**2.3. Normal morphology and distribution of C cells**

C cells represent the neuroendocrine cells that produce calcitonin in the thyroid gland. Since their discovering by Baber in 1876, these cells have been given many different terms. However, the two most common denominations employed are *C cells* and *parafollicular cells*. The name "C cell" was coined by Pearse in 1966 and it is reflective of calcitonin production; this term is preferred to that of "parafollicular cell" as these cells are not always related to follicles in parafollicular position and may, furthermore, be found outside the thyroid gland, unrelated to follicles, as occurs in the ultimobranchial glands in non-mammal vertebrates [11].

C cells are difficult to identify in formalin-fixed, paraffin-embedded sections stained with H&E. Generally, their nuclei are somewhat larger and paler than those of follicular cells. Therefore, the most reliable procedure for the identification of C cells involves the use of immunohistochemical procedures with antibodies for calcitonin (Figure 2).

C cells have variable shapes ranging from polygonal to spindled. The location of C cells relative to follicular cells also varies. They may be found between follicular cells or completely peripheral to the follicular epithelium. C cells are typically separated from the colloid by the cytoplasm of follicular cells and the basal aspects of C cells are usually in contact with follicular basal lamina. Occasionally, C cells emit cytoplasmic processes that surround adjacent follicular cells (see Figure 3). At ultrastructural level, many dense secretory granules are observed in the cytoplasm of C cells [13, 14].

Paracrine Regulation of Thyroid-Hormone Synthesis by C Cells 55

Weiler at al., 1989 [24, 25]

The ratio of C cells to follicular cells in the thyroid gland differs markedly among species. In humans, C cells comprise less than 1% of the total endocrine-cell population [21], while the proportion of C cells to follicular cells is at least 5% in rats [18]. Furthermore, C cell concentration may vary according to age. In humans, however, data are very contradictory: Wolfe et al. in 1974 found that the number of C cells per unit area appeared to decrease with age [15], while Gibson et al. in 1982 found an age-dependant increase in C cells [16]. In contrast, thyroid glands of adult rodents have clearly more numerous C cells than those of

The C cells share with other neuroendocrine cells the expression of different characteristic neuroendocrine markers, such as chromogranin, synaptophysin and NSE, with chromogranin A as the most reliable marker generally used to characterize cells of DNES (see Figure 4). Most of those neuroendocrine markers are shared with different populations

**7B2 protein** Human, rat, pig Marcinkiewicz et al., 1988 [26]

In addition to calcitonin, C cells may also contain many other regulatory peptides (Table 2), such as calcitonin gene-related peptide (CGRP) [30], katacalcin [31] or GRP [32]. Somatostatin has also been identified within C cells of most species. In the adult rabbits, bats and guinea pigs, most calcitonin-positive cells also contain somatostatin; however, in adult human and rat thyroid glands, only a small proportion of the calcitonin-positive cells are also somatostatin positive [33]. Similarly, peptides including neuromedin U [34] and helodermin-like peptide [35], have been demonstrated to colocalize with CT in normal C cells. Lately, a new generation of regulatory peptides, similar to those characteristically found in some hypothalamic nuclei, such as TRH [36], CART [37] or ghrelin [38], has

**Neuroendocrine markers Species Authors**

**NSE (***Neuron-Specific Enolase***)** Human Lloyd et al. 1983 [22] **PGP 9.5 (***Protein Gene Product 9.5***)** Human Thompson et al., 1983 [23] **Chromogranins (A, B)** Human O'Connor et al., 1983 [24, 25]);

**Synaptophysin** Human Weiler at al., 1989 [25] **Secretogranin-II/chromogranin C** Human Weiler at al., 1989 [25] **Secretoneurin** Human Schmid et al., 1995 [27] **MMP-2 and MMP-9** Human Tomita, 1997 [28] **SV2 (***Synaptic Vesicle Protein 2***)** Human Weiler at al., 1989 [25] **PC1 and PC2 (***Prohormone Convertases***)** Mouse Kurabuchi et al., 2002 [29] **Table 1.** Characteristic substances identified in different neuroendocrine cells, including C cells,

neonates [14, 18].

of nervous cells (Table 1).

according to a chronological order.

increased the long list of substances synthesized by C cells.

**2.4. Substances synthesized by C cells** 

**Figure 2.** Visualization of C cells using an immunohistochemical method for calcitonin. (A) Human thyroid gland; (B) Rat thyroid gland. C cells are more abundant in rat in comparison with the human thyroid gland. 200x.

**Figure 3.** Immunohistochemical demonstration of C cells in the normal rat thyroid gland. (A) Immunostaining for calcitonin followed by the PAS method for carbohydrates. (B) Double immunostaining for calcitonin (red) and somatostatin (brown). In Fig.A, the "parafollicular" ubication of C cells can be observed. In Fig. B, all C cells are immunostained for calcitonin but only very few of them are also immunopositive for somatostatin. This somatostatin-positive C cell exhibits a cytoplasmatic process surrounding neighbouring follicular cells. (x500).

The distribution of C cells within the thyroid lobes has also been reported to vary among species. In most species, C cells are located primarily in the centre of thyroid lobes, including humans [15, 16], mice [17], rats [18] and rabbits [17]. The area where C cells predominate has been termed as 'C-cell region' [19], which is also the place where the mostactive follicles of the gland predominate [20]. In general, portions of the thyroid containing the highest concentration of C cells correspond to the typical fusion sites of between the UBB and the medial thyroid anlage. As another reminder of their embryonic origin, C cells could also be located within the parathyroid gland and thymus in some species, such as rabbits and cats [17].

The ratio of C cells to follicular cells in the thyroid gland differs markedly among species. In humans, C cells comprise less than 1% of the total endocrine-cell population [21], while the proportion of C cells to follicular cells is at least 5% in rats [18]. Furthermore, C cell concentration may vary according to age. In humans, however, data are very contradictory: Wolfe et al. in 1974 found that the number of C cells per unit area appeared to decrease with age [15], while Gibson et al. in 1982 found an age-dependant increase in C cells [16]. In contrast, thyroid glands of adult rodents have clearly more numerous C cells than those of neonates [14, 18].
