PY2K function during fertilization

 描述（由申请人提供）：哺乳动物的受精开始于涉及精子蛋白IZUMO和卵母细胞蛋白JUNO的独特的细胞-细胞粘附事件，其使得融合蛋白相互作用以诱导精子和卵质膜的融合。目前接受的模型暗示配子融合自发发生，卵母细胞是被动的参与者。然而，我们最近发现，精子-卵母细胞接触诱导卵母细胞皮质中FAK家族成员PYK2的激活，并且PYK2是精子掺入所必需的，这表明卵母细胞是受精过程的积极参与者。我们提出的假设是精子-卵母细胞粘附导致精子和卵母细胞表面蛋白之间的反式相互作用，导致邻近卵母细胞皮质内PYK2的募集和激活。PYK2活性然后促进肌动蛋白聚合和微绒毛伸长，其增加膜接触面积，允许暂时融合孔变为永久性。PYK2还控制皮质肌动蛋白的重塑，以允许精子头部进入卵母细胞的细胞质区室。随后，我们提出PYK2活性通过卵母细胞皮质横向传播，以启动皮质肌动蛋白层的全局重构，这对于卵母细胞中Ca2+信号机制的优化至关重要。所提出的假设是重要的，因为它意味着精子和卵母细胞之间的"由外而内"的信号在受精卵发育中起着重要作用，在卵胞浆内单精子注射（ICSI）过程中，精子-卵母细胞表面相互作用不会发生。本提案的目的是鉴定启动PYK2信号传导的精子和卵母细胞表面蛋白，确定PYK2控制细胞骨架重塑的途径及其对卵母细胞Ca2+信号传导机制的影响，并确定人工激活PYK2是否可能改善使用小鼠和牛系统的ICSI结果，其中ICSI通常是无效的。目的1将确定精子和卵母细胞表面蛋白诱导PYK2激活受精。本研究旨在探索一种有效的人工诱导卵母细胞PYK 2活化的方法。目的2将阐明PYK2在精卵接触点诱导肌动蛋白丝组装和细胞突起伸长的途径。这项研究的这一部分将确定特定的蛋白质， 有针对性地增强卵母细胞中皮质肌动蛋白的重塑。第三个目标将定义PYK2在维持卵母细胞皮层内最佳Ca2+信号传导能力方面的具体作用。然后，实验将测试通过微球固定的精子蛋白人工激活PYK2是否可以改善小鼠和牛卵母细胞的ICSI结果。总之，本提案中提出的假设和目标代表了我们对卵母细胞激活的理解向前迈出的重要一步，并有可能大大提高用于提高家畜和人类生育力的辅助生殖技术的效率。

 DESCRIPTION (provided by applicant): Fertilization in mammals begins with a unique cell-cell adhesion event involving the sperm protein IZUMO and the oocyte protein JUNO that enables interaction of fusigenic proteins to induce fusion of the sperm and egg plasma membranes. The currently accepted model implies that gamete fusion occurs spontaneously and that the oocyte is a passive participant. However, our recent finding that sperm-oocyte contact induced activation of the FAK-family member PYK2 in the oocyte cortex, and that PYK2 was required for sperm incorporation, showed that the oocyte is an active participant in the fertilization process. Our proposed hypothesis is that sperm-oocyte adhesion results in trans-interactions between surface proteins on the sperm and oocyte that lead to recruitment and activation of PYK2 within the adjacent oocyte cortex. PYK2 activity then promotes actin polymerization and microvillus elongation that increases the area of membrane contact allowing transient fusion pores to become permanent. PYK2 also controls remodeling of the cortical actin later to allow the sperm head to enter the cytoplasmic compartment of the oocyte. Subsequently, we propose that PYK2 activity spreads laterally through the oocyte cortex to initiate global remodeling of the cortical actin layer, which is essential for optimization of the Ca2+ signaling machinery in the oocyte. The proposed hypothesis is significant because it implies that `outside-in' signaling between sperm and oocyte plays an important role in zygote development that is bypassed during the Intracytoplasmic Sperm Injection (ICSI) where sperm-oocyte surface interactions do not occur. The objective of this proposal is to identify sperm and oocyte surface proteins that initiate PYK2 signaling, define the pathway by which PYK2 controls cytoskeletal remodeling and its impact on oocyte Ca2+ signaling machinery, and establish whether artificial activation of PYK2 might improve the outcome of ICSI using the mouse and bovine systems where ICSI is usually inefficient. Aim 1 will identify the sperm and oocyte surface proteins required to induce PYK2 activation at fertilization. A microsphere-based immobilized ligand system will be tested to develop an effective way of inducing oocyte PYK2 activation artificially. Aim 2 will elucidate the pathway by which PYK2 induces actin filament assembly and cell process elongation at the point of sperm-oocyte contact. This part of the study will identify specific proteins that could be targeted pharmacologically to enhance cortical actin remodeling in oocytes. The third aim will define the specific role that PYK2 plays in maintaining optimal Ca2+ signaling capability within the oocyte cortex. Experiments will then test whether artificial activation of PYK2 by microsphere-immobilized sperm proteins can improve the outcome of ICSI in mouse and bovine oocytes. Together, the hypothesis and goals presented in this proposal represent a significant step forward in our understanding of oocyte activation, and have the potential to greatly improve the efficiency of Assisted Reproductive Techniques used to improve fertility in domestic animals and humans.