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

Abstract

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.

Chromosome segregation error during early cleavage in mouse pre-implantation embryo does not necessarily cause developmental failure after blastocyst stage

“…5b). Our results and the success of pregnancy after time-lapse imaging in the mouse/bovine embryo 12,25 suggest that karyotyping without biopsy can be applied to livestock embryos. In contrast, our technology involves injecting mRNA encoding fluorescent protein and laser irradiation for excitation, making it difficult to directly apply to human embryos.…”

Molecular Contribution to Embryonic Aneuploidy and Genotypic Complexity During Initial Cleavage Divisions of Mammalian Development

“…Although micronuclei-like structures have been detected in bovine embryos previously (Yao et al, 2018), their prevalence or whether they were associated with a particular stage of preimplantation development was not determined. To address this, we generated a large number (N=53) of bovine embryos by in vitro fertilization (IVF) and fixed them at the zygote to blastocyst stage to evaluate DNA integrity with DAPI and nuclear structure by immunostaining for the nuclear envelope marker, LAMIN-B1 (LMNB1; Fig.…”

“…While micronuclei-like structures have been previously detected in bovine embryos (Yao et al, 2018), we were able to assess their prevalence throughout preimplantation development and follow their fate in subsequent mitotic divisions. Of the cleavage-stage bovine embryos examined by immunostaining or live-cell imaging, over ~30% contained micro- or multi-nuclei and anaphase lagging of chromosomes was detected in certain embryos prior to micronuclei formation.…”

“…6A ). Unlike mouse embryos, which exhibit spatial separation of parental genomes by dual-spindle formation (Mayer, Smith, Fundele, & Haaf, 2000; Reichmann et al, 2018), bovine embryos normally undergo syngamy at the zygote stage (Yao et al, 2018). By avoiding syngamy and undergoing multipolar cytokinesis, zygotes differentially segregate entire parental genomes to daughter cells, helping to explain previous findings of heterogoneic divisions and the production of blastomeres with uniparental origins in both cattle and primates (Daughtry et al, 2019; Destouni et al, 2016; Middelkamp et al, 2020).…”

Three-dimensional live imaging of bovine embryos by optical coherence tomography

“…Bovine embryos for ET are usually evaluated by the IETS codes [6,7] whose accuracy relies on the proficiency of the operators. Sugimura et al [4,11] showed that high-quality bovine embryos could be selected using morphokinetic indicators such as timing, number of blastomeres at first cleavage, and number of blastomeres at the onset of the lag phase as well as by observing nuclear/chromosomal abnormalities of bovine embryos using live-cell imaging technology [19].…”