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Study on the regulatory mechanism of the lipid metabolism pathways during chicken male germ cell differentiation based on RNA-seq.

Zuo Q, Li D, Zhang L, Elsayed AK, Lian C, Shi Q, Zhang Z, Zhu R, Wang Y, Jin K, Zhang Y, Li B - PLoS ONE (2015)

Bottom Line: From the results of RNA-seq and the database analyses, we concluded that there are 328 genes in 27 lipid metabolic pathways continuously involved in lipid metabolism during the differentiation of ESC into SSC in vivo, including retinol metabolism.Expression of CYP26b1, in contrast, decreased throughout development.We conclude that the retinol metabolism pathway plays an important role in the process of chicken male germ cell differentiation.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, China.

ABSTRACT
Here, we explore the regulatory mechanism of lipid metabolic signaling pathways and related genes during differentiation of male germ cells in chickens, with the hope that better understanding of these pathways may improve in vitro induction. Fluorescence-activated cell sorting was used to obtain highly purified cultures of embryonic stem cells (ESCs), primitive germ cells (PGCs), and spermatogonial stem cells (SSCs). The total RNA was then extracted from each type of cell. High-throughput analysis methods (RNA-seq) were used to sequence the transcriptome of these cells. Gene Ontology (GO) analysis and the KEGG database were used to identify lipid metabolism pathways and related genes. Retinoic acid (RA), the end-product of the retinol metabolism pathway, induced in vitro differentiation of ESC into male germ cells. Quantitative real-time PCR (qRT-PCR) was used to detect changes in the expression of the genes involved in the retinol metabolic pathways. From the results of RNA-seq and the database analyses, we concluded that there are 328 genes in 27 lipid metabolic pathways continuously involved in lipid metabolism during the differentiation of ESC into SSC in vivo, including retinol metabolism. Alcohol dehydrogenase 5 (ADH5) and aldehyde dehydrogenase 1 family member A1 (ALDH1A1) are involved in RA synthesis in the cell. ADH5 was specifically expressed in PGC in our experiments and aldehyde dehydrogenase 1 family member A1 (ALDH1A1) persistently increased throughout development. CYP26b1, a member of the cytochrome P450 superfamily, is involved in the degradation of RA. Expression of CYP26b1, in contrast, decreased throughout development. Exogenous RA in the culture medium induced differentiation of ESC to SSC-like cells. The expression patterns of ADH5, ALDH1A1, and CYP26b1 were consistent with RNA-seq results. We conclude that the retinol metabolism pathway plays an important role in the process of chicken male germ cell differentiation.

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Morphological changes in chicken ESCs during in vitro induction by exogenous RA.D2: Note the few, small embryoid bodies. D4, D6. Embyroid bodies are more numerous and larger than in D2. D8. The embryoid bodies are dissociated. D10. SSC-like cells are present. Magnification: 400×.
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pone.0109469.g007: Morphological changes in chicken ESCs during in vitro induction by exogenous RA.D2: Note the few, small embryoid bodies. D4, D6. Embyroid bodies are more numerous and larger than in D2. D8. The embryoid bodies are dissociated. D10. SSC-like cells are present. Magnification: 400×.

Mentions: Retinol and its metabolites may regulate the chicken male germ cell differentiation process. To test this role, we applied RA (the retinol pathway activation agent) to ESCs and followed their subsequent development. ESCs were cultured on DMEM rich in glucose and containing 10% FBS and 10 μM RA. The morphology of the ESCs gradually changed, apparently differentiating into SSCs (Fig. 7). The SSC-like cells were labeled with the antibody specific for SSC, confirming that differentiation had occurred. Furthermore, we detected expression of the three key genes of the retinol metabolism pathways through qRT-PCR (Fig. 8).


Study on the regulatory mechanism of the lipid metabolism pathways during chicken male germ cell differentiation based on RNA-seq.

Zuo Q, Li D, Zhang L, Elsayed AK, Lian C, Shi Q, Zhang Z, Zhu R, Wang Y, Jin K, Zhang Y, Li B - PLoS ONE (2015)

Morphological changes in chicken ESCs during in vitro induction by exogenous RA.D2: Note the few, small embryoid bodies. D4, D6. Embyroid bodies are more numerous and larger than in D2. D8. The embryoid bodies are dissociated. D10. SSC-like cells are present. Magnification: 400×.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4320113&req=5

pone.0109469.g007: Morphological changes in chicken ESCs during in vitro induction by exogenous RA.D2: Note the few, small embryoid bodies. D4, D6. Embyroid bodies are more numerous and larger than in D2. D8. The embryoid bodies are dissociated. D10. SSC-like cells are present. Magnification: 400×.
Mentions: Retinol and its metabolites may regulate the chicken male germ cell differentiation process. To test this role, we applied RA (the retinol pathway activation agent) to ESCs and followed their subsequent development. ESCs were cultured on DMEM rich in glucose and containing 10% FBS and 10 μM RA. The morphology of the ESCs gradually changed, apparently differentiating into SSCs (Fig. 7). The SSC-like cells were labeled with the antibody specific for SSC, confirming that differentiation had occurred. Furthermore, we detected expression of the three key genes of the retinol metabolism pathways through qRT-PCR (Fig. 8).

Bottom Line: From the results of RNA-seq and the database analyses, we concluded that there are 328 genes in 27 lipid metabolic pathways continuously involved in lipid metabolism during the differentiation of ESC into SSC in vivo, including retinol metabolism.Expression of CYP26b1, in contrast, decreased throughout development.We conclude that the retinol metabolism pathway plays an important role in the process of chicken male germ cell differentiation.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, China.

ABSTRACT
Here, we explore the regulatory mechanism of lipid metabolic signaling pathways and related genes during differentiation of male germ cells in chickens, with the hope that better understanding of these pathways may improve in vitro induction. Fluorescence-activated cell sorting was used to obtain highly purified cultures of embryonic stem cells (ESCs), primitive germ cells (PGCs), and spermatogonial stem cells (SSCs). The total RNA was then extracted from each type of cell. High-throughput analysis methods (RNA-seq) were used to sequence the transcriptome of these cells. Gene Ontology (GO) analysis and the KEGG database were used to identify lipid metabolism pathways and related genes. Retinoic acid (RA), the end-product of the retinol metabolism pathway, induced in vitro differentiation of ESC into male germ cells. Quantitative real-time PCR (qRT-PCR) was used to detect changes in the expression of the genes involved in the retinol metabolic pathways. From the results of RNA-seq and the database analyses, we concluded that there are 328 genes in 27 lipid metabolic pathways continuously involved in lipid metabolism during the differentiation of ESC into SSC in vivo, including retinol metabolism. Alcohol dehydrogenase 5 (ADH5) and aldehyde dehydrogenase 1 family member A1 (ALDH1A1) are involved in RA synthesis in the cell. ADH5 was specifically expressed in PGC in our experiments and aldehyde dehydrogenase 1 family member A1 (ALDH1A1) persistently increased throughout development. CYP26b1, a member of the cytochrome P450 superfamily, is involved in the degradation of RA. Expression of CYP26b1, in contrast, decreased throughout development. Exogenous RA in the culture medium induced differentiation of ESC to SSC-like cells. The expression patterns of ADH5, ALDH1A1, and CYP26b1 were consistent with RNA-seq results. We conclude that the retinol metabolism pathway plays an important role in the process of chicken male germ cell differentiation.

Show MeSH
Related in: MedlinePlus