Modeling ovarian aging phenotype in mechanically tuned 3D matrices

项目摘要 卵巢老化与纤维化、女性激素水平变化和卵母细胞质量下降有关 量大，导致心血管疾病、骨质疏松、不孕不育等后遗症。还没 在绝经期，激素产生和卵母细胞质量有显著的年龄相关变化。卵巢 衰老是一个纤维化过程，涉及急剧的细胞外基质重塑，导致越来越刚性的细胞外基质。 微环境虽然已知ECM衍生的信号调节类固醇生成，但致病性的激素依赖性的激素。 基质力学和功能之间的关系以前没有得到证实。因此在 本文提出的研究，我将测试的假设，在卵泡微环境的机械变化 导致衰老小鼠模型中激素产生和卵子质量的逐渐下降。初步 研究，我们已经证明，3D打印明胶支架-这可以通过实验调整， 不同的刚性-支持卵泡的生存，生长和功能。在目标1中，我将实验性地 操纵3D打印明胶支架的刚度，以确定刚度对卵泡的影响。 结构和功能以及卵母细胞质量。具体来说，我假设我可以复制老年卵泡（即， 减少激素的产生和卵母细胞的质量）。 刚性支架相反，我将用培养在一个培养液中的老年动物的卵泡进行拯救实验。 更柔软的脚手架。在目标2中，所提出的实验将测试卵泡发育的可能机制。 机械传导在许多细胞类型中，Rho/ROCK信号传导负责转换机械信号 转化为生物反应。此外，在初步研究中，我们证明了Rho，磷酸化Rho和ROCK 存在于小鼠卵巢的离散卵泡中，表明Rho信号传导是一种可用的分子， 卵泡机械传导机制。我将分析Rho/ROCK信号在生殖年轻和 年龄较大的小鼠群体。此外，我将测试在3D打印支架中培养的卵泡中的Rho/ROCK信号传导， 不同的刚性和存在的途径抑制剂。这些研究将检验我的总体假设 卵巢基质的机械性质在年龄相关的卵巢功能障碍中起作用（卵巢功能减退）。 激素产生和卵质量），可能通过机械敏感性Rho/ROCK信号传导。而且这些 机械可调的3D打印支架代表了卵巢衰老的新型体外模型， 药物发现和开发的平台，可能会彻底改变与年龄相关的女性 不孕

PROJECT SUMMARY Ovarian aging is associated with fibrosis, a changing female hormone profile, and a decline in oocyte quality and quantity, resulting in sequelae such as cardiovascular disease, osteoporosis, and infertility. Even before menopause, there are significant age-related changes in hormone production and oocyte quality. Ovarian aging is a fibrotic process involving dramatic extracellular matrix remodeling, resulting in an increasingly rigid microenvironment. While it is known that ECM-derived signals regulate steroidogenesis, the causative relationship between matrix mechanics and function has not previously been demonstrated. Thus, in the studies proposed herein, I will test the hypothesis that mechanical changes in the follicle microenvironment cause a progressive decline in hormone production and egg quality in an aging mouse model. In preliminary studies, we have demonstrated that 3D-printed gelatin scaffolds – which can be experimentally tuned to different rigidities— support ovarian follicle survival, growth, and function. In Aim 1, I will experimentally manipulate the stiffness of the 3D printed gelatin scaffolds in order to define the effects of rigidity on follicle structure and function and oocyte quality. Specifically, I hypothesize that I can phenocopy older follicles (i.e. diminished hormone production and oocyte quality) by culturing follicles isolated from younger mice in more rigid scaffolds. Conversely, I will perform a rescue experiment with follicles from older animals cultured in a softer scaffold. In Aim 2, proposed experiments will test a possible mechanism of follicle mechanotransduction. In many cell types, Rho/ROCK signaling is responsible for converting mechanical cues into biological response. Moreover, in preliminary studies, we demonstrate that Rho, phospho-Rho, and ROCK are present in discrete follicles in the murine ovary, indicating that Rho signaling is an available molecular mechanism for follicle mechanotransduction. I will assay Rho/ROCK signaling in reproductively younger and older mouse cohorts. Additionally, I will test Rho/ROCK signaling in follicles cultured in 3D printed scaffolds of various rigidities and in the presence of pathway inhibitors. These studies will test my overarching hypothesis that the mechanical properties of the ovarian matrix play a role in age-related ovarian dysfunction (decline in hormone production and egg quality), possibly via mechanosensitive Rho/ROCK signaling. Moreover, these mechanically-tunable 3D-printed scaffolds represent novel in vitro models of ovarian aging and provide a platform for drug discovery and development that may revolutionize the treatment of age-related female infertility.