FSH Glycoforms and Ovarian Signaling Pathways

FSH 糖型和卵巢信号通路

基本信息

批准号：
10394339
负责人：
T. RAJENDRA KUMAR
金额：
$ 56.9万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-16 至 2026-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10394339
关键词：
Age Aging Anterior Pituitary Gland Antibodies Antral Binding Biochemical Biological Assay Biology of Aging Cell Culture Techniques Cell Line Collaborations Complex Coupled Culture Techniques Data Endocrinology Estrogens Estrus Expression Profiling Female Follicle Stimulating Hormone Follicle Stimulating Hormone Receptor Foundations GTP-Binding Proteins Gene Expression Gene Expression Profile Gene Proteins Genetic Engineering Genetic Models Germ Cells Glycoproteins Goals Gonadotropins Growth Growth and Development function Heterogeneity Hormone Receptor Hormone Responsive Hormones Human In Vitro Individual Infertility Knockout Mice Knowledge Lead Ligands Link Luteinizing Hormone Mammalian Cell Mass Spectrum Analysis Mediating Menopause Menstrual cycle Methods Mission Molecular Mus National Institute of Child Health and Human Development Oogenesis Ovarian Ovarian Follicle Ovarian Granulosa Cell Ovarian Stimulations Ovarian aging Ovary Pattern Pharmacology Phosphoproteins Pituitary Gland Polysaccharides Postmenopause Preparation Production Proteins Proteomics Protocols documentation Receptor Signaling Recombinant Follicle Stimulating Hormone Recombinants Regulation Role Signal Pathway Signal Transduction Signal Transduction Pathway Specificity Testing Thyrotropin Variant Woman age related aged base design egg experimental study folliculogenesis glycosylation granulosa cell hormonal signals improved in vivo insight mouse genetics mouse model novel novel therapeutics oocyte quality phosphoproteomics preservation protein complex protein expression receptor receptor-mediated signaling recruit reproductive reproductive senescence response sugar transcriptome sequencing young woman

项目摘要

PROJECT SUMMARY The long-term goal of this project is to study age-dependent mechanisms of follicle-stimulating hormone (FSH) actions in the ovary. FSH is a pituitary glycoprotein consisting of an α-and a β-subunit. Both the subunits are glycosylated with two N-linked sugar chains on each subunit. Glycosylation of FSH is estrous/menstrual cycle- and age-specific. Macro-heterogeneity results in FSH variants consisting of 2 sugar chains on the α subunit but either one or none on the β. These variants are known as hypo-glycosylated FSH glycoforms, FSH21, and FSH18, in contrast to the fully glycosylated FSH24. Interestingly, the abundance of hypo- and fully glycosylated FSH is age-dependent, with high levels of FSH21/18 glycoforms predominant in young women and FSH24 predominant in peri/post-menopausal women. This shift suggests a role of FSH24 in ovarian aging. In vitro studies and pharmacological rescue of Fshb null mice with recombinant hypo- and fully glycosylated FSH glycoforms indicate differences in regulation of known FSH-responsive ovarian genes and proteins downstream of FSH receptor (FSHR) signaling. However, the mechanisms by which these FSH glycoforms regulate ovarian signaling pathways in vivo are unknown. The central hypothesis is that age-specific FSH glycoforms act via FSHRs but regulate distinct downstream signaling cascades to elicit different gene/protein expression signatures in the ovary. This hypothesis will be tested using genetically engineered novel mouse models. In Aim 1, we will perform a trans-omics analysis by overlaying the gene, protein and phosphoprotein expression signatures in ovaries of Fshb null mice expressing individual FSH glycoforms to identify signaling networks regulated by each FSH glycoform. In Aim 2, we will test the hypothesis that the FSH glycoform specificity in FSHR-mediated signaling is achieved by recruitment of distinct protein complexes to activate different downstream gene/protein networks. Fshb null mice expressing individual FSH glycoform and His - tagged FSHRs in granulosa cells will be used. Pull-down experiments with an His tag-specific antibody followed by mass spectrometry analysis of ovarian proteins will allow us to identify the FSH glycoform-specific FSH receptor and receptor co-factor protein complexes in each case. In Aim 3, we will evaluate the direct effects of recombinant FSH glycoforms in secondary follicles obtained from reproductively young and old mice. Gene and protein expression profiling will be performed and how FSH glycoforms impact follicle growth and gamete quality in vitro will be determined. Successful completion of the proposed studies will advance our understanding of the mechanisms by which FSH glycoforms regulate selective recruitment of distinct FSHR - co-factor partner complexes to achieve FSHR-mediated signal transduction pathways in vivo in ovaries and provide a direct read out of FSH glycoform actions during in vitro folliculogenesis and oogenesis. Our mechanistic studies serve as the foundation for novel therapeutic opportunities to preserve ovarian function and will enable improved design of ovarian induction protocols, in alignment with the NICHD mission.

项目概要该项目的长期目标是研究促卵泡激素（FSH）的年龄依赖性机制 FSH 是一种垂体糖蛋白，由 α 和 β 亚基组成。每个亚基上有两个 N 连接糖链糖基化 FSH 是动情/月经周期的。宏观异质性导致 FSH 变异由 α 亚基上的 2 条糖链组成，但 β 上有一个或没有这些变体被称为低糖基化 FSH 糖型、FSH21 和 FSH18，与完全糖基化的 FSH24 不同。 FSH 具有年龄依赖性，年轻女性中以高水平的 FSH21/18 糖型为主，而 FSH24 这种转变在围绝经期/绝经后女性中占主导地位，表明 FSH24 在体外卵巢衰老中发挥作用。重组低糖基化和完全糖基化 FSH 的 Fshb 缺失小鼠的研究和药理学拯救糖型表明已知 FSH 反应性卵巢基因和蛋白质的调节差异 FSH 受体 (FSHR) 信号传导的下游然而，这些 FSH 糖型的机制。体内调节卵巢信号通路的机制尚不清楚，核心假设是年龄特异性 FSH。糖型通过 FSHR 发挥作用，但调节不同的下游信号级联以引发不同的基因/蛋白质卵巢中的表达特征将使用基因工程新型小鼠进行测试。在目标 1 中，我们将通过叠加基因、蛋白质和磷蛋白来进行跨组学分析。表达单个 FSH 糖型的 Fshb 缺失小鼠卵巢中的表达特征以识别信号传导在目标 2 中，我们将检验 FSH 糖型的假设。 FSHR 介导的信号传导的特异性是通过招募不同的蛋白质复合物来激活表达个体 FSH 糖型和 His 的不同下游基因/蛋白质网络。将使用 His 标签特异性抗体进行下拉实验。随后对卵巢蛋白进行质谱分析将使我们能够识别 FSH 糖型特异性在目标 3 中，我们将评估每种情况下的 FSH 受体和受体辅因子蛋白复合物。重组 FSH 糖型对年轻和年老小鼠的次级卵泡的影响。将进行基因和蛋白质表达谱分析，以及 FSH 糖型如何影响卵泡生长和体外配子质量将被确定，拟议研究的成功完成将推进我们的研究。了解 FSH 糖型调节不同 FSHR 选择性募集的机制 - 辅因子伴侣复合物在卵巢和体内实现 FSHR 介导的信号转导途径提供体外卵泡发生和卵子发生过程中 FSH 糖型作用的直接读数。机制研究为保护卵巢功能的新治疗机会奠定了基础并将改进卵巢诱导方案的设计，与 NICHD 的使命保持一致。