Heritable variation in maternally derived yolk androgens, thyroid hormones and immune factors.

Maternal reproductive investment can critically influence offspring phenotype, and thus these maternal effects are expected to be under strong natural selection. Knowledge on the extent of heritable variation in the physiological mechanisms underlying maternal effects is however limited. In birds, resource allocation to eggs is a key mechanism for mothers to affect their offspring and different components of the egg may or may not be independently adjusted. We studied the heritability of egg components and their genetic and phenotypic covariation in great tits (Parus major), using captive-bred full siblings of wild origin. Egg mass, testosterone (T) and androstenedione (A4) hormone concentrations showed moderate heritability, in agreement with earlier findings. Interestingly, yolk triiodothyronine hormone (T3), but not its precursor, thyroxine hormone (T4), concentration was heritable. An immune factor, albumen lysozyme, showed moderate heritability, but yolk immunoglobulins (IgY) did not. The genetic correlation estimates were moderate but statistically nonsignificant; a trend for a positive genetic correlation was found between A4 and egg mass, T and lysozyme and IgY and lysozyme, respectively. Interestingly, phenotypic correlations were found only between A4 and T, and T4 and T3, respectively. Given that these egg components are associated with fitness-related traits in the offspring (and mother), and that we show that some components are heritable, it opens the possibility that natural selection may shape the rate and direction of phenotypic change via egg composition.

Increased Thyroid Hormone Activation Accompanies the Formation of Thyroid Hormone-Dependent Negative Feedback in Developing Chicken Hypothalamus.

The hypothalamic-pituitary-thyroid axis is governed by hypophysiotropic TRH-synthesizing neurons located in the hypothalamic paraventricular nucleus under control of the negative feedback of thyroid hormones. The mechanisms underlying the ontogeny of this phenomenon are poorly understood. We aimed to determine the onset of thyroid hormone-mediated hypothalamic-negative feedback and studied how local hypothalamic metabolism of thyroid hormones could contribute to this process in developing chicken. In situ hybridization revealed that whereas exogenous T4 did not induce a statistically significant inhibition of TRH expression in the paraventricular nucleus at embryonic day (E)19, T4 treatment was effective at 2 days after hatching (P2). In contrast, TRH expression responded to T3 treatment in both age groups. TSHß mRNA expression in the pituitary responded to T4 in a similar age-dependent manner. Type 2 deiodinase (D2) was expressed from E13 in tanycytes of the mediobasal hypothalamus, and its activity increased between E15 and P2 both in the mediobasal hypothalamus and in tanycyte-lacking hypothalamic regions. Nkx2.1 was coexpressed with D2 in E13 and P2 tanycytes and transcription of the cdio2 gene responded to Nkx2.1 in U87 glioma cells, indicating its potential role in the developmental regulation of D2 activity. The T3-degrading D3 enzyme was also detected in tanycytes, but its level was not markedly changed before and after the period of negative feedback acquisition. These findings suggest that increasing the D2-mediated T3 generation during E18-P2 could provide the sufficient local T3 concentration required for the onset of T3-dependent negative feedback in the developing chicken hypothalamus.

Thyroid regulation in teleost embryonic and larval development - can it be a promise for aquaculture?

The role of thyroid hormones (THs) in fish development is often overlooked and particularly neglected for the embryonic and larval stages of fish. We used a set of experiments to elucidate the importance of TH in embryonic to early larval development in zebrafish. In the first set of experiments we used morpholino antisense oligonucleotides to knock down TH downstream genes and showed that the depletion of TH in early development resulted in severe deformities, developmental delay, and pigmentation, which could be quantitatively recovered by subsequent TH treatments. In the second set of experiments, blocking TH deposition into zebrafish eggs by treating parental fish with goitrogens resulted not only initial low TH content in eggs, but also subsequent loss of egg laying ability. Overall, these data strongly suggest a key role of TH in early development in fish, providing its worth to be investigated for effective use in reproduction and larviculture.

Thyroid hormone signaling in the Xenopus laevis embryo is functional and susceptible to endocrine disruption.

Thyroid hormone (TH) is essential for vertebrate brain development. Most research on TH and neuronal development focuses on late development, mainly the perinatal period in mammals. However, in human infants neuromotor development correlates best with maternal TH levels in the first trimester of pregnancy, suggesting that TH signaling could affect early brain development. Studying TH signaling in early embryogenesis in mammals is experimentally challenging. In contrast, free-living embryos, such as Xenopus laevis, permit physiological experimentation independent of maternal factors. We detailed key elements of TH signaling: ligands, receptors (TR), and deiodinases during early X. laevis development, before embryonic thyroid gland formation. Dynamic profiles for all components were found. Between developmental stages 37 and 41 (~48 h after hatching, coincident with a phase of continuing neurogenesis) significant increases in T(3) levels as well as in mRNA encoding deiodinases and TR occurred. Exposure of embryos at this developmental stage for 24 h to either a TH antagonist, NH-3, or to tetrabromobisphenol A, a flame retardant and known TH disruptor, differentially modulated the expression of a number of TH target genes implicated in neural stem cell function or neural differentiation. Moreover, 24-h exposure to either NH-3 or tetrabromobisphenol A diminished cell proliferation in the brain. Thus, these data show first, that TH signaling exerts regulatory roles in early X. laevis neurogenesis and second, that this period represents a potential window for endocrine disruption.

Dynamic mRNA distribution pattern of thyroid hormone transporters and deiodinases during early embryonic chicken brain development.

Maternal thyroid hormones (THs) are important in early brain development long before the onset of embryonic TH secretion, but information about the regulation of TH availability in the brain at these early stages is still limited. We therefore investigated in detail the mRNA distribution pattern of the TH activating type 2 and inactivating type 3 deiodinases (D2 and D3) and the TH transporters, organic anion transporting polypeptide 1c1 (Oatp1c1) and monocarboxylate transporter 8 (Mct8), in chicken embryonic brain as well as in retina and inner ear from day 3 to day 10 of development. Oatp1c1, Mct8 and D3 are expressed in the choroid plexus and its precursors allowing selective uptake of THs at the blood-cerebrospinal fluid-barrier with subsequent inactivation of excess hormone. In contrast, the developing blood-brain-barrier does not express Oatp1c1 or Mct8 but appears to be a site for TH activation by D2. Expression of D3 in several sensory brain centers may serve as protection against premature TH action. Expression of D2 and Mct8 but not D3 in the developing pituitary gland allows accumulation of active THs even at early stages. Mct8 is widely expressed in gray matter throughout the brain. This is the first comprehensive study on the dynamic distribution pattern of TH-transporters and deiodinases at stages of embryonic brain development when only maternal THs are available. It provides the essential background for further research aimed at understanding early developmental processes depending on maternal THs.

Interaction between ascites susceptibility and CO(2) during the second half of incubation of two broiler lines: the effect on post-hatch development and ascites mortality.

1. The aim of this study was to investigate if genetic predisposition to ascites interacts with changed incubation conditions, and how this might affect the post-hatch performance and ascites susceptibility. 2. An ascites sensitive (A) and resistant (E) broiler line were incubated under standard or high CO(2) conditions (up to 4%) from embryonic d 10 onwards. After hatch, chicks were exposed to cold from the 15th day of the rearing period to increase the incidence of ascites. 3. The A line had a higher post-hatch body weight from week three, higher blood pCO(2) from d 21, higher haematocrit at d 35 and d 42, and higher plasma corticosterone concentration from d 21 onwards, compared with the E line, regardless of incubation conditions, supporting the given selection criteria. Ascites mortality did not, however, differ between lines. 4. Incubation under high CO(2) conditions during the second half of incubation increased the ascites mortality, decreased body weight from week 4 onwards, affected venous blood pCO(2), decreased blood pO(2) from d 31, increased haematocrit at d 35 and d 42, and lowered the thyroxine and triiodothyronine concentrations at most sampling days. These effects were observed in both lines. The results suggested a metabolic programming of CO(2) incubated chickens which affected ascites susceptibility.

PTTG-binding factor (PBF) is a novel regulator of the thyroid hormone transporter MCT8.

Within the basolateral membrane of thyroid follicular epithelial cells, two transporter proteins are central to thyroid hormone (TH) biosynthesis and secretion. The sodium iodide symporter (NIS) delivers iodide from the bloodstream into the thyroid, and after TH biosynthesis, monocarboxylate transporter 8 (MCT8) mediates TH secretion from the thyroid gland. Pituitary tumor-transforming gene-binding factor (PBF; PTTG1IP) is a protooncogene that is up-regulated in thyroid cancer and that binds NIS and modulates its subcellular localization and function. We now show that PBF binds MCT8 in vitro, eliciting a marked shift in MCT8 subcellular localization and resulting in a significant reduction in the amount of MCT8 at the plasma membrane as determined by cell surface biotinylation assays. Colocalization and interaction between PBF and Mct8 was also observed in vivo in a mouse model of thyroid-specific PBF overexpression driven by a bovine thyroglobulin (Tg) promoter (PBF-Tg). Thyroidal Mct8 mRNA and protein expression levels were similar to wild-type mice. Critically, however, PBF-Tg mice demonstrated significantly enhanced thyroidal TH accumulation and reduced TH secretion upon TSH stimulation. Importantly, Mct8-knockout mice share this phenotype. These data show that PBF binds and alters the subcellular localization of MCT8 in vitro, with PBF overexpression leading to an accumulation of TH within the thyroid in vivo. Overall, these studies identify PBF as the first protein to interact with the critical TH transporter MCT8 and modulate its function in vivo. Furthermore, alongside NIS repression, PBF may thus represent a new regulator of TH biosynthesis and secretion.

Evaluation of dietary stevioside supplementation on anti-human serum albumin immunoglobulin G, Alpha-1-glycoprotein, body weight and thyroid hormones in broiler chickens.

Sixty male broiler chickens fed a diet supplemented with 130 mg/kg stevioside (S group) or an unsupplemented diet (C group) from day 1 of age onwards. On day 21 of age, ten birds from either the S (SH) or C (CH) group were injected subcutaneously with 100 µg human serum albumin (HSA) and ten others from either S (SP) or C (CP) group injected with 100 µl phosphate-buffered saline (PBS) in the same way. There were no significant effect of supplementation nor interaction with age on average body weights, T(3) and T(4) concentrations of non-injected chickens. After the primary immunization, α(1) -glycoprotein concentrations increased in all treatment groups except the CP group, and were significantly higher in the CH group in relation to the other groups. Fourteen and 18 days after the primary immunization, HSA injected chickens of both dietary treatments had significantly higher anti-HSA immunoglobulin G (IgG) levels than their PBS injected controls. No effect of stevioside supplementation was observed for IgG level. In conclusion, dietary stevioside inclusion can attenuate the pro-inflammatory response after stimulation of the innate immune response in broiler chickens.

Ontogenic expression profiles of thyroid-specific genes in embryonic and hatching chicks.

The last trimester of the embryonic life of chickens is marked by a steady increase in circulating thyroxine (T(4)) levels, reaching a maximum around hatching. We have measured thyroidal mRNA expression levels of several genes involved in the biosynthesis of T(4), namely sodium/iodine symporter (NIS), thyroglobulin (Tg), thyroid peroxidase (TPO), thyrotropin receptor (TSHR), and thyroid transcription factor 1 (TTF-1), during this period. Subsequently, we measured the expression of these genes in more detail during the entire hatching process and compared the gene expression profiles with concomitant changes in intrathyroidal and circulating thyroid hormone levels. We found that NIS and TPO mRNA expression increased significantly in the perinatal period, whereas Tg mRNA expression rose gradually throughout the last week of embryogenesis but was stable during hatching. TSHR and TTF-1 mRNA levels did not change significantly during the last week of embryonic development and hatching. Our results suggest that the elevated plasma T(4) levels observed in the developmental period studied are caused by an increased synthesis and secretion of T(4) by the thyroid gland. Augmented expression of Tg may play an important role in the increasing T(4) production during the last week of embryonic development, whereas increased NIS and TPO expression around hatching allows the thyrocytes to boost T(4) synthesis even further.

Chicken folliculo-stellate cells express thyrotropin receptor mRNA.

We investigated the presence of thyrotropin receptor (TSHR) mRNA in chicken pituitary and brain, and quantified the changes in its expression during the last week of embryonic development. We found that in the pituitary gland, TSHR mRNA co-localizes with folliculo-stellate cells but not with thyrotropic cells, suggesting the existence of a paracrine ultra-short thyrotropin feedback loop. TSHR mRNA was also present throughout the diencephalon and various other brain regions, which implies a more general function for thyrotropin in the avian brain. During late embryogenesis, when the activity of the hypothalamo-pituitary-thyroidal axis increases markedly, a significant rise in TSHR mRNA expression was observed in pituitary, which may signify an important change in pituitary ultra-short thyrotropin feedback regulation around the period of hatching.

The chicken embryo as a model for developmental endocrinology: development of the thyrotropic, corticotropic, and somatotropic axes.

The ease of in vivo experimental manipulation is one of the main factors that have made the chicken embryo an important animal model in developmental research, including developmental endocrinology. This review focuses on the development of the thyrotropic, corticotropic and somatotropic axes in the chicken, emphasizing the central role of the pituitary gland in these endocrine systems. Functional maturation of the endocrine axes entails the cellular differentiation and acquisition of cell function and responsiveness of the different glands involved, as well as the establishment of top-down and bottom-up anatomical and functional communication between the control levels. Extensive cross-talk between the above-mentioned axes accounts for the marked endocrine changes observed during the last third of embryonic development. In a final paragraph we shortly discuss how genomic resources and new transgenesis techniques can increase the power of the chicken embryo model in developmental endocrinology research.

Divergent selection for shape of growth curve in Japanese quail. 4. Carcase composition and thyroid hormones.

1. Changes in the relative weights of carcase, abdominal fat, breast and leg muscles, and plasma thyroid hormone concentrations occurring during the first 6 weeks of postnatal growth were analysed in males of HG and LG lines divergently selected for high and low relative body weight (BW) gain between 11 and 28 d of age, respectively, and constant adult BW. 2. The second week of postnatal life was a critical age at which the HG males exhibited a relatively faster growth in comparison to their LG counterparts and permanently exceeded LG males in the percentage by weight of carcase, breast and leg muscle. A higher production of muscle tissues was associated with lower accumulation of abdominal fat before sexual maturity. 3. In general, the plasma T(3) level of HG quail exceeded that of LG quail. Nevertheless, significant differences were found only at 14, 21 and 28 d of age, that is, in the period during which the highest inter-line differences in relative growth rate were noted. Also the T(3)/T(4) ratio followed a similar trend while plasma T(4) level showed no clear and consistent inter-line differences. 4. The results suggest that the selection for the shape of the growth curve, like the selection for body fat, modifies the carcase quality owing to shortening/prolongation of the acceleration growth phase. Individuals with a short acceleration phase of the growth curve are characterised by low carcase quality during the fattening period.

Endocrine disrupting polyhalogenated organic pollutants interfere with thyroid hormone signalling in the developing brain.

Persistent polyhalogenated organic pollutants are present worldwide and accumulate along the food chain. They interfere with human and animal health and are particularly harmful for pre- and perinatal neurodevelopment. The mechanisms behind the observed effects vary depending on the specific compound investigated. Co-planar polychlorinated biphenyls (PCBs) can act via the arylhydrocarbon receptor while many ortho-substituted PCBs disrupt intracellular Ca(2+) homeostasis. A common mechanism for a wide variety of PCBs is interference with thyroid hormone (TH) signalling in developing brain, by changing intracellular TH availability or by interacting directly at the level of the TH receptors. Studies on gene expression in cortex and cerebellum revealed both hypothyroid- and hyperthyroid-like effects. However, since THdependent gene expression plays a crucial role in the coordination of neuronal proliferation, migration, synaptogenesis, myelination, etc., both reduced/delayed and increased/premature expression may result in permanent structural changes in neuronal communication networks, leading to lifelong deficits in cognitive performance, motor functions, and psychobehavior. In a similar way, PCBs are able to interfere with estrogen- and androgen-dependent brain development and in some studies neurobehavioral outcome was shown to be gender-specific. Other persistent organohalogens like polychlorinated dibenzo-p-dioxins (PCDDs) and polybrominated diphenyl ethers (PBDEs) also act as endocrine disrupters in the developing brain. Several of the mechanisms involved are similar to those of PCBs, but each group also works via own specific pathways. The fact that persistent organohalogens can amplify the neurotoxic effects of other environmental pollutants, such as heavy metals, further increases their risk for human and animal neurodevelopment.

Peripheral ghrelin reduces food intake and respiratory quotient in chicken.

Ghrelin injection, either centrally or peripherally strongly stimulates feeding in human and rodents. In contrast, centrally injected ghrelin inhibits food intake in neonatal chickens. No information is available about the mechanism and its relationship with energy homeostasis in chicken. Since ghrelin is predominantly produced in the stomach, we investigated the effect of peripherally injected ghrelin (1 nmol/100g body weight) on food intake and energy expenditure as measured in respiratory cells by indirect calorimetry for 24h in one-week-old chickens. Plasma glucose, triglycerides, free fatty acids, total protein and T(3) were measured in a separate experiment until 60 min after injection. Food intake decreased until at least 1h after intravenous ghrelin administration. The respiratory quotient (RQ) in ghrelin-injected chickens was reduced until 14 h after administration whereas plasma glucose and triglycerides concentrations were not altered. Free fatty acids and total protein levels also remained unchanged. Ghrelin did not influence heat production and this was supported by the absence of changes in plasma T(3) levels when compared to the control values. In conclusion, peripheral ghrelin reduces food intake as well as RQ and might influence the type of substrate (macronutrient) that is used as metabolic fuel.

The chicken pituitary-specific transcription factor PIT-1 is involved in the hypothalamic regulation of pituitary hormones.

Pit-1 is a pituitary-specific POU-domain DNA binding factor, which binds to and trans-activates promoters of growth hormone- (GH), prolactin- (PRL) and thyroid stimulating hormone-beta- (TSHbeta) encoding genes. Thyrotropin-releasing hormone (TRH) is located in the hypothalamus and stimulates TSH, GH and PRL release from the pituitary gland. In the present study, we successfully used the cell aggregate culture system for chicken pituitary cells to study the effect of TRH administration on the ggPit-l* (chicken Pit-1), GH and TSHbeta mRNA expression in vitro. In pituitary cell aggregates of 11-day-old male broiler chicks the ggPit-l * mRNA expression was significantly increased following TRH administration, indicating that the stimulatory effects of TRH on several pituitary hormones are mediated via its effect on the ggPit-l* gene expression. Therefore, a semiquantitative RT-PCR method was used to detect possible changes in GH and TSHbeta mRNA levels. TRH affected both the GH and TSHbeta mRNA levels. The results of this in vitro study reveal that ggPit-1 * has a role in mediating the stimulatory effects of TRH on pituitary hormones like GH and TSHbeta in the chicken pituitary.

Role of spatiotemporal expression of iodothyronine deiodinase proteins in cerebellar cell organization.

Thyroid hormones (TH) play a crucial role in various developmental processes in all vertebrates. The expression of a number of thyroid hormone responsive genes is of critical importance in processes like cell maturation and migration. Since these genes are mostly regulated by binding of the receptor-active TH (T(3)) to the thyroid hormone receptor, the availability of this T(3) is indispensable for correct brain lamination. One important way to regulate local TH availability is via the ontogenetic changes in activating and inactivating iodothyronine deiodinases. The current study was set up to investigate the distribution of type I, type II and type III (D1, D2 and D3) iodothyronine deiodinase protein in the chicken cerebellum at two important developmental ages, namely embryonic day 18 when cerebellar cell migration is fully in progress, and 1 day posthatch, when cerebellar maturation is mostly finished. The results show that the deiodinase proteins are divergently expressed in the cerebellar cell population. D1 and D3 are expressed in the granule cells at E18, whereas D2 is found mostly in the molecular layer and the Purkinje cells at that time. One day posthatch, the expression of D1 is limited to the mature granule cells and that of D3 to the Purkinje cells exclusively, whereas D2 remains clearly present in the molecular layer. Comparison of the deiodinase protein distribution with the expression of TH-responsive proteins involved in cell migration (reelin, disabled protein 1 and tenascin-C) allows speculating about the effect of this spatiotemporal distribution pattern on cerebellar cell communicative pathways.

The release of growth hormone (GH): relation to the thyrotropic- and corticotropic axis in the chicken.

In the chicken and other avian species, the secretion of GH is under a dual stimulatory and inhibitory control of hypothalamic hypophysiotropic factors. Additionally, the thyrotropin-releasing hormone (TRH), contrary to the mammalian situation, is also somatotropic and equally important in releasing GH in chick embryos and juvenile chicks compared to the (mammalian) growth hormone-releasing hormone (GHRH) itself. Consequently, the negative feedback loop for GH release not only involves the insulin-like growth factor IGF-I but also thyroid hormones. In adult chickens, TRH does no longer have a clear thyrotropic activity, whereas its somatotropic activity depends on the feeding status of the animal. In addition, as in mammals, the secretion of GH and glucocorticoids is stimulated by ghrelin, a novel peptide predominantly synthesized in the gastrointestinal tract. Two chicken isoforms of the ghrelin receptor have been identified, both of which are highly expressed in the hypothalamus and pituitary, suggesting that a stimulatory effect may be directed at these levels. GH and glucocorticoids control the peripheral thyroid hormone function by down-regulating the hepatic type III deiodinating enzyme (D3) in embryos (GH and glucocorticoids) and in juvenile and adult chickens (GH). Moreover, glucocorticoids help to regulate T3-homeostasis in the brain during embryogenesis by stimulating the type II deiodinase (D2) expression. This way not only a multifactorial release mechanism exists for GH but also a functional entanglement of activities between the somatotropic-, thyrotropic- and corticotropic axis.

Interaction of PCBs with thyroid hormone levels and time of hatching in chicken embryos.

The effects of polychlorinated biphenyls (PCB 77, PCB 153, and the mixture Aroclor 1242) on circulating and intracellular thyroid hormone (TH) levels were studied during chicken embryonic development. We observed no influences of PCB 153 on TH availability. Aroclor 1242 caused a transient increase in the T(3) level in the cerebellum at day 16. Clear effects were only seen with PCB 77 around the period of hatching: a severely reduced TH peak, which normally coincides with the stage of internal pipping, and a considerable delay in the moment of hatching.

Neurotoxicity of polychlorinated biphenyls (PCBs) by disturbance of thyroid hormone-regulated genes.

PCBs are known as neurotoxic compounds. Part of this neurotoxicity could be due to an alteration of the expression of TH-regulated genes in brain. To identify such genes, brain protein extracts of hypo- and hyperthyroid as well as PCB-treated embryos were compared by fluorescent 2D-DIGE. In total, we observed 109 differentially expressed proteins, of which 17 differed with both PCB and hypo- or hyperthyroid treatment. It was found that the interaction of PCBs with the expression of TH-regulated genes is congener-specific and that both hyperthyroidism- and hypothyroidism-related effects occur.

Regulation of thyroid hormone availability by iodothyronine deiodinases at the blood-brain barrier in birds.

It is accepted that type II iodothyronine deiodinase (D2) is predominantly found in brain, where it maintains homeostasis of thyroid hormone (TH) levels. The current study describes the production of a polyclonal D2 antiserum and its use in the comparison of D2 protein distribution with that of type I (D1) and type III (D3) deiodinase protein in the chicken choroid plexus (CP). Immunocytochemistry showed high D2 protein expression in the epithelial cells of the CP, whereas the D1 and D3 proteins were absent. Furthermore, dexamethasone treatment led to an upregulation of the D2 protein in these cells.