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Maternally inherited rRNA triggers de novo nucleolus formation in porcine embryos

Published online by Cambridge University Press:  12 October 2018

Martin Morovic Open the ORCID record for Martin Morovic [Opens in a new window] ,
Olga Østrup ,
Frantisek Strejcek ,
Michal Benc ,
Matej Murin ,
Katarina Jedlickova ,
Alexandra Bartkova ,
Andrea Lucas-Hahn ,
Lazo Pendovski  and
Jozef Laurincik
Martin Morovic*
Affiliation:
Constantine the Philosopher University, Nitra, Slovak Republic
Olga Østrup
Affiliation:
Centre for Genomic Medicine, Copenhagen University Hospital, Copenhagen, Denmark
Frantisek Strejcek
Affiliation:
Constantine the Philosopher University, Nitra, Slovak Republic
Michal Benc
Affiliation:
Constantine the Philosopher University, Nitra, Slovak Republic Institute of Animal Science, Prague, Czech Republic
Matej Murin
Affiliation:
Constantine the Philosopher University, Nitra, Slovak Republic The Czech Academy of Sciences, Institute of Animal Physiology and Genetics, Libechov, Czech Republic
Katarina Jedlickova
Affiliation:
Constantine the Philosopher University, Nitra, Slovak Republic
Alexandra Bartkova
Affiliation:
Constantine the Philosopher University, Nitra, Slovak Republic
Andrea Lucas-Hahn
Affiliation:
Institute of Farm Animal Genetics (FLI), Mariensee, Neustadt, Germany
Lazo Pendovski
Affiliation:
Ss. Cyril and Methodius University in Skopje, Republic of Macedonia
Jozef Laurincik
Affiliation:
Constantine the Philosopher University, Nitra, Slovak Republic The Czech Academy of Sciences, Institute of Animal Physiology and Genetics, Libechov, Czech Republic
*
Author for correspondence: Martin Morovic. Department of Zoology and Anthropology, Faculty of Natural Sciences, Constantine the Philosopher University in Nitra, Nabrezie mladeze 91, 949 74, Nitra, Slovakia. Tel: +421 37 6408 712. Fax: +421 37 6408 556. E-mail: mmorovic@ukf.sk
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Summary

The 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.

Keywords

Embryonic genome activationNucleolusPorcine embryoRNA polymerase IrRNAPorcine embryo
Type
Research Article
Information
Zygote , Volume 26 , Issue 5 , October 2018 , pp. 395 - 402
Copyright
© Cambridge University Press 2018 

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