Increasing evidence reveals that a broad spectrum of environmental chemicals and pharmaceutical compounds cause female ovarian toxicity (ovotoxicity). The current gold standard of ovotoxicity testing largely relies on whole laboratory animals, but in vivo models are time consuming, costly, and present animal welfare concerns. We previously demonstrated that the 3D encapsulated in vitro follicle growth (eIVFG) is a robust in vitro model for ovotoxicity testing. However, the follicle preparation process is complex and highly dependent on technical skills. Here, we aimed to use vitrification method to cryopreserve murine immature follicles for a high-content eIVFG, chemical exposure, and ovotoxicity screening. Results indicated that a closed vitrification system combined with optimized vitrification protocols preserved mouse follicle viability and functionality and vitrified follicles exhibited comparable follicle and oocyte reproductive outcomes to freshly harvested follicles during eIVFG, including follicle survival and development, ovarian steroidogenesis, and oocyte maturation and ovulation. Moreover, vitrified follicles consistently responded to ovotoxic chemical, doxorubicin (DOX). We further used vitrified follicles to test the response of microcystins (MCs), an emerging category of environmental contaminants produced by cyanobacteria associated with harmful algal blooms (HABs), and found that different congeners of MCs exhibited differential ovotoxicities. In summary, our study demonstrates that vitrification enables a long-term-storage and ready-to-use ovarian follicle bank for high-throughput ovotoxicity screening, which identifies endocrine disrupting effects of MCs.

中文翻译：

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封闭式玻璃化系统使小鼠卵巢卵泡库能够进行高通量卵毒性筛查，从而识别微囊藻毒素的内分泌干扰活性。


越来越多的证据表明，多种环境化学物质和药物化合物会导致女性卵巢毒性（卵毒性）。目前卵毒性测试的黄金标准很大程度上依赖于整个实验动物，但体内模型耗时、昂贵，并且存在动物福利问题。我们之前证明，3D 封装体外卵泡生长 (eIVFG) 是用于卵毒性测试的稳健体外模型。然而，卵泡准备过程很复杂，并且高度依赖于技术技能。在这里，我们的目的是使用玻璃化方法冷冻保存小鼠未成熟卵泡，以进行高内涵 eIVFG、化学暴露和卵毒性筛查。结果表明，封闭式玻璃化系统与优化的玻璃化方案相结合，保留了小鼠卵泡的活力和功能，并且玻璃化卵泡在 eIVFG 期间表现出与新鲜收获的卵泡相当的卵泡和卵母细胞生殖结果，包括卵泡存活和发育、卵巢类固醇生成以及卵母细胞成熟和排卵。此外，玻璃化卵泡始终对卵毒性化学物质阿霉素（DOX）有反应。我们进一步使用玻璃化卵泡来测试微囊藻毒素（MC）的反应，微囊藻毒素是一种新兴的环境污染物，由与有害藻华（HAB）相关的蓝藻产生，并发现不同的微囊藻毒素同源物表现出不同的卵毒性。总之，我们的研究表明，玻璃化冷冻可以长期储存和即用型卵泡库，用于高通量卵毒性筛查，从而识别 MC 的内分泌干扰作用。