川中黑山羊和雷州山羊大小卵泡转录组学分析

doi:10.16431/j.cnki.1671-7236.2020.05.020

摘要/Abstract

摘要： 试验旨在通过比较两个品种山羊大小卵泡的mRNA表达图谱来挖掘影响山羊卵泡发育的基因，为进一步阐述山羊卵泡发育机制提供数据基础。使用氯前列醇钠对川中黑山羊和雷州山羊进行同期发情处理，屠宰后采集卵巢并在体视显微镜下分离单个卵泡（大卵泡>6 mm；小卵泡<3 mm），提取卵泡组织总RNA进行RNA-seq，利用生物信息学方法检测mRNA表达谱，筛选差异基因，并对其进行GO功能、KEGG通路富集分析。结果显示，川中黑山羊大小卵泡差异基因共4 451个，雷州山羊2 355个，二者共有差异基因1 771个。分析筛选出两个品种共有（INHBA、INHA、CYP19A1、KITLG、LHCGR和STAR）及各自特有（IGFBP6、BMP6和BMPR2）的已报道与卵泡发育相关的基因，此外还筛选出可能与这两种山羊繁殖性能相关的基因（GADD45B、TC2N和MSMO1）。GO功能分析显示，两个品种共有差异基因主要与各种物质的跨膜转运活性有关；KEGG通路分析显示，类固醇生成、细胞因子受体相互作用和cAMP信号通路在两个品种中均存在显著变化。类固醇生成过程中细胞色素P450代谢差异可能是川中黑山羊高产仔数的一个潜在因素。本研究结果为进一步探究山羊卵泡发育的基因功能及不同山羊品种繁殖性能差异提供参考。

关键词: 山羊; 卵泡; 转录组; 基因

Abstract: This study was aimed to excavate the genes affecting follicular development with the mRNA transcriptome difference between large and small follicles in two different goats,in order to provide a reference for further elucidating the mechanism of goat follicular development.Synchronization of estrus was carried out on the Chuanzhong Black and Leizhou goats with cloprostenol sodium.The single follicle (large follicle>6 mm,small follicle<3 mm) was isolated from ovaries which were collected from estrus goats mentioned above with the stereomicroscope.The total RNA was extracted from follicle tissues for RNA-seq,the profiles of mRNA expression were calculated with bioinformatics and differentially expressed genes (DEGs) were performed in GO function and KEGG pathway enrichment.The results showed that 4 451 DEmRNAs were obtained from different follicles in Chuanzhong Black goats,and 2 335 DEmRNAs were obtained from Leizhou goats,two goat breeds 1 771 genes.The common (INHBA,INHA,CYP19A1,KITLG,LHCGR and STAR) and unique (IGFBP6,BMP6 and BMPR2) genes related to follicular development were screened out.In addition,the new genes (GADD45B,TC2N and MSMO1) potentially affecting the reproductive performance in two goat breeds were also identified.GO function analysis showed that the DEGs in two goat breeds were mainly related to the transmembrane transport activity.KEGG pathway analysis revealed significant changes in steroidogenesis,cytokine-cytokine receptor interaction and cAMP signaling in both goat breeds.It was also found that the difference of cytochrome P450 metabolism in steroidogenesis when follicle developed could be a potential factor in the high litter size of Chuanzhong Black goats.The results provided a reference for further exploring the gene functions of follicle development and the research in reproductive performance differences of different goat breeds.

Key words: goats; follicle; transcriptome; gene

中图分类号:

S814.8

赵智锋, 邹娴, 柳广斌, 杨新月, 练志全, 郭勇庆, 刘德武. 川中黑山羊和雷州山羊大小卵泡转录组学分析[J]. 中国畜牧兽医, 2020, 47(5): 1459-1471.

ZHAO Zhifeng, ZOU Xian, LIU Guangbin, YANG Xinyue, LIAN Zhiquan, GUO Yongqing, LIU Dewu. Comparative Transcriptome Analysis Between Large and Small Follicles in Chuanzhong Black and Leizhou Goats[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(5): 1459-1471.

参考文献

[1] 曹俊国,陈敏,李文,等.哺乳动物卵泡发育调控分子机制研究进展[J].特产研究,2018,40(4):114-118. CAO J G,CHEN M,LI W,et al.Research progresses on the molecular mechanisms underlying mammalian follicle development[J].Special Wild Economic Animal and Plant Research,2018,40(4):114-118.(in Chinese)
[2] 孙理兰,谷朝勇,潘庆杰,等.原始卵泡形成与早期卵泡发育的遗传调控[J].生物技术通讯,2008,1:120-124. SUN L L,GU C Y,PAN Q J,et al.Genetic regulation of the primordial follicle formation and earliest follicular development[J].Letters in Biotechnology,2008,1:120-124.(in Chinese)
[3] KNIGHT P G,SATCHELL L,GLISTER C.Intra-ovarian roles of activins and inhibins[J].Molecular and Cellular Endocrinology,2012,359(1-2):53-65.
[4] DRUMMOND A E.TGFbeta signalling in the development of ovarian function[J].Cell and Tissue Research,2005,322(1):107-115.
[5] ZI X D,LU J Y,ZHOU H,et al.Comparative analysis of ovarian transcriptomes between prolific and non-prolific goat breeds via high-throughput sequencing[J].Reproduction in Domestic Animals,2018,53(2):344-351.
[6] LI J,LI Z,LIU S,et al.Transcriptome studies of granulosa cells at different stages of ovarian follicular development in buffalo[J].Animal Reproduction Science,2017,187:181-192.
[7] TERENINA E,FABRE S,BONNET A,et al.Differentially expressed genes and gene networks involved in pig ovarian follicular atresia[J].Physiological Genomics,2017,49(2):67-80.
[8] 李鹏飞,孟金柱,景炅婕,等.转录组测序筛选牛卵泡发育相关基因及其表达差异分析[J].中国农业科学,2018,51(15):187-195. LI P F,MENG J Z,JING J J,et al.Follicular development related genes screening and differential expressed analysis by transcriptome sequencing in bovine ovary[J].Scientia Agricultura Sinica,2018,51(15):187-195.(in Chinese)
[9] HATZIRODOS N,IRVING-RODGERS H F,HUMMITZSCH K,et al.Transcriptome profiling of granulosa cells of bovine ovarian follicles during growth from small to large antral sizes[J].BMC Genomics,2014,15:24.
[10] 吴阳升,林嘉鹏,汪立芹,等.绵羊小卵泡与中卵泡转录组差异特征分析[J].江苏农业学报,2016,32(4):832-842. WU Y S,LIN J P,WANG L Q,et al.Transcriptome profiling of ovine follicles during growth from small to middle antralsizes[J].Jiangsu Journal of Agricultural Sciences,2016,32(4):832-842.(in Chinese)
[11] WANG L J,SUN X W,GUO F Y,et al.Transcriptome analysis of the uniparous and multiparous goats ovaries[J].Reproduction in Domestic Animals,2016,51(6):877-885.
[12] HATZIRODOS N,HUMMITZSCH K,IRVING-RODGERS H F,et al.Transcriptome profiling of granulosa cells from bovine ovarian follicles during atresia[J].BMC Genomics,2014,15:40.
[13] 杨孟伯.大足黑山羊卵泡发育规律及发情周期中FSH、INH水平的变化[D].重庆:西南大学,2009. YANG M B.Study on the follicular wave of Dazu Black goat and concentrations changes of FSH and INH during the follicular development[D].Chongqing:Southwest University,2009.(in Chinese)
[14] 施阳阳.四川省山羊遗传资源发掘初报[D].成都:四川农业大学,2013. SHI Y Y.Preliminary study on genetic resources excavation of goats in Sichuan[D].Chengdu:Sichuan Agricultural University,2013.(in Chinese)
[15] 沙文锋,陈启康,朱娟,等.我国山羊品种资源的研究与利用进展[J].长江大学学报(自科版),2006,3(2):144-147. SHA W F,CHEN Q K,ZHU J,et al.Advances in the research and utilization of goat species resources in China[J].Journal of Yangtze University (Natural Science Edition),2006,3(2):144-147.(in Chinese)
[16] 中国畜禽遗传资源委员会.中国畜禽遗传资源志:羊志[M].北京:中国农业出版社,2011. CHINA NATIONAL COMMISSION OF ANIMAL GENETIC RESOURCES.Animal Genetic Resources in China:Sheep and Goats[M].Beijing:China Agricultrue Press,2011.(in Chinese)
[17] ALAVEZ R A,ARROYO L J,MONTES P R,et al.Short communication:Estrus synchronization using progestogens or cloprostenol in tropical hair sheep[J].Tropical Animal Health and Production,2014,46(8):1515-1518.
[18] 拓守珍,王忠明,竺秀芳,等.氯前列醇钠诱导母羊同期发情人工授精配种试验报告[J].中国畜牧杂志,2005,4:35-36. TUO S Z,WANG Z M,ZHU X F,et al.Experimental report on simultaneous estrus and artificial insemination of ewe induced by cloprostenol sodium[J].Chinese Journal of Animal Science,2005,4:35-36.(in Chinese)
[19] 李翠玲,李楠,黄章虎,等.水牛小腔卵泡分离方法的初步研究[J].中国畜牧兽医,2011,38(11):38-41. LI C L,LI N,HUANG Z H,et al.Studies on isolation of buffalo small antral follicles[J].China Animal Husbandry & Veterinary Medicine,2011,38(11):38-41.(in Chinese)
[20] ROSALES-TORRES A M,AVALOS-RODRIGUEZ A,VERGARA-ONOFRE M,et al.Multiparametric study of atresia in ewe antral follicles:Histology,flow cytometry,internucleosomal DNA fragmentation,and lysosomal enzyme activities in granulosa cells and follicular fluid[J].Molecular Reproduction and Development,2000,55(3):270-281.
[21] YANG M,HALL J,FAN Z,et al.Oocytes from small and large follicles exhibit similar development competence following goat cloning despite their differences in meiotic and cytoplasmic maturation[J].Theriogenology,2016,86(9):2302-2311.
[22] GALLEGO R I,PAI A A,TUNG J,et al.RNA-seq:Impact of RNA degradation on transcript quantification[J].BMC Biology,2014,12:42.
[23] VANDESOMPELE J,DE PRETER K,PATTYN F,et al.Accurate normalization of Real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[J].Genome Biology,2002,3(7):H34.
[24] 涂飞.排卵相关基因表达水平与湖羊繁殖性能的关系研究[D].南京:南京农业大学,2010. TU F.Study on the relationship between the reproductive ability of Hu sheep and the expression level of genes correlated with ovulation[D].Nanjing:Nanjing Agricultural University,2010.(in Chinese)
[25] PANWAR D,RAWAL L,SEHGAL N,et al.Cross talk between KGF and KITLG proteins implicated with ovarian folliculogenesisin Buffalo bubalusbubalis[J].PLoS One,2015,10(6):e127993.
[26] LEI M,CAI L,LI H,et al.Transcriptome sequencing analysis of porcine granulosa cells treated with an anti-inhibin antibody[J].Reproductive Biology,2017,17(1):79-88.
[27] LUO W,GUMEN A,HAUGHIAN J M,et al.The role of luteinizing hormone in regulating gene expression during selection of a dominant follicle in cattle[J].Biology of Reproduction,2011,84(2):369-378.
[28] WELT C K.Regulation and function of inhibins in the normal menstrual cycle[J].Seminars in Reproductive Medicine,2004,22(3):187-193.
[29] KEMPISTY B,JACKOWSKA M,WOZNA M,et al.Expression and cellular distribution of INHA and INHB before and after in vitro cultivation of porcine oocytes isolated from follicles of different size[J].Journal of Biomedicine and Biotechnology,2012,2012:742829.
[30] MENON K M,MENON B.Regulation of luteinizing hormone receptor expression by an RNA binding protein:Role of ERK signaling[J].The Indian Journal of Medical Research,2014,140(Suppl):S112-S119.
[31] 苗艳平,刘若男,魏彦辉,等.绵羊卵巢oar-miR-150靶向调节类固醇激素合成急性调节蛋白基因的表达[J].农业生物技术学报,2018,26(2):234-245. MIAO Y P,LIU R N,WEI Y H,et al.Target regulation of the expression of steroidogenic acute regulatory protein gene in sheep ovaries by oar-miR-150[J].Journal of Agricultural Biotechnology,2018,26(2):234-245.(in Chinese)
[32] ZHANG J,LIU Y,YAO W,et al.Initiation of follicular atresia:Gene networks during early atresia in pig ovaries[J].Reproduction,2018,156(1):23-33.
[33] NEUNZIG J,MILHIM M,SCHIFFER L,et al.The steroid metabolite 16(beta)-OH-androstenedione generated by CYP21A2 serves as a substrate for CYP19A1[J].The Journal of Steroid Biochemistry and Molecular Biology,2017,167:182-191.
[34] HUSSEIN M R.Apoptosis in the ovary:Molecular mechanisms[J].Human Reproduction Update,2005,11(2):162-177.
[35] DRIANCOURT M A,FAIR T,REYNAUD K.Oocyte apoptosis:When,how,why?[J].Contraception Fertilité Sexualité,1998,26(7-8):522-527.
[36] DRIANCOURT M A,REYNAUD K,CORTVRINDT R,et al.Roles of KIT and KIT LIGAND in ovarian function[J].Reviews of Reproduction,2000,5(3):143-152.
[37] KAMANGAR B B,GABILLARD J C,BOBE J.Insulin-like growth factor-binding protein (IGFBP)-1,-2,-3,-4,-5,and -6 and IGFBP-related protein 1 during rainbow trout post vitellogenesis and oocyte maturation:Molecular characterization,expression profiles,and hormonal regulation[J].Endocrinology,2006,147(5):2399-2410.
[38] TAMURA K,MATSUSHITA M,ENDO A,et al.Effect of insulin-like growth factor-binding protein 7 on steroidogenesis in granulosa cells derived from equine chorionic gonadotropin-primed immature rat ovaries[J].Biology of Reproduction,2007,77(3):485-491.
[39] MAZERBOURG S,MONGET P.Insulin-like growth factor binding proteins and IGFBP proteases:A dynamic system regulating the ovarian folliculogenesis[J].Frontiers in Endocrinology,2018,9:134.
[40] DIAZ P U,HEIN G J,BELOTTI E M,et al.BMP2,4 and 6 and BMPR1B are altered from early stages of bovine cystic ovarian disease development[J].Reproduction,2016,152(4):333-350.
[41] SHI J,YOSHINO O,OSUGA Y,et al.Growth differentiation factor 3 is induced by bone morphogenetic protein 6(BMP-6) and BMP-7 and increases luteinizing hormone receptor messenger RNA expression in human granulosa cells[J].Fertility and Sterility,2012,97(4):979-983.
[42] EDWARDS S J,READER K L,LUN S,et al.The cooperative effect of growth and differentiation factor-9 and bone morphogenetic protein (BMP)-15 on granulosa cell function is modulated primarily through BMP receptor Ⅱ[J].Endocrinology,2008,149(3):1026-1030.
[43] DE RESENDE L O,VIREQUE A A,SANTANA L F,et al.Single-cell expression analysis of BMP15 and GDF9 in mature oocytes and BMPR2 in cumulus cells of women with polycystic ovary syndrome undergoing controlled ovarian hyperstimulation[J].Journal of Assisted Reproduction and Genetics,2012,29(10):1057-1065.
[44] MAGIMAIDAS A,MADIREDDI P,MAIFREDE S,et al.GADD45B deficiency promotes premature senescence and skin aging[J].Oncotarget,2016,7(19):26935-26948.
[45] HAO X L,HAN F,ZHANG N,et al.TC2N,a novel oncogene,accelerates tumor progression by suppressing p53 signaling pathway in lung cancer[J].Cell Death and Differentiation,2018,26(7):1235-1250.
[46] BAI W,ZHANG C,CHEN H.Transcriptomic analysis of momordicacharantia polysaccharide on streptozotocin-induced diabetic rats[J].Gene,2018,675:208-216.
[47] SHISHIDO Y,BABA T,SATO T,et al.Differential lactate and cholesterol synthetic activities in XY and XX sertoli cells[J].Scientific Reports,2017,7:41912.
[48] 于敏,张双庆,闻镍,等.细胞色素P450酶系体外药物代谢研究方法进展[J].中国药事,2013,27(1):81-87. YU M,ZHANG S Q,WEN N,et al.In vitro research progress on cytochrome P450 for drug metabolism[J].Chinese Pharmaceutical Affairs,2013,27(1):81-87.(in Chinese)
[49] 宋复兴,王蓉,原永芳.药物代谢细胞色素P450酶学与研究方法应用进展[J].医学综述,2017,23(4):665-669. SONG F X,WANG R,YUAN Y F.Application progress in cytochrome P450 enzymes and research methods of drug metabolism[J].Medical Recapitulate,2017,23(4):665-669.(in Chinese)
[50] 徐彬,杨忠英,樊洪忠,等.细胞色素P450与药物代谢性相互作用[J].山西医药杂志,2015,44(12):1376-1380. XU B,YANG Z Y,FAN H Z,et al.Cytochrome P450 interacts with drug metabolism[J].Shanxi Medical Journal,2015,44(12):1376-1380.(in Chinese)