Intranuclear inclusions in pericytes of the hypothalamus of the rat

Summary This paper deals with the ultrastructure of two types of intranuclear inclusions, nuclear bodies and membranous lamellar bodies, present in hypothalamic pericytes of intact adult rats. The nuclear bodies exhibited simple and granular forms, whereas the membranous lamellar bodies were entirely made up of myelin-like membrane whorls.The occurrence of these bodies in nuclei of pericytes has never been previously reported. The origin and functional meaning of such structures is discussed in the light of recent ultrastructural and biochemical studies on nuclear inclusions.     

Thyroidectomy cells and their response to thyrotrophin releasing hormone (TRH) in the rat

Summary Thyroidectomy cells of the rat pituitary gland were studied by the peroxidase-antibody labeling procedure and by electron microscopy. Secretory granules accumulated in these cells in response to a short-term treatment with thyroxine, and the cells were then reactive to the peroxidase-antibody labeling procedure. An intravenous injection of synthetic thyrotrophin releasing hormone (TRH) to thyroxine-treated, thyroidectomized rats provoked an acute and active extrusion of secretory granules from the thyroidectomy cells. The secretory granules in these cells were mostly haloed after primary fixation in osmium tetroxide. It is concluded that TRH causes thyroidectomy cells to release their secretory granules, and presumably TSH, by the usual process of exocytosis or granule extrusion.This study was supported by USPHS Grant AM 12583.     

Histochemistry of glycogen in the inner ear

Synopsis The effect of fixation and processing upon the morphological appearance of glycogen within the outer hair cells of the guinea-pig was investigated using two methods. In each method, tissue was fixed for 12 h in cold phosphate-buffered 4% paraformaldehyde and eventually dehydrated in ethanol, embedded in Epon 812, and cut into 4 m sections. In procedure A, after complete processing, the sections were tained using the periodic acid-Schiff reaction (PAS) or the periodic acid-thiocarbo-hydrazide-osmium tetroxide (PATCO) reaction which resulted in the appearance of listinct, coarse granules in the cytoplasm of the outer hair cells. Diastase digestion on one of the two matched sections after Epon removal and prior to staining, confirmed the granules to be glycogen. In procedure B, after primary fixation, the tissue was post-fixed in 1% osmium tetroxide and then processed exactly as in procedure A. Here, unless the Epon and osmium was remoyed, there was no staining of the outer hair cell cytoplasm. However, after Epon removal there was diffuse, grainy appearance of the outer hair cell cytoplasm which we considered to be due to glycogen although diastase confirmation was not possible. We have concluded that osmium tetroxide (1) inhibits PAS or PATCO staining, (2) prevents diastase digestion, and (3) prevents the appearance by light microscopy of distinct granules of glycogen.     

Electron microscopic observations of the influence of different fixatives on the appearance of cellular ultrastructure

Summary The cells of the proximal convoluted tubules of the rat kidney, mouse hepatic parenchymal cells, and the juxtaglomerular cells (J. G. cells) of the afferent arteriole in rabbit kidney served as test tissues for a study of the modifications of structure produced by 14 different fixation procedures using osmium tetroxide, a chrome-osmium solution, glutaraldehyde, and formaldehyde as fixatives. The fixative solutions containing various buffers were used either alone (osmium tetroxide-containing compounds) or in various combinations (aldehyde fixation with postfixation in osmium tetroxide). The most conspicuous differences in appearance following different fixation procedures were noted in the cytosomes of renal proximal tubular cells and the granules in the J. G. cells. However, virtually all cytoplasmic organelles showed some modification of structure with different fixatives. The evidence indicated that the chemical composition of the buffer used with OsO₄ was an important factor which particularly affected the density of the cytosomal matrix in renal proximal tubular and J. G. cells. The density of the matrix of the cytosomes was, however, also influenced by the fixative itself as indicated by studies of aldehyde-fixed tissues. With these fixatives the ultrastructural appearance was not modified by the buffer. Clumping of nuclear chromatin along the nuclear membrane and around the nucleolus occurred following fixation in aldehyde and Dalton's chrome-osmium solution, whereas the chromatin was evenly distributed in the nucleus when buffered osmium tetroxide was utilized for fixation. A specific reaction of dichromate with the granule of the J. G. cells seemed to take place rendering these granules electron opaque, thereby facilitating their identification in the light and electron microscope. The findings were discussed with particular attention to the interaction of fixatives and buffers with various tissue components.Supported by grants from the Board of Swedish Life Insurance Companies, the Stiftelsen Therese och Johan Anderssons Minne, and grant No. AM-7919 from the National Institutes of Health, Bethesda, Maryland (USA).     

Ultrastructure of the oocyte and embryo of the calcified sponge,Petrobiona massiliana (Porifera,Calcarea)

Summary In the spongePetrobiona massiliana, a Calcarea related to pharetronid fossils, the oocyte and the embryo both receive an unusual amount of maternal nurse cells. Yolk granules are large and display a lamellar structure throughout the entire growth period, which allows them to be used as markers of the oocyte reserves. The cruciform cells (cellules en croix) of the embryo appear to degenerate at an early stage. These features are compared to those found in other Calcarea.     

Fine structure of the osteocyte capsule and of the wall of the lacunae in bone

Summary Bone tissue from femura of adult and old rats and from young mice, as well as from dogs, were fixed in osmium tetroxide, potassium permanganate, in an osmium tetroxide — potassium permanganate combination, and in glutaraldehyde followed by osmium tetroxyde — potassium permanganate. The results of the different fixatives were found to complement one another in such a way that existing controversies and uncertainties concerning the fine structure could be settled. This was especially true of the question whether or not the so-called capsule of the osteocyte contains collagen fibrils. Notwithstanding considerable variations in the structure of the capsule it was definitely shown that cross-banded fibrils are present in a mucopolysaccharide-containing ground substance. The material of the capsule corresponds, therefore, to the matrix of connective tissue in general, and its ground substance is, as in any connective tissue, the medium of transport between the blood and the tissue. In respect to the organic structures the intralacunar matrix is similar to the interlacunar mineralized matrix. In sections of demineralized bone, especially after osmium tetroxide fixation, the wall of the lacuna and canaliculi is marked by a dark line which is described as a special osmiophilic lamina. Since the same line, although thinner and less distinct, was found also in tissue fixed with agents other than osmic acid it was concluded that the osmiophilic lamina is a true structure which must be permeated by substances passing to and from the interlacunar matrix. The osmiophilic lamina belongs to a wider border zone which differs from the bulk of the mineralized matrix by its thinner and less tightly packed fibrils. Accordingly, the bone crystals were found to be less orderly arranged than those deeper inside the mineralized matrix. Bordering directly on the intralacunar pathway they were described as the coastal crystals and are believed to represent the labile bone minerals which are metabolically available without any change in the bone structure. The findings about the fine structure of the capsule of the osteocyte and of the wall of the lacunae were discussed in terms of the transport problems in bone. The osteocyte itself, by its fine structure and relationship to the intralacunar matrix seems to be engaged not only in the maintenance of the open pathways in bone but also in the transport mechanism itself.This investigation was carried out under the auspices of the United States Atomic Energy Commission and was supported, in part, by research career program award 5-K3-DE 7, 272 and research grants De-01406 and DE-01716 from the National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland.     

Elektronenmikroskopische Untersuchung mit Osmiumtetroxid fixierter globulÄrer Eiweisse

Summary Myoglobin, albumin, haemoglobin and -globulin were fixed with osmium tetroxide and studied in the electron microscope. The molecules are represented on the electron micrographs as electron-dense, roughly circular dots. Occasionally aggregations of molecules can be observed; in some cases a separation of the molecules into subunits occurs. It is interesting to note that, according to these results, albumin and -globulin have roughly spherical shapes. Though the possibility of artefacts must be considered the electron microscopical investigation of proteins fixed with osmium tetroxide contributes to the knowledge of their structure, and also helps in the interpretation of electron micrographs of cellular structures.     

The ultrastructure of the herring bodies in rats subjected to different experimental conditions

Summary A study of the morphology of Herring bodies of the posterior pituitary lobe of rats treated with colchicine and/or exposed to low temperatures has been performed. After treatment with colchicine (20 g in distilled water injected intracisternally) a predominance of Herring bodies with a large number of small synaptic-like vesicles surrounded by neurosecretory granules is found. Exposures to low temperature (4–6° C) result in an increase in the neurosecretory material and the Herring bodies show many neurosecretory granules of different densities. After treatment with colchicine and subsequent exposure to low temperatures, the Herring bodies are characterized by having a great number of autophagic bodies which become more numerous as the length of the exposure is increased; later autophagic vacuoles and lamellar bodies become evident.     

An electron microscopical study of the human neurohypophysis

Summary The human posterior pituitary obtained at operation was studied with the electron microscope.The fine structure of the human neurohypophysis was found to be basically similar to that of the other mammalian and cold-blooded vertebrates examined.In addition to the nerve fibres and their swellings two cell types were seen: the pituicytes and the cells in the perivascular spaces — the fibroblasts.The distribution, size and shape of the pituicytes was variable. Long pituicyte processes were seen (sometimes > 20 in length) running between nerve fibres and their swellings, and ending on or in the perivascular space.Several types of large nerve swellings were seen: a) those containing only elementary granules, mitochondria and small vesicles; b) modified swellings characterised by contorted mitochondria and mitochondrial fragments. The latter were often surrounded by single or multiple highly osmiophilic myelin-like membranes; c) multilamellar bodies characterised by concentric osmiophilic myelin-like membranes or lamellae apparently dividing the swellings into separate compartments. The small vesicles, appearing in considerable numbers in such swellings, are suggested to originate from the osmiophilic lamellae.While some true synaptic vesicles may occur in non-secretory nerve fibres, the small vesicles (250–600 Å) seen in most nerve fibre swellings were thought to be derived from elementary granules.Repeated swellings were seen in individual nerve fibres and their functional significance discussed.The electron-dense material within the elementary granules was variable and is suggested to be dependent on the functional state of the neurohypophysis rather than on hormone content. Internal structure was often seen in the elementary granules. The impression is gained that the osmiophilic content of the elementary granules was composed of microvesicles possibly representing molecular aggregations of the hormone-neurophysin complex.     

The effect of anisotonic media upon cellular ultra-structure in fresh and fixed rat brain

Summary When tissue slices or small blocks of unfixed rat cerebrum are incubated in various anisotonic physiological media, distinctive morphological changes are induced in glial cells, neurons, and endothelial cells. The variation in observed cellular swelling and shrinkage may be related to differences in ionic content of the cytoplasm of these cells. When HAA, glutal, and osmium tetroxide fixed tissue is incubated in this manner, only the cerebrum fixed in HAA responds to osmotic inequilibrium in a manner similar to unfixed tissue. Although HAA does not fix tissues very well, the permeability of plasma membranes in the brain appears to be less altered by HAA than by glutal or osmium tetroxide. The relationship of these findings to a demonstration of the extracellular space in HAA fixed tissues is discussed.This work was supported by grants (U-1293) from the Health Research Council of the City of New York and (NB 04161-02) from the National Institute of Neurological Disease and Blindness of the National Institutes of Health.     

The specific cytochemical demonstration in the electron microscope of periodate-reactive mucosubstances and polysaccharides containing vic-glycol groups

Summary A technique is described for the localization in the electron microscope of periodate-reactive mucosubstances and polysaccharides containing vic-glycol groups. In this technique the sugar residues are oxidized by periodic acid and the resulting aldehydes condensed with pentafluorophenylhydrazine under specified conditions. Further increase in specific electron density is achieved by treating the hydrazone end-product with ammonium sulphide followed by osmium tetroxide to form an osmium black.The technique has been applied to liver and small intestine cells in which glycogen, sialomucins and sulphated mucosubstances reacted especially strongly. A marked positive reaction has also been obtained from the interstitial cell matrix and from the Golgi apparatus and multivesicular bodies of the intestinal epithelial cells.The reaction can be prevented by the omission of the periodate oxidation and, if due to glycogen, by prior treatment with diastase.     

Ultrastructural evidence for a secretory function in the “gland cells” of the marine red algaBotryocladia pseudodichotoma (Rhodymeniaceae)

Summary Clusters of small, pyriform gland or vesiculate cells protrude from the inner cortex into the inflated bladders ofBotryocladia pseudodichotoma. These multinucleate cells contains a large, central vacuole surrounded by dense cytoplasm. The lobed chloroplasts lack phycobilisomes and contain concentric rather than parallel thylakoids. Irregularly shaped, osmiophilic bodies of undetermined composition are abundant in the cytoplasm and are also released into the vacuole. The cell is characterized by abundant Golgi apparatus. The Golgi bodies appear to form by coalescence of endoplasmic reticulum derived vesicles and tubules. The Golgi apparatus produces large, electron transparent vesicles that contain an osmiophilic, fibrous material. This material is secreted into a diffuse wall layer which appears to be continually sloughed. Thus, the vesiculate cells ofBotryocladia possess an ultrastructure characteristic of granulocrine secretory cells, and it is suggested that they produce the slime contained within the bladders of this genus.     

Granule formation in rat myeloid cells

Summary Rat bone marrow was fixed in glutaraldehyde, postfixed in osmium tetroxide, and processed for electron microscopy. The myeloid cells were arranged in order of maturation according to their successive compartments.On the basis of their differences in form, substructure, volume, and density five morphologically distinct types of developing granules are to be observed in neutrophil, two in eosinophil, and four in basophil, cells. Primordial granules appear in the interphase of the myeloblast, respectively in the early promyelocytes. The first granules in the neutrophils are pale, of homogeneous structure. These granules grow gradually denser with increasing condensation. In the myelocyte stage polymorphism is more pronounced. In the granulocytes, vacuoles and dense-cored vacuoles indicate the sites of granules. In the eosinophil line, the basophilic bodies decrease in number during differentiation. The eosinophil granules show fewer variations in the course of maturation than the neutrophils. The immature forms of the basophil granules are relatively large, pale, and of globular structure; they undergo condensation and show gradually higher density.Sites of granulogenesis in the rat are first of all the Golgi apparatus and, possibly, the cisternae in the endoplasmic reticulum. On occasion, bodies in a transitional stage between a mitochondrium and a granule can be observed, but whether they may have a bearing on the problem of granulogenesis is an open question.     

Segmental variations in the organization of the endoplasmic reticulum of the rat nephron

Summary The spatial organization of endoplasmic reticulum (ER) was examined in all segments of rat nephron. Tissues were fixed with glutaraldehyde, impregnated en bloc with osmium tetroxide, prepared for and examined by standard (80–100 kV) and high voltage (1 mEV) transmission electron microscopy.In all proximal tubule cells, ER forms a continuous and extensive network of canaliculi and abundant fenestrated saccules which surround mitochondria and cytoplasmic bodies; the cage-like structure of the fenestrated saccules was most evident around the spherical mitochondria of the S₃ segment. In the cells of the distal straight and convoluted tubules, the network consists mostly of canaliculi with rare non-fenestrated saccules. The ER network of canaliculi is particularly rich in intercalated cells, in contrast with its rudimentary appearance in the adjacent principal cells of the collecting tubule. In fact, in these cells there are few isolated ER cisternae and they are rarely impregnated. The nuclear envelope is well impregnated in most cells throughout the various segments. Segmental variations in ER organization and its relative abundance are most likely related to the well, established functional heterogeneity of the nephron segments. Moreover, the extensive and unique organization among mitochondria, ER and the basolateral membrane suggests that these three organelles function as a unit which is related to active electrolyte transport. In addition, because of its transepithelial organization, ER may well constitute a transcellular pathway for molecules.     

Three-dimensional architecture of the tubular endocytic apparatus and paramembranous networks of the endoplasmic reticulum in the rat visceral yolk-sac endoderm

The three-dimensional architecture of the tubular endocytic apparatus and the endoplasmic reticulum in the rat yolk-sac endoderm was investigated after loading with horseradish peroxidase-conjugated concanavalin A by intrauterine administration. After 30 min, small vesicles (50–150 nm in diameter), small tubules (80–100 nm in diameter) and large vacuoles (0.2–1.0 m in diameter) in the apical cytoplasm were labeled with the tracer, but lysosomes (1.0–3.5 m in diameter) in the supranuclear cytoplasm were not labeled until 60 min after loading. Stereo-viewing of the labeled small tubules in thick sections revealed that they were not isolated structures but formed three-dimensional anastomosing networks, which were also confirmed by scanning electron microscopy after maceration with diluted osmium tetroxide. Their earlier labeling with the endocytic tracer, localization in the apical cytoplasm and three-dimensional network formation indicated that the labeled small tubules represented tubular endosomes (tubular endocytic apparatus). These well-developed membranous networks provided by the tubular endosomes are suggested to facilitate the receptor-mediated endocytosis and transcytosis of the maternal immunoglobulin in the rat yolk-sac endoderm. Scanning electron microscopy further revealed lace-like networks of the smooth endoplasmic reticulum near the lateral plasma membrane. Their possible involvement in transport of small molecules or electrolytes is discussed.     

The ultrastructure of the corpus luteum of the guinea-pig

Summary An electron-microscopic investigation, based on the suggestion that differences seen in progesterone levels under differing hormonal conditions might be reflected in the ultrastructural organisation of the lutein cells of the guinea-pig was undertaken. Comparisons were made between corpora lutea taken from animals during the normal oestrous cycle, pregnancy and lactation, and after hysterectomy or hypophysectomy.The lutein cells from the oestrous cycle corpus luteum appeared to be of two types, light and dark. The former were more numerous. The main difference between them lay in the arrangement of the endoplasmic reticulum. Lutein cells from corpora lutea (with the exception of the old degenerating corpora lutea) all contained well-developed agranular endoplasmic reticulum, little granular endoplasmic reticulum, several electron-dense lipid granules, lysosomal bodies which ranged from small spherical bodies to large autophagic vesicles and mitochondria. The mitochondria were numerous, and in the corpus luteum of pregnancy, they were closely associated with the parallel arrays of granular endoplasmic reticulum.With minor exceptions, the lutein cells of the guinea-pig present a strikingly uniform picture despite their hormonal condition.The manner in which this uniformity of ultrastructure may be related to observed differences in progesterone levels in the corpus luteum of the guinea-pig is discussed.Meat and Livestock Commission (MLC) Scholar.The authors wish to thank Dr. J. S. Perry for doing the surgery involved in this work and for the specimens of corpora lutea of hysterectomy. They are also grateful to him for his helpful discussions and interest throughout.     

Remarks on the detection of osmium derivatives in tissue sections

Summary The reaction of substances with osmium tetroxide in tissue sections is evident when primary or secondary blackening occurs. Many substances are not colored, however, despite the fact that they may react with osmium tetroxide. Osmium derivatives bound in such structures may be either in an insufficient amount or in a higher valency state or both. For the demonstration of such weakly colored bound osmium derivatives some chemical reagents including those used in analytical chemistry were tested. Not all reagents used were equally effective. Tschugajev's reagent with thiourea is unreliable for in situ detection in tissue sections because the resulting colored complex is soluble and escapes from the section into solution. Yellow ammonium sulphide is not sensitive enough. N-phenyl-p-phenylene diamine, N-dimethyl-p-phenylene diamine, benzidine, o-dianisidine, N-diethyl-p-phenylene diamine sulphate, p-aminophenol, p-phenylene diamine, N-methyl-p-aminophenol sulphate, 2,4-diaminophenol hydrochloride, -naphthylamine, -naphthylamine, diphenylcarbazone, 8-amino-1-naphthol-3,6-disulphonic acid (H acid), aniline, tannin, proved to be the most suitable detecting reagents. By these reagents large quantities of osmium pigments could be demonstrated in many structures even after the most efficient lipid extraction methods.     

Development of the resting sporangia ofSynchytrium endobioticum,the causal agent of potato wart disease

Summary An ultrastructural study of the development of the resting sporangium ofSynchytrium endobioticum (Schilb.) Perc. infecting potato cells is presented. The resting sporangium is found to have a single large, centrally placed nucleus with a prominent nucleolus through its entirein situ development. The cytoplasmic organization of the resting sporangium is further characterized by numerous membrane-bound lipid bodies and osmiophilic bodies. The latter have a characteristic sieve-like appearance, probably because certain storage components have been extracted during preparation for electron microscopy. Because of the similar location and appearance of these osmiophilic bodies it is suggested that they are identical to what has earlier (based on light microscopy) been described as chromatin granules; and the ultrastructural studies presented here show that nucleolar discharge which was described from light microscopic observations as leading to chromatin granules in the cytoplasm, and finally forming the nuclei of the zoospores (bally 1912,curtis 1921,percival 1910) simply does not occur.The appearance of dense fibrillar-like structures on the sporangial surface at an early stage of resting sporangium development ultrastructurally distinguishes the resting sporangium from the zoosporangium. The development of the layered portion of the thick sporangial wall is shown to be due to the fusion of vacuoles containing pre-made wall fibrils with the cell membrane. It is suggested that the inner compact wall layer which is essentially substructureless is formed by the membrane itself.The characteristic wings of the matureS. endobioticum resting sporangium originate from the potato host cell wall. Remnants of host cell organelles in the outermost layer of the resting sporangium wall show that degradation of the host cell cytoplasm contributes to wall formation of the parasite.     

Ultrastructure of the pituitary gland (pars distalis) in sockeye salmon (Oncorhynchus nerka) during gonad maturation

Summary The fine structure of the various hormone-producing cell types (with the exclusion of the prolactin cells) in the pituitary gland (pars distalis) of migratory sockeye salmon is described. All fish were in an advanced stage of sexual maturation. In the proximal pars distalis five cell types were distinguished: growth hormone cells, ACTH cells, gonadotrops, vesicular cells, and chromophobe cells. Gonadotrops were also found throughout the rostral pars distalis. A conspicuous feature of the gonadotrops was the presence of two kinds of secretory inclusions: small electron-dense granules (200–375 m) and large, relatively electron-translucent globules (400–2 000 m). The large vesicular cells, so called because of their conspicuous vesicular endoplasmic reticulum, were numerous and often appeared to contain some small granules. It is argued that they may represent a second type of gonadotropic cell, which, in earlier stages of gonad development, contains many granules but becomes largely degranulated near the time of reproduction when the other gonadotrops (globular gonadotrops) abound. The chromophobes, which were smaller and far less abundant than the vesicular cells, also appeared to contain small granules (120–280 m). They are probably thyrotrops.The assistance of Mr. S. Killick, of the International Pacific Salmon Fisheries Commission, who helped in the collection of salmon, is gratefully acknowledged.     

Ultrastructural studies on the hypothalamic neurosecretory neurones of the rat

Summary The ultrastructural features of the paraventricular neurones of the non-treated rat are presented comparing them with those of the supraoptic neurones. No striking differences are seen between the general electron microscopic characteristics of the paraventricular and supraoptic neurones.The importance of adequate fixation to obtain good preservation of the neurones is emphasized, since inadequate fixation can cause e.g. artefactual appearance of dark neurones. The previously presented classification of the neurosecretory neurones into two categories (e.g. light and dark neurones) on the basis of the number of ribosomes is not considered justifiable, since their number can vary to a very great extent even within a single cell.The synthesis of neurosecretory products in the paraventricular neurones obviously follows the general mode of the synthesis of secretory proteins: ribosomes—RER—Golgi complex—secretory vesicles.On the basis of the localization of heavy metal deposits after osmium impregnation and demonstration of acid phosphatase the Golgi complex of the paraventricular neurones is found to be polarized. The direction of the polarity is discussed.The substructures of the dense cores of the neurosecretory granules and of the contents of the lysosomal dense bodies are nearly identical. Therefore it is considered impossible to determine positively the nature of the dark condensed material within the Golgi complex. The characteristics of the immature neurosecretory granules and the possibility of releasing neurosecretory products into the cytoplasm already within the perikarya are speculated.This study was supported by a grant from the Emil Aaltonen Foundation, Tampere. I express my best thanks to Docent Antti U. Arstila, Head of the Laboratory of Electron Microscopy, and Professor Urpo K. Rinne, Head of the Department of Neurology, for the guidance of this work.