Combined use of dbcAMP and IBMX minimizes the damage induced by a long‐term artificial meiotic arrest in mouse germinal vesicle oocytes
Combined use of dbcAMP and IBMX minimizes the damage induced by a long‐term artificial meiotic arrest in mouse germinal vesicle oocytes
复制标题
dbcAMP 和 IBMX 的联合使用可最大限度地减少小鼠生发囊卵母细胞长期人工减数分裂停滞引起的损伤
DOI：
10.1002/mrd.23315
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发表时间：
2020
影响因子：
2.5
通讯作者：
Cheng‐Guang Liang
中科院分区：
文献类型：
--
作者：
Xue‐Chen Wu;Zhe Han;Xin Hao;Yi‐Tong Zhao;Cheng‐Jie Zhou;Xin Wen;Cheng‐Guang Liang
磷酸二酯酶 (PDE) 介导的环磷酸腺苷 (cAMP) 活性降低可引发哺乳动物卵母细胞中的生发囊泡 (GV) 分解。长期将卵母细胞维持在 GV 阶段对于分析体外减数分裂恢复至关重要。 cAMP 调节剂毛喉素、cAMP 类似物二丁酰 cAMP (dbcAMP) 或 PDE 抑制剂米力农 (Mil)、西洛他唑 (CLZ) 和 3-异丁基-1-甲基黄嘌呤 (IBMX) 可以可逆地抑制分离卵母细胞中的减数分裂恢复。然而，这些化学物质在单独使用时会对卵母细胞的发育和成熟产生负面影响。在这里，我们使用 ICR 小鼠开发了一种模型，该模型可以维持 GV 期停滞，并对随后的卵母细胞和胚胎发育产生最小的毒性影响。我们确定了毛喉素、dbcAMP、Mil、CLZ、IBMX 及其组合用于抑制卵母细胞减数分裂恢复的最佳浓度。根据随后的发育潜力评估不良反应，包括洗脱后减数分裂恢复、首次极体挤出、早期凋亡、双链DNA断裂、线粒体分布、三磷酸腺苷水平和胚胎发育。与 50.0 μM dbcAMP 和 10.0 μM IBMX 组合孵育可有效抑制 GV 期卵母细胞减数分裂恢复，对随后的卵母细胞成熟和胚胎发育具有低毒性。这项工作提出了一种降低毒性的新方法，可以有效地将小鼠卵母细胞捕获并维持在 GV 阶段。
Phosphodiesterase (PDE)-mediated reduction of cyclic adenosine monophosphate (cAMP) activity can initiate germinal vesicle (GV) breakdown in mammalian oocytes. It is crucial to maintain oocytes at the GV stage for a long period to analyze meiotic resumption in vitro. Meiotic resumption can be reversibly inhibited in isolated oocytes by cAMP modulator forskolin, cAMP analog dibutyryl cAMP (dbcAMP), or PDE inhibitors, milrinone (Mil), Cilostazol (CLZ), and 3-isobutyl-1-methylxanthine (IBMX). However, these chemicals negatively affect oocyte development and maturation when used independently. Here, we used ICR mice to develop a model that could maintain GV-stage arrest with minimal toxic effects on subsequent oocyte and embryonic development. We identified optimal concentrations of forskolin, dbcAMP, Mil, CLZ, IBMX, and their combinations for inhibiting oocyte meiotic resumption. Adverse effects were assessed according to subsequent development potential, including meiotic resumption after washout, first polar body extrusion, early apoptosis, double-strand DNA breaks, mitochondrial distribution, adenosine triphosphate levels, and embryonic development. Incubation with a combination of 50.0 μM dbcAMP and 10.0 μM IBMX efficiently inhibited meiotic resumption in GV-stage oocytes, with low toxicity on subsequent oocyte maturation and embryonic development. This work proposes a novel method with reduced toxicity to effectively arrest and maintain mouse oocytes at the GV stage.