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Immunohistochemical study of C cell follicles in dog thyroid glands.

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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. (19771 Electron microscopical and immunohistochemical study on parafollicular cell complex with
reference to parafollicular cell as a paraneuron. Arch.
Histol. J pn., 40 /Suppl.):l33- 14 5.
60
Y. KAMEDA
Kameda, Y. (1981) Distribution of C-cells in parathyroid
gland IV and thymus IV of different mammals studied by
immunoperoxidase method using anti-calcitouinand antiC-thyroglobulin antisera. Kawasaki Med. J., 7 9 - 1 11.
Kameda. Y., T. Harada, K. Ito, and A. Ikeda (1979)Immunohistochemical study of the medullary thyroid carcinoma
with reference to C-thyroglobulin reaction of tumor cells.
Cancer, 44:2071-2082.
Kameda, Y., and A. Ikeda. (197th) The identification of a
specific fragment of dog thyroglobulin responsible for immunoreactivity to parafollicular cells. Endocrinology,
102:1702-1709.
Kameda. Y., and A. Ikeda, (1978b) Immunohistochemical
study of a large molecular fragment of thyroglobuliu in
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Kameda, Y . . and A. Ikeda (1979a)C cell (parafollicular cell)immuuoreactive thyroglobulin: Purification, identification and immunological characterization. Histochemistry, 60:155-168.
Kameda, Y., and A. Ikeda (1979b)lmmunochemical and immunohistochemical studies on the 2 7 s iodoprotein of dog
thyroid with reference to thyroglobulin-like reaction of
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241-247.
Kameda, Y., K. Ikeda, and A. Ikeda (1981) Uptake of
radioiodine in follicles of dog C-cell complexes studied by
autoradiograph and immunoperoxidase staining. Anat.
Rec., 200:461-470.
Kameda, Y., H. Shigemoto. and A. Ikeda (1980) Development and cytodifferentiation of C cell complexes in dog
fetal thyroids. Cell Tissue Res., 206:403-415.
Nunez. E.A., and M.D. Gershon (1976)Secretion by parafollicular cells beginning at birth: Ultrastructural evidence
from developing canine thyroid. Am. J. Anat. 147:
375-392.
ONeil. J.A., R.S. Birnbaum, A. Jacobsen, and B.A. Roos
(1981) A carbohydrate-containing form of immunoreactive calcitonin in transplantable rat medullary thyroid
carcinomas. Endocrinology, 108..1098-1100.
Potts Jr., J.T., H.D. Niall, H.T. Keutmann, L.J. Deftos. and
J.A. Parsons (1970) Calcitonin: Recent chemical and immunological studies. In: Calcitonin. Proceedings of the Second International Symposium. S. Taylor, ed. Heinemann. London, pp. 56-73.
Schurch, W., F. Babai, Y. Boivin. and M. Verdy (1977)
Light-electron microscopic and cytochemical studies on
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follicles in the dog thyroid Demonstration by in vivo perfusion. Anat. Rec., 168:69-78.
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