**1.2. Function**

T3 (3,5,3'-triiodine-thyronine) and T4 thyroid hormone (3,5,3',5'- tetraiodine-thyronine) synthesis reflects thyrocytes' follicular morphology and ultra-structure which can be divided into 3 stages (Figure 2 circles): stage 1, Tg synthesis and colloid secretion at

Thyroid Culture from Monolayer to Closed Follicles 349

intracellular level; stage 2, iodide accumulation and its organification regarding Tg in colloid at thyrocyte extracellular level; and stage 3, endocytosis and intracellular hormone

The first stage occurs at intracellular level, Tg is synthesised and glycosylated (*N*-osidic glycosylation) in RER and then glycosylation in culminates in the GC. Tg is transported in exocytic vesicles which emerge from the GC (Figure 2 green circle 1) and is released to the colloid [5]. Iodide accumulation takes place in the basal membrane via the sodium/iodide

The second stage happens at extracellular level. Thyroperoxidase (TPO) is found in the base of microvellosities anchored to the apical membrane which oxides iodide (Figure 2 blue) and thyroid oxidases 1 or 2 (Duox1 and 2) forming H2O2 (Figure 2 red). TPO fixes one or two iodines on specific Tg thyrosins in colloid (iodide organification), thereby forming monoand di-iodinethyrosins (Figure 2 circle 2). TPO couples the iodinethyrosins, producing iodine-thyronine or T3 and T4 hormones on Tg19S or thyroid prohormone (Figure 2 Tg-I); in

The third stage is intracellular. Tg19S is endocyted [10] (Figure 2 blue circle 3) and degraded in prelysosomes [18] and in lysosomes (Figure2 Pre, L), releasing the hormones which become diffused through the basal membrane to the blood stream [19] where they are

Due to the thyroid's morphological characteristics and its function, "it is an exquisitely regulated gland", [22]. Its function is essentially controlled by the hypothalamushypophysis and also by the nervous system and other thyroid systems [22]. Thyroid gland function and growth is controlled by TSH secreted by adenohypophysis thyreotropic cells. TSH secretion is stimulated by thyrotropin releasing hormones (TRH) secreted by the hypothalamus. TSH and TRH concentration in circulation are regulated by T3 and T4 concentration; thyroid and TSH concentrations are regulated by iodide concentration in the

TSH mainly activates the AMPc route which stimulates transcription factors (CREB, TTF-1 and -2, PAX8) and culminates by activating the transcription and expression of molecules implicated in T3 and T4 hormone synthesis (i.e. NIS in basal membrane, Tg in RER and its exocytosis, TPO and Douxs in the apical membrane and H2O2 formation). TSH's effect can

Normal iodide in circulation ranges from 10E-9 to 10E-7 M. Concentrations of this ion greater than 10E-5 inhibit T3 and T4 organification and synthesis during the first 48 h (called the Wolff-Chaikoff effect) [1], regardless of TSH concentration. Iodide organification inhibition directly depends on iodide intrathyroid accumulation [28]. This thyroid autoregulatory effect happens when inorganic iodine concentration in blood exceeds a set threshold (overload) and the gland blocks iodine's organic binding for 48 h [1]. The gland

transported by three families of blood proteins to an organism's cells [20,21].

blood stream obtained during daily intake [2,3,22,23,24,25].

be shown by increased T3 and T4 in the blood stream [26,27].

symporter (Figure 2 NIS) [13]. It then passes through colloid via

apical exchanger) [14] (Figure 2 P), regulated by the apical region's ClC-5

secretion [5,12].

pendrin (I-

channel [15].

symporter (NIS) or Na+/I-

/Cl-

both processes TPO reduces H2O2 [16,17].

**Figure 2. A.** A diagram of a thyroid follicle surrounded by capillaries (Cap); Co: colloid. **B.** A diagram of a thyrocyte showing the physiology of thyroid hormone synthesis. Iodide is captured in the basement membrane by the *sodium/*iodide symporter or Na+/I symporter (NIS) and rapidly transported to colloid, mainly via pendrin (P); it is used there for thyroid hormone synthesis. Tg is synthsised in the RER and *N*-osidic glycosylation culminates in the GC; Tg is secreted to colloid by exocytic vesicles (green circle 1). Once in colloid, thyroperoxidase (blue) with thyroid oxidase 1 or 2 (red) fixes iodide to Tg (red circle 2) forming iodine-thyronine on Tg (Tg-I). When thyroid hormones are required, Tg-I is endocyted by microvesicles or macrovesicles (blue circle 3) when TSH stimulates the thyroid. Tg-I becomes degraded by lysosomal enzymes (L) releasing T3 and T4 into the blood stream. N: nucleus; RER: rugose endoplasmic reticle; GC: Golgi complex; I- : ion iodide; NIS: Na+/I symporter; EeE: early endosome; Pre: prelysosome or late endosome; L: lysosome; BMs: basements membranes or basals laminas; T3 and T4: thyroid hormones. Diagram modified from Spinel (2003) [12].

intracellular level; stage 2, iodide accumulation and its organification regarding Tg in colloid at thyrocyte extracellular level; and stage 3, endocytosis and intracellular hormone secretion [5,12].

348 Thyroid Hormone

**Figure 2. A.** A diagram of a thyroid follicle surrounded by capillaries (Cap); Co: colloid. **B.** A diagram of a thyrocyte showing the physiology of thyroid hormone synthesis. Iodide is captured in the basement

colloid, mainly via pendrin (P); it is used there for thyroid hormone synthesis. Tg is synthsised in the RER and *N*-osidic glycosylation culminates in the GC; Tg is secreted to colloid by exocytic vesicles (green circle 1). Once in colloid, thyroperoxidase (blue) with thyroid oxidase 1 or 2 (red) fixes iodide to Tg (red circle 2) forming iodine-thyronine on Tg (Tg-I). When thyroid hormones are required, Tg-I is endocyted by microvesicles or macrovesicles (blue circle 3) when TSH stimulates the thyroid. Tg-I becomes degraded by lysosomal enzymes (L) releasing T3 and T4 into the blood stream. N: nucleus;

endosome; Pre: prelysosome or late endosome; L: lysosome; BMs: basements membranes or basals

laminas; T3 and T4: thyroid hormones. Diagram modified from Spinel (2003) [12].

symporter (NIS) and rapidly transported to

symporter; EeE: early

: ion iodide; NIS: Na+/I-

membrane by the *sodium/*iodide symporter or Na+/I-

RER: rugose endoplasmic reticle; GC: Golgi complex; I-

The first stage occurs at intracellular level, Tg is synthesised and glycosylated (*N*-osidic glycosylation) in RER and then glycosylation in culminates in the GC. Tg is transported in exocytic vesicles which emerge from the GC (Figure 2 green circle 1) and is released to the colloid [5]. Iodide accumulation takes place in the basal membrane via the sodium/iodide symporter (NIS) or Na+/I symporter (Figure 2 NIS) [13]. It then passes through colloid via pendrin (I- /Cl apical exchanger) [14] (Figure 2 P), regulated by the apical region's ClC-5 channel [15].

The second stage happens at extracellular level. Thyroperoxidase (TPO) is found in the base of microvellosities anchored to the apical membrane which oxides iodide (Figure 2 blue) and thyroid oxidases 1 or 2 (Duox1 and 2) forming H2O2 (Figure 2 red). TPO fixes one or two iodines on specific Tg thyrosins in colloid (iodide organification), thereby forming monoand di-iodinethyrosins (Figure 2 circle 2). TPO couples the iodinethyrosins, producing iodine-thyronine or T3 and T4 hormones on Tg19S or thyroid prohormone (Figure 2 Tg-I); in both processes TPO reduces H2O2 [16,17].

The third stage is intracellular. Tg19S is endocyted [10] (Figure 2 blue circle 3) and degraded in prelysosomes [18] and in lysosomes (Figure2 Pre, L), releasing the hormones which become diffused through the basal membrane to the blood stream [19] where they are transported by three families of blood proteins to an organism's cells [20,21].

Due to the thyroid's morphological characteristics and its function, "it is an exquisitely regulated gland", [22]. Its function is essentially controlled by the hypothalamushypophysis and also by the nervous system and other thyroid systems [22]. Thyroid gland function and growth is controlled by TSH secreted by adenohypophysis thyreotropic cells. TSH secretion is stimulated by thyrotropin releasing hormones (TRH) secreted by the hypothalamus. TSH and TRH concentration in circulation are regulated by T3 and T4 concentration; thyroid and TSH concentrations are regulated by iodide concentration in the blood stream obtained during daily intake [2,3,22,23,24,25].

TSH mainly activates the AMPc route which stimulates transcription factors (CREB, TTF-1 and -2, PAX8) and culminates by activating the transcription and expression of molecules implicated in T3 and T4 hormone synthesis (i.e. NIS in basal membrane, Tg in RER and its exocytosis, TPO and Douxs in the apical membrane and H2O2 formation). TSH's effect can be shown by increased T3 and T4 in the blood stream [26,27].

Normal iodide in circulation ranges from 10E-9 to 10E-7 M. Concentrations of this ion greater than 10E-5 inhibit T3 and T4 organification and synthesis during the first 48 h (called the Wolff-Chaikoff effect) [1], regardless of TSH concentration. Iodide organification inhibition directly depends on iodide intrathyroid accumulation [28]. This thyroid autoregulatory effect happens when inorganic iodine concentration in blood exceeds a set threshold (overload) and the gland blocks iodine's organic binding for 48 h [1]. The gland adapts once such 48 h have elapsed and organified iodide escapes, producing new hormones [29]; NIS expression becomes reduced at this time, as does iodide capture [30]. It has been suggested that there is a reduction in the function of the molecules implicated in organification and hormone formation: TPO, Duox 1 and 2, pendrin and Tg [30,31]. Thyrocytes in culture in the presence of 10-E3 M iodide reduce NIS expression and inhibit TPO and Tg synthesis [32]. Such reduction of NIS does not happen in hypothyroid mice, nor is Duox 1 and 2, TPO, pendrin and Tg gene expression modified [33]. An excess of iodide leads to iodide organification inhibition depending on TPO and not on NIS. TSH effects become reduced in the presence of strong concentrations of iodide, resulting in them adopting antagonic roles [34].

Thyroid Culture from Monolayer to Closed Follicles 351

/Cl-

some of the models which were described. Approaching the 1990s attempts were made to use very small fragments (less than 1mm3) in organ cultures (called mini organ cultures) which lasted 2 to 3 days without necrosis, exhibited iodide, sulphate and phosphate transport, synthesised a 19S Tg (normally glycosylated and iodised) [43] and were

Monolayer culture (better known as cell culture) mainly deals with a single cell type. This implies tissue dissociation by enzymatic digestion or mechanical action and the isolation of cellular types by different sepaproportion methods. Isolated cells are placed on different types of supports where they adhere and proliferate in a single layer until reaching confluence (called primary culture). Secondary culture consists of sowing cells removed from the primary culture in fresh recipients and so on. The term passage is used to indicate the number of successive secondary culture sowings, thus the 1st secondary culture is the 1st cell passage. Thyrocytes were first cultured in 1911 [45]. Using this dissociation technique and continuous shaking during culture has shown that sheep thyrocytes concentrate radioactive iodide and incorporate it in iodine-thyronine: MIT, DIT and T4 [46]. Isolated and small cells, aggregates of 10 to 15 thyrocytes, are obtained after dissociation with trypsin [47,48]. One of the greatest drawbacks is the loss of cultured thyrocytes' cellular polarity when one wishes to study thyroid physiology since such polarity is fundamental in conserving thyrocyte membrane domains, and thus the expression of domain-specific

Thyrocyte cultures were developed in dual chambers during the 1990s on cubic monolayers as *in vivo* with binding complexes in the lateral membranes' apical region, separating in thyrocytes' the apical membrane domains from the basolateral membrane domains, TSH favouring such cellular polarisation [50]*.* This model has demonstrated that ion flow is determined by thyrocytes' polarity, thereby corroborating the fact that ion channels are different in both thyrocytes membrane domains when thyrocytes' cubic form is conserved. A new channel has been described for the thyrocytes' apical membrane [51]; this new channel is CLC5 which is located in the apical region *in vivo* and it has been proposed that thyrocytes have a position in the apical membrane for controlling pendrin, the I-

The foregoing has shown the importance of conserving cell polarity and cubic form in

**Cell lines** are continually growing and indefinitely proliferating cell cultures because they have lost control over their own cell division, contrary to primary and secondary cell cultures which die after a finite number of passes or subcultures, as is genetically determined in normal cells. Thethyrocyte cell line was described [52,53]; Fischer rat thyroid cell line or FRTL is most used around the world as it has a more similar ultra-structure to thyrocytes and synthesised Tg. These have been very useful in studying gene expression,

cytoskeleton modification with different factors, iodide flow and that of other ions.

thyrocyte culture for studying the gland's physiology and biochemistry.

maintained for up to 7 days without cell death when coated with collagen [44]

**2.3. Isolation and monolayer culture or cell culture** 

molecules guaranteeing hormone synthesis [49].

transporter [15].
