FSH Glycoforms and Ovarian Signaling Pathways

项目总结 该项目的长期目标是研究卵泡刺激素(FSH)的年龄依赖性机制。 卵巢中的活动。卵泡刺激素是一种由α-和β-亚基组成的垂体糖蛋白。这两个亚单位都是 糖基化的在每个亚基上有两个N-连接的糖链。FSH的糖基化是发情/月经周期- 和特定的年龄。宏观异质性导致卵泡刺激素变异体在α亚单位上包含2个糖链，但 β上有一个或没有。这些变体被称为低糖基化的FSH糖形式，FSH21和 FSH18，而不是完全糖基化的FSH24。有趣的是，低糖和完全糖基化的丰度 FSH是年龄相关的，FSH21/18糖型在年轻女性中占主导地位，FSH24 主要见于围绝经期/绝经后妇女。这一转变表明FSH24在卵巢衰老中发挥了作用。离体 重组低糖和全糖化FSH对FSHB基因缺失小鼠的研究及药理挽救作用 糖形式表明对已知的FSH反应卵巢基因和蛋白的调控存在差异 FSH受体(FSHR)信号的下游。然而，这些FSH糖形成的机制 体内调控卵巢信号通路的研究尚不清楚。中心假设是特定年龄的FSH 糖形式通过FSHR发挥作用，但调节不同的下游信号级联以诱导不同的基因/蛋白 卵巢中的表情特征。这一假说将用基因工程的新型小鼠进行验证 模特们。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 FSHB基因缺陷小鼠卵巢表达单个FSH糖体以识别信号的表达特征 每种FSH糖体调控的网络。在目标2中，我们将检验FSH糖体的假设 FSHR介导的信号转导的特异性是通过招募不同的蛋白质复合体来激活的 不同的下游基因/蛋白质网络。表达单个FSH糖型和组氨酸的FSHB基因缺失小鼠 将使用颗粒细胞中标记的FSHR。His标签特异性抗体的下拉实验 随后对卵巢蛋白的质谱分析将使我们能够识别FSH糖体特异性 FSH受体和受体辅助因子蛋白复合体各1例。在目标3中，我们将评估直接 重组FSH糖体对年轻和老年生殖小鼠次级卵泡的影响。 将进行基因和蛋白质表达谱分析以及FSH糖形式如何影响卵泡生长和 配子在体外的质量将被确定。成功完成拟议的研究将会推动我们的 了解FSH糖型调节不同FSHR选择性招募的机制 辅因子伙伴复合体在体内实现FSHR介导的卵巢和卵巢信号转导通路 提供在体外卵泡发生和卵子发生过程中FSH糖形作用的直接读数。我们的 机制研究为保留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，以与NICHD任务保持一致。

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.