Uptake of radioiodine in follicles of dog C-cell complexes studied by autoradiograph and immunoperoxidase staining.код для вставкиСкачать
THE ANATOMICAL RECORD 200:461-470 (1981) Uptake of Radioiodine in Follicles of Dog C-Cell Complexes Studied by Autoradiograph and lmmunoperoxidase Staining YOKO KAMEDA, KAZUO IKEDA, AND AKIRA IKEDA Department ofAnatomy, Kawasaki Medical School, Kurashiki City, O k a y a m , 701 -01 Japan ABSTRACT C-cell complexes are special cell groups consisting of a mass of C-cells associated with other epithelial elements and cysts. They are remnants of ultimobranchial bodies retaining fetal characteristics. In the C-cell complexes there are follicular cells in various stages of differentiation, i.e., the cell clusters not yet organized into follicles, primordial follicles with small lumens and comparatively enlarged follicles storing plentiful amounts of colloid. They have a morphology similar to follicular cells of fetal thyroid glands and react to antiserum to 19s thyroglobulin. In order to determine whether or not the follicles in these complexes have the ability to incorporate radioiodine, autoradiography after a single injection of '''I was combined with immunoperoxidase staining using specific anti-calcitonin, anti-C-thyroglobulin, and anti-19s thyroglobulin antisera. The 19s-positive cells not yet organized into follicles did not take up radioiodine. Primordial follicles showed a heavy accumulation of silver grains over their follicular lumens storing new 1 9 s thyroglobulin as colloid. Comparatively enlarged follicles revealed a strong autoradiographic reaction and their labeling patterns were identical with those of typical thyroid follicles. These results confirm that the follicles in C-cell complexes, as well as thyroid follicles, can incorporate radioiodine and are related to thyroid hormone synthesis. That is, functional thyroid follicles can arise from the ultimobranchial bodies. In the thyroid gland of dogs there are C-cell complexes, special cell groups of C-cells (Kameda, 1971, 1976). They are remnants of ultimobranchial bodies and retain a n abundance of fetal characteristics for a very long time, showing a large number of undifferentiated cells and immature C-cells even in adult dogs (Kameda e t al., 1980; Kameda and Ikeda, 1980a). The complexes are composed of C-cells in various stages of differentiation, undifferentiated epithelial cells, and cysts of various sizes. In addition, many complexes contain colloid-containing small follicles similar in light and electron microscopic features to those of the definite thyroid gland that have developed from the midventral evagination of the pharynx (Kameda, 1971, 1973, 1977). The undifferentiated cells, which are peculiar embryonic cells, are characterized by a small amount of cytoplasm, a chromatin-dense nucleus, and a lack of immunoreactivity for antisera (Kameda, 1973; Kameda et al., 1980). The cells are more numerous in younger animals and, in fetal dogs, they occupy the greatest part of the complex. In a previous developmental and immunohistochemical study (Kameda et al., 1980), it was evidenced that the undifferentiated cells can, over an extended period of time, develop into clusters of follicular epithelial cells which proceed to form lumens and develop into structurally differentiated follicles. That is, not only C-cells but also follicular cells can differentiate from cells of ultimobranchial body origin. In contrast to thyroid follicular cells, the cells derived from ultimobranchial bodies develop very slowly; cell clusters not yet organized into follicles and primordial follicles with small lumens are recognizable even in the complexes of adult dogs (Kameda and Ikeda, 1980a). The clusters of follicular cells as well as the follicles that develop from them in the complexes can be stained immunocytochemically with antiserum against 19s thyroglob- 0003-276X/81/2004-0461$03.00 0 1981 ALAN R. LISS, INC. Received Odober 23, 1980;accepted January 8,1981. 462 Y. KAMEDA, K. IKEDA, AND A. IKEDA ulin (Kameda, 1977; Kameda et al., 1980; Kameda and Ikeda, 1980a). The presence of this particular glycoprotein is essential for the formation of thyroid hormones. The anti-19s thyroglobulin antiserum does, however, crossreact with inactive precursors and polymers of 19s thyroglobulin and further with the glycoprotein existing in thyroidal cysts. Therefore, positive immunocytochemical staining is not conclusive proof of the presence of 19s thyroglobulin and of thyroid hormone secretion. In order to determine whether the follicular cells derived from the ultimobranchial bodies possess the ability to synthesize and secrete thyroid hormones, the present study investigates by light microscopic autoradiography whether or not the follicles in complexes can incorporate lZ5I.The serial sections are also treated for distinct identification of constituent elements of complexes with immunoperoxidase method using specific anti-calcitonin, anti-C-thyroglobulin (C-Tg),and anti-19s thyroglobulin antisera, respectively. C-Tg is the largest molecular weight component (mol wt approx. 2,600,000) of thyroglobulin, which is composed of several components (Kameda and Ikeda, 1979b). Antiserum to C-Tg reacts to secretory granules of C-cells in addition to follicular cells and follicular colloid. The relationship between calcitonin and C-Tg has been described in the previous studies: (1)C-Tg and calcitonin antisera cross-react to a certain degree (Kameda and Ikeda, 1979b); 2) the reaction of anti-C-Tg antiserum in fetal C-cells appears at earlier stages and more strongly than that of anti-calcitonin antiserum (Kameda et al., 1980); 3) tumour 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).These data strongly suggest that the C-Tg molecule contains the specific peptide chain composition corresponding to the biosynthetic precursor of calcitonin. Furthermore, the biochemical nature of C-Tg in comparison with that of other thyroglobulin Fig. 1. Serial sections of C-cell complexes of a dog 8 hours after injection of Na1151(100 p,Ci/kg). Thyroid parenchyma in upper portion. F, follicles in thyroid; C, cyst in complex. x 100. a. Immunoperoxidase reaction for calcitonin. A greater portion of the complexes are occupied by C-cells in various stages of differentiation. Insertion: Higher magnification ( x 520)of the complex. The C-cells vary in size and in their immunoreactivity to anti-calcitonin antiserum. Undifferentiated cells (arrow) showing no immunoreaction are also observable. b. Immunoperoxidase reaction for 19s components has been reported in detail in other papers (Kameda, 1981a,b). MATERIALS AND METHODS Twelve young dogs of either sex, weighing 1t o 4 kg, were given a single intraperitoneal injection of 100 pCi carrier-free Na'251 (The Radiochemical Centre, Amersham, England) per kg body weight. The animals were killed 1,5,8,24,and 48 hours after the injection. The thyroid glands were fixed in Bouin's solution for 24-48 hours. The specimens were embedded in paraffin and cut into 5-7-pm-thick longitudinal total serial sections. Some sections were stained with hematoxylin-eosin or periodic acid-Schiff (PAS). For immunological staining, an unlabeled antibody-enzyme bridge technique was used as previously described (Kameda and Ikeda, 1978). Three specific antisera were employed: anti-porcine calcitonin antiserum, anti-dog CTg antiserum, and anti-dog 19s thyroglobulin antiserum. The preparation and serological studies of these antisera have been described previously (Kameda and Ikeda, 1979a,b). The sections were reacted with the following sequence of solutions after hydration with phosphate-buffered saline (PBS): an appropriate dilution of the primary antisera (anti-calcitonin, anti-C-Tg, and anti-19s antisera) for 5 hours, goat anti-rabbit globulin antiserum ( 1 : l O ) for 15 minutes, and rabbit anti-peroxidase antiserum (1:lO) for 15 minutes. After incubation in horseradish peroxidase solution (0.5 mg/100 ml) containing 0.1%bovine serum albumin, reaction products were developed with 3,3'-diaminobenzidine tetrahydrochloride (0.5 mg/ml) and HzO2(0.01%).Sections were washed three times for 7 minutes each with PBS between the steps. All reactions were carried out a t room temperature. Control reactions included replacing the primary antisera with normal (nonimmune) rabbit serum and absorbing primary antisera with excess of the respective antigens (extracted porcine calcitonin, dog C-Tg, and dog 19s thyroglobulin). thyroglobulin. No immunoreactive cells are detectable in complexes. Follicular cells in thyroid parenchyma reveal intense immunoreactivityand foamy substances in cyst are also reactive. c. Autoradiographic reaction and poststaining with hematoxylin-eosin. The follicular colloid in thyroid parenchyma shows an accumulation of silver grains. The smaller follicles reveal denser autoradiographic images. There are essentially no grains over the complexes.The cyst is devoid of silver grains. E9P 464 Y. KAMEDA, K. IKEDA, AND A. IKEDA Figure 2 a-c. DOG C-CELL COMPLEX FOLLICLE '%I UPTAKE 465 Fig. 2. A C-cell complex containing clusters of follicular cells 8 hours after injection of Nalz5I. Figures a to c are serial sections of the complex stained by three different methods. a. Immunoperoxidase reaction for calcitonin. The immunoreactivity of C-cells in complex is weaker than that of the cells in thyroid parenchyma. x 115. b. Immunoperoxidase reaction for 19s thyroglobulin. Small clusters of 19s-positive cells are distributed in the complex. Small follicles (F) in upper left. x 115. c. Autoradiographic reaction and poststaining with hematoxylin-eosin. Silver grains are localized over follicular lumens (F) at upper left, whereas not present over C-cell masses and over clusters of follicular cells (arrows). x 115.d. Higher magnification of the complex in b. The follicular cells are loaded with numerous reaction products for 19s thyroglobulin but not yet organized into follicles. x 580. e. Higher magnification of the complex in c. The clusters of follicular cells reveal more concentrated nuclei and smaller cytoplasm than C-cell masses. A small follicle (arrow) forming among follicular cells shows a distinct accumulation of silver grains, whereas the silver grains over the clusters of follicular cells and C-cells do not exceed those of the background. x 580. For autoradiography, the sections were coated with an aqueous dilution of melted Sakura NR-M2, dried, and stored in lighttight boxes containing Silica Gel. After 2- to 4-week exposure a t 4"C, radioautography was developed for 5 minutes in Konidol X, fixed in Fuji Super Fix, and stained with hematoxylineosin. cells revealed variable amounts of reaction products for calcitonin and C-Tg, representing various stages of differentiation (Fig. la). Mature C-cells with extensive cytoplasm were filled with numerous immunoreactive secretory granules. Immature C-cells contained sparse reaction products in the still-small amount of cytoplasm. The undifferentiated cells displayed scanty cytoplasm and chromatin-dense nuclei, and were more numerous in the complexes of younger animals. They were not stained with any antisera. In this type of complex, there were no cells showing the immunoreactivity for 1 9 sthyroglobulin (Fig. lb). The distribution of silver grains was not observed in the complexes (Fig. lc). That is, neither C-cells nor undifferentiated cells possessed the ability to concentrate radioiodine. Subsequently, the second type of complex was investigated. As the most immature form, there were complexes containing primitive follicular cells not yet organized into follicles RESULTS Eight out of the twelve dogs given a single injection of Na'251had one or more C-cell complexes. Two types of C-cell complexes were observed as described previously (Kameda, 1971; Kameda and Ikeda, 1980a).In one type of complex there were a large number of C-cells a t various developmental stages, undifferentiated cells, and various-sized cysts. A second type contained follicular cells in addition to these elements. When the first type of complex was stained with anti-calcitonin and anti-C-Tg antisera, C- 466 Y.KAMEDA, K. IKEDA, AND A. IKEDA Fig. 3. Autoradiograph of a C-cell complex containing primordial follicles and small follicles (Ff24 hours after injection of NalZ5I.Poststained with hematoxylin-eosin. Heavy accumulation of silver grains is obsemed over follicular lumens. x 230. (Fig. 2). The cells had a small amount of cytoplasm and chromatin-dense nuclei (Fig. Be). Although they were indistinguishable from undifferentiated cells with hematoxylin-eosin staining, their cytoplasm was filled with numerous reaction products after immunoperoxidase staining using slightly diluted anti-19s antiserum (Fig. 2b,d). The 19s-positive cells were arranged in small cell clusters and dispersed among C-cell masses in the complexes. (Fig. 2a,b). No significant incorporation of radioiodine into the 19s-positive cells was observed (Fig. 2c,e). In the complexes containing primordial follicles which represented small cavities storing new colloid, the follicles revealed a dense immunoreaction for 1 9 s thyroglobulin. Especially, colloid in follicular lumens was stained densely with all dilutions of anti-19s antiserum up to the marginal dilution. Heavy accumulation of silver grains was observed over the follicular colloid (Fig. 3); the labeling was correlated with the intensity of immunoperoxidase reaction. Autoradiographic reaction was specific for the follicular colloid. In the complexes containing comparatively enlarged follicles which were lined with a number of follicular cells and stored a plentiful amount of colloid in the follicular lumens, the follicles revealed the same immunoperoxidase and autoradiographic reactions as typical thyroid follicles (Fig. 4). The autoradiographic reaction of dog thyroid glands has been reported in the previous study (Kameda, 1981b). In complexes as well as in thyroids, the follicular cells were stained diffusely with slightly diluted anti-19s antiserum and colloid was stained more densely with the highly diluted antiserum (Fig. 4b). Homogeneous accumulation of silver grains was obtained over the follicular lumens (Fig. 4c). Among these, usually, smaller follicles accumulated more numerous Fig. 4. Serial sections of a C-cell complex containing comparatively enlarged follicles 24 hours after injection of NalZ5I.Congestion (arrows) which reveals a n excessive accumulation of blood owing to dilation of blood capillaries is seen in masses of C-cells. F, follicles. x 150. a. Immunoperoxidase reaction for C-Tg. Secretory granules of C-cells show strong immunoreaction. Using anti-C-Tg antiserum absorbed previously with a small quantity of 19s thyroglobulin, the reaction of colloid is denser than that of follicular cells. b. Immunoperoxidase reaction for 19s thyroglobd i n . Using the slightly diluted antiserum, the reaction of follicular cells is conspicuous. c. Autoradiographic reaction and poststaining with hematoxylin-eosin. Follicular colloid shows a heavy accumulation of silver grains. DOG C-CELL COMPLEX FOLLICLE 1251 UPTAKE 467 468 Y.KAMEDA, K. IKEDA, AND A. IKEDA silver grains. Ring reactions, i.e., relatively dark images in the shape of a ring over the peripheral region of the colloid, were observed over many lumens. The intensity of autoradiographic images increased with time and reached a peak a t 24 hours after the injection of 1251.At 48 hours after the injection, the intensity of reaction was similar to that after 24 hours. Thus, the follicles in complexes possessed the ability to accumulate radioiodine and the labeling pattern was identical with that of thyroid follicles. In this type of complex, most of C-cells were similar to ordinary thyroid C-cells, i.e., mature (Fig. 4a). They contained numerous reaction products for calcitonin and C-Tg in extensive cytoplasm. The C-cells revealed no autoradiographic reaction (Fig. 4c). DISCUSSION It is generally accepted by recent researchers that follicular cells and C-cells of thyroid glands are two distinct cell lines with different origins; i.e., follicular cells are derived from midline outpouching of the ventral pharyngeal floor and C-cells from the ultimobranchial bodies. However, we confirmed in a series of studies on C-cell complexes, remnants of ultimobranchial bodies, that the follicular cells are derived not only from the median thyroid primordium but also from the ultimobranchial bodies; i.e., the follicular cells as well as C-cells develop from undifferentiated cells within the complexes (Kameda et al., 1980; Kameda and Ikeda, 1980a,b). Transitional features of undifferentiated cells changing into primitive follicular cells were found in the C-cell complexes by immunoperoxidase and electron microscopic observations (Kameda, 1973; Kameda et al., 1980). Furthermore, follicular cells in various developmental stages are detectable in the complexes: 19s-positive cell clusters not yet organized into follicles,primordial follicles, and comparatively enlarged follicles. With the exception of maturing late, the follicular cells derived from ultimobranchial bodies represent the same characteristics as the cells from median thyroid primordium; both cells display the same light and electron microscopic features (Kameda, 1971,1973,1976)and are controlled by the pituitary gland (Kameda, 1974): Furthermore, they reveal the immunoreaction for 19s thyroglobulin (Kameda, 1977). Thyroglobulin is heterogeneous. In addition to the main protein component, 19s thyroglobulin, there are several components with slower and faster sedimentation coefficients. The slower components, 3-8S, 12s and 17-18S, are subunit precursors or noniodinated forms of 19s molecule. They are formed in rough endoplasmic reticulum and Golgi complexes of follicular cells (Nadler et al., 1964; Whur et al., 1969). Iodination of thyroglobulin takes place in the follicular lumens adjacent to the plasma membrane, resulting in a 19s molecule. The iodinated 19s molecules are accumulated in follicular lumens as colloid. The faster components, 27S, 32S, 37s and >37S, are usually accepted as polymers of 19s molecule (Salvatore et al., 1965; Van der Walt and Van Jaarsveld, 1972). They are formed by random noncovalent aggregation of 19s molecules during storage in follicular colloid (Frati et al., 1974). It is well known by biochemical experiments using sucrose density gradient ultracentrifugation that radioactive iodine is predominantly accumulated in 19s component fraction at all time intervals after injection and that its accumulation in faster and slower component fractions is very low (Robbinset al., 1966; Roche et al., 1968). The incorporation of radioiodine into each thyroglobulin component could be determined more accurately by the use of 2-16% polyacrylamide gradient gel electrophoresis after dogs were injected with Na'251 (Kameda, 1981b). When the distribution of radioactivity in each thyroglobulin component was determined along the gels, more than 90%of total thyroid radioiodine was observed in the 19s component band, and 27S, 32S, and 12s component bands also revealed weak radioactivities. No radioactivity was observed in the other component bands. Similarly, when the incorporation was determined by the gel autoradiography, a markedly strong autoradiographic reaction was obtained over 19s component band and the reaction conspicuously increased with time. The 27S, 32S, and 12s components also revealed weak autoradiographic reactions but little increase of the reaction with time. Thus, radioactive iodine is specifically incorporated into the 19s component; i.e., iodination, production of thyroid hormones (thyroxine and triiodothyronine), is specific for 19s component. In spite of being in various stages of maturation and having different functions, the antigenicity of each thyroglobulin component is very similar; the slower and faster components have an antigenic determinant in common with 19s component (Kameda and Ikeda, 1978, 1979a). The antisera to slower and faster components, as well as antiserum to 19s component, react to both follicular cells and luminal DOG C-CELL COMPLEX FOLLICLE 1251 UPTAKE colloid after immunoperoxidase staining. Therefore, it is impossible to determine by immunohistochemical methods alone whether or not the follicular cells derived from ultimobranchial bodies produce 19s component and are related to thyroid hormone synthesis. Autoradiographic technique is necessary to clarify this problem. The present light microscopic autoradiography clearly demonstrated that the follicles in complexes as well as thyroid follicles revealed a n accumulation of silver grains after dogs were injected with NalZ5I. The accumulation conspicuously increased with time and so-called ring reactions were observed over the peripheral region of follicular colloid. That is, the follicles in complexes possess the ability to accumulate radioiodine and their labeling pattern is identical with that of thyroid follicles. It is clear that the follicular cells in complexes produce 19s component to secrete thyroid hormones; the follicular cells derived from ultimobranchial bodies have a function completely identical with the cells from median thyroid primordium. The follicular cells derived from ultimobranchial bodies develop very slowly. In respect to immunoreaction and folliculogenesis, the follicular cells in complexes coincide with the cells of fetal thyroid glands (Kameda, 1973, 1977; Kameda et al., 1980; Kameda and Ikeda, 1980a). The present study further confirmed that their ability to incorporate lZ5Iwas also similar to that of fetal thyroid follicles described in rabbits and human (Olin et al., 1970; Roques et al., 1972). That is, in complexes the follicular cells remaining as cell clusters still lacked the ability to incorporate radioiodine. The iodine accumulation began synchronously with the formation of follicles; the onset of lZ5I collection and storage coincided with the appearance of the first follicles. The primordial follicles forming among cell clusters revealed a markedly heavy accumulation of silver grains. Along with the enlargement of follicular lumens, their autoradiographic images became similar to those of thyroid follicles of postnatal dogs. In complexes as well as in thyroids, silver grains were specifically located on follicular colloid. Among these, the smaller the follicles, the darker was the autoradiographic image. Especially, the reaction of primordial follicles was quite strong. It is sure that the colloid in primordial follicles is composed of newly synthesized 19s component. In fact, the new colloid represents markedly dense immunoreaction for 19s thyroglobulin. Increased size of 469 follicular lumens is reflected by increasing proportion of faster components in colloid, which are formed by polymerization of 19s molecules and reveal a low uptake of radioiodine, and by decreasing accumulation of silver grains on luminal colloid. ACKNOWLEDGMENTS This study was supported in part by a grant (No. 557012) from the Ministry of Education of Japan. LITERATURE CITED Frati, L., J. Bilstad, H. Edelhoch, J.E. Rall, and G. Salvatore (1974) Biosynthesis of the 27s thyroid iodoprotein. Arch. Biochem. Biophys., 162: 126-134. Kameda, Y. (1971) The occurrence of a special parafollicular cell complex in and beside the dog thyroid gland. Arch. Histol. Jpn., 33: 115-132. Eameda, Y. (1973) Electron microscopic studieson the parafollicular cells and parafollicular cell complexes in the dog. Arch. Histol. Jpn., 36: 89-105. Kameda, Y. (1974) Relationship between the thyroid parafollicular cells and pituitary gland. Arch. Histol. Jpn., 37; 225-244. Kameda, Y. (1976) Fine structural and endocrinological aspects of thyroid parafollicular cells. In: Chromaffin, Enterochromaffin and Related Cells. R.E. Coupland and T. Fujita, eds. Elsevier, Amsterdam, pp. 155-170. Kameda, Y. (1977) Electron microscopical and immunohistochemical study on parafollicular cell complex with reference to parafollicular cell as a paraneuron. Arch. Histol. Jpn., 40: Suppl. pp. 133-145. Kameda, Y. (1981a) Studies on C cell-immunoreactive thyroglobulin: Age difference, reduction and trypsin digestion. Biochim. Biophys. Acta In press. Kameda, Y. (1981b) Studies on C cell-immunoreactive thyroglobulin: Uptake of lz5I and iodine content. In press. 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 (1978) The identification of a specific fragment of dog thyroglobulin responsible for immunoreactivity to parafollicular cells. Endocrinology, 102: 1702-1709. Kameda, Y., and A. Ikeda (1979a) Immunochemical and immunohistochemical studies on the 275 iodoprotein of dog thyroid with reference to thyroglobulin-like reaction of the parafollicular cells. Biochim. Biophys. Acta, 577: 241-247. Kameda, Y., and A. Ikeda (1979b) C cell (parafollicular cell)immunoreactive thyroglobulin: Purification, identification and immunological characterization. Histochemistry, 60: 155-168. Kameda, Y., and A. Ikeda (1980a) Immunohistochemical study of the C-cell complex of dog thyroid glands with reference to the reactions of calcitonin, C-thyroglobulin and 19s thyroglobulin. Cell Tissue Res., 208: 405415. Kameda, Y., and A. Ikeda (1980b) Immunohistochemical reactions of C-cell complexes in dogs after induced hypercalcemia, antithyroid drug treatment and hypophysectomy. Cell Tissue Res., 208: 417432. 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. Nadler, N.J., B.A. Young, C.P. Leblond, and B. Mitmaker 470 Y. KAMEDA, K. IKEDA, AND A. IKEDA (1964)Elaboration of thyroglobulin in the thyroid follicle. Endocrinology, 74: 333-354. Olin, P., R. Ekholm, and S. Almqvist (1970) Biosynthesis of thyroglobulin related to the ultrastructure of the human fetal thyroid gland. Endocrinology, 87: 1000-1014. Robbins, J., G.Salvatore, G. Vecchio, and N. Ui (1966)Thyroglobulin and 273 iodoprotein. Iodination and ultracentrifugal heterogeneity. Biochim. Biophys. Acta, 127: 101-111. Roche, J.,G.Salvatore, L. Sena, S. Aloj, and I. Covelli (1968) Thyroid iodoproteins in vertebrates: Ultracentrifugal pattern and iodination rate. Comp. Biochem. Physiol., 27: 67-82. Roques, M., J. Torresani, M. Michel-Bechet, A. Jost, and S. Lissitzky (1972)Relationship between thyroglobulin syn- thesis, iodine metabolism, and histogenesis in the developing rabbit fetal thyroid gland. Gen. Comp. Endocrinol., 19: 457-472. Salvatore, G., G. Vecchio, M. Salvatore, H.J. Cahnmann, and J. Robbins (1965)27s thyroid iodoprotein. J. Biol. Chem., 240: 2935-2943. Van der Walt, B., and P. Van Jaarsveld (1972)Bovine 37s iodoprotein: Isolation and characterization. Arch. Biochem. Biophys., 150: 786-791. Whur, P.,A. Herscovics, and C.P. Leblond (1969)Radioautographic visualization of the incorporation of galactuse3H and m a n n ~ s e - ~by H rat thyroids in vitro in relation to the stages of thyroglobulin synthesis. J. Cell Biol., 43: 289-311.