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This Article Full Text Full Text (PDF) All Versions of this Article: 70/4/867    most recent biolreprod.103.021683v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal My Folders Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bjerregaard, B. Articles by Maddox-Hyttel, P. Search for Related Content PubMed PubMed Citation Articles by Bjerregaard, B. Articles by Maddox-Hyttel, P. Agricola Articles by Bjerregaard, B. Articles by Maddox-Hyttel, P.

Expression of Nucleolar-Related Proteins in Porcine Preimplantation Embryos Produced In Vivo and In Vitro¹

Department of Anatomy and Physiology,³ Royal Veterinary and Agricultural University, 1870 Frederiksberg C, Denmark Department of Biotechnology,⁴ Institute for Animal Science (FAL), Mariensee, 31535 Neustadt, Germany Constantin the Philosopher University,⁵ SK-949 74 Nitra, Slovak Republic Research Institute of Animal Production,⁶ SK-949 74 Nitra, Slovak Republic Section of Reproductive Biology,⁷ Department of Animal Breeding and Genetics, Danish Institute of Agricultural Sciences, 8830 Tjele, Denmark Precision Therapeutics,⁸ Pittsburgh, Pennsylvania 15213 Veterinary University,⁹ Vienna, A-1210 Wien, Austria

The expression of nucleolar-related proteins was studied as an indirect marker of the ribosomal RNA (rRNA) gene activation in porcine embryos up to the blastocyst stage produced in vivo and in vitro. A group of the in vivo-developed embryos were cultured with -amanitin to block the de novo embryonic mRNA transcription. Localization of proteins involved in the rRNA transcription (upstream binding factor [UBF], topoisomerase I, RNA polymerase I [RNA Pol I], and the RNA Pol I-associated factor PAF53) and processing (fibrillarin, nucleophosmin, and nucleolin) was assessed by immunocytochemistry and confocal laser-scanning microscopy, and mRNA expression was determined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). These findings were correlated with ultrastructural data and autoradiography following 20-min [³H]uridine incubation. Additionally, expression of the pocket proteins pRb and p130, which are involved in cell-cycle regulation, was assessed by semiquantitative RT-PCR up to the blastocyst stage. Toward the end of third cell cycle, the nuclei in non--amanitin-treated, in vivo-produced embryos displayed different stages of transformation of the nuclear precursor bodies (NPBs) into fibrillogranular nucleoli associated with autoradiographic labeling. However, on culture with -amanitin, NPBs were not transformed into a fibrillogranular nucleolus during this cell cycle, demonstrating that embryonic nucleogenesis requires de novo mRNA transcription. Moreover, immunolocalization of RNA Pol I, but not of UBF, and the mRNA expression of PAF53 and UBF were significantly reduced or absent after culture with -amanitin, indicating that RNA Pol I, PAF53, and presumably, UBF are derived from de novo embryonic transcription. Embryonic genomic activation was delayed in porcine embryos produced in vitro compared to the in vivo-derived counterparts with respect to mRNAs encoding PAF53 and UBF. Moreover, differences existed in the mRNA expression patterns of pRb between in vivo- and in vitro-developed embryos. These findings show, to our knowledge for the first time, a nucleolus-related gene expression in the preimplantation porcine embryo, and they highlight the differences in quality between in vivo and in vitro-produced embryos.

¹ Supported by grants from "Disease models, disease prevention and animal welfare improvement: the pig embryo as a model," Danish Research Agency (9901178); NATO (978658); Deutsche Forschungsgemeinschaft (DFG); and Bundesministerium für Wissenschaft und Verkehr der Republik Österreich (GZ 45.517/I-VI/B/7a/2002).

² Correspondence: Poul Maddox-Hyttel, Department of Anatomy and Physiology, Royal Veterinary and Agricultural University, Groennegaardsvej 7, DK-1870 Frederiksberg C, Denmark. FAX: 45 3528 2547; poh{at}kvl.dk

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