| description |
The development of a zygote from fertilization through implantation is orchestrated by a series of changes in gene expression at the RNA and protein levels. In the mouse model, the hexagonal-shaped free ribosomes that translate mRNAs into proteins are rare during cleavage and only become abundant at the morula-blastocyst stage. Thus, can we understand development by an analysis of transcripts as proxy for the proteins? This question led us to combine mass spectrometry with RNA sequencing, aiming to illuminate the relationship between the developmental proteome and transcriptome in mice. To obtain cellular material in sufficient numbers for a proteomic approach, we recovered B6C3F1 x CD1 fertilized oocytes in vivo after ovarian stimulation, and cultured them in KSOM(aa) medium under 5% CO2 in air at 37 degrees Celsius. We collected unfertilized oocytes and embryos from six preimplantation developmental stages (pronuclear oocyte, 2-cell embryo, 4-cell embryo, 8-cell embryo, advanced morula, and blastocyst) in triplicates (600 oocytes or embryos per replicate). The protein lysates of these samples were added with equimolar amounts of isotopically labeled F9 embryonal carcinoma cells, resulting in light/heavy (L/H) protein mixtures. These were analyzed using LTQ Orbitrap and Q-Exactive instruments to yield L/H ratios for each measured protein, which were compared to transcript levels measured by RNA sequencing using an Illumina HiSeq 2500 platform (100 oocytes or embryos per replicate). All statistical analysis was performed in R. Collectively, 6976 proteins were detected in at least the F9 cells (precondition for quantifying the L/H ratios in oocytes or embryos). In particular, 4991 proteins were detected in all developmental stages, and 1893 proteins were detected in all replicates. Spearman correlation analysis of each stage relative to unfertilized oocyte revealed different proteome and transcriptome developmental profiles. Furthermore, hierarchical clustering of the proteomes identified two main clusters (cluster 1: oocyte to 16-cell embryo; cluster 2: blastocyst) which are different from the well known clusters of the transcriptomes (cluster 1: oocyte to 2-cell embryo; cluster 2: 4-cell embryo to blastocyst). Functional analysis of the genes that are differently expressed across adjacent stages highlighted the developmental roles of DNA damage response and Wnt signaling among the proteins and metabolism and translation among the RNAs. In conclusion, our data provides new insight into the regulation of the transition from the differentiated oocyte into the embryo, focusing on the proteins that change in abundance from one embryonic stage to the next, suggesting important regulators of the corresponding morphogenetic transitions. |