Abstract
Chromatin looseness, which can be analyzed by fluorescence recovery after photobleaching (FRAP) using eGFP-tagged core histone proteins, is an important index of the differentiation potential of blastomere cells and embryonic stem cells. Whether chromatin looseness is a reliable index of the developmental potential of embryos during ontogenesis is not known. As a necessary first step toward answering this question, we investigated whether FRAP-analyzed embryos are capable of normal preimplantation and full-term development. All tested concentrations (50, 100, and 250 ng/μL) of microinjected eGFP-H2B mRNA were sufficient for detecting differences in chromatin looseness between male and female pronuclei. After FRAP analysis, most of the zygotes developed into blastocysts. Importantly, a considerable number of offspring developed from the FRAP analyzed zygotes (32/78; 41.0%) and grew into healthy adults. The offspring of zygotes injected with 250 ng/μL of eGFP-H2B mRNA and bleached using 110 μW laser power for 5 s were not genetically modified. Interestingly, bleaching using a 3-fold stronger laser intensity for a 6-fold longer time did not cause toxicity during preimplantation development, indicating that bleaching did not critically affect preimplantation development. Finally, we confirmed that similar results were obtained using two different types of confocal laser-scanning microscopes. This FRAP protocol would be useful for investigating the association between chromatin looseness and development.
Highlights
Ontogenesis begins from zygotes in which dynamic changes in the chromatin structure occur
The parental asymmetry of chromatin looseness was observed from the zygotes prepared using spermatozoa from various mice strains; the parental asymmetry of chromatin looseness was conserved in mice (Fig 1D and 1E)
We examined whether Fluorescence recovery after photobleaching (FRAP) analysis impairs embryo development
Summary
Ontogenesis begins from zygotes in which dynamic changes in the chromatin structure occur. Extensive alterations of epigenetic histone modifications and substitutions of histone variants in zygotic chromatin are thought to be important for subsequent development [1,2,3,4,5,6]. Euchromatin-enriched open chromatin has emerged as a new epigenetic factor and is extensively studied because of its association with cellular potency. Cells with greater differentiation potential have more open chromatin; upon cellular differentiation, this open chromatin structure changes to a closed state [7,8,9]. A higher mobility of histone proteins and pervasive transcription are thought to be indirect indices of open chromatin structure [8]
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