Novel insights into di­(2­ethylhexyl)phthalate activation: Implications for the hypothalamus­pituitary­thyroid axis.

Di (2­ethylhexyl) phthalate (DEHP), an environmental pollutant, is widely used as a plasticizer and causes serious pollution in the ecological environment. As previously reported, exposure to DEHP may cause thyroid dysfunction of the hypothalamic­pituitary­thyroid (HPT) axis. However, the underlying role of DEHP remains to be elucidated. The present study performed intragastrical administration of DEHP (150, 300 and 600 mg/kg) once a day for 90 consecutive days. DEHP­stimulated oxidative stress increased the thyroid follicular cavity diameter and caused thyrocyte oedema. Furthermore, DEHP exposure altered mRNA and protein levels. Thus, DEHP may perturb TH homeostasis by affecting biosynthesis, biotransformation, bio­transportation, receptor levels and metabolism through disruption of the HPT axis and activation of the thyroid­stimulating hormone (TSH)/TSH receptor signaling pathway. These results identified the formerly unappreciated endocrine­disrupting activities of phthalates and the molecular mechanisms of DEHP­induced thyrotoxicity.

Peripheral T3 signaling is the target of pesticides in zebrafish larvae and adult liver.

The intra-tissue levels of thyroid hormones (THs) regulate organ functions. Environmental factors can impair these levels by damaging the thyroid gland and/or peripheral TH metabolism. We investigated the effects of embryonic and/or long-life exposure to low-dose pesticides, ethylene thiourea (ETU), chlorpyrifos (CPF) and both combined on intra-tissue T4/T3 metabolism/signaling in zebrafish at different life stages. Hypothyroidism was evident in exposed larvae that showed reduced number of follicles and induced tshb mRNAs. Despite that, we found an increase in free T4 (fT4) and free T3 (fT3) levels/signaling that was confirmed by transcriptional regulation of TH metabolic enzymes (deiodinases) and T3-regulated mRNAs (cpt1, igfbp1a). Second-generation larvae showed that thyroid and TH signaling was affected even when not directly exposed, suggesting the role of parental exposure. In adult zebrafish, we found that sex-dependent damage of hepatic T3 level/signaling was associated with liver steatosis, which was more pronounced in females, with sex-dependent alteration of transcripts codifying the key enzymes involved in 'de novo lipogenesis' and ß-oxidation. We found impaired activation of liver T3 and PPARα/Foxo3a pathways whose deregulation was already involved in mammalian liver steatosis. The data emphasizes that the intra-tissue imbalance of the T3 level is due to thyroid endocrine disruptors (THDC) and suggests that the effect of a slight modification in T3 signaling might be amplified by its direct regulation or crosstalk with PPARα/Foxo3a pathways. Because T3 levels define the hypothyroid/hyperthyroid status of each organ, our findings might explain the pleiotropic and site-dependent effects of pesticides.

Indications, benefits and risks of transoral thyroidectomy.

The advancement of minimally invasive surgery in the field of endocrine surgery over the last 2 decades has fostered the attempt of natural orifice transluminal endoscopic surgery (NOTES) for thyroidectomy and parathyroidectomy via oral incisions. This technically demanding surgery is currently being evaluated in a number of specialised centres. The procedure has gained popularity worldwide and is performed in more than 50 centres. By retrieving information from published or presented articles and direct personal communications, this study reports several issues to enable and optimise correct patient and surgeon candidacy, present the advantages and prevent novel complications under the standards of open thyroid surgery. Not all patients are eligible for the transoral approach. Transoral endoscopic and robotic procedures were described and critically analysed in this study.

Thyroid Rosai-Dorfman disease with infiltration of IgG4-bearing plasma cells associated with multiple small pulmonary cysts.

BACKGROUND: Rosai-Dorfman disease (RDD) is a rare histiocytosis which involves principally lymph nodes. Thyroid involvement in RDD is a very rare situation, and lung involvement is even rarer. CASE PRESENTATION: We report the case of a 46-year-old woman presenting a painless mass in the right side of the neck and subacute dyspnoea. Computerised tomography (CT) scans of the neck and thorax showed a large thyroid mass causing tracheal stenosis and multiple cystic lesions in both lungs. Subtotal thyroidectomy with a tracheal segment resection and histological analysis confirmed the diagnosis of nodal and extranodal (thyroid, tracheal and probably lung) Rosai-Dorfman disease (RDD) with the presence of increased numbers of IgG4-bearing plasma cells. Clinical, functional and radiological follow up 4 years after surgery without medical treatment did not show any disease progression. CONCLUSIONS: This case report indicates a benign course of nodal RDD with thyroid and tracheal infiltration following surgical resection, association of typical histological signs of RDD (emperipolesis) with IgG4-related disease features, and that lung cysts might be a manifestation of RDD.

Loss of Primary Cilia Results in the Development of Cancer in the Murine Thyroid Gland.

Communications at the interface between the apical membrane of follicular cells and the follicular lumen are critical for the homeostasis of thyroid gland. Primary cilia at the apical membrane of thyroid follicular cells may sense follicular luminal environment and regulate follicular homeostasis, although their role in vivo remains to be determined. Here, mice devoid of primary cilia were generated by thyroid follicular epithelial cell-specific deletion of the gene encoding intraflagellar transport protein 88 (Ift88 ). Thyroid follicular cell-specific Ift88-deficient mice showed normal folliculogenesis and hormonogenesis; however, those older than 7 weeks showed irregularly dilated and destroyed follicles in the thyroid gland. With increasing age, follicular cells with malignant properties showing the characteristic nuclear features of human thyroid carcinomas formed papillary and solid proliferative nodules from degenerated thyroid follicles. Furthermore, malignant tumor cells manifested as tumor emboli in thyroid vessels. These findings suggest that loss-of-function of Ift88/primary cilia results in malignant transformation from degenerated thyroid follicles.

Amphibian (Rana nigromaculata)exposed to cyproconazole: Changes in growth index, behavioral endpoints, antioxidant biomarkers, thyroid and gonad development.

Pesticides are a major cause of reduction in the global population of amphibians. This study investigates the effect of varying concentrations of cyproconazole (1 and 10 mg/L) on Rana nigromaculata during a chronic 90 days exposure period. High levels of cyproconazole (10 mg/L) induced declined body weight, short snout-vent length, slow metamorphic development and abnormal behavioral endpoints in R. nigromaculata tadpoles. Tadpoles exposed to 10 mg/L did not survive beyond 42 days. Abnormal behaviors were observed more frequently with exposure to the highest concentration of cyproconazole. Compared with controls, the concentrations of dismutase (SOD), catalase (CAT) and glutathione (GSH) were significantly increased in tadpoles exposed to 1 mg/L cyproconazole. However, when the concentration of cyproconazole increased to 10 mg/L, concentrations of SOD, GSH and CAT activity began to decline. In addition, thyroid and gonad development were also affected at the gene and hormone level, with varied effects observed with different exposure levels and days. Exposure to cyproconazole at the lower level of 1 mg/L induced damage to histological structures of the thyroid gland. Stereoselective tissue distribution and bioaccumulation of cyproconazole was observed in tadpoles. The ranked order of bioaccumulation was: enantiomer -4 > 3> 2 > 1, with the level of cyproconazole highest in the gut. These findings reflect the toxicity of cyproconazole to R. nigromaculata and further our understanding of the effects of pesticide exposure on global amphibian population declines.

Maternal smoking and high BMI disrupt thyroid gland development.

BACKGROUND: Maternal lifestyle factors, including smoking and increased body weight, increase risks of adult diseases such as metabolic syndrome and infertility. The fetal thyroid gland is essential for the control of fetal metabolic rate, cardiac output, and brain development. Altered fetal thyroid function may contribute to increased disease onset later in life. Here, we investigated the impact of maternal smoking and high maternal weight on human fetal thyroid function during the second trimester. METHODS: Thyroid glands and plasma were collected from fetuses electively terminated in the second trimester (normally progressing pregnancies). Plasma total triiodothyronine (T3) and total thyroxine (T4) were measured by solid-phase extraction-liquid chromatography-tandem mass spectrometry. Fetal plasma thyroid-stimulating hormone (TSH) levels were measured using a multiplex assay for human pituitary hormones. Histology and immunolocalization of thyroid developmental markers were examined in thyroid sections. Transcript levels of developmental, functional, apoptotic, and detoxification markers were measured by real-time PCR. Statistical analyses were performed using multivariate linear regression models with fetal age, sex, and maternal smoking or maternal body mass index (BMI) as covariates. RESULTS: Maternal smoking was associated with significant changes in fetal plasma T4 and TSH levels during the second trimester. Smoke-exposed thyroids had reduced thyroid GATA6 and NKX2-1 transcript levels and altered developmental trajectories for ESR2 and AHR transcript levels. Maternal BMI > 25 was associated with increased fetal thyroid weight, increased plasma TSH levels, and abnormal thyroid histology in female fetuses. Normal developmental changes in AHR and ESR1 transcript expression were also abolished in fetal thyroids from mothers with BMI > 25. CONCLUSIONS: For the first time, we show that maternal smoking and high maternal BMI are associated with disturbed fetal thyroid gland development and endocrine function in a sex-specific manner during the second trimester. These findings suggest that predisposition to post-natal disease is mediated, in part, by altered fetal thyroid gland development.

An in vivo model for thyroid regeneration and folliculogenesis.

While thyroid is considered to be a dormant organ, when required, it can regenerate through increased cell proliferation. However, the mechanism for regeneration remains unknown. Nkx2-1(fl/fl);TPO-cre mouse thyroids exhibit a very disorganized appearance because their thyroids continuously degenerate and regenerate. In mouse thyroids, a cluster of cells are found near the tracheal cartilage and muscle, which are positive for expression of NKX2-1, the master transcription factor governing thyroid development and function. In the present study, we propose that this cluster of NKX2-1-positive cells may be the precursor cells that mature to become thyroid follicular cells, forming thyroid follicles. We also found that phosphorylation of AKT is induced by NKX2-1 in the proposed thyroid progenitor-like side-population cell-derived thyroid cell line (SPTL) cells, suggesting the possibility that NKX2-1 plays a role in differentiation through the modulation of AKT signaling. This study revealed that Nkx2-1(fl/fl);TPO-cre mice provide a suitable model to study in vivo regeneration and folliculogenesis of the thyroid.

Quantitative analysis of in-vivo responses of reproductive and thyroid endpoints in male goldfish exposed to monocrotophos pesticide.

Cross-regulation occurs at many points between the hypothalamic-pituitary-gonad (HPG) and hypothalamic-pituitary-thyroid (HPT) axes. Monocrotophos (MCP) pesticide could disrupt HPG and HPT axes, but its direct target within the endocrine system is still unclear. In the present study, hormone concentrations and transcriptional profiles of HPG and HPT genes were examined in male goldfish (Carassius auratus) exposed to 0, 4, 40, and 400 µg/L MCP for 2, 4, 8, and 12 d. In vivo data were analyzed by multiple linear regression and correlation analysis, quantitatively indicating that MCP-induced plasma 17ß-estradiol (E2) levels were most associated with alteration of cyp19a transcription, which was also a potential point indirectly modulated by the MCP-altered thyroid hormones (THs) status; disturbance of THs pathways was most related with effect of MCP on regulation of the hypothalamic-pituitary hormones involved in the thyroid system, and the increased E2 levels might enhance the impact of MCP on HPT axis by modulating hepatic deiodinase expression. Our finding, based on these correlational data, gave a whole view of the regulations, especially on the cross-talk between sex hormone and thyroid hormone pathways upon exposure to chemicals with unknown direct target in vivo, and cautions should be exercised when developing adverse outcome pathway networks for reproductive and thyroidal endocrine disruption.

Microcalcificaciones y focos ecogénicos puntiformes, ¿qué tan bien sabemos lo que estamos viendo? / Microcalcifications and punctate echogenic foci, how well do we know what we are seeing?

Resumen: La presencia de microcalcificaciones en nódulos tiroideos es un signo muy específico de malignidad, al corresponder a cuerpos de Psammoma. No existen suficientes estudios que demuestren una correlación entre su presencia histológica y su aspecto ecográfico real. Materiales y Métodos: Se seleccionaron todos los nódulos con tamaño mayor a 3 cm puncionados en el Hospital Clínico Universidad Católica entre los años 2010-2015 y se clasificó el aspecto ecográfico según la presencia de 3 tipos de focos ecogénicos con una definición más estricta a lo usual. Se correlacionó lo anterior con hallazgos en biopsias. Resultados: 44 nódulos correspondieron a cáncer papilar de tiroides. Hubo relación estadísticamente significativa entre una nueva definición ecográfica de las microcalcificaciones (focos ecogénicos puntiformes) y la presencia histológica de cuerpos de psamomma. Discusión: Habría una buena correlación entre una definición más estricta y la presencia real de microcalcificaciones en histología, mejorando la alta tasa de sobrediagnóstico advertido recientemente por algunos autores.

Abstract: The presence of microcalcifications in thyroid nodules is a very specific sign of malignancy, as it corresponds to Psammoma bodies. There are not enough studies that demonstrate a correlation between their histological presence and their actual ultrasound appearance. Materials and Methods: All nodules larger than 3 cm punctured at the Universidad Católica Clinical Hospital between 2010-2015 were selected, and the sonographic appearance was classified according to the presence of 3 types of echogenic foci according to a stricter definition than usual. The above was correlated with findings in biopsies. Results: 44 nodules corresponded to papillary thyroid cancer. There was a statistically significant relationship between a new ultrasound definition of the microcalcifications (punctate echogenic foci) and the histological presence of psamomma bodies. Discussion: There would be a good correlation between a stricter definition and the actual presence of microcalcifications in histology, improving the high rate of over diagnosis recently noticed by some authors.

Resveratrol has anti-thyroid effects both in vitro and in vivo.

Resveratrol is a natural polyphenol with antioxidant, anti-inflammatory, and antiproliferative properties. We have shown previously that resveratrol decreases sodium/iodide symporter expression and iodide uptake in thyrocytes, both in vitro and in vivo. In the present study, we further investigated the effects of resveratrol, with evaluation of the expression of additional thyroid-specific genes in the FRTL-5 rat thyroid cell line: thyroglobulin, thyroid peroxidase, TSH receptor, Nkx2-1, Foxe1 and Pax8. We observed decreased expression of these genes in FRTL-5 cells treated with 10 µM resveratrol. The effects of resveratrol was further evaluated in vivo using Sprague-Dawley rats treated with resveratrol 25 mg/kg body weight intraperitoneally, for 60 days. No clinical signs of hypothyroidism were seen, although the treated rats showed significant increase in thyroid size. Serum TSH and thyroid hormone levels were in the normal range, with significantly higher TSH seen in resveratrol-treated rats, compared with control rats. Histological and immunohistochemical analyses confirmed increased proliferative activity in the thyroid from resveratrol-treated rats. These data suggest that resveratrol acts as a thyroid disruptor and a goitrogen, which indicates the need for caution as a supplement and for therapeutic uses.

Natural history of thyroid cancer [Review].

Thyroid cancers have long been considered to arise in middle age and, after their repeated proliferation, resulting in further damage to the genome, they progress to more aggressive and lethal cancers. However, in 2014, some studies were reported that might lead to a marked change in our understanding of the natural history of thyroid cancer. A high prevalence of papillary carcinoma in the young suggested that the first initiation of thyroid cancer is likely to occur in the infantile period. Such a conclusion was also supported by a very slow growth rate of papillary microcarcinomas (PMCs) in an observation trial. The proliferation rate of PMCs was negatively correlated with the age, and surgery to remove PMCs did not contribute to reduce mortality from thyroid cancer. These findings strongly suggested the existence of self-limiting cancers, which are truly malignant but do not progress to lethal cancers, for the first time in human history. The early detection of self-limiting cancers results in overdiagnosis. Ultrasonographic screening of the thyroid in the young should be avoided. Lethal thyroid cancers, whose origin is still unknown, appear suddenly after middle age. In the elderly, thyroid cancers are a mixture of self-limiting and lethal cancers; thus, when thyroid cancer is detected, careful follow-up with examination of its growth rate is required.

Rtfc (4931414P19Rik) Regulates in vitro Thyroid Differentiation and in vivo Thyroid Function.

Thyroid is a one of the most important endocrine organs. Understanding the molecular mechanism underlying thyroid development and function, as well as thyroid diseases, is beneficial for the clinical treatment of thyroid diseases and tumors. Through genetic linkage analysis and exome sequencing, we previously identified an uncharacterized gene C14orf93 (RTFC, mouse homolog: 4931414P19Rik) as a novel susceptibility gene for familial non-medullary thyroid carcinoma, and demonstrated its function in promoting thyroid tumor. However, the role of RTFC in thyroid development and function remains unexplored. In this study, we found that knockout of Rtfc compromises the in vitro thyroid differentiation of mouse embryonic stem cells. In contrast, Rtfc-/- mice are viable and fertile, and the size and the morphology of thyroid are not affected by Rtfc knockout. However, female Rtfc-/- mice, but not male Rtfc-/- mice, display mild hypothyroidism. In summary, our data suggest the roles of Rtfc in in vitro thyroid differentiation of embryonic stem cells, and in vivo thyroid function.

Thyroid Hemiagenesis from Childhood to Adulthood: Review of Literature and Personal Experience.

Thyroid hemiagenesis (TH) is a rare congenital abnormality of the thyroid gland, characterised by the absence of one lobe. The true prevalence of this congenital abnormality is not known because the absence of one thyroid lobe usually does not cause clinical symptoms by itself. Between 1970 and 2010, 329 cases of TH have been reported. It is interesting to note that most cases have an agenesis of the left lobe (80% of cases) followed by the isthmus (44-50% of cases). Although the female to male ratio was 1:1.4 in 24,032 unselected 11-to 14-yr-old schoolchildren from South-eastern Sicily, several other reports have documented a higher prevalence in women, which may indicate a possible gender association. Most cases of TH are diagnosed when patients present a lesion in the functioning lobe. The functioning lobe of the thyroid gland can be a site of pathological changes similar to a normally developed gland and may present a spectrum of diseases like multinodular goiter, colloid goiter, follicular adenoma, thyroiditis, hypothyroidism and hyperthyroidism. In three of our patients, TH was associated with Hashimoto thyroiditis (n = 1) and with subclinical hypothyroidism (n = 2). The frequency of thyroid abnormalities in patients with TH varies with age, due to the longer exposure of the hemi-agenetic gland to TSH overstimulation in older patients. This could explain the controversy about the benign character of this anomaly. Other extrathyroidal lesions, such as parathyroid adenoma or hyperplasia, cervical thymic cysts, ectopic sublingual thyroid gland and thyroglossal duct cyst have been reported with TH. Therefore, systematic follow-up of all identified cases is recommended.

Effects of chlorpyrifos on in vitro sex steroid production and thyroid follicular development in adult and larval Lake Sturgeon, Acipenser fulvescens.

Chlorpyrifos is a widely used organophosphate pesticide that has previously been shown to enter waterways in biologically relevant concentrations and has the potential to disrupt both thyroid hormone and sex steroid biosynthesis in vertebrates. Because gonadal maturation and larval development in Lake Sturgeon, Acipenser fulvescens, potentially coincide with the application of chlorpyrifos we examined the effects of chlorpyrifos on both thyroid follicular development in larval Lake Sturgeon, and sex hormone synthesis in adult Lake Sturgeon. For the first time, the present study reports steroidogenesis from testicular and ovarian tissue in Lake Sturgeon using an established in vitro bioassay. Furthermore, incubating gonad tissue with 5, 500 or 2000ngmL(-1) chlorpyrifos revealed an inhibitory effect on testosterone synthesis in both testicular (control, 40.29pgmg(-1) tissue wet weight(-1)h(-1) compared to experimental, 21.84pgmg(-1) tissue wet weight(-1)h(-1)) and ovarian (control, 33.83pgmg(-1) tissue wet weight(-1)h(-1) compared to experimental, 15.19pgmg(-1) tissue wet weight(-1)h(-1)) tissue. In a second series of experiments, larval Lake Sturgeon were exposed to equivalent concentrations of chlorpyrifos as above for 10days (d) between hatch and the onset of exogenous feeding. Larvae from each treatment group were raised until 67days post hatch (dph) and growth rates were compared alongside key indicators of thyroid follicle growth. Chlorpyrifos treatment had no effect on the measured indicators of thyroid follicular development.

Effects of GH replacement therapy on thyroid volume and nodule development in GH deficient adults: a retrospective cohort study.

OBJECTIVE: Despite the well-known effects of GH/IGF1 signaling on the thyroid, few data are available on the risk of developing nodular goiter in hypopituitary subjects during GH replacement therapy (GHRT). We aimed to define the effects of GH therapy on thyroid volume (TV) and nodular growth. DESIGN: The records of 96 subjects (47 males and 49 females, median age 48 years) with GH deficit (GHD) were investigated. Seventy also had central hypothyroidism (CH). At the time of our retrospective evaluation, median treatment duration was 5 years. RESULTS: Pre-treatment TV was smaller in GHD patients than in healthy subjects (P=0.030). During GH treatment, TV significantly increased (P=0.016 for the entire group and P=0.014 in euthyroid GHD patients). Before starting GH therapy, 17 patients harbored thyroid nodules. During GH therapy, nodule size increased slightly in seven patients, and new thyroid nodules occurred in nine patients. Among the 79 patients without pre-existing thyroid nodules, 17 developed one or more nodules. There was no difference in the prevalence of CH in GHD patients with or without thyroid nodules (P=0.915; P=0.841, when patients with pre-therapy nodular goiter were excluded), the main predictor for nodule development being serum IGF1 (P=0.038). CONCLUSIONS: GHRT is associated with TV's increase in GHD patients. Thyroid nodules developed in 27% of patients, mainly in relation to pre-therapy IGF1 levels, independently of normal or impaired TSH stimulation.

Thyroid transcription factors in development, differentiation and disease.

Identification of the thyroid transcription factors (TTFs), NKX2-1, FOXE1, PAX8 and HHEX, has considerably advanced our understanding of thyroid development, congenital thyroid disorders and thyroid cancer. The TTFs are fundamental to proper formation of the thyroid gland and for maintaining the functional differentiated state of the adult thyroid; however, they are not individually required for precursor cell commitment to a thyroid fate. Although knowledge of the mechanisms involved in thyroid development has increased, the full complement of genes involved in thyroid gland specification and the signals that trigger expression of the genes that encode the TTFs remain unknown. The mechanisms involved in thyroid organogenesis and differentiation have provided clues to identifying the genes that are involved in human congenital thyroid disorders and thyroid cancer. Mutations in the genes that encode the TTFs, as well as polymorphisms and epigenetic modifications, have been associated with thyroid pathologies. Here, we summarize the roles of the TTFs in thyroid development and the mechanisms by which they regulate expression of the genes involved in thyroid differentiation. We also address the implications of mutations in TTFs in thyroid diseases and in diseases not related to the thyroid gland.

Molecular cloning and expression analysis of the GNAS gene in pig and porcine fibroblast cells.

The Alpha subunit of the stimulatory guanine nucleotide-binding protein (GNAS) is a complex imprinted gene. The major product of the GNAS gene is the α-subunit of the guanine nucleotide-binding protein (Gas), which plays a key role in multiple signal transduction pathways. Gas is required for the production of the receptor-stimulated intracellular cyclic adenosine monophosphate (cAMP). It has been demonstrated that an increase in the concentration of the intracellular second messenger cAMP promotes apoptosis in different tumor entities. Mutations of GNAS have also been identified in many tumors. This study aimed to investigate the expression pattern and the apoptosis effect in fibroblast cells for porcine GNAS. The results show that GNAS mRNA was detected in a wide range of tissues, especially in the longissimus dorsi muscle and thyroid gland. The developmental pattern of GNAS mRNA in the thyroid gland of Jinhua pigs was then examined; however, there was no significant difference (P > 0.05) among any of the stages. GNAS gene expression was relatively stable in the thyroid gland during the entire growth and development process. The developmental pattern of GNAS mRNA in the longissimus dorsi muscle was significantly different among the various developmental stages (P < 0.01). GNAS mRNA was strongly expressed at 60 days, 90 days, and 150 days after birth, whereas the expression level was very low during the embryo stages. Target RNA interference of GNAS in porcine fibroblast cells leads to lower mRNA expression of Bcl-2, Fas, and Caspase-3, which are recognized as apoptosis related markers.

Changes in the role of the thyroid axis during metamorphosis of the Japanese eel, Anguilla japonica.

To clarify the role of thyroid function during metamorphosis from leptocephalus to glass eel in the Japanese eel, we examined the histology of the thyroid gland and measured whole-body concentrations of thyroid hormones, thyroxine (T4) and triiodothyronine (T3), and thyroid stimulating hormone ß-subunit TSH (TSHß) mRNA expression levels in five stages of artificially hatched eels (leptocephalus, early-metamorphosis, late-metamorphosis, glass eel, and elver). During metamorphosis, the inner colloid of thyroid follicles showed positive immunoreactivity for T4, and both T4 and T3 levels were significantly increased, whereas a small peak of TSHß mRNA level was observed at the early-metamorphosis stage. Similarly, TSHß mRNA levels were highest in the glass eel stage, and then decreased markedly in the elver stage. In contrast to TSHß mRNA expression, thyroid hormones (both T4 and T3) increased further from the glass eel to elver stages. These results indicated that thyroid function in the Japanese eel was active both during and after metamorphosis. Therefore, the thyrotropic axis may play important roles not only in metamorphosis but also in subsequent inshore or upstream migrations.

Reciprocal epithelial:endothelial paracrine interactions during thyroid development govern follicular organization and C-cells differentiation.

The thyroid is a highly vascularized endocrine gland, displaying a characteristic epithelial organization in closed spheres, called follicles. Here we investigate how endothelial cells are recruited into the developing thyroid and if they control glandular organization as well as thyrocytes and C-cells differentiation. We show that endothelial cells closely surround, and then invade the expanding thyroid epithelial cell mass to become closely associated with nascent polarized follicles. This close and sustained endothelial:epithelial interaction depends on epithelial production of the angiogenic factor, Vascular Endothelial Growth Factor-A (VEGF-A), as its thyroid-specific genetic inactivation reduced the endothelial cell pool of the thyroid by > 90%. Vegfa KO also displayed decreased C-cells differentiation and impaired organization of the epithelial cell mass into follicles. We developed an ex vivo model of thyroid explants that faithfully mimicks bilobation of the thyroid anlagen, endothelial and C-cells invasion, folliculogenesis and differentiation. Treatment of thyroid explants at e12.5 with a VEGFR2 inhibitor ablated the endothelial pool and reproduced ex vivo folliculogenesis defects observed in conditional Vegfa KO. In the absence of any blood supply, rescue by embryonic endothelial progenitor cells restored folliculogenesis, accelerated lumen expansion and stimulated calcitonin expression by C-cells. In conclusion, our data demonstrate that, in developing mouse thyroid, epithelial production of VEGF-A is necessary for endothelial cells recruitment and expansion. In turn, endothelial cells control epithelial reorganization in follicles and C-cells differentiation.