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

 描述(由申请人提供) 内分泌干扰物(EDCs)，如双酚A和邻苯二甲酸酯，广泛存在于食物和水中，对人类健康具有毒理意义，特别是对育龄儿童和妇女。在过去的几十年里，包括美国在内的许多工业化国家都观察到了儿童青春期提前的显著趋势，最近人们怀疑暴露于EDCs可能是导致这一趋势的部分原因。然而，这种情况发生的机制尚不清楚。促性腺激素释放激素(GnRH)脉冲式释放的频率和幅度的增加被认为在人类和动物的青春期启动中起着核心作用。最近发现了促性腺激素释放激素上游的正负调控因子，称为Kispeptin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检验这一中心假设，即内分泌干扰物通过影响上游调节因子Kispeptin和GnIH来影响GnRH神经元的脉动性。由于GnRH神经元位于大脑深处，因此在体内监测GnRH神经元的表达、兴奋和同步在技术上一直是困难的。开发两个由短寿命荧光标记的GnRH组成的斑马鱼转基因株系将使这种可视化成为可能；第一个用于监测mrna表达，另一个用于监测兴奋，最终两者都将用于筛查。 寻找未来可能干扰内分泌的化合物。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，时间推移图像将用多光子荧光显微镜记录下来。最后，将通过在转基因鱼类品系上进行吗啡实验，研究Kispeptin和GnIH在EDC暴露幼虫中的作用。本课题的目的是了解内分泌细胞干扰促性腺激素释放激素的分子毒理学机制。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。该项目的PD/PI是具有动物饲料/食品安全临床诊断经验的委员会认证毒理学家Mukai博士。她将得到著名神经生物学家约瑟夫·费乔博士的指导，他一直在使用斑马鱼幼虫研究神经发生、神经动力学、神经回路和神经元再生。威斯康星大学麦迪逊分校的理查德·彼得森博士是生殖系统内分泌干扰物研究的专家，他将担任该项目的联合导师，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.