Animals
The animals, management, and sample collections were approved by the Ruakura Animal Ethics Committee (AEC 13574, 13934 and 14200) and Holstein-Friesian dairy cows used in this study were part of a larger experiment of an established fertility animal model (28, 29). The study was conducted in compliance with ARRIVE guidelines and all methods were performed in accordance with the relevant guidelines and regulations (30).
From the larger group of dairy cows, 52 cows were identified in 6 different groups based on fertility breeding value (BV), cow ovulation synchronization and postpartum anovulatory intervals (PPAI) which is the number of days from a cow calving to returning to oestrus (Table 1).
Table 1
classification of 52 dairy cows into 6 different groups by fertility breeding value (BV), ovulation synchronization and postpartum anovulatory intervals (PPAI).
Group | Fertility BV | Synchronization | PPAI (d = days) | Sample Quantity |
1 | High | no | 19-37d | 10 |
2 | High | no | 60-100d | 10 |
3 | Low | no | 19-37d | 7 |
4 | Low | no | 60-100d | 10 |
5 | Low | yes | 60-100d | 5 |
6 | Low | yes | 104-144d | 10 |
From these 6 groups, 20 cows were identified as being at two extreme diversities either high fertile, without synchronization and lowest PPAI – Group 1 (HF) (n = 10) and low fertile, without synchronization and highest PPAI – Group 4 (LF) (n = 10). These animals were kept similar in genetic characters for other key traits (e.g., body weight, milk production, and percentage of North American genetics) except from the fertility breeding values (FBV). Blood samples were collected into EDTA Vacutainer tubes (Greiner Bio-one, Kremsmunster, Austria) from a jugular vein straightaway or following a 48h oestrus synchronization with prostaglandins. The blood samples were placed immediately on ice and centrifuged at 1,500 × rcf for 12 min at 4°C, and the plasma aspirated and stored at − 80°C.
Extracellular vesicle isolation from plasma by ultracentrifugation
EVs were isolated from a total of 20 blood plasma samples using as established sequential centrifugation protocol as previously described (31, 32). Briefly, first the plasma was centrifuged at 2,000 x rcf for 30 min at 4°C and 12,000 x rcf for 30 min at 4°C to remove cellular debris and apoptotic bodies. Then the supernatant was filtered through a 0.22-µm filters (Corning Inc., Corning, NY) and ultracentrifuged at 100,000 x rcf for 2 hr at 4°C. Finally, the pellets containing the extracellular vesicles were resuspended in 500 µL of filtered Dulbecco’s Phosphate Buffered Saline (DPBS, pH 7.0–7.2; Gibco, Life Technologies Australia Pty Ltd) and stored at -80°C for further analysis.
sEV enrichment by size-exclusion chromatography columns (SEC)
Extracellular vesicles obtained from ultracentrifugation were fractionated using qEV original size exclusion columns (Izon Science, New Zealand). Individual 500 µL fractions were eluted from the column and collected in separate 1.5 mL microcentrifuge tubes (a total of 16 fractions), as per manufacturer’s instructions. Then the sEV characterization experiments were conducted for individual SEC fractions to identify sEV/exosome markers. The fractions were collected as follow s; 1–6 as void volume and particles > 200 nm, 7–10 as exosomal (EX) fractions (particles < 200 nm), and 11–16 as soluble proteins (non-EX) fractions. Separate columns were used per animal groups to maintain group heterogeneity. In between uses, the columns were flushed with 0.5 mL 1M NaOH solution, followed by 15–20 mL filtered DPBS.
Protein quantification
Quantification of protein concentration of SEC fractions was performed using Bicinchoninic Acid (BCA) assay (Sigma-Aldrich, St Louis, MO, USA) and bovine serum albumin (Sigma-Aldrich, St Louis, MO, USA) dilutions were used as standards.
Western Blot
Individual fractions resulting from SEC were analysed by western blot for the presence of EV markers and contaminating plasma proteins. Visualisation of the residual bovine serum albumin (BSA) and sEV/exosome proteins were conducted for the collected SEC fractions F7–F16. Equal volumes (10 µL) of sample from the individual SEC fractions F7–F16 were aliquoted for WB analysis, as previously described (33). Then the sEV/exosome protein enriched SEC fractions were selected as F7-F10 (EXO) and non-sEV/exosome protein (BSA) enriched SEC fractions as F11-F16 (non-EXO). Samples were dried in a vacuum concentrator (cat number 5305000380, Eppendorf Concentrator plus, Sydney, Australia) and resuspended to a final volume of 10.5 µl Briefly, 4× NuPAGE LDS sample buffer (NP0007, Thermofisher Scientific, Brisbane, Australia) and 10× NuPAGE sample reducing agent (NP0004, Thermofisher Scientific, Brisbane, Australia) were added to give a final concentration of 1×, and reduced for 10 min at 70°C, as per the manufacturer’s instructions. For visualisation of Flotillin-1 (FLOT-1), CD81 in pooled sEV-enriched (7–10) and non-sEV-enriched (11–16) fractions, the same procedure was followed as for BSA, as previously described (31). Samples were resolved by electrophoresis on NuPAGE™ 4 to 12%, Bis-Tris, 1.0 mm, Mini Protein Gels, 15-well (NP0336BOX, Thermofisher Scientific, Brisbane, Australia) with Chameleon® Duo Pre-stained Protein Ladder (928-60000, Li-COR, Mulgrave, Australia). The protein gel was transferred onto a polyvinylidene fluoride membrane (Bio-Rad Laboratories Pty Ltd., Sydney, Australia) using the Trans-Blot Turbo system. Membranes were briefly washed in phosphate buffered saline containing 0.1% Tween-20 (PBST) (Sigma-Aldrich (Merck), Melbourne, Australia), before blocking in 5 mL Odyssey Intercept blocking buffer (927-70001, Li-COR, Mulgrave, Australia) and 5 mL phosphate buffered saline (PBS) (Sigma-Aldrich (Merck), Melbourne, Australia) for 1 hr at RT. The primary antibody was diluted with 1:1 Odyssey Blocking buffer, PBS, and Tween-20 added to final concentration of 0.1%. Samples were incubated with primary antibody overnight; anti-BSA (1:5000 dilution, Rabbit polyclonal (ab192603, Abcam, Melbourne, Australia); recombinant anti-Flotillin-1 (1:1000 dilution, Rabbit monoclonal (ab133497, Abcam, Melbourne, Australia): anti-CD81 (1:500 dilution, Rabbit polyclonal (NBP1-77039, Novus Biologicals, LLC). The next day, membranes were washed four times in PBST for 5 min each, and the membranes were incubated with secondary antibody for 1 hr at RT in the dark with gentle rocking: Goat anti-Rabbit IgG (1:15,000 dilution, Li-COR, Mulgrave, Australia). The secondary antibody was diluted with 1:1 Odyssey Intercept blocking buffer, PBS, and Tween-20 added to a final concentration of 0.1%. The membranes were washed in PBST four times for 5 min each. Membranes were rinsed briefly in PBS and imaged with Li-COR Odyssey fluorescent scanner at 700 and 800 nm. All images were processed using Image Studio Lite v5.2 (Li-COR Biosciences, Lincoln, NE, USA). Contrast and brightness were adjusted equally across entire images to best visualise protein bands.
Transmission electron microscopy
Visualization of sEV particles from SEC fractions were conducted by JEOL 1400 transmission electron microscopy (JEOL, Sydney, Australia). sEV samples (5 µL) were added onto glow discharged copper grids (200 mesh) for 3 minutes. Next the grid was negatively stained with 1% uranyl acetate for 2 minutes, then briefly blotted with blotting paper to remove excess liquid. The samples were than visualized in JEOL 1400 transmission electron microscope operated at 100 kV, and images captured using a 2K TVIPS CCD camera (TVIPS, Gauting, Germany).
Nanoparticle tracking analysis
Based on the presence of exosomal markers, exosomal fractions 7–10 were pooled. Measurements of particle size and concentration were performed using a NanoSight NS500 instrument (NanoSight NTA 3.1 Build 3.1.46, Malvern Panalytical, Sydney, Australia) as previously described (14). Synthetic (latex) beads of size 100 nm were used to perform instrument calibration at a 1:250 dilution in deionized water as previously described (31).
Bovine endometrial epithelial and stromal cell culture
Bovine endometrial epithelial (bEEL) and stromal (bCSC) cell lines (34, 35) were a kind gift from Professor Michel A. Fortier (Université Laval, Québec). The cells were grown in RPMI media (Gibco, Thermo Fisher Scientific Australia Pty Ltd, Scoresby Vic) containing sEV/exosome depleted 10% fetal bovine serum (Bovorgen, Interpath Services Pty Ltd, Australia), and incubated at 37°C and 5% CO2. Experiments were conducted in media without fetal bovine serum. (27).
Functional studies of sEV on endometrial cells
For the initial cell culture, bCSC cells were grown in a seeding density of 8,000 cells per well and bEEL cells were grown in a seeding density of 35,000 cells per well. Then cells were incubated for 24 h grown in RPMI media (Gibco, Thermo Fisher Scientific Australia Pty Ltd, Scoresby Vic) containing 10% fetal bovine serum (Bovorgen, Interpath Services Pty Ltd, Australia). For the co-incubation experiment (treatment with sEV), FBS free RPMI media was used. Cells were incubated with RPMI media with no addition of sEV (No EXO control, for baseline measurements), or treated with HF-EXO (n = 10) or LF-EXO (n = 10) with 1 × 108 particles per well for 24 h to analyze gene expression. We performed 3 well replicates per individual cow (n = 20). sEV concentration for co-incubation (36) and incubation time (37) were chosen in reference to literature. Cell culture experiments were performed in triplicate per cell line. Cells and cultured media were collected and stored at − 80°C until required for further analyses.
RNA extraction and cDNA synthesis
RNA from the samples (bEEL and bCSC cells) were extracted accordingly to manufacturer’s protocol using a Rneasy Mini kit (Qiagen, Victoria, Australia). The concentration and purity of RNA was determined using a Nanodrop NanoDrop™ One Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific, Wilmington, Delaware). 500 ng of RNA was reverse transcribed into complementary DNA (cDNA) using RT2 First Strand Kit (Cat no. 330404; Qiagen, Victoria, Australia).
Eicosanoid enzymes and inflammatory mediator gene expression analysis
Gene expression was analyzed after 24 h of HF-EXO and LF-EXO treatments on bEEL and bCSC. Real-time PCR (RT-PCR) quantification of eicosanoid enzymes and inflammatory mediator gene expression was performed using a customized bovine RT2 Profiler PCR Array (Cat. no. 330171 CLAB39919; Qiagen, Victoria, Australia). The complete plate plan of the array is in supplementary file 1. The reaction mixture was prepared using the RT2 SYBR Green ROX qPCR Mastermix (Cat. no. 330523; Qiagen, Victoria, Australia) following the manufacturer’s instructions. Briefly, RT-PCR was performed using the Applied Biosystems® ViiA™ 7 Real-Time PCR System (Applied Biosystems®, Carlsbad, California). with an initial 2 min incubation at 50°C and 10 min incubation at 95°C followed by 40 cycles at 95°C for 15 s and 60°C for 60 s. The specificity of the RT-PCR products was confirmed by analysis of melting curves. Gene expression data which met the cycle threshold cut-offs (< 35) were analyzed. The endogenous control genes included on the array were Actin, beta (ACTB), TATA box binding protein (TBP), hypoxanthine phosphoribosyl transferase 1 (HPRT1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). PCR reproducibility, reverse transcription efficiency and the presence of genomic DNA contamination were verified before analyzing further. Housekeeper genes were not changed with treatment. Gene expression results were normalized to the endogenous control genes ACTB, TBP, HPRT1 and GAPDH. Real-time PCR data were analyzed using comparative CT method (27, 38, 39).
Statistical analysis
Data were imported into GraphPad Prism (version 9, GraphPad Inc., La Jolla, CA) and Mann-Whitney test was used to identify the statistically significant differences between control and each treatment. Data are presented as sample means ± SEM. For all statistical analyses, a P-value of < 0.05 was considered statistically significant.