Fine mapping of genome activation in bovine embryos by RNA sequencing

Abstract

During maternal-to-embryonic transition control of embryonic development gradually switches from maternal RNAs and proteins stored in the oocyte to gene products generated after embryonic genome activation (EGA). Detailed insight into the onset of embryonic transcription is obscured by the presence of maternal transcripts. Using the bovine model system, we established by RNA sequencing a comprehensive catalogue of transcripts in germinal vesicle and metaphase II oocytes, and in embryos at the four-cell, eight-cell, 16-cell, and blastocyst stages. These were produced by in vitro fertilization of Bos taurus taurus oocytes with sperm from a Bos taurus indicus bull to facilitate parent-specific transcriptome analysis. Transcripts from 12.4 to 13.7 × 10 3 different genes were detected in the various developmental stages. EGA was analyzed by (i) detection of embryonic transcripts, which are not present in oocytes; (ii) detection of transcripts from the paternal allele; and (iii) detection of primary transcripts with intronic sequences. These strategies revealed (i) 220, (ii) 937, and (iii) 6,848 genes to be activated from the four-cell to the blastocyst stage. The largest proportion of gene activation [i.e., (i) 59%, (ii) 42%, and (iii) 58%] was found in eight-cell embryos, indicating major EGA at this stage. Gene ontology analysis of genes activated at the four-cell stage identified categories related to RNA processing, translation, and transport, consistent with preparation for major EGA. Our study provides the largest transcriptome data set of bovine oocyte maturation and early embryonic development and detailed insight into the timing of embryonic activation of specific genes.

Positional changes of a pluripotency marker gene during structural reorganization of fibroblast nuclei in cloned early bovine embryos

“…Notably, GOF activation occurred during the same period of preimplantation development as major embryonic gene activation in IVF embryos at the 8-cell stage. 3,17 Apparently major reprogramming of gene expression signified by GOF activation was correlated with structural changes of nuclei in cloned embryos, which were strikingly similar with structural changes observed in IVF embryos. 11 Cloned embryos approaching the 8-cell stage adopted Table 2.…”

“…2 In bovine embryos major embryonic genome activation takes place at the 8-cell stage, where the largest proportion of genes becomes active during preimplantation development. 3 Some genes, however, are already activated earlier during minor genome activation. 4 The functional implications of nuclear architecture changes, which occur during this period, are currently not well understood.…”

“…8 Prior to major embryonic genome activation at the 8-cell stage we observed a peripheral, radial distribution of both chromosome territories 19 and 20. 3 After major embryonic genome activation a gene density-related radial distribution of chromosome territories, 19 (more central) and 20 (more peripheral), was established. 8 The goal of the present investigation was to explore a possible link between pluripotency gene activation and specific radial movements of such genes in nuclei of cloned bovine embryos with the help of a marker gene for pluripotency gene activation (GOF).…”

“…17 Although causal relationships between pluripotency gene activation and major embryonic genome activation are still not clear, we suggest a temporal correlation of GOF activation with major embryonic genome activation in cloned bovine embryos and that this event occurs in both cloned and IVF bovine embryos at the 8-cell stage. 3 This model system was employed for a quantitative 3D-FISH study to answer the question, whether Oct4/Pou5f1 promoter activation in nuclei of cloned bovine embryos is preceded or followed by changes of the radial nuclear positioning of GOF in comparison with the radial positioning of silent GOF in fibroblast nuclei. We considered 2 scenarios for a hypothetical repositioning of GOF.…”

Oocyte-associated transcription factors in reprogramming after somatic cell nuclear transfer: a review

“…The transcriptome analysis of the different developmental stages from oocyte to morula in both mouse and human reveal a sequential order of transcriptional changes in pathways of cell cycle, gene regulation, translation and metabolism, acting in a stepwise fashion from cleavage to morula [92]. In cattle, RNA sequencing analysis of transcripts in GV and M II oocytes, and in 4-, 8-, 16-cell and blastocyst stage embryos indicated that the largest proportion of gene activation was found in 8-cell embryos [93]. Cross-species comparisons between mouse and human showed that significant difference in developmental specificity and timing existed [92].…”