Cheetah interspecific SCNT followed by embryo aggregation improves in vitro development but not pluripotent gene expression

Abstract

The aim of this study was to evaluate the capacity of domestic cat (Dc, Felis silvestris) oocytes to reprogram the nucleus of cheetah (Ch, Acinonyx jubatus) cells by interspecies SCNT (iSCNT), by using embryo aggregation. Dc oocytes were in vitro matured and subjected to zona pellucida free (ZP-free) SCNT or iSCNT, depending on whether the nucleus donor cell was of Dc or Ch respectively. ZP-free reconstructed embryos were then cultured in microwells individually (Dc1X and Ch1X groups) or in couples (Dc2X and Ch2X groups). Embryo aggregation improved in vitro development obtaining 27.4, 47.7, 16.7 and 28.3% of blastocyst rates in the Dc1X, Dc2X, Ch1X and Ch2X groups, respectively (P!0.05). Moreover, aggregation improved the morphological quality of blastocysts from the Dc2X over the Dc1X group. Gene expression analysis revealed that Ch1X and Ch2X blastocysts had significantly lower relative expression of OCT4, CDX2 and NANOG than the Dc1X, Dc2X and IVF control groups. The OCT4, NANOG, SOX2 and CDX2 genes were overexpressed in Dc1X blastocysts, but the relative expression of these four genes decreased in the Dc2X, reaching similar relative levels to those of Dc IVF blastocysts. In conclusion, Ch blastocysts were produced using Dc oocytes, but with lower relative expression of pluripotent and trophoblastic genes, indicating that nuclear reprogramming could be still incomplete. Despite this, embryo aggregation improved the development of Ch and Dc embryos, and normalized Dc gene expression, which suggests that this strategy could improve full-term developmental efficiency of cat and feline iSCNT embryos.Reproduction (2015) 150 1-10

Applied Biotechnologies in the Conservation of Wild Felids: In Vitro Embryo Production and Cellular Regenerative Therapies

“…Moreover, it has been described that cheetah embryos generated by iSCNT have an aberrant expression of OCT4, SOX2, NANOG and CDX2 compared to domestic cat embryos generated by SCNT and IVF. This was in accordance with a reduced developmental competence of the cheetah cloned embryos, which might be due to an inadequate reprogramming of the cheetah donor cell by the cat oocyte [63].…”

“…On the other hand, several methods have been implemented to improve the low efficiency of the SCNT such as embryo aggregation. This consists of the in vitro culture of two or more zona-free embryos together in the well of the well system (WOW) [63]. It was reported that the aggregation of cloned cheetah embryos and cloned tiger embryos generated by iSCNT improves the in vitro development in these species.…”

“…It was reported that the aggregation of cloned cheetah embryos and cloned tiger embryos generated by iSCNT improves the in vitro development in these species. However, the developmental capacity of cheetah and tiger cloned embryos was reduced compared to domestic cat cloned embryos [63,64] ( Table 4 and Figure 1).…”

Efeito da técnica de coleta e do estágio do ciclo estral na recuperação de oócitos de boa qualidade em felinos e caninos domésticos

“…Por outro lado, os felinos domésticos ainda são descendentes próximos de outros felinos selvagens (Driscoll et al, 2007). Isto torna possível a utilização do gato doméstico como modelo experimental para os felídeos selvagens, que estão em sua maior parte ameaçados de extinção (Moro et al, 2015). Existem diversas biotécnicas reprodutivas que podem ser aplicadas para a preservação de espécies ameaçadas (Uchikura et al, 2010) e para a melhoria do desempenho reprodutivo (Luvoni et al, 2005).…”

Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality

“…Recently, our laboratory has demonstrated the advantage of zona free cloned embryo aggregation in the equine and the feline [ 8 – 10 ]. Embryo aggregation consists of placing more than one zona free embryos in the same well; some of the reported benefits in mammals are: improvement in embryo development in pig [ 6 ], equine [ 8 , 9 ], feline [ 10 ] and mouse [ 11 ], increasing the density of cells in the inner cell mass on cattle [ 12 ], blastocyst diameter in equine and in bovine [ 8 , 13 ], reduction in apoptosis in bovine and pigs [ 13 , 14 , 15 ], normalization of gene expression in bovine [ 15 ]and in some cases, it has also improved in vivo embryo development and early pregnancy rates in mouse [ 11 ], equine [ 8 ] and in cattle [ 13 , 16 , 17 ]. As a single clone may have epigenetic defects [ 15 ], embryo aggregation could compensate the reprogramming deficiencies through the interactions between blastomeres [ 11 ] or by paracrine pathways [ 11 ].…”