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

Abstract

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.

Thyrotrophin regulation of apical and basal exocytosis of thyroglobulin by porcine thyroid monolayers

“…The hormone can therefore be added to the basal medium, thus making contact only with the baso¬ lateral side of the epithelial layer where TSH receptors, connected with the adenylate cyclase complex, are present (Chambard, Verrier, Gabrion & Mauchamp, 1983). Under these conditions, TSH desensitization of thyroglobulin exocytosis occurs with physiological concentrations of TSH (25 pU/ml), as previously observed with follicles embedded in collagen gels (Chambard, Verrier, Gabrion & Mauchamp, 1984). However, the continuous secre¬ tion of thyroglobulin under chronic TSH stimu¬ lation can, in part, be responsible for the weaker response to the further acute stimulation by decreas¬ ing the intracellular pool of secretory vesicles.…”

Long-term iodination of thyroglobulin by porcine thyroid cells cultured in porous-bottomed culture chambers: regulation by thyrotrophin

“…Iodination of thyroglobulin was not detected in the absence of TSH, but from 10 pU TSH/ml, iodide uptake and incorporation into thyroglobulin were maximal. In previous reports, the same TSH concen¬ tration was sufficient to allow iodide uptake and iodination by follicles embedded in collagen gel (Chambard, Verrier, Gabrion & Mauchamp, 1984) or FRTL-5 lines (Marcocci, Luini, Santisteban & Grollman, 1987). Other studies, using different culture systems, reported that TSH stimulated iodide uptake and its incorporation into thyroglobulin but that maxi¬ mal responses were often obtained with TSH concen¬ trations as high as 100 pU/ml (Mauchamp et al 1979) or 200 pU/ml (Fayet et al 1982).…”

Tight junctions and apical/basolateral polarity

“…4e). However, exposure of this surface to collagen provokes the formation of a lumen at the core of the follicle, reversion of polarity, and migration of the TJ in the plane of the lateral membrane until it occupies the innermost end of the intercellular space [4,5,26,27,104]. HCO3-secreting cells of the cortical collecting duct of the kidney possess CI-/HCO3 exchangers in the apical domain and proton pumps in the basolateral membrane.…”