An ovary-on-a-chip for understanding early folliculogenesis and reproductive toxicology in a large mammalian model

项目摘要/摘要 体外卵泡发生的概述对于保持人类的生育能力和评估 化学污染物对卵巢健康的影响。尽管活的后代可以从卵母细胞中产生 回收的小鼠卵泡在体外生长，卵泡大小较大，卵泡发生持续时间长 哺乳动物可能需要更复杂的培养要求。家猫将被用作研究模型 因为该物种具有许多解剖和生殖特征，包括卵泡大小和 人类卵母细胞的核形态。计划中的项目将利用“芯片器官”。 微流控技术结合体细胞共培养及三维水凝胶的应用体会 简单地说，就是在“芯片上的卵巢”中的活体条件。具体地说，我建议将 用微管吸入法和原子力显微镜测定家猫卵巢硬度并鉴定仿生体 藻酸盐水凝胶的密度，为原始毛囊提供最佳的结构支持。我会决定 硬度和介质流速对体外卵泡发生的影响及串联质谱仪鉴定 以及培养中涉及卵泡激活的RNA测序技术、蛋白质和基因。比较 具有传统、静态文化的动态系统将使我更好地理解 结构刚性(水凝胶密度)、介质流动(微流控芯片)和旁分泌环境(产生 通过共培养)对体外卵泡存活和激活的影响(目标1)。然后我提议评估表观遗传的影响 (组蛋白甲基化，关键印迹基因)在该系统中的长期培养与传统的 培养，以及确定“芯片上的卵巢”在卵巢毒理学研究中的效用，使用活性的 概念验证研究中化疗环磷酰胺的形式(目标2)。总而言之，这些研究 提高对微环境对卵泡发育的贡献的理解，以实现AN的目标 提高生育能力保存和生殖毒理学的体外系统。

PROJECT SUMMARY/ABSTRACT Recapitulation of folliculogenesis in vitro is of interest for both preserving human fertility and assessing the influence of chemical contaminants on ovarian health. Although live offspring can be produced from oocytes recovered from murine follicles grown in vitro, the larger size and protracted duration of folliculogenesis of large mammals likely necessitates more complex culture requirements. The domestic cat will be used as a study model because this species shares a number of anatomical and reproductive characteristics including follicle size and oocyte nuclear configuration with the human. The proposed project will take advantage of ‘organ-on-chip’ microfluidic technology combined with somatic cell co-culture and my experience with 3-dimensional hydrogels to recapitulate, as possible, in vivo conditions in an “ovary-on-a-chip”. Specifically, I propose to quantify the rigidity of the domestic cat ovary via micropipette aspiration and atomic force microscopy and identify biomimetic densities of alginate hydrogel that provide optimal structural support to primordial follicles. I will determine the influence of rigidity and medium flow rate on in vitro folliculogenesis, and identify via tandem mass spectrometry and RNA sequencing technologies, proteins and genes involved in follicle activation in culture. Comparison of the dynamic system with traditional, static culture will allow me to improve understanding of the contributions of structural rigidity (hydrogel density), medium flow (microfluidic chip), and the paracrine environment (produced via co-culture) on follicle survival and activation in vitro (Aim 1). I then propose to evaluate the epigenetic impacts (methylation of histones, key imprinted genes) of long-term culture in this system compared with traditional culture, as well as determine the utility of the “ovary-on-a-chip” in ovarian toxicology studies, using the active form of the chemotherapeutic cyclophosphamide in proof-of-concept studies (Aim 2). Together, these studies advance understanding of contributions of the microenvironment on follicle development toward the goal of an in vitro system for enhancing fertility preservation and reproductive toxicology.