T3 Production Research Articles

Cold Adaptation and Thyroid Hormone Metabolism

Resting oxygen consumption and energy expenditure is sensitive to slight alterations in thyroid function. This means that timing and magnitude of cold adaptation would to some extent depend on thyroid function. Local thyroid hormone metabolism is important for energy expenditure and dissipation of heat in special tissues. Recruitment of brown adipocytes and upregulation of uncoupling protein 1 in mitochondria depends on high tissue T3 concentrations. Most of this T3 is derived from local 5' deiodination of T4. Brown fat is vital for cold exposed mice and rats, and may be important for temperature adaptation in human neonates. The role of thyroid hormone metabolism in adult human cold adaptation has not been finally clarified. Hypothetically, cold exposure may enhance T3 production by deiodination of T4 in skeletal muscle, which may enhance heat production in muscle via a change in muscle fiber type. Another hypothetical possibility is recruitment of brown adipocytes embedded in white adipose tissue in human adults. Understanding cold adaptation in human adults may lead to development of new drugs against obesity.

Type 2 iodothyronine deiodinase is the major source of plasma T3 in euthyroid humans

The relative roles of the types 1 and 2 iodothyronine deiodinases (D1 and D2) in extrathyroidal 3,5,3'-triiodothyronine (T3) production in humans are unknown. We calculated the rate of thyroxine (T4) to T3 conversion by intact cells transiently expressing D1 or D2 at low (2 pM), normal (20 pM), and high (200 pM) free T4 concentrations. Deiodinase activities were then assayed in cell sonicates. The ratio of T3 production in cell sonicates (catalytic efficiency) was multiplied by the tissue activities reported in human liver (D1) and skeletal muscle (D2). From these calculations, we predict that in euthyroid humans, D2-generated T3 is 29 nmol/d, while that of D1-generated T3 is 15 nmol/d, from these major deiodinase-expressing tissues. The total estimated extrathyroidal T3 production, 44 nmol/d, is in close agreement with the 40 nmol T3/d based on previous kinetic studies. D2-generated T3 production accounts for approximately 71% of the peripheral T3 production in hypothyroidism, but D1 for approximately 67% in thyrotoxic patients. We also show that the intracellular D2-generated T3 has a greater effect on T3-dependent gene transcription than that from D1, which indicates that generation of nuclear T3 is an intrinsic property of the D2 protein. We suggest that impairment of D2-generated T3 is the major cause of the reduced T3 production in the euthyroid sick syndrome.

Adaptive Activation of Thyroid Hormone and Energy Expenditure

The mechanisms by which thyroid hormone accelerates energy expenditure are poorly understood. In the brown adipose tissue (BAT), activation of thyroid hormone by type 2 iodothyronine deiodinase (D2) has been known to play a role in adaptive energy expenditure during cold exposure in human newborns and other small mammals. Although BAT is not present in significant amounts in normal adult humans, recent studies have found substantial amounts of D2 in skeletal muscle, a metabolically relevant tissue in humans. This article reviews current biological knowledge about D2 and adaptive T3 production and their roles in energy expenditure.

Influence of Bayakon Infusion on Thyroid Gland Function in Rats with Experimental Hypo- and Hyperthyroidism

The effect of bayakon preparation from the above-ground parts of Baikal monkshood (Aconitum baicalensis Turcz ex Rapaics) on thyroid gland function has been studied in rats with experimental hypo- and hyperthyroidism. The preparation corrects the production of thyroid hormones T3 and T4, as well as TTH secretion, to the normal levels.

Expression of thioredoxin in patients with Graves' disease

Thioredoxin (TRX), which is a stress-inducible protein with redox-active disulfide structures, has various biological activities by regulating DNA binding of transcription factors in cells. In Graves' disease that is among the common diseases of the thyroid, endogenous stresses that are induced by excess thyroid hormones or antibodies against thyroid stimulating hormone (TSH) receptors are responsible for the pathogenesis. The objective of this study was to examine the expression of TRX and to determine whether TRX is responsible for the pathogenesis of Graves' disease. The thyroid follicular cells were shown to express both TRX and vascular cell growth factors (VEGF) in all of the patients with Graves' disease by immunohistochemistry. In contrast, the expression of TRX or VEGF was not found in any of the normal thyroids. Serum levels of TRX were significantly elevated in patients with Graves' disease regardless of their thyroid function compared to those in healthy donors (122+/-16 versus 37+/-5 ng/ml, p<0.0001). Consecutive administration of iodine resulted in not only a reduction in serum levels of free triiodothyronine (T3) but also an increase in serum levels of TRX in the patients. These findings suggest that release of intracellular TRX from thyroid follicular cells in response to iodine resulted in suppression of T3 production. Taken together, TRX is highly produced under stress in Graves' disease and involved in regulating production of thyroid hormones. The investigation of biological behavior of this molecule may greatly help to understand pathogenesis of Graves' disease functions.

Developmentally defined regulation of thyroid hormone metabolism by glucocorticoids in the rat

Glucocorticoids are known regulators of thyroid function in vertebrates. In birds they have clear tissue-specific and age-dependent effects on thyroid hormone metabolism. In mammals, however, few studies exist addressing these aspects using an in vivo model system. We therefore set out to examine the acute effects of a single dose of dexamethasone (DEX) on plasma 3,5,3'-tri-iodothyronine (T(3)) and thyroxine (T(4)) levels, as well as on the activity of the different deiodinases in liver, kidney and brain in the developing rat. In 20-day-old fetuses (E20), glucocorticoids had no effects on circulating thyroid hormone levels despite their clear effects on hepatic and renal deiodinases, thereby indicating that under these conditions circulating thyroid hormone levels are more dependent on thyroidal secretion than on peripheral deiodination. In contrast, in 5-day-old rat pups, DEX did not seem to have any effects on hepatic and renal T(3) production (via the type I deiodinase), whereas type III deiodinase (D3) activity in both these tissues increased significantly. These observations therefore suggested that the DEX-induced increase in circulating T(3) levels is a direct consequence of the increase in plasma T(4) levels. In 12-day-old pups (P12), however, the main effect of glucocorticoids on circulating levels was by increasing inner ring deiodination T(3) through induction of D3 in both liver and kidney. Finally, in the brain, glucocorticoids stimulated thyroid hormone activity only during a short period of time (between E20 and P12) that largely overlaps with the transient window in time during which brain development is thyroid hormone sensitive. This was in contrast to the E20 and P12 brain, where the glucocorticoid-induced changes in type II deiodinase and D3 seemed to favor a status quo in local T(3) availability.

The effects of 17β-estradiol injections on thyroid hormone deiodination pathways in liver and other tissues of female and male rainbow trout (Oncorhynchus mykiss) at different stages of sexual maturity

The effects of two intraperitoneal injections of 17β-estradiol (E2) over 7 days were studied on thyroid hormone plasma levels and on activities of thyroxine (T4), 3,5,3'-triiodothyronine (T3), and 3,3',5'-triiodothyronine (rT3) outer-ring deiodination (ORD) and inner-ring deiodination (IRD) pathways in various tissues of female and male rainbow trout (Oncorhynchus mykiss (Walbaum, 1792)). E2 administered to adult females and males at different stages of sexual maturity increased liver mass, depressed plasma T3 levels with no change in plasma T4 levels, and severely decreased liver T4ORD activity. E2 also modestly depressed hepatic rT3ORD activity, but only at low substrate levels, and had no consistent effect on the hepatic IRD pathways. There were no E2-induced changes in brain, gill, or heart deiodination, but E2 increased kidney T3IRD activity. In contrast, an all-female stock of trout with rudimentary ovaries responded to E2 with an increase in liver mass but without change in plasma T4 and T3 levels or liver and brain deiodination activities. In conclusion, the decrease in plasma T3 levels in both male and female adult E2-injected trout may be due to both decreased hepatic T3 production and increased renal T3 degradation. However, thyroidal responses to E2 depend on physiological/developmental state and were absent in a highly immature all-female trout stock.

Characterization of the Protein Dimerization Domain Responsible for Assembly of Functional Selenodeiodinases

Thyroid hormone metabolism is catalyzed by a small family of selenoenzymes. Type I deiodinase (D1) is the best characterized family member and is an integral membrane protein composed of two 27-kDa subunits that assemble to a functional holoenzyme after translation. To characterize the protein domain(s) responsible for this post-translational assembly event, we used deletion/truncation analysis coupled with immune depletion assays to map the dimerization domain of D1. The results of our studies show that a highly conserved sequence of 16 amino acids in the C-terminal half of the D1 subunit, -D148FL-YI-EAH-DGW163-, serves as the dimerization domain. Based on the high conservation of this domain, we synthesized a novel bait peptide-green fluorescent protein fusion probe (DDD(GFP)) to examine holoenzyme assembly of other family members. Overexpression of either the DDD(GFP) or an inert D1 subunit (M4) into SeD2 (accession number U53505)-expressing C6 cells specifically led to the loss of >90% of the catalytic activity. Catalytically inactive D2 heterodimers composed of SeD2: DDD(GFP) subunits were rescued by specific immune precipitation with anti-SeD2 IgG, suggesting that SeD2 requires two functional subunits to assemble a catalytically active holoenzyme. These findings identify and characterize the essential dimerization domain responsible for post-translational assembly of selenodeiodinases and show that family members can intermingle through this highly conserved protein domain.

Role of the Constitutive Androstane Receptor in Xenobiotic-Induced Thyroid Hormone Metabolism

The induction of hepatic drug metabolizing enzymes alters not only the metabolism of the xenobiotic substances that induce them but also the metabolism of various endogenous hormones. The xenobiotic receptor constitutive androstane receptor (CAR) (NR1I3) mediates the well-studied induction of CYP2B genes and other drug-metabolizing enzymes by phenobarbital (PB), an antiepileptic drug that has been shown to alter thyroid hormone (TH) levels. Here we show that CAR is required for PB-mediated disruption of TH homeostasis and the induction of thyroid follicular cell proliferation. Treatment with PB or the more potent and more effective CAR ligand 1, 4-bis-[2-(3, 5,-dichloropyridyloxy)] benzene resulted in universal induction of thyroid hormone glucuronidation and sulfation pathways in a CAR-dependent manner. This resulted in a decrease in serum T4 concentration and a concomitant increase in serum TSH levels. CAR activation also decreased serum T3 levels in mice in which T3 production was blocked. The increase in serum TSH levels resulted in the stimulation of thyroid-follicular cell proliferation. These results highlight the central role of the xenosensor CAR in drug-hormone interactions.

Type 2 iodothyronine selenodeiodinase is expressed throughout the mouse skeleton and in the MC3T3-E1 mouse osteoblastic cell line during differentiation.

Thyroid hormone affects multiple aspects of bone metabolism, but little is known about thyroid hormone deiodination in bone cells except that cultures of skeletal cells and bone organ express types 1 and 2 iodothyronine deiodinases (D1 and D2) mRNAs. In the present study, outer ring deiodination (ORD) activity was detected in bone extracts of multiple sites of the mouse skeleton, bone marrow, and the MC3T3-E1 osteoblastic cell line. In all tissues, ORD was detected using 125I-rT3 or 125I-T4 as substrates and was found to be 6-n-propylthiouracil insensitive, display a Michaelis constant (T4) of approximately 1 nM, increase about 3-fold in hypo- and virtually disappear in thyrotoxicosis. Extracts of calvaria had the lowest ORD activity, whereas tibial and femoral extracts had roughly three times as much. The absence of ORD activity in bone extracts from mice with targeted disruption of the Dio2 gene confirms the principal role of D2 in this tissue. In the MC3T3-E1 osteoblasts, D2 activity increased in a time-dependent manner after plating, and with the content of selenium in the media, reaching a maximum 5-7 d later as cells attained more than 90% confluence. In these cells D2 half-life is about 30-40 min, which is further accelerated by exposure to substrate and stabilized by the proteasome inhibitor, MG132. Treatment with vitamin D [1,25(OH)2VD]-induced D2 activity by 2- to 3-fold as early as 24 h, regardless of the level of cell confluence, but estradiol, PTH, forskolin, leptin, TNFalpha, TGFbeta, and dexamethasone did not affect D2. Given the role of D2 in other cell types and processes, it is likely that bone ORD not only plays a role in bone development and adult bone T3 homeostasis but also contributes to extrathyroidal T3 production and maintenance of serum T3.

Changes in thyroxine, 3,3',5-triiodothyronine and 3,3'5'-triiodothyronine content in the thyroid gland and in serum to thyroid tissue iodothyronine ratios during ontogenesis in the fetal pig

The concentrations of thyroxine (T4), 3,3',5-triiodothyronine (T3) and 3,3',5'-triiodothyronine (reverse T3; rT3) in thyroid gland tissue and serum of the fetal pig (n = 68) from day 39 to 113 of gestation were measured. Tracer quantities of iodothyronines, displaying the onset of thyroid hormone activity, were found in the thyroid tissue on day 39, i.e. before the appearance of a measurable quantity of iodothyronines in the serum. The T4 and T3 thyroidal content showed the first rise between days 56 and 76. Then, T3 was increasing sharply from day 92 till birth, while T4 content was decreasing from about day 76 to a low value between day 92 and 105, and then showing an increase shortly before birth. The rT3 content was the highest on day 39 and then it was steadily decreasing to reach a nadir on about day 76. Measurable amounts of thyroid hormones (TH) in the serum were observed not earlier than on day 46 of gestation. Near birth, the tissues of the pig fetus are in a milieu characterised by the highest blood TH concentrations. The serum to thyroid concentration ratio for rT3 and T4 was generally below 1.0 until the last trimester of gestation, when it was over 5.0 for rT3 and over 4.0 for T4. By contrast, the T3 serum to thyroid ratio was below 0.5 throughout the gestation. The results show that the fetal pig thyroid displays a low rT3 and T4 content, but the marked T3 elevation observed near term supports the view that a high production and secretion of T3 near term may be a critical factor for normal postnatal adaptation to extrauterine cooling in the pig.

Ontogenic Redistribution of Type 2 Deiodinase Messenger Ribonucleic Acid in the Brain of Chicken

Thyroid hormone is essential for brain development. T(4) has to be converted to T(3) for efficient binding to thyroid hormone receptors. Type 2 deiodinase (D2) is the key enzyme that allows T(3) generation in the brain. To elucidate the onset and localization of T(3) production in the brain, we studied the changes of D2 activity, mRNA content, and the distribution of D2 mRNA in the brain of chicken embryos before and after the onset of thyroid function. D2 activity was detectable in the brain at all stages studied from embryonic day (E)7 to E15 and increased significantly with time. The wild-type chicken D2 transcript was detectable at all those stages by RT-PCR. The amount of D2 mRNA in the brain increased approximately 14-fold from E10 to E17 as assessed by Northern blot. Week D2 hybridization signal could be detected by in situ hybridization at E8 in cell clusters throughout the brain, and its intensity markedly increased to E15. Interestingly, no D2 expression was detected in hypothalamic tanycytes at these embryonic stages. However, D2 hybridization signal was observed in the wall of the third ventricle of adult chicken posterior to the rostral pole of the median eminence in the location typical for tanycytes, whereas D2 signal in other localizations was decreased throughout the brain. Our data suggest that D2 contributes to T(3) content of the developing chicken brain even before the onset of thyroid function. Furthermore, redistribution of D2 mRNA expression was observed during the development of the chicken brain.

Nutrition, evolution and thyroid hormone levels – a link to iodine deficiency disorders?

An increased iodine requirement as a result of significant changes in human nutrition rather than a decreased environmental iodine supply is suggested to represent the main cause of the iodine deficiency disorders (IDD). The pathomechanism proposed is based on the fact that serum concentrations of thyroid hormones, especially of trijodothyronine (T3), are dependent on the amount of dietary carbohydrate. High-carbohydrate diets are associated with significantly higher serum T3 concentrations, compared with very low-carbohydrate diets. While our Paleolithic ancestors subsisted on a very low carbohydrate/high protein diet, the agricultural revolution about 10,000 years ago brought about a significant increase in dietary carbohydrate. These nutritional changes have increased T3 levels significantly. Higher T3 levels are associated with an enhanced T3 production and an increased iodine requirement. The higher iodine requirement exceeds the availability of iodine from environmental sources in many regions of the world, resulting in the development of IDD.

Triiodothyronine Deiodinating Activity in Brown Adipose Tissue after Short Cold Stimulation Test in Trained and Untrained Rats.

Interscapular brown adipose tissue (IBAT) activity is controlled by sympathetic nervous system, and factors that influence thermogenesis appear to be centrally connected to the sympathetic outflow to IBAT. Cold exposure produces a rise in BAT temperature, which is associated with an increased thyroid activity, elevated serum levels of 3,5,3'-triiodothyronine (T3), and an increased rate of T3 production. This study evaluated the effect of swimming training on 5'-triiodothyronine deiodinase (5'-D) activity in IBAT under normal environmental conditions and after short (30 min) cold exposure (TST stimulation test). 5'-D activity is lower in trained rats at basal condition, and TST increases 5'-D in IBAT of both untrained and trained rats. However, this increase is lower in trained rats. Training reduces the deiodinating activity in normal environmental conditions as well as after short cold exposure. Probably, other compensatory mechanisms of heat production are active in trained rodents.

Potent thermogenic action of triiodothyroacetic acid in brown adipocytes.

Triiodothyroacetic acid (TRIAC) is a triiodothyronine (T3) metabolite with high affinity for T3 nuclear receptors. We compared the thermogenic action of TRIAC versus T3 in brown adipocytes, by studying target genes known to mediate thermogenic action: uncoupling protein 1 (UCP-1), a marker of brown adipocytes, and type II-5'deiodinase (D2), which provides the T3 required for thermogenesis. TRIAC is 10-50 times more potent than T3 at increasing the adrenergic induction of UCP-1 mRNA and D2 activities. TRIAC action on UCP-1 is exerted at the transcriptional level. In the presence of an adrenergic stimulus, TRIAC is also more potent than T3, inducing lipoprotein lipase mRNA and 5 deiodinase (D3) activity and mRNA. Maximal effects occur at very low concentrations (0.2 nM). The greater potency of TRIAC is not due to preferential cellular or nuclear uptake. Therefore, TRIAC is a potent thermogenic agent that might increase energy expenditure and regulate T3 production in brown adipocytes.

Teores plasmáticos de hormônios, produção e composição do leite em sala de espera climatizada

Através do presente trabalho, avaliou-se a eficiência da climatização do ambiente da sala de ordenha (ventilação ou ventilação mais aspersão) sobre a produção e composição do leite (gordura, proteína, lactose) e os níveis plasmáticos de cortisol, triiodotironina (T3) e tiroxina (T4). Foram utilizadas 21 vacas em lactação, com produção média de 21 kg d-1 de leite, distribuídas em um delineamento inteiramente casualisado. O período experimental foi de 90 dias, entre os meses de agosto e outubro. Os tratamentos utilizados foram: 1) sala de espera sem climatização - controle (CONTR); 2) sala de espera com ventilação artificial (V) e 3) sala de espera com ventilação artificial mais aspersão (VA). Os dados referentes à produção de leite foram coletados diariamente em cada ordenha e, para análise da composição do leite, coletaram-se amostras semanais. Para a determinação dos hormônios - cortisol, T3 e T4, foram colhidas amostras de sangue, semanalmente, da veia coccídea. Mediante análises dos dados (ANOVA), verificou-se que não houve diferença entre os tratamentos para a produção de leite, que os animais do tratamento V apresentaram maior teor de gordura no leite e, ainda, que os animais do grupo CONTR apresentaram maiores concentrações hormonais de cortisol, T3 e T4, seguida pelo tratamento VA. Por fim, o tratamento VA proporcionou melhores condições de conforto aos animais, porém o tempo de permanência dos animais na sala de ordenha climatizada (30 min) foi, provavelmente, insuficiente para proporcionar aumento na produção de leite.

NTP Center for the Evaluation of Risks to Human Reproduction: phthalates expert panel report on the reproductive and developmental toxicity of di- n-octyl phthalate

The thyroid gland secretes primarily tetraiodothyronine (T4), and some triiodothyronine (T3). Under normal physiological circumstances, only one-fifth of circulating T3 is directly released by the thyroid, but in states of hyperactivation of thyroid-stimulating hormone receptors (TSHRs), patients develop a syndrome of relative T3 toxicosis. Thyroidal T4 production results from iodination of thyroglobulin (TG) at residues Tyr5 and Tyr130, whereas thyroidal T3 production may originate in several different ways. In this study, the data demonstrate that within the carboxyl-terminal portion of mouse TG, T3 is formed de novo independently of deiodination from T4. We found that upon iodination in vitro, de novo T3 formation in TG was decreased in mice lacking TSHRs. Conversely, de novo T3 that can be formed upon iodination of TG secreted from PCCL3 (rat thyrocyte) cells was augmented from cells previously exposed to increased TSH, a TSHR agonist, a cAMP analog, or a TSHR-stimulating antibody. We present data suggesting that TSH-stimulated TG phosphorylation contributes to enhanced de novo T3 formation. These effects were reversed within a few days after removal of the hyperstimulating conditions. Indeed, direct exposure of PCCL3 cells to human serum from two patients with Graves' disease, but not control sera, led to secretion of TG with an increased intrinsic ability to form T3 upon in vitro iodination. Furthermore, TG secreted from human thyrocyte cultures hyperstimulated with TSH also showed an increased intrinsic ability to form T3. Our data support the hypothesis that TG processing in the secretory pathway of TSHR-hyperstimulated thyrocytes alters the structure of the iodination substrate in a way that enhances de novo T3 formation, contributing to the relative T3 toxicosis of Graves' disease.

Thyroid hormones promote chondrocyte differentiation in mouse ATDC5 cells and stimulate endochondral ossification in fetal mouse tibias through iodothyronine deiodinases in the growth plate.

Thyroid hormones (THs), 3,3',5-triiodo-L-thyronine (T3) and L-thyroxine (T4), are important for the normal development of the growth plate (GP); congenital TH deficiency leads to severe dwarfism. In mouse chondrogenic cell line, ATDC5, T3 enhanced differentiation and increased Alizarin red staining, but did not affect Alcian blue staining. In organ-cultured mouse tibias, THs stimulated the cartilage growth, especially in the hypertrophic zone. Interestingly, T4 was as equally potent as T3 in organ-cultured tibias, which suggests that T4 is metabolized locally to T3, because T4 is a prohormone and must be converted to T3 for its activity. Two enzymes catalyze the conversion; type I deiodinase (D1) and type II deiodinase (D2). D1 has a ubiquitous distribution and D2, with a high affinity for T4, is present where the maintenance of intracellular T3 concentration is critical. Messenger RNAs (mRNAs) for D1 and D2 were detected in neonatal mouse tibias and ATDC5 cells. The enzyme activity was unaffected by the D1 inhibitor 6-propyl-2-thiouracil, suggesting that D2 mainly catalyzes the reaction. D2 mRNA was detected in differentiated ATDC5 cells. In organ-cultured mouse tibias, D2 activity was greater at later stages. In contrast, thyroid hormone receptors (TRs) were expressed in neonatal mouse tibias and ATDC5 cells, but their expression levels in ATDC5 cells were stable throughout the culture periods. Therefore, increased T3 production at later stages by D2 is likely to contribute to the preferential effects of THs in the terminal differentiation of GP. This article is the first to show that T4 is activated locally in GP and enhances the understanding of TH effects in GP.

Biochemistry, Cellular and Molecular Biology, and Physiological Roles of the Iodothyronine Selenodeiodinases

The goal of this review is to place the exciting advances that have occurred in our understanding of the molecular biology of the types 1, 2, and 3 (D1, D2, and D3, respectively) iodothyronine deiodinases into a biochemical and physiological context. We review new data regarding the mechanism of selenoprotein synthesis, the molecular and cellular biological properties of the individual deiodinases, including gene structure, mRNA and protein characteristics, tissue distribution, subcellular localization and topology, enzymatic properties, structure-activity relationships, and regulation of synthesis, inactivation, and degradation. These provide the background for a discussion of their role in thyroid physiology in humans and other vertebrates, including evidence that D2 plays a significant role in human plasma T3 production. We discuss the pathological role of D3 overexpression causing “consumptive hypothyroidism” as well as our current understanding of the pathophysiology of iodothyronine deiodination during illness and amiodarone therapy. Finally, we review the new insights from analysis of mice with targeted disruption of the Dio2 gene and overexpression of D2 in the myocardium.

Morphological and functional differentiation of human thyroid cells in collagen gel culture

Objective: cultured human thyroid cells in collagen gel culture were examined on cell morphology and the production of thyroglobulin (Tg), triiodothyronine (T3) and thyroxine (T4) which are components of functional differentiation. Methods: thyroid cells obtained from normal human thyroid tissues (four cases), follicular adenoma tissues (three cases), papillary carcinoma tissues (three cases), and follicular carcinoma tissue (one case), were cultured in collagen gel. Then these cultured cells were observed on cellular morphology and production of Tg, T3 and T4. Moreover, changes in morphological characteristics and production of Tg, T3 and T4 induced by addition of thyroid stimulating hormone (TSH) and epidermal growth factor (EGF) to medium in collagen gel culture were determined. Results: normal and tumor cells in collagen gel culture formed colonies and follicles with Tg production, similar to in vivo-like three-dimensional cellular structures and functions. Normal thyroid cells stimulated TSH induced more Tg and produced morphological changes, i.e. enlarged follicular lumens and increased the height of follicular cells, but did not promote cell proliferation. Reversely, normal thyroid cells stimulated with EGF promoted cell proliferation, but did not change morphological findings and did not increase production of Tg, T3 and T4. Conclusion: these findings suggest that collagen gel culture is useful for observing the effects of stimulation by cell growth factor on the morphological and functional differentiation of human thyroid cells.