3D Microtissues Mimic the Architecture, Estradiol Synthesis, and Gap Junction Intercellular Communication of the Avascular Granulosa.
3D Microtissues Mimic the Architecture, Estradiol Synthesis, and Gap Junction Intercellular Communication of the Avascular Granulosa.
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3D 微组织模仿无血管颗粒的结构、雌二醇合成和间隙连接细胞间通讯。
DOI：
10.1093/toxsci/kfab153
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Morgan,JeffreyR
中科院分区：
文献类型：
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作者：
Ip,BlancheC;Leary,Elizabeth;Knorlein,Benjamin;Reich,David;Van,Vivian;Manning,Joshua;Morgan,JeffreyR
人类持续暴露于数千种未经测试的化学物质，这些化学物质在卵巢卵泡液中被检测到，并可能破坏生殖健康。人卵泡颗粒细胞（GCs）是卵泡的功能单位，具有类固醇生成和信号传导活性，在卵母细胞发育中起关键作用。在卵泡进展过程中，GC增殖形成3D无血管结构，并建立间隙连接细胞间通讯（GJIC），这对维持最佳活力和功能至关重要。我们开发了一种高通量的人类GC体外平台，用于筛选可以影响人类颗粒细胞GJIC和雌二醇（E2）产生的化学物质。我们用人卵巢颗粒样肿瘤KGN细胞制造的颗粒层3D微组织是多细胞分层结构，模拟卵母细胞周围的无血管颗粒层。这些微组织强烈表达类固醇生成CYP19芳香化酶和GJIC细胞间膜通道，连接蛋白43。颗粒微组织产生E2的速率与先前报道的原发性人GC相当。E2的产生被CYP 19抑制剂来曲唑抑制，并被CYP 19激活剂双酚A（100 µM）和染料木素（100 µM）诱导。颗粒微组织显示出活跃的GJIC功能，如钙黄绿素荧光染料从微组织表面到核心的连接蛋白43依赖性扩散所示，使用高通量共聚焦显微镜结合我们的开源自动图像分析工具。总的来说，我们的3D人类颗粒筛选平台非常有希望用于预测和有效的玻璃体毒性测试，以筛选污染卵泡液并可能影响生育力的化学物质。
Humans are consistently exposed to thousands of untested chemicals that have been detected in the follicular fluid of the ovaries, and can disrupt reproductive health. Human granulosa cells (GCs) are the functional unit of the ovarian follicle with steroidogenic and signaling activities, and play a pivotal role in oocyte development. During follicle progression, GCs multiply to form a 3D avascular structure, and establish gap junction intercellular communication (GJIC) that is critical to maintaining optimal viability and function. We developed a high-throughputin vitroplatform of human GCs for the screening of chemicals that can impact GJIC and estradiol (E2) production of human granulosa. Our granulosa 3D microtissues fabricated with human ovarian granulosa-like tumor KGN cells are multicell-layered structures that mimic the avascular granulosa layers surrounding the oocyte. These microtissues robustly expressed the steroidogenic CYP19 aromatase enzyme and GJIC intercellular membrane channel, connexin 43. Granulosa microtissues produced E2 at rates comparable to primary human GCs as previously reported. E2 production was suppressed by the CYP19 inhibitor, letrozole, and induced by CYP19 activators, bisphenol A at 100 µM, and genistein at 100 µM. Granulosa microtissues displayed active GJIC function, as demonstrated by the connexin 43-dependent diffusion of calcein fluorescent dye from microtissue surface to the core using high-throughput confocal microscopy in conjunction with our open-sourced automated image analysis tool. Overall, our 3D human granulosa screening platform is highly promising for predictive and efficientin vitrotoxicity testing to screen for chemicals that contaminate follicular fluid and may affect fertility.