Introduction
Oocyte ageing induces several functional changes that affect oocyte quality and subsequent embryo development after fertilization (Zhang et al., Reference Zhang, Liu, Chen, Wu, Nie, Gao and Miao2017). Post-ovulatory ageing is associated with a range of oocyte defects, including abnormalities in the structures of the zona pellucida, oolemma, cortical granules, mitochondria, and meiotic spindles, in addition to chromosomal structural abnormalities (Cheng et al., Reference Cheng, Li, Tang, Hao, Wang, Sun, Wang, Zhang, Chen and Liu2017). It is also accompanied by various biochemical and molecular changes, such as the generation of reactive oxygen species (ROS), decrease in the activity of maturation promoting factor (MPF), decrease in the expression of anti-apoptotic factor BCL-2, and activation of caspase-3, in addition to epigenetic modifications (Shimoi et al., Reference Shimoi, Tomita, Kataoka and Kameyama2019; Xu et al., Reference Xu, Jiang, He, Liu, Yang, Chen, Wu, Geng and Li2019; Kasapoğlu and Seli, Reference Kasapoğlu and Seli2020; Kim et al., Reference Kim, Choi, Seo, Lee, Kong, Lee, Lee, Lee, Ko, Kim, Lee and Lee2020).
The addition of antioxidants during oocyte ageing has been shown to delay the process and improve the oocyte quality (Lord et al., Reference Lord, Nixon, Jones and Aitken2013). Plant polyphenolic compounds, including flavonoids, are natural antioxidants. Supplementation with quercetin and the phenolics from Kaempferia galanga rhizome to maturation medium during oocyte ageing has been reported to reduce oxidative stress in cells and help maintain the spindle shape and function, consequently delaying oocyte ageing and improving the quality of the aged oocytes (Wang et al., Reference Wang, Jo, Oh and Kim2017; Liang et al., Reference Liang, Lin, Zhang, Sun, Zhou, Schatten, Sun and Qian2018; Yao et al., Reference Yao, Jiang, Li, Gao, Xu and Kim2019).
Onion peel extract (OPE) contains notable amounts of quercetin and other flavonoids, displaying better antioxidant activity than onion flesh extract (Kim et al., Reference Kim, Seo, Choi, Kim, Kang, Chung, Lee and Lee2014). In women with obesity, the antioxidant properties of OPE inhibited the decrease in SOD activity and the production of ROS associated with obesity (Kim and Yim, Reference Kim and Yim2015). Such findings suggest that OPE may help to prevent the development of various common chronic diseases associated with oxidative stress (Masood et al., Reference Masood, Rehman, Ihsan, Shahzad, Sabir, Alam, Ahmed, Shah, Alghabari, Mehmood and Chung2021). It has also been shown that OPE exerts a hypocholesterolemic effect. In particular, OPE increased the faecal excretion of serum and hepatobiliary sterols in mice fed a high cholesterol diet (Kang et al., Reference Kang, Pichiah, Abinaya, Sohn and Cha2016). In addition, OPE has been recognized as a potential candidate for human infertility treatment because of its ability to improve the flagellar voltage-gated proton channel Hv1 in sperm and increase sperm motility (Chae et al., Reference Chae, Kang, Lee, Choi, Kim, Park, Kim and Lee2017).
However, despite these advantages, research on the effect of OPE in oocytes is still scarce. In this study, we examined the influence of OPE on the ageing process of mouse oocytes, including its effects on mitochondrial function, antioxidant activity, and apoptosis-related processes, to provide a theoretical basis for determining the mechanism by which OPE improves the quality of ageing oocytes.
Materials and methods
All chemicals and reagents used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated.
Preparation of OPE
The onion peels were washed in distilled water to remove the silt from the skin and dried in a drying oven with an air blast at 60°C. The onion peels dried to a constant weight were crushed into powder with a high-speed pulverizer, and the powder was screened through a 40-mesh sieve for later use. The prepared onion peel powder was wrapped tightly in a non-woven impregnated cloth and extracted using 60% ethanol at a 1:40 solid-to-liquid ratio for 2 h at 60°C. The extraction was repeated two more times, and the pooled extract was concentrated using rotary evaporation at 60°C. The concentrated extract was dried using a freeze dryer to produce OPE for later use.
OPE was dissolved in dimethyl sulphoxide (DMSO) to prepare a concentrated stock solution, and different concentrations of OPE (0, 50, 100, and 200 μg/ml) were dissolved in M16 (keeping the DMSO content <0.5%).
Oocyte collection, culture, and ageing
Kunming mice aged 6−8 weeks were selected. For the ageing group and OPE treatment group, 10 IU of pregnant mare serum gonadotropin (PMSG) was injected intraperitoneally (i.p.) at 15:00 h on the first day, and 10 IU of human chorionic gonadotropin (hCG) was injected at 15:00 h on the second day. At 7:00 h on the third day, the stage II (MII) oocytes were collected from the ampulla of the fallopian tube. The ageing group was placed in M16 for direct culture, and the OPE treatment group was placed in M16 with different concentrations of OPE for culture. For the control group (fresh oocytes), PMSG 10 IU was injected i.p. at 24:00 h on the first day, hCG 10 IU was injected i.p. at 24:00 h on the second day, and the MII phase oocytes were collected at 16:00 h on the third day, and cultured in M16. The correlation test was conducted at 21:00 h on the third day.
Measurement of mitochondrial membrane potential (ΔΨm), ROS, and GSH generation
To assess ΔΨm, denuded MII-stage oocytes were incubated with 2 μM JC-1 (Invitrogen, Waltham, MA, USA) for 1 h at 37.5°C in the dark. The average ΔΨm of oocytes was then calculated as the ratio of red fluorescence intensity (J-aggregates; corresponding to activated mitochondria) to green fluorescence intensity (J-monomers; corresponding to inactive mitochondria) using ImageJ software. The fluorescence intensity of the resulting oocytes was analyzed using a fluorescence microscope (Nikon Corp., Tokyo, Japan). ROS levels were measured using a 2′,7′-dichlorofluorescein (H2DCFDA; Thermo Fisher Scientific, Waltham, MA, USA) assay. Briefly, denuded MII-stage oocytes were cultured in 0.1% bovine serum albumin (BSA)-PBS containing 10 μM H2DCFDA for 15 min at 37.5°C in the dark and then visualized with excitation at 485 nm and emission at 535 nm. GSH levels were quantified using CellTracker™ Blue dye (4-chloromethyl-6,8-difluoro-7-hydroxycoumarin, CMF2HC; Invitrogen). Briefly, denuded MII-stage oocytes were incubated in 0.1% BSA-PBS medium containing 10 μM CMF2HC for 15 min at 37.5°C in the dark and visualized with excitation at 371 nm and emission at 464 nm. The fluorescence intensity (1 s after shutter opening with 10 ms exposure for H2DCFDA; 3 s after shutter opening with 100 ms exposure for CMF2HC) of the resulting oocytes was analyzed using fluorescence microscopy (Nikon Corp.) using ImageJ software.
RT-PCR
MII oocytes were harvested, and mRNA was extracted from each of the 15 oocyte pools using the DynaBeads mRNA Direct Kit (catalogue #61012; Dynal Asa, Oslo, Norway) according to the manufacturer’s instructions. cDNA was obtained by reverse transcription of mRNA using oligo(dT)12−18 primers and SuperScript III reverse transcriptase (Invitrogen). qRT-PCR was performed using a KAPA SYBR® FAST kit (#KK4601; Kapa Biosystem Ltd, Cape Town, South Africa), in which each reaction contained 10 μl SYBR Green, 1 μl each of forward and reverse primers, and 2 μl cDNA template (10 ng/μl), in a final reaction volume of 20 µl. The amplification run was programmed as follows: 95°C for 3 min, followed by 40 cycles of 95°C for 3 s, 60°C for 30 s, and 72°C for 20 s. The primers used to amplify each gene are shown in Table 1. The target genes were SOD1, CAT, and GPX3, and GAPDH was used as a reference gene. The mRNA quantitation data were analyzed using the 2−ΔΔCT method.
Table 1. Primer sequences used for RT-qPCR
Statistical analysis
Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by the least significant difference (LSD) test using SPSS software (version 19.0; SPSS, Inc., Chicago, IL). Figures were generated using the GraphPad Prism software package (version 6.01; GraphPad, La Jolla, CA). Data are expressed as the mean ± standard deviation (SD), and statistical significance was set at P < 0.05. The total number (N) of oocytes/embryos used in each group is shown in the data columns. Each experiment was repeated three times.
Results
OPE reduces the fragmentation rate in aged oocytes
To explore the influence of OPE on the morphological changes in ageing oocytes, the oocytes were treated with OPE at different concentrations, and the fragmentation of oocytes was analyzed after 16 h of in vitro ageing. Compared with the ageing control group, the rate of oocyte fragmentation increased significantly to 18.78 ± 2.65% upon treatment with 200 μg/ml OPE (Figure 1b) and decreased, albeit not significantly, to 5.36 ± 4.11% and 4.27 ± 2.87% when treated with OPE at 50 and 100 μg/ml, respectively (Figure 1b). These results indicated that OPE exhibits a certain protective effect on the morphological changes in ageing oocytes. Therefore, a concentration of 100 μg/ml OPE was used in subsequent experiments.
Figure 1. Effect of OPE on cellular fragmentation in aged oocytes. (a) Representative images of oocytes matured under different culture conditions. Oocytes were cultured in M16 medium for 16 h for ageing. (b) Oocyte fragmentation rate. Significant differences are represented with different letters (P < 0.05).
OPE reduces oxidative stress in aged oocytes
Oxidative stress is a key factor that affects oocyte senescence (Jiang et al., Reference Jiang, Yao, Zhao, Gao, Jin, Li, Yan and Xu2019; Sasaki et al., Reference Sasaki, Hamatani, Kamijo, Iwai, Kobanawa, Ogawa, Miyado and Tanaka2019). Therefore, we examined the effects of OPE on oxidative stress levels in ageing mouse oocytes by determining the relative levels of ROS and GSH. The addition of 100 μg/ml OPE significantly reduced the level of ROS (2.41 ± 1.43 vs. 6.84 ± 3.45; Figure 2b) and increased the level of GSH (0.99 ± 0.17 vs. 0.55 ± 0.21; Figure 2b) in ageing oocytes compared with the control, indicating that OPE can reduce oxidative stress in ageing oocytes and therefore protect then against the ravages of ageing.
Figure 2. Effects of OPE on oxidative stress in mouse aged oocytes. (a, c) Representative images of ROS and GSH fluorescence in MII oocytes. Oocytes were cultured in M16 medium for 16 h for ageing. (b) Quantitative analysis of ROS fluorescence intensity. (d) Quantitative analysis of GSH fluorescence intensity. Significant differences are represented with different letters (P < 0.05).
OPE increases ΔΨm in aged oocytes
The generation of oxidative stress is closely related to mitochondrial function (Babayev and Seli, Reference Babayev and Seli2015; Babayev et al., Reference Babayev, Wang, Szigeti-Buck, Lowther, Taylor, Horvath and Seli2016). Therefore, we determined the ΔΨm of oocytes using a JC-1 dye-based assay in which the ΔΨm activity is proportional to the red/green fluorescence ratio. Compared with the ageing group, OPE treatment significantly increased the ΔΨm activity of ageing oocytes (0.94 ± 0.23 vs. 0.62 ± 0.17; Figure 3b), although the potential did not reach that of the control (fresh oocyte) group. This result shows that OPE can increase the mitochondrial activity of oocytes to protect them against the ravages of ageing.
Figure 3. Effect of OPE on mitochondrial membrane potential in aged oocytes. (a) JC-1 staining for mitochondrial membrane potential activity. Green fluorescence indicates the JC-1 monomeric form (low intensity) and red fluorescence indicates the JC-1-aggregated form (high intensity). Oocytes were cultured in M16 medium for 16 h for ageing. (b) Quantitative analysis of JC-1 fluorescence intensity (red/green). Significant differences are represented with different letters (P < 0.05).
OPE reduces caspase-3 activity in aged oocytes
As caspase-3 is an important apoptosis marker, we measured the activity of caspase-3 in oocytes (Porter and Jänicke, Reference Porter and Jänicke1999). Aged oocytes showed significantly higher caspase-3 activity than fresh oocytes, indicating the induction of apoptosis. By contrast, the caspase-3 activity in OPE-treated aged oocytes was significantly lower than that in untreated aged oocytes and similar to that in fresh oocytes (1.21 ± 0.54 vs. 1.73 ± 0.37; Figure 4b).
Figure 4. Effects of OPE on caspase-3 activity in mouse aged oocytes. (a) Representative images showing caspase-3 activity in fresh, aged, and OPE-treated aged MII oocytes. (b) Quantified fluorescence intensity for caspase-3 in oocytes. Significant differences are represented with different letters (P < 0.05).
OPE increases the expression of oxidative stress-related genes in aged oocytes
Next, to evaluate the effect of OPE on the expression of oxidative stress-related genes in mouse oocytes, we determined the mRNA levels of GPX3, SOD1, and CAT genes. The results showed that the mRNA expression levels of SOD1 (0.93 ± 0.09 vs. 0.57 ± 0.20; Figure 5), CAT (0.84 ± 0.23 vs. 0.33 ± 0.08; Figure 5), and GPX3 (1.06 ± 0.10 vs. 0.53 ± 0.11; Figure 5) in oocytes from the OPE-treated group were significantly higher than those from the ageing group but did not show any significant difference compared with the control (fresh oocyte) group.
Figure 5. Effects of OPE on expression of oxidative stress-related genes in mouse aged oocytes. Significant differences are represented with different letters (P < 0.05).
Discussion
If the oocyte is not fertilized, it will rapidly age and lose its development ability. Adding antioxidants before ageing can alleviate this process and ensure its developmental ability. As mentioned above, OPE is rich in flavonoids and other substances with antioxidant activity, but its effect on oocytes has not yet been studied. In this study, the in vitro culture medium for mouse oocytes was supplemented with OPE at different concentrations. The results showed that treatment with 100 μg/ml OPE reduced the oocyte fragmentation rate during ageing, thereby maintaining the quality of oocytes. Additionally, OPE treatment reduced oxidative stress and apoptosis and improved the mitochondrial activity and expression of antioxidative stress response genes in oocytes. These results suggest that OPE protects ageing oocytes from oxidative stress-induced apoptosis.
Senescent oocytes typically exhibit cellular fragmentation, and the fragmented oocytes have decreased developmental potential (Zhang et al., Reference Zhang, ShiYang, Zhang, Miao, Chen, Cui and Xiong2019). To examine the effect of OPE on oocyte ageing, we first examined its effect on the fragmentation rate. The results showed that high concentrations (200 and 500 μg/ml) of OPE increased the fragmentation rate, and all oocytes died after treatment with 500 μg/ml OPE. This may be attributed to the openness being a mixture that causes a change in the osmotic pressure of the medium, causing irreparable damage to the oocytes (Goto et al., Reference Goto, Saito, Hiradate, Hara and Tanemura2019). However, there was no significant difference in oocyte fragmentation rate between the ageing group and the low OPE concentration (50 and 100 μg/ml) groups. Therefore, we chose 100 μg/ml OPE for subsequent experiments.
Oxidative stress caused by the generation of ROS is a key mediator of oocyte ageing (Yao et al., Reference Yao, Jiang, Liang, Shen, Gao, Xu and Kim2018; Soto-Heras and Paramio, Reference Soto-Heras and Paramio2020), and oxidative stress has been closely associated with oocyte fragmentation (Lin et al., Reference Lin, Ren, Wang, Huang, Wang, Wang and Greene2018; Jia et al., Reference Jia, Zhang, Zhou, Xu and Feng2019). An overabundance of ROS can compromise the molecular and structural defence mechanisms of the ageing oocyte, leaving it vulnerable to oxidative insults (Mihalas et al., Reference Mihalas, Redgrove, McLaughlin and Nixon2017). The present study shows that OPE, rich in antioxidants, can reduce oxidative stress during oocyte ageing by significantly decreasing ROS in cells, increasing the GSH level, and increasing the expression of antioxidant genes, which is similar to the study that OPE can reduce the ROS generated during obesity (Kim and Yim, Reference Kim and Yim2015). In summary, OPE has beneficial effects on ageing oocytes by alleviating oxidative stress.
Oocyte ageing is accompanied by decreased activity of the mitochondrial respiratory complex and ΔΨm (Miao et al., Reference Miao, Cui, Gao, Rui and Xiong2020; Soares et al., Reference Soares, Sousa, Fernandes, Ferreira, Almeida-Santos and Ramalho-Santos2020), and ΔΨm affects ATP synthesis during oxidative phosphorylation (Babayev and Seli, Reference Babayev and Seli2015; Al-Zubaidi et al., Reference Al-Zubaidi, Liu, Cinar, Robker, Adhikari and Carroll2019). Quercetin can promote the expression of PVT1 to inhibit the oxidative stress and apoptosis of H9C2 cells and alleviate the structural and functional dysfunction of mitochondria (Li et al., Reference Li, Liu, Tang, Yan, Chen, Li and Yan2021). Quercetin can also restore the damaged ΔΨm of ageing porcine oocytes and reduce the intracellular ROS level and cell apoptosis (Jiao et al., Reference Jiao, Wang, Jiang, Wen, Cong, Chen and He2022). Our results showed that ΔΨm of ageing oocytes decreased significantly, indicating that ATP synthesis and the energy supply to cells were decreased. However, the addition of OPE assuaged this downward trend. This is similar to the previous observation that quercetin can alleviate mitochondrial function damage (Li et al., Reference Li, Liu, Tang, Yan, Chen, Li and Yan2021). Combined with the results of oxidative stress, OPE can inhibit the decrease in ΔΨm caused by oxidative stress.
Caspase-3 is a frequently activated death protease that catalyzes the specific cleavage of many key cellular proteins (Papandile et al., Reference Papandile, Tyas, O’Malley and Warner2004; Nicholas et al., Reference Nicholas, Alberio, Fouladi-Nashta and Webb2005). It has been suggested that caspase-3 activity is related to oocyte quality, and caspase-3 activity was significantly increased in aged oocytes compared with young oocytes (Papandile et al., Reference Papandile, Tyas, O’Malley and Warner2004). Quercetin can inhibit chrome-induced cell apoptosis in the kidney and decrease the caspase-9 and caspase-3 protein and mRNA expression levels (Huang et al., Reference Huang, Ding, Ye, Wang, Yu, Yan, Liu and Wang2022). Consistent with these observations, caspase-3 activity increased significantly in ageing oocytes in the present study, but its activity decreased significantly after adding OPE. It shows that OPE can improve the quality of ageing oocytes.
SOD1, CAT, and GPX3 are antioxidant factors (Jia et al., Reference Jia, Zhang, Zhou, Xu and Feng2019). The increase in the ratio of SOD1 activity to GPX/CAT activity correlates with an increase in lipid damage, cell ageing, and/or cell death (Lin et al., Reference Lin, Ren, Wang, Huang, Wang, Wang and Greene2018). A recent study has shown that when the expression of GDF8 was inhibited, the maturation rate of buffalo oocytes and the activities of SOD, CAT, and GPX enzymes were significantly decreased (El-Magd et al., Reference El-Magd, Ghoniem, Helmy, Abdelfattah-Hassan, Saleh, Abd Allah, Essawi and Kahilo2019). Another study found that quercetin could upregulate the expression level of SOD2 and CAT in porcine ageing oocytes (Jiao et al., Reference Jiao, Wang, Jiang, Wen, Cong, Chen and He2022). In the present study, we found that the mRNA expression levels of SOD1, CAT, and GPX3 in ageing oocytes were significantly decreased, whereas the expression levels of these three genes were significantly increased after OPE treatment, which is similar to the reported effects of quercetin (Jiao et al., Reference Jiao, Wang, Jiang, Wen, Cong, Chen and He2022). This indicates that OPE can promote gene expression for antioxidant enzymes and delay the ageing of oocytes. In conclusion, OPE delayed mouse oocyte ageing by reducing oxidative stress and apoptosis and enhancing mitochondrial function.
Author contribution
Yong-Nan Xu conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, and reviewed drafts of the paper. Guo-Bo Han performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, and reviewed drafts of the paper. Ying-Hua Li analyzed the data, reviewed drafts of the paper, and contributed reagents/materials/analysis tools. Guan-Hao Li and Nam-Hyung Kim conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared visual material, and reviewed drafts of the paper. All authors read and approved the final manuscript.
Financial support
This study was supported by the Science and Technology Planning Project of the Guangdong Provincial Department of Science and Technology (project number 2021B1212040016); and by the Special Project in Key Areas of Biomedicine and Health of the Guangdong Provincial Department of Education (project number 2021ZDZX2046).
Competing interests
The authors declare none.
Ethical standard
All mouse care and protocols were used in accordance with the guidelines of the Animal Research Committee of Yanbian University, China (SYXK2020–0009).
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