Recombinant human CCL2/JE/MCP-1 chemokines were purchased from R&D Systems (Minneapolis, MN, USA). Unless otherwise stated, all chemicals and reagents used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Ovary pairs from prepubertal three-way cross pigs (mixed Yorkshire, Landrace, and Duroc breeds) obtained from a local slaughterhouse, were collected for follicular fluid analysis. Aspirated porcine follicles were divided into three groups: small (1–2 mm), medium (3–7 mm), and large (≥8 mm) (29, 30). To eliminate debris and blood, the pFF was centrifuged for 20 min at 94 g and 4°C. The supernatant was extracted using a 10 cc disposable syringe (Sofjec; Hwajin Medical, Chungnam, Korea) with a 0.2 μm syringe filter (Corning, NY, USA) and frozen at −80°C. A porcine CCL2/MCP-1 ELISA kit (ES2RB; Invitrogen, Carlsbad, CA, USA) was used to measure the CCL2 concentration in the pFF samples, according to the manufacturer's instructions. The CCL2 concentration was determined using the mean value of four ELISA experiments, performed in duplicates.

Porcine ovaries were collected from local slaughterhouses and transported within 60 min to a laboratory where they were stored at 37°C in 0.9% NaCl. Oocyte collection and IVM were conducted as previously reported (31). A 10 cc disposable syringe with an 18-gauge needle was used to aspirate the follicular fluid from antral follicles (3–6 mm in size). The fluid was collected in a 15 mL conical tube, and debris was allowed to settle for 5 min, while the temperature was maintained at 37°C. After the supernatant was removed, precipitates were washed twice in HEPES-buffered Tyrode's medium (TLH) containing 0.05% (w/v) polyvinyl alcohol (PVA). Using a stereomicroscope, only COCs from the follicular fluid containing three or more uniform layers of compact CCs and homogeneous ooplasm were chosen. Approximately 60 COCs were placed in each well of a 4-well dish (Nunc, Roskilde, Denmark). Each well contained 500 μL of maturation medium (TCM199; Invitrogen) comprising 0.1% (w/v) PVA, 0.6 mM cysteine, 0.91 mM sodium pyruvate, 10 ng/mL epidermal growth factor, 75 μg/mL kanamycin, 1 μg/mL insulin, 10 IU/mL equine chronic gonadotropin (eCG), and 10 IU/mL human chorionic gonadotropin (hCG) (Intervet, Boxmeer, Netherlands). PVA was replaced with purified 10% (v/v) pFF in a general maturation medium for IVM. The selected COCs were cultured in a humidified chamber at 39°C with 5% CO₂ and 95% air. COC cultures were maintained for 40–42 h. During the first 22 h, the COCs were maintained in a medium containing eCG and hCG, and during the subsequent 18–20 h, they were maintained in a hormone-free medium. Throughout the maturation period, CCL2 was added to each well at four different concentrations [0 (Control), 1, 10, and 100 ng/mL], determined as previously described (28).

We used RT-PCR to examine CCL2 expression in the three types of porcine follicular cells (oocytes, CCs, and GCs), before and after IVM. In general, IVM targets COCs, but we separately cultured it to confirm the mRNA levels of CCL2 in GCs according to the presence or absence of hormone treatment. First, we isolated mural GCs and immature COCs from pFF samples precipitate collected as described in 2.3. The mural GCs were classified under a microscope except for COCs (32). Here, we decided to refer to these mural GCs after being immediately isolated from pFF as 'GCs before IVM'. Then, oocytes and CCs were separated from immature COCs using 0.1% (w/v) hyaluronidase and sampled for experiments. After 40–42 h of IVM, mature CCs and mature oocytes in the metaphase II (MII) stage were obtained. GCs were cultured separately but under the same conditions that were used for IVM. Referring to the previously described method for in vitro culture of porcine GCs, GCs were cultured in medium containing eCG and hCG for the first 22 h, followed by culture in hormone-free medium for 18–20 h (33). After 40–42 h of culture, we decided to refer to these as “GCs after IVM”.

RNA was extracted using TRIzol reagent (TaKaRa Bio, Otsu, Shiga, Japan). Complementary DNA (cDNA) was synthesized using a reverse transcription master mix (Elpis Bio, Daejeon, Korea), in accordance with the manufacturer's instructions. RT-PCR was conducted using 1 μL cDNA template, 10 pmol CCL2 forward and reverse primers (Macrogen, Seoul, Korea), two units of Taq polymerase (Elpis Bio), 2 μL 10X PCR buffer (Elpis Bio), and 5 pmol deoxyribonucleoside triphosphate mix (iNtRON Biotechnology, Seongnam, Korea). For RT-PCR amplification of oocytes, the following cycling conditions were used: 33 or 36 cycles of denaturation at 95°C for 30 s, annealing at 59°C for 30 s, and extension at 72°C for 30 s. Supplementary Table 1 lists all primer sequences. The mRNA expression values were standardized to that of 18S ribosomal RNA (RN18S). The RT-PCR products were analyzed by electrophoresis at 100 V for 25 min on a 1.25% agarose gel stained with RedSafe nucleic acid staining solution (iNtRON Biotechnology). A Bio-1000F gel imager (Microtek International, Hsinchu, Taiwan) was used to capture images of the gel.

GCs, CCs, and oocytes before and after IVM were cultured as described in Section 2.4. COCs collected before and after IVM were separated from each other by physical pipetting using 0.1% (w/v) hyaluronidase. Immunostaining was performed as previously described (34). First, CCs and GCs before and after IVM were fixed in 4% paraformaldehyde in PBS for 10 min at 25°C (room temperature; RT). Oocytes before IVM and mature oocytes at the M II stage after IVM were fixed in 0.1% paraformaldehyde in PBS. Following fixation, all cells were washed thrice for 5 min with Dulbecco's phosphate-buffered saline (DPBS) containing CaCl₂, MgCl₂, and 0.1% (w/v) PVA and then permeabilized for 30 min with 0.5% Triton X-100. All cells were treated with Image-iT™ FX Signal Enhancer (Invitrogen, Carlsbad, CA, USA) for 30 min at RT to remove the non-specific background and then blocked for 30 min at RT with blocking buffer (Cell Signaling Technology, Beverly, MA, USA). After they were processed with the blocking buffer, the cells were incubated overnight at 4°C with the following primary antibodies: mouse anti-MCP-1 (cs-32771, 1:50 dilution in blocking buffer; Santa Cruz Biotechnology, CA, USA) and anti-CCR2 (NBP1-48338, Novus Biologicals, Littleton, CO, USA). The next day, all cells were washed thrice for 5 min with 0.1% PVA-PBS and then incubated for 1 h on a shaker with the following secondary antibodies: goat anti-mouse IgG (H + L) Alexa Fluor™ 488 (A11029, 1:200; Invitrogen, Carlsbad, CA, USA) and donkey anti-rabbit IgG (H + L) Alexa Fluor™ 594 (A21207, 1:200; Invitrogen). After removal of the secondary antibodies, all cells were washed thrice for 5 min with 0.1% PVA-PBS. The nuclei of all cells were counterstained with 10 μg/mL Hoechst-33342 for 10 min and mounted on glass slides using an anti-fade mounting medium (Molecular Probes, Eugene, OR, USA). A confocal laser microscope (Carl Zeiss, Thornwood, NY, USA) was used to examine the oocytes, and ZEN (blue edition) software was used to analyze all captured images. In negative controls, a blocking buffer made in the incubation medium was used instead of the primary antibody.

The rate of nuclear maturation was evaluated after 40–42 h of IVM with CCL2 supplementation. CCs were removed from the oocytes by gently pipetting 0.1% hyaluronidase into the IVM medium and washing in with TLH/PVA. Denuded oocytes from each group were collected, stained for 10 min with TLH/PVA containing 10 mg/mL Hoechst-33342, and then examined under a fluorescence microscope (Nikon, Tokyo, Japan). To assess meiotic maturation, oocytes were classified into the following stages: germinal vesicle (GV), metaphase I (MI), anaphase/telophase I (A/TI), and MII. Four stages were evaluated based on the previously reported morphology of stained nuclei (35). Oocytes that underwent an initial polar body extraction were considered mature. The experiment was performed in triplicate.

Mature oocytes were collected 42–44 h after IVM, and intracellular GSH and ROS levels were measured as previously described (36). In summary, intracellular GSH was detected in the ooplasm using CellTracker Blue 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin (CMF₂HC) (Invitrogen), and intracellular ROS levels were detected using the indicator 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) (Invitrogen). Twenty oocytes were placed in TLH/PVA supplemented with 10 μM CMF₂HC dye or 10 μM H₂DCFDA and then incubated for 30 min in the dark. After washing thrice with TLH/PVA, the dyed oocytes were placed into a 10 μL drop of TLH/PVA and then quantified using an epifluorescence microscope (TE300; Nikon) with an ultraviolet filter (370 nm for GSH; 460 nm for ROS). The fluorescence intensity was quantified using Image J software (NIH, Bethesda, MD, USA) and normalized to that of control oocytes. Three independent experiments were conducted (GSH samples, n = 60; ROS samples, n = 60 per group).

To analyze CCL2 expression patterns in the three types of follicular cells before and after IVM, oocytes, CCs, and GCs were collected from aspirated pFF and sampled immediately. CCs and oocytes were sampled after 40–42 h of IVM in a medium containing pFF. GCs were cultured separately under the same conditions. CCs and oocytes were separated from the 60 COCs and isolated from each other by gentle pipetting with 0.1% hyaluronidase. All cells were washed twice in DPBS, placed in 1.5 mL microfuge tubes, and frozen at −80°C until analysis. qPCR was performed to analyze the expression of CCL2 and 18 specific genes associated with particular functions, including the apoptosis-related genes: BCL2 associated X (BAX), BCL2 like 1 (BCL2L1), and caspase-3 (CASP3); the antioxidant-related genes: nuclear factor erythroid 2-related factor 2 (NRF2), superoxide dismutase 1 (SOD1), and superoxide dismutase 2 (SOD2); the cumulus expansion-/proliferation-related genes: pentraxin 3 (PTX3), TNF alpha induced protein 6 (TNFAIP6), CD44 molecule (Indian blood group) (CD44), and proliferating cell nuclear antigen (PCNA); the oocyte meiotic resumption-related gene: natriuretic peptide receptor 2 (NPR2); the developmental competence-related genes: zygote arrest 1 (ZAR1) and nucleophosmin/nucleoplasmin 2 (NPM2); and the CCL2 signaling pathway-related genes extracellular signal-regulated protein kinase 1 (ERK1), extracellular signal-regulated protein kinase 2 (ERK2), phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1), AKT serine/threonine kinase 1 (AKT1), and nuclear factor kappa B subunit 1 (NFKB1). The mRNA expression levels of CCL2 were analyzed in oocytes, CCs, and GCs before and after IVM. In CCs, we assessed the mRNA expression levels of BAX, BCL2L1, CASP3, NRF2, SOD1, SOD2, PTX3, TNFAIP6, CD44, PCNA, NPR2, ERK1, ERK2, PIK3R1, AKT1, and NFKB1, and in oocytes, we assessed the mRNA expression levels of BAX, BCL2L1, CASP3, NRF2, SOD1, PCNA, ZAR1, NPM2, ERK1, ERK2, PIK3R1, AKT1, and NFKB1. Supplementary Table 1 lists all primer sequences.

RNA extraction and cDNA synthesis were conducted as described in Section 2.4. qPCR was performed as previously described (37). The qPCR mixture for the CFX96 Touch RealTime PCR Detection System (Bio-Rad, Hercules, CA, USA), containing 1 μg synthesized cDNA, 2X SYBR Premix Ex Taq (TaKaRa Bio), and 10 pmol of specific primers (Macrogen). The reactions were run for 40 cycles of denaturation at 95°C for 15 s, annealing at 57°C for 15 s, and extension at 72°C for 24 s. Relative quantification was performed at a constant fluorescence intensity, using threshold cycle-based techniques. Relative mRNA expression (R) was determined using the following equation: R = 2 ^{−[ΔCt sample−ΔCt control]}. R-values for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in CCs and GCs and RN18S in oocytes were used to normalize the R values for each gene. Statistical analyses were conducted using at least three independent replicates.

Protein expression and activation levels were measured by western blotting using previously described techniques (38). After IVM with CCL2 supplementation, CCs, and oocytes were separated using 0.1% hyaluronidase and sampled. All samples were stored at −80°C until analysis. Total protein was extracted from CCs and oocytes using a 100 X Halt™ protease and phosphatase inhibitor single-use cocktail and 0.5 M ethylenediaminetetraacetic acid solution (Thermo Fisher Scientific, Waltham, MA, USA). Using bovine serum albumin as a standard, the protein concentrations were assessed using a Pierce™ bicinchoninic acid protein assay kit (Thermo Fisher Scientific), according to the manufacturer's instructions. Lysates were resolved using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). After washing thrice for 10 min with Tris-buffered saline–Tween 20 buffer (0.2 μM Tris, 1.37 M NaCl, and 0.05% Tween 20), the membranes were blocked for 5 min at RT using EveryBlot blocking buffer (Bio-Rad) and then incubated with primary antibodies overnight at 4°C. The following primary antibodies were used rabbit anti-ERK1/2 (1:1,000; 9102S; Cell Signaling Technology), rabbit anti-phospho ERK1/2 (p-ERK1/2) (1:1,000; 9101S; Cell Signaling Technology), and rabbit anti-GAPDH (1:1,000; 2118S; Cell Signaling Technology). The following day, the membranes were washed thrice with Tris-buffered saline–Tween 20 buffer for 10 min on a shaker at 100 revolutions per min and then incubated with horseradish peroxidase-conjugated secondary antibodies (anti-rabbit) for 1.5 h at RT. A chemiluminescent substrate kit (4:1 mixture of SuperSignal West Pico PLUS chemiluminescent substrate and SuperSignal West Femto Maximum Sensitivity Substrate; Thermo Fisher Scientific) was used to visualize the target proteins. The protein bands were detected using a Lumino Graph II (ATTO Corporation), and the optical densities of the target proteins were determined using CS Analyzer 4 software. The expression levels of all proteins were normalized to that of GAPDH. All experiments were performed at least thrice.

After IVM, CCs and oocytes were isolated from each other, as described above. MII oocytes from each group were selected and washed with a calcium-free TLH/PVA medium. PA was performed using an established procedure (39). The MII oocytes were washed twice with a 280 mM mannitol solution containing 0.01 mM CaCl₂ and 0.05 mM MgCl₂. The MII oocytes were loaded between the electrodes of the chamber in 2 mL of a 260 mM mannitol solution containing 0.001 mM CaCl₂ and 0.05 mM MgCl₂. An electrical pulsing machine (LF101; Nepa Gene, Chiba, Japan) was connected to the chamber, and the MII oocytes were stimulated by two 60s 120 V/mm direct -current pulses. After PA, the activated oocytes from each group were immediately transferred to a porcine zygote medium containing 5 μg/mL cytochalasin B, which served as an IVC culture medium (40). The oocytes were incubated at 39°C in a humidified atmosphere of 5% CO₂ and 95% N₂ for 4 h. After rinsing twice in a fresh IVC medium, the oocytes were placed into droplets of 25 μL fresh IVC medium (10 oocytes per droplet) covered with mineral oil. Embryos were cultured for 7 d at 39°C in a humidified incubator with 5% O₂, 5% CO₂, and 90% N₂. The IVC culture medium was refreshed after 48 h (Day 2) and 96 h (Day 4) with 10% fetal bovine serum. The experiment was repeated thrice.

The cleavage of PA embryos was assessed on Day 2. Normally cleaved embryos were classified into the 2–3 cell, 4–5 cell, and 6–8 cell embryo stages, excluding 1-cell embryos and fragmented embryos. Blastocyst formation was evaluated on Day 7, similar to a previous study (41). The blastocysts were divided into three groups based on their morphology: early, expanded, or hatched. To calculate the total cell number, on Day 7, blastocysts were rinsed in TLH/PVA, and fixed in 4% paraformaldehyde in PBS/PVA and then stained for 10 min with 5 g/mL Hoechst-33342. Blastocysts fixed in 100% glycerol droplets were mounted on glass slides and counted manually using a fluorescence microscope (TE300, Nikon) with an ultraviolet filter (370 nm). The experiment was repeated thrice.

All experiments in this study were conducted at least thrice. For each replicate, ovaries obtained on the same day from the same slaughterhouse were used. Data were statistically analyzed using GraphPad Prism (GraphPad Software, San Diego, CA, USA) and SPSS version 12.0 (SPSS, IBM, Armonk, NY, USA). Following a one-way analysis of variance (ANOVA) test, Duncan's multiple range test was used to examine percentage data (rates of nuclear maturation and embryonic development) and average data (ELISA data, intracellular GSH and ROS levels in oocytes, and the total number of cells in blastocysts). An unpaired two-tailed Student's t-test was used to examine average data (Western blotting and qPCR of follicular cells before and after IVM) between two groups. Data are presented as the mean ± standard error of the mean. Differences were considered statistically significant at p < 0.05.

ELISA was performed to determine the concentration of CCL2 in follicular fluids produced by porcine follicles of three different sizes (small, medium, and large). The maturation medium, M199 was used as a negative control. CCL2 was detected in pFF from follicles of all sizes. The CCL2 concentration in pFF increased in proportion to follicle size. The CCL2 concentration in the large group (39.0 ± 0.3 pg/mL) was significantly higher than that in the small group (13.7 ± 0.9 pg/mL) and the medium group (24.2 ± 2.6 pg/mL). The CCL2 concentrations determined using a standard curve are shown in Table 1.

RT-PCR and qPCR were performed to analyze the expression of CCL2 in porcine follicular cells (oocytes, CCs, and GCs), before and after IVM. CCL2 was expressed in GCs, before and after IVM. In contrast, in oocytes and CCs, CCL2 was expressed only in the IVM group. After IVM, CCL2 was weakly expressed in oocytes (Figure 1A). The mRNA levels of CCL2 were quantified using qPCR. As shown in Figure 1B, these levels increased significantly in all three porcine follicular cell types after IVM.

The localization of CCL2 and its receptor, CCR2 in oocytes, CCs, and GCs were determined before and after IVM. The expression of CCL2 and CCR2 was observed in all cell types. In CCs and GCs, CCL2 and CCR2 were expressed in both the nucleus and cytoplasm, before and after IVM, and no significant difference was observed after IVM. Interestingly, in oocytes, CCL2 was strongly expressed in the zona pellucida before IVM (Figure 2A), but after IVM, it was strongly expressed in the zona pellucida and at the edge of the oocyte cytoplasm (Figure 2B). CCR2 exhibited high levels of overall expression in the oocyte cytoplasm both before and after IVM.

To investigate the effect of CCL2 treatment on the nuclear maturation of porcine oocytes during IVM, mature oocytes were evaluated at four stages: GV, MI, A/TI, and MII. After 42 h of IVM, the group supplemented with 100 ng/mL CCL2 exhibited a significantly (p < 0.05) higher M II rate (91.6 ± 3.6%) than the control group (81.8 ± 1.8%). In contrast, no significant differences were found between the other CCL2-treated groups (1 ng/mL: 87.6 ± 2.1%; 10 ng/mL: 91.3 ± 3.4%) and the control group (Table 2).

To investigate the effect of CCL2 treatment on oocyte cytoplasmic maturation during IVM, M II stage oocytes from each group were stained to evaluate intracellular GSH and ROS levels. Intracellular GSH levels were significantly (p < 0.05) increased in all CCL2-treated groups, compared to the control group. Intracellular ROS levels were significantly (p < 0.05) decreased in all CCL2-treated groups (Figures 3A, B). Therefore, CCL2 supplementation during IVM significantly affected the intracellular GSH and ROS levels in mature oocytes.

To investigate the effects of CCL2 treatment on porcine COCs during IVM, qPCR was used to analyze the expression of various genes in CCs and oocytes. To examine the effect of CCL2 on the expression of apoptosis-, antioxidant-, cumulus expansion-, and proliferation-related genes, the mRNA expression levels of BAX, BCL2L1, CASP3, NRF2, SOD1, SOD2, PTX3, TNFAIP6, CD44, and PCNA were assessed in CCs and oocytes. The mRNA expression of NPR2, a gene related to the resumption of meiosis in oocytes, was confirmed in CCs, and that of ZAR1 and NPM2, which are maternal-effect genes, was confirmed in oocytes.

Among apoptosis-related genes, the pro-apoptotic gene BAX exhibited a significantly (p < 0.05) decreased level of mRNA expression in CCs and oocytes in all CCL2-treated groups. CASP3 levels were significantly (p < 0.05) decreased in CCs in the 100 ng/mL treatment group, compared. CASP3 levels were also, significantly (p < 0.05) decreased in oocytes in all treatment groups, compared to control. Among antioxidant-related genes, the levels of SOD1 and SOD2 were significantly (p < 0.05) increased in CCs in the 100 ng/mL group, compared to the control group. The level of NRF2 was significantly (p < 0.05) increased in oocytes in the 10 ng/mL group. Among cumulus expansion- and proliferation-related genes, PTX3 levels were significantly (p < 0.05) increased in CCs in the 10 ng/mL group, compared to the control. CD44 levels were significantly (p < 0.05) increased in CCs in all treatment groups. PCNA levels were not significantly different in any of the treatment groups compared to those in the control. However, PCNA levels in oocytes were significantly (p < 0.05) increased in the 10 ng/mL group compared to those in the 100 ng/mL group.

The level of NPR2, a gene associated with oocyte meiotic resumption, was significantly (p < 0.05) decreased in CCs in the 100 ng/mL CCL2 treatment group, compared to the control. Levels of the maternal-effect gene, ZAR1 in oocytes exhibited no significant differences between any treatment groups and the control. In contrast, the expression of the maternal-effect gene NPM2 was significantly increased in oocytes treated with 10 ng/mL CCL2 (Figures 4A, B).

qPCR was used to assess the mRNA expression of several CCL2 signaling pathway-related genes, including ERK1, ERK2, PIK3R1, AKT1, and NFKB1, in CCs and oocytes after IVM.

The level of ERK1 in CCs and oocytes was significantly (p < 0.05) increased in the 10 ng/mL CCL2 treatment group, compared to that in the control. The levels of AKT1 and NFKB1 in oocytes were significantly (p < 0.05) increased in the 1 ng/mL treatment group, compared to those in the control (Figures 5A, B).

The mRNA expression of ERK1 was significantly increased in CCs and oocytes in the 10 ng/mL CCL2-treated group, compared to that in the control. Therefore, the protein expression of ERK1/2 was also analyzed.

In CCs, the ratio of phosphorylated ERK1/2 (p-ERK1/2) to total ERK1/2 (t-ERK1/2) was significantly (p < 0.01) increased only in the 10 ng/mL treatment group, compared to that in the control (Figure 6A). In contrast, in oocytes, there was no significant difference between the protein expression ratios of t-ERK1/2 to GAPDH, p-ERK1/2 to GAPDH, and p-ERK1/2 to t-ERK1/2 (Figure 6B).

After PA, the cleavage rate was significantly higher (p < 0.05) in the 100 ng/mL CCL2-treated group than in the control group (p < 0.05; Figure 7A). In contrast, the blastocysts formation rate was significantly higher in the 10 ng/mL CCL2-treated group than in the control group (p < 0.05; Figure 7B). However, the patterns of these two results were inconsistent. The rate of early blastocysts among PA embryos was significantly increased in the 100 ng/mL CCL2-treated group (p < 0.05), whereas the rate of hatched blastocysts in the 10 ng/mL CCL2-treated group was significantly higher than that in the control and other CCL2-treated groups (p < 0.05). However, the total cell number in blastocysts was not significantly different than in the control (Table 3).