title: Nuclear remodelling and early development in cryopreserved, porcine primordial germ cells following nuclear transfer into in vitro-matured oocytes.
abstract: Nuclear transfer was conducted in the pig using, as karyoplasts, primordial germ cells which had been cryopreserved. The cytoplasts were presumptive S- or MII-phase, in vitro-matured oocytes, which had been enucleated mechanically. Enucleation was effective in 94.3% of cases. Karyoplasts were introduced into the perivitelline space, in close contact with the cytoplasts, and the complexes fused by electrical stimulation and activation. Activation was successful in 82-88% of nonmanipulated, pulsed oocytes, and in 55% of germ cell-oocyte complexes. The reconstituted embryos were examined for nuclear remodelling and cleavage in vitro. Nuclear swelling was more prominent when MII-phase cytoplasts, rather than S-phase, cytoplasts, were used. After 24 h in culture, the cleavage rate was not significantly different whether blastomeres or primordial germ cells were used as karyoplasts, and whether MII-phase or S-phase cytoplasts were used. However, after 72 h in culture, the developmental rate was higher when MII-phase cytoplasts (75%) were used for the recipients of blastomeres compared with S-phase cytoplasts (38.5%, p < 0.05). Similar tendencies were observed with germ-cell nuclear transfer when inositol was used as medium for electrofusion (60% vs 27.8%, p < 0.05). Furthermore, when MII-phase cytoplasts were used, the nuclear transferred embryos derived from blastomeres developed at a significantly 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 or incubation in vitro to the 4-cell stage, following nuclear transfer to enucleated, activated (S-) or MII-phase oocytes. This experimental system may help to elucidate events in the early development of pig embryo following nuclear transfer using germ cells as karyoplasts.
authors.affiliation: Department of Development and Signalling, Babraham Institute, Cambridge, United Kingdom.
authors.forename: L
authors.initials: L
authors.lastname: Liu
authors.forename: R M
authors.initials: RM
authors.lastname: Moor
authors.forename: S
authors.initials: S
authors.lastname: Laurie
authors.forename: E
authors.initials: E
authors.lastname: Notarianni
mesh_terms: D000818:Animals; D001757:Blastomeres / Q000166:cytology / Q000502:physiology*; D002453:Cell Cycle; D002467:Cell Nucleus / Q000502:physiology*; D015925:Cryopreservation; D005314:Embryonic and Fetal Development; D005260:Female; D005854:Germ Cells / Q000166:cytology / Q000502:physiology*; D007294:Inositol; D008297:Male; D008561:Membrane Fusion; D008938:Mitosis; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology / Q000502:physiology*; D010053:Ovary / Q000166:cytology; D011247:Pregnancy; D016196:S Phase; D013552:Swine
chemical_list: D007294:Inositol