The factors mediating the accumulation of thyroglobulin are of great importance to the understanding of the pathogenesis of human and experimentally induced colloid goiters. To elucidate further the underlying cellular mechanism, thyroid fragments from newborn rats were incorporated into semisolid alginate beads and were cultured as three-dimensional organoids for up to 21 d. In five parallel cultures, the medium contained either no supplements (group A), Nal (group B), thyroid-stimulating hormone (TSH) (group C), Nal plus TSH in the same concentrations as B and C (group D), or Nal and TSH (as in group D) plus methimazole (MMI, group E). The thyroid organoids maintained morphological intergrity, functional activity, and ability to proliferate in vitro. Addition of iodine to the cultures significantly increased mean (±SEM) follicular diameters from 19.5±0.7 μm in controls to 33.9±2.2 μm (p<0.0001) when Nal was added alone (group B), and 30.4±1.7 μm (p<0.0001) when combined with TSH (group D). The effect of Nal on follicular size was abolished by MMI (group E, follicular diameter 23.5±1.3 μm). The results presented support the recent finding, using a rat colloid goiter model, that not only TSH but also iodine organification or its inhibition are important factors in modulating follicular morpholog

Büchler, Markus

Bürgi, Ulrich

Bürgi-Saville, Mary

Gebauer, Mathias

Gerber, Hans

Glaser, Christine

Marti, Ulrich

Peter, Hans-Jakob

Ruchti, Charles

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165Iodine Organification and Follicular Size/Glaser et al.Vol. 11, No. 2Inhibition of Iodine Organification and Regulationof Follicular Size in Rat Thyroid Tissue In VitroChristine Glaser,5 Ulrich Marti,1,2 Mary Elizabeth Bürgi-Saville,1 Charles Ruchti,4Mathias Gebauer,3 Markus W. Büchler,5 Hans Gerber,2 Ulrich Bürgi,1 and Hans-Jakob Peter31Endocrine and Diabetes Division; 2Department of Clinical Chemistry; 3Division of Medicine, Anna Seiler;4Department of Pathology, 5Department of Visceral Surgery, University Hospital, Bern, SwitzerlandEndocrine, vol. 11, no. 2, 165–170, October 1999 0969–711X/99/11:165–170/$11.50 © 1999 by Humana Press Inc. All rights of any nature whatsoever reserved.Received June 29, 1999; Revised July 26, 1999; Accepted July 26, 1999.Author to whom all correspondence and reprint requests should beaddressed: U. Marti, Endocrine and Diabetes Division, UniversityHospital Bern, Freiburgstrasse 15, CH-3010 Bern, Switzerland. E-mail:ulrich.marti@dkf2.unibe.chThe factors mediating the accumulation of thyroglo-bulin are of great importance to the understanding ofthe pathogenesis of human and experimentallyinduced colloid goiters. To elucidate further theunderlying cellular mechanism, thyroid fragmentsfrom newborn rats were incorporated into semisolidalginate beads and were cultured as three-dimensionalorganoids for up to 21 d. In five parallel cultures, themedium contained either no supplements (group A),NaI (group B), thyroid-stimulating hormone (TSH)(group C), NaI plus TSH in the same concentrations asB and C (group D), or NaI and TSH (as in group D) plusmethimazole (MMI, group E). The thyroid organoidsmaintained morphological integrity, functional activ-ity, and ability to proliferate in vitro. Addition of iodineto the cultures significantly increased mean (±SEM)follicular diameters from 19.5 ± 0.7 µm in controls to33.9 ± 2.2 µm (p < 0.0001) when NaI was added alone(group B), and 30.4 ± 1.7 µm (p < 0.0001) when com-bined with TSH (group D). The effect of NaI on folli-cular size was abolished by MMI (group E, folliculardiameter 23.5 ± 1.3 µm). The results presented sup-port the recent finding, using a rat colloid goiter model,that not only TSH but also iodine organification or itsinhibition are important factors in modulating follicu-lar morphology.Key Words: Follicular diameter; alginate culture; thy-roid gland; baby rat.IntroductionThe pathogenesis of colloid goiter has been a subject ofdebate for many years. In the 1920s, it was proposed thatcolloid-rich goiters developed in hyperplastic glands, afterthe removal of the goitrogenic stimulus (1,2). This conceptwas generally accepted for 50 yr (3,4). However, itexplained neither the observation that colloid accumu-lation occurs in most goiters exposed to chronicallyincreased thyroid-stimulating hormone (TSH) (which isstill a causative factor of many endemic goiters) northe coexistence of microfollicular areas containing littlecolloid and macrofollicular colloid-rich areas in simplegoiters (5–8).Later, it became apparent that the supply of thyroidiodine was also an important factor in the development ofcolloid goiters. It was reported that thyroglobulin accumu-lated in the thyroid glands of rats and mice injected withTSH and, at the same time, fed a high iodine diet (9). Fur-thermore, it could be shown that colloid goiters could beinduced in rats and mice by treatment with 5,5-diphenyl-2-thiohydantoin (DPTH) (10). This substance raises TSH byincreasing fecal loss of thyroid hormones and inhibitingthe conversion of thyroxine (T4) to triiodothyronine (T3),without influencing iodine uptake/organification by thethyroid gland (3,11). By contrast, when iodine organi-fication was blocked by methimazole (MMI) in DPTH-treated rats, hyperplastic, microfollicular goiters developed(10). These reports clearly established the idea that colloidaccumulation not depends on intense TSH stimulation butalso on thyroidal iodine uptake.The extracellular matrix (ECM) and its specializedform, the basement membrane (BM), play very importantroles in the replication, organization and biological func-tion of cells (for a review, see ref. 12). Therefore, it wouldseem possible that variations in the ECM/BM might beinvolved in disease states of tissues, particularly those inwhich alterations in tissue structure are involved, e.g., ingoiter or tumor growth. The ECM/BM in the thyroid hasreceived relatively little attention. Recently, we describedthe distribution patterns of various ECM/BM components(laminin, collagen IV, perlecan, fibronectin, osteonectinand some laminin variants) in normal and toxic adenomatoushuman thyroid tissue (13). Although there were no striking165166 EndocrineIodine Organification and Follicular Size/Glaser et al.differences in the composition of the ECM/BM in theadenomas studied, there was a quite marked production offibronectin in the capsules surrounding the adenomas (13).In a further report, we were able to show the ability ofthe alginate gel culture system to retain ECM and follicularstructure of rat thyroid tissue. In addition, iodine organi-fication was maintained in the cultured organoids (14).The present study was undertaken with two aims: first,to confirm the influence of iodine supply on colloid accu-mulation previously demonstrated in vivo (9,10) in adefined in vitro model using thyroid fragments from new-born rats; and, second, to study the distribution patterns ofsome ECM/BM components under different experimentalconditions.ResultsMorphological FindingsTable 1 summarizes the measurements of folliculardiameter from the five groups studied. Follicular diameterwas significantly increased in groups B (NaI) and D (NaI/TSH). This increase in follicular diameter was apparentlyinhibited when iodine uptake by the cells was blocked bythe addition of MMI to the culture medium (group E).These differences in follicular size are illustrated in Fig. 1,which shows immunohistochemical staining for thyroglo-bulin in the five groups. In cultures treated with NaI (groupB) or NaI plus TSH (group D), large follicles are presentfilled with thyroglobulin containing colloid. In group E,which was treated with MMI, the follicles are small andthe colloid is almost absent.BM components collagen IV, fibronectin, perlecan, andlaminin were investigated by immunohistochemical stain-ing; Fig. 2 shows an example of the results obtained forlaminin. Staining for this BM component was observedaround individual follicles in all five groups, and there wasno apparent difference in the intensity of the stainingamong the groups. Similar results were found for the otherBM components (data not shown).5-Bromo-2-deoxyuridine (BrdU) incorporation andimmunostaining showed that all groups contained divid-ing cells (data not shown).DiscussionSome years ago, in an in vivo model, it was shown thatDPTH induces large colloid-rich goiters by increasing fecalloss of thyroid hormones and inhibiting conversion of T4 toT3 (10), thereby increasing endogenous TSH and inducinggoiters without impairing iodine binding. DPTH goiterstreated with MMI were microfollicular, although serum TSHwas increased to the same level as in the goiters treated withDPTH alone. These findings supported our hypothesis thatthe degree of iodine organification and not TSH is the deci-sive factor in determining thyroid follicular size (9,10).The results of the present study confirm our earlier invivo experiments (9,10). Contrary to the classical concept(2) that colloid goiters are only produced from originallyhyperplastic microfollicular goiters when the goitrogenicstimulus ceases, we have shown, in vitro, that elevated TSHand colloid accumulation are not mutually exclusiveeffects. Tissue treated with iodine (group B) has folliculardiameters nearly twice as large as those of the control group(Table 1). The simultaneous exposure of the tissue to TSHand iodine (group D) demonstrates that TSH has no addi-tional effect on the follicular diameters. However, the inhi-bition of iodine organification by MMI reverses theiodine-induced increase in follicular diameter (group E).TSH alone dose not significantly affect the folliculardiameter (group C). The BrdU incorporation experimentconfirms that the cells, in all groups, are dividing in thealginate beads. It is now clear that iodine organification orits inhibition is also an important factor influencingwhether thyroid tissue becomes micro- or macrofollicular.Immunohistochemical studies on the basement mem-brane components laminin, fibronectin, perlecan, and col-lagen IV have confirmed their presence and normaldistribution in the alginate-embedded tissue. However,there was no obvious difference between the groups.Although iodine can affect follicular size, these results sug-gest that none of the culture media supplements used inthis study affect the BM composition.This in vitro model will facilitate further investigationsinto the regulation of follicle size and thyroglobulin accu-mulation, under defined in vitro conditions.Table 1Mean Follicular Diameters (±SEM)GroupA B C D ETreatment Control NaI TSH NaI/TSH NaI/TSH/MMIn 124 136 58 146 103Folliculardiameter (µm) 19.5 ± 0.7 33.9 ± 2.2a 23.6 ± 1.4b 30.4 ± 1.7a 23.5 ± 1.3b,cap < 0.0001 vs control.bp < 0.0001 vs B.cp < 0.0001 vs D. (All other statistical differences are not significant.)167Iodine Organification and Follicular Size/Glaser et al.Vol. 11, No. 2Fig. 1. Immunohistochemical staining for thyroglobulin in each of the treated groups: (A) Control group A, no supplements;(B) group B (1 µM NaI); (C) group C (1 U/L TSH); (D) group D (1µM NaI/1 U/L TSH); and (E) group E (1 µM NaI/1 U/L TSHand 1 mM MMI). In cultures treated with NaI (B) or NaI plus TSH (D), large follicles filled with thyroglobulin-containingcolloid are present. In group E, which was treated with MMI in addition to NaI and TSH, follicles are small and the colloid isalmost absent.168 EndocrineIodine Organification and Follicular Size/Glaser et al.Fig. 2. Immunohistochemical staining for laminin in each of the tested groups: (A) Control group A, no supplements; (B)group B (1 µM NaI); (C) group C (1 U/L TSH); (D) group D (1µM NaI/1 U/L TSH); and (E) group E (1 µM NaI/1 U/L TSHand 1 mM MMI). The BM component laminin is expressed and shows a normal distribution. However there is no obviousdifference among the groups.169Iodine Organification and Follicular Size/Glaser et al.Vol. 11, No. 2Materials and MethodsCulture of Thyroid OrganoidsNewborn Wistar rats weighing approx 25 g and not olderthan 48 h (from the Institute of Pathophysiology, Univer-sity of Bern, Switzerland) were used. They were killed byCO2 exposure in a closed chamber and were then thy-roidectomized under aseptic conditions. Five independentexperiments with a total of 50 animals were performed. Allanimal experimentation was conducted with the higheststandards of animal care, according to the ethical principlesand guidelines of the Swiss Academy of Medical Sciencesand approved by governmental authorities.The thyroid tissue was cultured in a semisolid alginatebead system composed exclusively of polymerized algi-nate (Keltone LV, Kelco, Chicago, IL), which allowsthree-dimensional growth of follicles, cell clusters, andorganoids, and itself contains no ECM components(13,15). To initiate the cultures, each thyroid lobe wassliced into four pieces of about 1 mm side length, and thetissue was left in Coon’s F-12 medium, supplemented withpenicillin (100 U/mL), streptomycin (100 µg/mL), L-glutamine (200 nM) (Seromed, Biochrom KG, Berlin,Germany), 5% newborn calf serum (Seromed), insulin (1.7µM), transferrin (55.6 nM), hydrocortisone (8.8 nM),glycin-L-histidyl-L-lysine (29.7 nM), and somatostatin (6.1nM) (Sigma Division of Fluka, Buchs, Switzerland) with-out TSH for 24 h. The tissue was then incorporated intoalginate beads approx 5 mm in diameter as previouslydescribed (13,14). The beads were randomly distributedamong five groups (A–E). Group A was the control groupin which no additions were made to the medium. In groupsB–E the culture medium was supplemented as follows: B,NaI (1 µM); C, TSH (1U/L, Thytropar, Rover, Fort Wash-ington, PA); D, NaI and TSH of the same concentrations asgroups B and C; E, NaI, TSH, and MMI (1 mM, Fluka AG,Buchs SG, Switzerland). The thyroid organoids were cul-tured for up to 21 d, during which the culture medium waschanged every 2 to 3 d. In two of the five experiments, cellproliferation was documented by the incorporation ofBrdU (Sigma, St. Louis, MO) into newly synthesized DNAduring S-phase of the proliferating thyroid tissue in cul-ture. BrdU can later be visualized in paraffin sections ofcell clusters and organoids by indirect immunohistochem-istry (16,17). BrdU was added to the culture medium at aconcentration of 0.06 mg/mL for the last 14 h before har-vesting the cells.At the end of the culture period, the beads were fixed in4% phosphate-buffered formalin, pH 7.4, containing 7 mMCaCl2. The alginate was then dissolved in a sodium citratebuffer (150 mM NaCl, 55 mM Na citrate, 20 mM EDTA,pH 5.1). The cell clusters and organoids released were pro-cessed into a cell block using a Shandon cytoblock kit(Shandon, Pittsburgh, PA) and embedded in paraffin forfurther analyses, as previously described (13,14).Morphological and Immunohistochemical StudiesTwo-micrometer paraffin sections were stained withperiodic acid Schiff and hematoxylin for morphologicaland morphometric analyses. Follicle diameters were mea-sured with a test screen (linear test circle series) accordingto Weibel (18). At least 58 follicles from each group wereevaluated. For the first three experiments, photographswere taken of each section (magnification ×273), and allfollicles visible in the photographs were measured. For thelast two experiments, a Visopan 314 projection microscope(Reichert-Jung, Vienna, Austria; magnification ×500) wasused with the same test screen as in the first three experi-ments. Mean and standard error of the mean (SEM) of fol-licular diameters were calculated (Table 1).Alkaline phosphatase immunohistochemical staining ofparaffin sections was used to study the distribution of ECMcomponents. An indirect immunohistochemical methodwas performed as previously described (14) using rabbitpolyclonal primary antibodies to EHS laminin (Sigma),human fibronectin (Dako A/S, Glostrup, Denmark), mousecollagen IV (Becton Dickenson), perlecan (kindly providedby Prof. J. Hassel, University of Pittsburgh, Pittsburgh, PA),and a swine antirabbit alkaline phosphatase–conjugatedsecondary antibody (Dako).Thyroglobulin was also localized using the same method,with a rabbit polyclonal human thyroglobulin antibody(Dako) crossreacting with rat thyroglobulin and the samesecondary antibody as just described.BrdU was visualized using a triple antibody methodwith a mouse monoclonal bromodeoxyuridine antibody(Dako) followed by rabbit antimouse immunoglobulins(Dako) and finally mouse monoclonal alkaline phos-phatase antialkaline phosphatase (Dako). The second andthird steps were repeated, and the sections were thenexposed to the substrate solution for 25 min, as previouslydescribed (14). The slides were then rinsed overnight inrunning tap water, counterstained with hematoxylin, andsealed with Entellan (Merck, Darmstadt, Germany).Statistical AnalysesResults of the measurements of follicular diameters arepresented as means ± SEM (Table 1). Statistical differencesamong the groups were tested by one-way analysis of vari-ance using the Bonferroni/Dunn post-hoc test.AcknowledgmentsThis work was supported by grants no. 31-34416.92 and32-039355.93 from the Swiss National Science Foundation.References1. Marine, D. (1928) In: Comtes Rendues de la ConférenceInternationale du Goiter 68–82, Hans Huber Verlag: Bern,Switzerland.2. Marine, D. (1923) Western Reserve University Bulletin. Gra-ham, W. (ed.) 7-123, Cleveland, OH.170 EndocrineIodine Organification and Follicular Size/Glaser et al.3. Astwood, E. B., Bissel, A., and Hughes, A. M. (1945) Endocri-nology 37, 456–481.4. Follis, R. H. Jr. (1959) Proc. Soc. Exp. Biol. Med. 100, 203–206.5. Walthard, B. (1969) In: Spezielle Pathologische Anatomie.Doerr, W., Seifert, O., and Uehlinger, E. (eds.). Springer Verlag:New York.6. Studer, H., Kohler, H., and Buergi, H. (1974). In: Thyroid.Handbook of Physiology. Greer, M. A. and Solomon, D. H.,(eds.). American Physiological Society: Washington, DC.7. Ramelli, F., Studer, H., and Bruggisser, D. (1982). Am. J.Pathol. 109, 215–223.8. Studer, H., and Gerber, H. (1991). In: Werner’s and Ingbar’s,The Thyroid. Braverman, L. E. and Utiger, R., (eds.). JBLippincott: Philadelphia.9. Gerber, H., Studer, H., Conti, A., Engler, H., Kohler, H., andHaeberli, A. (1981). J. Clin. Invest. 68, 1338–1347.10. Gerber, H., Huber, G., Peter, H. J., Kämpf, J., Lemarchand-Beraud, T., Fragu, P., and Stocker, R. (1994). Endocrinology135, 2688–2699.11. Tsukui, T., Aizawa, T., Yamada, T., and Kawabe, T. (1978).Endocrinology 102, 1662–1669.12. Hay, E. D. (1991). Cell Biology of the Extracellular Matrix, 2nded., Plenum: New York.13. Bürgi-Saville, M. E., Gerber, H., Peter, H. J., Paulsson, M.,Aeschlimann, D., Glaser, C., Kämpf, J., Ruchti, C., Sidiropoulos,I., and Bürgi, U. (1997). Thyroid 7, 347–356.14. Bürgi-Saville, M. E., Reut, B., Gerber, H., Peter, H. J., Paulsson,M., Kämpf, J., Simon, F., Marti, U., and Bürgi, U. (1998).Thyroid 8, 1147–1155.15. Hauselmann, H. J., Aydelotte, M. B., Schumacher, B. L.,Kuettner, K. E., Gitelis, S. H., and Thonar, E. J. (1992). Matrix12, 116–129.16. Ellwart, J. and Dormer, P. (1985). Cytometry 6, 513–520.17. Magaud, J. P., Sargent, I., Clarke, P. J., French, M., Rimokh,R., and Mason, D. Y. (1989). J. Histochem. Cytochem. 37,1517–1527.18. Weibel, E. R. (1979). Practical Methods for Biological Mor-phometry, vol. 1. Academic: London.

Inhibition of iodine organification and regulation of follicular size in rat thyroid tissue in vitro

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Inhibition of iodine organification and regulation of follicular size in rat thyroid tissue in vitro

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