Corpus luteum mechanical regulation in a tissue-engineered model of ovarian aging

描述(申请人提供)：卵巢老化永久改变女性荷尔蒙特征，导致不孕、骨质疏松症和心血管疾病等后遗症。在绝经前，荷尔蒙分泌的年龄相关变化发生在月经周期的两个阶段。与年轻女性相比，年龄较大的女性黄体期激素水平较低，黄体(CL)产生黄体激素。不幸的是，与年龄相关的CL功能障碍的机制尚不清楚。卵巢衰老是一个纤维化的过程，涉及剧烈的间质重塑，从而增加微环境的刚性。虽然已知ECM来源的信号调节CL类固醇的生成，但微环境刚性和激素产生之间的因果联系以前还没有被证明。因此，在这里提出的研究中，我们将检验这样的假设，即卵巢物理环境的变化会在衰老的小鼠模型中导致黄体期激素的进行性下降。在初步研究中，我们在一种新型的3D海藻酸盐系统中培养了小鼠CLS，该系统可以调整到不同的机械刚性。在目标1中，拟议的实验将操纵藻酸盐的刚性，并确定对CL结构和类固醇生成的影响。具体地说，实验将通过在更坚硬的藻酸盐条件下培养年轻动物的腺体来复制较老的CLS。相反，将用培养在不那么僵硬的藻酸盐环境中的老年动物的CLS进行救援实验。此外，还将通过竞争性抑制整合素与游离RGD多肽的结合来探讨ECM粘附性与机械力的关系。在这项提议的第二个目的中，实验将测试黄体细胞机械转导的可能机制。在许多细胞类型中，Rho/ROCK信号在感知物理环境中起主要作用，并且是ECM-整合素结合的已知效应器。此外，Rho/ROCK信号可能是细胞外基质刚性、整合素结合和黄体细胞结构功能之间的关键环节。实验将评估Rho/ROCK信号在年轻和老年动物队列中的作用。此外，我们将使用我们的3D水凝胶系统和几种途径抑制剂来测试在不同硬度的CLS中培养的Rho/ROCK信号。通过利用对卵巢微环境进行生物工程的能力，我们可以确定年龄、组织刚性和激素产生的交叉点的调节机制。在这样做的过程中，我们将测试一种新的女性生殖衰老理论，并确定可能在体内调节的机制，以改善老化、绝经前妇女的黄体功能。 公共卫生相关性：随着年龄的增长，卵巢激素的变化对女性的健康起着重要作用，增加了患心血管疾病、骨质疏松症和不孕不育的风险。这个项目的目标是了解年龄相关的卵巢纤维化如何改变荷尔蒙的产生。确定卵巢纤维化的后果将改善老年绝经前妇女激素相关问题的诊断和治疗。

DESCRIPTION (provided by applicant): Ovarian aging permanently alters the female hormone profile, causing sequellae such as infertility, osteoporosis, and cardiovascular disease. Before menopause, age-related changes in hormone production occur during both phases of the menstrual cycle. When compared to younger counterparts, older women have lower levels of luteal phase hormones, which are produced by the corpus luteum (CL). Unfortunately, the mechanism of age-related CL dysfunction is unknown. Ovarian aging is a fibrotic process involving dramatic stromal remodeling, which increases the microenvironment rigidity. While it is known that ECM- derived signals regulate CL steroidogenesis, the causative link between microenvironment rigidity and hormone production has not previously been shown. Thus, in the studies proposed herein, we will test the hypothesis that changes in the ovarian physical environment cause progressive luteal phase hormone decline in an aging mouse model. In preliminary studies, we have cultured murine CLs in a novel 3D alginate system, which can be tuned to different mechanical rigidities. In Aim 1, proposed experiments will manipulate alginate rigidity and define the effects on CL structure and steroidogenesis. Specifically, experiments will phenocopy older CLs by culturing glands from younger animals in more rigid alginate conditions. Conversely, a rescue experiment will be performed with CLs from older animals cultured in less rigid alginate environments. In addition, the relationship of ECM adhesivity and mechanical force will be explored by competitively inhibiting integrin binding with free RGD peptides. In the second Aim of this proposal, experiments will test a possible mechanism of luteal cell mechanotransduction. In many cell types, Rho/ROCK signaling plays a chief role in sensing the physical milieu and is a known effector of ECM-integrin engagement. Moreover, Rho/ROCK signaling could be the critical link between ECM rigidity, integrin binding, and luteal cell structure-function. Experiments will assess Rho/ROCK signaling in younger and older animal cohorts. Additionally, we will test Rho/ROCK signaling in CLs cultured in various rigidities using our 3D hydrogel system and several pathway inhibitors. By harnessing the ability to bioengineer the ovarian microenvironment, we can identify regulatory mechanisms at the intersection of age, tissue rigidity, and hormone production. In so doing, we will test a novel theory of female reproductive aging and identify mechanisms that may be regulated in vivo to improve luteal function in the aging, premenopausal women. PUBLIC HEALTH RELEVANCE: Changes in ovarian hormones play an important role in women's health as they age, increasing the risk for cardiovascular disease, osteoporosis and infertility. The goal of this project is to understand how age-related ovarian fibrosis alters hormone production. Determining the consequences of ovarian fibrosis will improve the diagnosis and treatment of hormone-related problems in aging, premenopausal women.