To characterize nongenomic progestin receptors via knockouts in zebrafish

通过敲除斑马鱼来表征非基因组孕激素受体

• 批准号: 8574602
• 负责人: Yong Zhu
• 金额: $ 31.55万
• 依托单位: EAST CAROLINA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8574602
• 关键词: Agonist; Animals; Binding; Biological Models; Cell Nucleus; Cells; Contraceptive methods; Coupled; Cyclic AMP; Cyclin B; Dactinomycin; Disease; Fertility; Fishes; Funding; GTP-Binding Proteins; Genetic Transcription; Genomics; Germ Cells; Goals; Human; In Vitro; Incubated; Infertility; Knock-out; Laboratories; Lead; MAP Kinase Gene; Malignant Neoplasms; Mammals; Meiosis; Membrane; Membrane Proteins; Mental disorders; Methods; Modeling; Nuclear; Oocytes; Outcome; Paracrine Communication; Pathway interactions; Physiological; Pregnancy; Pregnancy Maintenance; Progesterone Receptors; Progestins; Proteins; Receptor Signaling; Role; Sex Behavior; Signal Pathway; Signal Transduction; Staging; Steroid Receptors; Steroids; Transcript; Transcription Coactivator; Universities; Vertebrates; Zebrafish; behavior change; computerized data processing; cost; cost effective; disorder control; functional loss; gain of function; inhibitor/antagonist; loss of function; mutant; non-genomic; novel; nuclease; oocyte maturation; overexpression; protein activation; public health relevance; receptor; receptor expression; response; sperm cell; steroid hormone

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.

描述(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、抗细胞凋亡和性行为改变，都通过非基因组机制在几秒钟或几分钟内迅速发生。这一途径不需要存在细胞核或基因转录。类固醇的非基因组信号通路目前还知之甚少，主要是由于围绕负责受体的身份的争议。类固醇激素孕激素的快速作用是这些非基因组类固醇作用的典范。最近，一些候选蛋白质，膜孕激素受体(MPR)，核孕激素受体和孕激素受体膜组分被认为是包括人类在内的脊椎动物的非基因组孕激素受体。然而，还需要进一步的研究来确定它们的生理功能、它们的信号和潜在的相互作用。目前还不清楚哪一个是 或者，是否需要一组互补蛋白来进行非基因组信号传递。我们将使用功能丧失的方法来研究这些所谓的非基因组孕激素受体在斑马鱼基因敲除模型中的作用和非基因组孕激素信号转导。我们将使用TALEN(转录激活物样效应核酸酶)产生基因敲除，这是一种简单、经济有效和高效的基因敲除方法。此后，我们将研究基因敲除对动物生育力、成熟能力以及生殖细胞中卵母细胞成熟、精子激活和非基因组孕激素信号的影响。这项拟议的研究将是第一次建立和表征所谓的非基因组孕激素受体的敲除模型。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，并开发用于控制生育或治疗不孕不育的特定拮抗剂和激动剂。我们的短期目标是创造 基因修饰模型以解决最具争议性的问题，即MPR是否是负责非基因组孕激素信号转导的受体，以及单个或多个类别的受体是否参与非基因组孕激素信号转导。