Pan Bo, Qin JianPeng, Du KunLin, Zhang LuYao, Jia GongXue, Ye JiangFeng, Liang QiuXia, Yang QiEn, Zhou GuangBin
State Key Laboratory of Swine and Poultry Breeding Industry, Key Laboratory of Livestock and Poultry Multiomics, Ministry of Agriculture and Rural Affairs, Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, College of Animal Science and Technology, Sichuan Agricultural University, Sichuan, Chengdu 611130, PR China.
Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Qinghai, Xining 810001, PR China; University of Chinese Academy of Sciences, Beijing 100049, PR China; Qinghai Key Laboratory of Animal Ecological Genomics, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Qinghai, Xining 810001, PR China.
J Adv Res. 2024 Sep 2. doi: 10.1016/j.jare.2024.08.040.
Developmental competence of oocytes matured in vitro is limited due to a lack of complete understanding of metabolism and metabolic gene expression during oocyte maturation and embryo development. Conventional metabolic analysis requires a large number of samples and is not efficiently applicable in oocytes and early embryos, thereby posing challenges in identifying key metabolites and regulating their in vitro culture system.
To enhance the developmental competence of sheep oocytes, this study aimed to identify and supplement essential metabolites that were deficient in the culture systems.
The metabolic characteristics of oocytes and embryos were determined using ultrasensitive metabolomics analysis on trace samples and single-cell RNA-seq. By conducting integrated analyses of metabolites in cells (oocytes and embryos) and their developmental microenvironment (follicular fluid, oviductal fluid, and in vitro culture systems), we identified key missing metabolites in the in vitro culture systems. In order to assess the impact of these key missing metabolites on oocyte development competence, we performed in vitro culture experiments. Furthermore, omics analyses were employed to elucidate the underlying mechanisms.
Our findings demonstrated that betaine, carnitine and creatine were the key missing metabolites in vitro culture systems and supplementation of betaine and L-carnitine significantly improved the blastocyst formation rate (67.48% and 48.61%). Through in vitro culture experiments and omics analyses, we have discovered that L-carnitine had the potential to promote fatty acid oxidation, reduce lipid content and lipid peroxidation level, and regulate spindle morphological grade through fatty acid degradation pathway. Additionally, betaine may participate in methylation modification and osmotic pressure regulation, thereby potentially improving oocyte maturation and early embryo development in sheep.
Together, these analyses identified key metabolites that promote ovine oocyte maturation and early embryo development, while also providing a new viewpoint to improve clinical applications such as oocyte maturation or embryo culture.
由于对卵母细胞成熟和胚胎发育过程中的代谢及代谢基因表达缺乏全面了解，体外成熟卵母细胞的发育能力受到限制。传统的代谢分析需要大量样本，且在卵母细胞和早期胚胎中应用效率不高，这给识别关键代谢物及其体外培养系统的调控带来了挑战。
为提高绵羊卵母细胞的发育能力，本研究旨在识别并补充培养系统中缺乏的必需代谢物。
采用超灵敏代谢组学分析微量样本和单细胞RNA测序来确定卵母细胞和胚胎的代谢特征。通过对细胞（卵母细胞和胚胎）及其发育微环境（卵泡液、输卵管液和体外培养系统）中的代谢物进行综合分析，我们识别出体外培养系统中关键的缺失代谢物。为评估这些关键缺失代谢物对卵母细胞发育能力的影响，我们进行了体外培养实验。此外，还采用组学分析来阐明潜在机制。
我们的研究结果表明，甜菜碱、肉碱和肌酸是体外培养系统中关键的缺失代谢物，补充甜菜碱和L-肉碱显著提高了囊胚形成率（分别为67.48%和48.61%）。通过体外培养实验和组学分析，我们发现L-肉碱有可能促进脂肪酸氧化、降低脂质含量和脂质过氧化水平，并通过脂肪酸降解途径调节纺锤体形态等级。此外，甜菜碱可能参与甲基化修饰和渗透压调节，从而潜在地改善绵羊卵母细胞的成熟和早期胚胎发育。
这些分析共同识别出了促进绵羊卵母细胞成熟和早期胚胎发育的关键代谢物，同时也为改善卵母细胞成熟或胚胎培养等临床应用提供了新的视角。
