INTRODUCTION
Oocyte in vitro maturation (IVM) is an important technology to generate mature oocytes and embryos for human infertility treatment and animal reproduction. However, it is generally accepted that the quality and developmental potential of IVM oocytes is lower than those of their in vivo counterparts, probably partly due to insufficient cytoplasmic maturity [1]. Mitochondria are the most abundant organelles in cells, and they are thought to play important roles in determining oocyte cytoplasmic maturation [2]. It is well established that mitochondria are not only the cells’ energy generators, but also the major sites of reactive oxygen species (ROS) production. Nevertheless, excessive accumulation of ROS can cause oxidative stress, which may subsequently lead to mitochondrial dysfunction, decreasing the developmental competence of in vitro maturated oocyte [3]. Therefore, mitigating oxidative stress and strengthening mitochondria function is an effective strategy for improving the developmental competence of in vitro matured oocyte.
It is generally known that porcine oocytes contain a high content of intracellular lipids that are primarily stored in lipid droplets as triglycerides. Endogenous triglycerides are integral for energy metabolism during porcine oocyte IVM. However, the excessive lipid accumulation in oocytes is harmful to oocyte development in vitro [4]. Lipid metabolism via the fatty acid oxidation pathway produces adenosine triphosphate in mitochondria, which is important for oocyte maturation and embryo development [5]. Thus, regulation of lipid metabolism is beneficial for promoting mammalian oocytes IVM and embryo development [6].
Pyrroloquinoline quinone (PQQ), a water soluble, thermo-stable triglyceride-quinone, is identified initially as a mammalian vitamin-like redox cofactor [7]. It has been shown that PQQ is important for mammalian growth, development, reproduction and immune function [8]. PQQ can promote the expression of PGC-1α, which is a critical factor in regulating mitochondrial biogenesis and oxidative metabolism [9]. PQQ is also a powerful antioxidant protecting mitochondria against oxidative stress-induced lipid peroxidation, protein carbonyl formation and inactivation of the mitochondrial respiratory chain [10]. In addition to regulate mitochondrial biogenesis and mitigate oxidative stress-induced mitochondrial dysfunction, some studies indicated that PQQ could also enhance lipid β-oxidation, reduce lipid content and droplet size in animal liver and adipocytes [11, 12]. However, the beneficial effects and underlying mechanisms of PQQ during IVM on porcine oocyte maturation quality and their developmental competence have not been determined. Therefore, we investigated the effects of PQQ supplementation during porcine oocyte IVM on cumulus expansion, nuclear maturation, mitochondrial function, oxidative stress levels, lipid metabolism and developmental ability of embryos.
MATERIALS AND METHODS
Chemicals
All the chemicals and reagents used in this study were purchased from Sigma Aldrich (St. Louis, MO, USA) unless stated otherwise.
Animal studies
The protocols for the animal studies were approved by and performed in accordance with the requirements of Ethics Committee of Medicine and Life Sciences of Inner Mongolia Minzu University (approval no. NMD-DW-2024-11-58).
Oocyte collection and in vitro maturation
Porcine ovaries were obtained from a local slaughterhouse and transferred immediately to the laboratory in sterile 0.9% saline at 32°C to 35°C within 2 h. Follicular fluid containing cumulus oocyte complexes (COCs) was aspirated from about 3 to 6 mm antral follicles with an 18-gauge needle attached to a 10 mL disposable syringe. The COCs with compact cumulus cells and homogeneous cytoplasm of oocytes were collected and washed, and approximately 10 COCs were cultured in 50 μL of IVM medium covered with mineral oil for 42 h at 38.5°C with 5% CO2 in a humidified atmosphere. The IVM medium contained tissue culture medium 199 (TCM-199; Invitrogen, Waltham, MA, USA) supplemented with 10 IU /mL pregnant mare serum gonadotropin, 10 IU/mL human chorionic gonadotropin, 10 ng /mL epidermal growth factor, and 10% (v/v) porcine follicular fluid. During IVM, IVM medium was supplemented with different concentrations of PQQ (0 nM, 200 nM, 400 nM, 800 nM or 1,600 nM) depending on experimental design.
Cumulus expansion assessment
The degree of cumulus expansion was evaluated after IVM by microscopic examination. As shown in Figure 1C, grade 0: no expansion. Grade 1: minimum expansion, with spherical and compacted cumulus cells around the oocyte. Grade 2: outermost layer expansion of cumulus cells. Grade 3: the expansion of all layers of cumulus cells, except the corona radiata. Grade 4: complete expansion of all cumulus cell layers. Cumulus expansion index (CEI) was calculatedby by add up the scores of each COC (score 0 to 4) and dividing by the total number of COCs, as described previously [13].
Evaluation of oocyte nuclear maturation
After IVM, oocytes were mechanically separated from surrounding cumulus cells by repeated pipetting. Denuded oocytes were fixed in 4% paraformaldehyde for 20 min and permeabilized with Triton X-100 (0.5%) for at least 30 min at room temperature, then stained with 10 μg/mL 4’,6-diamidino-2-phenylindole for 5 min. After washing, denuded oocytes were mounted on a sliding glass with a coverslip and examined under a fluorescence microscope (Eclipse Ci-s; Nikon, Tokyo, Japan). We measured oocyte maturation by the percentage of oocytes at metaphase II (MII) stage.
Assessment of mitochondrial abundance and activity
Mitochondrial abundance and activity were evaluated with MitoTracker green (Molecular Probes, Eugene, OR, USA) and MitoTracker Red CMXRos (Beyotime, Shanghai, China), respectively. After IVM, oocytes were incubated with 200 nM MitoTracker green or 200 nM MitoTracker Red CMXRos in TCM199-based medium for 30 min at 37°C in the dark. Oocytes stained with MitoTracker green were washed three times in phosphate-buffered saline, and mounted and examined under a fluorescence microscope (Eclipse Ci-s; Nikon). Oocytes stained with MitoTracker Red CMXRos were washed three times in DPBS, and then fixed in 4% (w/v) paraformaldehyde for 15 minutes at 37°C. After fully washing, oocytes were mounted and examined under a fluorescence microscope (Eclipse Ci-s; Nikon). ImageJ software was used to analyze fluorescence intensity.
Mitochondrial membrane potential measurement
Mitochondrial membrane potential (MMP) was detected using a MitoProbe JC-1 Assay kit (Beyotime) according to the manufacturer’s instructions. Briefly, denuded oocytes were incubated in culture medium at 37°C for 30 min with JC-1. After fully washing, the oocytes were mounted on glass slides and then imaged by the fluorescence microscope (Eclipse Ci-s; Nikon). ImageJ software was used to analyze fluorescence intensity. MMP was calculated as the ratio of red fluorescence intensity to green fluorescence intensity.
Detection of intracellular reactive oxygen species and glutathione levels
Levels of ROS and glutathione (GSH) were measured as described by Wang et al [14]. Briefly, after IVM, matured COCs were denuded. Subsequently, denuded matured oocytes were stained for 30 min in the TCM199 medium containing 10 μM DCFH-DA or 10 μM CMF2HC at room temperature. After washing, oocytes were imaged by the fluorescence microscope (Eclipse Ci-s; Nikon). ImageJ software was used to analyze fluorescence intensity.
Detection of lipid droplet and fatty acid contents
Lipid droplet and fatty acid analysis were performed using lipid droplets green fluorescence staining kit assay (BODIPY 493/503) and fatty acid green fluorescence probe (BODIPY 500/510 C1, C12), respectively. Briefly, after IVM, oocytes were fixed in 4% paraformaldehyde for 15 min at room temperature. The fixed oocytes were washed three times in Dulbecco’s phosphate buffered saline (DPBS)-polyvinyl alcohol (PVA) and treated with BODIPY 493/503 (1:1000, Beyotime) or BODIPY 500/510 C1, C12 (6 μM, Beyotime) for 1 h at room temperature in the dark. After fully washing, oocytes were mounted on glass slides and then imaged by the fluorescence microscope (Eclipse Ci-s; Nikon). ImageJ software was used to analyze fluorescence intensity.
Parthenogenetic activationand culture in vitro
After IVM, COCs were denuded with 0.1% hyaluronidase (w/v) by pipetting; then, parthenogenetic activation (PA) was induced with two direct-current pulses of 1.2 kV/cm for 60 μs in activation medium containing 0.3 M mannitol, 0.05 mM CaCl2·2H2O, 0.1 mM MgCl2·6H2O, and 0.5 mM 4- (2-Hydroxyethyl)-1-piperazineethanesulfonic acid and 0.1% bovine serum albumin (BSA) (w/v). The oocytes were washed three times in PZM-3 medium supplemented with 2 mM 6-dimethylaminopurine and 4 mg/mL BSA and incubated in the same medium for 4 h. Next, activated oocytes thoroughly washed and cultured in in vitro culture (IVC) medium for 7 days at 38.5°C in an atmosphere of 5% CO2 and 100% humidity. The day of PA was considered Day 0. Cleavage and blastocyst formation rate were evaluated on Day 2 and Day 7, respectively. Total cell number per blastocyst was assessed by 10 μg/mL Hoechst 33342 for 10 min to label the nuclei and washed three times with 1% PVA/DPBS (w/v), and then mounted onto glass slides. Images were captured by the fluorescence microscope (Eclipse Ci-s; Nikon).
Quantitative real time polymerase chain reaction analysis
A CellAmpTM Direct SYBR RT-qPCR kit (Takara, Shiga, Japan) was used to produce cDNA according to the manufacturer’s instructions. Briefly, 50 oocytes of each group were lysed in Cell Lysis solution consisting of 48 μL Cell Lysis II Buffer and 2 μL DNase I at room temperature for 5 min. Reverse transcription was performed using a one-step procedure. Briefly, mixtures of each reaction containing 2 μL cell lysate, 4 μL 5× CellAmp Buffer II, 1 μL PrimeScript RT Enzyme Mix, 1 μL RT Primer Mix and 12 μL RNase Free H2O were added to a nuclease-free microfuge tube, and then incubated at 37°C for 30 minutes, and at 85°C for 5 seconds. Complementary DNA (cDNA) was stored at −20°C until used. The cDNA was quantified by qPCR using an ABI-Stepone Plus instrument (Applied Biosystems, Carlsbad, CA, USA). The amplification protocol included an initial denaturation step for 30 seconds at 95°C followed by 40 cycles consisting of denaturation for 5 seconds at 95°C, annealing for 15 seconds at 60°C. The 2−ΔΔCt method was used to analyzed the gene expression level after the melting-curve analysis was completed. The expression levels of the target genes were then normalized to the expression level of GAPDH in each sample. The primer used were listed in Table 1. Each experiment was repeated independently three times.
Statistical analysis
Each experiment was repeated at least three times, and experimental data are presented as means±standard error of the mean. Percentage data were subjected to arcsine transformation before statistical analysis. SPSS Statistics 18.0 was used for statistical analyses. To evaluate cumulus expansion and oocyte nuclear maturation, the one-way analysis of variance and Tukey’s test were utilized. All the other data were compared using the Student’s t-test. The differences were considered significant at p<0.05.
RESULTS
Effects of various concentrations of pyrroloquinoline quinone supplementation during in vitro maturation on cumulus expansion and oocyte nuclear maturation in porcine cumulus oocyte complexes
To evaluate the effect of PQQ on cumulus expansion and oocyte nuclear maturation, COCs were cultured in IVM medium supplemented with 0 nM (control), 200 nM, 400 nM, 800 nM or 1,600 nM PQQ. The results showed that 800 nM PQQ supplementation significantly increased the CEI compared to the control and other PQQ treatment groups (Figures 1A, 1C; p<0.05). Supplementation with 800 nM PQQ during IVM also significantly increased oocyte maturation rate (Figures 1B, p<0.05). Therefore, 800 nM was selected as the optimum dose of PQQ, and subsequent experiments were conducted with the control and 800 nM PQQ groups.
Effect of pyrroloquinoline quinone (800 nM) supplementation during in vitro maturation on mitochondrial function of porcine oocytes
To determine the effects of PQQ on mitochondrial function, after IVM, we measured mitochondrial abundance, mitochondrial activity, MMP levels, and transcript abundance of genes related to mitochondrial biogenesis in porcine oocytes. The mitochondrial content, and mitochondrial activities and MMP were significantly higher in PQQ-supplemented oocytes than in control oocytes (Figures 2A–2F; p<0.05). Furthermore, we also found that 800 nM PQQ supplementation significantly increased mitochondrial biogenesis-related genes expression (PGC-1a, NRF1, NRF2 and TFAM; Figure 2G; p<0.05).
Effect of pyrroloquinoline quinone (800 nM) supplementation during in vitro maturation on oxidative stress levels of porcine oocytes
To examined the effect of PQQ on oxidative stress, after IVM, we measured ROS and GSH levels, and transcripts abundance of genes related to antioxidative stress in porcine oocytes. Results showed that 800 nM PQQ supplementation significantly reduced ROS levels and increased GSH levels in oocytes compared to the control (Figures 3A–3D; p<0.05). Furthermore, we also found that 800 nM PQQ supplementation significantly increased antioxidant-related genes expression (SOD1, SOD2, GPX and CAT; Figure 3E; p<0.05).
Effect of pyrroloquinoline quinone (800 nM) supplementation during in vitro maturation on lipid metabolism of porcine oocytes
To test whether PQQ affected lipid metabolism, after IVM, we detected lipid droplets and fatty acids content, and transcript abundance of genes related to lipid metabolism in porcine oocytes. Our results revealed that 800 nM PQQ supplementation significantly decreased the fluorescence intensity of lipid droplets and fatty acids compared to the control (Figures 4A–4D; p<0.05). In addition, 800 nM PQQ supplementation significantly upregulated lipolysis (ATGL and HSL) and β-oxidation (CPT1B and CPT2)-related mRNA expression levels compared to the control (Figure 4E, p<0.05).
Effect of pyrroloquinoline quinone (800 nM) supplementation during in vitro maturation on the developmental competence of porcine parthenogenetic activation embryos
To further evaluate the oocyte developmental capacity, after IVM, PA was performed, cleavage rate, blastocyst formation rates, and the total cell numbers in the blastocyst were then examined. Results demonstrated that cleavage rate, blastocyst rates, and the total cell numbers in the blastocyst were significantly higher in the 800 nM PQQ-treated group than that in the control group (Figures 5A–5E; p<0.05).
DISCUSSION
The present study investigated whether the effects of PQQ supplementation during IVM on porcine oocyte maturation and embryo developmental competence. We found that adding PQQ to an IVM medium significantly improved cumulus expansion, oocyte maturation rate, cleavage rate, blastocyst rate, and the total cell numbers in the blastocyst after partheno-genetic activation. At the same time, the expressions of genes related to antioxidant, mitochondrial biogenesis and lipid metabolism in porcine oocyte were significantly increased. Moreover, PQQ treatment decreased oxidative stress, lipid droplets and fatty acids content, improved mitochondrial abundance, metabolism activity and membrane potential. These results indicated PQQ can positively influence porcine oocyte maturation and regulate mitochondrial function and lipid metabolism.
Cumulus expansion degree and nuclear maturation rate are considered as the key indicators of oocyte maturation quality. Our data showed that PQQ supplementation elevated the rate of MII oocytes and CEI, indicating the beneficial effects of PQQ on the oocyte maturation. Although previous studies have shown that antioxidants, such as quercetin, cysteamine, carnitine, vitamin C, resveratrol or Tannin, may improve CEI and promote cytoplasmic maturation, these antioxidants, unlike PQQ, have no significant effects on nuclear maturation rate and the total cell numbers in the blastocyst [15, 16].
Mitochondrion is an important organelle located in the cytoplasm and the functional state of the mitochondria is critical for providing energy to drive the normal oocyte development [17]. The number of mitochondria, mitochondrial membrane potential and its metabolism activity are closely associated with increased oocyte quality [18–20]. Our results showed that PQQ supplementation during IVM improved mitochondrial abundance, mitochondrial activity and MMP. Furthermore, we also found that PQQ upregulated mitochondrial biogenesis-related transcript expressions (PGC-1α, NRF1, NRF2 and TFAM). PGC-1α is known to be a central transcriptional coactivator inducing mitochondrial biogenesis. NRF1 and NRF2 regulate expression of the nuclear-encoded respiratory chain components and TFAM. TFAM is major regulator of mitochondrial DNA transcription and replication. Numerous studies have shown that the increased expression of PGC-1α enhanced mitochondrial biogenesis by modulating the gene expression of NRF1, NRF2 and TFAM, resulting in improved mitochondrial function [21–23]. Similarly, previous studies showed that some bioactive substances (Mogroside V, notoginsenoside R1, Nobiletin) supplementation to IVM medium promoted mitochondrial biogenesis and increased mitochondrial activity and MMP in porcine oocytes [24–26]. Thus, our data demonstrated that PQQ exerted a beneficial effect on oocyte maturation and subsequent embryonic development by modulating the mitochondrial biogenesis and function.
It is believed that IVC conditions induce excessive generation of ROS in oocytes due to the exposure to environmental stressors. Excessive generation of ROS will cause DNA fragmentation, spindle abnormalities, mitochondrial dysfunction and apoptosis, ultimately impairing oocyte maturation quality and reducing embryo development [27, 28]. Notably, PQQ supplementation significantly reduced ROS levels in porcine oocyte. This may have been due to the fact that PQQ upregulated the expression levels of genes related to antioxidant stress (SOD1, SOD2, GPX and CAT). SOD, GPX and CAT are key enzymatic antioxidants within cells, which convert oxidants into compounds that are harmless to the cell. Moreover, PQQ supplementation also increased the levels of GSH, while decreasing intracellular ROS levels. These results are similar to those of some previous reports using antioxidants, like resveratrol, melatonin or luteolin supplementation to IVM medium [29–31]. GSH is a key marker to evaluate the cytoplasmic maturation of oocytes [32]. Increased levels of intracellular GSH can scavenge ROS and increase mitochondrial activity, thereby improving oocyte maturation quality and their developmental capacity [33]. Furthermore, some studies have showed that antioxidants supplementation during oocytes IVM can prevent mitochondrial dysfunction by reducing ROS levels [34, 35]. Importantly, in this study, PQQ-supplemented oocytes not only reduced ROS levels, but also increased GSH levels, mitochondrial abundance, MMP and mitochondrial activity compared to the control, indicating that PQQ supplementation improved mitochondrial content and function partly due to its antioxidant activity. Our findings suggest that PQQ promotes porcine oocyte maturation and early embryonic development by mitigating oxidative stress.
It has been proven that pig oocytes contain large quantities of fatty acids that are stored as neutral lipids in the form of lipid droplets [36]. Fatty acids are generated by lipolysis and transported into the mitochondria, where they are further utilized by β-oxidation for energy production [37]. Previous studies showed that inhibition of fatty acids β-oxidation during IVM decreases the developmental competence of oocyte in both laboratory and livestock species [38, 39], whereas stimulation of fatty acids β-oxidation during IVM enhances the developmental competence of oocyte [40]. These results indicate that fatty acids β-oxidation during oocyte IVM is essential for oocyte maturation and subsequent embryo development. HSL and ATGL are key enzymes that modulate triglycerides degradation within cells. ATGL catalyzes triglycerides hydrolysis to diglycerides and free fatty acids, and HSL subsequently hydrolyzes diacylglycerols and produces monoacylglycerol and free fatty acids. CPT1B and CPT2 are key enzymes that maintain the transport of fatty acids into the mitochondria to β-oxidation. More importantly, this study showed that PQQ supplementation during IVM significantly reduced the levels of lipid droplets and fatty acids in porcine oocytes and increased the expression levels of genes related to lipolysis (HSL, ATGL) and β-oxidation (CPT1B, CPT2). These results demonstrate the beneficial effects of PQQ on cytoplasmic maturation of porcine oocytes through enhancement of lipid metabolism. Although some bioactive substances, such as Mogroside V, notoginsenoside R1 and Nobiletin, may promote mitochondrial function in porcine oocytes, these bioactive substances, unlike PQQ, have not been shown definitively to have beneficial effects on lipid metabolism [24–26].
CONCLUSION
Our findings demonstrate that the addition of PQQ to IVM medium is an effective approach to improve oocyte maturation quality and subsequent embryo development by alleviating oxidative stress and enhancing mitochondrial function and lipid metabolism. In addition, this study helps raise awareness of the beneficial effects of PQQ on oocyte maturation and early embryo development using a porcine model that may offer a potential strategy for treating female infertility.
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