Resveratrol Can Revert Adverse Effects of Oocyte aging in Pig

Resveratrol Can Revert Adverse Effects of Oocyte aging in Pig | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Original article Resveratrol Can Revert Adverse Effects of Oocyte aging in Pig Benazir Abbassi, Dong Yan, Rong Rui This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-120271/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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 fluorescence 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 significantly lower than control but higher than untreated aged groups. Our findings 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. Veterinary Epidemiology Animal Science oocyte aging reactive oxygen species oxidative stress resveratrol anti-oxidant apoptosis Figures Figure 1 Figure 1 Figure 1 Figure 2 Figure 2 Figure 2 Figure 3 Figure 3 Figure 3 Figure 4 Figure 4 Figure 4 Figure 5 Figure 5 Figure 5 Figure 6 Figure 6 Figure 6 Figure 7 Figure 7 Figure 7 Figure 8 Figure 8 Figure 8 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 ), modification 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 significantly 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 significant 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 flow 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 efflux (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 significantly 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 first 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 quantified 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 fluorescence 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 dichlorofluorescein (DCF) fluorescence assay as previously described (Gupta et al., 2010). Briefly, 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 reflect the fluorescence intensity of ROS (Excitation wavelength: 450- 490 nm and Emission wavelength: 515-565 nm). The results were immediately observed and photographed using an epifluorescence microscope. The mean values of fluorescent oocytes were further processed by using Image J 6.0 software. Background fluorescent values were subtracted from the final 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 fluorescence, 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 fluorescence, respectively. The fluorescence microscope (Olympus, Tokyo, Japan) with the same scan settings for each sample was used to measure the fluorescence intensity of each oocyte. Image J software was used to analyze the normal fluorescence pixel intensities of each oocyte. The ratio of green to red fluorescence 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 significant difference (LSD) test at 1% and 5% probability levels. The P-value of <0.05 was considered as a significant difference while, P <0.01 was considered as highly significant whereas, P <0.001 was considered as extremely significant. Results 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 significant difference in the first polar body extrusion rate (pbI) between the 2 µmol/L RES and the control group. However, no significant difference in first 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 significantly 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 significant decrease in perivitaline spaces after24h and 48h of aging. However, differences were non-significant 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 significantly higher than thecontrol group; while the expression of Bcl-2 was significantly lower (Figure 3). However, the expression of caspase-3 and Bax in 24 and 48 h REStreated groups was significantly decreased thanuntreatedaged groups. Moreover, the expression of Bcl-2 in the RES treated group was significantly lower than the control group but significantly higher than untreated aged groups. Effect of RES on the expression of antioxidant enzymes Our results showed that RES could significantly 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 significantly 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 significantly increased, but there was no significant 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 significantly decreased in 24 and 48 h aged groups whereas RES treatment significantly improved the GSH activity in 24 and 48 h aged groups (Fig 5). However, this difference was non-significant as compared to the control group. Effect of RES on ROS in aged oocytes Our results revealed that ROS levels significantly increased in 24 and 48 h aged groups compared with control, while the oocytes treated with resveratrol (both at 24 and 48 h) showed significantly higher ROS levels than those of the control group, but significantly 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 fluorescence. 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 significant tendency of keeping mitochondrial membrane potential in its normal state during the oocyteaging process (Fig: 7 and 8). Discussion Effect of RES on GSH enzyme activity, antioxidant genes, and ROS Our study revealed that under in vitro conditions, RES could significantly 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 significantly lowered the GSH contents than the control group (P <0.05), but still it was significantly higher than its untreated counterparts. Our findings 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 significantly 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 inflammatory 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 significantly reduced the ROS level in aged oocytes (24 and 48 h) which is consistent with earlier findings (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). The primary antioxidant enzymes are SOD, catalase (CAT), and glutathione peroxidase (GSH-Px). 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 disulfide 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 significantly 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 significantly increased the expression CAT, SOD1, and GSH Px in48h aged group than the untreated aging group. Our findings 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 findings as mentioned above . Furthermore, the first polar body extrusion rate of the 2 μmol/L RES group was also significantly 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 significant decrease in perivitaline spaces during 24h and 48h. However, differences were non-significant 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 findings. 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 beneficial 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 specific 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 (Zhang et al., 2017). 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 confirmed 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 up-regulates 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 significantly lower in the RES treated group than the control but was significantly higher as compared to untreated aged groups. Our findings 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 efficiency can be assessed by examining the mitochondrial membrane potential state. In this regard, fluorescence 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 significantly 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 B cl -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. Funding This research supported by the Doctoral Program of the Ministry of Education of China (20130097110020) and PAPD of Jiangsu Province. Author Contributions B. A., conceived the idea and perform experiment and wrote the draft of the manuscript. D. 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Tables Table1 .The details of RT-PCR primer pairs used for analysis of pro-apoptotic genes Target gene Sequence Accession Number Size (bp) GAPDH F: 5'-GTCGGTTGTGGATCTGACCT-3' NM_001206359 207 R: 5'-TTGACGAAGTGGTCGTTGAG-3' Caspase-3 F: 5'-CGTGCTTCTAAGCCATGGTG-3' NM_214131 186 R: 5'-GTCCCACTGTCCGTCTCAAT-3' Bcl-2 F:5'-AGGGCATTCAGTGACCTGAC-3' NM_214285 193 R: 5'-CGATCCGACTCACCAATACC-3' Bax F:5'-TGCCTCAGGATGCATCTACC-3' XM_003127290 199 R: 5'-AAGTAGAAAAGCGCGACCAC-3' CAT F-5'-AACTGTCCCTTCCGTGCTA-3' XM_021081498 195 R-5'-CCTGGGTGACATTATCTTCG-3' GSH-PX F-5'-CAAGTCCTTCTACGACCTCA-3' AJ010340 210 R-5'-GAAGCCAAGAACCACCAG-3' SOD1 F-5'-ACCTGGGCAATGTGACTG-3' NM_001190422 197 R-5'-TCCAGCATTTCCCGTCT-3' Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-120271","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Original article","associatedPublications":[],"authors":[{"id":5708565,"identity":"f9e4cedb-225d-4c8a-bd83-7fb4a1a9d30b","order_by":0,"name":"Benazir Abbassi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIiWNgGAWjYBACAzgpwQNiScgxMPCQqMWYSC0McC0MiQ2EtJiz9x78+KPgHgP/bBDjj0X6huNnDz74wGAnp9uAXYtlz7lkCQmDYgaJO+eSpXnbJHI3nMlLNpzBkGxsdgCHw27kGEgYGCQwMAAZ0owNQC0HcsykeRgOJG7DpeX+G+MfCUAt8jdyjH/++CORbnD+DQEtN3jMJA4AtQCtM5PgYZNIADHwarHsyTGzbDBI4DG8cy7NGugXw5k33hgbzjDA7Rdz9jPGN3/8SZCTu917GMiok+c7n2P44EOFnRwuLTCAiAsFsEoDXAqxAfkGUlSPglEwCkbBSAAASrBbNPmTagcAAAAASUVORK5CYII=","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Benazir","middleName":"","lastName":"Abbassi","suffix":""},{"id":5708566,"identity":"e299a71e-c933-46ba-b477-517089740c3c","order_by":1,"name":"Dong Yan","email":"","orcid":"","institution":"Nanjing Agricultural University - Weigang Campus: Nanjing Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dong","middleName":"","lastName":"Yan","suffix":""},{"id":5708567,"identity":"3dd91d13-0dcd-422c-a992-02e7cc3d1c66","order_by":2,"name":"Rong Rui","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rong","middleName":"","lastName":"Rui","suffix":""}],"badges":[],"createdAt":"2020-12-02 12:49:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-120271/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-120271/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":3996963,"identity":"927b0247-ccb5-430b-8a44-2f20e064b1e2","added_by":"auto","created_at":"2020-12-03 18:42:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":275392,"visible":true,"origin":"","legend":"Effect of RES treatment on cumulus spread and first polar body (pbI) extrusion rate in porcine oocytes A. 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5","display":"","copyAsset":false,"role":"figure","size":104942,"visible":true,"origin":"","legend":"The effect of RES on glutathione (GSH) activity in aging oocytes","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-120271/v1/21bc92c1ace62fd745b173a5.png"},{"id":3996958,"identity":"6667196b-7871-4ace-b128-85a3606217ca","added_by":"auto","created_at":"2020-12-03 18:42:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":104942,"visible":true,"origin":"","legend":"The effect of RES on glutathione (GSH) activity in aging oocytes","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-120271/v1/1147fb72a74014ee1a6ff448.png"},{"id":3996949,"identity":"6af6e307-504d-4c42-a6da-7cd66b4cda91","added_by":"auto","created_at":"2020-12-03 18:42:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":104942,"visible":true,"origin":"","legend":"The effect of RES on glutathione (GSH) activity in aging oocytes","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-120271/v1/58cb2e433cee894d079bf395.png"},{"id":3996968,"identity":"5c272dcd-883b-4e8b-ad53-954064005bb2","added_by":"auto","created_at":"2020-12-03 18:42:30","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":352221,"visible":true,"origin":"","legend":"A: The effect of RES on ROS levels in aging oocytes,bar=280 µm,B: Relative ROS fluorescence intensity* means significant difference (P\u003c0.05).","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-120271/v1/7ab0f850d164b637b34fe4c1.png"},{"id":3996959,"identity":"60621c4f-add7-4163-8ca7-ba9a881d929a","added_by":"auto","created_at":"2020-12-03 18:42:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":352221,"visible":true,"origin":"","legend":"A: The effect of RES on ROS levels in aging oocytes,bar=280 µm,B: Relative ROS fluorescence intensity* means significant difference (P\u003c0.05).","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-120271/v1/d476d28ed6a6854f8fa19b30.png"},{"id":3996950,"identity":"9b3c9c39-3f69-4e23-980d-2fefd26353fb","added_by":"auto","created_at":"2020-12-03 18:42:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":352221,"visible":true,"origin":"","legend":"A: The effect of RES on ROS levels in aging oocytes,bar=280 µm,B: Relative ROS fluorescence intensity* means significant difference (P\u003c0.05).","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-120271/v1/9a694b60a78173a0ac62e3e0.png"},{"id":3996969,"identity":"cb02e43c-74bd-4e95-a777-6f782799eddc","added_by":"auto","created_at":"2020-12-03 18:42:30","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":358281,"visible":true,"origin":"","legend":"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 fluorescence, which corresponds to activated mitochondria (J-aggregates), to green fluorescence, which corresponds to less-activated mitochondria (J-monomers). Fluorescence emitted from each oocyte was analyzed using the Image J software. ","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-120271/v1/f5bff5c161ae9ee9e6a8fd78.png"},{"id":3996960,"identity":"319a80ef-c5a6-44c5-a548-cd0d92bf2f4c","added_by":"auto","created_at":"2020-12-03 18:42:25","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":358281,"visible":true,"origin":"","legend":"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 fluorescence, which corresponds to activated mitochondria (J-aggregates), to green fluorescence, which corresponds to less-activated mitochondria (J-monomers). Fluorescence emitted from each oocyte was analyzed using the Image J software. ","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-120271/v1/9668e4712abed84597494f8e.png"},{"id":3996951,"identity":"accd5bbb-4853-47bd-a32e-5488985b93ca","added_by":"auto","created_at":"2020-12-03 18:42:24","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":358281,"visible":true,"origin":"","legend":"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 fluorescence, which corresponds to activated mitochondria (J-aggregates), to green fluorescence, which corresponds to less-activated mitochondria (J-monomers). Fluorescence emitted from each oocyte was analyzed using the Image J software. ","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-120271/v1/7ec6b93e7687f7425c34589a.png"},{"id":3996970,"identity":"1605854f-b4f1-47c4-a889-bee06d513b49","added_by":"auto","created_at":"2020-12-03 18:42:30","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":71483,"visible":true,"origin":"","legend":"Effect of RES on mitochondrial membrane potential in aged oocytes (*P \u003c 0.05 Values represents the means ± SEM from at least three separate experiments).","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-120271/v1/56f6a86f005dabd1988daae3.jpeg"},{"id":3996961,"identity":"5f7a241e-6c0a-4fa3-bd70-e5f29273eece","added_by":"auto","created_at":"2020-12-03 18:42:25","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":71483,"visible":true,"origin":"","legend":"Effect of RES on mitochondrial membrane potential in aged oocytes (*P \u003c 0.05 Values represents the means ± SEM from at least three separate experiments).","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-120271/v1/cac0ec1f37461d39c719428f.jpeg"},{"id":3996952,"identity":"d50e3133-1432-4fa5-8a8c-0e39537d6b61","added_by":"auto","created_at":"2020-12-03 18:42:24","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":71483,"visible":true,"origin":"","legend":"Effect of RES on mitochondrial membrane potential in aged oocytes (*P \u003c 0.05 Values represents the means ± SEM from at least three separate experiments).","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-120271/v1/cc2634eedeef05beb8bb8554.jpeg"},{"id":13623289,"identity":"0302529d-947a-45ac-a843-b3a1540c9ccc","added_by":"auto","created_at":"2021-09-17 07:17:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4795674,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-120271/v1/919d1ff5-e712-4e40-8fb1-572a05a141b6.pdf"}],"financialInterests":"","formattedTitle":"Resveratrol Can Revert Adverse Effects of Oocyte aging in Pig","fulltext":[{"header":"Introduction","content":" \u003cp\u003eThe 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 \u003cem\u003ein vitro\u003c/em\u003e fertilization (IVF) (Ma et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2005a\u003c/span\u003e; Mukherjee et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The quality of an oocyte is affected by structural and functional changes induced by aging such as chromosome and spindle anomalies (Saito et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1993\u003c/span\u003e), cortical granule exocytosis (D\u0026iacute;az and Esponda, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), lower fertilization rates (Goud et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), zona pellucida (ZP) hardening(Diaz and Esponda, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), and abnormal or retarded development of embryos/fetuses (Kosubek et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Tar\u0026iacute;n et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). 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, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), chromosomal abnormalities (Mailhes et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), modification of poly(A) tails (deadenylation) of genes responsible for maternal effects (Dankert et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e)as well as epigenetic alteration (Huang et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Trapphoff et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). 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, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOxidative stress is strongly associated with a declined in oocyte quality because it significantly reduces the glutathione (GSH) contents and enhances the accumulation of reactive oxygen species (ROS). The ROS such as superoxide anions (O\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e), hydroxyl radicals (OH\u003csup\u003e\u0026minus;\u003c/sup\u003e), and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) are released during normal metabolic (intermediate steps of oxygen reduction) processes (Arain et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Kim et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The mitochondrion is the major cell organelle responsible for ROS production (Inoue et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). 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, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). 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, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). 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., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Lord and Aitken, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The mitochondria as the major \u0026ldquo;energy generators\u0026rdquo; have a significant 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., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), which results in decreased ATP synthesis, altered mitochondrial membrane potential, oxidative stress and early onset of apoptosis (Babayev et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The excessive accumulation of ROS can affect the permeability of mitochondrial membranes to open mPTP and promote the flow of calcium ions (Zheng et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2004\u003c/span\u003e)which subsequently induces the release of cytochrome C and caspase 3 activation leading to apoptosis (Suzuki et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Verhagen et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe apoptotic activation is mainly induced by the glutathione efflux (Fico et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), and leads to several morphological changes including cell shrinkage and progressive DNA and cell membrane damage, ultimately leading to cell death (Papaliagkas et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Therefore, one of the major challenges in reproductive embryology is to prevent oocytes' degeneration to maintain their developmental competences (Hoffmann et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).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, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). 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\u0026nbsp;\u0026micro;M Resveratrol significantly reduced the levels of intracellular ROS while increasing GSH contents during \u003cem\u003ein vitro\u003c/em\u003e maturation (Kim et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Kwak et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite 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 \u003cem\u003ein vitro\u003c/em\u003e 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.\u003c/p\u003e "},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eOocytes collection and maturation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePig ovaries were collected from a local slaughterhouse and immediately placed in sterile physiological saline at 37\u0026deg;C and delivered to the laboratory within 2 h. The ovaries were washed twice in the sterile condition under pre-warmed PBS at 37\u0026deg;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 \u0026ge;3 layers of cumulus cells were selected for the experiment and randomly divided into 4 treatment groups by adding 0, 1, 2, and 4 \u0026mu;mol/L RES and transferred to TCM-199 culture medium. The cells were cultured in a CO2 incubator at 38.5\u0026deg;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 \u0026mu;mol/L RES was found as an optimum level for porcine oocytes, so further treatment was carried out by using only this level.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro aging and \u003c/strong\u003e\u003cstrong\u003eRES\u003c/strong\u003e\u003cstrong\u003e treatment of porcine oocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResveratrol was diluted with dimethyl sulfoxide (DMSO) to a 10 mmol/L concentrated stock solution and stored at -20 \u0026deg;C. The resveratrol concentrated stock solution was added to the TCM199 culture solution to adjust the concentration to 2 \u0026mu;mol/L. The COCs were cultured for 44 h as a control group, aged 24 and 48 h as \u003cem\u003ein vitro\u003c/em\u003e aging groups, placed in a mature medium containing 2 \u0026mu;mol/L RES, and then aged for 24 and 48 h, as a RES treatment group. The first polar body (pbI) of the oocyte was observed by microscopy and the change of the perivitaline space of the oocyte was detected.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFresh COCs were collected and washed twice with DPBS solution and stored at \u0026minus;80 \u0026deg;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\u0026rsquo;s instructions. The RNA thus extracted was quantified using nano-Drop and stored at \u0026minus;80 \u0026deg;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\u0026rsquo;s protocol.\u003c/p\u003e\n\u003cp\u003eThe 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 fluorescence 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\u0026nbsp;\u003cem\u003eGAPDH, Caspase-3, Bcl-2, Bax\u003c/em\u003e,\u0026nbsp;\u003cem\u003eCAT\u003c/em\u003e,\u0026nbsp;\u003cem\u003eGSH-PX\u003c/em\u003e, and\u0026nbsp;\u003cem\u003eSOD1\u003c/em\u003e\u0026nbsp;were synthesized by Nanjing Qingke Biotechnology Co., Ltd. The details of primers are presented in\u0026nbsp;Table1. The results of quantitative real-time PCR (qPCR) were analyzed using\u003c/p\u003e\n\u003cp\u003ethe 2\u003csup\u003e-\u003c/sup\u003e\u003csup\u003e△△\u003c/sup\u003e\u003csup\u003eCt\u003c/sup\u003e method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOocyte reactive oxygen species (ROS) assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe intracellular ROS level was determined by 2,7 dichlorofluorescein (DCF) fluorescence assay as previously described (Gupta et al., 2010). Briefly, oocytes were incubated with 10 mM DCF for 20 min at 37\u003csup\u003eo\u003c/sup\u003eC followed by washing thrice in TCM199. It took 1 h or less to reflect the fluorescence intensity of ROS (Excitation wavelength: 450- 490 nm and Emission wavelength: 515-565 nm). The results were immediately observed and photographed using an epifluorescence microscope. The mean values of fluorescent oocytes were further processed by using Image J 6.0 software. Background fluorescent values were subtracted from the final values before analyzing for the statistical difference between the groups. The experiment was replicated 3 times with 20\u0026ndash;25 oocytes each time.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of intracellular GSH contents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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\u0026rsquo;s instructions. A total of 50 oocytes from each group were mixed with 30 \u0026micro;l 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\u0026deg;C repeatedly for 3 times. Then the mixture was centrifuged at 10,000 rpm for 10 min at 4\u0026deg;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).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMitochondrial membrane potential assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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 10\u0026mu;l MJC-1 in 100\u0026mu;L working solution at 38.5\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e for20 min. To remove surface fluorescence, 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 fluorescence, respectively. The fluorescence microscope (Olympus, Tokyo, Japan) with the same scan settings for each sample was used to measure the fluorescence intensity of each oocyte. Image J software was used to analyze the normal fluorescence pixel intensities of each oocyte. The ratio of green to red fluorescence pixels was used to analyze mitochondrial membrane potential.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical\u003c/strong\u003e\u003cstrong\u003e analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEach 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 significant difference (LSD) test at 1% and 5% probability levels. The P-value of \u0026lt;0.05 was considered as a significant difference while, P \u0026lt;0.01 was considered as highly significant whereas, P \u0026lt;0.001 was considered as extremely significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eEffect of \u003c/strong\u003e\u003cstrong\u003eRES\u003c/strong\u003e\u003cstrong\u003e on\u0026nbsp;aged oocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effect of treatment of RES (0, 1, 2, and 4 \u0026micro;mol/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 \u0026micro;mol/L RES treated group was better than the control and 4 \u0026micro;mol/L RES treated groups (Fig. 1). Moreover, we observed a significant difference in the first polar body extrusion rate (pbI) between the 2 \u0026micro;mol/L RES and the control group. However, no significant difference in first polar body extrusion rate (pbI) in 1 and 4 \u0026micro;mol/L RES treated groups was observed as compared to control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of \u003c/strong\u003e\u003cstrong\u003eRES\u003c/strong\u003e\u003cstrong\u003e on perivitaline space of aged oocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTreatment with resveratrol significantly 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 significant decrease in perivitaline spaces after24h and 48h of aging. However, differences were non-significant as compared to the control group (Fig 2B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of \u003c/strong\u003e\u003cstrong\u003eRES \u003c/strong\u003e\u003cstrong\u003eon the expression of apoptotic genes \u003c/strong\u003e\u003cstrong\u003ein\u003c/strong\u003e\u003cstrong\u003e aged oocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur results revealed that the relative mRNA expression of \u003cem\u003ecaspase-3\u003c/em\u003e and \u003cem\u003eBax\u003c/em\u003e in aged oocyte groups was significantly higher than thecontrol group; while the expression of Bcl-2 was significantly lower (Figure 3). However, the expression of \u003cem\u003ecaspase-3\u003c/em\u003e and \u003cem\u003eBax \u003c/em\u003ein 24 and 48 h REStreated groups was significantly decreased thanuntreatedaged groups. Moreover, the expression of \u003cem\u003eBcl-2\u003c/em\u003e in the RES treated group was significantly lower than the control group but significantly higher than untreated aged groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of \u003c/strong\u003e\u003cstrong\u003eRES\u003c/strong\u003e\u003cstrong\u003e on\u003c/strong\u003e\u003cstrong\u003e the \u003c/strong\u003e\u003cstrong\u003eexpression of antioxidant enzymes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur results showed that RES could significantly 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 significantly 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 significantly increased, but there was no significant difference as compared with the control group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of \u003c/strong\u003e\u003cstrong\u003eRES \u003c/strong\u003e\u003cstrong\u003eon GSH enzyme activity of aged oocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResults showed that the activity of GSH was significantly decreased in 24 and 48 h aged groups whereas RES treatment significantly improved the GSH activity in 24 and 48 h aged groups (Fig 5). However, this difference was non-significant as compared to the control group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of \u003c/strong\u003e\u003cstrong\u003eRES \u003c/strong\u003e\u003cstrong\u003eon ROS in aged oocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur results revealed that ROS levels significantly increased in 24 and 48 h aged groups compared with control, while the oocytes treated with resveratrol (both at 24 and 48 h) showed significantly higher ROS levels than those of the control group, but significantly lower than those of untreated aged groups (Fig 6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of \u003c/strong\u003e\u003cstrong\u003eRES\u003c/strong\u003e\u003cstrong\u003e on mitochondrial membrane potential \u003c/strong\u003e\u003cstrong\u003eof\u003c/strong\u003e\u003cstrong\u003e oocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the mitochondrial membrane potential, we analyzed the ratio of green/red fluorescence. 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 significant tendency of keeping mitochondrial membrane potential in its normal state during the oocyteaging process (Fig: 7 and 8).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cstrong\u003eEffect of \u003c/strong\u003e\u003cstrong\u003eRES\u003c/strong\u003e\u003cstrong\u003eon GSH enzyme activity, antioxidant genes, and ROS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur study revealed that under\u0026nbsp;\u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003econditions, RES could significantly increase the GSH contents in 24 h aged oocytes as compared with the control group (P \u0026lt; 0.001). Although, RES at 48 h of aging significantly lowered the GSH contents than the control group (P \u0026lt;0.05), but still it was significantly higher than its untreated counterparts. Our findings 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\u0026nbsp;\u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003eaging 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\u0026nbsp;the\u003cem\u003e in vitro\u003c/em\u003e\u0026nbsp;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\u0026nbsp;\u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003eoocyte maturation (Matos et al., 1995). Similarly, our study revealed the potential of RES to significantly 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe apoptosis is a very complex process being affected by many factors such as inflammatory 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 significantly reduced the ROS level in aged oocytes (24 and 48 h) which is consistent with earlier findings (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).\u003c/p\u003e\n\u003cp\u003eThe primary antioxidant enzymes are SOD, catalase (CAT), and glutathione peroxidase (GSH-Px). Moreover, O\u003csub\u003e2\u003c/sub\u003e is converted by SOD to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 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 disulfide and triggers reduction to GSH by glutathione reductase (Wu, 2013). When Cu\u003csup\u003e+2\u003c/sup\u003e or Fe\u003csup\u003e+2\u003c/sup\u003e are available, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 \u003c/sub\u003ereacts 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 significantly 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).\u003c/p\u003e\n\u003cp\u003ePrevious 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 \u0026lt;0.01). However, expression of CAT and SOD1 genes was lower than that of the control group (P \u0026lt;0.05), but still higher than their untreated counterparts. Moreover, RES significantly increased the expression CAT, SOD1, and GSH Px in48h aged group than the untreated aging group. Our findings revealed that RES can effectively mediate oxidative stress induced by the aging oocytes during \u003cem\u003ein vitro\u003c/em\u003e 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of resveratrol on perivitaline space in aged oocyte\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRES 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 \u0026mu;mol/L and 2 \u0026mu;mol/L) which is consistent with earlier findings as mentioned above\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eFurthermore, the first polar body extrusion rate of the 2 \u0026mu;mol/L RES group was also significantly 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 significant decrease in perivitaline spaces during 24h and 48h. However, differences were non-significant compared with the control group\u0026nbsp;(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 findings. Similarly, RES supplementation into the \u003cem\u003ein vitro\u003c/em\u003e maturation medium improved the quality of bovine embryos and increased their resistance against oxidative damage during cryopreservation (Abdel-Wahab et al., 2012). The beneficial effects of RES might depend upon its ability to improve oocyte quality therefore, it can be concluded that RES (2\u0026mu;mol/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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of resveratrol on the expression of apoptotic genes of aged oocyte\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApoptosis 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 specific 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\u003csup\u003e+2\u003c/sup\u003e which may lead to oocyte fragmentation and accelerates aging(Ma et al., 2005a).\u003c/p\u003e\n\u003cp\u003eFurthermore, 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 (Zhang et al., 2017). 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 confirmed 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 up-regulates 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 significantly lower in the RES treated group than the control but was significantly higher as compared to untreated aged groups. Our findings 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMitochondrial membrane potential\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMitochondria are responsible to maintain cellular metabolic functions and its physiological efficiency can be assessed by examining the mitochondrial membrane potential state. In this regard, fluorescence 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 significantly 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 \u003cem\u003eB\u003c/em\u003e\u003cem\u003ecl\u003c/em\u003e\u003cem\u003e-2\u003c/em\u003e 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 \u003cem\u003ein vivo\u003c/em\u003e studies are required to elucidate its potential mechanism of action.\u003c/p\u003e\n\u003cp\u003eOur 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 \u003cem\u003eBcl 2\u003c/em\u003e while down-regulating the mRNA expression of \u003cem\u003eBax \u003c/em\u003eand \u003cem\u003eCaspase 3\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorresponding author will provide the data on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research supported by the Doctoral Program of the Ministry of Education of China (20130097110020) and PAPD of Jiangsu Province.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eB. A., conceived the idea and perform experiment and wrote the draft of the manuscript. D. Y., perform statistical analysis of the data. R. R., supervise the experiment and improve the final draft of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Doctoral Program of the Ministry of Education of China (20130097110020) and PAPD of Jiangsu Province.\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003eAbdel-Wahab, A.M., Zullo, G., Boccia, L., Blasi, M.D., Gasparrini, B., 2012. 132 Resveratrol during in vitro culture improves cryotolerance of in vitro produced bovine embryos. Reproduction Fertility \u0026amp; Development 25, 213-214.\u003c/p\u003e\n\u003cp\u003eArain, M.A., Mei, Z., Hassan, F., Saeed, M., Alagawany, M., Shar, A., Rajput, I., 2018. Lycopene: a natural antioxidant for prevention of heat-induced oxidative stress in poultry. 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Aging 10.\u003c/p\u003e\n\u003cp\u003eLiang, S., Guo, J., Choi, J.-W., Shin, K.-T., Wang, H.-Y., Jo, Y.-J., Kim, N.-H., Cui, X.-S., 2017. Protein phosphatase 2A regulatory subunit B55\u0026alpha; functions in mouse oocyte maturation and early embryonic development. Oncotarget 8, 26979.\u003c/p\u003e\n\u003cp\u003eLiu, M., Yu, Y., Ye, X., Ming, Z., Qiang, Z., Keefe, D.L., Lin, L., 2013. Resveratrol protects against age-associated infertility in mice. Human Reproduction, 3.\u003c/p\u003e\n\u003cp\u003eLiu, Y., Fiskum, G., Schubert, D., 2002. Generation of reactive oxygen species by the mitochondrial electron transport chain. Journal of neurochemistry 80, 780-787.\u003c/p\u003e\n\u003cp\u003eLord, T., Aitken, R.J., 2013. Oxidative stress and ageing of the post-ovulatory oocyte. Reproduction 146, R217-R227.\u003c/p\u003e\n\u003cp\u003eMa, W., Zhang, D., Hou, Y., Li, Y.-H., Sun, Q.-Y., Sun, X.-F., Wang, W.-H., 2005a. Reduced expression of MAD2, BCL2, and MAP kinase activity in pig oocytes after in vitro aging are associated with defects in sister chromatid segregation during meiosis II and embryo fragmentation after activation. Biology of Reproduction 72, 373-383.\u003c/p\u003e\n\u003cp\u003eMa, W., Zhang, D., Hou, Y., Li, Y.-H., Sun, Q.-Y., Sun, X.-F., Wang, W.-H., 2005b. Reduced Expression of MAD2, BCL2, and MAP Kinase Activity in Pig Oocytes after In Vitro Aging Are Associated with Defects in Sister Chromatid Segregation During Meiosis II and Embryo Fragmentation After Activation1. Biology of Reproduction 72, 373-383.\u003c/p\u003e\n\u003cp\u003eMailhes, J.B., Young, D., London, S., 1998. Postovulatory ageing of mouse oocytes in vivo and premature centromere separation and aneuploidy. Biology of reproduction 58, 1206-1210.\u003c/p\u003e\n\u003cp\u003eMatos, D.G.D., Furnus, C.C., Moses, D.F., Baldassarre, H., 1995. Effect of cysteamine on glutathione level and developmental capacity of bovine oocyte matured in vitro. Molecular Reproduction \u0026amp; Development 42, 432-436.\u003c/p\u003e\n\u003cp\u003eMccord, J.M., 2000. The evolution of free radicals and oxidative stress. American Journal of Medicine 108, 652-659.\u003c/p\u003e\n\u003cp\u003eMeng, Q., Guo, T., Li, G., Sun, S., He, S., Cheng, B., Shi, B., Shan, A., 2018. Dietary resveratrol improves antioxidant status of sows and piglets and regulates antioxidant gene expression in placenta by Keap1-Nrf2 pathway and Sirt1. Journal of Animal Science \u0026amp; Biotechnology 9, 147-159.\u003c/p\u003e\n\u003cp\u003eMovahed, A., Yu, L., Thandapilly, S.J., Louis, X.L., Netticadan, T., 2012. Resveratrol protects adult cardiomyocytes against oxidative stress mediated cell injury. Archives of Biochemistry \u0026amp; Biophysics 527, 74-80.\u003c/p\u003e\n\u003cp\u003eMukherjee, A., Malik, H., Saha, A.P., Dubey, A., Singhal, D.K., Boateng, S., Saugandhika, S., Kumar, S., De, S., Guha, S.K., 2014. Resveratrol treatment during goat oocytes maturation enhances developmental competence of parthenogenetic and hand-made cloned blastocysts by modulating intracellular glutathione level and embryonic gene expression. Journal of assisted reproduction and genetics 31, 229-239.\u003c/p\u003e\n\u003cp\u003eNutt, L.K., Gogvadze, V., Uthaisang, W., Mirnikjoo, B., Orrenius, S., 2005. Indirect effects of Bax and Bak initiate the mitochondrial alterations that lead to cytochrome c release during arsenic trioxide-induced apoptosis. Cancer Biology \u0026amp; Therapy 4, 459-467.\u003c/p\u003e\n\u003cp\u003ePapaliagkas, V., Ilonidis, G., Anogianaki, A., Anogeianakis, G., 2007. The proteins and the mechanisms of apoptosis. Aristotle University of Thessaloniki.\u003c/p\u003e\n\u003cp\u003eParborell, F., 2002. Effects of a Gonadotropin-Releasing Hormone Agonist on Rat Ovarian Follicle Apoptosis: Regulation by Epidermal Growth Factor and the Expression of Bcl-2-Related Genes. Biology of Reproduction 67, 481-486.\u003c/p\u003e\n\u003cp\u003ePascuali, N., Scotti, L., Abramovich, D., Irusta, G., Di Pietro, M., Bas, D., Tesone, M., Parborell, F., 2015. Inhibition of platelet-derived growth factor (PDGF) receptor affects follicular development and ovarian proliferation, apoptosis and angiogenesis in prepubertal eCG-treated rats. Mol Cell Endocrinol 412, 148-158.\u003c/p\u003e\n\u003cp\u003ePervaiz, S., Holme, A.L., 2009. Resveratrol: its biologic targets and functional activity. Antioxidants \u0026amp; redox signaling 11, 2851-2897.\u003c/p\u003e\n\u003cp\u003eRamalho-Santos, J., Varum, S., Amaral, S., Mota, P.C., Sousa, A.P., Amaral, A., 2009. Mitochondrial functionality in reproduction: from gonads and gametes to embryos and embryonic stem cells. Human reproduction update 15, 553-572.\u003c/p\u003e\n\u003cp\u003eRedza-Dutordoir, M., Averill-Bates, D.A., 2016. Activation of apoptosis signalling pathways by reactive oxygen species. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 1863, 2977-2992.\u003c/p\u003e\n\u003cp\u003eSaito, H., Koike, K., Saito, T., Nohara, M., Kawagoe, S., Hiroi, M., 1993. Aging changes in the alignment of chromosomes after human chorionic gonadotropin stimulation may be a possible cause of decreased fertility in mice. Hormone Research in Paediatrics 39, 28-31.\u003c/p\u003e\n\u003cp\u003eSuzuki, Y., Imai, Y., Nakayama, H., Takahashi, K., Takio, K., Takahashi, R., 2001. A serine protease, HtrA2, is released from the mitochondria and interacts with XIAP, inducing cell death. Molecular cell 8, 613-621.\u003c/p\u003e\n\u003cp\u003eTar\u0026iacute;n, J.J., P\u0026eacute;rez-Albal\u0026aacute;, S., Aguilar, A., Mi\u0026ntilde;arro, J., Hermenegildo, C., Cano, A., 1999. Long-term effects of postovulatory aging of mouse oocytes on offspring: a two-generational study. Biology of reproduction 61, 1347-1355.\u003c/p\u003e\n\u003cp\u003eTherond, P., 2006. Oxidative stress and damages to biomolecules (lipids, proteins, DNA). Annales Pharmaceutiques Fran\u0026ccedil;aises 64, 383-389.\u003c/p\u003e\n\u003cp\u003eTilly, J.L., Tilly, K.I., Kenton, M.L., Johnson, A.L., 1995. Expression of members of the bcl-2 gene family in the immature rat ovary: equine chorionic gonadotropin-mediated inhibition of granulosa cell apoptosis is associated with decreased bax and constitutive bcl-2 and bcl-xlong messenger ribonucleic acid levels. Endocrinology, 1.\u003c/p\u003e\n\u003cp\u003eTong, Z., Xie, Y., He, M., Ma, W., Zhou, Y., Lai, S., Meng, Y., Liao, Z., 2017. VDAC1 deacetylation is involved in the protective effects of resveratrol against mitochondria-mediated apoptosis in cardiomyocytes subjected to anoxia/reoxygenation injury. Biomedicine \u0026amp; Pharmacotherapy 95, 77-83.\u003c/p\u003e\n\u003cp\u003eTrapphoff, T., Heiligentag, M., Dankert, D., Demond, H., Deutsch, D., Fr\u0026ouml;hlich, T., Arnold, G., Gr\u0026uuml;mmer, R., Horsthemke, B., Eichenlaub-Ritter, U., 2016. Postovulatory aging affects dynamics of mRNA, expression and localization of maternal effect proteins, spindle integrity and pericentromeric proteins in mouse oocytes. Human Reproduction 31, 133-149.\u003c/p\u003e\n\u003cp\u003eVerhagen, A.M., Ekert, P.G., Pakusch, M., Silke, J., Connolly, L.M., Reid, G.E., Moritz, R.L., Simpson, R.J., Vaux, D.L., 2000. Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. cell 102, 43-53.\u003c/p\u003e\n\u003cp\u003eWang, F., Tian, X.Z., Zhang, L., He, C.J., Ji, P.Y., Li, Y., Tan, D.X., Liu, G.S., 2014. Beneficial effect of resveratrol on bovine oocyte maturation and subsequent embryonic development after in?vitro fertilization. Fertility \u0026amp; Sterility 101, 577-586.e571.\u003c/p\u003e\n\u003cp\u003eWang, J., Hao, L., Wang, Y., Qin, W., Wang, X., Zhao, T., Liu, Y., Sheng, L., Du, Y., Zhang, M., Lu, Q., 2015. Inhibition of poly (ADP-ribose) polymerase and inducible nitric oxide synthase protects against ischemic myocardial damage by reduction of apoptosis. Mol Med Rep 11, 1768-1776.\u003c/p\u003e\n\u003cp\u003eWang, T., Gao, Y.-Y., Chen, L., Nie, Z.-W., Cheng, W., Liu, X., Schatten, H., Zhang, X., Miao, Y.-L., 2017. Melatonin prevents postovulatory oocyte aging and promotes subsequent embryonic development in the pig. Aging (Albany NY) 9, 1552.\u003c/p\u003e\n\u003cp\u003eWang, T., Han, J., Duan, X., Xiong, B., Cui, X.-S., Kim, N.-H., Liu, H.-L., Sun, S.-C., 2016. The toxic effects and possible mechanisms of Bisphenol A on oocyte maturation of porcine in vitro. Oncotarget 7, 32554.\u003c/p\u003e\n\u003cp\u003eWenzel, E., Soldo, T., Erbersdobler, H., Somoza, V., 2005. Bioactivity and metabolism of trans‐resveratrol orally administered to Wistar rats. Molecular Nutrition \u0026amp; Food Research 49.\u003c/p\u003e\n\u003cp\u003eWu, C.C., Huang, Y.S., Chen, J.S., Huang, C.F., Su, S.L., Lu, K.C., Lin, Y.F., Chu, P., Lin, S.H., Sytwu, H.K., 2015. Resveratrol Ameliorates Renal Damage, Increases Expression of Heme Oxygenase-1, and Has Anti-Complement, Anti-Oxidative, and Anti-Apoptotic Effects in a Murine Model of Membranous Nephropathy. Plos One 10.\u003c/p\u003e\n\u003cp\u003eWu, J.Q., 2013. Free radicals, antioxidant defense systems, and schizophrenia. Progress in Neuropsychopharmacology \u0026amp; Biological Psychiatry 46, 200-206.\u003c/p\u003e\n\u003cp\u003eYew, N., Mellini, M.L., Martinez, C.K., Woude, G.F.V., 1992. Meiotic initiation by the mos protein in Xenopus. Nature 355, 649-652.\u003c/p\u003e\n\u003cp\u003eZhang, X., Liu, X., Chen, L., Wu, D.Y., Miao, Y.L., 2017. Caffeine delays oocyte aging and maintains the quality of aged oocytes safely in mouse. Oncotarget 8, 20602.\u003c/p\u003e\n\u003cp\u003eZheng, Y., Shi, Y., Tian, C., Jiang, C., Jin, H., Chen, J., Almasan, A., Tang, H., Chen, Q., 2004. Essential role of the voltage-dependent anion channel (VDAC) in mitochondrial permeability transition pore opening and cytochrome c release induced by arsenic trioxide. Oncogene 23, 1239-1247.\u003c/p\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable1\u003c/strong\u003e.The details of RT-PCR primer pairs used for analysis of pro-apoptotic genes\u003c/p\u003e\n\u003ctable border=\"1\" width=\"0\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"132\"\u003e\n\u003cp\u003eTarget gene\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"293\"\u003e\n\u003cp\u003eSequence\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"126\"\u003e\n\u003cp\u003eAccession Number\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"93\"\u003e\n\u003cp\u003eSize (bp)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"132\"\u003e\n\u003cp\u003e\u003cem\u003eGAPDH\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"293\"\u003e\n\u003cp\u003eF: 5'-GTCGGTTGTGGATCTGACCT-3'\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"126\"\u003e\n\u003cp\u003eNM_001206359\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"93\"\u003e\n\u003cp\u003e207\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"293\"\u003e\n\u003cp\u003eR: 5'-TTGACGAAGTGGTCGTTGAG-3'\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"132\"\u003e\n\u003cp\u003e\u003cem\u003eCaspase-3\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"293\"\u003e\n\u003cp\u003eF: 5'-CGTGCTTCTAAGCCATGGTG-3'\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"126\"\u003e\n\u003cp\u003eNM_214131\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"93\"\u003e\n\u003cp\u003e186\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"293\"\u003e\n\u003cp\u003eR: 5'-GTCCCACTGTCCGTCTCAAT-3'\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"132\"\u003e\n\u003cp\u003e\u003cem\u003eBcl-2\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"293\"\u003e\n\u003cp\u003eF:5'-AGGGCATTCAGTGACCTGAC-3'\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"126\"\u003e\n\u003cp\u003eNM_214285\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"93\"\u003e\n\u003cp\u003e193\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"293\"\u003e\n\u003cp\u003eR: 5'-CGATCCGACTCACCAATACC-3'\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"132\"\u003e\n\u003cp\u003e\u003cem\u003eBax \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"293\"\u003e\n\u003cp\u003eF:5'-TGCCTCAGGATGCATCTACC-3'\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"126\"\u003e\n\u003cp\u003eXM_003127290\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"93\"\u003e\n\u003cp\u003e199\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"293\"\u003e\n\u003cp\u003eR: 5'-AAGTAGAAAAGCGCGACCAC-3'\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"132\"\u003e\n\u003cp\u003e\u003cem\u003eCAT\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"293\"\u003e\n\u003cp\u003eF-5'-AACTGTCCCTTCCGTGCTA-3'\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"126\"\u003e\n\u003cp\u003eXM_021081498\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"93\"\u003e\n\u003cp\u003e195\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"293\"\u003e\n\u003cp\u003eR-5'-CCTGGGTGACATTATCTTCG-3'\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"132\"\u003e\n\u003cp\u003e\u003cem\u003eGSH-PX\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"293\"\u003e\n\u003cp\u003eF-5'-CAAGTCCTTCTACGACCTCA-3'\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"126\"\u003e\n\u003cp\u003eAJ010340\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"93\"\u003e\n\u003cp\u003e210\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"293\"\u003e\n\u003cp\u003eR-5'-GAAGCCAAGAACCACCAG-3'\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"132\"\u003e\n\u003cp\u003e\u003cem\u003eSOD1\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"293\"\u003e\n\u003cp\u003eF-5'-ACCTGGGCAATGTGACTG-3'\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"126\"\u003e\n\u003cp\u003eNM_001190422\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"93\"\u003e\n\u003cp\u003e197\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"293\"\u003e\n\u003cp\u003eR-5'-TCCAGCATTTCCCGTCT-3'\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e "}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"oocyte aging, reactive oxygen species, oxidative stress, resveratrol, anti-oxidant, apoptosis ","lastPublishedDoi":"10.21203/rs.3.rs-120271/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-120271/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis 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 \u003cem\u003ein vitro\u003c/em\u003e medium supplemented with four levels (0, 1, 2, and 4\u0026nbsp;\u0026micro;mol/L) of RES. Based on initial screening; optimum RES level (2\u0026nbsp;\u0026micro;mol/L) was used to evaluate its potential effects on oocytes aged for 24 and 48 hours. Our results revealed that aged oocytes (24\u0026nbsp;h and 48\u0026nbsp;h) treated with RES showed higher (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) ROS fluorescence intensity than the control group, but lower (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than untreated aged groups. The GSH content in untreated aged groups (24\u0026nbsp;h and 48\u0026nbsp;h) was lower (P\u0026thinsp;\u0026lt;\u0026thinsp;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\u0026thinsp;\u0026lt;\u0026thinsp;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\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than the control group but lower than untreated groups. Furthermore, the expression of Bcl-2 in the RES treated group was significantly lower than control but higher than untreated aged groups. Our findings 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.\u003c/p\u003e","manuscriptTitle":"Resveratrol Can Revert Adverse Effects of Oocyte aging in Pig","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-12-03 18:42:21","doi":"10.21203/rs.3.rs-120271/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6d4b8465-1334-4744-9fc0-fa93087e3330","owner":[],"postedDate":"December 3rd, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":1323779,"name":"Veterinary Epidemiology"},{"id":1323780,"name":"Animal Science"}],"tags":[],"updatedAt":"2020-12-03T18:42:23+00:00","versionOfRecord":[],"versionCreatedAt":"2020-12-03 18:42:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-120271","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-120271","identity":"rs-120271","version":["v1"]},"buildId":"369fNeqWncA4NS6XSWjrt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}