Extracellular Vesicles Secreted by Pre-Hatching Bovine Embryos Produced In Vitro and In Vivo Alter the Expression of IFNtau-Stimulated Genes in Bovine Endometrial Cells

Abstract

The embryo-maternal interaction occurs during the early stages of embryo development and is essential for the implantation and full-term development of the embryo. In bovines, the secretion of interferon Tau (IFNT) during elongation is the main signal for pregnancy recognition, but its expression starts around the blastocyst stage. Embryos release extracellular vesicles (EVs) as an alternative mechanism of embryo-maternal communication. The aim of the study was to determine whether EVs secreted by bovine embryos during blastulation (D5-D7) could induce transcriptomic modifications, activating IFNT signaling in endometrial cells. Additionally, it aims to assess whether the EVs secreted by embryos produced in vivo (EVs-IVV) or in vitro (EVs-IVP) have different effects on the transcriptomic profiles of the endometrial cells. In vitro- and in vivo-produced bovine morulae were selected and individually cultured for 48 h to collect embryonic EVs (E-EVs) secreted during blastulation. E-EVs stained with PKH67 were added to in vitro-cultured bovine endometrial cells to assess EV internalization. The effect of EVs on the transcriptomic profile of endometrial cells was determined by RNA sequencing. EVs from both types of embryos induced several classical and non-classical IFNT-stimulated genes (ISGs) and other pathways related to endometrial function in epithelial endometrial cells. Higher numbers of differentially expressed genes (3552) were induced by EVs released by IVP embryos compared to EVs from IVV (1838). Gene ontology analysis showed that EVs-IVP/IVV induced the upregulation of the extracellular exosome pathway, the cellular response to stimulus, and the protein modification processes. This work provides evidence regarding the effect of embryo origin (in vivo or in vitro) on the early embryo-maternal interaction mediated by extracellular vesicles.

Keywords: bovine embryos; embryo-maternal communication; endometrial bovine cells; extracellular vesicles; interferon tau.

Figure 1

Size distribution (X-axis) and concentration (Y-axis; (10⁹ particles × mL)) of EVs released by bovine embryos produced in vitro (A) or in vivo (B) during blastulation (morula-blastocyst). The graphics represent the average of three technical replicates, based on the diameter and concentration distribution of nanoparticles.

Figure 2

EVs secreted by bovine embryos during blastulation (morula to blastocyst). (a,b): representative images of EVs isolated from culture medium of embryos produced in vivo and vitro, respectively. Arrows are pointing identified EVs in the representative pictures.

Figure 3

Fluorescence images of epithelial endometrial cells incubated with EVs secreted during the blastulation of bovine embryos produced in vivo (EVs-IVV) or in vitro (EVs-IVP). (a) EVs released from embryos produced in vitro. (b) EVs released from embryos produced in vivo. (c) Negative control (PBS/PHK67). Cells were incubated with stained vesicles for 24 h. Arrows highlight the green dots in the cell cytoplasm, indicating internalized EVs stained with PHK67 dye.

Figure 4

Analysis of deregulated genes in bovine epithelial endometrial cells due to the effect of extracellular vesicles released during blastulation by bovine embryos produced in vivo (IVV) or in vitro (IVP). (a,b): Volcano plot showing the comparison between EVs from IVV embryos and PBS (negative control) and between EVs from IVP embryos and PBS (negative control), respectively. (c): Number of deregulated genes (up or down), comparing EVs from in vitro produced embryos with PBS (IVP-control), and EVs from in vivo produced embryos with PBS (IVV-control). (d). Venn diagram of differentially expressed genes from the analyses. Venn diagram was constructed using Venny 2.1 https://bioinfogp.cnb.csic.es/tools/venny/; accessed on 6 December 2022.

Figure 5

Heat map profile of differentially expressed genes related to IFNT signaling and endometrial function. Three clusters were defined: Cluster 1 includes genes associated with sex steroid signaling and interferon signaling; Cluster 2 includes solute and water transport secretory activity, proliferation activity, and interferon signaling; Cluster 3 includes extracellular matrix remodeling, proliferation activity and interferon signaling.

Figure 6

Effect of extracellular vesicles (EVs) secreted by bovine embryos produced in vivo (IVV) or in vitro (IVP) on the gene expression pattern of epithelial endometrial cells.

Figure 7

Fold change graphic comparing the fold change in RNA seq and RT qPCR of each evaluated gene. Data on Y-axis represent the log of fold change obtained from PCR analysis (red) and RNAseq (green). A Pearson correlation analysis showed a similar fold change tendency for most of the genes except MX2 in the EVs-IVP group.

Figure 8

Illustration of experimental design. Extracellular vesicles (EVs) were isolated from a culture media of in vitro (IVP)- or in vivo (IVV)-derived bovine embryos. Morulae were selected on Day 5 of development and cultured individually until Day 7. Culture media were collected from embryos that developed to blastocyst stage. Isolated EVs were stained with PKH67 and added to in vitro-cultured bovine endometrial cells to assess EV internalization 24 h later. Cells were harvested 48 h after the addition of EVs for gene expression analysis. Cells treated with PBS were used as a negative control.