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  • 1
    Electronic Resource
    Electronic Resource
    Flow sorting of X and Y chromosome-bearing mammalian sperm: Activation and pronuclear development of sorted bull, boar, and ram sperm microinjected into hamster oocytes (1988)
    New York, NY : Wiley-Blackwell
    Gamete Research 21 (1988), S. 335-343 
    Details
    ISSN: 0148-7280
    Keywords: flow cytometry ; sperm separation ; DNA ; sex ratio ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Flow cytometric techniques were used to measure relative DNA content of X and Y chromosome-bearing bull, boar, and ram sperm populations and to separate the two sex-determining populations. Neat semen was prepared for flow cytometric analysis by washing, light sonication, and staining with 9 μM Hoechst 33342. Computer analysis of the bimodal histograms showed mean X-Y DNA differences of 3.9, 3.7, and 4.2% for bull, boar, and ram, respectively. Flow cytometric reanalysis of sorted bull, boar, and ram sperm showed purities greater than 90%. Bull, boar, and ram sperm nuclei were microinjected into hamster oocytes. Microinjected sperm were either unsorted, sorted, unsorted plus dithio-threitol (DTT) exposure, or sorted plus DTT exposure. Following microinjection, eggs were incubated 3 hr, fixed, and stained. A total of 579 eggs was observed for sperm activation (decondensation or formation of a male pronucleus). A lower percentage of sorted than unsorted (3 vs. 23%) boar sperm was activated (P 〈.05). However, sorted and unsorted DTT-exposed boar sperm or sorted and unsorted bull or ram sperm, regardless of DTT treatment, did not differ significantly. Sorted sperm nuclei of both rams and bulls exhibited higher activation rates than sorted boar sperm (P 〈.05). Treatment of sperm with DTT increased the activation rate (P 〈 .05) for sorted boar sperm but not for bull or ram sperm. These data represent the first separation of bull, boar, and ram X and Y chromosome-bearing sperm populations and the first evidence that sperm of domestic animals sorted on the basis of DNA by flow cytometric procedures have the ability to decondense and to form pronuclei upon injection into a hamster egg.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1120210402
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  • 2
    Electronic Resource
    Electronic Resource
    Morphological and ultrastructural characterization of mammalian spermatozoa processed for flow cytometric DNA analyses (1984)
    New York, NY : Wiley-Blackwell
    Gamete Research 10 (1984), S. 339-351 
    Details
    ISSN: 0148-7280
    Keywords: spermatozoa ; flow cytometry ; DNA staining ; nuclear morphology ; ultrastructure ; mammals ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The morphological and ultrastructural changes that occur during preparation of porcine, bovine, and murine spermatozoa for flow cytometric quantification of the relative DNA content of the X- and Y-chromosome-bearing sperm populations were examined. Ejaculated spermatozoa from the boar and bull were washed using a series of dimethyl sulfoxide (DMSO) solutions prior to fixation, whereas the epididymal mouse spermatozoa were washed only in phosphate-buffered saline (PBS). Spermatozoa from all three species were then fixed in ethanol and processed for fluorochrome staining by a treatment regimen consisting of sulfhydryl reduction and proteolysis. The processed sperm nuclei were stained for DNA with the fluorochrome, 4′-6-diamidino-2-phenylindole (DAPI) before quantification by flow cytometry. Scanning and transmission electron micrographs of sperm heads taken at various steps of the preparation and staining procedures show 1) that the rigorous washing procedure disrupted the plasma and outer acrosomal membranes, 2) that ethanol fixation resulted in removal of the outer membranes and disintegration of the nuclear envelope, and 3) that thiol and proteolysis treatment removed the remaining cellular organelles including the tail and rapidly induced partial decondensation of the tightly packed chromatin. Sequential micrographs showed that the nuclear matrix of all three species increased in thickness about twofold during the preparation and staining. Consequently, the harsh procedures currently used for quantitative staining of DNA for high-resolution flow cytometric analyses destroy most cellular organelles and thereby prevent simultaneous characterization of DNA content and other sperm cell constituents.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1120100402
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    Paper (German National Licenses)
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  • 3
    Electronic Resource
    Electronic Resource
    Flow sorting of X and Y chromosome-bearing spermatozoa into two populations (1987)
    New York, NY : Wiley-Blackwell
    Gamete Research 16 (1987), S. 1-9 
    Details
    ISSN: 0148-7280
    Keywords: flow cytometry ; DNA ; sperm separation ; fluorescent stain ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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 〈 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.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1120160102
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    Paper (German National Licenses)
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  • 4
    Electronic Resource
    Electronic Resource
    Attempted sexing of bovine spermatozoa by fractionation on a Percoll density gradient (1988)
    New York, NY : Wiley-Blackwell
    Gamete Research 20 (1988), S. 83-92 
    Details
    ISSN: 0148-7280
    Keywords: spermatozoal ; separation ; flow cytometry ; semen sexing ; electron microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Bovine spermatozoa were fractionated on Percoll density gradients into two major subpopulations of motile spermatozoa and a minor fraction containing mostly nonmotile spermatozoa with abnormal morphology. Fractionation required the addition of bovine serum albumin and a continuous Percoll gradient buffered with sodium bicarbonate. It is postulated that, under suitable ionic conditions, the binding of bovine serum albumin to spermatozoa amplifies subtle differences between subpopulations. These studies were directed toward separating Y- and X-bearing spermatozoa. However, when the subpopulations were evaluated by flow cytometry, their Y:X ratios were similar to that of an unfractionated control.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1120200108
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    Paper (German National Licenses)
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