Distribution of microtubules and microfilaments in thyroid follicular epithelial cells of normal,TSH-treated,aged, and hypophysectomized rats

Summary We investigated the distribution of microtubules and microfilaments in rat thyroid follicular epithelial cells by applying an immunofluorescence technique with monoclonal antibodies against tubulin and by staining sections with rhodamine-phalloidin. In normal thyroid cells, microtubules run longitudinally from the apical region to the basal region intersecting with each other. In addition, intense labelling with tubulin antibodies was observed in the apical part of the cell. The ultrastructural examinations showed that microtubules often run along the apical plasma membrane. Dot-like labelling with anti-tubulin antibodies was often observed in the perinuclear space, but no microtubules were recognized in the nucleus. Microfilaments bound to rhodamine-phalloidin were distributed mainly beneath the apical plasma membrane, and the portion along the basolateral membrane was scarcely positive. The apical pole of the follicle cell was also decorated by anti-microtubule-associated protein-2 (MAP-2). After TSH stimulation, the intensity of immunocytochemical staining against tubulin was remarkably increased in the cytoplasm. Simultaneously, at the apical region, the staining intensity of rhodamine-phalloidin was increased. Microtubules and microfilaments appeared in the pseudopods after TSH injection. In hypophysectomized or aged rats, thyroid follicular epithelial cells decreased in height, and both immunofluorescent labelling against tubulin and rhodamine-phalloidin labelling were markedly decreased. These results indicate that the distribution and polymerization of microtubules and microfilaments in thyroid follicular epithelial cells vary with the functional stage.     

Immunocytochemical localization of calspectin (a non-erythroid spectrin-like protein) in thyroid glands of normal and TSH-treated rats

The localization of calspectin (fodrin, a non-erythroid spectrin-like protein), which is known to bind calmodulin and F-actin, was detected in the thyroid gland of normal and TSH-treated rats by means of light-microscopic immunocytochemistry. Calspectin was demonstrated in the cytoplasm of the follicle epithelial cells especially along the baso-lateral plasma membrane in normal rats. In TSH-treated animals, in addition to the baso-lateral plasma membrane region, the apical plasma membrane region of the follicle epithelial cells also showed positive reaction to the immunostaining. These results suggest that calspectin, in conjugation with calmodulin and actin, play a role in the secretory activities including reabsorption activity of colloid of the follicle epithelial cell.     

Immunocytochemical localization of calspectin (a non-erythroid spectrin-like protein) in thyroid glands of normal and TSH-treated rats

Summary The localization of calspectin (fodrin, a nonerythroid spectrin-like protein), which is known to bind calmodulin and F-actin, was detected in the thyroid gland of normal and TSH-treated rats by means of light-microscopic immunocytochemistry. Calspectin was demonstrated in the cytoplasm of the follicle epithelial cells especially along the baso-lateral plasma membrane in normal rats. In TSH-treated animals, in addition to the baso-lateral plasma membrane region, the apical plasma membrane region of the follicle epithelial cells also showed positive reaction to the immunostaining. These results suggest that calspectin, in conjugation with calmodulin and actin, play a role in the secretory activities including reabsorption activity of colloid of the follicle epithelial cell.Supported by grants from the Ministry of Education, Science and Culture, Japan     

Thyrotropin stimulation of cultured thyroid cells increases steady state levels of the messenger ribonucleic acid for thyroid peroxidase

We have examined the effect of TSH on thyroid peroxidase (TPO) mRNA levels in dog thyroid cell primary cultures. Freshly dispersed dog thyroid cells were cultured for up to 5 days in the absence or presence of 5 mU/ml bovine TSH. At the outset of culture, and at daily intervals thereafter, total cytoplasmic RNA was extracted and applied to Nytran paper using a slot-blot apparatus. A nick-translated cDNA fragment of the porcine TPO gene was used to probe these filters. Autoradiographs were quantified by densitometry. Nonspecific binding was negligible as determined using a pUC18 probe. During the first 2 days of culture, TPO mRNA levels declined irrespective of whether or not TSH was present in the medium. TSH did not affect this decline. Between 3 and 5 days of culture, TPO mRNA levels in control (no TSH) cells increased to 3 times the initial level (expressed relative to cellular DNA). However, during the same period TSH stimulated TPO mRNA levels 8-fold above the initial level. To confirm that the signal with the cDNA probe was actually that of dog TPO mRNA, cellular RNA (day 4 of culture) was subjected to Northern blot analysis using the same cDNA probe. Specific bands of 2.9 kilobases were detected corresponding to the known size of TPO mRNA in pig thyroid tissue. The signal of this 2.9 kilobase species was enhanced by TSH. In conclusion, the data indicate that chronic TSH stimulation raises steady state levels of TPO mRNA and provide an explanation, at least in part, for the mechanism by which TSH enhances TPO bioactivity in thyroid tissue.     

Hormonal regulation of thyroid peroxidase in normal and transformed rat thyroid cells

The hormonal induction of thyroid peroxidase (TPO) mRNA is studied in the functional rat thyroid cell line FRTL-5 and compared to the induction of thyroglobulin (TG) mRNA and I- uptake. TPO and TG mRNAs are regulated by TSH and by insulin-like growth factor I (IGF-I) and/or insulin. However, while TPO is more sensitive to TSH regulation (5- to 6-fold increase vs. 2- to 3-fold increase by IGF-I), TSH and IGF-I are equally potent in increasing TG mRNA levels (3- to 4-fold). Regulation of I- uptake appears to be different: thus TSH greatly (15-fold) increases I- uptake, while IGF-I or insulin are completely ineffective. TPO and TG mRNAs and I- transport display different sensitivity to transformation of rat thyroid cells. Thus, when another differentiated rat thyroid cell line, the PC cells, are transformed by human c-myc (PC myc), TPO and TG mRNAs are both present at normal levels, while I- uptake is slightly decreased; in the PC cells transformed by polyomavirus middle-T-antigen (PC PyMLV) TPO mRNA is undetectable and I- uptake is greatly decreased, while TG mRNA is present at normal levels. All three differentiated functions are switched off in PC cells transformed by the cooperation of c-myc and polyomavirus middle-T-antigen (PC myc + PyMLV).     

Variations in immunocytochemical localization of cathepsin B and thyroxine in follicular cells of the rat thyroid gland and plasma TSH concentrations over 24 hours

Summary Immunocytochemical localization of cathepsin B and thyroxine (T4) in follicular cells of the rat thyroid gland and plasma concentrations of thyroid stimulating hormone (TSH) were examined at six evenly spaced times over 24 h. By light- and electron microscopy, immunodeposits for cathepsin B were localized in cytoplasmic granules of various sizes, whereas those for T4 were detected mainly in larger granules of the cells and in the colloid lumen. The size and location of cytoplasmic granules showing immunoreactivity for cathepsin B and T4 in the cells varied over 24 h, corresponding to a change in plasma TSH concentrations. These immunopositive large granules appeared in the apical cytoplasm at 12.00 h, when the level of TSH was highest. At 20.00 h when the level of TSH was lowest, T4-positive granules almost disappeared, and cathepsin B-positive small granules were abundantly seen in the basal region. From 00.00 h to 08.00 h, these positive granules changed in the same manner as those seen from 12.00 h to 20.00 h, associated with an increase in plasma TSH levels. These results suggest that newly formed colloid droplets migrate from the apical to the basal regions. Cathepsin B may play a role not only in the degradation of thyroglobulin but in the maturation of thyroid hormones during the migration of the granules.     

Immunocytochemical demonstration of caldesmon and actin in thyroid glands of rats

Summary The distribution of caldesmon (a calmodulin-binding, F-actin interacting protein; Sobue et al. 1982) and actin was studied in the rat thyroid gland by means of light-microscopic immunocytochemistry, and the fine-structural distribution of actin filaments was examined by use of heavy meromyosin (HMM). Caldesmon and actin were demonstrated in the apical cytoplasm of almost all the follicle epithelial cells in normal as well as TSH-treated animals. Immunoreactivities for both caldesmon and actin showed almost the same pattern in localization. The smooth muscle cells of the blood vessels were also positive for caldesmon and actin. By electron microscopy, numerous actin filaments decorated by HMM and running perpendicularly or randomly to the apical surface were recognized in the apical cytoplasm of the follicle epithelial cell. These results suggest that caldesmon and actin, in conjugation with calmodulin, play a role in the regulation of cellular activity such as exocytosis and endocytosis in the apical portion of the follicle epithelial cell.This study was supported by grants from the Ministry of Education, Science and Culture, Japan     

Cyclic localization change of Golgi apparatus in Sertoli cells induced by mature spermatids in rats

Previously we reported that the intracellular localization of the Golgi apparatus of rat Sertoli cells changes during the seminiferous epithelial cycle, and that the cyclic changes seem to be correlated to specific generations of germ cells. To ascertain which generations of germ cells are responsible for the cyclic changes, we determined the relative volume of the Golgi apparatus within the basal, mid, and apical cytoplasm of Sertoli cells in testes with and without mature spermatids. In normal adult rats, the Golgi apparatus was usually localized exclusively in the basal cytoplasm, whereas at stages VII-IX it increased remarkably in mid and apical cytoplasm, with a concomitant decrease in the basal cytoplasm. In young adult testes without spermatids at steps 15-19 of spermiogenesis (2nd layer spermatids), the Golgi apparatus was localized in the basal cytoplasm throughout the seminiferous epithelial cycle. Orchiopexy maintained for 35 days following 60 days of cryptorchidism allowed germ cells to regenerate to spermatids at steps 1-14 of sperminogenesis (1st layer spermatids), but failed to change the intracellular localization of the Golgi apparatus in Sertoli cells. At 50 days after orchiopexy, when all generations of germ cells appeared in the tubules, the cyclic changes in localization of the Golgi apparatus were restored similar to those in normal adult testes. These findings indicate that the cyclic change in localization of the Golgi apparatus in Sertoli cells is evoked by the presence of 2nd layer spermatids.     

Thyrotropin chronically regulates the pool of thyroperoxidase and its intracellular distribution: A quantitative confocal microscopic study

The regulation of thyroperoxidase (TPO) expression and of its intracellular distribution was studied in porcine thyroid cells cultured on porous bottom filters. Cells were cultured for 18 days in the absence or in the presence of thyrotropin (TSH) and with or without iodide. Microsomes were purified and analyzed by electrophoresis. TPO was detected by immunoblotting with polyclonal anti-porcine TPO antibodies and quantified by scanning the bands. The amount of TPO was increased 2-fold by TSH. High concentrations of iodide (1–50 μM, added daily) decreased the level of TPO. Confocal microscopy served to determine the intracellular localization of TPO and its quantitative distribution. Intracellular and surface-located TPO was detected by fluorescein-labeled antibodies on saponin-treated cells. Quantitative confocal microscopy showed that TSH increased the total amount of TPO 2-fold as for immunoblotting. The highest amount of TPO was found in the perinuclear area and between the nucleus and the Golgi apparatus. Only 4% of TPO was present on the apical surface and about 1% on the basolateral membrane; the remainder (about 95%) was inside the cells. TSH did not change these relative contents. TSH modified the intracellular distribution of the enzyme, increasing the TPO pool from the perinuclear area to apical membrane. This domain could be a site of storage of TPO. Adding a physiological concentration of iodide (0.5 μM, daily) did not influence the intracellular distribution of TPO. We concluded that chronic TSH stimulation (1) increased 2-fold the pool of TPO but did not change the relative proportion of TPO inside the cells and on the apical surface, and (2) modified the intracellular distribution of vesicular TPO, the major part of which was accumulated in the perinuclear and cytoplasmic area under the subapical domain of the polarized cells. J. Cell. Physiol. 174:160–169, 1998. © 1998 Wiley-Liss, Inc.     

Regulation of thyroid peroxidase activity by thyrotropin, epidermal growth factor and phorbol ester in porcine thyroid follicles cultured in suspension

The activity of thyroid peroxidase (TPO) in porcine follicles cultured for 96 h in suspension with five hormones (5H) still attained over 50% of that in the freshly isolated follicles. On the other hand, the activity in those cultured with 5H + TSH (6H) was several times higher than that cultured with 5H after 96 h, although an initial decrease of TPO activity during the first 24 h of culture was observed in both conditions. The ability of follicles to metabolize iodide (uptake and organification) when cultured with 6H for 96 h was also several times higher than that of those cultured with 5H. The half-maximal dose of TSH for stimulation of TPO activity and iodide metabolism was 0.03-0.04 mU/ml and the effect was mediated by cAMP. These results indicate that in porcine thyroid follicles in primary suspension culture, TPO activity as well as the ability of iodide metabolism is induced by chronic TSH stimulation. In addition, epidermal growth factor (EGF, 10(-9)M) and phorbol 12-myristate 13-acetate (PMA, 10(-8) M) completely inhibited TSH stimulation on both activities and also basal (5H) activity of iodide metabolism.     

Fine structural aspects of the black thyroid induced by minocycline, and the effects of a low iodine diet, propylthiouracil, thyroxine tablet and TSH, on the black discoloration of the rat thyroid

Fine structural aspects of the effect of minocycline, an antibiotic of the tetracycline group, on the rat thyroid were studied. In all the rats administered minocycline (100 mg/kg/day) for 21 days, diffuse black discoloration of the thyroid gland occurred. However, when the rats were fed on a low iodine diet, given propylthiouracil (PTU) or thyroxine tablet with minocycline the black pigmentation of the thyroid gland did not take place. On the other hand, black discoloration of the thyroid was accelerated in the rats administered TSH and minocycline simultaneously. Ultrastructurally, numerous dense bodies containing highly electron-dense deposits were seen in the supranuclear region of the follicular epithelial cells of the black thyroid. These dense bodies, which showed positive acid phosphatase activity, are considered to be lysosomes containing minocycline or its derivatives. It is speculated that minocycline is taken up into follicular epithelial cells with iodine, and that the black discoloration of the thyroid gland is intimately related to iodine metabolism.     

Effects of inorganic iodide, epidermal growth factor and phorbol ester on hormone synthesis by porcine thyroid follicles cultured in suspension.

Porcine thyroid follicles cultured in suspension for 96 h synthesized and secreted thyroid hormones in the presence of thyrotropin (TSH). The secretion of newly synthesized hormones was assessed by determining the contents of thyroxine (T4) and triiodothyronine (T3) in the media and by paperchromatographic analysis of 125I-labelled hormones in the media where the follicles were cultured in the presence and absence of inhibitors of hormone synthesis. The hormone synthesis and secretion was modified by exogenously added NaI (0.1-100 microM). The maximal response was obtained at 1 microM. Thyroid peroxidase (TPO) activity in the cultured follicles with TSH for 96 h was dose-dependently inhibited by NaI. One hundred microM of NaI completely inhibited TSH-induced TPO activity. Moreover, both epidermal growth factor (EGF: 10(-9) and 10(-8) M) and phorbol 12-myristate 13-acetate (PMA: 10(-8) and 10(-7) M) inhibited de novo hormone synthesis. An induction of TPO activity by TSH was also inhibited by either agent. These data provide direct evidences that thyroid hormone synthesis is regulated by NaI as well as TSH at least in part via regulation of TPO activity and also that both EGF and PMA are inhibitory on thyroid hormone formation.     

Regulation of thyroperoxidase, thyroglobulin and iodide levels in sheep thyroid cells by TSH, tumor promoters and epidermal growth factor

Using sheep thyroid cells in culture, we have studied the effects of thyroid stimulating hormone (TSH), epidermal growth factor (EGF) and the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) on the activity and expression of both thyroglobulin (Tg) and thyroid peroxidase (TPO) and on the ability of cells to trap and organify iodide. Using Western blotting techniques, we found that TSH increased the absolute cellular levels of Tg. The optimum TSH concentration for Tg mRNA production was between 0.1-1.0 mU/ml. Thyroglobulin mRNA levels were stimulated by TSH but detectable levels were also present in cultures grown in its absence containing cortisol, insulin, transferrin, somatostatin and glycyl-lysyl-histidyl acetate. Unlike Tg, TPO protein levels were found to be completely dependent upon TSH. A time course of TSH stimulation of TPO mRNA showed increases after 8 h of TSH stimulation, whereas induction of Tg mRNA by TSH was seen at 24 h. Iodide trapping and organification were also TSH-dependent processes, showing maximum activities at 300-500 muU/ml of TSH. The addition of 10 nM TPA caused a biphasic decrease in radiolabeled pertechnetate uptake, with complete inhibition being seen at 14 h. Inhibition of iodide organification occurred more rapidly. TPA and EGF (1 nM) reduced the amount of newly synthesized Tg in TSH-stimulated cells by 50% but the absolute amount of Tg within the cells was not markedly inhibited at these early times.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endogeneous peroxidase activity in the frog thyroid gland

Summary The fine structural localization of the endogeneous peroxidase activity in the thyroid of the young frog was studied. The reaction product for peroxidase was observed over the peripheral luminal colloid and apical region of the follicular epithelial cell. Most apical small granules and some parts of Golgi lamellae and a few Golgi vesicles were specifically stained. The cisternae of rough endoplasmic reticulum and the nuclear cisternae did not demonstrate any positive reaction for peroxidase activity with difference from that of various cells of mammalia. In this study, only mature peroxidase seems to be positive for its reaction and the enzyme in the rough endoplasmic reticulum is considered to be too immature to react for DAB method in the frog thyroid cell. The relationship between the localization of peroxidase reaction and the site of the iodination of thyroglobulin was discussed.     

In vitro and in vivo regulation of thyrotropin receptor mRNA levels in dog and human thyroid cells.

Regulation of thyrotropin (TSH) receptor (TSHr) mRNA accumulation as compared with two other thyroid differentiation markers (thyroglobulin and thyroperoxidase (TPO] has been investigated by Northern blot. In dogs in vivo, chronic stimulation of the thyroid TSHr mRNA although it increased the levels of thyroglobulin and TPO mRNA. In dogs treated with thyroxin, the quiescent thyroids expressed normal levels of TSHr and TPO mRNA but depressed levels of thyroglobulin mRNA. In primary cultures of dog thyrocytes, dedifferentiation of the cells by treatment with epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate led to decreased TSHr mRNA levels and nearly abolished thyroglobulin and TPO gene expression. However, TSHr mRNA was always present, compatible with the fact that these cells, when treated by TSH, reexpress differentiation. Treatment of the cells with TSH or forskolin transiently increased the TSHr mRNA level after 20 h, an effect inhibited by cycloheximide. This up-regulation was confirmed at the protein level: forskolin-treated cells showed an enhanced cAMP response to TSH and an increased binding of labeled TSH to their membranes. Long term TSH treatment led to a slight down-regulation of TSHr mRNA in dog thyrocytes, but in human thyroid cells no marked down-regulation was observed.     

Iodide transport in primary cultured thyroid follicle cells: evidence of a TSH-regulated channel mediating iodide efflux selectively across the apical domain of the plasma membrane.

The transport of iodide was studied in porcine thyroid follicle cells cultured in bicameral chambers. The continuous layer of polarized follicle cells, joined by tight junctions, formed a diffusion barrier between the two compartments (apical and basal) of the culture chamber. Uptake and efflux of 125I- at either surface (apical and basolateral) of the cells were thus possible to determine. Protein binding of iodide was inhibited by methimazole (10(-3) M) in all experiments. Radioiodide was taken up by the cells from the basal medium in a thyroid-stimulating hormone (TSH)-dose dependent manner with a maximal cell/medium ratio of 125I- of about 50 in cultures prestimulated with 0.1 to 1 mU/ml for 2 days. This uptake was inhibited by perchlorate and ouabain. In contrast, 125I- was not taken up from the apical medium. In preloaded cells, iodide efflux was rapidly (within 1-2 min) and dose-dependently (0.1-10 mU/ml) stimulated by TSH. Bidirectional measurements revealed that TSH stimulated iodide efflux in apical direction, leaving efflux in basal direction unchanged. In experiments with continuous uptake of label from the basal compartment, the TSH-stimulated efflux in apical direction had a duration of 4 to 6 min and resulted in a reduction in the cellular content of radioiodide by up to 80%. Decreased levels of cellular 125I- remained for at least 15 min after TSH addition. From our observations we conclude that the TSH-regulated uptake and efflux of iodide take place at opposite surfaces of the porcine thyroid follicle cell. Acutely stimulated iodide efflux is not the result of an increased permeability for iodide in the entire plasma membrane but only in the apical domain of this membrane. This implicates the presence of an iodide channel mediating TSH-stimulated efflux across the apical plasma membrane of the follicle cell. The mechanism is suggested to facilitate a vectorial transport of iodide in apical direction, i.e., to the lumen of the intact follicle.     

Effects of thyroidectomy, propylthiouracil, and thyroxine on pituitary content and immunocytochemical staining of thyrotropin (TSH) and thyrotropin releasing hormone (TRH)

Previous studies have demonstrated immunocytochemical staining for beta chains of thyroid stimulating hormone (TSH-beta) in rough endoplasmic reticulum of pituitary cells hypertrophied after thyroidectomy ("thyroidectomy cells") (Moriarty CG(1976): J Histochem Cytochem (24:846; Moriarty GC, Tobin RB (1976): J Histochem Cytochem 24:1140). Here we report the localization of thyrotropin releasing hormone (TRH) in serial sections of the same pituitaries to determine if it could be found at similar sites. No staining for TRH was found in hypertrophied TSH cells formed 42 days after the surgery, or after 14, 34, and 70 days of propylthiouracil (PTU) treatment. The loss in immunostaining in the PTU-treated rats was correlated with radioimmunoassay (RIA) measurements that showed a 65% reduction in anterior pituitary TRH content after 34, 70, and 98 days of PTU treatment (from 22.9--7.8 pg/mg wet wt) and a 50% reduction in TSH content after 34 days of treatment. When thyroxine was administered to hypothyroid rats for 3 days before death, our previous studies had demonstrated intense staining for TSH in granules inside the rough endoplasmic reticulum. In this study, the radioimmunoassay showed that TSH content rose dramatically in the hypothyroid animals treated with PTU for 77 days and thyroxine for 2 days before death (from 8.5--64.1 mU/mg wet wt); however, the rise in TRH content was minimal (5.8--9.8 pg/mg wet wt). The immunocytochemical stain for TRH correlated well with the RIA showing a weak reaction mainly on small granules in the cytoplasm. No reaction for TRH was found in rough endoplasmic reticulum. These results suggest that TRH and TSH storage sites are dissimilar in the hypothyroid rat. The presence of stain for TRH in granules in the cytoplasm suggests that it might play a role in the storage or packaging of TSH. Its absence in profiles of rough endoplasmic reticulum staining intensely for TSH suggests that it is not synthesized at this site. No definite conclusions about its origin can be drawn at this time.     

生长抑素在大鼠乳腺组织中的分布和定位

目的研究生长抑素在大鼠乳腺组织中的分布和定位。方法本实验应用即用型快速免疫组化方法对处女期、妊娠6 d、12 d、18 d和泌乳6 d、12 d、18 d的SD大鼠的乳腺进行生长抑素检测。结果发现从处女期到泌乳期大鼠乳腺组织中均有生长抑素的表达,且主要分布于上皮细胞的胞质和腺泡的分泌物中。结论大鼠乳腺上皮细胞的胞质和腺泡的分泌物中有生长抑素的分布。