Tatsuma Yao

Heterochromatin Dynamics during the Differentiation Process Revealed by the DNA Methylation Reporter Mouse, MethylRO.

Summary In mammals, DNA is methylated at CpG sites, which play pivotal roles in gene silencing and chromatin organization. Furthermore, DNA methylation undergoes dynamic changes during development, differentiation, and in pathological processes. The conventional methods represent snapshots; therefore, the dynamics of this marker within living organisms remains unclear. To track this dynamics, we made a knockin mouse that expresses a red fluorescent protein (RFP)-fused methyl-CpG-binding domain (MBD) protein from the ROSA26 locus ubiquitously; we named it MethylRO (methylation probe in ROSA26 locus). Using this mouse, we performed RFP-mediated methylated DNA immunoprecipitation sequencing (MeDIP-seq), whole-body section analysis, and live-cell imaging. We discovered that mobility and pattern of heterochromatin as well as DNA methylation signal intensity inside the nuclei can be markers for cellular differentiation status. Thus, the MethylRO mouse represents a powerful bioresource and technique for DNA methylation dynamics studies in developmental biology, stem cell biology, as well as in disease states.

Live-cell imaging of nuclear–chromosomal dynamics in bovine in vitro fertilised embryos

Nuclear/chromosomal integrity is an important prerequisite for the assessment of embryo quality in artificial reproductive technology. However, lipid-rich dark cytoplasm in bovine embryos prevents its observation by visible light microscopy. We performed live-cell imaging using confocal laser microscopy that allowed long-term imaging of nuclear/chromosomal dynamics in bovine in vitro fertilised (IVF) embryos. We analysed the relationship between nuclear/chromosomal aberrations and in vitro embryonic development and morphological blastocyst quality. Three-dimensional live-cell imaging of 369 embryos injected with mRNA encoding histone H2B-mCherry and enhanced green fluorescent protein (EGFP)-α-tubulin was performed from single-cell to blastocyst stage for eight days; 17.9% reached the blastocyst stage. Abnormalities in the number of pronuclei (PN), chromosomal segregation, cytokinesis, and blastomere number at first cleavage were observed at frequencies of 48.0%, 30.6%, 8.1%, and 22.2%, respectively, and 13.0%, 6.2%, 3.3%, and 13.4%, respectively, for abnormal embryos developed into blastocysts. A multivariate analysis showed that abnormal chromosome segregation (ACS) and multiple PN correlated with delayed timing and abnormal blastomere number at first cleavage, respectively. In morphologically transferrable blastocysts, 30–40% of embryos underwent ACS and had abnormal PN. Live-cell imaging may be useful for analysing the association between nuclear/chromosomal dynamics and embryonic development in bovine embryos.

Quantitative Assessment of Embryo Quality Based on a Live-Cell Imaging Technique

Abstract The quality of embryos and the choice of the one with the best quality is an urgent issue in assisted reproductive technologies (ART) and the animal breeding industry. Although morphological assessments, such as Gardner criteria, are widely used, their objectivity and quantitation remain controversial. As the cellular structure and metabolism change dynamically during preimplantation development, it is desirable to monitor the whole process spatiotemporally based on multidimensional image information. We have developed a novel live-cell imaging methodology for the preimplantation embryo that enables the monitoring of the various events continuously taking place from fertilization to the blastocyst stage. One of advantage of this methodology is that we can see the effect of phenomena observed during imaging on the developmental potential to term, as cellular damage is suppressed in our system. Moreover, it allows the automatic extraction of various parameters from image data using customized algorithms developed by us. Therefore, we consider that this methodology will be a powerful tool for the quantitative analysis of the quality of embryos, and for the determination of the reasons for the attenuation of embryo quality.