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

本研究的目的是：(1)研究着床前胚胎中谷胱甘肽（GSH）的调节机制；(2)探讨谷胱甘肽对小鼠囊胚发育的调控作用；(3)改善研究环境和本科理科教育。研究者早期的研究表明，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。谷胱甘肽是细胞内主要的抗氧化剂，已被证明在防止活性氧、防止毒物、调节细胞分化和触发其他细胞类型的凋亡方面发挥作用。与所有其他动物细胞不同，卵裂期着床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吸收谷胱甘肽，并且对活性氧和谷胱甘肽消耗化学物质非常敏感。因此，卵裂期胚胎似乎在调节细胞总谷胱甘肽的能力方面存在独特的缺陷。由于这些原因，研究这种缺陷是如何和为什么发生的，以及它对分化有什么影响是很有趣的。本基金中描述的研究将调查以下问题：1)为什么卵裂期胚胎不能合成谷胱甘肽；2)这种合成谷胱甘肽的能力在什么时间和哪些细胞中开始；为什么，3)通常在其他细胞中诱导合成谷胱甘肽的处理可以在胚胎中诱导合成谷胱甘肽；4)改变胚胎的谷胱甘肽状态会对分化产生什么影响？该研究将在小鼠胚胎从未受精的卵母细胞发育到囊胚阶段的头4天进行。收集胚胎并使用高效液相色谱（HPLC）分析氧化和谷胱甘肽的总量。编码用于合成谷胱甘肽的酶的基因的表达将通过逆转录酶-聚合酶链反应和免疫印迹法进行研究。胚胎将用氧化剂和消耗谷胱甘肽的化学物质处理，胚胎中不同类型细胞的恢复能力将用高效液相色谱法和荧光显微镜测量。此外，这些治疗对基因表达的影响也将被确定。胚胎将用增加谷胱甘肽含量的化学物质或导致胚胎中谷胱甘肽氧化的化学物质处理，并使用荧光显微镜测量对细胞数量、细胞分化和凋亡的影响。本项目旨在进一步研究谷胱甘肽调控机制在小鼠胚胎早期发育过程中具有独特特征和重要作用的假设。提高对谷胱甘肽调节的基本过程和谷胱甘肽在分化中的作用的理解，对于科学探究技术的发展（用于生产基因工程动物的胚胎干细胞的发育等）、胚胎死亡的原因的理解以及对其他生物过程（信号转导、氧化还原调节、细胞凋亡等）的进一步理解具有重要意义。本科生将积极参与研究，在科学会议上展示他们的研究成果，并在科学期刊上发表他们的研究成果。

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.