PARENT SESSION
Preimplantation Embryo/Fetal Development

THE ACTIVATION OF RIBOSOMAL RNA GENES IN IN VIVO PORCINE EMBRYOS.

Viuff, Dorthe¹, Greve, Torben¹, Holm, Peter², Callesen, Henrik², Hyttel, Poul³, Thomsen, Preben ³, ^{1 2 3}

ABSTRACT- In porcine embryos, nucleoli are first observed during the 3rd postfertilization cell cycle. It is unknown, however, when the rRNA genes are activated and how their transcripts and argyrophilic nucleolar proteins (ANP) are organized.This was investigated by simultaneous visualization of the rRNA genes and the rRNA by fluorescent in situ hybridization using a porcine 28S rDNA probe and subsequent visualization of ANP by silver staining on extracted and fixed embryonic nuclei.The first experiment aimed at detecting at which embryonic stage the first sign of rRNA synthesis occurred. For the 2-cell embryos (n=24) 4 small foci of FITC labelling representing the rRNA genes on chromosome 8 and 10 were revealed. No specific silver staining was observed. For the 4-cell embryos (n=66) all except 6 showed FITC and silver labelling resembling the situation in the 2-cell embryos, whereas the last 6 embryos displayed medium to large round clusters of FITC labelling in some or all of the nuclei colocalized with silver staining. The 5-8 cell embryos (n=22) and the blastocysts (n=16) showed up to 4 large clusters of FITC labelling colocalized with silver staining. Thus, rRNA synthesis started at the 4-cell stage. In the second experiment we addressed the question at which time during the 3rd cycle the rRNA synthesis starts by fixing 4-cell embryos at either 10, 20 or 30 h post cleavage (hpc). At 10 and 20 hpc the embryos (n=24) showed the same FITC pattern as seen in the 2-cell embryos. At 30 hpc the collected embryos (n=6) displayed medium to large round clusters of FITC labelling co-localized with foci of silver labelling, demonstrating a detectable transcription. In the third experiment we inhibited rRNA transcription by Actinomycin D (10g/ml for 15 h). All treated 4-cell embryos (n=37) showed only small foci of FITC labelling in the nuclei whereas the control 4-cell embryos (30 hpc, n=11) displayed medium to large clusters of FITC labelling. Thus the rRNA detected in the 4-cell embryos is of embryonic origin. In conclusion, the embryonic rRNA transcription is initiated towards the end of the 3rd cell cycle and is associated with occurence of silver staining around the rRNA genes.

KEY WORDS: PORCINE, rRNA, EMBRYO, ACTIVATION