| Hedgehog(Hh)pathway regulates many developmental processes.The core components of the pathway include ligand Hh,receptors Patched(Ptch)and Smoothened(Smo),transcription factor Gliotactin1-3(Gli1-3).In vertebrates,Hh comprises Desert hedgehog(Dhh),Indian hedgehog(Ihh)and Sonic hedgehog(Shh);Ptch includes Pcth1 and Ptch2.In mammals,previous studies have shown that Hh pathway plays an important role in the differentiation and development of gonadal cells,such as germ cells(SC)and Leydig cells(LC).In fish,Hh signaling pathway has been mainly studied in embryonic development and other tissues of zebrafish(Danio rerio),while its function in gonadal cells is blank.On the other hand,the proliferation and differentiation of Leydig stem cell(SLC)directly affect the formation and maintenance of LC,which are the principal androgen-producing cells in male.However,to date,our knowledge about the mechanism of SLC proliferation and differentiation is limited in mammals and almost blank in fish.It has been confirmed that Hh receptor agonist plays an important role in the proliferation and differentiation of mammalian SLC,but its role in fish SLC remains obscure.The Nile tilapia(referred to as Nile tilapia(Oreochromis niloticus)unless specified in this study)is a worldwide farmed fish,which has the advantages of fast growth,strong disease resistance and adaptability,short maturation time and short spawning cycle,it is an excellent model for the study of fish reproductive physiology.Moreover,in our previous study,a stem Leydig cells line derived from a 3-month-old tilapia testicular culture has been successuflly developed,which was designated as TSL.It has retained stable proliferation with 90 passages without showing growth senescence.Importantly,upon induction in the differentiated medium containing adult tilapia testicular extract and human chorionic gonadotropin,they could undergo differentiation into functional LCs capable of 11-ketotestosterone(11-KT)production.Therefore,the TSL provides us an excellent model to explore the molecules and mechanisms underlying the proliferation and differentiation of SLC.So far,the expression patterns of Hh pathway components in tilapia and its role in different gonadal cells(especially SLC)have not been reported.In view of this,on the one hand,the c DNA sequences and expression patterns of Hh signaling pathway components from tilapia and their roles in the proliferation and survival of different gonadal cells were studied.On the other hand,TSL combining with an in vitro endogenous LC-depleted testicular organ culture system by ethane dimethanesolfonate(EDS)treatment were used to address the action of Smo signaling in SLC.The details are as follows:1 Identification of Hh signaling pathway components in tilapia and their roles in different gonadal cells(1)c DNA cloning and sequence analysisUsing RT-PCR,we successfully obtained the c DNA sequences of tilapia dhh,ptch1,ptch2 and smo completely open reading frames,encoding 458,1551,1491 and 825 amino acid residues,respectively.The results of protein structure,phylogenetic tree and gene syntenic analysis showed that tilapia Dhh,Ptch1,Ptch2 and Smo are homologous molecules of mammliam.(2)Expression patternsRT-PCR results showed that dhh,ptch1,ptch2 and smo were all expressed in gonad.The results of fluorescence in situ hybridization showed that dhh,ptch1,ptch2 and smo were expressed in all germ cells except sperm in testicular germ cells.In testicular somatic cells,dhh was mainly expressed in Sertoli cells(SC),ptch1,ptch2 and smo were expressed in LC and SC.In ovarian germ cells,dhh was mainly expressed in oogonia,ptch1,ptch2 and smo were expressed in all germ cells except IV oocytes.In ovarian somatic cells,dhh,ptch1 and ptch2 were expressed in all somatic cells,while smo was only expressed in granulosa cells.It is suggested that Hh pathway plays a role in the differentiation and development of different cells in tilapia testis and ovary.(3)The role in different gonadal cells.In the culture system of testis and ovary tissue in vitro,the proliferation activity and apoptosis of different gonadal cells were detected by Ed U incorporation and TUNEL methods after treatment with Smo agonist(SAG)or inhibitor(cyclopamine),respectively.The results showed that SAG significantly promoted the proliferation of testicular germ cells,cyclopamine significantly promoted the apoptosis of testicular LC and SC,while SAG and cyclopamine had no significant effect on the proliferation and apoptosis of ovarian germ cells and somatic cells.It is suggested that Hh pathway plays an important role in the proliferation of testicular germ cells and the survival of LC and SC.2.The role and mechanism of Smo in the proliferation and differentiation of tilapia SLC(1)The role of Smo in the proliferation and survival of SLCRT-PCR result showed that Smo was expressed in TSL.The effect of Smo on the proliferation and survival of SLC was detected by microscopic observation and cell counting.The results showed that SAG significantly promoted the proliferation of TSL compared with the control group(p<0.01).Treatment of TSL with cyclopamine inhibited proliferation and resulted in apoptosis.It is suggested that Smo plays an important role in the proliferation and survival of SLC.(2)The role of Smo in SLC differentiationThe role of SAG in the differentiation of TSL was studied by detecting the ability of SAG to induce TSL to differentiate into mature LC and the ability of SAG to promote the differentiation of SLC in testicular organ cultures.The results showed that after treating TSL with SAG-containing medium for 28 days,the cells expressed Star2 and Cyp11b2 and produced 11-KT,while the control cells did not detect the expression of Star2,Cyp11b2 and the production of 11-KT.EDS was used to eliminate the endogenous LC in testicular organ cultures,and then the testes tissue were cultured in medium containing SAG.The results show that on the 14 th day of culture,the level of11-KT reached 58 pg/m L in SAG-treated group,while the control group failed to detect the production of 11-KT.These data indicate that Smo can promote the differentiation of SLC.(3)The mechanism of Smo in the proliferation and differentiation of SLCThe Hh pathway general transcription factors Gli1 and Gli2 inhibitors were used to treat TSL and cultured testicular tissue to identify downstream signaling molecules and target genes that Smo-mediated SLC proliferation and differentiation.On the one hand,the Gli1 inhibitor(GANT58)or Gli1/2 inhibitor(GANT61)were added to the medium containing SAG to treat TSL.The results showed that GANT61 significantly promoted cell apoptosis(p<0.01),while GANT58 group was significantly different from the control group(p>0.05).It is suggested that Smo may mediate the proliferation and differentiation of SLC through Gli1/2.On the other hand,EDS was used to eliminate the endogenous LC in testicular organ cultures,and then the testes tissue were cultured in medium containing SAG and GANT61.The results showed that GANT61 inhibited the production of 11-KT.In addition,after SAG treated TSL for 48 h,Real-time PCR(q PCR)detected the expression of gli1 and gli2.The results showed that the expression of gli1 was significantly up-regulated(p<0.05),while the expression of gli2 did not change significantly compared with the control(p>0.05).These data indicate that Smo may mediate the proliferation and differentiation of SLC through Gli1/2,and gli1 is its downstream target gene.These data firstly suggest that the Hh pathway plays an important role in testicular germ cell proliferation and somatic cell survival in teleost fish.In addition,the role and mechanism of Smo in SLC were studied.This study not only enriches our understanding of the role of Hh signaling pathway in different gonad cells of teleost fish,but also promotes our understanding of the regulation mechanism of SLC proliferation and differentiation,and provide a new idea for the regulation of androgen level in fish and a reference for the treatment of androgen deficiency related diseases caused by the disorder of LC development. |
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