Jane C. Kaltenbach

Thyroxine concentrations in blood serum and pericardial fluid of metamorphosing tadpoles and of adult frogs

Thyroxine (T4) was measured by double antibody radioimmunoassay in blood serum and pericardial fluid of individual tadpoles and frogs of Rana catesbeiana. The pattern of change of mean T4 values during tadpole growth and metamorphosis was similar in serum and pericardial fluid. In growing tadpoles, stages V–XVIII, all T4 concentrations were below the limit of detection (<0.20 μg/dl). Mean T4 values first became detectable in early metamorphic stages, XIX, XX; they reached a peak (0.94 μg/dl serum) at stages XXI, XXII, XXIII. Differences in mean values between these three stages were not significant. At the end of metamorphosis, stages XXIV, XXV, mean T4 concentrations dropped to levels similar to those of stages XIX, XX. However, in adult summer frogs mean T4 values for serum and pericardial fluid were somewhat higher than mean values at the end of metamorphosis. Despite this general developmental pattern, T4 concentrations in some individual animals at each stage, including metamorphic stages, were below the limit of detection (<0.20 μg/dl).

Thyrotropin-releasing hormone (TRH): Immunohistochemical distribution in tadpole and frog brain ☆

The anatomical localization of immunoreactive TRH (IR-TRH) was demonstrated by the peroxidase-antiperoxidase technique in the brain and pituitary gland of larval and adult Rana catesbeiana. In the adult frog main sites of IR-TRH are perikarya and neuronal fibers in the preoptic and infundibular nuclei of the hypothalamus and in the amygdala and diagonal band of Broca of the telencephalon. In addition, TRH-positive neuronal fibers and endings were found in the septum, pallium, and brain stem as well as in the preoptico-hypophyseal tract, the external zone of the median eminence (which matures during late larval stages), and the pars nervosa; fibers were less extensive in the pars intermedia, and were absent from the pars distalis. In early larval stages, the magnocellular nucleus of the posterior preoptic area is the main site of immunoreactive perikarya. During late stages the extensive adult pattern of distribution of IR-TRH becomes established. The study represents the first immunohistochemical demonstration of IR-TRH in larval anurans, and serves as a basis for clarification of the neuroendocrine regulation of metamorphosis.

Direct action of thyroxine on skin of the adult newt.

Abstract Thyroxine-cholesterol and cholesterol control pellets were implanted subcutaneously in thyroidectomized, adult newts, Diemictylus viridescens . No molting of the skin was induced by cholesterol control implants or by thyroxine-cholesterol implants containing less than 0.02 μg thyroxine. Localized molting of the skin was induced by 39% of the implants containing approximately 0.02 to 0.96 μg thyroxine. Generalized molting of the skin over the entire surface of the body was induced by 37% of such implants and by all implants containing larger amounts of thyroxine. The occurrence of local molts immediately above and adjacent to thyroxine implants in thyroidectomized newts is attributed to a direct action of thyroxine upon the skin. It is therefore concluded that thyroid hormone probably acts directly upon the skin to cause molting in normal, adult newts.

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.

Histology and lectin-binding patterns in the skin of the terrestrial horned frog Ceratophrys ornata

The terrestrial horned frog, Ceratophrys ornata, lives on a wet substratum and absorbs water through the ventral epidermis; water is lost by evaporation from the dorsal skin. Thus, this species may be useful as a model for determining whether or not skin histology and lectin-binding patterns, indicative of glycoconjugates, are related to skin functions such as osmoregulation and water balance. With this in mind, a histological and lectin-histochemical study was carried out on dorsal and ventral skin of aquatic tadpoles and of a young terrestrial frog of C. ornata. Sections of skin were stained with various dyes to demonstrate general histological features and with two horseradish peroxidase (HRP)-conjugated lectins, Ulex europaeus agglutinin (UEA 1) and soybean agglutinin (SBA) which bind to specific terminal sugar residues of glycoconjugates, namely L-fucose and N-acetyl-D-galactosamine or D-galactose, respectively. In early stage tadpoles both lectin-binding patterns were similar in the bilaminar epidermis of dorsal and ventral skin (i.e., each lectin stained the apical cell layer). However, metamorphic changes resulted in a young frog with typical adult-type skin composed of a stratified squamous epidermis and three distinct types of glands containing glycoconjugates in their secretions. Strikingly different lectin-binding patterns were evident in the epidermis from dorsal and ventral regions of the body. The epidermis from the dorsal region was stained by both lectins; in contrast, that from the ventral region although stained strongly by HRP-SBA, did not react with HRP-UEA 1 indicating that few, if any, fucose residues were present in the ventral epidermis. These findings, as suggested in the discussion, indicate that different glycoconjugate patterns in dorsal and ventral skin may be associated with the regulation of water balance in the frog.

Direct action of thyroxine analogues on molting in the adult newt

Abstract Thyroxine analogue-cholesterol pellets were implanted subcutaneously in thyroid-ectomized, adult newts, Diemictylus viridescens, in order to determine if specific analogues have molt-inducing activity. Many of the analogues tested, depending upon their concentration in the implants, caused generalized or localized molting or both. The occurrence of a local molt in the region of a pellet is attributed to the direct action of the implanted thyroxine analogue upon the skin. Any chemical changes which may have taken place in the analogue must have occurred locally within the peripheral tissues around the implant. Analogues with acetic, propionic, and acrylic acid side chains and those with three iodine atoms bad as much, if not more, molt-inducing activity as thyroxine; those with two iodine atoms had somewhat less activity. Meta- and ortho-thyroxine and single-ring compounds likely to condense to biologically active diphenyl ethers were only effective when tested in relatively high concentrations, while thyronine and iodinated benzene rings unlikely to condense were inactive. In conclusion, the experiments demonstrate the ability of certain thyroxine analogues to induce molting in adult, thyroidectomized newts by a direct, local action upon the skin.

Apoptosis in Microciona prolifera Allografts

remain open under apoptotic conditions, to study the dynamics of gap junctional coupling during cell death, and to determine which molecules may pass from a dying cell to its neighbors to trigger the apoptotic process. Conversely, it is important to recognize that gap-junction-mediated cell death may result from the depletion of essential molecules (e.g., ATP) passing from the healthy cell to its dying neighbor. Previous studies of single Xenopus oocytes have shown that microinjection of cytochrome c induces apoptotic cell death, accompanied by a progressive loss of membrane potential, activation of caspase 3, and DNA fragmentation (6). In the present study we have shown that injection of cytochrome c into one oocyte of a Cx38-coupled pair causes death in both the injected and noninjected cells over a period of 2–3 h (Fig. 1a,b). Pairs of oocytes preinjected with an antisense oligonucleotide to Cx38 did not exhibit bystander cell killing following cytochrome c injection; although the cell into which cytochrome c had been injected did die, its paired neighbor did not. Similarly, in cases where the cells were electrically coupled, but where the cytochrome c injected cell lysed in less than 40 min, the noninjected cell did not die (Fig. 1a,b, left pair). This result suggests that the intercellular channels joining the cytoplasm of the coupled cells must remain intact for longer than 40 min for bystander cell death to occur. It also indicates that bystander killing is not mediated by contact or by extracellular toxins, since the surviving cell continued to be in close apposition to the dying cell but remained intact. Vehicle injections failed to induce cell death in either injected or noninjected cells of electrically coupled pairs. Dual electrode voltage-clamp using GeneClamp 500 amplifi ers controlled by pClamp 8 software (Axon Instruments, Foster City, CA) was used to monitor gap junctional coupling between cell pairs after injecting one of the cells with cytochrome c. The two cells were voltage-clamped to the same potential (40 mV), voltage steps were applied to the noninjected cell, and the current responses of both cells were recorded (7). As shown in Figure 1 (c,d), cells remained electrically coupled despite the gradual death of the injected cell. In addition, we found that the loss of membrane potential resulting from cytochrome c injection reported by Bhuyan et al. (6) in single oocytes could be seen also in cell pairs. After injecting one cell with cytochrome c, both cells underwent

Histochemical patterns in the tadpole tail during normal and thyroxine-induced metamorphosis. I. Alkaline phosphatase, acid phosphatase, esterase, and aminopeptidase.

Abstract The distribution of alkaline phosphatase, acid phosphatase, esterase, and aminopeptidase was demonstrated by histochemical methods in sections of both growing and resorbing tails of Rana pipiens tadpoles. In general, main sites of localization of each of the enzymes were the epidermis and connective tissue, but not striated muscle. In addition, alkaline phosphatase, acid phosphatase, and esterase were present in the ependyma of the spinal cord and the endothelium of various blood vessels. The latter two enzymes were also found in spinal ganglia and, along with aminopeptidase, in the notochord. As the tail shortened during both normal and thyroxine-induced metamorphosis, activity of these enzymes increased; it became particularly prominent in the epidermis and in phagocytes in the connective tissue. These data on the differential distribution of four hydrolytic enzymes in the R. pipiens tail support histochemical and biochemical studies in other species.

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. J. Morphol., 2011.

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 α‐L‐fucose, SBA for N‐acetyl‐D‐galactosamine, WGA for N‐acetyl‐β‐D‐glucosamine, PNA for β‐galactose, and Con A for α‐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. J. Morphol., 2008.