Apoptosis in larval and frog skin of Rana pipiens, R. catesbeiana, and Ceratophrys ornata.

Apoptosis (programmed cell death) occurs during normal development of anurans in organs such as gills, gut, and tail. For example, apoptotic cells have been reported in the luminal epithelium along the length of the digestive tract of both larvae and frogs; however, timing of the peak number of such cells varies in different species. The purpose of the present study was to ascertain whether apoptosis also varies by species during metamorphic restructuring of the skin (as larval epithelium is replaced by adult epidermis). To determine this, cross-sections of dorsal skin from representative larval stages and frogs of Rana pipiens, R. catesbeiana, and Ceratophrys ornata were incubated with monoclonal antibody against active caspase-3, one of the main enzymes in the apoptotic cascade. We observed apoptotic cells in the epidermis of the skin of the three species and found that such cells were more numerous in larval stages than in frogs and more abundant in the two ranid species than in C. ornata. These results contribute to our understanding of metamorphic changes in anuran skin.

Apoptosis in the digestive tract of herbivorous Rana pipiens larvae and carnivorous Ceratophrys ornata larvae: an immunohistochemical study.

The lifespan of herbivorous Rana pipiens larvae is ∼3 months, while that of carnivorous Ceratophrys ornata larvae is only about 2 weeks. During metamorphic climax, the larval gut shortens dramatically, especially in R. pipiens, and its luminal epithelium is replaced by adult-type epithelium. To determine when programmed cell death occurs during the metamorphic restructuring of the gut, we prepared cross-sections of the stomach, small intestine, and large intestine from representative larval stages and from juvenile frogs of both species. The sections were incubated with monoclonal antibody against active caspase-3, one of the key enzymes in the apoptotic cascade. We observed apoptosis in some luminal epithelial cells in each of the three regions of the larval gastrointestinal tract of both species. However, apoptotic cells appeared earlier in larval stages of R. pipiens than C. ornata and few were seen in juvenile frogs of either species. The results demonstrate the occurrence of apoptosis in the metamorphic remodeling of the gut of both R. pipiens larvae and C. ornata larvae.

Metamorphic changes in localization of sugars in skin of the leopard frog, Rana pipiens.

A lectin histochemical study was carried out to determine the distribution of specific sugars in glycoconjugates within an important osmoregulatory organ, amphibian skin. Paraffin sections were made of Rana pipiens skin from dorsal and ventral regions of aquatic larvae in representative developmental stages as well as from several body regions of semiaquatic adult frogs. Sections were incubated with horseradish peroxidase (HRP)-conjugated lectins, which bind to specific terminal sugar residues of glycoconjugates. Such sites were visualized by DAB-H2O2. The following HRP-lectins were used: UEA-1 for alpha-L-fucose, SBA for N-acetyl-D-galactosamine, WGA for N-acetyl-beta-D-glucosamine, PNA for beta-galactose, and Con A for alpha-mannose. We found that lectin binding patterns in larvae change during metamorphic climax as the skin undergoes extensive histological remodeling; this results in adult skin with staining patterns that are specific for each lectin and are similar in all body regions. Such findings in R. pipiens provide additional insight into the localization of molecules involved in osmoregulation in amphibian skin.

Cyclosporin A suspends transplantation reactions in the marine sponge Microciona prolifera.

Sponges are the simplest extant animals but nevertheless possess self-nonself recognition that rivals the specificity of the vertebrate MHC. We have used dissociated cell assays and grafting techniques to study tissue acceptance and rejection in the marine sponge Microciona prolifera. Our data show that allogeneic, but not isogeneic, cell contacts trigger cell death and an increased expression of cell adhesion and apoptosis markers in cells that accumulate in graft interfaces. Experiments investigating the possible existence of immune memory in sponges indicate that faster second set reactions are nonspecific. Among the different cellular types, gray cells have been proposed to be the sponge immunocytes. Fluorescence confocal microscopy results from intact live grafts show the migration of autofluorescent gray cells toward graft contact zones and the inhibition of gray cell movements in the presence of nontoxic concentrations of cyclosporin A. These results suggest that cell motility is an important factor involved in sponge self/nonself recognition. Communication between gray cells in grafted tissues does not require cell contact and is carried by an extracellular diffusible marker. The finding that a commonly used immunosuppressor in human transplantation such as cyclosporin A blocks tissue rejection in marine sponges indicates that the cellular mechanisms for regulating this process in vertebrates might have appeared at the very start of metazoan evolution.

Glycoconjugate localization in larval and adult skin of the bullfrog, Rana catesbeiana: a lectin histochemical study.

This study investigates whether or not the distribution of specific glycoconjugates within the skin is related to the regulation of water balance in the aquatic larvae and semiaquatic adults of the bullfrog, Rana catesbeiana. A lectin histochemical study was carried out on paraffin sections of dorsal and ventral skin from tadpoles in representative stages as well as from adult frogs. Sections were stained with the following horseradish peroxidase (HRP)-conjugated lectins, which bind to specific terminal sugar residues of glycoconjugates: UEA 1 for alpha-L-fucose, SBA for N-acetyl-D-galactosamine, WGA for N-acetyl-B-D-glucosamine, and PNA for beta-galactose. Results indicate that lectins serve as markers for specific skin components (e.g., a second ground substance layer within the dermis was revealed by positive UEA 1 staining). Moreover, each lectin has a specific binding pattern that is similar in dorsal and ventral skin; the larval patterns change as the skin undergoes extensive histological and physiological remodeling during metamorphic climax. These findings enhance our understanding of glycoconjugates and their relationship to skin structure and function-in particular, to the regulation of water balance in R. catesbeiana.