5 Nucleolar Development Requires Transcriptional Activity During Porcine Embryonic Genome Activation

Abstract

The development of a functional nucleolus accompanying the major embryonic genome activation (EGA) is considered a marker for embryo quality and viability. However, the use of this marker is limited by the lack of accurate knowledge of the biology of embryonic nucleologenesis. The objective of this study was to elucidate the role of RNA polymerase I (RPI) and total transcriptional activity, reflecting EGA, for nucleologenesis in in vivo-developed porcine embryos. Late 4-cell-stage embryos were cultured in the absence (control) or presence of actinomycin D (AD; 0.2 �g mL-1, 3 h for RPI inhibition; 2.0 �g mL-1, 3 h for total transcriptional inhibition). Late 2-cell-stage embryos were cultured to the late 4-cell stage with 0.2 �g mL-1 AD (long-term inhibition) to prevent EGA. Embryos were fixed at the late 4-cell stage and processed for RT-PCR (de novo synthesized rRNA), autoradiography (ARG, following culture with 3H-uridine for the last 20 min before fixation), TEM, FISH (probe-labeling rRNA and rDNA), silver staining of nucleolar proteins, and immunofluorescence for RPI. Control embryos displayed typical extranucleolar and nucleolar ARG labeling, fibrillo-granular nucleoli, and focal RPI localization signaling de novo rRNA synthesis in functional nucleoli, confirmed by RT-PCR. All nuclei showed large FISH clusters (rRNA and rDNA) that in 88% of the cases were co-localized with large foci of silver-stained nucleolar proteins. After RPI inhibition, only extranucleolar ARG labeling was detected and, instead of fibrillo-granular nucleoli, a segregated dense-fibrillar component and a granular component, but no fibrillar centers, were observed. RPI was dispersed in all nuclei, the number of nuclei presenting large FISH clusters decreased to 40%, and only 42% of nuclei showed nucleolar proteins localized to large foci. After total transcriptional inhibition and long-term inhibition, the nuclei did not display any ARG labeling and presented inactive nucleolus precursor bodies indicating lack of rRNA (RT-PCR) and total RNA synthesis. However, 40% of the nuclei in both groups presented large FISH clusters of rRNA. This rRNA is considered as partially processed residues of maternally inherited molecules, and their clustering is most likely independent of EGA. Inhibition of transcriptional activity at the time of EGA caused the dispersion of RPI (de novo synthesized) but did not influence the localization of silver-stained nucleolar proteins to large foci (41%). On the other hand, EGA inhibition caused the lack of RPI labeling and hampered the localization of nucleolar proteins to foci. Differences between these 2 groups could be due to the activation of RNA polymerase II before the 3-h AD treatment. In conclusion, RPI transcription and de novo protein synthesis are required for formation of functional nucleoli. However, the clustering of maternally inherited nucleolar transcripts is independent on transcriptional activity at the time of EGA. Failure in constituent RNA polymerase activation during EGA leads to pattern-specific changes in nucleologenesis, which may serve as a marker for early embryo quality.

The embryonic nucleologenesis during inhibition of major transcriptional activity in bovine preimplantation embryos

“…For the first time ever this finding was confirmed by experiments from this group. We, as the first, have confirmed the selective inhibition of RNA pol I by this concentration and time interval outcoming from the previous experiments by bovine (Svarcova et al 2007a) and swine embryos (Svarcova et al 2007b). As was proved earlier, for future successful development, embryos necessarily need ribosomes (Laurencik et al 2003).…”

“…The importance of EGA in further embryo development, including the importance of de novo nucleolar formation related to particular activation of RNA pol I transcription (Laurincik et al 2000(Laurincik et al , 2003Pivko et al 2004;Svarcova et al 2007bSvarcova et al , 2008, was reported in previous studies from this group. On the other hand, as was shown by specific inhibition transcriptional activation of RNA pol II and III, activity of RNA pol I is not sufficient for formation of fully functional nucleoli in early embryos (Svarcova et al 2007a).…”

“…The absence of FC residues in this group and the poor labeling in nucleoplasm proved the inherence of late processing machinery proteins (nucleolin and nucleophosmin) participating in this process. It demonstrates the maternal background of nucleologenesis (Svarcova et al 2007b;Kovalská et al 2010). As detected in the 12 hours group, this condition is lethal, stops all transcription and leads to apoptosis with significant signs on nuclear membrane and fragmented chromatin.…”

“…These groups showed a loss of transcriptional activity as proved by 3 H uridine labeling. Any structures resembling nucleoli or less DFC and GC have not been observed Svarcova et al 2007b). Moreover, the inherence of the pre-rRNAs, which are actively associated with rRNA processing machinery during nucleologenesis, are, by absence of transcription of RNA pol II and III, also missing .…”

“…Until this period the proteosynthesis is based on ribosomes inherited from the oocyte. The proof of maternally inherited rRNA and its role in embryonic nucleolar formation was first demonstrated by Svarcova et al (2007b).…”