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Donald P. Evenson - One of the best experts on this subject based on the ideXlab platform.
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boar fertility and sperm Chromatin Structure status a retrospective report
Journal of Andrology, 2009Co-Authors: Bradley A Didion, Kay Kasperson, Regina Wixon, Donald P. EvensonAbstract:Little information exists about boar sperm Chromatin quality and fertility within a commercial setting. The objective of this report is to provide information about boar sperm Chromatin integrity and its relationship to fertility. The sperm Chromatin Structure assay (SCSA) was used retrospectively to characterize sperm from 18 sexually mature boars having fertility information. Boar fertility was defined by farrow rate (FR) and average total number of pigs born (ANB) per litter of gilts and sows mated to individual boars. Fertility data was compiled for 1867 matings across the 18 boars. The SCSA uses flow cytometry to evaluate the structural integrity of sperm nuclear DNA. The SCSA parameters measured in this retrospective analysis were the percentage DNA fragmentation index (%DFI) and standard deviation of the DNA fragmentation index (SD DFI). The %DFI and SD DFI showed the following significant negative correlations with FR and ANB; %DFI vs FR, r = -0.55, P < .01; SD DFI vs FR, r = -0.67, P < .002; %DFI vs ANB, r = -0.54, P < .01; and SD DFI vs ANB, r = -0.54, P < .02. Although more information is required to better understand the relationship between DFI and boar fertility, this report suggests that the SCSA assay may be an important assay for identification of boars having potential for lowered fertility.
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relationship between the outcomes of assisted reproductive techniques and sperm dna fragmentation as measured by the sperm Chromatin Structure assay
Fertility and Sterility, 2003Co-Authors: Kjersten L Larsoncook, John D Brannian, Keith A Hansen, Kay Kasperson, Edward T Aamold, Donald P. EvensonAbstract:Abstract Objective To investigate how moderate and/or high levels of DNA fragmentation (DFI), as measured by the sperm Chromatin Structure assay (SCSA), affect either IVF or IVF with intracytoplasmic sperm injection (ICSI) fertilization, cleavage, blastulation, implantation, and pregnancy. Design: Retrospective clinical study. Setting: Academic human reproduction laboratory. Patient(s): Eighty-nine couples undergoing IVF with conventional fertilization or ICSI. Intervention(s): Sperm Chromatin Structure assay testing (SCSA) of semen aliquot taken from ejaculate used for assisted reproductive technology (ART). Main outcome measure(s): Related DFI to conventional semen parameters and cycle-specific outcomes after ART. Result(s): No patients achieved clinical pregnancy if SCSA values exceeded the DFI (27%, P P P r 2 ) between SCSA parameters and conventional parameters exceeded 0.29. Conclusion(s): Sperm Chromatin Structure assay identified thresholds for negative pregnancy outcome after ART not identified using conventional semen parameters. This is the first study analyzing the clinical value of sperm DFI to [1] include a large number of ART patients (n = 89), [2] perform SCSA analysis on a semen aliquot from the ejaculate used for ART, and [3] examine how the extent (moderate and high DFI) of DFI influenced ART outcomes.
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density gradient centrifugation and glass wool filtration of semen remove spermatozoa with damaged Chromatin Structure
Human Reproduction, 1999Co-Authors: K L Larson, Lorna K. Jost, John D Brannian, B K Timm, Donald P. EvensonAbstract:: The ability of double-layered density gradient centrifugation (DGC) or glass wool filtration (GWF) of semen to remove spermatozoa with damaged Chromatin Structure was assessed by the flow cytometric sperm Chromatin Structure assay (SCSA), which measures the susceptibility to sperm nuclear denaturation in situ. Ejaculates from 26 men attending a university-affiliated assisted reproduction laboratory were processed by DGC and GWF. Unprocessed, DGC- and GWF-processed specimens were assessed by the SCSA and by conventional semen parameters. Changes in Chromatin Structure were compared with conventional semen parameters. Both sperm preparation techniques yielded sperm suspensions with improved sperm Chromatin Structure as well as motility (%), forward progression (1-4) and viability (%). DGC was superior to GWF in the efficiency of recovering motile, morphologically normal, mature sperm suspensions. However, GWF produced improved Chromatin integrity (SDalpha(t)) and viability. Moderate correlations between SCSA and conventional sperm parameters were observed. Nevertheless, the SCSA provides additional information about the biochemical integrity of sperm DNA and may be used in future studies to provide insight into assisted reproduction technology outcomes not explained by conventional sperm parameters.
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effects of x irradiation on mouse testicular cells and sperm Chromatin Structure
Environmental and Molecular Mutagenesis, 1995Co-Authors: Brian L Sailer, Lorna K. Jost, K R Erickson, M A Tajiran, Donald P. EvensonAbstract:The testicular regions of male mice were exposed to x-ray doses ranging from 0 to 400 rads. Forty days after exposure the mice were killed and the testes and cauda epididymal sperm removed surgically. Flow cytometric measurements of acridine orange stained testicular samples indicated a repopulation of testicular samples indicated a repopulation of testicular cell types following x-ray killing of stem cells. Cauda epididymal sperm were analyzed by the sperm Chromatin Structure assay (SCSA), a flow cytometric measurement of the susceptibility of the sperm nuclear DNA to in situ acid denaturation. The SCSA detected increased susceptibility to DNA denaturation in situ after 12.5 rads of x-ray exposure, with significant increases following 25 rads. Abnormal sperm head morphology was not significantly increased until the testes were exposed to 60 rads of x-rays. These data suggest that the SCSA is currently the most sensitive, noninvasive method of detecting x-ray damage to testicular stem spermatogonia. 47 refs., 5 figs.
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Flow cytometric evaluation of boar semen by the sperm Chromatin Structure assay as related to cryopreservation and fertility.
Theriogenology, 1994Co-Authors: Donald P. Evenson, L. H. Thompson, Lorna K. JostAbstract:Boar semen from a heterospermic mating trial and semen cryopreserved by various methods were evaluated by the flow cytometric sperm Chromatin Structure assay (SCSA), which measures the susceptibility of sperm nuclear DNA to acid-induced denaturation in situ. Spermatozoa were treated with a pH 1.4 buffer and then stained with the metachromatic dye acridine orange. Acridine orange intercalated into double-stranded DNA (native) fluoresces green while single-stranded DNA (denatured) fluoresces red when excited with 488 nm light. The ratio of red to total fluorescence provides an index of normality/abnormality. The SCSA data on neat boar semen or semen in either Kiev-Merck or Pursel-Johnson extender and frozen directly on dry ice blocks or plunged into LN2 did not differ within individual boars. Therefore, Chromatin Structure, as measured by the SCSA, was not influenced differently by these 2 methods of semen cryopreservation. When semen from 6 boars was mixed in equal sperm numbers in six 3-way combinations and inseminated into at least 3 Duroc gilts per combination, 4 of the 6 combinations yielded 2 litters, while the remaining 2 combinations yielded 3 litters. The SCSA correctly predicted both the high and low fertility boars based on a ratio of offspring as deviated from the theoretical percentage. Thus, the SCSA was found to be a valuable adjunct method for evaluating boar cemen quality.
I I Kireev - One of the best experts on this subject based on the ideXlab platform.
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insights into interphase large scale Chromatin Structure from analysis of engineered chromosome regions
Cold Spring Harbor Symposia on Quantitative Biology, 2010Co-Authors: Andrew S Belmont, Paul Sinclair, Qian Bian, I I Kireev, Yan Hu, Wei WuAbstract:How Chromatin folds into mitotic and interphase chromosomes has remained a difficult question for many years. We have used three generations of engineered chromosome regions as a means of visualizing specific chromosome regions in live cells and cells fixed under conditions which preserve large-scale Chromatin Structure. Our results confirm the existence of large-scale Chromatin domains and fibers formed by the folding of 10 and 30 nm Chromatin fibers into larger, spatially distinct domains. Transcription at levels within several fold of the levels measured for endogenous loci occur within these large-scale Chromatin Structures on a condensed template linearly compacted several hundred fold to one thousand fold relative to B-form DNA. However, transcriptional induction is accompanied by a several fold decondensation of this large-scale Chromatin Structure that propagates hundreds of kb beyond the induced gene. Examination of engineered chromosome regions in mouse ES and differentiated cells suggests a surprising degree of plasticity in this large-scale Chromatin Structure, allowing long-range DNA interactions within the context of large-scale Chromatin fibers. Recapitulation of gene specific differences in large-scale Chromatin conformation and nuclear positioning using these engineered chromosome regions will facilitate identification of cis and trans determinants of interphase chromosome architecture.
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large scale Chromatin Structure of inducible genes transcription on a condensed linear template
Journal of Cell Biology, 2009Co-Authors: I I Kireev, Matt Plutz, Nazanin Ashourian, Andrew S BelmontAbstract:The Structure of interphase chromosomes, and in particular the changes in large-scale Chromatin Structure accompanying transcriptional activation, remain poorly characterized. Here we use light microscopy and in vivo immunogold labeling to directly visualize the interphase chromosome conformation of 1–2 Mbp Chromatin domains formed by multi-copy BAC transgenes containing 130–220 kb of genomic DNA surrounding the DHFR, Hsp70, or MT gene loci. We demonstrate near-endogenous transcription levels in the context of large-scale Chromatin fibers compacted nonuniformly well above the 30-nm Chromatin fiber. An approximately 1.5–3-fold extension of these large-scale Chromatin fibers accompanies transcriptional induction and active genes remain mobile. Heat shock–induced Hsp70 transgenes associate with the exterior of nuclear speckles, with Hsp70 transcripts accumulating within the speckle. Live-cell imaging reveals distinct dynamic events, with Hsp70 transgenes associating with adjacent speckles, nucleating new speckles, or moving to preexisting speckles. Our results call for reexamination of classical models of interphase chromosome organization.
Andrew S Belmont - One of the best experts on this subject based on the ideXlab platform.
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insights into interphase large scale Chromatin Structure from analysis of engineered chromosome regions
Cold Spring Harbor Symposia on Quantitative Biology, 2010Co-Authors: Andrew S Belmont, Paul Sinclair, Qian Bian, I I Kireev, Yan Hu, Wei WuAbstract:How Chromatin folds into mitotic and interphase chromosomes has remained a difficult question for many years. We have used three generations of engineered chromosome regions as a means of visualizing specific chromosome regions in live cells and cells fixed under conditions which preserve large-scale Chromatin Structure. Our results confirm the existence of large-scale Chromatin domains and fibers formed by the folding of 10 and 30 nm Chromatin fibers into larger, spatially distinct domains. Transcription at levels within several fold of the levels measured for endogenous loci occur within these large-scale Chromatin Structures on a condensed template linearly compacted several hundred fold to one thousand fold relative to B-form DNA. However, transcriptional induction is accompanied by a several fold decondensation of this large-scale Chromatin Structure that propagates hundreds of kb beyond the induced gene. Examination of engineered chromosome regions in mouse ES and differentiated cells suggests a surprising degree of plasticity in this large-scale Chromatin Structure, allowing long-range DNA interactions within the context of large-scale Chromatin fibers. Recapitulation of gene specific differences in large-scale Chromatin conformation and nuclear positioning using these engineered chromosome regions will facilitate identification of cis and trans determinants of interphase chromosome architecture.
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large scale Chromatin Structure of inducible genes transcription on a condensed linear template
Journal of Cell Biology, 2009Co-Authors: I I Kireev, Matt Plutz, Nazanin Ashourian, Andrew S BelmontAbstract:The Structure of interphase chromosomes, and in particular the changes in large-scale Chromatin Structure accompanying transcriptional activation, remain poorly characterized. Here we use light microscopy and in vivo immunogold labeling to directly visualize the interphase chromosome conformation of 1–2 Mbp Chromatin domains formed by multi-copy BAC transgenes containing 130–220 kb of genomic DNA surrounding the DHFR, Hsp70, or MT gene loci. We demonstrate near-endogenous transcription levels in the context of large-scale Chromatin fibers compacted nonuniformly well above the 30-nm Chromatin fiber. An approximately 1.5–3-fold extension of these large-scale Chromatin fibers accompanies transcriptional induction and active genes remain mobile. Heat shock–induced Hsp70 transgenes associate with the exterior of nuclear speckles, with Hsp70 transcripts accumulating within the speckle. Live-cell imaging reveals distinct dynamic events, with Hsp70 transgenes associating with adjacent speckles, nucleating new speckles, or moving to preexisting speckles. Our results call for reexamination of classical models of interphase chromosome organization.
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ectopic histone h3s10 phosphorylation causes Chromatin Structure remodeling in drosophila
Development, 2008Co-Authors: Huai Deng, Andrew S Belmont, Melissa J Blacketer, Jack Girton, Jorgen Johansen, Kristen M JohansenAbstract:Histones are subject to numerous post-translational modifications that correlate with the state of higher-order Chromatin Structure and gene expression. However, it is not clear whether changes in these epigenetic marks are causative regulatory factors in Chromatin Structure changes or whether they play a mainly reinforcing or maintenance role. In Drosophila phosphorylation of histone H3S10 in euchromatic Chromatin regions by the JIL-1 tandem kinase has been implicated in counteracting heterochromatization and gene silencing. Here we show, using a LacI-tethering system, that JIL-1 mediated ectopic histone H3S10 phosphorylation is sufficient to induce a change in higher-order Chromatin Structure from a condensed heteroChromatin-like state to a more open euchromatic state. This effect was absent when a `kinase dead9 LacI-JIL-1 construct without histone H3S10 phosphorylation activity was expressed. Instead, the `kinase dead9 construct had a dominant-negative effect, leading to a disruption of Chromatin Structure that was associated with a global repression of histone H3S10 phosphorylation levels. These findings provide direct evidence that the epigenetic histone tail modification of H3S10 phosphorylation at interphase can function as a causative regulator of higher-order Chromatin Structure in Drosophila in vivo.
Sarah C R Elgin - One of the best experts on this subject based on the ideXlab platform.
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(CT)n (GA)n repeats and heat shock elements have distinct roles in Chromatin Structure and transcriptional activation of the Drosophila hsp26 gene.
Molecular and Cellular Biology, 2015Co-Authors: Lori L Wallrath, Howard Granok, Sarah C R ElginAbstract:Previous analysis of the hsp26 gene of Drosophila melanogaster has shown that in addition to the TATA box and the proximal and distal heat shock elements (HSEs) (centered at -59 and -340, relative to the start site of transcription), a segment of (CT)n repeats at -135 to -85 is required for full heat shock inducibility (R.L. Glaser, G.H. Thomas, E.S. Siegfried, S.C.R. Elgin, and J.T. Lis, J. Mol. Biol. 211:751-761, 1990). This (CT)n element appears to contribute to formation of the wild-type Chromatin Structure of hsp26, an organized nucleosome array that leaves the HSEs in nucleosome-free, DNase I-hypersensitive (DH) sites (Q. Lu, L.L. Wallrath, B.D. Allan, R.L. Glaser, J.T. Lis, and S.C.R. Elgin, J. Mol. Biol. 225:985-998, 1992). Inspection of the sequences upstream of hsp26 has revealed an additional (CT)n element at -347 to -341, adjacent to the distal HSE. We have analyzed the contribution of this distal (CT)n element (-347 to -341), the proximal (CT)n element (-135 to -85), and the two HSEs both to the formation of the Chromatin Structure and to heat shock inducibility. hsp26 constructs containing site-directed mutations, deletions, substitutions, or rearrangements of these sequence elements have been fused in frame to the Escherichia coli lacZ gene and reintroduced into the D. melanogaster genome by P-element-mediated germ line transformation. Chromatin Structure of the transgenes was analyzed (prior to gene activation) by DNase I or restriction enzyme treatment of isolated nuclei, and heat-inducible expression was monitored by measuring beta-galactosidase activity. The results indicate that mutations, deletions, or substitutions of either the distal or the proximal (CT)n element affect the Chromatin Structure and heat-inducible expression of the transgenes. These (CT)n repeats are associated with a nonhistone protein(s) in vivo and are bound by a purified Drosophila protein, the GAGA factor, in vitro. In contrast, the HSEs are required for heat-inducible expression but play only a minor role in establishing the Chromatin Structure of the transgenes. Previous analysis indicates that prior to heat shock, these HSEs appear to be free of protein. Our results suggest that GAGA factor, an abundant protein factor required for normal expression of many Drosophila genes, and heat shock factor, a specific transcription factor activated upon heat shock, play distinct roles in gene regulation: the GAGA factor establishes and/or maintains the DH sites prior to heat shock induction, while the activated heat shock factor recognizes and binds HSEs located within the DH sites to trigger transcription.
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gaga factor and the tfiid complex collaborate in generating an open Chromatin Structure at the drosophila melanogaster hsp26 promoter
Molecular and Cellular Biology, 2002Co-Authors: Boris A Leibovitch, Lawrence R Benjamin, Yingyun Liu, David S Gilmour, Sarah C R ElginAbstract:The upstream regulatory region of the Drosophila melanogaster hsp26 gene includes two DNase I-hypersensitive sites (DH sites) that encompass the critical heat shock elements. This Chromatin Structure is required for heat shock-inducible expression and depends on two (CT)n*(GA)n elements bound by GAGA factor. To determine whether GAGA factor alone is sufficient to drive formation of the DH sites, we have created flies with an hsp26/lacZ transgene wherein the entire DNA segment known to interact with the TFIID complex has been replaced by a random sequence. The replacement results in a loss of heat shock-inducible hsp26 expression and drastically diminishes nuclease accessibility in the Chromatin of the regulatory region. Chromatin immunoprecipitation experiments show that the decrease in TFIID binding does not reduce GAGA factor binding. In contrast, the loss of GAGA factor binding resulting from (CT)n mutations decreases TFIID binding. These data suggest that both GAGA factor and TFIID are necessary for formation of the appropriate Chromatin Structure at the hsp26 promoter and predict a regulatory mechanism in which GAGA factor binding precedes and contributes to the recruitment of TFIID.
Lee W Kraus - One of the best experts on this subject based on the ideXlab platform.
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parp 1 regulates Chromatin Structure and transcription through a kdm5b dependent pathway
Molecular Cell, 2010Co-Authors: Raga Krishnakumar, Lee W KrausAbstract:PARP-1 is an abundant nuclear enzyme that regulates gene expression, although the underlying mechanisms are unclear. We examined the interplay between PARP-1, histone 3 lysine 4 trimethylation (H3K4me3), and linker histone H1 in the Chromatin-dependent control of transcription. We show that PARP-1 is required for a series of molecular outcomes at the promoters of PARP-1-regulated genes, leading to a permissive Chromatin environment that allows loading of the RNA Pol II machinery. PARP-1 does so by (1) preventing demethylation of H3K4me3 through the PARylation, inhibition, and exclusion of the histone demethylase KDM5B; and (2) promoting the exclusion of H1 and the opening of promoter Chromatin. Upon depletion of PARP-1, these outcomes do not occur efficiently. Interestingly, cellular signaling pathways can use the regulated depletion of PARP-1 to modulate these Chromatin-related molecular outcomes. Collectively, our results help to elucidate the roles of PARP-1 in the regulation of Chromatin Structure and transcription.
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nad dependent modulation of Chromatin Structure and transcription by nucleosome binding properties of parp 1
Cell, 2004Co-Authors: Miyoung Kim, Steven Mauro, Nicolas Gevry, John T Lis, Lee W KrausAbstract:PARP-1 is the most abundantly expressed member of a family of proteins that catalyze the transfer of ADP-ribose units from NAD+ to target proteins. Herein, we describe previously uncharacterized nucleosome binding properties of PARP-1 that promote the formation of compact, transcriptionally repressed Chromatin Structures. PARP-1 binds in a specific manner to nucleosomes and modulates Chromatin Structure through NAD+-dependent automodification, without modifying core histones or promoting the disassembly of nucleosomes. The automodification activity of PARP-1 is potently stimulated by nucleosomes, causing the release of PARP-1 from Chromatin. The NAD+-dependent activities of PARP-1 are reversed by PARG, a poly(ADP-ribose) glycohydrolase, and are inhibited by ATP. In vivo, PARP-1 incorporation is associated with transcriptionally repressed Chromatin domains that are spatially distinct from both histone H1-repressed domains and actively transcribed regions. Thus, PARP-1 functions both as a structural component of Chromatin and a modulator of Chromatin Structure through its intrinsic enzymatic activity.