CYTOPLASMIC TRANSFER IN COMBINATION WITH INTERSPECIES NUCLEAR TRANSFER: PRESENCE OF FOREIGN MITOCHONDRIA, PATTERN OF DISTRIBUTION AND EFFECT ON IN VITRO EMBRYONIC DEVELOPMENT

M.J. SANSINENA, J. LYNN, R.S. DENNISTON AND R.A. GODKE

ZYGOTE

CAMBRIDGE UNIV PRESS

Año: 2010 vol. 25 p. 1 - 10

0967-1994

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 mitochondrial mismatch between donor cell and recipient oocyte could cause the embryonic developmental arrest. A series of experiments were 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 analyzed by MitoTracker staining. In addition, the effect of transferring caprine ooplasm (iOT) into the bovine enucleated oocytes used in iSCNT was analyzed. 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 introduction of foreign mitochondria. The distribution pattern of transferred mitochondria most commonly remained in a distinct cluster after 12, 74 and 144 hours 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.