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W Steven Ward

Publications and source records attributed to W Steven Ward.

10 recordsLinked to original sources
PubMed2006

Topoisomerase II-mediated breaks in spermatozoa cause the specific degradation of paternal DNA in fertilized oocytes.

We have demonstrated that mouse spermatozoa can cleave their DNA into 50-kb fragments when treated with Triton X-100, MnCl(2), and CaCl(2). This cleavage, which is termed sperm chromatin fragmentation (SCF), is mediated by topoisomerase IIB (TOP2B) following stimulation by a factor in the epididymal fluid, most likely a nuclease, and can be at least partially religated by EDTA. When the protamines are removed, this DNA breakage is followed by digestion of the DNA by a nuclease(s). We tested whether the oocyte could repair TOP2B-induced sperm DNA breaks and whether partial religation by EDTA would allow spermatozoa to fertilize the oocytes normally. Oocytes injected with untreated spermatozoa developed normally. However, oocytes injected with spermatozoa treated with MnCl(2) and CaCl(2) to induce SCF, with or without subsequent EDTA treatment, failed to develop. In both of these treatment groups, the maternal pronuclei developed normally and replicated their DNA. However the paternal pronuclei did not replicate their DNA and this DNA began to disappear 6 h postinjection, which corresponded approximately to the time at which maternal DNA replication was initiated. These data suggest that when TOP2B is induced to cleave sperm DNA before fertilization, the paternal DNA is subsequently degraded by a highly regulated mechanism that does not affect the maternal chromatin. Furthermore, partial religation by EDTA of TOP2B-induced breaks prevents neither the inhibition of DNA synthesis nor DNA degradation.

Animals↗
PubMed2006

Topoisomerase IIB and an extracellular nuclease interact to digest sperm DNA in an apoptotic-like manner.

We previously demonstrated that mammalian spermatozoa contain a nuclease activity that cleaves DNA into loop-sized fragments. We show here that this activity is mediated by a nuclear matrix-associated topoisomerase IIB (TOP2B) interacting with an extracellular Mn2+/Ca2+-dependent nuclease. Together, these enzymes cleave all of the DNA into fragments of 50 kb, and this cleavage can be reversed by EDTA. If dithiothreitol is included, the nuclease digests the DNA, and if the protamines are removed the DNA is completely digested. A similar, TOP2B-mediated, chromatin fragmentation, which is reversible, followed by digestion of the DNA by an intracellular nuclease occurs in somatic cells during apoptosis. The extracellular location of the sperm nuclease made it possible to reconstitute the fragmentation activity in isolated spermatozoa, thus allowing us to identify two novel aspects of the mechanism. First, the fragmentation of all of the DNA to 50 kb by TOP2B required the addition of the extracellular nuclease or factor. Second, the subsequent, complete digestion of the DNA by the nuclease could be inhibited by etoposide, suggesting that the nuclease digestion requires TOP2B religation of the cleaved DNA. These data are the first demonstration of an active TOP2B in spermatozoa, suggesting this inert chromatin may be more active than previously thought. They also show that the unique chromatin structure of spermatozoa may provide an important model to study the regulated degradation of chromatin by TOP2B and associated nucleases.

Animals↗
PubMed2004

A model for the function of sperm DNA degradation.

In this review, we present our recent evidence suggesting, but not yet proving, that mammalian spermatozoa contain a mechanism by which they can digest their own DNA when exposed to a stressful environment. We discuss our recent data that demonstrate that when mammalian spermatozoa are treated in a variety of ways, the paternal chromosomes in the zygote, or the sperm DNA itself, are degraded into large, chromosome-sized fragments. These published data support the existence of nuclease activity in spermatozoa. We suggest that this nuclease activity is part of a mechanism the spermatozoon uses when it encounters a stressful environment to prevent fertilisation and to avoid the transmission of potentially damaged DNA to the embryo. We propose a model based on sperm chromatin structure by which this nuclease can digest the highly condensed sperm chromatin.

Chromatin↗
PubMed2003

Ability of hamster spermatozoa to digest their own DNA.

Mammalian sperm chromatin is bound by protamines into highly condensed toroids with approximately 50 kilobases (kb) of DNA. It is also organized into loop domains of about the same size that are attached at their bases to the proteinaceous nuclear matrix. In this work, we test our model that each sperm DNA-loop domain is condensed into a single protamine toroid. Our model predicts that the protamine toroids are linked by chromatin that is more sensitive to nucleases than the DNA within the toroids. To test this model, we treated hamster sperm nuclei with DNase I and found that the sperm chromatin was digested into fragments with an average size of about 50 kb, by pulse-field gel electrophoresis (PFGE). Surprisingly, we also found that spermatozoa treated with 0.25% Triton X-100 (TX) and 20 mM MgCl2 overnight resulted in the same type of degradation, suggesting that sperm nuclei have a mechanism for digesting their own DNA at the bases of the loop domains. We extracted the nuclei with 2 M NaCl and 10 mM dithiothreitol (DTT) to make nuclear halos. Nuclear matrices prepared from DNase I-treated spermatozoa had no DNA attached, suggesting that DNase I digested the DNA at the bases of the loop domains. TX-treated spermatozoa still had their entire DNA associated with the nuclear matrix, even though the DNA was digested into 50-kb fragments as revealed by PFGE. The data support our donut-loop model for sperm chromatin structure and suggest a functional role for this type of organization in that sperm can digest its own DNA at the sites of attachment to the nuclear matrix.

Animals↗
PubMed2002

Expression of foreign DNA is associated with paternal chromosome degradation in intracytoplasmic sperm injection-mediated transgenesis in the mouse.

The efficiency of intracytoplasmic sperm injection (ICSI)-mediated transgenesis is often limited by poor embryo development. Because our previous work indicated that impairment of embryo development is frequently related to chromosomal abnormalities, we hypothesized that foreign DNA and/or conditions used to enhance integration of the DNA might induce chromosome damage. Therefore, we examined the chromosomes of mouse embryos produced by transgenesis with the EGFP gene. Spermatozoa were processed with three methods that cause membrane disruption: freeze-thawing, Triton X-100, or Triton X-100 followed by a sucrose wash. Membrane-disrupted spermatozoa were mixed with EGFP plasmids and injected into metaphase II oocytes. Three endpoints were evaluated: paternal chromosomes of the zygote, embryo capacity to develop in vitro, and expression of the transgene at the morula/blastocyst stage. In all pretreatments, we observed a significant decrease (approximately 2-fold) in the frequency of normal karyoplates when spermatozoa were incubated with exogenous DNA as compared with the treatment when no DNA was added. As predicted, embryo development was correlated with the integrity of the paternal chromosomes of the zygote. Searching for the possible mechanism of chromosome degradation, we used the ion chelators EGTA and EDTA and found that they neutralize the harmful effect of the transgene and stabilize the paternal chromosomes. In the presence of chelating agents, however, the number of embryos expressing EGFP produced with ICSI-mediated transgenesis decreased significantly. The results suggest that treatment of spermatozoa with exogenous DNA leads to paternal chromosome degradation in the zygote. Furthermore, the mechanisms of disruption of paternal chromosomes and the integration of foreign DNA may be closely related.

Animals↗
PubMed2002

Sperm nuclear halos can transform into normal chromosomes after injection into oocytes.

Mouse sperm nuclei extracted with an ionic detergent and 2 M NaCl retain their overall morphology, but upon subsequent reduction of the protamine disulfides they lose all elements of chromatin structure except the organization of DNA into loop that are anchored to the nuclear matrix. These DNA loops appear as a halo surrounding the nuclear matrix, and nuclei extracted in this manner are, therefore, called nuclear halos. Here, we report that sperm nuclear halos injected into oocytes can form pronuclei, then transform into chromosomes with normal morphology. This suggests that sperm nuclear halos retain all the information necessary for normal chromosomal organization, and that micromanipulation of these extracted sperm nuclei can be accomplished without major DNA damage.

Animals↗
PubMed2002

Novel assay for determining DNA organization in human spermatozoa: implications for male factor infertility.

OBJECTIVES: To present a novel assay for determining DNA organization in fertile human spermatozoa and establish morphometric parameters for these samples. The three-dimensional organization of DNA within a cell nucleus is intimately related to cellular function. For example, it has recently been demonstrated that normal sperm DNA organization may be necessary for successful in vitro fertilization in the mouse. METHODS: Semen from 12 fertile volunteers was tested for sperm DNA organization using our nuclear matrix stability assay. Sperm DNA then underwent computerized digital image analysis and standards of normal were established. RESULTS: Sperm DNA organization was constant in all samples tested. Normal parameters established included mean nuclear matrix diameter (9.17 +/- 1.59 microm), mean DNA halo diameter (20.56 +/- 2.53 microm), mean halo area (66.88 +/- 7.92 microm(2)), and mean nuclear matrix area (32.98 +/- 4.3 microm(2)). CONCLUSIONS: This assay may be used to determine DNA organization in a semen sample. Defining sperm DNA organization may be important clinically, because normal DNA organization is necessary for normal cellular function.

Adult↗
PubMed2002

Combination of dithiothreitol and detergent treatment of spermatozoa causes paternal chromosomal damage.

Treatment of spermatozoa with either the nonionic detergent Triton X-100 (TX) or dithiothreitol (DTT) has been suggested to confer enhanced success on intracytoplasmic sperm injection (ICSI) in mice and humans. Here, we attempted to use both reagents together, to our knowledge for the first time, and found that this caused severe chromosomal breaks in paternal pronuclei. We documented this effect further by treating mouse spermatozoa with several combinations of DTT with and without detergent. Spermatozoa were treated with vigorous pipetting to induce membrane disruption or with TX or the ionic detergent mixed alkyltrimethylammonium bromide (ATAB). Swim-up spermatozoa were used as controls. In each treatment, two samples were tested, with or without the addition of DTT during the treatment procedure. In all samples with DTT, protamine reduction was confirmed by the decondensation assay. Sperm nuclei obtained after different treatments were injected into oocytes for cytogenetic analysis, and paternal and maternal chromosomes of the zygote were visualized and examined. We found that the numbers of normal paternal karyoplates resulting from ICSI with spermatozoa treated with either DTT (87%, 153/176), TX (79%, 112/142), or ATAB (85%, 99/116) alone were similar to swim-up controls (92%, 103/112). However, only 22% (23/103) and 40% (59/149) of examined metaphases were scored as normal in TX + DTT or ATAB + DTT treatments, respectively. Spermatozoa in which the membranes were disrupted by vigorous pipetting in the presence of DTT had a slightly reduced frequency of normal chromosomes (61%, 64/104), whereas those without DTT were normal (79%, 125/159). However, this difference was not statistically significant. When spermatozoa were treated with TX + DTT in the presence of EGTA or a mixture of EGTA and EDTA, the frequency of normal chromosomes was 39% (45/114) and 47% (38/81), respectively, suggesting that endogenous sperm nucleases may play a role in chromosomal damage. Our results indicate that simultaneous treatment of spermatozoa with detergent and DTT induces extensive chromosomal breakage and, therefore, should not be attempted in ICSI.

Animals↗
PubMedDate not supplied

An endogenous nuclease in hamster, mouse, and human spermatozoa cleaves DNA into loop-sized fragments.

Recent work from our laboratory provided evidence for the existence of a nuclease in hamster spermatozoa. This endogenous nuclease cleaves sperm chromatin at the bases of DNA loop domains into large fragments with an average size of roughly 50 kb. Here, we demonstrate that this sperm nuclease is present in the sperm nucleus and that it is activated by the presence of both calcium and magnesium much more efficiently than with either ion alone, resulting in DNA degradation in 30 minutes. We also show that similar nucleases are present in mouse and human spermatozoa. The human nuclease can be activated by freeze-thawing spermatozoa in noncryoprotective media. The activity of the sperm nuclease in all 3 species resembles that of a group of somatic cell DNAses that also require both calcium and magnesium and that digest the chromatin into loop-sized fragments during apoptosis.

Animals↗