Zinc Iodide-osmium Tetroxide Research Articles

Zinc iodide-osmium tetroxide impregnation of the "tubulo-vesicular system" in Tomes' process of the rat incisor ameloblast.

Zinc iodide-osmium tetroxide (ZIO) is a nonspecific but selective impregnation method that visualizes a tubulo-vesicular system in cells. The detailed structure and three-dimensional distribution of this ZIO-impregnated system was studied in the Tomes' process of secretory ameloblasts in the rat incisor. The ZIO-impregnated system consisted of an extensive array of smooth membrane-bound thick and thin tubules and vesicles. The interconnected thick and thin tubules formed a complex "core network" in the central cytoplasm of Tomes' process that enmeshed and often surrounded individual secretory granules. From the core network, radial branches extended toward the smooth cell membrane of the interdigitating portion of Tomes' process. Although the core network and branches frequently appeared connected to the secretory granules and the cell membrane, stereo-pair electron microscopy failed to show conclusive evidence of such continuity. However, many coated vesiclelike structures were attached to the core network and its branches. No special relationship was found between interrod and rod secretory sites and the tubulo-vesicular network. In thick sections, the ZIO-impregnated tubulo-vesicular network occupied a considerable volume of cytoplasm. The vinblastine-labile nature of this network as demonstrated previously (Nanci et al., 1987) indicated that the system undergoes rapid and extensive turnover. Considering the dynamic nature and sheer volume of the tubulo-vesicular system, we propose that it be regarded as a major cell organelle.

An evaluation of myelomeres and segmentation of the chick embryo spinal cord

We have investigated whether the neuromeres of the developing chick spinal cord (myelomeres) are manifestations of intrinsic segmentation of the CNS by studying the patterns of cell proliferation and neuronal differentiation. Treatment of 2-day embryos with colchicine does produce exaggerated myelomeres, in confirmation of Källén (Z. Anat. Entwickl.-Gesch. 123, 309-319, 1962). However, this does not imply that myelomeres are segmental proliferation centres: the undulations caused by colchicine are irregular alongside the unsegmented mesoderm, and another mitotic inhibitor, bromodeoxyuridine, has no such effects. In contrast to lower vertebrate embryos, there is no evidence for segmental groups of primary motor neurons in the chick: the earliest motor neurons express cholinesterase, and project their axons into the adjacent sclerotome, at random positions in relation to the somite boundaries. The population of motor neurons projecting HRP-labelled axons into a single somite lies out of phase with both myelomere and somite, and is placed symmetrically about the anterior half-sclerotome. The earliest intrinsic spinal cord neurons, as stained with zinc iodide-osmium tetroxide or anti-68 x Mr neurofilament antibody, show no segmental patterns of differentiation. We conclude that, in contrast to the rhombomeres of the developing hindbrain, myelomeres are not matched by segmental groupings of differentiating nerve cells, and result from mechanical moulding of the neuroepithelium by the neighbouring somites.

A Cytochemical Ultrastructural Study of the Lysosomal System of Different Species of Malaria Parasites

We have used ultrastructural techniques in different malarial species to demonstrate a lysosomal system. First, we have tried to localize acid phosphatase, a typical lysosomal label. Its activity was localized in the endoplasmic reticulum and in endocytic vesicles, and in dense-cored vesicles near the digestive vacuoles, especially in Plasmodium falciparum (FCR3 strain). Then, we have studied the different cellular compartments of the malarial parasite by the zinc iodide-osmium tetroxide technique that heavily contrasted the cellular compartments of the parasite. This experiment led to the observation of a profound rearrangement of the endoplasmic reticulum, especially in P. berghei. A very atypical but functional Golgi apparatus was demonstrated in all the growing stages of the parasite and lysosome-like vesicles were observed, showing a structure very similar to those of the coated vesicles of a true Golgi complex. The presence of these organelles are in favor of the existence of a lysosomal system and of the endogenicity of some enzymes involved hemoglobin degradation.

Golgi complex and lysosomes in rabbit derived Pneumocystis carinii

The ultrastructural morphology of Pneumocystis carinii obtained from nonimmunosuppressed rabbit is described in details. Golgi complex and primary lysosomes of P carinii are described here for the first time. They are easily revealed by the zinc iodide-osmium tetroxide cytochemical reagent. Thiamine pyrophosphatase and beta-glycerophosphatase activities are found in the parasite but cytidine 5' monophosphatase activity is not observed. A weak thiamine pyrophosphatase activity is detected in Golgi vesicles. An endomembranous saccular structure, present from the intracystic body stage to the precystic stage, apparently plays the role of secondary lysosome. A second type of endomembranous saccular structure, only present in the well developed trophozoitic and precystic forms is also described. The presence of carbohydrates in the cell wall of the parasite was demonstrated by periodic acid-thiosemicarbazide-silver proteinate staining and lectin concanavalin A labeling. The development of Golgi vesicles preceded the transition from double-layered to three-layered parasite stages.

Human gallbladder mucosa infrastructure: Evidence of intraepithelial nerve structures

The zinc iodide-osmium tetroxide (ZIO) fixative-staining method was used along with topographical ultrastructure to investigate cholecystectomized human gallbladders under light and electron microscopic techniques. This method delineated neuronal structures which may be involved in controlling the functions of the gallbladder epithelium. Three epithelial cell types were described in the surface epithelium: 1) Columnar clear cells; 2) dark, tuft osmiophilic cells; and 3) basal clear cells with electron-dense granules and showing intense ZIOphilic staining properties. While mucous granules were delineated in the first two cell types, the columnar epithelial clear cells are of uncertain function(s) and content but are probably absorptive cells. The small basal clear cells displaying intense ZIOphilia are associated with intraepithelial nerve endings. These nerve structures may have a sensory and/or motor function(s); they were detected throughout the gallbladder epithelial lining and mucosa.

Ultrastructure of the salt glands of the mangrove, Avicennia marina (Forssk.) Vierh., as indicated by the use of selective membrane staining

Each salt-excreting gland of the mangrove Avicennia marina (Forsskål) Vierh. consists of two to four collecting cells, one stalk cell, and eight to twelve excretory cells. Differential membrane staining by zinc iodide-osmium tetroxide (as a post-fixative) or phosphotungstic acid (as a section-stain) was used to characterise the ultrastructure of the glands. A large amount of tubular endoplasmic reticulum was found in the stalk and excretory cells of the gland, but not in the collecting cells. The ultrastructural arrangement of the endoplasmic reticulum indicates that salt is loaded from the apoplasm into the endoplasmic reticulum of the symplasm at the base of the stalk cell, traverses both cell types in the endoplasmic reticulum, and is excreted at the outer edge of the gland by an eccrine-type mechanism. Increasing development of the tubular endoplasmic reticulum accompanied differentiation of the gland cells.

The morphology of multivesicular bodies in soybean protoplasts and their role in endocytosis

Multivesicular bodies (MVBs) in soybean protoplasts are distinct organelles (generally 250–500 nm in diameter) consisting of a limiting membrane and a number of smaller internal vesicles (generally 40–100 nm in diameter). MVBs of soybean protoplasts are morphologically similar to MVBs of animal cell systems. They can have tubular protuberances which extend from the main body of the organelle and a lamellar plaque on the cytoplasmic surface of their limiting membrane. In addition, the internal vesicles can be labeled by a zinc iodide-osmium tetroxide postfixation and may form via invagination of the limiting membrane. The MVBs of soybean protoplasts are a major compartment in the endocytotic pathway. They accumulate, over time, exogenously applied cationized ferritin and may deliver it to the major lysosomal or lytic compartment of the plant cell, namely, the vacuoles.

Ultracytochemical evidence for the presence of GERL in pinealocytes of the Mongolian gerbil (Meriones unguiculatus).

Ultracytochemical reactions for the demonstration of acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase, as well as zinc iodide-osmium tetroxide impregnation, revealed the existence of GERL (Golgi apparatus-Endoplasmic Reticulum-Lysosomes) in pinealocytes of the Mongolian gerbil (Meriones unguiculatus). The spatial arrangement of this structure was studied on thick sections using a goniometric stage. Although it was not possible to determine whether GERL in pinealocytes belongs to the Golgi apparatus or to endoplasmic reticulum, it can be concluded that its presence in studied cells signifies that they are considerably more active synthetically than has been believed to date.

Morphological Alteration of the ZIO-Positive Small Vesicles in Rat Hypothalamus Induced by Hyperosmolarity

The zinc iodide-osmium tetroxide (ZIO) staining method was applied to the hypothalamo-hypophyseal system of the rat. Almost all small vesicles about 50 nm in diameter were selectively impregnated with ZIO, whereas large dense vesicles about 100 nm in diameter were negative for the staining. Endogenous catecholamine exhaustion induced by combined treatment of reserpine and α-methyl p-tyrosine to the animal did not affect the population of ZIO-positive small vesicles in the arcuate nucleus and the median eminence. These findings suggest that catecholamine itself Is not involved in ZIO reaction in the small vesicles but rather suggest ZlO-positive material is a carrier protein common to various kinds of neurotransmitters. Exposure of the hypo-thalamic tissue to hypertonic fixative and staining solution gave rise to two kinds of ZIO-positive small vesicles; one was spherical (S type) and the other was flat (F type) in shape. The appearance of two kinds of vesicles might reflect the functional difference of these vesicles.

Early and late sarcoplasmic reticulum changes in doxorubicin cardiomyopathy. An ultrastructural investigation with the zinc iodide-osmium tetroxide (ZIO) technique.

The sequence of myocardial changes in the mouse induced by doxorubicin (Dx) treatment (10 mg/kg i.v.) has been investigated by electron microscopy with the help of the zinc iodide-osmium tetroxide (ZIO) technique. Accumulation of ZIO-reactive material, possibly oxidized glutathione and other disulfides, in the sarcoplasmic reticulum (S.R.) is among the earliest (1 h after Dx injection), more prominent and persistent findings (up to 100 days). It may have a pathogenic relationship with a number of functional and morphologic changes occurring in myocardial cells, including impairment of calcium transport and contractility, S.R. dilation up to extensive vacuolization, as well as inhibition of DNA, RNA and protein synthesis leading to atrophy and disruption of sarcomeres. The latter finding, first appearing in a few cells 4 to 7 days after Dx and progressively increasing in severity and extension during the next 3 months, may represent a key factor in the evolution of chronic cardiomyopathy to cardiac insufficiency. In most cells, only a minority of mitochondria showed obvious ultrastructural lesions, which were first observed 24 h after treatment and disappeared by the end of the first month, when no more mitochondrial damage was found outside degenerating cells. The myocardium of mice receiving multiple Dx injections (4 mg/Kg, 10 times, or 9 mg/Kg, 5 times) showed the same changes observed in animals treated with a single dose, though they were more severe and extensive.

An electron microscope thick section study of endomembrane organization in the myxamoebae ofPhysarum polycephalum

The post-fixation ofPhysarum polycephalum myxamoebae with a zinc iodide-osmium tetroxide mixture resulted in the accumulation of an electron dense deposit in the lumen of the nuclear envelope, the endoplasmic reticulum, and most of the cisternae of the Golgi apparatus. In double-stained thin sections of impregnated myxamoebae the preservation of other cytoplasmic organelles was excellent. Examination of thick sections (0.25 μm) at 120 kV revealed the complexity of the endomembrane system and continuities between both the ER and the nuclear envelope, and the ER and the Golgi apparatus. No direct continuities were observed between the cisternae of the Golgi apparatus and the nuclear envelope. A three dimensional view of membrane organization was obtained from stereopairs, while tilting the sections at much steeper angles revealed whether any of the apparent continuities seen were real or were simply the result of overlap. A morphologically distinct region of the ER, which bears similarities to the GERL region described in other organisms, was found in continuity with the remaining ER, the Golgi apparatus, and the nuclear envelope.

Tritrichomonas foetus: Cytochemical visualization of the endoplasmic reticulum-Golgi complex and lipids

The zinc iodide-osmium tetroxide technique was used to analyze the distribution of the endoplasmic reticulum-Golgi complex system of Tritrichomonas foetus. Interconnections between the cisternae of the endoplasmic reticulum as well as between cisternae of the Golgi complex were observed. The nuclear pores, as well as fenestrations in the Golgi complex, were evident. Three to four profiles of the endoplasmic reticulum were seen in the proximal marginal lamellae, related to the attachment of the recurrent flagellum to the protozoan body. No reaction product was seen in the costae, microtubules, glycogen particles, or hydrogenosomes. Imidazole-buffered osmium tetroxide solution was used to visualize lipids. Electron-dense materials were seen either free in the cytoplasm or within membrane-bounded cytoplasmic vesicles. A high contrast of some membranes, mainly of those which enclosed the hydrogenosomes, was observed in unstained sections.

Segmentation in the vertebrate nervous system.

Although there is good evidence that growing axons can be guided by specific cues during the development of the vertebrate peripheral nervous system, little is known about the cellular mechanisms involved. We describe here an example where axons make a clear choice between two neighbouring groups of cells. Zinc iodide-osmium tetroxide staining of chick embryos reveals that motor and sensory axons grow from the neural tube region through the anterior (rostral) half of each successive somite. 180 degrees antero-posterior rotation of a portion of the neural tube relative to the somites does not alter this relationship, showing that neural segmentation is not intrinsic to the neural tube. Furthermore, if the somitic mesoderm is rotated 180 degrees about an antero-posterior axis, before somite segmentation, axons grow through the posterior (original anterior) half of each somite. Some difference therefore exists between anterior and posterior cells of the somite, undisturbed by rotation, which determines the position of axon outgrowth. It is widespread among the various vertebrate classes.

Stimulation of nodal and terminal sprouting of mouse motor nerves by gangliosides

Treatment of mice with daily intraperitoneal gangliosides (50 mg/kg) enhanced both the number and mean length of nodal sprouts 4 days after partial denervation of the gluteus maximus muscle. Terminal sprouting in the soleus muscle 14 days after botulinum toxin paralysis was also enhanced by gangliosides, as assessed by zinc iodide-osmium tetroxide staining. Functional recovery from paralysis, assessed electrophysiologically, was simultaneously accelerated; this may have resulted from the enlarged terminal area, rather than reflecting an additional action of gangliosides upon synaptogenesis.

Connections between dictyosomes, ER and GERL in cotyledons of mung bean (Vigna radiata L.)

The connections and structural inter-relations of dictyosomes and endoplasmic reticulum (ER) in cotyledons of germinating mung beans were studied using thick (0.3 μm) sections of aldehyde fixed, zinc iodide-osmium tetroxide (ZIO) impregnated tissue. The sections were examined by conventional (100 kV), rather than high voltage, transmission electron microscopy. Continuity of cisternal ER with tubular ER was confirmed and a direct connection of tubular ER totrans dictyosome cisternae was observed as were GERL networks associated withtrans dictyosome cisternae. Dictyosomes also gave rise to an extensive system of very fine tubules (10–20 nm diam) which have not been described previously in plant tissue. These tubules, which originated at thetrans dictyosome face, extended throughout the cytoplasm and were found connected to cisternal ER and tubular ER. The implications of these observations are discussed with regard to present ideas concerning endomembrane flow and protein sorting by the Golgi apparatus.

Synaptic and Synaptoid Vesicles Constitute a Single Category of Inclusions. New Evidence From ZIO Impregnation

Parallel observations on central synaptic and neurohaemal terminals of the same types of neurosecretory fibres in the polychaete annelid Nereis diversicolor reveal that their respective populations of inclusions exhibit identical, highly distinctive patterns of affinity for the zinc iodide-osmium tetroxide (ZIO) reagent. The method highlights the duality of possible secretory inclusions in nerve terminals. Many typical synaptic/synaptoid vesicles have ZIO-positive contents, but intermingle with unreactive vesicles. Both positively and negatively reacting vesicles contribute to the unusual dense clusters associated with sites of release of neurochemical mediators, characteristic of polychaete nervous systems. Fewer dense-cored synaptic/synaptoid vesicles have reactive cores. The larger ‘storage granules’ typically have unreactive contents, but dense deposits form within a small minority. A possible cytophysical, in contradistinction from a cytochemical, basis of affinity for ZIO is discussed. The results further support the postulated fundamental identity of synaptic and synaptoid vesicles.

Cell Reactivity Pattern of the Guinea Pig Cecal Mucosa Evidenced by the ZIO Technique

The zinc iodide-osmium tetroxide (ZIO) technique is presently employed to study both, neural and non neural tissues. Precipitates depends on cell types and possibly cell metabol ism as well.Guinea pig cecal mucosa, already known to be composed of epithelium with cells at different maturation stages and lamina propria which i s formed by morphologically and functionally heterogeneous cell population, was studied to determine the pat tern of ZIO impregnation. For this, adult Guinea pg cecal mucosa was fixed with buffered 1.2 5% g 1 utara 1 dehyde before incubation with ZIO for 16 hours, a t 4°C in the dark. Further steps involved a quick sample dehydration in graded ethanols, embedding in Epon 812 and sectioning to observe the unstained material under a phase contrast light microscope (LM) and a transmission electron microscope (TEM).

The Golgi apparatus in developing endosperm of wheat (Triticum aestivum L.)

The ultrastructure and distribution of the Golgi apparatus in developing wheat endosperm was investigated using a zinc iodide-osmium tetroxide staining complex in conjunction with low and high voltage electron microscopy. Dictyosomes were numerous in starchy endosperm and aleurone at 15 days after anthesis, and during the period of rapid storage protein deposition 25 d after anthesis. Fewer dictyosomes were seen in maturing endosperm. Two types of vesicles were associated with the dictyosomes; small, heavily-stained vesicles were sited at the ends of fine tubules which extend from the cisternae, and larger less-stained vesicles were associated with the periphery of the cisternae. Stereo-pairs of micrographs up to 1 μm thick were taken to demonstrate the interconnections between cisternal and tubular endoplasmic reticulum. Elements of tubular ER were closely associated with dictyosomes, but connections were not observed. These results are discussed in relation to the transport of endosperm storage proteins from their site of synthesis on the cisternal ER to their site of storage, the protein bodies.

The distribution of nodal sprouts in a paralysed or partly denervated mouse muscle

The right gluteus maximus muscles of young adult mice were paralysed with botulinum toxin for up to 21 days or partly denervated by spinal root section for up to 63 days; the intramuscular and extramuscular nerves were then examined in the electron microscope in thin sections of tissue fixed conventionally or stained with zinc iodide-osmium tetroxide. Contralateral muscles were also examined as controls. The distribution of nodal sprouts in the nerves of the paralysed or partly denervated muscles was determined by calculating the mean ratios of unmyelinated to myelinated axons in nerve profiles containing different numbers of myelinated axons (intact or degenerating) and comparing these with control ratios. In paralysed muscles there was a significantly higher proportion of nerve profiles containing one or two unmyelinated axons alongside a single myelinated axon. Nerve profiles containing two or more myelinated axons did not show any increase in the proportion of unmyelinated axons. Thus, there is probably nodal sprouting in paralysed muscles which is restricted to the most distal nodes of the intramuscular nerves. In muscles partly denervated for 8 days there were significant increases in the proportion of unmyelinated axons in nerve profiles which had contained up to 5–10 myelinated axons. After 21 days of partial denervation, similar increases may have occurred in the larger intramuscular nerve profiles and after 63 days there were large increases in the proportion of unmyelinated axons in the main intramuscular nerve branches and in the extramuscular nerve. Nodal sprouting in response to partial denervation is therefore localised initially to the smaller, more distal nerve branches; at later times, some sprouts probably grow slowly in a disto-proximal direction along denervated Schwann cell pathways. The existence of nodal sprouts in paralysed muscles and their restricted distribution in paralysed and partly denervated muscles suggest that the nodal sprouting stimulus is produced by the muscle and acts only at distal nodes.

Tubulo-vesicular structures in the basal cytoplasm of gut basal-granulated cells revealed by zinc iodide-osmium tetroxide staining.

Zinc iodide-osmium tetroxide staining was applied to the duodenum of a human fetus for the purpose of investigating the fine structure of basal-granulated cells. A positive reaction was demonstrated within a peculiar network consisting of fine tubulo-vesicular structures in the basal cytoplasm. The possibility was discussed that these structures represent a device by which the granule membrane returns to the Golgi complex after the exocytotic release of gut hormones.