TRANSFECTION OF THE INNER CELL MASS AND LACK OF A UNIQUE DNA-SEQUENCEAFFECTING THE UPTAKE OF EXOGENOUS DNA BY SPERM AS SHOWN BY DIDEOXY SEQUENCING ANALOGS

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 du e to unique DNA sequences. Methods: Mouse blastocysts at the hatching stage were exposed to migrating human sperm cells carrying exogenous D NA fragments synthesized from the E6-E7 conserved gene regions of huma n papillomavirus (HPV) types 16 and 18. After an interaction period of 2 hs the transfected blastocysts were washed several times to remove extraneous sperm and the blastocysts were dissected into groups of cel ls derived from the inner cell mass and trophoblasts. The cells were a nalyzed 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 a nd added with a S-35-radiolabeled G, A, Z or C sequencing mixture. The second fraction was similarly treated but the formalin step was omitt ed from the treatment After an hour of incubation at 37 degrees C, the sperm specimens were washed several rimes by centrifugation and DNA e xtracted by the GeneReleaser method. The extracted DNA were processed on sequence gels, and the autoradiographs analyzed. Results: Mouse bla stocysts transfected by carrier sperm with DNA from HPV types 16 and 1 8 showed localization of the HPV DNA to both the inner cell mass and t rophoblast cells. Negative controls consisting of untreated human sper m and untreated mouse blastocysts did not reveal any evidence of HPV D NA. The positive sperm control generated expected DNA fragments from H PV types 16 and 18. In the second experiment, the intensities of the D NA fragments in the G, A, T, and C columns from low to high molecular weights were not different from the positive control bands. Band inten sities of the four sequencing columns were similar: Formalin pretreatm ent of the sperm inhibited uptake of the DNA fragments from the smalle st 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 m echanism in the transference process. The results showed that DNA frag ments terminating in any of the four nucleotides were equally taken up by the sperm cell. Fragments of DNA produced by the sequencing reacti on failed to identify a unique DNA sequence that would facilitate or i nhibit the sperm from taking Icp exogenous DNA.