Mammalian parathyroids: morphological and functional implications.

Fixation with aldehydes is achieved either by immersion or perfusion. The parenchyma of parathyroid glands fixed by immersion consists of dark cells containing a lot of membranes of these organelles which are concerned with hormone secretion, light cells which are poor in these organelles, intermediate forms between the two, and multinuclear syncytial cells. They have been attributed to represent different functional stages of secretory activity, the dark cell being in an active form, the light cell in a resting form. Studies of the parathyroids of mice, rats, rabbits, cats, dogs, pigs, cattle, sheep, goats, and horses employing various fixation protocols clearly demonstrate that light cell variants and multinuclear syncytial cells are formed during improper immersion fixation as a result of membrane disintegration. Parathyroids fixed by perfusion or by immersion in an appropriate fixation medium comprise only one cell type which correspond to the dark chief cell. Parathyroid cells are polar cells bearing some of the rough endoplasmic reticulum in the basal pole, the rest of it, the Golgi complex, and secretory granules in the apical pole. The secretory product is released by exocytosis at the apicolateral domain of the plasma membrane into the intercellular space. Secretory activity can be altered experimentally, leading to drastic changes in the amount of cell membrane related to hormone synthesis, intracellular transport, exocytic release, and secretion coupled membrane retrieval. The sensitive reaction of parathyroid cells to both the mode of fixation and to fixation media demands careful evaluation of the fixation protocol. This and the polarity of parathyroid cells have to be borne in mind for estimating secretory activity on the basis of morphological criteria.

The biological significance of storage granules in rat parathyroid cells.

Both prosecretory and storage granules are concomitantly formed at the trans Golgi network including the innermost Golgi cisterna. Prosecretory granules develop into small secretory granules that release their contents by exocytosis finely regulated by a complex mechanism for maintaining calcium homeostasis. In the rat parathyroid cells, storage granules are large secretory granules storing parathyroid hormone for an emergency supply. The hormone is rapidly discharged by exocytosis when serum calcium concentration is decreased. The granules are constantly produced even under conditions of low serum calcium concentration in the regions of 8 mg/dl. The granule content is constantly hydrolyzed when not discharged, leading to a decreased core and finally to the formation of vacuolar bodies. The fate of the vacuolar bodies is unknown. Hypercalcemic conditions accelerate hydrolysis. The threshold value of calcium concentration required for the release of storage granule contents is between 8.0 and 7.5 mg/dl and that of calcium concentration for accelerating degradation of storage granules is about 11.5 mg/dl. Sympathetic stimulation causes storage granules to be discharged regardless of hypercalcemia or hypocalcemia. Parasympathetic stimulation accelerates hydrolysis. The degradation of storage granules seems to be closely associated with an intracellular regulatory mechanism for parathyroid hormone secretion.

Freeze-fracture replica studies of tight junctions in normal human bronchial epithelium.

Using freeze-fracture techniques, tight junctional networks were observed in the human normal bronchial epithelium. They were morphologically classified into three types: type I was a loosely interconnected, most complicated network consisting of 7-11 roughly parallel wavy strands and situated between ciliated cells; type II was a randomly anastomosing, simple network made up of 2-4 strands and present between goblet cells; type III was an irregularly anastomosing network composed of 4-7 strands and located between a ciliated cell and a goblet cell. Type III junctions, when a goblet cell was strongly bulged, were located on the swollen ridge, the upper surface of which was separated by a deep groove from the bulged apical surface, around the lateral surface of the cell at the level of the luminal surface. The possible relation between the orientation of strands of these networks and extra- or intracellular stress was discussed.

Electron-microscopic studies on the threshold value of calcium concentration for the release of storage granules and the acceleration of their degradation in the rat parathyroid gland.

To determine both a threshold value of calcium concentration (CC) for the release of storage granules and that for the acceleration of degradation of these granules, the rat parathyroid glands were perfused in situ with HEPES-Ringer solutions containing different concentration of Ca2+ for 10 min. With perfusates containing 0.83-1.21 mM Ca2+ (equivalent to 8-11 mg/dl serum calcium), the number of type-I storage granules (large core) [NSG-I] and that of type-II storage granules (small core) [NSG-II] remained unchanged. With perfusates containing 0.83 mM Ca2+ (7.5 mg/dl) or less, however, both NSG-I and NSG-II decreased remarkably and the former was larger than the latter. On the contrary, with perfusates containing 1.27 mM Ca2+ (11.5 mg/dl) or more, NSG-II increased and the ratio of NSG-I to NSG-II was changed reversely. We concluded that a threshold value of CC required for the release of storage granules may be present between 0.88 and 0.83 mM Ca2+ (8 and 7.5 mg/dl) and that a threshold value of CC for accelerating the transformation of type-I granules into type-II, the degradation of storage granules, may be situated at about 1.27 mM Ca2+ (11.5 mg/dl). Additionally, it was suggested that both pro-secretory and storage granules are not only formed at the innermost Golgi cisterna but also at the trans-Golgi network.

Correlated histochemical and electron microscopic studies of the esophageal epithelium in the salamander, Hynobius nebulosus.

The esophageal epithelium of the adult salamander, Hynobius nebulosus, was studied by histochemistry, including periodic acid-Shiff (PAS), alcian blue both at pH 2.5 and pH 1.0, and alcian blue (pH 2.5)-PAS with or without neuraminidase-digestion, and scanning and transmission electron microscopy. The epithelium was columnar in type, comprising partly pseudostratified and partly two-layered architecture; it consisted mainly of ciliated cells, goblet cells, and basal cells. The ciliated cells consisted of two types, light and dark: both types, especially the latter, frequently contained numerous small mucous granules in their apical portion. Ciliated-mucous cells were also occasionally present. Immature ciliated cells were frequently found. Goblet cells were divided into two types: Type I cells possessed electron lucent mucous granules, which frequently contained dense specific inclusions, and frequently bulged into the lumen; Type II cells had moderately electron-dense mucous granules with no inclusions and a conical apex which did not exceed the level of the lumen. The Type I cells were closely distributed throughout the esophagus, while the Type II were mainly dispersed in the cranial portion, remarkably decreasing in the caudal portion. Correlated histochemical and electron microscopic observations suggested that, in the Type I cell, mucous granules contain acid mucosubstances, while in the Type II, they possess neutral mucosubstances, and that in the Type I cell mucous granules consist of sialic acid-containing glycoproteins and their swollen portions are more highly sulfated than the non-swollen ones.

Freeze-fracture replica studies of effects of ACTH treatment and hypophysectomy on the cell surface of the rat adrenal inner cortex.

The morphological change of the cell surface in the inner zone of the adrenal cortex was studied in intact, hypophysectomized, and ACTH-treated hypophysectomized rats, using freeze-fracture replicas. In both intact and hypophysectomized ACTH-treated animals, the P fracture face of the plasma membranes of the cortical cells facing capillaries shows groups of characteristic finger-shaped microvilli, which arise horizontally from the edges of a small cavity on the cell surface to form various configurations ranging from incompletely organized, flower bud-like to complicated, arboraceous ones. In the non-treated hypophysectomized animals, however, the microvilli in such groups decrease in number and shorten in length but increase in width, looking like a blade of a prickly-pear cactus or an irregular-shaped plate. On the other hand, presumptive exocytotic and endocytotic specializations of intra-membraneous and experimental animals.

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

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.

Ultrastructural changes in development and aging of the interrenal cell of the salamander, Hynobius nebulosus.

The fine structure of interrenal cells in the salamander, Hynobius nebulosus, from prometamorphic larvae to its adult stage, was observed with electron and light microscopes. In all the animals examined the interrenal cell clusters were located at the medial edge of the ventral surface along the total length of each kidney, suggestive of a primitive nature. In larval salamanders, the interrenal cells contained small to moderate numbers of lipid droplets, and their cytoplasm was filled with a tubular network of smooth endoplasmic reticulum (SER) and numerous mitochondria with tubulo-vesicular cristae. In the adult and young adult salamanders, however, most interrenal cells were filled with lipid droplets, so that both tubular SER and mitochondria were decreased in amount in inverse proportion to the increased lipid droplets. These mitochondria frequently contained a crystalloid structure composed of closely packed tubules which were continuous with the cristae. These findings suggest that the interrenal cells in the larvae are more active than those in the adult or young adult salamanders. In addition, the interrenal cells at the end of metamorphosis contained a greater number of enlarged mitochondria with loosely distributed tubular cristae and with a less dense matrix and more numerous membrane-bounded dense bodies, 0.1-0.3 micron in diameter, than those at the prometamorphic stage. These findings suggest that the cells are their most active at this stage. In the prometamorphic larvae, bundles of filaments frequently occurred in the cytoplasm, especially around the nucleus. These filaments gradually decreased in number with the advance of age, and in young adult salamanders they appeared only occasionally.

Effects of short-term treatment with calcium on the parathyroid gland of the rat, under particular consideration of the alteration of storage granules.

Short-term effects of CaCl2-treatment on parathyroid cells of the rat, especially on their storage granules, were studied at the ultrastructural level. After an injection of 4% CaCl2, serum calcium levels (SCL) rapidly increased from 9.1 mg/dl (controls) to a maximum of 14.9 mg/dl at 20 min. At 5 min after the injection, the number of type-I storage granules (large core) [NSG-I] and that of type-II storage granules (small core) [NSG-II] remained unchanged, in spite of elevated SCL (12.4 mg/dl). As soon as SCL rose to 13.2 mg/dl at 7.5 min, NSG-I gradually decreased to a minimum at 30 min; in contrast, NSG-II gradually increased to a maximum at 30 min. Vacuolar bodies also increased together with the augmentation of type-II storage granules. The average diameter of the core of the storage granules decreased significantly after the injection. Protein A-gold method for immunocytochemistry showed that the cores of these granules contain parathormone. Acid-phosphatase activity was occasionally found in storage granules of both types, especially in those of type II. It is concluded that type-I storage granules may be transformed into vacuolar bodies via type-II granules as a result of hydrolysis, and that these processes may be accelerated during hypercalcemia.

Effects of pilocarpine treatment and of electrical stimulation of the vagus nerve on the rat parathyroid gland, with special reference to the alteration of storage granules.

Effects of pilocarpine treatment and of electrical vagal stimulation on the rat parathyroid were studied ultrastructurally. The number of type I storage granules with a narrow halo (NSG-I) and that of type II storage granules having a wide halo (NSG-II) were calculated. After pilocarpine treatment, NSG-I gradually decreased and reached a minimum at 30 min; in contrast, NSG-II gradually increased and reached a maximum at 20 min, but thereafter it slightly decreased and instead vacuolar bodies increased. Excluding these alterations, the ultrastructure of parenchymal cells showed no remarkable changes. Electrical vagal stimulation furthermore confirmed these results. Acid phosphatase activity was occasionally found in storage granules of both types in control and experimental rats. It was concluded that storage granules normally may be transformed from type I into type II and finally into vacuolar bodies as a result of hydrolysis, and that these processes may be accelerated by parasympathetic stimulation.

Endothelial tubuloreticular structures in intracranial germinomas.

In studying three human intracranial germinomas tubuloreticular structures were observed within the cisterns of granular endoplasmic reticulum (RER), as well as occasionally within dilated perinuclear spaces of capillary endothelial cells. These tubuloreticular structures seen as a network of branching, convoluted, tubular profiles appear to originate from amorphous material. The development of these structures could be classified into three stages. In Stage I, the precursor substance appears as dense amorphous material within the cisterns of RER. Stage II is marked by the transformation of the amorphous material to coarse particulate material which aggregates to form tubular units. During the first and second stages, the distended RER that participates in the formation of these structures is accompanied by numerous attached ribosomes and is closely associated with mitochondria. In Stage III, the tubular units fuse with one another to form the tubuloreticular structure. In this third stage both ribosomes and mitochondria are almost absent. As a result of the almost complete disappearance of these organelles at this time, both the attached ribosomes and mitochondria may play an important role in the synthesis of the precursor substance as well as in its transformation to the tubuloreticular structure.

Effects of epinephrine treatment on the rat parathyroid gland, with special reference to the frequency of storage granules.

Effects of epinephrine treatment on the rat parathyroid gland were studied morphologically. The mean number of storage granules per cell section (NSG) was rapidly decreased as early as 5 min after an injection of epinephrine and seemed to reach a minimum between 5 and 30 min. During this period, serum calcium levels (SCL) gradually rose and reached a maximum at 30 min. The ultrastructure of chief cells in these epinephrine-injected rats showed no marked difference as compared with that in control rats. In slightly hypocalcemic rats, induced previously by 2% EDTA-treatment, NSG was more rapidly decreased. It was suggested that storage granules may be released promptly by epinephrine treatments in spite of high SCL and that they are more promptly released under hypocalcemia.

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

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.

Intercellular junctions of the hen parathyroid gland. A freeze-fracture study.

The fine structure of the intercellular junctions of the hen parathyroid gland was studied using freeze-fracture replicas and thin sections. In the conventional thin sections, desmosomes, intermediate junctions (maculae adherentes) and gap junctions were observed, and in the lanthanum-fixed sections, tight junctions (maculae occludentes) were demonstrated as well. In the freeze-fracture replicas, desmosomes, gap junctions, tight junctions and combination forms of gap and tight junctions occurred, but intermediate junctions were not identified. Junctional complexes (zonulae occludentes) were not encountered in any preparations. The gap junctions varied in size and shape; they ranged from irregularly shaped, minute assemblages of particles to large aggregations of a round or elliptic outline. Both the tight junctions and the combination forms of gap and tight junctions also exhibited a variety of shape and dimension, and, depending on the form of the tight junctional strands, they were classified into three types: type I consisted of a simple line of strands; type II consisted of a closed network of strands; and type III consisted of an open network of strands. The combination forms were more numerous than the typical tight junctions. The possible significance of these junctions is discussed in relation to the function of the parathyroid parenchymal cell.

Freeze-fracture replica studies of the human adrenal cortex, with special references to microvillous projections.

Freeze-fracture replicas of human adrenocortical cells revealed en face fractures (both P and E faces) of the cell surface to be classified into three patterns: (1) the fracture face with no microvillous projections; (2) the fracture face mainly with short microvillous projections which arise horizontally from the edges of the small cavity on the cell surface to form a flower bud-like configuration on the P face; (3) the fracture face mainly with varying-sized, longer microvillous projections which arise almost vertically from the cell surface and show no specific organization. The former two fracture faces were supposed to correspond to the plasma membranes facing the interstitium including pericapillary spaces, since they had no gap junctions and frequently appeared along sinusoidal capillaries. On the other hand, the latter fracture face seemed to be equivalent to the plasma membranes facing intercellular spaces, since it frequently showed gap junctional specialization of intramembranous particles and usually both the P and E fracture faces of plasma membrane on the same surface. En face fractures of these gap junctions were various in shape and size. Tight junctions and their complex form with gap junctions were not encountered. Cell organelles and the nucleus had more numerous intramembranous particles on the P fracture face than on the E fracture face.

Electron microscopic studies on localization of phosphatases in the laying hen parathyroid.

Localization of phosphatases in the parathyroid of laying hens was examined by electron microscopy. Activities of both alkaline phosphatase and adenosine triphosphatase were intensive on the apposed plasma membranes between contiguous chief cells, but weak or almost lacking on those facing the interstitial connective tissue, and this finding differed from previous data in mammals. This difference seemed to be associated with the fact that in the parenchymal cells of the hens there was found a narrow, delicate filament-rich zone in the peripheral cytoplasm along the basal lamina. Activities of both thiamine pyrophosphatase and inosine diphosphatase were seen in most of the Golgi cisternae having serpentine tubular profiles, and this indicated that the latter cisterna belong to the Golgi apparatus. Acid phosphatase activities were mainly demonstrated in lysosomal dense bodies, including autophagic vacuoles, as well as in most of the lipofuscin granules, and only occasionally encountered in the Golgi apparatus, including the thick membranous cisternae, in contrast with findings in mammals. The reason for this weak activity in this organelle was discussed in relation to calcium metabolism, secretory products, and lysosomes in the laying hen.

Electron-microscopic studies on the relationship between the frequency of parathyroid storage granules and serum calcium levels in the rat.

In the rat parathyroid, the mean number of storage granules (NSG) per chief cell has been electron-microscopically studied and correlated with the mean serum calcium level (SCL). In animals given 4% CaCl2 plus vitamin D2 for 3 days. SCL is significantly elevated and NSG is increased. When these animals are injected with 2% EDTA, SCl is lowered to 8 mg/dl, but NSG is not affected; in those injected with 4% EDTA, however, SCL declines to a minimum (5.8 mg/dl) after 30 min, and NSG is also decreased. Control SCL are 8.9 mg/dl. These results indicate that storage granules may not be released until SCL is depressed to a certain level. In rats 3 weeks after castration, the chief cells show hyperplastic changes and SCl is at a low concentration (8.0 mg/dl). NSG, however, remains almost within control limits. Castrated animals injected with 4% EDTA show a hypocalcemia and a decrease in NSG, but NSG gradually recovers over a period of 6 h. These data suggest that storage granules can be produced even under lower calcium concentrations. It is concluded that storage granules may be constantly produced and stored, and are released only as an emergency supply of hormone.