The Aging Pituitary-Gonadal Axis

描述(由申请人提供)：需要五年的支持来研究老化的垂体性卵巢轴。我们试图通过卵泡刺激素(FSH)来了解垂体腺-卵巢的交流。众所周知，与年轻女性相比，35岁以上的女性生育率会下降。不孕症诊所报告说，老年妇女对外源性FSH制剂的反应性降低。这个项目将集中在老化的卵巢对两种主要的人(H)FSH糖体相对丰度的变化的反应。经典的同时含有α亚基和β亚基低聚糖的hFSH被命名为四糖基化的hFSH，而一种仅含有α亚单位低聚糖的新型hFSH糖体被命名为二糖基化的hFSH。21-24岁的女性表达的二糖化hFSH多于四糖化的hFSH，围绝经期女性表达的二糖化hFSH略少，而绝经后的女性主要表达四糖化的hFSH。项目1将研究月经周期中与增龄相关的hFSH糖体相对丰度的变化，并研究多糖调节FSH受体结合和激活的机制。项目2将比较卵巢中各种信号转导试验中hFSH糖体的活性，以确定增强二糖化hFSH生物活性的可能机制。我们还将研究这两种糖形式对骨吸收的不同影响。项目3将建立小鼠模型，以检验这两种hFSH糖体对生殖功能都是必需的假设。一个双hFSH糖基化突变体将取代正常的mFSH基因。这条线将与卵泡刺激素缺失的小鼠交叉，看看它是否能拯救女性不孕症。纯化的hFSH糖体也将在体内使用FSH缺失的小鼠进行测试。威斯康星州立大学FSH工艺核心实验室(核心B)将向所有项目提供表征良好的纯化hFSH糖形式。最初的产品将是二糖基化的hFSH和四糖基化的hFSH，这是所有科学项目都感兴趣的，但从其他来源得不到。由于天然来源的丰度较低，将首先表达重组二糖基化的hFSH。核心B将表征每个被占据的N-糖基化位点的糖链集落，从而提供具有已知糖基化差异而不是假设的糖基化差异的完全表征的糖型。CORE B还将为项目调查人员提供细胞培养和检测服务。休斯敦州立大学生物信息学核心(核心C)将提供一个数据共享平台，所有调查人员都可以通过互联网随时访问。这项研究的结果将是更好地理解卵巢反应随年龄增长而降低的机制，这可能会导致开发出更有效的FSH制剂来治疗不孕症。尽管目前可用的制剂在年轻女性中效果良好，但它们在老年女性中越来越无效，需要更高的剂量和更长的服药时间，但产生的卵子却更少。

DESCRIPTION (provided by applicant): Five years of support are requested to study the aging pituitary ovarian axis. We seek to understand pituitary-ovarian communication via follicle-stimulating hormone (FSH). It is well known that in women over the age of 35 fertility declines compared with younger women. Infertility clinics report decreased responsiveness of older women to exogenous FSH preparations. This project will focus on the response of the aging ovary to a change in relative abundance of two major human (h) FSH glycoforms. The classic hFSH possessing both alpha subunit and beta subunit oligosaccharides is designated tetra-glycosylated hFSH and a novel hFSH glycoform possessing only alpha subunit oligosaccharides is designated di-glycosylated hFSH. Women aged 21-24 express more di-glycosylated hFSH than tetra-glycosylated hFSH, perimenopausal women express slightly less di-glycosylated hFSH, while post-menopausal women express primarily tetra-glycosylated hFSH. Project 1 will investigate the changes in relative abundance of hFSH glycoforms during the menstrual cycle that are associated with increasing age and study mechanisms for glycan modulation of FSH receptor binding and activation. Project 2 will compare the activities of hFSH glycoforms in a variety of signal transduction assays in the ovary in order to identify possible mechanisms that enhance the biological activity of di-glycosylated hFSH. The differential effects of both glycoforms on bone resorption will also be studied. Project 3 will create mouse models to test the hypothesis that both hFSH glycoforms are necessary for reproductive function. A double hFSH¿ glycosylation mutant will replace the normal mFSH¿ gene. This line will be crossed with FSH¿ null mice to see if it can rescue female infertility. Purified hFSH glycoforms will also be tested in vivo using FSH null mice. The WSU FSH process core laboratory (Core B) will provide well characterized purified hFSH glycoforms, to all projects. The initial products will be di-glycosylated hFSH and tetra-glycosylated hFSH, which are of interest to all the scientific projects, but are not available from other sources. Recombinant di-glycosylated hFSH will be expressed first, due to low abundance in natural sources. Core B will characterize glycan populatlons at each occupied N-glycosylation site, thereby providing fully characterized glycoforms with known, rather than assumed glycosylation differences. Core B will also provide cell culture and assay services to project investigators. The WSU bioinformatics core (Core C) will provide a data-sharing platform readily accessible to all investigators via the internet. The outcome of this research will be a better understanding of the mechanisms for reduced ovarian responsiveness with aging that may lead to the development of more effective FSH preparations for treating infertility. While currently available preparations work well in young women, they become increasingly ineffective in older women, requiring higher doses and prolonged administration yet producing fewer oocytes.