Nuclear remodelling 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, 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.
Liu
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Department of Development and Signalling, Babraham Institute, Cambridge, United Kingdom.
Moor
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RM
Laurie
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Notarianni
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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
D007294:Inositol