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

项目概要/摘要-项目1（Kumar） 项目1的长期目标是研究卵泡刺激素（FSH）在卵巢癌中的作用机制。 生殖腺和性腺外组织。两个FSH亚基都被两个N-连接的 每个亚基上的糖链。已知FSH的糖基化具有发情/月经周期特异性和年龄特异性。 先前的研究发现FSH变体由α上的2个糖链组成，但β上有一个或没有 亚单位这些变体被称为低糖基化FSH糖型，并命名为FSH 21和FSH 18， 与完全糖基化的FSH 24相反。最重要的是，低-（FSH 21或FSH 18）与完全- 糖基化FSH形式（FSH 24）是年龄依赖性的，高水平的FSH 21/18糖型主要存在于 年龄小，卵巢功能最佳，FSH 24水平高，主要存在于绝经后/绝经后 女性，并可能导致与年龄相关的骨质流失。然而，不同的体内生物学功能 FSH糖型在体内的这些变化比率在衰老的卵巢和骨生理学中是未知的。 此外，是否记录的FSH对骨的作用是通过真正的卵巢FSH受体介导的， 从未被测试过中心假设是雌激素调节年龄依赖性糖基化开关 对FSH的作用，以及由此产生的更高丰度的完全糖基化FSH 24的转变介导骨特异性作用 通过破骨细胞上的FSH受体。这一假设将使用基因工程新小鼠进行测试。 模型在目标1中，我们将通过以下方法从年轻和老年雌性小鼠垂体中纯化GFP标记的促性腺激素： 流式分选并进行RNA-Seq分析，以鉴定年龄特异性和雌激素依赖性糖基化 途径酶。此外，我们将HFSHB 24转基因导入促性腺激素特异性Esr 1 条件无效背景，并确定雌激素信号转导的丧失是否导致 FSH糖型。在目标2中，我们将工程化表达FSH 18或FSH 21低表达的Fshb敲除小鼠。 糖基化形式和Tet-inducible HFSHB 24转基因。这种遗传策略可以让我们暂时 在所需的时间调节FSH糖型的年龄特异性变化比率，并系统地测试体内 在缺乏内源性激素的情况下，FSH糖型比例变化在卵巢生理学中的生物学作用 小鼠FSH。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， 明确测试FSH在骨中破骨细胞中的直接细胞自主作用。成功完成 项目1的研究可能揭示了FSH上年龄依赖性N-糖基化开关的新现象， 靶组织特异性（卵巢与骨）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质流失。项目1的三个目标是 很好地整合到整体P01中，其测试了中心假设，即在面对衰老的卵巢时， 从低糖基化FSH转变为全糖基化FSH，损害生殖潜力， 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.