FSH Glycoforms and Ovarian Signaling Pathways

FSH 糖型和卵巢信号通路

• 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
• 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

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.

项目摘要 本项目的长期目标是研究卵泡刺激素（FSH）的年龄依赖性机制 在卵巢中的作用。FSH是一种垂体糖蛋白，由α和β亚基组成。这两个小组都是 每个亚基上有两个N连接的糖链糖基化。FSH的糖基化是发情/月经周期- 和特定年龄。宏观异质性导致FSH变体由α亚基上的2条糖链组成， 在β上有一个或没有。这些变体被称为低糖基化FSH糖型，FSH 21， FSH 18，与完全糖基化的FSH 24相反。有趣的是，低糖基化和完全糖基化的 FSH具有年龄依赖性，高水平的FSH 21/18糖型主要见于年轻女性，FSH 24 主要见于绝经后/绝经后妇女。这种变化表明FSH 24在卵巢衰老中的作用。体外 用重组低糖基化和全糖基化FSH对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的调节差异 FSH受体（FSHR）信号传导的下游。然而，这些FSH糖型 在体内调节卵巢信号通路是未知的。中心假设是年龄特异性FSH 糖型通过FSHRs发挥作用，但调节不同的下游信号级联反应以引发不同的基因/蛋白质 卵巢中的表达特征。这一假设将使用基因工程新小鼠进行测试。 模型在目标1中，我们将通过覆盖基因、蛋白质和磷蛋白来进行反式组学分析。 在表达单个FSH糖型的Fshb缺失小鼠的卵巢中的表达特征以鉴定信号传导 由每个FSH糖型调节的网络。在目标2中，我们将检验FSH糖型 FSHR介导的信号传导的特异性是通过募集不同的蛋白质复合物来激活 不同的下游基因/蛋白质网络。Fshb基因敲除小鼠表达单个FSH糖型和His - 将使用颗粒细胞中标记的FSHR。用His标签特异性抗体的下拉实验 随后对卵巢蛋白质进行质谱分析将使我们能够鉴定FSH糖型特异性 FSH受体和受体辅因子蛋白复合物。在目标3中，我们将评估直接 重组FSH糖型在从生殖的年轻和年老小鼠获得的次级卵泡中的作用。 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， 将测定体外配子质量。若能顺利完成建议的研究， 了解FSH糖型调节不同FSHR选择性募集的机制- 辅助因子配偶体复合物，以在卵巢中体内实现FSH受体介导的信号转导途径， 提供体外卵泡发生和卵子发生期间FSH糖型作用的直接读数。我们 机制研究为保护卵巢功能的新治疗机会奠定了基础 并将使卵巢诱导方案的设计得到改进，与NICHD的使命保持一致。