Resveratrol Can Revert Adverse Effects of Oocyte aging in Pig

This study was aimed to evaluate the potential of resveratrol (RES) to alleviate the adverse effects of aging in porcine oocytes through alleviating oxidative stress and up-regulating anti-apoptotic genes. Porcine oocytes were cultured in an in vitro medium supplemented with four levels (0, 1, 2, and 4 µmol/L) of RES. Based on initial screening; optimum RES level (2 µmol/L) was used to evaluate its potential effects on oocytes aged for 24 and 48 hours. Our results revealed that aged oocytes (24 h and 48 h) treated with RES showed higher (P < 0.05) ROS uorescence intensity than the control group, but lower (P < 0.05) than untreated aged groups. The GSH content in untreated aged groups (24 h and 48 h) was lower (P < 0.05) than RES treated groups but both groups showed higher levels than the control. Similarly, the relative expression of antioxidant genes (CAT, GSH-Px, and SOD1) in RES treated groups was lower (P < 0.05) than the control group, but higher than untreated aged groups. Moreover, the relative mRNA expression of caspase-3 and Bax in RES treated groups was higher (P < 0.05) than the control group but lower than untreated groups. Furthermore, the expression of Bcl-2 in the RES treated group was signicantly lower than control but higher than untreated aged groups. Our ndings revealed that the RES can increase the activity of antioxidant enzymes while decreasing the level of ROS in porcine aged oocytes. Moreover, RES showed its potent anti-apoptotic effects and potentially resisted against the decline in mitochondrial membrane potential in aged oocytes.


Introduction
The quality of oocyte is the key factor which controls the viability of embryos for successful reproductive technologies like somatic cell nuclear transfer (SCNT), intra-cytoplasmic sperm injection (ICSI), and in vitro fertilization (IVF) (Ma et al., 2005a;Mukherjee et al., 2014). The quality of an oocyte is affected by structural and functional changes induced by aging such as chromosome and spindle anomalies (Saito et al., 1993), cortical granule exocytosis (Díaz and Esponda, 2004), lower fertilization rates (Goud et al., 1999), zona pellucida (ZP) hardening (Diaz and Esponda, 2004), and abnormal or retarded development of embryos/fetuses (Kosubek et al., 2010;Tarín et al., 1999). The exact molecular mechanism of reduced oocyte competence due to postovulatory aging is not fully understood yet. However, major factors that mediate time-dependent degradation in oocyte competence include oxidative stress (Lord and Aitken, 2013), chromosomal abnormalities (Mailhes et al., 1998), modi cation of poly(A) tails (deadenylation) of genes responsible for maternal effects (Dankert et al., 2014)as well as epigenetic alteration (Huang et al., 2007;Trapphoff et al., 2016). Therefore, it is imperative to better understand the various molecular pathways responsible for the postovulatory aging process to devise effective strategies to delay oocyte aging and increase the time required for performing normal reproductive functions (Lord and Aitken, 2013;Wang et al., 2017).
Oxidative stress is strongly associated with a declined in oocyte quality because it signi cantly reduces the glutathione (GSH) contents and enhances the accumulation of reactive oxygen species (ROS). The ROS such as superoxide anions (O − 2 ), hydroxyl radicals (OH − ), and hydrogen peroxide (H 2 O 2 ) are released during normal metabolic (intermediate steps of oxygen reduction) processes (Arain et al., 2018;Kim et al., 2019). The mitochondrion is the major cell organelle responsible for ROS production (Inoue et al., 2003;Liu et al., 2002). A dynamic balance is required between ROS production and antioxidant enzymes to ensure proper cellular homeostasis including cell proliferation, host defense, signal transduction, and gene expression (Droge, 2002). The oxidative stress disrupts this delicate balance through the overproduction of ROS which cannot be scavenged by the antioxidant defense system. Excessive load of ROS results in pro-apoptotic signaling subsequently leading to the activation of cell apoptosis (Redza-Dutordoir and Averill-Bates, 2016). Post-ovulatory aging is associated with excessive accumulation of ROS leading to oxidative stress, which exposes aged oocytes to the risk of apoptosis or improper embryonic development after fertilization (Liang et al., 2017;Lord and Aitken, 2013). The mitochondria as the major "energy generators" have a signi cant role in regulating proper function and survival of oocytes, following ovulation. However, being a chief source of ROS production, mitochondria are susceptible to ROS-induced damage (Ramalho-Santos et al., 2009), which results in decreased ATP synthesis, altered mitochondrial membrane potential, oxidative stress and early onset of apoptosis (Babayev et al., 2016;Wang et al., 2016). The excessive accumulation of ROS can affect the permeability of mitochondrial membranes to open mPTP and promote the ow of calcium ions (Zheng et al., 2004)which subsequently induces the release of cytochrome C and caspase 3 activation leading to apoptosis (Suzuki et al., 2001;Verhagen et al., 2000).
The apoptotic activation is mainly induced by the glutathione e ux (Fico et al., 2008), and leads to several morphological changes including cell shrinkage and progressive DNA and cell membrane damage, ultimately leading to cell death (Papaliagkas et al., 2007). Therefore, one of the major challenges in reproductive embryology is to prevent oocytes' degeneration to maintain their developmental competences (Hoffmann et al., 2001).To avoid oxidative damage by maintaining a robust antioxidant defense system in the oocyte, supplementation of exogenous antioxidants is envisaged as the most effective strategy. Resveratrol (RES) is a natural polyphenolic compound with excellent antioxidant and free radical scavenging capacity. It is associated with reduced ROS accumulation, scavenge superoxide radicals, inhibit lipid peroxidation, and regulate the expression of antioxidant cofactors and enzymes (Pervaiz and Holme, 2009). Moreover, RES has shown to prevent mitochondrial damage in cardio-myocytes through the up-regulation of the deacetylation of apoptotic proteins. Studies have revealed that treatment of porcine oocytes with 2 µM Resveratrol signi cantly reduced the levels of intracellular ROS while increasing GSH contents during in vitro maturation (Kim et al., 2018;Kwak et al., 2012).
Despite the exceptional antioxidant ability of RES, no study is available on the potential effects of RES on aged oocytes. Moreover, the mechanism of action of RES to protect oocytes against oxidative damage for optimal in vitro fertilization is still unclear. Therefore, this study was conducted to evaluate the effect of RES on pig oocytes during aging and to provide mechanistic insights regarding its potential protection of oocytes against ROS attack. We hypothesized that the resveratrol can maintain the quality of oocytes by scavenging free radicals produced during oocyte aging.

Materials And Methods
Oocytes collection and maturation Pig ovaries were collected from a local slaughterhouse and immediately placed in sterile physiological saline at 37°C and delivered to the laboratory within 2 h. The ovaries were washed twice in the sterile condition under pre-warmed PBS at 37°C, and the cumulus-oocyte complexes (COCs) having 3 to 6 mm diameter follicles were collected by aspiration. Under the stereo-microscope, the COC containing ≥3 layers of cumulus cells were selected for the experiment and randomly divided into 4 treatment groups by adding 0, 1, 2, and 4 μmol/L RES and transferred to TCM-199 culture medium. The cells were cultured in a CO2 incubator at 38.5°C with 5% CO2 gas phase and saturated humidity for 44 h, and the optimum concentration of resveratrol was screened out. According to the experiment, the culture was carried out for 44 h, and after aging, continued for further 24 and 48 h. As 2 μmol/L RES was found as an optimum level for porcine oocytes, so further treatment was carried out by using only this level.
In vitro aging and RES treatment of porcine oocytes Resveratrol was diluted with dimethyl sulfoxide (DMSO) to a 10 mmol/L concentrated stock solution and stored at -20 °C. The resveratrol concentrated stock solution was added to the TCM199 culture solution to adjust the concentration to 2 μmol/L. The COCs were cultured for 44 h as a control group, aged 24 and 48 h as in vitro aging groups, placed in a mature medium containing 2 μmol/L RES, and then aged for 24 and 48 h, as a RES treatment group. The rst polar body (pbI) of the oocyte was observed by microscopy and the change of the perivitaline space of the oocyte was detected.
RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR) Fresh COCs were collected and washed twice with DPBS solution and stored at −80 °C until the RNA was extracted. The total RNA was isolated from the oocytes by using the Trizol RNA extraction kit (Takara, Bio Inc., Tokyo, Japan) according to the manufacturer's instructions. The RNA thus extracted was quanti ed using nano-Drop and stored at −80 °C until further use. After that, cDNA was synthesized from 2 ug of total RNA using the Revert AidTM First Strand cDNA synthesis kit (Fermentas, St. Leon Rot, Germany) according to the manufacturer's protocol.
The levels of relevant mRNA, products of antioxidant-related genes, and apoptosis-related genes were determined by qRT-PCR using a One-Step SYBR Prime Script RT-PCR kit (TaKaRa Bio Inc., Tokyo, Japan) in a Light Cycler instrument (Roche, Mannheim, Germany). The levels of accumulated uorescence were analyzed using the second derivative method after the melting-curve analysis and the expression levels of the target genes were normalized using GAPDH as a control. The primer pairs for GAPDH, Caspase-3, Bcl-2, Bax, CAT, GSH-PX, and SOD1 were synthesized by Nanjing Qingke Biotechnology Co., Ltd. The details of primers are presented in Table1. The results of quantitative real-time PCR (qPCR) were analyzed using the 2 -△△Ct method.

Oocyte reactive oxygen species (ROS) assay
The intracellular ROS level was determined by 2,7 dichloro uorescein (DCF) uorescence assay as previously described (Gupta et al., 2010). Brie y, oocytes were incubated with 10 mM DCF for 20 min at 37 o C followed by washing thrice in TCM199. It took 1 h or less to re ect the uorescence intensity of ROS (Excitation wavelength: 450-490 nm and Emission wavelength: 515-565 nm). The results were immediately observed and photographed using an epi uorescence microscope. The mean values of uorescent oocytes were further processed by using Image J 6.0 software. Background uorescent values were subtracted from the nal values before analyzing for the statistical difference between the groups. The experiment was replicated 3 times with 20-25 oocytes each time.

Determination of intracellular GSH contents
The contents of total glutathione (T-GSH) were examined through an enzymatic method by using a GSH/GSSG assay kit (Beyotime Institute of Biotechnology, China) according to the manufacturer's instructions. A total of 50 oocytes from each group were mixed with 30 µl of protein scavenger M solution supplied with the kit and vortexed thoroughly for 5 min, then the mixture was frozen at liquid nitrogen for 2 min and thawed in a water bath at 37°C repeatedly for 3 times. Then the mixture was centrifuged at 10,000 rpm for 10 min at 4°C and placed on ice for 5 min using a 96-well plate, the samples or standard in the sequence were added and mixed accordingly. Immediately absorbance was observed at 405nm with a microplate reader, for 25 min, with a reading recorded for every 5 min. A standard curve was developed for the determination of the GSH content of each sample. The GSH concentration was calculated by dividing the total concentration of each sample by the total number of oocytes present in the sample (pmol/oocyte).

Mitochondrial membrane potential assay
The mitochondrial membrane potential assay kit with JC-1 (Beyotime Institute of Biotechnology, China) was used for the evaluation of mitochondrial membrane potential of the oocytes. The oocytes were exposed to 10μl MJC-1 in 100μL working solution at 38.5°C in 5% CO 2 for20 min. To remove surface uorescence, oocytes were treated with washing buffer and then mounted on glass slides using D-PBS for microscopy. Laser excitation was set at 488nm for green and 525nm for red uorescence, respectively. The uorescence microscope (Olympus, Tokyo, Japan) with the same scan settings for each sample was used to measure the uorescence intensity of each oocyte. Image J software was used to analyze the normal uorescence pixel intensities of each oocyte. The ratio of green to red uorescence pixels was used to analyze mitochondrial membrane potential.

Statistical analysis
Each treatment group had a minimum of 3 replicates and the data thus obtained were analyzed by the Statistical Package for Social Sciences (SPSS) software (version18.0) by using One-Way analysis of variance (ANOVA). The treatment means were compared by the least signi cant difference (LSD) test at 1% and 5% probability levels. The P-value of <0.05 was considered as a signi cant difference while, P <0.01 was considered as highly signi cant whereas, P <0.001 was considered as extremely signi cant.

Effect of RES on aged oocytes
The effect of treatment of RES (0, 1, 2, and 4 µmol/L) on the proliferation of cumulus cells was observed after 44 h under a microscope. The results revealed that cumulus diffusion in 1 and 2 µmol/L RES treated group was better than the control and 4 µmol/L RES treated groups (Fig. 1). Moreover, we observed a signi cant difference in the rst polar body extrusion rate (pbI) between the 2 µmol/L RES and the control group. However, no signi cant difference in rst polar body extrusion rate (pbI) in 1 and 4 µmol/L RES treated groups was observed as compared to control.

Effect of RES on perivitaline space of aged oocytes
Treatment with resveratrol signi cantly increased the perivitaline spaces in oocytes aged for 24h and 48h group compared with the control group and resveratrol treated groups (Fig 2). Furthermore, oocytes treated with RES showed a signi cant decrease in perivitaline spaces after24h and 48h of aging.
However, differences were non-signi cant as compared to the control group ( Fig 2B).

Effect of RES on the expression of apoptotic genes in aged oocytes
Our results revealed that the relative mRNA expression of caspase-3 and Bax in aged oocyte groups was signi cantly higher than thecontrol group; while the expression of Bcl-2 was signi cantly lower ( Figure 3). However, the expression of caspase-3 and Bax in 24 and 48 h REStreated groups was signi cantly decreased thanuntreatedaged groups. Moreover, the expression of Bcl-2 in the RES treated group was signi cantly lower than the control group but signi cantly higher than untreated aged groups.

Effect of RES on the expression of antioxidant enzymes
Our results showed that RES could signi cantly increase the relative expression of different antioxidant genes in aged oocytes. In this experiment, the expression of CAT and SOD-1 gene in RES treated groups was signi cantly higher than untreated aged groups, although the expression of CAT and SOD-1 gene was lower than that of the control group (Fig 4). Likewise, the expression of the GSH-Px gene in RES treated oocytes (aged for 24 and 48h) was signi cantly increased, but there was no signi cant difference as compared with the control group.

Effect of RES on GSH enzyme activity of aged oocytes
Results showed that the activity of GSH was signi cantly decreased in 24 and 48 h aged groups whereas RES treatment signi cantly improved the GSH activity in 24 and 48 h aged groups (Fig 5). However, this difference was non-signi cant as compared to the control group.

Effect of RES on ROS in aged oocytes
Our results revealed that ROS levels signi cantly increased in 24 and 48 h aged groups compared with control, while the oocytes treated with resveratrol (both at 24 and 48 h) showed signi cantly higher ROS levels than those of the control group, but signi cantly lower than those of untreated aged groups (Fig 6).

Effect of RES on mitochondrial membrane potential of oocytes
To evaluate the mitochondrial membrane potential, we analyzed the ratio of green/red uorescence. Compared to the oocytes treated with RES, the oocytes aged for 24 and 48h showed high ratios while fresh oocytes displayed the lowest values. These results suggested that RES possess a signi cant tendency of keeping mitochondrial membrane potential in its normal state during the oocyteaging process ( Fig: 7 and 8).

Discussion
Effect of RESon GSH enzyme activity, antioxidant genes, and ROS Our study revealed that under in vitro conditions, RES could signi cantly increase the GSH contents in 24 h aged oocytes as compared with the control group (P < 0.001). Although, RES at 48 h of aging signi cantly lowered the GSH contents than the control group (P <0.05), but still it was signi cantly higher than its untreated counterparts. Our ndings are in agreement with earlier studies that RES can induce progesterone secretion in bovine oocytes and reduce intracellular ROS levels while increasing GSH contents (Wang et al., 2014). Furthermore, (Boerjan and De Boer, 1990) also reported that in vivo aging of mouse oocytes was associated with the decreased cytoplasmic concentration of total glutathione (GSH plus GSSG) contents suggesting a concomitant decline in GSH/GSSG ratio. The GSH maintains the dynamic balance of the cell membranes by protecting the proteins from oxidative damage. During the in vitro maturation of bovine oocytes, the addition of cysteamine increases GSH content and blastocyst rate (Izumi et al., 2013)indicating its potential to regulate GSH to improve in vitro oocyte maturation (Matos et al., 1995). Similarly, our study revealed the potential of RES to signi cantly increase the GSH contents in aged oocytes with remarkable effects on the 48h aging group indicating alleviation of oxidative damage to facilitate proper oocytes development.
The apoptosis is a very complex process being affected by many factors such as in ammatory response and oxidative stress etc. The ROS induced oxidative stress can adversely affect a variety of biological processes including sperm capacitation, ovulation, and corpus luteum production, and can also trigger oocyte apoptosis. The accumulation of ROS has serious manifestation regarding the quality and aging of oocytes. Also, uncontrolled and excessive production of free radicals may harm DNA, proteins, and lipids, which can severely compromise cell health and contribute to disease development (Birben et al., 2012;Mccord, 2000;Therond, 2006). Our results showed that RES signi cantly reduced the ROS level in aged oocytes (24 and 48 h) which is consistent with earlier ndings (Liang et al., 2018). Outcomes in this study indicate the potential of RES to inhibit oocyte aging by reducing ROS levels owing to its reported antioxidant (De?La?Lastra and Villegas, 2007), anti-apoptosis (Jong-Wook et al., 2001), and anti-aging (Liang et al., 2018)activities. Moreover, RES has also shown to reduce lipid peroxidation by eliminating free radicals, and thus achieve the effect of protecting cells (Wenzel et al., 2005).
Moreover, O 2 is converted by SOD to H 2 O 2 , which is decomposed to water and oxygen by CAT, preventing the production of hydroxyl radicals. Additionally, GSH-Px converts peroxides and hydroxyl radicals into nontoxic forms by oxidizing reduced glutathione (GSH) into glutathione disul de and triggers reduction to GSH by glutathione reductase (Wu, 2013). When Cu +2 or Fe +2 are available, H 2 O 2 reacts with these ions to form unstable hydroxyl radicals. Previous studies have shown that RES can increase the activity of various antioxidant enzymes such as CAT, SOD, and GSH-Px in cells (Meng et al., 2018;Wu et al., 2015).
When low-dose of RES was used to treat cardio-myocytes, the catalytic activity of CAT and SOD increased signi cantly with no effect on glutathione activity. Moreover, SOD can reduce intracellular superoxide levels and potentially resist against cell apoptosis, membrane permeability changes, and mitochondrial dysfunction (Movahed et al., 2012).
Previous studies in our laboratory have demonstrated that RES can eliminate mitochondrial injury while relieving oocyte aging and improving the expression of sirtuin-1 (sirt1) and quality of aged porcine oocytes (Ma et al., 2005b). Similarly, in the present study RES treatment increased the expression of GSH-Px gene in 24 h aged oocytes as compared to the control and untreated aged groups (P <0.01). However, expression of CAT and SOD1 genes was lower than that of the control group (P <0.05), but still higher than their untreated counterparts. Moreover, RES signi cantly increased the expression CAT, SOD1, and GSH Px in48h aged group than the untreated aging group. Our ndings revealed that RES can effectively mediate oxidative stress induced by the aging oocytes during in vitro culture through increasing the antioxidant enzyme activity. Moreover, our study provides mechanistic insights into the potential antioxidant effects of RES to alleviate the adverse effects of oocyte aging and regulation of aging in porcine oocytes.
Effect of resveratrol on perivitaline space in aged oocyte RES can accelerate the formation of bovine oocyte polar bodies and increases the maturation rate of bovine oocytes (Wang et al., 2014). Progesterone causes elongation of the Mos poly-A tail via cytoplasmic polyadenylation, and this polyadenylation increases the rate of Mos translation leading to the accumulation of Mos protein (Gebauer et al., 1994). Mos protein is essentially required for the initiation of oocyte germinal vesicle breakdown (Yew et al., 1992). In our study, we observed an increase in cumulus spread after treatment of oocytes with different concentrations of RES (1 μmol/L and 2 μmol/L) which is consistent with earlier ndings as mentioned above. Furthermore, the rst polar body extrusion rate of the 2 μmol/L RES group was also signi cantly higher than other groups, indicating the potential of RES to increase oocyte maturation rate (Fig 1B). During this study, we found that oocytes treated with RES showed a signi cant decrease in perivitaline spaces during 24h and 48h. However, differences were non-signi cant compared with the control group (Fig 2).A full expansion of cumulus cells is mandatory for the proper maturation of the oocyte. Moreover, feeding RES to young mice for a longer period has shown to enhance the number and quality of oocytes (Liu et al., 2013)which is consistent with our ndings. Similarly, RES supplementation into the in vitro maturation medium improved the quality of bovine embryos and increased their resistance against oxidative damage during cryopreservation (Abdel-Wahab et al., 2012). The bene cial effects of RES might depend upon its ability to improve oocyte quality therefore, it can be concluded that RES (2μmol/L) can inhibit the increase of perivitaline space of oocytes aged for 24h and 48h, indicating its ability to alleviate the adverse effects of oocyte aging by improving the quality which is necessary for its development during fertilization.

Effect of resveratrol on the expression of apoptotic genes of aged oocyte
Apoptosis is one of the exogenous (mediated by death receptors) and mitochondria guided endogenous pathways. Both of these pathways participate in the activation of certain members of the Caspase family to trigger apoptosis. The proteins involved in the mitochondria-mediated endogenous pathway are mainly the Bcl-2 family, which includes both anti-apoptotic and pro-apoptotic proteins. The anti-apoptotic proteins (Bcl-XL, Bcl-2, and Mcl-1) potentially inhibit the activation of the Caspase family and block the transduction of apoptotic signals while pro-apoptotic proteins (Bcl XS, Bak, Bax, and Bad) promote and initiate an apoptotic response. Caspase 3 acts as a key effector in the process of apoptosis and directly hydrolyzes speci c substrates. It can damage cellular DNA and promote apoptosis (Wang et al., 2015). When there is apoptosis, Bax acts on the outer membrane mitochondrial of cell causing the release of mitochondrial cytochrome C (Cytc) that activates Caspase 3 and triggers Caspase cascade. The nucleated cytoskeleton recombines and degrades cytoskeletal structure (Nutt et al., 2005). However, Bcl 2 inhibits Cytc and Caspase causing an anti-apoptotic effect. Previous studies have shown that activated Caspase 3 promotes the further release of Ca +2 which may lead to oocyte fragmentation and accelerates aging (Ma et al., 2005a).
Furthermore, oocyte aging can lead to a series of morphological changes, such as increased perivitaline space, loose chromosome distribution, premature separation of sister chromatids, spindle abnormalities, cortical granule exocytosis, zona pellucida sclerosis, and cytoplasmic fragmentation . Although many molecules, such as Bcl-2 (Hsu and Hsueh, 2000), Bcl-X (Parborell and F., 2002), Bax (Tilly et al., 1995) and caspases (Flaws et al., 1995;L. and K., 1998), have been implicated in the regulation of ovarian apoptosis. Bax has been con rmed to be involved in follicles. The recruitment of Bax, which is knocked out, limits the expression of Bax and leads to an increase in the number of ovarian oocytes (Pascuali et al., 2015). Deacetylated Sirt1 and Sirt 3 inhibit the apoptotic pathway by affecting mPTP pores of the mitochondrial membrane (Tong et al., 2017). Furthermore, down-regulation of caspase 3 upregulates the expression of the anti-apoptotic protein such as Bcl-2 that subsequently inhibits apoptosis. We observed lower expression of Bax and Caspase 3 in RES treated aged oocytes in the present study.
Moreover, relative mRNA expression of Bcl-2 was signi cantly lower in the RES treated group than the control but was signi cantly higher as compared to untreated aged groups. Our ndings suggested that RES can effectively inhibit mitochondrial apoptotic pathway through down-regulation of the Bax and Caspase 3 while up-regulating Bcl 2 in aged oocytes, which subsequently alleviates adverse effects of aging in porcine oocytes.

Mitochondrial membrane potential
Mitochondria are responsible to maintain cellular metabolic functions and its physiological e ciency can be assessed by examining the mitochondrial membrane potential state. In this regard, uorescence probes like JC-1 tend to accumulate in the mitochondrial matrix (by forming IJ-aggregates) and produce red excitation light when the mitochondrial membrane potential is maintained high. However, if mitochondrial membrane potential is maintained low, JC-1 cannot accumulate in the mitochondrial matrix, hence forms monomers and generates green excitation light. During the present study, inclusion of RES in the oocyte culture medium maintained the mitochondrial membrane potential of aged oocytes in a state consistent with non-aged counterparts. Furthermore, RES signi cantly increased the expression of BCL-2 in 24 and 48h aged oocytes which subsequently can regulate the mitochondrial apoptotic pathway by controlling the permeability of the outer mitochondrial membrane (Brunelle and Letai, 2009). Consequently, the RES regulated follicular development primarily through increased expression of mitochondrial related Bcl-2 gene which might have played its role in the maintenance of mitochondrial membrane potential in its normal position in 24 and 48h aged oocytes. However, further in vivo studies are required to elucidate its potential mechanism of action.
Our study concluded that RES can effectively alleviate the adverse effects of oocyte aging by increasing the expression of antioxidant enzymes while decreasing the ROS level. Moreover, the RES treatment resisted against the decline in mitochondrial membrane potential in aged oocytes. Additionally, RES showed potent anti-apoptotic effects by potentially up-regulating the Bcl 2 while down-regulating the mRNA expression of Bax and Caspase 3.

Declarations Ethics approval and consent to participate
This animal study (short title: Resveratrol effect on oocyte aging) was carried out in strict accordance with the recommendation of the National Ethical commission (Nanjing, Jiangsu P.R. China). All experiments and procedures compile with the guideline and were approved by the local ethical committee of the Nanjing Agricultural University (Jiangsu Province P.R. China) with respect to animal experimentation and care of animal under study.

Consent for publication
Not applicable.

Availability of data and materials
Corresponding author will provide the data on request.

Competing Interest
The authors declare that they have no competing interests.  Effects of RES on the relative expression of Caspase-3, Bcl-2 and Bax genes in porcine oocytes at different aging time (* indicates P<0.05; **indicates P<0.01 and ***) Relative mRNA expression of antioxidant enzymes in aged oocyte treated with RES, * means signi cant difference (P<0.05).

Figure 4
Relative mRNA expression of antioxidant enzymes in aged oocyte treated with RES, * means signi cant difference (P<0.05).

Figure 7
Mitochondrial membrane potentials in porcine oocyte cultured in the absence or presence of Resveratrol.
(A) control (B) aged 24h (C) aged 24h+RES (D) aged 48h (E) aged 48h+RES. Membrane potential was calculated as the ratio of red uorescence, which corresponds to activated mitochondria (J-aggregates), to green uorescence, which corresponds to less-activated mitochondria (J-monomers). Fluorescence emitted from each oocyte was analyzed using the Image J software.

Figure 7
Mitochondrial membrane potentials in porcine oocyte cultured in the absence or presence of Resveratrol.