Ribosomal ribonucleic acid is transcribed at the 4-cell stage in in vitro-produced bovine embryos.
Ribosomal RNA, rRNA genes, and silver-staining nucleolar proteins were visualized in in vitro-produced bovine embryos from the 2-cell stage to the blastocyst using a sequential fluorescent in situ hybridization (FISH) and a silver-staining procedure. At FISH, the rRNA was differentiated from the signal of the rRNA genes through comparison of RNase- and non-RNase-treated embryos. Both RNase- and non-RNase-treated 2-cell embryos revealed up to 10 small clusters of fluorescein isothiocynate (FITC) labeling in interphase nuclei. The RNase-treated 4-cell embryos displayed the same FITC pattern as the 2-cell embryos. In the non-RNase-treated 4-cell embryos, in contrast, the clusters were larger and included numerous small spots. In 2-cell as well as 4-cell embryos, almost all FITC-labeled clusters colocalized with silver-stained spots. In the RNase-treated 8- to 16-cell embryos, up to 10 clusters of FITC labeling were organized as one or more large spots surrounding a central faint but homogeneously labeled area. The non-RNase-treated 8- to 16-cell embryos displayed similar complexes, but the central areas consisted of small labeled spots. In 8- to 16-cell embryos, all FITC-labeled clusters were again colocalized with silver-stained areas. In the blastocysts, 1-6 big clusters of FITC labeling colocalized with silver staining. In the RNase-treated blastocysts, the FITC labeling was typically located at the edges of the silver-stained areas, whereas in the non-RNase-treated blastocysts, the FITC labeling totally covered the silver-stained areas. In conclusion, there is a close association between the rRNA genes and silver-staining nucleolar proteins in in vitro-produced bovine embryos from the second cell cycle, i.e., the 2-cell stage; the first rRNA is apparently transcribed during the third cell cycle, and during the fourth cell cycle the molecular composition of functional nucleoli is established.
Department of Clinical Studies, Reproduction, Agricultural University, DK-1879 Frederiksberg C, Denmark. dv@kvl.dk
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Viuff
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Hyttel
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Avery
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Vajta
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Greve
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Callesen
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Thomsen
D000818:Animals; D001755:Blastocyst / Q000378:metabolism*; D002417:Cattle / Q000196:embryology*; D005307:Fertilization in Vitro; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D017404:In Situ Hybridization, Fluorescence; D012335:RNA, Ribosomal / Q000235:genetics*; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012339:RNA, Ribosomal, 28S / Q000235:genetics; D012340:RNA, Ribosomal, 5.8S / Q000235:genetics; D012260:Ribonucleases / Q000494:pharmacology; D016622:Silver Staining; D014158:Transcription, Genetic
D005456:Fluorescent Dyes; D012335:RNA, Ribosomal; D012337:RNA, Ribosomal, 18S; D012339:RNA, Ribosomal, 28S; D012340:RNA, Ribosomal, 5.8S; D012260:Ribonucleases; D016650:Fluorescein-5-isothiocyanate