The Aging Pituitary/Gonadal Axis

项目概要/摘要-总体 这个综合计划项目是根据发现的天然存在的，部分糖基化的， 卵泡刺激素（FSH）糖型。这些糖型中有两种具有3 4个N-聚糖，表现出更大的生物活性，在年轻人中比完全糖基化FSH更丰富， 妇女此外，低糖基化FSH变体表现出年龄依赖性降低， 众所周知的女性生育能力下降。项目的组织反映了对以下方面重要性的认识： 在以前的资助期间，受生物化学的启发， FSH糖型理解的进展，现在，反过来，这将激发在 信号传导、运输和结构生物学水平。在项目1（库马尔），强大的遗传模型，现在使它 可以独立地研究每种特定的糖型，通过将纯化的糖型制剂注射到 Fshb缺失小鼠以及表达FSH18、FSH21或FSH24的转基因小鼠。单细胞研究进展 RNA测序与特异性表达绿色荧光蛋白的转基因系偶联， 促性腺激素允许评价超过50种负责N-连接寡糖的酶 合成.由于FSH的非卵巢靶点引起了接受IVF的妇女的担忧和困惑， 人们设计了几种小鼠模型来解决相互矛盾的实验结果所提出的问题， 从FSHR基因的缺失开始，这将有效地消除所有形式的推定FSHR蛋白， 包括选择性剪接。在项目2（Davis）中，涉及原代培养物中FSH糖型信号的研究 的颗粒细胞，侧重于卵巢靶细胞的不同反应偏向激动剂FSH 18，FSH 21， FSH 24。两个主要的糖型调节差异转录因子（TF）CREB和YAP 1将具有其各自的功能。 在颗粒细胞中具有特征的下游通路。年轻和高龄小鼠的颗粒细胞和 妇女将提供与翻译工作有关的基本信息。FSH和FSHR之间的接口， 单体和寡聚体受体形式，将在项目3（乔纳斯）研究。激活cAMP蓄积 现在已知涉及FSHR动态，将进行评估，包括内化。FSH糖型是 预期在不同程度上改变FSHR构象，导致偏向激动剂信号。项目 4（Bousfield）将FSHR掺入脂质纳米盘中，以提供评估糖型-FSHR的手段。 膜相互作用，使用低温电子显微镜（cyro-EM）。这已经用LH和TSH完成了 受体（LHR和TSHR），这些研究揭示了配体结合位点发生了重大旋转， 在受体激活过程中将其从细胞膜上剥离。FSH寡糖的位置在 激素的背面，远离受体界面，这表明与细胞的相互作用 膜或膜蛋白。总之，这些项目整合了FSH糖型信号， 从完整生物体到糖基化激素本身的重叠水平。

Project Summary/Abstract - Overall This integrated program project was pursuant to the discovery of naturally occurring, partially glycosylated follicle-stimulating hormone (FSH) glycoforms by the Bousfield laboratory. Two of these glycoforms possessed 3 of 4 N-glycans, exhibited greater biological activity, and were more abundant than fully-glycosylated FSH in young women. Furthermore, hypo-glycosylated FSH variants exhibited an age-dependent decrease concomitant with a well-known decline in fertility in women. Projects are organized to reflect the recognition of the importance of the development of numerous mouse genetic models in previous funding periods inspired by biochemical advances in understanding of the FSH glycoforms, and which now, will inspire in turn, such studies at the signaling, trafficking, and structural biology levels. In Project 1(Kumar), powerful genetic models now make it possible to study each specific glycoform independently, both by injection of purified glycoform preparations into Fshb-null mice as well as in transgenic mice expressing FSH18, FSH21, or FSH24. Advances in single-cell RNA-sequencing coupled with a transgenic line that expresses green fluorescent protein specifically in gonadotropes permit evaluation of the more than 50 enzymes responsible for N-linked oligosaccharide synthesis. Since non-ovarian targets of FSH have raised concerns and confusion for women undergoing IVF, several mouse models were devised to address questions raised by conflicting experimental outcomes, beginning with deletion of the Fshr gene, which would effectively eliminate all forms of putative FSHR proteins, including alternative splicing. In Project 2(Davis), studies involving FSH glycoform signals within primary cultures of granulosa cells, focus on the disparate responses of ovarian target cells to biased agonists FSH18, FSH21, and FSH24. Two major, differentially glycoform-regulated transcription factors (TF), CREB and YAP1, will have their downstream pathways characterized in granulosa cells. Granulosa cells of young and advanced age mice and women will provide basic information relevant to translational efforts. The interface between FSH and FSHR, both monomeric and oligomeric receptor forms, will be studied in Project 3(Jonas). Activation of cAMP accumulation is now known to involve FSHR dynamics that will be evaluated, including internalization. FSH glycoforms are anticipated to alter FSHR conformation to varying degrees, leading to biased agonist signals. Project 4(Bousfield) will incorporate FSHR into lipid nanodiscs to provide a means to evaluate glycoform-FSHR- membrane interactions, using cryogenic electron microscopy (cyro-EM). This has been done with LH and TSH receptors (LHR & TSHR) and those studies have revealed the ligand binding site undergoes a major rotation, lifting it away from the membrane, during receptor activation. The location of FSH oligosaccha-rides is on the back side of the hormone, away from the hormone-receptor interface, which suggests interaction with the cell membrane or membrane proteins. Taken together, the projects integrate FSH glycoform signaling at overlapping levels ranging from the intact organism to the glycosylated hormone itself.