Are thyroid hormones mediators of incubation temperature-induced phenotypes in birds?

Incubation temperature influences a suite of traits in avian offspring. However, the mechanisms underlying expression of these phenotypes are unknown. Given the importance of thyroid hormones in orchestrating developmental processes, we hypothesized that they may act as an upstream mechanism mediating the effects of temperature on hatchling phenotypic traits such as growth and thermoregulation. We found that plasma T3, but not T4 concentrations, differed among newly hatched wood ducks (Aix sponsa) from different embryonic incubation temperatures. T4 at hatching correlated with time spent hatching, and T3 correlated with hatchling body condition, tarsus length, time spent hatching and incubation period. In addition, the T3 : T4 ratio differed among incubation temperatures at hatch. Our findings are consistent with the hypothesis that incubation temperature modulates plasma thyroid hormones which in turn influences multiple aspects of duckling phenotype.

Characterization of the neuropeptide Y system in the frog Silurana tropicalis (Pipidae): three peptides and six receptor subtypes.

Neuropeptide Y and its related peptides PYY and PP (pancreatic polypeptide) are involved in feeding behavior, regulation of the pituitary and the gastrointestinal tract, and numerous other functions. The peptides act on a family of G-protein coupled receptors with 4-7 members in jawed vertebrates. We describe here the NPY system of the Western clawed frog Silurana (Xenopus) tropicalis. Three peptides, NPY, PYY and PP, were identified together with six receptors, namely subtypes Y1, Y2, Y4, Y5, Y7 and Y8. Thus, this frog has all but one of the ancestral seven gnathostome NPY-family receptors, in contrast to mammals which have lost 2-3 of the receptors. Expression levels of mRNA for the peptide and receptor genes were analyzed in a panel of 19 frog tissues using reverse transcriptase quantitative PCR. The peptide mRNAs had broad distribution with highest expression in skin, blood and small intestine. NPY mRNA was present in the three brain regions investigated, but PYY and PP mRNAs were not detectable in any of these. All receptor mRNAs had similar expression profiles with high expression in skin, blood, muscle and heart. Three of the receptors, Y5, Y7 and Y8, could be functionally expressed in HEK-293 cells and characterized with binding studies using the three frog peptides. PYY had the highest affinity for all three receptors (K(i) 0.042-0.34 nM). Also NPY and PP bound to the Y8 receptor with high affinity (0.14 and 0.50 nM). The low affinity of NPY for the Y5 receptor (100-fold lower than PYY) differs from mammals and chicken. This may suggest a less important role of NPY on Y5 in appetite stimulation in the frog compared with amniotes. In conclusion, our characterization of the NPY system in S. tropicalis with its six receptors demonstrates not only greater complexity than in mammals but also some interesting differences in ligand-receptor preferences.

Lessons from evolution: developmental plasticity in vertebrates with complex life cycles.

Developmental plasticity is the property of a given genotype to produce different phenotypes in response to the environmental conditions experienced during development. Chordates have two basic modes of development, direct and indirect. Direct development (mode of humans) was derived evolutionarily from indirect development (mode of many amphibians), the major difference being the presence of a larval stage with indirect development; larvae undergo metamorphosis to the juvenile adult. In amphibians, environmental conditions experienced during the larval stage can lead to extreme plasticity in behaviour, morphology and the timing of metamorphosis and can cause variation in adult phenotypic expression (carry-over effects, or developmental programming). Hormones of the neuroendocrine stress axis play pivotal roles in mediating environmental effects on animal development. Stress hormones, produced in response to a deteriorating larval habitat, accelerate amphibian metamorphosis; in mammals, stress hormones hasten the onset of parturition and play an important role in pre-term birth caused by intra-uterine stress. While stress hormones can promote survival in a deteriorating larval or intra-uterine habitat, costs may be incurred, such as reduced growth and size at metamorphosis or birth. Furthermore, exposure to elevated stress hormones during the tadpole or foetal stage can cause permanent neurological changes, leading to altered physiology and behaviour later in life. The actions of stress hormones in animal development are evolutionarily conserved, and therefore amphibians can serve as important model organisms for research on the mechanisms of developmental plasticity.

Distribution and acute stressor-induced activation of corticotrophin-releasing hormone neurones in the central nervous system of Xenopus laevis.

In mammals, corticotrophin-releasing hormone (CRH) and related peptides are known to play essential roles in the regulation of neuroendocrine, autonomic and behavioural responses to physical and emotional stress. In nonmammalian species, CRH-like peptides are hypothesized to play similar neuroendocrine and neurocrine roles. However, there is relatively little detailed information on the distribution of CRH neurones in the central nervous system (CNS) of nonmammalian vertebrates, and there are currently no comparative data on stress-induced changes in CRH neuronal physiology. We used a specific, affinity-purified antibody raised against synthetic Xenopus laevis CRH to map the distribution of CRH in the CNS of juvenile South African clawed frogs. We then analysed stress-induced changes in CRH immunoreactivity (CRH-ir) throughout the CNS. We found that CRH-positive cell bodies and fibres are widely distributed throughout the brain and rostral spinal cord of juvenile X. laevis. Strong CRH-immunoreactivity (ir) was found in cell bodies and fibres in the anterior preoptic area (POA, an area homologous to the mammalian paraventricular nucleus) and the external zone of the median eminence. Specific CRH-ir cell bodies and fibres were also identified in the septum, pallium and striatum in the telencephalon; the amygdala, bed nucleus of the stria terminalis and various hypothalamic and thalamic nuclei in the diencephalon; the tectum, torus semicircularis and tegmental nuclei of the mesencephalon; the cerebellum and locus coeruleus in the rhombencephalon; and the ventral horn of the rostral spinal cord. To determine if exposure to an acute physical stressor alters CRH neuronal physiology, we exposed juvenile frogs to shaking/handling and conducted morphometric analysis. Plasma corticosterone was significantly elevated by 30 min after exposure to the stressor and continued to increase up to 6 h. Morphometric analysis of CRH-ir after 4 h of stress showed a significant increase in CRH-ir in parvocellular neurones of the anterior preoptic area, the medial amygdala and the bed nucleus of the stria terminalis, but not in other brain regions. The stress-induced increase in CRH-ir in the POA was associated with increased Fos-like immunoreactivity (Fos-LI), and confocal microscopy showed that CRH-ir colocalized with Fos-LI in a subset of Fos-LI-positive neurones. Our results support the view that the basic pattern of CNS CRH expression arose early in vertebrate evolution and lend further support to earlier studies suggesting that amphibians may be a transitional species for descending CRH-ergic pathways. Furthermore, CRH neurones in the frog brain exhibit changes in response to a physical stressor that parallel those seen in mammals, and thus are likely to play an active role in mediating neuroendocrine, behavioural and autonomic stress responses.

Developmental expression and hormonal regulation of glucocorticoid and thyroid hormone receptors during metamorphosis in Xenopus laevis.

Corticosteroids, the primary circulating vertebrate stress hormones, are known to potentiate the actions of thyroid hormone in amphibian metamorphosis. Environmental modulation of the production of stress hormones may be one way that tadpoles respond to variation in their larval habitat, and thus control the timing of metamorphosis. Thyroid hormone and corticosteroids act through structurally similar nuclear receptors, and interactions at the transcriptional level could lead to regulation of common pathways controlling metamorphosis. To better understand the roles of corticosteroids in amphibian metamorphosis we analyzed the developmental and hormone-dependent expression of glucocorticoid receptor (GR) mRNA in the brain (diencephalon), intestine and tail of Xenopus laevis tadpoles. We compared the expression patterns of GR with expression of thyroid hormone receptor beta (TRbeta). In an effort to determine the relationship between nuclear hormone receptor expression and levels of ligand, we also analyzed changes in whole-body content of 3,5,3'-triiodothyronine (T(3)), thyroxine, and corticosterone (CORT). GR transcripts of 8, 4 and 2 kb were detected in all tadpole tissues, but only the 4 and 2 kb transcripts could be detected in embryos. The level of GR mRNA was low during premetamorphosis in the brain but increased significantly during prometamorphosis, remained at a constant level throughout metamorphosis, and increased to its highest level in the juvenile frog. GR mRNA level in the intestine remained relatively constant, but increased in the tail throughout metamorphosis, reaching a maximum at metamorphic climax. The level of GR mRNA was increased by treatment with CORT in the intestine but not in the brain or tail. TRbeta mRNA level increased in the brain, intestine and tail during metamorphosis and was induced by treatment with T(3). Analysis of possible crossregulatory relationships between GRs and TRs showed that GR mRNA was upregulated by exogenous T(3) (50 nM) in the tail but downregulated in the brain of premetamorphic tadpoles. Exogenous CORT (100 nM) upregulated TRbeta mRNA in the intestine. Our findings provide evidence for tissue-specific positive, negative and crossregulation of nuclear hormone receptors during metamorphosis of X. laevis. The synergy of CORT with T(3) on tadpole tail resorption may depend on the accelerated accumulation of GR transcripts in this tissue during metamorphosis, which may be driven by rising plasma thyroid hormone titers.

Roles of corticotropin-releasing factor, neuropeptide Y and corticosterone in the regulation of food intake in Xenopus laevis.

In mammals, hypothalamic control of food intake involves counterregulation of appetite by an orexigenic peptides such as corticotropin-releasing factor (CRF), and orexigenic peptides such as neuropeptide Y (NPY). Glucocorticoids also stimulate food intake by inhibiting CRF while facilitating NPY actions. To gain a better understanding of the diversity and evolution of neuroendocrine feeding controls in vertebrates, we analysed the effects of CRF, NPY and glucocorticoids on food intake in juvenile Xenopus laevis. We also analysed brain CRF and NPY mRNA content and plasma corticosterone concentrations in relation to nutritional state. Intracerebroventricular (i.c.v.) injection of ovine CRF suppressed food intake while CRF receptor antagonist alpha helical CRF(9-41) significantly increased food intake relative to uninjected and placebo controls. By contrast, i.c.v. injection of frog NPY and short-term corticosterone treatment increased food intake. Semi-quantitative reverse transcription-polymerase chain reaction analyses showed that CRF and NPY mRNA fluctuated with food intake in the brain region containing the mid-posterior hypothalamus, pretectum, and optic tectum: CRF mRNA decreased 6 h after a meal and remained low through 31 days of food deprivation; NPY mRNA content also decreased 6 h after a meal, but increased to prefeeding levels by 24 h. Plasma corticosterone concentration increased 6 h after a meal, returned to prefeeding levels by 24 h, and did not change with prolonged food deprivation. This postprandial increase in plasma corticosterone may be related to the subsequent increase in plasma glucose and body water content that occurs 24 h postfeeding. Overall, our data support the conclusion that, similar to other vertebrates, CRF is anorexigenic while NPY is orexigenic in X. laevis, and CRF secretion modulates food intake in the absence of stress by exerting an inhibitory tone on appetite. Furthermore, the stress axis is activated in response to food intake, but in contrast to mammals and birds is not activated during periods of food deprivation.

Corticotropin-releasing hormone-binding protein: biochemistry and function from fishes to mammals.

Corticotropin-releasing hormone (CRH) plays multiple roles in vertebrate species. In mammals, it is the major hypothalamic releasing factor for pituitary adrenocorticotropin secretion, and is a neurotransmitter or neuromodulator at other sites in the central nervous system. In non-mammalian vertebrates, CRH not only acts as a neurotransmitter and hypophysiotropin, it also acts as a potent thyrotropin-releasing factor, allowing CRH to regulate both the adrenal and thyroid axes, especially in development. The recent discovery of a family of CRH-like peptides suggests that multiple CRH-like ligands may play important roles in these functions. The biological effects of CRH and the other CRH-like ligands are mediated and modulated not only by CRH receptors, but also via a highly conserved CRH-binding protein (CRH-BP). The CRH-BP has been identified not only in mammals, but also in non-mammalian vertebrates including fishes, amphibians, and birds, suggesting that it is a phylogenetically ancient protein with extensive structural and functional conservation. In this review, we discuss the biochemical properties of the characterized CRH-BPs and the functional roles of the CRH-BP. While much of the in vitro and in vivo data to date support an 'inhibitory' role for the CRH-BP in which it binds CRH and other CRH-like ligands and prevents the activation of CRH receptors, the possibility that the CRH-BP may also exhibit diverse extra- and intracellular roles in a cell-specific fashion and at specific times in development is also discussed.

Sublethal effects of chronic exposure to an organochlorine compound on northern leopard frog (Rana pipiens) tadpoles.

Global contamination with organochlorine compounds (OCs) has posed developmental and reproductive problems in wildlife worldwide. However, little is known about the impact of OCs or other pollutants on amphibians, despite mounting concerns about amphibian population declines and developmental deformities in the wild. Wildlife populations may be affected critically by sublethal impacts of anthropogenic disturbances, yet little research has focused on such effects in amphibians. In the current study, northern leopard frog (Rana pipiens) tadpoles were chronically exposed to a polychlorinated biphenyl (PCB) congener, 77-TCB, and effects on behavior, morphology, competitive performance, and corticosterone content were determined. R. pipiens activity levels and feeding rates were decreased by 77-TCB exposure, but morphology of mouthparts and body proportions were unaffected. 77-TCB enhanced growth and altered competitive interactions between R. pipiens and wood frog (Rana sylvatica) tadpoles. R. pipiens tadpoles exposed to 77-TCB showed decreased whole-body corticosterone content compared to controls both before and after injection with adrenocorticotropic hormone (ACTH). All of the factors examined in the current study play critical roles in tadpole development, growth, survivorship, and eventual reproductive success, suggesting negative population-level consequences for amphibians in PCB-contaminated habitats.

Biochemical characterization and expression analysis of the Xenopus laevis corticotropin-releasing hormone binding protein.

Corticotropin-releasing hormone (CRH) plays a key role in the regulation of responses to stress. The presence of a high affinity binding protein for CRH (CRH-BP) has been reported in mammals. We have characterized the biochemical properties and expression of CRH-BP in the South African clawed frog, Xenopus laevis. Apparent inhibition constants (K(i[app])) for different ligands were determined by competitive binding assay. Xenopus CRH-BP (xCRH-BP) exhibited a high affinity for xCRH (K(i[app])=1.08 nM) and sauvagine (1.36 nM). Similar to rodent and human CRH-BPs, the frog protein binds urotensin I and urocortin with high affinity, and ovine CRH with low affinity. RT-PCR analysis showed that xCRH-BP is expressed in brain, pituitary, liver, tail, and intestine. Brain xCRH-BP mRNA is expressed at a relatively constant level throughout metamorphosis and increases slightly in the metamorphic frog. By contrast, the gene is strongly upregulated in the tail at metamorphic climax. Thus, regulation of xCRH-BP gene expression is tissue specific. Because xCRH-BP binds CRH-like peptides with high affinity the protein may regulated, the bioavailability of CRH in amphibia as it does in mammals.

Basic transcription element-binding protein (BTEB) is a thyroid hormone-regulated gene in the developing central nervous system. Evidence for a role in neurite outgrowth.

Thyroid hormone (3,5,3'-triiodothyronine; T(3)) is essential for normal development of the vertebrate brain, influencing diverse processes such as neuronal migration, myelin formation, axonal maturation, and dendritic outgrowth. We have identified basic transcription element-binding protein (BTEB), a small GC box-binding protein, as a T(3)-regulated gene in developing rat brain. BTEB mRNA levels in cerebral cortex exhibit developmental regulation and thyroid hormone dependence. T(3) regulation of BTEB mRNA is neural cell-specific, being up-regulated in primary cultures of embryonic neurons (E16) and in neonatal astrocytes (P2), but not in neonatal oligodendrocytes (P2). T(3) rapidly up-regulated BTEB mRNA in neuro-2a cells engineered to express thyroid hormone receptor (TR) beta1 but not in cells expressing TRalpha1, suggesting that the regulation of this gene is specific to the TRbeta1 isoform. Several lines of evidence support a transcriptional action of T(3) on BTEB gene expression. Overexpression of BTEB in Neuro-2a cells dramatically increased the number and length of neurites in a dose-dependent manner suggesting a role for this transcription factor in neuronal process formation. However, other T(3)-dependent changes were not altered; i.e. overexpression of BTEB had no effect on the rate of cell proliferation nor on the expression of acetylcholinesterase activity.

Evolution of the corticotropin-releasing hormone signaling system and its role in stress-induced phenotypic plasticity.

Developing animals respond to variation in their habitats by altering their rates of development and/or their morphologies (i.e., they exhibit phenotypic plasticity). In vertebrates, one mechanism by which plasticity is expressed is through activation of the neuroendocrine system, which transduces environmental information into a physiological response. Recent findings of ours with amphibians and of others with mammals show that the primary vertebrate stress neuropeptide, corticotropin-releasing hormone (CRH), is essential for adaptive developmental responses to environmental stress. For instance, CRH-dependent mechanisms cause accelerated metamorphosis in response to pond-drying in some amphibian species, and intrauterine fetal stress syndromes in humans precipitate preterm birth. CRH may be a phylogenetically ancient developmental signaling molecule that allows developing organisms to escape deleterious changes in their larval/fetal habitat. The response to CRH is mediated by at least two different receptor subtypes and may also be modulated by a secreted binding protein.

The molecular basis of thyroid hormone-dependent central nervous system remodeling during amphibian metamorphosis.

Tadpole metamorphosis involves a coordinated series of changes in virtually every tissue of the body. This developmental process is induced by the single morphogen, thyroid hormone (TH). The amphibian central nervous system (CNS) is a primary target for TH, and it undergoes dramatic morphological and cytoarchitectural changes in response to the hormone. TH acts by regulating gene expression and its actions in metamorphosis are thought to result from its ability to induce tissue-specific genetic programs. Receptors for TH are ligand-dependent transcription factors whose mRNA expression is upregulated by TH during metamorphosis (receptor autoinduction). Studies on the tadpole CNS have identified four general classes of early TH response genes. These genes code for: (1) transcription factors, that are likely to be required for the expression of downstream genes (i.e. secondary response genes), (2) cellular enzymes, which carry out hormone conversions, energy transformations and may possibly mediate extranuclear effects of TH on neural cells, (3) cytoskeletal elements required for axonal development, and (4) secreted signaling molecules that control the production of TH. Recent studies suggest a critical, evolutionarily conserved role for the TH-induced transcription factor genes in controling neural cell proliferation and differentiation.

Hormonal correlates of environmentally induced metamorphosis in the Western spadefoot toad, Scaphiopus hammondii.

Tadpoles of several amphibian species have been shown to accelerate metamorphosis when their ponds dry. To understand the proximate mechanisms that mediate the developmental response to pond drying, I analyzed changes in endocrine activity in tadpoles of the Western spadefoot toad (Scaphiopus hammondii) exposed to experimental water volume reduction in the laboratory. Tadpoles exposed to a declining water level accelerated metamorphosis compared with tadpoles raised in a constant high water environment. The acceleration of development was associated with the precocious elevation of whole-body contents of the hormones that control metamorphosis, the thyroid hormones thyroxine (T4) and triidothyronine (T3), and the interrenal steroid corticosterone (CORT). The precocious activation of the thyroid system preceded external morphological change (i.e., increase in hind limb length, developmental stage) by 3 days. To test if tadpoles are capable of responding rapidly to water volume reduction, mid-prometamorphic tadpoles (Gosner Stage 37-38) were raised in a constant high water environment (10 L) and then transferred to either 1 or 10 L. Tadpoles transferred to 1 L exhibited significant metamorphic changes by 48 h after transfer. In addition, dramatic elevations in whole-body T4, T3, and CORT contents were evident at this time point. Thus, the metamorphic response to pond drying is likely driven by the activation of the thyroid and interrenal axes, the hormones of which control metamorphosis. Furthermore, this response is rapid, occurring within 48 h after exposure to the desiccating environment.

Environmental stress as a developmental cue: corticotropin-releasing hormone is a proximate mediator of adaptive phenotypic plasticity in amphibian metamorphosis.

Environmentally induced phenotypic plasticity allows developing organisms to respond adaptively to changes in their habitat. Desert amphibians have evolved traits which allow successful development in unpredictable environments. Tadpoles of these species can accelerate metamorphosis as their pond dries, thus escaping mortality in the larval habitat. This developmental response can be replicated in the laboratory, which allows elucidation of the underlying physiological mechanisms. Here I demonstrate a link between a classical neurohormonal stress pathway (involving corticotropin-releasing hormone, CRH) and the developmental response to habitat desiccation. Injections of CRH-like peptides accelerated metamorphosis in western spadefoot toad tadpoles. Conversely, treatment with two CRH antagonists, the CRH receptor antagonist alpha-helical CRH(9-41) and anti-CRH serum, attenuated the developmental acceleration induced by habitat desiccation. Tadpoles subjected to habitat desiccation exhibited elevated hypothalamic CRH content at the time when they responded developmentally to the declining water level. CRH injections elevated whole-body thyroxine, triiodothyronine, and corticosterone content, the primary hormonal regulators of metamorphosis. In contrast, alpha-helical CRH(9-41) reduced thyroid activity. These results support a central role for CRH as a neurohormonal transducer of environmental stimuli into the endocrine response which modulates the rate of metamorphosis. Because in mammals, increased fetal/placental CRH production may initiate parturition, and CRH has been implicated in precipitating preterm birth arising from fetal stress, this neurohormonal pathway may represent a phylogenetically ancient developmental regulatory system that allows the organism to escape an unfavorable larval/fetal habitat.

Thyroid hormone-dependent gene expression program for Xenopus neural development.

Although thyroid hormone (TH) plays a significant role in vertebrate neural development, the molecular basis of TH action on the brain is poorly understood. Using polymerase chain reaction-based subtractive hybridization we isolated 34 cDNAs for TH-regulated genes in the diencephalon of Xenopus tadpoles. Northern blots verified that the mRNAs are regulated by TH and are expressed during metamorphosis. Kinetic analyses showed that most of the genes are up-regulated by TH within 4-8 h and 13 are regulated by TH only in the brain. All cDNA fragments were sequenced and the identities of seven were determined through homology with known genes; an additional five TH-regulated genes were identified by hybridization with known cDNA clones. These include five transcription factors (including two members of the steroid receptor superfamily), a TH-converting deiodinase, two metabolic enzymes, a protein disulfide isomerase-like protein that may bind TH, a neural-specific cytoskeletal protein, and two hypophysiotropic neuropeptides. This is the first successful attempt to isolate a large number of TH-target genes in the developing vertebrate brain. The gene identities allow predictions about the gene regulatory networks underlying TH action on the brain, and the cloned cDNAs provide tools for understanding the basic molecular mechanisms underlying neural cell differentiation.

Pancreatic hormones differentially regulate insulin-like growth factor (IGF)-I and IGF-binding protein production by primary rat hepatocytes.

We investigated the influence of and interactions among pancreatic hormones on the secretion of insulin-like growth factor-I (IGF-I) and IGF-binding proteins (IG-FBPs) by treating primary hepatocytes from young male Long-Evans rats with insulin or glucagon in combination with rat GH (rGH). The concentration of IGF-I secreted into the medium was estimated by radioimmunoassay after formic acid-acetone cryoextraction, and secreted IGFBPs were analysed by Western ligand blot and immunoblot; accumulation of IGF-I mRNA was analysed by Northern blot. Both insulin (0.1-100 nmol/l) and rGH (0.5, 5 and 50 pmol/l) produced a dose-dependent stimulation of IGF-I secretion over a 24-h incubation period. In contrast, glucagon (0.1-100 nmol/l) inhibited IGF-I production in a dose-related manner. Glucagon (10 nmol/l) also inhibited IGF-I secretion stimulated by rGH (5 pmol/l) and insulin (10 nmol/l). Northern blot analysis of total RNA isolated from rat hepatocytes revealed that rGH (5 pmol/l) elevated IGF-I mRNA levels, glucagon (10 nmol/l) alone had no effect on this parameter, but glucagon significantly reduced IGF-I transcript accumulation in response to rGH. IGFBPs secreted by rat hepatocytes run in two molecular weight ranges on SDS-PAGE: approximately 25 kDa (IGFBP-4) and approximately 29-31 kDa (IGFBP-1 and -2); the predominant hormonally regulated IGFBP was identified as IGFBP-1. Insulin produced a dose-dependent inhibition of production of IGFBP-1, while glucagon was stimulatory; when given together at an equivalent concentration (1 nmol/l), the effects of insulin were dominant to glucagon on IGFBP-1. These observations provide support for significant opposite roles for the pancreatic hormones, insulin and glucagon, in the regulation of liver IGF-I and IGFBP-1 production. As the production of pancreatic hormones is influenced by nutritional status, these polypeptides may mediate the effects of changing nutritional state on the hormonal control of protein anabolism and glucose homeostasis by directly influencing the circulating level of liver-derived IGF-I and its binding proteins.

Acceleration of anuran amphibian metamorphosis by corticotropin-releasing hormone-like peptides.

Despite substantial information on the role of the pituitary-thyroid and pituitary-interrenal axes in controlling amphibian metamorphosis, the hypothalamic hormones responsible for controlling the activity of these axes have not been identified. The mammalian thyrotropin-releasing hormone (TRH) does not regulate the thyroid axis of tadpoles; however, corticotropin releasing hormone (CRH) stimulates the release of thyrotropin from bullfrog tadpole pituitary glands in vitro and may thus function as a central regulator of the thyroid axis during metamorphosis. I tested the possibility that a CRH-like peptide is involved in controlling amphibian development by treating tadpoles of two anuran species, the western spadefoot toad Scaphiopus hammondii, and the North American bullfrog, Rana catesbeiana, with neuropeptides and monitoring their effects on metamorphosis. Injection of spadefoot toad tadpoles with ovine (o) CRH (2 micrograms/animal every other day for 3 weeks) or the amphibian CRH-like peptide sauvagine (SV) significantly decreased their time from hatching to metamorphic climax (Gosner stage 42; frontlimb emergence) and their body weight and body length at climax compared with vehicle-injected controls; whereas, TRH had no effect and arginine vasotocin produced a small but significant lengthening of the larval period but did not alter body size at climax. In an acute response experiment, S. hammondii tadpoles (in Gosner stages 36-38--late prometamorphosis) treated with oCRH or SV (2 micrograms/animal) exhibited significantly elevated whole-body thyroxine (T4) content at 2 and 6 hr after injection; whereas, treatment with TRH (2 micrograms/animal) did not significantly alter whole-body T4. R. catesbeiana tadpoles treated with oCRH or SV (surgical implantation of ELVAX pellets impregnated with 100 micrograms peptide and injections of peptides at 5 micrograms/animal once every 3 days) exhibited accelerated spontaneous and triiodothyronine (T3)-induced metamorphosis as assessed by changes in tail height, hind limb development, and body weight; TRH had no effect. Injections of a pool of antisera generated against CRH-like peptides (rat/human CRH, oCRH, SV) slowed T3-induced metamorphosis when compared with normal serum-injected controls. These results support the hypothesis that a CRH-like peptide(s) is involved in the central control of metamorphosis of anuran amphibians, and may act, at least in part, through stimulation of the thyroid axis.

Thyroidal inhibition of chicken pituitary growth hormone: alterations in secretion and accumulation of newly synthesized hormone.

Hypothyroidism reduces GH synthesis and release in several mammalian species, in which thyroid hormone directly stimulates GH gene transcription. In contrast, hypothyroidism stimulates GH secretion in birds, in which thyroid hormone directly inhibits pituitary GH release. We have, therefore, investigated the effects of thyroid status on the accumulation of newly synthesized GH in the pituitaries of 8- to 10-week-old Leghorn cockerels in vitro and in vivo. The incorporation of [35S]methionine into immunoprecipitable GH ([35S] GH) was increased, over a 4-h incubation period, in glands from birds made hypothyroid by injections of methimazole (50 mg/kg day for 10 days) in comparison with glands from vehicle-injected controls. Treatment with tri-iodothyronine (T3, 100 micrograms/kg per day for 10 days) in vivo did not significantly alter the accumulation of [35S]GH in vitro but did block the release of [35S]GH in response to a GH secretagogue (thyrotrophin-releasing hormone; exposure to 280 nmol/l for 30 min) and reduced immunoassayable pituitary GH content. Pretreatment of glands from euthyroid birds with T3 (100 nmol/l) in vitro (for 20 h) reduced the basal accumulation of [35S]GH as well as that induced by another GH secretagogue (GH-releasing factor; 100 nmol/l) during a 6-h labelling period. These results show that, unlike the generally stimulatory action of thyroid hormone in mammals, in birds, T3 exerts a direct inhibitory effect on the accumulation of newly synthesized pituitary GH.

Comparative survey of blood thyroxine binding proteins in turtles.

The nature of plasma thyroxine (T4) binding activity was surveyed in turtles; binding to [125I]T4 was measured on polyacrylamide gel electrophoresis--PAGE--and on minicolumns of Sephadex G-25. An electrophoretically distinct T4 binding protein was identified in all 8 species of Pseudemys studied and in 3 other genera (Chrysemys, Deirochelys, and Emyoidea) of the same family, Emydidae. Levels of this binding activity were highly variable among individuals, but they consistently showed a similar low relative mobility (Rf) compared to albumin, and a relatively low capacity was indicated by displacement with unlabeled T4. Two emydids (Terrapene, Clemmys) showed a similar slow migrating binding peak, but binding activity was low and not as easily displaced by unlabeled T4. T4 binding to albumins was minimal in most of these emydid species, even when binding to the higher affinity, low capacity component was low or displaced by unlabeled T4 (2.5 micrograms/ml). In contrast, there was no clear evidence for a similar high affinity, low capacity binding protein in any of the other 19 species representing 13 genera of 8 families from two suborders. In these species, binding activity on Sephadex G-25 was typically low and binding on PAGE was associated largely with albumin; binding levels for albumins were highly variable. In several nonemydids (from distant lineages), binding activity on Sephadex was elevated and PAGE showed a second binding protein distinct from albumin, but it had high capacity (not readily saturable). Thus, an evolutionary divergence in T4 transport proteins is suggested within Chelonia.

The role of hormone binding in the cold suppression of hormone stimulation of the pituitary, thyroid, and testis of the turtle.

The ability of hormones to bind to their functional receptors on turtle (Pseudemys scripta) endocrine target tissues in the cold was tested by treating tissues with secretagogues at low temperatures (5-15 degrees) and then following subsequent target stimulation in the absence of secretagogue at a warm temperature (28 degrees). Administration of thyrotropin-releasing hormone (TRH), corticotropin-releasing hormone, and growth hormone-releasing hormone to pituitaries at low temperatures (20 degrees or below) suppressed responses in growth hormone (GH) and thyrotropin (TSH) secretion and there was little or no response in pituitaries subsequent to warming. In contrast, gonadotropin-releasing hormone treatment of pituitaries, TSH treatment of thyroid glands, and gonadotropin (FSH and LH) treatment of testes in the cold (down to 5 degrees) was followed by a large response in the target glands (secretion of LH, thyroxine, and testosterone (T), respectively) following warming. Additional studies with FSH and LH showed that these hormones can bind to testes rapidly (within 5 min) at low temperatures where no acute response is observed, although the dose sensitivity and the extent of this priming in the cold are less than at warm temperatures. Thus, postreceptor events may be more important than binding per se for temperature effects on hormone responses of tissues, but even this component of cell function varies among tissues. The effects of a receptor-independent secretagogue (tetraethylammonium chloride), which causes cell depolarization by blocking K+ efflux, were also blocked at low temperatures in thyrotropes and somatotropes but not in gonadotropes. Rapid depressions in TSH and GH secretions following cooling of TRH-stimulated pituitaries and of T secretion in LH-stimulated testes provide further evidence for cold sensitivity of postreceptor processes in these tissues.