Effects of elevated corticosterone on humoral innate and antibody-mediated immunity in southern leopard frog (Lithobates sphenocephalus) tadpoles.

As a free-living larval stage of a vertebrate, tadpoles are good subjects for the study of the development of physiological systems and the study of evolutionarily conserved, context-dependent responses to variable environments. While the basic components of innate and adaptive immune defenses in tadpoles are known, the impact of glucocorticoids on immune defenses in tadpoles is not well-studied. We completed four experiments to assess effects of elevation of corticosterone on humoral innate defenses and antibody-mediated immunity in southern leopard frog tadpoles (Lithobates sphenocephalus). To test humoral innate defense within the tadpoles exposed to short-term and long-term elevation of glucocorticoids, we exposed tadpoles to exogenous corticosterone for different lengths of time in each experiment (0-84 days). We used bacterial killing assays to assess humoral innate immune defense. To test antibody-mediated immune responses, we again exposed tadpoles to exogenous corticosterone, while also exposing them to Aeromonas hydrophila. We used A. hydrophila ELISA comparing IgM and IgY responses among groups. Plasma from corticosterone-dosed tadpoles killed more A. hydrophila than control tadpoles each following a short-term (14 day) and long-term (56 day) exposure to exogenous corticosterone. Conversely, corticosterone-dosed tadpoles had significantly lower IgM and IgY against A. hydrophila after 12 weeks. Our fourth experiment revealed that the lower IgY response is a product of weaker, delayed isotype switching compared with controls. These results show that elevated corticosterone has differential effects on innate and acquired immunity in larval southern leopard frogs, consistent with patterns in more derived vertebrates and in adult frogs.

Vitellogenin of the northern leopard frog (Rana pipiens): development of an ELISA assay and evaluation of induction after immersion in xenobiotic estrogens.

An immunoassay for leopard frog (Rana pipiens) vitellogenin was developed for studying endocrine disruption. Male frogs were injected with estradiol-17ß to stimulate vitellogenin for purification. SDS-PAGE revealed high amounts of a 170-180 kDa protein, which was confirmed to be vitellogenin by Western blotting. Vitellogenin was purified by DEAE chromatography and used to generate a polyclonal antibody. A competitive ELISA was developed for leopard frog vitellogenin with a detection limit of 6.0 ng mL(-1) and a working range of 20-1000 ng mL(-1). The intra-assay coefficient of variation averaged 5.47% for control sera and 9.71% for estrogen-treated sera. The inter-assay coefficient of variation averaged 8.21% for control sera and 9.93% for estrogen-treated sera. Recovery of purified vitellogenin averaged 95.2%. Vitellogenin was measured in male frogs immersed in the estrogenic compound diethylstilbestrol (DES) for various times and doses. Serum vitellogenin was detected within five days after immersion in 1.0 mg L(-1) DES and levels continued to increase through 20 d. In a 20-day dose-response experiment, serum vitellogenin was detected in frogs immersed in 0.01 mg L(-1) DES and vitellogenin concentration increased with dose. Immersion of frogs in one of several xenobiotic estrogens (nonylphenol, octylphenol, bisphenol-A) for 20 d did not increase vitellogenin for any treatment, suggesting that this frog may be less sensitive than fish to endocrine disruptors. Vitellogenin induction in R.pipiens may be a useful amphibian model system for field studies of endocrine disruption, due to its broad geographic range.

Immunomodulation in post-metamorphic northern leopard frogs, Lithobates pipiens, following larval exposure to polybrominated diphenyl ether.

Pollutants and disease are factors implicated in amphibian population declines, and it is hypothesized that these factors exert a synergistic adverse effect, which is mediated by pollutant-induced immunosuppression. Polybrominated diphenyl ethers (PBDEs) are ubiquitous pollutants that can exert immunotoxicity, making them of interest to test effects on amphibian immune function. We orally exposed Lithobates (Rana) pipiens tadpoles to environmentally realistic levels (0-634 ng/g wet diet) of a pentabromodiphenyl ether mixture (DE-71) from as soon as they became free-swimming through metamorphic climax. To assess adaptive immune response in juvenile frogs, we used an enzyme-linked immunosorbent assay to measure specific IgY production following immunization with keyhole limpet hemocyanin (KLH). Specific KLH antibody response was significantly decreased in juvenile frogs that had been exposed to PBDEs as tadpoles. When assessing innate immune responses, we found significantly different neutrophil counts among treatments; however, phagocytic activity of neutrophils was not significantly different. Secretion of antimicrobial skin peptides (AMPs) nonsignificantly decreased with increasing PBDE concentrations, and no significant effect of PBDE treatment was observed on efficacy of AMPs to inhibit chytrid fungus (Batrachochytrium dendrobatidis) growth. Our findings demonstrate that environmentally realistic concentrations of PBDEs are able to alter immune function in frogs; however, further research is needed to determine how these alterations impact disease susceptibility in L. pipiens.

Transformations of selected carotenoids in plasma, liver, and ocular tissues of humans and in nonprimate animal models.

PURPOSE: To determine the stereochemistry of carotenoids in human ocular tissues in comparison with plasma and liver and to elucidate the possible transformations of dietary (3R,3'R,6'R)-lutein and (3R,3'R)-zeaxanthin in the eye. Similarly, to characterize the carotenoid profiles in the eye tissues, plasma, and liver of quails and frogs to determine whether these can serve as appropriate nonprimate animal models for metabolic studies. METHODS: Configurational isomers of carotenoids and their nondietary by-products from pooled human plasma, liver, retinal pigment epithelium (RPE-choroid), ciliary body, iris, and lens were characterized and quantified by high-performance liquid chromatography (HPLC) on a chiral column. Carotenoids and their nondietary by-products in pooled extracts from quail and frog plasma, liver, retina, RPE-choroid, iris, and lens were similarly characterized and quantified. RESULTS: (3R,3'R,6'R)-lutein, (3R,3'R)-zeaxanthin, (3R,3'S; meso)-zeaxanthin, (3R,3'S,6'R)-lutein (3'-epilutein), 3-hydroxy-beta, epsilon -carotene-3'-one, and 5Z- and all-E-lycopene were detected in nearly all human ocular tissues examined. (3R,3'S; meso)-zeaxanthin was not detected in the human plasma and liver but was present in human macula, retina, and RPE-choroid. (3S,3'S)-zeaxanthin was detected in human macula in minute quantities. The carotenoid profiles in quail and frog ocular tissues were somewhat similar to those in humans, with the exception that lycopene was absent. Frog retina, plasma, and liver revealed the presence of (3S,3'S)-zeaxanthin. CONCLUSIONS: The most likely transformations of carotenoids in the human eye involve a series of oxidation-reduction and double-bond isomerization reactions. Quail and frog appear to possess the appropriate enzymes for conversion of dietary (3R,3'R,6'R)-lutein and (3R,3'R)-zeaxanthin to the same nondietary by-products observed in humans and thus may serve as excellent nonprimate animal models for metabolic studies.

Prolactin-like biological activity in the pituitary glands of the marsupial Monodelphis domestica and of the amphibian Rana pipiens detected by a colorimetric Nb2 lymphoma cell proliferation assay.

An inexpensive and reliable colorimetric microplate version of the Nb2 lymphoma cell proliferation bioassay for prolactin (PRL) was developed and optimized. The useful range of the assay is between 0.1 and 12.8 ng/ml in terms of rat pituitary PRL. The assay can accommodate up to 20 microl sample/well. The physiological relevance of the assay was verified by measuring thyrotropin-releasing hormone (TRH)-induced secretion of PRL in pituitary cultures and in serum samples of neonatal rats. Through the use of the colorimetric Nb2 assay, PRL-like bioactivities were demonstrated in pituitary extracts of the marsupial, Monodelphis domestica (1.47 ng PRL/microg protein) and of the amphibian, Rana pipiens (1.86 ng PRL/microg protein). Marsupial and amphibian PRLs are predicted to have low specific activities in the Nb2 assay. Since the PRL values were calculated in terms of a rat PRL standard, they probably underestimate the amounts of PRL present. Parallel dose-response curves were obtained with these pituitary extracts and standard rat PRL over a wide range of dilutions. The Nb2 bioassay may serve as a tool for the purification of PRL from these species. The colorimetric version of the Nb2 bioassay may be a useful alternative to traditional Nb2 assays that rely on direct cell count or [3H]thymidine uptake.

Influence of androgen on the GnRH-stimulated secretion and biosynthesis of gonadotropins in the pituitary of juvenile female bullfrogs, Rana catesbeiana.

The influence of androgen on pituitary sensitivity to gonadotropin-releasing hormone (GnRH) was investigated in juvenile female bullfrogs. Newly metamorphosed bullfrogs were treated in vivo for 7 days or their pituitaries were treated in vitro for 24 hr with 5 alpha-dihydrotestosterone (DHT). Pituitary sensitivity to GnRH was assessed by incubating glands with 100 ng/ml GnRH. The secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) was quantified by separate radioimmunoassays. Gth biosynthesis was quantified using immunoprecipitation to measure the incorporation of [35S]methionine. Prior exposure to DHT, in vitro or in vivo, significantly elevated the GnRH-induced secretion of LH and FSH. However, DHT had a differential influence on the two gonadotropins; both the basal and the GnRH-induced secretion of LH was enhanced, whereas only the GnRH-induced secretion of FSH was elevated. DHT did not significantly alter Gth biosynthesis. Treating older frogs (5 months postmetamorphic) in vivo with DHT (for 7 days) combined with a GnRH agonist (GnRHa) for 3 days enhanced the GnRH-stimulated Gth biosynthesis compared to treatment with either DHT or GnRHa alone. Therefore, while DHT may act on the pituitary to enhance gonadotropin secretion in response to GnRH, this action does not result in a concomitant increase in Gth biosynthesis. Juvenile female bullfrogs may require an increase in both GnRH and DHT in order to stimulate Gth biosynthesis.

Evaluation of a radioimmunoassay for the measurement of amphibian parathyroid hormone.

A commercial mid-region to carboxyl-region parathyroid hormone (PTH) radioimmunoassay (RIA) for measurement of PTH concentration in serum of humans was evaluated for its ability to detect amphibian PTH. The recovery of known amounts of human PTH added to aliquots of pooled frog serum demonstrated that frog serum does not interfere with the RIA system. The mean intraassay coefficient of variation was 0.11. During the spring and summer removal of the parathyroid glands from frogs resulted in significant decreases in serum calcium levels which the RIA revealed were associated with significant decreases in PTH levels. In the winter the parathyroid glands of frogs showed histological changes associated with inactivity. Also, parathyroidectomy of winter frogs did not affect serum calcium concentrations, and PTH levels of experimental and control animals were below or near the level of detection.

Effect of background color and low temperature on skin color and circulating alpha-MSH in two species of leopard frog.

Circulating levels of alpha-melanocyte stimulating hormone (alpha-MSH) in two species of leopard frog, Rana pipiens and R. chiricahuensis, were measured by radioimmunoassay to reveal the correlation between skin color change induced by background color and by low temperature. High levels of alpha-MSH were found in both species of frog on a black background, but R. chiricahuensis had eight times higher levels than R. pipiens, R. chiricahuensis also exhibited the ability to darken its ventral surface, whereas the ventral surface of R. pipiens remained white. Neither skin color nor plasma alpha-MSH of R. pipiens was affected by cold. Low temperature did, however, darken dorsal and ventral skin of R. chiricahuensis in vivo, which corresponded to increased levels of plasma alpha-MSH. Dorsal and ventral skin of R. chiricahuensis, in vitro, darken in a dose-dependent manner to alpha-MSH, but not to cold.

Fixation-associated quantitative variations of DNA fluorescence observed in flow cytometric analysis of hemopoietic cells from adult diploid frogs.

We have examined, by flow cytometry, the apparent DNA content of frog blood cells that had been fixed with either 50% ethanol, 70% ethanol, or 66% methanol, before being stained with either mithramycin, propidium iodide, or Hoechst 33258. After 50% ethanol fixation, regardless of the dye used, the DNA content of the hemopoietic cells appeared unimodal, but after either 70% ethanol or 66% methanol fixation, it appeared bimodal. Cell sorting revealed that the lower and upper modes are represented by erythrocytes (RBCs) and leukocytes (WBCs), respectively. In amphibians, the chromatin of metabolically inactive RBCs is highly condensed relative to the chromatin of metabolically active WBCs. The bimodal distribution of DNA contents seen with 66% methanol and 70% ethanol, but not 50% ethanol, seems to reflect this disparity in the degree of chromatin condensation existing between the RBCs and WBCs. This, in turn, implies that the accessibility of fluorescent DNA dyes to the chromatin of fixed frog hemopoietic cells, especially of RBCs, can be affected by the concentration of alcohol used for their fixation.

Relative oxidation of glutamate and glucose by vertebrate erythrocytes.

1. Lungfish erythrocytes (RBC), unlike those in other species of fishes, oxidize endogenous glutamate at a higher rate than they oxidize glucose. This pattern closely resembles that found in invertebrates. 2. The exogenous glutamate oxidation rate of RBC from most species of fish as well as other groups of vertebrates is approximately equal to or less than that observed for glucose. 3. These findings suggest that the nucleated RBC of most vertebrates appear to rely less on the TCA cycle for energy production and more on the glycolytic metabolism of carbohydrates. 4. The RBC of lungfish are also distinguished from RBC of other vertebrates by their relatively higher permeability to exogenous substrates. For example, chicken RBC have a glucose accumulation rate which is approximately one third that observed for lungfish RBC at twice the medium glucose concentration. 5. The unique characteristics of lungfish RBC may be related to their adaptation to the high concentrations of urea produced during estivation.

Role of sodium on H+ excretion in the integument of the leopard frog Rana pipiens.

1. The northern leopard frog, Rana pipiens, pipiens, in contrast to the southern leopard frog, Rana pipiens, berlandieri, did not demonstrate any significant H+ excretion across its integument even during a challenge of chronic metabolic acidosis. Likewise, no increase in the number of H+ secreting mitochondria-rich cells were observed in the northern frogs. 2. Under normal acid-base conditions in the southern frogs, H+ excretion was found to be dependent on mucosal sodium concentrations, whereas during chronic metabolic acidosis, H+ excretion was independent of mucosal sodium concentrations, but was amiloride sensitive. 3. High salinity adapted southern frogs, under normal and acidotic conditions, had enhanced H+ excretion rates as compared to the control non-salt adapted frogs. 4. Blood analyses demonstrated that significant acid-base changes were the result of systemic acidosis and not due to salt adaptations. Blood Na+ and K+ concentrations were also efficiently maintained during salt adaptations or chronic metabolic acidosis. 5. The results suggest that H+ excretion in epithelia can be influenced by the sodium transport state of the cell and the systemic acid-base profile. Models are proposed explaining these relationships.

Plasma immunoreactive calcitonin in the frog (Rana pipiens).

1. Plasma immunoreactive calcitonin (iCT) and ionic calcium [( Ca]i) were measured in intact frogs (Rana pipiens) within complete 24 hr light-dark cycles over an 18 month period. 2. Plasma iCT exhibits an annual periodicity about the annual mean of 10.0 ng/ml, with an amplitude of 5.4 ng/ml that peaks in October. 3. Within an annual cycle, a significant inverse association exists between the basal monthly levels of plasma iCT and [Ca]i for animals maintained in freshwater control conditions. 4. When subjected to a high calcium environment during the latter half of the year, plasma [Ca]i and iCT were elevated above control levels but exhibited independent cyclic patterns. 5. A distinct seasonal response of increased iCT in a high calcium environment may be related to the secretory activity of the ultimobranchial glands and physiological responsiveness to other calcemic hormones; e.g. parathyroid hormone and vitamin D.

Immunofluorescent detection and localization of thyroxine in blood of adult amphibians.

Indirect immunofluorescent staining was used to detect and localize thyroxine (T4) in blood smears from different species of adult amphibians, namely, Rana pipiens, Rana catesbeiana, Bufo marinus, Xenopus laevis, and Notopthalmus viridescens. Fluorescence, indicative of T4, was observed in both plasma and erythrocytes (RBC) from all individuals of the five species studied. It was weak and diffuse in the plasma and in the cytoplasm of the RBC but was intense in the nuclei (especially the nuclear perimeter) of these cells. The finding of intracellular T4 suggests that thyroid hormone may be of some physiological importance in adult amphibians.

The genome of frog erythrocytes displays centuplicate replications.

Seven nuclear lines derived from erythrocyte nuclei of Rana pipiens were produced by serial nuclear transplantation into oocytes and eggs. Even at the termination of the experiments, embryos and tadpoles developed in the eighth transplant generations. Thus, there was no evidence that the mitotic progeny of the erythrocyte nuclei lost their ability to replicate their genomes and continue cell cycling. We conclude that the genome of noncycling and terminally differentiated erythrocytes maintains its potential for widespread replication and extensive reversal of gene function in excess of a hundred (centuplicate) cell cycles.

Enzymological studies of melanotropins.

The relative stability of natural melanotropins and related synthetic analogues to serum and purified proteolytic enzymes was studied. Both alpha- and beta-MSH were rapidly inactivated by frog serum, but much more slowly by rat serum. beta-MSH was more stable than alpha-MSH to serum inactivation. Both alpha- and beta-MSH were rapidly inactivated by alpha-chymotrypsin and trypsin. The synthetic analogues, [Nle4, D-Phe7]-alpha-MSH and [Cys4, Cys10]-alpha-MSH, were totally resistant to inactivation by frog and rat serum enzymes. [Nle4, D-Phe7]-alpha-MSH was resistant to inactivation by alpha-chymotrypsin and trypsin, whereas [Cys4, Cys10]-alpha-MSH was partially resistant to these enzymes under similar conditions. Melanotropin analogues resistant to inactivation by serum enzymes may prove useful in a variety of physiological studies wherein natural melanotropins would be rapidly inactivated.

A simple method for obtaining blood samples from mature frogs.

A technique was developed for obtaining periodic blood samples from mature frogs. Up to 150 microliter of blood was collected at weekly intervals. Anesthetization was not required. The data compiled with this technique indicated that activity-induced hyperglycemia was minimal.

Inheritance of enzymes and blood proteins in the leopard frog, Rana pipiens: three linkage groups established.

Individuals from natural populations of the leopard frog, Rana pipiens, were analyzed for electrophoretic differences in blood proteins and enzymes from an amputated digit. The proteins examined represent products of 72 loci. Presumptive heterozygotes at multiple loci were selected for experimental crosses. Mendelian inheritance of 18 protein variations were demonstrated in the offspring. Tests for linkage or independent assortment were performed for 75 locus pairs. Three linkage groups were established. Linkage group 1 contains two loci, aconitase-1 (Acon 1) and serum albumin (Alb), with a 19% recombination frequency between them. Linkage group 2 contains four loci, glyoxalase (Gly), acid phosphatase-1 (Ap1), acid phosphatase-2 (AP2), and esterase-5 (Est5). The data show the relationships Gly-21.1%-AP1-0%-AP2-6.3%-Est5, and Gly-25.6%-Est5. Linkage group 3 consists of four closely linked esterase loci. The data, Est1-5.1%-Est6, Est6-1.8%-Est10-1.9%-Est4 and Est6-3.0%-Est4, do not establish a complete order but suggest that Est10 is between Est4 and Est6. These results, with data demonstrating apparent independent assortment of 67 other locus pairs, provide a foundation for establishing the frog genetic map.