GLUTS AND GLUCOSE TRANSPORT IN THE MOUSE BLASTOCYST

描述（由申请人提供）：本提案的目的是确定自噬（一种高度保守的细胞过程）是否在哺乳动物着床前发育中发挥作用，以及代谢诱导的该事件改变是否导致异常胚胎生长和发育。我们在最后一个赠款周期建立的胚胎葡萄糖转运异常的小鼠模型，包括糖尿病和遗传性模型，都经历了胚泡凋亡增加，并导致显著的生长缺陷，有些伴有特定的先天性畸形。在缺乏自噬蛋白的胚胎中也观察到类似的生长异常，最近的其他研究表明，发育过程中的基础自噬对于平衡增殖和凋亡以应对营养供应至关重要。我们的初步数据表明，葡萄糖转运缺陷，无论是基因敲除小鼠，反义小鼠还是在受精后前96小时暴露于异常条件的对照组，都持续到成年期，这表明生长缺陷可能是从发育的最早阶段“编程”到胚胎的。我们推测，自噬可能在这一过程中发挥重要作用，通过调节蛋白质的降解，也调节凋亡。在这种情况下，葡萄糖转运的变化导致胚泡期胚胎自噬的变化可能会编程发育和妊娠结果。这些研究具有高度创新性，因为它们是第一批研究自噬在哺乳动物胚胎发育和妊娠结局中的作用的研究。我们的理由是，与葡萄糖可用性波动相关的不良妊娠结局可能是可以避免的和/或通过改变自噬模式可逆的。这些研究旨在验证这一假设。具体目标1检查了野生型植入前胚胎中自噬的基线量，并确定体外操作该过程是否会导致改变妊娠结局的胚泡异常。具体目标2评估了在葡萄糖转运蛋白缺陷的小鼠模型中操纵自噬的效果，以及这些变化是否影响胚胎发育和能力。特定目标3研究了GLUT 8在小鼠胚泡自噬中可能发挥的作用。这项研究的结果将建立异常蛋白质降解的作用，在哺乳动物植入前发育过程中改变自噬状态。过量或不足的自噬模型导致胚胎死亡和异常神经上皮，然而，这种发育损伤的病因和时间尚不清楚。这些拟议的实验将试图探索这一现象，并可能为治疗干预提供思路，以提高糖尿病患者，复发性妊娠丢失以及接受IVF的患者的妊娠率。公共卫生相关性：在这个提议中，我们假设小鼠胚泡中的基础自噬受代谢环境变化的调节，并且自噬的改变可能导致不良的妊娠结局。来自糖尿病小鼠的囊胚表现出自噬途径的变化，葡萄糖摄取减少的遗传模型也是如此。通过调节这些模型中植入前期的自噬速率，然后将这些胚胎转移到代孕小鼠中，确定这些操作的结果。

DESCRIPTION (provided by applicant): The objective of this proposal is to determine whether autophagy, a highly conserved cellular process, plays a role in mammalian preimplantation development and whether metabolism-induced alterations to this event result in abnormal embryo growth and development. Our mouse models of abnormal embryonic glucose transport, both diabetic and genetic, which we established during the last grant cycle, all experience increased blastocyst apoptosis and result in significant growth deficiencies, some accompanied by specific congenital malformations. Similar growth abnormalities are seen in embryos lacking autophagy proteins as well, and other recent studies suggest that basal autophagy during development is critical to balancing proliferation and apoptosis in response to nutrient supply. Our preliminary data suggest that defects in glucose transport, whether gene knockout mice, antisense mice or controls exposed to abnormal conditions during the first 96 hours post fertilization, persist into adulthood, suggesting that growth deficiencies may be "programmed" in to the embryos from the earliest stages of development. We hypothesize that autophagy may play an important role in this process by regulating the degradation of proteins and also modulating apoptosis. In this case, changes in glucose transport leading to changes in autophagy in the blastocyst stage embryo may program development and pregnancy outcomes. These studies are highly innovative since they are some of the first to examine the role of autophagy in mammalian embryo development and pregnancy outcome. Our rationale is that poor pregnancy outcomes related to fluctuations in glucose availability may be avoidable and/or reversible by changing the pattern of autophagy. These studies aim to test this hypothesis. Specific Aim 1 examines the baseline amount of autophagy in wildtype preimplantation embryos and determines if in vitro manipulation of this process results in blastocyst abnormalities which change pregnancy outcome. Specific Aim 2 evaluates the effect of manipulating autophagy in established mouse models of glucose transporter deficiencies and whether these changes affect embryo development and competence. Specific Aim 3 investigates the possible role GLUT8 may play in autophagy in the mouse blastocyst. The results of this study will establish the role of abnormal protein degradation seen in altered autophagic states during mammalian preimplantation development. Models of excess or deficient autophagy result in embryonic lethality and abnormal neuroepithelium, however, the etiology and timing of this developmental insult are unknown. These proposed experiments will attempt to explore this phenomenon and perhaps provide ideas for therapeutic interventions to improve pregnancy rates in patients with diabetes, recurrent pregnancy loss as well as patients undergoing IVF. PUBLIC HEALTH RELEVANCE: In this proposal, we hypothesize that basal autophagy in the murine blastocyst is regulated by metabolic milieu changes and can alterations in autophagy can result in poor pregnancy outcomes. Blastocysts from diabetic mice exhibit changes in autophagic pathways, as do genetic models of decreased glucose uptake. By modulating the rate of autophagy during the preimplantation period in these models and then transferring these embryos into surrogate mice, the outcomes of these manipulations are determined.