Reproduction in Domestic Animals

Epigenetics of Foetal Development in Clones 305genes in the placentas of dead clones rather than thematernal-specific expression that is expected if placentalXCI is normal. Interestingly, the placentas ofsurviving clones, unlike those of dead clones, had onlyone active X chromosome, suggesting that aberrantpatterns of XCI might have contributed to foetal death(Xue et al. 2002). Aberrations in X-linked gene expressioncould have severe consequences for placentaldevelopment (Hemberger 2002).Trophectoderm and placental defects in SCNT embryosRecent molecular evidence supports the hypothesis thatthe placental lineage is especially vulnerable to problemsarising from reprogramming of the somatic nucleus afterNT. Anomalies in the trophectoderm of cloned embryoshave been described as early as the blastocyst stage.SCNT bovine blastocysts have been found to have alower ratio of trophectoderm-to-ICM cells comparedwith fertilized ones (Koo et al. 2002). Furthermore, TP-1 (also known as IFN-tau), the gene specificallyexpressed by the trophoblast for maternal recognitionof pregnancy, was abnormally expressed in SCNTbovine blastocysts (Wrenzycki et al. 2001). In theplacentas or placentomes of SCNT bovine foetusesaberrant gene expression has been detected as early asday 25 and throughout gestation to term (Hashizumeet al. 2002; Hill et al. 2002). Furthermore, 60 proteinswere differentially expressed in term placentas of clonedcalves compared with fertilized controls (Kim et al.2005). The majority of the cloned mouse embryos diebefore a functional placenta can develop (Jouneau et al.2006) showing phenotypic defects during gastrulation(E7–E8) characterized by an abnormal ratio between theembryonic and extraembryonic portions of the embryo.The abnormal phenotypes were not fully rescued withthe addition of ESCs or ICM cells from normal embryosbut were rescued by the addition of tetraploid cells.Gene expression in cloned mice derived from nuclei ofdifferent cell types shown some cell type-specific effects,but most of the abnormalities were independent ofdonor cell type and seemed to be a consequence of theNT procedure, with at least 4% of genes expressed in theplacenta showing dysregulation (Humpherys et al.2002).Strategies to Increase the Pregnancy Rates andReduce Foetal Abnormalities After SCNTFrom a practical point of view SCNT pregnanciespresent different level of problems: (i) negligible reprogrammingerrors, without any obvious physiologicalconsequences; (ii) small errors, where favourable environmentalconditions or veterinary care can compensatefor negative effects; (iii) serious reprogramming errorsresulting in placental or foetal failures and loss ofpregnancy.Currently there is a lack of diagnostic methods topredict the expected level of problems in individualembryos and for their pre-selection prior to ET.However, there are several different practical strategieswhich might increase the live birth rates of SCNTpregnancies:Modification of reprogramming success of both foetal andplacental genesThis strategy includes efforts to find the best combinationof many variables during SCNT. The main effortsare focused on the core elements of the SCNT procedure:the donor cells, the recipient cytoplast, and certainmodifiers of the reprogramming.The ‘best’ donor cellFinding of cell types more amenable for reprogrammingwould improve the SCNT, although the existing data isstill controversial concerning the effect of cell type onthe reprogramming – as approximately 200 cell typescould be tested, the task is rather big.Improving donor cell culture methods and using lowpassage number of such cells could help to avoid geneticaberrations and mutations accumulated during cellculture – although long term cultured cells can be usedas well for SCNT (Kubota et al. 2000) and telomereshortening seems not to be a limiting factor in SCNT(Lanza et al. 2000). The genetic origin of the cells is animportant factor, as it was studied in mouse in details(Wakayama et al. 1999; 2001; Rideout et al. 2000;Eggan et al. 2001). However, there is almost no dataon the effect of genetic component in domestic animals.The cell cycle status of the donor cells plays animportant role, and several studies reported on thepositive effect of serum starvation induced G0 (Wilmutet al. 1997) or cell culture confluency induced G1 stages(Cibelli et al. 1998a, 1998b, 2002; Wells et al. 2003). Thecell cycle must be in harmony with the state of therecipient cytoplast (Campbell et al. 1996; 2002; Wilmutet al. 2002) (see below).The ‘best’ recipient cytoplastThe origin of the recipient oocyte can be in vivo derivedor in vitro matured. The later ones in farm animals aremuch less expensive if slaughterhouse ovaries areavailable. In mouse there is evidence, that naturallyovulated oocytes are better quality than superovulatedones (Hiiragi and Solter 2005), but in farm animals thischoice would increase the cost substantially. The geneticorigin of the recipient oocyte also plays a role, accordingto mouse data (Wakayama et al. 1999; Gao et al. 2004),but there is very limited information on this issue inother species. Genotype in that case is probably importantdue to variations in the amount of maternal-originreprogramming factors available in the cytoplast. Furthermore,individual differences among oocyte donorsprobably exist, but scientific examination of such factorsis rather difficult, due to the low number of observationspossible when the oocyte donors are not used repetitively.Usually cytoplasts for SCNT are generated byenucleation of non-activated Metaphase II stage oocytes(Wilmut et al. 1997, 2002; Cibelli et al. 2002), butTelophase II stage can be also used as recipient withsimilar efficacy (Bordignon and Smith 1998; Baguisiet al. 1999), and recently it was demonstrated, that theuse of mitotic zygotes is also a viable option (Gredaet al. 2006; Schurmann et al. 2006; Egli et al. 2007).Ó 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag