Study on the Function of Glycolysis in Inducing Chicken PGCLC in vitro Formation

doi:10.13560/j.cnki.biotech.bull.1985.2020-1323

Abstract

Abstract:

Studies have shown that glycolysis is mainly involved in cell reprogramming and maintaining cell totipotency,but its role in germ cell differentiation is still poorly understood. This study aims to explore the function of glycolysis system in the formation of Primordial Germ Cell(PGC)based on the in vitro model of inducing PGC differentiation in chicken Embryonic Stem Cell(ESC)induced by Bone Morphogenetic Protein 4(BMP4),and to lay a theoretical foundation for analyzing the molecular mechanism of PGC formation in chickens from the metabolic level. The expression variations of glycolysis related genes of Pgk1(phosphoglycerate kinase 1),Hk1(hexokinase 1),Pkm2(pyruvate kinase M2),Ldha(lactate dehydrogenase A),Glut1(glucose transporter type 1),Pfkp(phosphofructokinase,platelet,)and Aldoc(aldolase,fructose-bisphosphate C)were detected by qRT-PCR during BMP4 induction with glycolysis inhibitor VK3 and activator DASA58. Cell morphology was observed under different treatments. Flow cytometry was used to analyze the percentage of DDX4 positive cells(PGC like cells,PGCLC)on the day 6 after induction. qRT-PCR results showed that during the differentiation of chicken ESC into PGC-like induced by BMP4,the glycolysis related genes were significantly down regulated,and the glycolysis pathway was inhibited;while the glycolysis pathway was significantly inhibited after adding VK3,and was significantly activated after adding DASA58. Morphological observation of cells showed that the number of embryoid bodies increased significantly after adding VK3 compared with the normal induction process,but decreased significantly after adding DASA58. Analysis by flow cytometry showed that the proportion of DDX4 positive cells increased after VK3 addition,and decreased after adding DASA58. In sum,the results indicate that inhibition of glycolysis may promote the formation of chicken PGC-like in vitro,that is,glycolysis is suppressed in the process of chicken PGCs formation.

Key words: glycolysis, chicken, embryonic stem cells, primordial germ like cells

ZHANG Chen, ZUO Qi-sheng, ZOU Yi-chen, ZHAO Juan-juan, ZHANG Ya-ni, LI Bi-chun. Study on the Function of Glycolysis in Inducing Chicken PGCLC in vitro Formation[J]. Biotechnology Bulletin, 2021, 37(6): 163-170.

Figures/Tables 5

Table 1 Primers used for qRT-PCR

引物名称 Primer name	5' 端引物 Primer sequence(5')	3' 端引物 Primer sequence(3')
Hk1	CCTCTTGGCTTCACATTC	TTCACAGTTTGGGTCTTCAT
Pkm2	GGCACCCACGAGTATCAT	CATTGTCCAGCGTCACTTT
Pfkp	GCCACAACAAACCTATAA-CA	ATCAAAGGCAGACGAACA
Ldha	TGGGCATCCATCCTCTGA	CCTGCTTGTGAACCTCCT
Glut1	TGTTTGGCTTGGACTTGAT	TCTTGAGGACGCTCTTGG
Hif1a	TTGACAAGGCATCCATTA	TCCTCAGAAAGCACCATA
Aldoc	CTGACGACGGCACTCCTTT	GACAGCCCATCCAGACCCT
Pgk1	AGGGCTGCATCACCATTA	CCACCTCCAGTGCTAACG

Fig. 1 Expression of glycolysis-related genes in the formation of PGC-like

Fig. 2 Effects of glycolysis inhibitors/activators on glycolysis-related genes during the induction in vitro

Fig.3 Effect of glycolysis inhibitor/activator on embryoid body formation during the induction in vitro A:Observation of cell morphology. B:The histogram shows the number of embryoid bodies

Fig. 4 Effect of glycolysis inhibitor/activator on PGC-like formation during the induction in vitro A:Proportion of positive cells with flow cytometry. B:Histogram showed the proportion of positive cells

References 35

[1]	Brinster RL, Harstad H . Energy metabolism in primordial germ cells of the mouse[J]. Experimental Cell Research, 1977, 109(1):111-117. doi: 10.1016/0014-4827(77)90050-7 URL
[2]	Koppenol WH, Bounds PL, Dang CV. Otto Warburg’s contributions to current concepts of cancer metabolism[J]. Nature Reviews Cancer, 2011, 11:325-337. doi: 10.1038/nrc3038 URL
[3]	Zheng J. Energy metabolism of cancer:Glycolysis versus oxidative phosphorylation(Review)[J]. Oncology Letters, 2012, 4(6):1151-1157. pmid: 23226794
[4]	Hayashi Y, Otsuka K, Ebina M, et al. Distinct requirements for energy metabolism in mouse primordial germ cells and their reprogramming to embryonic germ cells[J]. Proceedings of the National Academy of ences of the United States of America, 2017, 114(31):8289.
[5]	Kanatsu-Shinohara M, Tanaka T, Ogonuki N, et al. Myc/Mycn-mediated glycolysis enhances mouse spermatogonial stem cell self-renewal[J]. Genes & Development, 2016, 30(23):2637. doi: 10.1101/gad.287045.116 URL
[6]	Folmes CL, Nelson T, Martinez-Fernandez A, et al. Somatic oxidative bioenergetics transitions into pluripotency-dependent glycolysis to facilitate nuclear reprogramming[J]. Cell Metabolism, 2011, 14(2):264-271. doi: 10.1016/j.cmet.2011.06.011 URL
[7]	冯科珂, 刘晶锦, 姚其正. VK₃及其类似物的抗肿瘤作用[J]. 国外医药:抗生素分册, 2009, 30(4):171-177.
	Feng KK, Liu JJ, Yao QZ. The antineoplastic effect of vitamin K₃ and its derivates [J]. World Notes on Antibiotics, 2009, 30(4):171-177.
[8]	Chen J, Jiang Z, Wang B, et al. Vitamin K₃ and K₅ are inhibitors of tumor pyruvate kinase M2[J]. Cancer Letter, 2012, 316(2):204-10. doi: 10.1016/j.canlet.2011.10.039 URL
[9]	Wu FY, Liao WC, Chang HM. Comparison of antitumor activity of vitamins K₁, K₂ and K₃ on human tumor cells by two(MTT and SRB)cell viability assays[J]. Life Science, 1993, 52(22):1797-804.
[10]	Hitomi M, Yokoyama F, Kita Y, et al. Antitumor effects of vitamins K₁, K₂ and K₃ on hepatocellular carcinoma in vitro and in vivo[J]. International Journal of Oncology, 2005, 26(3):713-720.
[11]	Giannoni E, Taddei ML, Morandi A, et al. Targeting stromal-induced pyruvate kinase M2 nuclear translocation impairs oxphos and prostate cancer metastatic spread[J]. Oncotarget, 2015, 6(27):24061-24074. pmid: 26183399
[12]	Palsson-McDermott EM, Curtis AM, Goel G, et al. Pyruvate kinase M2 regulates Hif-1α activity and IL-1β induction and is a critical determinant of the warburg effect in LPS-activated macrophages[J]. Cell Metabolism, 2015, 21(1):65-80. doi: 10.1016/j.cmet.2014.12.005 URL
[13]	施青青, 张振韬, 李鹏程, 等. BMP4诱导鸡胚胎干细胞向雄性生殖细胞分化的研究[J]. 畜牧兽医学报, 2013, 44(11):1749-1757.
	Shi QQ, Zhang ZT, Li PC, et al. Study on differentiation of chicken embryonic stem cells to male germ cells by BMP4[J]. Chinese Journal of Animal and Veterinary Sciences, 2013, 44(11), 1749-1757.
[14]	于丹, 王莹, 高原, 等. Akt/GSK-3β/Snail通路在TGF-β_1诱导A549/DDP细胞上皮间质转化中的作用[J]. 中国病理生理杂志, 2018(6):1124-1128.
	Yu D, Wang Y, Gao Y, et al. Effect of Akt/GSK-3β/Snail signaling pathway on EMT in A549/DDP cells mediated by TGF-β1[J]. Chinese Journal of Pathophysiology, 2018(6):1124-1128.
[15]	杨海燕, 孙敏, 田智泉, 等. 鸡X期胚盘细胞分散培养与整胚培养比较初探[J]. 中国家禽, 2010, 32(7):14-17.
	Yang HQ, Sui M, Tian ZQ, et al. Preliminary comparison of dispersed culture and whole embryo culture of chicken blastodermal cells on X period[J]. China Poultry, 2010, 32(7):14-17.
[16]	靳锴, 汪怡临, 左其生, 等. 睾丸注射Mx-NA双基因制备抗病转基因鸡的研究[J]. 中国家禽, 2014, 36(14):8-12.
	Jin K, Wang YL, Zuo QS, et al. Generation of disease-resistant transgenic chicken by testis-mediated injection method of combined Mx-NA gene[J]. China Poultry, 2014. 36(14):8-12.
[17]	Yeung SJ, Pan J, Lee MH. Roles of p53, MYC, and HIF-1 in regulating glycolysis:the seventh hallmark of cancer[J]. Cellular and Molecular Life Sciences, 2008, 65:3981-3999. doi: 10.1007/s00018-008-8224-x pmid: 18766298
[18]	Hsu PP, Sabatini DM. Cancer cell metabolism:Warburg and beyond[J]. Cell, 2008, 134:703-707. doi: 10.1016/j.cell.2008.08.021 URL
[19]	Graziano F, Ruzzo A, Giacomini E, Ricciardi T, et al. Glycolysis gene expression analysis and selective metabolic advantage in the clinical progression of colorectal cancer[J]. Pharmacogenomics, 2017, 17(3):258-264.
[20]	Kakinuma Y, Miyauchi T, Suzuki T, et al. Enhancement of glycolysis in cardiomyocytes elevates endothelin-1 expression through the transcriptional factor hypoxia-inducible factor-1 alpha[J]. Clinical Science, 2002, 103(Suppl 48):210S-214S. doi: 10.1042/CS103S210S URL
[21]	Zhou W, Choi M, Margineantu D, et al. HIF1α induced switch from bivalent to exclusively glycolytic metabolism during ESC-to-EpiSC/hESC transition[J]. EMBO Journal, 2014, 31(9):2103-2116. doi: 10.1038/emboj.2012.71 URL
[22]	Taper HS, de Gerlache J, Lans M, et al. Non-toxic potentiation of cancer chemotherapy by combined C and K3 vitamin pre-treatment[J]. International Journal of Cancer, 1987, 40:575-579. pmid: 3666992
[23]	Taper HS, Keyeux A, Roberfroid M. Potentiation of radiotherapy by nontoxic pretreatment with combined vitamins C and K3 in mice bearing solid transplantable tumor[J]. Anticancer Research, 1996, 16:499-503.
[24]	Taper HS, Jamison JM, Gilloteaux J, et al. Inhibition of the development of metastases by dietary vitamin C:K3 combination[J]. Life Science, 2004, 75:955-967.
[25]	De Loecker W, Janssens J, Bonte J, et al. Effects of sodium ascorbate(vitamin C)and 2-methyl-1, 4-naphtho-quinone(vitamin K3)treatment on human tumor cell growth in vitro, II:synergism with combined chemotherapy action[J]. Anticancer Research, 1993, 13:103-106.
[26]	Sakagami H, Satoh K, Hakeda Y, et al. Apopto-sis-inducing activity of vitamin C and vitamin K[J]. Cellular and Molecular Biology, 2000, 46:129-143. pmid: 10726979
[27]	Jamison JM, Gilloteaux J, Taper HS, et al. Evaluation of the in vitro and in vivo antitumor activities of vitamin C and K-3 combinations against human prostate cancer[J]. Journal of Nutrition, 2001, 131:158S-160S. doi: 10.1093/jn/131.1.158S URL
[28]	Buc Calderon P, Cadrobbi J, Marques C, et al. Potential therapeutic application of the association of vitamins C and K3 in cancer treatment[J]. Current Medicinal Chemistry, 2002, 9:2271-2285. doi: 10.2174/0929867023368674 URL
[29]	Verrax J, Stockis J, Tison A, et al. Oxidative stress by ascorbate/menadione association kills K562 human chronic myelogenous leukaemia cells and inhibits its tumour growth in nude mice[J]. Biochem Pharmacol, 2006, 72:671-680. doi: 10.1016/j.bcp.2006.05.025 URL
[30]	Verrax J, Vanbever S, Stockis J, et al. Role of glycolysis inhibition and poly(-ADP-ribose)polymerase activation in necrotic-like cell death caused by ascorbate/menadione-induced oxidative stress in K562 human chronic myelogenous leukemia cells[J]. International Journal of Cancer, 2007, 120:1192-1197. doi: 10.1002/(ISSN)1097-0215 URL
[31]	Kim YH, Heo JS, Han HJ. High glucose increase cell cycle regulatory proteins level of mouse embryonic stem cells via PI3-K/Akt and MAPKs signal pathways[J]. Journal of Cellular Physiology, 2006, 209:94-102. doi: 10.1002/(ISSN)1097-4652 URL
[32]	Crespo FL, Sobrado VR, Gomez L, et al. Mitochondrial reactive oxygen species mediate cardiomyocyte formation from embryonic stem cells in high glucose[J]. Stem Cells, 2010, 28:1132-1142.
[33]	Khoo MLM, McQuade LR, Smith MSR, et al. Growth and differentiation of embryoid bodies derived from human embryonic stem cells:Effect of glucose and basic fibroblast growth factor[J]. Biology of Reproduction, 2005, 73:1147-1156. doi: 10.1095/biolreprod.104.036673 URL
[34]	Chae HD, Lee MR, Broxmeyer HE. 5-Aminoimidazole-4-carboxyamide ribonucleoside induces G₁/S arrest and Nanog downregulation via p53 and enhances erythroid differentiation[J]. Stem Cells, 2012, 30(2):140-149. doi: 10.1002/stem.778 URL
[35]	Hayashi Y, Otsuka K, Ebina M, et al. Distinct requirements for energy metabolism in mouse primordial germ cells and their reprogramming to embryonic germ cells[J]. Proceedings of the National Academy of ences of the United States of America, 2017, 114(31):8289.