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Melatonin ameliorates ochratoxin A-induced oxidative stress and apoptosis in porcine oocytes
Environmental Pollution xxx (xxxx) xxx
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Melatonin ameliorates ochratoxin A-induced oxidative stress and apoptosis in porcine oocytes* Mei Lan, Yu Zhang, Xiang Wan, Meng-Hao Pan, Yao Xu, Shao-Chen Sun* College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 August 2019 Received in revised form 8 October 2019 Accepted 9 October 2019 Available online xxx
Melatonin is a hormone which is generated from pineal gland, and it is responsible for the regulation of wake-sleep cycle. Melatonin is a well-known antioxidant and free radical scavenger to protect against multiple type of tissue damage. While ochratoxin A (OTA) is a mycotoxin found widely in contaminated food and foodstuffs, which causes nephrotoxicity, hepatotoxicity, immunotoxicity, and reproductive damage in humans and animals. In present study we report the toxicity of OTA on porcine oocyte quality and the protective effects of melatonin on OTA-exposed oocytes. Using transcriptome analysis our results show that OTA exposure alters the expression of multiple genes in oocytes, indicating its effect on oocyte maturation. The cellular changes following OTA treatment are examined, and the results show that OTA adversely affects oocyte polar body extrusion, which is confirmed by the delay of Cdc2-mediated cell cycle progression. OTA exposure also disrupts meiotic spindle formation, which is confirmed by altered phosphorylated MAPK expression. RNA-seq screening and further fluorescence staining results show that OTA induces aberrant mitochondria distribution and oxidative phosphorylation defects, which then causes oxidative stress, followed by early apoptosis and autophagy. Treatment with melatonin significantly ameliorates oxidative stress and apoptosis, which further protects cell cycle and spindle formation in OTA-exposed oocytes. Collectively, these results show the protective effects of melatonin against defects induced by OTA during porcine meiotic oocyte maturation. © 2019 Elsevier Ltd. All rights reserved.
Keywords: Ochratoxin A Melatonin Apoptosis Oxidative stress Oocyte
1. Introduction Ochratoxin A (OTA), a mycotoxin produced by Aspergillus spp, is one of the most commonly detected mycotoxins in food and foodstuffs such as coffee beans, fermented tea, and cereals (Brennan et al., 2017). Besides causing losses in the livestock economy, OTA bioaccumulation through the food chain results in immunomodulatory, hepatotoxic, nephrotoxic, genotoxic, and reproductive damage in animals and humans (Bhat et al., 2018; Malir et al., 2013). Previous studies showed that exposure to mycotoxins activates endocrine disrupted chemical (EDC) secretion, which results in functional decline of reproductive organs, including sub-fertility or infertility (Eze et al., 2018). The presence of OTA in drinking water or intravenous injection of OTA decreases oocyte fertilization rates and has deleterious effects on mouse early embryonic development (Huang and Chan, 2016). OTA exposure
* This paper has been recommended for acceptance by Wen Chen. * Corresponding author. E-mail address: [email protected] (S.-C. Sun).
potentially affects mouse sperm motility via the AMPK and PTEN signaling pathways in vitro (Lu et al., 2018). Studies have been carried out for decades to elucidate the mechanism of mycotoxin toxicities. Mycotoxins such as HT-2, citrinin, or deoxynivalenol may cause excess oxidative stress in cells, and when the reactive oxygen species (ROS) reach a maximum resistance threshold, it will result in organelle damage or even cell death (Huang and Chan, 2017; Lan et al., 2018; Zhang et al., 2017). Oxidative stress also plays critical roles in the toxicity of OTA, since it has been reported that OTA affects anti-oxidant self-defense in the gut, kidney (Marin et al., 2017), and embryo (Hsuuw et al., 2013). Our previous study provides preliminary data which shows that OTA affects oocyte maturation, and porcine oocytes is more sensitive than mouse oocytes (Lu et al., 2018). However, the causes and mechanism of OTA toxicity on porcine oocyte quality are still unclear. Moreover, there is still no effective approach to reduce the OTA toxicity in vivo. Melatonin, a well-known antioxidant and free radical scavenger, has been reported to protect against multiple type of tissue damage (Tan et al., 1993). Melatonin is generated from pineal gland and is the in vivo hormone responsible for the regulation of wakefulness.
https://doi.org/10.1016/j.envpol.2019.113374 0269-7491/© 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Lan, M et al., Melatonin ameliorates ochratoxin A-induced oxidative stress and apoptosis in porcine oocytes, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113374
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Field studies have shown that melatonin can protect the heart from reperfusion injury by inhibiting the mitochondrial permeability transition pore (MPTP) opening and cardiolipin peroxidation (Petrosillo et al., 2009). Melatonin treatment may function as a potential adjuvant therapy to alleviate inflammatory bowel disease (Liu et al., 2017). And melatonin works against cancer cell proliferation, motility, and invasiveness by acting on the members of MAPK family (Mortezaee, 2018). Moreover, studies have revealed that melatonin plays an important role in the regulation of the reproductive system via reducing oxidative stress (Lan et al., 2018) and modifying follicle and ovary steroidogenesis (Tamura et al., 1998). In RFR (radiofrequency radiation)-exposed mice, melatonin inhibits pre-meiotic spermatogenesis arrest in male germ cells through its anti-oxidative potential and ability to improve DNA repair pathways, leading to normal sperm count and morphology (Pandey and Giri, 2018). Our previous study also showed that in di2-ethylhexyl phthalate (DEHP)-exposed porcine oocytes, melatonin supplementation ameliorated oocyte maturation through its rescue of oxidative stress-mediated apoptosis and autophagy (Zhang et al., 2018). In this study, based on transcriptome analysis, we show the expression of several cellular process-related genes were altered after OTA exposure in porcine oocytes, which are confirmed from cellular analyses, showing cell cycle delay, disruption of the cytoskeleton and mitochondria distribution, and oxidative stress and apoptosis. These results illustrate the toxic effects and possible mechanism of OTA on porcine oocytes and also show that melatonin administration is effective for improving maturation of OTAexposed oocytes.
glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 50 mg/ml of streptomycin, 75 mg/ml of penicillin, 0.5 mg/ml of LH, 0.5 mg/ml of FSH, 10 ng/ml of epidermal growth factor (EGF) and 10% pFF]. Then selected COCs were cultured with different treatment in a four-well dish (Nunc, Roskilde, Denmark) containing 500 ml of IVM medium covered with 200 ml mineral oil at 38.5 C in a humidified atmosphere of 5% CO2 incubator for IVM. After a certain time of culture, COCs were treated with 0.02% (w/v) hyaluronidase at 37 C for 5 min. Gently blow off the surrounding cumulus cells. After 3e4 times of rinsing, the exposed oocytes were collected for subsequent analysis.
2. Materials and methods
2.4. RNA-seq (transcriptome sequencing)
2.1. Antibodies and chemicals
Transcriptome sequencing is based on the Illumina Hiseq sequencing platform to study all mRNA transcribed by cells at a certain time. After cultured for 27 h, the total RNA was extracted from the porcine oocytes of control group and OTA group, respectively. The quality of RNA was detected by using Agilent bioanalyzer 2100. RNA-seq transcriptome library was prepared following NEB Next Ultra RNA library Prep Kit for Illumina. After mixing libraries based on their effective concentration and the required sequencing data volume, Illumina HiSeq platform is used for high through-put sequencing and biological information analyzing. The original image data were analyzed using Bcl2fastq (v2.17.1.14) for base calling and preliminary quality analysis. Then functional-enrichment analysis including Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) pathways were compared with the whole-transcriptome background.
Melatonin was purchased from Sigma Chemical Company (M5250, St. Louis, MO, USA). Ochratoxin A was purchased from J&K Scientific chemical company (356520, Beijing, China). Mouse monoclonal anti-a-tubulin-FITC antibody (F2168), phalloidin-TRITC (P1951) and Hoechst 33342 (B2261) were purchased from Sigma (St. Louis, MO, USA). Rabbit monoclonal anti-microtubuleassociated protein 1 light chain 3 (LC3A) antibody was purchased from Abcam (ab128025, Cambridge, UK). Alexa Fluor 594 goat antimouse antibody, Alexa Fluor 488 and 594 goat anti-rabbit antibodies were from Invitrogen (Carlsbad, CA, USA). Rabbit monoclonal anti-p-MAPK antibody (4370), rabbit monoclonal anti-pcdc2 antibody (28439), rabbit monoclonal anti-p-53 antibody (2524), rabbit monoclonal anti-caspase3 antibody (9662), rabbit monoclonal anti-LC3B antibody (3868) and rabbit anti-GAPDH antibody (5174) were purchased from Cell Signaling Technology (Devers, MA, USA). Basic maturation culture medium was tissue culture medium (TCM-199) (St. Louis, MO, USA). 2.2. COCs collection and culture All experiments were approved by the Animal Care and Use Committee of Nanjing Agriculture University and were performed in accordance with Animal Research Institute Committee guidelines (SYXK-Su-2017-0027). Pig ovaries obtained from slaughterhouses were transported to the laboratory within 2 h in aseptic saline containing 800IU/ml gentamicin at 25e30 C. After washing with aseptic phosphate buffer saline twice, the cumulus oocyte complex was sucked out of the follicles (3e6 mm) with 20-gauge needles. Oocytes with intact and compact cumulus mass were selected and washed three times with in vitro maturation medium [supplemented with 0.1% polyvinyl alcohol (PVA), 3.05 mM D-
2.3. OTA and melatonin treatment OTA was dissolved in DMSO to 20 mM reserve solution and then was diluted in culture medium to produce a final concentration of 5 mM, 8 mM, 9 mM and 10 mM, respectively. The final concentration of DMSO was less than 0.1%. The collected COCs were incubated in a preheated medium and cultured in 38.5 C/5% CO2 incubator. After 27 h culture, oocytes developed to MI stage, and after 52 h of culture, oocytes developed to MII stage. According to the maturation of oocytes, the final concentration of OTA toxin in treatment group was 8 mM. Melatonin was dissolved in absolute ethanol to 0.1 M and then was diluted in 8 mM OTA contaminated culture medium to yield a final concentration of 1 109 M, 1 107 M, 1 105 M, 1 103 M. The final concentration of the ethanol was less than 0.1% of the culture medium.
2.5. Immunofluorescence staining and confocal microscopy Oocytes were immobilized in 4% paraformaldehyde for 30 min at room temperature. Then oocytes were permeabilized with 0.5% triton x-100 at room temperature for 4e6 h. Followed by blocking in 1% BSA-supplemented phosphate-buffered saline (PBS) for 1 h, oocytes were stained with different first antibodies (1:700 for a-tubulin; 5 mg/ml for Phalloidin-TRITC; 1:500 for LC3A) for 2 h at room temperature or 4 C overnight, respectively. After washing 3 times in PBS, the samples were then incubated with second antibody according to the species of the first antibody (1:500 for Alexa Fluor 488 goat anti-rabbit antibody; 1:500 for Alexa Fluor 594 goat anti-mouse antibody) at room temperature for 1 h. Oocytes were finally stained with Hoechst 33342 (10 mg/ml in PBS) for 10 min at room temperature, then were observed on sealing tablets examined with a laser scanning confocal microscope (Zeiss LSM 700 META; Jena, Germany).
Please cite this article as: Lan, M et al., Melatonin ameliorates ochratoxin A-induced oxidative stress and apoptosis in porcine oocytes, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113374
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2.6. Mitochondria detection by Mito-Tracker
2.10. RNA extraction and quantitative real time PCR
Mito-Tracker Red CMXRos (1:200) (Cat #M7512, Invitrogen, Eugene, OR, USA) was used to detect the mitochondria distribution in embryos which were incubated in M16 medium with at 37 C for 30 min. After the incubation, the embryos were washed 3 times with the M2 culture medium and then examined the live embryos with confocal laser-scanning microscope (Zeiss LSM 700 META, Germany).
A total of 60 oocytes after 27 h culture in different groups were collected, respectively. Total RNA was extracted from the oocytes using Qiagen RNeasy Mini Kit (Qiagen, Toronto, Canada), then it was reversed to cDNA and stored at 20 C until use. Each PCR reaction system is 20 mL, consisted of 10 mL of Advanced SYBR Green PCR Master Mix, 6.8 mL of water, 2 mL of cDNA sample, and 1.2 mL specific primers. Gene expression was determined using a One Step SYBR PrimeScript RT-PCR System (Roche Applied Science, Mannheim, Germany). And gene expression levels were analyzed using the 2△△Ct method after the melting-curve analysis was completed. The primers are listed in Table S1.
2.7. Reactive oxygen species (ROS) level detection Reactive oxygen species include superoxide radicals, hydrogen peroxide and its downstream products, peroxides and hydroxides, etc. Excessive oxidative stress often leads to cell senescence and apoptosis To analysis the levels of intracellular reactive oxygen species (ROS) in living oocyte, a Reactive Oxygen Species Assay Kit (DCFH-DA, Beyotime Institute of Biotechnology, China) was applied to detect ROS as green fluorescent signals, Denuded oocytes were incubated with 10 pM DCFH-DA (1:800) in DPBS containing 0.1% BSA for 30 min at 38.5 C. Then after two times washes in preheated DPBS, oocytes were placed on glass slides, The fluorescent signal in of each oocyte was examined immediately by the scanning settings (OLYMPUS CKX53, Japan). The fluorescence pixel intensities were analyzed using Image J software (version 1.50; National Institutes of Health, Bethesda, MD, USA).
2.11. Statistical analysis For each treatment, at least three biological replicates were performed. And for each group, no less than 30 oocytes were examined with results expression as means ± SEMs. Statistical analysis was performed using GraphPad Prism software (version 5.0, Graph Pad Prism software Inc., San Diego, CA). The homogeneity of variance was test by one-way ANOVA. If F statistics <0.05, then we used Games-Howell to compared the differences between groups. If F statistics >0.05, then Dunnett’s t-test will be applied. P < 0.05 is considered statistically significant. 3. Results
2.8. Annexin-V staining In normal cells, phosphatidylserine only distributes in the medial side of the membrane lipid bilayer, when cell apoptosis occurs, the membrane phosphatidylserine rolls from the inside of the lipid membrane to the outside. Annexin-V is a phospholipid binding protein with high affinity to phosphatidylserine. Therefore bind to the cell membrane of the early apoptotic cells. For AnnexinV staining, after washing twice in PBS, the viable oocytes were stained for 10 min in the dark with 90 pl of binding buffer containing 10 pl of Annexin-V-FITC (Vazyme Biotech Co., Ltd, Nanjing, China). Then after two times washes in DPBS, oocytes were moved to the glass slides. Fluorescent signal of oocytes was examined immediately by the scanning settings (Zeiss LSM 700 META; Jena, Germany). 2.9. Western blot analysis A total of 120 porcine oocytes in each group were collected (17 h, 27 h, respectively) and lysed in sample buffer (SDS sample buffer with 2-mercaptoethanol), and were boiled at 90 C for 10 min. The protein maker and the sample were added to the sample pore in sequence using SDS-polyacrylamide gel electrophoresis (PAGE). After electrophoretic separation, proteins were transferred onto a PVDF membrane (Millipore, Billerica, MA, USA). Membranes were blocked in 5% (w/v) BSA in Tris buffered saline (TBS) containing 0.1% (w/w) Tween 20 (TBST) for 1 h at room temperature. After a brief wash in TBST, the membrane was incubated with a rabbit monoclonal anti-p53 antibody (1:1000), anti-p-CDC2 antibody (1:1000), anti-p-MAPK-antibody (1:2000), anti-LC3B antibody (1:1000), anti-caspase3 antibody (1:1000), anti-GAPDH antibody (1:2000) at 4 C overnight. After washing three times in TBST (10 min each), membranes were then incubated for 1 h with conjugated goat antirabbit IgG (Santa Cruz, Texas, USA) antibodies (1:5000) in TBST. Then after washing five times in TBST (5 min each), membrane was exposed to an enhanced chemiluminescence reagent (EMD Millipore, Billerica, MA, USA), and protein bands were visualized by detection system (Tanon-3900, China).
3.1. Effects of melatonin on meiotic maturation in OTA-exposed oocytes We first examined the effects of OTA on porcine oocyte maturation, and we found that the cumulus granulosa cells in the OTA treatment group diffused to a reduced degree or exhibited no proliferation after being cultured for 52 h, and most oocytes failed to extrude their first polar bodies (Fig. 1A). In addition, as shown in Fig. 1B, the effects of OTA on oocyte maturation are dosedependent. In the control group, first polar body (PBI) discharge occurred in most oocytes (86.81 ± 2.42%, n ¼ 240), but this significantly decreased upon OTA treatment at 5 mM (72.57 ± 2.49%, n ¼ 220, p < 0.01), 8 mM (48.1 ± 1.64%, n ¼ 240, p < 0.001), 9 mM (30.57 ± 2.70%, n ¼ 220, p < 0.01), and 10 mM (15.37 ± 1.41%, n ¼ 220, p < 0.001). Melatonin was then added to examine its effects on oocyte maturation following OTA exposure. As shown in Fig. 1C, melatonin (107 M) significantly increased the proportion of cumulus dilatation and PBI in OTA-exposed oocytes (70.69 ± 1.06%, n ¼ 250, p < 0.01). Therefore, we used this concentration (107 M) for the following experiments. These results suggest that melatonin can protect oocyte meiotic maturation from OTA exposure. To explore the functional way of OTA toxicity on oocyte maturation, the difference in general gene expression after OTA treatment by RNA-seq was first analyzed. As shown in Fig. 2A, after 27 h of culture, transcriptome analysis found 1268 genes with significantly changed expression due to OTA treatment compared with the control group. Among these changed genes, 1120 (88.3%) were up-regulated, which was confirmed by the cluster analysis (Fig. 2B). The number of differentially expressed genes for each GO term, including relevant biological processes, cellular components, and molecular functions was then analyzed. The histogram showed that OTA mainly influenced the function of catalytic, cellular, and metabolic processes (Fig. 2C). The KEGG pathway functional enrichment database shown in Fig. 2D indicated that the differentially expressed genes (up-regulation) were mostly involved in important biochemical, metabolic, and signal transduction pathways. And the KEGG enrichment analysis also indicated that there
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Fig. 1. The effects of OTA exposure on gene expression and oocyte maturation. (A) Oocyte maturation after OTA treatment and melatonin (MT) supplement. (B) Rate of different concentrations of OTA treatment on the polar body extrusion in porcine oocytes. **, p < 0.01; ***, p < 0.001. (C) The rate of polar body extrusion after melatonin supplement.
are several genes down-regulated in oocytes after OTA exposure, which included the MAPK signaling pathway (Fig. 2E). However, it seems that melatonin protects oocytes from OTA-exposure from cellular level since the RNA-seq analysis indicated that the expression of only 156 genes was altered (Fig. S1A), and few overlap genes were found (4 genes) (Fig. S1B). The cluster analysis and gene KEGG enrichment results also confirm this (Figs. S1C and S1D). These results provided the general information about the functional pathway for the OTA toxicity on porcine oocyte quality. 3.2. Effects of melatonin on CDC2 phosphorylation and cell cycle progression in OTA-exposed oocytes We next explored how OTA affects oocyte maturation. Cluster analysis showed that several important cell cycle-related genes, such as Cdc6, Cdc25B, and Cdk7, were up-regulated, indicating that OTA may affect cell cycle progression in oocytes (Fig. 3A). Therefore, the proportion of oocytes in different meiosis stages following OTA treatment was investigated. As shown in Fig. 3B, after 27 h culture, oocytes treated with OTA were substantially in the GV (germinal vesicular) or GVBD (germinal vesicle breakdown) stages. The ratios were significantly higher than those of the control group: GV (2.5 ± 0.28%, n ¼ 100 vs 38.03 ± 1.43%, n ¼ 110, P < 0.01) and GVBD (9.38 ± 1.86%, n ¼ 100 vs 51.45 ± 4.26%, n ¼ 110, P < 0.05). A large proportion of oocytes in the control group (87.99 ± 2.43%, n ¼ 100) were at the metaphase I (MI) stage, whereas few oocytes in the OTA-treated group (10.51 ± 4.14%, n ¼ 110, P < 0.01) reached MI.
According to our results above, we chose 107 M melatonin for the following experiments due to its effects for the increased oocyte maturation under OTA exposure. Melatonin supplementation significantly alleviated the GV-GVBD block in OTA-exposed oocytes; there was a significant decrease in the percentage of GV (15.11 ± 6.17%, n ¼ 100) and GVBD (27.02 ± 3.13%, n ¼ 100) oocytes. We counted oocyte stage at 54 h. Similarly to the 27 h culture, there was still a large proportion of oocytes arrested at GV/GVBD in OTAtreated oocytes compared to the control group (1.33 ± 0.72%, n ¼ 100 vs 33.32 ± 0.54%, n ¼ 100, P < 0.01). The number of oocytes that developed to metaphase II (MII) in the OTA group was significantly lower than in the control group (83.26 ± 1.01%, n ¼ 100 vs 45.46 ± 5.32%, n ¼ 100, P < 0.01). Melatonin supplementation increased the percentage of MII oocytes significantly over OTAexposed oocytes (63.16 ± 3.40%, n ¼ 1000) (Fig. 3B). Next the mechanism of OTA exposure on oocyte cell cycle progression was explored. We checked p-CDC2 and p53 levels in different groups of oocytes. As shown in Fig. 3C, western blot results showed that p-CDC2 increased significantly in the OTA group compared to the control group (1, n ¼ 120 vs 1.45 ± 0.21, n ¼ 120, P < 0.05), and melatonin relieved this tendency (1.27 ± 0.13, n ¼ 120, p < 0.05) (Fig. 3D). While p53 levels significantly decreased in the OTA-exposed group compared to the control group (1, n ¼ 120 control vs 0.70 ± 0.04, n ¼ 120, P < 0.01), melatonin administration considerably increased p53 compared with the OTA-exposed group (0.95 ± 0.11, n ¼ 120, P < 0.05) (Fig. 3C and D). These results demonstrate that OTA exposure delays meiotic
Please cite this article as: Lan, M et al., Melatonin ameliorates ochratoxin A-induced oxidative stress and apoptosis in porcine oocytes, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113374
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Fig. 2. Transcriptome analysis for the effects of OTA exposure on oocyte maturation. (A) The number of differentially expressed genes and volcano plot analysis after OTA treatment. (B) The cluster analysis for the differentially expressed genes after OTA treatment. (C) The differential gene GO enrichment analysis for the differentially expressed genes after OTA treatment. (D) The rich factor by the up-regulation gene KEGG enrichment analysis for the differentially expressed genes after OTA treatment. (E) The rich factor by the down-regulation gene KEGG enrichment analysis for the differentially expressed genes after OTA treatment.
Please cite this article as: Lan, M et al., Melatonin ameliorates ochratoxin A-induced oxidative stress and apoptosis in porcine oocytes, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113374
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Fig. 3. The effects of melatonin on the cell cycle progression in the OTA-exposed oocytes. (A) The cluster analysis for the differentially expressed genes related with cell cycle after OTA treatment. (B) The cell cycle progression after treatment with OTA or melatonin supplement after culture for 27 h and 54 h, the time points when most oocytes reached MI stage and MII stage. *, p < 0.05; **, p < 0.01. (C) Western blot result of the protein expression of phosphorylated CDC2 and p53 after OTA treatment and melatonin supplement. (D) The band intensity analysis for the phosphorylated CDC2 and p53 after OTA treatment and melatonin supplement. *, p < 0.05; **, p < 0.01.
progression via arresting the G2 to M transition, while melatonin promotes cell cycle progression in OTA-exposed oocytes.
3.3. Effects of melatonin on spindle morphology in OTA-exposed oocytes To explore how OTA disturbs oocyte maturation, the subcellular spindle formation after 27 h of culture was then examined, when most oocytes are at MI, using confocal microscopy. As shown in Fig. 4A, most oocytes in the control group displayed typical barrel spindles and well-arranged chromosomes on the intermediate plate, while in the OTA treatment group, spindle morphology was completely destroyed and the chromosomes showed severe misalignment. Statistical analysis showed that the proportion of oocytes with abnormal spindle shape in OTA-treated cells was significantly higher than that of the control group (4.81 ± 0.92%, n ¼ 90 vs. 25.82 ± 1.89%, n ¼ 90, P < 0.01) (Fig. 4B). Melatonin supplementation rescued the spindle defects caused by OTA, and the proportion of damaged spindles was notably decreased (16.51 ± 1.48%, n ¼ 90, P < 0.05) (Fig. 4A and B). We then examined the level of p-MAPK, the key molecule in MAPK pathway which is associated with microtubule organization was examined. These results showed that p-MAPK was significantly decreased in the OTA group compared to controls (1.00 vs 0.44 ± 0.04, n ¼ 120, P < 0.01). Melatonin ameliorated the effects of OTA exposure, with the pMAPK protein level increasing to 0.85 ± 0.07 compared with the OTA treatment group (P < 0.05) (Fig. 4C and D).
3.4. Effects of melatonin on mitochondria function and oxidative stress in OTA-exposed oocytes We then explored the possible mechanism of OTA toxicity on porcine oocytes. The cluster analysis from RNA-seq results indicated that several important genes related to energy metabolism were up-regulated, indicating that OTA might affect mitochondria function (Fig. 5A). Enrichment of the KEGG pathway analysis for oxidative phosphorylation showed that genes related with NADH dehydrogenase, Cytochrome c reductase, Cytochrome c oxidase, Ftype ATPase, and V-type ATPase were altered (Fig. S2A). Moreover, TCA cycle-related genes and Complex-I (CxI), III, IV and V, which are related to mitochondria function, were also altered, further confirming the effects of OTA exposure on oocyte mitochondria-related gene expression (Figs. S2B and S2C). Mito-Tracker was utilized to track the distribution of mitochondria in oocytes. As shown in Fig. 5B, in the control oocytes, the mitochondria are distributed uniformly in the cytoplasm, while OTA treatment caused the mitochondria to cluster. In the melatonin-supplemented oocyte group, mitochondria distribution was normal, similar with the control oocytes. Oxidative stress is considered an important factor in mycotoxin toxicity, while melatonin is known to be involved in redox homeostasis. Because mitochondria dysfunction generally induces oxidative stress, we next explored ROS levels after 27 h of culture to find whether melatonin could alleviate oxidative stress in OTAexposed oocytes. As predicted, OTA induced ROS generation compared to control groups (5.44 ± 0.80, n ¼ 30, vs 30.68 ± 1.29,
Please cite this article as: Lan, M et al., Melatonin ameliorates ochratoxin A-induced oxidative stress and apoptosis in porcine oocytes, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113374
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Fig. 4. The effects of melatonin on the spindle organization in the OTA-exposed oocytes. (A) The spindle morphology after OTA treatment and melatonin supplement. Green, atubulin; blue, chromatin. Bar ¼ 5 mm. (B) The rate of abnormal spindle after OTA treatment and melatonin supplement. (C) The protein expression of p-MAPK after OTA treatment and melatonin supplement. (D) The band intensity analysis for p-MAPK after OTA treatment and melatonin supplement. *, p < 0.05; **, p < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
n ¼ 32, P < 0.001); however, melatonin administration significantly decreased ROS production (18.04 ± 0.75, P < 0.05) (Fig. 5C). The expression of genes associated with oxidative stress was evaluated, and, as shown in Fig. 5D, SOD1 (1.00 vs 1.53 ± 0.13, P < 0.05 vs 0.99 ± 0.10) and SOD2 (1.00 vs 1.45 ± 0.05, P < 0.05 vs 1.12 ± 0.13) were significantly increased in oocytes exposed to OTA compared with controls and melatonin groups. We also found that compared with control and OTA groups, melatonin improved the expression of GPX (1.00 vs 0.80 ± 0.04 vs 1.29 ± 0.08, P < 0.05) and CAT (1.00 vs 0.80 ± 0.08, vs 1.44 ± 0.08, P < 0.05). These results indicate that melatonin protects against mitochondria dysfunction and the resulting oxidative stress in oocytes exposed to OTA. 3.5. Effects of melatonin on apoptosis and autophagy levels in OTAexposed oocytes The cluster analysis from RNA-seq results also indicated that several important apoptosis-related genes were up-regulated (Fig. 6A); moreover, the analysis from the enrichment of the KEGG pathway showed that DNA replication-related gene expression was altered (Fig. S3A). Since DNA damage induces apoptosis, these transcriptome analysis results indicate that OTA might cause
apoptosis in oocytes. The early apoptosis in different groups of oocytes was detected by Annexin-V staining. As shown in Fig. 6B, the green ring of the external cellular membrane was Annexin-V positive. In the OTA-treatment group, the percentage of AnnexinV positive oocytes was significantly higher than that of the control group (7.63 ± 1.35%, n ¼ 40 vs 27.31 ± 2.03%, n ¼ 40, P < 0.05) while melatonin blocked the early apoptosis of oocytes induced by OTA, reducing apoptosis to 12.25 ± 1.71% (n ¼ 40, P < 0.05) (Fig. 6B). Excessive oxidative stress often leads to apoptosis, which further induces protective autophagy. Analysis from KEGG pathway enrichment showed that proteasome-related gene expression was altered (Fig. S3B). Because ubiquitination is important for autophagy, LC3, a marker of autophagy in oocytes was then stained. As shown in Fig. 6C, OTA stimulated more autophagy vacuoles in oocytes and markedly increased the autophagy rate compared to the control group, showing with stronger fluorescence intensity (6.72 ± 0.68%, n ¼ 60 vs 44.77 ± 4.33% n ¼ 60, P < 0.01). Melatonin supplementation yielded fewer autophagy oocytes, showing with similar fluorescence intensity compared with the control group (9.45 ± 1.76%, n ¼ 60, P < 0.01) (Fig. 6C). Caspase3 and LC3 protein expression were then examined, and the results showed a similar tendency via fluorescence staining (Fig. 6D). OTA significantly
Please cite this article as: Lan, M et al., Melatonin ameliorates ochratoxin A-induced oxidative stress and apoptosis in porcine oocytes, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113374
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Fig. 5. The effects of melatonin on the mitochondria distribution and ROS level in the OTA-exposed oocytes. (A) The cluster analysis for the differentially expressed genes related with oxidative phosphorylation after OTA treatment. (B) The mitochondria distribution pattern after OTA treatment or melatonin supplement. Red, Mito-tracker; blue, DNA. Bar ¼ 20 mm. (C) The ROS generation after OTA treatment or melatonin supplement. Green, ROS. Bar ¼ 20 mm. The fluorescence intensity analysis for the ROS signal was confirmed this. (D) The relative mRNA expression of oxidative stress-related genes after OTA treatment or melatonin supplement. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
increased the levels of Caspase3 (1, n ¼ 120 vs 1.36 ± 0.09, n ¼ 120, P < 0.01) and LC3B (1, n ¼ 120 vs 2.33 ± 0.29, n ¼ 120, P < 0.01); while melatonin reduced expression of Caspase3 (0.88 ± 0.05, n ¼ 120) and LC3B (1.34 ± 0.14, n ¼ 120) (Fig. 6E). The expression of genes associated with apoptosis and autophagy was then evaluated. As shown in Fig. 6F, compared with the OTA group, after melatonin administration, the expression of anti-apoptotic gene Bcl-2 increased markedly (1 vs 1.10 ± 0.10 vs 1.80 ± 0.23, P < 0.05), and the expression of pro-apoptotic gene Bax decreased (1 vs 1.59 ± 0.11 vs 0.80 ± 0.11, P < 0.05). Melatonin ameliorated autophagy-related gene expression in OTA-exposed oocytes, as shown with Atg7 (1 vs 1.54 ± 0.15 vs 1.17 ± 0.15, P < 0.05) and Lc3 (1 vs 1.54 ± 0.24 vs 1.13 ± 0.11, P < 0.05). These results indicate the protective role of melatonin on OTA-induced apoptosis and autophagy.
4. Discussion As a widespread toxin in nature, OTA has been gaining attention for its toxic effects on the animal reproductive system. In this study, we used transcriptome analysis to screen the potential mRNA expression level changes, and then elucidated the cellular mechanism of OTA toxicity on porcine oocytes. Finally, we propose an innovative solution to mitigate OTA toxicity and report that melatonin can reduce the oxidative stress induced by OTA, thereby reducing the abnormal cytoskeleton dynamics, autophagy, and apoptosis caused by oxidative stress, restore cell cycle progression, and ultimately improve porcine oocyte quality (Fig. 7). RNA-seq was first used to show altered expression of more than 1000 genes in oocytes after OTA exposure, indicating that OTA widely affects transcription and translation in the oocytes.
Please cite this article as: Lan, M et al., Melatonin ameliorates ochratoxin A-induced oxidative stress and apoptosis in porcine oocytes, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113374
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Fig. 6. The effects of melatonin on the apoptosis and autophagy in the OTA-exposed oocytes. (A) The cluster analysis for the differentially expressed genes related with apoptosis after OTA treatment. (B) The early apoptosis examined by Annexin-V staining after OTA treatment or melatonin supplement. Green, Annexin-V. Bar ¼ 20 mm. The percentage of early apoptosis also confirmed this. *, p < 0.05. (C) The autophagy level after OTA treatment or melatonin supplement. Green, LC3; blue, DNA. Bar ¼ 20 mm. The rate of autophagy analysis also confirmed this. **, p < 0.01. (D) The protein expression of Caspase 3 and LC3 after OTA treatment or melatonin supplement. (E) The relative band intensity analysis for the Caspase 3 and LC3 after OTA treatment or melatonin supplement. *, p < 0.05. (F) The relative mRNA expression of Bcl-2, Bax, Atg7 and Lc3 after OTA treatment or melatonin supplement. *, p < 0.05; **, p < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Amplification of cumulus cells and polar body extrusion are two important markers for oocyte maturation (Rosa et al., 2018), and the results show that cumulus granulosa cell diffusion/proliferation was disturbed, together with the failure of polar body extrusion, these indicated that OTA is toxic to oocyte maturation. We also demonstrated that melatonin could alleviate the meiotic maturation defects caused by OTA, and interestingly, its effects are not through changes in mRNA levels, since only about 100 genes were altered, which is far lower than the changes from OTA exposure. This indicates that melatonin, a pineal gland hormone, may not act through RNA. We next explored the functional mechanism of OTA toxicity on oocytes. From deep analysis of RNA-seq results, cell cycle-related
genes are a large proportion of the genes altered following OTA exposure. Comparing the proportions of oocytes at different stages after 27 h and 54 h indicated that OTA may delay meiotic progression via arresting the G2 to M transition. CDC2, an evolutionarily conserved serine/threonine-specific protein kinase, is essential in the transition from G2 to M, and it is negatively regulated by phosphorylation of two residues located in its ATP-binding site (Pandey and Giri, 2018). p53 protein, a transcription factor that acts as a checkpoint in the cell cycle, examines sites of DNA damage in G1 to monitor genomic integrity. If damage occurs, p53 blocks DNA replication to provide sufficient time for DNA repair (Kannappan et al., 2018). The transcriptome analysis results show that DNA duplication-related genes were altered. Upregulation of
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Fig. 7. Diagram of the effects of melatonin on OTA-exposed oocytes.
p-CDC2 and downregulation of p-53 was found following OTA exposure, indicating that OTA blocks cell cycle progression during oocyte maturation. Similar results were also reported for other toxic environmental chemicals such as ethylhexyl phthalate (Zhang et al., 2018), benzoapyrene (Miao et al., 2018), and zearalenone (Sambuu et al., 2011), which all caused maturation blocks in oocytes. Melatonin administration restored cell cycle progression, indicating that melatonin is a potential candidate to improve the oocyte maturation in OTA-exposed oocytes. During the cell cycle, the dynamics of the cytoskeleton play major roles, such as chromosome congression/segregation and cytokinesis, and proper spindle formation is important for this (Zhu et al., 2018). Our results show that OTA negatively affects spindle organization and chromosome alignment, and melatonin protects the oocytes from these spindle defects and chromosome misalignments. OTA may affect spindle formation through the MAPK signaling pathway, since from transcriptome analysis it was a main pathway affected by OTA exposure. Western blot results were consistent with the RNA-seq analysis, showing that phosphorylated MAPK decreased after OTA treatment increased after melatonin administration. MAPK was shown to play a key role in facilitating and stabilizing the spindle pole in oocyte meiotic processes (Zhu et al., 2018). These results are consistent with our previous studies which showed that melatonin might play a pivotal role in cytoskeletal modulation (Lan et al., 2018; Sripathi et al., 2017). Therefore, these results indicate that melatonin protects oocytes from OTA exposure through its effects on the cell cycle and spindle formation. We then tried to find out the mechanism for the toxic effects of OTA on oocytes. In addition to cell cycle-related genes, the results showed that a large proportion of oxidative phosphorylation and TCA cycle-related genes were altered, which are all related to mitochondria dysfunction. Mito-tracker staining confirmed this result, showing aberrant mitochondria distribution. Aberrant mitochondria distribution could lead to mitochondria dysfunction. For example, in the MII oocytes of diabetic mice, mitochondria showed clustering distribution or homogeneous distribution, which are all abnormal (Wang et al., 2009). Mitochondria dysfunction generally induces oxidative stress, which has the capacity to cause a decline in levels of critical cell cycle factors such as maturation-promoting factor (MPF). Several studies have
confirmed that oxidative stress is induced by mycotoxins, such as aflatoxin (Hou et al., 2013), zearalenone (Qin et al., 2015) and deoxynivalenol (Waskiewicz et al., 2014). We therefore hypothesized that superfluous oxidative stress was the reason for cell cycle delay and abnormal cytoskeletal dynamics in OTA-exposed oocytes. Our results showed that OTA induces ROS generation, and oxidative stress-related genes, such as SOD1 and SOD2, also exhibited altered RNA expression, indicating strong oxidative stress occurs in the cytoplasm and mitochondria. On the other hand, several studies have indicated a relationship between melatonin and mitochondria, since it was found that melatonin membrane receptors are present in mitochondria (Tan and Reiter, 2019). While melatonin supplementation depressed ROS generation, GPX and CAT mRNA levels were markedly increased, which suggests that melatonin can not only react directly with ROS, but also enhance the production of glutathione and catalase, thus further reducing intracellular ROS levels in OTA-exposed oocytes. This result is similar to previous research showing that melatonin pre-treatment could protect golden hamster germ cell from dexamethasone-induced oxidative stress (Tanabe et al., 2015), and a study which showed that melatonin also restored glutathione expression levels in rat pups exposed to plumbum (Bazrgar et al., 2015). Therefore, these results indicate that melatonin protects oocytes from OTA exposure through its effects on mitochondria dysfunction-induced oxidative stress. Oxidative stress generally leads to apoptosis, while autophagy reduces apoptosis through ubiquitination. Apoptosis and autophagy are generally activated by adverse environmental factors such as nutrient deprivation (Pang et al., 2014), infection (Hacker, 2014), radiation (Alan Mitteer et al., 2015), and toxic chemical substances (Liu et al., 2015). Similarly, after OTA treatment, apoptosis and ubiquitination-related gene expression were changed in our results. The intensive early apoptosis signals and elevated numbers of autophagy vacuoles were detected in OTAexposed oocytes, and the results showed that Caspase 3 and LC3 protein expression decreased while apoptosis-related gene Bcl-2 and Bax were also altered. Bcl-2 exerts an anti-apoptotic function through inhibition of mitochondrial cytochrome c release (Murphy et al., 2000). Bax is a key component for cellular induced apoptosis through mitochondrial stress, which increases the membrane’s permeability, leads to the release of cytochrome c from mitochondria, and activates the caspase activation pathway for apoptosis (Narita et al., 1998). These results demonstrate that melatonin can inhibit Caspase3-mediated apoptosis via upregulating Bcl-2 and down-regulating Bax. This indicates that OTA exposure caused mitochondria dysfunction and induced apoptosis/autophagy. As expected, melatonin could inhibit apoptosis and autophagy remarkably. We next tested autophagyrelated genes Atg7 and Lc3. Atg7 and Lc3 were originally identified as critical proteins for autophagy (Codogno and Meijer, 2005). Similar to the trend of reducing apoptosis, melatonin could inhibit autophagy via down-regulating Atg8 and Lc3 gene expression. This effect of melatonin modulating apoptosis and autophagy has been proposed in other models. For example, melatonin attenuated MPTP-induced neurotoxicity via preventing CDK5-mediated autophagy and SNCA/a-synuclein aggregation (Su et al., 2015). Melatonin ameliorated apoptosis and autophagy in liver cancer cells (Ordonez et al., 2015), as well as mouse smooth muscle actinpositive cells exposed to carbon tetrachloride-induced fibrosis (San-Miguel et al., 2015). These results demonstrate the superiority of melatonin for decreasing apoptosis and autophagy. 5. Conclusions In summary, our results demonstrate that OTA exposure causes
Please cite this article as: Lan, M et al., Melatonin ameliorates ochratoxin A-induced oxidative stress and apoptosis in porcine oocytes, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113374
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mitochondria dysfunction and induces oxidative stress-related apoptosis/autophagy, which further affects spindle formation and cell cycle progression in porcine oocytes. Melatonin, due to its antioxidant properties, is a promising pharmacological agent for its protective functions against OTA toxicity. Contributions ML and SCS designed the study. ML performed the majority of the experiments. YZ, XW, MHP and YX contributed to the regents and materials. ML and SCS analyzed the data. ML and SCS wrote the manuscript. Funding This work was supported by the National Key Research and Development Program (2018YFC1003802), China; National Natural Science Foundation of China (31622055, 31571547); the Fundamental Research Funds for the Central Universities (KYTZ201602, KJYQ201701), China. Declaration of competing interest There is no conflict of interest to declare. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.envpol.2019.113374. References Alan Mitteer, R., Wang, Y., Shah, J., Gordon, S., Fager, M., Butter, P.P., Jun Kim, H., Guardiola-Salmeron, C., Carabe-Fernandez, A., Fan, Y., 2015. Proton beam radiation induces DNA damage and cell apoptosis in glioma stem cells through reactive oxygen species. Sci. Rep. 5, 13961. Bazrgar, M., Goudarzi, I., Lashkarbolouki, T., Elahdadi Salmani, M., 2015. Melatonin ameliorates oxidative damage induced by maternal lead exposure in rat pups. Physiol. Behav. 151, 178e188. Bhat, P.V., Anand, T., Mohan, M.T., Khanum, F., 2018. Restorative effect of l-Dopa treatment against Ochratoxin A induced neurotoxicity. Neurochem. Int. 118, 252e263. Brennan, K.M., Oh, S.Y., Yiannikouris, A., Graugnard, D.E., Karrow, N.A., 2017. Differential gene expression analysis of bovine macrophages after exposure to the penicillium mycotoxins citrinin and/or ochratoxin A. Toxins (Basel) 9, e366. Codogno, P., Meijer, A.J., 2005. Autophagy and signaling: their role in cell survival and cell death. Cell Death Differ. 12 (Suppl. 2), 1509e1518. Eze, U.A., Huntriss, J., Routledge, M.N., Gong, Y.Y., 2018. Toxicological effects of regulated mycotoxins and persistent organochloride pesticides: in vitro cytotoxic assessment of single and defined mixtures on MA-10 murine Leydig cell line. Toxicol. In Vitro 48, 93e103. Hacker, G., 2014. ER-stress and apoptosis: molecular mechanisms and potential relevance in infection. Microb. Infect. 16, 805e810. Hou, Y.J., Zhao, Y.Y., Xiong, B., Cui, X.S., Kim, N.H., Xu, Y.X., Sun, S.C., 2013. Mycotoxin-containing diet causes oxidative stress in the mouse. PLoS One 8, e60374. Hsuuw, Y.D., Chan, W.H., Yu, J.S., 2013. Ochratoxin a inhibits mouse embryonic development by activating a mitochondrion-dependent apoptotic signaling pathway. Int. J. Mol. Sci. 14, 935e953. Huang, C.H., Chan, W.H., 2017. Protective effects of liquiritigenin against citrinintriggered, oxidative-stress-mediated apoptosis and disruption of embryonic development in mouse blastocysts. Int. J. Mol. Sci. 18, e2538. Huang, F.J., Chan, W.H., 2016. Effects of ochratoxin a on mouse oocyte maturation and fertilization, and apoptosis during fetal development. Environ. Toxicol. 31, 724e735. Kannappan, R., Mattapally, S., Wagle, P.A., Zhang, J., 2018. Transactivation domain of p53 regulates DNA repair and integrity in human iPS cells. Am. J. Physiol. Heart Circ. Physiol. 315, h512eh521. Lan, M., Han, J., Pan, M.H., Wan, X., Pan, Z.N., Sun, S.C., 2018. Melatonin protects against defects induced by deoxynivalenol during mouse oocyte maturation. J. Pineal Res., e12477 Liu, F., Du, K.J., Fang, Z., You, Y., Wen, G.B., Lin, Y.W., 2015. Chemical and biological insights into uranium-induced apoptosis of rat hepatic cell line. Radiat. Environ. Biophys. 54, 207e216. Liu, G., Jiang, Q., Chen, S., Fang, J., Ren, W., Yin, J., Yao, K., Yin, Y., 2017. Melatonin alters amino acid metabolism and inflammatory responses in colitis mice.
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Melatonin ameliorates ochratoxin A-induced oxidative stress and apoptosis in porcine oocytes* Mei Lan, Yu Zhang, Xiang Wan, Meng-Hao Pan, Yao Xu, Shao-Chen Sun* College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 August 2019 Received in revised form 8 October 2019 Accepted 9 October 2019 Available online xxx
Melatonin is a hormone which is generated from pineal gland, and it is responsible for the regulation of wake-sleep cycle. Melatonin is a well-known antioxidant and free radical scavenger to protect against multiple type of tissue damage. While ochratoxin A (OTA) is a mycotoxin found widely in contaminated food and foodstuffs, which causes nephrotoxicity, hepatotoxicity, immunotoxicity, and reproductive damage in humans and animals. In present study we report the toxicity of OTA on porcine oocyte quality and the protective effects of melatonin on OTA-exposed oocytes. Using transcriptome analysis our results show that OTA exposure alters the expression of multiple genes in oocytes, indicating its effect on oocyte maturation. The cellular changes following OTA treatment are examined, and the results show that OTA adversely affects oocyte polar body extrusion, which is confirmed by the delay of Cdc2-mediated cell cycle progression. OTA exposure also disrupts meiotic spindle formation, which is confirmed by altered phosphorylated MAPK expression. RNA-seq screening and further fluorescence staining results show that OTA induces aberrant mitochondria distribution and oxidative phosphorylation defects, which then causes oxidative stress, followed by early apoptosis and autophagy. Treatment with melatonin significantly ameliorates oxidative stress and apoptosis, which further protects cell cycle and spindle formation in OTA-exposed oocytes. Collectively, these results show the protective effects of melatonin against defects induced by OTA during porcine meiotic oocyte maturation. © 2019 Elsevier Ltd. All rights reserved.
Keywords: Ochratoxin A Melatonin Apoptosis Oxidative stress Oocyte
1. Introduction Ochratoxin A (OTA), a mycotoxin produced by Aspergillus spp, is one of the most commonly detected mycotoxins in food and foodstuffs such as coffee beans, fermented tea, and cereals (Brennan et al., 2017). Besides causing losses in the livestock economy, OTA bioaccumulation through the food chain results in immunomodulatory, hepatotoxic, nephrotoxic, genotoxic, and reproductive damage in animals and humans (Bhat et al., 2018; Malir et al., 2013). Previous studies showed that exposure to mycotoxins activates endocrine disrupted chemical (EDC) secretion, which results in functional decline of reproductive organs, including sub-fertility or infertility (Eze et al., 2018). The presence of OTA in drinking water or intravenous injection of OTA decreases oocyte fertilization rates and has deleterious effects on mouse early embryonic development (Huang and Chan, 2016). OTA exposure
* This paper has been recommended for acceptance by Wen Chen. * Corresponding author. E-mail address: [email protected] (S.-C. Sun).
potentially affects mouse sperm motility via the AMPK and PTEN signaling pathways in vitro (Lu et al., 2018). Studies have been carried out for decades to elucidate the mechanism of mycotoxin toxicities. Mycotoxins such as HT-2, citrinin, or deoxynivalenol may cause excess oxidative stress in cells, and when the reactive oxygen species (ROS) reach a maximum resistance threshold, it will result in organelle damage or even cell death (Huang and Chan, 2017; Lan et al., 2018; Zhang et al., 2017). Oxidative stress also plays critical roles in the toxicity of OTA, since it has been reported that OTA affects anti-oxidant self-defense in the gut, kidney (Marin et al., 2017), and embryo (Hsuuw et al., 2013). Our previous study provides preliminary data which shows that OTA affects oocyte maturation, and porcine oocytes is more sensitive than mouse oocytes (Lu et al., 2018). However, the causes and mechanism of OTA toxicity on porcine oocyte quality are still unclear. Moreover, there is still no effective approach to reduce the OTA toxicity in vivo. Melatonin, a well-known antioxidant and free radical scavenger, has been reported to protect against multiple type of tissue damage (Tan et al., 1993). Melatonin is generated from pineal gland and is the in vivo hormone responsible for the regulation of wakefulness.
https://doi.org/10.1016/j.envpol.2019.113374 0269-7491/© 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Lan, M et al., Melatonin ameliorates ochratoxin A-induced oxidative stress and apoptosis in porcine oocytes, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113374
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Field studies have shown that melatonin can protect the heart from reperfusion injury by inhibiting the mitochondrial permeability transition pore (MPTP) opening and cardiolipin peroxidation (Petrosillo et al., 2009). Melatonin treatment may function as a potential adjuvant therapy to alleviate inflammatory bowel disease (Liu et al., 2017). And melatonin works against cancer cell proliferation, motility, and invasiveness by acting on the members of MAPK family (Mortezaee, 2018). Moreover, studies have revealed that melatonin plays an important role in the regulation of the reproductive system via reducing oxidative stress (Lan et al., 2018) and modifying follicle and ovary steroidogenesis (Tamura et al., 1998). In RFR (radiofrequency radiation)-exposed mice, melatonin inhibits pre-meiotic spermatogenesis arrest in male germ cells through its anti-oxidative potential and ability to improve DNA repair pathways, leading to normal sperm count and morphology (Pandey and Giri, 2018). Our previous study also showed that in di2-ethylhexyl phthalate (DEHP)-exposed porcine oocytes, melatonin supplementation ameliorated oocyte maturation through its rescue of oxidative stress-mediated apoptosis and autophagy (Zhang et al., 2018). In this study, based on transcriptome analysis, we show the expression of several cellular process-related genes were altered after OTA exposure in porcine oocytes, which are confirmed from cellular analyses, showing cell cycle delay, disruption of the cytoskeleton and mitochondria distribution, and oxidative stress and apoptosis. These results illustrate the toxic effects and possible mechanism of OTA on porcine oocytes and also show that melatonin administration is effective for improving maturation of OTAexposed oocytes.
glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 50 mg/ml of streptomycin, 75 mg/ml of penicillin, 0.5 mg/ml of LH, 0.5 mg/ml of FSH, 10 ng/ml of epidermal growth factor (EGF) and 10% pFF]. Then selected COCs were cultured with different treatment in a four-well dish (Nunc, Roskilde, Denmark) containing 500 ml of IVM medium covered with 200 ml mineral oil at 38.5 C in a humidified atmosphere of 5% CO2 incubator for IVM. After a certain time of culture, COCs were treated with 0.02% (w/v) hyaluronidase at 37 C for 5 min. Gently blow off the surrounding cumulus cells. After 3e4 times of rinsing, the exposed oocytes were collected for subsequent analysis.
2. Materials and methods
2.4. RNA-seq (transcriptome sequencing)
2.1. Antibodies and chemicals
Transcriptome sequencing is based on the Illumina Hiseq sequencing platform to study all mRNA transcribed by cells at a certain time. After cultured for 27 h, the total RNA was extracted from the porcine oocytes of control group and OTA group, respectively. The quality of RNA was detected by using Agilent bioanalyzer 2100. RNA-seq transcriptome library was prepared following NEB Next Ultra RNA library Prep Kit for Illumina. After mixing libraries based on their effective concentration and the required sequencing data volume, Illumina HiSeq platform is used for high through-put sequencing and biological information analyzing. The original image data were analyzed using Bcl2fastq (v2.17.1.14) for base calling and preliminary quality analysis. Then functional-enrichment analysis including Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) pathways were compared with the whole-transcriptome background.
Melatonin was purchased from Sigma Chemical Company (M5250, St. Louis, MO, USA). Ochratoxin A was purchased from J&K Scientific chemical company (356520, Beijing, China). Mouse monoclonal anti-a-tubulin-FITC antibody (F2168), phalloidin-TRITC (P1951) and Hoechst 33342 (B2261) were purchased from Sigma (St. Louis, MO, USA). Rabbit monoclonal anti-microtubuleassociated protein 1 light chain 3 (LC3A) antibody was purchased from Abcam (ab128025, Cambridge, UK). Alexa Fluor 594 goat antimouse antibody, Alexa Fluor 488 and 594 goat anti-rabbit antibodies were from Invitrogen (Carlsbad, CA, USA). Rabbit monoclonal anti-p-MAPK antibody (4370), rabbit monoclonal anti-pcdc2 antibody (28439), rabbit monoclonal anti-p-53 antibody (2524), rabbit monoclonal anti-caspase3 antibody (9662), rabbit monoclonal anti-LC3B antibody (3868) and rabbit anti-GAPDH antibody (5174) were purchased from Cell Signaling Technology (Devers, MA, USA). Basic maturation culture medium was tissue culture medium (TCM-199) (St. Louis, MO, USA). 2.2. COCs collection and culture All experiments were approved by the Animal Care and Use Committee of Nanjing Agriculture University and were performed in accordance with Animal Research Institute Committee guidelines (SYXK-Su-2017-0027). Pig ovaries obtained from slaughterhouses were transported to the laboratory within 2 h in aseptic saline containing 800IU/ml gentamicin at 25e30 C. After washing with aseptic phosphate buffer saline twice, the cumulus oocyte complex was sucked out of the follicles (3e6 mm) with 20-gauge needles. Oocytes with intact and compact cumulus mass were selected and washed three times with in vitro maturation medium [supplemented with 0.1% polyvinyl alcohol (PVA), 3.05 mM D-
2.3. OTA and melatonin treatment OTA was dissolved in DMSO to 20 mM reserve solution and then was diluted in culture medium to produce a final concentration of 5 mM, 8 mM, 9 mM and 10 mM, respectively. The final concentration of DMSO was less than 0.1%. The collected COCs were incubated in a preheated medium and cultured in 38.5 C/5% CO2 incubator. After 27 h culture, oocytes developed to MI stage, and after 52 h of culture, oocytes developed to MII stage. According to the maturation of oocytes, the final concentration of OTA toxin in treatment group was 8 mM. Melatonin was dissolved in absolute ethanol to 0.1 M and then was diluted in 8 mM OTA contaminated culture medium to yield a final concentration of 1 109 M, 1 107 M, 1 105 M, 1 103 M. The final concentration of the ethanol was less than 0.1% of the culture medium.
2.5. Immunofluorescence staining and confocal microscopy Oocytes were immobilized in 4% paraformaldehyde for 30 min at room temperature. Then oocytes were permeabilized with 0.5% triton x-100 at room temperature for 4e6 h. Followed by blocking in 1% BSA-supplemented phosphate-buffered saline (PBS) for 1 h, oocytes were stained with different first antibodies (1:700 for a-tubulin; 5 mg/ml for Phalloidin-TRITC; 1:500 for LC3A) for 2 h at room temperature or 4 C overnight, respectively. After washing 3 times in PBS, the samples were then incubated with second antibody according to the species of the first antibody (1:500 for Alexa Fluor 488 goat anti-rabbit antibody; 1:500 for Alexa Fluor 594 goat anti-mouse antibody) at room temperature for 1 h. Oocytes were finally stained with Hoechst 33342 (10 mg/ml in PBS) for 10 min at room temperature, then were observed on sealing tablets examined with a laser scanning confocal microscope (Zeiss LSM 700 META; Jena, Germany).
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2.6. Mitochondria detection by Mito-Tracker
2.10. RNA extraction and quantitative real time PCR
Mito-Tracker Red CMXRos (1:200) (Cat #M7512, Invitrogen, Eugene, OR, USA) was used to detect the mitochondria distribution in embryos which were incubated in M16 medium with at 37 C for 30 min. After the incubation, the embryos were washed 3 times with the M2 culture medium and then examined the live embryos with confocal laser-scanning microscope (Zeiss LSM 700 META, Germany).
A total of 60 oocytes after 27 h culture in different groups were collected, respectively. Total RNA was extracted from the oocytes using Qiagen RNeasy Mini Kit (Qiagen, Toronto, Canada), then it was reversed to cDNA and stored at 20 C until use. Each PCR reaction system is 20 mL, consisted of 10 mL of Advanced SYBR Green PCR Master Mix, 6.8 mL of water, 2 mL of cDNA sample, and 1.2 mL specific primers. Gene expression was determined using a One Step SYBR PrimeScript RT-PCR System (Roche Applied Science, Mannheim, Germany). And gene expression levels were analyzed using the 2△△Ct method after the melting-curve analysis was completed. The primers are listed in Table S1.
2.7. Reactive oxygen species (ROS) level detection Reactive oxygen species include superoxide radicals, hydrogen peroxide and its downstream products, peroxides and hydroxides, etc. Excessive oxidative stress often leads to cell senescence and apoptosis To analysis the levels of intracellular reactive oxygen species (ROS) in living oocyte, a Reactive Oxygen Species Assay Kit (DCFH-DA, Beyotime Institute of Biotechnology, China) was applied to detect ROS as green fluorescent signals, Denuded oocytes were incubated with 10 pM DCFH-DA (1:800) in DPBS containing 0.1% BSA for 30 min at 38.5 C. Then after two times washes in preheated DPBS, oocytes were placed on glass slides, The fluorescent signal in of each oocyte was examined immediately by the scanning settings (OLYMPUS CKX53, Japan). The fluorescence pixel intensities were analyzed using Image J software (version 1.50; National Institutes of Health, Bethesda, MD, USA).
2.11. Statistical analysis For each treatment, at least three biological replicates were performed. And for each group, no less than 30 oocytes were examined with results expression as means ± SEMs. Statistical analysis was performed using GraphPad Prism software (version 5.0, Graph Pad Prism software Inc., San Diego, CA). The homogeneity of variance was test by one-way ANOVA. If F statistics <0.05, then we used Games-Howell to compared the differences between groups. If F statistics >0.05, then Dunnett’s t-test will be applied. P < 0.05 is considered statistically significant. 3. Results
2.8. Annexin-V staining In normal cells, phosphatidylserine only distributes in the medial side of the membrane lipid bilayer, when cell apoptosis occurs, the membrane phosphatidylserine rolls from the inside of the lipid membrane to the outside. Annexin-V is a phospholipid binding protein with high affinity to phosphatidylserine. Therefore bind to the cell membrane of the early apoptotic cells. For AnnexinV staining, after washing twice in PBS, the viable oocytes were stained for 10 min in the dark with 90 pl of binding buffer containing 10 pl of Annexin-V-FITC (Vazyme Biotech Co., Ltd, Nanjing, China). Then after two times washes in DPBS, oocytes were moved to the glass slides. Fluorescent signal of oocytes was examined immediately by the scanning settings (Zeiss LSM 700 META; Jena, Germany). 2.9. Western blot analysis A total of 120 porcine oocytes in each group were collected (17 h, 27 h, respectively) and lysed in sample buffer (SDS sample buffer with 2-mercaptoethanol), and were boiled at 90 C for 10 min. The protein maker and the sample were added to the sample pore in sequence using SDS-polyacrylamide gel electrophoresis (PAGE). After electrophoretic separation, proteins were transferred onto a PVDF membrane (Millipore, Billerica, MA, USA). Membranes were blocked in 5% (w/v) BSA in Tris buffered saline (TBS) containing 0.1% (w/w) Tween 20 (TBST) for 1 h at room temperature. After a brief wash in TBST, the membrane was incubated with a rabbit monoclonal anti-p53 antibody (1:1000), anti-p-CDC2 antibody (1:1000), anti-p-MAPK-antibody (1:2000), anti-LC3B antibody (1:1000), anti-caspase3 antibody (1:1000), anti-GAPDH antibody (1:2000) at 4 C overnight. After washing three times in TBST (10 min each), membranes were then incubated for 1 h with conjugated goat antirabbit IgG (Santa Cruz, Texas, USA) antibodies (1:5000) in TBST. Then after washing five times in TBST (5 min each), membrane was exposed to an enhanced chemiluminescence reagent (EMD Millipore, Billerica, MA, USA), and protein bands were visualized by detection system (Tanon-3900, China).
3.1. Effects of melatonin on meiotic maturation in OTA-exposed oocytes We first examined the effects of OTA on porcine oocyte maturation, and we found that the cumulus granulosa cells in the OTA treatment group diffused to a reduced degree or exhibited no proliferation after being cultured for 52 h, and most oocytes failed to extrude their first polar bodies (Fig. 1A). In addition, as shown in Fig. 1B, the effects of OTA on oocyte maturation are dosedependent. In the control group, first polar body (PBI) discharge occurred in most oocytes (86.81 ± 2.42%, n ¼ 240), but this significantly decreased upon OTA treatment at 5 mM (72.57 ± 2.49%, n ¼ 220, p < 0.01), 8 mM (48.1 ± 1.64%, n ¼ 240, p < 0.001), 9 mM (30.57 ± 2.70%, n ¼ 220, p < 0.01), and 10 mM (15.37 ± 1.41%, n ¼ 220, p < 0.001). Melatonin was then added to examine its effects on oocyte maturation following OTA exposure. As shown in Fig. 1C, melatonin (107 M) significantly increased the proportion of cumulus dilatation and PBI in OTA-exposed oocytes (70.69 ± 1.06%, n ¼ 250, p < 0.01). Therefore, we used this concentration (107 M) for the following experiments. These results suggest that melatonin can protect oocyte meiotic maturation from OTA exposure. To explore the functional way of OTA toxicity on oocyte maturation, the difference in general gene expression after OTA treatment by RNA-seq was first analyzed. As shown in Fig. 2A, after 27 h of culture, transcriptome analysis found 1268 genes with significantly changed expression due to OTA treatment compared with the control group. Among these changed genes, 1120 (88.3%) were up-regulated, which was confirmed by the cluster analysis (Fig. 2B). The number of differentially expressed genes for each GO term, including relevant biological processes, cellular components, and molecular functions was then analyzed. The histogram showed that OTA mainly influenced the function of catalytic, cellular, and metabolic processes (Fig. 2C). The KEGG pathway functional enrichment database shown in Fig. 2D indicated that the differentially expressed genes (up-regulation) were mostly involved in important biochemical, metabolic, and signal transduction pathways. And the KEGG enrichment analysis also indicated that there
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Fig. 1. The effects of OTA exposure on gene expression and oocyte maturation. (A) Oocyte maturation after OTA treatment and melatonin (MT) supplement. (B) Rate of different concentrations of OTA treatment on the polar body extrusion in porcine oocytes. **, p < 0.01; ***, p < 0.001. (C) The rate of polar body extrusion after melatonin supplement.
are several genes down-regulated in oocytes after OTA exposure, which included the MAPK signaling pathway (Fig. 2E). However, it seems that melatonin protects oocytes from OTA-exposure from cellular level since the RNA-seq analysis indicated that the expression of only 156 genes was altered (Fig. S1A), and few overlap genes were found (4 genes) (Fig. S1B). The cluster analysis and gene KEGG enrichment results also confirm this (Figs. S1C and S1D). These results provided the general information about the functional pathway for the OTA toxicity on porcine oocyte quality. 3.2. Effects of melatonin on CDC2 phosphorylation and cell cycle progression in OTA-exposed oocytes We next explored how OTA affects oocyte maturation. Cluster analysis showed that several important cell cycle-related genes, such as Cdc6, Cdc25B, and Cdk7, were up-regulated, indicating that OTA may affect cell cycle progression in oocytes (Fig. 3A). Therefore, the proportion of oocytes in different meiosis stages following OTA treatment was investigated. As shown in Fig. 3B, after 27 h culture, oocytes treated with OTA were substantially in the GV (germinal vesicular) or GVBD (germinal vesicle breakdown) stages. The ratios were significantly higher than those of the control group: GV (2.5 ± 0.28%, n ¼ 100 vs 38.03 ± 1.43%, n ¼ 110, P < 0.01) and GVBD (9.38 ± 1.86%, n ¼ 100 vs 51.45 ± 4.26%, n ¼ 110, P < 0.05). A large proportion of oocytes in the control group (87.99 ± 2.43%, n ¼ 100) were at the metaphase I (MI) stage, whereas few oocytes in the OTA-treated group (10.51 ± 4.14%, n ¼ 110, P < 0.01) reached MI.
According to our results above, we chose 107 M melatonin for the following experiments due to its effects for the increased oocyte maturation under OTA exposure. Melatonin supplementation significantly alleviated the GV-GVBD block in OTA-exposed oocytes; there was a significant decrease in the percentage of GV (15.11 ± 6.17%, n ¼ 100) and GVBD (27.02 ± 3.13%, n ¼ 100) oocytes. We counted oocyte stage at 54 h. Similarly to the 27 h culture, there was still a large proportion of oocytes arrested at GV/GVBD in OTAtreated oocytes compared to the control group (1.33 ± 0.72%, n ¼ 100 vs 33.32 ± 0.54%, n ¼ 100, P < 0.01). The number of oocytes that developed to metaphase II (MII) in the OTA group was significantly lower than in the control group (83.26 ± 1.01%, n ¼ 100 vs 45.46 ± 5.32%, n ¼ 100, P < 0.01). Melatonin supplementation increased the percentage of MII oocytes significantly over OTAexposed oocytes (63.16 ± 3.40%, n ¼ 1000) (Fig. 3B). Next the mechanism of OTA exposure on oocyte cell cycle progression was explored. We checked p-CDC2 and p53 levels in different groups of oocytes. As shown in Fig. 3C, western blot results showed that p-CDC2 increased significantly in the OTA group compared to the control group (1, n ¼ 120 vs 1.45 ± 0.21, n ¼ 120, P < 0.05), and melatonin relieved this tendency (1.27 ± 0.13, n ¼ 120, p < 0.05) (Fig. 3D). While p53 levels significantly decreased in the OTA-exposed group compared to the control group (1, n ¼ 120 control vs 0.70 ± 0.04, n ¼ 120, P < 0.01), melatonin administration considerably increased p53 compared with the OTA-exposed group (0.95 ± 0.11, n ¼ 120, P < 0.05) (Fig. 3C and D). These results demonstrate that OTA exposure delays meiotic
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Fig. 2. Transcriptome analysis for the effects of OTA exposure on oocyte maturation. (A) The number of differentially expressed genes and volcano plot analysis after OTA treatment. (B) The cluster analysis for the differentially expressed genes after OTA treatment. (C) The differential gene GO enrichment analysis for the differentially expressed genes after OTA treatment. (D) The rich factor by the up-regulation gene KEGG enrichment analysis for the differentially expressed genes after OTA treatment. (E) The rich factor by the down-regulation gene KEGG enrichment analysis for the differentially expressed genes after OTA treatment.
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Fig. 3. The effects of melatonin on the cell cycle progression in the OTA-exposed oocytes. (A) The cluster analysis for the differentially expressed genes related with cell cycle after OTA treatment. (B) The cell cycle progression after treatment with OTA or melatonin supplement after culture for 27 h and 54 h, the time points when most oocytes reached MI stage and MII stage. *, p < 0.05; **, p < 0.01. (C) Western blot result of the protein expression of phosphorylated CDC2 and p53 after OTA treatment and melatonin supplement. (D) The band intensity analysis for the phosphorylated CDC2 and p53 after OTA treatment and melatonin supplement. *, p < 0.05; **, p < 0.01.
progression via arresting the G2 to M transition, while melatonin promotes cell cycle progression in OTA-exposed oocytes.
3.3. Effects of melatonin on spindle morphology in OTA-exposed oocytes To explore how OTA disturbs oocyte maturation, the subcellular spindle formation after 27 h of culture was then examined, when most oocytes are at MI, using confocal microscopy. As shown in Fig. 4A, most oocytes in the control group displayed typical barrel spindles and well-arranged chromosomes on the intermediate plate, while in the OTA treatment group, spindle morphology was completely destroyed and the chromosomes showed severe misalignment. Statistical analysis showed that the proportion of oocytes with abnormal spindle shape in OTA-treated cells was significantly higher than that of the control group (4.81 ± 0.92%, n ¼ 90 vs. 25.82 ± 1.89%, n ¼ 90, P < 0.01) (Fig. 4B). Melatonin supplementation rescued the spindle defects caused by OTA, and the proportion of damaged spindles was notably decreased (16.51 ± 1.48%, n ¼ 90, P < 0.05) (Fig. 4A and B). We then examined the level of p-MAPK, the key molecule in MAPK pathway which is associated with microtubule organization was examined. These results showed that p-MAPK was significantly decreased in the OTA group compared to controls (1.00 vs 0.44 ± 0.04, n ¼ 120, P < 0.01). Melatonin ameliorated the effects of OTA exposure, with the pMAPK protein level increasing to 0.85 ± 0.07 compared with the OTA treatment group (P < 0.05) (Fig. 4C and D).
3.4. Effects of melatonin on mitochondria function and oxidative stress in OTA-exposed oocytes We then explored the possible mechanism of OTA toxicity on porcine oocytes. The cluster analysis from RNA-seq results indicated that several important genes related to energy metabolism were up-regulated, indicating that OTA might affect mitochondria function (Fig. 5A). Enrichment of the KEGG pathway analysis for oxidative phosphorylation showed that genes related with NADH dehydrogenase, Cytochrome c reductase, Cytochrome c oxidase, Ftype ATPase, and V-type ATPase were altered (Fig. S2A). Moreover, TCA cycle-related genes and Complex-I (CxI), III, IV and V, which are related to mitochondria function, were also altered, further confirming the effects of OTA exposure on oocyte mitochondria-related gene expression (Figs. S2B and S2C). Mito-Tracker was utilized to track the distribution of mitochondria in oocytes. As shown in Fig. 5B, in the control oocytes, the mitochondria are distributed uniformly in the cytoplasm, while OTA treatment caused the mitochondria to cluster. In the melatonin-supplemented oocyte group, mitochondria distribution was normal, similar with the control oocytes. Oxidative stress is considered an important factor in mycotoxin toxicity, while melatonin is known to be involved in redox homeostasis. Because mitochondria dysfunction generally induces oxidative stress, we next explored ROS levels after 27 h of culture to find whether melatonin could alleviate oxidative stress in OTAexposed oocytes. As predicted, OTA induced ROS generation compared to control groups (5.44 ± 0.80, n ¼ 30, vs 30.68 ± 1.29,
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Fig. 4. The effects of melatonin on the spindle organization in the OTA-exposed oocytes. (A) The spindle morphology after OTA treatment and melatonin supplement. Green, atubulin; blue, chromatin. Bar ¼ 5 mm. (B) The rate of abnormal spindle after OTA treatment and melatonin supplement. (C) The protein expression of p-MAPK after OTA treatment and melatonin supplement. (D) The band intensity analysis for p-MAPK after OTA treatment and melatonin supplement. *, p < 0.05; **, p < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
n ¼ 32, P < 0.001); however, melatonin administration significantly decreased ROS production (18.04 ± 0.75, P < 0.05) (Fig. 5C). The expression of genes associated with oxidative stress was evaluated, and, as shown in Fig. 5D, SOD1 (1.00 vs 1.53 ± 0.13, P < 0.05 vs 0.99 ± 0.10) and SOD2 (1.00 vs 1.45 ± 0.05, P < 0.05 vs 1.12 ± 0.13) were significantly increased in oocytes exposed to OTA compared with controls and melatonin groups. We also found that compared with control and OTA groups, melatonin improved the expression of GPX (1.00 vs 0.80 ± 0.04 vs 1.29 ± 0.08, P < 0.05) and CAT (1.00 vs 0.80 ± 0.08, vs 1.44 ± 0.08, P < 0.05). These results indicate that melatonin protects against mitochondria dysfunction and the resulting oxidative stress in oocytes exposed to OTA. 3.5. Effects of melatonin on apoptosis and autophagy levels in OTAexposed oocytes The cluster analysis from RNA-seq results also indicated that several important apoptosis-related genes were up-regulated (Fig. 6A); moreover, the analysis from the enrichment of the KEGG pathway showed that DNA replication-related gene expression was altered (Fig. S3A). Since DNA damage induces apoptosis, these transcriptome analysis results indicate that OTA might cause
apoptosis in oocytes. The early apoptosis in different groups of oocytes was detected by Annexin-V staining. As shown in Fig. 6B, the green ring of the external cellular membrane was Annexin-V positive. In the OTA-treatment group, the percentage of AnnexinV positive oocytes was significantly higher than that of the control group (7.63 ± 1.35%, n ¼ 40 vs 27.31 ± 2.03%, n ¼ 40, P < 0.05) while melatonin blocked the early apoptosis of oocytes induced by OTA, reducing apoptosis to 12.25 ± 1.71% (n ¼ 40, P < 0.05) (Fig. 6B). Excessive oxidative stress often leads to apoptosis, which further induces protective autophagy. Analysis from KEGG pathway enrichment showed that proteasome-related gene expression was altered (Fig. S3B). Because ubiquitination is important for autophagy, LC3, a marker of autophagy in oocytes was then stained. As shown in Fig. 6C, OTA stimulated more autophagy vacuoles in oocytes and markedly increased the autophagy rate compared to the control group, showing with stronger fluorescence intensity (6.72 ± 0.68%, n ¼ 60 vs 44.77 ± 4.33% n ¼ 60, P < 0.01). Melatonin supplementation yielded fewer autophagy oocytes, showing with similar fluorescence intensity compared with the control group (9.45 ± 1.76%, n ¼ 60, P < 0.01) (Fig. 6C). Caspase3 and LC3 protein expression were then examined, and the results showed a similar tendency via fluorescence staining (Fig. 6D). OTA significantly
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Fig. 5. The effects of melatonin on the mitochondria distribution and ROS level in the OTA-exposed oocytes. (A) The cluster analysis for the differentially expressed genes related with oxidative phosphorylation after OTA treatment. (B) The mitochondria distribution pattern after OTA treatment or melatonin supplement. Red, Mito-tracker; blue, DNA. Bar ¼ 20 mm. (C) The ROS generation after OTA treatment or melatonin supplement. Green, ROS. Bar ¼ 20 mm. The fluorescence intensity analysis for the ROS signal was confirmed this. (D) The relative mRNA expression of oxidative stress-related genes after OTA treatment or melatonin supplement. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
increased the levels of Caspase3 (1, n ¼ 120 vs 1.36 ± 0.09, n ¼ 120, P < 0.01) and LC3B (1, n ¼ 120 vs 2.33 ± 0.29, n ¼ 120, P < 0.01); while melatonin reduced expression of Caspase3 (0.88 ± 0.05, n ¼ 120) and LC3B (1.34 ± 0.14, n ¼ 120) (Fig. 6E). The expression of genes associated with apoptosis and autophagy was then evaluated. As shown in Fig. 6F, compared with the OTA group, after melatonin administration, the expression of anti-apoptotic gene Bcl-2 increased markedly (1 vs 1.10 ± 0.10 vs 1.80 ± 0.23, P < 0.05), and the expression of pro-apoptotic gene Bax decreased (1 vs 1.59 ± 0.11 vs 0.80 ± 0.11, P < 0.05). Melatonin ameliorated autophagy-related gene expression in OTA-exposed oocytes, as shown with Atg7 (1 vs 1.54 ± 0.15 vs 1.17 ± 0.15, P < 0.05) and Lc3 (1 vs 1.54 ± 0.24 vs 1.13 ± 0.11, P < 0.05). These results indicate the protective role of melatonin on OTA-induced apoptosis and autophagy.
4. Discussion As a widespread toxin in nature, OTA has been gaining attention for its toxic effects on the animal reproductive system. In this study, we used transcriptome analysis to screen the potential mRNA expression level changes, and then elucidated the cellular mechanism of OTA toxicity on porcine oocytes. Finally, we propose an innovative solution to mitigate OTA toxicity and report that melatonin can reduce the oxidative stress induced by OTA, thereby reducing the abnormal cytoskeleton dynamics, autophagy, and apoptosis caused by oxidative stress, restore cell cycle progression, and ultimately improve porcine oocyte quality (Fig. 7). RNA-seq was first used to show altered expression of more than 1000 genes in oocytes after OTA exposure, indicating that OTA widely affects transcription and translation in the oocytes.
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Fig. 6. The effects of melatonin on the apoptosis and autophagy in the OTA-exposed oocytes. (A) The cluster analysis for the differentially expressed genes related with apoptosis after OTA treatment. (B) The early apoptosis examined by Annexin-V staining after OTA treatment or melatonin supplement. Green, Annexin-V. Bar ¼ 20 mm. The percentage of early apoptosis also confirmed this. *, p < 0.05. (C) The autophagy level after OTA treatment or melatonin supplement. Green, LC3; blue, DNA. Bar ¼ 20 mm. The rate of autophagy analysis also confirmed this. **, p < 0.01. (D) The protein expression of Caspase 3 and LC3 after OTA treatment or melatonin supplement. (E) The relative band intensity analysis for the Caspase 3 and LC3 after OTA treatment or melatonin supplement. *, p < 0.05. (F) The relative mRNA expression of Bcl-2, Bax, Atg7 and Lc3 after OTA treatment or melatonin supplement. *, p < 0.05; **, p < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Amplification of cumulus cells and polar body extrusion are two important markers for oocyte maturation (Rosa et al., 2018), and the results show that cumulus granulosa cell diffusion/proliferation was disturbed, together with the failure of polar body extrusion, these indicated that OTA is toxic to oocyte maturation. We also demonstrated that melatonin could alleviate the meiotic maturation defects caused by OTA, and interestingly, its effects are not through changes in mRNA levels, since only about 100 genes were altered, which is far lower than the changes from OTA exposure. This indicates that melatonin, a pineal gland hormone, may not act through RNA. We next explored the functional mechanism of OTA toxicity on oocytes. From deep analysis of RNA-seq results, cell cycle-related
genes are a large proportion of the genes altered following OTA exposure. Comparing the proportions of oocytes at different stages after 27 h and 54 h indicated that OTA may delay meiotic progression via arresting the G2 to M transition. CDC2, an evolutionarily conserved serine/threonine-specific protein kinase, is essential in the transition from G2 to M, and it is negatively regulated by phosphorylation of two residues located in its ATP-binding site (Pandey and Giri, 2018). p53 protein, a transcription factor that acts as a checkpoint in the cell cycle, examines sites of DNA damage in G1 to monitor genomic integrity. If damage occurs, p53 blocks DNA replication to provide sufficient time for DNA repair (Kannappan et al., 2018). The transcriptome analysis results show that DNA duplication-related genes were altered. Upregulation of
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Fig. 7. Diagram of the effects of melatonin on OTA-exposed oocytes.
p-CDC2 and downregulation of p-53 was found following OTA exposure, indicating that OTA blocks cell cycle progression during oocyte maturation. Similar results were also reported for other toxic environmental chemicals such as ethylhexyl phthalate (Zhang et al., 2018), benzoapyrene (Miao et al., 2018), and zearalenone (Sambuu et al., 2011), which all caused maturation blocks in oocytes. Melatonin administration restored cell cycle progression, indicating that melatonin is a potential candidate to improve the oocyte maturation in OTA-exposed oocytes. During the cell cycle, the dynamics of the cytoskeleton play major roles, such as chromosome congression/segregation and cytokinesis, and proper spindle formation is important for this (Zhu et al., 2018). Our results show that OTA negatively affects spindle organization and chromosome alignment, and melatonin protects the oocytes from these spindle defects and chromosome misalignments. OTA may affect spindle formation through the MAPK signaling pathway, since from transcriptome analysis it was a main pathway affected by OTA exposure. Western blot results were consistent with the RNA-seq analysis, showing that phosphorylated MAPK decreased after OTA treatment increased after melatonin administration. MAPK was shown to play a key role in facilitating and stabilizing the spindle pole in oocyte meiotic processes (Zhu et al., 2018). These results are consistent with our previous studies which showed that melatonin might play a pivotal role in cytoskeletal modulation (Lan et al., 2018; Sripathi et al., 2017). Therefore, these results indicate that melatonin protects oocytes from OTA exposure through its effects on the cell cycle and spindle formation. We then tried to find out the mechanism for the toxic effects of OTA on oocytes. In addition to cell cycle-related genes, the results showed that a large proportion of oxidative phosphorylation and TCA cycle-related genes were altered, which are all related to mitochondria dysfunction. Mito-tracker staining confirmed this result, showing aberrant mitochondria distribution. Aberrant mitochondria distribution could lead to mitochondria dysfunction. For example, in the MII oocytes of diabetic mice, mitochondria showed clustering distribution or homogeneous distribution, which are all abnormal (Wang et al., 2009). Mitochondria dysfunction generally induces oxidative stress, which has the capacity to cause a decline in levels of critical cell cycle factors such as maturation-promoting factor (MPF). Several studies have
confirmed that oxidative stress is induced by mycotoxins, such as aflatoxin (Hou et al., 2013), zearalenone (Qin et al., 2015) and deoxynivalenol (Waskiewicz et al., 2014). We therefore hypothesized that superfluous oxidative stress was the reason for cell cycle delay and abnormal cytoskeletal dynamics in OTA-exposed oocytes. Our results showed that OTA induces ROS generation, and oxidative stress-related genes, such as SOD1 and SOD2, also exhibited altered RNA expression, indicating strong oxidative stress occurs in the cytoplasm and mitochondria. On the other hand, several studies have indicated a relationship between melatonin and mitochondria, since it was found that melatonin membrane receptors are present in mitochondria (Tan and Reiter, 2019). While melatonin supplementation depressed ROS generation, GPX and CAT mRNA levels were markedly increased, which suggests that melatonin can not only react directly with ROS, but also enhance the production of glutathione and catalase, thus further reducing intracellular ROS levels in OTA-exposed oocytes. This result is similar to previous research showing that melatonin pre-treatment could protect golden hamster germ cell from dexamethasone-induced oxidative stress (Tanabe et al., 2015), and a study which showed that melatonin also restored glutathione expression levels in rat pups exposed to plumbum (Bazrgar et al., 2015). Therefore, these results indicate that melatonin protects oocytes from OTA exposure through its effects on mitochondria dysfunction-induced oxidative stress. Oxidative stress generally leads to apoptosis, while autophagy reduces apoptosis through ubiquitination. Apoptosis and autophagy are generally activated by adverse environmental factors such as nutrient deprivation (Pang et al., 2014), infection (Hacker, 2014), radiation (Alan Mitteer et al., 2015), and toxic chemical substances (Liu et al., 2015). Similarly, after OTA treatment, apoptosis and ubiquitination-related gene expression were changed in our results. The intensive early apoptosis signals and elevated numbers of autophagy vacuoles were detected in OTAexposed oocytes, and the results showed that Caspase 3 and LC3 protein expression decreased while apoptosis-related gene Bcl-2 and Bax were also altered. Bcl-2 exerts an anti-apoptotic function through inhibition of mitochondrial cytochrome c release (Murphy et al., 2000). Bax is a key component for cellular induced apoptosis through mitochondrial stress, which increases the membrane’s permeability, leads to the release of cytochrome c from mitochondria, and activates the caspase activation pathway for apoptosis (Narita et al., 1998). These results demonstrate that melatonin can inhibit Caspase3-mediated apoptosis via upregulating Bcl-2 and down-regulating Bax. This indicates that OTA exposure caused mitochondria dysfunction and induced apoptosis/autophagy. As expected, melatonin could inhibit apoptosis and autophagy remarkably. We next tested autophagyrelated genes Atg7 and Lc3. Atg7 and Lc3 were originally identified as critical proteins for autophagy (Codogno and Meijer, 2005). Similar to the trend of reducing apoptosis, melatonin could inhibit autophagy via down-regulating Atg8 and Lc3 gene expression. This effect of melatonin modulating apoptosis and autophagy has been proposed in other models. For example, melatonin attenuated MPTP-induced neurotoxicity via preventing CDK5-mediated autophagy and SNCA/a-synuclein aggregation (Su et al., 2015). Melatonin ameliorated apoptosis and autophagy in liver cancer cells (Ordonez et al., 2015), as well as mouse smooth muscle actinpositive cells exposed to carbon tetrachloride-induced fibrosis (San-Miguel et al., 2015). These results demonstrate the superiority of melatonin for decreasing apoptosis and autophagy. 5. Conclusions In summary, our results demonstrate that OTA exposure causes
Please cite this article as: Lan, M et al., Melatonin ameliorates ochratoxin A-induced oxidative stress and apoptosis in porcine oocytes, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113374
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mitochondria dysfunction and induces oxidative stress-related apoptosis/autophagy, which further affects spindle formation and cell cycle progression in porcine oocytes. Melatonin, due to its antioxidant properties, is a promising pharmacological agent for its protective functions against OTA toxicity. Contributions ML and SCS designed the study. ML performed the majority of the experiments. YZ, XW, MHP and YX contributed to the regents and materials. ML and SCS analyzed the data. ML and SCS wrote the manuscript. Funding This work was supported by the National Key Research and Development Program (2018YFC1003802), China; National Natural Science Foundation of China (31622055, 31571547); the Fundamental Research Funds for the Central Universities (KYTZ201602, KJYQ201701), China. Declaration of competing interest There is no conflict of interest to declare. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.envpol.2019.113374. References Alan Mitteer, R., Wang, Y., Shah, J., Gordon, S., Fager, M., Butter, P.P., Jun Kim, H., Guardiola-Salmeron, C., Carabe-Fernandez, A., Fan, Y., 2015. Proton beam radiation induces DNA damage and cell apoptosis in glioma stem cells through reactive oxygen species. Sci. Rep. 5, 13961. Bazrgar, M., Goudarzi, I., Lashkarbolouki, T., Elahdadi Salmani, M., 2015. Melatonin ameliorates oxidative damage induced by maternal lead exposure in rat pups. Physiol. Behav. 151, 178e188. Bhat, P.V., Anand, T., Mohan, M.T., Khanum, F., 2018. Restorative effect of l-Dopa treatment against Ochratoxin A induced neurotoxicity. Neurochem. Int. 118, 252e263. Brennan, K.M., Oh, S.Y., Yiannikouris, A., Graugnard, D.E., Karrow, N.A., 2017. Differential gene expression analysis of bovine macrophages after exposure to the penicillium mycotoxins citrinin and/or ochratoxin A. Toxins (Basel) 9, e366. Codogno, P., Meijer, A.J., 2005. Autophagy and signaling: their role in cell survival and cell death. Cell Death Differ. 12 (Suppl. 2), 1509e1518. Eze, U.A., Huntriss, J., Routledge, M.N., Gong, Y.Y., 2018. Toxicological effects of regulated mycotoxins and persistent organochloride pesticides: in vitro cytotoxic assessment of single and defined mixtures on MA-10 murine Leydig cell line. Toxicol. In Vitro 48, 93e103. Hacker, G., 2014. ER-stress and apoptosis: molecular mechanisms and potential relevance in infection. Microb. Infect. 16, 805e810. Hou, Y.J., Zhao, Y.Y., Xiong, B., Cui, X.S., Kim, N.H., Xu, Y.X., Sun, S.C., 2013. Mycotoxin-containing diet causes oxidative stress in the mouse. PLoS One 8, e60374. Hsuuw, Y.D., Chan, W.H., Yu, J.S., 2013. Ochratoxin a inhibits mouse embryonic development by activating a mitochondrion-dependent apoptotic signaling pathway. Int. J. Mol. Sci. 14, 935e953. Huang, C.H., Chan, W.H., 2017. Protective effects of liquiritigenin against citrinintriggered, oxidative-stress-mediated apoptosis and disruption of embryonic development in mouse blastocysts. Int. J. Mol. Sci. 18, e2538. Huang, F.J., Chan, W.H., 2016. Effects of ochratoxin a on mouse oocyte maturation and fertilization, and apoptosis during fetal development. Environ. Toxicol. 31, 724e735. Kannappan, R., Mattapally, S., Wagle, P.A., Zhang, J., 2018. Transactivation domain of p53 regulates DNA repair and integrity in human iPS cells. Am. J. Physiol. Heart Circ. Physiol. 315, h512eh521. Lan, M., Han, J., Pan, M.H., Wan, X., Pan, Z.N., Sun, S.C., 2018. Melatonin protects against defects induced by deoxynivalenol during mouse oocyte maturation. J. Pineal Res., e12477 Liu, F., Du, K.J., Fang, Z., You, Y., Wen, G.B., Lin, Y.W., 2015. Chemical and biological insights into uranium-induced apoptosis of rat hepatic cell line. Radiat. Environ. Biophys. 54, 207e216. Liu, G., Jiang, Q., Chen, S., Fang, J., Ren, W., Yin, J., Yao, K., Yin, Y., 2017. Melatonin alters amino acid metabolism and inflammatory responses in colitis mice.
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Please cite this article as: Lan, M et al., Melatonin ameliorates ochratoxin A-induced oxidative stress and apoptosis in porcine oocytes, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113374