基于Label-free技术分析牛卵泡蛋白质组分及关键调控蛋白
|
摘要
旨在研究牛卵泡发育过程中蛋白质组表达变化并筛选卵泡发育关键调控蛋白,利用非标记(label-free)定量蛋白质组学技术对牛卵泡颗粒细胞(granulesa cells, GCs)蛋白质组分进行比较分析。采集牛发情周期优势卵泡(dominant follicles, DF)和从属卵泡(subordinate follicles, SF),分别分离GCs,并提取总蛋白,胰蛋白酶酶解,液相色谱-串联质谱(LC-MS/MS)进行蛋白质组分分析,数据库检索分析DF和SF中蛋白质表达情况,并应用生物信息学方法筛选牛卵泡发育关键调控蛋白。结果表明:本试验从30 321个肽段中共成功鉴定出3 409种蛋白质(FDR≤0.01),其中,DF中表达2 895种蛋白质,SF中表达3 102种蛋白质,获得差异表达蛋白质(差异倍数>2,P<0.05) 259种,17种差异表达蛋白质可能与牛卵泡优势化过程相关,SERPINB2可能调控牛卵泡闭锁。该研究筛选获得的牛卵泡差异表达蛋白质和特异表达蛋白质,丰富了牛卵泡发育调控理论,为进一步研究卵泡闭锁及优势化奠定基础。
The purpose of this study was to investigate changes in proteome expression and screen key regulatory proteins during bovine follicular development, and protein components of GCs(granulesa cells)were analyzed by label-free quantitative proteomics technology.DF(dominant follicles)and SF(subordinate follicles)during the bovine estrous cycle were collected, GCs were separated, total proteins were extracted, proteins were digested with trypsin and analyzed by LC-MS/MS, the expression of proteins in DF and SF were analyzed after database retrieval, and key regulatory proteins were screened by bioinformatics method. In this study, 3 409 proteins were successfully identified from 30 321 peptide fragments(FDR≤0.01), of which 2 895 were DF proteins and 3 102 were SF proteins, 259 differentially expressed proteins were identified(Fold change>2,P<0.05), 17 differential expressed proteins might be related to the process of bovine follicular dominance, and SERPINB2 might regulate bovine follicular atresia.The results enriched the studies on DF and SF proteome and laid a foundation for further studies on follicular atresia and dominance theory by identify differential expressed proteins and specific expression proteins.
The purpose of this study was to investigate changes in proteome expression and screen key regulatory proteins during bovine follicular development, and protein components of GCs(granulesa cells)were analyzed by label-free quantitative proteomics technology.DF(dominant follicles)and SF(subordinate follicles)during the bovine estrous cycle were collected, GCs were separated, total proteins were extracted, proteins were digested with trypsin and analyzed by LC-MS/MS, the expression of proteins in DF and SF were analyzed after database retrieval, and key regulatory proteins were screened by bioinformatics method. In this study, 3 409 proteins were successfully identified from 30 321 peptide fragments(FDR≤0.01), of which 2 895 were DF proteins and 3 102 were SF proteins, 259 differentially expressed proteins were identified(Fold change>2,P<0.05), 17 differential expressed proteins might be related to the process of bovine follicular dominance, and SERPINB2 might regulate bovine follicular atresia.The results enriched the studies on DF and SF proteome and laid a foundation for further studies on follicular atresia and dominance theory by identify differential expressed proteins and specific expression proteins.
引文
[1] FORTUNE J E,RIVERA G M,EVANS A C O,et al.Differentiation of dominant versus subordinate follicles in cattle[J].Biol Reprod,2001,65(3):648-654.
[2] ADAMS G P,MATTERI R L,KASTELIC J P,et al.Association between surges of follicle-stimulating hormone and the emergence of follicular waves in heifers[J].J Reprod Fertil,1992,94(1):177-188.
[3] FIELD S L,DASGUPTA T,CUMMINGS M,et al.Cytokines in ovarian folliculogenesis,oocyte maturation and luteinisation[J].Mol Reprod Dev,2014,81(4):284-314.
[4] SUNDERLAND S J,CROWE M A,BOLAND M P,et al.Selection,dominance and atresia of follicles during the oestrous cycle of heifers[J].J Reprod Fertil,1994,101(3):547-555.
[5] JOHNSON A L.Intracellular mechanisms regulating cell survival in ovarian follicles[J].Anim Reprod Sci,2003,78(3-4):185-201.
[6] KAFI M,MESBAH S F,DAVOODIAN N,et al.Fine structures of the oocyte in relation to serum,follicular fluid steroid hormones and IGF-I in the ovulatory-sized follicles in one-humped camel (Camelus dromedarius)[J].Avicenna J Med Biotechnol,2014,6(1):57-61.
[7] BABITHA V,PANDA R P,YADAV V P,et al.Amount of mRNA and localization of vascular endothelial growth factor and its receptors in the ovarian follicle during estrous cycle of water buffalo (Bubalus bubalis)[J].Anim Reprod Sci,2013,137(3-4):163-176.
[8] PEDERSEN H G,WATSON E D,TELFER E E.Analysis of atresia in equine follicles using histology,fresh granulosa cell morphology and detection of DNA fragmentation[J].Reproduction,2003,125(3):417-423.
[9] WONGSRIKEAO P,KANESHIGE Y,OOKI R,et al.Effect of the removal of cumulus cells on the nuclear maturation,fertilization and development of porcine oocytes[J].Reprod Domest Anim,2005,40(2):166-170.
[10] MARX H,MINOGUE C E,JAYARAMAN D,et al.A proteomic atlas of the legume Medicago truncatula and its nitrogen-fixing endosymbiont Sinorhizobium meliloti[J].Nat Biotechnol,2016,34(11):1198-1205.
[11] WEBB-ROBERTSON B J,WIBERG H K,MATZKE M M,et al.Review,evaluation,and discussion of the challenges of missing value imputation for mass spectrometry-based label-free global proteomics[J].J Proteome Res,2015,14(5):1993-2001.
[12] 李鹏飞,孟金柱,郝庆玲,等.PDF2和ODF1转录组测序筛选牛卵泡发育相关基因[J].畜牧兽医学报,2018,49(2):300-309.LI P F,MENG J Z,HAO Q L,et al.Screening and analysing of genes associated with follicular development in bovine ODF1 and PDF2 transcriptome[J].Acta Veterinaria et Zootechnica Sinica,2018,49(2):300-309.(in Chinese)
[13] SHAMANNA R A,HOQUE M,LEWIS-ANTES A,et al.The NF90/NF45 complex participates in DNA break repair via nonhomologous end joining[J].Mol Cell Biol,2011,31(23):4832-4843.
[14] CHENG S B,JIANG X,DING C F,et al.Expression and critical role of interleukin enhancer binding factor 2 in hepatocellular carcinoma[J].Int J Mol Sci,2016,17(8):1373.
[15] YAN C,THEODORESCU D,YE R D,et al.RAL GTPases:biology and potential as therapeutic targets in cancer[J].Pharmacol Rev,2018,70(1):1-11.
[16] GUO S K,SHEN M F,YAO H W,et al.Enhanced expression of TGFBI promotes the proliferation and migration of glioma cells[J].Cell Physiol Biochem,2018,49(3):1138-1150.
[17] SIGURDSON A J,BRENNER A V,ROACH J A,et al.Selected single-nucleotide polymorphisms in FOXE1,SERPINA5,FTO,EVPL,TICAM1 and SCARB1 are associated with papillary and follicular thyroid cancer risk:replication study in a German population[J].Carcinogenesis,2016,37(7):677-684.
[18] WANG Q C,LIN L C,REINKING B E,et al.Essential roles of an intercalated disc protein,mXinβ,in postnatal heart growth and survival[J].Circ Res,2010,106(9):1468-1478.
[19] COLLINS R J,JIANG W G,HARGEST R,et al.EPLIN:a fundamental actin regulator in cancer metastasis?[J].Cancer Metastasis Rev,2015,34(4):753-764.
[20] NEIDERT N,VON EHR A,Z?LLER T,et al.Microglia-specific expression of Olfml3 is directly regulated by transforming growth factor β1-induced Smad2 signaling[J].Front Immunol,2018,9:1728.
[21] DOOKERAN K A,ZHANG W,STAYNER L,et al.Associations of two-pore domain potassium channels and triple negative breast cancer subtype in The Cancer Genome Atlas:systematic evaluation of gene expression and methylation[J].BMC Res Notes,2017,10(1):475.
[22] LEE N H,CHO A,PARK S R,et al.SERPINB2 is a novel indicator of stem cell toxicity[J].Cell Death Dis,2018,9(7):724.
[23] BABU P S,KRISHNAMURTHY H,CHEDRESE P J,et al.Activation of extracellular-regulated kinase pathways in ovarian granulosa cells by the novel growth factor type 1 follicle-stimulating hormone receptor.Role in hormone signaling and cell proliferation[J].J Biol Chem,2000,275(36):27615-27626.
[24] PALMERINI M G,NOTTOLA S A,TUNJUNG W A S,et al.EGF-FSH supplementation reduces apoptosis of pig granulosa cells in co-culture with cumulus-oocyte complexes[J].Biochem Biophys Res Commun,2016,481(1-2):159-164.
[25] SUN Y,LIU W Z,LIU T,et al.Signaling pathway of MAPK/ERK in cell proliferation,differentiation,migration,senescence and apoptosis[J].J Recept Signal Transduct Res,2015,35(6):600-604.
[26] ZHANG L,YUE T,JIANG J.Hippo signaling pathway and organ size control[J].Fly(Austin),2009,3(1):68-73.
[27] HALDER G,JOHNSON R L.Hippo signaling:growth control and beyond[J].Development,2011,138(1):9-22.
[28] HERGOVICH A.Mammalian hippo signalling:a kinase network regulated by protein-protein interactions[J].Biochem Soc Trans,2012,40(1):124-128.
[29] SHIMADA T,FOURNIER A E,YAMAGATA K.Neuroprotective function of 14-3-3 proteins in neurodegeneration[J].BioMed Res Int,2013,2013:564534.
[30] RéGER B,PéTERFALVI á,LITTER I,et al.Challenges in the evaluation of D-dimer and fibrinogen levels in pregnant women[J].Thromb Res,2013,131(4):183-187.
[31] TASHEVA E S,MAKI C G,CONRAD A H,et al.Transcriptional activation of bovine mimecan by p53 through an intronic DNA-binding site[J].Biochim Biophys Acta,2001,1517(3):333-338.
[32] LEE H W,PARK Y M,LEE S J,et al.Alpha-smooth muscle actin (ACTA2) is required for metastatic potential of human lung adenocarcinoma[J].Clin Cancer Res,2013,19(21):5879-5889.
[33] HRISTOVA I.Role of heat shock proteins (Hsp) in human and mammalian fertilization and pregnancy.Part I[J].Akush Ginekol (Sofiia),2012,51(5):45-49.
[34] HRISTOVA I.Role of heat shock proteins (Hsp) in human and mammalian fertilization and pregnancy.Part II[J].Akush Ginekol (Sofiia),2012,51(6):37-40.
[2] ADAMS G P,MATTERI R L,KASTELIC J P,et al.Association between surges of follicle-stimulating hormone and the emergence of follicular waves in heifers[J].J Reprod Fertil,1992,94(1):177-188.
[3] FIELD S L,DASGUPTA T,CUMMINGS M,et al.Cytokines in ovarian folliculogenesis,oocyte maturation and luteinisation[J].Mol Reprod Dev,2014,81(4):284-314.
[4] SUNDERLAND S J,CROWE M A,BOLAND M P,et al.Selection,dominance and atresia of follicles during the oestrous cycle of heifers[J].J Reprod Fertil,1994,101(3):547-555.
[5] JOHNSON A L.Intracellular mechanisms regulating cell survival in ovarian follicles[J].Anim Reprod Sci,2003,78(3-4):185-201.
[6] KAFI M,MESBAH S F,DAVOODIAN N,et al.Fine structures of the oocyte in relation to serum,follicular fluid steroid hormones and IGF-I in the ovulatory-sized follicles in one-humped camel (Camelus dromedarius)[J].Avicenna J Med Biotechnol,2014,6(1):57-61.
[7] BABITHA V,PANDA R P,YADAV V P,et al.Amount of mRNA and localization of vascular endothelial growth factor and its receptors in the ovarian follicle during estrous cycle of water buffalo (Bubalus bubalis)[J].Anim Reprod Sci,2013,137(3-4):163-176.
[8] PEDERSEN H G,WATSON E D,TELFER E E.Analysis of atresia in equine follicles using histology,fresh granulosa cell morphology and detection of DNA fragmentation[J].Reproduction,2003,125(3):417-423.
[9] WONGSRIKEAO P,KANESHIGE Y,OOKI R,et al.Effect of the removal of cumulus cells on the nuclear maturation,fertilization and development of porcine oocytes[J].Reprod Domest Anim,2005,40(2):166-170.
[10] MARX H,MINOGUE C E,JAYARAMAN D,et al.A proteomic atlas of the legume Medicago truncatula and its nitrogen-fixing endosymbiont Sinorhizobium meliloti[J].Nat Biotechnol,2016,34(11):1198-1205.
[11] WEBB-ROBERTSON B J,WIBERG H K,MATZKE M M,et al.Review,evaluation,and discussion of the challenges of missing value imputation for mass spectrometry-based label-free global proteomics[J].J Proteome Res,2015,14(5):1993-2001.
[12] 李鹏飞,孟金柱,郝庆玲,等.PDF2和ODF1转录组测序筛选牛卵泡发育相关基因[J].畜牧兽医学报,2018,49(2):300-309.LI P F,MENG J Z,HAO Q L,et al.Screening and analysing of genes associated with follicular development in bovine ODF1 and PDF2 transcriptome[J].Acta Veterinaria et Zootechnica Sinica,2018,49(2):300-309.(in Chinese)
[13] SHAMANNA R A,HOQUE M,LEWIS-ANTES A,et al.The NF90/NF45 complex participates in DNA break repair via nonhomologous end joining[J].Mol Cell Biol,2011,31(23):4832-4843.
[14] CHENG S B,JIANG X,DING C F,et al.Expression and critical role of interleukin enhancer binding factor 2 in hepatocellular carcinoma[J].Int J Mol Sci,2016,17(8):1373.
[15] YAN C,THEODORESCU D,YE R D,et al.RAL GTPases:biology and potential as therapeutic targets in cancer[J].Pharmacol Rev,2018,70(1):1-11.
[16] GUO S K,SHEN M F,YAO H W,et al.Enhanced expression of TGFBI promotes the proliferation and migration of glioma cells[J].Cell Physiol Biochem,2018,49(3):1138-1150.
[17] SIGURDSON A J,BRENNER A V,ROACH J A,et al.Selected single-nucleotide polymorphisms in FOXE1,SERPINA5,FTO,EVPL,TICAM1 and SCARB1 are associated with papillary and follicular thyroid cancer risk:replication study in a German population[J].Carcinogenesis,2016,37(7):677-684.
[18] WANG Q C,LIN L C,REINKING B E,et al.Essential roles of an intercalated disc protein,mXinβ,in postnatal heart growth and survival[J].Circ Res,2010,106(9):1468-1478.
[19] COLLINS R J,JIANG W G,HARGEST R,et al.EPLIN:a fundamental actin regulator in cancer metastasis?[J].Cancer Metastasis Rev,2015,34(4):753-764.
[20] NEIDERT N,VON EHR A,Z?LLER T,et al.Microglia-specific expression of Olfml3 is directly regulated by transforming growth factor β1-induced Smad2 signaling[J].Front Immunol,2018,9:1728.
[21] DOOKERAN K A,ZHANG W,STAYNER L,et al.Associations of two-pore domain potassium channels and triple negative breast cancer subtype in The Cancer Genome Atlas:systematic evaluation of gene expression and methylation[J].BMC Res Notes,2017,10(1):475.
[22] LEE N H,CHO A,PARK S R,et al.SERPINB2 is a novel indicator of stem cell toxicity[J].Cell Death Dis,2018,9(7):724.
[23] BABU P S,KRISHNAMURTHY H,CHEDRESE P J,et al.Activation of extracellular-regulated kinase pathways in ovarian granulosa cells by the novel growth factor type 1 follicle-stimulating hormone receptor.Role in hormone signaling and cell proliferation[J].J Biol Chem,2000,275(36):27615-27626.
[24] PALMERINI M G,NOTTOLA S A,TUNJUNG W A S,et al.EGF-FSH supplementation reduces apoptosis of pig granulosa cells in co-culture with cumulus-oocyte complexes[J].Biochem Biophys Res Commun,2016,481(1-2):159-164.
[25] SUN Y,LIU W Z,LIU T,et al.Signaling pathway of MAPK/ERK in cell proliferation,differentiation,migration,senescence and apoptosis[J].J Recept Signal Transduct Res,2015,35(6):600-604.
[26] ZHANG L,YUE T,JIANG J.Hippo signaling pathway and organ size control[J].Fly(Austin),2009,3(1):68-73.
[27] HALDER G,JOHNSON R L.Hippo signaling:growth control and beyond[J].Development,2011,138(1):9-22.
[28] HERGOVICH A.Mammalian hippo signalling:a kinase network regulated by protein-protein interactions[J].Biochem Soc Trans,2012,40(1):124-128.
[29] SHIMADA T,FOURNIER A E,YAMAGATA K.Neuroprotective function of 14-3-3 proteins in neurodegeneration[J].BioMed Res Int,2013,2013:564534.
[30] RéGER B,PéTERFALVI á,LITTER I,et al.Challenges in the evaluation of D-dimer and fibrinogen levels in pregnant women[J].Thromb Res,2013,131(4):183-187.
[31] TASHEVA E S,MAKI C G,CONRAD A H,et al.Transcriptional activation of bovine mimecan by p53 through an intronic DNA-binding site[J].Biochim Biophys Acta,2001,1517(3):333-338.
[32] LEE H W,PARK Y M,LEE S J,et al.Alpha-smooth muscle actin (ACTA2) is required for metastatic potential of human lung adenocarcinoma[J].Clin Cancer Res,2013,19(21):5879-5889.
[33] HRISTOVA I.Role of heat shock proteins (Hsp) in human and mammalian fertilization and pregnancy.Part I[J].Akush Ginekol (Sofiia),2012,51(5):45-49.
[34] HRISTOVA I.Role of heat shock proteins (Hsp) in human and mammalian fertilization and pregnancy.Part II[J].Akush Ginekol (Sofiia),2012,51(6):37-40.