Project 1: Functional Analysis of Age-Specific FSH Analogs Using Genetically Altered Mice

项目摘要/摘要--项目1(库马尔) 项目1的长期目标是研究卵泡刺激素(FSH)在糖尿病中的作用机制。 老年女性的性腺和性腺外组织。FSH的两个亚基都被两个N-连接的糖基化 每个亚基上都有糖链。已知FSH的糖基化与发情/月经周期和年龄有关。 以前的研究发现，促卵泡激素在α上有2个糖链，但在β上有一个或没有 亚单位。这些变体被称为低糖基化的FSH糖型，并被指定为FSH21和FSH18， 与完全糖基化的FSH24形成对比。最重要的是，次-(FSH21或FSH18)与完全- 糖基化FSH形式(FSH24)是年龄相关的，高水平的FSH21/18糖形式在 卵巢功能最佳的年轻年龄和高水平的FSH24主要出现在围绝经期/绝经后 并可能导致与衰老相关的骨质流失。然而，独特的体内生物学功能 其中，体内FSH糖型的变化在卵巢和骨生理学中的变化是未知的。 此外，已有文献记载的FSH对骨骼的作用是否通过真正的卵巢FSH受体介导 从未经过测试。中心假设是雌激素调节年龄依赖的糖基化开关。 FSH及由此引起的高丰度全糖基化FSH24的改变介导了骨特异性作用 通过破骨细胞上的FSH受体。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，我们将通过以下方法从年轻和老年雌性小鼠脑垂体中纯化GFP标记的促性腺激素 流式分选和执行RNA-Seq分析以确定年龄特异性和雌激素依赖的糖基化 途径酶。此外，我们将在促性腺激素特异性ESR1中引入HFSHB24转基因 条件零背景，并确定雌激素信号的丢失是否会导致 卵泡刺激素糖体。在目标2中，我们将设计表达FSH18或FSH21亚基的FSHB缺失小鼠。 糖基化形式和Tet诱导的HFSHB24转基因。这种遗传策略将允许我们在时间上 在所需时间调节FSH糖型的年龄变化比率，并在体内进行系统的测试 非内源性条件下卵泡刺激素糖体比例变化在卵巢生理中的生物学作用 小鼠卵泡刺激素。在目标3中，我们将通过CRE-lox遗传方法选择性地删除破骨细胞中的FSHR，以 明确地测试FSH在骨骼中破骨细胞中的直接细胞自主作用。成功完成 项目1的研究可能揭开一种新的现象，即FSH上的年龄依赖性N-糖基化转换导致 靶组织特异性(卵巢与骨骼)的改变，并可能导致新的治疗选择 用于保护卵巢功能和干预老年妇女的骨丢失。项目1的三个目标是 很好地整合到整体P01中，该P01测试了中心假设，即面对衰老的卵巢， 从低糖基化的卵泡刺激素转变为完全糖基化的卵泡刺激素，会损害生殖潜力，并有助于 其他生理变化，如骨质流失。

PROJECT SUMMARY/ABSTRACT – Project 1(Kumar) The long-term goal of Project 1 is to study the mechanisms of follicle-stimulating hormone (FSH) actions in gonads and extra-gonadal tissues in the aging female. Both the FSH subunits are glycosylated with two N-linked sugar chains on each subunit. It is known that glycosylation of FSH is estrous/menstrual cycle- and age-specific. Previous studies identified FSH variants consisting of 2 sugar chains on the α but either one or none on the β subunit. These variants are known as hypoglycosylated FSH glycoforms and designated as FSH21, and FSH18, in contrast to the fully glycosylated FSH24. Most importantly, the ratio of hypo- (FSH21 or FSH18) to fully- glycosylated FSH forms (FSH24) is age-dependent, with high levels of FSH21/18 glycoforms predominant in young age for optimal ovarian function and high levels of FSH24 predominantly present in peri/post-menopausal women and may contribute to the aging-associated bone loss. However, the distinct in vivo biological functions of these changing ratios of FSH glycoforms in vivo are unknown in ovarian and bone physiology in aging. Moreover, whether the documented FSH actions on bone are mediated via the bona-fide ovarian FSH receptors has never been tested. The central hypothesis is that estrogen regulates an age-dependent glycosylation switch on FSH and the resulting shift in higher abundance of fully-glycosylated FSH24 mediates bone-specific actions via FSH receptors on osteoclasts. This hypothesis will be tested using genetically engineered novel mouse models. In Aim 1, we will purify GFP-tagged gonadotropes from young and old age female mouse pituitaries by flow sorting and perform RNA-Seq analysis to identify age-specific and estrogen-dependent glycosylation pathway enzymes. Additionally, we will introduce an HFSHB24 transgene onto gonadotrope-specific Esr1 conditional null background and determine whether loss of estrogen signaling leads to changes in abundance of FSH glycoforms. In Aim 2, we will engineer Fshb null mice expressing either a FSH18 or a FSH21 hypo- glycosylated form and a Tet-inducible HFSHB24 transgene. This genetic strategy will allow us to temporally regulate at desired times, the age-specific changing ratios of FSH glycoforms and test systematically the in vivo biological actions of changing ratios of FSH glycoforms in ovarian physiology in the absence of endogenous mouse FSH. In Aim 3, we will selectively delete Fshr in osteoclasts by a Cre-lox genetic approach to unequivocally test the direct cell-autonomous actions of FSH in osteoclasts in the bone. Successful completion of Project 1 studies may unravel a novel phenomenon of age-dependent N-glycosylation switch on FSH resulting in alterations in target tissue specificity (ovary versus bone) and may potentially lead to new therapeutic options for preserving ovarian function and intervention of bone loss in aging women. The three Aims of Project 1 are well integrated into the overall P01 which tests the central hypothesis that in the face a senescing ovary, the switch from hypo-glycosylated to fully-glycosylated FSH, compromises reproductive potential and contributes to other physiological changes, such as bone loss.