202 INNER CELL MASS EXCHANGE IN SHEEP EMBRYOS

Abstract

The presence of a developmental axis in the mammalian oocyte/embryo is still a controversial issue (Plusa 2005 Nature 17, 391–395). However, pre-established or not, mammalian blastocysts display a clear asymmetry with distinct embryonic and abembryonic poles. The present emphasis on ‘mosaic’ development in mammalian embryos is in contrast with classical embryological work, aimed at cell lineage analysis, where manipulation procedures severely perturbed the natural blastocyst asymmetry (Gardner 2001 RBM Online 4, 46–51). However, all of the experimental work thus far has been carried out on mouse embryos. In our work, we designed experiments to determine whether sheep embryos subjected to inner cell mass (ICM) transfer retain normal developmental competence. In vitro-derived sheep blastocysts (Ptak et al. 2003 Biol. Reprod. 69, 278–285) were manipulated with a Narishige micromanipulator fitted to a inverted Nikon microscope. ICMs were dissected with a blade, and the trophoblastic vesicle and ICMs were cultured in SOFaa plus 10% FCS. After re-expansion, trophoblastic vesicles were injected with ICMs by means of a bevelled pipette and cultured overnight with SOFaa plus 10% FCS. From a total of 35 blastocysts used, 25 re-expanded following injection, and 20 of them showed ICMs adherent to the trophoblast. Seven blastocysts were transferred into synchronized ewes 7 days after estrus, and monitored every month with an Aloka linear probe (7–5 MHz; Aloka Co., Ltd., Tokyo, Japan). Twenty-one in vitro-produced (IVP) embryos were transferred as a control. Three ewes receiving ICM-exchanged blastocysts were pregnant at the first scanning, and all delivered normal offspring (two female and one male lamb; weight: 3.54 ± 0.358 kg). These data demonstrate that dramatic alteration of the blastocyst structure does not compromise its developmental potential. Our efficiency in terms of offspring is lower compared with control IVP embryos, and also compared to data obtained in mice (Papaioannou 1982 J. Embryol. Exp. Morph. 68, 199–209), but technical improvements are expected to reduce such a gap. In conclusion, we demonstrated the feasibility of ICM/trophoblastic exchange in sheep blastocysts; these results might have important application for technologies like somatic cell nuclear transfer (SCNT). Common features of SCNT clones are placental abnormalities in early (DeSousa et al. 2001 Biol. Reprod. 65, 23–30) and late pregnancies (Loi et al. 2006 Theriogenology 65, 1110–1121). The transfer of ICM from cloned embryos to normal trophoblastic vesicles, although ineffective in cattle (Murakami et al. 2006 Cloning Stem Cells 8, 51–69), might be worth trying on sheep, a species where post-natal mortality in clones is a serious issue.

Table 1.  Development to term of manipulated and cloned embryos

Part of this work was supported by EUROSTELLS-European Science Foundation.