Novel regulation of early follicle formation

早期卵泡形成的新调控

• 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
• 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' 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

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（SC）向分化的细胞（SC）转化的基本机制是： 原始卵泡（PF）形成过程中的卵泡细胞（GCs）知之甚少。卵巢早衰的女性 失败（POF）、特纳综合征或FSH受体失活突变是不育的， 非生长型PF。因此，对PF形成的基础研究对于了解其分子基础是必要的 正常和有缺陷的卵泡形成我们提出了令人信服的证据，（1）骨形态发生蛋白2 (BMP2，SC衍生的配体）、GDF 9（卵母细胞衍生的配体）和雌二醇-17（E2，卵巢激素）间。 作用于促进胎儿卵巢中PF的形成，（2）GDF 9的旁分泌作用需要SC的BMP 2引发， 和（3）钙粘蛋白2（CDH 2）的失活干扰PF的形成。这些重要的发现使我们 假设BMP 2和GDF 9在时空上促进SC向GC的转变， 与卵母细胞组装。E2通过上调BMP 2的合成来调节BMP 2的作用 配体和受体。具体的目的（图1）是（1）确定BMP 2介导的原发性高血压的机制， 卵泡（PF）形成。目的是：（1）确定BMP 2是否调节SC的转化 （2）确定GDF 9的作用是否是SC向GC过渡所必需的或促进SC向GC的过渡，以及（3） 确定GDF 9是否促进卵母细胞-颗粒细胞粘附和组装。我们将敲除BMP 2或GDF 9 在E15仓鼠卵巢中，通过小分子药物抑制ALK 2/3的表达或阻断其作用或改变ALK 2/3活性， 抑制剂或shRNA。(2)确定雌二醇-17（E2）是否调节BMP 2-ALK 3/ALK 2系统。客观 E2是否通过调节BMP 2的表达和作用影响PF的形成。我们会击倒 或通过小分子抑制剂或shRNA在仓鼠胎儿卵巢中阻断BMP 2作用或改变ALK 2/3活性， 并在卵巢重组体中研究E2诱导SC向GC转化的机制。(3)分析基因 在PF形成期间受到BMP 2或GDF 9影响的网络。目的是鉴定和分析基因 在PF形成过程中，BMP 2或GDF 9靶向SC中的网络和相应通路。我们将 使用RNAseq分析来确定暴露于BMP 2的卵巢SC中的差异表达基因（DEG） 或GDF 9。高严格性截止值沿着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， 卵泡发育和功能，以及它们的缺陷引起卵巢功能障碍的可能性。生物 验证将使用目标1中概述的方法完成。这项研究将提供新的信息， 了解PF期间未分化SC向GC转变的基本机制- 形成，并可能揭示更好地了解PF形成缺陷的分子基础。