Table_1_The Exploration of miRNAs From Porcine Fallopian Tube Stem Cells on Porcine Oocytes.XLSX

Fallopian tube is essential to fertilization and embryonic development. Extracellular vesicles (EVs) from Fallopian tube containing biological regulatory factors, such as lipids, proteins and microRNAs (miRNAs) serve as the key role. At present, studies on oocytes from porcine oviduct and components from EVs remain limited. We aim to explore the effect of EVs secreted by porcine fallopian tube stem cells (PFTSCs) on oocyte. When the fifth-generation PFTSCs reached 80–90% of confluency, the pig in vitro maturation medium was utilized, and the conditioned medium collected for oocyte incubations. To realize the functions of EVs, several proteins were used to determine whether extracted EVs were cell-free. Field emission scanning electron microscope and nanoparticle tracking analyzer were used to observe the morphology. By next generation sequencing, 267 miRNAs were identified, and those with higher expression were selected to analyze the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment maps. The selected miR-152-3p, miR-148a-3p, miR-320a-3p, let-7f-5p, and miR-22-3p, were predicted to target Cepb1 gene affecting MAPK pathway. Of the five miRNAs, miR-320a-3p showed significant difference in maturation rate in vitro maturation. The blastocyst rate of pig embryos was also significantly enhanced by adding 50 nM miR-320a-3p. In vitro culture with miR-320a-3p, the blastocyst rate was significantly higher, but the cleavage rate and cell numbers were not. The CM of PFTSCs effectively improves porcine oocyte development. The miRNAs in EVs are sequenced and identified. miR-320a-3p not only helps the maturation, but also increases the blastocyst rates.

输卵管（Fallopian tube）对受精及胚胎发育至关重要。源自输卵管的细胞外囊泡（Extracellular vesicles, EVs）携带有脂质、蛋白质、微小RNA（microRNAs, miRNAs）等生物调控因子，发挥核心调控作用。当前针对猪输卵管来源卵母细胞及细胞外囊泡组分的研究仍较为匮乏。本研究旨在探究猪输卵管干细胞（porcine fallopian tube stem cells, PFTSCs）分泌的细胞外囊泡对猪卵母细胞的影响。当第5代PFTSCs汇合度达到80%~90%时，采用猪体外成熟培养基培养细胞，收集条件培养基用于卵母细胞孵育实验。为验证所提取的细胞外囊泡无细胞污染，通过多种蛋白标志物检测进行确认。利用场发射扫描电子显微镜与纳米颗粒追踪分析仪观察细胞外囊泡的形态特征。通过下一代测序（next generation sequencing）共鉴定出267种miRNAs，筛选其中高表达的miRNAs进行基因本体（Gene Ontology, GO）与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路富集分析。筛选得到的miR-152-3p、miR-148a-3p、miR-320a-3p、let-7f-5p及miR-22-3p，经生物信息学预测可靶向调控Cepb1基因，进而影响MAPK通路。在上述5种miRNAs中，miR-320a-3p在卵母细胞体外成熟过程中的成熟率存在显著差异。添加50 nM的miR-320a-3p可显著提升猪胚胎的囊胚形成率。采用miR-320a-3p进行体外培养时，猪胚胎的囊胚率显著升高，但卵裂率与细胞数无明显变化。猪输卵管干细胞的条件培养基可有效促进猪卵母细胞的发育成熟。本研究对细胞外囊泡中的miRNAs完成了测序与鉴定，其中miR-320a-3p不仅可助力卵母细胞成熟，还能提升囊胚形成率。