抑制糖酵解活性限制雄性原始生殖细胞的增殖
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摘要
目的:雄性原始生殖细胞在植入生殖嵴后,会从有丝分裂退出进入静息状态,在这一过程中伴随着细胞内代谢状态的改变,本研究旨在体解析原始生殖细胞增殖的改变与细胞代谢之间的因果关系。方法:通过体内Brdu掺入实验明确不同时间点雄性生殖细胞的增殖状态;分析比较增殖状态和静息状态原始生殖细胞糖酵解相关基因的表达;利用腹腔注射HK2特异性抑制剂2-Deoxy-D-glucose (2-DG),构建糖酵解抑制小鼠模型;通过免疫荧光与qPCR分析抑制糖酵解后原始生殖细胞的表型。结果:免疫荧光结果显示雄性生殖细胞增殖停滞从E13.5开始,至E15.5完全停滞;qPCR和Western Blot显示在此过程中HK2的表达是逐渐降低的;在E11.5抑制小鼠胚胎中的糖酵解过程,可以在E13.5检测到雄性PGCs增殖下降,并且可以抑制多能性基因如Sox2、Oct4的表达。结论:研究发现,E11.5-E13.5雄性原始生殖细胞内增殖与多能性的维持需要糖酵解。改变胚胎糖酵解水平可以影响原始生殖细胞增殖分化进程。
Objective: Male primordial germ cells will exit from mitosis and enter a cell cycle arrest after implantation of genital ridge, accompanied by changes in intracellular metabolic states. Our work is to explore causal relationship between primordial germ cells proliferation and cell metabolism. Methods: The proliferation states of male primordial germ cells at different time points was determined by BrdU incorporation assay. The expression of glycolysis related genes of primordial germ cells in the proliferative state and cell cycle arrest state was compared by qPCR. A mouse model of glycolysis inhibition was established by intraperitoneal injection of HK2-specific inhibitor 2-DG, and phenotype was analyzed by immunofluorescence and q PCR. Results: The results of immunofluorescence showed that the cell cycle arrest of male germ cells start at E13.5 and completely arrest at E15.5. qPCR and Western Blot showed that Hk2 was decreased during this process. Inhibition of Hk2 in mouse embryos at E11.5, the proliferation of male germ cells was significantly inhibited at E13.5, and the expression levels of pluripotent genes such as Sox2 and Oct4 were decreased. Conclusion: This study found that in E11.5-E13.5, glycolysis is required for proliferation and maintenance of pluripotency in male primordial germ cells. Changing the level of embryonic glycolysis can affect the proliferation and differentiation of primordial germ cells.
Objective: Male primordial germ cells will exit from mitosis and enter a cell cycle arrest after implantation of genital ridge, accompanied by changes in intracellular metabolic states. Our work is to explore causal relationship between primordial germ cells proliferation and cell metabolism. Methods: The proliferation states of male primordial germ cells at different time points was determined by BrdU incorporation assay. The expression of glycolysis related genes of primordial germ cells in the proliferative state and cell cycle arrest state was compared by qPCR. A mouse model of glycolysis inhibition was established by intraperitoneal injection of HK2-specific inhibitor 2-DG, and phenotype was analyzed by immunofluorescence and q PCR. Results: The results of immunofluorescence showed that the cell cycle arrest of male germ cells start at E13.5 and completely arrest at E15.5. qPCR and Western Blot showed that Hk2 was decreased during this process. Inhibition of Hk2 in mouse embryos at E11.5, the proliferation of male germ cells was significantly inhibited at E13.5, and the expression levels of pluripotent genes such as Sox2 and Oct4 were decreased. Conclusion: This study found that in E11.5-E13.5, glycolysis is required for proliferation and maintenance of pluripotency in male primordial germ cells. Changing the level of embryonic glycolysis can affect the proliferation and differentiation of primordial germ cells.
引文
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[26]Chandel NS,Jasper H,Ho TT,et al.Metabolic regulation of stem cell function in tissue homeostasis and organismal ageing[J].Nat Cell Biol,2016,18:823-832
[27]H N Xu,G Zheng,J Tchou,et al.Characterizing the metabolic heterogeneity in human breast cancer xenografts by 3D high resolution fluorescence imaging[J].Springerplus,2013,2(1):73
[28]Vander Heiden MG,Cantley LC,Thompson CB.Understanding the Warburg effect:the metabolic requirements of cell proliferation[J].Science,2009,324(5930):1029-1033
[29]Moussaieff A,Rouleau M,Kitsberg D,et al.Glycolysis-mediated changes in acetyl-CoA and histone acetylation control the early differentiation of embryonic stem cells[J].Cell Metab,2015,21(3):392-402
[30]Tischler J,Gruhn WH,Reid J,et al.Metabolic regulation of pluripotency and germ cell fate throughα-ketoglutarate[J].EMBO J,2019,38(1)
[31]Pang YY,Wang T,Chen FY,et al.Glycolytic inhibitor 2-deoxy-dglucose suppresses cell proliferation and enhances methylprednisolone sensitivity in non-Hodgkin lymphoma cells through down-regulation of HIF-1αand c-MYC[J].Leuk Lymphoma,2015,56(6):1821-1830
[2]Mochizuki K,Tando Y,Sekinaka T et al.SETDB1 is essential for mouse primordial germ cell fate determination by ensuring BMPsignaling[J].Development,2018,145(23)
[3]Weber S,Eckert D,Nettersheim D,et al.Critical function of AP-2gamma/TCFAP2C in mouse embryonic germ cell maintenance[J].Biol Reprod,2010,82:214-223
[4]Kumar DL,De Falco T.Of Mice and Men:In Vivo and In Vitro Studies of Primordial Germ Cell Specification[J].Semin Reprod Med,2017,35(2):139-146
[5]Ewen KA,Koopman P.Mouse germ cell development:from specification to sex determination[J].Mol Cell Endocrinol,2010,323(1):76-93
[6]Morgan CT,Noble D,Kimble J,et al.Mitosis-meiosis and sperm-oocyte fate decisions are separable regulatory events[J].Proc Natl Acad Sci USA,2013,110(9):3411-3416
[7]Lin YT,Capel B.Cell fate commitment during mammalian sex determination[J].Curr.Opin.Genet.Dev,2015,32:144-152
[8]Spiller CM,Bowles J.Sex determination in mammalian germ cells[J].Asian J.Androl,2015,17:427-432
[9]Spiller C,Koopman P,Bowles J.Sex Determination in the Mammalian Germline[J].Annu Rev Genet,2017,51:265-285
[10]Koubova J,Hu YC,Bhattacharyya T,et al.Retinoic acid activates two pathways required for meiosis in mice[J].PLoS Genet,2014,10:e1004541
[11]Yao X,Tang F,Yu M,et al.Expression profile of Nanos2 gene in dairy goat and its inhibitory effect on Stra8 during meiosis[J].Cell Prolif,2014,47:396-405
[12]Barrios F,Filipponi D,Pellegrini M,et al.Opposing effects of retinoic acid and FGF9 on Nanos2 expression and meiotic entry of mouse germ cells[J].Cell Sci,2010,123:871-880
[13]Rossitto M,Philibert P,Poulat F,et al.Molecular events and signalling pathways of male germ cell differentiation in mouse[J].Semin Cell Dev Biol,2015,45:84-93
[14]Teng H,Sui X,Zhou C,et al.Fatty acid degradation plays an essential role in proliferation of mouse female primordial germ cells via the p53-dependent cell cycle regulation[J].Cell Cycle,2016,15(3):425-431
[15]Te Slaa T,Chaikovsky AC,Lipchina I,et al.alpha-Ketoglutarate accelerates the initial differentiation of primed human pluripotent stem cells[J].Cell Metab,2016,24:485-493
[16]Pengchun Yu,Kerstin Wilhelm,Alexandre Dubrac,et al.FGF-dependent metabolic control of vascular development[J].Nature,2017,545:224-228
[17]Aduma N,Izumi H,Mizushima S,et al.Knockdown of DEAD-box helicase 4(DDX4)decreases the number of germ cells in male and female chicken embryonic gonads.[J].Reprod Fertil Dev,2019,31(5):847-854
[18]Carey BW,Finley LW,Cross JR,et al.Intracellularα-ketoglutarate maintains the pluripotency of embryonic stem cells[J].Nature,2015,518:413-416
[19]Kim H,Jang H,Kim TW,et al.Core Pluripotency Factors Directly Regulate Metabolism in Embryonic Stem Cell to Maintain Pluripotency[J].Stem Cells,2015,33(9):2699-2711
[20]Peng M,Yin N,Chhangawala S,et al.Aerobic glycolysis promotes Thelper 1 cell differentiation through an epigenetic mechanism[J].Science,2016,354(6311):481-484
[21]Esteban-Martínez L,Sierra-Filardi E,Mc Greal RS,et al.Programmed mitophagy is essential for the glycolytic switch during cell differentiation[J].EMBO J,2017,36(12):1688-1706
[22]Zhong D,Xiong L,Liu T,et al.The glycolytic inhibitor2-deoxyglucose activates multiple prosurvival pathways through IGF1R[J].J Biol Chem,2009,284(35):23225-23233
[23]Gustin SE,Stringer JM,Hogg K,et al.FGF9,activin and TGFβpromote testicular characteristics in an XX gonad organ culture model[J].Reproduction,2016,152:529-543
[24]Bowles J,Feng CW,Spiller C,et al.FGF9 suppresses meiosis and promotes male germ cell fate in mice[J].Dev Cell,2010,19(3):440-449
[25]Dawson EP,Lanza DG,Webster NJ.Delayed male germ cell sexspecification permits transition into embryonal carcinoma cells with features of primed pluripotency[J].Development,2018,145(6)
[26]Chandel NS,Jasper H,Ho TT,et al.Metabolic regulation of stem cell function in tissue homeostasis and organismal ageing[J].Nat Cell Biol,2016,18:823-832
[27]H N Xu,G Zheng,J Tchou,et al.Characterizing the metabolic heterogeneity in human breast cancer xenografts by 3D high resolution fluorescence imaging[J].Springerplus,2013,2(1):73
[28]Vander Heiden MG,Cantley LC,Thompson CB.Understanding the Warburg effect:the metabolic requirements of cell proliferation[J].Science,2009,324(5930):1029-1033
[29]Moussaieff A,Rouleau M,Kitsberg D,et al.Glycolysis-mediated changes in acetyl-CoA and histone acetylation control the early differentiation of embryonic stem cells[J].Cell Metab,2015,21(3):392-402
[30]Tischler J,Gruhn WH,Reid J,et al.Metabolic regulation of pluripotency and germ cell fate throughα-ketoglutarate[J].EMBO J,2019,38(1)
[31]Pang YY,Wang T,Chen FY,et al.Glycolytic inhibitor 2-deoxy-dglucose suppresses cell proliferation and enhances methylprednisolone sensitivity in non-Hodgkin lymphoma cells through down-regulation of HIF-1αand c-MYC[J].Leuk Lymphoma,2015,56(6):1821-1830