226 GREATER DYSREGULATION OF GENE EXPRESSION IN PRE-IMPLANTATION CLONED OVINE COMPARED WITH CLONED BOVINE CONCEPTUSES

Abstract

In our experience, the cloning of sheep by somatic cell nuclear transfer has been less successful than with cattle (5 v. 10% live births from embryo transfer). Here, we compare data collected over many years on the pre-implantation development of nuclear transfer (NT) ovine and bovine embryos with contemporary in vitro produced (IVP) embryos at the elongation (bovine Day 16–18 v. ovine Day 14) and allantois formation (bovine Day 26/27 v. ovine Day 21) stages. Sheep NT conceptuses were generated from three fibroblast types (skin, kidney, and lung) derived from a Day 100 female fetus. Bovine NT embryos were generated from a granulosa cell line derived from a mature dairy cow. The expression of a subset of genes (IGF2, H19, IGF2R, IGFBP-2, PEG1/Mest and trophoblast-expressed genes such as IFN-τ, TKDP-1, COX-2, and placental lactogen) was examined in both species at the two stages of development, using either Northern or slot-blot analysis. Pairwise comparisons between groups were carried out using Student's t-test. The proportion of ovine NT embryos that developed into blastocysts from the three cell types combined was 11% (117/1052) compared with 79% (86/109) for bovine NT from the one cell line. After transfer to synchronized recipients, 50–60% developed to the elongated conceptus stage for both species. Day 14 ovine NT (n = 19) and IVP (n = 26) conceptus lengths were similar whereas Day 16 bovine NT (n = 9) lengths were slightly shorter than those of IVP (n = 3) conceptuses (P > 0.05). However, only 30% (16/52) of ovine NT embryos developed to Day 21 compared with 64% (9/14) for Day 26/27 bovine NT. Bovine NT and IVP embryo and allantoic lengths were not significantly different; however, both lengths were shorter in ovine NT than in IVP controls (P < 0.05 and < 0.005, respectively). In each species, there was considerable individual variation in expression levels of most genes at each stage, probably due to variation in developmental stage of individuals even from the same day of gestation. The difference may be due to the timing of onset or termination of gene expression. In Day 16–18 bovine NT conceptuses, only the mean level of PEG1/Mest expression was significantly different (P < 0.05) from that of IVP conceptuses. In contrast, mean expression levels for TKDP-1 (P < 0.0001) and COX-2 (P < 0.05) were higher in ovine Day 14 NT, and PEG1/Mest was higher (P < 0.05) in Day 21 NT when compared with contemporary IVP controls. H19 and IGF2 expression was coordinately regulated in bovine NT and in ovine and bovine IVP conceptuses (IGF2 and H19 mRNA abundance was directly correlated). This coordinate regulation was disrupted in certain Day 21 ovine NT conceptuses, where high H19 levels occurred in the presence of low levels of IGF2, but not the converse. Thus, greater dysregulation in gene expression and loss of coordinate regulation of two key imprinted genes may explain the lower survival rate of ovine NT embryos to the early fetal stage when compared with bovine NT embryos. The effect of donor cell type on the species difference cannot be discounted.

This work was supported by FRST C10X0303 programme.