All chemicals and reagents used in this work were purchased from MilliporeSigma unless otherwise specified.

Oocyte IVM was performed according to a previously reported method (27). Briefly, porcine ovaries and uteri including the oviducts were obtained from a slaughterhouse in Daejeon City and transported to the laboratory in normal saline solution containing 75 mg/ml penicillin and 50 mg/ml streptomycin. The temperature of the solution was maintained between 34–36°C. Cumulus-oocyte complexes (COCs) were taken from antral follicles (3–8 mm in diameter) with an 18-gauge needle attached to a 10 ml syringe. COCs with three or more layers of cumulus cells and homogeneous ooplasm were selected and washed three times in HEPES-buffered Tyrode's media containing 0.05% (w/v) polyvinyl alcohol (PVA). COCs (n=50) were in vitro matured in 500 µl of maturation medium consisting of bicarbonate-buffered tissue culture medium 199 (TCM-199; Gibco, Thermo Fisher Scientific, Inc.), 10% (v/v) porcine follicular fluid, 0.91 mM sodium pyruvate, 0.57 mM L-cysteine, 10 ng/ml epidermal growth factor and 1 µg/ml insulin with 10 IU/ml equine chorionic gonadotropin (eCG), 10 IU/ml human chorionic gonadotropin (hCG) and 75 µg/ml kanamycin in 4-well dishes (SPL Life Sciences). COCs were incubated at 38.5°C in 5% CO₂ in humidified air incubator (Astec Co., Ltd.) for 22 h and then were cultured for an additional 22 h in a hormone-free IVM medium (24).

The post-ovulatory oviducts of multiparous female pigs (sows) were obtained from a local butcher and transported to the laboratory according to the aforementioned method. pOECs were isolated by mechanically scraping the oviduct with a slide and then centrifuged three times at 700 × g for 5 min at room temperature in 10 ml of DMEM supplemented with 10% (v/v) FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% (v/v) antibiotic-antimycotic solution (Gibco; Thermo Fisher Scientific, Inc.). pOECs were cultivated in a 100 mm Falcon tissue culture plate with a culture medium at 39°C and 5% CO₂ in a humidified environment. On day 3 of the initial culture, the pOEC outgrowths were examined, after which they were grown for a further 7 days. pOECs were trypsinized in 0.05% trypsin-EDTA, washed in phosphate-buffered saline (PBS) thrice and grown in DMEM without FBS for 48 h to obtain the conditioned medium for isolating OEC-E (24).

The supernatant was used to separate EVs using the Total EV Isolation kit after being centrifuged at 2,000 × g for 30 min at room temperature to remove cells and cell debris from the conditioned medium (Invitrogen; Thermo Fisher Scientific, Inc.). The kit reagent was combined with the supernatant, stirred vigorously, and incubated overnight at 2–8°C. The EV pellets were then frozen at −80°C until future investigations. The EV pellet was resuspended in modified Tris-buffered medium (mTBM) for experimental purposes, and the final protein concentration was adjusted to 50 ng/ml using a NanoDrop 2000 (Thermo Scientific; Thermo Fisher Scientific, Inc.). The EVs were visualized using transmission electron microscopy (TEM) after negative staining with 2% uranyl acetate as described previously (24).

A ZetaView PMX 110 (Particle Metrix GmbH) instrument was used for NTA as previously described (28). Briefly, 1 ml of diluted EVs pellet (in 1X PBS) was loaded into the device to measure each sample at 11 different positions and two reading cycles per position. After an automated analysis and outlier removal, the mean, median, and mode sizes (indicated as diameter) and the concentrations were calculated using ZetaView SP7 software (version 8.05.14; Particle Metrix) and Microsoft Excel 365 (version 2205, Microsoft Corporation). Device calibration was performed using 100 nm polystyrene particles (Thermo Fisher Scientific, Inc.).

In vitro matured oocytes with the extruded first polar body were washed twice with mTBM, before being cultured in fresh droplets of mTBM (15 oocytes/50 µl) at 39°C in a humidified atmosphere of 5% CO₂. In the OEC EVs group, mTBM contained EVs protein of 50 ng/ml (EVs-mTBM), while the control group was EVs-free. Chilled pig semen was commercially obtained from Darby Genetics Inc., South Korea. Oocytes were co-incubated with 5.0×10⁵ sperm/ml for 20 min. After co-incubation, the attached sperm were discarded from the zona pellucida (ZP) by gentle micro pipetting and the oocytes were washed twice in mTBM. Oocytes were then incubated in mTBM or EVs-mTBM without sperm for an additional 6 h in the same culture conditions. IVF conditions with either OEC-EVs-supplemented or plain culture mediums were compared for polyspermy, cortical granules' reaction, and mitochondrial staining. In vitro embryonic development was monitored after culturing the IVF oocytes in PZM-5 medium (Functional Peptides Research Institute Co. Ltd.) for 7 days at 38.5°C in 5% CO₂ and 5% O₂ in a humidified incubator.

After 18–20 h of IVF, the embryos were fixed using 3.7% (w/v) paraformaldehyde (PFA) for 30 min and washed in PBS/PVA three times at room temperature. The embryos were stained with 10 µg/ml Hoechst 33342 in PBS/PVA for 30 min at room temperature. After staining, the embryos were washed three times in PBS/PVA. The embryos were examined for penetration and pronucleus (PN) formation using a fluorescence microscope. Embryos with one nucleus and polar body (Fig. 1A) were considered non-penetrated. Embryos with one female PN and one male PN in the cytoplasm were considered to be monospermic (Fig. 1B). Embryos with more than one nuclear staining (i.e., sperm or male PNs) were considered to be polyspermic (Fig. 1C).

After OEC-EV isolation, the OEC EVs were mixed with the lipophilic PKH67 dye according to the manufacturer's instructions, and OEC-EVs were isolated to remove the excess free PKH67 dye according to the manufacturer's protocol (29,30). EVs were then supplemented with sperm or oocytes for 6 h to monitor their uptake using a confocal microscope. For negative control staining, the plain conditioned medium was mixed with PKH67 and processed through the same EV labeling procedure.

Three times, in vitro matured oocytes, were washed with PBS containing 0.1% (PBS/PVA). The OEC EVs group oocytes were treated for 20 min with OEC EVs in mTBM and the control group was kept in mTBM without EVs. The oocytes were then fixed using 3.7% (w/v) PFA at room temperature for 30 min, followed by three 10 min washes in PBS/PVA per oocyte. This was followed by a 1% Triton X-100 treatment for 30 min and three 10-min washes in PBS/PVA. Oocytes were then cultured for 30 min at room temperature in FITC-labeled Peanut Agglutinin (PNA) diluted 1:500 in PBS in a dark box. Following staining, the oocytes were washed for 10 min in PBS/PVA three times. Finally, the nuclei were stained with DAPI for 10 min, mounted on slides, and images were acquired using a DMi8 confocal microscope (Leica Microsystems GmbH).

After 18–20 h, the IVF embryos were washed with PBS/PVA three times and were then cultivated for 30 min at 37°C in 500 nM MitoTracker Red CMXRos (cat. no. M7512; Invitrogen; Thermo Fisher Scientific, Inc.) in a dark box. After staining, the embryo was washed three times for 10 min in PBS/PVA. Each embryo was fixed in 3.7% PFA at room temperature for 30 min, followed by three 10 min washes in PBS/PVA. Finally, the nuclei were stained using DAPI for 10 min at room temperature, mounted on slides, and images were acquired using a DMi8 confocal microscope (Leica Microsystems GmbH).

Lieven's test and Kolmogorov-Smirnov test were used to confirm the homogeneity of variance and the normality of distribution, respectively. At least 10–20 replicates were performed per experiment. For data normalization, the mean expression level value was calculated from the control group, and the means were normalized to an arbitrary unit (1-fold) by dividing the values against the control group. The image intensities were analyzed using ImageJ (version 1.53k; National Institutes of Health). Data were analyzed using unpaired Student's t-test using SPSS software (SPSS 22.0; IBM Corp). P<0.05 was considered to indicate a statistically significant difference.

OEC confluent cells were achieved as reported previously (24) and the expression of oviduct epithelial cells' specific gene expression [oviduct-specific glycoprotein (OVGP1)] and proteomics were performed for further confirmation. OEC-EVs were isolated, visualized, and characterized using TEM and NTA, the mean diameter of the isolated EVs was 146.2±57.2 nm, and the mean concentration was 5.3×10⁹ particles/ml (Fig. 1A-C).

Sperm penetration into the oocyte was detected 18–20 h after IVF. Overall, non-penetration and non-fertilized oocytes (Fig. 2A), penetrated and monospermic fertilized embryos (Fig. 2B), and penetrated and polyspermic abnormally fertilized embryos (Fig. 2C) were observed. The penetration rate did not differ between the OEC-EV and control groups (74.4±1.5 vs. 74.2±2.3, P=0.27). However, the polyspermy rate was significantly lower in the OEC-EV group when compared with the control group (32.9±2.5 vs. 43.8±3.1; P=0.0026; Table I).

The cleavage rate differed significantly between the OEC-EV and the control groups (67.6±2.5 vs. 57.3±1.9; P=0.0028). Furthermore, the OEC-EV group had significantly more embryos than the control group (16.4±1.2 vs. 10.2±0.8; P=0.0001; Table II).

In the OEC-EV group, it was observed that OEC-EVs attached to the heads and tails of sperm (Fig. 3) and OEC-EVs that entered the oocyte through the cell membrane (Fig. 4), which was not observed in the control group.

The CG distribution in the control and OEC-EV groups after FITC-PNA staining were presented (Fig. 5A). The OEC-EV group had significantly higher fluorescence intensity values than those in the control group (3.56±0.47 vs. 2.15±0.24; P<0.05; Fig. 5B).

Mitochondrial activity was detected in both control and OEC-EV-supplemented groups using MitoTracker Red staining (Fig. 6A). The fluorescence values of active mitochondria were higher in the OEC-EV group compared with those in the control group (8.14±0.34 vs. 5.96±0.38; P=0.00007; Fig. 6B).