Laboratório de Fisiologia e Ciências Ômicas, Departamento de Zootecnia, Universidade Federal do Ceará, Fortaleza, Brazil.
Instituto de Reproducción Animal Uruguay, Fundación IRAUy, Montevideo, Uruguay.
Reprod Domest Anim. 2022 Jul;57(7):784-797. doi: 10.1111/rda.14122. Epub 2022 Apr 24.
The present study was conducted to characterize the major proteome of preimplantation (D6) ovine embryos produced in vitro. COCs were aspirated from antral follicles (2-6 mm), matured and fertilized in vitro and cultured until day six. Proteins were extracted separately from three pools of 45 embryos and separately run in SDS-PAGE. Proteins from each pool were individually subjected to in-gel digestion followed by LC-MS/MS. Three 'raw files' and protein lists were produced by Pattern Lab software, but only proteins present in all three lists were used for the bioinformatics analyses. There were 2,262 proteins identified in the 6-day-old ovine embryos, including albumin, zona pellucida glycoprotein 2, 3 and 4, peptidyl arginine deiminase 6, actin cytoplasmic 1, gamma-actin 1, pyruvate kinase, heat shock protein 90 and protein disulfide isomerase, among others. Major biological processes linked to the sheep embryo proteome were translation, protein transport and protein stabilization, and molecular functions, defined as ATP binding, oxygen carrier activity and oxygen binding. There were 42 enriched functional clusters according to the 2,147 genes (UniProt database). Ten selected clusters with potential association with embryo development included translation, structural constituent of ribosomes, ribosomes, nucleosomes, structural constituent of the cytoskeleton, microtubule-based process, translation initiation factor activity, regulation of translational initiation, cell body and nucleotide biosynthetic process. The most representative KEEG pathways were ribosome, oxidative phosphorylation, glutathione metabolism, gap junction, mineral absorption, DNA replication and cGMP-PKG signalling pathway. Analyses of functional clusters clearly showed differences associated with the proteome of preimplantation (D6) sheep embryos generated after in vitro fertilization in comparison with in vivo counterparts (Sanchez et al., 2021; https://doi.org/10.1111/rda.13897), confirming that the quality of in vitro derived blastocysts are unlike those produced in vivo. The present study portrays the first comprehensive overview of the proteome of preimplantational ovine embryos grown in vitro.
本研究旨在对体外生产的植入前（D6）绵羊胚胎的主要蛋白质组进行特征分析。从窦卵泡（2-6 毫米）中抽吸 COC，在体外成熟和受精，并培养至第 6 天。从三个 45 个胚胎的池中分别提取蛋白质，并分别在 SDS-PAGE 中运行。将每个池的蛋白质单独进行胶内消化，然后进行 LC-MS/MS。Pattern Lab 软件生成了 3 个“原始文件”和蛋白质列表，但仅使用 3 个列表中都存在的蛋白质进行生物信息学分析。在 6 日龄绵羊胚胎中鉴定出 2262 种蛋白质，包括白蛋白、透明带糖蛋白 2、3 和 4、肽基精氨酸脱亚氨酶 6、细胞质肌动蛋白 1、γ-肌动蛋白 1、丙酮酸激酶、热休克蛋白 90 和蛋白二硫键异构酶等。与绵羊胚胎蛋白质组相关的主要生物学过程是翻译、蛋白质运输和蛋白质稳定，以及分子功能，定义为 ATP 结合、氧载体活性和氧结合。根据 2147 个基因（UniProt 数据库），有 42 个富集的功能簇。与胚胎发育有潜在关联的 10 个选定簇包括翻译、核糖体结构成分、核糖体、核小体、细胞骨架结构成分、微管为基础的过程、翻译起始因子活性、翻译起始调节、细胞体和核苷酸生物合成过程。最具代表性的 KEEG 途径是核糖体、氧化磷酸化、谷胱甘肽代谢、缝隙连接、矿物质吸收、DNA 复制和 cGMP-PKG 信号通路。功能簇的分析清楚地显示了与体外受精产生的植入前（D6）绵羊胚胎蛋白质组相关的差异，与体内对应物相比（Sanchez 等人，2021 年；https://doi.org/10.1111/rda.13897），证实体外衍生的囊胚的质量与体内产生的囊胚不同。本研究描绘了体外生长的植入前绵羊胚胎蛋白质组的首次全面概述。
