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.

项目摘要该项目的长期目标是研究刺激叶龙（FSH）的年龄依赖性机制卵巢的作用。 FSH是由α和β-亚基组成的Pitutai糖蛋白。两个亚基都是每个亚基上用两个N连接的糖链糖基化。 FSH的糖基化是发情/月经周期的和特定年龄。宏观结构性导致FSH变体由α亚基上的2个糖链组成，但 β上的一个或一个。这些变体被称为低糖基化的FSH糖基型，FSH21和 FSH18，与完全糖基化的FSH24相反。有趣的是，低糖基化和完全糖基化的抽象 FSH是年龄依赖性的，具有高水平的FSH21/18糖剂，主要是年轻女性和FSH24 在春季/绝经后妇女中占主导地位。这种转变表明FSH24在卵巢衰老中的作用。体外用重组低糖基化FSH对FSHB无效小鼠的研究和药物营救糖型表示已知的FSH响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。但是，这些FSH糖型的机制体内调节卵巢信号通路是未知的。中心假设是特定年龄的FSH 糖型通过FSHR作用，但调节不同的下游信号级联反应引起不同的基因/蛋白卵巢中的表达特征。该假设将使用一般设计的新型鼠标进行检验型号。在AIM 1中，我们将通过覆盖基因，蛋白质和磷蛋白来进行跨词分析表达单个FSH糖型的FSHB无效小鼠的卵巢中的表达特征以识别信号传导由每个FSH糖型调节的网络。在AIM 2中，我们将测试FSH糖型的假设 FSHR介导的信号传导的特异性是通过募集不同蛋白质复合物激活而实现的不同的下游基因/蛋白质网络。 FSHB无效的小鼠表达单独的FSH糖型及其 - 将使用颗粒细胞中标记的FSHR。带有特定标签的抗体的下拉实验然后进行卵巢蛋白的质谱分析将使我们能够识别FSH糖型特异性在每种情况下，FSH受体和受体co因子蛋白复合物。在AIM 3中，我们将评估直接重组FSH糖型在从生殖的年轻小鼠中获得的二次卵泡中的影响。将进行基因和蛋白质表达分析，以及FSH糖型如何影响叶片生长和将确定体外配子质量。成功完成拟议的研究将推动我们的了解FSH糖剂调节不同FSHR的选择性募集的机制 - 共同因素伴侣复合物以在卵巢中实现体内FSHR介导的信号传输途径在体外卵泡发生和卵子发生过程中，提供了直接读取的FSH糖型作用。我们的机械研究是保存卵巢功能的新型治疗机会的基础并将与NICHD任务保持一致，以改进卵巢诱导协议的设计。