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Regulation of Ribosomal RNA Synthesis During the Final Phases of Porcine Oocyte Growth¹

Department of Anatomy and Physiology,³ Royal Veterinary and Agricultural University, 1870 Frederiksberg, Denmark Department of Biotechnology,⁴ Institute for Animal Science (FAL), Mariensee, 31535 Neustadt, Germany Institute of Experimental Medicine⁵ and Institute of Animal Physiology and Genetics,⁶ Academy of Sciences of Czech Republic, Prague, Czech Republic, Constantin the Philosopher University,⁷ Nitra, Slovak Republic Research Institute of Animal Production,⁸ Nitra, Slovak Republic

In porcine oocytes, acquisition of meiotic competence coincides with a decrease of general transcriptional activity at the end of the oocyte growth phase and, specifically, of ribosomal RNA (rRNA) synthesis in the nucleolus. The present study investigated the regulation of rRNA synthesis during porcine oocyte growth. Localization and expression of components involved in regulation of the rRNA synthesis (the RNA polymerase I-associated factor PAF53, upstream binding factor [UBF], and the pocket proteins p130 and pRb) were assessed by immunocytochemistry and semiquantitative reverse transcription-polymerase chain reaction and correlated with ultrastructural analysis and autoradiography following [³H]uridine incubation in growing and fully grown porcine oocytes. In addition, meiotic resumption, ultrastructure, and expression of p130, UBF, and PAF53 were analyzed in growing and fully grown porcine oocytes cultured with 100 µM butyrolactone I (BL-I), a potent inhibitor of cyclin-dependent kinases, to gain insight concerning the regulation of rRNA transcription during meiotic arrest. Immunocytochemical analysis demonstrated that p130 became colocalized with UBF and PAF53 and that the intensity of the PAF53 labeling decreased toward the end of the oocyte growth phase. These data suggest that the decrease in rRNA synthesis is regulated through inhibition of UBF by p130 as well as by decreased availability of PAF53. Moreover, expression of mRNA encoding PAF53 was decreased at the end of the oocyte growth phase. At the morphological level, these events coincided with inactivation of the nucleolus, as visualized by the transformation of the fibrillogranular nucleolus to an electron-dense fibrillar sphere with remnants of the fibrillar centers at the surface. Meiotic inhibition with 100 µM BL-I had a detrimental effect on the ability of porcine oocytes to resume meiosis and on nucleolus morphology, resulting in a lack of RNA synthetic capability as the fibrillar components, where rRNA transcription and initial processing occur, condensed or even disintegrated.

¹ Supported by grants from the Danish Research Agency (9901178), NATO (978658), the Deutsche Forschungsgemeinschaft (DFG), the Grant Agency of the Academy of Sciences of the Czech Republic (IAA5039202) to P.H., and an institutional grant (AV0Z5039906) to P.H.

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