Reproduction in Domestic Animals

410 B ObackReprogramming Ability of the Recipient CellWhile there are over 200 donor cell types to choosefrom, choices for the recipient cell for NT are muchmore limited. The most commonly used type is harvestedas non-activated MI oocytes from follicles ofslaughtered adult animals. The maturation into MIIoocytes can occur in vivo or in vitro and both types havebeen successfully used for cloning. In mice, naturallyovulated oocytes improve cloning efficiency comparedwith superovulated ones (Hiiragi and Solter 2005);however, using them in mono-ovulatory species, suchas cattle, is impractical. In contrast to the donor, geneticfactors should be less important for choosing therecipient cell because its nuclear genome is removedprior to NT. However, genotype affects transcriptomeand proteome of the oocyte. The fact that hybrid vigouris beneficial for recipient cells (Gao et al. 2004) and thatearly transcriptional activation varies in clones reconstructedwith oocytes from different mouse strainsemphasizes the importance of the recipient cell’s genotypeduring cloning (Vassena et al. 2008). Furthercomparative studies are needed to fully evaluate theinfluence of oocyte source and maturation method onlivestock cloning efficiency. Cytoplasts prepared fromartificially activated MII oocytes (early telophase or TII)have also sometimes been used to clone goats (Baguisiet al. 1999) and cattle (Bordignon and Smith 1998;Kurosaka et al. 2002; Bordignon et al. 2003). Presently,there is no evidence that MII and TII cytoplasm differ intheir reprogramming ability.In early mammalian cloning experiments, fertilizedoocytes (zygotes) were successfully used as recipientswith two-cell stage donor cells in mouse (McGrath andSolter 1984; Robl et al. 1986), cattle (Prather and First1990) and pig (Prather et al. 1989). Once more advancedembryonic or even somatic donors were used, enucleatedzygotes repeatedly failed to support early developmentof the reconstituted NT embryo (Wakayama et al.2000). This 20-year-old perception that SCNT successcritically depended on the use of unfertilized oocyteschanged radically when the first cloned cattle (Schurmannet al. 2006) and mice (Greda et al. 2006; Egli et al.2007) were born from NT with somatic and embryonicdonors, respectively, into zygotes.Recipient zygotes can be either in interphase or inmitosis. Interphase zygotes were first reproducibly usedfor cloning mice in 2006 (Greda et al. 2006), perhapspartially confirming much earlier attempts (Illmenseeand Hoppe 1981). They succeeded by developing a newmicromanipulation technique for enucleation. The classicalway of enucleating (McGrath and Solter 1983),removes intact pronuclei including all nuclear componentsand some surrounding cytoplasm. In contrast, thenew technique appears to more selectively remove onlythe pronuclear envelope and its attached chromatin,while leaving other pronuclear components, such asnucleoli, behind. These zygotic cytoplasts supportedfull-term development after NT of eight-cell donor cells.It would be informative to determine which factorsremain in the zygotic cytoplasm after selective enucleationand if these factors would be sufficient to alsoreprogram somatic donor nuclei. However, the latterexperiment is technically challenging because of the cellcycle incompatibility between somatic donor andrecipient cell. Donor–recipient cell cycle coordinationis important to maintain normal ploidy and promotereprogramming, both of which are linked to the level ofcyclin B ⁄ Cdk 1 complex activity (M-Cdk, formerlymaturation-promoting factor) in the recipient (Obackand Wells 2002). Interphase zygotes contain lowM-Cdk-activity, thus an introduced donor nucleus ofany cell cycle stage no longer undergoes efficientchromatin remodelling, completely abolishing developmentpast the eight-cell stage (Tani et al. 2001). In orderto circumvent this problem for SCNT, zygotes have tobe at a stage when they contain enough M-Cdk-activityto disassemble the donor nuclear envelope and allowearly embryo development to proceed. This wasachieved by using zygotes which are either just exiting(i.e. early telophase) or entering mitosis (i.e. metaphase),and thus contain enough residual or newly synthesizedM-Cdk-activity, respectively. With early bovine zygotes,enucleated just 4 h after insemination, post-implantationdevelopment of SCNT embryos was improvedcompared with using chemically activated MII cytoplasts(Schurmann et al. 2006). Pregnancy establishmentwas significantly higher and this initial difference translatedinto about twofold higher development into calvesat weaning (Schurmann et al. 2006). There are severalreasons why sperm-activated oocytes might have beenbeneficial. Firstly, sperm is the natural agent of activation,inducing long-lasting Ca 2+ -oscillations that moreclosely resemble natural fertilization than the singlelarge Ca 2+ -rise triggered by artificial activation (Fissoreet al. 1992). Secondly, sperm delivers factors to the eggthat may improve early embryonic development (Krawetz2005). These include: (i) the centrosome, which isusually removed together with its associated componentsduring enucleation; (ii) some 3000 differentmRNAs and (iii) micro-RNAs, which do not code forproteins but play a role in controlling gene activity.Thirdly, there may be early sperm-derived transcriptsarising in the time interval between sperm–egg fusionand enucleation. Although there is no direct evidence forsuch early paternal transcription, it has been shown thatsome embryonic genome activation occurs shortly afterfertilization in cattle (Memili et al. 1998). The nature ofthese early transcripts and whether they come from thematernal and ⁄ or paternal genome is not known. Allthese molecules may participate in chromatin remodelling,pronuclear formation, establishment of imprints orembryonic genome activation (Krawetz 2005). Theincreased reprogramming ability of early zygotes clearlywarrants a more detailed analysis into their RNA andprotein composition.Soon after the cattle study, the birth of cloned miceafter NT into mitotically arrested late zygotes confirmedtheir suitability as NT recipients (Egli et al. 2007). Usingmetaphase-arrested zygotes before their first cleavage,cloned offspring were obtained after NT with mitoticallyarrested zygotes, 2-, 8- and ES-cells, but not somaticdonors. Because a direct comparison between metaphase-arrestedoocyte and zygote recipients, using donorcells of the same type and cell cycle stage, has not yetÓ 2008 The Author. Journal compilation Ó 2008 Blackwell Verlag