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Mercury (Hg) is a neurotoxicant known to cause developmental and behavioral abnormalities in vertebrates. Increasing evidence suggests that Hg can also disrupt endocrine functions and endocrine-dependent processes. For example, dietary Hg has been shown to delay tail resorption during metamorphic climax in amphibians, a process mediated by thyroid hormones. However, a direct link between Hg, hormone disruption, and developmental delays in amphibians has not been explored. Therefore, we examined the effects of dietary Hg (0.01, 2.5, and 10 μg/g total Hg, dry wt) on thyroid hormone concentrations, development, growth, performance, and survival of wood frogs (Rana sylvatica). Tadpoles accumulated Hg in a concentration-dependent manner; total Hg concentrations in tadpoles at the beginning of metamorphic climax (Gosner stage 42) were 0.03, 1.06, 3.54 μg/g, dry wt, for control, low, and high Hg diets, respectively. During metamorphic climax, tadpoles eliminated 35% of the inorganic Hg from their tissues but retained most of their accumulated methylmercury. Contrary to our predictions, we found no effect of Hg on the duration of tadpole development, size at metamorphosis, tail resorption time, or hopping performance. Consistent with the lack of effects on development, we also detected no differences in whole-body thyroid hormone concentrations among our dietary treatments. Our results, when compared with the effects of Hg on other amphibians, suggest that amphibian species may differ substantially in their sensitivity to dietary Hg, emphasizing the need for data on multiple species when establishing toxicity benchmarks.

Ground-dwelling birds in perchlorate-contaminated areas are exposed to perchlorate ion, a known thyroid disruptor, and might be vulnerable to the developmental effects of perchlorate-induced hypothyroidism. We hypothesized that perchlorate-induced hypothyroidism would alter the expression of thyroid-responsive genes involved in thyroid hormone (TH) regulation and in the development of target organ function. Japanese quail chicks were exposed to 2000 mg/L ammonium perchlorate in drinking water for 7.5 weeks beginning on day 5 posthatch. Hypothyroidism was evident after 2 weeks of exposure as lower plasma THs and lower TH content in exposed chicks than in controls. The degree of hypothyroidism was increased at 7.5 weeks, as indicated by significant thyroid gland hypertrophy and sustained changes in thyroid function. After 2 weeks of exposure, hypothyroidism increased type 2 5'-deiodinase (D2) mRNA level and decreased Spot 14 (SP14) mRNA level in the liver, whereas D2 mRNA and RC3 mRNA levels in brain were not affected. After 7.5 weeks of exposure, mRNA levels in the exposed group did not differ from those in controls in either the liver or brain, suggesting the responsiveness of these genes to THs decreased during development. These results suggest that the brain, but not the liver, was protected from the effects of hypothyroidism, probably by changes in D2 activity at the protein level and/or regulation of TH entry and exit from the brain. We concluded that perchlorate exposure caused hypothyroidism in young Japanese quail and affected the expression of thyroid-responsive genes during early posthatch development.

Much of the research on sublethal, adverse effects of mercury (Hg) has focused on impairment of neurological function and reproduction in fish and fish-eating vertebrates. Here we examined the associations between Hg and endocrine function (adrenocortical responses and plasma thyroid hormone concentrations) of insectivorous tree swallow nestlings adjacent to a Hg-contaminated river and nearby reference rivers in Virginia. Nestlings from the contaminated sites had blood Hg concentrations that exceeded those from the reference sites by more than an order of magnitude (354 +/- 22 vs 17 +/- 1 ppb wet weight). A regression of age and Hg concentrations suggested dietary Hg at the contaminated sites exceeded the nestlings' capacity to eliminate Hg through deposition into growing feathers. Although blood Hg concentrations among nestlings at the contaminated sites were lower than those typically associated with abnormal behavior or altered physiology in young birds, adrenocortical responses, plasma triiodothyronine, and thyroxin concentrations were suppressed, relative to reference levels, by the end of the nestling period. These results suggest that (1) Hg may disrupt endocrine systems of terrestrial avian young and (2) adverse effects of Hg on endocrine systems may be most evident once endocrine axes are fully developed.

Perchlorate, a known thyroid disruptor, is deposited in eggs of exposed female birds, raising concerns that the embryos from these eggs may become hypothyroid, which may in turn affect the development and function of thyroid-dependent organs. We hypothesized that exposure to ammonium perchlorate (AP) would decrease hen and embryonic thyroid function and affect the expression of thyroid-responsive genes in embryonic brain and liver. Laying Japanese quail hens were treated with 2000 mg/l or 4000 mg/l AP in drinking water. Thyroid status and expression of thyroid-responsive genes were examined in the embryos from eggs of exposed hens. Perchlorate exposure led to hypothyroidism in hens from both treatment groups; egg production was decreased in the high dosage group only. Embryos from eggs of perchlorate-exposed hens had hypertrophied thyroid glands and significantly lower thyroidal hormone storage, indicating hypothyroidism in these embryos. The embryonic hypothyroidism was associated with decreased embryonic growth, delayed hatching and greater mortality during hatching. The mRNA level of type 2 deiodinase (D2) in the liver of embryos from eggs of perchlorate-exposed hens was increased compared to the control embryos, a compensatory response that increases the production of metabolically active T(3). However, the mRNA levels of D2 and RC3 in the brain were not affected. These results suggest that the embryonic brain is protected from hypothyroidism by other mechanisms known to influence hormone entry into and exit from the brain. Our study shows that maternal perchlorate exposure led to embryonic hypothyroidism and may have interfered with embryonic development.

Polychlorinated biphenyls (PCBs) decrease thyroid function in laboratory rodents by inducing activity of a liver enzyme, uridine diphosphate-glucuronosyltransferase (UDP-GT), thereby increasing thyroxine (T4) clearance. This loss of T4 can lead to hypothyroidism. In this study, an assay was validated for measuring UDP-GT activity toward T4 in Japanese quail. UDP-GT induction by Aroclor 1254 was evaluated in quail, and responses of quail and mice were compared. In Experiment 1, Japanese quail and Balb/c mice were dosed orally with vehicle or Aroclor 1254 (250 or 500mg/kg) and sacrificed 5days later. In Experiment 2, Japanese quail were dosed orally with vehicle or Aroclor 1254 (500mg/kg) and sacrificed 5 or 21days later. UDP-GT capacity (pmol T4 glucuronidated by the liver/minper g body weight) increased with PCB exposure with all doses and exposure times in both species. Plasma T4 tended to decrease (not significant) with both PCB doses and exposure times in quail and was significantly decreased with both doses in mice. Quail did not become hypothyroid at either dose or exposure time. In contrast, mice did become hypothyroid after a 5-day exposure. It is unclear how PCBs affect the hypothalamic-pituitary-thyroid (HPT) axis in quail, but activation of the HPT axis appears to be inhibited in mice. We believe this is the first demonstration of a T4-specific, avian UDP-GT response to PCBs. However, this avian response was less than that in mice with equivalent doses of PCBs. Thus, thyroid function in birds appears to be less vulnerable to PCBs than in mammals.

This article reviews thyroid function and its hypothalamic-pituitary-thyroid (HPT) axis control in birds with emphasis on the similarities and differences in thyroid function compared to mammals and other vertebrate classes. Thyroid hormones are important in metabolism and the thermogenesis required for homeothermy in birds, as in mammals, the other homeothermic class of vertebrates. Thyroid hormones play important roles in development and growth in birds, as is the case for all vertebrate classes. The developmental effects of thyroid hormones in birds are presented in the context of differences in precocial and altricial patterns of development and growth with emphasis on oviparous development. The sections on thyroid hormone actions include discussion of effects on the development of a number of tissue types as well as on seasonal organismal processes and interactions of the thyroid axis with reproduction. The current picture of how environmental chemicals may disrupt avian thyroid function is relatively limited and is presented in the context of the assessment endpoints that have been used to date. These endpoints are categorized as thyroid and HPT axis endpoints versus target organ endpoints. The final section discusses two recommended assay protocols, the avian two-generation toxicity assay and the avian one-generation assay, and whether these protocols can evaluate thyroid disruption in birds.

Precocial and altricial modes of avian development are characterized by different degrees of maturation and physiological capabilities at hatching. In precocial birds, thyroid function and its control are well developed during the latter part of incubation and hatchlings exhibit metabolic responses to cooling and relatively mature sensory and locomotor capabilities. In altricial birds, thyroid function shows little maturation until after hatch as also is the case for thermoregulatory, sensory, and motor functions. This review describes the patterns of precocial and altricial thyroid development, their hypothalamic-pituitary control, extrathyroidal control of hormone activation and deactivation, and target tissue effects during development. Our knowledge is greatest for precocial galliform birds although the organismal picture of thyroid development has been investigated in several altricial avian species.

Precocial and altricial modes of avian development are characterized by different degrees of maturation and physiological capabilities at hatching. In precocial birds, thyroid function and its control are well developed during the latter part of incubation and hatchlings exhibit metabolic responses to cooling and relatively mature sensory and locomotor capabilities. In altricial birds, thyroid function shows little maturation until after hatch as also is the case for thermoregulatory, sensory, and motor functions. This review describes the patterns of precocial and altricial thyroid development, their hypothalamic-pituitary control, extrathyroidal control of hormone activation and deactivation, and target tissue effects during development. Our knowledge is greatest for precocial galliform birds although the organismal picture of thyroid development has been investigated in several altricial avian species.

Perchlorate, the oxidizer component in most solid rocket propellant formulations, is known to inhibit the uptake of iodide into the thyroid gland, thereby reducing production of the thyroid hormones, triiodothyronine and thyroxine (T4). Thyroid hormones regulate metabolism in endothermic organisms and are responsible for maintenance of homeothermic body temperatures. Little is known about the effects of perchlorate on metabolic capacity. The objectives of the present study were to determine if subchronic (51 d; 0, 1, and 10 mg/kg/d) and chronic (180 d; 0.75 mg/kg/d) perchlorate exposure in adult male prairie voles (Microtus ochrogaster) would alter resting metabolic rates as a result of decreased circulating thyroid hormone concentrations and to determine if perchlorate exposure disrupts thermogenesis in mammals exposed to cold stress. Voles exposed to perchlorate for 51 or 180 d experienced no significant alterations in resting metabolic rates at any point during the exposure period. Additionally, the treatment had no effect on peak metabolic rates or plasma thyroid hormone concentrations. However, thyroid gland T4 concentrations were significantly lower in perchlorate-exposed voles than in controls, indicating that thyroid gland T4 content may be a more sensitive endpoint than other thyroid variables for assessing perchlorate exposure. Overall, the present study did not provide evidence for energetic alterations associated with perchlorate exposure at concentrations that are higher than those typically found in groundwater or surface water in the environment.

The effects of a wide range of ammonium perchlorate (AP) concentrations in drinking water on thyroid function in bobwhite quail chicks was investigated at 2, 4, and 8 weeks of exposure. We measured plasma thyroid hormones (THs) to evaluate organismal thyroid status, thyroid weights to evaluate hypothalamic-pituitary-thyroid (HPT) axis activation, and thyroidal TH content to assess TH stores. At the highest AP exposures plasma THs were decreased, thyroid glands were hypertrophied, and thyroidal TH content was decreased. As in our previous studies, thyroidal thyroxine (T(4)) content was the most sensitive indicator of decreased thyroid function; plasma T(4) and thyroid weight were much less sensitive. The lack of sensitivity of these variables appears to result from cyclic patterns of thyroid responses involving the HPT axis and intermittent release of stored THs from the thyroid gland. With sustained AP exposure (8 weeks), at the lowest range of AP concentrations used, chicks showed adaptation in thyroid function that fully compensated for the initial (2 week) effects of AP. At the intermediate AP concentrations there was partial compensation for the initial AP effects. At the highest AP exposures used, thyroid function was very low throughout the study, with no indication of compensatory responses. The capability of chicks to increase some aspects of their thyroid function adaptively in response to some levels of sustained AP exposure is contrary to the common generalization that developing animals are most vulnerable to environmental contaminants.

Bobwhite quail chicks were used to investigate ammonium perchlorate (AP; NH4ClO4) effects on thyroid function and growth. Beginning at 3 to 4 d posthatch, we evaluated organismal thyroid status (circulating hormones), activation of the hypothalamic-pituitary-thyroid axis (thyroid wt) and thyroidal hormone content over a wide range of AP concentrations (50 microg/L - 4,000 mg/L) in drinking water, for relatively short (2-week) and longer (8-week) exposures. Thyroidal thyroxine (T4) content, the most sensitive index of decreased thyroid function, decreased markedly in response to increasing perchlorate exposure. Thyroid weight and plasma T4 were less sensitive indicators and similar in their ability to detect thyroid changes. Growth measurements (body wt and skeletal growth) were very insensitive indices. Because thyroids contain large hormone stores, with low exposures or short time periods, these stores can be used to maintain circulating hormones, at least temporarily. Most depletion of thyroidal T4 occurred during the first two weeks of AP exposure. Subsequent decreases were at a slower rate presumably because thyrotropin stimulation of the thyroids at least partially compensated for some of the perchlorate effect. Additional studies of the interactions between AP concentration and exposure time are needed for understanding the complex nature of thyroid responses to perchlorate.

Poor reproductive success, developmental abnormalities, and behavioral alterations in fish-eating birds in some Great Lakes areas have led to more than 35 years of toxicological studies and residue monitoring of herring gull (Larus argentatus) populations. Polyhalogenated aromatic hydrocarbons (PHAHs), especially polychlorinated biphenyls (PCBs), are widespread contaminants in the Great Lakes ecosystem. The introduction of regulations and elimination of point sources since the 1970s have resulted in decreased PHAHs in fish-eating bird eggs and tissues. PCB exposure is associated with thyroid disruption (hypothyroidism) in mammals, but much less is known of PCB effects on avian thyroid function. Our 1998-2000 studies of herring gulls from the Great Lakes show that both pipping embryos and prefledglings from highly contaminated sites have marked depletion of thyroid gland hormone stores compared with similarly aged gulls at the reference sites. However, organismal hypothyroidism was not apparent in many embryo and chick collections where severe depletion of thyroid gland hormone was observed. Adults, sampled at two high PCB sites and a low PCB site in the Great Lakes and the maritime reference colony in 2001, showed no differences in organismal thyroid status across sites, but gulls from the high sites had enlarged thyroid glands and depressed thyroid gland hormone stores. Here we discuss the evidence that ecological exposure to PHAHs are responsible for thyroid deficiencies in gulls and that during development these deficiencies lead to developmental abnormalities in young gulls from highly contaminated Great Lakes sites.

The purpose of this experiment was to determine the effects of Aroclor 1242, a mixture of polychlorinated biphenyls (PCBs), on plumage characteristics and molt in the American kestrel, Falco sparverius. Several characteristics of plumage, including color and molt schedule, are modulated by hormonal signals and hence may be modified by endocrine-active contaminants. If so, the functions of plumage (e.g., communication for mating or territorial defense) may be compromised by exposure to such compounds. Captive American kestrels were fed Aroclor 1242 at 0, 6.0, and 60.0 ppm (n = 6 males and 6 females per treatment) mixed in their normal diet. Concentrations of plasma estradiol and thyroxine were measured weekly from the beginning of treatment. Measured plumage characteristics included width of the black subterminal band on the tail, color (a composite index of hue and saturation), reflectance from 230 to 800 nm. pattern of feather loss and regrowth on the tail and wing, and timing of onset and duration of molt. Aroclor 1242 depressed plasma thyroxine. Plasma estradiol levels remained low due to the phase of the breeding cycle. Treatments did not disrupt the measured plumage characteristics. This may be due to timing or dose of exposure or to genetic factors.

We examined the proximate causes of delayed breeding and alloparental behavior in the cooperatively breeding red-cockaded woodpecker by measuring plasma concentrations of testosterone (T) and prolactin (PRL) in female breeders, male breeders, and male helpers during different stages of the reproductive cycle. Male breeders and helpers have low T during the prebreeding period, highest T during copulation, and low concentrations of T during the egg-laying/incubation and nestling provisioning stages. Helpers appear physiologically capable of reproducing; their T concentrations equal that of male breeders. Helpers unrelated to the breeding female have higher T than helpers sharing a territory with their mother. Sexual inactivity by male helpers might be explained by behavioral suppression resulting from interactions of male helpers with the breeding pair that somehow differ in accordance with the helper's relatedness to the breeding female (e.g., female breeders are generally unreceptive to courtship from male helpers and least receptive to related helpers). Female breeder, male breeder, and male helper PRL levels did not differ and increased from the prebreeding stage through the copulation and egg-laying/incubation stages. During the nestling provisioning stage, male breeder and helper PRL declined, while female PRL continued to increase. Based on these results, we conclude that the physiological bases of alloparental behavior have not diverged from those that mediate parental behavior in this species., (Copyright 2001 Academic Press.)

This study addressed the role of testosterone (T) in the development of sexually dimorphic behavior in the green anole lizard, Anolis carolinensis. We documented the pattern of endogenous T concentrations during ontogeny and we determined the behavioral effects of experimentally elevated T in juvenile males and females. T concentrations were measured in the plasma of hatchlings from eggs incubated in the laboratory, in juveniles of all sizes sampled in the field, and in the yolks of freshly laid eggs in the laboratory and were compared to plasma T in adult females (measured in this study) and adult males. There were no sex differences in plasma T in hatchling and small juvenile (<26-mm snout-vent length, SVL; <14 days old) males and females, concentrations of which in both sexes tended to decline over the 14-day posthatching period. Plasma T sharply increased in juvenile males, but not females, after approximately 14 days posthatching (>25-mm SVL), and it became significantly higher after approximately 38 days posthatching (>30-mm SVL). Plasma T for juvenile males was within the range detected in breeding adult females, but it was 20- to 45-fold lower than that of adult males, breeding or postbreeding. All eggs contained detectable yolk T, but eggs that gave rise to males contained nearly twice as much yolk T as those that gave rise to females. We do not know whether this yolk T comes from the mother, embryo, or both. In behavior trials conducted in the laboratory, juveniles (36- to 42-mm SVL) with T implants, regardless of whether they were male or female, had increased activity levels compared to juveniles with blank implants, due to increased rates of nearly every behavior monitored. These results are discussed in the context of the organization-activation theory of sexual differentiation and the particular life history of A. carolinensis., (Copyright 2001 Academic Press.)

We investigated the development of thyroid function during the transition to endothermy in red-winged blackbirds (Agelaius phoeniceus). Thermoregulatory capabilities of blackbirds improve markedly over their relatively short nestling period (10-12 days), with the most striking improvements occurring between days 6 and 8. We hypothesized that the development of endothermy in these birds is dependent in part on the development of thyroid function. We assessed thyroid development by measuring changes in thyroid gland histology and plasma concentrations of thyroxine (T4) and triiodothyronine (T3) during the nestling period. To gain insight into the role of thyroid maturation in the context of thermoregulation, we compared plasma thyroid hormone profiles in nestlings exposed to cold temperatures to those maintained at thermoneutral temperatures. The overall size of the thyroid (as cross-sectional area) increased during nestling development, with the fastest growth occurring just before the development of endothermy. By day 8, it reached the size typical of that in adults. Follicular cell height of the thyroid glands increased in nestlings up to day 6 and then decreased for the rest of the nestling period. The mean area of individual follicles increased up to day 8 of nestling life and then decreased. Individual nestlings were capable of strong endothermic responses at 7 to 8 days of age and had significantly decreased plasma T4 concentrations following cold exposure, suggesting increased T4 to T3 deiodination to maintain the plasma concentrations of the more metabolically active T3. The patterns of plasma T4 and T3 during nestling development were consistent with those of nestlings of other altricial species of birds that have been studied. Overall, the patterns of thyroid development observed were consistent with the hypothesis that the functional development of the thyroid is critical to the development of endothermic capabilities and that thyroid hormones play a role in endothermic responses to cold temperatures., (Copyright 1999 Academic Press.)

We addressed the relationship between the thyroid status of hens and the thyroid hormone content of their eggs, as well as the influences of egg hormones on embryonic development. Methods for measuring thyroid hormones in egg yolk were verified by demonstrating consistency in the recovery of yolk thyroid hormones following a methanol/chloroform extraction and in the measurement of thyroid hormones by RIA for a range of hormone concentrations in yolk extracts. Untreated hens produced eggs with yolk thyroxine (T4) concentrations that were low relative to plasma T4, but yolk triiodothyronine (T3) concentrations comparable to those of plasma. Hens dosed twice daily with T4 (1 or 3x the daily thyroid secretion rate, TSR, of T4 per dose) had significantly higher plasma and egg yolk T4 concentrations than did control hens dosed with saline. In general, the T4 concentration of egg yolk varied with the thyroid status of the hen. When the relationship between each hen's plasma T4 and the yolk T4 concentration of her eggs was examined, hens appeared to regulate T4 deposition into yolk at "levels" characteristic of the "levels" of thyroid status produced by the different doses of T4. Embryonic pelvic cartilage, a thyroid hormone-responsive tissue, showed enhanced growth and differentiation in embryos from eggs of hens given the highest dose of T4. Specifically, alkaline phosphatase activity (a marker of differentiation) and pelvic cartilage wet and dry weights were significantly greater in embryos from high T4 eggs (hens on the 3x TSR dose) than those in controls. However, embryos from high T4 eggs did not differ in general body growth (body weight, length, and general morphology) or hatchability compared to controls. In a single T3 experiment, hens were dosed twice daily with 1 microg T3. The embryos from eggs of these hens had accelerated differentiation/maturation of pelvic cartilages (sampled at Day 12) compared to those from control eggs; body growth did not differ from that of controls.

In this study, we compare the ontogenic patterns for thyroid hormones, growth hormone (GH), and insulin-like growth factor-I (IGF-I) in altricial European starlings and precocial Japanese quail and examine the effects of feed restriction on these species. The most marked difference in development between the altricial and precocial birds was with respect to plasma thyroid hormone patterns. In the starling, circulating concentrations of triiodothyronine (T3) and thyroxine (T4) were very low in embryos, then increased progressively after hatching to peak at 10-11 days of age. In contrast, in quail, in which other studies have shown that most thyroid maturation occurs during the embryonic and peri-hatch periods, the circulating concentrations of T3 and T4 showed little posthatch ontogenic change. Plasma concentrations of both GH and IGF-I showed similar patterns in both species with a posthatch rise (peak at 3 days in starlings and 8 days in quail), followed by a decline. Food restriction to maintain body weight resulted in decreased plasma concentrations of T3 and IGF-I in both species. After return to ad libitum feeding, plasma T3 and IGF-I increased in both early and late restricted starlings and in late restricted quail. Although both species responded to food restriction with similar patterns of endocrine change, age-related differences in the magnitude of hormonal responses were observed.

The control of both metabolism and developmental events by thyroid hormones involves a variety of metabolic modifier effects. These actions of thyroid hormones are mediated by nuclear thyroid hormone receptors that have their highest affinity for triiodothyronine (T3). Thyroid hormone deiodinations can have activating or inactivating effects, with the production of T3 (the most active form) and its degradation being of key importance. At the organismic level, the development of thermogenic metabolic responses necessary for homeothermy in birds is correlated with the pattern of thyroid development and the extrathyroidal deiodinations of thyroid hormones. At the cellular level, deiodination effects on cellular T3 availability can protect T3 supply for critical developmental processes (e.g., in brain) or potentially play a role in the differentiation and maturation of various hormone-responsive tissues. Thyroid hormones, both alone and through interactions with other hormones and growth factors, also can influence intermediary metabolic responses that, in turn, play roles in growth and development.

The role of glucocorticoids and their interaction with the glucocorticoid receptor (GR) during embryonic growth and development has been a topic of interest and research for many years. Glucocorticoids are known to be teratogenic, and administration of these compounds during pregnancy produces cleft palate in the offspring. In the mouse, induction of cleft palate correlates with the level of palatal glucocorticoid receptor (GR) expression. However, the specific patterns of GR expression during critical stages of palatal morphogenesis remain to be examined. In the present study, GR expression was evaluated in the developing palates of C57BL/6N mouse embryos on gestation days (GD) 12, 13, 14, and 15 by both in situ hybridization and immunohistochemistry. On GD 12, GR mRNA was locally expressed in the region of palatal shelf outgrowth in both mesenchymal and epithelial cells, and GR was expressed uniformly throughout the developing shelf. A similar pattern of distribution occurred on GD 13 as the shelf became larger and elevated. By GD 14, GR was regionally expressed with higher levels in epithelial cells relative to mesenchyme. Chondrogenic regions strongly expressed GR. In different regions of oral and nasal epithelia localized patterns of expression were noted and may be related to differentiated state. Correspondingly, GR mRNA was expressed in epithelia and became regional in mesenchyme with abundant mRNA in regions of bone formation. On GD 15, oral and nasal epithelial cells showed mRNA, but mesenchymal levels were low except for chondrogenic regions. This pattern correlated with immunohistochemical localization of GR on GD 15. During palatogenesis the expression of mRNA correlated with immunodetectable GR peptide. GR localized initially within regions of active morphogenesis and subsequently within differentiating cells. This specificity of spatial and temporal expression supports the idea that GR is involved in regulation of regional growth and differentiation during palatogenesis.

Plasma thyroid hormone concentrations, liver weight, body weight, and hepatic capability for triiodothyronine (T3) production were measured at 8-h intervals on Days 19 through 21 in embryos and chicks from lines selected for high (HW) and low (LW) body weight at 8 wk of age. Embryos were categorized as not having entered the perinatal period (nonpipped, NP) or as being in one of three perinatal stages: embryos that had pipped into the air cell (AC), embryos pipped through the shell (TS), and chicks within 24 h of hatching (H). Both plasma thyroxine (T4) and T3 peaked at the TS stage in embryos of both lines. Embryos had higher concentrations of both hormones and hatched earlier in the LW than in the HW line. This association and evidence from other studies is suggestive that T3 may be playing a role in line differences in hatching time. Hepatic 5'deiodinase (5'D) activity was higher in LW than HW embryos and chicks at the AC, TS, and H stages although the opposite had been the case prior to the perinatal period (NP stage). Hepatic 5'D increased at each stage from AC to TS to H. When the patterns of liver growth and body weight were considered, this pattern and the differences between the lines were exaggerated further. Comparison of the plasma and 5'D patterns during the perinatal period suggests that substrate (T4) supply may be more important than 5'D capability of the liver in determining the pattern of T3 supply during some of these stages.

Intestinal 5'deiodinase activity (5'D) in adults from lines of chickens selected for high (HW) and low (LW) juvenile body weight was studied in vitro using reverse T3 substrate. This avian intestinal 5'D activity is similar in its biochemical characteristics to the hepatic 5'D activity of mammals and birds. Adult 5'D activity was higher in line HW than LW; a pattern opposite to that prior to hatching. Previous studies have shown that thyroid hormones are important in triggering differentiation of the intestine during the latter part of embryonic life. Our studies indicate that the intestinal tissue is capable of producing T3 that may be important in stimulating differentiation of the intestine itself, in preparation for posthatching food assimilation. The intestines of LW embryos, which hatch earlier than HW embryos, show both more rapid maturation, as indicated by intestinal alkaline phosphatase activity (a marker of differentiation), and higher 5'D activity. Increases in intestinal 5'D in the HW line at hatch may be important in the differential intestinal growth that leads to higher relative intestinal weights in HW than LW chicks after hatch. The magnitude of 5'D activity and the relative size of the intestine suggest that this organ could be an important contributor to plasma T3.

1. Assay conditions for measuring hepatic 5'-deiodinase (5'D) activity at initial velocity, using reverse T3 as substrate, have been validated for adult chicken liver. 2. The characteristics of hepatic 5'D activity in adult chickens from lines selected for high (HW) and low (LW) juvenile body weight are similar to those in mammals and in the other birds that have been investigated. 3. Chickens from the HW line have significantly higher specific activity of hepatic 5'D, and thus potentially higher T3 production, than those from either the LW line or the F1 cross (HL) between the HW and LW lines.

Using rT3 as substrate, an in vitro 5'D assay was validated for use with liver tissue from adult Japanese quail, by defining conditions under which activity is proportional to enzyme (protein) concentration and is linear with incubation time. Activity was measured as the release of 125I from labeled rT3. Using validated assay conditions we found the following 5'D characteristics: maximal activity from 10 to 50 mM dithiothreitol (cofactor), an apparent Km of 0.52 microM rT3, pH optimum of 7.6-8.5, complete inhibition by 1 mM propylthiouracil and by 1.0 mM iopanoic acid, and substrate "preference" of rT3 greater than T4 greater than T3. Based on these characterizations the quail hepatic 5'D activity is like the Type I 5'D activity found in mammalian liver and kidney and embryonic chicken liver. To determine how previous unvalidated assays, that used high tissue and relatively low substrate (T4) concentrations, influenced 5'D studies we reevaluated 5'D development using an assay validated for each developmental stage with rT3 as substrate. We found extreme quantitative differences in the activities measured and in the proportional relationships between stages, and only limited qualitative similarity in the pattern of 5'D development when unvalidated T4 assay results were compared with validated rT3 assay results. Our data in this paper show good correspondence between whole liver 5'D activity per unit body weight and plasma T3/T4 ratios for the developmental stages sampled.

Adult ring dove hepatic 5'-deiodinase (5'D) activity was studied in vitro using reverse T3 (rT3) as substrate. A previous study of ring dove hepatic deiodinase in our laboratory did not include characterization studies and was unsuccessful in relating the pattern of 5'D development to that of plasma T3. In the present study we established valid assay conditions and characterized the hepatic 5'D in doves to provide a comparison with other avian species and 5'D activity is proportional to enzyme concentration (as postmitochondrial fraction (PMF) protein) over the range measured (0-0.163 mg PMF protein/ml, representing 0-2.19 mg original tissue/ml). Activity was linear with time from 5 to 30 min of incubation at 0.163 mg PMF protein/ml. Our 5'D assays used 20 mM DTT; activity was maximal from 5 to 30 mM DTT. Using the validated conditions the following characteristics were found: the apparent Km was 0.44 microM rT3, Vmax was 255 pM rT3 degraded/min-mg PMF protein, and activity was completely inhibited by 1 mM PTU. Activity was maximal at pH 8.04 and at 37.5 degrees (although this did not differ from activity at 41.5 degrees, the body temperature of doves). In summary, this study demonstrates conditions that measure 5'D at initial velocities in dove liver and demonstrates that the hepatic 5'D enzyme in ring doves is similar to the deiodinase activity in liver of galliform birds and mammals.

Japanese quail (Coturnix japonica) were used to study the effects of different egg iodide (I) availabilities on thyroid function during development. Low (less than 50 micrograms 1/kg feed in the maternal diet) and high (1200 micrograms 1/kg feed) I availability were compared to control levels (800 micrograms 1/kg feed), a standard supplementation for game bird feed. We measured thyroid gland content of I, triiodothyronine (T3) and thyroxine (T4), plasma concentrations of T3 and T4, hepatic 5' monodeiodinase (5'-D) activity, and the response of the thyroid gland to thyrotrophin (TSH) stimulation. Embryos, on day 14 of the 16.5-17 day incubation period, and 1-day chicks were used for most studies but thyroid gland hormone content and plasma hormone concentrations were determined for more stages. With high I, thyroidal I content was elevated but thyroidal T4 and T3 were not different from controls. Plasma T3 and T4, the thyroid gland response to TSH stimulation, and hepatic 5'-D activity did not differ between control and high I. Reduced body weight occurred with high I. In general, thyroid gland weight was not altered, but some high I birds exhibited thyroid hypertrophy and altered thyroid gland function. With low I availability, thyroid gland contents of I and T4 were reduced but thyroidal T3 content was maintained. The thyroid gland response to TSH stimulation, plasma thyroid hormone concentrations, and the developmental patterns of plasma thyroid hormones, hepatic 5'-D activity, body weight and thyroid weight were not different between control and low I groups. Developing Japanese quail exhibit excellent ability to adjust thyroid function over a wide range of I availabilities. Regulation appears to occur at the level of thyroid hormone synthesis in the thyroid gland, which allows most aspects of thyroid dynamics to remain unchanged in the maintenance of circulating thyroid hormone concentrations.

Thyroid function was studied in Japanese quail, Coturnix japonica, and Ring doves, Streptopelia risoria, when both were fed the same dietary iodine (I; 930 micrograms I/kg). We also compared thyroid function in groups of doves receiving low I (less than 100 micrograms I/kg) or moderate I (930 micrograms I/kg). We measured thyroid gland (TG) weight, TG stable I content, TG 125I uptake, and 125I labeling of thyroid hormones. Triiodothyronine (T3) and thyroxine (T4) concentrations in TGs and serum were also determined. Our results indicate that doves and quail receiving the same dietary I show similar serum T3 (the presumed metabolically active hormone) and TG functional state but that there are some differences between the species in the way in which this functional state is achieved. We also assessed the effects of differences in I availability on thyroid function in doves. With low dietary I doves show decreases in some measures of thyroid function (reduced serum T4 and TG-hormone stores) compared to doves with moderate I but maintain a comparable level of serum T3. This regulation of T3 appears to be independent of serum T4 or TG-hormone stores.

Water deprivation (WD) resulted in increased serum osmotic pressure (OP) and decreased body weight (WB); adrenal aldosterone content did not change. Adrenal corticosterone content tended to be elevated during early WD, indicating a stress response, but tended to decrease after seven days of WD, suggesting adrenal fatigue. During water restriction (WR), after the period of weight loss, adrenal corticosterone content and serum OP were elevated. As the birds began to gain weight, aldosterone levels did not change but adrenal corticosterone content and serum OP approached control values, suggesting that the birds were beginning to adapt to the WR. Adrenal sensitivity to ACTH was indicated by the elevated adrenal aldosterone and corticosterone content after ACTH injection.

The patterns of thyroid development in precocial Japanese quail and altricial Ring doves are described and compared. Thyroid development can be divided into two phases: The first is characterized by increasing functional capacity of the thyroid gland but low circulating concentrations of thyroid hormones; during the second phase there are further increases in thyroid gland activity as well as a shift toward much higher levels of thyroid activity in the periphery. In Japanese quail, the first phase occurs during the latter half of embryonic life, and there is an abrupt transition to the second phase beginning with a perinatal hormone peak. In Ring doves the first phase continues into the first few days of the posthatching period, and the transition to the second phase of higher serum hormones is gradual and lasts until about 6-8 days of age. In addition to the release of hormones from the thyroid gland, serum binding proteins and peripheral tissue 5'-monodeiodinase (which converts thyroxine to triiodothyronine, the metabolically active hormone) play roles in controlling the balance of thyroid hormone availability to the tissues. The potential roles of peripheral deiodinases in hormone dynamics are discussed in relation to tissue and organ growth and maturation during the first phase and in relation to whole organism metabolism with continued growth during the second phase.

We have developed a dilution method for avian urine and modified existing analytical methods for ammonia, urea, uric acid and total nitrogen to permit determination of all these compounds on aliquots of the same urine sample. The proportions of urinary nitrogen in these compounds were: uric acid 55-72%, ammonia 11-21% and urea 2-11%. This ranking of proportions was consistent for four different dietary protein-water availability regimes. There were no significant differences in the proportions of these nitrogenous compounds due to either dietary protein intake or changes in water availability.

Japanese quail, a species with a precocial pattern of development, were used as a model for studying the ontogeny of thyroid responses to TSH. Thiourea was administered to embryos early in incubation, and the effects were assessed by measuring thyroid 32P uptake, serum thyroid hormones, thyroid weights, and body weights. Thyroid stimulation after thiourea treatment indicated that maturation of thyroid-pituitary negative feedback occurs between days 9 and 10 of the 16.5-day incubation period. Since thyroid function continues to increase faster than thyroid size, hypothalamic or higher control of the pituitary appears to dominate over thyroid-pituitary feedback effects for the remainder of the embryonic period. TSH administration to embryos and chicks resulted in similar time-course and dose-response characteristics, as judged by the thyroid 32P uptake response. Single injections of TSH into 14-day-old embryos resulted in parallel increases in serum T3 and T4, with essentially no change in the T3 to T4 ratio. In contrast, the posthatching response of both 1-day-old chicks and adults to TSH involved significant increases in only T4 and resulted in decreased serum T3 to T4 ratios. Thus, there are developmental changes in the hormonal response to TSH. These results indicate that TSH effects on the embryonic thyroid cannot account for the increase in the T3 to T4 ratio during the perinatal hormone peak. Our data are consistent with the idea that T3 produced peripherally by monodeiodination of T4 accounts for the perinatal change in the thyroid hormone ratio.

Single injections of thyrotropin (TSH) increase serum T4 and thyroidal 32P uptake but not thyroidal 125I uptake regardless of dosage, exposure time or age. Chronic TSH exposure, with 3 or more days of injection, does increase thyroidal 125I uptake. Studies using iodine (I) supplementation indicated that the increased thyroidal radioiodine uptakes seen with chronic TSH administration were not due to an I deficiency in the thyroid resulting from high hormone release. Labeled and unlabeled experiments comparing the effects of single vs. multiple injections of TSH were used to describe the effects of TSH on hormone release, hormone production and thyroidal I uptake.

Binding studies were conducted using in vitro-labeled quail liver nuclei to identify and characterize receptors for L-triiodothyronine (T3). Saturation binding experiments were analyzed by Scatchard analysis and indicated a single class of high-affinity, limited-capacity T3 binding sites. These receptors exhibited binding specificity as demonstrated by competition experiments between labeled T3 and unlabeled thyroid hormones or hormone analogs. Binding specificity was virtually identical in quail and rat liver nuclei. Thus, apparent differences between mammals and birds, in regard to the biological potency of T4 versus T3, are not apparent at the level of the nuclear T3 receptor.

Chickens from lines selected for high (HA) and low (LA) antibody response to sheep red blood cells (SRBC) were fed ad libitum either a diet containing 0 (control) or .1% thiouracil (TF) throughout two trials. Chicks were injected intravenously with .1 ml of .25% SRBC at 37 days of age in Trial 1, and a booster of the same dosage was given to half of these chicks at 61 days of age. Antibody titers were measured 5 and 3 days after primary and secondary inoculations. In Trial 2, primary inoculations of SRBC were given at 21 and 38 days of age, and chicks were bled 3, 5, 7 and 10 days after inoculation. TF chicks had lower body weights and higher feed efficiencies than controls. Plasma thyroxine (T4) concentrations were similar for both lines, but line by diet interactions were present for plasma triiodothyronine (T3) and T3/T4 ratios. When fed the control diet, T3 concentrations and T3/T4 ratios were higher for line HA than line LA, but the pattern was reversed when thiouracil was fed. Antibody titers to SRBC in both trials were higher for HA than LA chicks, but were similar for TF and control chicks. Persistence of elevated titers appeared to be greater in TF than control chicks. There were no differences between TF and control chicks for heterohphil/lymphocyte ratios.

Ring doves (altricial development) and Japanese quail (precocial development) were used as models to compare differences in serum free hormone concentrations and the binding of thyroid hormones to serum protein fractions in adults, and the pattern of free thyroid hormones in the serum of altricial vs. precocial young. Total and free hormones were determined directly by RIA; free hormones also were determined by equilibrium dialysis. Binding protein fractions were identified by electrophoresis of serum preincubated with labelel hormones. Albumin bound the largest proportion of T4 in serum to both species; albumin also bound the largest proportion of T3 in doves, but globulin bound the largest proportion in quail. There were significant differences between species in the proportional binding of both thyroid hormones by different protein fractions at physiological pH. Electrophoretic separations at alkaline pH significantly altered hormone binding by different protein fractions from that at physiological pH. These data explain some conflicting results in the literature on thyroid hormone-binding proteins in different species. Free T4 and free T3 were below the sensitivity limits of the assays during the perinatal period in doves. After hatching, serum free T4 rose more rapidly than total T4. After day 12, hormone concentrations decreased, with a proportionately greater change in free T4 than in total T4. Serum free T3 concentrations were variable, but did not change significantly during development. These results demonstrate that the pattern of serum free thyroid hormones, like that of total hormones, is markedly different in altricial than in precocial development.

We compared the developmental pattern of thyroid function and its pituitary control in precocial Japanese quail and altricial Ring doves. We measured thyroid hormone (TH) content of thyroid glands (TG), TH concentrations in the serum, relative pituitary gland thyrotropin (PG-TSH) content, and the ability of the TG to respond to exogenous TSH as indicated by increases in TG-3',5'-monophosphate (cAMP) content or in serum TH concentrations. In embryonic quail there is considerable maturation of thyroid function prior to hatching. TG-TH content is low but detectable in Day 8 embryos; TG-TH content increases 300x between Day 8 and hatching (16.5-day incubation). Pituitary TSH is detectable by bioassay in quail on embryonic Day 8, with higher levels found closer to hatching. The TG of 8-day embryos responds to TSH injection by increased TG-cAMP content but the serum TH response to TSH does not appear until Day 9. Serum TH concentrations suggest that the TG is under pituitary control during the latter part of incubation. In doves most of the development of thyroid function and the maturation of its pituitary control occur after hatching and thus thyroid functional development is much later in doves than in quail. TG-TH content is extremely low in embryos and nestlings up to 3 days after hatching, increases slowly in nestlings up to Day 10, then increases sharply. Serum TH content is very low in embryos and rises steadily in nestlings to plateau after about Day 8. Pituitary TSH content, estimated by a quail bioassay, is undetectable in embryos and nestlings until Day 4 but increases thereafter. The TG does not respond (based on serum TH concentration) to TSH injection through the day of hatching (Day 16; mean incubation period of 16.5 days), but an increase in serum TH occurs in Day 2 nestlings in response to TSH injection. The magnitude of this response continues to increase during the first week after hatching.

Two forms of iodothyronine 5'-monodeiodinase (5'-D) were studied in liver homogenates from adult and developing quail. The influence of fasting in adults and corticosterone treatment in embryonic quail on 5'-D also were examined. Liver homogenates were assayed for 5'-D activity in the presence of abundant substrate (T4) and cofactor (dithiothreitol; DTT). Generation of T3 during a 15 min incubation at 37 degrees C was assessed by an ethanol-based RIA. In adults, both Type I [the fraction of activity inhibited by propylthiouracil (PTU)] and a putative Type II (the PTU-insensitive fraction) were present in liver homogenates. Type II activity typically comprised about 30% of Total activity. Type I activity first appeared on day 15 of the 16.5 day incubation period, increased 20-fold to peak at hatching, then gradually declined to reach adult levels by 21 days of age. Type II activity was present at all developmental stages and was highest during the perinatal period. Corticosterone treatment in vivo on day 13 of development induced increases in both Type I and Type II activities in liver homogenates 24- and 48-h after treatment. This study demonstrates that in avian liver a putative Type II 5'-D activity (generally considered to be lacking in mammalian liver) is present and may be important in the maintenance of minimal concentrations of tissue T3 during fasting. Both types of 5'-D contribute to the developmental pattern of serum T3 concentrations. Type II comprises a large proportion of total activity during late embryonic life; Type I becomes predominant at the beginning of the perinatal period.(ABSTRACT TRUNCATED AT 250 WORDS)

Technological advances, especially radioimmunoassays, have led to good descriptive information about the timing and pattern of development of the thyroid gland (TG) and circulating thyroid hormone (TH). Immunocytochemical studies in combination with more traditional techniques such as gland ablation and hormone replacement have revealed the time of appearance of each hormone in the axis, the relative amounts of hormone present in each gland at different developmental stages, and a general picture of the pattern of maturation of the hypothalamic-pituitary-thyroid axis. Studies of the pituitary control of the TG remain limited by the lack of adequately specific techniques for measuring avian thyroid-stimulating hormone. Our understanding of peripheral TH dynamics is progressing as a result of iodothyronine deiodinase assays but full understanding will require more elaborate studies that adequately characterize the enzyme system(s) in each case. Molecular techniques have made powerful strides in identifying the gene responsible for producing a protein that appears to be the triiodothyronine receptor. Receptor assays have revealed the pattern of changes in receptor-binding capacities during development but have not yet revealed how binding of hormone to the receptor triggers cellular activity. Molecular genetic techniques are being used to reveal the mechanisms whereby some examples of developmental events (e.g., malic enzyme synthesis) are induced by TH. Although it is not yet possible to assess the value of molecular studies in this area for developing practical applications, these techniques are revealing the fundamental biological roles of TH in growth and development.

Thyroid growth, thyroid function and body growth are markedly different in developing precocial Japanese quail and altricial Ring doves despite comparability in incubation period, hatchling size, adult body weight and adult serum thyroid hormone concentrations. In quail thyroid activity is high during the perinatal period, declines shortly after hatching, then gradually attains adult function. In contrast, in doves, there is no perinatal peak of thyroid activity. Thyroid function is low at hatching and increases steadily during the first week. Serum hormone concentrations vary around a mean similar to that of adults during the remainder of the nestling and fledgling periods.

The enzymatic nature of 5'-monodeiodination (5'-D) in avian liver homogenates was demonstrated by abolishment of activity by iopanoic acid (IOP). T3 production from T4 was dependent on enzyme and substrate concentrations, incubation time, incubation temperature, and pH. Two pathways of 5'-D activity were present in avian liver and exhibited characteristics similar to those described in mammalian tissues. Type II activity was identified as propylthiouracil (PTU)-insensitive activity. Type I (PTU-sensitive) was determined by difference between Total and Type II. Km values were 1.58 microM T4 for Total activity and 0.90 nM T4 for Type II, corresponding to the characteristics of the mammalian pathways. The effects of goitrogens on avian hepatic 5'-D were equivalent to those reported for the mammalian enzyme.

The development of thyroid function in altricial ring doves was assessed by measuring thyroid hormone concentrations in the serum, hormone content of the thyroid glands and hepatic 5'-monodeiodinase activity. Thyroid function is low at hatching and increases during the first eight days while the nestlings are ectothermic and completely dependent on parental care. The rate of increase of serum hormone concentrations slows after Day 8; hormone concentrations are stable by Day 15 and for the remainder of the nestling and early fledgling periods while locomotor ability, feeding self-sufficiency and thermoregulatory ability are maturing. Increases in serum hormone concentrations precede increases in thyroidal hormone content. T3/T4 ratios in serum are much higher than those of stored hormones in the thyroid. Deiodination of T4 to T3 is important in T3 production throughout development but most so in the early nestling stages.

Japanese quail were used as a model for studying the role of binding proteins in determining free T4 (FT4) and free T3 (FT3) concentrations during development. Adults were used to characterize thyroid hormone binding; developmental stages studied were late embryonic, perinatal, hatchling, and juvenile. Total and free hormones were determined directly by RIA, and free hormones were determined indirectly by equilibrium dialysis. Binding proteins were identified by electrophoresis of serum preincubated with labeled hormones. Serum FT4 and FT3 concentrations in adult quail were equivalent to those in humans. T4 bound principally to albumin and secondarily to prealbumin; T3 bound principally to alpha-globulin and secondarily to albumin and gamma-globulin. A specific T4-binding globulin, as in mammals, was not present. The relative affinity of stripped serum was greater for T4 than for T3. In late embryos, FT4 concentrations rise as a result of a marked increase in total T4 (TT4) and modest increases in binding proteins. The perinatal peak in FT4 reflects the perinatal surge of TT4 without a change in binding proteins. From days 1-6 posthatching, FT4 decreases as a consequence of TT4 decreasing faster than the decrease in binding. In juveniles, FT4 concentrations stabilize as increases in TT4 are paralleled by increases in serum binding. T3 binding shows few significant differences from adult values during development, so FT3 concentrations follow closely the pattern of TT3 changes. These results demonstrate that developmental changes in serum binding proteins play a significant role in determining the pattern of free thyroid hormones, especially for FT4, by modulating the total hormone concentrations controlled by the hypothalamic-pituitary axis.

Lines of chickens divergently selected for high (HW) or low (LW) 56-day posthatch body weight were evaluated for growth, development of small intestine, liver and pectoral muscle, and plasma T3 and T4 concentrations. Measurements were taken at 11,17, and 20 days of incubation and 5, 12, 20, 31, and 61 days posthatch. HW chicks and their organs were consistently larger than LW chicks and their organs from day 17 of incubation onward, and followed growth patterns characteristic of these lines. Significant line by age interactions indicated that chickens divergently selected for juvenile body weight had different temporal patterns for plasma T3 and for T4 from 11 days of incubation to 61 days posthatch. There were no differences between males and females within each line for either T3 or T4. There were no simple relationships between the patterns of plasma concentrations of either T3 or T4 and the pattern of growth of the body or the organs studied.