Preimplantation development is a period of dynamic epigenetic change that begins with remodeling of egg and sperm genomes, and ends with implantation. During this time, parental-specific imprinting marks are maintained to direct appropriate imprinted gene expression. We previously demonstrated that H19 imprinting could be lost during preimplantation development under certain culture conditions. To define the lability of genomic imprints during this dynamic period and to determine whether loss of imprinting continues at later stages of development, imprinted gene expression and methylation were examined after in vitro preimplantation culture. Following culture in Whitten's medium, the normally silent paternal H19 allele was aberrantly expressed and undermethylated. However, only a subset of individual cultured blastocysts (∼65%) exhibited biallelic expression, while others maintained imprinted H19 expression. Loss of H19 imprinting persisted in mid-gestation conceptuses. Placental tissues displayed activation of the normally silent allele for H19, Ascl2, Snrpn, Peg3 and Xist while in the embryo proper imprinted expression for the most part was preserved. Loss of imprinted expression was associated with a decrease in methylation at the H19 and Snrpn imprinting control regions. These results indicate that tissues of trophectoderm origin are unable to restore genomic imprints and suggest that mechanisms that safeguard imprinting might be more robust in the embryo than in the placenta.

Germ cells undergo epigenetic modifications as they develop, which suggests that they may be ideal donors for nuclear transfer (cloning). In this study, nuclei from confirmed embryonic germ cells were used as donors to determine whether they are competent for cloning and at which stage they are most competent. Embryos cloned from migrating 10.5-days-postcoitum (dpc) primordial germ cells (PGCs) showed normal morphological development to midgestation but died shortly thereafter. In contrast, embryos cloned from later-stage germ cells were developmentally delayed at midgestation. Thus, donor germ cell age inversely correlated with the developmental stage attained by cloned embryos. The methylation status of the H19-and Snrpn-imprinting control regions in germ cell clones paralleled that of the donors, and revealed that demethylation, or erasure of imprints, was already initiated in PGCs at 10.5 dpc and was complete by 13.5 dpc. Similarly, clones derived from male 15.5-dpc germ cells showed increased methylation correlating with the initiation of de novo methylation that resets imprints at this stage, and clones from neonatal germ cells showed nearly complete methylation in the H19 imprinting control region. These results indicate that the epigenetic state of the donor nucleus is retained in cloned embryos, and that germ cells are therefore inadequate nuclear donors for cloning because they are either erasing or resetting epigenetic patterns.S omatic cell cloning has been successfully used to produce live cloned offspring in a variety of mammals (1-3). Although the rate of obtaining healthy offspring is very low, such successes illustrate the capacity of the egg cytoplasm and the donor somatic cell nucleus to support embryonic development.As part of the normal course of development, embryonic germ cells are dynamically reprogrammed during germ cell migration and differentiation. In this context, reprogramming is defined as a stepwise process whereby the somatic-like epigenetic pattern, hypothesized to be characteristic of the earliest detectable primordial germ cells (PGCs), undergoes erasure and is transformed into the sex-specific pattern of mature germ cells (4, 5). This process is exemplified by reprogramming of methylation associated with imprinted genes during germ-cell development; the somatic methylation imprints associated with early PGCs are erased around 11.5 days postcoitum (dpc) in both male and female PGCs and are reestablished in a sex-specific manner with paternal methylation imprints acquired early and maternalspecific methylation obtained late in the gametogenic process (6-10).Because embryonic germ cells by their very nature are in the process of reprogramming their genome, cells with different epigenetic patterns can be obtained and, in this study, were isolated by using a germ cell marker, EMA-1. Before migration into the genital ridges (10.5 dpc), PGCs are thought to possess a somatic-like epigenetic pattern, whereas, after migration (11.5 dpc and onward), germ cells are in various stages of r...