Development of a High-Throughput Platform for Screening Ovotoxic Chemicals (K01ES030014)

项目总结 越来越多的证据表明，广泛的工业和环境化学品都可能导致 女性卵巢毒性(卵毒性)，并增加激素失衡、卵巢衰竭和不孕症的风险 在人类和其他哺乳动物物种中。卵巢是女性主要的生殖器官和功能 合成和分泌类固醇激素，支持卵母细胞成熟以排卵、受精和 怀孕了。卵子毒性可以通过两种方式来衡量，从发育中的卵泡的健康状况和 配子和性腺产生荷尔蒙的能力。不幸的是，缺乏最佳的体外模型 使目前测试化学物质卵毒性的黄金标准依赖于实验室动物的使用。 然而，体内模型既耗时又昂贵，而且对动物有害。在我们之前的研究中，我们有 用藻酸盐水凝胶包埋法培养小鼠和人腔前卵泡 卵巢外的卵泡生长称为包裹式体外卵泡生长(EIVFG)。EIVFG 维持卵泡的三维结构，支持卵泡生长、激素分泌和卵母细胞 成熟和排卵，为体外卵毒性试验提供了一种有前景的模型。但是，此方法是 相对缓慢，需要多轮卵泡分离、培养和化学暴露才能完成 剂量-反应和时间-过程实验，使当前的eIVFG方案不是最佳的，特别是在 在不可预测和新出现的环境威胁的情况下，需要进行快速筛查。在本建议中 项目中，我们假设我们可以使用冷冻保存技术和共聚焦高内容成像 系统，开发一个高通量的卵毒化学物质筛选平台。在目标1中，我们将把 通过玻璃化冷冻分离卵泡，建立一个长期、即用的卵泡存储平台。 在目标2中，我们将使用ImageXpress共焦高内容成像系统来开发高通量 卵泡监测、评估和分析平台。我们期望这些拟议的研究将使我们能够 获得快速可靠的证据，以了解环境污染物对女性的影响 生殖系统，为开展预防和保护活动提供指导，并尽量减少 对动物的依赖和利用。

PROJECT SUMMARY Increasing evidence shows that a broad spectrum of both industrial and environmental chemicals can cause female ovarian toxicity (ovotoxicity) and increase the risk of hormonal imbalance, ovarian failure, and infertility in both humans and other mammalian species. The ovary is the primary female reproductive organ and functions to synthesize and secret steroid hormones and to support the oocyte maturation for ovulation, fertilization, and pregnancy. Ovotoxicity can be measured in two ways, from the health of the developing ovarian follicle and gamete and from the ability of the gonads to produce hormones. Unfortunately, the lack of optimal in vitro models makes the current gold standard for testing the ovotoxicity of chemicals rely on the use of laboratory animals. However, in vivo models are time-­consuming, costly, and harmful to animals. In our previous studies, we have used an alginate hydrogel-­based encapsulation method to grow both mouse and human preantral follicles outside of the context of the ovary, which is termed encapsulated in vitro follicle growth (eIVFG). The eIVFG maintains the 3D architecture of the follicles and supports follicle growth, hormone secretion as well as oocyte maturation and ovulation, suggesting a promising model for in vitro ovotoxicity testing. However, this method is relatively slow, and multiple rounds of follicle isolation, culture, and chemical exposure are necessary to complete dose-­response and time-­course experiments, making the current eIVFG scenario not optimal, particularly when a rapid screening is required in the case of unpredictable and emerging environmental threats. In this proposed project, we hypothesize that we can use cryopreservation technology and a confocal high-­content imaging system to develop a high-­throughput platform for screening ovotoxic chemicals. In Aim 1, we will bank the isolated ovarian follicles through vitrification for establishing a long-­term and ready-­to-­use follicle storage platform. In Aim 2, we will use the ImageXpress confocal high-­content imaging system to develop a high-­throughput platform for follicle monitoring, assessing, and analysis. We expect that these proposed studies will allow us to obtain rapid and reliable evidences to understand the impacts of environmental contaminants on the female reproductive system, to provide guidance to conduct preventive and protective activities, as well as to minimize the dependence and use of animals.