Journal of Assisted Reproduction and Genetics

, Volume 14, Issue 2, pp 120–124 | Cite as

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

  • Milagros Cabrera
  • Philip J. Chan
  • Theresa H. Kalugdan
  • Alan King


Purpose: The purpose of this study was to determine whether exogenous DNA internalized into blastocysts after transference from DNA-carrier sperm are localized at the inner cell mass or trophoblast cells and to identify differences in uptake of exogenous DNA fragments by sperm due to unique DNA sequences.

Methods: Mouse blastocysts at the hatching stage were exposed to migrating human sperm cells carrying exogenous DNA fragments synthesized from the E6–E7 conserved gene regions of human papillomavirus (HPV) types 16 and 18. After an interaction period of 2 hr, the transfected blastocysts were washed several times to remove extraneous sperm and the blastocysts were dissected into groups of cells derived from the inner cell mass and trophoblasts. The cells were analyzed by polymerase chain reaction (PCR) for the presence of HPV DNA fragments. In the second part of the experiment, thawed donor (N=10) sperm cells were pooled, washed, and divided into two fractions. The first (control) fraction was added with formalin and further divided and added with a35S-radiolabeled G, A, T, or C sequencing mixture. The second fraction was similarly treated but the formalin step was omitted from the treatment. After an hour of incubation at 37°C, the sperm specimens were washed several times by centrifugation and DNA extracted by the GeneReleaser method. The extracted DNA were processed on sequence gels, and the autoradiographs analyzed.

Results: Mouse blastocysts transfected by carrier sperm with DNA from HPV types 16 and 18 showed localization of the HPV DNA to both the inner cell mass and trophoblast cells. Negative controls consisting of untreated human sperm and untreated mouse blastocysts did not reveal any evidence of HPV DNA. The positive sperm control generated expected DNA fragments from HPV types 16 and 18. In the second experiment, the intensities of the DNA fragments in the G, A, T, and C columns from low to high molecular weights were not different from the positive control bands. Band intensities of the four sequencing columns were similar. Formalin pretreatment of the sperm inhibited uptake of the DNA fragments from the smallest to the largest DNA molecules.

Conclusions: Exogenous DNA taken into blastocysts are localized to both the inner cell mass and trophoblast cells. Only live sperm exhibited the capacity to carry various sizes of exogenous DNA, suggesting the involvement of active cell membrane mechanism in the transference process. The results showed that DNA fragments terminating in any of the four nucleotides were equally taken up by the sperm cell. Fragments of DNA produced by the sequencing reaction failed to identify a unique DNA sequence that would facilitate or inhibit the sperm from taking up exogenous DNA.

Key words

spermatozoa inner cell mass blastocysts human papillomavirus sequencing reactions 


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  1. 1.
    Brackett BG, Baranska W, Sawicki W, Koprowsky H: Uptake of heterologous genome by mammalian spermatozoa and its transfer to ova through fertilization. Proc Natl Acad Sci USA 1971;68:353–357PubMedCrossRefGoogle Scholar
  2. 2.
    Lavitrano M, Camaioni A, Fazio VM, Dolci S, Farace MG, Spadafora C: Sperm cells as vectors for introducing foreign DNA into eggs: Genetic transformation of mice. Cell 1989;57:717–723PubMedCrossRefGoogle Scholar
  3. 3.
    Gagne MB, Pothier F, Sirard M-A: Electrophoresis of bovine spermatozoa to carry foreign DNA in oocytes. Mol Reprod Dev 1991;29:6–15PubMedCrossRefGoogle Scholar
  4. 4.
    Camaioni A, Russo MA, Odorisio T, Gandolfi F, Fazio, VM, Siracusa G: Uptake of exogenous DNA by mammalian spermatozoa: Specific localization of DNA on sperm heads. J Reprod Fert 1992;96:203–212CrossRefGoogle Scholar
  5. 5.
    Francolini M, Lavitrano M, Lamia CL, French D, Frati L, Cotelli F, Spadafora C: Evidence for nuclear internalization of exogenous DNA into mammalian sperm cells. Mol Reprod Dev 1993;34:139–149CrossRefGoogle Scholar
  6. 6.
    Chan PJ, Kalugdan T, Su BC, Whitney EA, Perrott W, Tredway DR, King A: Sperm as a noninvasive gene delivery system for preimplantation embryos. Fertil Steril 1995;63:1121–1124PubMedGoogle Scholar
  7. 7.
    Chan PJ, Seraj IM, Kalugdan TH, King A: Blastocysts exhibit preferential uptake of DNA fragments from the E6-E7 conserved region of the human papillomavirus. Gynecol Oncol 1995;58:194–197PubMedCrossRefGoogle Scholar
  8. 8.
    Sarkar FH, Crissman JD: Detection of human papillomavirus DNA sequences by polymerase chain reaction. Biotechniques 1990;9:180–185PubMedGoogle Scholar
  9. 9.
    Barbosa M, Schlegel R: The E6 and E7 genes of HPV 18 are sufficient for inducing two stage in vitro transformation of human keratinocytes. Oncogene 1989;4:1529–1532PubMedGoogle Scholar
  10. 10.
    Ting Y, Manos, MM: Detection and typing of genital human papillomavirus.In PCR Protocols: A Guide to Methods and Applications, MA Innis, DH Gelfnad, JJ Sninsky, TJ White (eds). San Diego, Academic Press, 1990, pp. 356–367Google Scholar
  11. 11.
    Lauria A, Gandolfi F: Recent advances in sperm cell mediated gene transfer. Mol Reprod Dev 1993;36:255–257PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Publishing Corporation 1997

Authors and Affiliations

  • Milagros Cabrera
    • 1
  • Philip J. Chan
    • 1
    • 2
  • Theresa H. Kalugdan
    • 1
  • Alan King
    • 1
  1. 1.Department of Gynecology and ObstetricsLoma Linda University School of MedicineLoma Linda
  2. 2.Department of Physiology and PharmacologyLoma Linda University School of MedicineLoma Linda

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