the active frogs was found as long as 2-3 weeks, while that of thrombocytes lasted for several months. In September the hibernating cell populations are formed and cell proliferation ceases. This process is suggested to be regulated by some complex centralized mechanisms rather than by a direct action of temperature. The size of circulating populations of both the cell types decreases during hibernation, part of the cells is deposited outside of circulation. The life span of cells rises considerably, their renewal begins only in spring. The ability of cells of the neutrophilic lineage to proliferate is preserved at low temperatures and is realized in pathological conditions. The similarities in seasonal adaptations are stated between amphibians and hibernating mammals at the level of cellular populations.     

Relations Between Hibernation and Ovarian Functions in a Temperate Zone Frog,Rana temporaria

Adult female frogs were exposed to artificial hibernation in a refrigerator, at about 1–3°C, 1–2 months earlier than in nature, isolated or together with males. Ovarian functional state at the beginning of hibernation was assessed from ovarian biopsies taken in mid-September. All frogs ovulated during hibernation which was interrupted in mid-May, but in seven out of 19 frogs ovulation was incomplete. The number of non-ovulated oocytes varied from a minor fraction to about two thirds of the complement of vitellogenic oocytes, indicating that the follicle size reached at the time of induced hibernation was at the critical level for successful maturation. This critical size was about half the normal oocyte volume at the onset of natural hibernation. There was no vitellogenic growth during hibernation, which presumably constitutes the period of follicle maturation. In five of the frogs, oogenesis sensu stricto had occurred during the period of hibernation, some oogenic events being completely finished at autopsy in mid-May and others still progressing.     

Functional analysis for gut microbes of the brown tree frog (Polypedates megacephalus) in artificial hibernation

Background

Annual hibernation is an adaptation that helps many animals conserve energy during food shortage in winter. This natural cycle is also accompanied by a remodeling of the intestinal immune system, which is an aspect of host biology that is both influenced by, and can itself influence, the microbiota. In amphibians, the bacteria in the intestinal tract show a drop in bacterial counts. The proportion of pathogenic bacteria is greater in hibernating frogs than that found in nonhibernating frogs. This suggests that some intestinal gut microbes in amphibians can be maintained and may contribute to the functions in this closed ecosystem during hibernation. However, these results were derived from culture-based approaches that only covered a small portion of bacteria in the intestinal tract.

Methods

In this study, we use a more comprehensive analysis, including bacterial appearance and functional prediction, to reveal the global changes in gut microbiota during artificial hibernation via high-throughput sequencing technology.

Results

Our results suggest that artificial hibernation in the brown tree frog (Polypedates megacephalus) could reduce microbial diversity, and artificially hibernating frogs tend to harbor core operational taxonomic units that are rarely distributed among nonhibernating frogs. In addition, artificial hibernation increased significantly the relative abundance of the red-leg syndrome-related pathogenic genus Citrobacter. Furthermore, functional predictions via PICRUSt and Tax4Fun suggested that artificial hibernation has effects on metabolism, disease, signal transduction, bacterial infection, and primary immunodeficiency.

Conclusions

We infer that artificial hibernation may impose potential effects on primary immunodeficiency and increase the risk of bacterial infections in the brown tree frog.

     

Nuclear changes and morphology of the epidermis in the hibernating frog

Cytochemical changes of chromatin and DNA in frog epidermal cells were correlated with some morphological features to investigate the skin physiology during hibernation in comparison with the active period. The epidermal cells of hibernating frogs showed less condensed chromatin in all the layers; a greater loss of DNA was found during the transition from the middle to the superficial layer. In the germinative layer, a lesser frequency of hyperdiploid cells and a remarkably low amount of mitoses were detected; this is accompanied by the increase of epidermal thickness and the presence of two layers of cornified cells. The slowing of tissue differentiation and cell renewal kinetics during hibernation can be related to lowered activity of the frog skin. Further, the smaller intercellular spaces as well as the scarcity of puffed ER and vacuoles may be indicative of a lower ion transport in epidermal cells during hibernation.     

Changes in the composition of the lysosomes in hepatocytes cultured in vitro in modelling pathological states

The monolayer cultures of newborn rats hepatocytes were treated with substrate starvation (20 min.) anoxia accompanied with substrate starvation (1 h.), following rehabilitation (1 h.) and cooling (4 degrees C, 30 min). After the neutral red staining we have examined the alterations in lysosomes quantity and the development of phagocytosis; we also tested the hepatocytes distribution due to the amount of the second lysosomes per cell. We have shown that a short time pathological treatment caused a decrease in large lysosomes (1-3 microns) amount in significant portion of hepatocytes. There were neither increase in phagosomes amount per cell nor alterations in the quantity of cells, containing phagosomes. However a long time pathological treatment caused the increase in phagosomes and cells. We propose that initial stage of lysosomal apparatus alterations, connected with reduction of CAMP level, is accompanied by a stamping of large lysosomes.     

Tumor necrosis factor-alpha-associated lysosomal permeabilization is cathepsin B dependent

Cathepsin B (Cat B) is released from lysososomes during tumor necrosis factor-alpha (TNF-alpha) cytotoxic signaling in hepatocytes and contributes to cell death. Sphingosine has recently been implicated in lysosomal permeabilization and is increased in the liver by TNF-alpha. Thus the aims of this study were to examine the mechanisms involved in TNF-alpha-associated lysosomal permeabilization, especially the role of sphingosine. Confocal microscopy demonstrated Cat B-green fluorescent protein and LysoTracker Red were both released from lysosomes after treatment of McNtcp.24 cells with TNF-alpha/actinomycin D, a finding compatible with lysosomal destabilization. In contrast, endosomes labeled with Texas Red dextran remained intact, suggesting lysosomes were specifically targeted for permeabilization. LysoTracker Red was released from lysosomes in hepatocytes treated with TNF-alpha or sphingosine in Cat B(+/+) but not Cat B(-/-) hepatocytes, as assessed by a fluorescence-based assay. With the use of a calcein release assay in isolated lysosomes, sphingosine permeabilized liver lysosomes isolated from Cat B(+/+) but not Cat B(-/-) liver. C(6) ceramide did not permeabilize lysosomes. In conclusion, these data implicate a sphingosine-Cat B interaction inducing lysosomal destabilization during TNF-alpha cytotoxic signaling.     

Functional analysis for gut microbes of the brown tree frog (<Emphasis Type="Italic">Polypedates megacephalus</Emphasis>) in artificial hibernation

Background

Annual hibernation is an adaptation that helps many animals conserve energy during food shortage in winter. This natural cycle is also accompanied by a remodeling of the intestinal immune system, which is an aspect of host biology that is both influenced by, and can itself influence, the microbiota. In amphibians, the bacteria in the intestinal tract show a drop in bacterial counts. The proportion of pathogenic bacteria is greater in hibernating frogs than that found in nonhibernating frogs. This suggests that some intestinal gut microbes in amphibians can be maintained and may contribute to the functions in this closed ecosystem during hibernation. However, these results were derived from culture-based approaches that only covered a small portion of bacteria in the intestinal tract.

Methods

In this study, we use a more comprehensive analysis, including bacterial appearance and functional prediction, to reveal the global changes in gut microbiota during artificial hibernation via high-throughput sequencing technology.

Results

Our results suggest that artificial hibernation in the brown tree frog (Polypedates megacephalus) could reduce microbial diversity, and artificially hibernating frogs tend to harbor core operational taxonomic units that are rarely distributed among nonhibernating frogs. In addition, artificial hibernation increased significantly the relative abundance of the red-leg syndrome-related pathogenic genus Citrobacter. Furthermore, functional predictions via PICRUSt and Tax4Fun suggested that artificial hibernation has effects on metabolism, disease, signal transduction, bacterial infection, and primary immunodeficiency.

Conclusions

We infer that artificial hibernation may impose potential effects on primary immunodeficiency and increase the risk of bacterial infections in the brown tree frog.

     

Mechanisms of cellular detoxication, nuclear aluminum concentration and hepatocyte protection after experimental overload in rats]

A single injection of a massive dose of Al-gluconate (4 mg.kg-1) into the saphenous vein of the rat did not provoke any ultrastructural damage in the liver cells, from 2 min. to 8 days after the injection. Al hepatocytes overload appeared only in nuclei and not in nuclei and not in lysosomes, contrarily to chronic intoxications. Nuclear Al concentration concerned only a small fraction of the quantity injected; another part was sequestered by the macrophage system after precipitation in the blood as phosphates chemically transformed during their incorporation in lysosomes. This effective detoxication mechanism which functioned probably after a first absorption by the hepatocyte, was likely to depend upon the form of gluconate and would explain the resistance of liver cells.     

The Lucke Frog Kidney Tumor and its Herpesvirus

Northern leopard frogs are afflicted with a spontaneous malignantneoplasm of the mesonephros. A herpesvirus is invariably associatedwith tumors obtained from frogs hibernating 30 days or longerand in tumors of frogs taken from breeding ponds. Tumors obtainedfrom prehibernating frogs do not have viruses as detected byelectron microscopy but virus particles are found in some tumorswithin 7 days after the onset of hibernation. Tumors of frogsmaintained at warm temperature in the laboratory do not haveviruses and tumors of frogs maintained at cold temperaturesin the laboratory do contain viruses. However, the productionof viruses in the laboratory follows a distinctly slower chronologythan that which occurs in nature. Injection of cell fractionscontaining the herpesvirus into frog embryos induces tumorsnear the time of metamorphosis in many experimental animals.Embryos injected with tumor extracts not containing herpesvirusesdo not develop tumors. Environmental and laboratory observationsare discussed which may relate to natural transmission of thispresumed viral oncogenic agent.     

Changes in activity of the organon vasculosum laminae terminalis in the annual cycle in Rana temporaria.

In 70 sexually mature male and femal Rana temporaria frogs captured in natural habitat, mean nuclear volumes for the cells of the pars ependymalis and pars parenchymalis of the organon vasculosum laminae terminalis (OVLT) were determined in seven characteristic stages in life. The mean nuclear volume for the cells of the pars ependymalis and pars parenchymalis of the OVLT showed distinct annual fluctuation. Maximum nuclear volume of the cells in both investigated parts of the OVLT were observed during the breeding period (Ist decade of April), and minimum volume of the nuclei of the pars ependymalis at the beginning of hibernation (IIIrd decade of October), and in the pars parenchymalis near the end of active life (Ist decade of September).     

Tilorone-induced lysosomal storage mimicking the features of mucopolysaccharidosis and of lipidosis in rat liver

This ultrastructural and histochemical study deals with the lysosomal storage phenomena occurring in the rat liver after repeated oral administration of tilorone, an agent with anti-tumor and anti-viral activities. In the sinusoidal endothelium and in Kupffer cells, the lysosomes were changed into large vacuoles which contained material with the histochemical characteristics of acid glycosaminoglycans. The alterations closely resembled those previously observed in the splenic red pulp of tilorone-treated rats. In hepatocytes, the lysosomes were converted into large multilamellated inclusions indicating storage of polar lipids. The results show that, in the rat liver, tilorone induces cellular alterations mimicking those of inherited mucopolysaccharidoses and lipidoses. After discontinuing drug treatment the two storage phenomena gradually faded at different rates: The lipidosis disappeared within 2 to 4 weeks, whilst mucopolysaccharidosis-like changes were still found 15 weeks after drug withdrawal. The occurrence of lipidosis is not surprising, since by its molecular structure tilorone can be regarded as belonging to the group of amphiphilic cationic drugs which often have this side effect. Much more surprising is the occurrence of mucopolysaccharidosis-like alterations. The exact biochemical identification of the polyanionic storage material and the molecular mechanisms responsible for this drug side effect remain to be established.     

Subcellular localization of ferritin and iron taken up by rat hepatocytes.

The subcellular localization of ferritin and its iron taken up by rat hepatocytes was investigated by sucrose-density-gradient ultracentrifugation of cell homogenates. After incubation of hepatocytes with 125I-labelled [59Fe]ferritin, cells incorporate most of the labels into structures equilibrating at densities where acid phosphatase and cytochrome c oxidase are found, suggesting association of ferritin and its iron with lysosomes or mitochondria. Specific solubilization of lysosomes by digitonin treatment indicates that, after 8 h incubation, most of the 125I is recovered in lysosomes, whereas 59Fe is found in mitochondria as well as in lysosomes. As evidenced by gel chromatography of supernatant fractions, 59Fe accumulates with time in cytosolic ferritin. To account for these results a model is proposed in which ferritin, after being endocytosed by hepatocytes, is degraded in lysosomes, and its iron is released and re-incorporated into cytosolic ferritin and, to a lesser extent, into mitochondria.     

Hepatic gluconeogenesis and mitochondrial function during hibernation

1. The aim of these studies was to investigate a mitochondrial basis for changes in gluconeogenesis during hibernation. 2. State 3 respiration rates in liver mitochondria from hibernating ground squirrels were reduced by 62-66%. The limiting reaction appeared to be electron transport, particularly in respiratory complex III. 3. The mitochondrial ATP + ADP + AMP content was reduced by 29% during hibernation; cellular adenine nucleotide content was unchanged. 4. Pyruvate carboxylation in intact mitochondria was decreased 75% during hibernation, although total pyruvate carboxylase activity was not lower. 5. Rates of gluconeogenesis in intact hepatocytes isolated from hibernators were lower than in cells from non-hibernators.     

Functional similarity of the P-2 form of primary sleep and hypobiosis in the frog Rana temporaria (based on data from luminescent microspectral analysis of the cytoplasm of neurosecretory neurons)

Using luminescent microspectral analysis of preparations stained by acridine orange, studies have been made on the ratio between single- and double-stranded parts in ribosomal RNA from the cytoplasm of neurosecretory neurones of the preoptic nucleus of the frog Rana temporaria. The animals were investigated in active period, during P-2 form of the primary sleep, in hypobiosis, and after injection of the active factor (peptide fraction with a molecular mass 1-10,000 Da) extracted from the small intestine of the ground squirrel Citellus undulatus during winter hibernation. It was shown that unlike actively awake frogs, animals from other experimental series exhibited similar changes, i.e. the decrease in the affinity of acridine orange to single- and double-stranded parts of rRNA and the decrease in the value of alpha which reflects the ratio of single- and double-stranded parts. It was also demonstrated that injection of the active factor from hibernating ground squirrels to frogs results in a condition which is rather similar to a natural resting form (P-2) of the primary sleep in cold-blooded vertebrates.     

Comparison of K(+)-p-nitrophenyl phosphatase activity in the urinary bladder of the frog Rana esculenta during hibernation and active life.

We have studied effects of hibernation on the frog urinary bladder, an organ involved in water and ion transepithelial transport and taking part in osmoregulation. We have demonstrated K(+)-p-nitrophenyl phosphatase activity (an enzyme involved in ion and water transport) both in active and hibernating frogs. Most of the reaction product deposition was found on basolateral membranes of granular cells of the urinary bladder epithelium during all seasons. Therefore, it seems likely that this organ, unlike organs studied previously (skin, kidney and lung), maintains its function in the osmoregulatory process during hibernation.     

Uptake of mannose-terminated glycoproteins in isolated rat liver cells. Evidence for receptor-mediated endocytosis in hepatocytes.

Even though most of the hepatic binding capacity for mannose-terminated glycoproteins has previously been shown to reside in the hepatocytes (not in the non-parenchymal cells), detailed evidence for the specific uptake of mannose-terminated ligands has been lacking. In the present studies, yeast invertase, a large glycoprotein (Mr 270 000) containing about 50% mannose, was shown to be taken up into hepatocytes by receptor-mediated endocytosis. The uptake was saturable and could be specifically inhibited by mannosides or by a Ca2+ chelator. The asialo-glycoprotein receptor was not involved. The low-Mr (13 000) ligand ribonuclease B, which contains a single high-mannose glycan, was not taken up by hepatocytes; however, it was taken up as fast as invertase by non-parenchymal liver cells. After injection of 131I-invertase into a rat in vivo, about one-half of the labelled protein was recovered in the hepatocytes. On a per-cell basis, each endothelial cell contained 3-4 times as much radioactivity as did the hepatocytes. On fractionation of hepatocytes in sucrose gradients, invertase showed a different intracellular distribution from that of asialo-fetuin, in that invertase moved much faster into that region of the gradient where the lysosomes were recovered. This indicates that invertase and asialo-fetuin are not transported intracellularly by identical mechanisms.     

The reorganization of the actin cytoskeleton of nuclear erythrocytes and leukocytes in fish, frogs and birds during cell migration

Changes in the spatial organization of actin filaments of nuclear erythrocytes and leukocytes during their migration in fish, frogs and birds have been studied by the method of confocal laser scanning microscopy. It has been shown that, during movement of cells, the reorganization of cytoskeleton microfilaments in erythrocytes is similar to that in leukocytes. During migration, red blood cells of amphibious and birds form pseudopodia filled with bunches in parallel laid actin filaments. Erythrocytes in fish do not form pseudopodia. Similar to leukocytes change in the structure of the actin cytoskeleton in nuclear erythrocytes determines the ability of red blood cells to reactions of migration and phagocytosis.     

Large intestine bacterial flora of nonhibernating and hibernating leopard frogs (Rana pipiens).

The bacteria in the large intestines of 10 northern leopard frogs (Rana pipiens) were enumerated and partially characterized. Four nonhibernating frogs were collected in the summer, four hibernating frogs were collected in the winter, and two frogs just emerged from hibernation were collected in the spring. All frogs had about 10(10) bacteria per g (wet weight) of intestinal contents and about 10(9) bacteria per g (wet weight) of mucosal scraping, although the counts from the winter frogs were slightly less than those from the other two groups of frogs. Another group of 14 summer frogs, after treatment to induce hibernation, showed a drop in bacterial counts accompanied by a change in the composition of the flora. In most frogs, Bacteroides was the dominant organism. Other bacteria repeatedly isolated at high dilutions were strict anaerobes, including butyrigenic and acetogenic helically coiled bacteria; fusobacteria; and acetogenic, small, gram-positive bacilli. These data indicate that the intestinal flora of frogs is similar to that of mammals and birds and that this flora can be maintained at temperatures close to freezing.     

Interaction between Karyolysus sp. and rock lizard liver cells during hibernation]

During the rock lizard hibernation, no nuclear division occurs in the exoerythrocytar trophozoites of Karyolysus sp., found in hepatocytes or Kupffer's cells. In addition, organelles of the apical complex are seen persisting in these trophozoites, unlike the situation routinely observed in the majority of other intracellular sporozoans. Thus, the parasites under study can be compared with hypnozoites of other coccidia. The material being examined from natural rather than experimental conditions, during lizards' hibernation, the dynamics of host-parasite interrelations can be followed that involves the appearance in the infected cell of autophagous vacuoles, changes in mitochondrial structure and pattern of endoplasmic reticulum, the connection of the system of lamellar channels with the space of the parasitophorous vacuole. The results of the present observations may suggest, first, that during the host hibernation, exoerythrocytar trophozoites of Karyolysus being intracellular in their spatial distribution, are able to obtain nutrients from the outer, extracellular space, through the system of lamellar channels; second, that these channels can represent intercellular connections, which makes one consider the exoerythrocytar trophozoites as intercellular rather than intracellular parasites.