Global analyses and potential effects of extracellular vesicles on the establishment of conceptus implantation during the peri-implantation period

Abstract

Intrauterine extracellular vesicles (EVs) are involved in establishing proper conceptus-endometrial communication, which is essential for conceptus implantation and subsequent successful placentation. Despite several studies on intrauterine EVs, the composition and quantitative changes in conceptus and endometrial EVs, as well as the effects of intrauterine EVs on endometrial epithelial cells (EECs) during the peri-implantation period, have not been well characterized. To elucidate global changes in proteins in EVs extracted from uterine flushings (UFs) during the pre-implantation (P17), just-implantation (P20), and post-implantation (P22) periods, the datasets of the proteome iTRAQ analysis were compared among P17, P20, and P22 EVs. These analyses revealed that the composition and function of proteins in the EVs changed dramatically during peri-implantation in cattle. Notably, intrauterine P17 EVs affected the high expression of "Developmental Biology" and "morphogenesis of an endothelium" compared with those in P20 and P22 EVs. Furthermore, P20 EVs had the functions of the high expression of "mitochondrial calcium ion homeostasis" and "Viral mRNA Translation" compared with those in P17 EVs. Transcripts extracted from EECs treated with P17, P20, or P22 EVs were subjected to RNA-seq analysis. These analyses identified 60 transcripts in EECs commonly induced by intrauterine EVs recovered from P17, P20, and P22, a large number of which were associated with "type I interferon signaling pathway". Collectively, these findings reveal the presence and multiple functions of EVs that are potentially implicated in facilitating conceptus implantation into the uterine epithelium during the peri-implantation period.

Keywords: Cow; Endometrial epithelial cells; Extracellular vesicles (EVs); Implantation; Pregnancy.

Fig. 1.

Characterization of EVs isolated from UFs on gestational days 17, 20, and 22. (A) Western blot analysis showed the presence of CD63 and HSP70 in pellets isolated from P17, P20, and P22 bovine UFs. Three independent experiments were performed, and a representative one is shown. (B) Transmission electron microscopy analysis revealed the presence of 50–150 nm vesicles in P17 UFs, consistent with those of EVs. Scale bar = 200 nm.

Fig. 2.

Proteins in P17 EVs compared with those in P20 and P22 EVs. (A) Heatmap analysis of proteins in P17, P20, and P22 EVs. (B) Venn diagram shows the number of highly expressed proteins with more than 1.5-fold changes in P17 EVs, among which, 40 proteins (red rectangle) were highly expressed compared with those in P20 and P22 EVs in common, and 56 proteins (red rectangle) were highly expressed compared with those in P22 EVs, respectively. (C) 40 highly expressed proteins in P17 EVs compared with those in P20 and P22 EVs in common were functionally classified by the biological process in GO terms and enriched pathway analyses. (D) 56 highly expressed proteins in P17 EVs compared with those in P22 EVs were functionally classified by the biological process in GO terms and enriched pathway analyses. (E) Venn diagram shows the number of highly expressed proteins with more than 1.5-fold changes in P20 and P22 EVs, from which 67 proteins (red rectangle) were highly expressed in P20 EVs compared with those in P17 EVs. (F) 67 highly expressed proteins in P20 EVs compared with those in P17 EVs were functionally classified by the biological process in enriched GO terms and enriched pathway analyses.

Fig. 3.

Upregulated transcripts in bovine EECs treated with intrauterine EVs during the peri-implantation period. (A) Biplot of transcripts in bovine endometrial epithelial treated with P17, P20, or P22 EVs, produced by the Partial least squares-discriminant analysis (PLS-DA). (B) The dot plot shows the factor loadings of PLS-DA. (C) RNAs were extracted from EECs incubated without (Intact) or with P17, P20, or P22 EVs (10 µg) for 48 h, and then were subjected to RNA-seq analysis. GAPDH mRNA served as internal control for RNA integrity. Data were obtained from three independent in vitro culture experiments. (D) Venn diagram shows the number of upregulated genes with 2.0-fold changes among P17, P20, and P22 EVs’ treatment groups compared with the control group, of which, 60 transcripts (red rectangle) were found in common among P17, P20, and P22 EVs’ treatment groups, and 135 transcripts (red rectangle) and 53 transcripts (red rectangle) were identified in only P17 or P20 EVs’ treatment group, respectively. (E) 60 transcripts found in common among P17, P20, and P22 EVs’ treatment groups were functionally classified by the biological process in enriched GO terms and enriched pathway analyses. (F) 135 upregulated transcripts in EECs treated with P17 EVs were functionally classified by the biological process in GO terms and enriched pathway analyses. (G) 53 upregulated transcripts in EECs treated with P20 EVs were functionally classified by the biological process in GO terms and enriched pathway analyses.

Fig. 4.

Downregulated transcripts in bovine EECs treated with intrauterine EVs during the peri-implantation period. (A) Venn diagram shows the number of downregulated genes with 2.0-fold changes among P17, P20, and P22 EVs’ treatment groups compared with the control group, of which, 46 transcripts (red rectangle) and 20 transcripts were identified in only P17 or P22 EVs’ treatment group, respectively. (B) 46 downregulated transcripts in EECs treated with P17 EVs were functionally classified by the biological process in GO terms and enriched pathway analyses. (C) 20 downregulated transcripts in EECs treated with P22 EVs were functionally classified by the biological process in GO terms and enriched pathway analyses.