RUI: Regulation and Role of Glutathione During Preimplantation Development of the Mouse Embyro

Gardiner本研究项目的目的是（1）研究谷胱甘肽（GSH）在植入前胚胎中的调节机制;（2）研究GSH在控制小鼠囊胚发育中的作用;（3）改善该机构的研究环境和本科科学教育。 研究人员的早期研究表明，植入前小鼠胚胎是一个有趣的和有用的系统，用于研究调节谷胱甘肽（GSH）的过程和GSH在分化中的作用。谷氨酸是主要的细胞内抗氧化剂，并且已显示在保护免受活性氧、保护免受毒物、调节细胞分化和触发其他细胞类型的凋亡中具有作用。与所有其他动物细胞不同，卵裂期植入前胚胎不能重新合成谷胱甘肽。此外，胚胎不能从细胞外来源摄取谷胱甘肽，并且对活性氧和谷胱甘肽消耗化学物质非常敏感。因此，似乎卵裂期胚胎在调节总细胞谷胱甘肽的能力方面是独特缺陷的。由于这些原因，研究这种缺陷如何以及为什么发生以及它对分化有什么影响是有意义的。本研究将探讨以下问题：1）为什么卵裂期胚胎不能合成GSH，2）GSH合成能力开始的时间和细胞，以及为什么，3）GSH合成可以通过通常在其他细胞中诱导的治疗在胚胎中诱导，以及4）改变胚胎的GSH状态对分化有什么影响？ 这项研究将在小鼠胚胎从未受精卵母细胞发育到胚泡阶段的前四天进行。 将采集胚胎，并使用高效液相色谱法（HPLC）分析氧化型和还原型谷胱甘肽的总量。将用逆转录酶-聚合酶链反应和蛋白质印迹法研究编码用于合成谷胱甘肽的酶的基因的表达。胚胎将用氧化剂和谷胱甘肽消耗化学品处理，胚胎中不同细胞类型的恢复能力将用HPLC和荧光显微镜测量。此外，还将确定这些处理对基因表达的影响。胚胎将被处理的化学物质，增加谷胱甘肽的量或化学物质，导致谷胱甘肽在胚胎中的氧化和细胞数量的影响，细胞分化和凋亡将使用荧光显微镜测量。该项目旨在进一步研究的叠加假说，谷胱甘肽调节机制具有独特的功能和重要作用，在小鼠胚胎的早期发育。提高对调节GSH的基本过程和GSH在分化中的作用的理解，对于科学研究技术的发展具有重要意义（开发用于生产基因工程动物的胚胎干细胞等），了解胚胎死亡的原因，并进一步了解其他生物过程（信号转导，氧化还原调节，细胞凋亡等）。本科生将积极参与研究，并将在科学会议上展示他们的结果，并在科学期刊上发表他们的研究。

GardinerThe objectives of this research project are (1) to investigate the mechanisms regulating glutathione (GSH) in the preimplantation embryo; (2) to investigate the role of GSH in the control of development of the mouse blastocyst; and (3) to improve the research environment and undergraduate science education at this institution. The investigator's earlier studies indicate that the preimplantation mouse embryo is an intriguing and useful system for investigating processes that regulate glutathione (GSH) and the role of GSH in differentiation. Glutathione is the major intracellular antioxidant and has been shown to have roles in protection from reactive oxygen species, protection from toxicants, regulation of cell differentiation, and triggering of apoptosis in other cell types. Unlike all other animal cells, the cleavage stage preimplantation embryo cannot synthesize glutathione de novo. In addition, the embryo cannot take up glutathione from extracellular sources and is exquisitely sensitive to reactive oxygen species and glutathione-depleting chemicals. Therefore, it appears that the cleavage stage embryo is uniquely deficient in its ability to regulate total cellular glutathione. For these reasons, it is of interest to study how and why this deficiency occurs, and what effect it has on differentiation. The studies described in this grant will investigate the following questions: 1) why is the cleavage stage embryo incapable of synthesizing GSH, 2) at what time and in which cells does this GSH synthetic capability begin and why, 3) can GSH synthesis be induced in the embryo by treatments that typically induce it in other cells, and 4) what effect will altering the GSH status of the embryo have on differentiation? The research will be conducted on mouse embryos during the first four days of development from the unfertilized oocyte to the blastocyst stage. Embryos will be collected and analyzed for the total amount of oxidized and reduced glutathione using high performance liquid chromatography (HPLC). The expression of genes that code for enzymes used to synthesize glutathione will be investigated with reverse transcriptase - polymerase chain reactions and western blotting. Embryos will be treated with oxidants and GSH-depleting chemicals and the ability of the different cell types in the embryo to recover will be measured with HPLC and fluorescence microscopy. Furthermore, the effects of these treatments on gene expression will also be determined. The embryo will be treated with chemicals that increase the amount of GSH or chemicals that lead to oxidation of glutathione in the embryo and the effects on cell number, cell differentiation, and apoptosis will be measured using fluorescence microscopy.This project is designed to further investigate the overlying hypothesis that glutathione regulatory mechanisms have unique features and important roles during the early development of the mouse embryo. Improving the understanding of the basic processes regulating GSH and the role of GSH in differentiation has significance for development of techniques for scientific inquiry (development of embryonic stems cells for production of genetically engineered animals, etc.), for understanding causes of embryonic mortality, and for furthering the understanding of other biological processes (signal transduction, redox regulation, apoptosis, etc.). Undergraduate students will be active participants in the research and will present their results at scientific meetings and publish their studies in scientific journals.