Novel regulation of early follicle formation

早期卵泡形成的新调控

基本信息

批准号：
10207699
负责人：
SHYAMAL K. ROY
金额：
$ 31.76万
依托单位：
UNIVERSITY OF NEBRASKA MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-01 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10207699
关键词：
ACVR1 gene Address Affect BMP2 gene Basic Science Bioinformatics Biological Biological Models Cadherins Candidate Disease Gene Cell Adhesion Cell Communication Coupled Defect Development Diagnosis Epithelial Estradiol Event Experimental Models Exposure to FOXL2 gene Female Fertility Follicle Stimulating Hormone Receptor GDF9 gene Genes Germ Cells Goals Hamsters Human In Vitro Infertility Lead Life Ligands Light Mammals Mediating Meiosis Mesenchymal Molecular Mutation Oocytes Ovarian Ovarian Diseases Ovarian Follicle Ovarian hormone Ovary Pathway interactions Premature Ovarian Failure Primordial Follicle Probability Process Recombinants Regulation Role Small Interfering RNA Somatic Cell System Transitional Epithelium Turner&apos s Syndrome Undifferentiated Validation Woman base bone morphogenic protein cell assembly differential expression fetal folliculogenesis granulosa cell improved in vivo knock-down novel ovarian dysfunction paracrine receptor reproductive small hairpin RNA small molecule inhibitor transcriptome sequencing vascular endothelial cadherin-2

项目摘要

The fundamental mechanism responsible for the transition of undifferentiated somatic cells (SCs) into gran- ulosa cells (GCs) during primordial follicle (PF) formation is poorly understood. Women with premature ovarian failure (POF), Turner Syndrome or inactivating mutation of FSH receptor are infertile and have defective and non-growing PFs. Therefore, the basic research on PF formation is necessary to understand the molecular basis of normal and defective folliculogenesis. We present compelling evidence that (1) bone morphogenic protein 2 (BMP2, a SC-derived ligand), GDF9 (an oocyte-derived ligand) and estradiol-17 (E2, an ovarian hormone) inter- act to promote PF formation in fetal ovaries, (2) the paracrine actions of GDF9 require BMP2 priming of SCs, and (3) inactivation of cadherin 2 (CDH2) interferes with PF formation.19 These important findings lead us to hypothesize that BMP2 and GDF9 spatio-temporally promote the transition of SCs into GCs and their assembly with the oocytes. E2 modulates the actions of BMP2 by upregulating the synthesis of BMP2 ligand and receptors. The specific aims (Fig. 1) are (1) Determine the mechanism of BMP2-mediated pri- mordial follicle (PF) formation. The objectives are, (1) To determine if BMP2 regulates the transition of SCs into GCs, (2) To determine if GDF9 action is required for or facilitates the transition of SCs to GCs, and (3) To determine if GDF9 facilitates oocyte-granulosa cell adhesion and assembly. We will knockdown BMP2 or GDF9 expression or block their actions or alter ALK2/3 activities in E15 hamster ovaries in vitro by small molecule inhibitors or shRNA. (2) Determine if estradiol-17 (E2) regulates BMP2-ALK3/ ALK2 system. The objective is to determine if E2 affects PF formation by regulating the expression and action of BMP2. We will knockdown or block BMP2 action or alter ALK2/3 activities by small molecule inhibitors or shRNA in hamster fetal ovaries, and in ovary recombinants to determine the mechanism of E2-induced SC to GC transition. (3) Analysis of gene networks affected by BMP2 or GDF9 during PF formation. The objective is to identify and analyze gene networks and corresponding pathways in SCs that are targeted by BMP2 or GDF9 during PF formation. We will use RNAseq analysis to determine differentially expressed genes (DEGs) in SCs of ovaries exposed to BMP2 or GDF9. High stringency cut off along with bioinformatics analysis will be used to select candidate genes for biological validation and will be prioritized based on their role in mesenchymal-epithelial transition (MET), in follicular development and functions, and the probability of their defects causing ovarian dysfunction. Biological validation will be accomplished using approaches outlined in Aim 1. This study will provide novel information to understand the basic mechanism responsible for the transition of undifferentiated SCs into GCs during PF for- mation, and may shed light to better understand the molecular basis of defects in PF formation.

未分化体细胞 (SC) 转变为粒细胞的基本机制原始卵泡（PF）形成过程中的ulosa细胞（GC）知之甚少。卵巢早衰的女性失败（POF）、特纳综合征或 FSH 受体失活突变导致不孕，并且有缺陷和非生长 PF。因此，有必要对PF形成进行基础研究，了解其分子基础正常和有缺陷的卵泡发生。我们提出了令人信服的证据表明 (1) 骨形态发生蛋白 2 （BMP2，一种 SC 衍生配体）、GDF9（一种卵母细胞衍生配体）和 estradiol-17（E2，一种卵巢激素）促进胎儿卵巢中 PF 的形成，(2) GDF9 的旁分泌作用需要 BMP2 启动 SC， (3) 钙粘蛋白 2 (CDH2) 失活会干扰 PF 形成。19 这些重要发现使我们得出假设 BMP2 和 GDF9 时空促进 SC 向 GC 的转变及其与卵母细胞的组装。 E2 通过上调 BMP2 的合成来调节 BMP2 的作用配体和受体。具体目标（图1）是（1）确定BMP2介导的优先机制普通卵泡（PF）的形成。目标是，(1) 确定 BMP2 是否调节 SC 的转变 (2) 确定 GDF9 行动是否需要或促进 SC 向 GC 的过渡，以及 (3) 确定 GDF9 是否促进卵母细胞-颗粒细胞粘附和组装。我们将击倒 BMP2 或 GDF9 通过小分子在 E15 仓鼠卵巢中表达或阻断其作用或改变 ALK2/3 活性抑制剂或shRNA。 (2)确定雌二醇17(E2)是否调节BMP2-ALK3/ALK2系统。目标目的是确定E2是否通过调节BMP2的表达和作用来影响PF的形成。我们将击倒或通过小分子抑制剂或 shRNA 在仓鼠胎儿卵巢中阻断 BMP2 作用或改变 ALK2/3 活性，并在卵巢重组体中确定 E2 诱导 SC 向 GC 转变的机制。 (3)基因分析 PF 形成过程中受 BMP2 或 GDF9 影响的网络。目的是识别和分析基因 PF 形成过程中 BMP2 或 GDF9 靶向的 SC 网络和相应通路。我们将使用 RNAseq 分析确定暴露于 BMP2 的卵巢 SC 中的差异表达基因 (DEG) 或 GDF9。高严格截断和生物信息学分析将用于选择候选基因生物学验证，并将根据其在间充质-上皮转化 (MET) 中的作用进行优先排序，卵泡发育和功能，以及其缺陷导致卵巢功能障碍的可能性。生物验证将使用目标 1 中概述的方法来完成。这项研究将为了解 PF 期间未分化 SC 转变为 GC 的基本机制化，并可能有助于更好地理解 PF 形成缺陷的分子基础。