Ultrastructural localization of calcitonin, somatostatin and serotonin in parafollicular cells of rat thyroid

Summary Three hormones were demonstrated in ultrathin sections of the rat thyroid using immunocytochemical methods with either a PAP complex or a protein A-gold complex as the tabel. In control rats, calcitonin was found to be present in all parafollicular cells and somatostatin in occasional cells. In rats pretreated with 5-hydroxytryptophan, serotonin was detected in all parafollicular cells as well. In serial ultrathin sections, the three hormones were seen to be localized in the same secretory granules.     

Ultrastructural immunocytochemical localization of calcitonin in the C cells of the rat thyroid

Using unlabeled antibodies and peroxidase-anti-peroxidase complexes, calcitonin was localized at the ultrastructural level in rat thyroid C cells. Calcitonin was present mainly in secretory granules of the cells. A less intense positive reaction was noted in the cytoplasm surrounding the secretory granules.     

Ultrastructural immunocytochemical localization of calcitonin in the C cells of the rat thyroid

Summary Using unlabeled antibodies and peroxidase-anti-peroxidase complexes, calcitonin was localized at the ultrastructural level in rat thyroid C cells. Calcitonin was present mainly in secretory granules of the cells. A less intense positive reaction was noted in the cytoplasm surrounding the secretory granules.     

Ultrastructural localization of calcitonin in control and stimulated thyroid C cells of the rat using protein A-gold immunocytochemical technique

Using anti-human calcitonin serum and a protein A - gold technique, calcitonin was localized at the ultrastructural level in control and calcium gluconate-stimulated thyroid C cells of the rat. In control rats calcitonin was detected within a majority of the secretory granules while in experimental animals it was demonstrated also within prosecretory granules present in Golgi apparatus.     

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

Summary 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.Supported in part by grants from the Italian Ministero della Pubblica Istruzione and Consiglio Nazionale delle Ricerche     

Ultrastructural localization of calcitonin in C-cells of dog thyroid; an immunocytochemical study

Summary Light microscopic observations of normal rat peritoneal mast cells and ultrastructural observations of human mast cells from lesions of nodular mastocytosis indicated that structural damage results in a pronounced increase in percentage of cells with a positive dopa reaction. Recent studies have indicated that the dopa reaction in mast cells is peroxidase-dependent. Enhancement of the dopa reaction by structural damage (latency) is probably related to increased substrate-enzyme interaction. Ultrastructural localization of dopa melanin to mast cell granules and the high percentage of mast cells showing a positive dopa reaction after structural damage is evidence against the possibility that dopa melanin is formed in mast cells by phagocytized enzyme.Supported by USPHS Grant Tl Am 5220, and by The General Research Support Fund, Boston City Hospital.     

Light- and electron-microscopical localization of calcitonin,calcitonin gene-related peptide,somatostatin and C-terminal gastrin/cholecystokinin immunoreactivities in rat thyroid

Summary Parafollicular C cells of the rat thyroid contain several immunoreactive peptides including calcitonin (CT), calcitonin gene-related peptide (CGRP), somatostatin and a C-terminal gastrin/CCK immunoreactive epitope as shown at the light-and electron-microscopical levels. Adult thyroid C cells are strongly immunoreactive to CT and most of the cells also react strongly with CGRP antisera and weakly with a gastrin/CCK antiserum. The latter antiserum may cross-react with CGRP. This cross-reactivity probably only occurs at very high concentrations of CGRP observed in adult thyroid C cells, but not in intrathyroidal CGRP-containing nerves, nor in early neonatal C cells. In neonatal rats, somatostatin immunoreactive C cells are numerous and most of these cells are also CT and CGRP immunoreactive. In contrast, only few C cells display somatostatin immunoreactivity in adult rat thyroids. Sequential staining experiments revealed that some thyroidal C cells simultaneously express all four types of immunoreactivity. At the electron microscopical level, all of these immunoreactivities were observed in secretory granules of C cells. Double- and triple-staining experiments, moreover, documented that some peptides are co-localized in the same granules.     

Light- and electron-microscopical localization of calcitonin, calcitonin gene-related peptide, somatostatin and C-terminal gastrin/cholecystokinin immunoreactivities in rat thyroid

Parafollicular C cells of the rat thyroid contain several immunoreactive peptides including calcitonin (CT), calcitonin gene-related peptide (CGRP), somatostatin and a C-terminal gastrin/CCK immunoreactive epitope as shown at the light- and electron-microscopical levels. Adult thyroid C cells are strongly immunoreactive to CT and most of the cells also react strongly with CGRP antisera and weakly with a gastrin/CCK antiserum. The latter antiserum may cross-react with CGRP. This cross-reactivity probably only occurs at very high concentrations of CGRP observed in adult thyroid C cells, but not in intrathyroidal CGRP-containing nerves, nor in early neonatal C cells. In neonatal rats, somatostatin immunoreactive C cells are numerous and most of these cells are also CT and CGRP immunoreactive. In contrast, only few C cells display somatostatin immunoreactivity in adult rat thyroids. Sequential staining experiments revealed that some thyroidal C cells simultaneously express all four types of immunoreactivity. At the electron microscopical level, all of these immunoreactivities were observed in secretory granules of C cells. Double- and triple-staining experiments, moreover, documented that some peptides are co-localized in the same granules.     

Ontogeny and ultrastructure of somatostatin and calcitonin cells in the thyroid gland of the rat

Summary Calcitonin cells are relatively numerous in the thyroid gland of the rat. In contrast, somatostatin cells are very scarce except at the time of birth and a few days thereafter, when they are conspicuously numerous. Somatostatin cells of the thyroid gland, which are ultrastructurally similar to somatostatin cells in gut and pancreas, also contain immunoreactive calcitonin. It is not clear whether somatostatin cells in the rat thyroid gland produce calcitonin or accumulate calcitonin from the environment.     

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.     

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.     

Single cellular origin of somatostatin and calcitonin in the rat thyroid gland

Summary Immunostaining of thin serial paraffin sections has shown that somatostatin is present in the same parafollicular cells as calcitonin in the adult rat thyroid gland. The number of cells containing both peptides is much smaller than the number containing calcitonin but not somatostatin.     

Hybridocytochemical detection of mRNA for calcitonin,CGRP, NPY and somatostatin in thyroid parafollicular (C) cells in three rodent species

The present study was aimed at hybridocytochemical (HCC) detection and interspecies comparison of mRNA for calcitonin (CT), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and somatostatin (SS) in thyroid C cells of two rodent families of wild Microtidae: pine voles and common voles and also of laboratory Muridae, Wistar rats. Studies were performed on adult males. The HCC method in situ and immunomax technique were used to detect mRNA. DNA oligonucleotide probes labeled with digoxigenin were used in the HCC method. The obtained results were compared to the results of immunocytochemical (ICC) examinations, where rabbit or mouse antibodies against human CT, SS, NPY and rat CGRP, as well as chromogranin A were performed. In the present study, HCC reaction has demonstrated the presence of mRNA for CT and CGRP in all thyroid C cells in all the species examined. However, mRNA for NPY and SS was observed in very few C cells in rat and in many more C cells in the two species of wild rodents. The distribution of the positive cells corresponded with that of ICC detected 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     

Differential changes in calcitonin, somatostatin and gastrin/cholecystokinin-like immunoreactivities in rat thyroid parafollicular cells during ontogeny

Immunocytochemical quantitative studies on the development of rat thyroid calcitonin (C) cells have been performed. In neonatal rat pups up to day 6 nearly 90% of all calcitonin cells are also somatostatin immunoreactive and 45% of these cells also show immunoreactivity to a C-terminal gastrin/cholecystokinin antiserum (CCK-4-like immunoreactivity). Already at day 8 the frequency of somatostatin immunoreactive calcitonin cells has dropped to 25%, whereas half of the calcitonin cells still display CCK-4-like immunoreactivity. In adult rats, less than 1% of the calcitonin cells are somatostatin immunoreactive, whereas 90% of the calcitonin cells display CCK-4-like immunoreactivity. These data show that between day 6-8 a pronounced change in the peptide repertoire of rat thyroid C cells occur and that these cells, prior to this time, mainly contain calcitonin and somatostatin immunoreactivity and after this time mainly contain calcitonin and CCK-4-like immunoreactivity. The time course of the change in the C cell peptides is similar to that observed with changes in transitory peptides of neonatal rat pancreas and duodenum, suggesting that possible hormonal mechanisms during this period act to change the peptide repertoire of several endocrine cell types simultaneously. It is possible that many of the transitory peptides exert actions in the developing individual that are necessary for growth and differentiation. Interestingly, many of these transitory peptides reappear in tumours, where theoretically they could exert similar actions.     

Differential changes in calcitonin,somatostatin and gastrin/cholecystokinin-like immunoreactivities in rat thyroid parafollicular cells during ontogeny

Summary Immunocytochemical quantitative studies on the development of rat thyroid calcitonin (C) cells have been performed. In neonatal rat pups up to day 6 nearly 90% of all calcitonin cells are also somatostatin immunoreactive and 45% of these cells also show immunoreactivity to a C-terminal gastrin/cholecystokinin antiserum (CCK-4-like immunoreactivity). Already at day 8 the frequency of somatostatin immunoreactive calcitonin cells has dropped to 25%, whereas half of the calcitonin cells still display CCK-4-like immunoreactivity. In adult rats, less than 1% of the calcitonin cells are somatostatin immunoreactive, whereas 90% of the calcitonin cells display CCK-4-like immunoreactivity. These data show that between day 6–8 a pronounced change in the peptide repertoire of rat thyroid C cells occur and that these cells, prior to this time, mainly contain calcitonin and somatostatin immunoreactivity and after this time mainly contain calcitonin and CCK-4-like immunoreactivity. The time course of the change in the C cell peptides is similar to that observed with changes in transitory peptides of neonatal rat pancreas and duodenum, suggesting that possible hormonal mechanisms during this period act to change the peptide repertoire of several endocrine cell types simultaneously. It is possible that many of the transitory peptides exert actions in the developing individual that are necessary for growth and differentiation. interestingly, many of these transitory peptides reappear in tumours, where theoretically they could exert similar actions.     

Simultaneous localization of calcitonin mRNA and peptide in a medullary thyroid carcinoma

We report the visualization of calcitonin gene expression products at the mRNA and peptide levels on the same section of a medullary thyroid carcinoma by combined in situ hybridization and immunohistochemistry. mRNA detection was accomplished by hybridization with radioactively labeled antisense RNA probes followed by autoradiography and immunohistochemically using the avidin-biotin complex method. Best results were obtained when in situ hybridization preceded immunohistochemistry, as determined by quantitative analysis of the autoradiographs. When immunohistochemistry was performed prior to in situ hybridization, the RNase inhibitor heparin had to be added to the antibodies to retain hybridizable mRNA. The intensity of the two reactions varied in individual cells, indicating a functional heterogeneity of tumor cells with regard to calcitonin mRNA content and storage of the related immunoreactive peptide. These results, in combination with elevated serum calcitonin levels, suggest significant differences in the rate of secretion of individual tumor cells. Simultaneous localization of mRNA and its peptide within the same cell may, therefore, provide further insight into gene expression and secretory activity at the single cell level.