IVF culture conditions promote single nucleolus formation and elevate rRNA associated signals during mouse...

The plasticity of mammalian embryonic nucleolus in response to post fertilization environmental cues during preimplantation period remains poorly characterized. In this study, using ICR mice model, we investigated nucleolar morphology and ribosomal RNA (rRNA) levels in embryos generated by in vitro fertilization (IVF) and subsequently cultured entirely in vitro, in comparison with embryos fertilized and developed in vivo (IVD). Nucleolar morphology and rRNA associated signal were assessed by immunostaining with mouse monoclonal NOPP140 and Y10b antibodies, respectively. To functionally investigate the relationship between nucleolar structure and ribosome biogenesis, embryos were treated with CX-5461, a specific RNA polymerase I inhibitor. Quantitative analysis revealed no significant difference in nucleoli number at the zygote and 2-cell stage. In contrast, from the 4-cell to blastocyst stage, IVF embryos exhibited significantly higher percentage of single nucleolus (SN) in individual blastomere than IVD embryos (P < 0.05). Correspondingly, rRNA level was significantly higher in IVF embryos compared to IVD (P < 0.05). CX-5461 treatment reduced Y10b signal intensity and increased SN morphology significantly (P < 0.05), suggesting a relationship between rRNA level and nucleolar morphology. Taken together, these results suggest that the IVF culture environment, in which both fertilization and development occur entirely in vitro, is associated with changes in single nucleolus formation and rRNA associated signal during the preimplantation stage.