Onset of nucleolar and extranucleolar transcription and expression of fibrillarin in macaque embryos developing in vitro

Specific aims were to characterize the onset of nucleolar and extranucleolar transcription and expression of the nucleolar protein fibrillarin during preimplantation development in vitro in macaque embryos using autoradiographic and immunocytochemical techniques. Autoradiography was performed on whole embryos cultured with [3H]uridine for assessment of nucleolar (rRNA) and extranucleolar (mRNA) transcription. Expression of fibrillarin was immunocytochemically assessed in whole embryos using a primary antibody against fibrillarin and a fluorescein isothiocyanate-conjugated secondary antibody. Extranucleolar incorporation of [3H]uridine was first detected in 2-cell embryos cultured 6-10 h with [3H]uridine. Culture with alpha-amanitin prevented incorporation of label in 2-cell embryos, and treatment with ribonuclease reduced the signal to background levels, indicating that [3H]uridine was incorporated into mRNA and not rRNA or DNA. Nucleolar incorporation of [3H]uridine was not evident in pronucleate-stage or 2- to 5-cell embryos, but it was detected in one 6-cell embryo and in all 8-cell to blastocyst-stage embryos. Fibrillarin was first expressed in some 6- to 7-cell embryos, but it was consistently expressed in all 8-cell embryos. Fibrillarin was localized to the perimeter of the nucleolar precursor bodies, forming a ring that completely encapsulated these structures. Fibrillarin was not expressed in 8- to 16-cell embryos cultured with alpha-amanitin, indicating that it is transcribed, rather than recruited, at the 8-cell stage. In conclusion, in in vitro-fertilized macaque embryos developing in vitro, extranucleolar synthesis of mRNA is initiated at the 2-cell stage while the onset of nucleolar transcription occurs at the 6- to 8-cell stage, coincident with expression of fibrillarin.