Abstract
Zygotes are totipotent cells that have the ability to differentiate into all cell types. It is believed that this ability is lost gradually and differentiation occurs along with the progression of preimplantation development. Here, we hypothesized that the loose chromatin structure is involved in the totipotency of one-cell stage embryos and that the change from loose to tight chromatin structure is associated with the loss of totipotency. To address this hypothesis, we investigated the mobility of eGFP-tagged histone H2B (eGFP-H2B), which is an index for the looseness of chromatin, during preimplantation development based on fluorescent recovery after photobleaching (FRAP) analysis. The highest mobility of eGFP-H2B was observed in pronuclei in 1-cell stage embryos and mobility gradually decreased during preimplantation development. The decrease in mobility between the 1- and 2-cell stages depended on DNA synthesis in 2-cell stage embryos. In nuclear transferred embryos, chromatin in the pseudopronuclei loosened to a level comparable to the pronuclei in 1-cell stage embryos. These results indicated that the mobility of eGFP-H2B is negatively correlated with the degree of differentiation of preimplantation embryos. Therefore, we suggest that highly loosened chromatin is involved in totipotency of 1-cell embryos and the loss of looseness is associated with differentiation during preimplantation development.
Highlights
Embryos at the 1-cell stage are totipotent and this totipotency is gradually lost during preimplantation development
The complementary RNA (cRNA) encoding eGFP-tagged histone H2B was injected into the cytoplasm of 1-cell stage embryos 2 h post-insemination. eGFP fluorescence was clearly observed in nuclei throughout preimplantation development (Supplemental Fig. 1A). eGFP-H2B appeared to be deposited in the nucleosomes, since the eGFP and DAPI signals were well-merged; the intense eGFP signal was observed in heterochromatin regions where DNA is condensed and the signal intensity of DAPI was high
This treatment cleared up the fluorescence signal from the nucleus, as well as the cytoplasm, of the embryos which had been injected with eGFP, the signal in the nucleus, but not nucleolar precursor body (NPB), remained in embryos injected with eGFP-H2B (Supplemental Fig. S2)
Summary
Embryos at the 1-cell stage are totipotent and this totipotency is gradually lost during preimplantation development. When the embryos reach the blastocyst stage, blastomeres are differentiated into 2 types of cell lineages: inner cell mass (ICM) and trophectoderm (TE). The former is pluripotent, while the latter is differentiated and its developmental potential is restricted to form the placenta. 1-cell stage embryos have totipotency and the highest plasticity. The mechanisms underlying these characteristics in 1-cell stage embryos and their loss during preimplantation development remain unclear
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