ECM proteins involved in cell migration and vessel formation compromise bovine cloned placentation

Abstract

Advances in Artificial Reproductive Technologies (ARTs) in bovine embryos to produce cloned pregnancies have been developed in the last years, however high pregnancy losses rates still present. Those rates are associated to placental morphology alterations that are majorly focused on extracellular matrix (ECM) alterations and consequently placentome hyperplasia, increased trophoblast cell migration and vascular defects. Herein, we aimed to search, at protein level, pathways altered by ART that can modify the placental development harmony. For this, we used 4-month-old control (n = 3), SDS-decellularized (n = 3) and cloned (n = 3) cotyledons for proteomic analysis. Samples were grouped by condition and were washed, lysed, urea-reduced, acetone-precipitated, DTT-educed, iodoacetamide-alkylated, trypsin digested, and C-18 column purified. At the end, 3 μg protein were loaded in Orbitrap Fusion Lumos spectrometer (ThermoScientific). Generated spectra were exported to MaxQuant software (v1.6.10.43) to produce the protein list of each sample, and the LFQ intensity were statistically analyzed by Inferno software (v.1.1.6970). After this, proteins related to ECM and cellular junction ontologies were filtered and manually annotated using DAVID Bioinformatics Resources 6.8. From 2577 identified protein sequences by MaxQuant software, 165 (7.1%) were filtered by selected ontologies. We found 10 proteins (B2M, COL6A6, FERMT3, LGALS3BP, NIBAN2, PDLIM5, PON1, PRP9, RASIP1 and SPARC) upregulated in clone, when compared to control condition. The ten pathways that enriched more proteins were: focal adhesion, ECM-receptor interaction, PI3K-Akt signaling pathway, protein digestion and absorption, amoebiasis, pathways in cancer, small cell lung cancer, platelet activation, regulation of actin cytoskeleton, and proteoglycans in cancer. Functionally, detected proteins, signaling pathways and ontologies are orchestrated to permit the binucleated trophoblastic cells migration and blood vessels modelling. In conclusion, the cloned condition presents the same mechanisms as control one, however overexpression of some specific ECM proteins could be responsible to exacerbate those mechanisms and can explain all morphophysiological alterations presented in cloned pregnancies associated to high pregnancies losses rates in this condition.