Demonstration of sugar residues in the ultimobranchial tubule and thyroid C-cells of the rat using peroxidase labelled lectins.

The distribution of various sugars, that contribute to the composition of the carbohydrate components of the glycoprotein in the ultimobranchial tubule and the thyroid C-cells has been studied, using eight peroxidase-labelled lectins (Dolichos biblorus, Glycine max, Bauhinia purpurea, Helix pomatia, Arachis hipogaea, Ulex europaeus I, Limax flavus and Sophora japonica). The results obtained seem to show that there are receptors for Dolichos biflorus, Glycine max, Bauhinia purpurea, Helix pomatia, Arachis hipogaea, Ulex europaeus I and Limax flavus in the ultimobranchial tubule, suggesting the existence of N-acetylgalactosamine, galactose, L-fucose and sialic acid. However, the C-cells show only a positive reaction with Ulex europaeus I indicating the existence of fucosyl residues in those cells. All these findings support the contention that the ultimobranchial body might contribute decisively not only to the formation of C-cells but also, partially, to the constitution of certain follicular cells.

Immunohistochemical and morphometric analysis of calcitonin distribution in anuran ultimobranchial glands.

The presence and distribution of immunoreactive calcitonin (iCT) in the anuran (Rana pipiens) ultimobranchial gland was examined with a specific antiserum to salmon calcitonin. Improved resolution of the distribution of iCT within individual cells allowed recognition of a iCT-negative [iCT(-)] early maturing cell, the primary iCT-positive [iCT-(+)] secretory cell with accumulation of iCT within the basal and apical cytoplasm, an iCT(-) cell that lacks a basal accumulation of secretory material, and a iCT(-) cell that borders the central lumen. A morphometric analysis was made of the entire glandular basal secretory surface area and a calculation was made of the relative area that contained iCT(+) secretory material (expressed as a basal index (BI)). For frogs kept in fresh water, the total basal area of a single gland was 0.335 mm2, with a BI of 0.72 and a total iCT(+) secretory surface of 0.241 mm2. A second group of frogs kept in a high calcium medium for 12 days had a BI of 0.74 with no significant increase in parenchyma volume or total basal area and therefore no increase in the total iCT(+) secretory area. A third group was kept in a high calcium medium for 12 days and given a single oral dose of vitamin D3 (500 IU/g body wt). The BI of this group remained unchanged at 0.75; however, there was a significant glandular hypertrophy concomitant with an increase in secretory surface area to 0.462 mm2. This resulted in a 43% increase in the iCT(+) secretory area to 0.345 mm2. A model is proposed to explain the increase in iCT in the gland in response to a vitamin D and high calcium challenge.

Localization of a salmon calcitonin-like molecule in one type of ultimobranchial cells in the freshwater turtle Pseudemys scripta.

Immunohistochemical methods using affinity adsorbed antibodies raised against the three families of calcitonins (CT) were applied to ultimobranchial (UB) cells in situ to investigate the nature of the Chelonian calcitonin molecule and its distribution in the ultimobranchial bodies of the freshwater turtle, Pseudemys scripta. In this species, the UB glands were present on both sides and consisted of scattered cell clumps between epithelial vesicular structures. The neighboring parathyroid tissue also contained two components, the majority being composed of similar vesicles, with occasional solid cell cords evenly distributed. Calcitonin immunoreactivity was found in the cell clumps of the UB gland and in the cell cords of the associated parathyroid, but not in the epithelial component lining the vesicles or in the amorphous material which sometimes filled the lumen. Turtle calcitonin was exclusively of the salmon type, as determined by the negative results obtained in situ after the use of antibodies raised against human and porcine molecules. The salmon-like calcitonin content of the ultimobranchial area was estimated as 15.2 ng; however, the molecule was undetectable in the circulation. In this work we localize the quantitate a salmon-like CT molecule in one type of ultimobranchial and parathyroid cell of a reptile for the first time.

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.

Hypocalcemic potency of the ultimobranchial gland in some urodelan amphibians.

The hypocalcemic potencies of the ultimobranchial glands of two urodelans, Onychodactylus japonicus and Hynobius nigrescens, were studied; according to the rat bioassay, their calcitonin values (MRC) were 30 and 18 mU/kg body wt, respectively. Various organs other than the ultimobranchial gland were also assayed in rats to see whether they had any hypocalcemic potency. However, the ultimobranchial gland was the only organ examined with detectable hypocalcemic potencies in these urodelans. The hypocalcemic potencies of urodelan ultimobranchial glands are one order lower than those reported in the other vertebrate classes, contrasting with the potencies of anuran ultimobranchial glands. The biological significance of this low potency is discussed.

Identification of both human and salmon calcitonin-like molecules in birds suggesting the existence of two calcitonin genes.

To investigate whether human calcitonin (hCT) is preserved during the evolution of vertebrates, we studied extracts of avian (pigeon and chicken) thyroid and ultimobranchial glands (UBG) which have previously been reported to contain salmon calcitonin (sCT)- like molecules. A sensitive and specific radioimmunoassay for hCT, employing two antisera reacting with different regions of the molecule, was used in combination with high performance liquid chromatography (HPLC). We found that extracts of thyroid and UBG from pigeons and chickens contain, in addition to an immunoreactive sCT-like molecule (which is the major immunoreactive form), an hCT-like molecule comprising from 0.4 to 3.75% of the calcitonin content. The extracts produced full displacement of 125I-labelled hCT from both antisera and gave a parallel displacement curve. With HPLC, we found an immunoreactive hCT peak which was 5 ml earlier than the insulin marker and an immunoreactive sCT peak which was 12 ml later than the insulin marker. These results demonstrate the presence of two calcitonins in birds, and suggest the existence of two genes with different degrees of expression.

Fluorescence microscopy of the avian (Gallus domesticus) carotid body.

Sterba's technique to detect neurosecretory substances was applied to carotid body tissue, surrounding branchial derivatives, ganglion nodosum and the carotid arterial wall. Mainly a yellowish-green, a yellowish-red and a bright yellow fluorescence was observed as well as greenish-yellow fibres with varicosities and terminal knobs. The metachromatic reaction of the technique was briefly discussed.

[Calcitonin in the ultimobrancial body of Anguilla (Anguilla anguilla L.): cytologic localization by indirect immunofluorescence using human anti-salmon-calcitonin antibodies]. / Calcitonine du corps ultimobranchial d'anguilles (Anguilla anguilla L.): localisation cytologique par immunofluorescence indirecte à l'aide d'un anticorps humain anti-calcitonine de saumon.

The localization of intracellular calcitonin has been achieved by immunofluorescence in the cytoplasm of all cells forming the epithelium of the ultimobranchial body of eels, using a human antiserum against synthetic Salmon calcitonin I. The specificity of the reaction is demonstrated by inhibition with synthetic salmon calcitonin (S.C.T.); the fluorescence is not inhibited by synthetic human calcitonin (H.C.T.).

Histology, innervation and histochemistry of the UB gland in the Mexican cave fish Anoptichthys jordani Hubbs et Innes (Teleostei: Characidae).

The histomorphological investigations carried out on the ultimobranchial body of the Teleost Anoptichthys jordani Hubbs and Innes have enabled us to establish that it is an epithelial multifollicular body which lies within the transverse septum, beneath the musculature of the oesophagus and caudal to the Sinus venosus. Its parenchyma is surrounded by an extensive nerve network revealed by the Bodian silver technique. This nerve network and the occurrence of MAO activity in the ultimobranchial tissue indicate that it possesses a sympathetic innervation. The follicular epithelium sometimes acquire a pseudostratified condition in some follicles and sometimes in multistratified in others. In this latter case the presence of two main types of cell was noticed (light cells and dark cells). In these cells by the fluorescence microscopic analysis was revealed an intensely green fluorescence due to the presence of an aromatic monoamine, dopamine. This conclusion was further confirmed by the agreement between the argyrophilic cells and the fluorescent cells after silver staining. The possible partecipation of dopamine in the elaboration of polypeptide factor (calcitonin) is suggested. A notable analogy from the structural point of view to the calcitonin-producing cells of higher Vertebrates is also confirmed by the markedly argyrophilia of the main cells.