Solid cell nests of the thyroid gland. Optic and immunohistochemical study at autopsies.

In order to establish the prevalence of solid cell nests (SCN) in adult thyroids, we studied 100 consecutive glands at necropsy. These were serially sectioned and stained with routine and immunoperoxidase techniques in order to detect calcitonin, carcinoembryonic antigen, thyroglobulin and keratin. SCN may be considered as normal thyroid gland components, and they share with C cells a common origin in the ultimobranchial body.     

Postnatal fate of the ultimobranchial remnants in the rat thyroid gland

The ultimobranchial follicles (UBFs) are considered embryonic remnants from the ultimobranchial body (UBB). They are follicular structures that vary in size and appearance depending on the age of the rat. The main objective of this article was to study the progressive changes in shape, size, and frequency of the UBFs in the postnatal rat, from birth to old‐age. To accomplish that objective, a systematic morphometric and incidental study of the UBF has been carried out in 110 Wistar rats of different ages and both sexes, divided into three groups: 1) young rats (5–90‐day‐old); 2) adult rats (6–15‐month‐old), and 3) old rats (18–24‐month‐old). The glands were serially sectioned and immunostained for calcitonin at five equidistant levels. According to our results, UBFs were observed in all thyroid glands but a more exhaustive sampling was occasionally necessary in male rats. In young rats, immature UBFs predominantly appeared whereas in adult rats, mature UBFs with cystic appearance and variable luminal content prevailed. We frequently found spontaneous anomalous UBFs in old rats, which we have termed as “ultimobranchial cystadenomata.” Additionally, in young rats, UBF areas significantly increased with age and they were larger when compared to that of normal thyroid follicles. Likewise, in adult rats, UBFs were significantly larger than normal thyroid follicles but only in female rats. In general, UBFs in females were also significantly larger than those found in male rats. Finally, all these differences related to UBFs together with a higher incidence in females of UB cystadenomata suggest a sexual dimorphism in regard to the destiny of these embryonic remnants during postnatal thyroid development. J. Morphol., 2013. © 2013 Wiley Periodicals, Inc.     

Evidence of the occurrence of calcitonin cells in the ultimobranchial follicle of the rat postnatal thyroid.

A study on thyroid glands of Wistar rats of ages ranging from 1 to 120 days was carried out. The glands were serially sectioned and stained for calcitonin using the peroxidase antiperoxidase method. All the thyroids contained ultimobranchial follicles (UBF) located partially embedded among the usual follicles but in a 5-day-old rat this structure showed an unusual position in the interstitium of connective tissue between the cartilage of the trachea and the thyroid gland. We have observed in the wall of that UBF the presence not only of resting C cells but also mitotic figures of C cells. Furthermore, on the opposite side of the same UBF an active area of formation of thyroid follicles was found. These observations provided the first evidence of the contribution of the UBF in the formation of C cells during the postnatal life of the rat. Furthermore, it is suggested that some C cells may share a common origin with ultimobranchially derived follicular cells.     

Origin of cholinesterase-containing follicle cells and parafollicular cells of the developing thyroid gland in the rat

Summary The location of cholinesterase-containing cells in the thyroid gland and its precursors (median thyroid primordium and ultimobranchial bodies) has been investigated light-microscopically in rat embryos from the 13 to the 20th day of gestation.From the 13th to the 16th day of gestation the median thyroid primordium and the ultimobranchial bodies are distinct from each other. Cholinesterase-containing cells are found in both. On the 17th–18th day of gestation the reacting ultimobranchial cells spread into the median thyroid primordium where they take up a parafollicular position. At the 19th–20th day of gestation the distribution of cholinesterase-containing cells is as in the adult rat. The results seem to show that cholinesterase-containing follicular cells derive from the median thyroid primordium and cholinesterase-containing parafollicular cells from the ultimobranchial body.     

An ultrastructural study of the cysts in chicken ultimobranchial glands,with special reference to C-cells

Summary The ultimobranchial glands of the chicken were examined by electron microscopy and immunocytochemistry using a calcitonin antiserum. Electron microscopy confirmed the presence of C-cells, containing numerous secretory granules storing calcitonin, in the luminal lining of cyst-like structures found in these glands. These cells were furnished with prominent microvillar projections at their luminal surface, and the cytoplasm of the apical region was filled with fibril material. Furthermore, the cells contained prominent junctional complexes and desmosomes at their apico-lateral surfaces. In these C-cells, secretory granules were concentrated near the lumen and some were attached to the apical cell membrane. The luminal content of the cysts had a colloid-like and flocculent appearance, and was frequently seen attached to the cytoplasmic projections or apical cell membrane of the C-cells. Since the cysts progressively increase in volume and number with age, it is suggested that they may partly play a role in the storage of excess or unneeded hormonal products.     

Morphological and histochemical changes of ultimobranchial follicles of the rat thyroid in the course of postnatal life.

Morphological and histochemical changes of ultimobranchial follicles of thyroid have been investigated in rats from newborn to 18 months of age. The first well-delimited ultimobranchial follicles, though with no lumen, were detected in the thyroid gland of 10-day-old rats. At 30 days of age, follicles possessing regular lumina were present in the thyroid. With age, the follicles gradually increased in volume assuming extreme dimensions in adult age. The follicles displayed varying shapes from simple cysts to bizarre forms. From the age of 50 days the cells of the follicular wall are separated from the cell debris contained in the lumen. The latter gave a PAS positive reaction. The cells of the ultimobranchial follicles did not exhibit argyrophilia and metachromasia showing that they differ considerably from the C-cells likewise of ultimobranchial origin, which are known to give marked argyrophilic and metachromatic reactions.     

Expression of the epithelial marker E-cadherin by thyroid C cells and their precursors during murine development.

Studies of chick-quail chimeras have reported that avian ultimobranchial C cells originate from the neural crest. It has consequently been assumed, without much supporting evidence, that mammalian thyroid C cells also originate from the neural crest. To test this notion, we employed both Connexin43-lacZ and Wnt1-Cre/R26R transgenic mice, because their neural crest cells can be marked. We also examined the immunohistochemical expression of a number of markers that identify migratory or postmigratory neural crest cells, namely, TuJ1, neurofilament 160, nestin, P75NTR, and Sox10. Moreover, we examined the expression of E-cadherin, an epithelial cell marker. At embryonic day (E)10.5, the neural crest cells densely populated the pharyngeal arches but were not distributed in the pharyngeal pouches, including the fourth pouch. At E11.5, the ultimobranchial rudiment formed from the fourth pouch and was located close to the fourth arch artery. At E13.0, this organ came into contact with the thyroid lobe, and at E13.5, it fused with this lobe. However, the ultimobranchial body was not colonized by neural crest-derived cells at any of these developmental stages. Instead, all ultimobranchial cells, as well as the epithelium of the fourth pharyngeal pouch, were intensely immunoreactive for E-cadherin. Furthermore, confocal microscopy of newborn mouse thyroid glands revealed colocalization of calcitonin and E-cadherin in the C cells. The cells, however, were not marked in the Wnt-Cre/R26R mice. These results indicated that murine thyroid C cells are derived from the endodermal epithelial cells of the fourth pharyngeal pouch and do not originate from neural crest cells.     

Innervation of the C cells of chicken ultimobranchial glands studied by immunohistochemistry, fluorescence microscopy, and electron microscopy

Innervation of the ultimobranchial glands in the chicken was investigated by immunohistochemistry, fluorescence microscopy and electron microscopy. The nerve fibers distributed in ultimobranchial glands were clearly visualized by immunoperoxidase staining with antiserum to neurofilament triplet proteins (200K-, 150K- and 68K-dalton) extracted from chicken peripheral nerves. The ultimobranchial glands received numerous nerve fibers originating from both the recurrent laryngeal nerves and direct vagal branches. The left and right sides of the ultimobranchial region were asymmetrical. The left ultimobranchial gland had intimate contact with the vagus nerve trunk, especially with the distal vagal ganglion, but was somewhat separated from the recurrent nerve. The right gland touched the recurrent nerve, the medial edge being frequently penetrated by the nerve, but the gland was separated from the vagal trunk. The left gland was innervated mainly by the branches from the distal vagal ganglion, whereas the right gland received mostly the branches from the recurrent nerve. The carotid body was located cranially near to the ultimobranchial gland. Large nerve bundles in the ultimobranchial gland ran toward and entered into the carotid body. By fluorescence microscopy, nerve fibers in ultimobranchial glands were observed associated with blood vessels. Only a few fluorescent nerve fibers were present in close proximity to C cell groups; the C cells of ultimobranchial glands may receive very few adrenergic sympathetic fibers. By electron microscopy, numerous axons ensheathed with Schwann cell cytoplasm were in close contact with the surfaces of C cells. In addition, naked axons regarded as axon terminals or "en passant" synapses came into direct contact with C cells. The morphology of these axon terminals and synaptic endings suggest that ultimobranchial C cells of chickens are supplied mainly with cholinergic efferent type fibers. In the region where large nerve bundles and complex ramifications of nerve fibers were present, Schwann cell perikarya investing the axons were closely juxtaposed with C cells; long cytoplasmic processes of Schwann cells encompassed large portions of the cell surface. All of these features suggest that C-cell activity, i.e., secretion of hormones and catecholamines, may be regulated by nerve stimuli.     

Pulmonary immunoreactive calcitonin in the African green monkey (Cercopithecus aethiops): Anatomic distribution

The hormone calcitonin, which occurs predominantly within the C cells of the mammalian thyroid gland, is also found within the pulmonary endocrine cells of the epithelium of the tracheobronchial tree. A study was made of the distribution of immunoreactive calcitonin (iCT) in the African green monkey. Using two different region-specific antisera, the total respiratory iCT comprised 2.5% and 5.8% of the total thyroid iCT. The mean concentration of iCT in the right lung exceeded that in the left, and the mean concentration of the right middle or right upper lobe exceeded that of all other lobes. Embryologically, the ultimobranchial bodies contribute their iCT-producing C cell primordia to the thyroid gland near the level of the primitive laryngotracheal cleft and shortly after the early arborization of the bronchial tree. In monkeys and most other mammals, the right main stem bronchus is larger and develops earlier than the left. The data suggest an early migration of cells from the ultimobranchial bodies to the bronchi, eventually giving rise to the iCT-containing pulmonary endocrine cells.     

Unpaired Ultimobranchial Glands of the African Lungfish, Protopterus dolloi

The ultimobranchial glands of juvenile African lungfish (Protopterus dolloi) (14 individuals; total body length 25-205 mm) were immunohistochemically examined. In individuals larger than 36 mm, one ultimobranchial gland was close to the left afferent branchial arteries. The topography of the ultimobranchial gland was similar to that of salamanders and sharks, but not to teleosts. With body growth, the ultimobranchial gland was vascularized and the parenchymal cells were gradually immunostained with anti-calcitonin antibody. In all individuals examined, the ultimobranchial gland existed only on the left side of the pharynx. These observations are discussed from a phylogenetic viewpoint.     

Ultimobranchial body and parathyroid gland of the frog, Rana tigrina in response to calcitonin administration

The effects of salmon calcitonin (0.25 MRC mU/g body wt) on the serum calcium and phosphate levels as well as on the activity of ultimobranchial body and parathyroid glands was investigated in the frog, Rana tigrina for 15 days. The hormone evokes hypocalcemia (on day 1 and day 3) which is followed by a significant hypercalcemia on day 10. Thereafter, the level of calcium decreases again on day 15. Calcitonin induces hypophosphatemia (on day 3 and day 5). Thereafter, hyperphosphatemia is recorded on day 10. By day 15 normal serum phosphate value is achieved. After treatment with calcitonin, the ultimobranchial body becomes inactive and the parathyroid glands get activated.     

Expression and localization of prohormone convertase PC1 in the calcitonin-producing cells of the bullfrog ultimobranchial gland.

We examined the expression and localization of the prohormone convertases, PC1 and PC2, in the ultimobranchial gland of the adult bullfrog using immunohistochemical (IHC) and in situ hybridization (ISH) techniques. In the ultimobranchial gland, PC1-immunoreactive cells were columnar, and were present in the follicular epithelium. When serial sections were immunostained with anti-calcitonin, anti-CGRP, anti-PC1, and anti-PC2 sera, PC1 was found only in the calcitonin/CGRP-producing cells. No PC2-immunopositive cells were detected. In the ISH, PC1 mRNA-positive cells were detected in the follicle cells in the ultimobranchial gland. No PC2 mRNA-positive cells were detected. RT-PCR revealed expression of the mRNAs of PC1 and the PC2 in the ultimobranchial gland. However, very little of the PC2 mRNA is probably translated because no PC2 protein was detected either by IHC staining or by Western blotting analysis. We conclude that the main prohormone convertase that is involved in the proteolytic cleavage of procalcitonin in the bullfrog is PC1.     

Isolation and determination of the amino acid sequence of chicken calcitonin I from chicken ultimobranchial glands

A radioimmunoassay for chicken calcitonin in chicken ultimobranchial glands was established utilizing a rabbit antiserum against eel calcitonin. This assay method, which is about 100 times as sensitive as the usual bioassay for hypocalcemic activity, was used for monitoring chicken calcitonin during its purification. The immunoreactivity in chicken ultimobranchial extract was separated by SP-Sephadex C-25 chromatography into two fractions. Chicken calcitonin I, which was occurred in the major immunoreactive fraction, was further purified to homogeneity as shown by reverse phase HPLC. In the end, 39 nmol of chicken calcitonin I was obtained from 3,384 chickens following a 12,000-fold purification. The complete amino acid sequence of purified chicken calcitonin I was determined to be H-Cys-Ala-Ser-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Ly s-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly-Thr-Pro-NH2 and confirmed by synthesis. The specific biological activity of chicken calcitonin I (4,500 MRCU/mg) was identical to that of eel calcitonin, which has the highest specific biological activity among the calcitonins so far isolated. Chicken calcitonin I resembled the calcitonins from the ultimobranchial glands both of salmon and eel in sequence, biological activity, and immunological property.     

Calcitonin cells and unusual follicles of the thyroid of the indian grey mongoose, Herpestes edwardsi (Geoffroy).

Histological preparations of thyroid, parathyroid and thymus glands of Herpestes edwardsi were examined for calcitonin cells. They reveal that (1) the thyroid calcitonin cells are oval, rounded and rarely elongated in shape; these cells and their nuclei are distinctly larger than those of the follicular cells and their nuclei; (2) calcitonin cells are unevenly distributed in the thyroid, with the result that certain portions of the thyroid are completely devoid of these cells; (3) on an average, calcitonin cells are in a ratio of 10-15 cells/100 follicular cells; (4) the parathyroid and thymus glands do not display calcitonin cells, and (5) the thyroid gland displays unusual follicles of two categories, (a) follicles with ciliated epithelial cells and (b) follicles with squamous epithelium.     

Difference between the thyroid gland of normal and hypomyelinated jimpy mice--a light microscopic study

A light microscopic quantitative analysis was performed on normal and jimpy male mice for studying the difference between the structures of the thyroid glands of the two animals. The results of this analysis showed that the thyroid gland of the normal mice consisted of numerous homogenous round follicles with cuboidal follicular cells, separated by thin interlobular and interfollicular connective tissue and a few adipose tissue. The thyroid gland of jimpy mice consisted of a few, small follicles surrounded by columnar follicular cells and intraepithelial capillaries, separated by thick connective tissue and abundant adipose tissue. The number of thyroid follicles are significantly less in the jimpy mice than in the normal mice. Another striking difference is that almost every follicular cell surrounding the follicular lumen of jimpy mice is accompanied by an intraepithelial capillary. In addition, the ratio of the number of intraepithelial capillaries to the number of the thyroid follicular cells are significantly higher in the jimpy mice than in the normal mice. The S-follicles or ultimobranchial cysts of the thyroid gland are well developed in the jimpy mice. The parafollicular cells are normal in appearance. Morphological evidence suggested that the thyroid follicular cells of the jimpy mice are very active in the transport, synthesis and release of thyroglobulin, and secretion of thyroid hormones. But owing to the significantly decreased number of thyroid follicles, the inadequate secretion of the thyroid hormones result in the hypothyroidism and the hypomyelination of the jimpy mice.     

Overexpression and purification of recombinant eel calcitonin and its phylogenetic analysis

Calcitonin (CT), a peptide hormone that is widely used for the treatment of osteoporosis, Paget's disease, hypercalcemic shock and chronic pain in terminal cancer patients, is produced by the para-follicular cells of the thyroid gland in mammals and by the ultimobranchial gland of birds and fish. Fish calcitonin, like eel calcitonin (eCT), is more potent and longer lasting than human CT and is one of the many bioactive peptides that require C-terminal amidation for full biological activity. In this study we describe the over-expression and over-production of C-terminal amidated eCT in recombinant Streptomyces avermitilis. A phylogenetic analysis was performed with all the known CT amino acid sequences.     

Immunohistochemical study of the C-cell complex of dog thyroid glands with reference to the reactions of calcitonin,C-thyroglobulin and 19S thyroglobulin

Summary Continued from the previous study in fetal animals (Kameda et al. 1980), the development and maturation of C-cell complexes in postnatal dogs from newborn to adult were investigated by use of an immunoperoxidase method using antisera to calcitonin, C-thyroglobulin (C-Tg) and 19S thyroglobulin, respectively. The younger the animals were, the more numerous were undifferentiated cells and high columnar epithelial cells in the complexes. With increasing age, the constituent elements of the complexes progressively differentiated. In one type of complex there are a large number of C-cells in various developmental stages, as well as undifferentiated cells and cysts. C-cell complexes composed mostly of mature C-cells were regarded as the more highly differentiated structures of this type. A second type contains follicular cells in various stages of differentiation in addition to undifferentiated cells and C-cells, i.e., 19S-positive cell masses not yet organized into follicles, primordial follicles with small lacunae and comparatively larger follicles. The follicular cells in the complexes were similar with respect to immunoreaction and folliculogenesis to the cells of fetal thyroids, but they developed very slowly. In conclusion, the present study indicates that follicular thyroid cells can differentiate within C-cell complexes, i.e., they develop from cells of ultimobranchial body origin.     

Studies of the ultimobranchial gland of a teleost, Esomus danrica (Ham.)

The author describes the histophysiology of the ultimobranchial gland of E. danrica. The gland is the site of synthesis and storage of the polypeptide calcitonin, which has a hypocalcaemic effect. However, the sexual dimorphism of the ultimobranchial gland suggests that it also plays a role in reproduction, particularly in ovarian maturation.     

Ultimobranchial gland of the domestic fowl

Summary The ultimobranchial gland (UBG) of birds is particularly rich in calcitonin, the hypocalcaemic hypophosphataemic hormone, that is secreted by the C-cells of the mammalian thyroid. The principal cells of the UBG have a striking resemblance with the mammalian C-cells, i.e., they possess small intracytoplasmic dense-core secretory granules, 150–300 nm in diameter. The gland also contains a second, morphologically distinct, endocrine cell type with larger granules, 500–800 nm in diameter. A sensitive immunocytochemical reaction was developed with the use of antibodies against salmon calcitonin. By means of this technique the presence of calcitonin-immunoreactive molecules was demonstrated in both secretory cell types of the UB gland of the chicken. This gland can thus be considered as a homogeneous calcitonin-producing tissue. Whether the secretory products are identical is discussed and differences in the secretory pathways are suggested.     

Embryonic induction and development of the ultimobranchial body in the embryogenesis of amphibia

At early embryonal and larval stages of development 7 species of amphibia have been studied. The ultimobranchial anlage and processes resulted in formation of the secretory follicle are investigated. Dynamics on changes of amount of the gland cells and the first appearance of capillaries are analyzed. In Anura and Urodela the anlage of the ultimobranchial gland develops from the epithelial lining of the pharynx behind the last branchial pocket rudiment. The gland is asymmetric and can be laid either in the right or in the left side of the body. Death of calcitonin-secreting cells is compensated at the expense of repeated anlage of follicles from the pharyngeal epithelium. The newly formed follicles can either incorporate into the existing gland, or form independent follicles. For amphibia formation of the capillary network around the gland after beginning of the follicular secretion is specific. Owing to these data, it is possible to conclude that the stimulus for the gland to become overgrown with capillaries is the beginning of calcitonin secretion.