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

Download Full-text

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

Reproduction Fertility and Development ◽

10.1071/rd15300 ◽

2017 ◽

Vol 29 (3) ◽

pp. 630 ◽

Cited By ~ 8

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.

Download Full-text

Evidence that single-stranded DNA breaks are a normal feature of koala sperm chromatin, while double-stranded DNA breaks are indicative of DNA damage

Reproduction ◽

10.1530/rep-09-0021 ◽

2009 ◽

Vol 138 (2) ◽

pp. 267-278 ◽

Cited By ~ 32

Author(s):

Yeng Peng Zee ◽

Carmen López-Fernández ◽

F Arroyo ◽

Stephen D Johnston ◽

William V Holt ◽

...

Keyword(s):

Dna Damage ◽

Comet Assay ◽

Dna Fragmentation ◽

Sperm Nucleus ◽

Severe Form ◽

Sperm Chromatin ◽

Dna Breaks ◽

Single Stranded Dna ◽

Double Stranded Dna ◽

Dispersion Test

In this study, we have used single and double comet assays to differentiate between single- and double-stranded DNA damage in an effort to refine the interpretation of DNA damage in mature koala spermatozoa. We have also investigated the likelihood that single-stranded DNA breakage is part of the natural spermiogenic process in koalas, where its function would be the generation of structural bends in the DNA molecule so that appropriate packaging and compaction can occur. Koala spermatozoa were examined using the sperm chromatin dispersion test (SCDt) and comet assays to investigate non-orthodox double-stranded DNA. Comet assays were conducted under 1) neutral conditions; and 2) neutral followed by alkaline conditions (double comet assay); the latter technique enabled simultaneous visualisation of both single-stranded and double-stranded DNA breaks. Following the SCDt, there was a continuum of nuclear morphotypes, ranging from no apparent DNA fragmentation to those with highly dispersed and degraded chromatin. Dispersion morphotypes were mirrored by a similar diversity of comet morphologies that could be further differentiated using the double comet assay. The majority of koala spermatozoa had nuclei with DNA abasic-like residues that produced single-tailed comets following the double comet assay. The ubiquity of these residues suggests that constitutive alkali-labile sites are part of the structural configuration of the koala sperm nucleus. Spermatozoa with ‘true’ DNA fragmentation exhibited a continuum of comet morphologies, ranging from a more severe form of alkaline-susceptible DNA with a diffuse single tail to nuclei that exhibited both single- and double-stranded breaks with two comet tails.

Download Full-text

P–103 Novel sperm preparation techniques compared with conventional preparation method

Human Reproduction ◽

10.1093/humrep/deab130.102 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

N Vahidi ◽

F S Amjadi ◽

F Kalat. sabz ◽

Z Zandie ◽

N Narimani

Keyword(s):

Dna Damage ◽

Zeta Potential ◽

Dna Fragmentation ◽

Sperm Parameters ◽

Sperm Chromatin ◽

P Value ◽

Sperm Preparation ◽

Sperm Dna Damage ◽

Sperm Dna ◽

Preparation Techniques

Abstract Study question Which sperm preparation technique separate the best quality sperm? Summary answer Microfluidic method improved the sperm parameters and decreased sperm DNA damage. What is known already About 40% of infertility issues are due to male factor. One of the known causes of male infertility is associated with low sperm parameters and high level DNA fragmentation. Sperm preparation techniques in ICSI procedures is used in order to obtain the best-quality sperm. Study design, size, duration The present study was designed to compare Microfluidic, Zeta potential, Magnetic Activated Cell Sorting (MACS) and Swim-up methods for sperm preparation and the effect of these methods on semen parameters and sperm DNA integrity in infertile men (n = 25) with a mean age of 38. Participants/materials, setting, methods In this study, each sample was divided into 4 groups, one part for preparing by Microfluidic method, one of them for preparing by Swim -up method, the other one was prepared by MACS and the last one was prepared by zeta potential. Then sperm count, viability, motility and morphology were assessed according to WHO 2010. DNA damage were assessed by Sperm DNA Fragmentation assay and sperm chromatin packaging assessed by CMA3 staining test Main results and the role of chance Sperm parameters including viability, motility, and morphology in the Microfluidic method were significantly improved and sperm DNA damage were significantly lower than three other methods (P-value <0.05). The sperm parameters and sperm DNA damage after preparation by MACS and Zeta potential methods were not significantly different however in the Swim-up method sperm parameters were lower than three other methods (P-value <0.05). Limitations, reasons for caution The fertilization and pregnancy rate of the resulting embryos are not available. Wider implications of the findings: Our results showed that Microfluidic can be an effective way to improve sperm quality of infertile male compared to conventional preparation methods. We also found instead of the MACS method, we can use the Zeta potential method according to their costs, for sperm preparation during ART cycle. Trial registration number *

Download Full-text

Assessment of avian sperm DNA fragmentation using the sperm chromatin dispersion assay

Reproduction Fertility and Development ◽

10.1071/rd19420 ◽

2020 ◽

Vol 32 (10) ◽

pp. 948 ◽

Cited By ~ 2

Author(s):

Stephen D. Johnston ◽

Carmen López-Fernández ◽

Eloise Pappin ◽

Alexandra Hampe ◽

Robert Doneley ◽

...

Keyword(s):

Dna Fragmentation ◽

Dynamic Assessment ◽

Bird Species ◽

Sperm Chromatin ◽

Species Variation ◽

Sperm Dna Fragmentation ◽

Simple Method ◽

Sperm Dna Damage ◽

Sperm Dna ◽

Dispersion Test

Herein we report a simple method for assessing avian sperm DNA fragmentation (SDF) using the sperm chromatin dispersion test (SCDt). The presence of sperm DNA damage was confirmed indirectly by correlating results of the SCDt determined in three bird species with results of a corresponding neutral comet assay (r=0.99; P<0.005). Frozen–thawed spermatozoa of each species were also incubated at 37°C for 5h and the within- and between-species variation of SDF, as an indicator of sperm DNA longevity, examined. The dynamic assessment of SDF using the SCDt revealed species and individual bird (rooster and turkey) differences in sperm DNA longevity.

Download Full-text

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

Archivio Italiano di Urologia e Andrologia ◽

10.4081/aiua.2013.1.8 ◽

2013 ◽

Vol 85 (1) ◽

pp. 8 ◽

Cited By ~ 12

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.

Download Full-text

A cost for high levels of sperm competition in rodents: increased sperm DNA fragmentation

Proceedings of The Royal Society B Biological Sciences ◽

10.1098/rspb.2015.2708 ◽

2016 ◽

Vol 283 (1826) ◽

pp. 20152708 ◽

Cited By ~ 5

Author(s):

Javier delBarco-Trillo ◽

Olga García-Álvarez ◽

Ana Josefa Soler ◽

Maximiliano Tourmente ◽

José Julián Garde ◽

...

Keyword(s):

Dna Damage ◽

Sperm Competition ◽

Dna Fragmentation ◽

Sperm Chromatin ◽

Dna Integrity ◽

Sperm Dna Fragmentation ◽

Sperm Dna Damage ◽

Sperm Dna Integrity ◽

Sperm Dna ◽

Physical And Chemical

Sperm competition, a prevalent evolutionary process in which the spermatozoa of two or more males compete for the fertilization of the same ovum, leads to morphological and physiological adaptations, including increases in energetic metabolism that may serve to propel sperm faster but that may have negative effects on DNA integrity. Sperm DNA damage is associated with reduced rates of fertilization, embryo and fetal loss, offspring mortality, and mutations leading to genetic disease. We tested whether high levels of sperm competition affect sperm DNA integrity. We evaluated sperm DNA integrity in 18 species of rodents that differ in their levels of sperm competition using the sperm chromatin structure assay. DNA integrity was assessed upon sperm collection, in response to incubation under capacitating or non-capacitating conditions, and after exposure to physical and chemical stressors. Sperm DNA was very resistant to physical and chemical stressors, whereas incubation in non-capacitating and capacitating conditions resulted in only a small increase in sperm DNA damage. Importantly, levels of sperm competition were positively associated with sperm DNA fragmentation across rodent species. This is the first evidence showing that high levels of sperm competition lead to an important cost in the form of increased sperm DNA damage.

Download Full-text

Ejaculate Oxidative Stress Is Related with Sperm DNA Fragmentation and Round Cells

International Journal of Endocrinology ◽

10.1155/2015/321901 ◽

2015 ◽

Vol 2015 ◽

pp. 1-6 ◽

Cited By ~ 25

Author(s):

Valeria Maria Iommiello ◽

Elena Albani ◽

Alessandra Di Rosa ◽

Alessandra Marras ◽

Francesca Menduni ◽

...

Keyword(s):

Oxidative Stress ◽

Dna Damage ◽

Dna Fragmentation ◽

Sperm Quality ◽

Reproductive Capacity ◽

World Health ◽

Sperm Chromatin ◽

Sperm Dna Fragmentation ◽

Sperm Dna Damage ◽

Sperm Dna

Oxidative stress (OS) plays an essential role in male infertility aetiology by affecting sperm quality, function, and also the integrity of sperm DNA. The assessment of oxidative stress in semen may be an important tool to improve the evaluation of sperm reproductive capacity. The purpose of this study was the evaluation of any possible relation between the unbalance of oxidative stress caused by superoxide anion in the ejaculate with the presence of sperm DNA fragmentation and high concentration of round cells. 56 semen samples from males from couples suffering from infertility were evaluated according to World Health Organisation (WHO) 2010 guidelines. Oxidative stress levels from N1 (low) to N4 (high) were assessed in ejaculates using oxiSperm; DFI (sperm DNA fragmentation index) as assessed by the SCSA (Sperm Chromatin Structure Assay) was used for evaluation of sperm chromatin integrity. Our data show that high oxidative stress (N3-N4 levels) correlated positively with aDFI≥30%(P=0.0379)and round cells≥1.500.000/mL(P=0.0084). In conclusion, OS increases sperm DNA damage. Thus evaluation of semen OS extent of sperm DNA damage in infertile man could be useful to develop new therapeutic strategies and improve success of assisted reproduction techniques (ART).

Download Full-text

Microscopic Varicocelectomy Significantly Decreases the Sperm DNA Fragmentation Index in Patients with Infertility

BioMed Research International ◽

10.1155/2014/695713 ◽

2014 ◽

Vol 2014 ◽

pp. 1-4 ◽

Cited By ~ 21

Author(s):

Teoman Cem Kadioglu ◽

Emin Aliyev ◽

Murad Celtik

Keyword(s):

Dna Damage ◽

Dna Fragmentation ◽

Sperm Chromatin ◽

Sperm Dna Fragmentation ◽

Sperm Dna Damage ◽

Infertile Men ◽

Fragmentation Index ◽

Sperm Dna ◽

Sperm Dna Fragmentation Index ◽

Dna Fragmentation Index

Background. Varicocele is associated with high levels of DNA damage in spermatozoa due to oxidative stress and elevated levels of sperm DNA fragmentation, which has been currently proposed to be an essential additional diagnostic test to be recommended for patients with clinical varicocele. The aim of this study was to evaluate the parameters of semen and the DNA fragmentation index (DFI) in patients with varicocele before and after varicocelectomy.Methods. The details of 92 consecutive patients were retrospectively analyzed from January 2010 to December 2012. The sperm samples were evaluated according to the World Health Organization Guidelines. Sperm DNA damage, characterized as DFI, was evaluated by sperm chromatin structure assay using flow cytometry.Results. There was a statistically significant improvement in the semen concentration, the total motile count, the total normal sperm count, and the sperm DNA fragmentation index (DFI; the percentage of sperm with denatured DNA) after varicocelectomy. There was a large decrease in DFI from a preoperative mean of 42.6% to a postoperative mean of 20.5% (P<0.001). A higher preoperative DFI was associated with a larger decrease in postoperative DFI, and significant negative correlations were observed between the DFI and sperm motility (r=-0.42,P<0.01).Conclusion. Our data suggest that varicocelectomy can improve multiple semen parameters and sperm DNA damage in infertile men with varicocele. The patients with preoperative defects in those parameters showed greater improvement postoperatively. Further research in this area is needed to understand the exact mechanisms of DNA damage in infertile men with varicocele.

Download Full-text