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Supplement: To Characterize Nongenomic Progestin Receptors via Knockouts in Zebrafish

补充：通过敲除斑马鱼来表征非基因组孕激素受体

基本信息

批准号：
9545944
负责人：
Yong Zhu
金额：
$ 1.5万
依托单位：
EAST CAROLINA UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-07-01 至 2021-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9545944
关键词：
ADAMTS1 gene ADAMTS9 gene Affect Anovulation CRISPR/Cas technology Cells Data Development Disease Disintegrins Drug Targeting Enzyme Activation Enzymes Event Extracellular Matrix Family Fertility Fishes Foundations Future Gene Expression Gene Targeting Genes Gonadotropins Heat-Shock Response Hormonal Human Infertility Knock-out Knockout Mice Malignant Neoplasms Mammals Matrix Metalloproteinases Mediating Mental disorders Metalloproteases Modeling Molecular Nuclear Oocytes Outcome Ovulation Peptide Hydrolases Pharmacotherapy Phenotype Process Progesterone Receptors Progestins Proteins Rattus Regulation Resources Rupture Signal Pathway Signal Transduction Signaling Protein Steroid Receptors Steroids Thrombospondins Time Tissue-Specific Gene Expression Tissues Transcript Zebrafish experimental study genome-wide hormone regulation infertility treatment member new therapeutic target non-genomic novel overexpression ovulation disorders reproductive transcription factor transcriptome sequencing

项目摘要

Ovulation is an indispensable reproductive event; yet our understanding of the molecular mechanisms that control ovulation is still incomplete. Activation of proteases leading to extracellular matrix disassociation is an essential step for follicle rupture and ovulation to proceed. Involvement of several metalloproteinase families including members of ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs), MMPs (matrix metalloproteinases), and ADAM (a disintegrin and metalloproteinase) in ovulation and tissue remodeling has been suggested. However, the molecular mechanisms regulating key proteinases and identities of essential enzymes that are responsible for the recurring tissue remodeling process have not been established. Our preliminary data suggest that a member of ADAMTS family, ADAMTS9, is likely a downstream target of nuclear progestin receptor (PGR), and is mainly responsible for ovulation. We will use CRISPR/Cas9 to generate global and conditional knockouts of ADAMTS9, and characterize these knockouts to determine whether knockout ADAMTS9 affects ovulation and fertility in zebrafish. If ADAMTS9 is a key downstream enzyme of PGR that is important for ovulation, we should be able to rescue PGR knockout anovulation phenotype by overexpressing ADAMTS9 in PGR knockout fish. In addition, we propose to determine the expression, regulation and functions of ADAMTS9 during ovulation in zebrafish, in order to elucidate the molecular mechanisms and signaling pathways for ovulation. We will determine expression of both transcript and protein of ADAMTS9 during the development of oocytes, and in the follicular cells of preovulatory oocytes around the ovulation in zebrafish. We will also determine whether the expression of ADAMTS9 is hormonally regulated by gonadotropins and/or progestin. We will then determine whether PGR regulates ADAMTS9 directly or indirectly via other transcriptional factors. For the first time, the regulation and functions of ADAMTS9, particularly in the follicular cells of oocytes, will be comprehensively determined in a vertebrate model. The ADAMTS9 knockout models will be very valuable resource for future studies. The functions of ADAMTS9 in ovulation and fertility will also be established in a zebrafish model, which will provide a foundation to study the functions and regulation of ADAMTS9 in mammals, as well as the development of new drug targets and novel non-steroid treatments for infertility, tissue remodeling, and cancer.

排卵是一项不可或缺的生殖活动；但我们对分子生物学的理解控制排卵的机制仍然不完整。激活蛋白水解酶导致细胞外基质解离是卵泡破裂和排卵进行的重要步骤。几个金属蛋白酶家族的参与，包括ADAMTS(一种去整合素)成员和带有凝血酶反应蛋白基序的金属蛋白酶)、基质金属蛋白酶(MMPs)和ADAM (一种去整合素和金属蛋白酶)与排卵和组织重塑有关。然而，调节关键蛋白水解酶的分子机制和必需的负责复发组织重塑过程的酶还没有被已经成立了。我们的初步数据表明，ADAMTS家族的一个成员，ADAMTS9，可能是一个核孕激素受体(PGR)下游靶点，主要负责排卵。我们将使用CRISPR/Cas9生成ADAMTS9的全局和条件敲除，以及鉴定这些基因敲除，以确定敲除ADAMTS9是否影响排卵和斑马鱼的繁殖力。如果ADAMTS9是PGR的关键下游酶，那么它对排卵，我们应该能够通过过度表达来挽救PGR基因敲除的无排卵表型在PGR基因敲除鱼中发现了ADAMTS9。此外，我们建议确定表达、调控以及ADAMTS9在斑马鱼排卵中的作用，以阐明其分子机制。排卵的机制和信号通路。我们将确定两个转录本的表达 ADAMTS9在卵母细胞发育过程中和卵泡细胞中的表达。斑马鱼排卵前后的排卵前卵母细胞。我们还将确定是否 ADAMTS9的表达受促性腺激素和/或孕激素的调节。到时候我们会的确定PGR是否直接或通过其他转录因子间接调节ADAMTS9。首次研究了ADAMTS9的调控和功能，特别是在卵泡细胞中。卵母细胞，将在脊椎动物模型中进行全面测定。ADAMTS9基因淘汰赛模型将是未来研究的非常有价值的资源。ADAMTS9在排卵中的作用并将在斑马鱼模型中建立生育力，为下一步的研究提供基础 ADAMTS9在哺乳动物中的功能、调控及新药开发不孕不育、组织重塑和癌症的靶点和新的非类固醇疗法。

项目成果

期刊论文数量（24）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Metalloproteases in gonad formation and ovulation.

DOI：
10.1016/j.ygcen.2021.113924
发表时间：
2021-12-01
期刊：
General and comparative endocrinology
影响因子：
2.7
作者：
Zhu Y
通讯作者：
Zhu Y

Nuclear progestin receptor (pgr) knockouts in zebrafish demonstrate role for pgr in ovulation but not in rapid non-genomic steroid mediated meiosis resumption.

斑马鱼核孕激素受体 (pgr) 敲除证明了 pgr 在排卵中的作用，但在快速非基因组类固醇介导的减数分裂恢复中没有作用。

DOI：
10.3389/fendo.2015.00037
发表时间：
2015
期刊：
Frontiers in endocrinology
影响因子：
5.2
作者：
Zhu Y;Liu D;Shaner ZC;Chen S;Hong W;Stellwag EJ
通讯作者：
Stellwag EJ

Subfertility and reduced progestin synthesis in Pgrmc2 knockout zebrafish.

Pgrmc2 敲除斑马鱼的生育力低下和孕激素合成减少。

DOI：
10.1016/j.ygcen.2019.113218
发表时间：
2019
期刊：
General and comparative endocrinology
影响因子：
2.7
作者：
Wu,Xin-Jun;Williams,MarcusJermaul;Patel,PujanRameshkumar;Kew,KimberlyAnn;Zhu,Yong
通讯作者：
Zhu,Yong

Reduced Vitellogenesis and Female Fertility in Gper Knockout Zebrafish.