THE ANATOMICAI, RECORD 204:55-60 (19x2) lmmunohistochemical Study of C Cell Follicles in Dog Thyroid Glands YOKO KAMEDA Department of A n a t o m y , Kali,a,saki M 4 i c n l School.Kzirashiki City,Okayama, 701-01Japan ABSTRACT In dog thyroid glands there are C cell follicles which are lined solely by C cells and which accumulate a colloidlike substance in the luminal cavities. In order to clarify the properties of' the colloidlike substance secreted by C cells, the C cell follicles were stained with PAS reaction and immunoperoxidase method using anticalcitonin, anti-C-thyroglobulin, and anti-19s-thyroglobulin antisera, respectively. The colloidlike substance was PAS positive and revealed the strong immunoreaction for C-thyroglobulin but a faint reaction for calcitonin. It was nonreactive with anti-19-thyroglobulin antiserum. These results confirm that C cells synthesize the glycoprotein immunoreactive to anti-C-thyroglobulin antiserum in addition to calcitonin and can store it in the follicular lumens. Thyroid parafollicular (C)cells synthesize and secrete calcitonin, a polypeptide hormone which lowers plasma calcium. They have no ability to incorporate radioiodine and are not functionally related to thyroid hormone synthesis (Kameda et al., 1981). However, several electron microscopic studies have reported that in dog thyroid glands there are small follicles composed solely of C cells; the follicles store a colloidlike substance in the luminal cavities, and C cells lining such follicles exhibit microvilli protrusions into the cavities and tight junctions at the apical lateral surface (Teitelbaum e t al., 1970; Nunez and Gershon, 1976; Kameda, 1977). Thus, C cells can form follicles and store secretory products into the follicular lumens, as well as thyroid follicular cells. I t is considered that C cells synthesize a glycoprotein resembling thyroglobulin in addition to calcitonin. In order to clarify the properties of the colloidlike substance secreted by C cells, the present study investigates the C cell follicles of dog thyroids with periodic acidSchiff reaction and immunoperoxidase staining using anticalcitonin, anti-C-thyroglobulin (C-Tg), and anti-19s-thyroglobulin antisera, respectively. Thyroglobulin, which is a glycoprotein essential for the formation of thyroid hormones, is composed of several components. In addition to the main protein component, 19s-thyroglobulin, there are several components with slower and faster sedimentation coefficients. The antiserum generated against C-Tg, a thyroglobulin component having the largest molec- 0003-276X/82/2041-0055$02.00 0 1982 AIJAN R. LISS, INC. ular weight (mol wt approx. 2,600,000), specifically reacts to secretory granules of C cells in addition to luminal colloid and follicular cells (Kameda and Ikeda, 1978a, 1979a). MATERIALS AND METHODS Sixty dogs of either sex and various ages from newborn to adult were used. The thyroid glands were fixed in Bouin's solution for 24-48 hours and embedded in paraffin. One lobe from each animal was cut into 5-10-pm-thick longitudinal total serial sections. The other lobe was cut in 5-pm nonserial sections. Some sections were stained with periodic acid-Schiff (PAS) reaction or hematoxylin-eosin. The immunohistochemistry was carried out according to the unlabeled antibody-enzyme bridge technique as previously described (Kameda and Ikeda, 1978a). Three specific antisera were employed: anti-porcine calcitonin, anti-dog C-Tg, and anti-dog 19s-thyroglobulin antisera. The preparation and serological studies for each antiserum have been described previously (Kameda and Ikeda, 1979a,b).Control reactions included replacing the primary antisera with normal (nonimmune) rabbit serum and absorbing the primary antisera with an excess of the respective antigens (extracted porcine calcitonin, dog C-Tg, and dog 19s-thyroglobulin). RESULTS In dog thyroid glands, the C cells are grouped mostly in cell clusters or, rarely, are distriI k ~ r i \ r dI t'1)iu.m 1 2 1982 ncceptcd Rl'i, 25 I Y X L 56 Y. KAMEDA buted as single cells in para- and inter-follicular positions. C cell follicles occasionally occurred in the areas in which C cells were abundantly distributed, forming large cell groups. They were more frequently observed in adult dogs than in young dogs. Each follicle was lined solely by C cells which were single cuboidal or columnar (Fig. 1).The luminal cavities, ordinarily round or oval in shape, were small and measured approximately 10-30 pm in diameter. The larger luminal cavities reaching 50 pm in diameter were occasionally observed (Fig. 2 ) .The lumens contained a colloidlike substance which stained with PAS reaction in much the same way as the colloid in typical thyroid follicles (Figs. 1, 2). The amounts of colloidlike substance varied from lumen to lumen. In most of the C cell follicles, the colloidlike substance occupied only a part of the luminal cavities and located primarily at the periphery. The lumens filled with the colloidlike substance, however, were rarely observed. Subsequently, the C cell follicles were stained with immunoperoxidase method using three specific antisera, anticalcitonin, anti-C-Tg, and anti-19s-thyroglobulin antisera. The C cells forming follicles had the same staining properties to each antiserum as the cells in clusters. With anticalcitonin antiserum, C cells were specifically stained; their cytoplasm was filled with numerous reaction products (Figs. 3, 4). The thyroid follicular cells, colloid, or surrounding fibrovascular connective tissue was nonreactive to the antiserum. The colloidlike material in the C cell follicles revealed the irregularity of the staining reaction with anticalcitonin antiserum. I t was usually stained weakly with the antiserum. However, in some cases the colloidlike material represented the considerable immunoreactivity for calcitonin (Fig. 3), whereas in others it revealed no reactivity (Fig. 4). With anti-C-Tgantiserum, C cells in addition to follicular cells and follicular colloid were stained densely. The reaction patterns of anti- Figs. 1, 2. C cell follicles of dog thyroid glands stained with PAS reaction. Their lumens contain the accumulation of small amounts of colloidlike substance (arrows) which is densely PAS-positive. F, thyroid follicles. x 1,200 Fig. 1. A C cell follicle is covered with single columnar C cells and is very small compared with typical thyroid follicles. Fig. 2. A C cell follicle showing a comparatively large luminal cavity. 57 C CELL FOLLICLE IN DOG THYROID C-Tg antiserum to C cells were completely identical with those of anticalcitonin antiserum; the secretory granules of C cells specifically reacted to the antiserum (Figs. 5, 6). The colloidlike substance in the C cell follicles constantly gave a strong immunoreaction for C-Tg (Figs. 5 , 6). The typical thyroid follicles revealed the different immunoreaction for 19s-thyroglobulin owing to dilutions of the antiserum. The cytoplasm of follicular cells was stained densely with slightly diluted anti-19s-thyroglobulin antiserum, whereas the colloid in follicular lumens was stained with the highly diluted antiserum (Figs. 7, 8). The peripheral regions of colloid and the colloid in the small primordial follicles, consisting of newly synthesized 19sthyroglobulin, were stained densely with all dilutions of the antiserum up to the marginal dilution. With any dilutions of anti-1%thyroglobulin antiserum, the colloidlike substance in C cell follicles revealed no immunoreaction (Figs. 7,8).In addition, C cells did not react to the antiserum. Figs. 3, 4. C cell follicles of dog thyroids stained by immunoperoxidase method using anticalcitonin antiserum. The C cells are filled with numerous immunoreactive secretory granules. The colloidlike substance (arrows)in C cell follicles varies in the immunoreactivity. F, thyroid follicles. x 1,200 DISCUSSION Thyroid C cells, like follicular cells, have an ability to form follicles and function, accumulating the secretory products into the follicular lumens. In the present study, the immunocytochemical reactions of the C cell follicles for calcitonin, C-Tg, and 19s-thyroglobulin, respectively, were investigated for the first time, and the properties of the secretory products stored in the follicular cavities were clarified. The C cells forming follicles had the same morphologic and immunostaining properties as the cells in solid clusters; the secretory granules were stained densely with both anticalcitonin and anti-C-Tg antisera. The secretory products in the follicular lumens were strongly PAS-positive and resembled the colloid in typical thyroid follicles. The colloidlike substance gave constantly the strong immunoreaction for C-Tg, though it was weakly reactive or occasionally even nonreactive with anticalcitonin antiserum. Thus, it is clear that C cells synthesize Fig. 3. The colloidlike substance showing the considerable immunoreactivity for calcitonin. Fig. 4. The colloidlike substance showing no immunoreactivity. Figs. 5, 6. C cell follicles of dog thyroids stained by immunoperoxidase method using anti-C-Tg antiserum. Since the antiserum was previously absorbed with 19s-thyroglobulin. the immunoreactivities of follicular cells and luminal colloid are weak. The secretory granules of C cells are stained densely. F. thyroid follicles. x 1,200 Fig. 5. Colloidlike substance (arrow) in C cell follicle reveals the strong immunoreaction for C-Tg. Fig. 6. There are two C cell follicles. Colloidlike substance (arrows)is intensely immunoreactive. Figs. 7.8. C cell follicles of dog thyroids stained by immunoperoxidase method using anti-19s-thyroglobulin antiserum in different dilutions. C cells are completely devoid of immunoreaction. F, thyroid follicles. x 1,200 Fig. 7. Using the slightly diluted antiserum (1:40). cytoplasm of follicular cells and periphery of colloid are stained densely. Colloidlike substance (arrow) in C cell follicle reveals no immunoreactivity. Fig. 8. Using the highiy diluted antiserum (1:2.000), the immunoreaction of colloid becomes distinct, but the colloidlike substance (arrow)in C cell follicle is still nonreactive. C CEI,I, FOLLICLE I N DOG THYROID the glycoprotein immunoreactive to the C-Tg antiserum in addition to calcitonin and can store it in the follicular lumens. The previous several light and electron microscopic studies have noted the presence of significant amounts of carbohydrate-containing material in the secretory granules of C cells by the PAS method or the phosphotungstic acid method (Gabe, 1959; Schurch et al., 1977). The C cell follicles were always small in size and accumulated a small amount of colloidlike substance in the luminal cavities, in contrast to typical thyroid follicles filled with colloid and large in size. The follicle structures are necessary for synthesis and secretion of thyroid hormones by follicular cells because the iodination of thyroglobulin occurs at the apical cell surface and newly iodinated thyroglobulin matures in the follicular lumens. On the other hand, C cell follicles are not thought to be necessary structures for the function of C cells, since most of C cells remain in solid clusters or are distributed singly. Probably, excess secretory products of C cells are temporarily stored in the lumens. Several studies have suggested that anti-CTg antiserum reacts to the biosynthetic precursors of calcitonin in the C cells: 1) C-Tg and calcitonin antisera cross-react to a certain degree (Kameda and Ikeda, 1979a); 2) the reaction of anti-C-Tg antiserum in fetal C cells appears at earlier stages and more strongly than that of anticalcitonin antiserum (Kamedaet al., 1980); 3) tumor cells in medullary thyroid carcinoma, a distinct neoplasm derived from C cells, reveal a far stronger immunoreaction for C-Tg than for calcitonin (Kameda et al., 1979). Recent biochemical studies have reported that higher molecular weight precursors of calcitonin contain covalently bound carbohydrates (O’Neilet al., 1981: Jacobs et al., 1981)as well as the precursors of ACTH and vasopressin. It is considered that the colloidlike substance in the C cell follicles consists of precursors for calcitonin. Unlike the colloid in typical thyroid follicles, the colloidlike substance in C cell follicles was completely devoid of immunoreaction for 19sthyroglobulin. The glycoprotein secreted by C cells is not related to 19s-thyroglobulin and not involved in the synthesis of thyroid hormones, though it reveals the immunoreaction for C-Tg, a thyroglobulin component. Several data support the idea that C-Tg itself is not synthesized by the C cells and that glycosylated precursors of calcitonin cross-react to anti-C-Tg antiserum. First, C-Tg has common 59 antigenic determinant sites with 19s molecules; anti-C-Tg antiserum forms a long precipitin line with 19s-thyroglobulin in immunodiffusion tests and it produces the same immunoperoxidase reaction to follicular cells and follicular colloid as does anti-19s-thyroglobulin antiserum (Kameda and Ikeda, 1979a). However, both colloidlike substance in C cell follicles and secretory granules of C cells reveal no immunoreactivity for 19s-thyroglobulin. Second, C-Tg has a huge molecular weight, approximately 2,600,000, whereas higher molecular weight precursors of calcitonin reported are far smaller, i.e., 17,000 (Jacobset al., 1981), or 9,600 (O’Neil et al., 1981) molecular weights, respectively. Calcitonin is composed of a single chain of 32 amino acids with a 1, 7-intrachain disulphide bridge. There is a large difference in the amino acid sequence of calcitonin from various animals (Potts et al., 1970).Therefore, the crossreactivity of antiserum to calcitonin is limited to the C cells synthesizing structurally similar calcitonin. For example, the antiserum to human calcitonin cross-reacts strongly with C cells of rabbits, rats, and guinea pigs, but faintly with the cells of dogs, cats, and goats: The antiserum to procine calcitonin cross-reacts strongly with the C cells of dogs, cats, and goats, but very weakly with those of rabbits, rats, and guinea pigs (Kameda, 1981). In contrast to the antiserum to calcitonin, antiserum to dog C-Tg shows a high degree of crossreactivity to the C cells of most of mamalian species; the cells of rats, rabbits, hamsters, mice, cats, lions, goats, cows, and human, etc., are stained densely with anti-dog C-Tg antiserum (Kamedaand Ikeda, 1978b).Anti-C-Tg antiserum may react to the carbohydrate moieties of glycosylated precursors of calcitonin, whose sequence seems to be similar in various animal species. ACKNOWLEDGEMENTS This study was supported by a grant (No. 56570016) from the Ministry of Education of Japan. LITERATURE CITED Gahe. P.M. (1959) Donnees histochimiques sur les cellules parafolliculaires de la glande thyroide du chien. Acta Anat., 38:332-344. Jacobs. J.W., P.K. Lund, J.T. Potts Jr.. N.H. Bell, and J.F. Hahener (1981) Procalcitonin is a glycoprotein. J . Biol. Chem., 256:2803-2807. Kameda. Y. 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