Goitrogen Treatment Research Articles

Interaction between thyroid hormones and gonadotropin inhibitory hormone in ex vivo culture of zebrafish testis: An approach to study multifactorial control of spermatogenesis

Reproduction is under multifactorial control of neurohormones, pituitary gonadotropins, as well as of local gonadal signaling systems including sex steroids, growth factors and non-coding RNAs. Among the factors, gonadotropin-inhibitory hormone (Gnih) is a novel RFamide neuropeptide which directly modulates gonadotropin synthesis and release from pituitary, and in the gonads, Gnih mediated inhibitory actions on gonadotropin response of zebrafish spermatogenesis. Thyroid hormones are peripheral hormones which are also known to interact with reproductive axis, in particular, regulating testicular development and function. This study investigated the interaction between Gnih and thyroid hormones in zebrafish spermatogenesis using in vivo and ex vivo approaches. Three experimental groups were established: “control” (non-treated fish), “methimazole” and “methimazole + T4”. Fish were exposed to goitrogen methimazole for 3 weeks; T4 (100 μg/L) was added in the water from the second week only in the “reversal treatment” group. After exposure, testes were dissected out and immediately incubated in Leibovitz's L-15 culture medium containing hCG, Gnih or hCG + Gnih for 7 days. Germ cell cysts and haploid cell population were evaluated by histomorphometry and flow cytometry, respectively. Our results showed that hypothyroidism affected germ cell development in basal and gonadotropin-induced spermatogenesis, in particular, meiosis and spermiogenesis. Hypothyroid testes showed lower amount of spermatozoa, and decreased potency of hCG. We also showed that goitrogen treatment nullified the inhibitory actions of Gnih on the gonadotropin-induced spermatogenesis. This study provided evidences that thyroid hormones are important regulatory factors for hCG- and Gnih-mediated functions in zebrafish spermatogenesis.

Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN-ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma.

Background: Thyroid tumor progression from well-differentiated cancer to poorly differentiated thyroid carcinoma (PDTC) and anaplastic thyroid carcinoma (ATC) involves step-wise dedifferentiation associated with loss of iodine avidity and poor outcomes. ALK fusions, typically STRN-ALK, are found with higher incidence in human PDTC compared with well-differentiated cancer and, as previously shown, can drive the development of murine PDTC. The aim of this study was to evaluate thyroid cancer initiation and progression in mice with concomitant expression of STRN-ALK and inactivation of the tumor suppressor p53 (Trp53) in thyroid follicular cells. Methods: Transgenic mice with thyroid-specific expression of STRN-ALK and biallelic p53 loss were generated and aged on a regular diet or with methimazole and sodium perchlorate goitrogen treatment. Development and progression of thyroid tumors were monitored by using ultrasound imaging, followed by detailed histological and immunohistochemical evaluation. Gene expression analysis was performed on selected tumor samples by using RNA-Seq and quantitative RT-PCR. Results: In mice treated with goitrogen, the first thyroid cancers appeared at 6 months of age, reaching 86% penetrance by the age of 12 months, while a similar rate (71%) of tumor occurrence in mice on regular diet was observed by 18 months of age. Histological examination revealed well-differentiated papillary thyroid carcinomas (PTC) (n = 26), PDTC (n = 21), and ATC (n = 8) that frequently coexisted in the same thyroid gland. The tumors were frequently lethal and associated with the development of lung metastasis in 24% of cases. Histological and immunohistochemical characteristics of these cancers recapitulated tumors seen in humans. Detailed analysis of PDTC revealed two tumor types with distinct cell morphology and immunohistochemical characteristics, designated as PDTC type 1 (PDTC1) and type 2 (PDTC2). Gene expression analysis showed that PDTC1 tumors retained higher expression of thyroid differentiation genes including Tg and Slc5a5 (Nis) as compared with PDTC2 tumors. Conclusions: In this study, we generated a new mouse model of multistep thyroid cancer dedifferentiation with evidence of progression from PTC to PDTC and ATC. Further, PDTC in these mice showed two distinct histologic appearances correlated with levels of expression of thyroid differentiation and iodine metabolism genes, suggesting a possibility of existence of two PDTC types with different functional characteristics and potential implication for therapeutic approaches to these tumors.

SAT-045 Transient Developmental Hypothyroidism Results in Dysregulation of Neural Progenitors and Birth Defect Formation in Rats

Cortical heterotopias are clusters of ectopic neurons in the brain, and are commonly associated with pharmaceutical resistant epilepsy in patients. We have previously characterized the robust penetrance of a cortical heterotopia in a rat model, induced by thyroid hormone (TH) disruption during gestation. However, how maternal TH insufficiency results in this birth defect is unknown. Here we first determined the developmental window susceptible to endocrine disruption, and describe a cellular mechanism responsible for its formation. We show that five days of maternal goitrogen treatment (10 ppm propylthiouracil) during the perinatal period (GD19-PN2) induces a periventricular heterotopia in 100% of the offspring; this period corresponds to the second trimester in humans. Beginning in the perinatal brain, neurons begin to aggregate near the ventricles of treated animals. In parallel, cellular changes of the ventricular epithelium were observed, including abnormal cell adhesion and altered progenitor cell morphology. As the ventricular epithelium is juxtaposed to both the cerebrospinal fluid and vasculature, two sources of brain THs, this stem cell niche may be especially susceptible to TH disruption. We are now performing RNA sequencing of the ventricular epithelium in both euthyroid and hypothyroid neonates to investigate how THs are affecting this cell population. This work highlights the spatiotemporal vulnerabilities of the developing brain, and suggests that maintenance of euthyroidism across pregnancy is critical to children’s health. This work does not reflect US EPA policy.

A transient window of hypothyroidism alters neural progenitor cells and results in abnormal brain development

Cortical heterotopias are clusters of ectopic neurons in the brain and are associated with neurodevelopmental disorders like epilepsy and learning disabilities. We have previously characterized the robust penetrance of a heterotopia in a rat model, induced by thyroid hormone (TH) disruption during gestation. However, the specific mechanism by which maternal TH insufficiency results in this birth defect remains unknown. Here we first determined the developmental window susceptible to endocrine disruption and describe a cellular mechanism responsible for heterotopia formation. We show that five days of maternal goitrogen treatment (10 ppm propylthiouracil) during the perinatal period (GD19-PN2) induces a periventricular heterotopia in 100% of the offspring. Beginning in the early postnatal brain, neurons begin to aggregate near the ventricles of treated animals. In parallel, transcriptional and architectural changes of this region were observed including decreased Sonic hedgehog (Shh) expression, abnormal cell adhesion, and altered radial glia morphology. As the ventricular epithelium is juxtaposed to two sources of brain THs, the cerebrospinal fluid and vasculature, this progenitor niche may be especially susceptible to TH disruption. This work highlights the spatiotemporal vulnerabilities of the developing brain and demonstrates that a transient period of TH perturbation is sufficient to induce a congenital abnormality.

The Role of Maternal Thyroid Hormones in Avian Embryonic Development

During avian embryonic development, thyroid hormones (THs) coordinate the expression of a multitude of genes thereby ensuring that the correct sequence of cell proliferation, differentiation and maturation is followed in each tissue and organ. Although THs are needed from the start of development, the embryonic thyroid gland only matures around mid-incubation in precocial birds and around hatching in altricial species. Therefore, maternal THs deposited in the egg yolk play an essential role in embryonic development. They are taken up by the embryo throughout its development, from the first day till hatching, and expression of TH regulators such as distributor proteins, transporters, and deiodinases in the yolk sac membrane provide the tools for selective metabolism and transport starting from this level. TH receptors and regulators of local TH availability are expressed in avian embryos in a dynamic and tissue/cell-specific pattern from the first stages studied, as shown in detail in chicken. Maternal hyperthyroidism via TH supplementation as well as injection of THs into the egg yolk increase TH content in embryonic tissues while induction of maternal hypothyroidism by goitrogen treatment results in a decrease. Both increase and decrease of maternal TH availability were shown to alter gene expression in early chicken embryos. Knockdown of the specific TH transporter monocarboxylate transporter 8 at early stages in chicken cerebellum, optic tectum, or retina allowed to reduce local TH availability, interfering with gene expression and confirming that development of the central nervous system (CNS) is highly dependent on maternal THs. While some of the effects on cell proliferation, migration and differentiation seem to be transient, others result in persistent defects in CNS structure. In addition, a number of studies in both precocial and altricial birds showed that injection of THs into the yolk at the start of incubation influences a number of parameters in posthatch performance and fitness. In conclusion, the data presently available clearly indicate that maternal THs play an important role in avian embryonic development, but how exactly their influence on cellular and molecular processes in the embryo is linked to posthatch fitness needs to be further explored.

Mouse Model of Poorly Differentiated Thyroid Carcinoma Driven by STRN-ALK Fusion

Chromosomal rearrangements of the ALK gene, which lead to constitutive activation of ALK tyrosine kinase, are found in various cancers. In thyroid cancers, ALK fusions, most commonly the STRN-ALK fusion, are detected in papillary thyroid cancer and with higher frequency in poorly differentiated and anaplastic thyroid cancers. Our aim was to establish a mouse model of thyroid-specific expression of STRN-ALK and to test whether this fusion drives the development of thyroid cancer with a propensity for dedifferentiation. Transgenic Tg-STRN-ALK mice with thyroglobulin-controlled expression of STRN-ALK were generated and aged with or without goitrogen treatment. Thyroids from these mice were subjected to histologic and immunohistochemical analysis. Transgenic mice with thyroid-specific expression of STRN-ALK developed poorly differentiated thyroid tumors by the age of 12 months in 22% of mice without goitrogen treatment and in 36% of mice with goitrogen treatment. Histologically and immunohistochemically, the tumors resembled poorly differentiated thyroid cancers in humans, demonstrating a solid growth pattern with sheets of round or spindle-shaped cells, decreased expression of thyroglobulin, and a tendency to lose E-cadherin. In this study, we report a novel mouse model of poorly differentiated thyroid cancer driven by the STRN-ALK oncogene with phenotypic features closely recapitulating human tumor, and with a more pronounced phenotype after additional thyroid-stimulating hormone stimulation.

Effects of thyroid endocrine manipulation on sex-related gene expression and population sex ratios in Zebrafish

Thyroid hormone reportedly induces masculinization of genetic females and goitrogen treatment delays testicular differentiation (ovary-to-testis transformation) in genetic males of Zebrafish. This study explored potential molecular mechanisms of these phenomena. Zebrafish were treated with thyroxine (T4, 2nM), goitrogen [methimazole (MZ), 0.15mM], MZ (0.15mM) and T4 (2nM) (rescue treatment), or reconstituted water (control) from 3 to 33days postfertilization (dpf) and maintained in control water until 45dpf. Whole fish were collected during early (25dpf) and late (45dpf) testicular differentiation for transcript abundance analysis of selected male (dmrt1, amh, ar) and female (cyp19a1a, esr1, esr2a, esr2b) sex-related genes by quantitative RT-PCR, and fold-changes relative to control values were determined. Additional fish were sampled at 45dpf for histological assessment of gonadal sex. The T4 and rescue treatments caused male-biased populations, and T4 alone induced precocious puberty in ∼50% of males. Male-biased sex ratios were accompanied by increased expression of amh and ar and reduced expression of cyp19a1a, esr1, esr2a, and esr2b at 25 and 45dpf and, unexpectedly, reduced expression of dmrt1 at 45dpf. Goitrogen exposure increased the proportion of individuals with ovaries (per previous studies interpreted as delay in testicular differentiation of genetic males), and at 25 and 45dpf reduced the expression of amh and ar and increased the expression of esr1 (only at 25dpf), esr2a, and esr2b. Notably, cyp19a1a transcript was reduced but via non-thyroidal pathways (not restored by rescue treatment). In conclusion, the masculinizing activity of T4 at the population level may be due to its ability to inhibit female and stimulate male sex-related genes in larvae, while the inability of MZ to induce cyp19a1a, which is necessary for ovarian differentiation, may explain why its “feminizing” activity on gonadal sex is not permanent.

Transient Maternal Hypothyroxinemia Potentiates the Transcriptional Response to Exogenous Thyroid Hormone in the Fetal Cerebral Cortex Before the Onset of Fetal Thyroid Function: A Messenger and MicroRNA Profiling Study

Thyroid hormone (TH) is essential for brain development both before and after birth. We have used gene expression microarrays to identify TH-regulated genes in the fetal cerebral cortex prior to the onset of fetal thyroid function to better understand the role of TH in early cortical development. TH levels were transiently manipulated in pregnant mice by treatment with goitrogens from gestational day (GD) 13-16 and/or by injection of TH 12 h before sacrifice on GD 16. The transcriptional response to exogenous TH in the GD 16 fetal cortex was potentiated by transient goitrogen treatment, suggesting that the hypothyroxinemic brain is a different substrate upon which TH can act, or that robust compensatory mechanisms are induced by transient hypothyroxinemia. Several known TH-responsive genes were identified including Klf9, and several novel TH-responsive genes such as Appbp2, Ppap2b, and Fgfr1op2 were identified in which TH response elements were confirmed. We also identified specific microRNAs whose expression in the fetal cortex was affected by TH treatment, and determined that Ppap2b and Klf9 are the target genes of miR-16 and miR-106, respectively. Thus, a complex redundant functional network appears to coordinate TH-mediated gene expression in the developing brain.

Iodine-Deficiency-Induced Long Lasting Angiogenic Reaction in Thyroid Cancers Occurs Via a Vascular Endothelial Growth Factor–Hypoxia Inducible Factor-1–Dependent, But Not a Reactive Oxygen Species–Dependent, Pathway

In the thyroid, iodine deficiency (ID) induces angiogenesis via a tightly controlled reactive oxygen species (ROS)-hypoxia inducible factor-1 (HIF-1)-vascular endothelial growth factor (VEGF) dependent pathway (ROS-HIF-VEGF). Deficient iodine intake may be associated with increased thyroid cancer incidence. The hypothesis of this work is to test whether ID affects the angiogenic processes in thyroid malignant cells by altering the ROS-HIF-VEGF pathway. Goiters were obtained in RET/PTC3 transgenic and wild-type (wt) mice and ID was induced in three thyroid carcinoma cell lines (TPC-1, 8305c, and R082-w1). Thyroid blood flow, VEGF mRNA and protein, and HIF-1α protein expression were measured. The role of HIF-1 and of ROS was assessed using echinomycin and N-acetylcysteine (NAC), respectively. The goitrogen treatment increased the thyroid blood flow in wt and RET/PTC3 mice. Compared with wt mice, basal VEGF expression was higher in RET/PTC3 mice and increased with goitrogen treatment. In the three cell lines, ID induced marked increases in VEGF mRNA, and moderate increases in HIF-1α protein expression that were not transient as in normal cells. ID-induced VEGF mRNA expression was fully (8305c), partially (TPC-1), or not (R082-w1) blocked by echinomycin. NAC had no effect on ID-induced VEGF mRNA and HIF-1α protein expression in the three cell lines. ID induces a long lasting angiogenic phenotype in thyroid cancer cells that occurs through VEGF induction via a pathway partially mediated by HIF-1, but not by ROS. These results suggest that, in contrast with normal cells, ID-induced angiogenesis in cancer cells occurs via alternative and likely less controlled routes, thereby leading to uncontrolled growth.

Molecular cloning and regulation of mRNA expression of the thyrotropin β and glycoprotein hormone α subunits in red drum, Sciaenops ocellatus

Full-length cDNAs for thyrotropin β (TSHβ) and glycoprotein hormone α (GSUα) subunits were cloned and sequenced from the red drum (Sciaenops ocellatus). The cDNAs for TSHβ (877 bp) and GSUα (661 bp) yielded predicted coding regions of 126 and 94 amino acid proteins, respectively. Both sequences contain all invariant cysteine and putative glycosylated asparagines characteristic of each as deduced by comparison with other GSUα and TSHβ sequences from representative vertebrate species. Multiple protein sequence alignments show that each subunit shares highest identity (79% for the TSHβ and 86% for the GSUα) with perciform fish. Furthermore, in a single joint phylogenetic analysis, each subunit segregates most closely with corresponding GSUα and TSHβ subunit sequences from closely related fish. Tissue-specific expression assays using RT-PCR showed expression of the TSHβ subunit limited to the pituitary. GSUα mRNA was predominantly expressed in the pituitary but was also detected in the testis and ovary of adult animals. Northern hybridization revealed the presence of a single transcript for both TSHβ and GSUα, each close in size to mRNA transcripts from other species. Dot blot assays from total RNA isolated from S. ocellatus pituitaries showed that in vivo T3 administration significantly diminished mRNA expression of both the TSHβ and GSUα subunits and that goitrogen treatment caused a significant induction of TSHβ mRNA only. Both TSHβ and GSUα mRNA expression in the pituitary varied significantly in vivo over a 24-h period. Maximal expression for both TSHβ and GSUα occurred during the early scotophase in relation to a peak in T4 blood levels previously documented. These results suggest the production of TSH in this species which may serve to drive daily cycles of thyroid activity. Readily quantifiable, variable, and thyroid hormone-responsive pituitary TSH expression, coupled with previously described dynamic daily cycles of circulating T4 and extensive background on the growth, nutrition, and laboratory culture of red drum, suggests that this species will serve as a useful model for experimental studies of the physiological regulation of TSH production.

Impact of connexin32 deletion on E7 or RET/PTC3 oncogene-driven growth and neoplastic transformation of the thyroid gland

Connexins (Cx) form gap junctions and allow direct cell-to-cell communication. Cx through gap junctions or by themselves play regulatory roles on cell growth and differentiation. Using genetically modified mice, we previously found that Cx32 acts as a down-regulator of growth in normal thyroid gland. In this study, we examined the impact of Cx32 ablation on oncogene-driven thyroid growth and neoplastic transformation. Cx32 knockout (Cx32-KO) mice were crossed with transgenic mice expressing, selectively in the thyroid gland, either the E7 or RET/PTC3 (RP3) oncogene. As already described, Cx32-KO mice had no detectable thyroid alteration in physiological conditions and mice expressing E7 or RP3 exhibited time-dependent thyroid hypertrophy and variable changes in expression of differentiation. The thyroid of E7 mice evolved towards a large colloid goitre whereas RP3 mice developed a hyperplastic thyroid of variable size, and the largest glands (about 40% of total) represented a profound tissue remodeling with proliferative papillary formations. E7-induced thyroid hypertrophy was reduced by about 40% in Cx32-KO mice as compared with wild-type (WT) littermates. On the contrary, thyroid hypertrophy induced by thyrotropin stimulation (in response to goitrogen treatment) was enhanced by about 40% in Cx32-KO mice as compared with WT mice. Thyroid hypertrophy of RP3 mice and the proportion of glands showing extensive tissue remodeling were drastically reduced in mice devoid of Cx32. Our data show that Cx32, which negatively controls thyroid growth activated by thyrotropin via the cAMP pathway, would act as a positive effector of thyroid growth triggered by oncogenes acting through other signaling cascades.

Pten Loss in the Mouse Thyroid Causes Goiter and Follicular Adenomas: Insights into Thyroid Function and Cowden Disease Pathogenesis

Inactivation and silencing of the tumor suppressor PTEN are found in many different epithelial tumors, including thyroid neoplasia. Cowden Disease patients, who harbor germ-line PTEN mutations, often display thyroid abnormalities, including multinodular goiter and follicular adenomas, and are at increased risk of thyroid cancer. To gain insights into the role PTEN plays in thyroid function and disease, we have generated a mouse strain, in which Cre-mediated recombination is used to specifically delete Pten in the thyrocytes. We found that Pten mutant mice develop diffuse goiter characterized by extremely enlarged follicles, in the presence of normal thyroid-stimulating hormone and T4 hormone levels. Loss of Pten resulted in a significant increase in the thyrocyte proliferative index, which was more prominent in the female mice, and in increased cell density in the female thyroid glands. Surprisingly, goitrogen treatment did not cause a substantial increase of the mutant thyroid size and increased only to some extent the proliferation index of the female thyrocytes, suggesting that a relevant part of the thyroid-stimulating hormone-induced proliferation signals are funneled through the phosphatidylinositol-3-kinase (PI3K)/Akt cascade. Although complete loss of Pten was not sufficient to cause invasive tumors, over two thirds of the mutant females developed follicular adenomas by 10 months of age, showing that loss of Pten renders the thyroid highly susceptible to neoplastic transformation through mechanisms that include increased thyrocyte proliferation. Our findings show that constitutive activation of the PI3K/Akt cascade is sufficient to stimulate continuous autonomous growth and provide novel clues to the pathogenesis of Cowden Disease and sporadic nontoxic goiter.

Expression of sodium-iodide symporter mRNA in the thyroid gland of Xenopus laevis tadpoles: developmental expression, effects of antithyroidal compounds, and regulation by TSH

The uptake of iodide represents the first step in thyroid hormone synthesis by thyroid follicular cells and is mediated by the sodium-iodide symporter (NIS). In mammals, expression of NIS is stimulated by TSH and transcription of the NIS gene involves regulation by the thyroid-specific transcription factors Pax8 and Nkx2.1. In this study, we examined the mRNA expression of NIS, Pax8 and Nkx2.1 in the thyroid gland of Xenopus laevis tadpoles by semi-quantitative reverse transcriptase (RT)-PCR. During spontaneous metamorphosis, NIS mRNA expression was low in premetamorphic tadpoles, increased throughout prometamorphosis, and peaked at climax stage 60. Analysis of TSH beta-subunit (TSHbeta) mRNA in the pituitary of the same tadpoles revealed a close temporal relationship in the expression of the two genes during metamorphosis, suggesting a regulatory role of TSH in the developmental expression of NIS. Treatment of tadpoles with goitrogenic compounds (sodium perchlorate and ethylenethiourea) increased TSHbeta mRNA expression (approximately twofold) and caused thyroid gland hyperplasia, confirming that feedback along the pituitary-thyroid axis was operative. Analysis of gene expression in the thyroid gland revealed that goitrogen treatment was correlated with increased expression of NIS mRNA (approximately 20-fold). In the thyroid gland organ culture experiments, bovine TSH (bTSH; 1 mU/ml) strongly induced NIS mRNA expression. This effect was mimicked by co-culture of thyroid glands with pituitaries from stage 58 tadpoles and by agents that increase intracellular cAMP (forskolin, dibutyryl-cAMP). In addition, it could be shown that thyroid glands of X. laevis tadpoles express Pax8 and Nkx2.1 mRNA in a developmentally regulated manner and that ex vivo treatment of thyroid glands with bTSH, forskolin, and cAMP analogs increased the expression of Pax8 and Nkx2.1 mRNA. This is the first report on developmental profiles and hormonal regulation of thyroid gland gene expression in amphibian tadpoles and, together, results reveal a critical role of TSH in the regulation of NIS mRNA expression in the thyroid gland of X. laevis tadpoles.

Evidence for processing of compact insoluble thyroglobulin globules in relation with follicular cell functional activity in the human and the mouse thyroid

Thyroglobulin (Tg) is stored within the follicular lumen mainly in a soluble form, but globules made of insoluble multimers are also present and considered to be a mechanism to store prohormone at high concentration. We investigated the immunohistochemical properties of these intrafollicular globules and their possible processing by thyroid cells upon stimulation in the human and in the mouse. Human thyroids (normal, Graves' disease and hot adenomas) and thyroids from old ICR mice without or with goitrogenic treatment were processed for light microscopy. Immunohistochemistry for Tg with a polyclonal antibody and two monoclonal antibodies, one specific for thyroxine-rich-iodinated Tg and the other recognizing Tg independently of its iodine level, staining with periodic-acid-schiff, and binding of lectins specific for mannose and sialic acid were performed on all tIssue sections. Intrafollicular globules were quantified, with distinction between 'active' or 'hot' and 'hypofunctioning' or 'cold' follicles. In normal human and old mouse thyroids, the intrafollicular globules were strongly stained with PAS, but negative for the three anti-Tg antibodies and the two lectin-binding assays, while the surrounding soluble Tg was positive. In normal human tIssue, globules were more frequent in 'hypofunctioning' than in 'active' follicles. They were exceptional in Graves' disease and hot adenomas. In old mice, Tg globules were more frequent in 'cold' than in 'hot' follicles. Along with the goitrogen treatment, they became fewer, fragmented and more often present in follicles with a 'hot' aspect. Upon TSH stimulation, thyrocytes become able to process colloid globules suggesting that this stock of Tg can be used in vivo for thyroid hormone synthesis.

Prenatal diagnosis and early in utero management of fetal dyshormonogenetic goiter

We present a case of a fetal dyshormonogenetic goiter diagnosed by ultrasound examination at 24 weeks of gestation, in a woman with no past history of thyroid disease or goitrogen treatment and with normal thyroid tests, including absence of auto-antibodies. In this situation, fetal goiter may only be associated with fetal hypothyroidism, therefore cord blood sampling was not performed but early treatment was initiated. Amniotic fluid instillation of thyroid hormone led to a rapid decrease in amniotic fluid volume and a clear reduction in thyroid goiter. However, fetal thyroid volume did not totally normalise, and cord blood analysis at birth showed elevated fetal TSH level. As prenatal treatment of fetal hypothyroidism remains controversial in euthyroid mothers, the main objective is to prevent obstetrical complications of large goiters. Therefore, in some selected cases with no maternal history of thyroid disease and normal thyroid function tests, cordocentesis is not necessary to confirm fetal thyroid status or to adjust fetal treatment.

Evidence for co-ordinated changes between vascular endothelial growth factor and nitric oxide synthase III immunoreactivity, the functional status of the thyroid follicles, and the microvascular bed during chronic stimulation by low iodine and propylthiouracyl in old mice.

Vasoactive and angiogenic factors are involved in the autocrine/paracrine thyroid regulation of microvascular bed during goiter development. In the thyroid of old mice, the presence of slowly functioning ('cold') follicles allowed us to study the microvascular regulation of each follicle in correlation with the immunohistochemical expression of vascular endothelial growth factor (VEGF) and nitric oxide synthase III (NOSIII). Mice aged 20 months did or did not receive a goitrogenic treatment (low iodine diet and propylthiouracyl), and their thyroids were processed for light and electron microscopy, and for autoradiography. The relative volumes (Vv) of the capillaries, the number of vessels per follicular area, the mean capillary area and the number of [(3)H]thymidine labeled nuclei were measured separately for 'hot' and 'cold' follicles. Already in control mice, the capillary bed surrounding 'hot' follicles was significantly larger than that seen around 'cold' follicles, because of larger diameters and twice the number of capillaries. This difference persisted whatever the length of the stimulatory treatment. During this treatment, the Vv of the capillaries increased to a larger extent around 'hot' follicles than around 'cold' ones. All vascular changes around 'cold' follicles were less extended and the increase in the capillary diameter was delayed. In control mice, the 'cold' follicles were negative for NOSIII and positive for VEGF while 'hot' follicles were positive for both. During stimulation, all follicles became progressively NOSIII positive. These data support the concept of 'angio-follicular units' in the thyroid and demonstrate their differential regulation in chronic stimulation during which local secretion of VEGF and NO is clearly involved.

Transformation of skin from larval to adult types in normally metamorphosing and metamorphosis-arrested salamander, Hynobius retardatus

Transformation of skin from larval to adult types in a salamander, Hynobius retardatus, which had been reported to show neotenic reproduction in a specific environment, was examined morphologically in normally metamorphosing, precociously metamorphosing and metamorphosis-arrested larvae. Typical larval skin was composed of an epidermis constituted by three types of cells such as apical, Leydig, and basal cells. The Leydig cells were larval specific, and thus disappeared and were replaced by adult epidermal cells during the metamorphosis. Disappearance of the Leydig cells was accomplished by apoptosis as confirmed by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end-labeling method and electron microscopy during the normal metamorphosis and precocious metamorphosis induced by exogenously applied triiodothyronine. Typical adult skin was composed of epidermis constituted by stratified squamous cells and of dermis mainly occupied with two types of dermal glands, mucous and serous glands. When the metamorphosis was arrested by different procedures (thyroidectomy, hypophysectomy, goitrogen treatment, and rearing at low temperature), the larval-specific Leydig cells fully remained in the epidermis, suggesting that the disappearance of these depended on the thyroid activity. Contrary to this, dermal glands behaved differently from the Leydig cells, though they developed and differentiated from epidermal basal cells and constituted the same skin. Those in the metamorphosis-arrested (thyroidectomized, hypophysectomized, or goitrogen-treated) larvae, except in the larvae reared at 4° C, appeared a little later than in the controls. Thus, the aged, metamorphosis-arrested larvae had skin which consisted of larval type epidermis (Leydig cells) and adult type dermis (mucous and serous glands).

Increase of basic fibroblast growth factor (FGF) and FGF receptor messenger RNA during rat thyroid hyperplasia: temporal changes and cellular distribution

Goitre was induced in adult rats by acute (1 or 2 weeks) or chronic (4 or 10 weeks) administration of methimazole together with a low iodine diet. Involution of thyroid growth was then observed at 16 weeks, 4 weeks after withdrawal of goitrogens and reversion to a normal diet. Experimental animals quickly became hypothyroid compared with controls and exhibited thyroid hyperplasia (control (n = 10): total serum thyroxine (T4) 66 +/- 4 nmol/l, thyroid weight 5 +/- 1 mg/100 g body weight, means +/- S.D.; experimental (n = 10): T4 undetectable, thyroid weight 27 +/- 4 mg/100 g body weight after 2 weeks of treatment). Thyroid growth rate subsequently slowed between 2 and 10 weeks. Messenger RNA for basic fibroblast growth factor (basic FGF) and for the high-affinity FGF receptor, was compared in the thyroids and livers of control and goitrous rats by ribonuclease protection assay. Low levels of mRNA for basic FGF and its receptor were detectable in thyroids from control rats at all times, while none was detected in the livers from any animal. Basic FGF and receptor mRNAs increased, and were detected at greatest abundance in hyperplastic thyroids at 1 and 2 weeks respectively, during goitre formation, but subsequently declined in parallel with thyroid growth rate at 4 and 10 weeks. When quantified by radioimmunoassay, basic FGF extracted from thyroids was fivefold greater than in controls after 1 week of goitrogen treatment (control (n = 4): 24 +/- 9 pmol/micrograms DNA; goitre (n = 4): 100 +/- 16 pmol/micrograms DNA; P < 0.05). Basic FGF and FGF receptor mRNAs localized by in situ hybridization predominantly to the epithelial cell population within follicles. Localization by immunohistochemistry demonstrated that basic FGF was present in the thyroids of control rats, and was largely associated with the basement membrane of follicles. During thyroid hyperplasia, increased basic FGF immunoreactivity appeared over the cytoplasm of follicular epithelial cells and was lost from the extracellular matrix. Thyroid involution following removal of goitrogen/low iodine treatment was associated with a decrease in mRNA for basic FGF or its receptor, and a loss of immunoreactive basic FGF from the cytoplasm of follicular cells. These results suggest that autocrine expression of basic FGF and FGF receptor could contribute to thyroid hyperplasia in rats.

Selenium deficiency aggravates the necrotizing effects of a high iodide dose in iodine deficient rats

The effect of selenium deficiency associated with various iodide intake was investigated in rats in order to better understand its possible role in the etiopathogeny of myxedematous cretinism. Groups of rat pups were fed from birth a low selenium diet (Se-) and submitted to goitrogenic treatment (1% perchlorate in water) for one month. Some animals were refed iodide after perchlorate withdrawal. The gland morphology was analyzed in correlation with the glutathione peroxidase (GPX) activity and the thyroid hormone plasma levels. In all Se- rats, the GPX activity was strongly reduced as compared to selenium sufficient (Se+) animals (P < 0.01). Goitrous rats were hypothyroid whatever the selenium intake. After iodide refeeding, plasma T4 and T3 levels were increased by 160% in Se- rats and by respectively 330% and 580% in Se+ rats. The thyroid morphology was different according to the selenium intake: necrotic cells were about three times more numerous in Se- than in Se+ rats (P < 0.01) and the inflammatory reaction was increased. These experimental data demonstrate the detrimental role of selenium deficiency in one experimental case of thyroid disease. Such reduction of cell defences could contribute to the thyroid failure of African myxedematous cretins.

Selenium deficiency aggravates the necrotizing effects of a high iodide dose in iodine deficient rats.

The effect of selenium deficiency associated with various iodide intake was investigated in rats in order to better understand its possible role in the etiopathogeny of myxedematous cretinism. Groups of rat pups were fed from birth a low selenium diet (Se-) and submitted to goitrogenic treatment (1% perchlorate in water) for one month. Some animals were refed iodide after perchlorate withdrawal. The gland morphology was analyzed in correlation with the glutathione peroxidase (GPX) activity and the thyroid hormone plasma levels. In all Se- rats, the GPX activity was strongly reduced as compared to selenium sufficient (Se+) animals (P < 0.01). Goitrous rats were hypothyroid whatever the selenium intake. After iodide refeeding, plasma T4 and T3 levels were increased by 160% in Se- rats and by respectively 330% and 580% in Se+ rats. The thyroid morphology was different according to the selenium intake: necrotic cells were about three times more numerous in Se- than in Se+ rats (P < 0.01) and the inflammatory reaction was increased. These experimental data demonstrate the detrimental role of selenium deficiency in one experimental case of thyroid disease. Such reduction of cell defences could contribute to the thyroid failure of African myxedematous cretins.