CHARACTERIZATION OF OXYGEN & CALCIUM FLUXES FROM EARLY MOUSE EMBRYOS & OOCYTES

氧气的特性

• 批准号: 6319679
• 负责人: DAVID KEEFE
• 金额: $ 1.87万
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-12-01 至 2000-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6319679
• 关键词: CHARACTERIZATION; OXYGEN; CALCIUM; FLUXES; EARLY

Despite the importance of the mammalian embryo to clinical and biomedical sciences, the physiology of pre-implantation embryos and oocytes is largely unexplored. For example, although calcium is known to participate in the early events of fertilization (1) and also plays a brief but critical role at each cleavage (2, 3), the regulation of transmembrane calcium flux during the interim between cleavages or during blastocoel formation is unknown. One reason for this gap in our knowledge lies in the difficulty in studying a single oocyte or embryo. The goal of the project reported here is to take advantage of new techniques for monitoring physiological parameters from individual cells and begin to characterize changes in embryo physiology during development. This work is part of larger study on the viability of the pre-implantation embryo. Measurements taken using calcium and oxygen selective, self-referencing electrodes demonstrate that developing embryos exhibit measurable net oxygen influx and calcium efflux at all stages examined. In particular, oxygen influx measured from blastocysts is 3 times that measured from earlier stages. These data are consistent with oxygen consumption measurements obtained from groups of embryos by using ultra-microfluorescence techniques (4). The increase in oxygen consumption coincident with blastocyst formation is thought to reflect an increased metabolic demand generated by the pumping of sodium and potassium ions that drives the enlargement of the blastocoel (5). In contrast to oxygen influx, calcium efflux was relatively constant (~22 fmol . cm-2 . sec-1) throughout the developmental series. Mouse embryos are known to be able to develop from embryos through the blastocyst stage in the absence of external calcium (6). We have not yet determined the physiological mechanism underlying this net efflux. We could be measuring a steady-state loss of calcium from the internal stores or, as is more likely, an efflux linked to calcium uptake from the medium via channels. As the probe technique is based on considerable signal averaging, with both high and low pass filters (7) rapid channel events occurring over periods of a second or less may not be recorded. The increase in oxygen influx in the absence of a change in calcium efflux that we observed in blastocyst-stage embryos demonstrates that the physiological state of the embryo changes markedly between the 16- cell stage and blastocyst formation. Characterization of the transmembrane movement of other ion species will continue to improve our understanding of the physiological mechanisms underlying early embryo development. This study demonstrates the utility of the noninvasive probes for the characterization of the early embryo. References 1. Jaffe, L.A.1996. Pp. 367-378 in molecular Biology of Membrane Transport Disorders, S.G. Shultz et al, eds, Plenum Press, New York. 2. Stachecki, J.J. and D. R. Armant. 1996. Dev. 122: 2485-2496. 3. Stricker, S.A. 1995. Dev. Biol. 170: 496-518. 4. Houghton, F. D., J. G. Thompson, C. J. Kennedy, and H. J. Leese. 1996. Mol. Reprod. Dev. 44: 476-485. 5. Biggers, J.D., Bell, J.E. & Benos, D.J.. 1988. Am J. Physiol. 255: C419-C432. 6. Santals, J., M. Grossmann, and J. Egozcue. 1996. Hum Reprod Update 2: 257-261. 7. Smith, P. J. S., R. H. Sanger, and L. F. Jaffe. 1994. Meth. Cell Biol. 40: 115-134.

尽管哺乳动物胚胎对临床和 生物医学科学、植入前胚胎的生理学和 卵母细胞很大程度上尚未被探索。 例如，虽然钙是已知的 参加受精的早期活动 (1) 并玩耍 在每次裂解中发挥短暂但关键的作用 (2, 3)， 分裂之间的过渡期间的跨膜钙通量或 囊胚腔形成期间尚不清楚。 造成这一差距的原因之一是 我们的知识在于研究单个卵母细胞或 胚胎。 此处报告的项目的目标是利用 监测个体生理参数的新技术 细胞并开始表征胚胎生理学的变化 发展。 这项工作是关于可行性的更大规模研究的一部分 植入前胚胎。 使用钙和 氧选择性自参考电极表明 发育中的胚胎表现出可测量的净氧流入和钙 检查所有阶段的外流。 特别是，测量的氧气流入量 囊胚的测量值是早期阶段测量值的 3 倍。 这些 数据与获得的耗氧量测量结果一致 使用超微荧光技术对胚胎组进行观察 (4)。 耗氧量的增加与囊胚同时发生 形成被认为反映了代谢需求的增加 由钠离子和钾离子的泵浦产生，驱动 囊胚腔增大 (5)。 与氧气流入相比， 钙流出量相对恒定（~22 fmol . cm-2 . sec-1） 贯穿整个发展系列。 众所周知，小鼠胚胎是 能够从胚胎发育到胚泡阶段 缺乏外部钙 (6)。 我们还没有确定 这种净流出的生理机制。 我们可以是 测量内部储存的钙的稳态损失，或者， 更有可能的是，流出与培养基中钙的吸收有关 通过渠道。 由于探测技术基于大量信号 平均，具有高通和低通滤波器 (7) 快速通道 发生在一秒或更短时间内的事件可能不会被记录。 在钙没有变化的情况下氧气流入量的增加 我们在囊胚期胚胎中观察到的外排表明 胚胎的生理状态在16- 细胞阶段和囊胚形成。 的表征 其他离子种类的跨膜运动将继续改善 我们对早期生理机制的理解 胚胎发育。 这项研究证明了该方法的实用性 用于表征早期胚胎的非侵入性探针。 参考文献 1. Jaffe，L.A.1996。 页码。 367-378 分子生物学 膜运输障碍，S.G. Shultz 等人编辑，Plenum Press， 纽约。 2.斯塔切基，J.J.和 D. R. 阿曼特。 1996. 开发。 122： 2485-2496。 3.史翠克，S.A. 1995。生物。 170：496-518。 4. 霍顿 (F. D.)、J. G. 汤普森 (J. G. Thompson)、C. J. 肯尼迪 (C. J. Kennedy) 和 H. J. Leese。 1996.摩尔。重现。 开发。 44：476-485。 5. 比格斯，J.D.，贝尔，J.E. & Benos, D.J.. 1988. Am J. Physiol。 255：C419-C432。 6. 檀香， J.、M. Grossmann 和 J. Egozcue。 1996. 嗡嗡声再现更新 2： 257-261。 7. 史密斯 (P. J. S.)、R. H. 桑格 (R. H. Sanger) 和 L. F. 贾菲 (L. F. Jaffe)。 1994年。 方法。 细胞生物学。 40：115-134。