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Biomedical subjects

D Pinkel

Publications and source records attributed to D Pinkel.

At least 145 records · Page 8Linked to original sources
PubMed1987

Flow sorting of X and Y chromosome-bearing spermatozoa into two populations.

The only established difference on which to base the separation of X and Y chromosome-bearing spermatozoa is chromosomal constitution. This difference is quantifiable both from chromosome morphology (karyotype) and from DNA content. Flow cytometric techniques were used to measure relative DNA content of the X and Y populations and to flow-sort spermatozoa from Chinchilla laniger. Epididymal spermatozoa were recovered in PBS, fixed in 80% ethanol, treated with papain and dithioerythritol, and stained for DNA with Hoechst 33342. Sperm nuclei were analyzed and sorted on an EPICS V flow cytometer/cell sorter, modified specifically for spermatozoa. Two clearly resolved peaks (coefficient of variation less than 1.5%) with approximately 7.5% difference in DNA content between X and Y chromosome-bearing spermatozoa were evident. Sperm nuclei were sorted from a portion of the X and Y peaks at a rate of 55 nuclei/sec for each population. Purities of individual X and Y populations averaged 95% as determined by reanalysis of the sorted populations. Successful sorting of Chinchilla X and Y chromosome-bearing spermatozoa into separate populations may aid in the identification of a biochemical marker that could be used to discriminate between the two sperm populations and lead to a practical procedure for sexing spermatozoa.

Animals↗
PubMed1987

Incidence of chromosome aberrations in mammalian sperm stained with Hoechst 33342 and UV-laser irradiated during flow sorting.

The separation of two sperm populations is possible using the technique of flow sorting, provided that a significant difference exists in the DNA content of X- and Y-bearing sperm. In order to ascertain whether or not chromosome damage was induced in sorted sperm, chromosome preparations were made from isolated sperm that had been microinjected into hamster eggs. While egg chromosomes exhibited a low frequency of chromosome aberrations, ranging from 4 to 7%, a large proportion of sperm cells exhibited chromosome damage. Between 29% of unstained and unsorted sperm and 38% of stained and unsorted sperm exhibited some type of chromosomal abnormality and this proportion increased to 50% in sorted sperm. If only damaged sperm nuclei are considered, the two unsorted sperm groups had a mean of 0.6 breaks, 0.8 triradial exchanges, and 0.2 quadriradial exchanges per nucleus. However, sorted sperm, which were stained with a fluorochrome and exposed to UV-laser irradiation, exhibited a mean of 2.9 breaks, 2.6 triradial, and 1.9 quadriradial exchanges per nucleus in which damage occurred. These observations indicate that the treatments and manipulations to which sperm nuclei are subjected during flow sorting cause chromosomal aberrations, and that exposure of the cells to UV-laser irradiation contributes substantially to the chromosome damage observed.

Animals↗
PubMed1987

Chromosome-specific alpha satellite DNA from human chromosome 1: hierarchical structure and genomic organization of a polymorphic domain spanning several hundred kilobase pairs of centromeric DNA.

The human alpha satellite repetitive DNA family is organized as distinct chromosome-specific subsets localized to the centromeric region of each chromosome. Here, we report he isolation and characterization of cloned repeat units which define a hierarchical subset of alpha satellite on human chromosome 1. This subset is characterized by a 1.9-kb higher-order repeat unit which consists of 11 tandem approximately 171-bp alpha satellite monomer repeat units. The higher-order repeat unit is itself tandemly repeated, present in at least 100 copies at the centromeric region of chromosome 1. Using pulsed-field gel electrophoresis we estimate the total array length of these tandem sequences at the centromere of chromosome 1 to be several hundred kilobase pairs. Under conditions of high stringency, the higher-order repeat probe hybridizes specifically to chromosome 1 and can be used to detect several associated restriction fragment length DNA polymorphisms. As such, this probe may be useful for molecular and genetic analyses of the centromeric region of human chromosome 1.

Chromosome Mapping↗
PubMed1986

Modification of a laser-based flow cytometer for high-resolution DNA analysis of mammalian spermatozoa.

Modification of a Coulter EPICS V orthogonal laser-based flow cytometer/cell sorter allows resolution of X and Y mammalian sperm populations based on DNA content. The modification consists of beveling the sample injection tube situated in the flow chamber, adding a second fluorescence detector directly forward along the laser beam axis, and routing the collected fluorescence through an optical fiber bundle to one of the existing photomultiplier tubes. The X and Y chromosome-bearing spermatozoa from the bull, boar, and ram can be resolved using this system.

Animals↗
PubMed1986

Orientation measurements of microsphere doublets and metaphase chromosomes in flow.

Obtaining information about the shape of particles from slit-scan profiles is facilitated if the particles are oriented. Elongated particles orient in the nozzle of flow cytometers, but orientation may be disrupted before the particles get to the point of measurement. We have used our slit-scan flow cytometer to investigate the orientation of microsphere doublets in a liquid jet in air, in flow across a glass surface, and in a 200-microns-square capillary tube as a function of distance from the flow chamber nozzle. Particles were classified as being oriented if there was a centrally located dip in the slit-scan profile. Essentially all the doublets in the jet were oriented, and no disorientation was noted over the distances measured (up to 10 mm from the nozzle). Particle orientation was maintained for 80 microns in flow across a glass surface. In the capillary-type flow chamber, essentially all of the particles were oriented at the tube entrance and for several millimeters into the tube. There then occurred a region where particle tumbling started and progressively fewer doublets met the orientation criteria. The distance to where tumbling began could be estimated by calculating the length required to establish the parabolic flow profile in the tube. Finally, the fraction of oriented particles reached a constant value that did not change with increased distance into the tube. When sample was injected off axis (i.e., halfway between the chamber center and the chamber walls), particle tumbling began closer to the tube entrance.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Line↗
PubMed1986

Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization.

This report describes the use of fluorescence in situ hybridization for chromosome classification and detection of chromosome aberrations. Biotin-labeled DNA was hybridized to target chromosomes and subsequently rendered fluorescent by successive treatments with fluorescein-labeled avidin and biotinylated anti-avidin antibody. Human chromosomes in human-hamster hybrid cell lines were intensely and uniformly stained in metaphase spreads and interphase nuclei when human genomic DNA was used as a probe. Interspecies translocations were detected easily at metaphase. The human-specific fluorescence intensity from cell nuclei and chromosomes was proportional to the amount of target human DNA. Human Y chromosomes were fluorescently stained in metaphase and interphase nuclei by using a 0.8-kilobase DNA probe specific for the Y chromosome. Cells from males were 40 times brighter than those from females. Both Y chromosomal domains were visible in most interphase nuclei of XYY amniocytes. Human 28S ribosomal RNA genes on metaphase chromosomes were distinctly stained by using a 1.5-kilobase DNA probe.

Aneuploidy↗
PubMed1986

Assessment of spermatozoal function using dual fluorescent staining and flow cytometric analyses.

Spermatozoa from bulls, boars, dogs, horses, mice, and men were examined using a fluorogenic stain consisting of the membrane-permeant substrate carboxyfluorescin diacetate (CFDA) and the relatively membrane-impermeant nuclear stain propidium iodide (PI). Three distinct populations of spermatozoa were discernible in samples from each species upon microscopic examination. Individual spermatozoa, presumed to be viable because of their motility, retained products of the fluorescein chromophore throughout the cell. A second population of spermatozoa in which the nuclei stained red with PI retained the green fluorescein fluorophore mainly in the acrosome. A third population, presumed to be degenerate spermatozoa, possessed only red fluorescent nuclei. These populations were quantified using dual parameter flow cytometry in 14 samples of cryopreserved bovine spermatozoa for which fertility and seminal quality data were available. Flow cytometric analyses were highly correlated with other seminal quality measurements. Sequential flow cytometric analyses provided the ability to rapidly quantitate changes in specific fluorescently stained populations. The ability to make rapid quantitative measurements should allow development of new and presumably more reliable information on the functional aspects of spermatozoa.

Acrosome↗
PubMed1985

The LLNL high-speed sorter: design features, operational characteristics, and biological utility.

This report describes a dual-beam, high-speed sorter (HiSS) with a droplet production rate of 220,000 s-1 now in routine use at the Lawrence Livermore National Laboratory. The system can process and sort objects at rates in excess of 20,000 s-1. We report here on the development of HiSS, describe its operational characteristics, and evaluate its utility for analysis and purification of human chromosomes, human erythrocytes, and living Chinese hamster ovary cells.

Cell Separation↗
PubMed1985

Flow cytometric determination of the proportions of X- and Y-chromosome-bearing sperm in samples of purportedly separated bull sperm.

A rapid assay for determining the proportions of X- and Y-chromosome-bearing sperm in semen samples would benefit research aimed at sex ratio control through sperm separation. It also would be of value for quality control should a separation technique be developed. Flow cytometric methods capable of measuring sperm DNA content precisely enough to resolve and quantify the X and Y populations in many mammalian species have been developed. They are effective for fresh and cryopreserved sperm of most domestic animals. Results are reported of flow cytometric analyses of bull sperm samples from seven commercial and academic sources after processing with procedures purported to separate the X and Y populations. In no case was enrichment of either sperm population observed. Breeding trials carried out by the sources of two of the sets of samples showed these procedures were ineffective in altering the sex ratio.

Animals↗
PubMed1985

Measurement of sister chromatid exchanges at very low bromodeoxyuridine substitution levels using a monoclonal antibody in Chinese hamster ovary cells.

A monoclonal antibody to bromodeoxyuridine (BrdUrd) incorporated into DNA allowed visualization of sister chromatid exchanges (SCE) when as little as 0.6% of the thymine in a single DNA strand has been substituted. Measurement of the SCE frequency as a function of BrdUrd substitution in a normal Chinese hamster ovary cell line showed a plateau of six SCEs per cell for substitution levels up to at least 20%. A clear elevation in frequencies was noted at 60% substitution. However, in the mutant line EM9, previously shown to have a highly elevated frequency of SCE, the level of exchanges declined continuously as the percentage of BrdUrd substitution decreased. At 0.6% substitution, the frequency of SCE was still 4-fold higher than that of the parental cells. The antibody procedure described here should be useful in evaluating the extent to which SCEs induced by mutagenic agents result from interactions between the DNA damage caused by the agent and the BrdUrd routinely used for measuring SCE.

Animals↗
PubMed1983

Bacterial characterization by flow cytometry.

Bacteria were analyzed in a dual-beam flow cytometer after double staining with the fluorescent dyes chromomycin A3 and Hoechst 33258, which bind preferentially to DNA that is rich in guanine-cytosine and adenine-thymine, respectively. The measurements were indicative of the cellular DNA content and base composition, cell concentration, and proliferative state of the population. The ratio of the chromomycin A3 signal to the Hoechst 33258 signal increased with the guanine-cytosine content of the cellular DNA for the six cultured species measured, following expectation. Bacteria in urine from patients with urinary tract infections were characterized without interference from host cell DNA, debris, or other particulates.

Bacteria↗
PubMed1983

Quantification of the X- and Y-chromosome-bearing spermatozoa of domestic animals by flow cytometry.

The relative content of DNA in spermatozoa presumed to be the X- and Y-chromosome-bearing gametes from bulls, boars, rams and rabbits and the amount of DNA in spermatozoa of cockerels was determined by flow cytometry. Differences in the relative content of DNA and proportions of the presumed X- and Y-sperm populations in cryopreserved semen from Holstein, Jersey, Angus, Hereford and Brahman bulls were also determined. Spermatozoa were washed by centrifugation using a series of dimethyl sulfoxide solutions made in isotonic sodium citrate, fixed in ethanol, treated with papain and dithioerythritol to loosen the chromatin structure and remove cellular organelles, and stained quantitatively for DNA with the fluorochrome 4'-6-diamidino-2-phenylindole (DAPI). Approximately 5000 stained sperm nuclei, which were nonviable due to the removal of other cellular organelles during the washing procedure, were measured for DNA in an epi-illumination flow cytometer. A single distinct peak for cockerel spermatozoa and two symmetrical, overlapping peaks for species with X- and Y-spermatozoa were seen. This and other evidence strongly supports the interpretation that the peaks represent the X- and Y-sperm populations. The content of DNA in sperm nuclei from cockerels, bulls, boars, rams and rabbits, as determined by fluorescence flow cytometry, corresponded to biochemical estimates of DNA per sperm cell. Analyses of the bimodal histograms by computer-fitting two Gaussian distributions to the data showed the means of the peaks differed by 3.9, 3.7, 4.1 and 3.9% for bulls, boars, rams and rabbits, respectively. In four replicate analyses of semen from 25 bulls representing 5 breeds, the average population of sperm nuclei in the Y-peaks ranged from 49.5 to 50.5% for all breeds. The X-Y peak differences did not vary within each breed, but were significantly different when the breeds were compared. Spermatozoa from Jersey bulls had larger X-Y peak differences (P less than 0.001) than spermatozoa from Holstein, Hereford, and Angus bulls; spermatozoa from Brahman bulls had smaller X-Y differences (P less than 0.004). It is suggested from the evidence obtained in these studies that flow cytometry can be used to assess the proportion of X- and Y-spermatozoa in semen of domestic animals and is thereby applicable to verification of the effectiveness of enrichment techniques for X- or Y-spermatozoa.

Animals↗
PubMed1983

Radiation-induced DNA content variability in mouse sperm.

Mouse sperm collected from the cauda epididymidis 35 days after acute testicular X-ray exposure and fluorescently stained for DNA show dose-dependent increases in the coefficient of variation (CV) of flow cytometrically obtained fluorescence distributions. By comparing dose-response curves obtained with three protocols which overcome the optical and cytochemical difficulties of sperm measurement in different ways we conclude the response is due to X-ray-induced DNA content variability. In the range between 0 and 600 rad the dose dependence of the square of CV of the DNA content variability, delta CV2D, is described by delta CV2D = Bx + Cx2, with 0 less than or equal to B less than or equal to 0.23 X 10(-2) and C = (0.44 +/- 0.06) X 10(-4). The dose x is measured in rad and delta CVD is expressed in percent. Computer modeling of the shapes of the fluorescence distributions show that at 600 rad 30 to 40% of the sperm have abnormal DNA content. Some have errors as large as two whole chromosomes, but it is not clear whether they are due to whole chromosome nondisjunction or a finer fragmentation of the genome. Exposures to benzo(a)pyrene and mitomycin C cause no detectable DNA content variability. We conclude mouse sperm DNA content measurements are not sensitive to small amounts of aneuploidy and as such will only be useful in detecting agents that produce substantial DNA content variability. Another animal with a smaller number of chromosomes might be more favorable. These sperm measurement techniques may find additional application in other areas of reproductive biology, such as the determination of the relative numbers of X and Y chromosome-bearing sperm in semen that may be artificially enriched in one population.

Animals↗
PubMed1982

Sex preselection in mammals? Separation of sperm bearing Y and "O" chromosomes in the vole Microtus oregoni.

The two sex determining sperm populations of the vole Microtus oregoni were separated according to DNA content by use of flow sorting instrumentation. Although the sperm were not viable, they should be useful for addressing the question of haploid expression of genes linked to sex chromosomes and for efficiently searching for biochemical markers that differentiate the two populations.

Animals↗
PubMed1982

High resolution DNA content measurements of mammalian sperm.

The high condensation and flat shape of the mammalian sperm nucleus present unique difficulties to flow cytometric measurement of DNA content. Chromatin compactness makes quantitative fluorescent staining for DNA difficult and causes a high index of refraction. The refractive index makes optical measurements sensitive to sperm head orientation. We demonstrate that the optical problems can be overcome using the commercial ICP22 epiillumination flow cytometer (Ortho Instruments, Westwood, MA) or a specially built cell orientating flow cytometer (OFCM). The design and operation of the OFCM are described. Measurements of the angular dependence of fluorescence from acriflavine stained rabbit sperm show that it is capable of orienting flat sperm with a tolerance of +/- 7 degrees. Differences in the angular dependence for the similarly shaped bull and rabbit sperm allow discrimination of these cells. We show that DNA staining with 4-6 diamidino-2-phenylindole (DAPI) or an ethidium bromide mithramycin combination allows resolution of the X and Y populations in mouse sperm. They have also been successful with sperm from the bull, ram, rabbit, and boar. Reliable results with human sperm are not obtained. The accuracy of the staining and measurement techniques are verified by the correct determination of the relative DNA content of these two populations in sperm from normal mice and those with the Cattanach [7 to X] translocation. Among the potential uses of these techniques are measurement of DNA content errors induced in sperm due to mutagen exposure, and assessment of the fractions of X and Y sperm in semen that may have one population artifically enriched.

Acriflavine↗