Ooplasm transfer and interspecies somatic cell nuclear transfer: heteroplasmy, pattern of mitochondrial migration and effect on embryo development.
Although interspecies somatic cell nuclear transfer (iSCNT) has potential applications in the conservation of exotic species, an in vitro developmental block has been observed in embryos produced by this approach. It has been suggested that mitochondrial mismatch between donor cell and recipient oocyte could cause embryonic developmental arrest. A series of experiments was conducted to investigate the effect of mixed mitochondrial populations (heteroplasmy) on early development of iSCNT-derived cloned embryos. The effect of combining the techniques of ooplasm transfer (OT) and somatic cell nuclear transfer (SCNT) was examined by monitoring in vitro embryonic development; the presence and pattern of migration of foreign mitochondria after OT was analysed by MitoTracker staining. In addition, the effect of transferring caprine ooplasm (iOT) into the bovine enucleated oocytes used in iSCNT was analysed. There was no significant effect of the sequence of events (OT-SCNT or SCNT-OT) on the number of fused, cleaved, blastocyst or hatched blastocyst stage embryos. MitoTracker Green staining of donor oocytes used for OT confirmed the introduction of foreign mitochondria. The distribution pattern of transferred mitochondria most commonly remained in a distinct cluster after 12, 74 and 144 h of in vitro culture. When goat ooplasm was injected into bovine enucleated oocytes (iSCNT), there was a reduction (p < 0.05) in fusion (52 vs. 82%) and subsequent cleavage rates (55 vs. 78%). The procedure of iOT prior to iSCNT had no effect in overcoming the 8- to 16-cell in vitro developmental block, and only parthenogenetic cow and goat controls reached the blastocyst (36 and 32%) and hatched blastocyst (25 and 12%) stages, respectively. This study indicates that when foreign mitochondria are introduced at the time of OT, these organelles tend to remain as distinct clusters without relocation after a few mitotic divisions. Although the bovine cytoplast appears capable of supporting mitotic divisions after iOT-iSCNT, heteroplasmy or mitochondrial incompatibilities may affect nuclear-ooplasmic events occurring at the time of genomic activation.
Sansinena
Marina J
MJ
Facultad de Ciencias Agrarias, Universidad Católica Argentina, Cap. Gral. R. Freire 183, CABA 1426, Argentina. msansinena@uca.edu.ar
Lynn
John
J
Bondioli
Kenneth R
KR
Denniston
Richard S
RS
Godke
Robert A
RA
D000818:Animals; D001755:Blastocyst / Q000166:cytology; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism; D019976:Cloning, Organism / Q000379:methods; D003593:Cytoplasm / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D047108:Embryonic Development; D005260:Female; D016678:Genome; D006041:Goats; D008928:Mitochondria / Q000378:metabolism*; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology* / Q000378:metabolism