Detection of DNA Damage Caused by Cryopreservation using a Modified SCGE in Large Yellow Croaker, Pseudosciaena crocea

Abstract

We used single-cell gel electrophoresis (SCGE) to detect the integrity of sperm DNA of the teleost large yellow croaker, Pseudosciaena crocea, cryopreserved with Cortland solution and a range of 5% to 30% DMSO concentrations in order to test how sperm cryopreservation affected the DNA stability of nuclei. Electrophoresis was conducted for 60 min at 130 mA and 15 V. The comet images were analyzed with software CometScore 1.5, and parameters such as comet length, tail length and percentage DNA in the tail were obtained. Then the comet rate and damage coefficient were calculated. Results demonstrated that there were no significant differences in motility, comet rate and damage coefficient between fresh sperm and cryopreserved sperm stored in 5%, 10%, 15% and 20% DMSO, while the sperm cryopreserved with 25% and 30% DMSO had a lower motility, higher comet length and damage coefficients than those of fresh sperm. There was a positive correlation between comet rate of cryopreserved sperm and the concentration of DMSO. Our results demonstrate that toxicity of the cryoprotectant is the main cause of DNA damage in cryopreserved sperm nuclei.

References

  1. 1.

    Beirão, J., Zilli, L., Vilella, S., Cabrita, E., Schiavone, R., Herraez, M. P. (2012) Improving sperm cryopreservation with antifreeze proteins: Effect on gilthead seabream (Sparus aurata) plasma membrane lipids. Biol. Reprod. 86, 59.

    Article  Google Scholar 

  2. 2.

    Cabrita, E., Robles, V., Rebordinos, L., Sarasquete, C., Herráez, M. P. (2005) Evaluation of DNA damage in rainbow trout Oncorhynchus mykiss and gilthead sea bream Sparus aurata cryopreserved sperm. Cryobiology 50, 144–153.

    CAS  Article  Google Scholar 

  3. 3.

    Chen, A. (1998) Detection of DNA damage on single cell using single cell gel electrophoresis. Forensic Medical Science (section of Clinical Biochemistry and Laboratory Medicine) 19, 145–146.

    Google Scholar 

  4. 4.

    Dahms, H. U., Gao, Q. F., Hwang, J. S. (2007) Optimized maintenance and larval production of the bryozoan Bugula neritina (Bryozoa) in the laboratory. Aquaculture 265, 169–175.

    Article  Google Scholar 

  5. 5.

    Gi, B. K., Richard, F. L. (2004) Effects of genotoxic compounds on DNA and development of early and late grass shrimp embryo stages. Mar. Environ. Res. 57, 329–338.

    Article  Google Scholar 

  6. 6.

    Giovannelli, L., Decorosi, F., Dolara, P., Pulvirenti, L. (2003) Vulnerability to DNA damage in the aging rat Substantia nigra: a study with the comet assay. Brain Res. 969, 244–247.

    CAS  Article  Google Scholar 

  7. 7.

    Goerin, P. L. (1995) Handbook of Experimental Pharmacology. New York: Springer-Verlag, pp. 187–213.

    Google Scholar 

  8. 8.

    Gwo, J. C., Arnold, C. R. (1992) Cryopreservation of Atlantic croaker spermatozoa: evaluation of morphological changes. J. Exp. Zool. 264, 444–453.

    CAS  Article  Google Scholar 

  9. 9.

    Hagedorn, M., Carter, V. L. (2011) Zebrafish reproduction: revisiting in vitro fertilization to increase sperm cryopreservation success. Plos One 6, 1–9.

    Article  Google Scholar 

  10. 10.

    Han, L. L., Yuan, Z., Dahms, H. U., Li, Q. Y., Zhang, Q. Z., Wu, R. J., Tan, J., Zou, X. Y., Hou, L. (2012) Molecular cloning, characterization and expression analysis of a C-type lectin (AJCTL) from the sea cucumber Apostichopus japonicus. Immunology Letters 143, 137–145.

    CAS  Article  Google Scholar 

  11. 11.

    Kim, J. H., Jeon, H. J., Baek, J. M., Han, K. N., Dahms, H. U. (2013) EDCs-induced glucocorticoid receptor related genes expression of the river pufferfish, Takifugu obscurus. Aquacul. Res. 44, 985–994.

    CAS  Article  Google Scholar 

  12. 12.

    Kim, J. H., Jung, S. J., Dahms, H. U., Lee, W. O., Ryu, K. M., Han, K. N. (2013) Osmoregulation related gene expression of the anadromous river pufferfish Takifugu obscurus under sudden salinity changes. J. Fish Physiol. Biochem. 40, 205–219.

    Google Scholar 

  13. 13.

    Koppen, G., Toncelli, T. M., Triest, L., Verschaeve, L. (1999) The comet assay: a tool to study alterations of DNA integrity in developing plant leaves. Mech. Age Dev. 110, 13–24.

    CAS  Article  Google Scholar 

  14. 14.

    Labbe, C., Martoriati, A., Devaux, A., Maisse, G. (2001) Effect of sperm cryopreservation on sperm DNA stability and progeny development in rainbow trout. Mol. Reprod. Dev. 60, 397–404.

    CAS  Article  Google Scholar 

  15. 15.

    Lin, D. J., You, Y. L. (2002) Physiological characteristics and cryopreservation of Pseudosciaena crocea (Richardson) sperm. J. Trop. Oceanography 21, 69–75.

    Google Scholar 

  16. 16.

    Lin, D. J., You, Y. L., Chen, B. Y. (2006) Variation of the motility and ultrastructure of the frozenthawed sperm in teleost, Pseudosciaena crocea (Richardson). J. Fujian Normal Univ. (Nat. Sci. Ed.) 22, 71–76.

    Google Scholar 

  17. 17.

    Lu, H., Zhang, L., Zhang, N., Tang, J., Ding, X. P., Tang, Y. (2002) Detection of DNA damage of human sperm using single cell electrophoresis. Zhonghua Nan Ke Xue 8, 416–418.

    CAS  PubMed  Google Scholar 

  18. 18.

    Maluf, S. W., Erdtmann, B. (2000) Follow-up study of the genetic damage in lymphocytes of pharmacists and nurses handling antineoplastic drugs evaluated by cytokinesis-block micronuclei analysis and single cell gel electrophoresis assay. Mutant Res. 471, 21–27.

    CAS  Article  Google Scholar 

  19. 19.

    Rossa, G. M., McMillan, T. J., Wilcox, P., Collins, A. R. (1995) The single cell microgel electrophoresis assay (comet assay): technical aspects and applications: Report on the 5th LH Gray Trust Workshop, Institute of Cancer Research 1994. Mutant Res. 337, 57–60.

    Article  Google Scholar 

  20. 20.

    Singh, N. P., McCoy, M. T., Tice, R. R., Schneider, E. L. (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 175, 184–191.

    CAS  Article  Google Scholar 

  21. 21.

    Song, B., Zheng, L. K., Deng, L. X., Zhang, Q. (2002) Freezing effect on sperm DNA. Zhonghua Nan Ke Xue 4, 253–254.

    Google Scholar 

  22. 22.

    Stavreva, D. A., Ptacek, O., Plewa, M. J., Gichner, T. (1998) Single cell gel electrophoresis analysis of genomic damage induced by ethyl methanesulfonate in cultured tobacco cells. Mutant Res. 422, 323–330.

    CAS  Article  Google Scholar 

  23. 23.

    Suquet, M. D., Dreanno, C., Petton, B., Normant, Y., Omnes, M. H., Billard, R. (1998) Long-term effects of the cryopreservation of turbot Psetta maxima spermatozoa. Aquatic Living Resour. 11, 45–48.

    Article  Google Scholar 

  24. 24.

    Tiersch, T. R., Figiel Jr., C. R., Wayman, W. R., Williamson, J. H., Carmichael, G. J., Gorman, O. T. (2000) Cryopreservation of sperm of the endangered razorback sucker. In: Tiersch, T. R., Mazik. P. M. (eds) Cryopreservation in Aquatic Species. World Aquaculture Society, Baton Rouge, Louisiana, pp. 117–122.

    Google Scholar 

  25. 25.

    Xu, D. X., Shen, H. M., Wang, J. N. (2000) Detection of DNA strand breakage in human spermatozoa by use of single-cell gel electrophoresis. China Medical Board NY 17, 281–284.

    CAS  Google Scholar 

  26. 26.

    Xu, X. R., Zhu, J. Q., Ye, T., Wang, C. L., Zhu, Y. F., Dahms, H. U., Jin, F., Yang, W. X. (2013) Improvement of single-cell alkaline comet assay gel electrophoresis (SCGE) procedure using sperm DNA of large yellow croaker Pseudosciaena crocea. Aquatic Biol. 18, 293–295.

    Article  Google Scholar 

  27. 27.

    Yang, J. Y., Li, L., Peng, Y., Du, S. J. (2004) DNA damage caused by low dose methotrexate and protective effect of folinic acid. Carcinogenesis, Teratogenesis & Mutagenesis 16, 24–26.

    Google Scholar 

  28. 28.

    Ye, T., Zhu, J. Q., Yang W. X., Wei, P., Wu, X. F. (2009) Sperm cryopreservation in Sparus macrocephalus and DNA damage detection with SCGE. Zool. Res. 30, 151–157.

    CAS  Article  Google Scholar 

  29. 29.

    Zilli, L., Schiavone, R., Zonno, V., Storelli, C., Vilella, S. (2003) Evaluation of DNA damage in Dicentrarchus labrax sperm following cryopreservation. Cryobiology 47, 227–235.

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank the following projects for their support: K. C. Wong Magna Fund in Ningbo University, The Scientific and Technical Project of Zhejiang Province, National Natural Science Foundation of China (Grant Nos. 41276151, 31272642 and 31072198).

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Correspondence to Hans-Uwe Dahms or Fan Jin or Wan-Xi Yang.

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Xu, XR., Tan, FQ., Zhu, JQ. et al. Detection of DNA Damage Caused by Cryopreservation using a Modified SCGE in Large Yellow Croaker, Pseudosciaena crocea. BIOLOGIA FUTURA 65, 405–413 (2014). https://doi.org/10.1556/ABiol.65.2014.4.5

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Keywords

  • Pseudosciaena crocea
  • sperm
  • cryopreservation
  • genetic damage
  • single-cell gel electrophoresis