Barton L. Gledhill

Quantification and classification of human sperm morphology by computer-assisted image analysis.

A quantitative, semi-automated method for classifying human sperm based on objective measurements of head shapes and sizes has been developed. Air-dried smears of semen from eight healthy men were stained with the Feulgen reaction and 283 sperm were selected as prototypic examples of the 10 morphology classes used in our classification system. Sperm heads were imaged through a microscope (NA = 1.3), sampled at 0.125-micron intervals, and measured on an image analysis system. Measurements included stain content, length, width, perimeter, area, and arithmetically derived combinations. Additionally, each sperm image was optically sectioned at right angles to its major axis to give a measure of lengthwise heterogeneity of shape. Linear stepwise discriminant analysis was used to identify the more powerful parameters and to create a model employing eight parameters. The jackknifed classification procedure distinguished normal from abnormal sperm with 95% accuracy and correctly assigned 86% of the sperm to one of 10 shape classes. Most of the misclassification errors occurred among closely related classes. The results demonstrate the ability of automated image analysis to classify individual sperm into clinically familiar shape categories.

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.

Cytometric analysis of shape and DNA content in mammalian sperm.

Male germ cells respond dramatically to a variety of insults and are important reproductive dosimeters. Semen analyses are very useful in studies on the effects of drugs, chemicals, and environmental hazards on testicular function, male fertility and heritable germinal mutations. Sperm were analyzed by flow cytometry and slit-scan flow analysis for injury following the exposure of testes to mutagens. The utility of flow cytometry in genotoxin screening and monitoring of occupational exposure was evaluated. The technique proved valuable in separation of X- and Y-chromosome bearing sperm and the potential applicability of this technique in artificial insemination and a solution, of accurately assessing the DNA content of sperm were evaluated-with reference to determination of X- and Y-chromosome bearing sperm.

Sex chromosome ratios determined by karyotypic analysis in albumin-isolated human sperm**Work performed under the auspices of the Office of Health and Environmental Research, United States Department of Energy, by the Lawrence Livermore National Laboratory under contract number W-7405-ENG-48.

Human sperm that had been processed for Y-enrichment (male sex preselection) according to a currently favored albumin density gradient procedure were analyzed karyotypically for the proportion of X and Y chromosomes with the use of the human sperm/hamster egg system. This method allows direct inspection of haploid chromosome complements from human sperm. In 290 albumin-isolated sperm from six men, there were 57.2% X- and 42.8% Y-bearing chromosome complements; 201 unprocessed concurrent control sperm from five of the men had 50.2% X and 49.8% Y complements. The observed shift in sex chromosome ratio in processed samples, a decrease in Y-bearing sperm, was not statistically different from that of unprocessed controls (P = 0.13) but was significantly different when compared with the theoretic X/Y ratio of 50/50 (P = 0.016). A total of 3187 historical control karyotypes were also reviewed, with an overall sex chromosome ratio (X/Y) of 49.8/50.2. The control groups did not differ significantly from the expected 50/50. The Y-enrichment of processed sperm was not confirmed.