维甲酸诱导精原干细胞分化过程中DNA甲基转移酶表达研究
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摘要
精原干细胞(spermatogonial stem cells,SSCs)是雄性哺乳动物体内能进行自我更新并通过精子发生将亲代遗传信息传递给子代的一类细胞。多项研究表明,维甲酸(retinoic acid,RA)可诱导SSCs分化,启动减数分裂。目前,关于维甲酸诱导SSCs体外分化的分子机制已取得一定进展,但此过程的DNA甲基化调控机制尚未探索。DNA甲基转移酶(DNA methyltransferases,Dnmts)催化DNA甲基化,研究SSCs分化前后Dnmts的表达变化将有助于本研究从表观遗传层面理解维甲酸诱导的SSCs分化过程。因此,在本研究中,首先通过两步酶消化法和免疫磁珠法从小鼠睾丸组织中分离纯化SSCs并建立细胞系。该细胞系在体外传代超过60代,并且表达Oct4、Plzf、Etv5、Dazl和Mvh等标志物。然后,本研究通过探索维甲酸诱导条件,建立了SSCs的体外分化体系。经维甲酸处理后,对SSCs自我更新起决定作用的转录因子Plzf及其共表达基因Oct4的表达下降,而分化相关基因(Stra8,c-Kit)表达上调。最后,本研究对SSCs分化前后Dnmts表达进行检测。检测显示与正常组SSCs比较,维甲酸处理组SSCs中Dnmt1、Dnmt3a表达下调。该发现初步表明Dnmt1、Dnmt3a在维甲酸诱导SSCs体外分化过程中起调控作用,为阐述维甲酸如何诱导SSCs分化的分子机制提供了新的证据。
Spermatogonial stem cells(SSCs) are a kind of cells in male mammals, which could undergo self-renewal and pass genetic information to the next generation through spermatogenesis. Several studies reported that retinoic acid(RA) could induce differentiation between SSCs and initiate meiosis. The molecular mechanism of RA-induced SSCs differentiation in vitro has been researched for a few years and some progress has been made, whereas the DNA methylation switches are still elusive. DNA methyltransferase(Dnmts) catalyzed DNA methylation. Thus, it is important to investigate the expression of Dnmts during SSCs differentiation, for it could help us to understand the process in epigenetic way. In this study, we first isolated SSCs by two-step enzymatic digestion and magnetic activated cell sorting(MACS) from mouse testis and successfully established in vitro propagation of SSCs. The SSCs in vitro were subcultured over 60 times and expressed SSCs markers(Oct4, Plzf, Etv5, Dazl and Mvh). Then,we treated SSCs with RA after exploring the condition, and successfully induced SSCs differentiation in vitro.Results showed that the expression of transcription factor Plzf and its coexpression gene Oct4, which play a decisive role in the self-renewal of SSCs, decreased, while the expression of differentiation related gene(Stra8,c-Kit) increased after RA-treated SSCs. Last, we investigated the expression of Dnmts in SSCs after RA treatment,and found that Dnmt1 and Dnmt3a were significantly down-regulated. Collectively, these findings suggested that Dnmt1 and Dnmt a might play a regulatory role in RA-induced SSCs differentiation in vitro, and provide new evidence for revealing the functions of RA in SSCs differentiation.
Spermatogonial stem cells(SSCs) are a kind of cells in male mammals, which could undergo self-renewal and pass genetic information to the next generation through spermatogenesis. Several studies reported that retinoic acid(RA) could induce differentiation between SSCs and initiate meiosis. The molecular mechanism of RA-induced SSCs differentiation in vitro has been researched for a few years and some progress has been made, whereas the DNA methylation switches are still elusive. DNA methyltransferase(Dnmts) catalyzed DNA methylation. Thus, it is important to investigate the expression of Dnmts during SSCs differentiation, for it could help us to understand the process in epigenetic way. In this study, we first isolated SSCs by two-step enzymatic digestion and magnetic activated cell sorting(MACS) from mouse testis and successfully established in vitro propagation of SSCs. The SSCs in vitro were subcultured over 60 times and expressed SSCs markers(Oct4, Plzf, Etv5, Dazl and Mvh). Then,we treated SSCs with RA after exploring the condition, and successfully induced SSCs differentiation in vitro.Results showed that the expression of transcription factor Plzf and its coexpression gene Oct4, which play a decisive role in the self-renewal of SSCs, decreased, while the expression of differentiation related gene(Stra8,c-Kit) increased after RA-treated SSCs. Last, we investigated the expression of Dnmts in SSCs after RA treatment,and found that Dnmt1 and Dnmt3a were significantly down-regulated. Collectively, these findings suggested that Dnmt1 and Dnmt a might play a regulatory role in RA-induced SSCs differentiation in vitro, and provide new evidence for revealing the functions of RA in SSCs differentiation.
引文
Busada J.T.,Chappell V.A.,Niedenberger B.A.,Kaye E.P.,Keiper B.D.,Hogarth C.A.,and Geyer C.B.,2015,Retinoic acid regulates Kit translation during spermatogonial differentiation in the mouse,Dev.Biol.,397(1):140-149
Duester G.,2013,Retinoid signaling in control of progenitor cell differentiation during mouse development,Semin Cell Dev.Biol.,24(10-12):694-700
Falciatori I.,Lillard-Wetherell K.,Wu Z.,Hamra F.K.,and Garbers D.L.,2008,Deriving mouse spermatogonial stem cell lines,In:Hou S.X.,and Singh S.R.(eds.),Germline Stem Cell Methods in Molecular Biology,Humana Press,New York USA,pp.181-192
Fu X.F.,Cheng S.F.,Wang L.Q.,Yin S.,De Felici M.,and Shen W.,2015,DAZ family proteins,key players for germ cell development,Int.J.Biol.Sci.,11(10):1226-1235
Kanatsu-Shinohara M.,Ogonuki N.,Inoue K.,Miki H.,Ogura A.Toyokuni S.,and Shinohara T.,2003,Long-term proliferation in culture and germline transmission of mouse male germline stem cells,Biol.Reprod.,69(2):612-616
Kanatsu-Shinohara M.,Ogonuki N.,Iwano T.,Lee J.,Kazuki Y.Inoue K.,Miki H.,Takehashi M.,Toyokuni S.,Shinkai Y.Oshimura M.,Ishino F.,Ogura A.,and Shinohara T.,2005Genetic and epigenetic properties of mouse male germline stem cells during long-term culture,Development,132(18):4155-4163
Kanatsu-Shinohara M.,and Shinohara T.,2013,Spermatogonial stem cell self-renewal and development,Annu.Rev.Cell Dev.Biol.,24:163-187
Kaneda M.,Okano M.,Hata K.,Sado T.,Tsujimoto N.,Li E.,and Sasaki H.,2004,Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting,Nature,429(6994):900-903
Kato Y.,Kaneda M.,Hata K.,Kumaki K.,Hisano M.,Kohara Y.,Okano M.,Li E.,Nozaki M.,and Sasaki H.,2007,Role of the Dnmt3 family in de novo methylation of imprinted and repetitive sequences during male germ cell development in the mouse,Hum.Mol.Genet.,16(19):2272-2280
Livak K.J.,and Schmittgen T.D.,2001,Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))Method,Methods,25(4):402-408
Oatley J.M.,and Brinster R.L.,2006,Spermatogonial stem cells,In:Abelson J.N.,and Simon M.I.(eds.),Methods in Enzymol,California Instutute of Technology,Pasadena,USA,pp.259-282
Reda A.,Hou M.,Winton T.R.,Chapin R.E.,Soder O.,and Stukenborg J.B.,2016,In vitro differentiation of rat spermatogonia into round spermatids in tissue culture,Mol.Hum.Reprod.,22(9):601-612
Riboldi M.,Rubio C.,Pellicer A.,Gil-Salom M.,and Simon C.,2012,In vitro production of haploid cells after coculture of CD49f+with Sertoli cells from testicular sperm extraction in nonobstructive azoospermic patients,Fertil.Steril.,98(3):580-590
Shiura H.,Ikeda R.,Lee J.,Sato T.,Ogonuki N.,Hirose M.,Ogura A.,Ogawa T.,a nd Abe K.,2013,Generation of a novel germline stem cell line expressing a germline-specific reporter in the mouse,Genesis,51(7):498-505
Tseng Y.T.,Liao H.F.,Yu C.Y.,Mo C.F.,and Lin S.P.,2015,Epigenetic factors in the regulation of prospermatogonia and spermatogonial stem cells,Reproduction,150(3):R77-R91
Van Pelt A.M.,and De Rooij D.G.,1990,Synchronization of the seminiferous epithelium after vitamin A replacement in vitamin A-deficient mice,Biol.Reprod.,43(3):363-367
Wang S.,Wang X.,Ma L.,Lin X.,Zhang D.,Li Z.,Wu Y.,Zheng C.,Feng X.,Liao S.,Feng Y.,Chen J.,Hu X.,Wang M.,and Han C.,2016,Retinoic acid is sufficient for the in vitro induction of mouse spermatocytes,Stem.Cell Reports,7(1)80-94
Yeh J.R.,Zhang X.,and Nagano M.C.,2007,Establishment of a short-term in vitro assay for mouse spermatogonial stem cells,Biol.Reprod.,77(5):897-904
Zhou Q.,Li Y.,Nie R.,Friel P.,Mitchell D.,Evanoff R.M.,Pouchnik D.,Banasik B.,Mc Carrey J.R.,Small C.,and Griswold M.D.,2008,Expression of stimulated by retinoic acid gene 8(Stra8)and maturation of murine gonocytes and spermatogonia induced by retinoic acid in vitro,Biol Reprod,78(3):537-545
Duester G.,2013,Retinoid signaling in control of progenitor cell differentiation during mouse development,Semin Cell Dev.Biol.,24(10-12):694-700
Falciatori I.,Lillard-Wetherell K.,Wu Z.,Hamra F.K.,and Garbers D.L.,2008,Deriving mouse spermatogonial stem cell lines,In:Hou S.X.,and Singh S.R.(eds.),Germline Stem Cell Methods in Molecular Biology,Humana Press,New York USA,pp.181-192
Fu X.F.,Cheng S.F.,Wang L.Q.,Yin S.,De Felici M.,and Shen W.,2015,DAZ family proteins,key players for germ cell development,Int.J.Biol.Sci.,11(10):1226-1235
Kanatsu-Shinohara M.,Ogonuki N.,Inoue K.,Miki H.,Ogura A.Toyokuni S.,and Shinohara T.,2003,Long-term proliferation in culture and germline transmission of mouse male germline stem cells,Biol.Reprod.,69(2):612-616
Kanatsu-Shinohara M.,Ogonuki N.,Iwano T.,Lee J.,Kazuki Y.Inoue K.,Miki H.,Takehashi M.,Toyokuni S.,Shinkai Y.Oshimura M.,Ishino F.,Ogura A.,and Shinohara T.,2005Genetic and epigenetic properties of mouse male germline stem cells during long-term culture,Development,132(18):4155-4163
Kanatsu-Shinohara M.,and Shinohara T.,2013,Spermatogonial stem cell self-renewal and development,Annu.Rev.Cell Dev.Biol.,24:163-187
Kaneda M.,Okano M.,Hata K.,Sado T.,Tsujimoto N.,Li E.,and Sasaki H.,2004,Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting,Nature,429(6994):900-903
Kato Y.,Kaneda M.,Hata K.,Kumaki K.,Hisano M.,Kohara Y.,Okano M.,Li E.,Nozaki M.,and Sasaki H.,2007,Role of the Dnmt3 family in de novo methylation of imprinted and repetitive sequences during male germ cell development in the mouse,Hum.Mol.Genet.,16(19):2272-2280
Livak K.J.,and Schmittgen T.D.,2001,Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))Method,Methods,25(4):402-408
Oatley J.M.,and Brinster R.L.,2006,Spermatogonial stem cells,In:Abelson J.N.,and Simon M.I.(eds.),Methods in Enzymol,California Instutute of Technology,Pasadena,USA,pp.259-282
Reda A.,Hou M.,Winton T.R.,Chapin R.E.,Soder O.,and Stukenborg J.B.,2016,In vitro differentiation of rat spermatogonia into round spermatids in tissue culture,Mol.Hum.Reprod.,22(9):601-612
Riboldi M.,Rubio C.,Pellicer A.,Gil-Salom M.,and Simon C.,2012,In vitro production of haploid cells after coculture of CD49f+with Sertoli cells from testicular sperm extraction in nonobstructive azoospermic patients,Fertil.Steril.,98(3):580-590
Shiura H.,Ikeda R.,Lee J.,Sato T.,Ogonuki N.,Hirose M.,Ogura A.,Ogawa T.,a nd Abe K.,2013,Generation of a novel germline stem cell line expressing a germline-specific reporter in the mouse,Genesis,51(7):498-505
Tseng Y.T.,Liao H.F.,Yu C.Y.,Mo C.F.,and Lin S.P.,2015,Epigenetic factors in the regulation of prospermatogonia and spermatogonial stem cells,Reproduction,150(3):R77-R91
Van Pelt A.M.,and De Rooij D.G.,1990,Synchronization of the seminiferous epithelium after vitamin A replacement in vitamin A-deficient mice,Biol.Reprod.,43(3):363-367
Wang S.,Wang X.,Ma L.,Lin X.,Zhang D.,Li Z.,Wu Y.,Zheng C.,Feng X.,Liao S.,Feng Y.,Chen J.,Hu X.,Wang M.,and Han C.,2016,Retinoic acid is sufficient for the in vitro induction of mouse spermatocytes,Stem.Cell Reports,7(1)80-94
Yeh J.R.,Zhang X.,and Nagano M.C.,2007,Establishment of a short-term in vitro assay for mouse spermatogonial stem cells,Biol.Reprod.,77(5):897-904
Zhou Q.,Li Y.,Nie R.,Friel P.,Mitchell D.,Evanoff R.M.,Pouchnik D.,Banasik B.,Mc Carrey J.R.,Small C.,and Griswold M.D.,2008,Expression of stimulated by retinoic acid gene 8(Stra8)and maturation of murine gonocytes and spermatogonia induced by retinoic acid in vitro,Biol Reprod,78(3):537-545