GLUTS AND GLUCOSE TRANSPORT IN THE MOUSE BLASTOCYST

描述(申请人提供)：本提案的目的是确定自噬这一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，以及新陈代谢诱导的这一事件的变化是否会导致胚胎生长和发育异常。我们在上一次资助周期中建立了糖尿病和遗传性胚胎葡萄糖转运异常的小鼠模型，所有小鼠都经历了胚泡凋亡增加，并导致显著的生长缺陷，其中一些伴有特殊的先天性畸形。在缺乏自噬蛋白的胚胎中也可以看到类似的生长异常，最近的其他研究表明，发育过程中的基础自噬对于平衡营养供应的增殖和凋亡至关重要。我们的初步数据表明，葡萄糖运输方面的缺陷，无论是基因敲除小鼠、反义小鼠还是受精后96小时内暴露在异常条件下的对照组，都会持续到成年，这表明生长缺陷可能从发育的最早阶段就被编程到胚胎中。我们推测，自噬可能通过调节蛋白质的降解和调节细胞凋亡在这一过程中发挥重要作用。在这种情况下，葡萄糖转运的变化导致胚泡阶段胚胎自噬的变化可能会规划发育和妊娠结果。这些研究具有很高的创新性，因为它们是首次研究自噬在哺乳动物胚胎发育和妊娠结局中的作用。我们的理论基础是，通过改变自噬的模式，与葡萄糖供应波动相关的不良妊娠结局可能是可以避免和/或可逆的。这些研究旨在检验这一假设。具体目标1检查野生型植入前胚胎的自噬基线数量，并确定体外操作这一过程是否会导致胚泡异常，从而改变妊娠结局。具体目的2评估在已建立的葡萄糖转运蛋白缺乏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。特定目的3研究GLUT8在小鼠囊胚自噬中可能发挥的作用。这项研究的结果将确定在哺乳动物植入前发育过程中改变的自噬状态中出现的异常蛋白质降解的作用。自噬过多或过少的模型会导致胚胎死亡和神经上皮细胞异常，然而，这种发育损害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想法，以提高糖尿病患者、反复妊娠丢失患者以及接受试管受精的患者的怀孕率。公共卫生相关性：在这项建议中，我们假设小鼠胚泡中的基础自噬受到代谢环境变化的调节，自噬中的变化可能会导致不良的妊娠结局。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。在这些模型中，通过调节植入前的自噬速率，然后将这些胚胎转移到代孕小鼠，这些操作的结果就被确定了。

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.