Organisation of the cytoskeleton during in vitro maturation of horse oocytes

Meiotic maturation of mammalian oocytes is a complex process during which microfilaments and microtubules provide the framework for chromosomal reorganisation and cell division. The aim of this study was to use fluorescence and confocal laser scanning microscopy to examine changes in the distribution of these important cytoskeletal elements and their relationship to chromatin configuration during the maturation of horse oocytes in vitro. Oocytes were cultured in M199 supplemented with pFSH and eLH and, at 0, 12, 24, and 36 hr after the onset of culture, they were fixed for immunocytochemistry and stained with markers for microtubules (a monoclonal anti-alpha-tubulin antibody), microfilaments (AlexaFluor 488 Phalloidin) and DNA (TO-PRO(3)). At the germinal vesicle stage, oocyte chromatin was amorphous and poorly condensed and the microfilaments and microtubules were distributed relatively evenly throughout the ooplasm. After germinal vesicle breakdown, the microtubules were aggregated around the now condensed chromosomes and the microfilaments had become concentrated within the oocyte cortex. During metaphase I, microtubules were detected only in the meiotic spindle, as elongated asters encompassing the aligned chromosomes, and, as maturation progressed through anaphase-I and telophase-I, the spindle assumed a more eccentric position and gradually rotated to assist in the separation of the homologous chromosomes and in the subsequent formation of the first polar body. During metaphase II, the meiotic spindle was a symmetrical, barrel-shaped structure with two poles and with the chromosomes aligned along its midline. At this stage, microtubules were found intermingled with chromatin within the polar body and, although, the bulk of the microfilaments remained within the oocyte cortex, a rich domain was found overlying the spindle. Thus, during the in vitro maturation of horse oocytes both the microfilament and microtubular elements of the cytoskeleton were seen to reorganise dramatically in a fashion that appeared to enable chromosomal alignment and segregation.     

The distribution and requirements of microtubules and microfilaments in bovine oocytes during in vitro maturation

Microtubules and microfilaments are major cytoskeletal components and important modulators for chromosomal movement and cellular division in mammalian oocytes. In this study we observed microtubule and microfilament organisation in bovine oocytes by laser scanning confocal microscopy, and determined requirements of their assembly during in vitro maturation. After germinal vesicle breakdown, small microtubular asters were observed near the condensed chromatin. The asters appeared to elongate and encompass condensed chromatin particles. At the metaphase stage, microtubules were observed in the second meiotic spindle at the metaphase stage. The meiotic spindle was a symmetrical, barrel-shaped structure containing anastral broad poles, located peripherally and radially oriented. Treatment with nocodazole did not inhibit germinal vesicle breakdown. However, progression to metaphase failed to occur in oocytes treated with nocodazole. In contrast, microfilaments were observed as a relatively thick uniform area around the cell cortex and overlying chromatin following germinal vesicle breakdown. Treatment with cytochalasin B inhibited microfilament polymerisation but did not prevent either germinal vesicle breakdown or metaphase formation. However, movement of chromatin to the proper position was inhibited in oocytes treated with cytochalasin B. These results suggest that both microtubules and microfilaments are closely associated with reconstruction and proper positioning of chromatin during meiotic maturation in bovine oocytes.     

The role of microtubules and microfilaments in the micronucleus ofParamecium bursaria during mitosis

Summary A unique spindle apparatus develops during mitosis in the micronucleus ofParamecium bursaria. During interphase the micronucleus contains short microtubule profiles and clumps of condensed chromatin. Throughout mitosis the nuclear envelope remains intact. During prophase, cup-shaped structures termed microlamellae develop in close association with regions of condensed chromatin. Each micromella consists of an outer sublamella, an inner sublamellae, and ring-shaped structures termed microsepta that join the two sublamellae. Microtubules elongate parallel to the division axis. During metaphase, the microlamellae appear to act as kinetochorelike structures that aid in the alignment of the chromosomes. The microlamellae appear conical and join to a meshwork of microfilaments at their apices. Further toward the polar regions the microfilaments join with microtubules that converge and terminate near the nuclear envelope. During metaphase-anaphase and anaphase the chromosomes are apparently moved by the microfilaments pulling on the kinetochorelike microlamellae. Also during metaphase-anaphase, extranuclear microtubules join the nuclear envelope of the micronucleus to microtubule elements of the cell cortex. By anaphasetelophase, microlamellae and the microfilament meshwork degenerate and microtubules represent the only spindle elements. The evidence of this report supports the hypothesis that microfilaments can participate with microtubules in the movement of chromosomes.This report is part of a Ph.D. Thesis presented by the senior author at Fordham University.     

Ultrastructural observations on the granular leucocytes of the tuatara Sphenodon punctatus (gray)

The granular leucocytes of an active, mature female tuatara, Sphenodon punctatus (Gray) were examined in the electron microscope. Eosinophils contained a lobulated nucleus, homogeneous, dense, irregularly shaped granules, assorted smaller granular inclusions, mitochondria and beta-glycogen. Endoplasmic reticulum, Golgi complexes and ribosomes were scanty. Immature neutrophils (myelocytes) were regular in outline and contained a compact nucleus. In the adjacent centrosomal region were paired centrioles with connected microtubules, and Golgi complexes. Ovoid electron-dense granules, mitochondria, lipid droplets and numerous microfilaments arranged randomly or in bundles, lay in the cytoplasm. Mature neutrophils were often highly irregular in outline, had a segmented nucleus and contained possibly a second type of granular inclusion. The basophils were regular in outline with a compact nucleus. Numerous ovoid homogeneous, electron-dense granules, mitochondria, beta-glycogen particles and some microfilaments were seen in the cytoplasm. The granules in many basophils appeared 'altered' or degenerate and most of these contained microtubules. The cytology of the granulocytes of the tuatara is compared with that in other vertebrates.     

Ultrastructural observations on the erythrocytes and thrombocytes of the tuatara, Sphenodon punctatus (gray)

The erythrocytes of Sphenodon punctatus (Gray) are nucleated, ellipsoidal and flattened, and contain 55--65 microtubules in their marginal band. The thrombocytes are also flattened, ellipsoidal, nucleated cells and in electron-microscopic preparations occurred in aggregrates. The thrombocytes appeared to be 'activated' and possessed many pseudopodia which were devoid of organelles. The latter were concentrated in the perinuclear region and were encircled by a ring of microtubules. The organelles included ribosomes, mitochondria, membrane--bound dense material and numerous actin-like microfilaments. Cytoplasmic vacuoles contained a moderately dense, filamentous material and/or spheroidal electron-dense inclusions, beta-glycogen particles were scattered in the general cytoplasm and were most concentrated in the pseudopodia. The erythrocytes and thrombocytes of S. punctatus are compared with those in other vertebrates.     

Organelle movement and fibrillar elements of the cytoskeleton in the angiosperm pollen tube

Summary Continuous observation of organelles and other cytoplasmic inclusions in the older stretches of living pollen tubes of Iris pseudacorus shows that in the more attentuated parts of the protoplast they move along single, mainly longitudinally oriented fibrils, corresponding to those previously isolated from other species and shown to contain bundles of uniformly polarised actin microfilaments. The traffic associated with each fibril is unidirectional, but organelles move along them independently, sometimes with conspicuously different velocities. Larger columns of cytoplasm passing along the tube are associated with several such fibrils, as revealed in occasional discontinuities and also in columns isolated from the tube in suitable medium without fixation. The dimensions of the individual fibrils suggest that the bundles of actin microfilaments are not likely to be enclosed in a unit membrane corresponding to a tonoplast. If so, the nature of the continuous cavities traversed by numerous fibrils in the older parts of the pollen tube requires reappraisal, since these are more likely to be volumes of attentuated cytoplasm comparable with that of the central cavity of the sieve tube than vacuoles of the normal plant-cell type.     

Intranuclear microfilament bundles in the ependymal cells of the third ventricle of the rat

Summary Intranuclear microfilament bundles were observed in ependymal cells of the third ventricle of the rat. They appeared as single, cylindricallyshaped fibrillar bands up to 4.9 m in length. In cross sections of bundles, the microfilaments exhibited an apparently regular arrangement. They were separated by a distance of 4 to 5 nm, measuring approximately 11 nm from centre to centre. One bundle consisted of 55 to 88 microfilaments. The intranuclear bundles terminated at the inner membrane of the nucleus. They exhibited close spatial relationship to perinuclear chromatin, chromatin centres, intrachromatin granules and fibrillar structures. The average ratio of nuclear sections with and without bundles was 115.Research fellow of the Alexander von Humboldt FoundationThis research was partly carried out at the Department of Histology and Embryology, Academy of Medicine at Poznan, Poland, and was supported in part by a grant from the Polish Academy of Science     

Nuclear grooves in cytologic preparations. A study of the utility of this feature in the diagnosis of papillary carcinoma

The sensitivity and specificity of nuclear grooves and inclusions for papillary carcinoma was investigated in 32 touch preparations and 69 fine needle aspiration cytologic preparations of the thyroid. Ultrastructurally, these grooves and inclusions are cytoplasmic invaginations into the nucleus. Overall, 100% of the papillary carcinomas contained nuclear grooves while only 70% contained inclusions. Grooves, however, could be seen in 70% of nonpapillary neoplasms and in 56% of nonneoplastic conditions of the thyroid, albeit generally fewer in number and often not as distinct. Inclusions were present in 13% of nonpapillary neoplasms and were absent in nonneoplastic conditions. Some nuclei on cytologic preparations contain lines that are probably artifacts of chromatin alignment and do not represent true nuclear grooves. Since such lines may be indistinguishable from true grooves, grooves should be used cautiously and in conjunction with other criteria in the diagnosis of papillary carcinoma.     

Covering common ground: F-actin-dependent transport of plant viral protein inclusions reveals a novel mechanism for movement utilized by unrelated viral proteins

Plant viruses are composed of diverse genomes (e.g., RNA or DNA) encoding proteins that vary widely in sequence. It is becoming clear, however, that some apparently unrelated viral proteins have similar functions. The P6 protein encoded by Cauliflower mosaic virus (CaMV) and the 126-kDa protein encoded by Tobacco mosaic virus (TMV) are examples of this convergence in protein function. Although having no apparent sequence similarity, both proteins are pathogenicity determinants during infection, are components of novel intracellular cytoplasmic inclusions and suppress RNA silencing. Here we review our recent results demonstrating an additional novel convergent activity between these proteins: both proteins traffic along the actin cytoskeleton (microfilaments). We also discuss results showing a unique property of the P6 protein: a non-mobile strong association with microtubules. Lastly, we discuss the potential mechanism by which the P6 and 126-kDa proteins traffic along microfilaments. We provide new results suggesting that actin filament polymerization-driven movement does not support 126-kDa protein transport, thus leading to a focus on myosins as the driving force for this movement.Key words: actin polymerization, cytoskeleton, cauliflower mosaic virus, microfilaments, microtubules, myosin, tobacco mosaic virus, virus movement, intracellular transport     

Effect of anisotonic NaCl treatment on cellular ultrastructure of V79 Chinese hamster cells. Part 1. Mitotic cells

The ultrastructural modifications produced by anisotonic NaCl treatment of Chinese hamster mitotic cells were observed at three NaCl concentrations which have been frequently used in radiosensitization studies: 0.05, 0.5 and 1.5 M. After exposure to 0.05 M NaCl, many well-spread chromosomes are visible. The chromatin fibres are well dispersed and membraneous material is associated with the chromosomes. After hypertonic treatment with 0.5 M NaCl, the chromosomes have a uniform, structureless appearance with some coalescing into larger anaphase-like masses. At 1.5 M NaCl, large scale cellular dehydration is apparent, and filamentous structures such as microfilaments are tightly constricted. The degree of chromosome staining is also reduced below the level of the cytoplasm. After both hypo- and hypertonic NaCl treatment the chromosomes appear swollen relative to untreated cells, but hypertonic treatment causes chromosome clumping and dissociates chromatin. Conformational changes in the chromatin may restrict the capacity for DNA repair and be related to cellular radiosensitivity.     

Organization of actin microfilaments in the apical border of oviduct ciliated cells

Actin microfilaments were localized in quail oviduct ciliated cells using decoration with myosin subfragment S1 and immunogold labeling. These polarized epithelial cells show a well developed cytoskeleton due to the presence of numerous cilia and microvilli at their apical pole. Most S1-decorated microfilaments extend from the microvilli downward towards the upper part of the ciliary striated rootlets with which they are connected. From the microvillous roots, a few microfilaments connect the proximal part of the basal body or the basal foot associated with the basal body. Microfilament polarity is shown by S1 arrowheads pointing away from the microvillous tip to the cell body. Furthermore, short microfilaments are attached to the plasma membrane at the anchoring sites of basal bodies and run along the basal body. The polarity of these short microfilaments is directed from the basal body anchoring fibers downward to the cytoplasm. At the cell periphery, microfilaments from microvillous roots and ciliary apparatus are connected with those of the circumferential actin belt which is associated with the apical zonula adhaerens. Together with the other cytoskeletal elements, the microfilaments increase ciliary anchorage and could be involved in the coordination of ciliary beating. Moreover, microvilli surrounding the cilia probably modify ciliary beating by offering resistance to cilium bending. The presence of microvilli could explain the fact that mainly the upper part of the cilia appanars to be involved in the axonemal bending in metazoan ciliated cells.     

Cytological changes in the testes of vitamin-A-deficient rats. II. Ultrastructural study of the seminiferous tubules.

Ultrastructural study confirmed that, in rats, vitamin A deficiency initially caused the sloughing of some spermatids and spermatocytes into the lumina of the seminiferous tubules around day 3 following the initial decrease of body weight. From days 5 to 10, a considerable number of spermatocytes and spermatids, which still remained in the epithelium, underwent necrosis. Several stages of dying spermatocytes and abnormal spermatids were observed. The latter were distinguished by the presence of chromatin aggregating along the nuclear envelopes and highly vacuolated mitochondria. These cells range from single to multinucleate forms. They were incapable of differentiating further into spermatozoa and ultimately degenerated. Within the same period, Sertoli cells exhibited numerous darkly stained lysosome-like inclusions, and the upper part of their cytoplasm appeared as irregular processes, some of which were broken off and resulted in the thinning of the epithelium. From days 10 to 20, the remaining germ cells comprised mainly spermatogonia and few abnormal spermatocytes. The latter appeared enlarged and were very lightly stained. Their nuclei exhibited unusual blocks of heavily condensed chromatin amidst very highly dispersed chromatin fibers. Though their number was reduced, most of the spermatogonia appeared unaltered. Processes of Sertoli cells became even more irregular and were interrupted at certain sites by large empty spaces. Darkly stained inclusions in their cytoplasm were fewer than observed earlier.     

蒙古黄鼠（Citellus dauricus）松果腺的超微结构观察

The distal part of pineal gland of the Mongolian ground squirrel was ultrastructurally studied. The gland was composed of low electron-dense parenchymal cells, among which glial cells, pigment cells, blood vessels and neural elements were occasionally interspersed. The pinealocytes contained numerous mitochondria, lysosomes, microtubules, microfilaments, Golgi apparatus and free ribosomes, as well as less prominent profiles of rough- and smooth-surfaced endoplasmic reticula and some cilia, centrioles, synaptic ribbons and few subsurface cisterns. Some pinealocytes were vacuolated. The content of the vacuoles released into the extracellular space by exocytosis could be observed. The gap junctions between pinealocytes were also observed. Of particular interest was that many mitochondria "fused together" and formed gap junction-like structure in about five percent pinealocytes. The pigment cell has a amorphous nucleus which contains many aggregated chromatin, its cell membrane has a few microvilli projecting into a central lumen, these features may indicated that this kind of cell differs either from the pinealocyte or astrocyte. There are axo-axonic synapses or axo-dendritic synapses between neuron processes or between neuron processes and pinealocytes.     

Cytoskeletal influence on merocyanine 540 receptors in the plasma membrane of erythroleukemic cells

When human erythroleukemic cells are induced to differentiate in vitro, the lipids in the plasma membrane that bind the fluorescent dye merocyanine 540 are redistributed into a cap at one pole of the cell. This capping phenomenon can also be observed in uninduced cells that have been incubated with cytochalasin B, an agent which disrupts actin-containing microfilaments or with local anesthetics which act on both microfilaments and microtubules. Colchicine which acts on microtubules, however, has no effect. This suggests that the uniform distribution seen in uninduced cells is maintained by the cytoskeletal microfilaments and that loss of these structures leads to spontaneous redistribution of merocyanine 540-binding sites.     

Localization of actin and microfilament-associated proteins in the microvilli and terminal web of the intestinal brush border by immunofluorescence microscopy.

Indirect immunofluorescence microscopy was used to localize microfilament-associated proteins in the brush border of mouse intestinal epithelial cells. As expected, antibodies to actin decorated the microfilaments of the microvilli, giving rise to a very intense fluorescence. By contrast, antibodies to myosin, tropomyosin, filamin, and alpha-actinin did not decorate the microvilli. All these antibodies, however, decorated the terminal web region of the brush border. Myosin, tropomyosin, and alpha-actinin, although present throughout the terminal web, were found to be preferentially located around the periphery of the organelle. Therefore, two classes of microfilamentous structures can be documented in the brush border. First, the highly ordered microfilaments which make up the cores of the microvilli apparently lack the associated proteins. Second, seemingly less-ordered microfilaments are found in the terminal web, in which region the myosin, tropomyosin, filamin and alpha-actinin are located.     

Ultrastructure of testicular biopsy from an XX male

Ultrastructural study of testicular biopsy specimens from an XX male showed hyalinized seminiferous tubules and tubules containing only mature Sertoli cells. These cells possessed large lipid inclusions as well as microfilament bundles which were perpendicular to the basement membrane and parallel to one another. The basal lamina was thickened and composed of several parallel layers with myofibroblast layers between them. The interstitium showed nodular to diffuse Leydig cell hyperplasia. Four types of Leydig cells were found: 1) normal Leydig cells with crystals of Reinke; 2) cells with abundant microcrystalline inclusions as well as microfilaments and concentric cisternae of smooth endoplasmic reticulum; 3) vacuolated cells containing numerous large lipid droplets; 4) immature Leydig cells. The different ultrastructural abnormalities found in the Sertoli and Leydig cells might be considered as the histological expression of a tubular-interstitial dysgenesis which is reflected in the high levels of gonadotropins and low levels of testosterone.     

Stabilizing microtubules with taxol increases microfilament stability during freezing of rye root tips

We have used double fluorescence labelling to investigate the effect of freezing on microtubules and microfilaments in root-tip cells of rye (Secale cereale L. cv Rymin). Freezing to -5°C (which does not kill these cells) caused partial depolymerization of both, but microfilaments were more resistant than microtubules. When microtubules were stabilized against freeze-induced depolymerization by pre-treating seedlings with taxol, microfilaments exhibited enhanced stability as well. Almost all the frozen cells containing taxol-stabilized microtubules also contained microfilaments. When seedlings were treated with the microtubule-destabilizing drug APM prior to freezing, microfilaments became more susceptible to freeze-induced depolymerization than in controls. These data suggest a physical interaction between microtubules and microfilaments in these cells.     

CALCIUM OXALATE CRYSTALS IN THE GREEN ALGA SPIROGYRA SP. (ZYGNEMATALES, CHLOROPHYTA)

Specimens of an unidentified species of the freshwater green alga Spirogyra were found to have abundant cruciate cellular inclusions up to 34 micrometers long. A crystalline nature was shown by birefringence in polarized light. Despite their large size and complex shape, these inclusions did not occur free in the large central vacuole. Instead, they were associated with cytoplasmic strands that spanned the space between gyres of the parietal spiral chloroplasts and with strands that suspended the nucleus in a cytoplasmic embayment of the central vacuole. Some crystals moved directionally along the cytoplasmic strands, and their movement was arrested by cytochalasin B, suggesting that actin microfilaments had a role in crystal movement. Solubility tests showed that the inclusions were composed of calcium oxalate; they dissolved rapidly in weak hydrochloric acid without effervescence, but they were not soluble in concentrated acetic acid or sodium hypochlorite. A colorimetric enzymatic test for oxalate was used to demonstrate microscopically the presence of oxalate and to quantify the amounts. The calcium oxalate crystals were surrounded by a water-soluble organic matrix that retained the shape of the crystal even after demineralization. Scanning electron microscopy was used to examine the morphology of isolated crystals.     

Nature of the nuclear inclusions formed by PQBP1, a protein linked to neurodegenerative polyglutamine diseases

PQBP1, for polyglutamine tract-binding protein-1, has been linked to progressive neurodegenerative diseases, such as spinocerebellar ataxia, that are caused by the expansion of a polyglutamine repeat in a key regulatory protein. The overexpression of PQBP1 results in the formation of nuclear inclusions, reminiscent of the protein aggregates that are detected in polyglutamine diseases. We show here that the occurrence of PQBP1-induced nuclear inclusions is dramatically increased by the co-expression of the pre-mRNA splicing factor SIPP1, a protein ligand of PQBP1. These nuclear inclusions did not co-localise with nuclear structures such as nucleoli, coiled bodies, PML bodies, speckles and stress bodies, and were not associated with (in)active chromatin or with nucleic acids. Site-directed mutagenesis showed that the facilitation in the formation of the nuclear inclusions required multiple independent interaction sites between SIPP1 and PQBP1. Moreover, the nuclear inclusions were highly dynamic and their formation did not require energy. Our data suggest that the SIPP1-PQBP1-induced nuclear inclusions are distinct from the protein aggregates that are associated with polyglutamine diseases and represent dynamic nucleoplasmic heteropolymers of SIPP1 and PQBP1.