Morphological characterization of the anuran integument of the Proceratophrys and Odontophrynus genera (Amphibia, Anuran, Leptodactylidae).

The morphological characteristics of the leptodactylid integument of Proceratophrys and Odontophrynus genera were investigated by means of stereoscopic, low vacuum scanning electron and light microscopy. The integument surface of Proceratophrys boiei, Proceratophrys laticeps and Proceratophrys appendiculata exhibited several projections, while the integument of Odontophrynus americanus had rounded elevations with smooth profile. Light microscopic observations showed the basic integument morphology for all anurans, i.e., an epidermis and a dermis, which is subdivided into a spongious layer and a compact layer. The epidermis is formed by basal, intermediary and cornified layers. However, in Proceratophrys genus the cornified layer had an irregular outline, while in O. americanus the external surface was smooth. In the spongious dermis, mucous and venom exocrine glands were observed, but in O. americanus an exclusive glandular type with apocrine secretory pattern was identified. The integument morphology showed peculiar characteristics that may be helpful for genus distinction. Thus, morphological methods may be considered as an efficient means to characterize and to differentiate anuran genera.

Dermatan sulfate is the major metachromatic glycosaminoglycan in the integument of the anuran Bufo ictericus.

Glycosaminoglycans from the ventral and dorsal integuments of the anuran Bufo ictericus were characterized based on biochemical and histochemical methods. Dermatan sulfate is the major metachromatic glycosaminoglycan found in these tissues, but small amount of heparan sulfate was also detected. The average molecular mass of the dermatan sulfate is approximately 20 kDa, similar to the glycosaminoglycan isolated from mammalian skin. In addition, the amphibian integument contains high amounts of hyaluronic acid, especially in the ventral area. We also observed that the glycosaminoglycans occur in the anuran integument as irregular deposits through the spongious dermis and in the mast cells, as revealed by histochemical analysis using Alcian blue, dimethylmethylene blue and toluidine blue stains. The concentration and composition of glycosaminoglycans found in the amphibian integument resemble those from mammalian skin except for the higher concentration of hyaluronic acid in the amphibian tissue. Possibly, this observation indicates that the function of the sulfated glycosaminoglycan in these tissues has been preserved during evolution, although the amphibian integument and the human skin have their own particular physiology.

Morphology of Bufo ictericus integument (Amphibia, Bufonidae).

Bufo ictericus integument was investigated by stereoscopic, low vacuum scanning electron microscopy, transmission electron microscopy and light microscopy. The studies revealed, that the dorsal integument surface is rougher than ventral. Three types of projections are visualized: larger rounded verrucae, smaller conical cornified tubercles, and conical short spines. Prominent verrucae are observed on the dorsal surface, being flatter on the ventral surface. The tubercles are visualized only on the dorsal surface. The verrucae are separated by grooves that may contribute spreading and retention of the glandular secretion upon the integument. The pattern of the epidermal grooves is also important for water distribution, protecting the animal against desiccation. The epidermis is composed of a stratified epithelium with intraepithelial blood vessels, where keratinocytes predominate, but flask cells, and Merkel cells also occur. In the spongious dermis, cutaneous glands are visualized. The compact dermis is a series of alternating layers of bundles of collagenous fibers, and between spongious and compact dermis there are basophilic areas that correspond to Eberth-Katschenko layer. The dorsal and the ventral surfaces of B. ictericus are morphologically distinct. The integument structure is related to the physiology of each surface and represents an adaptation to habitat, reflecting a lifestyle of the animal.

Temperature induced alterations in the liver of wall lizard (Hemidactylus frenatus): morphological and biochemical parameters.

Wall lizards (Hemidactylus frenatus) were adapted to 20, 25, and 30 degrees C, and the liver was examined using standard transmission electron microscopy (TEM) and biochemical analysis. Peroxisomes were visualized after using the 3,3'-diaminobenzidine (DAB) technique. Catalase, uricase and protein content were determined biochemically. The hepatocytes of animals adapted to higher temperature displayed larger lipid inclusions than those of animals adapted to lower temperature. Rough endoplasmic reticulum was better developed in the animals kept at low temperature (20 and 25 degrees C) than in the animals held at 30 degrees C. Cytoplasmic crystalline structures were visualized, and better developed in the hepatocytes at 25 degrees C. Peroxisomes varied with the temperature, being more frequent in the animals kept at 20 degrees C, while the bigger ones prevailed in the animals kept at 30 degrees C. The higher catalase activity at higher temperature was correlated to an increase in staining intensity of DAB-incubated peroxisomes as visualized cytochemically in TEM. The biochemical results confirmed the cytochemical reaction observed by TEM. The hepatocytes of the animals at 30 degrees C showed a reduction in the number of peroxisomes, however, at this temperature the largest peroxisomes with a stronger reaction to DAB and a higher activity of catalase predominate. In contrast, the uricase activity showed no significant variation in relation to adaptation temperature. Overall, these data show the morphological and functional plasticity of hepatocytes to temperature adaptation of H. frenatus.

Bidder's organ of Bufo ictericus: a light and electron microscopy analysis.

Male toads of the Bufonidae Family have rudimentary ovaries designated Bidder's organs, and if the testes are removed this organ develops into a functional ovary, representing a morphological strategy for the reproduction of the species. The Bidder's organ of Bufo ictericus was examined using routine and histochemical techniques by light microscopy and transmission electron microscopy. Each Bidder's organ presented a typical ovarian morphology, being composed of a cortex and a medulla. Bidderian follicles in different stages of development were visualized in the cortex, where they are better developed. The germ cells exhibit a large oocyte with a round-shaped nucleus. The Bidderian follicles are supported by a loose net of reticular fibers. In the medullar region, collagen fibers were immersed in the matrix rich in blood vessels that also contained a small quantity of neutral glycoproteins rich in hexose and/or sialic acid and carboxylated polymers with a characteristic distribution of glycosaminoglycans. The oocyte and the follicular cells were separated by a narrow space containing microvilli. The oocyte exhibit a well developed smooth endoplasmic reticulum, a poorly developed Golgi apparatus, and occasional lysosomes. Concentric cisternal complexes are often visualized; however, their morphological significance remains unclear. The peroxisomes display a fine granular matrix without a crystalline core, with a weak 3,3'-diaminobenzidine-reaction. Intimate association between peroxisomes, peroxisomes and lipid inclusions was observed in the oocyte, suggesting its participation in yolk metabolism.

Histopathologic analysis of hamster hepatocytes submitted to experimental infection with Leishmania donovani.

Hepatocytes from different vertebrates are used increasingly as models of environmentally driven cell structure plasticity and for the investigation of ultrastructural pathological patterns induced by cell injury. The present study was carried out to assess the morphological changes in hamster hepatocytes subjected to chronic infection by amastigote forms of Leishmania donovani. Liver fragments were processed for routine light and transmission electron microscopy. For cytochemical visualization of peroxisomes, liver slices were incubated in alkaline 3,3'-diaminobenzidine (DAB) medium at pH 10.0. The results showed that the presence of Leishmania donovani induced distinct ultrastructural changes in the liver acinus (zone 2). The significant pathological changes in hepatocytes consisted of disruption of the endomembrane system and alterations of both the peroxisomal compartment and the distribution of hepatic glycogen. Particularly, hepatic peroxisomes exhibited different shapes and sizes, with modifications of the peroxisomal matrix, including absence of the catalase reaction. These observations suggest an adaptive response of hepatocytes, with cytological reorganization after parasitic infection. The presence of DAB-negative peroxisomes could be morphological evidence of a metabolic disturbance of this organelle. The parasitic infection, through deregulation of the cytokene network, is probably responsible for those structural alterations, since similar changes have been observed in vivo and in vitro.

Electron microscopical investigation on aldrin-induced hepatocyte pathology in Rana catesbeiana, with special emphasis on peroxisomes.

We examined the effect of aldrin on hepatocyte ultrastructure in liver of Rana catesbeiana. The frogs were experimentally exposed to chemical substance and liver fragments processed for routine transmission electron microscopy. Hepatic peroxisomes were visualized after incubation with alkaline 3,3'-diaminobezidine (DAB) method. Ultrastructural analysis revealed progressive hepatocyte changes induced by this drug. After 2-weeks, in the hepatocytes the nuclear envelop and the cisternae of both smooth and rough endoplasmic reticulum (SER und RER, respectively) were unusually enlarged. Reduction of glycogen granules associated with an increased frequency of lysosomes was observed. Normal appearing peroxisomes were present in clusters. Lipid droplets were also visualuzed. After 4-weeks, there was a new increase of glcogen associated with a great number of mitochondria and peroxisomes. Moreover, SER und RER were still dilated. Intracellular lipid inclusions became more abundant. These results suggest that the aldrin 250 induces ultrastructural changes in the hepatocyte of Rana catesbeiana.

[Effect of starvation on the ultrastructure of hepatocytes of Hemidactylus frenatus (Lacertilia: Gekkonidae) with special emphasis on peroxisomes]. / Der Einfluss von Nahrungsentzug auf die Ultrastruktur der Hepatocyten von Hemidactylus frenatus (Lacertilia: Gekkonidae) mit besonderer Berücksichtigung der Peroxisomen.

The influence of starvation on hepatocyte ultrastructure of Hemidactylus frenatus (Lacertilia: Gekkonidae) was investigated with special emphasis on peroxisomes. Wall lizards (Hemidactylus frenatus) were sacrificed after different periods of starvation and their livers were processed for standard transmission electron microscopy. Peroxisomes were demonstrated by means of the 3,3'-diaminobenzidine (DAB) cytochemical technique. A control group consisted of individuals which were fed "ad libitum" with Tenebrio molitor larvae. After a 7-day period of starvation the ultrastructural observation of hepatocytes disclosed a marked reduction of glycogen and lipid inclusions associated with fragmentation of the endoplasmic reticulum (ER). In later stages of starvation (14 and 25 days) ER proliferation and partial reconstruction of glycogen aggregations were observed. Increasing numbers of peroxisomes were arranged either in clusters (14 days) or in close association with mitochondria, lipid droplets and elongated crystalloid structures (25 days). Particularly noteworthy is the increasing cytochemical response of these organelles to the DAB reaction, suggesting greater metabolic activity of catalase. These data suggest that morphological and functional plasticity of hepatocytes may contribute to adaptation of Hemidactylus frenatus to prolonged starvation.

Ultrastructural and histochemical study of collagen fibres types I and III in the venom gland of Bothrops jararaca during secretory cycle.

Fragments of snake (Bothrops jararaca) venom gland were analysed by light and transmission electron microscopy in order to characterize the changes in collagen fibres types I and III in the intertubular gland septa during the secretory cycle. The snakes were sacrificed at 45 days (unmilked group), 6 h, 4 and 8 days after manual extraction of the venom. The fragments were fixed, processed according to standard histologic technique for embedding in paraffin, and stained with haematoxylin-eosin and Gomori's trichrome and submitted to Gomori's silver impregnation technique and picrosirius-polarization method. For transmission electron microscopy the fragments were fixed and processed for embedding in Spurr's medium. At the 45th day (the gland at rest), when the secretory activity was at a minimum, the septa were narrow and filled with densely packed collagen fibrils. At 6 h, the septa were enlarged and exhibited wide spaces filled with finely granular Alcian Blue-positive material. Until the 8th day, the septa were narrower and the histologic aspect resembled that of the gland at rest. The results demonstrated structural modifications in the glandular septa according to the different periods of the secretory cycle. These modifications can be associated with the transformation in the secretory epithelium during the venom synthesis cycle.

The first report of the occurrence of cilium in fat-storing cells in the reptilian liver (Eumeces algeriensis, Daudin 1802).

Fat-storing cells in the liver of an adult Schneider's skink were investigated by means of transmission electron microscopy. The fat-storing cells were localized in the perisinusoidal space or in the hepatic "recessus". They revealed a single cilium originating from one of the paired centrioles and projecting into the perisinusoidal space. The functional significance of a single cilium in the fat-storing cell is still unclear.