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

描述（由申请人提供）：卵巢老化永久性地改变女性激素谱，导致不孕、骨质疏松、心血管疾病等后遗症。在绝经前，与年龄相关的激素分泌变化发生在月经周期的两个阶段。与年轻女性相比，老年女性的黄体激素水平较低，黄体激素是由黄体（CL）产生的。不幸的是，与年龄相关的CL功能障碍的机制尚不清楚。卵巢老化是一种纤维化过程，涉及剧烈的间质重塑，这增加了微环境的刚性。虽然已知ECM衍生的信号调节CL甾体生成，但微环境刚性和激素产生之间的因果关系尚未被证实。因此，在本文提出的研究中，我们将在衰老小鼠模型中验证卵巢物理环境变化导致黄体期激素进行性下降的假设。在初步研究中，我们在一种新的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度。在Aim 1中，拟议的实验将操纵海藻酸盐的硬度，并确定对CL结构和甾体生成的影响。具体地说，实验将通过在更严格的海藻酸盐条件下培养年轻动物的腺体来观察年老的CLs。相反，救援实验将使用在不那么严格的藻酸盐环境中培养的老年动物的CLs进行。此外，将通过竞争性抑制整合素与游离RGD肽的结合来探索ECM粘附性与机械力的关系。在本提案的第二个目的，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 -整合素参与的已知效应体。此外，Rho/ROCK信号可能是ECM刚性、整合素结合和黄体细胞结构功能之间的关键联系。实验将评估年轻和老年动物群体中的Rho/ROCK信号。此外，我们将使用我们的3D水凝胶系统和几种途径抑制剂测试不同硬度培养的CLs中的Rho/ROCK信号传导。通过利用生物工程卵巢微环境的能力，我们可以确定年龄、组织硬度和激素产生交叉的调节机制。在此过程中，我们将测试女性生殖衰老的新理论，并确定可能在体内调节的机制，以改善衰老，绝经前妇女的黄体功能。

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.