OOCYTE CORTICAL MATURATION AND GRANULE ACTIVATION

卵母细胞皮质成熟和颗粒激活

• 批准号: 3324656
• 负责人: THOMAS W DUCIBELLA
• 金额: $ 13.13万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3324656
• 关键词: calcium; calcium binding protein; calcium flux; egg /ovum; endoplasmic reticulum; fertilization; fluorescence microscopy; granule; imaging /visualization /scanning; in vitro fertilization; inositol phosphates; laboratory mouse; meiosis; microinjections; oogenesis; protein kinase C

Studies in a number of mammals indicate that 1-5% of fertilized eggs develop abnormally into triploid embryonic abortuses. In the human this is primarily due to fertilization by more than one sperm. The egg's primary means of preventing polyspermy is the fertilization-associated release of cortical granules (CGs) whose contents biochemically modify the egg's zona pellucida making it less penetrable to sperm. In antral follicle oocytes (pre-metaphase II) which have not completed cytoplasmic maturation, one major cause of polyspermy appears to be their incompetence to undergo CG release. Preliminary evidence indicates that this incompetence involves the activation mechanism which includes intracellular calcium (Ca) and protein kinase C (PKC). The long-range goals of this proposal are to determine the causes for this incompetence in terms of Ca and PKC regulation as well as more accurately assess when competence develops in the mouse oocyte. The following are specific aims: 1. To determine when the oocyte develops the ability to undergo sperm- induced CG release. At appropriate stages of meiotic maturation, sperm- induced CG release will be assayed by % CG loss [quantitative image analysis(QIA)], by biochemical analysis of the zona (protein ZP2), and by % monospermic (vs polyspermic) fertilization. 2. To analyse the oocyte's competence to store and respond to intracellular Ca during meiotic maturation. The development of cellular components involved in calcium regulation will be analyzed with specific fluorescent probes and QIA. The ability of the developing oocyte to undergo CG loss in response to (microinjected) intracellular calcium will be assessed. 3. To analyse the oocyte's competence to undergo CG release in response to protein kinase C activators during meiotic maturation. At appropriate stages of maturation, the ability of PKC agonists to induce CG release will be investigated by QIA and ZP2 analysis. 4. To test for the synergism of Ca and PKC agonists in activating the pathways leading to CG loss. Synergism will be investigated in mature eggs and, if found, its development will be traced during meiotic maturation. These studies should result in 1) a more detailed explanation of the mechanism of egg and CG activation, 2) an identification of the stages of oocyte maturation which are most susceptable to polyspermic fertilization, 3) at least one likely scientific explanation for the origin of abnormal triploid abortuses observed in some mammalian species.

对许多哺乳动物的研究表明，1-5% 的受精卵 异常发育成三倍体胚胎流产。 在人类中这是 主要是由于多个精子受精所致。 鸡蛋是初级的 预防多精受精的方法是与受精相关的释放 皮质颗粒（CG），其内容物通过生化改变卵子的透明带 透明，使其不易被精子穿透。 在尚未完成的窦卵泡卵母细胞（中期 II 前）中 细胞质成熟，多精受精的主要原因之一似乎是它们 没有能力进行CG发布。 初步证据表明 这种无能涉及激活机制，其中包括 细胞内钙 (Ca) 和蛋白激酶 C (PKC)。 远距离的 该提案的目标是确定这种无能的原因 Ca 和 PKC 调节的术语以及更准确地评估何时 小鼠卵母细胞的能力得到发展。 具体目标如下： 1. 确定卵母细胞何时具备接受精子的能力 诱导CG释放。 在减数分裂成熟的适当阶段，精子 诱导的 CG 释放将通过 CG 损失百分比进行测定 [定量图像 分析（QIA）]，通过透明带（蛋白质 ZP2）的生化分析，并通过 单精（与多精）受精百分比。 2. 分析卵母细胞的储存和反应能力 减数分裂成熟过程中的细胞内 Ca。 蜂窝技术的发展 将用特定的方法分析参与钙调节的成分 荧光探针和 QIA。 发育中的卵母细胞的能力 因（微注射）细胞内钙的反应而经历 CG 损失 进行评估。 3. 分析卵母细胞响应 CG 释放的能力 减数分裂成熟过程中的蛋白激酶 C 激活剂。 在适当的时候 在成熟阶段，PKC 激动剂诱导 CG 释放的能力将 通过 QIA 和 ZP2 分析进行研究。 4. 测试 Ca 和 PKC 激动剂在激活 导致 CG 损失的途径。 将在成熟卵中研究协同作用 如果发现的话，它的发育将在减数分裂成熟过程中被追踪。 这些研究应该导致 1) 更详细的解释 卵子和 CG 激活的机制，2) 阶段的识别 最容易受多精受精影响的卵母细胞成熟， 3）对于异常的起源至少有一种可能的科学解释 在一些哺乳动物物种中观察到的三倍体流产。