Table_3_In Vivo and In Vitro Matured Oocytes From Mice of Advanced Reproductive Age Exhibit Alternative Splicing Processes for Mitochondrial Oxidative Phosphorylation.docx

The mean age of women seeking infertility treatment has gradually increased over recent years. This has coincided with the emergence of in vitro maturation (IVM), a method used in assisted reproductive technology for patients with special requirements. However, when compared with conventional in vitro fertilization, IVM is associated with poor embryonic development potential and low live birth rates, thus limiting the widespread application of this technique. In this study, we performed RNA-sequencing transcriptomic assays and identified a total of 2,627 significant differentially expressed genes (DEGs) between IVM oocytes and in vivo matured oocytes from mice of advanced reproductive age. Next, Kyoto Encyclopedia of Genes and Genomes pathway analysis was used to identify the potential functions of the DEGs. The most significantly enriched pathway was oxidative phosphorylation (OXPHOS). In addition, we constructed a protein-protein interaction network to identify key genes and determined that most of the hub genes were mtDNA-encoded subunits of respiratory chain complex I. Antioxidant supplementation lead to an increase in ATP production and reduced the gene expression profile of the OXPHOS pathway in the IVM group. Moreover, alternative splicing (AS) events were identified during in vivo or in vitro oocyte maturation; data showed that skipped exons were the most frequent type of AS event. A number of genes associated with the OXPHOS pathway exhibited alterations in AS events, including Ndufa7, Ndufs7, Cox6a2, Ndufs5, Ndufb1, and Uqcrh. Furthermore, the process of IVO promoted the skipping of exon 2 in Ndufa7 and exon 3 in Ndufs7 compared with the IVM oocytes, as determined by semi−quantitative RT−PCR. Collectively, these findings provide potential new therapeutic targets for improving IVM of aged women who undergo infertility treatment.

近年来，寻求不孕不育治疗的女性平均年龄呈逐渐上升趋势。这与体外成熟（in vitro maturation, IVM）技术的出现不谋而合——该技术是辅助生殖技术中针对有特殊需求患者的治疗手段。然而，与传统体外受精相比，IVM存在胚胎发育潜能不佳、活产率较低的问题，因此限制了该技术的广泛应用。本研究通过RNA测序转录组分析，在高龄生殖年龄小鼠的IVM卵母细胞与体内成熟卵母细胞（in vivo matured oocytes, IVO）之间，共鉴定出2627个显著差异表达基因（differentially expressed genes, DEGs）。随后，我们利用京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路分析对差异表达基因的潜在功能进行注释，其中富集程度最高的通路为氧化磷酸化（oxidative phosphorylation, OXPHOS）。此外，我们构建了蛋白质-蛋白质相互作用网络以筛选关键基因，发现多数枢纽基因均为呼吸链复合体I的线粒体DNA编码亚基。补充抗氧化剂可提升IVM组卵母细胞的ATP生成水平，并下调其氧化磷酸化通路的基因表达谱。本研究还在体内或体外卵母细胞成熟过程中鉴定到可变剪接（alternative splicing, AS）事件，结果显示外显子跳跃是最常见的可变剪接类型。包括Ndufa7、Ndufs7、Cox6a2、Ndufs5、Ndufb1及Uqcrh在内的多个与氧化磷酸化通路相关的基因均发生了可变剪接事件。进一步通过半定量RT-PCR验证发现，与IVM卵母细胞相比，体内成熟卵母细胞可促进Ndufa7基因第2外显子以及Ndufs7基因第3外显子的跳跃。综上，本研究结果为改善不孕不育治疗中高龄女性的IVM技术提供了潜在的新型治疗靶点。