Sample preparation
The animal experiment has been described in Wang et al[41]. All experiments involving animals were carried out in accordance with regulations (No. 5 proclamation of the Standing Committee of Hubei People’s Congress) approved by the Standing Committee of the Hubei People's Congress in P. R. China. All animal sample collection procedures were approved by the Ethics Committee of Huazhong Agricultural University. The animals were housed at Jingpin farm of Huazhong Agricultural University (Wuhan, China) and were slaughtered at the slaughterhouse of Huazhong Agricultural University. Before every gilt was slaughtered, a warm shower to relax the pigs, then they were stunned with low-voltage electric shock to reduce the pain and were exsanguinated by puncturing carotid artery to death.
Nine purebred Yorkshire gilts with similar weights (120 ± 10 kg), ages (8 months) and genetic background were selected in this study. After estrus, gilts were artificially inseminated twice using extended semen from one boar (the breeding pig farm of Huazhong Agricultural University). Uteri were obtained from pigs slaughtered on days 10, 13 and 18 of pregnancy, and ULF was collected by flushing with 30 mL phosphate buffer saline (PBS; pH 7.4). The presence of filamentous conceptuses in the flush of the uterine horns was used to confirm pregnancy. For characterization of exosomes from porcine ULF, three additional nonpregnant gilts with similar weights, ages and genetic background (see above) were slaughtered and the uteri were collected. Exosomes were obtained by using the above method. At the same time, these uteri were used for the separation of endometrial epithelial cells.
ULF exosome isolation
The ULF was clarified by centrifugation (2000 g for 30 min at 4°C), filtered through a 0.22 μm filter (MILLEX-GP, USA) to remove impurity. 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). Exosomes were re-suspended in 200 μL PBS after washing with PBS (130,000 g for 2 h). Exosomes were stored at –80°C until the RNA was isolated.
Western blot (WB)
The exosomal proteins were extracted by DNA/RNA/protein Isolation Kit (catalog number: R6734-02). 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 CD9 (catalog number: 20597-1-AP, Proteintech), rabbit anti HSP70 (catalog number: GB11241, servicebio) and Mouse anti GAPDH (catalog number: 60004-1-Ig, Proteintech) overnight at 4°C with a final dilution of 1:1000 (v/v). After three times of washing, the membranes were incubated with goat anti-rabbit secondary antibodies (catalog number: GB23303, Sevicebio) or goat anti-mouse secondary antibodies (catalog number: GB23301, Sevicebio) 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).
Transmission electron microscope (TEM)
The morphology of isolated exosomes was visualized with high-resolution transmission electron microscope (Hitachi HT7700 ) based on a previous method [42]. In brief, the resuspended exosomes were placed on carbon-coated copper grid, and then subjected to standard uranyl acetate staining and dried overnight.
Nanoparticle tracking analysis (NTA)
The size distribution and concentration of isolated exosomes were analyzed by NTA with Zeta View (Particle Metrix). Isolated exosomes were diluted with particle-free PBS at the ratio of 1:200 and added into the chamber. The results of size distribution and vesicle concentrations were assessed with the software Zeta View.
Cell culture
The uterus of non-pregnant gilts was opened longitudinally on a sterile clean bench. 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 (catalog number: 17100017, Gibco) 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 pellets mainly containing endometrial epithelial cells (EECs) were suspended in Dulbecco’s modified Eagle’s medium/F-12 (DMEM/F12; 1:1) medium (Gibco, NY, USA) 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. Isolation and culture of porcine primary EECs referred to pervious research [41, 43].
Porcine trophectoderm cells (PTr2) 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 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) [44].
PKH67 staining and high-content screening (HCS)
Isolated exosomes were stained with PKH67 green fluorescent mini kit (catalog number: MINI67-1KT, Sigma-Aldrich) following the manufacturer’s instructions. Briefly, exosomes and PBS (negative control) were respectively added to 1 mL Diluent C, and then incubated with 4 ul PKH67 for 4 min. At last, the mixture was closed with 2 mL 0.5% bovine serum albumin (BSA), washed once with PBS, and then ultracentrifuged at 130,000 g for 2 h at 4°C.
After incubation with PKH67-labeled exosomes or negative control for 24 h, EECs and PTr2 cells were imaged by a high-content screening confocal microscope (PerkinElmer, USA). The number of fluorescent spots was counted with Opera Phenix TM High Content System as described in a previous study [45].
RNA extraction and small RNA sequencing
The exosomal RNAs were extracted by miRNeasy Serum/plasma Kit (catalog number: 217184, Qiagen, German) according to manufacturer’s protocol. The quantity and quality of RNA were assessed with the Agilent 2100 Bioanalyzer (Agilent Technologies, USA). Nine small RNA libraries were generated under standard procedures at Beijing Genomics Institute (BGI, China) according to a previous study [46]. Sequencing of the libraries was carried out with the BGISEQ-500 platform (BGI, China; http://www.seq500.com/en/).
Prediction of novel miRNAs, novel piRNAs and miRNA target genes
Before data analysis, clean reads were obtained by eliminating low-quality reads, adaptors, and other contaminants. Clean reads were then mapped to reference genome and other sRNA databases by Bowtie2 [47]. Because each unique small RNA was supposed to be mapped to only one category, we followed a precedence rule of MiRbase > pirnabank > snoRNA (human/plant) > Rfam > other sRNA. Piano [48] and miRNADeeps2 [49] were used to predict novel piRNAs and miRNAs, respectively. Prediction of the target genes of miRNAs was carried out by RNAhybrid and miRnada.
Differentially expressed (DE) small RNAs
The expression levels of small RNAs were calculated by using Transcript Per Kilobase Million (TPM) as described in a previous report [50]. Small RNAs with expression value >28 TPM in at least in one of the nine libraries were selected for subsequent data analysis [51]. The P-values calculated for every small RNA were adjusted to Q-values for multiple testing corrections by two alternative strategies. To improve the accuracy of the identification of DE small RNAs, the miRNAs with expression value >28 TPM in at least one of the libraries and P-values < 0.05 between two groups were selected as DE small RNAs.
Real time quantitative PCR (RT-qPCR)
First-strand cDNAs of small RNAs were synthesized by Mir-X™ miRNA First Strand Synthesis kit (TaKaRa, Dalian, China) according to the manufacturer’s instructions. The qPCR was carried out by using SYBR Green PCR Master Mix (TaKaRa, Dalian, China) and LightCycler 480II Real-Time PCR System. The forward primers of small RNAs were designed based on the mature sequences of small RNAs (Additional file 1: Table S1), and the reverse primer was a universal primer supplied by the above kit. The expression levels of small RNAs were normalized with U6 to obtain the relative expression by comparative CT method.
Hierarchical cluster analysis (HCA)
The expression values of the miRNAs (>28 TPM in at least one library) were normalized by using the Z-score. HCA was conducted using the R package.
Principal component analysis (PCA)
The expression values of the small RNAs (> 28 TPM in at least one library) were normalized by using the lg (TPM). PCA was performed using the imageGP (http://www.ehbio.com/ImageGP/index.php/Home/Index/PCAplot.html).
Functional analyses of the predicted target genes
Functional analyses of the predicted target genes were conducted on the basis of Kyoto Encyclopedia of Genes and Genomes database (KEGG) and Gene Ontology slim database (GO-slim) by using KOBAS (http://kobas.cbi. pku.edu.cn/) and PANTHER (http://www.pantherdb.org/), respectively. An enriched functional class with a P-value <0.05 was defined as a significantly enriched target gene candidate.
Time-series expression analysis
The profiles of DE miRNAs over time were visualized by Short Time-series Expression Miner v 1.3.8 (STEM, http://www.cs.cmu.edu/~jernst/stem/).
Statistical analysis
Statistical significance was assessed by two sets of comparisons using the Bonferroni t-test in SAS 9.1, and shown as * p < 0.05 and ** p < 0.01. Correlation tests were performed to calculate the correlation between the results of RNA-seq and qPCR.