Register      Login
Reproduction, Fertility and Development Reproduction, Fertility and Development Society
Vertebrate reproductive science and technology
RESEARCH ARTICLE

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

K. Pollock A D , J. Gosálvez B , F. Arroyo B , C. López-Fernández B , M. Guille C , A. Noble C and S. D. Johnston A
+ Author Affiliations
- Author Affiliations

A School of Agriculture and Food Science, The University of Queensland, Gatton, Qld 4343, Australia.

B Department of Biology, Genetics Unit, The Autonomous University of Madrid, Madrid, 20849, Spain.

C European Xenopus Resource Centre, The University of Portsmouth, King Henry 1 Street, Portsmouth, PO1 2DY, UK.

D Corresponding author. Email: kate.pollock@uqconnect.edu.au

Reproduction, Fertility and Development 27(8) 1168-1174 https://doi.org/10.1071/RD14070
Submitted: 22 February 2014  Accepted: 13 April 2014   Published: 3 June 2014

Abstract

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.

Additional keywords: African clawed frog, halomax, sperm DNA fragmentation.


References

Alvarez, J. G., and Gosálvez, J. (2012). Role of protamine disulfide cross-linking in counteracting oxidative damage to DNA. In ‘Studies on Men’s Health and Fertility’. (Eds A. Agarwal, R. J. Aitken and J. G. Alvarez.) pp. 221–235. (Humana Press: New York.)

Beesley, S. G., Costanzo, J. P., and Richard, E. L. (1998). Cryopreservation of spermatozoa from freeze-tolerant and -intolerant anurans. Cryobiology 37, 155–162.
Cryopreservation of spermatozoa from freeze-tolerant and -intolerant anurans.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaK1cvktlWqsA%3D%3D&md5=c346eae0bf3284b2189433be9f8a04b0CAS | 9769166PubMed |

Browne, R. K., Clulow, J., Mahony, M., and Clark, A. (1998). Successful recovery of motility and fertility of cryopreserved can toad (Bufo marinus) sperm. Cryobiology 37, 339–345.
Successful recovery of motility and fertility of cryopreserved can toad (Bufo marinus) sperm.Crossref | GoogleScholarGoogle Scholar | 9917350PubMed |

Browne, R. K., Clulow, J., and Mahony, M. (2002a). The short-term storage and cryopreservation of spermatozoa from hylid and myobatrachid frogs. Cryo Letters 23, 129–136.
| 1:CAS:528:DC%2BD38XktlOnsb4%3D&md5=5adc318cbf342a4b0af8b2f7ef8ff40cCAS | 12050781PubMed |

Browne, R. K., Davis, J., Pomering, M., and Clulow, J. (2002b). Storage of cane toad (Bufo marinus) sperm for 6 days at 0°C with subsequent cryopreservation. Reprod. Fertil. Dev. 14, 267–273.
Storage of cane toad (Bufo marinus) sperm for 6 days at 0°C with subsequent cryopreservation.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD38jgsFKjsg%3D%3D&md5=b6d2d7675e51297ec90daae264834881CAS | 12467350PubMed |

Burlibasa, L., and Gavrila, L. (2011). Amphibians as model organisms for studying environmental genotoxicity. Appl. Ecol. Env. Res. 9, 1–15.

Cortés-Gutiérrez, E. I., Crespo, F., Gosálbez, A., Dávila-Rodríguez, M. I., López-Fernández, C., and Gosálvez, J. (2008a). DNA fragmentation in frozen sperm of Equus asinus: Zamorano-Leonés, a breed at risk of extinction. Theriogenology 69, 1022–1032.
DNA fragmentation in frozen sperm of Equus asinus: Zamorano-Leonés, a breed at risk of extinction.Crossref | GoogleScholarGoogle Scholar | 18367243PubMed |

Cortés-Gutiérrez, E. I., Dávila-Rodríguez, M. I., López-Fernández, C., Fernández, J. L., and Gosálvez, J. (2008b). Alkali-labile sites in sperm cells from Sus and Ovis species. Int. J. Androl. 31, 354–363.
Alkali-labile sites in sperm cells from Sus and Ovis species.Crossref | GoogleScholarGoogle Scholar | 17651406PubMed |

Cortés-Gutiérrez, E. I., Dávila-Rodríguez, M. I., Fernández, J. L., Gosálvez, J., Johnston, S. D., and López-Fernández, C. (2009). Mapping alkali-labile sites in mammalian spermatozoa. In ‘Animal Reproduction: New Research Developments’. (Ed. L. T. Dahnof.) pp. 219–231. (Nova Publishers: New York.)

Cortés-Gutiérrez, E. I., Dávila-Rodríguez, M. I., López-Fernández, C., Fernández, J. L., Crespo, F., and Gosálvez, J. (2014a). Localisation of alkali-labile sites in donkey (Equus asinus) and stallion (Equus caballus) spermatozoa. Theriogenology 81, 321–325.
Localisation of alkali-labile sites in donkey (Equus asinus) and stallion (Equus caballus) spermatozoa.Crossref | GoogleScholarGoogle Scholar | 24182740PubMed |

Cortés-Gutiérrez, E. I., Dávila-Rodríguez, M. I., Cerda-Flores, R. M., Fernández, J. L., López-Fernández, C., Aragón, T., and Gosálvez, J. (2014b). Localisation and quantification of alkali-labile sites in human spermatozoa by DNA breakage detection – fluorescence in situ hybridisation. Andrologia , .
Localisation and quantification of alkali-labile sites in human spermatozoa by DNA breakage detection – fluorescence in situ hybridisation.Crossref | GoogleScholarGoogle Scholar | 24576285PubMed |

D’Occhio, M. J., Hengstberger, K. J., and Johnston, S. D. (2007). Biology of sperm chromatin structure and relationship to male fertility and embryonic survival. Anim. Reprod. Sci. 101, 1–17.
Biology of sperm chromatin structure and relationship to male fertility and embryonic survival.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXntFyhs7s%3D&md5=b296405a84268d0873e1ec438d47f8beCAS | 17303352PubMed |

Enciso, M., Sarasa, J., Agarwal, A., Fernández, J. L., and Gosálvez, J. (2009). A two-tailed comet assay for assessing DNA damage in spermatozoa. Reprod. Biomed. Online 18, 609–616.
A two-tailed comet assay for assessing DNA damage in spermatozoa.Crossref | GoogleScholarGoogle Scholar | 19549437PubMed |

EXRC (2012). ‘Holding and Producing Conditions’. (European Xenopus Resource Centre, University of Portsmouth: United Kingdom.) Available at http://www.port.ac.uk/research/exrc/holdingandproducingconditions/ [Accessed 1 June 2013].

Fernández, J. L., Muriel, L., Rivero, M. T., Goyanes, V., Vazquez, R., and Alvarez, J. G. (2003). The sperm chromatin dispersion test: a simple method for the determination of sperm DNA fragmentation. J. Androl. 24, 59–66.
| 12514084PubMed |

Gosálvez, J., López-Fernández, C., and Fernández, J. L. (2011a). Sperm chromatin dispersion test: technical aspects and clinical applications. In ‘Sperm Chromatin: Biological and Clinical Applications in Male Infertility and Assisted Reproduction’. (Eds A. Zini and A. Ashok.) pp. 151–170. (Springer: New York.)

Gosálvez, J., López-Fernández, C., Fernández, J. L., Gouraud, A., and Holt, W. V. (2011b). Relationships between the dynamics of iatrogenic DNA damage and genomic design in mammalian spermatozoa from eleven species. Mol. Reprod. Dev. 78, 951–961.
Relationships between the dynamics of iatrogenic DNA damage and genomic design in mammalian spermatozoa from eleven species.Crossref | GoogleScholarGoogle Scholar | 21919111PubMed |

Johnston, S. D., MacCallum, C., Blyde, D., McClean, R., Lisle, A., and Holt, W. V. (2006). An investigation into the similarities and differences governing the cryopreservation success of koala (Phascolarctos cinereus: Goldfuss) and wombat (Vombatus ursinus: Shaw) spermatozoa. Cryobiology 53, 218–228.
An investigation into the similarities and differences governing the cryopreservation success of koala (Phascolarctos cinereus: Goldfuss) and wombat (Vombatus ursinus: Shaw) spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28Xps1OmsLk%3D&md5=72c328320891ba8158d19837ac08b6b3CAS | 16889764PubMed |

Johnston, S. D., López-Fernández, C., Gosálbez, A., Zee, Y. P., Holt, W. V., Allen, C., and Gosálvez, J. (2007). The relationship between sperm morphology and chromatin integrity in the koala (Phascolarctos cinereus) as assessed by the sperm chromatin dispersion test (SCDt). J. Androl. 28, 891–899.
The relationship between sperm morphology and chromatin integrity in the koala (Phascolarctos cinereus) as assessed by the sperm chromatin dispersion test (SCDt).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXhtlCqtr%2FP&md5=9151758c46c9ab3737ccc9542ed3a7cbCAS | 17609294PubMed |

Johnston, S. D., López-Fernández, C., Gosálbez, A., Holt, W. V., and Gosálvez, J. (2009). Directional mapping of DNA nicking in ejaculated and cauda epididymidal spermatozoa of the short-beaked echidna (Tachyglossus aculeatus: Monotremata). Reprod. Fertil. Dev. 21, 1008–1014.
Directional mapping of DNA nicking in ejaculated and cauda epididymidal spermatozoa of the short-beaked echidna (Tachyglossus aculeatus: Monotremata).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhtlCjs7jM&md5=7c607d9431f43905db60b1d80ae8d26aCAS | 19874725PubMed |

Johnston, S. D., Satake, N., Zee, Y., López-Fernández, C., Holt, W. V., and Gosálvez, J. (2012). Osmotic stress and cryoinjury of koala sperm: an integrative study of the plasma membrane, chromatin stability and mitochondrial function. Reproduction 143, 787–797.
Osmotic stress and cryoinjury of koala sperm: an integrative study of the plasma membrane, chromatin stability and mitochondrial function.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38XovFahs7g%3D&md5=8221dad67111368482043da08e083d65CAS | 22499893PubMed |

Kouba, A. J., and Vance, C. K. (2009). Applied reproductive technologies and genetic resource banking for amphibian conservation. Reprod. Fertil. Dev. 21, 719–737.
Applied reproductive technologies and genetic resource banking for amphibian conservation.Crossref | GoogleScholarGoogle Scholar | 19567216PubMed |

López-Fernández, C., Crespo, F., Arroyo, F., Fernández, J. L., Arana, P., Johnston, S. D., and Gosálvez, J. (2007). Dynamics of sperm DNA fragmentation in domestic animals – II. The stallion. Theriogenology 68, 1240–1250.
Dynamics of sperm DNA fragmentation in domestic animals – II. The stallion.Crossref | GoogleScholarGoogle Scholar | 17919715PubMed |

López-Fernández, C., Pérez-Llano, B., García-Casado, P., Sala, R., Gosálbez, A., Arroyo, F., Fernández, J. L., and Gosálvez, J. (2008). Sperm DNA fragmentation in a random sample of Spanish boar livestock. Anim. Reprod. Sci. 103, 87–98.
Sperm DNA fragmentation in a random sample of Spanish boar livestock.Crossref | GoogleScholarGoogle Scholar | 17174491PubMed |

López-Fernández, C., Gage, M. J. G., Arroyo, F., Gosálbez, A., Larrán, A. M., Fernández, J. L., and Gosálvez, J. (2009). Rapid rates of sperm DNA damage after activation in tench (Tinca tinca: Teleostei, Cyprinidae) measured using a sperm chromatin dispersion test. Reproduction 138, 257–266.
Rapid rates of sperm DNA damage after activation in tench (Tinca tinca: Teleostei, Cyprinidae) measured using a sperm chromatin dispersion test.Crossref | GoogleScholarGoogle Scholar | 19494044PubMed |

Maselli, V., Polese, G., Rippa, D., Ligrone, R., Rastogi, R. K., and Fulgione, D. (2010). Frogs, sentinels of DNA damage induced by pollution in Naples and the neighbouring provinces. Ecotoxicol. Environ. Saf. 73, 1525–1529.
Frogs, sentinels of DNA damage induced by pollution in Naples and the neighbouring provinces.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXht1Wmu7fP&md5=4672181396cbb188ca0efa79c0208cf0CAS | 20684845PubMed |

Michael, S. F., and Jones, C. (2004). Cryopreservation of spermatozoa of the terrestrial Puerto Rican frog, Eleutherodactylus coqui. Cryobiology 48, 90–94.
Cryopreservation of spermatozoa of the terrestrial Puerto Rican frog, Eleutherodactylus coqui.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXht1yiurg%3D&md5=0ae19ecafff4a5742fe5130847d37d29CAS | 14969686PubMed |

Portas, T., Johnston, S. D., Hermes, R., Arroyo, F., López-Fernández, C., Bryant, B., Hildebrandt, T. B., Goritz, F., and Gosálvez, J. (2009). Frozen–thawed rhinoceros sperm exhibit DNA damage shortly after thawing when assessed by the sperm chromatin dispersion assay. Theriogenology 72, 711–720.
Frozen–thawed rhinoceros sperm exhibit DNA damage shortly after thawing when assessed by the sperm chromatin dispersion assay.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhtVSit7vK&md5=89f147669375bd6273b4368ac9bd7e65CAS | 19560805PubMed |

Sargent, M. G., and Mohun, T. J. (2005). Cryopreservation of sperm of Xenopus laevis and Xenopus tropicalis. Genesis 41, 41–46.
Cryopreservation of sperm of Xenopus laevis and Xenopus tropicalis.Crossref | GoogleScholarGoogle Scholar | 15645449PubMed |

Valencia, L. C., Garcia, A., Ramirez-Pinilla, M. P., and Fuentes, J. L. (2011). Estimates of DNA damage by the comet assay in the direct-developing frog Eleutherodactylus johnstonei (Anura, Eleutherodactylidae). Genet. Mol. Biol. 34, 681–688.
Estimates of DNA damage by the comet assay in the direct-developing frog Eleutherodactylus johnstonei (Anura, Eleutherodactylidae).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38XhtlOqsbs%3D&md5=9a63dca8ada53ff39df304f6b5d2d3aeCAS | 22215974PubMed |

Wang, M. Z., and Jia, X. Y. (2009). Low levels of lead exposure induce oxidative damage and DNA damage in the testes of the frog Rana nigromaculata. Ecotoxicology 18, 94–99.
Low levels of lead exposure induce oxidative damage and DNA damage in the testes of the frog Rana nigromaculata.Crossref | GoogleScholarGoogle Scholar | 18766441PubMed |

Wolf, D. P., and Hedrick, J. L. (1971). Molecular approach to fertilization – II. Viability and artificial fertilization of Xenopus laevis gametes. Dev. Biol. 25, 348–359.
Molecular approach to fertilization – II. Viability and artificial fertilization of Xenopus laevis gametes.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaE3MXkslejt7w%3D&md5=533bf31a6e668a0bb9b769c480d4546cCAS | 5567826PubMed |

Yokota, T., Takamune, K., and Katagiri, C. (1991). Nuclear basic proteins of Xenopus laevis sperm: their characterisation and synthesis during spermatogenesis. Dev. Growth Differ. 33, 9–17.
Nuclear basic proteins of Xenopus laevis sperm: their characterisation and synthesis during spermatogenesis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK3MXks1yrur0%3D&md5=c98aaa49d8aeb277c71397f8d0722e44CAS |

Zee, Y. P., López-Fernández, C., Arroyo, F., Johnston, S. D., Holt, W. V., and Gosálvez, J. (2009). 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 138, 267–278.
Evidence that single-stranded DNA breaks are a normal feature of koala sperm chromatin, while double-stranded DNA breaks are indicative of DNA damage.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXptlemt7g%3D&md5=c156b21f658db7393b236854839ecd0dCAS | 19494045PubMed |