Reprogramming of primordial germ cells begins before migration into the genital ridge, making these cells inadequate donors for reproductive cloning

Abstract

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...

Very small embryonic-like stem cells in adult tissues—Potential implications for aging

“…Trophoectoderm is responsible for formation of the placenta and the remaining part of the blastocyst, while ICM will give rise to the embryo including epiblast. Epiblast stem cells (EPSC) have been also demonstrated to preserve pluripotency as well as retain germ lineage potential (Matsui et al, 1992;Shamblott et al, 1998;Yamazaki et al, 2003). EPSC express following antigens related to their pluripotent stage: SSEA-1 (mice), SSEA-3/4 (human), Oct-4 and Nanog.…”

Gamete imprinting: setting epigenetic patterns for the next generation

“…Thus, the timing was similar to that noted in the methylation studies described above. Other investigators have examined PGC reprogramming by analysing embryos derived by somatic cell nuclear transfer using PGC nuclei at different stages of development; these experiments also provide evidence that methylation imprints are erased between 10.5 and 12.5 days of gestation (Lee et al 2002;Yamazaki et al 2003).…”

Bone marrow‐derived very small embryonic‐like stem cells: Their developmental origin and biological significance

“…Namely, in cultures freshly isolated from embryos, PGC proliferate for a few days only, and then disappear either because they differentiate or die (De Felici and McLaren, 1983). Furthermore, whereas the nuclei of migrating PGC at 8.5-9.5 dpc can be successfully used as donors for nuclear transfer, nuclei from PGC at 11.5 dpc and later are incompetent to support full-term development (Yamazaki et al, 2003). This finding is somehow intriguing, taking in consideration that PGC are the population of stem cells that carries "developmental totipotency" for oocytes and sperm.…”

“…To explain this phenomenon at the molecular level, it is known that the pluripotency of PGC nuclei depends on the methylation status of genomic imprinted genes (e.g., H19, Igf-2, Igf-2R, Snrpn; Mann, 2001;Yamazaki et al, 2003). PGC until 9.5 dpc display a somatic imprint (paternal and maternal pattern of methylation) of H19, Igf-2, Igf-2R, Snrpn-that is crucial to maintain their pluripotency.…”