Immunocytochemical studies on thyroid parafollicular cells in postnatal development of the rat

Studies were performed on Wistar strain rats aged 1-720 days. Immunocytochemical reactions were used to detect calcitonin, somatostatin, calcitonin gene-related peptide (CGRP), cholecystokinin, serotonin, neuron-specific enolase (NSE), secretory protein-I, chromogranin and Ca-binding protein. In the parafollicular cells of the rat, the presence of calcitonin, somatostatin, CGRP, NSE and secretory protein-I could be demonstrated. The number of parafollicular cells increased with the age of animals, and the increase was particularly pronounced in the early postnatal period and after the first year of age. The number of somatostatin-immunoreactive cells decreased after birth and increased again after the first year of age. The number of calcitonin-immunoreactive cells increased in the early postnatal period independently of the increase in parafollicular cell number, forming frequently tumor-like outgrowths in 2-year-old animals. A small proportion of these outgrowths contained no calcitonin even if they did contain somatostatin, CGRP and NSE immunoreactivity. Evident changes in immunoreactivity in the first days after birth may reflect the sudden change in environment and may be associated with growth and differentiation. In any period of life, CGRP- and NSE-immunoreactive cells have constituted the most numerous groups and, therefore, the respective antigens seem to represent the most suitable markers of parafollicular cells in the rat.     

Effect of hypercalcemia on parafollicular cells in the rat thyroid gland

Hypercalcemia was induced in rats by the administration of A.T.10. We then determined the levels of total and ionized calcium and calcitonin in the serum, as well as performed ultrastructural observations and histochemical investigations of the calcitonin and neuron-specific enolase immunoreactivities in the stimulated parafollicular cells. The main aim of the study was to apply histochemical procedures to determine the immunoreactions of calcitonin gene-related peptide (CGRP), somatostatin and secretory protein-I in stimulated parafollicular cells. Immunoreactions of CGRP and calcitonin decreased strikingly in A.T.10-treated animals, whereas no visible changes were noted in somatostatin immunoreactivity. In the case of secretory protein-I, an insignificant increase of its immunoreactivity was observed in the treated animals. The cytophysiological significance of these results is discussed.     

Effect of hypercalcemia on parafollicular cells in the rat thyroid gland

Summary Hypercalcemia was induced in rats by the administration of A.T.10. We then determined the levels of total and ionized calcium and calcitonin in the serum, as well as performed ultrastructural observations and histochemical investigations of the calcitonin and neuron-specific enolase immunoreactivities in the stimulated parafollicular cells. The main aim of the study was to apply histochemical procedures to determine the immunoreactions of calcitonin gene-related peptide (CGRP), somatostatin and secretory protein-I in stimulated parafollicular cells. Immunoreactions of CGRP and calcitonin decreased strikingly in A.T.10-treated animals, whereas no visible changes were noted in somatostatin immunoreactivity. In the case of secretory protein-I, an insignificant increase of its immunoreactivity was observed in the treated animals. The cytophysiological significance of these results is discussed.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Electron-immunocytochemical localization of calcitonin and calcitonin-gene-related peptide in human c cells of the thyroid

A preembedding immunocytochemical technique enabled us to demonstrate normal human parafollicular (C) cells at the electron-microscopic level. The normal human C cells had numerous large secretory granules with a diameter of approximately 200 nm, well-developed rough endoplasmic reticulum and Golgi complex in their cytoplasm. Calcitonin immunoreactivity and calcitonin-gene-related peptide (CGRP) immunoreactivity were present only in the C cells whose secretory granules were heavily labeled. Both calcitonin and CGRP immunoreaction deposits were seen in the cytosol but not in the cisterna of endoplasmic reticulum, Golgi apparatus or mitochondrial matrix. The two peptides produced from a single calcitonin gene were stored in the secretory granules of the C cells.     

Ultrastructural immunocytochemical localization of neuron-specific enolase in parafollicular cells of the rat thyroid

Summary Using pre- and post-embedding procedures, neuron-specific enolase and calcitonin were localized in rat thyroid parafollicular cells by light and electron microscopy. Peroxidase-antiperoxidase (PAP), biotin-avidin (ABC) and protein A — colloidal gold techniques were used. In paraffin sections neuron-specific enolase was demonstrated in all calcitonin-storing parafollicular cells in rats aging 1 to 180 days. The post-embedding procedure failed to detect neuron-specific enolase in ultrathin sections, but the enzyme could be demonstrated using a preembedding procedure. Neuron-specific enolase was localized exclusively within the cytosol of parafollicular cells, while calcitonin was localized within secretory granules applying either post- or pre-embedding incubation techniques.Supported by Sonderforschungsbereich 232     

Species differences in the immunoreactive patterns of calcitonin gene-related peptide in the pancreas

Summary In the pancreas, calcitonin gene-related peptide (CGRP) immunoreactivity has been described in nerve fibers and in distinct types of islet cells. This unique, apparently species-specific cell-type expression prompted the present investigation to clarify further the pattern of CGRP immunoreactivity in different mammalian species (i.e., different strains of rats, mice, guinea pigs, rabbits, cats, dogs, pigs, and humans) commonly used for functional and anatomical studies of the pancreas by means of immunohistochemistry using three different CGRP antibodies. In each species, CGRP-immunoreactive neurites innervate the exocrine and endocrine compartments, the vasculature, and the intrapancreatic ganglia, where they form dense networks encircling unstained cell bodies. The only exception is the pig pancreas, where the islets appear to be devoid of immunoreactive fibers. The overall density of immunoreactive pancreatic axons in different species is as follows: rat, mouse, and rabbit>guinea pigpig and cat> >dog and human. CGRP-immunoreactive endocrine cells appear to be restricted to the rat pancreas, where they form a subpopulation of somatostatin-containing D cells. In contrast, in mouse, guinea pig, cat, dog, and human pancreas, a homogeneous staining of the core of the islets, where insulin-producing B cells are located, was visualized in sections incubated with the rabbit CGRP antiserum at 4°C, but not at 37°C (an incubation temperature that does not affect the islet cell staining in the rat nor the fiber labeling in any species). Furthermore, the staining of islet B cells was not reproductible with all the CGRP antibodies used, all of which comparably stain nerve fibers in each species, and islet D cells in the rat. Immunoreactive islet cells were not visualized in pig and rabbit pancreas. These results are consistent with the hypothesis that the expression of CGRP in nerve fibers is a common feature of mammalian pancreas, whereas its expression in endocrine cells appears to be restricted to the D cells of the rat pancreas.     

Intramitochondrial helical filaments in medullary tubules of the rat kidney

Characteristic helical filaments were frequently found within dilated intracristal spaces of the mitochondria in epithelial cells of renal medullary tubules of normal rats of both sexes. These filaments had a helical structure with right-handed rotation. The approximate dimensions were 4 nm in thickness, 13 nm in helical diameter, and 16 nm in pitch. The filaments were common in all strains of rats examined in the present study but not in animals of other species including mice, guinea pigs, golden hamsters, Mongolian gerbils, house musk shrews, and rabbits. In Sprague-Dawley strain rats, the filaments were found not only in animals of all ages but also in 6-week-old germ-free animals and fetuses at the 18th day of gestation. Even in the same rats, the helical filaments were completely absent in the cortical tubules.     

Immunocytochemical characterization of two thyroid medullary carcinoma cell linesin vitro

Summary The immunocytochemical characterization of cell lines originating from thyroid medullary carcinoma, i.e. human TT cells and rat rMTC 6-23 cells, was undertaken. The immunocytochemical studies were supplemented by ultrastructural studies, including ultrastructural immunocytochemistry, and by radioimmunological estimation of calcitonin secretion to the medium. In rMTC 6-23 cells (subcultures 24 to 30), no hormone presence was demonstrated immunocytochemically, which corresponded to the absence of secretory granules at the ultrastructural level. Of various proteins sought, only neuron-specific enolase could be demonstrated. Nevertheless, the cells secreted calcitonin into the medium. TT cells (passages 145 to 160) produced secretory granules. The granules contained calcitonin, calcitonin gene-related peptide, somatostatin, neurotensin, met-enkephalin, leu-enkephalin, gastrin releasing peptide, parathyroid hormone-related protein, functional proteins of the chromogranin group and synaptophysin. Other functional proteins found in the cytosol of TT cells included non-specific enolase, calbindin and tyrosine hydroxylase. Receptor for calcitriol was localized in the cell nucleus. Marker proteins were localized in the cytosol (carcinoembryonic antigen) and in the cell skeleton (-tubulin, cytokeratin). Following changes in ionized calcium levels in the medium, changes in calcitonin secretion and in immunocytochemical detectability of some hormones and functional proteins were observed. TT cells demonstrated the expression of numerous hormones and functional proteins associated with calcitonin secretion. Further, the cells in their ultrastructure, immunocytochemical and secretory characteristics, resemble more closely normal parafollicular cells of the thyroid and, in our opinion, represent a more appropriate model for functional studies.     

ULTRASTRUCTURAL LOCALIZATION OF CALCITONIN IN THE PARAFOLLICULAR CELLS OF PIG THYROID GLAND WITH CYTOCHROME c-LABELED ANTIBODY FRAGMENTS

Parafollicular cells in mammalian thyroid glands are thought to be responsible for the secretion of calcitonin. In this study, calcitonin was localized in pig thyroid gland by an indirect immunocytochemical technique using rabbit antiserum directed against synthetic porcine calcitonin for the first step, and sheep Fab fragments prepared against rabbit Fab and coupled to cytochrome c for the second step. The antigenic determinants of calcitonin were present only in the parafollicular cells, whose secretory granules were heavily labeled. Labeling of the cytoplasmic matrix is thought to indicate a possible leakage of the polypeptide from the granules. A striking observation was the complete absence of labeling in the cisternae of the rough-surfaced endoplasmic reticulum and of the Golgi apparatus. It is concluded that the secretory granules of parafollicular cells contain calcitonin; the mechanism of synthesis of this peptide is not clearly understood.     

Alteration of parafollicular (C) cells activity in the experimental model of hypothyroidism in rats

Our previous study has shown the alteration of C cells activity in rats with experimental model of hyperthyroidism. The aim of the present study was the evaluation of parafollicular cells activity in rats with hypothyroidism evoked by propylthiouracil (PTU) given in drinking water over 21 days. Histological, ultrastructural and immunocytochemical studies using specific antibodies against calcitonin and CGRP were performed on thyroid glands taken from experimental and control groups of rats. Moreover, in all animals the calcitonin plasma levels were evaluated by radioimmunoassay. After chronic administration of PTU, thyroid image showed predominant microfollicular hyperplasia and attenuated density of parafollicular cells. The intensity of immunocytochemical reactions for CT and CGRP were weaker in the majority of C cells in comparison to the control rats, in which strong immunocytochemical reaction was observed. Examination in the electron microscope reveals the features of hypoactivity both in follicular and parafollicular cells, in which the quantity and electron density of secretory granules were smaller in comparison to the control group. These microscopic changes were accompanied by a significant decrease of calcitonin plasma concentration. Alteration of C cells activity in the experimental model of hypothyroidism, accompanied by microfollicular hypertrophy, may point to the mutual cooperation between parafollicular and follicular cells.     

Chemical coding of endocrine cells of the airways: presence of helodermin-like peptides

Summary The epithelium of the airways is rich in endocrine cells containing serotonin and/or a wide variety of regulatory peptides. These cells usually occur in clusters in the lungs but are also found scattered in the larynx and trachea. In the present study, endocrine cells in the airways of mouse, rat, hamster, guinea pig, pig, sheep and squirrel monkey were examined for the presence of serotonin, helodermin-like peptides and other regulatory peptides using immunocytochemistry and radioimmunoassay. In addition, we looked for the protein gene product 9.5 (PGP), which occurs in many peptide hormone-producing endocrine cells in the body. Both clustered and scattered endocrine cells in the airways were found to display coexistence of serotonin and peptides, such as a helodermin-like peptide, calcitonin and calcitonin gene-related peptide (CGRP). The PGP-immunoreactive cells were numerous and included elements containing serotonin and/or regulatory peptides. An additional PGP-immunoreactive endocrine cell population lacked serotonin and regulatory peptides. Helodermin-immunoreactive material was demonstrated in endocrine cells of the airways in the mouse and hamster but not in any of the other species studied. Serotonin was an endocrine cell constituent in all the species studied. Calcitonin and CGRP could be demonstrated by immunocytochemistry in the mouse, rat, and hamster, but not in the guinea pig, sheep, pig and monkey. In the hamster airways double immunostaining indicated that the helodermin-like peptide occurred in a subpopulation of the CGRP- and serotonin-containing cells. Most of the CGRP-containing cells stored serotonin; some of them also contained calcitonin. The chemical coding of these cells resembled that of the thyroid C cells.     

Co-occurrence of immunoreactive calcitonin and calcitonin gene-related peptide in neuroendocrine cells of rat lungs

Summary Neuroendocrine cells of the lung, occurring singly or in clusters known as neuroepithelial bodies, contain a variety of biologically active compounds, including several neuropeptides. We have investigated the localization of calcitonin and calcitonin gene-related peptide (CGRP) within single and grouped neuroendocrine cells in the respiratory epithelium of rats by an immunohistochemical double-staining technique which uses specific antisera raised in heterogeneous animal species. Calcitonin- and CGRP-immunoreactivities were nearly totally co-localized in both single neuroendocrine cells and neuroepithelial bodies. CGRP-immunoreactivity was also present in neurons in the jugular, nodose and dorsal root ganglia. The calcitonin-immunoreactivity in neuroendocrine cells, as in thyroid parafollicular (C) cells, was abolished by preincubation of the anticalcitonin serum with synthetic calcitonin. The CGRP-immunoreactivity in neuroendocrine cells and in the neuronal cells was abolished by preincubation of anti-CGRP serum with synthetic CGRP. Thus, while the calcitonin gene is expressed exclusively or predominantly as either calcitonin or CGRP in all other tissues except thyroid C-cells, our results strongly suggest that both peptides are expressed in the rat bronchopulmonary neuroendocrine cells.     

Uptake and retention of3H-estradiol by gonadotrophs and lactotrophs in the pituitary glands of the guinea pig,hamster and gerbil

Summary Nuclear uptake and retention of³H-estradiol by luteinizing hormone (LH) and prolactin (PRL) cells was examined in three species of rodents (guinea pigs, hamsters and gerbils) using the combined techniques of immunocyto-chemistry and autoradiography. Castrated animals were injected with³H-estradiol and decapitated 1.5 h later. The pituitary glands were processed for thaw-mount autoradiography followed by conventional immunocytochemical staining for LH and PRL.³H-estradiol accumulated in more than 80% of the anterior pituitary cells in the gerbils, while only 33 and 22% of the cells accumulated³H-estradiol in the hamsters and guinea pigs, respectively. A varying percentage of immunoreactive LH and PRL cells in all three species were found also to contain binding sites for estradiol. Some LH and PRL cells in hamsters and guinea pigs and only some in PRL cells of gerbils were found to be devoid of grains. Quantitative analysis revealed that the number of grains per nucleus differed considerably from cell to cell. LH cells of guinea pigs accumulated much larger amounts of³H-estradiol than did the PRL cells, while the LH cells in the hamsters and gerbils accumulated only slightly more³H-estradiol than the PRL cells.These results confirm the previous observations in rats and baboons that demonstrated tremendous species differences in percentage of cells in the anterior pituitary gland that accumulated³H-estradiol. Also, these data suggest that there are functionally heterogeneous cell types among the LH and PRL cells in hamsters, guinea pigs and gerbils as has been previously demonstrated in rats and baboons.     

Production of monoclonal antibodies against a novel glycoprotein synthesized and secreted by dog thyroid C-cells.

A monoclonal antibody (MAb) that reacted only with thyroid C-cells was raised against cell suspensions from dog thyroid glands, to examine a glycoprotein secreted by C-cells. After chronically-induced hypercalcemia and administration of an anti-thyroid drug, reaction products for the antibody markedly decreased in C-cells, coinciding with alterations in calcitonin immunoreactivity. The antigen recognized by the MAb appears to be a secretory protein. The MAb reacted with C-cells from a wide variety of mammalian species, including rats, mice, hamsters, cattle, cats, rabbits, and monkeys. Furthermore, tumor cells of human medullary thyroid carcinoma, which is derived from C-cells, were immunoreactive to the MAb. Exceptionally, C-cells from guinea pigs and pigs were not stained with the MAb. No crossreactivity was observed in any of the dog tissues examined. Immunoblot analysis demonstrated that the MAb recognized a single prominent band at a molecular weight of approximately 79,000. The 79 KD band reacted with various digoxigenin-labeled lectins, including GNA, DSA, SNA, and MAA; it is a glycoprotein containing mannose, N-acetylglucosamine, and sialic acid. Dog thyroid C-cells were also densely stained with these lectins. The results indicate that thyroid C-cells synthesize and secrete a specific glycoprotein in addition to peptide hormones.     

Practitioner's guide to pocket pet and rabbit theriogenology

This paper provides a review of reproductive characteristics of hamsters, guinea pigs, gerbils, hamsters, rats, and mice, as well as rabbits and chinchillas. It is not intended to be a source of the facts of reproduction (which can be readily found in many references) but rather to highlight some of the unique characteristics of the various species and common problems that clients may present to theriogenologists. This paper includes information regarding sexing, mating, gestation, parturition, lactation, and comments regarding spays and neuters.     

Parafollicular cells of rabbit thryoid store both calcitonin and somatostatin and resemble gut D cells ultrastructurally.

Both calcitonin and somatostatin have been detected immunohistochemically in rabbit parafollicular cells; only calcitonin has been found in the same cells of the dog, guinea-pig and man. Large amounts of a peptide radioimmunochemically identical with synthetic somatostatin have been detected in extracts of rabbit thyroid. The ultrastructural and staining features of rabbit parafollicular cells differ from those of parafollicular cells in other species, while resembling in part those of somatostatin D cells scattered in the rabbit stomach.     

Immunohistochemical study of a large molecular fragment of thyroglobulin in parafollicular cells

Thyroglobulin-like immunoreactivity of the parafollicular cells was studied by an immunoperoxidase bridge technique using antisera against dog thyroglobulin fragments. 1. The dog parafollicular cells were specifically stained by anti-peak I (27S and larger components fraction) antiserum absorbed with peak II (19S fraction). By this method, they were easily distinguishable from the non-reactive follicular cells and colloid droplets. More sensitive staining of the parafollicular cells was possible with anti-peak I' (larger components fraction) antiserum. The staining reactions indicated that the antigenic material responsible for immunoreactivity of the parafollicular cells was due to larger molecular components of thyroglobulin corresponding to 32S, 37S or greater than 37S, and was not due to either the 19S thyroglobulin or to the 27S iodoprotein. 2. A conspicuous decrease of the immunoreactive material in the parafollicular cells occurred in the dog after both chronically induced hypercalcemia and antithyroid drug treatment. This coincided with movement of secretory granules containing calcitonin as shown by staining with silver impregnation, HCl-basic dye, and lead-hematoxylin. 3. The antisera against larger molecular components of dog thyroglobulin showed a high degree of cross-reactivity to the parafollicular cells of most of the mammalian species investigated; rats, rabbits, hamsters, mice, cats, lions, goats, cows, and human.     

Participation of capsaicin-sensitive neurons in the cardiovascular effects of neurotensin in guinea pigs

Intravenous (IV) infusions of neurotensin (NT) in anesthetized guinea pigs elicited dose-dependent pressor effects and tachycardia. Both effects were significantly reduced or abolished in guinea pigs given a chronic treatment with the neurotoxin capsaicin. In guinea pig isolated atria NT evoked a positive inotropic and chronotropic effect. Both effects were completely abolished in atria derived from capsaicin-treated guinea pigs. The positive inotropic and chronotropic effects of NT in guinea pig atria were mimicked by capsaicin and calcitonin gene-related peptide (CGRP). These results were interpreted as an indication that NT produces its cardiovascular effects in guinea pigs by activating capsaicin-sensitive sensory neurons.     

Colocalization of immunoreactivities to serotonin, calcitonin and CGRP in endocrine pulmonary cells of the iberian lizard Podarcis hispanica (Reptilia)

The coexistence of immunoreactivities to serotonin (5HT), calcitonin (CT) and calcitonin gene-related peptide (CGRP) was studied in pulmonary endocrine cells of the Iberian lizard by immunocytochemistry and in semithin/thin sections under light and electron microscope. Immunostaining of serial sections revealed coexistence of 5HT/CT/CGRP immunoreactivities in some cells, while in others only 5HT/CT or CGRP immunoreactivities were found. Appropriate absorption controls excluded crossreactivity between the antisera used. Ultrastructurally, cells immunoreactive to 5HT/CT and CGRP share similar features, with round or slightly ovoid secretory granules of mean diameter from 165 to 180 nm. The possible functional significance of the copresence of 5HT, CT and CGRP is discussed.