Zinc Iodide-osmium Research Articles

Pollen grain development and male sterility in the perfect flowers of Maytenus obtusifolia Mart. (Celastraceae).

Perfect flowers of Maytenus obtusifolia have partial sterility of pollen grains, resulting in collapsed and developed free microspores. However, the cellular events resulting in partial male sterility have not been determined. In pistillate flowers of this species, male sterility is related to the premature programmed cell death (PCD) in tapetum and sporogenic cells. The process occurs through autophagy via macroautophagy and massive autophagy and is associated with sporophytic cytoplasmic male sterility (CMS). Here, we characterised the development of pollen grains and investigated the cellular events that result in tapetum cells and free microspores PCD in perfect flowers, using light and transmission electron microscopy combined with the TUNEL (Terminal deoxynucleotidyl transferase mediated dUDP end-Labeling) assay and the ZIO (Zinc iodide-osmium tetroxide) method. Pollen grain development in perfect flowers was divided into eight developmental stages based on the characteristics of the pollen grains. Tapetum cells undergo PCD at the free microspore stage, through a macroautophagic process, by formation of autophagosomes and by autophagosomes giving rise to lytic vacuoles at maturity. In the final stage of PCD, massive autophagy occurs by rupture of the tonoplast. The development of viable and inviable microspores diverges at the vacuolated microspore stage, when PCD occurs in some free microspores, causing interruption of pollen development through necrosis. These events result in the observed partial male sterility. Viable microspores undergo mitosis and develop into tricellular pollen grains. Male sterility in hermaphrodite individuals is here interpreted as gametophytic CMS.

Different subsets of Langerhans cells in human uterine tubes and uterus.

Langerhans cells (LC) are antigen-presenting cells present in tissues with high antigenic exposure. Their role in the upper female reproductive tract is not fully understood. This study aims to determine the distribution and morphology of LC in the normal and post-partum human uterine tubes and uterus by staining with the specific LC markers, CD1a and zinc iodide-osmium (ZIO), and to determine their association with helper and cytotoxic T cells. Normal and post-partum uterine tube and uterine specimens were stained with CD1a and ZIO and their morphology and distribution noted. Double immune staining with CD1a-CD4 and CD1a-CD8 in post-partum uterine tube were also done. It was noted that CD1a-positive cells were significantly fewer and smaller in diameter than ZIO-positive cells in the uterine tube and both types of cells were significantly more prevalent in post-partum tubes. Perivascular clusters of ZIO-positive cells were seen in the post-partum tubes. Close association of CD1a-positive cells with CD4- and CD8-positive T cells was noted in the post-partum uterine tube. In the uterus, scanty CD1a-positive cells were present in the surface and glandular epithelium and endometrial stroma. ZIO-positive cells were absent. This study suggests that CD1a-positive and ZIO-positive cells may be different subsets of LC that are needed for presentation of antigen to immunocompetent cells. Their respective functions are yet to be determined.

Morphological study of dendritic cells in human cervix by zinc iodide osmium method.

Dendritic cells (DCs) are a heterogeneous population of antigen presenting cells that have been identified in several tissues including the female reproductive organs. The aim of the present study is to demonstrate the morphological differences of dendritic cells in normal human exocervix using the Zinc Iodide Osmium (ZIO) procedure. Normal cervical tissues obtained from nine patients who underwent abdominal hysterectomies for various ailments were processed for histochemical study. Six microns thick serial sections were taken and viewed under a light microscope. The diameters of the cells were measured under a magnification of 40x using the Cellsens image analysing software and analysed using SPSS version 16. In the normal human exocervix, a greater density of ZIO-positive DCs was noted in the epithelium and subepithelium and their distribution was not uniform. In some areas of epithelium, the ZIO-positive cells in the basal layer showed a typical dendritic morphology, while the cells in the intermediate and superficial layers were nondendritic polygonal cells. Intraepithelial capillaries were noted, which were surrounded by ZIO-positive nondendritic polygonal cells. There was significant difference in the mean diameters of typical DCs (8.61±1.86 μm) and nondendritic polygonal cells (10.97±1.93 μm). In the subepithelium the DCs had typical morphology and their distribution varied. ZIO positive DCs were noted in the epithelium and cervical glands of endocervix also. In conclusion, the human cervix has different subsets of ZIO positive DCs with varied distribution. Their functional role has yet to be defined.

A Note on Langerhans Cells in the Oesophagus Epithelium of Domesticated Mammals

Using the zinc-iodide osmium tetroxide (ZIO) method, TEM and immunohistochemistry (for CD1a and langerin), the study demonstrates Langerhans cells in the oesophageal epithelium of domesticated mammals (herbivores: horse, cattle, goat; omnivores: pig, dog, laboratory rat; carnivores: cat), although with variations between the species. The ZIO method and TEM showed this cell type in the cat and, sporadically, in the horse; CD1a (+) Langerhans cells were demonstrated in the ovine, porcine and murine oesophagus. Positive staining for langerin was detected in single cells of the caprine, canine, murine and feline oesophagus and more distinct in almost all the cell layers of the equine and porcine oesophagus epithelium. The findings are discussed comparing specifically the results for CD1a and langerin, whereby the latter C-type lectin may be of importance in species with a rather thick oesophagus epithelium, such as that present in the plantivorous and most of the omnivorous animals, where antigenic pressure is reduced.

Leaf glands act as nectaries in Diplopterys pubipetala (Malpighiaceae)

Leaf glands of Diplopterys pubipetala were studied with light and electron microscopy. Aspects of their secretion, visitors and phenology were also recorded. Glands occur along the margin, at the apex and at the base of the leaf blade. All the glands begin secretion when the leaf is still very young, and secretion continues during leaf expansion. The highest proportion of young leaves coincides with the beginning of flowering. The glucose-rich secretion is collected by Camponotus ants, which patrol the newly formed vegetative and reproductive branches. All the glands are sessile, partially set into the mesophyll, and present uniseriate epidermis subtended by nonvascularised parenchyma. The glands at the apex and base are larger and also consist of vascularised subjacent parenchyma. The cytoplasm of epidermal and parenchyma cells has abundant mitochondria, polymorphic plastids filled with oil droplets and a few starch grains. Golgi bodies and endoplasmic reticulum are more abundant in the epidermal cells. The parenchyma cells of the subjacent region contain chloroplasts and large vacuoles. Plasmodesmata connect all the nectary cells. The zinc iodide-osmium tetroxide (ZIO) method revealed differences in the population of organelles between epidermal cells, as well as between epidermal cells and parenchyma cells. Ultrastructural results indicate that leaf glands of D.pubipetala can be classified as mixed secretory glands. However, the secretion released by these glands is basically hydrophilic and composed primarily of sugars, hence these glands function as nectaries.

Fine ultrastructure of chromaffin granules in rat adrenal medulla indicative of a vesicle-mediated secretory process

Observation by transmission electron microscopy, coupled with morphometric analysis and estimation procedure, revealed unique ultrastructural features in 25.94% of noradrenaline (NA)-containing granules and 16.85% of adrenaline (A)-containing granules in the rat adrenal medulla. These consisted of evaginations of the granule limiting membrane to form budding structures having different morphology and extension. In 14.8% of NA granules and 12.0% of A granules, outpouches were relatively short, looked like small blebs emerging from the granule surface and generally contained electron-dense material. A proportion of 11.2% of NA granules and 4.9% of A granules revealed the most striking ultrastructural features. These secretory organelles presented thin, elongated, tail-like or stem-like appendages, which were variably filled by chromaffin substance and terminated with spherical expansions of different electron density. A cohort of vesicles of variable size (30-150 nm in diameter) and content was found either close to them or in the intergranular cytosol. Examination of adrenal medullary cells fixed by zinc iodide-osmium tetroxide (ZIO) revealed fine electron dense precipitates in chromaffin granules, budding structures as well as cytoplasmic vesicles. These data indicate that a common constituent is revealed by the ZIO histochemical reaction in chromaffin cells. As catecholic compounds are the main tissue targets of ZIO complexes, catecholamines are good candidates to be responsible for the observed ZIO reactivity. This study adds further to the hypothesis that release of secretory material from chromaffin granules may be accomplished by a vesiclular transport mechanism typical of piecemeal degranulation.

Ultrasound thermal damage to rabbit corneas after simulated phacoemulsification

To determine rabbit cornea thermal tolerance and evaluate the effects of ultrasound (US) on this tissue after applying defined US heat doses. Eye Clinic; Anatomy Histology and Forensic Medicine, University of Florence, Florence, Italy. Hyperthermia was induced in rabbit corneas using US, simulating a phacoemulsification procedure. The US power was set at 100% in continuous mode, and temperature values were reached within 10 seconds of the onset of US treatment. Corneal surface temperatures were continuously monitored and recorded by thermographic registration. The eyes of 16 rabbits were examined: 4 controls, 8 treated at 40 degrees C for 10 seconds, 8 treated at 50 degrees C for 10 seconds, and 12 treated at 60 degrees C for 10 seconds. All 32 corneal buttons were removed and prepared for light microscopic evaluation with hematoxylin and eosin staining, trichromic staining, and zinc iodide-osmium tetroxide solution. The 12 corneas treated at 60 degrees C for 10 seconds were also processed for immunohistochemical analysis. Corneas at 40 degrees C for 10 seconds were grossly and histologically normal and were not different from control corneas. Corneas at 50 degrees C for 10 seconds showed initial stromal damage with collagen disorganization, mild stromal edema, and initial signs of keratocyte damage. Half of the corneas at 60 degrees C for 10 seconds were examined at time 0 and the other half after 1 week. At time 0, massive corneal damage with epithelial cell edema, collagen disorganization, severe stromal edema, intrastromal vacuole formation, plump keratocyte nuclei, and endothelial cell detachment were found, as was a severely impaired nerve plexus. At 1-week follow-up, corneas showed persistent stromal and endothelial cell edema with an increase activated keratocytes and mitotic features in the stroma and the epithelial layer. Rabbit corneas showed a considerable tolerance to US damage up to 50 degrees C. Higher thermal doses produced severe histological damage, even though corneas showed a considerable plasticity due to their regenerative capacity.

Modulation of smooth muscle cell function: Morphological evidence for a contractile to synthetic transition in the rat ventral prostate after castration

In this study, we evaluated the involvement of rat ventral prostate smooth muscle cells (SMC) in secretory activity and whether this function is modulated after castration. Cell morphology was examined at both light and electron microscopy levels and the organelles involved in secretory function were labeled by the zinc-iodide-osmium (ZIO) method at the ultrastructural level and their volume density was determined by stereology. Castration resulted in marked changes of the SMC, which adopted a spinous aspect and abandoned the layered arrangement observed in the prostates of non-castrated rats. The volume density of ZIO reactive organelles increased progressively after castration, reaching significantly higher levels 21 days after castration. Since previous studies have demonstrated that SMC express SMC markers (even 21 days after castration) and are able to respond to adrenergic stimulation, we concluded that differentiated SMC are able to shift from a predominantly contractile to a more synthetic phenotype without changing their differentiation status.

Structure and division of the Golgi complex in Trichomonas vaginalis and Tritrichomonas foetus

We present observations on the fine structure and the division process of the Golgi complex in the protists Trichomonas vaginalis and Tritrichomonas foetus, parasites of the urogenital tract of humans and cattle, respectively. The Golgi in trichomonads is a prominent structure, associated with striated parabasal filaments to which this organelle seems to be connected. We followed by immunofluorescence and electron microscopy the Golgi in interphasic and mitotic cells. Ultrastructural studies were performed using fast-freezing fixation, immunocytochemistry using antisera to the known adhesins AP65 and AP51, cytochemistry (acid phosphatase, Ca++-ATPase, zinc iodide-osmium tetroxide technique (ZIO), for analysis of distribution of the endoplasmic reticulum and Golgi complex, and Thiéry's techniques), routine and serial thin-sections. Three-dimensional reconstruction, NBD-ceramide, fluorescent lectin (WGA) and nocodazole treatments were also used. We demonstrate that: (1) the Golgi in trichomonads is a single-copy organelle; (2) presents a fenestrated structure; (3) is formed by 8-12 saccules; (4) is connected to the parabasal filaments by thin filamentous bridges; (5) by cytochemistry, presents a positive reaction for the lectin WGA, Ca++-ATPase, acid phosphatase, ZIO and Thiéry's techniques; (6) does not appear to break down at any point of the cell cycle; (7) elongates during the cell cycle by lateral growth; (8) is labeled by anti-glutamylated tubulin antibodies, but it is not fragmented by nocodazole treatment; (9) before mitosis, the already elongated Golgi ribbon undergoes progressive medial fission, cisternae by cisternae, starting at the cisternae adjacent to the cell surface and ending with the cis-most cisternae; (10) the Golgikinesis originates two small Golgi ribbons; (11) the Golgi is intensely labeled with the antisera to the AP65 and AP51 adhesins in T. vaginalis, thus seeming to be a key station in the production of adhesins.

Zinc iodide-osmium tetroxide (ZIO) reactive sites in the extrafloral nectary of Citharexylum mirianthum Cham. (Verbenaceae)

This paper reports on a study of the zinc iodide-osmium tetroxide method (ZIO) applicability to formaldehyde-glutaraldehyde prefixed extrafloral nectary tissues of Citharexylum mirianthum Cham. (Verbenaceae). The ZIO solution impregnates the dictyosome stacks and adjacent vesicles, smooth endoplasmic reticulum, nuclear envelope, multivesicular bodies, and peroxisomes. The use of this method greatly facilitates the observation and recognition of organelles in each nectary region. It also allows the correlation between structure and function in nectariferous cells.

Chromoplasts Are Formed in Vicia faba Suspensor Cells

In Vicia faba, the plastids of the suspensor have a structure that is unique for this tissue. Their development was followed from 4 to 15 days after pollination (dap), when the suspensor has started to degenerate. The appearence of the plastids changes from small and rounded to large and more or less amoeboid. A distinct peripheral reticulum and ribosomes are present throughout plastid development. At 11 dap, most plastids have formed one or more prolamellar bodies. After zinc iodide–osmium tetroxide staining, the plastids also show strongly electron‐scattering bundles of membrane tubules, sometimes with cross‐linking between individual thylakoids. At 13–14 dap, the thylakoid system includes large electron‐transparent spaces that are traversed by undulating membranes. Such a structure is characteristic of chromoplasts containing carotenoid crystals. In the light microscope, crystals are seen as bright red inclusions in the plastids.

Intraepidermal free nerve fiber endings in the hairless skin of the rat as revealed by the zinc iodide-osmium tetroxide technique.

The nerve fiber distribution in the epidermis of the hairless rat skin was studied light microscopically by means of zinc iodide-osmium tetroxide staining. Two different morphological types of free nerve fiber endings could be detected: clusters of relatively thick nerve fibers stretched up through the spinous layer up to the granular layer sending off terminal branches. In addition, many solitary thin varicose nerve fibers were seen within the epidermis. The observed discrepancies in nerve fiber diameters appeared to be larger than those reported for human intraepidermal nerve fibers in recent immunohistochemical studies. Moreover, dendritic cells, most probably representing Langerhans cells, could be selectively stained. These cells appeared to be in a close location to thin varicose nerve fibers. Both types of demonstrated free nerve endings have to be functionally connected with different sensoric functions. Possibly, a subpopulation of the thin nerve fibers might possess primarily a nociceptive task, whereas the thick ones have most probably to be regarded as mechanoreceptive. The nerve fibers innervating dendritic cells appear to be identical to the peptidergic ones which may regulate the antigen-presenting capacity of these cells. Due to its selectivity for intraepidermal nerve fibers, the used method might supplement immunohistochemical procedures in a helpful manner.

Characteristic intraepidermal nerve fibre endings of the intervibrissal fur in the mystacial pad of the rat: morphological details revealed by intravital methylene blue staining and the zinc iodide-osmium tetroxide technique

Characteristic intraepidermal nerve fibre endings of the intervibrissal fur in the mystacial pad of the rat: morphological details revealed by intravital methylene blue staining and the zinc iodide-osmium tetroxide technique

Ultrastructural characterization of the interstitial cells of Cajal.

Recent studies on the interstitial cells of Cajal (ICC) have determined ultrastructural criteria for the identification of these previously enigmatic cells. This review deals with the electron microscopic findings obtained by the author's research group in different tissue regions of the gut in mice, rats and guinea-pigs, comparing these with reports from other groups in different species and in humans. ICC are characterized by the following morphological criteria: numerous mitochondria, abundant intermediate filaments and large gap junctions which connect the cells with each other and with smooth muscle cells. Due to their location in the gut and the specific species, the ICC are markedly heterogeneous in appearance, ranging from cells closely resembling smooth muscle cells to those similar to fibroblasts (Table 1). Nevertheless, the above-mentioned morphological features are shared by all types of ICC and serve in identifying them. Recent discoveries on a significant role of c- kit in the maturation of the ICC and their specific immunoreactivity to anti-c-Kit antibody have confirmed the view that the ICC comprise an independent and specific entity of cells. This view is reinforced by the findings of the author's group that the ICC characteristically possess vimentin filaments and are stained with the zinc iodide-osmium tetroxide method which provides a staining affinity similar to methylene blue, the dye used in the original work by Cajal, (1911). Developmental studies indicate that the ICC are derived from a non-neuronal, mesenchymal origin. This paper further reviews advances in the physiological studies on the ICC, in support of the hypothesis by THUNEBERG (1982) that they function as a pacemaker in the digestive tract and a mediator transmitting impulses from the nerve terminals to the smooth muscle cells.

The nuclear reticulum in placental cells ofLilium regale is a part of the endomembrane system

Placental cells line the ovarian transmitting tract inLilium regale and produce exudates for secretion. Sections through the highly lobed nuclei of these cells reveal the presence of membrane profiles which form vesicles with varying dimensions in cross section. Computer reconstruction of the nucleus reveals that the vesicle profiles form a complex reticulum of tubular cisternae, which spans the whole nucleus, enclosing a maze of continuous lumen space. Connections between the vesicles and the inner nuclear envelope are visible at various points along the nuclear envelope. This complex network of tubules which constitutes the reticulum arises from the inner nuclear membrane. The nuclear reticulum dramatically increases the inner-envelope surface area, comprising 82% of the total membrane perimeter of inner nuclear envelope and nuclear reticulum. The inner nuclear envelope invaginates into the nucleus forming the nuclear reticulum and the outer nuclear envelope evaginates into the endoplasmic reticulum (ER), indicating that there is a continuity between the lumens of the nuclear reticulum and the ER. The nuclear reticulum is labelled with zinc iodide-osmium tetroxide, a staining pattern identical to that seen in the ER. Positive reaction to the zinc iodide-osmium tetroxide indicates that the nuclear reticulum is a site for Ca2+ deposition. The nuclear reticulum forms an extension of the endomembrane system which reaches deep into the nucleoplasm. The lumenal continuity of this system means that there is a channel for communication from the cytoplasm into the nucleoplasm, and that this channel sequesters calcium.

Investigation of the Ultrastructurel of Different Cells in Lymph Nodules by Using Zinc Iodide Osmium Tetroxide Technique

The zinc iodide -osmium tetroxide (ZIO) fixation/staining method was applied for neurocytological study and also to examine several other tissue samples including human blood and bone marrow on nerve endings in the median eminence, epidermal langerhans cells of lymphoid tissue. Although precise specificity can not be attributed to the staining reaction. Interesting staining patterns for different cell types in lymph node were observed by one of the ZIO staining solutions. The significance of ZIO positivity is briefly discussed.

Apposition of enteric nerve fibers to plasma cells and immunoblasts in the mouse small bowel

Light (LM) and electron microscopy (EM) were used to investigate the structural relationship between enteric nerves and the population of immune cells in the mouse small bowel. By LM, the osmium-zinc iodide procedure was used for visualizing nerve fibers; the incidence of nerve-plasma cell contacts in the mucosa and submucosa was calculated to be ~4 times and, respectively, 3 times greater than would be expected by chance alone ( P<0.0001). EM revealed close, synaptic-like contacts between axonal varicosities and plasma cells or B immunoblasts. The results presented here provide systematic quantitative evidence that a structural foundation for communication between nerve fibers and B cells exists in the mouse small bowel.

Observation of the continuity of Golgi apparatus with endoplasmic reticulum induced by brefeldin A studied by zinc iodide-osmium staining in tobacco protoplasts

Observation of the continuity of Golgi apparatus with endoplasmic reticulum induced by brefeldin A studied by zinc iodide-osmium staining in tobacco protoplasts

The innervation of taste buds in the soft palate and circumvallate papilla of the rat as revealed by the zinc iodide-osmium tetroxide technique.

The taste buds in the soft palate and the circumvallate papillae of the rat were investigated by the zinc iodide-osmium tetroxide technique. In addition, electron micrographs of taste buds stained with this method were presented for the first time. Differences in taste bud structures were found between the examined regions. The taste buds of the soft palate showed a complicated plexus of intragemmal nerve fibers. Some fibers exhibited terminal polymorphic swellings. Single branches could be traced close to the space of the taste pore. In the soft palate, the taste bud cells remained unstained, whereas in the circumvallate papillae of the tongue, a subpopulation of taste bud cells could be selectively stained and the intragemmal nerve fibers were characterized by large varicosities. The morphological dissimilarities between the taste buds of the investigated regions might be explained by their functional characteristics, or possibly their varying affinities to the taste qualities. Electron microscopic investigation of the stained circumvallate papillae revealed that the electron-dense reaction product had primarily accumulated in a subpopulation of light cells. Dark cells exhibited only a slight labelling. In detail, the precipitate was found loosely distributed in the cytoplasm as well as the nuclei of the cells, and particularly concentrated at the membranes of light vacuoles, this probably being profiles of dilated endoplasmic reticulum. A few roundish accumulations of precipitate were seen in the cytoplasm of taste bud cells, which showed no intensive light microscopic staining. Labelled material was also found within the taste pores outside the apical processes of the cells. The present findings indicate that the zinc iodide-osmium tetroxide technique is applicable to neuroanatomical studies of taste buds.

Light and electron microscopic examination of exocrine pancreas using zinc iodide-osmium tetroxide technique.

Zinc iodide-osmium tetroxide (ZIO) fixation/staining technique is a metallophilic technique which has been used for the examination of various tissues and cell types. We examined the ZIO (+) cell types in rat exocrine pancreas to obtain further evidence for the significance of the reaction. Among mostly non-reactive pancreatic acinar cells there were ZIO (+) acinar cells of varying staining intensity. Zymogenic granules and centroacinar cells were completely non-reactive. Our electron microscopic findings support the view that the reactivity of the technique used is cell specific but not cell type or organelle specific.