|
|
|
| Screening and Functional Analysis of SNP and InDel in Porcine (Sus scrofa) Follicular Fluid Exosomes Based on RNA-seq Technology |
| LIU Yang-Guang, ZHU Cui-Yun, WU Ping, YE Hai-Bo, WANG Qian-Qian, ZHANG Xiao-Dong*, YIN Zong-Jun* |
| College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China |
|
|
|
|
Abstract Exosomes are a type of extracellular vesicle. In the follicular microenvironment, exosomes serve as important mediators of cellular communication and are capable of regulating follicular growth and development through the genes they carry; however, information regarding the genes present in follicular fluid exosomes remains unclear. Therefore, this study aims to elucidate the genetic diversity of follicular fluid exosomes and to analyse the regulatory functions of exosomes in follicular growth and development, ovulation, and atresia, thereby providing a theoretical basis for breeding and reproductive research in domestic pigs (Sus scrofa). This study found that follicular fluid exosomes could promote ovarian proliferation and the secretion of steroid hormones in pigs. Through transcriptomic sequencing, 44 955 SNP/InDel mutations were identified in follicular fluid exosome samples, the majority of which were SNP variants, totalling 43 752. Only 2 430 of these were homozygous SNP and InDel mutations. Analysis of the mutation types in the sample revealed a total of 2.30×104 transitions SNPs and 2.08×104 transversions SNPs, with a ratio of 1.11 between the two, indicating a significant difference. Annotation of mutation locations revealed that SNP and InDel primarily occurred in intergenic and intron regions, followed by exonic regions and the 、3'UTR and 5'UTR, distributed across 1 073 genes. Analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment revealed that genes with mutations in exonic regions were primarily involved in pathways such as cellular oxidative stress, translation, autophagy, and cell cycle regulation. Furthermore, 10 genes associated with the regulation of follicular development were identified, such as β-actin (ACTB), tumour protein p53 (TP53), cathepsin B (CTSB) and lamin B1 (LMNB1). The SNP/InDel data obtained in this study may provide theoretical support for future research into the application of follicular fluid exosomes in swine breeding.
|
|
Received: 27 November 2025
|
|
|
|
Corresponding Authors:
*xdzhang1983@ahau.edu.cn; yinzongjun@ahau.edu.cn
|
|
|
|
[1] 李志杰, 赵莹, 马鑫, 等. 2023. 动物卵泡液外泌体分离方法及主要生殖功能研究进展[J]. 中国畜牧兽医, 50(04): 1480-1488.
(Li Z J, Zhao Y, Ma X, et al.2023. Research progress on isolation methods and main reproductive functions of exosomes from animal follicular fluid[J]. China Animal Husbandry & Veterinary Medicine, 50(04): 1480-1488.)
[2] 刘阳光, 章会斌, 文浩宇, 等. 2024. 猪卵泡液外泌体处理卵巢颗粒细胞的SNP/InDel筛选分析[J]. 畜牧兽医学报, 55(02): 576-586.
(Liu Y G, Zhang H B, Wen H Y, et al.2024. SNP/InDel screening analysis of porcine ovarian granulosa cells treated with follicular fluid exosomes[J]. Acta Veterinaria Et Zootechnica Sinica, 55(02): 576-586.)
[3] 孙雪, 李胜杰, 姜鹏, 等. 2021. 利用RNA-Seq技术分析草鱼生长性状相关基因和SNP标记[J]. 水产学报, 45(03): 333-344.
(Sun X, Li S J, Jiang P, et al.2021. RNA sequencing to identify genes and SNP markers associated with growth traits in Ctenopharyngodon idella[J]. Journal of Fisheries of China, 45(03): 333-344.)
[4] 王泳, 那日苏, 梁建勇, 等. 2025. 槲皮素对蒙古羊卵泡发育、卵泡液微环境及卵巢类固醇生成相关基因表达的影响[J]. 中国畜牧兽医, 52(03): 1150-1158.
(Wang Y, Na Y S, Liang J Y, et al.2025. Effects of quercetin on follicular development, follicular fluid microenvironment and ovarian steroidogenesis related gene expression in Mongolian sheep[J]. China Animal Husbandry & Veterinary Medicine, 52(03): 1150-1158.)
[5] 闫成琪, 赵源, 田慧彬, 等. 2026. 基因组选择技术在家畜育种中的应用[J]. 农业生物技术学报, 34(03): 480-490.
(Yan C Q, Zhao Y, Tian H B, et al.2026. Application of genomic selection techniques in livestock breeding[J]. Journal of Agricultural Biotechnology, 34(03): 480-490.)
[6] 姚博元, 杨志文, 孙亚朋, 等. 2025. 基于RNA-Seq对猪心组织新转录本解析及可变剪接和SNP分析[J]. 畜牧兽医学报, 56(04): 1664-1675.
(Yao B Y, Yang Z W, Sun Y P, et al.2025. RNA-Seq analysis of novel transcripts, alternative splicing, and SNP in porcine heart tissue based on RNA-Seq Technology[J]. Acta Veterinaria Et Zootechnica Sinica, 56(04): 1664-1675.)
[7] 郁霞青, 宋影春, 李丹. 2019. TDG介导的去甲基化在细胞发育分化及肿瘤发生中的研究进展[J]. 同济大学学报(医学版), 40(04): 516-522.
(Yu X Q, Song Y C, Li D.2019. Research progress of TDG-mediated demethylation in cell development, cell differentiation and tumorigenesis[J]. Journal of Tongji University(Medical Science), 40(04): 516-522.)
[8] 张志浩, 鲁立刚, 张子敬, 等. 2025. 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, 52(06): 2691-2704.
(Zhang Z H, Lu L G, Zhang Z J, et al.2025. Study on the role of miRNA from uterine exosomes in embryo development and implantation of Xianan cattle[J]. China Animal Husbandry & Veterinary Medicine, 52(06): 2691-2704.)
[9] 赵辉, 李启寨, 李俊, 等. 2006. 相邻碱基组分与产生SNP的转换或颠换在植物基因组中的研究[J]. 中国科学C辑:生命科学, (01): 1-8.
(Zhao H, Li Q Z, Li J, et al. 2006. The conversion or transversion of adjacent base components and the generation of SNP research in plant genomes[J]. Science in China Series C Life Sciences, (01): 1-8.)
[10] 周明夏, 张蕾, 章敬旗, 等. 2022. 开产前和产蛋高峰期泰州鹅卵巢转录组SNP分析[J]. 扬州大学学报(农业与生命科学版), 43(04): 36-44.
(Zhou M X, Zhang L, Zhang J Q, et al.2022. SNP screening and analysis between pre-egged and peak laying Taizhou geese' ovaries based on RNA-Seq[J]. Journal of Yangzhou University (Agricultural and Life Science Edition), 43(04): 36-44.)
[11] Auton A, Brooks L D, Durbin R M, et al.2015. A global reference for human genetic variation[J]. Nature, 526(7571): 68-74.
[12] Basuino L, Silveira C F Jr.2016. Human follicular fluid and effects on reproduction[J]. JBRA Assisted Reproduction, 20(1): 38-40.
[13] Biglari-Zadeh G, Sargazi S, Mohammadi M, et al.2023. Relationship between genetic polymorphisms in cell cycle regulatory gene TP53 and polycystic ovarian syndrome: A case-control study and in silico analyses[J]. Biochemical Genetics, 61(5): 1827-1849.
[14] Cao J, Huo P, Cui K, et al.2022. Follicular fluid-derived exosomal miR-143-3p/miR-155-5p regulate follicular dysplasia by modulating glycolysis in granulosa cells in polycystic ovary syndrome[J]. Cell Communication and Signaling : CCS, 20(1): 61.
[15] Chen X, Cao M, Yuan C, et al.2024. Follicular fluid exosomes inhibit expression of BTG2 and promote glucose uptake in granulosa cells by delivering miR-21-5p[J]. Theriogenology, 218: 45-55.
[16] Dai S, Wang H, Lin Z.2021. ACTB mutations analysis and genotype-phenotype correlation in becker's nevus[J]. Biomedicines, 9: 12-21.
[17] Fiscus J, Fraison É, Renault L, et al.2024. Metabolic signature of follicular fluid in infertility-related diseases: A narrative review[J]. Reproductive Biomedicine Online, 48(6): 103762.
[18] Gao Z, Zhang Y, Li Z, et al.2022. Genomic breed composition of Ningxiang pig via different SNP panels[J]. Journal of Animal Physiology and Animal Nutrition, 106(4): 783-791.
[19] Huang P, Chen S, Cai J, et al.2025. Circular RNA expression profiling and the potential role of hsa_circ_0005379 in decreased ovarian reserve[J]. Experimental and Therapeutic Medicine, 30(6): 224.
[20] Huang W, Zhang X, Li A, et al.2018. Genome-wide analysis of mRNAs and lncRNAs of intramuscular fat related to lipid metabolism in two pig breeds[J]. Cellular Physiology and Biochemistry: International Journal of Experimental Cellular Physiology, Biochemistry, and Pharmacology, 50(6): 2406-2422.
[21] Kim E, Ra K, Lee M S, et al.2023. Porcine follicular fluid-derived exosome: The pivotal material for porcine oocyte maturation in lipid antioxidant activity[J]. International Journal of Molecular Sciences, 24: 12-28.
[22] Li J, Guo H, Tian M, et al.2025. Exosome-derived lncRNA LIPE-AS1 enhances oocytes maturation and ameliorates diminished ovarian reserve via the miR-330-5p/HDAC3 axis[J]. Journal of Biochemical and Molecular Toxicology, 39(10): e70519.
[23] Li Y, Luo X, Peng X, et al.2023. Development of SNP and inDel markers by genome resequencing and transcriptome sequencing in radish (Raphanus sativus L.)[J]. BMC Genomics, 24(1): 445.
[24] Martínez-Giner M, Noguera J L, Balcells I, et al.2013. Selection of internal control genes for real-time quantitative PCR in ovary and uterus of sows across pregnancy[J]. PLOS ONE, 8(6): e66023.
[25] Mcgee E A, Hsueh A J.2000. Initial and cyclic recruitment of ovarian follicles[J]. Endocrine Reviews, 21(2): 200-214.
[26] Soares M, Almiñana C, Ferreira A F, et al.2025. Age and physical activity modulate miRNAs contained in human follicular fluid extracellular vesicles[J]. Reproduction (Cambridge, England), 170: 5-20.
[27] Voskarides K, Giannopoulou N.2023. The role of TP53 in adaptation and evolution[J]. Cells, 12(3): 3-14.
[28] Wang M, Sun Y, Yuan D, et al.2023. Follicular fluid derived exosomal miR-4449 regulates cell proliferation and oxidative stress by targeting KEAP1 in human granulosa cell lines KGN and COV434[J]. Experimental Cell Research, 430(2): 113735.
[29] Wang S, Li Z, Guo X, et al.2018. Comparative analysis of microsatellite, SNP, and InDel markers in four Rhododendron species based on RNA-seq[J]. Breeding Science, 68(5): 536-544.
[30] Wang X, Lu X, Wang J, et al.2016. Mining and analysis of SNP in response to salinity stress in upland cotton (Gossypium hirsutum L.)[J]. PLOS ONE, 11(6): e0158142.
[31] Westermann A J, Vogel J.2021. Cross-species RNA-seq for deciphering host-microbe interactions[J]. Nature Reviews Genetics, 22(6): 361-378.
[32] Xie S, Su Y, Zhang J, et al.2024. Upregulation of miRNA-450b-5p targets ACTB to affect drug resistance and prognosis of ovarian cancer via the PI3K/Akt signaling pathway[J]. Translational Cancer Research, 13(9): 4800-4812.
[33] Xu C, Zhang W, Jiang Y, et al.2023. Genome-wide detection and analysis of copy number variation in Anhui indigenous and western commercial pig breeds using porcine 80K SNP beadchip[J]. Genes, 14: 3-15.
[34] Yan X, Liu H, Hu J, et al.2022. Transcriptomic analyses of the HPG axis-related tissues reveals potential candidate genes and regulatory pathways associated with egg production in ducks[J]. BMC Genomics, 23(1): 281.
[35] Yebra M J, Bhagwat A S.1995. A cytosine methyltransferase converts 5-methylcytosine in DNA to thymine[J]. Biochemistry, 34(45): 14752-14757.
[36] Yuan C, Cao M, Chen L, et al.2023. Follicular fluid exosomes inhibit BDNF expression and promote the secretion of chemokines in granulosa cells by delivering miR-10b-5p[J]. Theriogenology, 199: 86-94.
[37] Yuan C, Chen X, Shen C, et al.2022. Follicular fluid exosomes regulate oxidative stress resistance, proliferation, and steroid synthesis in porcine theca cells[J]. Theriogenology, 194: 75-82.
[38] Zhao Y, Wang K, Wang W L, et al.2019. A high-throughput SNP discovery strategy for RNA-seq data[J]. BMC Genomics, 20(1): 160. |
|
|
|
