AN ELECTRON MICROSCOPE STUDY OF THE GLAND CELLS OF THE MINK ENDOMETRIUM

Portions of mink endometrium in delayed implantation, early postimplantation, and pseudo pregnancy were fixed in buffered osmium tetroxide with sucrose, or potassium permanganate. After rapid dehydration the portions of endometrium were embedded in either methacrylate or epoxy resin. Examination of the cells from the body of the glands of the endometrium of delayed implantation revealed the presence of prominent terminal bars, numerous secretion granules, and membrane discs in the apical region of the cell. In the supranuclear and infranuclear regions, mildly dilated cisternae of the endoplasmic reticulum were present, and in many cells unusually large mitochondria were seen. Numerous changes were noted in the gland cells of the post implantation stage. The endoplasmic reticulum in the basal region was extensively dilated, and the nuclei were situated more centrally. Giant mitochondria were no longer present. The large secretion granules were not present, but smaller granules were seen, especially in the Golgi region. Some of the Golgi cisternae were dilated and the pattern of parallel membranes was consequently less distinct. It is suggested that gland cells in the postimplantation and pseudopregnancy stages exhibit evidence of greater secretory activity than those in the delayed implantation stage.     

AN ELECTRON MICROSCOPE STUDY OF THE DEVELOPMENT OF A MOUSE HEPATITIS VIRUS IN TISSUE CULTURE CELLS

Samples taken at different intervals of time from suspension cultures of the NCTC 1469 line of mouse liver—derived (ML) cells infected with a mouse hepatitis virus have been studied with the electron microscope. The experiments revealed that the viruses are incorporated into the cells by viropexis within 1 hour after being added to the culture. An increasing number of particles are found later inside dense cytoplasmic corpuscles similar to lysosomes. In the cytoplasm of the cells from the samples taken 7 hours after inoculation, two organized structures generally associated and never seen in the controls are observed: one consists of dense material arranged in a reticular disposition (reticular inclusion); the other is formed by small tubules organized in a complex pattern (tubular body). No evidence has been found concerning their origin. Their significance is discussed. With the progression of the infection a system of membrane-bounded tubules and cisternae is differentiated in the cytoplasm of the ML cells. In the lumen of these tubules or cisternae, which are occupied by a dense material, numerous virus particles are observed. The virus particles which originate in association with the limiting membranes of tubules and cisternae are released into their lumen by a "budding" process. The virus particles are 75 mµ in diameter and possess a nucleoid constituted of dense particles or rods limiting an electron transparent core. The virus limiting membrane is sometimes covered by an outer layer of a dense material. In the cells from the samples taken 14 to 20 hours after inoculation, larger zones of the cell cytoplasm are occupied by inclusion bodies formed by channels or cisternae with their lumens containing numerous virus particles. In the samples taken 20 hours or more after the inoculation numerous cells show evident signs of degeneration.     

A STUDY OF THE STRUCTURE OF SPLENIC SINUSES IN MAN AND IN THE ALBINO RAT WITH THE LIGHT MICROSCOPE AND THE ELECTRON MICROSCOPE

The splenic sinuses in the spleens of 5 human beings and 7 albino rats have been studied in the light microscope and electron microscope after fixation in Dalton's fluid and Palade's fluid and embedding in n-butyl methacrylate. Splenic sinuses are tortuous vascular channels of large but variable diameter which represent the first venous vessels in the spleen and make up almost the entire red pulp in man and in rats. These vessels are composed of reticulo-endothelial cells flattened to endothelial form and sheathed by a netted reticulum. The luminal surface of the endothelium is made highly irregular by delicate and variable cytoplasmic protrusions, slender corridors separating adjacent endothelial cells, anastomotic openings to other sinuses, bulgings of entire cells, and even thrusts of endothelium spanning the sinai lumen. The supporting reticulum presents a well developed latticed appearance in tangential sections of sinuses, but in most cuts is punctate or linear. The reticulum is composed of strands without limiting membranes, which, in substance, are amorphous and resemble basement membrane. Material identical in appearance to the substance of the reticulum may be present in the endothelium, suggesting that the reticulum is formed by endothelial cells. The endothelium also contains deposits of presumed ferritin and hemosiderin. The extreme luminal bulgings of endothelium suggest production of circulating monocytes or lymphocytes by detachment of endothelial cells. Sinuses are patent and collapsed to varying degrees. Patent sinuses are separated by collapsed sinuses and these collapsed sinuses appear to constitute splenic (Billroth) cords.     

AN ELECTRON MICROSCOPE STUDY OF SILVER NITRATE REDUCTION IN LEAF CELLS

Brown , W. V., H. Mollenhauer , and C. Johnson . (U. Texas, Austin.) An electron microscope study of silver nitrate reduction in leaf cells. Amer. Jour. Bot. 49(1): 57–63. Illus. 1962.—As reported earlier in many studies, AgNO₃ is reduced quickly by the living chloroplasts of angiosperms. Electron microscope study has resolved the conflict of opinions concerning the exact location of the silver particles. Reduction of AgNO₃, as indicated by location of silver particles, occurs within the chloroplasts but not within the grana or pure stroma; it appears to be associated with the intergranal (also called stroma) lamellae. Silver particles are formed also at the surfaces of the cell wall, both in the middle lamella and at the inner surface, and also within plasmodesmata. It is concluded that chlorophyll is probably not involved directly in the reduction. There is some slight support for the popular hypothesis that ascorbic acid may be the chief reducing agent.     

LOCALIZATION OF TRITIATED NOREPINEPHRINE IN VASCULAR SYMPATHETIC AXONS OF THE RAT INTESTINE AND MESENTERY BY ELECTRON MICROSCOPE RADIOAUTOGRAPHY

The distribution of infused tritiated norepinephrine (NE-³H) in small mesenteric arteries and intestinal arterioles in rats was investigated with electron microscopic radioautography. Silver grains, indicating the presence of the tritium label on the sections, were found lying mainly over axon bundles, but some were present over collagen and smooth muscle cells. Axons with the highest concentrations of silver grains had been sectioned at points where they were naked of Schwann cell sheath, were dilated into varicosities, and contained small granular vesicles. This finding was taken as confirmatory circumstantial evidence that the small granular vesicles were the sites of uptake and storage of NE. The short interval between the start of infusion and the fixation of the tissue appeared to rule out any process other than a direct uptake of NE by the peripheral axons. If axonal sites of uptake of NE-³H correspond to sites of release of NE, then the evidence suggests that such sites of release are widespread over the terminal part of the axon and are not confined to those parts of the axon which are in close contact with smooth muscle cells. Since the fixation and embedding procedures will remove NE which is not strongly bound to tissues, the localization of NE-³H in the radioautographs does not necessarily correspond to the distribution of all the NE present in vivo.     

A LIGHT AND ELECTRON MICROSCOPE STUDY OF THE MORPHOLOGICAL CHANGES INDUCED IN RAT LIVER CELLS BY THE AZO DYE 2-ME-DAB

The cytological changes induced in rat liver cells by the aminoazo dye 2-Me-DAB have been examined by light and electron microscopy. It is observed that this non-carcinogenic compound duplicates most of the morphological alterations produced by other hepatotoxins, some of which, such as the closely related aminoazo dye 3'-Me-DAB, are potent carcinogens. These non-specific effects involve both the granular and agranular forms of the endoplasmic reticulum as well as the glycogen content of hepatic cells. The arrays of cisternal profiles of the granular reticulum in normal hepatic cells become disorganized and the dispersed cisternae often appear fragmented and irregular. Large cytoplasmic inclusions, consisting of loosely organized tubules and vesicles, are also observed which result from a hypertrophy of the agranular reticulum. The glycogen in the cells progressively decreases in amount. The most specific effect of 2-Me-DAB is to induce an increase in the number of mitochondria per cell. Many of these organelles are characterized by the presence of a median double membrane continuous with the inner limiting membrane of the mitochondrial envelope. Evidence is presented in favor of the view that this partition is directly related to the phenomenon of mitochondrial division. It was noted also in the course of the experiment that an increasing number of cells appear which stain quite intensely with methylene blue and appear denser than normal under electron microscopy. The significance of these cells is not known.     

ELECTRON MICROSCOPE STUDIES ON THE DICTYOSOMES AND ACROBLASTS IN THE MALE GERM CELLS OF THE CRICKET

The dictyosome (Golgi body) in the secondary spermatocyte of the cricket appears in electron micrographs as a duplex structure composed of (a) a group of parallel double-membraned lamellae and (b) a group of associated vacuoles arranged along the compact lamellae in a chain-like fashion. This arrangement of ultramicroscopic structure for the dictyosomes is strikingly comparable to that described for the Golgi apparatus of vertebrates. Accordingly, the two are considered homologous structures. Associated with the duplex structure of the dictyosomes is a differentiated region composed of small vacuoles. This is thought to represent the pro-acrosome region described in light microscope preparations. In the spermatid the dictyosomes fuse, giving rise to the acroblast. Like the dictyosomes, the acroblasts are made up of double-membraned lamellae and associated vacuoles. In addition, a differentiated acrosome region is present which, in some preparations, may display the acrosome vacuole and granule. Both the dictyosomes and acroblasts are distinct from mitochondria.     

A STUDY OF CHROMOSOMES WITH THE ELECTRON MICROSCOPE

Amphibian lampbrush chromosomes and meiotic prophase chromosomes of various insects and plants consist of a bundle of microfibrils about 500 A thick. These fibrils are double, being made of two closely associated fibrils 200 A thick. Fragments of interphase nuclei contain a mass of fibrils 200 A thick. Ultrathin sections through nuclei in prophase or interphase show sections of these double or single fibrils cut at various angles. A comparison of sections with the methacrylate left in and sections that were shadowed after removing the methacrylate suggests that the OsO₄ reacts only with the outer part of the fibrils either because it does not penetrate, or as a result of a chemical difference of the inner core and the outside of the fibril. It is suggested that in analogy to the structure of the tobacco mosaic virus the chromosomal microfibril may have an inner core of DNA surrounded by a shell of protein.     

AN ELECTRON MICROSCOPE STUDY OF MATURE AND DIFFERENTIATING PANETH CELLS IN THE RAT, ESPECIALLY OF THEIR ENDOPLASMIC RETICULUM AND LYSOSOMES

In an electron microsope study, the morphology of mature Paneth cells from the small intestine of adult rats is compared with that of differentiating Paneth cells from young rats 2 to 4 weeks old. All mature cells exhibit a marked polarity similar to that of other exocrine gland cells and contain a well developed endoplasmic reticulum, an elaborate Golgi complex, and numerous large secretory granules; they also possess an abundance of lysosomes. The most conspicuous occurrence in the process of differentiation is the development of the endoplasmic reticulum. The most immature Paneth cells possess an endoplasmic reticulum of the vesicular type, which, during maturation, is replaced by the characteristic lamellated ergastoplasm of the mature cell. At a certain stage of differentiation the cavities of the developing cisternae show numerous communications with the perinuclear space, suggesting an outgrowth of the ergastoplasm from the nuclear envelope. Furthermore, the cavities and the perinuclear space at this particular stage contain a material which shows a remarkable intrinsic periodicity. An identical periodicity was exhibited by material contained in Golgi cisternae and secretory granules. Lysosomes are also present in the differentiating cells.     

THE STIMULATION OF ARGININE PHOSPHOKINASE IN LOBSTER AXONS BY GABA

GABA at physiological concentrations has been shown to stimulate the synthesis of arginine phosphate in lobster walking nerves. This effect can be observed both in intact axons and in axonal homogenates. The stimulatory activity of GABA on the synthesis of arginine phosphate appears to be on the V_max of arginine phosphokinase whereas in the reverse reaction the effect is referable to the K_m of ADP.     

ELECTRON MICROSCOPE STUDIES OF THE MICROVILLI OF HELA CELLS

Microvilli of HeLa cells cultured in vitro were preserved for electron microscopic examination at different stages of routine cultivation procedures. By a double-embedding technique, vertical sectioning for electron microscopy was possible. It revealed that, although the microvilli were present on all sides of the cell in the dispersed stage and in the attached stage, they were not present on the bottom of the cell when it was stretched on the surface of the dish. When the cells were grown in dense colonies, they were found on top of each other, and microvilli were present on all sides, except on the bottom surface of those cells in contact with the dish. We achieved a more dramatic demonstration of the microvilli by developing a surface-replica technique which retains their spatial arrangement and permits characterization of the distribution of their number, length, and diameter.     

ELECTRON MICROSCOPE RADIOAUTOGRAPHIC IDENTIFICATION OF SEROTONIN-SYNTHESIZING CELLS IN THE MOUSE GASTRIC MUCOSA

This study correlates the fine structure of mouse gastric endocrine cells with their ability to synthesize serotonin (5-HT) from 5-hydroxytryptophan (5-HTP). Mice were sacrificed 2 hr after the intravenous injection of 5-HTP-³H or 5-HT-³H. Their stomachs were processed for light- and electron microscope radioautography in a manner which retained labeled 5-HT while washing out other labeled substances. Stomachs from additional mice were incubated in vitro with 5-HT-³H and processed similarly. All morphologic types of mouse gastric endocrine cells exhibited a similar facility to incorporate exogenous 5-HTP and to convert it to 5-HT which was bound intracellularly. Differences in densities of silver grains observed over endocrine cells suggested that individual endocrine cells indeed varied in their ability to synthesize and/or to bind 5-HT; such variations, however, were not reflected by differences in fine structure, with the exception that endocrine cells with few granules always contained little newly synthesized 5-HT. The newly synthesized 5-HT was associated with the intracellular granules. The gastric endocrine cells were not labeled by exogenous 5-HT-³H, whereas mast cells were labeled by either 5-HT-³H or 5-HTP-³H administration. The findings of the present study support the position that the gastric endocrine cells represent a single cell type, at least in respect to serotonin metabolism—that the argyrophil or argentaffin reactivity of these cells merely reflects their amine content at a given time.     

AN ELECTRON MICROSCOPE STUDY OF THE EPITHELIUM IN THE NORMAL MATURE AND IMMATURE MOUSE CORNEA

1. An electron microscope study at high resolution of the corneal epithelium of the normal mature and immature mouse revealed new information regarding the submicroscopic appearance of these cells. 2. Two thin dense lines separated by a less dense area constituted the structure of the limiting surface membrane of epithelial cells; the thickness of this membrane was about 80 A. 3. Some differences in the appearance of the cytoplasm and mitochondria of cells from the immature mouse cornea and the appearance of the cytoplasm and mitochondria of cells from the adult mouse cornea were observed. 4. The basement membrane appeared as a dense band about 600 A wide separating the basal epithelial cells from the substantia propria. Suggestions of periodicity were seen in some phosphotungstic acid-treated specimens. 5. Round bodies believed to be bacteria were seen on the surface of the outer epithelial cells in the adult mouse cornea but not in the immature, unopened eye.     

AN ELECTRON MICROSCOPE STUDY OF THE ENDOPLASMIC RETICULUM IN NEWT NOTOCHORD CELLS AFTER DISTURBANCE WITH ULTRASONIC TREATMENT AND SUBSEQUENT REGENERATION

Ultrasonic treatment of the tails of Triturus alpestris tadpoles, at intensities of 8 to 15 watts/cm², at 1 megacycle/sec., for 5 minutes, disrupted the epidermis and caused pycnosis in individual cells of the muscle and neural tube, but caused no damage to the notochord that could be detected by light microscopy. Electron microscopy showed that this ultrasonic treatment disordered nearly all the endoplasmic reticulum (ER) of the notochord cells into irregularly rounded vesicles, but within 3 hours after treatment some parallel arrays of normal endoplasmic reticulum were seen near, and continuous with, the outer nuclear membrane. In addition, a re-ordering of the previously disordered ER took place throughout the cytoplasm, in some cases. A classification was made of the state of the ER as shown in electron micrographs of material fixed immediately, 3, and 24 hours after treatment. This showed that more than half the total endoplasmic reticulum in notochord cells was normal again by 24 hours after treatment.