In mammals, cloning by nuclear transfer (NT) into an enucleated oocyte is a very inefficient process, even if it can generate healthy adults. We show that blastocysts derived from embryonic stem (ES) donor cells develop at a high rate, correctly express the pluripotential marker gene Oct4 in ICM cells and display normal growth in vitro. Moreover, the majority of them implant in the uterus of recipient females. We combine embryological studies, gene expression analysis during gastrulation and generation of chimaeric embryos to identify the developmental origin (stage and tissue affected) of NT embryo mortality. The majority died before mid-gestation from defects arising early, either at peri-implantation stages or during the gastrulation period. The first type of defect is a non-cell autonomous defect of the epiblast cells and is rescued by complementation of NT blastocysts with normal ES or ICM cells. The second type of defect affects growth regulation and the shape of the embryo but does not directly impair the initial establishment of the patterning of the embryo. Only chimaeras formed by the aggregation of NT and tetraploid embryos reveal no growth abnormalities at gastrulation. These studies indicate that the trophoblast cell lineage is the primary source of these defects. These embryological studies provide a solid basis for understanding reprogramming errors in NT embryos. In addition, they unveil new aspects of growth regulation while increasing our knowledge on the role of crosstalk between the extra-embryonic and the embryonic regions of the conceptus in the control of growth and morphogenesis.

Aims/hypothesis We used the GK/Par rat, a spontaneous model of type 2 diabetes with early defective beta cell neogenesis, to determine whether the development of GK/Par offspring in a non-diabetic intrauterine/postnatal environment would prevent the alteration of fetal beta cell mass (BCM) and ultimately decrease the risk of diabetes later in adult life. Methods We used an embryo-transfer approach, with fertilised GK/Par ovocytes (oGK) being transferred into pregnant Wistar (W) or GK/Par females (pW and pGK). Offspring were phenotyped at fetal age E18.5 and at 10 weeks of age, for BCM, expression of genes of pancreatic progenitor cell regulators (Igf2, Igf1r, Sox9, Pdx1 and Ngn3), glucose tolerance and insulin secretion. Results (1) Alterations in neogenesis markers/regulators and BCM were as severe in the oGK/pW E18.5 fetuses as in the oGK/pGK group. (2) Adult offspring from oGK transfers into GK (oGK/pGK/sGK) had the expected diabetic phenotype compared with unmanipulated GK rats. (3) Adult offspring from oGK reared in pW mothers and milked by GK foster mothers had reduced BCM, basal hyperglycaemia, glucose intolerance and low insulin, to an extent similar to that of oGK/pGK/sGK offspring. (4) In adult offspring from oGK transferred into pW mothers and milked by their W mothers (oGK/pW/sW), the phenotype was similar to that in oGK/pGK/sGK or oGK/pW/sGK offspring. Conclusions/interpretation These data support the conclusion that early BCM alteration and subsequent diabetes risk in the GK/Par model are not removed despite normalisation of the prenatal and postnatal environments, either isolated or combined.

The gene encodes one of the 'core' transcription factors necessary to establish and maintain pluripotency in mammals. Its function depends on its precise level of expression, so its transcription has to be tightly regulated. To date, few conserved functional elements have been identified in its 5' regulatory region: a distal and a proximal enhancer, and a minimal promoter, epigenetic modifications of which interfere with expression and function in -derived cell lines. Also, its permanent inactivation in differentiated cells depends on methylation of its promoter. However, little is known about the epigenetic regulation of expression in the embryo itself. We used the rabbit blastocyst as a model to analyze the methylation dynamics of the 5' upstream region, relative to its regulated expression in different compartments of the blastocyst over a 2-day period of development. We evidenced progressive methylation of the 5' regulatory region and the first exon accompanying differentiation and the gradual repression of Methylation started in the early trophectoderm before complete transcriptional inactivation. Interestingly, the distal enhancer, which is known to be active in naïve pluripotent cells only, retained a very low level of methylation in primed pluripotent epiblasts and remained less methylated in differentiated compartments than the proximal enhancer. This detailed study identified CpGs with the greatest variations in methylation, as well as groups of CpGs showing a highly correlated behavior, during differentiation. Moreover, our findings evidenced few CpGs with very specific behavior during this period of development.

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