Effect of endocrine disrupting compounds on gonadotropin-releasing hormone and its mechanisms

 描述（由申请人提供） 内分泌干扰化合物（EDCs），如双酚A和邻苯二甲酸酯，普遍存在于食物和水中，具有毒理学人类健康问题，特别是在发育中的儿童和育龄妇女中。最近，在包括美国在内的许多工业化国家，暴露于内分泌干扰物被怀疑是过去几十年在儿童中观察到的青春期提前发病的显著趋势的部分原因。促性腺激素释放激素（GnRH）脉冲释放的频率和幅度增加被认为在人类和动物青春期的启动中具有核心作用。最近发现了GnRH的上游正性和负性调节因子，称为kisspeptin和促性腺激素抑制激素（GnIH）。在这个拟议的项目中，早期发育阶段的斑马鱼将被用来测试的核心假设，内分泌干扰物影响上游调节因子，kisspeptin和GnIH的影响GnRH神经元的脉动。由于GnRH神经元位于大脑深处，因此在技术上很难监测体内GnRH神经元的表达、兴奋和同步。两个斑马鱼转基因品系的发展，包括短寿命荧光标记的GnRH将允许这样的可视化;第一个监测mRNA的表达，另一个监测兴奋，两者最终将允许用于筛选 潜在的内分泌干扰化合物。这些转基因鱼苗将用双酚A和邻苯二甲酸二（2-乙基己基）酯处理，并将用多光子荧光显微镜记录延时图像。最后，kisspeptin和GnIH的作用将在转基因鱼系中通过吗啉实验用EDC暴露的幼虫进行研究。本项目的目的是了解内分泌干扰物干扰GnRH的分子毒理学机制。该项目的广泛和长期目标是开发高通量筛选化合物的工具，以保护人类的生殖健康。 本项目的PD/PI，Motoko Mukai博士，DVM，Ph.D.是一名委员会认证的毒理学家，在动物饲料/食品安全方面具有临床诊断经验。她将接受约瑟夫·费特乔博士的指导，他是一位知名的领先神经生物学家，一直在使用斑马鱼幼虫研究神经发生，神经动力学，神经回路和神经元的再生。来自麦迪逊的威斯康星州大学的Richard Peterson博士是研究内分泌干扰物对生殖系统影响的专家，他将担任该项目的共同导师，提供分子毒理学指导。

 DESCRIPTION (provided by applicant) Endocrine disrupting compounds (EDCs), such as Bisphenol A and phthalates, are found ubiquitously in food and water and are of toxicological human health concern especially in developing children and women in their child-bearing age. Exposures to EDCs have recently been suspected to be part of the cause for a significant trend of early puberty onset observed in children over the past few decades in many industrialized countries including the U.S. However the mechanism of how this may occur is not well understood. Increased frequency and amplitude of the pulsatile release of gonadotropin releasing hormone (GnRH) is considered to have a central role in initiating puberty in humans and animals. There has been recent discovery of upstream positive and negative regulators of GnRH, called kisspeptin and gonadotropin inhibitory hormone (GnIH). In this proposed project, early developmental stage zebrafish will be used to test the central hypothesis that endocrine disrupting chemicals affect the pulsatility of GnRH neurons by affecting upstream regulators, kisspeptin and GnIH. It has been technically difficult to monitor the expression, excitation, and synchronization of GnRH neurons in vivo due to its location deep inside the brain. Development of two zebrafish transgenic lines consisting of short-life fluorescence-labeled GnRH will allow such visualization; first one to monitor mRNA expression and the other to monitor excitation, and both will ultimately allow for use in screening for potential endocrine disrupting compounds in the future. These transgenic fish larvae will be treated with Bisphenol A and Bis(2-ethylhexyl) phthalate and time-lapse images will be recorded with a multiphoton fluorescence microscope. Lastly, roles of kisspeptin and GnIH will be investigated with the EDC exposed larvae by morpholino experiments in the transgenic fish lines. Goal of this project is to understand the molecular toxicology mechanism of GnRH disruption by EDCs. Broad and long term goal of this project is to develop tools for high-throughput screening of chemical compounds in order to protect reproductive health in humans. The PD/PI of this project, Dr. Motoko Mukai, DVM, Ph.D. is a board certified toxicologist with clinical diagnostic experiences in animal feed/food safety. She will be mentored by Dr. Joseph Fetcho, a well- established leading neurobiologist, who has been using zebrafish larvae to study neurogenesis, neurodynamics, neural circuits, and regeneration of neurons. Dr. Richard Peterson from University of Wisconsin, Madison, expert in a study of endocrine disruptor on reproduction system will serve as a co-Mentor on this project for molecular toxicology guidance.