THE EFFECT OF DIOXINS ON GLUCOSE HOMEOSTASIS IN MURINE TESTES

描述(申请人提供)：二恶英是一类剧毒和广泛分布的环境危害，与男性生殖细胞功能异常有关，甚至与父亲接触相关的不良妊娠结局有关。其中许多影响需要几年时间才能显现。研究人员试图在细胞和分子水平上表征二恶英结合的毒性，但到目前为止，其机制仍不完全清楚。本研究的目的是确定二恶英对雄配子的作用机制是否涉及芳香烃受体(AhR)和葡萄糖转运之间的通讯。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，或许是干细胞中的基因表达。这些变化可能要到比接触更晚的时候才会显现出来，因此可能解释了父亲对后代和怀孕结果的影响。以前的研究表明，AhR受体，也被称为“二恶英受体”，可能在身体其他各种组织的上皮细胞中葡萄糖利用减少的过程中发挥作用。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发挥着重要作用，某些细胞信号蛋白/受体在精子发生的不同阶段对这些生殖细胞中的葡萄糖稳态起着调节作用。我们实验室以前的研究表明，葡萄糖稳态中断对小鼠的精子发生、精子活力和受精能力有几种功能性损害。此外，我们还建立了用于睾丸切片的激光显微切割方案，并成功地验证了我们的技术。我们假设，接触二恶英会通过激活AhR受体和随后破坏葡萄糖转运体(GLUT)亚型来扰乱精子发生，从而对基本的细胞功能和发育产生不利影响。我们针对以下具体目标来研究这一假说。具体目的1：睾丸中的哪些细胞类型，体细胞和生精生殖细胞阶段表达芳烃受体？特异性目的2.TCDD暴露对睾丸GLUT8、GLUT9a和GLUT9b蛋白和mRNA表达的影响？运输机的位置改变了吗？具体目标3：缺乏AhR表达是否会消除TCDD在睾丸中的作用？AhR缺乏会影响睾丸中葡萄糖转运蛋白的表达吗？！提出这一建议的理由是，确定对男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父系传播畸形和男性不育中的作用。如果成功地完成了这些目标，我们将阐明芳烃受体激活的一种新的主要下游效应。外源性二恶英激活AhR，导致睾丸细胞间葡萄糖利用率下降，将突出一种可能的作用机制，可能有助于我们进一步了解某些与父亲的有毒暴露有关的生殖结果。！！

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. ! !