Novel regulation of early follicle formation

早期卵泡形成的新调控

• 批准号: 10669562
• 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
• 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/10669562
• 关键词: ACVR1 gene; Address; Affect; BMP2 gene; Basic Science; Bioinformatics; Biological; Biological Models; Bone Morphogenetic Proteins; Cadherins; Candidate Disease Gene; Cell Adhesion; Cell Communication; Coupled; Defect; Development; Diagnosis; Epithelium; 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; 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; Turner' s Syndrome; Undifferentiated; Validation; Woman; candidate selection; cell assembly; differential expression; fetal; folliculogenesis; gene network; granulosa cell; improved; in vivo; knock-down; novel; ovarian dysfunction; paracrine; receptor; reproductive; small hairpin RNA; small molecule inhibitor; spatiotemporal; transcriptome sequencing

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.

未分化的体细胞(SCs)转化为粒细胞的基本机制是 原始卵泡(PF)形成过程中的卵泡细胞(GC)还知之甚少。卵巢早熟妇女 卵巢功能衰竭(POF)、Turner综合征或FSH受体失活突变是不育和有缺陷的 不增长的PFS。因此，对PF形成的基础研究对于理解PF的分子基础是必要的。 正常的和有缺陷的卵泡发生。我们提出了令人信服的证据：(1)骨形态发生蛋白2 (BMP2，SC来源的配体)，GDF9(卵母细胞来源的配体)和雌二醇-17(E2，一种卵巢激素)。 (2)GDF9的旁分泌作用需要BMP2启动SCs， 和(3)钙粘蛋白2(CDH2)的失活干扰PF的形成。19这些重要的发现引导我们 假设BMP2和GDF9在时空上促进干细胞向GCs的转化 与卵母细胞组装。E2通过上调BMP2的合成来调节BMP2的作用 配体和受体。其具体目的(图1)是：(1)确定BMP2介导的Pri-2的机制。 毛囊(PF)的形成。目的是：(1)确定BMP2是否调节SCs的转变 向GC过渡，(2)确定SCs向GC过渡是否需要或促进GDF9行动，以及(3) 确定GDF9是否促进卵母细胞-颗粒细胞的黏附和组装。我们将击倒BMP2或GDF9 小分子在体外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)测定雌二醇对BMP2-ALK3/ALK2系统的调节作用。目标是 目的是确定雌激素是否通过调节BMP2的表达和作用来影响PF的形成。我们将会击倒 或通过小分子抑制剂或shRNA阻断BMP2的作用或改变ALK2/3的活性， 并在卵巢中重组，以确定E2诱导SC向GC转化的机制。(3)基因分析 在PF形成过程中受BMP2或GDF9影响的网络。目的是鉴定和分析基因 在PF形成过程中被BMP2或GDF9靶向的干细胞中的网络和相应的通路。我们会 应用RNAseq分析确定BMP2暴露的卵巢干细胞差异表达基因(Deg) 或GDF9。将使用高严格性截断和生物信息学分析来选择候选基因 生物学验证，将根据它们在间充质-上皮转化(MET)中的作用优先考虑 卵泡的发育和功能，以及其缺陷导致卵巢功能障碍的可能性。生物学 验证将使用目标1中概述的方法来完成。这项研究将为 了解未分化干细胞转化为GC的基本机制。 这可能有助于更好地理解PF形成缺陷的分子基础。