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.     

Immunohistochemical, electron microscopic and morphometric studies of estrogen-induced rat prolactinomas after bromocriptine treatment

To clarify the effects of bromocriptine on prolactinoma cells in vivo, immunohistochemical, ultrastructural and morphometrical analyses were applied to estrogen-induced rat prolactinoma cells 1 h and 6 h after injection of bromocriptine (3 mg/kg of body weight). One h after treatment, serum prolactin levels decreased markedly. Electron microscopy disclosed many secretory granules, slightly distorted rough endoplasmic reticulum, and partially dilated Golgi cisternae in the prolactinoma cells. Morphometric analysis revealed that the volume density of secretory granules increased, while the volume density of cytoplasmic microtubules decreased. These findings suggest that lowered serum prolactin levels in the early phase of bromocriptine treatment may result from an impaired secretion of prolactin due to decreasing numbers of cytoplasmic microtubules. At 6 h after injection, serum prolactin levels were still considerably lower than in controls. The prolactinoma cells at this time were well granulated, with vesiculated rough endoplasmic reticulum and markedly dilated Golgi cisternae. Electron microscopical immunohistochemistry revealed positive reaction products noted on the secretory granules, Golgi cisternae, and endoplasmic reticulum of the untreated rat prolactinoma cells. However, only secretory granules showed the positive reaction products for prolactin 6 h after bromocriptine treatment of the adenoma cells. An increase in the volume density of secretory granules and a decrease in the volume densities of rough endoplasmic reticulum and microtubules was determined by morphometric analysis, suggesting that bromocriptine inhibits protein synthesis as well as bringing about a disturbance of the prolactin secretion.     

Quantitative analyses of atrial myoendocrine cells and plasma atrial natriuretic peptides (ANP) of the rat with special reference to the twenty-four-hour variations in secretory granules and plasma ANP concentrations

Summary Subcellular structures of atrial myoendocrine cells in the rat heart and plasma concentrations of atrial natriuretic peptides (ANP) were examined at six evenlyspaced time points over 24 h, using morphometric techniques and radioimmunoassay.Myofibrils and mitochondria of the cells occupied 73.3% of the cytoplasm; 2% of the cytoplasm was occupied by secretory granules, rough endoplasmic reticulum and Golgi complexes, structures characteristic of endocrine cells. Plasma ANP concentration was maximal at 08.00 h, when the individual volume of secretory granules was minimal. The numerical density of secretory granules was increased at 12.00 h. The plasma ANP concentration was minimal at 20.00 h, when the numerical density was minimal and the individual volume was maximal. The fluctuation in plasma ANP concentrations over 24 h was thus parallel to that in the numerical densities of secretory granules and inverse to that in individual volumes.These results suggest that in rats the secretory activity of atrial myoendocrine cells increases at the beginning of the resting period, whereas it decreases at the beginning of the active phase.     

Cell-free systems to study vesicular transport along the secretory and endocytic pathways

Proteins bound for the cell surface, lysosomes, and secretory storage granules share a common pathway of intracellular transport. After their synthesis and translocation into the endoplasmic reticulum, these proteins traverse the secretory pathway by a series of vesicular transfers. Similarly, nutrient and signaling molecules enter cells by endocytosis, and move through the endocytic pathway by passage from one membrane-bound compartment to another. Little is known about the mechanisms by which proteins are collected into transport vesicles, or how these vesicles form, identify their targets, and subsequently fuse with their target membranes. An important advance toward our understanding these processes has come from the establishment of cell-free systems that reconstitute vesicular transfers in vitro. It is now possible to measure, in vitro, the transport of proteins from the endoplasmic reticulum to the Golgi, between Golgi cisternae, and the formation of transport vesicles en route from the trans Golgi network to the cell surface. Along the endocytic pathway, cell-free systems are available to study clathrin-coated vesicle formation, early endosome fusion, and the fusion of late endosomes with lysosomes. Moreover, the selective movement of receptors between late endosomes and the trans Golgi network has also been reconstituted. The molecular mechanisms of vesicular transport are now amenable to elucidation.     

THE FINE STRUCTURE OF BRUNNER''S GLANDS IN THE MOUSE

Examined with the electron microscope, the secretory cells of the submucosal glands of Brunner in the mouse present a curious combination of the fine-structural features of both serous and mucus-secreting cells. The cells have numerous mitochondria, abundant basal ergastoplasm, dense secretory granules that bear a superficial resemblance to pancreatic zymogen granules, and an unusually extensive Golgi apparatus. The prominence of the lamellar, vesicular, and vacuolar elements of the Golgi complex facilitates detailed observation of these components. More evident than in other glandular cells, aggregates of small vesicles appear to represent the transitional elements and are vehicles for transport of the product between the ergastoplasm and the Golgi complex. The numerous vesicular evaginations of smooth-surfaced regions on cisternae of the rough-surfaced endoplasmic reticulum and accumulations of innumerable vesicles of similar size in the area between the nearest profiles of the ergastoplasm and the Golgi complex support this contention. The cytological characteristics and physiologic properties of Brunner's glands in various species are discussed briefly. It is concluded that the submucosal glands of the mouse are excellent material for exploration of the ultrastructural correlates of both protein and carbohydrate secretion, and it is suggested that their secretion may have functions other than those generally attributed to them, namely, chemical and mechanical protection of the duodenal surface epithelium.     

Radioautographic characterization of successive compartments along the rough endoplasmic reticulum-Golgi pathway of collagen precursors in foot pad fibroblasts of [3H]proline-injected rats

Young rats given an intravenous injection of [3H]proline were killed at successive times from 4 to 80 min later. Fibroblasts from the front foot pad were radioautographed ; silver grains were counted over several of the organelles and the results were expressed as percent radiolabel per unit volume. These percentages reached a peak over rough endoplasmic reticulum cisternae at 4 min, intermediate vesicles and tubules at 10 min, spherical distensions of cis-side Golgi saccules at 20 min, cylindrical distensions of trans-side saccules between 40 and 60 min, and secretory granules at 60 min. It is proposed that the succession of peaks corresponds to the migration pathway of collagen precursor proteins within fibroblasts; that is, the proteins synthesized in rough endoplasmic reticulum are delivered by intermediate vesicles and/or tubules to the spherical distensions of cis-side saccules, somehow pass from there to the cylindrical distensions of trans-side saccules and, finally, are carried by secretory granules to the extracellular space.     

Immunocytochemical localization of renin in juxtaglomerular cells

The involvement of various organelles in the synthesis, transport, and packaging of renin in the juxtaglomerular cells of newborn mice has been investigated by immunocytochemistry with the protein A-gold technique. Highly specific rabbit antibodies against mouse submandibular renin were used. Mild fixation and embedding in glycol methacrylate allowed enough sensitivity to identify a steep gradient of labeling from rough endoplasmic reticulum to Golgi complex to secretory granules. Routine fixation and embedding in Epon produced labeling differentials that allowed delineation of hitherto ill-defined types of secretory granules and vacuoles. The classical pattern of synthesis, transport, and packaging of secretory proteins involves the rough endoplasmic reticulum and Golgi complex and seems to apply to renin secretion. Immunoreactive renin is packaged as rhomboid crystals at the trans face of the Golgi complex. The limiting membrane of these rhomboids fuses to form coalescing protogranules where the crystals eventually yield their individuality maturing into secretory granules. Vacuoles containing a flocculent material, with or without a dense core, show significant immunocytochemical labeling. These vacuoles are not associated with the Golgi complex but occupy cytoplasmic areas well endowed with rough endoplasmic reticulum. As judged from their morphological features and their immunoreactivity, the vacuoles do not seem to follow the sequence of events typical of protogranules and coalescing protogranules. They possibly represent a parallel pathway of renin synthesis and transport, involving the nuclear envelope and bypassing the Golgi complex.     

Mechanisms of protein yolk synthesis and deposition in crustacean oocytes

Summary Electron microscope studies on the oocytes of several crustacean species demonstrate that the protein yolk arises within vesicular and lamellar forms of the rough-surfaced endoplasmic reticulum. The vesicular form of the endoplasmic reticulum may have its origin from a blebbing process of the outer layer of the nuclear envelope. Disc-shaped granules, representing precursor elements of the yolk granules, appear within the vesicular and lamellar profiles of endoplasmic reticulum. Autoradiographic results suggest that the ribosomes attached to the endoplasmic reticulum take part in the biosynthesis of yolk proteins. Numerous disc-shaped granules accumulate within the cisternae of the endoplasmic reticulum, but eventually they undergo a transformation into a finely granular yolk granule. Thus, both the origin and growth of protein yolk granules occur within membranes constituting the endoplasmic reticulum. The results provide evidence that intra-ooplasmic synthesis of yolk protein occurs in these oocytes.This investigation was supported by research grants (HD-00699; GM-09229) and a Career Development Award (GM-11,524) from the National Institutes of Health, U.S. Public Health Service.     

The fine structural localization of peroxidase activity in digestive organs of rats and mice

Summary An endogenous peroxidase activity is demonstrated in acinar cells of the salivary gland and epithelial cells of the colonic crypt of normal rats and mice using electron microscopic histochemistry. The main site of the enzymatic activity is cisternae of the rough endoplasmic reticulum including those of the nuclear envelope, while the intensity of the activity is greatly variable among cell types. Some vesicular and cisternal elements of the Golgi apparatus and secretory granules exhibit the reaction, but it is not consistent in all cells with the peroxidase-positive endoplasmic reticulum. It is very interesting that the peroxidase activity is positive in the rough endoplasmic reticulum-Golgi complex-secretory granule system (EGG system) of the cells located at the beginning and the end of the digestive tract. This suggests a peroxidase-dependent anti-infectious mechanism.Some large and small membrane-limited non-secretory granules and mitochondria also reacted.     

Stereological study of B-cell mitochondria in alloxan-treated mice.

Using stereological techniques, including semi-automatic image analysis, the B-cell mitochondria were studied in the pancreatic islets from one group of control mice and two groups of mice killed 10 min and 60 min, respectively, after alloxan administration. Ten min following alloxan the mitochondrial volume and envelope surface densities, the mean mitochondrial volume and surface area, and the area of mitochondrial profiles were significantly increased, whereas the mitochondrial numerical density was not significantly altered. At the 60 min observation time the mitochondrial volume density, the mean mitochondrial volume and surface areas, and the area of mitochondrial profiles were significantly decreased, whereas the mitochondrial envelope surface was not significantly altered. The findings indicate a rapid swelling, followed by disintegration of the mitochondria in the B-cells of alloxan-treated mice, thereby supporting our view that mitochondrial lesions play a primary role in the development of alloxan diabetes. These lesions are believed to be due to ionic alterations in the B-cells ("Pi-pH hypothesis").     

Quantitative study of hepatocytes from minipigs and minipig fetuses exposed to ethanol in vivo

The effect of ethanol on hepatocytes from pregnant minipigs and their half-term fetuses was studied with the aid of morphometric methods. In the pregnant minipigs the hepatocytes of the ethanol-treated animals showed a significant increase in the volume density of mitochondria, autophagic vacuoles, Golgi complexes and smooth endoplasmic reticulum, and a significant decrease of glycogen. In the half-term fetuses the hepatocytes of ethanol-exposed animals showed no significant change in the volume density of mitochondria, peroxisomes, autophagic vacuoles, Golgi complexes, smooth endoplasmic reticulum or glycogen, and no significant change in the surface density of granular endoplasmic cisternae. The present investigation indicates that in the maternal hepatocyte certain cytoplasmic components are quantitatively changed by ethanol, whereas the volume and surface densities of identical components in the fetal hepatocyte are unaffected. The significance of these results is discussed.     

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.     

Intracellular pathway and kinetics of protein secretion in the coagulating gland of the mouse

The coagulating gland of male rodents is part of the prostatic complex. Various mechanisms of secretion have been postulated, in part because organelles commonly involved in the secretory process possess unusual features, such as extreme distension of the rough endoplasmic reticulum. In the present study, the pathway, kinetics, and mode of secretion in the coagulating gland of the mouse were studied by electron microscope autoradiography at intervals between 5 min and 8 h after administration of 3H-threonine. The percentage of grains associated with the rough endoplasmic reticulum was initially high and generally decreased throughout the experiment, while a pronounced rise in the proportion of grains associated with the Golgi apparatus and secretory granules was observed 6 h after injection of precursor. In addition, there was a smaller elevation in the percentage of grains over the Golgi apparatus and secretory granules between 1 and 4 h, and radioactive material first reached the lumen of the gland 4 h after injection of the precursor. Although the general pathway of intracellular transport of secretory protein resembles that in other cells, the results indicate that there are several unusual aspects to the secretory process in the coagulating gland. First, the rate of transport was markedly slower than in most other exocrine gland cells, since the bulk of the labeled protein did not reach the Golgi apparatus and secretory granules until 6 h after administration of precursor. This reflected prolonged retention of secretory products in the endoplasmic reticulum. Second, in addition to the major bolus of labeled material that traversed the cells at about 6 h, a smaller wave of radioactivity appeared to pass through the Golgi apparatus and secretory granules and reach the lumen earlier, within the first few hours after the injection. Finally, the primary mode of secretion in the coagulating gland appears to be merocrine because the secretory granules contained much labeled protein.     

Alkaline phosphatase biosynthesis in the endoplasmic reticulum and its transport through the Golgi apparatus to the plasma membrane: cytochemical evidence

Enzyme induction of HeLa cell placental alkaline phosphatase with various agents such as prednisolone, sodium butyrate, hyperosmolality (NaCl), or combination of these inducers resulted in the appearance of enzyme activity in the rough endoplasmic reticulum, nuclear envelope, Golgi apparatus, and plasma membrane. In the Golgi apparatus, intense reaction product deposits tended to be concentrated on its trans side, with small vesicles and granules also being positively stained. Inhibition of protein synthesis with cycloheximide was followed by the disappearance of enzyme activity from these cytoplasmic organelles but not from the plasma membrane. Treatment with monensin, a secretory protein transport inhibitor, uniformly increased activity in the rough endoplasmic reticulum while causing marked dilatation of the intensely positive Golgi cisternae. These results suggest that intracellular alkaline phosphatase is newly synthesized in the endoplasmic reticulum and then passes en route through the Golgi apparatus to the plasma membrane. Accordingly, the present system could represent the biosynthesis, transport, and incorporation of the model cell surface enzyme protein to add to the vesicular stomatitus virus glyco-1 (VSV-G) protein and acetylcholine receptor model systems for studying the dynamics of cell surface protein genesis, transport, and membrane integration.     

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.     

Effects of starvation on the ultrastructure of the mouse parathyroid gland

Ultrastructural changes of the parathyroid glands of starved mice were examined. The parathyroid glands of the starved mice showed a decrease in the volume of Golgi complexes and storage granules and an increase in the volume of lipid droplets, and contained more heterogeneously dense bodies and multivesicular bodies compared with that of the control mice. In addition, the volume of mitochondria, cisternae of granular endoplasmic reticulum and secretory granules and the number of prosecretory granules appeared to be decreased compared to those of the control mice. Myelin-like structures were observed in the parathyroid glands of the starved mice. The results of our study provide support for the hypothesis that starvation exerts an inhibitory influence not only on the synthesis but also on release of parathyroid hormone.     

BETA GRANULE FORMATION IN ISOLATED ISLETS OF LANGERHANS : A Study By Electron Microscopic Radioautography

The distribution of radioautographic grains over organelles within the beta cells of rat islets of Langerhans was investigated at various times after pulse labeling of the isolated islets with tritium-labeled amino acids. Ten minutes after the start of labeling most of the grains were situated over the endoplasmic reticulum and cytoplasm; by contrast, 60 min from the start of labeling the majority of the grains were associated with the beta granules. At 20, 30, and 45 minutes after pulse labeling the proportion of grains associated with the Golgi complex was increased two- to three-fold over the 10- or 60-minute values. The distribution of radioautographic grains over granules in the intact cells did not suggest that the electron-lucent type of secretory granules were precursors of the electron-opaque granules. Furthermore, studies of the pattern of grains over granules isolated by centrifugation 60 min after pulse labeling showed no preferential labeling of the electron-lucent type of granule. It is concluded that labeled amino acids are incorporated initially in the endoplasmic reticulum, and that the label subsequently appears in the beta granules. The Golgi complex participates either in the formation of the beta granule or in the translocation of the granule through the cytoplasm of the cell.     

Cytochemical studies of GERL and its role in secretory granule formation in exocrine cells

Synopsis the structure and cytochemistry of GERL was studied in several different exocrine secretory cells, including the exorbital lacrimal gland, parotid, lingual serous (von Ebner's), submandibular, and sublingual salivary glands, and exocrine pancreas of the rat; the lacrimal, parotid and pancreas of the guinea-pig; and the lacrimal gland of the monkey. GERL was morphologically and cytochemically similar in all cell types studied. It was located in the inner Golgi region and consisted of cisternal and tubular portions. Immature secretory granules were in continuity with GERL through multiple tubular connections. Modified cisternae of endoplasmic reticulum, with ribosomes only on one surface, closely paralleled parts of GERL. GERL and immature granules were intensely reactive for acid phosphatase activity, while the inner Golgi saccules were reactive for thiamine pyrophosphatase and nucleoside diphosphatase activities. In the rat exorbital lacrimal and parotid glands, reaction product for endogenous peroxidase, a secretory enzyme, was present in the endoplasmic reticulum, Golgi saccules, immature and mature secretory granules. GERL was usually free of reaction product or contained only a small amount. The widespread occurrence of GERL in secretory cells, and its intimate involvement with the formation of granules, suggest that it is an integral component of the secretory process.     

Morphology of the mammotrophs and gonadotrophs in the anterior pituitary gland of rats with protein-calorie malnutrition

The structure of the anterior pituitary gland of rats with protein-calorie malnutrition was studied at the light and electron microscopic levels. Male Sprague-Dawley rats were fed a low-protein diet from 20 to 50 days of age. The hypophyses from these animals, along with those from age-matched controls, were then removed and fixed for light microscopic immunocytochemical analyses, using anti-rat PRL or anti-ovine LH beta serum, or for routine ultrastructural study. The overall number of mammotrophs and gonadotrophs appeared to be unaffected by the deficiency in dietary protein. However, the nuclei, in both cell types, were significantly smaller in the malnourished rats. Cytoplasmic volume was also apparently less than in control rats, leading to the "crowding" of adjacent cell types. Additionally, in the malnourished rats, the mammotrophs assumed a less polygonal profile, while the gonadotrophs, which were more irregularly shaped in the central zone, rarely displayed the negative image of a Golgi complex. Ultrastructurally, the mammotrophs in the experimental rats had a less extensively developed granular endoplasmic reticulum and Golgi complex. The number of secretary granules was not different; however, their size was markedly less and their shape was round to ovoid, rather than pleomorphic. Two types of gonadotrophs were distinguished, based on the classification of Kurosumi et al. ('76). The type-I cell (former "FSH" cell) had a smaller Golgi complex, less vesicular endoplasmic reticulum and smaller secretory granules. The major alteration in the type-II gonadotroph (former "LH" cell) was the smaller size of the secretory granules. The morphological changes found in this study support our previous physiological observations (Herbert, '80), which demonstrate that prolactin and gonadotrophin secretion are severely impaired in rats suffering from protein-calorie malnutrition.