PY2K function during fertilization

 描述(申请人提供)：哺乳动物受精始于一种独特的细胞-细胞黏附事件，涉及精子蛋白Izumo和卵母细胞蛋白Juno，它能使融合蛋白相互作用，诱导精子和卵子质膜融合。目前公认的模型表明，配子融合是自发发生的，卵母细胞是一个被动的参与者。然而，我们最近发现，精子-卵母细胞接触诱导了卵母细胞皮质中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-卵母细胞黏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PYK2在邻近的卵母细胞皮质内招募和激活。PYK2的活性随后促进了肌动蛋白聚合和微绒毛的伸长，从而增加了膜接触的面积，使瞬间的融合毛孔成为永久性的。PYK2还控制稍后皮质肌动蛋白的重塑，使精子头进入卵母细胞的细胞质隔间。随后，我们认为PYK2活性通过卵母细胞皮质横向扩散，启动皮质肌动蛋白层的整体重塑，这对于优化卵母细胞中的钙信号机制是必不可少的。提出的假说意义重大，因为它暗示精子和卵母细胞之间的“由外向内”信号在受精卵发育中发挥着重要作用，而在精子-卵母细胞表面不发生相互作用的胞质内单精子注射(ICSI)过程中，这种信号被绕过了。本研究的目的是确定启动PYK2信号转导的精子和卵母细胞表面蛋白，明确PYK2控制细胞骨架重塑的途径及其对卵母细胞钙信号转导机制的影响，并确定人工激活PYK2是否可以改善ICSI的结局，在小鼠和牛系统中ICSI通常是无效的。目的1将确定在受精时诱导PYK2激活所需的精子和卵母细胞表面蛋白。一种基于微球的固定化配体系统将被测试，以开发一种有效的人工诱导卵母细胞PYK2激活的方法。目的2阐明PYK2在精子-卵母细胞接触点诱导肌动蛋白微丝组装和细胞突起延长的途径。这部分研究将确定特定的蛋白质可能是 在药理学上靶向促进卵母细胞皮质肌动蛋白重塑。第三个目标将确定PYK2在维持卵母细胞皮质内最佳钙信号转导能力方面所扮演的特定角色。然后，实验将测试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，这项建议中提出的假设和目标代表着我们在理解卵母细胞激活方面向前迈出的重要一步，并有可能极大地提高用于提高家畜和人类生育能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.