THE EFFECT OF DIOXINS ON GLUCOSE HOMEOSTASIS IN MURINE TESTES

描述（由申请人提供）：二恶英是一类剧毒且广泛分布的环境危害物质，与男性生殖细胞功能异常有关，甚至与父亲接触二恶英导致的不良妊娠结果有关。其中许多影响需要数年才能显现出来。研究人员试图在细胞和分子水平上描述二恶英结合的毒性，但迄今为止，其机制仍不完全清楚。本研究的目的是确定二恶英暴露对雄性配子的作用机制是否涉及芳烃受体（AhR）和葡萄糖运输之间的通信。此外，我们试图确定二恶英暴露是否会影响精子发生不同阶段的生殖细胞和干细胞的基因表达。这些变化可能要比暴露时间晚得多才会显现出来，因此可以解释父亲对后代和妊娠结局的影响。先前的研究表明，AhR受体，也被称为“二恶英受体”，可能在降低身体其他组织上皮细胞对葡萄糖的利用方面发挥作用。我们的初步数据表明，葡萄糖转运蛋白在睾丸和精子发生中起着重要作用，某些细胞信号蛋白/受体在精子发生的各个阶段调节这些生殖细胞的葡萄糖稳态中起作用。我们实验室之前的研究表明，葡萄糖稳态紊乱会对小鼠的精子发生、精子活力和受精能力造成一些功能损害。此外，我们已经建立了一个激光显微解剖方案的睾丸部分，并已成功地验证了我们的技术。我们假设二恶英暴露通过激活AhR受体破坏精子发生，随后葡萄糖转运蛋白（GLUT）异构体的破坏对基本细胞功能和发育产生不利影响。我们针对以下具体目标来调查这一假设。特异性目标1：睾丸中哪些细胞类型（体细胞和生精生殖细胞阶段）表达芳烃受体？具体目标2。睾丸中GLUT8、GLUT9a和GLUT9b的蛋白和mRNA表达是否受TCDD影响？传送器的位置改变了吗？特异性目标3：缺乏AhR表达是否会消除TCDD在睾丸中的作用？AhR缺乏是否影响睾丸中葡萄糖转运蛋白的表达？！ 这一建议的基本原理是，确定雄性生殖细胞的毒性损伤机制将有助于更好地理解环境毒素在畸形和男性不育的父系传播中的作用。如果成功完成这些目标，我们将阐明芳基烃受体激活的一个新的主要下游效应。二恶英对AhR的外源性激活，导致睾丸细胞中葡萄糖利用率的降低，这可能会突出一种可能的作用机制，从而进一步了解与父亲接触有毒物质有关的某些生殖结果。！ ！

DESCRIPTION (provided by applicant): Dioxins represent a class of highly toxic and widely dispersed environmental hazards that have been implicated in aberrations in male reproductive cell function and even adverse pregnancy outcomes associated with paternal exposure. Many of these effects take several years to manifest. Investigators have attempted to characterize the toxicity of dioxin binding at a cellular and molecular level, but to date the mechanism remains incompletely understood. The objective of this study is to determine whether the mechanism of action of dioxin exposure on male gametes involves communication between the aryl hydrocarbon receptor (AhR) and glucose transport. In addition we seek to determine if dioxin exposure affects gene expression in the germ cells and perhaps stem cells at different stages of spermatogenesis. These changes may not manifest until much later relative to exposure and thus may explain the paternal effects on offspring and pregnancy outcome. Previous studies suggest that the AhR receptor, also known as the "dioxin receptor," may play a role in decreased glucose utilization in epithelial cells in various other tissues of the body. Our preliminary data show that glucose transporters play a significant role in the testes and on spermatogenesis and that certain cell signaling proteins/receptors play a role in modulating glucose homeostasis in these germ cells at the various stages of spermatogenesis. Previous study in our lab has shown several functional detriments to spermatogenesis, sperm motility, and fertilization capability in mice affected with disruptions in glucose homeostasis. Moreover we have established a Laser Microdissection protocol for testis sections and have successful validate our techniques. We hypothesize that dioxin exposure disrupts spermatogenesis through activation of the AhR receptor and subsequent disruption of glucose transporter (GLUT) isoforms adversely affects essential cellular function and development. We address the following specific aims to investigate this hypothesis. SPECIFIC AIM 1: Which cell types, somatic and spermatogenic germ cell stages, in the testes express the Arylhydrocarbon Receptor? SPECIFIC AIM 2. Are protein and mRNA expression of GLUT8, GLUT9a and GLUT9b in the testes affected by exposure to TCDD? Is the location of the transporters altered? SPECIFIC AIM 3: Does a lack of AhR expression eliminate the effect of TCDD in the testes? Does AhR deficiency affect glucose transporter expression in the testes? ! The rationale for this proposal is that identifying the mechanism of toxic injury to male germ cells will lead to a better understanding the role of environmental toxins in paternal transmission of malformations and male infertility. If successful in completing these aims, we will have elucidated a novel major downstream effect of activation of the Aryl hydrocarbon receptor. Exogenous activation of the AhR by dioxins, resulting in decrease glucose utilization among cells in the testes would highlight a likely mechanism of action that could further our understanding of certain reproductive outcomes that have been linked to paternal toxic exposures. ! ! PUBLIC HEALTH RELEVANCE: Dioxins represent a class of highly toxic and widely dispersed environmental hazards that are the unintentional byproducts of many industrial processes. Sperm cell production is known to be adversely affected by dioxin exposure, perhaps contributing to low sperm counts and poor function of the sperm cells. This research will help us better understand how such environmental exposures may affect the male reproductive tract.