205 RNA-SEq PROFILING OF BOVINE PRE-IMPLANTATION EMBRYOS

Abstract

In order to provide a comprehensive view of the transcriptome changes during the earliest stages of bovine development, we sequenced the total RNA content of bovine oocytes, 4-cell, 8-cell, and 16-cell embryos and the inner cell mass and trophoblast envelope of expanded blastocysts on the Illumina Genome Analyzer IIx. For each experiment pools of in vitro matured oocytes from the German Simmental cows were fertilized by sperm of a single bull, and 10 oocytes or embryos per developmental stage were collected to generate total RNA pools used for sequencing. Synthesis of cDNA was initiated directly in the cell lysate in order to avoid any losses during RNA preparation and was random primed in order to capture all RNA species. Amplified cDNA and unstranded sequencing libraries were prepared using kits from Nugen (Ovation RNA-Seq, Nugen, San Carlos, CA, USA). Biological replicates were generated by inseminating the oocytes with sperm from the distant breeds Jersey (n = 3) and Brahman (n = 3). This cross-breeding design allowed tracking of single sequencing reads back to the maternal or paternal genome, where breed-specific SNP are present in the expressed transcripts. The analysis of this dataset resulted in monitoring of zygotic genome activation and parent-specific expression for single transcripts, a catalogue of splicing isoforms, novel transcripts, and non-coding RNAs and differentially expressed genes between the single developmental stages. Using the program DESEqn, 2784 genes showed differential expression between any of the stages at a false discovery rate of 1%. Specifically, we found 200 genes differentially expressed between immature and matured oocytes, 209 genes between matured oocytes and 4-cell embryos, 580 genes between the 4-cell and 8-cell stage, 567 genes between the 8-cell and 16-cell stage, 987 genes between the 16-cell stage and the inner cell mass, and 1569 genes between the 16-cell and the trophoblast. Functional analysis revealed stage-specific functions of the differentially expressed genes. In summary, by fully exploiting the single-nucleotide resolution of the RNA-Seq method, this dataset provides an invaluable resource for the study of zygotic genome activation, imprinting, transcript annotation, and gene expression in the earliest developmental stages of bovine embryos.