蛋白质代谢通路对鸡雄性生殖细胞分化的调控

doi:10.3864/j.issn.0578-1752.2016.24.013

摘要/Abstract

摘要： 【目的】探究蛋白质代谢在鸡雄性生殖细胞分化过程中的作用机制，为完善鸡胚胎干细胞（embryonic stem cell，ESC）体外向雄性生殖细胞诱导分化体系研究提供依据。【方法】采用流式分选的方法获取高纯度的ESC、原始生殖细胞（primordial germ cells，PGCs）和精原干细胞（spermatogonia stem cell，SSCs），分别提取细胞的总RNA，采用RNA-Seq方法对3种细胞的转录本进行深度测序，然后进行WEGO（web gene ontology）和KEGG（kyoto encyclopedia of genes and genomes）通路富集分析，筛选出鸡雄性生殖细胞分化过程中参与蛋白质代谢的关键通路及其关键基因，RT-qPCR (Real time Quantitative PCR) 检测部分关键差异基因的表达变化，并与RNA-Seq（RNA sequencing）结果进行比较分析，同时分别从体内和体外水平对关键基因NOS2进行抑制，观察各分组不同天数的细胞形态变化及检测NOS2和C-kit、Cvh、Stra8、Dazl、integrin α6和integrin β1等生殖标记基因表达变化情况。【结果】在雄性生殖细胞分化的整个阶段，有697个差异基因参与生物代谢，显著富集于精氨酸-脯氨酸代谢通路、酪氨酸代谢通路以及色氨酸代谢通路，在这3条通路上筛选出NOS2、FAH和IDO等关键性基因，这些基因的在ESCs向SSCs分化过程中表达变化趋势与其在RNA-Seq中的结果一致。体内抑制NOS2基因后，NOS2及C-kit、Cvh、Stra8、Dazl、integrin α6和integrin β1等生殖标记基因在空白组和对照组之间无显著性的差异，而在抑制剂组中，NOS2及C-kit、Cvh、Stra8、Dazl、integrin α6和integrin β1等生殖标记基因的mRNA表达量均出现了降低；而体外抑制NOS2基因后，对照组中的ESCs在2、4、6、8和10d内细胞不断增殖，但是未出现类胚体；RA诱导组中，2d出现小的类胚体，4d类胚体增大，且数量增多，6d类胚体边缘开始出现破裂，8d类胚体解体，10d出现类精原样细胞；抑制剂组中，ESCs在2、4、6、8和10d内无类胚体出现，且相较于对照组细胞增殖缓慢；RA+抑制剂组中，2和4d内无类胚体出现，细胞增殖缓慢，6d出现小的类胚体，8d类胚体数量少量增多，且体积稍显增大，10d类胚体开始裂解。并且经过抑制剂的抑制后，NOS2及C-kit、Cvh、Stra8、Dazl、integrin α6和integrin β1等生殖标记基因的表达量在RA诱导组、抑制剂组和RA+抑制剂组中相对于对照组均呈显著性或极显著性的下调趋势。【结论】基于RNA-Seq技术及生物信息学方法筛选出ESCs向雄性生殖细胞分化过程中精氨酸-脯氨酸代谢通路及关键基因NOS2的基础上，通过对NOS2基因在鸡体内和体外的抑制，发现NOS2在被抑制后，ESCs向雄性生殖细胞分化的过程受到抑制。说明了精氨酸-脯氨酸代谢通路及关键基因NOS2对ESCs向雄性生殖细胞分化过程中起到重要的调节作用。

关键词: RNA-Seq, 鸡胚胎干细胞, 原始生殖细胞, 精原干细胞, 雄性生殖细胞, 一氧化氮合成酶2, 抑制剂, 分化

Abstract: 【Objective】 The aim of this study was to explore the regulatory mechanism of protein metabolism during the differentiation process of chicken male germ cells and provide a basis for improving the induction system of chicken embryonic stem cells (ESCs) differentiation to male germ cells in vitro. 【Method】RNA sequencing was performed using FACS-sorted cells from ESCs, PGCs(primordial germ cells) and SSCs(spermatogonial stem cells), and enrichment analysis, WEGO (Web Gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes), were carried out to find out the relevant pathways and the key genes, the expression level of which was analyzed by qRT-PCR. Moreover, NOS2 both in vitro and in vivo with NOS2 inhibitor was inhibited, and the morphologic changes of ESCs were observed and the mRNA expressions of NOS2 and other germ genes, C-kit, Cvh, Stra8, Dazl, integrin α6 and integrin β1 were detectedin different groups and in different days with RT-qPCR. 【Result】 Final results showed that 697 differentially expressed genes were involved in biological metabolism and significantly enriched in arginine-proline metabolic pathway, tyrosine metabolic pathway and tryptophan metabolic pathway and screened some key genes, like NOS2, FAH and IDO. It was found that the expression trends of NOS2, FAH and IDO were the same as that of RNA-Seq. In inhibitory experiment in vivo, the mRNA expression of NOS2, C-kit, Cvh, Stra8, Dazl, integrin α6 and integrin β1 between blank group and control group showed no significant difference. However, in inhibited group, NOS2, C-kit, Cvh, Stra8, Dazl, integrin α6 and integrin β1 expressions were down-regulated inordinately. Moreover, in inhibitory experiment in vitro, ESCs always proliferated on the 2, 4, 6, 8 and 10d, but disappeared the embryonic bodies in the control group. In induced group, small embryonic bodies appeared on the 2d and became bigger and increased on the 4d. Embryonic bodies started to burst in edges on the 6d, break up on the 8d and appeared spermatogonia-like cells. In inhibited group, no embryonic body appeared in the whole process and ESCs proliferated more slow than the control group. In induced-inhibited group, no embryonic body appeared on the 2d and 4d and ESCs proliferated slowly. Small embryonic bodies appeared on the 6d and the number and volume increased slightly on the 8d. On the 10d, the embryonic bodies started to break up. In vitro, NOS2, C-kit, Cvh, Stra8, Dazl, integrin α6 and integrin β1 expressions in induced group, inhibited group and induced-inhibited group were significantly down-regulated compared with the control group. 【Conclusion】In this study, based on the screening of arginine-proline metabolic pathway and NOS2 with RNA-Seq and Bioinformatics, it was found that the process of ESCs differentiation to male germ cell was inhibited after the inhibition of NOS2，which suggested that arginine-proline metabolic pathway and NOS2 has an important regulatory effect on differentiation of ESCs to male germ cells.

Key words: RNA-Seq, ESCs, PGCs, SSCs, male germ cells, NOS2, inhibitor, differentiation

李东，汤贝贝，王颖洁，纪艳芹，王飞，路镇宇，王曼，张亚妮，李碧春. 蛋白质代谢通路对鸡雄性生殖细胞分化的调控[J]. 中国农业科学, 2016, 49(24): 4814-4823.

LI Dong, TANG Bei-bei, WANG Ying-jie, JI Yan-qin, WANG Fei, LU Zhen-yu, WANG Man, ZHANG Ya-ni, LI Bi-chun. Regulatory Study of Protein Metabolism During the Differentiation Process of Chicken Male Germ Cells[J]. Scientia Agricultura Sinica, 2016, 49(24): 4814-4823.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2016.24.013

https://www.chinaagrisci.com/CN/Y2016/V49/I24/4814

参考文献

[1] 孙敏, 施青青, 李碧春. 胚胎干细胞诱导分化为雄性生殖细胞的研究进展. 生命科学，2012, 24(1):37-42.

SUN M, SHI Q Q, LI B C. Research progress on differentiation of embryonic stem cells into male germ cells. Chinese Bulletin of Life Sciences, 2012, 24(1): 37-42. (in Chinese)

[2] 韩明权, 刘嘉湘, 高虹, 陈善香, 朱晏伟, 许玲. 益肺抗瘤饮对实验性肺癌细胞周期及核酸和蛋白质合成的影响. 中西医结合学报，2003, 1(3):205-208.

HAN M Q, LIU J X, GAO H, CHEN S X, ZHU Y W, XU L. Effects of Yifei Kangliu Qral Liquid on cell cycle and protein-nucleic acid synthesis of experimental lung cancer. Journal China of Integrative Medicine, 2003, 1(3):205-208. (in Chinese)

[3] FEI T, XIA K, LI Z W, ZHOU B, ZHU S S, CHEN H, ZHANG J P, CHEN Z, XIAO H S, HAN J D J, CHEN Y G. Genome-wide mapping of SMAD target genes reveals the role of BMP signaling in embryonic stemcell fate determination. Genome Research, 2010, 20( 1) : 36-44.

[4] CHEN W, JIA W W, WANG K, ZHOU Q, LENG Y, DUAN T, KANG J H. Retinoic acid regulates germ cell differentiation in mouse embryonic stem cells through a Smad-dependent pathway. Biochemical and Biophysical Research Communications, 2012, 418(3): 571-577.

[5] NISHIMURA Y, KURISAKI A, NAKANISHI M, OHNUMA K, NINOMIYA N, KOMAZAKI S, ISHIURA S, ASASHIMA M. Inhibitory Smad proteins promote the differentiation of mouse embryonic stem cells into ependymal-like ciliated cells. Biochemical and Biophysical Research Communications, 2010, 401(1): 1-6.

[6] HELLINGMAN C A, DAVIDSON E N, KOEVOET W, VITTERS E L, VAN DEN BERG W B, VAN OSCH G J V M, VAN DER KRAAN P M. Smad signaling determines chondrogenic differentiation of bone- marrow-derived mesenchymal stem cells: inhibition of Smad1/5/8P prevents terminal differentiation and calcification. Tissue Engineering: Part A, 2011, 17(7-8): 1157-1167.

[7] TAN B, YIN Y L, KONG X F, LI P, LI X L, GAO H J, LI X G, HUANG R L, WU G Y. L-Arginine stimulates proliferation and prevents endotoxin-induced death of intestinal cells. Amino Acids, 2009, 38(4): 1227-1235.

[8] SULTAN M, SCHULZ M H, RICHARD H, MAGEN A, KLINGENHOFF A, SCHERF M, SEIFERT M, BORODINA T, SOLDATOV A, PARKHOMCHUK D, SCHMIDT D, O'KEEFFE S, HAAS S, VINGRON M, LEHRACH H, YASPO M L. A global view of gene activity and alternative splicing by deep sequencing of the human transcriptome. Science, 2008, 321(5891): 956-960.

[9] TANG F C, BARBACIORU C, BAO S Q, LEE C, NORDMAN E, WANG X H, LAO K Q, SURANI M A. Tracing the derivation of embryonic stem cells from the inner cell mass by single-cell RNA-Seq analysis. Cell Stem Cell, 2010, 5(6): 397-398.

[10] TRAPNELL C, WILLIAMS B A, PERTEA G, MORTAZAVI A, KWAN G, VAN BAREN M J, SAIZBERG S L,WOLD B J, PACHTER L. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nature Biotechnology, 2010, 28: 511-515.

[11] TANG F C, BARBACIORU C, NORDMAN E, LI B, XU N L, BASHKIROV V I, LAO K Q, SURANI M A. RNA-Seq analysis to capture the transcriptome landscape of a single cell. Nature Protocols, 2010, 5: 516-535.

[12] HAMBURGER V, HAMILTON H L. A series of normal stages in the development of the chicken. Morphology, 1951, 88(1): 49-92.

[13] 李碧春, 陈国宏, 赵东伟, 王克华, 钱菊芬. 鸡胚PGCs迁移与性腺发育关系的研究. 扬州大学学报, 2002, 23(1): 18-26.

LI B C, CHEN G H, ZHAO D W, WANG K H, QIAN J F. Relationship between PGCs migration and gonad development in the earlu chicken embryo. Journal of Yangzhou University (Agricultural and Life Science Edition), 2002, 23(1): 18-26. (in Chinese)

[14] 孙敏, 施青青, 傅德智, 阴彦辉, 张亚妮, 李碧春. 鸡胚ESCs 和SSCs 特定基因表达差异的研究. 生物技术, 2011, 21(3): 16-19.

SUN M, SHI Q Q, FU D Z, YIN Y H, ZHANG Y N, LI B C. Study of the expression of gene cell differentiation associated gene on chicken ESC and SSCs. Biotechnology, 2011, 21(3): 16-19. (in Chinese)

[15] 汪怡临, 靳锴, 蒋舒颖, 赵瑞丰, 左其生, 李东, 王颖洁, 张蕾, 张亚妮, 李碧春. 不同注射部位和剂量对鸡胚发育的影响. 中国家禽, 2014, 36(17): 7-10.

WANG Y L, JIN K, JIANG S Y, ZHAO R F, ZUO Q S, LI D, WANG Y J, ZHANG L, ZHANG Y N, LI B C. Effects of different injection location and dose on chicken embryos. China Poultry, 2014, 36(17): 7-10. (in Chinese)

[16] 陈兵, 易斌, 鲁开智. Smad 蛋白家族调控细胞分化的研究进展. 医学研究生学报, 2013, 26 (5): 544-547.

Chen B, Yi B, Lu K Z. Advances in researches on Smad proteins in cell differentiation. Journal of Medical Postgraduates, 2013, 26 (5): 544-547. (in Chinese)

[17] ANDREA V C, DIANA J L. Wnt and Bmp fit germ cells to a T. Developmental Cell. 2013, 27(5): 485–487.

[18] SUGIMOTO R, NABESHIMAB Y, YOSHIDA S. Retinoic acid metabolism links the periodical differentiation of germ cells with the cycle of Sertoli cells in mouse seminiferous epithelium. Mechanisms of Development, 2012, 128(11-12): 610–624.

[19] SUBRAMANIAM K, SEYDOUX G. nos-1 and nos-2, two genes related to Drosophilananos, regulate primordial germ cell development and survival in Caenorhabditis elegans. Development, 1999, 126: 4861-4871.

[20] GALLO C M, WANG J T, MOTEGI F, SEYDOUX G. Cytoplasmic partitioning of P granule components is not required to specify the germline in C. elegans. Science, 2010, 330(6011): 1685-1689.

[21] ZINI A, DE LAMIRANDE E. Low levels of nitric oxide promote human sperm capacitation in vitro. Journal of Andrology, 1995, 16 (5): 424-431.

[22] ZINI A, O’BRYAN M K, SCHLEGEL P N. Nitric oxide synthase activity in human seminal plasma. Urology, 2001, 58 (1) :85-89.

[23] HERRERO M B, DE LAMIRANDE E, GAGNON C. Nitric oxide regulates human sperm capacitation and protein-tyrosine phosphorylation in vitro. Biology of Reproduction, 1999, 61(3): 575-581.

[24] MITROPOULOS D, DELICONSTANTINOS G. Nitric oxide synthase and xanthine oxidase activities in the spermatic vein of patients with varicocele: apotential role for nitric oxide and peroxynitrite in sperm dysfunction. The Journal of Urology, 1996, 156(6): 1952-1958.

[25] YUMIKO S. Function of Nanos2 in the male germ cell lineage in mice. Cellular and Molecular Life Sciences, 2010, 67(22): 3815-3822.

[26] BARRIOS F, FILIPPONI D, PELLEGRINI M, PARONETTO M P, SIEN S D, GEREMIA R, ROSSI P, DE FELICI M, JANNINI E A, DOLCI S. Opposing effects of retinoic acid and FGF9 on Nanos2 expression and meiotic entry of mouse germ cells. Journal of Cell Science, 2010, 123: 871-880.

[27] CHILDS A J, COWAN G, KINNELL H L, ANDERSON R A, SAUNDERS P T K. Retinoic acid signalling and the control of meiotic entry in the human fetal gonad. PLoS One, 2011, 6(6): e20249.

[28] TSUDA M, SASAOKA Y, KISO M, ABE K, HARAGUCHI S, KOBAYASHI S, SAGA Y. Conserved role of nanos proteins in germ cell development. Science, 2003, 301(5637): 1239-1241.

[29] NAKAMURA S, KOBAYASHI K, NISHIMURA T, HIGASHIJIMA S, TANAKA M. Identification of germline stem cells in the ovary of the teleost medaka. Science, 2010, 328: 1561-1563.

[30] KÖPRUNNER M, THISSE C, THISSE B, RAZ E. A zebrafish nanos-related gene is essential for the development of primordial germ cells. Genes & Development, 2001, 15: 2877-2885.

[31] 刘秀红, 李宁. 肝病与肝癌的新疗法：干细胞研究的最新进展. 北京医学, 2013, 35(9): 813-814.

LIU X H, LI N. New therapy of Liver disease and liver cancer: the latest progress in the study of stem cells. Beijing Medical Journal, 2013, 35(9): 813-814. (in Chinese)

[32] Jang Y Y, Collector M I, Baylin S B, Deihl A M, Sharkis S J. Hematopoietic stem cells convert into liver cells within days without fusion. Nature Cell Biology, 2004; 6: 532-539.

[33] Takikawa O, Kuroiwa T, Yamazaki F, Kido R. Mechanism of interferon-gamma action. Characterization of indoleamine 2,3- dioxygenase in cultured human cells induced by interferon-gamma and evaluation of the enzyme-mediated tryptophan degradation in its anticellular activity. Journal of Biological Chemistry, 1988, 263(4): 2041-2048.

[34] Viola A, Bronte V. Metabolic mechanisms of cancer induced- inhibition of immune responses. Seminars in Cancer Biology, 2007, 17(4): 309-316.