Until now, no primate animals have been successfully cloned to birth with somatic cell nuclear transfer (SCNT) procedures, and little is known about the molecular events that occurred in the reconstructed embryos during preimplantation development. In many SCNT cases, epigenetic reprogramming of the donor nuclei after transfer into enucleated oocytes was hypothesized to be crucial to the reestablishment of embryonic totipotency. In the present study, we focused on two major epigenetic marks, DNA methylation and histone H3 lysine 9 (H3K9) acetylation, which we examined by indirect immunofluorescence and confocal laser scanning microscopy. During preimplantation development, 67% of two-cell- and 50% of eight-cell-cloned embryos showed higher DNA methylation levels than their in vitro fertilization (IVF) counterparts, which undergo gradual demethylation until the early morula stage. Moreover, whereas an asymmetric distribution of DNA methylation was established in an IVF blastocysts with a lower methylation level in the inner cell mass (ICM) than in the trophectoderm, in most cloned blastocysts, ICM cells maintained a high degree of methylation. Finally, two donor cell lines (S11 and S1-04) that showed a higher level of H3K9 acetylation supported more blastocyst formation after nuclear transfer than the other cell line (S1-03), with a relatively low level of acetylation staining. In conclusion, we propose that abnormal DNA methylation patterns contribute to the poor quality of cloned preimplantation embryos and may be one of the obstacles to successful cloning in primates.
Evidence from epidemiological studies has proved that periconceptional use of folic acid (FA) can significantly reduce the risk of neural tube defects (NTDs). However, it is hard to explore when and how FA plays roles in neurogenesis and brain development in vivo, especially in human or other nonhuman primate systems. Primate embryonic stem cell (ESC) lines are ideal models for studying cell differentiation and organogenesis in vitro. In the present study, the roles of FA in neural differentiation were assessed in a rhesus monkey ESC system in vitro. The results showed no significant difference in the expression of neural precursor markers, such as nestin, Sox-1, or Pax-6, among neural progenitors obtained from different FA concentrations or with the FA antagonist methotrexate (MTX). However, FA depletion decreased cell proliferation and affected embryoid body (EB) and neural rosette formation, as well as neuronal but not neuroglia differentiation. Our data imply that the ESC system is a suitable model for further exploring the mechanism of how FA works in prevention of NTDs in primates.
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