CYTOCHEMICAL LOCALIZATION OF ENDOGENOUS PEROXIDASE IN THYROID FOLLICULAR CELLS

Endogenous peroxidase activity in rat thyroid follicular cells is demonstrated cytochemically. Following perfusion fixation of the thyroid gland, small blocks of tissue are incubated in a medium containing substrate for peroxidase, before being postfixed in osmium tetroxide, and processed for electron microscopy. Peroxidase activity is found in thyroid follicular cells in the following sites: (a) the perinuclear cisternae, (b) the cisternae of the endoplasmic reticulum, (c) the inner few lamellae of the Golgi complex, (d) within vesicles, particularly those found apically, and (e) associated with the external surfaces of the microvilli that project apically from the cell into the colloid. In keeping with the radioautographic evidence of others and the postulated role of thyroid peroxidase in iodination, it is suggested that the microvillous apical cell border is the major site where iodination occurs. However, that apical vesicles also play a role in iodination cannot be excluded. The in vitro effect of cyanide, aminotriazole, and thiourea is also discussed.     

Ultrastructural pathology of the thyroid folliculo-papillary and follicular carcinoma

Twenty cases of thyroid carcinoma (10 follicular and 10 folliculo-papillary) were ultrastructurally studied. In the follicular carcinoma the most striking features were: microfollicular cavities with microvilli from the apical surface of the tumorous cells, intracellular microlumens, swollen mitochondria sometimes containing electrondense bodies and tightly packed filaments. In the solid sheaths light and dark cells were present. Golgi complexes were disposed in small dense cristae. The nuclei were large, round, oval or with a folded appearance. In the folliculo-papillary carcinoma were found nuclei with an irregular shape containing stage I and stage II inclusions, dilated endoplasmic sacks, closely packed, sometimes dystrophic mitochondria, dense bodies or tightly packed parallel filaments and numerous phagolysosomes. The peroxidase activity wa present as black precipitates in the nuclear envelope or around colloid droplets. The acid phosphatase activity was found as unhomogeneous precipitates inside the lysosomes. From this study it could be concluded that the follicular and folliculo-papillary carcinomas have some common ultrastructural features; the ultrastructural and cytoenzymological patterns suggest marked alteration of the synthesis, storage and secretion of thyroid hormones.     

THE ANATOMY OF SECRETION IN THE FOLLICULAR CELLS OF THE THYROID GLAND : II. The Effect of Acute Thyrotrophic Hormone Stimulation on the Secretory Apparatus

This paper reports a study by phase contrast and electron microscopy of changes observed in the thyroid gland of the rat at 1, 2, 12, and 24 hours following an injection of thyrotrophic hormone. Examination by phase contrast microscopy reveals that follicular cells contain numerous colloid droplets 1 and 2 hours after injection. By 12 and 24 hours, the colloid droplets are no longer present, and individual follicles appear to be subdividing into smaller units. The droplets are assumed to contain newly synthesized colloid, and their development was studied by electron microscopy. During the period of active secretion, increase in number of the Golgi vesicles leads to enlargement of this organelle. At its periphery small colloid droplets appear to form from large Golgi vesicles. As they form, their content becomes more adielectronic, and fine dense particles less than 75 A in diameter appear in their matrix. Small- and medium-sized droplets lying in the apical region of the cell contain numerous dense particles scattered in their moderately adielectronic content. Large, mature droplets in the same region have a relatively dielectronic content resembling follicular colloid and no longer contain dense particles. The follicular cells appear to utilize apical pseudopodia to release the content of mature droplets into the follicular lumen. Other droplet-like inclusions occur in follicular cells, but they do not seem to be directly concerned with secretion.     

LOCALIZATION OF ESTERASE AND ACID PHOSPHATASE IN GRANULES AND COLLOID DROPLETS IN RAT THYROID EPITHELIUM

Droplets which stain like colloid occur in the cytoplasm of the thyroid follicular epithelium of the rat following stimulation of the gland by thyroid-stimulating hormone (TSH). The occurrence of droplets was remarkably reduced when the lumen became depleted of colloid. Acid phosphatase and esterase were localized in the thyroid droplets and, in addition, in granules largely around the nucleus. Stimulation by TSH resulted in an increase in the number of droplets containing enzyme. Twenty-four hours after hypophysectomy, enzyme-associated granules were localized at the basal end of the cell and droplets were absent. Intravenous injection of TSH resulted in formation of droplets at the apical end of the cell and migration of enzyme-associated granules toward the apical end of the cell. The droplets were first observed approximately 10 minutes after TSH administration and at this time did not appear to contain enzyme. Within 15 minutes many droplets contained enzyme. The granules were largely localized near the nucleus on its apical side 30 minutes after a dose of 25 milliunits of TSH, but were less well localized following one-tenth this dose. These results indicate that the epithelial cell of the thyroid gland contains preformed hydrolytic enzymes associated with granules (lysosomes). When the gland is stimulated by TSH, droplets are formed from colloid derived from the lumen (phagosomes), and hydrolytic enzymes are transferred from granules to the droplets. The droplets may be intracellular organelles for hydrolysis of colloid and liberation of thyroxine prior to the release of thyroxine into the blood.     

RADIOAUTOGRAPHIC STUDY OF IN VIVO AND IN VITRO INCORPORATION OF FUCOSE-3H INTO THYROGLOBULIN BY RAT THYROID FOLLICULAR CELLS

The incorporation of fucose-³H in rat thyroid follicles was studied by radioautography in the light and electron microscopes to determine the site of fucose incorporation into the carbohydrate side chains of thyroglobulin, and to follow the migration of thyroglobulin once it had been labeled with fucose-³H. Radioautographs were examined quantitatively in vivo at several times after injection of fucose-³H into rats, and in vitro following pulse-labeling of thyroid lobes in medium containing fucose-³H. At 3–5 min following fucose-³H administration in vivo, 85% of the silver grains were localized over the Golgi apparatus of thyroid follicular cells. By 20 min, silver grains appeared over apical vesicles, and by 1 hr over the colloid. At 4 hr, nearly all of the silver grains had migrated out of the cells into the colloid. Analysis of the changes in concentration of label with time showed that radioactivity over the Golgi apparatus increased for about 20 min and then decreased, while that over apical vesicles increased to reach a maximum at 35 min. Later, the concentration of label over the apical vesicles decreased, while that over the colloid increased. Similar results were obtained in vitro. It is concluded that fucose, which is located at the end of some of the carbohydrate side chains, is incorporated into thyroglobulin within the Golgi apparatus of thyroid follicular cells, thereby indicating that some of these side chains are completed there. Furthermore, the kinetic analysis demonstrates that apical vesicles are the secretion granules which transport thyroglobulin from the Golgi apparatus to the apex of the cell and release it into the colloid.     

Localization of thyroglobulin antigenicity in rat thyroid sections using antibodies labled with peroxidase or (125)I-radioiodine

In the hope of localizing thyroglobulin within focullar cells of the thyroid gland, antibodies raised against rat thyroglobulin were labeled with the enzyme horseradish peroxidase or with (125)I-radioiodine. Sections of rat thyroids fixed in glutaraldehyde and embedded in glycol methacrylate or Araldite were placed in contact with the labeled antibodies. The sites of antibody binding were detected by diaminobenzidine staining in the case of peroxidase labeling, and radioautography in the case of 125(I) labeling. Peroxidase labeling revealed that the antibodies were bound by the luminal colloid of the thyroid follicles and, within focullar cells, by colloid droplets, condensing vacuoles, and apical vesicles. (125)I labeling confirmed these findings, and revealed some binding of antibodies within Golgi saccules and rough endoplasmic reticulum. This method provides a visually less distinct distribution than peroxidase labeling, but it allowed ready quantitation of the reactions by counts of silver grains in the radioautographs. The counts revealed that the concentration of label was similar in the luminal colloid of different follicles, but that it varied within the compartments of follicular cells. A moderate concentration was detected in rough endoplasmic reticulum and Golgi saccules, whereas a high concentration was found in condensing vacuoles, apical vesicles, and in the luminal colloid. Varying amounts of label were observed over the different types of colloid droplets, and this was attributed to various degrees of lysosomal degradation of thyroglobulin. It is concluded that the concentration of thyroglobulin antigenicity increases during transport from the ribosomal site of synthesis to the follicular colloid, and then decreases during the digestion of colloid droplets which leads to the release of the thyoid hormone.     

APPEARANCE AND FUNCTION OF ENDOGENOUS PEROXIDASE IN FETAL RAT THYROID

Iodination within the thyroid follicle is intimately associated with a thyroid peroxidase. In order to locate the in vivo site of iodination, the initial cytochemical appearance of this enzyme has been determined in fetal rat thyroid and its presence correlated with the onset of iodinated thyroglobulin synthesis. Peroxidase first appears in follicular cells during the 18th day of gestation. It is seen first in the perinuclear cisternae, the cisternae of the endoplasmic reticulum, and within the inner few Golgi lamellae. These organelles presumably represent sites of peroxidase synthesis. During the 19th and 20th days of gestation, there is a tremendous increase in peroxidase activity. In addition to the stained sites described, there are now many peroxidase-positive apical vesicles in the follicular cells. Newly forming follicles stain most conspicuously for peroxidase, the reaction product being heavily concentrated at the external surfaces of apical microvilli and in the adjacent colloid. Iodinated thyroglobulin becomes biochemically detectable in thyroids during the 19th day of gestation and increases greatly during the 20th day. The parallel rise in peroxidase staining that just precedes, and overlaps, the rise in iodinated thyroglobulin, suggests that apical vesicles and the apical cell membrane are the major sites of iodination within the thyroid follicle.     

Changes in colloid droplets and dense bodies in rat thyroid follicular cells during 24 hours: fine structural and morphometric studies

Two changes in cytoplasmic granules, corresponding to colloid droplets and dense bodies, in rat thyroid follicular cells during 24 hr were analyzed morphometrically in the apical, intermediate, and basal parts of the cells. The volume and surface densities of the cytoplasmic granules and their volumes and surface areas in the apical and intermediate parts varied bimodally over a 24-hr day, being high at 1200 and 0000 hr and low at other times. In apical and intermediate regions, colloid droplets were predominant at 1200 and 0000 hr, whereas the cytoplasmic granules became heterogeneously or homogeneously electron-dense at other times. In the basal part, these morphometric parameters were not synchronized with those in the other two regions, showing lower values. The basal cytoplasmic granules were heterogeneously or homogeneously electron-dense. Small, homogeneously dense granules appeared in both the apical and intermediate parts and in the basal part with a certain time lag. These granules were often in contact with colloid droplets in the lumenal two parts at 1200 and 0000 hr. Their numerical densities were low in the apical part, but high in the intermediate and basal parts. These results suggest that newly formed colloid droplets migrate from the apical through the intermediate to the basal part, changing in appearance as they go. Moreover, it seems likely that small, homogeneously dense granules are a final form of cytoplasmic granule. They may be reused for degradation of colloidal protein.     

The significance of paravacuolar granules of the thyroid. A histologic, cytologic and ultrastructural study

The presence of the so-called "paravacuolar granules" in thyroid follicular cells has been associated with increased metabolic activity of the gland, regressive changes, degeneration, phagocytic activity and benign papillary hyperplasia. During the course of a review of the intraoperative cytologic preparations and corresponding histologic sections from 73 thyroid cases, the presence of granules within follicular cells was noted in 25 cases (18 adenomatous or colloid goiters, 3 follicular adenomas, 2 papillary carcinomas, 1 follicular carcinoma and in thyroid tissue surrounding a follicular adenoma in 1 case). Histochemical and ultrastructural studies showed the granules to consist of lysosomes containing hemosiderin or lipofuscin pigments. These findings indicate that the presence of paravacuolar granules in thyroid cells is a common nonspecific finding that simply reflects: (1) the erythrophagocytic capability of the follicular epithelial cells, which results in the accumulation of iron within lysosomes, and (2) the accumulation of lipofuscin pigments within lysosomes as a result of degradation of endogenous cellular material.     

Immunocytochemical studies on differentiation of the thyroid gland in rabbit fetuses and chick embryos

Summary The morphogenesis of the thyroid gland in rabbit fetuses and chick embryos was investigated using the PAS stain and an immunoperoxidase method with anti-19S-thyroglobulin antiserum. In rabbit fetuses, the reaction for precursor components was firstly detected in the apical portions of follicular cells, arranged in clusters but not yet forming follicles, at 16 days of gestation. Although the first primordial follicles storing colloid droplets were observed on day 18, a drastic increase of follicle formation, the true onset of thyroid function, did not occur until day 22. The colloid in primordial follicles revealed very strong immunoreactivity for 19S-thyroglobulin. The follicles gradually increased in size with age. At 25 days of gestation the cytoplasm of follicular cells was stained densely by slightly diluted 19S-thyroglobulin antiserum, whereas the colloid was stained with highly diluted antiserum; these immunoreactions of follicular cells and colloid were comparable to those of postnatal animals. In chick embryos, significant numbers of primordial follicles were observed throughout the whole thyroid parenchyma at 9 days of incubation. On day 12, the follicles stored more PAS-positive and immunoreactive colloid. At 14 days of incubation follicles with enlarged follicular lumina, having an immunoreactivity similar to mature rollicles, became increasingly common.     

Light and electron microscopic immunocytochemical localization of thyroglobulin in the thyroid gland of the anadromous sea lamprey,Petromyzon marinus L., during its upstream migration

Summary Antibodies made against thyroglobulin (TG) were used in an immunocytochemical study for the light and electron microscopic localization of TG in the thyroid gland of the anadromous sea lamprey, Petromyzon marinus, during its upstream migration. TG was found in the follicular lumen and in some colloid droplets within the follicular cells. Except for an immunoreactive product observed in a small portion of the interstitial connective tissue, the location of TG in the lamprey was similar to that in the thyroid of the rat.Supported by National Research Council of Canada Grant no. A5945 to J.H.Y. We thank Dr. F.W.W. Beamish and Mr. R. Robinson who helped in the capture of the lamprey     

Investigations on the thyroid gland of embryonic larval and adult Ichthyophis glutinosus and Ichthyophis kohtaoensis (Gymnophiona,amphibia)

Summary Different developmental stages of two species of the genus Ichthyophis have been investigated. In the late embryo the follicular cells of the thyroid gland exhibit various degrees of cytodifferentiation. Well differentiated cells show a polar organization and contain numerous granular inclusions, but a colloid-containing lumen is rare. Most cells at this stage contain large lipid inclusions. In young and older larvae the cells contain well-developed rough ER and Golgi systems, numerous mitochondria, and abundant granular and vesicular inclusions. Tentative identifications were made of primary lysosomes, secondary lysosomes, residual bodies, and two types of small apical vesicles—containing resorbed colloid or transporting material into the follicular lumen. In the larvae the number of apical microvilli is relatively high. The thyroid cells of the older larvae seem to contain more granular and vesicular inclusions than those of the younger larvae. In the adult the size of the follicles greatly increases, the height of the epithelium decreases, microvilli become rare, residual bodies are more frequent, and the small primary lysosomes are replaced by larger ones. Colloid droplets have been found only rarely in the cytoplasm of the thyroid cells of adult animals. In the immediate neighbourhood of the follicular epithelium, profiles of nerve fibres were found in all animals. Radioiodide investigations—measurements of conversion ratio and thyroid uptake factor—show, if compared with the results of corresponding studies in other amphibians, only relatively small differences between the larvae on the one hand and larvae and adults on the other. The absolute counts of the thyroid region are lowest in the adult and highest in the older larvae, shortly before metamorphosis. Furthermore our results indicate, on the basis of four animals tested, that in Ichthyophis the activity of the thyroid gland is temperature dependent. The results in Ichthyophis show that the classical stages of metamorphosis, in other amphibians characterized among other things by different levels of thyroid activity, are very indistinct in this animal.We gratefully acknowledge the financial support of the Deutsche Forschungsgemeinschaft (We 380/5; Sto 76/4).     

Inhibition of thyroid secretion by sodium fluoride in vitro

NaF mimicked the activation by thyrotropin of iodide binding to proteins and of glucose C-I oxidation but not the accumulation of intracellular colloid droplets or the stimulation of secretion in dog thyroid slices in vitro. On the contrary, NaF inhibited the two latter thyrotropin effects. The inhibitory action of F⁻ was partially relieved by the addition of glucose to the medium; it was mimicked by sodium oxamate. These data suggest that NaF depresses the endocytosis of colloid and thyroid secretion by inhibiting aerobic glycolysis in the follicular cell. NaF inhibited the activation of colloid droplet accumulation and secretion by N⁶,O^2′-dibutyryl-adenosine 3′,5′-monophosphate (dibutyryl cyclic AMP) and the accumulation of cyclic AMP in thyrotropin-stimulated slices. This suggests an inhibition at the level of both cyclic AMP accumulation and cyclic AMP action. The inhibition by NaF and sodium oxamate of colloid droplet formation and thyroid secretion but not of glucose C-I oxidation in stimulated slices further confirms our conclusion that the latter effect is not merely a consequence of the activation by thyrotropin of colloid endocytosis.     

Response of the thyroid gland of the lizard Podarcis sicula to endothelin-1

Although endothelins were originally discovered as peptides with vasoconstrictor activity, recent studies have indicated a number of endothelin (ET) induced hormonal functions in various tissues. We have studied the interaction of ET-1 with thyroid gland of the lizard Podarcis sicula. The effects of ET-1 administration on the plasma levels of the thyroid hormones (T3 and T4) and TSH were stimulatory. Morphological changes in the thyroid after treatment with ET-1 were also detected: the height of the epithelial cells slightly increased and the apical surface acquired microvilli protruding into the follicular lumen. The colloid filled up the lumen and showed a rich peripheral vacuolation. In conclusion, a modulatory role in the control of the reptilian thyroid gland is suggested for ET-1. This is the first report on the interaction of ET-1 with the thyroid gland of reptilian.     

Pigmentation and dysfunction of Gunn rat thyroid

The thyroid gland of homozygous Gunn rats is moderately enlarged and displays a brownish-black discoloration. Light microscopic examination discloses that the follicular cells are filled with brown granules, which are shown, under the electron microscope, to be modified colloid droplets. Most of them possess a strong acid phosphatase and a mild peroxidase activity and contain a melanin-like pigment, according to histochemical analysis. In comparison with normal Wistar rats, Gunn rats possess significantly higher plasma thyroxine and lower triiodothyronine as well as an increased plasma TSH level. The soluble protein content of the thyroid is reduced in the Gunn rat, as is the total intrathyroid iodine content. The hyperthyroxinaemia of homozygous Gunn rats is due to a hereditary deficiency in hepatic glucuronyl transferase activity. The excess circulating thyroxine is of little functional importance because it is firmly bound to plasma proteins. But Gunn rats have a slight hypothyroid goitre for reasons not yet elucidated. The functional as well as morphological data at present available suggest a modified thyroid iodine metabolism and an altered composition of the thyroglobulin which may induce abnormalities in colloid proteolysis. The observed pigment may result from peroxidation of tyrosine. These alterations are probably independent of the sole enzymatic deficiency so far encountered in these animals and may probably be ascribed to a primary enzymatic defect in the thyroid gland itself.     

Immunohistochemical localization of NPY, VIP and 5-HT in the thyroid gland of the lizard, Podarcis sicula

The thyroid gland of the lizard Podarcis sicula was immunohistochemically studied in adult male specimens using specific antibodies against NPY, VIP and 5-HT and the avidin-biotin peroxidase complex (ABC) procedure to localize the three peptides. Fine beaded VIP-immunoreactive nerve fibers ran between the follicles, and VIP-immunoreactivity was evenly distributed in the apical cytoplasm of follicular cells. NPY-immunoreactive fibers were found around the follicles, and, in the cells, immunoreactivity was localizated only in the cellular apices. Immunoreactivity to 5-HT was observed in the colloid, with a concentration in the follicular lumen exceeding that in the follicular cells. In fact, most follicles showed immunoreactivity in the cytoplasmic bridges formed between the apical portion of the follicular cells and the colloid.     

A BIOCHEMICAL AND RADIOAUTOGRAPHIC ANALYSIS OF PROTEIN SECRETION BY THYROID LOBES INCUBATED IN VITRO

In this study we analyzed several aspects of protein secretion by thyroid follicular cells. The study was carried out on intact thyroid lobes obtained from newborn rats and incubated in vitro. The fate of leucine-³H incorporated into protein within follicular cells of untreated and thyrotropic hormone (TSH)-treated lobes was traced by quantitative electron microscope radioautography. Our findings indicate that protein synthesized by the rough-surfaced endoplasmic reticulum during a pulse exposure to leucine-³H is released relatively slowly by this organelle. Approximately 1 hr after onset of the pulse, a peak of radioactive protein appears in the Golgi region. The significance of this peak is not clear. Newly synthesized secretory protein passes through the apex of follicular cells without being concentrated or temporarily stored there in the form of large secretory droplets. Passage probably takes place via small vesicles which are intermingled among diverse small vesicles at the apex of the cells as well as in the Golgi region. Exposure of the lobes to TSH in the incubation medium for 45 or 90 min does not stimulate incorporation of leucine-³H into protein. Acute stimulation with TSH does, however, modify the movement of secretory protein within the exocrine secretory apparatus of the follicular cell. It accelerates the arrival of the protein at the apex of follicular cells, and it accelerates the release of the protein into the follicular lumen.     

Bimodal variations in subcellular structures of rat thyroid follicular cells during 24 hours: fine structural and morphometric studies

To clarify 24-hr variations in rat thyroid follicular cells under physiological conditions, their subcellular structures were examined at six evenly spaced times during 24 hr by using a morphometric technique. The volume, surface, and numerical densities of subcellular structures varied distinctly over each 24-hr period, with a bimodal pattern. The cellular and nuclear volumes varied also bimodally over 24 hr. A decrease in the surface density of the apical plasmalemma at 1200 and 0000 hr coincided with an increase in volume density of cytoplasmic granules representing colloid droplets and dense bodies. Most granules (colloid droplets) appearing at these times were reduced in electron density. At other times, especially at 1600 and 0400 hr, morphometric parameters of rough endoplasmic reticulum (rER), Golgi complex, and subapical vesicles were prominently increased, although values for rER did not peak at 1600 hr. At these times, the volume densities of cytoplasmic granules, most of which were heterogeneous and of homogeneous electron density, were decreased. These findings coincided with immediate and subsequent reactions of follicular cells after injection of thyroid-stimulating hormone (TSH). From the evidence, it seems likely that variations in follicular cells over a 24-hr period reflect variations in blood TSH concentration. The total membrane areas of membrane components in follicular cells were calculated from the morphometric measurements. These areas fluctuated unimodally during 24 hr over a 65% range. This suggests that the membranes in follicular cells are subjected to cyclic degradation and regeneration during each 24-hr period.     

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.