TY - JOUR
AU1 - Miao, Yilong
AU2 - Zhou, Changyin
AU3 - Bai, Qingyun
AU4 - Cui, Zhaokang
AU5 - ShiYang, Xiayan
AU6 - Lu, Yajuan
AU7 - Zhang, Mianqun
AU8 - Dai, Xiaoxin
AU9 - Xiong, Bo
AB - Abstract STUDY QUESTION Does melatonin restore the benzo(a)pyrene (BaP)-induced meiotic failure in porcine oocytes? SUMMARY ANSWER Melatonin effectively inhibits the increased reactive oxygen species (ROS) level and apoptotic rate in BaP-exposed porcine oocytes to recover the meiotic failure. WHAT IS KNOWN ALREADY BaP, a widespread environmental carcinogen found in particulate matter, 2.5 µm or less (PM2.5), has been shown to have toxicity at the level of the reproductive systems. BaP exposure disrupts the steroid balance, alters the expression of ovarian estrogen receptor and causes premature ovarian failure through the rapid depletion of the primordial follicle pool. In addition, acute exposure to BaP has transient adverse effects on the follicle growth, ovulation and formation of corpora lutea, which results in transient infertility. STUDY DESIGN, SIZE, DURATION Porcine oocytes were randomly assigned to control, BaP-exposed and melatonin-supplemented groups. BaP was dissolved in dimethylsulphoxide and diluted to a final concentration of 50, 100 or 250 μM with maturation medium, respectively. Melatonin was dissolved in the absolute ethanol and diluted with maturation medium to a final concentration of 1 nM, 100 nM, 10 μM and 1 mM, respectively. The in vitro cultured oocytes from each group after treatment were applied to the subsequent analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS Acquisition of oocyte meiotic competence was assessed using immunostaining, fluorescent intensity quantification and/or immunoblotting to analyse the cytoskeleton assembly, mitochondrial integrity, cortical granule dynamics, ovastacin distribution, ROS level and apoptotic rate. Fertilization ability of oocytes was examined by sperm binding assay and IVF. MAIN RESULTS AND THE ROLE OF CHANCE BaP exposure resulted in the oocyte meiotic failure (P = 0.001) via impairing the meiotic apparatus, showing a prominently defective spindle assembly (P = 0.003), actin dynamics (P < 0.001) and mitochondrion integrity (P < 0.001). In addition, BaP exposure caused the abnormal distribution of cortical granules (P < 0.001) and ovastacin (P = 0.003), which were consistent with the observation that fewer sperm bound to the zona pellucida surrounding the unfertilized BaP-exposed eggs (P < 0.001), contributing to the fertilization failure (P < 0.001). Conversely, melatonin supplementation recovered, at least partially, all the meiotic defects caused by BaP exposure through inhibiting the rise in ROS level (P = 0.015) and apoptotic rate (P = 0.001). LIMITATIONS, REASONS FOR CAUTION We investigated the negative impact of BaP on the oocyte meiotic maturation in vitro, but not in vivo. WIDER IMPLICATIONS OF THE FINDINGS Our findings not only deeply clarify the potential mechanisms of BaP-induced oocyte meiotic failure, but also extend the understanding about how environmental pollutants influence the reproductive systems in humans. STUDY FUNDING/COMPETING INTERESTS This study was supported by the National Natural Science Foundation of China (31571545) and the Natural Science Foundation of Jiangsu Province (BK20150677). The authors have no conflict of interest to disclose. benzo(a)pyrene, oocyte meiotic competence, melatonin, meiotic apparatus, cortical granule, ovastacin, oxidative stress Introduction With the acceleration of urbanization, the issue of environmental pollution is becoming a major source of health risk in many developing countries (Nancy et al., 2015; Fu et al., 2017). In recent years, most of Chinese cities have suffered from haze, one of the manifestations of the environmental degradation (Zhang and Hughes, 2017). This phenomenon has attracted a great deal of attention because of its toxicity and persistence, which threatens people’s health and causes diseases (Dockery et al., 2013; Nancy et al., 2015; Zhang and Hughes, 2017). Haze contains a variety of harmful chemicals, one of which is benzo(a)pyrene (BaP), a strong carcinogenic threat (Dockery et al., 2013; Zhang and Hughes, 2017). BaP is produced when the organic matters contained in the automobile exhaust and the fossil fuels undergo incomplete combustion and are exposed to high temperature. When BaP is released into the air, it adsorbs onto suspended particulate matter (PM) and moves to the atmosphere (Lybarger et al., 1999). After inhalation, a BaP’ metabolite, BaP-7,8–9,10 diol epoxide (BPDE), binds covalently to DNA to stably form a primary lesion termed a DNA adducts (Baird et al., 2005). DNA adducts are believed to induce mutagenicity and tumourigenicity which has been widely found in many somatic cells (Baird et al., 2005; Vineis and Husgafvel-Pursiainen, 2005). In addition to DNA-adducts, the production of reactive oxygen species (ROS) is likewise another molecular mechanism through which BaP induces cytotoxicity. Excessive ROS promote carcinogenic, teratogenic and mutagenic potential in cells which contributes to DNA damage with the formation of 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dGuo) lesions (Sobinoff et al., 2010; Keating et al., 2011). BaP metabolites involved in redox cycle generate the superoxide anion radicals ( O2−) and hydrogen peroxide (H2O2) (Bengtsson et al., 1983). Genetic mutations resulting from oxidative stress due to BaP exposure play a vital role in lung carcinogenesis (Zhou et al., 2009). Studies of the effects of BaP on the ovarian function have identified this chemical as a potent ovotoxicant (Binnemann, 1979; Valentovic et al., 2006). BaP exposure is correlated with the premature ovarian failure through the rapid depletion of the primordial follicle pool (Borman et al., 2000), and leads to the reduced conception rates in IVF (Rekhadevi et al., 2014). Furthermore, BaP metabolites compromise the normal follicle growth by promoting the formation of follicle atresia through induction of apoptosis (Sadeu et al., 2011a,b). Moreover, acute exposure to BaP has a transient adverse effect on the ovulation and formation of corpora lutea, which results in transient infertility (Sobinoff et al., 2012). However, the molecular mechanisms regarding how BaP influences the oocyte maturation has not yet been determined. Melatonin (N-acetyl-5-methoxytrypt amine) is produced by the pineal gland and many other organs (Reiter et al., 2014b). It has been documented that melatonin and its metabolites are powerful antioxidants and free radical scavengers (Galano et al., 2013; Manchester et al., 2015) that protect cells from oxidative stress and apoptosis (Reiter et al., 2013; Tamura et al., 2017). Of interest is that melatonin is found in human follicular fluid (Reiter et al., 2014a,b) where free oxygen radicals are in high concentrations during follicular growth (Zhang et al., 2017b). Melatonin protects the ovary including the oocytes against the oxidative stress, thereby ensuring the normal development of follicles (Tamura et al., 2013). In the present study, we document that BaP exposure impairs the porcine oocyte meiotic progression by inducing a high level of ROS and apoptosis, which compromises cytoskeleton structures, mitochondria integrity and cortical granule distribution. In addition, BaP exposure weakens the fertilization potential of porcine oocytes via disrupting the critical fertilization regulator ovastacin. Conversely melatonin effectively suppresses the oxidative stress and restores BaP-induced meiotic failure in porcine oocytes. Materials and Methods Antibodies Mouse monoclonal anti-α-tubulin FITC antibody, anti-phalloidin-TRITC antibody, anti-acetyl-α-tubulin (Lys-40) antibody and lens culinaris agglutinin (LCA)-FITC were purchased from Sigma (St. Louis, MO, USA); rabbit polyclonal anti-human ovastacin antibody was obtained from Dr Jurrien Dean; FITC-conjugated goat anti-mouse IgG (H + L) and TRITC-conjugated goat anti-mouse IgG (H + L) were purchased from Zhongshan Golden Bridge Biotechnology Co., Ltd (Beijing, China). Oocyte collection and in vitro maturation For in vitro maturation (IVM), ovaries were collected from prepubertal gilts at a local abattoir and transported to the laboratory in a 0.9% NaCl solution containing penicillinG (75 mg/ml) and streptomycin sulphate (50 mg/ml). Cumulus–oocyte complexes (COCs) were aspirated from medium-sized follicles (3–6 mm in diameter) with a 20-gauge needle fixed to a 20 ml disposable syringe. Overall, 20 COCs in each group were cultured in a 100 μl of maturation medium which consists of TCM-199 (Gibco, Grand Island, NY, USA) containing 0.055% d-glucose, 0.01% Na-pyruvate, 0.1% polyvinyl alcohol (PVA), 0.0075% penicillin and 0.005% streptomycin to different time points at 38.5°C in an atmosphere of 5% CO2 with saturated humidity. Cumulus-free oocytes were used in the subsequent experiments. BaP and melatonin treatment BaP (Sigma, St. Louis, MO, USA) was dissolved in dimethylsulphoxide and diluted to a final concentration of 50, 100 or 250 μM with maturation medium, respectively. Melatonin (Sigma, St. Louis, MO, USA) was dissolved in the absolute ethanol and diluted with maturation medium to a final concentration of 1 nM, 100 nM, 10 μM and 1 mM, respectively. The final concentrations of solvent in the culture medium were not more than 0.1%. Immunofluorescent and confocal microscopy Denuded oocytes (DOs) were fixed in 4% paraformaldehyde (PFA) in PBS for 1 h at room temperature. Oocytes were washed three times in PBS, and then rehydrated and transferred to the permeabilization solution (1% Triton X-100, 20 mM HEPES, pH 7.4, 3 mM MgCl2, 50 mM NaCl, 300 mM sucrose, 0.02% NaN3 in PBS) for 8–12 h. After blocking with 3% BSA for 1 h at room temperature, oocytes were incubated with anti-α-tubulin-FITC antibody (1:200), anti-acetylated tubulin antibody (1:100), anti-phalloidin-TRITC antibody (1:200) or anti-ovastacin antibody (1:100) at 4°C overnight, followed by incubation with an appropriate secondary antibody for 1 h and counterstaining of propidium iodide (PI) for 10 min at room temperature. Finally, oocytes were mounted on glass slides and observed under a laser-scanning confocal fluorescent microscope (Zeiss LSM 700 META). MitoTracker Red CMXRos (ThermoFisher, USA) was used to label the mitochondria. Oxidation-sensitive florescent probe dichloroflorescein (DCFH) (Beyotime, China) was applied to determine the ROS level. Annexin-V staining kit (Vazyme, China) was used to evaluate the apoptosis of the oocytes. For the quantification of fluorescence intensity, the images from both control and treated oocytes were acquired by performing the same immunostaining procedure and setting up the same parameters of confocal microscope. Then Image J software (National Institutes of Health, USA) was used to measure the fluorescence intensity of region of interest in the images. Western blotting analysis A total of 100 porcine oocytes was collected and lysed in 4× NuPAGE™ LDS sample buffer (ThermoFisher, USA) containing protease inhibitor, and then separated on 10% Bis-Tris precast gels and transferred onto polyvinylidene difluoride (PVDF) membranes. The blots were blocked in Tris-buffered saline Tween 20 (TBST) containing 5% low fat dry milk for 1 h at room temperature and then incubated with anti-acetylated tubulin antibody (1:1000) or anti-Gapdh (1:5000) antibody overnight at 4°C. After washing in TBST, the blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. Chemiluminescence was detected with ECL Plus (GE Healthcare, USA) and protein bands were visualized by Tanon-3900 (Tanon, China). Sperm binding assay The spermatozoa were suspended in the fertilization medium to a concentration of 1 × 106 cells/ml and capacitated by 1 h of incubation at 38.5°C. A 50 μl of sperm sample was added to the fertilization droplets containing the oocytes, giving a final sperm concentration of 0.25 × 106 cells/ml, and then incubated for 1 h. Sperm binding to matured oocytes or two-cell embryos from control and BaP-exposed groups was observed using the control two-cell embryos as a negative wash control. Samples were fixed in 4% PFA for 30 min, stained with Hoechst 33342. Bound sperm were quantified from z projections acquired by the confocal microscope, and results reflect the mean ± S.E.M. from at least three independently obtained samples, each containing 10–12 porcine oocytes/embryos. IVF The spermatozoa were suspended in the fertilization medium to a concentration of 1 × 106 cells/ml and capacitated by 1 h of incubation at 38.5°C. A 50 μl of sperm sample was added to the fertilization droplets containing 30–35 matured oocytes, giving a final sperm concentration of 0.25 × 106 cells/ml, and then incubated for 6 h. After fertilization, oocytes were washed three times and cultured with 500 μl of porcine zygote medium in 4-well dishes at 38.5°C, 5% CO2. Cleavage formation was evaluated on Day 2 after IVF and designated as the successful fertilization. Statistical analysis Data were presented as mean percentage (mean ± SEM) of at least three independent experiments and analysed by one-way ANOVA, followed by Fisher’s least significant difference (LSD) post hoc test, which was provided by SPSS16.0 statistical software (IBM, USA). The level of significance was accepted as P < 0.05. Results Effects of different concentrations of BaP and melatonin on porcine oocyte maturation To test the influence of BaP on the porcine oocyte meiotic progression, increasing concentrations of BaP (10, 50 or 100 μM) were supplemented to the culture medium. As shown in Fig. 1A, BaP exposure remarkably brought about oocyte meiotic arrest by displaying the poor expansion of cumulus cells surrounding COCs and the decreased rate of oocytes that reached MII stage. Quantitative analysis showed that treatment with different concentrations of BaP (10, 50 or 100 μM) led to a reduction of polar body extrusion (PBE) in varying degree in oocytes cultured for 44 h in vitro, and supplementation with 50 or 100 μM BaP significantly decreased the PBE rate from 66% in controls to 32 and 23%, respectively (66.3 ± 2.28%, n = 112, control vs 32.6 ± 2.84%, n = 109, 50 μM BaP, P = 0.001 vs 23.3 ± 1.45%, n = 121, 100 μM BaP, P < 0.001; Fig. 1B). Treatment of 50 μM BaP was used for subsequent studies because this concentration not only caused meiotic arrest but also allowed a proportion of oocytes to develop to MII stage for other investigations. To determine whether melatonin could alleviate the impaired oocyte maturation caused by BaP exposure, we simultaneously supplemented different concentrations of melatonin (1 nM, 100 nM, 10 μM, 1 mM) with 50 μM BaP in the culture medium. Expectedly, 1 mM melatonin improved the expansion of cumulus cells surrounding COCs and increased the proportion of PBE in BaP-exposed oocytes to the control comparable level (56.6 ± 1.32%, n = 131 vs 69.5 ± 1.8%, n = 119, P = 0.051; Fig. 1A and C). Taken together, these observations indicate that melatonin is able to, at least partially, recover the failure of porcine oocyte meiotic progression induced by BaP exposure. Figure 1 View largeDownload slide Effects of different concentrations of BaP and melatonin on the porcine oocyte maturation. (A) Representative images of oocyte meiotic progression in control, BaP-exposed and melatonin-supplemented oocytes. Cumulus cell expansion of cumulus oocyte complexes (COCs) and polar body extrusion (PBE) of denuded oocytes (DOs) were imaged by the confocal microscopy. Scale bar, 150 μm (a–c); 80 μm (d–f); 20 μm (g–i). (B) The rate of PBE was recorded in control and different concentrations of BaP-exposed groups (50 μM, 100 μM and 250 μM) after culture for 44 h in vitro. (C) The rate of PBE was recorded in control and different concentrations of melatonin-supplemented groups (1 nM, 100 nM, 10 μM and 1 mM) after culture for 44 h with 50 μM BaP in vitro. *P < 0.05, **P < 0.01, ***P < 0.001. Figure 1 View largeDownload slide Effects of different concentrations of BaP and melatonin on the porcine oocyte maturation. (A) Representative images of oocyte meiotic progression in control, BaP-exposed and melatonin-supplemented oocytes. Cumulus cell expansion of cumulus oocyte complexes (COCs) and polar body extrusion (PBE) of denuded oocytes (DOs) were imaged by the confocal microscopy. Scale bar, 150 μm (a–c); 80 μm (d–f); 20 μm (g–i). (B) The rate of PBE was recorded in control and different concentrations of BaP-exposed groups (50 μM, 100 μM and 250 μM) after culture for 44 h in vitro. (C) The rate of PBE was recorded in control and different concentrations of melatonin-supplemented groups (1 nM, 100 nM, 10 μM and 1 mM) after culture for 44 h with 50 μM BaP in vitro. *P < 0.05, **P < 0.01, ***P < 0.001. Melatonin restores the spindle/chromosome defects in BaP-exposed porcine oocytes In most cases, oocyte meiotic arrest is associated with the activation of spindle assembly checkpoint (SAC) induced by the abnormal spindle assembly. To confirm this possibility in BaP-exposed oocytes, spindle morphologies were observed by immunostaining with anti-α-tubulin-FITC antibody and chromosome alignment was visualized by counterstaining with PI. In controls, oocytes displayed a typical barrel-shape spindle apparatus with a well-aligned chromosome on the equatorial plate (Fig. 2A). By contrast, a higher incidence of abnormal spindles with misaligned chromosomes were observed in BaP-exposed oocytes (19.2 ± 2.6%, n = 90 vs 47.7 ± 2.3%, n = 87, P = 0.003, spindle; 12.7 ± 1.9%, n = 90 vs 67.4 ± 2.7%, n = 87, P < 0.001, chromosome; Fig. 2A–C), but reduced to a level indistinguishable from controls when supplemented with melatonin (25 ± 2.5%, n = 93, P = 0.092 spindle; 31.3 ± 2.6%, n = 93, P = 0.061, chromosome; Fig. 2A–C). Consistently, BubR1, an integral part of SAC, was present on the kinetochores in BaP-exposed oocytes instead of control and melatonin-supplemented oocytes at metaphase I stage (Fig. 2D), indicative of SAC activation. Figure 2 View largeDownload slide Effects of melatonin on the spindle/chromosome defects in BaP-exposed porcine oocytes. (A) Representative images of spindle morphologies and chromosome alignment in control, BaP-exposed and melatonin-supplemented oocytes. Scale bar, 5 μm. (B) The rate of aberrant spindles was recorded in control, BaP-exposed and melatonin-supplemented oocytes. (C) The rate of misaligned chromosomes was recorded in control, BaP-exposed and melatonin-supplemented oocytes. (D) Localization of BubR1 in control, BaP-exposed and melatonin-supplemented oocytes at metaphase I stage. Scale bar, 5 μm. **P < 0.01, ***P < 0.001. Figure 2 View largeDownload slide Effects of melatonin on the spindle/chromosome defects in BaP-exposed porcine oocytes. (A) Representative images of spindle morphologies and chromosome alignment in control, BaP-exposed and melatonin-supplemented oocytes. Scale bar, 5 μm. (B) The rate of aberrant spindles was recorded in control, BaP-exposed and melatonin-supplemented oocytes. (C) The rate of misaligned chromosomes was recorded in control, BaP-exposed and melatonin-supplemented oocytes. (D) Localization of BubR1 in control, BaP-exposed and melatonin-supplemented oocytes at metaphase I stage. Scale bar, 5 μm. **P < 0.01, ***P < 0.001. Melatonin elevates the reduced acetylation level of α-tubulin in oocytes exposed to BaP The defects of spindle apparatus prompted us to further test the microtubule dynamics in BaP-exposed oocytes. Because it has been shown that acetylated α-tubulin is an indicator of the stabilized microtubules in both mitotic and meiotic cells (Miao et al., 2017), we thus assessed the microtubule stability in oocytes exposed to BaP. We observed that BaP exposure significantly reduced the signals of acetylated α-tubulin compared to the controls (12.4 ± 1.8, n = 112 vs 39.8 ± 1.3, n = 101, P < 0.001; Fig. 3A and B), which was further confirmed by the western blotting analysis (Fig. 3C). This suggests that BaP exposure renders microtubules less stable and hence impairs the spindle assembly. Whereas melatonin supplementation increased the acetylated α-tubulin to the level indistinguishable from controls (36.8 ± 1.5, n = 107 vs 39.8 ± 1.3, n = 101, P = 0.178; Fig. 3A–C). Figure 3 View largeDownload slide Effects of melatonin on the acetylation level of α-tubulin in BaP-exposed porcine oocytes. (A) Representative images of acetylated α-tubulin (Ac-Tub) in control, BaP-exposed and melatonin-supplemented oocytes. Scale bar, 5 μm. (B) Quantitative analysis of the fluorescence intensity of acetylated α-tubulin in control, BaP-exposed and melatonin-supplemented oocytes. ***P < 0.001. (C) The acetylation levels of α-tubulin in control, BaP-exposed and melatonin-supplemented oocytes were examined by western blotting. Figure 3 View largeDownload slide Effects of melatonin on the acetylation level of α-tubulin in BaP-exposed porcine oocytes. (A) Representative images of acetylated α-tubulin (Ac-Tub) in control, BaP-exposed and melatonin-supplemented oocytes. Scale bar, 5 μm. (B) Quantitative analysis of the fluorescence intensity of acetylated α-tubulin in control, BaP-exposed and melatonin-supplemented oocytes. ***P < 0.001. (C) The acetylation levels of α-tubulin in control, BaP-exposed and melatonin-supplemented oocytes were examined by western blotting. Melatonin rescues the actin dynamics in BaP-exposed oocytes During oocyte maturation, actin filaments play a critical part in the asymmetric spindle positioning and cortical polarization. To ask whether the effect of BaP on the oocyte meiotic progression involves in the actin dynamics, phalloidin was used to label the F-actin. In control oocytes, actin was concentrated evenly on the plasma membrane with strong signals (Fig. 4A). In striking contrast, BaP-exposed oocytes exhibited a discontinuous distribution of actin filaments that could be partially rescued by the supplementation with melatonin (3.3 ± 1.6%, n = 87, P < 0.001 vs 84.9 ± 2.8%, n = 90 vs 44.5 ± 1.7%, n = 91, P < 0.001; Fig. 4A and B). Correspondingly, quantitative analysis of fluorescence intensity revealed that actin signals were significantly decreased in BaP-exposed oocytes, but rose to the control level after supplementation with melatonin (48.5 ± 1.3, n = 87, P < 0.001 vs 13.6 ± 1.3, n = 90 vs 39.6 ± 1.9, n = 91, P = 0.083; Fig. 4C), suggesting that melatonin prevents the actin dynamics from damage induced by BaP exposure. Figure 4 View largeDownload slide Effects of melatonin on the actin dynamics in BaP-exposed porcine oocytes. (A) Representative images of actin filaments in control, BaP-exposed and melatonin-supplemented oocytes. Scale bar, 20 μm. (B) Mis-localization rate of actin in control, BaP-exposed and melatonin-supplemented oocytes. (C) The fluorescence intensity of actin signals was measured in control, BaP-exposed and melatonin-supplemented oocytes. ***P < 0.001. DIC, differential interference contrast. Figure 4 View largeDownload slide Effects of melatonin on the actin dynamics in BaP-exposed porcine oocytes. (A) Representative images of actin filaments in control, BaP-exposed and melatonin-supplemented oocytes. Scale bar, 20 μm. (B) Mis-localization rate of actin in control, BaP-exposed and melatonin-supplemented oocytes. (C) The fluorescence intensity of actin signals was measured in control, BaP-exposed and melatonin-supplemented oocytes. ***P < 0.001. DIC, differential interference contrast. Melatonin recovers the integrity of cortical granules and mitochondria in BaP-exposed oocytes Both cortical granule (CG) and mitochondrial distributions are the critical indicators of cytoplasmic maturation of oocytes, we then tested their changes in the presence of BaP. As shown in Fig. 5A and B, in oocytes cultured with BaP, the distribution of CGs exhibited higher frequency of abnormal patterns than that in control and melatonin-supplemented oocytes (2.7 ± 0.2%, n = 69, P < 0.001 vs 68.5 ± 1.4%, n = 77 vs 29.6 ± 1.3%, n = 78, P < 0.001). The measurement of fluorescence intensity of CG signals had an obvious decline in BaP-exposed oocytes compared to the control group (10.5 ± 1.1, n = 77 vs 34.5 ± 1.5, n = 69, P < 0.001; Fig. 5C), and this decline was rescued by the supplementation of melatonin (25.9 ± 1.4, n = 78, P = 0.032; Fig. 5C). Figure 5 View largeDownload slide Effects of melatonin on the localization of cortical granules in BaP-exposed porcine oocytes. (A) Representative images of cortical granule (CG) localization in control, BaP-exposed and melatonin-supplemented oocytes. Scale bar, 20 and 40 μm. (B) Mis-localization rate of CGs in control, BaP-exposed and melatonin-supplemented oocytes. (C) The fluorescence intensity of CGs was measured in control, BaP-exposed and melatonin-supplemented oocytes. ***P < 0.001. Figure 5 View largeDownload slide Effects of melatonin on the localization of cortical granules in BaP-exposed porcine oocytes. (A) Representative images of cortical granule (CG) localization in control, BaP-exposed and melatonin-supplemented oocytes. Scale bar, 20 and 40 μm. (B) Mis-localization rate of CGs in control, BaP-exposed and melatonin-supplemented oocytes. (C) The fluorescence intensity of CGs was measured in control, BaP-exposed and melatonin-supplemented oocytes. ***P < 0.001. Most mitochondria aggregated around lipid droplets in control porcine oocytes (Fig. 6A), but lost the specific localization in BaP-exposed oocytes (Fig. 6A). The mis-localization rate was increased from 5% in controls to 69% in BaP-exposed oocytes, but reduced to 38% in melatonin-supplemented oocytes (5.6 ± 1.7%, n = 79, P < 0.001 vs 69.2 ± 1.9%, n = 81 vs 38.7 ± 2.2%, n = 80, P = 0.003; Fig. 6B). The quantification of fluorescence intensity showed that signals of mitochondria prominently reduced in BaP-exposed oocytes in comparison with the controls (9.4 ± 1.1, n = 81 vs 27.4 ± 1.6, n = 79, P < 0.001; Fig. 6C). Conversely, this defect was prevented by the treatment with melatonin (20.4 ± 1.8, n = 80, P = 0.01; Fig. 6C). Figure 6 View largeDownload slide Effects of melatonin on the distribution of mitochondria in BaP-exposed porcine oocytes. (A) Representative images of mitochondria in control, BaP-exposed and melatonin-supplemented oocytes. Scale bar, 20 μm. (B) Abnormal rates of mitochondrion distribution in control, BaP-exposed and melatonin-supplemented oocytes. (C) The fluorescence intensity of mitochondrion signals was recorded in control, BaP-exposed and melatonin-supplemented oocytes. ***P < 0.001. Figure 6 View largeDownload slide Effects of melatonin on the distribution of mitochondria in BaP-exposed porcine oocytes. (A) Representative images of mitochondria in control, BaP-exposed and melatonin-supplemented oocytes. Scale bar, 20 μm. (B) Abnormal rates of mitochondrion distribution in control, BaP-exposed and melatonin-supplemented oocytes. (C) The fluorescence intensity of mitochondrion signals was recorded in control, BaP-exposed and melatonin-supplemented oocytes. ***P < 0.001. Melatonin rescues the distribution of ovastacin in BaP-exposed oocytes Next, we examined the localization and protein level of ovastacin, the first identified component of CGs in mammals which is responsible for the post-fertilization cleavage of sperm recognizing site ZP2 to block sperm binding and polyspermy, after BaP exposure. Abnormal distribution of ovastacin was observed in BaP-exposed oocytes by showing the loss of localization and much lower signals of fluorescence intensity than those in control oocytes (16.7 ± 1.0, n = 90 vs 7.7 ± 1.9, n = 97, P = 0.003; Fig. 7A and B), suggesting that sperm binding site might be prematurely lost in BaP-exposed unfertilized oocytes. In contrast, after supplementation with melatonin, the defects of both CGs and ovastacin were improved tocomparable control levels (10.4 ± 1.5, n = 88 vs 16.7 ± 1.0, n = 90, P = 0.014; Fig. 7A and B). Figure 7 View largeDownload slide Effects of melatonin on the exocytosis of ovastacin and sperm binding to the zona pellucida in BaP-exposed porcine oocytes. (A) Representative images of ovastacin localization in control, BaP-exposed and melatonin-supplemented oocytes. Scale bar, 20 μm. (B) The fluorescence intensity of ovastacin was measured in control, BaP-exposed and melatonin-supplemented oocytes. (C) Eggs and two-cell embryos from control, BaP-exposed and melatonin-supplemented oocytes were incubated with capacitated sperm for 1 h to perform the sperm binding assay. Scale bar, 20 μm. (D) The number of sperm binding to the control, BaP-exposed and melatonin-supplemented eggs was counted. **P < 0.01, ***P < 0.001. Figure 7 View largeDownload slide Effects of melatonin on the exocytosis of ovastacin and sperm binding to the zona pellucida in BaP-exposed porcine oocytes. (A) Representative images of ovastacin localization in control, BaP-exposed and melatonin-supplemented oocytes. Scale bar, 20 μm. (B) The fluorescence intensity of ovastacin was measured in control, BaP-exposed and melatonin-supplemented oocytes. (C) Eggs and two-cell embryos from control, BaP-exposed and melatonin-supplemented oocytes were incubated with capacitated sperm for 1 h to perform the sperm binding assay. Scale bar, 20 μm. (D) The number of sperm binding to the control, BaP-exposed and melatonin-supplemented eggs was counted. **P < 0.01, ***P < 0.001. To ask whether abnormal ovastacin distribution would lead to the sperm binding defect in BaP-exposed oocytes, sperm binding assay was performed using both unfertilized eggs and two-cell embryos. The sperm head was counterstained with Hoechst to count the number of sperm binding to the zona pellucida. In control unfertilized eggs, zona pellucida supported the binding of numerous sperm, and in control two-cell embryos, zona pellucida no longer supported any sperm binding due to the loss of sperm binding site following fertilization (Fig. 7C). However, in BaP-exposed unfertilized eggs, the number of sperm binding to the zona pellucida was remarkably reduced compared to the controls which was restored by the supplementation of melatonin (183 ± 1.5, n = 59, P < 0.001 vs 55.6 ± 1.9, n = 63 vs 108 ± 1.3, n = 61, P = 0.038; Fig. 7C and D). Melatonin improves the fertilization ability of BaP-exposed oocytes The observed defective sperm binding in BaP-exposed oocytes prompted us to further examine their fertilization potential. As shown in Fig. 8A, most of control oocytes were able to be fertilized and develop to two-cell embryos, while BaP-exposed oocytes had significantly decreased fertilization rates (62.5 ± 1.1%, n = 140 vs 27.3 ± 1.3%, n = 133, P < 0.001; Fig. 8B). Meanwhile, in the melatonin-supplemented oocytes, the rates of fertilization were elevated to the control level (47.1 ± 1.5%, n = 149, P = 0.026; Fig. 8B). Figure 8 View largeDownload slide Effects of melatonin on the fertilization ability of BaP-exposed porcine oocytes. (A) Representative images of fertilized eggs in control, BaP-exposed and melatonin-supplemented groups. Scale bar (left to right), 150, 50 and 20 μm. (B) IVF rate was recorded in control, BaP-exposed and melatonin-supplemented oocytes. ***P < 0.001. Figure 8 View largeDownload slide Effects of melatonin on the fertilization ability of BaP-exposed porcine oocytes. (A) Representative images of fertilized eggs in control, BaP-exposed and melatonin-supplemented groups. Scale bar (left to right), 150, 50 and 20 μm. (B) IVF rate was recorded in control, BaP-exposed and melatonin-supplemented oocytes. ***P < 0.001. Melatonin suppresses oxidative stress induced by BaP exposure in porcine oocytes BaP is known to induce oxidative stress in various cells to impair the normal cellular functions, we thus tested if the defects observed in BaP-exposed porcine oocytes were also mediated by this mechanism. To verify this assumption, we compared the ROS levels between control and BaP-exposed oocytes. In control oocytes, the signals were hardly detected (Fig. 9A). However, BaP exposure considerably increased the green signals in the cytoplasm of oocytes (Fig. 9A). Consistently, the fluorescent intensity of ROS was apparently increased in BaP-exposed oocytes compared to controls (0.9 ± 1.1, n = 103 vs 38.8 ± 1.1, n = 98, P < 0.001; Fig. 9B). Treatment with melatonin effectively reduced the level of ROS (7.1 ± 1.8, n = 110, P = 0.015; Fig. 9B). Figure 9 View largeDownload slide Effects of melatonin on the reactive oxygen species (ROS) level and early apoptosis in BaP-exposed porcine oocytes. (A) Representative images of ROS levels in control, BaP-exposed and melatonin-supplemented oocytes. Scale bar, 20 and 40 μm. (B) The fluorescence intensity of ROS in control, BaP-exposed and melatonin-supplemented oocytes were measured by the confocal microscopy using identical settings and parameters. (C) Representative images of apoptotic oocytes in control, BaP-exposed and melatonin-supplemented groups. Scale bar, 20 μm. (D) The rate of early apoptosis was recorded in control, BaP-exposed and melatonin-supplemented oocytes. ***P < 0.001. Figure 9 View largeDownload slide Effects of melatonin on the reactive oxygen species (ROS) level and early apoptosis in BaP-exposed porcine oocytes. (A) Representative images of ROS levels in control, BaP-exposed and melatonin-supplemented oocytes. Scale bar, 20 and 40 μm. (B) The fluorescence intensity of ROS in control, BaP-exposed and melatonin-supplemented oocytes were measured by the confocal microscopy using identical settings and parameters. (C) Representative images of apoptotic oocytes in control, BaP-exposed and melatonin-supplemented groups. Scale bar, 20 μm. (D) The rate of early apoptosis was recorded in control, BaP-exposed and melatonin-supplemented oocytes. ***P < 0.001. Because the high level of ROS always induces apoptosis, we next examined the early apoptosis in oocytes by Annexin-V staining. The result revealed that the fluorescent signals were rarely observed in control oocytes (Fig. 9C), but it was clearly found on the plasma membrane of BaP-exposed oocytes. The rate of apoptotic oocytes was dramatically higher in BaP-exposed group than that in controls (0.03 ± 0.1, n = 109 vs 23.4 ± 1.7, n = 111, P < 0.001; Fig. 9D), but rescued in melatonin-supplemented group (7.1 ± 1.0, n = 107, P = 0.001; Fig. 9D). Discussion BaP, a ubiquitous environmental pollutant and carcinogen, does a great damage to the pulmonary cells in both animals and humans (Tao et al., 2012; Callén et al., 2014). Furthermore, BaP has been reported to disrupt the normal ovarian functions (Rekhadevi et al., 2014). It has been shown that significantly increased levels of BaP are found in the follicular fluid of women exposed to mainstream and/or sidestream cigarette smoke (Neal et al., 2008; Sadeu et al., 2011a,b). In addition, BaP is present in polluted air, petroleum products, charbroiled foods, contaminated water and incomplete combustion of fossil fuels (Ramesh et al., 2004). Clearly, humans are continuously exposed to BaP and it is of importance to determine the specific contribution of this pollutant to the failure of oocyte development. To investigate the potential impact of BaP exposure on the oocyte meiotic progression, we selected pig oocytes as a model because they are physiologically more similar to human oocytes than mouse oocytes, and thus the data could provide a more solid basis for human reproductive research. We first examined the effect of BaP exposure on the first polar body extrusion, a critical indicator for oocyte maturation, in porcine oocytes. With the use of increasing concentrations of BaP, the exposed oocytes displayed a remarkably reduced rate of polar body extrusion with poor expansion of cumulus cells, suggesting that BaP exposure perturbs the normal meiotic progression. This is further evidenced by the observations that spindle morphologies and chromosome alignment were severely perturbed in the BaP-exposed oocytes. The spindle abnormalities prompted us to assess the microtubule dynamics. Tubulin acetylation is a post-translational modification that occurs on Lys-40 of the α-tubulin subunit (Piperno et al., 1987; Bulinski et al., 1988) and is abundant in stable microtubules but is absent in dynamic subcellular structures (Robson and Burgoyne, 1989). Additionally, it has been recently reported that acetylated α-tubulin is found in stabilized microtubules in mouse oocytes (Dai et al., 2016; Zhang et al., 2017b). Our data illustrated that BaP exposure remarkably decreased the acetylation level of α-tubulin, indicating that the loss of microtubule stability might be one of the leading causes resulting in the disorganized spindle assembly. The actin filament is another indispensable component of cytoskeleton whose dynamics has been shown to be critical for the polar body extrusion and meiotic progression during meiosis (Field and Lénárt, 2011). Our findings validated that the compromised actin dynamics might be an important reason leading to the porcine oocyte meiotic arrest when exposed to BaP. Because mitochondria represent the primary source of ATP production within oocytes (Dumollard et al., 2007) and are critical for normal oocyte maturation, mitochondrial distribution was examined in BaP-exposed oocytes. Our findings revealed that the mitochondrial integrity shown by the localization and signal intensity was considerably impaired, suggesting that BaP exposure does compromise the functionality of the mitochondria in porcine oocytes. Mammalian cortical granules are oocyte-specific and membrane-bound vesicles that form a uniform layer in the subcortical region of fully grown eggs to exert functions during fertilization and prevention of polyspermy (Burkart et al., 2012). The normal distribution of CGs is often regarded as a sign of oocyte cytoplasmic maturation. We then tested if BaP exposure would perturb the CG dynamics. Our data revealed that CGs were absent from the subcortex of BaP-exposed oocytes, indicating that cytoplasmic maturation is impaired as a result of BaP exposure. Ovastacin, a mammalian CG component, is responsible for post-fertilization cleavage of ZP2 at the N-terminus to definitively block sperm binding to the zona pellucida surrounding fertilized eggs (Burkart et al., 2012). In line with the defect of cortical granule dynamics, the localization of ovastacin was disrupted and the amount was reduced in BaP-exposed oocytes. Meanwhile, much less sperm binding to BaP-exposed eggs were observed. Finally, we found that BaP-exposed porcine oocytes exhibited the significantly higher level of ROS and signs of early apoptosis. Melatonin partially restored all the defects related to the meiotic arrest and fertilization failure we observed in BaP-exposed oocytes via inhibition of ROS levels. Collectively, we provide several lines of evidence demonstrating that melatonin improves the quality of oocytes exposed to BaP by promoting meiotic progression and maintaining fertilization ability through inhibiting BaP-induced oxidative stress. Authors’ roles Y.M. and B.X. designed the research; Y.M., C.Z., Q.B., Z.C., X.S., Y.L., M.Z. and X.D. performed the experiments; Y.M. and B.X. analysed the data; Y.M. and B.X. wrote the article. Funding National Natural Science Foundation of China (31571545) and the Natural Science Foundation of Jiangsu Province (BK20150677). Conflict of interest The authors have no conflict of interest to disclose. Acknowledgements We would like to thank Dr Jurrien Dean for providing antibodies and Dr Russel Reiter for comments on the article. References Baird WM, Hooven LA, Mahadevan B. Carcinogenic polycyclic aromatic hydrocarbon‐DNA adducts and mechanism of action. Environ Mol Mutagen  2005; 45: 106– 114. 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TI - The protective role of melatonin in porcine oocyte meiotic failure caused by the exposure to benzo(a)pyrene
JF - Human Reproduction
DO - 10.1093/humrep/dex331
DA - 2018-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/the-protective-role-of-melatonin-in-porcine-oocyte-meiotic-failure-U0fBf6o7l4
SP - 116
EP - 127
VL - 33
IS - 1
DP - DeepDyve
ER -