Activation of ribosomal RNA genes in porcine embryos produced in vitro or by somatic cell nuclear transfer.

The onset of ribosomal RNA (rRNA) synthesis occurs during the second half of the third cell cycle, that is, at the four-cell stage, in porcine embryos developed in vivo. In the present study the onset of rRNA synthesis was investigated in porcine embryos produced in vitro (IVP) or by somatic cell nuclear transfer (SCNT) using fluorescence in situ hybridization (FISH) with an rDNA probe and subsequent visualization of the nucleolar proteins by silver staining. In the 205 IVP embryos investigated, all two-cell embryos (n = 34) were categorized as transcriptionally inactive. At the late four-cell stage (n = 45), 38% of the embryos contained 1-3 nuclei with signs of rRNA transcription, indicating an asynchronous transcription initiation. This pattern continued in the following stages, as 78% (n = 47), 47% (n = 42) and 83% (n = 37) of the embryos revealed a mixture of transcriptionally inactive and active cells at the eight-cell, 16-cell and blastocyst stage, respectively. In the 143 SCNT embryos investigated, all two-cell embryos (n = 34) and early four-cell embryos (n = 12) were also transcriptionally inactive. At the late four-cell stage (n = 33) and at the eight-cell stage (n = 24) there were equal proportions of transcriptionally active and inactive embryos and essentially all embryos that developed to the 16-cell stage (n = 21) and further to the blastocyst stage (n = 19) contained only transcriptionally active cells. In conclusion, porcine embryos produced in vitro had an asynchronous pattern of rRNA transcription initiation when compared to SCNT and in vivo developed porcine embryos.

Transcription of ribosomal RNA genes is initiated in the third cell cycle of bovine embryos.

Transcription from the embryos own ribosomal genes is initiated in most species at the same time as the maternal-embryonic transition. Recently data have indicated that a minor activation may take place during the third embryonic cell cycle in the bovine, one cell cycle before the major activation of the embryonic genome. In the present study, ribosomal RNA (rRNA) transcription was investigated by visualization of the rRNA by fluorescent in situ hybridization, and subsequent visualization of the argyrophilic nucleolar proteins by silver staining. A total of 145 in vivo developed and 200 in vitro produced bovine embryos were investigated to allow comparison of transcription initiation. Signs of active transcription of rRNA were observed in the third cell cycle in 29% of the in vitro produced embryos (n = 35) and in 58% of the in vivo developed embryos (n = 11). Signs of active transcription of rRNA were not apparent in the early phase of the fourth cell cycle but restarted later on. All embryos in the fifth or later cell cycles were all transcribing rRNA. The signs of rRNA synthesis during the third and fourth embryonic cell cycle could be blocked by actinomycin D, which is a strong inhibitor of RNA polymerase I. In conclusion, rRNA transcription is initiated during the third cell cycle at a low level in both in vivo developed and in vitro produced bovine embryos. Transcription seems to be interrupted during the G1 phase of the fourth cell cycle, but reinitiates in the late half of the cycle and persists during subsequent cell cycles.

Few polyploid blastomeres in morphologically superior bovine embryos produced in vitro.

Morphologically inferior bovine embryos developed in vivo have been shown by karyotyping to have a higher rate of chromosomally abnormal cells than morphologically normal embryos. The objective of this study was to re-examine this finding using interphase cytogenetics. A total of 155 IVP Day 8 bovine blastocysts were graded by their morphology (excellent, good, or poor) and timing of development (hatched, expanded, or non-expanded), and afterwards analysed for chromosome abnormalities by fluorescence in situ hybridization using differentially labelled probes for chromosomes 6 and 7. The overall frequency of diploid embryos was 7%, and did not differ according to grading. Although the frequency of mixoploidy was not correlated to the morphological grading, the blastocysts with excellent morphology displayed fewer polyploid nuclei in comparison to blastocysts with good (P=0.05) or poor morphology (P=0.01). There were however also prominent exceptions showing that a blastocyst with an excellent morphology can display a high degree of polyploidy. The results further demonstrate that the morphologically normal embryos contain a higher number of cells and develop more rapidly than the morphologically inferior embryos.