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Pre-implantation embryos produced by somatic cell nuclear transfer (SCNT) have varied developmental potentials. The majority of SCNT blastocysts do not develop to term, and the mechanisms inhibiting development are still largely unknown. Aggregation of cloned embryos has been attempted to compensate for the developmental deficiency of individual cloned embryos. In this report, we investigated the impact of aggregation of bovine cloned embryos at the four-cell stage on in vitro development and gene expression of the embryos. Cell numbers and development rate of aggregated (NTagg) and non-aggregated (NT) blastocysts were characterized and compared. The blastocyst formation after aggregation was modeled using the binominal distribution. The results indicate that aggregation enhances the blastocyst formation but does not increase the overall blastocyst rate. Additionally, utilizing microarray gene chip analysis 8.8% of 8,059 genes analyzed were differentially expressed between NTagg and NT blastocysts, with more than 80% of the differentially expressed genes up-regulated in the NTagg blastocysts. Up-regulated genes include those involved in transcription, biosynthesis and signaling such as TDGF1, HNFA, CAV1, GLU5, and CD81. Our results indicate that aggregation of bovine cloned embryos at an early stage promotes the in vitro development of the resulting pre-implantation embryos.

Reproductive efficiency using somatic cell nuclear transfer (SCNT) technology remains suboptimal. Of the various efforts to improve the efficiency, chromatin transfer (CT) and clone-clone aggregation (NTagg) have been reported to produce live cloned animals. To better understand the molecular mechanisms of somatic cell reprogramming during SCNT and assess the various SCNT methods on the molecular level, we performed gene expression analysis on bovine blastocysts produced via standard nuclear transfer (NT), CT, NTagg, in vitro fertilization (IVF), and artificial insemination (AI), as well as on somatic donor cells, using bovine genome arrays. The expression profiles of SCNT (NT, CT, NTagg) embryos were compared with IVF and AI embryos as well as donor cells. NT and CT embryos have indistinguishable gene expression patterns. In comparison to IVF or AI embryos, the number of differentially expressed genes in NTagg embryos is significantly higher than in NT and CT embryos. Genes that were differentially expressed between all the SCNT embryos and IVF or AI embryos are identified. Compared to AI embryos, more than half of the genes found deregulated between SCNT and AI embryos appear to be the result of in vitro culture alone. The results indicate that although SCNT methods have altered differentiated somatic nuclei gene expression to more closely resemble that of embryonic nuclei, combination of insufficient reprogramming and in vitro culture condition compromise the developmental potential of SCNT embryos. This is the first set of comprehensive data for analyzing the molecular impact of various nuclear transfer methods on bovine pre-implantation embryos. Mol. Reprod. Dev. 75: 744–758, 2008. © 2007 Wiley-Liss, Inc.