Signal Transduction at Fertilization

描述（由申请人提供）：本项目的目的是了解卵被激活以开始发育的信号机制。目前的更新申请，为30-34年，解决了垂体的促黄体激素（LH）如何作用于卵巢，使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在女性的卵巢中储存，在减数分裂前期停滞数十年。然后在周围卵泡的促黄体激素信号的作用下，减数分裂恢复，成熟卵子排卵。在小鼠中的研究表明，窦卵泡中的减数分裂停滞由颗粒细胞产生的环GMP维持，并通过间隙连接扩散到卵母细胞中; LH信号降低间隙连接通透性和颗粒细胞产生的cGMP，从而降低卵母细胞中的cGMP。cGMP减少导致减数分裂细胞周期抑制的释放。尽管了解这一级联，但许多重要问题仍然存在。该提案通过研究LH信号传导如何降低利钠肽受体2（NPR 2）的鸟苷酸环化酶活性（目的1和2）以及LH信号传导如何通过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NPR 2调节位点来降低NPR 2鸟苷酸环化酶活性。目的2将探讨如何LH激活G蛋白和EGF受体启动鸟苷酸环化酶活性的NPR 2的下降。目的3研究哪些cGMP磷酸二酯酶具有降低卵泡中cGMP的功能，以及它们是否受到LH的刺激。使用的方法包括从小鼠和大鼠中分离和培养卵泡和颗粒细胞、共聚焦显微镜、免疫沉淀、蛋白质印迹、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泡中的cGMP和钙以及体外酶活性测定。从这些研究中得出的结论也将适用于了解妇女减数分裂的调节。体外卵母细胞成熟，其中LH受体刺激在体外进行，是人类体外受精方法的一个新兴组成部分，特别是对于患有多囊卵巢综合征的患者以及与来自癌症患者的卵巢组织的冷冻保存相结合。拟议的研究将有助于了解生物化学和细胞生物学的基础临床进展，在体外成熟。

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.