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.

中文翻译：

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dbcAMP和IBMX的组合使用可将小鼠生发囊泡卵母细胞中长期人工减数分裂停滞所引起的损害降至最低。

磷酸二酯酶（PDE）介导的环磷酸一腺苷（cAMP）活性降低可引发哺乳动物卵母细胞的生小泡（GV）分解。长期将卵母细胞维持在GV阶段对体外减数分裂的恢复进行分析至关重要。cAMP调节剂福司高林，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）可在分离的卵母细胞中可逆地抑制减数分裂恢复。但是，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小鼠建立了一个模型，该模型可以维持GV阶段停滞，对随后的卵母细胞和胚胎发育的毒性最小。我们确定了福司可林，dbcAMP，Mil，CLZ，IBMX，及其抑制卵母细胞减数分裂恢复的组合。根据随后的发展潜力评估不良反应，包括冲洗后减数分裂恢复，第一极体挤出，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50.0μMdbcAMP和10.0μMIBMX一起孵育可有效抑制GV期卵母细胞的减数分裂恢复，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具有降低的毒性的新方法，以有效地阻滞和维持小鼠卵母细胞在GV阶段。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50.0μMdbcAMP和10.0μMIBMX一起孵育可有效抑制GV期卵母细胞的减数分裂恢复，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毒性降低的新方法，可以有效地将小鼠卵母细胞停滞并维持在GV阶段。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50.0μMdbcAMP和10.0μMIBMX一起孵育可有效抑制GV期卵母细胞的减数分裂恢复，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具有降低的毒性的新方法，以有效地阻滞和维持小鼠卵母细胞在GV阶段。