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The effects of IAM38 blocking or CD4 blocking on the binding of exogenous DNA in rabbit sperm

  • Shun Zhang
  • Xiaocan Lei
  • Peng Huo
  • Qingyou Liu
  • Xiaomei Guan
  • Jianrong Jiang
  • Deshun Shi
  • Fenghua Lu
Original Article
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Abstract

The binding of exogenous DNA to sperm is a key process for sperm-mediated gene transfer; however, the underlying molecular mechanisms have yet to be elucidated. In the present study, we aimed to identify the DNA binding proteins (DBPs) in rabbit sperm and to gain further understanding of the molecular mechanism of sperm and exogenous DNA interaction. Native polyacrylamide gel electrophoresis was used for separating free sperm proteins and complexes of DNA fragment/sperm proteins. A distinct band was found after Coomassie blue staining, and seven potential proteins were identified by mass spectrometry analysis. An analysis of the physical/chemical properties of the seven proteins revealed that the sperm inner acrosomal membrane protein IAM38 (IAM38) matched the features of the DBPs. Western blotting analysis showed that the IAM38 and CD4 were present in the sperm but not in the seminal plasma. Blocking of the IAM38 impaired the DNA-binding capacity of the sperm. Blocking the CD4 decreased the DNA-uptake capacity of the sperm but did not influence the DNA-binding capacity of the sperm. Moreover, the EGFP-positive embryos and EGFP-positive blastocysts were also decreased after IAM38 blocking or CD4 blocking in comparison with the control group. In conclusion, our results imply that foreign DNA first binds to the transmembrane IAM38 of the sperm plasma membrane and then forms the complex of DNA/IAM38/CD4 with CD4 to complete the transportation of exogenous DNA into the nucleus of sperm.

Keywords

Gene transfer DNA binding proteins Rabbit sperm IAM38 CD4 

Notes

Acknowledgements

This research was supported by China Natural Science Foundation (No. 31460282), Guangxi Science Foundation (No. 2017GXNSFBA198189), the Improving the Basic Ability of Young and Middle-aged University Teachers in Guangxi (No. 2017KY0477) and Guangxi Bagui Scholar Program.

Funding

This research was supported by China Natural Science Foundation (No. 31460282), Guangxi Science Foundation (No. 2017GXNSFBA198189), the Improving the Basic Ability of Young and Middle-aged University Teachers in Guangxi (No. 2017KY0477) and Guangxi Bagui Scholar Program.

Compliance with ethical standards

Conflict of interest

The authors have declared that no competing interests exist.

Supplementary material

11033_2018_4466_MOESM1_ESM.docx (216 kb)
Supplementary material 1 (DOCX 215 KB)

References

  1. 1.
    Zani M, Lavitrano M, French D, Lulli V, Maione B, Sperandio S, Spadafora C (1995) The mechanism of binding of exogenous DNA to sperm cells: factors controlling the DNA uptake. Exp Cell Res 217:57–64CrossRefGoogle Scholar
  2. 2.
    Shamila Y, Mathavan S (2000) Sperm/DNA interaction: DNA binding proteins in sperm cell of silkworm Bombyx mori. Mol Reprod Dev 56:289–291CrossRefGoogle Scholar
  3. 3.
    Spadafora C (2008) Sperm-mediated ‘reverse’ gene transfer: a role of reverse transcriptase in the generation of new genetic information. Hum Reprod 23:735–740CrossRefGoogle Scholar
  4. 4.
    Lavitrano M, French D, Zani M, Frati L, Spadafora C (1992) The interaction between exogenous DNA and sperm cells. Mol Reprod Dev 31:161–169CrossRefGoogle Scholar
  5. 5.
    Burkin HR, Miller DJ (2000) Zona pellucida protein binding ability of porcine sperm during epididymal maturation and the acrosome reaction. Dev Biol 222:99–109CrossRefGoogle Scholar
  6. 6.
    Green GR, Poccia DL (1988) Interaction of sperm histone variants and linker DNA during spermiogenesis in the sea urchin. Biochemistry 27:619–625CrossRefGoogle Scholar
  7. 7.
    Lavitrano M, Maione B, Forte E, Francolini M, Sperandio S, Testi R, Spadafora C (1997) The interaction of sperm cells with exogenous DNA: a role of CD4 and major histocompatibility complex class II molecules. Exp Cell Res 233:56–62CrossRefGoogle Scholar
  8. 8.
    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 38:1621–1628CrossRefGoogle Scholar
  9. 9.
    DeRouchey J, Parsegian VA, Rau DC (2010) Cation charge dependence of the forces driving DNA assembly. Biophys J 99:2608–2615CrossRefGoogle Scholar
  10. 10.
    DeRouchey JE, Rau DC (2011) Role of amino acid insertions on intermolecular forces between arginine peptide condensed DNA helices: implications for protamine-DNA packaging in sperm. J Biol Chem 286:41985–41992CrossRefGoogle Scholar
  11. 11.
    Parrish JJ (2014) Bovine in vitro fertilization: in vitro oocyte maturation and sperm capacitation with heparin. Theriogenology 81:67–73CrossRefGoogle Scholar
  12. 12.
    Bredderman PJ, Foote RH, Yassen AM (1964) An improved artificial vagina for collecting rabbit semen. J Reprod Fertil 7:401–403CrossRefGoogle Scholar
  13. 13.
    Coward K, Kubota H, Parrington J (2007) In vivo gene transfer into testis and sperm: developments and future application. Arch Androl 53:187–197CrossRefGoogle Scholar
  14. 14.
    Tsai C, Smider V, Hwang BJ, Chu G (2012) Electrophoretic mobility shift assays for protein-DNA complexes involved in DNA repair. Methods Mol Biol 920:53–78CrossRefGoogle Scholar
  15. 15.
    Laniel MA, Beliveau A, Guerin SL (2001) Electrophoretic mobility shift assays for the analysis of DNA-protein interactions. Methods Mol Biol 148:13–30PubMedGoogle Scholar
  16. 16.
    Yu Y, Vanhorne J, Oko R (2009) The origin and assembly of a zona pellucida binding protein, IAM38, during spermiogenesis. Microsc Res Tech 72:558–565CrossRefGoogle Scholar
  17. 17.
    Yu Y, Xu W, Yi YJ, Sutovsky P, Oko R (2006) The extracellular protein coat of the inner acrosomal membrane is involved in zona pellucida binding and penetration during fertilization: characterization of its most prominent polypeptide (IAM38). Dev Biol 290:32–43CrossRefGoogle Scholar
  18. 18.
    Lin YN, Roy A, Yan W, Burns KH, Matzuk MM (2007) Loss of zona pellucida binding proteins in the acrosomal matrix disrupts acrosome biogenesis and sperm morphogenesis. Mol Cell Biol 27:6794–6805CrossRefGoogle Scholar
  19. 19.
    Broux B, Markovic-Plese S, Stinissen P, Hellings N (2012) Pathogenic features of CD4+ CD28 T cells in immune disorders. Trends Mol Med 18:446–453CrossRefGoogle Scholar
  20. 20.
    Eldor J (1995) The possible roles of sperm and sperm antibodies in the pathogenesis and treatment of AIDS. Med Hypotheses 44:155–158CrossRefGoogle Scholar
  21. 21.
    Guo MW, Watanabe T, Mori E, Mori T (1995) Molecular structure and function of CD4 on murine egg plasma membrane. Zygote 3:65–73CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology, State Key Laboratory for Conservation and Utilization of Subtropical Agro-BioresourcesGuangxi UniversityNanningChina
  2. 2.Department of Reproductive Medical CenterThe Affiliated Hospital of Guilin Medical UniversityGuilinChina
  3. 3.School of Public Health of Guilin Medical UniversityGuilinChina

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