66 Unexpected Protein Dynamics During the Oocyte-to-Embryo Transition in Mice: a Mass Spectrometry and RNA Sequencing Tandem Study
66 Unexpected Protein Dynamics During the Oocyte-to-Embryo Transition in Mice: a Mass Spectrometry and RNA Sequencing Tandem Study
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66 小鼠卵母细胞到胚胎转变过程中意外的蛋白质动态：质谱和 RNA 测序串联研究
DOI：
10.1071/rdv30n1ab66
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Taher L
中科院分区：
文献类型：
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作者：
Israel S;Ernst M;Psathaki OE;Drexler HC;Casser E;Suzuki Y;Makalowski W;Fuellen G;Boiani M;Taher L
从受精到着床，受精卵的发育是由RNA和蛋白质水平上基因表达的一系列变化精心安排的。在小鼠中，将mrna转化为蛋白质的六角形游离核糖体在卵裂期间很少见，仅在桑葚胚期变得丰富。因此，通过分析转录本作为蛋白质的代理，我们能在多大程度上理解发育？这个问题促使我们将质谱法与RNA测序相结合，其长期目标是阐明小鼠胚胎中转录组、蛋白质组和形态发生转变之间的功能联系。为了获得足够数量的细胞材料用于蛋白质组学方法，我们在卵巢刺激后在体内回收B6C3F1 × CD1受精卵母细胞，并将其培养在空气中5% CO2的KSOM（aa）培养基中。我们收集了6个着床前发育阶段的未受精卵母细胞和胚胎（原核卵母细胞、2细胞胚胎、4细胞胚胎、8细胞胚胎、晚期桑葚胚和囊胚），分成3个重复（每个重复600个卵母细胞或胚胎）。这些样品的蛋白裂解物加入等量的同位素标记的F9胚胎癌细胞，得到轻/重（L/H）蛋白混合物。使用LTQ Orbitrap （Thermo Fisher Scientific, Waltham， MA， USA）和Q-Exactive （Thermo Fisher Scientific）仪器对这些进行分析，以产生每个测量蛋白的L/H比率，并将其与使用Illumina HiSEqn 2500平台（每个重复100个卵母细胞或胚胎；Illumina Inc., San Diego， CA， USA）的RNA测序测量的转录物水平进行比较。所有统计分析均在R （https://www.r-project.org/）中进行。总共在至少F9细胞中检测到6976个蛋白（定量卵母细胞或胚胎的L/H比的先决条件）。特别是在所有发育阶段均检测到4991蛋白，在所有重复中均检测到1893蛋白。与未受精卵母细胞相关的Spearman相关分析揭示了不同的蛋白质组和转录组发育特征。此外，蛋白质组的分层聚类鉴定出2个主要簇（簇1：卵母细胞到16细胞胚胎；簇2：囊胚），这与众所周知的转录组簇（簇1：卵母细胞到2细胞胚胎；簇2:4细胞胚胎到囊胚）不同。对相邻阶段表达不同的基因的功能分析强调了DNA损伤反应和Wnt信号在蛋白质和rna代谢和翻译中的发育作用。我们的数据为从分化的卵母细胞向胚胎过渡的调控提供了新的见解，突出了与mrna相比，在连续发育阶段定量变化的蛋白质所具有的不同基因身份和不同生物学过程。在发育生物学中，小鼠作为模型系统的地位丰富于蛋白质维度，在使用转录物动力学作为蛋白质动力学的代理时需要谨慎。
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.