Expression of fibrillarin in bovine oocytes and preimplantation embryos developing in vitro

Studies in somatic cells have demonstrated the participation of some defined nucleolar specific proteins in pre-rRNA synthesis and processing. Fibrillarin, a 34 kd protein localized to the dense fibrillar component of the nucleolus, is involved in the earliest cleavage step of pre-rRNA processing. Expression of fibrillarin has not been characterized during the course of bovine preimplantation development. Specific aims of this study were to characterize the expression of fibrillarin in bovine oocytes and preimplantation embryos developing in vitro. Oocytes and embryos were labeled with an anti-fibrillarin primary antibody and an FITC-conjugated goat antihuman secondary antibody and dual stained with Hoechst 33342 DNA dye. In germinal vesicle (GV) stage oocytes, obtained from 2 to 6 mm follicles, fibrillarin appeared as several large distinct fluorescent spheres within the nucleoplasm, but was not strictly localized to the nucleolus. Fibrillarin was not detected in metaphase oocytes. Expression of fibrillarin was first detected in embryos at the pronucleate stage, appearing as small spots or distinct fluorescent spheres randomly distributed within the nucleoplasm; this pattern persisted in 2- and 4-cell stage embryos. Beginning at the 8-cell stage, fibrillarin signals appeared as distinct fluorescent rings encapsulating each nucleolus. From the 16-cell through the blastocyst stage, a branching immunofluorescent pattern was observed in association with fully reticulated nucleoli. Expression of fibrillarin was absent in 8-cell, but not pronucleate stage embryos cultured with alpha-amanitin, indicating that fibrillarin is initially recruited from maternal stores in pronucleate stage embryos, but then subsequently transcribed beginning at the 8-cell stage, coincident with the onset of nucleolar, transcription. Results from this study provide further insight into the organization and development of the embryonic nucleolus during bovine preimplantation development, and may have important implications for assisted reproductive technologies, including nucleus transfer.