Proteomic characterization of eight-day-old equine blastocysts produced in vivo

Abstract

Early embryo development is dependent on molecular signatures of the gametes, gene expression and maternal-embryo interactions. The proteome of the embryo reflects its developmental state and defines its fate. The present study aimed to describe the proteome of 8-day old equine blastocysts produced in vivo. Follicular dynamics of eight mares were monitored by ultrasonography. When a preovulatory follicle was confirmed, mares received GnRH and were inseminated. After eight days, four embryos were recovered. Proteins were extracted from the embryos, pooled and subjected to mass spectrometry. Four bioinformatic platforms were used to increase the accuracy of the protein identification as they have different capacities and coverages. Overall, there was a list of 4,014 proteins, but there were overlaps among the bioinformatic search engines. After elimination of redundancies (at 3 levels, based on identifiers, peptides and cross-bank mapping), a final count of 1,977 proteins were identified reliably in the samples of equine embryos. There were 1,413 proteins identified by the Peaks program, 316 by Proteome Discoverer, 165 by SearchGUI, and 83 proteins using the PepExplorer program. The low number of overlaps observed for PepExploreris due to the nature of that particular search, which used peptides sequenced but not identified by the Peaks program. Biological processes of the embryo proteins identified included cellular and metabolic processes. Cellular components involved the cell, organelle and protein-containing complex. Molecular functions of embryo proteins included binding, catalytic activities and structural molecule activity. Six pathways relevant to embryo development (FDR < 0.05, Panther platform) related to the TCA cycle, pyruvate metabolism, glycolysis, purine metabolism, and the pentose phosphate pathway. Proteins of the 8-day old equine embryos were associated with pathways of cell-cell communication and extracellular matrix remodeling and pathways related to intracellular remodeling, such as cytoskeleton regulation by Rho GTPase. In summary, the current approach based on mass spectrometry and a combination of search engines allowed the identification of 1,977 proteins in 8-day-old equine embryos. This is, to date, the largest atlas of proteins from embryos of the equine species. Knowledge of protein expression by the embryo helps to understand its physiology, metabolic events and future identification of biomarkers for health.