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.     

Quantitative aspects of membrane shifts in rat parathyroid cells initiated by decrease in serum calcium

The secretory activity of parathyroid glands in rats was stimulated by decreasing the serum Ca++ concentration through constant intravenous infusion of EGTA. The morphometric analysis of the nuclear and cytoplasmic volume and of the surface area of the rough endoplasmic reticulum, Golgi complex, secretory granules and plasma membrane revealed a membrane shift from secretory granules and Golgi complex to the plasma membrane within 1 hr of calcium depression. Subsequently, between 1 and 3 hr of calcium depression, the membrane shift was from the plasma membrane to the Golgi complex. It is considered likely that these membrane shifts are related to a rise in release of parathyroid hormone by exocytosis and a subsequent increase in retrieval of plasma membrane by endocytosis—probably through the compartment of coated pits and coated and uncoated vesicles.     

Morphology of the granular secretory glands in skin of poison-dart frogs (Dendrobatidae)

The granular glands of nine species of dendrobatid frogs were examined using light and electron microscopy. The glands are surrounded by a discontinuous layer of smooth muscle cells. Within the glands proper the secretory cells form a true syncytium. Multiple flattened nuclei lie at the periphery of the gland. The peripheral cytoplasm also contains mitochondria, rough surfaced endoplasmic reticulum, the Golgi apparatus, and an abundance of smooth endoplasmic reticulum. Centrally, most of the gland is filled with membrane-bound granules surrounded by amorphous cytoplasm. Few other organelles are found in this region. Early in the secretory cycle, the central part of the gland is filled with flocculent material which appears to be progressively partitioned off by membranes to form the droplet anlage. As granules form, the structure of the contents becomes progressively more vesicular. Dense vesicles, which bud off from the Golgi apparatus, fuse with the granular membrane during the development of granules, and might contain enzymes involved in toxin synthesis. The granules at this point resemble multivesicular bodies. Their structure is similar in all species of dendrobatid frogs even though the different frogs secrete substances of different chemical structure and toxicity.     

Fine structure of chick embryonic parathyroid glands cultured on media with different concentrations of calcium.

An electron microscopical study of parathyroid glands from 13-day old chick embryos cultured for 2 days on media with different concentrations of calcium was conducted. The same as in non-cultured embryonic glands, the cells in all cultures contained a moderate amount of rough endoplasmic reticulum and a large Golgi complex with many cisternae, vacuoles, vesicles, coated vesicles and small prosecretory granules. In addition, large secretory granules, which are very rare in the non-cultured controls appeared frequently in the cultures and were especially numerous in the glands cultured on high-calcium medium. The fact that the amount of secretory granules varied according to the concentration of calcium in the medium is interpreted as indicating that the rate of parathyroid hormone secretion in the embryo is already responsive to variations in the concentration of calcium. To the extent that in vitro results may be accepted as representative of what happens in vivo these results support the idea that the embryonic gland may be controlled by variations in the calcemia as the adult gland does.     

Ultrastructural evaluation of the parathyroid glands of young cats with experimental hyperparathyroidism

Summary Kittens with an immature skeleton fed exclusively a calcium-deficient beef heart diet for 1 to 14 weeks developed hypocalcemia, parathyroid hyperplasia, and generalized osteitis fibrosa. Initial ultrastructural changes in the stimulated parathyroid glands were observed after 1 week and consisted of increased cytoplasmic volume, aggregation of the granular endoplasmic reticulum, and enlargement of the Golgi apparatus associated with increased numbers of prosecretory granules. After 3 weeks, chief cell hyperplasia was first observed and the numbers of mature secretory granules were greatly reduced. The hyperactive chief cells remained degranulated until the termination of the experiment. Stimulated chief cells accumulated glycogen in excess of controls and contiguous chief cells had intricately interdigitated plasma membranes. The parathyroid glands of experimental cats after being fed the low calcium diet for 14 weeks contained primarily hyperactive and vacuolated light chief cells with an occasional transitional water-clear cell.This investigation was supported in part by the Morris Foundation and grant GM 1052, National Institutes of Health, United States Public Health Service. The authors wish to acknowledge the technical assistance of Mr. D. Q. Davis and Mr. O. R. Kindig.     

Ultrastructure of the parathyroid gland of the japanese lizard in the spring and summer season

The ultrastructure of the parathyroid glands of adult Japanese lizards (Takydromus tachydromoides) in the spring and summer season was examined. The parenchyma of the gland consists of chief cells arranged in cords or solid masses. Many chief cells contain numerous free ribosomes and mitochondria, well-developed Golgi complexes, a few lysosome-like bodies, some multivesicular bodies and relatively numerous lipid droplets. The endoplasmic reticulum is mainly smooth-surfaced. Cisternae of the rough endoplasmic reticulum are distributed randomly in the cytoplasm. Small coated vesicles of 700-800 Å in diameter are found occasionally in the cytoplasm, especially in the Golgi region. The chief cells contain occasional secretory granules of 150-300 nm in diameter that are distributed randomly in the cytoplasm and lie close to the plasma membrane. Electron dense material similar to the contents of the secretory granules is observed in the enlarged intercellular space. These findings suggest that the secretory granules may be discharged into the intercellular space by an eruptocrine type of secretion. Coated vesicles (invaginations) connected to the plasma membrane and smooth vesicles arranged in a row near the plasma membrane are observed. It is suggested that such coated vesicles may take up extracellular proteins. The accumulation of microfilaments is sometimes recognized. Morphological evidence of synthetic and secretory activities in the chief cells suggests active parathyroid function in the Japanese lizard during the spring and summer season.     

Development of serous cutaneous glands in Scinax nasica (Anura,Hylidae): patterns of poison biosynthesis and maturation in comparison with larval glands in specimens of other families

We examined the development of serous (poison) cutaneous glands in larval and juvenile Scinax nasica (Hylidae) at the ultrastructural level. We describe the biosynthesis and maturation of the cutaneous poison in comparison with the corresponding processes in representatives of Discoglossidae, Leptodactylidae, Pelobatidae and Pipidae. Serous biosynthesis in S. nasica starts in discrete adenoblasts and continues in the syncytial secretory unit. Biosynthetic processes involve rough endoplasmic reticulum and the Golgi apparatus, that releases membrane-bounded material, varying from fine grained to flocculent. During the post-Golgian secretory phase, this material undergoes initial maturation, and two products are formed: dense granules and larger vesicles holding a thin substance that will later be structured into a three-dimensional, honeycomb-like net. Both the secretory granules and vesicles change into glomerular-like aggregates of bowed, rod-shaped subunits (modules). In adult frogs, formation of dense granules is bypassed. The modular granule substructure seems to be related to the merocrine release of small amounts of poison, involved in regulating skin homeostasis. Comparison with maturational changes in larval glands of species representing four anuran families discloses similar patterns in the Leptodactylidae, but production of opaque homogeneous granules occurs in the Discoglossidae, clear vesicles in the Pelobatidae and aggregates of dense bars in the Pipidae.     

Freeze-fracture study of the rat parathyroid gland under hypo- and hypercalcemic conditions,with special reference to secretory granules

Summary Freeze-fracture images of the parenchymal cells in the parathyroid gland of rats were observed after vitamin D₂ plus calcium chloride-suppression and EGTA-activation of secretion. In cells of the suppressed glands, large bulges protruded from the Golgi cisternae, and large granules with a stalk, which are identified as storage granules, suggest that, during maturation, some storage granules may be connected by long tubules with the Golgi cisternae and supplied with secretory products from the Golgi cisternae via these tubules.In the activated glands, presumptive exocytotic and endocytotic specializations of intramembranous particles of the parenchymal cell plasma membrane were frequently observed. In addition, elevations and complementary shallow depressions of various shape and extent were occasionally encountered in the intercellular space. From their morphological characteristics it was concluded that these originated from secretory granule cores, which are discharged from the parenchymal cells into the intercellular space by exocytosis, and it was suggested that discharged granule cores may retain their spherical shape until they fuse to form a flat conglomerate.     

Electron microscopic study of the parathyroid gland of the calcium-treated hamster subjected to hypergravity environment

The ultrastructure of the parathyroid glands of calcium-treated golden hamsters subjected to 5 gravity environment was studied. In the calcium-treated animals exposed to hypergravity environment, the Golgi complexes and cisternae of the granular endoplasmic reticulum were significantly decreased compared with those of the animals exposed to hypergravity environment only and appeared to increase compared with those of the calcium-treated animals, but were almost similar to those of the control animals. In addition, many chief cells contained some prosecretory granules in the Golgi areas, some secretory granules situated close to the plasma membrane and many lysosomes. The morphology of the parathyroid glands in the calcium-treated animals exposed to hypergravity environment resembled that of the control animals. These results suggest that the parathyroid glands suppressed by treatment of calcium and stimulated in response to hypergravity environment may indicate the secretory activity of the parathyroid glands of the control animals.     

Ultrastructure of the anuran pars intermedia following severance of hypothalamic connection

Summary The ultrastructure of the pars intermedia of Rana catesbeiana tadpoles was studied following isolation from the hypothalamus, in vivo after sectioning of the pituitary stalk, and in vitro after implantation of the pituitary into a piece of tail fin. Both experimental procedures were followed by rapid and sustained skin darkening. Pituitaries from normal light and dark adapted tadpoles served as controls.In 4-hour disinhibited glands, melanotrophs revealed hyperactive Golgi bodies, colloid vesicles (1–2 microns) in close proximity to axon terminals, and no apparent loss of secretory granules. At 24 hours extracellular colloid adjacent to axon terminals was found, and extensive arrays of RER appeared in the melanotrophs. Obvious granule loss from secretory cells occurred within a week, by which time the cytoplasm was occupied by large cisterns of SER and RER and abundant free ribosomes. Dense core vesicles (600–900 Å) in aminergic nerve terminals disappeared shortly after isolation of the pituitary from the hypothalamus, and only decreasing numbers of translucent vesicles (200–300 Å) were found.The functional significance of these changes is discussed, with particular emphasis on the mode of acute hormone release.This study was carried out in the Department of Anatomy, Albert Einstein College of Medicine, New York. I am greatly indebted to Professor Berta Scharrer and Professor William Etkin for their sponsorship, guidance and encouragement. Warm thanks are due to Mrs. Sarah Wurzelmann and Mr. Stanley Brown for their technical assistance. Support from U.S.P.H.S. grants NIH-NB 00840, NIH R01 Am 3984 and NSF Grant 12353 is gratefully acknowledged.     

Structure and ultrastructure of leaf and calyx glands in Galphimia brasiliensis (Malpighiaceae)

The present study describes the anatomical structure of calyx and leaf glands in Galphimia brasiliensis and analyzes the mechanism of secretion. The glands are marginal and suprabasal, cup-shaped, sessile, and scarcely visible with the naked eye. Light microscopy reveals the following features: a thin, smooth cuticle; unistratified secretory cells; subglandular parenchyma; and vascular bundle supply composed of phloem and xylem with abundant druses of calcium oxalate. Transmission electron microscopy reveals the presence of secretory cells with conspicuous nuclei, dense cytoplasm, lipid droplets, numerous vesicles, mitochondria, Golgi, rough endoplasmic reticulum (RER), and elongated plastids with osmiophilic contents. The secretion reaches the apoplastic space and accumulates beneath the cuticle. Finally, the viscous, translucent exudate is eliminated by mechanical rupture of the cuticle. Histochemical analysis confirms that lipids are the main constituent. Small amounts of polysaccharides were also identified.     

Cytochemical studies on the internal polarity of the golgi apparatus and the relationship between this organelle and GERL

Summary Cytochemical studies were performed to clarify the occurrence of an internal polarity of the Golgi apparatus and the relationship between this organelle and GERL in many kinds of cells having different morphologies and functions. The fine structural localizations of thiamine pyrophosphatase (TPPase) and acid phosphatase (AcPase) were examined in anterior pituitary cells, thyroid epithelial cells, gastric chief and parietal cells, duodenal absorptive epithelial cells, hepatocytes, adrenal cortical and medullary cells of mice, and thyroid epithelial cells of domestic fowls. TPPase activity is usually localized in the cisternae of 1–3 stacks and vesicles on the trans-side of the Golgi apparatus of all the cells examined, and in some immature secretory granules of anterior pituitary cells and of gastric chief cells. Rigid lamellae and multivesicular bodies are rarely positive to this reaction, in several kinds of cells. AcPase activity was usually demonstrable in the cisternae of 1–3 stacks and vesicles on the trans-side of the Golgi apparatus, and also in rigid lamellae, coated vesicles, multivesicular bodies and lysosomes in all varieties of cells studied. Some immature secretory granules are positive to the AcPase reaction in anterior pituitary cells and gastric chief cells. The areas positive for both enzyme activities were partially or almost completely overlapping in all the cells examined, though there were minor variations among them. The grades of overlap are classified into three types. Prolonged osmication was performed on thyroid epithelial cells, duodenal absorptive epithelial cells, hepatocytes, adrenal cortical cells, Leydig cells, the epithelial cells of the vas deferens and the theca cells of mice. Cisternae of 1–3 stacks on the cis-side of the Golgi apparatus of all the cells examined were stained with osmium tetroxide. In all these cells we observed that the Golgi apparatus has an internal polarity and that GERL is a part of this organelle in cytochemical respects.This study was supported by grants from the Japan Ministry of Education     

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.     

Quantitative morphological assessment of parathyroid activity in response to variations in calcium concentration in vitro

Isolated parathyroid glands from gerbils incubated at low (0.5 mM), normal (1.25 mM) or high (3 mM) calcium concentration for 1 or 6 h were studied with regard to the following stereological parameters: the volume densities of the nucleus, mitochondria, endoplasmic reticulum, Golgi zone, lipid bodies, lysosomes, secretory granules and storage granules, and the surface densities of the cell membrane and endoplasmic reticulum. In glands incubated for 1 h no significant differences were seen between the experimental groups in any of the parameters investigated, however, the volume density of the Golgi zone and the surface density of the cell membrane were significantly increased in glands incubated for 6 h at low calcium concentration, compared with those incubated at high calcium concentration. The enlargement of the Golgi zone in stimulated glands was also evident when the two glands from the same animal incubated at different calcium concentrations were compared. The data suggest that the early response of parathyroid glands to altered concentration of extracellular calcium includes functional activities associated with the Golgi zone.     

Ultrastructural study on the effects of hypophysectomy on the golden hamster parathyroid gland.

The ultrastructure of the parathyroid glands of hypophysectomized golden hamsters was studied. In the parathyroid glands of hypophysectomized animals the Golgi complexes and secretory granules were significantly decreased and large vacuolar bodies were significantly increased compared with those of the control animals. In addition, the chief cells contained a few prosecretory granules in the Golgi areas and a few secretory granules were present in the peripheral cytoplasm. These results suggest that the synthesis and release of parathyroid hormone may be suppressed in the parathyroid glands of the hypophysectomized animals.     

Effects of pinealectomy on the ultrastructure of the golden hamster parathyroid gland

Ultrastructural changes of the parathyroid glands of pinealectomized golden hamsters were investigated. The main changes in the parathyroid glands 1 hour and 1 day after pinealectomy compared with the control and sham-operated groups were an increase of the Golgi complexes, cisternae of the granular endoplasmic reticulum and large vacuolar bodies. In addition, many chief cells contained numerous prosecretory granules in the Golgi areas and many secretory granules in the peripheral cytoplasm. The morphology of the parathyroid glands 7 and 30 days after pinealectomy resembled that of the control parathyroid glands. These results suggest that pinealectomy affects the secretory activity of the parathyroid gland.     

Age-related changes of the ultrastructure in the cardiomyopathic hamster (UM-X7.1 Syrian hamster) parathyroid gland

We qualitatively and quantitatively investigated parathyroid glands of the UM-X7.1 cardiomyopathic hamster at 1, 2, 6 and 12 months of age to compare them with those of the normal hamster. We found that at 1 month of age in the UM-X7.1 hamster, the Golgi apparatus, lipid droplets and secretory granules decreased. There were no significant differences between the UM-X7.1 hamster and the control hamster at 2 months of age. At 6 months of age, the Golgi apparatus, rER and the secretory granules significantly increased in the UM-X7.1 hamster. At 12 months of age, the Golgi apparatus and lysosomes increased, while the secretory granules decreased. Ultrastructurally, we consider that in the UM-X7.1 hamster, the synthesis and release of the parathyroid at 6 months of age may be activated by an excessive amount of circulating catecholamine, and the functional activity of the parathyroid glands at 12 months of age may be depressed by the increased plasma calcium level. These findings suggest that the activities of the synthesis and release of the parathyroid hormone were the highest at 6 months of age in the UM-X7.1 hamster.     

Lectin-labeled membrane is transferred to the Golgi complex in mouse pituitary cells in vivo

Labeling of the Golgi complex with the lectin conjugate wheat germ agglutinin-horseradish peroxidase (WGA-HRP), which binds to cell surface membrane and enters cells by adsorptive endocytosis, was analyzed in secretory cells of the anterior, intermediate, and posterior lobes of mouse pituitary gland in vivo. WGA-HRP was administered intravenously or by ventriculo-cisternal perfusion to control and salt-stressed mice; post-injection survival times were 30 min-24 hr. Peroxidase reaction product was identified within the extracellular clefts of anterior and posterior pituitary lobes through 24 hr but was absent in intermediate lobe. Endocytic vesicles, spherical endosomes, tubules, dense and multivesicular bodies, the trans-most saccule of the Golgi complex, and dense-core secretory granules attached or unattached to the trans Golgi saccule were peroxidase-positive in the different types of anterior pituitary cells and in perikarya of supraoptico-neurohypophyseal neurons; endoplasmic reticulum and the cis and intermediate Golgi saccules in the same cell types were consistently devoid of peroxidase reaction product. Dense-core secretory granules derived from cis and intermediate Golgi saccules in salt-stressed supraoptic perikarya likewise failed to exhibit peroxidase reaction product. The results suggest that in secretory cells of anterior and posterior pituitary lobes, WGA-HRP, initially internalized with cell surface membrane, is eventually conveyed to the trans-most Golgi saccule, in which the lectin conjugate and associated membrane are packaged in dense-core secretory granules for export and potential exocytosis of the tracer. Endoplasmic reticulum and the cis and intermediate Golgi saccules appear not to be involved in the endocytic/exocytic pathways of pituitary cells exposed to WGA-HRP.     

Subcellular Fractionation of Prohormone Processing Products in the Bag Cell Neurons

Abstract: Multiple biologically active peptides arising from a common prohormone are sorted into distinct classes of dense core vesicles within the bag cell neurons of Aplysia californica . In this study, pulse-chase analysis, combined with subcellular fractionation on Percoll gradients, are used to define the location of the prohormone processing events within the secretory pathway. Initial cleavage of the prohormone occurs in a light cellular compartment associated with the Golgi apparatus. The amino-terminal processing intermediate then accumulates in a denser compartment containing small dense cores enclosed in membranous sacs, as well as larger immature vesicles. After 4 h, amino-terminai products are found primarily in a much denser compartment which consists of large and small dense core vesicles. These large and small vesicles can be separated from each other using Percoll gradient centrifugation and are found to be enriched in amino- and carboxy-terminal products, respectively. Lastly, membrane association experiments suggest differential binding to membranes, or integral membrane proteins, as a possible mechanism for sorting of amino- and carboxy-terminal products.     

Toxoplasma gondii Rab6 mediates a retrograde pathway for sorting of constitutively secreted proteins to the Golgi complex

Toxoplasma gondii relies on protein secretion from specialized organelles for invasion of host cells and establishment of a parasitophorous vacuole. We identify T. gondii Rab6 as a regulator of protein transport between post-Golgi dense granule organelles and the Golgi. Toxoplasma Rab6 was localized to cisternal rims of the late Golgi and trans-Golgi network, associated transport vesicles, and microdomains of dense granule and endosomal membranes. Overexpression of wild-type Rab6 or GTP-activated Rab6(Q70L) rerouted soluble dense granule secretory proteins to the Golgi and endoplasmic reticulum and augmented the effect of brefeldin A on Golgi resorption to the endoplasmic reticulum. Parasites expressing a nucleotide-free (Rab6(N124I)) or a GDP-bound (Rab6(T25N)) mutant accumulated dense granule proteins in the Golgi and associated transport vesicles and displayed reduced secretion of GRA4 and a delay in glycosylation of GRA2. Activated Rab6 on Golgi membranes colocalized with centrin during mitosis, and parasite clones expressing Rab6 mutants displayed a partial shift in cytokinesis from endodyogeny (formation of two daughter cells) to endopolygeny (multiple daughter cells). We propose that Toxoplasma Rab6 regulates retrograde transport from post-Golgi secretory granules to the parasite Golgi.