Thyroid hormones are involved in many physiological processes, during growth, development, behaviour, stress. Their actions are mediated by TH receptors (TR-alpha and TR-beta), which are members of the nuclear receptor (NR) superfamily and function as ligand-activated transcription factors. In amphibians, TR-alpha is expressed shortly after hatching and is maintained at a relatively constant level throughout tadpole life and metamorphosis. Then amphibian metamorphosis is dependent on thyroid hormone (TH) changes, which induces the suite of molecular and cellular changes that cause a tadpole to transform into a frog.
Hormones other than TH play important roles in amphibian metamorphosis, in part by modifying the production and actions of TH. Corticosteroids (CS), hormones produced by adrenocortical cells (interrenal glands in frogs and in fish), synergize with TH at target tissues to promote morphogenesis [1,2]. The production of CS changes with development, rising throughout metamorphosis and reaching a peak at metamorphic climax . Like TH, CS actions are mediated by NRs encoded by two different genes: the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR).
In the current study we examine molecular and physiological mechanisms involved in TH and CS axes regulation. We investigate the synergy between TH and CS not only during amphibian metamorphosis, but also in fish. Indeed TH play important role in fish development. TH level is especially high in the eggs and larvae of several fish species, including the Japanese flounder (
Due to their aqueous exposure, fish and amphibians can be used as indicators for ecotoxicological studies and for detection of endocrine disruptors (ED)
2. Thyroid hormone and corticosteroid endocrine systems: current knowledge
2.1. Thyroid hormone
Thyroid hormones (thyroxine T4 and tri-iodothyronine T3) play an important role in development, differentiation, and metabolism . The lack of T3 in early human development results in growth disturbances and severe mental retardation, a disease called cretinism. TH action is also primary for developmental changes in the nervous system that occur during amphibian metamorphosis. Later in life, T3 plays an important role in metabolic balance . T3 action is mediated by nuclear T3 receptors (TRs) that can bind T3 with high affinity . TRs belong to the nuclear receptor superfamily that also includes the receptors for retinoids, vitamin D, fatty acids, and prostaglandins, as well as “orphan receptors” with no identified ligands [10-13]. TR is encoded by two separate genes, which are designated TR-alpha and TR-beta, located in different chromosomes (17 and 3, respectively, in humans). Like other nuclear receptors, TRs have modular structures with six regions (A–F) and three functional domains.
TR is considered as a transcription factor: it regulates target genes expression directly through DNA response elements. The thyroid hormone response element (TRE) is composed of repeated DNA sequences . Although TRs can bind to TREs as monomers or homodimers, the major form of TR bound to the TRE is the heterodimer with Retinoid X Receptor (RXR). An important property of TRs is their ability to bind TREs constitutively independent of ligand occupancy [8,10,12,13]. Unliganded TR generally represses basal transcription. Ligand binding triggers a conformational change in the TR, resulting in activated transcription of its target gene. In the past few years, great progress in biochemical, functional, and structural studies has clarified the molecular mechanism of TR action.
A classical vertebrate model for thyroid hormone action in development is the amphibian tadpole. Thyroid hormone controls amphibian metamorphosis and thus plays an important role in the developmental changes in the nervous system that occur during metamorphosis. In anuran amphibians, thyroid function regulates the metamorphic process so these are one of the most commonly used
Although TH effects have been mainly studied in mammals and amphibians for metamorphosis process, more and more data show that TH play important role in fish development. TH level is especially high in the eggs and larvae of several fish species . In zebrafish, the thyroid gland begins to develop during early embryogenesis and begins to be active around 55 hours post fertilization (hpf) . Before this developmental stage, TH comes from the maternal stock in the egg . The TH receptors (TR-alpha and TR-beta) are both present in prehatch fish embryos, and allow TH functions. Prehatch embryos possess all TH function components: TR-alpha and TR-beta and TH from the maternal stock . TH are synthesized and secreted by the thyroid gland after TSH stimulation. TSH is produced by thyrotropes present in the fish adenopituitary. Terminal differentiation of thyrotropes in the zebrafish adenopituitary occurs around 48hpf. The thyroid gland is active later, about 55 h post fertilization. Concerning the thyroid gland tissue organization, the zebrafish thyroid gland derives from precursor cells located in the endoderm prior to pharynx formation. During two morphogenetic phases, the thyroid primordium first adopts a position close to the cardiac outflow tract, with the first differentiated thyroid follicle, that grows afterwards along the ventral pharyngeal midline. The thyroid gland in the adult zebrafish is a loose aggregation of follicles close to the ventral aorta.
Zebrafish genome encodes two TR-alpha genes and one TR-beta gene, which are expressed at different developmental stages, suggesting that they have different function during development (for review see reference 4). Essner
Corticosteroids are implicated in many physiological process including osmoregulation, respiration, immunity, reproduction, growth and metabolism. Like thyroid hormone, corticosteroids production and action has been studied in amphibian models, for their implication in the positive control of metamorphosis . In bony fishes, corticosteroids are secreted from the interrenal tissue located in the head kidney region. Cortisol is the major corticosteroid in teleost fish and its release involves the coordinated activation of the hypothalamus-pituitary-interrenal (HPI) axis. The key mediators include the release of corticotrophin-releasing factor (CRF) from the hypothalamus, and stimulating the release of adrenocorticotropic hormone (ACTH) from the pituitary. Circulating ACTH binds to melanocortin receptor 2 (MC2R) on the steroidogenic cells and activates the signalling pathway leading to cortisol biosynthesis .
In bony fishes, the corticosteroid receptor is a ligand-dependant transcription factor, with two major classes of receptors: the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR). Cortisol is the physiological ligand for GR. The molecular characterization of fish GR began with the cloning of rainbow trout (
Little is known about GR and MR function in fish development. Recently, zebrafish has been used to investigate the role of corticosteroid signalling in development. It was shown that both of these receptors are present during embryogenesis . Indeed, during embryogenesis, GR transcripts drops from 1.5 to 25 hpf, and then increases after hatching to the level at 1.5 hpf and was significantly higher at 25 hpf, and this level was maintained until 6 days. This study suggested a more important role for this receptor after hatch in zebrafish. It was also shown that cortisol synthesis occurred only after hatching and that maternal cortisol contributes to early developmental programming . Further, Pikulkaew
In mammals and non-mammalian vertebrates such as amphibians, the major mineralocorticosteroid is aldosterone. However, aldosterone is not detected in fish, and deoxycorticosterone (DOC) is considered as MR ligand in fish. Like aldosterone, DOC is a selective MR agonist, that does not activate trout GR. Cortisol is also a high-affinity ligand for MR. Fish MR is distributed beyond the tissue involved in salt and water balance, especially in gills and intestine, and MR mRNA is also high in rainbow trout brain. The role of MR and its ligand remains less clear than GR, especially concerning its implication during development. MR transcripts continuously increased between 1.5 and 97 hpf and remained at the same high level at 6 days. MR has been suggested as responsible for corticosteroid signalling just after hatching, since it could be activated by maternal cortisol .
2.3. Relationship between TH and CR
The corticosteroid and thyroid hormone receptors possess equivalent transactivation domains and have some structural functional similarity [10,29], suggesting that these nuclear receptors may enhance transcription of target genes by similar mechanisms as summarized above. The thyroid and corticosteroid systems interact at multiple levels to influence several physiological processes like development, growth or behaviour.
Thus, the hypothalamo-pituitary-interrenal axis modulates the thyroid axis in fishes and other vertebrates. Indeed heterologous CRH potently stimulated the release of TSH from cultured pituitary cells
Conversely, T3 seems to be involved in corticosteroid receptors regulation. Recently, Terrien
Other studies have suggested a coincident expression and synergetic action of TH and corticosteroids in other vertebrate models [34-37]. Indeed, corticosteroids and thyroid hormones act synergistically during some physiological processes such as amphibian metamorphosis, which is one of the most relevant biological models the most studied for TH and CR crossregulation . Thus, it was shown that corticosteroids can synergize with thyroid hormone to accelerate tadpole metamorphosis, whether corticosteroids increase T3 binding capacity , or corticosteroids can increase the conversion of active T3 from T4, and decrease the degradation of T3 [38,39]. Moreover, corticosterone treatment upregulates TR-beta expression in the intestine of premetamorphic tadpoles and in tail explants cultures . It is also known that GR suppresses TSH expression .
Like in fish models, TH seems to regulate CS in amphibians. Krain and Denver  showed that T3 upregulated the glucocorticoid receptor expression in tadpoles tail, and this regulation might be consistent with a physiological regulatory relationship, given the developmental pattern of thyroid hormone production and GR mRNA in the tail. In contrast, T3 has been shown to downregulate GR expression in the brain.
The coincident increase in cortisol and TH during flounder metamorphosis  and the regulation of GR mRNA expression after T3 treatment in
3. Use of aquatic organisms to investigate endocrine disruption
Many natural or synthetic chemicals are now routinely observed in water. Evidence revealed that these compounds might interfere with the endogenous endocrine systems of wildlife and humans. Thus, it is now essential to monitor their presence in the environment. Aquatic organisms as amphibians and fish models (zebrafish and medaka) are species widely used in ecotoxicology and for the development of transgenic techniques. These techniques allow development of new tools to detect and screen chemicals in the environment. The zebrafish has numerous technical advantages, so that it can be considered as a model organism: its complete embryogenesis occurs during the first 72 hours post-fertilization and most of the internal organs develop rapidly in the first 24-48 hours. They are easy to observe because embryos are transparent, which allows to easily track their development and expression of fluorescent proteins in transgenic fishes
Zebrafish embryo bioassay has been extensively employed in drug and chemical screening [43,44] and the advantages promoting the use of the embryo assay for those purposes should also promote its use for endocrine disruptors phenotypic screening. Zebrafish embryos and larvae express hormones and receptors, and they possess all molecular actors to respond to exposure to endocrine disruptors.
Moreover, transgenesis in zebrafish is fast and routinely used. For these reasons, the zebrafish is now used to develop simple, rapid, cost-effective and innovative methods for screening environmental pollutants [45,46]. Some stable transgenic zebrafish lines have recently been used for screening chemicals that can mimic the action of estrogens , and for developing automated image acquisition and analysis in 96-well plates . Recently, a fluorescent transient fluorescent transgenic zebrafish model has been developed to easily and rapidly screen compounds capable of disrupting thyroid function .
Another fish species, the medaka (
Finally, amphibians are also used as indicators for ecotoxicological potencies of several environmental stressors. The aquatic larvae are continuously exposed to chemicals compounds present in water because the eggs are lacking a protective eggshell or membrane. After hatching, the skin of amphibians’ larvae is still very permeable, allowing an easy penetration of all compounds leading to high bioavailability and bioaccumulation of endocrine disruptors. This development stage of amphibians is the most sensitive and the most used to study effects of environmental pollutants. Thus, Fini
4. Impact of endocrine disruptors on thyroid hormone and corticosteroids systems in fish
Many natural and man-made chemicals (plasticizers, pesticides, detergents and pharmaceuticals) interfere with the endocrine system and can result in adverse health effects in humans, mammals and fish. Wildlife living in or in closer association with the aquatic environment are especially impacted by these endocrine disruptors, because water act as sinks for chemical discharges. Thus, fish and amphibians are the main potential targets for endocrine disruption at multiple levels, either direct or indirect, through ingestion and accumulation of endocrine disruptor, the exposition or through the food chain. Chronic exposure to endocrine disruptors, such as the oestrogenic compounds used in birth control, can feminize male fish and decrease their capacity to reproduce. In the opposite, masculinised female specimens were found in effluent containing androgenic chemicals . Endocrine disruption on thyroid hormone and corticosteroids in fish was also studied.
Endocrine disruptors may impact corticosteroid signalling system in a direct manner (competition with endogenous ligand) or indirect manner (alteration in accessory proteins, kinases, cytoskeleton...). Among corticosteroids disruptors, chemicals including Polychlorinated Biphenyls (PCB) and heavy metals were the most studied. For example, it has been shown that Arsenic affects GR signalling and one mechanism involves the downregulation of GR content in trout hepatocytes . Another study showed that Copper exposure during 5 days
Cadmium is another metal that is widely distributed in the aquatic environment and is toxic to fish at sublethal concentrations . Due to its long half-life and low excretion rate, Cadmium can also accumulate in various organs, primarily within the liver, kidney and reproductive and respiratory systems in fish . This metal is known to disrupt head kidney corticosteroid production in fish. Vijayan
Because Polybrominated Diphenyl Ethers (PBDE), used as flame retardants, are similar in structure to thyroxine T4 and tri-iodothyronine T3 , several teams have studied their effect on thyroid function. Biologic effects of PBDEs in rodent are similar to those of PCB, increasing risks for reproductive and endocrine disruption. In 2011, Yu
Due to its structural homology with thyroid hormone, Bisphenol A (BPA) is also frequently studied as endocrine disruptor for thyroid function. Is has been shown that BPA can interact with TR and it can be considered as a TR antagonist [60,61]. First, transgenic
Because of the close relationship between thyroid and corticosteroid axis in fish and amphibians, alteration of one of these axes could affect the other one. Indeed endocrine disruption due to chemicals present in the environment affects these two endocrine systems, separately or concomitant. Further studies have to investigate the final effect of disruption of one of these endocrine axes on the other one.
Nuclear receptor crossregulation are important mechanisms for amplifying hormone signals, regulating hormone activity through negative feedback, and coordinating hormone action in a temporal and tissue-specific-manner. In this chapter, we were interested in crossregulation between thyroid hormone and corticosteroids. These two endocrine systems are keys actors of many physiological processes. Their coincident expression and synergetic action were studied in different models (amphibian metamorphosis, stress response in fish). Corticosteroids are known to synergize with thyroid hormone to promote metamorphosis, and links between the thyroid and corticosteroid axes are present at multiple levels. Understanding the interactions between TH and CS will allow us to better understand the effects of endocrine disruptors.
The use of fish species as model organism for research on endocrine disruption is interesting the identification of potential new endocrine disruptors because of endocrine system and hormone signalling pathways are sufficiently similar to other vertebrates. In this context, we should observe more and more studies leading to a large development of screening tools based on these aquatic animals in a next future. However the consequences of the molecular, physiological or organisms’ effects for the population may be different between species. Confirmation in higher vertebrates or in humans of the effects observed in fish is necessary if we want to clearly identify new endocrine disruptors. For instance, the use of aquatic organisms in endocrine disruption studies is relevant, because of their closeness with water-soluble chemicals. And the vast technical possibilities offered by the zebrafish and the medaka models for functional genomics studies justify their use in ED research.