不同等级牛卵丘-卵母细胞复合体体外成熟后卵母细胞发育能力差异的机制分析

doi:10.11843/j.issn.0366-6964.2025.09.025

摘要/Abstract

摘要： 旨在探究不同等级牛卵丘-卵母细胞复合体(cumulus-oocyte complexes, COCs)体外成熟后卵母细胞发育能力差异的分子机制。本试验以屠宰场卵巢来源的牛COCs为研究对象，参照国际胚胎技术学会手册根据卵丘细胞层数、卵丘细胞紧密程度和卵母细胞细胞质外观将牛COCs分为一二级(较好)和三四级(较差)两组，每组3次重复，每个重复组含100枚以上COCs，进行体外成熟培养。本研究通过Smart-seq2技术对成熟前后的一二级和三四级COCs的卵母细胞进行了转录组测序并对差异基因进行了分析验证，利用非靶向代谢组学技术对不同级别COCs的培养液进行了差异代谢物分析。结果显示：1)三四级COCs的卵母细胞体外成熟、受精后的卵裂率和囊胚率(60.59%±6.54%，22.11%±2.79%)显著低于一二级(78.09%±3.01%，32.56%± 4.33%，P＜0.05)；2)Smart-seq测序结果显示，体外成熟前一二级和三四级COCs的卵母细胞中共筛选出541个显著差异表达基因(DEGs)，体外成熟22~24 h(体外成熟后)一二级和三四级卵母细胞中共筛选出860个显著DEGs，GO和KEGG富集分析结果显示，这些DEGs主要与糖代谢、氧化磷酸化信号通路相关；3)数字PCR结果显示，体外成熟前后三四级COCs的卵母细胞中糖酵解相关基因PKM和电子传递链复合物I相关抑制基因NDUFA4L2的拷贝数水平均显著低于一二级(P＜0.01)，体外成熟后三四级卵母细胞中电子供体生成相关基因IDH3α的拷贝数水平显著低于一二级(P＜0.05)；同样体外成熟前后三四级COCs的卵丘细胞中NDUFA4L2 mRNA表达水平显著低于一二级(P＜0.05)，但是PKM和IDH3α mRNA表达水平却显著高于一二级(P＜0.05，P＜0.01)；4)代谢物分析结果显示，不同级别COCs的培养液中共鉴定出69种显著上调差异代谢物，71种显著下调差异代谢物，功能富集分析发现差异代谢物主要富集在三羧酸循环(TCA)、糖酵解/糖异生等代谢通路上，进一步对差异代谢物(α-D-葡萄糖、丙酮酸、乳酸)进行相对丰度分析发现三四级COCs成熟液中α-D-葡萄糖含量显著高于一二级(P＜0.01)。5)在三四级COCs体外成熟液中添加糖酵解产物丙酮酸钠能够显著提高三四级COCs体外成熟、受精后的卵裂率(72.54%±4.79% vs. 61.71%±4.75%)和囊胚率(30.63%±4.15% vs. 22.72%±2.39%，P＜0.05)。综上，本研究表明三四级COCs的卵母细胞体外成熟后发育能力差可能是由于其本身糖酵解活性较弱，加之其外围卵丘细胞能量底物供给不足，导致卵母细胞成熟时能量不足，发育能力受限；通过添加丙酮酸钠能够明显改善三四级COCs的卵母细胞体外成熟后发育能力，研究结果为提高COCs利用率和胚胎生产效率提供了理论依据。

关键词: 牛, 卵丘卵母细胞复合体, 体外成熟, 糖代谢, 丙酮酸钠

Abstract: This study aimed to investigate the molecular mechanisms underlying the differences in developmental competence of oocytes from different grades of bovine cumulus-oocyte complexes (COCs) after in vitro maturation. Bovine COCs derived from ovaries of bovine from slaughterhouse were classified into two groups (grades I-II (better)and grades III-IV (poor)) based on cumulus cell layers, compactness, and cytoplasm appearance according to the International Embryo Technology Society manual. The experiment was divided into 3 replicates, each replicate group contained more than 100 COCs. After in vitro maturation, transcriptome analysis of oocytes was performed using Smart-seq2 technology and the differentially expressed genes (DEGs) were validated. Non-targeted metabolomics was employed to analyze differential metabolites in the culture medium of different COC grades. The results showed that: 1) Grade III-IV COCs exhibited significantly lower cleavage rate (60.59%±6.54%) and blastocyst rate (22.11%±2.79%) compared to grade I-II COCs (78.09%±3.01%, 32.56%±4.33%, P<0.05); 2) Smart-seq2 analysis identified 541 significant DEGs between grade I-II and grade III-IV oocytes before maturation, and 860 DEGs after 22-24 h of maturation (after in vitro maturation). GO and KEGG enrichment analyses revealed that these DEGs were primarily associated with glucose metabolism and oxidative phosphorylation pathways; 3) Digital PCR demonstrated significantly lower copy numbers of glycolysis-related gene PKM and electron transport chain complex I inhibitor gene NDUFA4L2 in grade III-IV oocytes compared to grade I-II (P<0.01). After maturation, grade III-IV oocytes showed significantly lower copy numbers of electron donor generation-related gene IDH3α compared to grade I-II (P<0.05). Interestingly, grade III-IV cumulus cells exhibited significantly lower NDUFA4L2 mRNA expression (P<0.05) but higher PKM and IDH3α mRNA expression than grade I-II (P<0.05, P<0.01); 4) Metabolite analysis identified 69 significantly upregulated and 71 downregulated metabolites in different grades of bovine cumulus-oocyte complexes culture medium. Further functional enrichment analysis showed these metabolites were primarily involved in tricarboxylic acid cycle (TCA), and glycolysis/gluconeogenesis pathways. Relative abundance analysis of key metabolites (α-D-glucose, pyruvate, lactate) revealed significantly higher α-D-glucose content in grade III-IV maturation medium (P<0.01); 5) Supplementation of sodium pyruvate, a glycolysis product, significantly improved cleavage rate (72.54%±4.79% vs. 61.71%±4.75%) and blastocyst rate (30.63%±4.15% vs. 22.72%±2.39%, P<0.05) in grade III-IV COCs. This study suggests that the poor developmental competence of grade III-IV COCs after in vitro maturation may result from insufficient glycolytic activity in oocytes coupled with insufficient energy substrate supply from surrounding cumulus cells. The addition of sodium pyruvate significantly improved the developmental competence of grade III-IV COCs, which provide a theoretical basis for enhancing COC utilization and embryo production efficiency.

Key words: bovine, cumulus-oocyte complex, in vitro maturation, glucose metabolism, sodium pyruvate

中图分类号:

S823.3

王蕊, 衡诺, 胡樱凡, 王欢, 朱妮, 何维, 轩秀丽, 胡智辉, 熊铿, 巩建飞, 郝海生, 朱化彬, 赵善江. 不同等级牛卵丘-卵母细胞复合体体外成熟后卵母细胞发育能力差异的机制分析[J]. 畜牧兽医学报, 2025, 56(9): 4432-4451.

WANG Rui, HENG Nuo, HU Yingfan, WANG Huan, ZHU Ni, HE Wei, XUAN Xiuli, HU Zhihui, XIONG Keng, GONG Jianfei, HAO Haisheng, ZHU Huabin, ZHAO Shanjiang. Mechanisms of Developmental Competence Differences of Oocytes from Different Grades of Bovine Cumulus-Oocyte Complexes after in Vitro Maturation[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(9): 4432-4451.

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