TGF-β/Smad信号通路响应光周期变化参与调控斑马鱼卵巢发育
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摘要
为研究不同光周期条件下转化生长因子-β超家族/Samd (TGF-β/Smad)信号通路在斑马鱼(Danio rerio)卵巢中的作用,利用荧光定量PCR技术检测连续黑暗(0 L∶24 D, DD)、自然光照(14 L∶10 D, LD)和连续光照(24 L∶0 D, LL)条件下斑马鱼卵巢中TGF-β/Smad信号通路中相关基因的相对表达量,并利用免疫组化技术检测不同光周期条件下p-Smad2在斑马鱼卵巢中的定位情况。结果显示,在不同光周期条件下,TGF-β/Smad信号通路中信号分子在斑马鱼卵巢中的表达模式不尽相同,其中配体(tgfb3)、受体(tgfbr2a,tgfbr2b,tgfbr1b)和下游蛋白激酶(smad2和smad3a)的表达趋势一致,即在不同光周期处理3 d后,上述6个基因的相对表达量DD组最高,LL组最低,但处理7 d后则呈相反趋势。在斑马鱼卵黄发生前期至充分生长未成熟期的卵母细胞中均检测到p-Smad2蛋白信号,但光周期处理后未影响p-Samd2在斑马鱼卵巢中的定位。结果表明,光周期变化可能通过改变tgfb3/受体/蛋白激酶基因的表达模式影响斑马鱼的卵巢发育。
To study the role of TGF-β/Smad signaling pathway in zebrafish(Danio rerio) ovary under different photoperiod conditions, we detected the relative expression of relevant genes in TGF-β/Smad signaling pathway of zebrafish ovary under continuous dark(0 L∶24 D, DD), natural photoperiod(14 L∶10 D, LD) and continuous light(24 L∶0 D, LL) using quantitative real-time PCR. The location of p-Smad2 in zebrafish ovary was detected by immunohistochemical technology. The results show that the expression patterns of genes involved in TGF-β/Smad signaling pathway were different in zebrafish ovary under different photoperiod conditions, and the expression profiles of ligand(tgfb3), receptor(tgfbr2 a, tgfbr2 b and tgfbr1 b) and downstream protein kinase(smad2 and smad3 a) were consistent. The relative expression levels of the above six genes were the highest in DD group and the lowest in LL group after 3-day treatment of different photoperiods, and an opposite trend was observed after 7 d. The p-Smad2 protein signal was detected in the development of zebrafish oocytes from previtellogenic stage(PV) to full-grown immature stage(FG).Photoperiod treatment did not affect the localization of p-Samd2 in zebrafish ovary. The results suggest that change of photoperiod might affect the ovarian development of zebrafish by changing the expression pattern of genes of tgfb3/receptor/protein kinase.
To study the role of TGF-β/Smad signaling pathway in zebrafish(Danio rerio) ovary under different photoperiod conditions, we detected the relative expression of relevant genes in TGF-β/Smad signaling pathway of zebrafish ovary under continuous dark(0 L∶24 D, DD), natural photoperiod(14 L∶10 D, LD) and continuous light(24 L∶0 D, LL) using quantitative real-time PCR. The location of p-Smad2 in zebrafish ovary was detected by immunohistochemical technology. The results show that the expression patterns of genes involved in TGF-β/Smad signaling pathway were different in zebrafish ovary under different photoperiod conditions, and the expression profiles of ligand(tgfb3), receptor(tgfbr2 a, tgfbr2 b and tgfbr1 b) and downstream protein kinase(smad2 and smad3 a) were consistent. The relative expression levels of the above six genes were the highest in DD group and the lowest in LL group after 3-day treatment of different photoperiods, and an opposite trend was observed after 7 d. The p-Smad2 protein signal was detected in the development of zebrafish oocytes from previtellogenic stage(PV) to full-grown immature stage(FG).Photoperiod treatment did not affect the localization of p-Samd2 in zebrafish ovary. The results suggest that change of photoperiod might affect the ovarian development of zebrafish by changing the expression pattern of genes of tgfb3/receptor/protein kinase.
引文
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[29]ITOH S,TEN DIJKE P.Negative regulation of TGF-βreceptor/Smad signal transduction[J].Curr Opin Cell Biol,2007,19(2):176-184.
[30]MIYAZAWA K,MIYAZONO K.Regulation of TGF-βfamily signaling by inhibitory smads[J].Cold Spring Harbor Perspect Biol,2016,9(3):a022095.
[31]DONG C M,LI Z R,ALVAREZ R,et al.Microtubule binding to smads may regulate TGFβactivity[J].Mol Cell,2000(5):27-34.
[32]WANG Y,GE W.Spatial expression patterns of activin and its signaling system in the zebrafish ovarian follicle:evidence for paracrine action of activin on the oocytes[J].Biol Reprod,2003,69(6):1998-2006.
[2]SOKOLOWSKA E,KLESZCZYNSKA A,NIETRZEBA M,et al.Annual changes in brain concentration of arginine vasotocin and isotocin correspond with phases of reproductive cycle in round goby,Neogobius melanostomus[J].Chronobiol Int,2015,32(7):917-924.
[3]HANSEN T J,FJELLDAL P G,FOLKEDAL O,et al.Effects of light source and intensity on sexual maturation,growth and swimming behaviour of Atlantic salmon in sea cages[J].Aquacult Env Interac,2017,9:193-204.
[4]TARANGER G L,AARDAL L,HANSEN T,et al.Continuous light delays sexual maturation and increases growth of Atlantic cod(Gadus morhua L.)in sea cages[J].ICES J Mar Sci,2006,63(2):365-375.
[5]LEE C H,PARK Y J,LEE Y D.Effects of photoperiod manipulation on gonadal activity of the damselfish,Chromis notata[J].Dev Reprod,2017,21(2):223-228.
[6]TAKEUCHI Y,HADA N,IMAMURA S,et al.Existence of a photoinducible phase for ovarian development and photoperiodrelated alteration of clock gene expression in a damselfish[J].Comp Biochem Physiol A,2015,188:32-39.
[7]PARMEGGIANI A,GOVONI N,ZANNONI A,et al.Effect of photoperiod on endocrine profiles and vitellogenin expression in European eels Anguilla anguilla during artificially induced ovarian development[J].Theriogenology,2015,83(4):478-484.
[8]IMSLAND A K,JONASSEN T M,HANGSTAD T A,et al.The effect of continuous light and compressed photoperiods on growth and maturation in lumpfish Cyclopterus lumpus[J].Aquaculture,2018,485:166-172.
[9]LUO K X.Signaling cross talk between TGF-beta/Smad and other signaling pathways[J].Cold Spring Harb Perspect Biol,2017,9(1):a022137.
[10]詹绪良,吴金英,李文笙.TGF-β系统及其在硬骨鱼中的研究进展[J].南方水产科学,2013,9(3):90-96.
[11]ZHU B,PARDESHI L,CHEN Y Y,et al.Transcriptomic analysis for differentially expressed genes in ovarian follicle activation in the zebrafish[J].Front Endocrinol(Lausanne),2018,9:593.
[12]KOHLI G,HU S Q,CLELLAND E,et al.Cloning of transforming growth factor-β1(TGF-β1)and its type II receptor from zebrafish ovary and role of TGF-β1 in oocyte maturation[J].Endocrinology,2003,144(5):1931-1941.
[13]KOHLI G,CLELLAND E,PENG C.Potential targets of transforming growth factor-beta1 during inhibition of oocyte maturation in zebrafish[J].Reprod Biol Endocrinol,2005,3:53.
[14]SLOIN H E,RUGGIERO G,RUBINSTEIN A,et al.Interactions between the circadian clock and TGF-βsignaling pathway in zebrafish[J].PLoS One,2018,13(6):e0199777.
[15]GAST H,GORDIC S,PETRZILKA S,et al.Transforming growth factor-beta inhibits the expression of clock genes[J].Ann NY Acad Sci,2012,1261(1):79-87.
[16]LIU K C,LIN S W,GE W.Differential regulation of gonadotropin receptors(fshr and lhcgr)by estradiol in the zebrafish ovary involves nuclear estrogen receptors that are likely located on the plasma membrane[J].Endocrinology,2011,152(11):4418-4430.
[17]SELMAN K,WALLACE R A,SARKA A,et al.Stages of oocyte development in the zebrafish,Brachydanio rerio[J].J Morphol,1993,218(2):203-224.
[18]WANG Y,GE W.Developmental profiles of activin betaA,betaB,and follistatin expression in the zebrafish ovary:evidence for their differential roles during sexual maturation and ovulatory cycle[J].Biol Reprod,2004,71(6):2056-2064.
[19]CHU Y L,XU Y R,YANG W X,et al.The role of FSH and TGF-βsuperfamily in follicle atresia[J].Aging,2018,10(3):305-321.
[20]WANG D,WANG W,LIANG Q,et al.DHEA-induced ovarian hyperfibrosis is mediated by TGF-βsignaling pathway[J].J Ovarian Res,2018,11(1):6.
[21]BASU M,BHATTACHARYA R,RAY U,et al.Invasion of ovarian cancer cells is induced by PITX2-mediated activation of TGF-βand Activin-A[J].Mol Cancer,2015,14(1):162.
[22]HATZIRODOS N,BAYNE R A,IRVING-RODGERS H F,et al.Linkage of regulators of TGF-βactivity in the fetal ovary to polycystic ovary syndrome[J].FASEB J,2011,25(7):2256-2265.
[23]KNIGHT P G,GLISTER C.TGF-beta superfamily members and ovarian follicle development[J].Reproduction,2006,132(2):191-206.
[24]ROSAIRO D,KUYZNIEREWICZ I,FINDLAY J A.Transforming growth factor-beta:its role in ovarian follicle development[J].Reproduction,2008,136(6):799-809.
[25]LIU X,ANDOH K,ABE Y,et al.A comparative study on transforming growth factor-beta and activin A for preantral follicles from adult,immature,and diethylstilbestrol-primed immature mice[J].Endocrinology,1999,140(6):2480-2485.
[26]HELDIN C H,MOUSTAKAS A.Signaling receptors for TGF-βfamily members[J].CSH Perspect Biol,2016,8(8):a022053.
[27]TAN Q,BALOFSKY A,WEISZ K,et al.Role of activin,transforming growth factor-βand bone morphogenetic protein 15 in regulating zebrafish oocyte maturation[J].Comp Biochem Physiol A,2009,153(1):18-23.
[28]CHEN W T,LIU L,GE W.Expression analysis of growth differentiation factor 9(Gdf9/gdf9),anti-müllerian hormone(Amh/amh)and aromatase(Cyp19a1a/cyp19a1a)during gonadal differentiation of the zebrafish,Danio rerio[J].Biol Reprod,2017,96(2):401-413.
[29]ITOH S,TEN DIJKE P.Negative regulation of TGF-βreceptor/Smad signal transduction[J].Curr Opin Cell Biol,2007,19(2):176-184.
[30]MIYAZAWA K,MIYAZONO K.Regulation of TGF-βfamily signaling by inhibitory smads[J].Cold Spring Harbor Perspect Biol,2016,9(3):a022095.
[31]DONG C M,LI Z R,ALVAREZ R,et al.Microtubule binding to smads may regulate TGFβactivity[J].Mol Cell,2000(5):27-34.
[32]WANG Y,GE W.Spatial expression patterns of activin and its signaling system in the zebrafish ovarian follicle:evidence for paracrine action of activin on the oocytes[J].Biol Reprod,2003,69(6):1998-2006.