Release of an endothelial cell growth factor from cultured porcine thyroid follicles

It has been proposed from in vivo studies that thyroid angiogenesis during thyroid enlargement may be due to paracrine mitogenic factors released by epithelial thyroid cells. To study this paracrine growth regulating communication between thyroid cells and endothelial cells in vitro, culture medium from isolated porcine thyroid follicles was investigated for a growth promoting effect on porcine aortal endothelial cells. Serum-free conditioned medium (CM) from thyroid follicles in suspension culture contains a dose-related mitogenic activity which stimulates endothelial cell growth up to 197%. Stimulation of the thyroid follicles with TSH (1 mU/ml) significantly reduced the mitogenic activity for endothelial cells in CM to 131%. Thyroid hormones had no influence on mitogenic activity in CM. When follicles were treated with iodide (20 microM) during CM production, no proliferation of endothelial cells was observed by this CM. In contrast, CM from epidermal growth factor-treated thyroid follicles significantly enhanced the mitogenic activity for endothelial cells up to 235%. The mitogenic activity was precipitable by saturated ammonium sulfate, showed high affinity to heparin by chromatography on heparin-sepharose, and was abolished after treatment of CM with trypsin. On gel electrophoresis the heparin-binding fraction showed a double band with a mol wt of 15 and 15.5 k. These data show a paracrine mitogenic activity on endothelial cells released by thyroid follicles which is regulated by TSH, epidermal growth factor, and iodide in parallel with the direct effect of these substances on thyroid cell growth. The data suggest that the mitogenic factor is a polypeptide, which belongs to the heparin-binding growth factors.     

Relation of structural polarity to responses of cyclic 3', 5'-adenosine monophosphate accumulation and thyroid hormone release in cultured human thyroid follicles

The relationship of structural polarity to functional activities was examined in cultured human thyroid follicles, which were isolated from the thyroid gland of patients with Graves' disease by collagenase treatment. Structural polarity was examined morphologically by electron microscopy, while the functional response to bovine TSH was examined by measuring intracellular cAMP accumulation and T3 release. In freshly isolated thyroid follicles, structural polarity was normal and TSH induced significant cAMP accumulation but no significant release of T3. After culture for 5 days the structural polarity of thyroid follicles became inverted in the absence of thyroid stimulators, but normal polarity was retained in the presence of TSH or dibutyryl cAMP [Bu)2 cAMP). The response to TSH of cAMP accumulation increased markedly after culture in either the presence or absence of TSH, suggesting that cAMP accumulation in response to TSH is not related to structural polarity. In contrast, thyroid follicles cultured without thyroid stimulators showed no significant T3 release in response to TSH, whereas those cultured with TSH or (Bu)2 cAMP showed significant T3 release in response to TSH. These data indicate that in cultured human thyroid follicles, the responses to TSH of cAMP accumulation and T3 release are not always correlated. Among many other explanations, the results were at least compatible with the idea that normal structural polarity is necessary for thyroid hormone release in response to TSH.     

Microscopic distribution of iodine radioisotopes in the thyroid of the iodine deficient new-born rat: insight concerning the Chernobyl accident.

Thyroid cancer markedly increased in children exposed to iodine radioisotopes following the Chernobyl accident. This increase exceeded predictions based on dose estimates to the whole organ. We sought to investigate whether iodine deficiency may have influenced the pattern of microscopic distribution of radioiodines, which may be important to interpretation of the observed effects. Iodine-deficient new-born rats were injected with iodine-129 (129I) and the microscopic distribution in the thyroid tissue was studied at 24 hr and at one week after administration, using secondary ion mass spectrometry (SIMS). Twenty-four hr after administration, SIMS images showed large differences in 129I uptake among thyroid follicles, with more than a factor ten variation in the local concentration. In addition, the distribution of 129I inside follicles varied with time. At 24 hr, the highest concentration was found at the periphery of the colloid, close to the thyroid cells. There also was enhanced concentration of 129I at one pole of follicles. Distribution inside follicles was homogeneous at 7 days. Conclusions: 1/Dosimetric models, which assume uniform iodine uptake by thyroid follicles, give an oversimplified picture of radiation dosimetry in cases involving iodine deficiency, which induces patchy tissue irradiation. 2/The dynamic pattern of iodine distribution within thyroid follicles suggests that decay events from short-lived iodines will occur closer to thyroid cells than events resulting from iodine-131.     

Difference between the thyroid gland of normal and hypomyelinated jimpy mice--a light microscopic study

A light microscopic quantitative analysis was performed on normal and jimpy male mice for studying the difference between the structures of the thyroid glands of the two animals. The results of this analysis showed that the thyroid gland of the normal mice consisted of numerous homogenous round follicles with cuboidal follicular cells, separated by thin interlobular and interfollicular connective tissue and a few adipose tissue. The thyroid gland of jimpy mice consisted of a few, small follicles surrounded by columnar follicular cells and intraepithelial capillaries, separated by thick connective tissue and abundant adipose tissue. The number of thyroid follicles are significantly less in the jimpy mice than in the normal mice. Another striking difference is that almost every follicular cell surrounding the follicular lumen of jimpy mice is accompanied by an intraepithelial capillary. In addition, the ratio of the number of intraepithelial capillaries to the number of the thyroid follicular cells are significantly higher in the jimpy mice than in the normal mice. The S-follicles or ultimobranchial cysts of the thyroid gland are well developed in the jimpy mice. The parafollicular cells are normal in appearance. Morphological evidence suggested that the thyroid follicular cells of the jimpy mice are very active in the transport, synthesis and release of thyroglobulin, and secretion of thyroid hormones. But owing to the significantly decreased number of thyroid follicles, the inadequate secretion of the thyroid hormones result in the hypothyroidism and the hypomyelination of the jimpy mice.     

Studies on the development of endocrine glands in fishes. V. Development of the thyroid gland in two species of teleosts.

In Channa punctatus the thyroid anlage is seen 40 h after hatching and is recognized as cells aggregating in the form of follicles with no colloid. During subsequent stages of development the thyroid follicles increase in number and in 5 days old larva an active division of cells and many follicles in the form of epithelial cords are seen. In 2 months old juvenile fish several thyroid follicles and lymphatic vessels are seen enclosed by connective tissue capsule. In Clarias batrachus the thyroid anlage is seen as a group of cells around tiny colloid droplets and is situated just below the bifuration of the 1st branchial artery. This stage is seen in 60 h old larva and is developed into thyroid follicles which is a long chain along the jugular vein and ventral aorta during latter stages of development. It is concluded that during early larval period of these fishes, the thyroid cells begin to trap iodine from the blood and synthesize it in the form of thyroid hormone which is stored as colloid and is also liberated in blood stream simultaneously. The compact nature of the thyroid gland in C. punctatus is because of development of connective sheath around follicles during early stages.     

Evidence of the occurrence of calcitonin cells in the ultimobranchial follicle of the rat postnatal thyroid.

A study on thyroid glands of Wistar rats of ages ranging from 1 to 120 days was carried out. The glands were serially sectioned and stained for calcitonin using the peroxidase antiperoxidase method. All the thyroids contained ultimobranchial follicles (UBF) located partially embedded among the usual follicles but in a 5-day-old rat this structure showed an unusual position in the interstitium of connective tissue between the cartilage of the trachea and the thyroid gland. We have observed in the wall of that UBF the presence not only of resting C cells but also mitotic figures of C cells. Furthermore, on the opposite side of the same UBF an active area of formation of thyroid follicles was found. These observations provided the first evidence of the contribution of the UBF in the formation of C cells during the postnatal life of the rat. Furthermore, it is suggested that some C cells may share a common origin with ultimobranchially derived follicular cells.     

Activities of oxidative enzymes in thyroid follicles of Xiphophorin fishes.

The activity of some intracellular oxidative enzymes was studied histochemically in the cells of the thyroid follicles of teleost fishes of the genus Xiphophorus. The experimental material consisted of animals of the red swordtail and Mexican swordtail breeds of Xiphophorus helleri and of melanotic Xiphophorus maculatus fishes. Observations were carried out on adult specimens of both sexes, including pregnant femals of Mexican swordtail. Moreover, immature Mexican swordtails of both sexes were examined. Thyroid follicles were found to be present in the subpharyngeal region of all fishes studied. The distribution of these follicles as well as their number and form depended on sex, age and on the analysed stage of prenancy. A smaller number and size of thyroid follicles were characteristic of immature specimens, whereas they were most numerous in the thyroids of pregnant fishes. The follicles were arranged in characteristic dense aggregations, especially in the melanotic platyfish. The follicular eipthelium in the fishes under study was usually cubical, but pregnant and non-pregnant adult females also contained a considerable number of larger follicles with flattened epithelium. Besides, thyroid follicles of multilayer epithelium were rather frequently encountered, especially in male fishes, irrespective of their age. The thyroid follicle cells of these fishes demonstrated invariably high activities of reduced NAD and NADP dehydrogenases and of beta-hydroxybutyrate dehydrogenase, and a low activity of succinat dehydrogenase. The intensities of alpha-glycerophosphate and lactate dehydrogenases and of cytochrome oxidase varied with sex, age and breed of the studied fishes. The immature and pregnant fishes showed the most clearly pronounced differences in the intensity of enzymic activity, the thyroid follicles of immature specimens revealing a high activity of lactate dehydrogenase and low activity of cytochrome oxidase, an inverse picture being seen in pregnant fishes. The adult forms of both sexes exhibited an enhanced activity of cytochrome oxidase and a decline in that of lactate dehydrogenase. The observed differences in the intensities of enzymic acitivities in the thyroids of the studied fishes are related with functions of this gland which in the period of growth are different from those in the period of sexual maturity, and certainly also with individual metabolic characteristics of the studied fishes.     

Functional properties of normal and inverted rat thyroid follicles in suspension culture

Rat thyroid follicles in both the normal and the inverted configuration have been cultured in suspension. It has been found that normal follicles (i.e., those having a structural organization similar to the follicles in vivo) express most functional properties of the thyroid gland. They trap iodide, synthesize, secrete, and iodinate thyroglobulin, and secrete thyroid hormone into the culture medium. On the contrary, in inverted follicles (i.e., those having a cell polarity reversed with respect to normal) we were unable to detect expression of some of these functions. We conclude that both the follicular organization and the proper cell polarity are needed for the full expression of thyroid functional properties.     

Morphological and histochemical changes of ultimobranchial follicles of the rat thyroid in the course of postnatal life.

Morphological and histochemical changes of ultimobranchial follicles of thyroid have been investigated in rats from newborn to 18 months of age. The first well-delimited ultimobranchial follicles, though with no lumen, were detected in the thyroid gland of 10-day-old rats. At 30 days of age, follicles possessing regular lumina were present in the thyroid. With age, the follicles gradually increased in volume assuming extreme dimensions in adult age. The follicles displayed varying shapes from simple cysts to bizarre forms. From the age of 50 days the cells of the follicular wall are separated from the cell debris contained in the lumen. The latter gave a PAS positive reaction. The cells of the ultimobranchial follicles did not exhibit argyrophilia and metachromasia showing that they differ considerably from the C-cells likewise of ultimobranchial origin, which are known to give marked argyrophilic and metachromatic reactions.     

C-cell follicles of canine thyroid glands studied by PAS reaction and electron microscopy

Summary Small follicles composed solely of C cells were occasionally observed in large C cell groups of dog thyroid glands. The lumina of C-cell follicles were filled with, or contained peripheral depositions of PAS-positive amorphous material, which was similar in ultrastructural features to thyroglobulin-containing colloid in typical thyroid follicles. This indicates that C cells, in addition to secreting calcitonin, produce a glycoprotein that can be stored in the lumina of the follicles.     

Calpain inhibitors regulate the conversion of thyroid gland follicles in rats and humans

Effects of calpain inhibitors on thyroid follicle conversion into monolayer was investigated. Human and rat primary thyroid cultures require the follicular structure after enzyme disaggregation of native tissue fragments. Removal of thyroid-stimulating hormone (TSH) from the culture medium causes migration of thyrocytes from follicles into monolayer, some differences were noted in conversion of rat and human thyroid follicles. The locomotion of rat thyrocytes is observed after preliminary incubation with TSH, but migration of thyrocytes from human thyroid follicles does not require such a preincubation. Phorbol esters induce migration of rat and human thyroid cells. Calpain inhibitors exert a significant influence on locomotion of the thyroid gland cells induced by the removal of TSH from the culture medium. Human thyrocyte migration is markedly inhibited by calpain inhibitor I or II. Likewise, addition of calpain inhibitor I into primary culture of rat thyrocytes decreased the number of migrating cells by 52%, and shortened average migration distance by 34%. Also, calpain inhibitors reduced the speed of phorbol ether-induced conversion of rat and human thyroid follicles into monolayer.     

Microvascularization of the thyroid glands in larval and adult Xenopus laevis‐ histomorphology and scanning electron microscopy of vascular corrosion casts

This study demonstrates gross arterial supply, venous drainage and microvascular patterns of larval and adult thyroid glands in the African Clawed Frog, Xenopus laevis by scanning electron microscopy of vascular corrosion casts and light microscopy of stained serial tissue sections. Results confirm published findings gained by microscopical dissections with respect to gross arterial supply. However, in adult frogs one rather than two thyroid veins are found. This study reveals for the first time that bilaterally located thyroid glands in premetamorphosis have immature capillary networks, lack a clear hierarchy of blood vessels, and show many signs of sprouting angiogenesis. During metamorphic climax, blood vessels gain a clear hierarchy and capillaries form closed networks around thyroid follicles. From climax onwards, non‐sprouting angiogenesis (intussusceptive microvascular growth) becomes the prevailing mode of angiogenesis intensifying follicle capillarization. Due to narrow interfollicular spaces, thyroidal arterioles remain superficial while draining venules are located interfollicularly. In contrast with the mammalian thyroid gland where most thyroid follicles have their own capillary beds, most thyroid follicles in Xenopus share their capillary beds with neighbouring follicles. Consequently, the concept of individual morphological and functional angiofollicular units applicable to adult mammalian and human thyroid glands is not applicable to larval and adult amphibian thyroid glands.     

The effects of fasting and refeeding on the thyroid follicles of young and aged mice--morphometric analysis

The population movements of thyroid follicles in young and aged mice subjected to acute fasting and refeeding were analyzed by a morphometric method. After fasting for three days, the size distribution of thyroid follicles from young mice did not change, while the follicles from aged mice became significantly bigger than in the control group. Subsequent refeeding for four days caused a decrement in follicular sizes in both young and aged thyroid. The activity of follicular epithelia was decreased by fasting, but accelerated by refeeding. The equation describing the relationship between epithelial cell numbers and follicle diameters from a young control mouse agreed well with the theoretical one, especially in small follicles. However, the equation for an aged mouse differed from the theoretical one. This suggests that the older the mouse, the greater the deviation of the constituent factor, e.g. that of follicle size or cell number in thyroid. The extent of deviation would be one of the characteristics of aging in multicellular organisms.     

In vitro studies of the thyroglobulin degradation pathway: endocytosis and delivery of thyroglobulin to lysosomes, release of thyroglobulin cleavage products--iodotyrosines and iodothyronines

Iodinated thyroglobulin stored in the thyroid follicular lumen is subjected to an internalization process and thought to be transferred into the lysosomal compartment for proteolytic cleavage and thyroid hormone release. In the present study, we have designed in vitro models to study: 1) the transfer of endocytosed thyroglobulin into lysosomes, and 2) the intracellular fate of free thyroid hormones and iodinated precursors generated by intralysosomal proteolysis of thyroglobulin. Open follicles prepared from pig thyroid tissue by collagenase treatment were used to probe the delivery of exogenous thyroglobulin to lysosomes via the differentiated apical cell membrane. Open follicles were incubated with pure [125I]thyroglobulin with or without unlabeled thyroglobulin in the presence or in the absence of chloroquine. Subcellular fractionation on a Percoll gradient showed that [125I]thyroglobulin was internalized and present in low (for the major part) and high density thyroid vesicles. In chloroquine-treated open follicles, we observed the appearance of a definite fraction of [125I]thyroglobulin in a lysosome subpopulation having the expected properties of phagolysosomes or secondary lysosomes. In contrast, in control open follicles, the amount of [125I]thyroglobulin or degradation products found in high density vesicles was lower and associated with the bulk of lysosomes, i.e., primary lysosomes. The content in thyroglobulin and degradation products of lysosomes at steady-state was analyzed by Western blot using polyclonal anti-pig thyroglobulin antibodies. Under reducing conditions, immunoreactive thyroglobulin species correspond to polypeptides with molecular weights ranging from 130,000 to less than 20,000. The presence of free thyroid hormones and iodotyrosines inside lysosomes and their intracellular fate was studied in dispersed thyroid cells labeled with [125I]iodide. Neo-iodinated [125I]thyroglobulin gave rise to free [125I]T4 which was secreted into the medium. In addition to released [125I]T4, a fraction of free [125I]T4 was identified inside the cells. Lysosomes isolated from dispersed thyroid cells did not contain significant amounts of free [125I]T4. The free intracellular [125I]T4 fraction seems to represent an intermediate 'hormonal pool' between thyroglobulin-bound T4 and secreted T4. Evidence for such a precursor-product relationship was obtained from pulse-chase experiments. In conclusion: 1) open thyroid follicles have the ability to internalize thyroglobulin by a mechanism of limited capacity and to address the endocytosed ligand to lysosomes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Calcitonin cells and unusual follicles of the thyroid of the indian grey mongoose, Herpestes edwardsi (Geoffroy).

Histological preparations of thyroid, parathyroid and thymus glands of Herpestes edwardsi were examined for calcitonin cells. They reveal that (1) the thyroid calcitonin cells are oval, rounded and rarely elongated in shape; these cells and their nuclei are distinctly larger than those of the follicular cells and their nuclei; (2) calcitonin cells are unevenly distributed in the thyroid, with the result that certain portions of the thyroid are completely devoid of these cells; (3) on an average, calcitonin cells are in a ratio of 10-15 cells/100 follicular cells; (4) the parathyroid and thymus glands do not display calcitonin cells, and (5) the thyroid gland displays unusual follicles of two categories, (a) follicles with ciliated epithelial cells and (b) follicles with squamous epithelium.     

Mixed follicles of the human thyroid gland

Mixed follicles are structures composed of squamous-like and follicular epithelia. Little attention has been generally paid to these peculiar follicles of the human thyroid; thus the aim of the present study was to investigate their prevalence and biological properties by means of systematic autopsy, histochemical and immunohistochemical surveys. Mixed follicles were found to be present in 54% and 81% of the patients with solid cell nests, as well as in 50% and 77% of the total number of the ultimobranchial nests, when one or two histological samples from each solid cell nest were examined, respectively. The follicular lumen of mixed follicles usually contained an eosinophilic and PAS-positive colloid-like material, although in 22% of the cases acid mucins sometimes intermixed with PAS-positive granular material and cell debris were also present within lumina. Follicular cells lining mixed follicles basically did not stain positively for calcitonin. The results indicate that mixed follicles are not rarely found in the human thyroid. The presence of intraluminal mucins and cell debris and the absence of calcitonin-containing cells in the follicular epithelium lining these peculiar follicles suggest that at least some thyroid follicular cells could originate from ultimobranchial tissue.     

Autoradiographic studies with 3H 1,25 dihydroxyvitamin D3 in thyroid and associated tissues of the neck region

Rats and mice fed a vitamin D-deficient or vitamin D-complete diet were injected with 3H 1,25 (OH)2 vitamin D3. Autoradiograms prepared from cross sections through the neck region revealed nuclear concentration of radioactivity strongest in parathyroid chief cells, occasionally in thyroid follicular epithelial and interfollicular cells, in the epithelium of tubular remnants of the ultimobranchial body, in epithelium of the esophagus, in chondrocytes of tracheal cartilage, and in myoepithelial cells of tracheal glands. In the thyroid, most of the follicle epithelial cells did not show nuclear concentration of radioactivity which occurred only occasionally and predominantly in follicles located in marginal positions. Thyroglobulin in lumina of thyroid follicles contained varying amounts of radioactivity that correspond to the diameter of the follicles, with relatively high amounts in large follicles and little or no radioactivity in small follicles. Competition with excess of unlabeled 1,25 (OH)2 vitamin D3 abolished nuclear radioactivity, but not the radioactivity in the colloid, while 25 (OH) vitamin D3 did not affect either. When a combination of autoradiography and immunohistochemistry was applied, follicular and parafollicular C-cells positive for calcitonin antibodies, did not show nuclear concentration of radioactivity. Tubular remnants of ultimobranchial bodies, however, showed distinct nuclear labeling, but did not stain, or only weakly stain, with antibodies to calcitonin. When 3H 25 (OH) vitamin D3 was injected, no nuclear concentration of radioactivity was noted in any of the tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of cellular prion protein expression by TSH in human thyrocytes

The cellular isoform of prion protein (PrP(C)) is a cell-surface glycosyl-phosphatidylinositol-anchored protein which is ubiquitously expressed on the cell membrane. It may function as a cell receptor or as a cell adhesion molecule. Thyroid follicles, obtained from patients with Graves' disease at thyroidectomy, were cultured in F-12/RPMI-1640 medium supplemented with 0.5% fetal bovine serum and bovine thyroid stimulating hormone (bTSH). Northern blot analyses revealed that bTSH increased the steady-state expression levels of PrP mRNA in a time- and dose-dependent manner. This increase was reproduced by dibutyryl-cAMP and 12-decanoylphorbol-13-acetate. The mRNA expression was greater in thyroid follicles in suspension culture than in thyrocytes cultured in a monolayer. These findings suggest that TSH stimulates PrP mRNA expression in thyrocytes through the protein kinase A and C pathways. The greater mRNA expression in thyroid follicles than in monolayer cells suggests that PrP(C) may be involved in structure formation or maintenance of thyroid follicles.     

Structural and ultrastructural differentiation of the thyroid gland during embryogenesis in the grass snake Natrix natrix L. (Lepidosauria, Serpentes)

The differentiation of the thyroid primordium of reptilian species is poorly understood. The present study reports on structural and ultrastructural studies of the developing thyroid gland in embryos of the grass snake Natrix natrix L. At the time of oviposition, the thyroid primordium occupied its final position in the embryos. Throughout developmental stages I-IV, the undifferentiated thyroid primordium contained cellular cords, and the plasma membranes of adjacent cells formed junctional complexes. Subsequently, the first follicular lumens started to form. The follicular lumens were of intracellular origin, as in other vertebrate species, but the mechanism of their formation is as yet unclear. At developmental stages V-VI, the thyroid anlage was composed of small follicles with lumens and cellular cords. Cells of the thyroid primordium divided, and follicles were filled with a granular substance. At developmental stage VI, the cells surrounding the follicular lumen were polarized, the apical cytoplasm contained dark granules and the Golgi complex and the rough endoplasmic reticulum (RER) developed gradually. Resorption of the colloid began at developmental stage VIII. At the end of this stage, the embryonic thyroid gland was surrounded by a definitive capsule. During developmental stages IX-X, the follicular cells contained granules and vesicles of different sizes and electron densities and a well-developed Golgi apparatus and RER. At developmental stage XI, most follicles were outlined by squamous epithelial cells and presented wide lumens filled with a light colloid. The Golgi complex and RER showed changes in their morphology indicating a decrease in the activity of the thyroid gland. At developmental stage XII, the activity of the embryonic thyroid gradually increased, and at the time of hatching, it exhibited the features of a fully active gland.