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Nucleocytoplasmic hybrid (cybrid) embryos result from the combination of the nucleus of one species, and the egg cytoplasm of another species. Cybrid embryos can be obtained either in the haploid state by the cross-fertilization or intra-cytoplasmic injection of an enucleated egg with sperm from another species, or in the diploid state by the technique of interspecies somatic cell nuclear transfer (iSCNT). Cybrids that originate from the combination of the nucleus and the cytoplasm of distantly related species commonly expire during early embryonic development, and the cause of this arrest is currently under investigation. Here we show that cells isolated from a Xenopus cybrid (Xenopus (Silurana) tropicalis haploid nucleus combined with Xenopus laevis egg cytoplasm) embryo are unable to proliferate and expand normally in vitro. We also provide evidence that the lack of nuclear donor species maternal poly(A)(+) RNA-dependent factors in the recipient species egg may contribute to the developmental dead-end of distantly-related cybrid embryos. Overall, the data are consistent with the view that the development promoted by one species' nucleus is dependent on the presence of maternally-derived, mRNA encoded, species-specific factors. These results also show that cybrid development can be improved without nuclear species mitochondria supplementation or replacement.
Figure 1. Defective in vitro expansion of cybrids cells. The concentration of control haploid [l]xl embryonic cells (A-C) in a given area of a culture dish visibly increased over time, while that of [l]xt cybrid cells (D-F) did not. Pictures in (B, E) and (C, F) were taken 5 and 13 d, respectively, after those in (A, D). Scale bar: 0.1 mm.
Figure 2. Improved cybrid embryo development by nuclear species maternal poly(A)+ RNA injection. The most developed [l]xt cybrid embryo that was obtained following injection at the 1-cell stage with (A) dH20, (B) X. laevis oocyte poly(A)+ RNA, or (C) X. tropicalis oocyte poly(A)+ RNA from one experiment are shown as an example. All three embryos had a rudimentary sucker (arrowhead), yet noticeable improvements in the development of the animal shown in (C) include a better blastopore closure (green dotted line), axis formation and elongation (red dotted line), muscular response, and an increased head size (yellow dotted line). Embryos are shown (anterior to the right; dorsal up) at 48 h post-fertilization, about 24 h before they were finally scored for inclusion in Table 2. Scale bar: 1 mm.
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