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.
Viuff
D
D
Department of Clinical Studies, Reproduction, Agricultural University, DK-1879 Frederiksberg C, Denmark. dv@kvl.dk
Hyttel
P
P
Avery
B
B
Vajta
G
G
Greve
T
T
Callesen
H
H
Thomsen
P D
PD
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