Gene expression and in vitro development of inter‐species nuclear transfer embryos

Abstract

This study examined the chromatin morphology, in vitro development, and expression of selected genes in cloned embryos produced by transfer of mouse embryonic fibroblasts (MEF) into the bovine ooplasm. After 6 hr of activation, inter-species nuclear transfer (NT) embryos (MEF-NT) had one (70%) or two pronuclei (20%), respectively. After 72 hr of culture in vitro, 62.6% of the MEF-NTs were arrested at the 8-cell stage, 31.2% reached the 2- to 4-cell stage, and only 6.2% had more than eight blastomeres, but none of these developed to the blastocyst stage. Whereas, 20% of NT embryos derived from bovine embryonic fibroblast fused with bovine ooplasm (BEF-NT) reached the blastocyst stage. Donor MEF nuclei expressing an Enhanced Green Fluorescent Protein (EGFP) transgene resulted in 1- to 8-cell stage MEF-NT that expressed EGFP. The expression of selected genes was examined in 8-cell MEF-NTs, 8-cell mouse embryos, enucleated bovine oocytes, and MEFs using RT-PCR. The mRNA for heat shock protein 70.1 (Hsp 70.1) gene was detected in MEF-NTs and MEF, but not in mouse embryos. The hydroxy-phosphoribosyl transferase (HPRT) mRNA was found in normal mouse embryos and MEF but not in MEF-NTs. Expression of Oct-4 and embryonic alkaline phospatase (eAP) genes was only detected in normal mouse embryos and not in the inter-species NT embryos. Abnormal gene expression profiles were associated with an arrest in the development at the 8-cell stage, but MEF-NT embryos appeared to have progressed through gross chromatin remodeling, typical of intra-species NT embryos. Therefore, molecular reprogramming rather than chromatin remodeling may be a better indicator of nuclear reprogramming in inter-species NT embryos.

Development, embryonic genome activity and mitochondrial characteristics of bovine–pig inter-family nuclear transfer embryos

“…The analysis of remodelling of the donor nucleus in the cytoplasm of a recipient oocyte from a different species indicates that the normal pattern of nucleus remodelling was followed as previously reported in different studies on mammalian NT and iSCNT embryos (Tarkowski & Balakier 1980, Dominko et al 1999, Arat et al 2003, Uhm et al 2007. Thus, the biochemical mechanism of this process is universal, and it works also in intra-class NT embryos derived from chicken blastodermal cells and rabbit oocytes .…”

“…Comparison of gene expression in 8-16-cell stage human-bovine or human-rabbit embryos and human IVF or NT embryos using single-embryo transcriptome profiling revealed general downregulation of human genes in the bovine and rabbit recipient cytoplasm (Chung et al 2009). These data and the absence of Alppl2 (eAp), Pou5f1 (Oct4) and Cdh1 (e-Cad) expression in mouse embryonic fibroblast (MEF)-bovine iSCNT embryos (Arat et al 2003, Kim et al 2004 suggests that bovine ooplasm does not reprogram properly donor nuclei from other species.…”

“…Indeed, very few data are available on embryonic gene function before and at the time of EGA in iSCNT embryos (Arat et al 2003, Bjerregaard et al 2007, Chung et al 2009, Hamilton et al 2004, Kim et al 2004, Li et al 2008, Wang et al 2009, and all show abnormal genome activation and gene expression profiles in the iSCNT embryos studied. It is likely that the ineffectiveness of the iSCNT approach may be linked to abnormal (or absence of) EGA in such embryos due to aberrant nucleuscytoplasm cross-talk.…”

Reprogrammationet épigenèse

“…Chez la souris, il a été montré que le noyau transféré maintient une activité transcriptionnelle de type somatique pendant plusieurs cycles de division [45]. Des profils d'expression anormaux ont par ailleurs été corrélés au blocage précoce (au stade de l'EGA) après transfert interspéci-fique de fibroblastes embryonnaires de souris dans des ovocytes bovins [46]. Les études conduites chez les ruminants montrent que, malgré ces deux points de contrôle, au passage desquels on peut imaginer que les clones mal « reprogrammés » ont arrêté leur développement, 50% des clones arrivant au stade blastocyste présentent une hypermé-thylation des cellules du trophectoderme, en comparaison d'embryons fécondés in vivo.…”

Epigenetic reprogramming of somatic cells by nuclear transplant in zebrafish

“…In transplants of mouse embryo fibroblasts to bovine oocytes, Hsp 70.1 was not silenced after nuclear transfer (Arat et al, 2003). In addition, cloned mouse embryos prepared with myoblast nuclei continue to express the glucose transporter GLUT4, which is normally expressed in muscle but not in early embryos (Gao et al, 2003).…”