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RESEARCH ARTICLE

66 Unexpected Protein Dynamics During the Oocyte-to-Embryo Transition in Mice: a Mass Spectrometry and RNA Sequencing Tandem Study

S. Israel A , M. Ernst C , O. E. Psathaki E , H. C. Drexler A , E. Casser A , Y. Suzuki F , W. Makalowski D , G. Fuellen C , M. Boiani A and L. Taher B
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- Author Affiliations

A Max Planck Institute for Molecular Biomedicine, Muenster, Germany;

B University Erlangen-Nuernberg, Erlangen, Germany;

C Institute for Biostatistics and Informatics in Medicine and Ageing Research, Rostock, Germany;

D Westfaelische Wilhelms-Universität, Muenster, Germany;

E University of Osnabrueck, Osnabrueck, Germany;

F University of Tokyo, Kashiwa-shi, Chiba-ken, Japan

Reproduction, Fertility and Development 30(1) 172-172 https://doi.org/10.1071/RDv30n1Ab66
Published: 4 December 2017

Abstract

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 mice, the hexagonal-shaped free ribosomes that translate mRNAs into proteins are rare during cleavage and only become abundant at the morula-blastocyst stage. Thus, how well 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, with a long-term aim to illuminate the functional interconnection between transcriptome, proteome, and morphogenetic transitions in mouse embryos. To obtain cellular material in sufficient numbers for a proteomic approach, we recovered B6C3F1 × CD1 fertilized oocytes in vivo after ovarian stimulation, and cultured them in KSOM(aa) medium under 5% CO2 in air. We collected unfertilized oocytes and embryos from 6 pre-implantation 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 labelled F9 embryonal carcinoma cells, resulting in light/heavy (L/H) protein mixtures. These were analysed using LTQ Orbitrap (Thermo Fisher Scientific, Waltham, MA, USA) and Q-Exactive (Thermo Fisher Scientific) instruments to yield L/H ratios for each measured protein, which were compared to transcript levels measured by RNA sequencing using an Illumina HiSEqn 2500 platform (100 oocytes or embryos per replicate; Illumina Inc., San Diego, CA, USA). All statistical analyses were performed in R (https://www.r-project.org/). 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 distinct proteome and transcriptome developmental profiles. Furthermore, hierarchical clustering of the proteomes identified 2 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 signalling among the proteins and metabolism and translation among the RNAs. Our data provide new insight into the regulation of the transition from the differentiated oocyte into the embryo, highlighting the different gene identities and different biological processes featured by the proteins that change quantitatively across consecutive developmental stages, compared with mRNAs. The status of the mouse as model system in developmental biology is enriched with a protein dimension, and caution is called for in the use of transcript dynamics as proxy for protein dynamics.


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