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  • 1
    Electronic Resource
    Electronic Resource
    Germ cell degeneration in normal and microwave-irradiated rats: Potential sperm production rates at different developmental steps in spermatogenesis (1984)
    New York, NY [u.a.] : Wiley-Blackwell
    The @Anatomical Record 209 (1984), S. 501-507 
    Details
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Germ cell degeneration in 14 normal and 14 microwave-irradiated, adult (400-500 gm), Sprague-Dawley rats was compared by evaluating potential sperm production rates at different developmental steps in spermatogenesis. Following 9 days of irradiation at 1.3 GHz (6 hours/day at 6.3 mW/gm using 1-μsec pulsewidth at 600 pulses/second) or sham treatment, rats were killed at 6.5, 13.0, 26.0, or 52.0 days following treatment. Testes were perfused with 2% glutaraldehyde, embedded in Epon, and sectioned at 0.5 μm for morphometric analyses. Plasma LH and FSH concentrations were determined by radioimmunoassay from blood collected on the day of death. Considering nuclear size, percentage of nuclei in the parenchyma, and life span of different cells, potential daily sperm production was determined for type B spermatogonia, preleptotene or pachytene primary spermatocytes, or spermatids with round nuclei. No differences (P 〉 .05) in parameters tested were found among time periods following irradiation. With the possible exception of sperm production per testis (P 〈 .05) based on pachytene spermatocytes, microwave irradiation had no effect on the parameters evaluated. No degeneration was detected in spermatogenesis when potential sperm production rates were determined either from type B spermatogonia to spermatids or from type B spermatogonia to a posttesticular approximation of sperm production rate. Thus, it appears that regulation of sperm production rates must take place during spermatogonial mitoses, since once the number of type B spermatogonia is determined, there is essentially no subsequent alteration in sperm production potential in normal or irradiated adult rats.
    Additional Material: 1 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/ar.1092090410
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  • 2
    Electronic Resource
    Electronic Resource
    Novel gene exon homologous to pancreatic phospholipase A2: Sequence and chromosomal mapping of both human genes (1989)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 39 (1989), S. 327-337 
    Details
    ISSN: 0730-2312
    Keywords: phospholipase A2 ; human genes ; pancreatic ; human chromosome mapping ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We described previously the cloning and DNA sequence of the human gene encoding pancreatic phospholipase A2 [DNA 5, 519]. When pancreatic phospholipase A2 (PLA2) cDNA was used to screen a human genomic library, two classes of clones were obtained. One class encoded the pancreatic enzyme, and a second class encoded one exon of an apparently related PLA2. No additional PLA2 gene exons displayed sufficient homology to be detected by the probe. A homologous sequence in both rat and porcine genomic DNA was detected by DNA blot hybridization, and the corresponding gene fragments were cloned and sequenced. Within the deduced amino acid sequences, the presence of known functional residues along with the high degree of interspecies conservation suggests the genes encode a functional PLA2 enzyme form. The encoded sequence lacks Cys11, as do the “type II” viperid venom and other nonpancreatic mammalian PLA2 enzymes. The sequence is distinct from porcine intestinal PLA2 and appears not to be a direct homolog of the recently published rabbit ascites and rat platelet enzymes. Hybridization of DNA probes containing sequences from these genes to genomic DNA blots of mouse/human somatic cell hybrids permitted chromosomal assignment for both. The pancreatic gene mapped to human chromosome 12, and the homologous gene mapped to chromosome 1.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240390312
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  • 3
    Electronic Resource
    Electronic Resource
    Inhibition of low density lipoprotein uptake in confluent endothelial cell monolayers correlates with a restricted surface receptor redistribution (1979)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 100 (1979), S. 481-495 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Binding of either low density lipoprotein (LDL) or Concanavalin A (ConA) to actively growing vascular endothelial cells is associated with a redistribution of the appropriate cell surface receptor sites which form patches and caps. This receptor lateral mobility is greatly restricted when endothelial cells reach confluence and adopt the configuration of a cell monolayer composed of closely apposed and non-overlapping cells. In this case, although the cells still exhibit specific LDL binding to the appropriate cell surface receptor sites, neither the binding of LDL nor of ConA induces a receptor redistribution. The lack of LDL receptor redistribution correlates with a marked decrease in the rate of LDL internalization. In contrast, no such a density-dependent changes are observed in cell types which grow on top of each other and form multiple cell layers at confluence. Thus, neither LDL nor ConA induced cap formation in either sparse or confluent smooth muscle cell cultures and the same rate of LDL internalization is observed at both cell densities. Similarly, adsorptive endocytosis of cationized LDL (which enters the cell independently of the LDL receptor sites) was not correlated with a detectable receptor redistribution, nor was it significantly affected by changes in cell density and spatial organization.The formation of a confluent cell monolayer resting on an underlying basement membrane might therefore provide, via a change in membrane dynamics, a mechanism whereby the endothelium of large blood vessels can function as a protective barrier against the high circulating levels of LDL in plasma.
    Additional Material: 1 Tab.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041000311
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  • 4
    Electronic Resource
    Electronic Resource
    Delayed processing/export of messenger RNA under conditions of reduced protein synthesis (1988)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 135 (1988), S. 115-121 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The rates of processing and export of a variety of nuclear RNA species into the cytoplasmic compartment were studied by determining the rates of incorportion of tritiated uridine into nuclear and cytoplasmic RNA species. In exponentially growing cells, the rates of nuclear processing/export varied by more than a factor of ten for the six different mRNA species that were examined. Differences in the rates did not appear to be correlated with either the number or the sizes of introns in the genes for the RNA species. When cells were maintained under conditions of reduced protein synthesis (starvation for isoleucine and glutamine or exposure to cycloheximide), the processing rates for each species decreased by a factor of about 3. The decrease was not caused by the inability of hnRNA to associate with proteins, since the nuclear RNP distribution appeared normal in amino acidstarved cells.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041350116
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  • 5
    Electronic Resource
    Electronic Resource
    Proliferative variability of endothelial clones derived from adult bovine aorta: Influence of fibroblast growth factor and smooth muscle cell extracellular matrix (1983)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 114 (1983), S. 7-15 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Four endothelial cell clones derived from adult bovine aorta were examined with respect to their proliferative characteristics In vitro. Three of these clones, derived in the absence of fibroblast growth factor (FGF), displayed variable basal proliferative rates. One of these non-FGF derived clones grew at a maximal rate which could not be further enhanced with FGF. The other two clones grew at a suboptimal rate which was stimulated by low doses of FGF (10-50 ng/ml) and inhibited by higher doses (100-250 ng/ml). The fourth clone, derived in the presence of FGF, was stimulated by FGF in a dose-dependent manner (10-250 ng/ml) and was not growth inhibited at high FGF concentrations (250-1,000 ng/ml). Growth of all four clones on extracellular matrix (ECM) derived from bovine aortic smooth muscle (BASM) cells was optimal in the absence of FGF. ECM-coated dishes also significantly increased the sensitivity of all clones by at least fivefold to mitogenic stimulation by serum. The proliferative lifespans of the clones ranged between 60 and 120 generations with the most actively proliferating clones attaining the greatest lifespan. Continuous subculture of two of the endothelial clones in the presence of FGF or on ECM-coated dishes did not induce a dependence of the cells on either factor for subsequent growth in its absence. The results indicate that aortic endothelial cells display considerable clonal variability in ther basal proliferative rate and in their response to FGF. This clonal variability is not observed when the cells are maintained on ECM-coated dishes derived from vascular smooth muscle cells.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041140103
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  • 6
    Electronic Resource
    Electronic Resource
    The movement characteristics of rabbit spermatozoa before and after activation (1981)
    New York, NY : Wiley-Blackwell
    Gamete Research 4 (1981), S. 275-282 
    Details
    ISSN: 0148-7280
    Keywords: spermatozoa ; activation ; oviduct ; sperm movements ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Spermatozoa were flushed with mineral oil from the lower isthmus of the rabbit oviduct at four hours postcoitus (pc) and 11 hours pc. Videotapes were made of sperm behavior in the native isthmic fluid and after dilution of the fluid with culture medium. The tapes showed that, initially, spermatozoa in the native isthmic fluid were virtually immotile, but immediately resumed movement on contact with the culture medium. Isthmic sperm motility then evolved over a five- to 10-minute interval into the characteristic biphasic pattern of activated movement. Cine films of isthmic spermatozoa taken with a high-speed camera were analyzed to determine flagellar beat frequency, maximum flagellar curvature, and swimming velocity. Progressiveness ratios and hydrodynamic power outputs were then calculated for individual spermatozoa. Two phases of activated sperm movement, a whiplash phase and a progressive phase, were identified and characterized. The power output of activated spermatozoa increased twentyfold in comparison with the preactivated state. The power output of activated spermatozoa did not differ between the two phases of activated movement.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1120040403
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  • 7
    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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  • 8
    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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  • 9
    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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  • 10
    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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