Age-Dependent N-Glycosylation of Follicle-Stimulation Hormone in Gonadotropes

项目总结 绝经过渡是一种复杂的、动态的生理衰老现象，常常导致 对生活方式的不良影响。近85%的绝经后女性会出现多种症状。 对绝经后妇女的临床症状的治疗是困难的，如果不了解潜在的生物学知识 机制和分子参与者，因此需要进一步研究。垂体源前叶 异二聚体糖蛋白激素卵泡刺激素(FSH)由α亚基和β亚基组成。 促卵泡刺激素亚基的N-糖基化发生显著的年龄依赖性变化。更高的全额比率 糖基化促性腺激素β亚单位在老年绝经后妇女与年轻生殖活跃妇女中的比较 在人类脑下垂体和尿液中发现了女性。促性腺激素水平显著升高，因为伴随着下降 卵巢雌激素和孕激素在更年期和高水平的老年特异性FSH可能是 在骨骼和脂肪等组织中有害，在这些组织中，FSH通过炎症途径发挥非经典作用。 这一观察结果意义重大，因为骨质疏松症和体重增加是最常见的两种 绝经后妇女所经历的症状。这个项目的重点是N-糖基化过程 垂体前叶促性腺激素细胞，并试图确定参与糖基化的分子参与者 高龄女性向更充分的糖基化表型转变，以及调节这一过程的因素。 我们最近发现了差异调控的N-糖基化酶在两个年轻的(~4- 通过RNA测序分析，获得了年龄较大(8个月)的雌性小鼠促性腺激素细胞。几个 糖基化途径编码基因(例如，Man2a1、B4galt5)在 老年小鼠的促性腺激素。我们推测这些N-糖基化酶编码基因可能 在老年繁殖后活跃的雌性小鼠中，促性腺激素β中与年龄相关的“糖基化转变”有一定的作用。 在特定的目标1中，我们将定义N-糖基化酶MAN2A1的表达随年龄的变化 和B4GALT4在幼年和老年雌性小鼠的促性腺激素细胞中(使用GFP标记的促性腺激素小鼠 对整个垂体和促性腺激素细胞进行免疫染色，并进行酶活性分析。 在特定的目标2中，我们将确定雌激素和孕激素受体信号在调节 年轻和老年雌性小鼠促性腺细胞N-糖基化途径编码酶的研究 促性腺激素特异性敲除ESR1或PGR并执行荧光激活细胞分选(FACS)， 垂体促性腺激素细胞的RNA测序、定量聚合酶链式反应、免疫染色和酶活性分析。最终， 这里提出的工作代表了一种新的遗传和生化方法，并提供了关于 生殖衰老过程中促卵泡刺激素βN-糖基化的调节及其可能的间接作用 众所周知的更年期症状的病因学，如骨质流失和肥胖。

PROJECT SUMMARY The menopausal transition represents a complex and dynamic physiological aging phenomenon and often leads to adverse life-style effects. Nearly 85% of post-menopausal women will experience multiple symptoms. Treatment of clinical symptoms in post-menopausal women is difficult without knowing the underlying biological mechanisms and molecular players and hence requires further investigation. The anterior pituitary-derived heterodimeric glycoprotein hormone, follicle-stimulating hormone (FSH) consists of an alpha and a beta subunit. The FSH subunit undergoes significant age-dependent changes in N-glycosylation. Higher ratios of fully glycosylated FSHβ subunit in older, post-menopausal women compared to younger, reproductively active women were identified in human pituitaries and urine. FSH levels rise significantly due to concomitantly declining ovarian estrogen and progesterone during menopause and high levels of old age specific FSH may be deleterious in tissues such as bone and adipose, where FSH acts noncanonically via inflammatory pathways. This observation is of great significance because osteoporosis and weight gain are two of the most common symptoms post-menopausal women experience. This project focuses on the N-glycosylation process within anterior pituitary gonadotrope cells and seeks to identify the molecular players involved in the “glycosylation shift” to a more fully glycosylated phenotype in older age females, as well as the factors that regulate this process. We have recently identified differentially regulated N-glycosylation enzymes expressed in both young (~4- months) and older age (8-month) female mouse gonadotrope cells via RNA-sequencing analysis. Several glycosylation pathway-encoding genes (for example, Man2a1, B4galt5) exhibited a significant upregulation in gonadotropes of older age mice. We hypothesize that these N-glycosylation enzyme- encoding genes may contribute to the age-related “glycosylation shift” in FSHβ seen in older, post-reproductively active female mice. In Specific Aim 1, we will define age-dependent changes in expression of N-glycosylation enzymes MAN2A1 and B4GALT4 in gonadotrope cells of young and old female mice (using a GFP-tagged gonadotrope mouse model) and immunostaining whole pituitary and gonadotrope cells, as well as performing enzyme activity assays. In Specific Aim 2, we will determine the role of estrogen and progesterone receptor signaling in regulation of the N- glycosylation pathway-encoding enzymes in gonadotrope cells of young and old female mice using gonadotrope-specific knockout of either Esr1 or Pgr and performing fluorescence-activated cell sorting (FACS), RNA-sequencing, qPCR, immunostaining, and enzyme activity assays in pituitary gonadotrope cells. Ultimately, the work proposed here represents a novel genetic and biochemical approach and provide new knowledge on the regulation of FSHβ N-glycosylation during reproductive aging and how this may indirectly contribute to the etiology of the well-known menopausal symptoms such as bone loss and adiposity.