DNA breakage detection-FISH (DBD-FISH) in human spermatozoa: technical variants evidence different structural features

Fernández, José Luís; Vázquez-Gundı́n, Fernando; Delgado, Arantza; Goyanes, Vicente; Ramiro-Díaz, J.; Torre, J. de la; Gosálvez, Jaime

doi:10.1016/s0027-5107(00)00079-8

Cited by 84 publications

(36 citation statements)

References 12 publications

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“…In any case, the recurrence of this associative phenomenon calls attention to the possible role of constitutive heterochromatin as a conductor of chromatin condensation whose role is 'paying the price' of a high incidence of DNA base change. This would also be a plausible explanation for the extremely high incidence of ALS in sperm nuclei associated with heterochromatic regions where a higher chromatin packaging is required (Singh et al, 1989;Fernández et al, 2000;Cortés-Gutiérrez et al, 2008).…”

Section: Discussionmentioning

confidence: 99%

Whole-comparative genomic hybridization in domestic sheep <i>(Ovis aries)</i> breeds

Dávila-Rodríguez

Cortés‐Gutiérrez

López‐Fernández

et al. 2009

Cytogenet Genome Res

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Whole-comparative genomic hybridization (W-CGH) allows identification of chromosomal polymorphisms related to highly repetitive DNA sequences localized in constitutive heterochromatin. Such polymorphisms are detected establishing competition between genomic DNAs in an in situ hybridization environment without subtraction of highly repetitive DNA sequences, when comparing two species from closely related taxa (same species, sub-species, or breeds) or somewhat related taxa. This experimental approach was applied to investigating differences in highly repetitive sequences of three sheep breeds (Castellana, Ojalada, and Assaf). To this end, W-CGH was carried out using mouflon (sheep ancestor) chromosomes as a common target to co-hybridize equimolar quantities of two genomic DNAs obtained from either Castellana, Ojalada or Assaf sheep breeds. The results showed that the amount of constitutive heterochromatin is greater in all pericentromeric heterochromatin regions of acrocentric chromosomes than in metacentric or sex chromosomes. Additionally, when W-CGH was performed using DNAs from the Iberian breeds Castellana and Ojalada, chromosomal pericentromeric regions revealed quantitatively and qualitatively a presence of DNA families similar to that obtained from any of the above-cited breeds. On the contrary, when the DNA used in W-CGH experiments was obtained from Assaf, as compared to either Castellana or Ojalada, two different pericentromeric DNA families of highly repetitive sequences could be detected. Lastly, sex chromosomes were shown to be homogeneous among all breeds and thus revealed no detectable constitutive heterochromatin. W-CGH results were confirmed using DNA breakage detection-FISH experiments (DBD-FISH) carried out on lymphocytes. As a whole, the results showed that two different repetitive DNA families are present in the pericentromeric heterochromatin of the sheep breeds studied here. Additionally, they suggest a differential presence of these distinct repetitive DNA families in Castellana and Ojalada breeds as compared to the Assaf breed. Finally, the results of W-CGH after using mouflon as the targeted chromosomes also show that the two DNA families are present in the ancestor.

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Section: Discussionmentioning

confidence: 99%

Whole-comparative genomic hybridization in domestic sheep <i>(Ovis aries)</i> breeds

Dávila-Rodríguez

Cortés‐Gutiérrez

López‐Fernández

et al. 2009

Cytogenet Genome Res

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“…The most common types of identified sperm DNA damage are: (i) single and double DNA strand breaks; (ii) the chemical modification of a base by, for example, oxidation or alkylation; (iii) inter-or intrastrand crosslinkage; and (iv) DNA-protein crosslinks. 57,58 A variety of tests have been introduced including terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling or TUNEL, 59 comet, 60 in situ nick translation, 61 DNA breakage detection-fluorescence in situ hybridisation, 62 sperm chromatin dispersion test 63 and sperm chromatin structure assay. 64 Some of these tests measure DNA damage directly, such as TUNEL, or comet at neutral pH; others measure DNA damage after denaturation steps, such as the sperm chromatin structure assay, sperm chromatin dispersion and comet at acid or alkaline pH.…”

Section: Morphology and Dna Statusmentioning

confidence: 99%

Semen analysis and sperm function testing

Franken¹,

Oehninger²

2011

Asian J Androl

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Despite controversy regarding the clinical value of semen analysis, male fertility investigation still relies on a standardized analysis of the semen parameters. This is especially true for infertility clinics in both developing and developed countries. Other optional tests or sophisticated technologies have not been widely applied. The current review addresses important changes in the analysis of semen as described in the new World Health Organization (WHO) manual for semen analysis. The most important change in the manual is the use of evidence-based publications as references to determine cutoff values for normality. Apart from the above mentioned changes, the initial evaluation and handling methods remain, in most instances, the same as in previous editions. Furthermore, the review evaluates the importance of quality control in andrology with emphasis on the evaluation of sperm morphology. WHO sperm morphology training programmes for Sub-Saharan countries were initiated at Tygerberg Hospital in 1995. The external quality control programme has ensured that the majority of participants have maintained their morphological reading skills acquired during initial training. This review reports on current sperm functional tests, such as the induced acrosome reaction, and sperm-zona pellucida binding assays, as well as the impact of sperm quality in terms of DNA integrity, and the relationship of sperm function tests to sperm morphology.

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“…Neither human lymphocytes under alkaline conditions nor sperm under neutral conditions show these DNA breaks, which may represent functional characteristics but not pre-existing SSB (Singh et al 1989). In addition to intracellular processes like DNA replication and DNA repair, which utilise DNA nicking enzymes (Fairbairn et al 1994;McPherson and Longo 1993) during spermatogenesis, the reason for this high number of ALS seems to be the high degree of chromatin condensation (Fernandez et al 2000;Singh et al 1989). Under alkaline conditions, nicks in the DNA seem to provide a starting point for DNA unwinding by transforming the breaks into single strands (Rydberg 1975;Vazquez-Gundin et al 2000).…”

Section: The Use Of Sperm and Testicular Cells In The Comet Assaymentioning

confidence: 99%

“…These include the sperm chromatin dispersion (SCD) and the DNA-breakage detection fluorescence in situ hybridisation (DBD-FISH) assays utilising like the comet assay agarose embedded cells but without applying an electrophoretic field (Fernandez et al 2000;Fernandez et al 2003). Other approaches like in situ nick translation (ISNT; Irvine et al 2000) and terminal deoxynucleotidyl transferase dUTP nick end-labelling (TUNEL; Gorczyca et al 1993) take advantage of enzymes, which are able to incorporate in situ marker-moleculelabelled deoxynucleotides onto the DNA to detect DNA damage very accurately.…”

Section: In Vitro Comet Assay With Spermmentioning

confidence: 99%

The comet assay in male reproductive toxicology

2007

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Due to our lifestyle and the environment we live in, we are constantly confronted with genotoxic or potentially genotoxic compounds. These toxins can cause DNA damage to our cells, leading to an increase in mutations. Sometimes such mutations could give rise to cancer in somatic cells. However, when germ cells are affected, then the damage could also have an effect on the next and successive generations. A rapid, sensitive and reliable method to detect DNA damage and assess the integrity of the genome within single cells is that of the comet or single-cell gel electrophoresis assay. The present communication gives an overview of the use of the comet assay utilising sperm or testicular cells in reproductive toxicology. This includes consideration of damage assessed by protocol modification, cryopreservation vs the use of fresh sperm, viability and statistics. It further focuses on in vivo and in vitro comet assay studies with sperm and a comparison of this assay with other assays measuring germ cell genotoxicity. As most of the de novo structural aberrations occur in sperm and spermatogenesis is functional from puberty to old age, whereas female germ cells are more complicated to obtain, the examination of male germ cells seems to be an easier and logical choice for research and testing in reproductive toxicology. In addition, the importance of such an assay for the paternal impact of genetic damage in offspring is undisputed. As there is a growing interest in the evaluation of genotoxins in male germ cells, the comet assay allows in vitro and in vivo assessments of various environmental and lifestyle genotoxins to be reliably determined.

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DNA breakage detection-FISH (DBD-FISH) in human spermatozoa: technical variants evidence different structural features

Cited by 84 publications

References 12 publications

Whole-comparative genomic hybridization in domestic sheep <i>(Ovis aries)</i> breeds

Whole-comparative genomic hybridization in domestic sheep <i>(Ovis aries)</i> breeds

Semen analysis and sperm function testing

The comet assay in male reproductive toxicology

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