Induction of IFNT-Stimulated Genes by Conceptus-Derived Exosomes during the Attachment Period

Abstract

Biochemical and/or physical communication between the conceptus and the uterine endometrium is required for conceptus implantation to the maternal endometrium, leading to placentation and the establishment of pregnancy. We previously reported that in vitro co-culture system with bovine trophoblast CT-1 cells, primary uterine endometrial epithelial cells (EECs), and uterine flushings (UFs) mimics in vivo conceptus attachment process. To identify molecules in UFs responsible for this change, we first characterized protein contents of UFs from day 17 cyclic (C17) and pregnant (P17) ewes through the use of two dimensional-Polyacrylamide Gel Electrophoresis (2D-PAGE), followed by Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) analysis. These analyses identified 266 proteins specific for P17 UFs, from which 172 proteins were identified as exosomal proteins. Among 172 exosomal proteins, 8 proteins that had been identified as exosomal proteins were chosen for further analysis, including macrophage-capping protein (CAPG), aldo-keto reductase family 1, member B1 protein (AKR1B1), bcl-2-like protein 15 (BCL2L15), carbonic anhydrase 2 (CA2), isocitrate dehydrogenase 2 (IDH2), eukaryotic translation elongation factor 2 (EEF2), moesin (MSN), and ezrin (EZR). CAPG and AKR1B1 were again confirmed in P15 and P17 UFs, and more importantly CAPG and AKR1B1, mRNA and protein, were found only in P15 and P17 conceptuses. Moreover, exosomes were isolated from C15, C17, P15, or P17 UFs. Only P15 and P17 exosomes, originated from the conceptus, contained interferon tau (IFNT) as well as CAPG and AKR1B1, and up-regulated STAT1, STAT2, MX1, MX2, BST2, and ISG15 transcripts in EECs. These observations indicate that in addition to endometrial derived exosomes previously described, conceptus-derived exosomes are present in UFs and could function to modify endometrial response. These results suggest that exosomes secreted from conceptuses as well as endometria are involved in cell to cell interactions for conceptus implantation to the maternal endometrium.

Fig 1. Use of 2D-PAGE to identify proteins specific to day 17 pregnant UFs.

Representative SYPRO Ruby stained 2D gel images of UFs from day 17 cyclic (left) or pregnant (right) ewes. Several spots designated by circle in day 17 pregnant UFs were up-regulated compared to those in day 17 cyclic UFs, which were subjected to LC-MS/MS analysis.

Fig 2. Levels of 8 exosomal transcripts in endometria and conceptuses.

The relative mRNA expression of 8 exosomal transcripts, CAPG (A), AKR1B1 (B), BCL2L15 (C), CA2 (D), IDH2 (E), EEF2 (F), MSN (G), EZR (H), in C15, C17, P15, or P17 endometria (grey bar) and P15 or P17 conceptuses (solid bar) (n = 3 each day). ACTB and GAPDH were used as internal controls for RNA integrity. Values represent mean ± SEM. *Statistically significant difference between conceptus and endometrium in each day (P<0.05).

Fig 3. Expression and localization of CAPG and AKR1B1 proteins in the conceptuses.

(A) The presence of CAPG or AKR1B1 proteins in C15, C17, P15, or P17 ovine endometria and P15 or P17 conceptuses was examined by western blot analysis. ACTB was used as an internal control. A representative data from three independent experiments containing protein samples from different animals was shown. Endo, endometrium; Con, conceptus. (B) Immunohistochemical detection of CAPG (a, c, and e) and AKR1B1 (b, d, and f) in P17 ovine uteri. Tissue sections were immunostained for CAPG using anti-CAPG antibody (a and c) or normal goat IgG (e) as a negative control. Boxed area in (a) is shown at a higher magnification (c). Tissue sections were immunostained for AKR1B1 using anti- AKR1B1 antibody (b and d) or normal rabbit IgG (f) as a negative control. Boxed area in (b) is shown at a higher magnification (d). Con, conceptus; LE, luminal epithelium; GE, glandular epithelium. Scale bar = 250 μm (a, b, e, and f), or 50μm (c and d), respectively.

Fig 4. Identification of exosomal CAPG and AKR1B1 in days 15 and 17 pregnant UFs.

(A) The presence of CAPG and AKR1B1 in C15, C17, P15, or P17 UFs (10 μg each) was examined by western blot analysis (n = 3 each day). (B) Transmission electron microscopy analysis revealed the presence of approximately 150 nm vesicles in UFs, consistent with exosomes. Scale bar = 200 nm (a) or 100 nm (b). (C) Nanoparticle tracking analysis (n = 3, triplet analysis in each sample) of P17 UF revealed that a range of exosomal size is 50 to 150 nm. Gray area in (C) represents an average from three samples, whereas black area represents SEM. (D) Western blot analysis showed the presence of CD63 and HSP70 in exosomes isolated from C15, C17, P15, or P17 UFs, and the presence of CAPG and AKR1B1 in exosomes isolated from days 15 and 17 pregnant UFs (n = 3 each day). In (A), (B), and (D), a representative one is shown.

Fig 5. Presence of IFNT in UFs and exosomes from days 15 and 17 pregnant ewes and the effect of those exosomes on EECs.

(A) The presence of IFNT in C15, C17, P15, or P17 UFs and exosomes was examined by western blot analysis (n = 3 each day), and IFNT was detected in P15 and P17 UFs. A representative of three independent experiments is shown. (B) Effects of exosomes on EECs. EECs were treated with or without C15, C17, P15, or P17 exosome (10 μg each), or P17 UF (10 μg proteins) for 48 h. RNA was extracted from EECs and subjected to real-time PCR analysis for STAT1, STAT2, BST2, MX1, MX2, and ISG15 transcript levels. ACTB and GAPDH mRNA were used as internal controls for RNA integrity. Values were from three independent experiments, each containing duplicates. Values represent mean ± SEM. *Statistically significant difference in mRNA levels vs. control (Ctrl) without exosomes or UFs treatment (P<0.05).