Marie-Christine Many

Cloning of two human thyroid cDNAs encoding new members of the NADPH oxidase family

Two cDNAs encoding NADPH oxidases and constituting the thyroid H2O2 generating system have been cloned. The strategy of cloning was based on the functional similarities between H2O2 generation in leukocytes and the thyroid, according to the hypothesis that one of the components of the thyroid system would belong to the gp91Phox/Mox1 gene family and display sequence similarities with gp91Phox. Screening at low stringency with a gp91Phox probe of cDNA libraries from thyroid cells in primary culture yielded two distinct human cDNA clones harboring open reading frames of 1551 (ThOX1) and 1548 amino acids (ThOX2), respectively. The encoded polypeptides display 83% sequence similarity and are clearly related to gp91Phox (53 and 47% similarity). The theoretical molecular mass of 177 kDa is close to the apparent molecular mass of 180 kDa of the native corresponding porcine flavoprotein and the protein(s) detected by Western blot in dog and human thyroid. ThOX1 and ThOX2 display sequence similarities of 53% and 61%, respectively, with a predicted protein ofCaenorhabditis elegans over their entire length. They show along their first 500 amino acids a similarity of 43% with thyroperoxidase. The corresponding genes of ThOX1 and ThOX2 are closely linked on chromosome 15q15.3. The dog mRNA expression is thyroid-specific and up-regulated by agents activating the cAMP pathway as is the synthesis of the polypeptides they are coding for. In human thyroid the positive regulation by cAMP is less pronounced. The proteins ThOX1 and ThOX2 accumulate at the apical membrane of thyrocytes and are co-localized with thyroperoxidase.

Tyrosine sulfation is required for agonist recognition by glycoprotein hormone receptors

The glycoprotein hormone receptors (thyrotrophin receptor, TSHr; luteinizing hormone/chorionic gonadotrophin receptor, LH/CGr; follicle‐stimulating hormone receptor, FSHr) constitute a subfamily of rhodopsin‐like G protein‐coupled receptors (GPCRs) with a long N‐terminal extracellular extension responsible for high‐affinity hormone binding. These ectodomains contain two cysteine clusters flanking nine leucine‐rich repeats (LRR), a motif found in several protein families involved in protein–protein interactions. Similar to the situation described recently in CCR5, we demonstrate here that the TSHr, as it is present at the cell surface, is sulfated on tyrosines in a motif located downstream of the C‐terminal cysteine cluster. Sulfation of one of the two tyrosines in the motif is mandatory for high‐affinity binding of TSH and activation of the receptor. Site‐directed mutagenesis experiments indicate that the motif, which is conserved in all members of the glycoprotein hormone receptor family, seems to play a similar role in the LH/CG and FSH receptors.

Direct toxic effect of iodide in excess on iodine-deficient thyroid glands: epithelial necrosis and inflammation associated with lipofuscin accumulation.

Involution of thyroid hyperplasia (induced by a low iodine diet and a goitrogen, propylthiouracil, PTU) was obtained in mice by administering a high or a moderate dose of iodide (HID or MID, respectively). In HID involuting glands, vasoconstriction was observed after 12 hr whereas necrosis and inflammation were very abundant as early as after 6 hr and maximal after 48 hr. They were not prevented by papaverine by which vasoconstriction was inhibited, but were inhibited by the continuation of PTU by which iodide oxidation and organification were inhibited. Lipofuscin inclusions in thyroid and inflammatory cells were always associated with necrosis. On the contrary, when involution was induced by MID or by HID + triiodothyronine (T3), or by T3 alone, neither necrosis nor inflammation occurred and apoptosis was the only mode of cell deletion. No lipofuscin inclusion occurred. Our results demonstrate that iodide in excess, after being oxidized or organified, is directly toxic for iodine-deficient thyroid cells. The presence of lipofuscin suggests that its toxicity is mediated by lipid peroxidation, a consequence of production of free radicals in excess.

Selenium deficiency and thyroid fibrosis. A key role for macrophages and transforming growth factor beta (TGF-beta).

Free radical damage and fibrosis caused by selenium deficiency are thought to be involved in the pathogenesis of myxoedematous cretinism. So far, no pathway explains the link between selenium deficiency and tissue fibrosis. Pharmacological doses of iodine induce necrosis in iodine-deficient thyroids. Necrosis is much increased if the glands are also selenium-deficient, which then evolve to fibrosis. This rat model was reproduced to explore the role of selenium deficiency in defective tissue repair. At first, proliferation indexes of epithelial cells and fibroblasts were comparable between selenium-deficient and control groups. Then, in selenium-deficient thyroids the inflammatory reaction was more marked being mainly composed of macrophages. The proliferation index of the epithelial cells decreased, while that of the fibroblasts increased. These thyroids evolved to fibrosis. TGF-beta immunostaining was prominent in the macrophages of selenium-deficient rats. Anti TGF-beta antibodies restored the proliferation indexes, and blocked the evolution to fibrosis. In selenium deficiency, an active fibrotic process occurs in the thyroid, in which the inflammatory reaction and an excess of TGF-beta play a key role.

Vitrification preserves proliferation capacity in human spermatogonia

STUDY QUESTION Does vitrification of human immature testicular tissue (ITT) have potential benefits for future fertility preservation? Does vitrification of human ITT have potential benefits in an in vivo murine xenotransplantation model? SUMMARY ANSWER Vitrification is able to maintain proliferation capacity in spermatogonial cells after 6 months of xenografting. WHAT IS KNOWN ALREADY Controlled slow-freezing is the procedure currently applied for ITT cryobanking in clinical practice. Vitrification has been proposed as a promising technique for long-term storage of ITT, with a view to preserving spermatogonial stem cells (SSCs) for future fertility restoration in young boys suffering from cancer. After vitrification of ITT, in vitro survival of SSCs was demonstrated, but their functionality was not evaluated. STUDY DESIGN, SIZE, DURATION Ten ITT pieces issuing from 10 patients aged 2-12 years were used. Fragments of fresh tissue (serving as controls) and fresh, frozen-thawed and vitrified-warmed testicular pieces xenografted to the scrotum of nude mice for 6 months were compared. MATERIALS, SETTING, METHODS Upon graft removal, histological and immunohistochemical analyses were performed to evaluate spermatogonia (SG) (MAGE-A4), intratubular proliferation (Ki67), proliferating SG and Leydig cells (3β-HSD). The entire piece of grafted tissue was assessed in each case. MAIN RESULTS AND THE ROLE OF CHANCE Seminiferous tubules showed good integrity after cryopreservation and xenografting for 6 months in all three groups. Survival of SG and their ability to proliferate was observed by immunohistochemistry in all grafted groups. SG were able to initiate spermatogenesis, but blockage at the pachytene stage was observed. The recovery rate of SG was 3.4 ± 3.8, 4.1 ± 7.3 and 7.3 ± 6.3%, respectively, for fresh, slow-frozen and vitrified-warmed tissue after 6 months of xenografting. LIMITATIONS, REASONS FOR CAUTION The study is limited by the low availability of ITT samples of human origin. The mouse xenotransplantation model needs to be refined to study human spermatogenesis. WIDER IMPLICATIONS OF THE FINDINGS The findings of the present study have potential implications for cryobanking of ITT and fertility preservation. Spermatogonial loss recorded after fresh ITT transplantation indicates that the avascular grafting technique needs to be optimized. There are so far no convincing data justifying modification of current clinical practice for ITT storage with slow-freezing, but this study demonstrates that it is worth pursuing optimization of ITT vitrification as an alternative for preservation of SSCs. STUDY FUNDING/COMPETING INTEREST(S) The present study was supported by a grant from the Fonds National de la Recherche Scientifique de Belgique (grant Télévie N° 7. 4.572.09.F). The authors declare that there is no conflict of interest.

Quantification of cells expressing the thyrotropin receptor in extraocular muscles in thyroid associated orbitopathy.

Background/aim: Thyroid associated orbitopathy (TAO) and Graves’ disease (GD) have an autoimmune pathogenesis, possibly related to the thyrotropin receptor (TSHR). The aim of this study was to determine whether TSHR immunoreactivity is correlated with disease severity or serum TSHR antibody (TRAB) levels. Methods: Orbital tissues from 30 patients with TAO were compared with those of 20 patients with strabismus and four with non-thyroid orbital inflammation. TSHR was detected by immunohistochemistry and TRAB were measured by radioreceptor assay. Results: No TSHR immunoreactivity was detected in the 24 control orbital tissues, whereas in all TAO biopsies elongated fibroblast-like cells, expressing TSHR, were present. These cells were located between the muscle cells, which were separated by oedema in the acute phase but fibrous tissue in the chronic phase of disease. Semi-thin sections showed numerous mast cells present in the chronic phase and in close contact with adipocytes. The number of TSHR immunostained cells was high in early disease, decreased with disease duration, and was positively correlated with TRAB levels at the onset of TAO. Conclusion: TSHR immunoreactivity was demonstrated specifically in TAO orbits which highlights the importance of TRAB early in the pathogenesis.

Recent insights into the cell biology of thyroid angiofollicular units.

In thyrocytes, cell polarity is of crucial importance for proper thyroid function. Many intrinsic mechanisms of self-regulation control how the key players involved in thyroid hormone (TH) biosynthesis interact in apical microvilli, so that hazardous biochemical processes may occur without detriment to the cell. In some pathological conditions, this enzymatic complex is disrupted, with some components abnormally activated into the cytoplasm, which can lead to further morphological and functional breakdown. When iodine intake is altered, autoregulatory mechanisms outside the thyrocytes are activated. They involve adjacent capillaries that, together with thyrocytes, form the angiofollicular units (AFUs) that can be considered as the functional and morphological units of the thyroid. In response to iodine shortage, a rapid expansion of the microvasculature occurs, which, in addition to nutrients and oxygen, optimizes iodide supply. These changes are triggered by angiogenic signals released from thyrocytes via a reactive oxygen species/hypoxia-inducible factor/vascular endothelial growth factor pathway. When intra- and extrathyrocyte autoregulation fails, other forms of adaptation arise, such as euthyroid goiters. From onset, goiters are morphologically and functionally heterogeneous due to the polyclonal nature of the cells, with nodules distributed around areas of quiescent AFUs containing globules of compact thyroglobulin (Tg) and surrounded by a hypotrophic microvasculature. Upon TSH stimulation, quiescent AFUs are activated with Tg globules undergoing fragmentation into soluble Tg, proteins involved in TH biosynthesis being expressed and the local microvascular network extending. Over time and depending on physiological needs, AFUs may undergo repetitive phases of high, moderate, or low cell and tissue activity, which may ultimately culminate in multinodular goiters.

Local Induction of Adiponectin Reduces Lipopolysaccharide-Triggered Skeletal Muscle Damage

Adiponectin (ApN) exhibits metabolic and antiinflammatory properties. This hormone is exclusively secreted by adipocytes under normal conditions. We have shown that ApN was induced in tibialis anterior muscle of mice injected with lipopolysaccharide (LPS) and in C2C12 myotubes cultured with proinflammatory cytokines. We hypothesized that muscle ApN could be a local protective mechanism to counteract excessive inflammatory reaction and oxidative damage. To test this paradigm, we examined whether muscles of ApN-knockout (KO) mice exhibit a higher degree of oxidative stress and apoptosis than wild-type mice when challenged by ip LPS and whether these abnormalities may be corrected by local administration of ApN. Eventually we investigated the effects of ApN in vitro. When compared with wild-type mice, ApN-KO mice exhibited myocyte degenerescence, especially after LPS. Myocytes of ApN-KO mice also displayed much stronger immunolabeling for markers of oxidative stress (peroxiredoxin-3/5 and heme oxygenase-1) as well as for a lipid peroxidation product (hydroxynonenal). Expression of TNF-α, caspase-6, a marker of apoptosis, and nuclear factor-κB was enhanced as well. Eventually muscle electrotransfer of the ApN gene, which did not induce any rise of systemic ApN, corrected all these abnormalities in LPS-injected ApN-KO mice. Likewise, ApN attenuated LPS-induced production of proinflammatory cytokines and activation of nuclear factor-κB in C2C12 cells. Thus, induction of ApN into skeletal muscle in response to an inflammatory aggression appears to be a crucial mechanism to counteract in an autocrine or paracrine fashion excessive inflammatory damage, oxidative stress, and subsequent apoptosis.

Adiponectin and Skeletal Muscle: Pathophysiological Implications in Metabolic Stress

Upregulation of muscular adiponectin could act as a local protective mechanism to counteract cellular damage in obesity by weakening inflammation, oxidative stress, and apoptosis. To test this hypothesis, adiponectin-knockout (KO) and wild-type (WT) mice were fed a Western diet (WD). WT mice under WD conditions displayed 63% higher adiponectin expression in myocytes than those under standard laboratory diet (SLD) conditions (P = 0.011). WD-fed KO mice exhibited approximately threefold larger myocyte degeneration than WT mice (P = 0.003). Even under SLD conditions, myotubes of KO mice displayed already moderate immunolabeling for markers of oxidative stress (peroxiredoxin-3/5) and for a lipid peroxidation product (hydroxynonenal). Expression of tumor necrosis factor-α (TNF-α) and caspase-6, a marker of apoptosis, was also present. After WD challenge, immunoreactivity for these markers was strong in muscle of KO mice, although it was detected to a lesser extent in WT mice. Activation of NF-κB and caspase-6 doubled in myocytes of WD-fed KO mice when compared to WT mice (P < 0.001). Furthermore, muscle electrotransfer of the adiponectin gene prevented these abnormalities in WD-fed KO mice. Finally, gene abrogation of the adiponectin receptor 1 (AdipoR1) by siRNA recapitulated a pro-inflammatory state in C2C12 myotubes. Thus, upregulation of muscular adiponectin may be triggered by obesity and be crucial locally to counteract oxidative stress, inflammation, and apoptosis. These effects operate in an autocrine/paracrine manner via AdipoR1 and down-regulation of NF-κB signaling.

A Coherent Organization of Differentiation Proteins Is Required to Maintain an Appropriate Thyroid Function in the Pendred Thyroid

CONTEXT Pendred syndrome is caused by mutations in the gene coding for pendrin, an apical Cl-/I- exchanger. OBJECTIVE To analyze intrathyroidal compensatory mechanisms when pendrin is lacking, we investigated the thyroid of a patient with Pendred syndrome. The expression of proteins involved in thyroid hormone synthesis, markers of oxidative stress (OS), cell proliferation, apoptosis, and antioxidant enzymes were analyzed. RESULTS Three morphological zones were identified: nearly normal follicles with iodine-rich thyroglobulin in the colloid (zone 1.a), small follicles without iodine-rich thyroglobulin in lumina (zone 1.b), and destroyed follicles (zone 2). In zones 1.a, dual oxidase (Duox) and thyroid peroxidase (TPO) were localized at the apical pole, OS and cell apoptosis were absent, but ClC-5 expression was strongly increased. In zones 1.b, Duox and TPO were aberrantly present and increased in the cytosol and associated with high OS, apoptosis, cell proliferation, and increased expression of peroxiredoxin-5, catalase, and dehalogenase-1 but moderate ClC-5 expression. CONCLUSION In conclusion, the absence of pendrin is accompanied by increased ClC-5 expression that may transiently compensate for apical iodide efflux. In more affected follicles, Duox and TPO are relocated in the cytosol, leading to abnormal intracellular thyroid hormone synthesis, which results in cell destruction presumably because intracellular OS cannot be buffered by antioxidant defenses.