夏南牛子宫外泌体miRNA在牛胚胎发育和着床中的作用研究

doi:10.16431/j.cnki.1671-7236.2025.06.022

摘要/Abstract

摘要： 【目的】探索超数排卵后获取不同胚胎数量的牛子宫冲胚液外泌体miRNA的表达差异，以明确子宫冲胚液外泌体miRNA在牛胚胎发育和着床中的调控机制。【方法】选取15头3～6岁、480～600 kg夏南牛进行超数排卵、同期发情和人工授精。在人工授精后第7天，冲洗牛子宫以获取囊胚，根据国际胚胎生产的平均水平5.5枚作为标准，选取获取的囊胚数量多的3头夏南牛(试验组)和获取的囊胚数量少的2头夏南牛(对照组)，收集子宫冲胚液进行外泌体分离与鉴定，并提取外泌体miRNA进行测序分析；对差异表达miRNA进行靶基因预测，并对靶基因进行GO功能和KEGG通路富集分析。随机选取4个miRNAs(bta-miR-2888、bta-miR-11987、bta-miR-2484、bta-miR-27a-3p)进行实时荧光定量PCR验证。【结果】5个样本的囊泡粒径为30～150 nm，符合外泌体的特征。共筛选到14个差异表达miRNAs，其中9个miRNAs表达上调，5个miRNAs表达下调。差异表达miRNAs共预测到8 703个靶基因。GO功能富集分析表明，靶基因主要富集在与发育过程调节、多细胞生物发育调控、调节运输、发育过程的正向调节、细胞连接等功能有关的条目。KEGG通路富集分析表明，显著富集的MAPK和Hedgehog信号通路等与胚胎发育和着床相关，提示子宫冲胚液外泌体可能参与调控胚胎发育和着床。4个miRNAs的实时荧光定量PCR结果与转录组测序结果相一致。【结论】本研究从夏南牛子宫冲胚液中分离得到了外泌体，丰富了外泌体研究的物种，为夏南牛后续外泌体研究提供了材料。共筛选出14个差异表达miRNAs，有8个miRNAs与胚胎发育和着床相关。子宫内外泌体中miRNA的变化可能影响牛胚胎发育和着床。

关键词: 夏南牛; 外泌体; miRNA; 胚胎发育; 胚胎着床

Abstract: 【Objective】 This study was aimed to explore the expression differences of miRNA in uterine flushing exosomes of cattle with different embryo numbers obtained after superovulation,so as to clarify the regulatory mechanism of miRNA in uterine flushing exosomes during embryo development and implantation in cattle.【Method】 Fifteen Xianan cattle aged 3-6 years with 480-600 kg were selected for superovulation,synchronized estrus and artificial insemination.On the 7th day after artificial insemination,the uterus in Xianan cattle was washed to obtain blastocysts.According to the international average embryo production level of 5.5 embryos as the standard,three Xianan cattle (experimental group) with the highest number of blastocysts obtained and two Xianan cattle (control group) with the lowest number of blastocysts obtained were selected.Uterine flush embryo fluid was collected for isolation and identification of exosomes,and exosomal miRNA was extracted for sequencing analysis.Target genes of differentially expressed miRNA were predicted,and GO function and KEGG pathway enrichment analysis on the target genes were performed.Four miRNAs (bta-miR-2888,bta-miR-11987,bta-miR-2484 and bta-miR-27a-3p) were randomly selected for Real-time quantitative PCR validation.【Result】 The vesicle diameters of 5 samples was 30-150 nm,which was consistent with the characteristics of exosomes.A total of 14 differentially expressed miRNAs were screened,with 9 miRNAs upregulated and 5 miRNAs downregulated.A total of 8 703 target genes were predicted from differently expressed miRNAs.GO function enrichment analysis indicated that the target genes were primarily enriched in terms related to functions such as the regulation of developmental process,regulation of multicellular organismal development,regulation of transport,positive regulation of developmental process,cell junction,and others.KEGG pathway enrichment analysis indicated that the significantly enriched signaling pathways such as MAPK and Hedgehog,which were related to embryonic development and implantation,suggesting that exosomes in uterine flush embryo fluid might be involved in the regulation of embryonic development and implantation.Real-time quantification PCR results of 4 miRNAs were consistent with RNA-Seq results.【Conclusion】 This study isolated exosomes from the uterine fluid flush in Xianan cattle,enriching the species investigated in exosome research,and providing valuable experience for future exosome research in Xianan cattle.There were 14 differentially expressed miRNAs were identified in this study,of which 8 miRNAs were associated with embryonic development and implantation.The results suggested that the change of exosome miRNA in uterus might influence embryonic development and implantation in cattle.

Key words: Xianan cattle; exosome; miRNA; embryo development; embryo implantation

中图分类号:

S814.8

张志浩, 鲁立刚, 张子敬, 王香南, 闵佳, 韩艺伟, 彭晟坤, 栾曼茹, 刘奥兵, 施巧婷, 王二耀. 夏南牛子宫外泌体miRNA在牛胚胎发育和着床中的作用研究[J]. 中国畜牧兽医, 2025, 52(6): 2691-2704.

ZHANG Zhihao, LU Ligang, ZHANG Zijing, WANG Xiangnan, MIN Jia, HAN Yiwei, PENG Shengkun, LUAN Manru, LIU Aobing, SHI Qiaoting, WANG Eryao. Study on the Role of miRNA from Uterine Exosomes in Embryo Development and Implantation of Xianan Cattle[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(6): 2691-2704.

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