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Biomedical Research 1, 237-241 (1980)
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CALCITONIN AND SOMATOSTATIN ARE LOCALIZED IN DIFFERENT CELLS IN THE CANINE THYROID GLAND 1 i l
2 YOSHISUKE KUSUMOTO Department of Anatomy, Niigata University School of Medicine, Asahimachi 1, Niigata 951, Japan
ABSTRACT Localization of calcitonin-like and somatostatin-like immunoreactivity in the puppy thyroid gland was examined by the indirect immunoperoxidase procedure. By the use of the double staining method for calcitonin and somatostatin and of alternate immunostaining in serial, adjacent sections, it has been revealed that calcitonin-like and somatostatin-like immunoreactivity occurs in separate cells.
KEY WORDS thyroid gland / parafollicular cells / calcitonin / somatostatin / immunohisto- chemistry
The localization of calcitonin in parafollicular stained alternately by indirect immunoperoxi- cells of the mammalian thyroid has been inves- dase procedure using anticalcitonin and antiso- tigated by immunohistochemical techniques (4, matostatin antisera. 10, 19, 26, 27). Since the immunohistochemical 2. The double staining method using 4-Cl- study by Hokfelt and his associates (8), soma- l-naphthol and 3, 3’-diaminobenzidine-tetrahy- tostatin has been reported to occur also in the drochloride (DAB) (20) was applied to single parafollicular cells (23, 28). In the thyroid of sections of 4-5 /am thickness. the rat (22) and the rabbit (3), it has been re- Synthetic human calcitonin and synthetic so- ported that calcitonin and somatostatin occur matostatin (Peptide Institute Protein Research in the same cells. The present paper will report Foundation, Osaka) were used as antigens. An- that these polypeptides are localized in separate ticalcitonin and antisomatostatin sera were cells using the puppy thyroid. Since the dis- raised in rabbits by injection of either antigen. covery of the parafollicular cells (l, 21), the These primary antisera were kindly supplied by thyroid of the dog, especially of young ones, Dr Y. Yamada, Shinrakuen Hospital, Niigata has been known to contain them numerously and Dr S. Ito, First Department of Internal and is favored material also in recent studies Medicine, Niigata University School of Med- (4, 10-15). Yet the relationship between cal- icine, respectively. Anticalcitonin serum dilut- citonin and somatostatin producing cells has ed 100 fold and antisomatostatin serum diluted not been examined in this species as far as we 1,000 fold gave the most favorable reactions. are aware. The specificity of the reactions was checked (1) by adding 10 pg/ml human calcitonin or so- matostatin to the primary antiserum before- MATERIALS AND METHODS hand, (2) by using a 100 fold or 1,000 fold di- In the present study, five puppies of both sexes luted normal rabbit serum instead of the pri- from 1 to about 6 months in age were used. mary antiserum, and (3) by the use of only a Tissue blocks of the thyroid were fixed in Bou- peroxidase-conjugated secondary antiserum. in’s fluid for 16-20 hr and embedded in paraf- fin. Sections, partly cut in continuous series, RESULTS were treated with the following methods: 1. Serial sections of 2 pm thickness were The imrnunoperoxidase method clearly demon- 238 Y. KUSUMOTO 'r,i:.~=:(<.-.>q_ qll 1,- " “in-~ \ . 4;.’_ J -. " "‘ .
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Il- 5-in.__ "Q B Fig. 1 Calcitonin-reactive (A) and somatostatin-reactive (B) cells in the puppy thyroid as dem- onstrated by immunoperoxidase reaction. In micrograph B, a somatostatin-reactive cell ex- tends a cytoplasmic process to interstitial space (arrow). Note that the former cells are by far more numerous than the latter cells.
l Calcitonin-reactive cells were distributed widely in the interfollicular and parafollicular areas either in groups or singly and were en- countered constantly and numerously in every section examined (Fig. IA). They were usually oval or polygonal in shape. On the other hand, somatostatin-reactive cells were distributed mostly in the parafol- l licular position. They were much fewer in num- ber than the calcitonin-reactive cells. More- F over, they varied conspicuously in distribution due to the portion of the thyroid cut: they were numerous in the core part of the organ and I Fig. 2 Calcitonin-reactive cells (grey) and somato- I statin-reactive cells (brown) shown by double stain- very scant in the periphery. Somatostatin-re- I ing using 4-Cl-l-naphthol and DAB as substrates active cells were generally polygonal in shape L and tended to be much more angular than the strated in the puppy thyroid that calcitonin-like calcitonin-reactive cells. Some of them extend- immunoreactivity and somatostatin-like immu- ed slender cytoplasmic processes to the inter- noreactivity were localized in specific cells stitial space (Fig. 1B). I which all fall into the category of parafollicular By the use of 4-Cl-1-naphthol for calcitonin cells. However, the cells immunoreactive for and .DAB for somatostatin, we could demon- calcitonin and somatostatin dilfered in popula- strate that both immunoreactivities, colored tion and cell shape from each other (Figs. l-3). grey and brown respectively, were localized in
I CALCITONIN AND SOMATOSTATIN IN THE CANINE THYROID 239
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I -I '-.._=Ii¢*_<§Z=I1.3II3'. - 3I'<}- 3 I I "3" 3- 3 - I,3-=""'I"=3'>'I . % A / / Y‘ /§\/ ‘. 2,-. J2 2 I . A - 3' -.- ‘ I.-r-:-::. I. A I=I.>;;:I- . _ -. -.I-.*. I \. >» -== ,. r -=1’: - I4 I. , 2 , Y Iv I i ‘i » er “*1 Fig. 3 Three consecutive sections of the puppy thyroid alternately stained with anticalcitonin (B) and antisomatostatin (A, C) serum. Identical cells on the adjacent sections are indicated by arrows and figures. Occurrence of calcitonin-like and somatostatin-like immunoreactivity in different cells is evident. 240 Y. KUSUMOTO different cell types. Alt.ernate use of the sub- 16,24), ACTH and gastrin in the gastrin cell strates could stain the cells reversely. In the of the rat stomach (17, 18), endorphin and glu- serial sections cut 2 ,um in thickness we detect cagon in the A cell of the rat pancreas (7). ed calcitonin-like and somatostatin-like immu- It seems worthwhile to mention that DeLellis noreactivity alternately and compared their lo- ez‘ al. (1978) have distinguished two types of calization in detail (Fig. 3, A—C). It could thus thyroid parafollicular cells containing what be confirmed that calcitonin and somatostatin they called type I and type II granules respec- occurred in separate cells. In addition to these tively, in man on the basis of size and electron cells, we observed parafollicular cells which density of secretory granules, although this find- were not reactive to either antiserum. These ing obtained in medullary carcinoma and cal- were ovoid cells of a considerable number, lo- citonin cell hyperplasia cannot be correlated cated mainly in interfollicular positions. directly with the present result in the dog (6). In the present study, we did not count the They showed calcitonin-like immunoreactivity frequency of the cell types mentioned above but in both types of granules by the electron micro- our rough estimation indicated that the cells scopic immunoperoxidase method. Though were decreased in number in the following order: they do not mention occurrence of somatosta- calcitonin-reactive cells, cells not reactive to ei- tin-like immunoreactivity in the medullary car- ther antiserum, and somatostatin-reactive cells. cinoma they examined, the cells possessing ‘the type I granules’ are very similar to the normal somatostatin cells of the pancreas as far as we DISCUSSION judge from the electron micrograph of the pa- The present study showed that somatostatin- per. As a matter of fact, there are reports on reactive cells were separate from calcitonin- the occurrence of somatostatin in medullary reactive cells in the puppy thyroid. As far as thyroid carcinoma (5,25). Yamada er al. (1977) we know, two papers are available on the rela- have reported on the occurrence of somatosta- tionship of somatostatin-reactive cells to calci- tin-like immunoreactive cells in the human, dog, tonin-reactive cells in the thyroid. Noorden er and rat thyroids though they have not precisely al. (1977) demonstrated that in the rat thyroid, examined the topographic relationship of local- somatostatin, when histochemically demonstra- ization between calcitonin and somatostatin ble, was in the same cells as those containing (28). calcitonin. In the rabbit thyroid, Bulfa er al. (1979) demonstrated that at least most, if not all, The author thanks Dr T. Iwanaga for his kind co- somatostatin-immunoreactive cells reacted with operation in this study. anticalcitonin antibodies and that approximately 80% of calcitonin-immunoreactive cells reacted Received for publication 6 Ma)’ I980 with antisomatostatin sera. Thus, the conclusion of these two papers is REFERENCES that all or a major portion of somatostat.in-im- 1. BABER E. C. (1876) Contributions to the minute munoreactive cells were identical with calcito- anatomy of the thyroid gland of the dog nin-immunoreactive cells. This conspicuously (abstract). P:-0c. Roy. Soc. 24, 240-241 contradicts the present results in the dog. Wheth- 2. BEGEOT M., DUBo1s M. P. and DUBOIS P. M. er this discrepancy is simply due to species (1978) Immunologic localization of tr- and ,8- endorphins and ,6’-lipotropin in corticotropic difference is unknown. cells of the normal and anencephalic fetal pitu- At any rate, the problem on the source cells itaries. Cell Tiss. Res. 193, 413-422 of the two biologically active messengers, calci- 3. BUFFA R., CI-IAYVIALLE J. A., FONTANA P., USEL- tonin and somatostatin, attracts attention with LINI L., CAPELLA C. and SOLCIA E. (1979) Par- regard to the ‘one cell-one hormone’ theory afollicular cells of rabbit thyroid store both cal- which was widely accepted until recently and citonin and somatostatin and resemble gut D represented the promotive moment of modern cells ultrastructurally. Histochem. 62, 281-288 endocrinology. Besides the thyroid, and setting 4. Bussoparr G. and PEARSE A.G. E. 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