Tissues collection

Nine purebred Yorkshire gilts with similar weights (120 ± 10 kg), ages (8 months) and genetic background were selected in this study. Gilts were artificially inseminated using extended semen from one boar (the breeding pig farm of Huazhong Agricultural University) at the onset of estrus (day 0) and again 12 h later. Uteri and embryos were obtained from pigs slaughtered on days 10, 13 and 18, and uterine flushing fluids were collected by flushing three times with 30 mL phosphate buffer saline (PBS; pH 7.4, 02–024-1ACS, Biological Industries, Israel). The pregnancy was confirmed by the size and morphology of conceptuses as follows: day 10 (spherical conceptuses with a diameter of 2–8 mm), day 13 (filamentous forms of conceptuses) and day 18 (trophoblast tissue and embryos with evident vascularization). The endometrial tissues from pregnancy gilts were used for subsequent analyses.

EVs isolation and preparation

The cell culture medium was clarified by centrifugation (2000 g for 30 min at 4 °C) to remove whole and dead cells. The supernatant was subjected to a second centrifugation at 10,000 g for 30 min to remove cells debris. The supernatants were filtered through a 0.22 μm filter (MILLEX-GP, USA) to remove bacteria and impurities. Then, these samples were ultracentrifuged at 130,000 g for 2 h at 4 °C (Beckman Optima XE-90, SW32 Ti rotor, Beckman Coulter. USA). EVs were re-suspended in 200 μL PBS (catalog number: 02–024-1ACS, Biological Industries) after washing with PBS (130,000 g for 2 h). EVs were stored at –80 °C until the RNA was isolated. Protein concentration in the final EV pellet was measured by BCA (Pierce, Thermo Fisher Scientific) according to the manufacturer’s instructions.

NTA

Isolated EVs were diluted with PBS at the ratio of 1:200 and added into the chamber. The size distribution of isolated EVs was analyzed by NTA with Zetasizer Nano ZS (Malvern Panalytical).

TEM

The morphology of isolated EVs was visualized with high-resolution transmission electron microscope (Hitachi HT7700) based on a previous method [22]. In brief, the resuspended EVs were placed on a carbon-coated copper grid, and then subjected to 2% phosphotungstic acid (DZ0035, Leagene, Beijing, China) staining for 2 min. The excess liquid was blotted off by filter paper, and grids were allowed to dry overnight.

Species conservation analysis of miRNAs

The sequences of miRNAs in different species were obtained through miRbase (https://www.mirbase.org/). The conservation analysis was performed by comparing mature sequences of miRNAs from different species.

EVs-free cell culture medium preparation

The serum following 1:4 dilution was ultra-centrifuged with an ultracentrifuge (1,300,000 g, 18 h) to remove EVs [23]. After centrifugation, the supernatant was used to prepare the complete medium.

Cell culture

Six additional non-pregnant gilts (day12 of estrous cycle) at a similar age (8 months) and weight (120 ± 10 kg) were slaughtered for in vitro culture of endometrial epithelial cells. Isolation and culture of porcine primary endometrial epithelial cells (EECs) referred to previous research [24]. The endometrium was separated and shredded with a sterile scissor. After washing twice with PBS, the tissue pieces were incubated at 37 °C for 2.5 h with collagenase I (Gibco, NY, USA) and shaken vigorously every half hour. Undigested tissue pieces were removed by screen filtration. Then, the filtrate was centrifuged at 500 g for 10 min to remove supernatant (fraction rich of endometrial stromal cells). The pellet (epithelial‐rich fraction) was resuspended twice in PBS and recentrifuged (500 g, 10 min) twice. The resultant pellets containing EECs were suspended in Dulbecco’s modified Eagle’s medium/F-12 (DMEM/F12; 1:1) medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin (Gibco) and cultured in 37 °C and 5% CO2 incubator. Endometrial stromal cells were further removed by 0.25% trypsin without edetic acid disodium salt (EDTA) after 2–4 d. The epithelial cells (purity > 95%) characterized for epithelium-specific cytokeratin positive were then trypsinized with 0.25% trypsin–EDTA and placed on Cell Culture Flask (Corning, NY, USA) for subsequent experiments [24].

Porcine trophectoderm cells (PTr2 cells) were kindly provided by Mr. Jiang zongyong, Guangdong Academy of Agricultural Sciences. PTr2 cells were established from dispersed cell culture of day 12 filamentous conceptus obtained from pigs. These cells have been previously characterized for SN1/38 (porcine trophectoderm-specific monoclonal antibody) positive, cytokeratin 7 positive, vimentin negative, express fibronectin and many of the integrin subunits present in porcine trophectoderm in vivo. Detailed methods have been published [25]. These cells were cultured in DMEM-F12 (Gibco) supplemented with insulin (0.1 Units/mL; Sigma-Aldrich, German), glutamine (2 mM, Sigma-Aldrich), 1% penicillin–streptomycin and 5% fetal bovine serum (Gibco).

The uptake of CM-Dil labelled EVs

To monitor EVs trafficking, EVs were labeled with CM-Dil fluorescent dye using the Celltracker CM-Dil kit (Yeasen). Briefly, EVs were mixed with 1 μM CM-Dil, and the EVs–dye suspension was incubated for 30 min in the 37 °C incubator. After CM-Dil staining, the EVs were washed in 35 ml PBS and collected by ultracentrifugation (130,000 × g for 70 min) at 4ºC. Finally, CM-Dil labeled EVs were resuspended in PBS and added to PTr2 cells. After 6 h or 12 h, the PTr2 cells were dyed with AbFluor™ 488-Phalloidin Kit (Abbkine) according to the manufacturer’s instructions. These PTr2 cells were observed via laser scanning confocal microscope (Zeiss LSM 800).

Co-culture assay

After transfection with FAM-miR-92b-3p mimic, EECs were co-cultured with PTr2 cells at a ratio of 1:1 using a trans-well plate (0.4 mm polycarbonate filter, Corning) for 24 h, with PTr2 cells placed in the lower chamber and EECs placed in the upper chamber. After washing with PBS twice, the appearance of FAM green fluorescence on PTr2 cells was examined.

RNA extraction and real time quantitative PCR (RT-qPCR)

The RNA was extracted from cells using Trizol (Invitrogen) as recommended by the manufacturer and the concentration and quality were measured by the NanoDrop 2000 (ThermoFisher, Waltham, USA). The complementary DNA (cDNA) was synthesized with a reverse transcription kit (Takara, Tokyo, Japan). Then, the Mix (Toyobo, Japan) and specific primers for every gene (Table S1) were used to perform RT-qPCR on a Real-time System (Roche, Basel, Switzerland). The expression levels of miR-92b-3p and genes were normalized with U6 and RPS20 to obtain the relative expression using the 2–ΔΔCt method, respectively.

Transfection

All RNA oligonucleotides were designed and synthesized by GenePharm (Shanghai, China) and are shown in Table S2. EECs or PTr2 cells were transfected with 100 nM miRNA agomirs in 6-well plates with Lipofectamine 2000 (Invitrogen, Waltham, USA) according to the manufacturer’s instructions.

Cell proliferation assay

The Cell Counting kit-8 (CCK-8, Dojindo, Shanghai, China) was used to measure cell proliferation after transfection 48 h following the manufacturer's instructions. The optical density (OD) at 450 nm of each well plate was determined using a microplate reader (Bio-Rad, CA, USA).

Cell migration assay

Cell migration was assessed by Transwell assay that had 12 mm polycarbonate membranes of 8.0 μm pore size (Corning, NY, USA). The cells were re-suspended with serum-free medium as a single-cell solution at 4 h post-transfection. About 2 × 105 cells were seeded on the upper champers, and complete Medium with10% FBS was added to the lower chamber as a chemoattractant. After 24 h of incubation at 37 °C with 5% CO2, cells which migrated to the lower chamber were fixed with 4% paraformaldehyde for 5 min, stained with 0.1% crystal violet for 5 min, rinsed third in PBS and subjected to OLYMPUS DP80 microscope (Tokyo, Japan). The number of migration cells was obtained by counting five fields per membrane and derived from three independent experiments.

Cell adhesion

Cell adhesion was detected with reference to the previous method [26]. Firstly, matrigel solution (Corning, USA) was 1:5 diluted by serum-free medium, and then they were added into 96-well plate (20 μl/well) for 1 h at 37 °C. Secondly, the density of cells was adjusted to 1 × 105/ml using serum-free medium, and then they were added into the same 96-well plate (100 μl/well), which was incubated at 37 °C with 5% CO2 for 1 h. After that, the medium was removed, and the non-adherent cells were washed away by PBS, followed by adding serum-free medium into 96-well plate (200 μl/well). Thirdly, CKK-8 solution was added to the 96-well plate (10 μl/well), incubating for 4 h. After that, the OD value of each well was detected by a microplate reader (λ = 450 nm). Finally, the rate of adherent cells (%) was estimated using the following formula: the rate of adherent cells (%) = (Experimental group – Negative control group) / (Negative control group) *100%

Western blot

The exosomal and cellular proteins were extracted by DNA/RNA/protein Isolation Kit (catalog number: R6734-02, OMEGA). The sample was denatured by heating, separated by SDS-PAGE and transferred to a PVDF (polyvinylidene fluoride) membrane. Next, the membranes were blocked with 5% skimmed milk powder and separately probed with rabbit anti TSG101 (catalog number: GB11618, servicebio), rabbit anti HSP70 (catalog number: GB11241, servicebio), rabbit anti TSC1 (catalog number: GB11882, servicebio), rabbit anti calnexin (catalog number: 10427–2-AP, Proteintech), rabbit anti GAPDH (catalog number: GB11002, servicebio), rabbit anti β-actin (catalog number: GB11001, servicebio) and mouse anti DKK3(catalog number: 66758–1-Ig, Proteintech) overnight at 4 °C with a final dilution 1:1000 (v/v) in 5% milk. After three times of washing, the membranes were incubated with goat anti-rabbit secondary antibodies (GB23303, Sevicebio, Wuhan, China) or goat anti-mouse secondary antibodies (GB23301, Sevicebio, Wuhan, China) with 1:2000 dilution (v/v) at 37 °C for 1.5 h. The images of membranes treated with ECL (enhance chemiluminescence) were captured by Western Blotting Detection System (Tiangen, Beijing, China).

Plasmid construct and dual-luciferase reporter assay

The miR-92b-3p target genes were predicted using a miRBase (http://www.mirbase.org/), TargetScan (http://www.targetscan.org/) and miRDB (http://www.mirdb.org/miRDB/). To construct reporters for luciferase assays, the fragment contained the binding sites of miR-92b-3p on the 3' UTR of TSC1 or DKK3 was cloned into the Pmir-GLO Vector (Promega, Madison, USA). The mutant of ssc-miR-92b-3p binding sites on the 3' UTR was generated using mutagenic primers (Table S3) to construct mutant vector (Pmir-GLO-TSC1-Mut or PmirGLO-DKK3-Mut). MiR-92b-3p and the dual-luciferase reporter vectors (PmirGLO-TSC1-WT, PmirGLO-TSC1-Mut, PmirGLO-DKK3-WT or PmirGLO-DKK3-Mut) were co-transfected into PTr2 cells. Treated cells were collected at 24 h post-transfection and the luciferase activity was detected with PerkinElmer 2030 Multilabel Reader (Promega, MDN, USA).

Intrauterine injection of mice

It is difficult to perform in vivo experiments in pigs, so we used a murine implantation model based on other studies focusing on porcine or human implantation [27,28,29]. The date of finding the vaginal suppository after mating was designated as the first day. The intrauterine injection surgery under general anesthesia was performed on the eight mice on day 3 of pregnancy in the evening according to previous studies [28, 29]. 5 μL of 10 μM antagomiR-92b-3p and inhibitor negative control (NC) were injected into the left and right uterine horns, respectively. Then the wounds were sutured and the mice were put under a 37 °C warmer till awakening from the anesthesia. To reduce the pain of the mice, temgesic was injected into the mice at 12, 24, and 48 h after surgery. On day 7, five mice were killed and their uteri were isolated to record the number of implanted embryos.

Statistical analysis

Data were expressed as means ± standard deviation (SD) derived from at least three independent experiments. Student's t-test (one tailed) was used to perform statistical analysis. P value < 0.05 was considered as statistically significant. * P < 0.05; ** P < 0.01.