Robert M Zucker

Publications of Robert M Zucker

• Darkfield-confocal microscopy detection of nanoscale particle internalization by human lung cells.

  Authors: Eugene A Gibbs-Flournoy, Philip A Bromberg, Thomas P J Hofer, James M Samet, Robert M Zucker

  Particle and fibre toxicology. 01/2011; 8(1):2.

  Concerns over the health effects of nanomaterials in the environment have created a need for microscopy methods capable of examining the biological interactions of nanoparticles (NP). Unfortunately,Concerns over the health effects of nanomaterials in the environment have created a need for microscopy methods capable of examining the biological interactions of nanoparticles (NP). Unfortunately, NP are beyond the diffraction limit of resolution for conventional light microscopy (~200 nm). Fluorescence and electron microscopy techniques commonly used to examine NP interactions with biological substrates have drawbacks that limit their usefulness in toxicological investigation of NP. EM is labor intensive and slow, while fluorescence carries the risk of photobleaching the sample and has size resolution limits. In addition, many relevant particles lack intrinsic fluorescence and therefore can not be detected in this manner. To surmount these limitations, we evaluated the potential of a novel combination of darkfield and confocal laser scanning microscopy (DF-CLSM) for the efficient 3D detection of NP in human lung cells. The DF-CLSM approach utilizes the contrast enhancements of darkfield microscopy to detect objects below the diffraction limit of 200 nm based on their light scattering properties and interfaces it with the power of confocal microscopy to resolve objects in the z-plane. Validation of the DF-CLSM method using fluorescent polystyrene beads demonstrated spatial colocalization of particle fluorescence (Confocal) and scattered transmitted light (Darkfield) along the X, Y, and Z axes. DF-CLSM imaging was able to detect and provide reasonable spatial locations of 27 nm TiO2 particles in relation to the stained nuclei of exposed BEAS 2B cells. Statistical analysis of particle proximity to cellular nuclei determined a significant difference between 5 min and 2 hr particle exposures suggesting a time-dependent internalization process. DF-CLSM microscopy is an alternative to current conventional light and electron microscopy methods that does not rely on particle fluorescence or contrast in electron density. DF-CLSM is especially well suited to the task of establishing the spatial localization of nanoparticles within cells, a critical topic in nanotoxicology. This technique has advantages to 2D darkfield microscopy as it visualizes nanoparticles in 3D using confocal microscopy. Use of this technique should aid toxicological studies related to observation of NP interactions with biological endpoints at cellular and subcellular levels.

• Evaluation and purchase of confocal microscopes: numerous factors to consider.

  Authors: Robert M Zucker, Michael Chua

  Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.]. 10/2010; Chapter 2:Unit2.16.

  The purchase of a confocal microscope is a difficult decision. Many factors need to be considered, which include hardware, software, company, support, service, and price. These issues are discussedThe purchase of a confocal microscope is a difficult decision. Many factors need to be considered, which include hardware, software, company, support, service, and price. These issues are discussed to help guide the purchasing process.

• Altered health outcomes in adult offspring of Sprague Dawley and Wistar rats undernourished during early or late pregnancy.

  Authors: Robert G Ellis-Hutchings, Robert M Zucker, Brian E Grey, Joel Norwood, Judy H Richards, Christopher Lau, John M Rogers

  Birth defects research. Part B, Developmental and reproductive toxicology. 10/2010; 89(5):396-407.

  Birth weight in humans has been inversely associated with adult disease risk. Results of animal studies have varied depending on species, strain, and treatment. We compared birth weight and adultBirth weight in humans has been inversely associated with adult disease risk. Results of animal studies have varied depending on species, strain, and treatment. We compared birth weight and adult health in offspring following 50% maternal undernutrition on gestation days (GD) 1-15 (UN1-15) or GD 10-21 (UN10-21) in Sprague Dawley and Wistar rats. Offspring from food-deprived dams were weighed and cross-fostered to control dams. Litters were weighed during lactation and initiating at weaning males were fed either control or a high-fat diet. Young and mature adult offspring were evaluated for obesity, blood pressure (BP), insulin response to oral glucose, and serum lipids. Nephron endowment, renal glucocorticoid receptor, and renin-aldosterone-angiotensin system components were measured. The UN10-21 groups had birth weights lower than controls and transient catch up growth by weaning. Neither strain demonstrated obesity or dyslipidemia following prenatal undernutrition, but long-term body weight deficits occurred in the UN groups of both strains. High-fat diet fed offspring gained more weight than control offspring without an effect of prenatal nutrition. Sprague Dawley were slightly more susceptible than Wistar rats to altered insulin response and increased BP following gestational undernutrition. Nephron endowment in Sprague Dawley but not Wistar offspring was lower in the UN10-21 groups. Glucocorticoid and renin-aldosterone-angiotensin system pathways were not altered. The most consistent effect of maternal undernutrition was elevated BP in offspring. Long-term health effects occurred with undernutrition during either window, but the UN10-21 period resulted in lower birth weight and more severe adult health effects.

• An integrated imaging approach to the study of oxidative stress generation by mitochondrial dysfunction in living cells.

  Authors: Wan-Yun Cheng, Haiyan Tong, Evan W Miller, Christopher J Chang, James Remington, Robert M Zucker, Philip A Bromberg, James M Samet, Thomas P J Hofer

  Environmental health perspectives. 04/2010; 118(7):902-8.

  The mechanisms of action of many environmental agents commonly involve oxidative stress resulting from mitochondrial dysfunction. Zinc is a common environmental metallic contaminant that has beenThe mechanisms of action of many environmental agents commonly involve oxidative stress resulting from mitochondrial dysfunction. Zinc is a common environmental metallic contaminant that has been implicated in a variety of oxidant-dependent toxicological responses. Unlike ions of other transition metals such as iron, copper, and vanadium, Zn(2+) does not generate reactive oxygen species (ROS) through redox cycling. To characterize the role of oxidative stress in zinc-induced toxicity. We used an integrated imaging approach that employs the hydrogen peroxide (H2O2)-specific fluorophore Peroxy Green 1 (PG1), the mitochondrial potential sensor 5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethylbenzimidazolylcarbocyanine iodide (JC-1), and the mitochondria-targeted form of the redox-sensitive genetically encoded fluorophore MTroGFP1 in living cells. Zinc treatment in the presence of the Zn(2+) ionophore pyrithione of A431 skin carcinoma cells preloaded with the H(2)O(2)-specific indicator PG1 resulted in a significant increase in H(2)O(2) production that could be significantly inhibited with the mitochondrial inhibitor carbonyl cyanide 3-chlorophenylhydrazone. Mitochondria were further implicated as the source of zinc-induced H(2)O(2) formation by the observation that exposure to zinc caused a loss of mitochondrial membrane potential. Using MTroGFP1, we showed that zinc exposure of A431 cells induces a rapid loss of reducing redox potential in mitochondria. We also demonstrated that zinc exposure results in rapid swelling of mitochondria isolated from mouse hearts. Taken together, these findings show a disruption of mitochondrial integrity, H(2)O(2) formation, and a shift toward positive redox potential in cells exposed to zinc. These data demonstrate the utility of real-time, live-cell imaging to study the role of oxidative stress in toxicological responses.

• Perturbation of retinoic acid (RA)-mediated limb development suggests a role for diminished RA signaling in the teratogenesis of ethanol.

  Authors: Corey S Johnson, Robert M Zucker, Edward Sidney Hunter, Kathleen K Sulik

  Birth defects research. Part A, Clinical and molecular teratology. 10/2007; 79(9):631-41.

  BACKGROUND: A proposed mechanism for ethanol teratogenicity entails ethanol-mediated reductions in retinoic acid (RA). This premise was investigated utilizing a mouse model, with limb reductionBACKGROUND: A proposed mechanism for ethanol teratogenicity entails ethanol-mediated reductions in retinoic acid (RA). This premise was investigated utilizing a mouse model, with limb reduction defects as the teratogenic end point. METHODS: Ethanol, Disulfiram, or BMS-189453 was administered to C57BL/6J mice on the 9(th) day of pregnancy. Forelimb morphology was assessed on gestation day 18 using Alcian blue and Alizarin red staining. Nile blue sulfate or LysoTracker Red (LTR) vital staining identified cell death in the limb bud. The ability of RA to prevent ethanol-induced cell death was assessed by coadministration followed by laser scanning confocal microscopic examination of LTR-staining. In situ hybridization and qPCR were used to examine gene expression in treated limb buds. RESULTS: Ethanol, Disulfiram, and BMS-189453 resulted in postaxial ectrodactyly, intermediate ectrodactyly, and other digital defects. Excessive Nile blue sulfate staining was evident in the presumptive AER following each of the three exposures. Ethanol-induced LTR staining was prevented by RA supplementation. Both in situ hybridization and qPCR illustrated decreases in Shh and Tbx5 in ethanol-exposed embryos as compared to control. CONCLUSIONS: Contrary to studies of prolonged RA deficiency, acute exposure to functional antagonists of RA results in limb defects that are morphologically similar to those caused by ethanol. The rescue of ethanol-induced cell death by RA and similar changes in Shh transcription further suggest that RA contributes to ethanol-induced limb dysmorphology. Moreover, the repression of key mediators of limb development soon after ethanol exposure adds to the existing knowledge of the pathogenic effects of ethanol.

• Reliability of confocal microscopy spectral imaging systems: use of multispectral beads.

  Authors: Robert M Zucker, Paul Rigby, Ian Clements, Wendy Salmon, Michael Chua

  Cytometry. Part A : the journal of the International Society for Analytical Cytology. 04/2007; 71(3):174-89.

  BACKGROUND: There is a need for a standardized, impartial calibration, and validation protocol on confocal spectral imaging (CSI) microscope systems. To achieve this goal, it is necessary to haveBACKGROUND: There is a need for a standardized, impartial calibration, and validation protocol on confocal spectral imaging (CSI) microscope systems. To achieve this goal, it is necessary to have testing tools to provide a reproducible way to evaluate instrument performance. METHODS: We evaluated fluorescent spectral beads (FocalCheck) from Molecular Probes/Invitrogen that consist of four pairs with emissions between 500 and 725 nm and a europium macrocycle quantum dye bead. These bead tools compliment our previously published protocol for testing spectral imaging systems that used an inexpensive multi-ion discharge lamp (MIDL) that contains Hg(+), Ar(+), and inorganic fluorophores that emits distinct, stable spectral features. RESULTS: We acquired the spectra of the FocalCheck beads on a Zeiss 510 Meta, a Leica TCS-SP1, a Leica SP2 AOBS, an Olympus FV 1000, and a Nikon C1Si confocal systems and a PARISS microscopic spectral system and of the europium beads on the Leica TCS-SP1 and PARISS spectral imaging systems. A lack of performance with some equipment between 650 and 750 nm was identified using the far red pair of the FocalCheck beads. The position of the slider in front of PMT 2 that reflects light into PMT 1 and PMT 3 affected the measurement of all bead intensities. Unmixing algorithms were used to separate beads with different fluorochromes and separate two fluorochromes on the same bead. CONCLUSIONS: The FocalCheck multi-spectral beads yielded similar profiles on four CSI systems and a PARISS spectral system. The utilization of the spectral FocalCheck beads is helpful to evaluate proper spectral performance, especially in the far red region. Europium beads provide a very narrow spectrum that can help to identify machines that have spectral problems. The dyes located on individual beads or mixed together in ring-core configuration can be used as test particles to demonstrate spectral unmixing with various algorithms.

• Whole insect and mammalian embryo imaging with confocal microscopy: morphology and apoptosis.

  Authors: Robert M Zucker

  Cytometry. Part A : the journal of the International Society for Analytical Cytology. 12/2006; 69(11):1143-52.

  BACKGROUND: After fluorochromes are incorporated into cells, tissues, and organisms, confocal microscopy can be used to observe three-dimensional structures. LysoTracker Red (LT) is aBACKGROUND: After fluorochromes are incorporated into cells, tissues, and organisms, confocal microscopy can be used to observe three-dimensional structures. LysoTracker Red (LT) is a paraformaldehyde fixable probe that concentrates into acidic compartments of cells and indicates regions of high lysosomal activity and phagocytosis, which both correlate to apoptosis activity. LT has been shown to be an indicator of apoptotic cell death which is correlated to other standard apoptotic assays. METHODS: The mammalian samples were stained with LT, fixed with paraformaldehyde/glutaraldehyde, dehydrated with methanol (MEOH), and cleared with benzyl alcohol/benzyl benzoate (BABB). Following this treatment, the tissues were nearly transparent. Mosquitoes were fixed with MEOH and stained with propidium iodide. Next the tissues were dehydrated with MEOH and cleared with BABB. RESULTS: Tissues as thick as 500 microm can be visualized after clearing with BABB. LT staining revealed apoptotic regions in mammalian limbs, fetuses, and embryos. Morphological observation of insect tissue consisted of combining autofluorescence with either nucleic acid staining (either propidium iodide or ethidium bromide). CONCLUSIONS: The use of BABB matches the RI of the tissue within the suspending medium. It helps in increasing the penetration of laser light in a confocal microscope by reducing the amount of light scattering artifacts and allows for the visualization of morphology in thick tissues. LT is a probe that stains the acid regions of tissues and cells and has been correlated to apoptosis. Morphological features of a tissue or organism (embryo, mosquito larvae) can be elucidated by fixation aldehydes, autofluorescence, and red-emitting probes. This sample preparation procedure with optimization of confocal laser scanning microscopy allowed for the detection and visualization of apoptosis in fetal limbs and embryos which were approximately 500-microm thick.

• Confocal laser scanning microscopy of whole mouse ovaries: excellent morphology, apoptosis detection, and spectroscopy.

  Authors: Robert M Zucker, Susan C Jeffay

  Cytometry. Part A : the journal of the International Society for Analytical Cytology. 09/2006; 69(8):930-9.

  BACKGROUND: Ovaries consist of numerous follicles, oocytes, and granulosa cells in different stages of development. Many of these follicles will undergo an apoptotic process during the lifetime ofBACKGROUND: Ovaries consist of numerous follicles, oocytes, and granulosa cells in different stages of development. Many of these follicles will undergo an apoptotic process during the lifetime of the animal. By using proper tissue preparation methods, the events within the whole ovary can be observed by using 3D confocal microscopy. METHODS: Whole ovaries were stained with LysoTracker Red (LT), fixed with 4% paraformaldehyde (PF) and 1% glutaraldehyde (Glut), dehydrated with methanol (MEOH), and cleared with benzyl alcohol and benzyl benzoate (BABB). Using this tissue preparation technique, the ovary becomes relatively transparent, allowing its morphology to be observed with confocal microscopes. A spectral imaging system (PARISS) located on a conventional microscope was used to interpret the LT dye spectra and fixation products in the tissues with different excitation wavelengths. RESULTS: Apoptosis in the follicle was detected as clusters of intensely stained granulosa cells located in close proximity to the oocytes. The fixation with Glut and PF preserved morphological details, increased tissue fluorescence, thus increased the signal to noise of the background image. CONCLUSIONS: Thick tissues can be imaged after they are properly stained, aldehyde fixed, and BABB cleared. LT intensely stained single cells or clusters of apoptotic cells in the follicles and the nucleolus. Spectral differences between LT as an indicator of apoptosis and Glut-PF fixation was used to visualize ovarian morphology and apoptosis. The PARISS spectrophotometer revealed spectral peaks for LT at 609.6 nm and for Glut-PF at 471.3 nm. The proper use of the spectra from these fluorescence molecules is the foundation for high quality morphological images of apoptosis. By sequentially imaging the two probes with a 488 nm laser and a 543/568 nm laser, there was a reduction in fluorescent cross talk and an increase in image quality.

• Quality assessment of confocal microscopy slide based systems: performance.

  Authors: Robert M Zucker

  Cytometry. Part A : the journal of the International Society for Analytical Cytology. 08/2006; 69(7):659-76.

  BACKGROUND: All fluorescence slide-based cytometry detections systems basically include the following components: (1) an excitation light source, (2) intermediate optics, and (3) a detection deviceBACKGROUND: All fluorescence slide-based cytometry detections systems basically include the following components: (1) an excitation light source, (2) intermediate optics, and (3) a detection device consisting of a CCD camera or a PMT. The optical principles employed is slide-based systems are similar to those of confocal microscopes (CLSM). METHODS: The following tests evaluated confocal equipment performance: dichroic reflectivity, field illumination, lens performance, laser power output, spectral registration, axial resolution, PMT reliability, and system noise. RESULTS: Quality assurance tests provide a basis to determine if the equipment is operating correctly. Laser power, PMTs function, dichroic reflection, spectral registration, axial registration, system noise and sensitivity, lens performance and laser stability were tested colocalization of UV and visible peaks of a bead should be less than 210 nm. Interference contrast optics decrease fluorescence resolution. CONCLUSIONS: QA tests that assess CLSM system performance are also applicable to other slide-based systems. By utilization this type of testing approach, the subjective nature of assessing the CLSM may be eliminated. These tests serve as guidelines for other investigators to ensure that their machines are providing data that is accurate with the necessary resolution, sensitivity and precision.

• Quality assessment of confocal microscopy slide-based systems: instability.

  Authors: Robert M Zucker

  Cytometry. Part A : the journal of the International Society for Analytical Cytology. 08/2006; 69(7):677-90.

  BACKGROUND: All slide-based fluorescence cytometry detections systems basically include an excitation light source, intermediate optics, and a detection device (CCD or PMT). Occasionally, thisBACKGROUND: All slide-based fluorescence cytometry detections systems basically include an excitation light source, intermediate optics, and a detection device (CCD or PMT). Occasionally, this equipment becomes unstable, generating unreliable and inferior data. METHODS: A number of tests have been devised to evaluate equipment performance and instability. The following four instability tests are described: galvanometer scanning, stage drift, correct wavelength spectral detection, and long-term laser power. RESULTS: Quality assurance tests revealed that a confocal microscope can become unstable in the following parameters, yielding inaccurate data: laser power, PMTs functionality, spectrophotometer accuracy, galvanometer scanning and laser stability, and stage drift. Long-term laser power stability has been observed to vary greatly. CONCLUSIONS: Confocal systems can become unstable in the following parameters: long-term laser power, galvanometer scanning, spectrophotometer accuracy, and stage stability. Instability in any of these parameters will affect image quality. Laser power fluctuations result from either a defective Acousto-optic tunable filter or improper heat dissipation. Spectrophotometer instability will generate unreliable spectra data, extra light reflections, and poor image quality. Galvanometer scanning instability yields poor image quality while microscope stage drift results in a sample going out of the plane of focus. With minor modifications, these tests may be applicable to other slide-based systems.

• Evaluation of confocal microscopy system performance.

  Authors: Robert M Zucker

  Methods in molecular biology (Clifton, N.J.). 02/2006; 319:77-135.

  The confocal laser scanning microscope (CLSM) has enormous potential in many biological fields. When tests are made to evaluate the performance of a CLSM, the usual subjective assessment isThe confocal laser scanning microscope (CLSM) has enormous potential in many biological fields. When tests are made to evaluate the performance of a CLSM, the usual subjective assessment is accomplished by using a histological test slide to create a "pretty picture." Without the use of functional tests, many of the machines could be working at suboptimal performance levels, delivering suboptimum performance and possibly misleading data. To replace the subjectivity in evaluating a confocal microscope, tests were derived or perfected that measure field illumination, lens clarity, laser power, laser stability, dichroic functionality, spectral registration, axial resolution, scanning stability, photomultiplier tube quality, overall machine stability, and system noise. These tests will help serve as a guide for other investigators to ensure that their machines are working correctly to provide data that are accurate with the necessary resolution, sensitivity, and precision. Utilization of this proposed testing approach will help eliminate the subjective nature of assessing the CLSM and allow different machines to be compared. These tests are essential if one is to make intensity measurements.

• Wavelength and alignment tests for confocal spectral imaging systems.

  Authors: Robert M Zucker, Jeremy M Lerner

  Microscopy research and technique. 01/2006; 68(5):307-19.

  Confocal spectral imaging (CSI) microscope systems now on the market delineate multiple fluorescent proteins, labels, or dyes within biological specimens by performing spectral characterizations.Confocal spectral imaging (CSI) microscope systems now on the market delineate multiple fluorescent proteins, labels, or dyes within biological specimens by performing spectral characterizations. However, we find that some CSI present inconsistent spectral profiles of reference spectra within a particular system as well as between related and unrelated instruments. We also find evidence of instability that, if not diagnosed, could lead to inconsistent data. This variability confirms the need for diagnostic tools to provide a standardized, objective means of characterizing instability, evidence of misalignment, as well as performing calibration and validation functions. Our protocol uses an inexpensive multi-ion discharge lamp (MIDL) that contains Hg+, Ar+, and inorganic fluorophores that emit distinct, stable spectral features, in place of a sample. An MIDL characterization verifies the accuracy and consistency of a CSI system and validates acquisitions of biological samples. We examined a total of 10 CSI systems, all of which displayed spectral inconsistencies, enabling us to identify malfunctioning subsystems. Only one of the 10 instruments met its optimal performance expectations. We have found that using a primary light source that emits an absolute standard "reference spectrum" enabled us to diagnose instrument errors and measure accuracy and reproducibility under normalized conditions. Using this information, a CSI operator can determine whether a CSI system is working optimally and make objective comparisons with the performance of other CSI systems. It is evident that if CSI systems of a similar make and model were standardized to reveal the same spectral profile from a standard light source, then researchers could be confident that real-life experimental findings would be repeatable on any similar system.

• Calibration and validation of confocal spectral imaging systems.

  Authors: Jeremy M Lerner, Robert M Zucker

  Cytometry. Part A : the journal of the International Society for Analytical Cytology. 12/2004; 62(1):8-34.

  BACKGROUND: Confocal spectral imaging (CSI) microscopic systems currently on the market delineate multiple fluorescent proteins, labels, or dyes within biological specimens by performing spectralBACKGROUND: Confocal spectral imaging (CSI) microscopic systems currently on the market delineate multiple fluorescent proteins, labels, or dyes within biological specimens by performing spectral characterizations. However, some CSI systems have been found to present inconsistent spectral profiles of reference spectra within a particular system and between related and unrelated instruments. This variability confirms that there is a need for a standardized, objective calibration and validation protocol. METHODS: Our protocol uses an inexpensive multi-ion discharge lamp (MIDL) that contains Hg(+), Ar(+), and inorganic fluorophores that emit distinct, stable, spectral features in place of a sample. We derived reference spectra from the MIDL data to accurately predict the spectral resolution, ratio of wavelength to wavelength, contrast, and aliasing parameters of any CSI system. We were also able to predict and confirm the influence of pinhole diameter on spectral profiles. RESULTS: Using this simulation, we determined that there was good agreement between observed and theoretical expectations, thus enabling us to identify malfunctioning subsystems. We examined eight CSI systems and one nonconfocal spectral system, all of which displayed spectral inconsistencies. No instrument met its optimal performance expectations. In two systems, we established the need for factory realignment that had not been otherwise recognized. CONCLUSIONS: We found that using a primary light source that emits an absolute standard "reference spectrum" enabled us to diagnose instrumental errors and measure accuracy and reproducibility under normalized conditions. With this information, a CSI operator can determine whether a CSI system is working optimally and make objective comparisons with the performance of other CSI systems. We determined that, if CSI systems were standardized to produce the same spectral profile of a MIDL lamp, researchers could be confident that the same experimental findings would be obtained on any CSI system.

• Methanol exposure during gastrulation causes holoprosencephaly, facial dysgenesis, and cervical vertebral malformations in C57BL/6J mice.

  Authors: John M Rogers, Kimberly C Brannen, Brenda D Barbee, Robert M Zucker, Sigmund J Degitz

  Birth defects research. Part B, Developmental and reproductive toxicology. 05/2004; 71(2):80-8.

  BACKGROUND: Exposure of pregnant outbred CD-1 mice to methanol during the period of gastrulation results in exencephaly, cleft palate, and cervical vertebra malformations [Rogers and Mole, TeratologyBACKGROUND: Exposure of pregnant outbred CD-1 mice to methanol during the period of gastrulation results in exencephaly, cleft palate, and cervical vertebra malformations [Rogers and Mole, Teratology 55: 364, 1997], while inbred C57BL/6J mice are sensitive to the teratogenicity of ethanol. C57BL/6J fetuses exhibit the holoprosencephaly spectrum of malformations after maternal exposure to ethanol during gastrulation, but the sensitivity of C57BL/6J mice to methanol-induced teratogenesis has not been previously described. METHODS: Pregnant C57BL/6J mice were administered two i.p. injections totaling 3.4 or 4.9 g/kg methanol or distilled water four hrs apart on gestation day 'GD' 7. On GD 17, litters were examined for numbers of live, dead and resorbed conceptuses, fetuses were weighed as a litter and examined externally, and all fetuses were double stained for skeletal analysis. RESULTS: No maternal intoxication was apparent, but the high dosage level caused a transient deficit in maternal weight gain. The number of live fetuses per litter was reduced at both dosages of methanol, and fetal weight was lower in the high dosage group. Craniofacial defects were observed in 55.8% of fetuses in the low dosage group and 91.0% of fetuses in the high dosage group, including micro/anophthalmia, holoprosencephaly, facial clefts and gross facial angenesis. Skeletal malformations, particularly of the cervical vertebrae, were observed at both dosages of methanol, and were similar to those previously reported in the CD-1 mouse following methanol exposure. CONCLUSIONS: The types of craniofacial malformations induced in the C57BL/6J mouse by methanol indicate that methanol and ethanol have common targets and may have common modes of action.

• Pathogenesis of methanol-induced craniofacial defects in C57BL/6J mice.

  Authors: Sigmund J Degitz, Robert M Zucker, Clinton Y Kawanishi, Gwen S Massenburg, John M Rogers

  Birth defects research. Part A, Clinical and molecular teratology. 05/2004; 70(4):172-8.

  BACKGROUND: Methanol administered to C57BL/6J mice during gastrulation causes severe craniofacial dysmorphology. We describe dysmorphogenesis, cell death, cell cycle assessment, and effects onBACKGROUND: Methanol administered to C57BL/6J mice during gastrulation causes severe craniofacial dysmorphology. We describe dysmorphogenesis, cell death, cell cycle assessment, and effects on development of cranial ganglia and nerves observed following administration of methanol to pregnant C57BL/6J mice on gestation day (GD) 7. METHODS: Mice were injected (i.p.) on GD 7 with 0, 2.3, 3.4, or 4.9 gm/kg methanol, split into two doses. In embryos of mice treated with 0 or 4.9 gm/kg methanol, we used histology and LysoTracker red staining on GD 8 0 hr through GD 8 18 hr to examine cell death and dysmorphogenesis, and we also evaluated cell-cycle distribution and proliferation using flow cytometry (FCM) and BrdU immunohistochemistry. On GD 10, we evaluated the effect of GD 7 exposure to 0, 2.3, 3.4, or 4.9 gm/kg methanol on cranial ganglia and nerve development using neurofilament immunohistochemistry. RESULTS: Methanol treatment on GD 7 resulted in reduced mesenchyme surrounding the fore- and midbrain, and in the first branchial arches, by GD 8 12 hr. There were disruptions in the forebrain neuroepithelium and optic pit. Neural crest cell emigration from the mid- and hindbrain region was reduced in methanol-exposed embryos. Methanol had no apparent effect on BrdU incorporation or cell-cycle distribution on GD 8. Cell death was observed in the hindbrain region along the path of neural crest migration and in the trigeminal ganglion on GD 8 18 hr. Development of the cranial ganglia and nerves was adversely affected by methanol. Development of ganglia V, VIII, and IX was decreased at all dosage levels; ganglion VII was reduced at 3.4 and 4.9 gm/kg, and ganglion X was reduced at 4.9 gm/kg. CONCLUSIONS: These results suggest that gastrulation-stage methanol exposure affects neural crest cells and the anterior mesoderm and neuroepithelium. Cell death was evident in areas of migrating neural crest cells, but only at time points after methanol was cleared from the embryo, suggesting an indirect effect on these cells. Birth Defects Research (Part A), 2004. Published 2004 Wiley-Liss, Inc.

• Developmental toxicity of methanol: Pathogenesis in CD-1 and C57BL/6J mice exposed in whole embryo culture.

  Authors: Sigmund J Degitz, John M Rogers, Robert M Zucker, E Sidney Hunter

  Birth defects research. Part A, Clinical and molecular teratology. 05/2004; 70(4):179-84.

  BACKGROUND: Methanol causes axial skeleton and craniofacial defects in both CD-1 and C57BL/6J mice during gastrulation, but C57BL/6J embryos are more severely affected. We evaluated methanol-inducedBACKGROUND: Methanol causes axial skeleton and craniofacial defects in both CD-1 and C57BL/6J mice during gastrulation, but C57BL/6J embryos are more severely affected. We evaluated methanol-induced pathogenesis in CD-1 and C57BL/6J embryos exposed during gastrulation in whole embryo culture. METHODS: Conceptuses with five to seven somites were exposed to 0, 1, 2, 3, 4, or 6 mg methanol/ml culture medium for 24 hr and embryonic morphology was assessed. Cell death was evaluated by histology and LysoTracker red staining, and cell-cycle distribution was evaluated by flow cytometry. RESULTS: In C57BL/6J embryos, craniofacial defects were observed at 3 mg methanol/ml and greater. The response for CD-1 embryos was different, with increased dysmorphology only at 6 mg/ml. However, protein content in CD-1 embryos was reduced at 3 mg methanol/ml and above, indicating growth retardation. Yolk sac toxicity occurred only at 6 mg methanol/ml in both strains. Methanol caused only small changes in cell-cycle distribution, while cell death was induced at 4 and 6 mg methanol/ml in both strains after 8 hr. The extent of cell death after 8 hr was greater in C57BL/6J embryos, and increased over time through 18 hr; in contrast, CD-1 embryos showed less cell death at 18 than at 8 hr, suggesting recovery. CONCLUSIONS: Cell death plays a prominent role in methanol-induced dysmorphogenesis, while cell-cycle perturbation may not. Differences in the extent of cell death between CD-1 and C57BL/6J embryos correlated with differences in the severity of dysmorphogenesis.

• A short history of the initial application of anti-5-BrdU to the detection and measurement of S phase.

  Authors: Robert C Leif, Jeanne H Stein, Robert M Zucker

  Cytometry. Part A : the journal of the International Society for Analytical Cytology. 04/2004; 58(1):45-52.

• Abnormal fertilization is responsible for reduced fecundity following thiram-induced ovulatory delay in the rat.

  Authors: Tammy E Stoker, Susan C Jeffay, Robert M Zucker, Ralph L Cooper, Sally D Perreault

  Biology of reproduction. 07/2003; 68(6):2142-9.

  Brief exposure to some pesticides, applied during a sensitive window for the neural regulation of ovulation, will block the preovulatory surge of LH and, thus, delay ovulation. Previously, we haveBrief exposure to some pesticides, applied during a sensitive window for the neural regulation of ovulation, will block the preovulatory surge of LH and, thus, delay ovulation. Previously, we have shown that a single i.p. injection of 50 mg/kg of thiram, a dithiocarbamate fungicide that decreases norepinephrine synthesis, on proestrus (1300 h) suppresses the LH surge and delays ovulation for 24 h without altering the number of oocytes released. However, when bred, the treated dams had a decreased litter size and increased postimplantation loss. We hypothesized that the reduced litter size in thiram-delayed rats was a consequence of altered oocyte function arising from intrafollicular oocyte aging. To test this hypothesis, we examined delayed oocytes, zygotes, and 2-cell embryos for evidence of fertilization and polyspermy. In addition, we used confocal laser-scanning microscopy to evaluate and characterize cortical granule localization in oocytes and release in zygotes, because the cortical granule response is a major factor in the normal block to polyspermy. Our results demonstrate that a thiram-induced, 24-h delay in ovulation alters the fertilizability of the released oocyte. Although no apparent morphological differences were observed in the unfertilized mature oocytes released following the thiram-induced delay, the changes observed following breeding include a significant decrease in the percentage of fertilized oocytes, a significant increase in polyspermic zygotes (21%), and a 10-fold increase in the number of supernumerary sperm in the perivitelline space. Importantly, all the polyspermic zygotes exhibited an abnormal pattern of cortical granule exudate, suggestive of a relationship between abnormal cortical reaction and the polyspermy in the delayed zygotes. Because polyspermy is associated with polyploidy, abnormal development, and early embryonic death, the observed polyspermy could explain the abnormal development and decreased litter size that we observed previously following thiram-delayed ovulation.

• Quantitative fluorescence of 5-FU-treated fetal rat limbs using confocal laser scanning microscopy and Lysotracker Red.

  Authors: Owen T Price, Christopher Lau, Robert M Zucker

  Cytometry. Part A : the journal of the International Society for Analytical Cytology. 06/2003; 53(1):9-21.

  BACKGROUND: LysoTracker Red (LT) is a paraformaldehyde fixable probe that concentrates into acidic compartments of cells and tissues. After cell death, a high level of lysosomal activity (acidicBACKGROUND: LysoTracker Red (LT) is a paraformaldehyde fixable probe that concentrates into acidic compartments of cells and tissues. After cell death, a high level of lysosomal activity (acidic enzyme) is expressed in tissues resulting from phagocytosis of apoptotic bodies by neighboring cells. LT was shown previously to be an indicator of cell death in a manner similar to other standard assays (Annexin, terminal dUTP nick end labeling, Nile blue sulfate, neutral red, and acridine orange). METHODS: LT fluorescence in fetal rat hindlimbs at gestational day 14 was measured 8 h after administration of the teratogen, 5-fluorouracil (5-FU), with the use of confocal laser scanning microscopy (CLSM). Four dose levels of 5-FU (0, 20, 30, and 40 mg/kg) were studied. The preparation technique involved staining with LT, paraformaldehyde fixation, methanol dehydration, and clearance with benzyl alcohol and benzyl benzoate. After this treatment, the limb was nearly transparent and ready for CLSM analysis. RESULTS: LT staining was observed in specific regions undergoing apoptosis in normal (control) hindlimbs. After 5-FU treatment, highly fluorescent regions appeared in the progress zone (PZ) of the limb. A dose-dependent response to 5-FU treatment was observed. Compared with controls, hindlimbs treated with 20, 30, and 40 mg/kg of 5-FU exhibited more fluorescence within the highly proliferative PZ. These results showed a dose-response relation between 5-FU exposure and LT uptake. CONCLUSIONS: We found that three-dimensional volumetric regions indicating a high level of fluorescence in the embryonic limb bud can be quantified with three different computer analysis programs. The combination of a sample preparation procedure that clears tissue, a CLSM technique that addresses the equipment variables, and an application of statistical population analysis procedures enabled the visualization and quantification of fluorescence in entire fetal rat hindlimbs that were approximately 500 microm in thickness.

• Glutathione (GSH) concentrations vary with the cell cycle in maturing hamster oocytes, zygotes, and pre-implantation stage embryos.

  Authors: Kurt A Zuelke, Susan C Jeffay, Robert M Zucker, Sally D Perreault

  Molecular reproduction and development. 02/2003; 64(1):106-12.

  Glutathione (GSH) is thought to play critical roles in oocyte function including spindle maintenance and provision of reducing power needed to initiate sperm chromatin decondensation. PreviousGlutathione (GSH) is thought to play critical roles in oocyte function including spindle maintenance and provision of reducing power needed to initiate sperm chromatin decondensation. Previous observations that GSH concentrations are higher in mature than immature oocytes and decline after fertilization, suggest that GSH synthesis may be associated with cell cycle events. To explore this possibility, we measured the concentrations of GSH in Golden Hamster oocytes and zygotes at specific stages of oocyte maturation and at intervals during the first complete embryonic cell cycle. Between 2 and 4 hr after the hormonal induction of oocyte maturation, GSH concentrations increased significantly (approximately doubling) in both oocytes and their associated cumulus cells. This increase was concurrent with germinal vesicle breakdown and the condensation of metaphase I chromosomes in the oocyte. GSH remained high in ovulated, metaphase II (MII) oocytes, but then declined significantly, by about 50%, shortly after fertilization, as the zygote progressed back into interphase (the pronucleus stage). GSH concentrations then plummeted by the two-cell embryo stage and remained at only 10% of those in MII oocytes throughout pre-implantation development. These results demonstrate that oocyte GSH concentrations fluctuate with the cell cycle, being highest during meiotic metaphase, the critical period for spindle growth and development and for sperm chromatin remodeling. These observations raise the possibility that GSH synthesis in maturing oocytes is regulated by gonadotropins, and suggest that GSH is more important during fertilization than during pre-implantation embryo development.