Study question Is there a specific splicing of transcripts associated with the progression through meiosis in human oocytes? Summary answer Single oocyte mRNA sequencing (scRNA-seq) reveals that several meiosis- and spindle-associated genes are specifically spliced during final meiotic maturation. What is known already Final meiotic maturation in human oocytes entails a fine regulation of the expression of maternal mRNAs, for both final oocyte growth and the initial phases of embryo development and is key to reproductive success. However, very little is known about the post-transcriptional mechanism regulating this process. Alternative splicing (AS) is the process of intron removal and exon-ligation and contributes to transcriptome complexity by generating different protein isoforms from the same gene. The analysis of oocytes’ mRNAs splicing patterns during meiotic maturation might give important information on the regulation of key transcripts responsible for the acquisition of oocyte competence. Study design, size, duration Thirty-four women undergoing oocytes donation cycles were enrolled in this study, and 40 oocytes, processed and analysed individually, were included. The following groups were compared: 10 immature germinal vesicles (GV), 10 GVs that failed to undergo GV breakdown (GVBD) after 30 hours of in vitro maturation (IVM) in G2plus media (FTM-GV), 10 matured oocytes after IVM (IVM-MII) and 10 in vivo matured MII oocytes (IVO-MII). Participants/materials, setting, methods Total RNA from each oocyte was extracted with PicoPure RNA Extraction kit, cDNA was generated and libraries from single oocytes were constructed using OvationSoLo RNA-Seq System and sequenced on a HiSeq 2500, with 2x150bp reads for alternative isoform analysis. Data were processed and analysed using the Bioconductor package DEXSeq, and exons with adjusted p-value<0.05 were considered significantly different. EGSEA package was used to perform pathway analysis. Main results and the role of chance We obtained high-quality data from each sample, with nearly 96% of the reads mapping to the human reference genome. We analysed specifically the genes that did not present differences in global mRNA expression, but presented differences in single exons, indicating a differential splicing pattern. When comparing GV and FTM-GV, only 3 differentially spliced genes (DSG) were found, indicating that the two types of cells present similar splicing patterns. Conversely, the comparison of GV vs IVM-MII and GV vs IVO-MII resulted in 550 genes and 846 genes with differentially spliced exons, respectively, with a 55% transcripts overlap between the two comparisons. Pathway enrichment analysis from the KEGG database performed on the common DSGs between GV and MIIs identified oocyte meiosis, progesterone-mediated oocyte maturation and cell cycle as containing most of the spliced genes. Genes with known involvement in chromosome segregation and spindle assembly such as CENPC and AURKA, and cell cycle regulators like CDK1 and BUB1were within the most relevant spliced transcripts in the MII group. Our findings demonstrate that transcripts important for the acquisition of oocyte meiotic competence are specifically processed when the final phases of maturation are triggered, specifically after GVBD. Limitations, reasons for caution The immature oocytes used in this study were obtained after hormonal stimulation and LH priming. We did not perform a functional analysis of the spliced exons, therefore no speculations on the differential protein functions can be performed. Wider implications of the findings The analysis of DSG between GV and MIIs identified specific spliced exons from genes mainly involved in different aspects of oocyte meiosis. Our findings highlight the key role of post-transcriptional events during the final phases of human meiosis. Trial registration number NA

中文翻译：

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O-313 人卵母细胞的减数分裂进程以特定减数分裂和纺锤体相关转录物的 mRNA 剪接事件为特征

研究问题是否存在与人类卵母细胞减数分裂进程相关的转录本的特定剪接？总结答案 单卵母细胞 mRNA 测序 (scRNA-seq) 揭示了几个减数分裂和纺锤体相关基因在最终减数分裂成熟期间被特异性剪接。众所周知，人类卵母细胞的最终减数分裂成熟需要对母体 mRNA 的表达进行精细调节，这对于最终卵母细胞的生长和胚胎发育的初始阶段都是如此，并且是生殖成功的关键。然而，关于调节这一过程的转录后机制知之甚少。选择性剪接 (AS) 是内含子去除和外显子连接的过程，通过从同一基因产生不同的蛋白质亚型来促进转录组的复杂性。对减数分裂成熟过程中卵母细胞 mRNA 剪接模式的分析可能会为负责获得卵母细胞能力的关键转录物的调控提供重要信息。研究设计、规模、持续时间 本研究招募了 34 名接受卵母细胞捐赠周期的女性，其中包括 40 个单独处理和分析的卵母细胞。比较了以下组：10个未成熟的生发囊泡（GV），10个在G2plus培养基（FTM-GV）中体外成熟（IVM）30小时后未能经历GV分解（GVBD）的GV，10个IVM后成熟的卵母细胞（ IVM-MII) 和 10 个体内成熟的 MII 卵母细胞 (IVO-MII)。参与者/材料、设置、方法使用 PicoPure RNA 提取试剂盒提取每个卵母细胞的总 RNA，生成 cDNA 并使用 OvationSoLo RNA-Seq 系统构建来自单个卵母细胞的文库，并在 HiSeq 2500 上进行测序，读取 2x150bp 用于替代异构体分析。使用 Bioconductor 软件包 DEXSeq 处理和分析数据，调整后的 p 值＜0.05 的外显子被认为存在显着差异。EGSEA 软件包用于进行通路分析。主要结果和机会的作用我们从每个样本中获得了高质量的数据，近 96% 的读数映射到人类参考基因组。我们专门分析了在整体 mRNA 表达中没有差异但在单个外显子中呈现差异的基因，表明存在差异剪接模式。在比较 GV 和 FTM-GV 时，只发现了 3 个差异剪接基因（DSG），表明这两种类型的细胞呈现出相似的剪接模式。相反，GV 与 IVM-MII 和 GV 与 IVO-MII 的比较分别导致 550 个基因和 846 个具有差异剪接外显子的基因，两个比较之间有 55% 的转录本重叠。来自 KEGG 数据库的通路富集分析对 GV 和 MII 之间的常见 DSG 进行了鉴定，发现卵母细胞减数分裂、孕酮介导的卵母细胞成熟和细胞周期包含大部分剪接基因。已知参与染色体分离和纺锤体组装的基因（例如 CENPC 和 AURKA）以及细胞周期调节因子（例如 CDK1 和 BUB1）位于 MII 组中最相关的剪接转录本中。我们的研究结果表明，当成熟的最后阶段被触发时，特别是在 GVBD 之后，对获得卵母细胞减数分裂能力很重要的转录物被专门处理。限制，谨慎的原因 本研究中使用的未成熟卵母细胞是在激素刺激和 LH 启动后获得的。我们没有对剪接的外显子进行功能分析，因此无法推测不同的蛋白质功能。研究结果的更广泛影响 GV 和 MII 之间的 DSG 分析确定了来自主要参与卵母细胞减数分裂不同方面的基因的特定剪接外显子。我们的研究结果强调了转录后事件在人类减数分裂最后阶段的关键作用。试用注册号 NA 限制，谨慎的原因 本研究中使用的未成熟卵母细胞是在激素刺激和 LH 启动后获得的。我们没有对剪接的外显子进行功能分析，因此无法推测不同的蛋白质功能。研究结果的更广泛影响 GV 和 MII 之间的 DSG 分析确定了来自主要参与卵母细胞减数分裂不同方面的基因的特定剪接外显子。我们的研究结果强调了转录后事件在人类减数分裂最后阶段的关键作用。试用注册号 NA 限制，谨慎的原因 本研究中使用的未成熟卵母细胞是在激素刺激和 LH 启动后获得的。我们没有对剪接的外显子进行功能分析，因此无法推测不同的蛋白质功能。研究结果的更广泛影响 GV 和 MII 之间的 DSG 分析确定了来自主要参与卵母细胞减数分裂不同方面的基因的特定剪接外显子。我们的研究结果强调了转录后事件在人类减数分裂最后阶段的关键作用。试用注册号 NA 研究结果的更广泛影响 GV 和 MII 之间的 DSG 分析确定了来自主要参与卵母细胞减数分裂不同方面的基因的特定剪接外显子。我们的研究结果强调了转录后事件在人类减数分裂最后阶段的关键作用。试用注册号 NA 研究结果的更广泛影响 GV 和 MII 之间的 DSG 分析确定了来自主要参与卵母细胞减数分裂不同方面的基因的特定剪接外显子。我们的研究结果强调了转录后事件在人类减数分裂最后阶段的关键作用。试用注册号 NA