Early steps of embryo implantation are regulated by exchange of extracellular vesicles between the embryo and the endometrium

Abstract

In early pregnancy, as the embryo arrives in the uterus, intensive communication between the embryo and uterus begins. Hundreds of molecules are known to be involved, but despite numerous findings, full understanding of the complexity of the embryo-maternal dialog remains elusive. Recently, extracellular vesicles, nanoparticles able to transfer functionally active cargo between cells, have emerged as important players in cell-cell communication, and as such, they have gained great attention over the past decade also in reproductive biology. Here, we use a domestic animal model (Sus scrofa) with an epitheliochorial, superficial type of placentation because of its advantage in studding uterine luminal fluid extracellular vesicles. We show that during early pregnancy, the uterine lumen is abundant with extracellular vesicles that carry a plethora of miRNAs able to target genes involved in embryonic and organismal development. These extracellular vesicles, upon the delivery to primary trophoblast cells, affect genes governing development as well as cell-to-cell signaling and interactions, consequently having an impact on trophoblast cell proliferation, migration, and invasion. We conclude that the exchange of a unique population of extracellular vesicles and their molecular cargo at the maternal-embryo interface is the key to the success of embryo implantation and pregnancy.

Keywords: embryo; extracellular vesicles; implantation; invasion; miRNA; migration; pregnancy; proliferation; transcriptome; trophoblast.

FIGURE 1

The uterine lumen of early pregnancy is abundant in EVs released by the both endometrium and conceptuses. (A) Immunolocalization of CD63+ cells in the endometrium (top) and conceptuses (bottom) during consecutive days (D) of pregnancy (D12, D14, and D16). Control staining was performed without primary antibodies. (B) Nanoparticle Tracking Analysis was used to measure the size and concentration of EVs derived from the porcine uterine lumen at D12, D14, and D16 of pregnancy. Representative profile of particle distribution (left) along with statistical analysis of particle concentration (middle) and particle size distribution (right) for all tested days of pregnancy are presented. *p = .0343 (one‐way ANOVA and Tukey's multiple comparisons test). (C) Transmission electron microscope images for EVs collected from uterine lumen on D12, D14, and D16 of pregnancy. Widefield (left) and close‐up (right) images are shown. (D) The protein concentration for EVs samples collected during pregnancy (D12, D14, and D16) supported by the detection of EV protein markers (CD63, HSP70, Syntenin, TSG101). AGO2 and Carleticulin were used as negative markers. *p = .0265 (one‐way ANOVA and Tukey's multiple comparisons test).

FIGURE 2

Uterine‐derived EVs affect trophoblast cells' migration, invasion, and proliferation. (A) The migration of pTr cells tested in the wound‐healing assay in time and dose‐dependent manner. After EVs treatment of pTr cells with three different doses (0.2%, 2%, 4%) for 6 h (top panel) or 12 h (bottom panel), scratch‐induced migration was observed for 24 h, and snap shots were taken every 3 h. Mobility ratio was calculated at each time point and presented (line graph). Representative images at time 0 and 18 h are shown. The area under the curve for each condition was calculated (column bar graph). *p = .0255; **p = .0018 (repeated measures one‐way ANOVA and Dunnett's multiple comparisons test). (B) The number of pTr cells invaded through Matrigel‐coated porous membrane. NCS was used as a chemoattractant. *p ≤ .05 (repeated measures one‐way ANOVA and Dunnett's multiple comparisons test). (C) Proliferation of pTr cells after treatment with 2% of EVs (vs. control). NCS was used as a positive control. *p = .0134; **p = .0096 (repeated measures one‐way ANOVA and Dunnett's multiple comparisons test).

FIGURE 3

Extracellular vesicles affect the transcriptome of pTr cells. (A) Heatmap for paired samples clustered according to differentially expressed genes after EVs treatment of pTr cells. Legend presents log2 fold change values (ctrl vs treatment). (B) Top five canonical pathways (top), molecular and cellular functions (middle) and physiological system development and function (bottom) significantly enriched in pTr cells after EV. Ratio denotes the number of significantly expressed genes compared to the total number of genes associated with the canonical pathway. p‐values were calculated with a right‐tailed Fisher's Exact Test. (C) The molecular activation prediction network created using Ingenuity knowledge base and differentially expressed genes in pTr cells after treatment with uterine‐derived EVs (vs. ctrl). Colors indicate predicted relationships between gene expression levels and biofunctions, and color intensities reflect the degree of gene expression or bio‐function activity (see prediction legend for details).

FIGURE 4

Uterine‐derived extracellular vesicles carry miRNA important for organismal and embryonic development. (A) Representative Bioanalyzer electropherograms of EVs RNA generated using the Agilent RNA 6000 Pico (upper) and small RNA (lower) Kits. Graphs show concentrations of total, small, and microRNA as well as % of miRNA in total RNA for EVs collected from uterine lumen during consecutive days (D) of pregnancy (D12, D14, and D16; one‐way ANOVA and Tukey's multiple comparisons test). (B) Circos plot showing the miRNA abundance in uterine‐derived EVs collected on D12, D14, and D16 of pregnancy. The color scale of the heatmap pictures the abundance level as shown in the legend. miRNAs differentially expressed between tested days are marked in bold (one‐way ANOVA and Tukey's multiple comparisons test; detailed results are available in Table S2). (C) Ten miRNAs detected in TLDA analysis were validated using RT‐qPCR. *p < .05; **p < .01; ***p < .001 (one‐way ANOVA and Tukey's multiple comparisons test; detailed results are available in Table S2). (D) Confocal microscopy images showing uptake of labeled EVs' RNA by pTr cells. RNA cargo carried by EVs was stained with thiazole orange (green). F‐actin was stained with Alexa Fluor 488 Phalloidin (red). Nuclei were stained with Hoechst 33342 (blue).

FIGURE 5

Predicted interaction of miRNAs carried by uterine EVs and mRNAs in conceptuses/trophoblasts at the peri‐implantation stage. (A) Top 5 subnetworks generated by clustering interactions of miRNAs detected in uterine‐derived EVs and genes differentially expressed in conceptuses on day (D) 15 of pregnancy (vs. D12). The miRNA names have different font size based on their abundance (log2 [mean × 100]) at D16 in uterine‐derived EVs. Each label is colored based on log2 fold change in gene expression in conceptuses at D15 (vs. D12). (B) Possible miRNA‐mRNA interactions that occurred after pTr cells treatment with EVs. The EVs' miRNA names have different font size based on their abundance (log2 [mean × 100]) at D16 of pregnancy. Each label is colored based on log2 fold change in gene expression in pTr cells after EVs treatment (vs. ctrl).