scholarly journals Validation of the sperm chromatin dispersion (SCD) test in the amphibian Xenopus laevis using in situ nick translation and comet assay

2015 ◽  
Vol 27 (8) ◽  
pp. 1168 ◽  
Author(s):  
K. Pollock ◽  
J. Gosálvez ◽  
F. Arroyo ◽  
C. López-Fernández ◽  
M. Guille ◽  
...  
Keyword(s):  
Dna Damage ◽  
Comet Assay ◽  
Xenopus Laevis ◽  
Semen Quality ◽  
Sperm Chromatin ◽  
Sperm Dna Fragmentation ◽  
Nick Translation ◽  
In Situ Nick Translation ◽  
Sperm Dna

The integrity of sperm DNA is becoming increasingly recognised as an important parameter of semen quality, but there are no published reports of this procedure for any amphibian. The primary aim of this study was to apply a modified sperm chromatin dispersion (SCD) test (Halomax) to an amphibian sperm model (African clawed frog; Xenopus laevis) and to validate the assay against in situ nick translation (ISNT) and the double-comet assay procedure. Inactivated spermatozoa were collected from fresh testes (n = 3). Sperm DNA fragmentation (SDF) for each sperm sample was conducted immediately following activation (T0) and again after 1 h (T1) and 24 h (T24) of incubation at room temperature in order to produce a range of spermatozoa with differing levels of DNA damage. The SCD procedure resulted in the production of three nuclear morphotypes; amphibian sperm morphotype 1 (ASM-1) and ASM-2 showed no evidence of DNA damage, whereas ASM-3 spermatozoa were highly fragmented with large halos of dispersed DNA fragments and a reduced nuclear core. ISNT confirmed that ASM-3 nuclei contained damaged DNA. There was a significant correlation (r = 0.9613) between the levels of ASM-3 detected by the SCD test and SDF revealed by the double-comet assay.

170 ASSESSMENT OF FRESH AND FROZEN - THAWED BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATUS) SPERM DNA FRAGMENTATION USING THE SPERM CHROMATIN DISPERSION TEST

2013 ◽  
Vol 25 (1) ◽  
pp. 233
Author(s):  
M. J. Sanchez-Calabuig ◽  
J. de la Fuente ◽  
P. Beltrán-Breña ◽  
E. Martinez-Nevado ◽  
J. F. Perez-Gutierrez ◽  
...  
Keyword(s):  
Bottlenose Dolphin ◽  
Sperm Chromatin ◽  
Sperm Dna Fragmentation ◽  
Nick Translation ◽  
Neutral Comet Assay ◽  
Sperm Dna ◽  
Dispersion Test ◽  
Dynamic Loss ◽  
Sperm Chromatin Dispersion Test

There has been significant development over the last 20 years to improve genetic management of the captive bottlenose dolphin (Tursiops truncatus) by means of genome resource banking and assisted reproduction. Although standard semen parameters have been analysed in some detail, very little is known about sperm DNA fragmentation (SDF) in this species. The aim of this study was to develop a sperm chromatin dispersion test (SCDt) for the bottlenose dolphin to establish the baseline level of SDF immediately after ejaculation and cryopreservation and to determine the dynamic loss of sperm DNA quality after ex vivo handing and incubation in conditions that mimic the female reproductive tract. Semen from 8 bottlenose dolphins was collected by manual stimulation. Initial validation of the SCDt was conducted by means of in situ nick translation and neutral comet assay using a proven fertile male. To investigate the dynamic loss of sperm chromatin (rate of sDF loss), thawed sperm samples were incubated at 37.9°C for up to 48 h, and aliquots of spermatozoa were assessed after 1, 4, 8, 24, and 48 h. Dolphin sperm nuclei with fragmented DNA exhibited large halos of dispersed DNA fragments, whereas those with nonfragmented DNA displayed small halos of nondispersed DNA. A high correlation (r2 = 0.82; P ≤ 0.01) was found between the respective assessments of the SCDt and the neutral comet assay. All nucleoids resulting in a large halo of dispersed chromatin were intensely positive to in situ nick translation. The level of sDF fragmentation observed immediately after ejaculation in fresh and frozen samples was relatively low (1–5%). After comparing different ejaculates of the same individual, differences were found. Chromatin stability was high during the first 48 h of ejaculation or post-thawing and incubation. Evaluation of the sDF dynamics of fresh and frozen–thawed spermatozoa revealed no significant increase in the baseline level of sDF or in the relative increase of DNA damage after 48 h of incubation. Our data suggest that cryopreservation does not induce a dramatic increase in sperm chromatin damage. Interestingly, sperm samples derived from aged animals resulted in an increased rate of DNA loss, which was observed after 60 min post-incubation.

scholarly journals Are semen quality parameters sufficient for biomonitoring spermatozoa DNA integrity and oxidatively damaged DNA

Biomonitoring ◽  
2015 ◽  
Vol 2 (1) ◽  
Author(s):  
Hueiwang Anna Jeng ◽  
Ruei-Nian Li ◽  
Wen-Yi Lin
Keyword(s):  
Dna Damage ◽  
Dna Fragmentation ◽  
Semen Quality ◽  
Quality Parameters ◽  
Semen Parameters ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Phase System ◽  
Sperm Dna Fragmentation ◽  
Sperm Dna

Abstract:The present study aimed to investigate the relationship between semen quality parameters and DNA integrity, and determine whether semen quality parameters could serve as a reliable biomarker for monitoring sperm DNA damage. Conventional semen parameters from a total of 202 male human subjects were analyzed. DNA fragmentation and 8-oxo-7,8-dihydro-2′- deoxyguanosine (8-oxoGuo) were used to assess sperm DNA integrity. DNA fragmentation was analyzed by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and sperm chromatin structure assay (SCSA), while 8-oxodGuo was quantified by the liquid chromatography/tandem mass spectrometry (LC-MS/MS) coupled with an on-line solid phase system. The levels of 8-oxodGuo levels in sperm were related to the percentages of DNA fragmentation measured by both the TUNEL and SCSA (r = 0.22, p = 0.048; r = 0.12, p = 0.039). Sperm vitality, motility and morphology from all of the participants exhibited a weak correlation with the levels of 8-oxodGuo and the percentages of DNA fragmentation. Semen quality parameters may be independent of the formation of DNA fragmentation and oxidative adducts in sperm. Semen quality parameters may be insufficient to monitor sperm DNA fragmentation and oxidative damage. DNA damage in sperm is recommended to be included in routine measurements.

scholarly journals Sperm DNA fragmentation induced by DNAse I and hydrogen peroxide: an in vitro comparative study among different mammalian species

Reproduction ◽  
2010 ◽  
Vol 140 (3) ◽  
pp. 445-452 ◽  
Author(s):  
Paola Villani ◽  
Patrizia Eleuteri ◽  
Maria Giuseppa Grollino ◽  
Michele Rescia ◽  
Pierluigi Altavista ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Dna Damage ◽  
Comet Assay ◽  
Chromatin Structure ◽  
Dnase I ◽  
Sperm Chromatin ◽  
Sperm Dna Fragmentation ◽  
Dna Breaks ◽  
Sperm Dna

Sperm DNA damage may have adverse effects on reproductive outcome. Sperm DNA breaks can be detected by several tests, which evaluate DNA integrity from different and complementary perspectives and offer a new class of biomarkers of the male reproductive function and of its possible impairment after environmental exposure. The remodeling of sperm chromatin produces an extremely condensed nuclear structure protecting the nuclear genome from adverse environments. This nuclear remodeling is species specific, and differences in chromatin structure may lead to a dissimilar DNA susceptibility to mutagens among species. In this study, the capacity of the comet assay in its two variants (alkaline and neutral) to detect DNA/chromatin integrity has been evaluated in human, mouse, and bull sperm. The hypothesis that chromatin packaging might influence the amount of induced and detectable DNA damage was tested by treating sperm in vitro with DNAse I, whose activity is strictly dependent upon its DNA accessibility. Furthermore, hydrogen peroxide (H2O2) was used to assess whether spermatozoa of the three species showed a different sensitivity to oxidative stress. DNAse I-induced damage was also assessed by the sperm chromatin structure assay and the TUNEL assay, and the performances of these two assays were compared and correlated with the comet assay results. Results showed a different sensitivity to DNAse I treatment among the species with human sperm resulting the most susceptible. On the contrary, no major differences among species were observed after H2O2 treatment. Furthermore, the three tests show a good correlation in revealing sperm with DNA strand breaks.

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 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 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.

scholarly journals The aging male: Relationship between male age, sperm quality and sperm DNA damage in an unselected population of 3124 men attending the fertility centre for the first time

2019 ◽  
Vol 90 (4) ◽  
pp. 254-259 ◽  
Author(s):  
Alessandro Colasante ◽  
Maria Giulia Minasi ◽  
Filomena Scarselli ◽  
Valentina Casciani ◽  
Vincenzo Zazzaro ◽  
...  
Keyword(s):  
Dna Damage ◽  
Sperm Morphology ◽  
Semen Quality ◽  
Sperm Quality ◽  
World Health ◽  
Semen Parameters ◽  
Sperm Dna Fragmentation ◽  
Sperm Dna Damage ◽  
Male Age ◽  
Sperm Dna

Objective: the aim of our study was to put forward insights to treat any possible correlation among sperm quality, sperm DNA damage and male age as they may have fertility implications for men who choose to delay fatherhood. Materials and methods: Our study is a non-interventional retrospective analysis of 3124 semen samples from patients that were investigated for the conventional semen parameters. Tunel test assay was set up for the evaluation of the sperm DNA fragmentation index (DFI). We applied the Kappa index to compare both the 1999 and the 2010 World Health Organization (WHO) reference criteria to evaluate the competence of such semen parameters categorization during the standard routine of our laboratory. Results: With regards to our findings, it is possible to underline a significant relationship between aging and semen volume (p = 0.001), motility (p = 0.009), semen viscosity (p < 0.003) and sperm DNA damage (p < 0.009). We found a trend when focusing on the semen concentration (p = 0.05). The analysis of sperm morphology did not show any influence with advancing age (p = 0.606). When comparing both the 1999 and the 2010 WHO scales we found no accordance in the appraisal of sperm morphology but a very good one in the evaluation of the other parameters. Conclusions: Conventional semen analysis represents the opportunity to draw up a proxy insight on the male fertility status even if semen quality can only indirectly assess the probability of pregnancy. Several studies have verified a decay in the male reproductive system, sperm quality and fertility with advancing age although the reported results are not yet conclusive. Our results substantially agree with those findings outlined in the literature. Moreover we find that the discrepancy between the two WHO reference scales would eventually lead to an improper diagnosis of infertility.

Detection of β Cell-Specific DNA Damage in Streptozotocin-Treated Rats by In Situ Nick Translation with Immunostaining of α Cells

Pancreas ◽  
1995 ◽  
Vol 10 (4) ◽  
pp. 354-359 ◽  
Author(s):  
Satoshi Shibata ◽  
Yoshihiro Asanuma ◽  
Kenji Koyama ◽  
Ken Saito
Keyword(s):  
Dna Damage ◽  
Nick Translation ◽  
Β Cell ◽  
In Situ Nick Translation

scholarly journals 45 DNA Fragmentation of Epididymal Freeze-Dried Ram Spermatozoa Impairs Embryo Development

2018 ◽  
Vol 30 (1) ◽  
pp. 162
Author(s):  
L. Palazzese ◽  
D. A. Anzalone ◽  
J. Gosálvez´ ◽  
P. Loi ◽  
J. Saragusty
Keyword(s):  
Dna Damage ◽  
Embryonic Development ◽  
Dna Fragmentation ◽  
Embryo Development ◽  
Blastocyst Stage ◽  
Sperm Chromatin ◽  
Sperm Dna Fragmentation ◽  
Strand Breaks ◽  
Sperm Dna ◽  
Freeze Dried

Sperm freeze-drying is a revolutionary technique that resolves many of the drawback of long-term storage under liquid nitrogen. The first significant result of this method was provided by Wakayama and Yanagimachi (1998 Nat. Biotechnol. 16, 639-641, 10.1038/nbt0798-639), demonstrating for the first time the birth of healthy offspring from epididymal freeze-dried (mouse) spermatozoa. Besides models in the mouse and rat, which are the first small mammals born from epididymal lyophilized sperm by intracytoplasmic sperm injection (ICSI), most studies in this field have used ejaculated sperm. In this work, aiming to repeat the result of Wakayama and Yanagimachi, we tried to apply this technique to epididymal spermatozoa from a large mammal (ram). Moreover, we checked the correlation between freeze-dried spermatozoa DNA integrity and embryo development. To do this, epididymal sperm from 4 rams was lyophilized in a medium containing trehalose, glucose, KCl, HEPES, and Trolox. To evaluate DNA damage and fragmentation at rehydration, part of the sperm was processed for sperm chromatin dispersion test (SCD) and two-tailed comet assay and the rest was used for ICSI. Compared with rams 1 and 3, rams 2 and 4 had higher rate of spermatozoa with intact DNA (median: 3.3% v. 16.5%, respectively), lower rate of single strand breaks (SSB; median: 94.2% v. 81.5%, respectively) and lower rate of double-strand breaks (DSB; median: 2.5% v. 2%, respectively). Embryo development following ICSI showed that blastocyst stage was reached only from rams that had sperm with more intact DNA: ram 2 (4.8%, n = 83) and ram 4 (6.3%, n = 64). Spermatozoa from rams 1 and 3 produced no blastocysts. This can be explained by the fact that rams 2 and 4 had higher rate of spermatozoa with intact DNA than rams 1 and 3. Definitively, the implication of sperm DNA damage in embryonic development should depend on the balance between the extent of sperm DNA fragmentation, the type of fragmentation (SSB or DSB), and the oocyte’s repair capacity. Rams 2 and 4 were the only rams that produced blastocyst probably because they had considerably more sperm with normal DNA; thus, it is important to select spermatozoa of the best quality to perform a good ICSI. Fragmentation of DNA due to the lyophilization process impairs embryonic development. To conclude, oocytes injected with epididymal freeze-dried ram spermatozoa can reach the blastocyst stage. These are preliminary results; more conclusive outcomes will be given following embryo transfer experiments that are now in progress.

scholarly journals Sperm DNA Integrity Assessment: A New Tool in Diagnosis and Treatment of Fertility

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Mona Bungum
Keyword(s):  
Semen Quality ◽  
Significant Proportion ◽  
Semen Parameters ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Sperm Dna Fragmentation ◽  
Integrity Assessment ◽  
Sperm Dna Integrity ◽  
Sperm Dna

Infertility affects 15% of all couples. Although male infertility factors with reduced semen quality are contributing to about half of all involuntary childlessness, the value of standard semen parameters in prediction of fertilityin vivoand choice of proper method for assisted reproduction is limited. In the search for better markers of male fertility, during the last 10 years, assessment of sperm DNA integrity has emerged as a strong new biomarker of semen quality that may have the potential to discriminate between infertile and fertile men. Sperm DNA Fragmentation Index (DFI) as assessed by the flow cytometric Sperm Chromatin Structure Assay (SCSA) can be used for evaluation of sperm chromatin integrity. The biological background for abnormal DFI is not completely known, but clinical data show that DFI above 30% is associated with very low chance for achieving pregnancy in natural way or by insemination, but notin vitro. Already when the DFI is above 20%, the chance of natural pregnancy may be reduced, despite other sperm parameters being normal. Thus this method may explain a significant proportion of cases of unexplained infertility and can be beneficial in counselling involuntary childless couples need ofin vitrofertilisation.

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