Analysis of microRNA expression profiles during the differentiation of chicken embryonic stem cells into male germ cells.

The differentiation of embryonic stem cells (ESCs) into germ cells in vitro could have very promising applications for infertility treatment and could provide an excellent model for uncovering the molecular mechanisms of germline generation. This study aimed to investigate the differentially expressed miRNAs (DEMs) during the differentiation of chicken ESCs (cESCs) into male germ cells and to establish a profile of the DEMs. Cells before and after induction were subjected to miRNA sequencing (miRNA-seq). A total of 113 DEMs were obtained, including 61 upregulated and 52 downregulated DEMs. GO and KEGG enrichment analyses showed that the target genes were enriched mainly in the MAPK signaling pathway, HTLV infection signaling pathway, cell adhesion molecule (CAM)-related pathways, viral myocarditis, Wnt signaling pathway, ABC transporters, TGF-ß signaling pathways, Notch signaling pathways and insulin signaling pathway. The target genes of the miRNAs were related to cell binding, cell parts and biological regulatory processes. Six DEMs, let-7k-5p, miR-132c-5p, miR-193a-5p, miR-202-5p, miR-383-5p and miR-6553-3p, were assessed by qRT-PCR, and the results were consistent with the results of miRNA-seq. Based on qRT-PCR and western blot verification, miR-383-5p and its putative target gene STRN3 were selected to construct an STRN3 3'-UTR dual-luciferase gene reporter vector and its mutant vector. The double luciferase reporter activity of the cotransfected STRN3-WT + miR-383-5p mimics group was significantly lower (by approximately 46%) than that of the other five groups (p < 0.01). There was no significant difference in luciferase activity among the other 5 groups. This study establishes a DEM profile during the process of cESC differentiation into male germ cells; illustrates the mechanisms by which miRNAs regulate target genes; provides a theoretical basis for further research on the mechanisms of the formation and regulation of male germ cells; and provides an important strategy for gene editing, animal genetic resource protection and transgenic animal production.

Wnt signaling pathway regulates differentiation of chicken embryonic stem cells into spermatogonial stem cells via Wnt5a.

In this study, we investigated the mechanism of signaling pathway-mediated differentiation of embryonic stem cells (ESCs) into spermatogonial stem cells (SSCs) in chicken. The Wnt signaling pathway was identified based on previous RNA Sequencing results and was proven a crucial signaling pathway that participates in the differentiation of ESCs into SSCs. In retinoic acid (RA) induction experiments in vitro, we found that Wnt signaling expression was inhibited by Wnt5a-shRNA, resulting in decreased expression of corresponding marker genes in SSCs, C-kit, Cvh, integrin α6 and integrin ß1, but it was significantly promoted by RA treatment. Immunofluorescence assay showed that percentage of C-kit, Cvh, and integrin α6 and integrin ß1-positive cells in RA treatment group and Wnt5a overexpression group was significantly higher than that in Wnt5a signaling interference group. Results of fluorescence-activated cell sorting analysis (FACS) also showed that proportion of germ-like cells was reduced by 14.3% (from 18.3% to 4.0%) at day 4 and 15.4% (from 18.6% to 3.2%) at day 12 after transfection, respectively. In experiments in vivo, shRNA-Wnt5a was stably expressed in fertilized chicken embryos and significantly reduced germ cell formation by 11.3% (from 21.7% to 10.4%) and 3.7% (6.4% from 10.1%). Results of quantitative PCR (qRT-PCR) and western blot assays showed that the expression of some specific germ cell marker genes, integrin α6 and integrin ß1, was significantly suppressed following Wnt5a signaling interference in vivo. Taken together, our study suggests that Wnt signaling pathway could regulate positively the differentiation of chicken ESCs into SSCs through Wnt5a.

Advances in Isolation and Culture of Chicken Embryonic Stem Cells In Vitro.

Chicken embryonic stem cells (cESCs) isolated from the egg at the stage X hold great promise for cell therapy, tissue engineering, pharmaceutical, and biotechnological applications. They are considered to be pluripotent cells with the capacity to self-renewal and differentiate into specialized cells. However, long-term maintenance of cESCs cannot be realized now, which impedes the establishment of cESC line and limits their applications. Therefore, the separation locations, isolation methods, and culture conditions especially the supplements and action mechanisms of cytokines, including leukemia inhibitory factor, fibroblast growth factor, transforming growth factor beta, bone morphogenic protein, and activin for cESCs in vitro, have been reviewed here. These defined strategies will contribute to identify the key mechanism on the self-renewal of cESCs, facilitate to optimize system that supports the derivation and longtime maintenance of cESCs, establish the cESC line, and develop the biobank of genetic resources in chicken.

JAK-STAT signaling regulation of chicken embryonic stem cell differentiation into male germ cells.

The Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling is crucial in chicken germ stem cell differentiation, but its role in the regulation of germ cell differentiation is unknown. To address this, cucurbitacin I or interleukin 6 was used to inhibit or activate JAK-STAT signaling during embryonic stem cells (ESCs) differentiation. The expression of downstream JAK-STAT signaling molecules was assessed by Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR). PAS, and immunohistochemical staining of frozen sections was used to determine the appearance of primordial germ cells (PGCs) and, later, spermatogonial stem cells (SSCs) during gonadal development. Inhibition of the JAK-STAT signaling resulted in decreased expression of JAK2 and STAT3 as well as of PGCs markers; moreover, the proportion of CVH and C-KIT positive cells as well as the yield of PGCs were remarkably decreased, and the gonad was smaller than that of control samples. Conversely, activation of JAK-STAT resulted in increased expression of JAK2 and STAT3 as well as that of PGC marker CVH. In addition, the proportion of CVH and C-KIT-positive cells as well as the PGC yield was increased, and the gonad was significantly larger than that from control samples. Collectively, our results suggested that JAK-STAT effectively promoted the formation of PGCs in the genital ridge during early embryogenesis in vivo and played a positive role in the regulation of ESC to SSC differentiation in vitro, with JAK2 and STAT3 functioning as pivotal factors for intracellular signal transduction.

Chicken embryonic stem cells and primordial germ cells display different heterochromatic histone marks than their mammalian counterparts.

BACKGROUND: Chromatin epigenetics participate in control of gene expression during metazoan development. DNA methylation and post-translational modifications (PTMs) of histones have been extensively characterised in cell types present in, or derived from, mouse embryos. In embryonic stem cells (ESCs) derived from blastocysts, factors involved in deposition of epigenetic marks regulate properties related to self-renewal and pluripotency. In the germ lineage, changes in histone PTMs and DNA demethylation occur during formation of the primordial germ cells (PGCs) to reset the epigenome of the future gametes. Trimethylation of histone H3 on lysine 27 (H3K27me3) by Polycomb group proteins is involved in several epigenome-remodelling steps, but it remains unclear whether these epigenetic features are conserved in non-mammalian vertebrates. To investigate this question, we compared the abundance and nuclear distribution of the main histone PTMs, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in chicken ESCs, PGCs and blastodermal cells (BCs) with differentiated chicken ESCs and embryonic fibroblasts. In addition, we analysed the expression of chromatin modifier genes to better understand the establishment and dynamics of chromatin epigenetic profiles. RESULTS: The nuclear distributions of most PTMs and 5hmC in chicken stem cells were similar to what has been described for mammalian cells. However, unlike mouse pericentric heterochromatin (PCH), chicken ESC PCH contained high levels of trimethylated histone H3 on lysine 27 (H3K27me3). In differentiated chicken cells, PCH was less enriched in H3K27me3 relative to chromatin overall. In PGCs, the H3K27me3 global level was greatly reduced, whereas the H3K9me3 level was elevated. Most chromatin modifier genes known in mammals were expressed in chicken ESCs, PGCs and BCs. Genes presumably involved in de novo DNA methylation were very highly expressed. DNMT3B and HELLS/SMARCA6 were highly expressed in chicken ESCs, PGCs and BCs compared to differentiated chicken ESCs and embryonic fibroblasts, and DNMT3A was strongly expressed in ESCs, differentiated ESCs and BCs. CONCLUSIONS: Chicken ESCs and PGCs differ from their mammalian counterparts with respect to H3K27 methylation. High enrichment of H3K27me3 at PCH is specific to pluripotent cells in chicken. Our results demonstrate that the dynamics in chromatin constitution described during mouse development is not universal to all vertebrate species.