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Background/Objectives: Induced pluripotent stem cells (iPSCs) reprogrammed from somatic cells into cells with most of the ESC (embryonic stem cell) characteristics show promise toward solving ethical problems currently facing stem cell research and eventually yield clinical grade pluripotent stem cells for therapies and regenerative medicine. In recent years, an increasing body of research suggests that the chemical induction of pluripotency (CIP) method can yield iPSCs in vitro, yet its application in avian species remains unreported. Methods: Herein, we successfully obtained stably growing chicken embryonic fibroblasts (CEFs) using the tissue block adherence method and employed 12 small-molecule compounds to induce chicken iPSC formation. Results: The final optimized iPSC induction system was bFGF (10 ng/mL), CHIR99021 (3 μM), RepSox (5 μM), DZNep (0.05 μM), BrdU (10 μM), BMP4 (10 ng/mL), vitamin C (50 μg/mL), EPZ-5676 (5 μM), and VPA (0.1 mM). Optimization of the induction system revealed that the highest number of clones was induced with 8 × 104 cells per well and at 1.5 times the original concentration. Upon characterization, these clones exhibited iPSC characteristics, leading to the development of a stable compound combination for iPSC generation in chickens. Concurrently, employing a deletion strategy to investigate the functionality of small-molecule compounds during induction, we identified CHIR99021 and BrdU as critical factors for inducing chicken iPSC formation. Conclusions: In conclusion, this study provides a reference method for utilizing small-molecule combinations in avian species to reprogram cells and establish a network of cell fate determination mechanisms. [ABSTRACT FROM AUTHOR]

The determination of sex in mammals is established and controlled by various complex mechanisms. In contrast, sex control in poultry remains an unresolved issue. In this study, RNA-sequencing was conducted for male gonads and ovarian tissues in chicken embryos of up to 18.5 days to identify metabolic factors influencing male and female sex differentiation, as well as gonadal development. Our results reveal that PKM2, a critical glycolysis-related protein, plays a significant role in chicken sex differentiation via PPARG, a crucial hormone gene. We propose that our discoveries bolster the notion that glycolysis and oxidative phosphorylation function as antecedent contributors to sexual phenotypic development and preservation. [ABSTRACT FROM AUTHOR]

As an RNA binding protein (RBP), DDX5 is widely involved in the regulation of various biological activities. While recent studies have confirmed that DDX5 can act as a transcriptional cofactor that is involved in the formation of gametes, few studies have investigated whether DDX5 can be used as a transcription factor to regulate the formation of primordial germ cells (PGCs). In this study, we found that DDX5 was significantly up-regulated during chicken PGC formation. Under different PGC induction models, the overexpression of DDX5 not only up-regulates PGC markers but also significantly improves the formation efficiency of primordial germ cell-like cells (PGCLC). Conversely, the inhibition of DDX5 expression can significantly inhibit both the expression of PGC markers and PGCLC formation efficiency. The effect of DDX5 on PGC formation in vivo was consistent with that seen in vitro. Interestingly, DDX5 not only participates in the formation of PGCs but also positively regulates their migration and proliferation. In the process of studying the mechanism by which DDX5 regulates PGC formation, we found that DDX5 acts as a transcription factor to bind to the promoter region of BMP4—a key gene for PGC formation—and activates the expression of BMP4. In summary, we confirm that DDX5 can act as a positive transcription factor to regulate the formation of PGCs in chickens. The obtained results not only enhance our understanding of the way in which DDX5 regulates the development of germ cells but also provide a new target for systematically optimizing the culture and induction system of PGCs in chickens in vitro. [ABSTRACT FROM AUTHOR]

The identification of accurate gene insertion sites on chicken sex chromosomes is crucial for advancing sex control breeding materials. In this study, the intergenic region NC_006127.4 on the chicken Z chromosome and the non-repetitive sequence EE0.6 on the W chromosome were selected as potential gene insertion sites. Gene knockout vectors targeting these sites were constructed and transfected into DF-1 cells. T7E1 enzyme cleavage and luciferase reporter enzyme analyses revealed knockout efficiencies of 80.00% (16/20), 75.00% (15/20), and 75.00% (15/20) for the three sgRNAs targeting the EE0.6 site. For the three sgRNAs targeting the NC_006127.4 site, knockout efficiencies were 70.00% (14/20), 60.00% (12/20), and 45.00% (9/20). Gel electrophoresis and high-throughput sequencing were performed to detect potential off-target effects, showing no significant off-target effects for the knockout vectors at the two sites. EdU and CCK-8 proliferation assays revealed no significant difference in cell proliferation activity between the knockout and control groups. These results demonstrate that the EE0.6 and NC_006127.4 sites can serve as gene insertion sites on chicken sex chromosomes for gene editing without affecting normal cell proliferation. [ABSTRACT FROM AUTHOR]

Simple Summary: Embryonic stem cells have unique pluripotency and the potential to differentiate into any cell type in the body. This characteristic gives them great potential for application in the field of animal husbandry. Scientists can better control the differentiation process of embryonic stem cells by studying the maintenance mechanism of their pluripotency. In animal breeding, the use of embryonic stem cells can achieve the protection of animal species resources and genetic improvement of important traits, helping to cultivate animal breeds with faster birth growth and better meat quality. This not only improves agricultural production efficiency but also reduces the dependence on animals, which is more in line with ethical requirements and human production needs. Embryonic stem cells (ESCs) are remarkably undifferentiated cells that originate from the inner cell mass of the blastocyst. They possess the ability to self-renew and differentiate into multiple cell types, making them invaluable in diverse applications such as disease modeling and the creation of transgenic animals. In recent years, as agricultural practices have evolved from traditional to biological breeding, it has become clear that pluripotent stem cells (PSCs), either ESCs or induced pluripotent stem cells (iPSCs), are optimal for continually screening suitable cellular materials. However, the technologies for long-term in vitro culture or establishment of cell lines for PSCs in livestock are still immature, and research progress is uneven, which poses challenges for the application of PSCs in various fields. The establishment of a robust in vitro system for these cells is critically dependent on understanding their pluripotency maintenance mechanisms. It is believed that the combined effects of pluripotent transcription factors, pivotal signaling pathways, and epigenetic regulation contribute to maintaining their pluripotent state, forming a comprehensive regulatory network. This article will delve into the primary mechanisms underlying the maintenance of pluripotency in PSCs and elaborate on the applications of PSCs in the field of livestock. [ABSTRACT FROM AUTHOR]

Simple Summary: In this study, we aimed to improve the culture system for chicken embryonic-derived pluripotent stem cells (PSCs). These cells have immense potential in various fields, such as growth and development studies, vaccine production, and preserving genetic resources. However, establishing a stable and efficient culture system for chicken PSCs remains a challenge. We investigated the effects of different signaling pathways and feeder layers on the derivation and maintenance of chicken PSCs. Our results show that using STO cells as feeder layers, along with specific signaling pathway inhibitors, allows for the efficient derivation of chicken PSC-like cells. These cells exhibit characteristics of pluripotency and can differentiate into various cell types. This research enhances our understanding of chicken PSC culture conditions and lays the groundwork for their biomedical and biotechnological applications. The utilization of chicken embryonic-derived pluripotent stem cell (PSC) lines is crucial in various fields, including growth and development, vaccine and protein production, and germplasm resource protection. However, the research foundation for chicken PSCs is relatively weak, and there are still challenges in establishing a stable and efficient PSC culture system. Therefore, this study aims to investigate the effects of the FGF2/ERK and WNT/β-catenin signaling pathways, as well as different feeder layers, on the derivation and maintenance of chicken embryonic-derived PSCs. The results of this study demonstrate that the use of STO cells as feeder layers, along with the addition of FGF2, IWR-1, and XAV-939 (FIX), allows for the efficient derivation of chicken PSC-like cells. Under the FIX culture conditions, chicken PSCs express key pluripotency genes, such as POUV, SOX2, and NANOG, as well as specific proteins SSEA-1, C-KIT, and SOX2, indicating their pluripotent nature. Additionally, the embryoid body experiment confirms that these PSC-like cells can differentiate into cells of three germ layers in vitro, highlighting their potential for multilineage differentiation. Furthermore, this study reveals that chicken Eyal–Giladi and Kochav stage X blastodermal cells express genes related to the primed state of PSCs, and the FIX culture system established in this research maintains the expression of these genes in vitro. These findings contribute significantly to the understanding and optimization of chicken PSC culture conditions and provide a foundation for further exploration of the biomedical research and biotechnological applications of chicken PSCs. [ABSTRACT FROM AUTHOR]

Simple Summary: In this study, we screened key signaling pathways regulating the formation of chicken PGCs using transcriptome sequencing and established a two-step induction model of PGCs in vitro to improve the optimization of their induction efficiency. Our results provide cellular materials for realizing the specific application of PGCs. In recent years, inducing pluripotent stem cells to differentiate into functional primordial germ cells (PGCs) in vitro has become an important method of obtaining a large number of PGCs. However, the instability and low induction efficiency of the in vitro PGC induction system restrict the application of PGCs in transgenic animal production, germplasm resource conservation and other fields. In this study, we successfully established a two-step induction model of chicken PGCs in vitro, which significantly improved the formation efficiency of PGC-like cells (PGCLCs). To further improve the PGC formation efficiency in vitro, 5025 differentially expressed genes (DEGs) were obtained between embryonic stem cells (ESCs) and PGCs through RNA-seq. GO and KEGG enrichment analysis revealed that signaling pathways such as BMP4, Wnt and Notch were significantly activated during PGC formation, similar to other species. In addition, we noted that cAMP was activated during PGC formation, while MAPK was suppressed. Based on the results of our analysis, we found that the PGC formation efficiency was significantly improved after activating Wnt and inhibiting MAPK, and was lower than after activating cAMP. To sum up, in this study, we successfully established a two-step induction model of chicken PGCs in vitro with high PGC formation efficiency, which lays a theoretical foundation for further demonstrating the regulatory mechanism of PGCs and realizing their specific applications. [ABSTRACT FROM AUTHOR]

To investigate the regulatory mechanism of the competing endogenous RNAs (ceRNAs) on the egg performance of Gaoyou ducks, full transcriptome sequencing was performed to analyze the ovarian tissues in Gaoyou ducks. The ducks were categorized into high- and low-yield groups based on the individual in-cage egg production records and the hematoxylin–eosin (HE) staining results. The differentially expressed genes (DEGs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) were further processed by GO (gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses. In total, 72 DEmRNAs; 23 DElncRNAs; 4 DEcircRNAs; and 5 signaling pathways, including the ovarian steroidogenesis, PI3K-Akt, hedgehog, tryptophan metabolism, and oocyte meiosis signaling pathways, were significantly enriched. These results suggest that they could be associated with the Gaoyou duck's ovarian function and affect the total egg production or double-yolked egg production. Furthermore, a coregulation network based on the related candidate ceRNAs across the high- and low-yield egg production groups was constructed. Our findings provide new insights into the mechanisms underlying the molecular regulation of related circRNA/lncRNA–miRNA–mRNA in the egg production and double-yolked egg traits of Gaoyou ducks. [ABSTRACT FROM AUTHOR]

Primordial germ cells (PGCs) are essential for the genetic modification, resource conservation, and recovery of endangered breeds in chickens and need to remain viable and proliferative in vitro. Therefore, there is an urgent need to elucidate the functions of the influencing factors and their regulatory mechanisms. In this study, PGCs collected from Rugao yellow chicken embryonic eggs at Day 5.5 were cultured in media containing 0, 5, 10, 20, 50, and 100 μg/mL insulin. The results showed that insulin regulates cell proliferation in PGCs in a dose-dependent way, with an optimal dose of 10 μg/mL. Insulin mediates the mRNA expression of cell cycle-, apoptosis-, and ferroptosis-related genes. Insulin at 50 μg/mL and 100 μg/mL slowed down the proliferation with elevated ion content and GSH/oxidized glutathione (GSSG) in PGCs compared to 10 μg/mL. In addition, insulin activates the PI3K/AKT/mTOR pathway dose dependently. Collectively, this study demonstrates that insulin reduces apoptosis and ferroptosis and enhances cell proliferation in a dose-dependent manner via the PI3K-AKT-mTOR signaling pathway in PGCs, providing a new addition to the theory of the regulatory role of the growth and proliferation of PGC in vitro cultures. [ABSTRACT FROM AUTHOR]

The role of gga-miR-31 in chicken germ cell differentiation and spermatogenesis is of significant importance. The transcriptional properties of gga-miR-31 are crucial in establishing the foundation for the formation of chicken spermatogonia stem cells and spermatogenesis. In this study, a series of recombinant vectors including varying lengths of the gga-miR-31 promoter were predicted and constructed. Through the utilization of the dual luciferase reporting system, the upstream −2180~0 bp region of gga-miR-31 was identified as its promoter region. Furthermore, it was predicted and confirmed that the activity of the gga-miR-31 promoter is increased by retinoic acid (RA). The binding of RA to the gga-miR-31 and Stra8 promoter regions was found to be competitive. Through the deletion of C-jun binding sites and the manipulation of C-jun expression levels, it was determined that C-jun inhibits the activity of the gga-miR-31 promoter. Furthermore, the combined treatment of C-jun and RA demonstrated that the positive regulatory effect of RA on the gga-miR-31 promoter is attenuated in the presence of high levels of C-jun. Overall, this study establishes a foundation for further investigation into the regulatory mechanisms of gga-miR-31 action, and provides a new avenue for inducing chicken embryonic stem cells (ESC) to differentiate into spermatogonial stem cells (SSC), and sperm formation. [ABSTRACT FROM AUTHOR]

Simple Summary: The present review illustrates the importance of chicken primordial germ cells to understanding early-stage development for better and more fertile species. By studying early-stage development, we can develop transgenic species to meet food and pharmaceutical demands. This review sheds light on the significance of chicken primordial germ cells and their uses in transgenesis. Chicken is one of the most widely consumed sources of protein globally. Primordial germ cells (PGCs) are the precursors for ova and sperm. One of the early embryogenesis events in most animals is the segregation of the somatic and germ lineages. PGC cultures occur in the germline, and PGCs are less studied in many species. It is relatively challenging to separate, cultivate, and genetically alter chicken without mutating the basic germline. The present study aims to gather previous research about chicken PGCs and provide a customized review of studies and developments in the field of PGCs, especially for avian species. Furthermore, we show that the propagation of chicken PGCs into embryonic germ cells that contribute to somatic tissues may be produced in vitro. Primordial germ cells offer an ideal system in developmental biology, as these cells play a vital role in the genetic modification and treatment of infertility. Cryopreservation helps to maintain genetic resources and sustainable production in the poultry industry. Keeping in mind the significance of cryopreservation for storage and gametogenesis, we discuss its role in the preservation of primordial germ cells. Transgenesis and genetic modifications in chicken lead to the development of various medicinal chicken varieties and aid in improving their production and quality for consumption purposes. Additionally, these characteristics open up new possibilities for modifying the chicken genome for agricultural and medical purposes. [ABSTRACT FROM AUTHOR]

Simple Summary: The formation of primordial germ cells is a knotty process involving both genetic and epigenetic factors. However, the exact mechanism is not clear. Here, we found a new lncRNA, which is highly expressed in the primordial germ cell. Histone H3K4 methylation could accelerate the formation of primordial germ cells by regulating this lncRNA. Our study provides a reference for exploring the formation mechanism of germ cells, breeding work, and the protection of endangered animals. Primordial germ cells are the ancestors of female and male cells. Current research has shown that long non-coding RNA (lncRNA) and Histone methylation are the pivotal epigenetic factors in the PGC formation. However, there are few studies on the regulatory mechanism of lncRNA in the formation of PGC. Here, we define the lncRNA highly expressed in chicken PGC, lncCPSET1 (chicken-PGC-specifically-expressed transcript 1) This study found that compared with the interference of lncCPSET1/histone methylase Mll2 alone, the PGC formation was severely inhibited with the interference of lncCPSET1 and histone methylase Mll2 jointly in vivo and in vitro. Studies on the transcription level of lncCPSET1 found that H3K4me2 and transcription factor Jun have a positive effect on the activation of lncCPSET1; while DNA hypomethylation inhibits the expression of lncCPSET1. In terms of mechanism, compared with DNA methylation, H3K4me2 dominates lncCPSET1 activation. H3K4me2 can be enriched in the lncCPSET1 promoter, change its chromosome conformation, recruit the transcription factor Jun, and activate the expression of lncCPSET1. Taken together, we confirmed the model that H3K4me2 rather than DNA hypomethylation mediates Jun to regulate lncCPSET1 transcription, which broadens the study of lncCPSET1 pre-transcriptional mechanism. [ABSTRACT FROM AUTHOR]

Simple Summary: Studies have shown that alternative splicing (AS) has been utilized in a wide variety of life processes. However, there are very few studies on AS during germ cell development. In this study, we preliminarily investigated the variation of variable shear events during the formation of chicken germ cells through the RNA-seq data analysis of embryonic stem cells (ESCs), gonad PGCs (gPGCs), and spermatogonia stem cells (SSCs), and the critical AS mode for several crucial stage-specific genes, which were identified during germ cell development. The results of this study lay a theoretical foundation for further analysis of the regulation mechanism of key genes involved in germ cell formation. Alternative splicing (AS) is a ubiquitous, co-transcriptional, and post-transcriptional regulation mechanism during certain developmental processes, such as germ cell differentiation. A thorough understanding of germ cell differentiation will help us to open new avenues for avian reproduction, stem cell biology, and advances in medicines for human consumption. Here, based on single-cell RNA-seq, we characterized genome-wide AS events in manifold chicken male germ cells: embryonic stem cells (ESCs), gonad primordial germ cells (gPGCs), and spermatogonia stem cells (SSCs). A total of 38,494 AS events from 15,338 genes were detected in ESCs, with a total of 48,955 events from 14,783 genes and 49,900 events from 15,089 genes observed in gPGCs and SSCs, respectively. Moreover, this distribution of AS events suggests the diverse splicing feature of ESCs, gPGCs, and SSCs. Finally, several crucial stage-specific genes, such as NANOG, POU5F3, LIN28B, BMP4, STRA8, and LHX9, were identified in AS events that were transmitted in ESCs, gPGCs, and SSCs. The gene expression results of the RNA-seq data were validated by qRT-PCR. In summary, we provided a comprehensive atlas of the genome-wide scale of the AS event landscape in male chicken germ-line cells and presented its distribution for the first time. This research may someday improve treatment options for men suffering from male infertility. [ABSTRACT FROM AUTHOR]

Simple Summary: Chicken embryonic fibroblasts (CEF) can be induced into iPSCs by Oct4, Sox2, Nanog and Lin28 (OSNL). However, the instability of this system leads to low induction efficiency, and the mechanism of reprogramming is still unclear. Therefore, it is vital to study the mechanism of reprogramming and optimize its induction system. In this study, we first note that glycolysis was activated during the reprogramming of CEF using the OSNL induction strategy, and that the further activation of endogenous pluripotent genes promoted the reprogramming process. An optimized OSNL reprogramming cocktail was created by introducing two small-molecule inhibitors (2i-SP), TGF-β inhibitor and MEK/ERK inhibitor, which we termed the "glycolysis activator", which improved the efficiency of iPSC formation. This study provides an enhanced theoretical basis and suggests further areas for avian cell and embryo engineering research. Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) in vitro. Previously, a lentivirus induction strategy of introducing Oct4, Sox2, Nanog and Lin28 (OSNL) into the iPSC process has been shown as a possible way to produce chicken iPSCs from chicken embryonic fibroblasts, but the induction efficiency of this method was found to be significantly limiting. In order to help resolve this efficiency obstacle, this study seeks to clarify the associated regulation mechanisms and optimizes the reprogramming strategy of chicken iPSCs. This study showed that glycolysis and the expression of glycolysis-related genes correlate with a more efficient reprogramming process. At the same time, the transcription factors Oct4, Sox2 and Nanog were found to activate the expression of glycolysis-related genes. In addition, we introduced two small-molecule inhibitors (2i-SP) as a "glycolysis activator" together with the OSNL cocktail, and found that this significantly improved the induction efficiency of the iPSC process. As such, the study identifies direct molecular connections between core pluripotency factors and glycolysis during the chicken iPSC induction process and, with its results, provides a theoretical basis and technical support for chicken somatic reprogramming. [ABSTRACT FROM AUTHOR]

Simple Summary: The potential of primordial germ cells (PGCs) for multidirectional differentiation, together with their unique regeneration ability, makes them one of the most promising seed cells in clinical medicine and tissue engineering research. However, not enough PGCs can be obtained to meet the demand, which limits their application. We defined a novel long noncoding RNA (lncRNA) mediated by epigenetics, which could activate the miR-6577-5p/Btrc pathway to promote the formation of PGCs. The technical system we have established is a useful tool to obtain sufficient PGCs for scientific research. Our study offers great theoretical and practical value in the production of transgenic animals or genomic imprinting in poultry. We believe that our study will help researchers in the fields of agricultural production, developmental biology, and cell biology. Although lncRNAs have been identified as playing critical roles in the development of germ cells, their potential involvement in the development of PGCs in chickens remains poorly understood. Differentially expressed lncRNAs (DELs) from previous RNA-seq of embryonic stem cells (ESCs), PGCs, and spermatogonial stem cells (SSCs) were analyzed by K-means clustering, from which a key candidate, lncRNA (lncRNA PGC regulator, LncPGCR) was obtained. We confirmed that LncPGCR plays a positive role in the development of PGCs by increasing the expression of the PGC marker gene (Cvh and C-kit), while downregulating the pluripotency-associated gene (Nanog) in vitro and in vivo. The activation and expression of LncPGCR are regulated by histone acetylation, and transcription factor TCF7L2. Mechanistically, a rescue assay was performed to further confirm that LncPGCR contributed to the development of PGCs by regulating the gga-miR-6577-5p/Btrc signaling pathway. Adsorption of gga-miR-6577-5p activated the WNT signaling cascade by relieving the gga-miR-6577-5p-dependent inhibition of Btrc expression. Taken together, our study discovered the growth-expedited role of LncPGCR in PGCs development, showing the potential LncPGCR/miR-6577-5p/Btrc pathway. The results and findings provide a novel insight into the development of PGCs. [ABSTRACT FROM AUTHOR]

Simple Summary: In this study, we explored the function and molecular mechanism of Lin28B in the formation of chicken primordial germ cells (PGCs) in detail. Our results indicate that Lin28B participates in the formation of PGCs through let-7a-3p, which set a theoretical foundation for improving the function and mechanism of the Lin28 family in the formation of PGCs. Lin28A and Lin28B are two homologues of the same family of RNA binding proteins (RBPs). The function and molecular mechanism of Lin28A in the formation of primordial germ cells (PGCs) are very clear, but the related research on Lin28B is rarely reported. Here, we found that the overexpression of Lin28B can promote the formation of PGC in vivo. Furthermore, the overexpression of Lin28B also resulted in the inhibition of totipotency gene expression and upregulated the PGCs marker genes, and a significant increase in the number of PGCs in genital ridge, as detected by Periodic Acid-Schiff(PAS) staining. However, the inhibited Lin28B expression showed completely opposite results, which were confirmed on the PGC induction model in vitro. Mechanistically, we found that the overexpression of Lin28B can inhibit the maturation of let-7a-3p, and the results of high-throughput sequencing indicated that let-7a-3p was a negative regulator of the formation process of PGCs. Therefore, we conclude that our results determine that Lin28B participates in the formation of PGCs through let-7a-3p, which set a theoretical foundation for improving the function and mechanism of Lin28 family in the formation of PGCs. [ABSTRACT FROM AUTHOR]

Simple Summary: The study of chicken embryonic stem cells (ESCs) and primordial germ cells (PGCs) showed the potential application of developmental biology and translational medicine. However, the difference between amphoteric ESCs and PGCs is still elusive, limiting the accuracy of correlative research. In this paper, chicken amphoteric ESCs and PGCs were isolated, separated, and sequenced to explore their dynamic transcriptomes. Our results provide a knowledge base of transcriptomes in chicken amphoteric ESCs and PGCs, which will help other researchers interested in studying relative biological processes. Chicken (Gallus gallus) pluripotent embryonic stem cells (ESCs) and primordial germ cells (PGCs) can be broadly applied in the research of developmental and embryonic biology, but the difference between amphoteric ESCs and PGCs is still elusive. This study determined the sex of collected samples by identifying specific sex markers via polymerase chain reaction (PCR) and fluorescence activated cell sorter (FACS). RNA-seq was utilized to investigate the transcriptomic profile of amphoteric ESCs and PGCs in chicken. The results showed no significant differentially expressed genes (DEGs) in amphoteric ESCs and 227 DEGs exhibited in amphoteric PGCs. Moreover, those 227 DEGs were mainly enriched in 17 gene ontology (GO) terms and 27 pathways according to Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Furthermore, qRT-PCR was performed to verify RNA-seq results, and the results demonstrated that Notch1 was highly expressed in male PGCs. In summary, our results provided a knowledge base of chicken amphoteric ESCs and PGCs, which is helpful for future research in relevant biological processes. [ABSTRACT FROM AUTHOR]

Simple Summary: The differentiation of germ cells plays an important role in sex differentiation in poultry. Therefore, it is necessary for us to explore the potential regulators in the process of germ cell development. In this study, RNA-seq was used to detect the expression profile of long non-coding RNAs (lncRNAs) in chicken embryonic stem cells (ESCs), primordial germ cells (PGCs) and spermatogonial stem cells (SSCs). The results showed that a total of 296, 280 and 357 differentially expressed lncRNAs (DELs) were screened in ESCs vs. PGCs, ESCs vs. SSCs and PGCs vs. SSCs, respectively. Functional analysis of the target genes of DELs showed that autophagy, Wnt/β-catenin, TGF-β, Notch and ErbB signaling pathways were involved in the differentiation process of male germ cells and, moreover, XLOC_612026, XLOC_612029, XLOC_240662, XLOC_362463, XLOC_023952, XLOC_674549, XLOC_160716, ALDBGALG0000001810, ALDBGALG0000002986, XLOC_657380674549, XLOC_022100 and XLOC_657380 were predicted to be the key lncRNAs in this process. Our findings could not only supply scientific data for constructing the gene regulatory network of germ cell development, but also provide new ideas for further optimizing the induction efficiency of germ cells in vitro. Germ cells have an irreplaceable role in transmitting genetic information from one generation to the next, and also play an important role in sex differentiation in poultry, while little is known about epigenetic factors that regulate germ cell differentiation. In this study, RNA-seq was used to detect the expression profiles of long non-coding RNAs (lncRNAs) during the differentiation of chicken embryonic stem cells (ESCs) into spermatogonial stem cells (SSCs). The results showed that a total of 296, 280 and 357 differentially expressed lncRNAs (DELs) were screened in ESCs vs. PGCs, ESCs vs. SSCs and PGCs vs. SSCs, respectively. Gene Ontology (GO) and KEGG enrichment analysis showed that DELs in the three cell groups were mainly enriched in autophagy, Wnt/β-catenin, TGF-β, Notch and ErbB and signaling pathways. The co-expression network of 37 candidate DELs and their target genes enriched in the biological function of germ cell development showed that XLOC_612026, XLOC_612029, XLOC_240662, XLOC_362463, XLOC_023952, XLOC_674549, XLOC_160716, ALDBGALG0000001810, ALDBGALG0000002986, XLOC_657380674549, XLOC_022100 and XLOC_657380 were the key lncRNAs in the process of male germ cell formation and, moreover, the function of these DELs may be related to the interaction of their target genes. Our findings preliminarily excavated the key lncRNAs and signaling pathways in the process of male chicken germ cell formation, which could be helpful to construct the gene regulatory network of germ cell development, and also provide new ideas for further optimizing the induction efficiency of germ cells in vitro. [ABSTRACT FROM AUTHOR]

To investigate the regulatory mechanism of the competing endogenous RNAs (ceRNAs) on the egg performance of Gaoyou ducks, full transcriptome sequencing was performed to analyze the ovarian tissues in Gaoyou ducks. The ducks were categorized into high- and low-yield groups based on the individual in-cage egg production records and the hematoxylin-eosin (HE) staining results. The differentially expressed genes (DEGs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) were further processed by GO (gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses. In total, 72 DEmRNAs; 23 DElncRNAs; 4 DEcircRNAs; and 5 signaling pathways, including the ovarian steroidogenesis, PI3K-Akt, hedgehog, tryptophan metabolism, and oocyte meiosis signaling pathways, were significantly enriched. These results suggest that they could be associated with the Gaoyou duck's ovarian function and affect the total egg production or double-yolked egg production. Furthermore, a coregulation network based on the related candidate ceRNAs across the high- and low-yield egg production groups was constructed. Our findings provide new insights into the mechanisms underlying the molecular regulation of related circRNA/lncRNA-miRNA-mRNA in the egg production and double-yolked egg traits of Gaoyou ducks.

This study was aimed at identifying the active control area of chicken DAZL gene core promoter, to screen optimum inducers of the DAZL gene, thus to enhance the differentiation of embryonic stem cells into spermatogonial stem cells. Fragments of chicken DAZL gene promoter were cloned into fluorescent reporter plasmids and transfected into DF-1 cells. Then Dual-Luciferase® Reporter Assay System was used to identify the activity of the DAZL gene under different inducers. Our studies showed that the DAZL core promoter region for the Suqin yellow chicken was -383 to -39 bp. The dual-luciferase® reporter showed that all-trans retinoic acid (ATRA), a retinoic acid receptor alpha agonist (tamibarotene/Am80), or estradiol (E2) could significantly enhance DAZL transcription. The in vitro inductive culture of chicken ESCs demonstrated that, with ATRA treatment, DAZL transcription peaked at 6 days and then decreased slowly; whereas, DAZL transcription was continuous and peaked at 10 days with Am80 treatment. E2 treatment significantly increased DAZL expression after 8 days. All three treatments were associated with the appearance of male germ cell (MGC)-like cells on day 10. These results provide the optimum inducer screening of the DAZL gene and lay the foundation for further screening of compounds that can induce the differentiation of ESCs into MGCs in vitro.