Study on the Regulatory Mechanism of the Lipid Metabolism Pathways during Chicken Male Germ Cell Differentiation Based on RNA-Seq

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.

Dissecting chicken germ cell dynamics by combining a germ cell tracing transgenic chicken model with single-cell RNA sequencing

“…Transcriptome analyses of chicken germ-cell developmental stages have been limited and fragmentary [31] , [88] , and these analyses have not been applied in vivo . The main reasons for these limitations are the lack of a technology to discriminate germ cells and the lack of sophisticated markers to isolate these cells.…”

MAPK8 regulates chicken male germ cell differentiation through JNK signaling pathway

“…Chen found that Hedgehog signaling through IHH signal transduction positively regulates differentiation from ESC to male germ cells; Zhang found JAK‐STAT signaling pathway plays a key role in the induction of differentiation of male germ cells in vitro by chicken ESCs in vitro. Zuo found that activation of retinol pathway plays an important role in the differentiation of ESC into SSCs by the addition of RA Regulation. Based on the preliminary microarray, we screened the JNK signaling pathway in the process of differentiation of chicken male germ cells .…”

Hsd3b2 associated in modulating steroid hormone synthesis pathway regulates the differentiation of chicken embryonic stem cells into spermatogonial stem cells

“…In our previous lab research, it was found that the lipid metabolism pathway could regulate the differentiation of male germ cells, and testosterone is an important regulatory factor in lipid metabolism regulation process. 13,14 To further determine the role of testosterone during the differentiation of ESCs to SSCs, we added 15 ng/mL testosterone in the RA induction system, and found that the treatment led to more embryoid bodies and SSCs-like ( Figure 1A). qRT-PCR results indicated that the expression of integrin β1, a marker gene of SSCs, was significantly up-regulated ( Figure 1C).…”