胚胎染色质开放性在草铵膦父系暴露致子代神经行为发育异常中的作用

Glufosinate ammonium (GLA) is a widely used herbicide in agriculture, covering all the composition of Chinese diet, which is closely related to the life and health. Although it has been reported that GLA might be potentially neurotoxic, whether parental exposure to GLA affects neurodevelopment in offspring remains poorly understood. In our pilot study, we found that male mice exposed to low dose GLA would have pups with abnormal learning, memory, and social interaction, as well as a decreased rate of preimplantation embryogenesis. So we speculated that paternal GLA exposure could disturb the early embryogenesis leading to subsequent neurodevelopmental abnormalities. In this project, we are going to administer male mice with different doses of GLA, and get the pups by natural mating and in vitro fertilization respectively. Phenotyping including neurobehavioral test, histopathology and molecular biology will be used to confirm the toxic effects of paternal GLA exposure on the neurodevelopment in offspring. Preimplantation embryos from zygote to blastocyst are collected to examine if it is disturbed in early embryogenesis. As regarding to the chromatin accessibility, we will perform both sequencing and bioinformatics to reveal the regulation of GLA on the transcription during embryogenesis. This project will open avenues for the study of transgenerational neurotoxicity of GLA, provide theoretical references for risk assessment of GLA, and contribute to improving the quality of life and health of reproduction.

已知草铵膦（GLA）是农业广泛使用的除草剂，覆盖我国居民膳食各个组成方面，与生活和健康密切相关。虽然GLA以往报道具有潜在神经毒性，但其亲代暴露是否影响子代神经行为发育尚不明确。我们前期研究发现，长期暴露于低剂量GLA雄鼠子代的学习、记忆和社交功能异常，胚胎分化率降低。由此推测，GLA组小鼠早期胚胎发育受到干扰进而导致后续神经行为发育异常。本项目通过构建不同剂量GLA雄性小鼠暴露模型，分别利用自然交配和体外受精方式得到子鼠，采用神经行为学测试和分子生物学等方法，确认GLA父系暴露对子鼠神经行为发育的毒作用；通过体内、外培养方式收集自受精卵至囊胚阶段胚胎，阐明早期胚胎发育所受干扰，并以染色质开放性为研究对象，结合测序和生信技术，揭示GLA父系暴露对早期胚胎发育过程基因转录活性的表观调控作用。本项目的开展为有效预防GLA的健康危害提供科学依据，为提高人口素质、促进人类生殖健康作出贡献。

草铵膦（glufosinate-ammonium，GLA）作为一种新型触杀型有机磷除草剂，广泛施用于农作物除草，可能导致生物体的慢性暴露。为了探究草铵膦父系暴露对子代小鼠神经行为发育的影响，本项目通过建立低剂量草铵膦暴露模型，检测草铵膦父系暴露后子代小鼠社交能力、学习记忆能力、焦虑样行为以及运动能力等神经行为改变，观察子代小鼠海马和初级躯体感觉皮层中兴奋/抑制性神经元和胶质细胞的形态数量及蛋白表达，并利用转录组测序对草铵膦暴露后的早期胚胎进行差异表达基因的鉴定和功能分析。研究发现草铵膦父系暴露导致子代雌性小鼠的社交新奇性降低、学习记忆能力下降，引起海马和初级躯体感觉皮层中神经元兴奋/抑制性活动失衡、胶质细胞活化、突触结构异常。转录组测序发现Ccl3、Ccl4等与细胞趋化因子相关的基因在草铵膦组二细胞期胚胎表达水平出现显著改变，免疫荧光结果同样显示CCL3和CCL4蛋白表达水平均显著升高，提示草铵膦父系暴露可能在胚胎早期发育阶段激活炎症和凋亡反应。借助miRNA显微注射技术对早期胚胎中炎症通路相关基因进行干预，结果发现干预组子代雌性小鼠学习记忆能力和社交新奇性均恢复正常。综上，本项目发现草铵膦父系暴露通过激活早期胚胎发育阶段神经炎症反应，导致子代雌性小鼠记忆损伤，为规范草铵膦的合理使用和制订更完善的公共卫生政策保障后代健康提供了科学依据。

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