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Günther Bechtner, Dorothee Schopohl, Michael Rafferzeder, Roland Gärtner and Ulrich Welsch

Bechtner G, Schopohl D, Rafferzeder M, Gärtner R, Welsch U, Stimulation of thyroid cell proliferation by epidermal growth factor is different from cell growth induced by thyrotropin or insulin-like growth factor I. Eur J Endocrinol 1996;134:639–48. ISSN 0804–4643

Isolated intact porcine thyroid follicles free of contaminating single cells were embedded in "Matrigel". which is a gel-forming basement membrane preparation containing mainly collagen type IV, laminin, heparan sulfate proteoglycans and entactin. Follicles were treated with different growth factors: thyrotropin (TSH), insulin-like growth factor I (IGF-I), epidermal growth factor (EGF) or transforming growth factor beta. Cell proliferation was quantified by counting cell numbers. Morphological studies were done by photodocumentation and analysis of histology by light and electron microscopy. The thyrocytes had the physiological polarity with follicular cell arrangement, microvilli at the apical membrane, desmosomes and tight junctions. The lumen contained colloid. Iodide organification (10.2 ± 2.1 vs 26.1 ± 5.8 pmol/106 cells; TSH 0.1 mU/ml) and release of thyroid hormones thyroxine, 1754 ± 207 vs 2890 ± 460 pg/106 cells; triiodothyronine. 164 ± 22 vs 412 ± 106 pg/106 cells; TSH, 1 mU/ml) were significantly stimulated by TSH. There was no basal growth rate in serum-free medium but proliferation was slightly stimulated with TSH (1 mU/ml;149 ± 19%) and in the same order of magnitude with IGF-I (10 ng/ml; 159 ± 23%) but without follicle neoformation. In contrast, EGF (1.0–5.0 ng/ml) induced thyrocyte proliferation dose dependently three- to sixfold. With EGF up to 2 ng/ml, buds of new follicles formed surrounding pre-existing follicles. With EGF higher than 3 ng/ml, typical papillary structures developed. Transforming growth factor beta inhibited this dedifferentiated growth. A migration of single cells into the gel was never observed. Thus, three-dimensional culture of isolated thyroid follicles in "Matrigel" provides a tool for investigating the regulation of follicular growth and neoformation close to the in vivo situation.

G Bechtner, Medizinische Klinik, Klinikum Innenstadt, Ludwig-Maximilians-Universität München, Ziemssenstraße 1, 80336 München, Germany

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Roland Gärtner, Günther Bechtner, Walter Greil, Klaus Horn and C. Renate Pickardt

Abstract. Human thyroglobulin (Tg) was isolated to apparent purity from various thyroid tissue samples obtained after surgery. Iodine and iodothyronine content of Tg was determined. Isoelectric focussing (IEF) was performed on thin layer agarose gels. Tg revealed a microheterogeneity of 6 bands in the range between pH 4.2 and 4.6. The intensity of the single bands depended on the iodothyronine content of Tg. With increasing degree of iodination, the bands with a lower pI (pI 4.35, 4.40) became more prominent, whereas the bands with higher pI (pI 4.55, 4.60) diminished. This typical change in microheterogeneity pattern could be confirmed by kinetic in vitro iodination and consecutive iodothyronine formation of low iodinated Tg (0.05% iodine). After in vitro desialylation, the bands shifted to a higher pH range (pH 4.60 to pH 4.90), but no reduction of the number of bands occurred. Even in desialylated Tg microheterogeneity is still dependent on iodine content. These results suggest, that the microheterogeneity of Tg is influenced, but not caused by different iodine and NANA content. Different polypeptide composition may be responsible for the microheterogeneity of Tg.

In thyroid diseases without disturbance in Tg synthesis (endemic, diffuse and nodular goitre, Graves' disease) variations in relative intensity of single bands could be related to differences in iodine content. In thyroid cyst fluid and cold nodules in addition to low iodinated Tg, further bands were found with pI-values comparable to desialylated Tg.

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Günther Bechtner, Bernhard Rieder, Ullrich Linsenmaier, J Kellermann, Walter Greil and Roland Gärtner

Immunoreactive basic fibroblast growth factor (bFGF) could be isolated from the cytosol preparation of isolated porcine thyroid follicles as well as in the conditioned medium from thyroid follicles in suspension culture. A double band with 16 500 and 15 500 D was detected on sodium dodecyl sulfate polyacrylamide gel electrophoresis. In dot blot and western blot the isolated peptide was immunoreactive with a specific anti-bovine bFGF antibody. For further biochemical characterization, bFGF was isolated from entire porcine thyroid glands by ammonium sulfate precipitation, cation exchange chromatography and heparin affinity chromatography. The material obtained from all three origins was identical concerning affinity to heparin and immunoreactivity with the specific anti-bovine bFGF antibody and induced neovascularization in the chorioallantois membranes of chick embryos. Amino acid sequence analysis of the 16-amino-terminal amino acids of the isolated bFGF was in accordance with the established complete 146-amino-acid bFGF molecule except that glycine in position 10 is replaced by phenylalanine. An additionally identified minor peptide presumably is an amino-terminaltruncated form of bFGF, missing the first 15 amino acids. We conclude that the physiological significance of bFGF released by thyroid cells may be the regulation of angiogenesis during thyroid development and goiter growth.