Expression of Nucleolar-Related Proteins in Porcine Preimplantation Embryos Produced In Vivo and In Vitro1 (original) (raw)

2004, Biology of Reproduction

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 laserscanning 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 [ 3 H]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-␣-amanitintreated, 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 1