College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China.
School of Basic Medical Sciences, Peking University, Beijing 100191, China.
Hum Reprod. 2018 Jan 1;33(1):116-127. doi: 10.1093/humrep/dex331.
Does melatonin restore the benzo(a)pyrene (BaP)-induced meiotic failure in porcine oocytes?
Melatonin effectively inhibits the increased reactive oxygen species (ROS) level and apoptotic rate in BaP-exposed porcine oocytes to recover the meiotic failure.
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.
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.
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.
褪黑素能否恢复苯并（a）芘（BaP）诱导的猪卵母细胞减数分裂失败？
褪黑素能有效抑制 BaP 暴露引起的猪卵母细胞中活性氧（ROS）水平和凋亡率的增加，从而恢复减数分裂失败。
BaP 是一种广泛存在于颗粒物（PM2.5）中的环境致癌物质，已被证明具有生殖系统毒性。BaP 暴露会破坏类固醇平衡，改变卵巢雌激素受体的表达，并通过快速耗尽原始卵泡池导致卵巢早衰而导致过早卵巢衰竭。此外，BaP 的急性暴露对卵泡生长、排卵和黄体形成有短暂的不良影响，导致暂时性不孕。
研究设计、规模、持续时间：猪卵母细胞被随机分配到对照组、BaP 暴露组和褪黑素补充组。BaP 溶解在二甲亚砜中，并分别用成熟培养基稀释至终浓度为 50、100 或 250 μM。褪黑素溶解在绝对乙醇中，并分别用成熟培养基稀释至终浓度为 1 nM、100 nM、10 μM 和 1 mM。处理后从每组中取出体外培养的卵母细胞进行后续分析。
参与者/材料、设置、方法：通过免疫染色、荧光强度定量和/或免疫印迹分析细胞骨架组装、线粒体完整性、皮质颗粒动力学、ovastacin 分布、ROS 水平和凋亡率来评估卵母细胞减数分裂能力。通过精子结合试验和 IVF 检查卵母细胞的受精能力。
BaP 暴露通过破坏减数分裂装置导致卵母细胞减数分裂失败（P = 0.001），表现出明显的纺锤体组装缺陷（P = 0.003）、肌动蛋白动力学（P < 0.001）和线粒体完整性（P < 0.001）。此外，BaP 暴露导致皮质颗粒（P < 0.001）和 ovastacin 异常分布（P = 0.003），这与观察到未受精的 BaP 暴露卵周围的透明带周围只有较少的精子结合一致，导致受精失败（P < 0.001）。相反，褪黑素补充通过抑制 ROS 水平的升高（P = 0.015）和凋亡率（P = 0.001），至少部分恢复了 BaP 暴露引起的所有减数分裂缺陷。
局限性、谨慎的原因：我们研究了 BaP 对体外卵母细胞减数分裂成熟的负面影响，但没有研究体内情况。
我们的研究结果不仅深入阐明了 BaP 诱导的卵母细胞减数分裂失败的潜在机制，还扩展了对环境污染物如何影响人类生殖系统的认识。
研究资金/利益冲突：本研究得到了国家自然科学基金（31571545）和江苏省自然科学基金（BK20150677）的支持。作者没有利益冲突需要披露。
