CHANGES IN FINE STRUCTURE AND ACID PHOSPHATASE LOCALIZATION IN RAT THYROID CELLS FOLLOWING THYROTROPIN ADMINISTRATION

Shortly after the administration of 1/40 unit thyrotropin to rats, 24 hours post-hypophysectomy, the following sequence of changes has been observed within thyroid follicular epithelial cells: (1) the appearance of apical cell surface activity consisting of pseudopods projecting into the follicular lumen; (2) apparent phagocytic engulfment of colloid droplets lacking indications of acid phosphatase activity; (3) close association and probable fusion of newly formed colloid droplets and dense granules, the latter cytochemically positive for acid phosphatase activity; (4) the appearance of presumptive acid phosphatase activity within colloid droplets; and, (5) further colloid droplet changes, viz., basipetal migration and decrease in size, accompanied by an increase in density and in demonstrable acid phosphatase activity. These changes appeared to represent the resorption and degradation of follicular colloid. Comparable results were obtained using intact and more heavily stimulated animals. Colloid biosynthesis was tentatively visualized in these cells as a separate mechanism involving small vesicles prominent in the Golgi region and beneath the apical plasma membrane of some, but not all, thyroid follicular cells in each specimen.     

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.     

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.     

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.     

RADIOAUTOGRAPHIC VISUALIZATION OF THE INCORPORATION OF GALACTOSE-3H AND MANNOSE-3H BY RAT THYROIDS IN VITRO IN RELATION TO THE STAGES OF THYROGLOBULIN SYNTHESIS

Rat thyroid lobes incubated with mannose-³H, galactose-³H, or leucine-³H, were studied by radioautography. With leucine-³H and mannose-³H, the grain reaction observed in the light microscope is distributed diffusely over the cells at 5 min, with no reaction over the colloid. Later, the grains are concentrated towards the apex, and colloid reactions begin to appear by 2 hr. With galactose-³H, the reaction at 5 min is again restricted to the cells but it consists of clumped grains next to the nucleus. Soon after, grains are concentrated at the cell apex and colloid reactions appear in some follicles as early as 30 min. Puromycin almost totally inhibits incorporation of leucine-³H and mannose-³H, but has no detectable effect on galactose-³H incorporation during the 1st hr. Quantitation of electron microscope radioautographs shows that mannose-³H label localizes initially in the rough endoplasmic reticulum, and by 1–2 hr much of this reaction is transferred to the Golgi apparatus. At 3 hr and subsequently, significant reactions are present over apical vesicles and colloid, while the Golgi reaction declines. Label associated with galactose-³H localizes initially in the Golgi apparatus and rapidly transfers to the apical vesicles, and then to the colloid. These findings indicate that mannose incorporation into thyroglobulin precursors occurs within the rough endoplasmic reticulum; these precursors then migrate to the Golgi apparatus, where galactose incorporation takes place. The glycoprotein thus formed migrates via the apical vesicles to the colloid.     

Electron microscopic study on the thyroid gland of the salamander Hynobius nebulosus in the breeding season

Summary The thyroid gland of adult salamanders, Hynobius nebulosus, in the breeding season was studied by electron microscopy. The follicular cells are different in cell height and fine structures; the taller cells with many cell organelles and granules and the lower cells with a few cell organelles and granules are both present in the same follicle. In the cytoplasm, three types of membrane-bounded granules, namely, cytosomes, colloid droplets, and vacuolar bodies and circular membrane complexes occur. The vacuolar bodies are subdivided into two types; the ordinary type having loosely distributed particles and the specific type containing tubules and/or closely packed filaments, crystalloid structures, except for the particles. The chromophobe colloids within the Bensley-cells correspond to extremely large, ordinary type vacuolar bodies, while the Langendorff-colloid cells possess increased numbers of granular cisternae of endoplasmic reticulum and a ribosome-rich, dense cytoplasmic matrix but not extremely large colloid. The intracytoplasmic circular membrane complexes appear in the Golgi area of cytosome-rich cells. It is suggested that they originate from the Golgi apparatus which was activated to produce many cytosomes. Intranuclear inclusions consisting of microtubules and filaments and tight junctions between two adjacent lateral plasma membranes are occasionally encountered.     

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.     

Urethral chromaffin cells

Summary The urethral mucosa of the rat, rabbit and guinea-pig was examined with both fluorescence and electron microscopy. Employing the former technique, numerous brightly fluorescing flask-shaped cells were observed amongst the basal cells of the urethral epithelium in all three species. In the electron microscope cells with a similar shape and distribution are distinguished by their content of membrane-limited dense granules, extensive Golgi membranes and bundles of filaments. In favourable planes of section short microvilli extend from the apical region of these cells which are joined to neighbouring urethral epithelial cells by zonulae occludentes. These fluorescent, granule-containing cells are classified as urethral chromaffin cells.Fluorescent nerves were not observed in relation to the urethral epithelium although the electron microscope revealed axons lying singly or in groups both beneath and between the urethral epithelial cells. Many of these axons appear varicose and contain small, agranular vesicles, a few large granulated vesicles and numerous mitochondria. Occasionally a vesicle-containing axon lay adjacent to a urethral chromaffin cell. While a direct autonomic innervation of these cells could not be discounted it is concluded that the majority of nerves probably perform a sensory function.     

ELECTRON MICROSCOPY OF THE HUMAN SYNOVIAL MEMBRANE

The structure of the lining cells at the surface of the synovial membrane facing the joint cavity has been studied by electron microscopy. The long cytoplasmic processes of these cells appear to be oriented toward the surface of the membrane, where they overlap and intertwine. The matrix of the lining cells contains dense material but no fibers with the periodicity of collagen. The lining cells are divided into two cell types or states of activity on the basis of their cytoplasmic contents. Type A is more numerous and contains a prominent Golgi apparatus, numerous vacuoles (0.4 to 1.5 microns in diameter) containing varying amounts of a dense granular material, many filopodia, mitochondria, intracellular fibrils, and micropinocytotic-like vesicles. Type B contains large amounts of ergastoplasm with fewer large vacuoles, micropinocytotic-like vesicles, and mitochondria. The probable functions of these cells are discussed in the light of current knowledge of the metabolism and function of the synovial membrane.     

THE FINE STRUCTURE OF BRUNNER''S GLANDS IN THE MOUSE

Examined with the electron microscope, the secretory cells of the submucosal glands of Brunner in the mouse present a curious combination of the fine-structural features of both serous and mucus-secreting cells. The cells have numerous mitochondria, abundant basal ergastoplasm, dense secretory granules that bear a superficial resemblance to pancreatic zymogen granules, and an unusually extensive Golgi apparatus. The prominence of the lamellar, vesicular, and vacuolar elements of the Golgi complex facilitates detailed observation of these components. More evident than in other glandular cells, aggregates of small vesicles appear to represent the transitional elements and are vehicles for transport of the product between the ergastoplasm and the Golgi complex. The numerous vesicular evaginations of smooth-surfaced regions on cisternae of the rough-surfaced endoplasmic reticulum and accumulations of innumerable vesicles of similar size in the area between the nearest profiles of the ergastoplasm and the Golgi complex support this contention. The cytological characteristics and physiologic properties of Brunner's glands in various species are discussed briefly. It is concluded that the submucosal glands of the mouse are excellent material for exploration of the ultrastructural correlates of both protein and carbohydrate secretion, and it is suggested that their secretion may have functions other than those generally attributed to them, namely, chemical and mechanical protection of the duodenal surface epithelium.     

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.     

The perinuclear space of pancreatic acinar cells and the synthetic pathway of zymogen in Scorpaena scrofa L.: ultrastructural aspects

Electron microscopic examination of exocrine pancreatic tissues from the fish Scorpaena scrofa L., probably captured while replenishing the acinar cells, shows two main functional cell morphologies of the same cell type. One cell functional aspect contains numerous well-contrasted small vesicles, the zymogenic vesicles. The other functional morphology is mainly represented by a few cells containing large apical zymogen vesicles with many empty RER cisterns. In our observations, the zymogenic vesicles are always studded with ribosomes. The main cytological finding is to report that zymogenic vesicles can be extruded from the perinuclear space and it confirms the suspected, synthetic activity of this cell compartment. The pool of zymogenic vesicles, maintaining their coat of ribosomes, then fuses and transfers their content into the cis Golgi complex network. Finally, the zymogen vesicles are produced following the classical secretory pathway from the trans Golgi saccular network into the supranuclear, apical region of the acinar cells where the largest vesicles concentrate their content until secretion.     

The digestive cells of the hepatopancreas in Aplysia depilans (Mollusca, Opisthobranchia): ultrastructural and cytochemical study

Digestive cells are the most abundant cell type in the digestive diverticula of Aplysia depilans. These are tall columnar or club shaped cells, covered with microvilli on their apical surface. A large number of endocytic vesicles containing electron-dense substances can be found in the apical zone, but the presence of many heterolysosomes of large diameter is the main feature of these cells. Glycogen particles and some lipid droplets were also observed. Peroxisomes with a circular or oval profile were common, but crystalline nucleoids were not detected in them, although a dense spot in the matrix was observed in a few cases. These organelles were strongly stained after cytochemical detection of catalase activity. The Golgi stacks are formed by 4 or 5 cisternae, with dilated zones containing electron dense material. Arylsulphatase activity was detected in the Golgi stacks and also in lysosomes. Cells almost entirely occupied by a very large vacuole containing a residual dense mass seem to be digestive cells in advanced stages of maturation. The observation of semithin and ultrathin sections indicates that these very large vacuoles are the result of a fusion among the smaller lysosomes. Some images suggest that the content of these large vacuoles is extruded into the lumen of the digestive diverticula.     

ULTRASTRUCTURAL CHANGES ASSOCIATED WITH UTILIZATION OF METABOLITE RESERVES AND TRICHOME DIFFERENTIATION IN THE PROTOCORM OF VANDA

Certain aspects of protocorm development in Vanda were examined ultrastructurally. The parenchymal cells of the protocorm accumulate substantial quantities of lipid, protein, and carbohydrate reserves which disappear gradually with the senescence of the parenchymatous region. The proteinaceous reserves appear initially as discrete bodies which become intimately associated with clusters of small tubules. The tubules eventually disperse throughout the cytoplasm and disappear following depletion of the protein bodies. The lipid reserves also appear as discrete bodies and are associated with an electron dense, laminated inclusion which appears to increase in size with the disappearance of the lipid bodies. While plastids in the meristematic cells differentiate a well-developed thylakoid system and contain little starch, those of the parenchymal cells contain large starch grains and numerous osmiophilic droplets and develop meager thylakoid systems. Membrane-bound crystalline structures of hexagonal and rhomboid cross section occur frequently in the cytoplasm of senescent parenchyma cells. Trichome initials, which differentiate from the epidermis, contain few conventional organelles and exhibit numerous membrane-bound structures containing many small crystalline inclusions. Numerous vesicles accumulate at the tips of the trichomes in spaces between the cell wall and the plasmalemma.     

On the histology and ultrastructure of the Harderian gland in rabbits

Summary The Harderian gland of rabbits has been studied with light and electron microscopes. The red part contains relatively wide alveoli with an irregular cuboidal epithelium. The stainability of the cytoplasm is poor. The white part has smaller alveoli with a low columnar epithelium. The cells show cytoplasmic basophilia removable with ribonuclease. The cytoplasm of both kinds of cells is very dense when examined in the electron microscope. The mitochondria show branched, closely packed cristae and a dense matrix. The Golgi apparatus displays few lamellae and rows of vacuoles. The endoplasmic reticulum is very finemeshed and partly associated with ribonucleoprotein particles. Both kinds of cells contain numerous lipid droplets, leaving vacuoles in the sections prepared for electron microscopy. They are fewer but distinctly larger in the red part. In both lobes pictures suggesting a secretion of lipid droplets have been observed. Cells showing signs of degeneration with subsequent discharge of the detritus have been observed in both lobes, but this process does not correspond to the holocrine secretion in sebaceous glands. Likewise, no apocrine secretion was observed.     

The ultrastructure of the zymogen cells in Hydra viridis

Summary The zymogenic secretory cells of Hydra viridis are scattered between the digestive muscle cells of the gastric region. The mature zymogenic cells are located along the apical surface of the gastrodermal epithelium and contain numerous spherical secretory droplets. They appear to differentiate from stem cells located near the mesoglea. These stem cells resemble epidermal interstitial cells and are filled with free ribosomes. They differ from the interstitial cell in that they usually possess a small amount of granular endoplasmic reticulum. During the process of differentiation they elaborate a highly organized system of granular endoplasmic reticulum. This system becomes dispersed into vesicles as the secretory product is synthesized. There is no indication that the Golgi apparatus participates directly in the formation of the secretory droplets, and there is no indication of a membrane bounding the mature secretory droplet.The fate of the zymogenic cell following the discharge of its secretory product was not determined. It is possible that these cells revert back to a stage resembling the stem cell before resynthesizing a new supply of secretion. In this case the normal secretory process would be very similar to the events described in the dedifferentiation of the zymogenic cells during regeneration.This work was supported by Grant number GB-3262 from the National science Foundation.     

FUNCTIONS OF COATED VESICLES DURING PROTEIN ABSORPTION IN THE RAT VAS DEFERENS

The role of coated vesicles during the absorption of horseradish peroxidase was investigated in the epithelium of the rat vas deferens by electron microscopy and cytochemistry. Peroxidase was introduced into the vas lumen in vivo. Tissue was excised at selected intervals, fixed in formaldehyde-glutaraldehyde, sectioned without freezing, incubated in Karnovsky's medium, postfixed in OsO₄, and processed for electron microscopy. Some controls and peroxidase-perfused specimens were incubated with TPP,¹ GP, and CMP. Attention was focused on the Golgi complex, apical multivesicular bodies, and two populations of coated vesicles; large (> 1000 A) ones concentrated in the apical cytoplasm and small (<750 A) ones found primarily in the Golgi region. 10 min after peroxidase injection, the tracer is found adhering to the surface plasmalemma, concentrated in bristle-coated invaginations, and within large coated vesicles. After 20–45 min, it is present in large smooth vesicles, apical multivesicular bodies, and dense bodies. Peroxidase is not seen in small coated vesicles at any interval. Counts of small coated vesicles reveal that during peroxidase absorption they first increase in number in the Golgi region and later, in the apical cytoplasm. In both control and peroxidase-perfused specimens incubated with TPP, reaction product is seen in several Golgi cisternae and in small coated vesicles in the Golgi region. With GP, reaction product is seen in one to two Golgi cisternae, multivesicular bodies, dense bodies, and small coated vesicles present in the Golgi region or near multivesicular bodies. The results demonstrate that (a) this epithelium functions in the absorption of protein from the duct lumen, (b) large coated vesicles serve as heterophagosomes to transport absorbed protein to lysosomes, and (c) some small coated vesicles serve as primary lysosomes to transport hydrolytic enzymes from the Golgi complex to multivesicular bodies.     

Ultrastructure of Follicular Epithelia in the Ovary of Lepidochitona cinerea (L.) (Mollusca: Polyplacophora)

In the sac-like ovary of the polyplacophoran mollusc, Lepidochitona cinerea , nutritive tissue arises from the ventral gonadal wall of the organ as prominent folds which support the oocytes during the various stages of their development. Each oocyte is enveloped by the follicular epithelium. Approximately twenty follicle cells surround one full-grown oocyte and by this late stage are connected to it and to each other by desmosomes. The follicle cells contain glycogen, Golgi dictyosomes, mitochondria, lipid droplets, numerous cisternae and vesicles of the rough endoplasmic reticulum, and various kinds of lysosomes. The nutritional function of these cells and their possible role forming the oocytic hulls is discussed.     

In vivo shedding of apical plasma membrane in the thyroid follicle cells of the mouse

Summary Clusters of luminal dense bodies, limited by a triple-layered membrane, were found in all follicle lumina in thyroid glands of mice. After thyroxine treatment the number of luminal dense bodies increased, especially in the periphery of the lumen, where the intraluminal bodies often displayed a striking resemblance to microvilli. In hyperplastic goiters, obtained by feeding mice with propylthiouracil, luminal dense bodies were replaced by intraluminal vesicles. During goiter involution the vesicles were gradually replaced by luminal dense bodies; the presence of intermediate forms suggests that vesicles and dense bodies are basically the same formations. Luminal dense bodies were observed in colloid droplets indicating their removal by endocytosis. As demonstrated by electron-microscopic cytochemistry, luminal dense bodies contain a membranebound peroxidase, and electron-microscopic autoradiography after administration of ¹²⁵I indicate that they possess an iodinating capacity.Our observations on mouse thyroid glands suggest that the luminal dense bodies, which appear as vesicles in hyperplastic glands, are formed by shedding of the apical plasma membrane of the follicle cell. The shedding process might be of importance for the turnover of plasma-membrane material.This study was supported by Grant No. 12X-537 from the Swedish Medical Research Council.