Suzanne Lake

Measurement of mammalian sperm deoxyribonucleic acid by flow cytometry. Problems and approaches.

Measurement of mammalian sperm deoxyribonucleic acid content is of importance in several areas of biomedical research. When measured in flow systems with orthogonal axes of illumination, flow and detection, an unexpected, distorted distribution consisting of a narrow peak with a lateral extension to the right is observed. Several lines of evidence lead to the conclusion that this effect is an optical-geometric artifact attributable to the flat shape and high index of refraction of mammalian sperm heads. This artifact disappears when an epiillumination flow system is used in which the optic axes for illumination and detection and the flow axis are all coincident. Other approaches also eliminate the artifact. The resulting coefficients of variation observed after acriflavine-Feulgen staining are 4-5%, short of the goal of 1.5% required to distinguish between human sperm bearing X and Y chromosomes and to develop a mutagen test system using mice.

Flow cytometry of DNA in mouse sperm and testis nuclei

Abstract Mutations that occur in spermatogenic cells may be expressed as changes in DNA content, but developmentally-dependent alteration of its staining properties complicates the quantitation fo DNA in individual germ cells. These alterations have been studied with flow cytometric techniques. Nuclei from mouse testis cells and sperm were stained by the acriflavine-Feulgen method. The fluorescence intensity frequency distribution of nuclei of testis cells was characterized by 2 major and 5 minor peaks. Nuclei sorted from the various peaks with a fluorescence-activated cell sorter were identified microscopically. These data were confirmed by generation of peaks with nuclei prepared from cell suspensions enriched in specific cell types. One of the major peaks corresponded to round spermatid nuclei. The other major peak, located at 0.6 of the fluorescence intensity of the round nuclei, corresponded to elongated spermatid nuclei. Purified nuclei of epididymal and vas deferens spermatozoa displayed asymmetric fluorescence distributions. A minor peak at 0.8 the intensity of the round spermatid nuclei was tentatively assigned to elongating spermatids. 2 of the minor peaks, located at 1.7 and 2.0 times the fluorescence intensity of the round nuclei, corresponded to clumps of 2 haploid and diploid nuclei. The additional peaks, located at 3.0 and 3.7 times the fluorescence intensity of round spermatid nuclei correspond to leptotene and zygotene spermatocytes and to late pachytene spermatocytes, respectively. These peaks contained clumps of nuclei. The homogeneity of the nuclei sorted from the peaks, as well as the relative sizes of the peaks, was enhanced when the nuclei were prepared from cells enriched in specific stages of development. The relative fluorescence intensities of the various testis nuclei were characteristic and repeatedly found but were not stoichiometric with the DNA content of the nuclei.

Flow cytometry of mammalian sperm progress in DNA and morphology measurement

Variability in DNA content and head shape of mammalian sperm are potentially useful markers for flow cytometric monitoring of genetic damage in spermatogenic cells. The high refractive index and extreme flatness of the sperm heads produce an optical effect which interferes with DNA measurements in flow cytometers which have dye excitation and fluorescence light collection normal to the axis of flow. Orientation of sperm in flow controls this effect and results in coefficients of variation of 2.5% and 4.2%, respectively, for DNA measurements of mouse and human sperm. Alternatively, the optical effect can be used to generate shape-related information. Measurements on randomly oriented sperm from three mammalian species using a pair of fluorescence detectors indicate that large shape differences are detectable. Acriflavine-Feulgen stained sperm nuclei are significantly bleached during flow cytometric measurements at power levels routinely used in many flow cytometers. Dual beam studies of this phenomenon indicate it may be useful in detecting abnormally shaped sperm.

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.

Increased variability in nuclear dna content of testis cells and spermatozoa from mice with irregular meiotic segregation.

Abstract Variability in DNA content to testis cells and sperm from F 1 hybrids between the laboratory mouse ( M. muscullus ) and the tobacco mouse ( M. poschiavinus ), has been determined by flow cytometry (FMC). The F 1 hybrid mouse is known to be heterozygous for seven metacentric chromosomes produced by Robertsonian fusion. Enriched populations of nuclei from late pachytene spermatocytes and round spermatids were obtained by velocity sedimentation. These nuclei, as well as epididymal sperm nuclei and spleen cells, were stained by the acriflavin-Feulgen technique for DNA and measured by FCM. Peaks in the fluorescence intensity frequency distributions resulting from these measurements were analyzed to determine their mean fluorescence intensities and their widths (coefficients of variation). Because mean intensities of corresponding cell types from M. musculus and the F 1 hybrids were identical, the average DNA contents were taken to be the same. The average coefficients of variation of the peaks to fluorescence from the pachytene, spermatid, and sperm nuclei and spleen cells from M. muscullus animals were about 5%. While the peaks of fluorescence from spleen cells and pachytene nuclei from f 1 hybrids also had average coefficients of variation of 5%, post-meiotic nuclei from spermatids and spermatozoa had coefficients of variationof 8%. From these results we conclude that, in these F 1 hybrids, abnormal meiotic segregation causes an increased variability of 6% in the amount of DNA in the spermatozoa.

Electrophoretic analysis of nuclear proteins isolated from sperm of the black abalone Haliotis crackeroidii.

Abstract Nuclear proteins isolated from spermatozoa of the abalone, Haliotis crackeroidii , after sonication and CTAB treatment were characterized electrophoretically. Mature, sonication-resistant sperm nuclei contain only protamine and lack detectable levels of somatic histone. The amino acid composition and size of this protamine suggests that its structure must be intermediate between fish and mammalian protamines.

Division delay in sea urchin embryos induced by a specific protease inhibitor

Abstract Continuous incubation of fertilized sea urchin ( L. pictus and S. purpuratus ) eggs in 10 −4 M l-chloro-3-tosylamido-7-amino-2-heptanone (TLCK), a specific inhibitor of trypsin and trypsinlike enzymes, results in a cumulative delay in the time of cell division. Compared with untreated eggs, TLCK-treated eggs require 10% more time to complete the second division and more than twice as much time to reach the prism stage. The TLCK-treated eggs also remain in metaphase longer, at least through the first two divisions. They do not differ morphologically from untreated eggs at the same division stage. The TLCK treatment has little direct effect upon incorporation of thymidine into a DNase-sensitive, TCA-insoluble fraction. Little or no division delay is elicited by l-tosylamido-2-phenylethyl-chloromethyl ketone, iodoacetic acid, soybean trypsin inhibitor, lima bean trypsin inhibitor, ovomucoid trypsin inhibitor and pancreatic trypsin inhibitor. The division delay is not due to inhibition of cortical granule proteases.

Evidence for a single kind of d-glucose binding site on renal brush borders

The binding of d-glucose to renal brush border was studied in a preparation isolated from rabbit kidney. The data is indicate only one kind of binding site for d-glucose. The initial rate of binding was too rapid to be determined either directly or by extrapolation to zero time. Estimations of the amount of d-glucose bound at equilibrium for various concentrations of d-glucose yielded a straight line in a kinetic plot. This indicated a single binding site on the brush borders with a Kdiss of 4.8·10−5 M and a binding capacity of 264.8 pmoles/mg of protein. This conclusion is further supported by demonstration of a requirement for Na+ at two different concentrations of glucose, 1 and 100 μM, in the presence of Tris buffer. This binding was inhibited by other sugars and by phrlorizin indicating binding to a specific site.

Biological availability of radionuclides produced by the Plowshare Event Schooner. I. Body distribution in domestic pigs exposed in the field.

Abstract The biological availability and the distribution of radionuclides were studied in young Yorkshire pigs exposed to radioactive fallout from the Plowshare Event Schooner. Eight pigs were exposed in metabolic cages at various distances from ground zero in the expected path of the cloud, so that they could inhale debris and eat contaminated food. Three days after the detonation, the two pigs that were in the most contaminated portion of the fallout field were removed to a field laboratory, anesthetized with sodium pentobarbital, killed by exsanguination, and dissected. The organs were immediately sealed in tuna cans (capacity approximately 200 cm3) and sent to the Laboratory at Livermore for analysis for gamma-emitting radionuclides. The air filters that sampled the air at the pig station during the period of exposure were also canned and analyzed in the same manner. The radionuclides detected in the air filters were 188W/188Re, 132Te, 196Au, 131I, 103Ru and 74As; the short-lived 99Mo, 203Pb and 198Pu, although detected in the tissues, were too low in concentration for quantitation because of decay. The major radionuclides found in the pig tissues were 99Mo, 188W/188Re, 187W, 132Te, 203Pb, 196Au, 198Au, 131I, 103Ru and 74As. Comparisons between lungs, blood, and other tissues suggested that the lungs retained small amounts of 203Pb, 198Au, 103Ru and 74As. There seemed to be no differential concentration within the lung except perhaps for 198Au, which was more highly concentrated in the posterior portions. Of the other nuclides detected in the tissues, 99Mo was accumulated in kidney, liver, and gall bladder, 188W/188Re in kidney and bone, 187W in kidney, 196Au in the gall bladder, 198Au in the salivary gland, 103Ru in kidney and 74As in kidney, all in much higher concentrations than in blood.

BIOLOGICAL AVAILABILITY OF RADIONUCLIDES PRODUCED BY THE PLOWSHARE EVENT SCHOONER. II. RETENTION AND EXCRETION RATES IN PECCARIES AFTER A SINGLE ORAL DOSE OF DEBRIS.

The biological availability of radionuclides produced by the Plowshare event Schooner was studied in two peccaries (Tuyussu tuujcu). Debris was obtained from a surfaceatmospheric debris separator and collector which was designed and fabricated at the Lawrence Radiation Laboratory. This device was located in the expected cloud path 0.9 miles from ground zero. The debris which contained particles ranging from 10 to 80 p in diameter was administered in a single oral dose to two peccaries. The animals were then analyzed daily with a solid-state detector for all the gamma-emitting radionuclides until the activities had reached very low levels (9 days). The daily collections of urine and feces were sealed in 200 cm3 tuna cans and analyzed with the same detector. The major radionuclides detected were 1ssW~88Re, l96Au 9 7 1311 103Ru, T4As, S8C0, S4Mn and The 18sW/188Re, IS6Au, Io3Ru, 58C0, 54Mn and were excreted mostly in the feces; some lS8W/ls8Re (16%), Ig6Au (7%), and Io3Ru (3%) were excreted in the urine. Large amounts of 1311 (39%) and 74As (43%) were excreted in the urine.