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The TSH-induced cyclic AMP response was studied using a 3-year-old ovine thyroid cell line TSH-independent for growth: OVNIS 5H. The kinetics of cyclic AMP production was followed both in cell layers and in cell culture media, with or without phosphodiesterase inhibitor. It is noteworthy that following the first wave in cyclic AMP obtained within minutes, we observed later a sustained exponential increase in cyclic AMP during the 5 days following TSH stimulation. A bioassay of TSH was derived allowing measurement of 1 microU/ml TSH from a crude bTSH preparation.

Porcine thyroid cells were cultured alone or in mixed cultures with mesenchymal cells. The formation of a basal lamina in vitro was investigated ultrastructurally. Follicular reassociation of thyroid cells occurred in both types of culture; however, it was followed by formation of the basal lamina only when mesenchymal cells were present. The present findings suggest an epithelial origin of the basal lamina resulting from an interaction with mesenchymal cells.

The hormonal regulation of thyroglobulin synthesis has been studied using two independent clones of the OVNIS 6H cell line. Insulin, hydrocortisone and TSH were able to stimulate thyroglobulin synthesis, whereas transferrin, somatostatin and glycyl-histidyl-lysine were without effect. Insulin stimulated thyroglobulin synthesis without affecting cAMP production. Hydrocortisone, when combined with insulin was a stimulator too; this stimulation was not accompanied by an increase in cAMP. TSH alone was unable to stimulate either cAMP or thyroglobulin synthesis. The stimulatory effect of TSH on thyroglobulin synthesis took place only when combined with insulin or insulin plus hydrocortisone, and was mediated by cAMP. Consequently, insulin and hydrocortisone stimulated thyroglobulin synthesis by cAMP-independent mechanisms, whereas TSH acted via the cAMP system. Forskolin mimicked TSH effects on cAMP and thyroglobulin synthesis. Calf serum inhibited cAMP and thyroglobulin production. Optimal cAMP and thyroglobulin synthesis as well as TSH responsiveness were obtained in serum-free medium supplemented with 5 micrograms/ml insulin, 100 nM hydrocortisone and 1 mU/ml TSH.

Porcine thyroid cells were cultured for 2 days with or without dibutyryl cyclic AMP or thyrotropin (TSH). Then they were isolated post-culture by a gentle treatment with a calcium chelating agent. Some characteristics of the iodide transport system were studied in these thyroid cell suspensions. Iodide influx is a saturable, temperature- and energy-dependent phenomenon. It is blocked by ouabaïn, N-ethylmaleimide, dinitrophenol, cardiotoxin, low Na+ concentration and harmaline. Only 3% of the intracellular iodide is trichloracetic-acid-insoluble at equilibrium. The apparent Michaelis constant (Km) of the transport system is 30 micronM. For monolayer cells, the decrease of C/M ratio, increase of apparent Km, and decrease of Vmax between day 0 (freshly isolated cells) and day 6, indicate a loss of iodide-trapping ability up to passive diffusion. To the contrary, high values of C/M and normal Km (30 micronM) are observed in TSH follicles from reassociated cells. At iodide equilibrium, thryotropin, prostaglandins E1 and E2 and long-acting thyroid stimulator (LATS), induce a fast release of iodide. This release is dose-dependent in the first 5 min. It has been used to develop a bioassay of TSH and a fast detection of LATS.

The major protein synthesized in vitro by the ovine thyroid cell line OVNIS 6H is the prothyroid hormone thyroglobulin. Purified from serum-free cell culture media using sucrose gradient centrifugation, the thyroglobulin dimer was analysed for iodine content and observed by electron microscopy. In their usual medium, the OVNIS 6H cells produce a very poorly iodinated thyroglobulin containing 0.05 I atom per molecule. When cultured with methimazole or propylthiouracil, two inhibitors of iodide organification, less than 0.007 I atom/molecules was found. These molecules purified from cell cultures were compared to those purified from ovine thyroid tissue containing 26 I atoms/mol. Despite large differences in iodine content, the three preparations all consist of 19 S thyroglobulin dimers with the classical ovoidal shape. The variability in size measurements remains in a 2% range for all thyroglobulin types. Consequently, no real significant variation can be found between the highly iodinated thyroglobulin isolated from tissue, and the poorly or non-iodinated thyroglobulins isolated from cells cultured with or without methimazole or propylthiouracil.

The effects of three putative growth factors and serum on [Me-3H]thymidine and Na125I incorporation into thyroid cell cultures have been examined. We found that serum and EGF could stimulate radioactively labelled thymidine incorporation into confluent cultures. However, both factors completely inhibited iodine uptake and organification at low concentrations. Insulin also stimulated [Me-3H]thymidine incorporation but had no adverse effect on thyroid differentiated function. TSH examined under the same conditions was not a growth factor but was essential to maintain differentiated functions. We conclude that thyroid growth and differentiation are not mutually exclusive processes. However, EGF and serum inhibit thyroid differentiated function at very low concentrations. Elucidation of the physiological role of these factors and their mechanism of action may lead to a greater understanding of thyroid hormone biosynthesis.

Administration of thyrotropin to porcine thyroid follicles, obtained in a serum-free chemically defined medium, provoked marked increases in the activities of several glycosyltransferases involved in protein N-glycosylation. The coincidence of these effects with a previously demonstrated enhancement of thyroglobulin production renders a relationship between these events likely. The most important stimulation was for peptide oligosaccharyltransferase (3-fold). Among the enzymes involved in the synthesis of the lipid oligosaccharide donor, Dol-P glycosyl- and mannosyltransferases were increased 1.5-fold, and Dol-P N-acetylglucosaminylphosphotransferase only 1.15-fold. As regards terminal glycosyltransferases, asialofetuin sialyltransferase was increased 2-fold and ovomucoid galactosyltransferase only 1.2-fold. There was a continuous release of the latter two enzymes into the culture medium.

The fate of aminopeptidase N (APN) was investigated by means of immunofluorescence in porcine thyroid-cell cultures. At day 1, control cells cultured without TSH formed flat, cylinder-like aggregates in which APN was mainly located on the lateral surface. At day 2, the fluorescence completely disappeared. At days 3 and 4, APN appeared in the apical pole of monolayer cells directed toward the medium and in intracellular organelles. The cilium in each cell was intensely labelled and located in the center of the apical pole. In thyrotropin-stimulated cell cultures, the fluorescence at days 1 and 2 was located over the entire plasma membrane. At days 3 and 4, morphological polarization of cells, reorganized into follicles, occurs, APN being located either in the apical or basolateral regions and in intracellular vesicles. Between day 4 and 6, segregation of the enzyme to the apical membrane occurred. Between day 6 and 14, fluorescence was generally retained in the apical region and in an intracellular pool. In growing cell cultures, after 2 to 5 subcultures, the cells exposed APN in a pattern similar to primary monolayers. After conversion of monolayer cells into follicles, the antigen was recovered in the apical pole of cells facing the follicular lumen.

The ultra-structural of a C-cell carcinoma of the thyroid confirms previous data: the cells are slender, with a large nucleus. The Golgi apparatus is well developed. Granules in striking abundance are seen in every part of the cytoplasm: their diameter is 85 - 250 mmu.

Eagle's minimum essential medium supplemented with 20 per cent newborn calf serum (N.C.S.) allows porcine thyroid cell survival but not cell growth in vitro. In NCTC 109 medium supplemented with 20 per cent N.C.S. these cells actively grow and may be serially propagated. Cell population doubling time expressed as DNA doubling value is 3.5 days at 37 degrees C in 95 per cent air-5 per cent CO2. Thyrotropin does not affect porcine thyroid cell multiplication in vitro but stimulates the plating efficiency in primary cultures to about 130 per cent of controls. Cell selection was obtained by replacing media with Earle's balanced salt solution. This operation provoked death of nearly all cells by day 18 but subsequent addition of growth medium resulted in proliferation of epithelial cell clones. From generation 2 to generation 8, cells produce thyroglobulin but they do not actively trap iodide nor form follicles when thyrotropin is added to the media. Cell selection, demonstration of growth, as well as freeze-storage techniques described in this paper permit selection and storage of porcine thyroid cells and the potential constitution of cell collections.