To characterize nongenomic progestin receptors via knockouts in zebrafish

描述（由申请人提供）：类固醇的许多重要反应，如减数分裂恢复、精子激活、GnRH释放、抗凋亡和性行为改变，通过非基因组机制在数秒或数分钟内迅速发生。该途径不需要细胞核或基因转录的存在。类固醇的非基因组信号通路知之甚少，主要是由于围绕负责受体的身份的争议。类固醇激素的快速作用是这些非基因组类固醇作用的模型。最近，几个候选蛋白，膜resistin受体（mPRs），核resistin受体，和resistin受体膜组件已被认为是脊椎动物，包括人类的非基因组resistin受体。然而，需要进一步的研究来确定它们的生理功能，它们的信号传导和潜在的相互作用。目前尚不清楚哪一个是 主要受体，或者，替代地，是否需要一组互补蛋白质用于非基因组信号传导。我们将使用功能丧失的方法来研究斑马鱼基因敲除模型中这些据称的非基因组类胡萝卜素受体的作用和非基因组类胡萝卜素信号传导。我们将使用TALEN（转录激活因子样效应物核酸酶）产生敲除，TALEN是一种简单、成本有效且高效的敲除方法。此后，我们将研究基因敲除对动物生育力，成熟能力，卵母细胞成熟，精子激活和生殖细胞中的非基因组蛋白信号转导的影响。这项拟议的研究将是第一个产生和表征敲除模型的声称非基因组resistin受体。我们的长期目标是确定脊椎动物中非基因组类固醇受体的功能和信号传导，并开发用于生育控制或治疗不孕症的特异性拮抗剂和激动剂。我们的短期目标是 基因修饰的模型来解决最激烈的争议问题，即mPR是否是负责非基因组白蛋白信号传导的受体，以及是否有单一或多种受体参与非基因组白蛋白信号传导。

DESCRIPTION (provided by applicant): Many important responses of steroids, such as meiosis resumption, sperm activation, GnRH releasing, antiapoptosis, and sexual behavior changes, occur rapidly within seconds or minutes via a nongenomic mechanism. This pathway does not require the presence of a nucleus or gene transcription. The nongenomic signaling pathways of steroids are poorly understood, mainly due to the controversies surrounding the identity of the responsible receptors. The rapid action of the steroid hormone progestin is a model for these nongenomic steroid actions. Recently, several candidate proteins, membrane progestin receptors (mPRs), nuclear progestin receptor, and progestin receptor membrane components have been suggested to be the nongenomic progestin receptor in vertebrates, including humans. However, further studies are required to determine their physiological functions, their signaling, and potential interactions. Currently, it is not clear which one is the primary receptor or, alternatively, whether a set of complementary proteins are needed for nongenomic signaling. We will use a loss-of-functional approach to study the roles and nongenomic progestin signaling of these purported nongenomic progestin receptors in zebrafish knockout models. We will generate knockouts using TALENs (transcriptional activator-like effector nucleases), a simple, cost- effective and highly efficient knockout method. Thereafter, we will examine effects of knockouts in animal fertility, maturation competency, and changes in oocyte maturation, sperm activation and nongenomic progestin signaling in the germ cells. This proposed study will be the first to generate and characterize knockout models for purported nongenomic progestin receptors. Our long- term goals are to determine the functions and signaling of nongenomic steroid receptors in vertebrates and to develop specific antagonists and agonists for fertility control or treatments of infertility. Our short-term goal is to generate genetically modified models to solve the most hotly contested issues, i.e. if mPRs are the receptors responsible for the nongenomic progestin signaling, and whether a single or multiple classes of receptors is/are involved in the nongenomic progestin signaling.