All chemicals and reagents were purchased from Sigma (St. Louis, MO, USA) unless stated otherwise.

Ovaries used in the experiment were obtained from pigs raised about 6 months and weigh approximately 120 kg. Prepubertal porcine ovaries were collected from a local slaughterhouse and transported to the laboratory in saline supplemented with 75 μg/mL penicillin G and 50 μg/mL streptomycin sulfate within 2 h at 30°C to 33°C. Cumulus-oocyte complexes (COCs) were aspirated from follicles with a diameter of 2 to 8 mm using an 18-gauge needle and a disposable 10 mL syringe. COCs were washed three times in tissue culture medium (TCM)-199–HEPES containing 0.1% (w/v) bovine serum albumin (BSA). Thereafter, COCs were matured in groups of 50 in 500 μL TCM-199 (Gibco, Grand Island, NY, USA) containing Earle’s salts, 0.57 mM cysteine, 10 ng/mL epidermal growth factor, 0.5 μg/mL follicle-stimulating hormone from porcine pituitary, 0.5 μg/mL luteinizing hormone from sheep pituitary, and 10% (v/v) porcine follicular fluid under mineral oil for 44 h at 38.8°C in 5% CO₂ in air. Various concentrations (0 [control group], 1, 10, 100, and 200 μM) of PD were added together with 200 μM H₂O₂. For the normal group, neither PD nor H₂O₂ was added. Each experiment was independently repeated six times, with 50 to 60 oocytes per experiment. All data are presented as the means±standard error of the mean.

The aim of PA is to simulate the fertilization process by artificial stimulation to activate oocytes under non-sperm conditions [7]. Without sperm intervention, it is more efficient in researching physiological events which occurs in oocyte itself during its activation [7]. Moreover, it allows us to investigate the role of maternal genomes in controlling early embryo development separately from paternal genomes [8]. The PA and subsequent embryo culture were performed as previously described [9]. Following maturation, cumulus cells were removed by pipetting in the presence of 1 mg/mL hyaluronidase for 2 to 3 min. PA was induced by treating oocytes with porcine zygote medium-5 containing 0.4% (w/v) BSA (in vitro culture [IVC] medium) and 5 μM Ca²⁺ ionomycin for 5 min. After 3 h of culture in IVC medium containing 7.5 μg/mL cytochalasin B, embryos were washed three times with IVC medium and cultured for 7 days in the same medium at 38.8°C in a humidified atmosphere of 5% CO₂ and 95% air. On day 5, half the medium was removed and replaced with PZM-5 containing 10% (v/v) fetal bovine serum. On day 7, blastocysts were washed in Dulbecco’s phosphate-buffered saline (DPBS), and either fixed in 3.7% (w/v) paraformaldehyde for 20 min and stored at 4°C, or lysed and snap-frozen in liquid nitrogen and stored at −80°C, depending on the experiment.

Dichlorohydrofluorescein diacetate (DCFHDA) and CellTracker Blue 4-chloromethyl-6,8-difluoro-7-hydroxy-coumarin (CMF₂HC) were used to determine the intracellular levels of ROS and GSH, respectively, as previously described [10,11] with slight modifications. Briefly, cumulus cells were removed from COCs by pipetting in the presence of 0.1% (w/v) hyaluronidase. Denuded oocytes were incubated in DPBS containing 50 μM DCFHDA or 100 μM CMF₂HC in the dark for 20 min at 38.8°C. Thereafter, oocytes were washed more than five times with DPBS containing 0.1% (w/v) BSA to completely remove excess dye and immediately analyzed by epifluorescence microscopy (Olympus, Tokyo, Japan). The ROS level was measured using excitation and emission wavelengths of 450 to 490 nm and 515 to 565 nm, respectively. The excitation and emission wavelengths of CMF₂HC are 371 and 464 nm, respectively. Grayscale images were acquired with a digital camera (Nikon, Tokyo, Japan) attached to the microscope, and mean grayscale values were calculated using ImageJ software (NIH, Bethesda, MD, USA). Background fluorescence values were subtracted from the final values before statistical analysis. Each experiment was independently repeated 6 to 7 times, with 20 to 30 oocytes per experiment.

Meiotic spindles and nuclei of oocytes were visualized after maturation. Cumulus cells were removed from porcine COCs matured for 44 h and then oocytes were fixed overnight at 4°C in 4.0% (w/v) paraformaldehyde prepared in phosphate-buffered saline (PBS). Fixed oocytes were incubated for 30 min at 38.8°C with 0.5% (v/v) Triton X-100. After blocking for 1 h with 1% BSA (w/v) prepared in PBS (blocking solution I), oocytes were incubated overnight at 4°C with a mouse monoclonal anti-α-tubulin-FITC antibody (diluted 1:200 in blocking solution I). Nuclei were stained with Hoechst 33342 (1 μg/mL) for 30 min. Finally, oocytes were washed three times with PBS containing 0.1% (w/v) BSA, mounted on glass slides, and observed under an inverted Olympus IX-71 microscope. To further investigate the effect of PD on spindle organization, spindles without abnormalities were classified as normal, whereas those in which chromosomes failed to align at the metaphase plate were classified as abnormal [12]. Each experiment was independently repeated three times, and at least 20 oocytes were examined per group.

On day 7 after PA, blastocysts were fixed overnight at 4°C with 4.0% (w/v) paraformaldehyde prepared in PBS, washed three times with PBS containing 0.1% BSA, and then incubated with 0.1% Triton X-100 at 38.8°C for 30 min. Blastocysts were incubated with fluorescein-conjugated dUTP and terminal deoxynucleotidyl transferase (In Situ Cell Death Detection Kit; Roche, Manheim, Germany) in the dark for 1 h at 38.8°C. Thereafter, nuclei were stained with Hoechst 33342 (1 μg/mL) for 30 min, and stained blastocysts were washed with PBS containing 0.1% BSA. Washed blastocysts were mounted on glass slides and examined under an inverted Olympus IX-71 fluorescence microscope. The experiment was independently repeated 7 to 8 times, and at least 10 to 20 blastocysts were examined per group.

mRNA was isolated from more than three biological replicates, with 30 to 40 oocytes per replicate, using a Dynabeads mRNA Direct Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. mRNA was collected in 10 μL elution buffer provided with the kit. Eluted RNA was reverse-transcribed into complementary DNA using an oligo (dT) 20 primer and SuperScript II reverse transcriptase (Invitrogen, USA) according to the manufacturer’s instructions.

The protocol used was basically the same as that described previously [13]. Real-time reverse transcription polymerase chain reaction (RT-PCR) was performed using the primer sets listed in Table 2 and a StepOnePlus Real-time PCR System (Applied Biosystems, Warrington, UK) with a final reaction volume of 20 μL containing SYBR Green PCR Master Mix (Applied Biosystems, UK). The PCR conditions were as follows: 10 min at 95°C, followed by 39 cycles of 15 s at 95°C and 60 s at 54°C or 60°C. Samples were then cooled to 12°C. Relative gene expression levels were analyzed by the 2^−ΔΔCt method [14] after normalization against the expression level of the housekeeping gene β-actin. The experiment was independently repeated five times.

The protocol was basically the same as that described previously [13]. In brief, oocytes (40 per sample) were solubilized in 20 μL of 1× sodium dodecyl sulfate (SDS) sample buffer (62.5 mM Tris-HCl, pH 6.8, containing 2% [w/v] SDS, 10% [v/v] glycerol, 50 μM dithiothreitol, and 0.01% [w/v] bromophenol blue or phenol red) and heated for 5 min at 95°C. Proteins were resolved on 5% to 12% Tris SDS-polyacrylamide gel electrophoresis gels for 1.5 h at 80 to 100 V. Samples were then transferred to Hybond-ECL nitrocellulose membranes (Amersham, Buckinghamshire, UK) at 300 mA for 2 h in transfer buffer (25 mM Tris, pH 8.5, containing 200 mM glycine and 20% [v/v] methanol). After blocking with 5% (w/v) nonfat milk prepared in PBS for 1 h, the membranes were incubated for at least 2 h with an rabbit anti-p44/42 mitogen-activated protein kinase (MAPK) or anti-phospho-p44/42 MAPK antibody both purchased from Cell Signaling Technology (diluted 1:500 in blocking solution [1× Tris-buffered saline, pH 7.5, containing 0.1% (v/v) Tween-20% and 5% (w/v) nonfat milk]). Thereafter, the membranes were washed three times in TBST (20 mM Tris-HCl, pH 7.5, containing 250 mM NaCl and 0.1% [v/v] Tween-20) and incubated for 1 h with mouse anti-rabbit IgG-horseradish peroxidase purchased from Santa Cruz Biotechnology (diluted 1:2,000 in blocking solution). After three washes with TBST, immunoreactive protein bands were visualized on X-ray films using the chemiluminescent reagent luminol (Invitrogen, USA) in a dark room. The experiment was independently repeated three times.

The general linear model procedure within the Statistical Analysis System (SAS User’s Guide, 1985; Statistical Analysis System Inc., Cary, NC, USA) was used to analyze data from all experiments. The paired Tukey’s multiple range test was used to determine significant differences. p-values less than 0.05 were defined as statistically significant.

To determine the optimal concentration of PD, porcine oocytes were matured for 44 h with 0, 1, 10, 100, and 200 μM PD (control, 1PD, 10PD, 100PD, and 200PD groups, respectively) in the presence of 200 μM H₂O₂. Oocytes in the normal group were matured in IVM medium without any supplements. Following PA, the percentage of cleaved oocytes on day 2 did not significantly differ between the groups (normal, 80.1%±2.5%; control, 78.6%±2.3%; 1PD, 74.8%±4.0%; 10PD, 78.4%± 4.0%; 100PD, 80.7%±3.6%; and 200PD, 81.9%±2.6%; Table 2). However, the percentage of oocytes that reached the blastocyst stage on day 7 was significantly higher in the normal and 10PD groups than in the control and 1PD groups, but did not significantly differ between these four groups and the 100PD and 200PD groups (normal, 38.9%±1.4%; control, 30.5%±3.3%; 1PD, 30.8%±3.4%; 10PD, 41.8%±2.9%; 100PD, 35.8 %±4.7%; and 200PD, 38.5%±3.8%; Table 2). Therefore, the normal, control, and 10PD groups were compared in subsequent experiments.

The effects of PD on the ROS and GSH levels were assessed by staining oocytes with DCFHDA and CMF₂HC, respectively (Figure 1A). The ROS level did not significantly differ between the three groups. The GSH level was significantly higher (p<0.05) in the normal and 10PD groups than in the control group.

Expression of the antioxidant genes SOD1, SOD2, nuclear factor erythroid 2-related factor 2 (Nrf2), and hemo oxygenase-1 [HO-1] was analyzed by real-time RT-PCR (Figure 1B). mRNA expression of SOD1 was significantly higher (p<0.05) in the normal group than in the control group, and was substantially higher in the 10PD group than in the control group; however, this difference was not significant. mRNA expression of SOD2 was significantly higher (p<0.05) in the 10PD than in the normal and control groups, but did not significantly differ between the latter two groups. mRNA expression of Nrf2 was significantly higher in the normal and 10PD groups than in the control group. The mRNA expression pattern of HO-1 was similar to that of Nrf2.

The percentage of oocytes with a normal meiotic spindle and normal chromosomal alignment was significantly higher in the normal (p<0.01) and 10PD (p<0.05) groups than in the control group, and was similar in the normal and 10PD groups (normal, 80.5%±3.1%; control, 56.8%±6.4%; and 10PD, 78.0%±4.1%; Figure 2).

Several studies have suggested that MAPK phosphorylation is an important marker to evaluate cytoplasmic maturation. Therefore, we investigated whether PD improves porcine oocyte maturation via the MAPK signal transduction pathway. Lysates from the normal, control, and 10PD groups were immunoblotted with an anti-phosphorylated MAPK antibody and subsequently re-probed with an anti-MAPK antibody to normalize the densitometric results. MAPK migrates as a doublet at 44 and 42 kDa, representing p44/42 MAPK (ERK1/2). The ratio of phosphorylated MAPK (phospho-p44/42 MAPK), which is the active form, to total MAPK was significantly lower (p<0.05) in the control group than in the normal and 10PD groups and was significantly higher (p<0.05) in the 10PD group than in the normal group (normal, 1.0±0.0; control, 0.8±0.1; and 10PD, 1.2±0.1; Figure 3).

To investigate whether PD treatment during IVM of porcine oocytes influences subsequent embryo development and quality, the total cell number and genomic DNA fragmentation in blastocysts were assessed (Figure 4A). The total cell number per blastocyst was significantly higher (p<0.05) in the 10PD group than in the control group, and was slightly lower in the control group than in the normal group; however, this difference was not significant (normal, 78.0±5.0; control, 73.2±4.1; and 10PD, 85.0±2.9; Figure 4B). The percentage of apoptotic cells in blastocysts determined by assessment of genomic DNA fragmentation was higher in the control group than in the normal and 10PD groups; however, this difference was not significant (normal, 2.6%±0.6%; control, 2.7%±0.3%; and 10PD, 1.8%±0.4%; Figure 4C).

Expression of the development-related genes (POU domain, class 5, transcription factor 1 [POU5F1], caudal type homeobox 2 [CDX2], Nanog homeobox [NANOG]) at the blastocyst stage was analyzed by real-time RT-PCR (Figure 5). mRNA expression of POU5F1 was slightly decreased by addition of H₂O₂ and increased by supplementation of PD, but did not significantly differ between the three groups. mRNA expression of CDX2 was significantly higher (p<0.05) in the 10PD group than in the normal and control groups, and was similar in the latter two groups. mRNA expression of NANOG was slightly lower in the control group than in the normal group, and was significantly higher (p<0.05) in the 10PD group than in the control group.