Assisted reproduction technology (ART) procedures are often impacted by post-ovulatory aging (POA), which can lead to reduced fertilization rates and impaired embryo development. This study used RNA sequencing analysis and experimental validation to study the similarities and differences between in vivo- and vitro-matured porcine oocytes before and after POA. Differentially expressed genes (DEGs) between fresh in vivo-matured oocyte (F_vivo) and aged in vivo-matured oocyte (A_vivo) and DEGs between fresh in vitro-matured oocyte (F_vitro) and aged in vitro-matured oocyte (A_vitro) were intersected to explore the co-effects of POA. It was found that "organelles", especially "mitochondria", were significantly enriched Gene Ontology (GO) terms. The expression of genes related to the "electron transport chain" and "cell redox homeostasis" pathways related to mitochondrial function significantly showed low expression patterns in both A_vivo and A_vitro groups. Weighted correlation network analysis was carried out to explore gene expression modules specific to A_vivo. Trait-module association analysis showed that the red modules were most associated with in vivo aging. There are 959 genes in the red module, mainly enriched in "RNA binding", "mRNA metabolic process", etc., as well as in GO terms, and "spliceosome" and "nucleotide excision repair" pathways. DNAJC7, IK, and DDX18 were at the hub of the gene regulatory network. Subsequently, the functions of DDX18 and DNAJC7 were verified by knocking down their expression at the germinal vesicle (GV) and Metaphase II (MII) stages, respectively. Knockdown at the GV stage caused cell cycle disorders and increase the rate of abnormal spindle. Knockdown at the MII stage resulted in the inefficiency of the antioxidant melatonin, increasing the level of intracellular oxidative stress, and in mitochondrial dysfunction. In summary, POA affects the organelle function of oocytes. A_vivo oocytes have some unique gene expression patterns. These genes may be potential anti-aging targets. This study provides a better understanding of the detailed mechanism of POA and potential strategies for improving the success rates of assisted reproductive technologies in pigs and other mammalian species.

Keywords: mitochondrial function; oocyte quality; post-ovulatory aging; post-transcriptional regulation.

辅助生殖技术（ART）程序常常受到排卵后老化（POA）的影响，这可能导致受精率降低和胚胎发育受损。本研究通过RNA测序分析和实验验证来研究体内成熟和体外成熟的猪卵母细胞在POA前后的相似性和差异性。新鲜的体内成熟卵母细胞（F_vivo）与老化的体内成熟卵母细胞（A_vivo）之间以及新鲜的体外成熟卵母细胞（F_vitro）与老化的体外成熟卵母细胞（A_vitro）之间的差异表达基因（DEGs）进行了交叉分析，以探索POA的共同效应。结果发现，“细胞器”，特别是“线粒体”是显著富集的Gene Ontology (GO) 术语。与线粒体功能相关的“电子传递链”和“细胞氧化还原稳态”通路的相关基因在A_vivo组和A_vitro组中均表现出低表达模式。进行了加权相关网络分析，以探索特定于A_vivo的基因表达模块。性状-模块关联分析表明，红色模块与体内老化最密切相关。红色模块中有959个基因，主要富集在“RNA结合”、“mRNA代谢过程”等GO术语以及“剪接体”和“核苷酸切除修复”通路中。DNAJC7、IK 和 DDX18处于基因调控网络的中心位置。随后，分别在生殖泡（GV）阶段和中期II（MII）阶段敲低了DDX18和DNAJC7的表达来验证它们的功能。在GV阶段敲低导致细胞周期紊乱并增加了异常纺锤体的比例，在MII阶段敲低则使抗氧化剂褪黑素的效果减弱，增加胞内氧化应激水平，并引起线粒体功能障碍。总之，POA影响卵母细胞的细胞器功能。A_vivo卵母细胞有一些独特的基因表达模式。这些基因可能是潜在的抗老化靶点。本研究为更详细地了解POA机制提供了理解，并且可能有助于改善猪及其他哺乳动物种类的辅助生殖技术成功率。

关键词：线粒体功能；卵母细胞质量；排卵后老化；转录后调控