Profile of cortisol, glycaemia, and blood parameters of American Bullfrog tadpoles Lithobates catesbeianus exposed to density and hypoxia stressors / Perfil de cortisol, glicemia, e parâmetros sanguineos de girinos de rã-touro Lithobates catesbeianus expostos a diferentes densidades e hipóxia

The aim of this study was to evaluate alterations to the physiological profile (cortisol, glycaemia, and blood parameters) of Lithobates catesbeianus caused by the stressors density and hypoxia. The organisms were in the prometamorphosis stage and exposed to different tadpole densities: 1 tadpole/L (T1), 5 tadpoles/L (T2), and 10 tadpoles/L (T3) for 12 days. The blood was collected through the rupture of the caudal blood vessel and collected under normoxia (immediate collection) and hypoxia (after 15 minutes of air exposure) conditions. Cortisol levels rose on the fourth and eighth days of treatment and returned to basal levels by the end of the experiment. The stressor mechanisms tested did not affect glycaemia. White blood cells (total number of lymphocytes, neutrophils, and eosinophils) showed a significant difference at the twelfth day of the experiment when compared with the start of the experiment. We concluded that, under controlled conditions, a density of up to 10 tadpoles/L and air exposure for 15 minutes did not cause harmful physiological alterations during the experimental period. The answer to these stressors maybe was in another hormonal level (corticosterone).(AU)

O objetivo deste estudo foi avaliar a resposta fisiológica (cortisol, glicemia e parâmetros sanguineos) de girinos de rã-touro (Lithobates catesbeianus) em diferentes densidades e após exposição aérea. Os animais utilizados no experimento estavam entre os estágios 31 a 39, na fase de pró-metamorfose sendo testados 1 girino/L (Tratamento 1), 5 girinos/L (Tratamento 2) e 10 girinos/L (Tratamento 3), conduzidos em 3 réplicas simultâneas durante 12 dias. O sangue foi retirado por rompimento do vaso caudal na condição de Normóxia - N (tempo zero) e Hipóxia - H (tempo de 15 minutos de exposição ao ar). Foi observado um aumento nos valores de cortisol, aos 4 e 8 dias de exposição aérea retornando aos valores basais ao final do experimento, apesar de não haver diferenças significativas. A glicemia não apresentou diferenças significativas quanto aos estressores aplicados. Os parâmetros hematológicos da série branca, principalmente, o número de linfócitos, neutrófilos e eosinófilos mostraram diferença significativa aos 12 dias de experimentação quando comparados com o momento zero; concluindo-se que, em condições controladas, o adensamento de até 10 girinos/litro e a exposição aérea por 15 minutos não apresentou danos aos girinos de rã-touro durante o período experimental. O padrão de resposta a estes estímulos talvez seja expresso em outro nível hormonal (corticosterona).(AU)

Identification of immunoreactive plasma and stomach ghrelin, and expression of stomach ghrelin mRNA in the bullfrog, Rana catesbeiana.

In this study, we established a radioimmunoassay (RIA) specific for ghrelin from the bullfrog Rana catesbeiana using a novel antibody raised against the C-terminal amino acid sequence of bullfrog ghrelin [13-28]. We also examined the distribution of ghrelin-producing cells in the stomachs of bullfrogs using this antibody and a cRNA probe specific for the bullfrog ghrelin gene. Ghrelin levels in plasma and stomach extracts were approximately 150 fmol/ml and 83-135 fmol/mg wet tissue, respectively. Reverse-phase high performance liquid chromatographic analysis, combined with bullfrog ghrelin RIA, revealed that ghrelin immunoreactivity in the stomach was composed of non-acylated ghrelin (des-acyl ghrelin) and several acylated forms of ghrelin bearing different fatty acid modifications, which could induce increases in intracellular Ca2+ in cells expressing the rat GH secretagogue receptor. In the stomach, the major storage form was acylated ghrelin. In bullfrog plasma, however, the majority of ghrelin immunoreactivity was des-acyl ghrelin and C-terminal fragments of frog ghrelin. Acylated ghrelin forms comprised only minor peaks. Ghrelin-immunopositive and ghrelin mRNA-expressing cells were observed within the mucosal layer of the stomach. Following starvation, significant increases in plasma ghrelin levels and stomach ghrelin mRNA levels were observed as early as 10 days after starvation. These results indicate that ghrelin is present in the stomach and plasma of the bullfrog, which can be detected with our novel antibody. Interestingly, the primary storage form of ghrelin in the stomach differed from the circulating form dominating in the plasma. Furthermore, increases in ghrelin levels in plasma and mRNA levels in the stomach after starvation suggest the possible involvement of ghrelin in energy homeostasis in the bullfrog.

Blood and urine physiological values in farm-cultured Rana catesbeiana (Anura: Ranidae) in Argentina

A total of 302 samples of healthy farm-cultured Rana catesbeiana specimens (9-21 months-old, 50- 350 g liveweight, 50% each sex) from the north-east of Argentina, were analyzed through spectrophotometry, electrophoresis, densitometry, refractometry and microscopy in order to obtain blood and urine normal values. Confidence intervals (p<0.05) for PCV (28.6-31.6%), RBC (0.40-0.44 T/L), MCV (686-732 fL), hemoglobin (6.41-7.20 g/dL), MCH (151-164 pg), MCHC (22.6-24.0%), WBC (18.7-22.3 G/L), neutrophils (58.4-63.4%), lymphocytes (23.9-29.8%), monocytes (2.1-3.8%), eosinophils (4.6-7.0%), basophils (2.9-4.1%), bleeding time (289-393s), coagulation time (452-696s), prothrombin time (76-128s), urinary density (1.0061-1.0089 g/mL), urinary pH (6,38-6.96), fibrinogen (0.59-0.99 g/dL), total protein (4.19-4.49 g/dL), albumin (1.49-1.67 g/dL), alpha-1 globulin (0.20-0.24 g/dL), alpha-2 globulin (0.48-0.54 g/dL), beta globulin (0.68-0.77 g/dL), gamma globulin (1.28-1.42 g/dL), albumin/globulin ratio (0.50-0.58), creatinine (4.09-5.56 mg/L), urea (76.1-92.4 mg/L), uric acid (11.5-15.4 mg/L), triglycerides (0.34-0.52 g/L), total cholesterol (0.56-0.67 g/L), HDL-C (0.03-0.05 g/L), LDL-C (0.34-0.44 g/L), alpha lipoprotein (6.01-8.67%), beta lipoprotein (91.3-93.9%), glucose (0.45-0.54 g/L), Na (116-121 meq/L), K (3.42-3.81 meq/L), Cl (100-116 meq/L), Ca (7.98-8.61 mg/dL), P (8.31- 9.36 mg/dL), Mg (2.26-2.55 mg/dL), Fe (105-178 ug/dL), ALP (144-170 IU/L), ALT (10.0-14.8 IU/L), AST (42.8-53.4 IU/L), GGT (7.8-10.6 IU/L), LDH (99-135 IU/L), CHE (151-185 IU/L) and CPK (365-500 IU/L), were obtained. Some parameter ranges were similar to those obtained in amphibians, birds or mammals; others were very different. These parameters are useful to evaluate sanitary, metabolic and nutritional state on captive bullfrogs

Con el propósito de obtener valores normales sanguíneos y urinarios, 302 muestras de ejemplares sanos de Rana catesbeiana del nordeste argentino (9-21 meses de edad, 50-350 g de peso vivo, 50% de cada sexo), fueron analizados por espectrofotometría, electroforesis, densitometría, refractometría y microscopía. Fueron obtenidos intervalos de confianza (p<0.05) para hematocrito (28.6-31.6%), eritrocitos (0.40-0.44 T/L), VCM (686-732 fL), hemoglobina (6.41-7.20 g/dL), HCM (151-164 pg), CHCM (22.6-24.0%), leucocitos (18.7-22.3 G/L), neutrófilos (58.4-63.4%), linfocitos (23.9-29.8%), monocitos (2.1-3.8%), eosinófilos (4.6-7.0%), basófilos (2.9-4.1%), tiempo de sangría (289-393s), tiempo de coagulación (452- 696s), tiempo de protrombina (76-128s), densidad urinaria (1.0061-1.0089 g/mL), pH urinario (6.38-6.96), fibrinógeno (0.59-0.99 g/dL), proteínas totales (4.19-4.49 g/dL), albúmina (1.49-1.67 g/dL), alfa-1 globulina (0.20-0.24 g/dL), alfa-2 globulina (0.48-0.54 g/dL), beta globulina (0.68-0.77 g/dL), gamma globulina (1.28-1.42 g/dL), relación albúmina/globulinas (0.50-0.58), creatinina (4.09-5.56 mg/L), urea (76.1-92.4 mg/L), ácido úrico (11.5-15.4 mg/L), triglicéridos (0.34-0.52 g/L), colesterol total (0.56-0.67 g/L), C-HDL (0.03-0.05 g/L), C-LDL (0.34-0.44 g/L), alfa lipoproteína (6.01-8.67%), beta lipoproteína (91.3-93.9%), glucosa (0.45-0.54 g/L), Na (116-121 meq/L), K (3.42- 3.81 meq/L), Cl (100-116 meq/L), Ca (7.98-8.61 mg/dL), P (8.31-9.36 mg/dL), Mg (2.26-2.55 mg/dL), Fe (105-178 ug/dL), ALP (144-170 IU/L), ALT (10.0-14.8 IU/L), AST (42.8-53.4 IU/L), GGT (7.8-10.6 IU/L), LDH (99-135 IU/L), CHE (151-185 IU/L) y CPK (365-500 IU/L). Algunos intervalos fueron semejantes a los obtenidos en anfibios, aves o mamíferos, pero otros resultaron muy diferentes. Estos parámetros son útiles para evaluar estados sanitario, metabólico y nutricional de la rana toro en cautiverio

Plasma retinoid profile in bullfrogs, Rana catesbeiana, in relation to agricultural intensity of sub-watersheds in the Yamaska River drainage basin, Québec, Canada.

Amphibian populations are decreasing globally and the causes are presently unclear. Retinoids have been extensively studied in other vertebrate classes where they are associated with pleiotropic effects such as susceptibility to disease (including cancer and parasitic infections), deformities and reproduction. To investigate the hypothesis that retinoid homeostasis is influenced by agricultural activities, blood samples were collected from adult bullfrogs, Rana catesbeiana, at each of six sub-watersheds chosen to represent a gradient of agricultural intensity within the Yamaska River drainage basin. Samples of surface water were collected at each of the study sites approximately 1 month after spraying and analyzed for 53 pesticides. Male body weight was significantly different (p<0.001) between study sites with the smallest bullfrogs captured from the Rivière à la Barbue sub-watershed associated with high agricultural intensity. A significant linear regression (p<0.001; R2=0.176) was obtained between plasma retinol and body weight. Plasma retinol concentrations were significantly different between study sites (p<0.001) being lowest at both Rivière Noire and Rivière à la Barbue. More than 60% of the land area in these sub-watersheds is under intensive corn-soya cultivation and surface water contained the highest concentrations of the herbicides atrazine, deethyl-atrazine, simazine, metolachlor, dimethenamide, chlopyralide, dicamba and bentazone. Plasma 13-cis-4-oxo-retinoic acid was significantly different (p<0.001) between sub-watersheds, however this effect was apparently unrelated to agricultural intensity. Plasma retinol was negatively correlated (p=0.026; r=-0.237) with plasma 13-cis-4-oxo-retinoic acid. These results suggest that retinoid homeostasis in bullfrogs may be influenced by agricultural practices.

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.

Regulation of c-erbA-alpha messenger RNA species in tadpole erythrocytes by thyroid hormone.

Putative thyroid hormone (TH) nuclear receptors have been detected in several tissues of Rana catesbeiana tadpoles. T3 receptor number (sites per nucleus) in red blood cells (RBCs) and tail increases substantially just before metamorphic climax or in response to exogenous TH; in contrast, receptor number in liver remains relatively constant. TH receptors in mammals and birds are thought to be encoded by a c-erbA gene. In the present study, two c-erbA cDNAs, one prepared from Xenopus laevis oocytes (XenTR alpha 1) and one prepared from Rana catesbeiana tail (RC12), were used to examine the c-erbA-related mRNA species in Rana catesbeiana tissues and determine their role in the TH induction of tadpole RBC receptor number. XenTR alpha 1 encodes a protein with T3-binding properties typical of TH receptors. RC12 is almost 99% homologous with XenTR alpha 1 at the amino acid level and contains all of the putative T3-binding region and most of the DNA-binding region. Using either cDNA as a probe, it was found that two major species of c-erbA-related mRNA species (2.6 and 4.0 kilobases) were clearly evident in tadpole RBCs, tail, and liver. A third, more diffuse band (approximately 5.0 kilobases) was observed in RBC and tail. In RBCs, but not in liver, the combined level of c-erbA-related mRNA species was increased during spontaneous metamorphosis or after administration of TH. Furthermore, the TH-induced increase in both c-erbA-related mRNA species and receptor number in RBCs was prevented if actinomycin-D was administered with TH.(ABSTRACT TRUNCATED AT 250 WORDS)

Erythroid heterokaryons: a system for investigating the functional role of trans-acting factors in developmental hemoglobin switching.

We have shown that erythroid cells from widely divergent species such as amphibians and mammals can be efficiently fused using either calcium phosphate bridges or polyethylene glycol. Transient heterokaryons of adult mouse erythroid (MEL) cells and Rana catesbeiana (bullfrog) tadpole erythroid cells produce adult Rana alpha globin mRNA and adult Rana hemoglobin (Hb) tetramers. Rana tadpole/adult Xenopus erythroid heterokaryons also exhibit this switch to adult Rana globin gene expression. These results indicate that trans-acting factors--and the globin gene regulatory mechanism of which they are a part--are conserved in vertebrate phylogeny. We also wish to know whether the reciprocal Hb switch occurs in each of these two types of heterokaryons, i.e., whether embryonic or fetal globin genes are reactivated in the adult nucleus. Experiments to answer these questions are in progress and are briefly discussed. The influence of stage of erythroid differentiation of the larval and adult donor cells on the cross-inductions is also being explored. These types of experiments should indicate which cells will be the best sources of stimulatory and inhibitory factors that are globin-gene specific. This system may be useful as an in situ assay for the function of purified trans-factors, which could be encapsulated within RBC ghosts and delivered via cell fusion.

Seasonal and stress related changes in plasma gonadotropins, sex steroids, and corticosterone in the bullfrog, Rana catesbeiana.

Studies of seasonal gonadal cycles combined with direct measurements of plasma levels of the two gonadotropins (FSH and LH), several gonadal steroids (estrogen, E; progesterone, P; testosterone, T; and 5 alpha-dihydrotestosterone, DHT), and the interrenal steroid corticosterone (B) were made in male and female bullfrogs in central California over a 5-year period (between 1976 and 1981). During the course of these studies, we discovered that levels of plasma gonadotropins and steroids are highly labile and particularly sensitive to the effects of captivity, especially in males. In animals captured and sampled repeatedly in the field over a 3-day period, hormone levels remained constant, but if held in collecting sacks, gonadotropin and gonadal steroids began to drop within 2--4 hr and usually reached "baseline" levels within 20 hr. This effect was apparent in all seasons, except occasionally in early spring when hormone levels dropped by only about half. Hormone levels were also generally depressed by the time commercially collected frogs reached local supply houses. Plasma B increased within 30 min of capture and remained high for days in captive animals. Blood samples taken from several hundred animals at the time of capture reveal pronounced seasonal cycles in all hormones measured. These changes are discussed in connection with other gross changes in gonadal condition and with regard to possible interactions among gonadotropins and steroids. Both sexes show a general elevation of hormone levels in spring and early summer, but the sexes differ somewhat both in timing and in magnitude of the changes, as well as in the nature of the dominant steroids. Pronounced "surges" in gonadotropins are evident around the time of gamate release in bot sexes, but the temporal pattern of these surges is not the same for ovulation and spermiation; an elevation in plasma P is associated with the periovulatory surge in gonadotropins. Results were not entirely consistent with expectation of pituitary--gonadal relationships. Levels of plasma gonadotropins and steroids did not show the reciprocal relationship expected from a simple negative feed-back between gonadal and pituitary secretion, nor did changes in gonadotropins and gonadal activities show the consistent positive correlation expected from a direct dependence of gonadal function on circulating gonadotropins. In females, plasma T, but not E, correlated with ovarian growth. Plasma T in females reached much higher levels than in males, but DHT was higher in males. Androgens were generally elevated during the period of sexual activity in males, but absolute levels did not correlate well with individual differences in sexual behavior. Thus, seasonal changes in testicular and ovarian activities cannot be accounted for solely by seasonal cycles in circulating gonadotropin levels.

Cathodal proteins from primitive (embryonic) red cells of amphibia. Isolation and characterization.

A group of abundant (15% of the soluble protein) nonhemoglobin proteins was isolated from the primitive (embryonic) red cells found in tadpoles, using the cationic properties of the proteins at pH 8.6 to separate them from hemoglobin and other red cell proteins. The cathodal proteins (CP) were resolved into five components, and the two most predominant proteins were separated and characterized. Purified CP-1b and CP-2 had an amino acid composition similar to that of unfractionated cathodal proteins and to each other, except for small variations in the lysine and half-cystine content. The molecular weight of the purified CP-1b and CP-2 was 13 to 14,000, determined by gel filtration chromatography and electrophoresis in the presence of sodium dodecyl sulfate. Cathodal proteins were immunologically related although there were quantitative differences in reactivity. The concentration of cathodal proteins in primitive (embryonic) red cells was 100 times that in definitive (adult) red cells coincided with the replacement of primitive red cells. The synthesis of the cathodal proteins appeared to continue throughout the life of the primitive red cells; when hemoglobin synthesis declined in primitive red cells, approximately half of the protein synthesized by the cells was cathodal protein. Although the function of the cathodal proteins is as yet unknown, the data suggest that the cathodal proteins are a unique characteristic of erythroid differentiation in early development.