绵羊超数排卵卵巢差异表达基因及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2023.07.015

摘要/Abstract

摘要： 【目的】筛选出影响经超数排卵处理的多浪羊卵巢卵泡发育的关键基因、生物过程及信号通路，为进一步了解多浪羊超数排卵状态下卵巢生长发育机制提供依据。【方法】对15只2~3岁身体状况良好的多浪羊进行超数排卵处理后，采集超数排卵中早期（第11天）、中期（第13天）、晚期（第15天）卵巢组织，利用高通量测序技术进行转录组测序，将测序得到的reads序列与参考基因组进行序列比对，筛选出差异表达基因；通过STRING数据库对差异表达基因进行蛋白互作网络分析，采用Cytoscape软件进行可视化编辑处理，利用eggNOG-mapper软件进行GO功能和KEGG通路富集分析。【结果】超数排卵卵巢第11和13天共筛选出223个差异表达基因，获得259条互作关系，差异表达基因显著注释在细胞生长、核糖核蛋白复合等条目中，其中显著差异表达的基因为ITGB3和SNAI1，未发现显著富集通路。超数排卵卵巢第13和15天共筛选出694个差异表达基因，获得785条互作关系，差异表达基因显著注释在DNA复制的启动、DNA复制、DNA新陈代谢过程等条目中，其中显著差异表达的基因主要有CASP3、NOTCH1、WNT2和RUNX2，存在2条富集通路：DNA复制和细胞周期信号通路。超数排卵卵巢第11和15天共筛选出238个差异基因，获得151条互作关系，差异表达基因主要注释在蛋白质水解、细胞内信号转导等条目中，其中显著差异表达的基因主要有PRKAR2B、AQP5及HEY2，存在2条显著富集通路：肥厚型心肌病（HCM）和扩张型心肌病（DCM）信号通路。利用GO功能和KEGG通路富集结果筛选出10个在绵羊卵巢卵泡发育过程中具有直接或间接作用的基因：ITGB3、SNAI1、CASP3、PRKAR2B、AQP5、NOTCH1、RUNX2、WNT2、DPP10和HEY2。【结论】本研究从超数排卵卵巢不同发育时期筛选出10个差异表达基因，其相关功能作用的发挥符合不同时期卵巢卵泡发育变化机制，表明这些基因可能是直接或间接影响卵巢卵泡发育的关键基因，为探究超数排卵卵巢卵泡发育机制提供了理论参考。

关键词: 绵羊; 超数排卵; 转录组测序; 差异表达基因; 卵巢卵泡

Abstract: 【Objective】 The purpose of this study was to screen out the key genes, biological processes and signaling pathways affecting the development of ovarian follicles in Duolang sheep treated with superovulation, so as to provide reference for further understand the mechanism of ovarian growth and development in Duolang sheep under superovulation.【Method】 After superovulation treatment of 15 Duolang sheep aged 2-3 years old and in good physical condition, the ovarian tissues at the early (11 d), middle (13 d) and late (15 d) stages of superovulation were taken out for transcriptome sequencing by high-throughput sequencing technology.The reads sequence obtained by sequencing was aligned with the reference genome to screen out the differentially expressed genes.The protein interaction network of the differentially expressed genes was analyzed by STRING database, and the Cytoscape software was used for visual editing.GO function and KEGG pathway enrichment analysis were performed using eggNOG-mapper software.【Result】 A total of 223 differentially expressed genes were screened between days 11 and 13 of supernumerary ovulation ovaries, and 259 interactions were obtained.The differentially expressed genes were significantly annotated in entries such as cell growth, ribonucleoprotein complex, etc.Among them, the genes with significant differential expression included ITGB3 and SNAI1, but no significant enrichment pathways were found.A total of 694 differentially expressed genes were screened between day 13 and 15 of supernumerary ovulation ovaries, and 785 interactions were obtained.The differentially expressed genes were significantly annotated in initiation of DNA replication, DNA replication and DNA metabolic processes, etc.The genes with significant differential expression mainly included CASP3, NOTCH1, WNT2 and RUNX2.There were 2 enriched pathways of DNA replication and cell cycle signalling pathway.A total of 238 differentially expressed genes were screened between days 11 and 15 of supernumerary ovulation ovaries, and 151 interactions were obtained.The differentially expressed genes were significantly annotated in the entries of protein hydrolysis, intracellular signaling, etc.The genes with significant differential expression mainly included PRKAR2B, AQP5 and HEY2.There were 2 significantly enriched pathways of hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) signaling pathways.GO function and KEGG pathway results were used to screen out 10 genes with direct or indirect functions in the development of ovarian follicles in sheep:ITGB3, SNAI1, CASP3, PRKAR2B, AQP5, NOTCH1, RUNX2, WNT2, DPP10 and HEY2.【Conclusion】 In this study, 10 differentially expressed genes were screened from different developmental stages of superovulation ovary, and found that their related functional roles were consistent with the mechanism of ovarian follicular development in different periods, indicating that these genes might be the key genes that directly or indirectly affect ovarian follicular development, in order to provide theoretical reference for exploring the mechanism of ovarian follicular development in superovulation.

Key words: sheep; superovulation; transcriptome sequencing; differentially expressed genes; ovarian follicles

中图分类号:

晏航, 郝文, 王成倩, 王惠娥. 绵羊超数排卵卵巢差异表达基因及生物信息学分析[J]. 中国畜牧兽医, 2023, 50(7): 2740-2754.

YAN Hang, HAO Wen, WANG Chengqian, WANG Huie. Differentially Expressed Genes and Bioinformatics Analysis of Superovulation Ovary in Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2740-2754.

参考文献

[1] 张焱, 张华.哺乳动物卵巢卵泡发育调控机制研究进展[J].生理学报, 2020, 72(1):63-74. ZHANG Y, ZHANG H.Research advances in regulating mechanisms of mammalian ovarian folliculogenesis[J].Journal of Physiology, 2020, 72(1):63-74.(in Chinese)
[2] WANG H, FENG X, MUHATAI G, et al.Expression profile analysis of sheep ovary after superovulation and estrus synchronisation treatment[J].Veterinary Medicine and Science, 2022, 8(3):1276-1287.
[3] 王忠, 张翠永, 周道, 等.利用同期发情超数排卵技术治疗繁殖障碍母猪的效果研究[J].湖南畜牧兽医, 2021, 1:10-11. WANG Z, ZHANG C Y, ZHOU D, et al.The effect of using simultaneous estrous superovulation technique in treating reproductive disorder sows[J].Hunan Animal Husbandry and Veterinary Medicine, 2021, 1:10-11.(in Chinese)
[4] HIRAIZUMI S, NISHINOMIYA H, OIKAWA T, et al.Superovulatory response in Japanese Black cows receiving a single subcutaneous porcine follicle-stimulating hormone treatment or six intramuscular treatments over three days[J].Theriogenology, 2015, 83(4):466-473.
[5] ARASHIRO E K N, PINTO P H N, LIMA J T M, et al.Three superovulation protocols for in vivo embryo production in Santa Inês sheep[J].Ciência Rural, 2022, 52(8):e20210308.
[6] 岳成广, 黄飞, 王杰, 等.比利时兰白花牛超数排卵实验[J].中国畜牧杂志, 2022, 58(9):199-203. YUE C G, HUANG F, WANG J, et al.Supercount ovulation experiment in Belgian orchid white flowered cattle[J].Chinese Livestock Journal, 2022, 58(9):199-203.(in Chinese)
[7] BETTERIDGE K J.A history of farm animal embryo transfer and some associated techniques[J].Animal Reproduction Science, 2003, 79(3-4):203-244.
[8] HASLER J F.Forty years of embryo transfer in cattle:A review focusing on the journal Theriogenology, the growth of the industry in North America, and personal reminisces[J].Theriogenology, 2014, 81(1):152-169.
[9] 宋天增, 李杰, 马金英, 等.miR-127-3p对C2C12细胞成肌分化和转录组的影响[J].西北农业学报, 2019, 28(1):8-16. SONG T Z, LI J, MA J Y, et al.Effect of miR-127-3p on myogenic differentiation and transcriptome in C2C12 cells[J].Northwest Agricultural Journal, 2019, 28(1):8-16.(in Chinese)
[10] ZHAO Z Q, WANG L J, SUN X W, et al.Transcriptome analysis of the Capra hircus ovary[J].PLoS One, 2015, 10(3):e121586.
[11] KULUS M, KRANC W, SUJKA-KORDOWSKA P, et al.The processes of cellular growth, aging, and programmed cell death are involved in lifespan of ovarian granulosa cells during short-term IVC-Study based on animal model[J].Theriogenology, 2020, 148:76-88.
[12] XU L, LIU C, YE Z, et al.Overexpressed LINC00467 promotes the viability and proliferation yet inhibits apoptosis of gastric cancer cells via raising ITGB3 level[J].Tissue and Cell, 2021, 73:101644.
[13] KULUS M, SUJKA-KORDOWSKA P, KONWERSKA A, et al.New molecular markers involved in regulation of ovarian granulosa cell morphogenesis, development and differentiation during short-term primary in vitro culture-Transcriptomic and histochemical study based on ovaries and individual separated follicles[J].International Journal of Molecular Sciences, 2019, 20(16):3966.
[14] FRANK J W.Characterization of integrins and osteopontin at the uterine-placental interface during pregnancy in pigs and sheep[D].Texas:Texas A&M University, 2015.
[15] TRIBULO C, AYBAR M J, SANCHEZ S S, et al.A balance between the anti-apoptotic activity of Slug and the apoptotic activity of msx1 is required for the proper development of the neural crest[J].Developmental Biology, 2004, 275(2):325-342.
[16] VEGA S, MORALES A V, OCAÑA O H, et al.Snail blocks the cell cycle and confers resistance to cell death[J].Genes & Development, 2004, 18(10):1131-1143.
[17] GUO C, MENG X, BAI J, et al.Expression and localization of transcription factors SNAIL and SLUG in mouse ovaries and pre-implantation embryos[J].Cell and Tissue Research, 2014, 358(2):585-595.
[18] ILHA G F, ROVANI M T, GASPERIN B G, et al.Lack of FSH support enhances LIF-STAT3 signaling in granulosa cells of atretic follicles in cattle[J].Reproduction, 2015, 150(4):395-403.
[19] 苗艳平.miR-150靶向STAR基因影响绵羊卵泡颗粒细胞的增殖与凋亡[D].保定:河北农业大学, 2018. MIAO Y P.miR-150 affects proliferation and apoptosis of ovine ovarian granulosa cells by targeting STAR gene[D].Baoding:Hebei Agricultural University, 2018.(in Chinese)
[20] BASAVARAJAPPA M S, KARMAN B N, WANG W, et al.Methoxychlor induces atresia by altering Bcl2 factors and inducing caspase activity in mouse ovarian antral follicles in vitro[J].Reproductive Toxicology, 2012, 34(4):545-551.
[21] VORONTCHIKHINA M A, ZIMMERMANN R C, SHAWBER C J, et al.Unique patterns of Notch1, Notch4 and Jagged1 expression in ovarian vessels during folliculogenesis and corpus luteum formation.[J].Gene Expression Patterns, 2005, 5(5):701-709.
[22] CHEN C, FU X, WANG L, et al.Primordial follicle assembly was regulated by notch signaling pathway in the mice[J].Molecular Biology Reports, 2014, 41(3):1891-1899.
[23] TERAUCHI K J, SHIGETA Y, IGUCHI T, et al.Role of Notch signaling in granulosa cell proliferation and polyovular follicle induction during folliculogenesis in mouse ovary[J].Cell and Tissue Research, 2016, 365(1):197-208.
[24] ZHANG M.P4 down-regulates Jagged2 and Notch1 expression during primordial folliculogenesis[J].Frontiers in Bioscience, 2012, 4(8):2731.
[25] LIU T E, WANG S, ZHANG L, et al.Growth hormone treatment of premature ovarian failure in a mouse model via stimulation of the Notch-1 signaling pathway[J].Experimental and Therapeutic Medicine, 2016, 12(1):215-221.
[26] 高可心.RUNX1和RUNX2在奶山羊卵巢颗粒细胞中的表达调控机理及功能研究[D].杨凌:西北农林科技大学, 2018. GAO K X.Regulation and function of runt-related transcription factors (RUNX1 and RUNX2) in goat granulosa cells[D].Yangling:Northwest A&F University, 2018.(in Chinese)
[27] GOMEZ B I, ALOQAILY B H, GIFFORD C A, et al.ASAS-SSR triennial reproduction symposium:Looking back and moving forward-How reproductive physiology has evolved:Wnts role in bovine folliculogenesis and estrogen production[J].Journal of Animal Science, 2018, 96(7):2977-2986.
[28] WANG H X, GILLIO-MEINA C, CHEN S, et al.The canonical Wnt2 pathway and FSH interact to regulate gap junction assembly in mouse granulosa cells[J].Biology of Reproduction, 2013, 89(2):39.
[29] 陈欣, 达赖, 付绍印, 等.绵羊卵泡发育过程中关键信号通路研究进展[J].中国畜牧兽医, 2021, 48(3):963-974. CHEN X, DA L, FU S Y, et al.Advances on critical signaling pathway in the development of sheep follicles[J].China Animal Husbandry & Veterinary Medicine, 2021, 48(3):963-974.(in Chinese)
[30] JOHNSON J, ESPINOZA T, MCGAUGHEY R W, et al.Notch pathway genes are expressed in mammalian ovarian follicles[J].Mechanisms of Development, 2001, 109(2):355-361.
[31] YOON H, JANG H, KIM E Y, et al.Knockdown of PRKAR2B results in the failure of oocyte maturation[J].Cellular Physiology & Biochemistry, 2018, 45(5):2009-2020.
[32] SU W, GUAN X, ZHANG D, et al.Occurrence of multi-oocyte follicles in aquaporin 8-deficient mice[J].Reproductive Biology and Endocrinology, 2013, 11(1):88.
[33] MŁOTKOWSKA P, TANSKI D, ELISZEWSKI M, et al.The expression profile of AQP1, AQP5 and AQP9 in granulosa and theca cells of porcine ovarian follicles during oestrous cycle and early pregnancy[J].Journal of Animal and Feed Sciences, 2018, 27(1):26-35.
[34] STAROWICZ A, GRZESIAK M, MOBASHERI A, et al.Immunolocalization of aquaporin 5 during rat ovarian follicle development and expansion of the preovulatory cumulus oophorus[J].Acta Histochemica, 2014, 116(3):457-465.
[35] 姚晔俪.卵泡刺激素对卵巢癌细胞Notch1/HEY表达的作用[D].上海:复旦大学, 2009. YAO Y L.The Effect of Follicles-stmulating homorne on the expression of Notch1/HEY in ovarian cancer cells[D].Shanghai:Fudan University, 2009.(in Chinese)
[36] SUN X F, SUN X H, CHENG S F, et al.Interaction of the transforming growth factor-beta and Notch signaling pathways in the regulation of granulosa cell proliferation[J]. Reproduction Fertility and Development, 2016, 28(12):1873-1881.
[37] KITAZAWA M, KUBO Y, NAKAJO K.Kv 4.2 and accessory dipeptidyl peptidase-like protein 10(DPP10) subunit preferentially form a 4:2(Kv4.2:DPP10) channel complex[J].Journal of Biological Chemistry, 2015, 290(37):22724-22733.
[38] 王朕华, 丰有吉, 王以政.钾离子通道与肿瘤细胞的关系[J].国外医学(肿瘤学分册), 2005, 32(10):17-20. WANG Z H, FENG Y J, WANG Y Z.Relationship between potassium channels and tumor cells[J]. International Journal of Oncology, 2005, 32(10):17-20.(in Chinese)
[39] 王晓明, 臧益民, 龚卫琴, 等.钾、氯离子通道阻断剂对staurosporine诱导心肌细胞凋亡的调节作用[J].第四军医大学学报, 2004, 9:783-787. WANG X M, ZANG Y M, GONG W Q, et al.Role of potassium and chloride channel blockers in apoptosis of cardiomyocytes induced by staurosporine[J].Journal of the Fourth Military Medical University, 2004, 9:783-787.(in Chinese)
[40] 吴进.人骨肉瘤细胞与电压门控性钾离子通道关系的研究[D].西安:第四军医大学, 2009. WU J.The relationship between human osteosarcoma cells and Voltage-gated potassium channels[D].Xi'an:The Fourth Military Medical University, 2009.(in Chinese)
[41] 刘玲, 詹平, 陈倩, 等.钙激活性中电导钾离子通道在人宫颈癌组织中的表达及其在细胞凋亡和细胞周期进展中的作用[J].中国现代医学杂志, 2010, 20(20):3097-3101. LIU L, ZHAN P, CHEN Q, et al.Intermediate-conductance-Ca²⁺-activated K⁺channels are over expressed in cervical cancer and involved in regulating cell apoptosis and cell cycle of human cervix carcinoma cell line HeLa[J].Chinese Modern Medical Journal, 2010, 20(20):3097-3101.(in Chinese)
[42] 宋鹏琰, 锡建中, 张振红, 等.绵羊miR-200b对卵泡颗粒细胞周期和凋亡的影响[J].畜牧兽医学报, 2021, 52(12):3471-3479. SONG P Y, XI J Z, ZHANG Z H, et al.Effect of sheep miR-200b on cell cycle and apoptosis of follicle granulosa cells[J].Chinese Journal of Animal and Veterinary Sciences, 2021, 52(12):3471-3479.(in Chinese)
[43] CHEN R, HU B, JIANG M, et al.Bioinformatic analysis of the expression and clinical significance of the DNA replication regulator MCM complex in bladder cancer[J].International Journal of General Medicine, 2022, 15:5465-5485.