Department of Animal Science, Federal University of Ceará, Fortaleza, Brazil.
Laboratory of Protein Chemistry and Biochemistry, University of Brasília, Brasília, Brazil.
Reprod Domest Anim. 2021 Apr;56(4):586-603. doi: 10.1111/rda.13897. Epub 2021 Feb 2.
The present study was conducted to decipher the proteome of in vivo-produced pre-implantation ovine embryos. Ten locally adapted Morana Nova ewes received hormonal treatment and were inseminated 12 hr after ovulation. Six days later, 54 embryos (morula and blastocyst developmental state) were recovered from eight ewes and pooled to obtain sufficient protein for proteomic analysis. Extracted embryo proteins were analysed by LC-MS/MS, followed by identification based on four database searches (PEAKS, Proteome Discoverer software, SearchGUI software, PepExplorer). Identified proteins were analysed for gene ontology terms, protein clusters and interactions. Genes associated with the ovine embryo proteome were screened for miRNA targets using data sets of TargetScan (http://www.targetscan.org) and mIRBase (http://www.mirbase.org) servers. There were 667 proteins identified in the ovine embryos. Biological processes of such proteins were mainly related to cellular process and regulation, and molecular functions, to binding and catalytic activity. Analysis of the embryo proteins revealed 49 enriched functional clusters, linked to energy metabolism (TCA cycle, pyruvate and glycolysis metabolism), zona pellucida (ZP), MAPK signalling pathway, tight junction, binding of sperm to ZP, translation, proteasome, cell cycle and calcium/phospholipid binding. Sixteen miRNAs were related to 25 pre-implantation ovine embryo genes, all conserved in human, bovine and ovine species. The interaction network generated by miRNet showed four key miRNAs (hsa-mir-106b-5p; hsa-mir-30-5p; hsa-mir-103a-5p and hsa-mir-106a-5p) with potential interactions with embryo-expressed genes. Functional analysis of the network indicated that miRNAs modulate genes related to cell cycle, regulation of stem cell and embryonic cell differentiation, among others. Retrieved miRNAs also modulate the expression of genes involved in cell signalling pathways, such as MAPK, Wnt, TGF-beta, p53 and Toll-like receptor. The current study describes the first major proteomic profile of 6-day-old ovine embryos produced in vivo, setting a comprehensive foundation for our understanding of embryo physiology in the ovine species.
本研究旨在破译体内生产的绵羊胚胎的蛋白质组。10 只当地适应的莫拉纳诺瓦母羊接受激素治疗，并在排卵后 12 小时接受授精。6 天后，从 8 只母羊中回收了 54 个胚胎（桑葚胚和囊胚发育状态），并将其汇集起来，以获得足够的蛋白质进行蛋白质组学分析。提取的胚胎蛋白通过 LC-MS/MS 进行分析，然后基于四个数据库搜索（PEAKS、Proteome Discoverer 软件、SearchGUI 软件、PepExplorer）进行鉴定。鉴定的蛋白质被分析了基因本体论术语、蛋白质簇和相互作用。使用 TargetScan（http://www.targetscan.org）和 mIRBase（http://www.mirbase.org）服务器的数据集筛选与绵羊胚胎蛋白质组相关的基因的 miRNA 靶标。在绵羊胚胎中鉴定出 667 种蛋白质。这些蛋白质的生物学过程主要与细胞过程和调节以及分子功能有关，与结合和催化活性有关。胚胎蛋白分析显示 49 个丰富的功能簇与能量代谢（TCA 循环、丙酮酸和糖酵解代谢）、透明带（ZP）、MAPK 信号通路、紧密连接、精子与 ZP 的结合、翻译、蛋白酶体、细胞周期和钙/磷脂结合有关。16 个 miRNA 与 25 个绵羊胚胎前植入基因有关，这些基因在人类、牛和绵羊物种中都是保守的。miRNet 生成的互作网络显示了 4 个关键 miRNA（hsa-mir-106b-5p；hsa-mir-30-5p；hsa-mir-103a-5p 和 hsa-mir-106a-5p）与胚胎表达基因的潜在互作。网络的功能分析表明，miRNAs 调节与细胞周期、干细胞和胚胎细胞分化调节等相关的基因。提取的 miRNAs 还调节细胞信号通路中基因的表达，如 MAPK、Wnt、TGF-β、p53 和 Toll 样受体。本研究描述了首例在体生产的 6 天龄绵羊胚胎的主要蛋白质组学图谱，为我们了解绵羊物种的胚胎生理学奠定了全面的基础。
