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Thyroid hormone receptor (TR) α and β mediate thyroid hormone action at target tissues. TR isoforms have specific roles in development and in adult tissues. The mechanisms underlying TR isoform-specific action, however, are not well understood. We demonstrate that posttranslational modification of TR by conjugation of small SUMO to TRα and TRβ plays an important role in triiodothyronine (T3) action and TR isoform specificity. TRα was sumoylated at lysines 283 and 389, and TRβ at lysines 50, 146, and 443. Sumoylation of TRβ was ligand-dependent, and sumoylation of TRα was ligand-independent. TRα-SUMO conjugation utilized the E3 ligase PIASxβ and TRβ-SUMO conjugation utilized predominantly PIAS1. SUMO1 and SUMO3 conjugation to TR was important for T3-dependent gene regulation, as demonstrated in transient transfection assay and studies of endogenous gene regulation. The functional role of SUMO1 and SUMO3 in T3 induction in transient expression assays was closely matched to the pattern of TR and cofactor recruitment to thyroid hormone response elements (TREs) as determined by ChIP assays. SUMO1 was required for the T3-induced recruitment of the co-activator CREB-binding protein (CBP) and release of nuclear receptor co-repressor (NCoR) on a TRE but had no significant effect on TR DNA binding. SUMO1 was required for T3-mediated recruitment of NCoR and release of CBP from the TSHβ-negative TRE. SUMO3 was required for T3-stimulated TR binding to the TSHβ-negative TRE and recruitment of NCoR. These findings demonstrate that conjugation of SUMO to TR has a TR-isoform preference and is important for T3-dependent gene induction and repression.

It has long been known that the avian brain is capable of light detection independently of the eyes. The photoreceptive molecule neuropsin (OPN5) was identified in mammalian and avian brains, and shown to respond to biologically relevant light wavelengths. Whether OPN5 is functionally involved in light detection is unknown. Daylength plays a critical role in regulating the neuroendocrine control of reproduction in birds. The presence of light during a 'photoinducible' phase of the circadian cycle, which occurs 12-16 h after dawn, results in marked changes in hypothalamic gene expression. These changes ultimately control gonadotropin release from the pituitary gland that, in turn, stimulates gonadal development. In this study, we first measured OPN5 expression in the mediobasal hypothalamus (MBH) in border canaries during the photoinducible period in relation to thyrotropin (TSH) β-subunit mRNA expression, which is implicated in the control of avian reproduction. Second, the knockdown of OPN5 via small interfering RNA antisense in the MBH revealed that there is an inhibitory input in the photoinduced regulation of TSHβ mRNA expression. Our data indicate that a decrease in OPN5 mRNA expression is associated with the facilitation in TSHβ mRNA expression in the MBH, a critical step for the light-induced increase in gonadal recrudescence. We hypothesise that the removal of an inhibitory input by OPN5 in birds may be a step that occurs during the photoinducible period. Given the distribution of OPN5 in the brain and periphery, this suggests a possible multifunctional role for light information in regulating other physiological processes., (© 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

In vertebrates, stress and thyroid systems interact closely, most likely because of the involvement of both systems in energy metabolism. However, studies on these interactions, especially during larval development, are scarce. Recently, cDNAs coding for corticotropin-releasing hormone (CRH) and CRH-binding protein (CRH-BP), two key players in the regulation of the neuroendocrine stress response, were characterized for the flatfish Senegalese sole (Solea senegalensis). To investigate the involvement of stress and thyroid systems in this species, the effects of food deprivation during early development of S. senegalensis were assessed. Growth was arrested in food-deprived post-larvae, which was also reflected by decreased carbon and nitrogen contents, indicating increased catabolism. Food deprivation induces chronic stress, as illustrated by enhanced whole-body cortisol levels, as well as an up regulation of crh and a decrease of crh-bp expression levels. Furthermore, whole-body total T3 concentrations of food-deprived post-larvae were reduced, although tshβ subunit expression levels remained unaffected. Our results show that food deprivation is a chronic stressor that induces energy-releasing catabolic processes that compensate for the reduced energy intake, and inhibits anabolic processes via the peripheral thyroid system., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Accumulating evidence indicates that a wide range of chemicals have the ability to interfere with the hypothalamic-pituitary-thyroid (HPT) axis. Novel endpoints should be evaluated in addition to existing methods in order to effectively assess the effects of these chemicals on the HPT axis. Thyroid-stimulating hormone subunit β (TSHβ) plays central regulatory roles in the HPT system. We identified the regulatory region that determines the expression level of zebrafish TSHβ in the anterior pituitary. In the transgenic zebrafish with EGFP driven by the TSHβ promoter, the similar responsive patterns between the expression levels of TSHβ:EGFP and endogenous TSHβ mRNA in the pituitary are observed following treatments with goitrogen chemicals and exogenous thyroid hormones (THs). These results suggest that the TSHβ:EGFP transgenic reporter zebrafish may be a useful alternative in vivo model for the assessment of chemicals interfering with the HPT system., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Thyroid hormones have a profound influence on human development and disease. The hypothalamic-pituitary-thyroid axis involves finely tuned feedback mechanisms to maintain thyroid hormone (TH) levels. Despite the important role of TH-negative feedback in regulating this axis, the mechanism by which this occurs is not clearly defined. Previous in vivo studies suggest separate roles for the two thyroid hormone receptor isoforms, THRA and THRB, in this axis. We performed studies using a unique pituitary thyrotroph cell line (TαT1.1) to determine the relative roles of THRA and THRB in the regulation of Tshb. Using chromatin immunoprecipitation assays, we found that THRB, not THRA, bound to the Tshb promoter. By selectively depleting THRB, THRA, or both THRA and THRB in TαT1.1 cells, we found that simultaneous knockdown of both THRB and THRA abolished T(3)-mediated down-regulation of Tshb at concentrations as high as 100 nm T(3). In contrast, THRA knockdown alone had no effect on T(3)-negative regulation, whereas THRB knockdown alone abolished T(3)-mediated down-regulation of Tshb mRNA levels at 10 nm but not 100 nm T(3) concentrations. Interestingly, chromatin immunoprecipitation assays showed that THRA becomes enriched on the Tshb promoter after knockdown of THRB. Thus, a likely mechanism for the differential effects of THR isoforms on Tshb may be based on their differential DNA-binding affinity to the promoter.

The pituitary is a complex gland comprising different cell types each secreting specific hormones. The extensive network of signaling molecules and transcription factors required for determination and terminal differentiation of specific cell types is still not fully understood. The SRY-like HMG-box (SOX) transcription factor Sox4 plays important roles in many developmental processes and has two homologs in zebrafish, Sox4a and Sox4b. We show that the sox4b gene is expressed in the pituitary anlagen starting at 24 h after fertilization (hpf) and later in the entire head region including the pituitary. At 48 hpf, sox4b mRNA colocalizes with that for TSH (tshβ), glycoprotein subunit α (gsuα), and the Zn finger transcription factor Gata2a. Loss of Sox4b function, using morpholino knockdown or expression of a dominant-negative Sox4 mutant, leads to a drastic decrease in tshβ and gsuα expression and reduced levels of gh, whereas other anterior pituitary gland markers including prl, slβ, pomc, and lim3 are not affected. Sox4b is also required for expression of gata2a in the pituitary. Knockdown of gata2a leads to decreased tshβ and gsuα expression at 48 hpf, similar to sox4b morphants. Injection of gata2a mRNA into sox4b morphants rescued tshβ and gsuα expression in thyrotrope cells. Finally, sox4b or gata2a knockdown causes a significant decrease of gonadotropin expression (lhβ and fshβ) at 4 d after fertilization. In summary, our results indicate that Sox4b is expressed in zebrafish during pituitary development and plays a crucial role in the differentiation of thyrotrope and gonadotrope cells through induction of gata2a expression in the developing pituitary.

Our previous epidemiological study indicated that excessive intake of iodine could potentially lead to hypothyroidism. In the present study, we aimed to investigate the time and dose effect of iodine intake on serum thyrotropin (thyroid-stimulating hormone, TSH) levels and to explore the non-autoimmune regulation of serum TSH by pituitary type 2 deiodinase (D2). A total of 360 Wistar rats were randomly divided into five groups depending on administered iodine dosages (folds of physiological dose): normal iodine (NI), 3-fold iodine (3HI), 6-fold iodine (6HI), 10-fold iodine (10HI) and 50-fold iodine (50HI). At 4, 8, 12 and 24 weeks after administration of sodium iodide, blood was collected for serum TSH measurement by chemiluminescent immunoassay. Pituitaries were also excised for measurement of TSHβ subunit expression, D2 expression and activity, monocarboxylate transporter 8 (MCT8) and thyroid hormone receptor β2 isoform (TRβ2) levels. The results showed that iodine intake of 10HI and 50HI significantly increased pituitary and serum TSH levels from 8 to 24 weeks (P < 0·05 v. NI). Excess iodine had no effect on D2 mRNA or protein expression; however, 10HI and 50HI administration significantly inhibited pituitary D2 activities from 8 to 24 weeks (P < 0·05 v. NI). Iodine had no effect on MCT8 or TRβ2 protein levels. We conclude that prolonged high iodine intake inhibits pituitary D2 activity and induces elevation of serum TSH levels. These findings may provide a potential mechanism of iodine excess-induced overt and subclinical hypothyroidism.

Thyrotropin-releasing hormone (TRH) is a major stimulator of thyrotropin-stimulating hormone (TSH) synthesis in the anterior pituitary, though precisely how TRH stimulates the TSHβ gene remains unclear. Analysis of TRH-deficient mice differing in thyroid hormone status demonstrated that TRH was critical for the basal activity and responsiveness to thyroid hormone of the TSHβ gene. cDNA microarray and K-means cluster analyses with pituitaries from wild-type mice, TRH-deficient mice and TRH-deficient mice with thyroid hormone replacement revealed that the largest and most consistent decrease in expression in the absence of TRH and on supplementation with thyroid hormone was shown by the TSHβ gene, and the NR4A1 gene belonged to the same cluster as and showed a similar expression profile to the TSHβ gene. Immunohistochemical analysis demonstrated that NR4A1 was expressed not only in ACTH- and FSH- producing cells but also in thyrotrophs and the expression was remarkably reduced in TRH-deficient pituitary. Furthermore, experiments in vitro demonstrated that incubation with TRH in GH4C1 cells increased the endogenous NR4A1 mRNA level by approximately 50-fold within one hour, and this stimulation was inhibited by inhibitors for PKC and ERK1/2. Western blot analysis confirmed that TRH increased NR4A1 expression within 2 h. A series of deletions of the promoter demonstrated that the region between bp -138 and +37 of the TSHβ gene was responsible for the TRH-induced stimulation, and Chip analysis revealed that NR4A1 was recruited to this region. Conversely, knockdown of NR4A1 by siRNA led to a significant reduction in TRH-induced TSHβ promoter activity. Furthermore, TRH stimulated NR4A1 promoter activity through the TRH receptor. These findings demonstrated that 1) TRH is a highly specific regulator of the TSHβ gene, and 2) TRH mediated induction of the TSHβ gene, at least in part by sequential stimulation of the NR4A1-TSHβ genes through a PKC and ERK1/2 pathway.

Pitx2 is a homeodomain transcription factor required in a dose-dependent manner for the development of multiple organs. Pitx2-null homozygotes (Pitx2(-/-)) have severe pituitary hypoplasia, whereas mice with reduced-function alleles (Pitx2(neo/neo)) exhibit modest hypoplasia and reduction in the developing gonadotroph and Pou1f1 lineages. PITX2 is expressed broadly in Rathke's pouch and the fetal pituitary gland. It predominates in adult thyrotrophs and gonadotrophs, although it is not necessary for gonadotroph function. To test the role of PITX2 in thyrotroph function, we developed thyrotroph-specific cre transgenic mice, Tg(Tshb-cre) with a recombineered Tshb bacterial artificial chromosome that ablates floxed genes in differentiated pituitary thyrotrophs. We used the best Tg(Tshb-Cre) strain to generate thyrotroph-specific Pitx2-deficient offspring, Pitx2(flox/-;)Tg(Tshb-cre). Double immunohistochemistry confirmed Pitx2 deletion. Pitx2(flox/-);Tg(Tshb-cre) mice have a modest weight decrease. The thyroid glands are smaller, although circulating T(4) and TSH levels are in the normal range. The pituitary levels of Pitx1 transcripts are significantly increased, suggesting a compensatory mechanism. Hypothyroidism induced by low-iodine diet and oral propylthiouracil revealed a blunted TSH response in Pitx2(flox/-);Tg(Tshb-cre) mice. Pitx1 transcripts increased significantly in control mice with induced hypothyroidism, but they remained unchanged in Pitx2(flox/-);Tg(Tshb-cre) mice, possibly because Pitx1 levels were already maximally elevated in untreated mutants. These results suggest that PITX2 and PITX1 have overlapping roles in thyrotroph function and response to hypothyroidism. The novel cre transgene that we report will be useful for studying the function of other genes in thyrotrophs.

Synthetic gestagens, including levonorgestrel (LNG), are active compounds in contraceptives, and several studies report their occurrence in surface waters. However, information about endocrine-disrupting effects in nontarget organisms is scarce. The present study investigated effects of LNG exposure on thyroid hormone-dependent metamorphosis of Xenopus laevis. Premetamorphic X. laevis tadpoles at Nieuwkoop and Faber (NF) stage 48 were exposed in a flow-through culture system to four LNG concentrations (10(-11), 10(-10), 10(-9), and 10(-8)M) over the period of metamorphosis. At NF 58 and 66, tadpoles were examined sex specifically. Developmental time and organismal responses were recorded and correlated with molecular and histopathological endpoints. Exposure to 10(-8)M LNG caused an inhibition of metamorphosis resulting in developmental arrest at early climax stages as giant tadpoles or tailed frogs. In brain-pituitary tissue of NF 58 tadpoles, gene expression of thyroid-stimulating hormone (β-subunit; TSHβ), TH receptor β (TRβ), and deiodinase type 3 (D3) was not changed. Instead, prolactin (PRL) messenger RNA (mRNA) was significantly increased by 10(-9)M LNG in females and by 10(-8)M LNG in both sexes. In NF 66 tadpoles, mRNA levels of TSHβ mRNA were significantly increased in the 10(-9) and 10(-8)M LNG treatment groups indicating a hypothyroid state. No changes of TRβ, D3, and PRL gene expression were detected. Histopathological evaluation of thyroid gland sections revealed no typical sign of hypothyroidism but rather an inactivated appearance of the thyroid. In conclusion, our data demonstrate for the first time a completely new aspect of thyroid system disruption caused by synthetic gestagens in developing amphibians.

Retinoid X receptor (RXR) signaling influences thyrotrope function. Synthetic RXR agonists, rexinoids, can cause central hypothyroidism. To test the hypothesis that endogenous rexinoids contribute to the TSH 'set point', TαT1 mouse thyrotrope cells were treated with a rexinoid antagonist, LG101208. Increasing concentrations of LG101208 significantly increased TSHβ mRNA levels, indicating that the rexinoid antagonist may interfere with RXR-signaling by an endogenous rexinoid in thyrotropes. When the same experiments were repeated in the presence of charcoal-stripped serum the effect of the rexinoid antagonist was lost. Pretreatment with the transcription inhibitor DRB blocked the increase of TSHβ mRNA levels by rexinoid antagonist, indicating the primary effect is at the level of gene transcription. Mice treated with LG101208 had higher levels of serum T4, T4/TSH ratios as well as pituitary α-subunit and TSHβ mRNA compared with vehicle treated mice. Hypothalamic TRH levels were unchanged. In summary, the rexinoid antagonist, LG101208, increases TSH subunit mRNA levels in thyrotrope cells and mouse pituitaries, primarily at the level of gene transcription. These data suggest that an "endogenous rexinoid" contributes to the TSH 'set point' in thyrotropes., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Expression of mammalian GH is normally restricted to somatotropes and somatolactotropes (somatotrope lineages) in the anterior pituitary. The basis for this restriction remains incompletely understood. Recent studies indicate that deoxyribonuclease I hypersensitive site I (HSI) of the hGH locus control region, located at -14.5 kb relative to the hGH-N promoter, acts as a potent long-range enhancer of hGH-N transcription. Here we report that HSI is also critical to somatotrope-restriction of hGH-N expression. Loss of HSI activity, either by direct inactivation of HSI or by interference with HSI-dependent downstream events, results in a relaxation of hGH-N cell-type specification with expansion of hGH-N expression to the full spectrum of Pit-1 positive pituitary cell types. These findings expand the defined roles for HSI of the hGH locus control region to include somatotrope lineage restriction as well as transcriptional enhancement of hGH-N gene expression.

When mutated in mammals, paired-like homeobox Prop1 gene produces highly variable pituitary phenotypes with impaired regulation of Pit1 and eventually defective synthesis of Pit1-regulated pituitary hormones. Here we have identified fish prop1 orthologs, confirmed their pituitary-specific expression, and blocked the splicing of zebrafish prop1 transcripts using morpholino oligonucleotides. Very early steps of the gland formation seemed unaffected based on morphology and expression of early placodal marker pitx. Prop1 knock-down reduced the expression of pit1, prl (prolactin) and gh (growth hormone), as expected if the function of Prop1 is conserved throughout vertebrates. Less expectedly, lim3 was down regulated. This gene is expressed from early stages of vertebrate pituitary development but is not known to be Prop1-dependent. In situ hybridizations on prop1 morphants using probes for the pan pituitary gene pitx3 and for the hormone gene markers prl, gh and tshβ, revealed abnormal shape, growth and cellular organization of the developed adenohypophysis. Strikingly, the effects of prop1 knock-down on adenohypophysis morphology and gene expression were gradually reversed during late development, despite persistent splice-blocking of transcripts. Therefore, prop1 function appears to be conserved between mammals and fish, at least for the mediation of hormonal cell type differentiation via pit1, but the existence of other fish-specific pathways downstream of prop1 are suggested by our observations., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Whereas it is well known that T3 inhibits TSHβ gene transcription, its effects on TSHβ mRNA stability and translation have been poorly investigated. This study examined these possibilities, by evaluating the TSHβ transcripts poly(A) tail length, translational rate and binding to cytoskeleton, in pituitaries of thyroidectomized and sham-operated rats treated with T3 or saline, and killed 30 min thereafter. The hypothyroidism induced an increase of TSHβ transcript poly(A) tail, as well as of its content in ribosomes and attachment to cytoskeleton. The hypothyroid rats acutely treated with T3 exhibited a reduction of TSHβ mRNA poly(A) tail length and recruitment to ribosomes, indicating that this treatment decreased the stability and translation rate of TSHβ mRNA. Nevertheless, acute T3 administration to sham-operated rats provoked an increase of TSHβ transcripts binding to ribosomes. These data add new insight to an important role of T3 in rapidly regulating TSH gene expression at posttranscriptional level., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

The hypothalamus-pituitary-thyroid (HPT) axis is an integrated hormone network that is essential for maintaining metabolic homeostasis. It has long been known that thyroid stimulating hormone (TSH), a central component of the HPT axis, can be made by cells of the immune system; however, the role of immune system TSH remains enigmatic and most studies have viewed it as a cytokine used to regulate immune function. Recent studies now indicate that immune system-derived TSH, in particular, a splice variant of TSHβ that is preferentially made by cells of the immune system, is produced by a subset of hematopoietic cells that traffic to the thyroid. On the basis of these and other findings, we propose the novel hypothesis that the immune system is an active participant in the regulation of basal metabolism. We further speculate that this process plays a critical role during acute and chronic infections and that it contributes to a wide range of chronic inflammatory conditions with links to thyroid dysregulation. This hypothesis, which is amenable to empirical analysis, defines a previously unknown role for the immune system in health and disease, and it provides a dynamic connection between immune-endocrine interactions at the organismic level.

Living organisms detect seasonal changes in day length (photoperiod) [1-3] and alter their physiological functions accordingly to fit seasonal environmental changes. TSHβ, induced in the pars tuberalis (PT), plays a key role in the pathway that regulates vertebrate photoperiodism [4, 5]. However, the upstream inducers of TSHβ expression remain unknown. Here we performed genome-wide expression analysis of the PT under chronic short-day and long-day conditions in melatonin-proficient CBA/N mice, in which the photoperiodic TSHβ expression response is preserved [6]. This analysis identified "short-day" and "long-day" genes, including TSHβ, and further predicted the acute induction of long-day genes by late-night light stimulation. We verified this by advancing and extending the light period by 8 hr, which induced TSHβ expression within one day. In the following genome-wide expression analysis under this acute long-day condition, we searched for candidate upstream genes by looking for expression that preceded TSHβ's, and we identified the Eya3 gene. We demonstrated that Eya3 and its partner Six1 synergistically activate TSHβ expression and that this activation is further enhanced by Tef and Hlf. These results elucidate the comprehensive transcriptional photoperiodic response in the PT, revealing the complex regulation of TSHβ expression and unexpectedly rapid response to light changes in the mammalian photoperiodic system.

Seasonal synchronization based on day length (photoperiod) allows organisms to anticipate environmental change. Photoperiodic decoding relies on circadian clocks, but the underlying molecular pathways have remained elusive [1]. In mammals and birds, photoperiodic responses depend crucially on expression of thyrotrophin β subunit RNA (TSHβ) in the pars tuberalis (PT) of the pituitary gland [2-4]. Now, using our well-characterized Soay sheep model [2], we describe a molecular switch governing TSHβ transcription through the circadian clock. Central to this is a conserved D element in the TSHβ promoter, controlled by the circadian transcription factor thyrotroph embryonic factor (Tef). In the PT, long-day exposure rapidly induces expression of the coactivator eyes absent 3 (Eya3), which synergizes with Tef to maximize TSHβ transcription. The pineal hormone melatonin, secreted nocturnally, sets the phase of rhythmic Eya3 expression in the PT to peak 12 hr after nightfall. Additionally, nocturnal melatonin levels directly suppress Eya3 expression. Together, these effects form a switch triggering a strong morning peak of Eya3 expression under long days. Species variability in the TSHβ D element influences sensitivity to TEF, reflecting species variability in photoperiodic responsiveness. Our findings define a molecular pathway linking the circadian clock to the evolution of seasonal timing in mammals.

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.

Although the regulation of thyroid stimulating hormone β-subunit gene (TSHβ) has been intensively studied, the functions of transcription factors involved are not fully understood. The authors found that the -615/-516 promoter region of the TSHβ interacts specifically with nuclear proteins derived from pituitary tissue or from cultured thyrotroph cells. The actual binding site at the nucleotide level, as revealed by DNase I protection assay, includes the consensus sequence for nuclear factor I (NFI). RT-PCR analysis indicated that NFI-B expression is restricted to thyrotroph cells in the anterior pituitary. EMSA and ChIP analysis showed that NFI-B binds most efficiently to the -588/-560 region of TSHβ promoter. The forced expressions of NFI-B markedly reduced TSHβ promoter activity and its mRNA expression. Furthermore, it was also shown that the -588/-560 region is involved in the insulin-mediated repression of the TSHβ. It was of particular interest to observe that NFI-B was recruited to the -588/-560 region of the TSHβ promoter in an insulin-dependent manner. Taken together, this study provides new insights of the delicate regulations of energy metabolism and hormonal homeostasis.

Background: Much is known about how genes regulated by nuclear receptors (NRs) are switched on in the presence of a ligand. However, the molecular mechanism for gene down-regulation by liganded NRs remains a conundrum. The interaction between two zinc-finger transcription factors, Nuclear Receptor and GATA, was described almost a decade ago as a strategy adopted by the cell to up- or down-regulate gene expression. More recently, cell-based assays have shown that the Zn-finger region of GATA2 (GATA2-Zf) has an important role in down-regulation of the thyrotropin gene (TSHβ) by liganded thyroid hormone receptor (TR)., Methodology/principal Findings: In an effort to better understand the mechanism that drives TSHβ down-regulation by a liganded TR and GATA2, we have carried out equilibrium binding assays using fluorescence anisotropy to study the interaction of recombinant TR and GATA2-Zf with regulatory elements present in the TSHβ promoter. Surprisingly, we observed that ligand (T3) weakens TR binding to a negative regulatory element (NRE) present in the TSHβ promoter. We also show that TR may interact with GATA2-Zf in the absence of ligand, but T3 is crucial for increasing the affinity of this complex for different GATA response elements (GATA-REs). Importantly, these results indicate that TR complex formation enhances DNA binding of the TR-GATA2 in a ligand-dependent manner., Conclusions: Our findings extend previous results obtained in vivo, further improving our understanding of how liganded nuclear receptors down-regulate gene transcription, with the cooperative binding of transcription factors to DNA forming the core of this process.

Seasonal reproduction depends on photoperiod-regulated activation or suppression of the gonadal axis. Recent studies in quail have identified long-day induced TSH-beta expression in the pars tuberalis (PT) as a rapid trigger of gonadal activation. Thyroid-stimulating hormone (TSH) induces type 2 deiodinase (Dio2) in the ependymal cell layer (EC) of the infundibular recess to stimulate the gonadal axis. A similar mechanism is proposed in sheep and mice, but the experimental data on the temporal patterns of induction and suppression of TSH-beta and Dio2 expression are incomplete. In the present study, we examined the expression of TSH-beta and Dio2 in hamsters transferred from short- to long-day conditions for 9 days, and demonstrate the induction of TSH-beta and Dio2 on day 8 after transition. These data demonstrate the close relationship between TSH-beta and Dio2 expression in the inductive pathway. The temporal expression of TSH-beta and Dio2 in the suppressive pathway was also examined by s.c. melatonin injection, which mimics the transition from long to short days. Importantly, Dio2 expression in the EC is suppressed on day 1 after the onset of injection, whereas TSH-beta expression in the PT was not suppressed until day 10. These data suggest that regulated transcription of TSH-beta is involved in the induction of the gonadal axis in mammals, whereas the suppression of this axis is mediated by different mechanisms.

Photoperiodic regulation of reproduction in birds and mammals involves thyrotropin beta-chain (TSHb), which is secreted from the pars tuberalis (PT) and controls the expression of deiodinase type 2 and 3 in the ependymal cell layer of the infundibular recess (EC) via TSH receptors (TSHRs). To analyze the impact of melatonin and the molecular clockwork on the expression of Tshb and Tshr, we investigated melatonin-proficient C3H wild-type (WT), melatonin receptor 1-deficient (MT1-/-) or clockprotein PERIOD1-deficient (mPER1-/-) mice. Expression of Tshb and TSHb immunoreactivity in PT were low during day and high during the night in WT, high during the day and low during the night in mPER1-deficient, and equally high during the day and night in MT1-deficient mice. Melatonin injections into WT acutely suppressed Tshb expression. Transcription assays showed that the 5' upstream region of the Tshb gene could be controlled by clockproteins. Tshr levels in PT were low during the day and high during the night in WT and mPER1-deficient mice and equally low in MT1-deficient mice. Tshr expression in the EC did not show a day/night variation. Melatonin injections into WT acutely induced Tshr expression in PT but not in EC. TSH stimulation of hypothalamic slice cultures of WT induced phosphorylated cAMP response element-binding protein in PT and EC and deiodinase type 2 in the EC. Our data suggest that Tshb expression in PT is controlled by melatonin and the molecular clockwork and that melatonin activates Tshr expression in PT but not in EC. They also confirm the functional importance of TSHR in the PT and EC.

Young birds, in their post-natal growth period, may reduce their growth and metabolism when facing a food shortage. To examine how such responses can be mediated by endocrine-related factors, we exposed Japanese quail chicks to food restriction for either 2 days (age 6-8 days) or 5 days (age 6-11 days). We then measured growth and resting metabolic rate (RMR), and circulating 3,3',5-triiodo-l-thyronine (T3) and 3,5,3',5'-tetraiodothyronine (T4) levels as well as expression patterns of genes involved in growth (insulin-like growth factor-I: IGF-I) and thyroid hormone signalling (thyroid-stimulating hormone-beta: TSHbeta, type II iodothyronine deiodinase: D2, thyroid hormone receptors isoforms: TRalpha and TRbeta). The food-restricted chicks receiving a weight-maintenance diet showed reductions in structural growth and RMR. Plasma levels of both T3 and T4 were reduced in the food-restricted birds, and within the 5 days food-restricted group there was a positive correlation between RMR and T3. IGF-I mRNA showed significantly higher abundance in the liver of ad libitum fed birds at day 8 compared with food-restricted birds. In the brain, TSHbeta mRNA level tended to be lower in food-restricted quails on day 8 compared with controls. Furthermore, TRalpha expression was lower in the brain of food-restricted birds at day 8 compared with birds fed ad libitum. Interestingly, brain D2 mRNA was negatively correlated with plasma T3 levels, tending to increase with the length of food restriction. Overall, our results show that food restriction produced significant effects on circulating thyroid hormones and differentially affected mRNA species in the thyroid hormone signalling pathway. Thus, we conclude that the effects of food restriction observed on growth and metabolism were partly mediated by changes in the endocrine-related factors investigated.

The POU-homeodomain transcription factor Pit-1 governs the pituitary cell-specific expression of Pit-1, GH, prolactin (PRL), and TSHbeta genes. Alternative splicing generates Pit-1beta, which contains a 26-amino acid beta-domain inserted at amino acid 48, in the middle of the Pit-1 transcription activation domain (TAD). Pit-1beta represses GH, PRL, and TSHbeta promoters in a pituitary-specific manner, because Pit-1beta activates these same promoters in HeLa nonpituitary cells. Here we comprehensively analyze the role of beta-domain sequence, position, and context, to elucidate the mechanism of beta-dependent repression. Repositioning the beta-motif to the Pit-1 amino terminus, hinge, linker, and carboxyl terminus did not affect its ability to repress basal rat (r) PRL promoter activity in GH4 pituitary cells, but all lost the ability to repress Ras-induced rPRL promoter activity. To determine whether beta-domain repression is independent of Pit-1 protein and DNA binding sites, we generated Gal4-Pit-1TAD, Gal4-Pit-1betaTAD, and Gal4-beta-domain fusions and demonstrated that the beta-motif is sufficient to actively repress VP16-mediated transcription of a heterologous promoter. Moreover, beta-domain point mutants had the same effect whether fused to Gal4 or within the context of intact Pit-1beta. Surprisingly, Gal4-beta repression lost histone deacetylase sensitivity and pituitary specificity. Taken together, these results reveal that the beta-motif is a context-independent, modular, transferable, and dominant repressor domain, yet the beta-domain repressor activity within Pit-1beta contains cell type, promoter, and Pit-1 protein context dependence.

Thyroid hormones (THs) regulate growth, morphological development, and migratory behaviors in teleost fish, yet little is known about the transcriptional dynamics of gene targets for THs in these taxa. Here, we characterized TH regulation of mRNAs encoding thyrotropin subunits and thyroid hormone receptors (TRs) in an adult teleost fish model, the fathead minnow (Pimephales promelas). Breeding pairs of adult minnows were fed diets containing 3,5,3'-triiodo-L-thyronine (T(3)) or the goitrogen methimazole for 10 days. In males and females, dietary intake of exogenous T(3) elevated circulating total T(3), while methimazole depressed plasma levels of total thyroxine (T(4)). In both sexes, this methimazole-induced reduction in T(4) led to elevated mRNA abundance for thyrotropin beta-subunit (tshbeta) in the pituitary gland. Fish treated with T(3) had elevated transcript levels for TR isoforms alpha and beta (tralpha and trbeta) in the liver and brain, but reduced levels of brain mRNA for the immediate-early gene basic transcription factor-binding protein (bteb). In the ovary and testis, exogenous T(3) elevated gene transcripts for tshbeta, glycoprotein hormone alpha-subunit (gphalpha), and trbeta, while not affecting tralpha levels. Taken together, these results demonstrate negative feedback of T(4) on pituitary tshbeta, identify tralpha and trbeta as T(3)-autoinduced genes in the brain and liver, and provide new evidence that tshbeta, gphalpha, and trbeta are THs regulated in the gonad of teleosts. Adult teleost models are increasingly used to evaluate the endocrine-disrupting effects of chemical contaminants, and our results provide a systemic assessment of TH-responsive genes during that life stage.

Increasing evidence suggests that the hypophyseal pars tuberalis (PT) plays a key role in the transduction of light/dark (melatonin) information to the endocrine system. It has been shown that PT-specific cells express melatonin receptors and thyrotropin hormone (TSH) subunits. However, these cells do not resemble thyrotrophs or any other of the pars distalis (PD) cells. There is evidence that PT-specific cells secrete a glycoprotein hormone designated as 'tuberalin'. We have identified a putative tuberalin of 21 kDa (tuberalin II) and have raised antibodies against it. To further investigate whether tuberalin II is a distinct secretory compound of the PT, absorption studies of antituberalin II with TSH or with an extract of the rat PD containing beta-TSH, beta-luteinizing hormone (LH) and the common alpha-subunit of glycoprotein hormones (GSU), were performed. Neither of the absorption tests abolished the immunoreactivity of the PT to antituberalin II, suggesting that tuberalin II is different from TSH or the other PD glycoprotein hormones. Double immunofluorescence analyses using antibodies against tuberalin II, beta-TSH and GSU revealed that in the developing and adult PT there are 3 populations of PT-specific cells expressing tuberalin II and GSU (type 1), beta-TSH and GSU (type 2) and tuberalin II, beta-TSH and GSU (type 3). This further indicates that tuberalin II and beta-TSH correspond to different compounds and that they may be expressed either by different cells types or coexpressed in a 3rd cell type. The distribution and temporal expression of tuberalin II, beta-TSH, beta-LH and GSU were investigated in the developing pituitary gland. At E14.5, tuberalin II and GSU were expressed by cells of the PT primordium but not by the PD and pars intermedia primordia. The onset of expression of beta-TSH, beta-LH and GSU in cells of the PD occurred about 1 day later, further indicating the distinct nature of tuberalin II and supporting the earlier view that the secretion of polypeptides from the fetal rat pituitary gland begins in PT-specific cells.

The small intestine has been shown to be an extra-pituitary site of thyroid stimulating hormone (TSH) production, and previous in vivo studies have shown that TSH synthesis localizes within areas of enteric virus infection within the small intestine; however, the cellular source of intestinal TSH has not been adequately determined. In the present study, we have used the murine MODE-K small intestinal epithelial cell line to demonstrate both at the transcriptional level and as a secreted hormone, as measured in a TSHbeta-specific enzyme-linked assay, that epithelial cells in fact respond to infection with reovirus serotype 3 Dearing strain by upregulating TSH synthesis. Moreover, sequence analysis of a PCR-amplified TSHbeta product from MODE-K cells revealed homology to mouse pituitary TSHbeta. These findings have direct functional implications for understanding a TSH immune-endocrine circuit in the small intestine., ((c) 2008 Wiley-Liss, Inc.)

Thyroid hormone (TH) is essential for normal brain development, and polychlorinated biphenyls (PCBs) are known to interfere with TH action in the developing brain. Thus, it is possible that the observed neurotoxic effects of PCB exposure in experimental animals and humans are mediated in part by their ability to interfere with TH signaling. PCBs may interfere with TH signaling by reducing circulating levels of TH, acting as TH receptor analogs, or both. If PCBs act primarily by reducing serum TH levels, then their effects should mimic those of low TH. In contrast, if PCBs act primarily as TH agonists in the developing brain, then they should mimic the effect of T(4) in hypothyroid animals. We used a two-factor design to test these predictions. Both hypothyroidism (Htx) and/or PCB treatment reduced serum free and total T(4) on postnatal d 15. However, only Htx increased pituitary TSHbeta expression. RC3/neurogranin expression was decreased by Htx and increased by PCB treatment. In contrast, Purkinje cell protein-2 expression was reduced in hypothyroid animals and restored by PCB treatment. Finally, PCB treatment partially ameliorated the effect of Htx on the thickness of the external granule layer of the cerebellum. These studies demonstrate clearly that PCB exposure does not mimic the effect of low TH on several important TH-sensitive measures in the developing brain. However, neither did PCBs mimic T(4) in hypothyroid animals on all end points measured. Thus, PCBs exert a complex action on TH signaling in the developing brain.

Thyroid hormones (TH) play a critical role in flatfish metamorphosis. Their levels are regulated by the pituitary-thyroid axis. The expression profile of thyroid stimulating hormone (TSH) beta subunit and thyroglobulin (Tg) was investigated using a real-time PCR approach. Both genes exhibited different expression patterns during larval development in Senegalese sole. TSH beta mRNAs reduced progressively at the commencement of metamorphosis. On the contrary, Tg transcripts increased sharply at the onset of metamorphosis and dropped after the metamorphosis climax. T4 levels, as determined by radioimmunoassay, clearly resembled the Tg expression profile with a peak at the metamorphosis climax. To investigate if such expression profiles were regulated by TH, premetamorphic larvae were exposed to the goitrogen thiourea (TU). TU-treated larvae were not able to complete metamorphosis. However, the addition of exogenous T4 enabled to revert this effect. Expression analysis showed higher mRNA levels of both TSH beta and Tg in TU-treated larvae in comparison to control larvae. Moreover, the TU+T4 treated larvae exhibited similar or lower mRNA levels than in the control. Present results demonstrate that TH mediate metamorphosis and down-regulate TSH beta and Tg at transcriptional level in Senegalese sole.

We cloned and sequenced full-length cDNAs encoding the beta subunit of thyroid-stimulating hormone (TSHbeta) from the pituitary of fathead minnow (Pimephales promelas) using 5'- and 3'-rapid amplification of cDNA ends (RACE). Three cDNA variants for TSHbeta with lengths of 1184-, 1093-, and 818-bp were identified. The cDNA variant of 1184-bp included 453-bp of open-reading frame and 610-bp of 3' UTR followed by a poly(A)site. This cDNA encodes 150 amino acids including a 19 residue signal peptide and a mature TSHbeta protein of 131 residues with sequence identities of 97-53% to other fishes and 42-39% to mammals. The 1093-bp cDNA variant was identical to the 1184-bp variant in the open-reading frame, but contained a deletion of 40-bp in the 3' UTR. The 818-bp cDNA variant, however, contained 498-bp of open-reading frame followed by 227-bp of 3' UTR and a poly(A)site. The deduced amino acid sequence for this cDNA variant showed 99.2% homology with the 1184- and 1093-bp variants of TSHbeta, but a single deletion of 332-bp nucleotides spanning the predicted stop codon and 3' UTR resulted in a deduced amino acid sequence with 15 additional residues on the C terminus. The presence of this 818-bp cDNA variant in the pituitary was further confirmed by PCR using primers developed to the 5' and 3' UTR. PCR and Southern blot analyses of genomic DNA suggested only one gene for TSHbeta. Sequencing of this gene revealed a hairpin loop structure of approximately 300-bp located in the 3' UTR and corresponding to the region of the 332-bp deletion in the 818-bp transcript.

At temperate latitudes, increases in day length in the spring promote the summer phenotype. In mammals, this long-day response is mediated by decreasing nightly duration of melatonin secretion by the pineal gland. This affects adenylate cyclase signal transduction and clock gene expression in melatonin-responsive cells in the pars tuberalis of the pituitary, which control seasonal prolactin secretion. To define the photoperiodic limits of the mammalian long day response, we transferred short day (8 h light per 24 h) acclimated Soay sheep to various longer photoperiods, simulating those occurring from spring to summer in their northerly habitat (57 degrees N). Locomotor activity and plasma melatonin rhythms remained synchronized to the light-dark cycle in all photoperiods. Surprisingly, transfer to 16-h light/day had a greater effect on prolactin secretion and oestrus activity than shorter (12 h) or longer (20 and 22 h) photoperiods. The 16-h photoperiod also had the largest effect on expression of circadian (per1) and neuroendocrine output (betaTSH) genes in the pars tuberalis and on kisspeptin gene expression in the arcuate nucleus of the hypothalamus, which modulates reproductive activity. This critical photoperiodic window of responsiveness to long days in mammals is predicted by a model wherein adenylate cyclase sensitization and clock gene phasing effects of melatonin combine to control neuroendocrine output. This adaptive mechanism may be related to the latitude of origin and the timing of the seasonal transitions.

Previously, we identified reduced nicotinamide adenine dinucleotide phosphate-dependent cytosolic T(3) binding protein in rat cytosol. Cytosolic T(3)-binding protein is identical to mu-crystallin (CRYM). Recently, CRYM mutations were found in patients with nonsyndromic hereditary deafness. Although it has been established that CRYM plays pivotal roles in reserving and transporting T(3) into the nuclei in vitro and has a clinical impact on hearing ability, the precise functions of CRYM remain to be elucidated in vivo. To further investigate the in vivo functions of CRYM gene products, we have generated mice with targeted disruption of the CRYM gene, which abrogates the production of CRYM. CRYM knockout loses the reduced nicotinamide adenine dinucleotide phosphate-dependent T(3) binding activity in the cytosol of the brain, kidney, heart, and liver. At the euthyroid state, knockout significantly suppresses the serum concentration of T(3) and T(4) despite normal growth, heart rate, and hearing ability. The disruption of the gene does not alter the expression of TSHbeta mRNA in the pituitary gland or glutathione-S-transferase alpha2 and deiodinase 1 mRNAs in either the liver or kidney. When radiolabeled T(3) is injected intravenously, labeled T(3) rapidly enters into and then escapes from the tissues in CRYM-knockout mice. These data suggest that because of rapid T(3) turnover, disruption of the CRYM gene decreases T(3) concentrations in tissues and serum without alteration of peripheral T(3) action in vivo.

For better understanding of phylogenetic diversity and evolution of pituitary thyroid-stimulating hormone (TSH) in birds, we have cloned the cDNAs encoding TSH beta subunit (TSHbeta), by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) from two species of domestic ducks, common duck (Tsaiya duck and Pekin duck) (Anas platyrhynchos domesticus) and mule duck (hybrid of male muscovy duck Cairina moschata and female A. platyrhynchos domesticus). The nucleotide sequences of isolated TSHbeta cDNAs of the two species of ducks are identical, with each including 66 bp of 5'-untranslated region (UTR), 402 bp of coding region, and 82 bp 3'-UTR followed by 18 bp poly A tract. The deduced TSHbeta subunit of the ducks contains 134 amino acids consisting of a putative signal peptide of 19 amino acids and a putative mature protein of 115 amino acids. However, the TSHbetas of common duck and mule duck differ from the TSHbeta of muscovy duck in one amino acid at position 97 of the mature protein: isoleucine for common duck and mule duck, and valine for muscovy duck. Our findings thus demonstrate that inter-genus variation of TSHbeta exists in Family Anatidae, and that TSHbeta gene in the mule duck is preferentially transcribed from the maternal genome rather than from the paternal genome. TSHbeta mRNA expressions were investigated by culturing common duck pituitaries with various doses of hormones. Thyrotropin-releasing hormone (TRH) stimulated, while thyroid hormones, triiodothyronine (T(3)) and thyroxine (T(4)), inhibited the TSHbeta mRNA levels, in dose-related manners. The findings thus support that the mode of regulation of TSH gene expression in hypothalamo-pituitary-thyroid axis in birds is similar to that in mammals. Cortisol and corticosterone decreased the steady-state TSHbeta mRNA levels at the pituitary level, in a dose-related manner, the first-time demonstration in vertebrates. The results may suggest that glucocorticoids exert suppressive action directly at pituitary level in modulation of steady-state TSHbeta mRNA level.

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.

The anterior pituitary is comprised of five major hormone-secreting cell types that differentiate during embryonic development in a temporally distinct manner. Microarrays containing 5,128 unique cDNAs expressed in the chicken neuroendocrine system were produced and used to identify genes with potential involvement in the onset of thyroid-stimulating hormone beta-subunit (TSHbeta), growth hormone (GH), and prolactin (PRL) mRNA during embryonic development. We identified 352 cDNAs that were differentially expressed (P < or = 0.05) on embryonic day 10 (e10), e12, e14, or e17, the period of thyrotroph, somatotroph, and lactotroph differentiation. Self-organizing maps were used to identify genes that may function to initiate hormone gene transcription. Consistent with cellular ontogeny, TSHbeta mRNA increased steadily between e10 and e17, GH mRNA increased between e12 and e17, and PRL mRNA did not increase until e17. Expression of 141 genes increased in a manner similar to TSHbeta mRNA, and 64 genes decreased between e10 and e17. Although genes with these expression profiles are likely involved in development of the pituitary gland as a whole, some of these could be specifically associated with thyrotroph differentiation. Similarly, the expression profiles of 69 and 61 genes indicate a potential involvement in the induction of GH and PRL mRNA, respectively. Quantitative real-time RT-PCR was used to confirm microarray results for 31 genes. This is the first study to evaluate changes in anterior pituitary gene expression during embryonic development of any species using microarrays, and numerous transcription factors and signaling molecules not previously implicated in pituitary development were identified.

According to outdated paradigms humic substances (HS) are considered to be refractory or inert that do not directly interact with aquatic organisms. However, they are taken up and induce biotransformation activities and may act as hormone-like substances. In the present study, we tested whether HS can interfere with endocrine regulation in the amphibian Xenopus laevis. In order to exclude contamination with phyto-hormones, which may occur in environmental isolates, the artificial HS1500 was applied. The in vivo results showed that HS1500 causes significant estrogenic effects on X. laevis during its larval development and results of semi-quantitative RT-PCR revealed a marked increase of the estrogenic biomarker estrogen receptor mRNA (ER-mRNA). Furthermore, preliminary RT-PCR results showed that the thyroid-stimulating hormone (TSHbeta-mRNA) is enhanced after exposure to HS1500, indicating a weak adverse effect on T3/T4 availability. Hence, HS may have estrogenic and anti-thyroidal effects on aquatic animals, and therefore may influence the structure of aquatic communities and they may be considered environmental signaling chemicals.

The functional development of pituitary cells depends on the expression of a combination of transcription factors and co-factors. Pituitary-specific transcription factor-1 (Pit-1) is required for the expression of growth hormone (GH), prolactin (PRL), and the thyroid-stimulating hormone beta subunit (TSH beta) and acts synergistically with the estrogen receptor (ER) and GATA-binding protein 2 (GATA-2) to induce PRL and TSH beta expression, respectively. The glycoprotein hormone alpha subunit (alpha SU) is the first hormone to be expressed during pituitary development. In addition to being expressed in follicle-stimulating hormone, luteinizing hormone (LH), and TSH cells, alpha SU is reported to co-localize with GH in pituitary cells. These findings have led to the suggestion that the expression of Pit-1 in cells of the alpha SU-based gonadotropin cell lineage might also lead to the expression of GH. In this study, we transfected HP 75 cells (derived from a human non-functioning pituitary adenoma that expressed alpha SU and LH beta) with Pit-1 by using an adenovirus FLAG-Pit-1 construct. Most of the transfected cells expressed GH mRNA, with fewer cells expressing PRL and TSH beta mRNA. The HP 75 cells expressed the genes for ER and GATA-2, thus allowing their expression of GH, PRL, and TSH beta mRNA in response to Pit-1. These results support the hypothesis that GH can be induced in cells that possess an active alpha SU gene and shed light on the basic molecular mechanism that drives the development of GH, PRL, and TSH beta expression in the alpha SU-based gonadotroph lineage.

We report the elk (Cervus elaphus) thyroid stimulating hormone (TSH) beta-subunit cDNA cloning, nucleotide and deduced amino acid sequences. The TSH beta-subunit cDNA was obtained by RT-PCR of polyadenylated pituitary RNA. The deduced elk TSH beta-subunit peptide chain shares between 93 to 99% sequence similarities with the reported TSH beta-subunit of a sub-set of related species. The TSH beta-subunit gene is expressed in the elk pituitary gland as a mature transcript of approximately 600 bases, which corresponds to the size of the mRNA expressed in the sheep pituitary gland. Seasonal expression of the pituitary gonadotropin genes was investigated by Northern blot analyses. Samples of elk pituitary glands collected during the breeding season showed elevated steady state levels of common alpha-subunit and FSH and LH beta-subunit gene expression, consistent with the seasonal reproductive cycling of this species. Samples collected before the breeding season demonstrated decreased expression of the gonadotropin genes. TSH, which is not directly tied to reproduction, had similar levels of expression, regardless of the animal's reproductive status.

Prolactin (PRL),Growth Hormone (GH), and Thyroid-stimulating Hormone-beta(TSHbeta), secreting by chicken anterior pituitary, are essential hormones affecting chicken laying ability, growth rate, and immunity via regulating chicken broodiness, development, and immune response. Avian pituitary-specific transcription factor (POU1F1) is one of important transcription factors of GH, PRL, and TSHbetagene. POU1F1 plays an important role in avian growth, development and reproduction via regulating the expression of these genes. The gene organization and the expression characters of poultry Pit-1 were discussed comparing with those of mammals, and the functions of POU1F1 were also elucidated in the review. Considering the biological importance of POU1F1 and the results got in our research, we suggested that Pit-1 be a candidate gene for growth, egg laying and broodiness research in poultry.

Leptin and neuropeptide-Y (NPY) are indicated to play a role in hypothalamo-pituitary-thyroid (HPT) axis in relation to regulation of energy homeostasis mediated through acting at hypothalamic synthesis and release of thyrotropin (TSH)-releasing hormone (TRH). Whether leptin and NPY also act at pituitary level in HPT axis remains unknown. This study aimed at investigating whether or not leptin and NPY exert actions at pituitary in modulation of transcript levels of TSHbeta and the common pituitary glycoprotein hormone alpha (PGHalpha) subunits. The dispersed pituitary cells from 6 wk old male Wistar rats were incubated with or without TRH, leptin or NPY of 10(-8) M and 10(-10) M for 6 h at 37 degrees C in medium-199 under aeration of 95% O2 and 5% CO2. The mRNA levels of TSHbeta and PGHalpha subunits of the incubated cells were measured by reverse transcription-polymerase chain reaction. The results revealed that leptin stimulated, while NPY inhibited, TSHbeta mRNA levels in a dose-related manner. Both leptin and NPY increased alpha subunit mRNA levels. It is demonstrated for the first time that both leptin and NPY exert a direct action at rat pituitary affecting steady-state levels of mRNA of TSHbeta and PGHalpha subunits. The present study supports that both leptin and NPY act at the pituitary as well besides the hypothalamus in HPT axis in relation to regulation of energy homeostasis.

We have recently reported a disease-causing substitution (+5G > C) at the donor site of NF-1 exon 3 that produces its skipping. We have now studied in detail the splicing mechanism involved in analyzing RNA-protein complexes at several 5' splice sites. Characteristic protein patterns were observed by pulldown and band-shift/super-shift analysis. Here, we show that hnRNP H binds specifically to the wild-type GGGgu donor sequence of the NF-1 exon 3. Depletion analyses shows that this protein restricts the accessibility of U1 small nuclear ribonucleoprotein (U1snRNA) to the donor site. In this context, the +5G > C mutation abolishes both U1snRNP base pairing and the 5' splice site (5'ss) function. However, exon recognition in the mutant can be rescued by disrupting the binding of hnRNP H, demonstrating that this protein enhances the effects of the +5G > C substitution. Significantly, a similar situation was found for a second disease-causing +5G > A substitution in the 5'ss of TSHbeta exon 2, which harbors a GGgu donor sequence. Thus, the reason why similar nucleotide substitutions can be either neutral or very disruptive of splicing function can be explained by the presence of specific binding signatures depending on local contexts.

Objective: Double pituitary adenomas are extremely rare. They can be divided into contiguous and clearly separated types. Most contiguous tumours are surgically removed as one tumour and the co-existence of different adenoma types can be confirmed by histological methods. In contrast, detailed preoperative neuroimaging studies can suggest the co-existence of separated multiple adenomas. In patients with multiple adenomas, surgical failure may result when one adenoma is missed during surgery. Among 600 surgical cases we encountered four patients with clearly separated double pituitary adenomas; all were highly suspect on preoperative MRI studies., Patients and Results: All four patients manifested acromegalic symptoms; one patient also exhibited hyperprolactinemia and two had familial pituitary adenomas unrelated to multiple endocrine neoplasia type I (MEN-1). All underwent transsphenoidal surgery and histology confirmed the diagnosis of GH-producing plus gonadotroph adenoma in two cases and of two GH-producing adenomas each in the other two patients., Conclusion: Although the pathogenesis of our double adenomas remains unknown, genetic abnormalities may be involved because two patients had familial pituitary adenomas unrelated to MEN-1. When preoperative MRI is suggestive of double adenomas, careful surgical exploration is necessary to avoid missing the other adenoma because the risk of surgical failure is high, especially in patients with functioning adenomas.

The alpha-subunit is common to the heterodimeric glycoprotein hormones and has been highly conserved throughout vertebrate evolution. In an effort to determine if wild-type and engineered human alpha analogs can serve as agonists or antagonists to the human thyroid-stimulating hormone (TSH) receptor (TSHR), a potent alpha mutant, obtained by replacing four amino acid residues with lysine (alpha4K), was assayed and compared with the wild-type alpha-subunit. When added to CHO cells expressing TSHR, alpha4K, and to a very limited extent the fused homodimer, alpha4K-alpha4K, but not alpha, exhibited agonist activity as judged by cAMP production. When yoked to TSHR to yield fusion proteins, neither alpha, alpha4K, alpha-alpha, nor alpha4K-alpha4K activated TSHR, although yoked alpha4K and alpha4K-alpha4K were weak inhibitors of TSH binding to TSHR. The yoked subunit-receptor complexes were, however, functional as evidenced by increased cAMP production in cells co-expressing human TSHbeta and alpha-TSHR, alpha4K-TSHR, alpha-alpha-TSHR, and alpha4K-alpha4K-TSHR. These results demonstrate that agonists to TSHR can be obtained with alpha-subunit analogs and suggest that rational protein engineering may lead to more potent alpha-based derivatives. The differences found between the experimental paradigms of adding free alpha analogs to TSHR and covalent attachment are attributed to con-formational constraints imposed by fusion of the alpha-subunit analog and receptor, and may suggest an important role for a free (C-terminal) alpha-carboxyl in the absence of the beta-subunit., (Copyright 2004 Humana Press Inc.)

We have cloned and characterized the full-length cDNA encoding thyroid-stimulating hormone beta-subunit (TSHbeta) from orange-spotted grouper Epinephelus coioides. It contains 913 nucleotides with an open reading frame encoding 146 amino acids with a 20 amino acid signal peptide. The grouper mature TSHbeta has 75, 70, 61, 59, 41, 42 and 40% identities to that of rainbow trout, Atlantic salmon, zebrafish, European eel, chicken, mouse and human, respectively. RT-PCR analysis indicated that the TSHbeta mRNA was expressed abundantly not only in pituitary but also in gonads. A more interesting finding is to reveal the differential TSHbeta expressions between the ovaries and the transitional gonads or testes in natural individuals of orange-spotted grouper and red-spotted grouper Epinephelus akaara, and in artificial sex reversal individuals of red-spotted grouper induced by MT feeding. In situ hybridization localization provided direct evidence that the TSHbeta was transcribed in the germ cells. In the growing oocytes, the TSHbeta transcripts were concentrated on the ooplasm periphery. In testicular tissues, the intensively expressed TSHbeta cells were found to be spermatogonia and spermatocytes in the spermatogenic cysts. This is the first report of a TSHbeta expressed in the gonads of any vertebrates in addition to the expected expression in the pituitary, and it expresses more transcripts in the gonads during sex reversal or testis than in the ovaries both in E. coioides and E. akaara. Importantly, the TSHbeta identification in germ cells allows us to further investigate the functional roles and the molecular mechanisms in gametogenesis of groupers, especially in sex reversal and in spermatogenesis.

The purposes of this study were: (1) to clone the cDNA encoding pituitary thyroid-stimulating hormone beta subunit (TSH beta) of the Japanese eel, Anguilla japonica, together with its genomic DNA sequence, for phylogenetic analysis, and to study the regulation of the TSH beta gene expression in cultured pituitaries; and (2) to investigate the transcript levels of pituitary TSH beta mRNA and the serum thyroxine profiles at different stages of ovarian development before and during silvering in the wild female eels. The maturity of female eels was divided into four stages, juvenile, sub-adult, pre-silver, and silver, based on skin color and oocyte diameter. The genomic DNA of the TSH beta subunit contains two introns and three exons, and the TSH beta protein possesses a putative signal peptide of 20 amino acids and a mature peptide of 127 amino acids. The amino acid sequence identities of TSH beta mature peptide of Japanese eel compared with those of teleosts and other vertebrates are: European eel (98.4%), salmonids (60.6-61.3%), carps (52.0-56.7%), sturgeon (48.4%), and tetrapods (42.9-45.2%). In in vitro studies of the regulation of TSH beta mRNA it was found that thyrotropin-releasing hormone increased while thyroxine decreased its expression. RT-PCR and real-time quantitative PCR analysis showed that the transcript levels of TSH beta subunit increased during eel silvering. The serum thyroxine levels also increased in parallel with TSH beta mRNA expression during silvering, supporting the hypothesis that the hypothalamus-pituitary-thyroid axis is correlated to silvering in the wild female Japanese eels.

The pars tuberalis (PT) of the pituitary represents an important target site for the time-pacing pineal hormone melatonin because it expresses a large number of mt1 receptors. Functional studies suggest that the PT mediates the seasonal effects of melatonin on prolactin (PRL) secretion. The aim of this study was the characterization of the phenotype of melatonin-responsive cells. Furthermore, we determined whether RORbeta, a retinoid orphan receptor present in the PT, was co-expressed in the same cells. We combined nonradioactive in situ hybridization (ISH) with hapten-labeled riboprobes for detection of the receptors and immunocytochemistry (ICC) for detection of alphaGSU (alpha-glycoprotein subunit), betaTSH, betaFSH, betaLH, GH, PRL, and ACTH. Expression of mt1 mRNA was found in small round cells, co-localized with alphaGSU and betaTSH. However, not all betaTSH-containing cells expressed mt1 mRNA. The distribution of mt1- and RORbeta-positive cells appeared to overlap, although more cells were labeled for RORbeta than for mt1. Gonadotrophs, as well as other pars distalis cell types, were never labeled for mt1 melatonin receptor. Therefore, this study identifies the "specific" cells of the PT as the mt1 melatonin receptor-expressing cells.