Article

Expression and knockdown analysis of glucose phosphate isomerase in chicken primordial germ cells.

WCU Biomodulation Major, Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, Korea.
Biology of Reproduction (Impact Factor: 4.03). 06/2012; 87(3):57. DOI: 10.1095/biolreprod.112.101345
Source: PubMed

ABSTRACT Glucose is an important monosaccharide required to generate energy in all cells. After entry into cells, glucose is phosphorylated to glucose-6-phosphate and then transformed into glycogen or metabolized to produce energy. Glucose phosphate isomerase (GPI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. Without GPI activity or fructose-6-phosphate, many steps of glucose metabolism would not occur. The requirement for GPI activity for normal functioning of primordial germ cells (PGCs) needs to be identified. In this study, we first examined the expression of chicken GPI during early embryonic development and germ cell development. GPI expression was strongly and ubiquitously detected in chicken early embryos and embryonic tissues at Embryonic Day 6.5 (E6.5). Continuous GPI expression was detected in PGCs and germ cells of both sexes during gonadal development. Specifically, GPI expression was stronger in male germ cells than in female germ cells during embryonic development and the majority of post-hatching development. Then, we used siRNA-1499 to knock down GPI expression in PGCs. siRNA-1499 caused an 85% knockdown in GPI, and PGC proliferation was also affected 48 h after transfection. We further examined the knockdown effects on 28 genes related to the glycolysis/gluconeogenesis pathway and the endogenous glucose level in chicken PGCs. Among genes related to glycolysis/gluconeogenesis, 20 genes showed approximately 3-fold lower expression, 4 showed approximately 10-fold lower, and 2 showed approximately 100-fold lower expression in knockdown PGCs. The endogenous glucose level was significantly reduced in knockdown PGCs. We conclude that the GPI gene is crucial for maintaining glycolysis and supplying energy to developing PGCs.

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    ABSTRACT: Glucose phosphate isomerase (GPI) involves in the reversible isomerization of glucose 6-phosphate to fructose 6-phosphate in glucose pathways. Since glucose metabolism is crucial for the proliferation and differentiation of embryonic stem and germ cells, reducing GPI expression may affect the characteristic features of these cells. MicroRNAs (miRNAs) have been shown to regulate genes. In this study, we investigated the regulation of chicken GPI by its predicted miRNAs. We determined the expression patterns of seven GPI 3'-untranslated region (GPI 3'UTR) targeting miRNAs, including the gga-miR-302, gga-miR-106, gga-miR-17-5p and gga-miR-20 clusters in chicken primordial germ cells (PGCs) compared with GPI mRNA. Among the miRNAs, gga-miR-302b, gga-miR-302d and gga-miR-17-5p were expressed lower than the expression of GPI. Remaining four miRNAs were expressed higher than the expression of GPI. Then, we cotransfected four candidate miRNAs gga-miR-302b, gga-miR-106, gga-miR-17-5p and gga-miR-20a, respectively with GPI 3'UTR into 293FT cells by dual fluorescence reporter assay. Overexpression of gga-miR-302b and gga-miR-17-5p in 293FT cells significantly downregulated GPI expression, whereas the other two miRNAs had no effect. Then, knockdown and overexpression of these four candidate miRNAs were performed by RNA interference assay to regulate GPI in PGCs. In the RNA interference assay, the expression of GPI was greatly regulated by gga-miR-302b and gga-miR-17-5p. Finally, we examined the effects of GPI regulation on PGCs proliferation and migration. Our results suggested that the regulation of GPI by gga-miR-302b and gga-miR-17-5p affects PGCs proliferation. However, regulation of GPI using these two miRNAs does not affect the migration of PGCs into embryonic gonads.
    Biology of Reproduction 07/2013; · 4.03 Impact Factor

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