Signal Transduction at Fertilization

描述（由申请人提供）：该项目的目的是了解激活卵子开始发育的信号机制。目前的更新申请，30-34年，解决了垂体的黄体生成素（LH）如何作用于卵巢导致卵母细胞进展到可以受精的阶段的问题。哺乳动物的卵母细胞储存在卵巢中，在女性的减数分裂前期被抑制数十年。然后，在卵泡周围的黄体生成素信号的响应下，减数分裂恢复，成熟卵子排卵。小鼠研究表明，由颗粒细胞产生并通过间隙连接扩散到卵母细胞的环状GMP维持了窦卵泡的减数分裂停滞；LH信号降低间隙连接的通透性和颗粒细胞产生cGMP，从而降低卵母细胞中的cGMP。cGMP的减少导致减数分裂细胞周期抑制的释放。尽管了解了这个级联，许多重要的问题仍然存在。本提案通过研究LH信号如何降低利钠肽受体2 （NPR2）的关酰环化酶活性（目的1和2），以及LH信号如何通过cGMP磷酸二酯酶活性降低cGMP（目的3），重点关注cGMP降低的关键事件。目的1将测试LH信号是否通过去磷酸化NPR2调控位点来降低NPR2胍基环化酶活性。目的2将研究g蛋白和EGF受体的LH激活如何启动NPR2的胍基环化酶活性的降低。目的3将研究哪些cGMP磷酸二酯酶降低卵泡内的cGMP，以及它们是否受到LH的刺激。使用的方法包括小鼠和大鼠卵巢卵泡和颗粒细胞的分离和培养，共聚焦显微镜，免疫沉淀，Western blotting，使用转基因小鼠和特异性酶抑制剂，使用光学传感器测量活卵巢卵泡中的cGMP和钙，以及体外酶活性测定。这些研究得出的结论也将适用于理解女性减数分裂的调控。体外卵母细胞成熟，其中在体外进行LH受体刺激，是人类体外受精方法的一个新兴组成部分，特别是对于多囊卵巢综合征患者和结合冷冻保存癌症患者的卵巢组织。提出的研究将有助于了解体外成熟临床进展的生物化学和细胞生物学基础。

DESCRIPTION (provided by applicant): The objective of this project is to understand the signaling mechanisms by which eggs are activated to begin development. The current renewal application, for years 30-34, addresses the question of how luteinizing hormone (LH) from the pituitary acts on the ovary to cause oocytes to progress to the stage at which they can be fertilized. Mammalian oocytes are stored in the ovary, arrested at meiotic prophase, for decades in women. Then in response to luteinizing hormone signaling in the surrounding follicle, meiosis resumes, and the mature egg is ovulated. Studies in mice have shown that meiotic arrest in antral follicles is maintained by cyclic GMP that is produced by the granulosa cells and diffuses into the oocyte through gap junctions; LH signaling decreases gap junction permeability and cGMP production by the granulosa cells, thus lowering cGMP in the oocyte. The cGMP decrease leads to the release of inhibition of the meiotic cell cycle. Despite knowledge of this cascade, many important questions still remain. This proposal focuses on the key event of the reduction in cGMP by investigating how LH signaling reduces the guanylyl cyclase activity of natriuretic peptide receptor 2 (NPR2) (aims 1 and 2), and how LH signaling lowers cGMP through activity of cGMP phosphodiesterases (aim 3). Aim 1 will test whether LH signaling decreases NPR2 guanylyl cyclase activity by dephosphorylating NPR2 regulatory sites. Aim 2 will investigate how LH activation of G-proteins and the EGF receptor initiates the decrease in the guanylyl cyclase activity of NPR2. Aim 3 will investigate which cGMP phosphodiesterases function to lower cGMP in the follicle, and whether they are stimulated by LH. The methods to be used include isolation and culture of ovarian follicles and granulosa cells from mice and rats, confocal microscopy, immunoprecipitation, Western blotting, use of transgenic mice and specific enzyme inhibitors, use of optical sensors to measure cGMP and calcium in live ovarian follicles, and assays of enzyme activities in vitro. The conclusions reached from these studies will also be applicable to understanding the regulation of meiosis in women. In vitro oocyte maturation, in which LH receptor stimulation is performed in vitro, is an emerging component of methods for human in vitro fertilization, particularly for patients with polycystic ovary syndrome and in combination with cryopreservation of ovarian tissue from cancer patients. The proposed studies will contribute to understanding of the biochemistry and cell biology underlying clinical advances in in vitro maturation.