*5.3.1. Functional study*

Follicles were cultured for 1 day before starting to analyse the effect of different iodide concentrations.

Follicles accumulated iodide linearly for 6 h in the presence of 10E-10 and 10E-7 M NaI; accumulation was at its maximum after the first 30 min with 10E-5 M and 10E-3 M NaI (Table 7); they became accumulated 100 times more than with 10E-10 M NaI in the presence of 10E-7 M NaI. Whilst high O/A percentages in follicles were maintained in the presence of 10E-10 and 10E-7 M NaI, organification began after 2 h with 10E-5 M, accounting for only 5% of accumulated iodide, these values being maintained for 6 h, whilst O/A proportion was zero for all times with 10E-3 M NaI (Table 7), even though accumulation could have been 100 times greater to that presented by follicles in the presence of 10E-7 M NaI [98].


culture.

*5.3.1. Functional study* 

concentrations.

presenting culture morphology.

not become reduced during the 9 days of culture.

maintained *in vitro,* being equivalent to the gland *in vivo*.

TSH [93] had a TEM image showing thyrocytes having the ultra-structure for cells in the process of cell death with lysed mitochondria, without RER or GC. Using this human follicle culture model enabled analyzing the effect of TSH [95] or cytokines [95,96] without

Our results showed that if culture was begun with closed follicles then extracellular matrix support elements were not required [56,57,79,80] nor was TSH for maintaining follicular cavity, as has been described in most pseudofollicle or follicle cultures reconstructed from monolayers, or similar structures called "follicles" [57,50,60,61,62,79,96], thereby demonstrating that if closed follicles are used from the start of culture they conserve their morphology, having the correct polarity as that of their thyrocytes in culture and have a binding complex (tight junction, belt desmosome and spot desmosome) in lateral membranes' apical part as well as *in vivo* [9]. Such closed follicles in culture responded to TSH, like other *in vitro* models [42] or like gland follicles in normal *in vivo* to TSH stimulus [3,25], epithelium thickness, RER and GC becoming increased, and follicular cavities becoming notably reduced [11,83,90]. Follicle response to the absence of TSH both *in vivo*  [24] and *in vitro* [42] was also comparable as these organelles became reduced. This effect became reverted *in vivo* when TSH was added, reactivating thyroid functions [11,33,97].

Pig closed and isolated hypothyroid follicles behaved like the gland *in vivo* when the hypothyroid effect was deleted [24], RER and GC increased, follicular epithelium became thin to cubic and culture time became faster in the presence of TSH, but follicular cavity did

Our results thus showed that long-term thyroid follicle function and morphology can be

The next section describes the effect of increasing doses of iodide on closed follicles in

**5.3. Closed follicle cultures reproduce the Wolff-Chaikoff effect described** *in vivo*

Follicles were cultured for 1 day before starting to analyse the effect of different iodide

Follicles accumulated iodide linearly for 6 h in the presence of 10E-10 and 10E-7 M NaI; accumulation was at its maximum after the first 30 min with 10E-5 M and 10E-3 M NaI (Table 7); they became accumulated 100 times more than with 10E-10 M NaI in the presence of 10E-7 M NaI. Whilst high O/A percentages in follicles were maintained in the presence of 10E-10 and 10E-7 M NaI, organification began after 2 h with 10E-5 M, accounting for only 5% of accumulated iodide, these values being maintained for 6 h, whilst O/A proportion was zero for all times with 10E-3 M NaI (Table 7), even though accumulation could have been 100 times greater to that presented by follicles in the presence of 10E-7 M NaI [98].

**Table 7.** Response of organification percentage regarding iodide accumulation (O/A proportion) by rat follicles cultured in the presence of increasing doses of iodide (NaI, M) and Na125I 5 Ci/mL. The follicles were cultured on agarose with 0.5% FCS for 1 day, medium was changed and culture began with NaI and TSH experimental points. Each value was the average of two samples or culture dishes expressed in %/dose/μg DNA.

Iodide accumulation and organification in rat follicles could be assimilated to biochemical reactions after 30 min with and without TSH and thus define a constant (Km) and maximum speed (Vmax). Accumulation apparent Km without TSH was 5x10E-6 M and 10E-7 M de NaI with TSH; iodide organification apparent Km was 5x10E-7 M and was not modified by the presence of TSH (Figure 8). Iodide accumulation by isolated follicles corresponded well with saturable iodide transport characteristics [15,99] and stimulated TSH thereby reducing Km but not Vmax (Figure 8). It should be stated that organification had to be inhibited and

**Figure 8.** Initial 125Iiodide accumulation (**A**) and organification (**B**) for follicles in culture measured following half an hour in the presence of increasing doses of 127-I Na and 5 Ci/mL 125-I Na expressed in log M with and without TSH. The follicles were cultured on agarose with 0.5% FCS for 1 day, the medium was changed and culture began with NaI and TSH experimental points. The results were the average of two samples and were expressed as percentage of maximum value.

the time between the end of the culture and the moment when accumulation was determined had to be reduced for measuring exact iodide transport parameters. The washing times meant that part of 125I concentrated in follicles would be released before radioactivity was determined and this would induce under-stimulation of accumulation and perhaps overstimulation of O/A percentage.

Thyroid Culture from Monolayer to Closed Follicles 371

Follicle culture in the presence of the same iodide dose with 0.1 mU/mL and without TSH led up to 8 h with 10E-10 M and 10E-5 M NaI and up to 48 h with 10E-7 M and 10E-3 M NaI. An iodide accumulation function directly proportional to NaI concentration was reproduced

Accumulation in follicles became saturated after 8 h in the presence of **10E**-**7 M** NaI with and without TSH, becoming slightly reduced at 12 and 24 h and increasing at 48 h; iodide accumulation with TSH was lower at 8, 12 and 24 h than without TSH (significant only at 12 h, p< 0.05). Percentage of the proportion O/A was maintained high values with and without

Iodide accumulation satuproportion was reproduced in follicles in the presence of **10E-5 M** NaI, whether with or without TSH. TSH significantly stimulated organification inhibited by 10E-5 M NaI (p< 0.05) at 30 min, even though O/A percentage was slightly higher than at

Iodide accumulation in follicles in the presence of **10E-3 M** NaI with and without TSH reached a *plateau* at 3 h (Table 8) and was greater in follicles without TSH than with TSH at 12, 24 and 48 h; however, this difference was only significant at 12 and 48 h (p< 0.05). Iodide accumulation became reduced with and without TSH at 48 h to 3 h values, even though without TSH this was greater but not significantly so. Iodide organification was zero with

Pig euthyroidic follicles had the same response as rat follicles when culturing for 48 h in the presence of increasing doses of iodide with or without TSH. Organification was zero at all

**M, NaI 10E-10 10E-7 10E-3 Culture TSH % O/A N % O/A N % O/A N**  0.5 - 30.4 ± 0.56 2 30.4 ± 0.63 2 0 3

3 - 67.0 ± 1.54 2 63.2 ± 9.14 2 0 3

12 - 71.8 ± 2.35 2 52.4 ± 2.63 1 0 3

24 - 59.7 ± 2.09 2 87.6 ± 1.97 2 0 4

euthyroidic pig 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 accumulated or organified (O/A) iodide expressed in

Pig hypothyroid follicles had a different response to the presence of increasing doses of iodide than rat and pig euthyroidic follicles (Table 10). They accumulated 40 to 100 times

+ 34.7 ± 9.21 2 72.7 ± 2.84 2 0 3

+ 79.5 ± 6.79 2 73.6 1 0 3

+ 78.9 ± 2.63 2 77.6 ± 3.15 2 0 3

+ 68.8 ± 7.13 2 85.7 ± 0.99 2 0 3

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

TSH, difference with and without TSH at 30 min and 48 h being significant (Table 8).

such strong dose of iodide with and without TSH at all times examined (Table 8).

times in the presence of 10-E3 M NaI with and without TSH (Table 9).

other times (but was not significant) (Table 8).

as in table 7.

**Table 9.** The effect of I-

%/dose/μg DNA per number (N) of culture dishes ± SD.

The presence of TSH for 30 min suppressed the inhibiting effect on iodide organification at 10E-5 M, but did not suppress such iodide effect on organification in the presence of 10E-3 M TSH (Table 8).


**Table 8.** The effect of I dose on the accumulation (A) and organificación (O) of the rat follicles. 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 0.1 mU/mL TSH (-) and doses of Na127I (NaI). Each value was the average of two samples expressed in μg/dose/μg DNA. This result was representative of 5 independent experiments. O/A: percentage of the proportion O/A ± SD. \*p< 0.05. ND: not determinated.

Follicle culture in the presence of the same iodide dose with 0.1 mU/mL and without TSH led up to 8 h with 10E-10 M and 10E-5 M NaI and up to 48 h with 10E-7 M and 10E-3 M NaI. An iodide accumulation function directly proportional to NaI concentration was reproduced as in table 7.

370 Thyroid Hormone

M TSH (Table 8).

**Table 8.** The effect of I-

washing times meant that part of 125I-

perhaps overstimulation of O/A percentage.

the time between the end of the culture and the moment when accumulation was determined had to be reduced for measuring exact iodide transport parameters. The

radioactivity was determined and this would induce under-stimulation of accumulation and

The presence of TSH for 30 min suppressed the inhibiting effect on iodide organification at 10E-5 M, but did not suppress such iodide effect on organification in the presence of 10E-3

**M, NaI 10E-10 10E-7 Culture TSH A O % O/A A O % O/A**  0.5 - 2,58 1,29 50 ± 5\* 360,9 156,34 43 ± 6\*

3 - 10,88 8,16 75 ± 10\* 902,26 525,24 58 ± 7

8 - 35,97 29,84 83 ± 11 2255,64 1602,81 71 ± 4\*

12 - ND 1849,62 1191,13 64 ± 9

24 - ND 1714,29 1421,05 83 ± 10

48 - ND 1917,29 1361,98 71 ± 10\*

+ 8,06 4,52 56 ± 2\* 676,69 406,02 60 ± 14\*

+ 14,84 11,81 79 ± 1\* 1308,27 1079,24 82 ± 1

+ 32,26 28,06 87 ± 23 1736,84 1371,98 79 ± 10\*

+ ND 1353,38 853,23 63 ± 13

+ ND 1759,4 1254,17 71 ± 8

+ ND 3969,92 3194,23 80 ± 6\* **M, NaI 10E-5 10E-3 Culture TSH A O % O/A A O % O/A**  0.5 - 14749,09 0 0 \* 233469,39 0 0

+ 12480,01 2,63 21 ± 16\* 244244,9 0 0

+ 17541,82 2987,27 17 ± 2 466938,78 0 0

+ 20770,91 4450,91 21 ± 2 790204,08 0 0

+ ND \* 1957551,02 0 0

+ ND \* 682448,98 0 0

+ ND 484897,96 0 0

dose on the accumulation (A) and organificación (O) of the rat follicles. Follicles

3 - 15272,73 1553,64 10 ± 6 520816,33 0 0

8 - 16495,45 2397,15 14 ± 5 574693,88 0 0

12 - ND \* 718367,35 0 0

24 - ND \* 3071020,41 0 0

48 - ND 664489,8 0 0

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 0.1 mU/mL TSH (-) and doses of Na127I (NaI). Each value was the average of two samples expressed in μg/dose/μg DNA. This result was representative of 5 independent

experiments. O/A: percentage of the proportion O/A ± SD. \*p< 0.05. ND: not determinated.

concentrated in follicles would be released before

Accumulation in follicles became saturated after 8 h in the presence of **10E**-**7 M** NaI with and without TSH, becoming slightly reduced at 12 and 24 h and increasing at 48 h; iodide accumulation with TSH was lower at 8, 12 and 24 h than without TSH (significant only at 12 h, p< 0.05). Percentage of the proportion O/A was maintained high values with and without TSH, difference with and without TSH at 30 min and 48 h being significant (Table 8).

Iodide accumulation satuproportion was reproduced in follicles in the presence of **10E-5 M** NaI, whether with or without TSH. TSH significantly stimulated organification inhibited by 10E-5 M NaI (p< 0.05) at 30 min, even though O/A percentage was slightly higher than at other times (but was not significant) (Table 8).

Iodide accumulation in follicles in the presence of **10E-3 M** NaI with and without TSH reached a *plateau* at 3 h (Table 8) and was greater in follicles without TSH than with TSH at 12, 24 and 48 h; however, this difference was only significant at 12 and 48 h (p< 0.05). Iodide accumulation became reduced with and without TSH at 48 h to 3 h values, even though without TSH this was greater but not significantly so. Iodide organification was zero with such strong dose of iodide with and without TSH at all times examined (Table 8).


Pig euthyroidic follicles had the same response as rat follicles when culturing for 48 h in the presence of increasing doses of iodide with or without TSH. Organification was zero at all times in the presence of 10-E3 M NaI with and without TSH (Table 9).

**Table 9.** The effect of I dose on the proportion accumulation/organificación pourcentage (% A/O) in euthyroidic pig 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 accumulated or organified (O/A) iodide expressed in %/dose/μg DNA per number (N) of culture dishes ± SD.

Pig hypothyroid follicles had a different response to the presence of increasing doses of iodide than rat and pig euthyroidic follicles (Table 10). They accumulated 40 to 100 times

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 10), such value becoming reduced with culture time.

Thyroid Culture from Monolayer to Closed Follicles 373

Hypothyroid follicles in the presence of 10-3 M NaI accumulated more iodide without TSH than with TSH at the beginning (30 min), even though proportion of O/A percentage was greater at 30 min than at other times; this effect (different to euthyroidic ones) was similar to that described *in vivo* in hypothyroid glands [28]. They also responded by reproducing the effect observed in animals suffering experimental goitre as, when excess iodide was added to food, they did not respond to TSH [24]. 50% of human endemic goitre has responded to an iodine-rich diet and some of these to TSH [25] as hypothyroid follicles in the presence of TSH and 10E-3 M NaI have responded by becoming less organified than in the absence of TSH. It should be stated that there is no consensus regarding what should be the correct dose for defining low, medium or high iodide concentration in human alimentation [25].

Closed and isolated follicles thus responded to 10E-5 M and 10E-3 M NaI inhibiting iodide organification in a similar way to that described for the gland *in vivo* in 1948 [1] (i.e.

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

**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)

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

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

reproducing the Wolff-Chaikoff effect).

follicular architecture for 48 h (Figure 9D).

ones cubic epithelium (IM. Scale bar: A, B, C 800 μm, D 200 μm).

1C), the same as isolated cells (Figure 10B) or those found in follicles.

*5.3.2. Morphological study* 

6C and 6D).


**Table 10.** The effect of I dose on the proportion accumulation/organificación pourcentage (% A/O) in 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 in %/dose/μg DNA per number (N) of culture dishes ± SD. ND: not determinated.

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 did not exceed 11% for accumulated iodide.

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

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 gland *in vivo* [101,102].

Hypothyroid follicles in the presence of 10-3 M NaI accumulated more iodide without TSH than with TSH at the beginning (30 min), even though proportion of O/A percentage was greater at 30 min than at other times; this effect (different to euthyroidic ones) was similar to that described *in vivo* in hypothyroid glands [28]. They also responded by reproducing the effect observed in animals suffering experimental goitre as, when excess iodide was added to food, they did not respond to TSH [24]. 50% of human endemic goitre has responded to an iodine-rich diet and some of these to TSH [25] as hypothyroid follicles in the presence of TSH and 10E-3 M NaI have responded by becoming less organified than in the absence of TSH. It should be stated that there is no consensus regarding what should be the correct dose for defining low, medium or high iodide concentration in human alimentation [25].

Closed and isolated follicles thus responded to 10E-5 M and 10E-3 M NaI inhibiting iodide organification in a similar way to that described for the gland *in vivo* in 1948 [1] (i.e. reproducing the Wolff-Chaikoff effect).
