The Aging Pituitary/Gonadal Axis

摘要 绝经过渡期开始缺乏合适的诊断标记物。过渡是 伴随着严重的发病率，包括生殖周期不规律，功能失调性子宫出血， 泌尿生殖器官改变、生育能力受损、骨量下降、血管舒缩症状和心理 减损。这个项目的长期目标是了解卵泡刺激素的作用。 (FSH)糖基化变异体hFSH21和hFSH24与衰老有关。年轻女性拥有更多的低糖 HFSH21高于完全糖基化的hFSH24，而老年女性拥有的hFSH21较少。因为发现了这两种糖类 在尿液中，两者都进入血液循环，影响性腺和非性腺靶点。在之前的资助中 期间我们发现hFSH21比hFSH24在结合FSHR、激活性腺靶方面比hFSH24更活跃 细胞在体外，并在体内刺激卵巢。相比之下，hFSH24比hFSH21在 激活破骨细胞分化。因此，我们的总体假设是，面对衰老的卵巢， 从hFSH21到hFSH24的额外转换进一步损害了生育能力，并可能导致骨质流失。每个人 项目将通过以下具体目标来测试这一假设的各个方面：1.确定 部分糖基化hFSH导致FSHR结合显著增加的机制(S)。我们的 工作假说是hFSH21和小分子结合改变了FSHR的构象，产生了更多的FSH 可与两种糖型结合的结合部位，从而提高生物活性。一个令人兴奋的元素是协作 超分辨率显微镜研究将直接监测FSHR齐聚。2.定义不同的 在传统的性腺靶点和骨骼中，由FSH糖类激活的信号通路 非传统的靶细胞。在这一目标下，项目2将比较小鼠和人的FSH糖形激活 颗粒细胞或细胞系，以及破骨细胞前体。我们的工作假设是hFSH21更活跃 在性腺靶细胞中，hFSH24高于hFSH24，而非性腺靶细胞则相反。与年龄相关的转变 因此，糖形比具有重要的生理后果。3.使用遗传模型研究 FSH在衰老卵巢和年龄相关性骨质丢失中的糖化形式。在这一目标下，FSH低糖基化 糖形式将由项目3评估其将FSHB基因缺失的雌性小鼠从不孕和 它们对骨质流失的影响。我们的工作假设是与年龄相关的从FSH21到FSH24的转变是有害的 对卵巢功能的影响，但促进破骨细胞激活。这些项目将得到两个项目的支持 科学核心。核心B将为所有项目提供关键的促卵泡激素糖体制剂。核心C将协助 分析质谱学数据，提供下一代测序(NGS)并分析NGS数据。 这些项目将共同提供有关FSH糖体在生殖衰老中的作用的新知识 可以用来诊断更年期转变的开始，并用证据来支持或驳斥 促卵泡激素在老年女性骨质丢失中的作用存在争议。

Abstract The beginning of the menopausal transition lacks suitable diagnostic markers. The transition is attended by significant morbidity including irregular reproductive cycles, dysfunctional uterine bleeding, urogenital changes, impaired fertility, declining bone mass, vasomotor symptoms, and psychological impairment. The long-term objective of this project is to understand the role of follicle-stimulating hormone (FSH) glycosylation variants hFSH21 and hFSH24 in aging. Young women possess more hypo-glycosylated hFSH21 than fully-glycosylated hFSH24, while older women possess less hFSH21. As both glycoforms are found in urine, both enter the circulation where they affect gonadal and non-gonadal targets. In the previous funding period we determined that hFSH21 is much more active than hFSH24 in binding FSHR, activating gonadal target cells in vitro, and stimulating the ovary in vivo. In contrast, hFSH24 is much more active than hFSH21 in activating osteoclast differentiation. Thus, our overall hypothesis is, in the face of a senescing ovary, the additional switch from hFSH21 to hFSH24 further compromises fertility and may contribute to bone loss. Each project will test various aspects of this hypothesis via the following specific aims: 1. Determine the mechanism(s) by which partial glycosylation of hFSH leads to significantly increased FSHR binding. Our working hypothesis is that hFSH21 and small molecule binding alter FSHR conformation, making more FSH binding sites available to both glycoforms, thus increasing bioactivity. An exciting element is collaborative super resolution microscopy studies that will monitor FSHR oligomerization directly. 2. Define the different signaling pathways activated by FSH glycoforms in traditional gonadal targets as well as in bone, a nontraditional target cell. Under this aim, Project 2 will compare FSH glycoform activation of murine or human granulosa cells or cell lines, and osteoclast precursors. Our working hypothesis is that hFSH21 is more active than hFSH24 in gonadal target cells, while the reverse is true for non-gonadal target cells. The age-related shift in glycoform ratio thus has important physiological consequences. 3. Use genetic models to study the role of FSH glycoforms in the aging ovary and age-related bone loss. Under this aim, FSH hypo-glycosylated glycoforms will be evaluated by Project 3 for their ability to rescue Fshb null female mice from infertility and their effects on bone loss. Our working hypothesis is the age-related shift from FSH21 to FSH24 has deleterious effects on ovarian function, yet promotes osteoclast activation. These projects will be supported by two scientific cores. Core B will provide critical FSH glycoform preparations to all projects. Core C will assist in analyzing mass spectrometry data, provide next generation sequencing (NGS) and analyze NGS data. Together, the projects will provide new knowledge about the role of FSH glycoforms in reproductive aging that can be used to diagnose the onset of the menopausal transition, and evidence to support or refute the controversial role of FSH in bone loss in aging women.