RII Track-4: Using the Cryopreservation and Microfluidic Technologies to Engineer Bottlenose Dolphin Ovarian Function for Testing Marine Contaminants' Female Reproductive Toxicity

项目的非技术描述宽吻海豚是美国沿海各州最常见的海洋哺乳动物，根据《海洋哺乳动物保护法》（MMPA）被指定为联邦保护物种。作为顶级捕食者之一，宽吻海豚具有广泛的富含脂质的鲸脂层，更容易受到脂溶性海洋污染物的生物积累。这些污染物中有许多是内分泌干扰化学物质（EDCs），如多氯联苯（PCBs）和邻苯二甲酸盐，它们会对海洋哺乳动物的雌性生殖功能产生不利影响。调查海洋污染物对雌性宽吻海豚繁殖影响的相关研究依赖于记录海豚是否能怀孕并产下可存活的幼崽。然而，EDC对卵巢及其相关功能的具体影响难以监测。本研究计划的中心目标是利用低温保存和三维微流体技术在体外设计宽吻海豚卵巢功能。拟议的研究将允许建立一个高通量卵巢毒性筛选平台，以确定海洋污染物对雌性宽吻海豚以及其他海洋哺乳动物的生殖毒性。项目技术说明卵巢是女性的主要生殖器官，在不同的发育阶段包含卵泡。卵泡的主要功能是分泌性类固醇激素，并支持其封闭的生殖细胞卵母细胞成熟、排卵和受精。本次研究的目的是访问俄勒冈健康与科学大学的宿主地点，了解卵巢组织冷冻保存和大型哺乳动物卵泡的体外生长。研究一的第一个研究目的是通过玻璃化冷冻保存小鼠卵巢卵泡，建立卵泡长期储存平台。PI将在体外培养卵泡并检查卵泡生殖结果，以研究玻璃化对卵泡和卵母细胞健康的影响，并确定玻璃化诱导的细胞损伤。第二个研究目标是低温保存宽吻海豚卵巢卵泡，并利用微流体技术对海豚卵巢功能进行体外工程化。我们将从新鲜死亡的搁浅宽吻海豚身上收集并冷冻保存卵巢，使用微流控平台来支持海豚卵泡的长期发育和成熟，并通过测试已知的卵巢有毒化学物质来验证该系统。建议的工作将有助于建立一个高通量平台，研究海洋污染物对雌性宽吻海豚的生殖毒性，并提高PI及其团队在海洋动物雌性生殖毒理学方面的研究能力。这项研究的广泛影响将产生一个可用于冷冻保存卵巢组织和其他细胞/组织类型的平台。微流体培养将为哺乳动物细胞/组织的体外三维培养提供一种新的方法。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Nontechnical description of the projectThe bottlenose dolphin is the most common marine mammal along the U.S. coastal states and is designated as a federally protected species under the Marine Mammals Protection Act (MMPA). As one of the apex predators and by having extensive lipid-rich blubber layers, the bottlenose dolphin is more susceptible to the bioaccumulation of lipid soluble marine contaminants. Many of these contaminants are endocrine disrupting chemicals (EDCs) such as polychlorinated biphenyls (PCBs) and phthalates, that can adversely impact the female reproductive functions of marine mammals. Studies related to investigating the effect of marine contaminants on female bottlenose dolphin reproduction are dependent on recording whether a dolphin can be pregnant and produce a viable calf. However, it is difficult to monitor the specific impact of EDC's on the ovary and its related functions. The central objective of this research proposal is to use the cryopreservation and 3D microfluidic technologies to engineer the bottlenose dolphin ovarian function in vitro. The proposed research will allow the establishment of a high-throughput ovarian toxicity screening platform to determine the reproductive toxicity of marine contaminants on female bottlenose dolphins as well as other marine mammals.Technical description of the projectThe ovary is the primary female reproductive organ and contains follicles at various developmental stages. The follicle primarily functions to secrete sex steroid hormones and to support its enclosed germ cell oocyte for maturation, ovulation, and fertilization. The goal of the proposed research is to visit the host site at Oregon Health and Science University to learn ovarian tissue cryopreservation and the in vitro growth of follicles from large mammalian species. The first research objective of research one is to cryopreserve mouse ovarian follicles through vitrification for establishing a long-term follicle storage platform. The PI will culture follicles in vitro and examine the follicle reproductive outcomes to investigate the effect of vitrification on follicle and oocyte health and also determine the vitrification-induced cell injury. The second research objective is to cryopreserve bottlenose dolphin ovarian follicles and use microfluidic technology to engineer dolphin ovarian function in vitro. We will collect and cryopreserve ovaries from fresh-dead, stranded bottlenose dolphins, use the microfluidic platform to support a long-term dolphin follicle development and maturation, and validate this system by testing known ovarian toxic chemicals. The proposed work will allow the development of a high-throughput platform to study the female reproductive toxicity of marine contaminants on female bottlenose dolphins and enhance PI and his team's research ability in female reproductive toxicology in marine animals. The broader impact of this study will result in a platform that can be used for cryopreserving ovarian tissues and for other cell/tissue types. Microfluidic culture will provide a novel method to three-dimensionally grow mammalian cells/tissues in vitro.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.