Cortical granules remaining after fertilization in Xenopus laevis

Eggs of Xenopus laevis were examined in an electron microscope at unfertilized egg, 1-cell, 2-cell, 32-cell, and blastula stages. Granules closely resembling cortical granules were observed within the “germinal plasm” as well as in the peripheral cytoplasm of all the eggs examined. A staining method was developed that makes it easier to count cortical granules in thick Epon sections. Light and electron microscope examinations revealed that granules remaining after fertilization possessed morphological characteristics wholly consistent with those of cortical granules of unfertilized eggs. These granules were confirmed to be true cortical granules.     

THE INFLUENCE OF ENVIRONMENT ON THE SHELL STRUCTURE OF STARCH GRANULES

It is well known from light microscope studies of potato starch that the granules formed in a constant environment (of light and temperature) have a ring formation indistinguishable from that of granules formed under field conditions. Electron microscope studies have confirmed that normal potato starch granules have a fine shell structure not usually resolved by the light microscope, and also that shells do not develop in barley granules grown in a constant environment. The paper presented here reports a further study of the dependence of shell formation on environment. Potatoes were grown in a constant environment and starch granules from the newly formed tubers were examined in the light microscope, and in the electron microscope after corrosion by acid. No difference between these granules and normal granules was observed; both wide (light microscope) rings and fine lamellae developed in both granules. Parallel studies were made on wheat starch granules. In this case, shells were not differentiated in granules that developed in a constant environment, but they could be produced at will by imposing a dark period. Thus, shell formation in potato granules must be controlled by an endogenous rhythm, whereas in wheat granules it must be controlled by external environment.     

IDENTIFICATION OF GLYCOGEN IN ELECTRON MICROGRAPHS OF THIN TISSUE SECTIONS

The electron microscopic appearance of glycogen has been studied in the organs of several animal species. Glycogen almost always appears as roughly circular granules from 150 to 400 A in diameter. The intrinsic electron density of glycogen varies from tissue to tissue; however, treatment with lead hydroxide as described by Watson deeply stains the granules. Glycogen pellets were isolated from some of the tissues studied by centrifugation. Such pellets were shown to be glycogen by chemical and histochemical criteria. When thin sections of the pellet are examined under the electron microscope they can be seen to consist of densely packed granules similar to those found in the intact tissues. Such pellets are also stained for electron microscopy by short exposure to lead hydroxide.     

CORTICAL GRANULES PERSISTING IN GERM CELLS OF XENOPUS LAEVIS AFTER THE CLEAVAGE STAGES

Cortical granules were demonstrated, in two successive Epon sections (0.7 μm thick) stained with PAS reagent and the triple staining method respectively, to persist beyond the cleavage stages of development to the tadpole stages in Xenopus laevis. They were also examined by electron microscope. The granules which are similar both cytochemically and ultrastructurally to the cortical granules of the unfertilized eggs were observed not only in germ cells, pPGCs and PGCs, but also in somatic cells at all the stages examined. An ultrastructural similarity between the granules found in the PGCs at the tadpole stages and chromatoid body was discussed.     

Mitochondrial granules of chondrocytes in cryosections of growth cartilage.

Fresh frozen specimens of growth cartilage have been dry-sectioned and examined in the electron microscope without any staining. Dense granules in the mitochondria of chondrocytes were observed near calcifying areas. This confirms the presence of these mineral granules in the original tissue because this technique avoids the preparative artefacts of conventional electron microscopy.     

Structure and secretion of the carapace in some living ostracodes

The carapaces of some living freshwater and brackish-water ostracode species have been studied by means of light microscope, scanning electron microscope. transmission electron microscope, electron probe analyzer and X-ray diffractometer, with emphasis placed on the 'pigment' granules. The granules were found to consist of calcite and apatitic calcium orthophosphate. They are located within the epidermis, as a layer which is attached to the internal side of the calcite layer. It is suggested that the main function of the granules is the construction of the new calcareous valve in the moulting process. The layer of granules probably recrystallizes to form the calcite crystals of the valves.     

Electron microscope tomography of Balbiani Ring hnRNP substructure

Three-dimensional (3-D) reconstructions, by electron microscope tomography, of selectively stained, contrast enhanced Balbiani Ring (BR) hnRNP granules reveal a complex spatial arrangement of RNA-rich domains. This particulate substructure was examined by volume rendering computer graphics. Modeling the arrangement of RNA-rich domains is made difficult by apparent structural flexibility and/or heterogeneity of composition. Formulation of a consensus 3-D arrangement of RNA-rich domains will require an expanded data base of reconstructed BR granules and the development of new image manipulation and analysis techniques. This study demonstrates the potential for ultra-structural cell biology of combining several new techniques: selective nucleic acid staining, electron spectroscopic imaging to enhance contrast, electron microscope tomography and volume rendering computer graphics.Abbreviations BR Balbiani Ring - EMT electron microscope tomography - ESI electron spectroscopic imaging - hnRNP heterogeneous nuclear ribonucleoprotein - OA-B osmium ammine-B - kb kilobases by P.B. Moens     

RAPID RELEASE OF THE ZYMOGEN GRANULE PROTEIN BY OSMIUM TETROXIDE AND ITS RETENTION DURING FIXATION BY GLUTARALDEHYDE

Fixation by osmium tetroxide and glutaraldehyde of zymogen granules isolated from rat parotid and pancreas was investigated. Protein determinations showed that osmium tetroxide caused rapid release of most of the soluble protein of the granule during fixation in buffered isotonic sucrose. Such granules when examined in the electron microscope after shadow casting appeared quite flat, indicating that most of the contents had indeed been removed. Numerous damaged membranes of the granules were also observed. In contrast, zymogen granules fixed by glutaraldehyde and shadow cast essentially retained the spherical shape and the protein contents. The application of the shadow-casting technique in quantitative studies on the protein content of zymogen granules is discussed.     

Ultrastructural organization of polytene chromosomes of Chironomus thummi salivary glands]

An electronmicroscopical mapping of a number of regions of the polytene chromosomes of Ch. thummi salivary glands (3rd chromosome, right arm of the 1st chromosome, centromere regions, puffs 1-A2e, 1-A3ij, III-A5c and others) was done by the method of oriented ultrastructural sections of the unsquashed polytene chromosomes. The banding pattern on the electron micrograph was similar to the observed with the light microscope. The difference was that some doublets appeared as single cavity-containing bands with the double structure only in short regions under the electron microscope. It was also difficult to distinguish single bands in those regions where heavy adjacent bands were connected by dens, protrusions and anastomoses. These connections were most pronounced in the regions of the centromerers which had "spongy" appearance on the electron micrographs. These pictures may be connected with small interbands between heavy bands. Thin bands and some broad bands were frequently dotted. The puffs examined contained mainly RNP granules 200-400 A in diameter and RNP fibrils; BR-1 and BR-2 contained granules 500 A, RNP fibrils and smaller granules (200-400 A). BR and puffs were characterized by loop-like structures composed of granules arranged along the central DNP fibril. Only fibrils were presented in small interbands (0.05 mk), while larger interbands could include a small number of granules similar to those observed in puffs. It was found that centromere, telomeres and some heavy bands formed characteristic contacts with the nuclear membrane.     

On argyrophil reactions of endocrine cells in the human fetal pancreas

Summary The endocrine cells in the pancreas of five human fetuses with gestational ages of 18–20 weeks were examined by light and electron microscopy with special regard to argyrophil reactions. B-cells and typical A and D-cells were easily identified electron microscopically on the basis of their typical secretory granules. In the Grimelius argyrophil silver stain, a concentration of silver grains over the less electron dense peripheral mantle of the A-cell secretory granules was observed by electron microscopy. In the Hellerström and Hellman modification of the argyrophil Davenport alcoholic silver stain, silver grains were concentrated over the internal structures of the D-cell secretory granules. With this stain an accumulation of silver grains was also seen at the surface of the A-cell secretory granules. The argyrophil reaction of the A-granules was less pronounced than in the D-cells. In addition to B-cells and A- and D-cells, two other types of endocrine cell were observed by electron microscopy. These cells were argyrophil with the silver impregnation method of Grimelius. The electron microscopic findings at least partly explain the frequent overlapping between the two staining methods observed at the light microscope level.This study was supported by the Swedish Medical Research Council (Project No. 102)     

Fine structure of the granulocytes occurring in the hypothalamic-hypophyseal ventricle and neurohypophysis of the hagfish,Paramyxine atami

Strange granulated cells in the hypothalamic-hypophyseal ventricle and neurohypophysis of the hagfish,Paramyxine atami, were examined under light and electron microscope. Based on pleomorphic electron dense granules scattered in the entire cytoplasm and eccentrically shifted nucleus, the cells in question were diagnosed as the extravascular granulocytes.     

Ultrastructural Localization of Calcium in the Presumptive Ectodermal Cells in Gastrulae of the Newt, Cynops pyrrhogaster, as Revealed by Cytochemistry and X-Ray Microanalysis

The ultrastructural localization of calcium in the presumptive ectodermal cells of gastrulae of the newt, Cynops pyrrhogaster , was examined by cytochemical methods and X-ray microanalysis (XMA). The cells were fixed with solutions that contained potassium oxalate, potassium ferricyanide and potassium pyroantimonate to preserve the localization of intracellular calcium in situ and for the analysis of electron density due to calcium. Electron-dense deposits associated with the localization of calcium were observed under the electron microscope. Specificially, pigment granules, round vacuoles, endoplasmic reticulum and mitochondria as well as the extracellular matrix were observed to contain calcium. In addition, XMA clearly demonstrated the localization of calcium in all of these electron-dense organelles and yolk granules.     

Silicon-containing granules of rat liver,kidney and spleen mitochondria

Summary Isolated rat liver mitochondria containing granule aggregates (25–75 nm in diameter) and small (5–10 nm) electron opaque granules were examined by electron probe X-ray microanalysis. The granule aggregates gave an intense Si signal, while the small granules gave both Si and P signals. Isolated mitochondria of rat liver, spleen and kidney, subjected to detergent solubilization and differential centrifugation, produced two granule fractions: (1) a 10,000g fraction consisting predominantly of granule aggregates (25–75 nm) composed of smaller granules (5–10 nm in diameter), and (2) a 10,000–30,000 g fraction of non-aggregated small granules (5–10 nm). Thin sections of isolated granule aggregates gave Si X-ray signals similar to those obtained from in situ granules. In addition S, Cl, Mg, Cr and Fe X-ray signals were observed. Cr occurred only in the large kidney granules, while Fe occurred in both fractions of the spleen and kidney granules. The presence of Si in the granules was confirmed by chemical analysis of the isolated granules and in vivo radiolabeling of the granules with ³¹Si and ⁶⁸Ge. Contamination within the electron microscope was eliminated by a liquid nitrogen anticontamination device.Supported by Grant GM-08229-13-15 from the National Institutes of Health, USPHSWe are grateful to the Perkin-Elmer Corporation and to their Western Branch Manager, Mr. Michael E. Mullen and Microscopist, Mr. Minoru Saito, for use of and assistance with the Hitachi H 500 transmission electron microscope with the scanning attachment, and to the Kevex Corporation for the use of the Kevex X-ray Spectrometer. We also wish to acknowledge Mary Louise Chiappino for her technical assistance in preparing the thin sections, the final micrographs and X-ray spectra photographs and Darlene Lum for technical assistance in the laboratory     

MICROINCINERATION, ELECTRON MICROSCOPY, AND ELECTRON DIFFRACTION OF CALCIUM PHOSPHATE-LOADED MITOCHONDRIA

Isolated rat liver mitochondria were incubated in vitro under conditions supporting the massive accumulation of calcium and phosphate. Samples were embedded, thin sectioned, and examined in the electron microscope. The intramitochondrial distribution of insoluble or structure-bound mineral substances was studied by electron microscopy coupled with recently developed techniques of high resolution microincineration. As shown previously, the ion-loaded mitochondria acquire large, internal granules which have inherent electron opacity indicative of high mineral content. Study of ash patterns in preselected areas of sections directly confirmed the high mineral content of the granules, and the appearance of the residues was consistent with the copresence in the granules of some organic material. Other mitochondrial structures were almost devoid of mineral. Thin sections of unincubated control mitochondria also were incinerated. They were found to contain appreciable amounts of intrinsic mineral, seemingly associated with membranes. The normal, dense matrix granules commonly seen in unaltered mitochondria could be seen in intact sections of these control preparations, but after burning no definite correspondence of any ash to the granules could be demonstrated. The normal granules perhaps do not contain mineral. Heating experiments on ash patterns of all the preparations demonstrated the thermal stability and crystallizability of the ash. The crystallized ash of the in vitro-produced dense granules was tentatively shown by electron diffraction to be β-tricalcium phosphate (whitlockite). This, together with evidence from the literature, suggests that the original, noncrystalline mineral may be a colloidal, subcrystalline precursor of calcium-deficient hydroxyapatite. Experiments were performed on synthetic calcium phosphates for comparison. Other possible applications of the microincineration techniques are briefly discussed.     

Transmission and scanning electron microscopic study of adepidermal granules of teleosts and amphibia

Summary Granules in the adepidermal space of larvae of Salmo irideus, Hynobius tokyoensis and Rhacophorus buergeri, were observed by transmission and scanning electron microscopy. Adepidermal granules of S. irideus were smooth and spherical structures, those of H. tokyoensis were smooth and spherical, or oval, while in R. buergeri these granules appeared as single or grouped tangled strand-like or starfish-like structures under the scanning electron microscope. These adepidermal granules were spread all over the basal lamina in every animal investigated. The different sizes of adepidermal granules of S. irideus and H. tokyoensis seen under the transmission electron microscope are not the result of differently sectioned faces of granules, but the granules themselves exhibit different sizes. The probable functions of these granules are discussed.     

Polyhydroxybutyrate particles in Synechocystis sp. PCC 6803: facts and fiction

Transmission electron microscopy has been used to identify poly-3-hydroxybutyrate (PHB) granules in cyanobacteria for over 40 years. Spherical inclusions inside the cell that are electron-transparent and/or slightly electron-dense and that are found in transmission electron micrographs of cyanobacteria are generally assumed to be PHB granules. The aim of this study was to test this assumption in different strains of the cyanobacterium Synechocystis sp. PCC 6803. Inclusions that resemble PHB granules were present in strains lacking a pair of genes essential for PHB synthesis and in wild-type cells under conditions that no PHB granules could be detected by fluorescence staining of PHB. Indeed, in these cells PHB could not be demonstrated chemically by GC/MS either. Based on the results gathered, it is concluded that not all the slightly electron-dense spherical inclusions are PHB granules in Synechocystis sp. PCC 6803. This result is potentially applicable to other cyanobacteria. Alternate assignments for these inclusions are discussed.     

Identification of enterochromaffin cells in adjacent Epon-embedded sections at light and electron microscopic levels

Summary Methods for light and electron microscopic comparison of individual argentaffin and argyrophil enterochromaffin cells (EC) in the sheep duodenal mucosa are described. These silver procedures were applied for light microscopy to Epon-embedded sections. The adjacent sections were examined with the electron microscope. The most specific characteristics of the argentaffin and argyrophil EC in electron microscopy are highly osmiophilic cytoplasmic granules. In one cell type these granules are smaller and more roundish than in the another type. These two cell types are stainable both by the argentaffin and argyrophil reactions. No essential difference can be observed in the localization of these elements. It is suggested that both cell types belong to the enterochromaffin system. Both silver methods are also suitable for the light microscopic identification of other intestinal structures in sections adjacent to that sectioned for electron microscopy.This work was supported by a grant from the Yrjö Jahnsson Foundation, Helsinki, Finland.The electron microscopic observations were carried out in the Electron Microscope Laboratory, University of Helsinki.     

Mast cell response during the early phase of tuberculosis: an electron-microscopic study.

Guinea pig lungs were infected with Mycobacterium tuberculosis by intratracheal route and examined under electron microscope to investigate the morphological alterations of the organisms, if any, and the response of the host tissue. The bacilli showed no changes in their morphology, while the host tissues revealed several cells containing many electron-dense intracytoplasmic granules. These cells were predominantly seen during the 1st week of infection. The electron-dense bodies of these cells may be the ones observed by earlier workers and suggested to be the altered forms of tubercle bacilli. The present investigation, however, revealed them to be the granules of the mast cells. These cells were observed to respond to tuberculous infection during the first few days by appearing in large numbers crowded with intracytoplasmic granules and soon disintegrating as the result of subsequent degranulation. The above observation is presented and its significance discussed.     

中华绒螯蟹窦腺神经末梢及X-器官神经分泌细胞的类型

在电子显微镜下观察了性未成熟的中华绒螯蟹黄蟹的窦腺及X－器官的超微结构。X－器官位于眼柄神经节终髓的腹外侧,与窦腺位置斜相对,窦腺主要由神经分泌细胞的末梢和胶质细胞组成。神经末梢含有大量的膜结构包围的颗粒、线粒体、粗面内质网和许多电子透明的小泡,末梢外周有时可见指状突起。依据颗粒的大小、形状、电子致密度以及胞质特征,可区分出6种类型的窦腺神经末梢及7种X－器官神经分泌细胞。观察了末梢中神经分泌颗粒的胞吐作用方式的释放过程,并且尝试对窦腺不同末梢中的颗粒及X－器官神经分泌细胞中的颗粒作了比较,发现二者之间具有较好的对应性,即电子致密度无大的变化,形态特征相似,只是大小稍有增加。     

Immunochemical and cytochemical studies of relaxin-containing cells in the guinea pig uterus

Uteri and ovaries from cycling, pregnant, and lactating guinea pigs were studied for immunolocalization of relaxin with the light microscope. Endometrial gland cells (EGC) from the same group of animals were examined in the electron microscope for the presence of secretory granules. Those EGC that exhibited high numbers of granules were stained either for relaxin with the protein A colloidal gold method or for carbohydrate with the thiocarbohydrazide technique. Relaxin was found in EGC from middle and late pregnant animals but was not detected in ovaries or uteri from cycling animals. While cytoplasmic granules were noted in most EGC from cycling animals examined, the number of granules was greatest in uteri from estrus and proestrus animals. Granules in EGC from estrus animals contained a carbohydrate-rich material but did not contain relaxin. Endometrial gland cells from animals in early to middle stages of pregnancy (days 15 and 30) contained limited numbers of granules, almost all of which contained carbohydrate. At day 45 of pregnancy, EGC containing many granules were noted. The majority of granules contained relaxin; however, a significant number of EGC contained carbohydrate-rich granules. Infrequently, EGC were noted that contained two populations of granules, and these two populations were assumed to be made up of relaxin-containing and carbohydrate-rich granules. EGC from animals on day 60 of pregnancy typically contained granules, and the majority of these contained relaxin. Carbohydrate-rich granules were observed in EGC of the day 60 animals but were smaller in diameter and were noted in much lower numbers than the relaxin-containing granules. Endometrial gland cells from lactating animals infrequently contained granules. These studies are consistent with the hypothesis that the uterus is the primary source of relaxin in the guinea pig and that relaxin plays an important role in pregnancy and parturition of this species. The observations implicate endometrial glands and their products in the physiology of the cycling animal as well as the pregnant and parturient animal.