Polarity reversal of inside-out thyroid follicles cultured within collagen gel: reexpression of specific functions

Isolated porcine thyroid cells cultured in suspension in Eagle Minimum Essential Medium supplemented with calf serum (5-20%) reorganize to form vesicles, i.e. closed structures in which all cells have an inverted polarity as compared to that found in follicles: the apical membranes are bathed by the culture medium. Under these conditions, cells neither concentrate iodide nor respond to acute thyrotropin (TSH) stimulation. When embedded in collagen gel, these vesicles undergo polarity reversal to form follicles. We describe here the change in the orientation of cell polarity and the subsequent reappearance of specific thyroid functions. Six hr after embedding, membrane areas in contact with collagen fibers show basal characteristics. At this time, cells begin to concentrate iodide and to respond to acute TSH stimulation (iodide efflux and increased cAMP levels). Most cells form follicles 24 hr after embedding, but 48 hr are required for the transformation of all vesicles into follicles. This occurs without opening of the tight junctions. Iodide organification is detected 24 hr after embedding, when periodic acid-Schiff positive material, identified as thyroglobulin by immunofluorescence, accumulates in the lumen. Iodide concentration and organification, as well as response to TSH stimulation reach maximal levels after 3 days in the collagen matrix. After a 5-day culture in the collagen matrix in the absence of TSH, cell activity can be stimulated by chronic treatment with low hormone concentrations (10-100 microU/ml). As shown with thyroid cells grown in monolayer on permeable substrates (Chambard M., et al., 1983, J. Cell Biol. 96, 1172-1177), iodide uptake and cAMP-mediated TSH responses are expressed when the halogen and the hormone have direct access to the basal membrane. Organification, on the contrary, requires a closed apical compartment.     

Polarity reversal of inside-out thyroid follicles cultured within collagen gel: structure of the junctions assessed by freeze-fracture and lanthanum permeability

The organization of tight junctional complexes (TJs) was studied in cultured porcine thyroid cells during the inversion of polarity induced by collagen-embedding of inside-out follicles, using freeze-fracture replicas and lanthanum penetration. During the early steps of polarity reversal, freeze-fractures showed that TJs generally persisted. They increased in width and progressively branched out into the basolateral surfaces, towards the basal pole. Later, the number of TJ strands decreased and gap junctions inserted within TJ networks were found between cells in reversed follicles, in the same manner as in typically polarized follicles, embedded in collagen or in suspension. The de novo formation of TJ complexes was rarely found in the reversing structures. Despite the heterogeneity of TJs assessed by freeze-fracture, impermeability to lanthanum tracer was noted in inside-out structures. During the reversal process, some TJs remained unstained, whereas others displayed permeability to lanthanum. This heterogeneity might be due to the "opening" of a small number of junctions (perhaps only one by aggregate). When the process was achieved after 48 hr in collagen, the tightness of the junctions was complete, confirmed by the absence of lanthanum in luminal cavities of newly formed follicles.     

Polarity reversal of inside-out thyroid follicles cultured on the surface of a reconstituted basement membrane matrix.

When cultured in suspension, epithelial thyroid cells organized into inside-out follicles. We studied the behavior of these structures after seeding on polystyrene, type I collagen, and reconstituted basement membrane (RBM) gel. When seeded on plastic, type I collagen or mixed type I collagen-RBM gel, inside-out follicles attached and spread, forming polarized cell monolayers. In contrast, on thick RBM gel, inside-out follicles attached penetrated into the gel, and reorganized into properly oriented follicular structures. Polarity of the cell layer was progressively inverted while, after adhesion, cells penetrated the soft RBM gel. In the process of reorientation, cells with hybrid polarity were observed. The fraction of the apical pole which was not yet in the gel showed an inside-out orientation, while a modified orientation was observed in contact with the RBM gel. Cells which had penetrated completely in the matrix formed a new apical pole and displayed an opposite orientation of their polarity. A continuous basement membrane was observed, lining the basal cell surface when native RBM gel was present in the substratum.     

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.     

Embedding in a collagen gel stabilizes the polarity of epithelial cells in thyroid follicles in suspension culture

Separated thyroid follicles are stable in suspension culture in Coon's modified Ham's F12 medium containing 0.5% calf serum. They resemble follicles in vivo except for the absence of a basal lamina. However, the epithelial cells reverse polarity and the follicles invert when the serum concentration is raised to 5%. A number of substances, especially components of extracellular matrix, were added to the medium to ascertain if they could stabilize the follicles against inversion in 5% serum. Cellular and plasma fibronectin, gelatin, heat-denatured collagen, methylcellulose and laminin did not stabilize. The addition to the medium of as little as 50 micrograms/ml of acid-soluble collagen prepared from calf skin or rat tail tendons resulted in the formation of small clouds of gel. Follicles embedded within the gel were stabilized. Follicles in the same dish but not embedded in the gel inverted. Stabilization was not specific for collagen, since follicles embedded in a plasma clot were also stabilized. A gel was not sufficient for stabilization, since embedding in an agarose gel did not stabilize. Ultrastructural studies indicate that adherence to a limited number of gelled fibers of collagen covering only a small fraction of the basal plasma membrane may be sufficient to stabilize and that a basal lamina formed in the presence of laminin but without added collagen does not stabilize.     

Quantitative study of the development of isolated mouse pre-antral follicles in collagen gel culture

Follicles were isolated from the ovaries of 10-day-old C57BL6/CBA F1 hybrids by mechanical and enzymic treatment, embedded in a collagen-gel matrix to maintain the 3 dimensional integrity of the follicle and cultured for up to 14 days. Gels were removed at various times during the culture period and prepared for histology. Follicles grew from unilaminar to multilaminar stages within 6 days of the culture period. A more detailed assessment of growth by counting follicles at different stages and measuring oocyte and follicle diameters showed that follicle growth was maintained for up to 14 days in culture. Initially the proportion of unhealthy follicles was high but this declined after 6 days in culture.     

Regulation of collagen synthesis in fibroblasts within a three-dimensional collagen gel

Fibroblasts cultivated within a three-dimensional collagen gel display an elongated, spindle-like morphology, reduce their proliferation rate, contact the gel to a very dense tissue, and modify their metabolic activity as compared to monolayer cultures. Collagen synthesis measured as protein-bound hydroxyproline is reduced to 5% of the values found in monolayer culture. The reduction involving type I and type III collagen is due to decreased de novo synthesis and not to enhanced degradation. Dot blot hybridization, Northern blot analysis, and in situ hybridization using collagen I- and III-specific cDNA probes demonstrate that reduced biosynthesis rates are reflected by a marked reduction of pro alpha 1 (I), pro alpha 2 (I), and pro alpha 1 (III) collagen mRNA indicating pretranslational regulation. A similar reduction was observed for actin mRNA whereas levels of tubulin mRNA were similar for fibroblasts in monolayer culture or cultivated within the three-dimensional collagen gels. The data suggest a specific reprogramming of various cellular activities in response to contact with the reconstituted extracellular matrix.     

Immunohistochemical localization of type IV collagen in mouse tooth germ: an ultrastructural study

Localization of type IV collagen was analyzed at the ultrastructural level in mouse embryonic molars by using a preembedding technique. Cryostat sections were incubated with type IV collagen antibody and then treated with the peroxidase-antiperoxidase complex. This antibody was visualized at the epithelio-mesenchymal interface. Labeling was intense and uniformly distributed throughout the basement membrane. However, it was mainly restricted to the lamina densa. No immunostaining was detectable in the lamina lucida but it was crossed by fine filaments that appeared as projections from the lamina densa to the epithelial cell plasma membrane. At the mesenchymal aspect of the basement membrane, projections of labeled material extended from the lamina densa in the underlying dental mesenchyme. At the presecretory stage of odontoblasts, these projections were in close connection with mesenchymal cell processes.     

Propranolol has direct antithyroid activity: Inhibition of iodide transport in cultured thyroid follicles

The effect of propranolol on the process of thyroid hormone formation was studied in a physiological culture system. Porcine thyroid follicles were preincubated with propranolol for 24 h. Iodide transport, iodine organification, and de novo thyroid hormone formation were measured by incubating these follicles with the mixture of carrier-free 0·1 μCi Na ¹²⁵I and 50 nM NaI for 2 to 6 h at 37°C. A concentration of propranolol greater than 100 μM inhibited iodide transport in a dose-dependent manner; this inhibition was non-competitive with iodide and independent of thyrotropin (TSH). Reduced iodine organification and thyroid hormone formation was seen with 150 μM propranolol or greater. The inhibitory action of propranolol was not caused by beta-blocking activity, since D -propranolol (devoid of beta-blocking activity) inhibited iodide transport, and other beta-blockers (metoprolol, atenolol, and labetalol) did not inhibit iodide transport. The inhibition of iodide transport was most likely caused by membrane stabilizing activity since quinidine, which possess the same membrane stabilizing activity as propranolol, also inhibited iodide transport. TSH-mediated cAMP generation and Na ⁺K⁺ ATPase activity, membrane functions for iodide transport, were unaffected by propranolol. Our study has shown, for the first time, that propranolol has a direct antithyroid action, namely inhibition of iodide transport in the intact thyroid follicle.     

Formation of capillary networks from bone marrow cultured in collagen gel

The growth of capillaries from mouse bone marrow was studied in collagen gel. When the culture contained sufficient bone marrow cells (more than 1 X 10(6) cells) and cell clusters, short capillaries with lumina appeared about one to two weeks after inoculation, following the proliferation of fibroblastic cells and hemopoietic cells. Four weeks after inoculation, these capillaries formed a network among hemopoietic cells and adipocytes. Electron microscopic observations revealed that these capillaries had thin walls and poorly developed basement membranes, similar to the sinusoids of bone marrow. These capillaries did not appear when the amount of inoculated bone marrow was reduced or dispersed to the point that the marrow cell clusters disappeared. The quantity of the inoculum and the clustering of cells, therefore, seems to play important roles in the appearance of the capillaries.     

Ultrastructure of intermediate stages in polarity reversal of thyroid epithelium in follicles in suspension culture

Separated thyroid follicles can be maintained in suspension culture in Coon's modified F-12 medium in 0.5% calf serum. If the serum concentration is raised to 5%, the follicles undergo inversion in 3-5 d. During the process of inversion, epithelial cells can be observed in intermediate stages of polarity reversal. The earliest ultrastructural changes recognized are surface changes in which tight junctions and microvilli appear at the lateral margins of the cell near the medium. Later, changes in the distribution of intracellular organelles occur. The Golgi apparatus shifts towards the end of the cell facing the medium, and lysosomes shift toward the luminal end of the cell. The right junctions and microvilli at the luminal end of the cell disappear sometime after the cytoplasmic organelles rearrange. The luminal colloid disappears only after the surface changes (loss of tight junctions and microvilli) occur at the luminal end of the cell. There appears to be some regulation of the order in which changes occur during polarity reversal of the thyroid epithelial cell.     

Morphogenesis of mouse mammary epithelial cells growing within collagen gels: ultrastructural and immunocytochemical characterization

Mammary epithelial cells from adult virgin mice have been cultured within collagen gels in totally serum-free medium containing either epidermal growth factor or the mammogenic hormones, progesterone and prolactin, or prolactin alone. The cellular organization, differentiation and cell-type composition of the colonies from the three culture conditions were assessed by transmission electron microscopy and light-microscope immunocytochemistry. The epithelial cells form branching duct-like structures and, when exposed to mammogenic hormones, assume a secretory morphology (including casein micelles) similar to that seen in the early to mid-pregnant mouse.     

Effect of certain factors on sheep oocyte maturation when cultured within intact follicles

The effect of the medium composition and cultivation time on the maturation of sheep oocytes cultivated in vitro within the intact follicles was studied. The highest incidence of maturation was observed in medium 199 with 20% of sheep serum taken from an animal on the 16th day of the sexual cycle in a gaseous mixture of the following composition: 5% O2, 5% CO2, 90% N2 during 24 hrs.     

Sporozoites of Eimeria acervulina within intestinal macrophages in normal experimental infections: an ultrastructural study.

Sporozoites of Eimeria acervulina were observed in macrophages of the intestinal epithelium 5 and 6 days post-infection. These sporozoites lay within a well developed parasitophorous vacuole, were normal in structure and showed no signs of development. Macrophages harbouring sporozoites showed considerable structural changes, most pronounced being an absence of lysosomes, an enlarged nucleolus and extensive proliferation of the Golgi complex and endoplasmic reticulum. Possible mechanisms of survival and transport of sporozoites to preferred sites of development are discussed.     

Mucin synthesis and secretion by cultured tracheal cells: effects of collagen gel substratum thickness

Summary We previously demonstrated that human tracheobronchial epithelial (TBE) cells synthesize mucin and form mucous granules in culture when they are maintained on a collagen gel (CG) substratum, but not on a plastic tissue culture surface or a thin collagen-coated surface (Wu et al., Am. J. Respir. Cell Mol. Biol., 3:467–478; 1990). This observation led us to examine the effects of CG thickness on cell growth and differentiation in primary human/monkey TBE cell cultures. Using the same CG preparation, culture dishes with different thicknesses of CG substratum were prepared. In general, equivalent degrees of cell attachment and proliferation were observed in all cultures maintained on a collagen gel, independent of the thicknesses of CG substratum. However, a greater degree of mucin synthesis and secretion by the cells was observed as the thickness of the CG substratum was increased. Cultures maintained on a thick collagen gel (1 mm) exhibited greater apical membrane complexity, more pseudostratification, and more mucous granules than did cultures maintained on a thin CG substratum. The optimal culture surface for airway mucous cell differentiation contains more than 1-mm thickness of collagen gel substratum.     

Morphological and functional differentiation of human thyroid cells in collagen gel culture

In order to study the expression of the morphological and functional characteristics of human thyroid cells, 3-dimensional cultures were carried out in collagen gel. This substrate allows the cells to retain their organization in follicles with a normal polarity. Cellular polarities appeared normal at the time of collagen embedding, but there was a delay of 4-5 days in culture before the maximal TSH stimulation of 125I- uptake and of cAMP accumulation occurred. In normal and adenoma-derived cells, 125I- uptake, which could be increased by TSH, was demonstrated. cAMP accumulated in the culture medium and thyroglobulin was secreted into the follicle lumen. Of the 4 differentiated carcinomas for which the 72-hr uptake of 125I- was measured, only 2 displayed slight 125I- uptake and response to TSH. Thus, human thyroid cells exhibit better morphological and functional differentiation in collagen gel culture than in monolayer culture. Furthermore, in a variety of pathological cases studied, the expression of specific characteristics in culture varied in a fashion similar to differences observed in vivo.     

Subculture of rabbit articular chondrocytes within a collagen gel: growth and analysis of differentiation

Primary cultures of rabbit articular chondrocytes have been subcultured within three-dimensional (3D) collagen gels. Under these conditions, the cells remained viable and divided, but with a lower proliferation rate than that observed in control monolayer cultures. Flow cytometric analysis of progression of the cells into the cell cycle has confirmed and extended these findings. Also the cellular volume was decreased in 3D-culture, being in the same range as thein vivo size of cartilage cells. Specific staining for proteoglycans and type II collagen immunolocalization on sections of gels showed the expression of differentiated phenotypes and revealed the accumulation of these matrix components in the immediate surroundings of the cells. The use of Ultroser G (a serum substitute) improved the conditions for 3D- culture of rabbit articular chondrocytes.     

Effect of calcitonin on fibroblasts and collagen formation in rabbits: an ultrastructural and scanning electron microscopic study

Transmission and scanning electron microscopic studies demonstrate the stimulatory effect of synthetic salmon calcitonin on the fine structure of fibroblasts and on collagen formation in cutaneous wounds experimentally induced in rabbits. Long-term administration of calcitonin enhances fibroblast growth and collagen synthesis. The fibroblasts hypertrophy and exhibit a highly developed rough endoplasmic reticulum (RER), several polyribosomes, large nuclei, hypertrophic Golgi complex, and many dense granules and lysosomes. Mitochondria are elongate and ramify; intracellular as well as extracellular synthesis of collagen increases. Fibrils appear tightly packed, in large heaps or spicula, with a characteristic periodicity and striation. Scanning electron micrographs of topography and relationships with collagen fibers and fibrils and cells surface changes demonstrate an extensive network of fine fibrils between collagen fibers, marked ruffling of cell membranes as well as numerous blebs on the cell surface. The latter are significant in collagen formation and egestion.     

Membrane recycling after exocytosis: An ultrastructural study of cultured chromaffin cells

When exocytosis of granule contents is induced by nicotine stimulation, glycoprotein III (a chromaffin granule membrane constituent) is exposed on the surface of cultured chromaffin cells, where it may be labeled with an immunocytochemical tracer. The subsequent fate of this glycoprotein after endocytosis was followed at the ultrastructural level using immunogold methods and was analyzed by morphometry. After stimulation exocytosis membranes newly inserted into the plasma membrane labeled with gold particles for glycoprotein III were found to be endocytosed via coated vesicles and finally found in organelles devoid of chromogranin A, the major secretory granule protein. At intervals between 30 min and 24 h after cell stimulation and immunolabeling, most labeled structures were identified by two different morphological approaches as prelysosomes and lysosomes. In contrast with results obtained on freshly isolated chromaffin cells, it is thus concluded that in cultured cells granule membrane recycling into new granules does not occur. It is suggested that the fate of granule membrane endocytosed after cell stimulation may be influenced by the external conditions to which cells are previously exposed.