The Aging Pituitary/Gonadal Axis

项目摘要/摘要--总体 这一综合计划项目是根据发现的自然产生的、部分糖基化的 Bousfield实验室生产的卵泡刺激素(FSH)糖型。其中两种糖型含有3 在4种N-糖链中，显示出更大的生物活性，并且在年轻人中比完全糖基化的FSH更丰富 女人。此外，低糖基化的FSH变异体表现出年龄依赖性的减少，伴随着 众所周知，女性生育率下降。组织项目是为了反映对以下问题重要性的认识 在生物化学的启发下，在以前的资助时期发展了大量的小鼠遗传模型 对卵泡刺激素糖体的了解的进展，现在，将反过来启发，在 信号、贩运和结构生物学水平。在项目1(Kumar)中，强大的遗传模型现在实现了 可以单独研究每种特定的糖形式，既可以通过将纯化的糖形式制剂注射到 FSHB缺失的小鼠以及表达FSH18、FSH21或FSH24的转基因小鼠。单细胞技术的研究进展 RNA测序与表达绿色荧光蛋白的转基因株相结合 促性腺激素允许评估50多种负责N-连接低聚糖的酶 综合。由于卵泡刺激素的非卵巢靶点引起了接受体外受精的女性的担忧和困惑， 设计了几个小鼠模型来解决相互矛盾的实验结果提出的问题， 从FSHR基因的缺失开始，这将有效地消除所有形式的假定FSHR蛋白， 包括可选的剪接。在项目2(Davis)中，涉及原代培养中FSH糖形信号的研究 关注卵巢靶细胞对偏向激动剂FSH18、FSH21和FSH21的不同反应。 FSH24。两种主要的糖形式调控转录因子(TF)CREB和YAP1将有各自的 下游通路以颗粒细胞为特征。幼龄和高龄小鼠的颗粒细胞和 妇女将提供与翻译工作相关的基本信息。FSH和FSHR之间的接口，两者都是 单体和寡聚受体形式，将在项目3(Jonas)中进行研究。激活cAMP积聚 现已知涉及将进行评估的FSHR动态，包括内部化。促卵泡刺激素糖体是 预计会不同程度地改变FSHR的构象，导致偏向的激动剂信号。项目 4(Bousfield)将把FSHR结合到脂质纳米盘中，以提供一种评估糖形式-FSHR-的手段 膜相互作用，使用低温电子显微镜(CERO-EM)。促黄体生成素和促甲状腺激素已经做到了这一点。 受体(LHR和TSHR)和那些研究揭示了配体结合部位经历了主要的旋转， 在受体激活的过程中，将其从膜上抬起。FSH寡糖的位置在 荷尔蒙的背面，远离荷尔蒙-受体的界面，这意味着与细胞的相互作用 膜或膜蛋白。总而言之，这些项目整合了FSH糖形信号 重叠的水平从完整的有机体到糖基化的激素本身。

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.