Histology and lectin-binding patterns in the digestive tract of the carnivorous larvae of the anuran, Ceratophrys ornata

Larvae of Ceratophrys ornata are carnivorous, have relatively short digestive tracts and continue to feed during metamorphic climax, in contrast to those of more typical herbivorous anuran larvae. The present study describes both histological and histochemical changes in the stomach, small intestine, and large intestine of C. ornata prior to and during metamorphic climax. Modifications in these organs were found to be similar to but less dramatic than those in herbivorous larvae. Luminal epithelial cells in the three regions develop vacuoles, suggesting degeneration, but sloughing of this epithelium, as occurs in herbivorous larvae, was not observed in C. ornata. Multicellular tubular glands develop gradually in the gastric area during the larval stages, gastric pits appear during metamorphic climax, and mucous neck cells are first visible in the juvenile. Goblet cells in the small and large intestine increase in number during larval life, as do the number of folds in the intestinal wall. Increase in diameter and thickness of the wall occurs in the stomach as well as in the small and large intestine. Such changes result in an adult-type digestive tract characteristic of frogs in general. Staining with two horseradish peroxidase conjugated lectins, soybean agglutinin (SBA) and Ulex europaeus agglutinin I (UEA I), demonstrated specific sites along the digestive tract of glycoconjugates with terminal sugars N-acetylgalactosamine and alpha-fucose, respectively. As metamorphic climax approaches, staining intensities decrease--thus providing evidence for metamorphic changes in the sugar moieties of glycoconjugates present in the digestive tract of carnivorous larvae.     

Adenohypophysial-thyroid activity of the tiger salamander, Ambystoma tigrinum, as a function of metamorphosis and captivity

The aim of this study was to determine the timing of adenohypophysial activation during metamorphosis of the tiger salamander, Ambystoma tigrinum. It consisted of two parts: 1) determination of plasma thyroid hormone concentrations and analysis of thyroid gland histology as a function of metamorphic stage and 2) analysis of the time-course of uptake of 125I by the thyroids during metamorphosis as an indicator of endogenous thyrotropin (TSH) levels. Significant increases in both triiodothyronine (T3) and thyroxine (T4) first were evident at the onset of metamorphic climax (stage II). Maximum levels of both hormones were not observed, however, until the completion of gill resorption (stage VII). No changes in thyroid histology were observed that could be unambiguously related to metamorphic transformation. The thyroids accumulated 125I in a slow but linear fashion in premetamorphic larvae (stage I). However, uptake exhibited a rapid peak during early climax (stage II), before maximum concentrations of thyroid hormones were observed. In addition, uptake was maintained above premetamorphic levels at stage VII, in conjunction with maximum levels of T4 and T3. Captivity alone produced a small but significant increase in plasma concentrations of T3. It produced no significant effect on either thyroid histology or uptake of 125I. These results indicate that adenohypophysial activation occurs rapidly and is maximal at the onset of metamorphic climax.     

Early larval development and metamorphosis in the summer flounder: changes in per cent whole-body water content and effects of altered thyroid status

At the end of premetamorphosis, summer flounder Paralichthys dentatus larvae had 84·1% whole-body water content (WBW), which decreased to the lowest levels (8·5%) at the start of metamorphic climax (MC). During mid- and late MC, %WBW was slightly higher (82·1%) then returned to the lowest levels at the juvenile stage. In fish treated with thyroxine (T₄-Na salt, 100 ng ml⁻¹) beginning at premetamophosis, %WBW never differed from controls of the same age throughout metamorphosis, despite an earlier start of metamorphic climax and transitional settling behaviour. This suggests that thyroid hormones do not mediate the drop in %WBW which accompanies natural metamorphosis. Thiourea (TU, 30 μg ml⁻¹) treatment of fish over the same period induced a developmental stasis in early MC which was accompanied by initially higher %WBW than controls at 33 days post-hatch, followed by a progressive decrease to abnormally low %WBW by 42 and 45 days post-hatch. Since concurrent treatment with TU+T₄ rescued the fish from both the TU-induced developmental stasis and abnormally low %WBW, these findings suggest that thyroid hormones, or thyroid hormone-mediated developmental progression, are necessary for regulating %WBW.     

TISSUE DEGRADATION PATTERNS OF AMPHIBIAN TADPOLE TAIL: HISTOLOGICAL AND BIOCHEMICAL STUDIES

The pattern of tissue degradation in the tadpole tail of the bullfrog, Rana catesbeiana , was studied histologically and biochemically. Tadpoles at four metamorphic stages (premetamorphic stage, stage XIII; prometamorphic stage, stage XVIII; early to middle climax stage, stage XXII; late climax stage, stage XXIII) were examined. In the histological study, tissues were stained by two different methods, i.e., Masson's trichrome stain and Van Gieson's stain. The results are summarized as follows: 1) Muscle tissues degenerated earlier thatn connective tissues; 2) Collagen density increased as tissue degraded; 3) At stage XXIII, degenerating and fragmented muscle tissues, surrounded by connective tissues, were seen, and 4) At the late climax stage, cell density was greatly increased as compared to preceding stages.
The biochemical studies are summarized as follows; 1) The general catabolic pattern of tail proteins seems to differ between the premetamorphic and late climax stages; 2) The total hydroxyproline content per unit wet weight of the tail increased significantly at the late climax stage as compared to the premetamorphic stage, supporting the histological observations, and 3) Collagen degradation products were obtained at the late climax stage on Sephadex G-75 columns.     

Developmental regulation of granule size and numbers in larval salivary glands of drosophila by steroid hormone ecdysone

The size and number of secretory granules in late larval salivary glands of Drosophila melanogaster have been related to interecdysial and early metamorphic development represented by well-known puffs in polytene chromosomes. Interecdysial period (puff stage 1 (PS1)) is characterized by presence of numerous small granules (11,000 per cell). The transition from PSI to early metamorphic phase (PS2 and upwards), induced by rapid elevation in endogenous steroid hormone ecdysone, is accompanied by continuous growth of granule diameter with concomitant reduction in their number per cell. In the PS4, just prior to secretion, approximately 3000 mature granules occur per cell. The mature state is associated with the change from hyperbolic to Gaussian distribution of granule number over their size range. Similar changes in secretory granule parameters were observed in interecdysial salivary glands explanted from 3rd instar larvae and cultured in vitro in medium containing 5x10(-6) m ecdysone.     

Serial passage of MC3T3-E1 cells alters osteoblastic function and responsiveness to transforming growth factor-beta1 and bone morphogenetic protein-2.

The murine-derived clonal MC3T3-E1 cell is a well-studied osteoblast-like cell line. To understand the effects of serial passages on its cellular function, we examined changes in cell morphology, gap junctional intercellular communication (GJIC), proliferation, and osteoblastic function between early passage (<20) and late passage (>65) cells. MC3T3-E1 cells developed an elongated, spindle shape after multiple passages. Intercellular communication decreased significantly (33%) in late vs. early passage cells. Transforming growth factor-beta1 (TGF-beta1) stimulated cell proliferation in early passage cells and induced c-fos expression, while it inhibited proliferation in late passage cells. Using alkaline phosphatase (ALP) activity and osteocalcin (OC) secretion as markers for osteoblastic function and differentiation, we demonstrated that both markers were significantly reduced after multiple cell passages. Bone morphogenetic protein-2 (BMP-2) significantly enhanced ALP activity and OC secretion in early passage cells while TGF-beta1 exerted an opposite effect. Both BMP-2 and TGF-beta1 had minimal effects on late passage cells. We conclude that serial passage alters MC3T3-E1 cell morphology, and significantly diminishes GJIC, osteoblastic function, TGF-beta1-mediated cell proliferation, and responsiveness to TGF-beta1 and BMP-2. Cell passage numbers should be clearly defined in functional studies involving MC3T3-E1 cells.     

In vivo effects of TGFbeta1 on the growth of gastric epithelium in suckling rats

As the content of Transforming Growth Factor-beta (TGFbeta) wanes in the milk of lactating rat, an increase in TGFbeta is observed in the gastric epithelia concomitant with differentiation of the glands upon weaning. Whereas TGFbeta has been shown to inhibit the proliferation of gastrointestinal cells in vitro, its functional significance and mechanisms of action have not been studied in vivo. Therefore, we administered TGFbeta1 (1 ng/g body wt.) to 14-day-old rats in which the gastric epithelium was induced to proliferate by fasting, and determined the involvement of signaling through Smads and the impact on epithelial cell proliferation and apoptosis. After the gavage, we observed the progressive increase of active TGFbeta1 while TbetaRII-receptor remained constant in the gastric mucosa. By immunohistochemistry, we showed Smad2/3 increase at 60 min (p<0.05) and Smad2 phosphorylation/activation and translocation to the nucleus most prominently between 0 and 30 min after treatment (p<0.05). Importantly, TGFbeta1 inhibited cell proliferation (p<0.05), which was estimated by BrDU pulse-labeling 12 h after gavage. Lower proliferation was reflected by increased p27(kip1) at 2 h (p<0.05). Also, TGFbeta1 increased apoptosis as measured by M30 labeling at 60 and 180 min (p<0.001), and by morphological features at 12 h (p<0.05). In addition, we observed higher levels of activated caspase 3 (17 kDa) from 0 to 30 min. Altogether, these data indicate a direct effect of TGFbeta1 signaling through Smads on both inhibiting proliferation, through alteration of cycle proteins, and inducing apoptosis of gastric epithelial cells in vivo. Further, the studies suggest a potential role for both milk and tissue-expressed TGFbeta1 in gastric growth during postnatal development.     

Histological examination of the effects of corticosterone in larvae of the western toad,Bufo boreas (Anura: Bufonidae), and the oriental fire-bellied toad,Bombina orientalis (Anura: Discoglossidae)

The effects of corticosterone (CORT)-treatment on various tissues were examined in two species of anuran larvae, the discoglossid Bombina orientalis, and the bufonid Bufo boreas. Corticosterone was administered directly into aquarium water for 15 days. After treatment, histological analyses were conducted on skin, gut, spleen, thymus, and neural and muscle tissue. Corticosterone treatment prevented sloughing of the skin, which resulted in a build-up of stratum corneum, and inhibited the development of gland nests and the subsequent formation of dermal granular and mucous glands in both species. Corticosterone treatment also decreased epithelial folding in the gut and caused vesiculation of the gut epithelial cells. The thymus of CORT-treated animals was significantly reduced in size (P < .05) and cell density (P < .05), and the spleen of CORT-treated animals was completely involuted. The brain and pituitary of CORT-treated animals had a decreased cell density (P < .05) and many pyknotic cells. An examination of muscle revealed that muscle fibers of CORT-treated animals had a decreased cross-sectional area (P < .05). The dose of CORT used (1.1 μM) was within the range used in other studies in the literature and resulted in tissue levels within the range experienced by larvae at metamorphic climax. Thus, this study is appropriate to address the histological effects of CORT in experimental manipulations and the role of increasing CORT at metamorphic climax. The data suggest that increasing endogenous CORT at metamorphosis may be involved in degeneration of larval tissue, prior to regeneration, which is stimulated by thyroid hormones. © 1995 Wiley-Liss, Inc.     

Larval development and metamorphosis of the olfactory and vomeronasal organs in the toad Rhinella (Bufo) arenarum (Hensel, 1867)

Jungblut, L.D., Pozzi, A.G. and Paz, D.A. 2010. Larval development and metamorphosis of the olfactory and vomeronasal organs in the toad Rhinella (Bufo) arenarum (Hensel, 1867). — Acta Zoologica (Stockholm) 92 : 305–315. The olfactory and the vomeronasal system are the two major chemosensory systems found in terrestrial vertebrates. Among tetrapods, amphibians are unique in having an aquatic larval stage, followed by metamorphosis to a terrestrial adult. In the present work, we studied the histological development of the olfactory and vomeronasal organ and associated multicellular glands of the toad Rhinella (Bufo) arenarum, from early poshatching larva to postmetamorphic toadlets. As in other bufonids, the olfactory epithelium of R. arenarum in larvae is divided into dorsal and ventral branches in the rostral and mid‐nasal regions. At metamorphic climax, the larval pattern changes drastically and the adult olfactory configuration develops. Bowman’s glands appear in the olfactory epithelium of R. arenarum at the onset of metamorphic climax. The vomeronasal epithelium develops early in larval development in R. arenarum, around the time of operculum development. Interestingly, a novel sensory epithelium develops in the floor of the principal chamber of R. arenarum at metamorphic climax. This novel sensory epithelium resembles larval sensory epithelium lacking Bowman’s glands, and suggests that these animals would be able to sense not only air‐borne, but also water‐borne odors during their adult terrestrial life.     

Reduction of EGF is associated with the delay of ulcer healing by cigarette smoking

Cigarette smoking is associated with peptic ulcer diseases. Smokers have lower levels of salivary epidermal growth factor (EGF) than nonsmokers. We investigated whether reduction of EGF is involved in the delay of gastric ulcer healing by cigarette smoking. Rats with acetic acid-induced ulcers were exposed to cigarette smoke (0, 2, or 4% vol/vol) 1 day after ulcer induction. EGF level was elevated 1 day after ulcer induction in salivary glands and serum, and 4 days after ulcer induction in the gastric mucosa. However, cigarette smoke depressed these beneficial effects and EGF mRNA expression in salivary glands and gastric mucosa. Cigarette smoke delayed gastric ulcer healing and reduced cell proliferation, angiogenesis, and mucus synthesis. Exogenous EGF (10 and 20 microg/kg i.v.) before smoke exposure reversed the adverse effects of cigarette smoke, whereas vascular endothelial growth factor level and nitric oxide synthase activity were unaffected. It is concluded that the detrimental effect of cigarette smoke on ulcer healing is a consequence of reduction of angiogenesis, cell proliferation, and mucus secretion through the depressive action on EGF biosynthesis and its mRNA expression in salivary glands and gastric mucosa.     

Gastric lipase and pepsinogen during the ontogenesis of rabbit gastric glands

In rabbit stomach, gastric lipase activity level was found to increase from birth to 30 days old (weaning), and then decreased. In contrast, pepsin activity only appeared between 30 to 45 days old, and increased till to the adult level. It was observed that maturation of gastric glands in cardial mucosa was a downward elongation process from the mitotic cell pool. These mitotic cells were always found in the neck of the gastric glands, corresponding to the bottom of the gland at 6 days old and to the mid-zone of the gland in adult. Location of rabbit gastric lipase (RGL) cells in cardial glands varied with age and was found along the pit of the gastric glands at 6 days old. The extent of this cellular location decreased with age, whereas a second RGL cell zone appeared below the mitotic cell area at 18 and 30 days old. At 45 days old, the pepsinogen cells appeared in the bottom of the gland, and consequently the RGL cells were located in the mid-zone of the gastric glands, between mitotic cells (neck of the gland) and pepsinogen cells (lower part of the gland). Ultrastructural study of cardial gastric glands revealed different morphologies of the secretion granules in the cells along the gastric glands. In 6-day-old rabbits, secretory granules were found uniformly electron dense in the bottom of the glands and were RGL-labeled by the immunogold technique. In the medium part of the glands, granules appeared biphasic, with a clear and a dense part, and RGL labeling was confined to the electron-dense part.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antigenic activation of Th1 cells in the gastric mucosa enhances dysregulated apoptosis and turnover of the epithelial cells

Colonization of Helicobacter pylori in the stomach leads to chronic gastritis with massive infiltration by Th1 cells. To assess a role played by those T cells in the remodeling of gastric epithelium, we activated gastric T cells utilizing mice with CD4 T cells bearing transgenic TCR with or without deficiency in either IL-4 or IFN-gamma or IL-12. Mice developed gastritis upon injection of an antigen into gastric mucosa. While neutrophil infiltration occurred even with a control antigen, infiltration by transgenic T cells was dependent on the specific antigen. The numbers of epithelial cells undergoing apoptosis and regeneration were increased in the mice with infiltrating T cells producing IFN-gamma and the alignment of those cells in the glands was markedly dysregulated. In contrast, mice deficient in Th1 response showed no increase in cell division and apoptosis of epithelial cells. Thus, Th1 type T cells infiltrating into gastric mucosa play an independent role in controlling turnover of epithelial cells.     

Role of salivary glands and epidermal growth factor (EGF) in gastric secretion and mucosal integrity in rats exposed to stress

EGF, produced mainly by salivary glands, inhibits gastric acid secretion, stimulates the proliferation of gastric mucosal cells and protects the mucosa against various ulcerogens, but its role in the pathogenesis of stress ulcerations is unknown. In this study, rats with intact or resected salivary glands were exposed to water immersion and restraint stress (WRS) without and with pretreatment with exogenous EGF or dimethyl PGE2 (dmPGE2) at doses which were shown previously to protect the mucosa against topical irritants. During 1.5-12 h of WRS, the formation of gastric ulcerations increased progressively with the duration of stress reaching peak after 6 h of stress and being significantly higher in rats with removed salivary glands than in intact animals. Gastric acid secretion and DNA synthesis in oxyntic mucosa declined with the duration of WRS, but after sialoadenectomy a significant increase in gastric acid secretion and a further decline in DNA synthesis were observed after WRS. EGF contents in the gastric lumen and the gastric mucosa were several times higher in rats subjected to stress than in control unstressed animals, indicating that stress causes an extensive release of EGF. Both exogenous EGF (17 nmol/kg/h) and dmPGE2 (143 nmol/kg) prevented, in part, the formation of gastric lesions, while inhibiting gastric acid secretion both in rats with intact or resected salivary glands. We conclude that water immersion and restraint stress is accompanied by an excessive release of EGF, which appears to attenuate gastric secretion, enhances the DNA synthesis and may limit the formation of stress-induced gastric ulcerations.     

Developmental changes in cell proliferation in the auditory midbrain of the bullfrog, Rana catesbeiana

We examined patterns of cell proliferation in the auditory midbrain (torus semicircularis) of the bullfrog, Rana catesbeiana, over larval and early postmetamorphic development, by visualizing incorporation of 5-bromo-2'-deoxyuridine (BrdU) in cycling cells. At all developmental stages, BrdU-labeled cells were concentrated around the optic ventricle. BrdU-labeled cells also appeared within the torus semicircularis itself, in a stage-specific manner. The mitotic index, quantified as the percent of BrdU-positive cells outside the ventricular zone per total cells available for label, varied over larval development. Mitotic index was low in hatchling, early larval, and late larval stages, and increased significantly in deaf period, metamorphic climax, and froglet stages. Cell proliferation was higher in metamorphic climax than at other stages, suggesting increased cell proliferation in preparation for the transition from an aquatic to an amphibious existence. The change in mitotic index over development did not parallel the change in the total numbers of cells available for label. BrdU incorporation was additionally quantified by dot-blot assay, showing that BrdU is available for label up to 72 h postinjection. The pattern of change in cell proliferation in the torus semicircularis differs from that in the auditory medulla (dorsal medullary nucleus and superior olivary nucleus), suggesting that cell proliferation in these distinct auditory nuclei is mediated by different underlying mechanisms.     

A comparison of Thai silk fibroin-based and chitosan-based materials on in vitro biocompatibility for bone substitutes

The novel hybrid scaffolds fabricated from silk fibroin, gelatin, low deacetylation degree chitosan and hydroxyapatite were investigated for their in vitro biocompatibility and osteoconductivity to mouse pre-osteoblast cell line (MC3T3-E1) and rat bone marrow-derived stem cells (MSC). We found that gelatin-conjugated silk fibroin films and scaffolds dominantly promoted cell adhesion and proliferation. Film and scaffold prepared from gelatin-conjugated silk fibroin with hydroxyapatite grown crystals effectively enhanced osteogenic differentiation of both cell types, as evaluated by alkaline phosphatase activity and calcium content. However the blend of hydroxyapatite/low deacetylation degree chitosan hybrid materials did not support cell growth. Furthermore, the blended hydroxyapatite in the bulk scaffold was found to be less effective for osteogenic differentiation than the scaffold with hydroxyapatite grown crystals. The comparative study between MC3T3-E1 and MSC showed that both cell types had similar trend of proliferation and osteogenic differentiation on the same material. Also, higher proliferative rate of MC3T3-E1 than MSC was observed.     

Role of capsaicin sensory nerves and EGF in the healing of gastric ulcer in rats

Accumulating evidence indicates that capsaicin sensitive afferent fibers play a pivotal role not only in gastroprotection but also in ulcer healing. Denervation of capsaicin sensitive afferent fibers exerts an adverse action on these effects. However, whether such an action is mediated through a depression on epidermal growth factor (EGF) is undefined. In this study, the effects of denervation of sensory neurons with capsaicin (100 mg/kg, s.c.) on acetic acid-induced chronic gastric ulcers and their relationship with the EGF expression in salivary glands, serum and gastric mucosa were investigated. Capsaicin significantly increased ulcer size, decreased gastric mucosal cell proliferation at the ulcer margin, angiogenesis in the granulation tissue and also gastric mucus content. Ulcer induction by itself dramatically elevated EGF levels in salivary glands and serum on day 1 and 4, and also in the gastric mucosa on day 4. However, capsaicin completely abolished these effects. It is concluded that stimulation of EGF expression in salivary glands and serum may be one of the mechanisms by which capsaicin sensitive nerves contribute to the gastroprotective and ulcer healing actions in the stomach.     

Conditioned medium from osteocytes stimulates the proliferation of bone marrow mesenchymal stem cells and their differentiation into osteoblasts

Osteocytes are the most abundant cells in bone and there is increasing evidence that they control bone remodeling via direct cell-to-cell contacts and by soluble factors. In the present study, we have used the MLO-Y4 cell line to study the effect of osteocytes on the proliferation, differentiation and bone-forming capacity of bone marrow mesenchymal stem cells (MSC). Conditioned media (CM) from osteocytic MLO-Y4 and osteoblastic MC3T3-E1 cell lines were collected and added on mouse bone marrow cultures, in which MSC were induced to osteoblasts. There was a significant increase in alkaline phosphatase activity and osteocalcin expression in the presence of MLO-Y4 CM. No such stimulus could be observed with MC3T3-E1 CM. There was almost 4-fold increase in bone formation and up to 2-fold increase in the proliferation of MSC with MLO-Y4 CM. The highly proliferating bone marrow cells were negative for ALP and OCN, suggesting that they could represent early osteoblast precursors. MLO-Y4 CM did not enhance the viability of mature osteoblasts nor protected them of apoptosis. This is the first study to describe soluble signals between osteocytes and osteoblasts and there most likely are several still unidentified or unknown factors in osteocyte CM. We conclude that osteocytes have an active stimulatory role in controlling bone formation.