Thyrotrophs Research Articles

Thyroid-stimulating hormone-thyroid hormone signaling contributes to circadian regulation through repressing clock2/npas2 in zebrafish

Thyroid-stimulating hormone (TSH) is important for the thyroid gland, development, growth, and metabolism. Defects in TSH production or the thyrotrope cells within the pituitary gland cause congenital hypothyroidism (CH), resulting in growth retardation and neurocognitive impairment. While human TSH is known to display rhythmicity, the molecular mechanisms underlying the circadian regulation of TSH and the effects of TSH-thyroid hormone (TH) signaling on the circadian clock remain elusive. Here we show that TSH, thyroxine (T4), triiodothyronine (T3), and tshba display rhythmicity in both larval and adult zebrafish and tshba is regulated directly by the circadian clock via both E′-box and D-box. Zebrafish tshba−/− mutants manifest congenital hypothyroidism, with the characteristics of low levels of T4 and T3 and growth retardation. Loss or overexpression of tshba alters the rhythmicity of locomotor activities and expression of core circadian clock genes and hypothalamic-pituitary-thyroid (HPT) axis-related genes. Furthermore, TSH-TH signaling regulates clock2/npas2 via the thyroid response element (TRE) in its promoter, and transcriptome analysis reveals extensive functions of Tshba in zebrafish. Together, our results demonstrate that zebrafish tshba is a direct target of the circadian clock and in turn plays critical roles in circadian regulation along with other functions.

PSAT253 Differentiation of Pituitary Hyperplasia From Adenoma in the Setting of Severe Hypothyroidism

Abstract Introduction Pituitary hyperplasia due to long-standing or profound primary hypothyroidism is a rare finding in children. It occurs because of constant stimulation of thyrotrope cells by a thyrotropin-releasing hormone secreted from the hypothalamus due to a lack of negative feedback from thyroid hormones. The clinical presentation and imaging can mimic a pituitary mass. Patients can present with features of hypothyroidism, hyperprolactinemia, and even visual field defects if the hyperplastic pituitary gland compresses the optic chiasm. This case highlights this uncommon finding and discusses subtle clinical signs of hypothyroidism in children. Case Presentation A 10-year-old boy was incidentally found to have a pituitary lesion (15 mm×14 mm) when an MRI of the brain was performed as a healthy volunteer screening procedure. History and physical exam revealed cold intolerance, lack of height growth for 2 years (crossing down from 75th to 25th percentile), and dry skin with the barely palpable thyroid gland. There was no family history of thyroid or auto-immune disease. Evaluation of pituitary hormones showed remarkably high thyroid-stimulating hormone (>500 mcIU/ml) in the setting of undetectable free thyroxine, positive thyroid peroxidase antibody, low insulin-like growth factor-1, mildly elevated prolactin (67.4 mcg/L), and mild macrocytic anemia. Conclusions Differentiation of pituitary hyperplasia from pituitary hormone-secreting or non-secreting adenomas is crucial, as the primary modality of treatment of pituitary hyperplasia secondary to primary hypothyroidism is thyroid hormone replacement and not surgery unless the patient has visual field defects necessitating of decompression of the optic chasm to avoid permanent vision loss. In general, pituitary hyperplasia is described as homogeneously enhancing with the "nipple sign," which is when the pituitary gland has smooth contours, but a central protrusion of a pituitary mass is present. Elevated TRH can also lead to lactotroph hyperplasia, causing increased prolactin. Growth hormone secretion may be reduced by hypothyroidism and/or by damaged somatotroph cells due to space-occupying pituitary hyperplasia. Macrocytic anemia has been reported in patients with hypothyroidism and is thought to be an adaptation to a decreased basal metabolism. Early recognition and treatment of hypothyroidism during childhood is essential for normal growth and development. In children with the prolonged disease, final height may be compromised, and academic performance and psychosocial well-being may decline. Gradual replacement of thyroid hormone to therapeutic doses by slowly escalating doses of levothyroxine may be beneficial to prevent complications of therapy such as pseudotumor cerebri or empty sella due to rapid shrinkage of lactotroph and thyrotrope cells. Previous studies have shown approximately 85% of those with pituitary hyperplasia due to primary hypothyroidism demonstrate decreased pituitary gland size after starting thyroxine replacement and recommend repeating pituitary MRI after 2-4 months. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.

Thyrotropinoma - the case report about one of the rarest type of pituitary adenomas

Pituitary tumours are a significant diagnostic problem in clinical practice. Considering all the rarity of thyrotropic hormone secreting adenoma it is a diagnostic and therapeutic challenge in part due to the lack of the preoperative standards. TSH-oma, besides hyperthyroidism symptoms, might be a cause of other endocrine disorders, most often dysfunction of other tropic axes and may cause neurological symptoms associated with sella expansion. We report a case of a 49-year-old male patient with macroadenoma, overt clinical hyperthyroidism, secondary adrenal insufficiency and hypogonadotropic hypogonadism. The diagnosis of thyrotropinoma was confirmed by blood tests that revealed elevated FT3 and FT4 levels together with high TSH. Thyrotropin-releasing hormone (TRH) test was performed and magnetic resonance imaging (MRI) imaging revealed the presence of macroadenoma. Histopathology examination confirmed the diagnosis of thyrotropic cell pituitary adenoma. The appropriate therapy for thyrotropin pituitary adenoma has not been elaborated in reports of this domain. Preoperatively the patient was prepared by long-acting somatostatin analogue (Sandostatin LAR) injections to reduce tumor size with good therapeutic effect afterwards underwent surgical transsphenoidal treatment successfully. However, specific treatment guidelines for pituitary thyrotropic tumors need to be elaborated more in details.

Multi-omic profiling of pituitary thyrotropic cells and progenitors

BackgroundThe pituitary gland is a neuroendocrine organ containing diverse cell types specialized in secreting hormones that regulate physiology. Pituitary thyrotropes produce thyroid-stimulating hormone (TSH), a critical factor for growth and maintenance of metabolism. The transcription factors POU1F1 and GATA2 have been implicated in thyrotrope fate, but the transcriptomic and epigenomic landscapes of these neuroendocrine cells have not been characterized. The goal of this work was to discover transcriptional regulatory elements that drive thyrotrope fate.ResultsWe identified the transcription factors and epigenomic changes in chromatin that are associated with differentiation of POU1F1-expressing progenitors into thyrotropes using cell lines that represent an undifferentiated Pou1f1 lineage progenitor (GHF-T1) and a committed thyrotrope line that produces TSH (TαT1). We compared RNA-seq, ATAC-seq, histone modification (H3K27Ac, H3K4Me1, and H3K27Me3), and POU1F1 binding in these cell lines. POU1F1 binding sites are commonly associated with bZIP transcription factor consensus binding sites in GHF-T1 cells and Helix-Turn-Helix (HTH) or basic Helix-Loop-Helix (bHLH) factors in TαT1 cells, suggesting that these classes of transcription factors may recruit or cooperate with POU1F1 binding at unique sites. We validated enhancer function of novel elements we mapped near Cga, Pitx1, Gata2, and Tshb by transfection in TαT1 cells. Finally, we confirmed that an enhancer element near Tshb can drive expression in thyrotropes of transgenic mice, and we demonstrate that GATA2 enhances Tshb expression through this element.ConclusionThese results extend the ENCODE multi-omic profiling approach to the pituitary gland, which should be valuable for understanding pituitary development and disease pathogenesis.Graphical abstract

Control of the Anterior Pituitary Cell Lineage Regulator POU1F1 by the Stem Cell Determinant Musashi.

The adipokine leptin regulates energy homeostasis through ubiquitously expressed leptin receptors. Leptin has a number of major signaling targets in the brain, including cells of the anterior pituitary (AP). We have previously reported that mice lacking leptin receptors in AP somatotropes display growth hormone (GH) deficiency, metabolic dysfunction, and adult-onset obesity. Among other targets, leptin signaling promotes increased levels of the pituitary transcription factor POU1F1, which in turn regulates the specification of somatotrope, lactotrope, and thyrotrope cell lineages within the AP. Leptin's mechanism of action on somatotropes is sex dependent, with females demonstrating posttranscriptional control of Pou1f1 messenger RNA (mRNA) translation. Here, we report that the stem cell marker and mRNA translational control protein, Musashi1, exerts repression of the Pou1f1 mRNA. In female somatotropes, Msi1 mRNA and protein levels are increased in the mouse model that lacks leptin signaling (Gh-CRE Lepr-null), coincident with lack of POU1f1 protein, despite normal levels of Pou1f1 mRNA. Single-cell RNA sequencing of pituitary cells from control female animals indicates that both Msi1 and Pou1f1 mRNAs are expressed in Gh-expressing somatotropes, and immunocytochemistry confirms that Musashi1 protein is present in the somatotrope cell population. We demonstrate that Musashi interacts directly with the Pou1f1 mRNA 3' untranslated region and exerts translational repression of a Pou1f1 mRNA translation reporter in a leptin-sensitive manner. Musashi immunoprecipitation from whole pituitary reveals coassociated Pou1f1 mRNA. These findings suggest a mechanism in which leptin stimulation is required to reverse Musashi-mediated Pou1f1 mRNA translational control to coordinate AP somatotrope function with metabolic status.

Somatostatin analogues in the therapy of neuroendocrine tumors: Indications, contraindications, side-effects

The presence of somatostatin receptors (SSTRs) is crucial in planning the therapy of patients with neuroendocrine tumors. This applies especially to patients in whom surgery has proven unsuccessful or there are contraindications for it. Increased SSTR expression has been observed in many cancers originating in the neuroendocrine system. Among them we distinguish anterior pituitary adenomas producing GH in excess and leading to the development of acromegaly, adenocorticotropic adenomas that autonomously synthesize ACTH, which leads to the development of ACTH-dependent Cushing’s syndrome (Cushing’s disease), as well as adenomas of the anterior pituitary from thyrotropic cells. Rich expression of these receptors has been confirmed in epithelial tumors of neuroendocrine origin in the gastrointestinal tract, pancreas and lungs. Somatostatin analogues, also called somatostatin receptor ligands, are effective in symptomatic therapy; they enable disease control, exhibit anti-proliferative effects and allow hormonal balance, which reduces mortality among patients and improves their quality of life. The antitumor effect of somatostatin analogues has been proven in in vitro and in vivo studies. In therapy they are usually well tolerated and safe. For many years, somatostatin analogues have maintained an important place in the treatment of neuroendocrine tumors and are still the subject of many studies. The aim of the study is to analyze, based on available literature, therapeutic indications for the use of somatostatin analogues, taking into account contraindications for therapy and its possible side effects.

SUN-LB130 Identification of IGSF1 Ligands

Immunoglobulin superfamily, member 1 (IGSF1), is an X-linked, type 1 transmembrane glycoprotein that is highly expressed in the anterior pituitary gland and testes. Mutations in the IGSF1 gene cause congenital central hypothyroidism, variable hypoprolactinemia, growth hormone dysregulation, and macroorchidism. Igsf1 knockout mice exhibit reduced pituitary TRH receptor (Trhr1) expression with an associated impairment in TRH-stimulated TSH secretion. The mechanism through which IGSF1 loss leads to reductions in Trhr1 levels is unresolved, at least in part because IGSF1’s cellular functions are unknown. The mature form of the IGSF1 protein consists of seven extracellular Ig loops, a single transmembrane domain containing a positively charged arginine, and a short intracellular carboxy-tail devoid of known functional motifs. Recently, IGSF1 was argued to be a member of the leukocyte receptor cluster (LRC) family. LRC proteins act as cell surface receptors for soluble or membrane-bound proteins. We therefore hypothesized that IGSF1 is a cell surface receptor for a presently undescribed ligand that regulates Trhr1 expression in pituitary thyrotrope cells. To identify candidate IGSF1 ligands, we implemented a new ligand trapping method, Ecto-Fc MS. We fused the extracellular (Ecto) domain of IGSF1 to the fragment crystallizable (Fc) region of human IgG, creating an Ecto-Fc fusion protein. Secreted IGSF1-Fc was purified and used as a ligand trap for bait proteins extracted from rat testes. The protein complexes were affinity purified with protein A beads, trypsin digested into peptides, subjected to orthogonal high-pH fractionation, and identified by tandem LC-MS/MS. More than 700 proteins were enriched in IGSF1-Fc preparations compared to an Fc-only negative control. Several secreted ligands and plasma-membrane proteins were identified, many of which are also expressed in pituitary thyrotrope cells. Identifying the ligand or ligands will enable us to determine IGSF1 function, and may lead to the discovery of novel causes of central hypothyroidism and macroorchidism.

Pituitary Hyperplasia, Hormonal Changes and Prolactinoma Development in Males Exposed to Estrogens-An Insight From Translational Studies.

Estrogen signaling plays an important role in pituitary development and function. In sensitive rat or mice strains of both sexes, estrogen treatments promote lactotropic cell proliferation and induce the formation of pituitary adenomas (dominantly prolactin or growth-hormone-secreting ones). In male patients receiving estrogen, treatment does not necessarily result in pituitary hyperplasia, hyperprolactinemia or adenoma development. In this review, we comprehensively analyze the mechanisms of estrogen action upon their application in male animal models comparing it with available data in human subjects. Sex-specific molecular targets of estrogen action in lactotropic (PRL) cells are highlighted in the context of their proliferative and secretory activity. In addition, putative effects of estradiol on the cellular/tumor microenvironment and the contribution of postnatal pituitary progenitor/stem cells and transdifferentiation processes to prolactinoma development have been analyzed. Finally, estrogen-induced morphological and hormone-secreting changes in pituitary thyrotropic (TSH) and adrenocorticotropic (ACTH) cells are discussed, as well as the putative role of the thyroid and/or glucocorticoid hormones in prolactinoma development, based on the current scarce literature.

Цитокариометрические и электронно-микроскопические исследования тиреотропов и гонадотропов в аденогипофизе северных морских котиков в постнатальном онтогенезе

Цитокариометрические и электронно-микроскопические исследования тиреотропов и гонадотропов в аденогипофизе северных морских котиков в постнатальном онтогенезе

A mechanistic base for alternative splicing of human immune system-derived thyroid stimulating hormone (TSH) β-subunit.

Abstract Thyroid stimulating hormone (TSH) is a glycoprotein hormone consisting of an α and a β chain. Within the hypothalamus-pituitary-thyroid axis, TSH is produced by thyrotropic cells in the anterior pituitary. Upon release into the circulation, TSH binds to receptors in the thyroid, resulting in the release of triiodothyronine (T3) and thyroxine (T4). T3 and T4 regulate nearly every aspect of mammalian physiology, including basal metabolism, growth and development, mood, and cognition. Although TSHβ has been known for years to be produced by cells of the immune system, the functional significance of that has remained unclear. My laboratory identified a splice variant of human TSHβ (TSHβv) that consists of a truncated but biologically active portion of TSHβ made from exon 3 with the deletion of exon 2. The signal peptide of TSHβv is coded for by a portion of the 3′ end of intron 2. TSHβv is primarily produced by bone marrow cells and peripheral blood leukocytes, particularly though not exclusively by cells of the myeloid/monocyte lineage. An unresolved question pertains to the molecular mechanisms that account for TSHβv alternative splicing, Here, a model is presented whereby TSHβv is made through a series of unique genetic reorganization steps. These findings have implications not only for understanding how the TSHβv isoform is made, but also for gaining insights into the involvement of immune system TSHβ in health and disease.

From Consternation to Revelation: Discovery of a Role for IGSF1 in Pituitary Control of Thyroid Function.

Immunoglobulin superfamily, member 1 (IGSF1) is a transmembrane glycoprotein highly expressed in the mammalian pituitary gland. Shortly after its discovery in 1998, the protein was proposed to function as a coreceptor for inhibins (and was even temporarily renamed inhibin binding protein). However, subsequent investigations, both in vitro and in vivo, failed to support a role for IGSF1 in inhibin action. Research on IGSF1 nearly ground to a halt until 2011, when next-generation sequencing identified mutations in the X-linked IGSF1 gene in boys and men with congenital central hypothyroidism. IGSF1 was localized to thyrotrope cells, implicating the protein in pituitary control of the thyroid. Investigations in two Igsf1 knockout mouse models converged to show that IGSF1 deficiency leads to reduced expression of the receptor for thyrotropin-releasing hormone (TRH) and impaired TRH stimulation of thyrotropin secretion, providing a candidate mechanism for the central hypothyroidism observed in patients. Nevertheless, the normal functions of IGSF1 in thyrotropes and other cells remain unresolved. Moreover, IGSF1 mutations are also commonly associated with other clinical phenotypes, including prolactin and growth hormone dysregulation, and macroorchidism. How the loss of IGSF1 produces these characteristics is unknown. Although early studies of IGSF1 ran into roadblocks and blind alleys, armed with the results of detailed clinical investigations, powerful mouse models, and new reagents, the field is now poised to discover IGSF1’s function in endocrine tissues, including the pituitary and testes.

Light Microscopic Evaluation of Acute and Chronic Hypophyseal Endocrinopathy in a Kaolin-Induced Hydrocephalus Model.

To demonstrate progression of acute and chronic endocrinopathies in a kaolin-induced hydrocephalus model using light microscopy. Adult male Sprague-Dawley rats (n = 48) were divided into six groups. Hydrocephalus was induced by intracisternal injection of kaolin solution in the acute and chronic kaolin groups, whereas an identical volume of sterile saline was injected into the sham groups. Somatotropic cell concentrations were lower in the kaolin groups compared with their controls, but there was no difference in somatotropic cell concentration between the acute and chronic kaolin groups. Corticotropic cell concentrations were higher in the acute kaolin and sham groups compared with acute controls. Thyrotropic cell numbers were higher in the acute sham and kaolin groups compared with their controls, and although thyrotropic cell concentations were higher in the acute kaolin group than the acute sham group. No differences were observed between the acute and chronic controls and sham and kaolin groups regarding mammotropicand gonadototropic cell concentations. Somatotropic cells are most affected by hydrocephalus that causes pituitary dysfunction, and this effect was more prominent under acute and chronic phases.

Analysis of influencing factor of newly diagnosed thyroid associated ophthalmopathy after radioiodine treatment of Graves disease in short term

Objective To analyze the relevant factor of newly diagnosed thyroid associated ophthalmopathy (TAO) after radioiodine treatment of Graves disease(GD) in short term. Methods A total of 19 patients with GD and TAO in three months after radioiodine treatment were selected as observation group, 64 patients with GD treated by radioiodine in the same period were selected as control group.The thyrotrophic receptor antibody(TRAb), thyroglobulin antibody (TGAb), thyroid peroxidase antibody(TPOAb), tumor necrosis factor alpha (TNF-α) and the changes of thyroid function before and after treatment were compared. Results The base line levels of TPOAb, TRAb and TNF-α had no statistically significant differences between the observation group and the control group, but the ascensional range became bigger after treatment(TPOAb: t=-5.632, P=0.000; TRAb: t=-3.284, P=0.002; TNF-α: t=-3.907, P=0.000). One month later, FT3 level increased in the observation group after treatment, but that had no significant change in the control group (t=9.475, P=0.000). After 3 months, the falling range of thyroid function had no statistically significant difference between the two groups, which suggested that the TAO was not related to hypothyroidism.The thyroid function, thyroid weight (t=0.211, P=0.833), rate of iodine intake (t=1.235, P=0.221), gender (χ2=1.554, P=0.213), age (t=0.154, P=0.878), course of disease before radioiodine treatment (Z=0.761, P=0.447) were irrelevant to the occurrence of TAO. Conclusion The rise of TRAb, TPOAb, TNF-α and thyroid function are related to TAO after radioiodine treatment.Identifying the risk factors to predispose patients with GD to develop TAO can dramatically alter the management of these patients. Key words: Graves′ disease; Thyroid Associated Ophthalmopathy; 131I treatment

Image analysis for TSH mRNA in situ hybridization in pituitary glands from rats with thyroid follicular cell hypertrophy after treatment with three different test compounds

The goal of this in situ hybridization and image analysis technique is to study the effects of new pharmacological/chemical entities on the thyroid and pituitary gland in rats, reveal the pathogenesis of thyroid follicular cell hypertrophy and to retrospectively exclude the risk of thyroid tumor development in humans.In the present study, we describe the increase of thyroid-stimulating hormone- (TSH-) beta subunit mRNA in the pars distalis of the pituitary gland and the quantitative measurement of TSH mRNA positive cells from rats of three 4-week toxicity studies treated with three different test compounds inducing thyroid follicular cell and hepatocellular hypertrophy in rats. Compared to immunohistochemistry (IHC), in situ hybridization (ISH) for TSH was found to be more sensitive.With this technique we are able to exclude a direct effect of the test compound on the thyroid gland by showing the activation of thyrotrope cells from the pituitary gland and therefore this technique retrospectively enables us to exclude a possible risk for humans at an early stage of drug development. Also in case blood serum samples for evaluation of TSH are not available anymore or hepatocellular hypertrophy is not present (close metabolic relationship between thyroid gland and liver in rodents), the described method allows retrospective investigations on thyroid follicular cell hypertrophy or hyperplasia. This can be of high relevance in human safety assessment for certain drugs in order to exclude a primary effect on the thyroid gland especially when it comes to thyroid neoplasia in rodents as previously described.

TRH Action Is Impaired in Pituitaries of Male IGSF1-Deficient Mice

Loss-of-function mutations in the X-linked immunoglobulin superfamily, member 1 (IGSF1) gene cause central hypothyroidism. IGSF1 is a transmembrane glycoprotein of unknown function expressed in thyrotropin (TSH)-producing thyrotrope cells of the anterior pituitary gland. The protein is cotranslationally cleaved, with only its C-terminal domain (CTD) being trafficked to the plasma membrane. Most intragenic IGSF1 mutations in humans map to the CTD. In this study, we used CRISPR-Cas9 to introduce a loss-of-function mutation into the IGSF1-CTD in mice. The modified allele encodes a truncated protein that fails to traffic to the plasma membrane. Under standard laboratory conditions, Igsf1-deficient males exhibit normal serum TSH levels as well as normal numbers of TSH-expressing thyrotropes. However, pituitary expression of the TSH subunit genes and TSH protein content are reduced, as is expression of the receptor for thyrotropin-releasing hormone (TRH). When challenged with exogenous TRH, Igsf1-deficient males release TSH, but to a significantly lesser extent than do their wild-type littermates. The mice show similarly attenuated TSH secretion when rendered profoundly hypothyroid with a low iodine diet supplemented with propylthiouracil. Collectively, these results indicate that impairments in pituitary TRH receptor expression and/or downstream signaling underlie central hypothyroidism in IGSF1 deficiency syndrome.

Embryonic exposure to excess thyroid hormone causes thyrotrope cell death

Embryonic exposure to excess thyroid hormone causes thyrotrope cell death

Relation between third trimester urinary iodine concentrations and newborn thyrotrophic hormone

Relation between third trimester urinary iodine concentrations and newborn thyrotrophic hormone Mohammad Reza Rezvanfar, Heidar Farahani, Mohammad Rafiei, Elaheh Salehi, Elham Ebrahimi, Rezvan Kazemi-Majd Abstract Background: The aim of the study was to evaluate the third trimester urinary iodine excretion and to assess its relation with newborns thyroid function. Materials and Methods: A total of 208 healthy third trimester pregnant women without previous history of thyroid disease were included in the study. Urinary iodine levels of mothers were measured and neonatal TSH levels were screened on the 3- 5th day following birth. Results: The median urinary iodine level in the mothers was 50μg/L. According to WHO criteria for iodine status:14.9%, 34.1% and 49.6% had severe, moderate and mild iodine deficiency, respectively, and only 1.4%, had the adequate iodine intake . In 6 neonate (2.8%) who their TSH level were between 5- 10 IU/ml, nobody had criteria for congenital hypothyroidism at recall visit (15th day). Our results showed the reverse relation between mean Neonatal TSH levels and mother’s third trimester iodine level (r= -0.19, p=0.006). Conclusion: Although all of mothers included in this study stated that they were using iodized salt, iodine deficiency was frequent . There was also a significant relation between mean neonatal TSH levels and mother’s third trimester iodine urinary level.Keywords: Pregnancy, Iodine status, Newborn, Thyroid function

Embryonic exposure to excess thyroid hormone causes thyrotrope cell death

Central congenital hypothyroidism (CCH) is more prevalent in children born to women with hyperthyroidism during pregnancy, suggesting a role for thyroid hormone (TH) in the development of central thyroid regulation. Using the zebrafish embryo as a model for thyroid axis development, we have characterized the ontogeny of negative feedback regulation of thyrotrope function and examined the effect of excess TH on thyrotrope development. We found that thyroid-stimulating hormone β subunit (tshb) and type 2 deiodinase (dio2) are coexpressed in zebrafish thyrotropes by 48 hours after fertilization and that TH-driven negative feedback regulation of tshb transcription appears in the thyroid axis by 96 hours after fertilization. Negative feedback regulation correlated with increased systemic TH levels from the developing thyroid follicles. We used a transgenic zebrafish that expresses GFP under the control of the tshb promoter to follow thyrotrope fates in vivo. Time-lapse imaging revealed that early exposure to elevated TH leads to thyrotrope cell death. Thyrotrope numbers slowly recovered following the removal of excess TH. These data demonstrate that transient TH exposure profoundly impacts the thyrotrope population during a critical period of pituitary development and may have long-term implications for the functional reserve of thyroid-stimulating hormone (TSH) production and the TSH set point later in life.

The CpG Island in the Murine Foxl2 Proximal Promoter Is Differentially Methylated in Primary and Immortalized Cells

Forkhead box L2 (Foxl2), a member of the forkhead transcription factor family, plays important roles in pituitary follicle-stimulating hormone synthesis and in ovarian maintenance and function. Mutations in the human FOXL2 gene cause eyelid malformations and premature ovarian failure. FOXL2/Foxl2 is expressed in pituitary gonadotrope and thyrotrope cells, the perioptic mesenchyme of the developing eyelid, and ovarian granulosa cells. The mechanisms governing this cell-restricted expression have not been described. We mapped the Foxl2 transcriptional start site in immortalized murine gonadotrope-like cells, LβT2, by 5’ rapid amplification of cDNA ends and then PCR amplified approximately 1 kb of 5’ flanking sequence from murine genomic DNA. When ligated into a reporter plasmid, the proximal promoter conferred luciferase activity in both homologous (LβT2) and, unexpectedly, heterologous (NIH3T3) cells. In silico analyses identified a CpG island in the proximal promoter and 5’ untranslated region, suggesting that Foxl2 transcription might be regulated epigenetically. Indeed, pyrosequencing and quantitative analysis of DNA methylation using real-time PCR revealed Foxl2 proximal promoter hypomethylation in homologous compared to some, though not all, heterologous cell lines. The promoter was also hypomethylated in purified murine gonadotropes. In vitro promoter methylation completely silenced reporter activity in heterologous and homologous cells. Collectively, the data suggest that differential proximal promoter DNA methylation may contribute to cell-specific Foxl2 expression in some cellular contexts. However, gonadotrope-specific expression of the gene cannot be explained by promoter hypomethylation alone.

EFFECTS OF PROLACTIN ON THYROIDECTOMIZED BUFO BUFO LARVAE

SUMMARY Young Bufa bufo larvae (Rossi's stage V to VII), were thyroidectomized by means of a single dose of I131 (20 μC). Starting on the 37th day after radioactive iodine treatment, these larvae were given a daily injection of 0.3 I.U. of pure ovine prolactin (Richter) for 14 days. As compared to untreated larvae, prolactin-treated ones underwent a statistically significant (P < 0.001) higher loss in body weight during their last week of life; they exhibited a less marked degranulation of both the acidophilic cells that are regarded as producing a prolactin-like hormone, and of the thyrotropic cells. The possibility of some direct inhibitory effect being exerted by prolactin on the pituitary cells in correlation with the incomplete development of the hypothalamo-hypophysial axis, due to thyroidectomy, is discussed. In the integument, exogenous prolactin seems to have enhanced the differences brought about by thyroidectomy with respect to normal larvae (i.e. further gland rudiment reduction and more unifo...