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  • Title: Nuclear transfer in sheep embryos: the effect of cell-cycle coordination between nucleus and cytoplasm and the use of in vitro matured oocytes.
    Author: Liu L, Dai Y, Moor RM.
    Journal: Mol Reprod Dev; 1997 Jul; 47(3):255-64. PubMed ID: 9170105.
    Abstract:
    The developmental ability of nuclear transplant sheep embryos derived from in vitro matured oocytes was studied by controlling cell-cycle coordination of donor embryonic nuclei and recipient cytoplasts. Oocytes were recovered from nonatretic antral follicles of adult sheep ovaries and cocultured with follicle shells in M199-based medium supplemented with gonadotrophins in a nonstatic system. Effective activation if IVM oocytes was obtained by applying two pulses of 1.0 kv/cm 22 min apart in inositol-based electroporation medium to oocytes matured in vitro for 27 hr. Synthesis of DNA (S-phase) was assessed by BrdU incorporation and was found to initiate around 5 hpa (hours postactivation) and to persist until 18 hpa. Mitotic blastomeres were induced by treating embryos with 6.6 microM nocodazole for 14-17 hr. Three types of transfers were compared directly: "S-->S," early embryonic nuclei (mostly in S-phase) were transferred to presumptive S-phase cytoplasts; "M-->MII," nocodazole-treated embryonic nuclei (most in M-phase) were transferred to MII-phase cytoplasts; and control (S-->MII), conventional nuclear transfer of fusion and activation simultaneously. The results showed that fusion and recovery rates did not differ among the three groups. However, after 6 days of in vivo culture, the morula and blastocyst formation rate was significantly higher for the M-->MII combination than for the control (28.3% vs. 8.1%, P < 0.05), while no significant differences in developmental rate were observed between S-->S and M-->MII, and between S-->S and control, though developmental rate was also increased for S-->S compared to control (20.9% vs. 8.1%, P > 0.05). Transfer of blastocysts derived from M-->MII or S-->S nuclear cytoplasmic reconstitution to synchronized recipient ewes resulted in the birth of lambs. These data suggest that in vitro matured oocytes can support full-term development of nuclear transplant sheep embryos when the cell cycle of nucleus and cytoplasm is coordinated, and that M-->MII nuclear transfer might be an efficient and simple way to improve the developmental competence of the reconstituted embryos.
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