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Spontaneous uptake of exogenous DNA by goat spermatozoa and selection of donor bucks for sperm-mediated gene transfer

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Abstract

Sperm-mediated gene transfer (SMGT) has been long heralded as a faster and cheaper alternative to more commonly used methods of producing transgenic animals. In this study, the capra semen ejaculates were pooled together and then incubated in vitro with DIG-labeled DNA. The binding and internalizing rates were observed by the in situ hybridization methods. We also compared the standard sperm parameters and the efficiencies of interaction with exogenous DNA of 60 individuals to select donor bucks for SMGT. It was showed that labeled exogenous DNA was detected in different localizations in spermatozoa but genuine DNA uptake, in contrast to mere binding, seems to be limited to the postacrosomal region. The removal of seminal plasma increased significantly (P < 0.01) the capability in picking up exogenous DNA. Use of frozen-thawed semen (without cryoprotectant agents) and Triton X-100 treatment also increased significantly (P < 0.01) the DNA-binding capacity, but reduced the sperm viability. The binding rates (the proportion of labeled-DNA positive spermatozoa to all the spermatozoa counted) of 60 buck individuals were in the range of 3.08–73.39%, and the internalizing rates (the proportion of DNaseI-treated labeled-DNA positive spermatozoa to all the spermatozoa counted) were 4.83–70.00%. About 8.34% (5/60) bucks showed high binding, but low internalizing ability. Chi-square test showed that there was significant difference among the breeds (x 2 = 26.515, P < 0.01). Eight individual bucks that demonstrated high DNA uptake were selected for SMGT. It was demonstrated that the goat spermatozoa was capable of spontaneous uptake of exogenous DNA. Seminal fluid inhibits DNA uptake and that membrane disruption increases DNA binding but greatly diminishes uptake.

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References

  1. Bacci ML (2007) A brief overview of transgenic farm animals. Vet Res Commun 31(Suppl 1):9–14

    Article  PubMed  Google Scholar 

  2. Zhuang SZ et al (2010) Transgenic RNAi-mediated reduction of LZP3 in Lagurus lagurus oocytes results in decreased fertilization ability in IVF. Mol Biol Rep 37(3):1253–1259

    Article  PubMed  CAS  Google Scholar 

  3. Brackett B, Boranska W, Sawicki W, Koprowski H (1971) Uptake of heterologous genome by mammalian spermatozoa and its transfer to ova through fertilization. Proc Natl Acad Sci USA 68(1):353–357

    Article  PubMed  CAS  Google Scholar 

  4. Maione B, Lavitrano M, Spadafora C, Kiessling AA (1998) Sperm-mediated gene transfer in mice. Mol Reprod Dev 50(4):406–409

    Article  PubMed  CAS  Google Scholar 

  5. Smith K, Spadafora C (2005) Sperm-mediated gene transfer: applications and implications. Bioessays 27(5):551–562

    Article  PubMed  CAS  Google Scholar 

  6. Lanes CFC, Sampaio LA, Marins LF (2009) Evaluation of DNase activity in seminal plasma and uptake of exogenous DNA by spermatozoa of the Brazilian flounder Paralichthys orbignyanus. Theriogenology 71(3):525–533

    Article  PubMed  CAS  Google Scholar 

  7. Lavitrano M et al (1989) Sperm cells as vector for introduction foreign DNA into eggs: genetic transformation of mice. Cell 57:717–723

    Article  PubMed  CAS  Google Scholar 

  8. Lavitrano M et al (2002) Efficient production by sperm-mediated gene transfer of human decay accelerating factor (hDAF) transgenic pigs for xenotransplantation. Proc Natl Acad Sci USA 99(22):14230–14235

    Article  PubMed  CAS  Google Scholar 

  9. Lazzereschi D et al (2000) Efficiency of transgenesis using sperm-mediated gene transfer: generation of hDAF transgenic pigs. Transplant Proc 32(5):892–894

    Article  PubMed  CAS  Google Scholar 

  10. Lavitrano M et al (2006) Sperm-mediated gene transfer. Reprod Fertil Dev 18(1–2):19–23

    Article  PubMed  CAS  Google Scholar 

  11. Lavitrano M et al (2003) Sperm mediated gene transfer in pig: selection of donor boars and optimization of DNA uptake. Mol Reprod Dev 64(3):284–291

    Article  PubMed  CAS  Google Scholar 

  12. Brinster RL, Sandgren EP, Behringer RR, Palmiter RD (1989) No simple solution for making transgenic mice. Cell 59(2):239–241

    Article  PubMed  CAS  Google Scholar 

  13. Honaramooz A, Behboodi E, Blash S, Megee SO, Dobrinski I (2003) Germ cell transplantation in goats. Mol Reprod Dev 64(4):422–428

    Article  PubMed  CAS  Google Scholar 

  14. Zhao Y et al (2010) Production of transgenic goats by sperm-mediated exogenous DNA transfer method. Asian-Aust J Anim Sci 23(1):33–40

    Google Scholar 

  15. Wang L, Fan J, Yu M, Zheng S, Zhao Y (2011) Association of goat (Capra hircus) CD4 gene exon 6 polymorphisms with ability of sperm internalizing exogenous DNA. Mol Biol Rep 38(3):1621–1628

    Article  PubMed  CAS  Google Scholar 

  16. Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 271–275

    Google Scholar 

  17. Zhao Y-J, Li F-B, Sun X-M, Zhou J-H, Wei H (2005) Age- and- breed-dependence of exogenous DNA binding to spermatozoa in goats. Acta Zoologica Sinica 51(6):1058–1066

    CAS  Google Scholar 

  18. Perry ACF et al (1999) Mammalian transgenesis by intracytoplasmic sperm injection. Science 284(5417):1180–1183

    Article  PubMed  CAS  Google Scholar 

  19. Garcia-Vazquez FA, Garcia-Rosello E, Gutierrez-Adan A, Gadea J (2009) Effect of sperm treatment on efficiency of EGFP-expressing porcine embryos produced by ICSI-SMGT. Theriogenology 72(4):506–518

    Article  PubMed  CAS  Google Scholar 

  20. Atkinson PW et al (1991) Association of exogenous DNA with cattle and insect spermatozoa invitro. Mol Reprod Dev 29(1):1–5

    Article  PubMed  CAS  Google Scholar 

  21. Lavitrano M, French D, Zani M, Frati L, Spadafora C (1992) The interaction between exogenous DNA and sperm cells. Mol Reprod Dev 31(3):161–169

    Article  PubMed  CAS  Google Scholar 

  22. Camaioni A et al (1992) Uptake of exogenous DNA by mammalian spermatozoa: specific localization of DNA on sperm heads. J Reprod Fertil 96(1):203–212

    Article  PubMed  CAS  Google Scholar 

  23. Wang HJ, Lin AX, Chen YF (2003) Association of rabbit sperm cells with exogenous DNA. Anim Biotechnol 14(2):155–165

    Article  PubMed  CAS  Google Scholar 

  24. Anzar M, Buhr MM (2006) Spontaneous uptake of exogenous DNA by bull spermatozoa. Theriogenology 65(4):683–690

    Article  PubMed  CAS  Google Scholar 

  25. Zani M et al (1995) The mechanism of binding of exogenous DNA to sperm cells: factors controlling the DNA uptake. Exp Cell Res 217(1):57–64

    Article  PubMed  CAS  Google Scholar 

  26. Cabrera M, Chan PJ, Kalugdan TH, King A (1997) Transfection of the inner cell mass and lack of a unique DNA sequence affecting the uptake of exogenous DNA by sperm as shown by dideoxy sequencing analogues. J Assist Reprod Genet 14(2):120–124

    Article  PubMed  CAS  Google Scholar 

  27. Gandolfi F (1998) Spermatozoa, DNA binding and transgenic animals. Transgenic Res 7(3):147–155

    Article  PubMed  CAS  Google Scholar 

  28. Spadafora C (1998) Sperm cells and foreign DNA: a controversial relation. Bioessays 20(11):955–964

    Article  PubMed  CAS  Google Scholar 

  29. Maione B, Pittoggi C, Achene L, Lorenzini R, Spadafora C (1997) Activation of endogenous nucleases in mature sperm cells upon interaction with exogenous DNA. DNA Cell Biol 16(9):1087–1097

    Article  PubMed  CAS  Google Scholar 

  30. Leboeuf B, Restall B, Salamon S (2000) Production and storage of goat semen for artificial insemination. Anim Reprod Sci 62(1–3):113–141

    Article  PubMed  CAS  Google Scholar 

  31. Zhao Y, Wang Y, Wang J, Li F, Wei H (2008) Effect of cryopreservation on the efficiencies of goat sperm in picking up exogenous DNA and resulted transgenic embryos. Acta Zoologica Sinica 54(6):1089–1097

    CAS  Google Scholar 

  32. Shim SW, Kim YH, Lee HT, Shim H (2008) Effects of sperm membrane disruption and electrical activation of oocytes on in vitro development and transgenesis of porcine embryos produced by intracytoplasmic sperm injection. Asian-Aust J Anim Sci 21(3):358–363

    CAS  Google Scholar 

  33. Wu Y, Liu CJ, Wan PC, Hao ZD, Zeng SM (2009) Factors affecting the efficiency of producing porcine embryos expressing enhanced green fluorescence protein by ICSI-mediated gene transfer method. Anim Reprod Sci 113(1–4):156–166

    Article  PubMed  CAS  Google Scholar 

  34. Shadanloo F et al (2010) Sperm status and DNA dose play key roles in sperm/ICSI-mediated gene transfer in caprine. Mol Reprod Dev 77(10):868–875

    Article  PubMed  CAS  Google Scholar 

  35. Canovas S, Gutierrez-Adan A, Gadea J (2010) Effect of exogenous DNA on bovine sperm functionality using the Sperm Mediated Gene Transfer (SMGT) technique. Mol Reprod Dev 77(8):687–698

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The work was supported by: national science and technology specific project, grant number: 2008ZX08008-004, the Fundamental Research Funds for the Central Universities, grant number: XDJK2010C092 and the national natural science foundation of China, grant number: 30600430. We thank Dr. S. G. Revell (Genus Breeding Ltd. Ruthin, Denbighshire LL15 2UP, UK) and Mrs. Aparna Udupi (Department of Genetics and Biotechnology, Research Centre Foulum, Aarhus University, DK-8830 Tjele, DK) for critical reading the manuscript. We also thank seven anonymous reviewers and editors for constructive and professional comments.

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Authors and Affiliations

  1. College of Animal Science and Technology, Chongqing Key Laboratory of Forage & Herbivore, Southwest University, Beibei, Chongqing, 400716, People’s Republic of China

    Yongju Zhao, Mingju Yu, Lingbin Wang, Yaofeng Li & Jingsheng Fan

  2. College of Veterinary Medicine, Nanjing Agricultural University, Weigang 1, Nanjing, Jiangsu, 210095, People’s Republic of China

    Qian Yang

  3. College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, People’s Republic of China

    Yaping Jin

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  1. Yongju Zhao
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  2. Mingju Yu
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Correspondence to Yongju Zhao.

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Zhao, Y., Yu, M., Wang, L. et al. Spontaneous uptake of exogenous DNA by goat spermatozoa and selection of donor bucks for sperm-mediated gene transfer. Mol Biol Rep 39, 2659–2664 (2012). https://doi.org/10.1007/s11033-011-1019-4

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  • DOI: https://doi.org/10.1007/s11033-011-1019-4

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