Immunocytochemical study of localization and traffic of thyroid peroxidase/microsomal antigen.

We studied the distribution of binding sites for anti-peroxidase monoclonal antibody and anti-microsomal antibodies on isolated human thyroid follicles and a human thyroid cell line. Both open follicles and cells were incubated first with antibodies at +4 degrees C, then with colloidal gold labelled protein A. The topography of the binding sites for monoclonal anti-peroxidase antibody corresponded closely to the expected cell surface distribution of endogenous thyroid peroxidase since labelling was observed at the apical cell surface of the follicles. Furthermore, labelling was restricted to the microvilli level; while smooth membrane territories were devoid of binding sites. In some cases, incubations at 4 degrees C were followed by warming the follicles and cells up to 37 degrees C for 20 minutes in order to study internalization of ligands. Ligands were then observed in intracellular organelles: endosomes and lysosomes. Essentially the same results were observed when human antibodies to the microsomal antigen were used. Controls with microsomal antibodies depleted in anti-peroxidase were negative. In conclusion these findings show that: 1) thyroid peroxidase is present in limited areas on the apical cell surface, 2) labelling of follicles and cells by the anti-microsomal antibodies had the same pattern of distribution as the monoclonal anti-peroxidase antibody, thus suggesting that they recognize the same apical antigens, and 3) TPO/MIC antigen traffics from the cell surface towards lysosomes when the cells are incubated at 37 degrees C.

Forskolin stimulates cAMP production and the onset of the functional differentiation in the fetal rat thyroid in vitro.

Forskolin, a diterpen stimulating the adenylcyclase induces in vitro an accumulation of cAMP in thyroid glands explanted from 17 day-old rat fetuses. Thyroid glands from 15 day-old fetuses exposed to forskolin exhibit, within 24 hr, an active folliculogenesis and 125I fixation in follicular cavities. The results indicate that cAMP probably activates the onset of both morphological and physiological maturation in the fetal rat thyroid.

Evidence for a role of the cytoskeleton in the in vitro folliculogenesis of the thyroid gland of the fetal rat.

Thyrotropic hormone (TSH) or cAMP accelerate the formation of follicular cavities in the explanted thyroid gland of the 15-day-old rat fetus. Cytochalasin B or vinblastine and nocodazole or colchicine, which disorganize microfilamental and microtubular structures respectively, inhibit or completely block in vitro-induced folliculogenesis. Exposure of the thyroid tissue to lumicolchicine, a structural isomer of colchicine deprived of antimicrotubular activity, does not inhibit the activation of folliculogenesis induced by TSH. These results are strong evidence for the supposition that microfilaments and microtubules are involved in the TSH-stimulated mechanisms resulting in thyroid folliculogenesis. Folliculogenesis requires the integrity of both microfilaments and microtubules.

Thyroid follicular morphogenesis mechanism: organ culture of the fetal gland as an experimental approach.

The morphological and physiological changes induced by organ culture and thyroid-stimulating hormone (TSH) stimulation in the rat fetal thyroid gland were studied. Organ culture increased Golgi activity which was further enhanced by TSH, subsequently facilitating the formation of intracellular lumina. TSH also raised the intracellular cAMP level. The intracellular lumina observed during follicular morphogenesis are structurally comparable to typical intracellular cavities formed in adult thyroid cells, which are considered as being the result of increased Golgi activity. The intracellular lumen, therefore, is probably not a physiologically significant step in thyroid morphogenesis.

Basement membrane maintenance in cultures of isolated thyroid follicles.

Follicles from adult rat thyroid glands were isolated by digestion with collagenase followed by sieving. Electron microscopic studies have shown that basement membrane was present around each one of the isolated follicles. Follicles were cultured in vitro with or without thyrotropin and fibronectin. When cultured in a medium free of thyrotropin, basement membrane remained around them, while it was lacking in presence of this hormone.

Critical study of endogenous peroxidase activity: its role in the morphofunctional setting of the thyroid follicle in the rat fetus.

Endogenous peroxidase is associated with thyroglobulin iodination. It first appears at 17 day of pregnancy in rat fetal thyroid and seems to be synthesized within the perinuclear cistern and the rough endoplasmic reticulum. Then, the enzyme is transferred by vesicles up to the follicular lumen where thyroglobulin iodination takes place. The different steps of this process can occur within a very short time, since peroxidase activity is already located in the apical plasma membrane of follicles formed as early as 17 days of pregnancy, which is consistent with autoradiographic data showing iodinated thyroglobulin at 17 days. It can be concluded that the follicle is functional as soon as it is formed.

Development of the thyroid gland in the rat fetus in vivo. An ultrastructural and radioautographic study.

A study of the differentiation of the thyroid follicles was carried out in rat fetuses between day 14 of pregnancy, when cellular polarization begins and day 18, when completely formed follicular lumina are differentiated. After day 18, only the shape and the size of the lumina undergo modifications. The mechanism of folliculogenesis involves the participation of several structures. At 14 days of pregnancy, adherens type contact zones with membrane densification appear between the cells. These junctional zones spread out and form infoldings which deeply penetrate the cytoplasm. Golgi vesicles migrate to these zones and they fuse among themselves and with the contact membranes : follicular cavities result which can be recognized as soon as day 16 of pregnancy. A radioautographic study showed the beginning of iodide organification at 17 days 11 hours of pregnancy. From this time onward silver grains were seen over the follicular cavity, even it is still incompletely formed.

[Critical study of extra-lysosomal acid phosphatase localizations in thyroid follicular cells by the Gomori reaction (author's transl)]. / Etude critique de localisations phosphatasiques acides extra-lysosomiales des cellules folliculaires thyroïdiennes par la réaction de Gömöri

With the Gömöri technique, lead precipitates have been found in thyroid follicle cells in unusual localizations such as apical hyaloplasm and microvilli; it has been established that they were actually significant for acid phosphatase activity: constant results in spite of repeated controls and several variations from the original cytochemical technique, allow to think that lead precipitates were not merely artefactual, but actually significant of enzymatic activity. However it is pointed to the fact that the origin of the enzyme has to be questioned; it is assumed that most likely acid phosphatase has diffused from its original lysosomal site. Such diffusion implies variations of the selective permeability of lysosomal membranes; inappropriate relation between the quantity of enzyme present in these organelles and the quantity of substrate used might also be considered, though changes in the amount (resp. concentration) of substrate remained ineffective and induced no modification in the localization of observed enzymatic activity. In addition, one point of interest is an obvious relation between the observed enzyme diffusion and the state of activity resp. rest of the cell; in the present state of investigations, this remains unexplained and likely related to factors escaping control during processing; moreover, no explanation can be provided for the fact that it revealed impossible to avoid such diffusion even by means of variations of the numerous parameters involved in the Gömöri technique. So that it finally appears necessary to remain on a critical position regarding the results at the ultrastructural level of this standardized technique, and there is no doubt it would reveal useful that several assumptions in the literature about extra lysosomal acid phosphatase activity should be reinvestigated with a similar critical purpose.