Effects of isolation and culture of turkey primary follicular oocytes on morphology and germinal vesicle integrity

A novel approach to the production of transgenic poultry is to use primary follicular oocytes (PFOs). However, fundamental information regarding the impact of isolation and culture procedures on PFO integrity is lacking. This study describes the isolation and culture of PFOs from mature turkeys and the effects of these procedures on PFO morphology and germinal vesicle (GV) integrity. To isolate PFOs, ovarian cortex was incubated in trypsin-EDTA alone or further incubated in collagenase plus hyaluronidase (CH). About 200 to 500 PFOs, ranging in size from less than 100 microns in diameter to 1,000 microns, were recovered from each ovary. The culture of PFOs less than 100 microns in diameter for 4 h resulted in blebbing of the oolemma followed by extrusion of ooplasm. Primary follicular oocytes 100 to 250 microns in diameter survived culture for 24 h whereas larger PFOs survived for up to 7 d. Those PFOs with intact granulosa cell investments survived longer than those fully or partially denuded of granulosa cells with CH. Co-culture of PFOs (100 to 250 microns in diameter) on a monolayer of granulosa cells derived from mature, yellow-yolk follicles augmented PFO survival rates. The rate of GV breakdown was not influenced by the isolation or culture of the PFO. These data provide the basis for developing procedures for the in vitro maturation and in vitro fertilization of isolated PFOs.     

Planimetry of bronchoalveolar macrophages. Importance of preparation and staining techniques.

Bronchoalveolar lavage seems a well-established, valuable research tool in the study of alveolar macrophages. The influence of fixation, cytocentrifugation and staining procedures on the cellular and nuclear size has been investigated by planimetry. As a reference, mean profile areas of 109 and 39 microns 2 for cell and nucleus, respectively, were measured for alveolar macrophages suspended in the hemocytometer. For comparison, stained Cytospin preparations were measured. Unfixed cells were compressed during cytocentrifugation. The cellular profile areas for Cytospin preparations increased about 15% and 70% after May-Grünwald-Giemsa and Feulgen staining, respectively. The nuclear area was approximately 25% larger for both staining procedures as compared to the hemocytometer values. When the cells had been fixed prior to cytocentrifugation, these differences were less conspicuous. No significant differences were observed after May-Grünwald-Giemsa staining, showing a cellular area of 114 microns 2 and a nuclear area of 45 microns 2. Depending on the staining procedure, low nucleus:cell ratios (31%) were observed after Feulgen staining, while higher values (about 43%) were measured after May-Grünwald-Giemsa staining, regardless of which fixation or centrifugation procedure had been followed. In conclusion, these findings indicate that fixation should be carried out in order to prevent cell changes resulting from cytocentrifugation. Moreover, different staining procedures considerably influence the measurement of cellular and nuclear profile areas and the determination of nucleus:cell ratios.     

The distribution of centrosomes in migrating endothelial cells during wound healing in situ.

We have examined the distribution of centrioles in rabbit thoracic aortic endothelial cells induced to migrate by wounding the endothelium in situ. Following denudation of the endothelium from a segment of the aorta with a balloon catheter, a wound edge was created from which endothelial cells began to migrate onto the denuded surface. In this in situ model of cell migration, the position of centrioles was determined in cells along the wound edge by immunofluorescence and antibodies which specifically label these cell organelles, and then they were classified in relation to the nucleus and the direction of cell migration as being oriented toward the wound, in the center, or away from wound. At time 0, as in normal unwounded adult rabbit aorta, no preferential orientation of centrioles was evident. Within 12 h after wounding, the centrioles in about 53% of endothelial cells near the wound edge were oriented toward the wound, while in less than 20% of the cells they were oriented away from wound. At 24 h, in cells up to 800 microns from the wound edge, centrioles in only about 10% of the endothelial cells were oriented away from wound, while in about 52% of cells they were found in the center and in 38% of the cells they remained oriented toward the wound. At 48 h, up to 2000 microns from the wound edge, the majority of endothelial cells had their centrioles in the center, possibly as a result of an increase in mitotic index as cells replicate to reestablish an intact endothelium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Initiation of DNA replication in chromosomes of Chinese hamster ovary cells

The initiation of DNA replication and the subsequent chain elongation were studied using Chinese hamster ovary cells synchronized at the beginning of S phase. The cells were synchronized by a combination of mitotic selection and treatment with 5-fluorodeoxyuridine (FdU). The use of this drug at a concentration of 10^–5 M was found to effectively prevent the leakage of cells into S phase. Reversal of the FdU block by supplying thymidine resulted in the synchronous onset of initiation at multiple sites in each cell. The length of the nascent chains, as determined by autoradiography and velocity sedimentation in alkaline gradients, increased linearly with time during the first twenty minutes of S phase after release. — We applied these procedures to study the effects of the length of an FdU block on the number of functional origins per cell, the rate of chain growth, and the rate of DNA synthesis per cell following reversal of the block. Although no change was noted in the rate of DNA synthesis in cells held at the beginning of S phase from 10.5 to 24 h after division, the rate of chain growth decreased from 0.94 to 0.28 microns per min. This decrease indicated that the number of functional origins increased markedly with length of FdU block. The calculated number of utilized origins per cell increased from 1,900 to 5,700. We also presented arguments that 1,900 origins per cell represents the approximate number of origins utilized by any cell held at the beginning of S phase for less than 10.5 h after division.     

Gravitational and magnetic activation methods applied to cultured cells (LK 35.2)

Monoclonal antibody 10.2-16 is directed toward the mouse class II major histocompatibility complex gene product 1-Ak expressed on the cell line LK35.2. Instead of activating cells by fluorophor we used (acrylamide-coated) heavy and magnetic microspheres of 0.6 micron in radius. These microspheres are chemically coupled (carbodiimide method) with the antibody toward the surface antigen. The cells are observed through a microscope with horizontal alignment, as they sediment in a (temperature controlled) tube with square cross-section. Stokes Law allows the determination of the density of cells (first alone) using viscosity and density of Dulbecco's modified Eagle's Medium together with the observed mean sedimentation velocity (66 microns/min) and a mean diameter of 10 microns. We found a density of 1.0558 +/- 0.0028 g/cm3 at 10 degrees C. Independently, thinly coated, heavy (and magnetizable) microspheres with the cited antibody are attached to cells and observed likewise. The increased sedimentation velocity permits us to show that the cells were fully covered with microspheres (290 per cell). A magnetic field gradient opposing gravity moved these cells against gravity with two different mean velocities, 340 microns/min and 850 microns/min. The higher velocity resulted in 290 particles per cell, the lower one in 130 particles per cell. The limits for the expansion of this method to smaller particle sizes (down to 10 nm) are evaluated.     

Size distribution of ferret luteal cells during pregnancy

Steroidogenic cells in the corpus luteum of the ferret (Mustela putorius) during early (Days 6 and 13) to midpregnancy (Day 24) were characterized using electron microscopy, immunocytochemical localization of neurophysin, and smears of dispersed cells obtained by dissociating luteal cells with collagenase. The latter were stained for 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity, and the diameters of the cells were determined with an ocular micrometer. Very small cells (less than 12 microns) stained negative for 3 beta-HSD, occurred in clumps of 5-50 cells, and were presumed to be primarily endothelial cells. 3 beta-HSD-positive cells covered a wide spectrum of sizes ranging from 14 to 56 microns and did not exist as two discrete populations. The ratio of small (less than 25 microns) to large (greater than 25 microns) cells was 1.86:1.0 on Day 6, with the 17- to 20-microns cell size class predominating. On the day of implantation (Day 13), about 75% of the cells ranged from 26 to 50 microns, with the 29-microns size predominating. By Day 24, the ratio of small-to-large cells had declined to 0.15. Nearly 90% of the cells were in the 26- to 56-microns range, the predominant size being 35 microns. All size classes of luteal cells stained negative for neurophysin on all 3 days of pregnancy studied. Luteal cells obtained on Days 6, 13, and 24 of pregnancy failed to reveal any evidence of mitosis after in vivo or in vitro colchicine treatment. We interpret these results as indicating that the 3 beta-HSD-positive luteal cells of ferrets progressively increase in size as small luteal cells complete their differentiation from granulosa cells and ultimately form larger luteal cells with somewhat different ultrastructural characteristics.     

Purification of Chlamydia trachomatis by a simple and rapid filtration method

A simple method for filter purification of Chlamydia trachomatis from cell culture is described. Crude homogenates of chlamydiae-infected cells were passed through a glass prefilter and a 0.6 microns pore diameter polycarbonate filter. The filtrate was then passed through a 0.2 microns pore diameter filter on which the chlamydiae were trapped. This filter was then back-washed to collect the organisms. These procedures removed cell debris and soluble protein, and yielded particles with a narrow size distribution. The mean yield of viable chlamydiae purified by filtration was 64% when the filters were washed at each stage of the process.     

Development of a novel microperfusion chamber for determination of cell membrane transport properties.

A novel microperfusion chamber was developed to measure kinetic cell volume changes under various extracellular conditions and to quantitatively determine cell membrane transport properties. This device eliminates modeling ambiguities and limitations inherent in the use of the microdiffusion chamber and the micropipette perfusion technique, both of which have been previously validated and are closely related optical technologies using light microscopy and image analysis. The resultant simplicity should prove to be especially valuable for study of the coupled transport of water and permeating solutes through cell membranes. Using the microperfusion chamber, water and dimethylsulfoxide (DMSO) permeability coefficients of mouse oocytes as well as the water permeability coefficient of golden hamster pancreatic islet cells were determined. In these experiments, the individual cells were held in the chamber and perfused at 22 degrees C with hyperosmotic media, with or without DMSO (1.5 M). The cell volume change was videotaped and quantified by image analysis. Based on the experimental data and irreversible thermodynamics theory for the coupled mass transfer across the cell membrane, the water permeability coefficient of the oocytes was determined to be 0.47 micron. min-1. atm-1 in the absence of DMSO and 0.65 microns. min-1. atm-1 in the presence of DMSO. The DMSO permeability coefficient of the oocyte membrane and associated membrane reflection coefficient to DMSO were determined to be 0.23 and 0.85 micron/s, respectively. These values are consistent with those determined using the micropipette perfusion and microdiffusion chamber techniques. The water permeability coefficient of the golden hamster pancreatic islet cells was determined to be 0.27 microns. min-1. atm-1, which agrees well with a value previously determined using an electronic sizing (Coulter counter) technique. The use of the microperfusion chamber has the following major advantages: 1) This method allows the extracellular condition(s) to be readily changed by perfusing a single cell or group of cells with a prepared medium (cells can be reperfused with a different medium to study the response of the same cell to different osmotic conditions). 2) The short mixing time of cells and perfusion medium allows for accurate control of the extracellular osmolality and ensures accuracy of the corresponding mathematical formulation (modeling). 3) This technique has wide applicability in studying the cell osmotic response and in determining cell membrane transport properties.     

Actin filaments, stereocilia, and hair cells of the bird cochlea. I. Length, number, width, and distribution of stereocilia of each hair cell are related to the position of the hair cell on the cochlea

Located on the sensory epithelium of the sickle-shaped cochlea of a 7- to 10-d-old chick are approximately 5,000 hair cells. When the apical surface of these cell is examined by scanning microscopy, we find that the length, number, width, and distribution of the stereocilia on each hair cell are predetermined. Thus, a hair cell located at the distal end of the cochlea has 50 stereocilia, the longest of which are 5.5 microns in length and 0.12 microns in width, while those at the proximal end number 300 and are maximally 1.5 microns in length and 0.2 micron in width. In fact, if we travel along the cochlea from its distal to proximal end, we see that the stereocilia on successive hair cells gradually increase in number and width, yet decrease in length. Also, if we look transversely across the cochlea where adjacent hair cells have the same length and number of stereocilia (they are the same distance from the distal end of the cochlea), we find that the stereocilia of successive hair cells become thinner and that the apical surface area of the hair cell proper, not including the stereocilia, decreases from a maximum of 80 microns2 to 15 microns2. Thus, if we are told the length of the longest stereocilium on a hair cell and the width of that stereocilium, we can pinpoint the position of that hair cell on the cochlea in two axes. Likewise, if we are told the number of stereocilia and the apical surface of a hair cell, we can pinpoint the location of that cell in two axes. The distribution of the stereocilia on the apical surface of the cell is also precisely determined. More specifically, the stereocilia are hexagonally packed and this hexagonal lattice is precisely positioned relative to the kinocilium. Because of the precision with which individual hair cells regulate the length, width, number, and distribution of their cell extensions, we have a magnificent object with which to ask questions about how actin filaments that are present within the cell are regulated. Equally interesting is that the gradient in stereociliary length, number, width, and distribution may play an important role in frequency discrimination in the cochlea. This conclusion is amplified by the information presented in the accompanying paper (Tilney, L.G., E.H. Egelman, D.J. DeRosier, and J.C. Saunders, 1983, J. Cell Biol., 96:822- 834) on the packing of actin filaments in this stereocilia.     

Implications of nuclear diameter in small cell lung carcinoma

The nuclear diameter of 5,117 malignant cells from 42 small cell lung carcinoma (SCLC) patients was assessed either on pretreatment tissue sections (35 cases) or cytologic smears (7 cases) by ocular micrometry. The SCLCs were subtyped as 30 oat cell carcinomas and 12 intermediate cell carcinomas according to the World Health Organization classification, based on the predominant histology of the tumor. The median number of nuclei measured from each patient was 110. All patients were treated identically by sequential hemibody and local irradiation combined with chemotherapy and had a median follow-up time of 310 days. The mean nuclear diameter (+/- standard error) obtained from tissue sections was 8.2 +/- 0.03 microns (median = 8.0), including 7.3 +/- 0.03 microns (median = 7.0) for oat cell cases and 9.5 +/- 0.06 microns (median = 9.0) for intermediate cell cases (P less than .001). In 28.6% of these patients, the nuclear diameter overlapped in the range of 8 microns to 9 microns between both subtypes. Comparisons between the nuclear diameter of primary and metastatic SCLC cells revealed no statistically significant differences. The nuclear diameter of malignant cells correlated with the mitotic index and stage of disease, but did not correlate with the other nuclear morphologic variables or with survival. The only identified prognostic factor was the stage of disease; these results indicate that the nuclear diameter of malignant cells should not be considered a prognosticator or a guide for therapy in SCLC patients.     

Newly assembled microtubules are concentrated in the proximal and distal regions of growing axons

We have investigated the sites of microtubule (MT) assembly in neurons during axon growth by taking advantage of the relationship between the proportion of tyrosinated alpha-tubulin (tyr-tubulin) in MTs and their age. Specifically, young (newly assembled) MTs contain more tyr-tubulin than older (more long-lived) MTs. To quantify the relative proportion of tyr-tubulin in MTs, cultured rat sympathetic neurons were permeabilized under conditions that stabilize existing MTs and remove unassembled tubulin. The MTs were then double-stained with antibodies to tyr-tubulin (as a measure of the amount of tyr-tubulin in MTs) and to beta-tubulin (as a measure of total MT mass), using immunofluorescence procedures. Cells were imaged with a cooled charge-coupled device camera and the relative proportion of tyr-tubulin in the MTs was quantified by computing the ratio of the tyr-tubulin fluorescence to the beta-tubulin fluorescence using a novel application of digital image processing and analysis techniques. The amount of tyr-tubulin in the MTs was highest in the cell body and at the growth cone; peak ratios in these two regions were approximately 10-fold higher than for the axon shaft. Moving out from the cell body into the axon, the tyr-tubulin content declined over an average distance of 40 microns to reach a constant low value within the axon shaft and then rose again more distally, over an average distance of 110 microns, to reach a peak at the growth cone (average axon length = 358 microns). These observations indicate that newly assembled MTs are concentrated in the proximal and distal regions of growing axons and therefore that the cell body and growth cone are the most active sites of MT assembly dynamics in neurons that are actively extending axons.     

A colloidal gold labeling technique for the direct determination of the surface area of eukaryotic cells

We have developed a colloidal gold labeling technique for the direct quantitation of the cell surface area. The method is based on coating the cell surface with [195Au] colloidal gold-protein complexes followed by morphometric determination of the labeling density (gold particles/micron2 cell surface) and radiometric determination of the total number of gold particles bound per cell. The ratio of both values directly gives the cell surface area. The accuracy of the method was shown using Staphylococcus aureus cells as a model system, where the cell surface area determined with our assay (4.0 microns2) corresponded well to the value calculated from the radius of the cells (3.6 microns2). In a more complex model system J-774 mouse macrophages were labeled with different amounts of [195Au] gold-protein complexes to show that the assay is independent of the degree of saturation of the cell surface binding sites. Both high (135 Au/microns2) and low (65 Au/microns2) labeling densities resulted in a surface area of about 1200 microns2. The technique finally was applied to L-929 fibroblasts to determine the increase of the cell surface area when the cells change from a spherical to a flat monolayer state. We found that the cell surface area increased 3-fold during the spreading process. The results show that the colloidal gold labeling technique allows the direct determination of the surface area of complex eukaryotic cells. The technique is suitable for the quantitation of changes in the surface architecture known to occur in different functional states of eukaryotic cells.     

Effect of vinblastine on the cell cycle and migration of ameloblasts of mouse incisors as shown by autoradiography using 3H-thymidine

The effects of vinblastine on the cell cycle and the migration of ameloblasts were studied in the lower incisors of mice by labelling the cells with 3H-thymidine ([3H]TdR) and radioautography. A group of mice received 2 micrograms/g of body weight vinblastine intraperitoneally and 6 hr after these animals and those of a control group were injected with 1 microCi/g body weight of [3H]TdR, and sacrificed at time intervals from 0.75 hr to 15 days. The generation time of ameloblasts in the progenitor compartment was 14.8 hr in animals treated with vinblastine and 17 hr in the controls, using the FLM curve method; with the grain dilution method the duration was respectively 29.25 hr and 25.96 hr. The thymidine labelling index of the treated animals was 50% higher than the controls. The velocity of ameloblast migration, determined either by the displacement of the most incisally labelled cell or by the grain dilution method, was lower in the experimental group (2.48 cell positions/hr and 9.18 microns/hr respectively) as compared with the control (3.21 cell positions/hr and 18.88 microns/hr respectively). The results on the ameloblast production rate are contradictory but the slowing down in the velocity of cell migration is compatible with a decrease of the rate of cell production in the progenitor compartment as a vinblastine effect.     

Heat response of human melanoma multicellular spheroids: comparisons with single cells and xenografted tumors

Multicellular spheroids were grown from cells derived directly from a human melanoma xenograft propagated in athymic mice. The histological appearance of the spheroids was similar to that of the parent xenograft. The spheroids were heated in culture medium (42.5-44.5 degrees C); growth delay and single cell survival measured in soft agar were used as end points. There was a good correlation between the results obtained with these two end points, indicating that growth delay depended mainly on cell survival. Large spheroids (200 +/- 12 microns in diameter) were found to be more heat sensitive than small ones (100 +/- 5 microns in diameter), probably because the physiological conditions in large spheroids were more favorable for cell inactivation. The cells were more resistant when heated as spheroids than as single cells. This effect was not a secondary effect of differences in cell-cycle distribution. Spheroids were also found to be more heat resistant than xenografted tumors. In the tumors, heat treatment caused vascular damage which resulted in delayed cell death due to hypoxia and/or nutrient deficiency. It is concluded that spheroids seem well suited for studies of primary heat-induced cytotoxic effects. However, they appear not to mirror the complex heat response of tumors since that response also includes secondary effects, related to heat-induced reduced perfusion.     

A comparative study in twelve mammalian species of volume densities, volumes, and numerical densities of selected testis components, emphasizing those related to the Sertoli cell

Morphometric studies were performed on 12 mammalian species (degu, dog, guinea pig, hamster, human, monkey, mouse, opossum, rabbit, rat, stallion, and woodchuck) to determine volume density percentage (Vv%), volume (V), and numerical density (Nv) of seminiferous tubule components, especially those related to the Sertoli cell, and to make species comparisons. For most species, measurements were taken both from stages where elongate spermatids were deeply embedded within the Sertoli cell and from stages near sperm release where elongate spermatids were in shallow crypts within the Sertoli cell. Montages, prepared from electron micrographs, were used to determine Vv% of Sertoli cell components in seminiferous tubules. Excluding the tubular lumen, the Sertoli cell occupied from a high of 43.1% (woodchuck) to a low of 14.0% (mouse) of the tubular epithelium. There was a strong negative correlation (r = -0.83; P less than 0.005) of volume occupancy of Sertoli cells with sperm production. Nuclear volume, as determined by serial reconstruction using serial thick sections, ranged from a high of 848.4 microns 3 (opossum) to a low of 273.8 microns 3 (degu). There was no correlation (r = 0.02) of nuclear volume with volume occupancy (Vv%) in the tubule. Sertoli cell volume was determined by point-counting morphometry at the electron-microscope level as the product of the nuclear size and points determined over the entire cell divided by points over the nucleus. Sertoli cell V ranged from 2,035.3 microns 3 (degu) to 7,011.6 microns 3 (opossum) and was highly correlated (r = 0.85; P less than 0.001) with nuclear size. However, there was no significant correlation between the Sertoli cell size (V) and volume occupancy (Vv%; r = 0.13) or sperm production (r = -0.21). Stereological estimates of the numerical density (Nv) of Sertoli cells ranged from a high of 101.9 x 10(6) (monkey) to a low of 24.9 x 10(6) (rabbit) cells per cm3 of testicular tissue. There was no correlation of numerical density of Sertoli cells with sperm production (r = 0.002). A negative correlation was, however, observed between the numerical density of the Sertoli cells and the Sertoli cell size (r = -0.79; P less than 0.002). Data from the present study are compared with those previously published. This is the first study to compare Sertoli cell morphological measurements using unbiased sampling techniques. Morphometric data are provided which will serve as a basis for other morphometric studies.     

Fine needle aspiration biopsy of the breast. Value of nuclear morphometry after different sampling methods

OBJECTIVE: To study the potential of nuclear morphometry in supporting the interpretation of fine needle aspiration biopsy (FNAB) samples of the breast fixed in 50% ethanol and centrifuged on slides. STUDY DESIGN: Computerized morphometry was used to outline the nuclei of breast epithelial cells in breast cancer, fibroadenoma and fibrocystic disease. The diagnoses were histologically confirmed. We applied 2 different sampling methods (measurements done on cell groups and on free cells). RESULTS: The mean nuclear area of cell groups of malignant samples (23) varied from 42 to 125 microns 2, in fibroadenomas from 30 to 50 microns 2 and in fibrocystic disease from 26 to 57 microns 2. The mean nuclear area of free cells varied as follows: cancer, 66-181 microns 2; fibroadenoma, 33-70 microns 2; fibrocystic disease, 35-60 microns 2. Apocrine metaplasia was excluded from comparison on a morphologic basis. CONCLUSION: The study suggests that if the mean nuclear area of cell groups is < 42 microns 2, the lesion is probably benign; if > 57 microns 2, and apocrine metaplasia is excluded, malignancy should be considered. The differential diagnosis between carcinoma and fibroadenoma could be based on free cells: mean area of free cell nuclei < or = 65 microns 2 suggested a benign lesion, and of > or = 71 microns 2 suggested a malignant lesion. Morphometric nuclear size features (exemplified by nuclear area) appeared efficient in distinguishing between malignant and benign lesions when measured from free cells and cell groups.     

Measurement of marine picoplankton cell size by using a cooled, charge-coupled device camera with image-analyzed fluorescence microscopy.

Accurate measurement of the biomass and size distribution of picoplankton cells (0.2 to 2.0 microns) is paramount in characterizing their contribution to the oceanic food web and global biogeochemical cycling. Image-analyzed fluorescence microscopy, usually based on video camera technology, allows detailed measurements of individual cells to be taken. The application of an imaging system employing a cooled, slow-scan charge-coupled device (CCD) camera to automated counting and sizing of individual picoplankton cells from natural marine samples is described. A slow-scan CCD-based camera was compared to a video camera and was superior for detecting and sizing very small, dim particles such as fluorochrome-stained bacteria. Several edge detection methods for accurately measuring picoplankton cells were evaluated. Standard fluorescent microspheres and a Sargasso Sea surface water picoplankton population were used in the evaluation. Global thresholding was inappropriate for these samples. Methods used previously in image analysis of nanoplankton cells (2 to 20 microns) also did not work well with the smaller picoplankton cells. A method combining an edge detector and an adaptive edge strength operator worked best for rapidly generating accurate cell sizes. A complete sample analysis of more than 1,000 cells averages about 50 min and yields size, shape, and fluorescence data for each cell. With this system, the entire size range of picoplankton can be counted and measured.     

Differential neuronal loss following early postnatal alcohol exposure

Neonatal rats were exposed to 6.6 g/kg of alcohol each day between postnatal days 4 and 10 while artificial-rearing procedures were used, in a manner which produced high peak and low trough blood alcohol concentrations each day. Gastrostomy controls were reared artificially with maltose/dextrin isocalorically substituted for alcohol in the milk formula, and suckle controls were reared normally by dams. The pups were sacrificed on day 10 and tissue sections (2 microns thick) were obtained in the sagittal plane through the cerebellum and in the horizontal plane through the hippocampal formation. Overall area measures were obtained for the hippocampus proper, area dentata, and cerebellum, along with areas of the cell layers of these regions. In the hippocampal formation, cell counts were made of the pyramidal cells of the hippocampus proper, the multiple cell types of the hilus, and the granule cells of the area dentata. In the cerebellum, cell counts of Purkinje cells, granule cells of the granular layer, granule cells of the external granular layer, and mitotic cells of the external granular layer were obtained from lobules I, V, VII, VIII, and IX. Alcohol selectively reduced areas and neuronal numbers in the cerebellum but had no significant effects on neuronal numbers in the hippocampal formation. Purkinje cells exhibited the greatest percent reductions, and cerebellar granule cells were significantly reduced in the granular layer but not in the external granular layer. All lobules showed these effects, but lobule I was significantly more affected than the other four lobules that were analyzed. The results demonstrate the differential vulnerability of selected neuronal populations to the developmental toxicity of alcohol exposure during the brain growth spurt.     

Measurement of marine picoplankton cell size by using a cooled, charge-coupled device camera with image-analyzed fluorescence microscopy.

Accurate measurement of the biomass and size distribution of picoplankton cells (0.2 to 2.0 microns) is paramount in characterizing their contribution to the oceanic food web and global biogeochemical cycling. Image-analyzed fluorescence microscopy, usually based on video camera technology, allows detailed measurements of individual cells to be taken. The application of an imaging system employing a cooled, slow-scan charge-coupled device (CCD) camera to automated counting and sizing of individual picoplankton cells from natural marine samples is described. A slow-scan CCD-based camera was compared to a video camera and was superior for detecting and sizing very small, dim particles such as fluorochrome-stained bacteria. Several edge detection methods for accurately measuring picoplankton cells were evaluated. Standard fluorescent microspheres and a Sargasso Sea surface water picoplankton population were used in the evaluation. Global thresholding was inappropriate for these samples. Methods used previously in image analysis of nanoplankton cells (2 to 20 microns) also did not work well with the smaller picoplankton cells. A method combining an edge detector and an adaptive edge strength operator worked best for rapidly generating accurate cell sizes. A complete sample analysis of more than 1,000 cells averages about 50 min and yields size, shape, and fluorescence data for each cell. With this system, the entire size range of picoplankton can be counted and measured.     

Influence of cell cycle and oncogene activity upon topoisomerase IIalpha expression and drug toxicity

The cell cycle, oncogenic signaling, and topoisomerase (topo) IIalpha levels all influence sensitivity to anti-topo II drugs. Because the cell cycle and oncogenic signaling influence each other as well as topo IIalpha levels, it is difficult to assess the importance of any one of these factors independently of the others during drug treatment. Such information, however, is vital to an understanding of the cellular basis of drug toxicity. We, therefore, developed a series of analytical procedures to individually assess the role of each of these factors during treatment with the anti-topo II drug etoposide. All studies were performed with asynchronously proliferating cultures by the use of time-lapse and quantitative fluorescence staining procedures. To our surprise, we found that neither oncogene action nor the cell cycle altered topo IIalpha protein levels in actively cycling cells. Only a minor population of slowly cycling cells within these cultures responded to constitutively active oncogenes by elevating topo IIalpha production. Thus, it was possible to study the effects of the cell cycle and oncogene action on drug-treated cells while topo IIalpha levels remained constant. Toxicity analyses were performed with two consecutive time-lapse observations separated by a brief drug treatment. The cell cycle phase was determined from the first observation, and cell fate was determined from the second. Cells were most sensitive to drug treatment from mid-S phase through G(2) phase, with G(1) phase cells nearly threefold less sensitive. In addition, the presence of an oncogenic src gene or microinjected Ras protein increased drug toxicity by approximately threefold in actively cycling cells and by at least this level in the small population of slowly cycling cells. We conclude that both cell cycle phase and oncogenic signaling influence drug toxicity independently of alterations in topo IIalpha levels.