NUCLEAR REMODELING AND EARLY DEVELOPMENT IN CRYOPRESERVED, PORCINE PRIMORDIAL GERM-CELLS FOLLOWING NUCLEAR TRANSFER INTO IN VITRO-MATURED OOCYTES

Nuclear transfer was conducted in the pig using, as karyoplasts, primo rdial germ cells which had been cryopreserved. The cytoplasts were pre sumptive S- or Mil-phase, in vitro-matured oocytes, which had been enu cleated mechanically. Enucleation was effective in 94.3% of cases. Kar yoplasts were introduced into the perivitelline space, in close contac t with the cytoplasts, and the complexes fused by electrical stimulati on and activation. Activation was successful in 82-88% of nonmanipulat ed, pulsed oocytes, and in 55% of germ cell-oocyte complexes. The reco nstituted embryos were examined for nuclear remodelling and cleavage i n vitro. Nuclear swelling was more prominent when Mil-phase cytoplasts , rather than S-phase, cytoplasts, were used. After 24 h in culture, t he cleavage rate was not significantly different whether blastomeres o r primordial germ cells were used as karyoplasts, and whether Mil-phas e or S-phase cytoplasts were used. However, after 72 h in culture, the developmental rate was higher when MB-phase cytoplasts (75%) were use d for the recipients of blastomeres compared with S-phase cytoplasts ( 38.5%, p<0.05). Similar tendencies were observed with germ-cell nuclea r transfer when inositol was used as medium for electrofusion (60% vs 27.8%, p<0.05). Furthermore, when Mil-phase cytoplasts were used, the nuclear transferred embryos derived from blastomeres developed at a si gnificantly higher rate than from primordial germ cells (37.5%, p<0.05 ). We conclude that cryopreserved primordial germ cells are competent to undergo nuclear remodelling and cleavage during 72 h of incubation in vitro to the 4-cell stage, following nuclear transfer to enucleated , activated (S-) or MII-phase oocytes. This experimental system may he lp to elucidate events in the early development of pig embryos followi ng nuclear transfer using germ cells as karyoplasts.