scholarly journals 215.Analysis of DNA damage induced by pro-oxidant treatment of mammalian spermatozoa in vitro

2004 ◽  
Vol 16 (9) ◽  
pp. 215
Author(s):  
L. E. Bennetts ◽  
R. J. Aitken
Keyword(s):  
Dna Damage ◽  
Oxidative Damage ◽  
Dna Sequences ◽  
Nuclear Dna ◽  
Quantitative Polymerase Chain Reaction ◽  
Mammalian Species ◽  
Dose Range ◽  
Tammar Wallaby ◽  
Sperm Chromatin ◽  
Sperm Dna

Defects in the male genome produced as a consequence of oxidative insult have been associated with decreased fertility levels, an elevated incidence of childhood cancer and dominant genetic disease in the offspring (1). The objective of this study was to determine the relative susceptibility of sperm DNA of different mammalian species to oxidative injury. We applied a highly sensitive quantitative PCR assay (QPCR) to measure gene-specific DNA damage in nuclear and mitochondrial compartments of spermatozoa treated with H2O2. Human, murine and tammar wallaby (Macropus eugenii) spermatozoa were treated with H2O2 (0–5�mM) over a 1�h period. After DNA purification, DNA damage was assessed in a nuclear and a mitochondrial fragment of DNA by quantitative polymerase chain reaction assay (QPCR). DNA damage was detected as a decrease amplification of the target sequences. In murine and human spermatozoa, mitochondrial DNA exhibited greater sensitivity to oxidative damage than nuclear DNA. Doses ranging from 0.25–5�mM H2O2 induced DNA damage of up to 0.65 lesions/10�kb in the mouse, and 1.42 lesions /10�kb in the human. No significant effect on DNA damage was observed over this dose range in the nuclear DNA fragments investigated in these species. In contrast, tammar wallaby spermatozoa were susceptible to DNA damage at the 5�mM H2O2 dose in both nuclear (0.51 lesions/10�kb) and mitochondrial (0.55 lesions/10�kb) genomes. This study is the first to compare DNA damage in specific DNA sequences in spermatozoa of different mammalian species. Nuclear DNA of the metatherian species, the tammar wallaby, was more susceptible to oxidative damage than that of the eutherian species. A major difference between metatherian and eutherian spermatozoa is that, in general, the former possess protamines that are not stabilised by disulfide cross-linkage. These findings therefore suggest that sperm chromatin packaging affects the susceptibility of sperm DNA to oxidative damage. (1) Sawyer and Aitken (2000) Reprod. Med. Rev. 8, 107–126.

scholarly journals The study of spermatic DNA fragmentation and sperm motility in infertile subjects

2013 ◽  
Vol 85 (1) ◽  
pp. 8 ◽  
Author(s):  
Giuseppina Peluso ◽  
Alessandro Palmieri ◽  
Pietro Paolo Cozza ◽  
Giancarlo Morrone ◽  
Paolo Verze ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Fragmentation ◽  
Nuclear Dna ◽  
Inverse Correlation ◽  
World Health ◽  
Sperm Chromatin ◽  
Sperm Dna Fragmentation ◽  
Sperm Dna Damage ◽  
Spermatozoa Motility ◽  
Sperm Dna

Introduction: Although the pathophysiology of the testicular damage associated with varicocele remains unclear, sperm DNA damage has been identified as a potential explanation for this cause of male infertility. The current study was designed to determine the extent of sperm nuclear DNA damage in patients with varicocele, and to examine its relationship with parameters of seminal motility. Materials and method: Semen samples from 60 patients with clinical varicocele and 90 infertile men without varicocele were examined. Varicocele sperm samples were classified as normal or pathological according to the 1999 World Health Organizzation guidelines. Sperm DNA damage was evalutated using the Halosperm kit, an improved Sperm Chromatin Dispersion (SCD) test. Results: The DNA fragmentation index (DFI: percentage of sperm with denatured nuclei) values was significantly higher in patients with varicocele, either with normal or abnormal (DFI 25.8 ± 3.2 vs 17.4 ± 2.8 - P < 0,01) semen profiles. In addition, an inverse correlation was found between spermatic motility and the degree of spermatic DNA fragmentation in patients with clinical varicocele. Conclusions: Varicocele is associated with high levels of DNA-damage in spermatozoa. In addition, in subjects with varicocele, abnormal spermatozoa motility is associated with higher levels of sperm DNA fragmentation. DNA fragmentation may therefore be an essential additional diagnostic test that should be recommended for patients with clinical varicocele.

The assessment of sperm DNA fragmentation in the saltwater crocodile (Crocodylus porosus)

2017 ◽  
Vol 29 (3) ◽  
pp. 630 ◽  
Author(s):  
S. D. Johnston ◽  
C. López-Fernández ◽  
F. Arroyo ◽  
J. L. Fernández ◽  
J. Gosálvez
Keyword(s):  
Dna Damage ◽  
Dna Fragmentation ◽  
Sperm Chromatin ◽  
Sperm Dna Fragmentation ◽  
Fragmented Dna ◽  
Sperm Dna ◽  
Saltwater Crocodile ◽  
Crocodylus Porosus ◽  
Dispersion Test ◽  
Sperm Nuclei

Herein we report a method of assessing DNA fragmentation in the saltwater crocodile using the sperm chromatin dispersion test (SCDt) after including frozen–thawed spermatozoa in a microgel (Halomax; Halotech DNA, Madrid, Spain). Following controlled protein depletion, which included a reducing agent, sperm nuclei with fragmented DNA showed a homogeneous and larger halo of chromatin dispersion with a corresponding reduced nucleoid core compared with sperm with non-fragmented DNA. The presence of DNA damage was confirmed directly by incorporation of modified nucleotides using in situ nick translation (ISNT) and indirectly by studying the correlation of the SCDt with the results of DNA damage visualisation using a two-tailed comet assay (r = 0.90; P = 0.037). Results of the SCDt immediately following thawing and after 5 h incubation at 37°C in order to induce a range of DNA damage revealed individual crocodile differences in both the baseline level of DNA damage and DNA longevity.

scholarly journals Relocalized redox-active lysosomal iron is an important mediator of oxidative-stress-induced DNA damage

2004 ◽  
Vol 378 (3) ◽  
pp. 1039-1045 ◽  
Author(s):  
Tino KURZ ◽  
Alan LEAKE ◽  
Thomas von ZGLINICKI ◽  
Ulf T. BRUNK
Keyword(s):  
Dna Damage ◽  
Free Radical ◽  
Oxidative Damage ◽  
Nuclear Dna ◽  
Membrane Integrity ◽  
Lysosomal Hydrolases ◽  
Free Radical Biol ◽  
Active Iron ◽  
Vacuolar Compartment ◽  
Redox Active

Oxidative damage to nuclear DNA is known to involve site-specific Fenton-type chemistry catalysed by redox-active iron or copper in the immediate vicinity of DNA. However, the presence of transition metals in the nucleus has not been shown convincingly. Recently, it was proposed that a major part of the cellular pool of loose iron is confined within the acidic vacuolar compartment [Yu, Persson, Eaton and Brunk (2003) Free Radical Biol. Med. 34, 1243–1252; Persson, Yu, Tirosh, Eaton and Brunk (2003) Free Radical Biol. Med. 34, 1295–1305]. Consequently, rupture of secondary lysosomes, as well as subsequent relocation of labile iron to the nucleus, could be an important intermediary step in the generation of oxidative damage to DNA. To test this concept we employed the potent iron chelator DFO (desferrioxamine) conjugated with starch to form an HMM-DFO (high-molecular-mass DFO complex). The HMM-DFO complex will enter cells only via fluid-phase endocytosis and remain within the acidic vacuolar compartment, thereby chelating redox-active iron exclusively inside the endosomal/lysosomal compartment. Both free DFO and HMM-DFO equally protected lysosomal-membrane integrity against H2O2-induced oxidative disruption. More importantly, both forms of DFO prevented H2O2-induced strand breaks in nuclear DNA, including telomeres. To exclude the possibility that lysosomal hydrolases, rather than iron, caused the observed DNA damage, limited lysosomal rupture was induced using the lysosomotropic detergent O-methyl-serine dodecylamine hydrochloride; subsequently, hardly any DNA damage was found. These observations suggest that rapid oxidative damage to cellular DNA is minimal in the absence of redox-active iron and that oxidant-mediated DNA damage, observed in normal cells, is mainly derived from intralysosomal iron translocated to the nucleus after lysosomal rupture.

scholarly journals Rapid rates of sperm DNA damage after activation in tench (Tinca tinca: Teleostei, Cyprinidae) measured using a sperm chromatin dispersion test

Reproduction ◽  
2009 ◽  
Vol 138 (2) ◽  
pp. 257-266 ◽  
Author(s):  
Carmen López-Fernández ◽  
Matthew J G Gage ◽  
Francisca Arroyo ◽  
Altea Gosálbez ◽  
Ana M Larrán ◽  
...  
Keyword(s):  
Dna Damage ◽  
Comet Assay ◽  
Dna Fragmentation ◽  
Living Organism ◽  
Sperm Chromatin ◽  
Tinca Tinca ◽  
External Fertilization ◽  
Sperm Dna Damage ◽  
Sperm Dna ◽  
Dispersion Test

Spermatozoal haplotypic DNA is prone to damage, leading to male fertility problems. So far, the assessment of sperm DNA breakage has been challenging because protamines render the nuclear chromatin highly compacted. Here, we report the application of a new test to quantify DNA fragmentation in spermatozoa of an externally fertilizing teleost fish. The sperm chromatin dispersion (SCD) test uses a species-specific lysing solution to generate controlled protein depletion that, followed by DNA-specific fluorescent labelling, allows an easy morphological discrimination between nuclei affected by DNA damage. Using tench (Tinca tinca) as our model, we first trialled the test against established, but more technically demanding, assays employing in situ nick translation (ISNT) and the comet assay. The SCD test showed high concordance with ISNT, comet assay measures and a chromatin-swelling test, confirming the application of this straightforward SCD technique to various aspects of reproductive biology. Second, we examined between-male variation in DNA damage, and measured changes through time following spermatozoal activation. Between-male variation in the basal levels of average DNA damage ranged from 0 to 20% of sperm showing damage, and all showed increases in DNA fragmentation through time (0–60 min). The rates of DNA damage increase are the fastest so far recorded in sperm for a living organism, and may relate to the external fertilization mode. Our findings have relevance for broodstock selection and optimizing IVF protocols routinely used in modern aquaculture.

scholarly journals Effects of gamma-radiation on DNA damage in onion (Allium cepa L.) seedlings

2019 ◽  
Vol 489 (2) ◽  
pp. 199-204
Author(s):  
A. Ya. Bolsunovsky ◽  
D. V. Dementyev ◽  
T. S. Frolova ◽  
E. A. Trofimova ◽  
E. M. Iniatkina ◽  
...  
Keyword(s):  
Dna Damage ◽  
Comet Assay ◽  
Gamma Radiation ◽  
Allium Cepa ◽  
Low Dose ◽  
Nuclear Dna ◽  
Biological Effects ◽  
Dose Range ◽  
Dna Breaks ◽  
Allium Test

The effect of -radiation on the level of nuclear DNA damage in onion seedlings (Allium-test) was studied using the comet assay. DNA breaks were first found in cells of onion seedlings exposed to low-dose radiation ( 0,1 Gy). Dose dependence of DNA damage parameters showed nonlinear behavior: a linear section in the low-dose region (below 0,1 Gy) and a dose-independent plateau in the dose range between 1 and 5 Gy. Thus, the comet assay can be used to estimate the biological effects of low-dose gamma-radiation on Allium cepa seedlings.

scholarly journals Species-Specific Differences in Sperm Chromatin Decondensation Between Eutherian Mammals Underlie Distinct Lysis Requirements

2021 ◽  
Vol 9 ◽  
Author(s):  
Jordi Ribas-Maynou ◽  
Estela Garcia-Bonavila ◽  
Carlos O. Hidalgo ◽  
Jaime Catalán ◽  
Jordi Miró ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Condensation ◽  
Proteinase K ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Dna Breaks ◽  
Chromatin Decondensation ◽  
The Third ◽  
Core Length ◽  
Sperm Dna

Sperm present a highly particular DNA condensation that is acquired during their differentiation. Protamines are key elements for DNA condensation. However, whereas the presence of protamine 1 (P1) is conserved across mammalian species, that of protamine 2 (P2) has evolved differentially, existing only few species that use both protamines for sperm DNA condensation. In addition, altered P1/P2 ratios and alterations in the expression of P1 have previously been associated to infertility and DNA damage disorders. On the other hand, different methods evaluating DNA integrity, such as Sperm Chromatin Dispersion (SCD) and Comet tests, need a previous complete DNA decondensation to properly assess DNA breaks. Related with this, the present study aims to analyze the resilience of sperm DNA to decodensation in different eutherian mammals. Sperm samples from humans, horses, cattle, pigs and donkeys were used. Samples were embedded in low melting point agarose and treated with lysis solutions to induce DNA decondensation and formation of sperm haloes. The treatment consisted of three steps: (1) incubation in SDS + DTT for 30 min; (2) incubation in DTT + NaCl for 30 min; and (3) incubation in DTT + NaCl with or without proteinase K for a variable time of 0, 30, or 180 min. How incubation with the third lysis solution (with or without proteinase K) for 0, 30, and 180 min affected DNA decondensation was tested through analyzing core and halo diameters in 50 sperm per sample. Halo/core length ratio was used as an indicator of complete chromatin decondensation. While incubation time with the third lysis solution had no impact on halo/core length ratios in species having P1 and P2 (human, equine and donkey), DNA decondensation of pig and cattle sperm, which only present P1, significantly (P < 0.05) increased following incubation with the third lysis solution for 180 min. In addition, the inclusion of proteinase K was found to accelerate DNA decondensation. In conclusion, longer incubations in lysis solution including proteinase K lead to higher DNA decondensation in porcine and bovine sperm. This suggests that tests intended to analyze DNA damage, such as halo or Comet assays, require complete chromatin deprotamination to achieve high sensitivity in the detection of DNA breaks.

Effects of cryostorage on human sperm chromatin integrity

Zygote ◽  
2012 ◽  
Vol 21 (4) ◽  
pp. 330-336 ◽  
Author(s):  
Adriana Fortunato ◽  
Rita Leo ◽  
Francesca Liguori
Keyword(s):  
Nuclear Dna ◽  
Tertiary Structure ◽  
Human Sperm ◽  
Blue Staining ◽  
Sperm Chromatin ◽  
Sperm Dna Damage ◽  
Human Fertility ◽  
Sperm Dna ◽  
Duration Of Storage ◽  
Male Patients

SummaryThe integrity of sperm chromatin structure has proven to be of great importance for human fertility. In this study, we investigated whether sperm cryopreservation has an effect on nuclear DNA tertiary structure, (i.e. condensation), measured by aniline blue staining, in 103 male patients who required consultation for hypo-fertility. Sperm DNA damage was significantly higher in patients showing oligospermia and severe morphological abnormalities than in native sperm populations. Furthermore we observed that chromatin decondensation was related to the cryostorage technique and to the duration of storage. This increase in decondensation was highly significant (P < 0.01) immediately after cryopreservation and from 90 days of cryostorage onwards. The possible mechanisms involved in sperm chromatin cryoinjury and the need to incorporate new methods for testing sperm nuclear structure alteration into the routine spermiogram are discussed.

scholarly journals Revisiting summer infertility in the pig: could heat stress-induced sperm DNA damage negatively affect early embryo development?

2017 ◽  
Vol 57 (10) ◽  
pp. 1975 ◽  
Author(s):  
Santiago T. Peña, Jr ◽  
Bruce Gummow ◽  
Anthony J. Parker ◽  
Damien B. B. P. Paris
Keyword(s):  
Dna Damage ◽  
Heat Stress ◽  
Oxidative Damage ◽  
Unsaturated Fatty Acids ◽  
Negative Impact ◽  
Early Embryo ◽  
Antioxidant Systems ◽  
Sperm Dna Damage ◽  
Sperm Dna ◽  
Boar Sperm

Temperature is a crucial factor in mammalian spermatogenesis. The scrotum, pampiniform plexus, and cremaster and dartos muscles in mammals are specific adaptations to ensure sperm production in a regulated environment 4−6°C below internal body temperature. However, the limited endogenous antioxidant systems inherent in mammalian spermatozoa compounded by the loss of cytosolic repair mechanisms during spermatogenesis, make the DNA in these cells particularly vulnerable to oxidative damage. Boar sperm is likely to be more susceptible to the effects of heat stress and thus oxidative damage due to the relatively high unsaturated fatty acids in the plasma membrane, low antioxidant capacity in boar seminal plasma, and the boar’s non-pendulous scrotum. Heat stress has a significant negative impact on reproductive performance in piggeries, which manifests as summer infertility and results in productivity losses that amount to millions of dollars. This problem is particularly prevalent in tropical and subtropical regions where ambient temperatures rise beyond the animal’s zone of thermal comfort. Based on preliminary studies in the pig and other species, this article discusses whether heat stress could induce sufficient DNA damage in boar sperm to significantly contribute to the high rates of embryo loss and pregnancy failure observed in the sow during summer infertility. Heat stress-induced damage to sperm DNA can lead to disrupted expression of key developmental genes essential for the differentiation of early cell lineages, such as the trophectoderm, and can distort the timely formation of the blastocyst; resulting in a failure of implantation and ultimately pregnancy loss. Confirming such a link would prompt greater emphasis on boar management and strategies to mitigate summer infertility during periods of heat stress.

scholarly journals Nuclear Integrity but Not Topology of Mouse Sperm Chromosome is Affected by Oxidative DNA Damage

Genes ◽  
2018 ◽  
Vol 9 (10) ◽  
pp. 501 ◽  
Author(s):  
Alexandre Champroux ◽  
Christelle Damon-Soubeyrand ◽  
Chantal Goubely ◽  
Stephanie Bravard ◽  
Joelle Henry-Berger ◽  
...  
Keyword(s):  
Dna Damage ◽  
Oxidative Dna Damage ◽  
Sperm Nucleus ◽  
Sperm Chromatin ◽  
Sperm Dna Fragmentation ◽  
Chromosome 2 ◽  
Mouse Sperm ◽  
Sperm Dna Damage ◽  
Chromosome Architecture ◽  
Sperm Dna

Recent studies have revealed a well-defined higher order of chromosome architecture, named chromosome territories, in the human sperm nuclei. The purpose of this work was, first, to investigate the topology of a selected number of chromosomes in murine sperm; second, to evaluate whether sperm DNA damage has any consequence on chromosome architecture. Using fluorescence in situ hybridization, confocal microscopy, and 3D-reconstruction approaches we demonstrate that chromosome positioning in the mouse sperm nucleus is not random. Some chromosomes tend to occupy preferentially discrete positions, while others, such as chromosome 2 in the mouse sperm nucleus are less defined. Using a mouse transgenic model (Gpx5−/−) of sperm nuclear oxidation, we show that oxidative DNA damage does not disrupt chromosome organization. However, when looking at specific nuclear 3D-parameters, we observed that they were significantly affected in the transgenic sperm, compared to the wild-type. Mild reductive DNA challenge confirmed the fragility of the organization of the oxidized sperm nucleus, which may have unforeseen consequences during post-fertilization events. These data suggest that in addition to the sperm DNA fragmentation, which is already known to modify sperm nucleus organization, the more frequent and, to date, the less highly-regarded phenomenon of sperm DNA oxidation also affects sperm chromatin packaging.

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