Study question How does cyclophosphamide (CPA) treatment impact at transcriptional level on mouse ovarian tissue? Summary answer Cultured murine ovarian tissue with CPA versus control showed up-regulated intrinsic and extrinsic apoptotic signaling pathways, associated with DNA damage, DNA repair and oxidative response. What is known already Alkylating chemotherapeutic treatment depletes the ovarian pool and induces infertility in women. The suggested mechanisms behind these adverse effects include apoptosis and/or over-activation of the dormant primordial follicle pool. However, there is a lack of knowledge about the pathways that lead to these outcomes and previous researches have been inconclusive. The investigation of changes in the ovarian transcriptomic profiling following the alkylating drug CPA treatment can be useful to identify new potential targets for fertility preservation in women treated for cancer. Study design, size, duration Controlled experimental study using 20 female B6CBA/F1 4-day-old mice. Ovaries were collected and randomly assigned to CPA (4-hydroperoxycyclophosphamide) treated group (n = 20) or control group (n = 20). Five ovaries/group were collected at 8, 12, 24 and 36 h to investigate the dynamic of the changes. RNA extraction and RNA sequencing analysis were carried out. Participants/materials, setting, methods Ovaries were cultured on Millicell cell culture inserts floating on 0.25 mL culture medium in a 24-well plate. Freshly prepared 4-hydroperoxycyclophosphamide solution was added to the wells of CPA group (final concentration = 5 µM). Equal amount of solvent was added to the wells of control group. Culture medium was refreshed at 48 h with culture medium only. RNA sequencing data were processed for subsequent differentially expressed genes (DEGs) and gene set enrichment analysis (GSEA). Main results and the role of chance At 8 h, CPA treatment induced the up-regulation of biological processes related to hypoxia, cell growth and embryonic organ development. At 12 h, DNA damage and the ovarian cell responses were evidenced by an increased activity of DNA damage response, DNA damage checkpoint, DNA repair (double-strand break, mismatch, single strand binding), stress-activated MAPK cascade, antioxidant activity and intrinsic apoptotic signaling pathway. The representative genes of these processes there were Bbc3, Bax, Trp73, Cdkn1a, Trp53inp1 and Mdm2. A dramatic increase in the number of DEGs was found at 24 h (8 h, n = 209; 12 h, n = 239; 24 h, n = 2013). Also at 24 h DNA repair, intrinsic and extrinsic apoptotic signaling pathways were the most representative processes evidenced by the addition of Rad9a, H2afx, Casp3, Bak1 and Casp8 genes to the above mentioned. Whereas, germ cell related genes Ybx2, Nobox and Ddx4 were all down-regulated. At 36 h, the number of DEGs (n = 3804) still increased, the up-regulated pathways were similar to 24 h, while meiosis and microtubule-based movements pathways were observed in the down-regulated set too. Limitations, reasons for caution Although the age of the mice chosen for the experiment ensured a high and representative content of primordial follicles in the ovary, whole ovaries were used for RNA sequencing analysis containing a heterogeneous composition of cells other than follicles. Wider implications of the findings Our results provide evidence of dynamic sequential changes in transcriptional level where apoptosis was involved in CPA-induced ovarian follicle depletion. Our research indicates a time frame before the occurrence of DNA definitive damage following CPA-treatment, where application of possible treatments in order to prevent the following apoptosis would be possible. Trial registration number Not Applicable

中文翻译：

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P-448 实验性卵巢培养模型中环磷酰胺处理诱导的基因转录变化

研究问题 环磷酰胺 (CPA) 治疗如何在转录水平上对小鼠卵巢组织产生影响？摘要答案 与对照相比，具有 CPA 的培养小鼠卵巢组织显示出上调的内在和外在凋亡信号通路，与 DNA 损伤、DNA 修复和氧化反应相关。已知的烷基化化学治疗会耗尽卵巢池并导致女性不育。这些不良反应背后的建议机制包括休眠的原始卵泡池的凋亡和/或过度激活。然而，对导致这些结果的途径缺乏了解，以前的研究也没有定论。研究烷基化药物 CPA 治疗后卵巢转录组谱的变化有助于确定接受癌症治疗的女性生育力保存的新潜在目标。研究设计、规模、持续时间 使用 20 只雌性 B6CBA/F1 4 日龄小鼠进行的对照实验研究。收集卵巢并随机分配到 CPA（4-氢过氧环磷酰胺）治疗组（n = 20）或对照组（n = 20）。在 8、12、24 和 36 小时收集 5 个卵巢/组以研究变化的动态。进行RNA提取和RNA测序分析。参与者/材料、设置、方法 卵巢在 24 孔板中漂浮在 0.25 mL 培养基上的 Millicell 细胞培养插管上培养。将新鲜制备的 4-氢过氧环磷酰胺溶液添加到 CPA 组的孔中（最终浓度 = 5 μM）。对照组各孔加入等量溶剂。仅用培养基在 48 小时更新培养基。处理 RNA 测序数据以用于随后的差异表达基因 (DEG) 和基因集富集分析 (GSEA)。主要结果和机会的作用 在 8 小时，CPA 处理诱导了与缺氧、细胞生长和胚胎器官发育相关的生物过程的上调。在 12 小时，DNA 损伤和卵巢细胞反应通过 DNA 损伤反应、DNA 损伤检查点、DNA 修复（双链断裂、错配、单链结合）、应激激活的 MAPK 级联、抗氧化活性和内在的凋亡信号通路。这些过程的代表基因有Bbc3、Bax、Trp73、Cdkn1a、Trp53inp1和Mdm2。在 24 小时发现 DEG 数量急剧增加（8 小时，n = 209；12 小时，n = 239；24 小时，n = 2013）。同样在 24 小时 DNA 修复时，内源性和外源性凋亡信号通路是最具代表性的过程，通过在上述过程中添加 Rad9a、H2afx、Casp3、Bak1 和 Casp8 基因来证明。而生殖细胞相关基因 Ybx2、Nobox 和 Ddx4 均被下调。在 36 小时，DEG 的数量（n = 3804）仍然增加，上调的途径与 24 小时相似，而在下调的集合中也观察到减数分裂和基于微管的运动途径。限制，谨慎的理由 尽管为实验选择的小鼠的年龄确保了卵巢中原始卵泡的高含量和代表性，但整个卵巢被用于 RNA 测序分析，其中包含除卵泡以外的细胞的异质组成。研究结果的更广泛意义 我们的结果提供了转录水平动态连续变化的证据，其中细胞凋亡参与了 CPA 诱导的卵巢卵泡耗竭。我们的研究表明，在 CPA 治疗后 DNA 确定性损伤发生之前的时间范围内，可能会应用可能的治疗方法来防止随后的细胞凋亡。试用注册号 不适用 研究结果的更广泛意义 我们的结果提供了转录水平动态连续变化的证据，其中细胞凋亡参与了 CPA 诱导的卵巢卵泡耗竭。我们的研究表明，在 CPA 治疗后 DNA 确定性损伤发生之前的时间范围内，可能会应用可能的治疗方法来防止随后的细胞凋亡。试用注册号 不适用 研究结果的更广泛意义 我们的结果提供了转录水平动态连续变化的证据，其中细胞凋亡参与了 CPA 诱导的卵巢卵泡耗竭。我们的研究表明，在 CPA 治疗后 DNA 确定性损伤发生之前的时间范围内，可能会应用可能的治疗方法来防止随后的细胞凋亡。试用注册号 不适用