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PubMed1987

Binding of morphologically abnormal sperm to mouse egg zonae pellucidae in vitro.

The ability of morphologically abnormal mouse sperm to bind to the zona pellucida of the egg was examined with techniques of fertilization in vitro. After incubation with sperm, cumulus-free ova were scored by either phase microscopy or scanning electron microscopy for the number and type of sperm bound. The percentages of abnormal sperm bound to zonae were compared to the percentages of abnormal sperm in the inseminating suspension. In general, all abnormal classes (except broadly spatulate sperm) bound to zonae at a frequency significantly lower than their representation in the inseminating suspension. However, when the percentage of abnormal sperm was quite high, no significant difference existed between frequencies of abnormal sperm bound and in the inseminating solution. The percentage of abnormal sperm bound did not increase significantly over time. Scanning electron microscopy studies demonstrated that the association of abnormal sperm with the zona pellucida varied according to sperm morphology. Normal and some abnormal sperm bound at an angle perpendicular to the zona, while more grossly abnormal sperm bound to ova tangentially.

Acrosome↗

PubMed1987

Influence of the calcium ionophore A23187 on rat egg behavior and cortical F-actin.

Rat eggs treated with the calcium ionophore A23187 and subjected to long-term observation by phase microscopy were found to undergo many developmental changes that are normally associated with fertilization. These included cortical granule exocytosis and the abstriction of the second polar body. In addition, time-lapse video microscopy revealed that, unlike untreated eggs, whose surfaces remained relatively immotile, the ionophore-treated eggs underwent a lengthy period of surface undulatory activity. Since all of these events were remarkably similar in timing and morphology to those seen in fertilized eggs, we conclude that A23187 is capable of activating rat eggs. Using NBD-phallacidin, the distribution of F-actin in ionophore-activated eggs was determined. During most of the postactivation period the eggs possessed an uninterrupted, uniform band of polymerized actin encompassing the entire cortex of the egg. However, during a discrete 1.5-h period after the formation of the second polar body, an area adjacent to the region of polar body abstriction exhibited more intense staining than the rest of the cortex. Cytochalasin B treatment caused a dramatic reduction and/or rearrangement in cortical NBD-phallacidin staining in activated eggs as compared to activated controls not exposed to the drug. We observed that all the developmental changes described above could be produced in the absence of exogenous calcium, suggesting that the rat egg possesses internal stores of calcium sufficient to elicit an activational response. We conclude that the ionophore-induced release of free calcium ions into the cytosol stimulates many of the developmental changes that are normally seen during fertilization. These results indicate that calcium influx and cytoskeletal activity are correlated during the activation of this animal egg.

Actins↗

PubMed2001

In vitro dorsal root ganglia and human prostate cell line interaction: redefining perineural invasion in prostate cancer.

BACKGROUND: Little is understood regarding mechanisms of perineural invasion in prostate cancer progression. We present a novel model system and data that indicate perineural invasion is an active, specific, and reciprocal interaction between nerves and prostate cancer cells. METHODS: Mouse dorsal root ganglia (DRG) and human prostate cancer cells (Du-145, LNCaP, PC3) and stromal cells (HTS-40F) were co-cultured in Matrigel matrix. Control cultures consisted of prostate cancer and stromal cells only and DRG only. Neurite outgrowth, cell colony growth, neurite-colony contact, and retrograde extension were quantitated with dark phase microscopy and image analysis (Optimas 6.1). RESULTS: Directional outgrowth of neurites was observed projecting into DU-145 colonies within 24 hr of co-culture. Cultures with the greatest number of DU-145 cells recruited significantly more neurites and established contact earlier, indicating this process was cell-seeding density dependent. Once neurite/DU-145 cell contact was established neurite growth diminished, suggesting an active neurite recruitment by DU-145 cells. Subsequent to neurite contact, DU-145 cells migrated along neurites in a retrograde fashion into the nerve/ganglion of origin (retrograde extension) establishing perineural invasion. In addition to perineural invasion, DU-145 colony growth was elevated in DRG co-cultures relative to DU-145-only control cell cultures. Similarly, the degree of neurite outgrowth was elevated in DRG-cell co-cultures relative to DRG-only control cultures. The same observations were made with LNCaP and PC3 cells, but interactions between stromal cells and nerves were not found. CONCLUSIONS: This study shows the utility of the prostate cancer/DRG in vitro system to study specific mechanism of prostate cancer cell-nerve interaction. Moreover, these data suggest that perineural invasion mechanisms involve active and reciprocal interactions between carcinoma cells and adjacent nerve/ganglions in prostate cancer progression.

Animals↗

PubMed1993

Insulin-induced circular membrane ruffling on rat 1 cells expressing a high number of human insulin receptors: circular ruffles caused by rapid actin reorganization exhibit high density of insulin receptors and phosphotyrosines.

The early effects of insulin on morphological changes were examined using Rat1 cells expressing approximately one million human insulin receptors (Rat1HIR cells). Insulin transiently induced circular membrane ruffling on Rat1HIR cells. The circular ruffles were detected by phase microscopy as well as by fluorescence microscopy when actin was stained with rhodamine-conjugated phalloidin. The circular ruffles were also visualized by fluorescence microscopy when stained with either anti-insulin receptor monoclonal antibody, alpha IR-1, or anti-phosphotyrosine antibody, followed by fluorescein isothiocyanate-labeled second antibodies. Control Rat1 cells or Rat1HIR A/K1018 cells expressing kinase-defective insulin receptors did not show any circular ruffles when treated with insulin. These results suggest that the circular ruffles are formed by actin reorganization induced by insulin and that this process requires the protein-tyrosine kinase activity of the receptor. Detection of insulin receptors and phosphotyrosines in the circular ruffles suggests the possibility that the insulin receptors in the ruffles may be highly active and phosphorylating either the receptors themselves or proteins associated with the ruffles. These results are consistent with our previous in vitro observations that a large aggregate form of the purified insulin receptor is a highly active protein-tyrosine kinase.

Actin Cytoskeleton↗

PubMed1994

Increased adhesion of neutrophils to retinal vascular endothelial cells exposed to hyperosmolarity.

Recent studies suggest that leukocytes may contribute to capillary occlusion and endothelial cell injury in diabetic retinopathy. The present study is an attempt to determine whether high glucose concentration/hyperosmolar conditions would increase neutrophil adhesion to retinal endothelial cell monolayers. Confluent monolayers of bovine retinal endothelial cells were incubated for 24 hr in 96-well microtiter plates in 5.5, 20, 50 or 100 mM glucose using 20, 50 or 100 mM mannitol as osmotic controls. After 24 hr the cells were observed by phase microscopy, washed, and then incubated with isolated human neutrophils for 20 min. Non-adherent neutrophils were washed from the monolayers, and the number of adherent neutrophils was determined using an Elisa assay for neutrophil elastase. Adhesion of neutrophils to confluent monolayers of human umbilical vein and bovine aortic endothelial cells at all levels of glucose were assayed in a similar fashion for comparison. Neutrophil adherence to bovine retinal endothelial cells was significantly increased (P < 0.05, n = 8; paired t-test) at all elevated glucose concentrations compared with adherence at control (5.5 mM) glucose concentration. The effect was also concentration dependent with 20, 50 and 100 mM glucose resulting in 35, 57 and 70% increases respectively over controls. However, mannitol at equal concentrations produced similar increases in neutrophil adherence, indicating that the increases in adhesion caused by elevated glucose concentration were due to hyperosmolarity and not some special effect of glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Schwann cells degrade myelin and proliferate in the absence of macrophages: evidence from in vitro studies of Wallerian degeneration.

Interruption of axonal continuity in peripheral nerve trunks leads to axonal and myelin breakdown and removal distal to the injury site, a process known as Wallerian degeneration. Clearance of axonal and myelin debris has been attributed to the cooperative actions of two cell types, the indigenous Schwann cells and macrophages recruited to the regions of tissue damage. Recent work in this area has suggested a limited role for Schwann cells in myelin degradation and has emphasized the role of macrophages, not only in myelin clearance but also in the stimulation of Schwann cell proliferation which also occurs during Wallerian degeneration. In this report, we demonstrate that rat Schwann cells are capable of substantial myelin degradation unaided by macrophages. Observations were made following excision of neuronal somata from well-myelinated rat dorsal root ganglion neuron/Schwann cell co-cultures. The various stages of myelin breakdown were observed by phase microscopy, Sudan black staining, or electron microscopy. The time course for breakdown of individual myelin internodes varied from 2 to 10 days after injury and was to some extent dependent upon the original internodal length. Additionally, we show that most Schwann cells involved in Wallerian degeneration in the absence of macrophages undergo cell division following degradation of myelin into granules visible by light microscopy. The co-cultures employed were essentially free of macrophages as assessed by immunostaining for the OX42, ED2, and ED1 macrophage markers. No macrophages were detected by light or electron microscopy in the vicinity of the identified Schwann cells and furthermore, macrophages/monocytes were rarely observed in uninjured co-cultures as assessed by fluorochrome-conjugated acetylated LDL labelling. These results provide evidence in support of the ability of Schwann cells to carry out degradation of short myelin segments and to proliferate without macrophage assistance during Wallerian degeneration in vitro.

Animals↗

PubMed1985

Differential proliferative responses of cultured Schwann cells to axolemma and myelin-enriched fractions. II. Morphological studies.

Axolemma-enriched and myelin-enriched fractions were prepared from bovine CNS white matter and conjugated to fluorescein isothiocyanate (FITC). Both unlabelled and FITC-labelled axolemma and myelin were mitogenic for cultured rat Schwann cells. Treatment of Schwann cells with the FITC-labelled mitogens for up to 24 h resulted in two distinct morphological appearances. FITC-myelin-treated cells were filled with numerous round, fluorescent-labelled intracellular vesicles, while FITC-axolemma-treated cells appeared to be coated with a patchy, ill-defined fluorescence, primarily concentrated around the cell body but extending onto the cell processes. These observations were corroborated under phase microscopy. Electron microscopy revealed multiple, membrane-bound, membrane-containing phagosomes within myelin-treated cells and to a far lesser extent in axolemma-treated cells. The effect on the expression of the myelin-mediated and axolemma-mediated mitogenic signal when Schwann cells were treated with the lysosomal inhibitors, ammonium chloride and chloroquine, was evaluated. The mitogenicity of myelin was reduced 70-80% by these agents whereas the mitogenicity of axolemma was not significantly altered under these conditions. These results suggest that axolemma and myelin stimulate the proliferation of cultured Schwann cells by different mechanisms. Myelin requires endocytosis and lysosomal processing for expression of its mitogenic signal; in contrast, the mitogenicity of axolemma may be transduced at the Schwann cell surface.

Animals↗

PubMed1983

Use of pancreatic beta cells in culture to identify diabetogenic N-nitroso compounds.

Epidemiological observations suggest that environmental factors play a role in the pathogenesis of insulin dependent diabetes mellitus (1). Several chemicals have been identified as specific beta cell toxins (2-4). We report here studies to determine the feasibility of using monolayer cultures of pancreatic beta cells from neonatal rat to screen potential diabetogenic chemicals. Cytotoxicity was monitored both by phase microscopy and the release of insulin into the culture medium. In comparative studies, cellular protein and release of 51chromium (51Cr) were measured after addition of test compounds to cultures of fibroblasts derived from pancreatic tissue. The nitrosoamides 1 methyl-l-nitrosourea (MNU), 1,3 bis (2-choroethyl) nitrosourea (BCNU), chlorozotocin (CLZ), and the beta cell toxin, streptozotocin (SZ), were examined. CLZ and SZ were more toxic to pancreatic beta cells than to fibroblasts. In contrast, MNU and BCNU damaged both beta cells and fibroblasts at identical concentrations. These results suggest that in vitro techniques can be used to identify chemicals that selectively injure beta cells. Although SZ-induced toxicity was ameliorated with addition of nicotinamide to cultures of beta cells, nicotinamide did not prevent damage caused by CLZ. This observation indicates different mechanisms of drug-induced cytotoxicity.

Animals↗

PubMed1986

Increased viability and differentiation of normal and dystrophic striated muscle in vitro.

Primary cultures of muscle from normal (line 412) and dystrophic (line 413) chick embryos were exposed to corticosterone-21-acetate (C-21-A) or sodium ibuprofen (Motrin) for 28 d after myotube formation. Ibuprofen (0.5 to 500 micrograms/ml) or C-21-A (0.4 to 40 micrograms/ml)-treated cultures were fixed and assessed semiquantitatively using phase microscopy. On this basis, ibuprofen (50 micrograms/ml) and C-21-A (40 micrograms/ml) seemed to be effective in maintaining both normal and dystrophic muscle cultures. Using ibuprofen and C-21-A at these concentrations, experiments were repeated and analyzed quantitatively. Ibuprofen maintained culture viability (up to 68% more myotubes than untreated controls) but had no significant effect on the number of striated cells. C-21-A effectively maintained culture viability (up to 73% increase) and strongly promoted the formation of striated cells in these cultures (up to a sixfold increase). Both normal and dystrophic cultures were affected similarly by these agents, but the dystrophic cultures showed more consistent if not more extensive improvements in the parameters examined here. Thus, it seems that ibuprofen and C-21-A may affect both normal and dystrophic muscle directly to maintain survival and even promote differentiation.

Animals↗

PubMed1976

Preparation of large numbers of uniform tracheal organ cultures for long term studies. I. Effects of serum on establishment in culture.

Rat tracheas were each sectioned into fourteen rings of equal size with a slicing device which holds evenly spaced razor blades in register. The razor blades were positioned to minimize shearing of tissues during sectioning so that there was no gross tissue disruption or cell death distant from cut edges. Hundreds of these fragments can be conveniently prepared for studies requiring replicate samples. The cultures can be established in McCoy's 5a (modified) medium with or without calf serum. Cultures grown in the presence of calf serum were compared with those grown in serum-free medium, using vital phase microscopy, transmission and scanning electron microscopy, and light microscopic autoradiography of thymidine incorporation. When there is calf serum in the medium, epithelization of the entire surface of the ring occurs rapidly with the cells flattening and migrating as a sheet of closely apposed cells. Until migration is complete, mitoses are limited to the original mucosa near the cut edge. Without calf serum, migration is slow. The cells do not flatten or become closely apposed. Mitoses appear later but are present on all areas of the surface before migration is complete. In both serum-containing and serum-free media, ciliated cells are included in the migrating population and differentiation into pseudostratified epithelium occurs on newly epithelialized surfaces. The differing pattern of mitotic activity makes culture in serum-containing media more suitable for studies of wound healing and culture in serum-free medium more useful for some cytotoxicity and carcinogenicity studies.

Animals↗

PubMed1987

Tumor cell detection method using complement-mediated cytolytic reaction and imaging sensor system.

A novel tumor-detection system consisting of complement-mediated cytolytic reaction and an image processing system was developed for the simple and rapid determination of tumor cells. The present system consists of a CCD image sensor, image memory board, personal computer, and microscope. When monoclonal antibody 3C4, which is specific to the guinea pig hepatoma L-10, was added to cell suspension, only L-10 cytolysis occurred. Cytolysis caused a decrease in brightness of the cells observed by phase-contrast microscopy. The phase contrast image of the cells before cytolysis was converted to a digitalized signal and stored in computer memory. After cytolysis, a brightness threshold above that of lysed cells was subtracted from the digitalized signal and compared to the signal stored before reaction. L-10 cells in mixed cell suspension were determined specifically by the system. Measurement time was only 2 sec and overall time, including reaction time, was approximately 30 min. Since this method does not require a cell washing process, automation of the whole system is possible.

Animals↗

PubMed1978

Plasma membrane vesiculation: a cellular response to injury.

The shedding of plasma membrane vesicles has been shown to result from exposure of monolayer cell cultures to formaldehyde and other sulfhydryl blocking agents. Incubation of cells in concentrations of these agents as low as 5 to 10 mM for intervals as brief as fifteen minutes is effective (Scott, 1976). Plasma membrane vesiculation has been shown to be an energy-dependent process that requires Ca++ and physiological temperature. Following plasma membrane vesiculation, cell monolayers appear intact by phase microscopy and show only slight evidence of cell injury by electron microscopy. In view of these observations, the question has been raised whether plasma membrane vesiculation is compatible with continued cell growth and metabolism. The experiments described in this paper were designed to answer these questions. We pulse exposed 3T3 mouse embryo cells to concentrations of formaldehyde, between 2.5 and 250 mM, for intervals 15, 30 or 60 min. Cell momolayers were then washed in a variety of different media in an attempt to reverse the effect of formaldehyde on cells. Cell monolayers were thereafter assayed for the shedding of plasma membrane vesicles and for their ability to transport 2-deoxy-D-glucose. Cells were also replated in serum-containing medium and their ability to grow was assayed over a seven day interval. The results show an inverse relationship between the shedding of plasma membrane vesicles and the ability of the cells to transport nutrients and to grow. We interpret these data to suggest that the process of plasma membrane vesiculation results from a form of cell injury which blocks cellular metabolism and growth.

Biological Transport, Active↗

PubMed1975

Granular cell tumor (myoblastoma) of the orbit.

An 8-year-old white boy had a tumor in the left orbit with clinical manifestations of muscular impairment, exophthalmos, and reduced visual acuity. Five months after exenteration of the orbit the boy was healthy without tumor recurrence. Based on our observations with electron, light, and phase microscopy, and histochemical studies, we conclude that this tumor is benign, of histiocytic origin, and we propose the term granular cell histiocytoma be used to describe this entity.

Cell Nucleolus↗

PubMed1977

Maintenance of neurons isolated in bulk from rat brain: incorporation of radiolabeled substrates.

Neurons isolated in bulk from 10- to 15-day-old rat brain can be maintained for 18--24 h. Phase microscopy shows that the cells remain morphologically intact during this time. As a criterion of viability, the incorporation of radiolabeled amino acids and uridine into trichloroacetic acid-insoluble material was selected. The cells are capable of the incorporation both after isolation and after maintenance. The uptake is constant with time, is proportional to substrate concentration, and is inhibited by puromycin and cycloheximide, but not by chloramphenicol. Thus the cells remain viable during this maintenance period. This system may provide a model for the study of the synthesis of specific neuronal components.

Amino Acids↗

PubMed1986

Growth cones isolated from identified Aplysia neurons in vitro: biochemical and morphological characterization.

The right upper quadrant (RUQ) cells (R3-R13) of Aplysia regenerating in dissociated cell culture form unusually large growth cones. The movement of these growth cones was observed by time-lapse phase microscopy and their ultrastructure was examined by transmission electron microscopy. Their behavior and ultrastructure have features that are typical of growth cones in vitro. Additionally, they contain neurosecretory granules similar to those found in these cells in vivo. Because RUQ growth cones are large, they can be isolated by manual dissection. RUQ cells were grown in the presence of [35S]methionine and the labeled proteins transported to the growth cones were analyzed by SDS-PAGE. These proteins were compared to those in RUQ cell bodies, RUQ neurites, and to those in the neurites and cell bodies of other identified neurons grown in vitro. Most proteins synthesized by RUQ cells in vitro are transported to their growth cones, including several glycoproteins and the precursor to the R3-R14 neuropeptide. Neuropeptides are also synthesized by a number of other Aplysia neurons growing in vitro. We examined R2, LPL1, R15, and left upper quadrant neurons and found that their precursor peptides, like those of R3-R14, are readily recognized as major cell-specific radiolabeled bands on SDS gels. The presence in regenerating growth cones of neuropeptides, neurosecretory granules, and glycoproteins known to be rapidly transported toward synapses in vivo supports the emerging view that the growth cone in vitro contains not only a motility apparatus but also a macromolecular assembly capable of forming an active synapse immediately upon or shortly after contacting targets.

Actins↗

PubMed1983

Nonasbestos pulmonary mineral fibers in the general population.

Total pulmonary nonasbestos mineral content was determined for a series of 20 patients who had no occupational dust exposure. To extract mineral fibers, lung tissue was dissolved in bleach and the treated sediment transferred to the electron microscope grid. Mineral fibers were identified using electron diffraction and energy dispersive x-ray spectroscopy. The mean number of nonasbestos fibers, 106 X 10(3)/g wet lung, was almost identical to the mean number of asbestos fibers, 102 X 10(3)/g. Thirteen different species or groups of nonasbestos minerals were found: apatite accounted for 18% of the total and talc for 16%. All other forms accounted for less than 8% each. Silica was found in every lung, and talc in 19 of 20 lungs. Of the fibers, 86% were shorter than 5 microns, and most of the fibers had aspect ratios less than 15. No correlations were seen between numbers or types of fibers and age, sex, or smoking. It is concluded that (1) substantial numbers of nonasbestos fibers are present in lungs of the general population. Most of these fibers are short and of low aspect ratio; and (2) by phase microscopy one is as likely to observe nonasbestos as asbestos fibers in these preparations, indicating that light microscopic methods are not suitable for this type of analysis. These data provide a baseline for comparison with patients believed to have a mineral fiber-related disease.

Adult↗

PubMed1978

Acquisition of synchronous beating between embryonic heart cell aggregates and layers.

Synchronous beating between chick embryonic heart cell aggregates and heart cell layers was used to study the relationship between intercellular adhesion and ionic coupling. Adhesion was measured by counting the proportion of aggregates which were not to be removed from cell layers by gentle washing after a 30 min incubation. Synchrony between bound aggregates and contiguous layers was assessed by phase microscopy. The first evidence of synchrony was seen 1.5 h after addition of aggregates to layers, following which there was an increase in the percentage of aggregates beating synchronously, reaching over 50% at 7 h and slowly increasing to a maximum of 65% by 24 h. Scanning electron microscopy and autoradiography of thymidine-labeled cells suggest that synchrony does not depend on cell movement at the interface between aggregate and layer. Acquisition of synchrony can be prevented completely by inhibiting protein synthesis, although pulsation of aggregates and layers continues in proportions unchanged from controls. After reversal of protein synthesis inhibition, synchrony is acquired at a rate and to an extent closely resembling that of newly adherent controls. These data indicate that ionic coupling is neither an inevitable nor an immediate consequence of adhesion. Since ionic coupling has been shown to correlate with the presence of gap junctions, the findings suggest that gap junctions are not involved in the initial events responsible for intercellular adhesion in vitro and that their formation following adhesion in this system may depend upon protein synthesis.

Animals↗

PubMed1986

A second growth state for Schizosaccharomyces pombe.

The kinetics of volume increase in individual cells of Schizosaccharomyces pombe were determined by phase microscopy at osmolalities lower than those reported in the literature. At the highest osmolality, 550 mmol/kg, all cells followed a biphasic pattern of growth, in which cell volumes increased to their maximum values approximately four-fifths of the way through the growth cycle. At lower osmolalities (400-420 mmol/kg), many or most of the cells followed a different growth pattern, with a linear increase in cell volume throughout the cycle. The following evidence indicates that a different regulatory mechanism is responsible for the linear growth pattern: (1) Regulation of cell length and diameter differed for the two cases. During biphasic growth, cell length also increased biphasically and cell diameters remained essentially constant during the cycle, whereas during linear growth, both cell length and diameter increased linearly until formation of the cell plate very late in cycle. (2) The two different growth states were observed for cells growing on two very different kinds of medium. (3) Frequency distributions of the two growth patterns showed that there were two distinct groups of growing cells, with and without a cell volume plateau; these results rule out a single growth state in which plateaus are graded from large to infinitesimally small. (4) Linear regressions fitted to the data for linear growth did not differ significantly from the theoretical model for linear growth without a terminal plateau. These results reveal the operation of a second regulatory system for cell growth in S. pombe at osmolalities closer to those in liquid medium. The occurrence of transitions between the two growth states in successive generations and the agreement between several growth parameters for the two modes suggest that the growth states are closely related.

Cell Cycle↗