Maternally inherited rRNA triggers de novo nucleolus formation in porcine embryos

Abstract

SummaryThe present study examines the role of RNA polymerase I (RPI)-mediated transcription, maternally inherited rRNA and nucleolar proteins in the resumption of fibrillogranular nucleoli during embryonic genome activation (EGA) in porcine embryos. Late 4-cell embryos were incubated in the absence (control) or presence of actinomycin D (AD) (0.2 μg/ml for inhibition of RPI; 2.0 μg/ml for inhibition of total transcription) and late 2-cell embryos were cultured to the late 4-cell stage with 0.2 μg/ml AD to block EGA. Embryos were then processed for reverse-transcriptase polymerase chain reaction (RT-PCR), and for autoradiography (ARG), transmission electron microscopy (TEM), fluorescence in situ hybridization (FISH), silver staining and immunofluorescence (for RPI). Embryos in the control group displayed extranucleolar and intranucleolar ARG labelling, and exhibited de novo synthesis of rRNA and reticulated functional nucleoli. Nucleolar proteins were located in large foci. After RPI inhibition, nucleolar precursors transformed into segregated fibrillogranular structures, however no fibrillar centres were observed. The localization of rDNA and clusters of rRNA were detected in 57.1% immunoprecipitated (IP) analyzed nucleoli and dispersed RPI; 30.5% of nuclei showed large deposits of nucleolar proteins. Embryos from the AD-2.0 group did not display any transcriptional activity. Nucleolar formation was completely blocked, however 39.4% of nuclei showed rRNA clusters; 85.7% of nuclei were co-localized with nucleolar proteins. Long-term transcriptional inhibition resulted in the lack of ARG and RPI labelling; 40% of analyzed nuclei displayed the accumulation of rRNA molecules into large foci. In conclusion, maternally inherited rRNA co-localized with rDNA and nucleolar proteins can initiate a partial nucleolar assembly, resulting in the formation of fibrilogranular structures independently on activation of RPI-mediated transcription.

Elimination of apoptotic cells by non-professional embryonic phagocytes can be stimulated or inhibited by external stimuli

“…The disruption of ribosome biogenesis resulted in a decrease in the proliferative activity of blastomeres: Mouse blastocysts exposed to actinomycin D for 24 h showed a signi cantly decreased mean number of cells when compared to controls. A similar cleavage-inhibiting effect of actinomycin D was documented in previous studies on in vitro cultured mouse, rabbit and bovine oocytes 10,29,30 , developing preimplantation embryos 31 and blastocysts 15,32 . Actinomycin D at nanomolar concentrations also inhibited proliferation in various cell lines and tissues 33,34 .…”

“…0.2 µg/ml) actinomycin D inhibits transcription through RNA polymerase I 9,10 , and at higher concentration (e.g. 2.0 µg/ml) it inhibits transcription through RNA polymerases I, II, and III 10 . The result is inhibition of protein synthesis.…”

“…In the current study, we used actinomycin D at 5 ng/ml (4 nM), i.e. at a concentration with a proven ability to selectively inhibit ribosomal RNA synthesis 10 and to trigger apoptotic pathway in a signi cant number of embryonic cells within 24 h 15 .…”