Embryonic Trophectoderm Secretomics Reveals Chemotactic Migration and Intercellular Communication of Endometrial and Circulating MSCs in Embryonic Implantation

Abstract

Embryonic implantation is a key step in the establishment of pregnancy. In the present work, we have carried out an in-depth proteomic analysis of the secretome (extracellular vesicles and soluble proteins) of two bovine blastocysts embryonic trophectoderm primary cultures (BBT), confirming different epithelial-mesenchymal transition stages in these cells. BBT-secretomes contain early pregnancy-related proteins and angiogenic proteins both as cargo in EVs and the soluble fraction. We have demonstrated the functional transfer of protein-containing secretome between embryonic trophectoderm and maternal MSC in vitro using two BBT primary cultures eight endometrial MSC (eMSC) and five peripheral blood MSC (pbMSC) lines. We observed that eMSC and pbMSC chemotax to both the soluble fraction and EVs of the BBT secretome. In addition, in a complementary direction, we found that the pattern of expression of implantation proteins in BBT-EVs changes depending on: (i) their epithelial-mesenchymal phenotype; (ii) as a result of the uptake of eMSC- or pbMSC-EV previously stimulated or not with embryonic signals (IFN-); (iii) because of the stimulation with the endometrial cytokines present in the uterine fluid in the peri-implantation period.

Keywords: cell migration; epithelial to mesenchymal transition; extracellular vesicles; mesenchymal stromal cells; trophectoderm.

Figure 1

Bovine blastocyst embryonic trophectoderm primary cultures release EV-cargo proteins in homogeneous populations of EVs and soluble proteins. Representative size exclusion chromatography (SEC)-elution profile of EVs from BBT-9 and BBT-18 analyzed by bead-assisted flow cytometry using anti-CD63 and anti-CD9 antibodies. Mean fluorescence intensity (MFI) relative to the negative control is plotted in the left y-axis (a). Protein concentration was analyzed by Nanodrop for each fraction and plotted on the right y-axis. (a). SEC-elution profile of EVs from BBT-9 and BBT-18 analyzed by Dot Blot using an anti-CD9 specific antibody (b). Fresh BBT-9-EVs or BBT-18-EVs isolated were negatively stained with uranyl acetate and visualized by TEM (c). Quantitative results including diameter, perimeter, and roundness of BBT-9-EVs and BBT-18-EVs (d).

Figure 2

BBT-9- and BBT-18-derived secretomes contain EV-cargo proteins and soluble proteins. Venn diagrams of overrepresented EV-cargo proteins (a), or soluble proteins (b) identified by iTRAQ-LC-MS/MS analysis, overexpression ≥ 1.9-fold has been considered in all comparative analyses. Characteristic EMT expression markers identified and overrepresented in BBT-9-secretome and epithelial marker CDH1 overrepresented in BBT-18-soluble fraction (c).

Figure 3

Bovine blastocyst embryonic trophectoderm primary cultures release soluble proteins (a) and EV-cargo proteins (b) associated with early pregnancy.

Figure 4

Maternal MSCs show chemotactic migration to BBT-9- or BBT-18-secretome. Analysis of the chemotactic effect on rose diagrams of eMSC and pbMSC without stimuli (control) or stimulated by EVs or soluble proteins from BBT-9- or BBT-18-secretome (a). The maximum Euclidean distance (MED) of eMSC or pbMSC towards EVs or soluble proteins from BBT-9 or BBT-18 was measured (mean ± SD). Different letters indicate a significant difference. * p < 0.05; ** p < 0.005; **** p < 0.0001. (b). The center of mass (COM): a strong chemotactic migration parameter for the analysis of the migrating response of eMSC and pbMSC towards EVs or soluble proteins from BBT-9 or BBT-18. Charts showing the trajectory plots marking each cell track with its endpoint (c), or the center of mass length (d), of eMSC and pbMSC without stimuli (control) or stimulated by EVs or soluble proteins from BBT-9 or BBT-18. The velocity of eMSC or pbMSC towards EVs or soluble proteins from BBT-9 or BBT-18 was measured (mean ± SD). Different letters indicate a significant difference (e).

Figure 5

Experimental design.

Figure 6

BBT secret implantation proteins on EVs, and their secretion pattern, which is altered after the uptake of MSC-EVs or the presence of uterine cytokines. SEC-elution profile of EVs from BBT-9 and BBT-18 for the different experimental conditions (Control, Activin A, Activin A+FLST, pbMSC-EVs, τ-pbMSC-EVs, eMSC-EVs, and τ-eMSC-EVs) were analyzed by Dot Blot using an anti-CD9 specific antibody. The positive EV fractions of each experimental condition were quantified by ImageJ software analysis of the dot blot results (a). Bead-assisted flow cytometry analysis of selected implantation proteins expressed in BBT-9- (b) and BBT-18-EVs (c) after cell stimulation.

Figure 7

Representation of the BBT-identified proteins associated with the main reproductive or EMT events.