The Role Of Retinoic Acid Signaling Pathway In The Proliferation And Differentiation Of Medaka Spermatogonial Stem Cells

Spermatogonial stem cells(SSCs)are germline stem cells located in the basement membrane of seminiferous tubules and developed from primordial germ cells.They are also the only adult stem cells in males that can transmit genetic information to the offspring.SSC can not only renew to maintain its constant number,but also sequentially differentiate to the differentiated spermatogonia,primary spermatocyte,secondary spermatocyte and finally spermatozoa.SSC proliferation and differentiation may affect the reproductin or fertility,which is of great biological significance.Retinoic acid(RA)plays an important role in spermatogenesis of vertebrate.In mammals,a large number of studies have confirmed that RA is a key factor regulating the initiation of meiosis.Recent studies have reported that retinoic acid is sufficient for inducing leptotene/zygotene spermatocytes from cultured mouse SSCs in vitro.In fish,studies have shown that RA is important in meiosis,but it remains elusive whether it mediates the initiation of meiosis or not.Hong et al successfully established medaka(Oryzias latipes)spermatogonia stem cell line SG3,which provides us with an excellent tool for fish SSC research.Synaptonemal complex protein 3(Scp3)is a meiotic specific protein,which expressed in spermatocyte in testis and can monitor the meiosis.Protamine is a basic protein expressed in mature spermatid which can monitor spermatid production.In this study,the three fluorescence reported plasmid p GRY contains three promoters,i.e.,elongation factor 1?(ef1?)promoter driven histone H2B-green fluorescent protein(H2B-GFP),scp3 promoter driven puromycin-red fluorescent protein(puro-RFP),protamine promoter driven yellow fluorescent protein(YFP).SG3 was transfected with p GRY and screened for stable transgenic cell SG3-p GRY.SG3-p GRY was treated with RA and Rar(?,?and?)pan-antagonist BMS493 to study the effect of RA signal on cell proliferation,on meiosis in 2D and 3D culture by the expression of red fluorescent protein,and on the expression of pluripotency and differentiation related genes.In mammals,retinoic acid receptors include Rar?,?and?.Only Rar?and?expressed in testis.Recent studies have shown that mouse SSC may be regulated by Rar?to response to RA,and to maintain the balance between undifferentiation and differentiation state.The expression of rar?and rar?in testis and SG3 of medaka remain to be explored.In this study,rar?and rar?of medaka were identified and cloned,and their expression in testis and SG3 were detected.The results are as follows:1.The role of RA signal pathway in the proliferation and differentiation of SG3(1)Establishment of SG3-p GRY and its differentiation potentialSG3 was transfected with a three-fluorescence-reported plasmid(p GRY)and screened for stable transgenic cell line SG3-p GRY by puromycin.SG3-p GRY in normal density with subcultured(2D culture)constitutively expressed green fluorescent protein,but did not express red and yellow fluorescent protein.SG3-p GRY in high density without subcultured in vitro for 24 d expressed strong red fluorescence protein.Immunofluorescence with Scp3 antibody showed that the red fluorescent protein could indeed reflect the expression of endogenous Scp3.It is suggested that p GRY transfection does not affect the differentiation potential of SG3 cells,and the expression of red fluorescent protein can be used to monitor the meiosis.In addition,Hong et al reported that SG3 has differentiation potential in vitro,but it has not been confirmed in vivo.In this study,we detected the differentiation ability of SG3-p GRY in vivo by germ cell transplantation.Busulfan(80 mg/kg/bw)was administered by intraperitoneal injection to clear endogenous germ cells in the male medaka,and then 1*10~5 SG3-p GRY cells were transplanted into the recipient fish testis.On the 10,30,44 and 70 d after transplantation,a large number of donor cells with green fluorescent protein were observed in the recipient fish testis,and some of them expressed red fluorescence at the same time,suggesting that SG3-p GRY can successfully colonize in the testis and has the potential of differentiation.(2)The effect of RA signal on the proliferation of SG3-p GRYUnder the condition of 2D,cells were treated with RA(12.5?M)and Rar pan-antagonist BMS493(10?M).The results of MTS showed that RA could inhibit cell proliferation(p<0.05),while BMS493 could promote cell proliferation(p<0.05).It is suggested that RA signal pathway can inhibit the proliferation of SSC.(3)The effect of RA signal on the differentiation of SG3-p GRY in 2D cultureThere were no obvious red fluorescence protein in RA and control group under the condition of 2D for 8 d,it is suggested that RA can not initiate SG3-p GRY meiosis.The results of real-time PCR showed that RA could significantly down-regulate the expression of pluripotency related genes pou5f3 and klf4(p<0.05)and up-regulate the expression of dazl(p<0.05)compared with the control,although there was no red fluorescence in 2D conditions for 48 h.RA had no significant effect on the expression of scp3,ckit,spo11,rec8a,rec8b(p>0.05),it is suggested that RA can mediate the differentiation of SG3-p GRY.(4)The effect of RA signal on the differentiation of SG3-p GRY in 3D cultureObvious red fluorescence was observed in RA,BMS493 and control group in the condition of 3D(Corning 96-well spheroid microplates)for 48 h.The results showed that meiosis is initiated in SG3-p GRY under 3D culture,and RA signal not be necessary for the initiation of meiosis.Compared with 2D conditions,the expressions of differentiation related genes were up-regulated in SG3-p GRY with 3D culture for 48 h(p<0.05).In addition,the expression of differentiation related genes in RA of 3D culture was higher than other groups(p<0.05).It is suggested that RA can promote the differentiation of SG3-p GRY.Taken together,RA signal pathway can promote the differentiation of medaka SSC under 2D and 3D culture conditions,but unlike mammals,RA is not a key factor to induce meiosis initiation.2.c DNA clone,identification and expression pattern of medaka retinoic acid receptor(1)c DNA sequence and sequence analysisOlrar?,Olrar?1 and Olrar?2 encode 421,498,483 amino acid residues(aa),respectively.The identity between them and mammalian amino acid sequences is as high as 60%.On the phylogenetic tree,Ol Rar?,Ol Rar?1 and Ol Rar?2 were orthologs of Rar?and Rar?in mammals,and Ol Rar?1 and Ol Rar?2 were paralogs.(2)The m RNA expression patternRT-PCR results showed that Olrar?,Olrar?1 and Olrar?2 were expressed in ovary,testis and SG3 of medaka.In situ hybridization results showed that Olrar?and Olrar?1were expressed in different stages of spermatogenic cells in testis,and Olrar?2 is undetected.The expression level of Olrar?in spermatogonia was significantly lower than other spermatogenic cells.Our study firstly demonstrates that RA plays an important role in the proliferation and differentiation of SSC cultured in 2D and 3D conditions,but it is not necessary for SSC to initiate meiosis in teleost fish.This study provides an important reference for the study of differentiation and development of SSC,and is helpful for the application of SSC in the field of reproductive medicine.