*5.3.2. Morphological study*

372 Thyroid Hormone

**Table 10.** The effect of I-

gland *in vivo* [101,102].

did not exceed 11% for accumulated iodide.

more in the three doses than euthyroidic follicles, presenting organification in the presence of 10E-3 M NaI with or without TSH. Iodide accumulation in these follicles in the presence of perchlorate (30 M) was only inhibited at 12 h in the presence of 10E-3 M NaI and at 3 h in the presence of 10E-7 M NaI (euthyroidic follicles in the presence of 30 M perchlorate inhibited iodide capture at all concentrations) and O/A percentages in follicles in the presence of 10E-7 M NaI were greater in the absence of TSH than in their presence (Table

**NaI 10E-10 M 10E-7 M 10E-3 M Culture TSH % O/A N % O/A N % O/A N**  0.5 - 77.9 ± 0.61 4 74.9 ± 0.63 4 6.3 ± 0.63 3

3 - 75.8 ± 0.49 4 68.7 ± 9.14 4 5.4 ± 0.63 3

12 - ND 87.3 ± 2.63 4 10.4 ± 0.63 3

24 - ND 44.3 ± 1.97 4 11.6 ± 0.63 4

pig hypothyroid follicles cultured. Follicles cultured on agarose with 0.5% FCS for 1 day; the medium was changed and culture began with different experimental points. The follicles were keep in culture along the time (culture in hours) with 5 µCi/mL Na125I, with (+) or without 1 mU/mL TSH (-) and doses of Na127I (NaI). Each value was the average of iodide accumulated or organified (O/A) iodide expressed

Iodide accumulation values in the presence of 10E-3 M NaI in hypothyroid follicles were 2 times greater in follicles in the presence of TSH than without TSH, had organification in similar proportions to euthyroidic follicles in the presence of 10E-5 M NaI. Percentage of the proporction O/A was higher at 30 min without TSH than with TSH; it was lower at the other times. Without TSH increased with culture time whilst values became reduced regarding time with TSH (Table 10). Organification in this case was not nil, but the organified iodine

Mouse follicles cultured 3 days in the presence of 10E-7 M NaI was more intense in the presence of reactive species at the boundary between apical membrane microvellosities and colloid and the oxide reduction system became completely closed down with an excess of iodide (10E-4 M NaI) [100]. This proved that an excess of iodide inhibited the enzymes responsible

Euthyroidic follicles accumulated iodide regarding medium constant proportion concentration. Accumulated iodide was organified in the presence of 10E-10 M and 10E-7 M NaI; organification was extremely reduced in the presence of 10E-5 M and zero with 10E-3 M NaI as described *in vivo* [1]. Accumulation was greater in follicles without TSH than with TSH in the presence of 10E-3 M NaI at 12, 24 and 48 h; this reduction was similar to that of the

for organification in closed follicles as we have thought should be in the gland *in vivo.*

in %/dose/μg DNA per number (N) of culture dishes ± SD. ND: not determinated.

+ 57.3 ± 0.78 4 71.6 ± 2.84 4 15.1 ± 0.63 3

+ 60.6 ± 0.68 4 57.2 ± 9.14 4 3.0 ± 0.63 3

+ ND 72.8 ± 3.15 4 3.8 ± 0.63 3

+ ND 28.7 ± 0.99 4 9.7 ± 0.63 3

dose on the proportion accumulation/organificación pourcentage (% A/O) in

10), such value becoming reduced with culture time.

Hypothyroid follicles conserved follicular architecture during 48 h of culture in the presence of different doses of iodide with and without TSH (Figures 9A, 9B and 9C). Rat follicles and pig euthyroidic follicles at all iodide doses in the presence or absence of TSH conserved follicular architecture for 48 h (Figure 9D).

**Figure 9.** Appearance of pig follicles cultured on agarose with 0.5% FCS for 1 day; the medium was changed and culture lasted 48 h. A. Hypothyroid s follicles in the presence of 10E-10M NaI. **B.** Hypothyroid follicles in the presence of 10E-7M NaI. **C**. Hypothyroid follicles in the presence of 10E-3M NaI. **D.** Euthyroidic follicles in the presence of 10E-3M NaI. The follicular architecture of the gland *in vivo* was conserved in all cultures; hypothyroids (A, B and C) had thin epithelium and euthyroidic (D) ones cubic epithelium (IM. Scale bar: A, B, C 800 μm, D 200 μm).

The Trypan blue exclusion exam of thyrocytes from follicles cultured with 10E-3 M NaI did not have an alteration to their membranes and excluded the stain, whether being pig euthyroidic follicles cultured 48 h (Figure 10A) or rat ones cultured for 6 days (Figure 10B). The cells of cell aggregates which did not have follicular structure became stained (Figure 1C), the same as isolated cells (Figure 10B) or those found in follicles.

The ultra-structure of thyrocytes in all treatments and times preserved cell polarisation and organelle distribution (Figure 11), like the gland *in vivo* (Figure 1C); endocytic vesicles can be seen in thyrocytes' apical region (Figure 11) like follicles after 3 days of culture (Figures 6C and 6D).

Thyroid Culture from Monolayer to Closed Follicles 375

in the absence of TSH (Figures 12A and 12C). This was also observed en thyrocytes from follicles cultured for 8 h with 10E-3 M NaI and TSH, but colloid droplets were only located in the apical region in this dose (Figure 12D). Thyrocytes also had endocytic vesicles in this dose without TSH but also located in cells' apical region (Figure 12C). Thyrocytes ultrastructure in the presence of strong concentrations of iodide did not have morphological modifications, or distribution of organelles regarding normal or non-stimulated cells *in vivo*

**Figure 12.** The ultra-structure of rat follicle thyrocytes cultured on agarose with 0.5% FCS for 1 day; the medium was changed and 8 h of culture involved different experimental points: A. 10-E10 M NaI. B. 10- E10 M NaI + 0.1 mU/mL TSH. C. 10-E3 M NaI. D. 10-E3 M NaI + 0.1 mU/mL TSH. A. The ultra-structure of thyrocytes in the absence of TSH was identical to that of thyrocytes from follicles cultured for 1 day. B. Lysosomes close to the nucleus and few *colloid droplets* located in the apical pole (insert) were observed in the presence of TSH. C. They were well conserved in the presence of TSH, even in this strong dose of NaI. Endocytic vesicles were present in such strong dose of iodide. D. Colloid droplets were also observed in the presence of TSH, having the same density as colloid and were located in the thyrocytes' apical pole. Follicle centre was narrow and had abundant microvellosities (TEM. A 8,720 X,

B 12,510 X, box 10,720 X, C 7,430 X, box 70,950 X, D 13,450 X).

(Figure 1C) or *in vitro* (Figures 6C and 6D)*.*

**Figure 10.** Trypan blue exclusion exam of culture aliquots for follicles cultured on agarose with 0.5% FCS 1 day; the medium was changed and culturing involved 10E-3 M NaI. **A.** 2 day rat follicle culture. **B.** 6 day pig euthyroid follicle culture. The thyrocytes from the follicles excluded the stain whilst isolated cells did not exclude it, whether separated from the follicle (A) or on the follicle (B) (IM, Scale bar A 60 μm, B 15 μm).

**Figure 11.** The ultra-structure of rat follicle rat thyrocytes cultured on agarose with 0.5% FCS for 1 day; the medium was changed and 48-h culture involved different experimental points: **A.** 10E-7 M NaI. **B.** 10E-3 M NaI + 0.1 mU/mL TSH. Thyrocytes kept their polarisation; microvellosities were in contact with electron-dense colloid. The binding complexes were located in the lateral membrane's apical region between cells. The RER was slightly vesiculated. Thyrocytes did not have cytological differences regarding iodide dose; follicular centres only became narrowed in the presence of TSH (B) (TEM. A 8,720 X, B 10,720 X)

Follicles in the presence of TSH narrowed their follicle centres at all iodide doses used (Figures 12B and 12D), but did not undergo any ultra-structural modification in strong iodide concentrations: 10E-5M or 10E-3 M NaI. Organelle distribution was comparable to normal gland *in vivo* (Figure 1C) or follicles in long-term culture (Figures 6C and 6D).

Lysosome fusion occurred in follicles in the presence of 10E-10 M NaI and 0.1 mU/mL TSH for 8 h (Figure 12B) while this did not happen in thyrocytes' apical region (Figure 12B insert) in the absence of TSH (Figures 12A and 12C). This was also observed en thyrocytes from follicles cultured for 8 h with 10E-3 M NaI and TSH, but colloid droplets were only located in the apical region in this dose (Figure 12D). Thyrocytes also had endocytic vesicles in this dose without TSH but also located in cells' apical region (Figure 12C). Thyrocytes ultrastructure in the presence of strong concentrations of iodide did not have morphological modifications, or distribution of organelles regarding normal or non-stimulated cells *in vivo* (Figure 1C) or *in vitro* (Figures 6C and 6D)*.*

374 Thyroid Hormone

bar A 60 μm, B 15 μm).

8,720 X, B 10,720 X)

**Figure 10.** Trypan blue exclusion exam of culture aliquots for follicles cultured on agarose with 0.5% FCS 1 day; the medium was changed and culturing involved 10E-3 M NaI. **A.** 2 day rat follicle culture. **B.** 6 day pig euthyroid follicle culture. The thyrocytes from the follicles excluded the stain whilst isolated cells did not exclude it, whether separated from the follicle (A) or on the follicle (B) (IM, Scale

**Figure 11.** The ultra-structure of rat follicle rat thyrocytes cultured on agarose with 0.5% FCS for 1 day; the medium was changed and 48-h culture involved different experimental points: **A.** 10E-7 M NaI. **B.** 10E-3 M NaI + 0.1 mU/mL TSH. Thyrocytes kept their polarisation; microvellosities were in contact with electron-dense colloid. The binding complexes were located in the lateral membrane's apical region between cells. The RER was slightly vesiculated. Thyrocytes did not have cytological differences regarding iodide dose; follicular centres only became narrowed in the presence of TSH (B) (TEM. A

Follicles in the presence of TSH narrowed their follicle centres at all iodide doses used (Figures 12B and 12D), but did not undergo any ultra-structural modification in strong iodide concentrations: 10E-5M or 10E-3 M NaI. Organelle distribution was comparable to

Lysosome fusion occurred in follicles in the presence of 10E-10 M NaI and 0.1 mU/mL TSH for 8 h (Figure 12B) while this did not happen in thyrocytes' apical region (Figure 12B insert)

normal gland *in vivo* (Figure 1C) or follicles in long-term culture (Figures 6C and 6D).

**Figure 12.** The ultra-structure of rat follicle thyrocytes cultured on agarose with 0.5% FCS for 1 day; the medium was changed and 8 h of culture involved different experimental points: A. 10-E10 M NaI. B. 10- E10 M NaI + 0.1 mU/mL TSH. C. 10-E3 M NaI. D. 10-E3 M NaI + 0.1 mU/mL TSH. A. The ultra-structure of thyrocytes in the absence of TSH was identical to that of thyrocytes from follicles cultured for 1 day. B. Lysosomes close to the nucleus and few *colloid droplets* located in the apical pole (insert) were observed in the presence of TSH. C. They were well conserved in the presence of TSH, even in this strong dose of NaI. Endocytic vesicles were present in such strong dose of iodide. D. Colloid droplets were also observed in the presence of TSH, having the same density as colloid and were located in the thyrocytes' apical pole. Follicle centre was narrow and had abundant microvellosities (TEM. A 8,720 X, B 12,510 X, box 10,720 X, C 7,430 X, box 70,950 X, D 13,450 X).

The thyrocytes from follicles cultured from 30 min up to 6 days in the presence of 10E-3 M NaI did not have cytotoxic signs and all follicular cells were viable, like normal animals' thyroid glands' *in vivo* when fed with excess iodide for 3 weeks [103].

Thyroid Culture from Monolayer to Closed Follicles 377

**Figure 13.** Indirect immunofluorescence of NIS symporter (green) expression and localisation and DAPIlabelled nuclei (blue) in follicles isolated from rats cultured for 1 day on agarose with 0.5% FCS for 1 day; the medium was changed and were cultured for 12 h (A, B, C and D) and for 48 h (E, F, G and H), with different experimental points: NaI with and without 0.1 mU/mL TSH. **A.** 10E-7 M NaI, **B.** 10E-7 M NaI + TSH. **C.** 10E-3 M NaI. **D.** 10E-3 M NaI + TSH. **E**. 10E-7 M NaI. **F.** 10E-7 M NaI + TSH. **G.** 10E-3 M NaI. **H.** 10E-3 M NaI + TSH. NIS symporter was located in the basolateral membranes in the presence of 10E-10 M NaI at 12 h (A and B). TSH intensified labelling (B). NIS symporter was located in cytoplasmatic vesicles near thyrocytes' basolateral membranes in the presence of 10E-3 M NaI at 12 h (C and D). There was more intense labelling in some thyrocytes' basolateral membranes in the presence of TSH (D). [104]

(laser intensity, detector gain, scanning time) had to be adjusted again to increase labelling intensity. NIS protein was mainly observed in cytoplasmatic vesicles in these follicles, being more intense than labelling without TSH (G). Basement membranes had exiguous NIS

The NIS symporter in follicles cultured for **48 h** in the presence of 10E-7 M NaI were located in vesicles and basolateral membranes (Figure 13E); TSH intensified such labelling (Figure 13F). NIS expression was very reduced regarding the other treatments in the presence of 10E-3 M NaI with and without TSH and microscope parameters had to be readjusted for observing fluorescence. NIS was located in cytoplasmatic vesicles in this strong dose of NaI and without TSH (Figure 13G). TSH was located in vesicles in the base region but labelling

NIS has normally been located in thyrocytes' basolateral members *in vivo* [13], and a reduction in its normal expression has been associated with escape from the Wolff-Chaikoff effect [30] following 48 h in the presence of strong iodide concentrations [29]. Being found in vesicles has reduced NIS in its normal position for thyrocytes from follicles in the presence of a strong dose

These results were similar in the FRTL5 cell line where the same dose did not alter NIS RNAm percentage, but protein became reduced by 50% and 78 % at 24 and 48 h, respectively [32]. NIS RNAm became reduced in dogs with goitre at 48 h with a comparable

transport for thyroid hormone production.

labelling (CM. Scale bar: 10 μm).

was less intense (Figure 13H) than with TSH.

of NaI [104] and has thus suppressed I-

Open human follicle culture in the presence of 10E-3 M NaI with and without TSH for 24 h had ultra-structural alterations related to cytotoxicity [92] involving free radical attack and lipid peroxidation. Excess iodide in pig follicles led to thyrocyte apoptosis because iodine production in lactone became reduced, but did not present morphology. Our results showed that conserving closed follicles did not lead to signs of cell death with 10E-5 M or 10E-3 M NaI or disorganisation or alteration of thyrocyte ultra-structure. The difference with [92] and [94] was that they were open follicle cultures and thyrocytes died simply because they were cultured on a support which inhibited cellular adhesion, like our cell aggregates.

Closed follicles were present (Figure 12), as described for the gland. There was apical membrane turnover between microvellosities which is important for maintaining Tg synthesis and its secretion to colloid [8]. Coated endocytic vesicles (Figure 12C insert) were also present in the base of microvellosities, like micro-endocytosis *in vivo* [5], and those stimulated by TSH formed pseudopods and colloid droplets (Figures 12B insert and 12C letter DC), called *in vivo* macro-endocytosis [10]. Thyrocyte fusion with prelysosomes or late endosomes from the lysosome route was also observed (Figure 12B arrows) for Tg degradation and thyroid hormones were formed *in vivo* [19] and *in vitro* [18].

Closed rat and pig euthyroidic follicles responded to increasing doses of iodide, as *in vivo*, thereby producing the Wolff-Chaikoff effect [1], and presented no modifications in thyrocytes' follicular architecture or ultra-structure, being comparable to a gland *in vivo*.

#### *5.3.2.1. Na+/I symporter determination*

Many thyrocyte culture studies have described reduced RNAm and NIS protein expression when maintained in the presence of strong iodide concentrations (10E-6 to 10E-4 M of iodide). We wanted to determine NIS in rat follicles cultured with strong iodide concentrations with and without TSH 0.1 mU/mL.

Follicles cultured at **12 h** in the presence of 10E-7 M NaI and without TSH had NIS in basolateral membranes (Figure 13A) and labelling was more intense in lateral membranes in the presence of TSH (Figure 13B). In the presence of 10E-3 M NaI NIS was mainly located in vesicles between nucleus and basolateral membranes (Figure 13C), and in the presence of TSH; as well as being presented in vesicles they were observed in basolateral membranes (Figure 13D). It could have been that inhibiting vesicular movement in the presence of 10E-3 M NaI in the apical region (Figures 11C and 11D), as well as inhibiting the movement of the vesicles forming in the basement region with NIS symporter for avoiding excessive iodide entry to thyrocytes.

The NIS symporter was located in the basolateral members in the presence of 10E-10 M NaI at 48 h (E and F), labelling being more intense in the presence of TSH. The NIS symporter was found in the cytoplasmatic vesicles with TSH (F). NIS symporter expression in the presence of 10E-3 M NaI for 48 h (G and H) was so low that confocal microscope parameters

*5.3.2.1. Na+/I-*

entry to thyrocytes.

The thyrocytes from follicles cultured from 30 min up to 6 days in the presence of 10E-3 M NaI did not have cytotoxic signs and all follicular cells were viable, like normal animals'

Open human follicle culture in the presence of 10E-3 M NaI with and without TSH for 24 h had ultra-structural alterations related to cytotoxicity [92] involving free radical attack and lipid peroxidation. Excess iodide in pig follicles led to thyrocyte apoptosis because iodine production in lactone became reduced, but did not present morphology. Our results showed that conserving closed follicles did not lead to signs of cell death with 10E-5 M or 10E-3 M NaI or disorganisation or alteration of thyrocyte ultra-structure. The difference with [92] and [94] was that they were open follicle cultures and thyrocytes died simply because they were

Closed follicles were present (Figure 12), as described for the gland. There was apical membrane turnover between microvellosities which is important for maintaining Tg synthesis and its secretion to colloid [8]. Coated endocytic vesicles (Figure 12C insert) were also present in the base of microvellosities, like micro-endocytosis *in vivo* [5], and those stimulated by TSH formed pseudopods and colloid droplets (Figures 12B insert and 12C letter DC), called *in vivo* macro-endocytosis [10]. Thyrocyte fusion with prelysosomes or late endosomes from the lysosome route was also observed (Figure 12B arrows) for Tg

Closed rat and pig euthyroidic follicles responded to increasing doses of iodide, as *in vivo*, thereby producing the Wolff-Chaikoff effect [1], and presented no modifications in thyrocytes' follicular architecture or ultra-structure, being comparable to a gland *in vivo*.

Many thyrocyte culture studies have described reduced RNAm and NIS protein expression when maintained in the presence of strong iodide concentrations (10E-6 to 10E-4 M of iodide). We wanted to determine NIS in rat follicles cultured with strong iodide

Follicles cultured at **12 h** in the presence of 10E-7 M NaI and without TSH had NIS in basolateral membranes (Figure 13A) and labelling was more intense in lateral membranes in the presence of TSH (Figure 13B). In the presence of 10E-3 M NaI NIS was mainly located in vesicles between nucleus and basolateral membranes (Figure 13C), and in the presence of TSH; as well as being presented in vesicles they were observed in basolateral membranes (Figure 13D). It could have been that inhibiting vesicular movement in the presence of 10E-3 M NaI in the apical region (Figures 11C and 11D), as well as inhibiting the movement of the vesicles forming in the basement region with NIS symporter for avoiding excessive iodide

The NIS symporter was located in the basolateral members in the presence of 10E-10 M NaI at 48 h (E and F), labelling being more intense in the presence of TSH. The NIS symporter was found in the cytoplasmatic vesicles with TSH (F). NIS symporter expression in the presence of 10E-3 M NaI for 48 h (G and H) was so low that confocal microscope parameters

cultured on a support which inhibited cellular adhesion, like our cell aggregates.

degradation and thyroid hormones were formed *in vivo* [19] and *in vitro* [18].

 *symporter determination* 

concentrations with and without TSH 0.1 mU/mL.

thyroid glands' *in vivo* when fed with excess iodide for 3 weeks [103].

**Figure 13.** Indirect immunofluorescence of NIS symporter (green) expression and localisation and DAPIlabelled nuclei (blue) in follicles isolated from rats cultured for 1 day on agarose with 0.5% FCS for 1 day; the medium was changed and were cultured for 12 h (A, B, C and D) and for 48 h (E, F, G and H), with different experimental points: NaI with and without 0.1 mU/mL TSH. **A.** 10E-7 M NaI, **B.** 10E-7 M NaI + TSH. **C.** 10E-3 M NaI. **D.** 10E-3 M NaI + TSH. **E**. 10E-7 M NaI. **F.** 10E-7 M NaI + TSH. **G.** 10E-3 M NaI. **H.** 10E-3 M NaI + TSH. NIS symporter was located in the basolateral membranes in the presence of 10E-10 M NaI at 12 h (A and B). TSH intensified labelling (B). NIS symporter was located in cytoplasmatic vesicles near thyrocytes' basolateral membranes in the presence of 10E-3 M NaI at 12 h (C and D). There was more intense labelling in some thyrocytes' basolateral membranes in the presence of TSH (D). [104]

(laser intensity, detector gain, scanning time) had to be adjusted again to increase labelling intensity. NIS protein was mainly observed in cytoplasmatic vesicles in these follicles, being more intense than labelling without TSH (G). Basement membranes had exiguous NIS labelling (CM. Scale bar: 10 μm).

The NIS symporter in follicles cultured for **48 h** in the presence of 10E-7 M NaI were located in vesicles and basolateral membranes (Figure 13E); TSH intensified such labelling (Figure 13F). NIS expression was very reduced regarding the other treatments in the presence of 10E-3 M NaI with and without TSH and microscope parameters had to be readjusted for observing fluorescence. NIS was located in cytoplasmatic vesicles in this strong dose of NaI and without TSH (Figure 13G). TSH was located in vesicles in the base region but labelling was less intense (Figure 13H) than with TSH.

NIS has normally been located in thyrocytes' basolateral members *in vivo* [13], and a reduction in its normal expression has been associated with escape from the Wolff-Chaikoff effect [30] following 48 h in the presence of strong iodide concentrations [29]. Being found in vesicles has reduced NIS in its normal position for thyrocytes from follicles in the presence of a strong dose of NaI [104] and has thus suppressed Itransport for thyroid hormone production.

These results were similar in the FRTL5 cell line where the same dose did not alter NIS RNAm percentage, but protein became reduced by 50% and 78 % at 24 and 48 h, respectively [32]. NIS RNAm became reduced in dogs with goitre at 48 h with a comparable

iodide dose [105]. This was perhaps presented by reduced AM2Pc levels at 48 h, as excess Iinhibited an increase in AMPc stimulated by TSH in hypophysectomised rats [106] and mice [34], which could have explained the low NIS level during Wolff-Chaikoff effect and in follicles at 48 h in the presence of excess iodide (Figure s13G and 13H). Excess I inhibited IP3 production and increased Ca2+ flow induced by TSH, which could have led to reduced peroxide production during Wolff-Chaikoff effect [107,108]. The organification observed in the presence of 10E-5 M (Table 8) did not completely inhibit TPO and had no effect on NIS in the presence of 10E-3 M NaI (Figures 13E and 13H), thereby demonstrating that organification depends on TPO and not on NIS as in cells transfected with the TPO gene [34]. TSH did not stimulate the organification of iodide captured in 10E-3 M but did so in 10E-5 M NaI (Tables 8 and 10), as it has been described that the effect of TSH on thyroid physiology becomes reduced in the presence of excess I- , meaning that antagonic roles are assumed *in vivo* [102].

Thyroid Culture from Monolayer to Closed Follicles 379

conserving the idea of Tg19S synthesis usually being glycosylated and iodised, as also T3 and T4 hormone synthesis. Follicular morphological conservation is necessary for reproducing the Wolff-Chaikoff effect *in vitro*, as has been described *in vivo.* NIS symporter

This culture may be used for obtaining follicles from pathologies of human tissue whose epithelium may be thin plate-like cells for *in vitro* studies in controlled and homologous conditions regarding the pathology *in vivo*. It will also enable studying normal or pathological thyroid's physiological, cellular and molecular mechanisms (for example CLC-

This work was supported by grants from the Colombian Science, Technology and Innovation Department (COLCIENCIAS) programmes Ecos-Nord and Jóvenes Investigadores P 2009- 0745, by the Universidad Nacional de Colombia's Research Department and Fundacion Instituto de Inmunología de Colombia (FIDIC). We would like to extend our most sincere thanks to Marcela Camacho, Marie France van den Hove, Thierry Pourcher, Jean-François Denef, Hernando Curtidor and Manuel E Patarroyo for their support and encouragement for

We would also like to thank the students who carried out and who are carrying out BSc, MSc and PhD thesis experiments: Sandra Perdomo, Gabriela Delgado, Cristina Zapata, Ricardo Cabezas, Alejandro Ondo, Leslie Leal, Oscar Vivas, Claudia Moreno, Eleonora Bernal, Anyela González, Mauren Ortíz, Carolina Ochoa and Luz Marina Porras; this work

[1] Wolff J, Chaikoff IL (1948). Plasma inorganic iodide as a homeostatic regulator of

[2] Wollman S (1980) Structure of the thyroid gland. In: Vissher M, editor. The thyroid

[3] Werner C, Ingbar H (1971) The thyroid. A fundamental and clinical text, pp.5-40.

localisation in thyrocytes depends on I and TSH concentration.

5 channel) in a homologous model of the gland *in vivo*.

Clara Spinel1,3, Magnolia Herrera3 and and Jhon Rivera2,3

*1Biology Department, Science Faculty, Universidad Nacional de Colombia, Colombia 2Chemistry Department, Science Faculty, Universidad Nacional de Colombia, Colombia* 

*Membrane Biophysics and Biology Group,* 

*3International Physics Center, Bogotá, Colombia* 

carrying out basic research in Colombia.

could not have been completed without their input.

thyroid function. J Biol Chem. 174:555-64.

gland. New York: Raven Press. pp. 1-19.

London: Harper & Row. 914p.

**Author details** 

**Acknowledgement** 

**7. References** 

TSH modulated relative NIS expression and its subcellular localisation in the thyrocytes of isolated and closed follicles *in vitro*. These results were similar to those found in FRTL5cells, where it has been demonstrated that *de novo* synthesis [32], half-life time, NIS targeting and/or retention regarding cytoplasmatic membrane requires TSH to be located throughout cell membrane, due to loss of polarity [109].

Thyrocyte disposition in follicles has not been necessary for iodide accumulation, since it has been present in foetal thyroids before follicular lumen formation [110] and also in primary cultures from normal thyrocytes [111] or goitre patients [112] and in the FRTL cell line [113]; however, these cultures have required TSH, hormones and other molecules for maintaining them. Nevertheless, isolating the colloidal cavity from the exterior must be ensured for iodide accumulation and incorporation in Tg, T3 and T4 hormone synthesis, as demonstrated with rat or pig isolated and closed follicle cultures.

Rat and pig follicles thus inhibited iodide organification in the presence of strong concentrations of iodide, i.e. performed the Wolff-Chaikoff effect. Neither thyrocytes' follicular architecture nor ultra-structure was modified and no sign of cell death was presented. The TSH and iodide effects observed *in vivo* during the Wolff-Chaikoff effect were reproduced.
