Ultrastructure of the water-clear cell in the parathyroid gland of SAMP6 mice

Although the parathyroid water-clear cell is very rare, it has clinical significance because of its association with parathyroid hyperplasia or adenoma. SAMP6, a substrain of senescence-accelerated mouse, was developed as an animal model for senile osteoporosis. We investigated the morphology of the parathyroid glands in SAMP6 and age-matched normal mouse SAMR1. The parathyroid water-clear cells, which contained numerous vacuoles and the crystalloid inclusions, were found in SAMP6 mice at 5, 8 and 12 months of age. It was noted that the number of water-clear cells increased with aging, which are fairly consistent with the change of the serum parathyroid hormone (PTH) level. We did not find any water-clear cells in the parathyroid glands of SAMR1 mice. The existence of water-clear cells may represent hyperfunction of the parathyroid glands in SAMP6.     

Morphological changes of skeletal muscle, tendon and periosteum in the senescence-accelerated mouse (SAMP6): a murine model for senile osteoporosis

SAMP6, a substrain of senescence-accelerated mouse, was developed as an animal model for senile osteoporosis. Previously we observed age-related changes of the bone in SAMP6. In the present study, we investigated the morphology of the skeletal muscle, tendon and periosteum in SAMP6 and age-matched normal mouse SAMR1. We did not find any significant differences between SAMR1 and SAMP6 at 1 and 2 months of age. As compared with SAMR1, the cross-sectional area of type I and type II muscle fibers of the soleus muscle were significantly low in SAMP6 at 8 months of age. The projections in the interface of the muscle-tendon junctions were significantly decreased in SAMP6 at 8 months of age. The number of fibroblasts and the diameter of the tendon collagen fibers in Achilles fiber were significantly reduced in SAMP6 at 8 months of age. The diameter of Sharpey's fiber reduced in SAMP6 at 5 and 8 months of age. Some chondrocytes in the insertions of Achilles tendon and some osteogenic cells in the periosteum showed degenerative changes in SAMP6 at 5 and 8 months of age. The pronounced degenerative changes were detected in the skeletal muscle, muscle-tendon junction, tendon, tendon-bone interface and periosteum in SAMP6 with age. These findings indicated the atrophy of skeletal muscle, degeneration of tendon and periosteum in SAMP6, which may be involved in the bone loss for senile osteoporosis.     

Ultrastructural changes in bones of the senescence-accelerated mouse (SAMP6): a murine model for senile osteoporosis

SAMP6, a substrain of senescence-accelerated mice, was developed as an animal model for senile osteoporosis. In the present study, we investigated the bone morphology, together with serum calcium and bone mineral density (BMD) in SAMP6 and age-matched normal mice SAMR1. We did not find any significant differences between SAMR1 and SAMP6 at 1 month of age with regard to the serum compositions and bone morphology. As compared with SAMR1, BMD, the femoral weight, femoral calcium and phosphorus levels were significantly reduced in SAMP6 at 2 and 5 months of age. The number of osteoblasts in trabecular bones was also significantly reduced. Swollen mitochondria and myelin-like structures were found in osteoblasts and osteocytes of SAMP6 mice at 2 and 5 months of age. There was a greater proportion of resting surface and less forming surface in the femoral endosteal surfaces of SAMP6 mice. The amount of trabecular bone in the lumbar vertebra and the distal metaphysis of the femur was reduced. The number of the mast cells in bone marrow of the tibia significantly increased in SAMP6 mice. These findings indicate that the lower bone mass in SAMP6 was due to the reduction in osteoblast formation and suggested that mast cells in bone marrows play a role in the pathogenesis of senile osteoporosis.     

Ferroptosis in a sarcopenia model of senescence accelerated mouse prone 8 (SAMP8)

As a systemic syndrome characterized by age-associated degenerative skeletal muscle atrophy, sarcopenia leads to a risk of adverse outcomes in the elderly. Age-related iron accumulation is found in the muscles of sarcopenia animal models and patients, but the role of iron in sarcopenia remains poorly understood. It has been recently found that iron overload in several diseases is involved in ferroptosis, an iron- dependent form of programmed cell death. However, whether this excess iron can result in ferroptosis in muscles is still unclear. In our present study, we found that ferric citrate induced ferroptosis in C2C12 cells, as well as impaired their differentiation from myoblasts to myotubes. Due to the decreased muscle mass and fiber size, 40-week-old senescence accelerated mouse prone 8 (SAMP8) mice were used as a sarcopenia model, in whose muscles the iron content and markers of ferroptosis were found to increase, compared to 8-week- old SAMP8 controls. Moreover, our results showed that iron overload upregulated the expression of P53, which subsequently repressed the protein level of Slc7a11 (solute carrier family 7, member 11), a known ferroptosis-related gene. The downregulation of Slc7a11 then induced the ferroptosis of muscle cells through the accumulation of lipid peroxidation products, which may be one of the causes of sarcopenia. The findings in this study indicate that iron plays a key role in triggering P53- Slc7a11-mediated ferroptosis in muscles, and suggest that targeting iron accumulation and ferroptosis might be a therapeutic strategy for treating sarcopenia.     

Effect of a low calcium diet on the ultrastructure of the parathyroid gland of chicks

Summary The effect of a low calcium diet on the ultrastructure of the parathyroid gland in the chick was examined. Two-week-old White Leghorn chicks were fed a low calcium diet (calcium content 0.63%) for two weeks. In these chicks, the parathyroid glands are grossly enlarged. Hypertrophy and hyperplasia of the chief cells are evident. The plasma membranes between adjacent cells are relatively straight but interdigitate in some places. Chief cells contain occasional membrane-limited secretory granules (150–350 m in diameter) and with contents of variable electron density. Secretory granules are distributed randomly but some are closely applied to the plasma membrane. There is an increase in the development of the rough-surfaced endoplasmic reticulum. The Golgi complex is enlarged and consists of cisternae arranged in concentric layers, smooth-surfaced and coated vesicles and condensing vacuoles. Dilatations of the cisternae at several points are observed. Mitochondria and filaments are also encountered. These morphological features suggest that low calcium intake stimulates the synthetic activity of the chief cells of the chick parathyroid.     

Immunocytochemical localization of parathormone in the mammalian parathyroid gland using the protein A-gold technique

Summary Electron-microscopic immunocytochemistry for the demonstration of parathormone in parathyroid chief cells was performed in adult male rats, gerbils, mice, and dogs, using the protein A-gold technique. Protein A-gold particles were detected over both large and small secretory granules in all the animals examined. In the former, they were concentrated not only over type-I granules with a large core, but also over type-II granules with a small core. They were also located over atypical granules, including heterogeneously dense granules, granules having vesicles in a finely particulate core, and distorted granules. All labelled secretory granules were characterized by the presence of a clear halo of varying width around the core. Occasionally, Golgi cisternae as well as Golgi vacuoles with a finely particular content were also labelled. The labelling of the secretory granules was strong in dogs, moderate in rats and gerbils, and weak in mice. In addition, it was more intense in the non-osmicated preparations than in the osmicated preparations. The frequency of both types of large granules showed species differences. The possible factors involved in these differences are discussed.     

The relation between solid cell nests and C cells of the thyroid gland

Summary Thyroid tissue of 300 routine autopsies was processed in a standardized manner. So-called solid cell nests (SCN) were found in 21 patients (7 %). These cases were investigated carefully by serial step sectioning. In order to explore the correlation of SCN to the C-cell system, the sections were stained by silver impregnation and the immunoperoxidase method. Morphometric analyses revealed a significant increase in the density of C cells in the proximity of the SCN. With progressive distance from the SCN, the C-cell density decreased and reached normal values. In 30 % of the cases argyrophilic and calcitonin-positive cells were found lying within the SCN. Occasionally, mixed follicles could be discerned: These were lined on the one side by a multilayered squamous epithelium, on the other side by normal monolayered cubic follicular epithelium, and contained a peculiar granular material. In one case, SCN were associated with intrathyroid portions of the parathyroids and adult adipose tissue, in a second case with adipose tissue only. Most probably SCN are vestiges of the ultimobranchial body and should be interpreted as such, despite the fact that other authors have expressed different views. The lack of disturbances in the calcium metabolism of the patients and the absence of medullary carcinoma in their family histories led us to interpret locally confined C-cell hyperplasia not as reactive nor premalignant, but rather as normal.     

Inefficient processing of human protein C in the mouse mammary gland

Vitamin K-dependent plasma protein, human Protein C (HPC) has been expressed in transgenic mice, using a 4.2kb mouse whey acidic protein (WAP) promoter, 9.0 kb HPC gene and 0.4 kb 3flanking sequences. Expression was mammary gland-specific and the recombinant human Protein C (rHPC) was detected in milk at concentrations of 0.1 to 0.7mg ml^–1. SDS-PAGE revealed that the single, heavy and light chains of rHPC migrated with increased electrophoretic mobility, as compared to HPC. Enzymatic deglycosylation showed that these molecular weight disparities are in part due to differential glycosylation. The substantial increase observed in the amount of single chain protein, as well as the presence of the propeptide attached to 20–30% of rHPC, suggest that mouse mammary epithelial cells are not capable of efficient proteolytic processing of rHPC. TheK _m of purified rHPC for the S-2366 synthetic substrate was similar to that of plasma-derived HPC, while the specific activity was about 42–77%. Amino acid sequence analyses and low anticoagulant activity of purified rHPC suggest that -carboxylation of rHPC is insufficient. These results show that proteolytic processing and -carboxylation can be limiting events in the overexpression of fully biologically active rHPC in the mouse mammary gland.     

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.     

Immunohistochemical identification of calcitonin gene-related peptide and substance P in nerves of the bovine parathyroid gland

Summary Although peptide neurotransmitters have been shown to modulate hormone secretion in many glands, there are very few studies of neurotransmitters in the parathyroid gland. Bovine parathyroid glands were collected at a local abattoir, fixed with paraformaldehyde, sectioned using a cryostat, and stained by indirect immunohistochemistry for calcitonin gene-related peptide and substance P. We were able to positively identify both neuropeptides. Nerve fibres containing calcitonin gene-related peptide and substance P were identified in contact with the tunica media of arteries and arterioles and dispersed throughout the stroma of the gland. While many of the fibres encircled parenchymal lobules, no intimate contact with the peripheral chief cells was observed. All immunoreactive fibres were found to contain both neuropeptides. Since calcitonin gene-related peptide and substance P are vasodilators, they may increase blood flow within the gland. In addition, the neuropeptides may diffuse from perilobular nerve fibres into the parenchyma, thereby modulating secretion of parathyroid hormone.     

Postnatal variations in the number and size of C-cells in the rat thyroid gland

The heterogeneous distribution of thyroid C-cells has until now hindered an objective evaluation of changes caused by age or experimental stimuli. To overcome this, a rigorous methodology has been designed to detect variations in shape, size, and number of C-cells throughout development. Using this methodology, we have demonstrated that C-cells do not significantly alter their shape with age. However, their volume increases gradually from 472 m³ in newborn rats to 1653 m³ in 120-day-old animals. Over the same time period, the mean number of C-cells within the thyroid gland increased 9-fold (from 1.6x10⁴ to 1.5x10⁵), and the number of C-cells per unit area decreased (from 6.15x10⁴/mm³ to 2.6x10⁴/mm³). We conclude that there are marked variations in size, total number, and number of C-cells per unit area in the rat thyroid gland after birth.     

Molecular characterization of calcitonin gene-related peptide (CGRP) in a rat medullary thyroid carcinoma cell line

The rat medullary thyroid carcinoma cell line, CA-77, is known to express the calcitonin gene and the cell line has been used for characterization of procalcitonin. The present investigations concentrate on a molecular characterization of the calcitonin gene-related peptide (CGRP) expressed by a subclone of this cell line. The investigations demonstrate that this subclone produces significantly more CGRP compared to calcitonin. Gel chromatography of cell extracts demonstrates heterogeneity for both CGRP and calcitonin, but a significant amount of immunoreactivity elutes corresponding to the elution position for synthetic CGRP and calcitonin, respectively. The gel chromatogram for CGRP demonstrates four immunoreactive peaks with K_d of 0.42, 0.53, 0.68, and 0.85. The immunoreactive peak with K_d 0.42 elutes corresponding to synthetic rat CGRP. The four immunoreactive peaks were characterized by high pressure liquid chromatography followed by sequence analysis and mass spectrometry. The immunoreactive peak with K_d 0.42 was identified as rat -CGRP as was the peak with K_d 0.53. The peak with K_d 0.68 was identified as 19–37 rat -CGRP and the peak with K_d 0.85 as 28–37 rat -CGRP. In summary, we find that the CA-77 cell line expresses large quantities of normally processed amidated -CGRP and specific fragments thereof. However, the cell line does not express detectable levels of rat β-CGRP. The findings indicate that the CA-77 cell line can be useful for studies of calcitonin/CGRP gene expression.     

Investigations on the thyroid gland of embryonic larval and adult Ichthyophis glutinosus and Ichthyophis kohtaoensis (Gymnophiona,amphibia)

Summary Different developmental stages of two species of the genus Ichthyophis have been investigated. In the late embryo the follicular cells of the thyroid gland exhibit various degrees of cytodifferentiation. Well differentiated cells show a polar organization and contain numerous granular inclusions, but a colloid-containing lumen is rare. Most cells at this stage contain large lipid inclusions. In young and older larvae the cells contain well-developed rough ER and Golgi systems, numerous mitochondria, and abundant granular and vesicular inclusions. Tentative identifications were made of primary lysosomes, secondary lysosomes, residual bodies, and two types of small apical vesicles—containing resorbed colloid or transporting material into the follicular lumen. In the larvae the number of apical microvilli is relatively high. The thyroid cells of the older larvae seem to contain more granular and vesicular inclusions than those of the younger larvae. In the adult the size of the follicles greatly increases, the height of the epithelium decreases, microvilli become rare, residual bodies are more frequent, and the small primary lysosomes are replaced by larger ones. Colloid droplets have been found only rarely in the cytoplasm of the thyroid cells of adult animals. In the immediate neighbourhood of the follicular epithelium, profiles of nerve fibres were found in all animals. Radioiodide investigations—measurements of conversion ratio and thyroid uptake factor—show, if compared with the results of corresponding studies in other amphibians, only relatively small differences between the larvae on the one hand and larvae and adults on the other. The absolute counts of the thyroid region are lowest in the adult and highest in the older larvae, shortly before metamorphosis. Furthermore our results indicate, on the basis of four animals tested, that in Ichthyophis the activity of the thyroid gland is temperature dependent. The results in Ichthyophis show that the classical stages of metamorphosis, in other amphibians characterized among other things by different levels of thyroid activity, are very indistinct in this animal.We gratefully acknowledge the financial support of the Deutsche Forschungsgemeinschaft (We 380/5; Sto 76/4).     

Discovery of novel,potent, and orally bioavailable pyrido[2,3-d][1]benzazepin-6-one antagonists for parathyroid hormone receptor 1

Structural modification of a 1,4-benzodiazepin-2-one-based PTHR1 antagonist 5, a novel type of PTHR1 antagonist previously synthesized in our laboratories, yielded compound 10, which had better chemical stability than compound 5. Successive optimization of the lead 10 improved aqueous solubility, metabolic stability, and animal pharmacokinetics, culminating in the identification of DS37571084 (12). Our study paves the way for the discovery of novel and orally bioavailable PTHR1 antagonists.     

Development and cytodifferentiation of C cell complexes in dog fetal thyroids

Summary The development of C-cell complexes was investigated in dog fetuses by an immunoperoxidase method with three specific antisera: anti-calcitonin, anti-C-thyroglobulin (C-Tg), and anti-19S thyroglobulin. Ultimobranchial bodies joined with the thyroid anlage and then dispersed into the parenchyma to form large C cell groups. Sparse reaction products of C-Tg initially appeared in C cells with small amounts of cytoplasm. Later at about day 39 of gestation, when the immunoreactivity of calcitonin and 19S thyroglobulin appeared weakly in C cells and follicular cells, C-cell complexes were identified as large cell masses containing numerous undifferentiated cells without no immunoreactivity for any of the antisera. As development proceeded, the undifferentiated cells developed progressively the morphology of C cells. In addition, the undifferentiated cells developed 19S thyroglobulin immunoreactivity, that is, within some of the complexes small clusters of cells filled with material immunoreactive for 19S thyroglobulin. They were not organized into follicles during the fetal period, and were very slow in development. Depending on the degree of development of the undifferentiated cells, several features of the complexes were noted. The present study indicates that not only C cells but also follicular thyroid cells appear to be derived from the ultimobranchial bodies.     

Identification of cancer stem-like cells in the C6 glioma cell line and the limitation of current identification methods

The cancer stem cell (CSC) hypothesis has provided insights into the initiation and recurrence of brain tumor. Specific identification and targeted elimination of these CSCs within the tumor mass represents a promising therapeutic strategy for refractory brain tumors. In this study, we attempted to identify CSCs in the rat C6 glioma cell line by three different identification methods. It is interesting to note that single-cell clonal analysis showed most C6 cells are cancer stem-like cells with characteristics of self-renewal, multilineage differentiation potentials in vitro, and tumorigenic capacity in vivo. It is surprising to note that CD133 failed to identify the total cancer stem-like cell population in the C6 line, since both CD133 (+) and CD133 (-) C6 cells have cancer stem-like cell fractions. Moreover, Hoechst 33342 staining, which is used in flow cytometry to isolate the side population (SP), was found to be harmful to C6 cells. Therefore, CD133 (-) and non-SP C6 cells may also harbor cancer stem-like cells. These results imply the limitation of using current identification methods in C6 line and underscore the importance of defining the genetic and molecular basis of CSCs.     

Observations on the fine structure,enzyme histochemistry,and innervation of parathyroid gland and ultimobranchial body of Chthonerpeton indistinctum (Gymnophiona,Amphibia)

Summary Fine structural and enzyme histochemical observations on ultimobranchial body and parathyroid gland of the caecilian Chthonerpeton are presented. The cell clusters and follicles of the ultimobranchial body consist mainly of granulated cells which are termed C-cells and obviously belong to the APUD cell series. In the larger follicles additional possibly exhausted degranulated cells and replacement cells occur. A rich supply of nerve fibres has been found in this gland. Frequently nerve terminals were observed to come into synaptic contact with the C-cells. Two categories of nerve fibres occur: a) fibres containing large polymorphic electron dense granules (probably purinergic fibres), b) fibres containing small electron transparent vesicles and a few electron dense granules (probably cholinergic fibres). The parathyroid gland consists of elongated cells (one cell type) poor in organelles and often containing fields of glycogen and lipid droplets. The cells are further characterized by fair amounts of lysosomal enzymes; they are interconnected by maculae adhaerentes and occludentes. No nerves and blood vessels have been found in the parathyroid gland of Chthonerpeton. This study has been supported by the Deutsche Forschungsgemeinschaft We 380/5.     

Immunocytochemical evidence for endogenous calcitonin and parathyroid hormone in osteoblasts from the calvaria of neonatal mice

Summary Immunoreactivities to endogenous calcitonin, endogenous parathyroid hormone, endogenous estradiol and estradiol receptors were studied in osteoblasts from the calvaria of neonatal mice by immunocytochemistry with the use of ultrathin sections obtained by cryo-ultramicrotomy. Tissues were fixed in glutaraldehyde, postfixed in osmium tetroxide and frozen in liquid nitrogen. Estradiol and estradiol receptors could not be detected in osteoblasts, whereas calcitonin- and parathyroid hormone-like immunoreactivities were observed in this cell type. Calcitonin and parathyroid hormone had similar subcellular localizations: immunoreactivities were observed at the plasma-membrane level, in the cytoplasmic matrix, and in the nucleus. These results provide immunocytological evidence for: 1) the internalization of calcitonin and parathyroid hormone in osteoblasts; 2) a direct participation of calcitonin and parathyroid hormone in the regulation of osteoblasts; 3) the absence of estradiol receptors and estradiol in osteoblasts.