Collection and in vitro maturation of porcine oocytes
Ovaries from pre-mature sows were obtained from local slaughterhouses (Farm Story Dodarm B&F, Umsung, Chungbuk, South Korea) and transferred to a laboratory at 38.5°C in saline supplemented with 75 mg/mL penicillin G and 50 mg/mL streptomycin sulfate. Follicles 3–6 mm in diameter were aspirated using an 18 mm needle attached to a 10 mL disposable syringe. Cumulus-oocyte complexes were selected based on the presence of at least three layers of cumulus cells and a uniformly granulated follicle. After rinsing three times with in-vitro maturation medium TCM-199 (11150-059; Gibco, Grand Island, NY, USA) supplemented with 0.1 g/L sodium pyruvate, 10 ng/mL epidermal growth factor, 10% (v/v) porcine follicular fluid, 10 IU/mL luteinizing hormone, and 10 IU/mL follicle-stimulating hormone, approximately 70 cumulus-oocyte complexes were each transferred to one well of a 4-well dish (SPL Life Sciences, Seoul, South Korea) containing 500 µL of maturation medium. The medium was covered with mineral oil and the dish was incubated at 38.5°C in a humidified atmosphere containing 5% CO2 for 44 h. During incubation, the rotenone treated groups were supplemented with rotenone at different concentrations (2, 3, and 5 µM). At a concentration of 3 µM, there was a significant difference in the experiment, and, therefore, this concentration was used in the subsequent experiments.
Assessment of cumulus cell expansion
After 44 h of IVM, CC expansion was measured using four reference grades. Considering the average CC expansion size in the control group, grades were divided into 0-200, 200–300, 300–400, and > 400 µm. Both the control and treatment groups were separated according to these criteria and the ratios were compared[35].
ROS measurements
Total ROS levels in mature oocytes were measured using 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA, Cat # D399, Molecular Probes, Eugene, OR, USA)[36]. Oocytes were incubated in phosphate-buffered saline (PBS)/PVA containing 10 µM H2DCF-DA at 38.5°C for 30 min. After incubation, the oocytes were washed three times with PBS/PVA. Fluorescence signals were captured as .jpg files using a digital camera (DP72; Olympus, Tokyo, Japan) connected to a fluorescence microscope (IX70; Olympus). Mitochondrial ROS generation was assessed using the MitoSOX™ mitochondrial peroxide indicator (Thermo Fisher). Oocytes were incubated in PZM-5 with 10 µM MitoSOX™ solutions at 38.5°C for 30 min. After incubation, oocytes were washed three times with PBS/PVA and fixed in 3.7% paraformaldehyde for 30 min at room temperature (20–25°C). Total and mitochondrial-derived ROS levels were quantified by analyzing the fluorescence intensity of oocytes using Fiji software (National Institutes of Health).
GSH measurements
To measure GSH levels, oocytes were incubated in PBS/PVA containing 10 µM 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin dye (CellTracker™ Blue CMF2HC Dye, Cat #C12881, Thermo Fisher Scientific, Waltham, USA) at 38.5°C for 30 min and then washed three times with PBS/PVA. Fluorescence signals were captured as .jpg files using a digital camera (DP72; Olympus, Tokyo, Japan) connected to a fluorescence microscope (IX70, Olympus). GSH levels were quantified by analyzing the fluorescence intensity of oocytes using Fiji software (National Institutes of Health).
ATP contents assay
ATP contents were measured using a luciferin-luciferase ATP assay system with a luminometer (CentroPRO LB 962), according to the manufacturer’s instructions (A22066, Molecular Probes). Briefly, ten oocytes were collected in a 0.2 mL centrifuge tube containing 20 µL of lysis buffer (20 mM Tris, 0.9% Nonidet-40, and 0.9% Tween 20) and homogenized in a vortex mixer until they were completely lysed. The standard reaction solution was prepared according to the manufacturer's instructions and placed on ice in the dark before use. Before measurement, the samples (5 µL) were added to 96-well plates and equilibrated for 10 s. Subsequently, 200-µL standard reaction solution was added to each well, and the light signal was integrated for 10 s after a 2 s delay. The light intensity in the control group was arbitrarily set to 1, and the light intensity in the treatment group was measured and expressed as relative values to the control group.
Immunofluorescence and confocal microscopy
After washing thrice with PBS/PVA, the oocytes were fixed in 3.7% formaldehyde solution at room temperature for 30 min, permeabilized with PBS/PVA containing 0.5% Triton X-100 at room temperature for 30 min and incubated in PBS/PVA containing 1.0% BSA at room temperature for 1 h. These oocytes were then incubated overnight at 4°C with anti‐TOM20 (Cat #sc-17764, Santa Cruz), anti‐PGC-1α (Cat #66369-1-Ig, Proteintech), anti‐SIRT1 (Cat #60303-1-Ig, Proteintech), anti-ubiquitin (Cat #ab19247, Abcam), anti‐LC3 (Cat #2775S, Cell Signaling Technology), anti‐PINK1 (Cat #ab23707, Abcam), anti-cytochrome C (Cat #ab110325, Abcam), or anti‐Caspase3 (Cat #C8487, Sigma-Aldrich) diluted in blocking solution. After washing three times with PBS/PVA, the oocytes were incubated at room temperature for 1 h with Alexa Fluor 488™ donkey anti-mouse IgG (H + L) or Alexa Fluor 546™ donkey anti-rabbit IgG (H + L). The oocytes were then stained with 10 µg/mL Hoechst 33342 for 10 min, washed thrice with PBS/PVA, mounted onto slides, and examined under a confocal microscope (Zeiss LSM 710 META). The images were processed using Zen software (version 8.0, Zeiss).
To detect total and active mitochondria, oocytes were first incubated with 500 nM MitoTracker Red CMXRos (Cat #M7512, Invitrogen) at 38.5°C for 30 min. After three washes with PBS/PVA, immunofluorescence staining for TOM20 was performed as described above. Total and active mitochondria were quantified by analyzing the fluorescence intensity of the oocytes using Fiji software (National Institutes of Health, Bethesda, MD, USA).
Western blot analysis
As previous describe[37], briefly a total of 100 porcine oocytes per group were lysed with 1 × sodium dodecyl sulfate sample buffer by heating at 98°C for 10 min. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. Next, the membranes were blocked in 5% (w/v) skim milk for 1 h and then incubated at 4°C overnight with anti-SIRT1 (Cat #60303-1-Ig, Proteintech), anti-ubiquitin (Cat #ab19247, Abcam), or GAPDH (Cat #sc-365062, Santa Cruz), followed by incubation at room temperature for 1 h with horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG (1:20000; Santa Cruz Biotechnology). The membrane was detected using Pierce ECL substrate (Thermo Fisher Scientific). Blots were visualized using a CCD camera and UVISoft software (UVITEC, Cambridge, UK).
mtDNA copy number measurement
Each pool of three oocytes was transferred to a 0.2 mL tube containing 8 µL lysis buffer (20 mM Tris, 0.4 mg/mL proteinase K, 0.9% Nonidet-40, and 0.9% Tween 20) at 65°C for 30 min, followed by 95°C for 5 min. The samples were diluted 1:25 in sterile ddH2O before analysis. Real-time PCR was performed using the WizPure™ qPCR Master (Super Green) Mix (Cat # W1731-8, Wizbiosolution). Amplification was conducted as follows: 95°C for 3 min, followed by 40 cycles at 95°C for 15 s, 60°C for 25 s, and 72°C for 10 s, with a final extension at 72°C for 5 min. The real-time PCR primer set was: ND1-F (5′- CCT ACT GGC CGT AGC ATT CC-3′), ND1-R (5′-GAG GAT GTG CCT GGT CGT AG -3′). The mitochondrial DNA copy number was analyzed using the \({2}^{-{\Delta }{\Delta }\text{C}\text{T} }\) method[38].
Evaluation of co-localization
Oocytes were stained with Mito-Tracker, cytochrome C, PINK1, or TOM20 according to the method described in 'Immunofluorescence and confocal microscopy'. The oocytes were observed using a laser scanning confocal microscope (Zeiss LSM 710 META, Germany). Co-localization of mitochondrial/cytochrome C and PINK1/TOM20 was evaluated using Pearson’s correlation coefficient.
Statistical analysis
Each experiment was repeated at least three times, and the representative images are shown in Figs. 1–7. All percentage data were subjected to arcsine transformation before statistical analysis and are presented as the mean ± standard error (SEM). Statistical significance was set at P < 0.05. All calculations were performed using SPSS software v.19 (SPSS Inc., Chicago, IL, USA).